The graft-vs-tumor effect is an important part of the curative potential of allogeneic transplantation. We characterized the effect of transplanted donor mononuclear cells on relapse- and event-free survival after allogeneic bone marrow transplantation (BMT). We studied 113 consecutive patients with hematologic malignancies who received non-T-cell-depleted BMT from human leukocyte antigen (HLA)-matched siblings. Most patients (n = 103) received busulfan (Myleran)-based conditioning, and all patients received standard short-course methotrexate and tacrolimus (Prograf) as graft-vs-host disease prophylaxis. Sixty-four patients had low-risk diagnoses (acute lymphoblastic leukemia/acute myeloid leukemia [first complete remission], myelodysplastic syndrome [refractory anemia/refractory anemia with ring sideroblasts], and chronic myeloid leukemia [first chronic phase]); 49 patients had high-risk diagnoses (all others). Cox regression analyses evaluated risk strata, age, gender, and the numbers of nucleated cells, CD3-positive T cells, CD34-positive hematopoietic cells, and type 2 dendritic cells (DC2) as covariates for event-free survival, relapse, and nonrelapse mortality. Recipients of larger numbers of DC2 cells had significantly lower event-free survival, a lower incidence of chronic graft-vs-host disease, and an increased incidence of relapse. Recipients of larger numbers of CD34-positive cells had improved event-free survival; recipients of fewer CD34-positive cells had delayed hematopoietic engraftment and increased death from infections. In conclusion, content of donor DC2 cells was associated with decreased chronic graft-vs-host disease and graft-vs-leukemia effects consistent with Th2/Tc2 polarization of donor T cells following allogeneic bone marrow transplantation. [ONCOLOGY 16(Suppl 1):19-26, 2002]
ABSTRACT: The graft-vs-tumor effect is an important part of the curative potential of allogeneic transplantation. We characterized the effect of transplanted donor mononuclear cells on relapse- and event-free survival after allogeneic bone marrow transplantation (BMT). We studied 113 consecutive patients with hematologic malignancies who received non-T-cell-depleted BMT from human leukocyte antigen (HLA)-matched siblings. Most patients (n = 103) received busulfan (Myleran)-based conditioning, and all patients received standard short-course methotrexate and tacrolimus (Prograf) as graft-vs-host disease prophylaxis. Sixty-four patients had low-risk diagnoses (acute lymphoblastic leukemia/acute myeloid leukemia [first complete remission], myelodysplastic syndrome [refractory anemia/refractory anemia with ring sideroblasts], and chronic myeloid leukemia [first chronic phase]); 49 patients had high-risk diagnoses (all others). Cox regression analyses evaluated risk strata, age, gender, and the numbers of nucleated cells, CD3-positive T cells, CD34-positive hematopoietic cells, and type 2 dendritic cells (DC2) as covariates for event-free survival, relapse, and nonrelapse mortality. Recipients of larger numbers of DC2 cells had significantly lower event-free survival, a lower incidence of chronic graft-vs-host disease, and an increased incidence of relapse. Recipients of larger numbers of CD34-positive cells had improved event-free survival; recipients of fewer CD34-positive cells had delayed hematopoietic engraftment and increased death from infections. In conclusion, content of donor DC2 cells was associated with decreased chronic graft-vs-host disease and graft-vs-leukemia effects consistent with Th2/Tc2 polarization of donor T cells following allogeneic bone marrow transplantation. [ONCOLOGY 16(Suppl 1):19-26, 2002]
The aggregate 5-year survival for all patients with leukemia or lymphomas undergoing autologous transplantation, or allogeneic transplantation from human leukocyte antigen (HLA)-matched siblings, remains approximately 50%.[1-7] A number of factors, including patient disease status, age, conditioning regimen, and (for allogeneic recipients) type of graft-vs-host disease prophylaxis, influence the probability of survival after transplantation.[1,8,9] The most significant factor in predicting relapse-free survival is the patient’s diagnosis (eg, chronic myeloid leukemia vs acute lymphoblastic leukemia) and disease status at the time of transplant (eg, chronic phase chronic myeloid leukemia vs blast crisis). The factors amenable to manipulation include the conditioning regimen, graft-vs-host disease prophylaxis, supportive care during the peritransplant period, and the quality and quantity of donor cells in the graft.
