Distinct populations of antigen-presenting cells are required for activation of suppressor and contrasuppressor T cells by type III pneumococcal polysaccharide

CELLULAR

IMMUNOLOGY

128,528-54 1 (1990)

Distinct Populations of Antigen-Presenting Cells Are Required for Activation of Suppressor and Contrasuppressor T Cells by Type III Pneumococcal Polysaccharide’ HELEN Departmenls

BRALEY-MULLEN

of Medicine and Microbiology, University ofMissouri School of Medicine, Columbia, Missouri 652 I2 Received

July 28,1989;

accepted

March

7, 1990

Type III pneumococcal polysaccharide (S3) coupled to spleen cells (S3-SC) has been shown to activate S3-specific Ts and Tcs in mice. Ts activation required I-J identity between carrier SC and Ts donors whereas I-A identity was required for Tcs activation. The carrier SC therefore presumably function as APC for Ts and Tcs activation by S3 since they are apparently not represented by APC present in the Ts and Tcs donors. The properties of the APC required for activation of S3-specific Ts and Tcs were determined by coupling S3 to various spleen cell subpopulations and assessing the ability of the various SfSC populations to activate Ts and Tcs. The results indicate that Ts and Tcs are preferentially activated when S3 is presented on distinct cell types. S3-specific Ts were activated when S3 was coupled to plastic adherent cells. These cells are nonadherent to anti-& and nonfunctional in cyclophosphamide (Cy)-treated mice and their function is eliminated following treatment of cells with either anti-I-A or anti-I-J and C. In contrast, S3-specific Tcs were activated when S3 was coupled to anti-Ig adherent SC which bear I-A and the B cell marker Jl Id. These cells are functional in Cy-treated mice and their function is resistant to treatment with anti-I-J and C. Thus presentation of S3 on distinct cell types results in the preferential activation of T cells having opposing immunoregulatory fUnCtiOII.

0 1990 Academic

Press, Inc.

INTRODUCTION It is well established that activation of some subsets of T cells requires recognition of antigen in association with self-Ia molecules expressed on antigen-presenting cells (APC)2 ( 1,2). Several reports indicate that APC with distinct phenotypes are involved in the activation of TH versus Ts cells. For example, Tn are activated when antigen is presented in the context of self-I-A or I-E molecules on B cells, macrophages, or dendritic cells ( 1, 2). In contrast, several studies have shown that Ts are activated when antigen is presented by a specialized subset of macrophages which express I-J molecules (or receptors for self-I-J) as well as I-A molecules (3- 10). APC for Ts have also been distinguished from APC for Tn in that the former have been reported to be ’ This research was supported by Grant ROl -CA25054 from the National Institutes of Health. * Abbreviations used: APC, antigen-presenting cells; S3, type III pneumococcal polysaccharide; Cy, cyclophosphamide; Tcs, contrasuppressor T cells; S3-SC, S3 coupled to spleen cells; V.V., Vicia villosa lectin; PFC, plaque-forming cells. 528

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absent or nonfunctional in mice treated with cyclophosphamide (Cy) and resistant to uv irradiation (4,5,8, 1 I). While APC are presumably also required for activation of other regulatory T cells such as contrasuppressor T cells (Tcs) ( 12, 13), few studies have yet addressed the nature of the APC involved in Tcs activation. Previous studies from this laboratory indicated that the type 2 antigen type III pneumococcal polysaccharide (S3) coupled to syngeneic spleen cells (S3-SC) could activate S3-specific Ts in Cy-pretreated mice (14). The same preparations of S3-SC injected into normal (non-Cy-treated) mice also activated Ts but Ts could be demonstrated only after removal of a second T cell subset, Tcs, which were also induced by S3-SC (15). Activation of S3-specific Ts required presentation of S3 on SC which were I-J compatible with the Cy-treated Ts donors ( 14) suggesting that Ts were activated by S3 together with self-I-J determinants. On the other hand, I-J compatibility was not required for activation of Tcs by S3 although S3 coupled to cells which were I-A incompatible with Tcs donors could not activate Tcs (unpublished results). These observations raised the interesting possibility that Ts and Tcs might be activated by antigen coupled to distinct cell types. Moreover the fact that some genetic identity between the carrier SC and the Ts or Tcs donors was required for activation of both T cell subsets suggested that the functional APC for Ts and Tcs activation in this system were the SC to which the S3 was physically coupled, i.e., the injected S3-SC were apparently not re-presented by APC aiready present in the Ts or Tcs donors. There is as yet little information available concerning the requirement for or role of APC or accessory cells for T cell activation by polysaccharide or type 2 antigens although accessory cells are known to be required for B cell activation by such antigens ( 16 18). The present study was therefore undertaken in order to determine some of the characteristics of the APC required for Ts and Tcs activation by S3-SC. The results show that activation of Ts requires presentation of S3 on SC which are plastic adherent and Ig-negative and express molecules recognized by both anti-I-A mAb and an anti-I-J alloantiserum. In contrast, activation of Tcs by S3-SC requires an Igpositive cell which is I-A+ and J 11d+ but does not express molecules reactive with anti-I-J serum. Thus Ts and Tcs which reciprocally regulate the immune response to S3 are activated by antigen presented by distinct subsets of APC. MATERIALS

