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P815 tumor-specific T suppressor cell and suppressor factor Maier, Tom 1981

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P815 TUMOR-SPECIFIC T SUPPRESSOR CELL AND SUPPRESSOR FACTOR by TOM MAIER B.S., The University of Washington, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1981 © Tom Maier In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of I C ^ 0 ia T c? ^ y The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date b*( . V- , DE-6 (2/79) ii ABSTRACT The work reported here involves studies of suppressor T cells (TgC) and their suppressor factor (SF) which specifically suppress the in vitro generation of cells cytotoxic for a syngeneic tumor, P815, in DBA/2 mice. This work can be divided into three sections: a) the immunogenetic properties and requirements of this TgC and SF, b) the Lyt phenotype of the TgC as well as that of the cells involved in the cytotoxic response to the syngeneic tumor, c) the properties of syngeneic and allogeneic antisera raised to the P815 specific SF. a) P815-antigen specific TgC and suppressive extracts . ' obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 tumors, were compared for their immunogenetic properties and require ments. It was shown that pretreatment of TgC populations with anti-la^ antiserum plus rabbit complement removed the suppressive activity. Similarly, absorption of the SF with anti-la^ antiserum removed the suppressive properties of the material. It was found that the TjC and SF were capable of specifically suppressing the anti-P815 response of B6D2F^ radiation chimeras possessing lymphoid cells of the b 12 H-2 or H-2 haplotype equally as well as they could suppress the response of H-2^ bearing cells. This indicates that the TgC and SF are not H-2 restricted with respect to K or D markers on responder cells in this system. b) T C were also identified in the spleens of DBA/2 mice injected s intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells was determined. DBA/2 allogeneic killer cells were identified as Lyt-l+2+, whereas the syngeneic effector cells were found to be predominantly Lyt-1 2+. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-l+2 cell is essential in this suppression. c) P815 tumor-specific SF was partially purified by passage of suppressive spleen extracts through an immunoadsorbent containing P815 membrane components. Antisera raised in syngeneic DBA/2 and allogeneic, C57BL/6, mice were tested. It was found that these antisera, but not their controls were capable of absorbing out the SF. The antisera were also capable, in the presence of complement, of eliminating TgC from suppressive spleen cell populations. However, the antisera were not capable of eliminating syngeneic tumor specific in vitro generated killer cells, indicating that the receptor molecules on suppressor and effector cells in this system are distinct from each other. Only the antisera "raised in syngeneic DBA/2 mice had any observable effect on P815 tumor growth in vivo. iv TABLE OF CONTENTS Page ABSTRACT ii LIST OF FIGURES v LIST OF TABLES vil ACKNOWLEDGEMENTS vliCHAPTER I - INTRODUCTION 2 CHAPTER II - CHARACTERIZATION OF THE H-2 PROPERTIES AND REQUIRMENTS OF P815 TUMOR-SPECIFIC SUPPRESSOR T CELLS AND THEIR SOLUBLE FACTOR 8 A. IntroductionB. Materials and Methods 10 C. Results 1D. Discussion CHAPTER III - THE LYT PHENOTYPE OF CELLS INVOLVED IN THE CYTOTOXIC RESPONSE TO SYNGENEIC TUMOR AND OF TUMOR-SPECIFIC SUPPRESSOR CELLS AND IMPROVED PROCEDURES IN THE GENERATION OF TUMOR-SPECIFIC SUPPRESSOR CELLS 33 A. Introduction 3B. Materials and Methods C. Results 43 D. Discussion ^ CHAPTER IV - IN VITRO AND IN VIVO EFFECTS OF SYNGENEIC AND ALLOGENEIC ANTISERA RAISED TO TUMOR-SPECIFIC SUPPRESSOR FACTOR FROM DBA/2 MICE 55 A. Introduction B. Materials and Methods ^ C. Results 68 oo D. Discussion CHAPTER V - SUMMARY DISCUSSION Literature Cited 94 100 V LIST OF FIGURES Figure Title Page 1 The ability of P815 suppressor cells to suppress the in vitro primary cytotoxic response of normal DBA/2 splenocytes to syngeneic mitomycin C-treated P815 tumor cells 19 2 The ability of anti-la^ alloantiserum plus rabbit C to eliminate P815 suppressor cells 20 3 The ability of DBA/2 P815 suppressor cells to suppress the cytotoxic response of splenocytes of H-2 radiation chimeras to P815 22 4 The ability of DBA/2 P815 suppressor cells to suppress the cytotoxic response of splenocytes from H-2 (A.TH) radiation chimeras to P815. 23 5 The ability of the suppressor factor isolated from P815 suppressor thymocyte populations to suppress the in vitro primary cytotoxic response of normal DBA/2 splenocytes to syngeneic mitomycin C-treated tumor cells . 24 6. The effect of anti-la antiserum adsorption on the suppressive activity of the suppressor factor isolated from P815 suppressor thymocyte populations... 26 7 The ability of P815 suppressor factor to suppress the cytotoxic response of splenocytes from H~2 radiation chimeras to P815 27 8 Flow diagram illustrating the preparation of P815 tumor-specific suppressor cells.. 40 9 The ability of splenic P815 suppressor cells to suppress the in vitro primary cytotoxic response of normal DBA/2 splenocytes to syngeneic mitomycin-C treated P815 tumor cells 44 10 The ability of nylon wool-passed splenic P815 suppressor cells to suppress the iri vitro primary cytotoxic response of normal DBA/2 splenocytes to syngeneic mitomycin-C treated P815 tumor cells 45 11 Time course of appearance and disappearance of antigen-specific suppressor cells in the spleen of DBA/2 mice injected intraperitoneally with P815 membrane extracts on day 0 46 vi LIST OF FIGURES Continued Figure Title Page 12 The effect of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rabbit C on DBA/2 allogeneic effector cells raised against C57BL/6, on a EL4 target 48 13 The effect of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rabbit C on DBA/2 effector cells primed against the syngeneic P815 mastocytoma 49 14 The effect of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rabbit C on P815-primed nylon wool-passed DBA/2 splenic suppressor cells 50 15 Flow diagram illustrating the preparation of P815 tumor-specific suppressor factor (P815-SF) 60 16 The ability of P815-specific suppressor factor to suppress the in vitro primary cytotoxic response of normal DBA/2J splenocytes to. syngeneic mitomycin C treated P815 tumor cells 69 17 Titration of immunoadsorbent purified P815-specific suppressor factor 70 18 Characterization of P815-SF by ELISA assay 75 19 The ability of anti-P815-SF antisera plus complement to eliminate P815-specific suppressor cells 77 20 Effect of anti--P815-SF antisera in vivo on P815 tumor growth in syngeneic DBA/2 mice. . 80 21 Effect of anti-P815*-SF antisera in vivo on L1210 tumor growth in syngeneic DBA/2 mice 83 22 The inability of anti-P815-SF antisera, in the presence of complement, to lyse 51Cr labeled P815 tumor cells. ... 85 23 Does anti-SF antisera have any activity directed towards P815 tumor membrane determinants? vii LIST OF TABLES Table Title Page I Specificity of cytotoxicity generated in vitro 37 II Anti-P815 antisera is able to react and bind to P815-specific suppressor factor 73 III Results of Students' t-test run on 3 separate experi ments testing 3 individual bleeds of mice immunized with P815-SF or control Balentl-"SF" 74 IV Anti-P815-SF antisera plus complement (C) treatment has no effect on the ability of specific cytotoxic effector T cells to lyse the appropriate target 79 V Effect of anti-P815-SF antisera jm vivo on survival time of DBA/2 mice after P815 tumor injection 81 VI Effect of anti-P815-SF antisera jLn vivo on survival time of DBA/2 mice after L1210 tumor injection 84 ACKNOWLEDGEMENTS I am very grateful to Dr. Levy for her valuable instructions, guidance, and enthusiasm throughout the course of my research. I thank Dr. Doug Kilburn for his very useful discussions of this work. I would like to thank Primrose Gontier for helping finish the typing of this thesis. 1 would also like to extend a special thanks to everyone in the lab for helping to make my stay in Vancouver so very enjoyable. iX FOR PE8BI 1 CHAPTER I INTRODUCTION 2 CHAPTER I Introduction It is now apparent that the immune response is the result of a complex series of ongoing interconnecting events which is only ini tiated by antigenic stimulation (1). Initially, the immune response was thought of as a physiological response of the body to antigen, in which the sole end product was antibody. Later, the two main limbs of the immune response were recognized (humoral and cell-mediated), and within the past two decades, the two lymphocyte subpopulations mediating these responses were defined. Thus it was realized that at.', least two types of interactions occurred between antigen and the two responding cell types, the T and B lymphocytes. This was followed by the realization that the immune response is regulated via T cell-B cell intreactions, and also T cell-macrophage, and T cell-T cell inter actions. It is now recognized that the T lymphocyte population is made up of distinct subsets which interact with each other as well as B cells and macrophages, to both amplify and inhibit a variety of immunological responses. T cells have demonstrated enhancing effects on the immune response by way of helper T cells (Tu) (2-10). rl T cells have also been found to inhibit the immune response by way of suppressor T cells (Tg) (11-44). Although some of these studies have demonstrated the existance of non-specific regulatory T cells, the antigen specific regulatory T cells are expecially intriguing because they appear to possess the specificity that usually marks the immune response. 3 One of the first observations of antigen-specific Tg were made regarding antibody formation in rats (45-47). This was quickly expanded to include several different antigenic systems (il-17,22-44, 48-58). From this and other work some general characteristics of antigen-specific Tg begin to emerge. First they show specific antigen or idiotype binding potential. Antigen specific Tg also express la determinants. This is usually I-J coded. The inhibitory activity of these cells may or may not by genetically restricted to self H-2 type. The antigen-specific T^ in many experimental systems have a surface phenotype of Lyt-1 2"*". However, there are also some reports of Tg having the Lyt-l+2 phenotype or even Lyt-l+2+. In many of the investigations concerning antigen-specific reg ulatory T cells, it has been shown that soluble factors derived from these cells can apparently duplicate their function (59-61). Thus, the antigen-specific factors once produced may work independantly of the T cells which produced them. This would have the effect of increasing the effective radius of the regulatory T cell, much in the same way that antibody functions with respect to the B cell. These .factors may also interact via network type interactions to induce the activation of other lymphocytes, which eventually leads to the final net effect on the immune response. This has been best studied in the antigen-specific suppression systems (60). As in the antigen-specific Tg investigations, the initial discovery of antigen-specific suppressor factor (SF) was made by Tada and col leagues (62-64). Again, this observation was quickly broadened to many other experimental systems (47,59-61,65-80). The antigen-specific 4 suppressor factors exhibit several physical and functional character istics in common. They are produced or extracted from antigen primed Tg. They are proteins with specific antigen (or idiotype) binding potential. Their molecular weight is usually between 35-75,000. They lack constant region immunoglobulin determinants but may carry variable (idiotypic) determinants of the immunoglobulin heavy chain. They have determinants which are encoded by the Ir region (usually I-J), and like the antigen-specific Tg they may or may not be genetically restricted in their effect to acting on cells of the same H-2 type. Finally, since T cells would be unlikely to express antigen-specific molecules unless they were receptors, antigen-specific suppressor factors are an obvious candidate for the antigen receptor of Tg. Several reviews have been published recently on antigen-specific T cell factors, and one is directed there for a more comprehensive examination of antigen-specific suppressor factors (59-61). The early investigations carried out by Takei and colleagues in this laboratory provided preliminary data on which the work in this thesis is based. They found that DBA/2 mice injected with syngeneic P815 mastocytoma cells 8 days previously, contained in their thymus P815-specific Tg (P815-Tg), which could inhibit the in vitro generation of P815-specific cytotoxic T cells (P815-Tc) (11,12). The experimental design for demonstrating this was as follows. The in vitro assay for cytotoxic T cells involved the culturing of "immune" DBA/2 splenocytes (taken 12 days following subcutaneous injection of a small number of P815 cells into the DBA/2 mice) with mitomycin-C treated P815 cells for 4 days prior to cell harvesting. Specific cytotoxicity was measured by a 5 standard 18 h Cr release assay. When thymocytes from DBA/2 mice, inoculated 8 days previously with P815, were cocultured in this system, the generation of cytotoxic T cells was greatly reduced as assessed by the reduction in ^"*"Cr release mediated by these cultures in comparison to appropriate.controls. This suppressive effect was shown to be antigen-specific. It was later shown that a P815-specific suppressor factor (P815-SF) could be isolated from sonicated P815-T "extract (65). This SF could replace the P815-Ts in the in vitro functional assay. The P815-SF was capable of suppressing the in vitro generation of syngeneic cells cytotoxic for P815 tumor cells if it was added during the first 30 h of culture. It was found to have a molecular weight in the range of 40-60,000, with an isoelectric point in the range of 4.6-4.9. The P815-SF could be removed by passage through immunoadsorbent columns prepared from membrane fragments of P815 but not by analogous columns prepared with L1210 (syngeneic leukemia of DBA/2) membrane fragments. The P815-SF was also not removed by passage through immunoadsorbent columns containing anti-mouse immunoglobulin antibody. The work reported here involved continued studies of these suppressor T cells and the suppressor factor extracted from them. Both the cell and factor are P815 specific and obtained from DBA/2 mice primed with P815 tumor cells or P815 membrane fragments. The function al assay involves the ability of either cells or their extracts to specifically inhibit a primary in vitro cytotoxic response of normal DBA/2 splenocytes to P815 tumor cells. The work in this report has been divided into three self-contained chapters for ease of reading. 6 Chapter II examines some of the immunogenetic properties and require ments of the P815-T . This work was carried out between 6/77 and s 9/78. Chapter III examines the Lyt phenotype of the P815-Tg as well as that of the cells involved in the cytotoxic response to the syngeneic P815 tumor. This investigation was carried out between 6/78 and 2/79. The work reported in Chapter IV examines the in vitro and in vivo effects of syngeneic DBA/2 and allogeneic C57BL/6 antisera raised to P815-SF. This work was done between 10/78 and 5/81. Chapter V is a summary discussion of the studies reported in the preceeding three chapters. 