Partial Chimerism Research Articles

Neutrophil antigen antibodies affect engraftment and secondary graft failure in hematopoietic progenitor cell transplantation.

Research is limited on the role of antibodies against human neutrophil antigen (HNA) in hematopoietic progenitor cell (HPC) transplantation outcomes. A retrospective review was conducted on medical records of patients at the NIH Clinical Center enrolled in six research protocols. This case-control study included 21 patients tested for HNA antibodies from January 2010 to March 2022 who underwent HPC transplantation. In addition, 42 patients following the same research protocols were randomly selected as a control group. The cumulative incidence of time to neutrophil engraftment was significantly impacted by the patients' anti-HNA status (p = .042), with the patients with anti-HNA experiencing delayed engraftment. Secondary graft failure occurred in 4 out of 42 patients (9.52%; 95% confidence interval [CI]: 3.7-22.1) of the control group, while 5 out of 9 patients (55.5%; 95% CI: 26.7-81.1) with anti-HNA experienced secondary graft failure (p = .005). Furthermore, patients with anti-HNA had a lower proportion (p = .008 for full and p = .002 for partial chimerism) and cumulative incidence (p = .016 for full and p = .010 for partial chimerism) of achieving donor chimerism compared to the control group. The study reveals a potential link between anti-HNA and HPC transplantation outcomes not previously reported. Patients with anti-HNA had a lower proportion and cumulative incidence of achieving donor chimerism. Additionally, anti-HNA status affected the time for neutrophil engraftment, with a slower rate of neutrophil engraftment and increased risk of secondary failure in patients with anti-HNA.

Prevention of acute graft-versus-host-disease by Withaferin a via suppression of AKT/mTOR pathway

Acute Graft versus Host Disease (aGVHD) is a frequent and serious complication in patients receiving allogeneic bone marrow transplantation (allo-BMT) and often requires rigorous prophylaxis. The current treatment regimens for aGVHD are associated with several side effects which necessitates the development of novel interventions that prevent aGVHD without precluding graft-versus-tumor effects. In the present study, we show that treatment of donor graft with plant steroidal lactone Withaferin A (WA) prior to transplantation markedly reduced aGVHD mediated damage in target organs without compromising the graft-versus.-tumor activity of the transplanted lymphocytes. WA abrogated post-transplant cytokine storm associated with allo-activation of donor lymphocytes. This was attributed to the ability of WA to inhibit early signaling events in T-cell activation including lymphoblast formation and activation of AKT/mTOR pathway. Mortality and morbidity related to allo-transplantation was significantly reduced in recipients of WA treated donor splenocytes compared to recipient of vehicle treated donor splenocytes. Further, WA treatment did not have any effect on reconstitution of lymphoid and myeloid lineages in recipients, resulting in stable and complete donor chimerism. In agreement with previous reports showing the effectiveness of WA in a mouse model of partial chimerism, our data further establishes that WA is able to attenuate aGVHD in an MHC-mismatched high dose chemo-conditioned murine model without compromising engraftment. This study provides compelling scientific basis for possible application of WA for prevention and treatment of aGVHD in patients receiving allo-BMT.

High Dose Hematopoietic Stem Cell Transplantation Leads to Rapid Hematopoietic and Microglia Recovery and Disease Correction in a Mouse Model of Hurler Syndrome

