Early transcriptional and epigenetic divergence of CD8+ T cells responding to acute versus chronic infection.

During a microbial infection, responding CD8+ T cells give rise to effector cells that provide acute host defense and memory cells that provide sustained protection. An alternative outcome is exhaustion, a state of T cell dysfunction that occurs in the context of chronic infections and cancer. Although it is evident that exhausted CD8+ T (TEX) cells are phenotypically and molecularly distinct from effector and memory CD8+ T cells, the factors regulating the earliest events in the differentiation process of TEX cells remain incompletely understood. Here, we performed single-cell RNA-sequencing and single-cell ATAC-sequencing of CD8+ T cells responding to LCMV-Armstrong (LCMV-Arm) or LCMV-Clone 13 (LCMV-Cl13), which result in acute or chronic infections, respectively. Compared to CD8+ T cells that had undergone their first division in response to LCMV-Arm (Div1ARM) cells, CD8+ T cells that had undergone their first division in response to LCMV-Cl13 (Div1CL13) expressed higher levels of genes encoding transcription factors previously associated with exhaustion, along with higher levels of Ezh2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2) complex, which mediates epigenetic silencing. Modulation of Ezh2 resulted in altered expression of exhaustion-associated molecules by CD8+ T cells responding to LCMV-Cl13, though the specific cellular and infectious contexts, rather than simply the level of Ezh2 expression, likely determine the eventual outcome. Taken together, these findings suggest that the differentiation paths of CD8+ T cells responding to acute versus chronic infections may diverge earlier than previously appreciated.

Delineating the transcriptional landscape and clonal diversity of virus-specific CD4+ T cells during chronic viral infection.

Although recent evidence indicates that CD4+ T cells responding to chronic viral infection are functionally heterogenous, our understanding of the developmental relationships between these subsets, and a determination of how their transcriptional landscape compares to their acute infection counterparts remains unclear. Additionally, whether cell-intrinsic factors such as TCR usage influence CD4+ T cell fate commitment during persistent infection has not previously been studied. Herein, we perform single-cell RNA sequencing (scRNA-seq) combined with single-cell T cell receptor sequencing (scTCR-seq) on virus-specific CD4+ T cells isolated from mice infected with chronic lymphocytic choriomeningitis virus (LCMV) infection. We identify several transcriptionally distinct states among the Th1, Tfh, and memory-like T cell subsets that form at the peak of infection, including the presence of a previously unrecognized Slamf7+ subset with cytolytic features. We further show that the relative distribution of these populations differs substantially between acute and persistent LCMV infection. Moreover, while the progeny of most T cell clones displays membership within each of these transcriptionally unique populations, overall supporting a one cell-multiple fate model, a small fraction of clones display a biased cell fate decision, suggesting that TCR usage may impact CD4+ T cell development during chronic infection. Importantly, comparative analyses further reveal both subset-specific and core gene expression programs that are differentially regulated between CD4+ T cells responding to acute and chronic LCMV infection. Together, these data may serve as a useful framework and allow for a detailed interrogation into the clonal distribution and transcriptional circuits underlying CD4+ T cell differentiation during chronic viral infection.

Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity.

Adaptive immune repertoires are composed by the ensemble of B and T-cell receptors within an individual, reflecting both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Here, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following lymphocytic choriomeningitis virus (LCMV) infection, CD8+ T cells with binding specificity restricted to two distinct LCMV peptides, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T-cell inflation, and regulation. Together, this dataset provides a resource for immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

Arenaviral infection causes bleeding in mice due to reduced serotonin release from platelets.

Bleeding correlates with disease severity in viral hemorrhagic fevers. We found that the increase in type I interferon (IFN-I) in mice caused by infection with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV; an arenavirus) reduced the megakaryocytic expression of genes encoding enzymes involved in lipid biosynthesis (cyclooxygenase 1 and thromboxane A synthase 1) and a thrombopoietic transcription factor (Nf-e2). The decreased expression of these genes was associated with reduced numbers of circulating platelets and defects in the arachidonic acid synthetic pathway, thereby suppressing serotonin release from δ-granules in platelets. Bleeding resulted when severe thrombocytopenia and altered platelet function reduced the amount of platelet-derived serotonin below a critical threshold. Bleeding was facilitated by the absence of the activity of the kinase Lyn or the administration of aspirin, an inhibitor of arachidonic acid synthesis. Mouse platelets were not directly affected by IFN-I because they lack the receptor for the cytokine (IFNAR1), suggesting that transfusion of normal platelets into LCMV-infected mice could increase the amount of platelet-released serotonin and help to control hemorrhage.

Single MHC-I Expression Promotes Virus-Induced Liver Immunopathology.

