Figure Legends
Fig. 1. Frequency of circulating Tfh cells was significantly higher in MS patients. Fresh PBMCs from HC and MS patients were surface stained with anti-CD3, anti-CD4, anti-CXCR5 and anti-PD-1 antibodies. a) Construction of fine singlet’s; representative example of by flow cytometry gating of total live PBMC by exclusion of debris, doublets and dead cells. b) gating strategy: CD3+CD4+ T cells(left panel) were defined by gating them on PBMCs after constructing fine singlets, CXCR5+PD-1+(right panel) cells defined as cTfh cells which were selected by gating from CD3+CD4+ T cells (left panel), c) percentage of CD4+ T cells in MS and HC were calculated and compared, d) percentage of cTfh cells in MS patients was calculated and compared with that of HC. **p =0.002. Each data point represents each individual.
Fig. 2. cTfh17 and cTfh17.1 cells were the major subtypes of cTfh cells that increased in MS patients. Fresh PBMCs from HC and MS patients were surface stained with anti-CD3, anti-CD4, anti-CXCR5 anti-PD-1, anti-CXCR3, anti-CCR6 antibodies a) gating strategy; CXCR3 and CCR6 were gated from CXCR5+PD-1+cells to delineate cTfh subtypes, where CXCR3+CCR6- defined as cTfh1, CXCR3-CCR6- as cTfh2, CXCR3-CCR6+ as cTfh17 and CXCR3+CCR6+ as cTfh17.1 cell, b-e) percentages of different subtypes of cTfh cells were calculated in MS patients and compared with those in HC, *p=0.02 and **p=0.005 respectively.
Fig. 3. Frequency of IL-21 was significantly increased in MS patients. Fresh PBMCs from 12 HC and 20 MS patients were surface stained with monoclonal antibodies with anti-CD3, anti-CD4, anti-CD8, anti-CXCR5 and anti-PD-1 antibodies. Intracellular cytokine staining was performed using anti-IL-21 antibody after stimulating 5 hours with PMA, ionomycin and golgistop, a-b) gating strategy; IL-21 positive population was selected from PBMCs, CD4+ T cells and CD8+ T cells, c) percentage of IL-21 secretion in PBMCs, CD3+ T, CD4+ T and CD8+ T cells from MS patients was calculated and compared with those in HC, ***p=0.0004 and 0.0009 respectively and **p=0.002, d) gating strategy: IL-21 positive cells were selected from CXCR5+PD-1+ Tfh cells and CD4+CXCR5- non-follicular cTh cells, e) average frequencies of IL-21 producing cTfh and non-follicular cTh cells were calculated and compared between HC and MS. **p=0.003 indicates the difference of IL-21 secreting cTfh cells between MS patients and HC. ****p<0.0001 represents the statistical analysis of IL-21 producing cTfh vs. non-follicular cTh cells in MS patients. Mean±SEM are shown.
Fig. 4. Increased frequency of IL-21 producing cTh17.1 cells were observed in MS patients. Fresh PBMCs from 12 HC and 20 MS patients were surface stained with aforementioned antibodies described in Fig. 2. ICS was additionally performed using anti-IFN-γ, anti-IL-17, anti-IL-4 and anti-IL-21 antibodies after stimulating 5 hours with PMA, ionomycin and golgistop a) gating strategy; IL-21 population was positively selected from different subsets of cTfh cells, b) frequencies of IL-21 production by different subtypes of cTfh cells were calculated in MS and compared with those of HC ***p=0.002, c) proportion of IL-17 producing cTfh17 cells in MS and HC were calculated and compared, d) percentages of IL-17 production by follicular and non-follicular cTh17 cells were calculated and compared in MS patients, e) percentage of IL-17 producing cTfh17.1 cells in MS patients was calculated and compared with that of HC, f) frequency of IFN-γ producing cTfh17.1 cells was calculated in MS patients and compared with that in HC. Mean±SEM are shown. Each data point represents each individual.
Fig. 5. Frequency of cTfreg cells was significantly reduced in MS patients. Fresh PBMCs from both HC and MS were surface stained with anti-CD3, anti-CD4, anti-CXCR5, anti-PD-1 and anti-CD25 antibodies. FoxP3 staining with anti-FoxP3 antibody was performed according to manufacturer’s protocol (eBioscience), a) gating strategy: cTfreg(right panel) cells were selected by gating FoxP3+CD25+ cells on CXCR5+PD-1+cTfh(left panel) cells, b) percentage of cTfreg cells was calculated in MS patients and compared with that of HC ****p<0.0001, c) percentage of non-follicular cTreg cells was calculated in MS patients and compared with that in HC. Mean±SEM are shown. Each data point indicates each individual.
Fig. 6. Frequency of IL-10 producing cTfreg cells was reduced in MS patients. Fresh PBMCs from 12 HC and 20 MS patients were surface stained with monoclonal antibodies as described in Fig 5. ICS was additionally performed using anti-IL-10 antibody after stimulating PBMCs with PMA, ionomycin and golgi stop for 5 hours, a-c) gating strategy: IL-10 positive cells were gated on PBMCs (top panel) , cTfreg cells (mid panel) and non-follicular cTreg cells (bottom panel)respectively, d) percentage of IL-10 secretion in the PBMC of MS patients was calculated and compared with those of HC **p=0.004, e) average frequencies of IL-10 producing cTfreg cells and non-follicular cTreg cells were calculated in MS patients and compared with those in HC, *p=0.02 represents the comparative analysis of IL-10 secretion by cTfreg cells between HC and MS, whereas *p=0.05 indicates the statistical difference of IL-10 production by non-follicular cTreg cells in HC vs. MS, and finally ***p= 0.0001 and ****p<0.0001 demonstrate the statistical difference of IL-10 secretion by follicular vs. non-follicular cTreg cells in HC and MS patients respectively. Mean±SEM are shown. Each data point indicates each individual.