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The absolute neutrophil count (ANC) and platelet count per microliter (log10 scale) from dogs receiving unrelated cord blood transplantation (CBT) on day 0 after 9.2 Gy TBI. (A) Double-unit CBT group. (B) Single-unit CBT group. Each line/symbol represents blood counts from individual dogs. ET, euthanized because of meeting defined criteria of poor condition after transplantation (for details, see Tables 3 and 4).
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Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5 x 10(7)/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome t...
Citations
... 28,29 Moreover, a graft-facilitating role for the non-engrafting unit has been suggested from findings in experimental models in mice and dogs. 30,31 In addition to this, it was shown that alloreactive CD4 + T cells directed against mismatched HLA class II molecules were capable of directly eliminating leukemic cells in adult patients. 32 As many unit-recipient HLA class II mismatches were present, we hypothesize that HLA class II-specific CD4 + T cells of the ultimately surviving unit elicit an immediate and targeted alloreactive immune response towards mismatched HLA class II molecules of the non-engrafting unit in the absence of ATG. ...
Double umbilical cord blood transplantation is increasingly applied in the treatment of adult patients with high-risk hematological malignancies and has been associated with improved engraftment as compared to single unit cord blood transplantation. The mechanism of improved engraftment is however still incompletely understood as only one unit survives. In this multicenter phase II study we evaluated engraftment, early chimerism, recovery of different cell lineages and transplant outcome in 53 patients receiving a double cord blood transplantation preceded by a reduced intensity conditioning regimen. Primary graft failure occurred in 1 patient. Engraftment was observed in 92% of patients with a median time to neutrophil recovery of 36 days (range, 15-102). Ultimate single donor chimerism was established in 94% of patients. Unit predominance occurred by day 11 post transplant and early CD4+ T-cell chimerism predicted for unit survival. Total nucleated cell viability was also associated with unit survival. With a median follow up of 35 months (range, 10-51), the cumulative incidence of relapse and non-relapse mortality at 2 years were 39% and 19%, respectively. Progression-free survival and overall survival at 2 years were 42% (95% confidence interval , 28-56) and 57% (95% confidence interval, 43-70), respectively. Double umbilical cord blood transplantation preceded by a reduced intensity conditioning regimen using cyclophosphamide/fludarabine/TBI 4 Gy results in a high engraftment rate with low non-relapse mortality. Moreover, prediction of unit survival by early CD4+ lymphocyte chimerism might suggest a role for CD4+ lymphocyte mediated unit-versus-unit alloreactivity. www.trialregister.nl NTR1573.
... Furthermore, in our study, there was no indication that DSA influenced unit dominance. Unit dominance is likely related to CB hematopoietic potential [25] and immune-mediated graft-versus-graft reactions [10,22,[26][27][28]. ...
The impact of human leukocyte antigen (HLA) donor-specific antibodies (DSA) upon cord blood (CB) engraftment is controversial. We evaluated the influence of pre-existing HLA-antibodies (HLA-Abs) on engraftment in 82 double-unit CB recipients (median age 48 years) transplanted for hematologic malignancies. Of 28 patients (34%) with HLA-Abs, 12 had DSA (median MFI 5,255, range 1,057-9,453). DSA patients had acute leukemia (n = 11) or myelodysplasia (n = 1) and all received either high-dose or reduced intensity (but myeloablative) conditioning. After myeloablative CBT (n = 67), sustained donor engraftment was observed in 95% without HLA-Abs (median 23 days), 100% with non-specific HLA-Abs (median 23 days), and 92% with DSA (median 31 days, p = 0.48). Of 6 patients with HLA-Abs to one unit, 3 engrafted with that unit and 3 with the other. Of 6 patients with HLA-Abs against both units, one had graft failure despite being 100% donor, and 5 engrafted with one unit. Successful donor engraftment is possible in patients with DSA after myeloablative double-unit CBT. Our data suggest potential deleterious effects of DSA can be abrogated in patients with hematologic malignancies.
... To this end, a recipient dog was given 9.2 Gy of total body irradiation (TBI), a myeloablative dose, and then infused with autologous day 14 SR1/UNC0638 expanded HSPCs, 1.7 3 10 7 total nucleated cells per kilogram. To evaluate reconstitution, absolute neutrophil counts (ANCs) were monitored daily until complete hematopoietic recovery for 84 d post-transplantation. Remarkably, transplantation of SR1/UNC0638 expanded cells led to full recovery of the recipient (Fig. 6E; Georges et al. 2010). These results are consistent with the notion that SR1/UNC0638 expansion at the very least sustains canine HSC activity during 14-d expansion of HSPCs. ...
