(33% {MUN/(HmWWW”!!!WHIWIHIHH(WNW “55‘s LIBRARY “anagram more 1 University This is to certify that the thesis entitled Cell Sorter Analysis and Separation of Lymphoid Populations From Murine Bone Marrow presented by Susan Marie Goe ser has been accepted towards fulfillment of the requirements for Master , Science degree 1n Major professor Date W" 0-7639 MSU LIBRARIES —— \— RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. CELL SORTER ANALYSIS AND SEPARATION OF LYMPHOID POPULATIONS FROM MURINE BONE MARROW By Susan Marie Goeser A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1982 ABSTRACT CELL SORTER ANALYSIS AND SEPARATION OF LYMPHOID POPULATIONS FROM MURINE BONE NARROW BY Susan Marie Goeser Discrete cell populations were sorted from the bone marrow of adult mice using right angle blue light scatter and forward red light scatter on an Orthocytofluorograf-SO. A population (I) which represents 34% of the total bone marrow is characterized by low for- ward and low right angle light scatter. A second population (ll) represents 56% of the total bone marrow and is characterized by high forward and high right angle light scatter. Population I was shown to be enriched for B lymphocytes responsive to SRBC when compared to whole bone marrow and Population II. Positive fluorescent labeling of surface immunoglobulins was demonstrated for B cells from Population I, but few if any slg+ B cells were found in Population II. The kinetics of the antibody response to SRBC indicate the most mature PFC lie within Population I. Population II was enriched for CFU-S response compared to whole bone marrow and Population I. We therefore conclude that Population I contains a more mature set of B lymphocytes precursors, while Population II is comprised of immature stem cell and pre-B cell like populations. To Scott, my parents, Mike and Kurt ACKNOWLEDGMENTS I would like to thank my advisor Dr. Harold Miller for his help, advice, and guidance during the course of my research. In addition special thanks go to Dr. Walt Esselman and Dr. Ron Patterson for their help and suggestions throughout my studies. To Dr. Marlize Correa, for her advice and friendship 90 my deepest thanks and appreciation, which are hardly adequate in view of the debt owed. I would also like to acknowledge my indebtedness and gratitude to my friends and colleagues, Dr. Don Salter, Paula Jardieu, Dr. Mike Zaroukian, Barbara Laughter, and Kathy Miller for their invaluable contributions and patience. Angie Bissallion deserves the roommate of the year award for going through the entire process with me. Finally, I would like to express my gratitude and appreciation to Scott Gilbertson, who made it all possible by his support and confidence. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES . LIST OF ABBREVIATIONS . INTRODUCTION LITERATURE REVIEW . Functional Tests for Stem Cells . Phenotypic Markers of Stem Cells . Culture Requirements for Stem Cells Proliferation of Stem Cells Stem Cell Activity in Mutant Mice . . . Anti- Stem Cell Activity of Rabbit Anti -Mouse Brain . Terminal Deoxynucleotidyl Transferase . . Site of B Lymphocyte Development . Surface lsotypes . . Functional Properties of TI and TD Responsive B Lymphocytes . . CBA/N Mice as a Model for B Lymphocyte Development . Role of the Spleen in B Cell Maturation . . Secretion of Heavy Chain by Pre-B Cells Clonable B Cells . . Fluorescent Activated Cell Sorting . MATERIALS AND METHODS . Mice . Irradiation Cell Preparation . Media Sorting Antigen . Hemolytic Plaque Forming Cell Assay CFU- S Assay Antisera . . . Fluorescent Labeling . Cytotoxicity of Thymocyte Absorbed Rabbit Anti-Mouse Brain . . . . . . . . . . . . . . . . Analysis of TDT+ Cells . Page vi Page RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Separation of Bone Marrow Populations . . . . . . . . . 33 CFU- S Potential of Sorted and Unsorted Bone Marrow . . . . . . 38 Cytotoxicity of Thymocyte Absorbed Rabbit Anti -Mouse Brain for CFU- S . . . . . . . . . . . . 38 PFC Response of Sorted and Unsorted Bone Marrow . . . . . . . AI Kinetics of PFC Responses of Sorted and Unsorted Bone Marrow . . . . . . . . . AI Limiting Dilution Analysis of Sorted and Unsorted Bone Marrow . . . . . . . . . . 42 Surface Labeling of Bone Marrow with Anti-Igm and Anti-IgD . . . . . . . . . . . . . . . . . . . . . . 45 Analysis of TDT+ Cells . . . . . . . . . . . . . . . . . . . . AS DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Table VI. LIST OF TABLES CFU-S of Adult Bone Marrow . Limiting Dilution of CFU-S . Effect of Thymocyte Absorbed Rabbit Anti-Mouse Brain on CRU-S . . . . . . . . . . Anti-SRBC Response of Adult Bone Marrow Before and After Sorting . Labeling of Sorted Populations With Anti-lgM and Anti-IgD . . . . . . . . . . Analysis of TDT+ Bone Marrow Cells . vi Page 39 39 AD 1.2 A8 A8 Figure VI. LIST OF FIGURES Two dimensional analysis of whole bone marrow on the basis of right angle blue light scatter versus forward red light scatter. RBC were removed by lysis with NHACI The left window containing Population I is 34% of the total bone marrow population. The right window, containing Population II, is 56% of the total bone marrow . . . . . . Analysis of Population I after sorting showed enrichment to 71% with a 2.6% contamination by Population II Analysis of Population II after sorting showed enrichment to 78% with a 5.5% contamination by Population I . . . . . . . . . . . Irradiated syngeneic mice were reconstituted i.v. with 5 x 107 thymocytes and 5 x 105 sorted or unsorted bone marrow cells. Direct and indirect PFC responses were assayed on day 7, 9, II and 13 . . . . . . . . . . Limiting dilution analysis of sorted and unsorted bone marrow vii Page 3A 3A 36 36 43 Ah CFU-S: CSF: FCS: FITC: lg: LPS: PBS: PFC: RAMB: slg: SRBC: TDT: TNP: Tl: TD: LIST OF ABBREVIATIONS Colony forming unit - stem cell Colony stimulating factor Fetal calf serum Fluorescein isothyocyanate Immunoglobulin Intravenous Lipopolysaccharide Phosphate buffered saline Plaque forming cell Rabbit anti-mouse brain antiserum Surface immunoglobulin Sheep red blood cell Terminal deoxynucleotidyl transferase Trinitrophenol T-independent T-dependent viii INTRODUCTION Mature B lymphocytes develop from the pluripotent stem cells found within the adult bone marrow. The exact sequence of ontogeny and requirements of this developmental process are not well character- ized, in spite of the vast amount of work done on this complex area. Stem cells within the bone marrow undergo proliferation and differen- tiation giving rise to precursor B cells (pre-B cells) which further develop into functionally mature B cells capable of undergoing antigen stimulation with subsequent antibody production. Membrane markers such as sIg, Ia, Qa, and Ly antigens have been used in the identifica- tion of some phases of the maturational sequence of B cell populations. In addition, the ability to respond to various classes of mitogens and thymic independent and dependent antigens has proven useful in the study of B cell development. The technological development of fluorescent activated cell sorting has facilitated the identification and purification of cell types representing discrete stages of B cell development. By the use of specific antibodies to surface determinants in conjugation with fluorescent probes, viable lymphocytes can be sorted and purified for further in vivo and in vitro study. In addition, intrinsic character- istics such as cell size and light scattering properties can be of use in the separation of discrete B cell populations. Once obtained, these purified B cell populations can be used in the study of B cell 2 development, including the development of memory cell populations and the mechanism of tolerance. By the use of light scatter properties, we have identified two separate populations within the bone marrow of adult mice that contain functional B lymphocytes and their precursors. These populations have been characterized with respect to slg phenotype, responsiveness to the thymic dependent antigen SRBC, and numbers of stem cells as identified by the CFU-S assay in spleens of irradiated mice. The first population, population I, contains B lymphocytes and their more immediate precursors, while the second population, population II, contains a predominance of more immature cells such as pluripotent hemopoietic stem cells, and slg- pre-B cells. A portion of the stem cells from this population carries the antigen recognized by thymocyte absorbed rabbit anti-mouse brain which is known to identify pluri- potent hemopoietic stem cells. This method of cell separation provides a new approach to the characterization of maturing lymphocyte populations, and will permit the use of purified lymphocyte pOpulations for cell surface market analysis. LITERATURE REVIEW Pluripotent hemopoietic stem cells are defined by their ability to generate all of the necessary cellular components of the lympho- hemopoietic systems, as well as others, while maintaining their capacity for extensive self replication. Murine fetal liver, neonatal spleen, and adult bone marrow all contain such stem cells. The stem cell population once regarded as homogeneous, more recently has been defined as a complex and heterogeneous group of cells, governed by equally complex mechanisms of proliferation and differentiation. Functional Tests for Stem Cells The stem cell, while lacking distinct morphological criteria, is readily defined by functional tests. The most widely used test for the evaluation