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Intraepithelial T cells (IETs) are in close contact with intestinal epithelial cells and the underlying basement membrane, and they detect invasive pathogens. How intestinal epithelial cells and basement membrane influence IET survival and function, at steady state or after infection, is unclear. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is constitutively expressed by intestinal epithelial cells and is important for protection from pathogenic bacteria. Here, we showed that at steady-state LIGHT, an HVEM ligand, binding to epithelial HVEM promoted the survival of small intestine IETs. RNA-seq and addition of HVEM ligands to epithelial organoids indicated that HVEM increased epithelial synthesis of basement membrane proteins, including collagen IV, which bound to β1integrins expressed by IETs. Therefore, we proposed that IET survival depended on β1integrin binding to collagen IV and showed that β1integrin–collagen IV interactions supported IET survival in vitro. Moreover, the absence of β1integrin expression by T lymphocytes decreased TCR αβ+IETs in vivo. Intravital microscopy showed that the patrolling movement of IETs was reduced without epithelial HVEM. As likely consequences of decreased number and movement, protective responses toSalmonella entericawere reduced in mice lacking either epithelial HVEM, HVEM ligands, or β1integrins. Therefore, IETs, at steady state and after infection, depended on HVEM expressed by epithelial cells for the synthesis of collagen IV by epithelial cells. Collagen IV engaged β1integrins on IETs that were important for their maintenance and for their protective function in mucosal immunity.

IL10 is attributed with immune-suppressive and anti-inflammatory properties, which could promote or suppress cancer in the gastrointestinal tract. Loss of IL10 exacerbates colonic inflammation, leading to colitis and cancer. Consistent with this, transfer of IL10-competent regulatory T cells (Treg) into mice with colitis or hereditary polyposis protects against disease, while IL10-deficient mice are predisposed to polyposis with increased colon polyp load. Little is known about the protective or pathogenic function of IL10 in cancers of the small intestine. We found CD4+ T cells and CD4+ Foxp3+ Tregs to be the major sources of IL10 in the small intestine and responsible for the increase in IL10 during polyposis in the APCΔ468 mouse model of hereditary polyposis. Targeted ablation of IL10 in T cells caused severe IL10 deficiency and delayed polyp growth. However, these polyps progressively lost cytotoxic activity and eventually progressed to cancer. Several observations suggested that the effect was due to the loss of IFNγ-dependent immune surveillance. IL10-incompetent CD4+ T cells failed to secrete IFNγ when stimulated with polyp antigens and were inefficient in T-helper-1 (TH1) commitment. By contrast, the TH17 commitment was unaffected. These findings were validated using mice whose T cells overexpress IL10. In these mice, we observed high intra-polyp cytotoxic activity and attenuation of polyposis. Thus, expression of IL10 by T cells is protective and required for immune surveillance in the small intestine. Cancer Immunol Res; 3(7); 806–14. ©2015 AACR.

Legends to Supplementary Figures

Representative analysis of bone marrow reconstitution efficiency. (A) FACS of total live MNC derived from the intestine of donor CD45.2+ mouse. (B) Frequencies of CD45.1+ and CD45.2+ cells in the intestine of a representative chimeric mouse. CD45.1 donor bone marrow cells were depleted of Lin+ cells and injected into retro-orbital sinus of lethally irradiated 2 month old CD45.2 APCdelta468 mice. To establish the level of bone marrow reconstitution, chimeric APCdelta468 mice were aged to 4 months of age and intestinal MNC were isolated and analyzed by flow cytometry for expression of CD45.2 and CD45.1.

APCdelta468 small intestine serial sections were stained for rabbit anti-mouse granzyme B or isotype control (rabbit IgG) and identified using anti-rabbit Alexa-Fluor 594 secondary antibody. Sections were counterstained with DAPI.

(A-D) Representative immunofluorescent staining images of B6 small intestinal and APCdelta468 polyp and marginal tissue. (A) 200X images showing CD4 (red), Foxp3 (green), and DAPI (blue). (B) 400X images showing Foxp3 (green), IL-10 (red), and DAPI (blue). (C) 400X images showing CD4 (green), IL-10 (red), double positive CD4+IL-10+ (yellow), and DAPI (blue).

Intra-polyp perforin (green) and granzyme B (red) immunofluorescent co-staining (yellow) on 4 um sections from APCdelta468 and IL2pIL-10 APCdelta468 mice; x400.

Colitis is characterized by an exacerbated intestinal immune response, but the genetic and other mechanisms regulating immune activation remain incompletely understood. In order to identify new pathways leading to colitis, we sought to identify genes with increased expression in the colons of patients that also are near loci identified by genome wide association studies (GWAS) associated with IBD risk. One such SNP, rs9557195 was of particular interest because it is within an intron of

Mucosal-associated invariant T (MAIT) cells are a subpopulation of T lymphocytes that respond to microbial metabolites. We performed single-cell RNA sequencing and metabolic analyses of MAIT cell subsets in thymus and peripheral tissues from mice and humans to define the heterogeneity and developmental pathway of these innate-like lymphocytes. We show that the predominant mouse subset, which produces IL-17 (MAIT17), and the subset that produces IFNγ (MAIT1), have greatly different transcriptomes and metabolic states in the thymus and periphery. A splenic MAIT subset has a transcriptome similar to circulating lymphocytes, and in mice these also are found in recent thymic emigrants, suggesting partially mature cells emigrate from the thymus. Human MAIT cells are predominantly MAIT1 cells, but have a different metabolism from their mouse counterparts with increased fatty acid uptake and storage. Although mouse and human subsets are similar in thymus, in the periphery they diverge, likely reflecting environmental influences.

