In our study we quantitatively and phenotypically analysed B- and T-cell subsets in peripheral blood by flow cytometry at days 180 and 360 after alloHSCT in acute leukemic patients (n=36). In addition, apoptosis of B-cell subsets was investigated after stimulation with CpG and CD40L. To address the B-cell milieu cytokines and chemokines were measured with Luminex technology.
Half of patients at day 180 and all patients at day 360 displayed fully restored absolute B-cell numbers, although CD27+ memory B-cell subsets remained diminished (cells/µl±SEM: healthy control (HC) 26±4, alloHSCT 4±1; p≤0.001). All B-cell subsets were characterized by an activated/ pro-apoptotic phenotype with an increased CD86 and Fas but reduced Baff-R expression at day 180. Accordingly, an inflammatory milieu was present with increased serum levels of TNFa, IFNa2, G-CSF, IP-10, MCP-1, MIP-1b and Eotaxin in patients compared to HC. While CD86 normalized at day 360 on most B-cell subsets, all subsets retained a strong pro-apoptotic phenotype with increased Fas and reduced Baff-R expression. Interestingly, stimulation of peripheral blood mononuclear cells with CpG and CD40L resulted in significantly increased percentages of apoptotic cells for the patients compared to HC (% of B cells±SEM: HC 11±2, alloHSCT 28±5 day180, 60±6 day 360; p≤0.001).
Regarding the lack of CD27+ memory B-cell recovery, we assumed a defective germinal center (GC) reaction. Supporting this suggestion we found (A) a reduced CXCR5 expression on naïve B cells, important for the entry into B-cell follicles (MFI±SEM: HC 5899±975, alloHSCT 3494±554; p≤0.01), (B) a low HLA-DR expression on naïve B cells, essential for antigen presentation (HC 20440±4742, alloHSCT 9150±2386; p≤0.01), (C) a normal number of CD27-IgD- double negative (DN) B cells, suggested to prematurely leave the GC reaction, (D) a remaining CD4+ T-cell deficiency (cells/µl±SEM: HC 972±77, alloHSCT 303±45; p≤0.001) and (E) a reduced percentage of circulating CXCR5+ follicular T helper cells, importantly involved in GC reactions (%±SEM: HC 6±2, alloHSCT 2±1).
We conclude that a sustained inflammatory milieu and possibly damage within lymphoid organs might contribute to a prolonged pro-apoptotic state of B-cell subsets and defects in the GC reaction long-term after allo-HSCT. Despite normal total B-cell numbers significant functional deficits exist more than one year after transplant, so that adoptive transfer of memory CD27+ B cells should be pursued.