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The accurate identification and prioritization of antigenic peptides is crucial for the development of personalized cancer immunotherapies. Publicly available pipelines to predict clinical neoantigens do not allow direct integration of mass spectrometry immunopeptidomics data, which can uncover antigenic peptides derived from various canonical and noncanonical sources. To address this, we present an end-to-end clinical proteogenomic pipeline, called NeoDisc, that combines state-of-the-art publicly available and in-house software for immunopeptidomics, genomics and transcriptomics with in silico tools for the identification, prediction and prioritization of tumor-specific and immunogenic antigens from multiple sources, including neoantigens, viral antigens, high-confidence tumor-specific antigens and tumor-specific noncanonical antigens. We demonstrate the superiority of NeoDisc in accurately prioritizing immunogenic neoantigens over recent prioritization pipelines. We showcase the various features offered by NeoDisc that enable both rule-based and machine-learning approaches for personalized antigen discovery and neoantigen cancer vaccine design. Additionally, we demonstrate how NeoDisc's multiomics integration identifies defects in the cellular antigen presentation machinery, which influence the heterogeneous tumor antigenic landscape., (© 2024. The Author(s).)

Adoptive cell therapy (ACT) using in vitro expanded tumor-infiltrating lymphocytes (TILs) has inconsistent clinical responses. To better understand determinants of therapeutic success, we tracked TIL clonotypes from baseline tumors to ACT products and post-ACT blood and tumor samples in melanoma patients using single-cell RNA and T cell receptor (TCR) sequencing. Patients with clinical responses had baseline tumors enriched in tumor-reactive TILs, and these were more effectively mobilized upon in vitro expansion, yielding products enriched in tumor-specific CD8 + cells that preferentially infiltrated tumors post-ACT. Conversely, lack of clinical responses was associated with tumors devoid of tumor-reactive resident clonotypes and with cell products mostly composed of blood-borne clonotypes that persisted in blood but not in tumors post-ACT. Upon expansion, tumor-specific TILs lost tumor-associated transcriptional signatures, including exhaustion, and responders exhibited an intermediate exhausted effector state after TIL engraftment in the tumor, suggesting functional reinvigoration. Our findings provide insight into the nature and dynamics of tumor-specific clonotypes associated with clinical response to TIL-ACT, with implications for treatment optimization., Competing Interests: Declaration of interests G.C. has received grants, research support, or has been coinvestigator in clinical trials by Bristol-Myers Squibb, Tigen Pharma, Iovance, F. Hoffmann-La Roche AG, and Boehringer Ingelheim. The Lausanne University Hospital (CHUV) has received honoraria for advisory services G.C. has provided to Genentech, AstraZeneca AG, and EVIR. G.C. has previously received royalties from the University of Pennsylvania for CAR-T cell therapy licensed to Novartis and Tmunity Therapeutics. D.D.L., S.B., A.H., and G.C. are inventors on patent applications filed by the Ludwig Institute for Cancer Research Ltd. on behalf of the University of Lausanne and the CHUV pertaining to the subject matter disclosed herein, and such patent applications have been licensed to Tigen Pharma SA. S.Z. is currently an employee of F. Hoffmann-La Roche. O.M. has consulting/advisory roles for Bristol Myers Squibb, MSD, Roche, Novartis, Amgen, Pierre Fabre, and Neracare; research grants from Bristol Myers Squibb, MSD, Amgen, and PCL; was a consultant advisor or paid speaker for Bristol Myers Squibb, MSD, Novartis, Pierre Fabre, Amgen, and Nektar; and has received research funding from Bristol Myers Squibb and Pierre Fabre and is the cofounder of a cell therapy company called Cellula., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Approaches to analyze and cluster T-cell receptor (TCR) repertoires to reflect antigen specificity are critical for the diagnosis and prognosis of immune-related diseases and the development of personalized therapies. Sequence-based approaches showed success but remain restrictive, especially when the amount of experimental data used for the training is scarce. Structure-based approaches which represent powerful alternatives, notably to optimize TCRs affinity toward specific epitopes, show limitations for large-scale predictions. To handle these challenges, TCRpcDist is presented, a 3D-based approach that calculates similarities between TCRs using a metric related to the physico-chemical properties of the loop residues predicted to interact with the epitope. By exploiting private and public datasets and comparing TCRpcDist with competing approaches, it is demonstrated that TCRpcDist can accurately identify groups of TCRs that are likely to bind the same epitopes. Importantly, the ability of TCRpcDist is experimentally validated to determine antigen specificities (neoantigens and tumor-associated antigens) of orphan tumor-infiltrating lymphocytes (TILs) in cancer patients. TCRpcDist is thus a promising approach to support TCR repertoire analysis and TCR deorphanization for individualized treatments including cancer immunotherapies., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

