Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood.

Cord blood (CB) and autologous mobilized peripheral blood stem/progenitor cells (PBSC) are now used widely for clinical transplantation. We characterized the short-term (<8 weeks) and long-term (>8 weeks) engraftment in NOD/SCID mice resulting from transplanted CD34+ cells from these two sources. We also quantified the frequency of long-term engrafting cells, and the average proliferative capacity of individual engrafting cells by a competitive repopulation assay with binomial variance-covariance modeling. We found that 0.5 million CD34+ CB cells were able to generate sustained, high-level, multilineage human hematopoiesis, whereas a sixfold higher number of CD34+ PBSC (3 million) from cancer patients undergoing chemotherapy generated comparable short-term, but much lower sustained multilineage human hematopoiesis after transplantation. In comparison to CD34+ cells from PBSC from cancer patients, long-term engrafting cells were approximately eightfold enriched in CB CD34+ cells, and each CB long-term engrafting cell had an approximately 15-fold higher multilineage proliferative capacity. Thus, the number and function of transplantable hematopoietic cells were remarkably different between these two sources of stem/progenitor cells.     

Multiple unit HLA-unmatched sex-mismatched umbilical cord blood transplantation for advanced hematological malignancy

We investigated the effect of multiple-unit umbilical cord blood (UCB) transplantation on engraftment in the setting of severe human leukocyte antigen (HLA) mismatch. Ten poor-risk adult patients with hematological malignancy received multiple unit, HLA-unmatched, sex-mismatched, unrelated UCB transplantation after a reduced intensity-conditioning regimen (RICR) with engraftment as the primary endpoint. The median age of the patients was 55 years with a range of 28-67. Patients received one unit of UCB per 10 kg of recipient body weight (5-7 units). The median number of nucleated cells and CD34(+) cells per kilogram of recipient body weight infused was 6.3 x 10(7) (range 3.8-10.0) (NC/kg) and 5.7 x 10(5) (range 1.1-11.9) (CD34/kg), respectively. Three patients expired before day 28 and were not evaluable for engraftment. Five of the remaining 7 patients showed increasing neutrophil counts. Fluorescent in situ hybridization (FISH) for the Y chromosome or HLA-typing showed only donor cells in the peripheral blood. After engraftment, HLA typing was done on 3 patients and their infused UCB units. All revealed the presence of a single HLA type concordant with one of the infused units. Moreover, the order of infusion did not influence which UCB unit engrafted. The engrafting UCB units were infused first or second in one case and fourth in the other two. One patient transplanted for refractory acute lymphoblastic leukemia (ALL) survives in continuous complete remission 4 years after transplant. He engrafted with one UCB unit, is fully hematologically reconstituted, has no evidence of graft-versus-host disease (GVHD), and takes no immunosuppressive medication. HLA typing reveals that the recipient and the engrafted cord blood match at only one HLA-B locus using conventional 6 antigen typing (A, B, and DR). Although engraftment was not accelerated, it did occur in the majority of evaluable patients. Long-term disease-free survivorship without debilitating GVHD is possible in patients with refractory hematological malignancy who receive unmatched multiple unit UCB.     

In vitro evaluation of graft-versus-graft alloreactivity as a tool to identify the predominant cord blood unit before double cord blood transplantation

The transplantation of two cord blood (CB) units obtained from unrelated donors (double CBT) is an effective strategy for adult patients with hematologic malignancies. Sustained hematopoiesis after double CBT is usually derived from a single donor, and only a few transplantation recipients displaying a stable mixed donor-donor chimerism have been reported. We investigated the mechanisms underlying single-donor predominance in double CBT by studying in?vitro the role of the graft-versus-graft cell-mediated immune effect in two-way mixed-lymphocyte culture, along with the contribution of differential hematopoietic progenitor (HP) potency in HP mixed cultures. Results for the two-way mixed-lymphocyte culture showed that despite the weak and variable alloantigen-specific cytotoxic potential displayed by CB mononuclear cells, an immune-mediated dominance for one of the two CB units was detected in the majority of experiments. Alloantigen-induced cytotoxic activity was directed toward both CB-HP and phytohemagglutinin (PHA)-activated T lymphoblastoid cells. The CB unit with the higher fold expansion of CD34(+) cells in single-expansion culture was prevalent in the HP mixed-expansion culture, as shown by DNA chimerism evaluation. Based on these data, we hypothesize that the dominant CB unit is able to develop prevalent cytotoxic activity toward activated lymphocytes of the other CB unit, thereby preventing them from exerting alloantigen-specific cytotoxic potential against both activated lymphocytes and HPs of the dominant unit. In accordance with this hypothesis, we propose the evaluation of?alloantigen-induced cytotoxic activity generated in two-way mixed-lymphocyte culture and directed toward PHA-activated T lymphoblastoid cells as a tool to identify the potentially predominant CB unit before double CBT.     

