Suppressor cells induced by total lymphoid irradiation affect proliferation and lymphokine production of murine T helper cell clones

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In1 J. Radiolion Oncology Lkl. Phys.. Vol. 13, pp. 61-68

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Copyright 0 1987 Pergamon Journals Ltd.

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??Original Contribution

SUPPRESSOR CELLS INDUCED BY TOTAL LYMPHOID IRRADIATION AFFECT PROLIFERATION AND LYMPHOKINE PRODUCTION OF MURINE T HELPER CELL CLONES TIMOTHY

P. MATE, M.D.*

AND NANCY H. RUDDLE,

PH.D.-F

Yale University School of Medicine, 333 Cedar St., New Haven, CT 065 10, U.S.A. A key response to antigen is the activation of helper T cells to release lymphokines which stimulate and effect the immune reaction. This T cell population can secrete many different factors with diverse, often multifunctional roles, such as amplifying T or B cell antigen responses or being effecters of cell mediated delayed type hypersensitivity. Among these lymphokines are gamma-interferon (-y-IFN), interleukin-2 (K-2), or T cell growth factor, and lymphotoxin (LT) which has cytotoxic activity against a variety of cells. Immune suppression in mice following total lymphoid irradiation (TLI) has been correlated with the presence in lympho-reticular tissues of an antigen non-specific, null suppressor cell. This study examined what effects radiation induced suppressor cells had upon the in vitro activation and lymphokine responses of the ovalbumin (OVA) specific T helper cell clone, 153E6, following antigen presentation. Splenocytes from TLI treated mice obtained early in the post-irradiation period exerted a pan-inhibitory effect upon OVA induced 15336 proliferation and its concomitant release of y-IFN, LT, and IL-2. As the interval from irradiation increased, splenocytes from TLI treated mice showed persistent suppression of 15336 proliferation and -+IFN release, but had rapidly diminishing effects on the T cell’s capacity to produce LT and IL-2. These findings suggest that suppressor cells induced by TLI have a marked inhibitory effect in viva upon T helper cell proliferative responses to antigen and the production of various T helper cell lymphokines necessary to mediate the immune response. Such processes could contribute to the immunosuppressive effects of extensive nodal irradiation. TLI, Suppressor cells, T cells, Lymphokines. INTRODUCTION

Total lymphoid irradiation (TLI) is a radiotherapy technique used as a treatment for Hodgkin’s disease,’ various autoimmune disorders,‘2,25,27,30and as an immunosuppressive modality for allograft transplantation.‘3 Following such irradiation, host T cell immunity is markedly impaired. Manifestations of this T cell impairment include diminished cutaneous hypersensitivity to chemical haptens, an increased susceptibility to opportunistic bacterial and viral infections,’ subnormal antibody responses against T cell dependent antigens,29 and decreased in vitro lymphocyte responses.2*‘2930In addition to the absolute T cell lymphopenia that results from this irradiation, the helper (OKT-4, Leu-3) to suppressor (OKT-8, Leu-2) T cell ratio declines resulting in increased suppressor cell influences. ’2~’ 5,3o When splenocytes from mice which have undergone extensive nodal irradiation are co-cultured in vitro with normal splenocytes, significant inhibition of the prolifer-

ative response to mitogenic or allogeneic stimulation results.20,24.26This suppression is attributed to a morphologically distinct but phenotypically null lymphocyte population residing in the spleen and other reticuloendothelial tissues after irradiation. 14,24*34 The suppressive activity of this null lymphocyte has been shown to be neither antigen nor major histocompatibility antigen restricted. Several key immune processes are dependent upon activated T cell proliferation and secretion of immunoregulatory lymphokines. These are discrete macromolecues but each lymphokine may have multifunctional roles and a given immune process may need the concerted effects of two or more of these factors.’ Following antigen presentation, T helper cells simultaneously release interleukin-2 (IL-2) or T cell growth factor and express increased numbers of IL-2 receptors on their cell surlace.‘6 This serves to maintain self proliferation, but in addition, IL-2 release stimulates T cell clonal expansion.5 Gamma-interferon (y-IFN) released by activated T cells has

A preliminary report of these findings was presented at the Annual Meeting of ASTRO, 1985, Miami, Florida. * Dept. of Therapeutic Radiology. t Dept. of Epidemiology and Public Health.

