Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103+CD39+ tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103+CD39+ CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103+CD39+ CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103+CD39+ CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies.

The tumor microenvironment of squamous cell carcinoma of the head and neck (SCCHN) has been shown to be immune suppressive. Therefore, strategies aimed at overcoming this issue could have a positive therapeutic impact. Hence, we investigated the expression of the known immune-modulatory proteins OX40, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in SCCHN on different T-cell subsets of tumor-infiltrating lymphocytes (TIL) to ascertain whether these proteins could potentially be targeted alone or in combination for future clinical trials. T cells from peripheral blood (PBL) and tumor were analyzed for the expression of OX40, PD-1 and CTLA-4 in 29 patients undergoing surgery. These proteins were all expressed significantly higher in T-cell subsets isolated from tumors compared with PBL of the same patient. OX40 expression was significantly greater in the TIL regulatory T-cell (Treg) population relative to conventional CD4 and CD8 TIL or the Treg isolated from PBL. PD-1 expression was increased in all T-cell subsets relative to PBL. CTLA-4 was also increased in all TIL subsets relative to blood, and similar to OX40, its highest level of expression was observed in the Treg TIL. The highest frequency of PD-1, CTLA-4 and OX40 triple-positive cells were found in the Treg population isolated from the tumor. We analyzed both human papilloma virus-positive and -negative patients and found similar levels and expression patterns of these two patient populations for all three proteins. These data suggest that there may be therapeutic advantages of targeting these pathways independently or in combination for patients with this disease.

Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared. This comparison revealed a potential role for IL-12 through increased expression of the IL-12R-signaling subunit (IL-12Rβ2) on T cells activated 3 days previously with Ag and anti-OX40. The temporal expression of IL-12Rβ2 on OX40-stimulated CD4 T cells was tightly regulated and peaked ∼4–6 days after initial activation/expansion, but before the beginning of T cell contraction. IL-12 signaling, during this window of IL-12Rβ2 expression, was required for enhanced T cell survival and survival was associated with STAT4-specific signaling. The findings from these observations were exploited in several different mouse tumor models where we found that the combination of anti-OX40 and IL-12 showed synergistic therapeutic efficacy. These results may lead to the elucidation of the molecular pathways involved with CD4 T cell survival that contribute to improved memory, and understanding of these pathways could lead to greater efficacy of immune stimulatory Abs in tumor-bearing individuals.

OX40 (CD134) is a potent costimulatory molecule found on the surface of activated CD4 + and CD8 + T cells. Immunotherapy with OX40 agonists administered in vivo has demonstrated efficacy in several murine tumor models. A phase I clinical trial is currently underway in patients with advanced cancer using a mouse anti-CD134 monoclonal antibody. Therapy with this antibody will likely be limited to one cycle because patients develop neutralizing human anti-mouse antibody (HAMA). Therefore, we developed a humanized OX40 agonist that links the extracellular domain of human OX40L to the Fc domain of human IgG 1 via a trimerizing isoleucine zipper domain (ILZ). Physical characterization by velocity sedimentation revealed that this novel construct, hFcILZOX40L, was assembled into hexamers in which the Fc domains formed three disulfidebonded dimers and the ILZ-OX40L domains formed two trimers. Trimerization of the ILZ domain was necessary to achieve appropriate assembly. In vitro biologic activity of the hFcILZOX40L hexamer was equivalent to the activity of agonist antibodies in plate-bound assays and was superior when the agonists were tested as soluble agents. Our ultimate goal is to use this recombinant molecule in a future clinical trial and we feel that the OX40L hexamer will have equivalent or superior agonist activity in vivo when compared to an anti-OX40 antibody.

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BackgroundWe examined the phenotype and function of lymphocytes collected from the peripheral blood (PBL) and tumor (TIL) of patients with two different solid malignancies: colorectal cancer liver metastases (CRLM) and ovarian cancer (OVC).MethodsTumor and corresponding peripheral blood were collected from 16 CRLM and 22 OVC patients; immediately following resection they were processed and analyzed using a multi-color flow cytometry panel. Cytokine mRNA from purified PBL and TIL CD4+ T cells were also analyzed by qPCR.ResultsOverall, we found similar changes in the phenotypic and cytokine profiles when the TIL were compared to PBL from patients with two different malignancies. The percentage of Treg (CD4+/CD25+/FoxP3+) in PBL and TIL was similar: 8.1% versus 10.2%, respectively in CRLM patients. However, the frequency of Treg in primary OVC TIL was higher than PBL: 19.2% versus 4.5% (p <0.0001). A subpopulation of Treg expressing HLA-DR was markedly increased in TIL compared to PBL in both tumor types, CRLM: 69.0% versus 31.7% (p = 0.0002) and OVC 74.6% versus 37.0% (p <0.0001), which suggested preferential Treg activation within the tumor. The cytokine mRNA profile showed that IL-6, a cytokine known for its immunosuppressive properties through STAT3 upregulation, was increased in TIL samples in patients with OVC and CRLM. Both TIL populations also contained a significantly higher proportion of activated CD8+ T cells (HLA-DR+/CD38+) compared to PBL (CRLM: 30.2% vs 7.7%, (p = 0.0012), OVC: 57.1% vs 12.0%, (p <0.0001)).ConclusionThis study demonstrates that multi-color flow cytometry of freshly digested tumor samples reveals phenotypic differences in TIL vs PBL T cell sub-populations. The TIL composition in primary and metastatic tumors from two distinct histologies were remarkably similar, showing a greater proportion of activated/suppressive Treg (HLA-DR+, CD39+, CTLA-4+ and Helios+) and activated cytotoxic T cells (CD8+/HLA-DR+/CD38+) when compared to PBL and an increase in IL-6 mRNA from CD4 TIL.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-014-0038-9) contains supplementary material, which is available to authorized users.

The ability of memory CD8+ T cells to rapidly proliferate and acquire cytolytic activity is critical for protective immunity against intracellular pathogens. The signals that control this recall response remain unclear. We show that CD40L production by memory CD8+ T cells themselves is an essential catalyst for secondary expansion when systemic inflammation is limited. Secondary immunization accompanied by high levels of systemic inflammation results in CD8+ T cell secondary expansion independent of CD4+ T cells and CD40-CD40L signaling. Conversely, when the inflammatory response is limited, memory CD8+ T cell secondary expansion requires CD40L-producing cells, and memory CD8+ T cells can provide this signal. These results demonstrate that vaccination regimens differ in their dependence on CD40L-expressing CD8+ T cells for secondary expansion, and propose that CD40L-expression by CD8+ T cells is a fail-safe mechanism that can promote memory CD8+ T cell secondary expansion when inflammation is limited.

BackgroundThere has been a dramatic increase in T cell receptor (TCR) sequencing spurred, in part, by the widespread adoption of this technology across academic medical centers and by the rapid commercialization of TCR sequencing. While the raw TCR sequencing data has increased, there has been little in the way of approaches to parse the data in a biologically meaningful fashion. The ability to parse this new type of 'big data' quickly and efficiently to understand the T cell repertoire in a structurally relevant manner has the potential to open the way to new discoveries about how the immune system is able to respond to insults such as cancer and infectious diseases.