Case summary
Supplementary Table 2 compares the characteristics of the AS patients and HCs in two phases: the primary screening phase and the expanded validation phase.
Primary screening phase
The 28 T lymphocyte and 12 B lymphocyte subset content was analyzed in the AS and HC groups at both the primary screening phase and expanded validation phase. There was a significant change in the percentage of T lymphocytes and B lymphocytes in the AS patients compared to the HCs. The percentage of Th1 cells (CD3+CD4+CXCR3+CCR4−CXCR5−), Tfh1 cells (CD3+CD4+CXCR3+CCR4−CXCR5+), Tc1 cells (CD3+CD8+CXCR3+CCR4−CXCR5−), memory B cells (CD3−CD19+CD27+CD24+CD38+IgD+IgM+), and non-switched B cells (CD3−CD19+CD24−CD27+CD38+IgD+IgM+) were found to be significantly higher in the AS patients. However, the percentage of Th2 cells (CD3+CD4+CXCR3−CCR4+CXCR5−CCR6−), Th17 cells CD3+CD4+CXCR3−CCR4−CXCR5−CCR6+), Tfh2 cells (CD3+ CD4+CXCR3−CCR4+CXCR5+), and B cells (CD3−CD19+) were found to be significantly lower in the AS patients. The statistical results are listed in Supplementary Table 3 (P < 0.05). Representative cytofluorometric gating strategy and detailed examples of analyses for the subsets have been represented in supplementary fig. 1.
Expanded validation phase
Cluster analyses of immunophenotypic parameters that were differentially expressed in the AS patients and HCs at the expanded validation phase are summarized in Fig. 1. The magnitude of parameter expression is color-coded with red for a relative increase in expression and blue for a relative decrease in expression. Strong cluster separation can be observed between AS and HC. Part of immune cells with a relative increase in expression (upper left quadrant and lower right quadrant) clearly separated from others with a relative decrease in expression (upper right quadrant and lower left quadrant). Supplementary Table 4 shows the statistical results of average frequencies and P values of a total of 21 immune cells with significant differences between the two groups.
T lymphocyte
The percentage of CD4+ T cells at different stages of differentiation were calculated, and significant differences between the AS patients and HCs are shown in Fig. 2. CCR7+ CD4+T cells including naïve CD4+T cells (CD3+CD4+CD45RA+CCR7+, Fig. 2a) and central memory CD4+T cells (CD3+CD4+CD45RA−CCR7+, Fig. 2c) were significantly increased in the AS group, but CCR7− CD4+T cells including terminally differentiated CD4+T cells (CD3+CD4+CD45RA+CCR7−, Fig. 2b), and effector memory CD4+T cells (CD3+CD4+CD45RA−CCR7−, Fig. 2d) were significantly decreased.
As shown in Fig. 2, the percentage of CD8+ T cells at different stages of differentiation was also calculated. Naïve CD8+T cells (CD3+CD8+CD45RA+CCR7+, Fig. 2e), central memory CD8+T cells (CD3+CD8+CD45RA−CCR7+, Fig. 2g), and effector memory CD8+T cells (CD3+CD8+CD45RA−CCR7−, Fig. 2h) were significantly increased in the AS group, but terminally differentiated CD8+T cells (CD3+CD8+CD45RA+CCR7−, Fig. 2f) were significantly decreased.
Simultaneously, we tested Th cells (Th1 cells, Th2 cells, Th17 cells), Tc cells (Tc1 cells, Tc2 cells, Tc17 cells), and Tfh cells (Tfh1 cells, Tfh2 cells, Tfh17 cells), and the results with significant differences are shown in Fig. 3. The percentage of CXCR3+T cells, including Th1 cells (CD3+CD4+ CXCR3+ CCR4−CXCR5−, Fig. 3a), Tfh1 cells (CD3+CD4+CXCR3+CCR4−CXCR5+, Fig. 3b), and Tc1 cells (CD3+CD8+CXCR3+ CCR4−CXCR5−, Fig. 3c), was found to be significantly lower in the AS group. However, the percentage of CCR6+ helper T cells, such as Th17 cells (CD3+CD4+CXCR3−CCR4−CXCR5−CCR6+, Fig. 3d) and Tfh17 cells (CD3+CD4+CXCR3−CCR4−CXCR5+CCR6+, Fig. 3e), were found to be significantly higher. The total Tc cells (CD3+CD8+) (Fig. 3f) were also significantly increased in the AS group.
