Of the immune synapse. A number of studies have previously considered
Of the immune synapse. A number of studies have previously considered differences in immune synapse formation and TCR signaling between Th1 and Th2 cells: most of these are compatible with the notion that the synapse leading to Th2 differentiation comprises a less tightly focused, lower avidity complex. Th2, but not Th1 cells, fail to cluster TCR at the cellcell buy BMS-214662 interface due to increased expression of cytotoxic T-lymphocyte antigen 4 (CTLA-4) [57]. In line with this, co-clustering of TCR with CD4 in lipid rafts is moreReynolds et al. BMC Biology 2014, 12:32 http://www.biomedcentral.com/1741-7007/12/Page 16 ofAsfc per 106 cells1,200 1,000 800 600 400 200 0 0.0 0.5 1.0 10 20 Peptide concentration ( /ml)Bof CD4+ cells binding tetramerIAg7(PLP56-70) pMHCII50 40 30 20 100 10 20 30 40IAg7(CLIP)1.16.ThCDTh25.1.tetramer concentration ( /ml)Th7.1.CCD3 FITCD1,sfc per 106 cells1,000 800 600 400 200 0 0.0 0.5 1.0 10 20 Peptide concentration ( /ml)pMHCIIEof CD4+ cells binding tetramerIAg7(PLP56-70)24.IAg7(CLIP)1.Littermate17.CD2.TCRV0 0 10 20 30 405.72 1.tetramer concentration ( /mlt)TCRVFCytokine concentration (pg/ml)70 60 50 40 30 20 10 0 IFNIL-4 IL-5 IL-9 IL-Figure 6 (See legend on next page.)Reynolds et al. BMC Biology 2014, 12:32 http://www.biomedcentral.com/1741-7007/12/Page 17 of(See figure on previous page.) Figure 6 Th2 derived TCR has low avidity. (A) ELISPOT assays for Th1 (open squares; n = 4), Th2 (open triangles; n = 3) and Th17 (open diamonds; n = 6) polarized, non-transgenic cell lines cultured for eight days detect IFN, IL-4 and IL-17 producing cells, respectively, in response to increasing concentration of PLP 56-70 peptide. (B) Th1 (open squares; n = 6), Th2 (open triangles; n = 6) or Th17 (open diamonds; n = 2) polarized, non-transgenic cell lines incubated with H2-Ag7/PLP 56-70 or control (H2-Ag7/CLIP 103-117) tetramers on Day 8 after primed draining lymph nodes (DLN) were incubated with peptide. Percentages shown are the difference between staining with H2-Ag7/PLP 56-70 and control (H2-Ag7/CLIP 103-117) tetramers. Data are representative of three separate experiments. (C) Th1 and Th2 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28250575 polarized cell lines (A, B) have similar levels of CD3 expression. (D) ELISPOT assays for unpolarized, Th2 cytokine producing TCR lines (filled triangles; n = 3) and IFN producing littermate control lines (filled squares; n = 5) cultured in the absence of polarizing cytokines through two successive 10-day cycles of re-stimulation with peptide, detect IL-4 and IFN- producing cells, respectively, in response to increasing concentration of PLP 56-70 peptide. (E) Unpolarized Th2 cytokine producing TCR lines (filled triangles; n = 6), TCR lines (filled circles; n = 6) and IFN producing littermate control lines (filled squares; n = 5) cultured through two successive 10-day cycles of re-stimulation with peptide, incubated with H2-Ag7/PLP 56-70 or control (H2-Ag7/CLIP 103-117) tetramers at the concentrations shown. Percentages shown are the difference between staining with H2-Ag7/PLP 56-70 and control (H2-Ag7/CLIP 103-117) tetramers. (F) Functional binding of tetramers determined by cytokine production from unpolarized TCR lines and littermate controls following incubation with plate bound H2-Ag7/PLP 56-70 tetramer. Cytokine concentration shown is a total minus background cytokine production for cells incubated with phosphate-buffered saline (PBS) only. The TCR transgenic line (black bars) does not make IFN while the littermate control line (white bars) d.