Reprint requests to: Timothy P. Mate, M.D. This study was supported by NIH grants CA-09259 and PO 1 CA-29606. Accepted for publication 13 August 1986. 61

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been shown to enhance antigen presenting cell and cytotoxic T cell function,7,23,3’and furthermore, y-IFN plays an important role in host bacterial and viral immune defense.6s7%3’ T cell derived lymphokines are necessary in other aspects of the immune response. Several distinct T helper cell derived factors (i.e., BSFpl) have been described which promote activated B cell proliferation and maturation into immunoglobulin secreting plasma cells.” Cofactors in this process appear to include interleukin- 1, IL-2, and T-IFN. Lymphotoxin (LT) is another lymphokine produced by T cells, which has been shown to correlate with delayed type hypersensitivity and autoimmune disorders, and is probably important in mediating the effector phase of the immune response in its cytotoxicity towards a variety of cells.3~‘7~‘s~‘9 The critical roles played by these lymphokines in the immune response led us to question what effects radiation induced suppressor lymphocytes might have on the secretion of T helper cell factors. We used splenocytes from total nodal body irradiated mice which have been reported to contain antigen non-specific suppressor cells’4,32,34to examine in vitro how these splenocytes affect cloned murine T helper cell antigen induced proliferation and release of y-IFN, IL-2, and LT. METHODS

AND MATERIALS

Mice Female 8-12 week-old C57B1/6J mice were used for all experiments. Irradiation procedure

Mice were anesthesized with ketamine and diazepam and irradiated in a jig designed to expose the spleen and major nodal bearing regions, while shielding the head, lungs, heart, and extremities as described by Slavin et aL2’A 250 kV orthovoltage Xray machine was used to deliver 0.13 mm Cu filtered 250 kV Xrays (HVL 0.55 mm Cu) at a dose rate of 90.3 cGy/min., calibrated at the midplane of the mouse. Whole body irradiation was delivered in the same fashion but with all lead shielding removed. Five fractions of total lymphoid irradiation (TLI) at 200 cGy daily were given, followed by two additional doses of whole body irradiation at the same dose. This combination of total lymphoid and whole body irradiation (TLBI) was shown by Waer et a1.32to be as effective in inducing antigen non-specific suppressor cells as more protracted courses of TLI alone. Twenty to twenty-five mice were irradiated together as a group and several groups were consecutively irradiated over a period of 6 months. From each individual group, 3-8 mice were killed at 10,24,37, and 64 days after irradiation. Age matched mice served as controls. Preparation and maintenance of T cell clones

Mice were injected subcutaneously in the tail with 50 ug of ovalbumin (OVA) in complete Freund’s adjuvant

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and 8 days later the draining peri-aortic and inguinal lymph nodes were removed and cloned by the technique as modified from Srendi et aZ.22One clone, 153E6, was used in most of the experiments described below. The chicken gamma globulin (CGG) specific T cell clone, 82F 12, has been described previously.3 Both T cell clones expressed the membrane determinants Lyt- 1+ by microcytotoxicity testing with anti-Lyt- 1+ monoclonal antibody, and L3T4+ by indirect immunofluorescence staining with GKl.5 (anti L3T4+) monoclonal antibody, and the antigen specific proliferation of both clones could be inhibited by this GKl.5 antibody. These characteristics are typical for T helper cells.4,28 The T cell cloned lines were maintained in 24 well flatbottom plates in Click’s EHAA medium supplemented with 1% antibiotic/antimycotic (A/A), 50 ug/ml garamycin, 10% heat inactivated fetal bovine serum (HIFBS) and 25 U/ml of IL-2 prepared from phorbol myristic acetate (PMA) induced EL-4 cells. Once weekly T cells were fed x-irradiated (2000 cGy) syngeneic spleen cells and antigen. They were maintained at 37°C in a humidified incubator with 10% C02. All cell lines were tested and found to be mycoplasma free. For all experiments, resting T cells were used which had not been fed x-irradiated splenocytes for 7 days, nor IL-2 and Ag for 4 days. Splenocyte preparation Immediately following cervical dislocation, spleens were removed aseptically from irradiated or control mice. These organs were gently teased, and passed thru stainless steel mesh to yield single cells suspensions. Dead lymphocytes and RBC’s were removed by gradient centrifugation. Cell viability was determined by trypanblue exclusion. T cell proliferation assays

