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Unlike other histological types of epithelial ovarian carcinoma, clear cell ovarian carcinoma (CCOC) has poor response to therapy. In many other carcinomas, expression of the hypoxia-related enzyme Carbonic anhydrase IX (CAIX) by cancer cells is associated with poor prognosis, while the presence of CD8 + tumor-infiltrating lymphocytes (TIL) is positively prognostic. We employed [ 18 F]EF5-PET/CT imaging, transcriptome profiling, and spatially-resolved histological analysis to evaluate relationships between CAIX, CD8, and survival in CCOC. Tissue microarrays (TMAs) were evaluated for 218 cases in the Canadian COEUR study. Non-spatial relationships between CAIX and CD8 were investigated using Spearman rank correlation, negative binomial regression and gene set enrichment analysis. Spatial relationships at the cell level were investigated using the cross K-function. Survival analysis was used to assess the relationship of CAIX and CD8 with patient survival for 154 cases. CD8 + T cell infiltration positively predicted survival with estimated hazard ratio 0.974 (95% CI 0.950, 1000). The negative binomial regression analysis found a strong TMA effect (p-value < 0.0001). It also indicated a negative association between CD8 and CAIX overall (p-value = 0.0171) and in stroma (p-value = 0.0050) but not in tumor (p-value = 0.173). Examination of the spatial association between the locations of CD8 + T cells and CAIX cells found a significant amount of heterogeneity in the first TMA, while in the second TMA there was a clear signal indicating negative spatial association in stromal regions. These results suggest that hypoxia may contribute to immune exclusion, primarily mediated by effects in stroma., (© 2023. The Author(s).)

Macroautophagy/autophagy is a critical regulator of adaptive T cell immunity and homeostasis. However, the role of T cell autophagy in regulating antitumor immune responses is less clear. In a recent study, we showed that deletion of the essential autophagy genes Atg5, Atg14 , or Atg16l1 in host tissues dramatically impairs growth of autophagy-competent syngeneic tumors. We further demonstrated that CD8 + T cells lacking Atg5 acquire an effector memory phenotype and produce more IFNG/IFN-γ (interferon gamma) and TNF/TNF-α (tumor necrosis factor). These phenotypic changes are accompanied by enhanced glucose metabolism that results in alterations in histone methylation, and upregulation of glycolytic and immune response genes. In accordance with this, we observed control of tumor growth in autophagy-competent mice after adoptive transfer with a sub-therapeutic dose of atg5 -/- T cells. Collectively, we discovered a unique, cell-autonomous role for T cell autophagy in the metabolic control of antitumor immunity.

The chemokine CXCL13 mediates recruitment of B cells to tumors and is essential for the formation of tertiary lymphoid structures (TLSs). TLSs are thought to support antitumor immunity and are associated with improved prognosis. However, it remains unknown whether TLSs are formed in response to the general inflammatory character of the tumor microenvironment, or rather, are induced by (neo)antigen-specific adaptive immunity. We here report on the finding that the TGFβ-dependent CD103 + CD8 + tumor-infiltrating T-cell (TIL) subpopulation expressed and produced CXCL13. Accordingly, CD8 + T cells from peripheral blood activated in the presence of TGFβ upregulated CD103 and secreted CXCL13. Conversely, inhibition of TGFβ receptor signaling abrogated CXCL13 production. CXCL13 + CD103 + CD8 + TILs correlated with B-cell recruitment, TLSs, and neoantigen burden in six cohorts of human tumors. Altogether, our findings indicated that TGFβ plays a noncanonical role in coordinating immune responses against human tumors and suggest a potential role for CXCL13 + CD103 + CD8 + TILs in mediating B-cell recruitment and TLS formation in human tumors., (©2019 American Association for Cancer Research.)

