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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.

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.

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.

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.

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.

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 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.

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.

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.

The costimulatory molecule 4-1BB and its ligand 4-1BBL can control adaptive immunity, but here we show that their interaction also suppressed myelopoiesis. We found that 4-1BBL was expressed on hematopoietic stem cells, differentiating common myeloid progenitors and granulocyte-macrophage progenitors, and 4-1BB was inducible on activated myeloid progenitors. Steady-state numbers of granulocyte-macrophage progenitors, myeloid-lineage cells and mature dendritic cells were higher in 4-1BB- and 4-1BBL-deficient mice, indicative of a negative function, and we confirmed that result with bone marrow chimeras and in vitro, where the absence of interactions between 4-1BB and 4-1BBL led to enhanced differentiation into dendritic cell lineages. The regulatory activity was mediated by 4-1BBL, with binding by 4-1BB inhibiting differentiation of myeloid progenitors. Thus, 4-1BB and 4-1BBL have a previously unknown function in limiting myelopoiesis and the development of dendritic cells.

The intestinal intraepithelial lymphocytes (IEL) represent multi-lineage T cell populations. In addition to a major gammadeltaTCR(+) T cell subset, many IEL express alphabetaTCRs and they can be separated into alphabeta sublineages. Some TCRalphabeta(+)IEL have characteristics in common with conventional TCRalphabeta(+)T cells whereas others share an unconventional phenotype with their TCRgammadelta(+) counterparts. Because the latter are enriched for autoreactive TCRs and can be generated in the absence of a thymus, it has long been postulated that some IEL subsets develop locally in the intestine. Several new data however, indicate that under physiological conditions, IEL require a thymic education that directs lineage commitment and functional differentiation. This review will discuss the contributions of the thymus in shaping the various intestinal IEL sublineages.

Mouse small intestine intraepithelial lymphocytes (IEL) that express alphabetaTCR and CD8alphaalpha homodimers are an enigmatic T cell subset, as their specificity and in vivo function remain to be defined. To gain insight into the nature of these cells, we performed global gene expression profiling using microarray analysis combined with real-time quantitative PCR and flow cytometry. Using these methods, TCRalphabeta(+)CD8alphaalpha IEL were compared with their TCRalphabeta(+)CD8beta(+) and TCRgammadelta(+) counterparts. Interestingly, TCRalphabeta(+)CD8alphaalpha IEL were found to preferentially express genes that would be expected to down-modulate their reactivity. They have a unique expression pattern of members of the Ly49 family of NK receptors and tend to express inhibitory receptors, along with some activating receptors. The signaling machinery of both TCRalphabeta(+)CD8alphaalpha and TCRgammadelta(+) IEL is constructed differently than other IEL and peripheral T cells, as evidenced by their low-level expression of the linker for activation of T cells and high expression of the non-T cell activation linker, which suppresses T cell activation. The TCRalphabeta(+)CD8alphaalpha IEL subset also has increased expression of genes that could be involved in immune regulation, including TGF-beta(3) and lymphocyte activation gene-3. Collectively, these data underscore the fact that, while TCRalphabeta(+)CD8alphaalpha IEL resemble TCRgammadelta(+) IEL, they are a unique population of cells with regulated Ag reactivity that could have regulatory function.

Intraepithelial lymphocytes (IELs) contain several subsets, but the origin of the T-cell receptor (TCR)alphabeta(+) CD8 alpha alpha(+) IELs has been particularly controversial. Here we provide a synthesis, based on recent work, that attempts to unify the divergent views. The intestine has a primordial function in lymphopoiesis, and precursors with the potential to differentiate into T cells are found both in the epithelium and underlying lamina propria. Moreover, the thymus has been reported to export cells to the intestine that are not fully differentiated. TCR alpha beta(+) CD8 alpha alpha(+) IELs can differentiate in the intestine from each of these sources, but in normal euthymic mice, the thymus appears to be the major source for TCR alpha beta(+) CD8 alpha alpha(+) IELs. This unique IEL subset is a self-reactive population that requires exposure to self-agonists for selection in the thymus, similar to other regulatory T-cell populations. IELs transition through a double-positive (DP) intermediate in the thymus, but they originate from a subset of the DP cells that can be identified by its expression of CD8 alpha alpha homodimers. The agonist-selected cells in the thymus are TCRbeta(+) but CD4 and CD8 double negative. The evidence suggests that reacquired expression of CD8 alpha alpha and downregulation of CD5 occur after thymus export, perhaps in the intestine under the influence of interleukin-15. As a result of agonist exposure, a new gene expression program is activated. Therefore, the increased understanding of the developmental origin of TCR alpha beta(+) CD8 alpha alpha(+) IELs may help us to understand how they participate in immune regulation and protection in the intestine.

