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The functional outcomes of the T cell’s interaction with the peptide:MHC complex can be dramatically altered by the introduction of a single amino acid substitution. Previous studies have described the varied effects of these altered peptide ligands (APL) on T cell responses. These outcomes of T cell interaction with an APL include the induction of clonal unresponsiveness (anergy) and inhibition of T cell responses (antagonism). The phenotype of peptide-induced anergy, i.e. low proliferation and low IL-2 production, has been extensively described, and a number of groups have demonstrated antagonism. However, the response of T cells to an agonist ligand after encountering an antagonistic stimulus has not been previously characterized. Here, we show that T cells post-antagonism fail to proliferate but produce large quantities of IL-2 upon stimulation with their wild type ligand. This unique phenotype is not due to differences in IL-2 receptor expression or rates of apoptosis, and cannot be overcome by the addition of recombinant IL-2. The response of CD4 T cells to agonist stimulation after encountering an antagonist is a novel phenotype, and is distinct from previously described forms of anergy.
A T cell response is elicited following interaction of the T cell receptor (TCR) with its cognate ligand. The outcome of this interaction can be affected by introducing changes in the immunogenic peptide sequence [1, 2]. Peptides in which amino acid substitutions have been made at a TCR contact residue are known as altered peptide ligands (APL). APL have an affinity for MHC similar to that of the wild type peptide and are classified based on the potency of the T cell response they elicit. Agonist peptides stimulate T cells to levels similar to that of the wild type peptide, while weak agonists stimulate suboptimal proliferative and cytokine responses. Partial agonists stimulate only a subset of effector functions . This class of APL has been used to induce anergy, a state of unresponsiveness defined by a lack of proliferation and IL-2 production in response to an immunogenic stimulus . An anergic phenotype can also be induced by lack of costimulation [3-5], a low dose agonist ligand , or unstable peptides containing substitutions at their MHC anchor residues [7, 8].
In addition to partial agonists, which have been shown to induce T cell anergy, another category of APL that dampens the T cell response is antagonists. Antagonist ligands have been defined in vitro by their inability to stimulate T cell responses when presented alone and their ability to inhibit the T cell response to wild type peptide when both the antagonist and agonist are present [2, 9, 10]. Epitopes behaving as antagonists have been identified in a number of infections, including HIV [11, 12], Hepatitis B and C [13, 14] and malaria . Additionally, antagonist ligands may play a role in maintaining peripheral tolerance .
Although the phenomenon of antagonism has been extensively described in a number of systems, the exact mechanism by which antagonism occurs remains unclear. Studies utilizing dual-receptor expressing T cells suggest that the mechanism is one of active inhibition, rather than a passive effect of competition [17-20]. Further studies have indicated that antagonism impacts the signaling cascades normally activated upon T cell stimulation, activating a negative feedback loop involving the phosphatase SHP-1[21-23].
The response of a T cell under antagonist conditions is similar to the response of anergic cells to wild type stimulation. In both these conditions, T cells fail to proliferate and secrete little or no IL-2. To define the fate of these cells following rechallenge with agonist ligand has not been considered. We examine the phenotype of previously antagonized T cells in response to wild type stimulation. We have found that the phenotypes of anergy and antagonism differ dramatically when cells are rechallenged with wild type ligand post-antagonism. Both groups fail to proliferate upon stimulation with wild type peptide, but we demonstrate that previously antagonized cells make significant amounts of cytokines including IL-2. This distinct difference in cytokine production is a stark and important contrast between the phenotypes of antagonism and anergy. Thus, we describe a unique response pattern of T cells, a phenotype distinct from previously described forms of anergy.
3.L2 TCR transgenic mice were used at 6-12 weeks of age. The 3.L2 TCR is specific for Hb64-76 presented by I-Ek . Mice were bred and housed in the Emory University Department of Animal Resources facility according to federal guidelines.
The wild type peptide, Hb64-76 (GKKVITAFNEGLK) and the antagonist 72I (GKKVITAFIEGLK) were purchased from Invitrogen (Carlsbad, CA). 72I is a single amino acid substitution, Asn (N)→Ile (I) at position 72.
