Epigenetic tuning of PD-1 expression improves exhausted T cell function and viral control.

PD-1 is a key negative regulator of CD8+ T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8+ T cells in chronic infection, creating a 'sweet spot' of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1-knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8+ T cell dysfunction while avoiding excess immunopathology.

Epigenomic Profiling of B Cell Subsets by CUT&Tag.

Epigenetic programs play a key role in regulating the development and function of immune cells. However, conventional methods for profiling epigenetic mechanisms, such as the post-translational modifications to histones, present several technical challenges that prevent a complete understanding of gene regulation. Here, we provide a detailed protocol of the Cleavage Under Targets and Tagmentation (CUT&Tag) chromatin profiling technique for identifying histone modifications in human and mouse lymphocytes.

Proteobacteria impair anti-tumor immunity in the omentum by consuming arginine.

Gut microbiota influence anti-tumor immunity, often by producing immune-modulating metabolites. However, microbes consume a variety of metabolites that may also impact host immune responses. We show that tumors grow unchecked in the omenta of microbe-replete mice due to immunosuppressive Tregs. By contrast, omental tumors in germ-free, neomycin-treated mice or mice colonized with altered Schaedler's flora (ASF) are spontaneously eliminated by CD8+ T cells. These mice lack Proteobacteria capable of arginine catabolism, causing increases in serum arginine that activate the mammalian target of the rapamycin (mTOR) pathway in Tregs to reduce their suppressive capacity. Transfer of the Proteobacteria, Escherichia coli (E. coli), but not a mutant unable to catabolize arginine, to ASF mice reduces arginine levels, restores Treg suppression, and prevents tumor clearance. Supplementary arginine similarly decreases Treg suppressive capacity, increases CD8+ T cell effectiveness, and reduces tumor burden. Thus, microbial consumption of arginine alters anti-tumor immunity, offering potential therapeutic strategies for tumors in visceral adipose tissue.

Type I interferons induce an epigenetically distinct memory B cell subset in chronic viral infection.

Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.

Early generation of a precursor CD8 T cell that can adapt to acute or chronic viral infection.

Virus specific PD-1+ TCF-1+ TOX+ stem-like CD8+ T cells are essential for maintaining T cell responses during chronic infection and are also critical for PD-1 directed immunotherapy. In this study we have used the mouse model of chronic LCMV infection to examine when these virus specific stem-like CD8+ T cells are generated during the course of chronic infection and what is the role of antigen in maintaining the stem-like program. We found that these stem-like CD8+ T cells are generated early (day 5) during chronic infection and that antigen is essential for maintaining their stem-like program. This early generation of stem-like CD8+ T cells suggested that the fate commitment to this cell population was agnostic to the eventual outcome of infection and the immune system prepares a priori for a potential chronic infection. Indeed, we found that an identical virus specific stem-cell like CD8+ T cell population was also generated during acute LCMV infection but these cells were lost once the virus was cleared. To determine the fate of these early PD-1+TCF-1+TOX+ stem-like CD8+ T cells that are generated during both acute and chronic LCMV infection we set up two reciprocal adoptive transfer experiments. In the first experiment we transferred day 5 stem-like CD8+ T cells from chronically infected into acutely infected mice and examined their differentiation after viral clearance. We found that these early stem-like CD8+ T cells downregulated canonical markers of the chronic stem-like CD8+ T cells and expressed markers (CD127 and CD62L) associated with central memory CD8+ T cells. In the second experiment, we transferred day 5 stem-like cells from acutely infected mice into chronically infected mice and found that these CD8+ T cells could function like resource cells after transfer into a chronic environment by generating effector CD8+ T cells in both lymphoid and non-lymphoid tissues while also maintaining the number of stem-like CD8+ T cells. These findings provide insight into the generation and maintenance of virus specific stem-like CD8+ T cells that play a critical role in chronic viral infection. In particular, our study highlights the early generation of stem-like CD8+ T cells and their ability to adapt to either an acute or chronic infection. These findings are of broad significance since these novel stem-like CD8+ T cells play an important role in not only viral infections but also in cancer and autoimmunity.

Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

CRISPR/Cas9 editing reveals IRF8 regulated gene signatures restraining plasmablast differentiation.

The transcription factor Interferon regulatory factor 8 (IRF8) is involved in maintaining B cell identity. However, how IRF8 regulates T cell independent B cell responses are not fully characterized. Here, an in vivo CRISPR/Cas9 system was optimized to generate Irf8-deficient murine B cells and used to determine the role of IRF8 in B cells responding to LPS stimulation. Irf8-deficient B cells more readily formed CD138+ plasmablasts in response to LPS with the principal dysregulation occurring at the activated B cell stage. Transcriptional profiling revealed an upregulation of plasma cell associated genes prematurely in activated B cells and a failure to repress the gene expression programs of IRF1 and IRF7 in Irf8-deficient cells. These data expand on the known roles of IRF8 in regulating B cell identity by preventing premature plasma cell formation and highlight how IRF8 helps evolve TLR responses away from the initial activation towards those driving humoral immunity.

The 'omics of obesity in B-cell acute lymphoblastic leukemia.

The obesity pandemic currently affects more than 70 million Americans and more than 650 million individuals worldwide. In addition to increasing susceptibility to pathogenic infections (eg, SARS-CoV-2), obesity promotes the development of many cancer subtypes and increases mortality rates in most cases. We and others have demonstrated that, in the context of B-cell acute lymphoblastic leukemia (B-ALL), adipocytes promote multidrug chemoresistance. Furthermore, others have demonstrated that B-ALL cells exposed to the adipocyte secretome alter their metabolic states to circumvent chemotherapy-mediated cytotoxicity. To better understand how adipocytes impact the function of human B-ALL cells, we used a multi-omic RNA-sequencing (single-cell and bulk transcriptomic) and mass spectroscopy (metabolomic and proteomic) approaches to define adipocyte-induced changes in normal and malignant B cells. These analyses revealed that the adipocyte secretome directly modulates programs in human B-ALL cells associated with metabolism, protection from oxidative stress, increased survival, B-cell development, and drivers of chemoresistance. Single-cell RNA sequencing analysis of mice on low- and high-fat diets revealed that obesity suppresses an immunologically active B-cell subpopulation and that the loss of this transcriptomic signature in patients with B-ALL is associated with poor survival outcomes. Analyses of sera and plasma samples from healthy donors and those with B-ALL revealed that obesity is associated with higher circulating levels of immunoglobulin-associated proteins, which support observations in obese mice of altered immunological homeostasis. In all, our multi-omics approach increases our understanding of pathways that may promote chemoresistance in human B-ALL and highlight a novel B-cell-specific signature in patients associated with survival outcomes.

IL-6/STAT3 Signaling Axis Enhances and Prolongs Pdcd1 Expression in Murine CD8 T Cells.

CD8 cytotoxic T cells are a potent line of defense against invading pathogens. To aid in curtailing aberrant immune responses, the activation status of CD8 T cells is highly regulated. One mechanism in which CD8 T cell responses are dampened is via signaling through the immune-inhibitory receptor Programmed Cell Death Protein-1, encoded by Pdcd1. Pdcd1 expression is regulated through engagement of the TCR, as well as by signaling from extracellular cytokines. Understanding such pathways has influenced the development of numerous clinical treatments. In this study, we showed that signals from the cytokine IL-6 enhanced Pdcd1 expression when paired with TCR stimulation in murine CD8 T cells. Mechanistically, signals from IL-6 were propagated through activation of the transcription factor STAT3, resulting in IL-6-dependent binding of STAT3 to Pdcd1 cis-regulatory elements. Intriguingly, IL-6 stimulation overcame B Lymphocyte Maturation Protein 1-mediated epigenetic repression of Pdcd1, which resulted in a transcriptionally permissive landscape marked by heightened histone acetylation. Furthermore, in vivo-activated CD8 T cells derived from lymphocytic choriomeningitis virus infection required STAT3 for optimal Programmed Cell Death Protein-1 surface expression. Importantly, STAT3 was the only member of the STAT family present at Pdcd1 regulatory elements in lymphocytic choriomeningitis virus Ag-specific CD8 T cells. Collectively, these data define mechanisms by which the IL-6/STAT3 signaling axis can enhance and prolong Pdcd1 expression in murine CD8 T cells.