AND METHODS

Mice CAF, mice were obtained from Jackson Laboratories mice, 8- 12 weeks old, were used for all experiments. Preparation

(Bar Harbor, ME). Female

of S3-SC

S3 was coupled to spleen cells (SC) with chromic chloride as previously described ( 19). Mock-SC were treated with chromic chloride in the absence of antigen. In experiments involving coupling of S3 to separated SC populations, the cell separations were performed prior to the coupling procedure. Antibody and C Treatment of SC For some experiments SC were treated with alloantisera or monoclonal antibodies and C as previously described (19). The anti-I-JK (BIO.A(3R) X A.BY anti-

530

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BRALEY-MULLEN

BIO.A(SR) and anti-I-JB (A/Sri X BIO.A(SR) anti-BIO.A(3R) alloantisera were obtained from the NIH Serum Repository. These reagents were extensively tested and shown to have the appropriate specificity before being released by the NIH and have also been found in our hands to be specifically reactive with Ts cells from mice having the I-JK (3R anti-5R) or I-JB (5R anti-3R) haplotypes, respectively. A.TH anti-A.TL (anti-IaK) alloantiserum was prepared and tested in our laboratory (20). The monoclonal antibodies HO 13.4 (anti-Thyl.2) (21), 10.2.16 (anti-I-AK) (22), M5/144.15-2 (anti-I-AbTd,q, I-EK,d) (23), Ky76 (24) (anti-I-JK) and WF8.Cl2.8 (25) (anti-I-JK) and J 1 Id (26) were used as ascites. The cell lines producing these monoclonal antibodies (mAb) were obtained from the American Type Culture Collection or were obtained from Dr. Vera Hauptfeld (Washington University School of Medicine, St. Louis, MO) (Ky76) or Dr. Carl Waltenbaugh (Northwestern University School of Medicine, Chicago, IL). (WFC 12.8). Alloantisera were used at a final concentration of 1: 10 and ascites at a final concentration of 1:50 or 1: 100. These antibody concentrations were shown to be optimal for depletion of the appropriate cells by functional assays and, except for the anti-I-J antibodies, by cytotoxicity assays. In agreement with previous reports (4,25,27), the anti-I-J alloantisera and mAbs did not kill a readily measurable proportion of spleen cells (< 10%) reflecting the low frequency of I-J+ cells in normal spleen cell populations. Separation of SC on Anti-Ig-Coated

Plates

Normal CAF, spleen cells were separated into B cell-enriched (anti-Ig adherent) and B cell-depleted (anti-Ig nonadherent) fractions using polystyrene petri plates coated with 100 pg/ml of affinity-purified rabbit anti-mouse Ig (28). In our hands this procedure resulted in a nonadherent cell population that was depleted of 85-90% of splenic B cells whereas the adherent population contained B cells contaminated by < 10% T cells. Approximately 20-30% of the cells were recovered as nonadherent cells and 25-35% as adherent cells. Separation of SC by Adherence to Plastic Petri Plates Normal CAFr spleen cells were fractionated into plastic adherent and nonadherent cell populations by incubating cells in loo-mm tissue culture plates (Corning 25020) (6-7 X 10’ cells/plate in 6-7 ml BSS/2% FCS) for 1.5-2 hr at 37°C ( 18). Nonadherent cells were removed by gently swirling the plates and plates were rinsed twice with BSS to remove loosely adherent cells (the rinses were discarded). Versene (5 ml/plate) was then added and plates were incubated at 37°C for 5-15 min. Adherent cells were collected, cells were washed once in BSS and counted. Generally 40-60% of the cells were recovered as nonadherent cells and 15-25% as adherent cells. Induction

and Assay of Ts

Mice were injected ip with 100 mg/kg cyclophosphamide (Cy) (Cytoxan; Mead Johnson & Co., Evansville, IN); 2 days later they were injected iv with 5 X 10’ MockSC or S3-SC prepared as described for each individual experiment. In some experiments (e.g., Table 5) Cy-treated mice were injected iv with 1 pg S3 together with 5 X 10’ mitomycin C-treated CAF, spleen cells treated as indicated in the Table. Eight days later, 5 X lo6 spleen cells from these donors were transferred iv to groups of

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normal CAF, recipients. Recipient mice were immunized 4-6 hr later with 0.6 pg S3 and S3-specific IgM PFC were enumerated 5 days later (14). S3-SC has been shown to activate S3-specific CD 8+ I-J+ Ts in both Cy-treated and normal mice ( 14, 15). However, when normal mice are injected with S3-SC, Ts can be detected only after removal of Tcs on Vicia vilfosa (V.v.)-coated plates ( 15). Because elimination of Tcs with Cy allows activation and detection of Ts without requiring cell separation on V.V. ( 15), this method of Ts induction was used for the studies reported herein. The activation requirements, specificity, and characteristics of the Ts were described in detail previously ( 14). Induction

