CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer.

Exhausted CD8 T (Tex) cells are a distinct cell lineage that arise during chronic infections and cancers in animal models and humans. Tex cells are characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression, metabolic dysregulation, poor memory recall and homeostatic self-renewal, and distinct transcriptional and epigenetic programs. The ability to reinvigorate Tex cells through inhibitory receptor blockade, such as αPD-1, highlights the therapeutic potential of targeting this population. Emerging insights into the mechanisms of exhaustion are informing immunotherapies for cancer and chronic infections. However, like other immune cells, Tex cells are heterogeneous and include progenitor and terminal subsets with unique characteristics and responses to checkpoint blockade. Here, we review our current understanding of Tex cell biology, including the developmental paths, transcriptional and epigenetic features, and cell intrinsic and extrinsic factors contributing to exhaustion and how this knowledge may inform therapeutic targeting of Tex cells in chronic infections, autoimmunity, and cancer.

Shared and distinct biological circuits in effector, memory and exhausted CD8+ T cells revealed by temporal single-cell transcriptomics and epigenetics.

Naïve CD8+ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff, Tmem and Tex populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets, including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2, as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator, Btg1, in establishing the Tex population. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections.

Epigenetic scarring of exhausted T cells hinders memory differentiation upon eliminating chronic antigenic stimulation.

Exhausted CD8 T cells (TEX) are a distinct state of T cell differentiation associated with failure to clear chronic viruses and cancer. Immunotherapies such as PD-1 blockade can reinvigorate TEX cells, but reinvigoration is not durable. A major unanswered question is whether TEX cells differentiate into functional durable memory T cells (TMEM) upon antigen clearance. Here, using a mouse model, we found that upon eliminating chronic antigenic stimulation, TEX cells partially (re)acquire phenotypic and transcriptional features of TMEM cells. These 'recovering' TEX cells originated from the T cell factor (TCF-1+) TEX progenitor subset. Nevertheless, the recall capacity of these recovering TEX cells remained compromised as compared to TMEM cells. Chromatin-accessibility profiling revealed a failure to recover core memory epigenetic circuits and maintenance of a largely exhausted open chromatin landscape. Thus, despite some phenotypic and transcriptional recovery upon antigen clearance, exhaustion leaves durable epigenetic scars constraining future immune responses. These results support epigenetic remodeling interventions for TEX cell-targeted immunotherapies.

Stat5 opposes the transcription factor Tox and rewires exhausted CD8+ T cells toward durable effector-like states during chronic antigen exposure.

Rewiring exhausted CD8+ T (Tex) cells toward functional states remains a therapeutic challenge. Tex cells are epigenetically programmed by the transcription factor Tox. However, epigenetic remodeling occurs as Tex cells transition from progenitor (Texprog) to intermediate (Texint) and terminal (Texterm) subsets, suggesting development flexibility. We examined epigenetic transitions between Tex cell subsets and revealed a reciprocally antagonistic circuit between Stat5a and Tox. Stat5 directed Texint cell formation and re-instigated partial effector biology during this Texprog-to-Texint cell transition. Constitutive Stat5a activity antagonized Tox and rewired CD8+ T cells from exhaustion to a durable effector and/or natural killer (NK)-like state with superior anti-tumor potential. Temporal induction of Stat5 activity in Tex cells using an orthogonal IL-2:IL2Rß-pair fostered Texint cell accumulation, particularly upon PD-L1 blockade. Re-engaging Stat5 also partially reprogrammed the epigenetic landscape of exhaustion and restored polyfunctionality. These data highlight therapeutic opportunities of manipulating the IL-2-Stat5 axis to rewire Tex cells toward more durably protective states.

Developmental Relationships of Four Exhausted CD8+ T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms.

CD8+ T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8+ T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at "re-invigorating" Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1+ progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.

MicroRNA-29a attenuates CD8 T cell exhaustion and induces memory-like CD8 T cells during chronic infection.

