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Suppression of a thymus dependent humoral response in mice by concanavalin A in vivo
CELLULAR
IMMUNOLOGY
18, 365-374 (1975)
Suppression of a Thymus
Dependent
by Concanavalin
Humoral
Response in Mice
A in Vivol
HARWOOD S. EGAN AND RICHARD D. EKSTEDT Department
of Microbiology,
Northwestern University-McGaw Chicago, Illinois 60611 Received February
Medical
Center,
19,1975
Mice treated with Concanavalin A prior to immunization with sheep erthyrocytes exhibit a markedly reduced plaque forming spleen cell response. This immunosuppressive effect could be reversed by using higher doses of antigen or priming the animals with nonimmunizing doses of antigen prior to Concanavalin A injection designed to either by-pass or enhance thymus derived lymphocyte functions. It was also demonstrated that Concanavalin A in vivo activated the thymus derived lymphocyte subpopulation in the spleen, and this activation was dose dependent and correlated with the immunosuppression observed. Animals injected with Concanavalin A at various times prior to whole body lethal irradiation would not support the plaque forming cell response of adoptively transferred normal syngeneic spleen cells. This effect was shown to be time and dose of Concanavalin A dependent. It was also shown that the route of injection of Concanavalin A prior to irradiation determined the results observed, in that the intravenous route resulted in the suppression of transferred cells, while the intraperitoneal route showed no effect. It is suggested that Concanavalin A induced immunosuppression of the humoral, thymus dependent immune response in mice results for the activation of a subpopulation of thymus derived suppressors cells, and that the effect is short lived, radiation resistant, and dose of Concanavalin A and antigen dependent. Previous studies in this laboratory have shown that mice injected intraperitoneally (ip) 2 or intravenously (iv) with 500 rg of Concanavalin A (Con A) 24-48 hr prior to immunization with lo8 sheep erythrocytes (SRBC), exhibited a markedly reduced direct (IgM) or indirect (IgG) plaque forming spleen cell (PFC) primary response (1). It was reported in the same paper, that the secondary response was also suppressed in mice injected with Con A 2 days prior to the second injection of antigen 7, 10, or 21 days after the first injection, but not when Con A was injected 2 days prior to the first injection of antigen. In the latter case, an enhancement of both the IgM and IgG response was observed, suggesting that Con A treatment was affecting an early event in the antibody response, possibly at the T cell recognition step, but did not appear to affect the priming of the animals to make a secondary response. Other parameters of timing, dose of Con A 1 This work was supported in part by United States Public Health Service Grants AI07716, RR-05370, and by Contract N00014-67-A-0356-0019 from the Office of Naval Research. 2 Abbreviations : ip, intraperitoneal; iv, intravenous ; Con A, Concanavalin A; PFC, plaque forming cells; T cell, thymus derived lymphocyte; B cell, bone marrow derived lymphocyte; RPMI, Rosewell Park Memorial Institute ; SRBC, sheep erythrocytes. 365 1975 by Academic Press, Copyright All rights o8 reproduction in any form
366
EGAN
AND
EKSTEDT
and antigen, routes of Con A treatment and immunization, and T-cell dependency of the antigen used, were also considered (1). In this paper, we present the results of further studies designed to elucidate the mechanism of immunosuppression in Con A treated mice to a thymus dependent antigen. These include attempts to overcome the suppression by using higher doses of antigen (2) or priming the animals with subthreshold doses of antigen to produce “educated” T-cells (3). In addition, attempts were made to correlate the in viva mitogenecity of Con A with its immunosuppression and to study the PFC response of normal syngeneic spleen cells in Con A treated and irradiated mice. The results were consistent with the hypothesis that Con A functions in viva in suppressing the antibody response of mice to a thymus dependent antigen by activating a subpopulation of suppressor T-cells and that the effect was radiation resistant, short lived, and dose of Con A dependent. MATERIALS
AND METHODS
Animals. The mice used in these experiments were adult (6-8 wk) BALB/c males, which were received at weekly intervals from Jackson Laboratories, Bar Harbor, Maine. They were housed ten per cage in air-conditioned animal quarters and allowed food (Purina mouse chow) and water ad lib. In some experiments CE7 B1/6J mice obtained from the same source were used. Previous experiments had shown that this strain of mice responded identically in every respect in this system. Concanavalin A. The Con A used in these studies was prepared in this laboratory from Jack bean meal (Nutritional Biochemicals, Cleveland, Ohio) by the method of Agrawal and Goldstein (4), and was comparable in purity to preparations (Grade IV) obtained from Sigma Chemical Co., St. Louis, MO. A single preparation, stored in 1.0 M NaCl at -2O”C, and diluted appropriately just prior to use, was used in all the experiments reported here. Spleen cell preparations and assays. The methods used for preparing single spleen cell suspensions and assaying for antibody forming cells by the micromodification of the Jerne localized ,hemolysis-in-gel technique are described in the literature (5) or in our previous publication (1). Cell cultures and [3H] thymidine uptake. Spleen cell suspensions (5 x 106/ml) from Con A treated and untreated mice in RPM1 1640 tissue culture medium (Grand Island Biological Co., Grand Island, NY) containing L-glutamine and supplemented with 5% (v/v) heat inactivated (56”C/30 min) fresh human serum from the same donor, and 100 units of a streptomycin-penicillin solution (GIBCO) were dispensed (3 ml) into 13 X 100 mm culture tubes (Falcon Co., Oxnard, Calif.) in triplicate. To test the in viva mitogenic activation of the spleen cells by Con A, 1 $.Zi [3H]thymidine/culture (sp act 1.9 CiJmmole, Schwartz-Mann, Orangeburg, New Jersey) was added at the beginning of the culture period, allowed to label for 24 hr, and terminated. The methods for harvesting the cultures and determining the incorporation of radioactivity into the acid insdluble fraction by scintillation counting has been described (6). Con A treatment, irradiation, cell transfer, and immunization. Following the procedure of Takahashi et al. (7)) recipient BALB/c mice, treated previously with 500 pg Con A iv, or normal mice, were exposed to whole body irradiation delivered from a Picker machine at 270 kV, 15 mA, with 1.3 mm copper and 1.0 mm
IMMUNOSUPPRESSION
TABLE
367
BY CON A
1
EFFECT OF ANTIGEN DOSE ON THE DIRECT PFC RESPONSE OF MICE TO SRBC FOLLOWING TREATMENT WITH Con A
PFC/lOG spleen cellsa
Treatmenta
725 f 80 f 772 f 407 f
10s SRBC Con A + lo* SRBC 109 SRBC Con A + lo* SRBC
63 12 50 78
0 Animals were injected iv with SRBC antigen alone or 24 hr after treatment with 500 fig Con A iv. b PFC response was measured 4 days following immunization. The results are expressed as the mean response f SEM of 15 mice in each group assayed individually.
