- Email: [email protected]
Con-A-induced suppressor activity of lymphocytes distinguished by the presence or absence of the Fc receptor
CELLULAR
IMMUNOLOGY
47, 90-99 (1979)
Con-A-Induced Suppressor Activity of Lymphocytes Distinguished by the Presence or Absence of the Fc Receptor’ ANNE
COOKE,
Department
LESLEY
HEPPELL,
of Immunology,
Middlesex Received
PATRICIA Hospital
HUTCHINGS,
Medical
November
School,
AND IVAN London
WIP
9PG,
M. ROITT England
14, 1978
Lymphocytes bearing a receptor for the Fc region of the IgG molecule (Fey+) were separated from those lacking the receptor (Fey-) by a two-step procedure involving adherence to antibody-coated bovine erythrocyte monolayers followed by rosetting with antibodycoated bovine erythrocytes and separation on a Ficoll-Metrizoate gradient. Using this procedure, we confirmed previous work showing that cytotoxicity induced by the graft versus host reaction was mediated by cells bearing the Fc receptor. Separated cell populations were cultured with Con-A for 48 hr and then tested for their ability to mediate nonspecific suppression of both primary and secondary responses to SRBC in vitro. Suppressor cells were generated from both Fey+ and Fey- cells following culture with Con-A. Therefore, no distinct functional differences in the ability to mediate nonspecific suppression can be attributed to subclasses of T-cells on the basis of possession of the FcR.
INTRODUCTION Receptors for the Fc2 portion of the IgG and IgM molecule have been detected on T-cells in both mouse and man (1,2). Functional differences have been attributed to subclasses of T-cells as defined by their Fc receptors. In the mouse, Stout and Herzenberg (3) have demonstrated that helper T-cells are Fc- and that while both Fc+ and Fc- cells respond to PHA stimulation in vitro, only Fc+ T-cells respond to Con-A (4) although subsequent recruitment of Fc- cells may occur.3 Conflicting results have been obtained regarding the relationship between T-cell cytotoxicity and the presence of an Fc receptor, some results indicating that the Fc+ cells are the cytotoxic effecters (5,6) while others attribute this function to i This work was supported by grants from the MRC and the Arthritis and Rheumatism Council. * Abbreviations used: B-cells, bone marrow-derived lymphocyte; T-cells, thymus-derived lymphocyte; SRBC, sheep red blood cells; FCS, fetal calf serum; PFC, plaque-forming cells; EA, antibody sensitized bovine erythrocyte; Fc+, Fc receptor-bearing lymphocyte; Fc-, Fc receptornegative lymphocyte; FcR, Fc receptor; Fey+, lymphocytes bearing Fc receptor for IgG; Fey-, lymphocytes not bearing Fc receptor for IgG; PBS, phosphate-buffered saline; PLL, poly-L-lysine; BSS, balanced salt solution. 3 We know that the rabbit antibovine antiserum used in our experiments is pure IgG, no IgM being present as detectable by gel diffusion. We therefore refer to our FcR-bearing lymphocytes as either Fey+ or Fey- in accord with the terminology used for human FcR-bearing lymphocytes. When we refer to other workers’ fmdings we use Fc+ or Fc- since it is not always clear whether there is the possibility of Fcp-bearing cells being present. 90 0008-8749/79/I 10090-10$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
FC RECEPTORS
AND
SUPPRESSOR
91
T-CELLS
both Fc+ and Fc- cells (7, 8). The suppressor T-cell in an antigen-specific system has been shown to be Fc- (9) while in a non-antigen-specific system the suppressor T-cells generated by a graft-versus-host response were shown to be Fc+ (10). In view of the finding that only Fc+ T-cells respond well to Con-A (3), we decided to investigate the possibility that the cells mediating nonspecific suppression following activation by Con-A (1 l- 13) were derived from an Fc+ subpopulation of T-cells. MATERIALS
AND METHODS
Mice
CBA (Ca) and (CBA x BALB/c)F, from our own colony were used, the original breeding stocks having been obtained from LAC, Carshalton. Immunization
Mice were primed by injections of 2 x 10RSRBC ip and used 10 days following the priming injections. Cell Cultures
Pooled mouse spleen cells (RBC lysed with Tris-buffered NH&l) were washed three times, separated into different cell populations, and cultured in petri dishes at lO’/ml in Eagle’s MEM, 5% FCS, 5 mM glutamine, nonessential amino acids and sodium pyruvate containing 4 pg/ml Con-A (ICN Pharmaceuticals Ltd.). All media and supplements were obtained from Flow Labs., Irvine, Scotland. Cultures were carried out in a humidified incubator at 37°C in an atmosphere of 5% CO, in air. After 48 hr the cells were then harvested, washed three times, and then cultured for a further 5 days with fresh spleen cells from either unprimed or primed mice to estimate their suppressor activity in an in vitro antibody response. In Vitro Antibody
Production
Mouse spleen cells were cultured in a modified Marbrook-Diener culture system (14), using 15 X IO6 fresh spleen cells to which 2 x lo6 suppressor cells and 2 x lo7 SRBC were added. The culture medium used was RPM1 1640 supplemented with 5% FCS, 5 mM glutamine, nonessential amino acids, sodium pyruvate, 100 pg/ml streptomycin, and 100 units/ml penicillin G. The cultures were harvested after 5 days and the number of antibody-forming cells was estimated using the modification of the hemolytic plaque assay described by Cunningham and Szenberg (15). All cultures were set up in triplicate or quadruplicate and results were expressed as arithmetic means +- standard deviation. Production
of Sensitized
Bovine Erythrocytes
(EA)
A 5% suspension of bovine erythrocytes (Tissue Culture Services, Slough) was incubated with a subagglutinating dose of the y-globulin fraction of a hyperimmune rabbit antibovine erythrocyte antiserum for 45 min at room temperature. Cells were washed three times with cold PBS and resuspended at 5 x 10Vml in PBS. Separation
of Fey+ from
(a) On poly-L-lysine
Fey-
(PLL)
Cells Plates (Modified
from
Kennedy
and Axelrad
(16)).
