RelB+ Steady-State Migratory Dendritic Cells Control the Peripheral Pool of the Natural Foxp3+ Regulatory T Cells.
ABSTRACT: Thymus-derived natural Foxp3+ CD4+ regulatory T cells (nTregs) play a key role in maintaining immune tolerance and preventing autoimmune disease. Several studies indicate that dendritic cells (DCs) are critically involved in the maintenance and proliferation of nTregs. However, the mechanisms how DCs manage to keep the peripheral pool at constant levels remain poorly understood. Here, we describe that the NF-?B/Rel family transcription factor RelB controls the frequencies of steady-state migratory DCs (ssmDCs) in peripheral lymph nodes and their numbers control peripheral nTreg homeostasis. DC-specific RelB depletion was investigated in CD11c-Cre?×?RelBfl/fl mice (RelBDCko), which showed normal frequencies of resident DCs in lymph nodes and spleen while the subsets of CD103- Langerin- dermal DCs (dDCs) and Langerhans cells but not CD103+ Langerin+ dDC of the ssmDCs in skin-draining lymph nodes were increased. Enhanced frequencies and proliferation rates were also observed for nTregs and a small population of CD4+ CD44high CD25low memory-like T cells (Tml). Interestingly, only the Tml but not DCs showed an increase in IL-2-producing capacity in lymph nodes of RelBDCko mice. Blocking of IL-2 in vivo reduced the frequency of nTregs but increased the Tml frequencies, followed by a recovery of nTregs. Taken together, by employing RelBDCko mice with increased frequencies of ssmDCs our data indicate a critical role for specific ssmDC subsets for the peripheral nTreg and IL-2+ Tml frequencies during homeostasis.
Project description:T cells proliferate vigorously following acute depletion of CD4+ Foxp3+ T regulatory cells [natural Tregs (nTregs)] and also when naive T cells are transferred to syngeneic, nTreg-deficient Rag1 -/- hosts. Here, using mice raised in an antigen-free (AF) environment, we show that proliferation in these two situations is directed to self ligands rather than food or commensal antigens. In both situations, the absence of nTregs elevates B7 expression on host dendritic cells (DCs) and enables a small subset of naive CD4 T cells with high self affinity to respond overtly to host DCs: bidirectional T/DC interaction ensues, leading to progressive DC activation and reciprocal strong proliferation of T cells accompanied by peripheral Treg (pTreg) formation. Likewise, high-affinity CD4 T cells proliferate vigorously and form pTregs when cultured with autologous DCs in vitro in the absence of nTregs: this anti-self response is MHCII/peptide dependent and elicited by the raised level of B7 on cultured DCs. The data support a model in which self tolerance is imposed via modulation of CD28 signaling and explains the pathological effects of superagonistic CD28 antibodies.
Project description:Skin-derived dendritic cells (DCs) are potent antigen-presenting cells with critical roles in both adaptive immunity and tolerance to self. Skin DCs carry antigens and constitutively migrate to the skin-draining lymph nodes (LNs). In mice, Langerin-CD11b- dermal DCs are a low-frequency, heterogeneous, migratory DC subset that traffics to LNs (Langerin-CD11b- migDCs). Here, we build on the observation that Langerin-CD11b- migDCs are Fms-like tyrosine kinase 3 ligand (Flt3L) dependent and strongly Flt3L responsive, which may relate them to classical DCs. Examination of DC capture of FITC from painted skin, DC isolation from skin explant culture, and from the skin of CCR7 knockout mice, which accumulate migDCs, demonstrate these cells are cutaneous residents. Langerin-CD11b- Flt3L-responsive DCs are largely CD24(+) and CX3CR1(low) and can be depleted from Zbtb46-DTR mice, suggesting classical DC lineage. Langerin-CD11b- migDCs present antigen with equal efficiency to other DC subsets ex vivo, including classical CD8? cDCs and Langerin+CD103+ dermal DCs. Finally, transcriptome analysis suggests a close relationship with other skin DCs, and a lineage relationship with other classical DCs. This work demonstrates that Langerin- CD11b- dermal DCs, a previously overlooked cell subset, may be an important contributor to the cutaneous immune environment.
