Abstract
This report defines a cell surface receptor (OX40) expressed on effector CD4 T cells, which when engaged in conjunction with a danger signal, rescues Ag-stimulated effector cells from activation-induced cell death in vivo. Specifically, three signals were necessary to promote optimal generation of long-lived CD4 T cell memory in vivo: Ag, a danger signal (LPS), and OX40 engagement. Mice treated with Ag or superantigen (SAg) alone produced very few SAg-specific T cells. OX40 ligation or LPS stimulation, enhanced SAg-driven clonal expansion and the survival of responding T cells. However, when SAg was administered with a danger signal at the time of OX40 ligation, a synergistic effect was observed which led to a 60-fold increase in the number of long-lived, Ag-specific CD4 memory T cells. These data lay the foundation for the provision of increased numbers of memory T cells which should enhance the efficacy of vaccine strategies for infectious diseases, or cancer, while also providing a potential target (OX40) to limit the number of auto-Ag-specific memory T cells in autoimmune disease.
A major paradigm in current immunological thought is the notion that Ag stimulation alone is not sufficient for the induction of potent and long-lasting immune responses. Support for this paradigm is revealed in studies that show two signals are necessary for T cell growth and cytokine production (1, 2). CD28 and its ligands, B7-1 and B7-2, have emerged as the second signal required for efficient activation of naive T cells (3, 4, 5, 6, 7). Recently, it has been shown that CD28 ligation on T cells during TCR stimulation in vivo leads to significant clonal expansion; however, the initial expansion is followed by profound deletion (8). Therefore, it was hypothesized that other signals were necessary to generate and maintain long-lived memory CD4 T cells. The existence of other signals is further supported by the observations that B7 transgenic mice do not spontaneously develop autoimmune disease (9), and CD28 knockout mice are capable of clearing certain pathogenic infections (10).
The danger theory proposed by Matzinger (11) addresses this very complex and controversial issue of immune activation and memory T cell generation . Her hypothesis suggests that a stimulus that facilitates some type of biologic “damage” is critical for the induction of a long-lasting immune response. Furthermore, it is thought that the “danger signal” can promote autoimmune disease under the appropriate conditions, such as mice immunized with adjuvants and myelin components in experimental autoimmune encephalomyelitis (EAE)4 (12). Elements of the danger signal include factors that promote destruction of tissue and/or necrotic death of cells (13). These are not the only situations that lead to damage, but they represent examples that can be found in nature. For example, it has been known for some time that bacterial LPS is very capable of promoting severe inflammation, leading to tissue destruction (14, 15). LPS can activate macrophages to produce large quantities of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) that attract and stimulate other inflammatory cell types including T cells (16, 17, 18).
LPS can interfere with peripheral tolerance (19), and recent data show that Ag-induced peripheral T cell deletion can be tempered in the presence of LPS stimulation (20). The rescuing effect of LPS occurs independently of CD28 ligation, but is profoundly dependent on TNF-α production. Thus, it is likely that stimuli similar to LPS are danger signals and may be responsible for interfering with tolerance induction. Nevertheless, optimal T cell immunity in vivo is multifactoral and loss of one signal or receptor function may be compensated for by alternate ones.
Other endogenous signals have been reported to influence peripheral deletion, especially those involving members of the TNF receptor family such as FAS, CD40, and 4-1BB (21). OX40 is a member of the TNF receptor family that is expressed primarily on activated CD4+ T cells, which when engaged induces a potent costimulatory signal (22, 23). OX40+ T cells have been found preferentially within the inflammatory compartments of patients and rodents with autoimmune disease and cancer (24, 25). There is little or no expression of OX40 on T cells in the periphery. Thus, it is possible that OX40 is exclusively expressed on Ag-stimulated T cells.
At the effector stage auto-Ag-specific T cells recognize Ag and produce large amounts of cytokines, leading to inflammation within a target organ (26). It has been hypothesized that a low percentage of the effector T cells that recognize Ag and produce cytokines will subsequently differentiate into memory T cells. Because, at this stage, the effector T cells become quite susceptible to activation-induced cell death (AICD) (27). Effector T cells are quite sensitive to OX40-specific costimulation as compared with naive T cells, although the combination of B7 and OX40L signals delivered to naive T cells was found to be synergistic (28). We hypothesized that a signal delivered through OX40 might inhibit AICD during Ag-specific stimulation of effector T cells, thereby increasing the number of effector T cells that survive and become memory cells. To test this hypothesis, we explored the expression of OX40 and the effects of OX40 engagement on effector T cells in a superantigen (SAg) model system, which is known to result in the deletion of SAg-specific effector T cells. The SAg staphylococcal enterotoxin A (SEA) system allows for convenient detection of SAg-specific T cells with an anti-Vβ3 TCR Ab. We also examined effector T cell survival in an adoptive T cell transfer model in which peptide-induced deletion has been observed (29).
