Decreased Expression of Indoleamine 2,3-Dioxygenase 1 (IDO 1) in Dendritic Cells From Patients with Immune Thrombocytopenia (ITP) Correlates with Impaired Regulatory T Cells Development

Abstract 2770

Recent studies suggest a bi-directional interaction between regulatory T cells (Tregs) and dendritic cells (DCs). Despite their role as inducers of immunity, DCs may also be critical in maintaining tolerance to self-antigens by inducing Tregs. Specifically, Indoleamine 2,3-dioxygenase 1 (IDO 1) expressing DCs expand Tregs. In turn, Tregs, which express CTLA4, engage its receptors B7-1(CD80)/B7-2(CD86), thus inducing the expression of IDO1 enzyme in DCs. IDO1 expression leads to tryptophan depletion and immunosuppressive kynurenine enrichment in the tissue. Both factors drive the de novo generation of Tregs from CD4⁺ T cells. In addition, Tregs may modulate the maturation and/or function of DCs, which are required for the activation of effector T cells. Tregs out compete with naïve T cells in aggregating around DCs. After forming aggregates, Tregs specifically downregulate the expression of CD80 and CD86 on DCs. The result of the interaction between Tregs and DCs is the impaired capacity of the antigen presenting cells to establish long lasting interactions with effector T cells. Therefore, given the crucial role of DCs and Tregs in initiating, regulating, maintaining or repressing immune responses, investigations on their interaction is of great importance for better elucidating the pathogenesis of ITP. Of note, in ITP this interaction and its pathogenetic role have never been investigated in depth. In this paper, we enumerated and functionally characterized Treg subsets, exploring whether in ITP abnormal Tregs interactions with DCs and/or effector T cells might play a pathogenetic role. Specifically, we studied whether, in ITP patients: 1) DCs maturation is differentially modulated by Tregs from ITP patients as compared with healthy donors; 2) the mechanism of Tregs generation is altered. Forty adult ITP patients, newly diagnosed (14 cases) or with persistent (20 cases) or chronic ITP (9 cases) were studied after informed consent. At the time of the study, patients with persistent or chronic ITP were out off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 53×10⁹/L (range 8–99). Allogeneic Mixed Leukocyte Reaction (MLR) was performed to test the suppressive activity of Tregs. To analyze the in vitro ability of highly purified CD4+CD25+ T cells to inhibit DCs maturation, normal immature CD14-derived DCs were cultured alone and with allogeneic CD4+CD25+ T cells from healthy subjects or ITP patients in the presence of Lipopolysaccaryde. CD80 and CD86 expression of CD14-derived DCs was then tested at flow cytometry. To evaluate the in vitro conversion of non Tregs into Tregs, CD4+CD25- T cells were cultured alone and with autologous mature CD14-derived DCs from ITP patients and healthy subjects. The percentages of CD4+CD25highFoxP3+ T cells were quantitated at flow cytometry. mRNA IDO1 expression of immature and mature CD14-derived DCs was then evaluated by Real-time RT-PCR. To determine IDO1 enzyme activity kynurenine levels were measured in the supernantants of mature CD14-derived DCs. We found that in ITP Tregs show lower ability to suppress T cell proliferation and to inhibit CD14-derived DCs maturation because they do not affect the expression of the costimulatory molecules CD80 and CD86. We show that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low/negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). In addition, in ITP patients we document that the low number of circulating Tregs may be due to the reduced ability of mature CD14-derived DCs to convert non-Treg cells (CD4+CD25-) into Tregs (CD4+CD25highFoxP3+ Tregs). This is related to reduced expression and activity of the IDO1 enzyme in mature CD14-derived DCs from ITP patients, since DCs expressing IDO1 favour Treg generation. In conclusion, taken together our data demonstrate that in ITP the cross-talk between Tregs and DCs is impaired and plays a pathogenetic role. As a consequence, we found the generation of more immunogenic DCs and defective Tregs.