European Journal of Haematology

ORIGINAL ARTICLE

Effect of ATG-F on B-cell reconstitution after hematopoietic stem cell transplantation Petra Roll, Khalid Muhammad, Gernot Stuhler, Ulrich Grigoleit, Hermann Einsele, Hans-Peter Tony €rzburg, Wu €rzburg, Germany Department of Medicine II, University of Wu

Abstract Antithymocyte globulin Fresenius (ATG-F) is used before hematopoietic stem cell transplantation to prevent graft rejection and graft-versus-host disease in patients with HLA-matched unrelated donors or mismatched volunteers. However, little is known about the effect of ATG-F on the reconstitution of B-cell subsets. Sixty-seven patients were longitudinally studied at day 15, day 30, and then monthly after hematopoietic stem cell transplantation. Conditioning regimes included ATG-F, which was infused at days 3, 2 and 1 at a dosage of 10 mg/kg/d. Twenty-seven patients received conditioning regimes without ATG. ATG-treated patients showed a significant delay of CD19+ B cells in the early recovery period. The absolute numbers of circulating CD19+ B cells were significantly lower (P < 0.05) up to 5 months posttransplantation compared to non-ATG patients. The recovery of the memory compartment was delayed in both groups and did not reach normal values 1-year post-transplantation. ATG-treated patient showed significantly lower absolute numbers of circulating CD27+ memory B cells in the first-month after transplantation compared to non-ATG patients. In conclusion, treatment with ATG in the conditioning regime of patients undergoing allogeneic hematopoietic stem cell transplantation leads to a significant delay of CD19+ B cells. Thus, ATG seems also to negatively influence B-cell immune reconstitution. Key words ATG; B cells; Stem cell transplantation €rzburg, Oberdu €rrbacher Str. 6, Correspondence Prof. Dr. Hans-Peter Tony, Department of Medicine II, University of Wu €rzburg, Germany. Tel: +49 (0)931/201 40100; Fax: +49 (0)931/201 640100; e-mail: [email protected] 97080 Wu Accepted for publication 16 January 2015

Allogeneic stem cell transplantation (ASCT) offers cure for patients with high-risk hematologic malignancies, but puts these patients at a significant risk of developing graft-versushost disease (GVHD) following allo-SCT. GVHD is the culmination of a complex interplay between host and donors factors that lead to massive tissue injury and impaired immunological recovery. GVHD remains important cause of mortality and morbidity after ASCT challenging the limits of this application (1, 2). Strategies to reduce the severity and risk of GVHD have most often used pharmacological immunosuppressants such as calcineurin inhibitor in combination with antimetabolites like methotrexate. Pregraft serotherapy directed against lymphocytes has been demonstrated to reduce the risk of GVHD (3). Different types of pregraft serotherapy have been used including monoclonal antibodies such as OKT3, Campath IG anti-CD52 or rabbit or horse polyclonal antibodies

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

doi:10.1111/ejh.12524

directed against thymocytes (4, 5). In preclinical studies, murine recipients of an allogeneic bone marrow who underwent conditioning with anti-T-cell antibodies and repeated low-dose irradiation were fully protected from GvHD. Targeted depletion of the donor T-cells containing the alloreactive T-cell subset, which induces a/c GvHD, has been explored in different clinical studies (2, 3, 6, 7). Antithymocyte globulin Fresenius (ATG-F) is a potent immunosuppressive agent. Addition of ATG to prophylaxis regimens decreases the incidence of GVHD (3) and allograft rejection (8). ATG leads to in vivo T-cell depletion activity via opsonizing lymphocytes for complement-dependent lysis or Fas/Fas ligand-mediated activation-induced cell death (8– 10). Although lymphocyte depletion by ATG constitutes the primary mechanism of immunosuppression, other mechanisms such as apoptotic induction and blocking adhesion molecules are also involved. T-cell depletion involved active

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Roll et al.

ATG-F influence B cell reconstitution

cell death demonstrated by annexin V binding and TUNEL assays (2, 11, 12). In addition, some major functional effects are also achieved by ATG such as modulation of leukocyte surface antigens and adhesion molecules. Clinical studies indicate an influence of ATG therapy on different immune effector cells including B cells; the course of this study was to analyze dynamic changes in peripheral B-cell reconstitution in patients undergoing allogeneic stem cell transplantation who received or did not received ATG. Sixty-seven patients using hematopoietic stem cells obtained from an HLA-identical sibling or an unrelated donor or mismatched relatives between 2006 and 2009 were studied, including a detailed molecular analysis of preswitch memory B cells in patients who underwent stem cell transplantation. We prospectively analyzed individual preswitch memory Bcells expressing Ig-VH3 gene arrangements with regard to their mutational frequency during immune reconstitution 1 year after allo-SCT. Patients and methods Patients’ characteristics and study design

Sixty-seven patients treated with allogeneic hematopoietic stem cell transplantation (Allo-HSCT) at the University hospital of W€urzburg between 2006 and 2009 were included in the study. Informed consent was obtained from all patients before entering the study in accordance with the protocol approved by the ethics committee of the University of W€urzburg, Germany. Of the 67 patients, 40 patients received stem cells from HLA-matched (n = 35) or mismatched (n = 5) unrelated donors. Conditioning regimes included anti-T-lymphocyte globulin (ATG, Fresenius, Graefelfing, Germany), which was infused at days 3, 2 and 1 at a dosage of 10 mg/kg/d (ATG group). The conditioning regimes were myeloablative in 16 patients, reduced intensity in 22 patients, and non-myeloablative in two patients, respectively. Twenty-seven patients received an allograft from a matched family donor and conditioning regimes did not include ATG (non-ATG group). The conditioning regimes were myeloablative in 11 patients, reduced intensity in 16 patients, respectively. In the ATG group, 33 patients were full-donor chimeras at the early time point and in the non-ATG group 20 patients, respectively. Four of 27 patients were excluded from the study at different time points as they experienced relapse of the original disease (median 6.8 months, 4–8.9 months). Five of 27 patients died during the observation period in a median time of 8 months (2.5–11.9). In the ATG group, 6/40 patients died in a median time of 4.2 months (3.3– 10.4) after transplantation. A detail on patient characteristics, diagnosis, conditioning regimes, GvHD prophylaxis, and incidence of acute and chronic GvHD is shown in Table 1.

