FACS analysis of the numbers of circulating memory Tfh cells and ICOS+ memory Tfh cells in individual subjects.

<p>PBMCs were isolated from individual subjects and stained with different fluorescent antibodies. The cells were gated sequentially on living lymphocytes, CD3+ and CD4+, and then on CXCR5+ and CD45RA- cells. The frequency of CD3+CD4+CXCR5+CD45RA- (memory) Tfh cells was determined. Subsequently, memory Tfh cells were gated on ICOS and the frequency of memory Tfh and ICOS+ memory Tfh cells in lymphocytes was analyzed and the numbers of each type of cells in total lymphocytes per liter were calculated. (A) Flow cytometry analysis. (B) The numbers of memory Tfh cells in the HC, MS patients pre- and post-treatment. (C) The numbers of memory Tfh cells in the MS-CR patients pre- and post-treatment. (D) The numbers of memory Tfh cells in the MS-PR patients pre- and post-treatment. (E) The numbers of ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (F) The numbers of ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (G) The numbers of ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. There was no significant difference in the numbers of PD-1+, PD-1+ICOS+ and CD40L+ memory Tfh cells between the patients and HC as well as in MS patients pre- and post-treatment (data not shown). The horizontal lines indicate the median values for each group.</p

Analysis of plasma and CSF IL-21 in MS patients before and after treatment.

<p>The levels of plasma and CSF IL-21 in individual subjects were tested by ELISA. (A) The levels of plasma IL-21 in the HC, MS patients pre- and post-treatment. (B) The levels of plasma IL-21 in the MS-CR patients pre- and post- treatment. (C) The levels of plasma IL-21 in the MS-PR patients pre- and post- treatment. (D) The levels of CSF IL-21 in the MS patients pre-treatment and NND patients. Data are expressed as the mean values of individual samples. The horizontal lines represented the median values.</p

Correlation analysis of the numbers of different subsets of circulating memory Tfh cells and the values of clinical measures in MS patients.

<p>(A-B) The numbers of ICOS+ and CCR7+ICOS+ memory Tfh cells were positively associated with the EDSS scores in MS patients. (C-D) The numbers of ICOS+ and CCR7+ICOS+ memory Tfh cells were positively associated with the levels of plasma IL-21. (E-F) The numbers of CCR7+ICOS+ memory Tfh cells were positively correlated with the levels of CSF IL-21 and CSF IgG. (G-H) The numbers of CCR7+ICOS+ memory Tfh cells were positively correlated with the levels of CSF MBP-Ab and CSF MOG-Ab.</p

The demographic and clinical features of 12 MS patients after treatment.

<p>Data shown are median and range, except specified. CR: Complete remission; CSF: Cerebrospinal fluid; PR: Partial remission; WBC: White blood cells. Normal values: CSF WBC, 0–8 ×10⁶/L; CSF proteins, 0.15–0.45 g/L; CSF IgG, 0–34 mg/l. *P<0.05 vs. the HC.</p><p>The demographic and clinical features of 12 MS patients after treatment.</p

FACS analysis of the numbers of circulating CCR7+ and CCR7+ICOS+ memory Tfh cells in individual subjects.

<p>After being stained with different fluorescent antibodies, the CD3+CD4+CXCR5+CD45RA- T cells were gated. Subsequently, memory Tfh cells were gated on CCR7 expression and the frequency of CCR7+, CCR7+ICOS+, CCR7- and CCR7-ICOS+ memory Tfh cells was analyzed by flow cytometry and the numbers of each type of cells were calculated. (A) Flow cytometry analysis. (B) The numbers of CCR7+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (C) The numbers of CCR7+ memory Tfh cells in the MS-CR patients pre- and post- treatment. (D) The numbers of CCR7+ memory Tfh cells in the MS-PR patients pre- and post-treatment. (E) The numbers of CCR7+ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (F) The numbers of CCR7+ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (G) The numbers of CCR7+ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. (H) The numbers of CCR7- memory Tfh cells in the HC, MS patients pre- and post-treatment. (I) The numbers of CCR7- memory Tfh cells in the MS-CR patients pre- and post- treatment. (J) The numbers of CCR7- memory Tfh cells in the MS-PR patients pre- and post-treatment. (K)The numbers of CCR7-ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (L) The numbers of CCR7-ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (M) The numbers of CCR7-ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. There was no significant difference in the numbers of CCR7-PD-1+, CCR7-PD-1+ICOS+, CCR7-CD40L+, CCR7+PD-1+, CCR7+PD-1+ICOS+, CCR7+CD40L+ memory Tfh cells between the patients and HC as well as in MS patients before and after treatment (data not shown). The horizontal lines indicate the median values for each group.</p

The demographic and clinical features of MS patients and HC.

<p>Data shown are median and range, except specified. ARR: Annual relapse rate; EDSS: Expanded disability status scale; MS: Multiple sclerosis; WBC: White blood cells. Normal values: WBC, 3.5–9.5 ×10⁹/L; Lymphocytes, 1.1–3.2 ×10⁹/L.</p><p>The demographic and clinical features of MS patients and HC.</p

The demographic and clinical features of MS patients and NND who received a lumbar puncture.

<p>Data shown are median and range, except specified. CSF: Cerebrospinal fluid; MBP-Ab: Antibodies against myelin basic protein; MOG-Ab: Antibodies against myelin oligodendrocyte glycopotein; NND: Non-inflammatory neurological diseases; OD: Optical density; WBC: White blood cell. Normal values: CSF WBC, 0–8 ×10⁶/L; CSF proteins, 0.15–0.45 g/L; CSF IgG, 0–34 mg/l; Plasma MBP-Ab < 0.75; CSF MBP-Ab < 0.65; Plasma MOG-Ab < 0.64; CSF MOG-Ab < 0.56. *P<0.05 vs. the HC.</p><p>The demographic and clinical features of MS patients and NND who received a lumbar puncture.</p