Project description:Langerin is a C-type lectin receptor that recognizes glycosylated patterns on pathogens. Langerin is used to identify human and mouse epidermal Langerhans cells (LCs), as well as migratory LCs in the dermis and the skin draining lymph nodes (DLNs). Using a mouse model that allows conditional ablation of langerin(+) cells in vivo, together with congenic bone marrow chimeras and parabiotic mice as tools to differentiate LC- and blood-derived dendritic cells (DCs), we have revisited the origin of langerin(+) DCs in the skin DLNs. Our results show that in contrast to the current view, langerin(+)CD8(-) DCs in the skin DLNs do not derive exclusively from migratory LCs, but also include blood-borne langerin(+) DCs that transit through the dermis before reaching the DLN. The recruitment of circulating langerin(+) DCs to the skin is dependent on endothelial selectins and CCR2, whereas their recruitment to the skin DLNs requires CCR7 and is independent of CD62L. We also show that circulating langerin(+) DCs patrol the dermis in the steady state and migrate to the skin DLNs charged with skin antigens. We propose that this is an important and previously unappreciated element of immunosurveillance that needs to be taken into account in the design of novel vaccine strategies.
Project description:Langerhans cells (LCs) constitute a subset of dendritic cells (DCs) that express the lectin langerin and that reside in their immature state in epidermis. Paradoxically, in mice permitting diphtheria toxin (DT)-mediated ablation of LCs, epidermal LCs reappeared with kinetics that lagged behind that of their putative progeny found in lymph nodes (LNs). Using bone marrow (BM) chimeras, we showed that a major fraction of the langerin(+), skin-derived DCs found in LNs originates from a developmental pathway that is independent from that of epidermal LCs. This pathway, the existence of which was unexpected, originates in the dermis and gives rise to langerin(+) dermal DCs (DDCs) that should not be confused with epidermal LCs en route to LNs. It explains that after DT treatment, some langerin(+), skin-derived DCs reappear in LNs long before LC-derived DCs. Using CD45 expression and BrdU-labeling kinetics, both LCs and langerin(+) DDCs were found to coexist in wild-type mice. Moreover, DT-mediated ablation of epidermal LCs opened otherwise filled niches and permitted repopulation of adult noninflammatory epidermis with BM-derived LCs. Our results stress that the langerin(+) DC network is more complex than originally thought and have implications for the development of transcutaneous vaccines and the improvement of humanized mouse models.
Project description:The control of peripheral immune responses by FOXP3+ T regulatory (Treg) cells is essential for immune tolerance. However, at any given time, Treg frequencies in whole blood can vary more than fivefold between individuals. An understanding of factors that influence Treg numbers and migration within and between individuals would be a powerful tool for cellular therapies that utilize the immunomodulatory properties of Tregs to control pathology associated with inflammation. We sought to understand how season could influence Treg numbers and phenotype by monitoring the proportion of natural thymus-derived Tregs (nTregs) defined as (CD3+CD4+CD25+FOXP3+CD127-/low ) cells as a proportion of CD4+ T cells and compared these to all FOXP3+ Tregs (allTregs, CD3+CD25+FOXP3+CD127-/low ). We were able to determine changes within individuals during 1 year suggesting an influence of season on nTreg frequencies. We found that, between individuals at any given time, nTreg/CD4+ T cells ranged from 1.8% in February to 8.8% in the summer where median nTreg/CD4 in January and February was 2.4% (range 3.75-1.76) and in July and August was 4.5% (range 8.81-3.17) p = 0.025. Importantly we were able to monitor individual nTreg frequencies throughout the year in donors that started the year with high or low nTregs. Some nTreg variation could be attributed to vitamin D status where normal linear regression estimated that an absolute increase in nTreg/CD4+ by 0.11% could be expected with 10 nmol increase in serum 25 (OH) vitamin D3 (p = 0.005, 95% CI: 0.03-0.19). We assessed migration markers on Tregs for the skin and/or gut. Here cutaneous lymphocyte associated antigen (CLA+) expression on CD25+FOXP3+CD4+/CD4+ was compared with the same population expressing the gut associated integrin, ?7. Gut tropic CD25+FOXP3+?7+Tregs/CD4+ had similar dynamics to nTreg/CD4+. Conversely, CD25+FOXP3+CLA+Tregs/CD4+ showed no association with vitamin D status. Important for cellular therapies requiring isolation of Tregs, the absolute number of ?7+CD4+CD25+FOXP3+Tregs was positively associated with 25(OH)vitamin D3 (R 2 = 0.0208, r = 0.184, p = 0.021) whereas the absolute numbers of CLA+CD4+CD25+FOXP3+Tregs in the periphery were not influenced by vitamin D status. These baseline observations provide new opportunities to utilize seasonal variables that influence Treg numbers and their migratory potential in patients or donors.
