MODULATION OF B CELL ACTIVATION AND EFFECTOR FUNCTION BY THE ARYL
HYDROCARBON RECEPTOR IN THE RAT
By
Shawna D’Ingillo

A THESIS
Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of
Laboratory Research in Pharmacology and Toxicology - Master of Science
2013

ABSTRACT
MODULATION OF B CELL ACTIVATION AND EFFECTOR FUNCTION BY THE ARYL
HYDROCARBON RECEPTOR IN THE RAT
By
Shawna D’Ingillo
Suppression of the humoral immune response by 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) occurs through activation of the aryl hydrocarbon receptor (AHR) and the B cell
has been identified as one of the most sensitive targets to TCDD. Despite decades of
studies in multiple species the exact mechanisms by which TCDD suppresses B cell
activation and the primary IgM antibody response has yet to be elucidated.

The

magnitude of AHR regulation on B cell activation and effector function is also unknown.
It was hypothesized that the targeted deletion of the AHR results in the loss of
sensitivity of the B cell to suppression by TCDD and conversely in the augmentation of
the primary IgM antibody response, which is mediated in part, through a greater
magnitude in B cell activation in the absence of TCDD. The expression of surface
markers CD80, CD86, and MHC II signify early activation events in the B cell and can
be used to evaluate the magnitude of activation. Likewise, IgM secretion can be used to
evaluate the magnitude in B cell effector function. Using a rat AHR knockout model, in
vitro LPS activation of B cells resulted in higher expression of CD80, CD86 and MHC II
in AHR knockout compared to wild type rats. Similarly, AHR knockout rats had an
increased primary IgM antibody response with LPS activation.

TCDD did not

significantly alter CD80, CD86, and MHC II expression or the IgM antibody response of
AHR knockout and wild type rats. This study has demonstrated that AHR is partly
responsible for regulating the magnitude of B cell activation and effector function.

Copyright by
SHAWNA D’INGILLO
2013

TABLE OF CONTENTS
LIST OF FIGURES……………………………………………………………………………. iv
LIST OF ABBREVIATIONS...………………………………………………………………... vii
Introduction…………………………………………………………………………….......... 1
A. Organization of the Immune System………………………………………………... 1
B. The Adaptive Immune System………………………………………………………. 2
C. TCDD and the AHR Pathway………………………………………………………... 7
D. B cells as a Sensitive Target to TCDD Mediated Immunotoxicity……………….. 9
E. AHR plays a role in development and homeostasis………………………………. 15
Experimental Design………………………………………………………………………….. 20
A. Specific Aim 1…………………………………………………………………………. 20
B. Specific Aim 2…………………………………………………………………………. 21
Materials and Methods……………………………………………………………………….. 22
A. Animals…………………………………………………………………………………. 22
B. Optimization of B Cell Activation…………………………………………...……… 22
-/C. AHR and Wild Type Rat Dosing and Spleen Collection………………………… 23
D. Lymphocyte Isolation and Culture…………………………………………………… 24
E. Flow Cytometry Analysis……………………………………………………………... 25
F. IgM ELISA……………………………………………………………………………… 26
G. Statistical Analysis…………………………………………………………………….. 27
Experimental Results…………………………………………………………………………. 28
A. Optimization of Culture and Activation Conditions………………………………… 28
B. Evaluation of Rat B cell Activation by the Measurement of Activation Markers... 32
I. AHR Deletion Effects on the B cell Activation Marker CD80……………... 34
II. AHR Deletion effects on CD86 Expression………………………………… 43
III. Assessing the role AHR has on regulating MHC II Expression……….. 51
C. Modulation in IgM Primary Antibody Response………………………………… 60
Discussion……………………………………………………………………………………... 72
Conclusion………….………………………………………………………………………….. 87
APPENDIX………….…………………………………………………………………………. 90
BIBLOGRAPHY……………………………………………………………………………….. 91

iv

LIST OF FIGURES

Figure 1. Optimization of primary Sprague-Dawley rat B lymphocyte culture and
activation conditions. Splenocytes were isolated from one rat and
erythrocytes were either lysed with ACK buffer or allowed to remain in
culture (non-lysed) and activated with 15μg/mL or 60μg/mL of LPS or
15μg/mL or 60μg/mL of PWM. Supernatant were collected at 72 hours (A)
and 96 hours (B) and an ELISA was performed to detect levels of secreted
IgM. C depicts an ELISA at three days of culture with non-lysed primary rat
B cells treated with 10, 15, 30, or 60μg/mL LPS. Panels A and B are results
from one rat with each treatment group plated in duplicate. In C, data
presented is the mean ±SEM from each treatment plated in triplicate. * P
<0.05 and ** P <0.01 compared to naïve……………………..……………… 30

Figure 2. Schematic representation of the flow cytometry gating for the
-/identification of B cells within AHR
and wild type splenocyte
cultures. Samples were first gated on single cells noted as the singlet gate
to eliminate doublets which produce artifacts in the sample analysis. This is
done by sorting the cells by their size which is forward scatter-height (FSCH) and forward scatter-area (FSC-A). Taking the singlet gate, a second
gate was added to exclude the analysis of dead cells and include only the
live cells by the live/dead dye and side scatter-area (SSC-A) which
determines cellular granularity; this gate is noted as the viable gate. The
lymphocytes were then identified in the viable gate by their size (FSC-A)
and granularity (SSC-A). The B cells were then identified from the
lymphocyte gate by the presence of intracellular IgM. The gating scheme
was applied to all groups analyzed by flow cytometry. “For interpretation of
the references to color in this and all other figures, the reader is referred to
the electronic version of this thesis.”………………………………………...... 33
+

Figure 3. Targeted deletion of AHR leads to a higher percentage of CD80 B cells
upon LPS activation compared to wild type in group one. Vehicle
control cells in group one were evaluated by flow cytometry for the basal
-/expression of CD80 in wild type and AHR B cells. Cells were also treated
with 15μg/mL LPS to activate B cells inducing an upregulation of CD80
expression. Comparisons were made between transformed percent positive
-/data for non-activated and LPS-activated wild type and AHR B cells. Data
is presented as the mean ± SEM from five rats per treatment group. *** P ≤
0.001 compared to non-activated within the genotype. C indicates P ≤
0.001 compared to LPS-activated wild type cells....………….…………...…35

v

+

Figure 4. LPS activation increases the percentage of CD80 B cells and the
deletion of AHR leads to additional increases in CD80 expression in
group two. Vehicle control sets in group two were evaluated by flow
-/cytometry for the basal expression of CD80 in wild type and AHR B cells.
Cells were also treated with 15μg/mL LPS to activate B cells inducing an
upregulation of CD80 expression. Comparisons were made between
transformed percent positive data for non-activated and activated wild type
-/and AHR B cells. Data is presented as the mean ± SEM from five rats per
treatment group. * P ≤ 0.05 compared to non-activated within the
genotype………………………………………………………………………..... 36
+

Figure 5. Deletion of AHR produces a higher percentage of CD80 B cells
compared to wild type with no significant TCDD suppression observed
in either genotype. Data presented are from group one. Wild type and
-/AHR rats were dosed with corn oil vehicle (VH) control and varying
concentrations of TCDD. Splenocytes from these rats were activated with
15μg/mL LPS for 72 hours and compared to a non-activated control.
+
-/Percentage of CD80 B cells in wild type (A) and AHR (B) were measured
+

-/-

by flow cytometry. The MFI of CD80 B cells in wild type (C) and AHR (D)
were obtained in the same flow cytometry analysis. Data is presented as the
mean ± SEM from five rats per treatment. ** P ≤0.01 and *** P ≤ 0.001
compared to non-activated B cells within the genotype. a indicates p ≤ 0.05
and b indicates P ≤ 0.01 compared to VH control within the genotype……38

Figure 6. TCDD was unable to suppress the upregulation in the percentage of
+
-/CD80 B cells in wild type and AHR rat B cells upon LPS activation.
-/-

Data presented are from group two. Wild type and AHR rats were dosed
with corn oil vehicle (VH) control and varying concentrations of TCDD and
splenocytes from these rats were treated with 15μg/mL LPS for 72 hours
+
and compared to a non-activated control. Percentage of CD80 B cells in
wild type (A) and or AHR
+

-/-

(B) were measured by flow cytometry. The MFI
-/-

of CD80 B cells in wild type (C) and or AHR (D) were obtained in the
same flow cytometry analysis. Data is presented as the mean ± SEM from
five rats per treatment group. * P ≤ 0.05 compared to non-activated B cells
within the genotype………………………………………………………………41

vi

+

Figure 7. Targeted deletion of AHR in rats leads to higher percentage of CD86 B
cells upon LPS activation compared to wild type in group one. Vehicle
treatment control in group one was evaluated by flow cytometry for the
-/basal expression of CD86 in wild type and AHR B cells. Cells were also
treated with 15μg/mL LPS to activate B cells inducing an upregulation of
CD86 expression. Comparisons were made between transformed percent
-/positive data for non-activated and activated wild type and AHR B cells.
Data is presented as the mean ± SEM from five rats per treatment group. ***
P ≤ 0.001 compared to non-activated within the genotype. a indicates P ≤
0.05 compared to LPS-activated wild type B cells…………………………...44
+

Figure 8. Activation with LPS leads to higher percentage of CD86 B cells but the
deletion of AHR in rats does not produce a further increase in CD86
expression of group two. Vehicle control treatment of group two was
evaluated by flow cytometry for the basal expression of CD86 in wild type
-/and AHR B cells. Cells were also treated with 15μg/mL LPS to activate B
cells inducing an upregulation of CD86 expression. Comparisons were
made between transformed percent positive data for non-activated and
-/LPS-activated wild type and AHR B cells. Data is presented as the mean
± SEM from five rats per treatment group. *** P ≤ 0.001 compared to nonactivated within the genotype..…………………………………………………45
-/-

Figure 9. LPS activation upregulates CD86 expression in wild type and AHR rat
B cells similarly and no significant dose dependent TCDD-mediated
suppression of CD86 was observed. Data presented are from group one.
-/Wild type and AHR rats were dosed with corn oil vehicle (VH) control and
varying concentrations of TCDD and splenocytes from these rats were
treated with 15μg/mL LPS for 72 hours and compared to a non-activated
+
-/control. Percentage of CD86 B cells in wild type (A) and AHR (B) were
+

measured by flow cytometry. The MFI of CD86 B cells in wild type (C) and
-/-

AHR (D) were obtained in the same flow cytometry analysis. Data is
presented as mean ± SEM from five rats per treatment group. *** P ≤ 0.001
compared to non-activated B cells within the genotype. a indicates P ≤ 0.05
and c indicates P ≤ 0.001 in comparison to vehicle control within the
genotype…………………………………….…………………………………… 46

vii

-/-

Figure 10. LPS activation upregulates CD86 expression in wild type and AHR rat
B cells similarly and no significant TCDD-mediated suppression of
CD86 was observed. Data presented are from group two. Wild type and
-/AHR rats were dosed with corn oil vehicle (VH) control and varying
concentrations of TCDD and splenocytes from these rats were treated with
15μg/mL LPS for 72 hours and compared to a non-activated control.
+
-/Percentage of CD86 B cells in wild type (A) and AHR (B) were measured
+

-/-

by flow cytometry. The MFI of CD86 B cells in wild type (C) and AHR (D)
were obtained in the same flow cytometry analysis. Data is presented as the
mean ± SEM from five rats per treatment group. *** P ≤ 0.001 compared to
non-activated B cells within the genotype…………………………………..... 49
+

Figure 11. LPS activation results in a down regulation of MHC II expression in
-/-

AHR B cells compared to LPS-activated wild type B cells. Vehicle
control in group one was evaluated by flow cytometry for the basal
-/expression of MHC II in wild type and AHR B cells. Cells were also
treated with 15μg/mL LPS to activate B cells to induce an upregulation of
MHCII expression. Comparisons were made between transformed percent
-/positive data for non-activated and LPS-activated wild type and AHR B
cells. Data is presented as the mean ± SEM from five rats per treatment
group. *** P ≤ 0.001 compared to non-activated within the genotype. a
indicates P ≤ 0.05 compared to LPS-activated wild type splenocytes……..52
-/-

Figure 12. Non-activated AHR rat B cells have a lower basal expression of MHC
II while the level of MHC II upon LPS activation is similar to LPSactivated wild type B cells. Vehicle control in group two was evaluated by
-/flow cytometry for the basal expression of MHC II in wild type and AHR B
cells. Cells were also treated with 15μg/mL LPS to activate B cells to
induce an upregulation of MHC II expression. Comparisons were made
between transformed percent positive data for non-activated and LPS-/activated wild type and AHR B cells. Data is presented as the mean ±
SEM from five rats per treatment group. *** P ≤ 0.001 compared to nonactivated within the genotype. a indicates P ≤ 0.05 compared to nonactivated wild type splenocytes………………………………………………...53

viii

Figure 13. Targeted deletion of AHR in rats leads to the down regulation of MHC II
when activated with LPS and TCDD produces opposing trends in the
-/percentage of MHC II of wild type and AHR rats. Data presented are
-/-

from group one. Wild type and AHR rats were dosed with corn oil vehicle
(VH) control and varying concentrations of TCDD and splenocytes from
these rats were treated with 15μg/mL LPS for 72 hours and compared to a
+
non-activated control. Percentage of MHC II B cells in wild type (A) and
AHR

-/-

+

(B) were measured by flow cytometry. The MFI of MHC II B cells in
-/-

wild type (C) and AHR (D) were obtained in the same flow cytometry
analysis. Data is presented as the mean ± SEM from five rats per treatment
group. * P ≤0.05, ** P ≤ 0.01, and *** P ≤ 0.001 compared to non-activated
B cells within the genotype. a indicates P ≤ 0.05 compared to vehicle control
within its respective genotype and activation set.……………………………56

Figure 14. Targeted deletion of AHR in rats leads to the down regulation of MHC
II when activated with LPS. Data presented are from group two. Wild
-/Type and AHR rats dosed with varying concentrations of TCDD and
splenocytes from these rats were activated with 15μg/mL LPS for 72 hours
+
and compared to a non-activated control. Percentage of MHC II B cells
-/-

in wild type (A) and AHR
+

(B) were measured by flow cytometry. The MFI
-/-

of MHC II B cells in wild type (C) and AHR (D) were obtained in the
same flow cytometry analysis. Data is presented as the mean ± SEM from
five rats per treatment group. *** P ≤ 0.001 compared to non-activated B
cells within the genotype. a indicates P ≤ 0.05 compared to vehicle control
within its respective genotype and activation set………………….............58
-/-

Figure 15. Activated AHR B cells upregulate intracellular IgM over activated wild
type B cells. Corn oil vehicle control in group one was evaluated by flow
cytometry for the basal expression of intracellular IgM in wild type and
-/AHR B cells. Comparisons were made between non-activated control and
splenocytes activated with 15μg/mL LPS which induces an IgM antibody
+
response. Data is presented as the mean percentage of IgM high B cells
from five rats per treatment…………………………………………………….. 61

ix

Figure 16. Targeted deletion of AHR in rat B cells results in an enhanced
response of intracellular IgM over activated wild type B cells. Corn oil
vehicle control in group one was evaluated by flow cytometry for the basal
-/expression of intracellular IgM in wild type and AHR B cells. Comparisons
were made between non-activated control and splenocytes activated with
15μg/mL LPS which induces an IgM antibody response. Data is presented
+
as the mean percentage of IgM high B cells from five rats per treatment..62

Figure 17. Targeted deletion of AHR results in an increase in the percentage of
+
intracellular IgM high B cells. Data presented are from group one. Wild
-/-

type and AHR rats were dosed with corn oil vehicle (VH) control and
varying concentrations of TCDD and splenocytes from these rats were
treated with 15μg/mL LPS for 72 hours and compared to a non-activated
+
control. Percentage of intracellular IgM high B cells in wild type (A) and
-/-

AHR (B) were measured by flow cytometry. Data is presented as the
mean ± SEM from five rats per treatment group. * P ≤0.05 and *** P ≤ 0.001
compared to non-activated B cells within the genotype…….……………….64

Figure 18. Targeted deletion of AHR produces an upregulation of intracellular
+
IgM high B cells and TCDD is unable to suppress this upregulation.
-/-

Data presented are from group two. Wild type and AHR rats were
dosed with corn oil vehicle (VH) control and varying concentrations of
TCDD and splenocytes from these rats were treated with 15μg/mL LPS for
72 hours and compared to a non-activated control. Percentage of
+
-/intracellular IgM high B cells in wild type (A) and AHR (B) were
measured by flow cytometry. Data is presented as the mean ± SEM from
five rats per treatment group. ** P ≤0.01 and *** P ≤ 0.001 compared to
non-activated B cells within the genotype…………………………………..65

x

-/-

Figure 19. LPS-activated AHR rat B cells secrete higher quantity of IgM over
their wild type counterparts with no TCDD-mediated suppression of
the antibody response. Data presented are from group one. Wild type and
-/AHR rats were dosed with corn oil vehicle (VH) control and varying
concentrations of TCDD and splenocytes from these rats were treated with
15μg/mL LPS for 72 hours and compared to a non-activated control.
-/Quantity of secreted IgM measured in ng/mL from wild type (A) and AHR
(B) activated B cells were measured by ELISA. Panel C compares the basal
-/secretion of IgM in vehicle treated wild type and AHR B cells obtained in
the same ELISA assay. Data is presented as the mean ± SEM from five rats
per treatment group. * P ≤ 0.05 and *** P ≤ 0.05 compared to non-activated
B cells within the genotype. a in panel B indicates P ≤ 0.05 compared to
vehicle control in its respective genotype and activation set. a in panel C
indicates P ≤ 0.05 compared to LPS-activated wild type……………………67

