Mixed Bone Marrow Chimeras Research Articles

MicroRNA-142 is critical for peripheral NK cell homeostasis and function.

Abstract MicroRNAs (miRNA) are small, non-coding RNAs that post-transcriptionally regulate the translation of their targeted mRNAs. Next-generation small RNA sequencing has identified a number of mature miRNAs that are highly expressed in murine NK cells, including miR-142. We hypothesized that miR-142-5p/3p regulated NK cell development, survival, or functionality, and investigated this concept by examining the NK cell compartment in miR-142−/− mice. Despite having normal NK precursor and NK cell frequencies and absolute numbers in the bone marrow (BM), miR-142−/− mice exhibited a marked reduction of NK cells in the spleen (5-fold), blood (5-fold), lymph node (10-fold), and liver (2-fold) compared to controls. The NK deficiency observed in miR-142−/− mice was not due to blocked maturation, as the frequencies of CD27/CD11b stage II, III, and IV NK cells were similar between wild-type and miR-142−/− mice. However, the absolute numbers of each stage were reduced in miR-142−/−, confirming that miR-142−/− mice display a global loss of NK cells in the periphery that is independent of NK cell maturation. Mixed BM chimera studies support a cell-intrinsic role for miR-142 in NK cell peripheral homeostasis. Furthermore, miR-142−/−NK cells do not signal through their IL-12, IL-15, and IL-18 cytokine receptors and thus fail to produce IFN-γ in response to cytokine stimulation thereby limiting their effector functions. miR-142−/− NK cells have a dramatically altered integrin expression profile compared to wild-type, which we hypothesize leads a failure of miR-142−/− NK cells to progress properly through the BM niche, resulting in the loss of NK cells in the periphery. Thus, miR-142 is required for normal NK cell peripheral homeostasis and function.

TGF-β-mediated inhibition of IFN-γ production by Mycobacterium tuberculosis-specific T cells in the infected lung

Abstract Tuberculosis (TB) is a major global health problem killing over one million people every year. The emergence of multi and extremely-drug resistant (MDR and XDR) strains of Mycobacterium tuberculosis (Mtb) has made treatment difficult and has spurred interest in developing host-directed immunotherapeutics. A critical roadblock is our failure to understand how Mtb escapes eradication despite an apparently robust adaptive immune response. Interferon-γ (IFN-γ) producing CD4 T cells are critical for immunity, yet their abundance in the blood does not correlate with protection to TB, suggesting that their activity may be blocked locally at the primary site of infection in the lung. A leading candidate for IFN-γ suppression in the lung is transforming growth factor-β (TGF-β), a multifunctional cytokine that is capable of inhibiting IFN-γ. TGF-β is produced in excess at sites of active Mtb infection in both murine and human TB. Neutralization of TGF-β improves T-cell responses of active TB patients in vitro, but the extent of its role during in vivo TB is unknown. Here, we generated mixed bone marrow chimeras using WT mice and mice with a T cell-specific conditional deletion of TGF-β receptor II (dLck-Cre:TGFbRIIfl/fl). Mtb-specific TGF-βRII knockout cells outcompeted their WT counterparts and exhibited increased IFN-γ production during Mtb infection. Furthermore, Mtb-infected dLck-Cre:TGFbRIIfl/fl mice had decreased bacterial loads early during infection. Finally, two pharmacological inhibitors of TGF-β signaling lowered the bacterial loads of lungs from Mtb-infected mice both early and late during infection. Targeting TGF-β may pave the way for new host-directed immunotherapeutics for TB.

Role of GITR and its ligand in sustaining T cell responses against influenza infection

