B-Cell Reconstitution Recapitulates B-Cell Lymphopoiesis Following Haploidentical BM Transplantation and Post-Transplant CY

LETTER TO THE EDITOR B-cell reconstitution recapitulates B-cell lymphopoiesis following haploidentical BM transplantation and post-transplant CY

Bone Marrow Transplantation (2015) 50, 317–319; doi:10.1038/ From week 9, the proportion of transitional B cells progressively bmt.2014.266; published online 24 November 2014 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 The treatment of many hematological diseases benefits from our polychromatic panel. At week 9, when a sufficient proportion myeloablative or non-myeloablative conditioning regimens of cells were available for the analysis, B cells were mostly naive followed by SCT or BMT. HLA-matched donors are preferred but and remained so for 26 weeks after haploBMT (Figure 1d). Despite not always available. Instead, haploidentical donors can be rapidly low, the proportion of memory B cells reached levels similar to identified. Unmanipulated haploidentical BMT (haploBMT) with that of marrow donors (Supplementary Figure 1D). non-myeloablative conditioning and post-transplant Cy has been To further investigate the steps of B-cell maturation, we developed to provide a universal source of BM donors.1 Cy, analyzed CD5, a regulator of B-cell activation, and CD21, a which depletes proliferating/allogeneic cells, prevents GVHD.1 component of the B-cell coreceptor complex, on transitional B Importantly, the infection-related mortality was remarkably low, cells. These surface markers characterize different stages of 5,6 5,6 suggesting effective immune reconstitution.1,2 However, a transitional B-cell development. Similarly to published data, detailed analysis in this regard is missing. Here we show the we observed three distinct transitional subsets during reconstitution: T1 (CD5+CD21−), T2 (CD5+CD21+) and the CD5−CD21+ subset dynamics of the B-cell compartment. − − Ten consecutive patients were treated with the haploBMT (Figure 2a). In addition, we report an additional CD5 CD21 stage, protocol established by Luznik et al.1 (see Supplementary Methods here named T0, which precedes the aforementioned T1 and T2. for details; Supplementary Figure 1A). Patient characteristics are The differentiation status of transitional B cells shortly after BMT listed in Supplementary Table 1. Blood and BM were obtained was largely different between BM donors and patients and from Humanitas Cancer Center. Peripheral blood (PB) and BM significantly changed over time. At week 5, these cells mostly were processed and frozen in liquid nitrogen according to the displayed a T0 and, in smaller proportion, a T1 phenotype standard procedures and prepared for flow cytometry, as (Figure 2a). T0 cells progressively decreased in favor of T2 and − described.3 Non-parametric Wilcoxon rank test was used to CD5 CD21+ cells. No significant difference was detected between compare distributions. Differences in the pie chart distributions patients and BM donors at 14 weeks post haploBMT (Figure 2a). were calculated with SPICE software (National Institute of Allergy Transitional B-cell subsets in patients underwent a maturation and Infectious Diseases, Bethesda, MD, USA) by using a permuta- process leading to the generation of mature naive B cells, as they tion test. P-values are two sided and were considered significant progressively upregulated naive markers, that is, IgD, IgM and when ⩽ 0.05. CD217 (Figure 2b and Supplementary Figure 2). Notably, the Ten patients who underwent haploBMT were followed for a surface expression of IgM at week 15 was significantly higher median duration of 24 weeks (range: 5–26 weeks). B cells compared with that of transitional B cells from donors (CD3−CD14−CD19+CD20+) were not detected up to 4 weeks after (Supplementary Figure 2). haploBMT, suggesting that cells transferred with the graft did not In summary, a detailed longitudinal analysis of markers persist (Figure 1a). Rather, B cells appeared at week 4–5, tended to associated with B-cell differentiation shows that B cells are increase over time from week 7 and, in some patients, reached generated de novo following haploBMT. The B-cell compartment levels similar to that of the donors (Figure 1a). Analysis of donor– is initially characterized by the presence of transitional B cells and recipient chimerism of PB leukocytes (day 120) and of purified is progressively repopulated by mature naive B cells. The CD3−CD56−CD14−CD19+ B cells revealed that 498% of recipient differentiation of transitional B cells is characterized by a process cells carried donor alleles (Figure 1b). FACS analysis of the HLA of progressive maturation identified by CD5 and CD21 expression, haplotypes in donor–recipient pairs mismatched for HLA-A*02 as previously suggested.5,6 In humans, CD5 is expressed by confirmed the donor origin of the reconstituting B cells immature/transitional B cells and is downregulated by mature − (Supplementary Figure 1B). Recovering B cells were almost naive B cells,8 whereas CD21 distinguishes CD21 T1 from CD21+ Ki-67− at week 8 post BMT (Supplementary Figure 1C), suggesting T2 transitional stage.5,6 T1 are considered as precursors of T2 B that increase in the B-cell numbers was not due to the cells.7 Recently, pre-naive B cells, which are phenotypically naive homeostatic proliferation of transferred B cells.4 The phenotype but retain CD5 expression, have been described.5 Others referred of reconstituting B cells since 5 weeks post BMT was to cells with a similar phenotype and functional capacity, but − largely different from that of BM B cells; indeed, the former negative for CD5, as T3 transitional B cells.9 CD21 T1 cells were population predominantly displayed a CD38brightCD10+ immature/ proposed as the first to develop in the circulation following BMT.6 ‘transitional’ phenotype (that is, precursor of CD38dimCD10− Here, we integrate these data by using a polychormatic flow mature B cells), whereas the latter was mature (Figure 1c). At cytometry approach, and report a new putative transitional B-cell − − later time points, transitional cells were replaced by mature B cells stage, named T0, defined as CD5 CD21 . These cells are the first to (Figure 1c). Together, these data further indicate that B-cell appear during B-cell recovery. The B-cell maturation process from recovery depends on a new maturation process originating from T0 to naive B cells takes ~ 6 months to complete in haploBMT donor B-cell precursors. patients. Letter to the Editor 318 The small sample size could not allow to identify associations well as the functional capacity of the recovering B cells in this type between the extent of B-cell reconstitution and post-transplant of transplant. Patients receiving allogeneic transplants are complications. Future studies will need to address this aspect, as routinely vaccinated for multiple pathogens starting at 6 months

