It » r l 7" I I' x | VFiFQIR .LLVLIIHIFI . ~ .2. 1'7...”m — nus-flrazzAny Michigan State University ‘ m N“. This is to certify that the thesis entitled SOLUBLE PRODUCT(S) FROM HUMAN LYMPHOCYTES ACTIVATED BY CONCANAVALIN A presented by Edward Frank Rosloniec Jr. has been accepted towards fulfillment of the requirements for M. S. degreein Microbiology Major professor Date /fl"7’f0 0-7 639 W: 25: per day per item RETURNING LIBRARY MATERIALS: M Place in book return to remove charge from circulation records in Ft ‘ 3 Dcpartment w 1"» A. ‘ . 'Y?37 Q' "’ '1. .‘A.\ u. -AEIN .1 ‘ J; : it MPHOCYTFS ; quanta '1' 'A 1:11,}. .f-L, . ' ‘ f‘f . v . m ' ’( 4’» a I I O A V «We SOLUBLE PRODUCT(S) FROM HUMAN LEMPHOCYTES ACTIVATED BY CONCANAVALIN A By. Edward Frank Rosloniec Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements . for the degree of MASTER OF SCIENCE ‘ i ‘ 1 a. ‘ .bepartment of Microbiology and Public Health ' -~R Y“ 1980 51M ‘ ABSTRACT SOLUBLE PRODUCT(S) FROM HUMAN LYMPHOCYTES ACTIVATED BY CONCANAVALIN A By Edward Frank Rosloniec Jr. Human lymphocytes activated by concanavalin A (Con A) secrete into their supernatant a factor(s) with varying activities. This supernatant, termed soluble immune response suppressor-human (SIRSH). contains suppressor and aug- mentative activities that are dependent on their specific assay and the method of factor(s) production. SIRSH produced by #8 hour Con A activation demonstrates optimal suppression of the mixed lymphocyte culture, yet will also stimulate blastogenesis in a 3 day mitogenesis assay. When 24'hour produced SIRSH is used, a decrease in suppressor activity (SF) and an increase in mitogenic activity (MF) becomes evident. SIRSH-SF and MF appear to be separate factors based upon serum requirements for production and temporal separation of production. SIRSH will also enhance Con A mitogenesis, as well as augment natural killer (NK) activity and lectin dependent cytotoxicity. After 3 days in culture, SIRSH treated cells demonstrate an increase in NK activity that kinetically parallels the MF blastogenesis. However, the relationship between MF and the NK augmentation is unclear. ACKNOWLEDGEMENTS The author wishes to express his appreciation to the following people: Dr. Donald Kaufman, M.D., my major professor, for his support. encouragement, and enthusiam for my professional academic development. His contributions to my career . have been invaluable. Judy Martin and Alison Ruhle, for their technical and emotional support. Diane Rosloniec, my typist, editor. critic, inspiration. part-time technician, and wife. The Department of Pediatrics and Human Development for financial support. _‘_"~f_ ii __—._.___.— .___.. 1... - . ’— TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . Discovery of the T Cell Role . . . . . . . . Antigen-Induced Suppressor Cells . . . . . . . Tolerance and Antigen-Induced Suppressor Cells . . . . . . . . . . . . . . . . . . Suppressor Cells Affecting Antigens Under Ir Gene Control . . . . . In Vitro Analysis of Ts Cell Activity . . . . The Macrophage and TS cells . . . . Soluble Factors from Antigen- -Induced Suppressor Cells . . . . . Mechanisms and Targets of Antigen- -Induced Suppression . . . . . . . . . . . . . . . Mitogen-Induced Suppressor Cells . . . . . . . History of Mitogens in Immunobiology . . . Mitogenic Induction of Suppression . . . . Concanavalin A- Induced Ts Cells . . . . Mechanisms of Mitogen- -Activated T Cell Suppression . . . . Concanavalin A Stimulation and Suppressor Factors I I I I I I I I I I l t I I The Relationship Between Immune Response Help and Suppression . . . . . . . . . . . . . Separation of T Helper and T Suppressor Cells Antigen and Mitogen-Induced Helper Factors . Soluble Factors Demonstrating Help and Suppression . . . . . . . . . . . . . . . LIST OF REFERENCES. . . . . . . . . . . . . . . . . iii Page vi \n c- c— H kit 10 12 15 18 18 21 24 26 30 31 34 37 41 iv INTRODUCTION . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS Human Peripheral Blood Lymphocyte Isolation Soluble Factor Production . . . . . . Allogeneic Mixed Lymphocyte Cultures . . . Mitogenic Assay . . . . . . . . . . T Lymphocyte Isolation . . . . . . . . . . Cellular Cytotoxicity Assay . . . Natural Killer and Lectin Dependent Cellular Cytotoxicity Assays . . . . . . Macrophage Isolation . . . . . Macrophage Depletion of HPBL . . Peroxidase Staining . . . . . . RESULTS . . . . . . . . . . . . . . . Activities of Con A Supernatants . . . . . Dissection of MF and SF MIC-Suppressor Activity . . Effect of SIRSH-MF on Mitogen Stimulation Sensitivity of MP to OCMethyl Mannoside . Responding Cell to SIRSH- -MF . Adherent Cell Requirement for MF Activity SIRSH and Natural Killer Activity . . Effect of SIRSH on Lectin Dependent Cytotoxicity . . . . . . . . . . . DISCUSSION REFERENCES Page -‘_ 0 LIST OF TABLES Page Multiple Activities of SIRSH . . . . . . . . . . 69 Separation of SIRSH Activities . . . . . . . . . 69 Serum Requirement of MF and SF Production . . . 71 Sensitivity of MF to 0‘ Methyl Mannoside . . . . 71 Comparison of MF Production and Target Cell . . 78 -.J-‘~—n‘ V A Effect of SIRSH on Target Cell 51Chromium Release . . . . . . . . . . . . . . . . . 78 1‘ :1 I. -' ’ WQW . . I.’IT‘vM-t ‘ LIST OF FIGURES Page SmSH Production I I D I I I I I I I I I I I I 55 Kinetic Analysis of SIRSH . . 6? Kinetic Analysis of SIRSH . . . . . . . . 67 Effect of SIRSH-MF on Supraoptimal Con A Stimulation . . . . . . . . . . . . . . . . 73 Effect of SIRSH-MF on Suboptimal Con A Stimulation . . . . . . . . . . . . . . . . 7h Proliferative Target Cell of SIRSH-MF . . . . 76 Requirement of Adherent Cells for MF Activity. 79 SIRSH Augmentation of NK Activity . . . . . . 81 Effect of SIRSH and Con A on NK Activity . . . 83 Effect of SIRSH on LDCC . . . . . . . . . . . 85 2 ,4 vi INTRODUCTION The basic concepts of immunology date back to ancient times whereas the science itself is barely 100 years old. Recorded practices of immunization for the prevention of small pox date back to the Chinese, circa 1000 A.D., but not until the late 19th century was the process of active immunization first examined by such prominent scientists as Pasteur and Koch. During this era the field of immunology emerged into two distinct but interdependent categories-- cellular and humoral immunity. . The theories of immune induction have undergone major changes within recent years. The fact that cellular (lymphocytic) stimulation is responsible for humoral (anti- ' body) immunity was only recognized 15 years ago. Initially specific antibody production was thought to be the result of simple contact between antigen and a predetermined responding cell (Burnet 1959). Soon after the discovery of the function of the thymus gland (Miller 1961), Burnet's clonal selection theory underwent a radical transformation with the recognition that a synergism wasoccurringmdthin the cellular response to antigen. That is, for most antigens the co—operative action of thymus-derived cells (T cells) with bursa or bone marrow-derived cells (B cells) is required ‘l' for immunoglobulin response to foreign materials (Claman, Chaperon, Treplett 1966). Subsequently the T cell was found to be the site of control for the B cell response (Miller and Mitchell 1969). It has now become clear that there are subsets of T lymphocytes that influence the activity of other T lymphocytes as well as B lymphocytes. T cells have been identified that are capable of helper activity (TH) (Miller and Mitchell 1969), suppressor activity (TS) (Gershon and Kondo 1970, Dutton 1975), and possibly enhancement or amplifer activity (TA) (Cantor and Weissman 1976). In addition, T cells have been implicated as effectors of an immunologic response in a process known as cell mediated immunity. These T cells are capable of lysing cells displaying foreign antigens, both recognizable antigens (T ) (Cerottini and Brunner Killer 197k) or antigens never before seen by an individual (natural killer) (Herberman et al. 1973, Oldham et al. 1973). Studies on the control of the immune response and the role of T cells, i.e. help and suppression, have progressed to the area of soluble mediators. These lymphocyte-produced soluble factors, or "lymphokines", are capable of replacing T cell function (Kindred et al. 1979), acting upon B cells (Gisler, Stabler, et al. 1973), or acting upon other T cells (Smith, Gillis, et al. 1979). They have been produced from antigen stimulated (Gershon and Kondo 1970, Kapp et al. 1973) as well as mitogen stimulated lymphocytes (Dutton 1972, K .I , >1‘d« I] ‘, .‘t ’ h .I .. .. . l ‘ . 0J1”. I I 5| . ‘ 1" 3 ,1 n5. * ,' {ich and Pierce 1973a). The specifity of these factors : vcovers a wide range with some showing activity across . 'ispecies barriers (Kaufman, Carnaud, et al. 1979) while many are active in only one antigen specific system. With the I '1 l H ' \ ever increasing recognition of immunological dysfunction in man, the theoretical implications of immune response. control research is enormous. Consequently, the following literature review and article will attempt a somewhat detailed overview of the research in immune response control, both cellular and sub-cellular. LITERATURE REVIEW Discovery of the T Cell Role The concept of T cell control of the immune response is still very new, nevertheless important advances have been made in a relatively short time. The discovery of the function of the thymus by Dr. Jacques F. A. P. Miller opened the door to T cell immunology (Miller 1961). Using neonatally thymectomized mice Miller demonstrated a general defect in the numbers and activity of the remaining lympho- cytes, including loss of allograft rejection. Although this observation has been shown to vary in different strains of mice, the advent of the congenitally athymic (nude) mouse has confirmed the initial results. The actual participatory role of the T cell in the immune response was not elucidated until years later. In 1969 it was demonstrated by two groups that thymus derived cells (T cells) do not produce antibody but are required to interact with precursors of antibody producing cells (B cells) to support antibody response to certain antigens (Miller and Mitchell 1969, Claman and Chaperone 1969). These T cells were consequently described as T helper cells (TH). Those early reports marked the beginning of investigations of T cell control of the immune response. Antigen-Induced Suppressor Cells Tolerance and Antigen—Induced Suppressor Cells The original investigation of suppressor cell activity was the result of a study on the induction of tolerance (Gershon and Kondo 1970). Animals are said to be immunologically tolerant when they have lost their capacity to produce an immune response to an antigen after previous exposure. Using thymectomized, lethally irradiated, bone marrow reconstituted mice (chimeras), it was shown that T cell—independent antibody responses to sheep red blood cells (SRBC) could be blocked by a previously injected large dose of SRBC. Reconstitution with as little as 15 X 106 thymus cells quickly restored the animals ability to respond to a subsequent challenge with SRBC. In contrast, when the chimeras were given thymocytes along with the bone marrow inoculum and the same antigenic treatment followed, no response to later antigenic challenge could be achieved. Even with a later injection of thymocytes no quick restora- tion occurred. The only antibody response observed to the later challenge of SRBC in the bone marrow-thymocyte reconstituted mouse was that of regenerating precursor B cells capable of T cell independent stimulation. Thus, the induction of tolerance as well as the induction of the successful immune response with thymus dependent B cells, seemed to require the co-operation of thymus derived cells. These findings had a major impact from two aspects. One, they indicated a specific separation of T cell mediated suppression of the immune response from antigen-induced B cell tolerance. Chimeras with or without thymocytes in the bone marrow reconstitution were equally "suppressed" by heavy antigen inoculation. Yet subsequent thymocyte inoculation quickly broke only the B cell tolerance. Secondly, the first indication of thymocyte-mediated supres- sion of antibody response was reported. This suppression of SRBC antigenic challenge (bone marrow and thymocyte reconstituted chimera) was resistant to a secondary thymocyte injection and found to be specific for the SRBC antigen. Challenges with horse red blood cells (HRBC) produced normal levels of antibody, specific for HRBC. In follow up experiments, Gershon and Kondo studied the effects of splenic adoptive transfer to thymus-deprived chimeras (Gershon and Kondo 1971). Donor as well as recipient mice were thymectomized, irradiated and reconsti- tuted with bone marrow. The variable in question was the presence or absence of thymocytes in the bone marrow injection and how they affected the induction of the tolerized state (donor), or modified the response by adoptively transferring prestimulated cells (recipient). It was found that the SRBC sensitized spleen cells of the thymocyte-bone marrow reconstituted donor not only did not cooperate with the normal thymocytes of the recipient animal when challenged with SRBC, but these same transferred cells prevented the *_—.