Gene Regulatory Circuits in Innate and Adaptive Immune Cells.

Cell identity and function largely rely on the programming of transcriptomes during development and differentiation. Signature gene expression programs are orchestrated by regulatory circuits consisting of cis-acting promoters and enhancers, which respond to a plethora of cues via the action of transcription factors. In turn, transcription factors direct epigenetic modifications to revise chromatin landscapes, and drive contacts between distal promoter-enhancer combinations. In immune cells, regulatory circuits for effector genes are especially complex and flexible, utilizing distinct sets of transcription factors and enhancers, depending on the cues each cell type receives during an infection, after sensing cellular damage, or upon encountering a tumor. Here, we review major players in the coordination of gene regulatory programs within innate and adaptive immune cells, as well as integrative omics approaches that can be leveraged to decipher their underlying circuitry.

Rebalancing TGFß1/BMP signals in exhausted T cells unlocks responsiveness to immune checkpoint blockade therapy.

T cell dysfunctionality prevents the clearance of chronic infections and cancer. Furthermore, epigenetic programming in dysfunctional CD8+ T cells limits their response to immunotherapies, including immune checkpoint blockade (ICB). However, it is unclear which upstream signals drive acquisition of dysfunctional epigenetic programs, and whether therapeutically targeting these signals can remodel terminally dysfunctional T cells to an ICB-responsive state. Here we innovate an in vitro model system of stable human T cell dysfunction and show that chronic TGFß1 signaling in posteffector CD8+ T cells accelerates their terminal dysfunction through stable epigenetic changes. Conversely, boosting bone morphogenetic protein (BMP) signaling while blocking TGFß1 preserved effector and memory programs in chronically stimulated human CD8+ T cells, inducing superior responses to tumors and synergizing the ICB responses during chronic viral infection. Thus, rebalancing TGFß1/BMP signals provides an exciting new approach to unleash dysfunctional CD8+ T cells and enhance T cell immunotherapies.

Developmental plasticity allows outside-in immune responses by resident memory T cells.

Central memory T (TCM) cells patrol lymph nodes and perform conventional memory responses on restimulation: proliferation, migration and differentiation into diverse T cell subsets while also self-renewing. Resident memory T (TRM) cells are parked within single organs, share properties with terminal effectors and contribute to rapid host protection. We observed that reactivated TRM cells rejoined the circulating pool. Epigenetic analyses revealed that TRM cells align closely with conventional memory T cell populations, bearing little resemblance to recently activated effectors. Fully differentiated TRM cells isolated from small intestine epithelium exhibited the potential to differentiate into TCM cells, effector memory T cells and TRM cells on recall. Ex-TRM cells, former intestinal TRM cells that rejoined the circulating pool, heritably maintained a predilection for homing back to their tissue of origin on subsequent reactivation and a heightened capacity to redifferentiate into TRM cells. Thus, TRM cells can rejoin the circulation but are advantaged to re-form local TRM when called on.

Beta cell-specific CD8+ T cells maintain stem cell memory-associated epigenetic programs during type 1 diabetes.

The pool of beta cell-specific CD8+ T cells in type 1 diabetes (T1D) sustains an autoreactive potential despite having access to a constant source of antigen. To investigate the long-lived nature of these cells, we established a DNA methylation-based T cell 'multipotency index' and found that beta cell-specific CD8+ T cells retained a stem-like epigenetic multipotency score. Single-cell assay for transposase-accessible chromatin using sequencing confirmed the coexistence of naive and effector-associated epigenetic programs in individual beta cell-specific CD8+ T cells. Assessment of beta cell-specific CD8+ T cell anatomical distribution and the establishment of stem-associated epigenetic programs revealed that self-reactive CD8+ T cells isolated from murine lymphoid tissue retained developmentally plastic phenotypic and epigenetic profiles relative to the same cells isolated from the pancreas. Collectively, these data provide new insight into the longevity of beta cell-specific CD8+ T cell responses and document the use of this methylation-based multipotency index for investigating human and mouse CD8+ T cell differentiation.

De Novo Epigenetic Programs Inhibit PD-1 Blockade-Mediated T Cell Rejuvenation.

Immune-checkpoint-blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA-methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells, and approaches to reverse these programs improved T cell responses and tumor control during ICB. These data establish de novo DNA-methylation programming as a regulator of T cell exhaustion and barrier of ICB-mediated T cell rejuvenation.

Functional T cells are capable of supernumerary cell division and longevity.

