Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

Neonatal-derived IL-17 producing dermal γδ T cells are required to prevent spontaneous atopic dermatitis.

Atopic Dermatitis (AD) is a T cell-mediated chronic skin disease and is associated with altered skin barrier integrity. Infants with mutations in genes involved in tissue barrier fitness are predisposed towards inflammatory diseases, but most do not develop or sustain the diseases, suggesting that there exist regulatory immune mechanisms to prevent aberrant inflammation. The absence of one single murine dermal cell type, the innate neonatal-derived IL-17 producing γδ T (Tγδ17) cells, from birth resulted in spontaneous, highly penetrant AD with many of the major hallmarks of human AD. In Tγδ17 cell-deficient mice, basal keratinocyte transcriptome was altered months in advance of AD induction. Tγδ17 cells respond to skin commensal bacteria and the fulminant disease in their absence was driven by skin commensal bacteria dysbiosis. AD in this model was characterized by highly expanded dermal αß T clonotypes that produce the type three cytokines, IL-17 and IL-22. These results demonstrate that neonatal Tγδ17 cells are innate skin regulatory T cells that are critical for skin homeostasis, and that IL-17 has dual homeostatic and inflammatory function in the skin.

A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3+ Treg cell development.

The neonatal thymus generates Foxp3+ regulatory T (tTreg) cells that are critical in controlling immune homeostasis and preventing multiorgan autoimmunity. The role of antigen specificity on neonatal tTreg cell selection is unresolved. Here we identify 17 self-peptides recognized by neonatal tTreg cells, and reveal ligand specificity patterns that include self-antigens presented in an age- and inflammation-dependent manner. Fate-mapping studies of neonatal peptidyl arginine deiminase type IV (Padi4)-specific thymocytes reveal disparate fate choices. Neonatal thymocytes expressing T cell receptors that engage IAb-Padi4 with moderate dwell times within a conventional docking orientation are exported as tTreg cells. In contrast, Padi4-specific T cell receptors with short dwell times are expressed on CD4+ T cells, while long dwell times induce negative selection. Temporally, Padi4-specific thymocytes are subject to a developmental stage-specific change in negative selection, which precludes tTreg cell development. Thus, a temporal switch in negative selection and ligand binding kinetics constrains the neonatal tTreg selection window.

γδTCR-independent origin of neonatal γδ T cells prewired for IL-17 production.

A classical view of T cell lineages consists of two major clades of T cells expressing either the αß or γδ T cell receptor (TCR). However, genome-wide assessments indicate molecular clusters segregating T cell subsets that are preprogrammed for effector function (innate) from those that mediate conventional adaptive response, regardless of the TCR types. Within this paradigm, γδ T cells remain the prototypic innate-like lymphocytes, many subsets of which are programmed during intrathymic development for committed peripheral tissue localization and effector responses. Emerging evidence for innate γδ T cell lineage choice dictated by developmental gene programs rather than the sensory TCR is discussed in this review.

Interleukin-17-Producing γδ T Cells Originate from SOX13+ Progenitors that Are Independent of γδTCR Signaling.

Lineage-committed αß and γδ T cells are thought to originate from common intrathymic multipotent progenitors following instructive T cell receptor (TCR) signals. A subset of lymph node and mucosal Vγ2+ γδ T cells is programmed intrathymically to produce IL-17 (Tγδ17 cells), however the role of the γδTCR in development of these cells remains controversial. Here we generated reporter mice for the Tγδ17 lineage-defining transcription factor SOX13 and identified fetal-origin, intrathymic Sox13+ progenitors. In organ culture developmental assays, Tγδ17 cells derived primarily from Sox13+ progenitors, and not from other known lymphoid progenitors. Single cell transcriptome assays of the progenitors found in TCR-deficient mice demonstrated that Tγδ17 lineage programming was independent of γδTCR. Instead, generation of the lineage committed progenitors and Tγδ17 cells was controlled by TCF1 and SOX13. Thus, T lymphocyte lineage fate can be prewired cell-intrinsically and is not necessarily specified by clonal antigen receptor signals.

