NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR.

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

Caspase-7 activates ASM to repair gasdermin and perforin pores.

Among the caspases that cause regulated cell death, a unique function for caspase-7 has remained elusive. Caspase-3 performs apoptosis, whereas caspase-7 is typically considered an inefficient back-up. Caspase-1 activates gasdermin D pores to lyse the cell; however, caspase-1 also activates caspase-7 for unknown reasons1. Caspases can also trigger cell-type-specific death responses; for example, caspase-1 causes the extrusion of intestinal epithelial cell (IECs) in response to infection with Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium)2,3. Here we show in both organoids and mice that caspase-7-deficient IECs do not complete extrusion. Mechanistically, caspase-7 counteracts gasdermin D pores and preserves cell integrity by cleaving and activating acid sphingomyelinase (ASM), which thereby generates copious amounts of ceramide to enable enhanced membrane repair. This provides time to complete the process of IEC extrusion. In parallel, we also show that caspase-7 and ASM cleavage are required to clear Chromobacterium violaceum and Listeria monocytogenes after perforin-pore-mediated attack by natural killer cells or cytotoxic T lymphocytes, which normally causes apoptosis in infected hepatocytes. Therefore, caspase-7 is not a conventional executioner but instead is a death facilitator that delays pore-driven lysis so that more-specialized processes, such as extrusion or apoptosis, can be completed before cell death. Cells must put their affairs in order before they die.

The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity.

Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4+ T cells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3-/- mice exhibited increased and prolonged CD4+ T cell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limit NF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4+ T cell signaling and metabolism.

GATA-3 controls the maintenance and proliferation of T cells downstream of TCR and cytokine signaling.

GATA-3 controls T helper type 2 (TH2) differentiation. However, whether GATA-3 regulates the function of mature T cells beyond TH2 determination remains poorly understood. We found that signaling via the T cell antigen receptor (TCR) and cytokine stimulation promoted GATA-3 expression in CD8(+) T cells, which controlled cell proliferation. Although GATA-3-deficient CD8(+) T cells were generated, their peripheral maintenance was impaired, with lower expression of the receptor for interleukin 7 (IL-7R). GATA-3-deficient T cells had defective responses to viral infection and alloantigen. The proto-oncoprotein c-Myc was a critical target of GATA-3 in promoting T cell proliferation. Our study thus demonstrates an essential role for GATA-3 in controlling the maintenance and proliferation of T cells and provides insight into immunoregulation.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Inflammasomes Coordinate Pyroptosis and Natural Killer Cell Cytotoxicity to Clear Infection by a Ubiquitous Environmental Bacterium.

Defective neutrophils in patients with chronic granulomatous disease (CGD) cause susceptibility to extracellular and intracellular infections. Microbes must first be ejected from intracellular niches to expose them to neutrophil attack, so we hypothesized that inflammasomes detect certain CGD pathogens upstream of neutrophil killing. Here, we identified one such ubiquitous environmental bacterium, Chromobacterium violaceum, whose extreme virulence was fully counteracted by the NLRC4 inflammasome. Caspase-1 protected via two parallel pathways that eliminated intracellular replication niches. Pyroptosis was the primary bacterial clearance mechanism in the spleen, but both pyroptosis and interleukin-18 (IL-18)-driven natural killer (NK) cell responses were required for liver defense. NK cells cleared hepatocyte replication niches via perforin-dependent cytotoxicity, whereas interferon-γ was not required. These insights suggested a therapeutic approach: exogenous IL-18 restored perforin-dependent cytotoxicity during infection by the inflammasome-evasive bacterium Listeria monocytogenes. Therefore, inflammasomes can trigger complementary programmed cell death mechanisms, directing sterilizing immunity against intracellular bacterial pathogens.

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX.

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.

Viral protease cleavage of MAVS in genetically modified mice with hepatitis A virus infection.

