Neonatal Transplantation Research Articles

Spectratyping analysis of the islet-reactive T cell repertoire in diabetic NOD Igμnull mice after polyclonal B cell reconstitution

BackgroundNon Obese Diabetic mice lacking B cells (NOD.Igμnull mice) do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age.MethodsWe have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igμnull mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes.ResultsWe found that B cell reconstitution of NOD.Igμnull mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11) and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20). These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igμnull mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19+ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens) analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro.ConclusionsDiabetes in NOD.Igμnull mice appears to be caused by a polyclonal repertoire of T cell accumulation in pancreas without much lymphoid organ involvement and is dependent on the help by B cells.

T lymphocytes derived from human cord blood provide effective antitumor immunotherapy against a human tumor

BackgroundAlthough the graft-versus-tumor (GVT) effect of donor-derived T cells after allogeneic hematopoietic stem cell transplantation has been used as an effective adoptive immunotherapy, the antitumor effects of cord blood (CB) transplantation have not been well studied.MethodsWe established the animal model by transplantation of CB mononuclear cells and/or tumor cells into NOD/SCID mice. The presence of CB derived T cells in NOD/SCID mice or tumor tissues were determined by flow cytometric and immunohistochemical analysis. The anti-tumor effects of CB derived T cells against tumor was determined by tumor size and weight, and by the cytotoxicity assay and ELISPOT assay of T cells.ResultsWe found dramatic tumor remission following transfer of CB mononuclear cells into NOD/SCID mice with human cervical tumors with a high infiltration of CD3+ T cells in tumors. NOD/SCID mice that receive neonatal CB transplants have reconstituted T cells with significant antitumor effects against human cervical and lung tumors, with a high infiltration of CD3+ T cells showing dramatic induction of apoptotic cell death. We also confirmed that T cells showed tumor specific antigen cytotoxicity in vitro. In adoptive transfer of CD3+ T cells into mice with pre-established tumors, we observed much higher antitumor effects of HPV-specific T cells by ELISPOT assays.ConclusionsOur results show that CB derived T lymphocytes will be useful for novel immunotherapeutic candidate cells for therapy of several tumors in clinic.

Origins of Neonatal Heart Transplantation: An Historical Perspective

Clinical heart transplantation was first accomplished by Christiaan Barnard in 1967. Infant heart transplantation was first attempted by Adrian Kantrowitz just 3 days later in New York. Sixteen years lapsed before neonatal transplantation was again attempted, first in London and then in Loma Linda, California in the summer and fall of 1984, respectively. The latter infant became known as "Baby Fae." Neither infant survived a month, but both produced an important impact through media exposure on the public consciousness. The idea of infant organ donation slowly developed traction. The first successful neonatal heart transplant occurred in Loma Linda on November 15, 1985. That recipient, born with hypoplastic left heart syndrome, is now 25 years old. Thereafter, the incidence of infant and pediatric heart transplantation gradually increased. Today, 350 to 450 transplants are reported annually to the Registry of the International Society for Heart and Lung Transplantation, 25% of which are in young infants. Norwood's staged-reconstruction for complex univentricular heart disease has markedly reduced the need for primary transplantation in very early life. Still, many potential young infant recipients are best treated in no other way.

Transplantation Tolerance Induced in Humans at the Fetal or the Neonatal Stage

Patients transplanted with HLA-mismatched stem cells from fetal livers develop transplantation tolerance to donor antigens. Engraftment needs no conditioning regimen prior to transplantation in neonates with severe combined immunodeficiency disease or in human fetal patients having not yet developed any immune maturity, especially T-cell differentiation. The chimeric patients have donor-derived T lymphocytes which progressively demonstrate positive interactions with other host cells. They also can be shown to be tolerant toward both host and donor antigens. The latter tolerance relies upon clonal deletion from the T-cell repertoire, and it results from the contact between thymocytes of donor origin and dendritic cells or macrophages also deriving from donor stem cells. The former tolerance does not imply clonal deletion of T-cells with host reactivity. Numerous T-cells recognizing the allogeneic, host-type antigens are identified in these patients, but these cells are anergized, following interaction with epithelial cells of the host thymus. Induction of transplantation tolerance at the fetal stage requires minimal engraftment only; in the future it will be possible to further amplify the clinical benefit, using additional cell transplants after birth.

