The aim of this study was to investigate the effects of natural killer (NK) cells on transplant outcomes in patients receiving G-CSF-mobilized PBSC grafts and G-CSF-primed BM grafts from HLA-haploidentical donors. Forty-one haploidentical allogeneic hematopoietic SCT patients were analyzed according to the NK cell concentration in relation to acute GVHD (aGVHD), chronic GVHD (cGVHD), TRM and leukemia-free survival. The patients receiving a higher dose of CD56bright NK cells (>1.9 × 106/kg) showed a higher incidence of grades II–IV aGVHD (hazard risk (HR), 2.872; P=0.022) and cGVHD (HR, 2.884; P=0.039). A higher CD56dim/CD56bri NK cell ratio (>8.0) was correlated with a decreased risk of III–IV aGVHD (HR, 0.290; P=0.065) and TRM (HR, 0.072; P=0.012), thereby increasing the rate of leukemia-free survival (HR, 0.174; P=0.007) after haploidentical transplantation without in vitro T-cell depletion. Our results suggest that a high allograft CD56dim/CD56bright NK cell ratio (>8.0) plays an important role in improving transplant outcomes. A higher dose of CD56bright NK cells might be a predictor for a higher incidence of GVHD.
Family HLA-mismatched/haploidentical transplantation is a feasible therapeutic approach for patients with otherwise lethal malignant hematological diseases who lack an HLA-matched donor.1, 2, 3, 4, 5, 6 We recently developed a new method for haploidentical transplantation without in vitro T-cell depletion.4, 5, 6 The strategy is based on sequential in vivo maneuvers that involve the recipient, donor T-cell function, and the dose of donor hematopoietic stem cells using a protocol designated GIACl: G, donor treatment with G-CSF to induce immunological tolerance; I, intensified immunological suppression to both promote engraftment and prevent GVHD; A, inclusion of anti-human thymocyte Ig to prevent GVHD and graft rejection; C, use a combination of G-CSF-primed BM harvests and G-CSF-mobilized PBSC harvests as the source of stem cell grafts. Promising results have been achieved in our institute by using this protocol.5, 6
Natural killer (NK) cells are large granular lymphocytes that take part in both innate and adaptive immunity and are regarded as the interface between these immune systems.7, 8 The NK cells are the first cells to recover after autologous or allogeneic BM or PBSC transplantation. The role of NK cells and their alloreactivity in allogeneic hematopoietic SCT (allo-HSCT) is currently controversial for different transplant settings.1, 9, 10, 11, 12, 13, 14 Our earlier study confirmed the deleterious role of a killer Ig-like receptor (KIR) ligand mismatch in this haploidentical GIAC protocol.15, 16 A further study also showed that the recovery of NK cells is, and can be used as, an indicator of the clinical outcome after unmanipulated haploidentical transplantation.9, 17 However, no data are currently available on the role of NK dose or its subsets in the grafts after unmanipulated haploidentical transplantation. Yamasaki et al.13 showed earlier that the CD16+CD56+ cell dose is inversely correlated with the incidence of GVHD when they studied 27 patients who received PBSCs from HLA-identical sibling donors, suggesting an important role for NK cells in the development of GVHD. This was confirmed by Kim et al.14 in 61 patients receiving G-CSF-mobilized PBSCs. It has also been shown that a higher NK cell dose in BM is associated with faster neutrophil recovery and a decreased incidence of chronic GVHD (cGVHD).18 Therefore, we hypothesized that the number of NK cells in G-CSF-primed BM harvest and G-CSF-mobilized PBSC harvest mixture grafts infused into haploidentical recipients could be associated with transplant outcomes. To test our hypothesis, the number of CD56bright and CD56dim NK cell subsets in the mixture grafts was correlated with clinical outcome after haploidentical transplantation without in vitro T-cell depletion.
Materials and methods
Patients with malignant hematological malignancies who were suitable for allo-HSCT and had no match to HLA-identical related or unrelated donors were candidates for haploidentical HSCT. Forty-one patients, who underwent haploidentical allo-HSCT between August 2003 and April 2005, were included in this study. All patients and donors gave written informed consent and the Institutional Review Board of Peking University Institute of Hematology approved this study.
