Follicular Helper T Cell Derived Small Extracellular Vesicles Exacerbate The Severity Of Collagen-Induced Arthritis By Enhancing B Cell Responses

Rheumatoid arthritis（RA）is a common autoimmune disease characterized by chronic inflammation of the joints and synovial membranes,which significantly impacts the life quality of patients.Follicular helper T cells（Tfh）,a subpopulation of CD4⁺T cells that specialize in helping B cells,play a crucial role in B cell differentiation,antibody production,and class switching.In the pathogenesis of RA,abnormally activated Tfh cells can regulate the autoantibody secretion of B cells,which further exacerbate the disease progression.Small extracellular vesicles（sEVs）are microvesicles secreted by cells that selectively carry specific components from their source cells and participate in intercellular signaling.However,it has not been reported whether Tfh cells can regulate the autoantibody production of B cells and thus influence the RA pathogenesis through sEVs.ObjectiveInvestigate the role of Tfh cell-derived sEVs（Tfh-sEVs）on B cell differentiation and antibody production.Search for critical functional molecules of Tfh-sEVs in regulating B cell responses and revealing their regulatory mechanisms.Observe the role of Tfh cell-derived sEVs in the progression of collagen-induced arthritis.Assess the potential application value of plasma Tfh-like-sEVs in RA.Methods1.Analysis of the role of Tfh cell-derived sEVs in B cell differentiation and antibody productionMice were immunized with ovalbumin（OVA）,spleen Tfh cells（CD4⁺CD19^-CXCR5⁺GITR^-）,non-Tfh cells（CD4⁺CD19^-CXCR5^-GITR^-）,and B cells were sorted by immunomagnetic beads combined with flow cytometry（FCM）.Tfh or non-Tfh cells were co-cultured with B cells,the percentages of germinal center（GC）-like B cells（CD19⁺Ig G1⁺GL-7⁺）and plasmablasts（CD19⁺CD138⁺）in culture system were measured by FCM.Enzyme-linked immunosorbent assay（ELISA）was performed to detect total Ig G and anti-OVA Ig G levels in culture supernatant.Culture Tfh cells in vitro,collect Tfh cell-culture supernatant（Tfh-SN）.sEVs-depleted supernatant(Tfh-SN^{sEVs-depleted})was prepared by anti-CD63 magnetic beads,B cells were cultured with this supernatant for 3 days.The percentages of GC-like B cells and plasmablasts cells in the culture system were measured by FCM.The culture supernatant was collected,and the total Ig G and anti-OVA Ig G levels were measured by ELISA.Tfh cell culture supernatants were collected,and Tfh-sEVs were prepared with ultracentrifugation and Exo Quick isolation kit.The sEVs morphology was observed by transmission electron microscopy,the sEVs-characteristic proteins were detected by Western blot,and the sEVs-surface molecules were detected by FCM.Red DIR fluorescent dye-labeled Tfh-sEVs were cultured with B cells,and the binding of sEVs to B cells was observed by confocal laser scanning microscopy（CLSM）.sEVs surface lymphocyte function-associated antigen 1（LFA-1）was blocked with blocking antibodies,and the role of LFA-1 on sEVs binding was analyzed.Tfh-sEVs were cultured with B cells,non-Tfh cell-derived sEVs（non-Tfh-sEVs）was used as a control,percentages of GC-like B cells and plasmablasts in the culture system were measured by FCM.The total Ig G and anti-OVA Ig G levels in the culture supernatant were measured by ELISA.Transfect Tfh cells with Rab27a（sEVs secretion-regulated protein）sh RNA lentiviral expression vector,observe changes in sEVs secretion of Tfh cells after knockdown the Rab27a expression.Adoptive transfer Rab27a knockdown Tfh(Tfh^{Rab27a sh RNA})cells into nude mice.8 days after OVA immunization,ELISA was performed to determine the level of serum anti-OVA Ig G in nude mice.Enzyme-linked immunospot assay（ELISPOT）was performed to determine the number of spleen OVA-specific antibody-secreting cells.FCM was performed to determine the percentages of germinal center B（GC B）cells and plasma or follicular B（FOB）cells/marginal zone B（MZB）cells ratio.Spleen paraffin sections were prepared,and HE staining was used to observe the formation of germinal center.2.Explore the role of CD40L on B-cell differentiation and antibody production regulated by Tfh-sEVs and its regulatory mechanismDetect the CD40L expression on Tfh-sEVs by Western blot and FCM.CD40L blocking antibody-pretreated Tfh-sEVs were cultured with B cells,percentages of GC-like B cells and plasmablasts in the culture system were detected by FCM.The total Ig G and anti-OVA Ig G levels in supernatant were detected by ELISA.Detect the expression of SNX27 on Tfh cells and their sEVs by Western blot.Observe the co-localization of SNX27 and CD40L in Tfh cells by CLSM,and Co-immunoprecipitation（Co-IP）was used to analyze the interaction between these two proteins.Transfect the mouse T-lymphoma cell line EL-4 with SNX27 sh RNA lentiviral expression vector,after knockdown the SNX27 expression,detect the CD40L expression on