Staphylococcal enterotoxin B induces anergy to conventional peptide in memory T cells

Cellular Immunology 222 (2003) 144–155 www.elsevier.com/locate/ycimm

Staphylococcal enterotoxin B induces anergy to conventional peptide in memory T cells Andrew R.O. Watson,a James N. Mittler,a and William T. Leea,b,* b

a The Department of Biomedical Sciences, The School of Public Health, The University at Albany, Albany, NY 12201-0509, USA The Laboratory of Clinical and Experimental Immunology and Endocrinology, The Wadsworth Center, Albany, NY 12201-2002, USA

Received 4 February 2003; accepted 29 April 2003

Abstract Microbial superantigens can alter host immunity through aberrant activation and subsequent anergy of responding naive T cells. We show here that the superantigen, staphylococcal enterotoxin B (SEB), directly induces tolerance in memory CD4 T cells. Murine naive and memory CD4þ T cells were labeled with the fluorescent dye CFSE and the cells were exposed to SEB before they were cultured with specific peptide antigen. Memory, but not naive, T cells became anergic and did not respond to their cognate peptide antigen. The extent and duration of T cell receptor (TCR) clustering was similar to promote naive T cell activation and memory T cell anergy, suggesting similar TCR–SEB interactions led to distinct intracellular signaling processes in the two cell types. Like SEB, soluble anti-CD3 mAb does not stimulate memory cell proliferation. However, unlike SEB, soluble anti-CD3 mAbs did not induce anergy to cognate peptide. Anergy was directly visualized in vivo. CD4þ memory T cells were identified in mice that had been administered SEB. The cells failed to proliferate in response to subsequent immunization with their cognate recall antigen. Hence, one mode of pathogen survival is the modulation of host immunity through selective elimination of memory T cell responses. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Clonal anergy; Signal transduction; T lymphocytes; Immune tolerance; Immunological memory

1. Introduction Immune lymphocytes must respond quickly and vigorously to foreign pathogens while recognizing and not responding to self-components. Secondary immune responses occur more quickly and are more intense than primary responses; this is largely attributable to the unique characteristics of memory lymphocytes (reviewed in [1–3]). In part, memory cells mediate more intense responses because they secrete a broader array of lymphokines, and because the interval between activation and lymphokine secretion is short [3]. More rapid secondary responses can also be partially attributed to a lower activation threshold for memory cells than for naive cells [4]. Easier activation occurs through multiple means, such as altered requirements for costimulation, increased adhesion marker expression and, possibly, an

* Corresponding author. Fax: 1-518-474-3181. E-mail address: [email protected] (W.T. Lee).

increased affinity or sensitivity of signaling through the antigen receptor [4]. Differences in requirements for cell activation suggest that naive and memory cells might also possess unique regulatory processes that control self/non-self recognition. Pathogenic organisms have developed numerous strategies to subvert the immune response and thus facilitate their survival. One such strategy is the production or induction of superantigens (reviewed in [5–7]). The best-described superantigens are bacterial exotoxins, such as enterotoxins produced by Staphylococcus aureus, and endogenously produced proteins of viral origin [8,9]. The superantigens can modulate immune responses through polyclonal T cell expansion, deletion, and tolerance. For example, in previous studies in vivo administration of staphylococcal enterotoxin B (SEB)1 resulted in clonal expansion and subsequent deletion of responding (mostly TCR Vb8þ ) T cells [10]. Further in 1 Abbreviations used: SEB, staphylococcal enterotoxin B; CFSE, 5(and-6)-carboxyfluorescein diacetate succinimidyl ester.

0008-8749/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0008-8749(03)00117-5

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

vitro studies showed that the remaining Vb8þ cells proliferated poorly upon re-stimulation with SEB, suggesting that they were inactivated (anergic) [10]. However, inactivation was associated with memory cell development in response to prior exposure to SEB [11]. Surprisingly, even memory cells from mice that had not been previously exposed to SEB also failed to proliferate when cultured with SEB. Hence, it is not clear whether SEB induced anergy or, alternatively, whether it promoted differentiation from a responsive (naive) to a nonresponsive (memory) cell. The antigen receptor on CD4 T cells can be triggered by a variety of agents, such as conventional peptide antigen, superantigen, or anti-TCR/CD3 antibodies. These stimuli perturb the TCR differently, and each can induce distinct responses from naive and from memory cells. Notably, soluble TCR/CD3-specific antibodies (presented by Fc receptor-bearing APCs) [12], and superantigens [11] induce vigorous proliferation by naive CD4 T cells but not by memory CD4 T cells. The reasons why the same stimulus elicits different responses from naive and memory CD4 cells are not clear. The reasons why the different TCR-engaging stimuli lead to different outcomes in memory cells are also not clear. The failure of memory cells to respond to anti-CD3 is a consequence of impaired or altered early TCR-mediated signal transduction [13], but the mechanism of SEB-induced hyporesponsiveness is not understood. In the present study we have examined naive and memory CD4 T cell responses to conventional peptide antigen, superantigen, and CD3-specific mAbs. Through direct assessment of cell division using flow cytometry, we show that memory cells do not proliferate when exposed to either SEB or anti-CD3. We also report that the two agents promote different biological outcomes. Exposure to SEB, but not anti-CD3, leads to clonal anergy, as SEB-exposed memory T cells fail to respond to an agonist peptide antigen. Further, we provide direct evidence for peptide-specific anergy in vivo following exposure to bacterial superantigens. Hence, our data show that memory immune responses can be selectively modulated during pathogen infection.