-—. co-operation of normal thymocytes with normal bone marrow derived cells. In contrast, when normal donor spleen cells (not tolerized by SRBC) were transferred, a significant augmentation of the antibody response occurred. The results of this study reinforced their previous results and convincingly indicated that the thymocytes were responsible for the "infectious immunological tolerance" or suppression of the anti-SRBC response. Only the prestimulated spleen cells of thymus-reconstituted donor mice produced an immunodepression by adoptive transfer to recipient mice. In addition, the effect of transferring unstimulated or immune competent thymus reconstituted spleen cells was in agreement with the findings of Mitchell and Miller (1969). The transfer of immune cells caused an increase in antibody production in the recipient animal. In fact, it was found that the higher the titer of antibody in the donor, the higher would be the antibody titer in the recipient. Thus, both augmentive (T helper cells) as well as suppressive (T suppressor cells) capabilities appear to reside with the thymocyte population. Finally, in support of T cell mediated suppression, it was demonstrated that anti thy-1 plus complement (C)treatment of donor spleen cells before adoptive transfer blocked the suppressor activity. The author also hypothesized that the T cells responsible for the decreased response may produce an "immunosuppresive substance". Although this was a strikingly bold hypothesis for that era in immunology, the current evidence for such a substance or substances is very strong and will be discussed in later portions of this review. Suppressor Cells Affecting Antigens Under Ir Gene Control Some investigators had previously recongnized that the response to certain antigens was under genetic control. The site of this control was mapped within the H—2 region of the major histocompatibility complex (MHC) of the mouse to a loci of genes named the immune response genes (Ir). Benacerraf and colleagues, incorporating the findings of Gershon and Kondo, made a detailed study of suppressor cells and their role in the immune response to GAT, an antigen under Ir gene control (Kapp, Pierce, et al. 1974). Different strains of mice, varying in their genetic make-up, are known to be either responders or nonresponders to soluble GAT as measured by a plaque forming cell response (PFC). If CAT is coupled to a carrier, such as methylated bovine serum albumin (MBSA), and injected into a nonresponder, anti-CAT antibody is produced (Kapp, Pierce, and Benacerraf 1973). Therefore, in order to examine the possibility of suppressor cells being responsible for the nonresponder state of some animals the following experimental scheme was used. Nonresponder mice were injected with soluble CAT, and as exPected no response was observed. These same animals were then challenged with GAT-MBSA, and in contrast to immunizing with GAT-MBSA only, the animals failed to produce —-v\.—-- --_A‘> antibody (Kapp, Pierce, and Benacerraf 197A). Previous exposure to uncoupled GAT had rendered the animals unrespon- sive to GAT-MBSA. Furthermore, adoptive transfer of nonresponder spleen cells from GAT immunized animals to unimmunized animals of the same strain also rendered the recipient animal PFC unresponsive to a GAT-MBSA challenge. Like Gershon and Kondo's suppressor cell, the adoptive suppression could be eliminated by anti thy-1 plus C indicating that a T cell was responsible for the suppressive activity. Benacerraf‘s group was able to characterize this suppressor T cell (TS) even further by use of the newly produced anti-murine lymphocyte (Ly) antisera. They found that the transferred TS cell could be abolished by anti Ly 1', 2+, 3+ antisera. placing the TS cell into a distinct T lymphocyte subset. It is important to note that not all nonresponders exhibit T cells. However, both Gershon and Kondo and S Benacerraf et al., have clearly shown that suppressor cells are involved in the immune response. Their results are very similiar (T cell role, effect on antibody production), yet their approaches are quite different (soluble antigen versus particulate antigen and chimeras). Their contri- butions have been of prime importance in generating much of the research that is responsible for the current level of understanding of immune response control. 10 In Vitro Analysis of TS Cell Activity The initial suppressor cell research was all performed in 1129. With recent technical advances in la xitgg cell culture, new experimental approaches have become available. One of the most popular techniques is that of Mishell and Dutton (1967) whereby lymphocytes can be successfully immunized in vitro, with the response measured in the plaque forming cell assay. This method was utilized by Eardley and Gershon (1976) in further analysis of the suppressor T cell. Using an approach similar to Gershon and Kondo (1971), in Vitro stimulation of murine spleen cells followed by cell transfer to a normal murine anti-SRBC cultures was used to carefully follow the activity of TS cells. Antigen dose (SRBC) was found to be important in producing the desired effect, i.e. low dose of antigen favored helper cell activity whereas high dose favored suppressor cell activity. Antigen specific suppressor cells were produced in 2 - 3 days and had to be added to the Mishell-Dutton culture within 48 hours of its initiation, or no effect was seen. In addition, suppressor activity of high antigen dose stimulated spleen cells was abolished by treatment with anti-theta (9) plus C, a murine T cell specific antisera. This same technique was also used to produce antigen specific TS cells to a soluble antigen - keyhole limpet hemocycanin (KLH) (Feldman and Kontiainen 1976). Again 11 it was concluded that the suppressor activity was T cell controlled (thymectomy and anti-lymphocyte serum methodology), and, by several macrophage (MU) depletion techniques, it was also concluded that MC presence was not necessary for TS cell induction. T suppressor cells have also been shown to be produced by alloantigen stimulation. When two genetically different lymphocytes from the same species, either human (Hirschberg and Thorsby 1977) or murine (Rich and David 1979), are mixed in xitrg in a manner that only allows one of the individual's cells to proliferate (one-way mixed lymphocyte culture-—MLC), non-antigen specific TS cells can be produced. These cells actively suppress the response of autologous lymphocytes to other allogeneic cells. The murine alloantigen activated TS cells display I-C and I-J gene products (immune response antigens - Ia) from the Ir genes of the H-2 histocompati- bility complex. The recognition of I-J subregion products on antigen non-specific TS cells indicates a new relationship between these TS cells and antigen-specific TS cells, that only express I-J. The human alloantigen-activated TS cells are also suspected of displaying Ia antigens, but because of the lack of specific antisera. confirmation of this is harder to achieve. The Macrophage and To Cells Feldman and Kontiainen (1976) indicated that the MD was not necessary for antigen induction of Ts cells, but 12 several investigators have suggested an active role for the MC in the actual suppression of an immune response. A requirement for the interaction of MC and T cell for active suppression has been demonstrated in man (Stobo 1977), rat (Lause 1979), and mouse (Klimpel and Henney 1978). Each group utilized a different experimental system of antigen- induced suppressor cells, yet all three arrived at the conclusion that the MC was important in achieving suppression of the immune responses, which were decreased mixed lympho— cyte reactions (MLR), unresponsiveness to mitogenic stimulation, and decreased cytotoxic effector cell development. Once again, as more information is gathered on immune response control, the more complex the entire system becomes. Soluble Factors from Antigen-Induced Suppresggr Cells Although soluble factor control of the immune response had been hypothesized for many years, the first description of an antigenic-induced antigen specific suppressor factor occurred in 1973 (Tada et al. 1973). The discovery of this factor followed an extensive study on specific T cell suppression of a homocytotrophic antibody response in the rat (Okumura and Tada 1971). The same cells responsible for decreasing the IgE response were subjected to repeated freezing and thawing or sonication. The resulting cell-free supernatants were then tested in 1139 on rats with persistant high IgE titers. The drop in IgE production was rapid and lasted, in some instances, up to 40 days. The Specificity .f‘ 13 and molecular characteristics of this soluble factor were further defined by absorption studies and enzyme sensitiv- ities (Okumura and Tada 1974). The soluble factor was completely absorbed by insoluble immunoabsorbents composed of either homologous antigen, carrier protein, or antigen specific hapten coupled to a heterologous carrier, whereas passage of the factor through an anti-rat immunoglobulin column left the inhibitory activity of the supernatant virtually intact. Furthermore, the suppressor molecule was not sensitive to digestion with nuclease. but was completely destroyed by trypsin or pronase, indicating its protein nature. The molecular weight, estimated by gel filtration, was found to be between 35,000 and 65,000, another indication that it was not an immunoglobulin. The evidence supporting the protein-nonimmunoglobulin nature of the suppressor factor created as many new problems to be solved as it did excitement for those that predicted its existence. The origin of its specificity, genes respon- sible for its production, and the possibility of it being the elusive T cell receptor are all questions under intensive investigation. Benacerraf's group, working with the suppres- sor cells of genetically nonresponders to GAT, were also able to demonstrate an antigen-specific suppressor factor (Kapp, Pierce, et al. 1976; Kapp, Pierce, and Benacerraf 1977). T cells from CAT-primed nonresponder mice produce a protein suppressor factor (GAT-TSF) with no immunoglobulin 14 determinants and a molecular weight in the range of 40,000 - 55,000 (Theze, Kapp, and Benacerraf 1977). The physical characteristics are very similar to that of Tada's factor, and both are also absorbed by antigen. Recently