Platelets modulate the CD4 T cell immune response during immune thrombocytopenia.

Abstract Introduction. The immunomodulatory functions of platelets in immune thrombocytopenia (ITP) have been scarcely investigated although they showed pro- or anti-inflammatory properties depending on the clinical setting, notably in autoimmune diseases. In ITP, T cell dysregulation is clearly established with a pro-inflammatory Th1/Th17 polarization and a decrease in the anti-inflammatory response mediated by regulatory T cells. To determine the potential effects of platelets on the CD4 T cell response, we performed platelet phenotyping and assessed their effect in vitro during coculture with CD4 T cells. Methods. The platelet phenotype of ITP patients (n=38) was compared according to disease activity (platelets <30 G/L, n=13, or platelets between 30-100 G/L, n=25) with that of healthy subjects (n=13). The expression of different markers was measured by flow cytometry to assess activation (CD62P, PAC-1), degranulation (CD63), antigenic presentation (HLA-DR), expression of costimulatory (CD40, CD40L, CD80, CD86) and inhibitory molecules (PDL1, GARP/TGF-β). The impact of platelets on CD4 T cells was determined by measuring their proliferation upon stimulation (CD3/CD28 microbeads) with or without platelets (25 platelets/1 lymphocyte). The phenotyping of CD4 T cell included the expression of activation markers (CD25), inhibitory molecules (CTLA-4, GARP/TGF-β), polarization (Th1/IFN-γ, Th2/IL-4, Th17/IL-17) and the induction of regulatory T cells (CD4+CD25HiFoxp3+). Results. The median age of ITP patients was 57 [Q1-Q3:40-74] years with a median platelet count of 41 [25-70] G/L. The female/male sex ratio was 1.9. ITP was considered primary in 92%, 3 patients also presenting antiphospholipid syndrome, Grave disease or Sjogren disease. ITP was newly-diagnosed in 37%, persistent in 13% and chronic in 50% cases. While the proportion of platelets expressing CD62P was similar between controls and patients, the frequency of platelets binding to PAC-1, i.e. with an activated conformation of GPIIb/IIIa, was higher in ITP patients (2.3% [1.7-3.5] vs. 0.8% [0.6-1.4], p<0.0001), and even higher in patients with platelet count <30 G/L (3.0% [2.3-4.1]). The frequency of degranulated platelets assessed by CD63 expression was increased in ITP patients (1.1% [0.7-1.9] vs. 0.4% [0.3-0.7], p<0.0001). The proportions of platelets expressing HLA-DR and costimulatory molecules such as CD40, CD40L, CD80 and CD86 were also higher in ITP as compared to controls (1.4% [0.9-2.8] vs. 0.4% [0.2-1.5], p=0.003; 0.8% [0.4-1.4] vs. 0.2% [0.2-0.5], p=0.0009; 1.9 [0.9-3.6] vs. 0.2 [0.1-0.2], p<0.0001; 3.1% [1.3-4.7] vs. 0.7% [0.6-1.0], p=0.005; 1.6% [1.0-2.9] vs. 0.4% [0.2-0.9], p=0.0008, respectively). The number of platelets expressing the inhibitory molecules PDL1, TGF-β or its anchoring protein GARP, was higher in ITP than in controls (1.2% [0.8-2.5] vs. 0.6% [0.5-1.1], p=0.005; 1.5% [0.9-2.0] vs. 0.6 [0.4-1.1], p=0.0001; 1.1% [0.8-1.7] vs. 0.6% [0.5-0.9], p=0.0007, respectively). Coculture of CD4 T cells with platelets led to an inhibition of their proliferation, that was higher in ITP than in controls (66.6% [53.2-77.3] vs. 47.4% [28.6-55.6], p=0.01). There was also an induction of Th17 cells in presence of platelets (1.9% [1.2-2.4] to 3.3% [2.0-4.6], p=0.01), while Th1 and Th2 percentages remained stable. Platelets also promoted the expression of inhibitory molecules by CD4 T cells, such as TGF-β (0.9% [0.7-1.2] to 3.9% [2.9-5.7], p=0.002) and CTLA-4 (3.9% [2-6] to 9.2% [6.6-24.1], p=0.001). Finally, platelets induced regulatory T cell differentiation (2.4% [1.1-4.0] to 7.7% [4.6-12.0], p=0.002). Conclusion. The proportions of activated platelets, together with platelets expressing pro- and anti-inflammatory markers are increased in ITP. In vitro, platelets promote a Th17 skewing. However, this is associated with increased expression of the inhibitory molecules CTLA-4 and TGF-β by CD4 T cells, as well as increased differentiation toward regulatory T cells, arguing for a global inhibitory effect, as supported by the inhibition of CD4 T cell proliferation. Given the reduced platelet count in ITP, this effect is most probably abrogated, suggesting that low platelet count might contribute to the pro-inflammatory CD4 T cell response in ITP. On the other hand, normalization of platelet count upon therapies might directly participate to remission by restoring immune tolerance.