PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Development of an optimized closed and semi-automatic protocol for Good Manufacturing Practice manufacturing of tumor-infiltrating lymphocytes in a hospital environment.

BACKGROUND AIMS: Several studies report on Good Manufacturing Process (GMP)-compliant manufacturing protocols for the ex vivo expansion of tumor-infiltrating lymphocytes (TILs) for the treatment of patients with refractory melanoma and other solid malignancies. Further opportunities for improvements in terms of ergonomy and operating time have been identified. METHODS: To enable GMP-compliant TILs production for adoptive cell therapy needs, a simple automated and reproducible protocol for TILs manufacturing with the use of a closed system was developed and implemented at the authors' institution. RESULTS: This protocol enabled significant operating time reduction during TILs expansion while allowing the generation of high-quality TILs products. CONCLUSIONS: A simplified and efficient method of TILs expansion will enable the broadening of individualized tumor therapy and will increase patients' access to state-of-the-art TILs adoptive cell therapy treatment.

Development and Optimization of a GMP-Compliant Manufacturing Process for a Personalized Tumor Lysate Dendritic Cell Vaccine.

With the emergence of immune checkpoint inhibitors and adoptive T-cell therapies, there is a considerable interest in using personalized autologous dendritic cell (DC) vaccines in combination with T cell-targeting immunotherapies to potentially maximize the therapeutic impact of DC vaccines. Here, we describe the development and optimization of a Good Manufacturing Practice (GMP)-compliant manufacturing process based on tumor lysate as a tumor antigen source for the production of an oxidized tumor cell lysate loaded DC (OC-DC) vaccine. The manufacturing process required one day for lysate preparation and six days for OC-DC vaccine production. Tumor lysate production was standardized based on an optimal tumor digestion protocol and the immunogenicity was improved through oxidation using hypochloric acid prior to freeze-thaw cycles resulting in the oxidized tumor cell lysate (OC-L). Next, monocytes were selected using the CliniMACS prodigy closed system and were placed in culture in cell factories in the presence of IL-4 and GM-CSF. Immature DCs were loaded with OC-L and matured using MPLA-IFNγ. After assessing the functionality of the OC-DC cells (IL12p70 secretion and COSTIM assay), the OC-DC vaccine was cryopreserved in multiple doses for single use. Finally, the stability of the formulated doses was tested and validated. We believe this GMP-compliant DC vaccine manufacturing process will facilitate access of patients to personalized DC vaccines, and allow for multi-center clinical trials.

A Phase I/II trial comparing autologous dendritic cell vaccine pulsed either with personalized peptides (PEP-DC) or with tumor lysate (OC-DC) in patients with advanced high-grade ovarian serous carcinoma.

BACKGROUND: Most ovarian cancer patients are diagnosed at a late stage with 85% of them relapsing after surgery and standard chemotherapy; for this reason, new treatments are urgently needed. Ovarian cancer has become a candidate for immunotherapy by reason of their expression of shared tumor-associated antigens (TAAs) and private mutated neoantigens (NeoAgs) and the recognition of the tumor by the immune system. Additionally, the presence of intraepithelial tumor infiltrating lymphocytes (TILs) is associated with improved progression-free and overall survival of patients with ovarian cancer. The aim of active immunotherapy, including vaccination, is to generate a new anti-tumor response and amplify an existing immune response. Recently developed NeoAgs-based cancer vaccines have the advantage of being more tumor specific, reducing the potential for immunological tolerance, and inducing robust immunogenicity. METHODS: We propose a randomized phase I/II study in patients with advanced ovarian cancer to compare the immunogenicity and to assess safety and feasibility of two personalized DC vaccines. After standard of care surgery and chemotherapy, patients will receive either a novel vaccine consisting of autologous DCs pulsed with up to ten peptides (PEP-DC), selected using an agnostic, yet personalized, epitope discovery algorithm, or a sequential combination of a DC vaccine loaded with autologous oxidized tumor lysate (OC-DC) prior to an equivalent PEP-DC vaccine. All vaccines will be administered in combination with low-dose cyclophosphamide. This study is the first attempt to compare the two approaches and to use NeoAgs-based vaccines in ovarian cancer in the adjuvant setting. DISCUSSION: The proposed treatment takes advantage of the beneficial effects of pre-treatment with OC-DC prior to PEP-DC vaccination, prompting immune response induction against a wide range of patient-specific antigens, and amplification of pre-existing NeoAgs-specific T cell clones. Trial registration This trial is already approved by Swissmedic (Ref.: 2019TpP1004) and will be registered at http://www.clinicaltrials.gov before enrollment opens.