both factors have been mapped to the I region of the murine H-Z, with Tada's mapping to the I-J subregion (Tada, Taniguichi, and David 1976). The most specific differences between the two factors are: (1) their specificity for antigen, i.e. KLH and CAT; and (2) their strain restrictions for activity. Syngeneic or allogeneic nonresponders are both responsive to GAT-TSF, whereas the factor described by Tada is only functional with mice syngeneic at the I-J subregion. Other suppressor factors have now also been reported by investigators using the antigen-induced TS cell model. Although most antigen specific suppressor factors have been shown to be similar in physical characteristics including molecular weight, substantial differences have become quite clear as to (1) the type of response they control; (2) expression of antigenic determinants; (3) and restrictions as to the strain that will accept the factor. Delayed hypersensitivity (Asherson and Zembala 1974), tumor rejection (Fujimoto, Greene, and Sehon 1975, Greene 1977) as well as antibody response all appear to have suppressor factor mediators capable of controlling their respective response. These factors all exhibit determinants 15 of the H-2 complex, yet whether or not they all bear products of the I-J region is yet to be determined. Mechanisms and Targets of Antigen-Induced Suppression Perhaps the least understood area of immune response control is that of how it is actually achieved. It is quite evident that suppression is mediated by cells and in some cases their soluble factors, yet the pathway leading to the suppressed response is unclear. In the case of TS cells, it is difficult to ascertain the difference between cell-cell contact and mediation by soluble factors that may only be active in close proximity. Attempts to identify the cell types involved by isolation of specific cell subsets must be viewed carefully. Lymphocytes are a heterogenous population of cells and require more than just one or two isolated cell types for a normal in 1112 immune response. Suppressor factor mechanisms have been less difficult to study in the pathway analysis of immune response control. One reason is that they may represent an intermediate step in the entire process. Treatment of a single cell population and addition of the treated cells to normal populations produces data with more reliable interpretations than in an isolated subpopulation study. However, the apparent heterogeneity of antigen-induced suppressor factors clouds the significance of generalized results from a study of one factor in one specific system. Lastly, purification of many factors has proven to be very difficult, an obvious drawback in the analysis of its interaction with the cell. 13‘ 16 The target cell of antigen-induced suppression has also varied from one experimental system to another. In most cases suppression is clearly mediated by T cells, yet MC involvement has been indicated in several studies. Asherson and Zembala, in the study of soluble factor suppres— sion of delayed hypersensitivity, have found that their factor is bound by MC (Zembala, Asherson, et al. 1975). Similarly, tumor-induced Ts cells in the mouse have been found to be absorbed by murine MC monolayers, but not on spleen cell monolayers (Argyris and Cotellessa 1979). Allogeneic or syngeneic MC monolayers will eliminate the TS cell activity, but the process requires viable monolayers. Yet in contrast to the apparent necessity on MC interaction, the carrier—specific factor described by Tada is bound by T cells which in turn become TS cells. The simplified model of activated T cell directly influencing the B cell has become increasingly complex. Many new models have been hypothesized incorporating soluble factor control, T cell suppression of T cell responses, and possible role of MB in the mechanisms of suppression. Some of the current hypotheses are: 1. TS cells regulate immune response by inactivation of the required helper cell activity. 2. TS cells are capable of producing a suppressor factor that non-specifically inactivates other cell types as well as T cells. 17 1 (TS cells and TH,cells are capable of regulating each other, with a role existing for both specific . cm~ soluble factors and direct cell influence from each. 4. ‘vf ch bee of these hypotheses still has some support in earrent research. (e(t. '1.' p / . l .-‘ ‘ b; I. e \ 1 1 :1 . . L’Cllic 1 1 . 15731.. ' - ‘ 2'" ‘ . . ’ m4; Kc- r 'v“$1‘* ~ , . «r. . “a 21.3.. ~ 1.’ . ‘ .I‘ ,9"! cell . .x'. ‘ ‘ w». . . 3 I . I ‘. ~‘ 5" a -1. 18 Mitogen Induced Suppressor Cells History of Mitogens in Immunobiology The potentiating effect of mitogens on lymphocytes was not recognized until the early 1960's (reviewed by Nowell 1976). Previous to this time small lymphocytes were classi- fied as non or rarely dividing cells with a very short in vitro culture time. Upon discovery that certain plant extracts (lectins), bacterial products, antibody reagents, and miscellaneous chemicals could stimulate these "non- dividing cells" to undergo blastogenesis and mitosis, a new era in the study of lymphocyte activity began. One of the most pursued aspects of mitogen stimulation was that of serving as a model for certain aspects of the immune response. In addition, with the recognition of major lymphocyte subpopulations (B cells and T cells) came the discovery that some mitogens will selectively stimulate B cells or T cells. One such mitogen is Concanavalin A. Concanavalin A (Con A) is a lectin extracted from the plant Canavalia ensiformis. It is a dimer of identical subunits with a molecular weight of 106,000 daltons (Cunningham, Sela, et al. 1976). This lectin has gained a prominent role in lymphocyte research because of its specific T cell stimulatory capacity. Following the reports of T cell mediated help and suppression in antigen stimula- tion, Con A has been extensively studied in creating models of TH and TS cells in the in vitro immune response. 19 Mitogenic Induction of Suppression Mitogen induced suppression of an immune response was first demonstrated in 31339 using the Mishell-Dutton culture system (Mishell and Dutton 1967). When Con A was added to freshly prepared murine spleen cells, there was a marked inhibition of the immune response to heterologous erythrocytes, haptenated SRBC, and haptenated KLH (Dutton 1972). Both the primary (IgM) and secondary (IgG) responses were affected as measured in the Jerne plaque assay. The degree of inhibition is markedly dose dependent and time dependent. A somewhat higher-than-optimal mitogenic dose of Con A results in optimal suppression, but increasing the dosage even higher actually decreases the degree of suppression (i.e. the number of PFC increases). Suppression of the immunoglobulin response is not immediate, and only becomes marked on day 3 or 4 of culture. Similarly, if the spleen culture is initiated 24 hours before the addition of Con A, the ability to induce suppression is lost and often stimulation of the PFC response is observed. Again, the cell responsible for this suppression is clearly a T cell. From the methodology of Gershon, adult thymectomized, irridated, bone marrow reconstituted mice do not exhibit Con A-induced suppression. Similarly the suppressor cell activity is absent from the spleens of congenitally athymic (nude) mice (Dutton 1973) and is progressively lost from the spleens of adult thymectomized mice (Kappler and Marrack 1975). 20 Con A induced suppression of immunoglobulin response was also extended to cell mediated immunity (CMI) studies. Utilizing the in 11322 MLR, it was found that the addition of mitogenic doses of Con A would inhibit the development of specific cytotoxic T lymphocytes (CTL) that would normally occur (Peavy and Pierce 1974). The kinetics were very similar to Dutton's observation in that the suppression was dose and time dependent, and the suppression became evident only in the late phase of exponential exPansion. The suppression was also definitely T cell mediated. One of the drawbacks of the antigen—induced suppressor assays was the difficulty in preforming experiments with human lymphocytes. In 3139 techniques were obviously not possible and in 31339 technology was less developed for human peripheral blood lymphocyte (HPBL) study. However, in xitgg stimulation of HPBL with mitogens was quite possible, opening the door to the analysis of suppressor cells in the human immune response. A short time after the report of Con A-activated TS cells in the mouse, Con A suppression of HPBL immunoglobulin production was demonstrated (Haynes and Fauci 1977). Using pokeweed mitogen (PWM) stimulation of HPBL polyclonal immunoglobulin production, suppression was found to be dose dependent and a function of T cell mediation. In addition Con A inhibition of CTL development in the human MLR was also found to occur (Mawas, Charmot, et al. 1977). 21 There are many similarities between antigen and mitogen-induced suppression of immune responses. Both have a specific dose dependency. Variances in the amount of antigen or mitogeriproduce suboptimal suppressions, and in some cases augmentation of the immune response. Suppression has been demonstrated in immunoglobulin response and CMI for both systems, indicating that T cell as well as B cell functions can be affected. Finally. mitogen as well as antigen-induced suppression is mediated by T cells. That suppression is not mediated by antigen (Gershon and Kondo 1971) or mitogen (Haynes and Fauci 1977) has been clearly demonstrated. Cell mediation is supported by the observation that increasing amounts of Con A results in less suppression (Dutton 1972). It appears that that the in 21322 mitogen model of Con A-induced suppression closely approximates the antigen-induced suppressor system. Concanavalin A-Induced TS Cells It was soon recognized that not only did the addition of Con A cause a decreased antibody response mediated by T cells, but that the suppressor activity could be transferred by these same cells to normal lymphocytes never been exposed to Con A (Dutton 1972, Rich and Pierce 1973b). As in Gershon's analysis of the antigen-induced TS cell, the trans- fer of prestimulated cells to normal ig 1139 or in xitgg immune responses enabled the properties and activities of the suppressive cell to be characterized. 