2. Materials and methods 2.1. Animals The BALB/c ByJ and DO11.10 [14] mice used in these experiments were bred and maintained at the Wadsworth Center Animal Core Facility under specific pathogen-free conditions. The majority of T cells in the DO11.10 mice are CD4þ cells which bear a TCR that recognizes a chicken ovalbumin-derived peptide, OVAð323–339Þ (hereafter referred to as OVA), presented

145

by I-Ad [14]. This TCR is encoded by transgenes encoding Vb8.2/Va13.1 chains and can be identified by the anti-clonotypic mAb, KJ1-26 [15]. Unless otherwise indicated, the experiments were performed using 10–12week-old mice. Both male and female mice were used in different experiments with no discernible differences in the results. All mice used in these studies were bred and maintained in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (Washington, DC). All experiments were approved by the Wadsworth Center IACUC. 2.2. Reagents and antibodies MAbs KJ1-26 (anti-DO11.10 clonotype) [15], C363.29B (anti-CD3
) [16], S4B6 (anti-IL-2), XMG1.2 (anti-IFN-c), and 23G2 (anti-CD45RB) [17], and F23.1 (anti-Vb8) [18] were prepared from the supernatants of hybridoma cell lines. MAbs directed against mouse CD69, CD25, and IL-4 were purchased from BDPharmingen (San Diego, CA). Affinipure F(ab0 )2 fragment of mouse anti-rat IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Chicken OVA peptide (OVA323–339 ) was synthesized and supplied by the Wadsworth Center Peptide Synthesis Core Facility. 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) and SEB (Toxin Technology, Sarasota, FL) were purchased. 2.3. Preparation of cells In all experiments, enriched populations of CD4þ T cells were prepared by negative selection procedures as previously described [19] and were 90–95% CD4þ and <3% sIgþ as determined by flow-cytometry. Naive and memory cells were separated based upon CD45RB expression using mAb 23G2 and MACS (Miltenyi Biotec, Auburn, CA) sorting to separate the CD45RBhi (naive) and CD45RBlo (memory) populations. Following separation, the sorting mAb was removed using a low pH buffer as described [20]. For most experiments OVA and SEB were presented to T cells using RT11-mB7 fibroblast cells [21]. This adherent cell line was originally obtained from Dr. C.T. Weaver (The University of Alabama at Birmingham, Birmingham, AL) and has previously been transfected to express both I-Ad and B7-1 molecules [21]. Cells bearing these molecules were positively selected in culture through G418 resistance. Alternatively, APCs were prepared by T cell depletion of splenocytes, as described [20]. For stimulation of T cells with anti-CD3
, the cells were pre-coated with mAb C363.29B [16] which was cross-linked using F(ab0 )2 fragment mouse anti-rat IgG to initiate cell activation.

146

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

2.4. Cell labeling and culture DO11.10 naive and memory cells (1  105 /well) were cultured in 96-well U-bottom plates with irradiated (10,000 rad) RT11-mB7 cells (2  105 /well) in 0.2 ml RPMI-1640 medium supplemented with 10% FBS, 50 lM 2-mercaptoethanol, 100 U/ml penicillin, 100 lg/ ml streptomycin, and 2 mM glutamine. Where indicated, SEB (15 lg/ml), OVAð323–339Þ (0.1 lg/ml), or soluble antiCD3 (20 lg/ml) was added to the cultures. For activation marker analysis, cells were collected for flow cytometry following 20 h of culture. For proliferation analysis, the cells were cultured for 3 days, followed by a 16-h pulse with [3 H]TdR (1 lCi/well). Cells were harvested using a 96-well automated harvester, and radioactivity was measured using a BetaPlate (Wallac, Gaithersburg, MD) . For measurement of cell division using CFSE [22], DO11.10 CD4 cells were labeled with 5 lM CFSE prior to separation into naive and memory populations. Irradiated RT11-mB7 cells (5  106 /well) were cultured in 24-well flat-bottom plates for 3 h at 37 °C to permit adherence. Non-adherent cells were removed, and remaining cells were pulsed with SEB (20 lg/ml), or OVAð323–339Þ (1.0 lg/ml) for 4 h prior to addition of the CFSE-labeled T cells. In some experiments, the T cells were removed and placed into secondary cultures which contained OVA (0.1 lg/ml) or ConA (1.0 lg/ml) and either T-depleted splenocytes as APCs or no APCs. At the indicated times, the T cells were analyzed by flow cytometry after staining with mAb KJ1-26 to identify the DO11.10 clonotype-bearing cells. Flow cytometry was performed using a FACS Calibur and CellQuest software (BD Biosciences, Mountain View, CA). Because CFSE, unlike [3 H]TdR, is a direct indicator of cumulative cell proliferation and because the measurements are independent of total cell numbers, it was not necessary to adjust the DO11.10 cell concentrations between the primary and secondary cultures. 2.5. Lymphokine analysis Percentages of lymphokine-producing cells were obtained via flow cytometry [23]. Briefly, CD4 cells were stimulated before a 5-h stimulation with PMA (20 ng/ ml) and ionomycin (1 lM) in the presence of monensin (2 lM). Cells were stained with mAb KJ1-26 to identify the antigen-specific T cells or mAb F23.1 to identify Vb8þ cells and were permeabilized to allow staining and detection with specific mAbs. 2.6. Adoptive transfer of DO11.10 cells The procedure was done as described by Kearney et al. [24] with some modifications. CFSE-labeled, naive and memory CD4þ T cells (1.5 106 ) were suspended in sterile

PBS and injected i.v. into 6- to 8-week-old BALB/c mice. Twenty-four hours after adoptive transfer, mice were immunized s.c. with PBS, OVA323–339 (150 lg) in PBS, or SEB (25 lg) in PBS. Where indicated, some mice received an additional injection 24 h later, of OVA323–339 (150 lg) in PBS. At 66 h after the initial injection, the axillary, brachial, and cervical lymph nodes were removed, pooled, and analyzed by flow cytometry.