PD-1 combination therapy with IL-2 modifies CD8+ T cell exhaustion program.

Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ stem-like CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. The generation of these qualitatively superior CD8+ T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1+TCF1+ stem-like CD8+ T cells, also referred to as precursors of exhausted CD8+ T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8+ T cells emerging from the stem-like CD8+ T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25-CD122-CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.

Persistent Antigen Harbored by Alveolar Macrophages Enhances the Maintenance of Lung-Resident Memory CD8+ T Cells.

Lung tissue-resident memory T cells are crucial mediators of cellular immunity against respiratory viruses; however, their gradual decline hinders the development of T cell-based vaccines against respiratory pathogens. Recently, studies using adenovirus (Ad)-based vaccine vectors have shown that the number of protective lung-resident CD8+ TRMs can be maintained long term. In this article, we show that immunization of mice with a replication-deficient Ad serotype 5 expressing influenza (A/Puerto Rico/8/34) nucleoprotein (AdNP) generates a long-lived lung TRM pool that is transcriptionally indistinct from those generated during a primary influenza infection. In addition, we demonstrate that CD4+ T cells contribute to the long-term maintenance of AdNP-induced CD8+ TRMs. Using a lineage tracing approach, we identify alveolar macrophages as a cell source of persistent NP Ag after immunization with AdNP. Importantly, depletion of alveolar macrophages after AdNP immunization resulted in significantly reduced numbers of NP-specific CD8+ TRMs in the lungs and airways. Combined, our results provide further insight to the mechanisms governing the enhanced longevity of Ag-specific CD8+ lung TRMs observed after immunization with recombinant Ad.

Adipocyte-mediated epigenomic instability in human T-ALL cells is cytotoxic and phenocopied by epigenetic-modifying drugs.

The world's population with obesity is reaching pandemic levels. If current trends continue, it is predicted that there will be 1.5 billion people with obesity by 2030. This projection is alarming due to the association of obesity with numerous diseases including cancer, with recent studies demonstrating a positive association with acute myeloid leukemia (AML) and B cell acute lymphoblastic leukemia (B-ALL). Interestingly, several epidemiological studies suggest the converse relationship may exist in patients with T cell acute lymphoblastic leukemia (T-ALL). To determine the relationship between obesity and T-ALL development, we employed the diet-induced obesity (DIO) murine model and cultured human T-ALL cells in adipocyte-conditioned media (ACM), bone marrow stromal cell-conditioned media, stromal conditioned media (SCM), and unconditioned media to determine the functional impact of increased adiposity on leukemia progression. Whereas only 20% of lean mice transplanted with T-ALL cells survived longer than 3 months post-inoculation, 50%-80% of obese mice with leukemia survived over this same period. Furthermore, culturing human T-ALL cells in ACM resulted in increased histone H3 acetylation (K9/K14/K18/K23/K27) and methylation (K4me3 and K27me3) posttranslational modifications (PTMs), which preceded accelerated cell cycle progression, DNA damage, and cell death. Adipocyte-mediated epigenetic changes in human T-ALL cells were recapitulated with the H3K27 demethylase inhibitor GSK-J4 and the pan-HDAC inhibitor vorinostat. These drugs were also highly cytotoxic to human T-ALL cells at low micromolar concentrations. In summary, our data support epidemiological studies demonstrating that adiposity suppresses T-ALL pathogenesis. We present data demonstrating that T-ALL cell death in adipose-rich microenvironments is induced by epigenetic modifications, which are not tolerated by leukemia cells. Similarly, GSK-J4 and vorinostat treatment induced epigenomic instability and cytotoxicity profiles that phenocopied the responses of human T-ALL cells to ACM, which provides additional support for the use of epigenetic modifying drugs as a treatment option for T-ALL.

Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors.

Germline mutations in ETV6 are associated with a syndrome of thrombocytopenia and leukemia predisposition, and ETV6 is among the most commonly mutated genes in leukemias, especially childhood B-cell acute lymphoblastic leukemia. However, the mechanisms underlying disease caused by ETV6 dysfunction are poorly understood. To address these gaps in knowledge, using CRISPR/Cas9, we developed a mouse model of the most common recurrent, disease-causing germline mutation in ETV6. We found defects in hematopoiesis related primarily to abnormalities of the multipotent progenitor population 4 (MPP4) subset of hematopoietic progenitor cells and evidence of sterile inflammation. Expression of ETV6 in Ba/F3 cells altered the expression of several cytokines, some of which were also detected at higher levels in the bone marrow of the mice with Etv6 mutation. Among these, interleukin-18 and interleukin-13 abrogated B-cell development of sorted MPP4 cells, but not common lymphoid progenitors, suggesting that inflammation contributes to abnormal hematopoiesis by impairing lymphoid development. These data, along with those from humans, support a model in which ETV6 dysfunction promotes inflammation, which adversely affects thrombopoiesis and promotes leukemogenesis.

Obesity-induced galectin-9 is a therapeutic target in B-cell acute lymphoblastic leukemia.

The incidence of obesity is rising with greater than 40% of the world's population expected to be overweight or suffering from obesity by 2030. This is alarming because obesity increases mortality rates in patients with various cancer subtypes including leukemia. The survival differences between lean patients and patients with obesity are largely attributed to altered drug pharmacokinetics in patients receiving chemotherapy; whereas, the direct impact of an adipocyte-enriched microenvironment on cancer cells is rarely considered. Here we show that the adipocyte secretome upregulates the surface expression of Galectin-9 (GAL-9) on human B-acute lymphoblastic leukemia cells (B-ALL) which promotes chemoresistance. Antibody-mediated targeting of GAL-9 on B-ALL cells induces DNA damage, alters cell cycle progression, and promotes apoptosis in vitro and significantly extends the survival of obese but not lean mice with aggressive B-ALL. Our studies reveal that adipocyte-mediated upregulation of GAL-9 on B-ALL cells can be targeted with antibody-based therapies to overcome obesity-induced chemoresistance.

Circulating Tregs Accumulate in Omental Tumors and Acquire Adipose-Resident Features.

Tumors that metastasize in the peritoneal cavity typically end up in the omental adipose tissue, a particularly immune-suppressive environment that includes specialized adipose-resident regulatory T cells (Treg). Tregs rapidly accumulate in the omentum after tumor implantation and potently suppress antitumor immunity. However, it is unclear whether these Tregs are recruited from the circulation or derived from preexisting adipose-resident Tregs by clonal expansion. Here we show that Tregs in tumor-bearing omenta predominantly have thymus-derived characteristics. Moreover, naïve tumor antigen-specific CD4+ T cells fail to differentiate into Tregs in tumor-bearing omenta. In fact, Tregs derived from the pretumor repertoire are sufficient to suppress antitumor immunity and promote tumor growth. However, tumor implantation in the omentum does not promote Treg clonal expansion, but instead leads to increased clonal diversity. Parabiosis experiments show that despite tissue-resident (noncirculating) characteristics of omental Tregs in naïve mice, tumor implantation promotes a rapid influx of circulating Tregs, many of which come from the spleen. Finally, we show that newly recruited Tregs rapidly acquire characteristics of adipose-resident Tregs in tumor-bearing omenta. These data demonstrate that most Tregs in omental tumors are recruited from the circulation and adapt to their environment by altering their homing, transcriptional, and metabolic properties.

Targeting BMI-1 in B cells restores effective humoral immune responses and controls chronic viral infection.

