Role of the autologous rosette-forming T cells in the concanavalin A-induced suppressor cell function

CELLULAR

IMMUNOLOeY

61, 273-279 (1981)

Role of the Autologous Rosette-Forming T Cells in the Concanavalin A-Induced Suppressor Cell Function RONALD

PALACIOS

Department of Immunobiology. Karolinska Institute, Wallenberglaboratory. S-104 05. Stockholm 50, Sweden Received October 16, 1980; accepted January 8. 1981 It was recently reported that the human autologous rosette-forming T cells (Tar cells) are devoid of Fc receptors for IgM and IgG but that they give rise in vitro to T/J and T-y cells and that these cells participate actively in feedback inhibition. We now investigated whether Tar cells participate in the concanavalin A-induced suppressorcell function, using two indicator systems, namely, mitogen- or alloantigen-induced DNA synthesis and mitogen-driven polyclonal immunoglobulin synthesis. When Tar cells were removed from peripheral blood T cells (T-Tar) there was no generation of suppression determined on both DNA synthesis and immunoglobulin production. When Tar cells were readded to T-Tar cells suppressor activity was restored. When purified Tar cells were activated by concanavalin A they showed the highest degree of suppression as compared to that exerted by total T cells or T-Tar cells. Tar cells lose their capacity to generate suppression when treated with mitomycin C before their activation; however, they are no longer sensitive to mitomycin C treatment once they have completed the period of activation. These results together with our previous findings showing that Tar cells give rise in vitro to TP and T-y cells after their activation by Con-A suggest that Tar cells participate in the Con-A-induced suppressor ceil function as precursors of the suppressor effector T cells.

INTRODUCTION The existence of subpopulations of lymphoid cells capable of suppressing various parameters of immune reactivity has been demonstrated to play a major role in the regulation of the immune response in animal models (l-3) and in humans (47). The experiments of Shou et al. (5) showed that a population of human peripheral blood mononuclear cells (MNC) could be activated by mitogenic doses of concanavalin A (Con-A) to manifest suppressor activity in vitro. Subsequently, it has been amply demonstrated that Con-A-induced human suppressor cells are capable of regulating proliferative responsesto soluble antigens (6), mitogens (5-8) and TNP-modified autologous cells (9), mixed lymphocite reaction (MLR) (5-8, 10) in alloantigenic systems, and polyclonal immunoglobulin synthesis stimulated by pokeweed mitogen (PWM) (7, 10, 11) as well as antibody production in response to specific antigens ( 12). Recently, it has been suggested by a number of different studies that there is heterogeneity among the lymphocytes capable of being activated by Con-A to function as suppressor cells (13, 14). Thus, such cells have been shown to differ in their sensitivity to X rays ( 15, 16) corticosteroids ( 10, 16) and mitomycin C (10, 11, 14, 16). Furthermore, there is evidence to indicate that both 273 0008-8749/81/100273-07$02.00/O Copyright 0 I98 I by Academic Press. Inc. All rights of reproduction in any form reserved

274

RONALD PALACIOS

T cells with and without receptors for the Fc portion of IgG are able to exert suppression once they have been activated by Con-A (8, 14-17). We have recently shown that Tar cells possesssurface and functional properties similar to that of post-thymic precursor cells (18-19). Thus, Tar cells are sensitive to hydrocortisone both in vitro and in vivo, bind peanut agglutinin, adhere to nylon wool and their binding to either SRBC or autologous erythrocytes resist the thephylline treatment. Tar cells being devoid of Fc receptors give rise in vitro to Tr cells and Tp cells after activation by Con-A or by the serum thymic factor. Among their functional properties Tar cells play a mandatory role in feedback inhibition, respond in the autologous mixed lymphocyte reaction, but are unresponsive in the allogeneic mixed lymphocyte reaction (( 18-20), R. Palacios, L. Llorente, A. RuizArguelles, and D. Alarcbn-Segovia, submitted for publication). We have extended our studies regarding the functional properties of Tar cells by investigating whether these cells participate in the generation of suppressor T cells by Con-A. The results presented here show that Tar cells participate in the Con-A-induced suppressor function most likely as precursors of the suppressor effector T cells. MATERIALS

