Phenotypic and functional characterization of the CD6-ALCAM T-cell co-stimulatory pathway after allogeneic cell transplantation

Abstract

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), expressed on antigen presenting cells, epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T-cell activation, proliferation, and trafficking. In this study we examined expression of CD6 by reconstituting T cells in 95 patients after allogeneic cell transplantation and evaluated the effects of itolizumab, an anti- CD6 monoclonal antibody, on T-cell activation. CD6 T cells reconstituted early after transplant with CD4 regulatory T cells (Treg)-expressing lower levels of CD6 compared to conventional CD4 T cells (Tcon) and CD8 T cells. After onset of acute graft-versus-host disease (aGvHD), CD6 expression was further reduced in Treg and CD8 T cells compared to healthy donors, while no difference was observed for Tcon. ALCAM expression was highest in plasmacytoid dendritic cells (pDC), lowest in myeloid dendritic cells (mDC) and intermediate in monocytes and was generally increased after aGvHD onset. Itolizumab inhibited CD4 and CD8 T-cell activation and proliferation in preGvHD samples, but inhibition was less prominent in samples collected after aGvHD onset, especially for CD8 T cells. Functional studies showed that itolizumab did not mediate direct cytolytic activity or antibody-dependent cytotoxicity in vitro. However, itolizumab efficiently abrogated the costimulatory activity of ALCAM on T-cell proliferation, activation and maturation. Our results identify the CD6-ALCAM pathway as a potential target for aGvHD control and a phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGvHD is currently ongoing (clinicaltrials gov. Identifier: NCT03763318).

Figures

Figure 1.

CD6 expression on T cells afer hematopoietic cell transplantation. (A) Percentage of CD6-positive cells and (B) CD6 median fluorescence intensity (MFI) on regulatory CD4 T cells (Treg, red boxes), conventional CD4 T cells (Tcon, blue boxes) and CD8 T cells (CD8, green boxes) in healthy donors (HD) and in patients at 1, 2, 3 and 6 months after hematopoietic cell transplantation (HCT). Box plots indicate median, Q1 and Q3 and min and max. (C) Representative gating strategy used to define T-cell subsets, based on the expression of CD45RA, CCR7 and CD95 markers. (D) Heat map summarizes the median CD6 MFI values in the different T-cell subsets in HD and in patients at 1, 2, 3 and 6 months after HCT. White stars show statistically significant differences between HD and samples after transplant (any P<0.05). (E) CD6 MFI on Treg, Tcon and CD8 T cells based on the expression of PD-1 in HD and in patients at 1, 2, 3 and 6 months after HCT. Statistically significant differences are noted: ****P<0.0001; ***P< 0.001; **P<0.01; *P<0.05; Wilcoxon rank-sum test.

Figure 2.

ALCAM expression on monocytes and dendritic cells afer hematopoietic cell transplantation. (A) Representative gating strategy used to define dentritic cell (DC) subsets, based on the expression of CD11c and CD123 in flow cytometry. (B) Percentage of ALCAM-positive cells and (C) levels of ALCAM expression (MFI) on CD14+ monocytes (grey boxes), myeloid DC (mDC, light-blue boxes) and plasmacytoid DC (pDC, green boxes) in healthy donors (HD) and in patients at 1, 2, 3 and 6 months after hematopoietic cell transplantation (HCT). Correlation of PD-L1 and ALCAM expression on (D) mDC and (E) pDC. Statistically significant differences are noted: ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; Wilcoxon rank-sum test.

Figure 3.

Expression of CD6 and ALCAM in patients afer hematopoietic cell transplantation with and without acute graf-versus-host disease. (A) Levels of CD6 expression (median fluorescence intensity [MFI]) in Treg, Tcon and CD8 T cells. (B) Levels of ALCAM expression (MFI) in CD4 regulatory T cells (Treg), conventional CD4 T cells (Tcon) and CD8 T cells. Before graft-versus-host disease (preGvHD) samples (green boxes) and after GvHD (postGvHD) samples (red boxes) from patients who developed acute GvHD are compared with noGvHD samples obtained at different times after transplant (grey boxes) and healthy donors (HD) (blue boxes). Statistically significant differences are noted: ***P<0.001; **P<0.01; *P<0.05; Wilcoxon rank-sum test. HD n=9, preGVHD n=30, postGVHD n=20, noGVHD 1 month (1m) n=38, 2 months (2m) n=40, 3 months (3m) n=43 and 6 months (6m) n=21.

Figure 4.

Inhibition of T-cell proliferation, activation and differentiation by itolizumab. Cryopreserved peripheral blood mononuclear cells (PBMC) obtained from healthy donors (HD) and patients before or after acute graft-versus-host disease (aGvHD) onset were stimulated with anti-CD3/CD2/CD28 beads in the presence of itolizumab or isotype control (cetuximab). (A) Proliferation of CD4 and CD8 T cells measured by CFSE dye dilution; (B) Activation of CD4 and CD8 T cells measured by expression of CD25; (C) maturation of CD4 and CD8 T cells measured by expression of CD45RO; (D) ALCAM expression is absent in resting T cells and is an additional marker of T-cell activation. (E) Percent inhibition induced by itolizumab, comparing activity against HD, pre- and postGvHD samples in CD4 and CD8 T cells. Percentage inhibition was calculated using the following formula: (% cells in isotype control - % cells in itolizumab)/% cells in isotype control. If no difference was observed between isotype control and itolizumab the percentage of itolizumab inhibition equals 0%. Statistically significant differences are noted: **P<0.01; *P<0.05; Wilcoxon rank-sum test. HD n=9, preGvHD n=7, postGvHD n=8.

Figure 5.

Testing itolizumab for complement-dependent cytotoxicity, antibody-dependent cytotoxicity and antibody direct cellular cytotoxicity. For complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADC), CD3+ T cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and cultured in the presence of medium + antibody + 25% of human serum (HS) and medium + antibody, respectively. Percentage cell lysis was calculated by combining the percentage of positive cells for 7-AAD or Annexin V or both. CDC activity was calculated by subtracting the values obtained in medium + antibody from the values obtained in the culture with medium + antibody + HS. ADC activity was calculated by subtracting the values obtained in the culture with medium alone from the values obtained in the culture with medium + antibody. CDC and ADC were assessed after 6 and 24 hours of culture. For antibody direct cellular cytotoxicity (ADCC) PBMC from HD were cultured in the presence of antibody for 6 hours. Both percentage cell lysis and CD107a expression on NK cells were evaluated after 6 hours of culture. The effects of itolizumab (red boxes) were compared to alemtuzumab (green boxes - positive control) and cetuximab (blue boxes - negative control). Values are expressed as mean and standard deviation (SD), paired t-test, 2 tails was used. Statistically significant differences are noted: ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; paired t-test. HD n=5.

Figure 6.

Itolizumab activity is dependent on the presence of ALCAM. T cells from healthy donors (HD) were stimulated with anti-CD3 antibody with or without recombinant human ALCAM Fc chimera (ALCAM-Fc) for 96 hours. Itolizumab or isotype control cetuximab were added at the start of each culture. (A) Proliferation of CD4 and CD8 T cells; (B) expression of CD25; (C) expression of CD45RO; (D) expression of ALCAM after stimulation with anti-CD3 antibody alone or in combination with ALCAM-Fc, in the presence of cetuximab (blue boxes) or itolizumab (red boxes). Statistically significant differences are noted: **P<0.01; *P<0.05; Wilcoxon rank-sum test. HD n=10.