Plastic petri dishes (Corning, 5 cm) were treated with 2 ml of a 0.025 mg/ml solution
92
COOKE
ET AL.
of PLL (Sigma, St. Louis, MO.) in PBS for 15 min at room temperature. The plates were washed three times with PBS and 2.5 x lo8 sensitized bovine erythrocytes in 2 ml of PBS were added immediately. The cells were allowed to settle for 30 min agitating every 15 min to allow the formation of even monolayers. Plates were washed well and 3 x IO7 spleen cells were added to each plate in 2 ml of BSS supplemented with 5% FCS. These were incubated at room temperature for 45 min and then gently filled with BSS, inverted, and left at room temperature for a further 45 min to allow unattached lymphocytes to fall off. These unattached cells (Fey-) were collected, and Fey+ cells bound to the EA monolayer were removed by vigorous washing. (6) On Ficoll-Metrizoate (Parish and Hayward (17)). This procedure was used to remove any remaining Fey+ cells from the Fey- population. Fey+ cells remaining within the Fey- population were rosetted by suspending the population at 1 x lo7 cells/ml in BSS supplemented with 5% FCS and mixing with EA at a 25: 1 ratio of sensitized erythrocytes:lymphocytes. Cells were spun at 15Og for 2 min, incubated 5 min on ice, and gently resuspended in BSS. Of the rosette suspension (2 x 10’ lymphocytes), 5 ml was layered onto Ficoll-Metrizoate (17) and centrifuged at 8OOg for 20 min at 20°C. Fey- cells were collected from the interface between the medium and the Ficoll-Metrizoate. Contamination of the Fey- cells with Fey+ was measured by further rosetting using the above procedure and was always less than 3%. Spleen cells mixed with unsensitized bovine erythrocytes and treated by this method gave a “mock” depleted population at the interface between the medium and Ficoll-Metrizoate which was used as a control in all experiments. Biological
Assessment of Purity
of the Fey+ and Fey- Populations
The purity of the separation was estimated by employing the system described by Fridman et al. (10) in which they find that only Fc+ cells mediate cytotoxicity if separation is efficient. Lethally irradiated (CBA x BALB/c)F, mice were reconstituted with 1 x lo8 CBA thymocytes, generating a graft-versus-host response with anti-H-2d specificity. Five days following the reconstitution, spleen cells were separated into Fey+ and Fey- populations. The specific cytotoxic activity of these populations against 51Cr-labeled P815Y (H-2d) targets were measured using a microtiter plate modification of the method of Brunner et al. (18). The percentage specific lysis, measured by Wr release, was calculated by: cpm experimental release - cpm background release x 100. cpm maximum release - cpm background release RESULTS Effect of Fey- and Fey+ Cells Stimulated Response in Vitro
with Con-A on the Primary
Immune
In the first series of experiments spleen cells were separated into Fey+ and Feypopulations. In these experiments the Fey- populations contained less than 3% Fey+ cells. Since it can be shown that loss of cells occurs on gradient separation, it was necessary to include mock depleted populations in all experiments to control for loss or enrichment of suppressor cells. All experiments therefore included two
FC RECEPTORS
AND
SUPPRESSOR
T-CELLS
93
94
COOKE ET AL.
controls: (i) unseparated spleen; (ii) “mock” depleted cells (see under Materials and Methods). In all experiments unseparated cells and mock depleted cells gave the same results. A typical experiment can be seen in Fig. 1. It is clear that both the Fey+ and Fey- populations are capable of suppressing the primary immune response in vitro following 48-hr incubation with Con-A, and that both the direct and indirect plaque responses were affected. The extent of suppression by Fey+ and Fey- cells was similar in most experiments but in two out of six experiments the direct response and the indirect response was suppressed to a significantly greater extent (P < 0.01) by the Fey+ population. The extent of suppression obtained by the Fey+, Fey-, and mock depleted populations was not significantly different from that observed by the unseparated spleen cells. Furthermore, the suppression seen is not due to the simple carry over of Con-A since cells cultured for 24 hr with Con-A are unable to suppress either a primary or a secondary response in vitro. For example, a typical result demonstrating the inability of cells cultured for 24 hr with Con-A to suppress the immune response in vitro is shown in Table 1. Effect of Fey- and Fey+ Cells Stimulated with Con-A on the Secondary Immune Response in Vitro
It seemed possible that differential suppressor activity of the Fey+ and Fey- cells might be expressed in their ability to suppress virgin or memory cells. Therefore, the second series of experiments was designed to test whether suppression of the secondary response was similar to that of the primary. From Table 2, it can be clearly seen that both the Fey+ and Fey- populations suppressed the direct and indirect plaque-forming cell response of the secondary immune response in vitro. In one out of five experiments the indirect response was suppressed to a significantly greater extent (P < 0.01) by the Fey+ than the Fey- cell populations. The direct response was suppressed to the same extent by both populations. We therefore conclude that both the Fey+ and Fey- populations were capable of being stimulated by Con-A to generate suppressor cells, and the suppression of the primary response in vitro was similar to that of the secondary response in vitro. Assessment of Effectiveness of Separation Procedure
It is important obtained.
to demonstrate
that a clear separation of Fey+ and Fey- cells was
TABLE
1
Effect of 24- and 48-hr Con-A Stimulated Cells on the Primary Immune Response in Vitro Cells cultured
Suppressor cells (2 x lO%ulture)
CBA spleen cells (15 x 103
None + None + None
Time cultured with Con-A (hr)
Antigen (2 x 10’ SRBCkulture)
24 48
a Values are arithmetic means of three cultures f SD.
+ + + + -
PFCkulture” Direct 2860 3560 4440 2740 160
k 2 2 2 r
880 1200 680 440 60
Indirect 3040 3980 5600 2840 520
k 640 +- 840 ‘- 500 -t 420 k 180
FC RECEPTORS
AND
SUPPRESSOR
TABLE Ability
of Cells
95
T-CELLS
2
Generated from Fey- and Fey+ Populations Cultured to Suppress the 2°C Immune Response in Vim
with
Con-A
PFClculture Antigen (2 x IO’ SRBClculture)
Suppressor cells (2 x I0”iculture)
Cells cultured Primed CBA spleen cells (IS x IO?