CD8 T cells mediate protection against intracellular pathogens and tumors. However, persistent antigen during chronic infections or cancer leads to T cell exhaustion, suboptimal functionality, and reduced protective capacity. Despite considerable work interrogating the transcriptional regulation of exhausted CD8 T cells (TEX), the posttranscriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses during chronic viral infection and antagonized exhaustion. miR-29a inhibited exhaustion-driving transcriptional pathways, including inflammatory and T cell receptor signaling, and regulated ribosomal biogenesis. As a result, miR-29a fostered a memory-like CD8 T cell differentiation state during chronic infection. Thus, we identify miR-29a as a key regulator of TEX and define mechanisms by which miR-29a can divert exhaustion toward a more beneficial memory-like CD8 T cell differentiation state.

Differential immune transcriptomic profiles between vaccinated and resolved HCV reinfected subjects.

Successive episodes of hepatitis C virus (HCV) infection represent a unique natural rechallenge experiment to define correlates of long-term protective immunity and inform vaccine development. We applied a systems immunology approach to characterize longitudinal changes in the peripheral blood transcriptomic signatures in eight subjects who spontaneously resolved two successive HCV infections. Furthermore, we compared these signatures with those induced by an HCV T cell-based vaccine regimen. We identified a plasma cell transcriptomic signature during early acute HCV reinfection. This signature was absent in primary infection and following HCV vaccine boost. Spontaneous resolution of HCV reinfection was associated with rapid expansion of glycoprotein E2-specifc memory B cells in three subjects and transient increase in E2-specific neutralizing antibodies in six subjects. Concurrently, there was an increase in the breadth and magnitude of HCV-specific T cells in 7 out of 8 subjects. These results suggest a cooperative role for both antibodies and T cells in clearance of HCV reinfection and support the development of next generation HCV vaccines targeting these two arms of the immune system.

Expansion of Unique Hepatitis C Virus-Specific Public CD8+ T Cell Clonotypes during Acute Infection and Reinfection.

Hepatitis C virus (HCV) infection resolves spontaneously in ∼25% of acutely infected humans where viral clearance is mediated primarily by virus-specific CD8+ T cells. Previous cross-sectional analysis of the CD8+ TCR repertoire targeting two immunodominant HCV epitopes reported widespread use of public TCRs shared by different subjects, irrespective of infection outcome. However, little is known about the evolution of the public TCR repertoire during acute HCV and whether cross-reactivity to other Ags can influence infectious outcome. In this article, we analyzed the CD8+ TCR repertoire specific to the immunodominant and cross-reactive HLA-A2-restricted nonstructural 3-1073 epitope during acute HCV in humans progressing to either spontaneous resolution or chronic infection and at ∼1 y after viral clearance. TCR repertoire diversity was comparable among all groups with preferential usage of the TCR-ß V04 and V06 gene families. We identified a set of 13 public clonotypes in HCV-infected humans independent of infection outcome. Six public clonotypes used the V04 gene family. Several public clonotypes were long-lived in resolvers and expanded on reinfection. By mining publicly available data, we identified several low-frequency CDR3 sequences in the HCV-specific repertoire matching human TCRs specific for other HLA-A2-restricted epitopes from melanoma, CMV, influenza A, EBV, and yellow fever viruses, but they were of low frequency and limited cross-reactivity. In conclusion, we identified 13 new public human CD8+ TCR clonotypes unique to HCV that expanded during acute infection and reinfection. The low frequency of cross-reactive TCRs suggests that they are not major determinants of infectious outcome.

The hedgehog pathway suppresses neuropathogenesis in CD4 T cell-driven inflammation.

