Decreased Expression of Indoleamine 2,3-Dioxygenase 1 in Dendritic Cells From Patients with Immune Thrombocytopenia Induces Impaired Regulatory T-Cell Development

Abstract 696

Regulatory T cells (Tregs) are a subset of T cells involved in the maintenance of peripheral self-tolerance. Specifically, Tregs modulate the maturation and/or function of dendritic cells (DCs). In turn, along with their immunogenic role, DCs are also critical in maintaining tolerance to self-antigens by inducing Tregs via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1-expressing DCs may play a role in preventing the initiation of autoimmune disorders.

Immune Thrombocytopenia (ITP) is an autoimmune disorder in which platelet surface proteins become antigenic and stimulate the immune system. However, in ITP, the interaction between DCs and Tregs has never been investigated, although decreased numbers of Tregs as well as altered DCs have been described. Therefore, in the present study, we investigated whether, in ITP: 1) IDO1 expression/activity is decreased in monocyte-derived DCs (Mo-DCs); 2) the mechanism of Tregs generation is impaired; 3) Mo-DCs maturation is abnormally modulated by Tregs.

We studied 54 patients with active ITP. Nineteen patients were newly diagnosed and 35 patients had persistent (25 cases) or chronic (10 cases) ITP. At the time of samples collection, all patients with persistent or chronic ITP were out of any treatment for at least two months and none of the patients received previous treatment with rituximab or were splenectomized.

We demonstrated that in mature Mo-DCs from ITP patients the absolute number of IDO1 transcripts was significantly reduced as compared with healthy donors (973.000±771.000 vs 2.409.000±1.592.000 (IDO1 copy number/ABL1 copy number)*10.000; p<0.04). Accordingly, when we evaluated kynurenine levels as an index of IDO1 enzyme activity, we found that kynurenine concentration in the supernatants from equal numbers of mature Mo-DCs was significantly lower in ITP patients (40.7±12.5 μM) as compared to healthy subjects (66.5±9.4 μM) (p<0.05). We therefore assessed whether in ITP Mo-DCs, which have IDO1 reduced expression and activity, are less efficient in generating Tregs in vitro. We found that after co-cultures of Mo-DCs from healthy subjects or ITP patients with autologous CD4⁺CD25⁻ T cells, the mean percentage of FoxP3⁺ cells (gated on CD4⁺CD25^high T cells) was significantly reduced in ITP patients (54.4±4.5%) in comparison with that of healthy individuals (88.3±5.8%; p<0.01). When we evaluated whether these in vitro generated Tregs possess suppressive activity, we demonstrated that the inhibition of the proliferative response of CD4⁺CD25⁻ cells in coculture with autologous in vitro generated CD4⁺CD25⁺ T cells (100:1 ratio) was significantly lower in ITP patients (25±4%) as compared with healthy controls (47±6%; p<0.05). To evaluate whether Tregs induce functional differences in Mo-DCs, we first assessed the production of cytokines in the supernatants of normal immature Mo-DCs co-cultured with highly purified circulating CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells from ITP patients or healthy controls. We found that in ITP patients the concentration of IL-10 was significantly reduced in the supernatant of Mo-DCs plus CD4⁺CD25⁺ T cells as compared with the normal counterparts (99±26 pg/mL vs 218±19 pg/mL; p<0.01). However, since IL-10 plays a central role in immune tolerance and can be produced by either DCs or T cells, we performed the intracytoplasmic staining of IL-10 to evaluate whether its secretion was due to Mo-DCs upon co-culture and/or produced by Tregs. The mean percentage of IL-10-producing Tregs was significantly lower in ITP patients (19.3±8.0 %) in comparison with that of normal individuals (34.9±6.6 %; p<0.04).We therefore analyzed the capacity of highly purified circulating Tregs of inhibiting Mo-DCs maturation. We found that, at variance with healthy controls, highly purified CD4⁺CD25⁺ T cells from ITP patients failed to inhibit the expression of CD80 and CD86 molecules on Mo-DCs.

In summary, this study demonstrates that in ITP low IDO1 expression/activity in DCs results in the reduced ability of generating Tregs. In turn, circulating Tregs with impaired IL-10 production show decreased capacity of inhibiting DCs maturation. Taken together these data suggest that in ITP the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role. As a consequence, DCs are less “tolerogenic” and Tregs are defective in their regulatory function.