Autophagy is a cell survival process essential for the regulation of immune responses to infections. However, the role of T cell autophagy in anti-tumor immunity is less clear. Here, we demonstrate a cell-autonomous role for autophagy in the regulation of CD8 + T-cell-mediated control of tumors. Mice deficient for the essential autophagy genes Atg5, Atg14, or Atg16L1 display a dramatic impairment in the growth of syngeneic tumors. Moreover, T cells lacking Atg5 have a profound shift to an effector memory phenotype and produce greater amounts of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α). Mechanistically, Atg5 -/- CD8 + T cells exhibit enhanced glucose metabolism that results in alterations in histone methylation, increases in H3K4me3 density, and transcriptional upregulation of both metabolic and effector target genes. Nonetheless, glucose restriction is sufficient to suppress Atg5-dependent increases in effector function. Thus, autophagy-dependent changes in CD8 + T cell metabolism directly regulate anti-tumor immunity., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Purpose: A fundamental challenge in the era of next-generation sequencing (NGS) is to design effective treatments tailored to the mutational profiles of tumors. Many newly discovered cancer mutations are difficult to target pharmacologically; however, T-cell-based therapies may provide a valuable alternative owing to the exquisite sensitivity and specificity of antigen recognition. To explore this concept, we assessed the immunogenicity of a panel of genes that are common sites of driver mutations in follicular lymphoma, an immunologically sensitive yet currently incurable disease., Experimental Design: Exon capture and NGS were used to interrogate tumor samples from 53 patients with follicular lymphoma for mutations in 10 frequently mutated genes. For 13 patients, predicted mutant peptides and proteins were evaluated for recognition by autologous peripheral blood T cells after in vitro priming., Results: Mutations were identified in 1-5 genes in 81% (43/53) of tumor samples. Autologous, mutation-specific CD8(+) T cells were identified in 23% (3/13) of evaluated cases. T-cell responses were directed toward putative driver mutations in CREBBP and MEF2B. Responding T cells showed exquisite specificity for mutant versus wild-type proteins and recognized lymphoma cells expressing the appropriate mutations. Responding T cells appeared to be from the naïve repertoire, as they were found at low frequencies and only at single time points in each patient., Conclusions: Patients with follicular lymphoma harbor rare yet functionally competent CD8(+) T cells specific for recurrent mutations. Our results support the concept of using NGS to design individualized immunotherapies targeting common driver mutations in follicular lymphoma and other malignancies. Clin Cancer Res; 22(9); 2226-36. ©2015 AACR., (©2015 American Association for Cancer Research.)

αE(CD103)β7 is a TGFβ-regulated integrin that mediates retention of lymphocytes in peripheral tissues by binding to E-cadherin expressed on epithelial cells. We recently reported that αE(CD103)β7 specifically demarcates intraepithelial CD8(+) tumor-infiltrating lymphocytes (CD8 TIL) in ovarian cancer and that CD103(+) TIL have a surface profile consistent with an active effector phenotype (HLA-DR(+), Ki67(+), and CD127(lo)). These findings led us to hypothesize that, over time, CD103-mediated retention of CD8 TIL within the tumor epithelium might result in chronic stimulation by tumor antigen, which in turn might lead to an exhausted phenotype. To investigate this possibility, we evaluated PD-1 expression in a large cohort of ovarian tumors (N = 489) with known CD103(+) TIL content. PD-1(+) cells were present in 38.5% of high-grade serous carcinomas (HGSC), but were less prevalent in other histologic subtypes. PD-1(+) TIL were strongly associated with increased disease-specific survival in HGSC (HR, 0.4864; P = 0.0007). Multicolor immunohistochemistry and flow cytometry revealed a high degree of PD-1 and CD103 coexpression, specifically within the CD8 TIL compartment. PD-1(+)CD103(+) CD8 TIL were quiescent when assessed directly ex vivo yet were capable of robust cytokine production after pharmacologic stimulation. Moreover, they showed negligible expression of additional exhaustion-associated markers, including TIM-3, CTLA-4, and LAG-3. Thus, as hypothesized, CD103(+) CD8 TIL express PD-1 and appear quiescent in the tumor microenvironment. However, these cells retain functional competence and demonstrate strong prognostic significance. We speculate that, after standard treatment, PD-1(+)CD103(+) CD8 TIL might regain functional antitumor activity, an effect that potentially could be augmented by immune modulation., (©2015 American Association for Cancer Research.)