HVEM is a TNF family receptor that binds a TNF protein and Ig family proteins. Using structural biology and mutagenesis, we identified HVEM muteins with selective ligand binding. We verified their function in vivo in models of inflammation and infection., HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM–LIGHT–CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation., Graphical Abstract

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM:LIGHT:CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knock-in mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

CD4 T helper (Th) cells reprogram to cytotoxic T lymphocytes (CTLs) in the small intestinal epithelium. In the present study, CD4 CTLs were evaluated for their potential to promote tolerance during steady state as well as their ability to fend off enteric pathogens during infection. The CD4 T cell adoptive transfer model of colitis was used to determine if CTLs can promote tolerance. Naïve CD4 T cells were differentiated towards a CTL, Treg, or inflammatory Th (Th1 or Th17) fate and transferred into RagKO recipients. Strikingly, the recipients that received CTLs, like the Treg condition, had minimal disease; in contrast, the Th conditions all exhibited pathology. To determine if the forced presence of Th cells exacerbates colitis disease, CD4 T cell adoptive transfers were performed using cells that were forced to stay Th or predisposed to becoming CTLs. Altogether, the conversion of Th to CTL mitigated disease pathology and promoted tolerogenic conditions. The protective role of CD4 CTLs to rapidly respond to enteric pathogens and contain infections was also examined. Using Salmonella as a bacterial model, the data show that pre-existing CD4 CTLs have the capacity to kill infected cells, thereby preserving the integrity of the barrier and preventing bacterial dissemination. Functionally, CD4 CTLs represent a strategy of protective tolerance by the immune system to fortify the epithelium with quiescent but primed CTLs that can provide rapid immunity during enteric infection. Supported by grants from the NIH (U01 AI125957, F31 DK124078)

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM–LIGHT–CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation. Supported grants from NIH (S10 OD020068, P30CA023100, P30 DK120515, S10RR027366, U01 AI125955, P01 DK46763), U.S. Department of Energy (DE-AC02-98CH10886, DE-AC02-06CH11357), National Center for Research Resources (P41RR012408), National Institute of General Medical Sciences (P41GM103473), Albert Einstein Cancer Center (P30CA013330), Eli Lilly Company, Albert Einstein Macromolecular Therapeutics Development Facility, Price Family Foundation, Albert Einstein Center for Experimental Therapeutics, Pamela and Edward S. Pantzer, and Academia Sinica, Taiwan.

SummaryIntraepithelial T cells (IET) provide continuous surveillance of the intestinal epithelium, but little was known about how epithelial-derived signals regulate the IET population. We show that epithelial expression of the herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily (TNFRSF), maintained the survival of small intestine IET, especially innate-like TCRαβ+ cells lacking CD4 and CD8β. Patrolling movement of all CD8α+ IET also was impaired in the absence of HVEM. HVEM-deficient epithelial cells exhibited downregulation of synthesis of basement membrane components, including collagen IV. Collagen IV supported IET survival in vitro via interactions with β1 integrins expressed by the IET; absence of β1 integrins decreased some IET subsets. Therefore, these data define a circuit whereby epithelial cells regulate intestine resident T lymphocyte populations through basement membrane synthesis.

Significance We show that distinct subsets of mast cells (MCs) expand with sequential oncogenic events in small bowel cancer. Mucosal mast cells (MMCs) previously detected early during Trichinella spiralis infection expand in adenomatous polyps in an IL-10–dependent manner. Connective tissue mast cells (CTMCs), earlier shown to expand during the resolution of inflammation following clearance of T. spiralis, are independent of IL-10 and associate with the transition of polyps to adenocarcinoma. IL-33 upregulates the CTMC lineage-specific protease murine mast cell protease 6 (mMCP6). Ablation of mMCP6 attenuates tumor growth. Thus, tissue sentinel cells respond to oncogenic events and cellular transformation in effect to help promote cancer. Delineating the types of MCs present at various stages of disease offers actionable cellular targets for therapeutic intervention in disease progression., Mast cells (MCs) are tissue resident sentinels that mature and orchestrate inflammation in response to infection and allergy. While they are also frequently observed in tumors, the contribution of MCs to carcinogenesis remains unclear. Here, we show that sequential oncogenic events in gut epithelia expand different types of MCs in a temporal-, spatial-, and cytokine-dependent manner. The first wave of MCs expands focally in benign adenomatous polyps, which have elevated levels of IL-10, IL-13, and IL-33, and are rich in type-2 innate lymphoid cells (ILC2s). These vanguard MCs adhere to the transformed epithelial cells and express murine mast cell protease 2 (mMCP2; a typical mucosal MC protease) and, to a lesser extent, the connective tissue mast cell (CTMC) protease mMCP6. Persistence of MCs is strictly dependent on T cell-derived IL-10, and their loss in the absence of IL-10–expressing T cells markedly delays small bowel (SB) polyposis. MCs expand profusely in polyposis-prone mice when T cells overexpress IL-10. The frequency of polyp-associated MCs is unaltered in response to broad-spectrum antibiotics, arguing against a microbial component driving their recruitment. Intriguingly, when polyps become invasive, a second wave of mMCP5+/mMCP6+ CTMCs expands in the tumor stroma and at invasive tumor borders. Ablation of mMCP6 expression attenuates polyposis, but invasive properties of the remaining lesions remain intact. Our findings argue for a multistep process in SB carcinogenesis in which distinct MC subsets, and their elaborated proteases, guide disease progression.

