The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8
T cells have been described in RA, their ...function in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8
T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB
CD8
subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK
CD8
memory subpopulation comprises smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB
CD8
T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB
CD8
cells are present in RA synovium. These findings suggest that cytotoxic CD8
T cells targeting citrullinated antigens contribute to synovitis and joint tissue destruction in ACPA+ RA.
While antibodies to citrullinated proteins have become a diagnostic hallmark in rheumatoid arthritis (RA), we still do not understand how the autoimmune T cell response is influenced by these ...citrullinated proteins. To investigate the role of citrullinated antigens in HLA-DR1- and DR4-restricted T cell responses, we utilized mouse models that express these MHC-II alleles to determine the relationship between citrullinated peptide affinity for these DR molecules and the ability of these peptides to induce a T cell response. Using a set of peptides from proteins thought to be targeted by the autoimmune T cell responses in RA, aggrecan, vimentin, fibrinogen, and type II collagen, we found that while citrullination can enhance the binding affinity for these DR alleles, it does not always do so, even when in the critical P4 position. Moreover, if peptide citrullination does enhance HLA-DR binding affinity, it does not necessarily predict the generation of a T cell response. Conversely, citrullinated peptides can stimulate T cells without changing the peptide binding affinity for HLA-DR1 or DR4. Furthermore, citrullination of an autoantigen, type II collagen, which enhances binding affinity to HLA-DR1 did not enhance the severity of autoimmune arthritis in HLA-DR1 transgenic mice. Additional analysis of clonal T cell populations stimulated by these peptides indicated cross recognition of citrullinated and wild type peptides can occur in some instances, while in others cases the citrullination generates a novel T cell epitope. Finally, cytokine profiles of the wild type and citrullinated peptide stimulated T cells unveiled a significant disconnect between proliferation and cytokine production. Altogether, these data demonstrate the lack of support for a simplified model with universal correlation between affinity for HLA-DR alleles, immunogenicity and arthritogenicity of citrullinated peptides. Additionally they highlight the complexity of both T cell receptor recognition of citrulline as well as its potential conformational effects on the peptide:HLA-DR complex as recognized by a self-reactive cell receptor.
It has been hypothesized that HLA class II alleles associated with rheumatoid arthritis (RA) preferentially present self-antigens altered by post-translational modification, such as citrullination. ...To understand the role of citrullination we tested four RA-associated citrullinated epitopes and their corresponding wild-type version for binding to 28 common HLA class II. Binding patterns were variable, and no consistent impact of citrullination was identified. Indeed, in one case citrullination significantly increased binding compared to the WT peptide, in another citrullination was associated with a reduction in promiscuity by 40%. For a more comprehensive analysis, we tested over 200 citrullinated peptides derived from vimentin and collagen II for their capacity to bind the RA-associated shared epitope alleles DRB1*01:01 and DRB1*04:01. The overall effect of citrullination on binding was found to be relatively minor, and only rarely associated with 3-fold increases or decreases in affinity. Previous studies have suggested that citrullination of MHC anchor residues, in particular P4, is associated with generation of novel RA-associated epitopes. However, analysis of the predicted MHC-binding cores of all peptides tested found that in modified peptides with increased binding affinity the citrullinated residue was predicted to occupy an anchor position in only a minority of cases. Finally, we also show that identification of citrullinated peptide binders could be facilitated by using the NetMHCIIpan 3.1 algorithm, representing citrullination as a wildcard. Our studies identify a total of 117 citrullinated peptides that bound RA-associated alleles with an affinity of 1000 nM or better.
(2008) that Def6-/- mice express enhanced amounts of IL-17 (and IL-21), we consistently observe the opposite results. ...MOG-primed Def6-/- mice are resistant to EAE development and display highly ...reduced amounts of IL-17 in the CNS and draining lymph nodes, consistent with defective MOG-peptide-induced proliferation and cytokine (IL-2, IFN-γ, IL-17) production by CD4+ T cells from these mice (unpublished data). Irrespective of the difference in experimental systems used in different studies, the resistance to EAE development puts into question the validity and physiological relevance of the observations in Def6-/- DO11.10 mice. ...although the biology of Def6 may be complex, it is hard for us to see how it could be a negative regulator of IL-17 in Def6trap/trap DO11.10 mice and, at the same time, a positive regulator of IL-17 in Def6-/- C57BL/6 mice (unless, of course, the DO11.10 transgene expression resulted in an artifact).