Background . Allogeneic hematopoietic stem cell transplant (HSCT) is a promising approach to halt disease progression and prevent or ameliorate neurological symptoms arising from select inherited metabolic disorders (IMDs). Donor-derived cells, including microglia, limit disease progression post-HSCT via production of normal enzyme in a process called cross-correction. A standard cell dose used in HSCT is sub-optimal, resulting in delayed hematopoietic recovery and slower correction of central nervous system (CNS) defects (Lund et al BBMT 2019). To address these limitations, we developed MGTA-456, a cell therapy that contains large numbers of CD34+ cells and has led to accelerated neutrophil recovery and 100% engraftment post-HSCT in patients with malignant and non-malignant diseases (Wagner et al Blood 2017; Orchard et al AAN 2019). We previously showed that MGTA-456 leads to faster hematopoietic and microglia recovery in the brains of NSG mice (Goncalves et al AAN 2019); however, the impact of cell dose on disease outcomes and mechanism of cross-correction are unknown. Here, we show that faster and greater hematopoietic and microglia recovery leads to rapid and complete resolution of disease endpoints in a mouse model of mucopolysaccharidosis I (Hurler syndrome) and that, mechanistically, donor engraftment in the brain is required for disease cross-correction. Results . To determine whether cell dose impacts microglial engraftment, CD45.2 mice were conditioned with a clinically-relevant, myeloablative dose of busulfan and transplanted with increasing doses of CD45.1 bone marrow cells, beginning with 0.3x106 cells/mouse (2x106 cells/kg) based on allometric scaling to model high dose cell therapies. A dose-dependent increase in microglia was observed as early as 1 week post-HSCT, where 10x106 cells led to a 26-fold higher number of donor microglia compared to 0.3x106 cells (p<0.01), an effect that was sustained through 16 weeks post-HSCT (p<0.001). Despite high donor chimerism in the periphery at all cell doses (75-99%), only partial chimerism was observed in the brain. At 16 weeks, donor microglia represented only 2% of microglia after transplant of 0.3x106 cells but this was increased to 35% of total microglia in the brain following transplant of 10x106 cells. These data indicate that while busulfan can facilitate a low level of microglia engraftment, this effect can be enhanced by transplant of high cell doses. To evaluate the impact of cell dose on disease outcomes, we transplanted a low (0.3x106) or high (10x106) cell dose of wild-type bone marrow cells into busulfan-conditioned Idua-/- mice, a model of Hurler syndrome. At 1 month post-HSCT, peripheral donor myeloid chimerism was >75% and >99% for 0.3x106 and 10x106 cells, respectively. In the brain, transplant of 10x106 cells led to significantly higher donor microglial engraftment versus 0.3x106 cells (Figure A). Notably, high cell dose resulted in significantly higher levels of IDUA enzyme in the brain (Figure B), reduced levels of β-hexosaminidase and glycosaminoglycan (GAG) substrate, and normalization of behavioral outcomes, including rotarod performance, to wild type levels (Figure C). In peripheral tissues, transplant of 10x106 cells, but not 0.3x106 cells, led to a reduction of GAGs to wild type levels as early as 1 week post-HSCT (p<0.01). To determine if donor engraftment in the brain is required for cross-correction, we transplanted 10x106 cells into mice conditioned with a myeloablative dose of treosulfan, which is not sufficient to condition the brain for microglia engraftment. Treosulfan conditioning, followed by high dose HSCT, led to >99% donor myeloid chimerism in the periphery but neither increased microglial levels nor corrected CNS defects (Figures A-C), suggesting that donor engraftment in the brain is required for disease modification. Long-term outcomes and impact on skeletal phenotype in this model will also be presented. Conclusions . We demonstrate that high dose HSCT leads to robust microglia engraftment in the brain and improved disease endpoints. These data suggest that strategies to increase cell dose, such as MGTA-456, may accelerate resolution of neurologic disease in patients with IMDs. Similar approaches, possibly coupled with gene modification technologies, could be used to improve microglial function in other neurodegenerative diseases where defective microglia have been implicated. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

All Hematopoietic Stem Cells Engraft in Submyeloablatively Irradiated Mice

Significant controversy exists regarding the impact of hematopoietic stroma damage by irradiation on the efficiency of engraftment of intravenously transplanted stem cells. It was previously demonstrated that in normal syngenic mice, all intravenously transplanted donor stem cells, present in the bone marrow, compete equally with those of the host. In this study, we comprehensively compared the blood cell production derived from transplanted donor stem cells with that from the host stem cells surviving various doses of submyeloablative irradiation. We compared the partial chimerism resulting from transplantation with theoretical estimates that assumed transplantation efficiencies ranging from 100% to 20%. The highest level of consensus between the experimental and the theoretical results was 100% for homing and engraftment (ie, the utilization of all transplanted stem cells). These results point to a very potent mechanism through which intravenously administered hematopoietic stem cells are captured from circulation, engraft in the hematopoietic tissue, and contribute to blood cell production in irradiated recipients. The damage done to hematopoietic stroma and to the trabecular bone by submyeloablative doses of ionizing radiation does not negatively affect the homing and engraftment mechanisms of intravenously transplanted hematopoietic progenitor and stem cells.

