POS0367 NLRC4 AND FC-γ-R CROSSTALK ON CD1C+ DENDRITIC CELLS DIFFERENTIALLY CONTRIBUTES TO RHEUMATOID ARTHRITIS IMMUNOPATHOLOGY

Background:Rheumatoid arthritis (RA) is an autoimmune disorder in which Th17 cells, B cells and inflammatory cytokines (1-3) contribute to joint tissue damage, however the role of specific myeloid populations to immunopathogenesis of RA remains unclear.Objectives:To address this question, we studied transcriptional, phenotypical and functional characteristics of monocytes (Mo), CD1c+ and CD141+ conventional dendritic cells (cDC) from RA patients.Methods:Frequencies and maturation patterns of Lin-CD14-HLADR+ plasmacytoid (CD11c-), CD1c+ and CD141+ cDC (CD11c+) subsets and CD14+ Mo from n=25 RA patients at baseline were analyzed by multicolor flow cytometry. In addition, longitudinal studies on the evolution of these populations after treatment initiation were conducted on a smaller group of RA patients. Moreover, CD1c+ and CD141+ cDC subsets and total Mo were sorted from the peripheral blood from n=4 untreated RA and healthy individuals and the synovial fluid from n=3 RA and chondrocalcinosis patients. Differential transcriptional patterns within each population were analyzed by RNAseq. Functional validation of targets were performed in vitro with cDC subsets isolated form the synoviual fluid of RA patients. Finally, silencing of expression of NLRC4 and NLRP3 on CD1c+cDCs was performed with specific siRNAs.Results:Both CD1c+ (p=0.0001) and CD141+ (p=0.0008) cDCs were significantly depleted from the blood and enriched in the synovial fluid from untreated RA patients, but proportions of CD1c+ cDCs were more significantly recovered after treatment initiation and associated with improved clinical parameters. In addition, specific increased expression levels of the IgG-Fc receptor CD64 on CD1c+ cDC was associated with higher DAS28 (p=0.0002). Moreover, differential transcriptional patterns of circulating CD1c+cDCs from RA patients were characterized by genes linked to toll-like receptor, Fc-receptor, inflammasome pathways and elevated CCR2 expression (p=0.016), while CD141+cDCs transcribed interferon-related genes. Importantly, CCR2+ CD64Hi CD1c+cDCs from the synovial fluid from RA patients transcribed proinflammatory cytokines such as IL1-β, CCL3 and IL-8, actively expressed the inflammasome mediator caspase 1 and were more effective activating pathogenic IFNγ+IL-17+ CD4+ T cells in vitro than CD141+ cDC (p=0.0019). These functional profiles could be artificially induced stimulating CD1c+ cDCs with dsDNA in the presence of IgGs and was dependent on caspase 1 and the NLRC4 inflammasome.Conclusion:Our data provides novel insights about specific activation and functional patterns on CD1c+cDC contributing to RA pathogenesis and identifies new sensors that could represent novel therapeutic target to treat RA.References:[1]Alvandpur N, Tabatabaei R, Tahamoli-Roudsari A, Basiri Z, Behzad M, Rezaeepoor M, et al. Circulating IFN-gamma producing CD4+ T cells and IL-17A producing CD4+ T cells, HLA-shared epitope and ACPA may characterize the clinical response to therapy in rheumatoid arthritis patients. Human immunology. 2020.[2]Nistala K, Adams S, Cambrook H, Ursu S, Olivito B, de Jager W, et al. Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(33):14751-6.[3]Chapuy-Regaud S, Nogueira L, Clavel C, Sebbag M, Vincent C, Serre G. IgG subclass distribution of the rheumatoid arthritis-specific autoantibodies to citrullinated fibrin. Clinical and experimental immunology. 2005;139(3):542-50.Disclosure of Interests:None declared

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Comparison of In Vitro and In Vivo Effects of Infliximab on Cytokine Production in Proliferating CD4+ T Cells in Patients with Rheumatoid Arthritis

Clinical Anti-Inflammatory & Anti-Allergy Drugs ◽

10.2174/2212703802666150127001412 ◽

2015 ◽

Vol 1 (2) ◽

pp. 122-128

Author(s):

Syuichi Koarada ◽

Yuri Sadanaga ◽

Natsumi Nagao ◽

Satoko Tashiro ◽

Rie Suematsu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

Cd4 T Cells ◽

Cytokine Production

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THU0046 A PIPELINE TO STUDY ANTIGEN-SPECIFIC CD4+ T CELLS IN RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.1093 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 235.1-236

Author(s):

