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The mechanisms underlying the efficacy of anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) therapy are incompletely understood. Here, by immune profiling responding PD-1 + CD8 + T (T Resp ) cell populations from patients with advanced melanoma, we identified differential programming of T Resp cells in response to combination therapy, from an exhausted toward a more cytotoxic effector program. This effect does not occur with anti-PD-1 monotherapy. Single-cell transcriptome and T cell receptor repertoire analysis was used to identify altered effector programming of expanding PD-1 + CD8 + T cell clones with distinct regulon usage, STAT1 and STAT3 utilization and antitumor specificity connected to interleukin (IL)-21 signaling in combination and anti-CTLA-4 monotherapy. Therapeutic efficacy of CTLA-4 blockade was lost in B16F10 melanoma models with either Il21r - deficiency or anti-IL-21 receptor blockade. Together, these results show how IL-21 signaling to T Resp is critical for anti-CTLA-4-based checkpoint therapies and highlight major signaling differences to anti-PD-1 monotherapy., Competing Interests: Competing interests: F.M. served as consultant and/or has received honoraria or travel support from Novartis, Roche, BMS, MSD, Pierre Fabre and Sunpharma. D.R.S. served as consultant and/or has received honoraria from Roche and BMS and travel support from Sunpharma and Sanofi. R.Z. has received honoraria from Novartis, INcyte, MNK and Sanofi. R.V. has received honoraria or travel support from Abbvie, AstraZeneca, Galapagos, Janssen-Cilag, Novartis, Pfizer and Roche, and research grant support from Novartis and Pfizer. J.W. consulted for and received

Hepatitis B virus (HBV)-specific CD8 +  T cells play a dominant role during acute-resolving HBV infection but are functionally impaired during chronic HBV infection in humans. These functional deficits have been linked with metabolic and phenotypic heterogeneity, but it has remained unclear to what extent different subsets of HBV-specific CD8 +  T cells still suppress viral replication. We addressed this issue by deep profiling, functional testing and perturbation of HBV-specific CD8 +  T cells during different phases of chronic HBV infection. Our data revealed a mechanism of effector CD8 +  T cell attenuation that emerges alongside classical CD8 +  T cell exhaustion. Attenuated HBV-specific CD8 +  T cells were characterized by cytotoxic properties and a dampened effector differentiation program, determined by antigen recognition and TGFβ signaling, and were associated with viral control during chronic HBV infection. These observations identify a distinct subset of CD8 +  T cells linked with immune efficacy in the context of a chronic human viral infection with immunotherapeutic potential., (© 2024. The Author(s).)

Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide 1,2 , in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes 3-7 . Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6 + CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6 + CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion., (© 2024. The Author(s).)