22 As was expected, the T cell was found to be responsible for the transfer of suppression (Dutton 1973, Rich and Pierce 1973). In a study of the tissue distribution of Con A-inducible TS cells, it appears that the majority of cells are found in the peripheral lymphoid tissue, with few being generated from thymus cells and none from bone marrow (Rich and Pierce 1973). While these observations are in agreement with the T cell nature of the suppressor cell, they also tend to indicate some in 111g relevance for the Con A suppressor cell model. Perhaps the most complete study of the effects of Con A on the immune response is that of Rich and Pierce. From their initial study on the effect of mitogen on anti- body response (Rich and Pierce 1973a), they have extended their model to TS cells affecting various cell-mediated immune responses (Rich and Rich 1975; Peavy and Pierce, 1974) and soluble factor control (Rich and Pierce 1974). Using murine spleen cells as a source of Con A—TS cells, they have been able to demonstrate suppression by means of cell transfer of both the primary and secondary 1g vitro IgG and IgM responses to SRBC (Rich and Pierce 1973b). This suppression must be initiated early in the immune response and is only evident during the late stages of the response. Kinetics of the Con A-TS cell are very similar to those of Dutton in which Con A was added directly to the PFC culture. This is also true of the Con A-activated T cell suppression of the ig vitro MLR and CTL development (Rich and Rich 1975, Peavy and Pierce 1974). In both instances Con 23 A-induced TS cells actively produced a suppressed response observable only in the late phase of exponential expansion. Interestingly enough the two suppressor systems seem to vary in their sensitivity to irradiation. The suppressor cell in the MLR is sensitive to mitomycin C treatment as well as irradiation, whereas the TS cell responsible for decreased CTL development is not sensitive to irradiation. The authors feel that this may represent the possibility of varying subsets of TS cells (Rich and Rich 1975). Although there have been no reports of TS cell subsets, this hypothesis would help to explain the relationship among other TS cell studies yielding different results. The comparison of mitogen and antigen-induced TS cells is not without its differences. Although Con A-Ts cells are + 3* (Jandinski, Cantor, et al. 1976), they also Ly 1‘, 2 in general do not display Ia determinants like the antigen- induced suppressor cells (Dugan, Miederhuber, et al. 1977). Their radiation sensitivities also vary. Con A-induced TS cells become insensitive to irradiation after stimulation (Rich and Pierce 1973b). This is not the case with the GAT-TS cell, which remains sensitive to irradiation at all times (Kapp, Pierce, et al. 1974). Whether these differ- ences represent varying subsets of TS cells as has been suggested or the inability of the mitogen-induced model to fit the antigen-suppressor system is unclear. Human peripheral blood lymphocytes have also been found to contain Con A-inducible TS cells. These cells are capable 24 of suppressing: (1) immunoglobulin production as measured by both polyclonal PFC response (Haynes and Fauci 1977) and radioimmunoassay (Schwartz, Shou, et al. 1977); (2) T and B cell responses to several mitogens (Shou, Schwartz, and Good 1976; Hubert, Delespesse, and Govaerts 1976) and (3) T cell responces to allogeneic stimulation (Shou, Schwartz and Good, 1976; Mawas, Charmot, et al. 1977; Hubert, Delespesse, and Govaerts 1976). As in the mouse the Con A-induced TS cell is generally non-antigen specific. Mechanisms of Mitogen—Activated T Cell Suppression As in the antigen system, the most confusing area in the Con A-TS cell model is how suppression is achieved. Discrepancies concerning TS cells as to sensitivity to irradiation, MC requirement, target cells, and necessity for DNA synthesis are all apparent in the current literature. There is virtually total agreement that the TS cell must be present early in the initiation of the response and that its effect is seen late, yet the pathways described for this suppression are variable. The MC requirement of Con A-TS cell induction is one such discrepancy. Dutton has reported that suppressive activity is developed equally well if adherent cells are removed (Dutton 1975). In contrast, a recent report has indicated that MC-T cell interactions are required for Con A induction of the TS cell (Raff, Cochrum, and Stobo 1978). The difference may lie in the type of suppressive assay used, 25 where Dutton was measuring PFC and Raff measured phyto— hemagglutinin (PHA) blastogenesis. In support of this, Fauci, studying the polyclonal PFC response of HPBL, has reported that removal of adherent cells did not affect the generation of TS cells. Whether or not the B cell is directly affected by the TS cell seems to be dependent on the type of antigen used. Con A-TS cells, capable of decreasing the primary response to SRBC, show no effect on lipopolysacchide (LPS) stimulation of murine B cells (Ehstedt, Waterfield, et al. 1977). This has been interpreted as indicating that the B cell was not directly affected by the TS cell. Yet when Con A-TS cells are assayed with a different T independent antigen, DNP-levan, suppression is observed comparable to that in the SRBC system (Shand and Ivanyi 1977). It was suggested that the strong LPS stimulation of B cells-may be covering up any observable suppressor activity. Regardless of the difference in results, both groups indicated that Con A-TS cells may also be acting upon TH cells and/or MC. The most likely exPlanation for the wide range of activities of virtually identical Con A activation of TS cell is that a heterogeneity of TS cells exist. Presently at least one laboratory has addressed this possibility (Haynes and Fauci 1978). Using several T cell isolation procedures, Con A-induced TS cells were found in disparate isolated fractions, inspiring the authors to conclude that T cells S appear to be heterogenous. Elli. 26 Concanavalin A Stimulation and Suppr§§§or Factors Con A stimulated lymphocytes also produce soluble factors capable of suppressing immune responses. These mediators appear to be different from antigen-induced soluble suppressor factors in several aspects. Although few mitogen-induced suppressor factors have been well character- ized, they all are generally non-antigen specific and some have demonstrated 1Q 3112 as well as in 11339 suppression. The first reported Con A-induced suppressor factor was that of Rich and Pierce as a result of their studies of Con A-activated TS cells in the mouse (Rich and Pierce 1974). This factor, termed soluble immune response suppressor (SIRS), is found in the supernatant of Con A-activated TS cells. It can be found as early as 6 hours after Con A stimulation with maximum production occurring at 24-48 hours. In comparing SIRS to Con A-TS cells, their kinetics are very similar. SIRS must be added within 24 hours of initiation of the SRBC-PFC culture but only need be present during the first 24 hours. As in the case of TS cells, no effect on normal PFC response is seen until day 5 when strong suppres- sion of both IgG and IgM production becomes evident. It is interesting to note that Con A-TS cells can be added as late as 48 hours after addition of antigen and still suppress the response, whereas SIRS must be added no later than 6 hours after. SIRS has suppressed the antibody response to all antigens tested but has no effect on a variety of T cell 27 responses $2,2l322 (Pierce, Tadakuma, et al. 1976). In the suppressed antibody response, DNA synthesis stops abruptly 24 hours before the apparent decrease in PFC begins. Interruption of the B cell exponential eXpansion to antibody secreting plasma cells appears to be the level at which the suppression occurs, although a case can be made for prevention of antibody secretion or production by plasma cells. Never- theless, both would indicate the susceptibility of B cells to physiological signals that may be capable of limiting or terminating an immune response. The target cell of SIRS appears to be the MC (Tadakuma and Pierce 1976). Only SIRS-treated MC are capable of suppressing the antibody response of normal spleen cells, and this suppression can be overcome by the addition of normal untreated MC. Furthermore, suppression of the B cell response by SIRS-treated MC does not require the presence of T cells. It appears that the MC directly influences the activity of the B cell in this suppressor system. The necessity of cell contact between MC and responding B cell was examined by membrane separation (Tadakuma and Pierce 1978). Suppression of the antibody response can still be achieved without cell contact. Since SIRS has no effect on the B cell alone, this observation prompted the investigators to conclude the existence of a MC secreted-B cell inactivating factor, and that this MC factor was responsible for the termination of antibody production. 28 Biochemically, SIRS is similar to a few other well characterized lymphokines (Tadakuma, Kuhner, et al. 1976). It exhibits migration-inhibition factor (MIF) activity and has yet to be physically separated from MIF. SIRS also resembles interferon type II. SIRS activity is abolished by heating at 70°C for 30 minutes, 80°C for 10 minutes, or treatment with the enzymes trypsin or chymotrypsin. Although SIRS is a glycoprotein, it contains no immunoglobulin determinants. Supporting the nonimmunoglobulin structure of SIRS is the estimation of its molecular weight between 48,000 and 67,000 daltons. In total, all activities and physical properties of SIRS are in agreement with those of MIF. While it is conceivable that two biological functions are being observed from the same molecule, more highly purified MIF does not appear to exert suppressor activity. Reinerstein and Steinberg have also reported production of a soluble suppressor factor from Con A—activated spleen cells (Reinerstein and Steinberg 1977). Their factor not only inhibits antibody production in the PFC assay, but also suppresses the 13 yigg antibody reSponse. It is similar to SIRS in that it is nonantigen Specific, but its molecular weight of<110,000 daltons clearly separates it from SIRS as well as MIF and interferon. Human lymphocytes have also been found to produce a soluble suppressor factor when activated with Con A (Kaufman, Carnaud, et al. 1979). This factor, termed SIRSH, suppresses 29 the T cell proliferation measured in the one way MLR. The Con A-T cells produced in the same system will suppress S CTL development as well as the MLR. Yet the soluble factor from the Con A-T cells only suppress the T cell prolifer- S ation, indicating that proliferation may not be necessary for CTL development. This suppressor factor is also non-antigen specific and will suppress the xenogeneic MLR as well. It was suggested by the authors that the differ- ence between the suppressor activity of their factor and the SIRS of Rich and Pierce may be due to the possibility of separate factors for different immunological responses. Further elucidation of SIRSH mechanisms and physical pro- perties would be necessary to support this hypothesis. There exists an even greater difference between antigen and mitogen-induced suppressor factors than between their respective suppressor cells. Although both classes of factors are protein in nature, their specificties vary greatly. Con A-activated suppressor factors show little or no antigen specificity or H-2 restriction unlike antigen— induced factors. Their molecular weights fall into similar ranges but in contrast to antigen-induced factors, no Con A suppressor factors have been found which contain Ia antigenic determinants. The relationship between these differently produced suppressor factors will only become apparent upon further purification and characterization of each. 30 The Relationship Between Immune Response Help and Suppression Early in the development of immune response research it was recognized that the dose of antigen or mitogen was important in producing the desired response, i.e. help or suppression (Gershon and Kondo 1970; Gershon, Miebhaber, and Ryu 1974; Miller and Mitchell 1969; Dutton 1975). Several investigators found that variances in help or suppression were related to the number of T cells used, thus forming the hypothesis that suppression was actually a condition of too much help. Similarly, help was then considered to be inadequate suppression. With the advent of new cell separation techniques, the one cell concept of help and suppression is being challenged. Size differences, surface markers, and radiation and chemical sensitivities have all been used to indicate that separate TH and TS populations exist. The controversy of separate cells for help and suppres- sion also extends to soluble factor control of immune responses. Some lymphokines exhibit help or suppression depending upon the immunological assay. Some antigen- induced suppressor factors have been shown to contain helper activity as well (Tada, Okumura, and Taniguichi 1973). Dissection of the activity on a molecular basis may be the only means of assigning specific helper and suppressor activity to either one or more factors. 