Anti-CD6 monoclonal antibody UMCD6 induces CD6 internalization, remodels costimulatory receptor availability, and modulates CD4+ T-cell activation.

INTRODUCTION: CD6 is an immunological synapse-associated receptor that integrates adhesive and signaling cues to shape T-cell activation. We previously showed that, in CD6-humanized mice, the anti-human CD6 monoclonal antibody UMCD6 suppresses disease severity and Th1/Th17 responses in models of autoimmunity, but its mechanisms of action in human T cells remained incompletely defined. METHODS: Primary human peripheral blood mononuclear cells and isolated T cells were used. UMCD6-induced changes in the surface expression, internalization, and localization of CD6, CD2, and CD28 on CD4+ T cells were analyzed by conventional and imaging flow cytometry. Functional effects of UMCD6 were assessed by measuring activation markers, intracellular IL-2 production, Ki-67 expression, and cytokine secretion following anti-CD3/CD28 stimulation. Proximal T-cell receptor signaling was evaluated by LAT phosphorylation and Indo-1-based calcium flux analysis, whereas PKC-θ phosphorylation was assessed by phospho-flow cytometry as a readout of downstream costimulatory signaling. RESULTS: UMCD6 induced remodeling of the T-cell surface receptor landscape, including reduced surface CD6 and altered surface expression of CD2 and CD28. Imaging flow cytometry demonstrated redistribution and internalization of CD6 and CD2, accompanied by more modest changes in CD28 localization. Limited colocalization of CD6 with either CD2 or CD28 suggested broader reorganization of the costimulatory receptor network rather than passive co-internalization. Functionally, UMCD6 suppressed activation-induced expression of CD25, CD69, and OX40, reduced intracellular IL-2 production, and decreased secretion of IL-2, TNF-α, IFN-γ, and IL-17A, with more pronounced effects in CD4+ than CD8+ T cells. UMCD6 preserved proximal T-cell receptor signaling, including LAT phosphorylation and calcium flux, but was associated with a modest reduction in the early peak fold induction of PKC-θ phosphorylation. DISCUSSION: These findings support a model in which UMCD6 remodels the availability and organization of costimulatory receptors rather than inducing simple co-internalization of CD6 with CD2 or CD28. This receptor reorganization, together with a modest reduction in PKC-θ-associated signal amplification, may contribute to the preferential suppression of CD4+ T-cell activation without broadly disrupting proximal T-cell receptor signaling.