Materials and Methods
Mice
B10.BR and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) or from the National Cancer Institute (Frederick, MD). DO11.10 transgenic mice were generously provided by Drs. Nancy Kerkvliet and Marc Jenkins (Oregon State University, Corvallis, OR and University of Minnesota, Minneapolis, MN, respectively) (30). All mice were maintained at Oregon State University under specific pathogen-free conditions in accordance with federal guidelines.
Reagents, Abs, and flow cytometry
SEA and LPS were purchased from Sigma (St. Louis, MO) and administered to mice as i.p. injections. Chicken OVA (Sigma) was solubilized in balanced salt solution and administered i.p. without adjuvant.
T cells were purified by nylon wool fractionation as described previously (31). Flow cytometry staining was conducted as described previously (8). Briefly, cells were blocked for nonspecific binding and incubated with biotinylated anti-OX40 (33) for 30 min on ice. The cells were washed twice and incubated with anti-CD4-PE (PharMingen, San Diego, CA), anti-TCR Vβ3-FITC (KJ25-607.7; see Ref. 32), and RED 613-conjugated streptavidin (Life Technologies, Grand Island, NY). After several washes, the cells were analyzed on an Epics XL flow cytometer (Coulter Electronics, Miami, FL).
Experimental design
The experiment in Fig. 2⇓ was set up as follows: One group was noninjected (day 0). The remaining groups received SEA and rat IgG (open circles); SEA and anti-OX40 (filled circles); SEA, LPS, and rat IgG (open squares); and SEA, LPS, and anti-OX40 (filled squares). On day 0, mice were given 0.15 μg of SEA and immediately afterward, an injection of 25 μg of anti-OX40 or rat IgG. On day +1, mice were given a second injection of anti-OX40 or rat IgG, followed immediately by 30 μg of LPS. On day +2, mice were given a final injection of anti-OX40 or rat IgG. The percent and number of SEA-specific T cells were determined.
The experiments in Tables I and II were set up as follows: On days 0 and +2, 500 μg of OVA in balanced salt solution was injected, followed immediately by injection of 50 μg of anti-OX40. On days +1 and +3, anti-OX40 was injected, and, immediately after, 50 μg of LPS was injected i.p. On day +4, a final injection of anti-OX40 was administered. On day 10 or day 62, T cells from lymph node (LN) and spleen were isolated and examined for OVA-specific T cells.
Results
Detection of OX40 expression on Ag-specific T cells
Expression of OX40 by Vβ3 T cells was examined after mice were stimulated with SEA. Mice were injected with LPS alone, SEA alone, or SEA and LPS, and T cells were analyzed at 12, 24, 48, and 72 h later. SEA induced OX40 expression on the SEA-specific CD4 Vβ3 T cells which peaked at 12 h and declined at 24 and 48 h (Fig. 1⇓ shows 48-h time point). Only a small percentage of SEA-specific CD8 T cells expressed OX40, and SEA-unreactive T cells (TCR Vβ14) from the same mice showed no increase (data not shown). We also tested whether or not a danger signal alone would be capable of enhancing OX40 surface expression on T cells. Mice were injected with a high dose of LPS and T cells were analyzed as described above. In the absence of SAg stimulation, OX40 expression was not up-regulated (data not shown and Fig. 1⇓). These data show that TCR stimulation is sufficient to induce OX40 surface expression on SAg-stimulated CD4 T cells in vivo.
OX40 expression on SAg-stimulated T cells is enhanced in the presence of a danger signal. A total of 15 B10.A mice was divided into four groups. The first group was left noninjected. The second group received 0.15 μg of SEA 12, 24, 36, 48, 60, or 72 h before analysis. The third group received 120 μg of LPS i.p. 12, 24, 36, or 48 h before analysis. The last group received SEA 36, 48, 60, or 72 h before analysis, and an additional injection of LPS 24 h after the SEA injection. The LN T cells from each mouse were purified and stained with Abs against OX40, Vβ3, and CD4 or CD8. Rat IgG staining was also done as a control. Analysis of stained cells was completed by flow cytometry. Histograms representing the expression of OX40 on CD4 Vβ3 T cells are shown for mice injected with SEA 48 h and LPS 24 h before analysis. The other time points are not shown. Similar data were generated by staining for OX40 with a soluble OX40-ligand fusion protein.