Abstract Members of the TNFR family play a critical role in determining the magnitude of T cell responses against viral infections. Previous studies using TCR transgenic models showed that GITR, an NF-κB activating TNFR family member, is required intrinsically on CD8 T cells for maximal response against influenza virus. However, it remains unknown how GITR affects the overall endogenous T cell responses during influenza infection. Here, we examined the effect of GITR on endogenous T cell responses against influenza virus in competitive mixed bone marrow chimeras. We found that GITR is intrinsically required for the accumulation of antigen-specific CD4 and CD8 T cells in the lung and secondary lymphoid organs during influenza infection. GITR affected influenza PA224-233-specific CD8 T cells more dramatically than NP366-374-specific CD8 T cells. GITR also had a small positive effect on the number of Tregs. Among different APC subsets in the lung and its dLN, we observed highest GITRL expression on inflammatory DCs and inflammatory macrophages in both organs. Interestingly, we observed peak GITRL expression on these inflammatory subsets on day 3 and day 5 post-influenza infection in the lung but earlier in the dLN. The finding that GITRL is expressed on inflammatory APCs in the lung at the time when T cells enter the lung raises the possibility that GITRL plays a role in the lung tissue and not just in lymphoid organs. We are currently investigating when and where T cells receive signals through GITR, whether early in the dLN or later in the lung during influenza infection. A more thorough understanding of GITR/GITRL axis will provide valuable insights for influenza vaccine development.

The transcriptional coactivator Bob1 promotes the development of follicular T helper cells via Bcl6.

Follicular T helper (Tfh) cells are key regulators of the germinal center reaction and long‐term humoral immunity. Tfh cell differentiation requires the sustained expression of the transcriptional repressor Bcl6; however, its regulation in CD4+ T cells is incompletely understood. Here, we report that the transcriptional coactivator Bob1, encoded by the Pou2af1 gene, promotes Bcl6 expression and Tfh cell development. We found that Bob1 together with the octamer transcription factors Oct1/Oct2 can directly bind to and transactivate the Bcl6 and Btla promoters. Mixed bone marrow chimeras revealed that Bob1 is required for the expression of normal levels of Bcl6 and BTLA, thereby controlling the pool size and composition of the Tfh compartment in a T cell‐intrinsic manner. Our data indicate that T cell‐expressed Bob1 is directly involved in Tfh cell differentiation and required for mounting normal T cell‐dependent B‐cell responses.

A Context-Dependent Role for IL-21 in Modulating the Differentiation, Distribution, and Abundance of Effector and Memory CD8 T Cell Subsets.

The activation of naive CD8 T cells typically results in the formation of effector cells (TE) as well as phenotypically distinct memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory, effector memory (TEM), and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using mixed bone marrow chimeras and adoptive transfer studies in which CD8 T cells either do or do not express IL-21R, we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals under competitive conditions also resulted in lower levels of TRM phenotype cells and reduced expression of granzyme B in the small intestine. IL-21 differentially promoted the expression of the chemokine receptor CX3CR1 and the integrin α4β7 on CD8 T cells primed in vitro and on circulating CD8 T cells in the mixed bone marrow chimeras. The requirement for IL-21 to establish CD8 TE/TEM and TRM subsets was overcome by acute lymphocytic choriomeningitis virus infection; nevertheless, memory virus-specific CD8 T cells remained dependent on IL-21 for optimal accumulation in lymphopenic environments. Overall, this study reveals a context-dependent role for IL-21 in sustaining effector phenotype CD8 T cells and influencing their migratory properties, accumulation, and functions.

Multiple Inflammatory Cytokines Converge To Regulate CD8+ T Cell Expansion and Function during Tuberculosis

The differentiation of effector CD8(+) T cells is a dynamically regulated process that varies during different infections and is influenced by the inflammatory milieu of the host. In this study, we define three signals regulating CD8(+) T cell responses during tuberculosis by focusing on cytokines known to affect disease outcome: IL-12, type I IFN, and IL-27. Using mixed bone marrow chimeras, we compared wild-type and cytokine receptor knockout CD8(+) T cells within the same mouse following aerosol infection with Mycobacterium tuberculosis. Four weeks postinfection, IL-12, type 1 IFN, and IL-27 were all required for efficient CD8(+) T cell expansion in the lungs. We next determined if these cytokines directly promote CD8(+) T cell priming or are required only for expansion in the lungs. Using retrogenic CD8(+) T cells specific for the M. tuberculosis Ag TB10.4 (EsxH), we observed that IL-12 is the dominant cytokine driving both CD8(+) T cell priming in the lymph node and expansion in the lungs; however, type I IFN and IL-27 have nonredundant roles supporting pulmonary CD8(+) T cell expansion. Thus, IL-12 is a major signal promoting priming in the lymph node, but a multitude of inflammatory signals converge in the lung to promote continued expansion. Furthermore, these cytokines regulate the differentiation and function of CD8(+) T cells during tuberculosis. These data demonstrate distinct and overlapping roles for each of the cytokines examined and underscore the complexity of CD8(+) T cell regulation during tuberculosis.