A central challenge in developing personalized cancer cell immunotherapy is the identification of tumor-reactive T cell receptors (TCRs). By exploiting the distinct transcriptomic profile of tumor-reactive T cells relative to bystander cells, we build and benchmark TRTpred, an antigen-agnostic in silico predictor of tumor-reactive TCRs. We integrate TRTpred with an avidity predictor to derive a combinatorial algorithm of clinically relevant TCRs for personalized T cell therapy and benchmark it in patient-derived xenografts., (© 2024. The Author(s).)

Expansion of antigen-experienced CD8 + T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer 1 . Interleukin-2 (IL-2) acts as a key regulator of CD8 + cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability 2,3 . 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 (PGE 2 ), a known negative regulator of immune response in the tumour microenvironment 4,5 , is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8 + TILs via the PGE 2 receptors EP2 and EP4. Mechanistically, PGE 2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγ c chain, resulting in defective assembly of IL-2Rβ-IL2Rγ c 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 PGE 2  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 PGE 2 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., (© 2024. The Author(s).)

T cells have the ability to eliminate infected and cancer cells and play an essential role in cancer immunotherapy. T cell activation is elicited by the binding of the T cell receptor (TCR) to epitopes displayed on MHC molecules, and the TCR specificity is determined by the sequence of its α and β chains. Here, we collect and curate a dataset of 17,715 αβTCRs interacting with dozens of class I and class II epitopes. We use this curated data to develop MixTCRpred, an epitope-specific TCR-epitope interaction predictor. MixTCRpred accurately predicts TCRs recognizing several viral and cancer epitopes. MixTCRpred further provides a useful quality control tool for multiplexed single-cell TCR sequencing assays of epitope-specific T cells and pinpoints a substantial fraction of putative contaminants in public databases. Analysis of epitope-specific dual α T cells demonstrates that MixTCRpred can identify α chains mediating epitope recognition. Applying MixTCRpred to TCR repertoires from COVID-19 patients reveals enrichment of clonotypes predicted to bind an immunodominant SARS-CoV-2 epitope. Overall, MixTCRpred provides a robust tool to predict TCRs interacting with specific epitopes and interpret TCR-sequencing data from both bulk and epitope-specific T cells., (© 2024. The Author(s).)

Adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell-intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8 + TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor-reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

We have previously shown that vaccination with tumor-pulsed dendritic cells amplifies neoantigen recognition in ovarian cancer. Here, in a phase 1 clinical study ( NCT01312376 /UPCC26810) including 19 patients, we show that such responses are further reinvigorated by subsequent adoptive transfer of vaccine-primed, ex vivo-expanded autologous peripheral blood T cells. The treatment is safe, and epitope spreading with novel neopeptide reactivities was observed after cell infusion in patients who experienced clinical benefit, suggesting reinvigoration of tumor-sculpting immunity., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objectives: Intradermal skin test (IDT) with mRNA vaccines may represent a simple, reliable, and affordable tool to measure T cell response in immunocompromised patients who failed to mount serological responses following vaccination with mRNA covid-19 vaccines., Methods: We compared anti-SARS-CoV-2 antibodies and cellular responses in vaccinated immunocompromised patients (n = 58), healthy seronegative naive controls (NC, n = 8), and healthy seropositive vaccinated controls (VC, n = 32) by Luminex, spike-induced IFN-γ Elispot and an IDT. A skin biopsy 24 h after IDT and single-cell RNAseq was performed in three vaccinated volunteers., Results: Twenty-five percent of seronegative NC had a positive Elispot (2/8) and IDT (1/4), compared to 95% (20/21) and 93% (28/30) in seropositive VC, respectively. Single-cell RNAseq data in the skin of VC showed a predominant mixed population of effector helper and cytotoxic T cells. The TCR repertoire revealed 18/1064 clonotypes with known specificities against SARS-CoV-2, among which six were spike-specific. Seronegative immunocompromised patients with positive Elispot and IDT were in 83% (5/6) treated with B cell-depleting reagents, while those with negative IDT were all transplant recipients., Conclusions: Our results indicate that delayed local reaction to IDT reflects vaccine-induced T-cell immunity opening new perspectives to monitor seronegative patients and elderly populations with waning immunity., Competing Interests: Declaration of Competing Interest Dr Pantaleo and Dr Fenwick report having a patent pending (application No. EP20205298.1) for a SARS-Cov2 neutralization assay. Dr Gottardo has received consulting income from Takeda, Ozette Technologies and declares ownership in Ozette Technologies. The research was conducted without any other commercial or financial relationships that could be construed as a potential conflict of interest to this study., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The success of cancer immunotherapy depends in part on the strength of antigen recognition by T cells. Here, we characterize the T cell receptor (TCR) functional (antigen sensitivity) and structural (monomeric pMHC-TCR off-rates) avidities of 371 CD8 T cell clones specific for neoantigens, tumor-associated antigens (TAAs) or viral antigens isolated from tumors or blood of patients and healthy donors. T cells from tumors exhibit stronger functional and structural avidity than their blood counterparts. Relative to TAA, neoantigen-specific T cells are of higher structural avidity and, consistently, are preferentially detected in tumors. Effective tumor infiltration in mice models is associated with high structural avidity and CXCR3 expression. Based on TCR biophysicochemical properties, we derive and apply an in silico model predicting TCR structural avidity and validate the enrichment in high avidity T cells in patients' tumors. These observations indicate a direct relationship between neoantigen recognition, T cell functionality and tumor infiltration. These results delineate a rational approach to identify potent T cells for personalized cancer immunotherapy., (© 2023. The Author(s).)