Major histocompatibility complex I (MHC-I) molecules present epitopes on the cellular surface of antigen-presenting cells to prime cytotoxic clusters of differentiation 8 (CD8)+ T cells (CTLs), which then identify and eliminate other cells such as virus-infected cells bearing the antigen. Human hepatitis virus cohort studies have previously identified MHC-I molecules as promising predictors of viral clearance. However, the underlying functional significance of these predictions is not fully understood. Here, we show that expression of single MHC-I isomers promotes virus-induced liver immunopathology. Specifically, using the lymphocytic choriomeningitis virus (LCMV) model system, we found MHC-I proteins to be highly up-regulated during infection. Deletion of one of the two MHC-I isomers histocompatibility antigen 2 (H2)-Db or H2-Kb in C57Bl/6 mice resulted in CTL activation recognizing the remaining MHC-I with LCMV epitopes in increased paucity. This increased CTL response resulted in hepatocyte death, increased caspase activation, and severe metabolic changes in liver tissue following infection with LCMV. Moreover, depletion of CTLs abolished LCMV-induced pathology in these mice with resulting viral persistence. In turn, natural killer (NK) cell depletion further increased antiviral CTL immunity and clearance of LCMV even in the presence of a single MHC-I isomer. Conclusion: Our results suggest that uniform MHC-I molecule expression promotes enhanced CTL immunity during viral infection and contributes to increased CTL-mediated liver cell damage that was alleviated by CD8 or NK cell depletion.

Induction of thymic atrophy and loss of thymic output by type-I interferons during chronic viral infection.

Type-I interferon (IFN-I) signals exert a critical role in disease progression during viral infections. However, the immunomodulatory mechanisms by which IFN-I dictates disease outcomes remain to be fully defined. Here we report that IFN-I signals mediate thymic atrophy in viral infections, with more severe and prolonged loss of thymic output and unique kinetics and subtypes of IFN-α/ß expression in chronic infection compared to acute infection. Loss of thymic output was linked to inhibition of early stages of thymopoiesis (DN1-DN2 transition, and DN3 proliferation) and pronounced apoptosis during the late DP stage. Notably, infection-associated thymic defects were largely abrogated upon ablation of IFNαßR and partially mitigated in the absence of CD8 T cells, thus implicating direct as well as indirect effects of IFN-I on thymocytes. These findings provide mechanistic underpinnings for immunotherapeutic strategies targeting IFN-1 signals to manipulate disease outcomes during chronic infections and cancers.

A multifunctional mouse model to study the role of Samd3.

The capacity to develop immunological memory is a hallmark of the adaptive immune system. To investigate the role of Samd3 for cellular immune responses and memory development, we generated a conditional knock-out mouse including a fluorescent reporter and a huDTR cassette for conditional depletion of Samd3-expressing cells. Samd3 expression was observed in NK cells and CD8 T cells, which are known for their specific function against intracellular pathogens like viruses. After acute viral infections, Samd3 expression was enriched within memory precursor cells and the frequency of Samd3-expressing cells increased during the progression into the memory phase. Similarly, during chronic viral infections, Samd3 expression was predominantly detected within precursors of exhausted CD8 T cells that are critical for viral control. At the functional level however, Samd3-deficient CD8 T cells were not compromised in the context of acute infection with Vaccinia virus or chronic infection with Lymphocytic choriomeningitis virus. Taken together, we describe a novel multifunctional mouse model to study the role of Samd3 and Samd3-expressing cells. We found that Samd3 is specifically expressed in NK cells, memory CD8 T cells, and precursor exhausted T cells during viral infections, while the molecular function of this enigmatic gene remains further unresolved.

Profiling the specificity of clonally expanded plasma cells during chronic viral infection by single-cell analysis.

Plasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine BM plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single-cell sequencing approach recovered full-length and paired heavy- and light-chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class-switching, and somatic variants. Furthermore, antibodies from the highly expanded and class-switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross-reactivity to nonviral and autoantigens. Integrating single-cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross-reactive antibodies.

Pre-existing chromatin accessibility and gene expression differences among naive CD4+ T cells influence effector potential.

CD4+ T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probe the heterogeneity present among naive CD4+ T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and chromatin landscape differences. Single-cell RNA sequencing shows that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a readout of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4+ T cells impact follicular helper T (TFH) cell versus non-TFH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4+ T cell chromatin landscapes that ultimately shape their effector potential.

Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation.

To gain insight into the signaling determinants of effector-associated DNA methylation programming among CD8 T cells, we explore the role of interleukin (IL)-12 in the imprinting of IFNg expression during CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promoter. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, results in stable demethylation of the IFNg locus that is commensurate with IFNg expression. IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent demethylation in an ex vivo human chimeric antigen receptor T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector DNA demethylation programming in CD8 T cells and serve as proof of concept that cytokines can guide induction of epigenetically regulated traits for T cell-based immunotherapies.