Characterization of long-term mixed donor�Cdonor chimerism after double cord blood transplantation

Double cord blood transplantation (DCBT) with two matched or partially matched cord blood units has been implemented successfully to circumvent the limitations of graft cell dose associated with single CBT. After DCBT, sustained haematopoiesis is derived almost exclusively from only one of the donated units. None the less, we previously observed two of six evaluable DCBT patients still having mixed donor–donor chimerism at 28 and 45 months post‐transplantation, respectively. In the present study we utilize flow cytometry techniques to perform the first thorough analysis of phenotype and functionality of cord blood units in patients with mixed donor–donor chimerism. Our results suggest that the two stable cord blood units are different phenotypically and functionally: one unit shows more naive T cells, lower T cell cytokine production and higher frequencies of natural killer cells, the other shows higher frequencies of well‐differentiated and functional lymphocytes. Additionally, in comparison with control patients having a single prevailing cord blood unit, the patients with donor–donor chimerism exhibit less overall T cell cytokine production and a smaller fraction of memory T cells. Furthermore, our results indicate that human leucocyte antigen‐C match of donor units may partly explain the development of a donor–donor mixed chimerism.     

Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation.

In an attempt to capture graft-versus-tumor effects without graft-versus-host disease (GVHD), the authors initiated a trial of nonmyeloablative allogeneic bone marrow transplantation (BMT) in patients with advanced hematologic malignancies, with the majority of patients having chemotherapy-refractory disease. Forty-two patients received an HLA-matched related donor BMT after a cyclophosphamide and antithymocyte globulin-based conditioning that also included thymic irradiation for patients who had not received prior mediastinal radiotherapy. Prophylactic donor leukocyte infusion (pDLI) at a dose of 1 x 10(7) CD3(+) cells per kilogram were given beginning 5 weeks post-BMT to 16 patients with mixed chimerism (MC) but without GVHD, whereas 26 patients did not receive pDLI, either because of GVHD or early relapse. Twelve of 16 patients (75%) receiving pDLI had T cell chimerism at the time of pDLI >/=40%. These patients, by day 100 post-BMT, either converted to full donor chimerism (FDC) (n = 10) or had an increase in or stable donor chimerism (n = 2) after pDLI. Four of 4 patients whose T cell chimerism was /=grade II acute GVHD, including 12 after BMT and 7 after pDLI. Approximately one third of patients, after having initial MC, eventually lost their donor graft. The authors conclude that (1) pDLI has the potential to convert MC to FDC; (2) sustained remissions can be achieved in patients with chemorefractory hematologic malignancies who receive a pDLI, albeit with a significant risk of acute GVHD; and (3) the degree of donor T cell chimerism at the time of pDLI is predictive of the fate of MC, ie, donor T cell chimerism >/=40% or 相似文献   

Double Umbilical Cord Blood Transplantation: A Study of Early Engraftment Kinetics in Leukocyte Subsets using HLA-Specific Monoclonal Antibodies

Single cord blood unit (CBU) predominance is usually established within the first month after double umbilical cord blood transplantation (UCBT). However, the kinetics of engraftment of the different leukocyte subsets and the mechanism of graft predominance is largely unknown. To investigate whether a differential engraftment might reveal a specific subset that could play a key role in the mechanism of graft predominance, we studied early engraftment kinetics of different leukocyte subpopulations by flow cytometry using human monoclonal antigen–specific human leukocyte antigen antibodies, directed against mismatched human leukocyte antigen-A or –B antigens between recipient and CBUs. Twenty-two patients, who had received a double UCBT preceded by a reduced-intensity conditioning regimen, were evaluated at days +11, +18, +25, and +32 posttransplantation. Single CBU predominance in the various leukocyte subsets was established within 18 days posttransplantation. CD4+ T cells of the dominant CBU showed early peripheral blood expansion. Moreover, chimerism in CD4+ and CD8+ T cell and natural killer cell subsets at day +11 was predictive of ultimate graft predominance. These findings show that engraftment kinetics of the various leukocyte subsets vary considerably after double UCBT and may suggest an important role for CD4+ T cells in a presumed alloreactive graft-versus-graft rejection.     