BACKGROUND & AIMS: Consistent with its relatively narrow host species range, hepatitis A virus (HAV) cannot infect C57BL/6 mice. However, in Mavs-/- mice with genetic deficiency of the innate immune signaling adaptor MAVS, HAV replicates robustly in the absence of disease. The HAV 3ABC protease cleaves MAVS in human cells, thereby disrupting virus-induced IFN responses, but it cannot cleave murine MAVS (mMAVS) due to sequence differences at the site of scission. Here, we sought to elucidate the role of 3ABC MAVS cleavage in determining HAV pathogenesis and host species range. METHODS: Using CRISPR/Cas9 gene editing, we established two independent lineages of C57BL/6 mice with knock-in mutations altering two amino acids in mMAVS ('mMAVS-VS'), rendering it susceptible to 3ABC cleavage without loss of signaling function. We challenged homozygous Mavsvs/vs mice with HAV, and compared infection outcomes with C57BL/6 and genetically deficient Mavs-/- mice. RESULTS: The humanized murine mMAVS-VS protein was cleaved as efficiently as human MAVS when co-expressed with 3ABC in Huh-7 cells. In embyronic fibroblasts from Mavsvs/vs mice, mMAVS-VS was cleaved by ectopically expressed 3ABC, significantly disrupting Sendai virus-induced IFN responses. However, in contrast to Mavs-/- mice with genetic MAVS deficiency, HAV failed to establish infection in Mavsvs/vs mice, even with additional genetic knockout of Trif or Irf1. Nonetheless, when crossed with permissive Ifnar1-/- mice lacking type I IFN receptors, Mavsvs/vsIfnar1-/- mice demonstrated enhanced viral replication coupled with significant reductions in serum alanine aminotransferase, hepatocellular apoptosis, and intrahepatic inflammatory cell infiltrates compared with Ifnar1-/- mice. CONCLUSIONS: MAVS cleavage by 3ABC boosts viral replication and disrupts disease pathogenesis, but it is not by itself sufficient to break the host-species barrier to HAV infection in mice. IMPACT AND IMPLICATIONS: The limited host range of human hepatitis viruses could be explained by species-specific viral strategies that disrupt innate immune responses. Both hepatitis A virus (HAV) and hepatitis C virus express viral proteases that cleave the innate immune adaptor protein MAVS, in human but not mouse cells. However, the impact of this immune evasion strategy has never been assessed in vivo. Here we show that HAV 3ABC protease cleavage of MAVS enhances viral replication and lessens liver inflammation in mice lacking interferon receptors, but that it is insufficient by itself to overcome the cross-species barrier to infection in mice. These results enhance our understanding of how hepatitis viruses interact with the host and their impact on innate immune responses.

IRF3-mediated pathogenicity in a murine model of human hepatitis A.

HAV-infected Ifnar1-/- mice recapitulate many of the cardinal features of hepatitis A in humans, including serum alanine aminotransferase (ALT) elevation, hepatocellular apoptosis, and liver inflammation. Previous studies implicate MAVS-IRF3 signaling in pathogenesis, but leave unresolved the role of IRF3-mediated transcription versus the non-transcriptional, pro-apoptotic activity of ubiquitylated IRF3. Here, we compare the intrahepatic transcriptomes of infected versus naïve Mavs-/- and Ifnar1-/- mice using high-throughput sequencing, and identify IRF3-mediated transcriptional responses associated with hepatocyte apoptosis and liver inflammation. Infection was transcriptionally silent in Mavs-/- mice, in which HAV replicates robustly within the liver without inducing inflammation or hepatocellular apoptosis. By contrast, infection resulted in the upregulation of hundreds of genes in Ifnar1-/- mice that develop acute hepatitis closely modeling human disease. Upregulated genes included pattern recognition receptors, interferons, chemokines, cytokines and other interferon-stimulated genes. Compared with Ifnar1-/- mice, HAV-induced inflammation was markedly attenuated and there were few apoptotic hepatocytes in livers of infected Irf3S1/S1Ifnar1-/- mice in which IRF3 is transcriptionally-inactive due to alanine substitutions at Ser-388 and Ser-390. Although transcriptome profiling revealed remarkably similar sets of genes induced in Irf3S1/S1Ifnar1-/- and Ifnar1-/- mice, a subset of genes was differentially expressed in relation to the severity of the liver injury. Prominent among these were both type 1 and type III interferons and interferon-responsive genes associated previously with apoptosis, including multiple members of the ISG12 and 2'-5' oligoadenylate synthetase families. Ifnl3 and Ifnl2 transcript abundance correlated strongly with disease severity, but mice with dual type 1 and type III interferon receptor deficiency remained fully susceptible to liver injury. Collectively, our data show that IRF3-mediated transcription is required for HAV-induced liver injury in mice and identify key IRF3-responsive genes associated with pathogenicity, providing a clear distinction from the transcription-independent role of IRF3 in liver injury following binge exposure to alcohol.