HBV-specific CD8+ T cells are only present at the low frequency during chronic infection. Thus, they are often undetectable by conventional ex vivo staining methods using peptide-loaded HLA class I tetramers. Detection sensitivity can be increased by magnetic bead-based enrichment strategies following staining with peptide-loaded HLA class I tetramers. Additionally, some downstream applications like e.g., single cell RNA sequencing of virus-specific CD8+ T cells may also require a pre-enrichment step to increase the frequency of the cells of interest. For this, peptide-loaded HLA class I tetramers-associated magnetic bead-based enrichment is also a suitable method., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background & Aims: Exhaustion of CD8 T cells has been suggested to inform different clinical outcomes in Crohn's disease, but detailed analyses are lacking. This study aimed to identify the role of exhaustion on a single-cell level and identify relevant CD8 T cell populations in Crohn's disease., Methods: Blood and intestinal tissue from 58 patients with Crohn's disease (active disease or remission) were assessed for CD8 T cell expression of exhaustion markers and their cytokine profile by highly multiplexed flow and mass cytometry. Key disease-associated subsets were sorted and analyzed by RNA sequencing. CD39 inhibition assays were performed in vitro., Results: Activated CD39 + and CD39 + PD-1 + CD8 T cell subsets expressing multiple exhaustion markers were enriched at low frequency in active Crohn's disease. Their cytokine production capacity was inversely linked to the Harvey-Bradshaw Index. Subset-level protein and transcriptome profiling revealed co-existence of effector and exhaustion programs in CD39 + and CD39 + PD-1 + CD8 T cells, with CD39 + cells likely originating from the intestine. CD39 enzymatic activity controlled T cell cytokine production. Importantly, transcriptional exhaustion signatures were enriched in remission in CD39-expressing subsets with up-regulation of TOX. Subset-level transcriptomics revealed a CD39-related gene module that is associated with the clinical course., Conclusions: These data showed a role for the exhaustion of peripheral CD39-expressing CD8 T cell subsets in Crohn's disease. Their low frequency illustrated the utility of single-cell cytometry methods for identification of relevant immune populations. Importantly, the link of their exhaustion status to the clinical activity and their specific gene signatures have implications for exhaustion-based personalized medicine approaches., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Recent findings indicate that many immunopathologies are at their roots a consequence of impaired immune responses ("too little" immunity) and not the result of primarily exaggerated immune responses ("too much" immunity). We have summarized this conceptional view as "IMPATH paradox." In this review, we will focus on impaired immune reactions in the context of CD8 + T-cell-mediated immunopathologies. In particular, we will exemplify this concept in two disease models: Virus-triggered primary hemophagocytic lymphohistiocytosis, an inflammatory syndrome caused by genetically impaired cytolytic functions of T cells, and viral hepatitis, where T-cell exhaustion is a major underlying mechanism for impaired effector functions. In both situations, T cells fail to eliminate the source of immune stimulation, which usually serves as an important negative feedback loop curtailing immune reactions. Persistent antigen presentation by APCs and/or infected cells results in continuous stimulation causing chronic inflammation and immunopathology mediated by residual T-cell functions. Hence, immune stimulation or reconstitution rather than immune suppression may be strategies for therapeutic interventions., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Immunization with two mRNA vaccine doses elicits robust spike-specific CD8 + T cell responses, but reports of waning immunity after COVID-19 vaccination prompt the introduction of booster vaccination campaigns. However, the effect of mRNA booster vaccination on the spike-specific CD8 + T cell response remains unclear. Here we show that spike-specific CD8 + T cells are activated and expanded in all analyzed individuals receiving the 3 rd and 4 th mRNA vaccine shots. This CD8 + T cell boost response is followed by a contraction phase and lasts only for about 30-60 days. The spike-specific CD8 + T memory stem cell pool is not affected by the 3 rd vaccination. Both 4 th vaccination and breakthrough infections with Delta and Omicron rapidly reactivate CD8 + T memory cells. In contrast, neutralizing antibody responses display little boost effect towards Omicron. Thus, COVID-19 mRNA booster vaccination elicits a transient T effector cell response while long-term spike-specific CD8 + T cell immunity is conserved to mount robust memory recall targeting emerging variants of concern., (© 2022. The Author(s).)

SARS-CoV-2 spike mRNA vaccines 1-3 mediate protection from severe disease as early as ten days after prime vaccination 3 , when neutralizing antibodies are hardly detectable 4-6 . Vaccine-induced CD8 + T cells may therefore be the main mediators of protection at this early stage 7,8 . The details of their induction, comparison to natural infection, and association with other arms of vaccine-induced immunity remain, however, incompletely understood. Here we show on a single-epitope level that a stable and fully functional CD8 + T cell response is vigorously mobilized one week after prime vaccination with bnt162b2, when circulating CD4 + T cells and neutralizing antibodies are still weakly detectable. Boost vaccination induced a robust expansion that generated highly differentiated effector CD8 + T cells; however, neither the functional capacity nor the memory precursor T cell pool was affected. Compared with natural infection, vaccine-induced early memory T cells exhibited similar functional capacities but a different subset distribution. Our results indicate that CD8 + T cells are important effector cells, are expanded in the early protection window after prime vaccination, precede maturation of other effector arms of vaccine-induced immunity and are stably maintained after boost vaccination., (© 2021. The Author(s).)

Genetic variants of the interferon lambda (IFNL) gene locus are strongly associated with spontaneous and IFN treatment-induced clearance of hepatitis C virus (HCV) infections. Individuals with the ancestral IFNL4-dG allele are not able to clear HCV in the acute phase and have more than a 90% probability to develop chronic hepatitis C (CHC). Paradoxically, the IFNL4-dG allele encodes a fully functional IFNλ4 protein with antiviral activity against HCV. Here we describe an effect of IFNλ4 on HCV antigen presentation. Only minor amounts of IFNλ4 are secreted, because the protein is largely retained in the endoplasmic reticulum (ER) where it induces ER stress. Stressed cells are significantly weaker activators of HCV specific CD8 + T cells than unstressed cells. This is not due to reduced MHC I surface presentation or extracellular IFNλ4 effects, since T cell responses are restored by exogenous loading of MHC with HCV antigens. Rather, IFNλ4 induced ER stress impairs HCV antigen processing and/or loading onto the MHC I complex. Our results provide a potential explanation for the IFNλ4-HCV paradox., (© 2021. The Author(s).)

