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In VivoAnalysis of a Superantigen-Induced T Cell Suppressor Factor
CELLULAR IMMUNOLOGY
167, 285–292 (1996)
Article No. 0037
In Vivo Analysis of a Superantigen-Induced T Cell Suppressor Factor HSIANG-LING HU,* WILLIAM D. CORNWELL,† THOMAS J. ROGERS,†
AND
YEE-SHIN LIN*
*Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan, Republic of China; and †Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Received June 19, 1995; accepted September 25, 1995
We have previously reported that the superantigen staphylococcal enterotoxin B (SEB) was able to suppress an immune response to sheep red blood cells when administered intravenously to mice. While the capacity of the superantigens to stimulate lymphocytes and accessory cell functions has been thoroughly examined, it is clear that these agents may also exhibit potent immunosuppressive activity both in vivo and in vitro. This SEB-induced immunosuppression was determined by our laboratories to be mediated by a population of T suppressor cells. The suppression may be due to the generation of inhibitory lymphokines, including IL-10 or transforming growth factor b, following superantigen stimulation. Alternatively, the immunomodulatory activity may be due to the activation of antigen-specific and/or genetically restricted suppressor cells by SEB. The mechanism of activity of these suppressor cells has not been fully defined. In this report we wished to determine whether a suppressor factor generated from SEB-activated T cells in vitro may be responsible for the inhibition of antibody or delayed-type hypersensitivity responses in vivo. We observed that both antibody and delayed-type hypersensitivity responses were inhibited following administration of the SEB-induced suppressor factor. The in vivo inhibitory activity of the SEB-induced suppressor factor was found to be genetically restricted at the ‘‘I– J’’ locus. In addition, monoclonal anti-I–J antibodies recognized the suppressor factor in a haplotype-specific fashion. These results show that the suppressive product of SEB-induced T cells possesses the ability to inhibit, in a genetically restricted fashion, both cellular and humoral immune responses. q 1996 Academic Press, Inc.
INTRODUCTION Staphylococcal enterotoxin B (SEB), a major cause of food poisoning, is one of a family of gram-positive bacterial exotoxins which possess properties of a superantigen. As a superantigen, SEB is capable of binding to major histocompatibility complex class II molecules and is recognized by T cells bearing certain T cell-recep-
tor (TCR) V b alleles. SEB is recognized by murine TCR V b alleles 7, 8.1, 8.2, and 8.3 and human TCR V b alleles 3, 12, 14, 15, 17, and 20 (1, 2). The capacity of the bacterial superantigens to stimulate lymphocyte and accessory cell function has been thoroughly examined. However, it should be appreciated that several of the gram-positive pyrogenic toxins, including SEA, SEB, SEE, SpEC and TSST1, have been found to exhibit potent immunosuppressive activity both in vivo and in vitro (3–10). This immunosuppression can be mediated via clonal deletion (2–4), the induction of anergy (5–8), or the activation of T suppressor (Ts) cells (9, 10) among T cells which bear the appropriate TCR V b alleles. It has been observed that SEA, SEB, TSST1, and SpEC are potent inducers of T suppressor cell activity (10). Suppressor cell function is believed to be mediated in part by the synthesis of cytokines with potential immunosuppressive activity. It is well established, for example, that transforming growth factor b (TGFb), IL-2, and IL-10 can inhibit certain responses either in vivo or in vitro. This is particularly true for TGFb, which has been found to suppress both cell-mediated and humoral immunity (11–13). Exposure of certain CD4-bearing T cell clones to IL-2, at critical times during T cell stimulation, can result in a failure of these T cells to respond to subsequent antigen stimulation (14–16). Finally, IL-10 inhibits the production of several cytokines including IL-1, IL-6, and TNFa, and results in reduced T cell responses due to a lack of macrophage costimulatory signals (17, 18). This form of ‘‘suppressor’’ cell function is not genetically restricted and may be masked by the production of several additional lymphokines with compensating immunoenhancing activities. It is clear that suppressor cell function may also be mediated by the generation of genetically restricted and/or antigen-specific inhibitory soluble cell products. Results from studies carried out in several experimental systems suggest that these antigen-specific Ts cells function as a circuit of at least three distinct Ts cell populations (19–21). The first Ts cell population (Ts1) is an early-acting inducer subset which expresses the
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0008-8749/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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CD5HICD80 phenotype and appears to be activated specifically by antigen. Activation of each of these populations results in the production and release of a soluble factor (TsF) capable of inducing a subsequent population of cells (Ts2 or Ts3). Ts2 typically exhibits the CD5HICD8/ phenotype, and the third subset (Ts3) is a CD5LOCD8/ suppressor-effector population and is believed to mediate the immunosuppression. Another feature of Ts cell activity in certain circumstances is the apparent genetic restriction at the controversial I–J locus (22). I–J is believed to be a homodimer protein expressed on the surface of murine Ts cells and may be a component of a soluble factor, TsF, produced by one or more Ts subsets. The exact nature of the I–J protein has yet to be determined. Genetic mapping studies have indicated that I–J should be located within the I region of H-2 (23, 24). However, the DNA sequence of this region is not apparently consistent with the existence of a structural gene within this locus (25). Our laboratory has observed that SEB induces Ts cells in murine splenocyte cultures. Multiple populations of SEB-induced Ts cells appear to be present following activation with this superantigen. The nature of the immunosuppressive activity of these cells is not fully understood and may be due, at least in part, to the production of TGFb, IL-10, or IL-2 (or other immunosuppressive lymphokines) known to be produced in response to stimulation by superantigens (26–29). We have determined that SEB also induces the production of soluble factors with genetically restricted and antigen-specific immunosuppressive activity in vitro (9, 10, 30–32). However, the nature of the immunosuppressive activity of the products of superantigen activation has not been fully defined. The purpose of this study was to characterize the effect of the SEB-induced soluble cell-free products on cellular and humoral immune responses in vivo. The results show that the SEB-induced TsF inhibits both the DTH response and the antibody response to sheep red blood cells (SRBCs). Finally, the results show that the kinetics of both activity and genetic restriction of the SEB-induced TsF are similar for both in vivo and in vitro immune responses. METHODS Mice C3H/HeN (I–Jk) mice were supplied by the National Cancer Institute (Frederick, MD) and the Jackson Laboratories (Bar Harbor, ME). The BALB/c (I–Jd), B10 (I–Jb), B10.D2 (I–Jd), B10.A (I–Jk), B10.A (2R) (I–Jk), B10.A (4R) (I–Jb), C3H/HeJ (I–Jk), C3H/HTG (I–Jd), C3H/SW (I–Jb), and C57BL/6 (I–Jb) were obtained from the Jackson Laboratories. Mice were used at 6– 12 weeks of age.
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Preparation of Anti-SEB and Anti-I–J Affinity Columns The anti-SEB affinity column was generated by coupling 30 mg each of 1FD7 and 2GD9 (33) monoclonal antibodies to a 10-ml bed volume of Affi-Gel 15 cation matrix (Bio-Rad, Hercules, CA) in 0.1 M 3-(N-morpholino)propane-sulfonic acid, pH 7.8, overnight. The column was run with a mobil phase of PBS, pH 7.2. A maximum volume of 5.0 ml was loaded onto this column, and the active fractions were pooled. The anti-I–Jd (WF18-2B12-15) and anti-I–Jb (WF940-5) affinity columns were coupled individually to 1ml bed volume of Affi-Gel 15. The antibodies were originally described by Dr. Carl Waltenbaugh (34) and obtained from Dr. H. Y. Lei (National Cheng Kung University Medical College, Tainan, Taiwan, Republic of China). The SEB-depleted factor containing supernatant was applied to the column in 0.5-ml portions. The filtrate fractions were collected and pooled. The bound material was eluted from the column with 0.2 M glycine, pH 2.9. The eluate fractions were immediately neutralized with 200 mM phosphate, pH 7.8, and active fractions were pooled and dialyzed with MEM (GIBCO Laboratories, Grand Island, NY). Generation of Delayed-Type Hypersensitivity (DTH) Mice were immunized iv with 0.2 ml of a 0.01% SRBC (Rockland, Gilbertsville, PA) solution in PBS. Four days later the mice were challenged in the footpad with 0.03 ml of a 20% SRBC solution. A DTH response was evaluated 24 hr later by measuring the increase in footpad thickness. Negative control mice were injected intravenously with PBS alone and challenged on Day 4 with 0.03 ml of a 20% SRBC solution. Four mice were analyzed for each group. The data shown represent the increase in footpad thickness relative to the negative control. Generation of an in Vivo antibody Response to SRBCs Mice were intravenously injected with 0.2 ml of 0.01% SRBCs in PBS. Five days later the mice were sacrificed by cervical dislocation. The spleen cells were isolated from each mouse individually and washed twice with DMEM (GIBCO) containing 10% FCS and once with MEM. The splenocytes from each mouse were resuspended to 5 ml volume with MEM. Plaque-forming cells (PFCs) were enumerated using a hemolytic plaque assay (35). Each spleen was assayed in triplicate, and four mice were used for each group. Determination of PFCs PFCs present in splenocyte preparations were enumerated by hemolytic plaque assay (35). The splenocytes were incubated with complement (guinea pig serum) and SRBCs at 377C for 30–40 min inside Cun-
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ANALYSIS OF SEB-INDUCED T CELL SUPPRESSOR FACTOR
FIG. 1. Inhibition of the DTH response in BALB/c (I–Jd) mice by SEB-induced T cell suppressor factor generated from BALB/c, C3H/HeN, and B10 mice. BALB/c mice were injected intravenously with PBS (positive control), 1.0 ml SEB-PSC factor, or 1.0 ml NONPSC factor. Ninety minutes later the mice were immunized intravenously with 0.2 ml of a 0.01% SRBC solution. Four days later the mice were challenged in the footpad with 0.03 ml of a 20% SRBC solution. Negative-control mice only received footpad injections on Day 4. After 24 h, the footpad size for each mouse was determined. The negative-control response (no SRBC challenge) ranged fro 0.037 to 0.059 mm. Four animals were used in each group (*P õ 0.001).