Ineffective antibody-mediated responses are a key characteristic of chronic viral infection. However, our understanding of the intrinsic mechanisms that drive this dysregulation are unclear. Here, we identify that targeting the epigenetic modifier BMI-1 in mice improves humoral responses to chronic lymphocytic choriomeningitis virus. BMI-1 was upregulated by germinal center B cells in chronic viral infection, correlating with changes to the accessible chromatin landscape, compared to acute infection. B cell-intrinsic deletion of Bmi1 accelerated viral clearance, reduced splenomegaly and restored splenic architecture. Deletion of Bmi1 restored c-Myc expression in B cells, concomitant with improved quality of antibody and coupled with reduced antibody-secreting cell numbers. Specifically, BMI-1-deficiency induced antibody with increased neutralizing capacity and enhanced antibody-dependent effector function. Using a small molecule inhibitor to murine BMI-1, we could deplete antibody-secreting cells and prohibit detrimental immune complex formation in vivo. This study defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection.

Cohesin Core Complex Gene Dosage Contributes to Germinal Center Derived Lymphoma Phenotypes and Outcomes.

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

Extrafollicular IgD+ B cells generate IgE antibody secreting cells in the nasal mucosa.

Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naïve-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naïve B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

Selective DNA Demethylation Accompanies T Cell Homeostatic Proliferation and Gene Regulation in Lupus-Prone lpr Mice.

Systemic lupus erythematosus (SLE) is characterized by increased DNA demethylation in T cells, although it is unclear whether this occurs primarily in a subset of SLE T cells. The process driving the DNA demethylation and the consequences on overall gene expression are also poorly understood and whether this represents a secondary consequence of SLE or a primary contributing factor. Lupus-prone lpr mice accumulate large numbers of T cells with age because of a mutation in Fas (CD95). The accumulating T cells include an unusual population of CD4-CD8-TCR-αß+ (DN) T cells that arise from CD8+ precursors and are also found in human SLE. We have previously observed that T cell accumulation in lpr mice is due to dysregulation of T cell homeostatic proliferation, which parallels an increased expression of numerous genes in the DN subset, including several proinflammatory molecules and checkpoint blockers. We thus determined the DNA methylome in lpr DN T cells compared with their CD8+ precursors. Our findings show that DN T cells manifest discrete sites of extensive demethylation throughout the genome, and these sites correspond to the location of a large proportion of the upregulated genes. Thus, dysregulated homeostatic proliferation in lpr mice and consequent epigenetic alterations may be a contributing factor to lupus pathogenesis.

Murine gammaherpesvirus infection is skewed toward Igλ+ B cells expressing a specific heavy chain V-segment.

One of the defining characteristics of the B cell receptor (BCR) is the extensive diversity in the repertoire of immunoglobulin genes that make up the BCR, resulting in broad range of specificity. Gammaherpesviruses are B lymphotropic viruses that establish life-long infection in B cells, and although the B cell receptor plays a central role in B cell biology, very little is known about the immunoglobulin repertoire of gammaherpesvirus infected cells. To begin to characterize the Ig genes expressed by murine gammaherpesvirus 68 (MHV68) infected cells, we utilized single cell sorting to sequence and clone the Ig variable regions of infected germinal center (GC) B cells and plasma cells. We show that MHV68 infection is biased towards cells that express the Igλ light chain along with a single heavy chain variable gene, IGHV10-1*01. This population arises through clonal expansion but is not viral antigen specific. Furthermore, we show that class-switching in MHV68 infected cells differs from that of uninfected cells. Fewer infected GC B cells are class-switched compared to uninfected GC B cells, while more infected plasma cells are class-switched compared to uninfected plasma cells. Additionally, although they are germinal center derived, the majority of class switched plasma cells display no somatic hypermutation regardless of infection status. Taken together, these data indicate that selection of infected B cells with a specific BCR, as well as virus mediated manipulation of class switching and somatic hypermutation, are critical aspects in establishing life-long gammaherpesvirus infection.