Influence of bone marrow graft lymphocyte subsets on outcome after HLA-identical sibling transplants

Experimental Hematology 29 (2001) 1347–1352

Influence of bone marrow graft lymphocyte subsets on outcome after HLA-identical sibling transplants Vanderson Rochaa,c, Marie-Vonique Carmagnatb, Sylvie Chevretc, Odile Flinoisb, Henrique Bittencourta, Hélène Esperoua, Federico Garniera, Patricia Ribauda, Agnès Devergiea, Gérard Sociéa, Liliane Dal’Cortivod, Jean-Pierre Marolleaud, Dominique Charronb, Eliane Gluckmana, and Claire Rabianb a Bone Marrow Transplant Unit, bImmunology Histocompatibility Laboratory and INSERM U396, cBiostatistics Department, dCellular Therapy Laboratory, Saint Louis Hospital, Paris, France

(Received 14 May 2001; revised 25 June 2001; accepted 26 July 2001)

Objective. The aim of this study was to analyze bone marrow lymphocyte subsets and CD34 cell dose and their influence on the outcomes of bone marrow transplantation. Materials and Methods. Forty-eight patients (median age 30 years, range 5–54) receiving HLA-identical sibling bone marrow transplantation for hematologic malignancies were analyzed. Results. Median number (range) of nucleated cells and CD34 cells infused were 2.4 (0.4–6.0)  108/kg and 3.5 (0.5–13.0)  106/kg, respectively. Probability of neutrophil recovery was 97%. In a multivariate analysis, time to neutrophil recovery was shortened when a higher number of CD3/CD8 cells was infused (1.0  107/kg) (hazard ratio [HR]  2.13, p  0.018); when the patient was female or had negative cytomegalovirus serology (HR  2.03, p  0.03; HR  0.41, p  0.009; respectively). The incidence of grade II to IV acute graft-vs-host disease (GVHD) was 47%. Infusion of 1  107 CD4 infused/kg increased the risk of acute GVHD (HR  2.86, p  0.03). Nineteen of 40 patients at risk experienced chronic GVHD, the risk of which was increased by diagnosis of chronic leukemia (p  0.03), 2.0  108 nucleated cells infused/kg (p  0.05), and a low number of all lymphocyte subsets, except CD19. Estimated 3-year survival rate was 54%. Risk of death was increased in patients receiving 3.5  106CD34 infused/kg (HR  0.37, p  0.02). Only six patients relapsed. Conclusions. A high cell dose of CD3/CD8 is associated with faster neutrophil recovery, whereas a high cell dose of CD4 cells increases the incidence of acute GVHD. A high number of nucleated cells and CD34cells infused was associated with decreased risk of chronic GVHD and improved survival, respectively. © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Allogeneic stem cell transplantation has been used to treat a wide variety of diseases, including hematologic malignancies, aplastic anemia, red cell disorders, and congenital immunodeficiency. The outcome of stem cell transplantation is influenced by patient-, disease-, donor-, and transplantrelated factors. Among those factors, bone marrow (BM) cell dose present in the graft is known to be associated with lower incidence of graft rejection and higher speed of en-

Offprint requests to: Vanderson Rocha, M.D., Clinical Research Unit, Pavillon Lailler, Hôpital Saint Louis, 1, av Claude Vellefaux, 75475 Paris, Cedex 10, France; E-mail: [email protected]

graftment, lower incidence of graft-vs-host disease (GVHD), and prolonged survival [1–8]. Donor T-cell subsets mediate the delicate balance between graft rejection and the development of GVHD after allogeneic stem cell transplantation. In murine marrow transplant models, both CD4 and CD8 cells of the donor can cause GVHD, but donor CD8 cells are at least five-fold more effective than donor CD4 cells in preventing marrow graft rejection mediated by recipient T cells [9]. In humans, removal of CD8 cells from the donor marrow is associated with an increased risk of graft failure in HLA-identical sibling recipients [10], whereas infusion of 1  106 CD8 donor cells/kg of recipient does not increase graft failure [11].