Effect of the Initial Hormonal Status of Recipient Animals on Morphological State and Endocrine Function of the Ovarian Tissue Transplantant

The investigation aim was comparative analysis morpho-functional characteris- tics of allografts of mature ovarian tissue and neonatal ovary depending on initial hormonal status of recipient animals. Rats' mature ovarian tissue and neonatal ovary were transplanted under renal capsule simultaneously with ovariectomy and in 2 months later. Data analysis indi- cated that development of ovarian tissue transplant was determined by its maturity and initial hormonal status of recipient. Mature ovarian tissue transplants preserve morphology and endo- crine function to the 60th day observation in recipient animals with initial hormonal status that corresponds to the norm. Whereas neonatal ovary transplants are developed to mature stage during its transplantation to recipients with hormonal status that corresponds to the ovariecto- mied animals, that is, low concentration of sex hormones and increased level of follicle- stimulating hormone.

Parental Decision Making Regarding Treatment of Hypoplastic Left Heart Syndrome

Parents of children diagnosed with a complex congenital heart anomaly such as hypoplastic left heart syndrome (HLHS) are faced with the challenge of choosing the best treatment option for their children. Parents must decide among options of compassionate care, palliative reconstructive surgery, or neonatal cardiac transplantation, and additionally with antenatal diagnosis must decide regarding continuation of pregnancy. With improved survival rates yet persistent morbidities following the Norwood surgical approach for HLHS, controversy remains whether parents should be counseled on all treatment options as evidenced by research that articulates the varied opinions of health care providers. The parents' perspective is less well researched. In this study, interview data from parents who chose the Norwood-Sano surgical option were analyzed to provide insight into the various treatment options they were given following diagnosis of HLHS in their children and how they came to choose the surgical palliation option.

Lacto-N-fucopentaose III, a Pentasaccharide, Prolongs Heart Transplant Survival

Lacto-N-fucopentaose III (LNFPIII) is a pentasaccharide containing the Lewis(x) trisaccharide that is found on schistosome eggs and in breast milk. LNFPIII conjugates suppress host immune responses and have therapeutic efficacy in mouse models of psoriasis and type 1 diabetes. We used nonvascularized neonatal ear-heart transplantation and heterotopic vascularized heart transplantation models to evaluate immunosuppressive effects of LNFPIII and subsequently analyzed the mechanism. We found that administration of LNFPIII conjugates prolonged median graft survival by 80% when 1-day-old DBA/2 hearts were transplanted into ears of B6 mice. A similar graft prolongation was observed in a fully vascularized heterotopic heart transplantation model (DBA/2 into B6), No prolongation was observed with carrier protein (human serum albumin [HSA] or dextran) alone. We found increased programmed death ligand 1 (PD-L1) expression on F4/80 macrophages, CD4+ T cells, and CD11b+ CD11c+ (myeloid) dendritic cells, and increased arginase1 and Ym1 expression, typical of alternatively activated macrophages, in the draining (cervical) lymph node cells. We found accumulation of Foxp3+ regulatory T cells (Tregs) in the lymph nodes draining donor hearts, suggesting a possible role of Treg induction in graft prolongation. Anti-PD-L1 antibody treatment abrogated LNFPIII-mediated the graft survival benefit and Treg accumulation. LNFPIII-treated macrophages had increased PD-L1 expression and significantly prolonged DBA/2 allograft survival when injected intraperitoneally into B6 recipient mice. LNFPIII prolongs fully allogeneic graft survival in both vascularized and nonvascularized allograft transplantation models. The mechanism of graft prolongation seems to involve both alternatively activated PDL-1 macrophages and recruitment of Foxp3+ Treg cells.