######### Host ***** Donor 800 3 9 PBMCs B cells 600 4 11 5 13 6 15

L 400 8 17 µ 300 Recipient PB 200 Donor BM Week 5 Week 8 Week 14 B cells/ Transitional 7.6% 100 98.2% 93.4% 44.4%

0 D89132226475116 10 1214 15 CD10 Weeks after BMT Mature: 92.4% 1.8% 6.6% 55.6% CD38 Mature Naive ********* ** ** 100

% of B cells 20

0 D89132226475116 10 1214 15 D 9 10 11 12 13 14 15 22 26 Weeks after BMT Figure 1. B-cell dynamics following haploBMT. (a) Absolute numbers (cells/μL) of circulating CD20+ B lymphocytes in transplanted patients at different time points after haploBMT compared with those of related BM donors (D). Single donor/recipient pairs are identiﬁed by different symbols, as depicted in the legend. Gray box indicates the range of peripheral B cells observed in healthy donors. (b) Proportions of donor- derived cells (gray) and residual host cells (black) in the PBMCs of the entire cohort of BM recipients (left pie, day 120) and in FACS-sorted B cells (right pie) from three BM recipients. (c) CD38 and CD10 expression in B cells in the donor BM and in the recipient. (d) Percent mature and naive B cells at different times post haploBMT. Data are shown as in a *,#Po0.05 vs D and week 4, respectively.

Recipient PB CD21 IgD IgM Week 5 Week 8 Week 14 Donor PB 258 93 2864 T1 T2 39% 24% 12% 28% 13% 32% 35% 10% week 5

CD5 8.2% - + 432 506 4827 T0 5 21 7.6% 38% 40% CD21 week 8 Pie arcs: CD5+ CD21+ - + T2 5 21 569 640 7890

T0 week 15

** Patient D CD3+ * *

*** Figure 2. B-cell recovery recapitulates B-cell ontogeny. (a) Representative example of CD5 and CD21 expression on CD38brightCD10+ transitional B cells (top) and proportions (pie chart at the bottom) of T0 (CD5−CD21−), T1 (CD5+CD21−), T2 (CD5+CD21+) and CD5−CD21+ B cells at different times post haploBMT. Arcs indicate the relative proportion of CD5+ (gray arc) and CD21+ cells (black arc). *Po0.05; **Po0.01; ***Po0.001. (b) CD21, IgD and IgM expression on transitional B cells identiﬁed as in a. Numbers in the plots indicate the median ﬂuorescence intensity. CD3+ T cells are used as negative control.

Bone Marrow Transplantation (2015) 317 – 319 © 2015 Macmillan Publishers Limited Letter to the Editor 319 after stem cell infusion. Our phenotypic data indicate that 3Department of Medical Biotechnologies and Translational Medicine triggering the B-cell pool with vaccination at this time would (BioMeTra), University of Milan, Milan, Italy and potentially induce Ab responses that are similar to those observed 4ImmunoTechnology Section, Vaccine Research Center, National in other transplantation settings. Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA E-mail: [email protected] or CONFLICT OF INTEREST [email protected] The authors declare no conﬂict of interest. 5These authors contributed equally to this work.

