Cytotherapy, 2014; 0: 1e14

Immune reconstitution in patients with Fanconi anemia after allogeneic bone marrow transplantation

MIRIAM PERLINGEIRO BELTRAME1, MARIESTER MALVEZZI1, CARMEM BONFIM2, DIMAS TADEU COVAS3, ALBERTO ORFAO4 & RICARDO PASQUINI5 1

Flow Cytometry Service Core, Clinics Hospital, Federal University of Paraná, Curitiba, PR, Brazil, 2Pediatric Bone Marrow Transplantation Division, Federal University of Paraná, Curitiba, Brazil, 3Regional Blood Center of Ribeirão Preto, Ribeirão Preto, Brazil, 4Cancer Research Center (IBMCC-CSIC/USAL), Department of Medicine, Cytometry Service and IBSAL, University of Salamanca, Salamanca, Spain, and 5Hematology Division, Federal University of Paraná, Curitiba, Brazil

Abstract Background aims. Fanconi anemia is an autosomal recessive or X-linked genetic disorder characterized by bone marrow (BM) failure/aplasia. Failure of hematopoiesis results in depletion of the BM stem cell reservoir, which leads to severe anemia, neutropenia and thrombocytopenia, frequently requiring therapeutic interventions, including hematopoietic stem cell transplantation (HSCT). Successful BM transplantation (BMT) requires reconstitution of normal immunity. Methods. In the present study, we performed a detailed analysis of the distribution of peripheral blood subsets of T, B and natural killer (NK) lymphocytes in 23 patients with Fanconi anemia before and after BMT on days þ30, þ60, þ100, þ180, þ270 and þ360. In parallel, we evaluated the effect of related versus unrelated donor marrow as well as the presence of graftversus-host disease (GVHD). Results. After transplantation, we found different kinetics of recovery for the distinct major subsets of lymphocytes. NK cells were the first to recover, followed by cytotoxic CD8þ T cells and B cells, and finally CD4þ helper T cells. Early lymphocyte recovery was at the expense of memory cells, potentially derived from the graft, whereas recent thymic emigrant (CD31þ CD45RAþ) and naive CD4þ or CD8þ T cells rose only at 6 months after HSCT, in the presence of immunosuppressive GVHD prophylactic agents. Only slight differences were observed in the early recovery of cytotoxic CD8þ T cells among those cases receiving a graft from a related donor versus an unrelated donor. Patients with GVHD displayed a markedly delayed recovery of NK cells and B cells as well as of regulatory T cells and both early thymic emigrant and total CD4þ T cells. Conclusions. Our results support the utility of post-transplant monitoring of a peripheral blood lymphocyte subset for improved follow-up of patients with Fanconi anemia undergoing BMT. Key Words: bone marrow, Fanconi anemia, immune system, transplantation

Introduction Proliferation and maturation of blood cells are tightly regulated processes that involve a large number of growth factors and cytokines (1,2). In addition, the bone marrow (BM) stroma, which comprises vascular and mesenchymal cells, is critical to provide the ideal microenvironment for proper hematopoiesis (3). Therefore, changes in cytokine levels and/or the hematopoietic microenvironment may lead to altered hematopoiesis, including BM aplasia (4). Fanconi anemia (FA) is an inherited disease characterized by BM failure (5). Patients with FA have a defect in DNA repair that progressively leads to the accumulation of chromosomal and genetic alterations. Apart

from anemia, patients with FA display congenital malformations, deafness and skin hyper-pigmentation (eg, “cafe au lait” spots), among other symptoms (6). Regarding outcome, FA is an unpredictable disease, with some patients having development of leukemia and solid tumors at different stages in life (7). Among the available therapeutic options, hematopoietic stem cell transplantation (HSCT) is the most effective approach, significantly extending patient lifespan (8e12). One of the major caveats associated with HSCT is the transient immune deficiency state that follows transplantation, which is associated with significant patient morbidity and mortality (13). The duration and severity of immune

Correspondence: Miriam Perlingeiro Beltrame, PhD, Flow Cytometry Service Core, Clinics Hospital, Federal University of Paraná, Rua Padre Camargo 280, 80060240 Curitiba, PR, Brazil. E-mail: [email protected] (Received 8 May 2013; accepted 28 February 2014) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2014.02.015