Nonablative neonatal bone marrow transplantation rapidly reverses severe murine osteopetrosis despite low-level engraftment and lack of selective expansion of the osteoclastic lineage

Infantile malignant osteopetrosis (IMO) is caused by lack of functional osteoclasts leading to skeletal abnormalities, blindness owing to compression of the optic nerves, bone marrow (BM) failure, and early death. In most patients, TCIRG1, a proton pump subunit essential for bone resorption, is mutated. oc/oc mice represent a model for IMO owing to a deletion in Tcirg1 and die around 4 weeks of age. To determine if hematopoietic stem cell transplantation without prior conditioning can reverse osteopetrosis, neonatal mice were transplanted intravenously with lineage-depleted BM cells. More than 85% of oc/oc mice transplanted with 5 × 10(6) cells survived long term with an engraftment of 3% to 5% in peripheral blood (PB). At 3 weeks, engraftment in the BM was 1% to 2%, but the cellularity had increased 60-fold compared with untreated oc/oc mice, and RANKL and macrophage colony-stimulating factor (M-CSF) expression in the BM was normalized. Histopathology and micro-computed tomography revealed almost complete reversal of osteopetrosis after 4 weeks. In vitro studies showed that bone resorption by osteoclasts from transplanted oc/oc mice was 14% of transplanted controls, and immunofluorescence microscopy revealed that resorption was mainly associated with osteoclasts of donor origin. Lineage analysis of BM, PB, and spleen did not provide any evidence for selective recruitment of cells to the osteoclastic lineage. The vision also was preserved in transplanted oc/oc mice, as determined by a visual tracking drum test. In summary, nonablative neonatal transplantation leading to engraftment of only a small fraction of normal cells rapidly reverses severe osteopetrosis in the oc/oc mouse model.

Вплив вихідного гормонального статусу тварини- реципієнта на морфологічний стан та ендокринну функцію алотрансплантата оваріальної тканини

The aim of investigation was a comparative analysis of morphofunctional characteristics of allografts of mature ovarian tissue and neonatal ovary depending on initial hormonal status of recipient animals. Rats' mature ovarian tissue and neonatal ovary were transplanted under renal capsule simultaneously with ovariectomy and 2 months later. Data analysis indicated that the development of ovarian tissue transplant was determined by its maturity and initial hormonal status of recipient. Mature ovarian tissue transplants preserve morphology and endocrine function to the 60th day after transplantation to the recipient animals with the initial hormonal status that corresponds to the norm. Neonatal ovary transplants are developed to the mature stage following transplantation to recipients with hormonal status that corresponded to the ovariectomized animals, that is, low concentration of sex hormones and an increased level of follicle-stimulating hormone.

Diffuse liver diseases in neonatal and pediatric liver transplant candidates: a pictorial essay

A wide spectrum of common and uncommon diffuse liver diseases affecting neonatal and pediatric liver transplant candidates is presented and analyzed using 16 and 64 multi-detector row helical CT (MDCT) and 1.5 T MRI fast imaging. Correlation of imaging findings and explanted liver or histology is illustrated in representative cases. Associated uncommon congenital anomalies are shown. In conclusion, in pediatric liver transplant candidates, 16-MDCT and 1.5 T fast MRI are useful for diagnosis and staging of liver disease, as well as for the evaluation of associated congenital anomalies.

Does ABO-incompatible and ABO-compatible neonatal heart transplant have equivalent survival?