COVID-19 can cause severe neurological symptoms, but the underlying pathophysiological mechanisms are unclear. Here, we interrogated the brain stems and olfactory bulbs in postmortem patients who had COVID-19 using imaging mass cytometry to understand the local immune response at a spatially resolved, high-dimensional, single-cell level and compared their immune map to non-COVID respiratory failure, multiple sclerosis, and control patients. We observed substantial immune activation in the central nervous system with pronounced neuropathology (astrocytosis, axonal damage, and blood-brain-barrier leakage) and detected viral antigen in ACE2-receptor-positive cells enriched in the vascular compartment. Microglial nodules and the perivascular compartment represented COVID-19-specific, microanatomic-immune niches with context-specific cellular interactions enriched for activated CD8 + T cells. Altered brain T-cell-microglial interactions were linked to clinical measures of systemic inflammation and disturbed hemostasis. This study identifies profound neuroinflammation with activation of innate and adaptive immune cells as correlates of COVID-19 neuropathology, with implications for potential therapeutic strategies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In chronic hepatitis C virus (HCV) infection, exhausted HCV-specific CD8 + T cells comprise memory-like and terminally exhausted subsets. However, little is known about the molecular profile and fate of these two subsets after the elimination of chronic antigen stimulation by direct-acting antiviral (DAA) therapy. Here, we report a progenitor-progeny relationship between memory-like and terminally exhausted HCV-specific CD8 + T cells via an intermediate subset. Single-cell transcriptomics implicated that memory-like cells are maintained and terminally exhausted cells are lost after DAA-mediated cure, resulting in a memory polarization of the overall HCV-specific CD8 + T cell response. However, an exhausted core signature of memory-like CD8 + T cells was still detectable, including, to a smaller extent, in HCV-specific CD8 + T cells targeting variant epitopes. These results identify a molecular signature of T cell exhaustion that is maintained as a chronic scar in HCV-specific CD8 + T cells even after the cessation of chronic antigen stimulation.

Emerging data indicate that SARS-CoV-2-specific CD8 + T cells targeting different viral proteins are detectable in up to 70% of convalescent individuals 1-5 . However, very little information is currently available about the abundance, phenotype, functional capacity and fate of pre-existing and induced SARS-CoV-2-specific CD8 + T cell responses during the natural course of SARS-CoV-2 infection. Here, we define a set of optimal and dominant SARS-CoV-2-specific CD8 + T cell epitopes. We also perform a high-resolution ex vivo analysis of pre-existing and induced SARS-CoV-2-specific CD8 + T cells, applying peptide-loaded major histocompatibility complex class I (pMHCI) tetramer technology. We observe rapid induction, prolonged contraction and emergence of heterogeneous and functionally competent cross-reactive and induced memory CD8 + T cell responses in cross-sectionally analyzed individuals with mild disease following SARS-CoV-2 infection and three individuals longitudinally assessed for their T cells pre- and post-SARS-CoV-2 infection. SARS-CoV-2-specific memory CD8 + T cells exhibited functional characteristics comparable to influenza-specific CD8 + T cells and were detectable in SARS-CoV-2 convalescent individuals who were seronegative for anti-SARS-CoV-2 antibodies targeting spike (S) and nucleoprotein (N). These results define cross-reactive and induced SARS-CoV-2-specific CD8 + T cell responses as potentially important determinants of immune protection in mild SARS-CoV-2 infection.