NOtIC? Unseparated Mock depleted FCY’ Fey NOW
a Values are arithmetic li Values are anthmettc * Significant difference
Experiment
1”
lhrect
+ + + + +
81043 2320 l4M)? II40 2080 4602
means of four cultures 2 SD. means of three cultures + SD. between Fey’ and Fey- suppression
Experiment
Indirect
2 3120 k 400 760 2 120 z 660 I60
6380 I660 ll6Ot 960 2250 4002
2D lndlrect
DlKCt
+ 1340 + IM) 800 z 480* 2 520 360
4520 1480 2OOil 1020 1460 360
e e k t k k
840 680 580 400 240 200
3400 II80 1440 780 1280 440
+ t + r k t
500 600 420 300 180 180
(P c 0.01).
The possibility that the Fey- population was contaminated with Fey+ cells whose Fc receptor had become lost or blocked during the separation procedure and could therefore not rosette with EA, was investigated by the following experiments: (i) Fey- cells were cultured overnight in an attempt to allow regeneration or unblocking of the FcR. No change in the number of contaminating Fey+ cells was observed (Table 3a). (ii) Unseparated spleen cells were incubated for 5 hr at O”C, room temperature, and 37°C to monitor possible loss of FcR. During this time there was no significant change in the percentage of rosetting cells (Table 3b). TABLE Effect (a) Culture
of Culture
3 on the FcR
of the Fey-
population Percentage of Fey+ in Fey- population”
No incubation overnight at 37°C
1.0 2 I.0 1.8 + 1.6
No incubation 48 hr at 37°C
0.5 f 0.7 1.0 2 0.5 (b) Culture Length of culture (hr)
Temperature (“C) 0 0 22 37 o Results
mean
None 5 5 5 t SD.
of unseparated
spleen
cells % Fey+
Experiment 58.8 57.2 56.0 54.5
? + 2 t
1 5.5 3.3 7.8 6.5
cells” Experiment 55.4 55.8 54.4 51.4
k k k 2
2 4.8 5.0 5.7 2.6
96
COOKE
ET AL.
(iii) Treatment of the Fey- population with trypsin (0.1 mg/ml) for 15 min at 37°C (Stout and Herzenberg (4)), conditions designed to release any complexes which could be blocking the FcR, did not lead to an increase in the number of contaminating Fey+ cells within the Fey- population. The possibility that the Fey+ population was contaminated with Fey- cells adhering nonspecifically to the sensitized erythrocyte monolayers was eliminated by the demonstration that less than 3% of whole spleen cells adhere nonspecifically to an unsensitized erythrocyte monolayer (Table 4). Also, the adherence of Feycells to a sensitized erythrocyte monolayer was no greater than to an unsensitized monolayer (Table 5) indicating that any adherence of Fey- cells to an antibody-sensitized monolayer is both nonspecific and low. To determine whether Fey+ cells could be generated from Fey- cells under our culture conditions, we incubated Fey- cells for 48 hr without Con-A. Table 3a shows that no increase in the number of Fey+ cells occurred under our culture conditions. Finally, we demonstrated the purity of the separation by a functional assay, following the procedure of Leclerc er al. (6) to generate alloantigen-activated thymus cells which we separated into Fey+ and Fey- populations. From Fig. 2 it can be seen that cytotoxic activity was found to reside only in the Fey+ population in agreement with the results of Leclerc et al. (6). This implies that there is very little contamination of the Fey- population with Fey+ cells. DISCUSSION The validity of functional studies on isolated populations of Fey+ and Fey- cells depends critically on the effectiveness of the initial separation techniques. In all experiments carried out the maximum contamination of Fey- with Fey+ cells was 3% by rosetting and less than 3% as measured by adherence to antibody-sensitized erythrocyte monolayers. Since we show that only 3% of the Fey- population adheres to the antibody-sensitized erythrocyte monolayer we conclude that the contamination of Fey+ cells by Fey- cells is minimal. Furthermore, it is probable that this 3% is an upper estimate of Fey+ contamination since a comparable percentage adhere nonspecifically to unsensitized erythrocyte monolayers. Contamination of the Fey- populations with Fey+ cells could also occur either by blocking of the Fc receptors by complexes or frank loss of the receptors during the separation procedures. However, strategies aimed at removal of bound complexes TABLE Nonspecific
Adherence
of Spleen
Cells
to Monolayers Number
Experiment
Number of cells added to plates
1 2 3 (1 The plate monolayers.
6 x IO7 6 x 10’ 6 x 10’ depletion
method
Adherent
of Unsensitized
Bovine
of cells recovered Nonadherent
1.3 x 10” 1.5 x 10” 0.9 x 10” was followed,
4
5.7 x 10’ 5.5 x 107 5.1 x 107 adding
spleen
Total
Erythrocytes” Adherent cells/ total recovered cells (%)
5.83 x lo7 5.65 x 10’ 5.19 x 107 cells to unsensitized
2.2 2.7 1.7 bovine
erythrocyte
FC RECEPTORS
AND
SUPPRESSOR
TABLE Adherence
of Fey-
Cells
to Sensitized
Antibody-sensitized monolayer Unsensitized monolayer
5
and Unsensitized Number
Number cells added to plate
Adherent
97
T-CELLS
Bovine
Erythrocyte
of cells recovered Nonadherent
Total
16 x IO<
0.26
x lOti
10.4 x 10”
16 x 10”
0.38 x 10”
9.9 x 10”
10.66
Monolayers” Adherent cells/ total recovered cells (%)
x 10”
2.5
10.28 x 10”
3.7
(I 16 x 10” Fey- cells were added either to antibody-sensitized or -unsensitized bovine erythrocyte monolayers and treated by the plate depletion method (see under Materials and Methods).
such as incubation of Fey- cells overnight at 37°C or brief treatment with trypsin did not increase the number of contaminating Fey+ cells; neither was there evidence for loss of receptors on incubation of unseparated spleen cells. We have also excluded the possibility that Fey+ cells were generated from Fey- cells following Con-A stimulation or culture for 2 days, suggesting that the two cell types are probably not simply at different stages of a common pathway but represent separate populations. That our procedures can lead to funtionally distinct populations is shown by our demonstration that cytotoxic activity arising during a graft-versus-host reaction resided primarily in the Fey+ population in accord with the experiments of Leclerc et al. (6) who separated the population by rosette depletion techniques. The two-step procedure which we have employed would appear to have some advantages over conventional methods of rosette depletion. (1) Nonspecific sticking of lymphocytes to unsensitized monolayers is very low, less than 3%, compared with 6-12% nonspecific losses through the Ficoll- Metrizoate.
