Mixed Bone Marrow Chimeric Mice Research Articles

MicroRNA-155 plays a critical role in the establishment of latency in murine γ-herpesvirus infection (VIR1P.1148)

Abstract Gammaherpesvirus infection in immunosuppressed populations is associated with severe disease. MicroRNA-155 (miR155) has been shown to play significant roles in the immune response, including in the formation of germinal centers (GC) and the development and maturation of T follicular helper cells (Tfh). We sought to determine the relative contribution of miR-155 in the T and B cell compartments to latent murine gammaherpesvirus infection. Mice deficient in miR-155 had a 100-fold decrease in latent viral load, measured by qPCR, and exhibited decreases in both follicular helper cells and germinal center B cells. However, infection of mixed bone marrow chimeric mice showed a similar latent viral burden in both WT and miR-155 deficient B cells. Therefore the role of miR-155 may not be B cell intrinsic, but is likely mediated in part due to the regulation that miR155 exerts on the T follicular helper cell population. Studies using a YFP marker virus also address roles for miR-155 in virus reactivation and maintenance of latency. Further elucidating the relative role of miR155 on these cell populations will provide key insights into the development of latency in this model system.

GITR sustains IL-2+ CD4 helper T cells to facilitate early control of a chronic viral infection (IRC7P.422)

Abstract CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during infection remain incompletely defined. Here we show that during lymphocytic choriomeningitis virus (LCMV) clone 13 infection, glucocorticoid-induced TNFR (GITR)-deficient mice exhibit defective CTL responses and viral control. Differences in CTL responses and viral control between GITR+/+ and GITR-/- mice were lost when CD4 T cells were depleted. Furthermore, mixed bone marrow chimeric mice and transfer of LCMV-specific CD4 or CD8 T cells demonstrated that these effects of GITR are CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IL-2+ Th1 cells to facilitate CTL expansion and early viral control. In vivo GITR-dependent activation of p65 NF-kB and S6 ribosomal protein was detected at day 3 p.i., a time at which GITR-L is maximally expressed on macrophages. Defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day 5 p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CTLs to between days 4-8 p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells, thereby enhancing LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.

Interleukin 21 signaling in B cells is required for efficient establishment of murine gammaherpesvirus latency.

The human gammaherpesviruses take advantage of normal B cell differentiation pathways to establish life-long infection in memory B cells. Murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice also leads to life-long infection in memory B cells. To gain access to the memory B cell population, MHV68 infected B cells pass through the germinal center reaction during the onset of latency and require signals from T follicular helper (TFH) cells for proliferation. Interleukin 21 (IL-21), one of the secreted factors produced by TFH cells, plays an important role in both the maintenance of the germinal center response as well as in the generation of long-lived plasma cells. Using IL-21R deficient mice, we show that IL-21 signaling is required for efficient establishment of MHV68 infection. In the absence of IL-21 signaling, fewer infected splenocytes are able to gain access to either the germinal center B cell population or the plasma cell population – the latter being a major site of MHV68 reactivation. Furthermore, the germinal center B cell population in IL-21R-/- mice is skewed towards the non-proliferating centrocyte phenotype, resulting in reduced expansion of infected B cells. Additionally, the reduced frequency of infected plasma cells results in a significant reduction in the frequency of splenocytes capable of reactivating virus. This defect in establishment of MHV68 infection is intrinsic to B cells, as MHV68 preferentially establishes infection in IL-21R sufficient B cells in mixed bone marrow chimeric mice. Taken together, these data indicate that IL-21 signaling plays multiple roles during establishment of MHV68 infection, and identify IL-21 as a critical TFH cell-derived factor for efficient establishment of gammaherpesvirus B cell latency.

