Protein tyrosine phosphatase PTPN22 is dispensable for dendritic cell antigen processing and promotion of T-cell activation by dendritic cells.
ABSTRACT: The PTPN22R620W single nucleotide polymorphism increases the risk of developing multiple autoimmune diseases including type 1 diabetes, rheumatoid arthritis and lupus. PTPN22 is highly expressed in antigen presenting cells (APCs) where the expression of the murine disease associated variant orthologue (Ptpn22R619W) is reported to dysregulate pattern recognition receptor signalling in dendritic cells (DCs) and promote T-cell proliferation. Because T-cell activation is dependent on DC antigen uptake, degradation and presentation, we analysed the efficiency of these functions in splenic and GM-CSF bone marrow derived DC from wild type (WT), Ptpn22-/- or Ptpn22R619W mutant mice. Results indicated no differential ability of DCs to uptake antigen via macropinocytosis or receptor-mediated endocytosis. Antigen degradation and presentation was also equal as was WT T-cell conjugate formation and subsequent T-cell proliferation. Despite the likely presence of multiple phosphatase-regulated pathways in the antigen uptake, processing and presentation pathways that we investigated, we observed that Ptpn22 and the R619W autoimmune associated variant were dispensable. These important findings indicate that under non-inflammatory conditions there is no requirement for Ptpn22 in DC dependent antigen uptake and T-cell activation. Our findings reveal that perturbations in antigen uptake and processing, a fundamental pathway determining adaptive immune responses, are unlikely to provide a mechanism for the risk associated with the Ptpn22 autoimmune associated polymorphism.
Project description:A C1858T single nucleotide polymorphism within PTPN22 (which encodes PTPN22<sup>R620W</sup>) is associated with an enhanced susceptibility to multiple autoimmune diseases including type 1 diabetes and rheumatoid arthritis. Many of the associated autoimmune diseases have an autoantibody component to their pathology. Fc receptors (FcRs) recognise autoantibodies when they bind to autoantigens and form immune complexes. After immune complex binding and receptor crosslinking, FcRs signal via Src and Syk family kinases, leading to antigen uptake, presentation and cytokine secretion. Ptpn22 encodes a protein tyrosine phosphatase that negatively regulates Src and Syk family kinases proximal to immunoreceptor signalling cascades. We therefore hypothesised that PTPN22 regulates immune complex stimulated FcR responses in dendritic cells (DCs). Bone marrow derived DCs (BMDCs) from wild type (WT) or Ptpn22<sup>-/-</sup> mice were pulsed with ovalbumin:anti-ovalbumin immune complexes (ova ICs). Co-culture with WT OT-II T cells revealed that ova IC pulsed Ptpn22<sup>-/-</sup> BMDCs have an enhanced capability to induce T cell proliferation. This was associated with an increased capability of Ptpn22<sup>-/-</sup> BMDCs to present immune complex derived antigens and to form ova IC dependent DC-T cell conjugates. These findings highlight PTPN22 as a regulator of FcR mediated responses and provide a link between the association of PTPN22<sup>R620W</sup> with autoantibody associated autoimmune diseases.
Project description:It is becoming clear that ionizing radiation positively influences certain immune parameters, which opens the possibility for combining radio- and immunotherapies in cancer treatment. The presence of functionally competent dendritic cells (DCs) is crucial in mounting a successful antitumor immune response. While it has been shown that DCs are relatively radioresistant, few and contradictory data are available on how ionizing radiation alters the functional integrity of these cells. Therefore, our objective was to investigate the effect of whole-body irradiation on the function of splenic DCs. C57Bl/6 mice were irradiated with 0.1, 0.25, and 2 Gy X-rays and changes in the phenotype of splenic DCs were compared to unirradiated controls. An increase was seen in DC surface markers influencing DC-T cell interactions. In vivo cytokine production was determined by direct intracellular cytokine staining. Irradiation with 2 Gy induced a 1.6-fold increase in IL-1? production, while the combination of irradiation and lipopolysaccharide (LPS) treatment induced a 3.9-fold increase, indicating a strong synergism between irradiation and LPS stimulation. Interaction of DCs with effector and regulatory T cells was investigated in a mixed lymphocyte reaction. While DCs from control animals induced stronger proliferation of regulatory T cells, DCs from animals irradiated with 2 Gy induced stronger proliferation of effector T cells. Antigen uptake and presentation was investigated by measuring the capacity of DCs to internalize and present ovalbumine (OVA)-derived peptides on their major histocompatibility complex (MHCI) molecules. Irradiation with 2 Gy did not influence antigen uptake or presentation, while low doses stimulated antigen uptake and reduced the level of antigen presentation. In conclusion, high-dose in vivo irradiation induced increased expression of T cell costimulatory markers, enhanced production of proinflammatory cytokines and a stronger stimulation of effector T cell proliferation than that of regulatory T cells. However, it did not influence DC antigen uptake or presentation. On the other hand, low-dose irradiation increased antigen uptake and lowered antigen presentation of DCs, indicating that low- and high-dose irradiation act on different pathways in DCs.