7 CHAPTER II CHARACTERIZATION OF THE H-2 PROPERTIES AND REQUIREMENTS OF P815 TUMOR-SPECIFIC SUPPRESSOR T CELLS AND THEIR SOLUBLE FACTOR 8 CHAPTER II Introduction There have been a number of reports showing that the lymphoid organs of tumor-bearing mice contain T cells that are capable of specifically suppressing the response of normal cells to that tumor (12,15,36,83-85). It has also been shown that, in some cases, it is possible to isolate from such populations of suppressor cells, soluble factor(s) also capable of inhibiting the specific anti-tumor response of normal lymphoid cells (65,76,86,87). Studies on the characterization of soluble specific suppressor factors in tumor-bearing (65,76,86) animals have shown that they do not appear to differ significantly from antigen-specific suppressive factors demonstrated in animals immunized with a variety of antigenic stimuli (59-61,77,81,82,88). Generally, suppressive factors have been found to have m.w. in the range of 30-75,000. Moreover, they have been shown to exhibit antigen specificity (65,75-78,87) and not to share antigenic similarities with the constant regions of immuno globulin (65,77,78,87). Studies with these factors have also shown that they can be removed by absorption with antisera raised to products of the major histocompatibility complex (MHC) excluding the K and the D regions (anti-la serum). Further analyses of these absorptions have shown that the subregion of the la that is expressed on the suppressor factor is encoded by the I-J region (77,81,82,86). A recent observation that treatment of tumor bearing animals with small amounts of anti-I-J antiserum caused a significant slowing in the growth of syngeneic tumor (89) indicate that elimination of I-J-9 bearing cells in vivo may be of a clinical significance. The present study was undertaken to determine whether suppressor cells elicited in tumor-bearing animals were capable of suppressing the primary in vitro cytotoxic response of normal cells to tumor antigens, and to compare the suppressor cells, thus generated, with the equivalent soluble factors in terms of la-bearing characteristics and genetic restrictions regarding their suppressive qualities. Materials and Methods  Mice and tumors. Female DBA/2, B6D2F1> B10.D2, BIO and C57BL/6 mice (6 to 10 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, Maine). A.TH animals were obtained from the Banting and Best Institute from Dr. T. Delovitch and were bred in our own animal unit. P815 mastocytoma and L1210 leukemia were obtained from Dr. Bruce Smith (Institute for Cancer Research, Philadelphia, Pennsylvania) and maintained as an ascites tumor as described previously (11,12). Culture system for generation of cytotoxic effector cells. Single-cell suspensions were prepared from spleens of normal DBA/2 mice by pressing the tissue through a stainless steel 60-gauge mesh in RPMI 1640 (Grand Island Biological Company, Grand Island, N.Y.) medium containing 10% heat-inactivated fetal calf serum (FCS), 10 mM HEPES and 5 x 10~5 mM of 2-mercaptoethanol. Gentamycin was also added to a final concentration of 50 g/ml. Suspended cells were washed through 3.0 ml of FCS, resuspended in medium, and counted for viable cells by using trypan blue. Tumor cells were washed twice in medium before resuspension in complete medium for counting. Specific cytotoxicity against either P815 or L1210 was generated in vitro by incubating 107 splenic lymphoid cells with 5 x 10^ mitomycin C-treated tumor cells in Linbro multiwell plates containing 24 flat bottom wells (No. 76-033-05) at 37°C in a humidified incubator with 5% C02 for 5 days. The total volume of each culture was 2.5 ml. After 11 5 days, cells were harvested by centrifugation, washed, and viable cells were counted by trypan blue exclusion and tested for cytotoxicity. Assay for suppressor cells and suppressor factors. When the assay was used for measuring suppressor cell activity, 5 x 10 normal splenic lymphocytes were incubated with 6 6 5 x 10 suppressor thymocytes; a control contained 5 x 10 normal splenic lymphocytes and 5 x 10^ normal thymocytes. On all occa sions, experiments were run at least in duplicate with lymphocytes from individual pools of cells in order to minimize errors incurred by inaccurate counts. Assay for thymic suppressor factor involved incubation of 107 splenic lymphocytes with various con centrations of the factor under the same conditions as stated above. When the degree of suppression was quantitated, cytotoxicity was tested at various effector to target cell ratios and the decrease in total lytic units was assessed. One lytic unit, which was defined as the number of effector cells required to lyse 4 50% of the 10 target cells in 18 hr incubation, was estimated by linear regression analysis of percentage of cytotoxicity vs logarithm of effector to target cell ratio. Total lytic units were calculated from cell recovery in cultures and percentage of suppression was estimated from the decrease in total lytic units. No significant differences in cell recovery between test cultures and controls were normally observed. 12 When thymocytes on their own, at a concentration of 107 per well were cultured with micomycin-treated P815, no detectable killer cells were generated, indicating that in the syngeneic killing system, thymocytes do not contain significant levels of precur-sors. This was also found to be the case when 5 x 10 thymocytes were cultured with 5 x 10, mitomycin-treated DBA/2 spleen cells or C57BL/6 chimeric cells. Cytotoxicity assay. Target tumor cells were labeled with "'"'"Cr-sodium chromate (New England Nuclear, Boston, Mass.) as described previously (11). 4 51 Various numbers of lymphocytes and 10 Cr-labeled target cells in RPMI 1640 culture medium were dispensed in the wells of multi-dish microculture plates (Linbro Chemical, 1S-FB-96-TC, New Haven, Conn.) and the final volume was adjusted to 0.20 ml. The plates were incubated for 18 hr in a CO2 enriched, humidified incubator at 37°C. The cells were then sedimented by centrifuging the plates at 200 x G for 5 min, 0.10 ml of the supernatant was removed, and its radioactivity was measured on a gamma counter (Beckman Biogamma). Percentage of specific cytotoxicity was cal culated as follows: Specific cytotoxicity (%) test release (CPM) - spontaneous release (CPM) x maximum.release (CPM) - spontaneous release (CPM) 4 Spontaneous release was measured by incubating 10 target cells 4 alone, and maximum release was tested by lysing 10 target cells 13 with 5% TRITON X 100 (Sigma Chemical Company, St. Louis, Mo.). Spontaneous release of P815 cells was 20 to 25% and that of L1210 cells was 15 to 20% of maximum release. Generation of suppressor cells. 3 DBA/2 mice were injected subcutaneously with 2 x 10 P815 cells in the right flank. They were sacrificed 8 days later and their thymocytes used as. a source of suppressor cells. This pro tocol had been shown previously (65) to.be reliable and reproducible for the generation of T lymphocytes specifically suppressive in the assay system. Preparation of suppressor factor. This method has been described in detail previously <(65). Briefly, suppressive thymocytes prepared as stated above, were disrupted by sonication (Biosonic 80) at 0°C for three 1-min bursts and the soluble material was subjected to preparative isoelectric focusing after ultracentrifugation at 60,000 x G for 60 min. By titration of individual fractions, from both normal and tumor bearer ex tracts, the specifically suppressive fraction was isolated. This material was stable at 4°C and active up to dilutions equivalent to 1/125 of a single thymus. Assays for this material were always run against controls containing equal concentrations of the equivalent fractions from normal thymocytes. Isoelectric focusing. Preparative isoelectirc focusing was run on a 110 ml LKB-focusing column through a 5 to 50% linear sucrose gradient and a 14 total of 1.6% LKB ampholines (1.2%, pH 4 to 6, and 0.4%, pH 5 to 7). Crude thymic extracts in volumes of 2.5 to 3.0 ml with 280 nm absorbance of about 10.0/ml were used. The electrode solutions consisted of 7.0 ml H20 and 3.0 ml 1 M NaOH (light electrode) and 15 g sucrose, 13 ml ^0 and 3 ml 1 M H^PO^ (dense electrode). Focusing was carried out at 4°C for 24 hr at 950 volts and 0.8 mA. After the focusing was complete, distilled water was pumped slowly into the column and 1.0 ml fractions of the focused materials were collected. These fractions were monitored for pH and absorbance at 280 nm. Preliminary testing had indicated that the suppressive material focused at between pH 4.6 and 4.7. Therefore, when fractions were being pooled for subsequent testing, both pH and absorbance at 280 nm were used as indices for cutting the fractions. Pooled samples were dialyzed against physiologic saline to eliminate the sucrose and ampholines. They were then concentrated down to their original volume with an Amicon ultrafilter with a UM10 membrane. Fractions were then dialyzed against RPMI 1640 after which their absorbance at 280 nm was recorded. They were then sterilized by Millipore filtration and stored at 4°C until they were titrated or used for further characterization studies. Preparation of alloantisera. The anti-la^ antiserum used here was prepared by immunizing B10 mice repeatedly with B10-D2 splenocytes. Animals were injected i.p. every week with 107 B10.D2 splenocytes for 8 weeks. Subsequently, they were bled from the retroorbital sinus on 15 alternate weeks and continually immunized over a period of 2 months. The anti-H-2^ antiserum thus prepared was 100% cyto toxic to P815 cells in the presence of rabbit complement (C) up to a final dilution of 1/1200. In order to render the anti serum specific for the la region of the H-2^ MHC it was absorbed exhaustively against P815 cells until it was no longer cytotoxic in the presence of rabbit C for these targets. The absorbed antiserum (anti-la'*) , when tested for direct killing of DBA/2 splenic lymphocytes killed 60 to 65% of these cells up to a final dilution of 1/80 but had no appreciable cytotoxicity for thymocytes at a dilution of 1/4. When tested by ^Cr release assay with LPS blasts, it caused 75% "^Cr release up to a 1/100 dilution. In tests reported here the anti-la'* antisera was used at a final concentration of 1/40 in the presence of a 1/15 dilution of rabbit C (Lowtox Cedar Lane Labs, Hornby, Ontario). t2 Antisera specific for H-2 (A.TH anti A.TL) was obtained from Dr. T. Delovitch (Banting and Best Institute). Antiserum to H-2^ haplotype was prepared by immunizing B10.D2 mice with B10 splenocytes as described above. Radiation chimeras. B6D2F^ mice were lethally irradiated with 900 R and recon stituted with 107 bone marrow cells from either DBA/2, C57BL/6 or A.TH donors. The donor cells had been treated with rabbit anti-purified brain associated thy-1 antigen. The antiserum was obtained from M. Letarte (Ontario Cancer Institute) and 16 was used at a dilution of 1/200 with a 1/15 dilution of rabbit C. Animals were kept for 8 weeks before they were used in any experiments. When these animals were sacrificed, their spleen cells were tested with the appropriate alloantisera plus C to ascertain that their cells were those characteristic of the donor allotype. Anti-la^ killing of suppressor cells. In experiments testing for the presence of la markers on suppressor cells, 4 x 107 tumor bearer or normal thymocytes were suspended in 1.0 ml of a 1/40 dilution of antiserum in complete medium and incubated for 45 min at 37°C after which 0.5 ml of a 1/5 dilution of rabbit C in the same medium was added and incubation was continued for another 45 min. Appropriate controls of C and medium only were run concurrently. Cells were washed, resuspended in medium, and counted for viability. In no instance were viable counts in cell suspensions containing the anti-la^ antiserum significantly different from those of the controls and in no instance were counts lower than 90% of the number of cells before treatment. These cells were sub sequently used in suppressor cell assays. Immunoadsorbent assays. The ability of anti-la^ to remove the suppressive factor from thymic extracts was tested by using an immunoadsorbent assay described previously (65). "Briefly, both normal.and suppressor isoelectric focused fractions were mixed with either the anti-17 la antiserum or a control of anti-H-2 antiserum, and incubated overnight at 4°C. These materials were then passed over immuno-adsorbent Sepharose 4B columns to which goat anti-mouse Ig had been attached by cyanogen bromide. The capacity of the column was sufficient to remove at least twice the amount of the mouse Ig added to the fractions. The material eluting from the columns was titrated for suppressive activity over dilutions ranging from 1/5 to 1/125 (these dilutions of eluted materials represent between 0.2 and 0.04 of a thymus equivalent). 18 Results The ability of suppressor cells generated as previously described (12,65) to suppress specifically the primary in vitro generation of DBA/2 cells cytotoxic for the syngeneic P815 mastocytoma is shown in Figure 1. A primary cytotoxic response was generated against either P815 or L1210 mitomycin-treated cells in the presence of either normal or suppressor thymocytes. As can be seen in Figure la, the response to P815 in the presence of suppressor thymocytes was signif icantly suppressed whereas the response to L1210 was not. It was thus shown that these suppressor cells that had been found previously (12) to suppress the generation of P815 cytotoxic cells in populations of splenocytes primed in vivo by exposure to P815, could also suppress the primary response in an analogous manner. These results are entirely reproducible, the only difference being in the relative numbers of lytic units between cultures. The data shown in Figure 1 are representative in that control cultures contain roughly twice the number of lytic units as do cultures containing suppressor cells. It has been reported by others that specific T suppressor cells bear detectable la antigens on their surface (81,90-94). Experiments were run to determine whether anti-la** alloantiserum plus rabbit C could eliminate suppressor cells in this system. The results of a repre sentative experiment are shown in Figure 2. Although the C control showed that suppressor cells were present in the treated population of thymocytes (Fig. 2b), the anti-la treatment removed suppressive activity so that the cytotoxicity generated in the test population was not significantly different from the control population contain-19 Figure 1. The ability of P815 suppressor cells to suppress the in vitro primary cytotoxic response of normal DBA/2 splenocytes to syngeneic mitomycin C-treated P815 tumor cells. Anti-P815: the cytotoxic response of cells primed in vitro with P815 cells on Cr labeled P815 cells in an 18 hr-assay. • • , primary culture of normal DBA/2 splenocytes and normal DBA/2 thymocytes (lytic units were 33.3); o o, primary culture of normal DBA/2 splenocytes and P815 suppressor thymocytes (lytic units were 15.4). Anti-L1210: the cytotoxjLj response of cells primed in  vitro with L1210 cells on Cr labeled L1210 cells in an 18 hr assay. • 1, primary culture of normal DBA/2 spleno cytes and normal DBA/2 thymocytes; 0 0, primary culture of normal DBA/2 splenocytes and P815 suppressor thymocytes. Lytic units for both cultures were 11.1. 