Correction: T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab

<p>Correction: T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab</p

T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab

<div><p>CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1) specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4⁺ helper T-cells (Th17) have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in <i>in vitro</i> experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15–35% of CD4⁺ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab’)2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an important target for modulating autoimmune diseases.</p></div

Mouse anti CD6 domain 1 antibody (m CD6D1 mAb) ameliorates EAE in mice.

<p>EAE disease was induced in C57BL/6 normal female mice using MOG_35-55 emulsified in CFA and pertussis toxin. Randomized mice were injected with 60 μg in 100 μl volume of either control m Iso Ab (shown in circles) or m CD6D1 mAb (shown as squares) between day 15 and day 27 (arrows indicate days of dosing). 7 animals were used in each group. (A) Clinical scores are represented as Mean ± SEM. Fig is representative of six independent studies (*p≤0.05). The statistical significance was determined using 2 way ANOVA followed by Sidak’s multiple comparison analysis. (B-C) Splenocytes of these mice were stimulated with plate bound anti-mouse CD3 mAb. (B) After 96 h, proliferation of these splenocytes was estimated using Alamar Blue reagent <b>**</b>p≤0.01. Statistical significance was determined using non-parametric unpaired t-test followed by Mann-Whitney test (C) Supernatants were collected after 72 h and 6 cytokines were analyzed using Cytokine bead array (CBA) kit. All cytokines except IL-12 showed significant difference using unpaired t-test (*p≤0.05). (D) m Iso Ab and m CD6D1 mAb treated EAE-induced mice were sacrificed on day 32 post immunization and transverse lumbar spinal cord sections were prepared (middle and lower panel). Naive mouse without EAE induction was used as additional control (top panel). Left panel showed light microscopy of sections stained with hematoxylin and eosin (H&E), while the right panel showed luxol fast blue staining for demyelination of neurons. Arrows in the middle panel show infiltrating lymphocytes among neutrophils migrating into the spinal cord in mice with disease and treated with m Iso Ab. Representative Fig shown.</p

Differential expression of genes in presence of Itolizumab at different phases relative to control (gene expression custom 15K).

<p>Differential expression of genes in presence of Itolizumab at different phases relative to control (gene expression custom 15K).</p

Itolizumab causes reduction in signature Th17 specific markers.

<p><b>(</b>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>(</b>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.</p

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

<p>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.</p

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

<p>(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.</p

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

<p>(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.</p

Differential expression profiles over time for Itolizumab.

<p>Scatter plots for all the 15K genes in the array for control group versus Itolizumab in early, middle (Average expression for days 6 and 8) or late phases (Average expression for days 10 and 14) are shown. Regression line represents R-Squared value between Itolizumab and Iso Ab groups. The colored region is > log₂ 1 over unstimulated in the control group. Green region depicts the genes down regulated by at least 20% in the Itolizumab arm over the Iso Ab group.</p