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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.

The paucity of pathogenic T cells in circulating blood limits the information delivered by bulk analysis. Toward diagnosis and monitoring of treatments of autoimmune diseases, we have devised single-cell analysis approaches capable of identifying and characterizing rare circulating CD4 T cells.

The paucity of pathogenic T cells in circulating blood limits the information delivered by bulk analysis. Toward diagnosis and monitoring of treatments of autoimmune diseases, we have devised single-cell analysis approaches capable of identifying and characterizing rare circulating CD4 T cells.

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.

Ulcerative colitis (UC) is an increasingly prevalent form of inflammatory bowel disease, characterized by chronic T cell mediated inflammation of colonic mucosa. UC is likely caused by a confluence of immune dysregulation, genetics, gut microbiome dysbiosis, and environmental factors. Mucosal associated invariant T (MAIT) cells are a sub-population of CD8+ T cells that recognize microbial derived vitamin B2 based antigens and are a significant population in homeostatic mucosa. MAITs are likely a heterogenous population and a subset of MAITs may “link” microbiome dysbiosis, environmental factors and chronic colonic inflammation. To assess the role of MAIT cells in UC, we have analyzed MAITs isolated from matched colonic biopsies, both uninflamed and inflamed mucosa, and blood from a cohort of UC patients. Utilizing MR1 tetramers and single cell analysis techniques, we confirmed a decrease in MAIT cell frequency in peripheral blood of UC patients relative to healthy donors. We have also found an increase in MAIT frequency in inflamed colonic mucosa of UC patients relative to uninflamed mucosa. Single cell gene expression profiling revealed tissue specific differences between blood MAIT cells and colonic MAIT cells as well as a trend for higher expression of activation and pro-inflammatory genes in MAITs isolated from inflamed mucosa. Most intriguingly, single cell TCR sequencing revealed the expansion of a subset of MAITs in inflamed colonic mucosa with a distinct TCRVb and CDR3b sequence. This suggests that a sub-population of MAIT cells in inflamed colonic mucosa are reacting to unique microbial ligands. A new cohort will extend these findings, but our preliminary results support a role for MAIT cells in the pathology of UC.

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.

According to the Centers for Disease Control and Prevention, one in six children and adolescents in the United States are obese. The obesity epidemic is caused by chronic excess energy intake and low energy expenditure. This energy imbalance creates excess adipose tissue that produces systemic inflammation in the body. Obesity is detrimental to barrier tissues that protect the body from foreign pathogens and environmental stressors. This metabolic disease disrupts normal homeostasis within the epithelium, which then results in complications that include more severe inflammatory bowel disease. We have identified that as obesity progresses, T cell numbers are reduced within the epithelial layer of the intestine and proliferation of these immune cells in the tissue becomes dysfunctional. Interestingly enough, we show that these same T cells are partially restored in number with a low-fat diet. We examined the relationship between obesity onset and the ability of intraepithelial lymphocytes to seed and proliferate in the intestine to maintain homeostasis in the barrier tissue. Here we demonstrate that obesity is particularly devastating in young mice that are still establishing lymphocyte populations in their epithelia. In addition, the long-term effects of chronic obesity from childhood to adulthood shows a dramatic reduction in the number of intraepithelial lymphocytes in the intestine. This study helps shed light on how epithelial barrier maintenance differs in early versus late onset of obesity and will direct research to explore alternative approaches to restore intestinal intraepithelial T cells and promote optimal barrier integrity 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.

Barrier tissues such as the skin and intestine provide vital protection to the organism from pathogens and exposure to the environment. Epithelia-resident γδ T lymphocytes maintain this protective barrier via the production of growth factors and cytokines. Obesity and type 2 diabetes disrupt normal homeostasis within the epithelium and result in complications that include delayed tissue repair. We have previously identified a reduction in the number of T cells within the epithelium of the skin and intestine of mice as obesity progresses. Here we find that obesity impairs the ability of lymphocytes to maintain homeostatic proliferation within epithelial tissues. Our studies show that this is particularly devastating in young mice that are still establishing lymphocyte populations in their epithelia. Using blocking studies and gene chip analyses we have identified inflammatory mediators that are involved in the negative impact that obesity has on intraepithelial lymphocytes. The mechanism involved is independent of diet as both mice on a high fat diet and genetically obese mice on a normal chow diet exhibit reduced and dysfunctional epithelial lymphocytes. Reducing chronic inflammation by weight loss or blocking inflammatory mediators begins to reestablish T cell homeostasis and improve tissue repair. This data identifies new therapeutic targets for improving tissue repair during obesity and type 2 diabetes via the restoration of normal cellular homeostasis and gene expression.

CDC estimate the rate of obesity is on the rise, currently impacting one-third of the U.S. population. Obese patients exhibit side effects that include type 2 diabetes, chronic wounds and an increased vulnerability to infection. It is critical to understand how obesity impacts immune responses that affect these processes and create effective vaccines or antiviral treatments for obesity. We examined PBMC from donors with a lean BMI 30. T cells were studied for responses to influenza virus A/PR/9/38 infection by monitoring upregulation activation markers and cytokine production, using flow cytometry. From these studies we have determined that obese donors have quantifiably fewer γδ T cells, which has implications for rapid immune responses to infection. γδ T cells are normally activated upon contact with infected influenza virus cells, however our findings show that IFN-γ production by γδ T cells is reduced in obese patients. In contrast there was no significant difference in the expression of cytotoxic markers such as granzyme B and NKG2D between lean and obese patients. Due to our finding, IL-2Ra is reduced on the cell surface and IL-2 is able to restore γδ T cell antiviral function,we hypothesize that γδ T cells in obese patients are lacking key T cell specific growth factor signals. Our findings show that gd homeostasis and antiviral function becomes impaired with obesity contributing to the increased susceptibility to infection found in obese subjects.