20 Figure 2. The ability of anti-la" alloantiserum plus rabbit C to eliminate P815 suppressor cells. Anti-la: the cytotoxic response of cells primed in_ vitro with P815 after treatment of thymocytes with anti-la anti serum plus C. 0 0, primary culture of normal DBA/2 splenocytes plus anti-la- treated normal thymocytes; •—-j*, primary culture of normal DBA/2 splenocytes plus anti-la treated suppressor thymocytes. Lytic units in both cultures were 19.2. C control: C control of the anti-la experiment 0 0, primary culture of normal DBA/2 splenocytes and C-treated normal thymocytes (lytic units were 24.4); • • , primary culture of normal DBA/2 splenocytes and C-treated P815 suppressor thymocytes. Lytic units for suppressor cell populations in this instance could not be calculated because 50% lysis of cells was not achieved in the assay. 21 ing anti-la-treated normal thymocytes (Fig. 2a). The potential of suppressor cells to operate across a histo compatibility barrier was tested by using spleen cells from B6D2 radiation chimeras that had been reconstituted with DBA/2, C57BL/6, or A.TH bone marrow cells, thus providing cells of various H-2 haplo-types rendered tolerant to H-2^ alloantigens. These were then cultured with suppressor cells generated in vivo in DBA/2 mice plus mitomycin C-treated P815 cells. The results are shown in Figures 3 and 4. In Figure 3, the suppressive activity of these cells when cocultured with spleen cells of either C57BL/6 or DBA/2 allotype was essentially the same, indicating that suppressor cells do not require K or D identity in order to carry out their suppressive effect. These experiments have been repeated a number of times and are totally reproducible. Similar experiments were run with chimeras reconstituted with A.TH cells (Fig. 4). This haplotype shares the D locus with DBA/2 t2 but differs at all other regions of the MHC (H-2 ). As can be seen, again there was no difference in the suppressive activity of the DBA/2 t2 d suppressor cells with either H-2 or H-2 splenocytes. All the spleen cells from the chimeras were tested with appropriate antisera to ensure that they were the haplotype of the donor cells. The comparative properties of the suppressor cells and the suppressive factor separated from the suppressor cell populations were studied. Figure 5 shows the ability of suppressor factor to suppress specifically the primary in vitro generation of DBA/2 cells cytotoxic for the syngeneic P815 mastocytoma. As can be seen in Figure 5a, the response to P815 in the presence of suppressor factor 22 Figure 3. The ability of DBA/2 P815 suppressor ceLls to suppress the cytotoxic response of splenocytes of H-2 radiation chimeras to P8T.5. b H-2 : the cytotoxic response of H-2 (C57BL/6 reconsti tuted) radiation chimera splenocytes^primed i_n vitro with P815. 0 0, primary culture of H-2 chimera splenocytes and normal DBA/2 thymocytes (lytic units were 69.0); • 1 primary culture of H-2 chimera splenocytes and P815 sup-pressgr thymocytes (lytic units were 35.7). H-2 : contrgl assay for suppressor cells. 0 0, primary culture of H-2 (DBA/2) chimera splenocytes and normal DBA/2 thymocytes (lytic units were 41.7); 9—:—f, primary culture of H-2 chimera splenocytes and P815 suppressor thymocytes (lytic units were 27.4). 23 ol I 1 • —i—" 1 1 • ' 12-5:1 25:1 50:1 100:1 12-5:1 25:1 50:1 100:1 EFFECTOR : TARGET RATIO J Figure 4. The ability of DBA/2 P815 suppressor cells to suppress the cytotoxic response of splenocytes from H-2 (A.TH) radiajj^on chimeras to P815. ^2 H-2 : the cytotoxic response of H-2 radiation chimera splenocytes primed in vitro with P815. 0 0, primary culture of H-2 chimera splenocytes and normal DBA/2 thymocytes (ly|£c units were 10.9); * primary culture of H-2 chimera splenocytes and P815 suppressor thymocytes (lytic units could not be calculated because of low^ levels of cytotoxicity) . H-2 : control assay^for suppressor cells. 0 0, primary culture of H-2 chimera splenocytes and^normal DBA/2 thymocytes; • • , primary culture of H-2 chimera splenocytes and P815 suppressor thymocytes (Lytic units could not be measured in this experiment). 24 I 12.5:1 25.1 50.1 100:i 12.5M 25:1 50M 100:1 Effector: Target Ratio Figure 5. The ability of the suppressor factor isolated from P815 suppressor thymocyte populations to suppress the in vitro primary cytotoxic response of normal DBA/2 splenocytes to syngeneic mitomycin C-treated tumor cells. The dilutions used in this assay represent approximately 1/25 of a single thymus. Anti-P815: the c^Jotoxic response of cells primed in vitro with P815 cells on Cr labeled P815 cells in an 18 hr-assay. • • , primary culture of normal DBA/2 splenocytes and . . normal DBA/2 thymocyte extract (lytic units were 59.1); 0 0, primary culture of normal DBA/2 splenocytes and P815 suppressor thymocyte extract (lytic units were 31.8). Anti-Ll210: the cytotoxjLj response of cells primed in vitro with L1210 cells on Cr labeled L1210 cells in an 18 hr-assay. • • , primary culture of normal DBA/2 spleno cytes and normal DBA/2 thymocyte extract (lytic units were 45.6) 0 0, primary culture of normal DBA/2 splenocytes and P815 suppressor thymocyte extract (lytic units were 44.8). 25 was significantly suppressed whereas the response the L1210 was not. Experiments were run to determine whether adsorbtion with anti-la^ alloantiserum could eliminate suppressor factor activity in our system. Partially purified factor and the equivalent fraction of normal thymocyte extracts were subjected to treatment with anti-la^ antiserum or anti-H-2^ antiserum and adsorbed on immunoadsorbent columns containing anti-mouse immunoglobulin. The eluted materials were titrated for suppressive activity. The results are shown in Figure 6. While suppressive activity was present in the anti-H-2 suppressor factor eluates, the suppressive material treated with anti-la^ was not, showing that it expresses determinants encoded by the la region of the MHC. The ability of the factor, when added to B6D2F^ C57BL/6 chimeric splenocytes, to suppress the generation of cells cytotoxic to P815, was also tested. The results (Fig. 7) show, as with the suppressor cells, that the suppressive activity of the factor with H-2^ cells was similar to that observed with the DBA/2 cells tested at the same time. 26 Figure 6. The effect of anti-la antiserum adsorption on the suppres sive activity of the suppressor factor isolated from P.815 suppressor -thymocyte populations. The dilutions used in this assav represent approximately 1/25 of a single thymus. Anti-la : the effect of adsorption of the suppressive factor with anti-la antiserum, o—-^o, primary culture of normal DBA/2 splenocytes and. anti-la adsorbed normal thymocyte extracts; •—primary culture of normal DBA/2 splenocytes and anti-la adsorbed suppressor thymocyte extracts. ^Lytic units in both cases were 57.1. Anti-H-2 : the effect of adsorption of the suppressive factor with anti-la antiserum, o—^o, primary culture of normal DBA/2 splenocytes and anti-la adsorbed normal thymocyte extract (lytic units were 76.9); • g, primary culture of normal DBA/2 splenocytes and anti-la adsorbed suppressor thymocyte extract (lytic units were 38.5). 27 pi • i i 1—i ' 1 12.5n 25:1 5CM 1OO0 1250 25;1 5CM 1000 EFFECTOR :TARGET RATIO Figure 7. The ability of P815 suppressor factor tg suppress the cyto toxic response of splenocytes from H-2 radiation chimeras to P815. The amount of factor used in these experiments was approximately 1/25 thymus equivalent. H-2 : the cytotoxic response of H-2 radiation chimera splenocytes primed in vitro with P815. 0 0, primary culture of H-2 chimera splenocytes and normal thymocyte extract (lytic units were 62.5); • •, primary culture of H-2 chimera splenocytes and suppressor factor (lytic units^were 16.1). H-2 : control assay^for suppressor factor, o o, primary culture of H-2 chimera splenocytes and normal thymocyte extract (lytic units were 48.8); • • , primary culture of H-2 chimera splenocytes and suppressor factor (lytic units were 33.9). 28 Discussion In previous studies on suppressor cells and the specific factors possibly released from them, it was demonstrated that such suppressive elements were capable of specifically suppressing the generation of cytotoxic cells in vitro by spleen cells from P815-primed animals (11,12,65). In this chapter, it was shown that P815-specific sup pressor cells and P815 specific suppressor factor are also capable of suppressing a primary in vitro cytotoxic response to P815 (Fig. 1,5). Thus, the state (primed or virgin) of the cell population on which it acts appears to be unimportant for the P815-specific suppressor cell or P815-specific suppressor factor to express their activity. The ability of alloantisera directed to the la region (the I-J subregion in most studies) to kill suppressor T cells has been reported by others (81,90-93). These studies involved mainly the character ization of T suppressor cells participating in the regulation of the antibody response to soluble antigens. The suppressor cells studied here are those involved in regulating the generation of cytotoxic cells. However, they also appear to possess surface markers recognized by anti-haplotype sera to the la region, so in this respect they do not differ from other populations of antigen-specific T suppressor cells. Since at this time it is not possible to develop an antiserum specific for the I-J subregion of the Ia^ haplotype, it was not possible to test the cells for this specificity although many other studies have shown that this subregion of the la is expressed on antigen-specific suppressor T cells. 29 In this chapter it was undertaken to determine whether the popu lation of T suppressor cells under study was H-2 restricted. Because the immune response being assessed for suppression involved the development of cells cytotoxic for a syngeneic tumor cell line, it was necessary to use spleen cells from radiation chimeras that were d b tolerant to normal H-2 alloantigens. Thus B6D2F^ animals (H-2 x H-2** F^) were irradiated and reconstituted with either parental bone marrow cells (C57BL/6 or DBA/2) or with A.TH cells (H-2't'2) that shares only the D locus with H-2^. Suppressor cells were raised in DBA/2 tumor bearers and then cultured in vitro with mitomycin C-treated P815 cells and chimeric spleen cells of either DBA/2, C57BL/6, or A.TH origin. The experiments, which were repeated three times showed unequivocally that the suppressor cells were capable of suppressing the anti-P815 cytotoxic response of spleen cells differing at the K (A.TH) or both the K and D locus (C57BL/6) and thus did not exhibit the classical form of H-2 restriction. This does not exclude the possibility that some form of la recognition was required since these chimeric cells had been influenced by the thymus of the irradiated B6D2 recipients, but does show that K or D identity is not essential for this type of interaction. The suppressor factor(s) isolated from the suppressor cell thymocyte population and partially purified by preparative isoelectric focusing appeared to share the same properties as the suppressor cells in that it could specifically suppress the primary in vitro cytotoxic response of DBA/2 spleen cells (Fig. 5) and could be specifically removed by anti-la** alloantiserum (Fig. 6) . In this 30 respect this factor is not different from factors described by others (77,81,82,86,89). As mentioned above, it was not possible to test the subregion specificity with the H-2^ system. The data reported so far on the H-2 restrictions observed in suppressive factors are somewhat equivocal. In a series of reports by Tada and his associates (78,95,96) involving suppressive factor(s) that control the antibody response, it was found that this factor required a syngeneic target cell (presumably a helper T cell) before it could effectively suppress the antibody response. Moorhead, who produced a specific suppressor factor in vitro that could suppress the devel opment of delayed-type hypersensitivity to DNFB in mice, found that this material was H-2 restricted and that identity at either the K or D end of the MHC was sufficient to permit suppression in recipient mice (79,80,97). A I^E class specific suppressor factor also shows syngeneic MHC restriction of action (66). Alternately, Waltenbaugh and his colleagues (98) in studying a soluble factor that suppresses the antibody response to the synthetic copolymer L-glutamyl^^-L- . tyrosine"^ (GT) showed that a suppressive factor, elicted in mice by injection of GT alone, could effectively suppress the response to GT coupled to carrier protein across allogeneic barriers. Similar MHC unrestricted activity is found in the GAT system (99). A SRBC specific suppressor factor of a T-hybrid line has also recently been shown to act across allogeneic barriers (100). Kontiainen and collegues also observed no H-2 restriction of their suppressor factors (101). The results here show that the suppressor cells under study (and the presumed equivalent factor), are capable of suppressing the 31 generation of cytotoxicity to P815 by histoincompatible cells. The results show clearly that the K or D gene products do not have to be shared by the suppressor cell (or its factor) and its target. Because radiation chimeras, in which the recipients were B6D2F^ animals, were used for testing restriction, the conclusions can only extend to a lack of restriction at the K and D loci of the MHC since thymic influence by the recipient may have influenced recognition mechanisms on the part of the chimeric cells. CHAPTER III THE LYT PHENOTYPE OF CELLS INVOLVED IN THE CYTOTOXIC RESPONSE TO SYNGENEIC TUMOR AND OF TUMOR-SPECIFIC SUPPRESSOR CELLS AND IMPROVED PROCEDURES IN THE GENERATION OF TUMOR-SPECIFIC SUPPRESSOR CELLS 33 CHAPTER III Introduction Antigenic determinants found on lymphocyte cell surface molecules can be used as markers for studying the relationship of the expression of these determinants and cellular function. Thus, in the last few years, many studies have examined cell surface antigenic determinants which could be used to study and characterize distinct subsets of T cells. One of the most widely studied antigenic systems of T cells has been the one determined by the Ly-1 and Ly-2 loci in the mouse (102). Since these antigens appear to be expressed selectively on various sub sets of undifferentiated and differentiated T cells it has been suggested that they be called Lyt antigen (103). The gene controlling the express ion of Lyt-1 antigen is found on chromosome 19, whereas the gene controll ing Lyt-2 expression is found on chromosome 6 (104). Two allelic forms, Lyt3 and Lyt*5, of the Lyt-1 and Lyt-2 antigens have been found and are designated Lyt-1.1, -2.1 and Lyt-1.2, -2.2 respectively (104). A major part of the Lyt work has been carried out in mice bearing the Lyt*3 allele. In such animals a number of basic observations have been made. Killer cells raised against allogeneic cells (allogeneic killer cells) have been designated as Lyt-1 2+ (105-107). Recently, it has been reported that allogeneic effector cells with this allele may + + be of the Lyt-1 2 phenotype (108-110). This apparent discrepancy may be accounted for possibly on a quantitative rather than qualitative basis. Killer cells raised against syngeneic tumor cells (syngeneic killer cells) have also been reported to express the Lyt-l+2+ phenotype (107,111,112). Helper cells have been described by a number of investigators as expressing the Lyt-l+2 phenotype (114-116). Antigen-specific suppress or cells isolated from antigen-primed mice have been identified as