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. In this study, 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, although all peptides elicited similar T cell response size on 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, whereas 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.

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

Comparison of the (A) stability or (B) affinity of various I-E^k binding peptides at 37 °C. The lines represent the fit of the data to (A) a single-exponential function or (B) a three-parameter sigmoid function from which the half-lives (t_1/2) or the IC₅₀ could be determined. (C) Peptide sequences are aligned according to their anchor positions (P1, P9). P1 and P9 pocket residues are in bold. Half-lives and IC₅₀ of I-E^k/peptide complexes are indicated (mean ± SEM). Reactions were performed in triplicate, and data series represent one of three independent experiments.

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

(A, B) Antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) at day 7 in LN from B10.BR mice immunized with (A) whole PCC protein or PBS, or (B) with the indicated peptide in adjuvant. All profiles are gated on B220⁻CD8⁻CD11b⁻ DAPI negative cells. Numbers above boxed areas indicate percent cells (± SEM) in profile. (C) Total number of antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) 7 days after immunization with whole PCC protein or the indicated peptide (filled bars) or PBS (grey bar). (D) Total number of antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) at indicated days after immunization with PCC_88-104 (filled bars) or PCC_103K (empty bars). Means ± SEM for at least three animals are shown. **, P ≤ 0.01 (unpaired Student’s t-test). Data shown are derived from at least three independent experiments (n≥3 mice per group).

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

Single-cell repertoire analysis of individual antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) sorted from mice immunized with the indicated peptide. (A) Each filled circle represents the sequence information of a single-cell. The y-axis represents the number of preferred CDR3 features known to be selected in the PCC response (TCR-α: E at α93; S at α95; CDR3α length of 8aa; and TCRJα 16, 17, 22 and 34. TCR-β: N at β100; A/G at β102; CDR3β length of 9aa; and TCRJβ 1.2 and 2.5). Middle row: percent cells (± SEM) with ≥6 preferred features, expressing a restricted TCR of the dominant clonotype. The numbers of sequences (derived from three individual mice per group) used in the analysis are displayed as the n on the x-axis. (B-D) The average numbers of preferred CDR3 features per responding CD4 T cell for the (B) whole TCR, (C) the TCRα chain only or (D) the TCRβ chain only are plotted against the half-life of the peptide for the MHC classII as determined in Fig.1. Each symbol represents one mouse. Linear regression analyses are shown with Spearman correlation coefficients (r) and significance (P). Data are representative from three independent experiments (n≥3 mice per group).

Figure 4. Peptide-MHCII stability skews TCRβ chain usage

(A) Relative abundance of antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) expressing Jβ1.2 or Jβ2.5 gene segments for the indicated peptide (mean ± SEM). (B) Amino acid sequences of CDR3β regions for antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) isolated from mice immunized with the indicated peptide. Each filled circle represents the sequence information of a single-cell. Percent cells (± SEM) expressing public clonotypes are shown. *, P ≤ 0.05, between PCC_103K and all other peptides (unpaired Student’s t-test). (C) Scatter plot showing the frequency of public TCRβ sequences within the responding CD4 T cell compartment as a function of pMHCII half-life as determined in Fig. 1. Each symbol represents one mouse. Linear regression analysis is shown with Spearman correlation coefficient (r) and significance (P). Data are representative from three independent experiments (n≥3 mice per group).