Next, we tested whether or not a danger signal in the presence of TCR stimulation in vivo could influence OX40 expression. Mice were injected with SEA and LPS, and after various times OX40 expression on Vβ3 and Vβ14 T cells was analyzed. It is shown that SEA and LPS increased OX40 expression on the SEA-specific CD4 T cells only (Fig. 1⇑). The levels of OX40 on the SEA-specific T cells was significantly higher at 48 h than with either reagent alone (Fig. 1⇑) and had prolonged expression compared with SEA (data not shown). As before, staining of Vβ14 T cells showed no evidence of up-regulation of OX40. Thus, we conclude that a danger signal and TCR stimulation synergized to enhance OX40 expression on SAg-specific CD4 T cells in vivo.
Evaluating the effects of OX40 ligation on SAg-stimulated T cells during clonal expansion
The SAg model in mice has been used to study central and peripheral T cell tolerance. Early after SAg injection into mice, SAg-activated peripheral CD4 and CD8 T cells expand 2- to 5-fold during a 48-h period (34). This expansion is followed by profound deletion of those same T cells. These measurements are possible because unactivated (Vβ14) and activated (Vβ3) T cells can be detected over time by analyzing the respective cell populations by flow cytometry.
The hypothesis that ligation of OX40 on SEA-activated T cells will influence peripheral deletion of T cells was tested. Mice were not injected or injected with SEA with a control IgG (SEA); SEA with LPS and control IgG (SEA/LPS); SEA with anti-OX40 (SEA/OX40); or SEA with LPS and anti-OX40 (SEA/LPS/OX40). On days 2, 5, 7, and 12 after SEA injection, the percent and absolute number of LN and spleen CD4 and CD8 T cells were examined for TCR Vβ3 expression (Fig. 2⇓; LN data not shown). As expected, there was an initial expansion of Vβ3+ spleen T cells in the SEA-injected mice by day 2. However, by day 5, the number of Vβ3 CD4 cells was lower than in noninjected mice, suggesting that deletion of T cells had occurred. The Vβ3 T cells remained low in number for the duration of the experiment. Vβ3 T cells from mice injected with SEA/LPS or SEA/OX40 had expanded on day 2 to a greater level in the LN (data not shown) than that observed in mice with SEA alone. Nevertheless, for splenic T cells, both treatments resulted in some degree of T cell rescue compared with mice injected with SEA alone, but there was still profound clonal deletion in all three groups (Fig. 2⇓). For example, by day 12, 1.12 × 106 and 1.53 × 106 Vβ3 splenic T cells were observed in SEA/OX40- and SEA/LPS-treated mice, respectively, compared with 0.19 × 106 splenic Vβ3 T cells in the mice receiving SEA alone.
OX40 receptor ligation and a danger signal enhance SAg-stimulated splenic CD4 T cell growth and survival in vivo. A total of 52 B10.A mice was put into five groups as indicated in Materials and Methods. One group was noninjected (day 0). The remaining groups received SEA and rat IgG (○); SEA and anti-OX40 (•); SEA, LPS and rat IgG (□); and SEA, LPS, and anti-OX40 (▪). On days 2, 5, 7, and 12 after SEA injection, the spleen and LNs (data not shown) were removed from three mice per group. Purified T cells were counted, stained with Abs against CD4 and Vβ3, and analyzed by flow cytometry. Each point represents the mean percentages (A) and numbers (B) ± SEM from at least three mice from one representative experiment of four performed.
