Factors regulating thymic dendritic cell homeostasis and function

The immune system must balance the generation of a diverse T cell repertoire responsive to various pathogens versus the prevention of tissue destruction by self-antigen-specific T cells. Thymic dendritic cells (DC) are known to eliminate self-antigen-specific thymocytes. Despite this, little is known about the factors regulating thymic DC homeostasis and antigen presentation, which were investigated in this study. In the first part of our study, it was hypothesized that factors within the thymus regulate thymic DC homeostasis. Specifically, the role of CD4+ and CD8+ single-positive thymocytes (SP) was investigated. In mice lacking SP, significantly fewer DC resided in the steady-state thymus, and those DC displayed a less mature phenotype (e.g. reduced expression of major histocompatibility [MHC] and T cell costimulatory molecules) and a markedly reduced T cell stimulatory capacity compared to wild-type thymic DC. When CD4+SP or CD8+SP were individually restored, thymic DC activation status was only recovered under conditions where antigen-specific interactions with SP occurred. During antigen-specific interactions, the restoration of thymic DC activation status depended on CD40 ligand from CD4+SP but not CD8+SP. Thus, thymic DC homeostasis is regulated by antigen-specific interactions with CD4+SP or CD8+SP by distinct molecular mechanisms. To promote thymic tolerance, thymic DC must acquire and present cognate antigens to thymocytes; for example, through "nibbling" of antigens from thymic epithelial cells (TEC). However, the mechanism by which this occurs is largely unknown. Using an in vitro co-culture system, thymic DC nibbling of MHC was investigated. Thymic DC efficiently nibbled MHC from TEC and DC, but not from B cells. It was hypothesized that the cell surface organization of MHC may regulate nibbling by thymic DC. Specifically, the role of lipid rafts was analyzed. Thymic DC acquired lipid rafts from TEC, DC, and B cells. Interestingly, when another B cell surface molecule, immunoglobulin M (IgM), was actively clustered to lipid rafts, nibbling of IgM occurred. These data suggest that cell surface organization of clustered molecules may facilitate acquisition by thymic DC via nibbling. Together, the studies presented herein provide novel insight into the factors regulating thymic DC function and T cell central tolerance.Doctor of Philosoph

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

Thymic dendritic cells (DC) delete self-Ag-specific thymocytes, and drive development of FoxP3-expressing immunoregulatory T cells. Unlike medullary thymic epithelial cells (mTEC), which express and present peripheral self-Ag, DC must acquire self-Ag to mediate thymic negative selection. One such mechanism entails the transfer of surface MHC-self peptide complexes from mTEC 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. Here intercellular MHC transfer by thymic DC subsets was studied using a MHC-mismatch-based in vitro system. Thymic conventional DC (cDC) subsets SIRPα+ and CD8α+ readily acquired MHC class I and II from TEC but plasmacytoid DC (pDC) 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 pDC 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

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 crosstalk 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 antigen expression in the thymus were also immature; notably, however, thymic cDC maturation was reestablished by an Ag-specific cognate interaction with CD4+ or CD8+ single-positive thymocytes (SP). Blockade of CD40 ligand during Ag-specific interactions with CD4SP but not CD8SP limited the effect on cDC maturation. Together, these novel findings demonstrate that homeostatic maturation and function of thymic cDC is regulated by feedback delivered by CD4SP and CD8SP via distinct mechanisms during a cognate Ag-specific interaction

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

Neonatal-derived IL-17 producing dermal gammadelta 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 gammadelta T (Tgammadelta17) cells, from birth resulted in spontaneous, highly penetrant AD with many of the major hallmarks of human AD. In Tgammadelta17 cell-deficient mice, basal keratinocyte transcriptome was altered months in advance of AD induction. Tgammadelta17 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 alphabeta T clonotypes that produce the type three cytokines, IL-17 and IL-22. These results demonstrate that neonatal Tgammadelta17 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

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 antibodies in protecting against recurring infections, but S. aureus modulates the B cell response through expression of Staphylococcal Protein A (SpA), a surface protein that drives polyclonal B cell expansion and induces cell death in the absence of co-stimulation. 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 (LLPCs). The absence of LLPCs was associated with a rapid decline in antigen-specific, class-switched antibody. In contrast, when previously inoculated mice were challenged with isogenic Δspa S. aureus, cells proliferated in the BM survival niches and sustained long-term antibody titers. The effects of SpA on PC fate were limited to the secondary response, as antibody levels and the formation of B cell memory occurred normally during the primary response in mice inoculated with either WT or Δspa S. aureus. Thus, failure to establish long-term protective antibody 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

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 (T1D). The factors that influence the efficacy of thymic negative selection, and the kinetics of thymic output of autoreactive T cells remain ill-defined. We investigated thymic production of β cell-specific T cells using a thymus transplantation model. Thymi from different aged NOD mice representing distinct stages of T1D, were implanted into NOD.scid recipients and the diabetogenicity of the resulting T cell pool examined. Strikingly, the development of diabetes-inducing β 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 β 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 multi-organ 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 to colonic microbiota-responsive T cells in NOD mice

Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes

CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ

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 crosstalk 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 antigen expression in the thymus were also immature; notably, however, thymic cDC maturation was reestablished by an Ag-specific cognate interaction with CD4+ or CD8+ single-positive thymocytes (SP). Blockade of CD40 ligand during Ag-specific interactions with CD4SP but not CD8SP limited the effect on cDC maturation. Together, these novel findings demonstrate that homeostatic maturation and function of thymic cDC is regulated by feedback delivered by CD4SP and CD8SP via distinct mechanisms during a cognate Ag-specific interaction