BackgroundDespite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear.MethodsTo better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+and CD4+EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+and CD4+TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+and CD4+tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival.ResultsWe found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1−and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+and PD-1hiCD39+T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hicompared with PD-1−or PD-1dimT cells, and in fact, it was the CD4+PD-1hisubpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hitumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+TILs and PD-1hiand CXCL13 by CD4+Tconv TILs, correlated with prolonged survival of patients with EC.ConclusionsOur results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hiand CD39 or PD-1hican be used to select and expand CD8+and CD4+tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+and CD4+lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity.
Purpose: Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability. Experimental Design: Peptides presented on HLA-I were identified from 9 patient-derived TCL with melanoma, gynecological, and head and neck cancer through proteogenomics. 507 candidate tumor antigens including nonC-TL, neoantigens, cancer-germline, or melanocyte differentiation antigens were tested for T-cell recognition of pre-existing responses in cancer patients. Donor peripheral blood lymphocytes (PBL) were in vitro sensitized against 170 selected nonC-TL to isolate antigen-specific T-cell receptors (TCRs) and evaluate their therapeutic potential. Results: We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor-reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor PBL against 170 selected nonC-TL, led to the identification of TCRs specific to three nonC-TL, two of which mapped to the 5’ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells. Conclusions: Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies.
T-cell receptor β analysis can be exploited to monitor changes in the clonotypic composition of tumor-infiltrating lymphocyte (TIL) during ex vivo expansion in IL2. Oligoclonal TILs are often outgrown by infrequent clonotypes with greater proliferative capacity, and this could impact the antitumor reactivity of the expanded TILs. See related article by Poschke et al., p. 4289
Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability. Using proteogenomics, we identified 517 nonC-TL from 9 patients with melanoma, gynecological, and head and neck cancer. We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor peripheral blood lymphocytes against 170 selected nonC-TL, led to the identification of T-cell receptors (TCRs) specific to three nonC-TL, two of which mapped to the UTR5 regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies.
<p>Tumor cell line HLA-I typing and tumor type</p>
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