CD6 Is a Modulator of Treg Differentiation and Activity

The imbalance of proinflammatory effector T cells (T_eff) and anti-inflammatory regulatory T cells (T_reg) is associated with autoimmune and inflammatory diseases. Clinical trials have demonstrated that T_regs can prevent and treat GVHD, without increasing the risk of relapse and infection (Guo et al., 2021). CD6, a costimulatory receptor is expressed on T cells including pathogenic T_eff cells at high levels (CD6^high), while Tregs are CD6^low. Studies have demonstrated that ex-vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998), highlighting the importance of CD6. Notably T cells from CD6knockout mice fail to differentiate to Th17 cells but were seen to skew to a T_reg phenotype more rapidly, highlighting the importance in determining T_eff/T_reg cell fate. Although the role of CD6 in T_eff development is well characterized, here we sought to investigate the role of CD6 in the development and activity of T_reg cells.

Itolizumab, a humanized anti-CD6 monoclonal antibody that modulates cell surface levels of CD6, was used to generate D-CD6^low (derived CD6 low) T_regs which are T_regs derived from CD6^low naïve T cells, after which itolizumab was removed and T_regs were differentiated using a standard protocol. T_regs derived from itolizumab-treated D-CD6^low cells had greater co-expression of FOXP3 and HELIOS (~2-fold) vs. T_regs derived from isotype-treated CD6^high (D-CD6^high derived from CD6^high naïve T cells)cells prior to entering a standard T_reg suppression assay. These T_regs were also assessed for FOXP3 and HELIOS at the end of the suppression assay and D-CD6^low T_regs cells maintained greater co-expression over those D-CD6^high T_regs. Markers of active T_regs GITR and CTLA4 were also observed at higher levels on D-CD6^low T_regs compared to D-CD6^high T_regs. Furthermore, these D-CD6^low T_regs possessed greater suppressive function, with increased inhibition of proliferation by stimulated T responder cells by at least 50% more than D-CD6^high T_regs; this was verified in three different donors. Similar observations were made when measuring levels of cytokine. D-CD6^low T_regs were able to suppress T responder's production of Th1 and Th17 relevant cytokines (TNFα, IL-17A, and IL-22) by 60-90% compared to D-CD6^high T_regs.

This is the first study to directly characterize the role of CD6 in the development and activity of T_reg cells. These data suggest that reduced levels of cell surface CD6 associated with itolizumab treatment facilitate the development of T_reg cells with greater stability and suppressive activity. Consequently, modulating the levels of CD6 may improve immune tolerance during autoimmune and inflammatory diseases. Further, targeting the CD6 pathway may improve ex vivo generation of T_regs for therapies. A phase III study using itolizumab as a first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT05263999).

Ampudia:Equillium: Current Employment, Current equity holder in publicly-traded company. Chu:Equillium: Current Employment, Current equity holder in publicly-traded company. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company.