SAg plus the combination of danger and OX40 engagement, resulted in enhanced expansion and a marked inhibition of peripheral T cell deletion. Instead of the expected decrease in the splenic Vβ3 T cells on day 5, we observed a 12-fold increase in the SEA/OX40/LPS-treated mice compared with SEA. This was true for both percentage and absolute number of Vβ3 T cells (Fig. 2⇑, A and B, respectively). The Vβ3 population continued to increase on day 7 (28.5-fold; Fig. 2⇑B) with an apparent plateau between days 7 and 12 (Fig. 2⇑B). There was also an increase in the CD8 Vβ3 T cell population but not to the same degree as the CD4 T cells, and no significant changes were observed in a SEA-nonspecific Vβ14 T cell population (data not shown). Collectively, these data show that in vivo engagement of OX40 in the presence of a danger signal block SAg-induced peripheral T cell deletion while promoting effector T cell expansion. There also appeared to be rescue of CD8 T cells even though their level of OX40 expression was less compared with the CD4 T cells (Fig. 3⇓). There was a peak of expansion on day 2, both in terms of percentages and absolute numbers, but thereafter the response waned. Nevertheless, there was significant CD8 survival when animals were treated with all three signals, which was somewhat perplexing since there was little OX40 expression on the SEA-activated CD8 T cells (data not shown).
OX40 receptor ligation and a danger signal enhance SAg-stimulated splenic CD8 T cell growth and survival in vivo. These data are taken from the experiment shown in Fig. 2⇑, except that cells were stained for CD8 Vβ3 expression. Day 0 represents noninjected mice. The remaining groups received SEA and rat IgG (○); SEA and anti-OX40 (•); SEA, LPS, and rat IgG (□); and SEA, LPS, and anti-OX40 (▪). Each point represents the mean percentage (A) and absolute number (B) ± SEM from at least three mice from one representative experiment of four performed.
Three signals induce T cell memory development
To characterize the effects of OX40 engagement during presentation of peptide Ag, we examined the well-characterized model D011.10 TCR transgenics for tracking peptide/MHC class II-activated CD4 T cells (29). T cells from the D011.10 TCR transgenic mice recognize OVA in the context of Iad and the OVA-specific T cells can be detected with the anti-idiotypic Ab KJ1–26. DO11.10 TCR transgenic T cells were transferred into five groups of mice and thereafter treated with OVA, OVA with anti-OX40 (OVA/OX40), ova with LPS (OVA/LPS), or all three (OVA/OX40/LPS) and compared with noninjected mice (no OVA). Seven days after Ag exposure, T cells were analyzed for the absolute number of DO11.10-bearing cells in the LN and spleen (Table I⇓). Injection of OVA alone did not increase the number of Ag-specific T cells at day 7 postimmunization. Based on previous experiments, we believe that inspection of earlier time points would have shown an increase in the DO11.10 T cell population (data not shown). Coinjection of LPS and OVA did rescue some of the DO11.10 T cells from deletion compared with the OVA group, but this effect was minimal compared with the number of Ag-specific T cells on day 7 in the OVA/OX40 group. There was a 17.7-fold increase in the number of splenic KJ1-26+ T cells in the OVA/OX40 mice compared with OVA alone. Furthermore, there was an even greater increase of DO11.10 T cells in the OVA/LPS/OX40 mice (33.8-fold over the OVA-alone mice). These observations held true for both splenic and LN T cells, but the enhancement was greater for the spleen population. Although the data show that OX40 had a significant effect on T cell survival, there continues to be additional benefit when LPS and OX40 are combined during a peptide Ag-specific T cell response.
OX40 costimulation and danger promote optimal clonal expansion of Ag-specific T cells in vivoa
The final experiments were designed to examine the long-term effects on Ag-specific T cells that have been activated via OX40 engagement in the presence of a danger signal. The DO11.10 T cells were adoptively transferred into thymectomized BALB/c recipients that were used to prevent peripheral T cell repopulation by the thymus. Mice were treated as described in Table I⇑ and tested for DO11.10-bearing T cells 62 days after exposure to Ag (Table II⇓). Our data show that Ag stimulation followed by OX40 engagement enhanced growth and long-term survival of the DO11.10 T cells. The addition of LPS further enhanced this effect. A 12.3-fold increase was observed with Ag and OX40 engagement as compared with Ag alone, and a striking 59.8-fold increase in long-term surviving T cells was observed when both anti-OX40 and LPS were used to stimulate the OVA-specific T cells. Phenotypic analysis of the OVA-specific T cells (KJ1-26+) in the OVA/LPS/OX40 mice revealed that they were small, resting memory cells as assessed by low forward scatter and up-regulation of CD44 expression (Fig. 4⇓). In contrast, the KJ1-26− cells had a broad range of CD44 expression (4-fold lower in mean channel fluorescence) than that observed for the OVA-specific T cells. Therefore, we conclude that the optimal development of long-lived memory T cells required three signals during effector T cell stimulation: Ag, OX40 costimulation, and danger (LPS).