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deficiency vary according to graft manipulation, the choice of the type of graft, the development of graft-versus-host disease (GVHD) and the level of residual thymic activity, among other variables (14,15). Assessment of lymphocyte-associated markers can be a useful tool in the clinical setting and is currently under research (i) to monitor response of patients with FA to therapy, (ii) to measure disease activity, and (iii) to predict chronic GVHD (16e19). Successful allogeneic HSCT (allo-HSCT) requires reconstitution of normal T-cell immunity; key factors involved in this process include thymic activity of the HSCT recipient, biological features of the allograft (eg, degree of histocompatibility, number and type of infused donor T cells) and preparative regimens. Specific suppression of allo-reactive T cells, without inhibiting the entire T-cell repertoire, remains an important goal of transplantation immunology (20e22). In the present study, we evaluated the number and immunophenotype of circulating T, B and natural killer (NK) cells and their subsets in peripheral blood (PB) samples from 23 patients with FA who underwent allogeneic BM transplantation (BMT) after a myeloablative conditioning regimen. Our goal was to better understand the kinetics of immune reconstitution in patients with FA who received an allogeneic BMT and to identify potential factors associated with normal versus altered immune recovery.

Methods Patients A total of 23 patients with FA (12 male and 11 female; age range, 4e21 years) who underwent allogeneic BMT between 2009 and 2011 were studied. Before entering the study, informed consent was obtained for all subjects, and the study was approved by the Institutional Review Board of the Federal University of Clinics Hospital (Curitiba, Brazil). Twelve patients (52%) received a transplant from related donors, whereas in the other 11 cases (48%) the graft was of unrelated origin. The conditioning regimen consisted of cyclophosphamide (60 mg/kg) alone for those cases that received a BMT from related donors, whereas patients who received a BMT from an unrelated donor were treated with a combination of cyclophosphamide (60 mg/kg), fludarabine (125 mg/m2) and thymoglobulin (5 mg/kg). For both groups, immunoprophylactic treatment consisting of cyclosporine and methotrexate (Table I) was given for a mean time of 12 months (range, 4e22 months). Successful engraftment was

Table I. Patient characteristics (n ¼ 23). Age at transplantation (y) Sex (male/female) Donor recipient histocompatibility HLA identical One (HLA-A, B or DR) mismatch Donor Related Unrelated Conditioning regimen FLU/ATG/CFA Cyclophosphamide FLU/TBI Immunoprophylaxis Cyclosporine þ methotrexate Mycofenolate þ cyclosporine þ CFA GVHD Acute GVHD Chronic GVHD Chimerism on day þ100 Complete Mixed Cause of death Acute GVHD Rejection

8 (4e21) 12/11 (54%/46%) 20/23 (87%) 3/23 (13%) 12/23 (52%) 11/23 (48%) 13/23 (56%) 9/23 (35%) 1/23 (4%) 21/23 2/23 13/22 5/22 8/21

(91%) (9%) (59%) (23%) (38%)

12/23 (52%) 11/23 (48%) 1/23 (4%) 1/23 (4%)

Results are expressed as number of cases and percentages in parentheses or as median (range). ATG, thymoglobulin, 5 mg/m2; CFA, cyclophosphamide, 60 mg/ kg; FLU, fludarabine, 125 mg/m2; HLA, human leukocyte antigen; TBI, total body irradiation, 200 cGy.

observed in 21 of 23 patients (91%) after a minimum follow-up of 1 year (range, 1e5 years). Overall, 13 patients had acute (n ¼ 5) and/or chronic (n ¼ 8) GVHD. Causes of death (n ¼ 2) were acute GVHD occurring on day þ30 in one patient and transplant rejection occurring on day þ375 after HSCT in the second patient (Table I). Complete chimerism was defined when 100% of donor cells were detected, indicating complete hematopoietic replacement by donor cells; in turn, mixed chimerism was defined when host cells were detected within specific cell populations (eg, the lymphocytes) at percentage values 10% and 95% of all cells (Table I); the presence of mixed chimerism did not influence therapy. No patient received Rituximab or manipulated (eg, in vitro T-celledepleted) bone marrow grafts; however, 56% of patients (Table I) received in vivo T-cell depletion with a low dose of rabbit thymoglobulin (5 mg/kg) during the conditioning regimen.

