Nonclassical antigen-processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4(+) T cells

Abstract

Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.

Figure 1

Characterization of NY-ESO-1-specific tumor-recognizing (TR-CD4) and non-tumor-recognizing (NTR-CD4) CD4⁺ T cell clones. (A) Recognition of DP04⁺NY-ESO-1^+/− melanoma lines was tested by ELISPOT assays. (B) DP04⁺ melanoma cells were pulsed overnight with NY-ESO-1_157–170 peptide and recognition by T cells was evaluated by intracellular IFN-γ staining. (C) HLA-restriction of SK37-recognition was determined using blocking antibodies by intracellular IFN-γ staining. A02-restricted NY-ESO-1_157–165-specific CD8⁺ T cell clone (ESO-CD8) was used as control tumor-recognizing T cells. (D) Antigen-specificity of tumor recognition. NY-ESO-1 (ESO), pan-MAGE (MAGE) or GFP-specific siRNA was electroporated into SK37. Recognition was evaluated by IFN-γ-ELISPOT assays. (E) Recognition of NY-ESO-1_157–170 (Peptide), NY-ESO-1 protein (Protein) and adenovirally-induced NY-ESO-1 (Adeno) in SK29 was tested by IFN-γ ELISPOT assays. (F) Recognition of mRNA-induced intracellular NY-ESO-1. HLA-DP04⁺ EBV-transformed B cells were electroporated with ESO or GFP mRNA. Reactivity of TR-CD4 was measured by IFN-γ-ELISPOT assays. Statistical significance was calculated by Student’s t-test and is shown as *:P ≤ 0.05; **:P ≤ 0.01; and *** P ≤ 0.001.

Figure 2

Determination of minimum epitopes for TR-CD4 and NTR-CD4. (A-C) Dose-dependence of NY-ESO-1 peptide recognition by TR-CD4 and NTR-CD4. Indicated peptides were pulsed overnight on SK29 at indicated concentrations. Recognition by TR-CD4 and NTR-CD4 was evaluated by IFN-γ ELISPOT assays. (D) Dose-dependence of NY-ESO-1 protein recognition. NY-ESO-1 protein was pulsed on DP04⁺ immature monocyte-derived DCs overnight at indicated concentrations. TR-CD4 and NTR-CD4 were stimulated with these DCs for 24 hours. IFN-γ production in the supernatant was measured by ELISA. (E) Recognition of overlapping NY-ESO-1 peptide-pulsed SK29 was evaluated by intracellular IFN-γ-staining. (F) Dose-dependence of NY-ESO-1_161–169 recognition. NY-ESO-1_161–169 was pulsed on SK29 at indicated concentrations. Recognition was evaluated by intracellular IFN-γ staining. All experiments were repeated at least twice with consistent results. Error bars indicate s.d. from duplicated wells.

Figure 3

Detection of TR-CD4 at the local tumor site. (A) Tumor-infiltrating lymphocytes from an ovarian cancer patient were stimulated with NY-ESO-1_157–170. After 20-days, NY-ESO-1_157–170-reactive CD4⁺ T cells were isolated and expanded. Recognition of SK37, NY-ESO-1_157–170, and NY-ESO-1_161–169 was tested by intracellular staining. Values in quadrants indicate percentages of cells. (B) TR-CD4 or NTR-CD4 cells were co-cultured with NY-ESO-1^+/− tumor single cell suspensions (TSC) obtained from DP04⁺ patients for 24 hours. IFN-γ level in the supernatant was measured by ELISA.

Figure 4

Effect of inhibitors for antigen degradation on the recognition of SK37 by TR-CD4. (A, B) SK37 was cultured for 16–20 hours with or without indicated proteasome inhibitors (A) or protease inhibitors (B) followed by fixing with paraformaldehyde and extensive washes. SK37 was co-cultured for 20 hours with TR-CD4. IFN-γ level in the supernatant was measured by ELISA. Results are shown as % inhibition of IFN-γ production compared to untreated target cells. Statistical significance was calculated by Student’s t-test and is shown as *:P ≤ 0.05 and **:P ≤ 0.01.

Figure 5

Effect of inhibitors for MHC-I antigen-processing pathways on the recognition of SK37 by TR-CD4. (A) SK37 was treated by the indicated inhibitors for 16–20 hours followed by fixing with paraformaldehyde and extensive washes. SK37 was co-cultured for 20 hours with TR-CD4. IFN-γ level in the supernatant was measured by ELISA. (B) Role of TAP in the presentation. Untreated parental SK37 and SK37 clones stably expressing ICP47 gene were used as stimulator cells. GM-CSF level in the supernatant was measured by ELISA. Statistical significance was calculated by Student’s t-test and is shown as *:P ≤ 0.05; **:P ≤ 0.01; and *** P ≤ 0.001.

Figure 6

Effect of inhibitors for previously characterized endogenous MHC-II presentation pathways on the recognition of SK37 by TR-CD4. SK37 was treated by the indicated inhibitors for 40–44 hours (A, C) or 16–20 hours (D) followed by fixing with paraformaldehyde and extensive washes. SK37 was co-cultured for 20 hours with TR-CD4. GM-CSF level in the supernatant was measured by ELISA. (A) Effect of an inhibitor for macroautophagy. (B) Effect of siRNA-mediated silencing of LAMP2. SK37 was electroporated with indicated siRNA and cultured for 3 days. (C) Effect of an endosomal/lysosomal recycling inhibitor. (D) Effect of vesicular transport and protein synthesis inhibitors. All experiments were repeated at least three times with consistent results. Error bars indicate s.d. from duplicated wells. Statistical significance was calculated by Student’s t-test and is shown as *:P ≤ 0.05 and **:P ≤ 0.01.