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The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states. High resolution characterisation of the virus specific T cell response to SARS CoV2 provides further understanding to the immune response to the infection. Here the authors apply a reverse phenotyping approach to interrogate the SARS-CoV-2 T cell compartment at single cell resolution." [ABSTRACT FROM AUTHOR]

The Epstein-Barr virus (EBV) establishes lifelong infections in > 90% of the human population. Although contained as asymptomatic infection by the immune system in most individuals, EBV is associated with the pathogenesis of approximately 1.5% of all cancers in humans. Some of these EBV-associated tumors have been successfully treated by the infusion of virus-specific T-cell lines. Recent sequence analyses of a large number of viral isolates suggested that distinct EBV strains have evolved in different parts of the world. Here, we assessed the impact of such sequence variations on EBV-specific T-cell immunity. With the exceptions of EBNA2 and the EBNA3 family of proteins, an overall low protein sequence disparity of about 1% was noted between Asian viral isolates, including the newly characterized M81 strain, and the prototypic EBV type 1 and type 2 strains. However, when T-cell epitopes including their flanking regions were compared, a substantial proportion was found to be polymorphic in different EBV strains. Importantly, CD4+ and CD8+ T-cell clones specific for viral epitopes from one strain often showed diminished recognition of the corresponding epitopes in other strains. In addition, T-cell recognition of a conserved epitope was affected by amino acid exchanges within the epitope flanking region. Moreover, the CD8+ T-cell response against polymorphic epitopes varied between donors and often ignored antigen variants. These results demonstrate that viral strain heterogeneity may impair antiviral T-cell immunity and suggest that immunotherapeutic approaches against EBV should preferably target broad sets of conserved epitopes including their flanking regions. [ABSTRACT FROM AUTHOR]

T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but function and specificity of the CD4+ component are still poorly defined. Here, we assessed the tumor-protective potential of different CD4+ T-cell specificities in a PTLD-SCID mouse model. Injection of different virus-specific CD4+ T-cell clones showed that single specificities were capable of prolonging mouse survival and that the degree of tumor protection directly correlated with recognition of target cells in vitro. Surprisingly, some CD4+ T-cell clones promoted tumor development, suggesting that besides antigen recognition, still elusive functional differences exist among virus-specific T cells. Of several EBV-specific CD4+ T-cell clones tested, those directed against virion antigens proved most tumor-protective. However, enriching these specificities in LCL-stimulated preparations conferred no additional survival benefit. Instead, CD4+ T cells specific for unknown, probably self-antigens were identified as principal antitumoral effectors in LCL-stimulated T-cell lines. These results indicate that virion and still unidentified cellular antigens are crucial targets of the CD4+ T-cell response in this preclinical PTLD-model and that enriching the corresponding T-cell specificities in therapeutic preparations may enhance their clinical efficacy. Moreover, the expression in several EBV-negative B-cell lymphoma cell lines implies that these putative autoantigen(s) might also qualify as targets for T-cell-based immunotherapy of virus-negative B cell malignancies. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans by latently infecting B cells, with occasional cycles of reactivation, virus production, and reinfection. Protective immunity against EBV is mediated by T cells, but the role of EBV-specific T helper (Th) cells is still poorly defined. Here, we study the Th response to the EBV lytic cycle proteins BLLF1 (gp350/220), BALF4 (gp110), and BZLF1 and show that glycoprotein-specific Th cells recognize EBV-positive cells directly; surprisingly, a much higher percentage of target cells than those expressing lytic cycle proteins were recognized. Antigen is efficiently transferred to bystander B cells by receptor-mediated uptake of released virions, resulting in recognition of target cells incubated with <1 virion/cell. T cell recognition does not require productive infection and occurs early after virus entry before latency is established. Glycoprotein-specific Th cells are cytolytic and inhibit proliferation of lymphoblastoid cell lines (LCL) and the outgrowth of LCL after infection of primary B cells with EBV. These results establish a novel role for glycoprotein-specific Th cells in the control of EBV infection and identify virion proteins as important immune targets. These findings have implications for the treatment of diseases associated with EBV and potentially other coated viruses infecting MHC class II-positive cells. [ABSTRACT FROM AUTHOR]