Circulating c-Met-Expressing Memory T Cells Define Cardiac Autoimmunity.

BACKGROUND: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)-expressing (c-Met+) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. METHODS: In-depth phenotyping of peripheral blood T cells, including c-Met+ T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. RESULTS: We show that c-Met+ T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met+ T cells are distinct from those of c-Met-negative (c-Met-) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met+ T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met+ T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met+ T cells. CONCLUSIONS: Our study demonstrates that the detection of circulating c-Met+ T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.

The low-density lipoprotein receptor-mTORC1 axis coordinates CD8+ T cell activation.

Activation of T cells relies on the availability of intracellular cholesterol for an effective response after stimulation. We investigated the contribution of cholesterol derived from extracellular uptake by the low-density lipoprotein (LDL) receptor in the immunometabolic response of T cells. By combining proteomics, gene expression profiling, and immunophenotyping, we described a unique role for cholesterol provided by the LDLR pathway in CD8+ T cell activation. mRNA and protein expression of LDLR was significantly increased in activated CD8+ compared to CD4+ WT T cells, and this resulted in a significant reduction of proliferation and cytokine production (IFNγ, Granzyme B, and Perforin) of CD8+ but not CD4+ T cells from Ldlr -/- mice after in vitro and in vivo stimulation. This effect was the consequence of altered cholesterol routing to the lysosome resulting in a lower mTORC1 activation. Similarly, CD8+ T cells from humans affected by familial hypercholesterolemia (FH) carrying a mutation on the LDLR gene showed reduced activation after an immune challenge.

Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells.

Voltage-gated hydrogen channel 1 (Hvcn1) is a voltage-gated proton channel, which reduces cytosol acidification and facilitates the production of ROS. The increased expression of this channel in some cancers has led to proposing Hvcn1 antagonists as potential therapeutics. While its role in most leukocytes has been studied in depth, the function of Hvcn1 in T cells remains poorly defined. We show that Hvcn1 plays a nonredundant role in protecting naive T cells from intracellular acidification during priming. Despite sharing overall functional impairment in vivo and in vitro, Hvcn1-deficient CD4+ and CD8+ T cells display profound differences during the transition from naive to primed T cells, including in the preservation of T cell receptor (TCR) signaling, cellular division, and death. These selective features result, at least in part, from a substantially different metabolic response to intracellular acidification associated with priming. While Hvcn1-deficient naive CD4+ T cells reprogram to rescue the glycolytic pathway, naive CD8+ T cells, which express high levels of this channel in the mitochondria, respond by metabolically compensating mitochondrial dysfunction, at least in part via AMPK activation. These observations imply heterogeneity between adaptation of naive CD4+ and CD8+ T cells to intracellular acidification during activation.

A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer.

Protective immunity relies upon differentiation of T cells into the appropriate subtype required to clear infections and efficient effector T cell localization to antigen-rich tissue. Recent studies have highlighted the role played by subpopulations of tissue-resident memory (TRM) T lymphocytes in the protection from invading pathogens. The intestinal mucosa and associated lymphoid tissue are densely populated by a variety of resident lymphocyte populations, including αß and γδ CD8+ intraepithelial T lymphocytes (IELs) and CD4+ T cells. While the development of intestinal γδ CD8+ IELs has been extensively investigated, the origin and function of intestinal CD4+ T cells have not been clarified. We report that CCR9 signals delivered during naïve T cell priming promote the differentiation of a population of α4ß7+ IFN-γ-producing memory CD4+ T cells, which displays a TRM molecular signature, preferentially localizes to the gastrointestinal (GI) tract and associated lymphoid tissue and cannot be mobilized by remote antigenic challenge. We further show that this population shapes the immune microenvironment of GI tissue, thus affecting effector immunity in infection and cancer.

Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation.

Cholesterol homeostasis has a pivotal function in regulating immune cells. Here we show that apolipoprotein E (apoE) deficiency leads to the accumulation of cholesterol in the cell membrane of dendritic cells (DC), resulting in enhanced MHC-II-dependent antigen presentation and CD4+ T-cell activation. Results from WT and apoE KO bone marrow chimera suggest that apoE from cells of hematopoietic origin has immunomodulatory functions, regardless of the onset of hypercholesterolemia. Humans expressing apoE4 isoform (ε4/3-ε4/4) have increased circulating levels of activated T cells compared to those expressing WT apoE3 (ε3/3) or apoE2 isoform (ε2/3-ε2/2). This increase is caused by enhanced antigen-presentation by apoE4-expressing DCs, and is reversed when these DCs are incubated with serum containing WT apoE3. In summary, our study identifies myeloid-produced apoE as a key physiological modulator of DC antigen presentation function, paving the way for further explorations of apoE as a tool to improve the management of immune diseases.

Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis.

Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene-leading to increased GCK activity-had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration.

Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4+ T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals.

Low-grade systemic inflammation associated to obesity leads to cardiovascular complications, caused partly by infiltration of adipose and vascular tissue by effector T cells. The signals leading to T cell differentiation and tissue infiltration during obesity are poorly understood. We tested whether saturated fatty acid-induced metabolic stress affects differentiation and trafficking patterns of CD4+ T cells. Memory CD4+ T cells primed in high-fat diet-fed donors preferentially migrated to non-lymphoid, inflammatory sites, independent of the metabolic status of the hosts. This was due to biased CD4+ T cell differentiation into CD44hi-CCR7lo-CD62Llo-CXCR3+-LFA1+ effector memory-like T cells upon priming in high-fat diet-fed animals. Similar phenotype was observed in obese subjects in a cohort of free-living people. This developmental bias was independent of any crosstalk between CD4+ T cells and dendritic cells and was mediated via direct exposure of CD4+ T cells to palmitate, leading to increased activation of a PI3K p110δ-Akt-dependent pathway upon priming.

CD31 signals confer immune privilege to the vascular endothelium.

Constitutive resistance to cell death induced by inflammatory stimuli activating the extrinsic pathway of apoptosis is a key feature of vascular endothelial cells (ECs). Although this property is central to the maintenance of the endothelial barrier during inflammation, the molecular mechanisms of EC protection from cell-extrinsic, proapoptotic stimuli have not been investigated. We show that the Ig-family member CD31, which is expressed by endothelial but not epithelial cells, is necessary to prevent EC death induced by TNF-α and cytotoxic T lymphocytes in vitro. Combined quantitative RT-PCR array and biochemical analysis show that, upon the engagement of the TNF receptor with TNF-α on ECs, CD31 becomes activated and, in turn, counteracts the proapoptotic transcriptional program induced by TNF-α via activation of the Erk/Akt pathway. Specifically, Akt activation by CD31 signals prevents the localization of the forkhead transcription factor FoxO3 to the nucleus, thus inhibiting transcription of the proapoptotic genes CD95/Fas and caspase 7 and de-repressing the expression of the antiapoptotic gene cFlar. Both CD31 intracellular immunoreceptor tyrosine-based inhibition motifs are required for its prosurvival function. In vivo, CD31 gene transfer is sufficient to recapitulate the cytoprotective mechanisms in CD31(-) pancreatic ß cells, which become resistant to immune-mediated rejection when grafted in fully allogeneic recipients.

Hepatocyte Growth Factor Receptor c-Met Instructs T Cell Cardiotropism and Promotes T Cell Migration to the Heart via Autocrine Chemokine Release.

Effector-T-cell-mediated immunity depends on the efficient localization of antigen-primed lymphocytes to antigen-rich non-lymphoid tissue, which is facilitated by the expression of a unique set of "homing" receptors acquired by memory T cells. We report that engagement of the hepatocyte growth factor (HGF) receptor c-Met by heart-produced HGF during priming in the lymph nodes instructs T cell cardiotropism, which was associated with a specialized homing "signature" (c-Met(+)CCR4(+)CXCR3(+)). c-Met signals facilitated T cell recruitment to the heart via the chemokine receptor CCR5 by inducing autocrine CCR5 ligand release. c-Met triggering was sufficient to support cardiotropic T cell recirculation, while CCR4 and CXCR3 sustained recruitment during heart inflammation. Transient pharmacological blockade of c-Met during T cell priming led to enhanced survival of heart, but not skin, allografts associated with impaired localization of alloreactive T cells to heart grafts. These findings suggest c-Met as a target for development of organ-selective immunosuppressive therapies.

Metabolic regulation of regulatory T cell development and function.

It is now well established that the effector T cell (Teff) response is regulated by a series of metabolic switches. Quiescent T cells predominantly require adenosine triphosphate-generating processes, whereas proliferating Teff require high metabolic flux through growth-promoting pathways, such as glycolysis. Pathways that control metabolism and immune cell function are intimately linked, and changes in cell metabolism at both the cell and system levels have been shown to enhance or suppress specific T cell effector functions. Furthermore, functionally distinct T cell subsets require distinct energetic and biosynthetic pathways to support their specific functional needs. In particular, naturally occurring regulatory T cells (Treg) are characterized by a unique metabolic signature distinct to that of conventional Teff cells. We here briefly review the signaling pathways that control Treg metabolism and how this metabolic phenotype integrates their differentiation and function. Ultimately, these metabolic features may provide new opportunities for the therapeutic modulation of unwanted immune responses.

