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Obesity impacts over 30% of the United States population, resulting in a wide array of complications. Included among these is the deterioration of the intestinal barrier, which has been implicated in type 2 diabetes and susceptibility to bacterial transepithelial migration. The intestinal epithelium is maintained by αβ and γδ intraepithelial T lymphocytes, which migrate along the epithelia, support epithelial homeostasis, and protect from infection. In this study, we investigate how obesity impacts intraepithelial lymphocyte (IEL) persistence and function in intestinal homeostasis and repair. Mice were fed a high-fat diet to induce obesity and to study immunomodulation in the intestine. There is a striking reduction in αβ and γδ IEL persistence as obesity progresses with a different mechanism in αβ versus γδ IEL populations. CD4+ and CD4+CD8+ αβ intraepithelial T lymphocytes exhibit reduced homeostatic proliferation in obesity, whereas both αβ and γδ IELs downregulate CD103 and CCR9. The reduction in intraepithelial T lymphocytes occurs within 7 wk of high-fat diet administration and is not dependent on chronic inflammation via TNF-α. Young mice administered a high-fat diet upon weaning exhibit the most dramatic phenotype, showing that childhood obesity has consequences on intestinal IEL seeding. Together, this dysfunction in the intestinal epithelium renders obese mice more susceptible to dextran sulfate sodium–induced colitis. Diet-induced weight loss restores IEL number and CD103/CCR9 expression and improves outcome in colitis. Together, these data confirm that obesity has immunomodulatory consequences in intestinal tissues that can be improved with weight loss.

The difficulty of studying small tissue samples and rare cell populations have been some of the main limitations in performing efficient translational studies of immune mediated diseases. Many of these conditions are grouped under the name of a single disease whilst there are strong suggestions that disease heterogeneity leads to variable disease progression as well as therapeutic responses. The recent development of single cell techniques, such as single cell RNA sequencing, gene expression profiling, or multiparametric cytometry, is likely to be a turning point. Single cell approaches provide researchers the opportunity to finally dissect disease pathology at a level that will allow mechanistic classifications and precision therapeutic strategies. In this review, we will give an overview of the current and developing repertoire of single cell techniques, the benefits and limitations of each, and provide an example of how single cell techniques can be utilized to understand complex immune mediated diseases and their translation from mouse to human.

The class II region of the major histocompatibility complex (MHC) locus is the main contributor to the genetic susceptibility to type 1 diabetes (T1D). The loss of an aspartic acid at position 57 of diabetogenic HLA-DQβ chains supports this association; this single amino acid change influences how TCRs recognize peptides in the context of HLA-DQ8 and I-Ag7 using a mechanism termed the P9 switch. Here, we built register-specific insulin peptide MHC tetramers to examine CD4+ T cell responses to Ins12-20 and Ins13-21 peptides during the early prediabetic phase of disease in nonobese diabetic (NOD) mice. A single-cell analysis of anti-insulin CD4+ T cells performed in 6- and 12-week-old NOD mice revealed tissue-specific gene expression signatures. TCR signaling and clonal expansion were found only in the islets of Langerhans and produced either classical TH1 differentiation or an unusual Treg phenotype, independent of TCR usage. The early phase of the anti-insulin response was dominated by T cells specific for Ins12-20, the register that supports a P9 switch mode of recognition. The presence of the P9 switch was demonstrated by TCR sequencing, reexpression, mutagenesis, and functional testing of TCRαβ pairs in vitro. Genetic correction of the I-Aβ57 mutation in NOD mice resulted in the disappearance of D/E residues in the CDR3β of anti-Ins12-20 T cells. These results provide a mechanistic molecular explanation that links the characteristic MHC class II polymorphism of T1D with the recognition of islet autoantigens and disease onset.

The difficulty of studying small tissue samples and rare cell populations have been some of the main limitations in performing efficient translational studies of immune mediated diseases. Many of these conditions are grouped under the name of a single disease whilst there are strong suggestions that disease heterogeneity leads to variable disease progression as well as therapeutic responses. The recent development of single cell techniques, such as single cell RNA sequencing, gene expression profiling, or multiparametric cytometry, is likely to be a turning point. Single cell approaches provide researchers the opportunity to finally dissect disease pathology at a level that will allow mechanistic classifications and precision therapeutic strategies. In this review, we will give an overview of the current and developing repertoire of single cell techniques, the benefits and limitations of each, and provide an example of how single cell techniques can be utilized to understand complex immune mediated diseases and their translation from mouse to human.