T-bet– and STAT4–dependent IL-33 receptor expression directly promotes antiviral Th1 cell responses

During infection, the release of damage-associated molecular patterns, so-called "alarmins," orchestrates the immune response. The alarmin IL-33 plays a role in a wide range of pathologies. Upon release, IL-33 signals through its receptor ST2, which reportedly is expressed only on CD4(+) T cells of the Th2 and regulatory subsets. Here we show that Th1 effector cells also express ST2 upon differentiation in vitro and in vivo during lymphocytic choriomeningitis virus (LCMV) infection. The expression of ST2 on Th1 cells was transient, in contrast to constitutive ST2 expression on Th2 cells, and marked highly activated effector cells. ST2 expression on virus-specific Th1 cells depended on the Th1-associated transcription factors T-bet and STAT4. ST2 deficiency resulted in a T-cell-intrinsic impairment of LCMV-specific Th1 effector responses in both mixed bone marrow-chimeric mice and adoptive cell transfer experiments. ST2-deficient virus-specific CD4(+) T cells showed impaired expansion, Th1 effector differentiation, and antiviral cytokine production. Consequently, these cells mediated little virus-induced immunopathology. Thus, IL-33 acts as a critical and direct cofactor to drive antiviral Th1 effector cell activation, with implications for vaccination strategies and immunotherapeutic approaches.

Disruption of IL-21 signaling affects T cell-B cell interactions and abrogates protective humoral immunity to malaria.

Interleukin-21 signaling is important for germinal center B-cell responses, isotype switching and generation of memory B cells. However, a role for IL-21 in antibody-mediated protection against pathogens has not been demonstrated. Here we show that IL-21 is produced by T follicular helper cells and co-expressed with IFN-γ during an erythrocytic-stage malaria infection of Plasmodium chabaudi in mice. Mice deficient either in IL-21 or the IL-21 receptor fail to resolve the chronic phase of P. chabaudi infection and P. yoelii infection resulting in sustained high parasitemias, and are not immune to re-infection. This is associated with abrogated P. chabaudi-specific IgG responses, including memory B cells. Mixed bone marrow chimeric mice, with T cells carrying a targeted disruption of the Il21 gene, or B cells with a targeted disruption of the Il21r gene, demonstrate that IL-21 from T cells signaling through the IL-21 receptor on B cells is necessary to control chronic P. chabaudi infection. Our data uncover a mechanism by which CD4+ T cells and B cells control parasitemia during chronic erythrocytic-stage malaria through a single gene, Il21, and demonstrate the importance of this cytokine in the control of pathogens by humoral immune responses. These data are highly pertinent for designing malaria vaccines requiring long-lasting protective B-cell responses.

GITR intrinsically sustains early type 1 and late follicular helper CD4 T cell accumulation to control a chronic viral infection.

CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR+/+ and GITR-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ+IL-2+ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.

Nfil3 is crucial for development of innate lymphoid cells and host protection against intestinal pathogens.

The bZIP transcription factor Nfil3 (also known as E4BP4) is required for the development of natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s). We find that Nfil3 plays a critical role in the development of other mucosal tissue-associated innate lymphocytes. Type 3 ILCs (ILC3s), including lymphoid tissue inducer (LTi)-like cells, are severely diminished in both numbers and function in Nfil3-deficient mice. Using mixed bone marrow chimeric mice, we demonstrate that Nfil3 is critical for normal development of gut-associated ILC3s in a cell-intrinsic manner. Furthermore, Nfil3 deficiency severely compromises intestinal innate immune defense against acute bacterial infection with Citrobacter rodentium and Clostridium difficile. Nfil3 deficiency resulted in a loss of the recently identified ILC precursor, yet conditional ablation of Nfil3 in the NKp46(+) ILC3 subset did not perturb ILC3 numbers, suggesting that Nfil3 is required early during ILC3 development but not for lineage maintenance. Lastly, a marked defect in type 2 ILCs (ILC2s) was also observed in the lungs and visceral adipose tissue of Nfil3-deficient mice, revealing a general requirement for Nfil3 in the development of all ILC lineages.

CD43-, but not CD43+, IL-10-producing CD1dhiCD5+ B cells suppress type 1 immune responses during Chlamydia muridarum genital tract infection.