BACKGROUND Antigen-specific regulation of autoimmune disease is a major goal. In seropositive rheumatoid arthritis (RA), T cell help to autoreactive B cells matures the citrullinated (Cit) ...antigen-specific immune response, generating RA-specific V domain glycosylated anti-Cit protein antibodies (ACPA VDG) before arthritis onset. Low or escalating antigen administration under “sub-immunogenic” conditions favors tolerance. We explored safety, pharmacokinetics, and immunological and clinical effects of s.c. DEN-181, comprising liposomes encapsulating self-peptide collagen II259-273 (CII) and NF-κB inhibitor 1,25-dihydroxycholecalciferol.METHODS A double-blind, placebo-controlled, exploratory, single-ascending-dose, phase I trial assessed the impact of low, medium, and high DEN-181 doses on peripheral blood CII-specific and bystander Cit64vimentin59-71–specific (Cit-Vim–specific) autoreactive T cell responses, cytokines, and ACPA in 17 HLA-DRB1*04:01+ or *01:01+ ACPA+ RA patients on methotrexate.RESULTS DEN-181 was well tolerated. Relative to placebo and normalized to baseline values, Cit-Vim–specific T cells decreased in patients administered medium and high doses of DEN-181. Relative to placebo, percentage of CII-specific programmed cell death 1+ T cells increased within 28 days of DEN-181. Exploratory analysis in DEN-181–treated patients suggested improved RA disease activity was associated with expansion of CII-specific and Cit-Vim–specific T cells; reduction in ACPA VDG, memory B cells, and inflammatory myeloid populations; and enrichment in CCR7+ and naive T cells. Single-cell sequencing identified T cell transcripts associated with tolerogenic TCR signaling and exhaustion after low or medium doses of DEN-181.CONCLUSION The safety and immunomodulatory activity of low/medium DEN-181 doses provide rationale to further assess antigen-specific immunomodulatory therapy in ACPA+ RA.TRIAL REGISTRATION Anzctr.org.au identifier ACTRN12617001482358, updated September 8, 2022.FUNDING Innovative Medicines Initiative 2 Joint Undertaking (grant agreement 777357), supported by European Union’s Horizon 2020 research and innovation programme and European Federation of Pharmaceutical Industries and Associations; Arthritis Queensland; National Health and Medical Research Council (NHMRC) Senior Research Fellowship; and NHMRC grant 2008287.
SLAT (SWAP-70-like adaptor protein of T cells) is an adaptor protein expressed in cells of the hematopoietic system. SLAT interacts with and alters the function of small GTPase Rac1 in fibroblasts. ...In these nonhematopoietic models, the SLAT-Rac interaction leads to changes in F-actin and causes cytoskeletal reorganization. In T cells, SLAT expression regulates the development of T helper cells through Cdc42- and Rac1-mediated activation of the NF-AT transcription factor. Here we show that SLAT is expressed in macrophages. Overexpression of SLAT in a macrophage cell line inhibits the IgG Fcγ receptor-mediated phagocytic ability of THP1 cells. In bone marrow-derived macrophages, SLAT protein is recruited to the early phagosomes formed via Fcγ receptor engagement. SLAT recruitment to the phagosome was most efficient when the macrophages express at least one isoform of Rac (Rac1 or Rac2), because SLAT recruitment was reduced in macrophages of Rac-deficient mice. Macrophages derived from animals lacking SLAT show an elevation in the rate of Fcγ receptor-mediated phagocytosis. The absence of SLAT is associated with an increase in the amount of F-actin formed around these phagosomes as well as an increase in the amount of Rac1 protein recruited to the phagosome. Our results suggest that SLAT acts as a gatekeeper for the amount of Rac recruited to the phagosomes formed by Fcγ receptor engagement and thus is able to regulate F-actin re-organization and consequently phagocytosis.
Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS ...remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4-PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4-PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.
Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of ...RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.
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•A cytoplasmic isoform of RARα (cRARα) is activated by the TCR and binds TCR-ZAP-70•RA counteracts cRARα for suboptimal T cell activation and enhanced Treg conversion•Effective cRARα-TCR signaling induces CRABP2 for RA translocation to the nucleus•Defects in CRABP2 expression reduce anti-pathogen immunity and autoimmune disease
Nuclear retinoic acid receptor alpha (RARα) regulates gene transcription upon the binding of retinoic acid (RA). Larange, Takazawa, Kakugawa, Thiault et al. identify an RARα isoform in the cytoplasm of T cells (cRARα) and reveal that cRARα binds the TCR-ZAP-70 complex, thereby regulating T cell proliferation and effector differentiation. RA counteracts TCR-engaged RARα, resulting in suboptimal TCR activation but enhanced Treg cell differentiation.
The guanine nucleotide exchange factor SLAT (SWAP-70-like adaptor of T cells) regulates T cell activation and differentiation by enabling Ca(2+) release from intracellular stores in response to ...stimulation of the T cell receptor (TCR). We found a TCR-induced association between SLAT and inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1). The N-terminal region of SLAT, which contains two EF-hand motifs that we determined bound Ca(2+), and the SLAT pleckstrin homology (PH) domain independently bound to IP3R1 by associating with a conserved motif within the IP3R1 ligand-binding domain. Disruption of the SLAT-IP3R1 interaction with cell-permeable, IP3R1-based fusion peptides inhibited TCR-stimulated Ca(2+) signaling, activation of the transcription factor NFAT (nuclear factor of activated T cells), and production of cytokines, suggesting that this interaction is required for optimal T cell activation. The finding that SLAT is an IP3R1-interacting protein required for T cell activation suggests that this interaction could be a potential target for a selective immunosuppressive drug.
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