The class II region of the Major Histocompatibility locus is the main contributor to the genetic susceptibility to type 1 diabetes (T1D). The loss of an aspartic acid at position 57 of diabetogenic HLA-DQβ chains supports this association; it influences the recognition of peptides in the context of HLA-DQ8, and I-A(g7) using a mechanism termed the P9 switch. Here, we built register-specific insulin MHC tetramers, Ins(12-20) and Ins(13-21), to examine anti-insulin CD4 T cell responses during the early pre-diabetic phase of disease in mice. A single cell gene expression analysis and single cell T cell receptor (TCR) sequencing of anti-insulin CD4 T cells performed in 6 and 12-week-old NOD mice, revealed tissue-specific gene expression signatures. TCR signaling and clonal expansion were found only in the islets of Langerhans. and produced either classical Th1 differentiation or an unusual Treg phenotype, independent of TCR usage. The early phase of the anti-insulin response was dominated by cells specific for Ins(12-20), the register that supports a P9 switch mode of recognition. The presence of the switch was demonstrated by TCR sequencing, re-expression, mutagenesis, and functional testing of αβ TCR pairs in vitro. The genetic correction of the β57 mutation resulted in the disappearance of D/E residues in the CDR3β of anti-Ins(12-20) T cells, and inability of cells normally activated by a switch to recognize the Ins(9-23) peptide. These results provide the first molecular mechanistic explanation that links the unique MHC class II polymorphism of T1D with the recognition of islet antigens and disease onset.

The nature of the endogenous ligands for natural killer T (NKT) cells has been debated for more than a decade. Because the mammalian glycosylceramide synthases are invertases, it is believed that in mammals all glycosylceramides are β anomers. However, the possibility that an alternative enzymatic pathway, an unfaithful enzyme, or unique physico-chemical environments could allow the production of small quantities of α anomers should be entertained. Classic biochemical and chemical analysis approaches are not well suited for this challenge as they lack sensitivity. Using a combination of biological assays and new technological approaches, we have unequivocally demonstrated that α glycosylceramides were constitutively produced by mammalian immune cells, loaded onto CD1d and presented to NKT cells both in the thymus and in the periphery. Their amount is controlled tightly by catabolic enzymes, and can be altered in vitro and in vivo to modify NKT cell behavior.

BACKGROUND Mammalian target of rapamycin (mTOR) inhibitors are approved to prevent allograft rejection and control malignancy. Unfortunately, they are associated with adverse effects, such as wound healing complications that detract from more extensive use. There is a lack of prospective wound healing studies to monitor patients treated with mTOR inhibitors, such as everolimus or sirolimus, especially in nondiabetics. METHODS Patients receiving everolimus with standard immunosuppressant therapy or standard immunosuppressant therapy without everolimus were administered 3-mm skin biopsy punch wounds in the left scapular region. Homeostatic gene expression was examined in the skin obtained from the biopsy and wound surface area was examined on day 7. Peripheral blood mononuclear cells were examined for cytokine production. RESULTS There are no significant changes in autophagy related 13, epidermal growth factor, insulin-like growth factor binding protein 3, IL-2, kruppel-like factor 4, and TGFB1 gene expression in the skin suggesting that there is little impact of everolimus on these genes within nonwounded skin. Peripheral blood T cells are more sensitive to cell death in everolimus-treated patients, but they retain the ability to produce proinflammatory cytokines required for efficient wound repair. Importantly, there is no delay in the closure of biopsy wounds in patients receiving everolimus as compared to those not receiving mTOR inhibition. CONCLUSIONS Everolimus treatment is not associated with impaired closure of skin biopsy wounds in kidney transplant recipients. These data highlight the importance of exploring whether larger surgical wounds would show a similar result and how other factors, such as diabetes, impact wound healing complications associated with mTOR suppression.