Lyt-1 2+ (115). Suppressor cells generated in vitro by concanavalin A were also found to be Lyt-1 2+, while the helper cells thus generated were Lyt-l+2~ (114,117). There have been fewer studies carried out on mice bearing the Lyt allele. In these studies allogeneic killer cells have been identified as Lyt-l+2+ (108,118). Killers of syngeneic tumor cells in C3H mice were found to be predominantly Lyt-1 2+ although it was indicated that a population of Lyt-1 2 cells might also be involved (119). In the case of suppressor cells, it was found that two distinct populations of cells were present, those which suppressed the delayed type hypersensi tivity reaction and were Lyt-l+2 and those which suppressed a humoral response and were Lyt-1 2+ (120). It has also been reported that in vitro-induced suppressor cells in CBA mice are Lyt-1 2+ (118), while the helper cells are Lyt-l+2+ (118,121). The present study was carried out in DBA/2 mice (Lyt-1.1, -2.1) and involved the response of their cells to the syngeneic mastocytoma P815. The Lyt phenotypes of in vitro-generated allogeneic killers, syngeneic killer cells, and in vivo-generated T suppressor cells were characterized using anti-Lyt-1.1 and anti-Lyt-2.1 serum. Materials and Methods Experimental Animals and Tumors Female DBA/2J mice (H-2d, Lyt-1.1, -2.1) and C57BL/6 mice (H-2*3, Lyt-1.2, -2.2) (The Jackson Laboratories, Bar Harbor, Maine) between the ages of 2 and 5 months of age were used exclusively. The tumor lines used were the P815 mastocytoma and L1210 leukemia, both syngeneic for DBA/2J mice, and both maintained and transplanted as ascites tumors in DBA/2J mice, or as frozen cultures maintained at -70°C in liquid nitrogen. The methods of tumor maintenance have been described elsewhere (11). EL-4 lymphoma, syngeneic for C57BL/6 mice was maintained similarly. Cells Single-cell suspensions were prepared from spleens of DBA/2 mice by pressing the tissue through a stainless steel 60-gauge mesh in RPMT 1640 (Grand Island Biological Company, Grand Island, N.Y.) medium containing 10% heat-inactivated fetal calf serum (FCS), 10 mM Hepes buffer, and 5 x 10 ^ mM of 2-mercaptoethanol. Gentamycin was also added to a final concentration of 50 yg/ml. Suspended cells were washed through 3.0 ml of FCS, resuspended in complete medium, and counted for viable cells by using trypan blue. Tumor cells were washed twice-: in medium before resuspen-sion in complete medium for counting. Culture system for the generation of cytotoxic effector cells  Allogeneic effectors The method for the jLn vitro generation of allogeneic killer cells have been described in detail elsewhere (122). 36 Briefly, 5 x 10 spleen cells from DBA/2J mice were cultured, for 4 days in the presence of.5 x 10 mitomycin-treated spleen cells from C57BL/6 mice in 24-well Linbro trays in 2.5 ml RPMI 1640 medium supplemented with 10% FCS, 10 mM Hepes buffer, 5 x 10 ^ M 2-mercaptoethanol, and 50 g/ml genta-mycin. Cells were harvested at 4 days, counted by trypan blue exclusion, and assayed for their ability to kill "^Cr-labeled EL4 cells in a standard 4 hr assay, using target: effector cells from 30:1 to 3.75:1. Syngeneic effectors The method by which a primary in vitro cytotoxic response to syngeneic tumor cells can be generated has been described else where (113).The method is basically that described above with the exception that 5 x 10^ DBA/2 spleen cells were cultured for 5 days with 5 x 10"* mitomycin-treated P815 cells. Harvested cells were run in the standard "'"'"Cr release assay for 18 hr on labeled P815 cells with effector: . target ratios of between 100:1 and 12.5:1. Previous work in this laboratory has demonstrated that the killer cells generated in this way are specific for the stimulator cells (P815 in this instance) (123,124). (Also see Table I). Cytotoxicity test This has been described in detail in Chapter II. Generation of suppressor cells In most of the previous work carried out in this laboratory, antigen-specific T suppressor cells involved in regulation of the cytotoxic response of syngeneic mice to the P815 tumor line (123,124). 37 Table I. Specificity of cytotoxicity generated in vitro In vitro culture ,. Cytotoxocity Assay Responding Cells Stimulating cells Target cells % cytotoxicity Normal DBA Spleen P815 P815 56.4 + 2.5 Normal DBA Spleen P815 L1210 6.8 + 2.6 Normal DBA Spleen P815 EL-4 5.7 + 3.0 Normal DBA Spleen C57BL/6 P815 0.7 + 0.6 Normal DBA Spleen C57BL/6 L1210 5.8 + 3.2 Normal DBA Spleen C57BL/6 EL-4 61.2 + 2.9 a51 Cr release after 18 hr incubation^ effector: target ratio of 100:1 for P815 stimulated cells. Cr release after 4 hr incubation, effector: target ratio of 715:1 for EL-4 stimulated cells. 38 have been obtained from the thymuses of animals into which 3 x 3 10 tumor cells had been injected subcutaneously 8-9 days previous ly (65). The suppressor cells used in this study were generated by the intraperitoneal injection of soluble membrane extracts of P815 cells. P815 cells, from mice bearing the tumor as ascites, were drawn from the peritoneal cavity, washed in PBS, and lysed by freezing and thawing three times in distilled water. Large membrane fragments and other cell debris were removed by centrifugation at 12,000g for 30 min. Membrane components were then pelleted by centrifuation at 105,000g for 120 min. The membrane pellet was resuspended in PBS and subjected to three 1-min bursts of ultrasound (Biosonic probe, 30 setting) at 0°C. The material was again centrifuged and the supernatant presumably containing solubilized membrane components and small membrane fragments was used. The protein content of this material was determined by the standard Lowry test. Suppressor cells in the spleens of DBA/2J mice were readily induced by intraperitoneal injection of between 80 and 200 yg protein per animal. Animals were sacrificed 4 to 5 days after antigen inection, and their spleens used as a source of suppressor cells. This method is an adaptation of that described recently for the generation of suppressor T cells to methylcholanthrene-induced sarcomas 0-25). Because the spleens of these animals contained both antigen-specific and nonspecific suppressor cells (see below), it was necessary to separate these two populations. Spleen cells were passed through nylon wool columns according to the method of Julius el: al (126). The modifications used here were that cells, after application to the column, were incubated for 30 min at 37°C and then eluted at 37°C. The columns were then incubated for a further 30 min at 4°C before a second elution in cold medium. The cells from both elutions were pooled and used as a source of antigen-specific suppressor cells. This procedure utilized columns which were fairly tightly packed, containing about 12-15 ml of nylon wool through which 20 ml of supplemented medium at 37°C had been passed 8 before use. A total of 2 x 10 spleen cells were applied to columns of this size and approximately 20% of the cells were recovered, by elution. The properties of the eluted cells were characteristic of T-cell-enriched populations in that they were 95% sensitive to anti-thy-1 serum plus complement and contained very few phagocytic cells (<1.0%). The generation of suppressor cells is illustrated in Figure 8. Assay for suppressor cells The method used to assay for suppressor cells has been described 6 in detail elsewhere.(123). Briefly, 5 x 10 normal spleen cells were cocultured with 5 x 10^ (or fewer in some cases) suppressor cell popu lations in the presence of 5 x 10^ mitomycin-treated P815 cells in 24-well Linbro trays in 2.5 ml of medium. Cells were cultured for 5 days before assay for generation of cells cytotoxic for P815. Controls in these studies constituted the use of 5 x 10 normal spleen cells plus 5 x 10 normal spleen or spleen-derived cells which had been treated in a manner analogous to the suppressor population. When the degree of suppression was quantitated, cytotoxicity 40 Preparation of P815 Tumor-Specific  Suppressor Cells (P815-T ) Injection of 80-200 vg of Soluble P815 Tumor Membrane Extract IP DBA/2J Mice 4 Days Splenocytes - Containing both Specific Suppressor Cells (T-Cells): And Non-Specific Suppressor Cells (Probably M0) Passed Through Nylon Wool Column (30' at 37°) Eluted with 37° media Further 30' at 4° Eluted with 4° media Eluted Enriched T-cell Population Containing P815~Specif ic T .. Figure 8. Flow diagram illustrating the preparation of P815 tumor-specific suppressor cells. 41 was tested at various effector to target ratios and the decrease in total lytic units was assessed. One lytic unit, which was defined as the number of effector cells required to lyse 50% 4 of the 10 target cells in 18 hr incubation, was estimated by linear regression analysis of percentage of cytotoxicity vs . logarithm of effector to target cell ratio. Total lytic units were caluclated from cell recovery in cultures, and percentage of suppression was estimated from the decrease in total lytic units. In all experiments, no significant differences in cell recovery between test cultures and controls were observed. In all suppression experiments reported here, except the time course one (Fig- H), a minimum of 37% suppression was seen. Anti-Lyt treatment of cells The cells tested for their Lyt phenotype in this study were: allogeneic killer cells, syngeneic killer cells, and syngeneic tumor specified suppressor cells. DBA/2J mice are of the Lyt-1.1 and Lyt-2.1 phenotype. The antisera used here were the kind gift of Dr. R. Nowinski (The Fred Hutchinson Cancer Research Center, Seattle, Washington) who had raised and characterized the antisera according the previously described procedures (127). The anti-Lyt-1.1 serum titered at 1:640 and the anti-Lyt-2.1 titered at 1:160 when titrated in the presence of rabbit comple ment on B6^Lyt-l.l and B6Lyt-2.1,-3.1 thymocytes, respectively. The anti-Lyt-1.1 serum had no effect on B6Lyt-2.1, -3.1 thymocytes, likewise, the anti-Lyt-2.1 serum had no effect on B6.Lyt-l.l thymocytes. In the experiments run here, higher levels of the 42 antisera were found to be necessary in order to kill the appropriate cells effectively. For both the allogeneic and syngeneic killer cells, the procedure was identical. Effector cells were harvested, counted, and distributed to small plastic test tubes at a total concentration of 3.0 x 107 cells per tube. These cells were centrifuged and the supernatant removed. Three hundred microliters of medium was added to each tube and 50 yl of either anti-Lyt-1.1, Lyt-2.1, or medium was added to the appropriate tube. The cells were mixed and incubated at room temperature for 45 min following which 50 yl of neat rabbit complement (Low-tox, Cedar Lane, Ontario, Canada) was added to each tube except the control, to which 50 yl of medium was added. Incubation was continued for another 45 min. Cells were washed three times in supplemented medium, counted by trypan blue exclusion, and subsequently tested for their cytotoxic activity. One population of a 1:1 mixture of anti-Lyt-1 and anti-Lyt-2-treated cells were also reconstituted and tested for cytotoxicity. In the assay of suppressor cells, larger cell numbers had to be treated because the cells were subsequently going into the large cultures for 5 days. For this reason, 6 x 10^ cells were treated, and double the amounts of all reagents were used. A protocol as described above was used, except that after treatment, washing, and counting, the cells were put into culture at a concentration of 5 x 106 with 5 x 106 normal spleen cells plus mitomycin-C treated P815 cells. 43 Results The spleen cells of DBA/2J mice, 4 days following intraperitoneal injection of P815 membrane extracts, were very suppressive when they were cocultured with 5 x 10^ DBA/2 spleen cells in the presence of mitomycin-treated P815 cells. The generation of cytotoxic cells in these cultures was markedly below that seen in control cultures (Fig. 9a). When these suppressor cells were assayed in culture with normal spleen cells plus mitomycin-treated L1210 cells, the generation of cells cytotoxic for this tumor were also markedly suppressed (Fig. 9b). Since these two systems do not cross react at this level, (see Fig.T) ,(12) it was concluded that intraperitoneal injection of membrane extracts of P815 cells could generate both specific and nonspecific suppressor cells. Since the most common nonspecific suppressor cell is an adherent cell.,- pro bably of the macrophage-monocyte series (18,128,129), an attempt to achieve specificity in the system by passing the spleen cells from antigen-injected animals through nylon wool columns was attempted. The cells eluted from these columns at either 37 or 4°C were tested for their suppressive activity'. Since both populations were found to contain specific suppressor cells, subsequent studies involved a pool of the two populations (Fig. 10). The time course of appearance and disappearance of the antigen-specific suppressor cells was examined. As can be seen in Figure li, DBA/2 splenocytes show antigen-specific suppression within 2 days following intraperitoneal injection of P815 membrane extracts. This suppression peaks at 4-5 days, then falls off rapidly to control levels by 7-9 days post injection. 44 SOI 100=1 125=1 EFFECTOR ^TARGET RATIO Figure 9. Cytotoxicity titrations of DBA/2 spleen cells primed against either P815 stimulator ^a) or L1210 stimulators (b). (•) Control containing 10 normal DBA/2 splenocyjzes in the priming culture; (o) cultureg containing 5 x 10 normal DBA/2 splenocytes and 5 x 10 splenocytes from mice inoculated 4 days previously with P815 membrane extracts (80 yg per mouse). 45 125:1 50:1 lOO'l 125:1 EFFECTOR;TARGET RATIO Figure 10. Cytotoxicity titrations of DBA/2 spleen cells primed against either P815 (a) or L1210 £b) stimulators. (•) Control culture gontaining 5 x 10 normal DBA/2 spleno cytes and 5 x 10 normal DBA/2 nylon wool-pgssed splenic lymphocytes; (o) cultures containing 5 x 10 normal DBA/2 splenocytes and 5 x 10 P815 primed DBA/2 nylon wool-passed splenic lymphocytes (cells eluted at 37 and 4 C were pooled). i i \ \ \ 2 4 6 8 10 Days aftar Lp. injection of P815 membrane extract Figure 11. Time course of appearance and disappearance of antigen-specific suppressor cells in the spleen of DBA/2 mice injected intraperitoneally with P815 membrang extracts on day 0. Control cultureg contained 5 x 10 normal DBA splenocytes and 5 x 10 normal DBA/2 nylon wool-passedgSplenic lymphocytes. Test cultures gontained 5 x 10 normal DBA/2 splenocytes and 5 x 10 P815 primed DBA/2 nylon wool-passed splenic lymphocytes. Results are plotted as MEAN % suppression caused by P815 injected spleen as compared to controls. 47 The Lyt phenotypes of a variety of effector cells in DBA/2 mice was investigated. Since previous findings by others had characterized a + + allogeneic killers in mice of the Lyt allele as Lyt-1 2 , it was considered important to test the antisera by determining its effect on allogeneic killer cells. The results are shown in Figure 12, in which it can be seen that both anti-Lyt-1.1 and anti-Lyt-2.1 plus complement effectively abrogated the allogeneic killer cells. Mixing of anti-Lyt-1.1 and anti-Lyt-2.1-treatedcells prior to the cytotoxicity assay did not reconstitute the response (data not shown). These results, therefore, are in agreement with the observations of others and indicate that the allogeneic killer cell in the DBA/2J animal + + expresses the Lyt-1 2 phenotype.