and Assay of Tcs

Mice were injected iv with 2.5-5 X 10’ S3-SC prepared as described in the individual experiments; 5-7 days later (15) spleen cells from these mice were separated on plates coated with the (V.V.) lectin (Sigma Chemical Co., St. Louis, MO) as described in detail previously ( 13, 15). Cells that adhered to V.V. (V.v.Ad) were mixed with an equal number (5 X 106) of Ts induced by injecting Cy-treated mice with S3-SC. Cells were injected into normal recipients which were immunized 4-6 hr later with 0.6 pg S3 (13, 15). Tcs activity is assessed by the ability of the V.v.Ad cells to counteract the suppression induced by S3-specific Ts ( 13, 15). The activation, specificity, and characteristics of Tcs were described in detail previously ( 13). Induction

and Assay of S3-Specijic Tolerance

Normal CAFl mice were injected iv with 2-3 X 10’ Mock- or SZSC; 3-7 days later mice were immunized with 0.6 pg S3 and PFC were determined 5 days later ( 19). Mitomycin

C Treatment

of SC

Spleen cells (lO’/ml in BSS) were incubated at 37°C for 40-45 min with 25 pg/ml mitomycin C (Sigma Chemical Co., St. Louis, MO) and washed three times before injection into mice. SC treated with mitomycin C proliferate minimally, if at all, to T or B cell mitogens and Tcs cannot be activated from mitomycin C-treated SC (29). RESULTS Characteristics

ofAPC Requiredfor

Activation of S3-Specific Ts

As mentioned in the Introduction, previous studies indicated that activation of Ts by S3-SC required carrier SC which were I-J compatible with the Cy-treated Ts donors ( 14) suggesting that the SC required for Ts activation might express I-J-encoded molecules. In order to assessthis possibility, spleen cells from CAFr (I-JK,D) mice were treated with anti-I-JK and complement (C) prior to coupling with S3. These cells were assessed for their ability to activate Ts in Cy-treated mice (Table 1, Experiments 1 and 2). Spleen cells from mice injected with S3 coupled to SC treated with an irrelevant anti-I-JB+C suppressed the S3 response after transfer to normal mice (line 1 vs line 2). In contrast, Ts were not induced when S3 was coupled to anti-I-JK+C-treated SC (lines 3 and 4) or to SC treated with a polyclonal anti-IaK serum which putatively has reactivity to I-J as well as I-A and I-E (lines 5 and 6, Experiment 1). These results indicate that molecules recognized by anti-I-JK alloantiserum are required for Ts acti-

532

HELEN BRALEY-MULLEN TABLE 1 Activation of Ts by S3-SC Requires I-J and I-A Bearing SC S3 PFC/spleenC

Donors given” Mock-SC s3-SC Mock-SC s3-SC Mock-SC s3-SC

Treatmentb of SC anti-I-Jr’ anti-I-JB anti-I-JK anti-I-JK anti-IaK anti-IaK

+C +C +C +C + C” + Cb

Expt 1 21,075 f 4090 7,012 f 1078 (67) 12,800 + 866 16,120&4065(O) 13,650d 13,460 -c 1660 (2)

Expt 2 6480+ 2950 + 5700+ 6100 + 7633 f 5720 +

963 55 l(54) 369 1257 (0) 626 980 (25)

’ CAF, mice were given 100 mg/kg Cy 2 days before receiving 5 X 10’ Mock- or S3-SC iv. SC were treated as indicated (see Materials and Methods) prior to coupling with 53. Eight days later 5 X lo6 spleen ceils from donor mice were transferred iv to normal CAF, recipients which were immunized 4-6 hr later with 0.6 rg S3 (14). * In Experiment 1 cells were treated with a polyclonal anti-IaK (A.TH anti-A.TL) and in Experiment 2 with a monoclonal anti-I-AK (10.2.16). ’ Mean IgM PFC/Spleen f SEM of 4-5 mice/group determined 5 days after immunization (% suppression relative to corresponding Mock-SC group). d Mean response of two mice.

vation. In order to determine if other Ia molecules reactive with the polyclonal antiIaK serum used in the above experiment might also be involved in Ts activation, SC from CAF] mice were treated with a monoclonal anti-I-AK ( 10.2.16) and C prior to coupling with S3. As shown in Table 1, Experiment 2, lines 5 and 6, spleen cells from mice given S3 coupled to SC which had been treated with 10.2.16 and C were unable to suppress the S3 response of recipient mice. Similar results were obtained using another monoclonal (M5/144.15.2) which reacts with both I-A and I-E molecules expressed by CAF, SC (23) (data not shown). The inability of S3 coupled to anti-I-JK or anti-I-AK+C-treated SC to activate Ts was not due to an inability of S3 to be coupled to such cell populations since all of these S3-coupled SC were effective for induction of S3-specific tolerance (Table 2). Moreover treatment of SC with antiI-JK and C has no effect on the ability of S3-SC to activate Tcs (see Table 6). Thus activation of Ts by S3-SC requires carrier SC that express I-A determinants and determinants recognized by anti-I-JK alloantiserum. Whether both of these determinants must be expressed by the same SC population was not determined in these experiments although experiments by others (4) indicate that I-J and I-A determinants are expressed on a single APC subset. It is not known whether the determinants recognized by the anti-I-JK alloantiserum are conventional I-J molecules identical to those expressed by the Ts cells or whether they might be receptors for self I-J or I-J interacting molecules which have been shown to be expressed by APC involved in Ts activation (10,30) and may be recognized by anti-I-J alloantisera (3 1). In this regard, treatment of carrier SC (APC) with anti-I-JK alloantiserum always eliminated the ability of S3-SC to activate Ts (as in Table 1) whereas treatment of SC with monoclonal antiI-JK and C gave less consistent results. Treatment of APC with monoclonal anti-I-JK WFC12.8 (25) and C eliminated the ability of S3-SC to activate Ts in only 1 of 5 experiments while treatment with Ky76 (24) and C eliminated APC activity in 2 of 5