Distinct mechanisms govern populations of myeloid-derived suppressor cells in chronic viral infection and cancer.

Myeloid-derived suppressor cells (MDSCs) are major negative regulators of immune responses in cancer and chronic infections. It remains unclear if regulation of MDSC activity in different conditions is controlled by similar mechanisms. We compared MDSCs in mice with cancer and lymphocytic choriomeningitis virus (LCMV) infection. Chronic LCMV infection caused the development of monocytic MDSCs (M-MDSCs) but did not induce polymorphonuclear MDSCs (PMN-MDSCs). In contrast, both MDSC populations were present in cancer models. An acquisition of immune-suppressive activity by PMN-MDSCs in cancer was controlled by IRE1α and ATF6 pathways of the endoplasmic reticulum (ER) stress response. Abrogation of PMN-MDSC activity by blockade of the ER stress response resulted in an increase in tumor-specific immune response and reduced tumor progression. In contrast, the ER stress response was dispensable for suppressive activity of M-MDSCs in cancer and LCMV infection. Acquisition of immune-suppressive activity by M-MDSCs in spleens was mediated by IFN-γ signaling. However, it was dispensable for suppressive activity of M-MDSCs in tumor tissues. Suppressive activity of M-MDSCs in tumors was retained due to the effect of IL-6 present at high concentrations in the tumor site. These results demonstrate disease- and population-specific mechanisms of MDSC accumulation and the need for targeting different pathways to achieve inactivation of these cells.

UTX promotes CD8+ T cell-mediated antiviral defenses but reduces T cell durability.

Persistent virus infections can cause pathogenesis that is debilitating or lethal. During these infections, virus-specific T cells fail to protect due to weakened antiviral activity or failure to persist. These outcomes are governed by histone modifications, although it is unknown which enzymes contribute to T cell loss or impaired function over time. In this study, we show that T cell receptor-stimulated CD8+ T cells increase their expression of UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome) to enhance gene expression. During chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, UTX binds to enhancers and transcription start sites of effector genes, allowing for improved cytotoxic T lymphocyte (CTL)-mediated protection, independent of its trimethylation of histone 3 lysine 27 (H3K27me3) demethylase activity. UTX also limits the frequency and durability of virus-specific CD8+ T cells, which correspond to increased expression of inhibitory receptors. Thus, UTX guides gene expression patterns in CD8+ T cells, advancing early antiviral defenses while reducing the longevity of CD8+ T cell responses.

A unified atlas of CD8 T cell dysfunctional states in cancer and infection.

CD8 T cells play an essential role in defense against viral and bacterial infections and in tumor immunity. Deciphering T cell loss of functionality is complicated by the conspicuous heterogeneity of CD8 T cell states described across experimental and clinical settings. By carrying out a unified analysis of over 300 assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) experiments from 12 studies of CD8 T cells in cancer and infection, we defined a shared differentiation trajectory toward dysfunction and its underlying transcriptional drivers and revealed a universal early bifurcation of functional and dysfunctional T cell states across models. Experimental dissection of acute and chronic viral infection using single-cell ATAC (scATAC)-seq and allele-specific single-cell RNA (scRNA)-seq identified state-specific drivers and captured the emergence of similar TCF1+ progenitor-like populations at an early branch point, at which functional and dysfunctional T cells diverge. Our atlas of CD8 T cell states will facilitate mechanistic studies of T cell immunity and translational efforts.

Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity.

Nucleoprotein (N) is an immunodominant antigen in many enveloped virus infections. While the diagnostic value of anti-N antibodies is clear, their role in immunity is not. This is because while they are non-neutralising, they somehow clear infection by coronavirus, influenza and LCMV in vivo. Here, we show that anti-N immune protection is mediated by the cytosolic Fc receptor and E3 ubiquitin ligase TRIM21. Exploiting LCMV as a model system, we demonstrate that TRIM21 uses anti-N antibodies to target N for cytosolic degradation and generate cytotoxic T cells (CTLs) against N peptide. These CTLs rapidly eliminate N-peptide-displaying cells and drive efficient viral clearance. These results reveal a new mechanism of immune synergy between antibodies and T cells and highlights N as an important vaccine target.

IL4I1 Accelerates the Expansion of Effector CD8+ T Cells at the Expense of Memory Precursors by Increasing the Threshold of T-Cell Activation.

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.

T Cell-Intrinsic CX3CR1 Marks the Most Differentiated Effector CD4+ T Cells, but Is Largely Dispensable for CD4+ T Cell Responses during Chronic Viral Infection.