T-cell–intrinsic Tif1α/Trim24 regulates IL-1R expression on TH2 cells and TH2 cell-mediated airway allergy

There is a paucity of new therapeutic targets to control allergic reactions and forestall the rising trend of allergic diseases. Although a variety of immune cells contribute to allergy, cytokine-secreting αβ(+)CD4(+) T-helper 2 (TH2) cells orchestrate the type-2-driven immune response in a large proportion of atopic asthmatics. To identify previously unidentified putative targets in pathogenic TH2 cells, we performed in silico analyses of recently published transcriptional data from a wide variety of pathogenic TH cells [Okoye IS, et al. (2014) Proc Natl Acad Sci USA 111(30):E3081-E3090] and identified that transcription intermediary factor 1 regulator-alpha (Tif1α)/tripartite motif-containing 24 (Trim24) was predicted to be active in house dust mite (HDM)- and helminth-elicited Il4(gfp+)αβ(+)CD4(+) TH2 cells but not in TH1, TH17, or Treg cells. Testing this prediction, we restricted Trim24 deficiency to T cells by using a mixed bone marrow chimera system and found that T-cell-intrinsic Trim24 is essential for HDM-mediated airway allergy and antihelminth immunity. Mechanistically, HDM-elicited Trim24(-/-) T cells have reduced expression of many TH2 cytokines and chemokines and were predicted to have compromised IL-1-regulated signaling. Following this prediction, we found that Trim24(-/-) T cells have reduced IL-1 receptor (IL-1R) expression, are refractory to IL-1β-mediated activation in vitro and in vivo, and fail to respond to IL-1β-exacerbated airway allergy. Collectively, these data identify a previously unappreciated Trim24-dependent requirement for IL-1R expression on TH2 cells and an important nonredundant role for T-cell-intrinsic Trim24 in TH2-mediated allergy and antihelminth immunity.

Requirement of CD30 expression on CD4 T cells in the pathogenesis of experimental autoimmune encephalomyelitis

CD30, a member of the tumor necrosis factor receptor superfamily, is expressed preferentially by effector or memory helper T cells. Here we show that experimental autoimmune encephalomyelitis (EAE) is ameliorated with markedly reduced induction of antigen-specific Th1 and Th17 cells in CD30 knockout mice. Passive EAE indicated that CD30 on non-hematopoietic parenchymal cell is not required and mixed bone marrow chimera experiments revealed that CD30 signaling on CD4 T cells amplified the development of antigen-specific and encephalitogenic CD4 T cells. Thus, CD30 expression on CD4 T cells is critically involved in the pathogenesis of central nervous system autoimmunity.

Intrinsic functional defects of type 2 innate lymphoid cells impair innate allergic inflammation in promyelocytic leukemia zinc finger (PLZF)–deficient mice

The transcription factor promyelocytic leukemia zinc finger (PLZF) is transiently expressed during development of type 2 innate lymphoid cells (ILC2s) but is not present at the mature stage. We hypothesized that PLZF-deficient ILC2s have functional defects in the innate allergic response and represent a tool for studying innate immunity in a mouse with a functional adaptive immune response. We determined the consequences of PLZF deficiency on ILC2 function in response to innate and adaptive immune stimuli by using PLZF(-/-) mice and mixed wild-type:PLZF(-/-) bone marrow chimeras. PLZF(-/-) mice, wild-type littermates, or mixed bone marrow chimeras were treated with the protease allergen papain or the cytokines IL-25 and IL-33 or infected with the helminth Nippostrongylus brasiliensis to induce innate type 2 allergic responses. Mice were sensitized with intraperitoneal ovalbumin-alum, followed by intranasal challenge with ovalbumin alone, to induce adaptive TH2 responses. Lungs were analyzed for immune cell subsets, and alveolar lavage fluid was analyzed for ILC2-derived cytokines. In addition, ILC2s were stimulated exvivo for their capacity to release type 2 cytokines. PLZF-deficient lung ILC2s exhibit a cell-intrinsic defect in the secretion of IL-5 and IL-13 in response to innate stimuli, resulting in defective recruitment of eosinophils and goblet cell hyperplasia. In contrast, the adaptive allergic inflammatory response to ovalbumin and alum was unimpaired. PLZF expression at the innate lymphoid cell precursor stage has a long-range effect on the functional properties of mature ILC2s and highlights the importance of these cells for innate allergic responses in otherwise immunocompetent mice.

