Circulating B-Lymphocyte Subpopulations as Novel Biomarker for Measuring Activity of Chronic Graft-Versus-Host Disease.

Abstract

Introduction: Chronic graft-versus-host-disease (cGvHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT) and a leading cause of non-relapse mortality due to profound immunodeficiency. The approach currently used to establish the diagnosis of cGvHD depends almost exclusively on the clinical history, physical examination, and histopathologic confirmation. Biology-based markers for confirmation of diagnosis or monitoring progression of cGvHD are urgently warranted. The pathogenesis of cGvHD is poorly understood. Mounting evidence implies a role of B-cells in this autoimmune-like disorder. In this study we analyzed whether changes in B-cell subpopulations in the peripheral blood (PB) of HSCT recipients could serve as a biomarker for immune-modulatory consequences of cGvHD.Methods: Within six months 70 consecutive patients (45 men, 25 women; median age 47.5 years) with complete donor cell engraftment a median of 45.5 (range, 5–149) months after allogeneic HSCT were enrolled including 21 never having cGvHD and 49 with cGvHD (35 active and/or progressive, 14 resolved at study entry). The latter received standard anti-infective prophylaxis and immunosuppressive therapy with a median duration of 36 (range, 3–104) months. A third of patients with active/progressive cGvHD had more than 2 organs involved. Evaluations in the study consisted of clinical parameters including cGvHD severity and treatment, serum immunoglobulin (Ig) levels, and infections. PB was analyzed by multiparameter flow cytometry to define B-lymphocytes (CD19+), which were further subdivided by staining for surface Ig (IgD+/M+) and the B-cell memory marker CD27+ and more immature B-lymphocytes (CD19+/CD21−).Results: No significant differences in absolute B, T, and NK-cell counts between the groups with and without cGvHD were seen. However, the relative number of both class-switched and non-class-switched memory B-cells was significantly lower (below 3%) in patients with active cGvHD compared to patients never having cGvHD (up to 7%) (p<0.001, c2 =22.0 for class-switched and p=0.002, c2 =9.2 for non-class-switched). Absolute cell counts confirmed these findings (10.3 vs. 27.2 cells/uL, p=0.02 for class-switched and 12.1 vs. 22.1 cells/ul, p=0.047 for non-class-switched). Significantly more patients with active cGvHD had elevated numbers of immature CD19+/CD21− B-cells (>15% cut-off) than the groups with resolved or never having cGvHD (37% vs 21% vs 9%, p=0.024). In patients with >15% immature CD19+/CD21− B-cells significantly more severe infections (according to NCI-CTC Infection module grade III–IV) were seen compared to patients with low CD19+/CD21− B-cell counts (p=0.002, c2 =9.5). In the group of active cGvHD 12/13 (92%) patients with >15% CD19+/CD21− B-cells compared to 4/22 (18%) patients with ≤ 15% CD19+/CD21− B-cells experienced severe infections (p<0.001, c2 =18.1). First results in an ongoing prospective study showed an increase of memory B-cell numbers in patients with decreasing cGvHD activity.Discussion: Monitoring of class-switched and non-class-switched memory B-cells may allow measuring of disease activity of chronic GvHD. In combination with assessment of immature CD19+/CD21− B-cells new insights into immune-modulatory consequences of cGvHD focusing on B-cell defects and activation status are possible.