Fetal immune suppression as adjunctive therapy for in utero hematopoietic stem cell transplantation in nonhuman primates

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow-derived, CD34+ -enriched donor cells. Animals treated postnatally received either donor-derived T cell-depleted or CD34+ -enriched marrow cells. Chimerism was determined by traditional and real-time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune-suppressed animals relative to controls (11.3% +/- 2.7% and 5.1% +/- 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence-activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at > or = 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune-suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6-month follow-up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long-term chimerism also was not improved with postnatal donor cell infusion.     

Phase II study of a moderate-intensity preparative regimen with allogeneic peripheral blood stem cell transplantation for hematologic diseases: the Texas Transplant Consortium experience.

Conventional preparative regimens for allogeneic stem cell transplantation are associated with excessive regimen-related toxicity (RRT) in some patients because of underlying comorbidities, advanced age, or prior treatment. We studied a preparative regimen designed to reduce RRT, yet allow for adequate engraftment and development of a graft-versus-malignancy effect. Thirty patients (median age, 57 years) were entered on study. Twenty-nine patientsreceived stem cells from HLA-identical siblings and 1 from a sibling mismatched for 1 antigen at the A locus. Sixteen patients had received previous stem cell transplants (6 allogeneic and 10 autologous). The preparative regimen consisted of fludarabine 30 mg/M2 per day IV on day -10 to day -5, busulfan 1 mg/kg per dose PO (n = 6) or 0.8 mg/kg per dose IV (n = 24) for 8 doses every 6 hours on day -6 to day -5, and horse-derived antithymocyte globulin 5 mg/kg per day IV (n = 12) or 15 mg/kg per day IV (n = 18) on day -4 to day -1. GVHD prophylaxis consisted of cyclosporine (CYA) 3 mg/kg BID PO starting on day -3 (n = 13) or CYA and methotrexate 15 mg/m2 IV on day +1 and 10 mg/m2 IV on day +3 and day +6 (n = 17). The median number of CD34 cells transplanted was 3.19 x 10(6)/kg. All patients demonstrated recovery of hematopoietic function. Twenty-six (89%) of 29 evaluable patients achieved greater than 90% donor cell chimerism before day 100. Three patients never achieved greater than 90% donor chimerism, and another 3 patients subsequently lost donor chimerism. All 6 of these patients had autologous reconstitution with progressive disease. RRT was minimal; 7 patients had greater than grade II nonhematologic toxicity and there were no toxic deaths attributable to the conditioning regimen. Transplantation-related mortality was 7% (95% confidence interval [CI], 6%-8%) at 3 months and 28% (95% CI, 23%-34%) at 12 months after transplantation. Non-relapse-related mortality was most often due to infection. Grade II or greater GVHD developed in 56% of evaluable patients, and all patients with disease response developed GVHD. Actuarial estimates of overall and disease-free survival at 12 months were 52% (95% CI, 43%-63%) and 30% (95% CI, 24%-37%), respectively. Although this preparative regimen allowed adequate engraftment with minimal RRT, GVHD and infectious complications caused significant morbidity and mortality. Further study to define appropriate patient populations for this regimen, while limiting GVHD and infection risks, is needed.     

Stable mixed hematopoietic chimerism after bone marrow transplantation for sickle cell anemia.