R. Kumar ◽

N. Yoosuf ◽

C. Gerstner ◽

S. Turcinov ◽

K. Chemin ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Tenascin C ◽

Tetramer Staining ◽

Tcr Repertoire ◽

Citrullinated Antigen

Background:Autoimmunity to citrullinated autoantigens forms a critical component of disease pathogenesis in rheumatoid arthritis (RA). Presence of anti-citrullinated protein antibodies (ACPAs) in patients has high diagnostic value. Recently, several citrullinated antigen specific CD4+T cells have been described. However, detailed studies of their T-cell receptor usage and in-vivo profile suffer from the disadvantage that these cells are present at very low frequencies. In this context, we here present a pipeline for TCR repertoire analysis of antigen-specific CD4+T cells from RA patients, including both citrulline and influenza (control) specificities using in-vitro peptide challenge induced-cell expansion.Objectives:To enable studies of the T cell repertoire of citrullinated antigen-specific CD4+T cells in rheumatoid arthritisMethods:Peripheral blood mononuclear cells (PBMCs) (n=7) and synovial fluid mononuclear cells (SFMCs) (n=5) from HLA-DR*0401-postive RA patients were cultured in the presence of citrullinated Tenascin C peptide cocktails or influenza peptides (positive control). Citrulline reactive cells were further supplemented with recombinant human IL-15 and IL-7 on day 2. All cultures were replenished with fresh medium on day 6 and rIL-2 was added every 2 days from then. Assessment of proportion of peptide-HLA-tetramer positive cells was performed using flow cytometry whereby individual antigen-specific CD4+T cells were sorted into 96-well plates containing cell lysis buffer, followed by PCR-based alpha/beta TCR sequencing. TCR sequencing data was demultiplexed and aligned for TCR gene usage using MiXCR. Some tetramer positive cells were sorted into complete medium containing human IL-2 and PHA for expansion of antigen-specific cells. Cells were supplemented with irradiated allogenic PBMCs (30 times number of antigen specific cells). Clones of antigen specific CD4+T cells were further subjected to tetramer staining to confirm expansion of cells.Results:As evidenced by increase in frequency of tetramer positive CD4+T cells, in vitro peptide stimulation resulted in expansion of both influenza specific (Fig. 1a) and citrullinated antigen specific (Fig. 1b) CD4+T cells. Polyclonal in-vitro expansion of tenascin C tetramer positive sorted cells followed by tetramer staining further confirmed antigen specificity and enrichment for antigen specific CD4+T cells after polyclonal stimulation (Fig.1c). TCR repertoire analysis in PB and SF dataset from the first patient showed clonal expansion of influenza specific cells in both sites. Synovial fluid had more diversity of expanding clones as compared to paired PB, with few expanded clones being shared among SF and PB. We observed a more diverse TCR repertoire in citrulline specific CD4+T cells. We also observed sharing of TCR alpha chains among different citrulline specific CD4+T cell clones.Fig. 1In-vitroexpansion of antigen specific CD4+T cells:Conclusion:This method provides a highly suitable approach for investigating TCR specificities of antigen specific CD4+T cells under conditions of low cell yields. Building on this dataset will allow us to assess specific features of TCR usage of autoreactive T cells in RA.PBMCs were cultured in presence of (a) influenza (HA, MP54) and (b) citrullinated tenascin peptides. The proportion of antigen specific CD4+T cells was assessed using HLA-class II tetramer staining. We observed an increase in frequency of (a) Infleunza specific cells (red dots in upper left and lower right quadrants) and (b) citrullinated tenascin C specific cells (red dots in lower right quadrant), at day 13 post culture as compared to day 3. (c) Sorting of citrullinated tenascin specific CD4+T cells, followed by PHA expansion resulted in visible increase in proportion of citrullinated tenascin specific CD4+T cells.Disclosure of Interests:Ravi kumar: None declared, Niyaz Yoosuf: None declared, Christina Gerstner: None declared, Sara Turcinov: None declared, Karine Chemin: None declared, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica

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Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.

Journal of Experimental Medicine ◽

10.1084/jem.176.5.1431 ◽

1992 ◽

Vol 176 (5) ◽

pp. 1431-1437 ◽

Cited By ~ 156

Author(s):

M Croft ◽

D D Duncan ◽

S L Swain

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cd4 Cells ◽

Peptide Antigen ◽

Naive T Cells ◽

Naïve T Cells ◽

Antigen Presenting

Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.

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Contact-dependent delivery of IL-2 by dendritic cells to CD4 T cells in the contraction phase promotes their long-term survival

Protein & Cell ◽

10.1007/s13238-019-00662-0 ◽

2019 ◽

Vol 11 (2) ◽

pp. 108-123

Author(s):

Dan Tong ◽

Li Zhang ◽

Fei Ning ◽

Ying Xu ◽

Xiaoyu Hu ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Cd4 T Cell ◽

Immune Memory ◽

Term Survival

Abstract Common γ chain cytokines are important for immune memory formation. Among them, the role of IL-2 remains to be fully explored. It has been suggested that this cytokine is critically needed in the late phase of primary CD4 T cell activation. Lack of IL-2 at this stage sets for a diminished recall response in subsequent challenges. However, as IL-2 peak production is over at this point, the source and the exact mechanism that promotes its production remain elusive. We report here that resting, previously antigen-stimulated CD4 T cells maintain a minimalist response to dendritic cells after their peak activation in vitro. This subtle activation event may be induced by DCs without overt presence of antigen and appears to be stronger if IL-2 comes from the same dendritic cells. This encounter reactivates a miniature IL-2 production and leads a gene expression profile change in these previously activated CD4 T cells. The CD4 T cells so experienced show enhanced reactivation intensity upon secondary challenges later on. Although mostly relying on in vitro evidence, our work may implicate a subtle programing for CD4 T cell survival after primary activation in vivo.