Multiparameter flow cytometric analysis of lymphocyte subsets PB samples were collected into tubes containing 7.5% K3 ethylenediaminetetra-acetic acid for both flow cytometry and complete blood cell count

Immune system in transplanted patients with FA analyses (ADVIA 2120, Siemens, New York, NY, USA); cell morphology was assessed visually by means of conventional microscopy. Flow cytometric characterization of PB lymphocytes was performed with the use of conventional stain-and-then-lyse sample preparation techniques. In brief, 100 mL of fresh PB was incubated for 15 min at room temperature in the dark, with pre-titrated saturating amounts of four-color combinations of fluorochrome-conjugated monoclonal antibodies: fluorescein isothiocyanate, phycoerythrin, fluorochrome peridin chlorophyll and allophycocyanin (Table II). For every PB sample, an additional aliquot containing an unstained PB sample was processed in parallel as negative control. Afterward, non-nucleated red cells were lysed with the use of FACS Lysing solution (Becton-Dickinson Biosciences; San Jose, CA, USA), according to the instructions of the manufacturer, and the remaining cells were sequentially centrifuged, washed twice in phosphate-buffered saline (pH 7.4) and resuspended in 500 mL of phosphate-buffered saline. Intracellular FOXP3 staining was performed with the use of the Fix&Perm (Invitrogen, Camarillo, CA, USA) reagent kit, after staining for cell surface membrane markers, as per the recommendations of the manufacturer. Data acquisition was performed in a FACSCalibur flow cytometer (Becton-Dickinson Biosciences) immediately after sample preparation was completed. For each sample aliquot, a minimum of 100,000 events was acquired through the use of CellQUEST software (Becton-Dickinson Biosciences). For data analysis, the Infinicity software program (Cytognos SL, Salamanca, Spain) was used. Absolute lymphocyte counts were calculated by means of a dual platform approach by multiplying the percentage of each lymphocyte subset obtained by flow cytometry by the absolute leukocyte count obtained for that specific PB sample in an ADVIA 2120 hematology analyzer. The following lymphocyte subsets were specifically identified: CD3þ T-lymphocytes, CD3þCD4þ T-helper cells, CD3þCD8þ cytotoxic T cells, CD4þCD45RAþCD31þ new thymic emigrant helper T cells, CD57þCD28eCD4þ or CD8þ replicative senescent T cells, CD27þCD45ROCD4þ or CD8þ naive T cells, CD27þCD45ROþCD4þ or CD8þ central memory T cells, CD27CD45ROþCD4þ or CD8þ effector T cells, CD27CD45ROeCD4þ or CD8þ late effector T cells, CD3þCD69þ activated T cells and CD45highSSClowCD19CD3CD69þactivated non-T and noneB lymphocytes (NK cells). In this panel, NK cells were identified after excluding CD3þ T cells and CD19þ B cells from the CD45highSSClow of total lymphocyte gate. CD4þCD25highCD127lowFOXP3þ regulatory T cells (Tregs), CD4þCD25highGITRþ

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Table II. Fluorochrome-conjugated antibody reagents used for the flow cytometric analysis of PB subsets of lymphocytes and the corresponding antibody panel. Monoclonal antibody Fluorochrome CD3 CD3 CD4 CD4 CD8 CD8 CD10 CD16 CD19 CD19 CD20 CD25 CD27 CD28 CD31 CD45 CD45RA CD45RO CD56 CD57 CD69 CD95 CD127 CD178 FOXP3 GITR TCRab TCRgd

FITC PECy5 PerCP APC PE APC PE PE PE APC FITC FITC FITC PE PE PerCP FITC PE PE FITC PerCP FITC PE PE APC PE FITC PE

Clone SK7 SK7 SK3 SK3 SK1 SK1 HI10 B-E16 HIB19 SJ25C1 L27 M-A251 L128 CD28.8 WM59 2D1 L48 UCHL1 MOC-1 TB01 FN50 DX2 HIL-7R-M21 Kay-10 PCH101 TNFRSF18 T10B9.1A-31 V65

Commercial source BD Biosciences IQ Productsa BD Biosciences BD Biosciences BD Biosciences BD Biosciences BD Biosciences IQ Products Biolegendb Invitrogen BD Biosciences BD Biosciences BD Biosciences EXBIOc BD Biosciences BD Biosciences BD Biosciences IQ Products IQ Products Serotecd BD Biosciences BD Biosciences BD Biosciences BD Biosciences eBiosciencese eBiosciences BD Biosciences BD Biosciences

Antibody panel FITC CD3 CD3 CD20 TCRab CD45RA CD57 CD27 CD3 CD95 CD25 CD25

PE CD8 CD16þ56 CD10 TCRgd CD31 CD28 CD45RO CD19 CD178 CD127 GITR

PerCP or PECy5 CD45 CD45 CD45 CD4 CD4 CD4 CD4 CD69 CD3 CD4 CD4

APC CD4 e CD19 CD8 CD8 CD8 CD8 e CD19 cyFOXP3 cyFOXP3

APC, allophycocyanin; BD, Becton-Dickinson; Cy, cytoplasmic staining; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, fluorochrome peridin chlorophyll; PECy5, phycoerythrin cyanin 5. a Groningen, the Netherlands. b San Diego, CA, USA. c Vestec, Czech Republic. d Oxfordshire, United Kingdom. e San Diego, CA, USA.