CD4+ T cells play critical roles during chronic viral infections, but the factors that regulate these responses remain incompletely defined. During chronic infection of mice with lymphocytic choriomeningitis virus clone 13 (LCMV13), the TNFR family member GITR plays a critical CD4+ T cell-intrinsic role in allowing T cell accumulation and viral control. Previously, RNA sequencing of GITR+/+ and GITR-/- T cells sorted from the spleen of mice at day 3 of LCMV13 infection identified the chemokine receptor CX3CR1 as increased by GITR signaling in CD4+ T cells. In this study, we evaluated the role of CX3CR1 on CD4+ T cells during LCMV13 infection. CX3CR1 expression is induced on Ag-specific CD4+ T cells upon Ag stimulation, and GITR signaling further increases the level of CX3CR1 expression. CX3CR1 marks the most differentiated T-bethi, Th1 effector population. Adoptively transferred CX3CR1-/- SMARTA cells had slightly reduced expression of T-bet and IFN-γ per cell compared with their CX3CR1+/+ counterparts but showed no deficit in accumulation in the spleen, lung, or liver. In mixed-radiation chimeras reconstituted with CX3CR1+/+ and CX3CR1-/- bone marrow, CX3CR1+/+ CD4+ T cells showed a marginal deficit in tissue-resident memory T cell numbers compared with the CX3CR1-/- T cells. CX3CR1 may limit acquisition of the tissue-resident memory T cell phenotype because of its effects on increasing T-bet expression, albeit these small effects are unlikely to be of major biological significance. Taken together, these studies show that CX3CR1 marks the most highly differentiated CD4+ Th1 effector population but is largely dispensable for CD4+ T cell responses during chronic viral infection.

Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection.

Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

Sphingosine kinase 2 restricts T cell immunopathology but permits viral persistence.

Chronic viral infections are often established by the exploitation of immune-regulatory mechanisms that result in nonfunctional T cell responses. Viruses that establish persistent infections remain a serious threat to human health. Sphingosine kinase 2 (SphK2) generates sphingosine 1-phosphate, which is a molecule known to regulate multiple cellular processes. However, little is known about SphK2's role during the host immune responses to viral infection. Here, we demonstrate that SphK2 functions during lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) infection to limit T cell immune pathology, which subsequently aids in the establishment of virus-induced immunosuppression and the resultant viral persistence. The infection of Sphk2-deficient (Sphk2-/-) mice with LCMV Cl 13 led to the development of nephropathy and mortality via T cell-mediated immunopathology. Following LCMV infection, Sphk2-/- CD4+ T cells displayed increased activity and proliferation, and these cells promoted overactive LCMV Cl 13-specific CD8+ T cell responses. Notably, oral instillation of an SphK2-selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Cl 13 persistence in mice. Thus, SphK2 is indicated as an immunotherapeutic target for the control of persistent viral infections.

A network of immune and microbial modifications underlies viral persistence in the gastrointestinal tract.

Many pathogens subvert intestinal immunity to persist within the gastrointestinal tract (GIT); yet, the underlying mechanisms that enable sanctuary specifically in this reservoir are unclear. Using mass cytometry and network analysis, we demonstrate that chronic LCMV infection of the GIT leads to dysregulated microbial composition, a cascade of metabolic alterations, increased susceptibility to GI disease, and a system-wide recalibration of immune composition that defines viral persistence. Chronic infection led to outgrowth of activated Tbet-expressing T reg cell populations unique to the GIT and the rapid erosion of pathogen-specific CD8 tissue-resident memory T cells. Mechanistically, T reg cells and coinhibitory receptors maintained long-term viral sanctuary within the GIT, and their targeting reactivated T cells and eliminated this viral reservoir. Thus, our data provide a high-dimensional definition of the mechanisms of immune regulation that chronic viruses implement to exploit the unique microenvironment of the GIT and identify T reg cells as key modulators of viral persistence in the intestinal tract.

Type I IFNs and CD8 T cells increase intestinal barrier permeability after chronic viral infection.

Intestinal barrier leakage constitutes a potential therapeutic target for many inflammatory diseases and represents a disease progression marker during chronic viral infections. However, the causes of altered gut barrier remain mostly unknown. Using murine infection with lymphocytic choriomeningitis virus, we demonstrate that, in contrast to an acute viral strain, a persistent viral isolate leads to long-term viral replication in hematopoietic and mesenchymal cells, but not epithelial cells (IECs), in the intestine. Viral persistence drove sustained intestinal epithelial barrier leakage, which was characterized by increased paracellular flux of small molecules and was associated with enhanced colitis susceptibility. Type I IFN signaling caused tight junction dysregulation in IECs, promoted gut microbiome shifts and enhanced intestinal CD8 T cell responses. Notably, both type I IFN receptor blockade and CD8 T cell depletion prevented infection-induced barrier leakage. Our study demonstrates that infection with a virus that persistently replicates in the intestinal mucosa increases epithelial barrier permeability and reveals type I IFNs and CD8 T cells as causative factors of intestinal leakage during chronic infections.