of stem cell numbers is that developed by Till and McCulloch (I), who defined the pluripotent stem cell in terms of its ability to produce colonies in the spleen of an irradiated, syngeneic reconstituted mouse. Each colony is the product of a single stem cell, and represents one CFU-S (colony forming unit). Following high enough levels of irradiation (900 rads) the stem cell compartment is abolished. Upon reconstitution with a source of stem cells, only those stem cells from the donor will contribute to lymphopoiesis and repopulation of the stem cell compartment. Early work of Wu gt §l_(2), using irradiation induced unique chromosome markers, showed that each 3 I. spleen colony was a clone and the progeny of a single CFU-S. They also showed that some, but not all individual CFU-S have the ability to differentiate into clones containing thymocyte and lymph node like cells, thereby showing indirect evidence for lymphoid cells descended from a single CFU-S. However, they did not confirm the presence of the markers in functional lymphocytes from individual colonies (3). Abramson g£_§l_(h) repeated this work with modified techniques, and were able to identify several distinct classes of stem cells within the bone marrow of adult mice. These include the pluripotent stem cell capable of generating all cell types, as well as stem cells restricted for myeloid, erythrocyte, granulocyte, and megakaryocyte development and stem cells restricted for lymphocyte development (A). In addition, they identified the presence of the chromosomal marker in functional lymphocytes from marked clones by mitogen stimulation with FHA and LPS, and by transfer of cells into irradiated secondary recipients with subsequent generation of chromosomally marked clones. Lala and Johnson (5) used fetal liver or adult bone marrow from CBA or CBA/T6T6 mice as the source of stem cells for chromosomal analysis of subsequent colonies to show that lymphocyte colony forming cells can be generated within a spleen colony by the initiating CFU-S. Phenotypic Markers of Stem Cells Murine stem cells from all tissue sources are lg-, Ia , and Thy-l- (6-9). Qa+ stem cells are found in adult bone marrow, but not in other sources of stem cells such as fetal liver or neonatal spleen. This suggests that Qa is acquired by functionally immature stem cells at a later stage of development (IO). Qa most closely resembles 5 products of the H2K and H20 genes, and there is evidence that Qa plays a role in self recognition. Qa is more restricted in its distribution and also arises at a much later stage of devel0pment than MHC products (ll, 12). Culture Requirements for Stem Cells Dexter §£_§l_(l3, 14) have deveIOped a culture system for the in vitro proliferation and maintenance of pluripotent stem cells over a period of several months, with greater than 15 doublings. The maintenance of stem cells in culture depends upon the establishment of a bone marrow derived adherent cell layer, which is made up of a heterogeneous cross section of the bone marrow stroma. This includes macrophages, endothial cells, reticular cells, and giant fat cells. The presence of these high lipid content cells appears to be vital for CFU-S proliferation. Without them, cultures of stem cells do not survive more than a few days. Culturing at 33°C leads to better maintenance of stem cell cultures as compared to culturing at 37°C. The intervals at which cultures are fed is crucial to triggering a high rate of proliferation among the stem cell population. It is thought that. feeding, with the removal of cells, stimulates the remaining stem cells to proliferate and repopulate the culture, thus insuring continued growth and maintenance of the cultures. Proliferation of Stem Cells The CFU-S population of adult bone marrow has a low rate of proliferation, with less than 10% in DNA synthesis under normal steady 6 state conditions. There is an increased rate of DNA synthesis, greater than 30%, under conditions of bone marrow regeneration follow- ing total body irradiation (15). With partial body irradiation, it is possible to eliminate the stem cell proliferation of one body site, while allowing the rest of the stem cell population to proliferate at the normal steady state rate. This is supported by experiments with phenylhydrazine treated mice. Following treatment of normal mice with phenylhydrazine, the CFU-S of the spleen proliferate very slowly, while those of the bone marrow divide rapidly (16). Studies with stem cells from aged mice as compared to those of young donors have shown that age related defects of the immune system are not due to dysfunction of the stem cell population (17, 18). In serial transfer studies, repopulation of the bone marrow and restora- tion of normal immune function by stem cells from aged donors was sustained over a period of A to S transfers (18). This represents a time span far greater than the normal life expectancy of the average mouse. However, the ability to repopulate the marrow fails with progressive transfers beyond this point, perhaps due to dilution of the CFU-S population, or dilution of necessary amplifier cells (19, 20). Fetal and embryonic tissues have shown a greatly increased capacity for transfer of hemopoietic activity as compared to adult bone marrow, indicating that the stem cell compartment is most active in the early stages of development (21). During adult life, a steady state condition exists, and unless triggered to divide and mature, by some as yet unknown mechanism, the stem cell compartment is largely quiescent (22). Depletion of the stem cell compartment by the use of cytotoxic 7 chemotherapy has been associated with the late onset of stem cell failure of some cancer patients, and could result in a permanent decrease in the proliferative capacity of the stem cell compartment (23). Treatment of adult mice with the drugs busulfan and L-phenyla- lanine to induce a transient cell depletion without drug induced mortality, showed that for as long as two years following drug treat- ment, there was a significant decrease in the stem cell proliferative capacity (2A, 25). Crude supernatant materials from bone marrow cultures can be separated into fractions containing either inhibitory or stimulatory activity for stem cell proliferation or differentiation (16). Density gradient fractionation of total bone marrow into discrete populations shows the inhibitory and stimulatory activities governing stem cell proliferation and differentiation do not lie in the same fraction that contains the stem cell population (26). This indicates that control of the stem cell population involves other cell types, and is not restricted to the stem cell population itself. The effects of both the stimulatory and inhibitory factors was reversible, and the maintenance of high concentrations of the factors is not necessary for continued inhibition or proliferation (26). These factors are not well characterized and involve many as yet unanswered questions. All of the cultures tested negative for colony stimulating factor (CSF). CSF is a Iymphokine known to stimulate proliferation and differentiation of hemopoietic stem cells in vitro (13). A rapid decline in stem cell proliferation within bone marrow cultures occurred if crude prepara- tions of exogenous CSF were added, perhaps due to a feedback mechanism controlling stem cell proliferation. 8 The role of lymphocytes in the maintenance and regulation of hemopoietic stem cells is not clear. It is known that cultures of T lymphocytes stimulated with either mitogens or alloantigens in vitro release CSF into the media. This can be used to stimulate CFU-S proliferation in bone marrow cultures (27-30). In addition, bone marrow cells and Spleen cells from mice treated with semi-allogenic stimulation have an increased level of CFU-S activity in vivo (28). Immunization with DNP-KLH increased the level of stem cell prolifera- tion and differentiation. The activity of CSF in the serum of these mice was also greatly increased (31). The effects are thought to be due to the production of CSF by stimulated thymocytes. Stem Cell Activity in Mutant Mice W/Wv mutant mice are known to be deficient in their CFU-S population (32). The defect of W/Wv mice can be cured by transplanta- tion of histocompatible normal hemopoietic stem cells from adult bone marrow, neonatal spleen, and fetal liver. The level of colony formation in the Spleens of W/Wv recipients and +/+ normal recipients is the same. The majority of the lymphoid cell population switches over to the donor cell type following reconstitution of irradiated W/Wv mice with chromosome marked +/+ bone marrow. W/Wv marrow cells injected into +/+ recipients do not form colonies in the spleen. The curing of the W/Wv defect by marrow tranSplants appears to involve the cooperation of stem cells and a Thy-l+ regulatory cell. In the absence of this Thy-l+ Cell, stem cell proliferation is extremely limited (3“). Sl/Sld mutant mice are defective in the splenic environment 9 necessary for stem cell proliferation in the spleen (33). All attempts at curing the Sl/Sld defect by +/+ normal marrow transplants have failed. Injected Sl/Sld marrow cells form colonies in the spleens of irradiated +/+ recipients and produced the same number of colonies as did bone marrow from +/+ donors. Sl/Sld stem cells are therefore repressed in their SI/Sld environment, but are fully functional in a normal +/+ environment. Where exactly the defect lies, and the precise cellular nature of the defect, is