The concerted actions of the CNS and the immune system are essential to coordinating the outcome of neuroinflammatory responses. Yet, the precise mechanisms involved in this crosstalk and their contribution to the pathophysiology of neuroinflammatory diseases largely elude us. Here, we show that the CNS-endogenous hedgehog pathway, a signal triggered as part of the host response during the inflammatory phase of multiple sclerosis and experimental autoimmune encephalomyelitis, attenuates the pathogenicity of human and mouse effector CD4 T cells by regulating their production of inflammatory cytokines. Using a murine genetic model, in which the hedgehog signalling is compromised in CD4 T cells, we show that the hedgehog pathway acts on CD4 T cells to suppress the pathogenic hallmarks of autoimmune neuroinflammation, including demyelination and axonal damage, and thus mitigates the development of experimental autoimmune encephalomyelitis. Impairment of hedgehog signalling in CD4 T cells exacerbates brain-brainstem-cerebellum inflammation and leads to the development of atypical disease. Moreover, we present evidence that hedgehog signalling regulates the pathogenic profile of CD4 T cells by limiting their production of the inflammatory cytokines granulocyte-macrophage colony-stimulating factor and interferon-γ and by antagonizing their inflammatory program at the transcriptome level. Likewise, hedgehog signalling attenuates the inflammatory phenotype of human CD4 memory T cells. From a therapeutic point of view, our study underlines the potential of harnessing the hedgehog pathway to counteract ongoing excessive CNS inflammation, as systemic administration of a hedgehog agonist after disease onset effectively halts disease progression and significantly reduces neuroinflammation and the underlying neuropathology. We thus unveil a previously unrecognized role for the hedgehog pathway in regulating pathogenic inflammation within the CNS and propose to exploit its ability to modulate this neuroimmune network as a strategy to limit the progression of ongoing neuroinflammation.

The long noncoding RNA Morrbid regulates CD8 T cells in response to viral infection.

The transcriptional programs that regulate CD8 T-cell differentiation and function in the context of viral infections or tumor immune surveillance have been extensively studied; yet how long noncoding RNAs (lncRNAs) and the loci that transcribe them contribute to the regulation of CD8 T cells during viral infections remains largely unexplored. Here, we report that transcription of the lncRNA Morrbid is specifically induced by T-cell receptor (TCR) and type I IFN stimulation during the early stages of acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. In response to type I IFN, the Morrbid RNA and its locus control CD8 T cell expansion, survival, and effector function by regulating the expression of the proapoptotic factor, Bcl2l11, and by modulating the strength of the PI3K-AKT signaling pathway. Thus, our results demonstrate that inflammatory cue-responsive lncRNA loci represent fundamental mechanisms by which CD8 T cells are regulated in response to pathogens and potentially cancer.

Selective expansion of high functional avidity memory CD8 T cell clonotypes during hepatitis C virus reinfection and clearance.

The dynamics of the memory CD8 T cell receptor (TCR) repertoire upon virus re-exposure and factors governing the selection of TCR clonotypes conferring protective immunity in real life settings are poorly understood. Here, we examined the dynamics and functionality of the virus-specific memory CD8 TCR repertoire before, during and after hepatitis C virus (HCV) reinfection in patients who spontaneously resolved two consecutive infections (SR/SR) and patients who resolved a primary but failed to clear a subsequent infection (SR/CI). The TCR repertoire was narrower prior to reinfection in the SR/SR group as compared to the SR/CI group and became more focused upon reinfection. CD8 T cell clonotypes expanding upon re-exposure and associated with protection from viral persistence were recruited from the memory T cell pool. Individual CD8 T cell lines generated from the SR/SR group exhibited higher functional avidity and polyfunctionality as compared to cell lines from the SR/CI group. Our results suggest that protection from viral persistence upon HCV reinfection is associated with focusing of the HCV-specific CD8 memory T cell repertoire from which established cell lines showed high functional avidity. These findings are applicable to vaccination strategies aiming at shaping the protective human T cell repertoire.

Signatures of protective memory immune responses during hepatitis C virus reinfection.