Figure 20. The targeted deletion of AHR leads to a greater quantity of IgM
secretion in LPS-activated B cells. Data presented are from group two.
-/Wild type and AHR rats were dosed with corn oil vehicle (VH) control and
varying concentrations of TCDD and splenocytes from these rats were
treated with 15μg/mL LPS for 72 hours and compared to a non-activated
control. Quantity of secreted IgM measured in ng/mL from wild type (A) and
-/AHR (B) activated B cells were measured by ELISA. Panel C compares
-/-

the basal secretion of IgM in vehicle treated wild type and AHR B cells
obtained in the same ELISA assay. Data is presented as the mean ± SEM
from five rats per treatment group. * P ≤ 0.05 and *** P ≤ 0.001 compared to
non-activated B cells within the genotype. In panel C, a indicates P ≤ 0.05
compared to LPS-activated wild type………………………………………….69

xi

LIST OF ABBREVIATIONS
ACK

Ammonium chloride potassium

AHR

Aryl hydrocarbon receptor

AP-1

Activating protein 1

APC

Antigen presenting cell

ARA9

Aryl hydrocarbon associated protein 9

ARNT

Aryl hydrocarbon receptor nuclear translocator

Bach2

BTB and CNC homology 2

BCL-6

B cell lymphoma 6

BCR

B cell receptor

bHLH

Basic helix loop helix

BLIMP-1

B lymphocyte maturation protein 1

BSA

Bovine serum albumin

CD

Cluster of differentiation

DLC

Dioxin like compound

DMSO

Dimethyl sulfoxide

DRE

Dioxin response element

ELISA

Enzyme linked immunoabsorbent assay

ERK

Extracellular signal regulated kinase

FACS

Fluorescence-activated cell sorting

Hsp90

Heat shock protein 90

xii

Ig

Immunoglobulin

JNK

Jun amino terminal kinase

LPS

Lipopolysaccharide

MAPK

Mitogen-activated protein kinase

MHC II

Major histocompatibility complex II

MyD88

Myeloid differentiation primary response protein 88

NF-κB

Nuclear factor kappa B

PAMPs

Pattern-associated molecular patterns

Pax5

Paired box 5

PBS

Phosphate buffered saline

PWM

Pokeweed mitogen

ROS

Reactive oxygen species

sRBC

Sheep red blood cell

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

TH cell

T helper cell

TLR

Toll-like receptor

XPB-1

X box paired protein 1

xiii

Introduction

A. Organization of the Immune System
The immune system plays a critical physiological role by protecting an organism
against disease-causing pathogens through highly complex and regulated processes.
Defense against an invading pathogen occurs through innate and adaptive divisions of
the immune system. The innate immune system is the first to respond to a pathogen
and does so in a non-specific manner through activation of the complement system,
inflammation, mucosal barriers, phagocytosis by macrophages and direct killing of the
pathogen by natural killer cells. This system is effective in the protection against an
invading pathogen but it is unable to adapt to the changing environment in which the
host resides. The adaptive immune system complements the innate immune system in
that it is able to adapt according to the pathogens present in the host’s external
environment and produce specific responses to these pathogens. In addition to highly
specific responses, the adaptive immune system elicits memory against the pathogen
so that subsequent responses will be faster and more effective.
The adaptive immune system can be further divided into cell-mediated and
humoral immunity. Generally speaking, cell-mediated immunity is mediated by T cells,
macrophages, and natural killer cells and protects the organism against cancer, viral
and bacterial diseases. Humoral immunity is mediated by B cells and protects the
organism through the production of immunoglobulins (Ig) also called antibodies, which
neutralize, destroy and activate the complement component of the innate immune
system. The cell mediated and humoral immune response are antigen specific and

1

several cell types such as antigen presenting cells (APCs), T-helper cells (TH cells), and
B cells are responsible for coordinating their effector functions for antigen destruction.
Macrophages and dendritic cells are APCs that scavenge their environment and upon
encountering an antigen, present it to T cells. If the T cell recognizes the antigen as
foreign, then they will either become activated into an effector T cell and secrete
cytokines or differentiate into a memory T cell that will respond to the same antigen
again upon subsequent exposure.

B cells recognize antigens in their environment

through the B cell receptor (BCR) which is a membrane bound form of antibody. Once
bound to the BCR, the antigen is internalized into endosomes that fuse with an acidic
lysosomze that contain proteases to degrade the antigen into peptide fragments. A
specialized presenting protein, major histocompatibility complex class II (MHC II) is then
transferred from the endoplasmic reticulum into the peptide fragment containing
endosome where the MHC II molecule is loaded with an antigen peptide fragment. This
peptide bound MHC II molecule then travels to the cell surface to present the B cell
recognized antigen to TH cells initiating their activation. This TH cell activation leads to
cytokine secretion initiating B cell survival and proliferation. It is the balance of these
several cell types that allows for specific and swift responses to antigens and any
imbalances in the interactions of these cells can lead to autoimmune diseases.

B. The Adaptive Immune System
The cells of the adaptive immune response arise from a common
lymphoid progenitor cell in the bone marrow, which have developed from hematopoietic
stem cells. It is the tight contact between the stromal cells of the bone marrow and the
2

lymphoid progenitors that gradually commit the cells to either the myeloid or lymphoid
development pathway. Cells committed to the myeloid pathway become macrophages,
granulocytes, mast cells, and denditric cells whereas cells committed to the lymphoid
pathway become T or B cells. The coordinated physical interaction between lymphoid
progenitors and stromal cells as well as the timed release or temporal kinetics of
cytokines and chemokines leads to the development of lymphocytes from hematopoietic
stem cells and gives rise to immature B cells. Next, the production of a functional BCR
by an immature B cell is a critical step in the development of a mature B cell. Before
migrating to secondary lymphoid tissue to mature, the BCR is tested for autoreactivity to
antigens recognized as self. If the BCR is found to be autoreactive it can be amended
by one of three different manners. The autoreactive B cells can undergo apoptosis by
clonal deletion, the BCR can be edited so it no longer recognizes self antigens or, the
BCR become anergic or non-responsive against self antigens (Hardy and Hayakawa,
2001). Once a functional BCR is in place, the immature B cells can then migrate from
the bone marrow to the spleen for maturation into a naïve B cell.
As mentioned humoral immunity is specific in its response and the recognition of
a specific antigen by a B cell is based on the structure of the antibody. Antibodies
consist as a heterodimer of two heavy chains linked by disulfide bonds to two light
chains and each of these chains contains a variable and a constant region. It is the
variable region of both chains that allow for the specificity in antigen binding. Antigen
specificity is made possible through a diversification processes known as VDJ
recombination which genetically recombines heavy and light chain genes resulting in
several isotypes of both chains. The light chain constant regions are homodimers of

3

either Ig kappa (Igκ) or Ig lambda (Igλ) isotypes where the heavy chain constant regions
are homodimers of either μ, δ, γ, α, ε isotypes. The heavy chain isotypes designate the
five classes of antibodies with μ gene producing IgM, δ producing IgD, γ producing IgG,
α producing IgA, and ε producing IgE. The first antibody to be produced in the primary
humoral immune response is IgM.

This antibody has a low binding affinity but is

secreted as a pentamer leading to a high avidity upon binding to antigens.

It is

predominately found in the bloodstream and lymph and is effective in activating the
complement system. IgD is found co-expressed with IgM and signals B cells to exit the
bone marrow upon maturation. IgA exists as either a monomer or dimer in epithelial
surfaces where complement and phagocytes are not present to neutralize antigens. IgE
only exists as a monomer and has high avidity for receptors on mast cells and functions
to mediate allergic and hypersensitivity responses. IgG is the only antibody that is able
to cross the placental barrier and is mostly found as a monomer. It primarily functions in
secondary immune responses but can also activate the complement system and other
immune cells. The multimerization of IgM and IgA is mediated through the polypeptide
Ig J-chain (IgJ). As mentioned before, the BCR is a membrane bound form of antibody
and each of these Ig isotypes can exist as a BCR. It is the combination of the highly
diverse light and heavy chains that give such a large gamut of structurally different Igs,
allowing for B cells to recognize and bind to such a wide diversity of antigens.
Before a B cell is able to differentiate into an antibody secreting plasma cell it
must first become activated. B cell activation can occur in two ways, either in a T celldependent or T cell-independent manner.

The focus of this thesis is on TH cell-

independent B cell activation and hence TH cell-dependent B cell activation will not be

4

discussed in detail. T cell-dependent B cell activation requires two signals, one signal
through engagement of the BCR and the second signal from CD40-CD40 ligand
interactions with cognate T cells. Many antigens that activate B cells in a TH cell
independent manner do so via the activation of toll-like receptors (TLRs) and crosslinking of several BCRs. Pokeweed mitogen (PWM) is a lectin derived from the
Phytolacca americana plant and activates B cells through cross-linking several BCRs
leading to the proliferation and differentiation into IgM antibody secreting plasma cells
(Bekeredjian-Ding et al., 2012).

TLRs recognize pathogen-associated molecular

patterns (PAMPs) in the outer membrane of gram-negative and gram-positive bacteria,
viruses, and fungi. Each TLR recognizes a specific PAMP molecule, for example TLR7
and 8 respond to single stranded RNA of viruses, TLR9 responds to unmethylated CpG
DNA,

TLR1

and

TLR2

respond

to

lipopeptides

in

bacterial

membranes.

Lipopolysaccharide is a component in the outer cell membrane of gram negative
bacteria and is recognized by TLR4. Stimulation of TLR4 in B cells leads to polyclonal
activation and the production of a primary IgM antibody response (Iwasaki and
Medzhitov, 2004). In both cases of LPS and PWM TH cell-independent B cell activation,
nuclear factor kappa B (NF-κB) and activating protein-1 (AP-1) signaling cascades
become activated and are integrated and

converge onto mitogen-activated protein

kinases (MAPK), extracellular signal regulated kinase (ERK), and Jun amino terminal
kinase (JNK) through the adaptor protein myeloid differentiation primary response
protein 88 (MyD88) (Iwasaki and Medzhitov, 2004; Jilling et al., 2006; Shapiro-Shelef
and Calame, 2005). The crosstalk between these pathways leads to an activated B
cell phenotype that can be characterized by the upregulation of cell surface proteins

5

Cluster of Differentiation 80 (CD80, B7.1), CD86 (B7.2), and MHC II (Kaminski et al.,
2012; Rodo et al., 2006). CD80, CD86, and MHCII surface proteins are B cell activation
markers and can be used to identify B cells that have become activated in response to a
stimulus.
Once activated, a B cell can differentiate into an antibody secreting plasma cell;
this process is tightly controlled by a bistable regulatory network of transcription factors
including B-cell lymphoma 6 (BCL-6), paired box 5 (Pax5), B lymphocyte induced
maturation protein-1 (BLIMP-1), and X-box binding protein 1 (XBP-1), and BTB and
CNC homology 2 (Bach2) (Bhattacharya et al., 2010; Calame, 2001; Sciammas et al.,
2011; Shapiro-Shelef and Calame, 2005; Zhang et al., 2010). Pax5 is required for the
commitment of a lymphoid progenitor to the B cell lineage during development as well
as positively regulating the expression of BCL-6 and Bach2 (Sciammas et al., 2011). In
addition, Pax5 also regulates the expression of XBP-1 which is required for MHC II
transcription (Ono et al., 1991; Reimold et al., 2001). The maintenance of a resting
naïve B cell phenotype is due to the repression of BLIMP-1 by Pax5 and BCL-6. Upon
activation by LPS, AP-1 can act upon this bistable switch to induce an all-or-none
response and induce the expression of BLIMP-1 (Zhang et al., 2013).

BLIMP-1 is

considered the master regulator in B cell differentiation and its expression leads to a
feed forward loop to suppress Pax5 and BCL-6 (Fairfax et al., 2008; Lin et al., 2002;
Shaffer et al., 2002). It is the rise in the transcriptional repressor BLIMP-1 and XBP-1
and fall in BCL-6 and Pax5 that ultimately lead to the terminal differentiation of B cells
into plasma cells.

6

C. TCDD and the AHR Pathway
Halogenated aromatic hydrocarbons and halogenated polycyclic aromatic
hydrocarbons are structurally related compounds comprised of dioxin and dioxin like
compounds (DLCs) (Poland and Knutson, 1982). They are ubiquitous and persistent
environmental contaminants produced as byproducts from the manufacturing of
herbicides, combustion of chlorinated materials, chlorinated bleaching of paper pulp
during paper manufacturing. Although to a lesser extent they are also found naturally as
a result of natural combustion such as volcanic eruptions and forest fires. Dioxins and
DLCs are highly lipophilic compounds and the greatest route of human exposure is diet
due to bioaccumulation in the food chain leading to lifelong exposures (El-Shahawi et
al., 2010; White and Birnbaum, 2009). The most well studied and potent dioxin is 2, 3,
7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) and it has been shown to elicit toxic effects in
several species. The health effects of TCDD exposure seen in several laboratory
species include metabolic perturbations, thymic involution, endocrine disruption, liver
and stomach hyperplasia, hepatic lesions, carcinogenesis, and respiratory irritation
(Rysavy et al., 2013; Safe, 1986). Many of these toxic effects of TCDD can also be seen
in humans including carcinogenesis, endocrine disruption, liver damage, and immune
suppression, but the most common human health effect is chloracne characterized by
the hyperkeratinization of the skin with dark cysts (Marinkovic et al., 2010).
Despite common species effects there is marked inter-species and inter-strain
variability in the response to TCDD exposure. The LD50 is an average lethal dose
required to kill 50% of a tested population and is often used as a measurement of acute
toxicity. It was found that the LD50 of TCDD has a broad range across species and can
7

vary from 1 μg/kg in guinea pigs (found to be the most sensitive) to 5mg/kg in hamsters
(found to be the most resistant) (Mcconnell, 1985).

Inter-strain differences in rat

species have also been found. The Long-Evans rat are one of the more sensitive strains
to TCDD with an LD50 of 10-20μg/kg and the Han/Wistar rat are exceedingly resistant to
TCDD as the LD50 is higher than 9600μg/kg (Tuomisto et al., 1999).The inter-species
and intra-species specific differences in the effects of TCDD has been attributed to a
difference in ligand affinity resulting from allelic differences in a genetic locus identified
as the Ah locus which encodes the aryl hydrocarbon receptor (AHR) (Poland and
Glover, 1980; Poland and Glover, 1974; Poland et al., 1994; Quintana and Sherr, 2013).
The toxicity seen upon exposure to TCDD are thought to be mediated via the AHR
(Rowlands and Gustafsson, 1997).
The AHR has been identified as a ligand activated transcription factor in the PerArnt-Sim (PAS) family containing a basic helix loop helix domain (bHLH) (Stevens et al.,
2009). The PAS domain in AHR contains two domains denoted as A and B and the
ligand binding activity of AHR is found in the PAS B domain (Hao and Whitelaw, 2013).
The PAS domain also allows for dimerization with the Aryl Hydrocarbon Receptor
Nuclear Translocator (ARNT) while the bHLH domain is responsible for DNA binding
and dimerization with its associated chaperone proteins (Stevens et al., 2009). In the
absence of bound ligand, AHR is found in the cytosol as a complex with two heat shock
protein 90 (HSP90), p23, aryl hydrocarbon associated protein 9 (ARA9), or AHR
Interacting Protein (AIP). It has been suggested that the role of ARA9 and HSP90 is to
maintain a proper folding configuration of AHR to enable binding as well as preventing
inappropriate nuclear translocation, while the role of p23 is to maintain the association
8

of the AHR-HSP90 complex (Stevens et al., 2009).

Once ligand activated, AHR

translocates to the nucleus where it dissociates from its cytosolic chaperone proteins
and dimerizes with ARNT forming a heterodimer that acts as a transcription factor. The
AHR/ARNT complex then binds to regulatory DNA sequences containing dioxin
response elements (DRE) leading to alterations in gene expression. In addition to being
a ligand activated transcription factor, the AHR has also been shown to function as an
E3 ubiquitin ligase for selective protein degradation (Nguyen et al., 2013). The most
well characterized alteration in gene expression is increased expression of phase I and
phase II xenobiotic metabolism enzymes such as the cytochrome P450s (CYP)
specifically CYP1A1, CYP1A2, and CYP1B1, gluthatione-S-transferase A2 (GSTA2),
and UDP-glucoronosyltranferases 1A1 and 1A6 (UGT1A1, UGT1A6) (Abel and
Haarmann-Stemmann, 2010). These AHR responsive genes have been characterized
as the AHR gene battery and CYP1A1 and CYP1B1 are often used as an indirect
biochemical endpoint of TCDD exposure (Hu et al., 2007; Kerkvliet, 2002).

D. B cells as a Sensitive Target to TCDD Mediated Immunotoxicity
Most of the toxic effects of TCDD are thought to be mediated by AHR and immune
suppression is one of the most sensitive end points of TCDD exposure in mouse and
human (Holsapple et al., 1991; Sulentic and Kaminski, 2011). One early study to
demonstrate the humoral immune system as a sensitive target to TCDD-mediated
immunosuppression was conducted by the Holsapple group. In this in vivo mouse
study, TCDD elicited a concentration-dependent suppression of the primary IgM
responses to sheep red blood cells (sRBC), LPS, and DNP-ficoll (Holsapple et al.,

9

1986). Additional mouse in vitro studies using separation and reconstitution techniques
showed that only cultures containing B cells from TCDD-dosed mice had a significant
suppression of the primary IgM antibody response (Dooley and Holsapple, 1988; Morris
and Holsapple, 1991). Another in vitro study also supports evidence that IgM secretion
from LPS-activated B cells is suppressed upon TCDD exposure in humans (Zhang et
al., 2010) In addition to these studies, several reports from our laboratory have also
suggested that B cells are one of the sensitive targets of TCDD-mediated
immunosuppression with respect to the primary IgM response. As mentioned, μ gene
expression produces the IgM antibody and in vitro TCDD treatment suppressed μ gene
expression in a concentration-dependent manner, while TCDD congeners of lower
affinity for AHR were unable to downregulate this expression, suggesting that the affinity
for AHR also affects the primary antibody response (Sulentic et al., 2000). Most studies
describing the effect of TCDD on the IgM response have been performed in mouse
models. However, studies conducted with purified human B cells have also shown that
TCDD-mediated IgM suppression occurs in a concentration-dependent manner and the
magnitude of this response is consistent with the effect seen in the mouse model (Lu et
al., 2010).
Additional evidence has also supported the notion that AHR is required for TCDD
mediated immunosuppression. In the absence of an exogenous ligand, activation of
primary lymphocytes increased expression of AHR, enhanced AHR-ARNT DNA binding,
and induced CYP1A1 expression (Crawford et al., 1997).

These effects are all

hallmarks of TCDD induced AHR activation and also explains the sensitivity of B cells to
TCDD. Few endogenous AHR ligands have been discovered which include bilirubin,

10

bilivirdin, lipoxin A4, and several tryptophan metabolites such as 6-formylindolo[3,2b]carbazole (FICZ) and 2-1(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid (ITE)
(Fujii-Kuriyama and Mimura, 2005; Wincent et al., 2009; Yoshida et al., 2012). It was
found that the endogenous AHR agonist ITE suppressed the primary antibody response
in purified mouse activated B cells (Yoshida et al., 2012).