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Assessment

Patients were longitudinally studied at day 15, day 30, and then monthly after hematopoietic stem cell transplantation up to 1 year. At each time point, immunophenotyping was performed and different B-cell subsets were analyzed. Immunophenotyping was performed at CD19+ B-cell levels of >1%. Flow cytometric analysis

Peripheral blood was collected from patients at indicated time points. Two hundred microliters of whole blood was used and after washing twice with PBS and albumin (2.5 g/ 500 mL) incubated in PBS with 10 lL mAbs for 15 min. Cells were then incubated with 2 mL Versa-Lyse (Beckman Coulter, Krefeld, Germany) for 10 min and washed with PBS. Five color staining was performed using FC500 flow cytometer (Beckman Coulter). Phenotypic analyses were performed using CD19 (PC7), CD27 (PE/ECD), anti-human IgD (FITC), and anti-human IgM (PC5), CD38 (PC5), CD10 (PE), and CD 20 (ECD). As isotype control antibodies were used as follows: Mouse IgG1/G2a (FITC/PE), Mouse IgG1 (PC5), Mouse IgG1 (ECD), Mouse IgG1 (PC7). All antibodies were from Beckman Coulter (Beckman Coulter). B cells were identified by forward versus side scatter gating on viable lymphocytes in combination with gating on CD19+ cells. Ten thousand CD19+ events were collected for each analysis. Frequencies of CD19+ cells were calculated using CXP software (Beckman Coulter). The lymphocyte blood count was obtained by automatically measuring the leukocyte and lymphocyte count and then multiplying with the frequency of each B-cell population. The combination of CD19/CD27/IgD/CD38 and CD10 was used to define na€ıve, transitional, and memory B cells. Memory B cells were defined as CD27+/IgD+ preswitch and CD27+/IgDpostswitch B cells. Na€ıve B cells were defined as CD19+/ CD38+/IgD+ B cells and transitional B cells as CD19+/ CD38 high/IgD+/CD10+ B cells. The gates were set according to the negative control for separating CD38-/IgD+ and CD38+/IgD+ B cells. Transitional B cells were additionally identified by color overlay of CD10+/IgD B cells. Single-cell sorting and sequencing

Single B-cell sorting was performed as previously described (13). Briefly, B cells were stained for specific surface markers using fluorescence-labeled antibodies. Preswitch memory B cells were identified as IgD+CD27+ population in live lymphocyte gated CD19+ cell population. Individual B cells were sorted into each well of a 96-well plate, which contained lysis buffer, consisting of dithiothreitol, bovine serum albumin, oligo (dT) 15 primer, RNasin, Triton X-100 (Titan One Tube RT-PCR System;

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Roll et al.

ATG-F influence B cell reconstitution

Table 1 Patient’s characteristic

Mean age (min–max) Male/female Type of diagnosis, n Acute myelogenous leukemia Acute lymphocytic leukemia Myelodysplastic syndrome Malignant lymphoma Multiple myeloma Myeloproliferative syndromes Aplastic anemia Conditioning regimes, n TBI based chemotherapy based HLA-identical family donor HLA-matched unrelated donors HLA-mismatched unrelated donors GvHD prophylaxis, n Cyclosporin (CSA)/mycophenolate mofetil (MMF) CSA MMF Tacrolimus/CSA/MMF Tacrolimus/CSA Incidence of acute GvHD, n Skin Grad I Grad II Grad III Chronic GvHD, n Skin grade I/II Skin grade III Gut grade I–II Liver

HLA-(mis)matched unrelated donors n = 40

Family donors n = 27

50 (19–74) years 28/12

49 (18–71) 17/10

12 4 1 10 4 6 3

15 1 4 2 4 1 –

10 30 – 35 5

7 20 27 – –

34 – 1 5 – 5 2 2 1 7 3 1 2 1

25 1 – – 1 7 5 2 – 12 9 – 2 –

Roche Diagnostics, Mannheim, Germany) and distilled H2O followed by cDNA synthesis. Ig-VH3 genes were amplified by nested PCRs using family-specific primers described previously (14). The products were further purified using a MinElute gel extraction kit (Qiagen, Hilden, Germany) and directly sequenced with BigDye Terminator Cycle Sequencing kit and 50 internal primer by ABI PRISM310 (Applied Biosystems, Weiterstadt, Germany). Mutational frequencies, mutational hotspots, and CDR3 lengths were analyzed by matching their closest germline counterparts using the online programme directory JOINSOLVER (15). Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Values were always compared with baseline. For correlation of different B-cell subsets between the 2 groups, a nonparametric Mann–Whitney U-test was used. Unpaired t-test and chisquare test were in mutational data analysis. P value

Effect of ATG-F on B-cell reconstitution after hematopoietic stem cell transplantation.

Antithymocyte globulin Fresenius (ATG-F) is used before hematopoietic stem cell transplantation to prevent graft rejection and graft-versus-host disea...
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