50 -
l I
40 % Specific
Lysis unseparated I
1:
rFc
10 L,
o
/Fct
0 25:l Effector:
50: 1
1W:l
Target Ratio
FIG. 2. Specific cytotoxicity of Fey+ and Feylymphocytes (CBA x BALB/c)F, mice reconstituted with 1 X 10” CBA thymocytes. days after reconstitution and cytotoxicity was measured by specific targets.
from spleens of irradiated Spleen cells were harvested 5 “Cr release from labeled P815Y
98
COOKE
ET AL.
(2) It is useful to include a separation step which does not depend upon cell size or density since centrifugation methods of rosette depletion must overcome the possibility that suppressor cells may be more or less dense than helper cells. (3) The plate method will enable us to study cells with FcR of different affinities since the erythrocytes can be sensitized at a range of antibody concentrations from an agglutinating dose to a concentration where the number of rosettes is practically undetectable but the number of lymphocytes adhering to the sensitized monolayers is still significant. (4) There is less possibility of weakly binding Fey+ cells falling off the monolayer than of fragile rosettes breaking up during the centrifugation procedure. Our present studies using the two-step technique show convincingly that both Fey+ and Fey- populations, when stimulated by Con-A, generate cells capable of suppressing both primary and secondary antibody responses to sheep erythrocytes in vitro. These results compare closely with the finding in the human that Con-A can stimulate both Tp and Ty cells to suppress immunoglobulin production induced by pokeweed mitogen in a human system (19, 20). In the light of Stout and Herzenberg’s (4) report that Con-A is mitogenic only for the Fey+ population, these findings might seem paradoxical were it not for the demonstration by Tse and Dutton (21) that functional activities induced by Con-A do not depend upon DNA synthesis. Furthermore, in the human, suppressor activity generated by Con-A treatment is associated with both the blast and the “nonblast” populations (22). Several mechanisms of suppression have been postulated, including direct cell-to-cell contact (23) and the operation of a variety of soluble products of different molecular weights (9, 24-27), some antigen-specific, the others not. It is perhaps worth noting that preliminary results (Cooke et al., unpublished) indicate that only the Fey+ cells make interferon, which among other properties, is known to inhibit antibody production by lymphocytes. It would seem likely that the variations in behavior of the Fey+ and Fey- populations in different suppressor systems will reflect the variety of mechanisms which come into play under different circumstances and that possession of the FcR is not a necessary prerequisite for suppressor activity. ACKNOWLEDGMENT We should
like
to thank
Christine
Meats
for typing
this manuscript,
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO.
Anderson, C. L., and Grey, H. M., J. Exp. Med. 139, 1175, 1974. Dickler, H. B., Advan. Zmmunol. 24, 167, 1976. Stout, R. D., and Herzenberg, L. A., J. Exp. Med. 142, 611, 1975. Stout, R. D., and Herzenberg, L. A., J. Exp. Med. 142, 1041, 1975. Stout, R. D., Waksal, S. D., and Herzenberg, L. A., J. Exp. Med. 144, 54, 1976. Leclerc, J. C., Plater, C., and Fridman, W. H., Eur. J. Immunol. 8, 543, 1977. Krammer, P. H., Elliot, D. E., and von Boehmer, H., Eur. J. Zmmunol. 6, 138, 1976. Hayry, P. K., and Andersson, L. C., Cell. Zmmunol. 25, 237, 1976. Okumura, K., Takemori, T., Tokuhisa, T., and Tada, T., J. Exp. Med. 146, 1234, 1977. Fridman, W. H., Fradelizi, D., Guimezanes, A., Plater, C., and Leclerc, J. C., Eur. J. Zmmunol. 549, 1977. 11. Dutton, R. W., J. Exp. Med. 136, 1445, 1972. 12. Dutton, R. W.,J. Exp. Med. 138, 1496, 1973.
8,
FC RECEPTORS 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
AND
SUPPRESSOR
T-CELLS
99
Rich, R. R., and Pierce, C. W., J. Exp. Med. 137, 649, 1973. Feldman, M., and Basten, A., J. Exp. Med. 134, 103, 1971. Cunningham, A. J., and Szenberg, A., Immunology 14, 599, 1968. Kennedy. J. C., and Axelrad, M. A., Immunology 20, 253, 1971. Parish, C. R., and Hayward, J. A., Proc. R. Sot. London Srr. A. 187, 65, 1974. Brunner, K. T., Mattel, J., Cerrottini, J. C., and Chapuis, B., Immunology 14, 181, 1968. Hayward, A. R., Layward, L., Lydyard, P., Moretta, L., Dagg, M., and Lawton, A. R., J. Immunol. 121, 1, 1978. Haynes, B. F., and Fauci, A. S., J. fmmunol. 121, 559, 1978. Tse, H. Y., and Dutton, R. W., J. Exp. Med. 146, 747, 1977. Sakane, T., and Green, I., J. Immunol. 119, 1169, 1977. Parkhouse, R. M. E., and Dutton, R. W., J. Immunol. 97, 663, 1966. Rich, R. R., and Pierce, C. W., J. Immunol. 112, 1360, 1974. Tadakuma, T., Kuhner, A. L., Rich, R. R., David, J. R., and Pierce, C. W., J. Immunol. 117, 323, 1976. Waldman, S. R., and Gottlieb, A. A., Cell. Immunol. 9, 142, 1973. Curtiss, L. K., and Edgington, T. S., J. fmmunol. 118, 1966, 1977.