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intravenously to BALB/c responder mice, and on the same day the mice were immunized with SRBCs. The positive-control mice received only the SRBC immunization. Four days after initiation of the immunization, the mice were challenged in the footpad with SRBCs, and the DTH response was measured 24 hr later. Figure 1 shows the results of these experiments. The mice which received the NON-PSC derived from either of the three strains produced a DTH response which was equivalent to the positive-control groups. However, the SEB-induced TsF generated from BALB/c cells suppressed the DTH response by 58% (P õ 0.001). Furthermore, the SEB-induced TsF generated from the C3H/ HeN and B10 strains did not suppress the DTH response significantly in the BALB/c responder mice. The SEB-induced TsF generated from C3H/HeN and BALB/c cells was analyzed under the same conditions as above using C3H/HeN mice as a source of target cells (Fig. 2). Again, the NON-PSC supernatant from either the C3H/HeN cells or the BALB/c cells did not reduce the DTH response below that of the positive-control groups. However, under these conditions the SEB-induced TsF generated from C3H/HeN cells suppressed the DTH response (P õ 0.001). Even though SEB-induced TsF from BALB/c cells was able to inhibit the DTH response in the BALB/c mice, there was no inhibition of the DTH response in C3H/HeN mice. To further investigate the genetic restriction of the
ningham chambers (35). Plaques observed in the SRBC monolayer were counted and reported as PFC values. Generation of SEB-Induced T Cell Suppressor Factor Spleen cells from naive mice were cultured with and without SEB (10 mg/ml) for 48 hr at a cell density of 1.5 1 10 7 cells/ml. The supernatants from both cultures were passed over an anti-SEB affinity column, and the filtrates were collected. The filtrate from the supernatant of the untreated culture was designated NON-PSC and the filtrate from the supernatant of the SEB-treated culture was designated SEB-PSC. RESULTS DTH Response of Mice Treated with SEB-Induced TsF We wished to determine whether the SEB-induced TsF produced from the SEB-stimulated Ts cells would inhibit a DTH response. Suppressor factor was generated from BALB/c (I–Jd), C3H/HeN (I–Jk), and B10 (I– Jb) mice. Splenocytes were cultured in the presence (SEB-PSC) or the absence (NON-PSC) of SEB for 2 days; the culture supernatants were collected and passed over anti-SEB affinity chromatography columns to remove SEB. The filtrates were then administered
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FIG. 2. Inhibition of the DTH response in C3H/HeN (I–Jk) mice by SEB-induced T cell suppressor factor generated from C3H/HeN and BALB/c mice. The procedure was described as in the legend to Fig. 1, except that C3H/HeN mice were used as responders. The negative-control repsonse (no SRBC challenge) varied from 0.06 to 0.135 mm. Four animals were used in each group (*P õ 0.001).
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FIG. 3. H-2 restriction in the suppression of a DTH response by SEB-induced TsF. The same procedure was used as described in the legend to Fig. 1. SEB-induced T suppressor cell factor was generated from BALB/c (I–Jd), B10 (I–Jb), and B10.D2 (I–Jd) mice. The recipient strains were B10 (top) and B10.D2 (bottom). Negative-control responses (no SRBC challenge) ranged from 0.055 to 0.09 mm. Four animals were used in each group (* P õ 0.001).
nously with SRBCs at initiation of the experiment as described above. Five days later, the spleen cells harvested from the responder mice were assayed for PFCs in a hemolytic plaque assay. As can be seen in Fig. 4, BALB/c and C3H.HTG SEB-induced suppressor factor, I–Jd restricted, was able to inhibit an antibody response 69% (P õ 0.05) and 75% (P õ 0.005), respectively, in BALB/c responder mice. However, the SEBinduced TsF generated from C3H/HeJ, B10.A, and B10.A(2R) mice, which exhibit the I–Jk haplotype, did not suppress the response in BALB/c mice. Furthermore, SEB-induced TsF generated from C57BL/6, B10.A(4R), and C3H.SW, which are I–Jb, was unable to inhibit the antibody response of BALB/c responder mice. Here again, NON-PSC TsF, generated from the strains of mice listed in Fig. 4, did not exhibit suppressive activity in any case. The activity of the SEB-induced TsF generated from each of the various mouse strains was also analyzed in C3H/FeJ (I–Kk) and C57BL/6 (I–Jb) responder mice. The results from these experiments are summarized in Table 1. The SEB-induced TsF generated from BALB/ c cells was unable to inhibit the generation of an antibody response in C3H/FeJ or C57BL/6 mice. However, the SEB-induced TsF from strains which are I–Jk restricted was capable of inhibiting the antibody response in C3H/FeJ mice but not in C57BL/6 mice. Finally, the SEB-induced TsF from I–Jb strains was able to suppress the antibody response in C57BL/6 animals but not in C3H/FeJ recipient mice.
SEB-induced TsF in vivo, the ability of SEB-induced TsF from BALB/c cells to inhibit a DTH response in B10 mice and B10.D2 mice was analyzed (Fig. 3). The results show that the BALB/c SEB-induced TsF, which is I–Jd restricted, was unable to inhibit the DTH response in B10 mice (Fig. 3A). On the other hand, this SEB-induced TsF was able to suppress the DTH response elicited in the B10.D2 mice (Fig. 3B; P õ 0.001). As expected, the B10 SEB-induced TsF suppressed the DTH response in B10 responder mice (Fig. 3A; P õ 0.001) and the B10.D2 SEB-induced TsF suppressed the response in B10.D2 responders (Fig. 3B; P õ 0.001). Under all conditions the NON-PSC supernatants did not alter the DTH response. I–J-Restricted Suppression of the Antibody Response of Mice Treated in Vivo with SEB-Induced TsF The effect of the SEB-induced TsF on the humoral immune response to SRBCs was also studied. Preparations of SEB-induced TsF from several mouse strains were inoculated into BALB/c responder mice. In addition, the responder mice were immunized intrave-
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FIG. 4. I-J-restricted suppreson of an in vivo anitbody response by SEB-induced Ts factor. BALB/c mice were injected intravenously with PBS (positive control), 0.2 ml SEB-PSC factor, or 0.2 ml NONPSC factor. Ninety minutes later the mice were immunized with 0.1 ml of 0.02% SRBC soltion. Five days later the splenocytes were assayed for the number of PFCs. The results are represented as the percentage suppression relative to the positive control. Four animals were used in each group (*P õ 0.05).
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TABLE 1 Inhibition of the Antibody Response to SRBCs in In Vivo by SEB-Induced Ts Cell Factor
Factor a
BALB/c (I–Jd)
C3H/FeJ (I–Jk)
C57BL/6 (I–Jb)
BALB/c (I–Jd) C3H/HTG (I–Jd) C3H/HeJ (I–Jk) B10.A (I–Jk) B10.A(2R) (I–Jk) C57BL/6 (I–Jb) B10.A(4R) (I–Jb) C3H/SW (I–Jb)
Yesb Yes No No No No No No
No No Yes Yes Yes No No No
No No No No No Yes Yes Yes
a
Suppressor factor was generated from various strains of mice and analyzed for activity in BALB/c, C3H/FeJ, or C57BL/6 mice. Recipient mice received 0.2 ml of SEB-induced suppressor factor or 0.2 ml of NON-PSC factor on Day 0 of the experiment. These mice received 0.2 ml of 0.01% SRBCs 90 min later. Five days later, the mice were sacrificed and the splenocytes analyzed for the number of PFCs by a hemolytic plaque assay (35). b ‘‘Yes’’ indicates that the SEB-induced suppressor factor was able to significantly inhibit an antibody response to SRBCs in the recipient mouse strain, and ‘‘No’’ indicates it was not.