Project description:Dendritic cells (DCs) are specialized antigen presenting cells that instruct T cell responses through sensing environmental and inflammatory danger signals. Maintaining the homeostasis of the multiple functionally distinct conventional dendritic cells (cDC) subsets that exist <i>in vivo</i> is crucial for regulating immune responses, with changes in numbers sufficient to break immune tolerance. Using <i>Ptpn22</i> <sup>-/-</sup> mice we demonstrate that the phosphatase PTPN22 is a highly selective, negative regulator of cDC2 homeostasis, preventing excessive population expansion from as early as 3 weeks of age. Mechanistically, PTPN22 mediates cDC2 homeostasis in a cell intrinsic manner by restricting cDC2 proliferation. A single nucleotide polymorphism, PTPN22<sup>R620W</sup>, is one of the strongest genetic risk factors for multiple autoantibody associated human autoimmune diseases. We demonstrate that cDC2 are also expanded in mice carrying the orthologous PTPN22<sup>619W</sup> mutation. As a consequence, cDC2 dependent CD4<sup>+</sup> T cell proliferation and T follicular helper cell responses are increased. Collectively, our data demonstrate that PTPN22 controls cDC2 homeostasis, which in turn ensures appropriate cDC2-dependent T cell responses under antigenic challenge. Our findings provide a link between perturbations in DC development and susceptibility to a broad spectrum of PTPN22<sup>R620W</sup> associated human autoimmune diseases.
Project description:An exclusive feature of dendritic cells (DCs) is their ability to cross-present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen-antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8?+ DCs showed sustained MHC class I-peptide specific CD8+ T-cell activation for more than 4 days. CD8?- DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8?- DCs were able to present antigen in MHC class II to specific CD4+ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T-cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T-cell activation and have distinct roles in antigen presentation to specific T cells in vivo.
Project description:To date, the intracellular signaling pathways involved in dendritic cell (DC) function are poorly understood. The antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been shown to affect maturation, function, and subsequent DC-mediated T cell responses of murine and human DCs. In experimental autoimmune encephalomyelitis (EAE), as prototype animal model for a T helper cell-mediated autoimmune disease, antigen presentation, cytokine production, and costimulation by DCs play a major role. We explore the role of Nrf2 in DC function, and DC-mediated T cell responses during T cell-mediated autoimmunity of the central nervous system using genetic ablation and pharmacological activation in mice and men to corroborate our data in a translational setting. In murine and human DCs, monomethyl fumarate induced Nrf2 signaling inhibits DC maturation and DC-mediated T cell proliferation by reducing inflammatory cytokine production and expression of costimulatory molecules. In contrast, Nrf2-deficient DCs generate more activated T helper cells (Th1/Th17) but fewer regulatory T cells and foster T cell proliferation. Transfer of DCs with Nrf2 activation during active EAE reduces disease severity and T cell infiltration. Our data demonstrate that Nrf2 signaling modulates autoimmunity in murine and human systems <i>via</i> inhibiting DC maturation and function thus shedding further light on the mechanism of action of antioxidative stress pathways in antigen-presenting cells.