Iminosugar Glucosidase Inhibitors Reduce Hepatic Inflammation in Hepatitis A Virus-Infected Ifnar1-/- Mice.

Iminosugar compounds are monosaccharide mimetics with broad but generally weak antiviral activities related to inhibition of enzymes involved in glycobiology. Miglustat (N-butyl-1-deoxynojirimycin), which is approved for the treatment of lipid storage diseases in humans, and UV-4 [N-(9-methoxynonyl)-1-deoxynojirimycin] inhibit the replication of hepatitis A virus (HAV) in cell culture (50% inhibitory concentrations [IC50s] of 32.13 µM and 8.05 µM, respectively) by blocking the synthesis of gangliosides essential for HAV cell entry. We used a murine model of hepatitis A and targeted mass spectrometry to assess the capacity of these compounds to deplete hepatic gangliosides and modify the course of HAV infection in vivo. Miglustat, given by gavage to Ifnar1-/- mice (4,800 mg/kg of body weight/day) depleted hepatic gangliosides by 69 to 75% but caused substantial gastrointestinal toxicity and failed to prevent viral infection. UV-4, similarly administered in high doses (400 mg/kg/day), was well tolerated but depleted hepatic gangliosides by only 20% after 14 days. UV-4 depletion of gangliosides varied by class. Several GM2 species were paradoxically increased, likely due to inhibition of ß-glucosidases that degrade gangliosides. Both compounds enhanced, rather than reduced, virus replication. Nonetheless, both iminosugars had surprising anti-inflammatory effects, blocking the accumulation of inflammatory cells within the liver. UV-4 treatment also resulted in a decrease in serum alanine aminotransferase (ALT) elevations associated with acute hepatitis A. These anti-inflammatory effects may result from iminosugar inhibition of cellular α-glucosidases, leading to impaired maturation of glycan moieties of chemokine and cytokine receptors, and point to the potential importance of paracrine signaling in the pathogenesis of acute hepatitis A. IMPORTANCE Hepatitis A virus (HAV) is a common cause of viral hepatitis. Iminosugar compounds block its replication in cultured cells by inhibiting the synthesis of gangliosides required for HAV cell entry but have not been tested for their ability to prevent or treat hepatitis A in vivo. We show that high doses of the iminosugars miglustat and UV-4 fail to deplete gangliosides sufficiently to block HAV infection in mice lacking a key interferon receptor. These compounds nonetheless have striking anti-inflammatory effects on the HAV-infected liver, reducing the severity of hepatitis despite enhancing chemokine and cytokine expression resulting from hepatocyte-intrinsic antiviral responses. We propose that iminosugar inhibition of cellular α-glucosidases impairs the maturation of glycan moieties of chemokine and cytokine receptors required for effective signaling. These data highlight the potential importance of paracrine signaling pathways in the inflammatory response to HAV and add to our understanding of HAV pathogenesis in mice.

T cells protect against hepatitis A virus infection and limit infection-induced liver injury.