Proliferation was measured by a 3H-thymidine (3HTdR) incorporation assay. Briefly, 1 X lo5 T cells were co-cultured in triplicate wells of a 96 well plate with l-4 x 10’ normal splenocytes (NSC) or splenocytes from TLBI (TLBI-SC) treated mice. Antigen was added to a final concentration of 250 ug/ml, in a final culture volume of 250 ul. The culture medium contained RPM1 1640 with 25 mM HEPES and 25 mM glutamine supplemented with 1% A/A, 10% HI-FBS and 5 X lop5 mM 2-mercaptoethanol and maintained at 37°C in 5% C02. After 24 hours of incubation, one uCi of 3H-TdR (5 Ci/ mM) was added to each well, and the cultures harvested 24 hr later onto glass fiber filters using a multi-channel harvester. Two mls of scintillation fluid were added to each sample and the ‘H-TdR uptake by the cells was quantitated with a liquid scintillation counter. The net increase in counts per minute (cpm) was obtained by subtracting the cpm’s obtained from cultures containing spleen and T cells, but no antigen. Spleen or T cells alone, with or without antigen, produced insig-

TLI/T helper cells 0 T. P. MATE

nificant counts. The data were expressed as the arithmetic mean of triplicate cultures. The percent suppression was calculated as follows: % suppression = 1 _ net cpm of co-cultures with TLBI-SC X 100 . net cpm of co-cultures with NSC Induction of lymphokines Twenty-four or forty-eight hour supematants (SNs) of cultures containing 1 X lo5 153E6 T cells plus OVA (250 ug/ml) with 4 X lo5 TLBI-SC or NSC were used as the source of lymphokines in these experiments. Culture conditions were as in the proliferation assay, but without ‘H-TdR. Gener all y the contents of 3-5 wells were pooled, centrifuged to remove cells and stored at 4°C until analysis which was generally within 3 days of collection. IL-2 was assayed in the 24 hour SNs, whereas 48 hour SNs were used as the sources for LT and T-IFN.

AND

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y-IFN assay y-IFN activity in a test SN was measured in duplicate by its ability to protect L929 fibroblasts from the cytopathic effects of vesicular stomatitis virus as described by Conta et al3 Titers are expressed in international reference units (IU/ml) and are determined by comparing test SNs with a cloned murine recombinant y-IFN* previously calibrated with an NIH reference standard. L T assay Serial dilutions of test SNs were assayed for LT content by their ability to inhibit the proliferation of mouse L929 fibroblasts, as described by Ruddle.” After 72 hours of exposure, surviving fibroblasts are enumerated? and cytotoxicity calculated as follows: % cytotoxicity

= 1

number of L929 cells surviving test conditions number of L929 cells surviving control conditions

-

x 100. IL-2 preparation and assay EL-4 cells ( 1 X 106/ml) in flasks were induced with 10 ng/ml of PMA in RPM1 1640/ 10% HI-FBS/AA. Twenty-four hours later the SNs were harvested, centrifuged, filtered, and frozen at -20°C until needed. IL-2 content was determined by using an IL-2 dependent HT-2 cell proliferation assay, whose growth is exquisitely sensitive to the presence of this T cell growth factor.8 Briefly, test SNs were diluted 1: 1 with medium containing 1 X lo4 HT-2 cells per well, in triplicate, on 96 well plates in a final culture volume of 200 ul in RPM1 1640/10% HIFBS/AA. At 24 hours, the cultures are pulsed with 1 uCi of 3H-TdR (5 Ci/mM), and 18 hours later the proliferation was measured by 3H-thymidine incorporation into the DNA. The data are expressed as net cpm, that is, cpm with IL-2 containing SN minus cpm with medium alone. The arithmetic mean of triplicate cultures was used. The percent suppression could be calculated as follows:

=l-

change cpm with SNs from co-cultures containing NSC

% inhibition

=l-

LT units/ml from co-cultures containing TLBI-SC x 100. LT units/ml from co-cultures containing NSC

Statistics The paired Student’s t test was used to test for significant differences in data. RESULTS

% inhibition change cpm with SNs from co-cultures containing TLBI-SC

The units of LT activity are then determined using probit paper to graph the percentage of cytotoxicity vs log, serial dilution of the sample. One U of LT activity results in 50% inhibition of L929 growth. To calculate the percent LT or @W suppression, the following formula was applied, using LT suppression as an example:

x 100.

