PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of wtpaEurope PMCEurope PMC Funders GroupSubmit a Manuscript
 
J Immunol. Author manuscript; available in PMC 2010 November 7.
Published in final edited form as:
PMCID: PMC2975033
EMSID: UKMS32489

Peptide-MHC Class II Complex Stability Governs CD4 T Cell Clonal Selection

Abstract

The clonal composition of the T cell response can affect its ability to mediate infection control or to induce autoimmunity but the mechanisms regulating the responding TCR repertoire remain poorly defined. Here, we immunized mice with wild-type or mutated peptides displaying varying binding half-lives with MHC class II molecules to measure the impact of peptide-MHC class II stability on the clonal composition of the CD4 T cell response. We found that while all peptides elicited similar T cell response size upon immunization, the clonotypic diversity of the CD4 T cell response correlated directly with the half-life of the immunizing peptide. Peptides with short half-lives focused CD4 T cell response toward high affinity clonotypes expressing restricted public TCR, while peptides with longer half-lives broadened CD4 T cell response by recruiting lower affinity clonotypes expressing more diverse TCR. Peptides with longer half-lives did not cause the elimination of high affinity clonotypes, and at a low dose, they also skewed CD4 T cell response toward higher affinity clonotypes. Taken collectively, our results suggest the half-life of peptide-MHC class II complexes is the primary parameter that dictates the clonotypic diversity of the responding CD4 T cell compartment.

Introduction

The random rearrangement of TCR genes in the thymus enables the adaptive immune system to recognize a variety of pathogen-derived molecules (1). Antigen-specific T cells are selected from this vast pool of diverse, naïve cells based on the ability of their surface TCR to recognize peptide-MHC class complexes. Several studies of infection in mice (2) and in monkeys (3, 4) have suggested that the nature of the responding T cell repertoire can be important for effective pathogen control. There is also evidence that the progression of autoimmune disease in the murine experimental autoimmune encephalomyelitis is linked to the presence of high avidity public clonotypes (5, 6). While these studies suggest that the nature of the TCR repertoire selected may be crucial for pathogen control and for the development of autoimmunity, the mechanisms governing the clonal composition of the T cell response remain poorly understood.

The recognition of peptide-MHC class II (pMHCII) complexes by the TCR lies at the center of the CD4 T cell response. pMHCII complexes are stabilized by the interaction of peptide anchor residues with polymorphic pockets and depressions in the MHC class II binding groove (7). Previous studies have shown that the stability of pMHCII complexes impact CD4 T cell response, and more particularly peptide immunogenicity (8), immunodominance (9) and CD4 T cell differentiation (10, 11). However, the importance of pMHCII stability for CD4 T cell clonal selection remains unclear.

The I-Ek restricted murine response to pigeon cytochrome c (PCC) (12, 13) provides an ideal experimental model to determine the mechanisms of clonal selection in vivo. Immunization of B10.BR mice with PCC protein induces Vα11Vβ3-expressing CD4 T cells with restricted CDR3 regions that confer specificity to one dominant epitope (PCC88-104) (13). In this model, clonal dominance is established during the first week of the primary T cell response (13) and is based on threshold levels of TCR-pMHCII affinity (14). Adjuvants have been shown to alter this TCR based selection (15) but the factors setting the affinity threshold during an immune response are poorly understood.

In the current studies, immunization with cytochrome c peptides mutated at different critical MHCII anchor residues revealed the importance of pMHCII stability for CD4 T cell clonal selection. Although both low and high stability peptides induced extensive clonal expansion of antigen-specific CD4 T cells, the diversity of the antigen-specific TCR repertoire correlated directly with pMHCII stability. Peptides lacking key anchor residues for I-Ek skewed the CD4 response toward high affinity clonotypes with limited TCR repertoire diversity. Increasing peptide stability broadened TCR repertoire diversity by recruiting lower affinity clonotypes without eliminating high affinity clonotypes. However, this increased TCR repertoire diversity was regulated by antigen dose, with low dose of high stability peptides favoring higher affinity clonotypes. Thus, pMHCII stability controls the clonal composition of the antigen-specific CD4 T cell response in a dose-dependent manner.

Materials and methods

Mice

B10.BR, B10.BR-Thy1.1 congenic and ANDαβ transgenic mice were maintained under pathogen-free conditions at The Medical College of Wisconsin (MCW). MCW and the Institutional Animal Care and Use Committee reviewed and approved all experiments.

Peptide Synthesis

Peptides were synthesized by standard solid-phase methods, purified by HPLC, and confirmed by mass spectrometry. Prior to labeling with NHS-LC-Biotin (Pierce) an aminohexanoic acid spacer (Novabiochem) was added to the amino terminus of the peptide resin. The labeled peptides were then deprotected and purified by HPLC.