31 Separation of T Helper and T Suppressor Cells One of the first observations made linking help and suppression was from a study of T cell regulation of T cell responses (Gershon, Liebhaber, and Ryu 1974). It was found that the response of parental thymocytes injected into an irradiated F1 mouse could be modulated by an injection of F thymocytes. The modulation of the response 1 seemed to be dependent on the number and activity of the parental cells in the presence of a constant number of F1 injected cells. The proliferation of a large dose of par- ental cells or parental cells that were highly responsive were suppressed by the F1 cells; low dose or poor repsonding cells were augmented. Therefore, it appeared that the same inoculum of F1 thymocytes was capable of performing TH or TS activity. This evidence was clearly not conclusive for the assumption that one cell type was capable of both activities, yet it did indicate that the same population of T cells was capable of responding differently in accor- dance with the immunological situation. Stronger evidence indicating that TH and TS cells were of the same population has also been reported (Rich and Pierce 1973b). Con A- activated TS cells in the mouse exhibit a dichotomy of activities when used in various quantities. High numbers of Con A-activated cells will suppress the SRBC-PFC response, whereas low numbers augment the same response. These findings are in total agreement with the supposition that suppression is too much help. 32 Present research, however, heavily favors the hypothesis that T and T are distinct T cell subsets (Gershon 1975). H S Suppressor T cells are more sensitive to irradiation (Dutton 1973; Eardley and Gershon 1976; Herschberg and Thorsby 1977) than TH cells. In the murine system, TS cells are Ly 2+, 3+ and TH cells are Ly 1+, regardless of antigen or mitogen induction (Cantor, Shen, and Boyse 1976; Jandinski, Cantor, et al. 1976). In man Con A-induced TS cells have been ascribed to a distinct T cell subset described as the TH + cell (Reinherz and Schlossman 1979). Both the 2 I and TH ‘ TH 2 2 T cell subsets are activated by Con A, but only the Th + cells become TS cells. Furthermore, the 2 induction of TS cells has been stated to be MC independent, whereas TH cell induction appears to be MC dependent (Eardley and Gershon 1976; Feldman and Kontiainen 1976). One of the most recent reports separating human lymphocytic helper and suppressor T cells suggests that the two have different receptors for the FC region of IgM (TR) and IgG (TX) respectively (Moretta, Ferrarini, et al. 1976). T3 cells were reported not to express T“ receptors, and vice-a-versa, and only T“ cells respond to PHA, whereas both responded equally to Con A. These findings indicated to the authors that T5 and T“ are distinct subsets of T cells. Furthermore, all the helper activity was found in the T“ fraction, and the suppressor, after interaction with immune complex, in the TI fraction (Moretta, Webb, et al., 33 1977). This study was also extended to the mouse with comparable results (Heijnen, Uytdehaag, et al. 1979). Antigen specific murine TH and TS were found to be TM+, T5- and Tg-, TJ+ respectively, but in this system T3 cells were active TS cells without the necessity of immune complex activation. Variances in TS cell induction may be due to the method of TS activation or represent the speculated heterogeneity of types of suppressor cells. Although this PC distinction of T cell subpopulations is attractive for theories on the switching phenomena of IgM to IgG in the primary and secondary antibody response and its relationship to help and suppression, it appears that it is not clearcut. Cultured TX cells can loose their IgG receptors and actually gain IgM receptors, becoming T“ cells (Pichler, Lum, and Groder 1978). Under some conditions an intermediate cell eXpressing both T3 and T“ can be isolated. In addition, TAL cells, after activation with Con A, have been reported to suppress the PWM induced differentiation of B cells to plasma cells (Hayward, Layword, et al. 1978). These 34 suppressor cells even behave as Con A-TS cells in their sensitivity to irradiation. Although neither hypothesis is firmly established, the evidence is stronger in support of distinct T and T H S populations. Reproducible observations of systems exhibiting only help or only suppression have helped to dispell the theory that suppression is too much help. In support of 34 separate cell types is the observation by Dutton that PHA induces suppressor cell activity but is unable to show any immune response stimulatory activity (Dutton 1973). Also, suppression can be reversed by adding increasing numbers of TH cells (Scavulli and Dutton 1975). If suppression was too much help, addition of more helper cells should result in more suppression. The question concerning T5 and T« cells representing suppression and help is unclear. PHA respon- siveness of the two fractions seems to indicate they are distinct populations, yet the ability of T5 to become T4 raises some questions as to the significance of the markers. Finally, the ability of TX as well as T“ cells to act as suppressor cells may well support the growing hypothesis of heterogeneity among suppressor cells. Antigen and Mitogen-Induced Helper Factors It is necessary to review some of the helper factors that have been described in order to better understand the relationship between immune response help and suppression. The argument that suppression is too much help has even weaker support from soluble factor research. Suppressor factors, such as SIRS, have been tested in low concentrations that should have exposed their helper activity, yet no help was observed as would be eXpected if a single mediator was responsible for both activities. In addition many helper factors have been reported that do not eXpress suppressor activity. 35 As in suppression, helper factors can be induced by antigen (Gisler, Stabler, et al. 1973; Farrar, Koopman, and Fuller-Bonar 1977; Woody, Zvaifler, et al. 1979) or mitogen (Gery, Gershon, and Waskman 1971; Watson, Aarden, et al. 1979; Kindred, Gosing-Schneider, and Corley 1979). Antigen-induced factors are generally antigen specific with exception of one helper factor reported to contain both a specific and nonspecific factor (Gisler, Stabler, et al. 1973). Mitogen-induced helper factors have been produced by several investigators and exhibit very little specifity for antigen. The majority of mitogen-induced helper factors have been produced by lymphocyte activation with Con A (Kindred, Gosing-Schneider; and Corley 1979; Watson, Aarden, et al. 1979; Primi, Hammerstrom, et al. 1979; Bernabe, Martinez- Alonso, and Coutinho 1979). These factors are produced under conditions very similar to that of Con A-induced suppressor factors, with the main difference being the amount of Con A used. Although potentiation of the antibody response is the most commonly reported activity, Con A induced helper factors affecting T cell function have also been described (Morgan, Ruscette, and Gallo 1976). At least one factor is believed to have activity for B and T cells (Watson, Aarden, et al. 1979). Generally they are all T cell products but their methods of activity vary widely. Some require the presence of T cells in order to stimulate antibody synthesis (Primi, Hammerstrom, et al. 1979) and 36 others affect only the IgM response (Kindred, Bosing- Sneider, and Corley 1979). Their nonspecificity for antigen is consistent with suppressor factors induced by Con A. However, it appears that a distinct difference exists between helper and suppressor factors and their activity toward target cells. Suppressor factors are absorbed by their target cell, whereas helper factors are not (Bernabe, Martinez-Alonso, Coutinho 1979: Primi, Lewis, and Goodman 1979). This in itself indicated a separation of helper and suppressor mechanisms in the immune response. Other mitogens have also been used to produce helper factors. Supernatants from PWM activated T cells will stimulate human tonsillar B cells to undergo blastogenesis and increased antibody synthesis (Insel and Merler 1977). This helper factor production is very similar to many Con A factors in that the amount of PWM used is important for the desired affect. Low doses of PWM favor helper factor production whereas high doses favor suppressor factor. PHA and LPS are also capable of activating human and murine mononuclear cells to produce helper factors (Gery, Gerhson, and Waksman 1972). This factor differs from the Con A factors in that it is produced by adherent cells, probably the MC (Gery and Waksman 1972), it potentiates the response of T cells to mitogens and does not require the presence of MC for the blastogenesis. This lymphocyte activating 37 factor (LAF) contains two active molecules, 13,000 and 85,000 daltons, with most of the activity residing in the 13,000 fraction (Blyden and Handschumacher 1977). Helper and suppressor factors demonstrate a more complete separation of their mechanisms and activities than their respective cell types. The existence of two distinct classes of factors aids the argument for the existence of two distinct cell types responsible for help and suppression, though the source of these factors has not been established. The different sensitivities of the cell types for varying quantities of mitogen also suggests that they are distinct subsets, yet not all helper and suppressor factors are T cell products. There is increasing recognition of multiple roles the MC may be playing in the immune response. Soluble Factors DemonstratingHelpand Suppression A few soluble factors systems contain helper as well as suppressor activity. With the exception of interferon, all of these appear to involve separate mediators for each activity. Mitogen (Primi, Lewis, and Goodman 1979), soluble antigen (Tada, Okumura, and Taniguchi 1973), and allogeneic stimulation (Rubin 1979) have all been shown to be capable of producing supernatants with helper and suppressor activity. Two of the investigating groups' findings are similar deSpite the difference in factor production. The Con A-induced helper and suppressor system exhibits help by rescuing tolerized cells and inducing polyclonal antibody 38 synthesis (Primi, Lewis, and Goodman 1979). Similarly the DNP-As induced factor of Tada also rescues tolerized rat cells allowing for specific IgE production (Tada, Okumura, Taniguchi 1973). The suppressor fractions of each are Specific for antibody production, and the activity of both the helper and suppressor is believed to be T cell mediated. The allogeneic helper and suppressor system is unique in that each factor is produced independently but their activities are indirectly antagonistic (Rubin 1979). Both affect the antibody production of murine B cells, apparently through different target cells. The suppressor factor utilizes the MC whereas the helper factor acts directly upon the B cell. When equal activities of helper and sup- pressor factors are used in the same PFC assay, no effect of either is observed, i.e. the result is a normal PFC response. These factors evidently work independently of each other in the control of the same immune response. The author also speculates that there is a temporal relationship between the appearance of the two factors, the helper factor appearing early after stimulation, 24-48 hours, and the suppressor peaking later, at 72 hours. Recently a new emphasis has been placed on the possible role of interferon (IF) in immune response control. Activation of lymphocytes by various means, including Con A, is known to cause the production of an IF known as immune or type II IF. There is a distinct molecular difference 39 between type I and type II IF, and the two are generally separated by their varying sensitivities to acid and heat. Type II IF is the only single mediator that has been impli- cated as possessing both helper and suppressor capabilities. Many of the