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BALB/c responders on Days 0, 1, 2, and 3 (Fig. 7). All groups of mice including the positive control animals received SRBCs on initiation of the experiment. The BALB/c SEB-induced TsF demonstrated significant suppressive activity when injected on initiation of the experiment (53% suppression; P õ 0.001) or on Day 1 of the experiment (35% suppression; P õ 0.001). When the SEB-induced TsF was injected on Days 2 and 3, suppression of a DTH response was not observed relative to that of the positive control. DISCUSSION The capacity of the bacterial superantigens to stimulate lymphocyte and accessory cell function has been thoroughly examined; however, it should be appreciated that these agents can exhibit potent immunosuppressive activity both in vivo and in vitro (36–42). Several of the gram-positive pyrogenic toxins, including SEA, SEB, SEE, SpEC, and TSST1 have been found to induce a state of immunosuppression (36–42). The retroviral superantigens have also been shown to in-
Fractionation and Analysis of SEB-Induced TsF In an effort to further characterize the SEB-induced TsF, supernatants containing the TsF were fractionated on an anti-I–Jd affinity column and assayed for the ability to suppress a DTH response to SRBCs (Fig. 5). The eluate from the anti-I–Jd column exhibited significant suppression of the DTH response in BALB/c responder mice (P õ 0.001), whereas the filtrate did not alter the response (Fig. 5A). The NON-PSC supernatant was also fractionated on the affinity column, and neither the filtrate nor the eluate was able to inhibit the DTH response in BALB/c responder mice. As a control, the BALB/c SEB-induced TsF was fractionated on an anti-I–Jb affinity column (Fig. 5B). In these experiments, the filtrate significantly suppressed the BALB/c DTH response (P õ 0.001), whereas the eluate did not exhibit detectable activity. Furthermore, the BALB/c NON-PSC supernatant, also fractionated on the anti-I–Jb affinity column, was unable to suppress the DTH response relative to that of the positive control. Additional studies showed that the BALB/c SEBinduced TsF anti-I–Jd column eluates exhibited suppressive activity in a dose-dependent manner (Fig. 6). These studies showed that doses as low as 0.01 ml (P õ 0.001) of column eluate exhibited significant inhibitory activity. Time Course Kinetics of BALB/c SEB-induced TsF We wished to determine whether the suppressive activity of BALB/c SEB-induced TsF could act early or late in an immune response. BALB/c SEB-induced TsF was generated and administered intravenously to
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FIG. 5. Affinity chromatography of the SEB-induced T cell suppressor factor. Suppressor factor generated from BALB/c (I–Jd) mice was passed over an anti-I–Jd (top) or an anti-I–Jb (bottom) affinity column. The filtrates and the eluates from these columns (0.5 ml) were injected intravenously into BALB/c mice. The procedure for assessing the DTH response is described in the legend to Fig. 1. The negative-control responses (no SRBC challenge) ranged from 0.067 to 0.091 mm. Four animals were used in each group (* P õ 0.001).
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FIG. 6. Dose-dependent activity of I–Jd suppressor factor in BALB/c mice. SEB-induced suppressor factor from BALB/c mice was generated and passed over an anti-I–Jd affinity column. The eluate from this column was injected intravenously (at the dose indicated) into BALB/c mice. The DTH response was measured as described for the previous figures. The negative-control response (no SRBC challenge) was 0.066 mm. Four animals were used in each group (* P õ 0.001).
hibit mixed-lymphocyte responses, antibody production, and graft rejection (43–47). In a similar fashion, exposure to the bacterial superantigens inhibit both in vivo and in vitro antibody responses and the first-set (but not second-set) graft rejection response (37). The mechanism of this immunosuppressive activity has not been fully defined. It has been observed that SEA, SEB, TSST1, and SpEC are potent inducers of T suppressor cell activity (38, 41, 48, 49). It is apparent that the suppressor cell activity induced with these toxins is due to a combination of multiple cell activities. We have previously reported results which show that Ts cells are induced following stimulation with SEB either in vivo or in vitro (9, 10, 30). We have determined that at least three distinct populations of SEB-induced Ts cells are induced by SEB. The first population is an early-acting inducer subset which possesses the CD5HICD80 surface marker phenotype (9). The other populations express the CD5HI CD8/ and CD5LO CD8/ phenotypes and can inhibit antibody responses when added either early or late in an ongoing immune response (9, 10). We have demonstrated that the SEB-induced CD5HI CD80 population produces a soluble factor with suppressive activity in vitro which we term SEB-induced TsF (31). This factor mediates suppression of an in vitro antibody response to SRBCs in an I–J-restricted manner and bears a determinant which is recognized by anti-I–J antibodies (31).
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The SEB-induced TsF resembles antigen-specific suppressor factors described by a variety of other investigators. The capacity of anti-I–J monoclonal and polyclonal antibodies to neutralize TsF activity has been demonstrated for suppressor factors induced by immunization with several antigens including azobenzenearsonate (ABA)- and (NP)-coupled syngeneic cells (50, 51), random copolymers of glutamic acid, alanine, and tyrosine (GAT) (52–55), Keyhole Limpet hemocyanin (KLH) (56), and SRBCs (57). Suppressor factors which have been partially purified by anti-I–J antibody affinity chromatography have been shown to suppress cellular and humoral responses to a variety of antigens in a specific and genetically restricted fashion (50–55, 57). In the present report we show that the SEB-induced TsF binds to anti-I–J antibodies in a genetically restricted fashion, and we observe that both DTH and antibody responses to sheep erythrocytes are altered by the factor in vivo. Our earlier in vitro studies (31), and the present studies carried out in vivo, show that the function of the SEB-induced TsF is restricted at the I–J locus. This would appear to be a somewhat unique observation, in that most of the work carried out in other experimental systems has suggested that suppressorinducer factor activity is not I–J restricted (56, 58). However, the suppressor-inducer factor observed in the response to KLH exhibits I–J restriction and functions by inducing a population of CD5HI CD8/ suppressoreffector cells (59). An explanation for the disparity in
FIG. 7. Time course of suppressor factor activity. SEB-induced suppressor factor was generated from BALB/c mice and passed over an anti-I–Jd affinity column, and the eluate (0.5 ml) was injected intravenously into BALB/c mice at various times after the mice were immunized with SRBCs. A DTH response was elicited and measured as described for the previous figures. The negative-control response (no SRBC challenge) was 0.043 mm. Four animals were used in each group (* P õ 0.001).
