Human P2Y11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death

Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4+ and CD8+ T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4+ and CD8+ T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y11 receptor. In a recombinant expression system, the coexpression of the human P2Y11 receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y11 receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y11 receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y11 receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation

Ex vivo modulation of intact tumor fragments with anti-PD-1 and anti-CTLA-4 influences the expansion and specificity of tumor-infiltrating lymphocytes

Checkpoint inhibition (CPI) therapy and adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL-based ACT) are the two most effective immunotherapies for the treatment of metastatic melanoma. While CPI has been the dominating therapy in the past decade, TIL-based ACT is beneficial for individuals even after progression on previous immunotherapies. Given that notable differences in response have been made when used as a subsequent treatment, we investigated how the qualities of TILs changed when the ex vivo microenvironment of intact tumor fragments were modulated with checkpoint inhibitors targeting programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Initially, we show that unmodified TILs from CPI-resistant individuals can be produced, are overwhelmingly terminally differentiated, and are capable of responding to tumor. We then investigate these properties in ex vivo checkpoint modulated TILs finding that that they retain these qualities. Lastly, we confirmed the specificity of the TILs to the highest responding tumor antigens, and identified this reactivity resides largely in CD39+CD69+ terminally differentiated populations. Overall, we found that anti-PD-1 will alter the proliferative capacity while anti-CTLA4 will influence breadth of antigen specificity

CD8+ T cells from patients with narcolepsy and healthy controls recognize hypocretin neuron-specific antigens

Autoreactive T cells are suspected to destroy hypocretin-producing neurons in narcolepsy. Here the authors detect CD8 T cells recognizing narcolepsy-related proteins in healthy individuals and in patients with narcolepsy, and show that the frequency of self-reactive CD8 T cells differs between patients and controls sharing the same HLA-II risk allele

Global network of computational biology communities: ISCB's regional student groups breaking barriers [version 1; peer review: Not peer reviewed]

Regional Student Groups (RSGs) of the International Society for Computational Biology Student Council (ISCB-SC) have been instrumental to connect computational biologists globally and to create more awareness about bioinformatics education. This article highlights the initiatives carried out by the RSGs both nationally and internationally to strengthen the present and future of the bioinformatics community. Moreover, we discuss the future directions the organization will take and the challenges to advance further in the ISCB-SC main mission: “Nurture the new generation of computational biologists”.Fil: Shome, Sayane. University of Iowa; Estados UnidosFil: Parra, Rodrigo Gonzalo. European Molecular Biology Laboratory; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fatima, Nazeefa. Uppsala Universitet; SueciaFil: Monzon, Alexander Miguel. Università di Padova; ItaliaFil: Cuypers, Bart. Universiteit Antwerp; BélgicaFil: Moosa, Yumna. University of KwaZulu Natal; SudáfricaFil: Da Rocha Coimbra, Nilson. Universidade Federal de Minas Gerais; BrasilFil: Assis, Juliana. Universidade Federal de Minas Gerais; BrasilFil: Giner Delgado, Carla. Universitat Autònoma de Barcelona; EspañaFil: Dönertaş, Handan Melike. European Molecular Biology Laboratory. European Bioinformatics Institute; Reino UnidoFil: Cuesta Astroz, Yesid. Universidad de Antioquia; Colombia. Universidad Ces. Facultad de Medicina.; ColombiaFil: Saarunya, Geetha. University of South Carolina; Estados UnidosFil: Allali, Imane. Universite Mohammed V. Rabat; Otros paises de África. University of Cape Town; SudáfricaFil: Gupta, Shruti. Jawaharlal Nehru University; IndiaFil: Srivastava, Ambuj. Indian Institute of Technology Madras; IndiaFil: Kalsan, Manisha. Jawaharlal Nehru University; IndiaFil: Valdivia, Catalina. Universidad Andrés Bello; ChileFil: Olguín Orellana, Gabriel José. Universidad de Talca; ChileFil: Papadimitriou, Sofia. Vrije Unviversiteit Brussel; Bélgica. Université Libre de Bruxelles; BélgicaFil: Parisi, Daniele. Katholikie Universiteit Leuven; BélgicaFil: Kristensen, Nikolaj Pagh. Technical University of Denmark; DinamarcaFil: Rib, Leonor. Universidad de Copenhagen; DinamarcaFil: Guebila, Marouen Ben. University of Luxembourg; LuxemburgoFil: Bauer, Eugen. University of Luxembourg; LuxemburgoFil: Zaffaroni, Gaia. University of Luxembourg; LuxemburgoFil: Bekkar, Amel. Universite de Lausanne; SuizaFil: Ashano, Efejiro. APIN Public Health Initiatives; NigeriaFil: Paladin, Lisanna. Università di Padova; ItaliaFil: Necci, Marco. Università di Padova; ItaliaFil: Moreyra, Nicolás Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentin

Detection of antigen-specific CD8 T cells in cancer, virus infections and autoimmunity