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T CELLS

TABLE 2 Spleen Cells Depleted of I-A+ or I-J+ Cells Are Capable of Inducing Tolerance S3 PFC/spleen” Cells injected”

Treatment

Mock-SC s3-SC Mock-SC s3-SC Mock-SC s3-SC Mock-SC s3-SC

anti-I-J + C anti-I-Jr’ + C anti-I-JK + C anti-I-J“ + C anti-IaK + C anti-IaK + C 10.2.16+C 10.2.l6+C

Expt 1 615Ok 1306 f 7983 f 1925 + 6200 + 1738 t

578 294 (79) 763 215 (76) 1026 223 (72) ND ND

Expt 2 6500’ 638 f 133 (90) 8,150* 604 1,125 k 263 (86) ND ND 13,725 + 3360 1,875 + 213(86)

y Normal CAF, mice received 2 X 10’ Mock- or S3-SC treated as indicated before coupling with S3 (Mock- or S3-SC were the same cells used for the experiments in Table 1). Three days later mice were immunized with 0.6 pg S3 and IgM PFC were determined 5 days later (19). * Mean PFC/Spleen + SEM of 3-4 mice (% suppression relative to Mock-SC-injected mice). ’ Mean response of two mice.

experiments (data not shown). The inability of monoclonal anti-I-J C 12.8 to eliminate APC function in the majority of experiments has also been reported by others (4) although in another system (5) the C12.8 mAb was effective for elimination of Ts for APC. The inconsistent results obtained using anti-I-J mAb may indicate that APC determinants recognized by anti-I-J alloantiserum are distinct (or more numerous) than those recognized by some anti-I-J mAb. It is also possible that the mAb are simply less potent than the alloantiserum although all of the anti-I-JK reagents used here can functionally inactivate Ts cells (4,24,25) (not shown). Ts Are Activated by S3 Presented on Plastic Adherent and Anti-Ig Nonadherent

SC

To further assessthe properties of the carrier SC required for Ts activation, S3 was coupled to SC that were enriched for or depleted of various cell types. In the first experiment (Table 3, lines l-6), S3 was coupled to anti-Ig nonadherent (B cell-depleted) or anti-Ig adherent (B cell-enriched) SC. S3-coupled unseparated SC (line 1 vs line 2) and anti-Ig nonadherent SC (line 3 vs line 4) were essentially equivalent in their ability to activate Ts. In marked contrast, when S3 was coupled to an enriched B cell population (line 5 vs line 6), Ts were not activated. In addition, S3-coupled B cell lymphoma cells (LK35) were unable to activate Ts (data not shown). Again this was not due to lack of coupling of S3 to B cells as all cell populations (including LK35) were equally effective for induction of tolerance (data not shown) and S3coupled anti-Ig adherent SC were capable of activating Tcs (Table 7). These results therefore suggest that B cells are not effective APC for activation of Ts by S3, a conclusion which is also consistent with the fact that B cells do not express the I-J molecules required for Ts activation. The activation of Ts by S3-coupled to anti-Ig-nonadherent SC could be due to coupling of S3 to either a macrophage or a T cell population, both of which can express I-J and have APC function. To determine which of these cell populations was

534

HELEN

BRALEY-MULLEN TABLE 3

SC Required for Activation of Ts by S3-SC Are Plastic Adherent and Anti-Ig Nonadherent Donors given” Experiment 1’

Mock-SC s3-SC Mock-SC 01Ig NA S3-SC a Ig NA Mock-SC (YIg Ad S3-SC a Ig Ad

Experiment 2’

Mock-SC s3-SC Mock-SC plastic NA S3-SC plastic NA Mock-SC plastic Ad S3-SC plastic Ad

S3 PFC/Spleen b 9,075 * 3,170 iz 9,500 f 2,863 + 8,244? 8,86 I +

1011 930(65) 888 212 (70) 789 946 (0)

8,517 * 4,530 f 8,438 + 9,110+ 11,250~ 5,170-t

670 630 (47) 1125 696(O) 1191 441(54)

0 See footnote a, Table I. b See footnote c, Table 1. ’ Normal CAF, spleen cells were separated on anti-Ig-coated plates (Experiment 1) or by adherence to plastic (Experiment 2) prior to coupling with S3 (see Materials and Methods). Donors were injected iv with 3-4 X 10’ Mock- or S3-SC.