aluminum filtration. With a source to target distance of 50 cm and at an exposure of 87.4 R/min, a total of 600 R was administered. Within 3 hr of irradiation, the mice were injected with 5 x lo7 lymphocytes from normal syngeneic mice and lo8 SRBC. The spleen cells from these animals were then assayed 7 days later for direct PFC. The effect of different doses of Con A given iv 24 hr prior to irradiation was also studied in similar experiments. RESULTS Reversal of the Immunosuppressive
Activity
of Con A In Vivo
Higher antigenic doses. Since our original hypothesis concerning the mode of action of Con A in viva was that it was in some manner affecting the interaction of T-cells with antigen, several experiments were done in attempts to enhance the antibody response of Con A treated mice to SRBC by altering this interaction. Sinclair (2) reported that large doses of SRBC obviated the need for helper T-cell function, and in effect were thymic independent. The results presented in Table 1 show that injecting a tenfold higher dose of SRBC into mice injected iv 24 hr ‘earlier with 500 pg Con A, significantly increased their PFC response. Animals receiving log SRBC without prior treatment with Con A did not give a greater PFC response than those given lo8 SRBC alone, since the latter dose has been shown to be optimal in this system (2). Priwzed mice. Golub (3) reported that priming mice with subthreshold doses of SRBC antigen at some time prior to an adequate immunizing dose, resulted in both an accelerated and enhanced primary (IgM) PFC response. This effect has been attributed to an increase in the number of responding “educated” T-cells that would encounter the antigen after the second injection. It was reasoned that in our system, if Con A were actually reducing the number of responding T-cells prior to SRBC injection, then increasing their number might reduce the suppressive effect of Con A. Table 2 shows the results of these experiments. Con A treated mice primed with lo5 SRBC 5 days prior to an immunizing dose of lo8 SRBC, displayed a significant increase in the PFC response as compared to Con A treated unprimed animals. Although the response of primed animals treated with Con A did not reach that of the group primed with antigen but not treated with Con A, it seems clear that under the conditions of these experiments, prior exposure of mice to a
368
EGAN AND EKSTEDT TABLE
2
EFFECT OF Con A ON THE DIKECT PFC RESPONSE OF PRIMED MICE TO SRBC Treatment0
105 SRBC 106 SRBC
PFC/lOB
lo* SRBC + lOa SRBC Con A + lo8 SRBC + Con A + lo8 SRBC
spleen cells”
117 & 26 2938 f 537 10% 5 800 f 178
n Mice were primed with 106 SRBC given ip 5 days before iv injection of lo8 SRBC immunizing dose. 500 pg of Con A was injected iv 24 hr prior to the immunizing injection. b 15 Animals in each group were assayed individually 3 days after the immunizing injection, and the results are expressed as in Table 1. 105 SRBC injected alone resulted in no detectable increase in PFC response above background.
subthreshold dose of antigen does significantly reduce the effect of subsequent treatment with Con A upon the response to an immunizing dose of SRBC. It should also be made clear that in these experiments, the animals injected with 10’ SRBC alone show a lower PFC response than a similar group shown in Table 1, since in this case, the PFC assays were done 3 days after immunization, when the response is just beginning to appear in unprimed animals (1). Efect of CON A In Vivo on Other Parameters of Immune Responsiveness While the above experiments suggested strongly that a T-cell function in viva was being affected by Con A, it was considered important to determine what effect injection of Con A was exerting upon the other known cellular activities important in eliciting an immune response. Since it is well recognized that Con A activates T-cells in vitro to blast transformation and mitosis, it seemed worthwhile to test whether Con A in viva also resulted in activation of T-cells. If this were the case, such activated cells might be refractory, for a period of time, to antigenic stimulation. Accordingly, groups of mice were injected iv with various doses of Con A. TwentyTABLE
3
INCORPORATION OF [~HITHYMIDINE BY SPLEEN CELLS FROM MICE TREATED WITH VARIOUS DOSES OF Con A PRIOR TO CELL CULTURING [aH]thymidine incorporation mean counts per minute (cpm)” f SEM Uninjected controls Con A 5 pg Con A 125 rg Con A 500 rg
1500 2600 5500 12,500
+ 400 f 400 f 500 f 3500
PFC response (% of control) b
100 86 f 16 62 f 24 16 f 12
0 Spleen cell cultures were set up as described in Materials and Methods 24 hr after the iv injection of Con A. Cultures were set up in triplicate and the counts per minute averaged in each case. Results from at least four experiments are expressed as Mean f SEM. “Four days following immunization with SRBC, spleens from other mice were assayed for direct PFC. Where indicated, Con A was injected iv 24 hr prior to immunization. For each experimental group, 15 animals were assayed individually and the results are expressed as the percent of the response observed with animals immunized but not treated with Con A.
~MMUN~SUPPRESSI~N
-4 -I Con A Injection-
369
BY coiv A
-7 COnfrOl Days Before Irradiation
FIG. 1. The effect of varying the time interval after Con A injection and prior to irradiation on the PFC response of adoptively transferred lymphocytes to SRBC. 500 pg of Con A was injected iv at the days indicated prior to 600 R of total body irradiation. 5 X lo7 Normal syngeneic spleen cells along with 108 SRBC were injected iv within 3 hr. Results represent the mean -C SEM from nine mice per point assayed individually for direct PFC response 7 days after transfer and immunization.
four hours later they were killed, their spleens removed, prepared, and cultured in the presence of [ 3H] thymidine for an additional 24 hr as described. Table 3 shows the results of these experiments, where it can be seen that there was a direct correlation between the amount of Con A injected and the degree of activation of spleen cells. Also shown in the table is the degree of suppression of PFC response in animals so treated, which also correlated with the dose of Con A. In other experiments not shown (8) treatment of the spleen cell suspensions with anti-theta or anti-thymocyte serum and complement prior to culture confirmed that the cells undergoing activation were of the T cell line. Allessandro et al. (9) have recently demonstrated a similar in &JO activation of spleen cells to develop cytotoxicity, another T cell mediated response, in mice injected with Con A. In view of these results, and with the recent evidence for the existence of a subpopulation of suppressor T cells which function to regulate the immune responses (10, ll), the possibility that Con A in tivo was activating a suppressor rather than inuctivating a helper T cell population was considered. It has also been suggested recently, by the results of Veit and Michael (12), that a soluble suppressor factor, present in the serum of normal and immunized mice, and presumably synthesized by an appropriate T cell subpopulation, mediates this effect, and attempts to characterize this factor have been reported (13). Adoptive Imwaune Response in Con A Treated-Irradiated
Mice
In order to gain some further evidence that a suppressor T-cell or soluble mediator might be involved in the immunosuppressive activity of Con A in vivo, the following experiments were done. Mice were injected iv with 500 pg of Con A and at different times thereafter subjected to 6OOR whole body X-irradiation. They were then injected with 5 X lo7 syngeneic normal spleen cells and lOa SRBC, iv within 3 hr, and assayed for PFC response 7 days later. It was reasoned that if the Con A activated a T-cell suppressor population of lymphocytes, which elaborated a soluble suppressor factor, this latter material should be radiation resistant and still capable of inhibiting normal transferred cells. It has also been reported recently
370
EGAN
AND
EKSTEDT
ae 0 5 125 500 pg Con A Injected I Day Prior to Irradiation FIG. 2. Effect of different doses of Con A, given iv 1 day prior to irradiation, on the subsequent PFC response of adoptively transferred spleen cells. The same procedure as in Fig. 1 was used, and the results represent the mean k SEM from nine mice per point.