T cell receptor (TCR) technologies, including repertoire analyses and T cell engineering, are increasingly important in the clinical management of cellular immunity in cancer, transplantation, and other immune diseases. However, sensitive and reliable methods for repertoire analyses and TCR cloning are still lacking. Here, we report on SEQTR, a high-throughput approach to analyze human and mouse repertoires that is more sensitive, reproducible, and accurate as compared with commonly used assays, and thus more reliably captures the complexity of blood and tumor TCR repertoires. We also present a TCR cloning strategy to specifically amplify TCRs from T cell populations. Positioned downstream of single-cell or bulk TCR sequencing, it allows time- and cost-effective discovery, cloning, screening, and engineering of tumor-specific TCRs. Together, these methods will accelerate TCR repertoire analyses in discovery, translational, and clinical settings and permit fast TCR engineering for cellular therapies., Competing Interests: The University of Lausanne and Ludwig Institute for Cancer Research have filed for patent protection on the technology described herein. R.G. is named as inventor on this patent. G.C. has received grants from Celgene, Boehringer-Ingelheim, Roche, Bristol Myers Squibb, Iovance Therapeutics, and Kite Pharma. The institution with which G.C. is affiliated has received fees for G.C.’s participation on advisory boards or for presentation at a company-sponsored symposium from Genentech, Roche, Bristol Myers Squibb, AstraZeneca, NextCure, Geneos Tx, and Sanofi/Avensis. The Center Hospitalier Universitaire Vaudois (CHUV) and the Ludwig Institute for Cancer Research have filed for patent protection on the technology related to T cell expansion. S.B., A.H., and G.C. are named as inventors on this patent. G.C. has patents in the domain of antibodies and vaccines targeting the tumor vasculature as well as technologies related to T cell engineering for T cell therapy. G.C. holds patents around antibodies and receives royalties from the University of Pennsylvania regarding technology licensed to Novartis., (© 2023 The Authors.)

The recognition of pathogen or cancer-specific epitopes by CD8 + T cells is crucial for the clearance of infections and the response to cancer immunotherapy. This process requires epitopes to be presented on class I human leukocyte antigen (HLA-I) molecules and recognized by the T-cell receptor (TCR). Machine learning models capturing these two aspects of immune recognition are key to improve epitope predictions. Here, we assembled a high-quality dataset of naturally presented HLA-I ligands and experimentally verified neo-epitopes. We then integrated these data in a refined computational framework to predict antigen presentation (MixMHCpred2.2) and TCR recognition (PRIME2.0). The depth of our training data and the algorithmic developments resulted in improved predictions of HLA-I ligands and neo-epitopes. Prospectively applying our tools to SARS-CoV-2 proteins revealed several epitopes. TCR sequencing identified a monoclonal response in effector/memory CD8 + T cells against one of these epitopes and cross-reactivity with the homologous peptides from other coronaviruses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The identification of patient-specific tumor antigens is complicated by the low frequency of T cells specific for each tumor antigen. Here we describe NeoScreen, a method that enables the sensitive identification of rare tumor (neo)antigens and of cognate T cell receptors (TCRs) expressed by tumor-infiltrating lymphocytes. T cells transduced with tumor antigen-specific TCRs identified by NeoScreen mediate regression of established tumors in patient-derived xenograft mice., (© 2021. The Author(s).)