Successful treatment of murine autoimmune cholangitis by parabiosis: Implications for hematopoietic therapy

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4−/−Tg).Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4−/−Tg mice with established disease at 8–9 weeks of age with C57BL/6 control mice. Such parabiotic “twins” had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4−/−Tg and CD8−/− mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8+ T effector cells in the presence of wild type CD4 cells was noted. In conclusion, “correcting” the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.

The T Cell Repertoire-Diversifying Enzyme TSSP Contributes to Thymic Selection of Diabetogenic CD4 T Cell Specificities Reactive to ChgA and IAPP Autoantigens.

Multiple studies highlighted the overtly self-reactive T cell repertoire in the diabetes-prone NOD mouse. This autoreactivity has primarily been linked to defects in apoptosis induction during central tolerance. Previous studies suggested that thymus-specific serine protease (TSSP), a putative serine protease expressed by cortical thymic epithelial cells and thymic dendritic cells, may edit the repertoire of self-peptides presented by MHC class II molecules and shapes the self-reactive CD4 T cell repertoire. To gain further insight into the role of TSSP in the selection of self-reactive CD4 T cells by endogenous self-Ags, we examined the development of thymocytes expressing distinct diabetogenic TCRs sharing common specificity in a thymic environment lacking TSSP. Using mixed bone marrow chimeras, we evaluated the effect of TSSP deficiency confined to different thymic stromal cells on the differentiation of thymocytes expressing the chromogranin A-reactive BDC-2.5 and BDC-10.1 TCRs or the islet amyloid polypeptide-reactive TCR BDC-6.9 and BDC-5.2.9. We found that TSSP deficiency resulted in deficient positive selection and induced deletion of the BDC-6.9 and BDC-10.1 TCRs, but it did not affect the differentiation of the BDC-2.5 and BDC-5.2.9 TCRs. Hence, TSSP has a subtle role in the generation of self-peptide ligands directing diabetogenic CD4 T cell development. These results provide additional evidence for TSSP activity as a novel mechanism promoting autoreactive CD4 T cell development/accumulation in the NOD mouse.

Foxo1 Is a T Cell-Intrinsic Inhibitor of the RORγt-Th17 Program.

An uncontrolled exaggerated Th17 response can drive the onset of autoimmune and inflammatory diseases. In this study, we show that, in T cells, Foxo1 is a negative regulator of the Th17 program. Using mixed bone marrow chimeras and Foxo1-deficient mice, we demonstrate that this control is effective in vivo, as well as in vitro during differentiation assays of naive T cells with specific inhibitor of Foxo1 or inhibitors of the PI3K/Akt pathway acting upstream of Foxo1. Consistently, expressing this transcription factor in T cells strongly decreases Th17 generation in vitro as well as transcription of both IL-17A and IL-23R RORγt-target genes. Finally, at the molecular level, we demonstrate that Foxo1 forms a complex with RORγt via its DNA binding domain to inhibit RORγt activity. We conclude that Foxo1 is a direct antagonist of the RORγt-Th17 program acting in a T cell-intrinsic manner.

Cell-Intrinsic gp130 Signaling on CD4+ T Cells Shapes Long-Lasting Antiviral Immunity.

The IL-6 cytokine family utilizes the common signal transduction molecule gp130, which can mediate a diverse range of outcomes. To clarify the role of gp130 signaling in vivo during acute viral infection, we infected Cd4-cre Il6st(fl/fl) mice, in which gp130 is conditionally ablated in T cells, with acute lymphocytic choriomeningitis virus. We found that by day 12, but not at day 8, after infection the number of virus-specific CD4(+) T cells was reduced in the absence of gp130, and this was sustained for up to 2 mo postinfection. Additionally, gp130-deficient T follicular helper cells had lower expression of Maf, IL-21, and ICOS, and this was accompanied by a reduction in the proportion of germinal center B cells and plasmablasts. Remarkably, at 2 mo postinfection the proportion of IgG2a/c(+) memory B cells and the systemic levels of lymphocytic choriomeningitis virus-specific IgG2 Abs were dramatically decreased, whereas there was a corresponding increase in IgG1(+) memory B cells and virus-specific IgG1 Abs. In the same animals gp130-deficient virus-specific CD8(+) T cells showed a reduced proportion of memory cells, which expressed lower levels of Tcf7, and displayed diminished recall responses on secondary infection. Mixed bone marrow chimeras revealed that the aforementioned gp130 effects on CD4(+) T cells were cell intrinsic. Overall, our data show that gp130 signaling in T cells influences the quantity and quality of long-lasting CD4(+) T cell responses as well as CD8(+) T cell- and Ab-mediated immunity after acute viral infection.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