The amount of IL-2 contained in the test SN can be determined by comparing the net proliferation to a linear curve plotting the log, serial dilutions of an IL-2 containing standard (i.e., a 10% EL-4 SN) versus the net cpm of HT-2 proliferation induced by that IL-2 dilution. In the standard, 1 U of IL-2 is that amount which supports 50% maximal HT-2 cell proliferation in 24 hours.

*Courtesy of P. Gray, Genentech, Inc., S. San Francisco, CA.

Eflect of total lymphoid-body irradiation (TLBI) on splenocyte count Ten days after the completion of TLBI, the spleens of irradiated mice were severely depleted of nucleated cells. The spleens gradually became repopulated as shown in Figure 1, but even at 64 days post-irradiation the number of splenocytes present were still significantly below normal. TLBI splenocytes inhibit antigen induced T cell proliferation Splenocytes from TLBI treated mice 10 days after irradiation were tested for their ability to inhibit the prolifert 2Bl Coulter Counter, Hialeah, FL.

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Fig. 1. The number of nucleated cells present in the spleen at various intervals following 1400 cGy TLBI. Unirradiated mice had a mean splenocyte count of 5 1.6 f 8.2 X lo6 (100%). A minimum of 15 irradiated and control mice were assayed at each time interval indicated, and shown are the means -t standard errors of the mean (SEM).

ative response of 153E6 T cells to their specific antigen, OVA. Graded numbers of l-4 X 1O5 TLBI splenocytes containing putative suppressor cells were co-cultured in triplicate with 1 X lo5 153E6 T cells plus OVA. Identically prepared co-cultures with splenocytes from unirradiated mice of similar age served as controls. The data in Figure 2 which represent the mean of several (4-6) experiments show when 1 X lo5 splenocytes from TLBI treated mice 10 days after irradiation were co-cultured with 1 X lo5 responder 153E6 T cells, the

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Fig. 2. Effect on the proliferative response to OVA of 1 X lo5 153E6 T helper cells in co-culture with graded numbers (X 10’) of syngeneic normal spleen cells (NSC) or splenocytes from mice 10 days post-irradiation (TLBI-SC). The net proliferative responses were normalized (100%) to the proliferation of T cells and antigen with 1 X 1O5NSC (69,860 + 19,344 cpm). The means f (SEM) for 4-6 proliferation experiments is shown.

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net proliferative response to antigen was nearly identical to control co-cultures containing T cells, OVA and splenocytes from unirradiated mice. Antigen alone, in the absence of co-cultured TLBI or normal splenocytes, failed to induce 153E6 T cell proliferation, confirming the requirement for antigen presenting cells. As the ratio of TLBI splenocytes to responder T cells in co-culture increased a suppressive effect upon 153E6 T cell proliferation became evident. When the number of TLBI splenocytes in co-culture was 4 X 1O5 the OVA specific 153E6 T cell proliferative response was only 20% of the T cell response observed when T cells were co-cultured with a comparable number of normal splenocytes. In similar experiments employing 1 X lo5 CGG specific 82F12 T cells in co-culture with graded number of TLBI or normal splenocytes, a similar dose-dependent effect was evident as shown in Figure 3. Although the absolute and normalized proliferative responses of the CGG specific 82F12 T cell line were significantly different from OVA specific 153E6 T cells, the relative suppressive effects of a 4:l TLBI to responder T cell ratio were quite similar for both T cell lines. Temporal eflects qj’TLBI splenocytes to inhibit T cell proliferation To determine if the suppressive effects of TLBI splenocytes varied with the interval following irradiation, the capacity of TLBI splenocytes from mice at different times after irradiation to inhibit OVA induced proliferation of 153E6 T cells was assessed. In these experiments 4 X lo5 splenocytes from TLBI treated mice at 10, 24, 39, and 64 days post-irradiation were co-cultured with 1 X lo5 153E6 T cells plus OVA and the net proliferative responses of such cultures compared with those to which normal age-matched splenocytes have been added.

