Lymphocyte Stimulation by CD3‐CD28 Enables Detection of Low T Cell Interferon‐γ and Interleukin‐4 Production in Rheumatoid Arthritis

Abstract

The analysis of cytokine production is increasingly important in defining the course of an immune response and in evaluating specific therapies of immune diseases. In rheumatoid arthritis (RA), a dysregulation in T1/T2 cell balance, as defined by the production of their specific cytokines, IFN-gamma and IL-4, respectively, is suggested. A predominance of T1-cell mediated macrophage activity in the joint plays a key role in the destruction of articular cartilage and subchondral bone, whereas local T2 cell activity, mitigating disease, fails. However, analysis of the cytokines defining both T cell subsets is difficult and spontaneous production is often below detection limits. Several stimuli are therefore used to increase cytokine production. In the present study we examined whether stimulation of peripheral blood T cells in the context of mononuclear cells (PB MNC) by CD3-CD28 is a reliable method for assessing IFN-gamma and IL-4 production and is representative for the spontaneous production of these cytokines. The production of IFN-gamma and IL-4 following CD3-CD28 stimulation of RA PB MNC correlated significantly in a ratio 1 : 1 with production following ionomycin-PMA stimulation. In samples with detectable spontaneous production of IFNgamma and IL-4, production following CD3-CD28 stimulation was significantly higher than in stimulated samples with undetectable spontaneous production. Moreover, in the case of spontaneous production there was a significant positive linear correlation between the CD3-CD28 stimulated and spontaneous IFNgamma and IL-4 production, although production of both cytokines was not equally enhanced. Serial sampling did not show significant daily or weekly variation in stimulated cytokine production. The results demonstrate that a pecific T-cell stimulation by CD3-CD28 is a reliable way to enhance IFN-gamma and IL-4 production above the detection limit and so measure the T1/T2 cell balance in RA.

Salivary gland and peripheral blood T helper 1 and 2 cell activity in Sjogren's syndrome compared with non-Sjogren's sicca syndrome

“…T cell-specific cytokine production was amplified by adding CD3 and CD28 monoclonal antibodies (CLB, Amsterdam, The Netherlands; 1.5 mg/ ml and 2 mg/ml, respectively). 12 After 48 hours of ex vivo culture, 10 , the culture media were harvested, rendered cellfree by centrifugation, frozen in liquid nitrogen, and stored at 220˚C. IFNc and IL4 levels were determined by enzyme linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Biosource, Etten-Leur, The Netherlands).…”

Interleukin 7 stimulates tumour necrosis factor alpha and Th1 cytokine production in joints of patients with rheumatoid arthritis

“…SFMC were cultured for five days in the absence or presence of different concentrations of IL7 (Genzyme, Cambridge, USA, specific activity 1×10 7 U/mg), antihuman IL12 (p40/p70) neutralising monoclonal antibody (clone C8.6, IgG1, BD/Pharmingen, San Diego, CA), or IgG1 isotype control (BD/Pharmingen, San Diego, CA), followed by specific T cell stimulation with CD3/CD28 for 24 hours (1 µg/ml; CLB-T3/4.E and CLB-CD28, respectively, CLB Reagentia, Amsterdam, The Netherlands). 23 Subsequently, cytokine production during this 24 hour period was determined by ELISA and by FACS analysis (see below).…”

Clinical efficacy of leflunomide in primary Sjögren's syndrome is associated with regulation of T-cell activity and upregulation of IL-7 receptor α expression

“…In addition, TNFα production was evaluated on this T-cell restimulation. We have previously determined that TNFα production quantified in this way is for more than 90% T cell derived 28. Leflunomide reduced IFNγ production, although not statistically significantly (median values from 11 785 to 4741 pg/ml, p=0.133).…”

“…PBMC were incubated in 24 wells plates (1×106 cells/ml; Nunc, Roskilde, Denmark) for 3 days in RPMI supplemented with penicillin, streptomycin and glutamine and 10% pooled fetal calf serum (Gibco BRL, Life Technologies). Because IFNγ (Th1) and IL-4 (Th2) were not detectable in unstimulated cultures of almost all patients, for detection of these cytokines, T-cell cytokine production was amplified by adding anti-CD3/CD28 monoclonal antibodies (CLB, Amsterdam, The Netherlands; 1.5 µg/ml and 2 µg/ml, respectively) 28. In addition, the ex-vivo production of IL-1β and TNFα was determined in unstimulated cultures.…”