Ratio, inside the presence of 0, 05 or 0 ng/ml of anti-CD3 and analysed for the percentage of activated T cells indicated by CD69 expression just after 12 h, working with flow cytometry. (a) Representative flow cytometry dot-plots of activated CD69-expressing T cells. Cells have been surface-stained for CD69 expression. Numbers represent the percentage of CD69-positive T cells within the gate. (b) Paired information from seven independent experiments, displaying the percentage of CD4+CD69+ T cells following co-culture with SD (open circles) or KD LCLs (black circles) in unique ratios and inside the presence of varying levels of anti-CD3. Each point inside the paired information represents the imply from the triplicate measurement for every single condition. SD: scrambled siRNA duplex, KD: CLEC16A-specific targeting siRNA duplex.102 0 0 102 103 IDO Inhibitor Molecular Weight 104105 FITC-A: CD4102 0 0 102 103 104105 FITC-A: CD4 CD0 102103 104 105 FITC-A: CD4(b) of T cells expressing CD80 70 60 50 40 30 200 Dose 0 ng/ml 005 ng/ml 03 ng/ml 0 ng/ml 005 ng/ml 03 ng/ml anti-CD3 1:4 1:two n=7 B:T cell ratio Scrambled duplex Knock downdetected as early as 12 h post-co-culture, peaking at 48 h, immediately after which they remained constant for no less than 72 h (information not shown). Therefore, all CD25 measurements were recorded at 12, 24 and 48 h, respectively. As anticipated, there was a important impact of escalating anti-CD3 concentration on the percentage of CD69+- and CD25+-activated T cells at all time-points and B : T cell ratios measured (Figs 3 and four, Supporting information Figs S3 and S4). Even so, the percentage of T cells expressing CD69 or CD25 following activation by the co-culture assay was not significantly diverse involving T cells co-cultured with CLEC16A KD or SD LCLs at any measured time-point (P 05). This remained true irrespective of the B : T cell ratio in which the LCLs and CD4+ T cells have been combined along with the anti-CD3 concentration used (threshold versus saturating levels) (Figs 3 and 4, Supporting info Figs S3 and S4). No T cell activation was observed within the manage wells lackingLCLs, ATM Inhibitor Storage & Stability reflected by the negligible percentage of T cells expressing either CD69 or CD25 (data not shown).CLEC16A knock-down doesn’t influence T cell proliferation within a T cell CL co-culture assayKeeping in mind that the ultimate immune end-point for activated T cells is their proliferation and clonal expansion, we asked regardless of whether the CLEC16A KD in LCLs could have an effect on T cell division in this experimental set-up. The extent of proliferation was determined in T cells co-cultured with KD and SD LCLs for 72 h inside the presence of 05 or 0 ng/ml of anti-CD3. As expected, a higher number of proliferating T cells was observed using the larger dose of anti-CD3, specifically when B and T cells had been co-cultured in a 1:2 ratio (Fig. 5a, P 01). However, no significant differences in cell division profiles have been observed in CD4+ T cells2013 British Society for Immunology, Clinical and Experimental Immunology, 175: 485H. Zouk et al.(a) AntiCD3 0 ng/ml APC-A: CD25 ten 105 4005 ng/ml APC-A: CD25 10503 ng/ml APC-A: CD25 105 104 103 102 0 0 102 103 104 105 FITC-A: CD4CDSD103 102 0 0 102 103 104 105 FITC-A: CD4 CD4 005 ng/ml 104 103 10102 0 0 102 103 104 105 FITC-A: CD4AntiCD3 APC-A: CD250 ng/ml 10403 ng/ml APC-A: CD25 105 104 103 102 0 0 102 103 104 105 FITC-A: CD4KDFig. four. Assessing T cell activation by CD25 expression 12 h soon after a T cell ymphoblastoid cell line (LCL) co-culture assay. CD4+ T cells were activated by co-culture with either SD or knock-down (KD)-transfected LCLs in.