3. Results 3.1. Memory CD4 T cells proliferate in response to peptide antigen but not to either superantigen or anti-CD3 DO11.10 mice were used as a source of both naive and memory CD4 T cells. The cells express a transgenic ab TCR that imparts specificity for both peptide antigen (OVA323–339 /I-Ad ) and SEB [14]. Clonotype-bearing memory CD4 T cells are found in the absence of intentional immunization because of environmental stimulation via a second, co-expressed TCR composed of the transgenic TCRb (Vb8.2) chain paired with an endogenous TCRa chain [20,25]. We have previously reported on the genesis and properties of the DO11.10 CD4 memory T cells [20,25]. We have shown that these cells are identical to conventional memory cells, typically defined and isolated based upon differential expression of CD45RB, CD44, or CD62L [26,27]. Although these memory cells were not generated in response to OVA, the cells are typical memory cells that can secrete memory-type lymphokines (e.g., IL-4, -5, -6, IFN-cÞ upon exposures to OVA [20]. In the present study, naive and memory DO11.10 cells were exposed to either OVA or SEB by presentation using adherent RT11-mB7 cells that express both MHC class II (I-Ad ) and B7-1 [21]. Exposure to anti-CD3 was done by incubation with rat anti-mouse CD3
followed by cross-linking with F(ab0 )2 fragment of mouse anti-rat IgG [28]. Responses by T cells via these modes of activation were comparable to those seen when T-depleted splenocytes were used to present any of the three stimuli (data not shown). Previous studies by ourselves and others have shown that naive and memory T cells can give disparate responses to various stimuli that signal through the TCR [11,28–31]. Notably, although naive CD4 cells proliferate vigorously when exposed to either soluble anti-CD3 mAbs or superantigens, memory cells are hyporesponsive to either agent [11,28]. Consistent with the results of these previous studies, memory cells from DO11.10 mice in the present study were not stimulated by either SEB or soluble anti-CD3 mAbs; however, they proliferate vigorously and secrete lymphokines when cultured with OVA (Table 1). In contrast, DO11.10 naive cells were able to respond to all three stimuli (Table 1). The observed differences in proliferation were not due to kinetic

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

147

Table 1 Activation responses by DO11.10 naive and memory T cellsa Naive T cells SEB

Anti-CD3

OVA

SEB

Anti-CD3

CD25 CD69

47.3 65.6 81.9

32.0 55.0 70.9

62.6 84.8 94.1

53.9 77.5 78.4

7.2 47.4 61.4

1.8 78.6 76.3

IL-2 IL-4 IFN-c

51.2 3.8 12.8

48.9 2.7 12.9

45.9 0.0 5.1

29.3 17.6 36.9

1.7 0.8 0.9

2.3 0.0 1.7

c

Proliferation Activation markersd Lymphokine secretione

Memory T cells

OVAb

a Proliferation, activation marker, and cytokine expression were measured in separate experiments, and the data are compiled. The data depicted are representative of several (>3) experiments measuring each function event. b Stimuli were OVA323–339 (0.1 lg/ml); SEB (15 lg/ml); and soluble anti-CD3 (20 lg/ml). All wells contained exogenous T-depleted splenocytes as APCs. c Incorporation of [3 H]TdR (cpm  103 ) added 16 h before the termination of 72-h cultures. d Percentage of KJ1-26þ cells expressing CD25 or CD69 after 20 h of culture. e Percentage of KJ1-26þ cells containing IL-2, IL-4, or IFN-c after 66 h of culture.

differences in cell growth. Naive and memory cells were pre-labeled with CFSE and cultured as above. OVAspecific T cells were identified using the anti-clonotype mAb, KJ1-26, and cumulative cell division was measured [32]. We observed multiple rounds of cell division by OVA-stimulated naive and memory cells, and by SEB and anti-CD3-treated naive cells. In contrast, cell division by SEB and anti-CD3-treated memory cells was impaired (Fig. 1). Thus, memory, but not naive, cells are selectively hyporesponsive to anti-CD3 antibody and superantigen yet are activated by conventional peptide antigen. Although neither SEB nor anti-CD3 stimulated memory T cell proliferation, it was not clear whether they were similar in other respects. We have previously characterized certain features of memory cells after exposure to SEB [33]. With respect to functional responses, SEB fails to stimulate memory cell lymphokine secretion and fails to provide help for B cell antibody production. However, memory cells are not ‘‘ignorant’’ of the superantigen, as SEB induces the expression of certain activation markers, such as CD25 and CD69 [33]. Likewise, when memory cells are stimulated with anti-CD3, activation markers are expressed, even though both proliferation and lymphokine secretion is impaired (Table 1). Hence, both SEB and anti-CD3 transduce signals through the TCR on memory cells.

Fig. 1. CD4 memory T cells respond to peptide antigen but not to superantigen or anti-CD3 mAbs. DO11.10 CD4þ naive and memory T cells were cultured for 72 h with adherent RT11-mB7 cells and either OVA323–339 or SEB, or with cross-linked soluble anti-CD3, as indicated. Cells were labeled with CFSE before culture and, after 72 h, cell division was indicated by decreased CFSE fluorescence intensity as assessed by flow cytometry. Data are gated to show CFSE staining on viable KJ1-26þ cells.

3.2. SEB induces anergy in memory but not naive T cells We have previously hypothesized that memory T cells, unlike naive cells, become anergic when exposed to superantigens, such as SEB [11]. In the present study, we have directly tested this hypothesis by examining proliferative responses to OVA by SEB-treated memory cells. A two-step culture was used to assay anergy. In a primary culture, T cells were stimulated

for various times before removal, washing, and addition to a secondary culture. Secondary cultures contained the stimulatory agonist, OVA. In the primary culture, antigen was presented using RT11-mB7 cells. These APCs are adherent and are not present in the secondary culture. In additional control experiments we confirmed that antigen was not carried from the

148

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

primary cultures into the secondary cultures (data not shown). To determine whether SEB induced anergy in memory cells, we labeled naive and memory DO11.10 T cells with CFSE and cultured them with SEB-pulsed RT11-mB7 cells for 18 h. The T cells were then removed, washed, and cultured with OVA-pulsed T-de-

Fig. 2. SEB induces anergy in CD4 memory T cells in vitro. (A) CFSE-labeled DO11.10 naive and memory cells were cultured with RT11-mB7 cells presenting the indicated stimulus for 18 h before the T cells were removed and re-cultured with T cell-depleted splenocytes and OVA for an additional 60 h before they were collected, stained with mAb KJ1-26, and analyzed by flow cytometry. Data are gated to show CFSE staining on viable KJ1-26þ cells. (B) CFSE-labeled BALB/c naive and memory cells were cultured with RT11-mB7 cells presenting the indicated stimulus for 18 h before the T cells were removed and re-cultured with T cell-depleted splenocytes and ConA for an additional 60 h before they were collected, stained with mAb F23.1, and analyzed by flow cytometry. Data are gated to show CFSE staining on viable Vb8þ cells (solid lines) and Vb8 cells (dashed lines).