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Fig. 3. The dose dependent effect of co-cultured TLBI-SC on the net proliferative response of 1 X lo5 82Fl2 T helper cells to CGG. The culture conditions were as described in Figure 2. The mean net proliferative response of the 82F12 T cells cocultured with 1 X lo5 NSC to CGG was 35,370 + 9731 cpm (100%).

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Splenocytes from TLBI treated mice 10 days after irradiation inhibited antigen induced T cell proliferation by approximately 75% as shown in Figure 4. A similar degree of T cell growth suppression was exerted by TLBI splenocytes obtained from mice 24 days following irradiation. As the interval between irradiation and assay increased, the inhibitory effects of TLBI splenocytes upon 153E6 T cell proliferation decreased, and at the 64th post-irradiation day there were no apparent suppressive effects.

The efects of TLBI splenocytes upon 153E6 T cell secretion of y-IFN, L T and IL-2 To determine whether TLBI splenocytes would affect the production of lymphokines from 156E6 T cells, cell free supernatants of 24 or 48 hour cultures containing 1 X lo5 153E6 T cells plus OVA with 4 X lo5 normal age matched control splenocytes or TLBI splenocytes were assayed for the amount of IL-2 (24 hr SNs) or y-IFN and LT (48 hr SNs) present. These cultures were identical to (but without 3H-TdR) and done concurrently with those experiments testing the effects of TLBI splenocytes at various post-irradiation intervals on 15386 T cell proliferation to antigen. Control (NSC) and test (TLBI) SNs from each experiment were assayed simultaneously for lymphokine (yIFN, LT or IL-2) activity within 3 days of collection. Control supernatants from cultures with 1 X lo5 T ceils, OVA and 4 X 1O5normal aged matched splenocytes contained a mean y-IFN activity of 12,240 +- 3340 units. These control supernatants also contained an average of 63.5 + 13.0 and 9.7 +- 1.2 units of LT and IL-2 activity, respectively. In the range of mouse ages used (8-15 weeks), splenocytes from different age unirradiated mice did not affect the lymphokine yield.

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Fig. 4. The capacity of 4 X lo5 co-cultured TLBI-SC at various intervals post-irradiation to suppress the proliferative response of 1 X lo5 153E6 T cells to OVA. The net proliferative response to OVA of T cells co-cultured with 4 X lo5 NSC was 82,227 + 4949 cpm, and was taken as normal (0% suppression). The mean + SEM of 4-6 experiments is shown.

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Fig. 5. Effects on the secretion of -/-IFN from 1 X 10’ OVA stimulated I53E6 T cells when co-cultured with 4 X lo5 TLBISC from mice at various intervals post-irradiation. Control supernatants from co-cultures containing the same number of T cells, OVA and 4 X IO5 NSC contained a mean -r-IFN activity of 12,240 f 3440 units, and was taken as normal (0% suppression). The mean + SEM suppression in T-IFN response in the presence of TLBI-SC for 4 experiments is given.

The production of IL-2, y-IFN, and LT by 153E6 resting T cells alone in culture without splenocytes plus or minus antigen was negligible, as was the secretion of these lymphokines by normal or TLBI splenocytes alone in culture. Therefore, we believe that the lymphokines contained in the co-culture supematants were derived predominantly from the antigen activated cloned T cells. For each experiment the amount of IL-2, r-IFN, or LT secreted by 153E6 T cells in co-culture with agematched splenocytes from unirradiated mice and antigen was taken as normal (0% suppression) and compared to the level of lymphokines produced by 153E6 T cells in co-culture with TLBI splenocytes. Splenocytes from TLBI treated mice 10 days following irradiation significantly impaired the secretion of -/-IFN by OVA stimulated 153E6 T cells as shown in Figure 5. This inhibitory effect decreased with time, but even at 64 days after irradiation splenocytes from TLBI treated mice could still exert a notable suppressive effect upon the secretion of r-IFN by 153E6 T cells. The ability of TLBI splenocytes at the 10th post-irradiation day to inhibit OVA induced 153E6 T cell LT and IL-2 secretion was also significant as shown in Figures 6 and 7. However at the 24th post-irradiation day and beyond, splenocytes from TLBI treated mice appeared to have little or no inhibitory effects upon the capacity of 153E6 T cells to secrete LT or IL-2. These latter findings appeared to be quite different from the pattern of consistent inhibition exerted by TLBI splenocytes upon antigen induced T cell proliferation and -/-IFN secretion. DISCUSSION A key response to antigen is the activation of helper T cells to release lymphokines which mediate the immune