AND METHODS

Cells’ separation. Peripheral blood mononuclear cells (MNC) were obtained from healthy adult volunteers as previously described (18-20). T cells and non-T cells were separated by rosetting MNC with sheep red blood cells (SRBC) also as previously described (18-20). Briefly, MNC suspended in RPM1 1640 medium supplemented with 20% of heat-inactivated fetal calf serum were incubated with SRBC at a ratio of I:70 at 37OC for 15 min. The cells’ mixture was centrifuged and incubated at 4°C for 18 hr. The cells were resuspended, layered on FicollHypaque density gradients and centrifuged at 4°C 1400 rpm, for 30 min. The rosetted cells in the pellet were resuspended and centrifuged again over FicollHypaque cushions at the same conditions as above. SRBC were lysed by hypotonic shock and the lymphocytes were washed two times with RPM1 medium. Total T cells included more than 95% of SRBC-rosetting cells and less than 2% of immunoglobulin-bearing cells identified with fluorescein-labeled F(ab’)2 fragments of goat anti-human immunoglobulin serum. Autologous rosette-forming T cells (Tar cells) were identified and isolated as previously described (18-20). Briefly, 2 X lo6 MNC were incubated with 0.05 ml of autologous serum diluted 1:5 in RPM1 medium at 4°C for 30 min. Autologous erythrocytes obtained from the same heparinized venous sample were washed three times with PBS, resuspended in RPM1 medium, and 16 X lo6 of them added to each tube. The cells’ mixture were incubated at 4°C overnight. After this, the cells were resuspended in cold RPM1 medium, layered on cold(4”C) Ficoll-Hypaque gradients, and centrifuged at 4°C 1400 rpm, for 40 min. The rosetted cells in the pellet were resuspendedagain in cold medium and centrifuged over Ficoll-Hypaque cushions at the same conditions as above. The rosetted cells were resuspended in warm (37°C) RPM1 medium, incubated at 37°C for 30 min, and centrifuged over Ficoll-Hypaque gradients at 1200 rpm for 30 min to rid the lymphocytes of the autologous erythrocytes. The cells at the interphase were collected, washed two times with RPM1 medium, and resuspended in RPM1 1640 medium (Gibco, Glas-