The optimal numbers for each cell type in the bone marrow or peripheral blood progenitor cell allograft are not known. A variety of studies support the role of larger numbers of CD34-positive cells in the graft in faster hematopoietic reconstitution after autologous and allogeneic transplantation.[10-15] Transplantation with fewer than 1 × 106 CD34-positive cells/kg from T-cell-depleted allogeneic bone marrow and peripheral blood stem cell grafts was associated with increased relapse and treatment-related mortality.[11,16] Recipients of T-cell-depleted bone marrow allografts have a decreased risk of developing graft-vs-host disease,[17,18] but experience delayed immune reconstitution and have an increased risk of infections, graft failure, and relapse.[19-22]
There are numerous commercially available devices to positively select CD34-positive hematopoietic progenitors and passively remove T cells. However, the higher rates of relapse after T-cell-depleted allografts are unacceptable. Indeed, a randomized, multicenter phase III study of CD34-positive cell selection in allogeneic peripheral blood stem cell transplantation was recently amended to exclude high-risk patients because of significantly higher relapse rates among recipients of CD34-positive selected grafts.
Dendritic cells have a specialized capacity to present peptide antigens to T cells and regulate the initiation of the immune response.[23,24] There is significant heterogeneity within the dendritic cell lineage. Dendritic cells develop from hematopoietic progenitor "stem" cells under the influence of cytokines that act during early myelomonocytic differentiation.[25-32] Phenotypic and functional analyses have demonstrated two general types of dendritic cells-type 1 (DC1) and type 2 (DC2)-that differ in surface marker expression as well as their functional effect on cognate T cells.
Studies of cytokines used to mobilize peripheral blood hematopoietic progenitors have indicated increased numbers of DC1 cells after administration of Flt3 and granulocyte-macrophage colony-stimulating factor (GM-CSF [Leukine]), while increased numbers of DC2 cells have been noted after treatment with granulocyte colony-stimulating factor (G-CSF [Neupogen]). DC1 cells promote Th1 immune responses in responding CD4-positive T cells characterized by enhanced interferon (INF)-gamma, tumor necrosis factor (TNF), and interleukin (IL)-12 synthesis. DC2 cells promote Th2 responses in CD4-positive T cells characterized by IL-4 and IL-10 synthesis, and inhibition of INF-gamma and TNF production in cognate T cells. The effects of GM-CSF and G-CSF on the content of different dendritic cell subtypes in the blood, and the functional differences between DC1 and DC2, offer potential therapeutic indications for these cytokines.
Dendritic Cells in Hematopoietic Progenitor Cell Transplantation
Dendritic cells play an important role in the antitumor effect of autologous and allogeneic transplantation. Antigen-primed dendritic cells have been used with promising results in adoptive vaccination against tumors.[34,35] Bone marrow contains monocytes and CD86-positive, CD34-positive progenitor cells that can differentiate into DC1 cells in the presence of TNF and GM-CSF,[36-38] as well as CD123bright dendritic cell progenitors that differentiate into DC2 cells in the presence of IL-3. Most studies involving hematopoietic progenitor cell transplantation to date have examined the role of DC1 cells as a method of adoptive immunotherapy for cancer following autologous hematopoietic progenitor cell transplantation.
One recent report, using a murine transplant model, indicated that only host CD11c-positive dendritic cells (DC1 cells) were necessary for the development of acute graft-vs-host disease. Donor dendritic cells did not appear to efficiently present host antigens via cross-priming in a way that led to the initiation of graft-vs-host disease. In this study, the possible effect of donor dendritic cells on inhibiting graft-vs-host disease was not examined. The potential role of increased numbers of donor DC2 cells in regulating graft-vs-host disease after G-CSF-mobilized allogeneic peripheral stem cell transplants has recently been recognized.