(108,118). An experiment was run concurrently to determine the Lyt phenotype of the cytotoxic effector to syngeneic tumor cells. The results are shown in Figure 13. In this case complement plus anti-Lyt-2.1 but not anti-Lyt-1.1 reduced the killing effiency of this population, indicating that the Lyt phenotype of this cell is Lyt-1 2+. Mixing of the two treated populations did not restore the response. Finally, the Lyt phenotype of the DBA/2 antigen-specific suppressor cell was investigated. The results are shown in Figure 14. In this instance, the anti-Lyt-1 antiserum eliminated suppressor cells, or their development, whereas the anti-Lyt-2 serum had no effect. In this case also, mixtures of anti-Lyt-1 and Lyt-2-treated cells did not reconstitute the response. It would therefore appear, in this system, that .the suppressor cell, its progenitor, or a cell vital in its development expresses the Lyt-1 2 phenotype. 48 i 3-75:1 75:1 15:1 30:1 EFFECTOR : TARGET RATIO I ! I -Figure 12. The effect of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rabbit C' on DBA/2 allogeneic effector .cells raised against C57BL/6, on a EL4 target. (o) Control culture; (t) anti-Lyt-1.1-treated effector cells; (A) anti-Lyt-2.1-treated effector cells. 49 100 -80 Control All 60 CYTOTOXIC 40 'anti Ly-1-1 < anti Ly-2-7 ^? ON 20 -• I 1X5:1 25:1 50:1 100:1 EFFECTOR: TARGET RATIO Figure 13. The effect of ariti-Lyt-1.1 and anti-Lyt-2.1 serum plus rabbit C' on DBA/2 effector cells primed against the syngeneic P815 mastocytoma. (o) Control; (•) anti-Lyt-1.1-treated effector cells; (A) anti-Lyt-2.1-treated effector cells. 50 1001 80 O I->• u QN 60r-40 20 • A Control 0 Suppressor Cell Control • Anti-Lyl-1 • A Anti-Ly 2~1 i • 12.5 1 25 1 50--1 100--1 EFFECTOR '. TARGET RATIO Figure 14. The effect of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rabbit C' on P815-primed nylon wool-passed DBA/2 splenic ^ suppressor cells. (A) ControlgCultures containing 5 x 10 normal DBA/2 cells plus 5 x 10 nylon wool-passed normal DBA/2 gplenocytes; (o) positive control culture containing 5 x 10 normal DBA/2 splenocytes plus 5 x 10 nylon wool-passedgP815 suppressors cells; (•) culture cgntaining 5 x 10 normal DBA/2 splenocytes plus 5 x 10 anti-Lyt-1.1-treated^ P815 suppressor cells; (A) culture cgntaining 5 x 10 normal DBA/2 splenocytes plus 5 x 10 anti-Lyt-2.1-treated P815 suppressor cells. The SEM in this experi ment was always <3.0% and therefore not shown. Cell recoveries of antiserum-treated suppressor T cell populat ions were as follows: anti-Lyt-1.1, 68.7%;" anti-Lyt-2.1, 70.3%.. These data are based on "estimates from the comple ment-treated control cells which were regarded as 100% recovery. Lytic units were: (A) 84.3, (o) 46.1, (•) 78.6, (A) 41.7. 51 Discussion In this chapter, a method for the in vivo induction of specific suppressor cells which inhibit the hi vitro generation of cells cyto toxic for a syngeneic tumor has been described. The method is similar to the method described by Fujimoto for the induction of the same type of suppressor cells with a different tumor (125). However, in the work shown here, the suppression was not specific when whole spleen cell populations of tumor antigen-injected mice were used, as indicated by their ability to suppress the jm vitro cytotoxic response to L1210 leukemia cells as well as P815 mastocytoma cells. Since it is well documented that antigen-stimulated spleen cells may contain a populat ion of nonspecific suppressor cells which exhibit the characteristics of adherence to plastic or nylon wool and phagocytic activity (18,128, 129), it was felt that the passage of the stimulated spleen cells through nylon wool might remove the nonspecific suppressive activity. This proved to be the case. That the antigen-specific suppressor cell was of thymic origin has been shown previously (12) and is implicit in the finding that the nylon wool nonadherent population (which is >95% thy-1 positive) contained the antigen-specific suppressor cell. The studies on the Lyt phenotypes of the various effector cells in this in vitro system were informative. The finding that the allo geneic killer cell in the DBA/2J mouse was Lyt-l+2"*" was not surprising, since it confirms the observations of others that, in mice bearing the Lyt-1.1 and 2.1 allele, the allogeneic killer cell is indeed Lyt-l+2+ (108,118). Very little information is available regarding syngenic killer and suppressor cells in mice expressing this allele. 52 The finding that the syngeneic killer differs from the allogeneic killer cell in expressing the Lyt-1 2+ phenotype is somewhat surprising. However, other investigators have reported that anti-tumor syngeneic killer cells in C3H mice were predominantly of the Lyt-1 2+ phenotype. These workers also noted the probable involvement of Lyt-l+2+ cells in the syngeneic system. In a recent report very applicable to this study, Mills and colleagues found that in DBA/2 mice, syngeneic tumor killers directed towards P815 tumor cells expressed'Lyt-1 antigen.(130). They however did not look at Lyt-2 antigen expression on these cells. Therefore, as far as expression of Lyt-1 on syngeneic killers goes, that study and the one reported here seem to be in conflict. One reason for this may be the different methods of generation of cytotoxic cells to P815. Syngeneic cytotoxic cells in the Mills study were generated in vitro (in the presence of IL-2) from P815 tumor bearing DBA/2 mice. Therefore, they were probably generating secondary cytotoxic killers as opposed to primary cytotoxic cells in this investigation. Thus, there may be a difference in the Lyt-1 antigen expression on primary vs secondary syngeneic killers in the DBA/2-P815 system. This of course leads to the recent findings that all T cells probably express Lyt-1 and Lyt-2, although in varying amounts (131). Therefore, it is impossible to conclude at this time that the syngeneic killer cell is Lyt-1 2+, since it may only be quantitative differences between it and the allogeneic killer in expression of the Lyt-1 alloantigen which appear to render them as distinct populations. The observation made here that anti-Lyt-1 antiserum effectively abrogated suppressor cell function, in the in vitro system used, is explicable in at least two possible ways. First, it has been reported previously that suppressor cells which lower DTH reactions in mice of a + -the Lyt allele are of the Lyt-1 2 phenotype (120). Second, the cell that was isolated from immune spleens may not be the effector cell for suppression but may induce the suppressors in culture. Evidence for a mechanism such as this has been presented for both humoral and cell-mediated responses (42-44, 132,133). In the model presented by Germain, Benacaraf and colleagues (22,23,44,60,134-136), the first cell in immune suppression is a Lyt-l"*", I-J+, antigen-binding "inducer" of suppression. This cell may "induce", in a cascade-like mechanism, other T cells to actively suppress in the same system. The 5-day culture period of the assay here might allow the induction of suppressors in situ under the influence of this Lyt-l+2 cell. At present, it can only be concluded that in a population of suppressor cells, or their precursors, an Lyt-l+2 cell is essential for the suppressive effect to the observed in in vitro cytotoxic assays. CHAPTER IV IN VITRO AND IN VIVO EFFECTS OF SYNGENEIC AND ALLOGENEIC ANTISERA RAISED TO TUMOR-SPECIFIC SUPPRESSOR FACTOR FROM DBA/2 MICE 55 CHAPTER IV Introduction It is now recognized that T lymphocytes are made up of distinct sub sets which interact with each other as well as B lymphocytes and macro phages to both amplify and inhibit a variety of immunological responses. Thus T cells have demonstrated enhancing effects on the immune response by way of T helper cells (2-10). T cells have also been found to in hibit the immune response by way of T suppressor cells (11-17,19~44,65). In many of the investigations concerning antigen-specific regu latory T cells, it has been shown that soluble factors derived from these cells can apparently duplicate their functions (14,59-61,67-74,76,77,82, 137-139). Little is known of these factors but, as indicated in Chap ter II, what is known shows they exhibit certain physical, chemical and antigenic properties in common. They are proteins with specific antigen binding potential, have molecular weights of about 50,000, have deter minants which are coded by the Ir region, lack constant region immuno globulin determinants, but may carry variable (idiotypic) determinants of the I heavy chain. All this leads to the intriguing possibility that these "factors" may in fact be the antigen receptors of these specific regulatory T cells. Yamauchi e_t al_ (125) have recently shown that injection of soluble S1509a tumor extracts into syngeneic A/J mice activates, in the spleen tumor-specific T suppressor cells. This has recently been extended to the syngeneic P815 tumor system in DBA/2J mice (Chapther III, 13). In this chapter, it is shown that by passing extracts of this suppressive population over P815 membrane - Sepharose columns, P815-specific suppressor factor can be eluted. This P815-specific suppressor factor was then injected into syngeneic and allogeneic groups of mice. The hope being that in the syngeneic mice the- umique determinants (idiotypic?), found in or near the antigen binding site of the P815-specific sup pressor factor, would elicit an antibody response in at least some of the animals. While both these and the ©onstant determinants on the suppressor factor might induce an antibody response in the allogeneic mice. Both groups of mice produced antisera which was capable of reacting in vitro specifically with the P815-specific suppressor factor, and with complement was able to abrogate the function of P815-specific T suppressor cells, but was unable to effect the action of P815-specific T cytotoxic effector cells. The antisera produced in syngeneic mice was also shown to be effective in vivo in slowing P815-tumor growth and prolonging survival times of DBA/2 mice injected with this tumor. 57 Materials and Methods  Mice and Tumors. Female DBA/2J and C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). Except for the prepa ration of anti-suppressor factor antisera, all mice were used between the ages of 2 and 5 months of age. The tumor lines used were the P815 mastocytoma and L1210 leukemia, both syngeneic for DBA/2J mice. They have both been maintained and trans planted as ascites tumors in DBA/2J mice (11), or as frozen cultures maintained in liquid nitrogen in this lab for the past 6 years. Balentl tumor, syngeneic to Balb/c mice, was also used and maintained as above. Cells. This has been described in detail in Chapter III. Culture system for generation of cytotoxic effector cells. The method by which a primary in vitro cytotoxic response to syngeneic tumor cells can be generated has been described pre viously • (123):. "Briefly, specif ic. cytotoxicity .against either P815 or L1210 was generated in vitro by incubating 5 x 10 spleen cells with 5 x 10^ mitomycin treated tumor cells in Linbro multiwell plates containing 24 flat bottom wells, at 37°C in a humidified incubator with 5% CO2 for 5 days. The total volume of each culture well was made up 2.5 ml with complete 1640 medium. After incubation, cells were harvested, counted,-resuspended'in complete: 1640 medium'at 'a concentration 58 of 10 /ml and titrated in quadruplicate with doubling dilutions starting at an effector:target ratio of 100:1 in a standard 18 h ^Cr release assay which has been described in previous chapters (II and III). The specific effector cells in this syngeneic tumor system have previously been shown to be T lymphocytes (11,13). Generation of suppressor cells. The method, for generation of tumor-spcific T suppressor cells, involved in the regulation of cytotoxic response of DBA/2J mice to the syngeneic P815 tumor, has been described previously (Chapter III). Briefly, suppressor cells used in this study were generated by the intraperitoneal injection of soluble membrane extracts of P815 cells. Suppressor cells were readily induced in the spleens of DBA/2J mice by intraperitoneal in jection of 150 Ug of protein per animal 4-5 days before sacrifice. Because the spleens of these animals contained both antigen-specific and non-specific suppressor cells (13) it was necessary to separate the two populations. Spleen cells were passed through nylon wool columns by a modified method of Julius et al (126). The method is basically that described before (Chapter III, 13) with a few modifications outlined below. In short, the cells, after application to the nylon wool column, were incubated for 60 min at room temperature (20°C) and then eluted with warmed 37°C complete 1640 medium. Approximately 15% of the cells were recovered by this elution process. The properties of the eluted cells were characteristic of T cell enriched populations in that 59 they were >95% sensitive to anti-Thy 1 serum plus complement and contained very few phagocytic cells (<1.0%). This popu lation was antigen-specific in its suppressive activity, and has been found repeatedly to contain cells, which under the conditions used here, were capable of suppressing the in vitro primary cytotoxic .response 30-70%. Preparation of suppressor factor. Antigen-specific T suppressor cells, obtained from spleens of DBA/2J mice injected intraperitoneally 4-5 days previously with P815 tumor membrane fragments were used for the preparation of P815 antigen-specific suppressor factor (P815-SF). Control factor preparations were made from normal DBA/2 splenocytes, as well as from mice injected with Balentl tumor membrane fragments (this preparation was termed Balentl-"SF" even though it had no demonstratable effect, suppressive or otherwise). The spleno cytes were sonicated for 3 one minute bursts (Biosonic 80, 60 watt setting) at 0°C. This material was then centrifuged for 30 min at 15,000 x G at 0°C. The supernatant was then taken and mixed with normal DBA/2J membrane fragments linked to Sepharose 4B. This step was taken to ensure that any material which non-specifically bound to DBA/2J membrane would be removed here. After 60 min at 0°C any non-bound material was eluted with ice cold phosphate buffered saline (PBS). This material was then mixed with rabbit anti-mouse immunoglobulin linked to Sepharose 4B. This was to remove any anti-body molecules which Preparation of P815 Tumor-Specific  Suppressor Factor (P815-SF) 60 IP Injection of 100-200 ug of Soluble P815 Membrane Extract into DBA/2J Mice 4 Days Splenocytes (Contain P815-specific T suppressor cells) Sonication + Centrifugation Supernate (Crude Extract) v Normal DBA/2 Membrane: Sepharose Column (60', 0°) Elute with PBS Rabbit Anti-MIg: Sepharose Column (60', 0°) Elute with PBS P815 Membrane: Sepharose Column (60', 0°) Wash with PBS Elute Absorbed Material with 2M NaCl si/ P815-Specific SF Figure 15. Flow diagram illustrating the preparation of P815 tumor-specific suppressor factor (P815-SF) 61 might bind in the following step. The capacity of the anti-mouse immunoglobulin column was sufficient to remove at least twice the amount of Ig found in the extracts. After 60 min at 0°C any non-bound material, was eluted with ice cold PBS. This material was then mixed with P815 membranes linked to Sepharose 4B. After 60 min at 0°C the beads were washed thoroughly with ice cold PBS. Bound material was eluted under midly dissociating conditions with 2M NaCl. The absorbance at 280 nm of this material was in all cases