IMMUNOLOGY
47, 90-99 (1979)
Con-A-Induced Suppressor Activity of Lymphocytes Distinguished by the Presence or Absence of the Fc Receptor’ ANNE
COOKE,
Department
LESLEY
HEPPELL,
of Immunology,
Middlesex Received
PATRICIA Hospital
HUTCHINGS,
Medical
November
School,
AND IVAN London
WIP
9PG,
M. ROITT England
14, 1978
Lymphocytes bearing a receptor for the Fc region of the IgG molecule (Fey+) were separated from those lacking the receptor (Fey-) by a two-step procedure involving adherence to antibody-coated bovine erythrocyte monolayers followed by rosetting with antibodycoated bovine erythrocytes and separation on a Ficoll-Metrizoate gradient. Using this procedure, we confirmed previous work showing that cytotoxicity induced by the graft versus host reaction was mediated by cells bearing the Fc receptor. Separated cell populations were cultured with Con-A for 48 hr and then tested for their ability to mediate nonspecific suppression of both primary and secondary responses to SRBC in vitro. Suppressor cells were generated from both Fey+ and Fey- cells following culture with Con-A. Therefore, no distinct functional differences in the ability to mediate nonspecific suppression can be attributed to subclasses of T-cells on the basis of possession of the FcR.
INTRODUCTION Receptors for the Fc2 portion of the IgG and IgM molecule have been detected on T-cells in both mouse and man (1,2). Functional differences have been attributed to subclasses of T-cells as defined by their Fc receptors. In the mouse, Stout and Herzenberg (3) have demonstrated that helper T-cells are Fc- and that while both Fc+ and Fc- cells respond to PHA stimulation in vitro, only Fc+ T-cells respond to Con-A (4) although subsequent recruitment of Fc- cells may occur.3 Conflicting results have been obtained regarding the relationship between T-cell cytotoxicity and the presence of an Fc receptor, some results indicating that the Fc+ cells are the cytotoxic effecters (5,6) while others attribute this function to i This work was supported by grants from the MRC and the Arthritis and Rheumatism Council. * Abbreviations used: B-cells, bone marrow-derived lymphocyte; T-cells, thymus-derived lymphocyte; SRBC, sheep red blood cells; FCS, fetal calf serum; PFC, plaque-forming cells; EA, antibody sensitized bovine erythrocyte; Fc+, Fc receptor-bearing lymphocyte; Fc-, Fc receptornegative lymphocyte; FcR, Fc receptor; Fey+, lymphocytes bearing Fc receptor for IgG; Fey-, lymphocytes not bearing Fc receptor for IgG; PBS, phosphate-buffered saline; PLL, poly-L-lysine; BSS, balanced salt solution. 3 We know that the rabbit antibovine antiserum used in our experiments is pure IgG, no IgM being present as detectable by gel diffusion. We therefore refer to our FcR-bearing lymphocytes as either Fey+ or Fey- in accord with the terminology used for human FcR-bearing lymphocytes. When we refer to other workers’ fmdings we use Fc+ or Fc- since it is not always clear whether there is the possibility of Fcp-bearing cells being present. 90 0008-8749/79/I 10090-10$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
FC RECEPTORS
AND
SUPPRESSOR
91
T-CELLS
both Fc+ and Fc- cells (7, 8). The suppressor T-cell in an antigen-specific system has been shown to be Fc- (9) while in a non-antigen-specific system the suppressor T-cells generated by a graft-versus-host response were shown to be Fc+ (10). In view of the finding that only Fc+ T-cells respond well to Con-A (3), we decided to investigate the possibility that the cells mediating nonspecific suppression following activation by Con-A (1 l- 13) were derived from an Fc+ subpopulation of T-cells. MATERIALS
AND METHODS
Mice
CBA (Ca) and (CBA x BALB/c)F, from our own colony were used, the original breeding stocks having been obtained from LAC, Carshalton. Immunization
Mice were primed by injections of 2 x 10RSRBC ip and used 10 days following the priming injections. Cell Cultures
Pooled mouse spleen cells (RBC lysed with Tris-buffered NH&l) were washed three times, separated into different cell populations, and cultured in petri dishes at lO’/ml in Eagle’s MEM, 5% FCS, 5 mM glutamine, nonessential amino acids and sodium pyruvate containing 4 pg/ml Con-A (ICN Pharmaceuticals Ltd.). All media and supplements were obtained from Flow Labs., Irvine, Scotland. Cultures were carried out in a humidified incubator at 37°C in an atmosphere of 5% CO, in air. After 48 hr the cells were then harvested, washed three times, and then cultured for a further 5 days with fresh spleen cells from either unprimed or primed mice to estimate their suppressor activity in an in vitro antibody response. In Vitro Antibody
Production
Mouse spleen cells were cultured in a modified Marbrook-Diener culture system (14), using 15 X IO6 fresh spleen cells to which 2 x lo6 suppressor cells and 2 x lo7 SRBC were added. The culture medium used was RPM1 1640 supplemented with 5% FCS, 5 mM glutamine, nonessential amino acids, sodium pyruvate, 100 pg/ml streptomycin, and 100 units/ml penicillin G. The cultures were harvested after 5 days and the number of antibody-forming cells was estimated using the modification of the hemolytic plaque assay described by Cunningham and Szenberg (15). All cultures were set up in triplicate or quadruplicate and results were expressed as arithmetic means +- standard deviation. Production
of Sensitized
Bovine Erythrocytes
(EA)
A 5% suspension of bovine erythrocytes (Tissue Culture Services, Slough) was incubated with a subagglutinating dose of the y-globulin fraction of a hyperimmune rabbit antibovine erythrocyte antiserum for 45 min at room temperature. Cells were washed three times with cold PBS and resuspended at 5 x 10Vml in PBS. Separation
of Fey+ from
(a) On poly-L-lysine
Fey-
(PLL)
Cells Plates (Modified
from
Kennedy
and Axelrad
(16)).
