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, 67 (4), 988-99

Circulating Follicular Helper-Like T Cells in Systemic Lupus Erythematosus: Association With Disease Activity

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Circulating Follicular Helper-Like T Cells in Systemic Lupus Erythematosus: Association With Disease Activity

Jin-Young Choi et al. Arthritis Rheumatol.

Abstract

Objective: To assess circulating follicular helper T (Tfh)-like CD4+ T cells in patients with systemic lupus erythematosus (SLE) and determine their relationship to disease activity.

Methods: Blood samples from patients with SLE, as well as blood samples from patients with Behçet's disease (BD) and healthy individuals as controls, were analyzed. In all samples, circulating Tfh-like cells were enumerated by flow cytometry, using, as markers, expression of CXCR5, inducible T cell costimulator (ICOS), and programmed death 1 (PD-1) protein, as well as secretion of interleukin-21 (IL-21). The frequency of circulating Tfh-like cells was compared to that of circulating plasmablasts (CD19+IgD-CD38+). In addition, the possible association of circulating Tfh-like cells with the SLE Disease Activity Index (SLEDAI) was evaluated.

Results: The subset of circulating Tfh-like T cells, identified as CXCR5(high) ICOS(high) PD-1(high) , was expanded in the blood of SLE patients compared to controls. Circulating Tfh-like cells were found to produce IL-21 and had lower expression of CCR7 as compared to that in circulating CXCR5(high) central memory T cells, thereby enabling their distinction. Expression of PD-1, but not ICOS or CXCR5, was significantly elevated in circulating Tfh-like cells from SLE patients compared to controls. PD-1 expression among CXCR5(high) circulating Tfh-like cells correlated with the SLEDAI, frequency of circulating plasmablasts, and anti-double-stranded DNA antibody positivity, but not with disease duration or past organ injury; rather, this cell profile appeared to be a reflection of current active disease.

Conclusion: Circulating Tfh-like cells are associated with disease activity in SLE, suggesting that their presence indicates abnormal homeostasis of T cell-B cell collaboration, with a causal relationship that is central to disease pathogenesis. These findings also suggest that circulating Tfh-like cells provide a surrogate for aberrant germinal center activity in SLE, and that their PD-1 expression offers a tool for measuring disease activity and monitoring the response to therapies.

