Circulating T follicular helper cells with increased function during chronic graft-versus-host disease

Abstract

Chronic graft-versus-host disease (cGVHD) remains a major late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Previous studies have established that both donor B and T cells contribute to immune pathology in cGVHD but the mechanisms responsible for coordinated B- and T-cell responses directed against recipient antigens have not been understood. T follicular helper cells (TFH) play an important role in the regulation of B-cell immunity. We performed extensive phenotypic and functional analysis of circulating TFH (cTFH) and B cells in 66 patients after HSCT. Patients with active cGVHD had a significantly lower frequency of cTFH compared with patients without cGVHD. This was associated with higher CXCL13 plasma levels suggesting increased homing of TFH to secondary lymphoid organs. In patients with active cGVHD, cTFH phenotype was skewed toward a highly activated profile with predominance of T helper 2 (Th2)/Th17 subsets. Activated cTFH in patients with cGVHD demonstrated increased functional ability to promote B-cell immunoglobulin secretion and maturation. Moreover, the activation signature of cTFH was highly correlated with increased B-cell activation and plasmablast maturation in patients after transplant. These studies provide new insights into the immune pathogenesis of human cGVHD and identify TFH as a key coordinating element supporting B-cell involvement in this disease.

Figure 1

Abnormal cTFH values and CXCL13 levels in patients with active cGVHD. (A) Frequency of cTFH (CXCR5⁺CD45RA⁻ within CD4⁺ T cells) in healthy donors (HD), patients with no cGVHD, active cGVHD, and resolved cGVHD. (B) Absolute number of cTFH (CXCR5⁺CD45RA⁻CD4⁺ T cells) in the different clinical groups. (C) CXCL13 plasma concentration (pg/mL) was measured by ELISA and plotted for each patient group. Data were log₁₀ transformed. Black bar represents median value for each group. The Wilcoxon rank-sum test was used. *P < .05; **P < 10⁻⁴. ns, not significant.

Figure 2

cTFH are activated and proliferate in patients with active cGVHD. (A) Frequency of ICOS^hiPD-1^hi in cTFH (CXCR5⁺CD45RA⁻CD4⁺ T cells) was determined in different patient groups. (B) Ki67 expression within cTFH (CXCR5⁺CD45RA⁻CD4⁺ T cells) was determined in different patient groups. (C) Positive correlation between proliferation and frequency of highly activated ICOS^hiPD-1^hi cTFH in the active cGVHD group. Black bar represents median value for each group. The Wilcoxon rank-sum test was used in panels A and B. The Spearman test was used in panel C. *P < .05; **P < 10⁻⁴. The gating strategy for identifying ICOS^hiPD-1^hi in cTFH is shown in supplemental Figure 1.

Figure 3

Function of cTFH subsets and relative distribution after HSCT. (A) IgM production by naive B cells (IgD⁺CD27⁻) after in vitro coculture with different subsets of cTFH (CXCR5⁺) and non-TFH (CXCR5⁻) cells. (B) IgG production by naive B cells (IgD⁺CD27⁻) after in vitro coculture with different subsets of cTFH (CXCR5⁺) and non-TFH (CXCR5⁻) cells. N = 3. Graphs represent mean and standard deviation (SD). (C) Ratio of (Th2+Th17)/Th1 cTFH subsets in different patient groups. Black bars represent median values for each group. (D) Whisker plots represent frequency of Th1 (blue) and Th17 (red) cTFH subsets in patients with different severity of cGVHD. Exact Wilcoxon rank-sum test was used. ns, not significant; *P < .05; **P < 10⁻⁴.

Figure 4

Increased cTFH function in patients with active cGVHD. Frequency of plasmablasts (CD27^hiCD38^hi B cells) was measured after naive B cells were cultured with cTFH in the presence of SEB for 6 days. Circulating TFH were purified by cell sorting (purity >95%) from fresh patient samples and cultured with autologous naive (IgD⁺CD27⁻) B cells (Auto B) or naive B cells from an allogeneic healthy donor (Allo B). Results are compared for cTFH obtained from patients with no cGVHD (n = 6, for the autologous condition; n = 5 for the allogeneic condition) or active cGVHD (n = 8 for the autologous condition; n = 5 for the allogeneic condition). Black bars represent median values for each group. Exact Wilcoxon rank-sum test was used. *P < .05.

Figure 5

cTFH signature correlates with plasmablast generation in patients with cGVHD (N = 38). (A) Correlation between number of activated cTFH (ICOS^hiPD-1^hi CXCR5⁺CD45RA⁻CD4⁺ T cells) and number of circulating CD27⁺ B cells in patient samples. (B) Correlation between cTFH proliferation (%Ki67) and percentage of plasmablasts (CD27^hiCD38^hi) in patient samples. (C) Correlation between proliferation (%Ki67) of CXCR3⁻ cTFH and percentage of plasmablasts (CD27^hiCD38^hi) in patient samples. (D) Correlation between number of activated cTFH (ICOS^hiPD-1^hi) and number of plasmablasts (CD27^hiCD38^hi) in patient samples. (E) Correlation between percentage of activated CXCR3⁻ cTFH (ICOS^hiPD-1^hi) and number of plasmablasts (CD27^hiCD38^hi) in patient samples. The Spearman test was used.