Ligation of nuclear retinoic acid receptors (RARs) by retinoic acid (RA) activates transcription and promotes cell differentiation. RARs can also participate in non-genomic functions in extra-nuclear spaces. Here we identify an alternative isoform of RARα expressed in the cytoplasm of T lymphocytes. Extranuclear RARα functions in T cell receptor (TCR) proximal signaling and NOTCH/c-MYC-driven proliferation. In contrast to nuclear RARα, RA negatively affects extra-nuclear RARα function in T cells. Upon activation, TCR signaling induces expression of the cellular retinoic acid binding protein 2 (CRABP2), which transports RA from the cytoplasm to the nucleus to activate nuclear RARα and at the same time sequesters RA away from extra-nuclear RARα thereby promoting TCR-driven proliferation. These data show that T cell proliferation and differentiation are reciprocally regulated by extra-nuclear and nuclear RARα and that activation-induced CRABP2 functions as a rheostat of TCR signaling via its dual control over the non-genomic and genomic actions of cytoplasmic and nuclear RARα.

Objective Up-regulation of glucose metabolism has been implicated not only in tumor cell growth but also in immune cells upon activation. However, little is known about the metabolite profile in rheumatoid arthritis (RA), particularly in fibroblast-like synoviocytes (FLS). This study was undertaken to evaluate whether changes in glucose metabolism in RA FLS could play a role in inflammation and joint damage. Methods Synovium and FLS were obtained from patients with RA and patients with osteoarthritis (OA). The rate of glycolysis after stimulation of FLS with lipopolysaccharide and platelet-derived growth factor BB was measured using glycolysis stress test technology. FLS function was evaluated using a glycolysis inhibitor, 2-deoxy-d-glucose (2-DG). After stimulation of the FLS, a migration scratch assay, MTT assay, and enzyme-linked immunosorbent assay were performed to measure the effect of 2-DG on FLS migration, viability of the FLS, and cytokine secretion, respectively. IRDye 800CW 2-DG was used to assess glucose uptake in the arthritic joints and stromal cells of mice after K/BxN mouse serum transfer. The mice were injected daily, intraperitoneally, with 3-bromopyruvate (BrPa; 5 mg/kg) to assess the effect of inhibition of glycolysis in vivo. Results Compared to human OA FLS, the balance between glycolysis and oxidative phosphorylation was shifted toward glycolysis in RA FLS. Glucose transporter 1 (GLUT1) messenger RNA (mRNA) expression correlated with baseline functions of the RA FLS. Glucose deprivation or incubation of the FLS with glycolytic inhibitors impaired cytokine secretion and decreased the rate of proliferation and migration of the cells. In a mouse model of inflammatory arthritis, GLUT1 mRNA expression in the synovial lining cells was observed, and increased levels of glucose uptake and glycolytic gene expression were detected in the stromal compartment of the arthritic mouse joints. Inhibition of glycolysis by BrPa, administered in vivo, significantly decreased the severity of arthritis in this mouse model. Conclusion Targeting metabolic pathways is a novel approach to understanding the mechanisms of disease. Inhibition of glycolysis may directly modulate synoviocyte-mediated inflammatory functions and could be an effective treatment strategy for arthritis.

THEMIS functions in the cytoplasm as an adaptor molecule which dampens TCR signaling. During thymic development, THEMIS sets the affinity threshold for activation, enabling the selection of conventional T cells in response to low affinity antigens. Although these events occur in the cytoplasm, THEMIS possess a nuclear localization signal (NLS) and a significant amount of THEMIS resides in the nucleus. To investigate the importance of THEMIS in the nucleus, we deleted the Themis NLS to force THEMIS in the cytoplasm. Surprisingly, T cell development was impaired similar to the Themis null mice, suggesting that THEMIS may have important roles in the nucleus as well. Alternatively, we established Themis mutant mice, in which THEMIS localized exclusively in the nucleus or where THEMIS was constantly present in both compartments. Normal conventional T cells were absent in both mutant mice. Since lack of THEMIS or the constant presence of THEMIS in the cytoplasm or nucleus or both, all resulted in aberrant T cell development it indicated that the translocation of THEMIS to or/and from the nucleus is essential for its function. To examine the role of THEMIS in T cells in the periphery, we generated mutant mice where THEMIS is expressed normal during thymic development but altered post-thymically. Adoptive transfer of naïve CD4 T cells with altered THEMIS expression into RAG deficient host induced enhanced sever colitis and furthermore, we observed that forced localization of THEMIS in the nucleus redirects the polarization of CD4 T cells toward the pathogenic TH17 helper lineage. These findings identify a dual function for THEMIS as a cytoplasmic adaptor during TCR signaling and a nuclear factor directing gene expression in thymocytes and mature T cells.