Differentiated somatic mammalian cells putatively exhibit species-specific division limits that impede cancer but may constrain lifespans1-3. To provide immunity, transiently stimulated CD8+ T cells undergo unusually rapid bursts of numerous cell divisions, and then form quiescent long-lived memory cells that remain poised to reproliferate following subsequent immunological challenges. Here we addressed whether T cells are intrinsically constrained by chronological or cell-division limits. We activated mouse T cells in vivo using acute heterologous prime-boost-boost vaccinations4, transferred expanded cells to new mice, and then repeated this process iteratively. Over 10 years (greatly exceeding the mouse lifespan)5 and 51 successive immunizations, T cells remained competent to respond to vaccination. Cells required sufficient rest between stimulation events. Despite demonstrating the potential to expand the starting population at least 1040-fold, cells did not show loss of proliferation control and results were not due to contamination with young cells. Persistent stimulation by chronic infections or cancer can cause T cell proliferative senescence, functional exhaustion and death6. We found that although iterative acute stimulations also induced sustained expression and epigenetic remodelling of common exhaustion markers (including PD1, which is also known as PDCD1, and TOX) in the cells, they could still proliferate, execute antimicrobial functions and form quiescent memory cells. These observations provide a model to better understand memory cell differentiation, exhaustion, cancer and ageing, and show that functionally competent T cells can retain the potential for extraordinary population expansion and longevity well beyond their organismal lifespan.

TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.

Microglia-targeted inhibition of miR-17 via mannose-coated lipid nanoparticles improves pathology and behavior in a mouse model of Alzheimer's disease.

Neuroinflammation and accumulation of Amyloid Beta (Aß) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aß accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aß burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs.

Effector CD8 T cells dedifferentiate into long-lived memory cells.

Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells while key effector genes remain demethylated, demonstrating that memory T cells arise from a subset of fate-permissive effector T cells.

Generating long-lived CD8(+) T-cell memory: Insights from epigenetic programs.

T-cell-based immunological memory has the potential to provide the host with life-long protection against pathogen reexposure and thus offers tremendous promise for the design of vaccines targeting chronic infections or cancer. In order to exploit this potential in the design of new vaccines, it is necessary to understand how and when memory T cells acquire their poised effector potential, and moreover, how they maintain these properties during homeostatic proliferation. To gain insight into the persistent nature of memory T-cell functions, investigators have turned their attention to epigenetic mechanisms. Recent efforts have revealed that many of the properties acquired among memory T cells are coupled to stable changes in DNA methylation and histone modifications. Furthermore, it has recently been reported that the delineating features among memory T cells subsets are also linked to distinct epigenetic events, such as permissive and repressive histone modifications and DNA methylation programs, providing exciting new hypotheses regarding their cellular ancestry. Here, we review recent studies focused on epigenetic programs acquired during effector and memory T-cell differentiation and discuss how these data may shed new light on the developmental path for generating long-lived CD8(+) T-cell memory.

Depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections.

Viruses such as influenza suppress host immune function by a variety of methods. This may result in significant morbidity through several pathways, including facilitation of secondary bacterial pneumonia from pathogens such as Streptococcus pneumoniae. PKH26-phagocytic cell labeling dye was administered intranasally to label resident alveolar macrophages (AMs) in a well-established murine model before influenza infection to determine turnover kinetics during the course of infection. More than 90% of resident AMs were lost in the first week after influenza, whereas the remaining cells had a necrotic phenotype. To establish the impact of this innate immune defect, influenza-infected mice were challenged with S. pneumoniae. Early AM-mediated bacterial clearance was significantly impaired in influenza-infected mice: ~50% of the initial bacterial inoculum could be harvested from the alveolar airspace 3 h later. In mock-infected mice, by contrast, >95% of inocula up to 50-fold higher was efficiently cleared. Coinfection during the AM depletion phase caused significant body weight loss and mortality. Two weeks after influenza, the AM population was fully replenished with successful re-establishment of early innate host protection. Local GM-CSF treatment partially restored the impaired early bacterial clearance with efficient protection against secondary pneumococcal pneumonia. We conclude that resident AM depletion occurs during influenza infection. Among other potential effects, this establishes a niche for secondary pneumococcal infection by altering early cellular innate immunity in the lungs, resulting in pneumococcal outgrowth and lethal pneumonia. This novel mechanism will inform development of novel therapeutic approaches to restore lung innate immunity against bacterial superinfections.

Adjunctive corticosteroid therapy improves lung immunopathology and survival during severe secondary pneumococcal pneumonia in mice.