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Fig. 6. The effects on LT secretion from OVA stimulated 153E6 T cells in co-culture with TLBI-SC. The average number of LT units/ml contained in control culture supernatants (described in Fig. 5) was 63.5 + 13.0 (0% suppression). Shown is the mean * SEM suppression in LT secretion from 153E6 T cells in the presence of TLBI-SC.

response by stimulating T or B cell growth and differentiation or being effecters of cell mediated immuSuch T helper cell derived lymphokines nity. ‘,5.7.10,‘8,3’ include interleukin-2, gamma interferon, lymphotoxin, and various B cell growth factors such as BSFp 1. When mice are subjected to extensive nodal irradiation a significant suppression of T cell mediated immunity results.2’,26This in part can be attributed to the appearance of an antigen non-specific suppressor ce11’4,20,24,26,34 arising in lympho-reticular tissues following irradiation. The mechanisms by which these radiation induced suppressor cells exert their inhibitory effects on target regulatory and effector T cells responsible for mediating the immune response have not been fully characterized. The object of this study was to investigate in mice how antigen stimulated proliferative responses and the secretion of helper and effector factors by cloned T helper cells might be altered in vitro by splenocytes containing putative suppressor cells induced by a combination of total nodal and whole body irradiation (TLBI). The results reported here show that splenocytes from C57B l/65 mice treated with TLBI inhibited the proliferative responses of two different antigen specific cloned murine T helper cell (L3T4+) lines, 82F12 and 153E6. The ability of TLBI splenocytes to suppress 15386 T cell OVA induced proliferation was most marked in the first l-3 weeks following irradiation, then gradually disappeared as the interval between irradiation and testing increased. The degree, time course, and antigen non-specific nature of this cloned T helper cell inhibition is quite similar to the previously characterized suppressive effects TLI or TLBI splenocytes have upon mitogenic and allogeneic induced proliferative responses of non-purified lymphocyte or splenocyte populations. “*20*26,32 In addition, our proliferation studies appear to provide

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indirect evidence that TLBI treated spleens contain cells capable of presenting antigen to T cells. Experiments by others 14,20*26,34 using lectin and allogeneic test systems, included normal bulk spleen populations as responders in which antigen presenting cells would have been included. From these latter experiments, one cannot determine if TLI treated spleens per se have antigen presenting capability. Our studies reported herein show that cultures containing 1 X 1O5cloned T cells and antigen with either 1 X lo5 normal or TLBI splenocytes are able to generate approximately equivalent proliferative responses (Figs. 2 and 3). Since the T cell clones used in our experiments have an obligatory antigen presenting cell requirement, we interpret these findings as evidence that TLBI spleens contain cells capable of presenting antigen to T cells. As the ratio of normal splenocytes in culture with T cells increased, the net 153E6 and 82F12 proliferative responses increased, suggesting enhanced antigen presentation. In contrast, as the ratio of TLBI splenocytes in co-culture increased the net 153E6 proliferative response clearly decreased in both absolute cpm and relative percent. Similarly, the 82F12 response was not enhanced. Assuming net proliferation is a balance between antigen presentation and suppression, we attribute these results to a net increase in suppressor cell activity, as the addition of TLBI splenocytes which contain only antigen presenting cells without the concommitant addition of suppressor cells would have been expected to enhance the proliferative response. It should be emphasized that other investigators have previously characterized and propagated natural suppressor cells from the spleens of TLI treated mice.14~20~24~26~34 In those studies with allogeneic or mitogen induced test systems, greater inhibition of proliferation was observed as the ratio of TLI splenocytes to responder cells in co-culture increased.‘4,20,26*34

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Fig. 7. Effects of TLBI-SC on the secretion of IL-2 from OVA stimulated 153E6 T cells. Control supematants (described in Fig. 5) contained an average of 9.7 f 1.2 units of IL-2 activity (0% suppression). The mean + SEM suppression in IL-2 secretion from 153E6 T cells in co-culture with TLBI-SC is given.