FOXP3þ Tregs, CD3þCD16þand/or CD56þNK/T lymphocytes, CD3þTCRabþ T cells, CD3þTCRgdþ T cells, CD19þCD20þ mature B lymphocytes, CD19þCD10þ immature B lymphocytes and CD3eCD16þCD56þ NK cells. In addition, we also

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Figure 1. Kinetics of recovery of different subsets of PB lymphocytes after BMT. In each panel, results are expressed as absolute number of lymphocytes per microliter of PB at seven different time points: before BMT (pre) and on days þ30, þ60, þ100, þ180, þ270 and þ360. Different panels show the distribution a distinct cell subsets: (A) CD3þT cells; (B) CD3þCD4þ helper T cells; (C) CD3þCD8þ cytotoxic T cells; (D) CD3þCD4CD8 cytotoxic natural effector T cells; (E) CD3þCD4þCD8þ cytotoxic T cells. Notched-boxes represent 25th and 75th percentile values; the line in the middle and vertical lines correspond to median values and the 10th and 90th percentile values, respectively. aP  0.05.

determined the number of T and B cells expressing Fas (CD95) and FasL (CD178): CD3þFasþ and CD3þFasLþ T cells, as well as CD19þFasþ and CD19þFasLþ B lymphocytes.

Statistical methods To evaluate the statistical significance of differences observed between groups, non-parametric Wilcoxon

Immune system in transplanted patients with FA

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Figure 2. Kinetics of recovery of different subsets of PB T cells after BMT. In each panel, results are expressed as absolute lymphocyte counts per microliter of PB before BMT (pre) and on days þ30, þ60, þ100, þ180, þ270 and þ360 after transplantation. In the different panels, the distribution of distinct subpopulations of T cells is shown. (A and B) CD31þCD45RAþCD4þ or CD8þ newly thymic emigrant CD4þ T-helper cells (A) and CD8þ cytotoxic T cells (B), respectively; (C and D) CD57þCD28eCD4þ or CD8þ senescent T-helper cells (C) and cytotoxic T cells (D), respectively; (EeH) CD27þCD45RO naive (E), CD27þCD45ROþ central memory (F), CD27CD45ROþ effector (G) and CD27CD45RO late effector (H) CD4þ T-helper cells, respectively; (IeL) CD27þCD45RO naive (E), CD27þCD45ROþ central memory (F), CD27-CD45ROþ effector (G) and CD27eCD45RO late effector (H) CD8þ cytotoxic T cells, respectively; (M and N) CD3þCD69þ activated T cells (M) and CD25highCD127lowFOXP3þCD4þ Tregs (N), respectively. Notched boxes represent 25th and 75th percentile values; the line in the middle and vertical lines correspond to median values and the 10th and 90th percentile values, respectively. aP 0.05.

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Figure 2. (continued).

and Mann-Whitney U tests were used. P values 0.05 were considered to be associated with statistical significance (Statistica v. 8.0 software, Stat Soft, OK, USA). Results One of the major risks associated with HSCT is the transient immune deficiency state that follows

transplantation, which leads to significant patient morbidity and mortality (13). Overall, CD3CD56þ NK cells were the first lymphoid cells to emerge in our cases after BMT, followed by CD3þCD8þ cytotoxic T cells, CD19þ B cells and finally CD3þCD4þ helper T cells. The kinetics observed for each of these specific lymphoid subsets is described in more detail below.

Immune system in transplanted patients with FA

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Figure 2. (continued).