Background and Aims: Dysregulated T cell responses contribute to the pathogenesis of inflammatory bowel disease [IBD]. Because vitamin D [vitD] deficiency is a risk factor for adverse disease outcomes, we aimed to characterize the impact of vitD on intestinal and peripheral T cell profiles., Methods: T cells were isolated from peripheral blood and intestinal biopsies of IBD patients, incubated with vitD and characterized by flow cytometry. To translate these in vitro findings to the clinic, serum vitD concentrations and clinical outcomes were correlated with T cell phenotype and function in a prospective patient cohort., Results: Incubation of peripheral and intestinal T cells with 1,25(OH)2-vitD resulted in strongly reduced frequencies of pro-inflammatory CD4+ and CD8+ T cells producing interferon γ [IFNγ], interleukin-17 [IL-17], IL-22, IL-9 and tumour necrosis factor [TNF]. Univariable analysis of 200 IBD patients revealed associations of vitD deficiency with non-compliant vitD intake, season of the year and anaemia in Crohn's disease [CD] as well as disease activity in ulcerative colitis [UC]. Ex vivo immunophenotyping revealed that CD4+ and CD8+ T cell subsets were not substantially altered in vitD-deficient vs vitD-sufficient patients while regulatory T cell frequencies were reduced in UC and non-smoking CD patients with vitD deficiency. However, normalization of serum vitD concentrations in previously deficient CD patients resulted in significantly reduced frequencies of CD4+ T cells producing IFNγ, IL-17 and IL-22., Conclusion: vitD exerts profound anti-inflammatory effects on peripheral and intestinal CD4+ and CD8+ T cells of IBD patients in vitro and inhibits TH1 and TH17 cytokine production in CD patients in vivo., (Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background & Aims: CD100 is constitutively expressed on T cells and can be cleaved from the cell surface by matrix metalloproteases (MMPs) to become soluble CD100 (sCD100). Both membrane-bound CD100 (mCD100) and sCD100 have important immune regulatory functions that promote immune cell activation and responses. This study investigated the expression and role of mCD100 and sCD100 in regulating antiviral immune responses during HBV infection., Methods: mCD100 expression on T cells, sCD100 levels in the serum, and MMP expression in the liver and serum were analysed in patients with chronic HBV (CHB) and in HBV-replicating mice. The ability of sCD100 to mediate antigen-presenting cell maturation, HBV-specific T cell activation, and HBV clearance were analysed in HBV-replicating mice and patients with CHB., Results: Patients with CHB had higher mCD100 expression on T cells and lower serum sCD100 levels compared with healthy controls. Therapeutic sCD100 treatment resulted in the activation of DCs and liver sinusoidal endothelial cells, enhanced HBV-specific CD8 T cell responses, and accelerated HBV clearance, whereas blockade of its receptor CD72 attenuated the intrahepatic anti-HBV CD8 T cell response. Together with MMP9, MMP2 mediated mCD100 shedding from the T cell surface. Patients with CHB had significantly lower serum MMP2 levels, which positively correlated with serum sCD100 levels, compared with healthy controls. Inhibition of MMP2/9 activity resulted in an attenuated anti-HBV T cell response and delayed HBV clearance in mice., Conclusions: MMP2/9-mediated sCD100 release has an important role in regulating intrahepatic anti-HBV CD8 T cell responses, thus mediating subsequent viral clearance during HBV infection., Lay Summary: Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. The clearance of HBV relies largely on an effective T cell immune response, which usually becomes dysregulated in chronic HBV infection. Our study provides a new mechanism to elucidate HBV persistence and a new target for developing immunotherapy strategies in patients chronically infected with HBV., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Chronic hepatitis B virus (HBV) infection is a major global health burden affecting around 257 million people worldwide. The consequences of chronic HBV infection include progressive liver damage, liver cirrhosis, and hepatocellular carcinoma. Although current direct antiviral therapies successfully lead to suppression of viral replication and deceleration of liver cirrhosis progression, these treatments are rarely curative and patients often require a life-long therapy. Based on the ability of the immune system to control HBV infection in at least a subset of patients, immunotherapeutic approaches are promising treatment options to achieve HBV cure. In particular, T cell-based therapies are of special interest since CD8 + T cells are not only capable to control HBV infection but also to eliminate HBV-infected cells. However, recent data show that the molecular mechanisms underlying CD8 + T-cell failure in chronic HBV infection depend on the targeted antigen and thus different strategies to improve the HBV-specific CD8 + T-cell response are required. Here, we review the current knowledge about the heterogeneity of impaired HBV-specific T-cell populations and the potential consequences for T cell-based immunotherapeutic approaches in HBV cure., (Copyright © 2019 Heim, Neumann-Haefelin, Thimme and Hofmann.)

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential 1-4 . This process, known as T cell exhaustion or dysfunction 1 , is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1) 5-8 . The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood 9-12 . Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein 13-15 . TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1 + self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.