Adoptive Transfer of Immature Dendritic Cells
A recently published study in normal donors evaluated the ability of immature dendritic cells to augment T-cell activation after in vitro exposure to antigen. Immature dendritic cells were derived from peripheral blood monocytes and then exposed to the influenza peptide MP and keyhole limpet hemocyanin. After return of these dendritic cells to the host, a marked decline in the frequency of MP-specific INF-gamma-producing cells-which persisted for 30 days-was noted. At the same time, the frequency of influenza-specific T cells was unchanged, suggesting that the decrease in MP-specific INF-gamma-producing cells was related to dendritic cell exposure to the antigen, rather than to a global reduction in influenza response.
Also, this reduction in MP-specific INF-gamma-producing cells was paralleled by an increase in the frequency of IL-10-producing MP-specific cells, and a reduction in the ability of CD8-positive MP-specific cells to kill target cells that had been loaded with MP peptides. There was no reduction in the overall frequency of MP-specific T cells after return of the immature dendritic cells to account for this change in antigen response.
We undertook a prospective analysis of the cellular constituents of the bone marrow allograft in order to help identify those factors that might predict relapse, graft-vs-host disease, and event-free survival among patients receiving transplants for hematologic malignancies. The study population included 64 patients with low-risk leukemia (chronic myeloid leukemia in first chronic phase, myelodysplastic syndromes of refractory anemia and refractory anemia with ringed sideroblasts, acute myeloid leukemia, and acute lymphoblastic leukemia in first complete remission) and 49 patients with higher-risk malignancies (all other chronic myeloid leukemia, myelodysplastic syndrome, acute myeloid leukemia, or acute lymphoblastic leukemia and any non-Hodgkin’s lymphomas, Hodgkin’s disease, chronic lymphocytic leukemia, or multiple myeloma).
The most frequent indication for transplant was chronic myeloid leukemia (40 patients), followed by acute myeloid leukemia (28 patients), myelodysplastic syndrome (16 patients), non-Hodgkin’s lymphoma (12 patients), and acute lymphoblastic leukemia (8 patients). The median ages of patients in the low-risk group (45 years) and the high-risk group (42 years) were similar. Male patients were more frequent in the high-risk group. 103 of 113 patients received busulfan (Myleran)-based conditioning: busulfan plus cyclophosphamide (Cytoxan, Neosar) (chronic myeloid leukemia and acute myeloid leukemia), busulfan/cyclophosphamide plus cytarabine (some chronic myeloid leukemia and acute myeloid leukemia), or busulfan/cyclophosphamide plus etoposide (multiple myeloma, non-Hodgkin’s lymphoma, and Hodgkin’s disease). Ten patients received total-body irradiation plus cyclophosphamide as pretransplant conditioning (acute lymphoblastic leukemia).
Significance of Disease Diagnosis and Status
The actuarial relapse-free survival rates of low-risk patients were 84%, 68%, and 58% at 100 days, 1 year, and 3 years posttransplant, respectively, vs 57%, 27%, and 14%, respectively, for patients in the high-risk group. A univariate analysis of clinical factors that were associated with relapse or death posttransplant showed that the high-risk stratum was the most significant factor associated with worse relapse-free survival (P < .0001).