Relationship of Targeted Busulfan Exposure to Engraftment in Children Undergoing Myeloablative Matched Sibling Donor Transplantation for Sickle Cell Disease.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) for sickle cell disease (SCD) provides curative therapy but carries significant risk of transplant-related morbidity and mortality, graft failure, and disease recurrence. Past trials of myeloablative HSCT with busulfan in SCD patients showed a 10–13% rate of rejection and 8–9% rate of partial chimerism with busulfan steady-state concentration (Css) targeted to 400–600 ng/ml (area under curve (AUC) 575–877 mmol•min/L). In thalassemia and other diseases, total systemic exposure to busulfan is a critical determinant of engraftment yet is limited by risk of toxicity. Therapeutic drug monitoring (TDM) is used to measure variability in individual patient pharmacokinetics and allows dose adjustments to achieve desired level of busulfan exposure. A retrospective analysis of busulfan AUC in SCD patients during HSCT was performed to determine if higher systemic busulfan levels resulted in more effective engraftment or increased toxicities.METHODS: A retrospective review of busulfan pharmacokinetics, engraftment status, and clinical toxicities in a cohort of SCD patients who received MSD bone marrow grafts at a single pediatric institution was performed. All patients received a myeloablative preparative regimen with busulfan (initially dosed for total 14 mg/kg), cyclophosphamide (200 mg/kg), antithymocyte globulin (90 mg/kg), and graft versus host disease prophylaxis with methotrexate and cyclosporine. Anticonvulsant prophylaxis with phenytoin or with levitiracetam plus lorazepam was given during busulfan administration. Busulfan was administered every 6 hours for 16 doses. Busulfan AUC was determined for the first dose and adjustments were made on subsequent doses if needed. Total AUC was calculated as a weighted average of total doses. Target AUC varied widely over the study period; however, for the last 3 years, the intended AUC has been targeted to 900–1100 mmol•min/L (Css 618–753 ng/ ml). Busulfan-associated toxicities recorded were: hepatic veno-occlusive disease (VOD), interstitial pneumonitis (IP), and seizure during or 1 day following busulfan administration.RESULTS: Twenty-seven consecutive hemoglobin SS SCD patients received HSCT with MSD between December 1993 and August 2007. All patients transplanted since May 1996 (n=25) had busulfan TDM performed. Median age was 8.8 years (range 3.3–17.4 years). Event-free survival was 96% with a median follow-up time of 3.9 years. Engraftment occurred in all cases with no subsequent graft rejection. Full donor chimerism (>95% donor leukocytes) was seen in 21 (84%) patients, and high partial donor chimerism (50–95% donor) in 4 (16%) at >12 months post-HSCT. Busulfan was administered orally in 18 (72%) and intravenously in 7 (28%). Dose adjustments were made in 17 patients (8 increased, 9 decreased). Median dose increase was 16.3% (range 9.7–76.5%), and median decrease was 14.3% (range 4.2–57.1%). Five (20%) patients had dose alterations >20% of initial dose (2 increased, 3 decreased). Median total dose received was 13.9 mg/kg (range 10.8–24.0). Median AUC for first dose was 968 mmol•min/L (range 595–1379) and median total AUC was 992 mmol•min/L (range 780–1305). Busulfan-associated toxicities were observed in 9 (36%) patients: 8 with mild-moderate VOD, 1 with IP. No seizures occurred during busulfan administration. Patients with busulfan-associated toxicity had mean total AUC of 1044.7 mmol•min/L (range 821–1305) versus 962.4 mmol•min/L (range 780–1184) in those without toxicity (p=0.185). Patients with partial donor chimerism had lower total AUC (mean 861.5 mmol•min/L, range 780–932) compared to those with full donor chimerism (mean 1017 mmol•min/L, range 885–1305; p=0.0379) (Figure). Since the implementation of a target busulfan AUC range of 900–1100 mmol•min/L, all patients (n=10) achieved full donor chimerism.CONCLUSIONS: We conclude that higher busulfan levels are associated with decreased incidence of partial donor chimerism in SCD patients during myeloablative HSCT with MSD bone marrow grafts. A target AUC range of 900–1100 mmol•min/L, attainable with TDM and dose adjustment, provides effective engraftment without increased risk of busulfan-related toxicity. [Display omitted]

Chimerism and liver transplant tolerance

Chimerism and tolerance in a recipient of a deceased-donor liver transplant. Alexander SI, Smith N, Hu M, Verran D, Shun A, Dorney S, Smith A, Webster B, Shaw PJ, Lammi A, Stormon MO. Complete hematopoietic chimerism and tolerance of a liver allograft from a deceased male donor developed in a 9-year-old girl, with no evidence of graft-versus-host disease 17 months after transplantation. The tolerance was preceded by a period of severe hemolysis, reflecting partial chimerism that was refractory to standard therapies. The hemolysis resolved after the gradual withdrawal of all immunosuppressive therapy. [Abstract reproduced by permission of N Engl J Med 2008;358:369–374]

Use of alemtuzumab as graft-versus-host disease prophylaxis in myeloablative unrelated donor transplants

