Immunosuppressive effects of a CD4 receptor down-modulator: in vitro study of the signal peptide-dependent ER translocation inhibitor cyclotriazadisulfonamide (CADA)

The small molecule cyclotriazadisulfonamide (CADA) was identified as an inhibitor of human immunodeficiency virus replication. It was shown that its antiviral potency is due to signal peptide-dependent inhibition of co-translational translocation of human CD4, the primary receptor for human immunodeficiency virus entry. The CD4 receptor plays an important role in the immune system, especially during activation of T cells by stabilizing the interaction between T cells and antigen presenting cells, by contributing to intracellular signaling and by enhancing T cell sensitivity. As CADA down-modulates the CD4 receptor, we were interested whether the compound could have some immunosuppressive potential. A possible application of CADA could then be in the prevention of allograft rejection after transplantation, as there is a need for new drugs that promote immune tolerance without the side effects observed with current immunosuppressive drugs. In this Ph.D. work we show that CADA has an in vitro immunosuppressive effect by inhibiting lymphocyte proliferation in the mixed lymphocyte reaction and after activation with CD3/CD28 beads or phytohemagglutinin. No significant inhibition of T cell activation by CADA was observed when CD4+ T cells were activated by superantigens, perhaps due to obviation of the CD4 receptor in this activation model. The immunosuppressive effect of CADA involves both CD4+ and CD8+ T cells and is probably initiated by direct down-modulation of the CD4 receptor, in combination with two newly identified targets of CADA, the CD8 receptor and the co-stimulatory molecule CD28. Immunosuppression by CADA is characterized by reduced expression of the T cell receptor complex members CD3 and TCRα/β, of the co-stimulatory molecules GITR, OX40 and 4-1BB, as well as of the α-chain of the IL-2 receptor also known as CD25. Diminished expression of CD25 is probably due to reduced intracellular phosphoSTAT5 levels in the presence of CADA and is likely involved in inhibition of lymphocyte proliferation. The level of soluble CD25, a marker of excessive T cell activation, and of several cytokines including IL-2, are decreased by treatment with CADA. As the sorting receptor sortilin was identified as an additional substrate of CADA and as it is involved in the secretion of several cytokines, we were interested whether CADA-induced down-modulation of sortilin could be involved in the immunosuppressive effect of CADA. The sortilin inhibitor AF38469 has no immunosuppressive effect in the mixed lymphocyte reaction, suggesting that sortilin is not essential for proper immune function and that down-modulation of sortilin does not contribute to the immunomodulatory effect of CADA. In vitro immunosuppression by CADA is less potent compared to the clinically used antiproliferative agent mycophenolate mofetil, but CADA is characterized by almost no in vitro cytotoxic and cytostatic effects, two promising characteristics for a new immunosuppressive agent. The immunosuppressive capacity of CADA in vitro however is more potent than that of Clenoliximab, a nondepleting anti-CD4 monoclonal antibody that reached phase II clinical trial for the treatment of rheumatoid arthritis. This Ph.D. work demonstrates a profound and consistent immunosuppressive profile of CADA in different in vitro T cell activation models and suggests that this compound has interesting characteristics for further exploration as a potential new immunosuppressive drug. Although the effects of CADA on intracellular T cell signaling and regulatory T cell function have not been elucidated yet, this study provides a solid in vitro foundation for future in vivo assessment of the immunosuppressive potential of CADA.

Jaar van publicatie:2019

Toegankelijkheid:Open