still unknown (34). Bone marrow cells from athymic, nude mice exhibit a decreased level of CFU-S response in the spleens of irradiated recipients (35). In addition, nude bone marrow has a reduced capacity to restore the anti-SRBC response of normal recipients. This indicates some type of thymus directed control over stem cell development, although the presence of the thymus is not an absolute requirement. There is evidence that nu/nu mice do have weakly Thy-l+ cells in the spleen (5-6% of the total spleen), although no function has been assigned to them (36). Anti-Stem Cell Activity of Rabbit Anti-Mouse Brain The anti-stem cell activity of thymocyte absorbed rabbit anti- mouse brain (absorbed RAMB) was first suggested by the work of Golub (37, 38). He found that the neuronal fraction of mouse brain contained an antigen also present on the cell surface of a majority of the CFU-S population (39). The serum obtained after immunization of rabbits with this crude antigen preparation contained activity towards the CFU-S population as identified in the colony forming unit assay in the spleens of irradiated mice. Proper absorptions are vital to the l0 establishment of the desired immunological activity. Unabsorbed RAMB has activity for thymocytes, erythrocytes, B lymphocytes, and CFU-C as well as CFU-S (38-Al). Absorptions of RAMB with brain tissue removed the anti-stem cell activity, while absorptions with adult murine thymocytes, spleen cells, erythrocytes, or liver cells did not effect the activity (A2). Absorptions of RAMB with brain tissue from rats and hamsters removed the activity as effectively as mouse brain tissue, indicating cross species recognition of the anti-stem cell antigen (AZ). This is in contrast to the results of Golub (38), who found a lack of cross species recognition of the anti-stem cell antigen. The stem cell antigen recognized by absorbed RAMB appears in the brain tissue after a postnatal stage of development. Brain tissue from I to 3 day old murine neonates does not remove the activity towards the CFU-S population of the bone marrow, whereas absorption with adult brain tissue does. It is of interest to note that fetal liver does not absorb out the anti-stem cell activity (A3). The development of the antigen recognized by absorbed RAMB appears to parallel the appearance of Thy-l antigen, although it is clear from cytotoxicity data that the two antigens are distinct. Treatment of adult bone marrow with anti-Thy-l and complement does not reduce the level of the CFU-S response (AA). The CFU-S response of adult bone marrow can be eliminated by anti-stem cell antibody administration both in vivo and in vitro (A3). The removal of the CFU-S response does not appear to have an absolute requirement for the presence of complement (A3). Maximal cell cyto- toxicity is obtained without the presence of exogenous complement ll during treatment of bone marrow cells with absorbed RAMB in media alone. Rather, the mechanism of CFU-S depletion following treatment with absorbed RAMB appears to involve opsonization of the antibody coated CFU-S population, followed by phagocytosis with radio- resistant macrophages (AA). Antibody coated CFU-S could give a normal level of response in the spleens of irradiated mice if the macrOphages were the first depleted by treatment with anti-macrophage antibody (AS). The numbers of CFU-S from regenerating marrow are also depleted by incubation with absorbed RAMB, to the same degree as the CFU-S population of normal marrow. In both cases there is a small percentage of the CFU-S population not affected by treatment with absorbed RAMB, no matter what the concentration of antibody used. Velocity sedimen- tation of cells from normal and regenerating marrow showed that all but a slowly sedimenting subpopulation of CFU-S were susceptible to inactivation by absorbed RAMB. This population was determined to be a noncycling population on the basis of radioactive suicide methods. It is similar in size in both normal and regenerating, irradiated bone marrow. This noncycling subpopulation does not express the antigen recognized by absorbed RAMB. However, expression of the antigen appears to be unrelated to a specific phase of cell cycle (A6). Therefore, CFU-S in all stages of cell cycle express the antigen identified by absorbed RAMB, although a small proportion that represent noncycling (GO/GI) cells do not express the antigen. Following in vivo treatment of mice with absorbed RAMB there is a rapid recovery of CFU-S numbers which may be due to an antibody resistant CFU-S population which is rapidly triggered to proliferate 12 and divide following depletion of the original CFU-S population. There would seem to be some as yet unknown mechanism for the rapid recovery of the stem cell population from the remaining stem cells (A6). Recent work by Cambier g£_§l (A7) reports the characterization of a large secretory cell which is brain antigen positive and found in murine bone marrow, spleen, and fetal liver. They are slg-, Ly 2-, Thy-1‘, and secrete a product of unknown function, that does not appear to be CSF. They resemble stem cells as defined by their size, surface phenotype, and resistance to cortisone, but their exact function is as yet unknown (A7). Terminal Deoxynucleotidyl Transferase Terminal deoxynucleotidyl transferase (TDT) is postulated to be a marker for primitive cells of the hemopoietic cell system. It is an unusual DNA polymerase that catalyzes the synthesis of deoxynucleotide sequences onto any 3' OH terminal segment of DNA without template requirement. This absence of template requirement distinguishes it from the replicative DNA polymarases. It has a relative mass of 32,000 daltons, with two peptide chains of 8000 daltons and 2A,000 daltons designated alpha and beta reSpectively (A8). TDT is found predominantly in the thymus and bone marrow of all species tested, with thymocytes having the highest concentration of all tissues tested. It is not found in circulating T lymphocytes. Thymocytes have an average of 21.6 units/108 cells, and bone marrow cells have an average of 11.7 units/108 cells (A9). Other tissues of normal young adult mice such as Spleen, heart, brain, and liver lack 13 detectable levels of TDT activity (50). It has been found in neo- plastic cells from both acute lymphoblastic leukemia and is considered to have potential use in the early diagnosis of leukemia (51). It has been found in T cell derived leukemia and lymphoma cell lines and in normal and neoplastic cells expressing B cell characteristics (52). The first TDT+ cells are detected in the fetal liver where they appear on approximately day 17 of gestation. Maximum levels of T0T+ cells are reached at A weeks of age with 67% of the total thymus expressing TDT. TDT+ cells first appear in the bone marrow on day 1 after birth, with a maximal level of 3.7% of the total bone marrow TDT+ at A weeks of age (53). Normal percentages of TDT+ cells are found in the bone marrow of athymic (nu/nu) mice and neonatally thymectomized mice (53, 5A). However, the Specific activity of TDT in these mice is low compared to that of normal mice. Following both in vivo and in vitro treatment with thymosin, the TOT activity of nu/nu or neonatally thymectomized bone marrow cells increased Significantly (55). Goldschneider g£_§l_ (56) suggest this is due to the induction of TDT+ cells from TDT- cells by treatment with thymosin. They maintain that these TDT+ cells are prothymocytes, which go on to become functional T lymphocytes. Fifty percent of the TDT+ cells in the bone marrow can be driven to express Thy-l antigen by in vitro incubation with thymopoietin (57). The decrease in TDT activity is interesting in that the pluri- potent stem cell population does not undergo a Significant decrease in population size with age. Because of its restricted distribution and unusual enzyme properties, it has been suggested that TDT may function as a somatic mutagen in early lymphocyte differentiation (58). 1A Therefore, Bollum (A9) has proposed that TDT may represent a marker for an immature, stem cell like population. The presence of TDT is detected by one of two ways. The first involves measuring the enzyme activity of radiolabeled cell extracts, as defined by units/IO8 cells, with units being moles of product formed/hour. The second method involves immunofluorescent labeling of fixed cells. The first method has a major disadvantage in that the cell extracts require large numbers of purified cells. Indirect immunofluorescence on the other hand provides a fast and efficient means of determining TDT+ cells either by immunofluorescent microscopy or by fluorescent activated cell sorting (56). Antibody to bovine TDT cross reacts with TDT from many species, including human and murine TDT, and is commercially available. Site of B Lymphocyte Development Mature 8 cells differentiate from B cell precursors that are found in the bone marrow of adult mice. These pre-B cells are distinct from the less differentiated pluripotent stem cells. While much work has been done on the maturation process a B lymphocyte undergoes, relatively little is known about the actual differentiation steps that precede the appearance of a functionally mature B cell. 3H-Thymidine, In vivo studies in which mice were injected with and the labeling of null, slg- marrow lymphocytes compared to that of slg+ B lymphocytes gave the first indirect evidence for the marrow as the site of B lymphocyte production in adults (60). Cell transfer experiments