Simultaneous inhibition of human CD4 and 4-1BB biogenesis suppresses cytotoxic T lymphocyte proliferation

The small molecule cyclotriazadisulfonamide (CADA) down-modulates the human CD4 receptor, an important factor in T cell activation. Here, we addressed the immunosuppressive potential of CADA using in vitro activation models. CADA inhibited lymphocyte proliferation in a mixed lymphocyte reaction, and when human PBMCs were stimulated with CD3/CD28 beads or phytohemagglutinin. The immunosuppressive effect of CADA involved both CD4+ and CD8+ T cells but was, surprisingly, most prominent in the CD8+ T cell subpopulation where it inhibited cell-mediated lympholysis. We discovered a direct down-modulatory effect of CADA on 4-1BB (CD137) expression, a survival factor for activated CD8+ T cells. More specifically, CADA blocked 4-1BB protein biosynthesis by inhibition of its co-translational translocation across the ER membrane in a signal peptide-dependent way. This study demonstrates that CADA, as potent down-modulator of human CD4 and 4-1BB, has promising in vitro immunomodulatory characteristics for future in vivo exploration as immunosuppressive drug. ### Competing Interest Statement The authors have declared no competing interest. * CADA : cyclotriazadisulfonamide CD : cluster of differentiation CTPS1 : cytidine triphosphate synthase 1 ER : endoplasmic reticulum hCD4 : human CD4 IL : interleukin Lck : lymphocyte C-terminal Src kinase mCD4 : murine CD4 MLR : mixed lymphocyte reaction MMF : mycophenolate mofetil PBMC : peripheral blood mononuclear cell PHA : phytohemagglutinin pSTAT5 : phosphorylated signal transducer and activator of transcription 5 sCD25 : soluble CD25 SP : signal peptide