In addition to the ITE-

mediated suppression of the antibody response, the mRNA levels of CYP1A1 were
found to be elevated in ITE treated purified mouse B cells. The mouse B cell lymphoma
cell lines CH12.LX and BCL-1 have been demonstrated to be a useful in vitro model to
elucidate the mechanism of TCDD mediated immunosuppressive effects as these cells
could be activated with LPS into IgM secreting plasma cells. They also allow for
determining the role AHR has in modulating the IgM antibody response as the CH12.LX
expresses AHR where as the BCL-1 cell line lacks AHR. Using these two cell lines, it
was found that LPS is able to induce a primary IgM response in both CH12.LX and
BCL-1 cells but treatment with TCDD only induced a concentration-dependent
suppression in the IgM response of CH12.LX cells (Sulentic et al., 1998). An AHR
murine knockout model supports this finding as the primary antibody response in AHR
-/-

knockout (AHR ) mice remained unaffected with TCDD treatment where as wild type
mice with the same genetic background demonstrated supression (Vorderstrasse et al.,
2001). These two pieces of evidence suggest that AHR is required for eliciting the toxic
effects of TCDD and may play a role in modulating the IgM antibody response.
Several studies have also identified that a window of sensitivity exists for the
humoral immune system suppression by TCDD. Time of addition studies conducted in
vivo showed that TCDD was unable to suppress the IgM response if added 3 hours
11

post LPS activation (Holsapple et al., 1986), and 24 hours post-sRBC stimulation
(Tucker et al., 1986). This window of sensitivity was also shown in vitro in CH12.LX
cells, where TCDD added 24 hours post-LPS activation was unable to elicit suppressive
effects on the IgM antibody response (Crawford et al., 2003). Based on the
establishment of a window of sensitivity it is likely that in addition to the alteration in
effector function, TCDD also alters early activation signaling events in the B cell.
B cell differentation into a plasma cell is partly dependent on a bistable
transcription factor network and much of the early focus was on elucidating mechanisms
by which TCDD mediated immunosuppression was dependent on gene network
expression modifications. Pax5 is responsible for the maintenance of the resting naïve
B cell and acts as a transcriptional repressor on BLIMP-1 (Yasuda et al., 2012). From
mouse in vitro and in vivo studies it has been determined that TCDD mediates
concentration- and dose-dependent increases in mRNA and protein expression of Pax5
(North et al., 2009; Schneider et al., 2008; Yoo et al., 2004). The TCDD-mediated
increase in Pax5 expression and its inverse relationship with BLIMP-1 led to additional
studies using CH12.LX cells and primary mouse splenocytes to evaluate the effects of
TCDD on BLIMP-1 and its target transcription factors. It was found that TCDD
suppressed mRNA and protein expression of BLIMP-1 in LPS-activated B cells (North
et al., 2009; Schneider et al., 2009). The study conducted by Schneider et al., also
determined that TCDD reduced BLIMP-1 binding to the promoter region of the Pax5
gene.

These studies along with others have also detected a reduction in AP-1

expression upon exposure of TCDD in LPS-activated B cells (Suh et al., 2002). Similar
to Pax5, LPS-activated CH12.LX cells showed TCDD elicited concentration-dependent

12

increases in Bach2 (De Abrew et al., 2011). With the recent advances in stochastic
modeling, it was hypothesized that these gene expression changes occur in an all-ornone fashion and modeling has predicted that TCDD induced modifications in AP-1 and
Bach2 expression can result from an all or none mode of suppression (Zhang et al.,
2013) .
Despite the alterations in the B cell differentiation regulatory gene network, the
mechanisms by which TCDD can affect the early events in B cell activation have yet to
be elucidated. The relationship between the key transcription factors AP-1 and BCL-6
that mediate B cell differentiation and upstream targets are currently being evaluated to
help link pertubations in the activation of early signaling events and pertubations in the
primary antibody response upon TCDD exposure. LPS activation of B cells is mediated
by TLR4 and the receptor signaling

converges on the JNK and ERK signalling

cascades. JNK has been shown to phosphorylate c-Jun which is a component of AP-1,
thereby inducing the transcription of AP-1 (Smeal et al., 1991). ERK has been shown to
regulate B cell differentiation by phosphorylating BCL-6 targeting it for proteosomal
degradation thus lifting its repression on AP-1 and BLIMP-1 (Niu et al., 1998; Yasuda et
al., 2011). TCDD has been shown to inhibit the phosphorylation of ERK and JNK in both
mouse and human in vitro studies suggesting that ERK and JNK activation was
inhibited (Lu et al., 2011; North et al., 2010). This bottom up approach is additional
evidence that TCDD plays a role in mediating alterations of the early events that occur
in B cell activation in addition to perturbing effector function.
Most of the knowledge of TCDD-mediated immunosuppression have been
obtained from studies that utilize mouse and human models but some studies have also

13

been conduced in several rat strains with similar findings to the mouse and human of
TCDD-mediated suppression of the primary antibody response. A study performed in
mouse and Fischer 344 rats observed that TCDD suppressed the primary antibody
response to TNP-LPS in both species (Smialowicz et al., 1996). While this study found
TCDD could mediate suppression of the antibody response in rats, it was noted that the
dose required to induce the immunosuppressive effects in the rat (30μg/kg TCDD) was
higher than the dose necessary to induce immunosupression in the mouse (10μg/kg
TCDD), suggesting that the mouse is more sensitive than the rat to TCDD. In female
Brown Norway rats, serum IgE and IgG in response to house dust mite allergens was
decreased upon exposure to TCDD (Luebke et al., 2001). TCDD-mediated suppression
has also been shown in Sprague-Dawley rats as the primary antibody response to
sRBC was significantly suppressed in rats dosed with 100μg/kg TCDD (Pazdernik and
Rozman, 1985). In addition, it has also been demonstrated in Wistar albino rats that
TCDD exposure decreased IgM secretion and the suppression of IgM secretion could
be partly recovered by the anti-oxidant curcumin, a spice found in turmeric (Çiftçi,
2011).
While TCDD-mediated suppression of the primary antibody response has been
shown in mouse, human and rat, the rat model is of particular interest as TCDDmediated enhancement of the primary antibody response have also been observed.
One of the first studies conducted in the rat compared the effects of TCDD between
mouse and Fischer 344 and Long-Evans rats by using the plaque forming cell (PFC)
assay, which measures IgM secreting cells. This study demonstrated TCDD decreased
the PFC response to sRBC in the mouse in a concentration-dependent manner;

14

however, in the Fischer 344 rat PFC response to sRBC was enhanced in a
concentration dependent manner with TCDD treatment and an increase in the PFC
response in Long-Evans rats treated with 1μg/kg TCDD. (Smialowicz et al., 1994).
Another early study performed in Fischer 344 rats demonstrated that TCDD enhanced
the humoral immune response to a Trichinella spiralis infection by observing an
increased number of encysted larvae; larvae encystment occurs after the parasite has
been destroyed in an antibody-dependent manner (Luebke et al., 1995). The IgG
antibody response has also been found to be enhanced by TCDD as IgG secretion in
response to sRBC was increased in a concentration-dependent manner in SpragueDawley rats treated with TCDD (Fan et al., 1996). An additional study which exposed
rats to TCDD over a longer time period has also confirmed the enhancement of the
primary antibody response in rats. In rats dosed with 0.1μg/kg TCDD for four weeks, it
was observed that TCDD increased the expression of Igλ1 and 2 chain C-regions which
are components of the Ig light chain (Son et al., 2003). Although, the mechanism by
which TCDD mediates enhancement and suppression of the antibody response is
unknown, it is clear that TCDD is capable of modulating B cell function in the rat.

E. AHR plays a role in development and homeostasis
Although several targets of B cell activation and effector function are known to be
affected by TCDD, the specific mechanisms by which TCDD elicits toxicity have not yet
been elucidated. In both rodents and humans, a range in sensitivity to the toxic effects
of TCDD has been observed and has been attributed in part to genetic variations such
as single nucleotide polymorphisms (SNPs) in AHR (Hao and Whitelaw, 2013; Nebert,

15

1989). Recent studies using microarray technology have allowed for the detection of
species differences across human, mouse, and rat species. In microarray comparisons
between primary mouse, rat, and human hepatocytes treated in vitro with TCDD, it was
found that AHR activation via TCDD elicits species specific alterarations in global gene
expression and the sensitivity to TCDD-mediated differential gene expression was
found markedly divergent among species (Black et al., 2012; Forgacs et al., 2013).
With the increasing evidence that TCDD produces differential toxic effects, several
AHR

-/-

animal models have been constructed to understand the function of AHR in

greater detail.
Using AHR

-/-

mouse models, it became evident that AHR also plays a role in

tissue development and homeostasis. AHR

-/-

mice on C57/B6 backgrounds with

targeted disruptions in exon 1 or exon 2 have been generated in three separate
laboratories (Esser, 2009). Using zinc finger technology, an AHR

-/-

rat has also recently

been created by the targeted deletion of exon 2 which contains the bHLH domain that is
involved in DNA binding and dimerization with ARNT (Harrill et al., 2013). The targeted
deletion of AHR exon 2 by zinc finger technology uses specifically engineered zinc
finger proteins that are coupled to the FokI restriction enzyme to induce DNA double
stranded breaks that are then repaired by homology directed repair (HDR) or
nonhomologous end joining (NHEJ) resulting in the deletion of specific areas in the
genome allowing for genomic disruptions (Urnov et al., 2010). The targeted deletion of
AHR in the knockout rat was confirmed at both the mRNA and protein level. Western
blot analysis of liver tissue detected the 96kDa AHR protein in wild type rats and the
16

same band was absent in the AHR

-/-

rats (Harril et al., 2013). Furthermore, a reduction

in AHR mRNA was detected by three separate qPCR assays (Harril et al., 2013).
Additionally, the wild type and AHR

-/-

rats received a single dose of 25μg/kg TCDD. In

wild type rats an increase in expression levels of CYP1A1, CYP1A2, and CYP1B1 was
dectected and this increase of metabolic enzyme expression was absent in the TCDD-/-

dosed AHR

rats (Harril et al., 2013).

Another hallmark of TCDD exposure is an

increase in liver weight and a decrease in thymic weight (Bunger et al., 2008). As
expected, wild type rats dosed with TCDD showed increases in liver weight and
decrease in thymic weight while AHR
changes.

-/-

rats displayed no liver or thymic weight

Taken together, the targeted deletion of AHR in rat has been confirmed

creating the AHR

-/-

rat animal model and this model is resistant to some of the known

toxic effects of TCDD.
-/-

Despite the AHR

rat resistance to TCDD the phenotypic characterizations of

this newly developed animal model have not yet been fully evaluated and phenotypic
characterizations in organ development has recently come to light. Comparisons
between the three AHR

-/-

mouse models have been made however since the focus of
-/-

this thesis is on B cell function in the AHR

-/-

rat, the AHR mouse model developed by

the Bradfield group will be used for comparisons in phenotype since both knockout
models contain the targeted deletion exon 2 in the AHR gene. In characterizing the
phenoytpe of the AHR

-/-

rat several phenotypic differences between the AHR

17

-/-

rat and

mouse were detected. In all three knockout mouse models, the most notable phenotype
is a reduction in liver weight which has been attributed to a patent ductus venosus that
results in a reduction of blood flow to the liver (Harstad et al., 2006; Schmidt et al.,
-/-

1996). The pefusion of AHR

mouse and rat with contrast media was used to

determine if a patent ductus venous was present in the AHR

-/-

rat and a distinct color
-/-

change of the liver as well as of outflow from the vena cava was seen in the AHR
-/-

with the color change and outflow absent in AHR

mouse (Harril et al., 2013). This data

indicates that the portal vasculature is unaffected in the AHR
display the patent ductus venous seen in the AHR
characteristic seen in the AHR

-/-

rat

-/-

-/-

mouse.

rat and they do not
Another phenotypic

mouse is the presence of a persistent hyaloid artery of

the eye, this characteristic is absent in the AHR
to immune function, it has been found in AHR

-/-

-/-

rat (Harril et al., 2013). With regards
mice that there is a decrease in the

percentage of mature B cells and an increase in the rise of B cell precursors compared
to wild type (Thurmond et al., 2000). Preliminary characterizations of the immune
-/-

system in male AHR

rats were evaluated by complete blood counts (CBC) and no

alterations in the number of red blood cells or white blood cells was detected in female
AHR

-/-

-/-

rats. However, male AHR

rats displayed a decrease in the number of platelets

and leukocytes with no alteration in platelet function detected, while the number of
erythrocytes, monocytes, neutrophils, and eosinophils remained unaffected. The one
effect of the targeted deletion of AHR that is specific to the AHR
18

-/-

rat are renal

abnormalities including hydronephrosis, hydroureters and bilateral lesions in the ureters
and renal pelvis. These tissue abnormalities are absent in the AHR

-/-

al., 2013). Histology performed on the urinary tract of the AHR

mouse (Harril et
-/-

rats detected

histopathological changes in the kidney and ureters and are thought to be secondary to
the hydronephrosis and hydroureters (Harril et al., 2013). Harril et al., suggests that the
renal abnormalities are due to a dysregulation in the alternative splicing of Wilm’s tumor
suppressor 1 (WT1) as the alternative splicing of WT1 mRNA has been shown to be
regulated by AHR and play a role in kidney development.

19

Experimental Design

The majority of TCDD-mediated immunosuppressive effects on B cell activation
and effector function that have been studied have been conducted with mouse and
human B cells. With both model systems in place the mechanism by which TCDD
mediates its immunosuppressive effects has yet to be determined. Using the newly
developed AHR knockout rat animal model may identify possible mechanisms that have
yet to be observed in the mouse and human. The purpose of this study was to evaluate
the involvement of AHR and understand the effects TCDD plays on the humoral
immune system with a focus on B cell activation and effector function using a AHR

-/-

rat

model. It was hypothesized that the targeted deletion of the aryl hydrocarbon receptor
(AHR) results in the loss of sensitivity of the B cell to suppression by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and conversely the augmentation of the primary
IgM antibody response, which is mediated in part, through a greater magnitude in B cell
activation in the absence of TCDD.

A. Specific Aim 1:
To identify whether the targeted deletion of AHR affects the magnitude of B cell
activation and reduces the sensitivity of the B cell to TCDD-mediated suppression by
detecting increases in surface activation marker CD80, CD86, and MHC II expression.
Activation with LPS upregulates the expression of CD80, CD86, and MHCII on the B
cell surface and signifies early B cell activation. The level of expression can provide
information concerning the magnitude of activation. To date, there has been no study
20

evaluating the effect of targeted deletion of AHR on rat B cell activation but it has been
shown that the targeted deletion in mice results in a down regulation of CD80 and CD86
in antigen presenting cells (Jux et al., 2009; Singh et al., 2011). Flow cytometry will be
used to identify the basal expression patterns and expression pattern changes upon
activation with LPS in wild type and AHR

-/-

rats dosed with the AHR agonist TCDD.

B. Specific Aim 2:
To evaluate whether the targeted deletion of AHR affects the magnitude of the
IgM antibody response and reduces the sensitivity of the B cell to TCDD-mediated
suppression by detecting increases in IgM. The downstream effector function of B cells,
signified by the primary IgM antibody response, may be indirectly related to the
magnitude of B cell activation. Increases in the expression of CD80, CD86, and MHCII
by the targeted deletion of AHR may suggest similar changes in magnitude of the IgM
-/-

antibody response. Wild type and AHR rats dosed with TCDD will be activated with
LPS to induce the IgM antibody response. IgM secretion in addition to intracellular IgM
will be evaluated by ELISA and flow cytometry, respectively, to determine the
magnitude to which targeted deletion of AHR can affect B cell effector function.

21

Materials and Methods
A. Animals
5-8 week old female Sprague-Dawley rats purchased from Charles River
(Wilmington, MA) were used in the optimization of B cell activation studies and
euthanized with 100mg/kg phenobarbitol. All animals were used in accordance to
the guidelines set by the Michigan State University Institutional Animal Care and Use
Committee. This study was performed in collaboration with the laboratory of Dr.
Russell Thomas at The Hamner Institute for Health Sciences, Institute for Chemical
-/-

Safety Sciences (Research Triangle Park, NC) and all AHR

rats and wild type

control rat spleens used in this study were received from that facility. Female AHR

-/-

and wild type control animals were housed and cared for as described previously
(Harril et al., 2013).

B. Optimization of B cell Activation
To determine the appropriate activation for rat B cells, preliminary studies using
Sprague-Dawley rats were performed to identify the conditions that will be used during
the study. AHR

-/-

rats and wild type controls were shipped from the Hamner Institute for

Health Sciences and to mimic shipping conditions spleens were placed in 5mL of RPMI
1640 supplemented with 10% bovine serum overnight at 4⁰C. Splenocytes were
isolated via aseptic mechanical disruption and split into two aliquots; one aliquot was
reserved for erythrocyte lysis by ammonium chloride potassium (ACK) buffer and is
referred to as the lysed group, while the second aliquot remained untouched and is
22

referred to the non-lysed group, each aliquot was put into a single cell suspension at
6

5x10 cells/mL. Lysed and non-lysed cells were plated in duplicate with a naïve control
and splenocytes were activated with either 15μg/mL pokeweed mitogen (PWM),
15μg/mL lipopolysaccharide (LPS), 30μg/mL PWM or 30μg/mL LPS. Cells were
cultured at 37⁰C with 5% CO2 in RPMI 1640 supplemented with 10% bovine serum and
100 units/mL penicillin, 100 units/mL streptomycin. Supernatants for IgM sandwich
enzyme linked immunoabsorbent assay (ELISA) were collected at days three and four
of culture and frozen at -80⁰C in bovine calf serum supplemented with 10% dimethyl
sulfoxide (DMSO) until ready for analysis.
To further evaluate the proper culture conditions for B cell activation the
concentrations of LPS used were expanded. For this preliminary study one rat spleen
was incubated overnight as above to mimic shipping conditions. Erythrocytes were not
lysed and single cell suspensions containing 5x106 cells/mL were plated in triplicate.
Cells were activated with 10, 15, 30, or 60μg/mL LPS; a non-activated naïve control
group was also included. Cells were cultured at 37⁰C with 5% CO2 for three days and
the supernatants were collected for IgM ELISA as described below.