A best evidence topic (BET) in cardiac surgery was written according to a structured protocol. The question addressed was whether ABO-incompatible (ABO-I) heart transplant recipients have a similar survival rate as an ABO-compatible (ABO-C) transplant in the pediatric population <1 year of age. Altogether more than 112 papers were found using the reported search, of which 10 represented the best evidence to answer the clinical question. Generally, ABO-I transplantation has been associated with the neonatal population because of the relative immaturity of the immune system for the first year of life. In the BET the search-term 'pediatric' was used as a method to ensure retrieval all relevant papers. However, the vast majority of the patients reviewed were <1 year of age with specific techniques undertaken to modulate preoperative, intraoperative and postoperative isohemagglutinin titer levels with rejection monitoring. Therefore, the BET conclusions should not be applied to the pediatric group as a broad age classification. Two large series are of particular interest. Patel et al. reviewed all primary heart transplantation recipients < or =1 year of age in the United Network for Organ Sharing/Organ Procurement and Transplantation Network (UNOS/OPTN) registry (ABO-I=35 vs. ABO-C=556). There was no difference in 30-day mortality (ABO-I=5.9% vs. ABO-C=8.8%; P=0.55); one-year mortality (ABO-I=16.6% vs. ABO-C=14.7%; P=0.77); graft rejection (ABO-I=1 vs. ABO-C=0); and graft failure (ABO-I=24% vs. ABO-C=24%; P>0.99). Three-year Kaplan-Meier survival was 70% (P=0.85). Propensity score adjusted analysis did not implicate ABO-I as a predictor of mortality [hazard ratio (HR)=3.6, confidence interval (CI): 0.2-49.0; P=0.33]. The ABO-I group demonstrated an increased need for pacemaker (ABO-I=3.1% vs. ABO-C=0.4%; P=0.03) and higher stroke rate (ABO-I=12.9% vs. ABO-C=1.3%; P<0.0001). Dipchand et al. published the results of the Toronto cohort from 1992 to 2006 (ABO-I=16 vs. ABO-C=38). The median age at transplantation (ABO-I=88 days vs. ABO-C=84 days; P=0.82) and the number of neonatal transplant recipients (ABO-I=17% vs. ABO-C=22%; P=0.59) was similar. The freedom from post-transplantation death or retransplantation was equivalent at one year (ABO-I=77% vs. ABO-C=84%) and seven years (ABO-I=74% vs. ABO-C=74%; P=0.87). No significant difference was observed for the five-year freedom from: rejection (ABO-I=60% vs. ABO-C=45%; P=0.41); renal dysfunction (ABO-I=67% vs. ABO-C=72%; P=0.70); allograft vasculopathy (ABO-I=87% vs. ABO-C=78%; P=0.45); and post-transplantation lymphoproliferative disorder (PTLD) (ABO-I=87% vs. ABO-C=86%; P=0.93). We conclude that ABO-I transplantation is comparable to ABO-C transplantation, with several retrospective papers concluding there is no difference in mortality, morbidity or graft failure in the pediatric population <1 year of age.

Hepatic failure, neonatal hemochromatosis and porto-pulmonary hypertension in a newborn with trisomy 21 - a case report

Liver failure in neonates is a rare but often fatal disease. Trisomy 21 is not usually associated with significant infantile liver disease. If present, hepatic dysfunction in an infant with Trisomy 21 is likely to be attributed to transient myeloproliferative disorder with hepatic infiltration by hematopoietic elements and may be associated with secondary hemosiderosis. A less commonly recognized cause of liver failure in neonates with Trisomy 21 is neonatal hemochromatosis (NH); this association has been reported in nine cases of Trisomy 21 in literature. NH is a rare, severe liver disease of intra-uterine onset that is characterized by neonatal liver failure and hepatic and extrahepatic iron accumulation that spares the reticuloendothelial system. NH is the most frequently recognized cause of liver failure in neonates and the commonest indication for neonatal liver transplantation. Although porto-pulmonary hypertension (PPH) has been reported as a complication of liver failure in adults and older children, this has not been reported in neonates with liver failure of any etiology. This is probably due to the rarity of liver failure in newborns, delayed diagnosis and high mortality. The importance of recognizing PPH is that it is reversible with liver transplantation but at the same time increases the risk of post-operative mortality. Therefore, early diagnosis of PPH is critical so that early intervention can improve the chances of successful liver transplantation. We report for the first time the association of liver failure with porto-pulmonary hypertension secondary to NH in an infant with Trisomy 21.