by Peavy and Pierce (14) that suppressor T-cells are highly resistant to irradiation. If, on the other hand, a T-cell helper population of lymphocytes was being affected by Con A in viva, repopulation of these animals with normal lymphocytes should allow an immune response to SRBC comparable to control animals which had been irradiated and repopulated, but not treated with Con A prior to irradiation, since helper cell function is radiation sensitive (15). The results of these experiments are shown in Fig. 1, where it is clear that the immune response of normal transferred cells was markedly and consistently suppressed in mice irradiated 24 hr after treatment with Con A. As the interval between Con A treatment and irradiation was extended, the effect became progressively less. In addition, Fig. 2 shows that decreasing the dose of Con A given 24 hr prior to irradiation also reduces the suppression of an adoptive immune response to SRBC. In a further series of experiments similar to those described above, the effect of injecting the Con A by the ip route was compared with that observed when the Con A was given iv. In our earlier studies (1) a comparable degree of immunosuppression was observed if both Con A and antigen were injected by the same route in normal mice. The marked inflammatory response observed in the peritoneal cavity of mice injected ip with Con A (8) made ‘it difficult to interpret the role of the activated macrophages under these conditions. In the adoptive immune response TABLE
4
EFFECT OF Con A INJECTED BY DIFFERENT ROUTES ON THE RESPONSE OF ADOPTIVELY TRANSFERRED SPLEEN CELLS IN IRRADIATED MICE~
Con A treatment Uninjected Con A iv Con A ip
Mean plaque forming cells/106 cells f SEM 290 f 22~ 260 f
10 8 30
70 of Control 100 7.6 90
a Con A was injected either iv or ip 24 hr prior to the irradiation of the treated mice. They were then reconstituted with 5 X lo7 syngeneic normal spleen cells injected iv, immunized iv with 108 SRBC, and assayed for direct PFC 7 days later. The results represent the mean f SEM for 15 animals in each group assayed individually.
IMMUNOSUPPRESSION
BY CON A
371
experiments reported here (Table 4)) it can be seen that a clear difference exists when mice were treated with Con A ip 24 hr prior to irradiation as compared to the iv route. No suppression was seen in the former, while a marked effect was seen in the latter case. In addition, these results suggest that the effect is probably not due to a persistence of active Con A in treated mice at the time of cell transfer, since if this were the case, it seems unlikely that the route of Con A injection, under the conditions of these experiments, would influence its rate of inactivation. DISCUSSION On the basis of our earlier investigations of the effect of Con A in tivo in suppressing the antibody response of mice to a thymus dependent antigen, it was postulated that the mechanism of this phenomenon involved the interaction of Con A with T-cells, which in some manner blocked their helper cell function in this system (1). That this hypothesis was at least partially correct was suggested when it was observed that Con A did not suppress the antibody response to a thymus independent antigen. It was further supported by the experiments described in this paper which demonstrated that the immunosuppressing effects of Con A were diminished by various ways which included either by-passing the need for helper cells or enhancing their function. Thus, using higher immunizing doses of SRBC, or priming the animals with subimmunizing doses of antigen, enhanced their subsequent PFC response, and reversed the immunosuppressing effect of Con A treatment. Although these results suggested that T-cell helper function may have been altered, no direct evidence was available to indicate that Con A was directly affecting T-cells in z&o. This was demonstrated in the experiments described here in which the [3H] thymidine incorporation into spleen cells from mice injected iv 24 hr previously with 500 PLgCon A was markedly increased as compared to animals not treated with Con A. Furthermore, treatment of these cells with either anti-theta or anti-thymus cell serum and complement prior to cell culturing markedly reduced the [9H] thymidine incorporation (8). It seemed possible on the basis of these results, that T-cells activated in tivo by Con A had become refractive to subsequent stimulation with SRBC. This insensitivity could have resulted from the augmentation of the metabolic processes evoked by Con A, which commit the cells to other activities channeled away from and incompatible with the immune response to antigen. Although the correlation between in vivo activation of spleen cells and immunosuppression elicited by Con A appeared clear-cut in the dose-response experiments (Table 3), other experiments attempting to demonstrate this correlation were not always as impressive. For example, [ 3H] thymidine incorporation into spleen cells from mice treated 48 hr previously with Con A was considerably less than at 24 hr, and yet the immunosuppression was still maximal at the longer interval (1). In addition, it had been shown in other experiments (8) that while immunosuppressing activity could be regenerated by a second injection of Con A 8 days after the first, the change in [3H] thymidine incorporation observed at this time was not as great as that seen 24 hr after the first injection of Con A. In a recent study of the in viva induction of lymphocyte-mediated cytotoxicity by Con A, Allesandro et al. (9) pointed out that although the degree of cytotoxicity correlated with DNA synthesis and blast transformation in vivo in their system, there
372
EGAN ANDEKSTEDT
was no indication that this activation was a prerequisite for the cytotoxicity observed. Since Con A is antigenic, the possibility also occurred to us that the suppression we were observing was the result of antigenic competition (15). This also seemed unlikely, since it was shown (8) that Con A which was mitogenically inactivated by heating or by mild acetylation, but which retained its antigenicity, did not suppress the PFC response of mice to SRBC. While the antigenicity of Con A may not account for its suppressing effect in z&o, the reduction in the response to SRBC we have demonstrated is very similar to the “nonspecific” suppression elicited by the injection of horse erythrocytes (16, 17) or allogeneic cells (18, 19) prior to immunization with SRBC. Moller (18) and Sjoberg (20) using the graft versus host (GVH) reaction have suggested that the inhibition may be due to an intense stimulation of T-cells resulting in a nonspecific suppressive effect on B-cells. The role of a unique subpopulation of “suppressor” T-cells, which has been recently postulated (10, 11, 21) remains to be clarified. Taussig (22) has recently reported the isolation of a T-cell