have Shown that embryonic yolk sac, fetal liver, neonatal spleen and adult bone marrow all have the ability to regenerate the B 15 lymphocyte population of irradiated recipients. As the fetal liver and neonatal spleen decline as sites of hemopoietic development after birth, the bone marrow takes over as the site of B lymphocyte deveIOp- ment (62, 63). The spleen and liver have the ability to revert back to sites of B lymphocyte production if bone marrow damages, arguing that the bone marrow is not the defacto site of lymphocyte production (6A, 65). By the use of immunofluorescent techniques, lymphocytes contain- ing ch, but lacking detectable slg have been identified in the fetal liver neonatal spleen, and adult bone marrow, but not in the adult spleen or lymph nodes, where slg+ lymphocytes are found (66, 10). CIgM+ lymphocytes can be identified by day 12 of gestation in murine fetal liver and are present at fairly constant levels in adult bone marrow, as determined on the basis of immunofluorescence. SIgM+ cells appear at day 17 of gestation in fetal liver (66). The chM+ cells of fetal liver and adult bone marrow are large in size and have a rapid labeling time of 1-2 hours in culture with 3H-Thymidine. SIgM+ cells of the bone marrow are smaller in size and label after a lag period of approximately A8 hours (67). This is consistent with the theory of a pool of rapidly proliferating large chM+ cells present in the adult bone marrow and fetal liver that give rise to a smaller sIgM+ cell which moves out of the marrow and matures into a functional B lymphocyte. Indeed, when 1A day slg- fetal liver is placed in culture, sIgM+ small lymphocytes appear within 3 days (10). 16 Surface Isotypes The next differentiation step involving slg is the acquisition of other surface isotypes. Abney §£_al_(68) have shown that expression of IgG and IgA precedes the expression of lgD. While lgM+ cells first appear in the fetal liver on day 17, 5190+ cells do not appear in the bone marrow until between birth and 3 days of age, with large numbers appearing between 2 and A weeks (69). Treatment with anti-mu anti- bodies beginning at birth prevents the development of B lymphocytes expressing any surface isotype. The bone marrow of these mice contain a population of chM+ cells which are analogous to pre-B cells. They can give rise to slgM+, LPS responsive lymphocytes if placed in culture (70, 71). This points to the need for slgM expression as a prequisite for further immunological development. Both slgM and slgD acquisition are antigen and T cell independent (72). The appearance of 5190 on B lymphocytes coincides with changes in the functional properties of these cells. Among the most important of the roles assigned to 5190 is its ability to confer resistance to tolerance induction. If 5190 is removed by capping or papain diges- tion, the cells are easily tolerized (73, 7A). Treatment with anti- delta antiserum to chronically suppress slgD expression in adult mice, renders these mice extremely susceptible to tolerance induction (75). It has been suggested that the interaction of antigen with slgM alone leads to tolerance, while binding of antigen to both slgM and slgD leads to a stimulatory signal, with subsequent antibody production (76). Therefore, upon acquisition of 5190, B lymphocytes are no longer considered susceptible to tolerance induction (7A). During ontogeny, spleen cells acquire resistance to tolerance before bone 17 marrow cells. Tolerance is easily induced in the neonate or in adult bone marrow cells, which have a predominance of slgM+, 5190- B cells. In contrast, the unprimed cells of the spleen are predominantly slgM+, slgD+ and are not easily put into a state of tolerance. Similarly, IgM responses are easier to tolerize than 196 responses (77, 78). However, tolerance can be induced in spleen cells both in vitro and in vivo, with an immature cell population emerging from the stem cell pool the target for tolerance induction and the mature B cell popula- tion remaining functionally intact (79). Functional Properties of TI and T0 Responsive B Lymphocytes The acquisition of responsiveness to thymic dependent (T0) and thymic independent (Tl) antigens and the development of B cell clonal diversity is a very orderly process, with the ability to respond to a given antigen appearing in an ordered sequence, as antigen responsive B cells mature during ontogeny. In addition, the heterogeneity of the antibody response as assayed by the variety of clones triggered by the same hapten, increases with age (80, 81). The functional maturity of B lymphocytes can be studied using three classes of antigen: TI-l antigens such as TNP-Brucellosus abortus and TNP-LPS, Tl-2 antigens such as TNP-Ficoll, and T0 antigens such as SRBC. B cell responses to TI-1 antigens are acquired first, by one week of age, with responses to Tl-2 antigens being acquired later, at 3-A weeks of age (82). The response of young spleen cells to TNP-Ficoll is improved by pretreatment of the cells with anti-Theta and complement, indicating the presence of a T cell suppressor population (83). Corvese §£_§l_ 18 (8A) have found the suppressor cell in the bone marrow not to be a mature T or B lymphocyte, or a mature macrophage, but rather an immature cell of the monocytic-myeloid lineage. This suppressor cell is responsible for depressing both the TNP-Ficoll and SRBC responses of adult bone marrow. The delayed appearance of TNP-Ficoll clones in the spleen is not due to the presence of a suppressor cell, but rather a difference in the ontogeny of TI-l and Tl-2 antigen responsiveness. Injection of LPS or Dextran Sulfate also failed to induce responsiveness to TNP-Ficoll at an earlier stage in ontogeny (82). The ability of immunocompetent B cells to respond to Tl antigens is also correlated with their susceptibility to tolerance induction. Thus TNP-LPS responsive clones from neonates are highly susceptible to tolerance induction in vitro, whereas TNP-Ficoll responsive clones are not (81). The addition of anti-delta antibodies to normal mouse spleen cells in culture blocked the response to TNP-Ficoll but did not effect the TNP-LPS or TNP-BA responses (85). The addition of anti-Lyb 5.1 and complement, which selectively removes B cells with a low ratio of slgM to 5190, removed the ability of spleen cells to respond to TNP-Ficoll but did not effect the response to TNP-LPS or TNP-BA (85). Studies in the CBA/N mouse suggest that Lyb 5.1 is a marker of a late developing subpopulation of B lymphocytes that is absent in CBA/N mice. Lyb 5.1 is not detected on spleen cells of normal mice until 2 weeks of age, with adult levels attained by 5 weeks of age (86). It is thought that Tl-l antigens such as TNP-LPS require only slgM for triggering, while TI-Z antigens such as TNP-Ficoll require 19 both slgM and $190, as do T0 antigens such as SRBC (87). TI-l antigens may bypass the requirement for lgD by binding to mitogen receptors on the cells, or Tl-l antigens with sufficiently high epitope density may trigger cells by cross linking of slgM receptors (85, 88, 89). There is a lack of agreement among investigators concerning this point however. Layton §£_§l_(90, 91) have found that acquisition of 5190 did not effect the level of response to the TI antigen Flu-Pol, in that both 5190- and slgD+ cells responded to Flu-Pol and LPS. The same degree of tolerance susceptibility was found in both 5190- and slgD+ populations from young (2% week) and adult spleen cells. Similarly, responses to T0 antigens such as DNP-KLH are not restricted to a slgD+ subset of cells, and resistance to tolerance induction is not conferred by the acquisition of $190. CBA/N Mice as a Model for B Lymphocyte Development The CBA/N mouse has an X-linked immune deficiency gene (512) that results in a defective B cell population (92). Male and female CBA/N mice and F1 males carrying the xid gene do not present normal immune functions. These include reduced or absent proliferative responses to LPS or anti-lg, failure to generate B cell colonies in soft agar, reduced antibody responses to infectious agents, low levels of serum 19M or 1963, and an increased susceptibility to tolerance induction (93-97). The pattern of response to TNP-LPS, TNP-Ficoll and SRBC is similar to that of immature, normal 8 lympho- cytes. CBA/N B cells will respond to TI-1 antigens but lack the ability to respond to TI-2 antigens (82, 98). In addition, there are fewer pre-B cells capable of responding to TNP-LPS than in normal mice 20 (99). In general, CBA/N mice give low primary responses to SRBC, with an almost nonexistent secondary response (100). Increasing the amount of T cell help in culture does not alter the responsiveness of the cells. However, in the splenic fragment assay, where T cell help is maximized, there is little difference in the ability of normal F 1 females and defective F males to respond to SRBC (96). 