Memory T cells are anergic to the superantigen staphylococcal enterotoxin B.

We have used staphylococcal enterotoxin B (SEB) to study the role of naive and memory T cells in the induction of peripheral tolerance. After administration of SEB to mice, the numbers of naive and memory T cells increase, as does the proportion of memory T cells, which are unresponsive to further stimulation with SEB in vitro. In addition, memory T cells generated in response to conventional antigen, which proliferate and provide help to B cells in the presence of the conventional antigen, fail to respond to superantigen. Hence, memory T cells, in general, are anergized by SEB. These results suggest that SEB-induced activation and anergy reflect the combined responses of naive and memory T cells. The differential activation vs. anergy of naive and memory T cells by superantigen may be related to cytokine production and may play an important role in the etiology of autoimmune diseases or immunodeficiency diseases such as acquired immune deficiency syndrome.


Summary
We have used staphylococcal enterotoxin B (SEB) to study the role of naive and memory T cells in the induction of peripheral tolerance. After administration of SEB to mice, the numbers of naive and memory T cells increase, as does the proportion of memory T cells, which are unresponsive to further stimulation with SEB in vitro. In addition, memory T cells generated in response to conventional antigen, which proliferate and provide help to B cells in the presence of the conventional antigen, fail to respond to superantigen. Hence, memory T cells, in general, are anergized by SEB. These results suggest that SEB-induced activation and anergy reflect the combined responses of naive and memory T cells. The differential activation vs. anergy of naive and memory T cells by superantigen may be related to cytokine production and may play an important role in the etiology of autoimmune diseases or immunodeficiency diseases such as acquired immune deficiency syndrome. p eripheral tolerance, due to either T cell deletion or inactivation (anergy), plays a major role in the suppression of autoimmunity (1). The mechanisms underlying induction of tolerance in peripheral T cells are incompletely understood, but significant progress has been made using model systems that use superantigens (2,3), such as Mls antigens or bacterial enterotoxins. Superantigens react with both class II antigens on APCs and external domains of specific TCR V/~ regions (2). In studies of peripheral tolerance using either Mls antigens or bacterial exotoxins, such as staphylococcal enterotoxin B (SEB), both T cell deletion and anergy follow an immune response (4,5). Hence, after administration of the superantigen, specific TCR VB-containing T cells rapidly proliferate, reflecting the superantigen-mediated mitogenic effects that are seen in vitro (4,5). After this expansion phase, T cells decline in number, presumably reflecting a deletion phase. Analyses of the remaining T cells indicate that they are unresponsive to restimulation by the superantigen in vitro (T cell anergy) (6,7).
We have used the SEB superantigen to examine the role of memory T (Try) cells during the induction of tolerance. Our data suggest that superantigen-induced T cell anergy may be due to the differentiation of SEB-reactive virgin T cells into anergic memory cells and/or the activation and subsequent deletion of virgin T cells. We favor the former possibility since all the anergic T cells displayed a memory phenotype as indicated by changes in expression of the CD45R isoform. Unexpectedly, our data also demonstrate that Tm cells are unresponsive to SEB, regardless of whether they are generated by prior exposure to SEB or to conventional an-tigens. Based on our results we suggest that: (a) T cell tolerance can be induced after memory cells have been generated (i.e., during a secondary response), and (b) naive and memory T cells are differentially stimulated by superantigens. The latter observation suggests the use of alternative signaling pathways by the T cells or, more likely, a different requirement for accessory signals by the APC.

Materials and Methods
Animals. Female BALB/c mice were bred and maintained in the mouse colony in the Department of Microbiology, University of Texas Southwestern Medical Center. All mice were used at 6-8 wk of age.
Antigens and Immunizations. KLH was obtained from Calbiochem-Behring Corp. (La Jolla, CA). For the generation of Tm cells, mice were primed by intraperitoneal injection with 100 #g of KLH in CFA 4-8 wk before experimentation. To induce T cell anergy, mice were primed by intravenous injection with 20 #g of SEB (Sigma Chemical Co., St. Louis, MO) in PBS 7 d before experimentation.

