| Part one The frequency and function of follicular helper T cells in children with Wiskott-Aldrich syndromeObjective:To analyze the impact of WAS gene mutation on the number and function of follicular helper T cells in children with Wiskott-Aldrich syndrome (WAS).Methods:Fresh blood samples of 35 WAS patients and 35 age-matched healthy children (HC) were collected. The frequency of circulating Tfh cells, subsets of circulating Tfh cells, and IL21-secreting Tfh were detected by flow cytometry (FCM). CD4+T cells of WAS patients and HC were sorted by FCM. The mRNA expression of Bcl6 and Blimp-1 in CD4+T cells of WAS patients and HC were detected by qPCR.Results:In WAS patients, we found that the frequency of circulating Tfh cells was significantly reduced (HC:7.0±0.6%; WAS:3.9±0.4%; P<0.0001), especially Thl-type (HC:37.8±1.6%; WAS:32.3±1.9%; P=0.02). The composition of CXCR5+cells within memory CD4+T cells was considerably decreased across all ages among WAS patients (HC: 21.Oil.4%; WAS:10.1±1.1%; P<0.0001). We also found circulating CXCR5+CD4+i T cells in WAS patients with high expression of inducible T cell costimulator (ICOS) as compared with control subjects (naive: HC:3.8±.3%; WAS 7.1±0.8%; P=0.001; PHA:HC:19±2.8%; WAS 28.6±2.6%; P=0.02). The frequency of IL21-secreting Tfh, IL21 and Blimp-1 mRNA expression in CD4+ T cells were not affected in WAS patients, while Bcl-6 expression was decreased in CD4+ T cells of WAS patients (HC:1.7±0.3%; WAS 0.5±0.1%; P=0.02).Conclusion:These findings suggest an essential role of WASp in the development of Tfh, and the decreased expression of Bcl-6 may be associated with decreased number of circulating Tfh cells.Part two Impact of WASp deficiency on secondary immune response of follicular helper T cellsObjective:To analyze the Impact of WASp deficiency on secondary immune response of follicular helper T cells in WAS ko mice.Methods:Six WAS ko mices and 6 WT mices were randomly chosen as immune group and NS control group respectively. Each group enrolled 3 mice. For immunization,400μL NS and 400μg NP-KLH was injected s.c. on the mouse flanks at dl and d30 separately. All of the animals were sacrificed 5 days after secondary immunization. Mouse spleen were harvested, blood was collected via cardiac puncture, and immune cells were isolated by sucrose density centrifugation using Lymphocyte Separation Media. The frequency of Tfh cells were detected by FCM.Results:In NS group, the percentage of CD4+CXCR5+ICOS+T cells (Blood, WT:2.3±0.3%; WAS 4.2±0.2%; P=0.01; Spleen, WT:4.1±0.9%; WAS 8.0±0.3%; P=0.02) and CD4+CXCR5+PD-1+T cells (Blood, WT:2.3±0.6%; WAS 5±0.3%; P=0.02; Spleen, WT:4.3±0.5%; WAS 8.6±0.3%; P=0.002) of peripheral blood and spleen cells in WAS ko mice was significantly increased as compared to WT. But there were no significant difference in the percentage of CD4+CD44hiCXCR5+ T cells of peripheral blood and spleen cells and the frequency of CD4+CXCR5+Bcl6+ T cells of spleen between WAS ko mice and WT. In NP-KLH group, the frequency of CD4+CD44hiCXCR5+ T cells (Blood, WT:4.9±0.1%; WAS 3.1±0.1%; P=0.001; Spleen, WT:6.7±0.3%; WAS 8.4±0.1%; P=0.008) of both peripheral blood and spleen and the frequency of CD4+CXCR5+Bcl6+ T cells (WT:6.8±0.5%; WAS 4.6±0.5%; P=0.04) of spleen were significantly reduced in WAS KO mice. Consistant with NS group, the percentage of CD4+CXCR5+ICOS+ T cells (Blood, WT:5.8±0.4%; WAS 3.2±0.5%; P=0.02; Spleen, WT:13.8±0.2%; WAS 16.7±0.5%; P=0.007) and CD4+CXCR5+PD-1+T cells (Blood, WT:3.9±0.5%; WAS 6.5±0.7%; P=0.04; Spleen, WT:14.2±0.5%; WAS 17.5±0.4%; P=0.008) of peripheral blood and spleen cells in WAS ko mice was significantly increased as compared with WT in both immune groups.Conclusion:WASp deficiency presents significant defects of secondary immune response of Tfh, probably because the WASp deficiency lead to the abnormal signal transfer between ICOS and Bcl6. |
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