functioning in Ts activation, SC were separated by adherence to plastic petri plates into adherent (macrophage-enriched) or nonadherent (macrophage-depleted) populations prior to coupling with S3 (Table 3, lines 7- 12). Although both cell populations were equivalent in their ability to induce tolerance to S3 (data not shown) only the S3coupled plastic adherent cells could activate Ts (Table 3, lines 11 and 12). In addition, treatment of SC with anti-Thy1.2 and C prior to coupling with S3 had no effect on the ability of such cells to activate Ts (data not shown). Overall these results suggest that the APC required for Ts activation by S3 are probably a subset of macrophages which express molecules reactive with anti-I-J alloantiserum. APC Which Function Mice

to Activate S3-Specific Ts Are Nonfunctional

in Cy-Treated

Previous studies have shown that I-J+ cells which function as APC for Ts activation are nonfunctional or absent in Cy-treated mice (4,5, 8, 11). To determine if SC from Cy-treated mice could function as carriers for activation of S3-specific Ts, Mock- or S3-SC were prepared using spleen cells from either normal mice or mice which had been given 20 mg/kg Cy 2 days previously (Table 4). Although S3-SC prepared using SC from Cy-treated mice were effective for induction of tolerance (Column A, Groups D vs C), these S3-SC were unable to activate Ts cells (Column B, Groups D and C vs Groups A and B). The S3-SC did not have to be capable of cell division since S3-SC prepared from normal mice could activate Ts following treatment of SC with mitomycin C (Column B, Groups E and F) or following 1500 R irradiation (data not shown). Thus, the APC which function in activation of S3-specific Ts are absent or nonfunctional in mice treated with Cy.

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TABLE 4 S3-SC Prepared Using SC from Cy-Treated Mice Do Not Activate Ts S3 PFC/spIeen a Donors given b A B C D E F

Mock-SC (normal SC) S3-SC (normal SC) Mock-SC (Cy SC) s3-SC (Cy SC) Mock-SC (mitomycin C) S3-SC (mitomycin C)

A

B

Tolerance’

Ts assayd

6,675 -c 930 1,175 + 52 (82) 10,138 + 923 980 f 167 (91) ND ND

6899 + 1248 3260 f 324 (53) 6931 f 573 6912+1199(l) 5600? 306 2565 f 366 (59)

0 Mean PFC/spleen -t SEM of four to five mice determined 5 days after immunization. b Mock- or S3-SC were prepared from SC of normal CAF, mice (A, B, E, F) or from SC of mice given 20 mg/kg Cy 2 days earlier (C, D). For groups E and F, SC were treated with mitomycin C after coupling with S3. ‘To assessthe ability of S3-SC to induce tolerance, 3 X IO7 Mock- or S3-SC were injected iv into normal CAF, mice which were immunized 4 days later with 0.6 PLgS3 (19). d SS-SC were assessedfor their ability to activate Ts by injecting 5 X IO’ Mock- or S3-SC iv into Cytreated CAF, mice. Eight days later, 5 X IO6 cells from donor mice were transferred iv to normal CAF, recipients which were immunized 4 hr later with 0.6 rg S3 (14).

APC Requirements for Activation

of T, by Soluble S3

In earlier studies, we showed that Ts could not be activated in Cy-treated mice using soluble S3 (14). This was surprising since subsequent studies showed that soluble S3 did activate S3-specific Ts in normal mice which could be detected following removal of Tcs (13). The results presented in Table 4 showing that S3-SC prepared from SC of Cy-treated mice were unable to activate Ts suggested that our earlier findings might be explained by an absence of APC required for Ts activation by soluble S3. To investigate this possibility, Cy-treated mice to be used as Ts donors were given normal SC together with soluble S3 (Table 5). As reported previously (14), S3 did not activate Ts in Cy-treated mice (Group A vs Group H). However, when the mice were given mitomycin C-treated normal SC together with soluble S3, cells from such mice could suppress the S3 response after transfer to normal recipients (Group B vs Groups A and H). The suppression was due to T cells since treatment of donor spleen cells with anti-Thy 1.2 and C at the time of cell transfer abrogated the suppression (data not shown). The spleen cells required for activation of Ts by soluble S3 had properties similar to those described above for Ts activation by S3-SC since Ts were not activated if the spleen cells were from Cy-treated donors (Group C, Experiment 1 in Table 5) or if the spleen cells were treated with anti-I-JK alloantiserum or monoclonal anti-I-AK and C prior to injection (with S3) into Cy-treated donors (Table 5, Groups D and F). The results of Group G in Experiment 2 of Table 5 also indicate that B cells are not required for Ts activation by soluble S3 since treatment of SC with J 11 d+C had no effect on Ts activation. These results suggest that activation of Ts by either soluble S3 or S3-SC requires presentation of the antigen on I-J+IA+ non-B cells that are functionally absent in Cy-treated mice.