population with nonspecific suppressing effects on the immune response, from a culture of antigen activated carrier T-cells, at a time when helper cell function had disappeared. The mechanism of antigenic competition is considered by some investigators (12, 17, 23) to be the result of the elaboration by T-cells of a soluble factor which temporarily suppresses further immunologic reactivity. This concept has been generated in part by evidence which shows that the suppressing influence of antigenic competition persists after lethal irradiation (18, 23, 24). In the investigation reported here we found that treatment of mice with Con A prior to irradiation markedly and consistently altered the ability of adoptively transferred normal lymphocytes to develop a primary PFC response to SRBC. It is unlikely, but cannot be ruled out at this time, that the pretreated animals would have an altered ability to localize the transferred cells and/or the antigen in the spleen. It appears more likely that some nonspecific humoral factor is produced after Con A injection, as is postulated in antigenic competition, and that this factor is resistant to irradiation. Rich and Perce (11) have reported that their “suppressor” T-cell population, induced by Con A in vitro, may elaborate a factor with immunosuppressing activity. In a more recent study, however, Peavy and Pierce (14) reported that mitogenic concentrations of Con A or small numbers of Con A activated spleen cells markedly inhibited the generation of cytotoxic lymphocytes in an ZN vitro system, and more important to our observations, after activation by Con A, the function of their suppressor T-cell population was highly resistant (2000 R) to radiation. What relationship their observations on the suppression of a cell mediated phenomenon in vitro with Con A have to our findings of the suppression of a humoral response in viva in animals irradiated after Con A injection, remains to be determined. The results of the experiments in which we altered the route of injection of Con A prior to irradiation and adoptive transfer of normal spleen cells into these animals are of considerable interest (Table 4). These results are in contrast to those reported previously by us (1) in that with the adoptive transfer system reported here, the route of injection of the Con A made a marked difference in the responsiveness observed, while in normal mice treated with Con A, the immunosuppression was marked if both Con A and antigen were injected by the same route, either iv or ip. It was not observed if the Con A was injected ip and the
IMMUh’OSUPPRESSION
BY CON i\
373
antigen given iv, however, Con A injected ip produces an intense inflammatory response and cellular exudate into the peritoneal cavity (8) and it was suggested earlier (1) that subsequent injection of antigen into this site resulted in its excessive destruction and removal, allowing little to escape in an appropriate form to the spleen, as had been suggested by previous studies of Perkins (25). While this mechanism may play some part in the suppression observed, it is likely that a more complex sequence of cell-cell interactions occur when antigen is introduced into such an inflammatory site containing significantly increased numbers of macrophages and lymphocytic cells activated by Con A. Pearson and Raffel (26) have shown that SRBC which have been degraded after ingestion by macrophages, lose activity as an antigenic stimulus for antibody formation, but progressively gain in their capacity to induce delayed hypersensitivity. Further investigation will be necessary to determine whether or not this type of immune deviation is occuring in our system. We have elected to use a relatively large dose of Con A (500 pg) in these experiments on the basis of our earlier studies (1) where the variability of the immunosuppression observed in this system with lower doses of Con A was rather large. The possibility was considered, early in the course of our adoptive transfer experiments, that residual active Con A present in animals after irradiation and cell transfer was affecting the transferred cells. Although this possibility cannot be ruled out at this time, it would seem unlikely on the basis of the marked difference in suppression observed when the route of Con A injection was altered. The possibility that Con A would be inactivated more readily when injected by the ip route seems to us to be an unsatisfactory explanation of the results observed. Attempts to transfer suppression with spleen cells or serum from Con A treated mice to normal mice in viva or in appropriate in vitro experiments are in progress. Whether the immunosuppression observed in viva in the experiments reported here are mediated by a soluble factor synthesized by activated suppressor T-cells, or due to direct interaction of these cells with appropriate B cells remains to be clarified. REFERENCES 1. Egan, H. S., Reeder,W. J., and Ekstedt, R. D., J. Immunol. 112, 63, 1974. 2. Sinclair, N. R. S. C. and Elliot, E. V., Immunology (London) 15, 32.5,1968. 3. Golub, E., Cell. Immunol. 3, 62, 1972. 4. Agrawal, B. B. L. and Goldstein, I. J., Biorkem. J. 986,23, 1965. 5. Mosier, D. E. and Coppelson,L. W., Proc. Nat. Acad. Sri. USA 61, 542, 1968. 6. Adler, W. H., Takiguchi, T., and Smith, R. T., In “In Vitro Methods in Cell Mediated Immunity.” (B. R. Bloom and P. R. Glade, Eds.), p. 433. Academic Press, New York, 1971. 7. Takahashi, T., Mond, M. J., Canswell, E. A., and Thorbecke, G. J., J. Imnzunol. 107, 1520, 1970.
8. Egan, H. S., Thesis, Northwestern University, Evanston, Illinois, 1974. 9. Allessandro, A., Waterfield, J. D., and Moller, G., J. Immunol. 113, 870, 1974. 10. Rich, R. R. and Pierce, C. W., J. Exfi. Med. 137, 649, 1973. 11. Rich, R. R. and Pierce, C. W., J. Immunol. 112, 1360,1974. 12. Veit, B. C. and Michael, J. G., J. Immunol. 111, 341, 1973. 13. Zembala, M., Asherson, G. L., Mayhew, B., and Krejci, J., Nature (London) 253, 14. Peavy, D. L. and Pierce, C. W., J. Exp. Med. 140, 356, 1974. 15. Taussig, M. J., Czdrr.Topics Immzmol. Microbial. 59, 125, 1972. 16. Radovich, J. and Talmage, D. W., Science 158, 512, 1967. 17. Gershon, R. K. and Kondo, K., J. Immunol. 106, 1524, 1971.
72, 1975.
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18. Moller, G., Immunology (London) 20, 597, 1971. 19. Gershon, R. K., Cohen, P., Hencin, R., and Liebhaber, S. A., J. Immunol. 108, 586, 1972. 20. Sjoberg, O., Immunology (London) 21, 351, 1971. 21. Reif, A. E. and Allen, T. M., Nature (London) 209, 521, 1966. 22. Taussig, M. J., Nature (London) 248, 236, 1974. 23. Moller, G. and Sjoberg, O., Cell. Immunol. 1, 110, 1970. 24. Waterston, R. H., Science 179, 1108, 1970. 25. Perkins, E. H., In “Mononuclear Phagocytes” (R. van Furth, Ed.), p. 526. Blackwell Pub]. Oxford, England 1971. 26. Pearson, M. N. and Raffel, S., J. Exp. Med. 133, 494, 1971.