Droplet microfluidics are characterized by the generation and manipulation of discrete volumes of solutions, generated with the use of immiscible phases. Those droplets can then be controlled, transported, analyzed or their content modified. In this wide droplet microfluidic toolbox, no means are available to generate, in a controlled manner, droplets co-encapsulating to aqueous phases. Indeed, current methods rely on random co-encapsulation of two aqueous phases during droplet generation or the merging of two random droplets containing different aqueous phases. In this study, we present a novel droplet microfluidic device to reliably and efficiently co-encapsulate two different aqueous phases in micro-droplets. In order to achieve this, we combined existing droplet microfluidic modules in a novel way. The different aqueous phases are individually encapsulated in droplets of different sizes. Those droplet populations are then filtered in order to position each droplet type towards its adequate trapping compartment in traps of a floating trap array. Single droplets, each containing a different aqueous phase, are thus paired and then merged. This pairing at high efficiency is achieved thanks to a unique combination of floating trap arrays, a droplet railing system and a droplet size-based filtering mechanism. The microfluidic chip design presented here provides a filtering threshold with droplets larger than 35 μm (big droplets) being deviated to the lower rail while droplets smaller than 20 μm (small droplets) remain on the upper rail. The effects of the rail height and the distance between the two (upper and lower) rails were investigated. The optimal trap dimensions provide a trapping efficiency of 100% for small and big droplets with a limited double trapping (both compartments of the traps filled with the same droplet type) of 5%. The use of electrocoalescence enables the generation of a droplet while co-encapsulating two aqueous phases. Using the presented microfluidic device libraries of 300 droplets, dual aqueous content can be generated in less than 30 min.

Mutation-derived neoantigens are now established as attractive targets for cancer immunotherapy. The field of adoptive T cell transfer (ACT) therapy was significantly reshaped by tumor neoantigens and is now moving towards the genetic engineering of T cells with neoantigen-specific T cell receptors (TCRs). Yet, the identification of neoantigen-reactive TCRs remains challenging and the process needs to be adapted to clinical timelines. In addition, the state of recipient T cells for TCR transduction is critical and can affect TCR-ACT efficacy. Here we provide an overview of the main strategies for TCR-engineering, describe the selection and expansion of optimal carrier cells for TCR-ACT and discuss the next-generation methods for rapid identification of relevant TCR candidates for gene transfer therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Arnaud, Bobisse, Chiffelle and Harari.)

In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1 mut ) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1 mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8 + T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53 -/- Brca1 -/- but not Brca1 +/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers., Competing Interests: Declaration of interests G. Coukos has received grants from Celgene, Boehringer-Ingelheim, Roche, BMS, Iovance Therapeutics, and Kite Pharma. The institution G. Coukos is affiliated with has received fees for G. Coukos’ participation on an advisory board or for presentation at a company-sponsored symposium from Genentech, Roche, BMS, AstraZeneca, NextCure, Geneos Tx, and Sanofi/Avensis. G. Coukos has patents in the domain of antibodies and vaccines targeting the tumor vasculature as well as technologies related to T cell expansion and engineering for T cell therapy. G. Coukos holds patents around antibodies and receives royalties from the University of Pennsylvania regarding technology licensed to Novartis. The remaining authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

CD8+ T cell recognition of peptide epitopes plays a central role in immune responses against pathogens and tumors. However, the rules that govern which peptides are truly recognized by existing T cell receptors (TCRs) remain poorly understood, precluding accurate predictions of neo-epitopes for cancer immunotherapy. Here, we capitalize on recent (neo-)epitope data to train a predictor of immunogenic epitopes (PRIME), which captures molecular properties of both antigen presentation and TCR recognition. PRIME not only improves prioritization of neo-epitopes but also correlates with T cell potency and unravels biophysical determinants of TCR recognition that we experimentally validate. Analysis of cancer genomics data reveals that recurrent mutations tend to be less frequent in patients where they are predicted to be immunogenic, providing further evidence for immunoediting in human cancer. PRIME will facilitate identification of pathogen epitopes in infectious diseases and neo-epitopes in cancer immunotherapy., Competing Interests: The authors declare no competing interests. G. Ciriello is a member of the advisory board of Cell Reports Medicine., (© 2021 The Author(s).)

