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Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. Although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals, the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence, we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions (DMRs) when compared to memory CD8+ T cells. Among the top demethylated DMRs in cytomegalovirus-specific CD8+ T cells was TBKBP1, coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells, we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with higher TBKBP1 expression at both mRNA and protein level, independent of alternative splicing of TBKBP1 transcripts. Notably, the distinct DNA methylation patterns in CD8+ T cell subsets was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively, our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies. Author summary: Human cytomegalovirus (CMV) is a herpesvirus that infects a significant portion of the global population. While it usually causes asymptomatic or mild infections, CMV can have severe consequences for individuals with a weakened immune system, such as stem cell or organ transplant recipients or individuals with HIV/AIDS. The immune response to CMV is characterized by expansion of virus-specific CD8+ T cells, which recognize and eliminate CMV-infected cells, thereby controlling the infection. Studies have shown that the pathogen-induced differentiation of naive to effector memory CD8+ T cells is accompanied by epigenetic changes. In order to identify the major genes involved in the functionality of CMV-specific CD8+ T cells, which are also regulated by DNA methylation, we compared the methylation profiles of CMV-specific CD8+ T cells with memory CD8+ T cells. As a result of this, we found that TBK-binding protein 1 (TBKBP1) plays a crucial role in the function of CMV-specific CD8+ T cells and enhances virus neutralization capacity upon overexpression, which has not been reported previously. This study opens the possibility of not only identifying unknown genes that contribute to the functionality of CMV-specific CD8+ T cells, but also potentially lead to improvements in adoptive T cell therapies. [ABSTRACT FROM AUTHOR]

Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Discussion: ARMATA does not only allow same day identification of antiviral Tcell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection. [ABSTRACT FROM AUTHOR]

Complications due to HCMV infection or reactivation remain a challenging clinical problem in immunocompromised patients, mainly due to insufficient or absent T‐cell functionality. Knowledge of viral targets is crucial to improve monitoring of high‐risk patients and optimise antiviral T‐cell therapy. To expand the epitope spectrum, genetically‐engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble (s)HLA‐A*11:01 and infected with an HCMV mutant lacking immune evasion molecules (US2‐6 + 11). More than 700 HLA‐A*11:01‐restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open‐reading‐frames (ORFs) were identified by mass spectrometry and screened for HLA‐A*11:01‐binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy HLA‐A*11:01+/HCMV+ donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti‐apoptotic protein UL36 and ORFL101C (A11SAL), were identified. Their HLA‐A*11:01 complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T‐cell responses against A11SAL, with T‐cell responses comparable to the immunodominant HLA‐A*02:01‐restricted HCMVpp65NLV epitope. A11SAL‐specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV‐infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically‐engineered DCs to identify naturally presented immunodominant HCMV‐derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells. Author summary: Cytomegalovirus (CMV) infection is a very common cause of complications in transplant recipients. These patients suffer from opportunistic viruses because their immune system is weakened by immunosuppressive drugs, which are required to avoid transplant rejection by T cells, a key lymphocyte population involved in antiviral defenses. The commonly used immunosuppressive drugs target gene products in a signaling pathway inside T lymphocytes involving two Nuclear Factors of Activated T cells (NFAT) called NFATc1 and NFATc2. We tested the effects of NFAT signaling on T cell responses to CMV in mice lacking either of these genes, or both of them. We show that the T cell responses to CMV are compromised in absence of NFATc1 in a peculiar manner. While early responses upon infection are robust, they are not maintained in the long term. A detailed analysis of these cells showed that cells lacking NFAT display a deficit in T-cell maturation upon the initial activation. Our data may have relevance for the design of future immunosuppressive drugs that may protect patients from organ rejection, while retaining antiviral immune defenses. [ABSTRACT FROM AUTHOR]

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS21 human cells. Our data show that the spike protein of SARSCoV- 2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. Although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals, the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence, we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions (DMRs) when compared to memory CD8+ T cells. Among the top demethylated DMRs in cytomegalovirus-specific CD8+ T cells was TBKBP1, coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells, we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with higher TBKBP1 expression at both mRNA and protein level, independent of alternative splicing of TBKBP1 transcripts. Notably, the distinct DNA methylation patterns in CD8+ T cell subsets was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively, our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chaudhry et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The potential of memory T cells to provide protection against reinfection is beyond question. Yet, it remains debated whether long-term T cell memory is due to long-lived memory cells. There is ample evidence that blood-derived memory phenotype CD8 + T cells maintain themselves through cell division, rather than through longevity of individual cells. It has recently been proposed, however, that there may be heterogeneity in the lifespans of memory T cells, depending on factors such as exposure to cognate Ag. CMV infection induces not only conventional, contracting T cell responses, but also inflationary CD8 + T cell responses, which are maintained at unusually high numbers, and are even thought to continue to expand over time. It has been proposed that such inflating T cell responses result from the accumulation of relatively long-lived CMV-specific memory CD8 + T cells. Using in vivo deuterium labeling and mathematical modeling, we found that the average production rates and expected lifespans of mouse CMV-specific CD8 + T cells are very similar to those of bulk memory-phenotype CD8 + T cells. Even CMV-specific inflationary CD8 + T cell responses that differ 3-fold in size were found to turn over at similar rates., (Copyright © 2022 by The American Association of Immunologists, Inc.)