7045 Background: Graft-versus-host disease(GvHD) remains a frequent, morbid, and costly complication of allogeneic stem cell transplant (alloSCT) when using unrelated donors(URD). Optimal prophylaxis remains uncertain. With standard post-alloSCT tacrolimus and mini-mtx the incidence of acute GvHD (aGvHD) approaches 50–75%. Severe aGvHD occurs in about 30%, with mortality >80%. T-cell depletion is commonly employed but associated with high risk of disease relapse and graft rejection. Methods: We report observations from 43 pts who received Alemtuzumab pre-alloSCT for GvHD prevention with myeloablative conditioning and URD in a phase II study. The accrual of >30pts allowed us to demonstrate with significance (90% power; 5% 1-sided α) that Alemtuzumab is associated with less aGvHD in this setting. Eligible pts had hematologic malignancies and were scheduled for alloSCT on protocol. There were 2 cohorts of patients: 1–10(A) were treated with 5 days of Alemtuzumab (20mg/d); 11–43(B) were treated with 3. Cohort B was created due to frequency and severity of infections. 25/43 (58%) had >CR2 or refractory disease at the time of conditioning. CMV PCR was obtained weekly with any positive prompting therapy. Results: Pts were evaluated for engraftment, presence of GvHD before and after day 100, and DFS at day 100. 2 had primary engraftment failure. One had a sustained partial chimerism. 33/43 (76.7%) treated were alive and evaluable at day 100: 2 died due to progressive disease, 6 of severe infections, 1 had a probable PE and 1 TTP. Only 1/43 had severe aGvHD; 7/43 had grade 1–2 (16.2%). 13/43 had any chronic GvHD (30%), 7/43 of these were extensive (16.3%). Two deaths are attributed to chronic GvHD. At this report 11/43 are alive at a median f/u of 25 months. 12/43 (27.9%) died of progressive disease. CMV reactivation occurred in 17/43, and was a major factor in death in 5. 70% of cohort A, vs 42% of cohort B succumbed to infections. Conclusions: Alemtuzumab given prior to alloSCT from URD is effective in preventing severe acute and chronic GvHD and does not compromise engraftment rates. The frequency and severity of viral infections require aggressive monitoring and therapy. Disease control is not adversely affected. Consideration should be given to the lower dose use as GvHD prophylaxis. No significant financial relationships to disclose.

Chimerism and Tolerance in a Recipient of a Deceased-Donor Liver Transplant

Complete hematopoietic chimerism and tolerance of a liver allograft from a deceased male donor developed in a 9-year-old girl, with no evidence of graft-versus-host disease 17 months after transplantation. The tolerance was preceded by a period of severe hemolysis, reflecting partial chimerism that was refractory to standard therapies. The hemolysis resolved after the gradual withdrawal of all immunosuppressive therapy.

Transient Mixed Chimerism after Stem Cell Transplantation Can Induce Durable Molecular Complete Remissions in Chronic Myelogenous Leukemia.

Three patients with Ph1 (+), BCR/ABL+ chronic myelogenous leukemia were allografted, two with unrelated compatible placental blood cells and one from an HLA compatible sibling. The three patients engrafted successfully, achieved mixed chimerism and all cleared the BCR/ABL fusion transcript. Despíte the fact that the three patients lost the partial chimerism, they have remained in complete molecular remissions 7 months, fourteen months and five years after the allografts. It is possible that the iatrogenic induction of a transient chimerism and in turn of a transient graft versus leukemia effect, might have a role in the induction of the sustained molecular remission of these three individuals with CML despite the fact that the three patients lost the graft; however other possible explanations can be offered. A longer follow up of the patients is mandatory to further clarify these observations.

A Novel Triple Umbilical Cord Blood Transplant (UCBT) Strategy To Promote NK Cell Immunotherapy (Unit 1) with a Fully Ablative Preparative Regimen Followed by a Double UCBT in Patients with Refractory AML.

Treatment options for refractory AML are usually ineffective. Previously, we tested adoptive transfer of haploidentical peripheral blood (PB) derived NK cells without transplantation and demonstrated correlation between in vivo NK cell expansion and those who achieved a complete remission. This therapy is limited by:the inability to expand NK cells in most patients,prolonged neutropenia in some patients andinconsistent efficacy.UCB, in contrast to adult PB, is rich in NK precursors with CD34+/CD7−, CD34+/CD7+ and CD34−/CD7+ phenotypes. We hypothesized that UCB-derived NK cells may show better in vivo expansion than adult derived NK cells after cytoreduction. Therefore, we tested our triple UCBT strategy in patients with refractory relapsed AML who were <45 years old, without active infection and eligible for myeloablative conditioning. The UCB NK product (unit 1) was CD3 depleted (using a CliniMacs system) and activated with IL-2 (1000U/ml for 16–20 hours). The UCB-derived NK cells (matched at 3 HLA loci and KIR-ligand mismatched when possible) were infused on day -12 after conditioning with cyclophosphamide 120 mg/kg, fludarabine 125 mg/m2 and TBI 1320 cGy on days -19 to -13. Subcutaneous IL-2 (10 MU) was was given on days -12, -10, -8, -6, -4 and -2 to facilitate in vivo NK cell expansion. On day 0, two UCB units (≥4/6 match) were transplanted for hematopoietic rescue and followed by mycophenolate mofetil and cyclosporine for GVHD prophylaxis. Compared to pre-treatment levels, endogenous IL-15 was markedly increased after the preparative regimen at the time of the NK UCB unit. The NK UCB units contained both precursor and mature NK cells. Three product samples were cultured for 28 days in limiting dilution on a murine stromal feeder, demonstrating cloning frequencies of 1:5, 1:9 and 1:12 infused UCB cells giving rise to NK progeny. Two of the 3 patients had partial chimerism derived from the NK product on day -1. Unexpectedly, these same two patients demonstrated prompt neutrophil engraftment on days 3 and 7 after hematopoietic stem cell rescue. In both instances, chimerism was achieved from the NK product. Of the non-NK and non-T cells in the NK UCB units from these 2 patients there were 9.6% and 5.3% CD34+/CD7− cells. In the third patient, the NK UCB unit had only 2% CD34+/CD7− cells and they did not contribute to neutrophil engraftment which occurred on day 36 after UCBT. Of note, 3/3 tolerated the NK infusion without toxicity and were leukemia-free at the time of engraftment. Two remain alive (one died of TRM) with one relapse before day 100. These data suggest UCB NK cells may be administered safely and, despite CD3 depletion and IL-2 activation (ex vivo and in vivo), provide long term engraftment potential that may dominate over unmanipulated UCB infused subsequently. In summary, UCB is a rich source of NK precursors capable of in vivo expansion which are potentially better suited than adult NK cells for use in treatment of patients with refractory AML.