IMMUNOLOGY
18, 365-374 (1975)
Suppression of a Thymus
Dependent
by Concanavalin
Humoral
Response in Mice
A in Vivol
HARWOOD S. EGAN AND RICHARD D. EKSTEDT Department
of Microbiology,
Northwestern University-McGaw Chicago, Illinois 60611 Received February
Medical
Center,
19,1975
Mice treated with Concanavalin A prior to immunization with sheep erthyrocytes exhibit a markedly reduced plaque forming spleen cell response. This immunosuppressive effect could be reversed by using higher doses of antigen or priming the animals with nonimmunizing doses of antigen prior to Concanavalin A injection designed to either by-pass or enhance thymus derived lymphocyte functions. It was also demonstrated that Concanavalin A in vivo activated the thymus derived lymphocyte subpopulation in the spleen, and this activation was dose dependent and correlated with the immunosuppression observed. Animals injected with Concanavalin A at various times prior to whole body lethal irradiation would not support the plaque forming cell response of adoptively transferred normal syngeneic spleen cells. This effect was shown to be time and dose of Concanavalin A dependent. It was also shown that the route of injection of Concanavalin A prior to irradiation determined the results observed, in that the intravenous route resulted in the suppression of transferred cells, while the intraperitoneal route showed no effect. It is suggested that Concanavalin A induced immunosuppression of the humoral, thymus dependent immune response in mice results for the activation of a subpopulation of thymus derived suppressors cells, and that the effect is short lived, radiation resistant, and dose of Concanavalin A and antigen dependent. Previous studies in this laboratory have shown that mice injected intraperitoneally (ip) 2 or intravenously (iv) with 500 rg of Concanavalin A (Con A) 24-48 hr prior to immunization with lo8 sheep erythrocytes (SRBC), exhibited a markedly reduced direct (IgM) or indirect (IgG) plaque forming spleen cell (PFC) primary response (1). It was reported in the same paper, that the secondary response was also suppressed in mice injected with Con A 2 days prior to the second injection of antigen 7, 10, or 21 days after the first injection, but not when Con A was injected 2 days prior to the first injection of antigen. In the latter case, an enhancement of both the IgM and IgG response was observed, suggesting that Con A treatment was affecting an early event in the antibody response, possibly at the T cell recognition step, but did not appear to affect the priming of the animals to make a secondary response. Other parameters of timing, dose of Con A 1 This work was supported in part by United States Public Health Service Grants AI07716, RR-05370, and by Contract N00014-67-A-0356-0019 from the Office of Naval Research. 2 Abbreviations : ip, intraperitoneal; iv, intravenous ; Con A, Concanavalin A; PFC, plaque forming cells; T cell, thymus derived lymphocyte; B cell, bone marrow derived lymphocyte; RPMI, Rosewell Park Memorial Institute ; SRBC, sheep erythrocytes. 365 1975 by Academic Press, Copyright All rights o8 reproduction in any form
366
EGAN
AND
EKSTEDT
and antigen, routes of Con A treatment and immunization, and T-cell dependency of the antigen used, were also considered (1). In this paper, we present the results of further studies designed to elucidate the mechanism of immunosuppression in Con A treated mice to a thymus dependent antigen. These include attempts to overcome the suppression by using higher doses of antigen (2) or priming the animals with subthreshold doses of antigen to produce “educated” T-cells (3). In addition, attempts were made to correlate the in viva mitogenecity of Con A with its immunosuppression and to study the PFC response of normal syngeneic spleen cells in Con A treated and irradiated mice. The results were consistent with the hypothesis that Con A functions in viva in suppressing the antibody response of mice to a thymus dependent antigen by activating a subpopulation of suppressor T-cells and that the effect was radiation resistant, short lived, and dose of Con A dependent. MATERIALS
AND METHODS
Animals. The mice used in these experiments were adult (6-8 wk) BALB/c males, which were received at weekly intervals from Jackson Laboratories, Bar Harbor, Maine. They were housed ten per cage in air-conditioned animal quarters and allowed food (Purina mouse chow) and water ad lib. In some experiments CE7 B1/6J mice obtained from the same source were used. Previous experiments had shown that this strain of mice responded identically in every respect in this system. Concanavalin A. The Con A used in these studies was prepared in this laboratory from Jack bean meal (Nutritional Biochemicals, Cleveland, Ohio) by the method of Agrawal and Goldstein (4), and was comparable in purity to preparations (Grade IV) obtained from Sigma Chemical Co., St. Louis, MO. A single preparation, stored in 1.0 M NaCl at -2O”C, and diluted appropriately just prior to use, was used in all the experiments reported here. Spleen cell preparations and assays. The methods used for preparing single spleen cell suspensions and assaying for antibody forming cells by the micromodification of the Jerne localized ,hemolysis-in-gel technique are described in the literature (5) or in our previous publication (1). Cell cultures and [3H] thymidine uptake. Spleen cell suspensions (5 x 106/ml) from Con A treated and untreated mice in RPM1 1640 tissue culture medium (Grand Island Biological Co., Grand Island, NY) containing L-glutamine and supplemented with 5% (v/v) heat inactivated (56”C/30 min) fresh human serum from the same donor, and 100 units of a streptomycin-penicillin solution (GIBCO) were dispensed (3 ml) into 13 X 100 mm culture tubes (Falcon Co., Oxnard, Calif.) in triplicate. To test the in viva mitogenic activation of the spleen cells by Con A, 1 $.Zi [3H]thymidine/culture (sp act 1.9 CiJmmole, Schwartz-Mann, Orangeburg, New Jersey) was added at the beginning of the culture period, allowed to label for 24 hr, and terminated. The methods for harvesting the cultures and determining the incorporation of radioactivity into the acid insdluble fraction by scintillation counting has been described (6). Con A treatment, irradiation, cell transfer, and immunization. Following the procedure of Takahashi et al. (7)) recipient BALB/c mice, treated previously with 500 pg Con A iv, or normal mice, were exposed to whole body irradiation delivered from a Picker machine at 270 kV, 15 mA, with 1.3 mm copper and 1.0 mm
IMMUNOSUPPRESSION
TABLE
367
BY CON A
1
EFFECT OF ANTIGEN DOSE ON THE DIRECT PFC RESPONSE OF MICE TO SRBC FOLLOWING TREATMENT WITH Con A
PFC/lOG spleen cellsa
Treatmenta
725 f 80 f 772 f 407 f
10s SRBC Con A + lo* SRBC 109 SRBC Con A + lo* SRBC
63 12 50 78
0 Animals were injected iv with SRBC antigen alone or 24 hr after treatment with 500 fig Con A iv. b PFC response was measured 4 days following immunization. The results are expressed as the mean response f SEM of 15 mice in each group assayed individually.