Focal liver diseases in neonatal and pediatric liver transplant candidates: a pictorial essay

The aim of this review is to present the wide spectrum of common and uncommon focal liver diseases affecting neonatal and pediatric liver transplant candidates, analyzed using ultrasonography (US), 16- or 64-multidetector row helical CT (MDCT) and 1.5-T magnetic resonance (MR) fast imaging. Correlation of imaging findings and explanted liver or histology is illustrated in representative cases. Associated uncommon congenital anomalies are shown.

B Cells Are Dispensable for Neonatal Transplant Tolerance Induction

Prior studies have demonstrated that neonatal B cells possess unique immunoregulatory properties. Specifically, neonatal B-1 cells produce interleukin (IL)-10 in response to toll-like receptor stimulation; this modulates innate and adaptive alloimmune responses. Here, we examine whether this process plays a critical role in neonatal transplant tolerance induction. We used a murine model of neonatal transplant tolerance induction, whereby female C57BL/6 mice injected with male spleen cells 3 days after birth acquire the ability to accept a male skin transplant in adulthood. We investigated the role of B cells in this model by using mice with targeted genetic B-cell deficiencies (including muMT, JH-/-, and XID mice). In addition, we examined the role of IL-10 in this model using IL-10-/- mice. Transplant tolerance induction was neither dependent on B cells nor on IL-10. Despite the role of neonatal B cells in suppressing innate and adaptive alloimmune responses, B cells are dispensable for neonatal transplant tolerance induction in this experimental model.

Hemodynamic failure as an indication to urgent liver transplantation in infants with giant hepatic hemangiomas or vascular malformations – Report of four cases

The aim of this study was to present acute hemodynamic failure as a rare indication for liver transplantation in neonates and infants with liver hemangiomatosis. We report four patients aged one to six months with giant liver hemangiomas, with huge arterio-venous shunting within these malformations. In three, many skin hemangiomas were found. All children developed right ventricular failure. In two, a trial of pharmacological reduction was attempted with corticosteroids and cyclophosphamide. In one patient, the arterio-venous fistulas were embolized without any improvement in hemodynamic status. Two children underwent rescue hepatic artery surgical ligation, which did not prevent heart and then multiorgan failure including liver failure. After unsuccessful conventional therapy, all infants were considered for urgent liver transplantation; in three cases, it was performed with a living-related donor, and in one case with a deceased donor. All patients are alive and well with the follow-up between nine and 37 months after transplantation. Liver transplantation should be considered as a rescue treatment in children with hepatic vascular malformations leading to hemodynamic insufficiency when conventional therapy is unsuccessful and multiorgan failure develops.

Intracranial Calcification after Cord Blood Neonatal Transplantation for Krabbe Disease

Infantile-onset Krabbe disease results from a deficiency of the lysosomal enzyme galactocerebrosidase and leads to death from profound central and peripheral demyelination. Neonatal hematopoietic cell transplantation may result in near-normal cognitive development and partial rescue of gross motor development. The long-term course of the disorder for treated patients seems to involve slowly progressive neurological impairment. We describe the detailed 3-year outcomes of this experimental procedure using umbilical cord blood in a prenatally-diagnosed newborn with Krabbe disease. Substantial perivascular calcifications and atrophy of the white matter developed in the first year post-transplantation. Despite persistent neuroradiological and electrophysiological evidence of leukodystrophy, at age 3 years she has had only mildly impaired non-motor development and moderately impaired motor skills. The cause of these severe white matter changes may have been due to ongoing Krabbe disease or to effects of the chemotherapy regimen or to an interaction of these factors. Extended long-term follow-up of children neonatally transplanted for Krabbe disease is needed before the full utility and limitations of neonatal transplantation can be determined.