Antagonism was performed as previously described by Sette and colleagues . Briefly, for antagonist conditions, cells were pre-pulsed with 1μM Hb64-76 for 2h at 37°C, washed twice in HBSS, then cultured with 10μM 72I or the indicated concentration of peptide for proliferation assays.
3.L2 spleen cells (2×106/well) were cultured ex vivo with 1μM Hb64-76, pre-pulse only (1μM Hb64-76 for 2h, at 37°C) or under antagonist conditions (1μM Hb64-76 pre-pulse, 10μM 72I continuously) with 10 pg/mL IL-2 for 7 days in 24-well plates. Live cells were purified over a Ficoll gradient (Mediatech) and were restimulated with irradiated syngeneic splenocytes (2000 rad) and the indicated concentrations of peptide. Culture media consisted of RPMI 1640 supplemented with 10% FBS, 2mM L-glutamine, .01M Hepes buffer, 100μg/mL gentamicin (Mediatech, Herndon, VA) and 5×10−5 M 2-mercaptoethanol (Sigma, St. Louis, MO).
Naïve 3.L2 splenocytes (3×105/well) or cultured T cells (5×104/well) and irradiated syngeneic splenocytes (2000 rad, 5×105/well) were cultured in 96-well plates with the indicated concentration of peptide at 37°C. Where indicated, recombinant IL-2 (BD Pharmingen, San Diego, CA) was added at 3.5ng/well. After 48h in culture, 0.4μCi/well of [3H] thymidine was added. After an additional 18h, cells were harvested on a FilterMate harvester (PerkinElmer Life and Analytical Sciences, Wellesly, MA) and analyzed on a Matrix 96 Direct Beta Counter (PerkinElmer).
After one week in culture under the indicated conditions, 3.L2 T cells (1×106/well) were stimulated with irradiated syngeneic splenocytes (2000 rad, 5×106/well) and the indicated concentration of peptide for 24h (IL-2) or 48h (IFN-γ). Supernatants were incubated in triplicate on microtiter plates that had been coated with 50μl of purified anti-IL-2 (5 μg/mL, clone JES6-1A12, BD Pharmingen) or anti-IFN-γ (5 μg/mL, clone R4-6A2, BD Pharmingen) overnight at 4°C. Recombinant IL-2 or IFN-γ (BD Pharmingen) was used as a standard. Captured cytokines were detected using biotinylated anti-IL-2 (JES6-5H4, BD Pharmingen; 100 μg/mL, 100 μl/well) or biotinylated anti-IFN-γ (clone XMG1.2, BD Pharmingen; 100 μg/mL, 100 μl/well) followed by alkaline phosphatase-conjugated avidin (Sigma, St. Louis, MO) and p-nitrophenylphosphate (pNPP) substrate (BioRad, Hercules, CA). Colorimetric change was measured at 405 nm on a Microplate Autoreader (Biotek Instruments, Winooski, VT).
3.L2 T cells were stained with anti-CD25 (IL-2Rα)-FITC (clone PC61), anti-Vβ8.3-PE (clone 1B3.3) and anti-CD4-APC (clone RM4-5; all BD Pharmingen) at 24h and 48h after restimulation. Alternatively, cells were stained with PE conjugated CD122 (clone TM-b1) or CD132 (clone 4G3). For intracellular cytokine staining, cells were stimulated for 6h, then fixed and permeabilized with Caltag Fix and Perm cell permeabilization kit per the manufacturers instructions (Caltag, San Diego, CA). Permeabilized cells were stained with anti-CD4-PerCP and anti-IL-2-APC (clone JES6-5H4) or anti-IL-4-APC (clone 11B11; all BD Pharmingen). Annexin V and 7-AAD staining was performed according to manufacturer’s instructions (BD Pharmingen). Flow cytometry was performed on a BD FACSCalibur (Franklin Lakes, NJ) and data were analyzed using FlowJo Software (Tree Star, San Carlos, CA). Data are gated on Vβ8.3+ or CD4+ cells as indicated.
Data were analyzed using GraphPad Prism. Statistical significance was determined by t test or ANOVA, as indicated in the figure legends.