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells.

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr(29). Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.

Self-recognition of the endothelium enables regulatory T-cell trafficking and defines the kinetics of immune regulation.

Localization of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells to lymphoid and non-lymphoid tissue is instrumental for the effective control of immune responses. Compared with conventional T cells, Treg cells constitute a minute fraction of the T-cell repertoire. Despite this numeric disadvantage, Tregs efficiently migrate to sites of immune responses reaching an optimal number for the regulation of T effector (Teff) cells. The array and levels of adhesion and chemokine receptor expression by Tregs do not explain their powerful migratory capacity. Here we show that recognition of self-antigens expressed by endothelial cells in target tissue is instrumental for efficient Treg recruitment in vivo. This event relies upon IFN-γ-mediated induction of MHC-class-II molecule expression by the endothelium and requires optimal PI3K p110δ activation by the T-cell receptor. We also show that, once in the tissue, Tregs inhibit Teff recruitment, further enabling a Teff:Treg ratio optimal for regulation.

Enhanced and aberrant T cell trafficking following total body irradiation: a gateway to graft-versus-host disease?

Pre-transplant conditioning regimens play a major role in triggering graft-versus-host disease (GVHD). This study investigated the effect of irradiation on donor T cell trafficking to lymphoid and non-lymphoid tissues by comparing the migration of carboxy-fluorescein diacetate succinimidyl ester-labelled, naïve donor T lymphocytes in vivo in irradiated and non-irradiated syngeneic mice recipients. Recruitment of adoptively transferred naïve T cells to secondary lymphoid organs was increased in irradiated mice and naïve T cells also aberrantly localized to non-lymphoid tissues. Irradiation also induced aberrant effector memory T cell migration into lymph nodes and their localization to homing-privileged non-lymphoid sites, such as the gut. The presence of a minor histocompatibility mismatch further enhanced the aberrant accumulation of T cells in both lymphoid and non-lymphoid tissue, whilst their migratory pattern was not modified as compared to fully matched irradiated recipients. These effects correlated with decreased permeability of, and the secretion of chemotactic factors by the endothelium. Our findings are consistent with the possibility that excessive, dysregulated extravasation of T cells induced by irradiation promotes the development of GVHD.

Distinct in vivo CD8 and CD4 T cell responses against normal and malignant tissues.

Normal tissue and tumour grafts expressing the same alloantigens often elicit distinct immune responses whereby only normal tissue is rejected. To investigate the mechanisms that underlie these distinct outcomes, we compared the responses of adoptively transferred HY-specific conventional (CD8 and CD4) or regulatory T (Treg) cells in mice bearing HY-expressing tumour, syngeneic male skin graft or both. For local T cell priming, T cell re-circulation, graft localization and retention, skin grafts were more efficient than tumours. Skin grafts were also capable of differentiating CD4 T cells into functional Th1 cells. Donor T cell responses were inversely correlated with tumour progression. When skin graft and tumour transplants were performed sequentially, contemporary graft and tumour burden enhanced CD8 but reduced CD4 T cell responses causing accelerated skin-graft rejection without influencing tumour growth. Although both skin grafts and tumours were able to expand HY-specific Treg cells in draining lymph node (dLN), the proportion of tumour-infiltrating Treg cells was significantly higher than that within skin grafts, correlating with accelerated tumour growth. Moreover, there was a higher level of HY antigen presentation by host APC in tumour-dLN than in graft-dLN. Finally, tumour tissues expressed a significant higher level of IDO, TGFß, IL10 and Arginase I than skin grafts, indicating that malignant but not normal tissue represents a stronger immunosuppressive environment. These comparisons provide important insight into the in vivo mechanisms that conspire to compromise tumour-specific adaptive immunity and identify new targets for cancer immunotherapy.

Suppression of the allogeneic response by the anti-allergy drug N-(3,4-dimethoxycinnamonyl) anthranilic acid results from T-cell cycle arrest.