Regulatory lymphocytes
We compared changes in the ratio in negative regulatory cells, such as Tregs and Bregs, between the AS and HC groups. The percentage of Tregs (CD3+CD4+CD25+CD127−) and immature Bregs (CD3−CD19+CD24+CD27−CD38+IgD+IgM+) was found to be significantly lower in the AS group than in the HC group (P < 0.0001). Supplementary Fig. 2 lists the scatter plot results. B10 cells (CD3−CD19+CD24+CD27+CD38−IgD+IgM+) were also shown to be decreased in the AS group, but the difference was not significant.
B lymphocytes
Figure 4 shows the percentage of B cells at different stages of differentiation. The proportion of total B cells (CD3−CD19+, Fig. 4a) and class-switched B cells (CD3−CD19+CD27+CD38−IgD−IgM−, Fig. 4b) were all significantly increased in the AS group. However, antibody-secreting phenotype B cells (CD19+ CD38+) including non-switched B cells (CD3−CD19+CD27+CD38+CD24−IgD+ IgM+, Fig. 4c), plasma cells (CD3−CD19+CD27+CD38+IgD−IgM−, Fig. 4d), memory B cells (CD3−CD19+CD24 +CD27+CD38+IgD+IgM+, Fig. 4e), and immature Bregs (CD3−CD19+IgD+IgM+ CD27−CD38+CD24+, Fig. 4f) were found to be significantly decreased in the AS group compared to the HC group.
The impact of Anbainuo therapy on lymphocyte subsets in AS
A total of 23 active-phase AS patients were included in this research. The mean age of the patients (M/F:19/4) was 30 years (range, 25–36 years), and mean disease duration was 8 years (range, 4.5–13.00 years) at baseline. Disease activity was indicated by CRP of 12.10 mg/L (range, 2.60–20.90 mg/L), ASDAS of 2.97 ± 1.02, and BASDAI of 4.25 ± 1.37 before treatment. After 12 weeks of Anbainuo therapy, CRP, ASDAS, and BASDAI all decreased significantly after treatment (P < 0.05), and the average values were 2.50 mg/L (range, 0.50–8.00 mg/L), 1.37 ± 1.04, and 1.69 ± 1.32, respectively, as shown in Supplementary Table 5.
After 12 weeks of Anbainuo therapy, the amount of some lymphocyte subsets in the peripheral blood of the AS patients changed significantly (Fig. 5). CD4+T cells and CD8+T cells were measured at different stages of differentiation, and comparisons were made between the AS and HC groups. As shown in Fig. 5, naïve CD4+ T cells (CD3+CD4+CD45RA+CCR7+, Fig. 5a) were decreased and effector memory CD8+ T cells (CD3+CD8+CD45RA−CCR7−, Fig. 5d) increased after Anbainuo therapy.
The number of regulatory lymphocytes detected in the blood of the AS patients changed significantly after Anbainuo treatment, with the percentage of Treg cells (CD3+CD4+CD25+CD127−, Fig. 5b) and B10 cells (CD3−CD19+CD24+CD27+ CD38−IgD+IgM+, Fig. 5c) increasing significantly but immature Bregs (CD3−CD19+CD24+ CD27CD38+IgD+IgM+, Fig. 5g) decreasing significantly.
Simultaneously, we measured the number of Th cells (Th1 cells, Th2 cells, Th17 cells), Tc cells (Tc1 cells, Tc2 cells, and Tc17 cells), and Tfh cells (Tfh1 cells, Tfh2 cells, and Tfh17 cells) before and after Anbainuo therapy. As shown in Fig. 5, the proportion of Tc1 cells (CD3+CD8+CXCR3+CCR4−CXCR5−, Fig. 5e) decreased, and the proportion of Tfh17 cells (CD3+CD4+CXCR3−CCR4−CXCR5+ CCR6+, Fig. 5f) increased after treatment.
However, apart from immature Bregs and B10 cells, the proportion of various B cell subtypes did not change significantly after treatment with Anbainuo.
Correlation between immune cells and disease activity
In order to understand whether disease activity of AS patients is related to immune cell imbalance, we analyzed the correlation between disease activity indicators (CRP and ASDAS) and frequency of immune cells. But only the frequency of Tc1 cells (CD3+CD8+CXCR3+CCR4−CXCR5−) was found to be negatively correlated with CRP level (r = − 0.182, P = 0.041).
To understand the correlation between changes in disease status (including CRP, BASDAI, and ASDAS) and changes in lymphocyte frequency after Anbainuo therapy, Spearman’s rank correlation analyses showed that the decrease in CRP was positively correlated with the increase in the frequency of Tregs (CD3+CD4+CD25+CD127−) following Anbainuo therapy for 12 weeks (r = 0.489, P = 0.018).