Abstract CD6 is a co-stimulatory receptor expressed on the surface of T cells that promotes immune synapse formation, T cell activation, and migration into tissues by engaging its ligand, ALCAM. CD6high cells have been described to be more pathogenic relative to CD6low cells. Furthermore, T cells in CD6 knockout mice have decreased proliferation and inflammatory responses. Hence, a therapeutic that can modulate surface levels of CD6 may have favorable outcomes for autoimmune/inflammatory conditions. We recently demonstrated that the anti-CD6 mAb, itolizumab, induces proteolytic cleavage of cell surface CD6. The goal of the work shown here is to characterize the mechanism of cleavage and the downstream functional consequences of reduced cell surface CD6. Treatment of PBMCs with itolizumab induces CD6 cleavage from the cell surface and a concomitant increase in the soluble form detected in the supernatant. Itolizumab-induced loss of CD6 was not observed with isolated T cells, however CD6 levels were reduced by 87% when T cells and monocytes were cocultured with itolizumab. The cleavage of CD6 is initiated through rapid cell-to-cell contact between T cells and monocytes via FcγRI. The dose-dependent reduction in surface levels of CD6 positively correlates with decreases in T cell activation markers such as CD25, PD-1, and CD71 (Pearson, p<0.001) and cytokine production including IL-2 (p<0.01) and TNFα (p<0.05). Itolizumab-treated cells are also less alloreactive as shown by a reduction in proliferation and cytokine production of responder cells in a mixed-lymphocyte reaction. This data further supports targeting CD6 as an effective means to inhibit pathogenic T cell activity in the treatment of autoimmune and inflammatory diseases.