Pharmacokinetics of a Test Dose of Intravenous Busulfan Guide Dose Modifications to Achieve an Optimal Area Under the Curve of a Single Daily Dose of Intravenous Busulfan in Children Undergoing a Reduced-Intensity Conditioning Regimen with Hematopoietic Stem Cell Transplantation

We studied 30 pediatric patients with malignant (n = 16) or nonmalignant (n = 14) conditions. The preparative regimen consisted of fludarabine, intravenous (IV) busulfan (Bu) for 2 daily doses, and antithymocyte globulin before stem cell transplantation. A test dose of IV Bu (0.8 mg/kg), anticipated to target an area under the concentration-time curve (AUC) of 800 to 1200 μmol · min, was followed later by 2 daily doses adjusted according to the pharmacokinetics (PK) to target an AUC of 3200 to 4800 μmol · min. The median test dose AUC was 953 μmol · min (range, 439-1315 μmol · min). The median AUC of single daily doses was 3798 μmol · min (range, 1511-7254 μmol · min). PK-based dose modification was required in 20 patients: 12 were adjusted to a higher dose, and in 8 the dose was decreased. Nausea and vomiting were noted in 15 patients. No patient developed hepatic veno-occlusive disease or seizures. Full donor chimerism was attained in 20 patients (mean of 24.5 days), 3 achieved partial chimerism, 5 did not engraft, and in 2 it is too early to assess chimerism. Acute graft-versus-host disease developed in 11 patients, grades I to II developed in 10 patients, and grade III developed in 1. Four patients died of infection and 5 of progressive disease. Thus, PK of a test dose of IV Bu provided information to adjust subsequent daily doses of IV Bu: this resulted in a regimen that was feasible, safe, and convenient for administration to children.

Donor Lymphocyte Infusion Management and Impact on Transplant Outcome after Reduced Intensity Conditioning Allogeneic Transplantation.

Management of donor lymphocyte infusions (DLI) after reduced intensity conditioning regimen allogeneic hematopoietic stem cell transplantation (RICT) remains not clear and needs prospective studies. We performed a retrospective analysis on 47 patients (29 males and 18 females) who received 94 DLI among 96 patients who underwent RICT in our institution. Twenty-four patients received 1 DLI (D) [mean dose: 0.3x108 CD3 /Kg (0.01–1)] and 70 escalating doses (ED) [2 DLI (n=10), 3 DLI (n=9), 4 DLI (n=1), 5 DLI (n=1), 6 DLI (n=1) and 8 DLI (n=1)] from 0.1 to 5.6x108 CD3/Kg. The diagnosis pretransplant was acute leukemias (n=10) and myelodysplasia (n=3), chronic myeloid leukemia (n=2), Hodgkin (n=7) and non Hodgkin lymphomas (n=6), multiple myeloma (n=14) and solid tumours (n=5). Twenty patients have already been transplanted before RICT and 9 patients were in complete remission (CR), 19 in partial response (PR), 18 in evolutive disease (EDis). As hematopoietic stem cells, 22 patients received peripheral blood and 25 bone marrow and as conditioning 26 patients received busulfan, fludarabine and anti-thymocytes globulines (ATG), 13 patients TBI 2 grays associated to fludarabine (n=8) and 6 grays associated with cyclophosphamide (n=5), 5 patients received cyclophosphamide and ATG and 3 patients aracytine, idarubicine and fludarabine. After transplant and before any DLI, 21 developped acute GVHD (11 grade I, 8 grade II and 2 grade III). The indications and results after DLI are given in the Table 1.Only 4 patients received DLI for two different indications within time: 2 for relapse firstly and partial chimerism secondly and 2 according to protocol requirement firstly and for relapse secondly. After DLI, we noted 18 acute GVHD (7 grade I, 4 grade II, 6 grade III and 1 grade IV), 9 were resolutive after specific therapy and 18 patients developed chronic GVHD (13 limited and 5 extensive). Probability of overall survival at 2 years of patients who underwent RICT including DLI (n=47) was significantly better than for patients undergoing RICT without DLI (n=57) [43% (95%CI 30.5–60.4) vs 31.5 (95%CI 18.8–52.8) (p=0.01)] but there was no difference when we consider EFS [20.7% (95%CI 11.6–36.8) vs 21.6% (11–42.5) (p=0.49)]. We performed for patients receiving DLI a multivariate analysis stratified on diagnosis studying sex, age, status at transplant and mean dose of infused lymphocytes and we demonstrated that lymphocyte dose had a significant negative impact on EFS (HR= 1.06 95% CI 1.01–1.10) (p=0.01). To better understand the real place of DLI within allogeneic immunotherapy against malignancies, we need more details about the results of DLI in retrospective analysis but principally prospective studies in the future.TABLE 1Indication of DLINb of patientsNb of DLIDLI (D)DLI (ED)Dose x 10 exp8 CD3/KgResponse to DLIEvolutive Disease N = 34232.2610131.46EDis51.6230.66CR61.5240.45PRMixed Chimerism N = 761.5510.7Total Donor11100.7Mixed ChimerismProtocol N = 611100.1CR31.6210.34PR22120.52EDis