A multicenter investigation of allogeneic bone marrow transplantation for children with sickle cell disease was conducted that included 27 European and North American transplant centers. Fifty-nine patients who ranged in age from 3.3 to 15.9 years (median, 10.1 years) received HLA-identical sibling marrow allografts between September 1991 and April 2000. Fifty-five patients survive, and 50 survive free from sickle cell disease, with a median follow-up of 42.2 months (range, 11.8 to 115 months) after transplantation. Of the 50 patients with successful allografts, 13 developed stable mixed donor-host hematopoietic chimerism. The level of donor chimerism, measured > or =6 months after transplantation in peripheral blood, varied between 90% and 99% in 8 patients. Five additional patients had a lower proportion of donor cells (range, 11% to 74%). Among these 5 patients, hemoglobin levels varied between 11.2 and 14.2 g/dL (median, 11.3 g/dL; mean, 12.0 g/dL). In patients who had donors with a normal hemoglobin genotype (Hb), the Hb S fractions were 0%, 0%, and 7%, corresponding to donor chimerism levels of 67%, 74%, and 11%, respectively. Among patients who had donors with sickle trait, the Hb S fractions were 36% and 37%, corresponding to donor chimerism levels of 25% and 60%, respectively. Thus, allograft recipients with stable mixed chimerism had Rb S levels similar to donor levels, and only 1 patient required a red blood cell transfusion beyond 90 days posttransplantation. None of the patients have experienced painful events or other clinical complications related to sickle cell disease after transplantation. These observations strongly suggest that patients with sickle cell disease who develop persistent mixed hematopoietic chimerism after transplantation experience a significant ameliorative effect.     

Pharmacokinetics and pharmacodynamics of anti-thymocyte globulin in recipients of partially HLA-matched blood hematopoietic progenitor cell transplantation.

Polyclonal anti-thymocyte globulin (ATG) administered before allogeneic blood hematopoietic progenitor cell transplantation reduces the risks of graft rejection and graft-versus-host disease, but may delay posttransplant immune reconstitution caused by delayed clearance of ATG from the blood. We studied graft-versus-host disease, infections, and the kinetics of immune reconstitution in 28 patients with very poor-risk hematologic malignancies who received lymphocyte-depleted, CD34(+) cell-enriched hematopoietic progenitor cell grafts from partially HLA-matched related donors (PMRD). The incidence of these clinical events was correlated with blood ATG levels in 19 transplant recipients who received rabbit ATG (r-ATG, thymoglobulin) during conditioning. Total r-ATG and the fraction of ATG antibodies that bind human cells (active ATG) were measured for up to 45 days posttransplantation using enzyme-linked immunosorbent assay and flow cytometry assays. Three patients received equine ATG (e-ATG; total dose of 60 mg/kg/day), 3 patients received 10 mg/kg r-ATG, and 22 patients received 6 mg/kg r-ATG during conditioning. All evaluable patients engrafted. Median numbers of blood CD4(+) and CD8(+) T cells at 100 days posttransplantation were 15 and 8 cells/microL, respectively. Acute graft-versus-host disease developed in 3 of 3 recipients of e-ATG and 1 of 25 recipients of r-ATG. Rapid T-cell reconstitution was seen only in younger patients. Overall mortality was 93% (26/28 patients) with poor immune reconstitution contributing to death in 21 of 28 patients. Recipients of 6 mg/kg r-ATG had peak levels of total and active r-ATG of 64+/-20 microg/mL and 9.2+/-5.8 microg/mL, respectively, with clearance of active r-ATG (t(1/2)6 days) to sub-therapeutic levels (<1 microg/mL) by a median of 15 days posttransplantation (range, 8-38 days). Delayed immune reconstitution is likely a consequence of ex vivo and in vivo purging of donor T cells in the graft coupled with inadequate thymic function rather than persistence of active r-ATG in the blood for months posttransplantation.     

Posttransplantation cyclophosphamide facilitates engraftment of major histocompatibility complex-identical allogeneic marrow in mice conditioned with low-dose total body irradiation.

