Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance

Abstract

Understanding the mechanisms underlying the poor immunogenicity of human self/tumor antigens is challenging because of experimental limitations in humans. Here, we developed a human-mouse chimeric model that allows us to investigate the roles of the frequency and self-reactivity of antigen-specific T cells in determination of the immunogenicity of an epitope (amino acids 209-217) derived from a human melanoma antigen, gp100. In these transgenic mice, CD8+ T cells express the variable regions of a human T cell receptor (hTCR) specific for an HLA-A*0201-restricted gp100(209-217). Immunization of hTCR-transgenic mice with gp100(209-217) peptide elicited minimal T cell responses, even in mice in which the epitope was knocked out. Conversely, a modified epitope, gp100(209-217(2M)), was significantly more immunogenic. Both biological and physical assays revealed a fast rate of dissociation of the native peptide from the HLA-A*0201 molecule and a considerably slower rate of dissociation of the modified peptide. In vivo, the time allowed for dissociation of peptide-MHC complexes on APCs prior to their exposure to T cells significantly affected the induction of immune responses. These findings indicate that the poor immunogenicity of some self/tumor antigens is due to the instability of the peptide-MHC complex rather than to the continual deletion or tolerization of self-reactive T cells.

Figure 1

Characterization of the human-mouse TCR–HLA-A2/K^b transgenic mouse model. (A) Illustration of a hTCR–HLA-A*0201–gp100_209–217 complex in the JR209-Tg mouse model. (B) hTCR Vβ8 and CD8 expression on lymphocyte-gated population from spleens of non–TCR-transgenic A2/Kb (A2/Kb) and JR209-Tg littermate mice. The percentage of hVβ8⁺CD8⁺ lymphocytes was approximately 0 in A2/Kb mice and 41 in JR209-Tg littermates. (C) Binding of HLA-A*0201–gp100_209–217 tetramer to the lymphocyte-gated population from spleens of A2/Kb (area under thick black line) and JR209-Tg (gray area) mice. M1 (tetramer positive gate) represented 42% of the gated population. (D) HLA-A*0201 expression on lymphocyte-gated population from splenocytes of C57BL/6 (gray area), A2/Kb (area under gray line), and JR209-Tg (area under thick black line) mice.

Figure 2

T cells from JR209-Tg mice are functional. (A) Expression of CD62L, CD44, CD69, and CD25 on freshly isolated (gray area) and ex vivo peptide-stimulated JR209-Tg (area under thick black line) CD8⁺ T cells. The florescence intensities of cells labeled with isotype Ab’s were less than 10² (not shown). (B) IFN-γ release in 24-hour coculture of 1 × 10⁵ naive (filled squares) or gp100_209–217 peptide–activated (open squares) CD8⁺ JR209-Tg T cells and 1 × 10⁵ A2/Kb splenocytes pulsed with titrated gp100_209–217 peptides. Data represented the mean of duplicate testing samples. (C) IFN-γ release in 24-hour coculture of 1 × 10⁵ freshly isolated (gray bars) or ex vivo peptide-stimulated (white bars) CD8⁺ JR209-Tg T cells and 1 × 10⁵ A2/Kb splenocytes pulsed with 1 μM of gp100_{209–217(2M)}, gp100_209–217, and gp100_154–162 (irrelevant control) peptide. (D) IFN-γ release in 24-hour coculture of 1 μM gp100_209–217 peptide–pulsed A2/Kb splenocytes and activated JR209-Tg T cells and their blockade by anti–HLA-A2 and anti-mCD8 mAb’s. Data represent the mean of duplicate testing samples.

Figure 3

Native gp100_209–217 peptide fails to activate JR209-Tg T cells in both gp100_209–217^WT and gp100_209–217^KO mice. (A) Ex vivo antigen-specific proliferative responses of freshly isolated splenocytes from JR209-Tg mice with and without gp100_209–217 epitope expression. CFSE-labeled splenocytes were cultured in media containing 1 μM of gp100_209–217 or gp100154–162 peptide for 48 hours before FACS. The dot plots represent 10,000 total events in each sample. (B) gp100_209–217 peptide–specific IFN-γ release in cells from draining lymph nodes (pooled from two mice in each group) after various doses of gp100_{209–217(2M)} and gp100_209–217 peptide immunization in JR209-Tg mice with (white bars) and without (black bars) the epitope expression. Draining lymph nodes were collected 7 days after immunization. One micromole of gp100_209–217 peptide was added to 1 × 10⁵ cells in 200 μl of culture media and incubated for 24 hours. IFN-γ concentrations in the supernatant were determined by ELISA. (C) In vivo antigen-specific proliferative responses of adoptively transferred freshly isolated splenocytes from JR209-Tg mice with and without gp100_209–217 epitope expression. CFSE-labeled splenocytes (1 × 10⁷) from JR209-Tg–gp100_209–217^WT or JR209-Tg–gp100_209–217^KO mice were intravenously injected into A2/Kb recipient mice, followed by immunization (into the footpad) with 100 μg of gp100_{209–217(2M)}, gp100_209–217 peptide, or PBS (in IFA). Four days after immunization, the cells of draining lymph nodes were pooled from two mice in each group and gated on hVβ8⁺CD8⁺ T cells for FACS analysis.

Figure 4

Native gp100_209–217–MHC-I complex is metastable. (A and B) Determination of peptide dissociation rates from target cell surface. Calculated amounts of gp100_209–217 (filled squares) and gp100_{209–217(2M)} (open squares) peptide bound to HLA-A2 molecules were plotted over time. Apparent k_off values were determined by fitting to a single exponential decay of all points above an undetectable concentration of peptide (1 × 10^–10 M using human T cell clone for A and 1 × 10^–9 M using JR209-Tg T cells for B). t_1/2 was determined from the relationship t_1/2 = 0.693/k_off. (C) Direct assay of peptide dissociation from purified peptide-MHC complexes using fluorescence anisotropy. Dissociation rates and t_1/2of gp100_209–217and gp100_{209–217(2M)} peptides are indicated. Experiments were repeated in triplicate; experimental errors for the reported parameters were 3% for gp100_209–217 and 7% for gp100_{209–217(2M)}. mA, millianisotropy.

Figure 5

Stable peptide–MHC-I complex is required for in vivo induction of antitumor responses in JR209-Tg mice. (A) Tumor-specific IFN-γ production by freshly isolated (gray bars) or ex vivo peptide-stimulated (white bars) CD8⁺ JR209-Tg T cells in 24-hour coculture with target cells from B16-A2/K^b, its parental B16 melanoma, and MC-38 murine adenocarcinoma. Data represent the mean of duplicate testing samples. (B) Treatment of B16-A2/K^b tumor in A2/Kb (open symbols) and JR209-Tg (filled symbols) mice by peptide immunization. One hundred micrograms of gp100_209–217 (circles), gp100_{209–217(2M)} (squares) peptides or PBS (triangles) in IFA was subcutaneously injected into mice 13 days after tumor inoculation. Five mice were in each group. *Significantly different (P < 0.05). **Not significantly different (P > 0.05). (C) Treatment of B16-A2/K^b tumor in A2/Kb transgenic mice receiving adoptive transfer of ex vivo immunized JR209-Tg splenocytes. Peptide-pulsed splenocytes were separated from JR209-Tg T cells for 24 hours before being cocultured overnight and then injected into tumor-bearing mice. In a control group, gp100_209–217 peptide–pulsed splenocytes were directly cocultured with JR209-Tg T cells overnight and injected into tumor-bearing mice. Seven mice were in each group.