Secondary bacterial pneumonia is a significant cause of morbidity and mortality during influenza, despite routine use of standard antibiotics. Antibiotic-induced immunopathology associated with bacterial cell wall lysis has been suggested to contribute to these poor outcomes. Using Streptococcus pneumoniae in a well-established murine model of secondary bacterial pneumonia (SBP) following influenza, we stratified disease severity based on pneumococcal load in the lungs via in vivo bioluminescence imaging. Ampicillin treatment cured mice with mild pneumonia but was ineffective against severely pneumonic mice, despite effective bacterial killing. Adjunctive dexamethasone therapy improved ampicillin-induced immunopathology and improved outcomes in mice with severe SBP. However, early dexamethasone therapy during primary influenza infection impaired lung adaptive immunity as manifest by increased viral titers, with an associated loss of its protective functions in SBP. These data support adjunctive clinical use of corticosteroids in severe cases of community-acquired pneumonia.

Inflammasome-independent role of the apoptosis-associated speck-like protein containing CARD (ASC) in the adjuvant effect of MF59.

Clinical studies have indicated that subvirion inactivated vaccines against avian influenza viruses, particularly H5N1, are poorly immunogenic in humans. As a consequence, the use of adjuvants has been championed for the efficient vaccination of a naïve population against avian influenza. Aluminum salts (alum) and the oil-in-water emulsion MF59 are safe and effective adjuvants that are being used with influenza vaccines, but the mechanism underlying their stimulation of the immune system remains poorly understood. It was shown recently that activation of a cytosolic innate immune-sensing complex known as "NLR-Pyrin domain containing 3" (NLRP3) inflammasome, also known as "cryopyrin," "cold-induced autoinflammatory syndrome 1" (CIAS1), or nacht domain-, leucine-rich repeat-, and PYD-containing protein 3 (Nalp3), is essential for the adjuvant effect of alum. Here we show that the inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), an adapter protein within the NLRP3 inflammasome, is a crucial element in the adjuvant effect of MF59 when combined with H5N1 subunit vaccines. In the absence of ASC, H5-specific IgG antibody responses are significantly reduced, whereas the responses are intact in NLRP3(-/-) and caspase-1(-/-) mice. This defect is caused mainly by the failure of antigen-specific B cells to switch from IgM to IgG production. We conclude that ASC plays an inflammasome-independent role in the induction of antigen-specific humoral immunity after vaccination with MF59-adjuvanted influenza vaccines. These findings have important implications for the rational design of next-generation adjuvants.

DNA hypomethylation promotes the expression of CASPASE-4 which exacerbates inflammation and amyloid-ß deposition in Alzheimer's disease.

Alzheimer's disease (AD) is the sixth leading cause of death in the USA. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release pro-inflammatory products such as IL-1ß which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed reduced representation bisulfite sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed promotes the generation of IL-1ß and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4 and CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-ß (Aß) and increased microglial production of IL-1ß in 5xFAD mice. Utilizing RNA-sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1ß from macrophages in response to cytosolic Aß through cleavage of downstream effector Gasdermin D (GSDMD). Therefore, here we unravel the role for CASP11 and GSDMD in the generation of IL-1ß in response to Aß and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential DNA methylation in AD microglia contributes to the progression of AD pathobiology. Thus, we identify CASP4 as a potential target for immunotherapies for the treatment and prevention of AD.

DNA hypomethylation promotes the expression of CASPASE-4 which exacerbates neuroinflammation and amyloid-ß deposition in Alzheimer's disease The Ohio State University College of Medicine.

Alzheimer's Disease (AD) is the 6th leading cause of death in the US. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release proinflammatory products such as IL-1ß which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed Reduced Representation Bisulfite Sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed, can be involved in generation of IL-1ß and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4, CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-ß (Aß) and increased microglial production of IL-1ß in 5xFAD mice. Utilizing RNA sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1ß from macrophages in response to cytosolic Aß through cleavage of downstream effector Gasdermin D (G SDMD). We describe a role for CASP11 and GSDMD in the generation of IL-1ß in response to Aß and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential methylation in AD microglia contributes to the progression of AD pathobiology, thus identifying CASP4 as a potential target for immunotherapies for the treatment of AD.

Aiolos represses CD4+ T cell cytotoxic programming via reciprocal regulation of TFH transcription factors and IL-2 sensitivity.

During intracellular infection, T follicular helper (TFH) and T helper 1 (TH1) cells promote humoral and cell-mediated responses, respectively. Another subset, CD4-cytotoxic T lymphocytes (CD4-CTLs), eliminate infected cells via functions typically associated with CD8+ T cells. The mechanisms underlying differentiation of these populations are incompletely understood. Here, we identify the transcription factor Aiolos as a reciprocal regulator of TFH and CD4-CTL programming. We find that Aiolos deficiency results in downregulation of key TFH transcription factors, and consequently reduced TFH differentiation and antibody production, during influenza virus infection. Conversely, CD4-CTL programming is elevated, including enhanced Eomes and cytolytic molecule expression. We further demonstrate that Aiolos deficiency allows for enhanced IL-2 sensitivity and increased STAT5 association with CD4-CTL gene targets, including Eomes, effector molecules, and IL2Ra. Thus, our collective findings identify Aiolos as a pivotal regulator of CD4-CTL and TFH programming and highlight its potential as a target for manipulating CD4+ T cell responses.