C. AHR

-/-

and Wild Type Rat Dosing and Spleen Collection

All dosing and tissue collection was performed by the laboratory of Dr. Russell
Thomas at The Hamner Institute for Health Sciences, Institute for Chemical Safety
Sciences. Female wild type and AHR

-/-

rats were dosed with TCDD at 3, 22, 100, 300,

23

or 1000 ng/kg/day for 5 days a week for four weeks. TCDD was suspended in corn oil
and control rats received this vehicle, the delivery volume was 5mL/kg and dosing
solutions were prepared as previously described (2006). After four weeks of dosing rats
were euthanized as described in Harril et al. 2013, the spleens were collected and cut
into two segments sagitally. One half of the spleen was shipped in 1640 RPMI mediate
supplemented with 10% bovine serum on wet ice overnight and received for immediate
processing the next day.

D. Lymphocyte Isolation and Culture
Spleens received from the Hamner Institute for Health Sciences in two separate
shipments and are denoted as group one and two respectively. Splenocytes were
isolated via mechanical disruption aseptically to produce a single cell suspension
7

(1x10 cells/mL) in RPMI 1640 supplemented with 10% bovine serum, 100 units/mL
penicillin, 100 units/mL streptomycin, and 2.1 g/L sodium bicarbonate. Splenocytes
6

were plated at 0.5x10

cells/well in a 48 well plate. Cells were treated with LPS

(15μg/mL) or vehicle to produce a non-activated control group and a LPS-activated
group. Cells were cultured at 37⁰C with 5% CO2.

After 72 hours, plates were

centrifuged at 3000 rpm for 5 minutes and the supernatant was collected in 200μL
aliquots and frozen at -80⁰C for the IgM ELISA assay. The remaining cells were
collected and washed once in protein free buffer 1 x Hank’s Balanced Salt Solution
(HBSS, pH 7.4; Invitrogen) for flow cytometry staining and analysis.

24

E. Flow Cytometry Analysis
Cells collected from group one were stained the same day while the cells
collected from group two were frozen in bovine calf serum with 10% DMSO at -80⁰C
until ready for assessment. Cells were first stained with Live/Dead Fixable Dead Cell
Stain Kit near-infrared (Invitrogen, Grand Island, NY) for 15 minutes then washed twice
with 1 x HBSS.

Prior to surface and intracellular protein marker staining, the surface

Fcγ receptor was blocked by incubation with mouse anti-rat CD32 (BD Pharmingen,
San Jose, CA). All antibodies used for surface and intracellular protein staining are
listed in appendix A and are conjugated to fluorescent dyes. All procedures were
performed in the dark.
All surface markers were stained simultaneously in fluorescence-activated cell
sorting (FACS) buffer which consisted of 1 x HBSS with 0.1% sodium azide (pH 7.6,
Sigma-Aldrich, St. Louis, MO)
Billerica, MA).

and 1% bovine serum albumin (BSA, Calibiochem,

In addition to the fluorescently labeled antibody for surface marker

staining, a non-fluorescent mouse anti-Rat IgM (BD Pharmingen, San Jose, CA) was
used to block the surface IgM for the detection of intracellular levels of IgM. All samples
were incubated with the antibodies for 20 minutes and washed three times with FACS
buffer. The cells were then incubated with CytoFix fixation buffer (BD Biosciences, San
Jose, CA) for 10 minutes and washed once with FACS buffer.
Intracellular IgM staining was performed after surface marker staining. Cells were
permeabilized by incubation in 1 x Perm/Wash solution (BD Biosciences, San Jose, CA)
for 30-60 minutes.

The antibody used for intracellular staining was diluted in
25

permeabilization buffer and incubated for 20 minutes. Cells were washed once with
FACS buffer then reconstituted in a final volume of 200μL for analysis. All samples
were processed with a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA)
and evaluated with FloJo (Tree Star Inc., Ashland, OR).

F. IgM ELISA
96-well Immunlon 4 HBX plates (Thermo Scientific, Waltham, MA) were
assembled and coated with purified mouse anti-Rat IgM antibody (BD Pharmingen, San
Jose, CA) at 1:100 in phosphate buffered saline (PBS) overnight at 4⁰C. The plate was
then blocked with PBS containing 3% BSA (3% BSA-PBS) for 30 minutes at 37⁰C while
the supernatants were allowed to thaw on ice. Supernatants were appropriately diluted
in PBS and incubated at 37⁰C for one hour, afterwards a secondary biotinylated mouse
anti-rat IgM antibody was added at a 1:2000 dilution in 3% BSA-PBS and allowed to
incubate for one hour at 37⁰C. Strepavidin peroxidase (Sigma-Aldrich, St. Louis, MO)
was diluted at 1:2000 in 3% BSA-PBS and incubated for 30 minutes at 37⁰C. ABTS
solution was prepared from ABTS tablets (Roche, Indianapolis, IN) and ABTS buffer
(Roche, Indianapolis, IN) based on the manufacturers protocol and added to the plate.
The plate was kinetically read at 405nm for 60 minutes at 60 second intervals with 3
seconds of shaking in between reads on a Bio-Teck Synergy HT (Winooski, VT) plate
reader. The plates were washed three times with PBS containing 0.05% Tween20
(Sigma-Aldrich, St. Louis, MO) and three times with distilled water between each

26

addition of samples and reagents. Quantification of samples was prepared from a
standard curve made from rat IgM standard (Rockland Immunochemicals, Gilbertsville,
PA).

G. Statistical Analysis
The mean was determined for each treatment group (n = 5) in individual
experiments, the error bars represent the ± SEM for each group. Homogenous data
was analyzed using a one-way analysis of variance (ANOVA) followed by a Dunnett’s
two tailed t-test to compare treatment groups to non-activated control groups when
significant differences were observed (p < 0.05). Two- way ANOVA was employed
when two variables were involved and followed by a Bonferroni’s post-hoc test to
determine significant differences among groups.

Data expressed as percent were

transformed by using Y = log(Y) prior to statistical analysis. All statistical analysis was
performed with GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA).

27

Results

A. Optimization of Culture and Activation Conditions
The first step in this study was to determine the optimal stimulus that would
induce B cell activation leading to an IgM antibody response in Sprague-Dawley rats.
To induce B cell activation and differentiation into IgM secreting plasma cells the B cell
polyclonal activators LPS and PWM were selected. To establish the optimal activator for
this study, rat splenocytes were treated with 15μg/mL and 30μg/mL of LPS and
15μg/mL and 30μg/mL of PWM and the quantity of secreted IgM was measured by
ELISA after 72 and 96 hours. The activator that yielded the highest IgM response was
15μg/mL LPS at both 72 and 96 hours of culture (Figure 1A and 1B). Rat B cells
responded modestly to both 15μg/mL and 30μ/mL PWM and secreted a lower quantity
of IgM in comparison to similar concentrations of LPS. Based on these data it was
determined that LPS would be used in subsequent experiments and the dose of LPS
was further optimized by expanding the LPS concentration response curve.

An

increase in the IgM antibody response of activated primary rat B cells was seen in
comparison to naïve (inactivated) cells; however, there was no significant differences in
IgM secretion observed with increasing concentrations of LPS above 10μg/mL (Figure
1C).
Red blood cells (RBCs) present in culture have been shown to enhance the
responses of B cells to activators and prior to beginning the study we wanted to
confirm this effect (Yachnin, 1972). Single cell suspensions of splenocytes were split
into two aliquots; one aliquot containing erythrocytes (non-lysed group), and in the

28

second aliquot the erythrocytes were removed by lysis with ACK buffer (lysed group).
The lysed group secreted more IgM compared to the non-lysed group at 72 and 96
hours (Figure 1A and 1B). This result is in contrast to previous studies that have
reported an enhanced B cell response to activators in the presence of erythrocytes.
Cell counts were performed at both 72 and 96 hours of culture and the lysed group
contained a large number of dead cells making quantification of IgM and comparisons
between naïve and activated B cells difficult. The removal of erythrocytes from
splenocyte preparations allowed for a higher level of IgM secretion but due to the cell
death that had taken place in the lysed naïve control, it was determined that primary cell
cultures of wild type and AHR

-/-

rat splenocytes would retain the erythrocytes.

29

Figure 1. Optimization of primary Sprague-Dawley rat B lymphocyte culture and activation conditions.
Splenocytes were isolated from one rat and erythrocytes were either lysed with ACK buffer or allowed to remain in culture
(non-lysed) and activated with 15μg/mL or 60μg/mL of LPS or 15μg/mL or 60μg/mL of PWM. Supernatant were collected
at 72 hours (A) and 96 hours (B) and an ELISA was performed to detect levels of secreted IgM. C depicts an ELISA at
three days of culture with non-lysed primary rat B cells treated with 10, 15, 30, or 60μg/mL LPS. Panels A and B are
results from one rat with each treatment group plated in duplicate. In C, data presented is the mean ±SEM from each
treatment plated in triplicate. * P <0.05 and ** P <0.01 compared to naïve.

30

Figure 1 (cont’d)

31

B. Evaluation of Rat B cell Activation by the Measurement of Activation Markers
Wild type and AHR

-/-

rat splenocytes were examined by flow cytometry and B cells

were identified using the gating scheme shown in Figure 2. First, splenocytes were
gated to identify single cells (singlet gate) to eliminate artifacts created by doublets in
the analysis. Within the singlet gate, splenocytes were gated on viability (viable gate) to
eliminate dead cells from analysis. Lymphocytes were identified within the viable gate
based on cell size and granularity.

The final gate identified B cells within the

lymphocyte gate by the presence of IgM. The two groups were analyzed separately to
prevent a loss in the detection of significant differences among the activated and TCDD
treatments since the two groups were shipped separately and may have been handled
under different shipping conditions. A second rationale for analyzing the groups
separately was that group one was processed immediately after collection for flow
cytometry analysis while the cells from group two were frozen at -80⁰C before
processing for flow cytometry.

32

Figure 2. Schematic representation
of the flow cytometry gating for
the identification of B cells within
-/AHR
and wild type splenocyte
cultures. Samples were first gated
on single cells noted as the singlet
gate to eliminate doublets which
produce artifacts in the sample
analysis. This is done by sorting the
cells by their size which is forward
scatter-height (FSC-H) and forward
scatter-area (FSC-A).
Taking the
singlet gate, a second gate was
added to exclude the analysis of
dead cells and include only the live
cells by the live/dead dye and side
scatter-area
(SSC-A)
which
determines cellular granularity; this
gate is noted as the viable gate. The
lymphocytes were then identified in
the viable gate by their size (FSC-A)
and granularity (SSC-A). The B cells
were then identified from the
lymphocyte gate by the presence of
intracellular IgM. The gating scheme
was applied to all groups analyzed by
flow cytometry. “For interpretation of
the references to color in this and all
other figures, the reader is referred to
the electronic version of this thesis.”

33

I.

AHR Deletion Effects on the B cell Activation Marker CD80
To evaluate the potential AHR-mediated alteration of CD80 expression in the rat,

basal levels of non-activated and LPS-activated vehicle control B cells from both group
one and group two were compared separately.

In group one, activation with LPS

+

significantly increased the percentage of CD80 B cells in both wild type (from 4.6% to
8.1%) and AHR

-/-

(from 5.1% to 16.1%) rats with the AHR

-/-

having a more robust

response to LPS activation when compared to wild type (Figure 3). In group two, LPS
+

activation of wild type B cells increased the percentage of CD80 cells from 5.7% to
-/-

13.5% but this upregulation of CD80 was not significant (Figure 4). AHR

had a more

+

robust response and significantly increased the percentage of CD80 cells from 6.7% in
non-activated B cells to 20.7% in LPS-activated B cells (Figure 4). The observed
+

-/-

increase in the percentage of CD80 cells of AHR

rats in group one was not seen in

group two.
In addition to evaluating the basal expression, the percentage and mean
fluorescence intensity (MFI) was also used to determine the density of CD80 expressed
-/-

on the surface in TCDD-dosed wild type and AHR

rat B cells. The percentage of

+

CD80 cells determines the number of B cells expressing CD80 on the surface within a
sample and the MFI determines the magnitude of surface expression of CD80 on a per
+

cell basis. In group one; LPS activation significantly increased the percentage of CD80

34

+

Figure 3. Targeted deletion of AHR leads to a higher percentage of CD80 B cells
upon LPS activation compared to wild type in group one. Vehicle control cells in
group one were evaluated by flow cytometry for the basal expression of CD80 in wild
-/type and AHR B cells. Cells were also treated with 15μg/mL LPS to activate B cells
inducing an upregulation of CD80 expression. Comparisons were made between
transformed percent positive data for non-activated and LPS-activated wild type and
-/AHR B cells. Data is presented as the mean ± SEM from five rats per treatment
group. *** P ≤ 0.001 compared to non-activated within the genotype. C indicates P ≤
0.001 compared to LPS-activated wild type cells.

35

+

Figure 4. LPS activation increases the percentage of CD80 B cells and the
deletion of AHR leads to additional increases in CD80 expression in group two.
Vehicle control sets in group two were evaluated by flow cytometry for the basal
-/expression of CD80 in wild type and AHR B cells. Cells were also treated with
15μg/mL LPS to activate B cells inducing an upregulation of CD80 expression.
Comparisons were made between transformed percent positive data for non-activated
-/and activated wild type and AHR B cells. Data is presented as the mean ± SEM from
five rats per treatment group. * P ≤ 0.05 compared to non-activated within the genotype.

36

B cells in wild type and AHR

-/-

rats in comparison to non-activated splenocytes of their

respective genotype (Figure 5A and 5B). AHR

-/-

B cells had an increased response to

+

LPS resulting in a higher percentage of CD80 cells when compared to wild type. In
wild type rats, the percentage of CD80

+

non-activated B cells was significantly

decreased at 300 and 1000ng/kg/day TCDD treatment compared to vehicle (Figure 5A).
The percentage of CD80

+

-/-

B cells from AHR

rats treated with 22ng/kg/day TCDD

decreased compared to the B cells from vehicle treated AHR

-/-

rats (Figure 5B). Even

though there is no significant dose-response in LPS-activated AHR

-/-

B cells there is an

overall trend towards a decrease with increasing doses of TCDD. The MFI in wild type
-/-

and AHR

rats of group one remained relatively unchanged upon LPS activation
+

(Figure 5C and 5D). A significant increase in the CD80 MFI was also observed in wild
type LPS-activated B cells treated with 300ng/kg/day TCDD (Figure 5C). Whereas in
-/-

the AHR

there is a bell shaped expression pattern with a significant increase peaking

at 100ng/kg/day TCDD then decreasing with increasing TCDD doses (Figure 5D). The
only significant genotype effect in the MFI was a reduction in LPS-activated AHR
cells at 1000ng/kg/day TCDD treatment (Figure 5D).

37

-/-

B

+

Figure 5. Deletion of AHR produces a higher percentage of CD80 B cells compared to wild type with no
significant TCDD suppression observed in either genotype. Data presented are from group one. Wild type and
-/AHR rats were dosed with corn oil vehicle (VH) control and varying concentrations of TCDD. Splenocytes from these
+

rats were activated with 15μg/mL LPS for 72 hours and compared to a non-activated control. Percentage of CD80 B
-/-

cells in wild type (A) and AHR
-/-

+

(B) were measured by flow cytometry. The MFI of CD80 B cells in wild type (C) and

AHR (D) were obtained in the same flow cytometry analysis. Data is presented as the mean ± SEM from five rats per
treatment. ** P ≤0.01 and *** P ≤ 0.001 compared to non-activated B cells within the genotype. a indicates p ≤ 0.05 and b
indicates P ≤ 0.01 compared to VH control within the genotype.

38

Figure 5 (cont’d)

39

Similar evaluations were made for group two.
percentage of CD80

+

No significant increase in the

B cells was observed in wild type upon activation with LPS

despite an overall trend towards an upregulation of CD80 (Figure 6A). However, LPS
+

-/-

activation led to significant increases in the percentage CD80 B cells of AHR

rats in

the vehicle, 22 and 100μg/kg/day TCDD treatments, when compared to non-activated B
cells of the same treatment (Figure 6B). It was also noticed that the overall response in
+

-/-

the percentage of CD80 B cells of AHR

rats in group two was more varied compared

to group one but the same trend towards a dose dependent decrease is still observed.
No significant alterations in the MFI were observed for non-activated and LPS-activated
wild type and AHR

-/-

B cells (Figure 6C and 6D). In contrast to group one, no TCDD
+

effects were detected in the percentage or MFI of CD80 B cells of wild type or AHR
rats.

40

-/-

+

Figure 6. TCDD was unable to suppress the upregulation in the percentage of CD80 B cells in wild type and
-/-

-/-

AHR rat B cells upon LPS activation. Data presented are from group two. Wild type and AHR rats were dosed with
corn oil vehicle (VH) control and varying concentrations of TCDD and splenocytes from these rats were treated with
+
15μg/mL LPS for 72 hours and compared to a non-activated control. Percentage of CD80 B cells in wild type (A) and or
-/-

+

-/-

AHR (B) were measured by flow cytometry. The MFI of CD80 B cells in wild type (C) and or AHR (D) were obtained
in the same flow cytometry analysis. Data is presented as the mean ± SEM from five rats per treatment group. * P ≤ 0.05
compared to non-activated B cells within the genotype.

41

Figure 6 (cont’d)

42

II.

AHR deletion effects on CD86 Expression
The expression of CD86 was evaluated in non-activated and LPS-activated

vehicle treated wild type and AHR

-/-

B cells by flow cytometry; this evaluation was done

for both groups. LPS activation induced a significant increase in the percentage of
+

CD86 B cells in wild type and AHR

-/-

rats of group one (Figure 7). This significant

upregulation was also detected in group two (Figure 8). A difference was observed
between the two genotypes of group one as the AHR

-/-

B cells had a stronger response
+

to LPS by the significant increased the percentage of CD86 B cells compared to LPS+

activated wild type B cells. No difference in the percentage of CD86 cells between the
two genotypes was observed for group two in either non-activated or LPS-activated B
cells.
As previously described for CD80, the percentage and MFI of CD86 was
evaluated to detect changes in the magnitude of activation in TCDD-dosed wild type
and AHR

-/-

rat B cells. In group one, activation with LPS upregulated the percentage of

+

CD86 B cells in wild type and AHR

-/-

rats (Figure 9A and 9B). Also observed in group

one is a significant increase in the CD86

+

-/-

MFI in both wild type and AHR

LPS-

activated B cells in comparison to non-activated cells within their respective genotype
(Figure 9C and 9D). No significant TCDD effects were observed in the percentage of
+

CD86 B cells of wild type and AHR
-/-

observed in LPS-activated AHR

-/-

rats however, a trend towards a decrease was

B cells (Figure 9A and 9B) . In group one LPS-

43

+

Figure 7. Targeted deletion of AHR in rats leads to higher percentage of CD86 B
cells upon LPS activation compared to wild type in group one. Vehicle treatment
control in group one was evaluated by flow cytometry for the basal expression of CD86
-/in wild type and AHR B cells. Cells were also treated with 15μg/mL LPS to activate B
cells inducing an upregulation of CD86 expression. Comparisons were made between
-/transformed percent positive data for non-activated and activated wild type and AHR
B cells. Data is presented as the mean ± SEM from five rats per treatment group. *** P
≤ 0.001 compared to non-activated within the genotype. a indicates P ≤ 0.05 compared
to LPS-activated wild type B cells.

