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

Increasing Viral Dose Causes a Reversal in CD8(+) T Cell Immunodominance during Primary Influenza Infection due to Differences in Antigen Presentation, T Cell Avidity, and Precursor Numbers

Fulltext embargoed for: 12 months post date of publicationT cell responses are characterized by the phenomenon of immunodominance (ID), whereby peptide-specific T cells are elicited in a reproducible hierarchy of dominant and subdominant responses. However, the mechanisms that give rise to ID are not well understood. We investigated the effect of viral dose on primary CD8(+) T cell (T(CD8+)) ID by injecting mice i.p. with various doses of influenza A virus and assessing the primary T(CD8+) response to five dominant and subdominant peptides. Increasing viral dose enhanced the overall strength of the T(CD8+) response, and it altered the ID hierarchy: specifically, NP(366-374) T(CD8+) were dominant at low viral doses but were supplanted by PA(224-233) T(CD8+) at high doses. To understand the basis for this reversal, we mathematically modeled these T(CD8+) responses and used Bayesian statistics to obtain estimates for Ag presentation, T(CD8+) precursor numbers, and avidity. Interestingly, at low viral doses, Ag presentation most critically shaped ID hierarchy, enabling T(CD8+) specific to the more abundantly presented NP(366-374) to dominate. By comparison, at high viral doses, T(CD8+) avidity and precursor numbers appeared to be the major influences on ID hierarchy, resulting in PA(224-233) T(CD8+) usurping NP(366-374) cells as the result of higher avidity and precursor numbers. These results demonstrate that the nature of primary T(CD8+) responses to influenza A virus is highly influenced by Ag dose, which, in turn, determines the relative importance of Ag presentation, T(CD8+) avidity, and precursor numbers in shaping the ID hierarchy. These findings provide valuable insights for future T(CD8+)-based vaccination strategies

Influenza A virus infection-induced macroautophagy facilitates MHC class II-restricted endogenous presentation of an immunodominant viral epitope

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed

Identification and characterization of a novel NY-ESO-1 T_CD8+ epitope.

<p><i>A</i>. PBMCs were collected from patient 8 on day 70 following vaccination with NY-ESO-1 ISCOMATRIX™ vaccine. These cells were cultured with a panel of overlapping NY-ESO-1 18 mer peptides and then tested for responsiveness to each peptide in an ICS assay for IFN-γ. Because the background to control peptides was negligible, the results from individual cultures were plotted as a single combined figure. <i>B, C</i>, T_CD8+ line expanded with NY-ESO-1_79–96 18 mer was tested under FCS-free condition for its reactivity to various HPLC-purified peptides (B) and the minimum peptide NY-ESO-1_88–96 at various peptide concentrations (<i>C)</i>. <i>D</i>, a panel of LCL lines sharing HLA alleles with patient 8 were pulsed with the minimum NY-ESO-1_88–96 peptide, extensively washed, co-cultured with NY-ESO-1_88–96-specific T_CD8+ line and followed with ICS.</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

NY-ESO-1_88–96-specific T cells are vaccine boosted and utilize polyclonal T cell receptors.

<p>PBMCs from patient 8 collected before (day 0) and after (day 70) vaccination with NY-ESO-1 ISCOMATRIX™ vaccine were expanded with 18 mer NY-ESO-1_79–96 and the T cells were assessed by ICS (A). A similar T cell line expanded from day 70 PBMC sample from patient 8 was first stimulated with NY-ESO-1_88–96 peptide, then split into multiple wells and stained with anti-CD8 and a panel of antibodies specific to various TCR Vβ families separately, followed with ICS for IFN-γ (B). Graph indicates the percentage of NY-ESO-1_88–96-specific (IFN-γ-producing) T cells expressing the indicated TCR Vβ families.</p

NY-ESO-1_88–96 is not naturally presented by melanoma cells.

<p><i>A</i>, NY-ESO-1_157–165– and NY-ESO-1_88–96–specific T_CD8+ lines were expanded from PBMCs collected from the previously reported patient 7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044707#pone.0044707-Davis1" target="_blank">[6]</a> and patient 8 with 18 mer peptides NY-ESO-1_157–174 and NY-ESO-1_79–96 respectively. These T cells were then co-incubated with tumor line (SK-MEL-8) with or without a 48 hr IFN-γ induction (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044707#s4" target="_blank">Materials and Methods</a></b> for details). The untreated SK-MEL-8 cells were also pulsed with both peptides followed by washing out excessive peptides to serve as a maximum antigen presentation control. Antigen-specific T cell activation was then revealed by tetramer and IFN-γ double staining. Percentage represents antigen-specific, IFN-γ-producing cells amongst total tetramer positive cells (note, the double negative cell population was not included in the percentage calculation). <i>B</i>, the same T_CD8+ lines used in A were also assessed for their affinity by peptide titration. Percentage represents Ag-specific T cells among total CD8⁺ T cells. Similar data were obtained from three similar experiments.</p

T_CD8+ response to HLA-B18/NY-ESO-1_88–96.

<p>Melanoma patients from three clinical trials (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044707#s4" target="_blank">Materials and Methods</a>) with detectable anti-NY-ESO-1 antibody responses and HLA-B18 expression were selected for the screen. T cells from PBMC samples post vaccination (or placebo controls that did not receive the NY-ESO-1 ISCOMATRIX™ vaccine but received diluents) were expanded with 18 mer NY-ESO-1_79–96 peptide for 12∼15 days and assessed with NY-ESO-1_88–96 in an ICS assay (only ICS results <0.1% are shown as negative “–” indicated by ‘*’). For patients who showed positive T_CD8+ response to this epitope (>0.1%, data not shown) in their post vaccination samples, pre- and post-vaccination PBMC samples were then expanded the same way side-by-side in a second screen intended to determine whether the response was a result of the vaccination. The peptide-specific T_CD8+ in the second screen were assessed with the specific HLA-B18/NY-ESO-1_88–96 tetramer. Tetramer results >0.1% of total CD8⁺ T cells with a discrete staining pattern are shown; and those results <0.1% are shown as “-”. Pre – pre-existed response; Boosted – vaccine-boosted response; ND – not determined, Pre-vac, prior to vaccination; Post-vac, after vaccination.</p

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