aluminum filtration. With a source to target distance of 50 cm and at an exposure of 87.4 R/min, a total of 600 R was administered. Within 3 hr of irradiation, the mice were injected with 5 x lo7 lymphocytes from normal syngeneic mice and lo8 SRBC. The spleen cells from these animals were then assayed 7 days later for direct PFC. The effect of different doses of Con A given iv 24 hr prior to irradiation was also studied in similar experiments. RESULTS Reversal of the Immunosuppressive
Activity
of Con A In Vivo
Higher antigenic doses. Since our original hypothesis concerning the mode of action of Con A in viva was that it was in some manner affecting the interaction of T-cells with antigen, several experiments were done in attempts to enhance the antibody response of Con A treated mice to SRBC by altering this interaction. Sinclair (2) reported that large doses of SRBC obviated the need for helper T-cell function, and in effect were thymic independent. The results presented in Table 1 show that injecting a tenfold higher dose of SRBC into mice injected iv 24 hr ‘earlier with 500 pg Con A, significantly increased their PFC response. Animals receiving log SRBC without prior treatment with Con A did not give a greater PFC response than those given lo8 SRBC alone, since the latter dose has been shown to be optimal in this system (2). Priwzed mice. Golub (3) reported that priming mice with subthreshold doses of SRBC antigen at some time prior to an adequate immunizing dose, resulted in both an accelerated and enhanced primary (IgM) PFC response. This effect has been attributed to an increase in the number of responding “educated” T-cells that would encounter the antigen after the second injection. It was reasoned that in our system, if Con A were actually reducing the number of responding T-cells prior to SRBC injection, then increasing their number might reduce the suppressive effect of Con A. Table 2 shows the results of these experiments. Con A treated mice primed with lo5 SRBC 5 days prior to an immunizing dose of lo8 SRBC, displayed a significant increase in the PFC response as compared to Con A treated unprimed animals. Although the response of primed animals treated with Con A did not reach that of the group primed with antigen but not treated with Con A, it seems clear that under the conditions of these experiments, prior exposure of mice to a
368
EGAN AND EKSTEDT TABLE
2
EFFECT OF Con A ON THE DIKECT PFC RESPONSE OF PRIMED MICE TO SRBC Treatment0
105 SRBC 106 SRBC
PFC/lOB
lo* SRBC + lOa SRBC Con A + lo8 SRBC + Con A + lo8 SRBC
spleen cells”
117 & 26 2938 f 537 10% 5 800 f 178
n Mice were primed with 106 SRBC given ip 5 days before iv injection of lo8 SRBC immunizing dose. 500 pg of Con A was injected iv 24 hr prior to the immunizing injection. b 15 Animals in each group were assayed individually 3 days after the immunizing injection, and the results are expressed as in Table 1. 105 SRBC injected alone resulted in no detectable increase in PFC response above background.
subthreshold dose of antigen does significantly reduce the effect of subsequent treatment with Con A upon the response to an immunizing dose of SRBC. It should also be made clear that in these experiments, the animals injected with 10’ SRBC alone show a lower PFC response than a similar group shown in Table 1, since in this case, the PFC assays were done 3 days after immunization, when the response is just beginning to appear in unprimed animals (1). Efect of CON A In Vivo on Other Parameters of Immune Responsiveness While the above experiments suggested strongly that a T-cell function in viva was being affected by Con A, it was considered important to determine what effect injection of Con A was exerting upon the other known cellular activities important in eliciting an immune response. Since it is well recognized that Con A activates T-cells in vitro to blast transformation and mitosis, it seemed worthwhile to test whether Con A in viva also resulted in activation of T-cells. If this were the case, such activated cells might be refractory, for a period of time, to antigenic stimulation. Accordingly, groups of mice were injected iv with various doses of Con A. TwentyTABLE
3
INCORPORATION OF [~HITHYMIDINE BY SPLEEN CELLS FROM MICE TREATED WITH VARIOUS DOSES OF Con A PRIOR TO CELL CULTURING [aH]thymidine incorporation mean counts per minute (cpm)” f SEM Uninjected controls Con A 5 pg Con A 125 rg Con A 500 rg
1500 2600 5500 12,500
+ 400 f 400 f 500 f 3500
PFC response (% of control) b
100 86 f 16 62 f 24 16 f 12
0 Spleen cell cultures were set up as described in Materials and Methods 24 hr after the iv injection of Con A. Cultures were set up in triplicate and the counts per minute averaged in each case. Results from at least four experiments are expressed as Mean f SEM. “Four days following immunization with SRBC, spleens from other mice were assayed for direct PFC. Where indicated, Con A was injected iv 24 hr prior to immunization. For each experimental group, 15 animals were assayed individually and the results are expressed as the percent of the response observed with animals immunized but not treated with Con A.
~MMUN~SUPPRESSI~N
-4 -I Con A Injection-
369
BY coiv A
-7 COnfrOl Days Before Irradiation
FIG. 1. The effect of varying the time interval after Con A injection and prior to irradiation on the PFC response of adoptively transferred lymphocytes to SRBC. 500 pg of Con A was injected iv at the days indicated prior to 600 R of total body irradiation. 5 X lo7 Normal syngeneic spleen cells along with 108 SRBC were injected iv within 3 hr. Results represent the mean -C SEM from nine mice per point assayed individually for direct PFC response 7 days after transfer and immunization.
four hours later they were killed, their spleens removed, prepared, and cultured in the presence of [ 3H] thymidine for an additional 24 hr as described. Table 3 shows the results of these experiments, where it can be seen that there was a direct correlation between the amount of Con A injected and the degree of activation of spleen cells. Also shown in the table is the degree of suppression of PFC response in animals so treated, which also correlated with the dose of Con A. In other experiments not shown (8) treatment of the spleen cell suspensions with anti-theta or anti-thymocyte serum and complement prior to culture confirmed that the cells undergoing activation were of the T cell line. Allessandro et al. (9) have recently demonstrated a similar in &JO activation of spleen cells to develop cytotoxicity, another T cell mediated response, in mice injected with Con A. In view of these results, and with the recent evidence for the existence of a subpopulation of suppressor T cells which function to regulate the immune responses (10, ll), the possibility that Con A in tivo was activating a suppressor rather than inuctivating a helper T cell population was considered. It has also been suggested recently, by the results of Veit and Michael (12), that a soluble suppressor factor, present in the serum of normal and immunized mice, and presumably synthesized by an appropriate T cell subpopulation, mediates this effect, and attempts to characterize this factor have been reported (13). Adoptive Imwaune Response in Con A Treated-Irradiated
Mice
In order to gain some further evidence that a suppressor T-cell or soluble mediator might be involved in the immunosuppressive activity of Con A in vivo, the following experiments were done. Mice were injected iv with 500 pg of Con A and at different times thereafter subjected to 6OOR whole body X-irradiation. They were then injected with 5 X lo7 syngeneic normal spleen cells and lOa SRBC, iv within 3 hr, and assayed for PFC response 7 days later. It was reasoned that if the Con A activated a T-cell suppressor population of lymphocytes, which elaborated a soluble suppressor factor, this latter material should be radiation resistant and still capable of inhibiting normal transferred cells. It has also been reported recently
370
EGAN
AND
EKSTEDT
ae 0 5 125 500 pg Con A Injected I Day Prior to Irradiation FIG. 2. Effect of different doses of Con A, given iv 1 day prior to irradiation, on the subsequent PFC response of adoptively transferred spleen cells. The same procedure as in Fig. 1 was used, and the results represent the mean k SEM from nine mice per point.