BACKGROUND & AIMS: Development of a vaccine against hepatitis C virus (HCV) has been hindered by our limited understanding of immune correlates of protection during real-life exposure to the virus. We studied the immune response during HCV reinfection. METHODS: We analyzed blood samples from participants in the Montreal Acute Hepatitis C Injection Drug User Cohort Study who were reinfected with HCV from 2009 to 2012. Five patients spontaneously resolved their second infection and 4 developed chronic infections. We monitored the phenotypic and functional dynamics of HCV-specific memory T cell responses in all subjects during natural re-exposure and re-infection. RESULTS: Populations of CD4(+) and CD8(+) T cells with HCV-specific polyfunctional memory were expanded in all 5 individuals who resolved 2 successive HCV infections. We detected CD127(hi) HCV-specific memory CD8(+) T cells before reinfection regardless of a subject's ability to clear subsequent infections. Protection against viral persistence was associated with the expansion of a CD127(neg), PD1(lo) effector memory T cells at the peak of the response. We also observed broadening of T-cell response, indicating generation of de novo T-cell responses. The 4 individuals who failed to clear their subsequent infection had limited expansion of HCV-specific CD4(+) and CD8(+) memory T cells and expressed variable levels of the exhaustion marker PD1 on HCV-specific CD8(+) T cells. Dominant epitope regions of HCV strains isolated from patients with persistent reinfection had sequence variations that were not recognized by the pre-existing memory T cells. CONCLUSIONS: Protection from persistent HCV reinfection depends on the magnitude, breadth, and quality of the HCV-specific memory T-cell response. Sequence homology among viruses and ability of T cells to recognize multiple strains of HCV are critical determinants of protective memory.

CD8+ T cell-derived Fgl2 regulates immunity in a cell-autonomous manner via ligation of FcγRIIB.

The regulatory circuits dictating CD8+ T cell responsiveness versus exhaustion during anti-tumor immunity are incompletely understood. Here we report that tumor-infiltrating antigen-specific PD-1+ TCF-1- CD8+ T cells express the immunosuppressive cytokine Fgl2. Conditional deletion of Fgl2 specifically in mouse antigen-specific CD8+ T cells prolongs CD8+ T cell persistence, suppresses phenotypic and transcriptomic signatures of T cell exhaustion, and improves control of the tumor. In a mouse model of chronic viral infection, PD-1+ CD8+ T cell-derived Fgl2 also negatively regulates virus-specific T cell responses. In humans, CD8+ T cell-derived Fgl2 is associated with poorer survival in patients with melanoma. Mechanistically, the dampened responsiveness of WT Fgl2-expressing CD8+ T cells, when compared to Fgl2-deficient CD8+ T cells, is underpinned by the cell-intrinsic interaction of Fgl2 with CD8+ T cell-expressed FcγRIIB and concomitant caspase 3/7-mediated apoptosis. Our results thus illuminate a cell-autonomous regulatory axis by which PD-1+ CD8+ T cells both express the receptor and secrete its ligand in order to mediate suppression of anti-tumor and anti-viral immunity.

Comparison of immune restoration in early versus late alpha interferon therapy against hepatitis C virus.

Early alpha interferon (IFN-alpha) therapy against hepatitis C virus (HCV) rescues polyfunctional, virus-specific memory CD8(+) T cells, but whether immune restoration is possible during late therapy remains controversial. We compared immune restoration of HCV-specific memory T cells in patients who cleared HCV infection spontaneously and following early or late IFN therapy. Multifunctional CD4(+) and CD8(+) memory T cells were detected in spontaneous resolvers and in individuals treated early following an acute infection. In contrast, limited responses were detected in patients treated during chronic infection, and the phenotype of HCV-specific cells was influenced by autologous viral sequences. Our data suggest that irreversible damage to the HCV-specific memory T-cell response is associated with chronic HCV infection.

Impact of IL10, MTP, SOD2, and APOE Gene Polymorphisms on the Severity of Liver Fibrosis Induced by HCV Genotype 4.

