Chemokine receptor CCR9 suppresses the differentiation of CD4+CD8αα+ intraepithelial T cells in the gut.

The chemokine receptor CCR9 equips T cells with the ability to respond to CCL25, a chemokine that is highly expressed in the thymus and the small intestine (SI). Notably, CCR9 is mostly expressed on CD8 but not on CD4 lineage T cells, thus imposing distinct tissue tropism on CD4 and CD8 T cells. The molecular basis and the consequences for such a dichotomy, however, have not been fully examined and explained. Here, we demonstrate that the forced expression of CCR9 interferes with the tissue trafficking and differentiation of CD4 T cells in SI intraepithelial tissues. While CCR9 overexpression did not alter CD4 T cell generation in the thymus, the forced expression of CCR9 was detrimental for the proper tissue distribution of CD4 T cells in the periphery, and strikingly also for their terminal differentiation in the gut epithelium. Specifically, the differentiation of SI epithelial CD4 T cells into immunoregulatory CD4+CD8αα+ T cells was impaired by overexpression of CCR9 and conversely increased by the genetic deletion of CCR9. Collectively, our results reveal a previously unappreciated role for CCR9 in the tissue homeostasis and effector function of CD4 T cells in the gut.

The molecular basis and cellular effects of distinct CD103 expression on CD4 and CD8 T cells.

Integrin CD103 mediates the adhesion and tissue retention of T cells by binding to E-cadherin which is abundant on epithelial cells. Notably, CD103 is highly expressed on CD8 T cells but conspicuously absent on most CD4 T cells. The mechanism controlling such lineage-specific expression of CD103 remains unclear. Using a series of genetically engineered mouse models, here, we demonstrate that the regulatory mechanism of CD103 expression is distinct between CD4 and CD8 T cells, and that the transcription factor Runx3 plays an important but not an essential role in this process. We further found that the availability of integrin ß7 which heterodimerizes with CD103 was necessary but also constrained the surface expression of CD103. Notably, the forced surface expression of CD103 did not significantly alter the thymic development of conventional T cells but severely impaired the generation of MHC-II-restricted TCR transgenic T cells, revealing previously unappreciated aspects of CD103 in the selection and maturation of CD4 T cells. Unlike its effect on CD4 T cell development, however, CD103 overexpression did not significantly affect CD4 T cells in peripheral tissues. Moreover, the frequency and number of CD4 T cells in the small intestine epithelium did not increase even though E-cadherin is highly expressed in this tissue. Collectively, these results suggest that most mature CD4 T cells are refractory to the effects of CD103 expression, and that they presumably utilize CD103-independent pathways to control their tissue retention and residency.

RORγt limits the amount of the cytokine receptor γc through the prosurvival factor Bcl-xL in developing thymocytes.

The cytokine receptor subunit γc provides critical signals for T cell survival and differentiation. We investigated the molecular mechanism that controls the cell surface abundance of γc during T cell development in the thymus. We found that the amount of γc was low on CD4+CD8+ double-positive (DP) thymocytes before their positive selection to become mature T cells. The transcription factor RORγt was abundant in immature DP thymocytes, and its loss resulted in an increase in the abundance of surface γc, specifically on preselection DP cells. Rather than directly repressing expression of the gene encoding γc, RORγt acted through the antiapoptotic protein Bcl-xL to reduce the abundance of surface γc, which resulted in decreased cytokine signaling and was associated with inhibition of cell metabolism and mitochondrial biogenesis. Accordingly, overexpression of Bcl-xL in RORγt-deficient thymocytes restored the amount of surface γc to that present on normal preselection DP cells. Together, these data highlight a previously unappreciated role for RORγt and Bcl-xL in limiting γc abundance at the cell surface and reveal a signaling circuit in which survival factors control cytokine signaling by limiting the abundance and surface distribution of a receptor subunit shared by several cytokines.

Out-sourcing for Trans-presentation: Assessing T Cell Intrinsic and Extrinsic IL-15 Expression with Il15 Gene Reporter Mice.

