Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming

Abstract

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8(+) T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4(+) Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8(+) T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8(+) T cells, indicated that elicitation of NY-ESO-1-specific CD8(+) T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Serological responses. (A) Serological responses were assessed by ELISA at baseline and at the indicated study week after vaccination. (B) The isotype of vaccine-induced IgG (week 12, serum dilution of 1:100) was assessed by ELISA using isotype-specific antibodies. (C) Linear B cell epitopes were determined by using patients' immune sera and a panel of 30-aa-long peptides spanning the protein sequence.

Fig. 2.

Assessment of CD4+ and CD8+ T cell responses after in vitro stimulation. (A) The presence of specific CD4+ and CD8+ T cells in stimulated cultures from vaccinated patients was assessed by intracellular staining with anti-IFN-γ monoclonal Ab after stimulation in the absence or presence of the NY-ESO-1 peptide pool. Numbers in the upper right quadrant are the percentages of IFN-γ-producing cells in CD4+ or CD8+ T cells. As an example, data are shown for one responder patient. (B) Summary of the results obtained for all patients. Values correspond to the percentage of CD4+ or CD8+ T cells specifically producing IFN-γ in response to stimulation with the peptide pool. (C) Cultures were stimulated with single peptides in the NY-ESO-1 pool. The proportion of IFN-γ-producing cells was assessed by intracellular staining as above. Symbols are as given in the Fig. 1 key.

Fig. 3.

Ex vivo assessment of CD4+ and CD8+ T cell responses. (A) Dot plots gated on CD3+ cells are shown for a high-responder patient. Numbers in the upper right quadrant are the percentages of IFN-γ-secreting cells among CD4+ T cells (Upper) and CD8+ T cells (Lower). (B) Summary of the results obtained for all patients. Symbols are as given in the Fig. 1 key. For each sample, values obtained in the absence of the NY-ESO-1 peptide pool were subtracted. Dashed lines represent the mean values of baseline samples plus three times their standard deviation. The baseline sample from patient N4, who had detectable NY-ESO-1-specific CD8+ T cells before vaccination as shown in Fig. 2 and Table 1, was excluded from this calculation. Frequencies of IFN-γ-secreting cells above this value were considered significant. (C) Phenotype and cytokine production of vaccine-induced CD4+ and CD8+ T cells.

Fig. 4.

Recognition of NY-ESO-1 by vaccine-induced T cells and Ab-aided cross-presentation. (A) Vaccine-induced CD4+ and CD8+ T cell populations enriched by cytokine-secretion-guided sorting were assessed by intracellular IFN-γ staining after stimulation in the absence or presence of the NY-ESO-1 peptide pool. (B) Recognition of autologous mDCs by CD4+ and CD8+ T cells was assessed after incubation with serial dilutions of rNY-ESO-1 or after electroporation with a plasmid encoding NY-ESO-1. (C) The MHC class I allele restricting antigen recognition by CD8+ T cells was determined upon transfection of COS-7 cells with plasmids encoding the alleles of the patient. Recognition of endogenous NY-ESO-1 was assessed upon cotransfection of COS-7 cells with plasmids encoding NY-ESO-1 and HLA-B35 or by transfecting the tumor cell lines NA8-MEL (NY-ESO-1−) and HT1080 (NY-ESO-1+) with a plasmid encoding HLA-B35. (D) Cross-presentation, by autologous mDCs, of rNY-ESO-1 (10 μg/ml) or immune complexes formed either with a murine NY-ESO-1-specific monoclonal Ab (ES121, 10 μg/ml) or with pre- and postimmune serum at the indicated dilution to vaccine-induced CD8+ T cells. In B and C, antigen recognition was assessed by measurement of IFN-γ secretion in the culture supernatant.