Personalized Dendritic Cell Vaccines-Recent Breakthroughs and Encouraging Clinical Results.

With the advent of combined immunotherapies, personalized dendritic cell (DC)-based vaccination could integrate the current standard of care for the treatment of a large variety of tumors. Due to their proficiency at antigen presentation, DC are key coordinators of the innate and adaptive immune system, and have critical roles in the induction of antitumor immunity. However, despite proven immunogenicity and favorable safety profiles, DC-based immunotherapies have not succeeded at inducing significant objective clinical responses. Emerging data suggest that the combination of DC-based vaccination with other cancer therapies may fully unleash the potential of DC-based cancer vaccines and improve patient survival. In this review, we discuss the recent efforts to develop innovative personalized DC-based vaccines and their use in combined therapies, with a particular focus on ovarian cancer and the promising results of mutanome-based personalized immunotherapies.

Lymphatic vessel density is associated with CD8+ T cell infiltration and immunosuppressive factors in human melanoma.

Increased density of tumor-associated lymphatic vessels correlates with poor patient survival in melanoma and other cancers, yet lymphatic drainage is essential for initiating an immune response. Here we asked whether and how lymphatic vessel density (LVD) correlates with immune cell infiltration in primary tumors and lymph nodes (LNs) from patients with cutaneous melanoma. Using immunohistochemistry and quantitative image analysis, we found significant positive correlations between LVD and CD8+ T cell infiltration as well as expression of the immunosuppressive molecules inducible nitric oxide synthase (iNOS) and 2,3-dioxygénase (IDO). Interestingly, similar associations were seen in tumor-free LNs adjacent to metastatic ones, indicating loco-regional effects of tumors. Our data suggest that lymphatic vessels play multiple roles at tumor sites and LNs, promoting both T cell infiltration and adaptive immunosuppressive mechanisms. Lymph vessel associated T cell infiltration may increase immunotherapy success rates provided that the treatment overcomes adaptive immune resistance.

Deciphering HLA-I motifs across HLA peptidomes improves neo-antigen predictions and identifies allostery regulating HLA specificity.

The precise identification of Human Leukocyte Antigen class I (HLA-I) binding motifs plays a central role in our ability to understand and predict (neo-)antigen presentation in infectious diseases and cancer. Here, by exploiting co-occurrence of HLA-I alleles across ten newly generated as well as forty public HLA peptidomics datasets comprising more than 115,000 unique peptides, we show that we can rapidly and accurately identify many HLA-I binding motifs and map them to their corresponding alleles without any a priori knowledge of HLA-I binding specificity. Our approach recapitulates and refines known motifs for 43 of the most frequent alleles, uncovers new motifs for 9 alleles that up to now had less than five known ligands and provides a scalable framework to incorporate additional HLA peptidomics studies in the future. The refined motifs improve neo-antigen and cancer testis antigen predictions, indicating that unbiased HLA peptidomics data are ideal for in silico predictions of neo-antigens from tumor exome sequencing data. The new motifs further reveal distant modulation of the binding specificity at P2 for some HLA-I alleles by residues in the HLA-I binding site but outside of the B-pocket and we unravel the underlying mechanisms by protein structure analysis, mutagenesis and in vitro binding assays.

Heterogeneity assessment of functional T cell avidity.

The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation.

Rapid and Continued T-Cell Differentiation into Long-term Effector and Memory Stem Cells in Vaccinated Melanoma Patients.

Purpose: Patients with cancer benefit increasingly from T-cell-based therapies, such as adoptive T-cell transfer, checkpoint blockade, or vaccination. We have previously shown that serial vaccinations with Melan-AMART-126-35 peptide, CpG-B, and incomplete Freund adjuvant (IFA) generated robust tumor-specific CD8 T-cell responses in patients with melanoma. Here, we describe the detailed kinetics of early- and long-term establishment of T-cell frequency, differentiation (into memory and effector cells), polyfunctionality, and clonotype repertoire induced by vaccination.Experimental Design: Twenty-nine patients with melanoma were treated with multiple monthly subcutaneous vaccinations consisting of CpG-B, and either the native/EAA (n = 13) or the analogue/ELA (n = 16) Melan-AMART-126-35 peptide emulsified in IFA. Phenotypes and functionality of circulating Melan-A-specific CD8 T cells were assessed directly ex vivo by multiparameter flow cytometry, and TCR clonotypes were determined ex vivo by mRNA transcript analyses of individually sorted cells.Results: Our results highlight the determining impact of the initial vaccine injections on the rapid and strong induction of differentiated effector T cells in both patient cohorts. Moreover, long-term polyfunctional effector T-cell responses were associated with expansion of stem cell-like memory T cells over time along vaccination. Dominant TCR clonotypes emerged early and persisted throughout the entire period of observation. Interestingly, one highly dominant clonotype was found shared between memory and effector subsets.Conclusions: Peptide/CpG-B/IFA vaccination induced powerful long-term T-cell responses with robust effector cells and stem cell-like memory cells. These results support the further development of CpG-B-based cancer vaccines, either alone or as specific component of combination therapies. Clin Cancer Res; 23(13); 3285-96. ©2016 AACR.