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

(A-D) Antigen-specific CD4 T cells were selected as CD8⁻B220⁻CD11b⁻ DAPI⁻ cells expressing Vα11, binding pMHCII tetramers and expressing high level of CD44. (A) Antigen-specific CD4 T cells (Vα11⁺pMHCIITet⁺CD44^hi) at day 7 in LN from B10.BR mice immunized with PBS or with the indicated peptide. Numbers beside boxed areas indicate percent cells in profile (mean ± SEM). (B) Total number of Vα11⁺pMHCIITet⁺CD44^hiCD62L^lo T cells (filled bars) or Vα11⁺Vβ3⁺CD44^hiCD62L^lo T cells (empty bars) after immunization with the indicated peptide. Means ± SEM for at least four animals are shown. **, P ≤0.01 (unpaired Student’s t-test). (C, D) Scatter plots showing the mean fluorescence intensity of (C) pMHCII tetramer staining or (D) Vα11 expression of antigen-specific CD4 T cells (Vα11⁺pMHCIITet⁺CD44^hiCD62L^lo) as a function of pMHCII half-life as determined in Fig. 1. Each symbol represents one mouse. Linear regression analysis is shown with Spearman correlation coefficient (r) and significance (P). (E-G) LN cells from B10.BR mice 7 days after immunization with the indicated peptide were restimulated with MCC_88-103 peptide (E-G) or PCC_103K (E) ex vivo and IFN-γ concentrations in supernatants collected after 4 days were measured by ELISA. (E) IFN-γ production upon restimulation with either 10μM MCC_88-103 (black bars) or 10μM PCC_103K peptide (grey bars) (mean ± SEM). (F) Percent maximal IFN-γ response induced by ex vivo restimulation with the indicated MCC_88-103 peptide concentration, as measured by the amount of cytokine produced at each peptide concentration divided by the amount of cytokine produced at the optimal peptide concentration (mean ± SEM). (G) Scatter plot showing the EC₅₀ expressed as a function of pMHCII half-life as determined in Fig. 1. Each symbol represents one mouse. Linear regression analysis is shown with Spearman correlation coefficient (r) and significance (P). Data shown are derived from at least three independent experiments (n≥3 mice per group).

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

10⁵ total splenocytes from AND TCRαβ mice were transferred into CD90.1⁺ syngeneic hosts and recipients were immunized with PCC_88-104 or PCC_103K. (A) Representative probability contours of Vβ3 staining versus CD90.2 for PCC_88-104 (left) and PCC_103K (right) immunized recipients. (B) Total number of PCC-specific transgenic effector AND TCRαβ CD4 T cells (Vα11⁺Vβ3⁺CD90.2⁺CD44^hi) in draining LN of recipients immunized with PCC_88-104 or PCC_103K (mean ± SEM). (C) Annexin V–PE staining of PCC-specific transgenic effector AND TCRαβ CD4 T cells (Vα11⁺Vβ3⁺CD90.2⁺CD44^hi) in draining LN (left and middle panel). Staurosporine-treated splenocytes (right panel) were used as positive control. Data shown are derived from two independent experiments (n≥3 mice per group).

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

(A) Antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) at day 7 in LN from mice immunized with decreasing dose of PCC_88-104 or PCC_103K peptides. Numbers above boxed areas indicate percent cells in representative profile (B) Total number of antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) after immunization with decreasing dose of PCC_88-104 or PCC_103K peptides. **, P ≤ 0.01 (unpaired Student’s t-test). (C) Use of preferred CDR3 features per cell in mice immunized with indicated dose of PCC_103K. Each dot represents the sequence information from a single-cell. Middle row: percent cells (± SEM) with ≥6 preferred features, expressing a restricted TCR of the dominant clonotype. The numbers of sequences (derived from three individual mice) used in the analysis are displayed as the n on the x-axis. (D) Relative abundance of antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) expressing Jβ1.2 or Jβ2.5 gene segments for indicated PCC_103K dose. **, P ≤0.01 (unpaired Student’s t-test). (E) Amino acid sequences of CDR3β regions for antigen-specific CD4 T cells (Vα11⁺Vβ3⁺CD44^hiCD62L^lo) isolated from mice immunized with indicated dose of PCC_103K. Percent cells (± SEM) expressing predominant clonotypes are shown. (F) Representative Tetramer staining of antigen-specific CD4 T cells (Vα11⁺pMHCIITet⁺CD44^hi) at day 7 in LN from B10.BR mice immunized with indicated dose of PCC_103K. Numbers beside boxed areas indicate percent cells in profile. (G) Mean fluorescence intensity of pMHCII tetramer staining for antigen-specific CD4 T cells (Vα11⁺pMHCIItet⁺CD44^hiCD62L^lo) after immunization with the indicated dose of PCC_103K. **, P ≤0.01 (paired Student’s t-test). Data shown are derived from three independent experiments (n≥3 mice per group).