Recovery of T-cell subsets after allo-BMT The total number of T cells was significantly decreased after transplantation. Pre-transplant levels were restored only by month þ3 (day þ100) after BMT. However, distinct kinetics were observed for the different T-cell subsets. Thus, we found a faster increase in the number of CD3þCD8þ cytotoxic T cells, which reached pre-transplant values at day þ60, remaining stable thereafter (Figure 1AeE). Conversely, CD3þCD4þ T-helper cells only reached pre-transplant levels at day þ180, increasing significantly thereafter (Figure 1B). Identical recovery kinetics were observed for CD3þCD4CD8 T cells (Figure 1D) and TCRgdþ T lymphocytes (data not shown). Of note, PB CD3þCD4-CD8e include both TCRabþ and TCRgdþ T cells involved in natural immunity; interestingly, several authors (23e25) have suggested that these cells may play an important role in the recovery of neutrophils and production of growth factors both during the very early post-BMT period, and, later on, during the immunodeficient state of chronic GVHD. In turn, CD3þCD4þCD8þ (CD4þCD8þ double-positive) T cells were already significantly increased versus pre-transplant levels on day þ180 (Figure 1E). Interestingly, a significant decrease in the frequency of PB T cells displaying a newly thymic emigrant phenotype (CD31þCD45RAþ) was also observed after transplantation both within the CD4þ T helper and the CD8þ cytotoxic T-cell compartments (Figure 2A,B). This reduction lasted up 3e4 months after transplantation, followed by a significant expansion of such T cells that reached pre-transplant levels by day þ270 (Figure 2A,B). As would be expected, a profile similar to that of the kinetics of recovery of newly thymic emigrant T cells was also observed for

the CD4þCD27þCD45RO naive T cells (Figure 2E) but not for the CD8þ naive T lymphocytes (Figure 2I). In turn, T cells expressing a replicative/senescent (CD4þCD57þCD28 or þ þ  CD8 CD57 CD28 ) phenotype were virtually undetectable before transplantation; of note, these late stage effector T cells became first detectable by day þ60, and they slowly but progressively increased throughout the following time points, particularly as regards T cells with a CD8þCD57þCD28 phenotype (Figure 2C,D). A pattern similar to that of replicative/senescent late-effector T cells was found for effector NK/T cells as well as for both memory effector (CD27CD45ROþ) and late effector (CD27CD45RO) CD4þ and CD8þ T cells (Figure 2G,H,K,L). Nevertheless, whereas the number of both subsets of effector CD8þ T cells and memory effector CD4þ T cells increased by day þ60 after transplantation, terminal effector CD4þ T cells showed a later recovery, being significantly increased only by day þ180 after transplantation. Regarding CD27þCD45ROþCD4þ central memory T-helper cells, there were no significant changes throughout the entire follow-up period (Figure 2F); in contrast, CD27þCD45ROþCD8þ central memory cytotoxic T cells increased from day þ180 onward (Figure 2J). Despite all the above changes, the overall number of CD69þ activated T cells remained virtually unchanged after transplantation, except for a significantly decreased number of theses cells observed on day þ100 (Figure 2M). In turn, CD25highCD127lowCD4þFOXP3þ (Figure 4N) Tregs started to recover at day þ60 after transplantation, reaching significantly increased numbers (twice pre-transplant values) from day þ270 onward (Figure 2N).

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Figure 3. Kinetics of recovery of different subsets of PB NK cells, B cells and apoptotic cells after BMT. (A and B) Total CD3CD16þCD56þ NK cells and CD45highSSClowCD3CD19CD69þ activated NK cells are displayed; (CeF) in turn, the distribution of CD19þCD10þ immature B cells (C), CD19þCD20þ total B cells (D), CD3þCD95þCD178þ apoptotic T cells (E) and CD19þCD95þCD178þ apoptotic B cells (F) are displayed, respectively. In each panel, results are expressed as absolute cell number per microliter of PB before BMT (pre) and on days þ30, þ60, þ100, þ180, þ270 and þ360 after transplantation. Notched boxes represent 25th and 75th percentile values; the line in the middle and the vertical lines correspond to median values and both the 10th and 90th percentile values, respectively. aP  0.05.

Recovery of NK cells after allo-BMT As mentioned above, NK cells peaked between day þ30 and day þ60, slightly decreasing thereafter

toward values that were still significantly increased versus pre-transplant levels (Figure 3A). Similarly, CD45highSSClowCD19CD3CD69þ NK cells had

Immune system in transplanted patients with FA

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F

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Figure 4. Kinetics of recovery of different subsets of T cells after BMT in patients with FA who are receiving an allo-HSCT from an unrelated versus a related donor. Distribution of the following T-cell populations is shown: total CD3þ T cells (A), CD3þCD4þ T-helper cells (B), CD3þCD8þþ cytotoxic T cells (C), CD4CD8 cytotoxic T cells (D), CD4þCD8þ cytotoxic T cells (E) and TCRgdþ T cells (F). In each panel, results are expressed as absolute cell number per microliter of PB before BMT (pre) and on days þ30, þ60, þ100, þ180, þ270 and þ360 after transplantation. Notched boxes represent 25th and 75th percentile values; small squares inside the notched boxes and the vertical lines correspond to median values and both the 10th and 90th percentile values, respectively. White boxes correspond to patients receiving a BMT from an unrelated donor; filled black boxes correspond to patients with a related donor. aP  0.05.