In the small intestine epithelium, the major TCRab population is the double negative (DNT) cells lacking the expression of either CD4 or CD8. In contrast to the conventional selected cells, the DNT cells undergo an agonist selection, which leads to their functional education and capacity to migrate to intestine. DNT cells also possess the capacity to reach the liver, where we observed a heterogeneity of the DNT cells populations, based on expression of ThPOK and Runx3. In the gut, the latter is only expressed by the DNT cells. GSEA analysis and cytokines intra-cellular staining have shown that ThPOK+ DNT cells and Runx3 DNT cells are strongly divergent in their biological function. ThPOK+ DNT cells signature is enriched in genes expressed by CD4 T helper cells with a strong production of the inflammatory cytokine Il-17 whereas Runx3+ DNT cells signature is enriched in gene upregulated in CD8 T cytotoxic cells. In the thymus, based on the expression of these two transcription factors, we also found distinct DNT populations. Adoptive transfer, as well as hanging drop fetal thymus culture have demonstrated that solely Runx3+ DNT cells in the thymus are the precursors of the Runx3+ DNT cells in the gut. Furthermore, we demonstrated in the thymus both DNT cells populations are selected upon an agonist interaction with a self-antigen presented by developing thymocytes, for the development of ThPOK+ DNT cells, and TECs for Runx3+ DNT cells. RNA-seq experiment of thymic DNT populations revealed that the agonist selection process results in their differential functional education since T helper pathways are upregulated in ThPOK+ DNT cells whereas pathways involved in function and differentiation of cytotoxic cells are upregulated in Runx3+ DNT cells.

CD4 T helper (Th) cells reprogram to cytotoxic T lymphocytes (CTLs) in the intestinal epithelium during steady state. The unique localization of mucosal CD4 CTLs, in the absence of a pathogenic threat, suggests local non-pathogen antigens and organ-specific properties direct the CTL conversion. Similar to myeloid antigen presenting cells, intestinal epithelial cells (IECs) constitutively express MHC class II (MHCII), indicating crosstalk with nearby CD4 T cells. IECs constantly absorb and process luminal digested proteins and present these dietary antigens to neighboring CD4 T cells on their basolateral side. We have generated solid evidence with murine conditional knock-out and bone marrow chimera models that MHCII+ IECs and the dietary epitopes they present are instrumental in the development of small intestinal CD4 CTLs in a homeostatic, non-inflammatory environment. These dietary antigen-specific CD4 CTLs accumulate over time as innate-like protective cells that fortify the epithelium with a pre-existing protective arm. As fully differentiated and functional resident T cells, CD4 CTLs are aptly designed to rapidly respond, independent of pathogen specificity, and contain infections. Using Salmonella as an enteric pathogen model, the data show that dietary antigen-specific CD4 CTLs have the capacity to rapidly and effectively kill infected epithelial cells, thereby preserving the integrity of the barrier and blocking systemic spreading of the pathogen. The double-pronged ability for CD4 CTLs to protect against invaders while also mitigating immunopathology, by differentiating inflammatory CD4 T cells into excellently trained CD4 CTLs, reveal previously unknown mechanisms of mucosal lymphocyte biology.

IL10 is attributed with immune-suppressive and anti-inflammatory properties, which could promote or suppress cancer in the gastrointestinal tract. Loss of IL10 exacerbates colonic inflammation, leading to colitis and cancer. Consistent with this, transfer of IL10-competent regulatory T cells (Treg) into mice with colitis or hereditary polyposis protects against disease, while IL10-deficient mice are predisposed to polyposis with increased colon polyp load. Little is known about the protective or pathogenic function of IL10 in cancers of the small intestine. We found CD4+ T cells and CD4+ Foxp3+ Tregs to be the major sources of IL10 in the small intestine and responsible for the increase in IL10 during polyposis in the APCΔ468 mouse model of hereditary polyposis. Targeted ablation of IL10 in T cells caused severe IL10 deficiency and delayed polyp growth. However, these polyps progressively lost cytotoxic activity and eventually progressed to cancer. Several observations suggested that the effect was due to the loss of IFNγ-dependent immune surveillance. IL10-incompetent CD4+ T cells failed to secrete IFNγ when stimulated with polyp antigens and were inefficient in T-helper-1 (TH1) commitment. By contrast, the TH17 commitment was unaffected. These findings were validated using mice whose T cells overexpress IL10. In these mice, we observed high intra-polyp cytotoxic activity and attenuation of polyposis. Thus, expression of IL10 by T cells is protective and required for immune surveillance in the small intestine. Cancer Immunol Res; 3(7); 806–14. ©2015 AACR.

The thymus plays a central role in self-tolerance, partly by eliminating precursors with a T cell receptor (TCR) that binds strongly to self-antigens. However, the generation of self-agonist-selected lineages also relies on strong TCR signaling. How thymocytes discriminate between these opposite outcomes remains elusive. Here, we identified a human agonist-selected PD-1+ CD8αα+ subset of mature CD8αβ+ T cells that displays an effector phenotype associated with agonist selection. TCR stimulation of immature post-β-selection thymocyte blasts specifically gives rise to this innate subset and fixes early T cell receptor alpha variable (TRAV) and T cell receptor alpha joining (TRAJ) rearrangements in the TCR repertoire. These findings suggest that the checkpoint for agonist selection precedes conventional selection in the human thymus.