Mucosal dendritic cells (DCs) in the intestine acquire the unique capacity to produce retinoic acid (RA), a vitamin A metabolite that induces gut tropism and regulates the functional differentiation of the T cells they prime. Here, we identified a stromal cell (SC) population in the intestinal lamina propria (LP), which is capable of inducing RA production in DCs in a RA- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent fashion. Unlike DCs, LP SCs constitutively expressed the enzymatic machinery to produce RA even in the absence of dietary vitamin A, but were not able to do so in germ-free mice implying regulation by microbiota. Interestingly, DCs promoted GM-CSF production by the SCs indicating a two-way cross-talk between both cell types. Furthermore, RA-producing LP SCs and intestinal DCs localized closely in vivo suggesting that the interactions between both cell types might have an important role in the functional education of migratory DCs and therefore in the regulation of immune responses toward oral and commensal antigens.

Summary: The intestinal intraepithelial lymphocytes (IELs) are mostly T cells dispersed as single cells within the epithelial cell layer that surrounds the intestinal lumen. IELs are, therefore, strategically located at the interface between the antigen-rich outside world and the sterile core of the body. The intestine of higher vertebrates has further evolved to harbor numerous commensal bacteria that carry out important functions for the host, and while defensive immunity can effectively protect against the invasion of pathogens, similar immune reactions against food-derived antigens or harmless colonizing bacteria can result in unnecessary and sometimes damaging immune responses. Probably as a result of this unique dilemma imposed by the gut environment, multiple subsets of IEL have differentiated, which all display characteristics of ‘activated yet resting’ immune cells. Despite this common feature, IELs are heterogeneous with regard to their phenotype, ontogeny, and function. In this review, we discuss the different subtypes of IELs and highlight the distinct pathways they took that led to their unique differentiation into highly specialized effector memory T cells, which provide the most effective immune protection yet in a strictly regulated fashion to preserve the integrity and vital functions of the intestinal mucosal epithelium.

Normal immune homeostasis of the intestine requires peaceful coexistence with commensal flora, combined with host defense against pathogens. Perhaps as a result of this unique dilemma, distinct populations of regulatory and effector T lymphocytes are found in the lamina propria and epithelium of the intestine. Here we summarize the properties and functions of these unusual T cells, and describe the molecular and cellular interactions that lead to their development and function. Some mucosal T cells, sometimes called type a, are conventional activated/memory T cells that have received instructions to migrate to the intestine during priming by dendritic cells in the mesenteric lymph node and elsewhere. Others, however, particularly subsets residing permanently in the epithelium, are intestine-specific T cell subpopulations generated by an atypical differentiation pathway.

Previous studies demonstrated that interleukin-10 (IL-10) overexpression decreases formation of early fatty-streak lesions in mice independent of lipoprotein levels. The present studies, using bone marrow transplantation, demonstrate that overexpression of IL-10 by T cells inhibits advanced atherosclerotic lesions in LDL receptor–null mice fed an atherogenic diet. In mice receiving bone marrow from the IL-10 transgenic mice compared with those receiving wild-type marrow, there was a 47% decrease in lesion size and a marked decrease in lesion complexity with an 80% reduction in the necrotic core. Accumulation of cholesterol and phospholipid oxidation products in the aorta was decreased by 50% to 80%, unrelated to plasma lipid or IL-10 levels. Our studies also provide insight into the mechanism of the IL-10–mediated decrease in lesion size. Although a strong influence toward a Th1 phenotype has previously been demonstrated in atherosclerotic models, T lymphocytes in the IL-10 transgenic (Tg) group revealed a marked shift to a Th2 phenotype, with decreased IFN-γ production and an increase in IL-10. Evaluation of specific immunoglobulin subclasses demonstrated a preponderance of IgG1isotype, characteristic of a Th2 influence on B cell immunoglobulin class-switching in the IL-10 Tg group. A major finding of these studies was altered monocyte/macrophage function in the IL-10 Tg group. Monocytes showed a decrease in activation resulting in decreased expression of IFN-γ. Furthermore, macrophage foam cells within lesions of the IL-10 Tg group exhibited markedly decreased apoptosis. These studies demonstrate that T lymphocyte IL-10 can influence the function of other immune cells to reduce the development of advanced atherosclerotic lesions in mice.

