Thymic Dendritic Cells Subsets Research Articles

Cellular and molecular mediators of thymic DC homeostasis and activation

Abstract Central tolerance in the thymus ensures that autoreactive thymocytes undergo negative selection or diversion to the regulatory T cell (Treg) lineage. Thymic antigen presenting cells (APCs) such as medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present myriad self-peptides to thymocytes to induce central tolerance. We and others have previously shown that thymic cDCs can be subdivided into activated CCR7+MHC-IIhi and resting CCR7− MHC-IIlo cDC1 and cDC2 subsets with distinct functional properties, suggesting the thymic DC compartment may be more heterogeneous than previously described. Here, we performed single-cell RNA sequencing (scRNAseq) of hematopoietic APCs isolated from adult mouse thymi to comprehensively identify thymic DC subsets with distinct gene expression profiles. Our data reveal 11 transcriptionally distinct cDC subsets. We identify 6 cDC1 subsets, 3 cDC2 subsets, 2 activated CCR7+ “cDC3” subsets distinguished by expression of cDC1 and cDC2 markers, and 4 plasmacytoid DC clusters. Through functional assays, we find that the XCR1+ cDC3s are the most efficient at presenting mTEC-derived peptides on MHC-I and MHC-II, while 2 distinct cDC2 subsets are the most efficient at generating Tregs in vitro. Additional scRNA-seq and flow cytometry analyses of thymic DCs from mice in which thymocyte maturation was blocked at different developmental stages reveal candidate cellular and molecular crosstalk signals that regulate homeostasis and/or activation of pDC and cDC subsets. Our findings reveal hitherto unknown functional heterogeneity within the thymic DC compartment and identify potential interactions that support DC homeostasis and activation which in turn could impact central tolerance. Supported by NIH P01 AI139449-02

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.

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

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

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Thymic-Derived Tolerizing Dendritic Cells Are up-Regulated upon Treatment with Intravenous Immunoglobulin or Splenectomy in a Murine Model of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet counts. ITP has a complex pathogenesis, in which both anti-platelet antibodies as well as T cells have been shown to be important. Initial management of newly diagnosed ITP may be either watchful waiting or pharmacologic intervention, such as glucocorticoids or Intravenous Immunoglobulin (IVIg), a blood product consisting of polyclonal immunoglobulin G (IgG) derived from thousands of donors. Second-line therapy may include dexamethasone, high-dose methylprednisolone, rituximab, thrombopoietin (TPO)-receptor agonists, or splenectomy. The working mechanism of IVIg is actively under investigation and is still a matter of debate, as various different working mechanisms have been suggested. One of them is that IVIg may shift the balance from a pro- to anti-inflammatory state through immunomodulating the activity of dendritic cells (DCs). To gain more insights into the role of DCs in ITP, upon IVIg treatment or splenectomy, we analyzed DC subsets in a murine model of ITP, which features both the antibody and T cell mediated thrombocytopenia. Severe combined immunodeficient (SCID) mice were administrated 4x104 splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 (or naïve control splenocytes) and the mice were treated with or without 1 g/kg IVIg twice a week. Also the same type of splenocytes were transferred into splenectomized SCID mice. Weekly platelet counts were assessed and after 4 weeks the mice were sacrificed and spleen and thymuses were harvested. Splenocytes and thymocytes were isolated and examined by flow cytometry for cross-presenting (XCR1+) and non-presenting tolerizing (SIRP alpha+) DCs. Without IVIg or splenectomy, both splenic DC subset numbers correlated positively with platelet counts and both the thymic DC subset numbers correlated negatively with platelet counts, indicating thymic retention of DC in a setting of thrombocytopenia. Interestingly, splenectomized SCID mice, apart from increased platelet counts, demonstrated a complete reversal of the DC pattern in the thymus, as thymic DC subsets correlated positively with platelet counts in splenectomized mice. Upon IVIg treatment, apart from a general increase in platelet counts, the splenic tolerizing DCs significantly increased in numbers. Moreover, the thymic retention of tolerizing DCs and thus the negative correlation with platelet counts (R2: 0.46, p<0.05) was fully abrogated upon IVIg treatment (R2: 0.02, NS). Overall, our results indicate that both splenectomy as well as IVIg treatment can immunomodulate thymic tolerizing DCs significantly, in a murine model of ITP. DisclosuresNo relevant conflicts of interest to declare.