Immunopathogenic and antibacterial effects of H3N2 influenza A virus PB1-F2 map to amino acid residues 62, 75, 79, and 82.

The influenza A virus protein PB1-F2 has been linked to the pathogenesis of both primary viral and secondary bacterial infections. H3N2 viruses have historically expressed full-length PB1-F2 proteins with either proinflammatory (e.g., from influenza A/Hong Kong/1/1968 virus) or noninflammatory (e.g., from influenza A/Wuhan/359/1995 virus) properties. Using synthetic peptides derived from the active C-terminal portion of the PB1-F2 protein from those two viruses, we mapped the proinflammatory domain to amino acid residues L62, R75, R79, and L82 and then determined the role of that domain in H3N2 influenza virus pathogenicity. PB1-F2-derived peptides containing that proinflammatory motif caused significant morbidity, mortality, and pulmonary inflammation in mice, manifesting as increased acute lung injury and the presence of proinflammatory cytokines and inflammatory cells in the lungs compared to peptides lacking this motif, and better supported bacterial infection with Streptococcus pneumoniae. Infections of mice with an otherwise isogenic virus engineered to contain this proinflammatory sequence in PB1-F2 demonstrated increased morbidity resulting from primary viral infections and enhanced development of secondary bacterial pneumonia. The presence of the PB1-F2 noninflammatory (P62, H75, Q79, and S82) sequence in the wild-type virus mediated an antibacterial effect. These data suggest that loss of the inflammatory PB1-F2 phenotype that supports bacterial superinfection during adaptation of H3N2 viruses to humans, coupled with acquisition of antibacterial activity, contributes to the relatively diminished frequency of severe infections seen with seasonal H3N2 influenza viruses in recent decades compared to their first 2 decades of circulation.

Fatal outcome of pandemic H1N1 2009 influenza virus infection is associated with immunopathology and impaired lung repair, not enhanced viral burden, in pregnant mice.

Pandemic A (H1N1) 2009 influenza virus (pH1N1) infection in pregnant women can be severe. The mechanisms that affect infection outcome in this population are not well understood. To address this, pregnant and nonpregnant BALB/c mice were inoculated with the wild-type pH1N1 strain A/California/04/09. To determine whether innate immune responses are associated with severe infection, we measured the innate cells trafficking into the lungs of pregnant versus nonpregnant animals. Increased infiltration of pulmonary neutrophils and macrophages strongly correlated with an elevated mortality in pregnant mice. In agreement with this, the product of nitric oxide (nitrite) and several cytokines associated with recruitment and/or function of these cells were increased in the lungs of pregnant animals. Surprisingly, increased mortality in pregnant mice was not associated with higher virus load because equivalent virus titers and immunohistochemical staining were observed in the nasal cavities or lungs of all mice. To determine whether exacerbated inflammatory responses and elevated cellularity resulted in lung injury, epithelial regeneration was measured. The lungs of pregnant mice exhibited reduced epithelial regeneration, suggesting impaired lung repair. Despite these immunologic alterations, pregnant animals demonstrated equivalent percentages of pulmonary influenza virus-specific CD8(+) T lymphocytes, although they displayed elevated levels of T-regulator lymphocytes (Tregs) in the lung. Also, pregnant mice mounted equal antibody titers in response to virus or immunization with a monovalent inactivated pH1N1 A/California/07/09 vaccine. Therefore, immunopathology likely caused by elevated cellular recruitment is an implicated mechanism of severe pH1N1 infection in pregnant mice.

Epigenetic remodeling by vitamin C potentiates plasma cell differentiation.

Ascorbate (vitamin C) is an essential micronutrient in humans. The severe chronic deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. From a micronutrient analysis, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique as other antioxidants failed to promote plasma cell differentiation. Ascorbate is especially critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. As a cofactor for epigenetic enzymes, ascorbate facilitates TET2/3-mediated DNA modification and demethylation of multiple elements at the Prdm1 locus. DNA demethylation augments STAT3 association at the Prdm1 promoter and a downstream enhancer, thus ensuring efficient gene expression and plasma cell differentiation. The results suggest that an adequate level of ascorbate is required for antibody response and highlight how micronutrients may regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.