1 Studies of the slg characteristics of adult CBA/N mice show that they lack a subpopulation of cells that appear during ontogeny in normal mice. This subpopulation in normal mice is characterized as having a low density of slgM, a high density of 5190, are Lyb 3+ and Lyb 5+. CBA/N mice instead have a subpopulation of B cells that exhibits a high density of slgM and a low density of $190, with an Lyb 3-, Lyb 5- phenotype (101). T cells from CBA/N mice are capable of helping B cells from normal mice respond to T0 antigens to the same extent as normal T cells. Mitogen responses and cytotoxic T cell activity are all normal, as are macrophage functions (93). The immune defect of CBA/N mice can be cured by reconstitution with normal B cells, indicating that the defect does not lie within the microenviron- ment (102). Fidler §£_al (103) have postulated that a maturational block exists within the CBA/N lymphocyte population. This is controlled by the xid_gene, such that an immature B cell population accumulates with the absence of a more mature population, such as is found in the normal adult. This block is not an absolute one, in that the ability of CBA/N B cells to respond to antigens previously unrecognized such as TNP-Ficoll is acquired between 18 and 2A months of age (103). This block in maturation prevents the development of a normal, functionally 21 mature B cell population in CBA/N mice. Role of the Spleen in B Cell Maturation The role of the spleen in the maturation of functional 8 cells is vital. In the absence of the spleen, especially in the neonatal stages of development, a significant, lifelong impairment of B cell maturation occurs. This is characterized by an increase in the total numbers of B lymphocytes in the lymph nodes, with a concurrent accumu- lation of less mature B lymphocytes as measured on the basis of an increased ratio of slgM/slgD, increased level of response to Dextran Sulfate, decreased level of response to LPS, increased numbers of slgM+, 5190. B cells, and an increased susceptibility to tolerance induction (6A, 10A, 105). Secretion of Heavy Chain by Pre-B Cells Recent work by Levitt and Cooper (106) indicates that u chain synthesis precedes light chain synthesis during 8 cell ontogeny. Pre-B cells isolated from murine fetal liver, which lacks mature B lymphocytes, had no slgM+ B cells and only u chains were present in the cytoplasm on the basis of immunofluorescent labeling of cyto- plasmic immunoglobulin. Pre-B cells producing u chains actively secrete it in soluble form. Similar results have been found in male patients with X-linked agammaglobunemia. These patients exhibit normal numbers of pre-B cells but lack mature B lymphocytes. Again there is active secretion of u chains in these patients (106). Similar results were found by Siden e£_§l_(111) using hybridization of cloned 19 DNA 22 to lg mRNA of pre-B cells. This confirms the hypothesis that synthesis of u chain alone is a characteristic phenotype of pre-B cells (107). At the level of the DNA, heavy chain gene rearrangement appears to precede that of light chain rearrangement during B cell deveIOpment. The regulatory process controlling commitment to a single alelle for both the heavy and light chain genes is still unknown. However, the development of Abelson Murine Leukemia Virus transformed cell lines that represent early stages of gene rearrangements in pre-B cells, should be useful in studying the B cell maturation processes at the level of the cells' DNA (108). It should be noted that immature B cells, like adult B cells, are greater than 90% kappa+ in their light chain expression, and that changes in kappa/lambda ratios do not occur during the maturation of B cells. B cells are committed to kappa or lambda expression at a very early stage of ontogeny, prior to anti- genic stimulation (109). The synthesis of J chains is also indicative of the stage of B cell maturation. Upon contact with antigen or mitogen, B cells change from synthesis of receptor IgM to synthesis of pentamer IgM. This is accompanied by an increase in the concentration of intracellular J chains (111). Plasmacytomas secreting pentamer lgM contain large amounts of J chain. Further evidence for the lack of J chain synthesis in unstimulated B cells comes from the analysis of mRNA with a J chain probe cloned from unstimulated B lymphocyte cDNA. No J chain specific RNA could be found in a lymphoma cell line representative of undifferentiated B lymphocytes, while 3 species of J chain RNA were identified in plasmacytoma cells representative of lgM secreting B cells (112). 23 Clonable B Cells The clg+/slg- B lymphocyte precursors mature to become colony forming B cells as measured by transfer to normal irradiated reci- pients or immunodeficient CBA/N recipients, and also under appropriate culture conditions in semisolid agar cultures (102, 113). In this culture system, first developed by Metcalf gt_§l_(llA), individual cells are dISpersed in semisolid agar and stimulated with mitogens. Helper or suppressor T cells are not needed. The influence of macro- phages, which can release inhibitory or enhancing substances can be overcome by manipulation of the culture conditions. Under appropriate culture conditions, colony numbers are consistent and directly proportional to the numbers of pre-B cells in culture. The cells that respond in this assay can be found in all lymphoid tissues and are heterogeneous with regard to size, bouyant density, capping of slg, slg density, and surface antigen characteristics (115-117). The ability to form colonies in semisolid culture is acquired simultaneously with the acquisition of slg. If cells lacking slg are placed in semisolid cultures, they will not mature sufficiently to acquire slg, and further develop into colonies (118). Both clonable, slg+ pre-B cells and those pre-B cells lacking slg express Lyb 2. This marker may therefore be of use in the detection and separation of those pre-B cells lacking slg expression, which are not detected in the colony forming assay. In addition, Lyb 2+, clg+ pre-B cells of adult bone marrow express Qa, but lack expression of la alloantigens (119). Qa antigen is expressed on a wide variety of mature and immature cells of the hemopoietic system (11). Ia- pre-B cells from fetal liver lack Lyb 2 2A and Qa determinants as determined on the basis of susceptibility to antibody and complement lysis. This suggests that expression of these two markers is acquired at a late stage of embryogenesis. It should be noted that lysis with complement and antigen specific anti- body does not always detect the presence of antigenic determinants. Clonable pre-B cells, i.e. 519+, Lyb 2+, Qa+ cells are la+, and the majority of clonable pre-B cells in adult bone marrow are killed by treatment with polyvalent anti-Ia antiserum and complement (72, 120, 121). This suggests that increasing maturity of the B cell population is accompanied by increased slg and la expression. Fluorescent Activated Cell Sorting With the development of fluorescent activated cell sorting, complex mixtures of cells from various tissues can be physically separated into highly purified fractions for a wide range of investi- gational purposes. This procedure has not only been of great use in the study of immunological phenomena, but also in the study of a wide variety of biological disciplines, in both functional and biochemical investigations. Some of these areas include cell cycle analysis, detection of malignant cell types on the basis of cell surface markers, chromosome analysis, correlation of cell surface markers with functional characteristics, kinetic analysis of membrane components and binding of surface receptors. The instruments in use today are refinements of the basic design developed by Bonner g£_al_(122) at Stanford in 1972. The following discussion centers upon the Orthocytofluorograf-SO, although the basic principles remain valid for other models. 25 A single cell suspension, filtered to remove debris and clumped cells, is driven under pressure to a quartz flow cell, where it enters a laminar and concentrically flowing sheath fluid, usually saline. These conditions, referred to as hydrodynamic focusing, result in essentially no mixing of the sample and sheath. The sample pressure determines the flow rate of the cells through the quartz cell. A low sample pressure reduces the diameter of the stream, causing the cells to be confined more rigorously to a Single path which is illuminated by the laser beam. This increases the accuracy of para- meter measurement (123). One or two laser beams can be used, with the beams brought into coincidence along the axis of excitation. The cells pass through the beams, blocking light, measured as axial light loss, scattering light, measured as right angle or forward light scattering, or emitting a fluorescent signal as a result of excitation of a bound fluorochrome or intrinsic autofluorescence. Axial light loss is detected by a PIN photodiode which produces a Signal proportional to the loss of light caused by blockage of the laser beam. The signal is directly routed through a preamplifier and fed into a signal processor. The fluorescence emissions and scattered light are focused onto fiber optics which are hooked to photomultiplier tubes. Signals from the photomultipliers are routed through preampli- fiers and fed into the signal processor, where analysis of pulse height, pulse area, or pulse width are determined as chosen by the operator. The cells exit the flow cell through a 75 micron orifice. The flow cell is physically coupled to a piezoelectric transducer, which 26 oscillates at a frequency of 32,000/second, breaking the jet into uniform droplets at a rate of 32,000/second. If the signal produced by a particular cell is within predetermined amplitude limits, a charging pulse is generated, after a delay corresponding to the transit time of the cell from the laser beam path to the point of droplet formation. The pulse produces a charge on the drop proportional to the applied voltage. The drops pass through a transverse electro- static field produced by two parallel