Expression analysis of surface molecules on human thymic dendritic cells with the 10th HLDA Workshop antibody panel.

Dendritic cells (DC) in the thymus have an important role in the establishment of central tolerance by promoting negative selection of autoreactive T cells and regulatory T-cell differentiation. Whereas human DC have recently been studied in various tissues in more detail, thymic DC subsets are still ill-defined. In the present work, we studied the binding of 71 monoclonal antibodies (mAb) submitted to the HLDA10 workshop to human CD123+ plasmacytoid DC and the two subsets of conventional DC (cDC, CD141+ and CD11b+) isolated from thymus tissue of infants undergoing corrective heart surgery. Within the panel, we found mAb binding to thymic pDC and both cDC subsets (for example, anti-Clec12A, TIM-3, Clec4A, CCR5, Axl, FLT3), but most of them additionally reacted with other thymic cell types. MAb directed to CD85h (ILT1) and the C-type lectin Clec7A (now CD369) reacted selectively with both cDC subsets, but not with other cells. Only one mAb directed to CD85g (ILT7) stained thymic pDC in a highly specific manner. Clec9A (DNGR1, now CD370) was the only tested HLDA10 antigen exclusively expressed on thymic CD141+ cDC. The present report summarizes all data obtained.

Transcriptional priming of intrathymic precursors for dendritic cell development

Specialized dendritic cells (DCs) within the thymus are crucial for the deletion of autoreactive T cells. The question of whether these cells arise from intrathymic precursors with T-cell potential has been hotly debated, and the regulatory pathways and signals that direct their development remain unclear. Here, we compared the gene expression profiles of thymic DC subsets with those of four early thymic precursor subsets: early T-cell precursors (ETPs), double-negative 1c (DN1c), double-negative 1d (DN1d) and double-negative 1e (DN1e) subsets. We found that the DN1d subset expressed Spi-B, HEBCan, Ccr7 and Ccr4, similar to thymic plasmacytoid DCs, whereas the DN1e subset expressed Id2, Ccr7 and Ccr4, similar to thymic conventional DCs. The expression of Ccr7 and Ccr4 in DN1d and DN1e cells suggested that they might be able to migrate towards the medulla (low in Dll proteins) and away from the cortex (high in Dll proteins) where early T-cell development occurs. We therefore assessed the sensitivity of developing DC precursors to Dll-Notch signaling, and found that high levels of Dll1 or Dll4 were inhibitory to DC development, whereas medium levels of Dll4 allowed DC development but not myeloid development. To evaluate directly the lineage potential of the ETP, DN1d and DN1e subsets, we injected them into nonirradiated congenic hosts intrathymically or intravenously, and found that they were all able to form medullary DCs in vivo. Therefore, DN1d and DN1e cells are transcriptionally primed to home to the thymus, migrate into DC-permissive microenvironments and develop into medullary DCs.

Regeneration of dendritic cells in aged mice

Age-related thymic involution causes a decreased output of thymocytes from the thymus, thereby resulting in impairment of T cell-mediated immunity. While alterations in the T cell and non-haematopoietic stromal compartments have been described, the effects of thymic involution on thymic dendritic cells (DC) are not clearly known. Thymic DC play an essential role in shaping T cell-mediated immune responses by deleting self-reactive thymocytes to establish central tolerance and by inducing regulatory T-cell (Treg) development. It is therefore important to assess the prevalence of and alterations to thymic DC with age, as this may impact on their function. We assessed the numbers and proportions of the three distinct subsets of thymic DC in ageing mice, and showed that these subsets are differentially regulated. This is expected as thymic DC subsets have different origins of development. We further assessed the responses of thymic DC in a regenerative environment, such as that induced by sex-steroid ablation (SSA), and clearly showed that, consistent with global thymus regrowth, all three DC populations increased in numbers and regained their relative proportions to thymocytes after an initial lag period. These findings are important for the clinical translation of thymic regenerative approaches, and indicate that SSA facilitates the maintenance of critical processes such as negative selection and Treg induction through promoting thymic DC regeneration.