44

+

Figure 8. Activation with LPS leads to higher percentage of CD86 B cells but the
deletion of AHR in rats does not produce a further increase in CD86 expression of
group two. Vehicle control treatment of group two was evaluated by flow cytometry for
-/the basal expression of CD86 in wild type and AHR B cells. Cells were also treated
with 15μg/mL LPS to activate B cells inducing an upregulation of CD86 expression.
Comparisons were made between transformed percent positive data for non-activated
-/and LPS-activated wild type and AHR B cells. Data is presented as the mean ± SEM
from five rats per treatment group. *** P ≤ 0.001 compared to non-activated within the
genotype.

45

-/-

Figure 9. LPS activation upregulates CD86 expression in wild type and AHR rat B cells similarly and no
significant dose dependent TCDD-mediated suppression of CD86 was observed. Data presented are from group
-/one. Wild type and AHR rats were dosed with corn oil vehicle (VH) control and varying concentrations of TCDD and
splenocytes from these rats were treated with 15μg/mL LPS for 72 hours and compared to a non-activated control.
+
-/+
Percentage of CD86 B cells in wild type (A) and AHR (B) were measured by flow cytometry. The MFI of CD86 B cells
-/-

in wild type (C) and AHR (D) were obtained in the same flow cytometry analysis. Data is presented as mean ± SEM
from five rats per treatment group. *** P ≤ 0.001 compared to non-activated B cells within the genotype. a indicates P ≤
0.05 and c indicates P ≤ 0.001 in comparison to vehicle control within the genotype.
46

Figure 9 (cont’d)

47

+

activated wild type B cells a significant increase was observed in the CD86 MFI at
1000ng/kg/day TCDD treatment (Figure 9C). However, in LPS-activated AHR
a significant decrease in the CD86

+

-/-

B cells

MFI was observed at 300 and 1000ng/kg/day

TCDD treatment (Figure 9D). These trends are not seen in the percentage or MFI of
+

CD86 B cells in wild type rats. In group two the same significant increase in both the
+

percentage (Figure 10A and 10B) and MFI (Figure 10C and 10D) of CD86 cells was
detected in the LPS-activated wild type and AHR

-/-

rats. No significant TCDD effect was

observed in group two and the trend towards a decrease in CD86 expression observed
in group one was absent.

48

-/-

Figure 10. LPS activation upregulates CD86 expression in wild type and AHR rat B cells similarly and no
significant TCDD-mediated suppression of CD86 was observed. Data presented are from group two. Wild type and
-/AHR rats were dosed with corn oil vehicle (VH) control and varying concentrations of TCDD and splenocytes from these
+

rats were treated with 15μg/mL LPS for 72 hours and compared to a non-activated control. Percentage of CD86 B cells
-/-

+

-/-

in wild type (A) and AHR (B) were measured by flow cytometry. The MFI of CD86 B cells in wild type (C) and AHR
(D) were obtained in the same flow cytometry analysis. Data is presented as the mean ± SEM from five rats per treatment
group. *** P ≤ 0.001 compared to non-activated B cells within the genotype.
49

Figure 10 (cont’d)

50

III.

Assessing the role AHR has on regulating MHC II Expression
To demonstrate that B cell activation is modulated by AHR, basal MHC II

expression and expression of MHC II upon LPS activation in vehicle treated rats were
evaluated. In contrast to what was previously observed with CD80 and CD86 in group
+

one, LPS activation resulted in a significant decrease in the percentage of MHC II B
cells in AHR

-/-

rats compared to wild type.

LPS activation resulted in a two-fold

decrease from 48% in wild type B cells to 22.6% in AHR

-/-

B cells of group one (Figure
+

11). In group two, LPS activation resulted in an increased percentage of MHC II B cell
in wild type and AHR

-/-

rats, no significant differences were observed between the two

genotypes. Also observed in group two was a significant decrease in the percentage of
MHC II

+

-/-

AHR

non-activated B cells compared to non-activated wild type B cells
+

(Figure 12). There was a two-fold decrease in the percentage of MHC II non-activated
B cells in the AHR

-/-

rats (4.6%) in comparison to non-activated wild type B cells (8.6%).

Interestingly, the difference between the two genotypes observed in group two was
detected in the non-activated B cells, whereas the genotype effect of group one was
seen in the LPS-activated B cells.
The percentage and MFI of MHC II
AHR

-/-

+

B cells were analyzed in wild type and

rats as previously done with CD80 and CD86. In contrast to the previous two

activation markers examined, MHC II displayed different trends between wild type and

51

+

Figure 11. LPS activation results in a down regulation of MHC II expression in
-/-

AHR B cells compared to LPS-activated wild type B cells. Vehicle control in group
one was evaluated by flow cytometry for the basal expression of MHC II in wild type and
-/AHR B cells. Cells were also treated with 15μg/mL LPS to activate B cells to induce
an upregulation of MHCII expression. Comparisons were made between transformed
-/percent positive data for non-activated and LPS-activated wild type and AHR B cells.
Data is presented as the mean ± SEM from five rats per treatment group. *** P ≤ 0.001
compared to non-activated within the genotype. a indicates P ≤ 0.05 compared to LPSactivated wild type splenocytes.

52

-/-

Figure 12. Non-activated AHR rat B cells have a lower basal expression of MHC
II while the level of MHC II upon LPS activation is similar to LPS-activated wild
type B cells. Vehicle control in group two was evaluated by flow cytometry for the
-/basal expression of MHC II in wild type and AHR B cells. Cells were also treated with
15μg/mL LPS to activate B cells to induce an upregulation of MHC II expression.
Comparisons were made between transformed percent positive data for non-activated
-/and LPS-activated wild type and AHR B cells. Data is presented as the mean ± SEM
from five rats per treatment group. *** P ≤ 0.001 compared to non-activated within the
genotype. a indicates P ≤ 0.05 compared to non-activated wild type splenocytes.

53

AHR

-/-

rat B cells. For group one, LPS activation significantly increased the percentage
+

of MHC II B cells in wild type and AHR

-/-

rats at all TCDD doses (Figure 13A and 13B).

+

+

The MHC II MFI differs from the percentage of MHC II cells in that LPS activation
significantly increased MHC II in wild type B cells treated with vehicle, 300 and1000
ng/kg/day TCDD doses (Figure 13A) and AHR

-/-

B cells treated with 100 and

1000ng/kg/day TCDD (Figure 13D). TCDD-mediated upregulation in the percentage of
+

MHC II B cells at 100ng/kg/day in LPS-activated AHR
+

-/-

in the MHC II MFI of the AHR

-/-

rats (Figure 13B). An increase

B cells was also observed at 100ng/kg/day treatment

(Figure13D). In wild type rats the only TCDD-mediated effects observed were in the
MFI of non-activated B cells treated with 22 and 100ng/kg/day TCDD (Figure 13C).
In group two wild type B cells, LPS activation resulted in an increase in the
+

percentage of MHC II cells (Figure 14A). No significant TCDD effects were detected;
however, a U-shaped dose-response curve was observed and not seen in any other
-/-

treatment (Figure 14A). The LPS-activated AHR

B cells had a similar increase in the

+

percentage of MHC II cells; although, the trend towards an increase with increasing
doses of TCDD observed in group one was not seen (Figure 14B). In contrast to wild
+

type, there was a significant decrease in the percentage of MHC II LPS-activated B
-/-

cells of AHR

rats when treated with 3, 22, and 1000ng/kg/day TCDD (Figure 14B).

54

+

The MHC II MFI for both wild type and knockout B cells increased with LPS activation
but remained unaltered upon TCDD exposure (Figure 14C and 14D).

55

Figure 13. Targeted deletion of AHR in rats leads to the down regulation of MHC II when activated with LPS and
-/TCDD produces opposing trends in the percentage of MHC II of wild type and AHR rats. Data presented are from
-/-

group one. Wild type and AHR rats were dosed with corn oil vehicle (VH) control and varying concentrations of TCDD
and splenocytes from these rats were treated with 15μg/mL LPS for 72 hours and compared to a non-activated control.
+
-/+
Percentage of MHC II B cells in wild type (A) and AHR (B) were measured by flow cytometry. The MFI of MHC II B
-/-

cells in wild type (C) and AHR (D) were obtained in the same flow cytometry analysis. Data is presented as the mean ±
SEM from five rats per treatment group. * P ≤0.05, ** P ≤ 0.01, and *** P ≤ 0.001 compared to non-activated B cells within
the genotype. a indicates P ≤ 0.05 compared to vehicle control within its respective genotype and activation set.

56

Figure 13 (cont’d)

57

Figure 14. Targeted deletion of AHR in rats leads to the down regulation of MHC II when activated with LPS. Data
-/presented are from group two. Wild Type and AHR rats dosed with varying concentrations of TCDD and splenocytes
from these rats were activated with 15μg/mL LPS for 72 hours and compared to a non-activated control. Percentage of
+
-/+
MHC II B cells in wild type (A) and AHR (B) were measured by flow cytometry. The MFI of MHC II B cells in wild type
-/-

(C) and AHR (D) were obtained in the same flow cytometry analysis. Data is presented as the mean ± SEM from five
rats per treatment group. *** P ≤ 0.001 compared to non-activated B cells within the genotype. a indicates P ≤ 0.05
compared to vehicle control within its respective genotype and activation set.
58

Figure 14 (cont’d)

59

C. Modulation of the IgM Primary Antibody Response
The second aim of this study was to determine the magnitude of change in the
IgM antibody response in the absence of AHR. Two approaches were taken; the level
of intracellular IgM and the level of IgM secreted into the external environment were
determined by flow cytometry and ELISA, respectively. It was observed during flow
+

cytometry analysis that a separate population of IgM B cells expressing high levels of
+

IgM was detected in both genotypes and this population was denoted as IgM high.
+

The IgM high B cell population was used for comparisons between treatment groups.
+

In group one, LPS activation increased the percentage of intracellular IgM high wild
type B cells from 1.79% (non-activated) to 3.28% (LPS) (Figure 15A and 15B),
suggesting the B cells were only modestly activated. However, in group one AHR

-/-

rats

+

the percentage of intracellular IgM high B cells increased from 1.83% (non-activated)
to 7.27% (LPS), a two-fold increase compared to the activated wild type rats (Figure
15B and 15D). In group two, similar observations were made but a more robust
response was seen in splenocytes from the LPS-activated AHR

-/-

rats with an increase

+

in intracellular IgM high B cells from 1.26 % (non-activated) to 9.28% (LPS) (Figure
16B and 16D). This is in contrast to group two wild type B cells which only increased in
the percentage of intracellular IgM

+

high cells from 1.03% (non-activated) to 3.95%

(LPS), a two-fold decrease in comparison to LPS-activated AHR
and 16C).
60

-/-

B cells (Figure 16A

Figure 15.
-/Activated AHR B cells
upregulate intracellular
IgM over activated wild
type B cells. Corn oil
vehicle control in group
one was evaluated by
flow cytometry for the
basal
expression
of
intracellular IgM in wild
-/type and AHR B cells.
Comparisons were made
between
non-activated
control and splenocytes
activated with 15μg/mL
LPS which induces an
IgM antibody response.
Data is presented as the
+
mean percentage of IgM
high B cells from five rats
per treatment.

61

Figure 16.
Targeted deletion of AHR
in rat B cells results in an
enhanced response of
intracellular
IgM
over
activated wild type B cells.
Corn oil vehicle control in
group one was evaluated by
flow cytometry for the basal
expression of intracellular
-/IgM in wild type and AHR
B cells. Comparisons were
made
between
nonactivated
control
and
splenocytes activated with
15μg/mL LPS which induces
an IgM antibody response.
Data is presented as the
+
mean percentage of IgM
high B cells from five rats
per treatment.

62

The magnitude of change in intracellular IgM in vehicle and TCDD treated wild
type and AHR

-/-

rats for group one and group two were examined next. In the

splenocytes of group one wild type rats, LPS activation moderately upregulated the
percentage of intracellular IgM

+

high B cells and no significant TCDD-mediated

suppression was observed; however, a trend towards a decrease was seen (Figure
17A). The targeted deletion of AHR in group one led to a significant increase in the
percentage of intracellular IgM

+

high B cells (Figure 17B).

immunosuppressive effects were detected in the AHR

-/-

No TCDD-mediated

B cells. These data show that

+

the percentage of rat intracellular IgM high B cells increased upon the targeted deletion
of AHR.
The same comparisons were made for vehicle and TCDD treated wild type and
AHR

-/-

+

rats of group two and the increase in the percent intracellular IgM high B cells

was found to be dissimilar to group one. Group two LPS-activated wild type B cells
+

significantly increased the percentage of IgM high B cells, which indicated that group
two responded more strongly to LPS

when compared to group one (Figure 18A).

Similar to group one, no significant TCDD-mediated suppression in IgM was observed
but a trend towards a decrease was observed at higher TCDD doses in group two. The
targeted deletion of AHR in the LPS-activated knockout B cells resulted in a two-fold
+

increase in the percentage of intracellular IgM high B cells compared to wild type
(Figure18B).

63

+

Figure 17. Targeted deletion of AHR results in an increase in the percentage of intracellular IgM high B cells.
-/-

Data presented are from group one. Wild type and AHR rats were dosed with corn oil vehicle (VH) control and varying
concentrations of TCDD and splenocytes from these rats were treated with 15μg/mL LPS for 72 hours and compared to a
+
-/non-activated control. Percentage of intracellular IgM high B cells in wild type (A) and AHR (B) were measured by flow
cytometry. Data is presented as the mean ± SEM from five rats per treatment group. * P ≤0.05 and *** P ≤ 0.001
compared to non-activated B cells within the genotype.

64

+

Figure 18. Targeted deletion of AHR produces an upregulation of intracellular IgM high B cells and TCDD is
-/-

unable to suppress this upregulation. Data presented are from group two. Wild type and AHR rats were dosed with
corn oil vehicle (VH) control and varying concentrations of TCDD and splenocytes from these rats were treated with
+
15μg/mL LPS for 72 hours and compared to a non-activated control. Percentage of intracellular IgM high B cells in wild
-/-

type (A) and AHR (B) were measured by flow cytometry. Data is presented as the mean ± SEM from five rats per
treatment group. ** P ≤0.01 and *** P ≤ 0.001 compared to non-activated B cells within the genotype.

65

The modifications in IgM secretion were also determined using an ELISA assay.
Activation with LPS in wild type B cells only produced a modest increase in secreted
IgM of group one and no TCDD-mediated suppression was observed (Figure 19A).
LPS activation in AHR

-/-

B cells resulted in a significant increase in secreted IgM when

compared to non-activated and no TCDD-mediated suppression of secreted IgM was
observed (Figure 19B). Similar to CD80, CD86 and MHC II expression, vehicle treated
non-activated and LPS-activated wild type and AHR
the basal quantity of secreted IgM in group one.

-/-

B cells were used to determine

AHR

-/-

B cells responded more

robustly to LPS compared to wild type which only moderately increased IgM secretion
when activated with LPS (Figure 19C). Also, a difference between the two genotypes
was observed as LPS-activated AHR

-/-

B cells secreted a greater quantity of IgM when

compared to LPS-activated wild type B cells.
Similar to the intracellular IgM results described, the quantity of IgM secretion in
group two was found dissimilar to group one. In group two, LPS-activated wild type B
cells significantly increased the secretion of IgM in rats treated with vehicle, 100 and
300ng/kg/day TCDD (Figure20A). No TCDD-mediated IgM suppression was observed;
however, a bell-shaped trend which peaked at 100ng/kg/day TCDD was detected. In
group two, LPS activation of AHR

-/-

B cells significantly increased IgM secretion except

for the 100ng/kg/day TCDD dose (Figure 20B). TCDD treatment did not lead to an

66

-/-

Figure 19. LPS-activated AHR

rat B cells secrete higher quantity of IgM over their wild type counterparts with
-/-

no TCDD-mediated suppression of the antibody response. Data presented are from group one. Wild type and AHR
rats were dosed with corn oil vehicle (VH) control and varying concentrations of TCDD and splenocytes from these rats
were treated with 15μg/mL LPS for 72 hours and compared to a non-activated control. Quantity of secreted IgM
-/measured in ng/mL from wild type (A) and AHR (B) activated B cells were measured by ELISA. Panel C compares the
-/-

basal secretion of IgM in vehicle treated wild type and AHR B cells obtained in the same ELISA assay. Data is
presented as the mean ± SEM from five rats per treatment group. * P ≤ 0.05 and *** P ≤ 0.05 compared to non-activated
B cells within the genotype. a in panel B indicates P ≤ 0.05 compared to vehicle control in its respective genotype and
activation set. a in panel C indicates P ≤ 0.05 compared to LPS-activated wild type.
67

Figure 19 (cont’d)

68

Figure 20. The targeted deletion of AHR leads to a greater quantity of IgM secretion in LPS-activated B cells. Data
-/presented are from group two. Wild type and AHR rats were dosed with corn oil vehicle (VH) control and varying
concentrations of TCDD and splenocytes from these rats were treated with 15μg/mL LPS for 72 hours and compared to a
-/non-activated control. Quantity of secreted IgM measured in ng/mL from wild type (A) and AHR (B) activated B cells
-/-

were measured by ELISA. Panel C compares the basal secretion of IgM in vehicle treated wild type and AHR B cells
obtained in the same ELISA assay. Data is presented as the mean ± SEM from five rats per treatment group. * P ≤ 0.05
and *** P ≤ 0.001 compared to non-activated B cells within the genotype. In panel C, a indicates P ≤ 0.05 compared to
LPS-activated wild type.

