DExD/H-box helicase 9 intrinsically controls CD8+ T cell-mediated antiviral response through noncanonical mechanisms.

Upon virus infection, CD8+ T cell accumulation is tightly controlled by simultaneous proliferation and apoptosis. However, it remains unclear how TCR signal coordinates these events to achieve expansion and effector cell differentiation. We found that T cell-specific deletion of nuclear helicase Dhx9 led to impaired CD8+ T cell survival, effector differentiation, and viral clearance. Mechanistically, Dhx9 acts as the key regulator to ensure LCK- and CD3ε-mediated ZAP70 phosphorylation and ERK activation to protect CD8+ T cells from apoptosis before proliferative burst. Dhx9 directly regulates Id2 transcription to control effector CD8+ T cell differentiation. The DSRM and OB_Fold domains are required for LCK binding and Id2 transcription, respectively. Dhx9 expression is predominantly increased in effector CD8+ T cells of COVID-19 patients. Therefore, we revealed a previously unknown regulatory mechanism that Dhx9 protects activated CD8+ T cells from apoptosis and ensures effector differentiation to promote antiviral immunity independent of nuclear sensor function.

RIG-I and MDA5 Protect Mice From Pichinde Virus Infection by Controlling Viral Replication and Regulating Immune Responses to the Infection.

RIG-I and MDA5 are major cytoplasmic innate-immune sensor proteins that recognize aberrant double-stranded RNAs generated during virus infection to activate type 1 interferon (IFN-I) and IFN-stimulated gene (ISG) expressions to control virus infection. The roles of RIG-I and MDA5 in controlling replication of Pichinde virus (PICV), a mammarenavirus, in mice have not been examined. Here, we showed that MDA5 single knockout (SKO) and RIG-I/MDA5 double knockout (DKO) mice are highly susceptible to PICV infection as evidenced by their significant reduction in body weights during the course of the infection, validating the important roles of these innate-immune sensor proteins in controlling PICV infection. Compared to the wildtype mice, SKO and DKO mice infected with PICV had significantly higher virus titers and lower IFN-I expressions early in the infection but appeared to exhibit a late and heightened level of adaptive immune responses to clear the infection. When a recombinant rPICV mutant virus (rPICV-NPmut) that lacks the ability to suppress IFN-I was used to infect mice, as expected, there were heightened levels of IFN-I and ISG expressions in the wild-type mice, whereas infected SKO and DKO mice showed delayed mouse growth kinetics and relatively low, delayed, and transient levels of innate and adaptive immune responses to this viral infection. Taken together, our data suggest that PICV infection triggers activation of immune sensors that include but might not be necessarily limited to RIG-I and MDA5 to stimulate effective innate and adaptive immune responses to control virus infection in mice.

BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8+ T cells.

During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.

Arenavirus Induced CCL5 Expression Causes NK Cell-Mediated Melanoma Regression.

Immune activation within the tumor microenvironment is one promising approach to induce tumor regression. Certain viruses including oncolytic viruses such as the herpes simplex virus (HSV) and non-oncolytic viruses such as the lymphocytic choriomeningitis virus (LCMV) are potent tools to induce tumor-specific immune activation. However, not all tumor types respond to viro- and/or immunotherapy and mechanisms accounting for such differences remain to be defined. In our current investigation, we used the non-cytopathic LCMV in different human melanoma models and found that melanoma cell lines produced high levels of CCL5 in response to immunotherapy. In vivo, robust CCL5 production in LCMV infected Ma-Mel-86a tumor bearing mice led to recruitment of NK cells and fast tumor regression. Lack of NK cells or CCL5 abolished the anti-tumoral effects of immunotherapy. In conclusion, we identified CCL5 and NK cell-mediated cytotoxicity as new factors influencing melanoma regression during virotherapy.

CD49a+CD49b+ NK cells induced by viral infection reflect an activated state of conventional NK cells.