soluble factors, both antigen and mitogen-induced, are suSpected of either being IF or containing IF in addition to other lymphokine mediators. Human type II IF will suppress the human MLC (Heron, Berg, and Cantill 1976). At certain concentrations, blastogenesis can actually be suppressed, yet with a definite simultaneous increase in CTL development. In addition B cell function can also be altered by type II IF. Decreases in antibody synthesis after treatment with IF has been reported both in humans and the mouse (Chester, Pauker, and Merigan 1973). Type II interferon will also augment natural killer (NK) activity. NK cells are lymphocytes found in the spleen or circulating blood of man and other animals. These cells, without prior eXposure, will lyse a variety of in zitrg and in yiyg tumor cell lines (Herberman et al. 1973; Oldham et al. 1973). Lysis is independent of C, antibody, and is non phogocytic (Kiessling, Klein, et al. 1975). Lysis is apparently achieved in the same manner that educated CTL lyse target cells displaying the sensitizing antigens. NK cells are nonadherent lymphocytes displaying a low density of T cell markers (Herberman, Djeu et al. 1979). They have no 40 surface membrane immunoglobulins, no C receptors, but are generally characterized by their receptors for the Fc region of IgG (West, Cannon, et al. 1977). The recent interest in NK cells is a result of several hypotheses relating NK activity to in yizg tumor surveillance. Although no hard evidence for this phenomena has been demonstrated in man, the advent of NK augmentation by IF has stimulated a great deal of interest in the area of cancer treatment. Lymphocyte proliferation is not involved in NK aug- mentation by IF (Droller, Borg, and Perlmann 1979). Although many in XEEEQ cell lines are known to produce IF of one type of another, IF doesn not make the target more susceptible to lysis. The increase in NK activity is apparently due to recruitment of pre-NK cells (Saksela, Timonen, and Cantell 1979), yet this isstill disputed. One reason is that NK activity can be augmented as quickly as one hour after a IF treatment. NK augmentation has been demonstrated in yivg as well as in vitrg (Senik, Gresser, et al. 1979). The in vivg increase is very similar to the in XEEEQ event in that it occurs quickly and apparently does not require lymphocyte proliferation. LIST OF REFERENCES LIST OF REFERENCES Argyris, B. F., and Cotellessa, A. Adsorption of Suppressor Cell Activity on Mouse Macrophage Monolayers. Trans- platation 28:372-376. 1979. Asherson, G. L., and Zembala, M. 'T Cell Suppression of Contact Sensitivity in the Mouse III. The Role of MO and the Specific Triggering of Nonspecific Sup: preision. European Journal of Immunology #:8OA-807. 197 . Bernabe, R. R., Martiwez-Alonso, C., and Coutinho, A. The Specificity of Nonspecific Concanavalin A-Induced Helper Factors. 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T cells are known to participate in the majority of antibody responses (11, 45), develop as specific cytotoxic killer cells (9), and control many aspects of the immune response. This role of T cells in control of the immune response, i.e. help and suppression, has progressed to a.major area of immunological study. Gershon and Kondo were first to describe T cell suppression of an immune response (19) and were instru- mental in initiating the hypothesis of cellular separation of help and suppression. It was also suggested by these authors that soluble mediators may play a role in T cell suppression. Dutton soon followed with the first descrip- tion of Con A induction of help and suppression in antibody production (14,15). The demonstration of Con A inducible TS and TH cells provided a new model for both the study of help and suppression and the existence of soluble mediators. Subsequently, several investigators have reported suppressor factors (36, 48, 49, 52), and helper factors (37, 59) produced from Con A activated T cells. Con A induced suppressor factors have been described that will affect MLC blastogenesis (36) and antibody 51 52 production (49, 52). One such soluble factor is SIRSH, described by Kaufman et al. SIRSH is a product of Con A ' activated human lymphocytes. Since it is asuppressor factor, it has been termed a soluble immune response suppressor'~ (SIRS) in reference to a similar murine factor described by Rich and Pierce (52), and has been designated SIRSH to indicate it is a product of human lymphocytes. The suppressor activity of SIRSH is Specific for the mixed lymphocyte culture, however, SIRSH was suspected of having more than just suppressor activity. Several investi- gators have reported mitogen induced supernatants that contain both suppressor and helper activities (16, 46,1:8). In some cases the activities are antagonistic and in others they appear to be independent of each other. Similarly, SIRSH has been found to be composed of a mitogenic factor as well as a suppressor factor. In addition, it will augment the cytotoxicities of natural killer cells and lectin dependent cellular cytotoxicity. The suppressor factor and mitogenic factor appear to act independently, but their relationship to the NK increase is unclear. Finally the relationship between the SIRSH soluble factor activities and the defect in lymphocyte function in auto- immune disorders will be discussed. MATERIALS AND METHODS Human Peripheral Blood Lymphocyte Isolation Venous human peripheral blood was collected in a sterile heparinized (Panheprin, Abbot Laboratories, Chicago, Illinois, 10 units/ml blood) syringe(Becton and Dickinson, Rutherford, New Jersey) from healthy volunteers. Five milliliters (ml) of blood was layered over 5.0 ml of Ficoll - Hypaque solution (FH) (specificy density 1.077; oral Hypaque diatrizoate solium, 34%, Winthrop Laboratories, New York, New York; Ficoll, 9%, Sigma Chemical Company, St. Louis, Missouri) in sterile polystyrene culture tubes (Falcon 2001, 17 X 100 mm) and centrifuged at 400 X g for 25 minutes (Sorvall GLC-2B with swinging bucket rotor, HL-4, Newtown, Connecticut) (6). In some cases the autologous plasma was removed from the top of the resulting differen- tial gradient, heat inactivated at 5600 for 45 minutes, and saved for later use. The middle band containing the mononuclear cells was collected, diluted 1:2 with RPMI—1640 (Grand Island Biological Company - GIBCO, Grand Island, New York) and centrifuged at 500 X g for 10 minutes. The pellet was resuspended and washed 3 times and finally resuspended in RPMI 1640 + 10% fetal bovine serum (GIBCO- mycoplasma and viral screened, Grand Island, New York) or 10% human plasma (autologous or pooled), supplemented with 53 54 '50 ug/ml gentomycin (Schering Corporation, Kenilowrth, New Jersey). Human peripheral blood lymphocytes (HPBL) isolated by this method yielded 1 - 2 X 106 lymphocytes per ml of blood with a purity and viability of mononuclear cells greater than 95% as measured respectively by Wright's Stain (DIF-QUIK, Harleco, Gibbstown, New Jersey) and trypan blue dye exclusion (GIBCO, Grand Island, New York). Soluble Factor Production Soluble immune response suppressor-human (SIRSH) factor was produced by incubation of HPBL at a concentration of 2 X 106 cells/ml in RPMI 1640 + 10% FCS with 10 ug/l X 106 cells of Concanavalin A (Con A, Miles—Yeda, 3X crystalized, lyophilized, Miles Laboratories, Elkhart, Indiana) for 24 - 48 hours at 3700 in a 5% CO2 humidified incubator (National Appliance, Portland, Oregon) (Figure 1) (36). At the end of the incubation period, the cells were pelleted and the supernatnat (SIRSH) collected- This crude supernatant was either stored at ~2000 or used immediately. Before measuring the activity of individual batches of SIRSH, the supernatant was depleted of Con A by 3 incubations with equal amounts of Sephadex G-50 (G-50 - 300, 100 - 300“ , Sigma Chemical Company, St. Louis, Missouri) for a minimum of 1 hour at 400 (2). This treatment, by means of [3H]--Con A, has been shown to be effective in removing all significant amounts of Con A. All fine particles of Sephadex were removed by high speed centrifugation (11,000 X g, Beckman J - 21, Palo Alto, 55 HPBL + Con-A (50 ug Con A/5 X 106 cells, 2 X 106/ml) 24-48 hrs., 37°C, 5% 002 - Spin Down Cells (TS) - Collect Supernatant ABSORB OUT Con-A WITH EQUAL VOLUMES OF SEPHADEX G-SO 3x, 1 Hr., 40 C FILTER STERILIZE AND ASSAY BIOCHEMICAL ANALYSIS EFFECT ON CMI Figure 1. SIRSH Production 56 California), and the resulting supernatant was sterilized by passage through a 0.2 um filter (Nalgene Filter Unit, Rochester, New York: Gelman 25mm Acrodisc, Ann Arbor, Michigan). SIRSH controls consisted of media plus Con A and HPBL without Con A, both absorbed with Sephadex c-50. Allogeneic Mixed Lymphocyte Cultures Allogeneic one-way mixed lymphocyte cultures (MLC) were performed in flat bottom microtiter trays (Falcon Micro Test II, Oxnard, California) (57). Equal numbers of responder and stimulator cells (1 X 105/well) in RPMI 1640 + 10% autologous (responder) or pooled human plasma, supple- mented with 200 mM L-glutamine, sodium pyruvate, 100X nonessential amino acids (GIBCO, Grand Island, New York), were co-cultured for 6 days at 37°C and 5% 002. Stimulator 6 cells were treated with 104g of Mitomycin C per 1 X 10 cells at 2 X 106 cells/ml (Boehringer Mannheim, West Germany) for 30 minutes in a 37°C water bath and then washed 3 times before use. On day 5.1 uCi of [EHJ-thymidine (specific activity 24 Ci/Mmol, methyl-3H, Amersham, England) was added to each well for an 18 hour overnight incubation. Cultures were harvested on day 6 by the collection of labeled cells on glass fiber filters (Whatman 934, AH grade, Clifton, New Jersey) through the use of a multiple sample harvester and isotonic saline (Otto Hiller Company, Madison, Wisconsin). The filters were allowed to air dry and the DNA solubilized with 0.5 molar Protosol at 60°C for 30 minutes (New England Nuclear, Boston, Massachusetts). Five milliliters of a 57 PPO-POPOP toluene based scintillation fluid was added to each sample and the incorporation of [EHJ-thymidine was determined by liquid scintillation counting for 10 minutes. (Packard Tri Carb, Scintillation Spectrometer, Model 3003: 0.4% 2,5 - diphenyloxazole, PPO, Research Products Inter- national-RPI, Elk Grove Village, Illinois: 0.01% 1,4 bis [E-(4-methyl-5-phenyloxazlyl{]-benzene, POPOP, RPI, Elk Grove Village, Illinois: dissolved in a toluene base, Malincrodt, Paris, Kentucky). SIRSH, SIRSH controls, or Con A was added at the beginning of the MLC culture (36), all in 0.1 ml aliquots. All tests were done in triplicate with values calculated as counts per minute (cpm), stimulation indexes (SI), or per cent suppression (%S), where SI : cpm stimulated or (R X SM) cpm cpm nonstimulated (R X RM) cpm (R X SM + SIRSH) - (R X RM) %S x 100 (R x SM) - (R x RM) (R - responder, S = stimulator, and M = mitocycin C treatment) (36)- Mitqgenic Assay Blastogenesis was measured in a 3 day micortiter assay. Cells (0.1 ml of 1 X 106 cells/ml) in supplemented RPMI 1640 + 10% FCS or 10% human plasma were distributed in flat bottom wells of a Falcon microtiter plate and stimulated with Con A(0ptimal dose 2ug/1 X 105 cells), phytohem- agglutinin (PHA, 1:200 dilution, reagent grade, Wellcome 58 Reagents, Beckenham, England), or pokeweed mitogen (PWM, l 20. GIBCO, Grand Island, New York). At the end of the second day, 1uCi of I?H] thymidine was added to each well and allowed to incubate overnight. Each well was then harvested using the multiple sample harvester and counted in a liquid scintillation counter as previously discribed (see "Allo- geneic Mixed Lymphocyte Cultures" in Materials and Methods). The mitogenic capabilities of SIRSH was assayed for by the same method, using 0.1 ml/105 cells of various dilutions of SIRSH or control SIRSH. In some cases the effect of SIRSH on mitogen stimulation was examined by adding both to the same microtiter well. The ability of80%. A comparison of the Con A and MF sensitivity to 0( MM appears to indicate that MF has a higher affinity for o< MM than Con A. Alternatively it could be argued that the concentration of MP is substantially lower than the concentration of Con A. Regponding Cell to SIRSH-MF Lymphocytes were separated into T cell and non-T cell fractions by means of PH centrifugation after the formation of E-rosettes in order to determine the responding cell type to SIRSH-MF. Cells separated by this procedure were tested for their quality by re-rosetting and blastogenesis with PHA, a predominantly T cell mitogen. The T cell isolated fraction was greater than 90% E-rosette positive as well as highly responsive to PHA (Figure 6). The non- resetting non-T cell fraction contained less than 5% E-rosette positive cells and was highly insensitive to PHA stimulation (Figure 6). The T cell lineage appears to be the major responding lymphocyte p0pulation to SIRSH-MF (Figure 6). Lymphocytes forming rosettes with SRBC respond to ME nearly as well as unfractionated cells, whereas the non-rosetting fraction responded minimally. A comparison of the effects of 24 hour and 48 hour produced SIRSH-MF on the isolated cell fractions supports both the time dependency of production of MF and -76 Unfractionated _ 60 Non T Cells VI. SO 3 T cells x 40 o p c H c o 13‘ m 30 H 3 E H 4.) m 20 - lO - SIRSH PHA .