Cyclophosphamide (Cy) has been studied extensively for its immunosuppressive properties and is frequently combined with total body irradiation (TBI) as conditioning prior to HLA-identical allogeneic blood or marrow transplantation (alloBMT) in humans. Because Cy is most effective at suppressing host-versus-graft reactions when the drug is given after the transplantation (Mayumi H et al. Transplant Proc. 1986;18:363-369), we investigated whether posttransplantation Cy could prevent rejection of allogeneic marrow in mice conditioned with low-dose TBI. In a mouse model, posttransplantation Cy reduced the dose of TBI required from 500 cGy to < or = 200 cGy for the engraftment of 10 million major histocompatibility complex (MHC)-identical marrow cells in 100% of recipients. In animals conditioned with low-dose TBI and posttransplantation Cy, donor chimerism was proportional to the dose of TBI, was present in multiple hematopoietic lineages, and was associated with the indefinite survival of donor-strain skin grafts. In contrast, animals conditioned with either TBI alone or posttransplantation Cy alone failed to achieve engraftment after alloBMT and contained antidonor cytotoxic T-cells. Although <5% donor chimerism could be induced without TBI by transplanting > or = 50 million MHC-identical cells and administering posttransplantation Cy, the addition of low-dose TBI reduced the dose of donor cells required for alloengraftment and increased long-term donor chimerism to >50%. These data demonstrate that low-dose TBI and posttransplantation Cy cooperate to prevent graft rejection following the transplantation of standard doses of MHC-identical marrow cells.     

G-CSF-mobilized peripheral blood mononuclear cells added to marrow facilitates engraftment in nonmyeloablated canine recipients: CD3 cells are required.

Stable mixed donor/host hematopoietic chimerism can be uniformly established in dogs conditioned with 200 cGy TBI before dog leukocyte antigen (DLA)-identical marrow transplantation and immunosuppressed with a short course of mycophenolate mofetil (MMF) and cyclosporine (CSP) after the transplantation. A further decrease in the TBI dose to 100 cGy or the elimination of MMF in this model results in graft rejection. Here we asked whetherthe addition of G-CSF-mobilized peripheral blood mononuclear cells (G-PBMC) to marrow grafts would enhance donor engraftment in dogs conditioned with 100 cGy TBI and given postgrafting immunosuppression with CSP alone. Using this model, 7 of 9 dogs given only marrow cells rejected their grafts within 8 to 17 weeks after transplantation. In contrast, the addition of unmodified G-PBMC to marrow grafts resulted in stable mixed donor/host chimerism in 5 of 8 dogs studied (P = .06). However, addition of the CD3-depleted fraction of G-PBMC, which contained both CD34 cells and CD14 cells, resulted in engraftment in only 1 of 7 recipients. We conclude that adding G-PBMC to marrow grafts replaced the requirement of MMF and 100 cGy of TBI, and that CD3 cells were required to facilitate engraftment of marrow cells in DLA-identical recipients, whereas the additional CD34 cells present in G-PBMC were not sufficient for this effect.     

Monitorowanie kinetyki zmian wczesnego chimeryzmu hematopoetycznego i charakterystyka wszczepienia molekularnego za pomocą metody STR-PCR u pacjentów poddanych alloHSCT

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for proportion of patients suffering from malignant and non-malignant hematological disorders. Successful transplantation is a process that requires the engraftment of pluripotent hematopoietic stem cells which can re-establish normal hemopoesis and immune system. Distinguishing between donor and host origin of bone marrow and blood cells is crucial for monitoring of engraftment process. One of the most useful tools for engraftment monitoring is the assessment of hematopoietic chimerism after alloSCT that describes the percentage of donor hematopoietic in a transplant recipient.Thirty eight adult patients after alloHSCT were included into the study. In total 43 allogeneic stem cell transplantations were performed. Hematopoietic chimerism was assessed by STR-PCR technique. The analysis of early chimerism were performed starting from 2^nd to 14th day on every 2 days than to 28 days weekly and on day +30 after alloHSCT.Early hematopoietic chimerism assessment demonstrated that the kinetics of chimerism in patients after alloSCT was compatible with linear trend (R²=0.996) and in patients after alloNMSCT was compatible with logarithmic trend (R²=0.959). The hematopoietic chimerism level was higher in alloSCT on day 2 the difference was statistically significant (p=0.0048).Molecular engraftment preceded hematological engraftment in patients after either myeloablative or non-myeloablative conditioning regiments (alloSCT patients p=1.44×10^?12, alloNMSCT p=2.12×10^?6).Earlier ME was observed in patients after alloHSCT and alloSCT who received more than 3×10⁶ CD34+ cells/kg (alloSCT p=0.0013, alloHSCT p=0.021). The difference was statistically significant.     

Purified canine CD34+Lin- marrow cells transduced with retroviral vectors give rise to long-term multi-lineage hematopoiesis.