Preparation and cell harvesting
The conditioning, mobilization and collection of stem cells, as well as GVHD prevention, were described earlier.5, 6 All patients received a myeloablative regimen, which included two 4 g/m2 doses of Ara-C on days 9 and 10, 12 mg/kg of orally administered BU in 12 doses over the course of days 6 through 8, two 1.8 g/m2 doses of CY on days 4 and 5, 250 mg/m2 Simustine (MeCCNU) on day 3, and 2.5 mg/kg rabbit anti-human thymocyte Ig (Thymoglobulin; Sangstat, Fremont, CA, USA) by i.v. administration each day on days 2 through 5.
Donors received 5 μg/kg rhG-CSF (Filgrastim) daily for 5–6 days. On the fourth day, BM cells were harvested. The target total nucleated cell count was 3.0 × 108 (median, 3.6 × 108; range, 0.24 × 108–8.16 × 108) cells/kg recipient weight. On the fifth and sixth days, PBPCs were collected. The target mononuclear cell count was 3.0 × 108 (median, 3.65 × 108; range, 2.77 × 108–11.7 × 108) cells/kg recipient weight. The fresh and unmanipulated BM and PBPCs were infused into the recipients on the day of collection.
The GVHD prophylaxis included CsA and short-term MTX with mycophenolate mofetil. CsA was started intravenously on day 9 at a dosage of 2.5 mg/kg and switched to an oral formulation as soon as the patient was able to take medication after the graft. The dosage was adjusted to blood levels. A half gram of mycophenolate mofetil was administered orally every 12 h from 9 days before transplantation until day 30 after transplantation. It was then given at 0.25 g twice a day for 1–2 months. The dosage of MTX was 15 mg/m2 and was administered intravenously on day 1; 10 mg/m2 was administered on days 3, 6 and 11 after transplantation. The diagnosis and grading of GVHD was performed according to the published criteria.19 Filgrastim (G-CSF) was given to all recipients subcutaneously at a dosage of 5 μg/kg per day from day 6 after transplantation until the neutrophil count reached 0.5 × 109 cells/l on 3 consecutive days. BM aspiration and cytogenesis studies were performed 1, 2 and 3 months after transplantation to assess the graft. To detect donor chimerism, HLA genotyping and DNA fingerprinting (STR) were performed. For each patient, at least two methods were used to confirm donor chimerism.
Cells from G-CSF-primed BM harvest and G-CSF-mobilized PBSC harvest were stained without further separation to minimize selective loss. MoAbs, CD16-FITC and CD56-PE, were used in combination with anti-CD45-Percp and anti-CD3-APC (BD Bioscience, Mountain View, CA, USA) in individual 4-color flow cytometry assays to analyze the immunophenotype lymphocytes in the allograft, including CD3+ T cells and CD56+ NK cells and its subsets CD56dim (CD56dimCD16bright) and CD56bright (CD56brightCD16low/neg) based on CD56 cell-surface density by FACS (Becton-Dickinson Flow Cytometer; San Jose, CA, USA) as described earlier.20 The grafts were analyzed for the total number of mononuclear cells, the CD34+ cell content and subsets of lymphoid cell populations (CD3+, CD4+, CD8+ cells) using a standardized Multi-Set Kit (Becton-Dickinson, San Jose, CA, USA).
HLA and KIR ligand typing and compatibility characterization of patient–donor pairs
All patients had allele-level molecular typing performed at HLA-A, -B, -C and -DRB1. Cases were divided into those KIR ligand (KIR-L) GVHD direction match or mismatch, as described by Ruggeri et al.,21 on the basis of known KIR-Ls (HLA-C alleles with Asn77-Lys80; HLA-C alleles with Ser77-Asn80; and HLA-Bw4 alleles).
The characteristics of patients with a low or high CD56dim/CD56bright ratio were compared using the χ2-test for categorical variables and the Mann–Whitney U-test for continuous variables. The associations between NK cells in the allograft and post transplant outcomes were analyzed by the Kaplan–Meier method or calculated using cumulative incidence curves to accommodate competing risks.22 The log-rank test was used in survival, and Gray's test was used in cumulative incidence analyses. The risk factors calculated for univariate analysis included the recipient and donor ages and sex; diagnosis; the status of KIR-L mismatch/match between donor and recipients, HLA mismatch; pre-transplantation risk category; the concentration of CD3+ T cells, CD34+ cells, and CD56+ NK cells and its subsets CD56bright and CD56dim; and the CD56dim/CD56bright NK cell ratio. The Cox regression model was not built because of the limited number of cases. All calculations were performed with SPSS 13.0 statistical software, and R software was used to calculate the cumulative incidence when considering the presence of competing risks.