charged deflection plates, where they are deflected into separate containers. The uncharged drops, carrying unwanted cells, are discharged into a waste vacuum. The charging pulse lasts for one or more droplet periods, centered upon the time a desired cell is expected to enter a droplet. The charging pulses are synchronized with the transducer to ensure uniform charging of all droplets. To reduce noise in any given channel, the axial signal serves in a triggering mode. Only when a signal is received from the axial PIN diode will the other channels be activated. This is referred to as axial gated triggering (12A). The required purity of the sorted (deflected) fractions sets a limit upon the rate of cell processing. Setting of suitably sort charge gates ensures the collection of only those droplets containing a single cell of the desired parameters. This reduces contamination of the sorted population, and increases the percentage of enrichment. When sorting cells for further functional assays, the viability and stability of the cells must be maintained at a high level. Cooling of the sample and deflected populations to A°C helps maintain viability as does selection of an appropriate media supplemented with serum. A 27 reduced flow rate also helps to prevent cell loss due to disintegra- tion. When a desired cell population represents a small fraction of the initial cell suspension it is helpful to remove nonessential cell types to facilitate the often lengthy process of sorting. Low levels of a particular cell type can often make obtaining enough cells for functional analysis difficult (125). Data can be collected in a variety of ways. Histograms provide a frequency distribution, while cytograms give a two dimensional plot of individual measurements. Simple x-y plotters are often integrated into the distribution analyzers to provide hardcopy data. More recently, computer systems for data acquisition, storage, and process- ing have been interfaced onto the cell sorter. This allows the processing of dual parameter analysis in the graphical form of iso- metric, profile and contour plots (126). MATERIALS AND METHODS Mice Adult C57BI/6 X C H/HE(BC3F1) female mice were obtained from 3 Cumberland View Farms, Cumberland View, Tennessee. Irradiation Mice were irradiated with 900 rads of total body irradiation from a cobalt source at the Department of Chemistry, Michigan State University. Irradiated mice were maintained on acid water, pH A. Prior to injection of cells, irradiated mice were allowed to rest for A-6 hours. Cell Preparation Bone marrow cells from the femurs of 10-16 week old mice were gently aspirated with a syringe and needles of increasing gauge (21- 27) to obtain a single cell suspension. Thymocytes were obtained by mincing of thymuses from 6 to 10 week old mice with forceps, followed by gentle aspiration with syringe and needles to obtain a single cell suspension. Cell suspensions were washed one time with media and resuspended in fresh media. Red blood cells in bone marrow suspensions were removed by lysis with Tris buffered NHACI, pH 7.2, followed by 3 washes with cold media. 28 29 Media CRML-1066 (Grand Island Biological Co., Grand Island, NY) buffered with 25 mM HEPES (Sigma Chemical Co., Saint Louis, M0) to pH 7.2, and supplemented with 0.15 mM L-asparagine, 0.2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 ug/ml streptomyecin and 10% fetal calf serum was used as medium for all cell preparations during sorting. Eagles MEM (Grand Island Biological Co.) buffered with 25 mM HEPES to pH 7.2 was used as medium in all fluorescent labeling experiments and cytotoxicity experiments. Sorting An Orthocytofluorograf-SO was used to sort discrete cell populations from adult bone marrow on the basis of right angle blue light scatter and forward red scatter using a A88 nm blue line from an Argon laser, and a 585 nm red line from a Helium-Neon laser. Sorted populations were collected in medium supplemented with 20-30% FCS at A°C. Sorted cells were periodically centrifuged at 1500 X g for AS minutes, and resuspended in fresh medium at A°C. Sorted populations had an average viability of greater than 95% as measured by trypan blue exclusion. During sorting, cells were run at a flow rate of approximately lOOO/second, with an anti-coincidence of less than 10%. Antigen Sheep erthrocytes (SRBC) were obtained from a single animal (Grand Island Biological Co.) and stored in Alsevers solution. Prior to injection, SRBC were washed 3 times with sterile PBS and resuspended 30 to a concentration of 5 X 108 cells/ml. Each mouse was injected i.v. with 108 SRBC in 0.2 ml of PBS. Hemolytic Plaque Forming Cell Assay Syngeneic, irradiated mice were injected i.v. with sorted or 7 thymocytes/mouse. After 2A unsorted bone marrow cells and 5 X 10 hours (day 1) mice were injected i.v. with 108 SRBC. Spleens were assayed on day 7, 9, 11, or 13 for both indirect and direct antibody reSponses to SRBC in a Jerne plaque assay as modified for use on glass slides as described elsewhere (127). The following modifications were used. ,All medium was buffered with 25 mM HEPES and adjusted to pH 7.2. Slides were incubated in a normal atmosphere 37°C humid incubator. Guinea pig complement (Grand Island Biological Co.) was tested for nonspecific cytotoxicity and used at a 1/20 dilution. CFU-S Assay S Irradiated, syngeneic mice were injected i.v. with either 10 sorted or unsorted bone marrow cells. Spleens were taken on day 7 and assayed in fresh Boulins fixative. Colony numbers were determined by macrosc0pic examination. Antisera All antibodies were aliquoted and frozen at -20°C. Repeated freeze thawing was avoided. The following sera were commercially ob- tained: FlTC-rabbit anti-mouse lgG (Miles-Veda, Elkhart, IN), FITC- goat anti-rabbit IgG (Miles-Yeda), rabbit anti-mouse lgM (Bionetics, 31 Kensington, MD), monoclonal anti-mouse Ing (mouse anti-lgD) (Becton- Dickinson, Mountain View, CA), and rabbit anti-mouse brain, unabsorbed (Bionetics). All antibodies were ultracentrifuged for 60 minutes at 100,000 X 9 prior to use to remove aggregrates. Fluorescent Labeling Bone marrow cells with RBC removed by NHhCl lysis were suspended to a concentration of 2 X 106 cells/ml in cold media. Anti-lgM (0.1 ml of a 1/50 dilution) or anti-IgD (0.05 ml of a 1/1000 dilution) were added to 0.5 ml of cell suspension and incubated one hour on ice, being shaken 2 times during that period. Cell suspensions were washed 3 times with cold media and resu5pended in 0.5 ml of media. FITC labeled antibody (0.1 ml of a 1/20 dilution) was added to both test samples and controls. Cells were incubated for one hour on ice, and shaken twice. Cell suspensions were washed 3 times and resuspended in 1 ml of medium. Fluorescent intensity was analyzed on the basis of green fluorescent intensity versus forward red scatter. Cytotoxicity of Thymocyte Absorbed Rabbit Anti-Mouse Brain Rabbit anti-mouse brain was absorbed 2 times with thymocytes and 2 times with agarose for 1 hour on ice for each absorption. The antiserum was then centrifuged at 20,000 X g for 30 minutes to remove particles, aliquoted and frozen until used. Absorbed rabbit anti- mouse brain (ARAMB) lost all activity for thymocytes as tested on the basis of cytotoxicity in the presence of complement and ARAMB. Treat- ment of sorted bone marrow populations with ARAMB and complement was 32 performed according to the method of Golub (37) as follows. A suspen- sion of cells (107 cells in 0.1 ml) and 0.1 ml of a 1/10 dilution of ARAMB were incubated on ice for 30 minutes. The cells were washed once and resuspended in 0.1 ml of media with 0.1 ml of a 1/5 dilution of agarose absorbed complement and incubated for 1 hour at 37°C. The cells were washed 3 times with medium, suspended to a concentration of 5 5 X 10 cells/ml and 0.2 ml were injected i.v. Spleens were removed and assayed on day 7 in Boulins fixative for the presence of colonies. Analysis of TDT+ Cells Rabbit anti-bovine TDT and FITC-F(ab)2 goat anti-rabbit IgG were obtained commercially (Bethsedea Research Lab, Gaithersburg, MD). A suSpension of sorted and unsorted bone marrow populations and spleen cells from A week old BC3F1 mice (5 X 106 cells in 1 ml) were fixed in 9 ml of absolute methanol at A°C for 30 minutes. The cells were washed 3 times with PBS and resuspended in 1 ml of PBS. Rabbit anti- bovine TDT and fixed cells (50 ul of anti-TDT in 0.2 ml of cells) were incubated for A5 minutes at room temperature, being shaken several times. The cells were washed 2 times with PBS, resuspended to 0.1 ml and 50 ul of FlTC-F(ab)2 goat anti-rabbit IgG was added. The cells were incubated for A5 minutes at room temperature and then washed 3 times with PBS. The cells were analyzed for fluorescent intensity on the basis of green fluorescence versus forward red scatter. RESULTS Separation of Bone Marrow Populations Selection of two discrete bone marrow populations was carried out using an Orthocytofluorograf-SO on the basis of right angle blue light scatter versus forward red light scatter. Typical results are presented in Figures 1 through IV. Population I is 3A% of the total bone marrow population and is characterized by low right angle blue and low forward red light scatter. After sorting, Population I is enriched to 71% purity with a 2.6% contamination of Population II. Population II is 56% of the total bone marrow population. It is characterized by high right angle blue and high forward red light scatter. Following sorting, Population II is enriched