Reply to the letter by PJ Shaw and M Bleakley

On behalf of our co-authors, we would like to thank Drs Shaw and Bleakley for their comments on the subject of CD34 selection in hematopoietic stem cell transplantation (HSCT) for metabolic disorders. The primary aim of our study was to demonstrate that bone marrow can be processed with the CliniMACS device, and that the final product can be safely used to perform HSCT in children.1 Drs Shaw and Bleakley confirm the feasibility and reproducibility of the procedure in patients with hemato-oncological diseases. We would emphasize that the goals of HSCT in lysosomal diseases are not only survival but also stabilization or enhancement of the neuropsychological function stemming from repopulation of the recipient with metabolically normal donor marrow-derived cells. This includes replacement of non-neuronal cells in the central nervous system. Unlike patients with hemato-oncological diseases, partial chimerism should not necessarily be considered a failure, since there are many Hurler patients with stable partial chimeric engraftment, who are experiencing benefits including neuropsychological developmental trajectories that are comparable to those observed in patients with full chimerism. To date, 11 patients with differing lysosomal diseases have been transplanted at the Center in Monza, six of them with MPS-IH. Among the five survivors with MPS-IH, two patients achieved mixed chimerism and one patient experienced full donor chimerism after their first HSCT. Two patients were full donor chimera after the second HSCT. All patients, including those with partial chimerism, have been continuing to show significant improvements in their medical conditions and neurocognitive status. We agree that TCD, though effective in reducing the risks of GvHD, could add to the risk of graft failure related to the underlying disease; however, GVHD is correlated with a poorer neuropsychological outcome in these patients.2 Therefore, we will continue to follow our procedure that includes TCD for these patients. Drs Shaw and Bleakley also comment about possible alternative strategies (eg to 'cytoreduce' patients prior to transplant). To our knowledge, there are no studies to address this issue in the unique field of lysosomal diseases. No less toxic and less resource-intensive ways than a second unrelated HSCT for Hurler patients who experienced rejection have been available till now. Recently, laronidase was approved in the United States and European Union for use in MPS-I patients. It is possible that enzyme replacement therapy with this agent may enhance both the safety and efficacy of HSCT in MPS-I patients when both therapies are used. This hypothesis is being investigated through specific pilot studies.

Correction of a large animal model of type I Glanzmann's thrombasthenia by nonmyeloablative bone marrow transplantation

Objective The purpose of this study was to determine if nonmyeloablative bone marrow transplantation would induce stable hematopoietic chimerism that would correct the bleeding diathesis associated with type I Glanzmann's thrombasthenia (GT). Methods Three young dogs (less than 12 weeks of age) with GT were transplanted with DLA-matched bone marrow from littermates. Recipients received a sublethal dose (200 cGy) of total-body irradiation (TBI) prior to infusion with bone marrow (1–4×10 8 cells/kg). Recipient dogs were immunosuppressed with cyclosporine (15 mg/kg) and mycophenolate mofetil (10 mg/kg). Chimerism was determined by quantitation of donor microsatellite repeat polymorphisms in peripheral blood DNA and by flow cytometry to detect the presence of glycoproteins IIb and IIIa on platelets. Platelet function was assessed by a clot retraction test. Results One dog died one week posttransplant due to hemorrhage. Another dog died four weeks posttransplant from an unrecognized congenital heart defect and complications due to canine distemper virus infection. At the time of death, microsatellite analysis indicated 35 to 50% chimerism. Flow cytometry showed 20% of circulating platelets positive for glycoproteins IIb and IIIa. The third dog is alive and doing well approximately two years posttransplant. Hematopoietic chimerism has been sustained at 35 to 60% with approximately 30% of the platelets positive for glycoproteins IIb and IIIa. Platelet function is normal based on clot retraction. The animal does not have clinical signs of bleeding. Conclusions These observations suggest that GT and perhaps other severe inherited platelet disorders can be corrected using nonmyeloablative bone marrow transplantation to establish partial chimerism with normal platelets in the platelet compartment.

