B Cell Deficiency and CD21low B Cells As Markers For Measuring Cgvhd Severity and Activity In a Large Patient Cohort
Authors: Kuzmina Zoya , Rose Jeremy , Rehman Najibah , Cowen Edward W. , Baird Kristin , Cole Kristen , Baruffaldi Judy , Gress Ronald E. , Pavletic Steven Z. , Hakim Frances T. 
Published: October 2019

Background
In chronic graft versus host disease (cGVHD) a role for B cells in disease pathogenesis is supported by the presence of autoantibodies and the therapeutic efficacy of B cell depletion. Active cGVHD is also associated with altered B cell homeostasis. Elevated levels of B cell activation factor (BAFF) are coupled to a high incidence of lymphopenia and an elevated frequency of an atypical population of CD21- B cells. In order to assess the utility of altered B cell populations as markers of cGVHD, we examined the frequency and clinical features of B lymphopenia and characterized atypical CD21- B cells in a large cohort of patients.

Patients and methods
Between October 2004 and May 2013, B cells (CD19+) were assessed in 260 patients consecutively enrolled in a cross-sectional natural history study of cGVHD (NCT00092235). We assessed B cell transitional, naïve, memory and plasmablast populations and the expression of markers, of antigen presentation (CD86, HLA-DR), tissue homing (CXCR3, CXCR4, CXCR5) and activation (CD95, CD11c, CD38, HLA-DR). Non-parametric Mann-Whitney comparisons of populations were used with Bonferroni correction for multiple comparisons.

Results
Of 260 patients examined, 44% (n=114) were severely B-lymphopenic (fewer than 100 B cells/µl; median 22). Compared to cGVHD patients with normal to elevated B cell numbers (median 563), these patients had reduced levels of immunoglobulin (IgM, IgG, IgA), decreased T subset and NK cells and significantly higher levels of BAFF (4.7 vs. 2.9 ng/ml, p=.003). B-lymphopenic patients had more active cGVHD (34% vs. 12%, p=.001), and had received more lines of systemic therapy (4.4 vs. 3.3, p<.0001), including prior anti-CD20 therapy for cGVHD (39% vs. 10%, p<.0001). These patients had shorter durations of cGVHD (2.5 years vs. 3.7, p=.007) and time from transplant (3.6 vs. 4.8 years, p=.007) Clinically they had a higher frequency of skin involvement (54% vs. 27%, p<.001), mainly erythematous (p<.001) and a lower Karnofsky score (p=.005).
Anti-CD20 can result in prolonged B cell deficiency. Excluding such patients from our analysis, we identified a significant expansion (>10%) of an atypical population of CD19+CD21low B cells in 40% of the patients. Consistent with previous reports, these high %CD21low patients were receiving more immunosuppression (p=.008), had lower Karnofsky scores (p=.036), and increased mortality (p=.018). To further characterize the potential role of CD21low cells in cGVHD, in 26 patients we then studied expression of markers associated with B cell maturation, activation, trafficking and capacity for antigen presentation. Comparing patients with more than 10% CD21low B cells (12) with those with less, the high %CD21low group had a lower frequency of naïve (IgD+CD27-) B cells (p=.006), but a higher frequency of IgD-CD27- B cells. The CD21low B cells, and in particular those that were IgD- and CD27+, showed evidence of prior activation: increased size (forward scatter), expression of CD95 and of the Toll receptor-inducible integrin CD11c. Chemokine receptor expression on CD21low B cells reflected an increased capacity to home to inflammatory sites. CXCR3 (receptor responding to interferon-induced chemokines) was increased, while CXCR4 and CXCR5 (supporting trafficking of naïve B cells to lymph nodes) were reduced. Finally, expression of CD86, a critical molecule for antigen presentation by B cells, was increased. All of these changes were more pronounced in the patient subset with a higher frequency of CD21lowCD27- cells: higher expression of CXCR3 (p=.027), CD11c (p=.001), CD95 (p=.001) and CD86 (p=.008), but decreased CXCR4+CXCR5+ (p=.035).

Conclusion
B-lymphopenia is common in cGVHD population and is associated with severe and active disease, thus posing important and frequently underappreciated limitations to studying B cells. Patients with severe cGVHD and circulating B cells show accumulation of CD21low B cells. Disease activity and “chronicity” can be related to B cells activation (CD95, CD11c), altered migration (downregulation of CXCR4/5, increased CXCR3) and an increased capacity to function as APC (CD86+). Alterations in B cell subpopulations may explain the role of B cells in cGVHD pathogenesis as well as provide noninvasive cellular marker for objective diagnosis and monitoring of cGVHD activity.

Disclosures:
No relevant conflicts of interest to declare.