The response of 3.L2 T cells to both Hb64-76 and the APL 72I has been previously described [24, 25]. As expected, the antagonist peptide alone induced minimal proliferation of 3.L2 T cells, whereas stimulation with the wild type peptide resulted in a dose dependent proliferative response (Fig. 1A, p=0.0184). Additionally, cells pre-pulsed with the wild type peptide were effectively antagonized (>95%) by addition of 72I (Fig. 1B). Thus the APL 72I acts as an antagonist, as it fails to stimulate proliferation when presented alone, and inhibits the T cell response to wild type peptide in a dose-dependent manner when both wild type and 72I are presented.
Although T cell antagonism has been extensively described [10, 17, 19, 26-28], the response of these antagonized cells to subsequent stimulation with wild type ligand has not yet been described. Previous studies have shown that under antagonist conditions, T cells exhibit a dose dependent decrease in proliferation to wild type stimulus [10, 17-19, 26-28], altered cytokine production [1, 29], and altered signaling [10, 12, 18]. In order to assess the effects of T cell antagonism on subsequent T cell function, we have characterized the proliferative and cytokine responses of previously antagonized T cells to rechallenge with wild type ligand.
After 7d in culture with either wild type peptide, pre-pulse alone or under antagonist conditions, cells were restimulated with wild type peptide. Cells cultured on wild type peptide show characteristic, dose-dependent proliferation upon restimulation with wild type peptide. However, cells cultured on the antagonist condition show significantly blunted proliferation relative to wild type cultured cells (Fig. 2A, p<0.05). Antagonized cells secrete near normal or slightly elevated levels of the effector cytokine IFN-γ after 48h of wild type stimulation (Fig. 2B, p=0.3878).
The failure of pre-pulse only and antagonized cells to proliferate in response to wild type stimulation suggests a lack of IL-2, as is the case in classical anergy. As expected, cells cultured on pre-pulse alone produce little IL-2 after 24h of stimulation (Fig. 3, prepulse vs. antagonist, p<0.001). The lack of IL-2 production in the pre-pulse only condition coupled with the hypoproliferative response defines these cells as anergic. This is consistent with previous data indicating that anergy can be induced by a low dose of agonist ligand . Antagonized cells, however, secrete significantly greater amounts of IL-2 relative to wild type cultured cells after 24h of restimulation with wild type peptide (Fig. 3, Hb64-75 vs. antagonist p<0.01). This increased production of IL-2 contrasts sharply with the lack of proliferation exhibited by the antagonized T cells. This phenotype is clearly distinct from previously described forms of anergy, in which IL-2 production in blunted or entirely absent.
The differences in IL-2 production in these groups do not appear to be due to cell death, as no significant differences in Annexin V or 7-AAD staining were observed at 48h. (Fig. 4A). Additionally, these cells have not been skewed to a Th2 phenotype, as they are not producing IL-4 as measured by cytokine capture ELISA or intracellular cytokine staining (data not shown). The disconnect between IL-2 production and proliferation that is observed in antagonized cells upon restimulation with wild type peptide suggests a disruption of the IL-2 signaling pathway. To address the mechanism of this unique phenotype, IL-2R levels were determined by flow cytometry. These studies indicated that expression of CD25, the high affinity IL-2R, is similar in both antagonized and wild type cultured cells across a range of doses at both 24h and 48h after restimulation (Fig. 4B, p=0.4112). Expression of CD122 and CD132 is also similar (data not shown), which implies that the disruption in the IL-2 pathway is not at the level of receptor expression.
Additionally, the anergic phenotype of cells cultured on pre-pulse alone can be overcome by the addition of exogenous IL-2 (Fig. 5; Hb64-76 vs. prepulse p>0.05), as has been described in other cases of low dose anergy . However, proliferation of antagonized cells cannot be rescued in this way (Fig. 5, Hb64-76 vs. antagonist p<0.05). These data further support the idea that a blockage in the IL-2 signaling pathway is responsible for the phenotype of hypoproliferation yet enhanced IL-2 production we have observed post-antagonism.
