Table 1.
Demographics of SLE patients and normal controls*.
Table 2.
Alteration of Tfh cells before and after MP treatment.
Figure 1.
Three tubes from one sample, stained with 1) CD4, CXCR5 isotype control and PD1, 2) CD4, CXCR5 and PD1 isotype control, and 3) CD4, CXCR5 and PD1 separately, were analyzed at the same time. PBMCs are gated on the forward scatter of living cells and then centered on CD4+ T cells (A,B). After setting the threshold using isotype control staining for CXCR5 (C) and PD1 (D), the proportion of Tfh cells is analyzed as the percentage of CXCR5+ PD1+ cells within total CD4+ T cells (E).
Figure 2.
Aberrant circulating CXCR5+ PD1+/CD4+ but not CCR6+/CD4+ T cells correlated with disease activity in SLE patients.
A. Elevated circulating CXCR5+ PD1+/CD4+ cells in SLE patients compared to normal controls. PBMCs were quantified by FACS, each symbol represented an individual sample and horizontal lines showed median values. Mann-Whitney U test was conducted to compare the data between two groups. B. Circulating CCR6+/CD4+ cell proportions enriched for Th17-like cells were compared between SLE patients and normal controls. C. Correlation between percentages of CXCR5+ PD1+/CD4+ cells and disease activity of 42 SLE patients. Pearson correlation was applied to depict linear relationships.
Figure 3.
Positive correlation of CXCR5+ PD1+/CD4+ cells with ANA and CD19+ CD138+ cells in SLE patients.
A. Percentages of circulating CXCR5+ PD1+/CD4+ cells were positively correlated with ANA values (Pearson r = 0.40, p<0.05) in 29 SLE patients. B. There were no association between percentages of CXCR5+ PD1+/CD4+ cells and circulating levels of IgG anti-dsDNA by Pearson correlation test. However, in those patients having high levels of anti-dsDNA antibodies (∼ 50%), a trend of correlation with CXCR5+ PD1+/CD4+ cells was observed. C. Circulating CD19+ CD138+ cells were elevated in SLE patients as compared with normal controls. Each symbol represented an individual sample, horizontal lines showed median values, and Mann-Whitney U test was applied. D. Percentages of CXCR5+ PD1+/CD4+ cells were positively correlated with percentages of CD19+ CD138+ cells in 23 SLE patients (Pearson r = 0.45, p<0.05).
Table 3.
Correlation between clinical manifestations and Tfh cells in SLE patients*.
Figure 4.
Decreased CXCR5+ PD1+/CD4+ cell production by dexamethasone.
A. Dexamethasone (Dex) reduced the proportion of IL-21 stimulated CXCR5+ PD1+/CD4+ cell. Each line represented the alterations of CXCR5+ PD1+/CD4+ cell percentages of an individual sample when cultured with medium control, IL-21 or IL-21+ Dex for 24 hours in vitro. Paired t-test was applied to compare results between two groups and Kruskal-Wallis test was used to determine the difference among three groups. Compared with control group, CXCR5+ PD1+/CD4+ cells were increased when cultured with IL-21 (p<0.01). Adding dexamethasone to IL-21 culture system significantly inhibited the production of CXCR5+ PD1+/CD4+ cells (p<0.0001). B. A similar tendency was observed when cells were cultured for 72 hours. Each line represented the alterations of CXCR5+ PD1+/CD4+ cell percentages of an individual sample. C. Dexamethasone reduced the proportion of CXCR5+ PD1+/CD4+ cell in a dose-dependent manner. Each line represented the changes of CXCR5+ PD1+/CD4+ cell percentages of an individual sample when cultured with different concentrations of dexamethasone for 24 hours in vitro and paired t-test was applied to compare results between two groups. D. Downregulation of CXCR5+ PD1+/CD4+ cell by dexamethasone was not a consequence of the decline in total cell numbers. Cells were equally divided into 3 groups to culture with different concentrations of dexamethasone. After 24 hours culturing, all the cells were harvested and counted by flow cytometry. There were no differences of total cell numbers among the groups (p>0.05). E. Increased cell apoptosis after dexamethasone treatment. PBMCs from 6 SLE patients were collected and cultured in the presence or absence of dexamethasone for 24 hours. Then cells were harvested and quantified by FACS. Data were shown as mean ± SEM. The percentages of Annexin V+ cells are much higher in CXCR5+ PD1+/CD4+ groups, especially for those treated with high dose dexamethasone.