Peripheral CD5+ B cells in antineutrophil cytoplasmic antibody-associated vasculitis

Abstract

Objective: CD5+ B cells have been conceptualized as a possible surrogate for Breg cells. The aim of the present study was to determine the utility of CD5+ B cells as biomarkers in antineutrophil cytoplasmic antibody-associated vasculitis (AAV).

Methods: The absolute and relative numbers (percentages) of CD5+ B cells (explanatory variables) were measured longitudinally during 18 months in 197 patients randomized to receive either rituximab (RTX) or cyclophosphamide (CYC) followed by azathioprine (AZA) for the treatment of AAV (Rituximab in ANCA-Associated Vasculitis [RAVE] trial). Outcome variables included disease activity (status of active disease versus complete remission), responsiveness to induction therapy, disease relapse, disease severity, and, in RTX-treated patients, relapse-free survival according to the percentage of CD5+ B cells detected upon B cell repopulation.

Results: CD5+ B cell numbers were comparable between the treatment groups at baseline. After an initial decline, absolute CD5+ B cell numbers progressively increased in patients in the RTX treatment arm, but remained low in CYC/AZA-treated patients. In both groups, the percentage of CD5+ B cells increased during remission induction and slowly declined thereafter. During relapse, the percentage of CD5+ B cells correlated inversely with disease activity in RTX-treated patients, but not in patients who received CYC/AZA. No significant association was observed between the numbers of CD5+ B cells and induction treatment failure or disease severity. The dynamics of the CD5+ B cell compartment did not anticipate disease relapse. Following B cell repopulation, the percentage of CD5+ B cells was not predictive of time to flare in RTX-treated patients.

Conclusion: The percentage of peripheral CD5+ B cells might reflect disease activity in RTX-treated patients. However, sole staining for CD5 as a putative surrogate marker for Breg cells did not identify a subpopulation of B cells with clear potential for meaningful clinical use. Adequate phenotyping of Breg cells is required to further explore the value of these cells as biomarkers in AAV.

Figure 1

Absolute and relative numbers of CD5+ B cells in patients with antineutrophil cytoplasmic antibody–associated vasculitis (AAV) treated with cyclophosphamide/azathioprine (CYC/AZA)–based and rituximab (RTX)–based regimens. Whole blood was obtained at different time points during a period of 18 months from patients with AAV who were treated with RTX (n = 99) or CYC/AZA (n = 98). Cells were stained for CD19 and CD5, and expression was determined by flow cytometry. Results are expressed in A, absolute numbers of CD19+CD5+ B cells/μl and B, percentages of CD5+ B cells within total CD19+ B cells. Groups were compared using repeated-measures analysis of variance with adjustment for multiple testing. Bars show the median and interquartile range. * = P < 0.05.

Figure 2

Percentage of CD5+ B cells in AAV patients treated with CYC/AZA or RTX during times of active disease and complete remission. Percentages of CD5+ B cells were serially obtained from patients treated with CYC/AZA or RTX who achieved and maintained complete remission with their original treatment until month 18 (nonrelapsers; RTX n = 52, CYC/AZA n = 50), and those who achieved complete remission with their original treatment but subsequently experienced a disease flare (relapsers; RTX n = 24, CYC/ AZA n = 20). The percentages of CD5+ B cells were compared within groups using Wilcoxon’s signed rank test. The × symbols represent values obtained from individual patients during active disease at baseline, circles represent values obtained during complete remission (e.g., first complete remission in all subgroups and at 18 months in nonrelapsers), and asterisks represent values obtained during disease flare (in relapsers). The remaining 51 subjects from the original cohort of 197 patients did not achieve complete remission and were therefore not included in this analysis. See Figure 1 for other definitions.

Figure 3

Percentage of CD5+ B cells prior to and during disease relapse in AAV patients treated with RTX or CYC/AZA. The percentage of CD5+ B cells was compared immediately before and during disease relapse in 37 patients with AAV according to treatment group (CYC/AZA n = 16, RTX n = 21). Symbols joined by lines represent individual patients. Results were compared using Wilcoxon’s signed rank test. See Figure 1 for other definitions.

Figure 4

Absolute and relative numbers of CD5+ B cells in RTX-and CYC/AZA-treated patients who developed either a relapsing or nonrelapsing disease course. The absolute numbers (A) and relative numbers (percentage) (B) of CD5+ B cells were measured longitudinally in 146 AAV patients over a period of 18 months according to relapsing phenotype (those without relapse during the study [nonrelapsers] and those with at least one relapse during the study [relapsers]) and treatment group (RTX nonrelapsers n = 52 and RTX relapsers n = 24; CYC/AZA nonrelapsers n = 50 and CYC/AZA relapsers n = 20). Bars show the median and interquartile range. The remaining 51 subjects from the original cohort of 197 patients did not achieve complete remission and were therefore not included in this analysis. See Figure 1 for other definitions.

Figure 5

Relapse-free survival in patients with AAV according to the kinetics of CD5+ B cell repopulation after RTX treatment. Kaplan-Meier estimates of the time to relapse from complete remission were determined according to the percentage of CD5+ B cells at the time of B cell redetection (n = 69) and reconstitution (n = 54) after RTX administration. A and B, The percentage of CD5+ B cells was analyzed as a dichotomous predictor of time to relapse (>30% CD5+ B cells [brown lines] versus ≤30% CD5+ B cells [purple lines]) during times of B cell redetection (>30% n = 32 and ≤30% n = 37) (A) and B cell reconstitution (>30% n = 15 and ≤30% n = 39) (B). C and D, The percentage of CD5+ B cells was analyzed as a categorical predictor of time to relapse (stratified in quartiles: first quartile = purple lines, second quartile = green lines, third quartile = brown lines, fourth quartile = yellow lines) during times of B cell redetection (first quartile n = 18, second–fourth quartiles n = 17 each) (C) and B cell reconstitution (first quartile n = 14, second quartile n = 13, third quartile n = 13, fourth quartile n = 14) (D). Bars show the 95% confidence intervals at 183, 274, and 365 days. Results were compared using the log rank test in A and B and the Cox proportional hazards method in C and D. See Figure 1 for other definitions.