Peptide dissociation assay

I-Ek molecules were expressed as described previously (14) and loaded with CLIP peptide (Anaspec) to stabilize the molecule. For dissociation assays, soluble I-Ek (8μM final concentration) was loaded with biotinylated peptide (160μM final concentration) in 50mM NaH2PO4 and 50mM sodium citrate (pH 5.3) and protease inhibitor for 72 h at 37°C. I-Ek/peptide complexes were purified by buffer exchange to PBS using a Centricon-30 spin column (Millipore). For dissociation, reaction complexes (400nM final concentration) were incubated with a 100-fold molar excess of unlabeled competitor peptide (MCC88-103) to prevent rebinding of dissociated peptide. At indicated time points, aliquots were removed and immediately put on ice. Remaining complexes were quantified with an Europium-labeled streptavidin based solid-phase immunoassay. Maxisorb microtiter plates (Nunc) were coated with 14.4.4 (anti-I-Ek) antibody. Complexes were captured for 4 h at 4°C and detected with Streptavidin-Europium. Plates were measured in a Wallac VICTOR counter (PerkinElmer Wallac). Data were normalized and expressed as the percentage of bio-peptide/I-Ek complex remaining relative to the complex at t = 0. Graphpad Prism 5.0 was used to fit the data to an exponential-decay model for the determination of complex half-life.

Competitive peptide binding assay

Soluble I-Ek (40nM final concentration) was incubated with biotinylated MCC88-103 peptide (1000nM final concentration) and various concentrations of unlabeled competitor peptide (ranging from 0-320μM) in 50mM NaH2PO4 and 50mM sodium citrate (pH 5.3) and protease inhibitor for 72 h at 37°C. Complexes were quantified as described above but were captured for 1 h at 37°C. Data were normalized and fit to a three-parameter sigmoid function. IC50 values were calculated using SigmaPlot 2000 software.

Immunization and adoptive transfer

Mice were immunized subcutaneously at the base of the tail with 60μg or the indicated dose of peptide in combination with monophosphoryl lipid A-based adjuvant (lab formulation based on procedures in ref (16)). For adoptive transfer, 105 total splenocytes from ANDαβ transgenic mice containing 104 naïve PCC-specific CD4 T cells were transferred intravenously into B10.BR-Thy1.1 congenic mice at the time of immunization.

Flow Cytometry

Cell suspensions from lymphoid tissues in PBS with 5% FCS were labeled for 45 min at 4°C at a density of 2.0×108 cells/ml with predetermined optimal concentrations of the following fluorophore-labeled monoclonal antibodies: FITC-conjugated anti-Vα11 (RR8.1), PE-conjugated anti-Vβ3 (KJ25; all produced in the lab); Cy7-APC -conjugated anti-CD44 (IM7), APC-conjugated anti-CD90.2 (30-H12), Cy5-PE-conjugated anti-B220 (6B2), anti-CD8α (53-6.7) and anti-CD11b (M1/70; all from Biolegend); APC-Alexa Fluor® 750 conjugated anti-CD44 (Pgp-1; eBioscience); APC conjugated anti-CD62L (MEL-14; BD Biosciences). PE-MCC/I-Ek tetramers (pMHCII tetramer), prepared as previously described (14), were incubated for 2 h at room temperature at a final concentration of 300nM. After staining, cells were suspended in 1.5μg/ml DAPI (Invitrogen) (for dead cells exclusion) for analysis. For annexin V staining, cells that had been incubated with anti–surface marker antibodies were washed and incubated with Annexin V-PE (BD Biosciences) and DAPI for 15 min at room temperature. As positive control, splenocytes were incubated with 5μM Staurosporine for 3 h. Data were collected with FACS Diva software (BD Biosciences) and were analyzed with FlowJo software (TreeStar). Profiles are presented as 5% probability contours with outliers.

Single-Cell Repertoire Analysis

Single cells with the appropriate surface phenotype were sorted for repertoire analysis on a FACS Aria and CloneCyt software (BD Biosciences). The synthesis of cDNA and amplification of TCR Vα11 and Vβ3 regions were carried out as previously described (13). Direct sequencing of the purified PCR products was carried out with a Vα11-specific or Vβ3-specific primer by a commercial vendor (IDT). The frequency of obtaining a sequenceable PCR product from single cells varies between TCR-α and TCR-β with minimal variation across different peptides.

Ex Vivo Restimulation and Cytokine ELISA

Cell suspensions from draining LN in RPMI supplemented with 10% fetal bovine serum, 50nM 2-ME, were plated in 24-well plates (3×106 cells/ml) and restimulated with titrated dilutions of MCC88-104 or PCC103K peptide for four days. IFN-γ levels in supernatants were measured by sandwich ELISA using mAb from Biolegend. Plates were read at 405nm by a VERSAmax microplate reader and data were analyzed with SoftMax Pro software (Molecular Devices). Detection limit was 15pg/ml.

Statistical analysis

Statistical differences between experimental groups were determined by the Student’s t-test. Correlations between parameters were assessed by the Spearman correlation analysis. Statistical analysis was conducted with Prism software (GraphPad Software, Inc.). P< 0.05 was considered statistically significant.

