Tetramer enrichment reveals the presence of phenotypically diverse hepatitis C virus-specific CD8+T cells in chronic infection

Virus-specific CD8+ T cells are rarely detectable ex vivo by conventional methods during chronic hepatitis C virus (HCV) infection. In this study, however, we were able to detect and characterize HCV-specific CD8+ T cells in all chronically HCV genotype 1a-infected, HLA-A*02:01-positive patients analyzed by performing major histocompatibility complex (MHC) class I tetramer enrichment. Two-thirds of these enriched HCV-specific CD8+ T-cell populations displayed an effector memory phenotype, whereas, surprisingly, one-third displayed a naive-like phenotype despite ongoing viral replication. CD8+ T cells with an effector memory phenotype could not expand in vitro, suggesting exhaustion of these cells. Interestingly, some of the naive-like CD8+ T cells proliferated vigorously upon in vitro priming, whereas others did not. These differences were linked to the corresponding viral sequences in the respective patients. Indeed, naive-like CD8+ T cells from patients with the consensus sequence in the corresponding T-cell epitope did not expand in vitro. In contrast, in patients displaying sequence variations, we were able to induce HCV-specific CD8+ T-cell proliferation, which may indicate infection with a variant virus. Collectively, these data reveal the presence of phenotypically and functionally diverse HCV-specific CD8+ T cells at very low frequencies that are detectable in all chronically infected patients despite viral persistence. IMPORTANCE In this study, we analyzed CD8+ T-cell responses specific for HLA-A*02:01-restricted epitopes in chronically HCV-infected patients, using MHC class I tetramer enrichment. Importantly, we could detect HCV-specific CD8+ T-cell populations in all patients. To further characterize these HCV-specific CD8+ T-cell populations that are not detectable using conventional techniques, we performed phenotypic, functional, and viral sequence analyses. These data revealed different mechanisms for CD8+ T-cell failure in HCV infection, including T-cell exhaustion, viral escape, and functional impairment of naive-like HCV-specific CD8+ T cells

Immunodominance of HLA-A2-restricted hepatitis C virus-specific CD8+ T cell responses is linked to naive-precursor frequency

The impact of naïve precursor frequency on human virus-specific CD8+ T cell immunodominance is not well understood. Using a recently developed MHC class I tetramer enrichment protocol, we found a conserved hierarchy and >10-fold difference in naïve precursor frequencies across three HLA-A2 restricted HCV-specific epitopes. Importantly, the NS31406 epitope with the highest naïve precursor frequency in healthy donors was also the most frequently targeted epitope in a large cohort of chronically HCV-infected patients, both ex vivo and after in vitro stimulation. These results indicate for the first time that immunodominance in a human viral infection is linked to naïve precursor frequency

TOX defines the degree of CD8+ T cell dysfunction in distinct phases of chronic HBV infection

Objective Chronic hepatitis B virus (HBV) infection is characterised by HBV-specific CD8+ T cell dysfunction that has been linked to Tcell exhaustion, a distinct differentiation programme associated with persisting antigen recognition. Recently, Thymocyte Selection-Associated High Mobility Group Box (TOX) was identified as master regulator of CD8+ T cell exhaustion. Here, we addressed the role of TOX in HBV-specific CD8+ T cell dysfunction associated with different clinical phases of infection. Design We investigated TOX expression in HBV-specific CD8+ T cells from 53 HLA-A*01:01, HLA-A*11:01 and HLA-A*02:01 positive patients from different HBV infection phases and compared it to hepatitis C virus (HCV)-specific, cytomegalovirus (CMV)-specific, Epstein-Barr virus (EBV)-specific and influenza virus (FLU)-specific CD8+ T cells. Phenotypic and functional analyses of virus-specific CD8+ T cells were performed after peptide-loaded tetramer-enrichment and peptide-specific expansion. Results Our results show that TOX expression in HBV-specific CD8+ T cells is linked to chronic antigen stimulation, correlates with viral load and is associated with phenotypic and functional characteristics of T-cell exhaustion. In contrast, similar TOX expression in EBV-specific and CMV-specific CD8+ T cells is not linked to T-cell dysfunction suggesting different underlying programmes. TOX expression in HBV-specific CD8+ T cells is also affected by targeted antigens, for example, core versus polymerase. In HBV-specific CD8+ T cells, TOX expression is maintained after spontaneous or therapy-mediated viral control in chronic but not self-limiting acute HBV infection indicating a permanent molecular imprint after chronic but not temporary stimulation. Conclusion Our data highlight TOX as biomarker specific for dysfunctional virus-specific CD8+ T cells in the context of an actively persisting infection

Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load

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

Immunodominance and functional alterations of tumor-associated antigen-specific CD8+T-cell responses in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide with a poor prognosis and limited therapeutic options. To aid the development of novel immunological interventions, we studied the breadth, frequency, and tumor-infiltration of naturally occurring CD8+ T-cell responses targeting several tumor-associated antigens (TAA). We used overlapping peptides spanning the entire alpha-fetoprotein (AFP), glypican-3 (GPC-3), melanoma-associated gene-A1 (MAGE-A1) and New York-esophageal squamous cell carcinoma-1 (NY-ESO-1) proteins and major-histocompatibility-complex-class-I-tetramers specific for epitopes of MAGE-A1 and NY-ESO-1 to analyze TAA-specific CD8+ T-cell responses in a large cohort of HCC patients. After nonspecific expansion in vitro, we detected interferon-γ (IFN-γ)-producing CD8+ T cells specific for all four TAA in the periphery as well as in liver and tumor tissue. These CD8+ T-cell responses displayed clear immunodominance patterns within each TAA, but no consistent hierarchy was observed between different TAA. Importantly, the response breadth was highest in early-stage HCC and associated with patient survival. After antigen-specific expansion, TAA-specific CD8+ T cells were detectable by tetramer staining but impaired in their ability to produce IFN-γ. Furthermore, regulatory T cells (Treg) were increased in HCC lesions. Depletion of Treg from cultures improved TAA-specific CD8+ T-cell proliferation but did not restore IFN-γ-production. Conclusion: Naturally occurring TAA-specific CD8+ T-cell responses are present in patients with HCC and therefore constitute part of the normal T-cell repertoire. Moreover, the presence of these responses correlates with patient survival. However, the observation of impaired IFN-γ production suggests that the efficacy of such responses is functionally limited. These findings support the development of strategies that aim to enhance the total TAA-specific CD8+ T-cell response by therapeutic boosting and/or specificity diversification. However, further research will be required to help unlock the full potential of TAA-specific CD8+ T-cell responses

Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient as a source of immune escape variants.

The origin of SARS-CoV-2 variants of concern remains unclear. Here, we test whether intra-host virus evolution during persistent infections could be a contributing factor by characterizing the long-term SARS-CoV-2 infection dynamics in an immunosuppressed kidney transplant recipient. Applying RT-qPCR and next-generation sequencing (NGS) of sequential respiratory specimens, we identify several mutations in the viral genome late in infection. We demonstrate that a late viral isolate exhibiting genome mutations similar to those found in variants of concern first identified in UK, South Africa, and Brazil, can escape neutralization by COVID-19 antisera. Moreover, infection of susceptible mice with this patient's escape variant elicits protective immunity against re-infection with either the parental virus and the escape variant, as well as high neutralization titers against the alpha and beta SARS-CoV-2 variants, B.1.1.7 and B.1.351, demonstrating a considerable immune control against such variants of concern. Upon lowering immunosuppressive treatment, the patient generated spike-specific neutralizing antibodies and resolved the infection. Our results suggest that immunocompromised patients could be a source for the emergence of potentially harmful SARS-CoV-2 variants

ERAP1 allotypes shape the epitope repertoire of virus-specific CD8+ T cell responses in acute hepatitis C virus infection

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

Mechanisms of CD8+ T cell failure in chronic hepatitis E virus infection

Background and aims In immunosuppressed patients, persistent hepatitis E virus (HEV) infection is common and may lead to cirrhosis and liver failure. HEV clearance depends on an effective virus-specific CD8+ T cell response, however, the knowledge gap of HEV-specific CD8+ T cell epitopes hindered analysis of the mechanisms of T cell failure in persistent infection thus far. Methods We comprehensively studied HEV-specific CD8+ T cell responses in 46 patients with self-limiting (n=34) or chronic HEV infection (n=12), by epitope-specific expansion, functional testing, ex vivo peptide HLA class I tetramer multi-parametric staining, and viral sequence analysis. Results We identified 25 HEV-specific CD8+ T cell epitopes restricted by 9 different HLA class I alleles. In self-limiting HEV infection, HEV-specific CD8+ T cells were vigorous, contracted after resolution of infection, and formed functional memory responses. In contrast, in chronic infection, the HEV-specific CD8+ T cell response was diminished, declined over time, and displayed phenotypic features of exhaustion. However, improved proliferation and interferon-γ production of HEV-specific CD8+ T cells and evolution of a memory-like phenotype was observed upon reduction of immunosuppression and/or ribavirin treatment and was associated with viral clearance. In one patient, mutational viral escape in a targeted CD8+ T cell epitope contributed to CD8+ T cell failure. Conclusion Chronic HEV infection is associated with HEV-specific CD8+ T cell exhaustion, indicating that T cell exhaustion driven by persisting antigen recognition also occurs in severely immunosuppressed hosts. Functional reinvigoration of virus-specific T cells is at least partially possible when antigen is cleared. In a minority of patients, viral escape also contributes to HEV-specific CD8+ T cell failure and thus needs to be taken into account in personalized immunotherapeutic approaches. Lay Summary In immunosuppressed patients, chronic HEV infection is common. For resolution of infection a functional HEV-specific CD8+ T cell response is essential, however, in immunosuppressed individuals CD8+ T cell exhaustion and viral escape contribute to CD8+ T cell failure

Spatiotemporal Correlations between Blood-Brain Barrier Permeability and Apparent Diffusion Coefficient in a Rat Model of Ischemic Stroke

Variations in apparent diffusion coefficient of water (ADC) and blood-brain barrier (BBB) permeability after ischemia have been suggested, though the correlation between ADC alterations and BBB opening remains to be studied. We hypothesized that there are correlations between the alteration of ADC and BBB permeability. Rats were subjected to 2 h of transient middle cerebral artery occlusion and studied at 3 and 48 h of reperfusion, which are crucial times of BBB opening. BBB permeability and ADC values were measured by dynamic contrast-enhanced MRI and diffusion-weighted imaging, respectively. Temporal and spatial analyses of the evolution of BBB permeability and ADC alteration in cortical and subcortical regions were conducted along with the correlation between ADC and BBB permeability data. We found significant increases in BBB leakage and reduction in ADC values between 3 and 48 h of reperfusion. We identified three MR tissue signature models: high Ki and low ADC, high Ki and normal ADC, and normal Ki and low ADC. Over time, areas with normal Ki and low ADC transformed into areas with high Ki. We observed a pattern of lesion evolution where the extent of initial ischemic injury reflected by ADC abnormalities determines vascular integrity. Our results suggest that regions with vasogenic edema alone are not likely to develop low ADC by 48 h and may undergo recovery