Although the negative selection of self-reactive B cells in the bone marrow of mammals has been clearly demonstrated, it remains unclear in models of gut-associated B cell lymphopoiesis, such as that of the chicken (Gallus gallus). We have generated chicken surface IgM–related receptors in which the diversity region of the lamprey variable lymphocyte receptor (VLR) has been fused to the C region of chicken surface IgM (Tμ). Expression of a VLR:Tμ receptor with specificity for PE supported normal development of B cells, whereas a VLR:Tμ receptor specific to hen egg lysozyme (a self-antigen with respect to chicken B cells) induced, in vivo, complete deletion of VLRHELTμ-expressing B cells. In ovo i.v. injection of PE resulted in deletion of VLRPETμ-expressing Β cells in the embryo spleen, demonstrating that negative selection was independent of the bursal microenvironment. Although chickens transduced with a murine CD8α:chicken Igα fusion protein contained B cells expressing mCD8α:chIgα, cotransfection of the mCD8α:chIgα construct, together with thymus leukemia Ag (a natural ligand for mCD8α), resulted in reduced levels of mCD8α:chIgα-expressing B cells in inverse proportion to the levels of thymus leukemia Ag–expressing cells. Deletion of mCD8α:chIgα-expressing cells was specific for B cells and required active signaling downstream of the mCD8α:chIgα receptor. Ag-mediated negative selection of developing chicken B cells can therefore occur independently of the bursal microenvironment and is dependent on signaling downstream of the BCR.

CD4 T cells adapt to their surroundings by differentiating into various T helper (Th) subsets. At steady state, regulatory CD4 T cells (Treg) and interleukin (IL)-17 producing CD4 T cells (Th17 cells) are present in the intestine and localize mainly to the lamina propria, whereas CD4 cytotoxic T lymphocytes (CTL) reside in the epithelium. A hallmark of CD4 CTL is the reactivation of the Cd8 locus and the re-expression of CD8a controlled by the Cd8 enhancer I (E8I). We showed before that CD4 Th cells can reprogram to CTL. Here, we elucidated a mechanism that drives the CTL reprogramming of CD4 Th cells. We identified a long noncoding RNA transcribed from the Cd8 locus, as the critical regulator of the reciprocal expression of T-BET and RUNX combined with the suppression of Foxp3 and Rorc in CD4 CTL precursors. We further found that the E8I region functions as is central coordinator of the RUNX3-controlled cytotoxic gene expression program marked by the re-expression of CD8a. Adoptive transfer of naïve E8I deletion mutant CD4 T cells to Rag1−/− recipient mice led to accelerated weight loss, and severe small and large intestine inflammation with excessive accumulation of pathogenic IL-17A and IL-17A/IFNg double producing CD4 T cells. Altogether, these findings indicate that the Cd8 locus controls the functional polarization of CD4 Th cells and furthermore, that the conversion to CTL not only enhances the direct protective capacity but also regulate the immune response and prevents excessive inflammation and immune pathology at the mucosal border of the intestine.

The most common cause of community-acquired pneumonia in the world is Streptococcus pneumoniae (S. pneumoniae). It is a serious and urgent issue to develop new therapies including non-antibiotic treatments for this pathogen. The herpes virus entry mediator (HVEM), a member of the tumor necrosis receptor super family 14 (TNFRSF14), has been reported to provide host innate immune defense in intestinal mucosal barriers against Citrobacter rodentium infection. Here we show using C10-Cre transgenic mice crossed to mice with a floxed Hvemallele that mice with pulmonary bronchiolar non-ciliated Clara cell-specific deletion of HVEM (HvemdelCC10) display higher bacterial burden and increased mortality after S. pneumoniae infection. Injection of an HVEM neutralizing antibody had a similar effect. This indicates that epithelial HVEM is also critical for pulmonary mucosal protection during the infection. In response to S. pneumonia, HVEM-deficiency led to reduced CXCL1 secretion, accompanied by impaired neutrophil recruitment to the lung. Likewise, deletion of part of the cytoplasmic tail of HVEM (HvemdelCT), causing loss of the ability of HVEM to signal, showed a similar susceptibility to S. pneumonia, indicating the importance of the intracellular signaling of HVEM in epithelial cells for the bacterial clearance. Taken together, these results reveal the protective role of HVEM in epithelial cells against S. pneumonia infection and suggest that activated HVEM signaling in epithelial cells could be used for the treatment of S. pneumoniae infection.