Coreceptor CD4 and CD8αβ double-negative (DN) TCRαβ(+) intraepithelial T cells, although numerous, have been greatly overlooked and their contribution to the immune response is not known. Here we used T cell receptor (TCR) sequencing of single cells combined with retrogenic expression of TCRs to study the fate and the major histocompatibility complex (MHC) restriction of DN TCRαβ(+) intraepithelial T cells. The data show that commitment of thymic precursors to the DN TCRαβ(+) lineage is imprinted by their TCR specificity. Moreover, the TCRs they express display a diverse and unusual pattern of MHC restriction that is nonoverlapping with that of CD4(+) or CD8αβ(+) T cells, indicating that they sense antigens that are not recognized by the conventional T cell subsets. The new insights indicate that DN TCRαβ(+) T cells form a third lineage of TCRαβ T lymphocytes expressing a variable TCR repertoire, which serve nonredundant immune functions.

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) share a developmental relationship with Foxp3(+) regulatory T cells (T(reg) cells). Here we show that a T(H)-17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-beta (TGF-beta). Like previously described T(H)-17 cells, the T(H)-17 cells that developed in the thymus expressed the transcription factor RORgamma t and the IL-23 receptor. These cells also expressed alpha(4)beta(1) integrins and the chemokine receptor CCR6 and were recruited to the lung, gut and liver. In the liver, these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus, T(H)-17 cells, like T(reg) cells, can be selected by self antigens in the thymus.

In a number of animal models of spontaneous autoimmune diabetes, pathogenesis has been highly correlated with autoreactive T-cell production of the type 1 cytokine interferon-gamma (IFN-gamma), while protection from disease was associated with type 2 cytokines such as interleukin (IL)-4. Curiously, in some models, diabetes is associated with unexpected cytokine patterns; for example, diabetes can develop in NOD mice lacking a functional IFN-gamma gene. In another situation, acceleration of diabetes occurs in transgenic mice with constitutive beta-cell expression of the type 2 cytokine IL-10. IL-10 has generally been associated with immunosuppression, including the modulation of class II expression on antigen-presenting cells and the generation of regulatory CD4 T-cells. Because it is possible that unregulated expression of any cytokine might lead to unphysiological effects in vivo, we tested the notion that an inducible T-cell-specific IL-10 transgene might yet mediate a more physiological protection from autoimmune diabetes. Our results show that indeed, regulated T-cell production of IL-10 does not accelerate diabetes and instead can provide significant protection from disease. These results help rectify the apparent discrepancies between the effect of IL-10 on various models of autoimmune diabetes.

Interleukin (IL)-10 is a pleiotropic cytokine which inhibits a broad array of immune parameters including T helper cell type 1 (Th1) cytokine production, antigen presentation, and antigenspecific T cell proliferation. To understand the consequences of altered expression of IL-10 in immune models of autoimmune disease, the response to infectious agents, and the response to tumors, we developed transgenic mice expressing IL-10 under the control of the IL-2 promoter. Upon in vitro stimulation, spleen cells from unimmunized transgenic mice secrete higher levels of IL-10 and lower amounts of IFN-γ than do controls, although no gross abnormalities were detected in lymphocyte populations or serum Ig levels. Transfer of normally pathogenic CD4+ CD45RBhigh splenic T cells from IL-10 transgenic mice did not cause colitis in recipient severe combined immunodeficiency mice. Furthermore, co-transfer of these transgenic cells with CD4+ CD45RBhigh T cells from control mice prevented disease. Transgenic mice retained their resistance to Leishmania major infection, indicating that their cell-mediated immune responses were not globally suppressed. Lastly, in comparison to controls, IL-10 transgenic mice were unable to limit the growth of immunogenic tumors. Administration of blocking IL-10 mAbs restored in vivo antitumor responses in the transgenic mice. These results demonstrate that a single alteration in the T cell cytokine profile can lead to dramatic changes in immune responses in a manner that is stimulus dependent. These mice will be useful in defining differences in inflammatory conditions and cellular immunity mediated by IL-10.

Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse T cells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to T cell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human T cells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response. Fil: Carrera Silva, Eugenio Antonio. University of Yale. School of Medicine; Estados Unidos de América; Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina; Fil: Chan, Pamela Y.. University of Yale. School of Medicine; Estados Unidos de América; Fil: Joannas, Leonel. University of Yale. School of Medicine; Estados Unidos de América; Fil: Errasti, Andrea Emilse. Universidad de Buenos Aires. Facultad de Medicina. Cátedra de Farmacología; Argentina; University of Yale. School of Medicine; Estados Unidos de América; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina; Fil: Gagliani, Nicola. University of Yale. School of Medicine; Estados Unidos de América; Fil: Bosurgi, Lidia. University of Yale. School of Medicine; Estados Unidos de América; Fil: Jabbour, Maurice. University Of Arizona; Estados Unidos de América; Fil: Perry, Anthony. Banner MD Anderson Cancer Center; Estados Unidos de América; Fil: Smith Chakmakova, Faye. Saint Joseph’s Hospital and Medical Center; Estados Unidos de América; Fil: Mucida, Daniel. La Jolla Institute for Allergy and Immunology; Estados Unidos de América; Fil: Cheroutre, Hilde. La Jolla Institute for Allergy and Immunology; Estados Unidos de América; Fil: Burstyn Cohen, Tal. Salk Institute for Biological Studies; Estados Unidos de América; Fil: Leighton, Jonathan A.. Mayo Clinic Arizona; Estados Unidos de América; Fil: Lemke, Greg. Salk Institute for Biological Studies; Estados Unidos de América; Fil: Ghosh, Sourav. University Of Arizona; Estados Unidos de América; Fil: Rothlin, Carla V.. University of Yale. School of Medicine; Estados Unidos de América

TCRαβ thymocytes differentiate into either CD8αβ(+) cytotoxic T lymphocytes or CD4(+) helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4(+) thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4(+) T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4(+) T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II-restricted CD4(+) cytotoxic T lymphocytes.

Regulatory T cells (Treg) express the forkhead box p3 (Foxp3) transcription factor and suppress pathological immune responses against self and foreign antigens, including commensal microorganisms. Foxp3 has been proposed as a master key regulator for Treg, required for their differentiation, maintenance, and suppressive functions. Two types of Treg have been defined. Natural Treg (nTreg) are usually considered to be a separate sublineage arising during thymus differentiation. Induced Treg (iTreg) originate upon T cell receptor (TCR) stimulation in the presence of tumor growth factor beta. Although under homeostatic conditions most Treg in the periphery are nTreg, special immune challenges in the intestine promote more frequently the generation of iTreg. Furthermore, recent observations have challenged the notion that Treg are a stable sublineage, and they suggest that, particularly under lymphopenic and/or inflammatory conditions, Treg may lose Foxp3 and/or acquire diverse effector functions, especially in the intestine, which may contribute to uncontrolled inflammation.

Recent advances in stem cell research have redefined previous concepts of hematopoietic hierarchy, lineage commitment, and cell fate. The immune system is comprised of several well-defined cell lineages of which many exhibit high levels of plasticity or capacity in changing their phenotype. The CD4 T helper cells provide a peculiar example of apparently defined cell subsets, at times described as lineages, but also highly sensitive to tissue environmental cues that may change their fate. The classical Th1/Th2 CD4 T cell differentiation referred to for many years as the main CD4 T cell fate dichotomy and the later additions of CD4 helper T cell variants, such as T helper 17 (Th17) and induced regulatory T cells (iTreg), have added complexity but also doubts on the accuracy of defining CD4 T cell subsets as fixed T cell lineages.

