A novel HLA-B18 restricted CD8+ T cell epitope is efficiently cross-presented by dendritic cells from soluble tumor antigen

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8<sup>+</sup>T cell epitope, NY-ESO-1<sub>88–96</sub> (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1<sub>157–165</sub> epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1<sub>88–96</sub> is much more efficiently cross-presented from the soluble form, than NY-ESO-1<sub>157–165</sub>. On the other hand, NY-ESO-1<sub>157–165</sub> is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A<sub>26–35</sub>; whereas NY-ESO-1<sub>88–96</sub> was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1<sub>88–96</sub> is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1<sub>88–96</sub> from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8<sup>+</sup>T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed

Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon

Cross-priming refers to the activation of naive CD8+ T cells by antigen-presenting cells that have acquired nominal antigens from another cell. The biological relevance of cross-priming of CD8+ T cells has recently been challenged (Zinkernagel, R. M., Eur. J. Immunol. 2002. 32: 2385-2392), on the basis that responses are weak or poorly quantitated, and the determinants recognized are undefined. Here we show that cross-priming is a robust process that elicits vigorous primary responses to multiple peptides in two well-defined systems. Our findings support the relevance of cross-priming in CD8+ T cell responses to viruses and tumor cells, and demonstrate that cross-priming elicits CD8+ T cells to determinants generated by the endogenous processing pathway

Characterization of antigen-specific CD8+ T lymphocyte responses in skin and peripheral blood following intradermal peptide vaccination

Immune responses to cancer vaccines are commonly tested by measuring cutaneous reactions to intradermal (i.d.) antigen. When well-characterized peptide epitopes are injected i.d., infiltrates of CD4+ and CD8+ T lymphocytes are frequently seen. In this study, we have further characterized T cells derived from vaccine-infiltrating lymphocyte (VIL) responses. We found that the infiltrates capable of producing IFN-gamma and cytolytic activity could recognize vaccine peptide, as well as antigen-positive melanoma cells. We studied antigen-specific T cell responses from VILs and peripheral blood in 10 patients who participated in a clinical trial. All patients received systemic Flt3 ligand (20 µg/kg/d) and i.d. peptides: Three NY-ESO-1 peptides, SLLMWITQCFL (157-167), SLLMWITQC (157-165), QLSLLMWIT (155-163); tyrosinase internal peptide YMDGTMSQV (368-376); Melan-A/MART-1 analogue peptide ELAGIGILTV (26-35, E27L substitution); and influenza matrix peptide GILGFVFTL (58-66). In 54 paired VIL and peripheral blood analyses, a good correlation was found between responses in skin and in blood. These cells could be rapidly expanded in a short-term assay and thus appear to be memory T cells. The demonstrated presence of antigen-specific T cells at vaccination sites validates this method of assessing the immune response to i.d. vaccines

NY-ESO-1_88–96 is directly presented by tumor cells expressing high level of immunoproteasome.

<p>Five melanoma lines, including SK-MEL-8, were left untreated or treated with either IFN-γ for 48 hrs or were further infected with rVV.NY-ESO-1 for 5 hrs. The tumor cells were then co-cultured with T cell lines specific for NY-ESO-1_157–174, NY-ESO-1_88–96 and Melan A26–35 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044707#pone-0044707-g003" target="_blank">Figure 3</a>. The purity of the T_CD8+ lines were 42%, 88% and 68% respectively (data not shown). The antigen presentation results are shown in A and the western blot results for LMP2, LMP7 and the loading control GAPDH for the corresponding tumor lines and the treatment conditions are shown in B. The FACS analysis results of the cell surface HLA molecules as Mean Channel Fluorescence intensity (MCF) are shown in C. The MCF values for HLA-A2 and B18 were about 100 for the FITC-conjugated secondary antibody alone; and those values for the All Class I group for the PE-conjugated secondary antibody alone were about 300. Similar data were obtained from three similar experiments.</p

NY-ESO-1_88–96 is cross-presented efficiently by DCs from soluble antigen.

<p>In A, MoDCs expressing both HLA-A2 and HLA-B18 were cultured for 7 days, and then incubated overnight under the indicated conditions before being co-cultured with the indicated T_CD8+ lines for 5 hrs in the presence of BFA. NY-ESO-1 specific T_CD8+ activation was assessed by tetramer and ICS. IFN-γ producing cells out of total antigen-specific (tetramer positive) T_CD8+ were converted to percentages of maximum activation induced by the respective minimum peptide (peptide activation of NY-ESO-1_157–174 T_CD8+ line and NY-ESO-1_79–96 T_CD8+ line were both 30% to 45% for all three experiments conducted, data not shown) and plotted as “% Maximum activation”. After data conversion, mean values and standard deviations were calculated from data obtained from three similar experiments. In B, one of the control experiments was shown for APCs that were either pulsed with the corresponding peptide or soluble NY-ESO-1 for one hour followed with BFA addition to demonstrate the nature of intracellular cross-presentation for both T_CD8+ epitopes without affecting extracellular peptide presentation. Similar results were obtained twice.</p