Intestinal epithelial cells (IEC) are a first barrier that segregates host and commensal bacteria to maintain intestinal homeostasis. Intestinal intraepithelial lymphocytes (IEL) are located beneath or between adjacent IEC and directly contact IEC. The herpes virus entry mediator (HVEM), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is highly expressed by IEC. Epithelial HVEM expression was previously reported as a regulator of innate immune defense during acute infections in the intestine (Shui et al., Nature, 2012). Here, we identify that HVEM signaling in IEC is important for the regulation of the gut microbiota at steady state. Mice with an epithelial-specific deletion of the gene encoding HVEM (HvemΔIEC) had significantly increased segmented filamentous bacteria (SFB) which caused an increase in Th17 cells in the ileum. Treatment with the antibiotic vancomycin eliminated SFB and decreased Th17 cells in HvemΔIEC mice. Additionally, mice with a deletion of the gene encoding CD160, which is a ligand for HVEM and is highly expressed by IEL, including intraepithelial innate lymphoid cells (ILC) and intraepithelial T cells, had increased SFB in the ileum. Our findings suggest that the interaction of CD160 expressed by IEL with HVEM expressed by IEC is important at steady state for shaping the microbiota in the intestine.

The CD4 T helper cell lineage is initially established and controlled in the thymus by the transcription factor, ThPOK. Sustained expression of ThPOK by mature T cells in the periphery maintains the CD4 T helper commitment and allows for plasticity and differentiation to different helper fates (Th1, Th2, Th17...) in response to stimulation with cognate antigens. Our group demonstrated before that in the intestine, repeated stimulation of mature CD4 T cells results in the loss of ThPOK expression followed by the reprogramming of the T helper cells to CD4 cytotoxic T lymphocytes (CTL), expressing notably CD8 T cells attributes (such as Runx3, granzyme B). Continuous activation is also characteristic of chronic viral infections. We therefore investigated if viral infections lead to the generation of reprogrammed CD4 CTL and moreover if these reprogrammed CD4 CTL play essential roles for the organism’s protection. Using the well-defined lymphocytic choriomeningitis virus (LCMV) acute (strain LCMV-Amstrong) versus chronic (LCMV-Clone 13) infection model, we observed that solely chronic infection enables activated CD4 T helper cells to lose ThPOK and convert to CTL with potent killer activity. Moreover, viral-induced CD4 CTL precursors produce various cytokines, notably Il-10, which is required for the induction of the reprogramming process. Finally, we engineered two unique mouse model systems, in which either all the CD4 T cells are forced to reprogram to CTL (gain of function) or else, prevented from reprogramming to CTL (loss of function). Using these models, we show that the reprogrammed CD4 CTL generated under chronic infection conditions, play critical roles in protective immunity.

Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αβ-expressing 'single-positive' thymocytes from CD4(+)CD8αβ(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.

During thymic development, thymocytes expressing a T cell receptor consisting of an alpha and beta chain (TCRαβ), commit to either the cytotoxic- or T helper-lineage fate. This lineage dichotomy is controlled by key transcription factors, including the T helper (Th) lineage master regulator, the Th-inducing BTB/POZ domain-containing Kruppel-like zinc-finger transcription factor, ThPOK, (formally cKrox or Zfp67; encoded by Zbtb7b), which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4(+) thymocytes and the Runt related transcription factor 3 (Runx3), which counteracts ThPOK in MHC class I restricted precursor cells and promotes the lineage commitment of CD8αβ(+) cytolytic T lymphocytes (CTL). ThPOK continues to repress the CTL gene program in mature CD4(+) T cells, even as they differentiate into effector Th cell subsets. The Th cell fate however is not fixed and two recent studies showed that mature, antigen-stimulated CD4(+) T cells have the flexibility to terminate the expression of ThPOK and functionally reprogram to cytotoxic effector cells. This unexpected plasticity of CD4(+) T cells results in the post-thymic termination of the Th lineage fate and the functional differentiation of distinct MHC class II-restricted CD4(+) CTL. The recognition of CD4 CTL as a defined separate subset of effector cells and the identification of the mechanisms and factors that drive their reprogramming finally create new opportunities to explore the physiological relevance of these effector cells in vivo and to determine their pivotal roles in both, protective immunity as well as in immune-related pathology.

Vitamin A is a multifunctional vitamin implicated in a wide range of biological processes. Its control over the immune system and functions are perhaps the most pleiotropic not only for development but also for the functional fate of almost every cell involved in protective or regulatory adaptive or innate immunity. This is especially key at the intestinal border, where dietary vitamin A is first absorbed. Most effects of vitamin A are exerted by its metabolite, retinoic acid (RA), which through ligation of nuclear receptors controls transcriptional expression of RA target genes. In addition to this canonical function, RA and RA receptors (RARs), either as ligand-receptor or separately, play extranuclear, nongenomic roles that greatly expand the multiple mechanisms employed for their numerous and paradoxical functions that ultimately link environmental sensing with immune cell fate. This review discusses RA and RARs and their complex roles in innate and adaptive immunity.

CD4 − CD8αβ − (double-negative or DN) T lymphocytes expressing a T cell receptor (TCR) composed of a variable α and β chain (DN TCRαβ + ) constitute a substantial population of mature T cells that reside mainly in the intestinal epithelium where they likely have a protective as well as regulatory role to preserve the integrity of this critical barrier. DN TCRαβ + intraepithelial T cells develop in the thymus as mature effector/memory cells with variable major histocompatibility complex (MHC) restriction ranging from classical MHC class I or II to various nonclassical MHC class I specificities. DN TCRαβ + T cells develop in the thymus from precursors that undergo positive selection in response to strong TCR-self-antigen/MHC complex interactions called self-agonist-dependent selection. This article will describe the developmental stages and agonist selection process in the thymus as well as the characteristics of mature DN TCRαβ + intraepithelial T cells that distinguish them from conventional naive CD4 and CD8αβ TCRαβ + cells that reside in lymphoid tissues. Finally, the MHC restriction and antigen specificity of DN TCRαβ + intraepithelial T cells will be discussed in light of their unique location within the mucosal epithelial barrier of the intestine.