Immunological memory allows for rapid and effective protective immunity to previously encountered pathogens. New insights in understanding specific memory differentiation and function have now indicated that in addition to providing enhanced immunity, an important purpose of immunological memory is to provide immediate protection at all sites of the body, including non-lymphoid tissues. Effector memory CD8 T cells have the capacity to reside long-term at epithelial surfaces, where they allow for rapid containment of the invading pathogens at the local entry site and prevent systemic spreading and excessive immune responses. The accumulation of tissue-specific memory T cell subsets, together with cross-reactivity of these antigen-experienced T cells even to unrelated pathogens, provides flexibility and expansion of their specificity repertoire that over time greatly surpasses that of the declining naive T cell populations. This review will discuss new insights into T cell memory. We will focus in particular on the generation and function of effector memory CD8 T cells at the intestinal mucosa, which represents one of the largest entry sites for pathogens.

Memory T cells are long-lived antigen-experienced T cells that are generally accepted to be direct descendants of proliferating primary effector cells. However, the factors that permit selective survival of these T cells are not well established. We show that homodimeric α chains of the CD8 molecule (CD8αα) are transiently induced on a selected subset of CD8αβ+T cells upon antigenic stimulation. These CD8αα molecules promote the survival and differentiation of activated lymphocytes into memory CD8 T cells. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8αα.

TheTCL1protooncogene is overexpressed in many mature B cell lymphomas, especially from AIDS patients. To determine whether aberrant expression promotes B cell transformation, we generated a murine model in which aTCL1transgene was overexpressed at similar levels in both B and T cells. Strikingly, transgenic mice developed Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL) with attendant Bcl-6 expression and mutated JHgene segments at a very high penetrance beginning at 4 months of age. In contrast, only one mouse developed a T cell malignancy at 15 months, consistent with a longer latency for transformation of T cells byTCL1. Activation of premalignant splenic B cells by means of B cell antigen receptor (BCR) engagement resulted in significantly increased proliferation and augmented AKT-dependent signaling, including increased S6 ribosomal protein phosphorylation. Transgenic spleen cells also survived longer than wild-type spleen cells in long-term culture. Together these data demonstrate thatTCL1is a powerful oncogene that, when overexpressed in both B and T cells, predominantly yields mature B cell lymphomas.

LIGHT, a member of the TNF family of cytokines (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed on T cells), is induced on activated T cells and mediates costimulatory and antitumor activity in vitro. Relatively little information is available on the in vivo effects of LIGHT expression, particularly within the T cell compartment. In this work, we describe transgenic mice that express human LIGHT under the control of the CD2 promoter, resulting in constitutive transgene expression in cells of the T lymphocyte lineage. LIGHT-transgenic animals exhibit abnormalities in both lymphoid tissue architecture and the distribution of lymphocyte subsets. They also show signs of inflammation that are most severe in the intestine, along with tissue destruction of the reproductive organs. These LIGHT-mediated effects were recapitulated when immune-deficient mice were reconstituted with bone marrow from LIGHT-transgenic donor mice. T cells in the LIGHT-transgenic mice have an activated phenotype and mucosal T cells exhibit enhanced Th1 cytokine activity. The results indicate that LIGHT may function as an important regulator of T cell activation, and implicate LIGHT signaling pathways in inflammation focused on mucosal tissues.

The development of lymphoid organs requires membrane-bound lymphotoxin (LT), a heterotrimer containing LTα and LTβ, but the effects of LT on T cell function have not been characterized extensively. Upon TCR cross-linking in vitro, splenocytes from both LTα−/− and LTβ−/− mice failed to produce IL-4 and IL-10 due to a reduction in NK T cells. Concordantly, LTα−/− and LTβ−/− mice did not respond to the lipoglycan α-galactosylceramide, which is presented by mouse CD1 to Vα14+ NK T cells. Interestingly, both populations of NK T cells, including those that are mouse CD1 dependent and α-galactosylceramide reactive and those that are not, were affected by disruption of the LTα and LTβ genes. NK T cells were not affected, however, in transgenic mice in which LT signaling is blocked, beginning on day 3 after birth, by expression of a soluble decoy LTβ receptor. This suggests that membrane-bound LT is critical for NK T cells early in ontogeny, but not for the homeostasis of mature cells.