69

Figure 20 (cont’d)

70

-/-

effect in AHR

B cells but an opposite trend occurred from wild type in that a U-shaped

trend was seen with the lowest IgM quantity at 100ng/kg/day TCDD. In comparing the
basal IgM secretion in vehicle treated wild type rats, the mean IgM secretion
significantly increased from 1114ng/mL to 1946ng/mL in LPS-activated B cells (Figure
20C). The vehicle treated AHR

-/-

B cells had a stronger response to LPS compared to

wild type as the mean IgM secretion significantly increased from 1429ng/mL to
2730ng/mL, a 1.9 fold change upon activation with LPS (Figure 20C). The targeted
deletion of AHR did result in a difference between the two genotypes as LPS-activated
AHR

-/-

B cells secreted a greater quantity of IgM compared to LPS-activated wild type B

cells.

71

Discussion
-/-

This AHR

rat study was conducted in collaboration with the group of Dr.

Russell Thomas at the Hamner Institute for Health Sciences and was primarily designed
as a model to evaluate the carcinogenic effects of TCDD. The doses of TCDD that
were selected by those at the Hamner Institute for Health Sciences were based on
previous

studies

that

demonstrated

increased

hepatocellular

adenoma

and

cholangiocarcinomas incidence in female Sprague-Dawley rats over a range from 22
ng/kg/day to 100ng/kg/day (2006). Although, the specific mechanism in which TCDD
induces liver tumors is unknown, it is hypothesized that inflammation is a key event in
the mode of action by which TCDD promotes liver cancers. Inflammation can result from
exposure to allergens, microbial infections, viral infections, and toxic chemicals. Chronic
inflammation has been associated with several steps in carcinogenesis including tumor
initiation and promotion, cancerous cell survival, proliferation, and metastasis (Reuter et
al., 2010). Inflammation results from the recruitment of several immune cell types, such
as neutrophils and monocytes, to the site of damage where they act to remove the
damaging stimulus. In the rat exposure to TCDD results in inflammatory cell infiltration
and exposure to 50μgkg TCDD has been demonstrated to increase liver inflammation in
Long-Evans and Hans/Wistar rats (Pohjanvirta et al., 1989; Pohjanvirta and Tuomisto,
1994). Inflammation can lead to the production of reactive oxygen species (ROS) which
is thought to be a potential link between inflammation and the development of cancer as
ROS can induce DNA damage which promotes neoplastic transformations (Reuter et
al., 2010). In primary Wistar rat hepatocytes, treatment of TCDD induced the production
of ROS and 8-oxo-Dg, a hallmark of oxidative DNA damage (Knerr et al., 2006). The
72

induction of DNA damage by ROS has been shown to be AHR-dependent as a study
performed in the mouse hepatoma cell line Hepa1c1c7, demonstrated that AHR
activation by the agonist B[a]P-7,8-dione increased 8-oxo-Dg production whereas
B[a]P-7,8-dione failed to induce the production of 8-oxo-Dg in Hepa1c1c2, a mouse
hepatoma cell line that is deficient in AHR (Park et al., 2009). The evidence that AHR
plays a role in carcinogenesis led to the development of the AHR

-/-

rat to further

evaluate the mechanism of TCDD-mediated tumor development.
While this study did not focus on the liver tumor promoting activities of TCDD, we
had the opportunity to use spleens from AHR

-/-

rats to assess TCDD-mediated

perturbations in several immune cell types with this study focusing on B cell activation
and effector function. Using flow cytometry, the resting B cell expressions of surface
markers CD80, CD86, and MHC II were evaluated to determine if the targeted deletion
of AHR affects the magnitude of B cell activation. No change was observed in the
+

percentage of CD80 B cells between non-activated wild type and AHR

-/-

B cells. It

+

was also determined that the percentage of CD86 B cells were similar between wild
type and AHR
AHR
AHR

-/-/-

-/-

rats. The expression of MHC II in non-activated B cells of wild type and

rats was similar in group one; however, MHC II expression in non-activated
B cells of group two was significantly decreased when compared to non-

activated wild type B cells. The difference in MHC II expression between the groups
may be due to individual or shipment variability as group two was found to be more

73

variable than group one. Taken together, the targeted deletion of AHR does not affect
the B cell in its resting state.
To determine whether the targeted deletion of AHR alters the magnitude of B cell
activation, comparisons were also made between LPS-activated wild type and LPSactivated AHR

-/-

B cells. LPS was a sufficient stimulus to induce B cell activation as the
+

+

percentage of CD80 , CD86 , and MHC II
type and AHR

-/-

+

cells were increased in LPS-treated wild

B cells when compared to non-activated wild type and AHR

-/-

B cells.

The targeted deletion of AHR led to a discrepancy in the expression of MHC II between
group one and two. There was no difference between LPS-activated wild type and LPSactivated AHR

-/-

+

B cells in group two; however, a decrease in the percentage of MHC II

B cells in the AHR

-/-

rat was observed in group one.

expression in the LPS-activated B cells of the AHR

-/-

The decrease in MHC II

rat is in contrast to another study

in which MHC II expression was upregulated in Langerhans cells isolated from AHR

-/-

mice (Jux et al., 2009). The targeted deletion of AHR produced differential effects on
MHC II expression in mice and rats and suggests that the transcription of MHC II is
regulated in a species specific manner. Due to the discrepancy in the effects of MHC II
expression in the LPS-activated B cell of the AHR

-/-

rat, no conclusion can be made as

to whether the targeted deletion of AHR alters the magnitude of MHC II expression. In
contrast to MHC II, clear alterations in the expression of CD80 and CD86 were
observed in the absence of AHR.

In LPS-activated B cells of AHR

74

-/-

rats, the

+

percentage of CD80 cells were upregulated compared to LPS- activated wild type B
cells.
AHR

-/-

Similarly, the percentage of CD86

+

cells were upregulated in LPS-activated

B cells compared to LPS-activated wild type B cells. The upregulation in CD80

and CD86 activation marker expression suggests that AHR plays a role in regulating the
magnitude of B cell activation. In the absence of AHR there is an augmentation of B
cell activation in the AHR

-/-

rat.

In determining whether the targeted deletion of AHR augments B cell effector
function, the IgM antibody response to LPS was compared in wild type and AHR

-/-

rats.

Using flow cytometry, it was observed that LPS treatment increased the percentage of
+

intracellular IgM high B cells of AHR

-/-

rats when compared to LPS-activated B cells of

wild type rats. In addition, IgM secretion as measured by ELISA was also increased in
the LPS-activated AHR

-/-

B cells, when compared to LPS-activated wild type B cells.

The targeted deletion of AHR results in an augmentation in B cell effector function in the
rat as demonstrated by the increase in intracellular IgM and IgM secretion in the AHR

-/-

rat. The augmentation of B cell activation and effector function in the absence of AHR
suggests that AHR regulates B cell activation and effector function at the transcriptional
level.
It is evident by the presence of the ductus venosus in the AHR
renal abnormalities in the AHR

-/-

-/-

mouse and the

rat that the AHR is an important ligand activated

transcription factor for developmental homeostasis. The developmental perturbations in
75

the AHR
ligand.

-/-

mouse and rat may arise from the lack of AHR activation by an endogenous

AHR activation by an endogenous ligand also suggests that AHR activation

may be responsible for regulating B cell homeostasis. Removal of the AHR in the rat
prevents the transcription factor from becoming activated by endogenous ligands such
that it leads to the increase in CD80, CD86, MHC II, and IgM expression. Essentially,
the augmentation of the B cell activation and effector function in the AHR

-/-

rat occurs

because the brake in the regulation of activation marker and IgM expression is absent.
Plasma cell development begins with the activation of signaling cascades
mediated through the engagement of TLR on the surface of the B cell. The early
signaling event within the B cell converges on several protein kinases, including JNK,
AKT, and ERK, which leads to activation of the bistable network of transcription factor
responsible for B cell differentiation.

JNK regulates the activity of AP-1 as JNK

phosphorylates c-Jun, a constituent of AP-1, to increase the transcriptional activity of
AP-1 (Smeal et al., 1991). ERK and AKT are also important controllers of plasma cell
development as they regulate the abundance and transcription of BCL-6 respectively
(Omori et al., 2006; Niu et al., 1998). B cell activation by LPS can converge on JNK,
-/-

ERK, and AKT signaling to initiate B cell differentiation. In addition, studies in AHR

mice have demonstrated that the deletion of AHR results in an increased sensitivity to
LPS (Sekine et al., 2009).

A plausible explanation for the augmentation in B cell

activation and IgM response in the AHR

-/-

rat may be an increase in the sensitivity of

the B cell to LPS which would allow for a more robust activation of JNK, ERK, and AKT.

76

The increased kinase activity may lead to further increases in the transcriptional activity
of AP-1 and decrease in BCL-6 levels such that plasma cell development is enhanced.
In addition to LPS, B cells can also become activated by cytokines secreted from
-/-

various immune cells such as macrophages and T cells. Studies performed in AHR

mice have demonstrated that cytokine secretion by macrophages is also regulated in an
AHR-dependent manner. It has been observed that LPS-activation of bone marrow
-/-

derived macrophages of AHR

mice resulted in an increased secretion of interleukin-1β

(IL-1β) when compared to wild type mice (Sekine et al., 2009).
-/-

conducted in AHR

A second study

mice had demonstrated that LPS-activation of bronchoalveolar

lavage cells, comprised of

99% macrophages, resulted in an increase in IL-6 secretion

compared to wild type mice (Thatcher et al., 2007). The cytokines IL-1β and IL-6 play
an important role in B cell function as IL-1β can promote B cell activation and IL-6 is an
important cytokine in promoting B cell differentiation. The deletion of AHR in the rat
may also affect macrophages and lead to an increase in IL-1β and IL-6 secretion. It may
be possible that the elevated IL-1β and IL-6 secretion by macrophages could promote
enhanced B cell activation and differentiation and be partially responsible for the
-/-

augmentation in B cell activation and effector function observed in the AHR
-/-

While both wild type and AHR

rat.

rat B cells were activated in response to LPS,

the magnitude of B cell activation and the primary antibody response were not as strong
when compared to other B cell studies previously reported by our laboratory. The
increased expression of rat CD80, CD86, and MHC II in response to LPS is lower
compared to previous mouse in vitro and in vivo studies from our laboratory (North et
77

al., 2009 et al. North 2010). Also, the increased expression of rat CD80 and CD86 in
response to LPS is lower compared to the increase of CD80 and CD86 in activated
primary human B cells previously reported by our laboratory (Lu et al., 2009; Lu et al.,
2011). The decrease in the magnitude of B cell activation in response to LPS in the rat
may be due to a reduction in the sensitivity of the rat B cell to LPS. It has been reported
that the sensitivity to LPS can vary among species with mouse being one of the most
responsive species to LPS while the rat has been found to be one of the least sensitive
species to LPS (Brade, 1999). It has also been reported that there are intra-strain
differences in the sensitivity to LPS with the Winstar rat being more resistant to LPS in
comparison to the Sprague-Dawley rat (Brade, 1999). While rats are one of the most
resistant strains to LPS, the strain used in this study (Sprague-Dawley) is one of the
more responsive strains within the rat species.
As mentioned, the response of B cells to LPS is mediated through activation of
TLR4 and species specific differences in tissue distribution and function may be partly
responsible for the reduced sensitivity to LPS in the rat. Since the LPS response in wild
-/-

type and AHR

rats was lower compared to what has been observed in the mouse it is

possible that the tissue distribution of TLR4 in the rat differs from the mouse. In the
mouse, lung, heart, leukocytes and muscle tissue express high levels of TLR4 and in
humans TLR4 expression in B cells is very low (Ganley-Leal et al., 2010; Rehli, 2002).
The rat B cell may have similar levels of TLR4 expression to that of the human B cell
leading to the reduction of sensitivity to LPS. Also, functional differences in TLR4 have
been determined between human and mouse (Werling et al., 2009). LPS treatment
increased TLR4 expression in human macrophages whereas LPS downregulated the
78

expression of TLR4 in murine macrophages (Rehli, 2002).

The species specific

functional differences shown in mouse and human suggest that there could be
additional functional differences of TLR4 in the rat.
While the magnitude of B cell activation and effector function was compared in
LPS-activated wild type and AHR

-/-

rats, this study also evaluated the sensitivity of the B

cell to TCDD to determine if TCDD alters B cell function in the presence and absence of
AHR. TCDD produced modest alterations in B cell activation of AHR

-/-

and wild type

rats but no common trend toward suppression or enhancement was observed between
the two groups and no single dose of TCDD elicited more effects than other doses
used. The TCDD-mediated alterations in B cell activation observed were mainly on the
expression of MHC II and TCDD-mediated upregulation in the percentage and MFI of
+

MHC II B cells in both wild type and knockout rats of group one. The upregulation of
MHCII observed in the rat is similar to previous reports that TCDD increases MHC II
expression in mouse denditric cells (Bankoti et al., 2010). However, in group two TCDD
+

treatment suppressed the percentage of MHC II B cells in AHR

-/-

rats. The variability

in the effect of TCDD on the expression of MHC II makes it difficult to conclude that
TCDD-mediates a specific effect on MHC II expression.
Despite the variability in the effect of MHC II, clear TCDD-mediated alterations
were made on the expression of CD80.
+

-/-

percentage of CD80 in wild type or AHR
was observed in the AHR

-/-

TCDD did not significantly affect the
rats; however, a trend towards a decrease

rats and a trend towards an increase was observed in wild

79

type rats.

The TCDD-mediated increase in CD80 of wild type rats has also been

demonstrated in a recent study conducted in wild type and AHR

-/-

mice in which TCDD-

mediated upregulation of CD80 and CD86 expression in dendritic cells of wild type mice
(Vogel et al., 2013). In this study, TCDD did not affect the expression of CD86 in wild
-/-

type or AHR

rats. The increase in CD80 expression of wild type rats demonstrated in

this study is in divergence from other studies performed previously by our laboratory in
which TCDD-mediated suppression of CD80 and CD86 in mice and humans (Lu et al.,
2011; North et al., 2009).

Though CD80 was moderately affected by TCDD, no

significant TCDD-mediated alterations in B cell activation were observed and suggest
that the doses selected were suboptimal for inducing immune effects. The doses of
TCDD that were selected were appropriate for a cancer model and it should be noted
that in vivo doses typically used for immunological studies are done in the μg/kg range
and are only given as a single dose rather than several ng/kg doses over the course of
several weeks. For example, studies conducted in the rat have demonstrated that
TCDD-mediated suppression of the IgM response resulted from single in vivo doses
ranging from 1-30μg/kg TCDD. The dosing scheme may have been a possibility for the
lack of significant TCDD effects on B cell activation.
Several studies have demonstrated significant TCDD-mediated effects on the
primary antibody response in the rat and interestingly both TCDD-mediated suppression
and enhancement of IgM has been observed. Reports from several laboratories have
demonstrated TCDD-mediated suppression of the primary antibody response in the
Fischer 344, Brown-Norway, Sprague-Dawley, and Wistar albino rat strains under a
variety of experimental conditions. It should be noted that although the B cell activators
80

and doses of TCDD used varied from study to study, the commonality among the
studies was that the rats were dosed with TCDD prior to being immunized. In the
Fischer 344 rat study, 30μg/kg TCDD was given as a single intraperitoneal injection and
seven days after dosing rats were immunized with TNP-LPS (Smialowicz et al., 1996).
Three days post immunization it was observed that the PFC response to TNP-LPS was
suppressed (Smialowicz et al., 1996). At five days post immunization with sRBC; the
Sprague-Dawley rat also demonstrated suppression in the PFC response when dosed
with a single intraperitoneal injection of 100μg/kg TCDD given one week prior to
immunization (Rozman et al., 1984). In the studies performed in the Brown-Norway rat,
TCDD was given as a single intraperitoneal dose seven days prior to sensitization to
house dust mite allergen (Luebke et al., 2001). Two week post sensitization, BrownNorway rats were challenged with house dust mite allergens and the IgG and IgE
antibody response was suppressed upon exposure to 10 and 30μg/kg TCDD (Luebke et
al., 2001). The study performed in the Wistar albino rat differs from the other rat strain
studies in that Wistar albino rats were orally gavaged with 2μg/kg/week TCDD for 8
weeks (Çiftçi 2011). TCDD-mediated a suppression of IgM in the Wistar albino rat and
this suppression could be partially recovered when the rats were also administered with
100mg/kg/day curcumin concurrently with TCDD (Çiftçi 2011). The dosing scheme in
this study differs from the other studies performed in the rat as lower doses of TCDD (31000ng/kg/day) were given over an extended period of time (4 weeks). Also, this study
differs from previous reports as LPS was used to activate B cells. Although the
experimental conditions were different from earlier studies performed in the rat, similar
results were observed as TCDD-mediated a trend towards suppression in the

81

percentage of IgM

+

high B cells of wild type rats.

The observed trend towards a

suppression of the IgM response was not significant which may have been due to low
doses of TCDD used in this study. To allow for the detection of significant TCDDmediated suppression of the IgM response to LPS in wild type rats, it would be
beneficial to increase the dose of TCDD as this would allow for a more accurate
estimation of TCDD-mediated alterations of the immune response in the rat.
It has also been demonstrated in Fischer 344, Long-Evans, and Sprague-Dawley
rats that TCDD treatment increased the primary antibody response under different
experimental conditions. Fischer 344 and Long-Evans rats were dosed with 1, 3, 10,
and 30μg/kg TCDD by a single intraperitoneal injection and immunized with sRBC one
week post TCDD treatment (Smialowicz et al, 1994). Four days after immunization it
was demonstrated that there was a dose-dependent increase in the PFC response to
sRBC starting at 3μg/kg TCDD in the Fischer 344 rat and an increase of the PFC
response at 1, 3, and 10μg/kg TCDD in the Long-Evans rat. (Smialowicz et al, 1994).
In the Sprague-Dawley study, rats were orally gavaged with 10, 20, and 40μg/kg TCDD
and immunized sRBC five days post TCDD treatment (Fan et al., 1996). At 7 and 14
days post immunization, TCDD-mediated a dose-dependent increase in serum IgG
levels with no change in IgM levels occurring in Sprague-Dawley rats (Fan et al., 1996).
While doses of TCDD and the B cell activator used in this study differed from the
previous reports it was also observed that TCDD treatment resulted in a trend toward an
+

-/-

increase in the percentage of IgM high B cells in AHR

rats, however the increase

was not significant. ELISA was also used to evaluate TCDD-mediated alterations in the
magnitude of B cell effector function and no TCDD effect was observed in wild type or
82

AHR

-/-

rats. The changes observed in the magnitude of the primary antibody response

were only observed when the treatments were evaluated by flow cytometry and this is
due to flow cytometry having a greater sensitivity in the detection of IgM compared to
ELISA. Although no significant effects of TCDD treatment were observed, the trend
-/-

towards TCDD-mediated enhancement of IgM in AHR

B cells suggests it is possible

that the deletion of AHR does not result in the loss of sensitivity of the B cell to TCDD.
As mentioned, the lack of a significant increase of the IgM antibody response in the
AHR

-/-

rats may be due to the lower doses of TCDD used in this study.