Allogeneic chimerism with low-dose irradiation, antigen presensitization, and costimulator blockade in H-2 mismatched mice

AbstractWe have previously shown that the keys to high-level nontoxic chimerism in syngeneic models are stem cell toxic, nonmyelotoxic host treatment as provided by 100-cGy whole-body irradiation and relatively high levels of marrow stem cells. This approach was unsuccessful in H-2 mismatched B6.SJL to BALB/c marrow transplants, but with tolerization, stable multilineage chimerism was obtained. Ten million B6.SJL spleen cells were infused intravenously into BALB/c hosts on day −10 and (MR-1) anti-CD40 ligand monoclonal antibody (mAb) injected intraperitoneally at varying levels on days −10, −7, −3, 0, and +3 and the BALB/c mice irradiated (100 cGy) and infused with 40 million B6.SJL/H-2 mismatched marrow cells on day 0. Stable multilineage chimerism at levels between 30% to 40% was achieved in the great majority of mice at 1.6 mg anti-CD40 ligand mAb per injection out to 64 weeks after transplantation, without graft-versus-host disease. The transplanted mice were also tolerant of donor B6.SJL, but not third-party CBA/J skin grafts at 8 to 9 and 39 to 43 weeks after marrow transplantation. These data provide a unique model for obtaining stable partial chimerism in H-2 mismatched mice, which can be applied to various clinical diseases of man such as sickle cell anemia, thalassemia, and autoimmune disorders.

Engraftment following low dose total body irradiation (Tbi) (200 Cgy)/fludarabine (Flu) Nonmyeloablative conditioning for allogeneic stem cell transplants (alloSCT)

We recently demonstrated that it is possible to achieve fast and stable engraftment with minimal organ toxicity using Flu/Bu/ATG conditioning for alloSCT. We have now evaluated the possibility of achieving similar results with Flu and low dose TBI (200cGy). Eleven patients underwent alloSCT from fully matched sibling donors following the TBI/Flu conditioning protocol. Six had AML (resist.dis./relapse—5, CR-1), one ALL (relapse), one NHL (resist.dis.) and 3 had genetic disease. Eight were male and 3 female, of median age 38 (11–63) years. The patients were transplanted with G-CSF mobilized PBSC (1–2 phereses). The graft consisted of 13.2 (3.1–18.5) × 108/kg MNC, 9.3 (1.7–13.5) × 106/kg CD34+ cells and 2.8 (1.9–4.8) × 108/kg CD3+ T cells. In the 9 patients who engrafted the ANC never dropped below 0.5 × 109/L and the plt. counts were never below 20 × 109/L. Eight of the patients had transient chimerism but lost the graft and died one month post alloSCT from his basic disease (AML). Two patients who did not engraft (NHL-1, AML-1) had persistent disease, and died. None of the patients suffered from mucositis, liver, renal, pulmonary or other organ toxicity. Grade I-II transient acute GVHD of the skin was observed in 6 patients. Donor lymphocyte infusion was administered to 2 patients because of relapse and partial chimerism, respectively. The relapsing patient developed grade IV acute GVHD and died. Seven of the 11 patients (acute leukemia-4, genetic disease-3) are alive with no GVHD and no toxicity, 5 (4-10) months post alloSCT. We conclude that low dose TBI (200cGy) may be substituted for Bu and ATG as it induces stable engraftment with mixed or complete chimerism with minimal organ toxicity following alloSCT from matched sibling donors.

Flow cytometric monitoring of red blood cell chimerism after bone marrow transplantation.

Chimerism after bone marrow transplantation (BMT) was investigated by flow cytometry analysis of red blood cells (RBCs) and of reticulocytes using a series of selected monoclonal or polyclonal antibodies directed against ABO, Rhesus, Kell, Duffy or MNSs antigens. The method allows the routine detection of less than 0.1% of positive cells in artificial mixed field populations. Blood samples from 135 patients undergoing BMT were investigated around days 15, 30, 45, 60, 90, 180, and then every 6 months after transplantation. Characteristic patterns showing expression of donor red blood cell antigens (expansion markers) and concomitant decrease of recipient specific antigens (depletion markers) within days 16-20 were observed for 125 successfully engrafted patients. Distinct patterns were obtained in 10 patients. A delay in engraftment was evidenced in four patients by the absence of chimerism during the first 6 months without any sign of relapse. Re-appearance of recipient RBCs and reticulocytes was observed in five patients; it was consistent with relapse that was later confirmed by clinical, haematological and cytogenetic studies. Finally, a stable and partial chimerism with 20% of RBCs expressing a marker from the recipient was observed in one patient without any sign of relapse. The reported investigation demonstrated that flow cytometry of RBCs and reticulocytes represents a powerful method to efficiently monitor bone marrow transplanted patients on a long-term basis.