to 78% purity with a 5.5% contamination of Population I. Viability of the sorted populations is high, averaging 95% or greater. Due to their apparent fragility and inability to withstand the stress present during the sorting process, many of the original bone marrow cells are lost. This problem is not so apparent with other, less fragile cell types such as spleen cells, thymocytes, and cell lines. It should be noted that following sorting, the majority of the cells from the sorted populations that are not included within the boundaries of the window, are clustered closely around the edges. Due to the physical stress placed upon the cells during the sorting 33 3A Figure 1: Two dimensional analysis of whole bone marrow on the basis of right angle blue light scatter versus forward red light scatter. RBC were removed by lysis with NHACI. Figure II: The left window containing Population I is 3A% of the total bone marrow population. The right window, contain- ing Population II, is 56% of the total bone marrow. RIGHT ANGLE BLUE LIGHT SCATTER RIGHT ANGLE BLUE LIGHT SCATTER "Whole Murine Bone Marr 35 FIGURE I OW V FORWARD RED LIGHT SCATTER FIGURE 11 Selection of Windows v FORWARD RED LIGHT SCATTER 36 Figure lIl: Analysis of Population I after sorting Showed enrichment to 71% with a 2.6% contamination by Population 11. Figure IV: Analysis of Population II after sorting showed enrichnmuwt to 78% with a 5.5% contamination by Population I. 37 FIGURE III Population I RIGHT ANGLE BLUE LIGHT SCATTER FORWARD RED LIGHT SCATTER FIGURE 13? Population II -., RIGHT ANGLE BLUE LIGHT SCATTER FORWARD RED LIGHT SCATTER 38 process, it is possible that the physical configuration of the cells changes somewhat. Upon resorting to check the purity of the sorted populations, this results in a slightly altered position of the cell relative to the original one within the window. CFU-S Potential of Sorted and Unsorted Bone Marrow Sorted and unsorted bone marrow cells were transferred to irradiated, syngeneic recipients. On day 7, the spleens were assayed in Boulins fixative for the presence of colonies. A two fold enrich- ment for the CFU-S response was found in Population II as compared to both whole unsorted bone marrow and Population I (Table I). There is no significant difference between whole unsorted bone marrow and Population I. The CFU-S response of Population II in a typical experiment is A5 colonies per spleen compared to 16 colonies per spleen for whole unsorted bone marrow, when 105 cells per mouse are injected i.v. A typical limiting dilution analysis of the CFU-S potential is shown in Table II. Cytotoxicity of Thymocyte Absorbed Rabbit Anti-Mouse Brain for CFU-S Thymocyte absorbed rabbit anti-mouse brain (RAMB) is known to have specificity directed towards pluripotent stem cells (38). Sorted and unsorted bone marrow were therefore treated with absorbed RAMB and complement to determine the cytotoxicity directed towards stem cells within Population I and Population II. A decrease in the CFU-S response of whole unsorted bone marrow and Population II was found (Table III). The CFU-S reSponse of whole bone marrow decreased from 39 Table l: CFU-S of Adult Bone Marrow. CFU-S/IO5 cells Bone Marrow1 Exp 1 Exp 2 Exp 3 Whole 16.3 11.92 20.2 :_2.1 16.6 :_2.h Population I 1A.9 i_1.9 16.6 :_2.1 13.0 :_1.8 Population II AS.1 :_2.9 37.0 :_A.2 36.8 :_l.9 Control3 0.67 i 0.75 0.20 :_0.h0 0.h0 + 0.A9 5 Irradiated synegeneic mice were reconstituted i.v. with 10 bone marrow cells per mouse. Results expressed as mean : standard deviation of 5 mice per group. 3 Control mice received no cells. Table II: Limiting Dilution of CFU-S. Bone Marrow] 105 5 x 10“ 10“ Whole 16.212.112 8.8: 1.6 0.80:0.80 Population I ND A.6 : 1.1 0.20 :_O.A5 Population II ND 25.6 :_l.8 5.2 :_1.8 Control3 O.A0 :_0.55 --- --- Irradiated syngeneic mice were injected i.v. with bone marrow cells. Results expressed as the mean :_standard deviation of 5 mice per group. 3 Control mice received no cells. A0 Table III: Effect of Thymocyte Absorbed Rabbit Anti-Mouse Brain on CFU-S. Treatment with ARAMB Bone Marrow and Complement Exp 1 Exp 2 Whole - 17.2 i 2.32 16.2 1 Whole + 3.2 :_1.3 3.0 1 Population I - 1A.6 : 1.8 11.6 1. Population I + 11.6 :_1.3 9.A : Population II - 33.6 :_2.7 35.A : Population II + 8.6 i 2.3 3.8 1; Control3 - 0.A0 _+_ 0.5 0.80 1’. C' Control“ - 17.3 :_2.2 17.2 i 5 Irradiated syngeneic mice were reconstituted i.v. with 10 bone marrow cells per mouse. Results expressed as the mean 1 standard deviation of 5 mice per group. 3 Control mice received no cells. 5 Complement control mice were injected with 10 whole bone marrow cells treated with complement alone. A1 17 to 3 colonies per spleen, while that of Population 11 decreased from 3A to 9 colonies per spleen for a typical experiment. Population I was not significantly effected by treatment with absorbed RAMB. Complement alone had no effect. Noncycling stem cells are not thought to express the antigen recognized by absorbed RAMB. The stem cells within Population I may represent these noncycling stem cells, while Population II contains a cycling stem cell population. PFC Response of Sorted and Unsorted Bone Marrow To examine the ability of sorted and unsorted bone marrow to respond to antigen stimulation, the PFC responses to the T dependent antigen SRBC were determined. Irradiated syngeneic mice were reconstituted with sorted and unsorted bone marrow and excess thymo- cytes. The PFC response per Spleen was measured on day 9. The highest level of both direct and indirect PFC responses are seen in Population I with 528 direct PFC/Spleen and 578 indirect PFC/spleen (Table IV). The level of direct and indirect reSponses of whole un- sorted bone marrow and Population II are not significantly different and are approximately two fold lower than those of Population I. Kinetics of PFC Responses of Sorted and Unsorted Bone Marrow As the maturity of a B lymphocyte is one of the factors deter- mining its ability to respond to antigenic stimulation, the kinetics of the PFC response were examined to determine the relative maturity of the responding cell population. The highest level of direct and indirect responses are seen in Population I from day 9 to day 13, with A2 A000 direct and 5800 indirect PFC on day 13 with 5 X 105 cells in- jected per mouse (Figure V). The direct and indirect PFC responses of both whole bone marrow and Population II are not Significantly different from day 7 to day 13. Both are approximately two fold lower than the level of response of Population I, from day 9 to day 13. Table IV: Anti-SRBC Response of Adult Bone Marrow Before and After Sorting. Bone Marrow1 Direct PFC Indirect PFC Whole 296 :2 1.42 358 1 57 Population I 528 :_73 578 :_82 Population II 190 :_58 - 212 1.63 Control3 35 :.AA A0 :_61 Irradiated syngeneic mice were injected i.v. with 5 X 105 bone marrow cells and 5 X 107 thymocytes. PFC responses of spleen cells were assayed on day 9. Results expressed as mean :_standard deviation. 3 Control mice received no cells. Limiting Dilution Analysis of Sorted and Unsorted Bone Marrow Limiting dilution analysis of antibody producing cells is a method used for the study of the frequency, function and potential of the least frequent cell type of a heterogeneous population of cells involved in the immune response. If the antibody response depends entirely upon the presence of the single most rare functional cell type, the distribution of immune responses among mice given limiting numbers of cells will conform to a Poisson distribution. If the cell population contains several types of immunocompetent cells capable of AB Irradiated syngeneic mice were reconstituted i.v. with 5 sorted or unsorted bone Figure V: 5 x 107 thymocytes and 5 x 10 Direct and indirect PFC responses were marrow cells. assayed on day 7, 9, 11 and 13. PFC (x103) PER 1511105 sous MARROW CELLS “T 3-1 2. DIRECT PFC RESPONSE AA FIGURE I KINETIC ANALYSIS OF PFC RESPONSE i000. WHOLE BONE MARROW POPULATION I POPULATION n CONTROL 11 13 DAY OF ASSAY INDIRECT PFC RESPONSE A5 participating in the immune response, a non-Poisson distribution would be seen. As seen in Figure V1, with the exception of the direct responses of Population I, a non-Poisson distribution profile is seen. This non-Poisson distribution of the PFC responses suggest that the potentially immunocompetent cells were heterogeneous. The PFC responses could therefore be the result of marrow cells responsive to SRBC in different stages of development (128). Surface Labeling of Bone Marrow with Anti-lgM and Anti-lgD SlgM and 5190 positive cells were assayed by indirect fluorescent labeling on the basis of green fluorescence versus forward red light scatter (Table V). The majority of slgM+ and slgD+ cells lie within Population I, with only a few found in Population II. Twenty-three percent of Population I are slgM+, and 7% are slgD+, while of Population II, 3% are slgM+ and 1% are $190+. The numbers presented here were obtained by setting windows around the positive fluorescent populations. Analysis of TDT+ Cells Sorted and unsorted bone marrow populations were assayed for the presence of terminal deoxynucleotidyl transferase (TDT) on the basis of positive immunofluorescence. TDT+ cells were found within Population II (10%) and whole bone marrow (A), but not within Popula- tion I or whole spleen cells (Table VI). TDT+ cells have been reported by other investigators to be present in small numbers in bone marrow of young mice, but absent from Spleen cells (53). The exact A6 Figure VI: Limiting dilution analysis of sorted and unsorted bone marrow. A7 Figure I! Olrect Reeponee tool- 3 4 eob ./ e .7’ o 00'- : // r: 3 'l o :' .’