IL-21 signaling modulates the differentiation, distribution, and abundance of CD8 T cell subsets (LYM7P.616)

Abstract The activation of naïve CD8 T cells typically results in the formation of effector (TE) cells as well as memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory (TCM), effector memory (TEM) and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using adoptive transfer studies and mixed bone marrow chimeras in which CD8 T cells either do or do not express the IL-21 receptor (IL-21R), we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals resulted in reduced accumulation of CD8 T cells in various tissues including the small intestine intraepithelial lymphocyte compartment. The reduced fraction of IL-21R-/- CD8 TRM cells in the small intestine also displayed markedly lower levels of the cytolytic effector molecule granzyme B. Notably, IL-21 signaling was associated with the expression of the chemokine receptor CX3CR1 and the integrin α4β7 on circulating CD8 T cells in the mixed bone marrow chimeras. Acute lymphocytic choriomeningitis virus infection overcame the necessity for IL-21 to establish CD8 TE/TEM and TRM subsets. Overall, this study demonstrates that IL-21 modulates the differentiation, accumulation, and function of effector-phenotype CD8 T cells in a context-dependent manner.

Regulation of virus-specific natural killer cell memory by IL-10 and TGF-β (CCR4P.207)

Abstract Natural killer (NK) cells are critical for host control of viral infections and are rapidly activated by pro-inflammatory cytokines. These activating cytokines are thought to be counter-balanced by regulatory cytokines to prevent excessive cytotoxicity or inflammation. This study investigates the role of the immunosuppressive cytokines transforming growth factor (TGF)-β and interleukin (IL)-10 in NK cell development and function, particularly in the generation of effector and memory responses against viral infection. We compared TGF-β-insensitive NK cells (using TGF-βRIIfl/fl x NKp46iCre mice) to NK cells from littermate control mice directly, as well as in mixed bone marrow chimera and adoptive transfer settings. Although they exhibited no obvious defect at steady state, TGF-β-insensitive NK cells revealed greater activation but decreased memory cell formation during mouse cytomegalovirus (MCMV) infection. Conversely, IL-10R-deficient mice at steady state contained greater percentages of mature NK cells in all organs. Similar to TGF-β-insensitive NK cells, IL-10R-deficient NK cells were also defective in generation of memory following MCMV infection. These studies reveal a novel and important role for immunosuppressive cytokines in regulating the magnitude of the effector response and in the optimal generation of long-lived NK cells following viral infection.

Rac GTPases have opposite roles in T cells and dendritic cells in mucosal immune function (MUC8P.730)

Abstract Variants in RAC1 and RAC2, which encode small Rho-family GTPases, are associated with risk for inflammatory bowel disease. We have shown reduced regulatory T-cell (Treg) proportions in Rac2 knockout (KO) mice with additional dendritic cells (DC)-specific deletion of Rac1 compared to WT. Since Rac is crucial to TCR signaling, we hypothesized that a T-cell specific deletion of Rac1 would also result in defects in Treg development. Treg proportions and susceptibility to dextran sulfate sodium(DSS)-induced intestinal inflammation were assessed in mice deficient in Rac2 in addition to either T cell- or DC-specific deletion of Rac1 (cDKO). Surprisingly, elevated Treg proportions were observed in T-cell specific cDKO mice compared to WT as well as higher Treg proportions within the cDKO compared to the WT compartment in mixed bone marrow chimera. Furthermore, cDKO naïve CD4+ T cells were able to differentiate into Tregs, unlike cDKO DCs, which led to defective Treg generation of WT naïve CD4+ T cells in in vitro Treg generation assays. In vivo Treg generation was reduced in DC-specific cDKO mice compared to WT controls with ovalbumin (OVA) feeding following transfer of CD4+ T cells transgenic for OVA. Lastly, exacerbated DSS colitis was observed in DC-specific, but not T-cell specific cDKO mice, compared to WT. In conclusion, deficiency of Rac proteins result in contrasting effects on mucosal immune function in DCs compared to T cells. The precise mechanism is under investigation.