The activation and expansion of effector CD8(+) T cells are essential for controlling viral infections and tumor surveillance. During an immune response, T cells encounter extrinsic and intrinsic factors, including oxidative stress, nutrient availability, and inflammation, that can modulate their capacity to activate, proliferate, and survive. The dependency of T cells on autophagy for in vitro and in vivo activation, expansion, and memory remains unclear. Moreover, the specific signals and mechanisms that activate autophagy in T effector cells and their survival are not known. In this study, we generated a novel inducible autophagy knockout mouse to study T cell effector responses during the course of a virus infection. In response to influenza infection, Atg5(-/-) CD8(+) T cells had a decreased capacity to reach the peak effector response and were unable to maintain cell viability during the effector phase. As a consequence of Atg5 deletion and the impairment in effector-to-memory cell survival, mice fail to mount a memory response following a secondary challenge. We found that Atg5(-/-) effector CD8(+) T cells upregulated p53, a transcriptional state that was concomitant with widespread hypoxia in lymphoid tissues of infected mice. The onset of p53 activation was concurrent with higher levels of reactive oxygen species (ROS) that resulted in ROS-dependent apoptotic cell death, a fate that could be rescued by treating with the ROS scavenger N-acetylcysteine. Collectively, these results demonstrate that effector CD8(+) T cells require autophagy to suppress cell death and maintain survival in response to a viral infection., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Purpose: Tumor-infiltrating lymphocytes (TIL), in particular CD8(+) T cells and CD20(+) B cells, are strongly associated with survival in ovarian cancer and other carcinomas. Although CD8(+) TIL can mediate direct cytolytic activity against tumors, the role of CD20(+) TIL is poorly understood. Here, we investigate the possible contributions of CD20(+) TIL to humoral and cellular tumor immunity., Experimental Design: Tumor and serum specimens were obtained from patients with high-grade serous ovarian cancer. CD8(+) and CD20(+) TIL were analyzed by immunohistochemistry and flow cytometry. Immunoglobulin molecules were evaluated by DNA sequencing. Serum autoantibody responses to the tumor antigens p53 and NY-ESO-1 were measured by ELISA., Results: The vast majority of CD20(+) TIL were antigen experienced, as evidenced by class-switching, somatic hypermutation, and oligoclonality, yet they failed to express the canonical memory marker CD27. CD20(+) TIL showed no correlation with serum autoantibodies to p53 or NY-ESO-1. Instead, they colocalized with activated CD8(+) TIL and expressed markers of antigen presentation, including MHC class I, MHC class II, CD40, CD80, and CD86. The presence of both CD20(+) and CD8(+) TIL correlated with increased patient survival compared with CD8(+) TIL alone., Conclusions: In high-grade serous ovarian tumors, CD20(+) TIL have an antigen-experienced but atypical CD27(-) memory B-cell phenotype. They are uncoupled from serum autoantibodies, express markers of antigen-presenting cells, and colocalize with CD8(+) T cells. We propose that the association between CD20(+) TIL and patient survival may reflect a supportive role in cytolytic immune responses., (©2012 AACR.)

