The Role And Mechanism Of Follicular Helper T Cells And B Cell Subsets In Advanced Non-segmental Vitiligo

Vitiligo is a common hypopigmentation disease characterised with the formation of depigmented patches on skin and/or mucosa,which results from the loss or dysfunction of melanocytes.As often develops in exposed parts such as the face,it severely affects the patients'normal daily life.Due to the characteristics of difficult treatment and easy recurrence,vitiligo has long received attention from dermatologists.However,the pathogenesis of vitiligo remains unclear.It is generally believed that vitiligo may be caused by the interaction between various pathogenic mechanisms,which leads to the destruction of melanocytes,and finally develops the same clinical symptoms.Recently,it is generally believed that cytotoxic T cells(CD8⁺T cells)are the main killer cells in the immunological study of vitiligo.Various studies have focused on the chemotaxis,induction,recruitment and arrest of CD8⁺T cells,which have made some progress.However,there are few studies exploring the role of humoral immunity in vitiligo.Some studies suggested that the follicular helper T cells(T follicular helper cell,Tfh)participates in the regulation of the humoral immune and plays an important role in the differentiation of B cells,whose excessive auxiliary can lead to autoimmune disease.In order to further understand the autoimmune mechanism of vitiligo,we conducted a study on the auxiliary role of Tfh cells in humoral immunity in patients with advanced non-segmental vitiligo.In the first part,we collected peripheral blood from 75 patients with advanced non-segmental vitiligo,and included 67 healthy subjects with similar age and gender composition as controls.The proportion of Tfh cells in peripheral blood mononuclear cells and the proportion of each Tfh cell subsets in CD4⁺CXCR5⁺Tfh cells were detected by flow cytometry.The results showed that the proportions of CD4⁺CXCR5⁺Tfh cells,CD4⁺CXCR5⁺PD-1⁺Tfh cells and CD4⁺CXCR5⁺ICOS⁺Tfh cells in peripheral blood of patients with advanced non-segment vitiligo were significantly increased.In CD4⁺CXCR5⁺Tfh cells,the proportion of CXCR3^-CCR6^-Tfh2 subsets decreased significantly,while the proportion of CXCR3^-CCR6⁺Tfh17subsets increased significantly.The results also showed a declined Tfh1/Tfh17 ratio,and an elevated(Tfh2+Tfh17)/Tfh1 ratio.Using flow cytometry to detect the functional changes of the characteristic cytokine IL-21 secreted by Tfh cells,we found that the proportion of Tfh cells expressing IL-21 was significantly increased.Through the detection of inhibitory T cells expressing transcription regulator Foxp3 in Tfh cells,we found that the proportion of CD4⁺CXCR5⁺Foxp3⁺Tfr cells and Tfr/Tfh ratio decreased significantly.The results suggested an active status of Tfh cells and an imbalance of Tfr/Tfh exist in advanced non-segmental vitiligo.We hypothesized that this may lead to the overactivity of the germinal center,which helps B cells secrete excessive antibodies to participate in humoral immunity.Therefore,we further analyzed the expression levels of various antibodies in the peripheral blood of advanced non-segmental vitiligo patients as well as the proportion of B cell subsets in mononuclear cells.The results showed that the expression levels of Ig M and Ig G3 were significantly decreased,while the Ig G1 levels were significantly increased.In addtion,there were no significant differences in the expression levels of Ig A,Ig E,total Ig G,Ig G2 and Ig G4.Also,there was no significant difference in the proportion of CD19⁺B cells and subgroups of B cells(naive B cells,memory untransformed B cells,memory transformed B cells and double negative B cells).However,the proportion of CD19⁺CD27^hiIg D^-antibody secreting B cells(Sab)was significantly increased.Further studies showed that the proportion of immature transitional CD19⁺CD24^hiCD38^hiBreg cells in patients with advanced non-segmental vitiligo was significantly increased,but the proportion of IL-10 secreting inhibitory Breg cells in mononuclear cells and the expression level of IL-10 in plasma were both significantly decreased.These results suggested that the excessive accumulation of transitional CD19⁺CD24^hiCD38^hiBreg cells may lead to the decrease of mature Breg cells that mainly secrete IL-10.Moreover,their inhibitory function in germinal response and autoimmune antibodies secretion is also weakened.In the second part,we first verified the increased expression level of IL-1?in peripheral circulation and local skin lesions of patients with advanced non-segment vitiligo by ELISA and immunohistochemical staining.Simultaneously,we found that IL-1?can also synergize with IL-12 to induce the differentiation of naive CD4⁺T cells into CD4⁺CXCR5⁺Tfh cells,suggesting that IL-1?is involved in the immune regulation of vitiligo.To further study the role of IL-1?in vitiligo,we used q PCR and Western blotting to detect the direct effects of IL-1?and IL-1?on the melanin-producing ability of melanocytes.The results revealed that IL-1?and IL-1?exert opposite effects on melanogenesis:IL-1?can promote melanocyte proliferation,and enhancing the expression of melanin-producing proteins such as MITF,TYR,TYRP1 and TYRP2.IL-1?showed no effect on the activity of melanocytes,but inhibited the expression of MITF,TYR,TYRP1 and TYRP2.Furthermore,IL-1?could not enhance the ROS production of melanocytes and keratinocytes,while keratinocytes could increase ROS production under the stimulation of IL-1?,which suggesting that IL-1?can aggravate oxidative stress and may be involved in the initiating environment of vitiligo autoimmune response.In this study,the proportions of Tfh cells and B-cell subsets in peripheral blood and the expression levels of various antibodies in patients with advanced non-segmental vitiligo were systematically detected for the first time.The induction of IL-1?on melanocytes and Tfh cells was analyzed,and it was found that IL-1?and IL-1?in IL-1 family exhibited opposite effects on melanin production.The above-mentioned experimental results further enriched the research on humoral immunity of advanced non-segmental vitiligo,and explained the possible mechanism of IL-1?in vitiligo,providing laboratory basis for further exploration of new therapeutic targets.