Epitope located N-glycans impair the MHC-I epitope generation and presentation.

The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells.

Identifying Individual T Cell Receptors of Optimal Avidity for Tumor Antigens.

Cytotoxic T cells recognize, via their T cell receptors (TCRs), small antigenic peptides presented by the major histocompatibility complex (pMHC) on the surface of professional antigen-presenting cells and infected or malignant cells. The efficiency of T cell triggering critically depends on TCR binding to cognate pMHC, i.e., the TCR-pMHC structural avidity. The binding and kinetic attributes of this interaction are key parameters for protective T cell-mediated immunity, with stronger TCR-pMHC interactions conferring superior T cell activation and responsiveness than weaker ones. However, high-avidity TCRs are not always available, particularly among self/tumor antigen-specific T cells, most of which are eliminated by central and peripheral deletion mechanisms. Consequently, systematic assessment of T cell avidity can greatly help distinguishing protective from non-protective T cells. Here, we review novel strategies to assess TCR-pMHC interaction kinetics, enabling the identification of the functionally most-relevant T cells. We also discuss the significance of these technologies in determining which cells within a naturally occurring polyclonal tumor-specific T cell response would offer the best clinical benefit for use in adoptive therapies, with or without T cell engineering.

Potential Cross-Talk between Alternative and Classical NF-κB Pathways in Prostate Cancer Tissues as Measured by a Multi-Staining Immunofluorescence Co-Localization Assay.

BACKGROUND: While the classical NF-κB/p65 pathway is known to be involved in prostate cancer progression and is associated with poor patient outcome, the role of the NF-κB /RelB alternative protein is not well defined. Here we analyzed the activation of both NF-κB pathways in prostate cancer tissues and correlate this activation with clinical features of the disease. METHODS: A multiple immunofluorescence technique was employed to concomitantly and quantitatively visualize the nuclear localization of p65 and RelB in 200 paraffin embedded samples. Epithelia were defined using appropriate fluorochrome markers and the resulting immunofluorescent signals were quantified with an automated scoring system. RESULTS: The nuclear frequency of p65 was found to be significantly increased in tumor tissues as compared with normal adjacent tissue, whereas the frequency for RelB was decreased (p < 0.001, Wilcoxon test). As previously reported, p65 nuclear frequency was associated with a risk of biochemical recurrence. Although, RelB nuclear frequency alone did not predict recurrence, the presence of activated RelB reduced the risk of recurrence associated with the activation of p65. CONCLUSION: For the first time p65/RelB co-distribution was assessed in prostate cancer tissues and suggested a negative crosstalk between the two NF-κB pathways in prostate cancer progression.

Quantitative TCR:pMHC Dissociation Rate Assessment by NTAmers Reveals Antimelanoma T Cell Repertoires Enriched for High Functional Competence.

Experimental models demonstrated that therapeutic induction of CD8 T cell responses may offer protection against tumors or infectious diseases providing that T cells have sufficiently high TCR/CD8:pMHC avidity for efficient Ag recognition and consequently strong immune functions. However, comprehensive characterization of TCR/CD8:pMHC avidity in clinically relevant situations has remained elusive. In this study, using the novel NTA-His tag-containing multimer technology, we quantified the TCR:pMHC dissociation rates (koff) of tumor-specific vaccine-induced CD8 T cell clones (n = 139) derived from seven melanoma patients vaccinated with IFA, CpG, and the native/EAA or analog/ELA Melan-A(MART-1)(26-35) peptide, binding with low or high affinity to MHC, respectively. We observed substantial correlations between koff and Ca(2+) mobilization (p = 0.016) and target cell recognition (p < 0.0001), with the latter independently of the T cell differentiation state. Our strategy was successful in demonstrating that the type of peptide impacted on TCR/CD8:pMHC avidity, as tumor-reactive T cell clones derived from patients vaccinated with the low-affinity (native) peptide expressed slower koff rates than those derived from patients vaccinated with the high-affinity (analog) peptide (p < 0.0001). Furthermore, we observed that the low-affinity peptide promoted the selective differentiation of tumor-specific T cells bearing TCRs with high TCR/CD8:pMHC avidity (p < 0.0001). Altogether, TCR:pMHC interaction kinetics correlated strongly with T cell functions. Our study demonstrates the feasibility and usefulness of TCR/CD8:pMHC avidity assessment by NTA-His tag-containing multimers of naturally occurring polyclonal T cell responses, which represents a strong asset for the development of immunotherapy.