BACKGROUND & AIMS: Hepatitis A virus (HAV) is a common cause of enterically transmitted viral hepatitis. In non-immune individuals, infection results in typically transient but occasionally fulminant and fatal inflammatory liver injury. Virus-specific T cell frequencies peak when liver damage is at its zenith, leading to the prevalent notion that T cells exacerbate liver disease, as suspected for other hepatotropic virus infections. However, the overall contribution of T cells to the control of HAV and the pathogenesis of hepatitis A is unclear and has been impeded by a historic lack of small animal models. METHODS: Ifnar1-/- mice are highly permissive for HAV and develop pathogenesis that recapitulates many features of hepatitis A. Using this model, we identified HAV-specific CD8+ and CD4+ T cells by epitope mapping, and then used tetramers and functional assays to quantify T cells in the liver at multiple times after infection. We assessed the relationships between HAV-specific T cell frequency, viral RNA amounts, and liver pathogenesis. RESULTS: A large population of virus-specific T cells accumulated within the livers of Ifnar1-/- mice during the first 1-2 weeks of infection and persisted over time. HAV replication was enhanced and liver disease exacerbated when mice were depleted of T cells. Conversely, immunization with a peptide vaccine increased virus-specific CD8+ T cell frequencies in the liver, reduced viral RNA abundance, and lessened liver injury. CONCLUSION: These data show that T cells protect against HAV-mediated liver injury and can be targeted to improve liver health. LAY SUMMARY: Hepatitis A virus is a leading cause of acute viral hepatitis worldwide. T cells were thought to contribute to liver injury during acute infection. We now show that virus-specific T cells protect against infection and limit liver injury.

LAG-3 Confers a Competitive Disadvantage upon Antiviral CD8+ T Cell Responses.

Ongoing clinical trials are evaluating the benefits of systemic blockade of lymphocyte activation gene-3 (LAG-3) signals to improve immunity to tumors. Those studies are founded on the well-established inhibitory role of LAG-3 in regulating CD8(+) T cells during chronic virus infection and antitumor responses. However, the T cell response in LAG-3-deficient mice is similar in size and function to that in wild type animals, suggesting LAG-3 has nuanced immune-regulatory functions. We performed a series of adoptive transfer experiments in mice to better understand the T cell-intrinsic functions of LAG-3 in the regulation of CD8(+) T cell responses. Our results indicate that LAG-3 expression by CD8(+) T cells inhibits their competitive fitness and results in a slightly reduced rate of cell division in comparison with LAG-3-deficient cells. This cell-intrinsic effect of LAG-3 was consistent across both acute and chronic virus infections. These data show that LAG-3 directly modulates the size of the T cell response and support the use of LAG-3 blockade regimens to enhance CD8(+) T cell responses.

IgG-Immune Complexes Promote B Cell Memory by Inducing BAFF.

Memory B cell responses are vital for protection against infections but must also be regulated to prevent autoimmunity. Cognate T cell help, somatic hypermutation, and affinity maturation within germinal centers (GCs) are required for high-affinity memory B cell formation; however, the signals that commit GC B cells to the memory pool remain unclear. In this study, we identify a role for IgG-immune complexes (ICs), FcγRs, and BAFF during the formation of memory B cells in mice. We found that early secretion of IgG in response to immunization with a T-dependent Ag leads to IC-FcγR interactions that induce dendritic cells to secrete BAFF, which acts at or upstream of Bcl-6 in activated B cells. Loss of CD16, hematopoietic cell-derived BAFF, or blocking IC:FcγR regions in vivo diminished the expression of Bcl-6, the frequency of GC and memory B cells, and secondary Ab responses. BAFF also contributed to the maintenance and/or expansion of the follicular helper T cell population, although it was dispensable for their formation. Thus, early Ab responses contribute to the optimal formation of B cell memory through IgG-ICs and BAFF. Our work defines a new role for FcγRs in GC and memory B cell responses.

Epigenetic Dysfunction in Turner Syndrome Immune Cells.

Turner syndrome (TS) is a chromosomal condition associated with partial or complete absence of the X chromosome that involves characteristic findings in multiple organ systems. In addition to well-known clinical characteristics such as short stature and gonadal failure, TS is also associated with T cell immune alterations and chronic otitis media, suggestive of a possible immune deficiency. Recently, ubiquitously transcribed tetratricopeptide repeat on the X chromosome (UTX), a histone H3 lysine 27 (H3K27) demethylase, has been identified as a downregulated gene in TS immune cells. Importantly, UTX is an X-linked gene that escapes X-chromosome inactivation and thus is haploinsufficient in TS. Mice with T cell-specific UTX deficiency have impaired clearance of chronic viral infection due to decreased frequencies of T follicular helper (Tfh) cells, which are critical for B cell antibody generation. In parallel, TS patients have decreased Tfh frequencies in peripheral blood. Together, these findings suggest that haploinsufficiency of the X-linked UTX gene in TS T cells underlies an immune deficit, which may manifest as increased predisposition to chronic otitis media.