536

HELEN

BRALEY-MULLEN TABLE 5

Activation of Ts in Cy-Treated Mice by Soluble S3 Requires I-A and I-J+ SC from Normal Mice S3 PFC/spleen” Donors given’

Expt 1,

cy+ 1 rgS3 cy+ lrlgS3+SC Cy + 1 pg S3 + SC (Cy donors) Cy+ lpgS3+SC(aI-JK+C) Cy+ lrgS3+SC(aI-J*+C) Cy+ lpgS3+SC(10.2.16+C) Cy+ lpgS3+SC(Jlld+C) No cells

12,678 6,480 10,139 15,595 5,660 11,075

+ 993 + 446 (49) + 653 (20) f 2057 (0) f 1397 (55) f 1068 (13) ND 13,660* 3174

Expt 2 12,169 + 1489 5,410 + 845 (55) ND 10,093 -+ 452(17) ND 10,050 + 1062 (17) 4,58 I + 483 (62) ND

a Mean PFC/Spleen + SEM of 4-5 mice/group determined 5 days after immunization (% suppression relative to Group A). b CAF, mice were given 100 mg/kg Cy 2 days before receiving 1 rg S3 f 5 X 10’ mitomycin C-treated CAF, SC. SC were treated with antibodies and C as indicated (D-G) or were derived from donors given 20 mg/kg Cy 2 days earlier(C). Eight days later 5 X lo6 SC from donor mice were transferred iv to normal CAF, recipients which were immunized 4-6 hr later with 0.6 pg S3.

APC Required for Activation Activation

of Tcs by S3-SC Difler from APC Required for Ts

Previous studies have shown that SS-SC can activate both Ts and Tcs in mice ( 15). Having established the properties of the carrier SC (APC) which function in Ts activation, it was of interest to determine if the carrier SC required for activation of Tcs by S3-SC had similar or distinct properties. Therefore, CAFi SC were treated with antiI-JK alloantiserum, monoclonal anti-I-AK, J 1 Id and C, or with C alone prior to coupling with S3. These S3-SC were injected into normal CAF, mice; 7 days later spleen cells from these mice were separated on V.v.-coated plates and the V.v.-adherent cells assessed for Tcs activity as described under Materials and Methods (Table 6). V.v.adherent cells from donors injected with S3-SC treated with C alone or with antiI-JK and C were able to abrogate the suppression of the S3 response induced by S3specific Ts (Groups C and D vs Group B, Experiment I), i.e., these cells had Tcs activity. In contrast, S3-SC treated with anti-I-AK and C (Group E) or Jl Id and C (Group F) did not activate Tcs. These results indicate that whereas the SC required for activation of Ts by S3-SC are functionally inactivated by anti-I-JK and C (Table I), SC required for activation of Tcs are not inactivated by this antibody. Moreover, since the mAb J 1 Id is cytotoxic for B cells (26), the results of Group F, Table 6 suggest that B cells are required for activation of Tcs but not for Ts (Tables 3 and 5). It should also be noted that each of the S3-SC preparations was adequately coupled with S3 as evidenced by the ability of each to induce S3-specific tolerance (data not shown). Requirement for Ig+ Cells for Tcs Activation To determine more directly whether B cells could present S3 for Tcs activation, CAFi spleen cells were separated into B cell-depleted or B cell-enriched fractions on anti-Ig-coated plates. These separated cells as well as unfmctionated SC were coupled

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537

T CELLS

TABLE 6 Activation of Tcs by S3-SC Requires SC which Bear I-A and J I Id

A B C D E F

Donors given”

Cells transferredb

Cy + Mock-SC cy + s3-SC s3-SC (C) S3-SC (anti-I-JK + C) S3-SC (anti-I-AK + C) S3SC(jlld+C)

A B V.v.Ad V.v.Ad V.v.Ad V.v.Ad

B+ B+ B+ B+

S3 PFC/spleen’ 14,100 5,533 11,377 12,255 5,440 5,244

of C of D of E of F

f 2091 -c 906 (61) +- 1243 (19) + 504(15) f 967 (62) f 299 (63)

a CAF, mice were given 100 mg/kg Cy 2 days before Mock- or S3-SC (A and B); 8 days later SC were transferred to recipient mice. In Groups C-F donors were not given Cy; these mice received 3 X 10’ S3-SC treated as indicated before coupling with S3. Five days later SC from these mice were separated on V.V. and V.v.Ad SC were transferred together with Ts to recipient mice. b 5 x lo6 spleen cells from each donor group were transferred to normal CAF, recipients which were immunized 4-6 hr later with 0.6 pg S3. In Groups C-F, mice received 5 X IO6 spleen cells from Group B mice (Ts) plus 5 X IO6 V.v.Ad cells from each respective donor group. ’ Mean PFC/spleen f SEM of four to five mice determined 5 days after immunization (W suppression relative to Group A).

with S3 and injected into normal CAF, mice. V.v.-adherent cells from these mice were assessed for Tcs activity as in the previous experiment (Table 7). As expected, V.v.-adherent cells from donors injected with S3 coupled to unseparated SC had Tcs activity (Group C vs Group B). In contrast to the experiments in which S3 coupled to anti-Ig nonadherent SC were found to activate Ts (Table 3), S3-coupled anti-Ig nonadherent cells were ineffective for activation of Tcs (Group D vs Group B). Conversely, whereas S3 coupled to anti-Ig adherent cells could not activate Ts (Table 3), such cells were as effective as S3-SC for activation of Tcs (Group E vs Group B). Cells

TABLE 7 Activation of Tcs by S3-SC Requires Presentation of S3 on Ig+ Cells PFC/spleen a

A B C D E F G

Donors given b

Cells transferred’

Cy + Mock-SC cy + s3-SC s3-SC S3-LUIg NA SC S3-cuIg Ad SC S3-a Ig NA SC + S3-a Ig Ad SC S3-SC (Cy donors)

A B B + V.v.Ad of C B + V.v.Ad of D B + V.v.Ad of E

9,817& 4,150f 11,270 + 4,828 + 9,789 f

B + V.v.Ad of F B + V.v.Ad ofG

10,780 f 408 (0) ND

’ See footnote c, Table 6. b See footnote a, Table 6 and footnote b, Table 3. ‘See footnote b, Table 6. d Mean response of two mice.

