Evidence for B Cell Exhaustion in Chronic Graft-versus-Host Disease

Abstract

Chronic graft-versus-host disease (cGvHD) remains a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). A number of studies support a role for B cells in the pathogenesis of cGvHD. In this study, we report the presence of an expanded population of CD19+CD21- B cells with features of exhaustion in the peripheral blood of patients with cGvHD. CD21- B cells were significantly increased in patients with active cGvHD compared to patients without cGvHD and healthy controls (median 12.2 versus 2.12 versus 3%, respectively; p < 0.01). Compared with naïve (CD27-CD21+) and classical memory (CD27+CD21+) B cells, CD19+CD21- B cells in cGvHD were CD10 negative, CD27 negative and CD20hi, and exhibited features of exhaustion, including increased expression of multiple inhibitory receptors such as FCRL4, CD22, CD85J, and altered expression of chemokine and adhesion molecules such as CD11c, CXCR3, CCR7, and CD62L. Moreover, CD21- B cells in cGvHD patients were functionally exhausted and displayed poor proliferative response and calcium mobilization in response to B-cell receptor triggering and CD40 ligation. Finally, the frequencies of circulating CD21- B cells correlated with cGvHD severity in patients after HSCT. Our study further characterizes B cells in chronic cGVHD and supports the use of CD21-CD27-CD10- B cell frequencies as a biomarker of disease severity.

Keywords: CD19+CD21−CD27−CD10− cells; CD21– B cells correlate with cGVHD severity; chronic graft-versus-host disease; exhausted B cells; stem cell transplantation.

Figure 1

B cell subsets in patients with or without chronic graft-versus-host disease (cGvHD) and healthy controls (HC). Box plots, showing medians, 25th and 75th percentiles and ranges, compare total lymphocyte count (A) and total CD19+ B cell count (B) in cGvHD patients compared to “no cGvHD” patients and HC. (C) Representative FACS plots demonstrating CD19+ B cell phenotype in a “no GvHD,” a cGvHD patient and a healthy control. Gated CD19+ B cells were examined for CD24 and CD38 expression; transitional (CD19+CD24hiCD38hi), naïve (CD19+CD24+CD38+) and memory (CD19+CD24hiCD38−) B cell subsets are presented. (D) Scatter plots compare the percentage of individual B cell subsets in patients with “no GvHD” (n = 23), with cGvHD (n = 32) and HC (n = 18).

Figure 2

CD19+CD21− B cells in patients with or without chronic graft-versus-host disease (cGvHD) and healthy controls (HC). (A) FACS plots showing CD21− B cell frequency in a representative “no GvHD” patient, GvHD patient, and a healthy control. Chronic GvHD patients (n = 32) have significantly higher percentage of circulating CD21− B cells compared with “no cGvHD” patients (n = 23) and HC (n = 18). (B,C) Color-coded gating on CD19+ B cells in representative “no GvHD” and active cGvHD patients; (B) cGvHD patient has higher frequencies of CD21− B cells (red) compared with the “no cGvHD” representative patient (yellow). (C) Gated CD19+ B cells from the same examples are shown; the majority of CD21− B cells in the “no cGvHD” group (yellow) fall within the CD24hiCD38hi transitional B cell and CD24−CD38hi plasmablast regions. Conversely, the majority of CD21− B cells in “cGvHD” patients (Red) fall within the CD24−CD38− exhausted B cell and CD24−CD38hi plasmablast regions. (D) Phenotypic characterization of transitional (yellow) and exhausted CD21− B cells (red) compared with CD21+ B cells (black) in a patient with cGVHD.

Figure 3

Expression of inhibitory and chemokine receptors on CD19+CD21−CD27−CD10− B cells. (A) FACS plots from a representative chronic graft-versus-host disease (cGvHD) patient show the expression of chemokine and inhibitory receptors on gated CD19+ B cells in relation to CD21. (B) Color-coded gating on CD27+CD21+ in B cells from a representative cGvHD patient. The expression of inhibitory and chemokine receptors on naïve (blue), classical memory (red), and CD21− B cells (black). (C) Inhibitory and homing receptor expression on CD21−CD27−CD10− B cells relative to naïve and classical CD27+ memory B cells. Plots depict individual median fluorescence intensity (MFI) values for each cell subpopulation from 22 cGvHD patients. (D) FACS plot show gating strategy on CD21−CD27+ activated memory B cells from a representative cGVHD patient (left). Scatter blot compare the frequency of activated memory B cells between the two post-transplant groups; p = ns. *Represents a p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

Proliferation of CD19+ B cell in response to B cell receptor (BCR) triggering and CD40L ligation. Carboxyfluorescein Succinimidyl Ester (CFSE)-stained peripheral blood mononuclear cells from healthy donors and patients with or without chronic graft-versus-host disease (cGvHD) were stimulated, anti-CD3/CD28 alone, or a combination of anti-BCR and anti-CD3/CD28 beads for 96 h. (A) Representative CFSE histograms comparing the proliferation of gated CD19+ B cells. (B) Comparison of B cell proliferation in 10 cGvHD patients, 7 no GvHD patients, and 10 healthy controls (HC). Chronic GvHD patients had the lowest proliferative potential in response to B cell stimulation compared with “no GvHD” patients and HC. (C) FACS plots of a representative cGvHD patient comparing the proliferation of CD27+ memory B cells and CD21+CD27− naïve B cells with CD21− B cells. (D) CD21− B cells proliferated significantly less than the rest of B cells (n = 8) when compared using non-parametric t-test p < 0.001.

Figure 5

Exhausted B cells (CD21−CD27−) fail to mobilize calcium (Ca2+) in response to B cell receptor (BCR) triggering. (A,B) Peripheral blood mononuclear cells from chronic graft-versus-host disease (cGVHD) patients were loaded with the calcium dye Fluo-4AM and stained with monoclonal antibodies against CD19, CD21, and CD27 (n = 10). Ca2+ mobilization was assessed by flow cytometry after gating on CD21−CD27− and CD21+CD27− B cell subsets. CD21−CD27− B cells had an attenuated Ca2+ response compared to CD21+CD27− B cells. (C) Median fluorescence intensity (MFI) of Fluo-4AM was significantly lower in CD21−CD27− B cells (n = 10); p = 0.005. (D) CD21− B cells from healthy donors did not have reduced Ca2+ mobilization in response to BCR stimulation when compared with CD21+ B cells (n = 8; p = 0.15).

Figure 6

CD21−CD27−CD10− B cells and severity of chronic graft-versus-host disease (cGvHD). Box plot depict the severity of cGvHD (moderate to severe) according to CD21− B cell frequencies in 32 patients with cGVHD.