Abstract
Acquired aplastic anaemia (AA) is caused by T-cells migrating to and attacking bone marrow (BM) in response to chemokines (e.g., CXCR4). We investigated CXCR4 expressions on circulating T-cell subsets, plasma IL-17A concentrations, and their correlations with AA manifestations. We enrolled 71 patients with acquired AA (36 severe AA cases [SAA] and 35 non-severe AA cases [NSAA]) and 42 healthy volunteers. We used flow cytometry and ELISA to measure circulating CD4+ and CD8+ T-cells, their CXCR4 expressions, and plasma IL-17A concentrations. Compared to the healthy controls, SAA patients had fewer peripheral CD4+ T-cells, more CD8+ T-cells, and a significantly decreased CD4+/CD8+ ratio which was positively correlated with AA manifestations. Patients with SAA or NSAA had higher proportions of CD4+CXCR4+ and CD8+CXCR4+ T-cells, which were negatively correlated with haemoglobin concentrations and absolute neutrophil counts. Patients with SAA or NSAA had higher plasma IL-17A concentrations, which were negatively correlated with AA manifestations and the CD4+/CD8+ ratio. IL-17A concentrations showed a very week correlation with CD4+CXCR4+ T-cells frequencies, and no correlation with CD8+CXCR4+ T-cells frequencies. Aberrant CXCR4 expression may allow circulating T-cells, especially CD8+ T-cells, to infiltrate BM during AA progression. Elevated IL-17A concentrations may contribute to AA progression outside of the CXCR4-SDF-1α axis.
Highlights
Further studies of BM from patients with AA are needed to validate our findings
Gating on the CD4+ or CD8+ T-cells allowed us to calculate the frequencies of circulating CD4+CXCR4+ or CD8+CXCR4+ T-cells (Fig. 3A)
The SAA group had the highest frequency of CD8+CXCR4+ T-cells (89.1 ± 7.7%), which was followed by that in the NSAA group (82.4 ± 12.8%) and the control group (74.6 ± 16.8%)
Summary
The whole blood was used for flow cytometry. The flow cytometry was performed after incubating 50 μL of whole blood with monoclonal antibodies for 30 min at 4 °C. The monoclonal antibodies targeted human CD3 (clone SK7, PerCP-Cy5-5), CD4 (clone RPA-T4, FITC), CD8 (clone SK2, PE), and CXCR4 (CD184, clone 12G5, APC), and were all from BD Biosciences (San Diego, USA). The IL-17A level was determined using a specific human IL-17A Platinum ELISA kit (Cat#BMS2016; Bender Med Systems, Burlingame, USA). The significance level was set at 5% for all statistical tests. The data were initially analysed using analysis of variance or the Kruskal-Wallis H test. If a significant result was observed, the Student-Newman-Keuls or Mann-Whitney tests were used to detect inter-group differences. Spearman’s correlation coefficient was used to test the correlations between pairs of two continuous variables
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