Expt 1 298 587(58) 816 (0) 786 (5 1) 1092 (1)

Expt 2 9,225d 4,475 f 720 (52) 8,889 f 645 (4) 2,670 f 877 (71) 9,250 + 1580 (0) 11,480 * 999 (0) 9,820 f 1200 (0)

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from donor mice given a mixture of S3-coupled adherent and nonadherent SC had Tcs activity comparable to that of mice given S3-anti-Ig adherent SC (Group F). Thus, the presence of S3-anti-Ig nonadherent cells which activate Ts did not interfere with the activation of Tcs by the S3-coupled anti-Ig-adherent cells. In addition, S3SC prepared using SC from Cy-treated mice could activate Tcs (Group G) whereas these cells did not activate Ts (Table 4). The results in Tables 6 and 7 clearly indicate that the carrier spleen cell population required for activation of Tcs by S3-SC is distinct from that which is required for Ts activation by S3-SC (Tables l-5). These results suggest that presentation of S3 on B cells results in Tcs activation whereas presentation of S3 on I-J+ plastic adherent cells (presumably macrophages) results in Ts activation. DISCUSSION Previous studies from this laboratory have established that S3-SC can activate both Ts and Tcs in mice ( 13- 15). The carrier SC to which S3 is coupled presumably function to present S3 to S3-specific Ts and Tcs since the carrier SC must derive from strains of mice which are I-J compatible with donors in which Ts are induced (14) or I-A compatible with donors in which Tcs are induced (unpublished results). The requirement for genetic identity between the carrier SC and the Ts or Tcs donors strongly suggests that the injected S3-SC are not re-presented by APC present in the donor animals. The results presented herein further establish the critical importance of carrier SC for activation of S3-specific Ts and Tcs and provide evidence that distinct subsets of SC are required for activation of these two T cell subsets. As was suggested by our earlier studies which demonstrated a requirement for I-J compatibility between the carrier SC and the Ts donors ( 14), the results presented here directly demonstrate that molecules reactive with anti-I-J alloantisera (discussed below) are required for activation of Ts by S3-SC (Table 1). Carrier SC (APC) required for activation of S3-specific Ts were also I-A+ (Table I), absent or nonfunctional in Cy-treated mice (Table 4) anti-Ig-nonadherent and plastic-adherent (Table 3). APC required for activation of some hapten- and protein-specific Ts subsets have also been shown to be I-A+ (4, 5, 8), reactive with anti-I-J sera (3-9) plastic or glass adherent (5, 30-33) and nonfunctional in Cy-treated mice (5, 8, 11, 33). Thus, the above-mentioned studies all support the conclusion that Ts are activated when antigen is presented by a subset of anti-I-J reactive adherent cells (presumably macrophages (3)) and that plastic nonadherent cells (B cells and T cells) are not efficient APC for Ts activation (Table 3 and references 30, 3 1, 33). Whereas the above-mentioned studies clearly suggest that B cells are not effective APC for Ts activation, a number of studies have demonstrated that B cells or plasma cells are involved in the activation of some subsets of Ts (34-39). However, in most of these studies the B cells must come from antigen-primed mice (34-38) and it has been suggested that the Ts are activated by B cell idiotype rather than by antigen (35, 37). Thus, these studies cannot be directly compared to the studies mentioned above which indicate that antiI-J-reactive adherent cells from unprimed mice function as APC for the activation of Ts by antigen. Indeed, in the systems in which B cells are known to be required for Ts activation, anti-I-J reactive adherent cells may also function to present antigen or B cell idiotype to the Ts (38,39). The nature of the molecules recognized by anti-I-J antibodies which are involved in I-J-restricted interactions among regulatory T cells and between regulatory T cells