Complications of hepatitis C virus (HCV) chronic infection cause ~400,000 deaths worldwide annually. One complication, liver fibrosis, is influenced by host genetic factors. Genes influencing fibrosis include immune, metabolic, oxidative stress, and viral entry genes, such as interleukin 10 (IL10), microsomal triglyceride-transfer protein (MTP), superoxide dismutase-2 (SOD2), and apolipoprotein E (APOE)-encoding genes, respectively. Thus, correlating variations in these genes with HCV-induced fibrosis represents an attractive biomarker for the prognosis of fibrosis severity in chronically infected patients. Here, we aimed to test whether polymorphisms in IL10, MTP, SOD2, and APOE genes correlated with the severity of fibrosis induced by HCV genotype 4 (HCV-gt4) in a cohort of chronically infected Egyptian patients. Our results demonstrate a significant association between the severity of fibrosis and specific SNPs in IL-10, SOD2, and ApoE-encoding genes. Haplotype-combination analysis for IL10, MTP, SOD2, and APOE showed statistically significant associations between specific haplotype combinations and fibrosis severity. Identifying biomarkers correlating with the severity of HCV-gt4-induced fibrosis would significantly impact precision prophylaxis and treatment of patients at risk.

Early interferon therapy for hepatitis C virus infection rescues polyfunctional, long-lived CD8+ memory T cells.

The majority of acute hepatitis C virus (HCV) infections progress to chronicity and progressive liver damage. Alpha interferon (IFN-alpha) antiviral therapy achieves the highest rate of success when IFN-alpha is administered early during the acute phase, but the underlying mechanisms are unknown. We used a panel of major histocompatibility complex class I tetramers to monitor the phenotypic and functional signatures of HCV-specific T cells during acute HCV infection with different infection outcomes and during early IFN therapy. We demonstrate that spontaneous resolution correlates with the early development of polyfunctional (IFN-gamma- and IL-2-producing and CD107a(+)) virus-specific CD8(+) T cells. These polyfunctional T cells are distinguished by the expression of CD127 and Bcl-2 and represent a transitional memory T-cell subset that exhibits the phenotypic and functional signatures of both central and effector memory T cells. In contrast, HCV-specific CD8(+) T cells in acute infections evolving to chronicity expressed low levels of CD127 and Bcl-2, exhibited diminished proliferation and cytokine production, and eventually disappeared from the periphery. Early therapeutic intervention with pegylated IFN-alpha rescued polyfunctional memory T cells expressing high levels of CD127 and Bcl-2. These cells were detectable for up to 1 year following discontinuation of therapy. Our results suggest that the polyfunctionality of HCV-specific T cells can be predictive of the outcome of acute HCV infection and that early therapeutic intervention can reconstitute the pool of long-lived polyfunctional memory T cells.

Viruses Teaching Immunology: Role of LCMV Model and Human Viral Infections in Immunological Discoveries.

Virology has played an essential role in deciphering many immunological phenomena, thus shaping our current understanding of the immune system. Animal models of viral infection and human viral infections were both important tools for immunological discoveries. This review discusses two immunological breakthroughs originally identified with the help of the lymphocytic choriomeningitis virus (LCMV) model; immunological restriction by major histocompatibility complex and immunotherapy using checkpoint blockade. In addition, we discuss related discoveries such as development of tetramers, viral escape mutation, and the phenomenon of T-cell exhaustion.

The 7th Canadian Symposium on Hepatitis C Virus: "Toward Elimination of HCV: How to Get There".

Hepatitis C virus (HCV) affects more than 268,000 people in Canada. Both the Canadian Institutes of Health Research and the Public Health Agency of Canada recognize the significant impact of HCV-related liver diseases and supported the establishment of a national hepatitis C research network, the Canadian Network on Hepatitis C (CanHepC). Interferon-free direct-acting antiviral regimens lead to more than 95% cure rates in almost all patients with well-tolerated short-course therapy. However, the goal of eliminating HCV in Canada cannot be fully realized until we overcome the financial, geographical, cultural, and social barriers that affect the entire continuum of care from diagnosis and linkage to care through treatment and prevention of new and reinfections. Current practices face difficulties in reversing HCV-induced immunological defects, expanding treatment to neglected communities, combating reinfections and co-infections, and expediting and simplifying the processes of diagnosis and treatment. As part of its knowledge translation mandate, CanHepC has organized the annual Canadian symposium on hepatitis C since 2012. The theme of this year's symposium, "Toward Elimination of HCV: How to Get There?" focused on identifying the requirements of our therapeutic strategies and health policies for the elimination of HCV in Canada.