Regulatory B (Breg) cells are known to modulate immune responses through predominantly interleukin-10 (IL-10)-dependent mechanisms and can be hypothetically divided into innate and adaptive subsets based on the nature of their activating signals. However, the specific role of different Breg subsets in modulating immune responses remains ambiguous. Here we have shown that Chlamydia induces IL-10-producing splenic B-cell populations consisting of CD43(+) and CD43(-) subsets of IgM(hi)IgD(lo) innate-like B (ILB) cells in vitro. While CD43(+)IL-10-producing B cells displayed innate type features and were readily induced by Chlamydia via Toll-like-receptor (TLR) signaling, CD43(-)IL-10-producing B cells required additional B-cell activating factor (BAFF)-mediated signals from dendritic cells (DCs) for their differentiation and activation, thereby classifying them as adaptive type Bregs. Importantly, CD43(-), but not CD43(+), IL-10-producing ILB cells displayed bona fide Breg activity by potently suppressing interferon-γ (IFN-γ) production in vitro in an IL-10-dependent manner. Furthermore, a novel CD43(-)CD1d(hi)CD5(+) IL-10-producing Breg population was predominantly induced by Chlamydia genital infection in vivo. Correspondingly, mixed bone marrow chimeric mice with B-cell-specific IL-10 deficiency exhibited significantly increased type 1 immune responses, decreased bacterial burden, and reduced oviduct pathology upon infection. Our data demonstrate for the first time a distinct role for CD43(-)CD1d(hi)CD5(+)-adaptive Bregs over CD43(+) innate counterparts in controlling mucosal responses against intracellular bacterial infection.

Murine autoimmune cholangitis requires two hits: cytotoxic KLRG1+ CD8 effector cells and defective T regulatory cells (BA13P.122)

Abstract Primary biliary cirrhosis (PBC) is an enigmatic disease mediated by autoimmune destruction of cholangiocytes in hepatic bile ducts. Early immunological events in PBC are poorly understood; clinical signs of disease occur very late in the pathological process. We have used our unique murine model of PBC in dominant-negative TGF-β receptor type II transgenic mice to delineate critical early immunopathological pathways, and previously showed that dnTGFβRII CD8 T cells alone transfer biliary disease. Herein we report significantly increased numbers of hepatic terminally differentiated (KLRG1+) CD8 T cells in dnTGFβRII compared to B6; these cells are the major cytotoxic CD8 subset. Unexpectedly, the disease is not simply CD8 T cell intrinsic since mixed bone marrow chimeric mice were protected from biliary disease. Adoptive transfer studies showed that co-transfer of B6 but not dnTGFβRII Tregs prevented dnTGFβRII CD8 T cell mediated cholangitis; the protection was associated with decreased numbers of hepatic KLRG1+ CD8 T cells and with elimination of hepatic dnTGFβRII CD8 mediated cholangiocyte cytotoxicity. DnTGFβRII Treg cells were inferior in suppressing effector CD8 T cells in vitro compared to wild type B6 Tregs. In conclusion, these results emphasize that autoimmune cholangitis requires defects in both the T effector and regulatory compartments, which has important implications for understanding the early pathogenesis of human PBC.

Transcription Factor IRF4 Regulates Germinal Center Cell Formation through a B Cell–Intrinsic Mechanism

In response to antigenic stimulation, mature B cells interact with follicular helper T cells in specialized structures called germinal centers (GCs), which leads to the development of memory B cells and Ab-secreting plasma cells. The transcription factor IFN regulatory factor 4 (IRF4) is essential for the formation of follicular helper T cells and thus GCs, although whether IRF4 plays a distinct role in GC B cells remains contentious. RNAseq analysis on ex vivo-derived mouse B cell populations showed that Irf4 was lowly expressed in naive B cells, highly expressed in plasma cells, but absent from GC B cells. In this study, we used conditional deletion of Irf4 in mature B cells as well as wild-type and Irf4-deficient mixed bone marrow chimeric mice to investigate how and where IRF4 plays its essential role in GC formation. Strikingly, GC formation was severely impaired in mice in which Irf4 was conditionally deleted in mature B cells, after immunization with protein Ags or infection with Leishmania major. This effect was evident as early as day 5 following immunization, before the development of GCs, indicating that Irf4 was required for the development of early GC B cells. This defect was B cell intrinsic because Irf4-deficient B cells in chimeric mice failed to participate in the GC in response to L. major or influenza virus infection. Taken together, these data demonstrate a B cell-intrinsic requirement for IRF4 for not only the development of Ab secreting plasma cells but also for GC formation.

