scholarly journals AB0094 FUNCTIONAL TREG CELLS MAY BE CONVERTED INTO T EFFECTOR PHENOTYPE ON EXPOSURE TO INFLAMMATORY MILIEU IN RHEUMATOID ARTHRITIS (RA) SYNOVIAL FLUID: IN-VITRO STUDY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1346.1-1347
Author(s):  
V. Kommoju ◽  
C. Kavadichanda ◽  
V. Negi
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Synovial Fluid ◽  
Cell Surface ◽  
Inflammatory Cytokines ◽  
Peripheral Blood ◽  
Treg Cells ◽  
Cell Surface Expression ◽  
Inflammatory Milieu

Background:The debate on functional versus numerical difference in T regulatory cell population among patients with Rheumatoid arthritis (RA) is not clear. Tregs expressing Inflammatory subset phenotype markers, such as Th1(cxcr3, Tbet) and Th17(ccr6,Rorϒ) are reported. (1) Though the reported numbers of synovial fluid tregs are higher in RA, (2) the fate of Tregs on entering the synovial inflammatory milieu from peripheral blood (PB) has not yet been investigated.Objectives:To compare Treg frequencies in PB and synovial fluid between osteoarthritis (OA) and RATo compare cytokine levels in PB and SF between OA and RATo study the effect of autologous synovial fluid on RA and OA Treg isolated form peripheral bloodMethods:The Peripheral Blood (PB) and synovial fluid (SF) of RA (n=80) and OA (n=30) patients were analyzed for CD4+T-cell subset frequencies and phenotypes by flow cytometry. Cytokine concentrations in plasma and SF were measured by cytometric bead array. Tregs from 5 RA-PB and 5 OA-PB were isolated and cultured in autologous synovial fluid for 24 hrs. Phenotypic expression of Th1 and Th17 chemokines on the cell surface were analyzed by flow cytometry and expression levels of T-bet, RORγ and FOXP3 in those Treg cells were measured with quantitative real-time PCR (RT-qPCR).Results:The PB and SF frequencies of Th1, Th17 and Tregs are shown in Table 1. The pro-inflammatory cytokines were high in the plasma and SF of RA but the anti-Inflammatory cytokines were similar (Fig 1.A&B). Treg cells were isolated from RA and OA PB and cultured in autologous SF for 24 hrs. RA Treg showed increased cell surface expression of CXCR3+ and CCR6+ (Fig 1C) and there was no difference in OA Treg. Gene expression studies showed an increased expression of T-bet, RORγ and decreased expression of Foxp3 in RA Tregs while there was no difference in OA Tregs before and after in-vitro culture (Fig 1D).Table 1.Tcell subsets in Rheumatoid Arthritis and Osteoarthritis.CD4 subtypeRAOAPBSFPBSFN=80N=30N=30N=30Th126.65 ± 5.5934.99 ± 1.3025.97 ± 7.2426.45 ± 1.87*Th25.19 ± 1.916.36 ± 1.735.24 ± 2.151.44 ± 0.10*Th1714.05 ± 3.2921.18 ± 2.0410.81 ± 2.78*2.45 ± 0.23*Treg10.68 ± 2.4712.53 ± 2.1011.162.9513.04 ± 2.57*P<0.05Conclusion:Tregs in RA may be converted to Th1 and Th17 phenotype on exposure to inflammatory cytokine in the synovial fluid, thus losing their regulatory functions. Understanding factors influencing stability of Treg cells may help improve future therapeutics.References:[1]Jinlin Miao & Ping Zhu. Functional Defects of Treg Cells: New Targets in Rheumatic Diseases, Including Ankylosing Spondylitis. Current Rheumatology Reports 2018; 20: 30[2]Penatti et al. Differences in serum and synovial CD4+ T cells and cytokine profiles to stratify patients with inflammatory osteoarthritis and rheumatoid arthritis. Arthritis Research & Therapy.2017; 19:103Acknowledgments:Department of Science and technology, India for the research grant.Disclosure of Interests:None declared

scholarly journals Therapeutic Effect of Exogenous Treg Cells on Collagen-Induced Arthritis and Rheumatoid Arthritis