Table 1 presents selected patient characteristics. We placed the patients into subgroups designated as ‘high’ or ‘low’ according to the cutoff of 8.0 for the CD56dim/CD56bright NK cell ratio in the allograft. We decided on this threshold on the basis of an identified correlation between the CD56dim/CD56bright ratio and transplant outcomes, and because a comparison of other patient characteristics among these groups revealed no significant differences. All patients achieved engraftment and complete donor chimerism after transplantation. As of 1 November 2007, there were 26 patients who were surviving without leukemia, 11 patients who had died of transplant-related complications and 4 patients who had relapsed on days 370, 365, 89 or 44. The median follow-up was 858 days (range, 43–1404 days). Twelve of the 41 patients had 0–1 acute GVHD (aGVHD), and grades II, III and IV aGVHD occurred in 17, 3 and 9 patients, respectively. Among 34 patients evaluated for cGVHD who survived longer than 100 days, 24 developed limited (17 patients) or extensive (7 patients) cGVHD.
We further analyzed the correlation between the CD56dim/CD56bright ratio and ANC or plt engraftment after transplantation. The P of ANC recovery at day 100 was 100% for both groups, with a median recovery time of 13 and 14 days for patients in the high and low CD56dim/CD56bright groups, respectively. The P of plt recovery at day 100 was 95% and 76.2% for patients in the high and low ratio groups, respectively (P=0.121). The median time to plt recovery was 18 and 19 days for the high and low ratio groups, respectively (P>0.05). No rejections occurred after transplantation in any of the 41 patients.
As shown in Table 2, the concentrations of CD3+ T cells were separated on the basis of the median concentration of the CD3+ T cells in the allograft. On the basis of their respective receiver operating characteristic curves and sensitivity/specificity curves, the optimal cutoff points for the concentration of CD34+ cells, NK cells and NK subsets CD56bright and CD56dim were 1.34 × 106/kg, 1.8 × 107/kg, 1.9 × 106/kg and 1.2 × 107/kg, respectively. In the univariate analysis, no statistically significant associations were observed between the development of aGVHD or cGVHD and the concentration of infused CD3+ cells (Table 2). The concentration of CD56bright was associated with increased II–IV aGVHD (hazard risk (HR), 2.872; P=0.022). A high concentration of NK cells (HR, 0.355; P=0.121) or CD56dim NK cells (HR, 0.316; P=0.084) or a high CD56dim/CD56bright NK cell ratio (HR, 0.290; P=0.065) tends to prevent the occurrence of III–IV aGVHD. A high concentration of CD56bright (HR, 2.884; P=0.039) and CD34+ cells (HR, 2.463; P=0.050) was associated with increased cGVHD. Patients in the ‘high’ CD56bright NK cell group had a higher cumulative incidence of II–IV aGVHD (85.2±6.8 vs 52.1±15.5%; P=0.015; Figure 1a) and cGVHD (89.4±7.0 vs 42.9±14.6%; P=0.025; Figure 1b) compared with those in the ‘low’ CD56bright NK cell group. Patients who received a high concentration of NK cells (>1.8 × 107/kg) or CD56dim NK cells (>1.2 × 107/kg) or a higher CD56dim/CD56bright NK cell ratio (>8.0) tended to have a lower incidence of III–IV aGVHD compared with those who received a low concentration of NK cells (<1.8 × 107/kg; 30.6±16.1 vs 47.2±11.1%; P=0.070) or CD56dim NK cells (<1.2 × 107/kg; 30.4±16.1 vs 49.8±11.8%; P=0.044) or a lower CD56dim/CD56bright NK cell ratio (30.20±14.9 vs 48.1±11.7%; P=0.013; Figure 2a). There was an increased incidence of cGVHD in patients who received a high concentration of CD34+ cells compared with those patients who received a low concentration of CD34+ cells (86.9±8.3 vs 53.8±13.8%; P=0.035).
