Efficient short-term expansion of human peripheral blood regulatory T cells for co-culture suppression assay.

Regulatory T cells (Tregs) are a small population of CD4+ lymphocytes and play a key role as suppressors of the immune system, a role that can be identified by employing a co-culture suppression assay. Conventional protocol requires a long period of in vitro expansion of Treg numbers; hence, this study describes an establishment of a co-culture suppression assay using a short-term expansion of peripheral blood (PB) Tregs and autologous T cells (Tconvs) IL-2-pre-cultured in parallel for the same length of time, thereby obviating the need of freeze/thawed autologous Tconvs. Tregs and Tconvs were isolated from PB mononuclear cells employing magnetic bead-aided depletion of CD8+ cells followed by cell sorting of CD4+ CD25high+CD127low- (Treg) and CD4+ CD25-CD127+ (Tconv) cell populations. Following a 3-day co-cultivation period under optimized conditions, Treg suppression activity was monitored by comparing using flow cytometry the number of carboxyfluorescein succinimidyl ester-labeled Tconvs to that of Treg-minus control. The assay allowed significant differentiation between Treg suppression activity of patients with active rheumatoid arthritis and those in remission. This method should be more convenient and time-saving than the conventional Treg suppression assay in current use.

Analgesic and Anti-Inflammatory Effects of 1% Topical Cannabidiol Gel in Animal Models.

Introduction: Cannabidiol (CBD), a phytocannabinoid isolated from cannabis plants, is an interesting candidate for studying its anti-inflammatory effects, especially in the pre-clinical and animal models. Its anti-inflammatory effects, such as reduction of edema and arthritis, have been demonstrated in animal models. However, topical CBD administration requires further evaluation of CBD dosage and efficacy in animal models and clinical settings. Methods: This in vivo study investigated the anti-inflammatory effects of topical CBD administration in an animal model. Scientific experiments, including the formalin test, writhing test, carrageenan-induced edema, histopathological examination, and detection of various proinflammatory mediators, were performed. Results: The anti-inflammatory effects in vivo after inflammation induction, represented by decreased times of paw licking, degree of paw edema, and decreased writhing response, showed that 1% of tropical CBD use had significantly comparable or better anti-inflammatory effects when compared with tropical diclofenac, an anti-inflammatory agent. Moreover, the anti-inflammatory effects were significant compared with the placebo. In addition, the histopathological examination showed that topical CBD drastically reduced leukocyte infiltration and the degree of inflammation. This study also showed that the levels of various proinflammatory mediators in the plasma of mice treated with topical CBD did not differ from those treated with diclofenac. Conclusions: The topical administration of 1% CBD gel is a potentially effective candidate for an anti-inflammatory agent. Candidate for an anti-inflammatory agent.

Autoimmunity to synovial extracellular matrix proteins in patients with postinfectious Lyme arthritis.

BACKGROUNDAutoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides, and therefore the reasons for these associations, has frequently remained elusive.METHODSUsing immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi-mimic (Bb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins.RESULTSOf 24 postinfectious LA patients, 58% had CD4+ T cell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Vα1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins. Patients with autoreactive T cell responses had significantly increased frequencies of HLA-DRB1*04 or -DRB1*1501 alleles and more prolonged arthritis. When tetramer reagents were loaded with ECM or corresponding Bb-mimic peptides, binding was only with the autoreactive T cells. A high percentage of ECM-autoreactive CD4+ T cells in synovial fluid were T-bet-expressing Th1 cells, a small percentage were RoRγt-expressing Th17 cells, and a minimal percentage were FoxP3-expressing Tregs.CONCLUSIONAutoreactive, proinflammatory CD4+ T cells and autoantibodies develop to ECM proteins in a subgroup of postinfectious LA patients who have specific HLA-DR alleles. Rather than the traditional molecular mimicry model, we propose that epitope spreading provides the best explanation for this example of infection-induced autoimmunity.FUNDINGSupported by National Institute of Allergy and Infectious Diseases R01-AI101175, R01-AI144365, and F32-AI125764; National Institute of Arthritis and Musculoskeletal and Skin Diseases K01-AR062098 and T32-AR007258; NIH grants P41-GM104603, R24-GM134210, S10-RR020946, S10-OD010724, S10-OD021651, and S10-OD021728; and the G. Harold and Leila Y. Mathers Foundation, the Eshe Fund, and the Lyme Disease and Arthritis Research Fund at Massachusetts General Hospital.

Inhibitory activity of FOXP3+ regulatory T cells reveals high specificity for displaying immune tolerance in remission state rheumatoid arthritis.

Immune regulation status may indicate immunological remission in rheumatoid arthritis (RA). This cross-sectional study aimed to determine the Regulatory T cell (Treg) properties, together with 14 plasma cytokines levels between active RA and clinical remission patients. Peripheral blood (PB) Foxp3+ Treg was collected from RA patients for determination of Treg inhibitory activity using a co-culture system. Other PB T cell types and plasma cytokines were determined by flow-cytometry. The Treg results were analyzed according to the disease activity score-28 (DAS28). Then sensitivity and specificity were calculated for the indication of the remission status. The number and inhibitory activity of Treg are higher in the clinical remission as compared to the active RA (p value < 0.0001). Also, Treg: CD4+CD25+CD127+ cell ratio demonstrates the similar result (p value < 0.05). Treg inhibitory activity is inversely correlated with the DAS28. Specificity and positive likelihood ratio of inhibitory activity for indicating remission status are 92.31% (95% CI 63.97-99.81) and 11.14 (95% CI 1.67-74.14), respectively. Treg inhibitory activity is a promising prognostic marker and probably represents the immunological remission status in RA.

Regulatory T Cell Suppressive Activity Predicts Disease Relapse During Disease-Modifying Anti-rheumatic Drug Dose Reduction in Rheumatoid Arthritis: A Prospective Cohort Study.

When the dose of conventional disease-modifying anti-rheumatic drugs (cDMARDs) is tapered in rheumatoid arthritis (RA) patients who achieve sustained remission, biomarkers for predicting disease relapse may be needed. A prospective, unblinded cohort study was conducted in nine RA patients with remission. Peripheral blood samples were collected at baseline and at 6, 12, and 24 weeks after cDMARD dose reduction (dose of combination regimens reduced to 50%) to determine the number of regulatory Foxp3+T cells (Tregs) and other T cell subpopulations as well as Treg suppressive activity. Additionally, plasma levels of 14 cytokines at each time-point were measured via flow cytometry. Univariate and multivariate analyses were performed to identify the factor(s) associated with RA relapse during the observational period. In univariate analysis, Treg suppression and DAS28 and VAS scores were associated with RA relapse after cDMARD dose tapering. However, in multivariate analysis, only Treg suppressive activity (<42%) was found to be an independent factor associated with RA relapse after cDMARD dose reduction to 50%. Of all patients who had ≥42% Treg suppressive activity during cDMAD reduction, three-fourth patients remained in the remission stage for 24 weeks. Treg suppressive activity (<42%) in RA patients with remission could be a potential biomarker for predicting RA relapse after cDMARD dose reduction, especially over a short-term period (24 weeks).