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.

According to the Center of Disease Control and Prevention, more than one-third of the U.S. adult population is obese. Obesity is caused by overnutrition and accumulation of excess body fat leading to a state of chronic inflammation that impacts the function of epithelial tissues such as the intestine. The intestine consists of villi that increase the overall surface area to digest food and absorb nutrients. The epithelial layer of the villi contains immune cells such as intraepithelial intestinal lymphocytes that are important in maintaining epithelial homeostasis and repairing damaged intestinal tissue. Mice were placed on a high fat diet to define the mechanism by which obesity impacts intestinal epithelia and barrier function. Our data show that obese mice exhibit an increase in the severity of DSS-induced colitis. Using immunofluorescent microscopy we observe an increase in intestinal villi length with a simultaneous reduction in IEL number. The lamina proprial macrophages become skewed toward M1 CD11b+ CD11c+ proinflammatory type macrophages. Short-term diet reduces the severity of colitis for obese mice suggesting that dieting improves intestinal homeostasis in obesity. These data show the detrimental effects of obesity in the small intestine and future studies should focus on finding therapeutic treatments to fully restore IEL number and function to promote optimal intestinal homeostasis and barrier function.

Intraepithelial lymphocytes in tissues such as the skin and intestine play key roles in maintaining barrier integrity via the production of growth factors and cytokines. Obesity and type 2 diabetes induce defects in tissue repair and barrier function causing chronic nonhealing wounds and more severe cases of colitis. Our studies show that as obesity and type 2 diabetes progress, animals exhibit a dramatic decline in the number of epithelial T cells due to impaired T cell homeostatic proliferation. To understand how obesity impacts gene expression in the epithelium, we performed gene chip analysis and qPCR on cells isolated from the epithelium of obese mice. Our studies show that the normal gene expression program in both epithelial cells and intraepithelial T cells becomes disrupted. While T cells exhibit a gene profile indicative of exhaustion with a reduced ability to make growth factors and cytokines, the neighboring epithelial cells downregulate cell adhesion molecules and members of the IL-1 family such as IL-1F6. Returning the animals to a low fat diet does not rapidly restore the ability of T cells to recover their numbers and improve their function. However, blocking the low level of chronic inflammation that occurs with obesity improves T cell function in repair. These data indicate that controlling the myriad of epithelial complications associated with obesity will require turning down chronic inflammation and restoring the normal cellular program in intraepithelial T cells so immune responses at the critical barrier tissues are preserved.

Obesity is an independent risk factor for severe and fatal influenza infection. Little is known about how obesity impacts influenza-specific T cell responses in humans. In healthy adults, γδ T cells are the major circulating population of T cells that rapidly respond to influenza virus infected APCs. The majority express a Vγ9Vδ2 TCR that recognizes phosphoantigens expressed by infected or transformed cells. To determine whether this anti-viral T cell population was impacted by obesity, human peripheral blood mononuclear cells were isolated from non-obese and obese donors and analyzed for homeostatic T cell numbers and function. We found the percentage of Vγ9Vδ2 T cells negatively correlates with increased body mass index (BMI), suggesting the severity of obesity impacts γδ T cell homeostasis. Furthermore, Vγ9Vδ2 T cells exhibit a skewed maturation, with a higher propensity toward a unique γδ T effector CD45RA+ memory cell population. We have identified that obese patients show reduced expression of the homing molecule CCR5 on γδ T cells as well as reduced IFN-γ and TNF-α production in response to influenza infection. This blunted Vγ9Vδ2 T cell response in obese patients is T cell intrinsic as function can be rescued using a strong TCR agonist. Our data show that one arm of the antiviral immune response to influenza is compromised by obesity. These studies support the concept that therapies should be developed to restore immune homeostasis in obese patients.

Within the epidermis of the skin resides a unique population of γδ T lymphocytes that play key roles in skin homeostasis and wound repair. During obesity and related metabolic disease the skin-resident γδ T lymphocytes become dysfunctional due the chronic inflammatory environment. This dysfunction contributes to the non-healing wounds associated with disease. We are now investigating how skin γδ T cell dysfunction impacts normal keratinocyte homeostasis. Interestingly, in lean mice we have determined that the number of keratinocytes in the epidermis is directly proportional to the number of closely neighboring γδ T cells. Once obesity and related metabolic disease ensues, the keratinocyte numbers resemble areas that are devoid of γδ T cells in lean mice and keratinocytes are unable to maintain normal homeostatic numbers. Furthermore, keratinocytes in obese mice exhibit diminished proliferation and accelerated differentiation. This results in a dysregulated epidermis in which fewer basal keratinocytes express the proliferation marker keratin 5 and more exhibit early expression of the differentiation marker keratin 1. Additionally, skin γδ T cells downregulate CD103 in obese mice, while keratinocytes alter expression of its ligand E-cadherin. Together these studies demonstrate that dysfunctional γδ T cells in the epidermis of obese mice contribute to impaired keratinocyte homeostasis ultimately resulting in compromised skin barrier function.

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.