by Peavy and Pierce (14) that suppressor T-cells are highly resistant to irradiation. If, on the other hand, a T-cell helper population of lymphocytes was being affected by Con A in viva, repopulation of these animals with normal lymphocytes should allow an immune response to SRBC comparable to control animals which had been irradiated and repopulated, but not treated with Con A prior to irradiation, since helper cell function is radiation sensitive (15). The results of these experiments are shown in Fig. 1, where it is clear that the immune response of normal transferred cells was markedly and consistently suppressed in mice irradiated 24 hr after treatment with Con A. As the interval between Con A treatment and irradiation was extended, the effect became progressively less. In addition, Fig. 2 shows that decreasing the dose of Con A given 24 hr prior to irradiation also reduces the suppression of an adoptive immune response to SRBC. In a further series of experiments similar to those described above, the effect of injecting the Con A by the ip route was compared with that observed when the Con A was given iv. In our earlier studies (1) a comparable degree of immunosuppression was observed if both Con A and antigen were injected by the same route in normal mice. The marked inflammatory response observed in the peritoneal cavity of mice injected ip with Con A (8) made ‘it difficult to interpret the role of the activated macrophages under these conditions. In the adoptive immune response TABLE
4
EFFECT OF Con A INJECTED BY DIFFERENT ROUTES ON THE RESPONSE OF ADOPTIVELY TRANSFERRED SPLEEN CELLS IN IRRADIATED MICE~
Con A treatment Uninjected Con A iv Con A ip
Mean plaque forming cells/106 cells f SEM 290 f 22~ 260 f
10 8 30
70 of Control 100 7.6 90
a Con A was injected either iv or ip 24 hr prior to the irradiation of the treated mice. They were then reconstituted with 5 X lo7 syngeneic normal spleen cells injected iv, immunized iv with 108 SRBC, and assayed for direct PFC 7 days later. The results represent the mean f SEM for 15 animals in each group assayed individually.
IMMUNOSUPPRESSION
BY CON A
371
experiments reported here (Table 4)) it can be seen that a clear difference exists when mice were treated with Con A ip 24 hr prior to irradiation as compared to the iv route. No suppression was seen in the former, while a marked effect was seen in the latter case. In addition, these results suggest that the effect is probably not due to a persistence of active Con A in treated mice at the time of cell transfer, since if this were the case, it seems unlikely that the route of Con A injection, under the conditions of these experiments, would influence its rate of inactivation. DISCUSSION On the basis of our earlier investigations of the effect of Con A in tivo in suppressing the antibody response of mice to a thymus dependent antigen, it was postulated that the mechanism of this phenomenon involved the interaction of Con A with T-cells, which in some manner blocked their helper cell function in this system (1). That this hypothesis was at least partially correct was suggested when it was observed that Con A did not suppress the antibody response to a thymus independent antigen. It was further supported by the experiments described in this paper which demonstrated that the immunosuppressing effects of Con A were diminished by various ways which included either by-passing the need for helper cells or enhancing their function. Thus, using higher immunizing doses of SRBC, or priming the animals with subimmunizing doses of antigen, enhanced their subsequent PFC response, and reversed the immunosuppressing effect of Con A treatment. Although these results suggested that T-cell helper function may have been altered, no direct evidence was available to indicate that Con A was directly affecting T-cells in z&o. This was demonstrated in the experiments described here in which the [3H] thymidine incorporation into spleen cells from mice injected iv 24 hr previously with 500 PLgCon A was markedly increased as compared to animals not treated with Con A. Furthermore, treatment of these cells with either anti-theta or anti-thymus cell serum and complement prior to cell culturing markedly reduced the [9H] thymidine incorporation (8). It seemed possible on the basis of these results, that T-cells activated in tivo by Con A had become refractive to subsequent stimulation with SRBC. This insensitivity could have resulted from the augmentation of the metabolic processes evoked by Con A, which commit the cells to other activities channeled away from and incompatible with the immune response to antigen. Although the correlation between in vivo activation of spleen cells and immunosuppression elicited by Con A appeared clear-cut in the dose-response experiments (Table 3), other experiments attempting to demonstrate this correlation were not always as impressive. For example, [ 3H] thymidine incorporation into spleen cells from mice treated 48 hr previously with Con A was considerably less than at 24 hr, and yet the immunosuppression was still maximal at the longer interval (1). In addition, it had been shown in other experiments (8) that while immunosuppressing activity could be regenerated by a second injection of Con A 8 days after the first, the change in [3H] thymidine incorporation observed at this time was not as great as that seen 24 hr after the first injection of Con A. In a recent study of the in viva induction of lymphocyte-mediated cytotoxicity by Con A, Allesandro et al. (9) pointed out that although the degree of cytotoxicity correlated with DNA synthesis and blast transformation in vivo in their system, there
372
EGAN ANDEKSTEDT
was no indication that this activation was a prerequisite for the cytotoxicity observed. Since Con A is antigenic, the possibility also occurred to us that the suppression we were observing was the result of antigenic competition (15). This also seemed unlikely, since it was shown (8) that Con A which was mitogenically inactivated by heating or by mild acetylation, but which retained its antigenicity, did not suppress the PFC response of mice to SRBC. While the antigenicity of Con A may not account for its suppressing effect in z&o, the reduction in the response to SRBC we have demonstrated is very similar to the “nonspecific” suppression elicited by the injection of horse erythrocytes (16, 17) or allogeneic cells (18, 19) prior to immunization with SRBC. Moller (18) and Sjoberg (20) using the graft versus host (GVH) reaction have suggested that the inhibition may be due to an intense stimulation of T-cells resulting in a nonspecific suppressive effect on B-cells. The role of a unique subpopulation of “suppressor” T-cells, which has been recently postulated (10, 11, 21) remains to be clarified. Taussig (22) has recently reported the isolation of a T-cell