Analysis of the Allograft Constituents
Three- and four-color flow cytometry was used to enumerate the frequency of T cells, B cells, monocytes, natural killer (NK) cells, dendritic cells, CD34-positive cells, and the subset of CD34-positive, CD38-negative cells in the graft using Paint-A-Gate (BD Immunocytometry Systems). The number of transplanted cells in each subset was determined by multiplying the percentage of nucleated (CD45-positive) cells with each phenotype by the number of nucleated cells in the graft. All cell transplant data are expressed as the number of cells transplanted per kilogram body weight of the recipient. The CD3-negative, CD4bright, CD8-negative, low side scatter phenotype was used as a surrogate marker for the population of DC2 cells in bone marrow.[27,29]
DC2 Cells in Allograft and Relapse-Free Survival
Analysis was limited to relapse-free survival in patients who received unmanipulated bone marrow allografts, with events defined as relapse or death. As expected, patients who received more CD34-positive cells in the allograft had improved relapse-free survival posttransplant (relative risk [RR] of 0.79 associated with an increase of 1 × 106 donor CD34-positive cells/kg in the graft [95% CI = 0.64-0.97], P = .028). The number of colony-forming units transplanted, the total number of nucleated cells transplanted, and numbers of donor T cells, B cells, NK cells, and monocytes were not significantly associated with the risk of relapse and/or death posttransplant. Surprisingly, the most significant cellular factor in predicting overall survival and relapse-free survival posttransplant was the content of donor CD4bright DC2 cells in the allograft. Larger numbers of donor cells with the DC2 phenotype (CD3-negative, CD4bright, CD8-negative, and low side scatter) were associated with increased mortality and relapse posttransplant (RR of 2.2 associated with an increase of 1 × 106 cells/kg [95% CI = 1.2-3.8], P = .0072).
A similar association of the DC2 cell content of the graft with decreased overall survival was also seen (RR of DC2 cell content for death of 2.1 [95% CI = 1.1-3.8], P = .015). The association of increased numbers of DC2 cells with relapse-free survival was present in the entire group of patients (n = 113) as well as in the subset of patients that received busulfan-based conditioning (n = 103) and the subset of chronic myeloid leukemia (first chronic phase) and acute myeloid leukemia (first complete remission) patients (n = 53). A particularly striking effect on enhanced relapse-free survival was seen among low-risk patients who received fewer CD4bright dendritic cells in their grafts (Figure 1A). Low-risk patients in group 1DC (< 0.6 × 106 CD4bright DC2 cells/kg) had a 3-year actuarial relapse-free survival of 80%, while those in group 3DC (> 0.9 × 106 CD4bright DC2 cells/kg) had a 3-year actuarial relapse-free survival of only 48% (Figure 1A, P = .009 comparing group 1DC and group 3DC).
An analysis of the 49 high-risk patients divided according to the content of CD4bright DC2 cells showed a trend toward better survival among recipients of fewer CD4bright DC2 cells (P = .09, Figure 1B). In contrast, low-risk patients who received larger numbers of CD34-positive cells had significantly improved relapse-free survival (Figure 1C). Transplantation with larger numbers of total nucleated cells was also associated with improved relapse-free survival among low-risk patients only (not shown), while the content of CD3-positive cells in the graft did not affect relapse-free survival in either low- or high-risk subsets (not shown).
DC2 Cell Content and Relapse-Free Survival
A multivariate Cox regression model was built using factors significantly associated (P < .05) with relapse-free survival in the univariate analyses. In addition, patient and graft characteristics hypothesized a priori as being potentially significant with respect to relapse-free survival were also included (age, number of nucleated cells transplanted, and number of transplanted donor T cells). Covariates independently associated with increased risk of death or relapse in all 113 patients included risk strata (RR 2.8, P = .0002), male gender (RR 1.8, P = .04), and number of donor CD4bright dendritic cells (RR 2.0, P = .03) in the bone marrow allograft (Table 1). In contrast, larger numbers of donor CD34-positive cells (RR 0.76, P = .027) were associated with improved relapse-free survival in the group of 113 patients (Table 1).
A separate analysis of the subset of 64 low-risk patients showed that CD4bright DC2 content remained a highly significant factor for worse relapse-free survival (RR of 21, P = .0008). Transplantation with larger numbers of donor nucleated cells (RR of 0.41, P = .05) and larger numbers of donor CD34-positive cells (RR of 0.71, P = .1) were also associated with improved relapse-free survival among low-risk patients (Table 1). In the subset of 49 high-risk patients, no covariate was significantly associated with relapse-free survival in the multivariate analysis. The use of conditional entry criteria for inclusion of covariates in the multivariate model (P values < .05 or < .1) did not alter the role of gender, risk, CD34-positive cell content, and CD4bright DC2 content as independently significant factors (data not shown). Multivariate analyses based upon overall survival as the clinical end point gave very similar results in multivariate analyses (data not shown).