In this study we have found that after stimulation under antagonist conditions, T cells respond to wild type antigen by producing high levels of IL-2 while also exhibiting a marked lack of proliferation. This lack of proliferation is similar in both anergized and antagonized cells, although these two groups of cells exhibit significant differences in their ability to produce and respond to IL-2. These findings indicate that antagonism is a distinctly different phenomenon than known forms of anergy.
Previous studies have described anergy induced by lack of costimulation [3-5, 30], low dose of agonist peptide , APL  and peptides containing substitutions at their MHC anchor residues [7, 8]. Lack of proliferation and IL-2 production are key characteristics of all of the forms of anergy. Our data show that antagonized cells exhibit a phenotype of hypoproliferation and normal IFN-γ production that is similar to anergic cells. In most forms of anergy, including that induced by low doses of wild type peptide, proliferation can be rescued by the addition of exogenous IL-2 [1, 3]. However, the lack of proliferation in antagonized cells is not reversible by the addition of exogenous IL-2. The failure of IL-2 to rescue the hypoproliverative phenotype post-antagonism is similar to APL induced anergy, suggesting that these phenotypes may share some mechanistic features. In most forms of anergy, for example the pre-pulse only condition, cells are unable to produce IL-2, but they remain able to respond to it . In contrast, antagonized cells secrete large quantities of IL-2, but have been rendered unresponsive to this growth factor. Furthermore, our data demonstrate that the presence of an antagonist is sufficient to at least partially overcome low dose induced anergy, suggesting that this phenotype is mechanistically distinct from low dose induced anergy.
The failure of antagonized cells to respond to IL-2 from both endogenous and exogenous sources is not due to differences in apoptosis or decreased expression of IL-2R, as we have found that all three chains of the high affinity IL-2R are expressed at similar levels in both antagonized and wild type cultured cells. These data suggest an intracellular disruption of the IL-2 signaling pathway as the mechanism mediating the phenotype post-antagonism.
Several pathogens have been shown to generate escape epitopes that function as T cell antagonists, including HIV [11, 12], Hepatitis B and C [13, 14] and malaria . In the context of one of these infections, the phenotype of T cells that encounter their cognate ligand after interacting with an antagonist mutant epitope could dramatically impact the course of the infection. The data presented herein would suggest that these T cells would produce large quantities of IL-2, perhaps expanding or activating regulatory T cells [31, 32], and thereby potentiating the efficacy of the pathogen’s escape strategy. Alternatively, this cytokine production could result in non-specific bystander activation, which could also result in subsequent failure to clear the infection and/or immunopathology. The phenotype that we have observed post-antagonism underscores the role of T cell epitope mutation in the escape of chronic infections from control by the immune system.
T cell unresponsiveness can result from many different pathways, as evidenced by the diverse mechanisms of anergy that have been previously described. In T cells that are anergic, the proliferative and IL-2 responses are significantly reduced. Similarly, T cell antagonism also results in a refractory state in which proliferation is blocked. Unlike classical anergy, however, antagonized cells retain their ability to secrete the growth factor IL-2, despite their inability to respond to it. This inability to respond is similar to APL induced anergy, as well as MHC variant peptide induced anergy, which has been shown to be mediated by the negative regulatory phosphatase SHP-1 . SHP-1 has also been shown to play a role in the early stages of antagonism [21, 25]. Furthermore, at least one report has demonstrated association of SHP-1 with the IL-2 receptor complex . Taken together, these data suggest that this phosphatase may play an important role in regulating the phenotype that we have described. In this way, the phenotype post-antagonism may be mechanistically similar to anergy induced by altered peptide ligands, as neither of these states can be overcome by IL-2.
The data presented herein demonstrate that the response of T cells post-antagonism is distinct from previously described forms of anergy, as antagonized cells respond to wild type stimulation by producing large quantities of IL-2, whereas anergized cells produce very little or no IL-2. The increased production of IL-2 by antagonized T cells in response to wild type stimulus contrasts sharply with the failure of these cells to proliferate. This cytokine hyperproduction coupled with the inability to proliferate represents a unique phenotype in T cell biology.
This work was supported by NIH R01 awards AI056017 and NS062358. The authors wish to thank the members of the Evavold Lab for critical reading of the manuscript.
Conflict of interest: The authors declare no financial or commercial conflict of interest.
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