Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Regulatory T cells promote innate inflammation after skin barrier breach via TGF-ß activation.

Regulatory T cells (Tregs) use multiple mechanisms to attenuate inflammation and prevent autoimmunity. Tregs residing in peripheral (i.e., nonlymphoid) tissues have specialized functions; specifically, skin Tregs promote wound healing, suppress dermal fibrosis, facilitate epidermal regeneration, and augment hair follicle cycling. Here, we demonstrated that skin Tregs were transcriptionally attuned to interact with their tissue environment through increased expression of integrin and TGF-ß pathway genes that influence epithelial cell biology. We identified a molecular pathway where skin Tregs license keratinocytes to promote innate inflammation after skin barrier breach. Using a single-cell discovery approach, we identified preferential expression of the integrin αvß8 on skin Tregs Upon skin injury, Tregs used this integrin to activate latent TGF-ß, which acted directly on epithelial cells to promote CXCL5 production and neutrophil recruitment. Induction of this circuit delayed epidermal regeneration but provided protection from Staphylococcus aureus infection across a compromised barrier. Thus, αvß8-expressing Tregs in the skin, somewhat paradoxical to their canonical immunosuppressive functions, facilitated inflammation acutely after loss of barrier integrity to promote host defense against infection.

Regulatory T cells in skin are uniquely poised to suppress profibrotic immune responses.

At the center of fibrosing diseases is the aberrant activation of tissue fibroblasts. The cellular and molecular mechanisms of how the immune system augments fibroblast activation have been described; however, little is known about how the immune system controls fibroblast function in tissues. Here, we identify regulatory T cells (Tregs) as important regulators of fibroblast activation in skin. Bulk cell and single-cell analysis of Tregs in murine skin and lungs revealed that Tregs in skin are transcriptionally distinct and skewed toward T helper 2 (TH2) differentiation. When compared with Tregs in lung, skin Tregs preferentially expressed high levels of GATA3, the master TH2 transcription factor. Genes regulated by GATA3 were highly enriched in skin "TH2 Treg" subsets. In functional experiments, Treg depletion resulted in a preferential increase in TH2 cytokine production in skin. Both acute depletion and chronic reduction of Tregs resulted in spontaneous skin fibroblast activation, profibrotic gene expression, and dermal fibrosis, all of which were exacerbated in a bleomycin-induced murine model of skin sclerosis. Lineage-specific deletion of Gata3 in Tregs resulted in an exacerbation of TH2 cytokine secretion that was preferential to skin, resulting in enhanced fibroblast activation and dermal fibrosis. Together, we demonstrate that Tregs play a critical role in regulating fibroblast activation in skin and do so by expressing a unique tissue-restricted transcriptional program that is mediated, at least in part, by GATA3.

Regulatory T cells in inflammatory skin disease: from mice to humans.

The skin is the largest organ in the body and one of the primary barriers to the environment. In order to optimally protect the host, the skin is home to numerous immune cell subsets that interact with each other and other non-immune cells to maintain organ integrity and function. Regulatory T cells (Tregs) are one of the largest immune cell subsets in skin. They play a critical role in regulating inflammation and facilitating organ repair. In doing so, they adopt unique and specialized tissue-specific functions. In this review, we compare and contrast the role of Tregs in cutaneous immune disorders from mice and humans, with a specific focus on scleroderma, alopecia areata, atopic dermatitis, cutaneous lupus erythematosus and psoriasis.

Adenosine 2a Receptor Signal Blockade of Murine Autoimmune Arthritis via Inhibition of Pathogenic Germinal Center-Follicular Helper T Cells.

OBJECTIVE: CD4 germinal center (GC)-follicular helper T (Tfh) cells are important in the pathogenesis of autoimmune arthritis. Previous studies have shown that adenosine 2a receptor (A2aR; Adora2a) signaling can divert CD4 T cells away from the GC-Tfh cell lineage during the primary response to foreign antigens. This study was undertaken to examine the effects of A2aR signaling on CD4 T cells during the recognition of self antigen in a murine model of autoimmune arthritis. METHODS: Wild-type and Adora2a-deficient mouse KRN T cell receptor-transgenic CD4 T cells specific for glucose-6-phosphate isomerase (GPI)/I-Ag7 were transferred into immunodeficient Tcra-/- I-Ag7 -expressing mice to induce arthritis. Recipients were then treated with either the selective A2aR agonist CGS-21680 (CGS) or phosphate buffered saline alone. Severity of disease, autoantibody titers, KRN T cell numbers and phenotype, and GPI-specific isotype class-switched plasmablasts were tracked. RESULTS: CGS treatment inhibited the development of arthritis and differentiation of KRN GC-Tfh cells, blocked the appearance of high-affinity GPI-specific and IgG1 isotype class-switched polyclonal plasmablasts, and led to a reduction in serum titers of anti-GPI IgG1. In addition, therapeutic administration of CGS after the onset of arthritis blocked further disease progression in association with reductions in the number of KRN GC-Tfh cells and anti-GPI IgG1 serum titers. CONCLUSION: Strong A2aR signaling diverts autoreactive CD4 T cell differentiation away from the GC-Tfh cell lineage, thus reducing help for the differentiation of dangerous autoreactive B cells that promote arthritis. These data in a mouse model of autoimmune arthritis suggest that A2aR and its downstream signaling pathways in CD4 T cells may be promising therapeutic targets for interfering with potentially dangerous autoreactive GC-Tfh cell differentiation.