Surviving Ag-stimulated T cells express a memory phenotype in vivo. KJ1-26+ T cells from mice injected with OVA/LPS/anti-OX40 from the experiment described in Table II⇓ were stained for CD44 expression and examined by flow cytometry. Histograms representing CD44 expression and forward scatter are shown for the KJ1-26+ or KJ1-26− T cell populations. Mean channel fluorescence (MCF) for each peak is listed. Data are representative of two separate experiments.
Optimal long-term memory T cell survival of Ag-activated CD4+ T cells is obtained when OX40 engagement occurs in a proinflammatory environmenta
Discussion
For years the mechanism of memory T cell acquisition has been vigorously studied and hotly debated (35, 36). Much emphasis has been dedicated to defining the phenotype, activation requirements, and life span of memory cells. Nevertheless, these parameters are not very well defined as compared with the data gathered on B cells. In part this is due to the fact that naive B cells are readily distinguished from memory cells by the type of surface Ig they express. Memory T cells do not change their Ag receptors nor does the affinity increase after antigenic challenge, which is in contrast to B cells. The data presented in this study are focused on the requirements necessary for optimal memory CD4 T cell generation.
Our data clearly show that two signals are better than one and that three signals are better than two. Nevertheless, what is key is that they are three different signals. Of course Ag is signal 1, signal 2 is a growth signal such as that observed with OX40, and signal 3 is a danger signal, which in this study is LPS. Although signals 2 and 3 are somewhat interchangeable, they seem to synergize when activated concomitantly during an antigenic response. For example, LPS can enhance growth at high doses (37), and OX40 can certainly increase survival without LPS (see Table II⇑); but when combined the greatest amount of long-term survival is observed. One important difference between costimulatory or growth signals and danger signals is that LPS alone can induce shock at very low doses, whereas high doses of anti-OX40 alone does not (our unpublished observations). These data suggest that CD4 T cell memory development can be obtained by more than one set of parameters.
We show that optimal T cell memory acquisition requires three signals at refined doses, but nevertheless suboptimal survival was obtained without LPS or without OX40 stimulation (Fig. 3⇑ and Table II⇑). Additionally, it is clear that LPS can synergize with CD40 stimulation to generate profound SAg-specific T cell survival; however, each signal individually with SAg is far less effective (our unpublished data). This latter model is totally dependent on CD28/B7 ligation for growth (8). Interestingly, two costimulatory signals through OX40 and CD40 (B7/CD28) were not sufficient to block peripheral deletion of SAg-stimulated T cells (data not shown), which strongly suggests that two costimulatory signals are not sufficient to substitute for one survival signal.
It is clear from our data that survival was not limited to CD4 T cells. For example we show that CD8 T cells can also be rescued from deletion even though OX40 expression was far less on activated CD8 T cells than that observed on activated CD4 T cells (Fig. 3⇑). One possibility is that OX40 ligation and Ag stimulation prime CD4 T cells to secrete large quantities of cytokines which activate bystander CD8 T cells to survive. Alternatively, it is possible that the low levels of OX40 on the surfaces of CD8 T cells were in enough quantity to be ligated and thereby promote rescue. Nevertheless, this is a very complex problem that is currently being investigated.
A role for cytokines in this system is likely to be paramount to the rescuing process. It was previously shown that LPS could rescue T cells from SAg-induced deletion through the action of TNF-α and a minor role for IFN-γ (20). It is still unclear how these cytokines promote long-term survival, especially in light of the fact that TNF-α has also been implicated in driving T cell death (38). This also seems to be the case for CD95 which has been shown to be a very important death signal in a variety of systems (39), but has also been shown to promote growth in others (40). Therefore, it is likely that the cytokine environment influences activated T cells to respond with a survival or death response depending on the variety and balance of cytokines. This has been clearly observed in other systems including Th1 and Th2 skewing.
Proinflammatory cytokines are not the only cytokines that promote T cell survival. Several ligands specific to members of the common γ chain family of receptors have also been implicated in mediating T cell survival (41, 42). Of these, IL-4, which is clearly not proinflammatory, can block Ag-induced death and spontaneous death of nonactivated “fresh” T cells. Oddly enough TNF-α does not seem to block death in vitro of these same cell types (data not shown). These data argue that the mechanism of survival induction is different between TNF-α and IL-4. IL-4-induced T cell survival is definitely dependent on the common γ chain for rescue (data not shown), whereas it is not clear whether TNF-α rescues Ag-activated T cells directly. For example, under the appropriate circumstances, activated T cells may bind TNF-α, which in turn may inhibit an activated death program. Alternatively, it is possible that TNF-α induces other factors to block various death pathway(s).