Results

Generation of a peptide hierarchy to sample the importance of pMHCII stability for CD4 T cell clonal selection

To examine the importance of pMHCII stability for CD4 T cell clonal selection, we mutated two well-characterized cytochrome c peptides (PCC88-104 and moth cytochrome c (MCC88-103)) at I-Ek anchor residues to produce peptides with altered binding strengths. PCC88-104 is known to raise a heteroclitic response to MCC88-103, which means that all T cell clones raised by immunization with PCC88-104 respond better to MCC88-103 (17). An important difference between MCC88-103 and PCC88-104 is that PCC88-104 has an alanine at position 103, which partially fills the P9 pocket (18) and reduces its binding affinity and stability to I-Ek (19). To produce a PCC88-104 variant with increased stability, an Ala→Lys substitution at P9 pocket residue (PCC103K) was synthesized. Most peptides that bind to I-Ek have a large hydrophobic residue filling the P1 pocket of I-Ek (Ile or Leu). To produce a MCC88-103 peptide variant with decreased stability, an Ile→Ala substitution at P1 (MCC95A) was synthesized. Purified I-Ek molecules were loaded with biotinylated peptide in vitro at endosomal pH and 37°C for 3 days and pMHCII half-lives were determined. PCC103K displayed the longest half-life (t1/2 = 229 h) and was 3 times more stable than MCC88-103 (t1/2 = 73 h) (Fig. 1, A and C). In contrast, peptides lacking one key anchor residue displayed reduced pMHCII stability. PCC88-104 displayed a half-life of only 5 h, nearly 50-fold less than its higher stability variant PCC103K. MCC95A displayed the shortest half-life of all peptides tested, (t1/2 = 0.6 h). The same hierarchy of binding was found when peptides were assayed by competition (Fig. 1, B and C). Collectively, our measurements of relative stability established the following hierarchy of peptides: MCC95A<PCC88-104<MCC88-103<PCC103K.

Figure 1
Generation of a peptide hierarchy to sample the importance of pMHCII stability for CD4 T cell clonal selection

Impact of pMHCII stability on the local accumulation of antigen-specific CD4 T cells

Subcutaneous immunization of B10.BR mice with 400μg of whole PCC protein triggers a PCC-specific CD4 T cell response that reaches a maximum population size in draining lymph nodes (LN) 7 days after immunization (13, 15) (Fig. 2A). When we compared the day 7 responses induced by an equivalent molar amount of peptides, we found that all four peptides elicited similar numbers of antigen-specific CD4 T cells (Vα11+Vβ3+CD44hiCD62Llo) regardless of their differences in pMHCII stability (Fig. 2B, C). Antigen-specific CD4 T cells accumulated earlier in draining LN of mice immunized with high stability peptides PCC103K (Fig. 2D), but the magnitude of the CD4 T cell response between PCC88-104 and PCC103K was similar at later time points. There was also an increase of Vα11+Vβ3+CD44hiCD62Lhi cells for all peptides at day 7, but their total number paralleled what was found in the CD44hiCD62Llo compartment (Fig. S1). Therefore, the level of accumulation of antigen-specific CD4 T cells in draining LN appears minimally impacted by pMHCII stability.

Figure 2
Impact of pMHCII stability on the local accumulation of antigen-specific CD4 T cells

Antigen-specific TCR repertoire diversity correlates directly with pMHCII stability

To determine the clonal composition of the responding T cells induced by peptides with varying binding half-lives with MHC class II molecules, we sorted single antigen-specific CD4 T cells (Vα11+Vβ3+CD44hiCD62Lhi cells) from draining LN 7 days after peptide immunization and sequenced their CDR3α and CDR3β regions using a single-cell RT-PCR approach. Previous studies have shown that the CD4 T cell response to whole PCC protein is dominated by clonotypes expressing TCR with eight preferred CDR3 features (13). Four preferred features have been defined in the TCRα chain: 1) glutamic acid at positions 93, 2) serine at positions 95, 3) a CDR3 length of eight amino acids and 4) preference for Jα16, 22, 34 or 17 gene segment. Similarly, four CDR3β features commonly found in PCC-specific CD4 T cells have been defined: 1) asparagine at position 100, 2) alanine or glycine at position 102, 3) a CDR3 length of nine amino acids and 4) preference for Jβ1.2 or Jβ2.5 gene segment. All four peptides favored antigen-specific CD4 T cells expressing six or more of these CDR3 features in their TCR (>70% of responders express six or more of the eight preferred features for all peptides, Fig. 3A). However, there was a significant inverse relationship between the average number of preferred TCR features expressed by the responding CD4 T cells and the pMHCII half-life (Spearman: r=−0.77 and P=0.003; Fig. 3B). This inverse correlation was evident for both the TCRα (Spearman: r=−0.61 and P=0.03; Fig. 3C) and TCRβ chain (Spearman: r=−0.74 and P=0.006; Fig. 3D). These data suggest that pMHCII stability determines the clonotypic diversity of the responding CD4 T cell compartment.

Figure 3
Antigen-specific TCR repertoire diversity correlates directly with pMHCII stability