Natural killer (NK) cells are important innate effectors that play a pivotal role in the defense against tumors and infections and participate in regulating adaptive immunity. Recent studies have revealed phenotypic and functional heterogeneity of NK cells. Here, using murine models of acute and chronic lymphocytic choriomeningitis virus infection, we observed that a CD49a+ CD49b+ NK cell subset emerged in the liver and other tissues, and underwent vigorous expansion following viral infection, before progressively decreasing in cell number. These viral infection-induced CD49a+CD49b+ NK cells displayed an activated and mature phenotype. Moreover, compared with liver-resident NK cells and conventional NK (cNK) cells, CD49a+CD49b+ NK cells showed increased functional competence, as evidenced by higher amounts of IFN-γ production and stronger cytotoxic capabilities during viral infection. Generation of these CD49a+CD49b+ NK cells was shown to be independent of the T-bet transcription factor. Adoptive transfer experiments revealed that cNK cells could convert into CD49a+CD49b+ NK cells following viral infection. Collectively, these results suggest that viral infection-induced CD49a+CD49b+ NK cells represent a transiently activated state of cNK cells.

Fragile X mental retardation protein protects against tumour necrosis factor-mediated cell death and liver injury.

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.

Modulation of the humoral immune response by constitutively active STAT6 expression in murine B cells.

The transcription factor STAT6 regulates gene expression in response to IL-4 and IL-13. To further investigate how activated STAT6 modulates B cells development and function in vivo, we characterized mice that express a constitutively active version specifically in B cells. CD19Cre_STAT6vt mice show spontaneous phosphorylation and nuclear translocation of STAT6 in B cells. About 80 genes were more than twofold up- or downregulated in splenic B cells from CD19Cre_STAT6vt as compared to control mice. B cell development, tissue localization, and populations of follicular and marginal zone B cells, B1 B cells, GC B cells, and plasma cells (PCs) appeared to be normal. However, the number of IgE+ and IgG1+ GC B cells and PCs as well as serum IgE and IgG1 levels were increased in CD19Cre_STAT6vt mice. Infection with Lymphocytic choriomeningitis virus associated with high levels of TNF and IFN-γ did not prevent the development of a significantly increased IgE and IgG1 response against the virus in CD19Cre_STAT6vt mice. These results suggest that prolonged STAT6 activation during chronic allergic inflammation may result in IgE responses during subsequent viral or bacterial infection that could further stimulate mast cell activation even in the absence of the initial allergic response.

The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors.

Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of "drug repurposing" strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections.

Restored immune cell functions upon clearance of senescence in the irradiated splenic environment.

Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent-associated secretory phenotype (SASP) and expression of p16INK4a in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16-3MR mice that allow elimination of p16INK4a -positive cells with exposure to ganciclovir, we show that impaired T-cell proliferation is partially reversed, mechanistically dependent on p16INK4a expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16INK4a expression. Our results provide molecular insight on how senescence-inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment.

Modulation of asymmetric cell division as a mechanism to boost CD8+ T cell memory.

Asymmetric partitioning of fate determinants is a mechanism that contributes to T cell differentiation. However, it remains unclear whether the ability of T cells to divide asymmetrically is influenced by their differentiation state, as well as whether enforcing asymmetric cell division (ACD) rates would have an impact on T cell differentiation and memory formation. Using the murine LCMV infection model, we established a correlation between cell stemness and the ability of CD8+ T cells to undergo ACD. Transient mTOR inhibition was proven to increase ACD rates in naïve and memory cells and to install this ability in exhausted CD8+ T cells. Functionally, enforced ACD correlated with increased memory potential, leading to more efficient recall response and viral control upon acute or chronic LCMV infection. Moreover, transient mTOR inhibition also increased ACD rates in human CD8+ T cells. Transcriptional profiling revealed that progenies emerging from enforced ACD exhibited more pronounced early memory signatures, which functionally endowed these cells with better survival in the absence of antigen exposure and more robust homing to secondary lymphoid organs, providing critical access to survival niches. Our data provide important insights into how ACD can improve long-term survival and function of T cells and open new perspectives for vaccination and adoptive T cell transfer therapies.

Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction.

T cells become dysfunctional when they encounter self antigens or are exposed to chronic infection or to the tumour microenvironment1. The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction2. However, the molecular mechanisms that underlie this dysfunction remain unclear. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. Overexpression of NR4A1 inhibits effector T cell differentiation, whereas deletion of NR4A1 overcomes T cell tolerance and exaggerates effector function, as well as enhancing immunity against tumour and chronic virus. Mechanistically, NR4A1 is preferentially recruited to binding sites of the transcription factor AP-1, where it represses effector-gene expression by inhibiting AP-1 function. NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy.

Immune Exhaustion: Past Lessons and New Insights from Lymphocytic Choriomeningitis Virus.