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already doubled their pre-transplant levels on day þ30, and they continued to increase until the end of the study (Figure 3B).

Recovery of B lymphocytes after allo-BMT B lymphocytes, including both CD19þCD10þ immature and CD19þCD20þ mature B cells, almost disappeared at day þ30; however, B-cell numbers slowly recovered, thereafter reaching significantly increased values compared with pretransplant levels from day þ180 and day þ270 onward, respectively (Figure 3C,D). The CD19þCD24þCD38þ transitional human Blymphocyte population reached initial pre-transplant values by day þ60 after transplantation, with such values being maintained until the end of the follow-up period (data not shown).

Expression of the CD95 and CD95L apoptosis-associated markers on T and B lymphocytes No significant changes were observed throughout the study as regards the number of CD95þ and CD95Lþ (CD178) T lymphocytes (Figure 3E). In turn, the percentage of CD19þ B lymphocytes coexpressing CD95 and CD178 was significantly increased versus re-transplant levels from day þ100 on (Figure 5F); of note, however, during this period (day þ100 to day þ360) the mean number of B cells expressing the CD95 and CD178 apoptotic markers always remained below 10 cells/mL (Figure 3F).

Immune reconstitution of patients with FA receiving donor-related versus donor-unrelated allogeneic BMT On grouping of patients with FA according to the type of donor (eg, related versus unrelated donor), no statistically significant differences were found for most of the subsets of lymphocytes analyzed at the distinct time points, with a few exceptions (Figure 4). These exceptions included the number of (i) total CD3þ T cells (303  408 versus 832  737  106 cells/L; P ¼ 0.02; Figure 4A), (ii) TCRgdþ T lymphocytes (15  22 versus 51  55  106 cells/L; P ¼ 0.04; Figure 4F), (iii) CD3þCD8þ (172  208 versus 541  546  106 cells/L; P ¼ 0.03; Figure 4C) cytotoxic T cells and (iv) CD3þCD4CD8 (14  21 versus 54  54  106 cells/L; P ¼ 0.03; Figure 4E) cytotoxic and regulatory T cells. Of note, these cell subsets were significantly higher at day þ30 after transplantation among those patients receiving a BMT from an unrelated donor versus those who received a BMT from a related donor.

Immune reconstitution of patients with FA with versus without GVHD On grouping patients with versus without GVHD, significant differences in the recovery of distinct cell populations were noted between day þ60 and day þ270. Thus, cases who had GVHD (acute and/ or chronic GVHD; n ¼ 13) showed a significantly delayed recovery of NK cells and B lymphocytes, both cell populations only reaching values similar to those of patients without GVHD by day þ360 (Figure 5). In addition, recovery of both CD4þ early thymic emigrant and total T cells, as well as of Tregs, was also delayed among cases with GVHD until day þ270 (Figure 5). One year after transplantation (day þ360), no significant differences were observed between cases with and without GVHD (Figure 5). Discussion Immune reconstitution after HSCT is increasingly recognized as a critical determinant of morbidity and mortality in HSCT recipients mainly because of opportunistic infections and GVHD. Previous studies have shown that post-transplant T cells derive from both mature T cells present in the donor graft and T cells derived from transplanted donor hematopoietic stem cells that develop de novo in the recipient (20). Whereas the former results in transient adoptive transfer of immunity as well as GVHD mediated by cells with alloreactivity against recipient antigen determinants, the latter would lead to longterm immune reconstitution (21). In this study, we analyzed in detail the immune reconstitution of PB-lymphocyte subsets from a relatively large series of patients with FA who received a BMT from related and unrelated donors and that were sequentially followed for a minimum period of 1 year after transplantation. Although there is limited information about the immune recovery of patients with FA after BMT, results similar to those reported in other hematological malignancies, reviewed in Porrata et al. (23), Peggs et al. (24) and Auletta et al. (25), were observed in our series. Overall, we found different kinetics of recovery after transplantation for the distinct major populations of lymphocytes, as also described previously by Zhu et al. (26). Briefly, NK cells were the first to recover, followed by cytotoxic CD8þ T cells and B cells, and finally the CD4þ T-helper cells. These results are in line with those also observed in cases of other maturation-associated subsets of lymphocytes. Thus, CD31þCD45RAþ recent emigrant (CD4þ or CD8þ) and naive CD4þ T cells only rose 6 months after BMT, with such increase occurring in the presence of immunosuppressive GVHD prophylactic agents. The parallel