pleted splenocytes for 60 h. Cell recovery was comparable regardless of the primary stimulus. In the secondary cultures, cell division was determined using flow cytometry to measure a step-wise reduction in CFSE fluorescence intensity. As shown in Fig. 1, naive and memory T cells proliferated in response to OVA. However, if memory cells were exposed to SEB prior to culture with the OVA-bearing APCs, no proliferation occurred (Fig. 2A). Similarly, we observed a marked diminution of secreted lymphokines when memory cells were exposed to SEB before being stimulated with OVA (Table 2). Naive and memory T cells were cultured with SEB or anti-CD3 for 18 h prior to washing and re-stimulation for 60 h with OVA. Cytokine production was then measured by intracellular staining for IL-2, IL-4, or IFN-c and flow cytometry. As previously reported, naive cells do not produce much IL-4 or IFN-c but they do produce copious amounts of IL-2 [19,34]. Memory T cells produce IL-2 as well as IL-4 and IFN-c [19,34]. However, if the cells are first exposed to SEB, then fewer memory cells produce lymphokines (Table 2). As described in more detail below, exposure to anti-CD3 did not lead to reduced production of cytokines by memory cells responding to OVA, even though anti-CD3 did not, itself, promote cytokine secretion (Table 1). Thus, SEB-exposed memory, but not naive, cells were anergic. Anergy was not merely a function of using cells from TCR transgenic mice. To further demonstrate that our conclusions are not limited to the transgenic mouse model, we measured SEB-induced anergy in memory cells obtained from conventional BALB/c mice. We have previously reported that memory cells from conventional mice do not respond to SEB [11,33]). However, because of the low frequency of individual antigen-specific T cells, we could not examine the effects of SEB exposure on subsequent responses to peptide. Hence, we used the mitogen, Con A, as a second stimulus and measured polyclonal proliferation of Vb8þ CD4þ T cells. Memory cells were isolated and were labeled with CFSE. The cells were then stimulated with SEB for 18 h before the SEB was removed and the cells were re-stimulated with Con A. When we examined the Vb8þ cells, we found that proliferation was markedly inhibited, similar to our observations with DO11.10 memory cells (Fig. 2B). However, we did observe that treatment with Con A led to a slight amount of proliferation, as compared to OVA stimulation. Further, there was marked proliferation of the Vb8 cells which did not bind SEB in the original cultures. The small amount of proliferation of the Vb8þ memory cells is likely a consequence of IL-2 secreted by the Con A-stimulated Vb8 cells in the same cultures. We have previously shown that exogenous IL-2 ablates the inhibitory effect of SEB on

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

149

Table 2 Intracellular lymphokine production after anergy induction in memory CD4 T cellsa Naive T cells

c

Lymphokine secretion

IL-2 IL-4 IFN-c

Memory T cells

Unstimulatedb

OVA

Anti-CD3

SEB

Unstimulated

OVA

Anti-CD3

SEB

58.3 0.0 0.0

85.3 0.7 1.2

50.4 2.2 0.0

51.9 2.6 2.9

33.0 4.7 10.4

38.7 3.9 11.9

29.8 2.6 10.1

10.1 0.9 0.9

a Lymphokine production was measured in both naive and memory CD4 T cells. The data depicted are representative of two separate experiments. b Primary stimuli were nothing (unstimulated), OVA323–339 (1.0 lg/ml) or SEB (20 lg/ml) presented by adherent RT11-mB7 cells, or stimulated with soluble anti-CD3 (1 lg/ml). After 18 h, the T cells were washed and re-stimulated with OVA323–339 (0.1 lg/ml) presented by exogenous T-depleted splenocytes. c Percentage of KJ1-26þ cells containing IL-2, IL-4, or IFN-c after 60 h of culture with OVA323–339 .

Fig. 3. SEB induces anergy in CD4 memory T cells in vivo. Purified CFSE-labeled DO11.10 naive and memory cells were injected into BALB/c mice, which were then immunized with the indicated agent 24 h later. LN cells were collected, stained with mAb KJ-126, and analyzed by flow cytometry to assess proliferation of the donor cells. Data are gated to show CFSE staining on viable KJ1-26þ cells. (A) Single injection and analysis 66 h later. (B) Twenty-four hours after the initial immunization the mice were immunized with OVA and proliferation was assessed 48 h later (72 h after the initial injection).

memory cells [33]. Still, the small degree of observed proliferation was markedly less than that of comparably treated naive Vb8þ cells or Vb8 memory cells exposed to Con A. Anergy was not limited to in vitro exposure to the superantigen or separation of the memory from the naive cells. Previous studies have shown that small numbers of indicator transgenic T cells, transferred into non-irradiated mice, can reliably represent the normal immune function of resident cells [24]. Using this previously described adoptive transfer model, we injected CFSE-labeled naive or memory DO11.10 CD4 T cells into BALB/c hosts. The recipient mice were injected with SEB. Lymph nodes were isolated 66 h later, and proliferation of the KJ1-26þ donor cells was determined using flow cytometry. As shown in Fig. 3A, naive DO11.10 cells proliferated vigorously in response to SEB but memory cells did not. Further, both naive and memory donor cells proliferated in vivo if the recipient mice were injected with soluble OVA. We then determined whether SEB induced anergy in vivo. After transfer of CFSE-labeled naive or memory cells, the recipient mice were injected first with SEB and then with soluble OVA 24 h later. Lymph nodes were collected 48 h later and proliferation was assessed in the KJ1-26þ population. Regardless of whether naive or memory cells were transferred, comparable numbers of DO11.10 cells were recovered from the isolated host lymph nodes 24 h after injection of SEB as compared to PBS (prior to initial cell proliferation) (0.5–2% of the total injected cell number (data not shown)). Further, at this time point there was little difference in DO11.10 cell recovery in mice that had received naive, as opposed to memory, cells. Of the donor cells that were present in the lymph nodes, it was evident that cell responses to SEB had occurred. Similar to what was observed in the in vitro cultures, naive cells proliferated (Fig. 3B). In contrast, OVA-specific proliferation was absent if the memory cells were first exposed to SEB (Fig. 3B). Hence, memory cells became anergic within a normal lymph node environment in which resident, SEB-stimulated naive cells were present.

