Anti-Human CD6 Recombinant Antibody (Itolizumab) (CAT#: TAB-247)

Figure 1 Increased expression of CD6 on T cells after activation in Th17 polarizing conditions.

(A) PBMCs were stimulated in Thnp or Th17pol conditions, supernatant was collected and analyzed for secreted IFN-γ (Th1 signature cytokine) and IL-17 (Th17 signature cytokine). Ratio of absolute concentration of IFN-γ and IL-17 in Th17pol and Thnp conditions (Th17pol: Thnp) is plotted across the days of analysis. (B) Absolute levels of IFN-γ and IL-17 on day 13 is compared between Thnp and Th17pol conditions. Data shown is mean ±SD for triplicate ELISA wells (*p≤0.05). (C) PBMCs were left unstimulated or stimulated in Thnp or Th17pol conditions. CD25 expression on CD4+ T cells was analyzed on Day 3. Percentage of cells are indicated in the quadrants. Dot plots are gated on lymphocyte scatter. (D) PBMCs were left unstimulated (shaded histogram) or stimulated in Thnp or Th17pol conditions. CD6 expression (using biotinylated Itolizumab as detection reagent) was analyzed on Day 9 and plotted as CD6 overlay histograms gated on lymphocyte scatter. The secondary alone histogram is also overlayed for reference. (E) CD6 molecules/cell (receptor density) in unstimulated, Thnp and Th17pol conditions on gated CD4+ T-cells is shown as a scatter plot using biotinylated Itolizumab as detection reagent (*p≤ 0.05). For Fig 1B and 1E, statistical significance was determined using non-parametric unpaired t-test followed by Mann-Whitney test. In panels A and B, data is representative of 3 independent similar experiments, panel C is representative from at least 3 independent experiments, panel D is representative data from 6 donors and panel E has data from 6 donors.

Bughani, U., Saha, A., Kuriakose, A., Nair, R., Sadashivarao, R. B., Venkataraman, R.,... & Pai, H. V. (2017). T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab. PloS one, 12(7), e0180088.

Figure 2 Itolizumab inhibits T-cell activation and proliferation in both Thnp and Th17pol.

(A) PBMCs were stimulated in Thnp or Th17pol conditions in presence of Itolizumab or isotype control mAb (Iso Ab) both at 40 μg/ml. On day 3, cells gated on lymphocyte scatter and CD4+T-cells were analyzed for CD25 expression. Percent CD4+CD25+ T-cells in stimulated PBMCs, is plotted as bar graphs. Data shown is mean±SD from 3 different donors (*p≤0.05). (B) PBMCs labelled with CFSE dye were stimulated in Thnp (a-c) or Th17pol (d-f) conditions in presence of Itolizumab (c and f) or Iso Ab (b and e). On day 3, cells were analyzed for CFSE dilution on cells gated on lymphocyte scatter and CD4+ T-cells. Percent cells are indicated in dot plots of forward scatter (FS) vs CFSE. Data is representative from 3 independent experiments. (C) As derived from panel B, fold reduction in percentage of proliferated T-cells upon Itolizumab treatment is compared with Iso Ab. Fold reduction is determined by calculating percent total divided (proliferated) cells (with in the rectangular gate in Fig 2B) in Iso Ab or Itolizumab / percent total divided (proliferated) cells in Iso Ab. Bar graphs show mean±SD from 3 independent experiments. For Fig 2A and 2C, statistical significance was determined using non-parametric unpaired t-test followed by Mann-Whitney test.

Bughani, U., Saha, A., Kuriakose, A., Nair, R., Sadashivarao, R. B., Venkataraman, R.,... & Pai, H. V. (2017). T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab. PloS one, 12(7), e0180088.

Figure 3 Itolizumab causes reduction in expression of IL-17 and IFN-γ cytokines in cells stimulated in Th17 polarizing conditions.