2019 ◽  
Author(s):  
Shutong Li ◽  
Hongxing Wang ◽  
Hui Wu ◽  
Guoqing Zhang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Synovial Fibroblast ◽  
Treg Cells ◽  
Cell Counting ◽  
Fibroblast Cells ◽  
Collagen Induced Arthritis ◽  
Gm Csf

Abstract Background Regulatory T (Treg) cells have anti-inflammatory and anti-autoimmune functions. The proportion and functions of Treg cells are perturbed in rheumatoid arthritis (RA) patients. Methods Human Treg cells were induced to amplify in vitro and cocultured with RA synovial fibroblast cells (RASFs). The proliferation and apoptosis of RASFs were determined by the cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Human Treg cells were also injected to collagen-induced arthritis (CIA) rats via the tail vein. Changes in lymphocyte subtypes and cytokines in the peripheral blood and spleen were observed by flow cytometry. Results After coculture with the Treg cells, the proliferation of RA synovial fibroblast cells decreased (p<0.01), and the rate of apoptosis increased (p=0.037). The human Treg cells were injected into the tail veins of collagen-induced arthritis (CIA) rats. The severity of the CIA was reduced (p<0.01) following the injection, the percentages of rat endogenous Treg cells in the peripheral blood and spleen increased significantly (p=0.007 and p<0.01, respectively), and the proportion of B cells decreased (p=0.031). The levels of interleukin IL-5 and IL-6 and the Th1/Th2 ratio in the peripheral blood were significantly decreased (p=0.013, 0.009 and 0.012, respectively). The number of NK cells and the levels of IL-4, IL-13, TNF-α, IFN-γ and GM-CSF in the peripheral blood and spleen did not change significantly. Conclusion These results suggest that exogenous Treg cells play a therapeutic role in RA and CIA. Treg cell treatment could serve as a therapy for RA.

scholarly journals Immunotherapeutic Targeting of TSLPR with Chimeric Antigen Receptor T Cells in AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2789-2789
Author(s):  
Lindsey F Call ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Cynthia Nourigat-Mckay ◽  
LaKeisha Perkins ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Car T Cells ◽  
Car T

Abstract Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML. We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells. In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. Disclosures Pardo: Hematologics, Inc.: Current Employment.

scholarly journals Characterization of CD30/CD30L+Cells in Peripheral Blood and Synovial Fluid of Patients with Rheumatoid Arthritis

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Alessandro Barbieri ◽  
Marzia Dolcino ◽  
Elisa Tinazzi ◽  
Antonella Rigo ◽  
Giuseppe Argentino ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Synovial Fluid ◽  
Peripheral Blood ◽  
Treg Cells ◽  
T Cell Subsets ◽  
Immune Effector ◽  
Effector Cells ◽  
Signalling System ◽  
Soluble Cd30

The CD30/CD30L signalling system has been implicated in the pathogenesis of several autoimmune and inflammatory conditions. In rheumatoid arthritis (RA), soluble CD30 (sCD30) levels reflect the recruitment of CD30+T cells into the inflamed joints and correlate with a positive response to immunosuppressive therapy. The aim of our report was to clarify the role of CD30/CD30L signalling system in the pathogenesis of RA. Our analysis of the CD30L+T cell subsets in peripheral blood (PB) and synovial fluid (SF) of RA patients and of the related cytokine profiles suggests the involvement of CD30/CD30L signalling in polarization of T cells towards a Th17 phenotype with proinflammatory features. Moreover, in RA SF nearly 50% of Treg cells express CD30, probably as an attempt to downmodulate the ongoing inflammation. We also show here that the engagement of CD30L on neutrophils stimulated with CD30/Fc chimera may play a crucial role in RA inflammation since activated neutrophils release IL-8, thus potentially amplifying the local inflammatory damage. In conclusion, the results obtained suggest that the complex CD30/CD30L signalling pathway is implicated in the pathogenesis and progression of RA synovitis through a concerted action on several immune effector cells.

Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis

2015 ◽  
Vol 75 (6) ◽  
pp. 1170-1176 ◽  
Author(s):  
Priscilla F Kerkman ◽  
Emeline Fabre ◽  
Ellen I H van der Voort ◽  
Arnaud Zaldumbide ◽  
Yoann Rombouts ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
B Cells ◽  
Peripheral Blood ◽  
Memory B Cells ◽  
Protein Antigens ◽  
High Frequencies ◽  
Culture Supernatants ◽  
Citrullinated Antigen

ObjectivesImmunity to citrullinated antigens is a hallmark of rheumatoid arthritis (RA). We set out to elucidate its biology by identifying and characterising citrullinated antigen-specific B cells in peripheral blood of patients with RA.MethodsDifferentially labelled streptavidin and extravidin tetramers were conjugated to biotinylated CCP2 or control antigens and used in flow cytometry to identify citrullinated antigen-specific B cells in peripheral blood. Tetramer-positive and tetramer-negative B cells were isolated by fluorescence activated cell sorting (FACS) followed by in vitro culture and analysis of culture supernatants for the presence of antibodies against citrullinated protein antigens (ACPA) by ELISA. Cells were phenotypically characterised by flow cytometry.ResultsBy combining differentially labelled CCP2 tetramers, we successfully separated citrullinated antigen-specific B cells from non-specific background signals. Isolated tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon in vitro stimulation. Phenotypic analyses revealed that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells and plasmablasts, whereas only few cells displayed a naïve phenotype. The frequency of tetramer-positive cells was high (up to 1/500 memory B cells with a median of 1/12 500 total B cells) and correlated with ACPA serum titres and spontaneous ACPA production in culture.ConclusionsWe developed a technology to identify and isolate citrullinated antigen-specific B cells from peripheral blood of patients with RA. Most cells have a memory phenotype, express IgA or IgG and are present in relatively high frequencies. These data pave the path for a direct and detailed molecular characterisation of ACPA-expressing B cells and could lead to the identification of novel therapeutic targets.

scholarly journals Chronic inflammation-induced senescence impairs immunomodulatory properties of synovial fluid mesenchymal stem cells in rheumatoid arthritis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hyeon-Jeong Lee ◽  
Won-Jae Lee ◽  
Sun-Chul Hwang ◽  
Yongho Choe ◽  
Saetbyul Kim ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Synovial Fluid ◽  
Cellular Senescence ◽  
Average Duration ◽  
Immunomodulatory Properties ◽  
Inflammatory Milieu

Abstract Background Although the immunomodulatory properties of mesenchymal stem cells (MSCs) have been highlighted as a new therapy for autoimmune diseases, including rheumatoid arthritis (RA), the disease-specific characteristics of MSCs derived from elderly RA patients are not well understood. Methods We established MSCs derived from synovial fluid (SF) from age-matched early (average duration of the disease: 1.7 years) and long-standing (average duration of the disease: 13.8 years) RA patients (E-/L-SF-MSCs) and then analyzed the MSC characteristics such as stemness, proliferation, cellular senescence, in vitro differentiation, and in vivo immunomodulatory properties. Results The presence of MSC populations in the SF from RA patients was identified. We found that L-SF-MSCs exhibited impaired proliferation, intensified cellular senescence, reduced immunomodulatory properties, and attenuated anti-arthritic capacity in an RA animal model. In particular, E-SF-MSCs demonstrated cellular senescence progression and attenuated immunomodulatory properties similar to those of L-SF-MSC in an RA joint-mimetic milieu due to hypoxia and pro-inflammatory cytokine exposure. Due to a long-term exposure to the chronic inflammatory milieu, cellular senescence, attenuated immunomodulatory properties, and the loss of anti-arthritic potentials were more often identified in SF-MSCs in a long-term RA than early RA. Conclusion We conclude that a chronic RA inflammatory milieu affects the MSC potential. Therefore, this work addresses the importance of understanding MSC characteristics during disease states prior to their application in patients. Graphical abstract

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