We did not analyze relapse because only four patients relapsed. As shown in Table 2, a high concentration of CD56+ cells or CD56dim NK cells and a high CD56dim/CD56bright ratio was associated with a decreased incidence of TRM and better survival. Patients who received a high concentration of NK cells (>1.8 × 107/kg) or CD56dim NK cells (>1.2 × 107/kg) tended to have a lower incidence of TRM compared with those who received a low concentration of NK cells (<1.8 × 107/kg; 9.5±6.4 vs 49±11.8%; P=0.007) or CD56dim NK cells (<1.2 × 107/kg; 9.1±6.1 vs 51.9±12.1%; P=0.003); therefore, those patients had better survival rates than others who received fewer NK cells (<1.8 × 107/kg; 81±8.6 vs 45±11.1%; P=0.008) or CD56dim NK cells (<1.2 × 107/kg; 81.8±8.2 vs 42.1±11.3%; P=0.005). Patients in the high ratio group received a significantly higher dose of CD56+ NK cells (P=0.003) and CD56dim NK cells (P=0.030), as well as a lower dose of CD56bright NK cells (P=0.001) compared with the low ratio group. The cumulative incidence of TRM in patients in the high ratio group was lower than that of patients in the low ratio group (5±4.9 vs 51.4±11.50%, P=0.001; Figure 2b). Furthermore, the highest 4-year cumulative incidence of leukemia-free survival was observed in patients in the high ratio group (85±8 vs 42.9±10.8%; P=0.002; Figure 2c).
We further analyzed the effect of III–IV aGVHD on clinical outcomes. Patients with III–IV aGVHD had a higher incidence of TRM (66.75±13.6 vs 10.9±5.9%; P<0.0001) and poorer survival (25±12.5 vs 79.3±7.5%; P=0.0002) compared with those with 0–II aGVHD.
The correlation of cell composition with transplant outcome after allo-HSCT has been extensively studied, particularly in HLA-matched settings.23, 24, 25, 26, 27, 28, 29 The NK cell concentration has been found to be an important factor influencing transplant outcome, although there is some controversy over the effect of NK cell dose on aGVHD, cGVHD and survival.10, 13, 14, 18, 30 Our earlier study showed the role of NK cells in this transplant protocol from the perspective of NK cell alloreactivity or, after transplantation, recovery kinetics,9, 15, 16, 17 but the role of allograft NK concentration in the clinical outcome for this protocol is unknown.
In the Perugia group's haploidentical transplant protocol, NK cell alloreactivity was associated with less aGVHD occurrence.1, 21 In accordance with the earlier study in HLA-matched HSCT,13, 14, 18 we found that the transplantation of a higher concentration of NK cells is associated with a lower incidence of III–IV aGVHD and TRM, and thus with better outcomes after the haploidentical GIAC protocol. However, no study has yet explored the association of NK subset cells in the allograft, particularly CD56dim and CD56bright, with clinical outcome. When we analyzed the NK cell subsets, we found that an increased number of CD56bright NK cells in the allograft was strongly associated with a higher occurrence of grades II–IV aGVHD and cGVHD. With the present knowledge, it was difficult to clarify the underlying mechanism explaining the relationship between CD56bright NK cell doses and GVHD after unmanipulated HLA-mismatched transplantation. Differences in GVHD prophylaxis and graft cellularity may partially explain the association between CD56bright NK cell doses and GVHD. In the Perugia group's haploidentical transplant protocol, T-cell depletion is vigorous, with the stem cell inoculums containing an average of 3 × 106 T cells/kg,21 as compared with an average of 2.07 × 108 T cells/kg in our protocol. Earlier studies have shown that the CD56bright NK cells are enriched in the T-cell region of resting lymph nodes, and the communication among CD56bright NK cells, DC cells and T cells is crucial for the interaction between adaptive and innate immunity. Therefore, more CD56bright NK cells might activate more DC and T cells, and then induce higher aGVHD and cGVHD occurrence in these unmodified haploidentical transplantations.31 Although obtained in a limited number of patients with a relatively short follow-up, the result of this study of haploidentical HSCT without in vitro T-cell depletion shows that CD56 bright NK cells are associated with higher aGVHD. Further prospective studies involving larger numbers of patients are necessary to confirm our observations. The immunoregulatory roles of NK cells under this unmodified haploidentical HSCT are being investigated at our institute.
