In Utero Hematopoietic Cell Transplantation Research Articles

Fetal allotransplant recipients are resistant to graft-versus-host disease

In utero hematopoietic cell transplantation (IUHCT) is an experimental treatment for congenital hemoglobinopathies, including Sickle cell disease and thalassemias. One of the principal advantages of IUHCT is the predisposition of the developing fetus toward immunologic tolerance. This allows for engraftment across immune barriers without immunosuppression and, potentially, decreased susceptibility to graft-versus-host disease (GVHD). We demonstrate fetal resistance to GVHD following T cell-replete allogeneic hematopoietic cell transplantation compared with the neonate. We show that this resistance is associated with elevated fetal serum interleukin-10 conducive to the induction of regulatory T cells (Tregs). Finally, we demonstrate that the adoptive transfer of Tregs from IUHCT recipients to neonates uniformly prevents GVHD, recapitulating the predisposition to tolerance observed after fetal allotransplantation. These findings demonstrate fetal resistance to GVHD following hematopoietic cell transplantation and elucidate Tregs as important contributors.

Sustained Engraftment in Fanconi Anemia (FA) Mice Using in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction FA is the most common inherited bone marrow failure syndrome caused by a mutation in one of 23 genes involved in DNA repair; the disease manifests as hematologic abnormalities by age seven with near uniform bone marrow failure in adult patients. The gold standard therapy is bone marrow transplantation (BMT), which portends best outcomes when performed at a young age from Human Leukocyte Antigen (HLA)-matched sibling donors. However, due to the underlying pathophysiology of the disease, FA patients are especially sensitive to the chemoradiation conditioning regimens used with BMT, leading to high short-term morbidity and an increased long-term cancer risk. Due to these challenges of conventional BMT in FA, IUHCT is an attractive non-myeloablative and non-immunosuppressive option capable of inducing donor-specific tolerance when in utero testing has identified pathogenic FA mutations. Here we analyzed whether non-conditioned IUHCT confers fetal engraftment as a potential therapy in the FA mouse disease model. Methods Fanconi anemia type C homozygous mice (Fancc-/-) underwent IUHCT on gestation day 14 (E14) via vitelline vein injections of 1x107 T-cell depleted bone marrow cells from green fluorescent protein (GFP)-expressing congenic donor mice. Littermate wild-type mice served as controls. Donor cell engraftment in peripheral blood and bone marrow was assessed by flow cytometry. To confirm long-term donor HSC engraftment, we performed primary and secondary transplantation of an equivalent number of BM mononuclear cells from either Fancc -/- or wild-type mice that had undergone IUHCT. Statistical analysis was performed via Welch's t-test. P values <0.05 were considered statistically significant; for graphical purposes ***p<0.001; **p<0.002; *p<0.033; nonsignificant, ns. Results Fancc-/- homozygous mice had significantly higher donor GFP+ cell engraftment than wild-type controls at one, three, and six months post-transplantation (Figure 1). This higher engraftment was maintained across cell types in peripheral blood including CD3+ T cells (0.7% vs 3.4%), B220+ B cells (1.2% vs 6.9%), GR1+ granulocytes (1.7% vs 10.6%), and CD11B+ myeloid cells (1.9% vs 11.5%). Analysis of the bone marrow of these mice at 6 months post-transplant similarly showed a trend toward increased donor cell engraftment of progenitor cell populations including LSK, HSPC, LTHSC, STHSC, MPP2, MPP3, MPP4, CLP, CMP, GMP, and MEP cells in Fancc -/- recipients compared to wild-type mice. At all timepoints after primary and secondary transplants, recipient mice engrafted with BM mononuclear cells from prenatally transplanted Fancc -/- mice had higher peripheral blood GFP+ chimerism than those engrafted with BM mononuclear cells from prenatally transplanted wild-type mice (Figure 2). Conclusion Fancc-/- homozygous mice have higher sustained donor cell engraftment compared with wild-type littermates, indicating successful engraftment and possible early selection for wild-type donor cells in this preclinical model. These initial studies support the potential of IUHCT as a therapy for FA and highlight the need for additional studies to identify approaches to enhance donor cell engraftment to more clinically relevant levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Experimental and clinical progress of in utero hematopoietic cell transplantation therapy for congenital disorders.