TLI/T helper cells 0 T. P. MATE AND N. H. RUDDLE

Our results are consistent with these observations, and therefore we conclude that the dose dependent difference in proliferation between cultures containing normal or TLBI splenocytes with cloned T cells and antigen, are due primarily to the presence of suppressor cells within the latter splenocyte population, and not due to limitations in antigen presenting cell function. Furthermore, our results also show that following TLBI, splenocytes from irradiated mice can exert a significant inhibitory effect in vitro upon the capacity of cloned 153E6 T helper cells to secrete -y-IFN, LT, and IL-2 in response to ovalbumin challenge. These in vitro findings suggest that suppressor cells induced by extensive nodal irradiation could influence T helper cell function in vivo by impairing T helper lymphocyte proliferative responses to antigen and down regulating the flow of r-II% and IL-2 necessary to recruit other T and B cells into the immune response, as well as inhibit the release of LT capable of mediating cytotoxicity. Such processes we believe could contribute to the state of depressed T cell mediated immunity found after extensive nodal irradiation,9,29 and especially the increased susceptibility to Herpes Zoster infections.6*7,9 Waldor et al. reported that the administration of GK 1.5 monoclonal antibody, which recognizes the L3T4+ determinant on murine T helper cells could reverse experimental allergic encephalomyelitis in mice.33 In a similar study, Wofsy and Seaman successfully treated autoimmunity in NZB/NZW mice, which is an animal model for systemic lupus, by the administration of this

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anti-L3T4+ monoclonal antibody.35 These studies provide evidence that unregulated T helper cell function may be the basis of certain autoimmune disorders. Autoimmunity in NZB/NZW mice can be prevented by TLI” and extended nodal irradiation has also been reported by Strober et a1.25 to be useful in the treatment of intractable lupus nephritis in man. In view of our findings, the success of extensive nodal irradiation as a treatment for lupus and other autoimmune disorders such as rheumatoid arthritis’2,25,27,30may be in part caused by the inhibition of T cell proliferation and their release of immunoregulatory and cytotoxic lymphokines. It was observed that TLBI splenocytes could suppress T cell proliferative responses to antigen for a prolonged period following irradiation (Fig. 4). The concommitant secretion +#N was also impaired for a long period, however there was a much earlier loss in the inhibition of T cell IL-2 and LT secretion (Figs. 5,6,7). These findings suggest that suppressor cells can affect antigen induced T cell proliferation and the production of its lymphokine repotoire to different degrees. The basis for these observations are not yet clear, but there have been suggestions in previous publications3 that non-co-ordinate production of T cell lymphokines can occur. Experiments which will analyze the effects of clonally derived suppressor cells or their factors upon the regulation of T cell’s lymphokine mRNAs will further ellucidate these mechanisms. Information obtained from such studies will add to our understanding of the molecular processes responsible for radiation induced immune suppression.

REFERENCES 1. Altman, A., Katz, D.H.: The biology of monoclonal lymphokines secreted by T cell lines and hybridomas. Adv. Immunol. 33: 73- 166,1982. 2. Blomgren, H., Baral, E., Jarstrand, C., Petrini, B., Strender, L., Wallgren, A., Wasserman, J.: Effects of external radiation therapy on the peripheral lymphocyte popuEflects of Radiation lation. In Immunopharmocologic Therapy, J.B. Dubois, B. Serrou, C. Rosenfield (Eds.). New York, NY, Raven Press. 198 1, pp. 299-3 19. 3. Conta, B.S., Powell, M.B., Ruddle, N.H.: Production of lymphotoxin, UN-gamma and IFN-alpha and beta by murine T cell lines and clones. J. Zmmunol. 130: 223 l-2235, 1983. 4. Dialynas, D.P., Quan, Z.S., Wall, F.A., Pierres, A., Quin-

tans, M., Loken, R., Pierres, M., Fitch, F.W.: Characterization of the murine T cell surface molecule designated L3T4, identified by monoclonal antibody GK1.5: Similarity of L3T4 to the human Leu-3/T4 molecule. J. Zmmunol. 131: 2445-245

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