Project description:Langerhans cells (LCs) are antigen-presenting cells that reside in the epidermis of the skin and traffic to lymph nodes (LNs). The general role of these cells in skin immune responses is not clear because distinct models of LC depletion resulted in opposite conclusions about their role in contact hypersensitivity (CHS) responses. While comparing these models, we discovered a novel population of LCs that resides in the dermis and does not represent migrating epidermal LCs, as previously thought. Unlike epidermal LCs, dermal Langerin(+) dendritic cells (DCs) were radiosensitive and displayed a distinct cell surface phenotype. Dermal Langerin(+) DCs migrate from the skin to the LNs after inflammation and in the steady state, and represent the majority of Langerin(+) DCs in skin draining LNs. Both epidermal and dermal Langerin(+) DCs were depleted by treatment with diphtheria toxin in Lang-DTREGFP knock-in mice. In contrast, transgenic hLang-DTA mice lack epidermal LCs, but have normal numbers of dermal Langerin(+) DCs. CHS responses were abrogated upon depletion of both epidermal and dermal LCs, but were unaffected in the absence of only epidermal LCs. This suggests that dermal LCs can mediate CHS and provides an explanation for previous differences observed in the two-model systems.
Project description:CCR7 is regarded as an essential chemokine receptor for cutaneous dendritic cell (DC) migration into the regional lymph nodes. However, complete migratory inhibition cannot be obtained in CCR7-deficient mice, suggesting that there exist other chemokine receptors involved in this process. Initially, we found that CXCR4 was highly expressed on migrated cutaneous DCs and that its ligand, CXCL12, was detected in the LYVE-1(+) lymphatic vessels in the skin. FITC-induced cutaneous DC migration into the draining lymph nodes was impaired by the specific CXCR4 antagonist 4-F-Benzoyl-TN14003. Among FITC(+) cells, Langerin(+) Langerhans cells and Langerin(-) (dermal) dDC subsets were detected as CD11c(high+)CD11b(int+) cells and CD11c(high+)CD11b(high+) plus CD11c(low+)CD11b(int+) cells, respectively, both of which were suppressed by CXCR4 antagonist. Moreover, in vivo contact hypersensitivity response was impaired by CXCR4 antagonist administered during the sensitization phase. The in vitro proliferative response to dinitrobenzene sulfonic acid of sensitized lymph node cells was inhibited by CXCR4 antagonist treatment. These findings demonstrated that CXCL12-CXCR4 engagement on cutaneous DCs plays a crucial role in the initiation of skin immune response by enhancing cutaneous DC migration.
Project description:BACKGROUND: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance. PRINCIPAL FINDINGS: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression. CONCLUSION: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.
Project description:The relationship between the TCR repertoires of natural regulatory T cells (nTregs) and conventional CD4(+) T cells (Tconv) capable of responding to the same antigenic epitope is unknown. In this study, we used TCR?-chain transgenic mice to generate polyclonal nTreg and Tconv populations specific for a foreign Ag. CD4(+) T cells from immunized 3.L2?(+/-) TCR?(+/-) Foxp3(EGFP) mice were restimulated in culture to yield nTregs (EGFP(+)) and Tconv (EGFP(-)) defined by their antigenic reactivity. Relative to Tconv, nTreg expansion was delayed, although a higher proportion of viable nTregs had divided after 72 h. Spectratype analysis revealed that both the nTreg and Tconv responses were different and characterized by skewed distributions of CDR3 lengths. CDR3 sequences from nTregs displayed a divergent pattern of J? usage, minimal CDR3 overlap (3.4%), and less diversity than did CDR3 sequences derived from Tconv. These data indicate that foreign Ag-specific nTregs and Tconv are clonally distinct and that foreign Ag-specific nTreg populations are constrained by a limited TCR repertoire.
Project description:OBJECTIVE:To assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model. DESIGN:Investigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13). SETTING:Charité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union). PARTICIPANTS:Recipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9). INTERVENTIONS:CD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×106 cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48. MAIN OUTCOME MEASURES:The primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio. RESULTS:For all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire. CONCLUSIONS:The application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies. TRIAL REGISTRATION:NCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).