150

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

3.3. The duration of TCR signaling is similar for both activation and anergy We next addressed the relationship between TCR signaling and anergy in memory cells. We first determined the time required for SEB to induce an anergic signal. DO11.10 memory T cells were cultured with SEB for varying time periods before they were washed and re-cultured with OVA. As indicated in Fig. 4A, the

Fig. 4. Proliferation and anergy require similar signaling times. CFSElabeled DO11.10 CD4þ (A,C) memory and (B,D) naive cells were cultured with (A,B) SEB or (C,D) OVA-pulsed RT11-mB7 cells for the indicated lengths of time. The cells were then removed and re-cultured, either (B,C,D) in the absence of additional stimulus, or (A) with APCs plus OVA for an additional 60 h, before the cells were collected, stained with mAb KJ1-26, and analyzed by flow cytometry. Data are gated to show CFSE staining on viable KJ1-26þ cells. Naive cells which received SEB and which were re-cultured with OVA, proliferated as in Fig. 2.

memory cells needed to be exposed to SEB for 10–12 h before they became unresponsive to OVA. In additional cultures, naive cells were exposed to SEB for various times before the superantigen was removed. The cells were then cultured in the absence of any stimulus for an additional 60 h before proliferation was assessed. As indicated in Fig. 4B, naive cells required 10–12 h of exposure to SEB before they became committed to proliferate. This is longer than the interval required for OVA-mediated proliferation. As we have reported previously ([35], also shown in Fig. 4C) commitment to

Fig. 5. Proliferation and anergy occur at the same SEB concentrations. Purified CFSE-labeled DO11.10 (A,C) memory and (B,D) naive cells were cultured with the indicated concentrations of SEB for 20 h. The T cells were then collected and re-cultured either (A,B) in the absence of further stimulation, or (C,D) with APCs plus OVA for an additional 60 h, before the cells were collected, stained with mAb KJ1-26, and analyzed by flow cytometry. Data are gated to show CFSE staining on viable KJ1-26þ cells.

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

proliferate in response to OVA occurs much more quickly (approximately 2 h). Similarly, only 2 h are needed for memory cells to become committed to proliferate in response to OVA (Fig. 4D). These data suggest that the different stimuli require different durations of TCR signaling. Further, activation and anergy occur over the same time frames, suggesting that anergy is not a consequence of a greater or lesser contact period between the APC and the responding T cell. 3.4. The extent of TCR occupancy does not alter the anergic signal The previous experiments directly showed that, not only do memory cells fail to proliferate when exposed to SEB, but they also become anergic and can no longer respond to an agonist peptide. We next examined whether the degree of TCR clustering determined whether memory cells became anergic. We asked whether altering the strength of the SEB-mediated signal could permit a memory response to OVA. DO11.10 naive and memory cells were cultured with varying amounts of SEB. At no dose did memory cells proliferate in response to the superantigen (Fig. 5A). The concentration of SEB in the cultures did influence the fraction of naive cells that could proliferate, with cell division beginning at 0.4 lg/ml and with most cells proliferating if exposed to 1 lg/ml of SEB (Fig. 5B). We cultured memory cells with similar doses of SEB for 20 h prior to washing and addition of OVA. When memory cells were exposed to very low doses of SEB in the initial cultures, they could

151

still proliferate when later cultured with OVA. However, beginning at the concentration of 0.4 lg/ml of SEB and clearly evident at 1 lg/ml, greater numbers of undivided cells were present in the secondary cultures (Fig. 5C). Even at very high doses of SEB in the primary culture, the memory cells remained unresponsive to OVA. Conversely, at no dose of SEB in the initial culture did naive cells lose the ability to subsequently respond to OVA (Fig. 5D). Hence, anergy and activation occur over the same range of SEB concentrations. Since we could not convert the anergic signal into an activating or even a ‘‘null’’ signal by altering the dose of SEB, we wished to determine whether we could convert a null signal into an anergic signal. As shown (Fig. 1), soluble anti-CD3 mAb does not stimulate memory cells to proliferate. A previous report suggested that soluble anti-CD3 did not impair a subsequent response to T cell mitogens [28]. We also wished to show whether antiCD3 elicited a similar response to peptide antigens. The DO11.10 naive and memory CD4 cells were labeled with CFSE prior to exposure to anti-CD3 for various times. The cells were then stimulated by OVA-bearing APCs and subsequent cell division was measured using flow cytometry. As shown in Fig. 6A, both naive and memory cells proliferated in response to OVA, regardless of whether or not they were previously exposed to antiCD3. Hence, in contrast to superantigens, anti-CD3 did not induce anergy in memory cells. To ensure that this distinction was not due to differences in the extent of TCR clustering, we cultured naive and memory cells with different concentrations of the anti-CD3 mAb for

Fig. 6. Anti-CD3 does not induce anergy in CD4 memory T cells. CFSE-labeled DO11.10 CD4þ naive and memory T cells were cultured with crosslinked, soluble anti-CD3 (A) for the indicated times, or else (B) they were cultured for 20 h with the indicated concentrations of anti-CD3. At the end of the primary culture, the T cells were collected and re-cultured with APCs plus OVA for 60 h before proliferation was assessed. Data are gated on viable KJ1-26þ cells. The unstimulated cells received no treatment in either culture.

152

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

20 h prior to addition of OVA. At no dose of anti-CD3 mAb did naive or memory cells lose the ability to proliferate in response to peptide antigen, nor did they become anergic (Fig. 6B). Hence, OVA, SEB, and antiCD3 induce functionally distinct outcomes in memory cells: activation, anergy, and an ‘‘ignorant’’ response, respectively. In contrast, these same stimuli all lead to activation of naive cells.