Peptide-MHCII Stability Skews TCR Usage

We have previously shown that, within the dominant clonotypes (clones expressing six or more of the eight preferred features), there were considerable variations in TCR binding properties that broadly correlated with J region usage. PCC-specific TCRβ cells expressing Jβ2.5 displayed overall lower affinity for pMHCII than cells expressing Jβ1.2 (14, 15). Similarly, different Jα genes pairing with the same TCRβ also conferred different binding kinetics (14). Hence, we next examined the prevalence of individual CDR3 features selected by low and high stability peptides. At the level of the CDR3α region, there were some variations predominantly based on Jα region usage (Fig. S2). All peptides selected similar frequencies of the canonical Jα but low stability peptides favored clonotypes expressing Jα16, while higher stability peptides favored dominant clonotypes expressing Jα22 (Fig. S2A). All other CDR3α features were highly restricted across all peptides except for the highest stability peptide PCC103K that selected a higher number of clonotypes with longer CDR3α (Fig. S2B) and more diverse amino acids at positions α93 and α95 (Fig. S2C and D, respectively). However, the most consistent and systematic change between low and high stability peptides was observed at the level of the CDR3β region (Fig. S3), particularly at the level of Jβ region usage (Figs. (Figs.4A4A and S3A). Similar to what was observed with the whole PCC protein, PCC88-104 peptide induced 3 times more Jβ1.2- than Jβ2.5-expressing clonotypes (Fig. 4A) (15). The lowest stability peptide MCC95A further exaggerated the frequency of Jβ1.2-expressing clonotypes (Ratio Jβ1.2/Jβ2.5 = 13±4, Fig. 4A). In contrast, higher stability peptides did not display this bias and recruited similar numbers of Jβ1.2- and Jβ2.5-expressing clonotypes (Fig. 4A). The CDR3β feature of G or A at β102 correlated with the Jβ region differences (Fig. S3B) while the CDR3β length of 9aa (Fig. S3C) and the selection for N at β100 (Fig. S3D) were similarly restricted across all peptides. Hence, pMHCII stability skews J region gene usage of the dominant antigen-specific CD4 T cell compartment.

Figure 4
Peptide-MHCII stability skews TCRβ chain usage

We have previously shown that the CD4 T cell response to PCC whole protein was dominated by Jβ1.2 clonotypes expressing one specific public (present in most of the mice) CDR3 rearrangement (SLNNANSDY or 5C.C7β chain) (15) that conferred high pMHCII binding affinity (14, 15). Next, we analyzed the CDR3β amino acid sequences selected by high and low stability peptides and divided the sequences based on their relative prevalence distinguishing public, recurrent (present in more than one mouse) and private (specific to individual mice) TCRβ chains (Figs. (Figs.4B4B and S4). Interestingly, the frequency of public TCRβ sequences in the antigen-specific CD4 T cell repertoire correlated inversely with the pMHCII stability (Spearman: r=−0.9 and P<0.0001; Fig. 4C). Responses to low and intermediate stability peptides MCC95A, PCC88-104 and MCC88-103 were dominated by clones expressing public TCRβ chains while response to the high stability peptide PCC103K was dominated by clones displaying private TCRβ chains (Figs. (Figs.4B4B and S4). When we compared the public TCRβ chains selected by individual peptides, we observed that low stability peptides predominantly selected the 5C.C7β chain (SLNNANSDY) and the related C.F6β chain (SLNSANSDY) (20) (Fig. 4B). Both 5C.C7β and C.F6β-expressing clonotypes were found in MCC88-103 immunized mice but this response was dominated by a third public CDR3β chain (SLNRGQDTQ) (Fig. 4B) that was also selected by low affinity PCC-specific CD4 T cells in mice immunized with PCC protein and IFA or Alum (15). Overall, our results demonstrate that pMHCII stability controls the clonotypic diversity of the CD4 T cell compartment and suggest that decreasing pMHCII stability focuses antigen-specific CD4 T cells toward public clonotypes expressing high affinity TCR.

Preferential accumulation of high affinity CD4 T cells with low stability peptides

Peptide-MHCII tetramer staining provides a distinct method to detect PCC-specific CD4 T cells in vivo (15, 21). Importantly, the measurement of pMHCII tetramer mean fluorescence intensity (MFI) at optimal concentrations also provides a reliable indicator of differential TCR-binding affinity (14, 15, 22-25). For three out of four peptides, the number of antigen-specific CD4 T cells detected by pMHCII tetramer staining was similar to the one estimated by V region-based staining (Fig. 5, A and B). However, for the highest stability peptide PCC103K, significantly fewer antigen-specific CD4 T cells were recognized by pMHCII tetramer staining (Fig. 5, A and B), suggesting the presence of lower affinity clonotypes that fall below the level of detection of pMHCII tetramer reagents (15). Consistent with the TCR repertoire studies, we observed an inverse relationship between the pMHCII tetramer staining mean fluorescence intensity (MFI) of the responding CD4 T cells and the pMHCII stability (Spearman: r=−0.6 and P=0.0019; Fig. 5C). The highest stability peptide PCC103K induced the antigen-specific T cell population with the lowest MFI intensity compared to all other peptides (Fig. 5C). For lower stability peptides (PCC88-104 and MCC95A), there was a significant increase in MFI corresponding to the increased presence of higher affinity cells (Fig. 5, A and C). This difference in pMHCII tetramer staining was not caused by a difference in TCR expression levels, as the Va11 staining MFI by Vα11+pMHCIITet+CD44hi cells were similar across all peptides (Fig. 5D).