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the genetic restriction in the function of these suppressor-inducer factors is not currently available. Studies of the I–J-restricted nature of Ts cells and their factors have been based in part on both polyclonal and monoclonal antibodies reactive with the I–J determinant. These antibodies have been generated by cross-immunization of several recombinant mouse strains, including B10.A(3R) [I–Jb], B10.A(5R) [I–Jk], B10.A(9R) [I–Jk], and B10.HTT [I–Js]. Early genetic analysis suggested that the recombination points of the B10.A(5R) and B10.A(9R) strains defined the centromeric (left) boundary, and the recombination points of the B10.A(3R) and B10.HTT strains defined the right boundary, of the I–J locus (23, 24). These studies led to the assumption that the B10.A(3R) mice were congenic with respect to the I–J locus with B10.A(5R) mice, and the same was true of the B10.A(9R) and B10.HTT strains. However, sequencing of the H-2 region believed to represent the I–J region of both B10.A(3R) and B10.A(5R) showed that the length of this region was insufficient for the presence of a structural gene (25). For this reason, the genetic basis for the I–J restriction and the location of the gene encoding the I–J determinant remain undefined. Nevertheless, it has been the experience of a great number of investigators that the monoclonal and polyclonal anti-I–J antibodies bind to and neutralize TsF activity generated in response to a variety of antigenic stimuli (reviewed in 26 and 58). Our studies show that the SEB-induced TsF must be administered early in the immune response (by Day 1) in order to exert suppressive activity. This is consistent with our earlier work which showed that the SEB-induced TsF inhibits the in vitro antibody response to sheep erythrocytes only when added in the first 2 days of the immune response (60). We have gone on to show that at least a part of the function of this TsF is to induce late-acting Ts populations which exhibit distinct surface phenotypes (60). The kinetics of the activity of the SEB-induced TsF is consistent with that of the activities of suppressor-inducer factors generated in response to administration of GAT, ABA, NP, and SRBCs (50–57). Most commonly the T cell responsible for the generation of these suppressor-inducer factors exhibits the CD5HI CD80 phenotype (9, 10, 52, 55). Our previous results show that the SEB-induced suppressor-inducer factor is the product of an I–J/ CD5HI CD80 population. It is now well known that superantigens may exert immunosuppressive activity through the induction of anergy or clonal deletion of responsive T cell populations (2–8). However, our previous studies have shown that the administration of microgram quantities of SEB to mice leads to an inhibition of the DTH response to sheep erythrocytes (30). These studies also showed that the inhibition in the DTH response is due to the generation of suppressor T cells (30). Indeed, naive mice receiving splenic or lymph node T cells from SEB-
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treated mice fail to manifest normal DTH responses (30). We find in the present report that suppressor factor generated in vitro by treatment with SEB also exerts suppressive activity for in vivo DTH and antibody responses. Taken together, these results suggest that the immunosuppressive activity widely reported for superantigens may also be due to the generation of antigen-specific and/or genetically restricted suppressor cell populations. We suggest that the activity of these suppressor cells is due, at least in part, to the generation of suppressor factors which bear the widely observed I–J determinant. REFERENCES 1. Gascoigne, N. R. J., Sem. Immunol. 5, 13, 1993. 2. White, J., Herman, A., Pullen, A. M., Kubo, R., Kappler, J. W., and Marrack, P., Cell 56, 27, 1989. 3. Wahl, C., Miethke, T., Heeg, K., and Wagner, H., Eur. J. Immunol. 23, 1197, 1993. 4. Kawabe, Y., and Ochi, A., Nature 349, 245, 1991. 5. O’Hehir, R. E., and Lamb, J. R., Proc. Natl. Acad. Sci. USA 87, 8884, 1990. 6. Hewitt, C. R. A., Lamb, J. R., Hayball, J., Hill, M., Owen, M. J., and O’Hehir, R. E., J. Exp. Med. 175, 1493, 1992. 7. Baschieri, S., Lees, R. K., Lussow, A. R., and MacDonald, H. R., Eur. J. Immunol. 23, 2661, 1993. 8. Kawabe, Y., and Ochi, A., J. Exp. Med. 172, 1065, 1990. 9. Holly, M., Lin, Y.-S., and Rogers, T. J., Immunology 64, 643, 1988. 10. Taub, D. D., Lin, Y.-S., and Rogers, T. J., J. Immunol. 144, 456, 1990. 11. Torre-Amione, G., Beauchamp, R. D., Koeppen, H., Park, B. H., Schreiber, H., Moses, H. L., and Rowley, D. A., Proc. Natl. Acad. Sci. USA 87, 1486, 1990. 12. Su, H. C., Leite-Morris, K. A., Braun, L., and Biron, C. A., J. Immunol. 147, 2717, 1991. 13. Ruegemer, J. J., Ho, S. N., Augustine, J. A., Schlager, J. W., Bell, M. P., McKean, D. J., and Abraham, R. T., J. Immunol. 144, 1767, 1990. 14. Wilde, D. B., Prystowsky, M. B., Ely, J. M., Vogel, S. N., Dialynas, D. P., and Fitch, F. W., J. Immunol. 133, 636, 1984. 15. Otten, G., Wilde, D. B., Prystowsky, M. B., Olshan, J. S., Rabin, H., Henderson, L. E., and Fitch, F. W., Eur. J. Immunol. 16, 217, 1986. 16. Otten, G., Herold, K. C., and Fitch, F. W., J. Immunol. 139, 1348, 1987. 17. Fiorentino, D. F., Zlotnik, A., Mosmann, T. R., Howard, M., and O’Garra, A., J. Immunol. 147, 3815, 1991. 18. Taga, K., and Tosato, G., J. Immunol. 148, 1143, 1992. 19. Green, D. R., Flood, P. M., and Gershon, R. K., Annu. Rev. Immunol. 1, 439, 1983. 20. Dorf, M. E., and Benacerraf, B., Annu. Rev. Immunol. 2, 127, 1984. 21. Asherson, G. L., Colizzi, V., and Zembala, M., Annu. Rev. Immunol. 4, 37, 1986. 22. Murphy, D. B., Annu. Rev. Immunol. 5, 405, 1987. 23. Murphy, D. B., Herzenberg, L. A., Okumura, K., Herzenburg, L., and McDievitt, H., J. Exp. Med. 144, 699, 1974. 24. Tada, T., Taniguchi, M., and David, C., J. Exp. Med. 144, 713, 1974.
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HU ET AL.
25. Kronenberg, M., Steinmetz, M., Kobori, J., Kraig, E., Kapp, J. A., Pierce, C. W., Sorensen, C. M., Suzuki, G., Tada, T., and Hood, L., Proc. Natl. Acad. Sci. USA 80, 5704, 1983. 26. Nagelkerken, L., Gollob, K. J., Tielemans, M., and Coffman, R. L., Eur. J. Immunol. 23, 2306, 1993. 27. Florquin, S., Amraoui, Z., Abramowicz, D., and Goldman, M., J. Immunol. 153, 2618, 1994. 28. Tarazona, R., Gonza´lez-Garci´a, A., Zamzani, N., Marchetti, P., Frechin, N., Gonzalo, J. A., Ruiz-Gayo, M., van Rooijen, N., Marti´nez-A., C., and Kroemer, G., J. Immunol. 154, 861, 1995. 29. McKnight, A. J., Shea, C. M., Wiens, G. R., Rimm, I. J., and Abbas, A. K., Immunology 81, 513, 1994. 30. Lin, Y.-S., Hu, S.-C., Jan, M.-S., and Rogers, T. J., Cell. Immunol. 132, 532, 1991. 31. Taub, D. D., Lin, Y.-S., Hu, S.-C., and Rogers, T. J., J. Immunol. 143, 813, 1989. 32. Taub, D. D., and Rogers, T. J., Cell. Immunol. 134, 216, 1991. 33. Lin, Y.-S., Largen, M. T., Newcomb, J. R., and Rogers, T. J., J. Med. Microbiol. 27, 263, 1988. 34. Waltenbaugh, C., J. Exp. Med. 154, 1570, 1981. 35. Cunningham, A., and Szenberg, A., Immunology 14, 599, 1968. 36. Smith, B., and Johnson, H., J. Immunol. 115, 575, 1975. 37. Pinto, M., Torten, M., and Birnbaum, S., Transplantation 25, 320, 1978. 38. Schlievert, P., Infect. Immun. 28, 876, 1980. 39. Chen, S., Tse, C., Bernstein, I., and Archer, D., Int. Arch. Allergy Appl. Immunol. 63, 470, 1980. 40. Torres, B., Yamamoto, J., and Johnson, H., Infect. Immun. 35, 770, 1982. 41. Donnelly, R., and Rogers, T., Cell. Immunol. 72, 166, 1982. 42. Kawaguchi-Nagata, K., Okamura, H., Shoji, K., Kanagawa, H., Semma, M., and Shinagawa, K., Microbiol. Immunol. 29, 183, 1985.
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43. Jacobsson, H., Lilliehook, B., and Blomgren, H., Scand. J. Immunol. 4, 181, 1975. 44. Lilliehook, B., Jacobsson, H., and Blomgren, H., Scand. J. Immunol. 4, 209, 1975. 45. Halle-Pannenko, O., Pritchard, L., Festenstein, H., and Berumen, L., J. Immunogenet. 13, 437, 1986. 46. Halle-Pannenko, O., Pritchard, L., Bruley-Rosset, M., Berumen, L., and Motta, R., Immunol. Rev. 88, 59, 1985. 47. Halle-Pannenko, O., Pritchard, L., and Bruley-Rosset, M., Eur. J. Immunol. 17, 1751, 1987. 48. Platsoucas, C., Oleszak, E., and Good, R., Cell. Immunol. 97, 371, 1986. 49. Carlsson, R., Hedlund, G., and Sjogren, H., Scand. J. Immunol. 25, 11, 1987. 50. Okuda, K., Minami, M., Sherr, D. H., and Dorf, M. E., J. Exp. Med. 154,468, 1981. 51. Sherr, D. H., Minami, M., Okuda, K., and Dorf, M. E., J. Exp. Med. 157, 515, 1983. 52. Pierres, M., Benacerraf, B., and Germain, R. N., J. Immunol. 123, 2756, 1979. 53. Theze, J., Waltenbaugh, C., Dorf, M. E., and Benacerraf, B., J. Exp. Med. 146, 287, 1977. 54. Theze, J., Waltenbaugh, C., Germain, R. N., and Benacerraf, B., Eur. J. Immunol. 7, 705, 1977. 55. Sorensen, C. M., and Pierce, C., J. Exp. Med. 156, 1691, 1982. 56. Tada, T., Taniguchi, M., and David, C. S., Cold Spring Harbor Symp. Quant. Biol. 41, 119, 1976. 57. Yamauchi, K., Murphy, D., Cantor, H., and Gershon, R. K., Eur. J. Immunol. 11, 905, 1981. 58. Dorf, M. E., and Benacerraf, B., Immunol. Rev. 83, 23, 1985. 59. Tada, T., Taniguchi, M., and David, C. S., J. Exp. Med. 144, 713, 1976. 60. Taub, D. D., Lin, Y.-S., and Rogers, T. J., Cell. Immunol. 131, 170, 1990.