Persistent infection by high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, which remains one of the most common cancers among women worldwide. In addition, there is a growing appreciation that high risk HPVs are associated with a number of other cancers including anogenital cancers as well as a subset of head and neck cancers. Recently, prophylactic HPV vaccines targeting the two most prevalent high risk HPVs (HPV16 and HPV18) have been deployed in large-scale vaccination campaigns. However, the extent to which these prophylactic vaccines confer protection against other high risk HPV genotypes is largely unknown and prophylactic vaccines have been shown to be ineffective against pre-existing infection. Thus there continues to be an urgent need for effective therapeutic vaccines against HPV. The E7 protein of HPV16 has been widely studied as a target for therapeutic vaccines in HPV-associated cancer settings because HPV16 is the most prevalent of the high risk HPV genotypes. However, HPV16 accounts for only about 50% of cervical cancers and there are at least 15 other high risk HPVs that are known to be oncogenic. We have developed a novel, broad-spectrum, therapeutic vaccine (Pentarix) directed at the E7 proteins from five of the most prevalent high-risk genotypes of HPV worldwide (HPV16, 18, 31, 45 and 52) that together account for more than 80% of all HPV-associated cancers. Pentarix is a recombinant protein-based vaccine that elicits strong, multi-genotype specific CD8 T cell immunity when administered to mice in combination with adjuvants comprised of agonists of the TLR3 or TLR9 family of innate immune receptors. Furthermore, large, established E7-expressing TC-1 tumors undergo rapid and complete regression after therapeutic vaccination of mice with Pentarix. Together, these data suggest that Pentarix may be of clinical value for patients with E7-positive, HPV-associated precancerous lesions or malignant disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The development of vaccines that elicit robust CD8(+) T cell immunity has long been a subject of intense investigation. Although whole exogenous protein has not historically been considered as useful for eliciting CD8(+) T cell immunity, we report herein that whole, protein antigen is capable of eliciting profound levels of CD8(+) T cell immunity if it is administered via repeated, daily subcutaneous immunization in combination with the TLR3 agonist poly(I:C). Mice immunized for four consecutive days with 100 μg of either whole exogenous OVA or whole HPV16 E7 protein combined with 10 μg of poly(I:C) mounted remarkable antigen-specific CD8(+) T cell responses as measured by tetramer staining and ELISPOT analysis of splenocytes and peripheral blood, with up to 30% of peripheral CD8(+) T cells being antigen specific within 7-8 days of vaccination. CD8(+) T cell immunity elicited using this vaccination approach was critically dependent upon cross presentation, as either whole protein or long synthetic peptides were highly effective immunogens whereas minimal peptide epitopes were not. Vaccine-induced CD8(+) T cells were also able to regress large, established tumors in vivo. Together these data suggest that 'cluster' vaccination with exogenous antigen combined with TLR3 agonist may constitute a profoundly important advancement in therapeutic vaccine design., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

Adoptive immunotherapy shows promise for the treatment of cancer; however, partial or mixed responses remain common outcomes due to the heterogeneity of tumours. We studied three murine mammary tumour lines that express an ovalbumin-tagged version of HER-2/neu and reproducibly undergo complete regression (CR), partial regression (PR), or progressive disease (PD) after adoptive transfer of ovalbumin-specific CD8(+) (OT-I) and CD4(+) (OT-II) T cells. The three tumour lines were implanted in immunocompetent C57Bl/6 host mice, and established tumours were treated by adoptive transfer of naive OT-I and OT-II T cells. Tumours of the CR and PR classes triggered almost indistinguishable T cell responses in terms of activation, proliferation, trafficking to the tumour site, infiltration of tumour stroma, and intratumoural T cell proliferation; however, tumours of the PR class showed reduced infiltration of tumour epithelium by donor T cells. PD responses were associated with early impairment of T cell activation and proliferation in draining lymph node, followed by negligible infiltration of tumour tissue by donor T cells. Histopathological determinants of outcome were investigated through an unsupervised analysis of 64 untreated tumours representing the three response classes. Tumours of the CR class had proportionately more stroma, which had a looser, more collagen-rich histological appearance. Thus, the amount and composition of tumour stroma distinguished successfully (CR) from unsuccessful (PR or PD) outcomes after adoptive T cell transfer, a finding that might facilitate the design of immunotherapy trials for human breast cancer.