Lymphocytic choriomeningitis virus (LCMV) is a paradigm-forming experimental system with a remarkable track record of contributing to the discovery of many of the fundamental concepts of modern immunology. The ability of LCMV to establish a chronic infection in immunocompetent adult mice was instrumental for identifying T cell exhaustion and this system has been invaluable for uncovering the complexity, regulators, and consequences of this state. These findings have been directly relevant for understanding why ineffective T cell responses commonly arise during many chronic infections including HIV and HCV, as well as during tumor outgrowth. The principal feature of exhausted T cells is the inability to elaborate the array of effector functions necessary to contain the underlying infection or tumor. Using LCMV to determine how to prevent and reverse T cell exhaustion has highlighted the potential of checkpoint blockade therapies, most notably PD-1 inhibition strategies, for improving cellular immunity under conditions of antigen persistence. Here, we discuss the discovery, properties, and regulators of exhausted T cells and highlight how LCMV has been at the forefront of advancing our understanding of these ineffective responses.

Cutting Edge: Early Attrition of Memory T Cells during Inflammation and Costimulation Blockade Is Regulated Concurrently by Proapoptotic Proteins Fas and Bim.

Apoptosis of CD8 T cells is an essential mechanism that maintains immune system homeostasis, prevents autoimmunity, and reduces immunopathology. CD8 T cell death also occurs early during the response to both inflammation and costimulation blockade (CoB). In this article, we studied the effects of a combined deficiency of Fas (extrinsic pathway) and Bim (intrinsic pathway) on early T cell attrition in response to lymphocytic choriomeningitis virus infection and during CoB during transplantation. Loss of Fas and Bim function in Bcl2l11-/-Faslpr/lpr mice inhibited apoptosis of T cells and prevented the early T cell attrition resulting from lymphocytic choriomeningitis virus infection. Bcl2l11-/-Faslpr/lpr mice were also resistant to prolonged allograft survival induced by CoB targeting the CD40-CD154 pathway. These results demonstrate that both extrinsic and intrinsic apoptosis pathways function concurrently to regulate T cell homeostasis during the early stages of immune responses and allograft survival during CoB.

DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection.

Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication associated with both a previously un-recognized DDX3 inhibitory role in type I interferon production in arenavirus infected cells and a positive DDX3 effect on arenavirus RNA synthesis that was dependent on its ATPase and Helicase activities. Our results uncover novel mechanisms used by arenaviruses to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses.

Antibodies to the Glycoprotein GP2 Subunit Cross-React between Old and New World Arenaviruses.

Arenaviruses pose a major public health threat and cause numerous infections in humans each year. Although most viruses belonging to this family do not cause disease in humans, some arenaviruses, such as Lassa virus and Machupo virus, are the etiological agents of lethal hemorrhagic fevers. The absence of a currently licensed vaccine and the highly pathogenic nature of these viruses both make the necessity of developing viable vaccines and therapeutics all the more urgent. Arenaviruses have a single glycoprotein on the surface of virions, the glycoprotein complex (GPC), and this protein can be used as a target for vaccine development. Here, we describe immunization strategies to generate monoclonal antibodies (MAbs) that cross-react between the glycoprotein complexes of both Old World and New World arenaviruses. Several monoclonal antibodies isolated from immunized mice were highly cross-reactive, binding a range of Old World arenavirus glycoproteins, including that of Lassa virus. One such monoclonal antibody, KL-AV-2A1, bound to GPCs of both New World and Old World viruses, including Lassa and Machupo viruses. These cross-reactive antibodies bound to epitopes present on the glycoprotein 2 subunit of the glycoprotein complex, which is relatively conserved among arenaviruses. Monoclonal antibodies binding to these epitopes, however, did not inhibit viral entry as they failed to neutralize a replication-competent vesicular stomatitis virus pseudotyped with the Lassa virus glycoprotein complex in vitro In addition, no protection from virus challenge was observed in in vivo mouse models. Even so, these monoclonal antibodies might still prove to be useful in the development of clinical and diagnostic assays.IMPORTANCE Several viruses in the Arenaviridae family infect humans and cause severe hemorrhagic fevers which lead to high case fatality rates. Due to their pathogenicity and geographic tropisms, these viruses remain very understudied. As a result, an effective vaccine or therapy is urgently needed. Here, we describe efforts to produce cross-reactive monoclonal antibodies that bind to both New and Old World arenaviruses. All of our MAbs seem to be nonneutralizing and nonprotective and target subunit 2 of the glycoprotein. Due to the lack of reagents such as recombinant glycoproteins and antibodies for rapid detection assays, our MAbs could be beneficial as analytic and diagnostic tools.