Figure 5
Preferential accumulation of high affinity CD4 T cells with low stability peptides

Because it is possible that differences in pMHCII tetramer staining intensities may not reflect in TCR affinity but differences in activation status (26), we also assessed the functional avidities of CD4 T cells responding to low and high stability peptides. The functional avidity of the responding populations was measured by assessing their ability to produce IFN-γ ex vivo in response to decreasing concentration of the high stability peptide MCC88-103. For all immunization conditions, we found that the maximal response was detected with 10−5 M peptide and that replacing MCC88-103 by PCC103K did not affect the magnitude of this response (Fig. 5E). When restimulated with optimal MCC88-103 peptide concentration, antigen-specific CD4 T cells induced by high stability peptides (PCC103K, MCC88-103) produced more IFN-γ than those elicited by low stability peptides (PCC88-104 and MCC95A) (Fig. 5E), but with lower peptide concentrations in culture, the magnitude of the IFN-γ response induced by high stability peptides decreased rapidly, showing a half-maximal response (EC50) between 0.6 × 10−6 M and 1 × 10−6 M (MCC88-103 and PCC103K, respectively; Fig. 5F). In contrast, antigen-specific CD4 T cells induced by low stability peptides displayed a superior EC50 between 0.8 × 10−7 M and 1 × 10−7 M (PCC88-104 and MCC95A, respectively; Fig. 5F). Consistent with the pMHCII tetramer binding assays, there was a direct relationship between the EC50 and the pMHCII stability (Spearman: r=0.5 and P=0.01; Fig. 5G). Together these data suggest that pMHCII stability alters the prevalence of high affinity cells within the antigen-specific CD4 T cell population.

Comparable Expansion of High Affinity Transgenic CD4 T cells by Low and High Stability Peptides

The low prevalence of T cells bearing high affinity TCR in response to high stability peptides could result from their elimination by apoptosis (5). To investigate this possibility, we used high affinity AND PCC-specific TCRαβ-transgenic CD4 T cells in adoptive-transfer experiments. AND CD4 T cells express the 5C.C7β chain paired with a Jα16-expressing TCRα chain (CDR3α: EASSGQKL) conferring high affinity for pMHCII binding (14) (Fig. S5). We transferred CD90.2-expressing transgenic cells into CD90.1 recipient mice and immunized recipients with either low stability peptide PCC88-104 or high stability peptide PCC103K. Seven days after immunization, comparable numbers of high affinity AND CD4 T cells were recovered from draining LN of recipients immunized with high or low stability peptides (Fig. 6, A and B). Furthermore, there was no evidence for increased apoptosis of AND cells in mice immunized with the highest stability peptide PCC103K (Fig. 6C). Hence, immunization with high stability peptides does not cause the elimination of high affinity clonotypes.

Figure 6
Comparable expansion of high affinity transgenic CD4 T cells by low and high stability peptides

Impact of pMHCII Stability on Clonal Selection is Dependent on Antigen Dose

Using whole PCC protein antigen, we had previously demonstrated that TCR-based selection was independent of antigen dose (14, 15). Because of the low stability of the PCC immunodominant peptide, it was important to readdress the importance of the antigen dose in the context of a higher stability peptide such as PCC103K. Decreasing 100-fold the dose of low stability peptide PCC88-104 significantly altered antigen-specific CD4 T cell clonal expansion (Fig. 7A, B). In contrast, a 10,000-fold reduction in the dose of high stability peptide PCC103K was necessary to observe a significant decrease in the number of antigen-specific CD4 T cells (Fig. 7A, B). To determine the clonal composition of the responding T cells induced by these lower doses of PCC103K, we isolated single antigen-specific CD4 T cells from mice immunized with 0.06μg peptides for repertoire analysis. At this dose, there was a significant increase in the number of clonotypes expressing eight preferred features (Figs. (Figs.7C7C and S6) and an increase in the prevalence of clonotypes expressing Jβ1.2 instead of Jβ2.5 (Fig. 7D). At the TCRβ chain peptidic level, we also observed an increased prevalence of high affinity public clonotypes (SLNNANSDY and SLNSANSDY) (Fig. 7E). Consistent with this TCR repertoire change, antigen-specific CD4 T cells induced at a low antigen dose had a greater pMHCII tetramer staining intensity than antigen-specific CD4 T cells induced at a higher dose (Fig. 7, F and G). Thus, pMHCII stability regulates the clonotypic diversity of the antigen-specific CD4 T cell response in a dose-dependent manner.

Figure 7
Impact of pMHCII stability on clonal selection is dependent on antigen dose

Discussion

Our studies revealed the remarkable adaptation of the antigen-specific CD4 T cell response and its TCR repertoire to pMHCII stability. By mutating key anchor residues, we generated four peptides with marked differences in their capacity to form stable pMHCII complexes. Despite these differences in pMHCII stability, all four peptides induced comparable clonal accumulation of antigen-specific CD4 T cells in vivo. There was, however, a direct correlation between pMHCII stability and the clonotypic diversity of the CD4 T cell response as measured by the prevalence of preferred CDR3 features or by the frequency of public TCRβ chains in the antigen-specific CD4 T cell repertoire. Lower stability peptides (MCC95A, PCC88-104) selected clonotypes expressing more restricted TCR with public TCRβ chains and displaying high avidity for their pMHCII ligand. In contrast, higher stability peptides (MCC88-103, PCC103K) selected clonotypes expressing less restricted TCR, more private TCRβ chains and displaying lower avidity for their pMHCII ligand. The antigen dose altered this clonal selection, and at a low dose, high stability peptides recruited more restricted TCR repertoire. Hence, our studies established that the clonotypic diversity of the antigen-specific CD4 T cell compartment that regulates the adaptive immune response is governed by pMHCII stability and the antigen dose in vivo.