PRECURSORS OF SUPPRESSOR T CELLS

275

gow, Scotland) supplemented with 10% heat-inactivated human AB serum, 20 pg/ml of gentamicin, and 0.4 mg of L-glutamine, which will be referred to as complete culture medium (CCM). Tar cells obtained had more than 90% of autologous rosette-forming T cells, less than 5% of Tp cells, and less than 1% of Ty cells identified by rosetting with IgG or IgM-coated ox erythrocytes. Total T cells were depleted of Tar cells by rosetting them with autologous erythrocytes and repeating the above procedure of gradient separation but collecting the cells at the interphase (T-Tar cells). Total T cells had 27% of Tar cells, whereas T-Tar cells included less than 2% of Tar cells. In order to obtain adherent cells, MNC suspended in RPM1 medium enriched with 10% human AB serum were incubated for 120 min at 37°C in plastic petri dishes. Nonadherent cells were removed by three washings; adherent cells were obtained by removing them from a plastic surface with a rubber policeman and resuspended in CCM. General experimental design. The various cell populations obtained as described above were treated or untreated with Con-A to activate suppressor cells in a first culture. These cells were then added to responder cells in a second assay culture to measure their suppressor ability. The responder cells in the second culture were always from the same donor. Two different assay systems were employed: first, proliferation in response to phytohemaggulitin (PHA, Wellcome Research Lab., Beckenham, England) or allogeneic cells (MLR); second, polyclonal immunoglobulin synthesis induced by pokeweed mitogen. Generation of suppressor cells with Con-A. In the first culture, 3 X lo6 of the following cell groups were supplemented with 5% of irradiated autologous adherent cells and suspended in 3 ml of CCM: total T cells, T cells depleted of Tar cells (T-Tar), T-Tar cells reconstituted with Tar cells((T-Tar) + Tar cells), and purified Tar cells. Each of the cell groups were treated with Con-A (12.5 pg/ml) or medium and incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 3 days. At the end of the culture period, the cells were washed four times with RPM1 medium. A portion of each of the cell populations was then treated with mitomycin C (45 pg/ml) at 37°C for 30 min, washed three times with PBS, adjusted at a concentration of lo6 cells/ml of CCM, and their suppressor activity in proliferative responsestested (detailed below). When testing for suppressor cell activity in PWMdriven immunoglobulin production the cells were treated as above except that they were not exposed to mitomycin C. Assay for suppressor cell activity. Responding cells were obtained 3 days later from a second bleeding of the same donor who provided the cells for the first culture. Two assay systems were used to detect suppressor cell activity. In the first, 1 X lo5 mitomycin C-treated Con-A-activated cells or their controls (mitomycintreated nonactivated cells) from the first culture were cocultured with fresh autologous responding MNC (1 X lo5 cells/well) in flat-bottom microtiter plates (A/ S Nunc LiterMed). Cultures were stimulated either with PHA( 1 pg/ml) or 1 X lo5 irradiated (2500 rad) allogeneic MNC and incubated at 37°C in a humidified atmosphere of 5% CO* in air. Proliferation of the responding cells was measured by the incorporation of [‘HI-thymidine (0.5 &i/well, The Radiochemical Centre, Amershan, England) during the last 18 hr of a 4-day culture -period for PHAinduced proliferation and a 6-day culture for the MLR. The cells were harvested with a multisample collector, placed in scintillation fluid, and counted in a liquid scintillation counter. The viability of the cells determined by trypan blue exclusion

276

RONALD PALACIOS

at the end of the culture period was similar between those cultures containing ConA-activated cells and those containing nonactivated cells. In the second assay system, inhibition of immunoglobulin synthesis was measured, here 1O6responding autologous MNC suspended in CCM were cocultured with 1O6 of each of the Con-A-activated cell populations or their respective controls in a final volume of 2 ml in 12 X 75-mm round-bottom plastic tubes (Falcon Plastics, Oxnard, Calif.). Polyclonal immunoglobulin production was induced by the addition of PWM (20 pg/ml) to the cultures which were incubated at 37°C for 7 days. At the end of the culture period, the tubes were centrifuged at 2000 rpm for 15 min, and the amount of immunoglobulins G, A, and M contained in the supernatants determined by nephelometry as described elsewhere (18, 21). Briefly, 0.1 ml of the supernatant was placed together with 0.2 ml of a 1:5 dilution of rabbit antiserum to human IgG, IgA, and IgM (Beringwereke, Marburg Lahn, West Germany) in plastic cuvettes with low background and read in a helium-neon laser nephelometer (Beringwerke). RESULTS Autologous rosette-forming T cells seem to play a mandatory role in the ConA-induced suppressor cell function. Actually, the results depicted in Table 1, in which 6 out of 10 independent experiments are presented, show that depletion of Tar cells from total T cells (T-Tar cells) produced a significantly lower suppressor activity when compared to that exerted by total T cells. Addition of Tar cells to a population of T-Tar cells (T-Tar) + Tar) restablished the suppressor activity which was higher but did not reach statistical significance when compared to that produced by total T cells. Furthermore, when the suppressor T cells were a purified population of Tar cells activated by Con-A, the suppressor activity was always the highest obtained compared to that exerted by total T cells or T-Tar cells. These findings occurred irrespective of the indicator system employed (Table 1). In order to determine whether Tar cells need to proliferate in order to generate suppression, the cells were treated with mitomycin C (as detailed before) before and after their activation by Con-A. After this, Tar cells were cocultured with fresh autologous MNC which were stimulated to synthesize immunoglobulins by PWM. The results indicate that Tar cells are sensitive to mitomycin C before they were activated by Con-A but this sensitivity is no longer apparent once Tar cells have completed their activation (data not given). DISCUSSION The experiments presented here demonstrate that the presenceof Tar cells seems to be essential to obtain suppressor activity induced by Con-A. Analogous results were obtained whether the indicator system was [3H]-thymidine uptake induced by PHA or alloantigens or PWM-driven immunoglobulin production. Tar cells seem to participate in the Con-A-induced suppressor cell assay as precursors of the suppressor effector T cells, since it was previously found that Tar cells being devoid of Fc receptors give rise in vitro to Tp and Ty cells after 24 hr or more of stimulation with Con-A (18, 19) R. Palacios, L. Llorente, A. RuizArguelles, and D. Alarcon-Segovia, manuscript submitted for publication). Also supporting the notion that Tar cells function as precursors of the suppressor effector