Human CD34+ cells have been shown to retain long-term hematopoietic engrafting potential in preclinical and clinical studies. However, recent studies of human and murine CD34- stem cells suggest that these are functionally important early progenitors. Using autologous transplantation, we investigated whether canine CD34 and CD34- marrow cells could be transduced and give rise to long-term hematopoiesis. CD34+Lin- and CD34-Lin- cell populations purified by fluorescence-activated cell sorting were separately cocultivated with retroviral vectors LN (CD34+Lin-) and LNY (CD34-Lin-), which carry the neomycin (neo) gene. After myeloablative total body irradiation (920 cGy), 3 dogs received transplants of both CD34+Lin- cells and CD34-Lin- cells and 2 dogs received only CD34-Lin- cells. Untransduced autologous marrow cells were given to ensure hematopoietic recovery. Using CFU-C assays, transduction efficiencies of CD34+Lin- cells ranged from 6% to 18% with no CFU-C formation from CD34-Lin- cells. PCR-based detection of the neo gene from WBCs was used to detect transduced cells weekly after transplantation. Additional PCR studies in 3 dogs given both CD34+Lin- and CD34-Lin- cells were performed on monocytes, granulocytes, and T cells (2 dogs, one at 7.5 months and the other at 9 months) and granulocytes (1 dog at 12 months). LN was detected up to 12 months posttransplantation in WBCs and mono-myeloid and lymphoid populations from 3 dogs receiving transplants of transduced CD34+Lin- cells. LNY was not detected at any time after transplantation in 5 dogs that received transduced CD34-Lin- cells. Whereas canine CD34+Lin- marrow cells contributed to long-term multilineage hematopoiesis, progeny of CD34-Lin- progenitor cells were not detected after transplantation in these experiments.     

Chimerism studies in HLA-identical nonmyeloablative hematopoietic stem cell transplantation point to the donor CD8(+) T-cell count on day + 14 as a predictor of acute graft-versus-host disease.

Chimerism analysis of hematopoietic cells has emerged as an essential tool in nonmyeloablative hematopoietic stem cell transplantation. We have investigated the development of donor chimerism in granulocytes and CD4(+) and CD8(+) T cells in blood and bone marrow of 24 patients with hematologic malignancies who received HLA-identical sibling peripheral blood stem cell grafts after conditioning with fludarabine and 2 Gy of total body irradiation. The T-cell chimerism of blood and bone marrow was tightly correlated. Complete donor chimerism was reached earlier in the granulocytes than in the T cells. Mixed T-cell chimerism was common at the time of onset of acute graft-versus-host disease (aGVHD), and both CD4(+) and CD8(+) donor T-cell chimerism increased with the occurrence of aGVHD grades II to IV (P =.0002 and P =.019, respectively). The rate of disappearance of recipient CD8(+) T cells was faster in patients with aGVHD grades II to IV than in patients without clinically significant aGVHD (P =.016). This observation indicates a role of graft-versus-lymphohematopoietic tissue reactions in creating complete donor T-cell chimerism. A donor CD8(+) T-cell count above the median on day +14 increased the risk of subsequent development of aGVHD grades II to IV (P =.003).     

Results of minimally toxic nonmyeloablative transplantation in patients with sickle cell anemia and beta-thalassemia.