population with nonspecific suppressing effects on the immune response, from a culture of antigen activated carrier T-cells, at a time when helper cell function had disappeared. The mechanism of antigenic competition is considered by some investigators (12, 17, 23) to be the result of the elaboration by T-cells of a soluble factor which temporarily suppresses further immunologic reactivity. This concept has been generated in part by evidence which shows that the suppressing influence of antigenic competition persists after lethal irradiation (18, 23, 24). In the investigation reported here we found that treatment of mice with Con A prior to irradiation markedly and consistently altered the ability of adoptively transferred normal lymphocytes to develop a primary PFC response to SRBC. It is unlikely, but cannot be ruled out at this time, that the pretreated animals would have an altered ability to localize the transferred cells and/or the antigen in the spleen. It appears more likely that some nonspecific humoral factor is produced after Con A injection, as is postulated in antigenic competition, and that this factor is resistant to irradiation. Rich and Perce (11) have reported that their “suppressor” T-cell population, induced by Con A in vitro, may elaborate a factor with immunosuppressing activity. In a more recent study, however, Peavy and Pierce (14) reported that mitogenic concentrations of Con A or small numbers of Con A activated spleen cells markedly inhibited the generation of cytotoxic lymphocytes in an ZN vitro system, and more important to our observations, after activation by Con A, the function of their suppressor T-cell population was highly resistant (2000 R) to radiation. What relationship their observations on the suppression of a cell mediated phenomenon in vitro with Con A have to our findings of the suppression of a humoral response in viva in animals irradiated after Con A injection, remains to be determined. The results of the experiments in which we altered the route of injection of Con A prior to irradiation and adoptive transfer of normal spleen cells into these animals are of considerable interest (Table 4). These results are in contrast to those reported previously by us (1) in that with the adoptive transfer system reported here, the route of injection of the Con A made a marked difference in the responsiveness observed, while in normal mice treated with Con A, the immunosuppression was marked if both Con A and antigen were injected by the same route, either iv or ip. It was not observed if the Con A was injected ip and the
IMMUh’OSUPPRESSION
BY CON i\
373
antigen given iv, however, Con A injected ip produces an intense inflammatory response and cellular exudate into the peritoneal cavity (8) and it was suggested earlier (1) that subsequent injection of antigen into this site resulted in its excessive destruction and removal, allowing little to escape in an appropriate form to the spleen, as had been suggested by previous studies of Perkins (25). While this mechanism may play some part in the suppression observed, it is likely that a more complex sequence of cell-cell interactions occur when antigen is introduced into such an inflammatory site containing significantly increased numbers of macrophages and lymphocytic cells activated by Con A. Pearson and Raffel (26) have shown that SRBC which have been degraded after ingestion by macrophages, lose activity as an antigenic stimulus for antibody formation, but progressively gain in their capacity to induce delayed hypersensitivity. Further investigation will be necessary to determine whether or not this type of immune deviation is occuring in our system. We have elected to use a relatively large dose of Con A (500 pg) in these experiments on the basis of our earlier studies (1) where the variability of the immunosuppression observed in this system with lower doses of Con A was rather large. The possibility was considered, early in the course of our adoptive transfer experiments, that residual active Con A present in animals after irradiation and cell transfer was affecting the transferred cells. Although this possibility cannot be ruled out at this time, it would seem unlikely on the basis of the marked difference in suppression observed when the route of Con A injection was altered. The possibility that Con A would be inactivated more readily when injected by the ip route seems to us to be an unsatisfactory explanation of the results observed. Attempts to transfer suppression with spleen cells or serum from Con A treated mice to normal mice in viva or in appropriate in vitro experiments are in progress. Whether the immunosuppression observed in viva in the experiments reported here are mediated by a soluble factor synthesized by activated suppressor T-cells, or due to direct interaction of these cells with appropriate B cells remains to be clarified. REFERENCES 1. Egan, H. S., Reeder,W. J., and Ekstedt, R. D., J. Immunol. 112, 63, 1974. 2. Sinclair, N. R. S. C. and Elliot, E. V., Immunology (London) 15, 32.5,1968. 3. Golub, E., Cell. Immunol. 3, 62, 1972. 4. Agrawal, B. B. L. and Goldstein, I. J., Biorkem. J. 986,23, 1965. 5. Mosier, D. E. and Coppelson,L. W., Proc. Nat. Acad. Sri. USA 61, 542, 1968. 6. Adler, W. H., Takiguchi, T., and Smith, R. T., In “In Vitro Methods in Cell Mediated Immunity.” (B. R. Bloom and P. R. Glade, Eds.), p. 433. Academic Press, New York, 1971. 7. Takahashi, T., Mond, M. J., Canswell, E. A., and Thorbecke, G. J., J. Imnzunol. 107, 1520, 1970.
8. Egan, H. S., Thesis, Northwestern University, Evanston, Illinois, 1974. 9. Allessandro, A., Waterfield, J. D., and Moller, G., J. Immunol. 113, 870, 1974. 10. Rich, R. R. and Pierce, C. W., J. Exfi. Med. 137, 649, 1973. 11. Rich, R. R. and Pierce, C. W., J. Immunol. 112, 1360,1974. 12. Veit, B. C. and Michael, J. G., J. Immunol. 111, 341, 1973. 13. Zembala, M., Asherson, G. L., Mayhew, B., and Krejci, J., Nature (London) 253, 14. Peavy, D. L. and Pierce, C. W., J. Exp. Med. 140, 356, 1974. 15. Taussig, M. J., Czdrr.Topics Immzmol. Microbial. 59, 125, 1972. 16. Radovich, J. and Talmage, D. W., Science 158, 512, 1967. 17. Gershon, R. K. and Kondo, K., J. Immunol. 106, 1524, 1971.
72, 1975.
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18. Moller, G., Immunology (London) 20, 597, 1971. 19. Gershon, R. K., Cohen, P., Hencin, R., and Liebhaber, S. A., J. Immunol. 108, 586, 1972. 20. Sjoberg, O., Immunology (London) 21, 351, 1971. 21. Reif, A. E. and Allen, T. M., Nature (London) 209, 521, 1966. 22. Taussig, M. J., Nature (London) 248, 236, 1974. 23. Moller, G. and Sjoberg, O., Cell. Immunol. 1, 110, 1970. 24. Waterston, R. H., Science 179, 1108, 1970. 25. Perkins, E. H., In “Mononuclear Phagocytes” (R. van Furth, Ed.), p. 526. Blackwell Pub]. Oxford, England 1971. 26. Pearson, M. N. and Raffel, S., J. Exp. Med. 133, 494, 1971.