Murine autoimmune cholangitis requires two hits: Cytotoxic KLRG1+ CD8 effector cells and defective T regulatory cells

Primary biliary cirrhosis (PBC) is an enigmatic disease mediated by autoimmune destruction of cholangiocytes in hepatic bile ducts. The early immunological events leading to PBC are poorly understood; clinical signs of disease occur very late in the pathological process. We have used our unique murine model of PBC in dominant-negative TGF-β receptor type II transgenic mice to delineate critical early immunopathological pathways, and previously showed that dnTGFβRII CD8 T cells transfer biliary disease. Herein we report significantly increased numbers of hepatic dnTGFβRII terminally differentiated (KLRG1+) CD8 T cells, a CD8 subset previously shown to be enriched in antigen specific cells during hepatic immune response to viral infections. We performed bone marrow chimera studies to assess whether dnTGFβRII CD8 mediated disease was cell intrinsic or extrinsic. Unexpectedly, mixed (dnTGFβRII and B6) bone marrow chimeric (BMC) mice were protected from biliary disease compared to dnTGFβRII single bone marrow chimerics. To define the protective B6 cell subset, we performed adoptive transfer studies, which showed that co-transfer of B6 Tregs prevented dnTGFβRII CD8 T cell mediated cholangitis. Treg mediated disease protection was associated with significantly decreased numbers of hepatic KLRG1+ CD8 T cells. In contrast, co-transfer of dnTGFβRII Tregs offered no protection, and dnTGFβRII Treg cells were functionally defective in suppressing effector CD8 T cells in vitro compared to wild type B6 Tregs. In vitro cholangiocyte cytotoxicity assays demonstrated significantly increased numbers of cytotoxic hepatic dnTGFβRII KLRG1+ CD8 cells compared to B6. Protection from disease by B6 Tregs was associated with elimination of hepatic dnTGFβRII CD8 mediated cholangiocyte cytotoxicity. These results emphasize that autoimmune cholangitis requires defects in both the T effector and regulatory compartments, and that an intrinsic T cell effector defect is not sufficient to mediate autoimmune biliary disease in the setting of intact immune regulation. These results have important implications for understanding the early pathogenesis of human PBC.

The Host Environment Is Responsible for Aging-Related Functional NK Cell Deficiency

NK cells play an important role in immunity against infection and tumors. Aging-related functional NK cell deficiency is well documented in humans and mice. However, the mechanism for this is poorly understood. Using an adoptive transfer approach in mice, we found that NK cells from both young and aged mice responded vigorously to priming by pathogen-derived products after being cotransferred into young mice. In contrast, NK cells from young mice responded poorly to priming by pathogen-derived products after being transferred to aged mice. In addition to defects in NK cell priming, maturation of NK cells under steady-state conditions is also impaired in aged mice, resulting in a decreased proportion of CD27(-) mature NK cells. We found that bone marrow from young and aged mice gave rise to CD27(-) mature NK cells similarly in young mixed bone marrow chimeric mice. Furthermore, by using a novel bone marrow transfer approach without irradiation, we found that after being transferred to aged mice, bone marrow from young mice gave rise to NK cells with maturation defects. Finally, we found that aging-related functional NK cell deficiency was completely reversed by injecting soluble IL-15/IL-15Rα complexes. In contrast, blockade of IL-10 signaling, which broadly augments inflammatory responses to pathogen-derived products, had little effect on aging-related defects in NK cell priming. These data demonstrate that the aged host environment is responsible for aging-related functional NK cell deficiency. Additionally, our data suggest that IL-15 receptor agonists may be useful tools in treating aging-related functional NK cell deficiency.