0301-472X/01 $–see front matter. Copyright © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(01)0 0 7 3 7 - 8

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However, after HLA-mismatched unrelated transplants, the number of CD8 cells needed to prevent rejection (at least 5  106/kg) is sufficient to cause an unacceptable risk of GVHD [12]. Recently, in T-depleted peripheral blood stem cell (PBSC) transplants, a higher CD3 cell dose was associated with decreased incidence of graft failure [13]. To study the lymphocyte composition in the BM graft and the quantitative influence of specific lymphocyte subsets (mainly T and B) on the outcomes of transplantation, we studied 48 patients undergoing non–T-depleted HLAidentical sibling bone marrow transplantation (BMT).

Patients and methods Patients and transplantation procedure Between May 29, 1995 and May 22, 1997, 48 patients with hematologic malignant diseases received an HLA-identical sibling BMT at Hospital Saint Louis (Paris, France). This report includes patients 1) with malignant diseases; 2) receiving unmanipulated BM grafts; 3) receiving from HLA-identical sibling donors; and 4) from whom a BM sample was available for flow cytometry. Table 1 shows patient, donor, and transplant characteristics. Collection and processing of BM BM cells were collected at the Saint Louis Hospital with the goal of having at least 2  108/kg nucleated cells. BM was harvested from both posterior iliac crests under general anesthesia. BM was collected into an anticoagulant medium consisting of acid citrate dextrose (ACD) (for BM collected between 1995 and 1998). Final harvested BM had a dilution of 1:3 in RPMI heparin and 1:10 in ACD. BM was concentrated by centrifugation on COBE-2991 (IBM) at 3,000 rpm/min for 5 minutes. The supernatant and part of red blood cells were discarded. Buffy-coat cells were resuspended in human albumin and infused. In the case of major ABO-incompatibility, red blood cells and plasma were separated from the graft before transplantation. Written informed consent to study BM samples was obtained from adult donors or guardians of children younger than 18 years. Flow cytometry and panel of antibodies Samples of buffy-coat BM cells were taken for phenotypic study of lymphocyte subpopulations and CD34 counts. Direct immunofluorescence staining was performed using labeled monoclonal antibodies. Lymphocyte subsets were analyzed by two- or three-color immunofluorescence on an FACScan analyzer (Becton-Dickinson). Precisely 100 L of sample was added to a premixed solution of directly conjugated monoclonal antibodies at the appropriate dilution. Erythrocytes then were removed by lysis through the addition of FACS lysing solution to the tubes. After 15 minutes of incubation at 4 C in the dark, flow cytometric analysis of 2  103 gated lymphocytes was performed. Results are expressed as percentage of positive cells gated and then as percentage of total mononuclear cell population. The conjugated monoclonal antibodies were purchased from Becton-Dickinson. The following antibodies and combinations were used: CD2 fluorescein isothiocyanate (FITC), CD19 phycoerythrin (PE), CD3 PerCP, CD4 FITC, CD8 PE, CD8 FITC, CD28 PE, anti-HLA class II DR PE, CD3/ CD8, CD8/CD28/CD3, CD3/DR, CD3/CD4 /CD8 , and CD 3 / CD16/CD56.

Table 1. Patient, donor, and transplant characteristics Characteristics Age (years) No. children Weight (kg) Gender Male Female Diagnosis Acute leukemia ALL AML Chronic leukemia CML CLL Other malignancies* Status at transplant† Early Intermediate Advanced Recipient’s CMV serology prior to BMT Negative Positive Donor’s CMV serology prior to BMT Negative Positive Donor’s age (years) ABO compatibility Matched Mismatched Conditioning CY  BU Ara-C, melphalan, and TBI CY  VP16 (associated with TBI or BU) Other association GVHD prophylaxis CsA CsA  MTX CsA  MTX  steroid

N  48 30 (5–54) 12 (25%) 60 (15–95) 26 (54%) 22 (46%)

12 (25%) 16 (33.5%) 13 (27%) 1 (2%) 6 (12.5%) 32 (66.5%) 10 (21%) 6 (12.5%) 18 (37.5%) 30 (62.5%) 20 (42%) 28 (58%) 33 (5–59) 21 (44%) 27 (56%) 21 (44%) 12 (25%) 10 (21%) 5 (11%) 1 (2%) 36 (75%) 11 (23%)