Reconstitution ratio is critical for alloreactive T cell deletion and skin graft survival in mixed bone marrow chimeras.

Although allogeneic bone marrow (BM) transplantation is an effective way to induce donor-specific tolerance, its clinical application is hampered because of the risk associated with vigorous myeloablative pretransplant conditioning. One approach to overcome this problem is to establish a lower chimeric state by mild myeloablation. It is not clear, however, whether there is a threshold in the extent of chimerism that is required for tolerance induction. In this study, we establish a mixed BM chimera system to examine the correlation between the reconstitution ratio of BM chimerism, donor-reactive T cell deletion, and skin graft acceptance using I-E alpha transgenic C57BL/6 mice as the BM and skin graft donors and Ly5 congenic C57BL/6 mice as the recipients. In this system, the class II MHC molecule I-E was the transplantation Ag, and the extent of I-E-reactive T cell deletion was determined by flow cytometry using a mAb specific for the V beta 11 TCR. The degree of BM chimerism was measured by examining the expression of donor-derived Ly5.2 and host-derived Ly5.1 on peripheral blood cells. Transplantation of I-E+ transgenic donor BM cells resulted in deletion of V beta 11+CD4+ T cells in recipient's PBL, and the extent of deletion was proportional to the degree of chimerism. When mice of different degrees of chimerism were tested for skin graft survival, we found that recipient mice with > 30% chimerism could accept skin grafts from I-E+ donor mice, whereas those with < 10% chimerism showed prolonged but not permanent graft survival. These findings revealed the sequence of events for induction of tolerance. First, the degree of BM chimerism determines the number of I-E+ cells in the thymus, which then elicits negative selection of I-E-reactive T cells in a form of clonal deletion. The extent of T cell deletion ultimately determines the mode of tolerance. These data provide experimental evidence for the potential use of partial chimerism by bone marrow transplantation for the induction of donor-specific tolerance in clinical settings.

Partial Chimerism After T-Cell-Depleted Allogeneic Bone Marrow Transplantation in Leukemic HLA-Matched Patients: A Cytogenetic Documentation

We evaluated serially by cytogenetics the blood and marrow chimerism of 38 leukemic recipients of HLA-matched bone marrow transplants (BMT) who were prepared by high doses of alkylating agents and fractionated total-body irradiation (2.2 Gy X 5). Donor or host mitoses were identified by examination of sex chromosomes in 32 patients or by evaluation of the polymorphism of other chromosomes after specific banding in six patients. Twenty-four patients were recipients of untreated BMT, and 14 were recipients of T-cell-depleted BMT. In the 24 patients who received untreated BMT, all showed successful engraftment, and only three had a transient mixed chimera. In the 14 recipients of T-cell-depleted BMTs, four rejected their grafts, and seven had mixed chimeras; these mixed chimeras were more frequent in blood lymphocytes than in marrow cells and could be detected up to 26 months after BMT. This high frequency of partial chimerism after T-cell-depleted BMT by comparison with a control group suggests that the donor's T cells play an important role in the eradication of host residual hematopoiesis after allogeneic BMT.

Partial chimerism after T-cell-depleted allogeneic bone marrow transplantation in leukemic HLA-matched patients: a cytogenetic documentation

We evaluated serially by cytogenetics the blood and marrow chimerism of 38 leukemic recipients of HLA-matched bone marrow transplants (BMT) who were prepared by high doses of alkylating agents and fractionated total- body irradiation (2.2 Gy X 5). Donor or host mitoses were identified by examination of sex chromosomes in 32 patients or by evaluation of the polymorphism of other chromosomes after specific banding in six patients. Twenty-four patients were recipients of untreated BMT, and 14 were recipients of T-cell-depleted BMT. In the 24 patients who received untreated BMT, all showed successful engraftment, and only three had a transient mixed chimera. In the 14 recipients of T-cell-depleted BMTs, four rejected their grafts, and seven had mixed chimeras; these mixed chimeras were more frequent in blood lymphocytes than in marrow cells and could be detected up to 26 months after BMT. This high frequency of partial chimerism after T-cell-depleted BMT by comparison with a control group suggests that the donor's T cells play an important role in the eradication of host residual hematopoiesis after allogeneic BMT.