4. Discussion By using CFSE to directly visualize cumulative cell division of antigen-specific CD4 T cells, we studied the responses made by naive and memory T cells to various TCR-ligating stimuli. We provide direct evidence that bacterial superantigens induce anergy selectively in memory T cells. Additionally, we confirm that, although neither superantigen nor soluble anti-CD3 mAbs stimulate memory cell proliferation, even though both agents induce vigorous responses by naive T cells, only superantigens prevent subsequent responses to recall peptide antigens. Hence, ligation of the TCR on memory cells may lead to three distinct outcomes depending upon the stimulus: activation (peptide antigen), anergy (superantigen), or ignorant responses (anti-CD3 mAbs). Our data strongly suggest that the exposure to superantigens via pathogen infection can modulate recall or post-vaccination immune responses. In part, we began this study to determine the similarity between memory cell responses to SEB and to soluble anti-CD3 mAbs. Since neither agent induces proliferation, it was important to discern whether they act in mechanistically similar ways. Clearly, both agents transduce signals through the TCR, which result in activation marker expression but which are insufficient to induce either proliferation or lymphokine secretion. Hence, both anti-CD3 and SEB promote partial signaling in memory cells, and complete signaling in naive cells. In our earlier report, we had speculated that SEB induced specific memory cell anergy [11]. Certainly, CD4 T cells from mice that had been previously exposed to superantigens did not proliferate when re-challenged in vitro. However, since we could not specifically examine subsequent responses to peptide antigen made by T cells of the same TCR Vb family recognized by SEB, we could not distinguish anergy induction from a simple failure to respond to the superantigen. Likewise, memory cells that had been previously exposed to anti-CD3 were able to proliferate when re-challenged with mitogens (Con A); however, peptide-specific recall responses were not tested [28]. The DO11.10 mouse model is useful for studying these different stimuli since this mouse has an abundance of peptide-specific memory CD4 cells. In this report, then, we have been able to test our earlier hypothesis and also provide the first direct evidence that

SEB does indeed induce anergy. We have also been able to confirm our results in conventional BALB/c mice through the analysis of responses to Con A. Since comparable results were obtained when using conventional mice and DO11.10 transgenic mice, our data indicate that superantigens prevent the response of memory cells to recall peptides, as exemplified by OVA323–339 . Although cells obtained from either DO11.10 or conventional mice behaved similarly in our studies, one additional consideration was potential heterogeneity in our memory cell populations. The cells that we used in our study were isolated based on low expression of CD45RB, a commonly used means of isolating memory cells. Further, the isolated cells were also CD62Llo and CD44hi , thus possessing a conventional memory cell phenotype [1,3,26]. Independent isolation of memory cells based on high expression of CD44, instead of low expression of CD45RB, resulted in similar functional characteristics in all experiments, including SEB-induced tolerance (WTL, unpublished observations). Based on these phenotypic characteristics, we would conclude that all or most of the cells we have isolated are conventional memory cells. However, even if CD45RBlo non-memory cells were present in our cell populations, they must also become anergic in response to SEB, since we did not observe subpopulations of cells that proliferated when exposed to SEB, or subsequently, to OVA. Further analysis of the DO11.10 CD45RBlo cells indicated that they did not express CD25 prior to cell activation and did not secrete IL-10 upon stimulation with SEB [33]. These observations suggest that there is not a preponderance of ‘‘regulatory’’ T cells in our cell populations and that anergy is directly imposed upon the cells rather than via exposure to IL-10. The different outcomes of anti-CD3 and SEB interactions on memory cells are not simply a matter of difference in the degree of TCR clustering or affinity since, over broad concentration ranges and culture, SEB still induced anergy and anti-CD3 did not. Studies have indicated that, when anti-CD3 is presented by FcRþ APCs, the presence of MHC class II is the critical element which determines whether memory T cells will proliferate [28,36]. Because MHC class II is not used to present anti-CD3, signaling by MHC class II through CD4 occurs independently of the TCR/CD3 complex. This signal through CD4 is inhibitory, although not tolerogenic [28]. We presume that SEB is dissimilar, since the superantigen is presented as a complex with MHC class II, and therefore MHC class II/CD4 signaling occurs in physical conjunction with TCR/CD3 signaling. This conclusion is supported by data from DO11.10  CD4= mice, for which we observed that memory cells respond well to both OVA and soluble anti-CD3 but remain unresponsive to SEB (WTL, unpublished observations). Instead, we favor the hypothesis that SEB-induced anergy occurs by alternative

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

signaling through the TCR. SEB begins to stimulate proliferation of naive cells at the same concentrations at which it causes memory cells to become anergic. Further, signaling, leading to memory cell commitment to anergy, occurs during the same time period in which naive cells become committed to proliferate. These data suggest that the same signaling processes lead to distinct outcomes. We also note that SEB-signaling requires prolonged contact between the T cell and APC relative to OVA-mediated signaling (12 h versus 2 h). We have previously reported that CD4 T cells require only a short exposure to OVA before they become committed to proliferate in an OVA-independent fashion [35]. In the current study we have confirmed this finding, and we provide evidence that the nature of the stimulus, rather than intrinsic T cell factors, determines the time period required to prime cells for proliferation (or anergy). We also extend our previous findings to show that naive and memory cells require similar time periods before they become committed to proliferate in the absence of continuous peptide stimulation. As noted above and as we have previously reported, the addition of IL-2 to SEB-treated memory cells enables proliferation in in vitro cultures [33]. Because SEB is a potent inducer of IL-2 secretion by naive T cells, one concern was that anergy only occurred under artificial conditions in which naive and memory cell populations were physically separated during in vitro culture. In an in vivo environment, exposure to superantigens might occur in lymphoid tissue in which both memory and IL2-secreting naive cells are present. Hence, it was important to show both that memory cells were initially hyporesponsive, and also that they were induced to anergy under conditions in which they might normally encounter superantigens. Correspondingly, in our adoptive transfer experiments, we observed that DO11.10 memory cells failed to respond to SEB, even in lymph nodes where robust host naive cell proliferation was occurring. Further, these memory cells became anergic, suggesting that host IL-2 concentrations surrounding the donor cells were insufficient to prevent tolerance. These adoptive transfer experiments strongly suggest that exposure to superantigens in vivo selectively ablates memory responses. Prior studies of superantigens had generally considered them as possible inducers of autoimmune disease due to their broad stimulation of naive T cells [7,37]. In this regard, it is worth noting that a number of studies have employed SEB to manipulate responses to myelin basic protein (MBP) in models of EAE [38–40]. The preponderance of these studies have shown that SEB can suppress EAE if the superantigen is administered before initial immunization with MBP. These data conform to our current understanding of SEB-induced tolerance; that is, that naive T cells become stimulated by SEB and then are deleted or become anergic. How-