*Other malignancies include myelodysplastic syndrome (n  3), hypereosinophilic syndrome (n  1), metaplasia agnogeneic (n  1), and nonHodgkin’s lymphoma (n  1). † International Bone Marrow Transplant Registry classification. Early  first remission, chronic phase, refractory anemia with or without ringed sideroblasts. Intermediate  subsequent remission, accelerated phase. Advanced  not in remission, blast phase, refractory anemia with excess blasts or with excess blasts in transformation. Hypereosinophilic syndrome and metaplasia agnogeneic were considered to be in the intermediate status group. ALL  acute lymphoblastic leukemia; AML  acute myeloid leukemia; BMT  bone marrow transplantation; BU  busulfan; CML  chronic myeloid leukemia; CLL  chronic lymphoid leukemia; CMV  cytomegalovirus, CsA  cyclosporin A; CY  cyclophosphamide; MTX  methotrexate; TBI  total body irradiation.

For CD34 analysis, dual platform was used. Briefly, one million BM cells were suspended in phosphate-buffered saline (PBS) and incubated for 10 minutes at room temperature with 20 L of HPCA2-PE and anti–CD45-FITC monoclonal antibodies (BectonDickinson, France). Isotype-matched antibodies (IgG1, IgG2a, and IgG2b FITC/PE conjugated; Becton-Dickinson, France) served as controls. After erythrocytes lysis and washing, 50,000 events were acquired for double-color analysis. CD34 cells were analyzed by

V. Rocha et al./Experimental Hematology 29 (2001) 1347–1352

gating whole nucleated cells excluding unlysed red cells, platelets, and debris. The proportion of CD34 cells was calculated following the subtraction of nonspecific events in the negative control. The absolute number of CD34 progenitors was determined by multiplying the frequency of positive cells by the viable nucleated cell number. Total CD34 obtained at the end of each processing corresponded to the CD34 cells infused to the patient. Endpoints Engraftment: Neutrophil engraftment was defined as the first of 3 consecutive days when the absolute neutrophil count was 0.5  109/L with evidence of donor hematopoiesis. Patients in whom no engraftment occurred were censored if they died before day 28, as were patients receiving a second transplant for nonengraftment. Acute and chronic GVHD: Patients were considered at risk of grade II to IV acute GVHD only if neutrophil engraftment occurred, whereas only those with sustained engraftment of donor hematopoiesis and surviving for more than 100 days after transplant were evaluated for development of chronic GVHD. Acute and chronic GVHD were evaluated according to previously described criteria [14,15]. Overall survival was the time between transplantation and death due to any cause. Data on patients were censored at the time of last follow-up visit. Relapse was indicated by morphologic evidence of leukemia in BM, cerebrospinal fluid, or peripheral blood or by cytogenetic recurrence of a neoplastic clone. It was considered as the time interval between BMT and relapse, with censoring at death in complete remission Statistical analysis Reference date of the analysis was January 1, 1999. Median duration of follow-up was 33 months (range 17–45). No patients were lost to follow-up. Time to acute and chronic GVHD, neutrophil engraftment, relapse, and overall survival were estimated by the Kaplan-Meier method. Univariate prognostic analyses used the log rank test, testing the influence on each endpoint of diagnosis (acute vs chronic vs other malignancies), recipient and donor gender (male vs female), recipient and donor age (children vs adults), recipient and donor cytomegalovirus (CMV) serology (negative vs positive), ABO compatibility, number of nucleated cells infused (median), and medians of colony-forming unit granulocyte-macrophage (CFU-GM), CD34, CD19, CD3, CD2, CD4, CD8, CD3/ CD8, CD28, CD8/CD28/CD3, HLA-DR, CD3/HLA-DR, CD3/ CD4 /CD8 cells infused/kg of recipient weight. Continuous covariates were encoded as binary covariates after dichotomization, using the median as the cutoff. Covariates in which the missing value rate was above 10% were not considered in the analyses. Multivariate prognostic analyses were performed for the main endpoints (engraftment, acute GVHD, and survival) using Cox models in which all covariates were introduced that were associated with the corresponding outcome at the 10% level by the log rank test. All p values were two sided, with p  0.05 indicating statistical significance. Statistical analysis used performed using the SAS software package (SAS Institute, Cary, NC, USA).