O.01; therefore, it has been difficult to quantify it other than by its biological properties. It was filter sterilized and stored at -70°C. This material which was eluted from the P815 column was then titrated for suppressive activity. Concentration when mentioned is quoted as spleen equivalents based on the volume of original spleen homogenates corrected for the change in volume following passage through the columns. Values for the eluted fractions are based on the assumption that all the activity was recovered. All the above-mentioned columns were prepared as described previously (65,140) by the cyanogen bromide method (141). The preparation of suppress or factor is illustrated in Figure 15. Assay for suppressor cells and suppressor factors. The method used to assay for suppressor cells has been described in detail elsewhere (123). Briefly, 5 x 10u normal spleen cells were cocultured with 5 x 10^ (or fewer in some cases) suppressor cell populations in the presence of 5 x 10^ mitomycin 62 C treated tumor cells, in 24 well Linbro trays in 2.5 ml of complete 1640 medium. Cells were cultured for 5 days before being assayed for cells cytotoxic to the tumor. Controls in these studies constituted the use of 5 x 10 normal spleen cells plus 5 x 10 normal spleen cells or spleen cells which had been treated in a manner analogous to the suppressor popu lation. Assay for the splenic suppressor factor activity involved incubation of 5 x 10^ normal spleen cells with various concen trations of the factor preparation, under the same conditions as outlined above. All experiments reported herein, using these assays were repeated at least three times on separate occasions (therefore separate preparations of both suppressor cells and factor). Preparation of antisera against P815-specific suppressor factor. The anti-P815-specific suppressor factor antisera (anti-P815-SF antisera) used in this study was prepared by immunizing syngeneic DBA/2J mice and allogeneic C57BL/6 mice repeatedly with P815-specific immunoadsorbent eluted suppressor factor (see above). Control material (Balentl-"SF") was prepared in parallel from DBA/2J mice injected 4-5 days previously with Balentl tumor membrane extracts. This material was injected into control groups of mice from the same batch. Animals were injected subcutaneously every 2 weeks with 0.1 ml (0.036 spleen equivalent) of either P815-specific suppressor factor or control Balentl-"suppressor factor" in complete Freund's adjuvant. After 63 4 months, the mice were bled from the retroorbital sinus once a month (one week after the last immunization) while maintaining the immunization schedule. The resulting antisera was inactivated at 56°C for 30 min, then tested for its ability to abrogate in the presence of complement the function of P815 specific T suppressor cells. In all experiments reported here the antisera from the mice within each group were pooled before testing. Each of the four groups contained at least 15 mice each. Anti-P815-SF antisera killing of cells. The cells tested for their expression of P815-specific suppressor factor determinants in this study were: syngeneic P815 tumor-specific suppressor T cells and syngeneic P815 tumor-cytotoxic T cells. In preliminary tests the two anti-P815-SF antisera pools were found to be effective, in conjunction with rabbit complement (Rabbit Low Tox Complement, Cedar Lane Labs, Hornby, Ontario), in eliminating suppressor cell function, at dilutions of up to 1:40. In experiments run here all the antisera was used at a final concentration of 1:10, in the presence of a 1:9 final concentration of complement. At this concentration no anti-normal DBA/2J or anti-tumor activity was detected in the anti-P815-SF antisera. In experiments testing for the presence of P815-specific suppressor factor determinants on suppressor cells, 2 x 107 splenic T suppressor cells, prepared from DBA/2J mice injected with tumor membrane extracts and 2 x 10^ splenocytes from normal 64 DBA/2 controls, were suspended in 0.40 ml of a 1:10 dilution of antisera and incubated for 45 min at room temperature. Then 50 yl of neat rabbit complement was added and incubation was continued for another 45 min. Appropriate controls of complement and medium only were run concurrently. Following incubation cells were washed twice in complete 1640 medium, and counted by trypan blue exclusion. In no instance were viable counts in cell suspensions containing the anti-P815-SF antisera significantly different from those of the controls, and in no instance were counts lower than 90% of the number of cells before treatment. These cells were subsequently used in suppressor cell assays. In experiments testing for the presence of P815-specific suppressor factor determinants on cytotoxic cells, the protocol was as described above except 1.6 x 10^ cells, harvested from 5 day in vitro cultures with mitomycin C treated tumor cells, were treated and subsequently tested in the cytotoxicity assay. Since this experiment essentially resulted in negative results, cyto toxic cells were also treated with monoclonal anti-Thy 1 antisera (kindly supplied by Dr. H.-S. Teh, U.B.C., and used at a dilution of 1:100) plus complement. Anti-SF antisera absorption of suppressor factor activity. The ability of anti-P815-SF antisera to remove the suppressive activity from P815-specific suppressor factor preparations was tested by an immunoadsorbent assay similar to the one described previously (Chapter 11,65). -In short, P815-specific suppressor 65 factor preparations or normal control material prepared in parallel were mixed with either the anti-P815-SF antisera or the control anti-Balentl-"SF" antisera, and incubated for 120 min at 0°C. These materials were then passed through immuno-adsorbent Sepharose 4B columns to which rabbit anti-mouse immuno globulin had been attached by cyanogen bromide treatment. The capacity of the column was sufficient to remove at least twice the amount of mouse immunoglobulin added to the suppressor factor preparations. The material eluted from the columns was titrated for suppressive activity over dilutions ranging from 1:25 to 1:300 (these dilutions of eluted material represent between 0.036 and 0.0033 spleen equivalents). ELISA assay. This test was carried out as described before.(142). Briefly, 0.2 ml of antigen (e.g. P815-SF in Fig 18) in pH 9.6 carbonate buffer was attached to substrate microtiter plates (Cooke Engineering Co., Alexandia, Va., No. 1-220-295) for 18 hr at 4°C. After washing with PBS-tween buffer, antisera to be tested for their activity against the coated antigens were added to the wells in 0.2 ml aliquots. Following incubation for 2 hr at room temperature and subsequent washing, the developing alkaline phosphatase-linked rabbit anti-mouse Ig (RaMI ) or goat anti-rabbit (GoRI ), at a dilution of 1:600 or 1:400 respectively, was added in 0.2 ml aliquot. After a further 2 h incubation and final washing with buffer, 0.2 ml of the enzyme substrate solution (Sigma-104-105, p-nitrophenyl phosphate 66 disodium) was added to each well and the enzyme substrate reaction was allowed to continue while color developed in the wells. The color change was followed on a Titertek Muliskan (Flow Laboratories). All tests were done at least in triplicate. Gontrols of normal mouse serum (NMS), normal rabbit serum (NRS) or antigen alone with no added serum were always run and the appropriate control response was subtracted to get the specific response. Antisera plus complement killing of P815 tumor cells. P815 tumor cells were labeled with "^Cr as described pre viously (Chapter II) . The "'"'"Cr labeled P815 tumor cells were resuspended at 2 x 10^/ml in complete medium. 100 ul (2 x 10^) of these cells were dispensed into the wells of multi-dish microculture plates. 50 ul of antisera plus 50 ul of 1:2 dilution of rabbit complement was then added to each well. Appropriate controls of complement and medium only were run concurrently. They were then incubated for 90 minutes at room temperature. 0.10 ml of the supernatant was removed, and its radioactivity was measured.on a gamma counter. In vivo effects of anti-P815-SF antisera. The in vivo experiments of anti-P815-SF antisera were carried out to determine their effect on P815 tumor growth in DBA/2 mice. Two days prior to injection of P815 tumor cells 50 ul of anti-P815-SF antisera, anti-Balentl-"SF" antisera, or PBS were in-3 jected i.v. into DBA/2 mice. Two-days later 2 .x 10. P815 tumor 67 cells were injected s.c. into these mice. Tumor growth was followed by two dimensional measurements with calipers. There were 7 mice/ groups, and these experiments were carried out on three separate occasions. Therefore, a total of 21 mice in each of the four groups. The combined results are reported here. Analysis of Data. On all occasions, experiments were repeated a minimum of three times. Within each experiment, all treatment groups were tested for cytotoxicity in quadruplicate over an effector:target ratio range of 100:1 to 12.5:1 in the ^Cr release assay. Quantification of cyto toxicity in lytic units was calculated as described previously (Chapters II and III) and total lytic units from recovered cells after in vitro culture is recorded in the figure or table legends. Statisti cal analysis of data from repeated experiments was done by Students' t-test. These results are shown in tables where appropriate. 68 Results Since it had been found (Chapter 111,13,125) that intraperitoneal in jection of solubilized tumor membrane antigens induce, in the spleen, T suppressor cells specific for that tumor, an attempt was made to prepare from these cells relatively pure specific suppressor factor. The relative purification of the P815-specific suppressor factor was achieved by passing crude spleen extracts, from DBA/2J mice previously injected intraperitoneally with solubilized P815 tumor extract, over immunoadsorbent columns made up of membrane extracts of P815 cells, and subsequently eluting from the column the P815-specific suppressor factor with 2M NaCl. Figure 16a shows,, that when added to in vitro cultures, the P815-specifIc suppressor factor significantly inhibits the generation of cytotoxic cells to P815 tumor cells when.compared to untreated control cultures. The specificity of the P815-specific suppressor factor is shown by its inability to affect the generation of cyto toxic cells to L1210, another tumor syngeneic to DBA/2J mice (Fig. 16b) . The P815-specific suppressor factor was found to be relatively stable when stored at -70°C, and was active at very high dilutions. Figure 17 shows a typical titration curve for this suppressor factor. While the suppressor factor was very inhibitory at high dilutions, at high concentrations it was not, in fact sometimes it was some what stimulatory. The reason for this is not understood, but this was a reproducible observation. The titration curve (Figure 17) may possibly be accounted for by the presence of P815-specific helper 69 Figure 16. The ability of P815-speeific suppressor factor to suppress the in vitro primary cytotoxic response of normal DBA/2J splenocytes to syngeneic mitomycin C treated P815 tumor cells. ,(a) Anti-P815: the cytotoxic activity of cells primed in vitro with P815 cells on Cr labelled P815 cells in an 18 h assay. (b) Anti-L1210: the cytotoxic activity of cells primed in vitro with L1210 cells on Cr labelled L1210 cells in an 18 h assay. • •, primary culture of normal DBA/2J splenocytes and 1/150 dilution (0.0071. spleen equivalent) of P815-specific suppressor factor. Total lytic units: (a) 7.35 (b) 29.30. • • , primary culture of normal DBA/2J splenocytes, no suppressor factor added. Total lytic units (a) 31.32 (b) 27.17 70 -40 \-1:150 1:600 1:1200 DILUTION Figure 17. Titration of immunoadsorbent purified P815-specific sup pressor factor. Cytotoxic values were calculated at an effector:target ratio of 50:1 and converted to % sup pression by comparison with equivalent cultures incubated in the presence of a control suppressor factor isolated in an analogous fashion from L1210 primed mice. 71 factor which could co-purify with the suppressor material. However, other investigators, working with suppressor factor producing hybri-domas have also found that high concentrations of hybridoma super natants are sometimes much less suppressive than are higher dilutions (143), so this may not be the explanation. While the methods used for the purification of the P815-specific suppressor factor were carried out to maximize the purity of the factor, it is quite probable that the material eluted from P815 columns contained a number of contaminants. Because of the exceedingly small amounts of protein in our factor preparations (<0.01 absorbance at 280 nm), at this time it is difficult to establish its degree of purity by conventional biochemical means. However, it appears that this material has properties analogous to those described previously (Chapter 11,65,123) for a suppressor factor isolated by similar methods from thymocytes of P815-bearing mice (Chapter 11,65). Taken together, these results show that a very inhibitory and highly specific suppressor factor can be prepared from spleens of DBA/2J mice previously injected intraperitoneally with soluble tumor membrane extracts. Since we had a relatively pure, specific suppressor factor, a program was set up to immunize groups of both syngeneic DBA/2J and allogeneic C57BL/6 mice with P815-specific suppressor factor. The hope was that in the syngeneic mice the unique determinants (idiotypic?), found in or around the antigen binding site of the P815-specific suppressor factor, would elicit an antibody response in at least some of the animals. While both these and the constant 72 determinants on the suppressor factor might induce an antibody response in the allogeneic mice. Control groups of mice were injected with material (Balentl-"SF") prepared in an identical fashion (i.e. final dilution off of P815 membrane columns) from DBA/2J mice, injected intraperitoneally with Balentl tumor extracts. All the results reported here are from antisera pooled within each of the four groups. The fact that the DBA/2J and C57BL/6 antisera thus prepared contained anti-P815-SF activity was shown by their ability to absorb out the inhibitory activity of the P815-specific suppressor material. A representative set of data is shown in Table II. P815-specific suppressor factor was mixed with either the anti-P815-SF antisera or anti-Balentl-"SF" antisera, and subsequently absorbed on immuno adsorbent columns containing anti-mouse immunoglobulin. The unattached eluted material was then tested for suppressive activity. While suppressive activity was still present in the control anti-Balentl-"SF" antisera treated eluates, the suppressive material treated with anti-P815-SF antisera lost its activity, thus showing that both DBA/2J anti-P815-SF antisera and C57BL/56 anti-P815-SF antisera recognize and can bind determinants expressed on the P815-specific suppressor factor. This experiment was run on three separate occasions and, the differences were all similar and significant in each case. The ability of the anti-P815-SF antisera and not the control anti-Balentl-"SF" antisera to bind to determinants expressed on P815-SF is shown using a different method in Figure 18. The ELISA assay was used to show that both.the DBA/2 and the C57BL/6 anti-P815-SF antisera reacted positively with P815-SF, while neither of Table II. Anti-P815 antisera is able to react and bind to P815-specific suppressor factor. Suppressor factor preparations were tested after reaction with various mouse antisera and absorption on insolubilized rabbit anti-mouse Ig. P815-Specifica SF added P815-Specific SFb Absorbed With . . % Cytotoxicity , 100:1 50:1 Total Lytic6 Units Pf + DBA anti-P815-SF 53.4± 3.1 33.8 ± 2.6 36.4 N.S. + DBA anti-Balentl-"SF" 43.0± 3.1 23.5 ± 0.9 23.1 <.005 + C57BL/6 anti-P815-SF 74.8 ± 5.8 35.3 ± 2.2 39.7 N.S. + C57BL/6 anti-Balentl-"SF" 49.1± 1.9 20.9 ±1.1 25.8 <.005 + - 41.1±3.9 17.5 ±1.4 21.7 <.005 — — 56.9 ±0.9 36.7 ±1.3 39.6 lSF added at the start of 5 day in vitro cultures for the generation of cytotoxic cells as described in Materials and Methods. SF was treated as outlined in next column prior to addition. A 1/150 dilution (0.0071 spleen equivalent) of SF was used. 