Low mucosal-associated invariant T-cell number in peripheral blood of patients with immune thrombocytopenia and their response to prednisolone

Abstract

Mucosal-associated invariant T (MAIT) cells help protect against certain infections and are related to some autoimmune diseases. Immune thrombocytopenia (ITP) is a relatively rare hematological autoimmune disease associated with low platelet count. We designed a cross-sectional study wherein we examined peripheral blood samples of patients with ITP for the number of MAIT cells (CD3+TCR-Vα7.2+CD161+IL-18Rα+ lymphocytes) and their CD4/8 subsets (by flow cytometry) and levels of cytokines (by multiplex assays). The study cohort included 18 patients with ITP and 20 healthy controls (HCs). We first compared the number of MAIT cells between HCs and patients with ITP and then performed subgroup analysis in patients with ITP. The number of total MAIT cells in patients with ITP was significantly lower than that in HCs (p < 0.0001), and the CD4-CD8+ subset of MAIT cells showed the same trend. Moreover, patients with ITP refractory to prednisolone exhibited a significantly lower number of total MAIT and CD4-CD8+ MAIT cells than patients sensitive to prednisolone. The number of total MAIT and CD4-CD8+ MAIT cells was not correlated with the response to thrombopoietin receptor agonist treatment or with Helicobacter pylori infection. We found no relation between cytokine levels and response to prednisolone treatment, although the levels of IP-10 and RANTES showed a correlation with the number of total MAIT and CD4-CD8+ MAIT cells. In conclusion, total MAIT and CD4-CD8+ MAIT cells in peripheral blood were decreased in patients with ITP, correlating with their response to prednisolone.

Fig 1. Number and rate in the blood CD3⁺ T cells of mucosal-associated invariant T (MAIT) cells in the peripheral blood of healthy controls (HCs) (n = 20) and patients with immune thrombocytopenia (ITP) (n = 18).

(A) Number and frequency in the blood CD3⁺ T cells of total MAIT cells in the peripheral blood of HCs and patients with ITP. We defined the total MAIT cells by gating CD3⁺TCR-Vα7.2⁺ lymphocytes and then gating the CD161⁺IL-18Rα⁺ subset. The number of MAIT cells of patients was significantly lower than that of HCs. (B) Number and frequency in the blood CD3⁺ T cells of CD4⁺CD8⁺ MAIT cells subset in the peripheral blood of HCs and patients with ITP. To distinguish the CD4 CD8 subset of MAIT cells, we gated CD161⁺TCR-Vα7.2⁺ lymphocytes and after that divided them into CD4⁺CD8⁺, CD4⁺CD8⁻, CD4⁻CD8⁻, and CD4⁻CD8⁺ subsets. (C) Number and frequency in the blood CD3⁺ T cells of CD4⁺CD8⁻ MAIT cells subset in the peripheral blood of HCs and patients with ITP. (D) Number and frequency in blood CD3⁺ T cells of CD4⁻CD8⁻ MAIT cells subset in the peripheral blood of HCs and patients with ITP. (E) Number and frequency in the blood CD3⁺ T cells of CD4⁻CD8⁺ MAIT cells subset in the peripheral blood of HCs and patients with ITP. This subset showed the same trend as total MAIT cells. Statistical significance is calculated by the Mann–Whitney U test, *p < 0.05, **p < 0.005, ***p < 0.0001.

Fig 2. Comparison of the number of CD4⁻CD8⁺ MAIT cells between the subgroups of patients with ITP based on the response to treatment or the presence of Helicobacter pylori infection.

(A) The number of total MAIT, CD4⁻CD8⁺ MAIT, and CD4⁻CD8⁻ MAIT cells in responders to prednisolone treatment [ITP patients achieving complete response (CR) and response (R)] (n = 7) and non-responders to prednisolone treatment [ITP patients exhibiting no response (NR) and loss of R] (n = 8). The non-responders exhibited a significantly lower number of total MAIT and CD4⁻CD8⁺ MAIT cells than the responders. (B) The number of total MAIT, CD4⁻CD8⁺ MAIT, and CD4⁻CD8⁻ MAIT cells in high responders to thrombopoietin receptor (TPO-R) agonist treatment [ITP patients achieving CR] (n = 11), and responders to TPO-R agonist treatment [ITP patients going no further than R] (n = 5). We found no significant changes in the number of total MAIT, CD4⁻CD8⁺ MAIT, or CD4⁻CD8⁻ MAIT cells between the subgroups. (C) The number of total MAIT, CD4⁻CD8⁺ MAIT, and CD4⁻CD8⁻ MAIT cells in patients with ITP with (n = 9, 8 of them were treated with prednisolone) or without (n = 9, seven of them were treated with prednisolone) H. pylori infection. We found no significant changes in the number of total MAIT, CD4⁻CD8⁺ MAIT, or CD4⁻CD8⁻ MAIT cells between the two subgroups. Statistical significance was calculated by the Mann–Whitney U test, *p < 0.05.

Fig 3. Receiver operating characteristic curve showing the cut-off value of the number of CD4⁻CD8⁺ MAIT cells in the peripheral blood for the corticosteroid-sensitive subgroup (CR and R) versus the corticosteroid-refractory subgroup (NR and loss of R) in patients with ITP.

The area under the curve was 0.875, and a cut-off value <600 cells/ml signaled a sensitivity and specificity of refractoriness to corticosteroid treatment of 87.5% and 85.7%, respectively.

Fig 4. Correlations between the number of CD4⁻CD8⁺ MAIT cells in peripheral blood and the levels of cytokines in patients with ITP (n = 15).

(A) Correlation between the number of total MAIT, CD4⁻CD8⁺ MAIT, and CD4⁻CD8⁻ MAIT cells and the concentration of IP-10 in the plasma of patients with ITP. IP-10 showed a negative correlation with the number of total MAIT and CD4⁻CD8⁺ MAIT cells. (B) Correlation between the number of total MAIT, CD4⁻CD8⁺ MAIT, CD4⁻CD8⁻ MAIT cells and the concentration of RANTES in the plasma of patients with ITP. RANTES also exhibited a negative correlation with the number of total MAIT and CD4⁻CD8⁺ MAIT cells. Spearman’s rank correlation coefficient was calculated, and hypothesis testing was performed to identify statistical significance.