MyD88 Signaling in CD4 T Cells Promotes IFN-γ Production and Hematopoietic Progenitor Cell Expansion in Response to Intracellular Bacterial Infection

Hematopoietic stem and progenitor cell (HSPC) phenotype and function can change in response to infectious challenge. These changes can be mediated by cytokines, IFNs, and pathogen-associated molecules, via TLR, and are thought to promote tailored immune responses for particular pathogens. In this study, we investigated the signals that activate HSPCs during ehrlichiosis, a disease characterized by profound hematopoietic dysfunction in both humans and mice. In a mouse model of ehrlichiosis, we observed that infection-induced proliferation of bone marrow HSPCs was dependent on IFN-γ signaling and was partially dependent on MyD88. However, MyD88 was not required in HSPCs for their expansion during infection, because similar frequencies of MyD88-deficient and wild-type HSPCs proliferated in mixed bone marrow chimeric mice. MyD88-deficient mice exhibited low serum and bone marrow concentration of IFN-γ compared with wild-type mice. We next identified CD4 T cells as the primary cells producing IFN-γ in the bone marrow and demonstrated a nonredundant role for CD4-derived IFN-γ in increased HSPCs. Using mixed bone marrow chimeric mice, we identified a requirement for MyD88 in CD4 T cells for increased T-bet expression, optimal IFN-γ production, and CD4 T cell proliferation. Our data demonstrate an essential role for CD4 T cells in mediating HSPC activation in response to bacterial infection and illustrate a novel role for MyD88 signaling in CD4 T cells in this process. These findings further support the idea that IFN-γ production is essential for HSPC activation and hematopoietic responses to infection.

DNAM-1 is required for expansion of effector NK cells and generation of memory NK cells during mouse cytomegalovirus infection (P6100)

Abstract Recent studies have demonstrated that natural killer (NK) cells have adaptive immune features including antigen-specific expansion and subsequent generation of a memory subset. However, the precise signaling pathways that promote the expansion and the generation of memory NK cells remain to be elucidated. Using mixed bone marrow chimeric mice, we investigated the role of DNAM-1, an activating NK cell receptor, in expansion and memory generation of NK cells in a mouse model of cytomegalovirus infection (MCMV). We demonstrated that DNAM-1-deficient MCMV-specific Ly49H+ NK cells exhibit not only a poor expansion following MCMV infection, but also defects in both memory NK cell generation and secondary expansion after re-challenge with MCMV, as compared with wild type Ly49H+ NK cells. The frequency of DNAM-1- NK cells bearing Ly49H gradually increase and the long-lived Ly49H+ NK cells do not express DNAM-1 in MCMV-infected wild type mice. Interestingly, DNAM-1-Ly49H+ effector NK cells are more proliferative and less sensitive to cell death than DNAM-1+Ly49H+ NK cells during MCMV infection. These observations suggest that cooperative signaling through DNAM-1 and Ly49H induces downmodulation of DNAM-1 on Ly49H+ NK cells, resulting in predominant expansion and persistence of DNAM-1-Ly49H+ NK cells during MCMV infection. Therefore, DNAM-1 is a key molecule for optimal effector response and memory generation of virus-specific NK cells.

Innate amplification of CD4 T cell responses during infection: NLRs and TLRs converge (P3342)

Abstract During infection T cells are activated in an antigen-specific manner and can then secrete effector cytokines in response to secondary interactions with antigen-presenting cells. Effector cytokines are critical for pathogen clearance, but are tightly controlled to limit host pathology. However, mounting evidence suggests alternative non-cognate pathways can induce these effector responses. Here, we examine the amplification of Th1 CD4 T cell production of IFNγ by innate stimuli to show that this non-specific response occurs in a broad variety of infectious and stimulatory contexts and requires cooperation of both Nod-like receptor and Toll-like receptor pathways. Using knock-out and mixed bone marrow chimeric mice we show a requirement for the inflammasome, T cell intrinsic IL18 receptor and extrinsic Toll-like receptors. This suggests a model wherein infected cells undergo inflammasome activation, and upon further innate stimulation, secrete cytokines to non-specifically amplify nearby activated T cell effector responses. The mechanisms of such a pathway could lead to a better understanding of immune-mediated clearance of pathogens, as well as immune-mediated host pathology.