Background & Aims: Endoplasmic reticulum aminopeptidase 1 (ERAP1) polymorphisms are linked with human leukocyte antigen (HLA) class I-associated autoinflammatory disorders, including ankylosing spondylitis and Behçet's disease. Disease-associated ERAP1 allotypes exhibit distinct functional properties, but it remains unclear how differential peptide trimming in vivo affects the repertoire of epitopes presented to CD8 + T cells. The aim of this study was to determine the impact of ERAP1 allotypes on the virus-specific CD8 + T cell epitope repertoire in an HLA-B*27:05 + individual with acute hepatitis C virus (HCV) infection., Methods: We performed genetic and functional analyses of ERAP1 allotypes and characterized the HCV-specific CD8 + T cell repertoire at the level of fine epitope specificity and HLA class I restriction, in a patient who had acquired an HCV genotype 1a infection through a needle-stick injury., Results: Two hypoactive allotypic variants of ERAP1 were identified in an individual with acute HCV infection. The associated repertoire of virus-derived epitopes recognized by CD8 + T cells was uncommon in a couple of respects. Firstly, reactivity was directed away from classically immunodominant epitopes, preferentially targeting either novel or subdominant epitopes. Secondly, reactivity was biased towards longer epitopes (10-11-mers). Despite the patient exhibiting favorable prognostic indicators, these atypical immune responses failed to clear the virus and the patient developed persistent low-level infection with HCV., Conclusions: ERAP1 allotypes modify the virus-specific CD8 + T cell epitope repertoire in vivo, leading to altered immunodominance patterns that may contribute to the failure of antiviral immunity after infection with HCV., Lay Summary: Endoplasmic reticulum aminopeptidase 1 (ERAP1) plays a key role in antigen presentation. Genetic variants of ERAP1 (leading to distinct allotypes) are linked with specific autoinflammatory disorders, such as ankylosing spondylitis and Behçet's disease. We found that ERAP1 allotypes modified the repertoire of virus-specific CD8 + T cell epitopes in a patient with hepatitis C virus, leading to an altered pattern of immunodominance that may have contributed to the failure of antiviral immunity in this patient., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Objective: A hallmark of chronic HBV (cHBV) infection is the presence of impaired HBV-specific CD8+ T cell responses. Functional T cell exhaustion induced by persistent antigen stimulation is considered a major mechanism underlying this impairment. However, due to their low frequencies in chronic infection, it is currently unknown whether HBV-specific CD8+ T cells targeting different epitopes are similarly impaired and share molecular profiles indicative of T cell exhaustion., Design: By applying peptide-loaded MHC I tetramer-based enrichment, we could detect HBV-specific CD8+ T cells targeting epitopes in the HBV core and the polymerase proteins in the majority of 85 tested cHBV patients with low viral loads. Lower detection rates were obtained for envelope-specific CD8+ T cells. Subsequently, we performed phenotypic and functional in-depth analyses., Results: HBV-specific CD8+ T cells are not terminally exhausted but rather exhibit a memory-like phenotype in patients with low viral load possibly reflecting weak ongoing cognate antigen recognition. Moreover, HBV-specific CD8+ T cells targeting core versus polymerase epitopes significantly differed in frequency, phenotype and function. In particular, in comparison with core-specific CD8+ T cells, a higher frequency of polymerase-specific CD8+ T cells expressed CD38, KLRG1 and Eomes accompanied by low T-bet expression and downregulated CD127 indicative of a more severe T cell exhaustion. In addition, polymerase-specific CD8+ T cells exhibited a reduced expansion capacity that was linked to a dysbalanced TCF1/BCL2 expression., Conclusions: Overall, the molecular mechanisms underlying impaired T cell responses differ with respect to the targeted HBV antigens. These results have potential implications for immunotherapeutic approaches in HBV cure., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background & Aims: Hepatitis D virus (HDV) superinfection in patients with hepatitis B virus (HBV) is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8 + T cells are thought to control the virus, little is known about which HDV epitopes are targeted by virus-specific CD8 + T cells or why these cells ultimately fail to control the infection. We aimed to define how HDV escapes the CD8 + T-cell-mediated response., Methods: We collected plasma and DNA samples from 104 patients with chronic HDV and HBV infection at medical centers in Europe and the Middle East, sequenced HDV, typed human leukocyte antigen (HLA) class I alleles from patients, and searched for polymorphisms in HDV RNA associated with specific HLA class I alleles. We predicted epitopes in HDV that would be recognized by CD8 + T cells and corresponded with the identified virus polymorphisms in patients with resolved (n = 12) or chronic (n = 13) HDV infection., Results: We identified 21 polymorphisms in HDV that were significantly associated with specific HLA class I alleles (P < .005). Five of these polymorphisms were found to correspond to epitopes in HDV that are recognized by CD8 + T cells; we confirmed that CD8 + T cells in culture targeted these HDV epitopes. HDV variant peptides were only partially cross-recognized by CD8 + T cells isolated from patients, indicating that the virus had escaped detection by these cells. These newly identified HDV epitopes were restricted by relatively infrequent HLA class I alleles, and they bound most frequently to HLA-B. In contrast, frequent HLA class I alleles were not associated with HDV sequence polymorphisms., Conclusions: We analyzed sequences of HDV RNA and HLA class I alleles that present epitope peptides to CD8 + T cells in patients with persistent HDV infection. We identified polymorphisms in the HDV proteome that associate with HLA class I alleles. Some variant peptides in epitopes from HDV were only partially recognized by CD8 + T cells isolated from patients; these could be mutations that allow HDV to escape the immune response, resulting in persistent infection. HDV escape from the immune response was associated with uncommon HLA class I alleles, indicating that HDV evolves, at the population level, to evade recognition by common HLA class I alleles., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection. IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development., (Copyright © 2018 American Society for Microbiology.)