Antigen-specific CD8 T cells perform immune surveillance of host environments through binding to antigen-derived peptide epitopes bound in context of major-histocompatibility complex class I (MHC class I). When CD8 T cells recognize a peptide epitope, or pMHC molecules, important cytotoxic and inflammatory effects can be induced, which have important functional consequences across cancer and autoimmunity. Furthermore, recognition can mediate protective function in secondary virus infections where resident memory CD8 T cells quickly respond to a previously encountered infectious agent. Identification of epitopes “seen” by antigen-specific CD8 T cells is therefore of interest to immunologists as it allows for longitudinal monitoring of immune function in health and disease. It enables rational vaccine design, where a known immunogenic epitope is included in the given vaccine. It enables the same developers to test and monitor the quality of their vaccination platform as T cell responses should be robust and memory long-lived. It enables clinicians to monitor CMV-specific CD8 T cells during bone-marrow transplantation. And it enables translational scientists to isolate and record T cell receptors that can be used to inform the development of a next generation of transgenic cellular therapies. Identification of peptide epitopes is therefore of interest to the science community.Manuscript 1. We establish that neoepitopes are detectable among expanded tumor-infiltrating lymphocyte (TILs) in TIL-infusion products used for treating metastatic melanoma patients with adoptive cell therapy (TIL-ACT), and that recognition of neoepitopes occur at higher CD8 T cell frequencies in responding than non-responding patients. Furthermore, neoepitope-specific CD8 T cells persists for years in responding patients, whereas the counterpart in non-responding patients appear more transient.Manuscript 2. In my second manuscript, I design a minimal panel of virus-derived epitopes frequently recognized by CD8 T cells in healthy and hepatitis donors. I do so across a wide range of HLA genotypes and viruses without the use of MHC-binding prediction. Instead I selected candidates from the Immune Epitope Database (IEDB). By also introducing a pre-stimulation step in our high-throughput pMHC multimer-binding assay I could furthermore assign a relative category of TCR-engagement, which correlated with the subsequent induction of activation markers CD69 and CD137. Such simultaneous capture of pMHC multimer binding and TCR-engagement is valuable, because it allows the investigator to selectively study multimer-binding in context of TCR-binding and activation.Manuscript 3. In my third and final manuscript, we attempted to map epitopes towards four EBV latent antigens and five historically relevant antigens associated with myelin sheets of the central nervous system. While we were blinded to all clinical information, we report an observed depletion of antigen-specific CD8 T cells specific for latent EBV and neuroantigens in context of A*02:01 and B*07:02. Such depletion was not present in context of A*01:01 or B*08:01, nor was the depletion observed for FLU- and CMV-specific multimers. Neuroantigen-specific CD8 T cells were furthermore characterized by being CD8low and almost exclusively of naïve/central memory phenotypes, whereas virus-specific CD8 T cells were CD8high and almost exclusively non-naïve.Collectively the reports enclosed provide epitope-targets and preliminary phenotypes for the further investigation of neoepitope-specific, virus-specific, and neuroantigen-specific CD8 T cells in health and disease

Simultaneous analysis of pMHC binding and reactivity unveils virus-specific CD8 T cell immunity to a concise epitope set

CD8 T cells provide immunity to virus infection through recognition of epitopes presented by peptide major histocompatibility complexes (pMHCs). To establish a concise panel of widely recognized T cell epitopes from common viruses, we combined analysis of TCR down-regulation upon stimulation with epitope-specific enumeration based on barcode-labeled pMHC multimers. We assess CD8 T cell binding and reactivity for 929 previously reported epitopes in the context of 1 of 25 HLA alleles representing 29 viruses. The prevalence and magnitude of CD8 T cell responses were evaluated in 48 donors and reported along with 137 frequently recognized virus epitopes, many of which were underrepresented in the public domain. Eighty-four percent of epitope-specific CD8 T cell populations demonstrated reactivity to peptide stimulation, which was associated with effector and long-term memory phenotypes. Conversely, nonreactive T cell populations were associated primarily with naive phenotypes. Our analysis provides a reference map of epitopes for characterizing CD8 T cell responses toward common human virus infections.</p

image_2_Human P2Y11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death.tif

<p>Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4⁺ and CD8⁺ T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4⁺ and CD8⁺ T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y₁₁ receptor. In a recombinant expression system, the coexpression of the human P2Y₁₁ receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y₁₁ receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y₁₁ receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y₁₁ receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation.</p

Neoantigen-reactive CD8+ T cells affect clinical outcome of adoptive cell therapy with tumor-infiltrating lymphocytes in melanoma

BACKGROUND. Neoantigen-driven recognition and T cell–mediated killing contribute to tumor clearance following adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs). Yet how diversity, frequency, and persistence of expanded neoepitope-specific CD8+ T cells derived from TIL infusion products affect patient outcome is not fully determined. METHODS. Using barcoded pMHC multimers, we provide a comprehensive mapping of CD8+ T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic melanoma patients who received ACT. RESULTS. We identified 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observed neoepitope-specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we found that the frequency of neoepitope-specific CD8+ T cells in TIL infusion products correlated with increased survival and that neoepitope-specific CD8+ T cells shared with the infusion product in posttreatment blood samples were unique to responders of TIL-ACT. Finally, we found that a transcriptional signature for lymphocyte activity within the tumor microenvironment was associated with a higher frequency of neoepitope-specific CD8+ T cells in the infusion product. CONCLUSIONS. These data support previous case studies of neoepitope-specific CD8+ T cells in melanoma and indicate that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8+ T cells. FUNDING. NEYE Foundation; European Research Council; Lundbeck Foundation Fellowship; Carlsberg Foundation