277

PRECURSORS OF SUPPRESSOR T CELLS TABLE 1 Role of Tar Cells in Con-A-Induced Suppressor Cell Function [‘H]Thymidine uptake Expt

Responding cells

I

MNC

2

PHA

MLR

130.2’ Nonactivated T T T-Tar (T-Tar) + Tar Tar

136.4 63.0 112.0 55.2 41.7

66.8b 69.3 31.8 58.7 23.4 19.9

5550 5600 2350 5300 2200 2050

85.6 66.3

83.5 88.0 50.3 71.9 47.5 39.8

4200 4350 2350 3950 2150 1950

128.6 120.3 67.7 103.0 60.1 48.3

80.8 73.5 36.2 69.1 33.4 26.5

4900 4950 2550 4400 2300 2050

MNC Nonactivated T T T-Tar (T-Tar) + Tar Tar

3

MNC Nonactivated T T T-Tar (T-Tar) + Tar Tar

4

MNC

Ig synthesis (ng/106 cells)

Cell Population added”

177.0 197.5 92.4

170.8

Nonactivated T T T-Tar (T-Tar) + Tar Tar

166.9 168.0 77.2 70.3 60.9

70.1 65.8 39.4 55.8 35.5 26.7

3850 3800 1450 3100 1350

139.9

1100

5

MNC

Nonactivated T T T-Tar (T-Tar) + Tar Tar

121.0 133.4 66.6 98.8 52.1 40.4

84.7 90.0 50.1 71.6 43.3 31.2

5100 5200 3550 4850 3350 3000

6

MNC

Nonactivated T T T-Tar (T-Tar) + Tar Tar

206.1 197.7 89.9 155.9 75.3 68.8

58.3 56.5 30.2 49.4 22.8 18.3

6200 6150 3950 5450 3600 2950

a Cells activated by Con-A were added to fresh autologous MNC (responding cells) and their inhibitory activity was determined in PHA- or alloantigen-induced proliferation and PWM-driven immunoglobulin production. ’ Mean cpm X lo-’ of triplicate cultures.

T cells are the findings that Tar cells lose their capacity to generate suppression if they are treated with mitomycin C before their activation by Con-A and that this sensitivity to mitomycin C is no longer apparent once Tar cells have completed the activation period. This indicates that Tar cells initially need to proliferate