Neonatal bone marrow transplantation without prior conditioning rapidly reverses osteopetrosis in oc/oc mice despite only minimal donor cell engraftment

Neonatal bone marrow transplantation without prior conditioning rapidly reverses osteopetrosis in oc/oc mice despite only minimal donor cell engraftment

Low-dose busulphan conditioning and neonatal stem cell transplantation preserves vision and restores hematopoiesis in severe murine osteopetrosis

Infantile malignant osteopetrosis is a fatal disease caused by lack of functional osteoclasts. In most of patients, TCIRG1, encoding a subunit of a proton pump essential for bone resorption, is mutated. Osteopetrosis leads to bone marrow failure and blindness due to optic nerve compression. Oc/oc mice have a deletion in Tcirg1 and die around 3 to 4 weeks, but can be rescued by neonatal stem cell transplantation (SCT) after irradiation conditioning. However, as irradiation of neonatal mice results in retinal degeneration, we wanted to investigate whether conditioning with busulphan prior to SCT can lead to preservation of vision and reversal of osteopetrosis in the oc/oc mouse model. Pregnant dams were conditioned with busulphan and their litters transplanted with 1 x 10(6) normal lineage-depleted bone marrow cells intravenously or intraperitoneally. Mice were followed in terms of survival and engraftment level, as well as with peripheral blood lineage analysis, bone and eye histopathology and a visual-tracking drum test to assess vision. Busulphan at 15 mg/kg was toxic to oc/oc mice. However, six of seven oc/oc mice conditioned with busulphan 7.5 mg/kg survived past the normal lifespan with 10% engraftment, correction of the skeletal phenotype, and normalization of peripheral blood lineages. Busulphan, in contrast to irradiation, did not have adverse effects on the retina as determined by histopathology, and 8 weeks after transplantation control and oc/oc mice retained their vision. Low-dose busulphan conditioning and neonatal SCT leads to prolonged survival of oc/oc mice, reverses osteopetrosis and prevents blindness even at low engraftment levels.

Neonatal Bone Marrow Transplantation Following Ex Vivo Gene Transfer and in Vivo Chemoselection for Hereditary Disorders

Transplantation of genetically engineered hematopoietic stem cells (HSC) holds promise for treatment of genetic and other disorders; however, several obstacles restrict the clinical application of this approach. These include leukemogenesis resulting from insertional mutagenesis, the lack of a competitive advantage for transplanted genetically engineered autologous HSCs, and the requirement for the construction and clinical approval of a gene transfer vehicle specific to each disorder. Allogenic transplantation approaches are further limited by a shortage of compatible donors, the toxicity of preparative regimens, immunosuppression, and graft versus host disease (GVHD).We have established a neonatal transplantation model to develop strategies to overcome these hurdles to HSC transplantation and gene therapy. We have uniquely combined allogenic transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of tolerance induction mechanisms to neo-antigens, and allogenic stem cell engraftment during immune ontogeny. In this model, bone marrow-derived HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O6-methylguanine-methyltransferase (MGMTP140K) and GFP (MAG vector). The MGMTP140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. This enables enrichment of donor cells at the stem cell level by in vivo chemoselection. Furthermore, in vivo administration of BG/BCNU may potentially deplete allo-reactive cells of donor and host origin, reducing the need for toxic ablative or immunosuppressive treatment.The in vivo selection strategy was first tested in neonates in the absence of MHC barriers. Syngenic whole bone marrow (WBM) was transduced with MAG and 5 × 105 cells were transplanted. Five weeks after transplantation and prior to chemoselection, 0.5% of cells expressed GFP in peripheral blood. Following two cycles of in vivo selection we observed 39.5% expression of GFP in peripheral blood. To assess the engraftment of fully MHC-mismatched HSC in neonates without in vivo selection, 107 B10.Br (H-2k) WBM was transplanted into C57BL/6 (H-2b) neonates using a non-myleoablative regimen. This resulted in 98% engraftment in adults without development of GVHD. To evaluate in vivo selection in an allogenic setting, 2 × 106 MAG transduced WBM from BALB/c (H-2d) adult mice were transplanted into C57BL/6 × BALB/c F1 (H-2b/d) neonatal mice. Despite having a graft versus host MHC disparity, we observed an increase from 4.7% (5 weeks after transplant) to 37.4% GFP expression following two cycles of in vivo chemoselection without evidence of GVHD.We have demonstrated successful engraftment and enrichment of both syngenic and allogenic HSC expressing MGMTP140K following neonatal transplantation and in vivo selection. Stable engraftment was achieved without myeloablation or post-transplant immunosuppression. No evidence of GVHD was observed after full MHC-mismatched transplantation. These results hold promise for the development of non-myeloablative allogenic transplant approaches using this in vivo selection strategy.