Long-lasting stem cell-like memory CD8+ T cells with a naïve-like profile upon yellow fever vaccination.

Efficient and persisting immune memory is essential for long-term protection from infectious and malignant diseases. The yellow fever (YF) vaccine is a live attenuated virus that mediates lifelong protection, with recent studies showing that the CD8(+) T cell response is particularly robust. Yet, limited data exist regarding the long-term CD8(+) T cell response, with no studies beyond 5 years after vaccination. We investigated 41 vaccinees, spanning 0.27 to 35 years after vaccination. YF-specific CD8(+) T cells were readily detected in almost all donors (38 of 41), with frequencies decreasing with time. As previously described, effector cells dominated the response early after vaccination. We detected a population of naïve-like YF-specific CD8(+) T cells that was stably maintained for more than 25 years and was capable of self-renewal ex vivo. In-depth analyses of markers and genome-wide mRNA profiling showed that naïve-like YF-specific CD8(+) T cells in vaccinees (i) were distinct from genuine naïve cells in unvaccinated donors, (ii) resembled the recently described stem cell-like memory subset (Tscm), and (iii) among all differentiated subsets, had profiles closest to naïve cells. Our findings reveal that CD8(+) Tscm are efficiently induced by a vaccine in humans, persist for decades, and preserve a naïveness-like profile. These data support YF vaccination as an optimal mechanistic model for the study of long-lasting memory CD8(+) T cells in humans.

Pulmonary sarcoid-like granulomatosis after multiple vaccinations of a long-term surviving patient with metastatic melanoma.

Autoimmune side effects are frequent in patients with cancer treated with immune checkpoint-targeting antibodies, but are rare with cancer vaccines. Here, we present a case report on a patient with metastatic melanoma who developed pulmonary sarcoid-like granulomatosis following repetitive vaccinations with peptides and CpG. Despite multiple metastases, including one lesion in the brain, the patient is alive and well more than 13 years after the diagnosis of metastatic disease. The strongly activated tumor-specific CD8(+) T cells showed robust long-term memory and effector functions. It is possible that long-term survival and adverse autoimmune events may become more common for vaccines inducing robust anticancer immune responses as were present in this patient.

The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo.

BACKGROUND: The involvement of NF-κB signaling in prostate cancer (PCa) has largely been established through the study of the classical p65 subunit. Nuclear localization of p65 in PCa patient tissues has been shown to correlate with biochemical recurrence, while in vitro studies have demonstrated that the classical NF-κB signaling pathway promotes PCa progression and metastatic potential. More recently, the nuclear location of RelB, a member of the alternative NF-κB signaling, has also been shown to correlate with the Gleason score. The current study aims to clarify the role of alternative NF-κB in PCa cells by exploring, in vitro and in vivo, the effects of RelB overexpression on PCa biology. METHODS: Using a lentivirus-expression system, we constitutively overexpressed RelB or control GFP into 22Rv1 cells and monitored alternative transcriptional NF-κB activity. In vivo, tumor growth was assessed after the injection of 22Rv1-derived cells into SCID mice. In vitro, the impact of RelB on 22Rv1 cell proliferation was evaluated in monolayer culture. The anchorage-independent cell growth of derived-22Rv1 cells was assessed by soft agar assay. Apoptosis and autophagy were evaluated by Western blot analysis in 22Rv1-derived cells cultured in suspension using poly-HEMA pre-coated dishes. RESULTS: The overexpression of RelB in 22Rv1 cells induced the constitutive activation of the alternative NF-κB pathway. In vivo, RelB expression caused a lag in the initiation of 22Rv1-induced tumors in SCID mice. In vitro, RelB stimulated the proliferation of 22Rv1 cells and reduced their ability to grow in soft agar. These observations may be reconciled by our findings that, when cultured in suspension on poly-HEMA pre-coated dishes, 22Rv1 cells expressing RelB were more susceptible to cell death, and more specifically to autophagy controlled death. CONCLUSIONS: This study highlights a role of the alternative NF-κB pathway in proliferation and the controlled autophagy. Thus, the interplay of these properties may contribute to tumor survival in stress conditions while promoting PCa cells growth contributing to the overall tumorigenicity of these cells.