/ a re :' ’1 . .0 O I e 5 .’ / ¢ 00 5 I [I e :' x' / .2 5° 5 ,' ,’ .-.-.; Whole : 5° ’0 [I eeeeeee: popu|."°n x z 4 :5 ,° // ---: Populetlen n e r’ ,. / i 30 5° ./. I, 5 / I 2 5° ’0 I, e O / : / , Io : ' , g.’ I 4’ 0’ - l 10‘ 10‘ 10‘ Log Number of Trensplented Celle Indirect Response 100- 3 / eoI- / e / : 80h / / e. 0 / o I / a. 70- .o . / . .0. l / e . // c 60'- .e. l / .0. I l 0 ..° / / g 50- I. // u .0. e / e- :0 / / P 40- . / O .0 / / 3 ..° /. / 301— .0. / .° ° / * ..° / / 201- ' / .0. o’// / 10*- ..e e I .° // ." 7 e v 1 10‘ 105 106 Log Number or Transplanted Celle A8 Table V: Labeling of Sorted Populations With Anti-lgM and Anti-lgD. Bone Marrow % IgM+ % IgD+ Whole] 12% #2 Population I 23% 7% Population II 3% 1% Sorted and unsorted bone marrow cells were fluorescently labeled with commercially prepared sera to detect the presence of slgM+ and slgD+ cells. Table VI: Analysis of TDT+ Bone Marrow Cells. % TDT+ Cells Whole1 A% Population I 0 Population II 10% Spleen 0 1 Sorted and unsorted bone marrow cells were methanol fixed and fluorescently labeled with commercially prepared sera to detect the presence of terminal deoxynucleotidyl transferase (TDT). A9 function is unknown, but it is thought to act as a somatic mutagen in the early stages of B lymphocyte development (58). Its presence serves as a marker for the identification of immature lymphocytes (A9). DISCUSSION Preparative cell sorting on the basis of forward red versus right angle blue light scatter parameters has permitted the analytical and functional characterization of two subpopulations of adult bone marrow. Population I represents a more mature slg+ B cell population, capable of responding to stimulation with SRBC, and containing low numbers of stem cells. Population II was determined to be less mature, as it contained increased numbers of stem cells and slg- cells. These pre-B like cells respond to SRBC in the PFC assay if given time to mature and develop within the spleens of irradiated recipients, as shown by kinetic analysis. The majority of the stem cells of Population II express the determinant recognized by thymocyte absorbed rabbit anti-mouse brain (RAMB), as determined by the decrease in the CFU-S response following treatment with absorbed RAMB and complement. Little effect was seen on the CFU-S response of Population 11 under similar conditions. It is thought that noncycling stem cells do not express the antigen recognized by absorbed RAMB (A6). The stem cells within Population I may therefore represent noncycling (GO/GI) stem cells, that are not actively proliferating and differentiating, while those of Population II are part of a cycling stem cell population. It is not known if the expression of this stem cell surface antigen has any functional significance in the regulation of the stem cell popula- tion (A0, A3). As stem cells are not a homogeneous population, but 50 51 rather a complex and heterogeneous mixture of cells, not all stem cells found in the bone marrow may express the antigenic determinant recognized by absorbed RAMB. Stem cells are traditionally thought of as null cells, due to the current lack of stem cell specific markers. While lacking such obvious and well characterized surface markers as Thy-l, slg, and Ia antigens (6-9), they may have unique and characteristic surface markers, which may be expressed at low levels and at Specific stages of stem cell ontogeny. Alternatively, they may express unique glycolipids with weak antigenic properties that render them difficult to detect. With the isolation of specific antigens characteristic of various stem cell populations, and the development of monoclonal antibodies towards them, cell populations purified to homogeneity on the basis of fluorescent activated cell sorting can be obtained for further functional studies. This will also enable the selection of antigenic determinants specific for a particular lineage of lymphocyte develop- ment. During sorting, a large percentage, between 25 and A0% of the original population are lost (unpublished results). This problem did not occur to as great an extent with spleen cells or thymocytes for example, which appear to be hardier cells. The viability, as measured by trypan blue exclusion of sorted populations, is high, averaging greater than 95%. However viability alone does not guarantee the ability of a cell to respond normally in a functional assay. The level of response may inadvertently be decreased due to the sorting process. Similarly, the sorting process may activate the bone marrow in some nonspecific manner, so that an increased level of response is seen. 52 The sorted populations are collected in medium supplemented with high levels of fetal calf serum, which can act as a nonspecific activator (129). While these conditions are necessary for maintaining high viability of sorted populations, the possibility of nonspecific activa- tion must be considered. The relative increase in the CFU-S response of Population II compared to Population I and whole unsorted bone marrow indicates this procedure can be used as an effective method for the enrichment of stem cells from lymphoid cells. There was no significant difference between Population I and whole bone marrow. As other cells within the bone marrow exercise control over stem cell proliferation and differen- tiation, preparative sorting of whole bone marrow into Populations I and Il may delete from one or the other populations as necessary for controlling cell type. One can propose that Population I does not contain cell types responsible for stem cell control. A residual amount of stem cells within Population I may therefore undergo a rapid proliferation following injection into irradiated recipients, similar to the results of Mickelem gt_§l_(22). Stem cells usually have a very low rate of proliferation. Following depletion of the stem cell population, recovery of stem cell numbers is rapid, especially if the depletion has been drastic. When recovery has reached a certain level, usually approximately 50% of a nondepleted control, the rate of proliferation slows. This may be due to a cell concentration feedback mechanism, mediated by some form of cell to cell communication (13, 15, 16). Suppressor cells, thought to be immature macrophages, have been found in the bone marrow of adult mice (8A). They are capable of 53 suppressing the antibody response to both T independent and T dependent antigens, but no evidence has been presented for a regulatory effect on stem cells in vivo. There is considerable evidence for the role of macrophages, or their products influencing lymphoid cell differentia- tion in vitro. Macrophages influence hemopoietic stem cell colony formation in vitro, cause the sequential transition of newly formed B cells into large blasts, and effect the development of 519- pre-B lymphocytes into slg+ B lymphocytes that are capable of forming colonies in soft agar (116, 130-132). While in vitro conditions such as these are often artificial, they serve to indicate an association between macrophages and developing B lymphocytes in intact bone marrow. The antibody responsive cells of either Population I or Popula- tion 11 appear to represent different stages of B lymphocyte develop- ment. The differential ability of these cells to respond to mitogens, SRBC, or endotoxin in the pre-B cell cloning assay in soft agar would provide additional information as to their stage of development, as different subsets of B lymphocytes grow under varied cloning condi- tions (11A-117). This assay system has proven highly useful in the delineation of the complex interactions 3 pre-B cell undergoes during its development, although its usefulness is limited by the purity of the cell populations under study. Further characterization of antigens such as Ia, Qa, and Ly markers that are present on the cell surface of these two populations are also of interest in the determination of the cellular state of development (7A, 118-121). Another series of functional classifications helpful in the determination of the functional maturity of B lymphocytes and their sequence of ontogeny, is their responsiveness to various classes of 5A T-independent antigens such as TNP-LPS, TNP-Brucella abortus, and TNP- Ficoll (82, 85, 87). Work with the immunodeficient CBA/N mouse has allowed the categorization of antigen responsiveness and the mechanism by which these antigens trigger B cells. The pattern of antigen responsiveness seen in CBA/N mice is similar to that seen in immature B lymphocytes, as is the slg and cell surface marker patterns (93-97). Further characterization of Population I and POpulation II with respect to T independent antigens should be of use in correlating immune responsiveness with surface membrane phenotypes. Since immature cells of the 8 cell lineage are easily put into a state of permanent and lifelong tolerance, the ability to tolerize cells of either Population I or Population II will give insight as to the functional status of these populations (77-79). The splenic frag- ment assay developed by Klinman provides a means of looking at the mechanism of lymphoid stem cell differentiation and the susceptibility of emerging lymphoid cells to tolerance induction at the level of a single cell, within a normalized environment (133). 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