Say it isn't pso: IL-25 drives skin inflammation.

IL-25 from keratinocytes mediates skin inflammation in psoriasis.

Relationship between CD4 Regulatory T Cells and Anergy In Vivo.

Selective suppression of effector CD4+ T cell functions is necessary to prevent immune cell-mediated damage to healthy tissues. This appears especially true during pregnancy or in individuals predisposed to autoimmunity. Foxp3+ regulatory T (Treg) cells and induction of anergy, an acquired state of T cell functional unresponsiveness in Foxp3- cells, have both been implicated as mechanisms to suppress dangerous immune responses to tissue-restricted self-Ags. Anergic CD4+ T cells and Treg cells share a number of phenotypic and mechanistic traits-including the expression of CD73 and folate receptor 4, and the epigenetic modification of Treg cell signature genes-and an interesting relationship between these two subsets has recently emerged. In this review, we will compare and contrast these two subsets, as well as explore the role of anergy in the generation of peripheral Treg cells.

CD4(+) T cell anergy prevents autoimmunity and generates regulatory T cell precursors.

The role of anergy, an acquired state of T cell functional unresponsiveness, in natural peripheral tolerance remains unclear. In this study, we found that anergy was selectively induced in fetal antigen-specific maternal CD4(+) T cells during pregnancy. A naturally occurring subpopulation of anergic polyclonal CD4(+) T cells, enriched for self antigen-specific T cell antigen receptors, was also present in healthy hosts. Neuropilin-1 expression in anergic conventional CD4(+) T cells was associated with hypomethylation of genes related to thymic regulatory T cells (Treg cells), and this correlated with their ability to differentiate into Foxp3(+) Treg cells that suppressed immunopathology. Thus, our data suggest that not only is anergy induction important in preventing autoimmunity but also it generates the precursors for peripheral Treg cell differentiation.

Cutting edge: type 1 diabetes occurs despite robust anergy among endogenous insulin-specific CD4 T cells in NOD mice.

Insulin-specific CD4(+) T cells are required for type 1 diabetes. How these cells are regulated and how tolerance breaks down are poorly understood because of a lack of reagents. Therefore, we used an enrichment method and tetramer reagents to track insulin-specific CD4(+) T cells in diabetes-susceptible NOD and resistant B6 mice expressing I-A(g7). Insulin-specific cells were detected in both strains, but they only became activated, produced IFN-γ, and infiltrated the pancreas in NOD mice. Unexpectedly, the majority of Ag-experienced cells in NOD mice displayed an anergic phenotype, but this population decreased with age as tolerance was lost. B6 mice expressing I-A(g7) were protected because insulin-specific cells did not become effector or anergic T cells but remained naive. These data suggest that NOD mice promote tolerance through anergy induction, but a small proportion of autoreactive T cells escape anergy to provoke type 1 diabetes.

Murine thymic selection quantified using a unique method to capture deleted T cells.

Thymic positive and negative selection events generate a T-cell repertoire that is MHC restricted and self-tolerant. The number of T cells undergoing positive and negative selection in normal mice has never been firmly established. We generated mice that lack the proapoptotic molecule Bim (bcl2l11) together with a Nur77(GFP) transgene, which allowed the identification and enumeration of T cells that would normally undergo clonal deletion. Using this method, we report the striking observation that six times more cells undergo negative selection than complete positive selection. Seventy-five percent of the negatively selected cells are deleted at the double positive stage in the thymic cortex, compared with 25% at the single positive stage in the medulla. The fact that more thymocytes are highly reactive to MHC than are weakly reactive is inconsistent with a random model of recognition and suggests that T-cell recognition is MHC biased. Furthermore, Bim(-/-) mice had an increased number of GFP(hi) cells in the peripheral lymphoid tissue and a corresponding increase in antigen experienced or anergic cell phenotype. Our data also show that the CD4+ T cells that are clonally deleted experienced only slightly stronger T-cell receptor signaling than those that developed into regulatory T cells.