IFN-λ exerts opposing effects on T cell responses depending on the chronicity of the virus infection.

IFN-λ induces an antiviral state in many cell types and may contribute to the overall inflammatory environment after infection. Either of these effects may influence adaptive immune responses, but the role of type 3 IFNs in the development of primary and memory T cell responses to infection has not been evaluated. In this study, we examined T cell responses to acute or persistent lymphocytic choriomeningitis virus infection in IFN-λR1-deficient mice. Following acute infection, we find that IFN-λR1-deficient mice produced normal levels of IFN, robust NK cell responses, but greater than normal CD4+ and CD8+ T cell responses compared with wild type BALB/c mice. There were more T cells that were IL-7R(hi) and, correspondingly, the IFN-λR-deficient mice showed a 2- to 3-fold increase in memory T cell number. The inhibitory effect of IFN-λR expression was independent of direct cytokine signaling into T cells. In contrast with acute infection, the IFN-λR-deficient mice generated markedly diminished T cell responses and had greater weight loss compared with wild type mice when confronted with a highly disseminating variant of lymphocytic choriomeningitis virus. These data indicate that IFN-λR limits T cell responses and memory after transient infection but augments T cell responses during persisting infection. Thus, the immune-regulatory functions for IFN-λR are complex and vary with the overall inflammatory environment.

B cell depletion curtails CD4+ T cell memory and reduces protection against disseminating virus infection.

Dynamic interactions between CD4(+) T cells and B cells are needed for humoral immunity and CD4(+) T cell memory. It is not known whether B cells are needed early on to induce the formation of memory precursor cells or are needed later to sustain memory cells. In this study, primary and memory CD4(+) T cells responses were followed in wild-type mice that were depleted of mature B cells by anti-CD20 before or different times after acute lymphocytic choriomeningitis virus infection. The Ab treatment led to a 1000-fold reduction in B cell number that lasted 6 wk. Primary virus-specific CD4(+) Th1 cells were generated in B cell-depleted mice; however, there was a decrease in the CD4(+)Ly6C(lo)Tbet(+) memory precursor population and a corresponding 4-fold reduction in CD4(+) memory cell number. Memory T cells showed impaired cytokine production when they formed without B cells. B cell depletion had no effect on established memory populations. During disseminating virus infection, B cell depletion led to sustained weight loss and functional exhaustion of CD4(+) and CD8(+) T cells, and prevented mice from resolving the infection. Thus, B cells contribute to the establishment and survival of memory CD4(+) T cells post-acute infection and play an essential role in immune protection against disseminating virus infection.

NK cells and their ability to modulate T cells during virus infections.

Natural killer (NK) cells are important in protection against virus infections, and many viruses have evolved mechanisms to thwart NK cell activity. NK cells respond to inflammatory signals at an early stage of virus infection, resulting in proliferation, cytokine production, and cytolytic activity that can reduce virus loads. Moreover, the rapid kinetics of the NK cell response enables NK cells to influence other populations of innate immune cells, affect the inflammatory milieu, and guide adaptive immune responses to infection. Early NK cell interactions with other leukocytes can have long-lasting effects on the number and quality of memory T cells, as well as impact the exhaustion of T cells during chronic infections. The ability of NK cells to modulate T cell responses can be mediated through direct T-NK interactions, cytokine production, or indirectly through dendritic cells and other cell types. Herein, we summarize our current understanding of how NK cells interact with T cells, dendritic cells, B cells, and other cell types involved in adaptive immune responses to virus infection. We outline several mechanisms by which NK cells enhance or suppress adaptive immune response and long-lived immunological memory.