IL-15 is a cytokine of the common γ-chain family that is critical for natural killer (NK), invariant natural killer T (iNKT), and CD8 memory T cell development and homeostasis. The role of IL-15 in regulating effector T cell subsets, however, remains incompletely understood. IL-15 is mostly expressed by stromal cells, myeloid cells, and dendritic cells (DCs). Whether T cells themselves can express IL-15, and if so, whether such T cell-derived IL-15 could play an autocrine role in T cells are interesting questions that were previously addressed but answered with mixed results. Recently, three independent studies described the generation of IL-15 reporter mice which facilitated the identification of IL-15-producing cells and helped to clarify the role of IL-15 both in vitro and in vivo. Here, we review the findings of these studies and place them in context of recent reports that examined T cell-intrinsic IL-15 expression during CD4 effector T cell differentiation.

CD4 effector T cell differentiation is controlled by IL-15 that is expressed and presented in trans.

T cells are both producers and consumers of cytokines, and autocrine cytokine signaling plays a critical role in T cell immunity. IL-15 is a homeostatic cytokine for T cells that also controls inflammatory immune responses. An autocrine role of T cell-derived IL-15, however, remains unclear. Here we examined IL-15 expression and signaling upon effector T cell differentiation in mice, and, surprisingly, found that CD4 T cells did not express IL-15. CD4 T cells lacked Il15 gene reporter activity, did not contain IL-15 transcripts, and did not produce IL-15Rα, the proprietary IL-15 receptor required for IL-15 trans-presentation. Moreover, IL-15 failed to inhibit Th17 cell differentiation and failed to generate Foxp3+ Treg cells in vitro. IL-2, which utilizes the same IL-2Rß/γc receptor complex, however, successfully did so. Exogenous IL-15 only exerted bioactivity and controlled T cell differentiation when it was trans-presented by IL-15Rα. Consequently, IL-15Rα-bound IL-15, but not free IL-15, suppressed Th17 cell differentiation and induced Treg cell generation. Collectively, these results reveal the absence of an IL-15 autocrine loop in CD4 T cells and strongly suggest that IL-15 trans-presentation by non-CD4 T cells is the primary mechanism via which IL-15 controls CD4 effector T cell differentiation.

Natural killer cells limit cardiac inflammation and fibrosis by halting eosinophil infiltration.

Myocarditis is a leading cause of sudden cardiac failure in young adults. Natural killer (NK) cells, a subset of the innate lymphoid cell compartment, are protective in viral myocarditis. Herein, we demonstrated that these protective qualities extend to suppressing autoimmune inflammation. Experimental autoimmune myocarditis (EAM) was initiated in BALB/c mice by immunization with myocarditogenic peptide. During EAM, activated cardiac NK cells secreted interferon γ, perforin, and granzyme B, and expressed CD69, tumor necrosis factor-related apoptosis-inducing ligand treatment, and CD27 on their cell surfaces. The depletion of NK cells during EAM with anti-asialo GM1 antibody significantly increased myocarditis severity, and was accompanied by elevated fibrosis and a 10-fold increase in the percentage of cardiac-infiltrating eosinophils. The resultant influx of eosinophils to the heart was directly responsible for the increased disease severity in the absence of NK cells, because treatment with polyclonal antibody asialogangloside GM-1 did not augment myocarditis severity in eosinophil-deficient ΔdoubleGATA1 mice. We demonstrate that NK cells limit eosinophilic infiltration both indirectly, through altering eosinophil-related chemokine production by cardiac fibroblasts, and directly, by inducing eosinophil apoptosis in vitro. Altogether, we define a new pathway of eosinophilic regulation through interactions with NK cells.

Activated T cells secrete an alternatively spliced form of common γ-chain that inhibits cytokine signaling and exacerbates inflammation.