'SF was mixed with the antisera then passed through an anti-mouse Ig column. Material which passed through was tested for suppressive ability. Control SF preparations were only passed through anti-mouse Ig column then tested. Cytotoxicity was tested by 18 h ~^Cr release assay. Numbers are % specific release ± S.E.M. Effector:Target ratio. 4 One lytic unit was defined as the number of effector cells required to lyse 50% of 10 target cells. Total lytic units were the number of lytic units found in the recovered cell population. Data were analysed by Students' t-test. N.S. - not significant. P values of <.05 were considered significant. Table III. Results of Students' t-test run on 3 separate experiments testing 3 individual bleeds of mice immunized with P815-SF or control Balentl-"SF'^ Differences between groups were calculated from data obtained in Cr release assay at effector-target ratios of 50:1 (levels at which killing is in the linear part of the curve). All groups were compared to the killing in cultures containing suppressor cells which had been treated only with C. Treatment of Suppressor Cells Pa Expt. 1 Expt. 2 Expt. 3 DBA anti-P815-SF <.025 <.005 <.005 DBA anti-Balentl-"SF" N.S. N.S. N.S. C57BL/6 anti-P815-SF <.05 <.05 <.005 C57BL/6 anti-Balentl-"SF" ' <.025 N.S. N.S. P values of <.05 are considered significant. 75 o J3 0 M < 0.8 0.6 0.4 0.2 '/10 V40 Vo40 Antisera Dilution Figure 18. Characterization of P815-SF by ELISA assay. P815-SF (0.0023 spleen equivalent) was used as antigen in the ELISA. Various antisera were added to determine what characteristics were expressed on the P815-SF. •-—• , DBA/2 anti-P815-SF; o o, DBA/2 anti-Balentl-"SF"; • •, C57BL/6'anti-P815-SF, • •, C57BL/6 anti-Balentl-"SF", k 4, anti-H-2 ; A A, anti-la ; x x, anti-MIg. Incubation of plate after addition of enzyme substrate was for 45 minutes. 76 the anti-Balentl-"SF" antisera reacted above background levels. These results confirm the results of the absorbtion experiments (Table II). Another important feature to note is which of the other antisera preparations reacts with the P815-SF. While both anti-HZ^ and anti-la** antisera react with P815-SF, anti-mouse Ig antisera does not. This shows that P815-SF has determinants coded by the la region of the H-2 major histocompatibility complex, but does not have "common" mouse Ig constant region determinants. These properties are the same as those described previously for P815-specific sup pressor factor isolated from thymocytes of P815 bearing mice (Chapter II, 65,21). The possibility that the activity of the P815-specific T suppressor cells could be abrogated by anti-P815-SF antisera plus complement was investigated. Prior to addition to normal DBA/2J spleen cells plus mitomycin C treated P815 cells at the start of in  vitro culture, the specific T suppressor cell population was treated with complement plus either anti-P815-SF antisera or anti-Balentl-"SF" antisera. Both of the anti-P815-SF antisera produced in the syngeneic and allogeneic mice were effective in eliminating the sup pressive effect normally produced by these cells (Fig. 19). Again, as with the adsorption of the P815-specific suppressor factor, neither of the anti-Balentl-"SF" antisera controls had an effect. With all the bleeds tested the DBA/2J anti-P815-SF antisera and the C57BL/6 anti-P815-SF antisera were approximately equally effective (Table III). The data presented in Figure 19 is from experiment 2. Therefore, it appears that in this system the suppressor cell, its progenator, 77 1? 5 1 25:1 50-1 12.5:1 251 50:1 EFFECTOR:TARGET RATIO •Figure 19. The ability of anti-P815-SF antisera plus complement to eliminate P815-specific suppressor cells. (a) DBA/2J anti-SF:- the cytotoxic response of cells primed in vitro with P815 after treatment of the suppress ive splenocytes with DBA/2 anti-SF antisera plus complement. (b) C57BL/6 anti-SF:- the cytotoxic response of cells primed in vitro with P815 after treatment of the suppress ive splenocytes with C57BL/6 anti-SF antisera plus comple ment . • ^ , primary culture of normal DBA/2J splenocytes plus anti-P815-SF and complement treated P815-specific suppressor splenocytes. Total lytic units (a) 101.02 (b) 90.13 • • , primary culture of normal DBA/2J splenocytes plus anti-Balentl-"SF" and complement treated P815 specific suppressor splenocytes. Total lytic units (a) 63.19 (b) 52.25 0 0, primary culture of normal DBA/2J splenocytes plus complement only treated P815-spcific suppressor spleno cytes. Total lytic units (a) and (b) 57.54. 78 or a cell vital for its development, expresses on its surface, sup pressor factor, or at least determinants common to it. The possibility that in vitro generated syngeneic cells cyto toxic for P815 could also be killed by these antisera was also in vestigated. In vitro generated cytotoxic T cells specific for either P815 or L1210 were treated with complement plus either anti-P815-SF antisera or anti-Balentl-"SF" antisera. As can be seen in Table IV, neither the syngeneic DBA/2J anti-P815-SF antisera nor the allogeneic C57BL/6 anti-P815-SF antisera had any effect on the killing carried out by the cytotoxic T cells. Therefore, showing that in the syngeneic P815 tumor system of DBA/2J mice the specific cytotoxic T cells lack determinants in common with those found on either the specific T suppressor cells or the specific suppressor factor as detected by the anti-SF antisera. In repeats of these experiments, no significant differences were found between groups on any occasions. Finally the in vivo effect of the anti-P815-SF antisera on P815 tumor growth in DBA/2 mice was assessed. Two days prior to injection with P815 tumor cells, DBA/2 mice were injected intravenously with 50 yl of one of the four antisera preparations on PBS. Figure 20 shows the growth of the tumor after injection subcutaneously. As can be seen the DBA/2 anti-P815-SF antiserum greatly slowed the growth of the P815 tumor. The other antisera preparations had no effect on tumor growth. These results are also born-out in the improved survival time of animals injected with DBA/2 anti-P815-SF antisera (Table V). The mice injected with DBA/2 anti-P815-SF antisera had a 48% increased survival time compared to those injected with PBS 79 Table IV. Anti-P815-SF antisera plus complement (C') treatment has no effect on the ability of specific cytotoxic effector T cells to lyse the appropriate target. Effectors Generated Cells Treated Withb % Cytotoxicity0 pe Against Antisera C 100:ld 50:ld P815 Anti-Thy 1 + 6.6 ± 3.3 2.5 ± 1.4 <.005 DBA anti-P815-SF + 40.5 ± 2.5 28.5 ± 4.7 N.S. DBA anti-Balentl-"SF" + 36.9 ± 4.7 29.8 ± 3.0 N.S. C57BL/6 anti-P815-SF + 45.9 ± 4.8 32.4 + 3.5 N.S. C57BL/6 anti-Balentl-"SF" + 45.0 ± 4.0 32.2 ± 3.8 N.S. - + 44.0 ± 5.2 31.7 ± 3.1 - - 45.2 ± 3.8 34.9 ± 2.3 25:ld 12.5:ld L1210 Anti-Thy 1 + 9.8 ± 2.7 1.2 ± 0.8 <.005 DBA anti-P815-SF + 40.5 + 3.9 24.1 ± 1.6 N.S. DBA anti-Balentl-"SF" + 42.7 ± 2.3 26.6 ± 2.1 N.S. C57BL/6 anti-P815-SF + 39.2 + 4.1 27.7 ± 2.7 N.S. C57BL/6 anti-Balentl-"SF" + 47.9 ± 3.2 21.5 ± 3.2 N.S. - + 39.2 ± 3.8 27.2 ± 1.0 - 39.8 ± 0.8 26.2 ± 2.2 aEffectors were generated in vitro by mixing DBA/2J spleen cells with the appropriate mitomycin C treated tumor cells and incubating them for 5 days as described in the Materials and Methods. ^The effector cells were treated with antisera plus complement then washed and tested for their ability to lyse the appropriate ^lcrlabelled targets as described in Materials and Methods. Control effector populations were treated with only complement or media alone. c 51 . Cytotoxicity was tested by 18 h Cr release assay. Numbers are % specific release ± S.E.M. dEffector:Target ratio. eData were analysedby Students, t-test. N.S. - not significant. In three experiments, no significant differences were found between any of the anti-SF groups. 80 255 SIZE OF 195 TUMOR 135 75 -2 0 11 15 19 21 / \ antisera iv 2M03 DAYS P815 sc Figure 20. Effect of anti-P815-SF antisera in vivo on P815 tumor growth^in syngeneic DBA/2 mice. Mice were injected with 2 x .10 P815 subcutaneously on day 0. Two days prior 50 yl of antisera had been injected intraveneously. Tumor size was measured in two dimensions with calipers. This figure shows the combined results of three separate experiments with a total of 21 mice/group. A A, DBA/2 anti-P815-SF; • •, C57BL/6 anti-P815-SF; A A, DBA/2 anti-Balentl-"SF"; • • , C57BL/6 anti-Balentl-"SF"; • • , PBS. 81 Table V. Effect of anti-P815-SF antisera in vivo on survival time of DBA/2 mice after P815 ..... a tumor injection . DBA/2 mice injected i.v. with 10 mean survival time (days)c 20 30 40 DBA/2 anti-P815-SF anitsera DBA/2 anti-Balentl-"SF" antisera C57BL/6 anti-P815-SF antisera C57BL/6 anti-Balentl-"SF" antisera PBS 38.1 ± 4.4 p<.005 29.0 ± 2.3 N.S. 27.2 ± 3.5 N.S. 29.5 ± 2.4 N.S. 25.7 ± 2.5 *Three separate experiments were run and the results were combined for this table. "T)BA/2 mice were injected i.v. wij^h 50 ul of antisera two days prior to P815 tumor challenge. 2 x 10 P815 tumor cells were injected s.c. on day zero. "The mean survival time of^individual groups (21 mice/group) of mice after injection of 2 x 10 P815 tumor cells s.c. 82 alone. Therefore, even though both DBA anti-P815-SF antisera and C57BL/6 anti-P815-SF antisera were reactive in vitro only the syngeneic DBA/2 preparation shows any effect in vivo. Experiments were also carried out in vivo with the anfc.i-P815-SF antisera to assess its effect on L1210 tumor growth in DBA/2 mice. The results shown in Figure 21 and Table VI show that neither the syngeneic DBA/2 anti-P815-SF nor the C57BL/6 anti-P815-SF antisera had a statistically significant effect on L1210 tumor growth in vivo. Since the preparation of the P815-SF used to induce the ariti-P815-SF antisera entailed a final elution step off a P815 membrane: Sepharose column, there is a possibility that the P815-SF material could also contain small amounts of P815 antigen which was shed off the column along with the bound suppressor factor. It is there fore possible that the antisera may contain anti-P815 antigen activity, which could interfere with the interpretation of some of the results reported here. Two different experiments were set up to explore this possibility. The first is shown in Figure 22. Here the two anti-P815-SF antisera were tested for their ability to lyse "^Cr labeled P815 tumor cells in the presence of complement. As can be seen neither the DBA/2 anti-P815-SF antisera nor the C57BL/6 anti-P815-SF antisera had any anti-P815 lytic ability. The second experiment, to determine if the two anti-P815-SF antisera contain any anti-P815 activity, was to react these antisera with P815 membrane extracts in an ELISA assay. It can be seen in Figure 23 that both DBA/2 and C57BL/6 anti-P815-SF antiserum contain 83 -2 0 8 9 10 11 12 13 antisera iv 2x103 DAYS j P815sc , ! i Figure 21. Effect of anti-P815-SF antisera in vivo on L1210 tumor growth^in syngeneic DBA/2 mice. Mice were injected with 2 x 10 L1210 subcutaneously on day 0. Two days prior 50 yl of antisera had been injected intraveneously. Tumor size was measured in two dimensions with calipers. This figure shows the combined results of two separate experiments with a total of 18 mice/group. A A, DBA/2 anti-P815-SF; •- •, C57BL/6 anti-P815-SF; • -A, DBA/2 anti-Balentl-"SF"; • • , C57BL/6 anti-Balentl-"SF"; • • , PBS. 84 Table VI. Effect of anti-P815-SF antisera in vivo om' survival time of DBA/2 mice after L1210 tumor injection . *• b DBA/2 mice injected i.v. with DBA/2 anti-P815-SF antisera DBA/2 anti-Balentl-"SF" antisera C57BL/6 anti-P815-SF antisera C57BL/6 anti-Balentl-"SF" antisera PBS mean survival time (days) 5 10 15 20 17.4 ± 1.9 N.S. 16.7 ± 1.3 N.S. 15.7 + 1.5 N.S. 16.5 ± 2.2 N.S. 16.7 + 2.1 Two separate experiments were run and the results were combined for this table. 'DBA/2 mice were injected i.v. witb^ 50 yl of antisera two days prior to L1210 tumor challenge. 2 x 10 L1210 tumor cells were injected s.c. on day zero. The mean survival time of^individual groups (18 mice/group) of mice after injection of 2 x 10 L1210 tumor cells s.c. 85 80 60 40 20 Vt J/64 '/256 Dilution of Antisera Figure 22. The inability of anti-P^15-SF antisera, in the presence g£ complement, to lyse Cr labeled P815 tumor cells. Cr labeled P815 tumor cells were incubated with various dilutions of antisera plus complement anti-P815-SF; o H-2 ; • •, anti-la o,dC57BL/6 anti-P815-SF; DBA/2 Hi, anti-86 '4 '/20 '/80 '/320 '^280 I I Dilution of Antisera Figure 23. Does anti-SF antisera have any activity directed towards P815 tumor membrane determinants? Crude P815 membrane extract was used as antigen in the ELISA assay to determine if anti-SF antisera has any activity directed agasint P815. • • , DBA/2 anti-P815-SF, 0 o, C57BL/6 anti-P815-SF, • •, rabbit anti-P815; P—rabbit anti-DBA/2. Incubation of plates after addition of enzyme substrate was for 90 minutes. only a very small amount of anti-P815 reactivity. It should be noted that the ELISA assay is a very sensitive method of detecting antibody reactivity, with only very small amounts (as little as 50 ng/ml) of specific antibody necessary to show a positive reaction in a very short time (142). Therefore the amount of anti-P815 reactivity found in the anti-P815-SF antisera preparations is a very small percentage of the total reactivity and probably plays no role in any of the experiments reported here, especially the ones mediated through comple ment . 