Dendritic cells (DC) in the gut promote immune tolerance by expressing retinal dehydrogenase (RALDH), an enzyme that promotes retinoic acid, which aids differentiation of Foxp3+ inducible regulatory T cells (iTreg) in the intestinal mucosa. How RALDH expression is regulated is unclear. We found that 4-1BB (CD137), a member of the TNFR family, together with CD103, marked mesenteric lymph node DC with the highest level of RALDH activity, and ligation of 4-1BB maintained RALDH expression in these gut DC. Moreover, 4-1BB signals synergized with those through TLR2 or GM-CSFR to promote RALDH activity in undifferentiated DC. Correspondingly, 4-1BB–deficient mice were impaired in their ability to generate iTreg in the GALT when exposed to oral Ag, and 4-1BB–deficient mesenteric lymph node DC displayed weak RALDH activity and were poor at promoting iTreg development. Thus, our data demonstrate a novel activity of 4-1BB in controlling RALDH expression and the regulatory activity of DC.

In diabetic patients and susceptible mice, insulin is a targeted autoantigen. Insulin B chain 9-23 (B:9-23) autoreactive CD4 T cells are key for initiating autoimmune diabetes in NOD mice; however, little is known regarding their origin and function. To this end, B:9-23–specific, BDC12-4.1 T-cell receptor (TCR) transgenic (Tg) mice were studied, of which, despite expressing a single TCR on the recombination activating gene–deficient background, only a fraction develops diabetes in an asynchronous manner. BDC12-4.1 CD4 T cells convert into effector (Teff) and Foxp3+-expressing adaptive regulatory T cells (aTregs) soon after leaving the thymus as a result of antigen recognition and homeostatic proliferation. The generation of aTreg causes the heterogeneous diabetes onset, since crossing onto the scurfy (Foxp3) mutation, BDC12-4.1 TCR Tg mice develop accelerated and fully penetrant diabetes. Similarly, adoptive transfer and bone marrow transplantation experiments showed differential diabetes kinetics based on Foxp3+ aTreg’s presence in the BDC12-4.1 donors. A single-specificity, insulin-reactive TCR escapes thymic deletion and simultaneously converts into aTreg and Teff, establishing an equilibrium that determines diabetes penetrance. These results are of particular importance for understanding disease pathogenesis. They suggest that once central tolerance is bypassed, autoreactive cells arriving in the periphery do not by default follow solely a pathogenic fate upon activation.

The intraepithelial lymphocytes (IELs) that reside within the epithelium of the intestine form one of the main branches of the immune system. As IELs are located at this critical interface between the core of the body and the outside environment, they must balance protective immunity with an ability to safeguard the integrity of the epithelial barrier: failure to do so would compromise homeostasis of the organism. In this Review, we address how the unique development and functions of intestinal IELs allow them to achieve this balance.

The antigen-rich environment of the gut interacts with a highly integrated and specialized mucosal immune system that has the challenging task of preventing invasion and the systemic spread of microbes, while avoiding excessive or unnecessary immune responses to innocuous antigens. Disruption of the mucosal barrier and/or defects in gut immune regulatory networks may lead to chronic intestinal inflammation as seen in inflammatory bowel disease. The T-cell populations of the intestine play a critical role in controlling intestinal homeostasis, and their unique phenotypes and diversities reflect the sophisticated mechanisms that have evolved to maintain the delicate balance between immune activation and tolerance at mucosal sites. In this article, we will discuss the specialized properties of mucosal T cells in the context of immune homeostasis and inflammation.

Innate lymphoid cells (ILC) are important regulators of early infection at mucosal barriers. They are known to be activated by cytokines and neuropeptides, but it remains to be determined if they integrate other signals in providing protection. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is expressed by all intestinal ILC subsets. Here, we show that HVEM signaling of ILC3 is important for host defense against oral infection with the enteric bacterial pathogen Yersinia enterocolitica (Y. enterocolitica). Surprisingly, IFNγ production by CCR6 negative ILC3 was strongly implicated in protection, likely because these cells were more numerous than other innate lymphocytes that produced IFNγ early after infection. We identified the TNF superfamily member LIGHT, as the ligand inducing HVEM signals in ILC. Therefore, our results demonstrate a novel role for LIGHT-HVEM signaling in regulating ILC3 IFNγ production and protection following infection.