Immune system in transplanted patients with FA

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B

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E

Figure 5. Recovery of different PB-lymphocyte subsets showing significantly different kinetics after BMT in cases with (black boxes) versus without (white boxes) GVHD. Distribution of CD4þ T-helper cells (A), CD45RAþCD31þ new thymic emigrant CD4þ T cells (B), CD3CD16þCD56þ NK cells (C), CD19þ B cells (D) and CD25highCD127lowFOXP3þ Tregs is expressed as absolute cell number per microliter of PB. Notched boxes represent 25th and 75th percentile values; the line in the middle and vertical lines correspond to median values and both the 10th and 90th percentile values, respectively. White boxes correspond to patients who showed no GVHD; filled black boxes correspond to cases who had GVHD. aP  0.05.

increase in recent thymic emigrant and naive CD4þCD27þCD45RO T-helper cells suggests that most probably, the latter cells were not the expanded

progeny of naive T cells present in the donor graft but rather correspond to newly produced T-helper lymphocytes, as also indicated by Skert et al. (27).

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Expression of CD31 on CD4þ T-helper cells has been associated with higher intracellular levels of T-cell receptor excision circles, with CD31 being therefore considered a surrogate phenotypic marker of thymic reconstitution in lymphopenic subjects, for example, after HSCT (28,29). In our series, this T-cell subset was fully recovered 9 months after transplantation. Although we detected release of new thymic emigrants as early as month þ3 after transplantation, several groups only found these cells around month þ6 (30,31). This difference could be related to the use of different surrogate markers and phenotypes for the definition of recent thymic emigrants, because in these earlier studies markers such as CD44, CD45RB and CD62L were used instead of CD31 (25e32). T cells expressing a CD57þCD28CD4þ or CD8þ phenotype have been shown to represent functionally senescent cells with low in vitro replication potential, reduced telomeres and an increased susceptibility to undergo apoptosis (34); overall, such functional properties correspond to those of end-stage terminal effector T cells. In the transplantation setting, these cells have been reported to be increased in patients with GVHD and cytomegalovirus infection (35e41). Unlike recent thymic emigrants, CD57þCD28 senescent CD4þ and CD8þ T cells were found to be already increased early during follow-up, in parallel to the recovery of both memory effector and terminal effector CD8þ and also CD4þ T lymphocytes, as well as cytotoxic TCRgdþ and CD4CD8 T lymphocytes and NK/ T cells (42e44). This increase of end-stage cytotoxic and helper T cells could be caused by the activation and differentiation of memory cells, because, in contrast to CD4þ naive and new thymic emigrant T cells, these terminal effector cells showed neither decreased nor increased levels throughout the whole follow-up period (45e48). Altogether, these results suggest that early recovery of NK cells (including activated CD69þ NK cells) and other cytotoxic T cells (and potentially also B lymphocytes) are at the expense of adoptive transfer of donor lymphocytes. In contrast, newly produced CD4þ T-helper cells would contribute to the recovery of the naive T-cell compartment at latter time points. These results are in line with previous studies showing early recovery of NK cells, which may even represent the majority of all PB mononuclear cells at the earliest time points after transplantation (32,33,40,45e47), whereas recovery of other B- and T-cell populations (particularly CD4þ T cells) is substantially delayed, their number tending to remain low for up to 12 months or even longer periods in patients with chronic GVHD (48,49). In contrast, the number of CD8þ cells usually returns to normal levels much faster (51), as also found here. Although B cells are known