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Article No. 0037
In Vivo Analysis of a Superantigen-Induced T Cell Suppressor Factor HSIANG-LING HU,* WILLIAM D. CORNWELL,† THOMAS J. ROGERS,†
AND
YEE-SHIN LIN*
*Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan, Republic of China; and †Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Received June 19, 1995; accepted September 25, 1995
We have previously reported that the superantigen staphylococcal enterotoxin B (SEB) was able to suppress an immune response to sheep red blood cells when administered intravenously to mice. While the capacity of the superantigens to stimulate lymphocytes and accessory cell functions has been thoroughly examined, it is clear that these agents may also exhibit potent immunosuppressive activity both in vivo and in vitro. This SEB-induced immunosuppression was determined by our laboratories to be mediated by a population of T suppressor cells. The suppression may be due to the generation of inhibitory lymphokines, including IL-10 or transforming growth factor b, following superantigen stimulation. Alternatively, the immunomodulatory activity may be due to the activation of antigen-specific and/or genetically restricted suppressor cells by SEB. The mechanism of activity of these suppressor cells has not been fully defined. In this report we wished to determine whether a suppressor factor generated from SEB-activated T cells in vitro may be responsible for the inhibition of antibody or delayed-type hypersensitivity responses in vivo. We observed that both antibody and delayed-type hypersensitivity responses were inhibited following administration of the SEB-induced suppressor factor. The in vivo inhibitory activity of the SEB-induced suppressor factor was found to be genetically restricted at the ‘‘I– J’’ locus. In addition, monoclonal anti-I–J antibodies recognized the suppressor factor in a haplotype-specific fashion. These results show that the suppressive product of SEB-induced T cells possesses the ability to inhibit, in a genetically restricted fashion, both cellular and humoral immune responses. q 1996 Academic Press, Inc.
INTRODUCTION Staphylococcal enterotoxin B (SEB), a major cause of food poisoning, is one of a family of gram-positive bacterial exotoxins which possess properties of a superantigen. As a superantigen, SEB is capable of binding to major histocompatibility complex class II molecules and is recognized by T cells bearing certain T cell-recep-
tor (TCR) V b alleles. SEB is recognized by murine TCR V b alleles 7, 8.1, 8.2, and 8.3 and human TCR V b alleles 3, 12, 14, 15, 17, and 20 (1, 2). The capacity of the bacterial superantigens to stimulate lymphocyte and accessory cell function has been thoroughly examined. However, it should be appreciated that several of the gram-positive pyrogenic toxins, including SEA, SEB, SEE, SpEC and TSST1, have been found to exhibit potent immunosuppressive activity both in vivo and in vitro (3–10). This immunosuppression can be mediated via clonal deletion (2–4), the induction of anergy (5–8), or the activation of T suppressor (Ts) cells (9, 10) among T cells which bear the appropriate TCR V b alleles. It has been observed that SEA, SEB, TSST1, and SpEC are potent inducers of T suppressor cell activity (10). Suppressor cell function is believed to be mediated in part by the synthesis of cytokines with potential immunosuppressive activity. It is well established, for example, that transforming growth factor b (TGFb), IL-2, and IL-10 can inhibit certain responses either in vivo or in vitro. This is particularly true for TGFb, which has been found to suppress both cell-mediated and humoral immunity (11–13). Exposure of certain CD4-bearing T cell clones to IL-2, at critical times during T cell stimulation, can result in a failure of these T cells to respond to subsequent antigen stimulation (14–16). Finally, IL-10 inhibits the production of several cytokines including IL-1, IL-6, and TNFa, and results in reduced T cell responses due to a lack of macrophage costimulatory signals (17, 18). This form of ‘‘suppressor’’ cell function is not genetically restricted and may be masked by the production of several additional lymphokines with compensating immunoenhancing activities. It is clear that suppressor cell function may also be mediated by the generation of genetically restricted and/or antigen-specific inhibitory soluble cell products. Results from studies carried out in several experimental systems suggest that these antigen-specific Ts cells function as a circuit of at least three distinct Ts cell populations (19–21). The first Ts cell population (Ts1) is an early-acting inducer subset which expresses the
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0008-8749/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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CD5HICD80 phenotype and appears to be activated specifically by antigen. Activation of each of these populations results in the production and release of a soluble factor (TsF) capable of inducing a subsequent population of cells (Ts2 or Ts3). Ts2 typically exhibits the CD5HICD8/ phenotype, and the third subset (Ts3) is a CD5LOCD8/ suppressor-effector population and is believed to mediate the immunosuppression. Another feature of Ts cell activity in certain circumstances is the apparent genetic restriction at the controversial I–J locus (22). I–J is believed to be a homodimer protein expressed on the surface of murine Ts cells and may be a component of a soluble factor, TsF, produced by one or more Ts subsets. The exact nature of the I–J protein has yet to be determined. Genetic mapping studies have indicated that I–J should be located within the I region of H-2 (23, 24). However, the DNA sequence of this region is not apparently consistent with the existence of a structural gene within this locus (25). Our laboratory has observed that SEB induces Ts cells in murine splenocyte cultures. Multiple populations of SEB-induced Ts cells appear to be present following activation with this superantigen. The nature of the immunosuppressive activity of these cells is not fully understood and may be due, at least in part, to the production of TGFb, IL-10, or IL-2 (or other immunosuppressive lymphokines) known to be produced in response to stimulation by superantigens (26–29). We have determined that SEB also induces the production of soluble factors with genetically restricted and antigen-specific immunosuppressive activity in vitro (9, 10, 30–32). However, the nature of the immunosuppressive activity of the products of superantigen activation has not been fully defined. The purpose of this study was to characterize the effect of the SEB-induced soluble cell-free products on cellular and humoral immune responses in vivo. The results show that the SEB-induced TsF inhibits both the DTH response and the antibody response to sheep red blood cells (SRBCs). Finally, the results show that the kinetics of both activity and genetic restriction of the SEB-induced TsF are similar for both in vivo and in vitro immune responses. METHODS Mice C3H/HeN (I–Jk) mice were supplied by the National Cancer Institute (Frederick, MD) and the Jackson Laboratories (Bar Harbor, ME). The BALB/c (I–Jd), B10 (I–Jb), B10.D2 (I–Jd), B10.A (I–Jk), B10.A (2R) (I–Jk), B10.A (4R) (I–Jb), C3H/HeJ (I–Jk), C3H/HTG (I–Jd), C3H/SW (I–Jb), and C57BL/6 (I–Jb) were obtained from the Jackson Laboratories. Mice were used at 6– 12 weeks of age.
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Preparation of Anti-SEB and Anti-I–J Affinity Columns The anti-SEB affinity column was generated by coupling 30 mg each of 1FD7 and 2GD9 (33) monoclonal antibodies to a 10-ml bed volume of Affi-Gel 15 cation matrix (Bio-Rad, Hercules, CA) in 0.1 M 3-(N-morpholino)propane-sulfonic acid, pH 7.8, overnight. The column was run with a mobil phase of PBS, pH 7.2. A maximum volume of 5.0 ml was loaded onto this column, and the active fractions were pooled. The anti-I–Jd (WF18-2B12-15) and anti-I–Jb (WF940-5) affinity columns were coupled individually to 1ml bed volume of Affi-Gel 15. The antibodies were originally described by Dr. Carl Waltenbaugh (34) and obtained from Dr. H. Y. Lei (National Cheng Kung University Medical College, Tainan, Taiwan, Republic of China). The SEB-depleted factor containing supernatant was applied to the column in 0.5-ml portions. The filtrate fractions were collected and pooled. The bound material was eluted from the column with 0.2 M glycine, pH 2.9. The eluate fractions were immediately neutralized with 200 mM phosphate, pH 7.8, and active fractions were pooled and dialyzed with MEM (GIBCO Laboratories, Grand Island, NY). Generation of Delayed-Type Hypersensitivity (DTH) Mice were immunized iv with 0.2 ml of a 0.01% SRBC (Rockland, Gilbertsville, PA) solution in PBS. Four days later the mice were challenged in the footpad with 0.03 ml of a 20% SRBC solution. A DTH response was evaluated 24 hr later by measuring the increase in footpad thickness. Negative control mice were injected intravenously with PBS alone and challenged on Day 4 with 0.03 ml of a 20% SRBC solution. Four mice were analyzed for each group. The data shown represent the increase in footpad thickness relative to the negative control. Generation of an in Vivo antibody Response to SRBCs Mice were intravenously injected with 0.2 ml of 0.01% SRBCs in PBS. Five days later the mice were sacrificed by cervical dislocation. The spleen cells were isolated from each mouse individually and washed twice with DMEM (GIBCO) containing 10% FCS and once with MEM. The splenocytes from each mouse were resuspended to 5 ml volume with MEM. Plaque-forming cells (PFCs) were enumerated using a hemolytic plaque assay (35). Each spleen was assayed in triplicate, and four mice were used for each group. Determination of PFCs PFCs present in splenocyte preparations were enumerated by hemolytic plaque assay (35). The splenocytes were incubated with complement (guinea pig serum) and SRBCs at 377C for 30–40 min inside Cun-
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FIG. 1. Inhibition of the DTH response in BALB/c (I–Jd) mice by SEB-induced T cell suppressor factor generated from BALB/c, C3H/HeN, and B10 mice. BALB/c mice were injected intravenously with PBS (positive control), 1.0 ml SEB-PSC factor, or 1.0 ml NONPSC factor. Ninety minutes later the mice were immunized intravenously with 0.2 ml of a 0.01% SRBC solution. Four days later the mice were challenged in the footpad with 0.03 ml of a 20% SRBC solution. Negative-control mice only received footpad injections on Day 4. After 24 h, the footpad size for each mouse was determined. The negative-control response (no SRBC challenge) ranged fro 0.037 to 0.059 mm. Four animals were used in each group (*P õ 0.001).