Anchor residues in peptides determine the stability of binding to major histocompatibility complex class II molecules. Kersh et al. have however shown that a single aa substitution at the P6 I-Ek anchor position of the hemoglobin peptide, that did not alter the peptide stability, had sufficient affect on the solvent exposed face of the pMHCII complex to modify CD4 T cell recognition (27). In our studies, substituting the P9 anchor residue of PCC88-104 or the P1 anchor residue of MCC88-103 leads to the predicted enhancement or attenuation of binding to I-Ek. Although we cannot exclude that these substitutions also produce some changes in the solvent exposed face of the pMHCII complex offered to the T cells, the remarkable correlations between the peptide binding half-lives with I-Ek and the complexity and avidity of the responding CD4 TCR repertoires strongly argue that pMHCII stability is the major determinant of the clonotypic diversity of the antigen-specific CD4 T cell compartment in our study. One of the surprising results derived from the peptide binding stability studies was that PCC103K displayed significantly longer pMHCII half-life than MC88-103 despite sharing the same core amino acid sequence and all MHC anchor residues indicating that positions outside the “formal” peptide binding groove could play a role in the overall binding energy of the peptide:MHC complex. It is possible that the lysine located outside of the binding groove in PCC103K at position P10 participates to the peptide anchoring to I-Ek. The P10 position has been found to play a role in complex stability in a number of studies (28, 29) and a structural correlate has been determined in that the side chain can lie on a shallow external shelf (28).

We have proposed that the expansion of antigen-specific CD4 T cells in vivo is limited by a TCR affinity threshold (14). One critical aspect of this model is that, above a threshold affinity, all clonotypes undergo similar clonal expansion regardless of further differences in their TCR binding properties. In the present studies, we identify pMHCII stability as a critical regulator of this affinity-based selection. Our results suggest that high stability peptides lower the CD4 T cell affinity-based selection threshold allowing the expansion of a large number of lower affinity clonotypes. Our results do not support the idea that peptides with long half-lives drive the apoptosis of high affinity CD4 T cells (5). Instead, consistent with our threshold selection model, we interpret the low prevalence of high affinity clonotypes in response to high stability peptides by the lack of proliferative advantage of CD4 T cells with high affinity TCR when the selection threshold is decreased. The discrepancy between the two models could come from the fact that Anderton et al. were studying the fate of autoreactive CD4 T cells in a EAE model. In this model, high affinity auto-reactive CD4 T cells ultimately disappear from the immune repertoire as the animal recovers (6). In contrast, high affinity antigen-specific CD4 T cells generated by immunization with foreign antigen in Ribi adjuvant preferentially develop into effector follicular T helper (TFH) cells (25), persist as memory TFH in draining LN (24) and dominate the response to secondary antigen challenge (12, 13). Hence, the elimination of high affinity auto-reactive CD4 T cells in the periphery may represent a form of peripheral tolerance and not a general mechanism of negative selection of high affinity clonotypes by strong peptide agonists.

How the antigen dose impacts the clonotypic composition of the CD4 T cell compartment is not well understood. Our previous studies using whole PCC protein immunization reported that antigen dose did not significantly impact CD4 T cell clonal selection (14, 15). Rees et al., using a peptide immunization model found that low antigen dose promoted the selection of high affinity CD4 T cells but this selection was only apparent by tetramer staining at later stages of the primary CD4 T cell response (30). Our current study shows that antigen dose does indeed impact the clonal composition of the effector CD4 T cell compartment and this impact can be seen at the peak of the primary immune response. This dose effect may not be as pronounced with lower stability peptides because the antigen-specific TCR repertoire obtained at high antigen dose is already very restricted and low stability peptides do not promote a CD4 T cell response at a very low dose (14, 15). The capacity of high stability epitopes to differentially expand high affinity CD4 T cells at low and high antigen doses may have important implications in defining the antigen-specific CD4 T cell repertoire to complex pathogens. The effective antigen dose presented to the antigen-specific CD4 T cells following infection is in large part determined by the capacity of the innate immune response to prevent the pathogen dissemination. A robust innate immune response would lower the antigen dose presented to CD4 T cells and thus favor the development of high affinity CD4 T cells. Consistent with this, we have previously reported that mice resistant to Leishmania major infection, that effectively prevent the parasite dissemination (31) very rapidly focus their parasite-specific CD4 T cell response toward high affinity clonotypes, while susceptible BALB/c mice that fail to prevent the parasite dissemination (31) recruit lower affinity parasite-specific CD4 T cells (32).

The emergence of high affinity clonotypes with similar TCR repertoire usage in situations of low antigen dose and low pMHCII stability suggests that pMHCII stability and antigen dose regulate one unique parameter that determines the affinity selection threshold. Henrickson et al. have shown that peptide-MHC class I stability and antigen dose ultimately determine the number of peptide-MHC class I complexes displayed by antigen-presenting dendritic cells in draining LN (33). Peptide-MHC class I complex density on the surface of DC appears to set a threshold for CD8 T cell activation (33). Peptide-MHCII density on the surface of the antigen-presenting cells may set a similar threshold for CD4 T cell activation by regulating the magnitude of TCR engagement (34-36). Since CD4 T cells required pMHCII contacts throughout their expansion phase (37, 38), decreasing pMHCII density on the surface of the antigen-presenting cells at the initiation of the CD4 T cell response (immunization with low antigen dose) or during CD4 T cell clonal expansion (immunization with low stability peptide) may set a higher activation threshold that favors the expansion of high affinity clonotypes.

