Cells were re-stimulated with PMA-Ionomycin for 5 hours and analyzed for appearance of intracellular cytokine IL-17A and IFN-. pol circumstances over unstimulated cells. Data represents 3 donors.(DOCX) pone.0180088.s002.docx (46K) GUID:?4DCFD198-38A3-415A-A700-866EFFD7B9DC S3 Fig: Compact disc6 receptor in lymphocytes isn’t internalised and it is occupied with Itolizumab. Individual PBMCs had been still left unstimulated or activated with soluble anti Compact disc3 0.5 ng/ml (OKT-3) in presence of Iso Ab or Itolizumab at 10 g/mL for 3 days. Post stimulation, cells were harvested and stained with anti CD6 Ab, MEM98 clone (A) and anti-human IgG, Fc specific (B). In panel A, since the CD6 receptor is occupied with Itolizumab, MEM 98 (commercially available FLJ34463 anti CD6 D1) could not bind in Itolizumab treated groups and hence no signal is Ciwujianoside-B observed. The positive signal with anti-human IgG, in Itolizumab treated group in panel B suggests that Itolizumab is occupying CD6 receptor on lymphocyte surface. Data is representative of at least 3 independent experiments.(DOCX) pone.0180088.s003.docx (82K) GUID:?4BA54DF9-C94A-4D50-8E8A-275BF6C22BB6 S4 Fig: Itolizumab inhibits IFN- and IL17-A expression in CD8+T cells. Human PBMCs were stimulated with anti-CD3 and anti-CD28 beads or soluble anti CD3 0.1 ng/ml (OKT3) and sol anti CD28 (10 ng/ml) in Th17pol conditions in presence of Itolizumab or Iso Ab at 40 g/mL. On day 6, cells were re-stimulated with PMA-Ionomycin for 5 hours and analyzed for expression of intracellular cytokine IFN- and IL-17A. Representative flow cytometry dot plots (gated on lymphocyte scatter and CD8+ lymphocytes) on day 6 are shown in Fig. Percent cells are indicated in the quadrants Itolizumab substantially inhibits IFN- and IL-17A expression in CD8+ lymphocytes. Data is representative of 2 independent experiments.(DOCX) pone.0180088.s004.docx (124K) GUID:?B3035D2C-FA68-4194-9F9F-98C73E106DCC S5 Fig: Itolizumab does not induce AICD in stimulated PBMC. (A) Human PBMCs were left unstimulated or stimulated with soluble anti CD3 0.5 ng/ml (OKT-3) in the presence of Iso Ab or Itolizumab at 10 g/mL for 3 days. Post incubation, cells were harvested and stained with anti CD3, Annexin V and 7-AAD. The % Annexin V positive, 7-AAD negative CD3+T cells has been plotted. The bar graphs show meanSD from 3 independent experiments. (B) Similar experiment as described in panel A with staining at different time points was done to analyse AICD across days. At each time point, cells were harvested and stained with CD3, Annexin V and 7-AAD. The % Annexin positive, 7-AAD negative CD3+ T cells has been plotted. Data is from one experiment.(DOCX) pone.0180088.s005.docx (110K) GUID:?7BB8CA4B-0DE6-4C1F-909A-614E92A30830 S6 Fig: Phenotyping of unstimulated human PBMCs using IL-17 and IFN- intracellular cytokine expression across days. (A) PBMCs were left unstimulated for 3 days and analysed for expression of intracellular cytokine IFN- and IL-17A. Representative flow cytometry dot plots (gated on lymphocyte scatter and CD3+ T-cells) is shown. Percent T-cells are indicated in the quadrants. (B) PBMCs were left unstimulated for 3, 6, 8 and 13 days. Cells were re-stimulated with PMA-Ionomycin for 5 hours and analyzed for expression of intracellular cytokine IFN- and IL-17A. Representative flow cytometry dot plots (gated on lymphocyte scatter and CD3+ T cells) across days are shown. Percent T-cells are indicated in the quadrants. In the panels, before gating on lymphocyte gate, total cells were selected and gated to get uniform event count display.(DOCX) pone.0180088.s006.docx (209K) GUID:?68E49638-6F11-4B94-9DC8-C50AEFAD33B6 S7 Fig: Lymphocyte gate based on SSC and FSC excludes 7AAD Ciwujianoside-B positive (dead) cells. PBMCs were left unstimulated or stimulated with soluble anti CD3 0.5 ng/ml (OKT-3) for 3 days / 6 days. Post incubation, cells were harvested and stained with 7-AAD. Panel A shows the representative lymphocyte gate that is put in all experiments. The dead cells (7-AAD positive) seen in green are excluded out of the gate. In panel B, no gate has been applied and positive signal is seen with 7-AAD, indicating the presence of dead Ciwujianoside-B cells. In panel C, lymphocyte gate has been applied and the cells do not stain positive for 7-AAD, indicating healthy cell population. Day3 7-AAD staining is a representative of 3 independent experiments and Day6 7-AAD staining is from a single experiment.(DOCX) pone.0180088.s007.docx (378K) GUID:?239E407D-DCDB-489E-A381-654661A6B34E S8 Fig: Coomassie Blue staining for F(ab)2.