Overall, 60 deaths had occurred among the 113 patients by the reference date: 21 of 64 low-risk patents (33%) and 39 of 49 high-risk patients (80%). Relapse was the most frequent primary cause of death, and fatal relapse occurred in 7 of 64 low-risk patients (11%) and 20 of 49 high-risk patients (41%) at a median of 331 days posttransplant. Thirteen deaths (3 relapse, 10 nonrelapse) occurred among patients transplanted with < 0.6 × 106 CD4bright DC2 cells/kg (group 1DC); 21 deaths (11 relapse, 10 nonrelapse) occurred in the group 2DC patients (³ 0.6 × 106, £ 0.9 × 106 CD4bright DC2 cells/kg); and 26 deaths (13 relapse, 13 nonrelapse) occurred in the group 3DC patients (> 0.9 × 106 CD4bright DC2 cells/kg).
The cumulative incidences of relapse in groups 1DC, 2DC, and 3DC were compared after stratification on risk. The incidence of relapse was 33% for low-risk patients who received the largest number of CD4bright DC2 cells vs 8% for low-risk patients who received the fewest dendritic cells (Figure 2A; P = .01; group 1DC vs group 3DC). The incidence of relapse was 48% among high-risk patients who received the highest and the intermediate numbers of CD4bright DC2 cells vs only 22% among high-risk patients who received the fewest CD4bright DC2 cells (Figure 2B, P = .01, group 1DC vs group 2DC or group 3DC).
A multivariate model utilizing the same covariates as in Table 1 evaluated relapse as a single clinical end point. Risk strata (RR of 3.8, P = .0023) and age (RR of 0.94, P = .003) were independently associated with relapse in the entire group of 113 patients. Among low-risk patients, relapse occurred less among recipients of a larger number nucleated cells (RR of 0.2, P = .03) and was more frequent among recipients of more CD4bright dendritic cells (RR of 114, P = .0074). Among high-risk patients, the only covariate significantly associated with relapse was age (RR of 0.92, P = .0028).
A multivariate analysis of factors associated with nonrelapse mortality indicated increased mortality with age and decreased nonrelapse mortality associated with larger numbers of CD34-positive cells in the allograft (Table 2).
DC2 Cell Content and Chronic Graft-vs-Host Disease
The incidence of acute graft-vs-host disease did not vary with DC2 cell content. Among the entire study population, 24 of 113 patients (21%) developed acute graft-vs-host disease at a median of 38 days posttransplant. Of 113 patients, 20 (18%) experienced grade 2-4 acute graft-vs-host disease, and 8 (7%) experienced grade 3/4 acute graft-vs-host disease. Twenty-one patients with acute graft-vs-host disease were treated with systemic steroids. No single donor factor or combination of factors (Table 1) was significantly associated (P < .05) with the risk of developing acute graft-vs-host disease in univariate or multivariate analyses (data not shown). In particular, the median number of CD4bright DC2 cells in patients who developed acute graft-vs-host disease (0.83 ± 0.33 × 106/kg) was not significantly different than the corresponding value of those patients who did not develop acute graft-vs-host disease (0.7 ± 0.4 × 106/kg, P = .2). The mean number of transplanted T cells was not significantly different for the 24 patients who developed acute
graft-vs-host disease (25.4 × 106/kg) vs the 89 patients who did not (24.2 × 106/kg, P = .6).