The common γ-chain (γc) plays a central role in signaling by IL-2 and other γc-dependent cytokines. Here we report that activated T cells produce an alternatively spliced form of γc mRNA that results in protein expression and secretion of the γc extracellular domain. The soluble form of γc (sγc) is present in serum and directly binds to IL-2Rß and IL-7Rα proteins on T cells to inhibit cytokine signaling and promote inflammation. sγc suppressed IL-7 signaling to impair naive T cell survival during homeostasis and exacerbated Th17-cell-mediated inflammation by inhibiting IL-2 signaling upon T cell activation. Reciprocally, the severity of Th17-cell-mediated inflammatory diseases was markedly diminished in mice lacking sγc. Thus, sγc expression is a naturally occurring immunomodulator that regulates γc cytokine signaling and controls T cell activation and differentiation.

Interleukin-6 expands homeostatic space for peripheral T cells.

T cell homeostasis and survival is dependent on interleukin-7 (IL-7). Immune activation, however, downregulates IL-7 receptor expression on T cells so that T cell survival during activation must be maintained independently of IL-7. The pro-inflammatory cytokine IL-6 shares common signaling pathways with IL-7 and can promote T cell survival in vitro. But whether IL-6 promotes T cell survival and homeostasis in vivo is not clear. Notably, IL-6 overexpression results in massive plasmacytosis and autoimmunity so that an IL-6 effect on in vivo T cell survival has remained untested. To overcome this limitation, here we generated IL-6 transgenic mice on an immunoglobulin heavy chain (IgH) deficient background which rendered them B cell deficient. Notably, such IgH(KO)IL6(Tg) mice were free of any signs of inflammation or autoimmunity and remained healthy throughout the course of analysis. In these mice, we found that IL-6 overexpression significantly increased peripheral T cell numbers, but importantly without increasing thymopoiesis. Moreover, IL-6 signaled T cells maintained their naïve phenotype and did not express activation/memory markers, suggesting that increased T cell numbers were due to increased T cell survival and not because of expansion of activated T cells. Mechanistically, we found that IL-6 signaling induced expression of pro-survival factors Mcl-1 and Pim-1/-2 but not Bcl-2. Thus, IL-6 is a T cell homeostatic cytokine that expands T cell space and can maintain the naïve T cell pool.

Pim1 permits generation and survival of CD4+ T cells in the absence of γc cytokine receptor signaling.

γ-Chain (γc) cytokine receptor signaling is required for the development of all lymphocytes. Why γc signaling plays such an essential role is not fully understood, but induction of the serine/threonine kinase Pim1 is considered a major downstream event of γc as Pim1 prevents apoptosis and increases metabolic activity. Consequently, we asked whether Pim1 overexpression would suffice to restore lymphocyte development in γc-deficient mice. By analyzing Pim1-transgenic γc-deficient mice (Pim1(Tg) γc(KO) ), we show that Pim1 promoted T-cell development and survival in the absence of γc. Interestingly, such effects were largely limited to CD4(+) lineage αß T cells as CD4(+) T-cell numbers improved to near normal levels but CD8(+) T cells remained severely lymphopenic. Notably, Pim1 over-expression failed to promote development and survival of any T-lineage cells other than αß T cells, as we observed complete lack of γδ, NKT, FoxP3(+) T regulatory cells and TCR-ß(+) CD8αα IELs in Pim1(Tg) γc(KO) mice. Collectively, these results uncover distinct requirements for γc signaling between CD4(+) αß T cells and all other T-lineage cells, and they identify Pim1 as a novel effector molecule sufficient to drive CD4(+) αß T-cell development and survival in the absence of γc cytokine receptor signaling.

CD8 lineage-specific regulation of interleukin-7 receptor expression by the transcriptional repressor Gfi1.