PBMCs were stimulated with anti-CD3 and anti-CD28 beads in Th17pol conditions in presence of Itolizumab or Iso Ab (both at 40 μg/ml). On days 3, 6, 8 and 13 cells stimulated in Th17pol conditions with Iso Ab or Itolizumab, were re-stimulated with PMA-Ionomycin for 5 hours and analyzed for expression of intracellular cytokine IFN-γ and IL-17A. (A) Representative flow cytometry dot plots (gated on lymphocyte scatter and CD3+ T-cells) on day 6 are shown. Percent T-cells are indicated in the quadrants. Data is representative of 2 independent experiments. (B) Percentage of IFN-γ+ and (C) Percentage of IL-17A+ T-cells in presence of Itolizumab or Iso Ab and in unstimulated cells are plotted across days as obtained from flow cytometry analysis. Data is representative of 2 independent similar experiments. In panel/data 3A-C, before gating on lymphocyte gate, total cells were selected and gated to get uniform event count display. To analyze the level of secreted cytokines, supernatants were collected from quadruplicate wells of PBMCs stimulated in Th17pol conditions in presence of Itolizumab or Iso Ab, prior to PMA-ionomycin restimulation. As evaluated by ELISA, secreted (D) IFN-γ and (E) IL-17 levels are plotted across days. The level of cytokine release from unstimulated cells was negligible and hence is not plotted in the graphs. In Panel D and E representative data is shown as mean ± SD. Statistical analysis was performed from triplicate ELISA wells from one of the 3 independent similar experiments, for each time point in control and treated groups (*p≤ 0.05). For Fig 3D and 3E statistical significance was determined by t-test. For (B-E) open triangle, circle and box indicate Itolizumab, Iso Ab and unstimulated cells respectively.

Bughani, U., Saha, A., Kuriakose, A., Nair, R., Sadashivarao, R. B., Venkataraman, R.,... & Pai, H. V. (2017). T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab. PloS one, 12(7), e0180088.

Figure 4 Itolizumab causes reduction in signature Th17 specific markers.

(A) PBMCs were stimulated in Th17pol conditions in presence of Itolizumab or Iso Ab (both at 40 μg/ml) and analyzed for expression of transcription factor pSTAT3. Day 3 post stimulation data is shown as histogram for pSTAT3 on gated CD4+ T-cells (with a prior gating on lymphocyte scatter) and is representative of 3 independent experiments. (B) Cells stimulated in Th17pol condition in presence of Itolizumab or Iso Ab were re-stimulated with PMA-Ionomycin for 5 hours and analyzed for expression of intracellular cytokine IL-17A and Th17 signature transcription factor RORγT. Day 6 representative dot plots of RORγT and IL-17A gated on lymphocyte scatter and CD3+ T-cells are shown. Percent cells are indicated in the plots. (C) Data from panel B is plotted as histogram overlays of RORγT MFI on gated CD3+ T-cells stimulated in Th17pol condition in presence of Iso Ab or Itolizumab. In panel B and C, data shown is representative of 2 independent similar experiments. (D) For the experiment, similar to the one explained in panel B, Day 6 representative dot plots of CCR6 and IL-17A gated on lymphocyte scatter and CD3+CCR6+ T-cells are shown. Percent T-cells are indicated in the plots. Data is representative of 2 different time points with similar results (on Day 6 and Day 10). In panel 4B-D before gating on lymphocyte gate, total cells were selected and gated to get uniform event count display.

Bughani, U., Saha, A., Kuriakose, A., Nair, R., Sadashivarao, R. B., Venkataraman, R.,... & Pai, H. V. (2017). T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab. PloS one, 12(7), e0180088.

Figure 5 Itolizumab but not its F(ab’)2 fragment inhibits T cell signaling, activation and proliferation.

(A) Human PBMCs were plated on ALCAM (10 μg/ml) coated plates for 40 minutes with or without Itolizumab (10 μg/ml), F(ab')2 fragment of Itolizumab (equimolar amount), or Iso Ab (10 μg/ml). CD6 was immune precipitated with either Itolizumab or Iso Ab and immune blotted for CD6, p-Tyr, Zap70, SLP76, phospho and total SHP1 and SHP2. Corresponding 10% input samples were run as negative controls. Representative blots from at least three independent experiments is shown here. (B-D) Human PBMCs were stimulated with 0.5 ng/ml of anti-CD3 antibody (OKT3), and treated with Itolizumab / Isotype antibody (10 μg/ml), or F(ab')2 at (equimolar amount) respectively, for 72 h. (B-C) CD25 was used as T cell activation marker in CD4+-gated cells using flow cytometer. B shows representative dot plot, while C is the quantification from 2 independent experiments. (D) T cell proliferation was estimated by cell titre glo reagent, and bar graph shows mean ± SD values from 2 independent experiments each done in triplicate *p≤0.05. For Fig 7D non-parametric one way ANOVA statistical analysis was used using Dunn's multiple comparison analysis.

Bughani, U., Saha, A., Kuriakose, A., Nair, R., Sadashivarao, R. B., Venkataraman, R.,... & Pai, H. V. (2017). T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab. PloS one, 12(7), e0180088.