Plastic petri dishes (Corning, 5 cm) were treated with 2 ml of a 0.025 mg/ml solution
92
COOKE
ET AL.
of PLL (Sigma, St. Louis, MO.) in PBS for 15 min at room temperature. The plates were washed three times with PBS and 2.5 x lo8 sensitized bovine erythrocytes in 2 ml of PBS were added immediately. The cells were allowed to settle for 30 min agitating every 15 min to allow the formation of even monolayers. Plates were washed well and 3 x IO7 spleen cells were added to each plate in 2 ml of BSS supplemented with 5% FCS. These were incubated at room temperature for 45 min and then gently filled with BSS, inverted, and left at room temperature for a further 45 min to allow unattached lymphocytes to fall off. These unattached cells (Fey-) were collected, and Fey+ cells bound to the EA monolayer were removed by vigorous washing. (6) On Ficoll-Metrizoate (Parish and Hayward (17)). This procedure was used to remove any remaining Fey+ cells from the Fey- population. Fey+ cells remaining within the Fey- population were rosetted by suspending the population at 1 x lo7 cells/ml in BSS supplemented with 5% FCS and mixing with EA at a 25: 1 ratio of sensitized erythrocytes:lymphocytes. Cells were spun at 15Og for 2 min, incubated 5 min on ice, and gently resuspended in BSS. Of the rosette suspension (2 x 10’ lymphocytes), 5 ml was layered onto Ficoll-Metrizoate (17) and centrifuged at 8OOg for 20 min at 20°C. Fey- cells were collected from the interface between the medium and the Ficoll-Metrizoate. Contamination of the Fey- cells with Fey+ was measured by further rosetting using the above procedure and was always less than 3%. Spleen cells mixed with unsensitized bovine erythrocytes and treated by this method gave a “mock” depleted population at the interface between the medium and Ficoll-Metrizoate which was used as a control in all experiments. Biological
Assessment of Purity
of the Fey+ and Fey- Populations
The purity of the separation was estimated by employing the system described by Fridman et al. (10) in which they find that only Fc+ cells mediate cytotoxicity if separation is efficient. Lethally irradiated (CBA x BALB/c)F, mice were reconstituted with 1 x lo8 CBA thymocytes, generating a graft-versus-host response with anti-H-2d specificity. Five days following the reconstitution, spleen cells were separated into Fey+ and Fey- populations. The specific cytotoxic activity of these populations against 51Cr-labeled P815Y (H-2d) targets were measured using a microtiter plate modification of the method of Brunner et al. (18). The percentage specific lysis, measured by Wr release, was calculated by: cpm experimental release - cpm background release x 100. cpm maximum release - cpm background release RESULTS Effect of Fey- and Fey+ Cells Stimulated Response in Vitro
with Con-A on the Primary
Immune
In the first series of experiments spleen cells were separated into Fey+ and Feypopulations. In these experiments the Fey- populations contained less than 3% Fey+ cells. Since it can be shown that loss of cells occurs on gradient separation, it was necessary to include mock depleted populations in all experiments to control for loss or enrichment of suppressor cells. All experiments therefore included two
FC RECEPTORS
AND
SUPPRESSOR
T-CELLS
93
94
COOKE ET AL.
controls: (i) unseparated spleen; (ii) “mock” depleted cells (see under Materials and Methods). In all experiments unseparated cells and mock depleted cells gave the same results. A typical experiment can be seen in Fig. 1. It is clear that both the Fey+ and Fey- populations are capable of suppressing the primary immune response in vitro following 48-hr incubation with Con-A, and that both the direct and indirect plaque responses were affected. The extent of suppression by Fey+ and Fey- cells was similar in most experiments but in two out of six experiments the direct response and the indirect response was suppressed to a significantly greater extent (P < 0.01) by the Fey+ population. The extent of suppression obtained by the Fey+, Fey-, and mock depleted populations was not significantly different from that observed by the unseparated spleen cells. Furthermore, the suppression seen is not due to the simple carry over of Con-A since cells cultured for 24 hr with Con-A are unable to suppress either a primary or a secondary response in vitro. For example, a typical result demonstrating the inability of cells cultured for 24 hr with Con-A to suppress the immune response in vitro is shown in Table 1. Effect of Fey- and Fey+ Cells Stimulated with Con-A on the Secondary Immune Response in Vitro
It seemed possible that differential suppressor activity of the Fey+ and Fey- cells might be expressed in their ability to suppress virgin or memory cells. Therefore, the second series of experiments was designed to test whether suppression of the secondary response was similar to that of the primary. From Table 2, it can be clearly seen that both the Fey+ and Fey- populations suppressed the direct and indirect plaque-forming cell response of the secondary immune response in vitro. In one out of five experiments the indirect response was suppressed to a significantly greater extent (P < 0.01) by the Fey+ than the Fey- cell populations. The direct response was suppressed to the same extent by both populations. We therefore conclude that both the Fey+ and Fey- populations were capable of being stimulated by Con-A to generate suppressor cells, and the suppression of the primary response in vitro was similar to that of the secondary response in vitro. Assessment of Effectiveness of Separation Procedure
It is important obtained.
to demonstrate
that a clear separation of Fey+ and Fey- cells was
TABLE
1
Effect of 24- and 48-hr Con-A Stimulated Cells on the Primary Immune Response in Vitro Cells cultured
Suppressor cells (2 x lO%ulture)
CBA spleen cells (15 x 103
None + None + None
Time cultured with Con-A (hr)
Antigen (2 x 10’ SRBCkulture)
24 48
a Values are arithmetic means of three cultures f SD.
+ + + + -
PFCkulture” Direct 2860 3560 4440 2740 160
k 2 2 2 r
880 1200 680 440 60
Indirect 3040 3980 5600 2840 520
k 640 +- 840 ‘- 500 -t 420 k 180
FC RECEPTORS
AND
SUPPRESSOR
TABLE Ability
of Cells
95
T-CELLS
2
Generated from Fey- and Fey+ Populations Cultured to Suppress the 2°C Immune Response in Vim
with
Con-A
PFClculture Antigen (2 x IO’ SRBClculture)
Suppressor cells (2 x I0”iculture)
Cells cultured Primed CBA spleen cells (IS x IO?