Interleukin-7 receptor α (IL-7Rα) is essential for T cell survival and differentiation. Glucocorticoids are potent enhancers of IL-7Rα expression with diverse roles in T cell biology. Here we identify the transcriptional repressor, growth factor independent-1 (Gfi1), as a novel intermediary in glucocorticoid-induced IL-7Rα up-regulation. We found Gfi1 to be a major inhibitory target of dexamethasone by microarray expression profiling of 3B4.15 T-hybridoma cells. Concordantly, retroviral transduction of Gfi1 significantly blunted IL-7Rα up-regulation by dexamethasone. To further assess the role of Gfi1 in vivo, we generated bacterial artificial chromosome (BAC) transgenic mice, in which a modified Il7r locus expresses GFP to report Il7r gene transcription. By introducing this BAC reporter transgene into either Gfi1-deficient or Gfi1-transgenic mice, we document in vivo that IL-7Rα transcription is up-regulated in the absence of Gfi1 and down-regulated when Gfi1 is overexpressed. Strikingly, the in vivo regulatory role of Gfi1 was specific for CD8(+), and not CD4(+) T cells or immature thymocytes. These results identify Gfi1 as a specific transcriptional repressor of the Il7r gene in CD8 T lymphocytes in vivo.

An in vivo IL-7 requirement for peripheral Foxp3+ regulatory T cell homeostasis.

All T cells are dependent on IL-7 for their development and for homeostasis. Foxp3(+) regulatory T cells (Tregs) are unique among T cells in that they are dependent on IL-2. Whether such IL-2 dependency is distinct from or in addition to an IL-7 requirement has been a confounding issue, particularly because of the absence of an adequate experimental system to address this question. In this study, we present a novel in vivo mouse model where IL-2 expression is intact but IL-7 expression was geographically limited to the thymus. Consequently, IL-7 is not available in peripheral tissues. Such mice were generated by introducing a thymocyte-specific IL-7 transgene onto an IL-7 null background. In these mice, T cell development in the thymus, including Foxp3(+) Treg numbers, was completely restored, which correlates with the thymus-specific expression of transgenic IL-7. In peripheral cells, however, IL-7 expression was terminated, which resulted in a general paucity of T cells and a dramatic reduction of Foxp3(+) Treg numbers. Loss of Tregs was further accompanied by a significant reduction in Foxp3(+) expression levels. These data suggest that peripheral IL-7 is not only necessary for Treg survival but also for upregulating Foxp3 expression. Collectively, we assessed the effect of a selective peripheral IL-7 deficiency in the presence of a fully functional thymus, and we document a critical requirement for in vivo IL-7 in T cell maintenance and specifically in Foxp3(+) cell homeostasis.

A locus on chromosome 1 promotes susceptibility of experimental autoimmune myocarditis and lymphocyte cell death.

We previously identified by linkage analysis a region on chromosome 1 (Eam1) that confers susceptibility to experimental autoimmune myocarditis (EAM). To evaluate the role of Eam1, we created a congenic mouse strain, carrying the susceptible Eam1 locus of A.SW on the resistant B10.S background (B10.A-Eam1 congenic) and analyzed three outcomes: 1) the incidence and severity of EAM, 2) the susceptibility of lymph node cells (LNCs) to Cy-enhanced cell death, and 3) susceptibility of lymphocytes to antigen-induced cell death. Incidence of myocarditis in B10.A-Eam1 congenic mice was comparable to A.SW mice, confirming that Eam1 plays an important role in disease development. Caspase 3, 8 and 9 activation in LNCs following Cy treatment and in CD4(+) T cells after immunization with myosin/CFA was significantly lower in A.SW than B10.S mice whereas B10.A-Eam1 congenic mice exhibited an intermediate phenotype. Our results show that Eam1 reduces lymphocyte apoptosis and increases susceptibility to EAM.

Genetic differences in bone marrow-derived lymphoid lineages control susceptibility to experimental autoimmune myocarditis.

Bone marrow (BM) transplantation has been used to study the cellular basis of genetic control of autoimmune diseases, but conclusions remain elusive due to the contradictory findings in different animal models. In the current study, we found that BM cells from myocarditis-susceptible A.SW mice can render irradiated, myocarditis-resistant B10.S recipient mice susceptible to myosin-induced myocarditis, indicating that hematopoietic cells express the genetic differences controlling susceptibility to autoimmune myocarditis. We then sought to differentiate the role of lymphoid vs nonlymphoid components of BM in the pathogenesis of myocarditis by comparing mixed chimeras receiving BM from A.SW wild-type or RAG(-/-) mice mixed with BM from B10.S wild-type mice. This experiment clearly demonstrated that T and B lymphocytes were indispensable for transferring the susceptible phenotype to disease-resistant recipients. Our findings significantly narrow the cellular expression of genetic polymorphisms controlling the EAM phenotype.