Results Patient, disease, and transplant characteristics Forty-eight patients, most of them adults, were analyzed with hematologic malignancies. Table 1 lists the main characteris-

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tics of patients, diseases, and transplants, including donor factors. Twenty-eight patients had acute leukemia, 14 chronic leukemia, and 6 other malignancies. Conditioning regimen varied according to disease and disease status; however, 21 patients (44%) received cyclophosphamide and busulfan as preparative regimen. As GVHD prophylaxis, all patients except one received cyclosporin A associated with methotrexate. Graft composition Table 2 shows the percentages and median counts of nucleated cells, lymphocytes subsets, and progenitors counts (CFU-GM and CD34 cells) present in the BM graft and infused per body weight of recipient. Engraftment Two patients died during the first 28 days after transplantation, without signs of engraftment. All but two of the remaining 46 patients achieved sustained donor engraftment. Two patients had graft rejection. Median time to neutrophil recovery was 22 days (range 11–34). At day 40 post transplant, estimate of neutrophil recovery was 97% 5% (95% confidence interval [CI]). In univariate prognostic analysis, the following factors were associated with shorter and higher probability of neutrophil recovery: 1) negative recip-

Table 2. Graft compostion of bone marrow cells and lymphocyte subsets

Type of cells infused Median no. of nucleated cells infused (108/kg) Median no. of CFU-GM infused (105/kg) Median no. of CD34 cells infused (106/kg) Lymphocyte subsets ( 107/kg) B cells CD19 T cells CD2 CD3 CD4 CD8 CD3/CD8 CD28 CD8/CD28/CD3 HLA-DR CD3/DR NK cells CD3 /CD16/ CD56* CD57* CD8/CD57* Other CD3/CD4 /CD8

*Only assessed in 22 patients.

Median (range)

Mean percentage (range)

2.4 (0.4–5.9)

—

0.86 (0.1–2.4)

—

3.5 (0.5–12.9) 4.08 (0.66–12.7)

1.45 (1.25–2.18) 19.4 (8–67)

0.48 (0.06–3.6)

3.05 (0.58–15.05)

2.4 (0.3–6.4) 2.2 (0.3–5.4) 1.2 (0.15–2.7) 1.2 (0.15–3.2) 1.1 (0.13–2.8) 1.8 (0.3–4.5) 0.65 (0.1–1.8) 1.8 (0.2–8.5) 0.29 (0.04–1.4)

10.64 (4.06–36.92) 9.89 (4.5–38.84) 5.14 (1.96–16.31) 5.23 (2.17–22.87) 4.54 (1.90–21.50) 8.46 (3.98–29.59) 3.04 (1.60–11.40) 8.78 (3.13–20.11) 1.83 (0.46–14.02)

1.4 (0.3–3.7) 0.1 (0.0–1.2) 0.08 (0.0–0.9)

6.47 (2.98–14.26) 0.71 (0.11–2.69) 0.49 (0.08–1.89)

0.09 (0.0–0.47)

0.53 (0.12–3.53)

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Figure 1. Influence of CD3/CD8 cell dose on probability of neutrophil recovery after HLA-identical bone marrow transplantation in patients with hematologic malignancies.

Figure 2. Influence of CD4 cell dose on time to acute GVHD grade II or higher after HLA-identical bone marrow transplantation in patients with hematologic malignancies.

ient CMV serology (p  0.008) and 2) infusion of 1  107 CD3/CD8 cells/kg (p  0.04) with a median time to neutrophil recovery of 21 days (range 11–28) compared to 24 days (range 13–34) in those patients receiving less (Fig. 1). There was a trend of faster neutrophil recovery in patients receiving 3  106/kg of CD3/HLADR (p  0.06), in female recipients (p  0.06) and in patients receiving 3.5  106/kg of CD34 cells (p  0.08). Finally, in a multivariate analysis, the following factors were associated with a rapid neutrophil recovery: negative CMV serology (HR  0.41, 95% CI  0.21–0.80, p  0.009); female recipient (HR  2.03, 95% CI  1.08–3.80, p  0.026), and a number of 1  107/kg CD3/CD8 cells infused (HR  2.13, 95% CI  1.13–4.00, p  0.018).