Preparation of Cells. In all experiments CD4 + T cells from
BALB/c mice (female, 4-8 wk) were prepared as previously described (16). Resulting populations were >90% CD4 +. Purified populations of Tm or virgin T (T~) cells were prepared from CD4 + splenic T cells by cell sorting using mAb 23G2 (anti-CD45RB) to separate CD45RB ~ (%) cells from CD45RB l~ (Tm) cells. APCs were prepared by T cell depletion of splenocytes using anti-Thy-l.2 and baby rabbit complement (BRC) (Pel-Freeze Biologicals, Rogers, AR) followed by anti-CD4 (mAb 2B6) plus anti-CD8, and by BKC. APCs were treated with mitomycin C ( Immunoglobulin Secretion Assays. The preparation of trinitrophenyl (TNP)-specific antigen-binding B cells (TNP-ABCs) and the T/B cognate interaction assay has been described in detail previously (16). Purified CD4 + splenic T cells from KLH-primed mice were sorted into CD45RB hi (%) and CD45RB J~ (Tin) cell populations using mAb 23G2 and FITC-MARK-1. T cells (10S/well) were cultured with TNP-ABCs (5 x 104/well) and the indicated stimuli for 7 d. Levels of secreted Ig were determined by radioimmunoassay (16).

Results and Discussion
SEB specifically interacts with V/38, 7, 11, and 17 (2). We have studied SEB-induced anergy by monitoring the expression of V/$8 on virgin CD45RB hi (T~) and memory CD45RB 1~ (Tm) CD4 + cells after in vivo administration of SEB. In normal mice, similar percentages of splenic T~ and Tm cells expressed VB8 (Tv --28%; Tm --27%), and there was a normal distribution of T~ and Tm cells within the V38 + cell population (Tv/Tm --2:1 [ Fig. 1 a]). After immunization with SEB, V38 + T cells rapidly proliferated and then disappeared (Fig. I b). This is reflected in both the V38 + T~ and Tm populations ( Fig. 1 c), and could be due to either activation and deletion of both T cell types or to the expansion of T~ cells followed by their differentiation into %, cells, which are then deleted. Regardless of the mechanism involved, there was an increase in the proportion of V•8 + Tm cells after immunization with SEB (V38 + Tv/Tm ---1:1 [ Fig. 1 d]). It should be noted that there was also an increase in the proportion of VB8-Tv cells after administration of SEB (V38-%/Tin = 3.7:1 [ Fig. 1 d]). Although there were modest variations in the V38-population from experiment to experiment, the values always fell within the range observed in normal mice. The V38-population shown in Fig. 1 d falls within this range. However, in all experiments the V38 + Tv/Tm ratio became 1:1 within 5 d after SEB administration. We next studied the T~ and Tm cells in functional assays. In accord with previous reports (5, 7), CD4 + cells from SEB-immunized mice were unresponsive to in vitro restimulation with SEB in the presence of mitomycin C-treated APCs, although these cells still responded to the polyclonal activators Con A and immobilized anti-CD3, albeit to a lesser degree than cells from control mice (Fig. 2 a). Based on a previous report, polyclonaUy activated T cells contain the same proportion of V38 + cells as do resting cells (17), suggesting that SEB-responsive VB8 + cells respond to Con A. When T~ and Tm cells from these mice were separated on the basis of CD45RB expression (18), the Tm cells were unresponsive to SEB and the residual response of the unfractionated T cells could be attributed to the T~ cells (Fig. 2 b). In comparing Fig. 2, a with b, it should be noted that the number of cells was two fold greater in the latter and that there was enrichment of the SEB-responsive (T,) cells in the experiment shown in Fig. 2 b. Primary stimulation with SEB (as opposed to conventional antigens) was not required for SEBmediated anergy of Tm cells. Thus, when CD4 + Tv and Tm cells from KLH-primed mice were examined for their ability to proliferate in response to conventional antigen (KLH) or to superantigen (SEB), the Tm cells were stimulated by KLH in a manner consistent with a recall response, but they proliferated poorly in response to SEB (Fig. 2 c). Since the frequency of KLH-responsive Tm cells is much lower than the frequency of SEB-reactive cells (0.025-0.05% vs. 20-30%) (3,19), the absolute response to KLH is significantly greater. The poor proliferative response of Tm cells to SEB was restored by the addition of Ib2, suggesting that Tm cells are 4~ la b deficient in their ability to produce Ib2 after stimulation with SEB. Since previous studies have shown that conventionally activated Tm cells secrete Ib2 (16,18), the failure of Tm cells to respond to SEB is not due to their inherent inability to secrete Ib2. In this regard, in all experiments, the SEB-induced proliferative response of Tm cells from KLH-primed animals was slightly higher than that of Tm cells from normal mice (Fig. 2, c and d), although it was markedly lower than that of Tv cells (Fig. 2 c). This enhanced proliferative response may be due to endogenous ID2 production by Tm cells previously activated by KLH or