In utero hematopoietic cell transplantation (IUHCT) is considered a potentially efficient therapeutic approach with relatively few side effects, compared to adult hematopoietic cell transplantation, for various hematological genetic disorders. The principle of IUHCT has been extensively studied in rodent models and in some large animals with close evolutionary similarities to human beings. However, IUHCT has only been used to rebuild human T cell immunity in certain patients with inherent immunodeficiencies. This review will first summarize the animal models utilized for IUHCT investigations and describe the associated outcomes. Recent advances and potential barriers for successful IUHCT are discussed, followed by possible strategies to overcome these barriers experimentally. Lastly, we will outline the progress made towards utilizing IUHCT to treat inherent disorders for patients, list out associated limitations and propose feasible means to promote the efficacy of IUHCT clinically.

Regulatory T Cells Prevent Donor Cell Rejection in a Mouse Model of Delayed Gestational Age in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor-specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). The window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges and exclude from treatment all fetuses evaluated after 14 weeks gestation. We have previously demonstrated that IUHCT results in the peripheral induction of traditional and non-traditional regulatory T cells that contribute to the suppression of GVHD (Riley et al. ASH 2016 Abstract #4540). We hypothesized that regulatory T cells also contribute to DST and, furthermore, may be used to expand the window of central tolerance induction to a later point in gestation.Methods: A mouse model of delayed gestational age IUHCT was developed in which 9.7x10^6 C57/Bl6 (B6) GFP T cell-depleted (TCD) bone marrow (BM) cells were injected intravenously into day of life 0 (P0) naive Balb/c pups via the facial vein. The ability of IUHCT-induced regulatory T cells to prevent donor cell rejection was assessed by injecting 9.7x10^6 B6 GFP TCD BM cells plus 5x10^6 CD4+ splenocytes harvested from 2-4 week old chimeric mice. Fetuses injected with 9.7x10^6 B6 GFP TCD BM cells at gestational day 14 (E14) served as positive controls for tolerance and engraftment. Peripheral blood (PB) was analyzed at 4, 8, 12, and 24 weeks of age for donor cell chimerism (%CD45+H2kb+GFP+/CD45+H2kd+) and for engraftment (defined as chimerism >1% at 4 weeks). Weight gain and histology of the skin, liver, lungs, and small bowel were analyzed to confirm the absence of GVHD. PB was analyzed at 6 months of age to confirm multilineage engraftment. An in vivo mixed lymphocyte reaction (MLR) was performed at 6 months of age to confirm long-term DST. B6-sensitized Balb/c mice served as positive controls for alloreactivity. Comparison of continuous data was performed using Student's t test. Binary data were compared using Fischer's exact test. All tests were two-sided with an alpha level set at 0.05 for significance.Results: Our model of delayed gestational age IUHCT was associated with donor cell rejection in 52 of 56 (92.9%) pups injected with TCD BM only. Co-injection of CD4+ chimeric splenocytes significantly improved engraftment (100% vs. 7.1%, p<0.001) and chimerism (7.56% vs. 0.46% at 4 weeks, p=0.003) compared to TCD BM alone. No difference in engraftment (100% vs. 90%, p=0.501) or chimerism (7.56% vs. 7.66% at 4 weeks, p=0.961) was observed compared to fetuses injected with TCD BM at E14 (Figures 1A and 1B). These trends persisted at subsequent time points. Pups injected with TCD BM plus CD4+ chimeric splenocytes displayed normal weight gain and no histologic evidence of GVHD. Survival to adulthood was 100%. Donor cells of all hematopoietic lineages were present in the PB at 6 months of age. On in vivo MLR, pups injected with TCD BM plus CD4+ chimeric splenocytes displayed significantly reduced T cell proliferation compared to B6-sensitized Balb/c mice and P0 pups injected with TCD BM only. T cell proliferation did not differ significantly from fetuses injected with TCD BM at E14 (Figure 1C).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and requires injection at an early gestational age to avoid donor cell rejection by the maturing fetal immune system. This study demonstrates an important role for regulatory T cell populations in the induction of DST following IUHCT. The adoptive transfer of regulatory T cells from a chimeric mouse to a naïve P0 pup inhibits donor cell rejection in our model of delayed gestational age IUHCT, allowing central tolerance induction and long-term engraftment to occur despite the increased maturity of the recipient immune system. These findings represent proof-of-principle that augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a later point in gestation, decreasing the technical difficulty of the procedure and allowing fetuses evaluated after 14 weeks to be eligible for treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Utero Stem Cell Transplantation in Patients with Alpha Thalassemia Major: Interim Results of a Phase 1 Clinical Trial