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intravenously to BALB/c responder mice, and on the same day the mice were immunized with SRBCs. The positive-control mice received only the SRBC immunization. Four days after initiation of the immunization, the mice were challenged in the footpad with SRBCs, and the DTH response was measured 24 hr later. Figure 1 shows the results of these experiments. The mice which received the NON-PSC derived from either of the three strains produced a DTH response which was equivalent to the positive-control groups. However, the SEB-induced TsF generated from BALB/c cells suppressed the DTH response by 58% (P õ 0.001). Furthermore, the SEB-induced TsF generated from the C3H/ HeN and B10 strains did not suppress the DTH response significantly in the BALB/c responder mice. The SEB-induced TsF generated from C3H/HeN and BALB/c cells was analyzed under the same conditions as above using C3H/HeN mice as a source of target cells (Fig. 2). Again, the NON-PSC supernatant from either the C3H/HeN cells or the BALB/c cells did not reduce the DTH response below that of the positive-control groups. However, under these conditions the SEB-induced TsF generated from C3H/HeN cells suppressed the DTH response (P õ 0.001). Even though SEB-induced TsF from BALB/c cells was able to inhibit the DTH response in the BALB/c mice, there was no inhibition of the DTH response in C3H/HeN mice. To further investigate the genetic restriction of the
ningham chambers (35). Plaques observed in the SRBC monolayer were counted and reported as PFC values. Generation of SEB-Induced T Cell Suppressor Factor Spleen cells from naive mice were cultured with and without SEB (10 mg/ml) for 48 hr at a cell density of 1.5 1 10 7 cells/ml. The supernatants from both cultures were passed over an anti-SEB affinity column, and the filtrates were collected. The filtrate from the supernatant of the untreated culture was designated NON-PSC and the filtrate from the supernatant of the SEB-treated culture was designated SEB-PSC. RESULTS DTH Response of Mice Treated with SEB-Induced TsF We wished to determine whether the SEB-induced TsF produced from the SEB-stimulated Ts cells would inhibit a DTH response. Suppressor factor was generated from BALB/c (I–Jd), C3H/HeN (I–Jk), and B10 (I– Jb) mice. Splenocytes were cultured in the presence (SEB-PSC) or the absence (NON-PSC) of SEB for 2 days; the culture supernatants were collected and passed over anti-SEB affinity chromatography columns to remove SEB. The filtrates were then administered
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FIG. 2. Inhibition of the DTH response in C3H/HeN (I–Jk) mice by SEB-induced T cell suppressor factor generated from C3H/HeN and BALB/c mice. The procedure was described as in the legend to Fig. 1, except that C3H/HeN mice were used as responders. The negative-control repsonse (no SRBC challenge) varied from 0.06 to 0.135 mm. Four animals were used in each group (*P õ 0.001).
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FIG. 3. H-2 restriction in the suppression of a DTH response by SEB-induced TsF. The same procedure was used as described in the legend to Fig. 1. SEB-induced T suppressor cell factor was generated from BALB/c (I–Jd), B10 (I–Jb), and B10.D2 (I–Jd) mice. The recipient strains were B10 (top) and B10.D2 (bottom). Negative-control responses (no SRBC challenge) ranged from 0.055 to 0.09 mm. Four animals were used in each group (* P õ 0.001).
nously with SRBCs at initiation of the experiment as described above. Five days later, the spleen cells harvested from the responder mice were assayed for PFCs in a hemolytic plaque assay. As can be seen in Fig. 4, BALB/c and C3H.HTG SEB-induced suppressor factor, I–Jd restricted, was able to inhibit an antibody response 69% (P õ 0.05) and 75% (P õ 0.005), respectively, in BALB/c responder mice. However, the SEBinduced TsF generated from C3H/HeJ, B10.A, and B10.A(2R) mice, which exhibit the I–Jk haplotype, did not suppress the response in BALB/c mice. Furthermore, SEB-induced TsF generated from C57BL/6, B10.A(4R), and C3H.SW, which are I–Jb, was unable to inhibit the antibody response of BALB/c responder mice. Here again, NON-PSC TsF, generated from the strains of mice listed in Fig. 4, did not exhibit suppressive activity in any case. The activity of the SEB-induced TsF generated from each of the various mouse strains was also analyzed in C3H/FeJ (I–Kk) and C57BL/6 (I–Jb) responder mice. The results from these experiments are summarized in Table 1. The SEB-induced TsF generated from BALB/ c cells was unable to inhibit the generation of an antibody response in C3H/FeJ or C57BL/6 mice. However, the SEB-induced TsF from strains which are I–Jk restricted was capable of inhibiting the antibody response in C3H/FeJ mice but not in C57BL/6 mice. Finally, the SEB-induced TsF from I–Jb strains was able to suppress the antibody response in C57BL/6 animals but not in C3H/FeJ recipient mice.
SEB-induced TsF in vivo, the ability of SEB-induced TsF from BALB/c cells to inhibit a DTH response in B10 mice and B10.D2 mice was analyzed (Fig. 3). The results show that the BALB/c SEB-induced TsF, which is I–Jd restricted, was unable to inhibit the DTH response in B10 mice (Fig. 3A). On the other hand, this SEB-induced TsF was able to suppress the DTH response elicited in the B10.D2 mice (Fig. 3B; P õ 0.001). As expected, the B10 SEB-induced TsF suppressed the DTH response in B10 responder mice (Fig. 3A; P õ 0.001) and the B10.D2 SEB-induced TsF suppressed the response in B10.D2 responders (Fig. 3B; P õ 0.001). Under all conditions the NON-PSC supernatants did not alter the DTH response. I–J-Restricted Suppression of the Antibody Response of Mice Treated in Vivo with SEB-Induced TsF The effect of the SEB-induced TsF on the humoral immune response to SRBCs was also studied. Preparations of SEB-induced TsF from several mouse strains were inoculated into BALB/c responder mice. In addition, the responder mice were immunized intrave-
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FIG. 4. I-J-restricted suppreson of an in vivo anitbody response by SEB-induced Ts factor. BALB/c mice were injected intravenously with PBS (positive control), 0.2 ml SEB-PSC factor, or 0.2 ml NONPSC factor. Ninety minutes later the mice were immunized with 0.1 ml of 0.02% SRBC soltion. Five days later the splenocytes were assayed for the number of PFCs. The results are represented as the percentage suppression relative to the positive control. Four animals were used in each group (*P õ 0.05).
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TABLE 1 Inhibition of the Antibody Response to SRBCs in In Vivo by SEB-Induced Ts Cell Factor
Factor a
BALB/c (I–Jd)
C3H/FeJ (I–Jk)
C57BL/6 (I–Jb)
BALB/c (I–Jd) C3H/HTG (I–Jd) C3H/HeJ (I–Jk) B10.A (I–Jk) B10.A(2R) (I–Jk) C57BL/6 (I–Jb) B10.A(4R) (I–Jb) C3H/SW (I–Jb)
Yesb Yes No No No No No No
No No Yes Yes Yes No No No
No No No No No Yes Yes Yes
a
Suppressor factor was generated from various strains of mice and analyzed for activity in BALB/c, C3H/FeJ, or C57BL/6 mice. Recipient mice received 0.2 ml of SEB-induced suppressor factor or 0.2 ml of NON-PSC factor on Day 0 of the experiment. These mice received 0.2 ml of 0.01% SRBCs 90 min later. Five days later, the mice were sacrificed and the splenocytes analyzed for the number of PFCs by a hemolytic plaque assay (35). b ‘‘Yes’’ indicates that the SEB-induced suppressor factor was able to significantly inhibit an antibody response to SRBCs in the recipient mouse strain, and ‘‘No’’ indicates it was not.