Naïve CD4 and CD8 T cells are selected in the thymus in response to low-avidity TCR interactions. Such weak TCR signals are crucial for installing a quiescent program that guides them to the lymph nodes yet allows for immune activation upon encounter of high-affinity ligands in the periphery. In contrast, agonist-selected T cells receive strong self-based TCR signals in the thymus that imprint them to guard the mucosal borders and maintain quiescence at steady state. Although the TCR signal strength is a decisive factor in instructing these different outcomes, the mechanism that generates protective self-specific T cells remains largely enigmatic. Previous work suggests that the self-specific double negative (DN) T cell lineage already diverges early during T cell development. We have explored the role of developmental timing by Cre-mediated deletion of components of the TCR signaling pathway. The Lck proximal promoter drives Cre expression before b-selection and the CD4 promoter drives Cre expression after b-selection but before positive selection. As TCR targets we investigated the lack of Themis expression, essential for the generation of conventional naïve T cells, but dispensable for agonist selection. Vice versa, we analyzed the requirement for Stim1 and Stim2, driving agonist selection, but not conventional selection. Both Lck prox Cre- and CD4 Cre-driven deletion of Themis lead to a drastic decrease in naïve T cells. However, DN T cells are particularly dependent on early strong TCR signals at the Pre-TCR stage, as only Lck prox Cre- but not CD4 Cre-driven deletion of Stim1/2 eliminates this subset. Our results suggest that strong signals at the Pre-TCR checkpoint are crucial for the generation of innate self-specific T cells.

Regulatory T cells (T(reg) cells) that express the transcription factor Foxp3 suppress the activity of other cells. Here we show that interleukin 10 (IL-10) produced by CD11b(+) myeloid cells in recombination-activating gene 1-deficient (Rag1(-/-)) recipient mice was needed to prevent the colitis induced by transferred CD4(+)CD45RB(hi) T cells. In Il10(-/-)Rag1(-/-) mice, T(reg) cells failed to maintain Foxp3 expression and regulatory activity. The loss of Foxp3 expression occurred only in recipients with colitis, which indicates that the requirement for IL-10 is manifested in the presence of inflammation. IL-10 receptor-deficient (Il10rb(-/-)) T(reg) cells also failed to maintain Foxp3 expression, which suggested that host IL-10 acted directly on the T(reg) cells. Our data indicate that IL-10 released from myeloid cells acts in a paracrine manner on T(reg) cells to maintain Foxp3 expression.

The epithelium of the gastrointestinal tract, which represents the greatest body surface area exposed to the outside environment, is confronted with a plethora of foreign and potentially harmful antigens. Consequently, the immune system of the gut faces the daunting task of distinguishing harmless dietary proteins and commensal bacteria from potentially dangerous pathogens, and of then responding accordingly. Mucosal T cells play a central role in maintaining barrier function and controlling the delicate balance between immune activation and immune tolerance. This review will focus on the unique features of mucosal T cell subsets that reside in the epithelium and lamina propria of the gut.

Colitis-associated cancer (CAC) is the most serious complication of inflammatory bowel disease. Proinflammatory cytokines have been suggested to regulate preneoplastic growth during CAC tumorigenesis. Interleukin 6 (IL-6) is a multifunctional NF-kappaB-regulated cytokine that acts on epithelial and immune cells. Using genetic tools, we now demonstrate that IL-6 is a critical tumor promoter during early CAC tumorigenesis. In addition to enhancing proliferation of tumor-initiating cells, IL-6 produced by lamina propria myeloid cells protects normal and premalignant intestinal epithelial cells (IECs) from apoptosis. The proliferative and survival effects of IL-6 are largely mediated by the transcription factor Stat3, whose IEC-specific ablation has profound impact on CAC tumorigenesis. Thus, the NF-kappaB-IL-6-Stat3 cascade is an important regulator of the proliferation and survival of tumor-initiating IECs.

Intestinal intraepithelial lymphocytes (IEL) bear a partially activated phenotype that permits them to rapidly respond to antigenic insults. However, this phenotype also implies that IEL must be highly controlled to prevent misdirected immune reactions. It has been suggested that IEL are regulated through the interaction of the CD8αα homodimer with the thymus leukemia (TL) antigen expressed by intestinal epithelial cells. We have generated and characterized mice genetically-deficient in TL expression. Our findings show that TL expression has a critical role in maintaining IEL effector functions. Also, TL deficiency accelerated colitis in a genetic model of inflammatory bowel disease. These findings reveal an important regulatory role of TL in controlling IEL function and intestinal inflammation.

Influenza is one of the most prevalent viral diseases in humans. For some high-risk human populations, including the infant, the elderly, and the immunocompromised, who may not benefit from active immunization, passive immunotherapy with antibodies reactive with all influenza A strains may be an alternative. In this study, we characterized several fully human monoclonal antibodies (MAb) reactive with M2e, which were generated from transchromosomic mice engineered to produce fully human antibodies following immunization with a consensus-sequence M2e peptide. The MAbs showed strong binding to M2e peptide and to virus infected MDCK cells. One MAb recognizing the highly conserved N-terminal portion of consensus M2e displayed high binding to the majority of M2e variants from natural viral isolates, including highly pathogenic avian strains, which were recently reported to infect humans. Passive immunotherapy with this MAb in mice resulted in significant reduction in virus replication in the lung and protection from lethal infection when administered either prophylactically or therapeutically. These results suggest the potential of the anti-M2e human MAb with broad binding spectrum as a universal passive immunotherapeutic agent to infection by influenza A virus.

The primary CD8+ T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8+ T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2Kb, resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F1 hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8+ T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33–41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8+ T cell responses.