In conclusion, we have demonstrated that pMHCII stability and antigen dose are important determinants of the clonotypic diversity of antigen-specific CD4 T cell responses. Our findings provide new insights into the molecular mechanism of CD4 T cell clonal selection during an immune response. Together with our previous studies (15), an important implication of our observations is that several parameters (antigen dose, nature of CD4 T cell epitope, adjuvant) in a protein subunit vaccine or immunotherapy have the potential to change the affinity and clonotypic diversity of antigen-specific CD4 T cell responses and may thereby affect the quality of the protective immune response.

Supplementary Material

Supplementary Figures

Acknowledgements

We would like to thank Dr Bonnie Dittel for helpful discussion, Dr Aniko Szabo for help with statistical analysis and Trudy Holyst for technical assistance with the reagents.

1This work was supported by NIH grant U19 A162627, the American Cancer Society and the MCW Cancer Center. C.B. was supported by a fellowship from FWF - the Austrian Science Fund.

3Abbreviations used in this paper

LN
lymph nodes
pMHCII
peptide-MHC class II
PCC
pigeon cytochrome c
MCC
moth cytochrome c
MFI
mean fluorescence intensity

Footnotes

Publisher's Disclaimer: This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the United States National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.

References

1. Nikolich-Zugich J, Slifka MK, Messaoudi I. The many important facets of T-cell repertoire diversity. Nat. Rev. Immunol. 2004;4:123–132. [PubMed]
2. Messaoudi I, Guevara-Patino JA, Dyall R, LeMaoult J, Nikolich-Zugich J. Direct link between mhc polymorphism, T cell avidity, and diversity in immune defense. Science. 2002;298:1797–1800. [PubMed]
3. Price DA, West SM, Betts MR, Ruff LE, Brenchley JM, Ambrozak DR, Edghill-Smith Y, Kuroda MJ, Bogdan D, Kunstman K, Letvin NL, Franchini G, Wolinsky SM, Koup RA, Douek DC. T cell receptor recognition motifs govern immune escape patterns in acute SIV infection. Immunity. 2004;21:793–803. [PubMed]
4. Price DA, Asher TE, Wilson NA, Nason MC, Brenchley JM, Metzler IS, Venturi V, Gostick E, Chattopadhyay PK, Roederer M, Davenport MP, Watkins DI, Douek DC. Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection. J. Exp. Med. 2009;206:923–936. [PMC free article] [PubMed]
5. Anderton SM, Radu CG, Lowrey PA, Ward ES, Wraith DC. Negative selection during the peripheral immune response to antigen. J. Exp. Med. 2001;193:1–11. [PMC free article] [PubMed]
6. Menezes JS, van den Elzen P, Thornes J, Huffman D, Droin NM, Maverakis E, Sercarz EE. A public T cell clonotype within a heterogeneous autoreactive repertoire is dominant in driving EAE. J. Clin. Invest. 2007;117:2176–2185. [PMC free article] [PubMed]
7. Stern LJ, Brown JH, Jardetzky TS, Gorga JC, Urban RG, Strominger JL, Wiley DC. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature. 1994;368:215–221. [PubMed]
8. Hall FC, Rabinowitz JD, Busch R, Visconti KC, Balmares M, Patil NS, Cope AO, Patel S, McConnell HM, Mellins ED, Sonderstrup G. Relationship between kinetic stability and immunogenicity of HLA-DR4/peptide complexes. Eur. J. Immunol. 2002;32:662–670. [PubMed]
9. Lazarski CA, Chaves FA, Jenks SA, Wu SH, Richards KA, Weaver JM, Sant AJ. The kinetic stability of MHC class II: Peptide complexes is a key parameter that dictates immunodominance. Immunity. 2005;23:29–40. [PubMed]
10. Constant SL, Bottomly K. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol. 1997;15:297–322. [PubMed]
11. Murray JS. How the MHC selects Th1/Th2 immunity. Immunol. Today. 1998;19:157–163. [PubMed]
12. McHeyzer-Williams MG, Davis MM. Antigen-specific development of primary and memory T cells in vivo. Science. 1995;268:106–111. [PubMed]
13. McHeyzer-Williams LJ, Panus JF, Mikszta JA, McHeyzer-Williams MG. Evolution of antigen-specific T cell receptors in vivo: Preimmune and antigen-driven selection of preferred complementarity-determining region 3 (CDR3) motifs. J. Exp. Med. 1999;189:1823–1837. [PMC free article] [PubMed]
14. Malherbe L, Hausl C, Teyton L, McHeyzer-Williams MG. Clonal selection of helper T cells is determined by an affinity threshold with no further skewing of TCR binding properties. Immunity. 2004;21:669–679. [PubMed]
15. Malherbe L, Mark L, Fazilleau N, McHeyzer-Williams LJ, McHeyzer-Williams MG. Vaccine adjuvants alter TCR-based selection thresholds. Immunity. 2008;28:698–709. [PMC free article] [PubMed]
16. Baldridge JR, Crane RT. Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines. Methods. 1999;19:103–107. [PubMed]