T-cell recognition of peptides incorporating nonsynonymous mutations, or neoepitopes, is a cornerstone of tumor immunity and forms the basis of new immunotherapy approaches including personalized cancer vaccines. Yet as they are derived from self-peptides, the means through which immunogenic neoepitopes overcome immune self-tolerance are often unclear. Here we show that a point mutation in a non-major histocompatibility complex anchor position induces structural and dynamic changes in an immunologically active ovarian cancer neoepitope. The changes pre-organize the peptide into a conformation optimal for recognition by a neoepitope-specific T-cell receptor, allowing the receptor to bind the neoepitope with high affinity and deliver potent T-cell signals. Our results emphasize the importance of structural and physical changes relative to self in neoepitope immunogenicity. Considered broadly, these findings can help explain some of the difficulties in identifying immunogenic neoepitopes from sequence alone and provide guidance for developing novel, neoepitope-based personalized therapies.

Among immune correlates of clinical responses, tumor-specific neoantigens took the spotlight as relevant targets for cancer immunotherapy. The implementation of pipelines for personalized cancer therapy remains challenging due to the privacy, that is patient-specificity, of neoantigens and the low-frequency of neoantigen-specific T cells in blood and tumor samples. To overcome these obstacles, recent developments in the field of biotechnology have allowed the multiplexed identification of neoepitope-specific T cells. This review addresses the pros and cons of conventional neoantigen screening methodologies and highlights the current as well as the prospective biotechnological opportunities in the field., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The main role of the human immune system is to eliminate cells presenting foreign antigens and abnormal patterns, while maintaining self-tolerance. However, when facing highly variable pathogens or antigens very similar to self-antigens, this system can fail in completely eliminating the anomalies, leading to the establishment of chronic pathologies. Prototypical examples of immune system defeat are cancer and Human Immunodeficiency Virus-1 (HIV-1) infection. In both conditions, the immune system is persistently exposed to antigens leading to systemic inflammation, lack of generation of long-term memory and exhaustion of effector cells. This triggers a negative feedback loop where effector cells are unable to resolve the pathology and cannot be replaced due to the lack of a pool of undifferentiated, self-renewing memory T cells. In addition, in an attempt to reduce tissue damage due to chronic inflammation, antigen presenting cells and myeloid components of the immune system activate systemic regulatory and tolerogenic programs. Beside these homologies shared between cancer and HIV-1 infection, the immune system can be shaped differently depending on the type and distribution of the eliciting antigens with ultimate consequences at the phenotypic and functional level of immune exhaustion. T cell differentiation, functionality, cytotoxic potential and proliferation reserve, immune-cell polarization, upregulation of negative regulators (immune checkpoint molecules) are indeed directly linked to the quantitative and qualitative differences in priming and recalling conditions. Better understanding of distinct mechanisms and functional consequences underlying disease-specific immune cell dysfunction will contribute to further improve and personalize immunotherapy. In the present review, we describe relevant players of immune cell exhaustion in cancer and HIV-1 infection, and enumerate the best-defined hallmarks of T cell dysfunction. Moreover, we highlight shared and divergent aspects of T cell exhaustion and T cell activation to the best of current knowledge., (Copyright © 2020 Vigano, Bobisse, Coukos, Perreau and Harari.)

In this study we present a novel microfluidic hydrodynamic trapping device to probe the cell-cell interaction between all cell samples of two distinct populations. We have exploited an hydrodynamic trapping method using microfluidics to immobilize a batch of cells from the first population at specific locations, then relied on hydrodynamic filtering principles, the flowing cells from the second cell population are placed in contact with the trapped ones, through a roll-over mechanism. The rolling cells interact with the serially trapped cells one after the other. The proposed microfluidic phenomenon was characterized with beads. We have shown the validity of our method by detecting the capacity of olfactory receptors to induce adhesion of cell doublets overexpressing these receptors. We report here the first controlled on-flow single cell resolution cell-cell interaction assay in a microfluidic device for future application in cell-cell interactions-based cell library screenings., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

Predictions of epitopes presented by class II human leukocyte antigen molecules (HLA-II) have limited accuracy, restricting vaccine and therapy design. Here we combined unbiased mass spectrometry with a motif deconvolution algorithm to profile and analyze a total of 99,265 unique peptides eluted from HLA-II molecules. We then trained an epitope prediction algorithm with these data and improved prediction of pathogen and tumor-associated class II neoepitopes.