We describe previously transfused patients with sickle cell disease (n = 6) and thalassemia (n = 1) who received nonmyeloablative hematopoietic stem cell transplantation (HCT) to induce stable (full or partial) donor engraftment. Patients were 3 to 20 years (median, 9 years) old. All 7 received pretransplantation fludarabine and 200 cGy of total body irradiation; 2 patients also received horse antithymocyte globulin. Patients received bone marrow (n = 6) or peripheral blood stem cells (n = 1) from HLA-identical siblings, followed by a combination of mycophenolate mofetil and cyclosporine or tacrolimus for postgrafting immunosuppression. After nonmyeloablative HCT, absolute neutrophil counts were <0.5 x 10(9)/L and <0.2 x 10(9)/L for a median of 5 days (range, 0-13 days) and 0 days (range 0-13 days), respectively. A median of 0 (range, 0-9) platelet transfusions were administered. No grade IV nonhematologic toxicities were observed. One patient experienced grade II acute graft-versus-host disease. Two months after transplantation, 6 of 7 patients had evidence of donor chimerism (range, 25%-85%). Independent of red blood cell transfusions, these 6 patients initially had increased total hemoglobin and hemoglobin A concentrations and a reduction of reticulocytosis and transfusion requirements. There were no complications attributable to sickle cell disease during the interval of transient mixed chimerism. However, after posttransplantation immunosuppression was tapered, there was loss of the donor graft, and all patients experienced autologous hematopoietic recovery and disease recurrence. One patient did not engraft. The duration of transient mixed chimerism ranged from 97 to 441 days after transplantation in patients 4 and 6, respectively, and persisted until immunosuppressive drugs were discontinued after transplantation. In summary, the nonmyeloablative HCT regimens described here produced minimal toxicity and resulted in transient donor engraftment in 6 of 7 patients with hemoglobinopathies. Although complications from the underlying hemoglobinopathies did not occur during the period of mixed chimerism, these results suggest that stable (full or partial) donor engraftment after nonmyeloablative HCT is more difficult to achieve among immunocompetent pediatric patients with hemoglobinopathies than among adults with hematologic malignancies, perhaps in part because recipients may have been sensitized to minor histocompatibility antigens of their donor by preceding blood transfusions.     

Risk Factor Analysis of Outcomes after Unrelated Cord Blood Transplantation in Patients with Hurler Syndrome

Allogeneic stem cell transplantation (SCT) is considered effective in preventing disease progression in patients with Hurler syndrome (HS). Unrelated umbilical cord blood (UCB) grafts are suggested as an alternative to bone marrow (BM) or peripheral blood stem cells (PBSC). We studied 93 HS patients receiving an UCB graft to analyze risk factors for outcomes. The median time from diagnosis to transplant was 4.6 months, median follow-up was 29 months, and median number of nucleated CB cells infused was 7.6 × 10⁷/kg. Most of the patients received 1 or 2 HLA disparate grafts, and the most frequently used conditioning regimen was cyclophosphamide + busulfan (Bu/Cy). All patients received anti-T cell antibody. At post transplant day +60, the cumulative incidence of neutrophil engraftment was 85%. A younger age at transplant and a higher CD34⁺ dose at infusion were favorably associated with engraftment. With the exception of 2 patients, all engrafted patients achieved full and sustained donor chimerism. The 3-year event-free survival (EFS) and 3-year overall survival (OS) rates were 70% and 77%, respectively. In a multivariate analyses, use of Bu/Cy and a shorter interval from diagnosis to transplant were predictors for improved EFS rate (82% for patients transplanted within 4.6 months after diagnosis compared to 57% for the rest). Improved outcomes from early transplantation and immediate availability of CB unit lead us to conclude that CB transplantation is a beneficial option, which should be considered expediently for children with HS.     

A novel route of transplantation of human cord blood stem cells in preimmune fetal sheep: the intracelomic cavity

The intracelomic route for in utero hematopoietic stem cell transplantation was evaluated in preimmune fetal sheep and the engraftment characteristics were defined. Twelve twin ovine fetuses (gestational age: 40-45 days) received intracelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspensions of the liver, spleen, bone marrow, and thymus by flow cytometry, cloning assays, and polymerase chain reaction (PCR) analyses of human beta2-microglobulin. Four fetuses (33%) aborted shortly after intracelomic transplantation and were not evaluable for engraftment. Engraftment was detected in four fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degrees of engraftment in these four fetuses ranged from 6%-22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (no. 435184) and 105 (no. 915293, no. 037568) days and one fetus delivered at term that received CD34-selected cord blood cells had human engraftment with 10%, 32%, 20%, and 10% CD45(+) cells in bone marrow, respectively. In six of eight fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta2-microglobulin, which also identified human cells in brain, spinal cord, heart, lung, and skeletal muscle. This preliminary study indicates that intracelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.     

The contribution of cytotoxic and noncytotoxic function by donor T-cells that support engraftment after allogeneic bone marrow transplantation.