The differential effects of TCDD on the primary antibody response may also be a
result of the rat strain used in each of the studies as not all rat strains are equally
sensitive to TCDD. The Hans-Wistar rat is one of the most resistant strains to TCDD
with an LD50 of over 9600μg/kg while the Long-Evans rat is one of the more sensitive
strains with an LD50 of 10-20μg/kg (Tuomisto et al., 1998). This study utilized the
Sprague-Dawley rat strain and this strain is known to be one of the more sensitive
strains to TCDD as the AHR cDNA sequence between the Long-Evans and SpragueDawley rats are similar while the cDNA sequence of AHR in the Hans-Wistar rat differs
(Tuomisto et al., 1998). In addition to the differences in AHR sequence between rat
-/-

strains and the modest upregulation of IgM in AHR

rat observed in this study, the toxic

effects of TCDD in the rat suggests that AHR may interact with other factors such as
NF-κB in eliciting the effects of TCDD-mediated toxicity as well as altering the
magnitude of B cell activation and effector function.

83

The NF-κB family of transcription factors regulate the expression of over 200 genes
that correspond to various immune functions such as inflammation, cell growth, and B
cell development (Aggarwal, 2004; Feng et al., 2004) and is composed of NF-κB
proteins such as p50 and Rel proteins that include p65 (RelA), RelB, and c-Rel. Many
proteins are involved in NF-κB signaling and the proteins involved in the canonical and
noncanonical pathways can be reviewed in (Gilmore, 2006). The involvement of NF-κB
in immune cell function has lead to the discovery that AHR associates with several
members of the NF-κB family including p65 and RelB in several cell types (Barouki et
al., 2007; Ruby et al., 2002). Some effects of NF-κB that have been demonstrated in
immune cell activation include studies in primary human dendritic cells by which
inhibition of NF-κB downregulated the expression of CD80, CD86, and MHC II
(Yoshimura et al., 2001).

Similar results were observed in primary human monocytes

in that blocking NF-κB translocation to the nucleus inhibited LPS-mediated induction of
CD80, CD86 and MHC II (Ardeshna et al., 2000).
A requirement of NF-κB transcriptional activity includes the association with coactivator proteins and it has been suggested that AHR and NF-κB share the
transcriptional co-activator steroid co-activator 1 (SRC-1) in addition to NF-κB and
ARNT sharing the co-activator p300/CBP (Tian et al., 2002).

The sharing of co-

activator proteins may create a competition between AHR and NF-κB for binding to
transcriptional co-activators.

The deletion of AHR in the rat may alleviate the

competition between AHR and NF-κB such that NF-κB can bind to SRC-1 and
p300/CBP to increase the expression of CD80, CD86, and MHC II compared to wild
type rats. Additional evidence for supporting competition between AHR and NF-κB was

84

found in a study using the Hepa1c1c7 cell line which demonstrated that AHR
suppressed NF-κB DNA binding and TCDD treatment enhanced the inhibition of AHR
on NF-κB, preventing the binding of NF-κB to its promoter regions within the DNA (Tian
et al., 1999).

However, a previous report demonstrated that TCDD induced DNA

binding of NF-κB family members p65, RelB, and c-Rel in CH12.LX cells which contain
AHR (Sulentic et al., 2000). The TCDD-mediated increase in binding of NF-κB in the
presence of AHR suggests that activation of NF-κB in B cells is AHR-independent. In
studies using AHR
-/-

activity in AHR

-/-

mice, it has been observed that there is an increase in NF-κB

mice compared to wild type (Thatcher et al., 2007). The augmentation

of the magnitude of B cell activation in the AHR

-/-

rat may suggest that AHR and NF-κB

interact in the rat and these interactions could account for the differences in what has
been observed in the wild type and AHR

-/-

B cells. In the presence of AHR, it may be

possible that AHR inhibits NF-κB activation by LPS such that B cell activation is
suppressed resulting in decreased expression of CD80, CD86, and MHC II which could
subsequently decrease the primary IgM antibody response. In contrast, the increased
CD80, CD86, and MHC II expression of the AHR

-/-

rat observed in this study may have

resulted from the AHR-mediated repression of NF-κB being lifted.
In addition to modifications to the magnitude of B cell activation, the suppression
of NF-κB by AHR could also mediate perturbations in the primary IgM antibody
response as NF-κB has been shown to interact with some of the transcription factors in
the bistable network of transcription factors that is required for B cell differentiation.
One mouse study has demonstrated that inhibition of NF-κB in LPS-activated B cells
85

abates BLIMP-1 expression (Morgan et al., 2009). Using a human pancreatic cell line,
another study observed that a significant downregulation of c-fos, a component of AP-1,
occurred upon the inhibition of NF-κB (Fujioka et al., 2004).

The AHR-mediated

suppression of NF-κB could potentially reduce the expression of transcription factors
that promote B cell differentiation leading to a reduction in plasma cell development
subsequently affecting the IgM response to LPS.
observed in the AHR

-/-

The increased IgM response

rat may have resulted from increased NF-κB activity which

upregulated AP-1 and BLIMP-1 expression to promote B cell differentiation.

The

evidence that NF-κB activity affects CD80, CD86 and MHC II expression and
transcription factors required for plasma cell development suggest that NF-κB could
serve as a link between B cell activation and effector function in the rat.

86

Conclusion

TCDD-mediated suppression of the immune response have been evaluated over
the course of several decades and studies have utilized a variety of models including
cell lines derived from several species, primary rat B cells, primary mouse B cells, and
primary human B cells.

The advance in scientific technology has allowed for the
-/-

creation of knockout animal models and the AHR

mouse has been an important tool in

determining the mechanism of TCDD-induced toxicity. Despite the multitude of models
available the mechanisms by which TCDD mediates its immunotoxic effects have not
yet been discovered. Also, the manner in which AHR is able to modulate B cell function
in response to TCDD is unknown. We had the opportunity to utilize spleens from the
newly developed AHR

-/-

rat as a novel model system to evaluate the role the AHR plays

in modulating B cell activation and effector function in the presence and absence of
TCDD.
This thesis demonstrates for the first time that the AHR is capable of regulating
the magnitude of B cell activation as the deletion of the AHR led to the increase in CD80
and CD86 when compared to wild type rats activated in vitro with LPS. MHC II was also
used to measure the magnitude of B cell activation but due to the variability between
groups it was excluded in the conclusion that the targeted deletion of AHR augments B
cell activation. It was also discovered that AHR augments the IgM antibody response to
LPS as B cells from AHR

-/-

+

rats have an increased percentage of IgM high B cells as

well as an increase in secreted IgM when compared to B cells of wild type rats.

87

-/-

The augmentation in B cell activation and effector function in the AHR

rat as

well as the sensitivity of the B cell to TCDD remaining intact are novel findings which
illustrate that AHR not only mediates TCDD-induced toxicity but in part is also
responsible for regulating B cell physiology.

The concomitant increase in B cell

activation marker expression and IgM response upon LPS activation in the absence of
AHR also suggests that these two B cell events are correlated with one another. While
the mechanisms in which the magnitude of B cell activation are correlated with the
magnitude of effector function was not determined, it would be of interest to evaluate
-/-

alterations of the MAPK and NF-κB pathway in the AHR

rat as the MAPK family

members JNK, ERK, and AKT in addition to NF-κB are known to interact with AHR as
well as regulating the expression of activation markers and transcription factors that are
responsible for B cell differentiation.
The NF-κB signaling cascade is composed of a number of proteins and future
studies could be performed to determine which members of the NF-κB pathway
associate with AHR in rat as well as the effects of NF-κB regulation on transcription in
the presence and absence of AHR. Future studies could also evaluate the possibility of
a direct effect of TCDD on NF-κB since this study has observed modest TCDDmediated alterations in B cell activation and effector function in the absence of AHR. In
addition to the future studies evaluating the effect of TCDD and the targeted deletion of
AHR on NF-κB, it would be interesting to repeat the studies conducted in this thesis
using an alternate B cell activator rather than LPS since the response to LPS was not
as robust when compared to the mouse and human studies performed previously in our
laboratory. It may be beneficial to activate the rat B cell using an alternate T-cell
88

independent activator or T-cell dependent method to allow for more accurate
comparisons between species. Moreover, the doses of TCDD used in this study were
suboptimal for producing significant changes in B cell activation and effector function
and it would be beneficial to use doses of TCDD that range from 30-1μg/kg which are
similar to the historical studies performed in the rat which have established TCDDmediated alterations in B cell effector function, this would allow for more accurate
comparisons of TCDD-mediated toxicity among species.
In summary, significant scientific advances in this study have been made not only
in addressing the question whether AHR modulates the magnitude of B cell activation
and effector function but also in providing new evidence of TCDD-mediated effects that
occur in the absence of AHR. The establishment of the novel AHR

-/-

rat model has led

to the discovery of species specific alterations in B cell activation and effector function
not previously observed in the mouse and human and can serve as a useful tool in the
exploration of new possibilities to determine the mechanism of TCDD-induced
immunotoxicity.

89

APPENDIX

90

APPENDIX

A. Antibodies
Target
CD80

Fluorophore
PE

Host
Mouse

Reactivity
Rat

CD86

Alexa Flour 647

Mouse

Rat

MHC II

PerCP

Mouse

Rat

IgM

None

Mouse

Rat

IgM

FITC

Mouse

Rat

91

Type
Monoclonal;
3H5
Monoclonal;
24F
Monoclonal;
Ox-6
Monoclonal;
G53-238
Monoclonal;
G53-238

Supplier
BD Pharmingen
Biolegend
BD Pharmingen
BD Pharmingen
BD Pharmingen

BIBLIOGRAPHY

92

BIBLIOGRAPHY
(2006). NTP technical report on the toxicology and carcinogenesis studies of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) (CAS No. 1746-01-6) in female Harlan SpragueDawley rats (Gavage Studies). Natl Toxicol Program Tech Rep Ser. 4-232.
Abel, J., and Haarmann-Stemmann, T. (2010). An introduction to the molecular basics
of aryl hydrocarbon receptor biology. Biol Chem 391(11), 1235-48.
Aggarwal, B. B. (2004). Nuclear factor-κB: the enemy within. Cancer cell 6(3), 203-8.
Ardeshna, K. M., Pizzey, A. R., Devereux, S., and Khwaja, A. (2000). The PI3 kinase,
p38 SAP kinase, and NF-kappaB signal transduction pathways are involved in the
survival and maturation of lipopolysaccharide-stimulated human monocyte-derived
dendritic cells. Blood 96(3), 1039-46.
Bankoti, J., Rase, B., Simones, T., and Shepherd, D. M. (2010). Functional and
phenotypic effects of AhR activation in inflammatory dendritic cells. Toxicol Appl
Pharmacol 246(1-2), 18-28.
Barouki, R., Coumoul, X., and Fernandez-Salgueroc, P. M. (2007). The aryl
hydrocarbon receptor, more than a xenobiotic-interacting protein. Febs Letters 581(19),
3608-15.
Bekeredjian-Ding, I., Foermer, S., Kirschning, C. J., Parcina, M., and Heeg, K. (2012).
Poke weed mitogen requires Toll-like receptor ligands for proliferative activity in human
and murine B lymphocytes. PLoS One 7(1), e29806.
Bhattacharya, S., Conolly, R. B., Kaminski, N. E., Thomas, R. S., Andersen, M. E., and
Zhang, Q. (2010). A bistable switch underlying B-cell differentiation and its disruption by
the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Sci 115(1),
51-65.
Black, M. B., Budinsky, R. A., Dombkowski, A., Cukovic, D., LeCluyse, E. L., Ferguson,
S. S., Thomas, R. S., and Rowlands, J. C. (2012). Cross-species comparisons of
transcriptomic alterations in human and rat primary hepatocytes exposed to 2,3,7,8tetrachlorodibenzo-p-dioxin. Toxicol Sci 127(1), 199-215.
Brade, H. (1999). Endotoxin in health and disease. CRC Press.
93

Bunger, M. K., Glover, E., Moran, S. M., Walisser, J. A., Lahvis, G. P., Hsu, E. L., and
Bradfield, C. A. (2008). Abnormal liver development and resistance to 2,3,7,8tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding
domain of the aryl hydrocarbon receptor. Toxicol Sci 106(1), 83-92.
Calame, K. L. (2001). Plasma cells: finding new light at the end of B cell development.
Nat Immunol 2(12), 1103-8.
Çiftçi, O. (2011). Curcumin prevents toxic effects of 2, 3, 7, 8-tetrachlorodibenzo-pdioxin (TCDD) on humoral immunity in rats. Food and Agricultural Immunology 22(1),
31-8.
Crawford, R. B., Holsapple, M. P., and Kaminski, N. E. (1997). Leukocyte Activation
Induces Aryl Hydrocarbon Receptor Up-Regulation, DNA Binding, and Increased
Cyp1a1Expression in the Absence of Exogenous Ligand. Molecular pharmacology
52(6), 921-7.
Crawford, R. B., Sulentic, C. E., Yoo, B. S., and Kaminski, N. E. (2003). 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) alters the regulation and posttranslational
modification of p27kip1 in lipopolysaccharide-activated B cells. Toxicol Sci 75(2), 33342.
De Abrew, K. N., Phadnis, A. S., Crawford, R. B., Kaminski, N. E., and Thomas, R. S.
(2011). Regulation of Bach2 by the aryl hydrocarbon receptor as a mechanism for
suppression of B-cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl
Pharmacol 252(2), 150-8.
Dooley, R. K., and Holsapple, M. P. (1988). Elucidation of cellular targets responsible
for tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of antibody responses:
The role of the B lymphocyte. Immunopharmacology 16(3), 167-80.
El-Shahawi, M. S., Hamza, A., Bashammakh, A. S., and Al-Saggaf, W. T. (2010). An
overview on the accumulation, distribution, transformations, toxicity and analytical
methods for the monitoring of persistent organic pollutants. Talanta 80(5), 1587-97.
Esser, C. (2009). The immune phenotype of AhR null mouse mutants: not a simple
mirror of xenobiotic receptor over-activation. Biochem Pharmacol 77(4), 597-607.
Fairfax, K. A., Kallies, A., Nutt, S. L., and Tarlinton, D. M. Plasma cell development:
from B-cell subsets to long-term survival niches2008, pp. 49-58.
94

Fan, F., Wierda, D., and Rozman, K. K. (1996). Effects of 2, 3, 7, 8-tetrachlorodibenzop-dioxin on humoral and cell-mediated immunity in Sprague-Dawley rats. Toxicology
106(1), 221-8.
Feng, B., Cheng, S., Pear, W. S., and Liou, H.-C. (2004). NF-kB inhibitor blocks B cell
development at two checkpoints. Med Immunol 3(1), 1.
Forgacs, A. L., Dere, E., Angrish, M. M., and Zacharewski, T. R. (2013). Comparative
analysis of temporal and dose-dependent TCDD-elicited gene expression in human,
mouse, and rat primary hepatocytes. Toxicol Sci 133(1), 54-66.
Fujii-Kuriyama, Y., and Mimura, J. (2005). Molecular mechanisms of AhR functions in
the regulation of cytochrome P450 genes. Biochem Biophys Res Commun 338(1), 3117.
Fujioka, S., Niu, J., Schmidt, C., Sclabas, G. M., Peng, B., Uwagawa, T., Li, Z., Evans,
D. B., Abbruzzese, J. L., and Chiao, P. J. (2004). NF-kappaB and AP-1 connection:
mechanism of NF-kappaB-dependent regulation of AP-1 activity. Mol Cell Biol 24(17),
7806-19.
Ganley-Leal, L. M., Liang, Y., Jagannathan-Bogdan, M., Farraye, F. A., and Nikolajczyk,
B. S. (2010). Differential regulation of TLR4 expression in human B cells and
monocytes. Mol Immunol 48(1-3), 82-8.
Gilmore, T. D. (2006). Introduction to NF-kappaB: players, pathways, perspectives.
Oncogene 25(51), 6680-4.
Hao, N., and Whitelaw, M. L. (2013). The emerging roles of AhR in physiology and
immunity. Biochem Pharmacol 86(5), 561-70.
Hardy, R. R., and Hayakawa, K. (2001). B cell development pathways. Annu Rev
Immunol 19(1), 595-621.
Harrill, J. A., Hukkanen, R. R., Lawson, M., Martin, G., Gilger, B., Soldatow, V.,
Lecluyse, E. L., Budinsky, R. A., Rowlands, J. C., and Thomas, R. S. (2013). Knockout
of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats
and mice. Toxicol Appl Pharmacol 272(2), 503-18.

95

Harstad, E. B., Guite, C. A., Thomae, T. L., and Bradfield, C. A. (2006). Liver
deformation in Ahr-null mice: evidence for aberrant hepatic perfusion in early
development. Mol Pharmacol 69(5), 1534-41.
Holsapple, M. P., Dooley, R. K., McNerney, P. J., and Ann McCay, J. (1986). Direct
suppression of antibody responses by chlorinated dibenzodioxins in cultured spleen
cells from (C57BL/6× C3H) F1 and DBA/2 mice. Immunopharmacology 12(3), 175-86.
Holsapple, M. P., Morris, D. L., Wood, S. C., and Snyder, N. K. (1991). 2,3,7,8tetrachlorodibenzo-p-dioxin-induced changes in immunocompetence: possible
mechanisms. Annu Rev Pharmacol Toxicol 31(1), 73-100.
Hu, W. Y., Sorrentino, C., Denison, M. S., Kolaja, K., and Fielden, M. R. (2007).
Induction of Cyp1A1 is a nonspecific biomarker of aryl hydrocarbon receptor activation:
Results of large scale screening of pharmaceuticals and toxicants in vivo and in vitro.
Molecular Pharmacology 71(6), 1475-86.
Iwasaki, A., and Medzhitov, R. (2004). Toll-like receptor control of the adaptive immune
responses. Nat Immunol 5(10), 987-95.
Jilling, T., Simon, D., Lu, J., Meng, F. J., Li, D., Schy, R., Thomson, R. B., Soliman, A.,
Arditi, M., and Caplan, M. S. (2006). The roles of bacteria and TLR4 in rat and murine
models of necrotizing enterocolitis. J Immunol 177(5), 3273-82.
Jux, B., Kadow, S., and Esser, C. (2009). Langerhans cell maturation and contact
hypersensitivity are impaired in aryl hydrocarbon receptor-null mice. J Immunol 182(11),
6709-17.
Kaminski, D. A., Wei, C., Qian, Y., Rosenberg, A. F., and Sanz, I. (2012). Advances in
human B cell phenotypic profiling. Front Immunol 3(302), 1-15.
Kerkvliet, N. I. (2002). Recent advances in understanding the mechanisms of TCDD
immunotoxicity. Int Immunopharmacol 2(2-3), 277-91.
Knerr, S., Schaefer, J., Both, S., Mally, A., Dekant, W., and Schrenk, D. (2006). 2,3,7,8Tetrachlorodibenzo-p-dioxin induced cytochrome P450s alter the formation of reactive
oxygen species in liver cells. Mol Nutr Food Res 50(4-5), 378-84.