278

RONALD PALACIOS

probably to differentiate into TP and Ty cells, both T-cell subpopulations with capacity to exert suppression (8, 14-l 7). The results described here are in agreement with those of other authors, who showed that a population of T cells depleted of Ty cells can generate in vitro Tr cells and their suppressor activity (8, 14-17), findings that are in accordance with those reported previously to take place in Tar cells, such as that Tar cells are devoid of Fc receptors and able to differentiate into Ty cells after stimulation with ConA or the serum thymic factor (18, 19). Recently, Sakane et al. (22) and Innes et al. (23) have described that the responding T cells in the autologous mixed lymphocyte reaction (AMLR) can be induced by Con-A to generate suppression, in agreement with the results presented here, since we have recently described that Tar cells are the responding cells in the AMLR (20). The possibility that Tar cells participate in these assays by activating other cells to become suppressors is no longer tenable, since it was found here that a purified population of Tar cells activated by Con-A generate suppressor cell activity. It is important to mention, that autoimmune diseasesin which defective Con-Ainduced suppressor cell function has been detected (24, 25) are also known to have altered numbers or functions of Tar cells (26, 27) which provides new insights in the cellular bases of the immunoregulatory aberrations that occur in these entities (26-28) and tools to improve our knowledge regarding the regulation of the immune response in man. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Gershon, R. K., Contemp. Top. Immunobiol. 3, 1, 1974. Pierce, C. W., and Kapp, J. A., Contemp. Top. Immunobiol. 5, 91, 1976. Nespoli, L., Milller, G., Waterfield, D., and Eksted, R., J. Exp. Med. 145, 631, 1977. Kurnick, J. T., Bell, C., and Grey, H. M., Scund. J. Immunof. 5, 771, 1976. Shou, L., Schwartz, S. A., and Good, R. A., J. Exp. Med. 143, 1100, 1976. Hubert, C., Delespesse,G., and Govaerts, A., Clin. Exp. Immunol. 26, 95, 1976. Haynes, B. F., and Fauci, A. S., J. Immunol. 120, 700, 1978. Sakane, T., and Green, I., J. Immunol. 119, 1169, 1977. Mayumi, M., Yoshida, T., Shinomiya, K., Nishikawa, S., Hirata, T., Izumi, T., and Mikawa, H., J. Immunol. 123, 772,1979. 10. Lobo, P. I., and Spencer, C. E., 1. C/in. Invest. 63, I 157, 1979. 11. Haynes, B. F., and Fauci, A. S., J. Immunol. 118, 2281, 1977. 12. Morimoto, C., Clin. Exp. Immunol. 32, 125, 1978. 13. Haynes, B. F., and Fauci, A. S., J. Immunol. 121, 559, 1978. 14. Herscowitz, H. B., Sakane, T., Steinberg, A. D., and Green, I., J. Immunol. 124, 1403, 1980. 15. Hayward, A. R., Lydyard, L., Lydyard, P. M., Moretta, L., Daggs, M., and Lawton, A. R., J. Immunol. 121, 1, 1978. 16. Lydyard, P. M., and Hayward, A. R., C/in. Exp. Immunol. 39, 496, 1979. 17. Haynes, B. F., Mann, D. L., Hemler, M. E., Schroer, J. A., Scelhamer, J. H., Strominger, J. L., Thomas, C. A., Mostowski, H. S., and Fauci, A. S., Proc. Nat. Acad. Sci. USA 77, 2914, 1980. 18. Palacios, R., Alarcbn-Segovia, D., Llorente, L., Ruiz-ArguelIes, A., and Diaz-Jouanen, E., Immunology 42, 127, 1981. 19. Palacios, R., Doctoral thesis, National University of Mexico, 1980. 20. Palacios, R., Llorente, L., Alarc&Segovia, D., Ruiz-Arguelles, A., and Diaz-Jouanen, E., J. Clin. Invest. 65, 1527, 1980. 21. Pryjma, J. L., Munoz, L., Galbraigh, R. M., Fudenberg, H. H., and Virella, G., J. Immunol. 124, 656, 1980. 22. Sakane, T., and Green, I., J. Immunol. 123, 584, 1979.

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23. Innes, J. B., Kuntz, M. M., Kim, Y. T., and Weklesr, M. E., J. C/in. Invest. 64, 1608, 1979. 24. Abdou, N. I., Sagawa, A., Pascual, E., Hebert, J., and Sadeghee, S., C/in. Immunol. Immunopathol. 6, 192, 1976. 25. Ruiz-Arguelles, A., Alar&n-Segovia, D., Llorente, L., and Del Giudice, J. A., Arthritis Rheum. 23, 1004. 1980. 26. Alar&n-Segovia, D., and Ruiz-Arguelles, A., Arthritis Rheum. 23, 143, 1980. 27. Palacios, R., Alarcbn-Segovia, D., Llorente. L., Ruiz-Arguelles, A., and Fisbein, E., J. C/in. Lab. Immunol., in press. 28. Alar&t-Segovia, D., and Palacios, R., Arthritis Rheum., in press. 29. Palacios, R., and Alarcbn-Segovia, D., Clin. Immunol. Immunopathol. 18, 362, 1981.