Causes of death, survival, and relapse Twenty-two patients died: 5 after relapse or progression of the disease and 17 of other transplant-related causes (acute GVHD 2, infection 12, hemorrhage 1, and acute respiratory distress syndrome 2). Kaplan-Meier estimate of overall survival at 3 years was 54% 14%. Factors individually influencing the risk of death were 2.0  108 nucleated cells/kg (p  0.05), 3.5  106 CD34 cells infused/kg (p  0.009), and 0.5  107 CD19 infused/kg (41% 20% vs 67% 19%, p  0.05). In multivariate analysis, CD34 cells dose 3.5  106 was associated with improved survival (HR  0.37, 95% CI  0.15–0.88, p  0.024). Six of 42 leukemia patients relapsed: 4 acute and 2 leukemia. The 3-year estimate of relapse was 26% 22%.

Acute and chronic GVHD Forty-four patients were available to determine the incidence of acute GVHD. The cumulative incidences at day 100 of grades II to IV or III to IV were 47% 15% and 11% 10%, respectively. In a univariate prognostic analysis, infusion of 1  107/kg CD4 increased the risk of grade II to IV (63% vs 30%) (Fig. 2). There was a trend for a higher incidence of acute GVHD in patients with a positive CMV serology prior to transplant (58% vs 33%; p  0.07) and patients receiving a higher cell dose of CD8 (1.2  108/kg; 59% vs 36%; p  0.06) and CD3 cells (2.0  108/kg; 60% vs 36%; p  0.08). In a multivariate analysis, the number of CD4 cells in the graft superior to 1  107/kg increased the risk of acute GVHD (HR  2.85, 95% CI  1.10–7.40, p  0.031). Forty patients were at risk of developing chronic GVHD. The incidence rate was 52% 16% at 3 years. Five patients presented limited chronic GVHD and 14 extensive chronic GVHD. The most striking finding was the association of low cell dose with increased incidence of chronic GVHD. In fact, the 3-year estimate probability of chronic GVHD was 43% for patients 2.0  108/kg nucleated cells compared to 70% for those patients receiving less. In consequence, all subsets of lymphocytes studied, except CD19, influenced the appearance of chronic GVHD (data not shown).

Discussion The immunologic role of lymphocytes present in BM graft on the outcome of stem cell transplantation has been widely studied in T-depleted stem cell transplantation. Many studies have demonstrated the influence of some lymphocyte subsets on the occurrence of graft failure [16–18], acute GVHD, and graft-vs-leukemia (GVL) effect [12]. However, most of these studies analyzed specific depletion of subsets in experimental situations [9] or in unrelated BMT [12] or HLA-matched or HLA-mismatched PBSC transplants [13,19]. We analyzed the composition of the BM graft and the influence of hematopoietic progenitors and different subsets of lymphocytes in 48 patients with malignancies receiving BM from an HLA-identical sibling donor. This situation offered an opportunity to study differences in the clinical manifestations of alloreactivity in a setting where histocompatibility differences were not an issue. The first objective of this study was to evaluate the BM graft content of lymphocyte subsets. We found in the BM graft that approximately 19% (median range 8–67%) of the nucleated cells were lymphocytes, similar to 16% (range 11–23%) reported in BM aspirates. However, the variability of the numbers in the infused BM graft was more prominent and could be explained by the dilution of peripheral blood