Introduction: In utero hematopoietic cell transplantation (IUHCT) has the potential to treat patients with blood disorders by taking advantage of bidirectional maternal-fetal tolerance during pregnancy. Although engraftment has only been seen in immunodeficient patients in the past, recent preclinical studies indicate that a higher dose of maternal cells should be given intravascularly to maximize engraftment in other settings. We hypothesized that: 1) fetuses will tolerate a maternal IUHCT without conditioning and develop low engraftment with sustained tolerance to maternal alloantigens; and 2) tolerant patients could receive a postnatal booster maternal transplant with minimal immunoablative conditioning and thereby achieve a cure. To test the first of these hypotheses in a controlled setting, we have initiated a phase 1 clinical trial of IUHCT in fetuses with alpha thalassemia major (ATM), who already require a fetal intervention (in utero transfusions (IUT)) to survive the pregnancy. We present interim results from the first two patients. Methods: After obtaining IND approval, we initiated the clinical trial in 2017 (NCT02986698). Fetuses with ATM between 18-26 weeks' gestation without other major anomalies were included. To date, we have evaluated 19 patients for possible inclusion: 15 patients terminated the pregnancy, 2 patients were outside the inclusion window and underwent IUT alone, and 2 have been transplanted and completed the 1 year follow up. For IUHCT, 300 ml of maternal bone marrow was harvested, CD34+ cells were selected and transplanted fresh into the fetus via the umbilical vein, along with a standard IUT. IUTs were repeated every 3 weeks until delivery. Maternal cell chimerism was detected using qRT-PCR for non-inherited maternal HLA-DR alleles on sorted cell populations in cord blood and after birth. Mixed lymphocyte reactions (MLRs) were performed by culturing CFSE-labeled host CD4 and CD8 responder cells with maternal (or third-party) derived antigen presenting cells (APC; direct pathway) or self APC/maternal lysate (indirect pathway). Results: Both fetuses were diagnosed with severe hydrops on ultrasound and received an initial IUT prior to the IUHCT. Patient 1 was transplanted at 23 weeks with 1x 108CD34+ cells/kg + 1 x 107T cells/kg and Patient 2 was transplanted at 25 weeks with 5x 107CD34+ cells/kg + 5 x 106T cells/kg; both received 4 more IUTs until birth. Prenatal hydrops resolved in both and neonatal hospitalizations were 16 and 13 days, respectively. Maternal chimerism was detected in both patients, but levels were higher and persisted throughout the year only in patient 1 (Fig 1A). We detected 70-fold higher levels of erythrocyte precursors (Fig 1B) but normal levels of CD34+ cells in the cord blood of patients compared to healthy controls, highlighting a potential barrier to engraftment in a disease with prenatal hypoxia. Patient 1 demonstrated sustained tolerance to maternal antigens by MLR throughout the year, whereas patient 2 showed reactivity against maternal antigens (Fig 1C). HLA antibodies were negative in both. Based on the finding of donor-specific tolerance in patient 1, we offered a booster transplant with maternal cells but the parents declined. Both patients are undergoing monthly transfusions and have had normal development and Bayley neurologic testing at 1 year. There were no unanticipated safety events in either mother/child pair. Conclusions: These results demonstrate the safety of IUHCT in fetuses with ATM and the potential of fetal transplantation in the second trimester to sustain tolerance to maternal cells after birth. Low engraftment levels are predicted in the setting of host hyperproliferation and lack of space-making conditioning, confirming the need for a two-step strategy (IUHCT and postnatal booster transplant) for a definitive cure. The earlier transplantation in patient 1 enabled a higher dose of cells and may explain the improved outcome observed. Implementation of earlier screening in at-risk patient groups could allow earlier diagnosis and enrollment in the future. Although parents of fetuses with ATM are commonly counseled for pregnancy termination due to concern for poor outcomes, our results indicate that fetal therapy with IUTs can result in survival to term and excellent quality of life. This strategy of maternal IUHCT could be employed to treat fetuses with other hematologic disorders in the future. Figure 1 Disclosures MacKenzie: Acrigen: Membership on an entity's Board of Directors or advisory committees; Ultragenyx: Research Funding. Vichinsky:Global Blood Therapeutics: Consultancy; Agios: Research Funding; Pfizer: Research Funding.