MHC-Matching at 5 Loci Results in Higher Levels of Engraftment of Allogeneic Cord Blood (CB) Hematopoietic Stem Cells (HSC) Following Neonatal Transplantation.

CB represents a rich source of transplantable HSC and its use in matched unrelated clinical HSC transplants (HSCT) is rapidly increasing, due largely to its potential for decreased GVHD compared to BM. Although CB use in adult recipients is increasing, the majority of CB transplants (Tx) have thus far been done in children, due to limited numbers of cells in single CB units. Over the past 20 years, we have used the human/sheep model to study human HSC from a variety of sources including CB. Although this model represents a biologically and possibly clinically relevant model of human hematopoiesis, some critical aspects of HSCT involving allogeneic donor cells may not be revealed by this xenogeneic model. Therefore, we developed the necessary methods to obtain and bank functionally viable CB from sheep at normal delivery in volumes sufficient to rescue lethally myeloablated neonatal sheep recipients, thus making it possible to begin performing allogeneic sheep-to-sheep CB HSCT. To this end, CB collected at birth (volume: 62±14ml; MNC: 5.2±0.6 x108; n=74) was used to Tx 3 week-old newborn lambs, with either a single or multiple (male plus Female) CB unit. Recipients were myeloablated with high dose chemotherapy (HDC) consisting of Etoposide plus Cytoxan, and Group I (n=3) was transplanted with 26±0.75×107 MNC/kg from a single cord while Group II (n=3) received the same number of cells pooled from 2 cords; Group III remained as a non-transplanted control. Prednisone was given in all transplanted animals until 5 weeks post-Tx. All the animals in Group III and one recipient in Group I died at about 23 days post-HDC. Engraftment of neutrophils and platelets occurred at approximately 35 days and 45 days respectively for Group I and 29 and 40 for Group II. Karyotypic analysis of donor cell engraftment in Group II revealed that when CB were pooled both were able to engraft but there was a gradual decline in the contribution to blood production of one of the transplanted cords with time post-Tx. Mild chronic GVHD occurred in all of the transplanted animals after withdrawal of the prednisone at day 210 after Tx. All the symptoms subsided after re-administration of prednisone. We therefore felt it was necessary for HSCT to develop reagents that would allow us to employ donor/recipient matched CB cells. After nearly 3 years of work, we now have the necessary MHC class I and II reagents for this purpose. Employing an in utero MHC typing strategy using amniotic fluid cells, we found that transplantation of CB HSC from donors matched at 5 loci into 60 day-old fetal sheep resulted in significantly higher donor cell activity than fetuses transplanted with HSC matched at 2 loci (3.% vs 14%). Long-term observation of these animals revealed that while the donor cell level remained higher in this group than in the group transplanted with HSC matched at 2 loci, by about 1 year post-HSCT, a decline in donor cell activity occurred. We have now performed a head-to-head comparison of human CB as a source of donor HSC for in utero HSCT (IU-HSCT) with matched and unmatched sheep CB. Our results thus far suggest that matched CB represents the more optimal source of HSC for IU-HSCT, and that the development of more specific antibodies that allow better matching may result in clinically useful levels of donor cell engraftment that will persist long-term without the previously observed decrease in donor cell activity. The similarity of our observations in this model to that of humans in kinetics of engraftment and GVHD development in HDC-treated sheep transplanted with allogeneic CB cells suggests that this model may be useful in developing clinically relevant strategies for the use of allogeneic CB in post-natal and IU-HSCT.