NOtIC? Unseparated Mock depleted FCY’ Fey NOW
a Values are arithmetic li Values are anthmettc * Significant difference
Experiment
1”
lhrect
+ + + + +
81043 2320 l4M)? II40 2080 4602
means of four cultures 2 SD. means of three cultures + SD. between Fey’ and Fey- suppression
Experiment
Indirect
2 3120 k 400 760 2 120 z 660 I60
6380 I660 ll6Ot 960 2250 4002
2D lndlrect
DlKCt
+ 1340 + IM) 800 z 480* 2 520 360
4520 1480 2OOil 1020 1460 360
e e k t k k
840 680 580 400 240 200
3400 II80 1440 780 1280 440
+ t + r k t
500 600 420 300 180 180
(P c 0.01).
The possibility that the Fey- population was contaminated with Fey+ cells whose Fc receptor had become lost or blocked during the separation procedure and could therefore not rosette with EA, was investigated by the following experiments: (i) Fey- cells were cultured overnight in an attempt to allow regeneration or unblocking of the FcR. No change in the number of contaminating Fey+ cells was observed (Table 3a). (ii) Unseparated spleen cells were incubated for 5 hr at O”C, room temperature, and 37°C to monitor possible loss of FcR. During this time there was no significant change in the percentage of rosetting cells (Table 3b). TABLE Effect (a) Culture
of Culture
3 on the FcR
of the Fey-
population Percentage of Fey+ in Fey- population”
No incubation overnight at 37°C
1.0 2 I.0 1.8 + 1.6
No incubation 48 hr at 37°C
0.5 f 0.7 1.0 2 0.5 (b) Culture Length of culture (hr)
Temperature (“C) 0 0 22 37 o Results
mean
None 5 5 5 t SD.
of unseparated
spleen
cells % Fey+
Experiment 58.8 57.2 56.0 54.5
? + 2 t
1 5.5 3.3 7.8 6.5
cells” Experiment 55.4 55.8 54.4 51.4
k k k 2
2 4.8 5.0 5.7 2.6
96
COOKE
ET AL.
(iii) Treatment of the Fey- population with trypsin (0.1 mg/ml) for 15 min at 37°C (Stout and Herzenberg (4)), conditions designed to release any complexes which could be blocking the FcR, did not lead to an increase in the number of contaminating Fey+ cells within the Fey- population. The possibility that the Fey+ population was contaminated with Fey- cells adhering nonspecifically to the sensitized erythrocyte monolayers was eliminated by the demonstration that less than 3% of whole spleen cells adhere nonspecifically to an unsensitized erythrocyte monolayer (Table 4). Also, the adherence of Feycells to a sensitized erythrocyte monolayer was no greater than to an unsensitized monolayer (Table 5) indicating that any adherence of Fey- cells to an antibody-sensitized monolayer is both nonspecific and low. To determine whether Fey+ cells could be generated from Fey- cells under our culture conditions, we incubated Fey- cells for 48 hr without Con-A. Table 3a shows that no increase in the number of Fey+ cells occurred under our culture conditions. Finally, we demonstrated the purity of the separation by a functional assay, following the procedure of Leclerc er al. (6) to generate alloantigen-activated thymus cells which we separated into Fey+ and Fey- populations. From Fig. 2 it can be seen that cytotoxic activity was found to reside only in the Fey+ population in agreement with the results of Leclerc et al. (6). This implies that there is very little contamination of the Fey- population with Fey+ cells. DISCUSSION The validity of functional studies on isolated populations of Fey+ and Fey- cells depends critically on the effectiveness of the initial separation techniques. In all experiments carried out the maximum contamination of Fey- with Fey+ cells was 3% by rosetting and less than 3% as measured by adherence to antibody-sensitized erythrocyte monolayers. Since we show that only 3% of the Fey- population adheres to the antibody-sensitized erythrocyte monolayer we conclude that the contamination of Fey+ cells by Fey- cells is minimal. Furthermore, it is probable that this 3% is an upper estimate of Fey+ contamination since a comparable percentage adhere nonspecifically to unsensitized erythrocyte monolayers. Contamination of the Fey- populations with Fey+ cells could also occur either by blocking of the Fc receptors by complexes or frank loss of the receptors during the separation procedures. However, strategies aimed at removal of bound complexes TABLE Nonspecific
Adherence
of Spleen
Cells
to Monolayers Number
Experiment
Number of cells added to plates
1 2 3 (1 The plate monolayers.
6 x IO7 6 x 10’ 6 x 10’ depletion
method
Adherent
of Unsensitized
Bovine
of cells recovered Nonadherent
1.3 x 10” 1.5 x 10” 0.9 x 10” was followed,
4
5.7 x 10’ 5.5 x 107 5.1 x 107 adding
spleen
Total
Erythrocytes” Adherent cells/ total recovered cells (%)
5.83 x lo7 5.65 x 10’ 5.19 x 107 cells to unsensitized
2.2 2.7 1.7 bovine
erythrocyte
FC RECEPTORS
AND
SUPPRESSOR
TABLE Adherence
of Fey-
Cells
to Sensitized
Antibody-sensitized monolayer Unsensitized monolayer
5
and Unsensitized Number
Number cells added to plate
Adherent
97
T-CELLS
Bovine
Erythrocyte
of cells recovered Nonadherent
Total
16 x IO<
0.26
x lOti
10.4 x 10”
16 x 10”
0.38 x 10”
9.9 x 10”
10.66
Monolayers” Adherent cells/ total recovered cells (%)
x 10”
2.5
10.28 x 10”
3.7
(I 16 x 10” Fey- cells were added either to antibody-sensitized or -unsensitized bovine erythrocyte monolayers and treated by the plate depletion method (see under Materials and Methods).
such as incubation of Fey- cells overnight at 37°C or brief treatment with trypsin did not increase the number of contaminating Fey+ cells; neither was there evidence for loss of receptors on incubation of unseparated spleen cells. We have also excluded the possibility that Fey+ cells were generated from Fey- cells following Con-A stimulation or culture for 2 days, suggesting that the two cell types are probably not simply at different stages of a common pathway but represent separate populations. That our procedures can lead to funtionally distinct populations is shown by our demonstration that cytotoxic activity arising during a graft-versus-host reaction resided primarily in the Fey+ population in accord with the experiments of Leclerc et al. (6) who separated the population by rosette depletion techniques. The two-step procedure which we have employed would appear to have some advantages over conventional methods of rosette depletion. (1) Nonspecific sticking of lymphocytes to unsensitized monolayers is very low, less than 3%, compared with 6-12% nonspecific losses through the Ficoll- Metrizoate.