Commensal bacteria and T cells control the generation of memory NK cells (P6103)

Abstract Previously, only T and B cells were thought able to generate long-lived memory cells; however, recent studies demonstrated that natural killer (NK) cells expressing the activating Ly49H receptor undergo clonal expansion, contraction, and generate long-lived memory cells after infection with mouse cytomegalovirus (MCMV). It is still unclear which subsets of NK cells preferentially give rise to memory NK cells. In this study, we show that expression of killer cell lectin-like receptor G1 (KLRG1) defines short-lived effector cells and precursor for long-lived memory cells. We found that sorted KLRG1-, but not KLRG1+ NK cells can generate memory cells. Interestingly, the percentage of KLRG1+ NK cells is significantly increased in Rag1-/- mice and Rag1-/- NK cells are largely impaired in the generation of memory cells. This phenotype was rescued by mixed bone marrow chimeric (wild-type:Rag1-/-) mice. Notably, the same results were also demonstrated in Tcra-/- or T cell-depleted mice. Meanwhile, the percentage of KLRG1+ NK cells was decreased in germ-free mice and antibiotic treatment enhanced memory generation. We further determined that steady-state cytokines induced by nucleotide-binding, oligomerization domain-containing protein (Nod) 1 and Nod2, but not Myd88 and Trif are responsible for the induction of KLRG1+ NK cells. Taken together, our findings demonstrate that commensal bacteria and T cells reciprocally regulate the generation of long-lived memory NK cells.

B Lymphocyte–Induced Maturation Protein-1 Contributes to Intestinal Mucosa Homeostasis by Limiting the Number of IL-17–Producing CD4+ T Cells

The transcription factor B lymphocyte-induced maturation protein-1 (Blimp-1) plays important roles in embryonic development and immunity. Blimp-1 is required for the differentiation of plasma cells, and mice with T cell-specific deletion of Blimp-1 (Blimp-1CKO mice) develop a fatal inflammatory response in the colon. Previous work demonstrated that lack of Blimp-1 in CD4(+) and CD8(+) T cells leads to intrinsic functional defects, but little is known about the functional role of Blimp-1 in regulating differentiation of Th cells in vivo and their contribution to the chronic intestinal inflammation observed in the Blimp1CKO mice. In this study, we show that Blimp-1 is required to restrain the production of the inflammatory cytokine IL-17 by Th cells in vivo. Blimp-1CKO mice have greater numbers of IL-17-producing TCRβ(+)CD4(+)cells in lymphoid organs and in the intestinal mucosa. The increase in IL-17-producing cells was not restored to normal levels in wild-type and Blimp-1CKO-mixed bone marrow chimeric mice, suggesting an intrinsic role for Blimp-1 in constraining the production of IL-17 in vivo. The observation that Blimp-1-deficient CD4(+) T cells are more prone to differentiate into IL-17(+)/IFN-γ(+) cells and cause severe colitis when transferred to Rag1-deficient mice provides further evidence that Blimp-1 represses IL-17 production. Analysis of Blimp-1 expression at the single cell level during Th differentiation reveals that Blimp-1 expression is induced in Th1 and Th2 but repressed by TGF-β in Th17 cells. Collectively, the results described here establish a new role for Blimp-1 in regulating IL-17 production in vivo.

The B10 Idd9.3 Locus Mediates Accumulation of Functionally Superior CD137+ Regulatory T Cells in the Nonobese Diabetic Type 1 Diabetes Model