The present studies were designed for investigation of the requirements for cytotoxic function in donor T-cells transplanted to support engraftment after infusion of allogeneic bone marrow. The experiments examined the capacity of donor CD8 T-cells lacking Fas ligand and/or perforin function to facilitate donor B6 congenic (B6-Ly5.1) BM engraftment across major histocompatibility complex class I/II barriers after transplantation. T-cell-depleted BM cells from B6-Ly5.1 donors were transplanted into sublethally irradiated (5.5 Gy) BALB/c recipients together with different lymphocyte populations from wild-type B6 (B6-wt) donors or donors lacking functional cytotoxic pathways. Early presence of lineage-committed donor progenitor cells was assessed by the presence of day 5 splenic colony-forming units-granulocyte-macrophage (CFU-GM). Recipients of BMT without donor T-cells did not demonstrate significant CFU-GM activity 5 days post-BMT. Lineage-committed progenitor cells in recipient spleens could be supported by addition to the BM of wild-type (B6-wt) and cytotoxically single- (perforin, B6-pko or FasL, B6-gld) or double-deficient (B6-cdd) CD8 T-cells. However, B220+-enriched B-cells could not support the presence of day 5 donor CFU-GM. For further assessment of the capacity of cytotoxically impaired T-cells to participate in the engraftment process, the ability of these and normal CD8 cells to support the homing of donor cells to the BM was examined after infusion of carboxyfluorescein diacetete succinimidyl ester-labeled progenitors. In a syngeneic model lacking resistance, cytotoxically impaired donor T-cells supported increased numbers of progenitor cells in the marrow equivalent to the support provided by wild-type donor T-cells. Examination of peripheral chimerism indicated that during the first month after B6-->BALB/c BMT, donor chimerism was detected in BMT recipients receiving unfractionated T-cells or CD8+ T-cells from B6-wt donors, and chimerism was maintained at least 80 days after BMT. In contrast, B6-cdd unfractionated or CD8+ T-cells failed to maintain long-term B6 donor chimerism in the host. Experiments with highly enriched populations of positively selected CD8+ T-cells from B6-pko, B6-gld, or B6-cdd donors demonstrated that although each of these T-cell populations could promote the initial presence of donor CFU-GM early post-BMT, B6-pko and B6-cdd CD8+ T-cell populations were not able to support long-term peripheral chimerism. These results demonstrate that donor T-cells lacking major cytotoxic effector pathways have functions that support initial donor progenitor cell presence in the host hematopoietic compartment after BMT. They also demonstrate that support of long-term donor BM engraftment requires CD8+ T-cells with intact cytotoxic, that is, perforin, function. Finally, syngeneic B6-->B6 BMT suggests activation of CD8+ T-cells posttransplantation apparently is required to support enhanced progenitor cell activity. This study provides new findings concerning the role of cytotoxic function in the process of facilitating allogeneic donor BM engraftment.     

In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells

In utero transplantation of hematopoietic stem cells is a promising treatment for immune and hematologic diseases of fetuses and newborns. Unfortunately, there are limited data from nonhuman primates and humans describing optimal transplantation conditions. The purpose of this investigation was to determine the effect of T-cell number on engraftment and the level of chimerism after in utero transplantation in nonhuman primates. CD34(+) allogeneic adult bone marrow cells, obtained from the sire after G-CSF and stem cell factor administration, were transplanted into female fetal recipients. The average CD34(+) cell dose was 3.0 x 10(9)/kg (range, 9.9 x 10(8) to 4.4 x 10(9)) and the T-cell dose ranged from 2.6 x 10(5) to 1.1 x 10(8)/kg. Chimerism was determined in peripheral blood subsets (CD2, CD13, and CD20) and in progenitor cell populations by using polymerase chain reaction. Chimerism was noted in seven of eight live-born animals. The level of chimerism in the progenitor population was related to the fetal T-cell dose (r = 0.64, p < 0.02). At the lowest T-cell dose (2.6 x 10(5)/kg), no chimerism was detected. As the T-cell dose increased to 10(6-7)/kg, the level of chimerism increased. Adjusting the T-cell dose to 1.1 x 10(8)/kg resulted in fatal graft-versus-host disease (GVHD). The results of this study emphasize the importance of T cells in facilitating donor cell engraftment and in producing GVHD in fetal nonhuman primates. Some animals achieved levels of chimerism in the marrow hematopoietic progenitor cell population that would likely have clinical relevance. However, the levels of chimerism in peripheral blood were too low for therapeutic benefit. Further studies are needed to test methods that are likely to enhance donor cell engraftment and peripheral blood levels of donor cells.