17. Solinger AM, Ultee ME, Margoliash E, Schwartz RH. T-lymphocyte response to cytochrome c. I. Demonstration of a T-cell heteroclitic proliferative response and identification of a topographic antigenic determinant on pigeon cytochrome c whose immune recognition requires two complementing major histocompatibility complex-linked immune response genes. J. Exp. Med. 1979;150:830–848. [PMC free article] [PubMed]
18. Fremont DH, Dai SD, Chiang H, Crawford F, Marrack P, Kappler J. Structural basis of cytochrome c presentation by IEk. J. Exp. Med. 2002;195:1043–1052. [PMC free article] [PubMed]
19. Krogsgaard M, Prado N, Adams EJ, He XL, Chow DC, Wilson DB, Garcia KC, Davis MM. Evidence that structural rearrangements and/or flexibility during TCR binding can contribute to T cell activation. Mol. Cell. 2003;12:1367–1378. [PubMed]
20. Fink PJ, Matis LA, McElligott DL, Bookman M, Hedrick SM. Correlations between T-Cell Specificity and the Structure of the Antigen Receptor. Nature. 1986;321:219–226. [PubMed]
21. Savage PA, Boniface JJ, Davis MM. A kinetic basis for T cell receptor repertoire selection during an immune response. Immunity. 1999;10:485–492. [PubMed]
22. Crawford F, Kozono H, White J, Marrack P, Kappler J. Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes. Immunity. 1998;8:675–682. [PubMed]
23. Reichstetter S, Ettinger RA, Liu AW, Gebe JA, Nepom GT, Kwok WW. Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response. J. Immunol. 2000;165:6994–6998. [PubMed]
24. Fazilleau N, Eisenbraun MD, Malherbe L, Ebright JN, Pogue-Caley RR, McHeyzer-Williams LJ, McHeyzer-Williams MG. Lymphoid reservoirs of antigen-specific memory T helper cells. Nat. Immunol. 2007;8:753–761. [PubMed]
25. Fazilleau N, McHeyzer-Williams LJ, Rosen H, McHeyzer-Williams MG. The function of follicular helper T cells is regulated by the strength of T cell antigen receptor binding. Nat. Immunol. 2009;10:375–384. [PMC free article] [PubMed]
26. Vollers SS, Stern LJ. Class II major histocompatibility complex tetramer staining: progress, problems, and prospects. 2008;123:305–313. [PubMed]
27. Kersh GJ, Miley MJ, Nelson CA, Grakoui A, Horvath S, Donermeyer DL, Kappler J, Allen PM, Fremont DH. Structural and functional consequences of altering a peptide MHC anchor residue. J. Immunol. 2001;166:3345–3354. [PubMed]
28. Yassai M, Afsari A, Garlie J, Gorski J. C-terminal anchoring of a peptide to class II MHC via the P10 residue is compatible with a peptide bulge. J. Immunol. 2002;168:1281–1285. [PubMed]
29. Zavala-Ruiz Z, Strug I, Anderson MW, Gorski J, Stern LJ. A polymorphic pocket at the P10 position contributes to peptide binding specificity in class II MHC proteins. Chem. Biol. 2004;11:1395–1402. [PubMed]
30. Rees W, Bender J, Teague TK, Kedl RM, Crawford F, Marrack P, Kappler J. An inverse relationship between T cell receptor affinity and antigen dose during CD4(+) T cell responses in vivo and in vitro. Proc. Natl. Acad. Sci. U. S. A. 1999;96:9781–9786. [PubMed]
31. Laskay T, Diefenbach A, Rollinghoff M, Solbach W. Early Parasite Containment Is Decisive for Resistance to Leishmania-Major Infection. Eur. J. Immunol. 1995;25:2220–2227. [PubMed]
32. Malherbe L, Filippi C, Julia V, Foucras G, Moro M, Appel H, Wucherpfennig K, Guery JC, Glaichenhaus N. Selective activation and expansion of high-affinity CD4(+) T cells in resistant mice upon infection with Leishmania major. Immunity. 2000;13:771–782. [PubMed]
33. Henrickson SE, Mempel TR, Mazo IB, Liu B, Artyomov MN, Zheng H, Peixoto A, Flynn MP, Senman B, Junt T, Wong HC, Chakraborty AK, von Andrian UH. T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation. Nat. Immunol. 2008;9:282–291. [PMC free article] [PubMed]
34. McNeil LK, Evavold BD. TCR reserve: A novel principle of CD4 T cell activation by weak ligands. J. Immunol. 2003;170:1224–1230. [PubMed]
35. McNeil LK, Evavold BD. Dissociation of peripheral T cell responses from thymocyte negative selection by weak agonists supports a spare receptor model of T cell activation. Proc. Natl. Acad. Sci. U. S. A. 2002;99:4520–4525. [PubMed]
36. Mirshahidi S, Ferris LCK, Sadegh-Nasseri S. The magnitude of TCR engagement is a critical predictor of T cell anergy or activation. J.Immunol. 2004;172:5346–5355. [PubMed]
37. Obst R, van Santen HM, Mathis D, Benoist I. Antigen persistence is required throughout the expansion phase of a CD4(+) T cell response. J. Exp. Med. 2005;201:1555–1565. [PMC free article] [PubMed]
38. Obst R, van Santen HM, Melamed R, Kamphorst A, Benoist C, Mathis D. Sustained antigen presentation can promote an immunogenic T cell response, like dendritic cell activation. Proc. Natl. Acad. Sci. U. S. A. 2007;104:15460–15465. [PubMed]