Donor cell engineering with GSK3 inhibitor–loaded nanoparticles enhances engraftment after in utero transplantation

AbstractHost cell competition is a major barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Here we describe a cell-engineering strategy using glycogen synthase kinase-3 (GSK3) inhibitor–loaded nanoparticles conjugated to the surface of donor hematopoietic cells to enhance their proliferation kinetics and ability to compete against their fetal host equivalents. With this approach, we achieved remarkable levels of stable, long-term hematopoietic engraftment for up to 24 weeks post-IUHCT. We also show that the salutary effects of the nanoparticle-released GSK3 inhibitor are specific to donor progenitor/stem cells and achieved by a pseudoautocrine mechanism. These results establish that IUHCT of hematopoietic cells decorated with GSK3 inhibitor–loaded nanoparticles can produce therapeutic levels of long-term engraftment and could therefore allow single-step prenatal treatment of congenital hematological disorders.

Simple Approach to Increase Donor Hematopoietic Stem Cell Dose and Improve Engraftment in the Murine Model of Allogeneic In Utero Hematopoietic Cell Transplantation.

The rationale for in utero hematopoietic cell transplantation (IUHCT) rests on exploitation of normal events during hematopoietic and immunologic ontogeny to allow allogeneic hematopoietic engraftment without myeloablative conditioning. Host hematopoietic competition is among the primary barriers to engraftment in IUHCT. In the murine model this can be partially overcome by delivery of larger donor cell doses, but volume is limiting. Enrichment of donor hematopoietic stem cells (HSCs) would seem to offer a more efficient approach, but such enriched populations have engrafted poorly in existing models of IUHCT. To increase HSC dose while maintaining the presence of accessory cells, we used a less stringent enrichment protocol of single-step lineage depleted cells alone (lin-) or in combination with whole donor bone marrow mononuclear cells. Our results confirm that increasing doses of HSCs in combination with bone marrow accessory cells can dramatically improve engraftment after IUHCT. This represents a practical and clinically applicable strategy to maximize the engraftment potential of the donor graft without risk of treatment-associated toxicity.

Preclinical Canine Model of Graft-versus-Host Disease after In Utero Hematopoietic Cell Transplantation

In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allogeneic engraftment and associated donor-specific tolerance without the need for toxic conditioning, as we have previously demonstrated in the murine and canine models. This strategy holds great promise in the treatment of many hematopoietic disorders, including the hemoglobinopathies. Graft-versus-host disease (GVHD) represents the greatest theoretical risk of IUHCT and has never been characterized in the context of IUHCT. We recently described a preclinical canine model of IUHCT, allowing further study of the technique and its complications. We aimed to establish a threshold T cell dose for IUHCT-induced GVHD in the haploidentical canine model and to define the GVHD phenotype. Using a range of T cell concentrations within the donor inoculum, we were able to characterize the phenotype of IUHCT-induced GVHD and establish a clear threshold for its induction between 3% and 5% graft CD3+ cell content. Given the complete absence of GVHD at CD3 doses of 1% to 3% and the excellent engraftment with the lowest dose, there is a safe therapeutic index for a clinical trial of IUHCT.

Acceptability of In Utero Hematopoietic Cell Transplantation for Sickle Cell Disease.

In utero hematopoietic cell transplantation (IUHCT) has curative potential for sickle cell disease (SCD) but carries a risk of fetal demise. We assessed the conditions under which parents of children with SCD and young adults with SCD would consider IUHCT in a future pregnancy, given a 5% fixed risk of fetal demise. Participants were randomized to consider a hypothetical cure rate (20%, 40%, or 70%). Subsequently, cure rate was either increased or decreased depending on the previous answer to reveal the lowest acceptable rate. Participants also completed the Pediatric Research Participation Questionnaire (PRPQ) and an omission scale. Overall, 74 of 79 (94%) participants were willing to consider IUHCT, and 52 (66%) participants accepted IUHCT at a cure rate of 40%, the estimated rate of therapeutic mixed chimerism. Participants with higher scores on the PRPQ perceived benefits scale were more likely to participate at lower cure rates (OR 1.08, p=0.007) and participants with a greater degree of omission bias were less likely to participate at lower cure rates (OR 0.83, p=0.04). Demographics and SCD severity were not significantly associated with acceptability of IUHCT. This study suggests that the majority of parents >and young adults would consider IUHCT under expected therapeutic conditions.