Absence of CD47 in vivo influences thymic dendritic cell subset proportions but not negative selection of thymocytes

CD47 is a ubiquitously expressed molecule which has been attributed a role in many cellular processes. Its role in preventing cellular phagocytosis has defined CD47 as an obligatory self-molecule providing a 'don't-eat-me-signal'. Additionally, CD47-CD172a interactions are important for cellular trafficking. Yet, the contribution of CD47 to T cell stimulation remains controversial, acting sometimes as a co-stimulator and sometimes as an inhibitor of TCR signalling or peripheral T cell responses. Most of the experiments leading to this controversy have been carried in in vitro systems. Moreover, the role of CD47 on thymocyte differentiation, which precisely relies on TCR signal strength, has not been evaluated. Here, we examine the in vivo role of CD47 in T cell differentiation using CD47-deficient mice. We find that, in the absence of CD47, thymocyte positive and negative selection processes are not altered. Indeed, our data demonstrate that the absence of CD47 does not influence the strength of TCR signalling in thymocytes. Furthermore, in agreement with a role for CD47-CD172a interactions in CD172a(+) dendritic cell migration, we report a reduced proportion of thymic dendritic cells expressing CD172a in CD47-deficient mice. As the total proportion of dendritic cells is maintained, this creates an imbalance in the proportion of CD172a(+) and CD172a(low) dendritic cells in the thymus. Together, these data indicate that the altered proportion of thymic dendritic cell subsets does not have a primordial influence on thymic selection processes.

Ex vivo characterization of human thymic dendritic cell subsets

Interactions between thymic dendritic cells (DC) and thymocytes are critical for proper development of T-cells. We identified human thymic DC populations on the basis of CD123, CD11c and CD14 expression. High levels of CD123 (IL-3R) and CD45RA defined the plasmacytoid DC (pDC) subset. Human thymic CD11c + DC expressed CD45RO and myeloid-related markers (CD13, CD33 and CD11b). CD11c + DC could be separated into two main subsets based on differential expression of CD14: CD11c + CD14 − and CD11c + CD14 + cells. Spontaneous production of IL-10 and IFN γ without exogenous stimulation, was observed in the three DC subsets. Important phenotype modifications were observed in pDC cultures supplemented with IL-3. A down-regulation of CD123 and appearance of myeloid markers such as CD11b and CD11c on CD45RA + cells was noticed within the first 48 h; at a later time there was a shift from CD45RA to CD45RO expression, as well as appearance of CD14 expression. CD11c + cells emerging in pDC culture did not express high levels of HLA-DR, CD83 and co-stimulatory molecules. This suggests an in vitro evolution of human thymic pDC toward a myeloid phenotype found in the CD11c + subset of thymic DC.

Differential susceptibility of human thymic dendritic cell subsets to X4 and R5 HIV-1 infection

Human thymus can be infected by HIV-1 with potential consequences on immune regeneration and homeostasis. We previously showed that CD4 thymocytes preferentially replicate CXCR4 tropic (X4) HIV-1 dependently on interleukin (IL)-7. Here we addressed the susceptibility of thymic dendritic cells (DC) to HIV-1 infection. We investigated the replication ability of CXCR4 or CCR5 (R5) tropic HIV-1 in thymic micro-explants as well as in isolated thymic CD11clowCD14- DC, CD11chighCD14+ DC and plasmacytoid DC subsets. Thymic tissue was productively infected by both X4 and R5 viruses. However, X4 but not R5 HIV-1 replication was enhanced by IL-7 in thymic micro-explants, suggesting that R5 virus replication occurred in cells other than thymocytes. Indeed, we found that R5 HIV-1 replicated efficiently in DC isolated from thymic tissue. The replicative capacity of X4 and R5 viruses differed according to the different DC subsets. R5 but not X4 HIV-1 efficiently replicated in CD11chighCD14+ DC. In contrast, no HIV-1 replication was detected in CD11clowCD14- DC. Both X4 and R5 viruses efficiently replicated in plasmacytoid DC, which secreted interferon-alpha upon HIV-1 exposure. Productive HIV-1 infection also caused DC loss, consistent with different permissivity of each DC subset. Thymic DC sustain high levels of HIV-1 replication. DC might thus be the first target for R5 HIV-1 infection of thymus, acting as a Trojan horse for HIV-1 spread to thymocytes. Furthermore, DC death induced by HIV-1 infection may affect thymopoiesis.

Construction and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virions.

Construction and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virions.