88 Discussion The results presented previously (Chapter II) show that intraperi toneal injection of solubilized P815 tumor membrane antigen into syn geneic DBA/2J mice induces, in the spleen, T suppressor cells specific for P815. It was shown in this chapter that a very inhibitory and relatively purified P815-specific suppressor factor could be prepared from these suppressive spleen cells. This P815-specific suppressor factor was injected into syngeneic DBA/2J and allogeneic C57BL/6 mice and the resulting antisera was shown to (a) interact with P815-specific suppressor factor by absorption studies and ELISA assays, (b) interact with P815-specific T suppressor cells by complement mediated killing studies, and (c) not to interact with P815-specific T cytotoxic cells by complement mediated killing studies, while (d) only the syngeneic antisera was shown to be effective in vivo. The results shown in Figure 16 indicate that a suppressor factor extracted from a suppressive cell population can duplicate the biol ogical function of these cells. In this case both the T suppressor cell and the suppressor factor presumed derived from it are able to specifically suppress the primary in vitro generation of cytotoxic T cells to P815 (8, Chapter II). These results plus those in Figure 18 show that this suppressor factor has properties analogous to those described previously for a suppressor factor isolated by similar methods from thymocytes of P815-bearing DBA/2 mice (9,21,65,123, Chapter II). It was then possible to use the P815-suppressor factor to prepare anti-P815-SF antisera. This was done by injecting both syngeneic 89 DBA/2 and allogeneic C57BL/6 mice with P815-SF. As Table II and Figures 18 and 19 show the resulting anti-P815-SF antisera could react with determinants expressed on the suppressor factor and the suppressor cell it is presumed derived from. The results shown in Table IV indicate that determinants expressed on P815 specific suppressor factor and P815-specific T suppressor cells are not ex pressed on P815-specific T cytotoxic cells. Since the preparation of the P815-SF used to induce the anti-P815-SF antisera entailed a final elution step off of a P815 membrane column, there is a possibility that the P815-SF material could also contain small amounts of P815 antigen which was shed off the column along with the bound suppressor factor. It is therefore possible that the antisera possibly contained anti-P815 antigen activity, which could interfere with the interpretation of some of the results reported here. The results shown in Figure 22 and 23 show that the amount of anti-P815 reactivity found in the anti-P815-SF antisera is at most a very small percentage of the total reactivity and probably plays no role in any of the experiments reported here, especially the ones mediated through complement. And of course, since the Balentl-"SF" was prepared in an identical fashion to P815-SF (i.e. final elution off of a P815 membrane column), the antisera prepared to it serves as an excellent internal control for these experiments. It should be restated here that at the concentration used in this work there was no detectable anti-normal DBA/2J activity found in any of the antisera preparations. Also it should be noted that the suppressor factor was prepared from DBA/2J cells primed in vivo, and during 90 preparation and prior to immunization into the appropriate syngeneic and allogeneic mice, was only suspended in PBS without fetal calf serum. Therefore, it is reasonable to assume that any antibodies being formed in immunized animals would be directed exclusively to antigens of DBA/2J origin. Thus, it seems probable, that the anti-P815-SF antisera from the syngeneic DBA/2J mice is directed to unique determinants (idiotypic?) expressed at or near the antigen binding site of the suppressor factor. The anti-P815-SF antisera from the allogeneic C57BL/6 mice would probably be directed to both receptor site determinants as well as the constant determinants. Similar conclusions were drawn by others using very similar protocols (26, 144-146). The observation that these antisera do not kill anti-P815 cyto toxic T cells but do kill P815-specific suppressor T cells, implies that these two cell types bear unrelated determinants. As mentioned in the introduction, it is very possible that the specific suppressor factors may be the receptors of specific suppressor T cells. There fore, since the antisera used contained anti-SF activity (Table II and Figure 18), it is possible that the determinants recognized on the suppressor T cells are associated with the receptor molecules of these cells. The fact that these antisera did not kill anti-P815 cytotoxic T cells implies the possibility that these two P815-specific T cell types may bear unrelated receptor determinants at least at the antigen binding site. Alternatively, the sensitivity of T cytotoxic cells to the anti sera plus complement treatment may reflect a lower density or dif-91 ferent arrangement of receptors on their surface when compared to T suppressor cells. It is becoming very apparent that differential activation of the various T cell subsets can be accomplished by varying the mode of immunization (30,125,147-152). It also seems there may be deter minants which selectively react with T helper cells and other deter minants which react with T suppressor•cells. This can be demonstrated most definitively with small chemically defined molecules in which various fragments of the whole molecule have been shown to selectively activate either T suppressor cells or T helper cells in certain strains of mice (20,153,157). Furthermore, Yamauchi et al., in experiments involving varying immunization protocols and blocking studies, have very recently shown that in one tumor system T sup pressor cells and T cytotoxic cells recognize different antigeneic determinants (125). In this chapter, these findings are extended by showng that antisera, from both syngeneic DBA/2J and allogeneic C57BL/6 mice, directed against P815-specific suppressor factor reacts with P815-specific T suppressor cells but not specific T cytotoxic cells to this same tumor. Finally the in. vivo effect of the anti-P815-SF antisera on P815 tumor growth in DBA/2 mice was assessed. As shown in Figure 20 and Table V only the DBA/2 antisera had any observable effect in vivo. This is in contrast to the in vitro work which showed that both the syngeneic DBA/2 anti-P815-SF and the allogeneic C57BL/6 anti-P815-SF antisera reacted with suppressor factor/cell determinants. The reason.for this is unknown. Although it may be related to the 92 fact that the allogeneic C57BL/6 anti-P815-SF antisera would likely react with many more determinants present on P815-SF and P815-specific suppressor T cells, than would the syngeneic DBA/2 anti-P815-SF antisera. Therefore, the allogeneic anti-P815-SF antisera may have much more complex activity in vivo than the syngeneic antisera. For example, the allogeneic anti-P815-SF antisera could possibly stimulate P815-specific suppressor T cell induction as well as inhibiting it in vivo. Thus, effectively "cancelling out" its effect in vivo. CHAPTER V SUMMARY DISCUSSION 94 CHAPTER V Summary Discussion The work reported in the preceeding three chapters involved studies of DBA/2 antigen-specific suppressor T cells and the antigen-specific suppressor factor derived from them. Both the suppressor cells and the suppressor factors specifically inhibit the in vitro generation of DBA/2 cytotoxic T cells for the syngeneic tumor, P815. In Chapter II P815-Tg and P815-SF were obtained from the thymuses of DBA/2 mice primed previously with P815 tumor cells subcutaneously. The experiments reported in that chapter showed that the P815-Tg and -SF both expressed Ia^ determinants and were not H-2 restricted in their ability to effectively suppress the in vitro response to P815 by radiation chimeras of a different H-2 haplotype. The P815-Tg used in experiments reported in Chapter III were prepared from the spleens of DBA/2 mice injected intraperitoneal^ with membrane fragments of the P815 tumor cell. Results in that chapter showed among other things that the DBA/2 P815-Tg expressed the cell surface + -  + phenotype of Lyt-1 2 , whereas the DBA/2 P815-TG was Lyt-1 2 . Chapter IV used P815-Te prepared as in Chapter III. P815-SF was then prepared from these suppressor T cells. Syngeneic DBA/2 and allo geneic C57BL/6 antisera was prepared against the P815-SF. Both of the anti-P815-SF antisera reacted in vitro with determinants expressed on the P815-SF and the P815-T , but did not react with the P815-T s' c generated in vitro. Only the DBA/2 anti-P815-SF antisera had any observable effect in vivo on P815 tumor growth in DBA/2 mice. 95 The studies of some of the genetic properties of the P815-T s in Chapter II indicate that this suppressor cell express la antigen on its surface. It was also shown that the P815-SF could be absorbed out with anti-la** antisera, and thus also expressed la coded deter minants. This last result was also shown in Chapter IV using a different method of preparing P815-SF. There the ELISA assay was used to show that P815-SF prepared from DBA/2 splenocytes expressed H-2** and more specifically la** coded determinants. In every system tested to date the antigen-specific Tg and the antigen-specific SF produced from it have been shown to express la determinants (59-61). Therefore, these results in the DBA/2-P815 system are not surprising and fit in nicely with the assumption that all antigen-specific suppressor T cells and their suppressive factors probably express determinants encoded within the I-J to IE/C region (59-61). Some of the immunogenetic requirements for the expression of P815-specific suppression were looked at in Chapter II. The results showed that the P815-T and the P815-SF derived from them, were s capable of suppressing the in vitro generation of cytotoxicity to P815 by histoincompatable cells. These results showed that the genes within the MHC do not have to be shared by the suppressor cell (or its factor) and its target. In the antigen-specific suppressor systems in which H-2 restriction has been addressed, the published results of others are somewhat equivocal. In most systems involving cell-mediated responses there has been no H-2 restriction found, at least for the first part of the systems activity (see below) (59-61). In one system studied by Moorhead of a antigen-specific SF which 96 suppresses contact sensitivity to DNP, it was found that the SF .. required homology at the K and/or D loci of the H-2 with the target cells for activity to be observed (79,80,97). The P815-SF clearly does not need this homology with its target to show suppressive activity. The work in Chapter III presents evidence of the Lyt phenotype of the cytotoxic T cell and the suppressor T cell in the P815 tumor system in DBA/2 mice. It was found that the primary in vitro generated DBA/2 P815-Tc were Lyt-1 2+. As was pointed out in the discussion in Chapter III, there may be a difference in the Lyt-1 antigen expression on primary vs. secondary syngeneic tumor-Tc in the DBA/2-P815 system (13,130), and also between syngeneic-T and allogeneic-^ in DBA/2 mice (13). Of course these results probably reflect a purely quantitative not qualitative difference, as all T cells probably express Lyt-1 antigens, although in varying amounts (131). Even though most antigen specific-Tg in other systems have been shown to express the surface phenotype of Lyt-1 2+ (114-118,120), it is not surprising that the DBA/2 P815-Tg were shown to be Lyt-l+2 . This is because there have been several reports in a variety of systems of antigen-specific Tg which are Lyt-l+2 (42-44, 120, 132,133). As was discussed in Chapter III, these cells are usually found to not be the actual effector cell for suppression but may "induce" other T cells to actively suppress (22,23,44,60,134-136). Thus, the Lyt-l+2 , Ia+, antigen-binding, H-2 unrestricted Tg^ -inducer is thought to produce a Ia+, antigen-binding, H-2 unrestricted SF, 97 which induces a second suppressor T cell, This Tg2 is Lyt-1 2-1", Ia+ anti-idiotypic, and H-2 restricted in activity. It produces an Ia+,anti-idiotypic, H-2 restricted SF2, which induces a third suppressor T cell, Tg3. Tg3 is Lyt-l~2t Ia+,. idiotype+, "final" effector cell, which may suppress in a non-specific fashion, possibly via an inter action with macrophage. Thus, it can be seen that both the P815-T and P815-SF from s the thymus or spleen of DBA/2 mice would fit nicely into the above mentioned model of suppression at the Tsx and SF^ stage. This may at first seem strange since the P815-Ts> which appears after intra peritoneal injection of P815 membrane fragments, does so about 4 days sooner than .the P815-Ts induced by P815 cells injected subcutaneously. One explanation for this may be that it takes several days for the antigen load produced by the subcutaneously injected live P815 to equal the amount injected directly via the intraperitoneal injection method. Thus the two P815-specific suppressor T cells may be identical and equivalent to the T ^- in other systems. It may be worth noting that 4-6 days after maximum suppression appears in the thymus, sup pression appears in the spleen and lymph nodes of tumor bearing mice (148). This may be an indication of the time it takes for the thymus P815-Tg to "induce" the full suppressor effector function in vivo. The 5 day culture period of the in vitro assay may also allow the "induction" of suppression through the influence of the Lyt-l+2" P815-T . s In the earlier studies of P815-T and P815-SF in DBA/2 mice the s suppressor cell and suppressor factor were obtained from the thymuses 98 of DBA/2 mice which had been primed previously with P815 tumor cells (123,124, Chapter II). The suppressive activity of these materials was assayed by their ability to specifically suppress the secondary in vitro cytotoxic response of primed DBA/2 splenocytes to mitomycin-C treated P815 cells. In the work reported in this thesis P815-T s and P815-SF, either prepared as above from thymocytes or from spleno cytes of DBA/2 mice which had been primed previously with P815 tumor membrane extract, could specifically inhibit the primary in vitro cytotoxic response of normal DBA/2 splenocytes to mitomycin-C treated P815. Therefore, the state (primed or virgin) of the cell population on which it acts appears to be unimportant for the P815-Tg or P815-SF activity. Since the number of P815-Tc is probably higher in the P815 primed DBA/2 spleen population than the normal DBA/2 spleen population (130), it would seem that the P815-Ts and P815-SF may act on the helper T cell population. Thus, inhibiting the T^ provided 2nd signal, which is probably necessary for the P815-Tc to differ entiate and become active effectors of cytotoxicity. While this study presents no evidence supporting this possibility, there has been good recent experimental evidence for SF effecting T^ activity directly (158). The results presented in Chapter IV show that anti-P815-SF antisera, raised both in syngeneic DBA/2 and allogeneic C57BL/6 mice, could absorb out the P815-SF. These antisera were also capable, in the presence of complement, of eliminating P815-T but not P815-T from s s DBA/2 mice. 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(1981) Biological properties of a purified antigen-specific suppressive glycoprotein. J. Supramol. Struct. _5:7. PUBLICATIONS Levy, J.G., T. Maier, and D.G. Kilburn. Further chara cterization of thymic suppressor cells and a factor that suppresses the generation of cells cytotoxic for a syngeneic tumor. J. Immunol., 122:766 (1979) Levy, J.G., D.G. Kilburn, and T. Maier. Regulation of the immune response to syngeneic tumor. j_n Regulation by T-Cells. Edited by D.G. Kilburn, J.G. Levy, and H.-S. Teh. pp. 131-153 (1979). Maier, T., J.G. Levy, and D.G. Kilburn. The Lyt phenotype of cells involved in the cytotoxic response to syngeneic tumor and of tumor specific suppressor cells. Cell. Immunol., 56:392 (1980). Maier, T., D.G. Kilburn, and J.G. Levy. Properties of syngeneic and allogeneic antisera raised to tumor-specific suppressor factor from DBA/2 mice. J. . Supramol. Struct. _5:52 (1981). Maier, T., D.G. Kilburn, and J.G. Levy. Properties of syngeneic and allogeneic antisera raised to tumor-specific suppressor factor from DBA/2 mice. Cancer Immunol. Immunotherapy (In Press). -M^i-e-Tv-I—ajrd-J-rGv---L-evy-;—-ffi—v-i-vo activity of syn^eneijc ^i^t^4^efi€4c-^t-+s-eru raised to-UmtQ-p-'Spocifie -s^ppe^m'-^a-ekQ-r—f-mtr-B-BA/2-- mice-;—(^u-bm44ie4— HoJtr, T • L<-*y • A«l-',- K>*M>r t-C-Ctc-W Uc\-tVo ,b-,~T. I^V.e^ ( T. 6-. U.uy ., K>; 6-. H, Ta^zrs, a^tL Wit o-£ v^o~> oclow<\ \ 

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