Characterizing and Augmenting Peripheral Tolerance in in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). Central deletion, however, is incomplete, and donor-reactive host T cells remain following allogeneic IUHCT. Furthermore, central deletion alone cannot explain the absence of GVHD either following IUHCT using non-T cell depleted bone marrow (BM) or in chimeric animals that undergo postnatal donor lymphocyte infusion (DLI) with subsequent conversion to >90% allogeneic donor cell engraftment (Hayashi et al., Blood 2002;100:804-12). Studies in both mice and humans have demonstrated the importance of regulatory T cells, including traditional CD25+FoxP3+ Tregs and CD49b+LAG-3+ Type 1 regulatory T (Tr1) cells, in maintaining tolerance following postnatal allogeneic transplantation. We hypothesized that peripheral regulatory T cells are increased in chimeric animals following IUHCT and that they contribute to the establishment of DST and prevention of GVHD.Methods: 10x106 BM cells from 6-8 week old C57Bl/6 (B6) FoxP3GFP mice (H2Kb, CD45.2+) were injected intravenously (IV) into gestational day 14 Balb/c FoxP3GFP fetal recipients (H2Kd, CD45.2+). Mice were sacrificed at 2, 4, 8, and 24 weeks of age. Splenocytes were analyzed for the expression of CD4, H2Kb, H2Kd, CD49b, LAG-3, CD25, and FoxP3 to determine the percentage of traditional Tregs and Tr1 cells within the CD4+ population as well as for the expression of intracellular IL-10 - a key mediator of regulatory T cell function. A GVHD model was developed in which 10x106 B6 BM cells were injected IV into day of life 0 (P0) naive Balb/c mice. The ability of IUHCT-induced regulatory T cells to suppress GVHD was assessed in two groups: 1) 10x106 B6 BM cells injected into P0 mice that had undergone IUHCT and 2) 10x106 B6 BM cells co-injected with 5x106 CD4+ splenocytes harvested from 2 week old chimeric mice into P0 naive Balb/c mice. To evaluate the role of regulatory T cells following DLI in chimeric mice, 30x106 B6CD45.1 (H2Kb, CD45.1+) splenocytes were injected IV into 4 week old chimeric recipients. Mice were sacrificed at 48 hours and 2 weeks post-DLI, and regulatory T cells were measured as a percentage of CD45.2+CD4+ cells in the spleen.Results: IUHCT was associated with a significant increase in Tr1 cells, predominantly of donor origin, compared to naive controls (Figure 1). There was a corresponding increase in intracellular IL-10 expression in donor CD4+ splenocytes in chimeric animals compared to naive B6 FoxP3GFP controls at 2 weeks of age (MFI 7.07 vs. 3.26, p=0.0017). Elevated levels of regulatory T cells following IUHCT also had a functional effect in the GVHD model (Figure 2). Mice that had undergone IUHCT (group 1) demonstrated a trend toward improved survival compared to naive mice when injected with B6 BM at P0. Naive mice injected at P0 with B6 BM plus CD4+ splenocytes from chimeric mice (group 2) demonstrated significant improvement in survival compared to naive recipients of B6 BM alone (p=0.049). Although we detected no increase in traditional Tregs after IUHCT alone, we noted a 3-fold increase in traditional Tregs of host origin in chimeric animals at 48 hours post-DLI (25.5% vs. 8.3%, p<0.001).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and preventing GVHD. We demonstrate an increase in nontraditional regulatory T cells following IUHCT which can minimize GVHD as well as an increase in traditional Tregs following DLI in an established model of complete allogeneic engraftment. These studies suggest an important role for regulatory T cell populations in the induction of tolerance following IUHCT. They are significant in light of a potential obstacle to the clinical translation of IUHCT: the window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges. Augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a technically feasible point later in gestation. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Enhanced Allogeneic Engraftment Achieved Via Prenatal Tolerance Induction and Postnatal Anti-CD45 Immunotoxin Conditioned Hematopoietic Cell Transplantation