50 -
l I
40 % Specific
Lysis unseparated I
1:
rFc
10 L,
o
/Fct
0 25:l Effector:
50: 1
1W:l
Target Ratio
FIG. 2. Specific cytotoxicity of Fey+ and Feylymphocytes (CBA x BALB/c)F, mice reconstituted with 1 X 10” CBA thymocytes. days after reconstitution and cytotoxicity was measured by specific targets.
from spleens of irradiated Spleen cells were harvested 5 “Cr release from labeled P815Y
98
COOKE
ET AL.
(2) It is useful to include a separation step which does not depend upon cell size or density since centrifugation methods of rosette depletion must overcome the possibility that suppressor cells may be more or less dense than helper cells. (3) The plate method will enable us to study cells with FcR of different affinities since the erythrocytes can be sensitized at a range of antibody concentrations from an agglutinating dose to a concentration where the number of rosettes is practically undetectable but the number of lymphocytes adhering to the sensitized monolayers is still significant. (4) There is less possibility of weakly binding Fey+ cells falling off the monolayer than of fragile rosettes breaking up during the centrifugation procedure. Our present studies using the two-step technique show convincingly that both Fey+ and Fey- populations, when stimulated by Con-A, generate cells capable of suppressing both primary and secondary antibody responses to sheep erythrocytes in vitro. These results compare closely with the finding in the human that Con-A can stimulate both Tp and Ty cells to suppress immunoglobulin production induced by pokeweed mitogen in a human system (19, 20). In the light of Stout and Herzenberg’s (4) report that Con-A is mitogenic only for the Fey+ population, these findings might seem paradoxical were it not for the demonstration by Tse and Dutton (21) that functional activities induced by Con-A do not depend upon DNA synthesis. Furthermore, in the human, suppressor activity generated by Con-A treatment is associated with both the blast and the “nonblast” populations (22). Several mechanisms of suppression have been postulated, including direct cell-to-cell contact (23) and the operation of a variety of soluble products of different molecular weights (9, 24-27), some antigen-specific, the others not. It is perhaps worth noting that preliminary results (Cooke et al., unpublished) indicate that only the Fey+ cells make interferon, which among other properties, is known to inhibit antibody production by lymphocytes. It would seem likely that the variations in behavior of the Fey+ and Fey- populations in different suppressor systems will reflect the variety of mechanisms which come into play under different circumstances and that possession of the FcR is not a necessary prerequisite for suppressor activity. ACKNOWLEDGMENT We should
like
to thank
Christine
Meats
for typing
this manuscript,
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO.
Anderson, C. L., and Grey, H. M., J. Exp. Med. 139, 1175, 1974. Dickler, H. B., Advan. Zmmunol. 24, 167, 1976. Stout, R. D., and Herzenberg, L. A., J. Exp. Med. 142, 611, 1975. Stout, R. D., and Herzenberg, L. A., J. Exp. Med. 142, 1041, 1975. Stout, R. D., Waksal, S. D., and Herzenberg, L. A., J. Exp. Med. 144, 54, 1976. Leclerc, J. C., Plater, C., and Fridman, W. H., Eur. J. Immunol. 8, 543, 1977. Krammer, P. H., Elliot, D. E., and von Boehmer, H., Eur. J. Zmmunol. 6, 138, 1976. Hayry, P. K., and Andersson, L. C., Cell. Zmmunol. 25, 237, 1976. Okumura, K., Takemori, T., Tokuhisa, T., and Tada, T., J. Exp. Med. 146, 1234, 1977. Fridman, W. H., Fradelizi, D., Guimezanes, A., Plater, C., and Leclerc, J. C., Eur. J. Zmmunol. 549, 1977. 11. Dutton, R. W., J. Exp. Med. 136, 1445, 1972. 12. Dutton, R. W.,J. Exp. Med. 138, 1496, 1973.
8,
FC RECEPTORS 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
AND
SUPPRESSOR
T-CELLS
99
Rich, R. R., and Pierce, C. W., J. Exp. Med. 137, 649, 1973. Feldman, M., and Basten, A., J. Exp. Med. 134, 103, 1971. Cunningham, A. J., and Szenberg, A., Immunology 14, 599, 1968. Kennedy. J. C., and Axelrad, M. A., Immunology 20, 253, 1971. Parish, C. R., and Hayward, J. A., Proc. R. Sot. London Srr. A. 187, 65, 1974. Brunner, K. T., Mattel, J., Cerrottini, J. C., and Chapuis, B., Immunology 14, 181, 1968. Hayward, A. R., Layward, L., Lydyard, P., Moretta, L., Dagg, M., and Lawton, A. R., J. Immunol. 121, 1, 1978. Haynes, B. F., and Fauci, A. S., J. fmmunol. 121, 559, 1978. Tse, H. Y., and Dutton, R. W., J. Exp. Med. 146, 747, 1977. Sakane, T., and Green, I., J. Immunol. 119, 1169, 1977. Parkhouse, R. M. E., and Dutton, R. W., J. Immunol. 97, 663, 1966. Rich, R. R., and Pierce, C. W., J. Immunol. 112, 1360, 1974. Tadakuma, T., Kuhner, A. L., Rich, R. R., David, J. R., and Pierce, C. W., J. Immunol. 117, 323, 1976. Waldman, S. R., and Gottlieb, A. A., Cell. Immunol. 9, 142, 1973. Curtiss, L. K., and Edgington, T. S., J. fmmunol. 118, 1966, 1977.