Eric Tran

STAT5 Is Essential for Akt/p70S6 Kinase Activity during IL-2-Induced Lymphocyte Proliferation

IL-2R activates two distinct signaling pathways mediated by the adaptor protein Shc and the transcription factor STAT5. Prior mutagenesis studies of the IL-2R have indicated that the Shc and STAT5 pathways are redundant in the ability to induce lymphocyte proliferation. Yet paradoxically, T cells from STAT5-deficient mice fail to proliferate in response to IL-2, suggesting that the Shc pathway is unable to promote mitogenesis in the genetic absence of STAT5. Here we show in the murine lymphocyte cell line Ba/F3 that low levels of STAT5 activity are essential for Shc signaling. In the absence of STAT5 activity, Shc was unable to sustain activation of the Akt/p70S6 kinase pathway or promote lymphocyte proliferation and viability. Restoring STAT5 activity via a heterologous receptor rescued Shc-induced Akt/p70S6 kinase activity and cell proliferation with kinetics consistent with a transcriptional mechanism. Thus, STAT5 appears to regulate the expression of one or more unidentified components of the Akt pathway. Our results not only explain the severe proliferative defect in STAT5-deficient T cells but also provide mechanistic insight into the oncogenic properties of STAT5 in various leukemias and lymphomas.

Tumor-Infiltrating T Cells Correlate with NY-ESO-1-Specific Autoantibodies in Ovarian Cancer

Background Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens. Methodology and Principal Findings We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-γ ELISPOT and MHC class I pentamer staining. Conclusion and Significance We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy.

Profound elevation of CD8+ T cells expressing the intraepithelial lymphocyte marker CD103 (αE/β7 Integrin) in high-grade serous ovarian cancer

INTRODUCTION Tumor-infiltrating CD8(+) T cells are strongly associated with survival in high-grade serous ovarian cancer, but their functional phenotype remains poorly defined. The mucosal integrin CD103 (alpha(E)/beta(7)) facilitates the infiltration of T cells into epithelial tissues, including gut and lung mucosa, solid organ allografts, and various epithelial cancers. We reasoned that CD103 might also be expressed by tumor-reactive T cells in ovarian cancer. METHODS Flow cytometry was used to assess the frequency and phenotype of CD103-expressing T cells in primary ascites fluid from 13 patients with high-grade serous ovarian cancer and 2 patients with recurrent disease. RESULTS We report that a subset of patients with advanced serous ovarian cancer have profoundly elevated frequencies of CD103-expressing CD8(+) cells in ascites (between 20% and 70% of CD8(+) cells in ascites were CD103(+)) and that CD103 expression correlated with levels of TGF-beta in ascitic fluid. Conversely, CD103 was not expressed on CD4(+) cells, even in those patients with very high frequencies of CD8(+)CD103(+) cells. CD8(+)CD103(+) cells were antigen-experienced (CD45RA(-)CD45RO(+)CD62L(lo)CCR7(-)) and of an intermediate (EM2) effector memory phenotype (CD27(+)CD28(-)). TCR repertoire analysis indicated significant skewing between CD8(+)CD103(-) and CD8(+)CD103(+) T cell subsets, suggesting the two populations contain distinct antigenic specificities. Lastly, HLA pentamer analysis revealed that one patient in the cohort harbored a high frequency of CD8(+) T cells in ascites that were specific for the tumor antigen NY-ESO-1, and that approximately 75% of these NY-ESO-1 specific CD8(+) T cells were CD103(+). CONCLUSIONS CD103(+) may be a marker of activated and tumor-reactive CD8(+) T cells in high-grade serous ovarian cancer.

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines.

Background Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment. Methodology/Principal Findings Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited. Conclusions/Significance The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.

An in vitro-transcribed-mRNA polyepitope construct encoding 32 distinct HLA class I-restricted epitopes from CMV, EBV, and Influenza for use as a functional control in human immune monitoring studies