Purpose: Characterization of tumor antigen-specific CD4 T-cell responses in healthy donors and malignant melanoma patients using an in vitro amplified T-cell library screening procedure., Patients and Methods: A high-throughput, human leukocyte antigen (HLA)-independent approach was used to estimate at unprecedented high sensitivity level precursor frequencies of tumor antigen- and neoantigen-specific CD4 T cells in healthy donors and patients with cancer. Frequency estimation was combined with isolation and functional characterization of identified tumor-reactive CD4 T-cell clones., Results: In healthy donors, we report frequencies of naïve tumor-associated antigen (TAA)-specific CD4 T cells comparable with those of CD4 T cells specific for infectious agents ( Tetanus toxoid ). Interestingly, we also identified low, but consistent numbers of memory CD4 T cells specific for several TAAs. In patients with melanoma, low frequencies of circulating TAA-specific CD4 T cells were detected that increased after peptide-based immunotherapy. Such antitumor TAA-specific CD4 T-cell responses were also detectable within the tumor-infiltrated tissues. TAA-specific CD4 T cells in patients displayed a highly polyfunctional state, with partial skewing to Type-2 polarization. Finally, we report the applicability of this approach to the detection and amplification of neoantigen-specific CD4 T cells., Conclusions: This simple, noninvasive, high-throughput screening of tumor- and neoantigen-specific CD4 T cells requires little biologic material, is HLA class II independent and allows the concomitant screening for a large number of tumor antigens of interest, including neoantigens. This approach will facilitate the immunomonitoring of preexisting and therapy-induced CD4 T-cell responses, and accelerate the development of CD4 T-cell-based therapies., (©2019 American Association for Cancer Research.)

We conducted a pilot clinical trial testing a personalized vaccine generated by autologous dendritic cells (DCs) pulsed with oxidized autologous whole-tumor cell lysate (OCDC), which was injected intranodally in platinum-treated, immunotherapy-naïve, recurrent ovarian cancer patients. OCDC was administered alone (cohort 1, n = 5), in combination with bevacizumab (cohort 2, n = 10), or bevacizumab plus low-dose intravenous cyclophosphamide (cohort 3, n = 10) until disease progression or vaccine exhaustion. A total of 392 vaccine doses were administered without serious adverse events. Vaccination induced T cell responses to autologous tumor antigen, which were associated with significantly prolonged survival. Vaccination also amplified T cell responses against mutated neoepitopes derived from nonsynonymous somatic tumor mutations, and this included priming of T cells against previously unrecognized neoepitopes, as well as novel T cell clones of markedly higher avidity against previously recognized neoepitopes. We conclude that the use of oxidized whole-tumor lysate DC vaccine is safe and effective in eliciting a broad antitumor immunity, including private neoantigens, and warrants further clinical testing., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Immunotherapy directed against private tumor neo-antigens derived from non-synonymous somatic mutations is a promising strategy of personalized cancer immunotherapy. However, feasibility in low mutational load tumor types remains unknown. Comprehensive and deep analysis of circulating and tumor-infiltrating lymphocytes (TILs) for neo-epitope specific CD8 + T cells has allowed prompt identification of oligoclonal and polyfunctional such cells from most immunotherapy-naive patients with advanced epithelial ovarian cancer studied. Neo-epitope recognition is discordant between circulating T cells and TILs, and is more likely to be found among TILs, which display higher functional avidity and unique TCRs with higher predicted affinity than their blood counterparts. Our results imply that identification of neo-epitope specific CD8 + T cells is achievable even in tumors with relatively low number of somatic mutations, and neo-epitope validation in TILs extends opportunities for mutanome-based personalized immunotherapies to such tumors.

Emerging clinical evidence on the role of the antitumor activity of the immune system has generated great interest in immunotherapy in all cancer types. Recent clinical data clearly demonstrated that human tumor cells express antigenic peptides (epitopes) that can be recognized by autologous tumor-specific T cells and that enhancement of such immune reactivity can potentially lead to cancer control and cancer regression in patients with advanced disease. However, in most cases, it is unclear which tumor antigens (Ags) mediated cancer regression. Mounting evidence indicates that numerous endogenous mutated cancer proteins, a hallmark of tumor cells, can be processed into peptides and presented on the surface of tumor cells, leading to their immune recognition in vivo as "non-self" or foreign. Massively parallel sequencing has now overcome the challenge of rapidly identifying the comprehensive mutational spectrum of individual tumors (i.e., the "mutanome") and current technologies, as well as computational tools, have emerged that allow the identification of private epitopes derived from their mutanome and called neoantigens (neoAgs). On this basis, both CD4(+) and CD8(+) neoantigen-specific T cells have been identified in multiple human cancers and shown to be associated with a favorable clinical outcome. Notably, emerging data also indicate that neoantigen recognition represents a major factor in the activity of clinical immunotherapies. In the post-genome era, the mutanome holds promise as a long-awaited 'gold mine' for the discovery of unique cancer cell targets, which are exclusively tumor-specific and unlikely to drive immune tolerance, hence offering the chance for highly promising clinical programs of cancer immunotherapy.