Persistence of EBV antigen-specific CD8 T cell clonotypes during homeostatic immune reconstitution in cancer patients.

Persistent viruses are kept in check by specific lymphocytes. The clonal T cell receptor (TCR) repertoire against Epstein-Barr virus (EBV), once established following primary infection, exhibits a robust stability over time. However, the determinants contributing to this long-term persistence are still poorly characterized. Taking advantage of an in vivo clinical setting where lymphocyte homeostasis was transiently perturbed, we studied EBV antigen-specific CD8 T cells before and after non-myeloablative lympho-depleting chemotherapy of melanoma patients. Despite more advanced T cell differentiation, patients T cells showed clonal composition comparable to healthy individuals, sharing a preference for TRBV20 and TRBV29 gene segment usage and several co-dominant public TCR clonotypes. Moreover, our data revealed the presence of relatively few dominant EBV antigen-specific T cell clonotypes, which mostly persisted following transient lympho-depletion (TLD) and lymphocyte recovery, likely related to absence of EBV reactivation and de novo T cell priming in these patients. Interestingly, persisting clonotypes frequently co-expressed memory/homing-associated genes (CD27, IL7R, EOMES, CD62L/SELL and CCR5) supporting the notion that they are particularly important for long-lasting CD8 T cell responses. Nevertheless, the clonal composition of EBV-specific CD8 T cells was preserved over time with the presence of the same dominant clonotypes after non-myeloablative chemotherapy. The observed clonotype persistence demonstrates high robustness of CD8 T cell homeostasis and reconstitution.

Large-scale independent validation of the nuclear factor-kappa B p65 prognostic biomarker in prostate cancer.

PURPOSE: Over the last decade, we and others have uncovered a robust association between the nuclear localisation of nuclear factor-kappa B (NF-κB) p65, prostate cancer (PCa) aggressiveness and biochemical recurrence (BCR). Our goal was to validate these results in a large independent cohort of PCa patients who underwent radical prostatectomy. EXPERIMENTAL DESIGN: A set of 1826 fully annotated prostate cancers treated by radical prostatectomy were analysed in a tissue microarray (TMA) format for NF-κB p65 immunohistochemistry-based protein expression. We performed standard Cox proportional hazard regression models for follow-up data, bootstrap procedure for model internal validation, Harrell's concordance index for model discrimination and graphical assessment of predicted versus actual outcomes for model calibration. RESULTS: We observed a significant association between an increase in the nuclear frequency of NF-κB p65 and Gleason score (P<0.001), overall BCR (P<0.001) and development of metastases (P=0.001). NF-κB was found to be an independent predictor of BCR (P<0.001, Cox regression). However its contribution to the predictive accuracy of a multivariate model, which included preoperative PSA, Gleason score, extraprostatic extension, lymph node invasion, seminal vesicle involvement and surgical margin status, was modest. CONCLUSIONS: Our study offers validating results linking NF-κB p65 with disease progression using a large cohort of European men. However, the contribution of NF-κB to a post-surgical predictive model appears modest. Further validating work should focus on evaluating the contribution of NF-κB p65 in pre-treatment models.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Impact of hemochromatosis gene (HFE) mutations on epithelial ovarian cancer risk and prognosis.

Cancer cells require large amounts of micronutrients, particularly iron, for their rapid growth and frequent divisions. Cellular iron uptake is regulated by the transferrin receptor and the hemochromatosis protein (HFE) system. Two frequent mutations in the HFE gene, H63D and C282Y, are associated with hemochromatosis type I, an inherited iron overload disease and, possibly, with cancer. In this study, we evaluated the frequency of the H63D and C282Y mutations in a cohort of 677 consecutive cases of woman with gynecological pathologies. Cases included 80 women with tumor-free pathologies normal ovary (NOV), 124 with benign ovarian tumors (BOV), 96 with epithelial ovarian cancer (EOC) tumors of low malignant potential (LPM), 264 with invasive tumors of the ovary (TOV) and 113 with endometrial cancer. We found that the C282Y allele frequency in EOC patients was higher than that in the control NOV group (5.8% vs. 1.3%, p < 0.001) and was associated with an increased risk of ovarian cancer (OR = 4.88; 95% CI 1.15-20.61; p = 0.018). The effect of the two HFE mutations on patient survival was also analyzed. Kaplan-Meier analyses did not find any significant association between the H63D allele and patient survival. However, EOC patients with at least one C282Y allele had a decreased overall survival compared to those with no C282Y allele (p = 0.001). These results indicate that the C282Y mutation may increase the risk of developing ovarian cancer and may be further associated with poor outcomes.