We tested the efficacy of CD8+ T cells lacking the Cbl-b gene against a panel of mammary tumor lines with different intrinsic sensitivities to T cells. Mice bearing established tumors expressing an ovalbumin-tagged version of HER-2/neu underwent adoptive transfer with Cbl-b-replete or -null CD8+ T cells from OT-I T cell receptor transgenic donor mice. In general, Cbl-b-null OT-I cells showed enhanced expansion, persistence, and capacity for tumor infiltration. This resulted in markedly enhanced efficacy against two tumor lines that normally demonstrate complete (NOP21) or partial (NOP23) regression. Moreover, a third tumor line (NOP6) that normally demonstrates progressive disease underwent complete regression in response to Cbl-b-null OT-I cells. However, a fourth tumor line (NOP18) was resistant to Cbl-b-null OT-I cells owing to a profound barrier to lymphocyte infiltration. Thus, Cbl-b-null CD8+ T cells are generally more efficacious but are nonetheless unable to mediate curative responses against all tumor phenotypes.

Immunohistochemical detection of increased levels of protein-associated nitrotyrosine has become widely used as a surrogate marker of in situ inflammation. However, the potential consequences of protein-associated nitrotyrosine formation in terms of cellular immune recognition has received surprisingly little attention. Using a well-defined I-E(K)-restricted epitope of pigeon cytochrome c, we previously demonstrated that conversion of a single tyrosine residue to nitrotyrosine can have a profound effect on recognition by CD4 T cells. In this study, we used the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein (gp33) to demonstrate that conversion of tyrosine to nitrotyrosine can also profoundly affect recognition of MHC class I-restricted epitopes. Conversion of the Y4 residue of the gp33 epitope to nitrotyrosine completely abrogated recognition by gp33-specific T cells from P14 TCR-transgenic mice. In contrast, CD8(+) T cells specific for "nitrated gp33" (NY-gp33) can be readily elicited in C57BL/6 mice after immunization with NY-gp33 peptide. Interestingly, T-T hybridomas specific for NY-gp33 peptide were found to fall into two distinct subsets, being specific for NY-gp33 presented in the context of either H-2D(b) or H-2K(b). This latter result is surprising in light of previous structural studies showing that Y4 comprises a critical TCR-contact residue when presented by H-2D(b) but that the same residue points downward into the peptide-binding groove of the MHC when presented by H-2K(b). Together, these results indicate that nitrotyrosine formation can impact T cell recognition both directly, through alteration of TCR-contact residues, or indirectly, through alterations in MHC-contact positions.

Purpose: In vitro studies suggest that ovarian cancer evades immune rejection by fostering an immunosuppressive environment within the peritoneum; however, the functional responses of ovarian cancer-specific T cells have not been directly investigated in vivo. Therefore, we developed a new murine model to enable tracking of tumor-specific CD8(+) T-cell responses to advanced ovarian tumors., Experimental Design: The ovarian tumor cell line ID8 was transfected to stably express an epitope-tagged version of HER-2/neu (designated Neu(OT-I/OT-II)). After i.p. injection into C57BL/6 mice, ID8 cells expressing Neu(OT-I/OT-II) gave rise to disseminated serous adenocarcinomas with extensive ascites. CD8(+) T cells expressing a transgenic T-cell receptor specific for the OT-I epitope of Neu(OT-I/OT-II) were adoptively transferred into tumor-bearing mice, and functional responses were monitored. Cytokine signaling requirements were evaluated by comparing the responses of wild-type donor T cells with those with genetic deletion of the interleukin (IL)-2/IL-15 receptor beta subunit (CD122) or the IL-2 receptor alpha subunit (CD25)., Results: On adoptive transfer into tumor-bearing hosts, wild-type OT-I T cells underwent a striking proliferative response, reaching peak densities of approximately 40% and approximately 90% of CD8(+) T cells in peripheral blood and ascites, respectively. OT-I cells infiltrated and destroyed tumor tissue, and ascites completely resolved within 10 days. By contrast, CD122(-/-) OT-I cells and CD25(-/-) OT-I cells proliferated in blood but failed to accumulate in ascites or tumor tissue or induce tumor regression., Conclusions: Contrary to expectation, advanced ovarian cancers can support extraordinary CD8(+) T-cell proliferation and antitumor activity through an IL-2/IL-15-dependent mechanism.