Involvement of Pro-Inflammatory Macrophages in Liver Pathology of Pirital Virus-Infected Syrian Hamsters.

New World arenaviruses cause fatal hemorrhagic disease in South America. Pirital virus (PIRV), a mammarenavirus hosted by Alston’s cotton rat (Sigmodon alstoni), causes a disease in Syrian golden hamsters (Mesocricetus auratus) (biosafety level-3, BSL-3) that has many pathologic similarities to the South American hemorrhagic fevers (BSL-4) and, thus, is considered among the best small-animal models for human arenavirus disease. Here, we extend in greater detail previously described clinical and pathological findings in Syrian hamsters and provide evidence for a pro-inflammatory macrophage response during PIRV infection. The liver was the principal target organ of the disease, and signs of Kupffer cell involvement were identified in mortally infected hamster histopathology data. Differential expression analysis of liver mRNA revealed signatures of the pro-inflammatory response, hematologic dysregulation, interferon pathway and other host response pathways, including 17 key transcripts that were also reported in two non-human primate (NHP) arenavirus liver-infection models, representing both Old and New World mammarenavirus infections. Although antigen presentation may differ among rodent and NHP species, key hemostatic and innate immune-response components showed expression parallels. Signatures of pro-inflammatory macrophage involvement in PIRV-infected livers included enrichment of Ifng, Nfkb2, Stat1, Irf1, Klf6, Il1b, Cxcl10, and Cxcl11 transcripts. Together, these data indicate that pro-inflammatory macrophage M1 responses likely contribute to the pathogenesis of acute PIRV infection.

Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment.

Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/ß receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNARfl/flxFoxp3YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.

Conserved and Differential Features of TNF Superfamily Ligand Expression on APC Subsets over the Course of a Chronic Viral Infection in Mice.

There is currently much interest in how different APC subsets shape the immune response. We recently described a division of labor between classical dendritic cells (cDC) and inflammatory monocyte-derived APC in provision of costimulatory ligands to T cells early during chronic lymphocytic choriomeningitis clone 13 (LCMV 13) infection in mice. At day 2 of LCMV 13 infection, cDC preferentially express CD80 and CD86, whereas TNF superfamily ligands GITRL, 4-1BBL, CD70, and OX40L are preferentially induced by type I IFN on inflammatory monocyte-derived APC, with minimal expression on cDC. In this study, we further investigate the expression of TNF and B7 family ligands on APC over the course of LCMV 13 infection. OX40L and 4-1BBL remain above baseline through the chronic stage of infection, with predominant expression on inflammatory APC compared with cDC in the spleen, partially blocked by anti-IFN-γR Ab pretreatment. Conversely, CD70, like GITRL, returns to baseline on the APC within a few days postinfection. In the lung, TNF family ligands were also preferentially expressed on inflammatory monocyte-derived APC. CD86 was generally higher on cDC than inflammatory APC in the spleen, but in the lung CD86 was highest on inflammatory APC. Moreover, in the spleen, CD80 levels on different APC subsets fluctuated over the course of the infection. We also show that LPS induction of TNF superfamily ligands is largely mediated through type I IFN. This study highlights the importance of IFNs and monocyte-derived APC in TNF superfamily ligand expression in both secondary lymphoid organs and tissues during chronic viral infection.

Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response.

CD4+ follicular regulatory T (Tfr) cells suppress B cell responses through modulation of follicular helper T (Tfh) cells and germinal center (GC) development. We found that Tfr cells can also promote the GC response through provision of interleukin-10 (IL-10) after acute infection with lymphocytic choriomeningitis virus (LCMV). Sensing of IL-10 by B cells was necessary for optimal development of the GC response. GC B cells formed in the absence of Treg cell-derived IL-10 displayed an altered dark zone state and decreased expression of the transcription factor Forkhead box protein 1 (FOXO1). IL-10 promoted nuclear translocation of FOXO1 in activated B cells. These data indicate that Tfr cells play a multifaceted role in the fine-tuning of the GC response and identify IL-10 as an important mediator by which Tfr cells support the GC reaction.