Telomerase (TERT) is overexpressed in 80% to 90% of primary tumors and contributes to sustaining the transformed phenotype. The identification of several TERT epitopes in tumor cells has elevated the status of TERT as a potential universal target for selective and broad adoptive immunotherapy. TERT-specific cytotoxic T lymphocytes (CTL) have been detected in the peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, but display low functional avidity, which limits their clinical utility in adoptive cell transfer approaches. To overcome this key obstacle hindering effective immunotherapy, we isolated an HLA-A2-restricted T-cell receptor (TCR) with high avidity for human TERT from vaccinated HLA-A*0201 transgenic mice. Using several relevant humanized mouse models, we demonstrate that TCR-transduced T cells were able to control human B-CLL progression in vivo and limited tumor growth in several human, solid transplantable cancers. TERT-based adoptive immunotherapy selectively eliminated tumor cells, failed to trigger a self-MHC-restricted fratricide of T cells, and was associated with toxicity against mature granulocytes, but not toward human hematopoietic progenitors in humanized immune reconstituted mice. These data support the feasibility of TERT-based adoptive immunotherapy in clinical oncology, highlighting, for the first time, the possibility of utilizing a high-avidity TCR specific for human TERT. Cancer Res; 76(9); 2540-51. ©2016 AACR., (©2016 American Association for Cancer Research.)

Immunotherapy is emerging as a promising anti-cancer curative modality. However, in contrast to recent advances obtained employing checkpoint blockade agents and T cell therapies, clinical efficacy of therapeutic cancer vaccines is still limited. Most vaccination attempts in the clinic represent "off-the shelf" approaches since they target common "self" tumor antigens, shared among different patients. In contrast, personalized approaches of vaccination are tailor-made for each patient and in spite being laborious, hold great potential. Recent technical advancement enabled the first steps in the clinic of personalized vaccines that target patient-specific mutated neo-antigens. Such vaccines could induce enhanced tumor-specific immune response since neo-antigens are mutation-derived antigens that can be recognized by high affinity T cells, not limited by central tolerance. Alternatively, the use of personalized vaccines based on whole autologous tumor cells, overcome the need for the identification of specific tumor antigens. Whole autologous tumor cells could be administered alone, pulsed on dendritic cells as lysate, DNA, RNA or delivered to dendritic cells in-vivo through encapsulation in nanoparticle vehicles. Such vaccines may provide a source for the full repertoire of the patient-specific tumor antigens, including its private neo-antigens. Furthermore, combining next-generation personalized vaccination with other immunotherapy modalities might be the key for achieving significant therapeutic outcome., (Copyright © 2015. Published by Elsevier B.V.)

In immune intervention trials, the comprehensive investigation of immunogenicity or T-cell epitope-mapping is challenging especially when a large set of epitopes needs to be screened and limited sample material is available. To this end, T-cell responses are often monitored using peptide pools. Here, we assessed the magnitude and sensitivity of detection of antigen-specific CD8 + and CD4 + T cells using a single peptide alone or mixed into large pools. Interestingly the magnitude of ex vivo anti-viral and anti-tumor T-cell responses was identical irrespective of the presence and number of irrelevant peptides, in different functional assays with PBMCs from healthy donors and cancer patients. Moreover, the presence of up to 300 irrelevant peptides did not affect the threshold of responsiveness of antigen-specific CD8 + T cells to single cognate peptides. These data demonstrate the relevance of using very large peptide pools for the sensitive and specific immune-monitoring of epitope-specific T cells in natural or immune-modulated context.