Obese patients are susceptible to increased morbidity and mortality associated with infectious diseases such as influenza A virus. γδ T cells and memory αβ T cells play key roles in reducing viral load by rapidly producing IFN-γ and lysing infected cells. In this article we analyze the impact of obesity on T lymphocyte antiviral immunity. Obese donors exhibit a reduction in γδ T cells in the peripheral blood. The severity of obesity negatively correlates with the number of γδ T cells. The remaining γδ T cells have a skewed maturation similar to that observed in aged populations. This skewed γδ T cell population exhibits a blunted antiviral IFN-γ response. Full γδ T cell function can be restored by potent stimulation with 1-Hydroxy-2-methyl-buten-4yl 4-diphosphate (HDMAPP), suggesting that γδ T cells retain the ability to produce IFN-γ. Additionally, γδ T cells from obese donors have reduced levels of IL-2Rα. IL-2 is able to restore γδ T cell antiviral cytokine production, which suggests that γδ T cells lack key T cell specific growth factor signals. These studies make the novel finding that the γδ T cell antiviral immune response to influenza is compromised by obesity. This has important implications for the development of therapeutic strategies to improve vaccination and antiviral responses in obese patients.

Epithelial cells provide an initial line of defense against damage and pathogens in barrier tissues such as the skin; however this balance is disrupted in obesity and metabolic disease. Skin gammadelta T cells recognize epithelial damage, and release cytokines and growth factors that facilitate wound repair. We report here that hyperglycemia results in impaired skin gammadelta T cell proliferation due to altered STAT5 signaling, ultimately resulting in half the number of gammadelta T cells populating the epidermis. Skin gammadelta T cells that overcome this hyperglycemic state are unresponsive to epithelial cell damage due to chronic inflammatory mediators, including TNFalpha. Cytokine and growth factor production at the site of tissue damage was partially restored by administering neutralizing TNFalpha antibodies in vivo. Thus, metabolic disease negatively impacts homeostasis and functionality of skin gammadelta T cells, rendering host defense mechanisms vulnerable to injury and infection.

The major gammadelta T cell subset in the human peripheral blood expresses the Vgamma9delta2 TCR and recognizes non-peptidic prenyl pyrophosphate antigens such as isopentylpyrophosphate (IPP). Upon activation the gammadelta T cells rapidly secrete antiviral cytokines similar to classical memory alphabeta T cells. Here we have investigated the ability of gammadelta T lymphocytes from human PBMC to become activated by influenza A virus infection. Vgamma9Vdelta2 T lymphocytes rapidly upregulate expression of CD25 and CD69 and produce IFN-gamma following influenza infection of PBMC. Moreover, the recognition is cross-reactive between various subtypes of influenza, but not with vaccinia virus. Vgamma9Vdelta2 T cell responses are potently reduced by the HMG-CoA reductase inhibitor mevastatin, which inhibits the mevalonate pathway and IPP synthesis. Our results indicate that influenza virus infection induces the rapid activation and function of Vgamma9Vdelta2 T lymphocytes in the peripheral blood via a mechanism that depends on the mevalonate pathway.

SummaryGlycosylceramides in mammalian species are thought to be present in the form of β-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylceramide synthase, both β-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.

Skin complications and chronic non-healing wounds are common in obesity, metabolic disease, and type 2 diabetes. Epidermal γδ T cells normally produce keratinocyte growth factors, participate in wound repair, and are necessary for keratinocyte homeostasis. We have determined that in γδ T cell-deficient mice, there are reduced numbers of keratinocytes and the epidermis exhibits a flattened, thinner structure with fewer basal keratinocytes. This is important in obesity, where skin-resident γδ T cells are reduced and rendered dysfunctional. Similar to γδ T cell-deficient mice, keratinocytes are reduced and the epidermal structure is altered in two obese mouse models. Even in regions where γδ T cells are present, there are fewer keratinocytes in obese mice, indicating that dysfunctional γδ T cells are unable to regulate keratinocyte homeostasis. The impact of absent or impaired γδ T cells on epidermal structure is exacerbated in obesity as E-cadherin localization and expression are additionally altered. These studies reveal that γδ T cells are unable to regulate keratinocyte homeostasis in obesity and that the obese environment further impairs skin structure by altering cell–cell adhesion. Together, impaired keratinocyte homeostasis and epidermal barrier function through direct and indirect mechanisms result in susceptibility to skin complications, chronic wounds, and infection.