B-cell reconstitution recapitulates B-cell lymphopoiesis following haploidentical BM transplantation and post-transplant CY
The treatment of many hematological diseases benefits from myeloablative or non-myeloablative conditioning regimens followed by SCT or BMT. HLA-matched donors are preferred but not always available. Instead, haploidentical donors can be rapidly identified. Unmanipulated haploidentical BMT (haploBMT) with non-myeloablative conditioning and post-transplant Cy has been developed to provide a universal source of BM donors. Cy, which depletes proliferating/allogeneic cells, prevents GVHD. Importantly, the infection-related mortality was remarkably low, suggesting effective immune reconstitution. However, a detailed analysis in this regard is missing. Here we show the dynamics of the B-cell compartment. Ten consecutive patients were treated with the haploBMT protocol established by Luznik et al. (see Supplementary Methods for details; Supplementary Figure 1A). Patient characteristics are listed in Supplementary Table 1. Blood and BM were obtained from Humanitas Cancer Center. Peripheral blood (PB) and BM were processed and frozen in liquid nitrogen according to the standard procedures and prepared for flow cytometry, as described. Non-parametric Wilcoxon rank test was used to compare distributions. Differences in the pie chart distributions were calculated with SPICE software (National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA) by using a permutation test. P-values are two sided and were considered significant when ⩽ 0.05. Ten patients who underwent haploBMT were followed for a median duration of 24 weeks (range: 5–26 weeks). B cells (CD3CD14CD19CD20) were not detected up to 4 weeks after haploBMT, suggesting that cells transferred with the graft did not persist (Figure 1a). Rather, B cells appeared at week 4–5, tended to increase over time from week 7 and, in some patients, reached levels similar to that of the donors (Figure 1a). Analysis of donor– recipient chimerism of PB leukocytes (day 120) and of purified CD3CD56CD14CD19 B cells revealed that 498% of recipient cells carried donor alleles (Figure 1b). FACS analysis of the HLA haplotypes in donor–recipient pairs mismatched for HLA-A*02 confirmed the donor origin of the reconstituting B cells (Supplementary Figure 1B). Recovering B cells were almost Ki-67 at week 8 post BMT (Supplementary Figure 1C), suggesting that increase in the B-cell numbers was not due to the homeostatic proliferation of transferred B cells. The phenotype of reconstituting B cells since 5 weeks post BMT was largely different from that of BM B cells; indeed, the former population predominantly displayed a CD38CD10 immature/ ‘transitional’ phenotype (that is, precursor of CD38CD10 mature B cells), whereas the latter was mature (Figure 1c). At later time points, transitional cells were replaced by mature B cells (Figure 1c). Together, these data further indicate that B-cell recovery depends on a new maturation process originating from donor B-cell precursors. From week 9, the proportion of transitional B cells progressively decreased (not shown), whereas that of mature B cells increased (Figure 1d). To further evaluate the differentiation of mature cells, we included markers of naivety (IgM and IgD) and memory (IgG) in our polychromatic panel. At week 9, when a sufficient proportion of cells were available for the analysis, B cells were mostly naive and remained so for 26 weeks after haploBMT (Figure 1d). Despite low, the proportion of memory B cells reached levels similar to that of marrow donors (Supplementary Figure 1D). To further investigate the steps of B-cell maturation, we analyzed CD5, a regulator of B-cell activation, and CD21, a component of the B-cell coreceptor complex, on transitional B cells. These surface markers characterize different stages of transitional B-cell development. Similarly to published data, we observed three distinct transitional subsets during reconstitution: T1 (CD5CD21), T2 (CD5CD21) and the CD5CD21 subset (Figure 2a). In addition, we report an additional CD5CD21 stage, here named T0, which precedes the aforementioned T1 and T2. The differentiation status of transitional B cells shortly after BMT was largely different between BM donors and patients and significantly changed over time. At week 5, these cells mostly displayed a T0 and, in smaller proportion, a T1 phenotype (Figure 2a). T0 cells progressively decreased in favor of T2 and CD5CD21 cells. No significant difference was detected between patients and BM donors at 14 weeks post haploBMT (Figure 2a). Transitional B-cell subsets in patients underwent a maturation process leading to the generation of mature naive B cells, as they progressively upregulated naive markers, that is, IgD, IgM and CD21 (Figure 2b and Supplementary Figure 2). Notably, the surface expression of IgM at week 15 was significantly higher compared with that of transitional B cells from donors (Supplementary Figure 2). In summary, a detailed longitudinal analysis of markers associated with B-cell differentiation shows that B cells are generated de novo following haploBMT. The B-cell compartment is initially characterized by the presence of transitional B cells and is progressively repopulated by mature naive B cells. The differentiation of transitional B cells is characterized by a process of progressive maturation identified by CD5 and CD21 expression, as previously suggested. In humans, CD5 is expressed by immature/transitional B cells and is downregulated by mature naive B cells, whereas CD21 distinguishes CD21 T1 from CD21 T2 transitional stage. T1 are considered as precursors of T2 B cells. Recently, pre-naive B cells, which are phenotypically naive but retain CD5 expression, have been described. Others referred to cells with a similar phenotype and functional capacity, but negative for CD5, as T3 transitional B cells. CD21 T1 cells were proposed as the first to develop in the circulation following BMT. Here, we integrate these data by using a polychormatic flow cytometry approach, and report a new putative transitional B-cell stage, named T0, defined as CD5CD21. These cells are the first to appear during B-cell recovery. The B-cell maturation process from T0 to naive B cells takes ~ 6 months to complete in haploBMT patients. Bone Marrow Transplantation (2015) 50, 317–319 © 2015 Macmillan Publishers Limited All rights reserved 0268-3369/15