Of particular interest in this regard is IL-6. IL-6 is involved in acute phase responses, B cell stimulation, T cell activation, hematopoiesis, and many other functions (43). In particular, however, IL-6 has been shown to block spontaneous death on resting T cells (44). This result is consistent with the fact that IL-6−/− mice have a diminished number of peripheral T cells (45). Therefore, it is possible that a cytokine like IL-6, which is induced by TNF-α, may mediate survival. Preliminary studies have shown that IL-6 does not block activation-induced death in vitro (data not shown), and others have shown that IL-6 administration in vivo only minimally affects T cell rescue from deletion (46). Once again, it may be that a mixture of cytokines in the right proportions can influence memory acquisition. Therefore, individually they are ineffective but in combination are substantially effective.
Other cytokines induced by TNF-α are also possibilities and include IL-1β and IL-12. By itself, IL-1β has been shown to block deletion (47), but it is not known whether this cytokine directly inhibits death by binding to T cells or inhibits deletion by an indirect method. Previous reports have shown that IL-12 can stimulate Th1 differentiation during a tolerogenic response but does not block deletion by itself (46, 48). Furthermore, it has been firmly established that OX40 stimulation potentiates cytokine production on effector T cells (27). Specifically, it has been shown that Th1 and Th2 cytokine production can be enhanced by cross-linking OX40 on Ag-stimulated T cells (23, 49). Finally, it is clear that resolving the survival mechanism in vivo will certainly involve cytokines and chemokines, surface receptors, non-T cells, and possibly factors from the endocrine system.
These data show that peptide-specific T cells are very responsive to OX40 ligation in combination with a danger signal, suggesting that this treatment may significantly improve the efficacy of vaccines designed to activate T cells. A major limiting factor in vaccine development has largely been the identification of a practical adjuvant that is efficient. Because most adjuvants would be too toxic for human use, due to massive inflammatory reactions, it is likely that a more refined targeted treatment will be necessary. The OX40 protein is an excellent target because it is expressed only on recently activated Ag-specific cells, which are primarily found at the site of inflammation (24, 50). Perhaps the danger mechanism described in this study (i.e., up-regulation of OX40 expression on Ag-activated T cells by LPS) helps explain the positive effects adjuvants exert on activated T cells and may lead to more defined approaches for vaccination protocols.
Collectively, these data suggest that costimulation in the absence of danger can lead to Ag-dependent clonal expansion, but does not elicit the same magnitude of long-term T cell survival when compared with the same response elicited in the presence of a danger signal. These data may help explain why B7 transgenic mice do not develop autoimmune diseases spontaneously, and why the same mice crossed with TNF-α transgenic mice develop autoimmune disease (9). Additionally, it is widely accepted that TNF-α is an important effector molecule in the development of EAE (51). Recently, it has also been shown that OX40 and OX40-ligand expression is found on T cells and activated macrophages, respectively, within the inflamed tissue of rodents with clinical signs of EAE (our unpublished data). Thus, it is possible that inflammatory cytokines like TNF-α promote the appropriate activation of autoreactive memory T cells via OX40 up-regulation.
Our data support a new model of T cell activation that incorporates three definable signals. In this study, we suggest that one set of optimal conditions for long-term survival of Ag-specific T cells requires three signals, which include an antigenic stimulus, OX40 stimulation, and activation by LPS.
Acknowledgments
We thank William Amberg and Dr. Barbara Smith of the Laboratory of Animal Resources for care of the animals used in this work at Oregon State University.
Footnotes
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↵1 This work was supported by the Oregon Research Medical Foundation and the Linus Pauling Institute and Grant AI42858A from the National Institutes of Health (to A.T.V.).
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↵2 J.M. and A.W. contributed equally to this work.
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↵3 Address correspondence and reprint requests to Dr. Anthony T. Vella, Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331. E-mail address: vellaa{at}bcc.orst.edu
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↵4 Abbreviations used in this paper: EAE, experimental autoimune encephalomyelitis; SAg, superantigen or peptide antigen; SEA, staphylococcal enterotoxin A; LN, lymph node.
- Received August 5, 1999.
- Accepted October 14, 1999.
- Copyright © 2000 by The American Association of Immunologists
References
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