It is true that the role of T cells for the development of GVHD cannot be neglected. However, in our transplant settings, T-cell dose in the allograft failed to associate with the development of GVHD analyzed by Huang et al.5, 32 and in this study. Several possible factors may be responsible for this. First, it has been suggested that beyond a critical threshold dose of T cells, perhaps 1 × 105/kg–1 × 106/kg of patient body weight, increasing the dose does not necessarily result in a higher incidence of GVHD.33, 34 Second, sequential immunosuppression was used to prevent both GVHD and rejection. CsA was infused from day 9 before transplantation and anti-human thymocyte Ig was used before transplantation. This helped to prevent graft rejection by suppressing or killing host lymphocytes. It may also have induced depletion of infused donor T lymphocytes in vivo and thus lowered the incidence of GVHD. Third, the immunosuppression regimen, using CsA, MTX, mycophenolate mofetil and anti-human thymocyte Ig, may have achieved efficient engraftment and prevented severe GVHD in haploidentical HSCT. Fourth, the grafts we used were mixtures of G-CSF-primed BM harvest and PBSCs, which were intended to combine the advantages of both elements. The mixture of G-CSF-primed BM harvest and PBSCs in different proportions could form new grafts maintaining the hyporesponsiveness and polarization potential of T cells.35
Highlighted by the results from our analysis, the CD56dim/CD56bright NK cell ratio had a significant impact on clinical outcome; a higher CD56dim/CD56bright ratio was associated with a lower incidence of III–IV aGVHD and could have beneficial effects for TRM and leukemia-free survival. Our earlier studies found that the concentration of CD56dim NK cells was positively associated with the absolute number of CD56bright NK cells on day 14 after transplantation.9 Cooper et al.7 provided evidence to support the idea that CD56bright NK cells are the major cytokine-producing subset of human NK cells with unique immunoregulatory roles in vivo. It is reasonable to conclude that the rapid expansion of donor-derived CD56bright NK cells may have particular implications for immune recovery because of their immunoregulatory roles and the ability to proliferate and engage in IFN-γ production on interaction with DCs and T cells.31 Therefore, in our transplant settings, the higher proportion of CD56dim NK cells in the allograft, which would be expressing intermediate affinity IL-2R and showing enhanced cytotoxicity activity without proliferation in response to high (nanomolar) concentrations of IL-2, may not only be involved in eliminating residual leukemic cells, but may also contribute to the recovery of CD56bright NK cells, all of which lead to the improvement of transplant outcomes. In addition, the association between the CD56dim/CD56bright ratio and clinical outcome emphasizes that the function of NK cells in the allograft may be important to transplantation. It is conceivable that differences in the proportion of cell subsets could compensate for differences in absolute numbers in the allograft, which affects the functional status of grafts and determines the clinical outcome.
However, we failed to show the deleterious role of KIR-L mismatch on GVHD after this unmanipulated haploidentical transplantation on this occasion, which had been reported earlier.15 Although KIR-L mismatch between donor and patients pairs had a trend toward a higher incidence of GVHD (data not shown), it could not reach a significant level in the univariate or multivariate analysis. The relatively small number of patients in this study compared with our previous work might be the major reason for this different result. Another possible factor to consider for the effect of NK cells on outcomes is the role of activating KIR and/or KIR B haplotypes, which has been shown to increase GVHD, fight viral infections and decrease relapse.36, 37
In summary, for the first time we demonstrate that a high concentration of CD56bright NK cells (>1.9 × 106/kg) in an allograft is associated with an increased risk of aGVHD and cGVHD, and that a higher CD56dim/CD56bright NK cell ratio (>8.0) is associated with an increased rate of leukemia-free survival and a decreased risk of III–IV aGVHD and TRM in patients with hematological malignancies after haploidentical transplantation without in vitro T-cell depletion. Therefore, careful monitoring of the number of CD56bright NK cells and the CD56dim/CD56bright NK cell ratio in allografts might predict the occurrence of early transplant-related events and improve transplant outcomes in the context of haploidentical HSCT with T-cell repletion.
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This work was supported by the National Natural Science Foundation of China (Grant no. 30800485), National Outstanding Young Scientist's Foundation of China (Grant no. 30725038), Hi-Tech Research and Development Program of China (no. 2006AA02Z4A0), Program for Innovative Research Team in University (IRT0702) and Beijing Nova program 2008605.
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