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Inflamed joints of patients with rheumatoid arthritis contain T cells that display in vitro proliferation to antigens present in autologous synovial fluid functional analysis on the basis of synovial-fluid-reactive T-cell clones and lines

Human Immunology ◽

10.1016/0198-8859(94)90028-0 ◽

1994 ◽

Vol 40 (4) ◽

pp. 291-298 ◽

Cited By ~ 16

Author(s):

Pieter C.M. Res ◽

Linda Struijk ◽

Angela Leow ◽

Mohammed R. Daha ◽

Peter C. van den Elsen ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Functional Analysis ◽

T Cells ◽

T Cell ◽

Synovial Fluid ◽

In Vitro Proliferation ◽

T Cell Clones ◽

Cell Clones

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Modification of host dendritic cells by microchimerism-derived extracellular vesicles generates split tolerance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618364114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 1099-1104 ◽

Cited By ~ 23

Author(s):

William Bracamonte-Baran ◽

Jonathan Florentin ◽

Ying Zhou ◽

Ewa Jankowska-Gan ◽

W. John Haynes ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Cd4 T Cells ◽

Extracellular Vesicle ◽

Membrane Microdomains ◽

Transplant Tolerance ◽

Split Tolerance ◽

Cross Dressing

Maternal microchimerism (MMc) has been associated with development of allospecific transplant tolerance, antitumor immunity, and cross-generational reproductive fitness, but its mode of action is unknown. We found in a murine model that MMc caused exposure to the noninherited maternal antigens in all offspring, but in some, MMc magnitude was enough to cause membrane alloantigen acquisition (mAAQ; “cross-dressing”) of host dendritic cells (DCs). Extracellular vesicle (EV)-enriched serum fractions from mAAQ+, but not from non-mAAQ, mice reproduced the DC cross-dressing phenomenon in vitro. In vivo, mAAQ was associated with increased expression of immune modulators PD-L1 (programmed death-ligand 1) and CD86 by myeloid DCs (mDCs) and decreased presentation of allopeptide+self-MHC complexes, along with increased PD-L1, on plasmacytoid DCs (pDCs). Remarkably, both serum EV-enriched fractions and membrane microdomains containing the acquired MHC alloantigens included CD86, but completely excluded PD-L1. In contrast, EV-enriched fractions and microdomains containing allopeptide+self-MHC did not exclude PD-L1. Adoptive transfer of allospecific transgenic CD4 T cells revealed a “split tolerance” status in mAAQ+mice: T cells recognizing intact acquired MHC alloantigens proliferated, whereas those responding to allopeptide+self-MHC did not. Using isolated pDCs and mDCs for in vitro culture with allopeptide+self-MHC–specific CD4 T cells, we could replicate their normal activation in non-mAAQ mice, and PD-L1–dependent anergy in mAAQ+hosts. We propose that EVs provide a physiologic link between microchimerism and split tolerance, with implications for tumor immunity, transplantation, autoimmunity, and reproductive success.

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IL-12hi Rapamycin-Conditioned Dendritic Cells Mediate IFN-γ–Dependent Apoptosis of Alloreactive CD4+ T Cells In Vitro and Reduce Lethal Graft-Versus-Host Disease

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2013.11.007 ◽

2014 ◽

Vol 20 (2) ◽

pp. 192-201 ◽

Cited By ~ 9

Author(s):

Elizabeth O. Stenger ◽

Brian R. Rosborough ◽

Lisa R. Mathews ◽

Huihui Ma ◽

Markus Y. Mapara ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Graft Versus Host Disease ◽

Cd4 T Cells ◽

Host Disease ◽

Graft Versus Host ◽

Ifn Γ

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The macrophage mannose receptor promotes uptake of ADAMTS13 by dendritic cells

Blood ◽

10.1182/blood-2011-09-377754 ◽

2012 ◽

Vol 119 (16) ◽

pp. 3828-3835 ◽

Cited By ~ 30

Author(s):

Nicoletta Sorvillo ◽

Wouter Pos ◽

Linda M. van den Berg ◽

Rob Fijnheer ◽

Luisa Martinez-Pomares ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Mannose Receptor ◽

Immune Recognition ◽

Binding Studies ◽

Macrophage Mannose Receptor ◽

Carbohydrate Recognition Domains

Abstract ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura. The formation of autoantibodies depends on the activation of CD4+ T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4+ T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. ADAMTS13 endocytosis was decreased in the presence of a blocking mAb directed toward the macrophage mannose receptor (MR). Furthermore, siRNA silencing of MR reduced the uptake of ADAMTS13 by DCs. In addition, in vitro binding studies confirmed the interaction of ADAMTS13 with the carbohydrate recognition domains of MR. The results of the present study indicate that sugar moieties on ADAMTS13 interact with MR, thereby promoting its endocytosis by APCs.

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