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and macrophages is not yet known ( 10,30). Several studies have suggested that macrophages involved in Ts activation express I-J encoded molecules (3-9) although these molecules may be distinct from those expressed by Ts (3, 40). More recently, Kuchroo et al. ( 10) have shown that the I-J-like molecules expressed by macrophages react with anti-I-J idiotype sera. These determinants have been termed I-J-interacting molecules (IJ-IM); IJ-IM are postulated to interact with the Ts I-J molecules during antigen presentation ( 10). Anti-I-J alloantisera such as those used in the present study may contain both anti-I-J and anti-I-J idiotype reactivities (3 1,33). Although further studies are required to define the carrier SC molecules required for Ts activation by S3-SC, the fact that anti-I-JK alloantiserum consistently eliminated APC function for Ts activation (Table 1) while two monoclonal anti-I-JK antibodies (Ky76 (24) and WFC12.8 (25)) did not (see Results) is consistent with the possibility that IJ-IM (10) may be the relevant molecules required for Ts activation by S3-SC. The major point, however, is that the molecules recognized by anti-I-J alloantiserum are expressed by the APC required for Ts activation (Table 1) but not by the APC required for Tcs activation (Table 6). Sensitivity of APC function to Cy also distinguishes the APC required for Ts and Tcs activation (Tables 4 and 7). Although the APC required for Ts activation have often been shown to be nonfunctional in Cy-treated mice (5, 8, 11, 33) the basis for this sensitivity is unknown It is possible that Cy may affect required macrophage differentiation processes (11) and/or the expression of I-J or IJ-IM on APC may be Cy-sensitive (5). It should be noted that whereas these and other studies indicate that presentation of antigen by anti-I-J-reactive adherent cells is required for Ts activation, other cell types can present the same antigens to result in immunologic tolerance. Thus, as demonstrated above, coupling of S3 to any of the spleen cell populations used in these studies was effective for inducing S3-specific tolerance (Tables 2 and 4 and data not shown). Studies by others have also demonstrated that there are distinct requirements for the induction of tolerance versus activation of Ts (32, 41). The fact that tolerance could be induced by all populations of S3-SC used in these studies provided a convenient and reliable means to determine that the antigen had been effectively coupled to each cell population. The major observation in these studies was the demonstration that distinct carrier SC (APC) were required for activation of Tcs and Ts by S3-SC. Thus, I-A+ anti-I-J reactive SC were required for Ts activation (Table 1) whereas I-A+ I-J- SC were required for Tcs activation (Table 6). In addition, S3-SC derived from Cy-treated donors could activate Tcs (Table 7) but not Ts (Table 4) and anti-Ig-adherent S3-SC (Table 7) bearing the B cell marker J 11d (Table 6) activated Tcs but not Ts (Tables 3 and 5). These studies are of particular significance as they directly show that association of antigen with distinct cell types can result in the preferential activation of T cell subsets having opposing immunoregulatory functions. The APC requirement for Tcs activation is not yet well understood, although some early studies showed that antigens coupled to specialized APC such as dendritic cells or Langerhans cells could lead to “suppressor cell resistant” immunity (42, 43) or could induce immunity rather than tolerance (44). While these results suggest that dendritic or Langerhans cells might function as APC for Tcs, those studies did not directly show that the antigen-coupled cells were functioning to present antigen to Tcs. In addition, antigen-antibody complexes coupled to peritoneal exudate cells

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(PEC) were recently shown to activate Tcs even when the PEC derived from allogeneic donors, i.e., activation of Tcs was not genetically restricted (45). While these results suggest, in contrast to our results, that activation of Tcs may not require antigen presentation by class II MHC-bearing APC, the possibility that the antigen-antibody complexes were represented by host I-A+ APC was not excluded (45). While our studies do not exclude the possibility that dendritic or Langerhans cells may be able to present antigens to Tcs in some situations, J 11d+ anti-Ig adherent B cells are clearly required for Tcs activation in this system (Tables 6 and 7). Whether other cell types such as Langerhans cells, dendritic cells, or macrophages (42-46) can also function as APC for the activation of some Tcs subsets or can present antigens other than S3 to Tcs is a possibility that requires further study. While the results presented here are, to our knowledge, the first to demonstrate that B cells can present antigen to Tcs, there is considerable evidence which shows that B cells play an important role in the activation of Tcs. For example, Tcs cannot be induced in mice which have been depleted of B cells by treatment from birth with anti-Q&I (43,47,48), and both the induction (49) and expression (29) of contrasuppression is restricted by genes mapping to the Ig heavy chain (Igh) locus. In addition, Tcs cannot be induced in immune defective (xid) CBA/N X Balbc F1 mice ( 13) which are unable to produce antibody to S3 due to the absence of the Lyb 5+ subset of B cells (50). However, S3-specific Tcs can be induced in xid mice in the presence of B cells from phenotypically normal Balbc X CBA/N F, mice (47). These results as well as studies demonstrating a role for Ig (45,46) or B cell idiotypes (5 1) in Tcs activation all indicate the importance of B cells for Tcs activation. The present studies significantly extend these previous observations by demonstrating that one important function of B cells is to present antigen to Tcs. Finally, the results presented here establish that S3-specific Ts and Tcs are activated when antigen is presented by distinct subsets of APC. These observations, if found to be applicable in other systems, could be of considerable significance in furthering our abilities to direct a particular immune response toward either immunity or suppression. It is also of interest that we have shown that S3-specific Ts and Tcs appear to be activated by antigen presented in the context of either a class II MHC gene product (I-A in the case of Tcs) or an I-J or IJ-IM molecule (the nature of which is as yet unknown ( 10, 27)). These results thus suggest that polysaccharide antigens may not differ substantially from proteins and other classical T cell-dependent antigens in the way they are initially presented to T cells. Whether or not polysaccharides also may undergo some type of intracellular processing prior to presentation is not known and is currently under investigation. ACKNOWLEDGMENTS The author thanks Patra Mierzwa for excellent technical assistance and Dale Melloway for preparing the manuscript.

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