Project description:Apoptotic cell death of Dendritic cells (DCs) is critical for immune homeostasis. Although intrinsic mechanisms controlling DC death have not been fully characterized up to now, experimentally enforced inhibition of DC-death causes various autoimmune diseases in model systems. We have generated mice deficient for Protein Phosphatase with EF-Hands 2 (Ppef2), which is selectively expressed in CD8+ DCs, but not in other related DC subtypes such as tissue CD103+ DCs. Ppef2 is down-regulated rapidly upon maturation of DCs by toll-like receptor stimuli, but not upon triggering of CD40. Ppef2-deficient CD8+ DCs accumulate the pro-apoptotic Bcl-2-like protein 11 (Bim) and show increased apoptosis and reduced competitve repopulation capacities. Furthermore, Ppef2 -/- CD8+ DCs have strongly diminished antigen presentation capacities in vivo, as CD8+ T cells primed by Ppef2 -/- CD8+ DCs undergo reduced expansion. In conclusion, our data suggests that Ppef2 is crucial to support survival of immature CD8+ DCs, while Ppef2 down-regulation during DC-maturation limits T cell responses.
Project description:The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103(+) dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3(+) dermal DCs (thought to be equivalent to murine CD103(+) dermal DCs) and DC-SIGN(+) DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3(+) dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3(+) dermal DCs and DC-SIGN(+) cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.
Project description:Multiple autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus, are associated with an allelic variant of protein tyrosine phosphatase nonreceptor 22 (PTPN22), which encodes the protein LYP. To model the human disease-linked variant LYP-R620W, we generated knockin mice expressing the analogous mutation, R619W, in the murine ortholog PEST domain phosphatase (PEP). In contrast with a previous report, we found that this variant exhibits normal protein stability, but significantly alters lymphocyte function. Aged knockin mice exhibited effector T cell expansion and transitional, germinal center, and age-related B cell expansion as well as the development of autoantibodies and systemic autoimmunity. Further, PEP-R619W affected B cell selection and B lineage-restricted variant expression and was sufficient to promote autoimmunity. Consistent with these features, PEP-R619W lymphocytes were hyperresponsive to antigen-receptor engagement with a distinct profile of tyrosine-phosphorylated substrates. Thus, PEP-R619W uniquely modulates T and B cell homeostasis, leading to a loss in tolerance and autoimmunity.
Project description:Dendritic cells (DCs) process and present self and foreign antigens to induce tolerance or immunity. In vitro models suggest that induction of immunity is controlled by regulating the presentation of antigen, but little is known about how DCs control antigen presentation in vivo. To examine antigen processing and presentation in vivo we specifically targeted antigens to the two major subsets of DCs using chimeric monoclonal antibodies. Unlike CD8+ DCs that express the cell surface protein CD205, CD8- DCs, which are positive for the 33D1 antigen, are specialized for presentation on MHC class II. This difference in antigen processing is intrinsic to the DC subsets and associated with increased expression of proteins associated with MHC processing. Experiment Overall Design: This study includes data from cell sort purified dendritic cells, B cells and CD4 and CD8 T cells. The genearray was performed to identify the transmembrane molecule recognized by the antibody 33D1. The antibody 33D1 binds specifically to CD8-CD11cHigh DCs in the spleen. Therfore the data set was reduced in this way that all molecules that are expressed either in CD8=CD11cHigh DCs, B cells and T cells were diminished of the CD8+CD11cHigh DC data set. This Genearray was also used to analyze MHC class I and MHC class II associated moelcules as the DC subsets differ in the antigen presentation. Each Series consists of 3 individuall samples
Project description:Inhibitory/repressor-receptors are upregulated significantly on activated T cells, and have been the molecules of attention as targets for inducing immune tolerance. Induction of effective antigen specific tolerance depends on concurrent engagement of the TCR and one or more of these inhibitory receptors. Here, we show, for the first time that dendritic cells (DCs) can be efficiently engineered to express multiple T cell inhibitory ligands, and enhanced engagement of T cell inhibitory receptors, upon antigen presentation, by these DCs can induce effective CD4+ T cell tolerance and suppress autoimmunity. Compared to control DCs, antigen presentation by DCs that ectopically express CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1, and HVEM-CRD1 respectively) individually (mono-ligand DCs) or in combination (multi-ligand DCs) causes an inhibition of CD4+ T cell proliferation and pro-inflammatory cytokine response, as well as increase in Foxp3+ Treg frequency and immune regulatory cytokine production. Administration of self-antigen (mouse thyroglobulin; mTg) loaded multi-ligand DCs caused hyporesponsiveness to mTg challenge, suppression of autoantibody production, and amelioration of experimental autoimmune thyroiditis. Overall, this study shows that engineered DC-directed enhanced concurrent activation of multiple T cell coinhibitory pathways is an effective way to induce self-antigen specific T cell tolerance to suppress ongoing autoimmunity.