In contrast to the lack of association of DC2 content with acute graft-vs-host disease, chronic graft-vs-host disease was less common among recipients of larger numbers of DC2 cells. Of 113 patients, 34 (30%) developed chronic graft-vs-host disease at a median of 182 days posttransplant. Nine patients had limited-grade and 25 patients had extensive-grade chronic graft-vs-host disease. Thirty-one patients received systemic corticosteroids for treatment of chronic graft-vs-host disease. In 82 patients who survived to day 100 and were evaluable for chronic graft-vs-host disease, a higher content of CD4bright dendritic cells was associated with a lower risk of chronic graft-vs-host disease (RR of 0.42 [95% CI = 0.17-1.05], P = .06]) (Figure 3).
The mean number of CD4bright DC2 cells among the 34 patients who developed chronic graft-vs-host disease was significantly lower than the corresponding value of those patients who did not develop chronic graft-vs-host disease (0.68 ± 0.37 × 106/kg vs 0.85 ± 0.4 × 106/kg, respectively, P = .03). Recipients of < 0.6 × 106/kg CD4bright DC2 cells/kg and recipients of 0.6-0.9 × 106/kg CD4bright DC2 cells/kg who survived to day 100 experienced higher incidences of chronic graft-vs-host disease (52% and 50%, respectively) than the 22% incidence of chronic graft-vs-host disease seen among recipients of > 0.9 × 106/kg CD4bright DC2 cells/kg (log-rank statistic 4, P = .04). The mean number of transplanted T cells was not significantly different for the 34 patients who developed chronic graft-vs-host disease (24.4 × 106/kg) vs the 48 patients who did not (26.7 × 106/kg, P = .3).
Our hypothesis was that significant interdonor variability in the cellular constituents of the graft would lead to quantitative and qualitative differences in hematopoietic engraftment, graft-vs-host disease, and relapse. The novel finding in this study was that larger numbers of CD4bright DC2 cells had an adverse effect on survival, and were associated with an increased incidence of posttransplant relapse and decreased chronic graft-vs-host disease. In contrast to previous reports of increased relapse associated with T-cell-depleted allografts, the number of transplanted T cells or various T-cell subsets were not significantly correlated with either survival or relapse. As predicted, the CD34-positive cell dose positively affected engraftment kinetics and posttransplant survival.[11,16,44] The independent association of CD4bright DC2 cells with event-free survival is supported by the lack of correlation between the numbers of transplanted CD34-positive cells and CD4bright DC2 cells (correlation coefficient [CC] 0.22), as well as the independent significance of nucleated cells, CD34-positive cells, and CD4bright DC2 cells as covariates in the multivariate analysis (Table 1).
A number of hypotheses may explain the role of donor CD4bright DC2 cells in influencing relapse and chronic graft-vs-host disease posttransplant. Larger numbers of CD4bright DC2 cells in allogeneic bone marrow grafts may shift the developing donor immune system toward a type 2 response characterized by decreased Th1 cellular immune responses, and a decreased graft-vs-leukemia effect.[33,45,46] Donor DC2 cells may capture alloantigen via apoptotic bodies of residual host hematopoietic cells and induce tolerance to minor histocompatability antigens in donor T cells (indirect antigen presentation or "cross-priming"), thereby inhibiting the graft-vs-leukemia effect.[47-49] Such an antigen-specific effect could occur as one of the initial events in the early posttransplant period or during subsequent de novo T-cell development of donor stem cells in the host thymic microenvironment.
In addition, donor DC2 progenitors may function as immunoregulatory cells that synthesize interferon and promote survival of mature DC2 cells, thus potentiating the effect of relatively small numbers of donor DC2 on immune responses of donor T cells. Finally, the association of higher numbers of CD4bright DC2 cells with less graft-vs-leukemia and chronic graft-vs-host disease may reflect the preexisting polarization of donor T cells toward Th2/Tc2 that is independent of any direct effect of donor dendritic cells on posttransplant immunity. The net effect of dendritic cells in regulating immune reconstitution posttransplant likely represents the competing effects of donor DC1 cells vs DC2 cells in the graft, as well as donor-derived dendritic cells that differentiate from CD34-positive cells under the influence of either GM-CSF/TNF (DC1) or IL-3/G-CSF (DC2).[36,37,52]
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