EL-4^{SNX27 sh RNA} cells and their sEVs by FCM and Western blot.EL-4^{SNX27 sh RNA} cells were treated with lysosomal function inhibitor Bafilomycin A1（BAF）,and the effect of BAF treatment on CD40L expression on EL-4^{SNX27 sh RNA} cells and their source sEVs was analyzed by FCM and Western blot.3.Explore the role of Tfh cell-derived sEVs in CIA progressionConstruct a CIA mouse model and observe the degree of redness and swelling of mice paws.Prepare the joint sections,and HE staining was performed to observe the damage of joints.The percentages of Tfh cells,GC B cells,and plasma cells in the spleen/lymph nodes of CIA mice were measured by FCM.The serum anti-chicken type II collagen（CII）Ig G level was measured by ELISA.Sort spleen Tfh cells and non-Tfh cells from CII immunized mice,construct a CIA mouse model and adoptive-transfer Tfh cells or non-Tfh cells into mice.Observe the degree of redness and swelling of mice paws,record the incidence,average number of paws affected,and arthritis score in each group.Prepare the joint sections,and HE staining was performed to observe the damage of joints.Percentages of Tfh cells,GC B cells,and plasma cells in spleen/lymph nodes of each group were measured by FCM.The anti-CII Ig G level in serum was measured by ELISA.To explore the role of Tfh cell-derived sEVs in the progression of CIA disease,we analyzed the sEVs secretion ability of spleen Tfh cells in CIA mice by Exo ELISA kit,and adoptive-transferred Tfh^{Rab27a sh RNA} cells into CIA mice,observe its effects on disease progression after the inhibition of sEVs secretion.4.Observe the effect of ICOS signaling on the secretion and function of Tfh-sEVsFCM was used to detect the ICOS levels on Tfh and non-Tfh cells in spleen and lymph nodes of CIA mice,as well as the the expression of ICOSL on GCB cells.Tfh cells or non-Tfh cells were cultured with anti-CD3 and anti-ICOS antibodies,culture supernatants were collected and the sEVs secretion ability of Tfh cells and non-Tfh cells was analyzed by Exo ELISA kit.Western blot was performed to detect the phosphorylation of AKT in Tfh cells stimulated with anti-CD3 and anti-ICOS antibodies.Tfh cells were treated with LY294002/Wortmannin for 24h under anti-CD3 and anti-ICOS antibody activation conditions,culture supernatants were collected,and the sEVs secretion ability of Tfh cells was analyzed by Exo ELISA kit.Stimulate Tfh cells with anti-ICOS antibody under anti-CD3 and anti-CD28antibody activation conditions for 48h,Tfh-sEVs were prepared,and the CD40L expression on Tfh cells and Tfh-sEVs were measured by Western blot.Culture this Tfh-sEVs with B cells.The percentages of GC-like B cells and plasmablasts in the culture system were measured by FCM.The total Ig G and anti-OVA Ig G levels in the supernatant were measured by ELISA.5.Assess potential values of plasma Tfh-like-sEVs in RA diseasePlasma was collected from RA patients and healthy subjects,ELISA was performed to detect the s CD40L level in plasma,the correlation of s CD40L with disease activity scores in 28 joints（DAS28）,rheumatoid factor（RF）and anti-cyclic citrullinated peptide（CCP）antibody levels was analyzed.sEVs were prepared from the plasma of RA patients and healthy subjects.Based on the characteristic of Tfh cell-surface molecules,we defined CD4⁺CXCR5⁺sEVs as Tfh-like-sEVs.The level of Tfh-like-sEVs and CD40L expression on Tfh-like-sEVs were measured by FCM,their correlation with DAS28,RF and anti-CCP antibody levels were also analyzed.Results1.In T-B cell co-culture system,compared to non-Tfh cells,percentages of GC-like B cells and plasmablasts in Tfh-B cell cultured system were significantly increased,and total Ig G and anti-OVA Ig G levels in which were also higher.Tfh-SN increased the percentages of GC-like B cells and plasmablasts in the B cell culture system,as well as the total Ig G and anti-OVA Ig G levels in the supernatant.When sEVs were depleted in Tfh-SN,the percentages of GC-like B cells and plasmablasts in the culture system decreased,the total Ig G and anti-OVA Ig G levels in the supernatant were also significantly reduced.Tfh-sEVs had a disc-like structure under electron microscopy,the particle size was ranging from 50-150 nm.CD63,CD9,HSP70,and TSG101 can be observed in Tfh-sEVs.Tfh-sEVs also expressed CD4,CXCR5,CD40L,ICOS,and LFA-1 on their surfaces.Red DIR fluorescent dye labelled Tfh-sEVs were added to B cell culture system,after 4h,84.95±3.00%B cells showed a red fluorescence.After blocking LFA-1 on sEVs,only 14.95±9.15%B cells showed a red fluorescence,the results suggested that Tfh-sEVs can bind to B cells in a LFA-1 dependent manner.After Tfh-sEVs treatment,the percentages of GC-like B cells and plasmablasts in B cell-culture system were significantly increased,and total Ig G and anti-OVA Ig G levels in which were also significantly increased.After Rab27a knockdown,the sEVs secretion ability of Tfh cells was significantly