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during the aspiration that can vary from 68% to 88% of peripheral blood contamination per volume of aspirate [20– 22] and the concentration of the graft. Another factor that can explain the variability of the number and type of BM lymphocytes infused in the graft is donor age [23]. Rego et al. [23] analyzed BM biopsies by flow cytometry and reported that, with age, there was a progressive decrease in the percentage of B cells and an increase of T cells, reaching similar proportions in the BM from adults. The percentage of natural killer cells did not change with age. The second finding of our study was the association between CD3/CD8 cell dose and probability and speed of engraftment. Recently, in T-depleted HLA-identical PBSC transplants, the number of CD3 cells present in the inoculum was the most critical factor for sustained engraftment [13]. Earlier studies showed that donor CD8 T cells prevent BM graft rejection in mice, primarily by generating cytotoxic T lymphocytes that eliminate immune cells of the recipient [9,24]. Recently, in an animal model two subsets of CD8 cells were identified as facilitating cells for engraftment, namely, CD8 TCR and CD8 TCR cells. The TCR subset seems to eliminate recipient T cells, whereas the TCR subset does not [18]. However, we did not specifically study this subset of CD8 cells in our study. We found that patients receiving 1  107 CD3/CD8 cells infused/kg had quicker and better engraftment compared to patients receiving less. Champlin et al. [10] described an increased rate of graft failure after CD8 removal from the BM graft in HLA-identical sibling transplants. Martin et al. [12] conducted a Phase I to II clinical trial to evaluate removal of CD4 cells and partial depletion of CD8 cells from donor marrow from 27 HLA-mismatched unrelated recipients. In this study, all patients receiving 3.9  106 CD8 cells/kg engrafted, whereas 4 of 7 patients who were given marrow containing 3.9  106 CD8 cells/kg had graft failure. After comparison with non–T-depleted unrelated BMT, they concluded that the increased incidence of graft failure was caused by depletion of CD8 cells from the donor marrow and not by undetected disparity for class I alleles [12]. Among patient- and transplant-related factors, we also found that a female recipient and a negative recipient CMV serology were associated with more rapid time to neutrophil recovery. CD34 cell dose was not associated with early neutrophil recovery, reinforcing results of previous studies; however, it was a very important factor for survival [25,26]. Our group recently analyzed the influence of CD34 cell dose on outcomes (including infectious episodes) of 212 patients undergoing BMT [27]. We confirmed that CD34 cell dose is not an important factor for early granulocyte recovery; however, it was important for rapid monocyte recovery and increasing risk of fungal infection after BMT [27]. GVHD is the most frequent clinical complication and remains the major cause of transplant-related mortality following BMT. Several risk factors for developing GVHD

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have been reported, such as HLA disparities, age, gender, GVHD prophylaxis, conditioning regimen, and cytomegalovirus infection [28,29]. Among these factors, the presence of T cells in the graft is important, because T-cell depletion of BMT leads to lower GVHD risk. However, the number of T cells infused with BMT is about 107/kg, and it is known that GVHD can be induced by a number of T CD3 cells as low as 1  106/kg and even fewer in HLA-mismatched situations [12,19]. Removal of CD4 (3 log depletion) in HLAmismatched unrelated donor transplantation did not decrease the incidence of acute GVHD [12]. Moreover, in HLA-identical peripheral stem cell transplantation, infusion of 108 T cells/kg does not increase the risk of acute GVHD [30–32]. In HLA-identical cord blood transplantation, acute GVHD is reduced when compared to BMT [33]. The number of cord blood T cells infused in the graft is about 106/kg. These examples considered together suggest that the number of T cells infused in the graft and the incidence of acute GVHD are influenced not only by the number of T cells infused, but also by the immune function of the lymphocytes, the source of the stem cell graft (BM, peripheral stem cells, or cord blood), and associated genetic disparities (HLA, minor antigens, cytokine polymorphisms). We found an association between the risk of acute GVHD and the number of CD4 cells infused, and a recipient positive CMV serology. Other known factors (such as age, female gender) did not influence the risk of acute GVHD. In a multivariate analysis, high CD4 cell dose was associated with increased risk of GVHD. The influence of CD8 and CD3 cell dose infused on the risk of GVHD was only observed in a univariate analysis. This finding should be confirmed in larger series. We and others have already observed that a high number of nucleated cells infused in the BM graft decreases the risk of chronic GVHD [7,25]. A possible explanation for this finding is that a high cell dose leads to a decreased incidence of early post-transplant infections, which may decrease GVHD [7]. However, it seems that the incidence of chronic GVHD is higher after allo-PBSC transplants when compared to BMT [30,31]. This observation suggests that the function of mobilized peripheral lymphocytes probably is a more important cause of chronic GVHD than the number of lymphocytes infused. In our series, the number of CD34 cells infused affected survival, as observed in previous series [25–27]. We conclude that a high cell dose of CD3/CD8 shortened neutrophil recovery, whereas a high cell dose of CD4 increased the incidence of acute GVHD. A high number of CD34 cells infused in the graft also increased overall survival, whereas a high nucleated cell dose reduced the incidence of chronic GVHD.

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