96

Lin, K. I., Angelin-Duclos, C., Kuo, T. C., and Calame, K. (2002). Blimp-1-dependent
repression of Pax-5 is required for differentiation of B cells to immunoglobulin Msecreting plasma cells. Mol Cell Biol 22(13), 4771-80.
Lu, H., Crawford, R. B., Kaplan, B. L., and Kaminski, N. E. (2011). 2,3,7,8tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation
of primary human B cells. Toxicol Appl Pharmacol 255(3), 251-60.
Lu, H., Crawford, R. B., Suarez-Martinez, J. E., Kaplan, B. L., and Kaminski, N. E.
(2010). Induction of the aryl hydrocarbon receptor-responsive genes and modulation of
the immunoglobulin M response by 2,3,7,8-tetrachlorodibenzo-p-dioxin in primary
human B cells. Toxicol Sci 118(1), 86-97.
Luebke, R., Copeland, C., and Andrews, D. (1995). Short Communication. Host
resistance to Trichinella spiralis infection in rats exposed to 2, 3, 7, 8tetrachlorodibenzo-p-dioxin (TCDD). Fund. Appl. Toxicol 24, 285-9.
Luebke, R. W., Copeland, C. B., Daniels, M., Lambert, A. L., and Gilmour, M. I. (2001).
Suppression of allergic immune responses to house dust mite (HDM) in rats exposed to
2,3,7,8-TCDD. Toxicol Sci 62(1), 71-9.
Marinkovic, N., Pasalic, D., Ferencak, G., Grskovic, B., and Stavljenic Rukavina, A.
(2010). Dioxins and human toxicity. Arh Hig Rada Toksikol 61(4), 445-53.
Mcconnell, E. E. (1985). Comparative Toxicity of Pcbs and Related-Compounds in
Various Species of Animals. Environ Health Persp 60(May), 29-33.
Moffat, I. D., Boutros, P. C., Chen, H., Okey, A. B., and Pohjanvirta, R. (2010). Aryl
hydrocarbon receptor (AHR)-regulated transcriptomic changes in rats sensitive or
resistant to major dioxin toxicities. BMC Genomics 11(1), 263.
Morgan, M. A., Magnusdottir, E., Kuo, T. C., Tunyaplin, C., Harper, J., Arnold, S. J.,
Calame, K., Robertson, E. J., and Bikoff, E. K. (2009). Blimp-1/Prdm1 alternative
promoter usage during mouse development and plasma cell differentiation. Mol Cell Biol
29(21), 5813-27.
Morris, D. L., and Holsapple, M. P. (1991). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) on humoral immunity: II. B cell activation. Immunopharmacology 21(3), 171-81.

97

Nebert, D. W. (1989). The Ah locus: genetic differences in toxicity, cancer, mutation,
and birth defects. Crit Rev Toxicol 20(3), 153-74.
Nguyen, N. T., Hanieh, H., Nakahama, T., and Kishimoto, T. (2013). The roles of aryl
hydrocarbon receptor in immune responses. Int Immunol 25(6), 335-43.
Niu, H., Ye, B. H., and Dalla-Favera, R. (1998). Antigen receptor signaling induces MAP
kinase-mediated phosphorylation and degradation of the BCL-6 transcription factor.
Genes Dev 12(13), 1953-61.
North, C. M., Crawford, R. B., Lu, H., and Kaminski, N. E. (2009). Simultaneous in vivo
time course and dose response evaluation for TCDD-induced impairment of the LPSstimulated primary IgM response. Toxicol Sci 112(1), 123-32.
Omori, S. A., Cato, M. H., Anzelon-Mills, A., Puri, K. D., Shapiro-Shelef, M., Calame, K.,
and Rickert, R. C. (2006). Regulation of class-switch recombination and plasma cell
differentiation by phosphatidylinositol 3-kinase signaling. Immunity 25(4), 545-57.
Ono, S. J., Liou, H. C., Davidon, R., Strominger, J. L., and Glimcher, L. H. (1991).
Human X-Box-Binding Protein-1 Is Required for the Transcription of a Subset of Human
Class-II Major Histocompatibility Genes and Forms a Heterodimer with C-Fos. Proc Natl
Acad Sci USA 88(10), 4309-12.
Park, J. H., Mangal, D., Frey, A. J., Harvey, R. G., Blair, I. A., and Penning, T. M.
(2009). Aryl hydrocarbon receptor facilitates DNA strand breaks and 8-oxo-2'deoxyguanosine formation by the aldo-keto reductase product benzo[a]pyrene-7,8dione. J Biol Chem 284(43), 29725-34.
Pazdernik, T. L., and Rozman, K. K. (1985). Effect of thyroidectomy and thyroxine on
2,3,7,8-tetrachlorodibenzo-p-dioxin-induced immunotoxicity. Life Sci 36(7), 695-703.
Pohjanvirta, R., and Tuomisto, J. (1994). Short-term toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin in laboratory animals: effects, mechanisms, and animal
models. Pharmacol Rev 46(4), 483-549.
Pohjanvirta, R., Kulju, T., Morselt, A. F., Tuominen, R., Juvonen, R., Rozman, K.,
Mannisto, P., Collan, Y., Sainio, E. L., and Tuomisto, J. (1989). Target tissue
morphology and serum biochemistry following 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) exposure in a TCDD-susceptible and a TCDD-resistant rat strain. Fundam Appl
Toxicol 12(4), 698-712.
98

Poland, A., and Glover, E. (1974). Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a
potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. Mol
Pharmacol 10(2), 349-59.
Poland, A., and Glover, E. (1980). 2,3,7,8-tetrachlorodibenzo-p-dioxin - Segregation of
Toxicity with the Ah Locus. Mol Pharmacol 17(1), 86-94.
Poland, A., and Knutson, J. C. (1982). 2,3,7,8-tetrachlorodibenzo-p-dioxin and related
halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu
Rev Pharmacol Toxicol 22, 517-54.
Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine
aryl hydrocarbon receptor. Mol Pharmacol 46(5), 915-21.
Quintana, F. J., and Sherr, D. H. (2013). Aryl hydrocarbon receptor control of adaptive
immunity. Pharmacol Rev 65(4), 1148-61.
Rehli, M. (2002). Of mice and men: species variations of Toll-like receptor expression.
Trends Immunol 23(8), 375-8.
Reimold, A. M., Iwakoshi, N. N., Manis, J., Vallabhajosyula, P., Szomolanyi-Tsuda, E.,
Gravallese, E. M., Friend, D., Grusby, M. J., Alt, F., and Glimcher, L. H. (2001). Plasma
cell differentiation requires the transcription factor XBP-1. Nature 412(6844), 300-7.
Reuter, S., Gupta, S. C., Chaturvedi, M. M., and Aggarwal, B. B. (2010). Oxidative
stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 49(11),
1603-16.
Rodo, J., Goncalves, L. A., Demengeot, J., Coutinho, A., and Penha-Goncalves, C.
(2006). MHC class II molecules control murine B cell responsiveness to
lipopolysaccharide stimulation. J Immunol 177(7), 4620-6.
Rowlands, J. C., and Gustafsson, J. A. (1997). Aryl hydrocarbon receptor-mediated
signal transduction. Crit Rev Toxicol 27(2), 109-34.
Ruby, C. E., Leid, M., and Kerkvliet, N. I. (2002). 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin
suppresses tumor necrosis Factor-α and Anti-CD40–Induced Activation of NF-κB/Rel in
dendritic cells: p50 Homodimer Activation is not affected. Mol Pharmacol 62(3), 722-8.

99

Rysavy, N. M., Maaetoft-Udsen, K., and Turner, H. (2013). Dioxins: diagnostic and
prognostic challenges arising from complex mechanisms. J Appl Toxicol 33(1), 1-8.
Safe, S. H. (1986). Comparative toxicology and mechanism of action of polychlorinated
dibenzo-p-dioxins and dibenzofurans. Annu Rev Pharmacol Toxicol 26(1), 371-99.
Schmidt, J. V., Su, G. H., Reddy, J. K., Simon, M. C., and Bradfield, C. A. (1996).
Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic
growth and development. Proc Natl Acad Sci USA 93(13), 6731-6.
Schneider, D., Manzan, M. A., Crawford, R. B., Chen, W., and Kaminski, N. E. (2008).
2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated impairment of B cell differentiation
involves dysregulation of paired box 5 (Pax5) isoform, Pax5a. J Pharmacol Exp Ther
326(2), 463-74.
Sciammas, R., Li, Y., Warmflash, A., Song, Y., Dinner, A. R., and Singh, H. (2011). An
incoherent regulatory network architecture that orchestrates B cell diversification in
response to antigen signaling. Mol Syst Biol 7(1), 495.
Sekine, H., Mimura, J., Oshima, M., Okawa, H., Kanno, J., Igarashi, K., Gonzalez, F. J.,
Ikuta, T., Kawajiri, K., and Fujii-Kuriyama, Y. (2009). Hypersensitivity of aryl
hydrocarbon receptor-deficient mice to lipopolysaccharide-induced septic shock. Mol
Cell Biol 29(24), 6391-400.
Shaffer, A. L., Lin, K. I., Kuo, T. C., Yu, X., Hurt, E. M., Rosenwald, A., Giltnane, J. M.,
Yang, L., Zhao, H., Calame, K., and Staudt, L. M. (2002). Blimp-1 orchestrates plasma
cell differentiation by extinguishing the mature B cell gene expression program.
Immunity 17(1), 51-62.
Shapiro-Shelef, M., and Calame, K. (2005). Regulation of plasma-cell development. Nat
Rev Immunol 5(3), 230-42.
Singh, K. P., Garrett, R. W., Casado, F. L., and Gasiewicz, T. A. (2011). Aryl
hydrocarbon receptor-null allele mice have hematopoietic stem/progenitor cells with
abnormal characteristics and functions. Stem Cells Dev 20(5), 769-84.
Smeal, T., Binetruy, B., Mercola, D. A., Birrer, M., and Karin, M. (1991). Oncogenic and
transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63
and 73. Nature 354(6353), 494-6.
100

Smialowicz, R. J., Williams, W. C., and Riddle, M. M. (1996). Comparison of the T cellindependent antibody response of mice and rats exposed to 2,3,7,8-tetrachlorodibenzop-dioxin. Fundam Appl Toxicol 32(2), 293-7.
Smialowicz, R. J., Riddle, M. M., Williams, W. C., and Diliberto, J. J. (1994). Effects of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on humoral immunity and lymphocyte
subpopulations: differences between mice and rats. Toxicol Appl Pharmacol 124(2),
248-56.
Son, W. K., Lee, D. Y., Lee, S. H., Joo, W. A., and Kim, C. W. (2003). Analysis of
proteins expressed in rat plasma exposed to dioxin using 2‐dimensional gel
electrophoresis. Proteomics 3(12), 2393-401.
Stevens, E. A., Mezrich, J. D., and Bradfield, C. A. (2009). The aryl hydrocarbon
receptor: a perspective on potential roles in the immune system. Immunology 127(3),
299-311.
Suh, J., Jeon, Y. J., Kim, H. M., Kang, J. S., Kaminski, N. E., and Yang, K. H. (2002).
Aryl hydrocarbon receptor-dependent inhibition of AP-1 activity by 2,3,7,8tetrachlorodibenzo-p-dioxin in activated B cells. Toxicol Appl Pharmacol 181(2), 116-23.
Sulentic, C. E., and Kaminski, N. E. (2011). The long winding road toward
understanding the molecular mechanisms for B-cell suppression by 2, 3, 7, 8tetrachlorodibenzo-p-dioxin. Toxicological Sciences 120(suppl 1), S171-S91.
Sulentic, C. E. W., Holsapple, M. P., and Kaminski, N. E. (1998). Aryl hydrocarbon
receptor-dependent suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin of IgM secretion
in activated B cells. Molecular Pharmacology 53(4), 623-9.
Sulentic, C. E. W., Holsapple, M. P., and Kaminski, N. E. (2000). Putative link between
transcriptional regulation of IgM expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin and
the aryl hydrocarbon receptor/dioxin-responsive enhancer signaling pathway. J
Pharmacol Exp Ther 295(2), 705-16.
Thatcher, T. H., Maggirwar, S. B., Baglole, C. J., Lakatos, H. F., Gasiewicz, T. A.,
Phipps, R. P., and Sime, P. J. (2007). Aryl hydrocarbon receptor-deficient mice develop
heightened inflammatory responses to cigarette smoke and endotoxin associated with
rapid loss of the nuclear factor-kappaB component RelB. Am J Pathol 170(3), 855-64.

101

Thurmond, T. S., Staples, J. E., Silverstone, A. E., and Gasiewicz, T. A. (2000). The aryl
hydrocarbon receptor has a role in the in vivo maturation of murine bone marrow B
lymphocytes and their response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl
Pharmacol 165(3), 227-36.
Tian, Y., Rabson, A. B., and Gallo, M. A. (2002). Ah receptor and NF-kappaB
interactions: mechanisms and physiological implications. Chem Biol Interact 141(1-2),
97-115.
Tian, Y., Ke, S., Denison, M. S., Rabson, A. B., and Gallo, M. A. (1999). Ah receptor
and NF-kappaB interactions, a potential mechanism for dioxin toxicity. J Biol Chem
274(1), 510-5.
Tucker, A. N., Vore, S. J., and Luster, M. I. (1986). Suppression of B cell differentiation
by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mol Pharmacol 29(4), 372-7.
Tuomisto, J. T., Viluksela, M., Pohjanvirta, R., and Tuomisto, J. (1999). The AH receptor
and a novel gene determine acute toxic responses to TCDD: segregation of the
resistant alleles to different rat lines. Toxicol Appl Pharmacol 155(1), 71-81.
Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., and Gregory, P. D. (2010).
Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11(9), 636-46.
Vogel, C. F., Wu, D., Goth, S. R., Baek, J., Lollies, A., Domhardt, R., Grindel, A., and
Pessah, I. N. (2013). Aryl hydrocarbon receptor signaling regulates NF-kappaB RelB
activation during dendritic-cell differentiation. Immunol Cell Biol 91(9), 568-75.
Vorderstrasse, B. A., Steppan, L. B., Silverstone, A. E., and Kerkvliet, N. I. (2001). Aryl
hydrocarbon receptor-deficient mice generate normal immune responses to model
antigens and are resistant to TCDD-induced immune suppression. Toxicol Appl
Pharmacol 171(3), 157-64.
Werling, D., Jann, O. C., Offord, V., Glass, E. J., and Coffey, T. J. (2009). Variation
matters: TLR structure and species-specific pathogen recognition. Trends Immunol
30(3), 124-30.
White, S. S., and Birnbaum, L. S. (2009). An overview of the effects of dioxins and
dioxin-like compounds on vertebrates, as documented in human and ecological
epidemiology. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 27(4), 197-211.
102

Wincent, E., Amini, N., Luecke, S., Glatt, H., Bergman, J., Crescenzi, C., Rannug, A.,
and Rannug, U. (2009). The Suggested Physiologic Aryl Hydrocarbon Receptor
Activator and Cytochrome P4501 Substrate 6-Formylindolo[3,2-b]carbazole Is Present
in Humans. J Biol Chem 284(5), 2690-6.
Yachnin, S. (1972). The potentiation and inhibition by autologous red cells and platelets
of human lymphocyte transformation induced by pokeweed mitogen concanavalin A,
mercuric chloride, antigen, and mixed leucocyte culture. Clin Exp Immunol 11(1), 10924.
Yasuda, T., Kometani, K., Takahashi, N., Imai, Y., Aiba, Y., and Kurosaki, T. (2011).
ERKs induce expression of the transcriptional repressor Blimp-1 and subsequent
plasma cell differentiation. Sci Signal 4(169), ra25.
Yasuda, T., Hayakawa, F., Kurahashi, S., Sugimoto, K., Minami, Y., Tomita, A., and
Naoe, T. (2012). B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression
of BLIMP1 through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma
Cell Differentiation. J Immunol 188(12), 6127-34.
Yoo, B. S., Boverhof, D. R., Shnaider, D., Crawford, R. B., Zacharewski, T. R., and
Kaminski, N. E. (2004). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the regulation
of Pax5 in lipopolysaccharide-activated B cells. Toxicol Sci 77(2), 272-9.
Yoshida, T., Katsuya, K., Oka, T., Koizumi, S., Wakita, D., Kitamura, H., and Nishimura,
T. (2012). Effects of AhR ligands on the production of immunoglobulins in purified
mouse B cells. Biomedical Research-Tokyo 33(2), 67-74.
Yoshimura, S., Bondeson, J., Foxwell, B. M., Brennan, F. M., and Feldmann, M. (2001).
Effective antigen presentation by dendritic cells is NF-κB dependent: coordinate
regulation of MHC, co-stimulatory molecules and cytokines. International immunology
13(5), 675-83.
Zhang, Q., Bhattacharya, S., Kline, D. E., Crawford, R. B., Conolly, R. B., Thomas, R.
S., Kaminski, N. E., and Andersen, M. E. (2010). Stochastic modeling of B lymphocyte
terminal differentiation and its suppression by dioxin. BMC Syst Biol 4, 40.
Zhang, Q., Kline, D. E., Bhattacharya, S., Crawford, R. B., Conolly, R. B., Thomas, R.
S., Andersen, M. E., and Kaminski, N. E. (2013). All-or-none suppression of B cell
terminal differentiation by environmental contaminant 2,3,7,8-tetrachlorodibenzo-pdioxin. Toxicol Appl Pharmacol 268(1), 17-26.
103