G9a and GLP are conserved protein methyltransferases that play key roles during mammalian development through mono- and dimethylation of histone H3 Lys 9 (H3K9me1/2), modifications associated with transcriptional repression. During embryogenesis, large H3K9me2 chromatin territories arise that have been proposed to reinforce lineage choice by affecting high-order chromatin structure. Here we report that in adult human hematopoietic stem and progenitor cells (HSPCs), H3K9me2 chromatin territories are absent in primitive cells and are formed de novo during lineage commitment. In committed HSPCs, G9a/GLP activity nucleates H3K9me2 marks at CpG islands and other genomic sites within genic regions, which then spread across most genic regions during differentiation. Immunofluorescence assays revealed the emergence of H3K9me2 nuclear speckles in committed HSPCs, consistent with progressive marking. Moreover, gene expression analysis indicated that G9a/GLP activity suppresses promiscuous transcription of lineage-affiliated genes and certain gene clusters, suggestive of regulation of HSPC chromatin structure. Remarkably, HSPCs continuously treated with UNC0638, a G9a/GLP small molecular inhibitor, better retain stem cell-like phenotypes and function during in vitro expansion. These results suggest that G9a/GLP activity promotes progressive H3K9me2 patterning during HSPC lineage specification and that its inhibition delays HSPC lineage commitment. They also inform clinical manipulation of donor-derived HSPCs.
... Ex vivo expansion of 1 of the 2 CBUs could potentially enhance engraftment by increasing the number of HSCs and hematopoietic progenitor cells, and the unexpanded unit provides a ready source of HSCs, should the exvivo expansion fail. To date, many preclinical studies have been performed, but none has truly mimicked the conditions under which these DCBT ex vivo expansion CB clinical trials are being conducted [4][5][6]. ...
Ex vivo expansion of cord blood (CB) hematopoietic stem cells and cotransplantation of 2 CB units (CBUs) could enhance the applicability of CB transplantation in adult patients. We report an immunodeficient mouse model for cotransplantation of ex vivo expanded and unexpanded human CB, showing enhanced CB engraftment and provide proof of concept for this transplantation strategy as a means of overcoming the limiting cell numbers in each CBU. CBUs were expanded in serum-free medium supplemented with stem cell factor, Flt-3 ligand, thrombopoietin, and insulin growth factor binding protein-2 together with mesenchymal stromal cell coculture. Unexpanded and expanded CB cells were cotransplanted by tail vein injection into 45 sublethally irradiated nonobese diabetic SCID-IL2γ(-/-) (NSG) mice. Submandibular bleeding was performed monthly, and mice were sacrificed 4 months after transplantation to analyze for human hematopoietic engraftment. Expansion of non-CD34(+) selected CB cells yielded 40-fold expansion of CD34(+) cells and 3.1-fold expansion of hematopoietic stem cells based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts exhibited 4.30% human cell repopulation, compared with 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis (P = .07). Ex vivo expanded grafts with lower initiating cell doses also showed equivalent engraftment to unexpanded grafts with higher cell dose (8.0% versus 7.9%; P = .93). In conclusion, ex vivo expansion resulted in enhanced CB engraftment despite eventual rejection by the unexpanded CBU.
... Reducing apoptosis within the MNC and CD34 + population of umbilical UCB units was considered worthy of study as delayed neutrophil engraftment is commonly observed following UCB transplantation, which is in part due to limited cell number in the small volume collected coupled with the need for cryopreservation(133,134). Importantly, our results suggest that strategies to reduce cold-induced apoptosis in UCB may allow for more widespread application of UCB, since apoptotic cells may be interfering with the success of HSCT as demonstrated in the second aspect of this thesis. Other methods to overcome the limited cell quantities available in UCB units have been developed, including simultaneous transplantation of two UCB units that are sufficiently matched for their HLA alleles(135,136), ex vivo expansion of CD34 + progenitors(137)(138)(139) and intrabone marrow transplantation(140,141). Even so, reducing cold-induced apoptosis will further minimize apoptotic cells that could interfere with engraftment, and greater understandings of the specific mechanisms responsible for cold-induced apoptosis are needed to focus future research efforts to optimize cryopreservation procedures.The results of the second aspect of this thesis suggest that reinfusion of PBSC grafts containing increased levels of apoptotic cells is associated with more prolonged delay of neutrophil recovery after autologous HSCT. ...