Local antigen and inflammation in the lung induce the preferential establishment and maintenance of CXCR6 and CD49a-expressing antigen-specific CD8 TRM cells in the lung parenchyma and airways (MUC1P.900)

Abstract Acting as sentinels capable of quickly organizing a secondary immune response upon pathogen challenge, resident memory CD8 T (TRM) populations are initially primed by microenvironment cues and APC licensing following acute infection of peripheral tissues. Despite their capacity for heterologous protection against influenza virus (flu) challenge, scant knowledge exists as to factors necessary to establish and maintain airway and lung parenchymal (LP) CD8 TRM cells. In contrast to mechanisms described for other tissues, lung CD8 TRM cell establishment required cognate antigen recognition once cells were recruited to the lung by local inflammation. Furthermore, this combination of local antigen and inflammation formed long-lasting TRM populations in the LP and airways that highly express the chemokine receptor CXCR6 and adhesion molecule CD49a. During a primary intranasal (IN) flu infection, CXCR6 is preferentially expressed on virus-specific CD8 T cells in the airways and LP by day 7 post-infection. Selectively, LP CD8 TRM cells highly express CXCR6, while systemically circulating flu-specific cells achieve only an intermediate expression level. Finally, use of mixed bone marrow chimeras show that wild-type virus-specific memory CD8 T cells predominate over CXCR6-/- cells in the airway and LP after IN infection with Sendai or flu virus. Thus, our data show that local antigen and inflammation coupled with CXCR6 expression is key to establish CD8 TRM cells in the LP and airways.

Role of miR-27 in controlling T cell immunity (IRM15P.605)

Abstract miR-23~27~24 clusters are highly enriched in regulatory cells while expressed at low levels in conventional T cells. However, despite a selective expression pattern of miR-23~27~24 clusters in T cells, to date, studies of this miRNA family have primarily focused on their role in tumorigenesis. Here, we show miR-23~27~24 clusters play a pivotal role in regulating T cell immunity. Particularly, our results have demonstrated that the majority of miR-23~27~24 cluster-dependent phenotypes could be attributed to a single member of this miRNA family, miR-27. Mice with T cell-specific overexpression of miR-27 spontaneously developed autoimmune phenotypes. On the other hand, in vitro polarization studies revealed that T cells with miR-27 overexpression exhibited impaired differentiation and effector function of multiple T helper cell lineages. Finally, our mixed bone marrow chimeras studies further demonstrated that miR-27 could control different T cell responses through either a T cell-intrinsic or -extrinsic manner. Collectively, Our results identify a new "immune regulatory" miRNA that can play multi-faceted roles in regulating T cell immunity.

MiR-146a as a negative regulator in controlling humoral immunity (IRM10P.612)

Abstract The germinal center (GC) reaction is the hallmark of B cell-mediated immune responses to T cell-dependent antigens Recently, a specialized immune population of follicular helper T (Tfh) cells has emerged as a central player in driving GC B cell response and promoting humoral immunity. It has been shown that many genes that are critically involved in the generation of GC B cells also play an indispensable role in the differentiation of Tfh cell population from naive T cells. Here, we show miR-146a is highly upregulated in both Tfh and GC B cells. Our analysis of mixed bone marrow chimeras as well as mice with cell-type-specific deletion of miR-146a demonstrated that elevated levels of miR-146a in both Tfh and B cells are pivotal to control corresponding responses during GC reactions in a cell intrinsic manner. Moreover, deletion of both miR-146a and its paralog, miR-146b resulted in further enhance phenotypes, suggesting that both miR-146 family members contribute to the negative regulation of humoral immunity. Finally, combining transcriptional profiling and biochemical approaches including HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation), several miR-146a targets with potential roles in controlling Tfh and GC B cell responses have been identified. In sum, our study identifies a miRNA family highly enriched in GC cell subsets with the capacity to control GC reaction by limiting differentiation of Tfh and GC B cells.