Memory CD8 + T cells are essential in orchestrating protection from re-infection. Hallmarks of virus-specific memory CD8 + T cells are the capacity to mount recall responses with rapid induction of effector cell function and antigen-independent survival. Growing evidence reveals that even chronic infection does not preclude virus-specific CD8 + T-cell memory formation. However, whether this kind of CD8 + T-cell memory that is established during chronic infection is indeed functional and provides protection from re-infection is still unclear. Human chronic hepatitis C virus infection represents a unique model system to study virus-specific CD8 + T-cell memory formation during and after cessation of persisting antigen stimulation., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Differentiation and fate of virus-specific CD8 + T cells after cessation of chronic antigen stimulation is unclear. Here we show that a TCF1 + CD127 + PD1 + hepatitis C virus (HCV)-specific CD8 + T-cell subset exists in chronically infected patients with phenotypic features of T-cell exhaustion and memory, both before and after treatment with direct acting antiviral (DAA) agents. This subset is maintained during, and for a long duration after, HCV elimination. After antigen re-challenge the less differentiated TCF1 + CD127 + PD1 + population expands, which is accompanied by emergence of terminally exhausted TCF1-CD127-PD1 hi HCV-specific CD8 + T cells. These results suggest the TCF1 + CD127 + PD1 + HCV-specific CD8 + T-cell subset has memory-like characteristics, including antigen-independent survival and recall proliferation. We thus provide evidence for the establishment of memory-like virus-specific CD8 + T cells in a clinically relevant setting of chronic viral infection and we uncover their fate after cessation of chronic antigen stimulation, implicating a potential strategy for antiviral immunotherapy.

The salivary glands (SGs) of virus-immune mice contain substantial numbers of tissue-resident memory CD8 + T cells (T RM cells) that can provide immunity to local infections. Integrins regulate entry of activated T cells into nonlymphoid tissues but the molecules that mediate migration of virus-specific CD8 + T cells to the SGs have not yet been defined. Here, we found that polyinosinic-polycytidylic acid (poly(I:C)) strongly promoted the accumulation of P14 TCR-transgenic CD8 + T RM cells in SGs in an α 4 β 1 integrin-dependent manner. After infection with lymphocytic choriomeningitis virus, accumulation of P14 T RM cells in SGs and intestine but not in kidney was also α 4 integrin dependent. Blockade of α 4 β 7 by monoclonal antibodies (mAbs) inhibited lymphocytic choriomeningitis virus-induced accumulation of P14 T RM cells in the intestine but not in SGs. In conclusion, our data reveal that α 4 β 1 integrin mediates CD8 + T RM accumulation in SGs and that poly(I:C) can be used to direct activated CD8 + T cells to this organ., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