to be significantly decreased (or even undetectable) during the first 2 months after BMT, we detected in our study the presence of immature CD19þCD10þ B cells early after transplantation (day þ60) (21,22). Of note, Tregs were significantly increased from day þ270 on. Tregs, particularly those coexpressing CD4þ and Foxp3þ, play a key role in the maintenance of tolerance after an allo-HSCT. Despite similar kinetics in patients with FA receiving a BMT from related and unrelated donors, differences were observed in the timing of early recovery of total T cells and CD8þ T cells (earlier among unrelated donors), with both cell populations reaching similar values in the two patient groups by day þ100. Such differences may be to the result of an increased T-cellemediated allo-reactivity among patients receiving a BMT from unrelated versus related donors. Similarly, cases with acute and/or chronic GVHD also showed a significantly delayed recovery of NK cells and B cells, as well as of thymic emigrant and total CD4þ T cells, which only reached those values observed among the other transplanted patients with FA after 9 and 12 months, depending on the specific cell population (NK cells plus B cells and both recently produced and total CD4þT lymphocytes, respectively). As previously suggested in the allo-BMT setting (50e52), such delayed recovery could be directly related to the underlying GVHD immunological response. In summary, in this study, which is based on the largest series of patients with FA undergoing BMT reported thus far, we show that the kinetics of recovery of the different populations of lymphocytes follows those patterns also described for patients with other hematological malignancies: early recovery of NK cells, followed by effector cytotoxic T cells and B cells, and finally, CD4þ T-helper cells. Interestingly, early recovery appears to be due to the expansion of memory cells potentially derived from the graft, whereas at later periods, recovery of recent thymic emigrants and naive T cells indicates recovery of T-cell production derived from the transplanted hematopoietic stem cells. In addition, our results also point out the existence of slight differences in the early recovery of cytotoxic T cells among cases receiving a graft from related versus unrelated donors; in contrast, clear differences existed among cases with GVHD versus cases without GVHD. These results point out the potential utility of post-transplant monitoring of PB-lymphocyte subsets for improved follow-up of patients undergoing BMT. In this regard, monitoring of the major PB NK-cell, B-cell and CD8þ T-cell subsets after day þ60, together with the evaluation of recent thymic emigrants and naive CD4þ T-helper cells at days þ100, þ180 and þ360 after BMT, would be particularly informative.

Immune system in transplanted patients with FA Acknowledgments We are grateful to Dr Rita Perlingeiro and Michael Kyba for critical review of the manuscript. We thank all personnel from the immunophenotype, hematology and biochemistry laboratories, the nursing team from the Bone Marrow Transplantation Unit and the student of scientific initiation Luana Wunsche de Almeida. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Kutler DI, Singh B, Satagopan J, Batish SD, Berwick M, Giampietro PF, et al. A 20-year perspective on the International Fanconi Anemia Registry (IFAR). Blood. 2003;1014: 1249e56. 2. Green AM, Kupfer GM. Fanconi anemia. Hematol Oncol Clin North Am. 2009;23:193e214. 3. Soulier J. Fanconi Anemia. Hematology. Washington (DC): American Society of Hematology Education Book; 2011. p. 492e7. 4. Shukla P, Ghosh K, Vundinti BR. Current and emerging therapeutic strategies for Fanconi anemia. HUGO J. 2012;6:1e8. 5. Svahn J, Dufour C. Fanconi anemia: learning from children. Pediatr Rep. 2011;3:18e20. 6. Colin AS, Eric Nisbet-Brown, David GN. Congenital bone marrow failure syndromes. Br J Haematol. 2000;111:30e42. 7. Medeiros CR, Bitencourt MA, Neto JZ, Bonfim CS, Funke VM, et al. Allogeneic hematopoietic stem cell transplantation from an alternative stem cell source in Fanconi anemia patients: analysis of 47 patients from a single institution. Braz J Med Biol Res. 2006;39:1297e304. 8. Dalle JH. HSCT for Fanconi anemia in children: factors that influence early and late results. Bone Marrow Transplant. 2008;42:S51e3. 9. Eiler ME, Frohnmayer D, Frohnmayer L, Larsen K, Olsen J, editors. In: Fanconi Anemia: Guidelines for Diagnosis and Management. 3rd edition. Eugene, Oregon: Fanconi Anemia Research Fund, Inc; 2008;32:33e46. 10. Myres KC, Davies SM. Hematopoietic stem cell transplantation for bone marrow failure syndromes in children. Biol Blood Marrow Transplant. 2009;15:279e92. 11. Pinto FO, Leblanc T, Chamousset D, Le Roux G, Brethon B, Cassinat B, et al. Diagnosis of Fanconi anemia in patients with bone marrow failure. Haematologica. 2009;94:487e95. 12. MacMillan M, Wagner JE. Haematopoeitic cell transplantation for Fanconi anaemia: when and how? Br J Haematol. 2010;149:14e21. 13. Schultz KR, Miklos DB, Fowler D, Cooke K, Shizuru J, Zorn E, et al. Toward biomarkers for chronic graft-versushost disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease, III: Biomarker Working Group Report. Biol Blood Marrow Transplant. 2006;12: 126e37. 14. Abrahamsen IW, Søme S, Heldal D, Egeland T, Kvale D, Tjønnfjord GE. Immune reconstitution after allogeneic stem cell transplantation: the impact of stem cell source and graftversus-host disease. Haematologica. 2005;90:86e93.

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