3b) in terms of a low production of IL-4

and IL-5 and high secretion of IL-10. No correlation was observed for Rucaparib manufacturer the individual donors between the levels of response to TG and TT (data not shown), indicating that the variability observed was restricted to TG, as the challenging antigen. To identify the source of IL-10 on day 1, PBMC were coated with bi-specific anti-CD45/anti-IL-10 beads before antigen stimulation to capture secreted cytokine at the cell surface. The CD4+ T cells and CD14-expressing monocytes were then examined by flow cytometry for the presence of released IL-10. Upon stimulation with TG, low IL-10 staining of most monocytes, indicated by a right shift of the cell profile, was consistently observed (Fig. 4a). On the other hand, IL-10 capture by CD4+ T cells was minimal (< 10 IL-10-bearing cells per 10 000 CD4+ T cells, Fig. 4b), consistent with a clonal response to the antigen. Counterstaining for memory and naive T cells, with anti-CD45RO

and anti-CD45RA, respectively, revealed that TG induced IL-10 production in a significant proportion of CD4+ memory T cells (3·1 ± 1·7 per 10 000 CD4+ T cells, P < 0·01, selleck chemicals Fig. 4c), whereas the numbers of cells producing IL-10 in response to TT and KLH were non-significant (0·38 ± 0·52 and 0·52 ± 0·43 cells per 10 000 CD4+ T cells, respectively). The corresponding numbers of naive CD4+ T cells producing IL-10 upon stimulation with TG, TT and KLH were 1·1 ± 0·61, 0·21 ± 0·37 and 1·8 ± 1·1 cells per 10 000 CD4+ T cells, respectively (Fig. 4d), and, as such, were non-significant. To address the question Etofibrate of whether TG-specific memory T cells were orchestrating the monocyte IL-10 response to TG, PBMC were depleted of CD3+ T cells or CD14+ monocytes (as appropriate control) and then stimulated with

either TG or TT. The IL-10 and TNF-α responses were examined at day 1 after stimulation. Depletion with the anti-CD3 beads removed 99·2 ± 0·4% of the T cells from the PBMC with quantitative recovery (116 ± 20%) of the monocytes, while CD14 depletion almost completely removed the monocytes (98·7 ± 2·4%), with a non-significant reduction (43·5 ± 22·5%) in the size of the T-cell population. Monocyte depletion abrogated TNF-α production, following TG stimulation, and markedly diminished (though only with borderline significance, P < 0·06) TNF-α secretion in response to TT (Fig. 5a). By contrast, T-cell depletion resulted in only non-significant reductions in TNF-α production upon stimulation with either antigen (Fig. 5a). Similarly, virtual ablation of IL-10 synthesis was observed upon CD14+ cell depletion, irrespective of the challenging antigen (Fig. 5b), confirming that monocytes were primarily responsible for this cytokine’s production on day 1. On the other hand, the effect of T-cell depletion on IL-10 production differed markedly for the two antigens. While TG-stimulated secretion of IL-10 was drastically reduced (P < 0·002) (Fig.