Previously we have shown that indoleamine 2,3-dioxygenase (IDO) and the tryptophan metabolite, 3-hydroxykynurenine (3HK) can prolong corneal allograft survival. IDO modulates the immune response by depletion of the essential amino acid tryptophan by breakdown to kynurenines, which themselves act directly on T lymphocytes. The tryptophan metabolite analogue N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA, 'Tranilast') shares the anthranilic acid core with 3HK. Systemic administration of DAA to mice receiving a fully MHC-mismatched allograft of cornea or skin resulted in significant delay in rejection (median survival of controls 12 days, 13 days for cornea and skin grafts, respectively, and of treated mice 24 days (P < 0.0001) and 17 days (P < 0.03), respectively). We provide evidence that DAA-induced suppression of the allogeneic response, in contrast to that induced by tryptophan metabolites, was a result of cell cycle arrest rather than T-cell death. Cell cycle arrest was mediated by up-regulation of the cell cycle-specific inhibitors p21 and p15, and associated with a significant reduction in interleukin-2 production, allowing us to characterize a novel mechanism for DAA-induced T-cell anergy. Currently licensed as an anti-allergy drug, the oral bioavailability and safe therapeutic profile of DAA make it a candidate for the prevention of rejection of transplanted cornea and other tissues.

The T-cell receptor is not hardwired to engage MHC ligands.

The bias of αß T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.

Arginine depletion as a mechanism for the immune privilege of corneal allografts.

The cornea is an immune privileged tissue. Since arginase has been found to modulate T-cell function by depleting arginine, we investigated the expression of arginase in the cornea and its possible role in immune privilege using a murine transplant model. We found that both the endothelium and epithelium of murine corneas express functional arginase I, capable of down-regulating T-cell proliferation in an in vitro culture system. The administration of the specific arginase inhibitor N-hydroxy-nor-L-Arg to recipient mice resulted in an accelerated rejection of allogeneic C57BL/6 (B6) corneal grafts. In contrast, in vivo blockade of arginase activity had no effect in altering the course of rejection of primary skin grafts that express little, if any, arginase. In addition, the inhibition of arginase did not alter systemic T-cell proliferation. These data show that arginase is functional in the cornea and contributes to the immune privilege of the eye, and that modulation of arginase contributes to graft survival.

Molecular mechanisms of induction of antigen-specific allograft tolerance by intranasal peptide administration.

We have previously shown that intranasal (i.n.) administration of a single MHC class II-restricted HY peptide to female mice induces tolerance to up to five additional epitopes expressed on test male grafts, a phenomenon known as linked suppression. In this study, we investigated the molecular mechanisms involved both in the induction phase following peptide administration and during linked suppression after grafting. We report that following initial i.n. administration, peptide is widely disseminated and is presented by functionally immature dendritic cells. These fail to cause optimal stimulation of the responding HY-specific CD4(+) T cells that express genes characteristic of regulatory T cells. Following i.n. peptide plus LPS administration, causing immunization, HY-specific CD4(+) T cells express genes characteristic of activated T cells. We further find that following male skin grafting, HY-specific CD8(+) T cells from peptide-treated tolerant mice display both quantitative and qualitative differences compared with similar cells from untreated mice that reject their grafts. In tolerant mice there are fewer HY-specific CD8(+) cells and they express several genes characteristic of exhausted T cells. Furthermore, associated with specific chemokine receptor and integrin expression, HY-specific CD8(+) T cells show more limited migration from the graft draining lymph node into other tissues.

Functional plasticity of antigen-specific regulatory T cells in context of tumor.

Although polyclonal regulatory T cells (Tregs) that once expressed Foxp3 (ex-Tregs) derived from Foxp3(+) Tregs have been described in homeostatic and autoimmune settings, little is known regarding the influence of the tumor environment on ex-Treg development. After adoptive transfer of HY-specific green Tregs (peripheral or thymic) to Rag2(-/-) B6 female mice bearing syngeneic HY-expressing MB49 tumors, a significant fraction rapidly lost expression of Foxp3. On the second transfer to a Rag2(-/-) B6 male environment, these ex-Tregs expanded strongly, whereas Tregs that maintained expression of Foxp3 expression did not. Both FACS and quantitative real-time-PCR analysis revealed that ex-Tregs upregulated genes characteristic of a Th1 effector-memory phenotype including IFN-γ and downregulated a panel of Treg-specific genes. Peripheral HY-specific green Tregs were adoptively transferred to Rag2(-/-) B6 male mice, to dissect the factors regulating ex-Treg differentiation. Development of ex-Tregs was more efficient in the mesenteric lymph node (mLN) than peripheral lymph node environment, correlating with a much greater level of IL-6 mRNA in mLN. In addition, the preferential development of ex-Tregs in mLN was significantly impaired by cotransfer of HY-specific naive CD4 T cells. Collectively, our study not only demonstrates the plasticity of Ag-specific Tregs in the context of the tumor environment, but also defines key molecular and cellular events that modulate ex-Treg differentiation.