ACKNOWLEDGEMENTS REFERENCES We would like to thank the patients for their participation to this study and the nurse 1 Luznik L, O'Donnell PV, Symons HJ, Chen AR, Leffell MS, Zahurak M et al. team of the Hematology and Bone Marrow Transplant Unit (Humanitas Research HLA-haploidentical bone marrow transplantation for hematologic malignancies Hospital, Rozzano, Milan, Italy) who performed blood draws and samples collection, using nonmyeloablative conditioning and high-dose, posttransplantation cyclo- Margaret Beddall and Pratip Chattopadhyay (Vaccine Research Center, National phosphamide. Biol Blood Marrow Transplant 2008; 14: 641–650. Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 2 Raiola A, Dominietto A, Varaldo R, Ghiso A, Galaverna F, Bramanti S et al. MD, USA) for Ab conjugation and validation, Achille Anselmo and Chiara Buracchi Unmanipulated haploidentical BMT following non-myeloablative conditioning (Flow Cytometric facility, Humanitas Clinical and Research Center) for help with cell and post-transplantation CY for advanced Hodgkin's lymphoma. Bone Marrow sorting, Barbara Cassani and Gaetano Bulfamante (Unit of Pathology of San Paolo Transplant 2014; 49: 190–194. Hospital, Milan, Italy) for the chimerism analyses and Anna Villa (CNR, Milan, Italy) and 3 Lugli E, Mueller YM, Lewis MG, Villinger F, Katsikis PD, Roederer M. IL-15 delays the members of the Mavilio laboratory for critical discussion. This work has been suppression and fails to promote immune reconstitution in virally suppressed supported by grants from the Fondazione Cariplo (Grant Ricerca Biomedica chronically SIV-infected macaques. Blood 2011; 118: 2520–2529. 2012/0683 to EL), by European Union (Marie Curie Career Integration Grant 322093 4 Cabatingan MS, Schmidt MR, Sen R, Woodland RT. Naive B lymphocytes undergo to EL), by Associazione Italiana per la Ricerca sul Cancro (AIRC) (MFAG 10607 to EL), by homeostatic proliferation in response to B cell deficit. J Immunol 2002; 169: the intramural program of the National Institutes of Allergy and Infectious Diseases to 6795–6805. MR and by intramural research and clinical funding programs assigned to DM and LC. 5 Lee J, Kuchen S, Fischer R, Chang S, Lipsky PE. Identification and characterization of Alessandra Roberto is a recipient of the ‘Guglielmina Lucatello e Gino Mazzega’ a human CD5+ pre-naive B cell population. J Immunol 2009; 182: 4116–4126. fellowship from the Fondazione Italiana per la Ricerca sul Cancro (FIRC). ClinicalTrials. 6 Suryani S, Fulcher DA, Santner-Nanan B, Nanan R, Wong M, Shaw PJ et al. gov identifier No. NCT02049424. Differential expression of CD21 identifies developmentally and functionally distinct subsets of human transitional B cells. Blood 2010; 115: 519–529. A Roberto1, L Castagna2, S Gandolfi2, V Zanon1, S Bramanti2, 7 Suryani S, Tangye SG. Therapeutic implications of advances in our understanding B Sarina2, R Crocchiolo2, E Todisco2, C Carlo-Stella2, P Tentorio1, of transitional B-cell development in humans. Expert Rev Clin Immunol 2010; 6: – I Timofeeva2, A Santoro2, S Della Bella1,3, M Roederer4, 765 775. 1,3,5 1,5 8 Sims GP, Ettinger R, Shirota Y, Yarboro CH, Illei GG, Lipsky PE. Identification and D Mavilio and E Lugli 105 1 characterization of circulating human transitional B cells. Blood 2005; : Unit of Clinical and Experimental Immunology, Humanitas Clinical 4390–4398. and Research Center, Rozzano, Italy; 9 Palanichamy A, Barnard J, Zheng B, Owen T, Quach T, Wei C et al. Novel human 2 Hematology and Bone Marrow Transplant Unit, Humanitas Cancer transitional B cell populations revealed by B cell depletion therapy. J Immunol Center, Humanitas Clinical and Research Center, Rozzano, Italy; 2009; 182:5982–5993.

Supplementary Information accompanies this paper on Bone Marrow Transplantation website (http://www.nature.com/bmt)