The depletion of NK cells prevents T cell exhaustion to efficiently control disseminating virus infection.

NK cells have well-established functions in immune defense against virus infections and cancer through their cytolytic activity and production of cytokines. In this study, we examined the frequency of NK cells and their influence on T cell responses in mice given variants of lymphocytic choriomeningitis virus that cause acute or persisting infection. We found increased frequencies of circulating NK cells during disseminating infection compared with uninfected or acutely infected mice. Consistent with recent reports, we observed that the depletion of NK cells in mice with disseminated infection increased peak numbers of virus-specific cytokine producing CD8(+) T cells and resulted in the rapid resolution of disseminated infection. Additionally, we show that NK cell depletion sustained T cell responses across time and protected against T cell exhaustion. The positive effects of NK cell depletion on T cell responses only occurred when NK cells were depleted within the first 2 d of infection. We find that the improved CD8(+) T cell response correlated with an enhanced ability of APCs from NK cell-depleted mice to stimulate T cell proliferation, independently of the effects of NK cells on CD4(+) T cells. These results indicate that NK cells play an integral role in limiting the CD8 T cell response and contribute to T cell exhaustion by diminishing APC function during persisting virus infection.

Differential T cell responses to residual viral antigen prolong CD4+ T cell contraction following the resolution of infection.

The contraction phase of the T cell response is a poorly understood period after the resolution of infection when virus-specific effector cells decline in number and memory cells emerge with increased frequencies. CD8(+) T cells plummet in number and quickly reach stable levels of memory following acute lymphocytic choriomeningitis virus infection in mice. In contrast, virus-specific CD4(+) T cells gradually decrease in number and reach homeostatic levels only after many weeks. In this study, we provide evidence that MHCII-restricted viral Ag persists during the contraction phase following this prototypical acute virus infection. We evaluated whether the residual Ag affected the cell division and number of virus-specific naive and memory CD4(+) T cells and CD8(+) T cells. We found that naive CD4(+) T cells underwent cell division and accumulated in response to residual viral Ag for >2 mo after the eradication of infectious virus. Surprisingly, memory CD4(+) T cells did not undergo cell division in response to the lingering Ag, despite their heightened capacity to recognize Ag and make cytokine. In contrast to CD4(+) T cells, CD8(+) T cells did not undergo cell division in response to the residual Ag. Thus, CD8(+) T cells ceased division within days after the infection was resolved, indicating that CD8(+) T cell responses are tightly linked to endogenous processing of de novo synthesized virus protein. Our data suggest that residual viral Ag delays the contraction of CD4(+) T cell responses by recruiting new populations of CD4(+) T cells.

A pathologically expanded, clonal lineage of IL-21 producing CD4+ T cells drives Inflammatory neuropathy.

Inflammatory neuropathies, which include CIDP (chronic inflammatory demyelinating polyneuropathy) and GBS (Guillain Barre Syndrome), result from autoimmune destruction of the peripheral nervous system (PNS) and are characterized by progressive weakness and sensory loss. CD4+ T cells play a key role in the autoimmune destruction of the PNS. Yet, key properties of pathogenic CD4+ T cells remain incompletely understood. Here, we use paired scRNAseq and scTCRseq of peripheral nerves from an inflammatory neuropathy mouse model to identify IL-21 expressing CD4+ T cells that are clonally expanded and multifunctional. These IL-21-expressing CD4+ T cells are comprised of two transcriptionally distinct expanded populations, which express genes associated with Tfh and Tph subsets. Remarkably, TCR clonotypes are shared between these two IL-21-expressing populations, suggesting a common lineage differentiation pathway. Finally, we demonstrate that IL-21 signaling is required for neuropathy development and pathogenic T cell infiltration into peripheral nerves. IL-21 signaling upregulates CXCR6, a chemokine receptor that promotes CD4+ T cell localization in peripheral nerves. Together, these findings point to IL-21 signaling, Tfh/Tph differentiation, and CXCR6-mediated cellular localization as potential therapeutic targets in inflammatory neuropathies.