153

ever, when SEB is administered after disease onset or during remission, EAE can either be exacerbated or reinitiated, respectively [38]. On the surface, these data suggest that antigen-experienced cells are activated, rather than tolerized, and appear to differ from the data presented in our current study. However, we believe that the recurrence of disease in these models reflect activation of effector, and not memory, cells. As noted above, effector, as opposed to resting memory, cells are easily activated by SEB. Hence, it is likely that the self-reactive cells in a chronic autoimmune condition, such as that modeled by EAE, do not become truly resting and, thus, do not become susceptible to superantigen-mediated tolerance. The summation of these studies suggest that superantigens have multiple effects, depending upon the responding cell. In naive cells, superantigens induce activation followed by tolerance; in effector cells, superantigens promote robust activation; and, in CD4 memory cells superantigens directly induce anergy. We also note that CD8 memory T cells respond well to SEB stimulation [41]. While memory CD4 and CD8 cells are clearly different in many respects, including having multiple distinct regulatory requirements, it is of interest to determine why these two cell types differ in their response to superantigens. Our data have implications for the better understanding of host–pathogen relationships. Superantigens may be encountered normally during infection, either as bacterial products or because viral infection may promote host cell surface expression of superantigens encoded by endogenous retroviruses [42,43]. Bacterial toxins have also recently been proposed as potential bioterrorism agents [42]. We suggest an additional consequence of exposure to superantigens, namely, the loss of pre-existing immunity through anergy of memory cells. Polyclonal activation of naive cells by superantigens might permit pathogens to escape specific immunity [7]. Similarly, we speculate that tolerance of broad families of memory cells would facilitate pathogen evasion or facilitate future infections. Obviously, as with the case for naive cell tolerance, such immune perturbation would require either the removal of immunity towards predominant epitopes on the pathogen, targeted by a specific TCR Vb family, or alternatively, the involvement of multiple superantigens. Consequently, exposure to superantigens would shape the memory repertoire and could subvert prior intentional vaccination.

Acknowledgments The authors acknowledge the Wadsworth Center Immunology Core and the Wadsworth Center Peptide Synthesis Core. We also acknowledge Mr. G. Pasos, Mr. K. Moynehan, and Ms. L. Cecchini for their expert technical assistance. We thank Drs. E. Vitetta,

154

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155

D. Murphy, and G. Winslow for many helpful discussions during the course of this work and for their critical reviews of the manuscript. This work was supported by National Institutes of Health Grants AI- 35583 and AG17158.

References [1] E.S. Vitetta, M.T. Berton, C. Burger, M. Kepron, W.T. Lee, X.M. Yin, Memory T and B cells, Annu. Rev. Immunol. 9 (1991) 193– 217. [2] R.W. Dutton, L.M. Bradley, S.L. Swain, T cell memory, Annu. Rev. Immunol. 16 (1998) 201–223. [3] S.L. Swain, Helper T cell differentiation, Curr. Opin. Immunol. 11 (1999) 180–185. [4] M. Croft, C. Dubey, Accessory molecule and costimulation requirements for CD4 T cell response, Crit. Rev. Immunol. 17 (1997) 89–118. [5] C.A. Janeway Jr., J. Yagi, P.J. Conrad, M.E. Katz, B. Jones, S. Vroegop, S. Buxser, T-cell responses to M1s and to bacterial proteins that mimic its behavior, Immunol. Rev. 107 (1989) 61–88. [6] A. Herman, J.W. Kappler, P. Marrack, A.M. Pullen, Superantigens: mechanism of T-cell stimulation and role in immune responses, Annu. Rev. Immunol. 9 (1991) 745–772. [7] J. Fraser, V. Arcus, P. Kong, E. Baker, T. Proft, Superantigens— powerful modifiers of the immune system, Mol. Med. Today 6 (2000) 125–132. [8] P. Marrack, J. Kappler, The staphylococcal enterotoxins and their relatives, Science 248 (1990) 705–711. [9] D.L. Woodland, M.P. Happ, K.J. Gollob, E. Palmer, An endogenous retrovirus mediating deletion of alpha beta T cells? Nature 349 (1991) 529–530. [10] Y. Kawabe, A. Ochi, Selective anergy of Vbeta8+, CD4þ T cells in staphylococcus enterotoxin B-primed mice, J. Exp. Med. 172 (1990) 1065–1070. [11] W.T. Lee, E.S. Vitetta, Memory T cells are anergic to the superantigen, staphylocoocal enterotoxin B, J. Exp. Med. 176 (1992) 575–580. [12] M. Luqman, K. Bottomly, Activation requirements for CD4þ Tcells differing in CD45r expression, J. Immunol. 149 (1992) 2300– 2306. [13] D.L. Farber, O. Acuto, K. Bottomly, Differential T cell receptormediated signaling in naive and memory CD4 T cells, Eur. J. Immunol. 27 (1997) 2094–2101. [14] K.M. Murphy, A.B. Heimberger, D.Y. Loh, Induction by antigen of intrathymic apoptosis of CD4þ CD8þ TCRlo thymocytes in vivo, Science 250 (1990) 1720–1722. [15] K. Haskins, R. Kubo, J. White, M. Pigeon, J. Kappler, P. Marrack, The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody, J. Exp. Med. 157 (1983) 1149–1169. [16] P. Portoles, J. Rojo, A. Golby, M. Bonneville, S. Gromkowski, L. Greenbaum, C.A. Janeway Jr., D.B. Murphy, K. Bottomly, Monoclonal antibodies to murine CD3 epsilon define distinct epitopes, one of which may interact with CD4 during T cell activation, J. Immunol. 142 (1989) 4169–4175. [17] M.L. Birkeland, J. Metlay, V.M. Sanders, R. Fernandez-Botran, E.S. Vitetta, R.M. Steinman, E. Pure, Epitopes on CD45R (T200) molecules define differentiation antigens on murine B and T lymphocytes, J. Mol. Cell. Immunol. 4 (1988) 71–85. [18] U.D. Staerz, H.G. Rammensee, J.D. Benedetto, M.J. Bevan, Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor, J. Immunol. 134 (1985) 3994–4000.