‘:=S: Figure 6. Proliferative Target Cell of SIRS -MF. Stimulation of lymphocytes separated by ability tU form E-rosettes. BlastOgenesis assayed in standard 3 day assay, with PHA at 1:200 and SIRSH at 1:3 final dilution. Error bars represent S.E.M. 77 the primarily T cell responding target (Table 5). MF from 24 hour SIRSH cultures clearly has a stronger MF stimulation in all fractionated and unfractionated cell groups studied. At least a 50% reduction in stimulation is observed when using 48 hour supernatants as a source of MF. and in the case of the non T cell fraction, virtually no stimulation occurs. Adherent lel Requirement for ME Activity The requirement of adherent cells for the blastogenic response to ME was examined by adherent cell depletion on plastic followed by passage over a nylon wool column. Lymphocytes prepared by this method contain less than 0.1% adherent cells as measured by peroxidase staining. When used in a PHA blastogenesis assay, the nonadherent cells exhibit a drastic decrease in 3H-thymidine uptake as compared to unfractionated mononuclear cells as has been reported by other investigators (Figure 7). Addition of 1000 adherent cells to 105 adherent depleted cells restores the lymphocyte response to PHA to greater than 50% of the unfractionated cells response. When nonadherent cells are used as responding cells in a MF blastogenesis assay, virtually no blastogenesis occurs. The addition of 1000 adherent cells restores approximately 50% of the original MF response of unfaction- ated cells. Adherent cells alone do not respond to MF. They were isolated from plastic dishes with a purity of 80% as 78 Table 5. Comparison of MP Prodpgtion and Target Cell Stimulation Index 24 Hr. SIRS 48 Hr. SIRS H H Unfractionated 26.814.8 10.512.7 Cells T Cells 11.012.1 6.011.2 Non T Cells 5.012.3 1.610.2 Effect of 24 and 48 hour produced SIRS -MF on lymphocyte fractions after SRBC rosetting. Values are mean SI from 3 day assays 1 S.E.M. Table 6. Effect of SIRSH on Target Ce1151Chromium Release Per Cent Release Expt. I Expt. II Expt. III Medium + K562 12.2% 16.3% 32.7% Control SIRSH + K562 11.3% 14.1% 34.1% SIRSH + K562 11.3% 12.9% 30.5% Mitogenic factor (MF) blastogenesis reported as the mean SI 1 S.E.M. of a 3 day assay. MLC suppression reported as the mean per cent 1 S.E.M. of a 6 day assay. 79 Unfractionated 35 Nonadherent Cells Nonadherent + m Adherent Cells 25 .— K (D 'U C. H C O H .p S, :3 15 — s H 4..) U) ‘ r 5 l i IKAVA PHA SIRSH-MF Figure 7. Requirement of.Adherent cells for MF Activity. Analysis of adherent cell role for blastOgenesis by SIRSH-MF in a 3 day assay. Error bars represent S.E.M. 80 measured by peroxidase staining, and a viability of >’90% as measured by trypan blue exclusion. The inability of the adherent cells to more completely restore the response to MF may reflect the selectiveness of T cells passing through the nylon wool column. SIRSEand Natural Ki1ler Activity The suppressor fraction of SIRSH has been previously described as having no effect on specific cytotoxic T lymphocyte development (Kaufman et al.). In light of the newly discovered mitogenic activity of SIRSH, the effect of SIRSH on other cytotoxic cells was examined. Natural killer (NK) cells are circulating lymphocytes that display a lbw density of T cell markers (3, 32). Modulation of NK activity has been reported by several investigators, and SIRSH also affects the activity of NK cells. When HPBL are incubated for 3 days in the presence of SIRSH, a significant increase in per cent cytolysis is observed in comparison to control cultures. This increase can be demonstrated against both an allogeneic target, the K652 human myeloid leukemia, and a xenogeneic target, the P815 murine mastocytoma (Figure 8). Interestingly enough the increase is higher against the P815 xenogeneic target, with the increases against both targets statistically significant at least the P<0.05 level. Addition of SIRSH to the effector target mixture has no affect on cytolysis. A kinetic analysis of the incubation 81 .mhmmmm thonOpOpho anon ma Mo .E.m.m psomopoos mama nopnm m .Asuomv Ho.ovo cos Aauomv no.ovo so pcmoamacwfim mama pmcfimmm ommopocH xz moms can mama so :mmHm no poouum Nwmx x Huom Huom V, 7n, : “v‘IVL- min + \ . , K cocamficmam Nomx unawmwm omdohocH 2 mmHm .mN0.0Vm #m mmHm .mHHoo x2 noLSHHSO zap m an mfiwha Haoo pomsmw .bpfi>flpo¢.xz mo coHpmpcmEmz< mama X HuOm I mmHm .m mpzwflm I o v stsfitoifig % 82 period required for the increase in NK activity revealed that 3 days gives the optimum increase, with 1 day yielding a slight increase, and 5 days of incubation producing no observable increase in cytolytic activity. Similarly, 24 hour SIRSH produces a more consistent increase in NK activity than 48 hour SIRSH. This SIRSH augmented NK activity is significant at effector to target ratios ranging from 100:1 to 12:1 (Figure 9). In addition, when 0.2 ug of Con A, a suboptimal mitogenic dose, is added in addition to the SIRSH, and even greater augmentation is observed (Figure 9). This amount of Con A is insufficient to produce a significant increase in per cent cytotoxicity after the same 3 day culture time. Yet the combined SIRSH-Con A augmentation is far greater than the additive effect of the separate SIRSH and separate Con A stimulation of NK activity. This augmentation of NK activity by SIRSH in many ways parallels the results obtained in the study of the MP of SIRSH. Both activities are optimal in 24 hour SIRSH, and the kinetics of each are very similar with peak.activity occurring on day 3 to 4. _ffect of SIRSH on Lectin Depengent Cytotoxicity After the observation that SIRSH will augment the sub0ptimal Con A induced cytotoxicity, the ability of SIRSH to affect lectin dependent cytoxicity (LDCC) was examined. Unlike the NK assays, these cytotoxic assays were performed 83 0’ s ’0 O O O O l .0 ‘0 O O O O '0 0' - o ,v‘ ° o O I 53 0' ¢’ g. ‘f' 9°. .—. i o {F 4’ SS 0 .4’ ' ’O l 12:1 25:1 50:1 104:1 EffectorzTarget Ratio Figure 9. Effect of SIRS and Con A on NK Activity. NK assay after 3 days in culture, no treatment (o-—-o), + 0.2ug Con A (o———o), + SIRSH 1:3 final dilution (e---o), and + SIRS + Con A (o---o). Data calculated by a linear regression model. SIRS , and SIRS + Con A augmentations significant at 100:1 anH 12:1 compgred to untreated at P<0.05, in 18 hour cytotoxicity assays. 84 withoutprior incubation. SIRSH as well as the mitogen was added directly to the freshly isolated HPBL-target cell mixture. - As in the NK augmentation, SIRSH also increases LDCC of PHA and Con A (Figure 10). Although the increase is less than that observed in SIRSH or SIRSH-Con A augmentation of NK activity, it is consistent and observable against both the P815 and K562 targets. The amount of lectin used alone, either PHA or Con A, increased the per cent cytoxicity in the range of 10 to 20% over that of lymphocytes without lectin. SIRSH has no affect on the release of 51Cr from target cells alone (Table 6), and it has very little but insignificant augmentative activity upon addition to the effector-target mixture without mitogen. 85 50 - x K562, 6:1 (E:T) m H 2, 30 H o .p a o lO-:.'—’-'- I l l I l l I 0.5 1.0 2.0 4.0 1.0 2.0 4.0 8.0 Con A (ug) PHA (ug) 50 x P815, 12:1 (E:T) U] H U) a H o .1.) a o 2.5 5.0 10.0 20.0 0.12 0.25 0.5 1.0 Con A (ug) PHA (ug) Figure 10. Effect of SIRS on LDCC. Fresh isolated HPBL used as effector cells, lectin alone (-——), lectin + SIRS (- --). Dotted line intersecting vertical axis indicages % cytotoxicity in the absence of lectin (18 hour cytotoxicity assay). DISCUSSION The supernatant from Con A activated lymphocytes contains several activities. Optimal suppression of the one way MLC can be demonstrated with SIRSH—SF produced from 48 hour cultures, whereas MF blastogenesis and the increase in NH and LDCC cytotoxicities is more prevalent when using 24 hour supernatants of SIRSH. Mitogen-induced immune response help and suppression has been previously demonstrated by other investigators both by simple addition of mitogen or by addition of mito- gen-activated cells (14,31,50,51 ). Similarly, several mitogen-induced supernatants have also been described that are capable of conferring only help or only suppression on some specific immunological reaction (37, 52, 59 ). Much of this work has aided the generally accepted conclusion that help and suppression exists as separate T cell subsets. Although the MF and SF fractions of SIRSH are equally produced by Con A, an apparent dose dependency separates the two as has been described for the Con A activation of TS and TH cells (15, 31, 46 ). This would seem to support the hypothesis that the MF activity and SF activity are the result of separate soluble mediators with separate cellular sources, yet several other theoretical models have not been 86 8? ruled out. Both factors could also be the product of one cell type that is responsible for soluble mediator control of immune responses. Alternatively, MF and SF may still be activities of the same complex molecule, as separation on a physical basis has not yet been achieved. Also, it may not be possible to term MF blastogenesis or NK augmentation true immunological helper activities until their mechanisms are more fully understood. The demonstration of two seemineg antagonistic mediators in the same Con A activated supernatant has been reported in other animal models (16, 46, 48, 58). Although none of these authors report the same activities found in SIRSH, they do indicate that a variety of mitogens can produce both helper and suppressor factors. Goodman and co-workers have reported that the supernatant from Con A activated murine spleen cells contains helper and suppressor factors that both affect antibody response. In addition their helper factor also stimulates a blastogenic response, as does the MP of SIRSH. This murine soluble factor system appears to parallel the SIRSH system closely. However, the effect of SIRSH on antibody synthesis is unknown at this time. In the initial kinetic analysis of the MLC suppression with SIRSH, it was convenient to speculate that the blasto- genesis at day 3 was suppressor cell proliferation. Further analysis of this MF 3 day response has indicated that it 88 is not TS cell proliferation. The maximum MF activity is found in 24 hour supernatants when the SF demonstrates only marginal activity. Also, lowering the Con A dose will produce