adjuvant. In addition, the SEBmediated response of activated Tm cells may explain why cultured CD45RB 1~ V~8 + T cell clones can proliferate when stimulated by SEB (20). We next examined the ability of Tv and Tm ceils to provide help to B cells that present SEB. We have shown previously that Tm cells help hapten-spedfic B cells to secrete Ig in the presence of the appropriate antigen (16). In contrast, Tv cells, which do proliferate in response to antigen, do not provide help to B cells unless they differentiate into Tm cells (16). We have extended these observations using SEB as the stimulating antigen. As shown in Fig. 3 II li " II Figure 2. Memory T cells from SEB-primed or normal animals are unresponsive to in vitro stimulation with SEK BALB/c mice were primed with KLH (intraperitoneally) in CFA 4-6 wk before use or were primed with 20/zg of SEB (intravenously) and used 7 d later. Purified splenic CD4 + T cells sorted into CD45RB hi (Tv) and CD45RB 1~ (Tin) popula-r/./ tions on the basis of CD45KB expression. Cells were cultured at 10S/well (except for b, where 2 x 10S/well were used). All wells contained 5 x 104/well APCs. After 3 d the wells were pulsed with [3H]TdR (1 #Ci/ well) and harvested 12 h later. Where indicated, SEB (15/~g/ml), Con A (5/zg/ml, KLH (15/~g/ml), or human rib2 (20 U/ml) were added to the cultures. Proliferation analyses of (a) CD4 § splenic T cells from normal (I-q) or SEB-primed (i) mice that were stimulated with SEB, Con 'rlfP-KI~I 8gB SEB A, or immobilized anti-CD3. Proliferation analyses of purified Tv ([-1) or Tm (ll) cells from (b) SEB-primed, (c) KLH-primed, or (d) unprimed mice that were stimulated with the indicated agents. In the absence of stimuhting agents or T cells (APC only), the total proliferation was <1,000 cpm/weU. In all panels, the data are an average cpm from duplicate wells from a representative experiment of four separate experiments. Although only a single data point is shown in these panels, broad cell and SEB doserange experiments were performed with similar results. The data points that are shown reflect these data. SEB or conventional antigen (TNP-KLH). Tm cells (from KLH-primed mice), which do provide help when stimulated with TNP-KLH, do not induce Ig secretion when stimulated with SEB, even when proliferation is restored by the addition of Ib2. Thus, proliferation of SEB-reactive Tm cells is insufficient to generate helper activity, either because SEB, unlike TNP-KLH, does not stimulate the secretion of the appropriate cytokines, or because it does not signal the B cells via its sIg receptor. We consider the latter hypothesis unlikely since it has been shown that activated T cells can help B cells in the absence of a signal mediated by sIg (21). Our observations suggest that SEB-induced T cell anergy follows primary stimulation of Tv cells and their differentiation into Tm cells. Tm cells then fail to proliferate or pro-vide help to B cells in the presence of SER It is possible that the activation requirements of Tm cells are more stringent than those of Tv cells. A foreign antigen, such as KLH, which requires processing and presentation in a conventional fashion, elicits an immune response by Tm cells, whereas SEB, which requires MHC class II + APCs, but not antigen processing (3), does not elicit a response. Unlike KLH, presentation of SEB may fail to induce APC-mediated costimulatory signals that are required by Tn~ but not % cells. In this regard anergy could be partially reversed in Tm calls by the addition of either II.-2, IL4, or IL-1 plus IL-6 (Fig. 4). These cytokines can promote T cell proliferation (22), and both Ibl and IL-6 are important cytokines typically produced by APCs that may act as "second signals" (23) in the activation of Tm cells. We suggest that the lack of such accessory signals may lead to tolerance even though Tm cells have already been generated by prior activation.
The processing of foreign antigen by APCs may dicit accessory signals that sdf-peptides do not. If this were the case, then one might speculate as to why T,, cells would be selectively nonresponsive or tolerized. Receptors on naive T cells that bind avidly to self-antigens are deleted in the thymus so that peripheral Tv cells do not recognize stir-antigens with high avidity (24). In addition, Tv calls are relatively nonfunctional but must differentiate into Tm cells before they can provide hdp to B cells. As Tm cells develop, they also express higher levels of adhesion molecules (25), which facilitate interactions with APCs. In a situation where an increase in adhesion leads to the increased binding of Tm ceUs to self-antigen on APCs, in the absence of appropriate accessory signals the Tm calls might be tolerized. Hence, a possible reason that the activation of Tm cells is more stringently regulated than that of Tv cells is to assure that low affinity binding of Tm cells to se/f-antigen prevents an autoimmune response.