reduced.Compared to Tfh^{NC sh RNA}cell-transferred mice,the size of spleen germinal center in Tfh^{Rab27a sh RNA}cell-transferred mice was reduced,the percentages of spleen GC B cells,plasma cells,and the ratio of FOB/MZB were decreased,the serum anti-OVA Ig G level and the number of spleen OVA-specific antibody-secreting cells were also significantly reduced.These results suggested that Tfh cells can promote B cell differentiation and antibody production through sEVs in vitro and in vivo.2.Tfh-sEVs expressed CD40L,the CD40L expression on Tfh-sEVs was significantly higher than that of non-Tfh-sEVs.After CD40L blocking on Tfh-sEVs,the ability of Tfh-sEVs to promote B cell differentiation into GC-like B cells and plasmablasts,as well as total Ig G and anti-OVA Ig G production,was significantly reduced.Tfh cells and Tfh-sEVs expressed SNX27.CLSM results showed that SNX27co-localized with CD40L in Tfh cells.Co-IP results showed that SNX27 bind to CD40L.Explore the role of SNX27 on CD40L expression in EL-4 cells,results showed that the CD40L expression on both EL-4^{SNX27 sh RNA}cells and their sEVs was significantly reduced.After treatment with the lysosomal function inhibitor BAF,the CD40L expression on EL-4^{SNX27 sh RNA}cells was restored,but the CD40L expression on their sEVs was still reduced,which suggested that SNX27 knockdown directly reduces the CD40L expression on sEVs when intracellular CD40L is stably expressed after blocking cytosolic lysosomal function.These results suggested that Tfh-sEVs promote B cell differentiation and antibody production in vitro dependent on CD40L molecule,and SNX27 is involved in the regulation of CD40L expression on sEVs.3.The percentage and number of spleen/lymph node Tfh cells in CIA mice were significantly increased,as well as the serum anti-CII Ig G level.After Tfh cell-adoptive transfer,CIA mice showed a higher incidence and arthritis score and more average number of paws affected.The numbers of spleen/lymph node Tfh cells,GC B cells,and plasma cells,as well as the serum anti-CII Ig G level,were also significantly increased in the Tfh cell-transferred group.The sEVs secretion ability of Tfh cells from CIA mice was significantly enhanced as compared to that from WT mice.After reducing the secretion of sEVs,CIA mice in the Tfh cell-transfer group showed a lower incidence and arthritis score and less average number of paws affected.Moreover,the number of spleen/lymph node plasma cells and level of serum anti-CII Ig G were also significantly decreased.These results indicated that Tfh cells can promote CIA disease progression,after reducing the secretion of sEVs,the effect of Tfh cells in promoting CIA disease progression was significantly diminished.4.In CIA mice,the ICOS expression on spleen Tfh cells was 4.5-fold higher than non-Tfh cells,the ICOS expression on lymph node Tfh cells was 5.3-fold higher than that non-Tfh cells.In addition,the ICOS expression on Tfh cells in spleen and lymph nodes of CIA mice was further increased compared to WT mice.In addition,during the progression of CIA,high level of ICOSL on GC B cells from spleen and lymph nodes was also observed.Under anti-CD3 and anti-ICOS antibody activation conditions,Tfh cells secreted more sEVs than non-Tfh cells.After anti-ICOS antibody stimulation,the AKT phosphorylation level in Tfh cells was increased.After treatment with AKT pathway inhibitor LY294002/Wortmannin,Tfh cells showed a reduced AKT phosphorylation level,a decreased Rab27a expression,and a reduced sEVs secretion.After stimulating Tfh cells with anti-ICOS antibody,the CD40L expression on Tfh cells and Tfh-sEVs was significantly increased,the ability of Tfh-sEVs to promote B cell differentiation into plasmablasts and total Ig G and anti-CII Ig G production was significantly enhanced.These results suggested that upregulated ICOS signalling can promote the sEVs secretion of Tfh cells during the pathogenesis of CIA.5.The plasma s CD40L level was significantly higher in RA patients than in healthy subjects,but the plasma s CD40L level in RA patients was not correlated with DAS28 scores,RF,and anti-CCP antibody levels.The percentage of plasma Tfh-like-sEVs and CD40L expression on Tfh-like-sEVs in RA patients were significantly higher than that in healthy subjects,which were positively correlated with DAS28 scores,RF,and anti-CCP antibody levels.ConclusionssEVs are important components of Tfh cells for B cell differentiation and antibody production,and CD40L is a functional molecule of Tfh-sEVs.SNX27 is involved in the regulation of CD40L expression on sEVs.During the pathogenesis of CIA,upregulated ICOS signalling can promote the sEVs secretion of Tfh cells,Tfh cells enhance B-cell immune responses by secreting sEVs,exacerbating the disease process.The level of plasma Tfh-like-sEVs and CD40L expression on Tfh-like-sEVs are expected to be evaluation indicators of RA disease activity...