About 500 million people all over the world are chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV) and are thus at high risk of developing liver fibrosis, cirrhosis or hepatocellular carcinoma. While in adults about 90% of acutely HBV-infected patients clear the virus, only 30% of acute HCV infections clear spontaneously. Several mechanisms contribute to the failure in viral clearance. The main factors responsible for the chronification of HBV and HCV infection are, on the one hand, viral escape mutations leading to lack of recognition by antiviral immune cells and, on the other hand, loss of antiviral effector functions of virus-specific CD8+ T cells, called T-cell exhaustion. This review focuses on the latter highlighting current knowledge about the heterogeneity of exhausted CD8+ T cells and the potential for re-invigoration of exhausted T-cell populations during chronic viral hepatitis. Although direct-acting antivirals successfully clear chronic HCV infection, there is still the need for a prophylactic vaccine to prevent primary infection. Moreover, a therapeutic strategy eliminating HBV infection still does not exist. A better understanding of T-cell exhaustion and the potential for functional recovery will help to develop new immunotherapeutic approaches for chronic viral hepatitis., (© 2017 S. Karger AG, Basel.)

Chronic infections promote the terminal differentiation (or "exhaustion") of T cells and are thought to preclude the formation of memory T cells. In contrast, we discovered a small subpopulation of virus-specific CD8(+) T cells that sustained the T cell response during chronic infections. These cells were defined by, and depended on, the expression of the transcription factor Tcf1. Transcriptome analysis revealed that this population shared key characteristics of central memory cells but lacked an effector signature. Unlike conventional memory cells, Tcf1-expressing T cells displayed hallmarks of an "exhausted" phenotype, including the expression of inhibitory receptors such as PD-1 and Lag-3. This population was crucial for the T cell expansion that occurred in response to inhibitory receptor blockade during chronic infection. These findings identify a memory-like T cell population that sustains T cell responses and is a prime target for therapeutic interventions to improve the immune response in chronic infections., (Copyright © 2016 Elsevier Inc. All rights reserved.)

CD8(+) T cells are the main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems, such as low infection rates, restrict mechanistic studies of HBV-specific CD8(+) T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2(hNTCP) cells that endogenously processed viral antigens and presented them to HBV-specific CD8(+) T cells. This induced cytolytic and noncytolytic CD8(+) T-cell effector functions and reduction of viral loads., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The thymus is a primary lymphoid organ responsible for production and selection of T cells. Nonetheless, mature T cells and in particular activated T cells can reenter the thymus. Here, we identified memory CD8(+) T cells specific for lymphocytic choriomeningitis virus or vaccinia virus in the thymus of mice long-time after the infection. CD8(+) T cells were mainly located in the thymic medulla, but also in the cortical areas. Interestingly, virus-specific memory CD8(+) T cells in the thymus expressed the cell surface markers CD69 and CD103 that are characteristic of tissue-resident memory T cells in a time-dependent manner. Kinetic analyses and selective depletion of peripheral CD8(+) T cells by antibodies further revealed that thymic virus-specific memory CD8(+) T cells did not belong to the circulating pool of lymphocytes. Finally, we demonstrate that these thymus-resident virus-specific memory CD8(+) T cells efficiently mounted a secondary proliferative response, exhibited immediate effector functions and were able to protect the thymus from lymphocytic choriomeningitis virus reinfection. In conclusion, the present study not only describes for the first time virus-specific memory CD8(+) T cells with characteristics of tissue-resident memory T (T(RM)) cells in a primary lymphoid organ but also extends our knowledge about local T-cell immunity in the thymus., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The salivary glands are important effector sites for IgA-mediated humoral immunity to protect oral surfaces. Within murine submandibular glands (SMG), we identified a memory CD8 T-cell population that exhibited a unique cell-surface phenotype distinct from memory CD8 T cells in spleen but similar to memory T cells resident in the intraepithelial lymphocyte compartment of the intestinal mucosa. In mice immune to lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus(VSV), virus-specific memory CD8 T cells with this unusual phenotype were present in SMG at remarkably high frequencies. LCMV-specific memory CD8 T cells in SMG showed potent functional activities in vivo, including cytokine-induced bystander proliferation, antigen-triggered IFNγ production, and viral clearance. Adoptive transfer experiments further revealed that the capacity to accumulate in SMG decreased during CD8 T-cell differentiation and that SMG CD8 T cells were poorly replenished from the circulation, indicating that they were tissue-resident. Moreover, they preferentially relocalized within their tissue of origin after adoptive transfer and antigen rechallenge, thus revealing an imprinted differentiation status. Accumulation of memory CD8 T cells within SMG did not require local antigen presentation but was promoted by the epithelial differentiation molecule E-cadherin intrinsically expressed by these CD8 T cells. This finding extends the epithelial-restricted function of E-cadherin to an impact on lymphocyte accumulation within epithelial tissues.