[19] W.T. Lee, X.-M. Yin, E.S. Vitetta, Functional and ontogenetic analysis of murine CD45Rhi and CD45Rlo CD4þ T cells, J. Immunol. 144 (1990) 3288–3295. [20] W.T. Lee, J. Cole-Calkins, N.E. Street, Memory T cell development in the absence of specific antigen priming, J. Immunol. 157 (1996) 5300–5307. [21] T. Zhou, C. Weaver, P.S. Linsley, J.D. Mountz, T cells of staphylococcal enterotoxin B-tolerized autoimmune MRL-lpr/lpr mice require co-stimulation through the B7-CD28/CTLA-4 pathway for activation and can be reanergized in vivo by stimulation of the T cell receptor in the absence of this co-stimulatory signal, Eur. J. Immunol. 24 (1994) 1019–1025. [22] A.B. Lyons, C.R. Parish, Determination of lymphocyte division by flow cytometry, J. Immunol. Methods 171 (1994) 131– 137. [23] C. Prussin, D.D. Metcalfe, Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies, J. Immunol. Methods 188 (1995) 117–128. [24] E.R. Kearney, K.A. Pape, D.Y. Loh, M.K. Jenkins, Visualization of peptide-specific immunity and peripheral tolerance induction in vivo, Immunity 1 (1994) 327–339. [25] W.T. Lee, V. Shiledar-Baxi, G.M. Winslow, D. Mix, D.B. Murphy, Self-restricted dual receptor memory T cells, J. Immunol. 161 (1998) 4513–4519. [26] W.T. Lee, E.S. Vitetta, The differential expression of homing and adhesion molecules on virgin and memory T cells in the mouse, Cell. Immunol. 132 (1991) 215–222. [27] D.N. Ernst, M.V. Hobbs, B.E. Torbett, A.L. Glasebrook, M.A. Rehse, K. Bottomly, K. Hayakawa, R.R. Hardy, W.O. Weigle, Differences in the expression profiles of CD45RB, Pgp-1, and 3G11 membrance antigens and in the patterns of lymphokine secretion by splenic CD4þ T cells from young and aged mice, J. Immunol. 145 (1990) 1295–1302. [28] D.L. Farber, M. Luqman, O. Acuto, K. Bottomly, Control of memory CD4 T cell activation: MHC class II molecules on APCs and CD4 ligation inhibit memory but not naive CD4 cells, Immunity 2 (1995) 249–259. [29] D.L. Farber, Cutting edge commentary: differential TCR signaling and the generation of memory T cells, J. Immunol. 160 (1998) 535–539. [30] J.A. Byrne, J.L. Butler, M.D. Cooper, Differential activation requirements for virgin and memory T cells, J. Immunol. 141 (1988) 3249–3257. [31] A. Lerner, T. Yamada, R.A. Miller, PgP-1hi T lymphocytes accumulate with age in mice and respond poorly to concanavalin A, Eur. J. Immunol. 19 (1989) 977–982. [32] W.T. Lee, W.J. Pelletier, Visualizing memory phenotype development after in vitro stimulation of CD4þ T cells, Cell. Immunol. 188 (1998) 1–11. [33] W.T. Lee, G.R. Thrush, E.S. Vitetta, Staphylococcal enterotoxin B induces the expression of activation markers on murine memory T cells in the absence of proliferation or lymphokine secretion, Cell. Immunol. 162 (1995) 26–32. [34] L.L. Carter, S.L. Swain, From naive to memory. Development and regulation of CD4+ T cell responses, Immunol. Res. 18 (1998) 1–13. [35] W.T. Lee, G. Pasos, L. Cecchini, J.N. Mittler, Requirements for continued antigen stimulation during CD4þ T cell clonal expansion, J. Immunol. 168 (2002) 1682–1689. [36] D.P. Metz, K. Bottomly, Function and regulation of memory CD4 T cells, Immunol. Res. 19 (1999) 127–141. [37] T. Renno, H. Acha-Orbea, Superantigens in autoimmune diseases: still more shades of gray, Immunol. Rev. 154 (1996) 175– 191. [38] M.R.P. Das, A. Cohen, S.S. Zamvil, H. Offner, V.K. Kuchroo, Prior exposure to superantigen can inhibit or exacerbate autoimmune encephalomyelitis: T-cell repertoire engaged by the

A.R.O. Watson et al. / Cellular Immunology 222 (2003) 144–155 autoantigen determines clinical outcome, J. Neuroimmunol. 71 (1996) 3–10. [39] L.M. Kuschnaroff, L. Overbergh, H. Sefriouni, H. Sobis, M. Vandeputte, M. Waer, Effect of staphylococcal enterotoxin B injection on the development of experimental autoimmune encephalomyelitis: influence of cytokine and inducible nitric oxide synthase production, J. Neuroimmunol. 99 (1999) 157–168. [40] J.M. Soos, M.G. Mujtaba, J. Schiffenbauer, B.A. Torres, H.M. Johnson, Intramolecular epitope spreading induced by staphylo-

155

coccal enterotoxin superantigen reactivation of experimental allergic encephalomyelitis, J. Neuroimmunol. 123 (2002) 30–34. [41] M.A. Coppola, M.A. Blackman, Bacterial superantigens reactivate antigen-specific CD8+ memory T cells, Int. Immunol. 9 (1997) 1393–1403. [42] M. Llewelyn, J. Cohen, Superantigens: microbial agents that corrupt immunity, Lancet Infect. Dis. 2 (2002) 156–162. [43] D.L. Woodland, Immunity and retroviral superantigens in humans, Trends Immunol. 23 (2002) 57–58.