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BALB/c responders on Days 0, 1, 2, and 3 (Fig. 7). All groups of mice including the positive control animals received SRBCs on initiation of the experiment. The BALB/c SEB-induced TsF demonstrated significant suppressive activity when injected on initiation of the experiment (53% suppression; P õ 0.001) or on Day 1 of the experiment (35% suppression; P õ 0.001). When the SEB-induced TsF was injected on Days 2 and 3, suppression of a DTH response was not observed relative to that of the positive control. DISCUSSION The capacity of the bacterial superantigens to stimulate lymphocyte and accessory cell function has been thoroughly examined; however, it should be appreciated that these agents can exhibit potent immunosuppressive activity both in vivo and in vitro (36–42). Several of the gram-positive pyrogenic toxins, including SEA, SEB, SEE, SpEC, and TSST1 have been found to induce a state of immunosuppression (36–42). The retroviral superantigens have also been shown to in-
Fractionation and Analysis of SEB-Induced TsF In an effort to further characterize the SEB-induced TsF, supernatants containing the TsF were fractionated on an anti-I–Jd affinity column and assayed for the ability to suppress a DTH response to SRBCs (Fig. 5). The eluate from the anti-I–Jd column exhibited significant suppression of the DTH response in BALB/c responder mice (P õ 0.001), whereas the filtrate did not alter the response (Fig. 5A). The NON-PSC supernatant was also fractionated on the affinity column, and neither the filtrate nor the eluate was able to inhibit the DTH response in BALB/c responder mice. As a control, the BALB/c SEB-induced TsF was fractionated on an anti-I–Jb affinity column (Fig. 5B). In these experiments, the filtrate significantly suppressed the BALB/c DTH response (P õ 0.001), whereas the eluate did not exhibit detectable activity. Furthermore, the BALB/c NON-PSC supernatant, also fractionated on the anti-I–Jb affinity column, was unable to suppress the DTH response relative to that of the positive control. Additional studies showed that the BALB/c SEBinduced TsF anti-I–Jd column eluates exhibited suppressive activity in a dose-dependent manner (Fig. 6). These studies showed that doses as low as 0.01 ml (P õ 0.001) of column eluate exhibited significant inhibitory activity. Time Course Kinetics of BALB/c SEB-induced TsF We wished to determine whether the suppressive activity of BALB/c SEB-induced TsF could act early or late in an immune response. BALB/c SEB-induced TsF was generated and administered intravenously to
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FIG. 5. Affinity chromatography of the SEB-induced T cell suppressor factor. Suppressor factor generated from BALB/c (I–Jd) mice was passed over an anti-I–Jd (top) or an anti-I–Jb (bottom) affinity column. The filtrates and the eluates from these columns (0.5 ml) were injected intravenously into BALB/c mice. The procedure for assessing the DTH response is described in the legend to Fig. 1. The negative-control responses (no SRBC challenge) ranged from 0.067 to 0.091 mm. Four animals were used in each group (* P õ 0.001).
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FIG. 6. Dose-dependent activity of I–Jd suppressor factor in BALB/c mice. SEB-induced suppressor factor from BALB/c mice was generated and passed over an anti-I–Jd affinity column. The eluate from this column was injected intravenously (at the dose indicated) into BALB/c mice. The DTH response was measured as described for the previous figures. The negative-control response (no SRBC challenge) was 0.066 mm. Four animals were used in each group (* P õ 0.001).
hibit mixed-lymphocyte responses, antibody production, and graft rejection (43–47). In a similar fashion, exposure to the bacterial superantigens inhibit both in vivo and in vitro antibody responses and the first-set (but not second-set) graft rejection response (37). The mechanism of this immunosuppressive activity has not been fully defined. It has been observed that SEA, SEB, TSST1, and SpEC are potent inducers of T suppressor cell activity (38, 41, 48, 49). It is apparent that the suppressor cell activity induced with these toxins is due to a combination of multiple cell activities. We have previously reported results which show that Ts cells are induced following stimulation with SEB either in vivo or in vitro (9, 10, 30). We have determined that at least three distinct populations of SEB-induced Ts cells are induced by SEB. The first population is an early-acting inducer subset which possesses the CD5HICD80 surface marker phenotype (9). The other populations express the CD5HI CD8/ and CD5LO CD8/ phenotypes and can inhibit antibody responses when added either early or late in an ongoing immune response (9, 10). We have demonstrated that the SEB-induced CD5HI CD80 population produces a soluble factor with suppressive activity in vitro which we term SEB-induced TsF (31). This factor mediates suppression of an in vitro antibody response to SRBCs in an I–J-restricted manner and bears a determinant which is recognized by anti-I–J antibodies (31).
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The SEB-induced TsF resembles antigen-specific suppressor factors described by a variety of other investigators. The capacity of anti-I–J monoclonal and polyclonal antibodies to neutralize TsF activity has been demonstrated for suppressor factors induced by immunization with several antigens including azobenzenearsonate (ABA)- and (NP)-coupled syngeneic cells (50, 51), random copolymers of glutamic acid, alanine, and tyrosine (GAT) (52–55), Keyhole Limpet hemocyanin (KLH) (56), and SRBCs (57). Suppressor factors which have been partially purified by anti-I–J antibody affinity chromatography have been shown to suppress cellular and humoral responses to a variety of antigens in a specific and genetically restricted fashion (50–55, 57). In the present report we show that the SEB-induced TsF binds to anti-I–J antibodies in a genetically restricted fashion, and we observe that both DTH and antibody responses to sheep erythrocytes are altered by the factor in vivo. Our earlier in vitro studies (31), and the present studies carried out in vivo, show that the function of the SEB-induced TsF is restricted at the I–J locus. This would appear to be a somewhat unique observation, in that most of the work carried out in other experimental systems has suggested that suppressorinducer factor activity is not I–J restricted (56, 58). However, the suppressor-inducer factor observed in the response to KLH exhibits I–J restriction and functions by inducing a population of CD5HI CD8/ suppressoreffector cells (59). An explanation for the disparity in
FIG. 7. Time course of suppressor factor activity. SEB-induced suppressor factor was generated from BALB/c mice and passed over an anti-I–Jd affinity column, and the eluate (0.5 ml) was injected intravenously into BALB/c mice at various times after the mice were immunized with SRBCs. A DTH response was elicited and measured as described for the previous figures. The negative-control response (no SRBC challenge) was 0.043 mm. Four animals were used in each group (* P õ 0.001).