Mutations in Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Btk is a critical component of signaling pathways regulating B cell development and function. We used a genetic approach to determine whether Btk is also limiting for these processes. One allele of a murine Btk transgene expressed a dosage of Btk (25% of endogenous levels in splenic B cells) sufficient to restore normal numbers of phenotypically mature conventional B cells in xid mice. 2,4,6-trinitrophenyl–Ficoll response, anti-IgM-induced proliferation, B1 cell development, and serum IgM and IgG3levels remained significantly impaired in these animals. B cells from Btk −/− transgenic mice also responded poorly to anti-IgM, indicating that the xid mutation does not create a dominant negative form of Btk. Response to 2,4,6-trinitrophenyl–Ficoll and B cell receptor cross-linking were increased 3- to 4-fold in xid mice homozygous for the transgene. These results demonstrate that Btk is a limiting component of B cell antigen receptor signaling pathways and suggest that B cell development and response to antigen may require different levels of Btk activity.

The B and T lymphocyte attenuator (BTLA) is an Ig super family member that binds to the herpes virus entry mediator (HVEM), a TNF receptor super family (TNFRSF) member. Engagement of BTLA by HVEM triggers inhibitory signals, although recent evidence indicates that BTLA also may act as an activating ligand for HVEM. In this study, we reveal a novel role for the BTLA-HVEM pathway in promoting the survival of activated CD8(+) T cells in the response to an oral microbial infection. Our data show that both BTLA- and HVEM-deficient mice infected with Listeria monocytogenes had significantly reduced numbers of primary effector and memory CD8(+) T cells, despite normal proliferation and expansion compared to controls. In addition, blockade of the BTLA-HVEM interaction early in the response led to significantly reduced numbers of antigen-specific CD8(+) T cells. HVEM expression on the CD8(+) T cells as well as BTLA expression on a cell type other than CD8(+) T lymphocytes, was required. Collectively, our data demonstrate that the function of the BTLA-HVEM pathway is not limited to inhibitory signaling in T lymphocytes, and instead, that BTLA can provide crucial, HVEM-dependent signals that promote survival of antigen activated CD8(+) T cell during bacterial infection.

The thymus leukemia (TL) antigen is a major histocompatibility complex-encoded nonclassical class I molecule. Here we present data demonstrating that expression of the TL antigen, unlike other class I molecules, is completely independent of the function of the transporter associated with antigen processing (TAP). The TL antigen is expressed by transfected TAP-2-deficient RMA-S cells when these cells are grown at 37 degrees C. In transfected RMA cells, the kinetics of arrival of TL antigen on the cell surface are similar to those of a classical class I molecule. The kinetics are not altered in TAP-deficient RMA-S cells, demonstrating that surface TL expression in TAP-deficient cells is not due to the stable expression of a few molecules that leak out by a TAP-independent pathway. Soluble TL molecules produced by Drosophila melanogaster cells are highly resistant to thermal denaturation, unlike peptide-free classical class I molecules synthesized by these insect cells. In addition, these soluble TL molecules are devoid of detectable bound peptides. The results demonstrate that the TL antigen is capable of reaching the surface without bound peptide, although acquisition of peptide or some other ligand through a TAP-independent pathway cannot be formally excluded. We speculate that the ability of the TL antigen to reach the cell surface, under conditions in which other class I molecules do not, may be related to a specialized function of the TL molecule in the mucosal immune system, and possibly in the stimulation of intestinal gamma delta T cells.

The immune system preserves and makes use of autoreactive lymphocytes with specialized functions. Here we showed that one of these populations, CD8alphaalpha(+)TCRalphabeta(+) intestinal intraepithelial lymphocytes (IELs), arose from a unique subset of double-positive thymocytes. This subset of cells was precommitted to preferentially give rise to CD8alphaalpha(+)TCRalphabeta(+) IELs, but they required exposure to self-agonist peptides. The agonist-selected TCRalphabeta(+) thymocytes are CD4 and CD8 double-negative, and their final maturation, including the induction of CD8alphaalpha expression, appeared to occur only after thymus export in the IL-15-rich environment of the gut. These developmental steps, including precommitment of immature thymocytes, TCR-mediated agonist selection, and postthymic differentiation promoted by cytokines, define a unique pathway for the generation of CD8alphaalpha(+)TCRalphabeta(+) IEL.

A simple and efficient method for direct in vitro synthesis of capped transcripts of cloned eukaryotic genes is described. As an example capped transcripts were made from a plasmid containing the human fibroblast interferon gene cloned under the control of a prokaryotic promoter. These transcripts were translated in vivo in Xenopus laevis oocytes and in vitro in reticulocyte and in wheat germ cell-free protein synthesizing systems.