Processing of tumor antigen differentially impacts the development of helper and effector CD4+ T-cell responses

Abstract

CD4(+) T cells contribute to the antitumor T-cell response as both effectors that promote tumor rejection and helpers that facilitate the activation of other antitumor effector cells, such as CD8(+) T cells. Maximal engagement of both effector and helper CD4(+) T-cell responses is a desirable attribute of cancer vaccines. We have employed the B16F10 murine melanoma model and a series of recombinant adenovirus (Ad) vaccines expressing mutant forms of the tumor antigen, dopachrome tautomerase, to investigate the relationship between antigen processing and the antitumor CD4(+) T-cell response. Our results have revealed an unexpected dichotomy in the generation of helper and effector CD4(+) T-cell responses where CD4(+) T effector responses are dependent upon protein processing and trafficking, whereas CD4(+) T helper responses are not. The results have important implications for strategies aimed at augmenting antigen immunogenicity by altering intracellular processing and localization.

Figure 1

Immunization with AdhDCTΔVYD abrogates CD4⁺ T cell–mediated tumor protection while T-cell responses to the known helper CD4 epitopes are maintained. (a) Schematic representation of the hDCTΔVYD variant (SS, endoplasmic reticulum signal sequence; TM, transmembrane domain; MTS, melanosomal transport signal). (b,c) T-cell responses to the known (b) CD8 epitopes (hDCT_180–188 and hDCT_342–351) and (c) CD4 epitopes (hDCT_89–101 and hDCT_242–254) were assessed via intracellular cytokine staining. Results are shown as mean ± SEM from 10 mice pooled from two separate experiments and represent the absolute number of antigen-specific cells per spleen (*P < 0.05 and **P < 0.0005 compared to AdhDCT; NS, not significant). (d–f) Mice (n = 10) were immunized intramuscularly with 10⁸ plaque-forming units of AdControl, AdhDCT, or AdhDCTΔVYD. (d) Wild-type mice, (e) CD8⁺ T cell–depleted wild-type mice, and (f) CD8-deficient mice were challenged subcutaneously with 2 × 10⁴ B16F10 cells 7 days after immunization. Depletion of CD8⁺ T cells was performed by intraperitoneal injection of purified 2.43 antibodies starting on 3 days and 1 day prior to tumor challenge and subsequently administered once a week until tumors developed. A rat IgG was used as control treatment.

Figure 2

Immunization with hDCT variants lacking mature N-linked glycosylation results in impaired CD4⁺ T cell–mediated tumor protection while T-cell responses to the known helper CD4 epitopes are maintained. (a) Western blot analysis reveals lack of mature N-linked glycosylation in hDCTΔVYD and hDCT-N178D protein variants. 293T cells were transfected for 24 hours with plasmid DNA expressing hDCT, hDCTΔVYD, or hDCT-N178D. Lysates were prepared in RIPA lysis buffer, and equal amounts of protein (~20 µg) were treated with the glycosidase enzymes EndoH_f or PNGaseF for 24 hours at 37 °C. The entire reaction was diluted in 2× gel loading buffer and resolved by 10% SDS-PAGE. DCT was probed for using α-TRP-2 (C-9) and developed by ECL, followed by reprobing with α-GAPDH as a loading control. (b) Schematic representation of the hDCT-N178D variant (SS, endoplasmic reticulum signal sequence; TM, transmembrane domain; MTS, melanosomal transport signal). (c,d) T-cell responses to the known (c) CD4 epitopes (hDCT_89–101 and hDCT_242–254) and (d) CD8 epitopes (hDCT_180–188 and hDCT_342–351) were assessed via intracellular cytokine staining. Results are shown as mean ± SEM from 10 mice pooled from two separate experiments and represent the absolute number of antigen-specific cells per spleen (NS, not significant). (e) CD8-deficient mice (n = 10–15) were immunized intramuscularly with 10⁸ plaque-forming units of AdControl, AdhDCT, or AdhDCT-N178D. Mice were challenged subcutaneously with 2 × 10⁴ B16F10 cells 7 days after immunization.

Figure 3

Immunization with AdhDCTΔSS, which is glycosylation-defective, but not AdhDCTΔMTS, abrogates CD4⁺ T cell–mediated tumor protection while T-cell responses to the known helper CD4 epitopes are maintained. (a) Schematic representation of the hDCTΔSS and AdhDCTΔMTS variants (SS, endoplasmic reticulum signal sequence; TM, transmembrane domain; MTS, melanosomal transport signal). (b) Western blot analysis reveals lack of mature N-linked glycosylation in hDCTΔSS variant, but not AdhDCTΔMTS variant. 293T cells were transfected with plasmid DNA expressing hDCT, hDCTΔSS, hDCTΔMTS, or GFP for 24 hours. Lysates were prepared in RIPA lysis buffer and equal amounts of protein (~20 µg) were treated with the glycosidase enzymes EndoH_f or PNGaseF for 24 hours at 37 °C. The entire reaction was diluted in 2× gel loading buffer and resolved by 10% SDS-PAGE. DCT was probed for using α-TRP-2 (C-9) and developed by ECL, followed by reprobing with α-GAPDH as a loading control. (c,d) T-cell responses to the known (c) CD4 epitopes (hDCT_89–101 and hDCT_242–254) and (d) CD8 epitopes (hDCT_180–188 and hDCT_342–351) were assessed via intracellular cytokine staining. Results are shown as mean ± SEM from 10 mice pooled from two separate experiments and represent the absolute number of antigen-specific cells per spleen (*P < 0.05 compared to AdhDCT; NS, not significant). (e) CD8-deficient mice (n = 10–15) were immunized intramuscularly with 10⁸ plaque-forming units of AdControl, AdhDCT, AdhDCTΔSS, or AdhDCTΔMTS. Mice were challenged subcutaneously with 2 × 10⁴ B16F10 cells 7 days after immunization.

Figure 4

Immunization of DCT-deficient mice with AdhDCTΔVYD abrogates CD4⁺ T cell–mediated tumor protection while T-cell responses to the known helper CD4 epitopes are maintained. (a,b) T-cell responses to the known (a) CD4 epitopes (hDCT_89–101 and hDCT_242–254) and (b) CD8 epitopes (hDCT_180–188 and hDCT_342–351) were assessed via intracellular cytokine staining. Results are shown as mean ± SEM from 10 mice pooled from two separate experiments and represent the absolute number of antigen-specific cells per spleen (*P < 0.05 and **P < 0.0005 compared to AdhDCT; NS, not significant). (c) DCT-deficient mice (n = 10–15) were immunized intramuscularly with 10⁸ plaque-forming units of AdControl, AdhDCT, or AdhDCTΔVYD. Mice were challenged subcutaneously with 2 × 10⁴ B16F10 cells 7 days after immunization. Depletion of CD8⁺ T cells was performed by intraperitoneal injection of purified 2.43 antibodies starting on 3 days and 1 day prior to tumor challenge and subsequently administered once a week until tumors developed. A rat IgG was used as control treatment.