Interest and activity in the areas of clinical immunotherapy and therapeutic vaccines are growing dramatically, thus there is a pressing need to develop robust tools for assessment of vaccine-induced immunity. CD8+ T cell immunity against specific antigens is normally measured by either flow cytometry using MHC tetramer reagents or via biological assays such as intracellular cytokine staining or ELISPOT after stimulation with specific peptide epitopes. However, these methodologies depend on precise knowledge of HLA-restricted epitopes combined with HLA typing of subjects. As an alternative approach, electroporation of antigen presenting cells (APC) with in vitro-transcribed mRNA (IVT-mRNA) encoding the antigen of interest bypasses the requirements for HLA typing and knowledge of specific epitopes. A current limitation of the IVT-mRNA technique is the lack of robust positive control RNAs to verify the efficacy of electroporation and to ensure that the electroporated APC retain the ability to stimulate T cells. Herein we describe an IVT-mRNA construct wherein all 32 HLA class I-restricted epitopes of the widely used CEF (Cytomegalovirus, Epstein-Barr Virus and Influenza Virus) positive control peptide pool have been genetically spliced together to generate a single polyepitope construct. Each epitope is flanked by three amino- and three carboxy-terminal amino acids from the original parent protein to facilitate proteolytic processing by the proteasome. Using cells obtained from a panel of normal healthy donors and cancer patients we report that dendritic cells, CD40-activated B cells, PHA blasts, and even tumor cells can be transfected with CEF polyepitope IVT-mRNA and can elicit robust CEF-specific responses from autologous T cells, as measured by IFN-gamma ELISPOT. Moreover, the response elicited by CEF IVT-mRNA-transfected APC was similar in magnitude to the response elicited by the complete pool of CEF minimal peptide epitopes, implying that the polyepitope parent protein encoded by the CEF mRNA was efficiently processed into individual epitopes by the proteolytic machinery of the APC. In summary, the CEF polyepitope IVT-mRNA described herein comprises a robust positive control for immunomonitoring studies requiring IVT-mRNA transfection and potentially provides a unique tool for assessing MHC class I processing regardless of HLA haplotype.

Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of neoantigen-reactive TILs could be enhanced by enriching T cells that express PD-1 and/or T cell activation markers followed by microwell culturing to avoid overgrowth of nonreactive T cells. In 6 patients with metastatic epithelial cancer, this method led to the detection of CD4+ and CD8+ T cells targeting 18 and 1 neoantigens, respectively, compared with 6 and 2 neoantigens recognized by CD4+ and CD8+ T cells, respectively, when using our standard TIL fragment screening approach. In 2 patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of 5 neoantigen-reactive T cell receptors (TCRs) against 5 different mutations from one patient and a highly potent MHC class II-restricted KRASG12V-reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated 3 MHC class II-restricted TCRs targeting the TP53G245S hot-spot mutation. In conclusion, this approach provides a highly sensitive platform to isolate clinically relevant neoantigen-reactive T cells or their TCRs for cancer treatment.

391. Mutated Tumor Neoantigens Are Recognized by Tumor Infiltrating Lymphocytes from Metastatic Ovarian Cancer

Somatic mutations expressed by the tumor can serve as neoantigens for autologous T cells. Tumor infiltrating lymphocytes (TIL) with varying degrees of neoantigen-reactivity infused for the treatment of melanoma resulted in 50% overall objective response and 20% complete response rates. A largely oligoclonal population of ERBB2IPE805G mutated neoantigen-specific T cells resulted in a long-term, ongoing partial regression of metastatic cholangiocarcinoma suggesting that infusion of selected mutation-reactive TIL could be efficacious in the treatment of common epithelial cancers. We now studied whether TIL obtained from metastatic ovarian cancer recognized tumor mutations. Exome and transcriptome sequencing was performed from resected metastatic ovarian cancer deposits in parallel with growth of TIL fragment cultures in interleukin-2. Long peptides and tandem minigenes encompassing all mutations were synthesized, introduced into autologous antigen presenting cells, co-cultured with individual TIL fragments and T-cell reactivity was determined by interferon-y ELISPOT and surface expression of 41BB. In the 8 ovarian tumors evaluated, there was a median of 227 mutations (range: 63-332) and an average of 94% of 24 fragments initiated (range: 58% - 100%) available for testing. Six of eight (75%) patients had T cell responses to mutated neoantigens at >5% of the fragment culture. Antigens identified to date were unique to each patient, i.e. no overlapping mutations between patients, and both CD4 and CD8 responses have been detected. One patient had a CD4 T cell response to p53G245S hotspot mutation, which opens opportunities for treatment of other cancer patients with TCR-transduced T cells because this mutation is present in 2.8% of all cancers. The average time from resection to identification of mutation-reactive T cell fragment culture was 8 weeks indicating that this strategy could be used for prospective therapy. In summary, mutation-specific T-cell responses were found in 6 of 8 patients with metastatic ovarian cancer, which opens the opportunity to use these cells for adoptive T cell treatment of advanced ovarian cancer.