Elevated Numbers of Immature/Transitional CD21⁻ B Lymphocytes and Deficiency of Memory CD27⁺ B Cells Identify Patients with Active Chronic Graft-versus-Host Disease

Time from HSCT until analysis of B cell subpopulations. On the X axis, the 3 patient groups consisting of never cGVHD (no), resolved (res.) and active (act.) cGVHD at the time of first flow cytometric exam are shown; on the Y axis, the months from day 0 of hematopoietic stem cell transplantation (HSCT) until first B cell analysis. Bars represent mean values in each group. P-value between patient groups never cGVHD and active cGVHD was calculated by two-tailed Students t-test.

Phenotype of circulating B cells after HSCT. Whole blood cells were stained with the indicated fluorochrome conjugated mAbs. Subsequently, red blood cells were lysed and the remaining nucleated cells were analyzed by flow cytometry. CD19⁺ peripheral blood B lymphocytes were gated and the differential expression of surface IgD, CD27 and CD21 was assessed in healthy donors (HD) and patients with active (active cGVHD) or without (no cGVHD) cGVHD. Results are expressed as two-parameter dot-plots and are representative for the individuals within the same group. Numbers indicate the percentage of positive cells in the respective area.

Significantly lower numbers of memory B lymphocytes in patients with active cGVHD. Whole blood cells were stained for the expression of CD19, IgD, and CD27 as exemplified in Figure 3. After HSCT, patients were assigned to various groups: 35 patients with active (act.–partial response or progression) and 14 with resolution (res.–complete response) of cGVHD according to published criteria. Another 21 patients had never experienced cGVHD (no) until study entry. In patients analyzed serially, only the first time point was included in this figure. Diamonds represent the percentage of non-class-switched (A) and class- switched (B) memory B lymphocytes for each patient. Statistical analysis was performed by the paired Student's t-test, and P values are indicated. The 2 outliers in A have been excluded from calculations.

Serial analyses of B cell subpopulations. On the X-axis, the time on study in months calculated from first assessment of B cell subsets is shown; on the Y-axis, the ratio formed between immature/transitional B lymphocytes and memory B cells is shown. A represents patients with active cGVHD not responding or progressing during study (n=12); B shows patients with active cGVHD achieving a partial overall response during study (n=13); and C shows patients without cGVHD (n=11).