Immunodominance is a complex phenomenon that relies on a mere numerical concept, while being potentially influenced at every step of the immune response. We investigated the mechanisms leading to the establishment of CTL immunodominance in a retroviral model and found that the previously defined subdominant Env-specific CD8(+) T cells are endowed with an unexpectedly higher functional avidity than is the immunodominant Gag-recognizing counterpart. This high avidity, along with the Env Ag overload, results in a supraoptimal TCR engagement. The overstimulation makes Env-specific T lymphocytes more susceptible to apoptosis, thus hampering their expansion and leading to an unintentional "immune kamikazing." Therefore, Ag-dependent, hyperactivation-induced cell death can be regarded as a novel mechanism in the establishment of the immunodominance that restrains and opposes the expansion of high-avidity T cells in favor of lower-affinity populations., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.

The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27(KIP1), an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27(KIP1) expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5'UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF-encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27(KIP1) expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27(KIP1) activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27(KIP1) activity can also be modulated by an uORF and mutations affecting uORF could change p27(KIP1) expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest-derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL., Design: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics., Results: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine., Conclusions: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients., (©2012 AACR.)

Context: Anecdotal evidence suggests a high incidence in Trentino, Italy, of head and neck paragangliomas (HNPGL), a rare autosomal dominant disease called paraganglioma type 1 syndrome and caused by germ-line mutations of the SDHD gene., Objective: The aim of this study was to investigate the origin, spread, and clinical expression of the disease in this geographic region., Design, Setting, and Participants: Trentino natives with HNPGL were recruited for establishing clinical expression of the disease, presence of a founder effect, and age of common ancestor. A large sample of the local population was recruited for determination of mutation prevalence and spread., Main Outcome Measures: SDHD genetic testing was offered to first-degree relatives, and clinical surveillance was offered to at-risk carriers. The hypothesis of a founder effect was explored by haplotype analysis, and time to the most recent common ancestor was estimated by decay of haplotype sharing over time., Results: A total of 287 of the 540 recruited individuals from 95 kindreds carried the SDHD c.341A>G p.Tyr114Cys mutation. The prevalent phenotype was bilateral or multiple HNPGL, with low prevalence of pheochromocytoma and malignant forms. Penetrance was high. A common ancestor was dated between the 14th and 15th century, with the mutation spreading from the Mocheni Valley, a geographic, cultural and, presumably, a genetic isolate to 1.5% of the region's population., Conclusions: A combination of particular demographic, geographical, and historical conditions has resulted in the oldest and largest SDHD founder effect so far characterized and has transformed a rare disease into an endemic disease with major public health implications.

Background: Pheochromocytoma (Pheo) is usually considered a sporadic disease. Recently, an increasing rate of genetically based tumors has been reported. However, the need for systematic screening of unsuspected germline mutations in apparently sporadic forms is still debated. This study aimed to assess the effective rate of germline mutations causing Pheo and Paraganglioma (PGL), and the role of systematic genetic screening., Methods: Demographics, clinical, and genetic evaluation were performed in a series of 71 patients with Pheo and/or PGL., Results: Twelve patients had evident inherited/familial disease at presentation: NF1 (n = 4); MEN2 (n = 4), and familial Pheo/PGL (n = 4). Among 59 patients with apparently sporadic disease, unsuspected germline mutations occurred in 8 cases: TMEM127 (n = 4), SDHB (n = 2), VHL (n = 1), SDHC (n = 1). No differences were found between hereditary and sporadic disease concerning age, sex, and tumor size; bilateral Pheo and/or PGL and recurrences occurred most often in hereditary disease., Conclusion: Hereditary Pheo and/or PGL are frequent (28.2%). Inheritance is evident at presentation only in 16.9% of cases; 13.6% of apparently sporadic variants are genetically determined. Despite increased costs, systematic genetic screening might be useful because it might lead to a stricter follow-up, early diagnosis of recurrences in index cases and presymptomatic detection of disease in relatives., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Hereditary pheochromocytoma (PCC) is often caused by germline mutations in one of nine susceptibility genes described to date, but there are familial cases without mutations in these known genes. We sequenced the exomes of three unrelated individuals with hereditary PCC (cases) and identified mutations in MAX, the MYC associated factor X gene. Absence of MAX protein in the tumors and loss of heterozygosity caused by uniparental disomy supported the involvement of MAX alterations in the disease. A follow-up study of a selected series of 59 cases with PCC identified five additional MAX mutations and suggested an association with malignant outcome and preferential paternal transmission of MAX mutations. The involvement of the MYC-MAX-MXD1 network in the development and progression of neural crest cell tumors is further supported by the lack of functional MAX in rat PCC (PC12) cells and by the amplification of MYCN in neuroblastoma and suggests that loss of MAX function is correlated with metastatic potential.