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ANALYSIS OF SEB-INDUCED T CELL SUPPRESSOR FACTOR
the genetic restriction in the function of these suppressor-inducer factors is not currently available. Studies of the I–J-restricted nature of Ts cells and their factors have been based in part on both polyclonal and monoclonal antibodies reactive with the I–J determinant. These antibodies have been generated by cross-immunization of several recombinant mouse strains, including B10.A(3R) [I–Jb], B10.A(5R) [I–Jk], B10.A(9R) [I–Jk], and B10.HTT [I–Js]. Early genetic analysis suggested that the recombination points of the B10.A(5R) and B10.A(9R) strains defined the centromeric (left) boundary, and the recombination points of the B10.A(3R) and B10.HTT strains defined the right boundary, of the I–J locus (23, 24). These studies led to the assumption that the B10.A(3R) mice were congenic with respect to the I–J locus with B10.A(5R) mice, and the same was true of the B10.A(9R) and B10.HTT strains. However, sequencing of the H-2 region believed to represent the I–J region of both B10.A(3R) and B10.A(5R) showed that the length of this region was insufficient for the presence of a structural gene (25). For this reason, the genetic basis for the I–J restriction and the location of the gene encoding the I–J determinant remain undefined. Nevertheless, it has been the experience of a great number of investigators that the monoclonal and polyclonal anti-I–J antibodies bind to and neutralize TsF activity generated in response to a variety of antigenic stimuli (reviewed in 26 and 58). Our studies show that the SEB-induced TsF must be administered early in the immune response (by Day 1) in order to exert suppressive activity. This is consistent with our earlier work which showed that the SEB-induced TsF inhibits the in vitro antibody response to sheep erythrocytes only when added in the first 2 days of the immune response (60). We have gone on to show that at least a part of the function of this TsF is to induce late-acting Ts populations which exhibit distinct surface phenotypes (60). The kinetics of the activity of the SEB-induced TsF is consistent with that of the activities of suppressor-inducer factors generated in response to administration of GAT, ABA, NP, and SRBCs (50–57). Most commonly the T cell responsible for the generation of these suppressor-inducer factors exhibits the CD5HI CD80 phenotype (9, 10, 52, 55). Our previous results show that the SEB-induced suppressor-inducer factor is the product of an I–J/ CD5HI CD80 population. It is now well known that superantigens may exert immunosuppressive activity through the induction of anergy or clonal deletion of responsive T cell populations (2–8). However, our previous studies have shown that the administration of microgram quantities of SEB to mice leads to an inhibition of the DTH response to sheep erythrocytes (30). These studies also showed that the inhibition in the DTH response is due to the generation of suppressor T cells (30). Indeed, naive mice receiving splenic or lymph node T cells from SEB-
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treated mice fail to manifest normal DTH responses (30). We find in the present report that suppressor factor generated in vitro by treatment with SEB also exerts suppressive activity for in vivo DTH and antibody responses. Taken together, these results suggest that the immunosuppressive activity widely reported for superantigens may also be due to the generation of antigen-specific and/or genetically restricted suppressor cell populations. We suggest that the activity of these suppressor cells is due, at least in part, to the generation of suppressor factors which bear the widely observed I–J determinant. REFERENCES 1. Gascoigne, N. R. J., Sem. Immunol. 5, 13, 1993. 2. White, J., Herman, A., Pullen, A. M., Kubo, R., Kappler, J. W., and Marrack, P., Cell 56, 27, 1989. 3. Wahl, C., Miethke, T., Heeg, K., and Wagner, H., Eur. J. Immunol. 23, 1197, 1993. 4. Kawabe, Y., and Ochi, A., Nature 349, 245, 1991. 5. O’Hehir, R. E., and Lamb, J. R., Proc. Natl. Acad. Sci. USA 87, 8884, 1990. 6. Hewitt, C. R. A., Lamb, J. R., Hayball, J., Hill, M., Owen, M. J., and O’Hehir, R. E., J. Exp. Med. 175, 1493, 1992. 7. Baschieri, S., Lees, R. K., Lussow, A. R., and MacDonald, H. R., Eur. J. Immunol. 23, 2661, 1993. 8. Kawabe, Y., and Ochi, A., J. Exp. Med. 172, 1065, 1990. 9. Holly, M., Lin, Y.-S., and Rogers, T. J., Immunology 64, 643, 1988. 10. Taub, D. D., Lin, Y.-S., and Rogers, T. J., J. Immunol. 144, 456, 1990. 11. Torre-Amione, G., Beauchamp, R. D., Koeppen, H., Park, B. H., Schreiber, H., Moses, H. L., and Rowley, D. A., Proc. Natl. Acad. Sci. USA 87, 1486, 1990. 12. Su, H. C., Leite-Morris, K. A., Braun, L., and Biron, C. A., J. Immunol. 147, 2717, 1991. 13. Ruegemer, J. J., Ho, S. N., Augustine, J. A., Schlager, J. W., Bell, M. P., McKean, D. J., and Abraham, R. T., J. Immunol. 144, 1767, 1990. 14. Wilde, D. B., Prystowsky, M. B., Ely, J. M., Vogel, S. N., Dialynas, D. P., and Fitch, F. W., J. Immunol. 133, 636, 1984. 15. Otten, G., Wilde, D. B., Prystowsky, M. B., Olshan, J. S., Rabin, H., Henderson, L. E., and Fitch, F. W., Eur. J. Immunol. 16, 217, 1986. 16. Otten, G., Herold, K. C., and Fitch, F. W., J. Immunol. 139, 1348, 1987. 17. Fiorentino, D. F., Zlotnik, A., Mosmann, T. R., Howard, M., and O’Garra, A., J. Immunol. 147, 3815, 1991. 18. Taga, K., and Tosato, G., J. Immunol. 148, 1143, 1992. 19. Green, D. R., Flood, P. M., and Gershon, R. K., Annu. Rev. Immunol. 1, 439, 1983. 20. Dorf, M. E., and Benacerraf, B., Annu. Rev. Immunol. 2, 127, 1984. 21. Asherson, G. L., Colizzi, V., and Zembala, M., Annu. Rev. Immunol. 4, 37, 1986. 22. Murphy, D. B., Annu. Rev. Immunol. 5, 405, 1987. 23. Murphy, D. B., Herzenberg, L. A., Okumura, K., Herzenburg, L., and McDievitt, H., J. Exp. Med. 144, 699, 1974. 24. Tada, T., Taniguchi, M., and David, C., J. Exp. Med. 144, 713, 1974.
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HU ET AL.
25. Kronenberg, M., Steinmetz, M., Kobori, J., Kraig, E., Kapp, J. A., Pierce, C. W., Sorensen, C. M., Suzuki, G., Tada, T., and Hood, L., Proc. Natl. Acad. Sci. USA 80, 5704, 1983. 26. Nagelkerken, L., Gollob, K. J., Tielemans, M., and Coffman, R. L., Eur. J. Immunol. 23, 2306, 1993. 27. Florquin, S., Amraoui, Z., Abramowicz, D., and Goldman, M., J. Immunol. 153, 2618, 1994. 28. Tarazona, R., Gonza´lez-Garci´a, A., Zamzani, N., Marchetti, P., Frechin, N., Gonzalo, J. A., Ruiz-Gayo, M., van Rooijen, N., Marti´nez-A., C., and Kroemer, G., J. Immunol. 154, 861, 1995. 29. McKnight, A. J., Shea, C. M., Wiens, G. R., Rimm, I. J., and Abbas, A. K., Immunology 81, 513, 1994. 30. Lin, Y.-S., Hu, S.-C., Jan, M.-S., and Rogers, T. J., Cell. Immunol. 132, 532, 1991. 31. Taub, D. D., Lin, Y.-S., Hu, S.-C., and Rogers, T. J., J. Immunol. 143, 813, 1989. 32. Taub, D. D., and Rogers, T. J., Cell. Immunol. 134, 216, 1991. 33. Lin, Y.-S., Largen, M. T., Newcomb, J. R., and Rogers, T. J., J. Med. Microbiol. 27, 263, 1988. 34. Waltenbaugh, C., J. Exp. Med. 154, 1570, 1981. 35. Cunningham, A., and Szenberg, A., Immunology 14, 599, 1968. 36. Smith, B., and Johnson, H., J. Immunol. 115, 575, 1975. 37. Pinto, M., Torten, M., and Birnbaum, S., Transplantation 25, 320, 1978. 38. Schlievert, P., Infect. Immun. 28, 876, 1980. 39. Chen, S., Tse, C., Bernstein, I., and Archer, D., Int. Arch. Allergy Appl. Immunol. 63, 470, 1980. 40. Torres, B., Yamamoto, J., and Johnson, H., Infect. Immun. 35, 770, 1982. 41. Donnelly, R., and Rogers, T., Cell. Immunol. 72, 166, 1982. 42. Kawaguchi-Nagata, K., Okamura, H., Shoji, K., Kanagawa, H., Semma, M., and Shinagawa, K., Microbiol. Immunol. 29, 183, 1985.
/ m4896$8030
01-04-96 20:07:33
cia
43. Jacobsson, H., Lilliehook, B., and Blomgren, H., Scand. J. Immunol. 4, 181, 1975. 44. Lilliehook, B., Jacobsson, H., and Blomgren, H., Scand. J. Immunol. 4, 209, 1975. 45. Halle-Pannenko, O., Pritchard, L., Festenstein, H., and Berumen, L., J. Immunogenet. 13, 437, 1986. 46. Halle-Pannenko, O., Pritchard, L., Bruley-Rosset, M., Berumen, L., and Motta, R., Immunol. Rev. 88, 59, 1985. 47. Halle-Pannenko, O., Pritchard, L., and Bruley-Rosset, M., Eur. J. Immunol. 17, 1751, 1987. 48. Platsoucas, C., Oleszak, E., and Good, R., Cell. Immunol. 97, 371, 1986. 49. Carlsson, R., Hedlund, G., and Sjogren, H., Scand. J. Immunol. 25, 11, 1987. 50. Okuda, K., Minami, M., Sherr, D. H., and Dorf, M. E., J. Exp. Med. 154,468, 1981. 51. Sherr, D. H., Minami, M., Okuda, K., and Dorf, M. E., J. Exp. Med. 157, 515, 1983. 52. Pierres, M., Benacerraf, B., and Germain, R. N., J. Immunol. 123, 2756, 1979. 53. Theze, J., Waltenbaugh, C., Dorf, M. E., and Benacerraf, B., J. Exp. Med. 146, 287, 1977. 54. Theze, J., Waltenbaugh, C., Germain, R. N., and Benacerraf, B., Eur. J. Immunol. 7, 705, 1977. 55. Sorensen, C. M., and Pierce, C., J. Exp. Med. 156, 1691, 1982. 56. Tada, T., Taniguchi, M., and David, C. S., Cold Spring Harbor Symp. Quant. Biol. 41, 119, 1976. 57. Yamauchi, K., Murphy, D., Cantor, H., and Gershon, R. K., Eur. J. Immunol. 11, 905, 1981. 58. Dorf, M. E., and Benacerraf, B., Immunol. Rev. 83, 23, 1985. 59. Tada, T., Taniguchi, M., and David, C. S., J. Exp. Med. 144, 713, 1976. 60. Taub, D. D., Lin, Y.-S., and Rogers, T. J., Cell. Immunol. 131, 170, 1990.
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