CD137 is a T cell costimulatory molecule encoded by the prime candidate gene (designated Tnfrsf9) in NOD.B10 Idd9.3 congenic mice protected from type 1 diabetes (T1D). NOD T cells show decreased CD137-mediated T cell signaling compared with NOD.B10 Idd9.3 T cells, but it has been unclear how this decreased CD137 T cell signaling could mediate susceptibility to T1D. We and others have shown that a subset of regulatory T cells (Tregs) constitutively expresses CD137 (whereas effector T cells do not, and only express CD137 briefly after activation). In this study, we show that the B10 Idd9.3 region intrinsically contributes to accumulation of CD137(+) Tregs with age. NOD.B10 Idd9.3 mice showed significantly increased percentages and numbers of CD137(+) peripheral Tregs compared with NOD mice. Moreover, Tregs expressing the B10 Idd9.3 region preferentially accumulated in mixed bone marrow chimeric mice reconstituted with allotypically marked NOD and NOD.B10 Idd9.3 bone marrow. We demonstrate a possible significance of increased numbers of CD137(+) Tregs by showing functional superiority of FACS-purified CD137(+) Tregs in vitro compared with CD137(-) Tregs in T cell-suppression assays. Increased functional suppression was also associated with increased production of the alternatively spliced CD137 isoform, soluble CD137, which has been shown to suppress T cell proliferation. We show for the first time, to our knowledge, that CD137(+) Tregs are the primary cellular source of soluble CD137. NOD.B10 Idd9.3 mice showed significantly increased serum soluble CD137 compared with NOD mice with age, consistent with their increased numbers of CD137(+) Tregs with age. These studies demonstrate the importance of CD137(+) Tregs in T1D and offer a new hypothesis for how the NOD Idd9.3 region could act to increase T1D susceptibility.

Pathogen-specific Th1 cells are activated by IL-1 family cytokines induced by innate stimulation. (164.2)

Abstract Upon antigen recognition, naïve CD4 T cells can develop into effector Th1 cells capable of trafficking to sites of infection and secreting pro-inflammatory cytokines, such as IFNγ. The mechanism of Th1 activation within tissues is often assumed to require additional TCR ligation. However, we have explored whether Th1 cells can be activated by innate stimulation in Salmonella and Chlamydia-infected mice. Using mixed bone marrow chimeric mice, our data show that bacterial-specific Th1 cells can be activated by PAMPs and that production of IFN-γ is dependent upon intrinsic expression of MyD88, but not intrinsic TLR4. Furthermore, loss of IL-18R and IL-33R reduces the ability of Th1 cells to produce IFN-γ, while IL-1R and IFN-γR are not required. Th1 cells were also activated in vitro by IL-1 family cytokines and IL-12, demonstrating that these are sufficient for reactivation. We are currently determining the minimal requirements for Th1 cell stimulation by IL-1 family cytokines and examining the role of this pathway in infection models. Understanding the mechanism and role of innate Th1 stimulation could have widespread implications for understanding antimicrobial and autoimmune pathways.

Imprinting CD103+ LP-DCs and the acquisition of luminal antigen is dependent upon CCR6 (71.12)

Abstract Dendritic cells (DCs) play a crucial role initiating and guiding immune responses. DCs are found within the gut associated lymphoid tissue, within the lamina propria (LP), and closely associated with the small intestinal epithelium. The epithelia associated DCs (EADC) are positioned to be imprinted by epithelial cells and to acquire luminal antigens, however the factors recruiting LPDCs to the epithelium are not known. We observed that CCL20 is expressed by small intestine villous enterocytes, and that the receptor for CCL20, CCR6, is expressed by small intestine EADCs and LPDCs. CCR6-/- mice had a diminished population of EADCs, but not LPDCs when compared with wildtype mice. Mixed bone marrow chimeric mice revealed that CCR6-/- LPDCs were less efficient at associating with the epithelium, demonstrating a requirement for CCR6 recruiting EADCs. Consistent with our observation that goblet cells (GC) act as passages delivering luminal antigens and GC antigens, LPDCs from CCR6-/- mice were inefficient at presenting luminal antigens to T-cells and rarely stained positive for the goblet cell protein cytokeratin 18. Moreover CD103+ LPDCs from CCR6-/- mice had decreased aldehyde dehydrogenase activity, consistent with the requirement for DC-epithelia associations to imprint DCs with a mucosal phenotype. These observations demonstrate an essential role for CCR6 in recruiting LPDCs to the epithelium where these DCs are imprinted and acquire luminal and GC antigens.