SOX4 controls invariant NKT cell differentiation by tuning TCR signaling.

Natural killer T (NKT) cells expressing the invariant T cell receptor (iTCR) serve an essential function in clearance of certain pathogens and have been implicated in autoimmune and allergic diseases. Complex effector programs of these iNKT cells are wired in the thymus, and upon thymic egress, they can respond within hours of antigenic challenges, classifying iNKT cells as innate-like. It has been assumed that the successful rearrangement of the invariant iTCRα chain is the central event in the divergence of immature thymocytes to the NKT cell lineage, but molecular properties that render the iTCR signaling distinct to permit the T cell lineage diversification remain obscure. Here we show that the High Mobility Group (HMG) transcription factor (TF) SOX4 controls the production of iNKT cells by inducing MicroRNA-181 (Mir181) to enhance TCR signaling and Ca2+ fluxes in precursors. These results suggest the existence of tailored, permissive gene circuits in iNKT precursors for innate-like T cell development.

Thymic Dendritic Cell Subsets Display Distinct Efficiencies and Mechanisms of Intercellular MHC Transfer.

Thymic dendritic cells (DC) delete self-antigen-specific thymocytes, and drive development of Foxp3-expressing immunoregulatory T cells. Unlike medullary thymic epithelial cells, which express and present peripheral self-antigen, DC must acquire self-antigen to mediate thymic negative selection. One such mechanism entails the transfer of surface MHC-self peptide complexes from medullary thymic epithelial cells to thymic DC. Despite the importance of thymic DC cross-dressing in negative selection, the factors that regulate the process and the capacity of different thymic DC subsets to acquire MHC and stimulate thymocytes are poorly understood. In this study intercellular MHC transfer by thymic DC subsets was investigated using an MHC-mismatch-based in vitro system. Thymic conventional DC (cDC) subsets signal regulatory protein α (SIRPα+) and CD8α+ readily acquired MHC class I and II from thymic epithelial cells but plasmacytoid DC were less efficient. Intercellular MHC transfer was donor-cell specific; thymic DC readily acquired MHC from TEC plus thymic or splenic DC, whereas thymic or splenic B cells were poor donors. Furthermore DC origin influenced cross-dressing; thymic versus splenic DC exhibited an increased capacity to capture TEC-derived MHC, which correlated with direct expression of EpCAM by DC. Despite similar capacities to acquire MHC-peptide complexes, thymic CD8α+ cDC elicited increased T cell stimulation relative to SIRPα+ cDC. DC cross-dressing was cell-contact dependent and unaffected by lipid raft disruption of donor TEC. Furthermore, blocking PI3K signaling reduced MHC acquisition by thymic CD8α+ cDC and plasmacytoid DC but not SIRPα+ cDC. These findings demonstrate that multiple parameters influence the efficiency of and distinct mechanisms drive intercellular MHC transfer by thymic DC subsets.

Staphylococcus aureus Protein A Disrupts Immunity Mediated by Long-Lived Plasma Cells.

Infection with Staphylococcus aureus does not induce long-lived protective immunity for reasons that are not completely understood. Human and murine vaccine studies support a role for Abs in protecting against recurring infections, but S. aureus modulates the B cell response through expression of staphylococcus protein A (SpA), a surface protein that drives polyclonal B cell expansion and induces cell death in the absence of costimulation. In this murine study, we show that SpA altered the fate of plasmablasts and plasma cells (PCs) by enhancing the short-lived extrafollicular response and reducing the pool of bone marrow (BM)-resident long-lived PCs. The absence of long-lived PCs was associated with a rapid decline in Ag-specific class-switched Ab. In contrast, when previously inoculated mice were challenged with an isogenic SpA-deficient S. aureus mutant, cells proliferated in the BM survival niches and sustained long-term Ab titers. The effects of SpA on PC fate were limited to the secondary response, because Ab levels and the formation of B cell memory occurred normally during the primary response in mice inoculated with wild-type or SpA-deficient S. aureus mutant. Thus, failure to establish long-term protective Ab titers against S. aureus was not a consequence of diminished formation of B cell memory; instead, SpA reduced the proliferative capacity of PCs that entered the BM, diminishing the number of cells in the long-lived pool.

RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion.

Plasmodium falciparum invasion of host erythrocytes is essential for the propagation of the blood stage of malaria infection. Additionally, the brief extracellular merozoite stage of P. falciparum represents one of the rare windows during which the parasite is directly exposed to the host immune response. Therefore, efficient invasion of the host erythrocyte is necessary not only for productive host erythrocyte infection, but also for evasion of the immune response. Host traits, such as hemoglobinopathies and differential expression of erythrocyte invasion ligands, can protect individuals from malaria by impeding parasite erythrocyte invasion. Here we combine RBC barcoding with flow cytometry to study P. falciparum invasion. This novel high-throughput method allows for the (i) direct comparison of P. falciparum invasion into different erythrocyte populations and (ii) assessment of the impact of changing erythrocyte population dynamics on P. falciparum invasion.

Cutting edge: Antigen-specific thymocyte feedback regulates homeostatic thymic conventional dendritic cell maturation.

Thymic dendritic cells (DC) mediate self-tolerance by presenting self-peptides to and depleting autoreactive thymocytes. Despite a significant role in negative selection, the events regulating thymic DC maturation and function under steady-state conditions are poorly understood. We report that cross-talk with thymocytes regulates thymic conventional DC (cDC) numbers, phenotype, and function. In mice lacking TCR-expressing thymocytes, thymic cDC were reduced and exhibited a less mature phenotype. Furthermore, thymic cDC in TCR-transgenic mice lacking cognate Ag expression in the thymus were also immature; notably, however, thymic cDC maturation was re-established by an Ag-specific cognate interaction with CD4+ or CD8+ single-positive thymocytes (SP). Blockade of CD40L during Ag-specific interactions with CD4 SP, but not CD8 SP, limited the effect on cDC maturation. Together, these novel findings demonstrate that homeostatic maturation and function of thymic cDC are regulated by feedback delivered by CD4 SP and CD8 SP via distinct mechanisms during a cognate Ag-specific interaction.

Thymic development of autoreactive T cells in NOD mice is regulated in an age-dependent manner.

Inefficient thymic negative selection of self-specific T cells is associated with several autoimmune diseases, including type 1 diabetes. The factors that influence the efficacy of thymic negative selection, as well as the kinetics of thymic output of autoreactive T cells remain ill-defined. We investigated thymic production of ß cell-specific T cells using a thymus-transplantation model. Thymi from different aged NOD mice, representing distinct stages of type 1 diabetes, were implanted into NOD.scid recipients, and the diabetogenicity of the resulting T cell pool was examined. Strikingly, the development of diabetes-inducing ß cell-specific CD4(+) and CD8(+) T cells was regulated in an age-dependent manner. NOD.scid recipients of newborn NOD thymi developed diabetes. However, recipients of thymi from 7- and 10-d-old NOD donor mice remained diabetes-free and exhibited a progressive decline in islet infiltration and ß cell-specific CD4(+) and CD8(+) T cells. A similar temporal decrease in autoimmune infiltration was detected in some, but not all, tissues of recipient mice implanted with thymi from NOD mice lacking expression of the autoimmune regulator transcription factor, which develop multiorgan T cell-mediated autoimmunity. In contrast, recipients of 10 d or older thymi lacked diabetogenic T cells but developed severe colitis marked by increased effector T cells reactive to intestinal microbiota. These results demonstrate that thymic development of autoreactive T cells is limited to a narrow time window and occurs in a reciprocal manner compared with colonic microbiota-responsive T cells in NOD mice.