The Role Of TSLP And CD8+ T Cells Participating In The Pathogenesis Of IgG4-related Disease

Objectives:To figure out the expression and functions of thymic stromal lymphopoietin(TSLP)in IgG4-related disease(IgG4-RD).Methods:Seventy-one treatment-naive IgG4-RD patients and forty-one healthy controls(HC)were enrolled.Plasma TSLP levels of IgG4-RD patients and HC were tested by Elisa(enzyme-linked immunosorbent assay),and its two receptors:TSLPR(TSLP receptor)and IL-7Ra(interleukin 7 receptor a)were detected by flow cytometry.Expressions of TSLP in involved organs were stained by immunohistochemistry,and immunofluorescence was performed to detect the location of TSLPR and immune cells.CD 19+B cells sorted by magnetic beads were stimulated with TSLP for 5 days,and proliferation,apoptosis,and B cell subsets were analyzed by flow cytometry,and IgG,IgG4 and IgE levels in the supernatant of B cells were detected by Elisa on day 7.B cells activated by TSLP for 3 days,were co-cultured with naive CD4+T cells for 5 days,and polarization of T cells were tested by flow cytometry.Activated signaling pathway was identified by RNA-sequencing and was verified by Western Blot.Anti-TSLP therapy was adapted in LatY136F knock-in mice,and inflammation and fibrosis of involved organs were analyzed by HE(hematoxylin-eosin staining)and Masson staining.Results:TSLP levels were increased in the plasma of IgG4-RD patients and were positively correlated with serum IgG,IgG1,IgG4,IgE levels and responder index(RI).TSLPR were upregulated in CD4+T cells and CD 19+B cells.Massive infiltration of TSLP was observed in the submandibular glands(SMGs)of IgG4-RD patients and immunofluorescence revealed that TSLPR was co-localized with CD 19+B cells.TSLP promoted B cell proliferation and expansion of plasmablasts,and TSLP-activated B cells polarized na?ve CD4+T cells into follicular helper T(Tfh)cells through OX40L.RNA-sequencing identified JAK(Janus kinase)-STAT(signal transducers and activators of transcription)signaling pathway in TSLP-activated B cells and it was verified by Western Blot.Western Bolt also revealed the upregulated levels of phosphorylation of JAK2 and Stat3 in B cells of patients compared with HC.Anti-TSLP therapy alleviated the inflammation of lung in LatY136F knock-in mice but did not relive the fibrosis in involved organs.Conclusions:Elevated TSLP expression in IgG4-RD promoted B cells proliferation and activated JAK-STAT signaling pathway.TSLP-activated B cells polarized naive CD4+T cells into Tfh cells through OX40L.Anti-TSLP therapy alleviated the inflammation of lung in LatY136F knock-in mice.TSLP was implicated in the pathogenesis of IgG4-RD and could be served as a potential therapeutic target.Objectives:To assess the expression and functions of CD8+ T cells in IgG4-related disease(IgG4-RD).Methods:The proportions of CD8+ T cells and NK cells in peripheral blood of IgG4-RD patients and HC(healthy controls),as well as secretion ability of IFN-γ,TNF-α,granzyme B(GZMB),and perforin 1(PFN1)in CD8+T cells and NK cells,were detected by flow cytometry.At the same time,the expressions of GSDM(gasdermin)protein family in the submandibular gland of patients with IgG4-RD or patients with chronic submandibular inflammation were analyzed by immunohistochemistry and Western Blot.The CD8+T cells and NK cells selected by magnetic beads were co cultured with epithelial cells of submandibular gland or pancreatic epithelial cells for 6 hours.Then the secretions of lactate dehydrogenase(LDH)in the culture supernatant were tested,and apoptosis of epithelial cells were measured by flow cytometry,and the GSDME fragment of epithelial cells were detected by Western Blot.The supernatant of CD8+T cells were further used to stimulate the epithelial cells of submandibular gland or pancreatic epithelial cells for 16 hours,and LDH secretion,apoptosis,and GSDME fragments of epithelial cells were detected.By adding functional inhibitors of IFN-y,TNF-α,and GZMB to the supernatant of CD8+T cells to analyze the impact of these factors on the apoptosis and LDH secretion of epithelial cells.The supernatant of pyroptotic epithelial cells were used to stimulate fibroblasts and collagen related genes were quantified by polymerase chain reaction(PCR).Furthermore,flow cytometry was used to detect the percentages,activation,and secretion abilities of CD8+T cells and NK cells in the spleen,liver,and lung of LatY136F knock-in mice and wild type(WT)mice.Results:The proportions of CD8+T cells in the peripheral blood of IgG4-RD patients were comparable with HC,but the functions of secreting IFN-γ,TNF-α,and GZMB in CD8+T cells were significantly increased.However,there were no significant difference in the proportions and secretion abilities of NK cells between IgG4-RD patients and HC.Immunohistochemistry showed that the infiltration of CD8+T cells in the submandibular gland of IgG4-RD patients were obviously increased.Western Blot showed that the expression levels of GSDME N terminal in the submandibular gland of IgG4-RD patients were higher than that of the control groups.CD8+T cells as well as its supernatant could apparently promote LDH secretion,apoptosis,and cleavage of GSDME in epithelial cells of submandibular gland and pancreatic epithelial cells.Inhibitor of IFN-γ significantly reduced the levels of LDH secretion and apoptosis of epithelium cells of the submandibular gland induced by the supernatant of CD8+T cells.The supernatant of pyroptotic epithelial cells obviously upregulated the expression of collagen genes of COL1A1 and ACTA2 in fibroblasts.Compared with WT mice,the proportions of CD8+T cells in the spleen of LatY136F knock-in mice were decreased,but the expression levels of CD69 were increased and the abilities of secreting TNF-α,GZMA,and GZMB were significantly increased.Conclusions:The abilities of secreting IFN-γ,TNF-α,and GZMB of CD8+T cells in peripheral blood of IgG4-RD patients were enhanced,and the expression levels of CD8+T cells and GSDME-N terminals in submandibular gland tissue of IgG4-RD patients were increased.CD8+T cells as well as its supernatants could promote the pyroptosis of epithelial cells,and functionally inhibition of IFN-γ in the supernatant of CD8+T cells significantly reduced the secretion levels of LDH and apoptosis of epithelial cells.The supernatant of pyroptotic epithelial cells enhanced the expression levels of collagen related genes in fibroblasts.