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.