Genetic complexity of autoimmune myocarditis.

Autoimmune myocarditis, a chronic stage of myocardial inflammation, occurs in a small subset of patients after acute cardiotropic viral infection and can lead to dilated cardiomyopathy (DCM). This disease can be recapitulated in susceptible mouse strains by infection with coxsackievirus B3, or by immunization with cardiac myosin or cardiac troponin I. The etiologies of myocarditis are multifactorial and genetically complex. Genetic linkage between susceptibility to myocarditis/DCM and the major histocompatibility complex (MHC) genes has been reported in both humans and experimentally induced mouse models. However, unlike other autoimmune diseases, the non-MHC genes seem to have greater impact than MHC genes on disease susceptibility. Several myocarditis-related non-MHC loci have been identified by our laboratory and others in different models. Most of these loci overlap with other autoimmune disease susceptibility loci, suggesting common or shared genetic traits influencing general autoimmunity. For example, we have demonstrated that Eam1 and Eam2 may influence disease susceptibility via regulating T cell apoptosis at different developmental stages. Blockade of signaling through specific genes, such as CTLA4, ICOS and PD-1, can either enhance or prevent the development of experimental autoimmune myocarditis, but it remains unclear whether functional polymorphisms in these genes are involved in predisposition to disease. In humans, mutations/deletions in immunologically important genes such as CD45, and genes encoding cardiac proteins, have been reported in patients with recurrent myocarditis or DCM. Identification of genetic polymorphisms controlling autoimmune myocarditis will help us understand the mechanisms underlying autoimmune diseases in general, thereby improving potential therapies in patients.

Discordant changes in cortical TrkC mRNA and protein during the human lifespan.

Neurotrophin-3 (NT-3) exerts its trophic effects in brain via tyrosine kinase receptor C (trkC) signaling. TrkC splice variants produce receptors with (full-length) and without (truncated) a tyrosine kinase domain. The relative abundance of trkC isoforms and the anatomical localization of trkC in the human prefrontal cortex (PFC) in relationship to development and maturation are currently unknown. We have examined the temporo-spatial expression of trkC protein and mRNA during the development of the human PFC. We have found two major isoforms, a full-length (150 kDa) and a truncated (50 kDa) form of the trkC protein in the human PFC. We report that the full-length form is expressed at low levels throughout development while the truncated form is expressed at moderate levels early in development and increases to reach mature levels by adolescence. In contrast, trkC mRNA levels are uniformly expressed throughout most of postnatal life, but decline in ageing. TrkC protein and mRNA are expressed in both pyramidal and non-pyramidal neurons; additionally, trkC protein is detected in glia and neuropil. Our results suggest that truncated trkC is prevalent in the human PFC and that neurons and glia may be responsive to NT-3 in the PFC throughout life.

Two autoimmune diabetes loci influencing T cell apoptosis control susceptibility to experimental autoimmune myocarditis.

The pathogenesis of immune-mediated myocarditis depends on genetic and environmental factors. To study the genetic mechanisms, we have developed a model of experimental autoimmune myocarditis in the A.SW mouse. Here we provide evidence that loci on murine chromosome 6, and possibly chromosome 1, are involved in regulating susceptibility. Moreover, these loci overlap with loci implicated in other autoimmune diseases including diabetes in the NOD mouse. These two loci also regulate apoptosis in thymocytes as well as peripheral T cells in the NOD mouse, and we report further that A.SW mice demonstrate the same characteristics in apoptosis. These results suggest that common pathogenetic mechanisms involving apoptosis of both thymic and peripheral T cells are shared by multiple autoimmune diseases.