Figures

Figure 1
Figure 1
Circulating CXCR5hiICOShiPD-1hi Tfh-like cells are expanded in the blood of patients with SLE compared to healthy controls and patients with Behçet's disease. A, CD4+TCRβ+ T cells were separated into naïve (CD45RA+) and activated (CD45RA) pools, with the latter subdivided into CXCR5hi and CXCR5lo subsets (left panel). Gates for the expression of PD-1 and ICOS were set using naïve CD4+ T cells from healthy controls (right panel). B, Contour plots of ICOS and PD-1 expression on CD45RA CXCR5hi cells from a representative healthy control (left panel) and an SLE patient (middle panel), compared to tonsillar cells from a non-SLE donor (right panel). Values are the percentage of ICOShiPD-1hi cells among CD4+ CXCR5hi cells. C, Percentage of ICOShiPD-1hi lymphocytes among CXCR5hi and CXCR5lo CD4+ T cells in patients with SLE (n = 49), Behçet's disease (BD; n = 28) and healthy controls (H; n = 16) (One-way ANOVA ***p < 0.001). D, Staining of Bcl6 in naïve (shaded) and Tfh cells (black line) from a tonsillar sample, compared to circulating CXCR5hiPD-1hi cells (dotted line) from a representative SLE patient. E, Correlation between the MFI of PD-1 and ICOS on CXCR5hi and CXCR5lo CD4+ T cells (Pearson r = 0.38, p = 0.03, left panel, and Pearson r = 0.10, p = 0.59, right panel, respectively).
Figure 2
Figure 2
Expression of the Tfh-cell surface markers CXCR5, PD-1, and ICOS on circulating CD4+ T lymphocytes. A and B, Percentage of activated CD45RACXCR5hi (A) and CD45RACXCR5lo (B) cells among the circulating CD4+TCRβ+ population in the blood of SLE patients (n = 49), in comparison to healthy controls (H; n = 16) and BD (n = 28) patients. C and D, MFI of ICOS (C) and PD-1 (D) expression on CXCR5hi and CXCR5lo CD4+ T cells from the blood of SLE patients compared to patients with BD and healthy controls. Data points represent individual subjects; horizontal lines represent the mean values with standard error of the mean (One-way ANOVA *p < 0.05; **p < 0.01; ***p < 0.001).
Figure 3
Figure 3
Association between Tfh-cell surface markers, SLEDAI, and circulating plasmablasts. A and B, Correlation between the MFI of PD-1 (A) and ICOS (B) on circulating CXCR5hi cells and SLEDAI (A, Spearman r = 0.43, p = 0.03 and B, Spearman r = 0.16; p = 0.52). C, Association between percentage of CXCR5hi cells among circulating CD4+TCRβ+ cells and SLEDAI (Spearman r = −0.21, p = 0.28). D, Correlation between MFI of PD-1 on the circulating CXCR5lo subset and SLEDAI (Spearman r = 0.43, p = 0.02). E, Percentage of circulating CD19+IgDCD38+ plasmablasts among total B cells in the blood of SLE patients (n = 49), healthy controls (H; n =16), and BD (n = 28) patients (One-way ANOVA, *p < 0.05). F, Percentage of circulating plasmablasts in patients with SLE, arranged according to therapy (Student’s t-test, n.s. = non significant). Horizontal lines represent the mean values with standard error of the mean. G and H, Correlation between percentage of circulating plasmablasts among circulating B cells in SLE and the PD-1 MFI on activated CXCR5hi and CXCR5lo cells, respectively (G, Spearman r = 0.34, p = 0.02; H, Spearman r = 0.32, p = 0.03). Data points represent individual subjects.
Figure 4
Figure 4. Analysis of CXCR5hi Th-cell subsets and correlation with SLEDAI
A-D, Percentage of CCR6+CXCR3(A), CCR6CXCR3+ (B), CCR6+CXCR3+ (C), and CCR6 CXCR3(D) cells among the circulating CD45RACXCR5hi CD4+ population in SLE patients (n = 49), in comparison to healthy controls (H; n = 16) and BD (n = 28) patients. (Student’s t-test, n.s. = non significant). Horizontal lines represent the mean values with standard error of the mean. E and F, Correlation between percentage of CCR6+CXCR3+ (E), and CCR6CXCR3(F) cells among circulating, activated CD45RACXCR5hi CD4+ cells and SLEDAI (E, Spearman r = −0.3, p = 0.03; F, Spearman r = 0.34, p = 0.01). Data points represent individual subjects.
Figure 5
Figure 5
Circulating CXCR5hiICOShiPD-1hi cTfh-like cells in SLE are dividing and robustly produce IL-21, in comparison to CXCR5hi central memory (Tcm) cells. A, Contour plots of CCR7 and PD-1 expression on naïve (left panel) and activated CD45RACXCR5hi (right panel) cells in the blood of a representative patient with SLE, demonstrating the gating strategy to determine PD-1lo Tcm and PD-1hi cells. B and C, The MFI of CCR7 (B) and the percentage of Ki-67+ cells (C) in each of the indicated subsets (Student’s t-test ***p < 0.001, ****p < 0.0001). D, Contour plots of intracellular IL-21 staining in circulating CXCR5hiPD-1lo Tcm populations and CXCR5hiPD-1hi cTfh-like populations from a representative patient with SLE (left and center panels) and the percentage of IL-21+ cells among seven SLE patients (right panel), compared to that among naïve CD45RA+ cells. Horizontal lines represent the mean values with standard deviation of the mean (right panel) (Student’s t-test *p < 0.05; **p < 0.01; ***p < 0.001).

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