Although double umbilical cord blood transplantation (dUCBT) in adult patients may be associated with less graft failure compared with single UCBT, hematopoietic recovery generally originates from a single cord blood unit (CBU). CBU predominance is still incompletely understood. We recently showed that blood CD4+ T-cell numbers rapidly increase after dUCBT, and early CD4+ T-cell chimerism predicts for graft predominance. Given the frequent HLA class II allele mismatches between CBUs in dUCBT, we hypothesized that alloreactive HLA class II-specific CD4+ T cells from the "winning" CBU may contribute to rejection of the "loser" CBU. We evaluated whether CD4+ T cells originating from the predominant (PD)-CBU would recognize HLA class II allele mismatches, expressed by the nonengrafting (NE)-CBU. Alloreactive effector CD4+ T cells toward 1 or more mismatched HLA class II alleles of the NE-CBU were detected in 11 of 11 patients, with reactivity toward 29 of 33 (88%) tested mismatches, and the strongest reactivity toward DR and DQ alleles early after dUCBT. Mismatched HLA class II allele-specific CD4+ T cells recognized primary leukemic cells when the mismatched HLA class II allele was shared between NE-CBU and patient. Our results suggest that cytotoxicity exerted by CD4+ T cells from the PD-CBU drives the rapid rejection of the NE-CBU, whose alloreactive effect might also contribute to graft-versus-leukemia.
BACKGROUND: Double-cord-blood transplantation (DCBT) in patients is typically accompanied by predominance of a single unit. The causative mechanism, however, is unknown. Identifying the dynamics of mixed donor chimerism in general and in specific subpopulations may help to resolve this question. We conducted studies in a mouse model to develop a new analytic method using anti-human HLA Class I allele–specific monoclonal antibodies (HLA-MoAbs) in flow cytometry.
STUDY DESIGN AND METHODS: Single-cord-blood transplantation or DCBT from HLA-mismatched donors was performed in NOD/SCID mice. Bone marrow (BM) and peripheral blood were collected from 3 to 20 weeks after transplantation. Donor chimerism was determined quantitatively within human platelets (hPLTs), human CD45+ (hCD45+) cells, and human myeloid and lymphocyte subsets by flow cytometry.
RESULTS: Both cord donors stably engrafted in NOD/SCID. The sensitivity to detect chimerism measured with all HLA-MoAbs was 1% (>10 cells/µL). In mouse BM, the percentage of human cells measured with hCD45+ versus HLA-MoAbs correlated excellently (r = 0.999). Donor origin could be defined with HLA-MoAbs for nearly all (>93.6%) human cells in mouse peripheral blood and BM in all lineages. Chimerism of hPLTs in peripheral blood correlated well with hCD45+ cells in BM enabling frequent measurement of chimerism from early after transplantation onward.
CONCLUSION: This approach using HLA-MoAbs enables longitudinal analysis of double-mixed human chimeric populations despite low absolute concentrations of human hematopoietic cell subsets in peripheral blood and BM in mice. Lacking reactivity with mouse cells, the HLA-MoAbs are suitable for use in other mouse models and in humans to identify the mechanisms involved in DCBT.
This review summarizes the current status of double-unit cord blood transplantation (CBT) to improve engraftment, reduce transplant-related mortality, and improve disease-free survival.
Transplantation of cord blood provides a potentially curative therapy for many patients without a suitably human leukocyte antigen-matched related or unrelated donor. Single-unit CBT outcomes have been compromised, however, in adults and larger children by limited cell dose. The introduction of double-unit CBT has improved engraftment and transplant-related mortality in adult patients transplanted for hematologic malignancies, with recent data also suggesting a protection against relapse. These improved outcomes are seen despite only a single unit being responsible for sustained donor hematopoiesis in nearly all patients. The study of double-unit CBT provides unique insights into transplant biology, with emerging data suggesting unit dominance is related to unit viability and unit-versus-unit immune interactions. Multiple unit CBT further serves as a platform to test novel graft manipulations.
The development of double-unit CBT now allows the majority of patients, regardless of size or racial/ethnic background, access to transplant therapy. Ongoing investigation will serve to further improve outcomes and expand the role of CBT in the future.