SIRSH with good MF activity and no SF activity., Thus, the suppressor activity appears to function separately from the mitogenic activity, again supporting the existence of two separate mediators. Serum requirement for factor production also indicates a separation of SF and MF. With the concentration of Con A constant, the absence of serum completely abrogates the 24 hour production of MF. while only mildly affecting the suppressor activity. Increasing the incubation time to 48 hours in the absence of serum did not restore MF activity. SIRSH-MF activity in some ways resembles other previously described enhancing factors. One such factor is lymphocyte activating factor (LAF) described by Gery, et al. (21, 22, 23). LAF is a soluble factor of 13,000 daltons produced generally by PHA stimulation (5). Like SIRS -MF, H it potentiates the mitogenic response of T cells to Con A, and has no apparent affect on B cells. Its principal source of production is adherent cells, presumably the ME. The principal differences between LAF and SIRSH-MF are their mode of production and cellular requirement for activity. LAF production by Con A is described as minimal whereas MF production is very good with Con A. Secondly MF requires the presence of adherent cells for the blastogenic 89 T cell response. LAF will stimulate T cells depleted of adherent cells, as would be expected for a product of adherent cells. One of the most interesting activities of SIRSH-MF. is its affect on supraoptimal Con A stimulation in a basic mitogen assay. Just as MF potentiates suboptimal Con A stimulation of HPBL, it also increases the blastogenic response of cells exposed to high doses of Con A. The most dramatic affect can be demonstrated at supra0ptimal doses closest to optimal mitogenic concentrations, yet a significant blastogenic increase by MP can still be demon- strated at 20 times the optimal mitogenic concentration of Con A. This observation combined with the extreme sensitivity of ME stimulation to CKMM indicates that MF may have a high affinity for glycoprotein cell surface receptors as Con A does. In addition, MF'S affect on high Con A stimulation suggests that it may share the same receptor or one in close proximity to the Con A binding site. Addition of constant amounts of MF to increasing amounts of Con A yields results suggestive of a competitive binding nature between MF and Con A. Augmentation of NK activity by SIRSH may be the most noteworthy in terms of biological importance. A few investigators have reported soluble factors produced by a variety of methods that are capable of augmenting cytolytic activity (35, 41, 47), yet SIRSH augmentation is unique in 90 that the optimal response requires 3 days of culture, in comparison to other enhancers of NK activity that only require a few hours (e.g. interferon). The NK activity of SIRSH treated cells is Significantly increased at effector to target ratios from 100:1 to 6:1. Before measuring the cytotoxicity of SIRSH treated cells and untreated cells, both were adjusted to the same cell concentration. There- fore, the increased activity of SIRSH treated cells does not simply represent increased cell numbers, although it may represent an increased percentage of NK cells or an increased efficiency of pre-existing NK cells. The simultaneous occurrence of a blastogenic response (MF) in tandem with NK augmentation makes the theoretical recruitment of more NK cells attractive, however, the relationship between the blastogenesis and augmented cytotoxicity is unclear. Saksela et al. have indicated that augmentation of NK activity by interferon (IF) is via recruitment of "pre-NK" cells, yet there is no blastogenesis associated with this IF enhancement (54). Another possibility is that SIRSH activates a sub- population of cells other than NK cells. Promonocytes have been shown to have NK-like activity and they also respond to IF augmentation (42). It is unlikely that the SIRS -NK H increase is the result of promonocyte activation, as these cells are primarily isolated from bone marrow, and the cells used in the SIRSH experiments were isolated from peripheral blood. 91 The combined effect of SIRSH and Con A on lymphocytes also increases NK cytotoxicity. Again, a large blasto- genesis accompanies the augmentation, yet this blastogenesis does not assure increased NK activity. The inability of Con A blastogenesis alone to augment NK activity is in agreement with other investigators (54). However when SIRSH is added in addition to Con A, the affect after 3 days in culture is an even larger increase in NK cytotoxicity than with SIRSH alone. This observation again indicates an intrinsic interaction between the SIRSH and Con A activities. The enhancing ability of SIRSH is not restricted to the lectin Con A. In a cytotoxic assay that is dependent on the cross linking of effector and target cell by lectins (LDCC) (24, 38, 43, 56). SIRSH will also increase this cytolytic capacity of fresh human lymphocytes. The addi- tion of PHA and Con A to effector-target cell mixtures results in an increased level of cellular cytotoxicity that is apparently dependent on a T cell type other than NK cells (3, 12, 32). When SIRSH is added at the same time as the- lectin, an even higher level of cytolysis is achieved. This affect can be demonstrated on both an allogeneic and xenogeneic target, and equally as well regardless of the lectin used. Addition of SIRSH without lectin has no significant affect on cytolysis. It is unlikely that this increase is related to the increase in NK activity since the 92 exposure time of the lymphocytes to SIRSH is only 18 hours, whereas the NK augmentation only becomes apparent after 2 to 3 days in culture with SIRS Finally, the SIRS - H' H LDCC increase is not caused by the direct affect of SIRS 51 H on Or release from the target cell. A primary concern when working with lectin or mitogen produced supernatants is effective removal of the lectin or mitogen. The most common way to remove Con A is by repeated extractions with Sephadex. The more cross linked the Sephadex, the more the sugar moieties are exposed and consequently the better removal of Con A (2). SIRSH is depleted of Con A by this method, and multiple extractions of SIRSH with Sephadex have been used to indicated that Con A is not responsible for the various activities of SIRSH. In addition, Con A has been added directly to several of the test systems in an attempt to demonstrate that Con A is not capable of some of the SIRSH activities. One example is the MLC suppression. Only highly mitogenic doses of Con A (3 ug or greater) will produce suppression of the MLC that is considered significant (2>20%). Doses in the lower range (0.2 ug or less) that are more likely to be found in SIRSH are highly stimulatory to the MLC. The affect of MF on Con A stimulation also contradicts the null hypothesis that Con A is responsible for MF's activity. It is unreasonable to believe that the addition of more Con A to lymphocytes already supra0ptimally stimulated with Con A will result in a higher degree of blastogenesis, yet MF will 93 do this. The third activity, increased cytotoxicity, is clearly not Con A mediated, as the addition of Con A alone has no significant affect on NK activity. Watson et al. have reported that Con A bound to 0(MM will precipitate out of solution at 40% (NHu) 2804 (59). Preliminary work on SIRSH-MF indicates that the MP is still in solution after treatment with 40% (NHu) 2SOL}. Recently several investigators have reported that IF will augment NK activity (13, 25, 27, 55). If has also been reported to suppress the MLC and antibody production of B cells (26). However, there are several reasons supporting the supposition that SIRSH activities are not the result of IF, especially IF type II. The two major differences are that IF suppresses blastogenesis in general and it is fast acting in its augmentation of NK activity. Although the majority of blastogenesis from MF is found in 24 hour supernatants, 48 hour MLC suppressive supernatants still contain significant MF blastogenic activity. This is contrary to what would be expected if the MLC suppression was interferon mediated.' NK augmentation by SIRSH is only optimal after 3 days in culture, whereas augmentation by IF is very rapid, occurring within hours after exposure. These two differences alone provide strong evidence that SIRSH activity is not IF activity. The affect of MF on Con A mitogenesis also suggest that SIRSH contains little or no IF. 94 Another important consideration when comparing lectin induced soluble factors to IF is the purity of the preparation of IF. This is especially true of immune IF or type II IF, which has not yet been pruified. It has even been suggested that preparations of type II IF may contain lymphokines (20), allowing for the possibility that some of the wide range affects of IF may be lymphokine mediated. Finally, while it is known that mitogen stimulation will elicit some IF production, significant amounts of IF have only been found after virus activation of lymphocytes. The theoretical implications of SIRSH have enormous impact in the area of autoimmunity. Within the last five years a great deal of research has been aimed at autoimmune disorders, utilizing both animal models and human lympho- cytes. One disease in specific, systemic lupus erythematosus (SLE), has been the objective of a majority of autoimmune research. SLE has been characterized by a number of lympho- cytic disorders, with one of the most recent being a defect in suppressor cells. The NZB mouse, a murine model for SLE, has been shown to steadily loose its TS cell activity as it matures, while simultaneously developing disease symptoms of SLE (18). As mature animals these mice also exhibit a deficiency in Con A inducible TS cells (39). Similarly, human lymphocytes from SLE patients are also deficient in Con A inducible TS cells (1, 7, 36). This suppression has been measured in a variety of assays '95 including blastogenesis and immunoglobulin synthesis. In addition to suppressor cell defects, recent reports have indicated defects in NK cell activity of SLE patients (29), and of other patients with related immunological disorders (53, 4). These defects in lymphocyte activity correspond very closely to the activities of SIRSH. The defect in TS cells and NK cells in SLE may be at the soluble factor level and not necessarily at the cellular level. Yet more importantly the lymphocyte defect in this and other immuno- logical diseases may be a more general defect rather than a selective one for one specific hypothetical cell type. This defect may be a general lack of T cell mediators, or lack of a generally active mediator such as MF. SIRS -MF H appears to enhance or potentiate virtually every response it is tested upon. Perhaps this factor should be referred to as a potentiating factor. A factor originating from amplifier-type cells, whose role is to boost specific immunological responses, responses weak or deficient in immunodisease states. Finally, even the selective higher increase in NK activity by SIRSH for the xenogeneic target may have a relationship to the disease state of SLE. Lymphocytes from SLE patients exhibit a defect in generation or expression of T killer cells for xenogeneic cells (10). The MLR proliferation is normal, yet a selective decrease in cytotoxicity exists. In summary, Con A activated HPBL secrete a factor(s) into their supernatant. This supernatant, SIRSH, is capable 96 of various activities including MLC suppression (SF), mitogenesis (MF), and augmented NK activity and LDCC. The MF activity is directed at T cells and requires adherent cells for expression of its activity. The MP and SF are independent activities that can be separated on a temporal and serum requirement basis. 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