INTRODUCTION: In utero hematopoietic cell transplantation (IUHCT) is a non-myeloablative, non-immunosuppressive transplant approach that takes advantage of the immunologic immaturity of the fetus to engraft donor cells across immune barriers and induce donor specific immune tolerance (DST). Though it has the potential to treat many congenital hematologic disorders, IUHCT is limited by levels of engraftment below those anticipated to be therapeutic for most target diseases, including sickle cell anemia. Thus, a likely initial clinical application of IUHCT is to induce DST to allow for non-myeloablative, non-immunosuppressive postnatal "booster" transplants to increase engraftment. We have previously validated this approach in murine sickle cell, beta-thalassemia, and non-diseased models using low dose total body irradiation and busulfan prior to postnatal bone marrow transplantation (BMT). Although encouraging, less toxic approaches that specifically target the hematopoietic system would make IUHCT + postnatal BMT more applicable. CD45 is a membrane glycoprotein expressed by all leukocytes and hematopoietic stem cells. Recent studies have demonstrated enhanced engraftment in a congenic postnatal transplant mouse model following pre-transplant conditioning with an anti-CD45-saporin (SAP) immunotoxin (Palchaudhuri et al., Nat Biotech 2016; 34:738-45). We hypothesized that mixed allogeneic chimerism achieved by IUHCT can be enhanced after birth by conditioning with an anti-CD45-SAP immunotoxin followed by a same-donor allogeneic BMT.METHODS: 5x106 BM mononuclear cells from C57Bl/6 mice (H2Kb, CD45.2+) were injected intravenously into gestational day 14 (E14) Balb/c fetuses (H2Kd, CD45.2+). Donor cell peripheral blood (PB) chimerism was confirmed at 4 weeks of age, at which time chimeric mice were injected via tail vein with PBS (control) or CD45.2-SAP. Six days following treatment, chimeric animals were intravenously injected with 30x106 B6Pep3b (H2Kb, CD45.1+) BM cells. Additional control groups consisted of recipients of IUHCT alone without a postnatal BMT and naïve 5 week-old Balb/c mice which did not undergo IUHCT and only received CD45.2-SAP + postnatal BMT. Following postnatal BMT, PB was assessed bi-weekly by flow cytometry for total donor chimerism and the contribution of prenatal and postnatal donor cells to engraftment. Mice were weighed before each bleeding and evaluated for signs of GVHD. Statistical comparison was performed using the Student’s t-test for 2 samples at each time point assuming unequal variances.RESULTS: There was no significant difference in initial donor cell chimerism levels at 4 weeks of age between treatment (IUHCT+CD45.2-SAP+BMT, 12.4±2.2%, n=5) and control (IUHCT alone, 9.3±2.7%, n=18; IUHCT+BMT, 10.0±2.6%, n=7) groups prior to therapy. In contrast to naïve non-chimeric Balb/c mice undergoing a postnatal BMT (n=10) which demonstrated no donor cell engraftment, recipients of IUHCT and a postnatal BMT (both PBS and CD45.2-SAP treated groups) demonstrated postnatal donor cell engraftment, supporting DST achieved by IUHCT. While postnatal BMT after IUHCT failed to demonstrate a significant enhancement of engraftment compared to IUHCT alone (4.4±1.3% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p = 0.7), low-level allogeneic chimerism following IUHCT was significantly enhanced to high-level chimerism following conditioning with CD45.2-SAP+BMT (55.7±9.8% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p=0.0001) (Fig. 1). The majority of donor cell chimerism resulted from the postnatal donor population in recipients of CD45.2-SAP, supporting a mechanism of enhanced engraftment via a competitive advantage following recipient CD45.2-SAP treatment (Fig. 2). Chimerism was stable and multilineage at 22 weeks indicating enhanced stem cell/early progenitor engraftment. All mice demonstrated appropriate weight gain without GVHD.CONCLUSIONS: Prenatal tolerance induction achieved by IUHCT followed by postnatal CD45-SAP conditioning and same-donor BMT results in enhanced stable multilineage allogeneic engraftment at therapeutic levels for many target diseases. This work supports a potentially less toxic approach for the strategy of prenatal tolerance induction to facilitate non-myeloablative, non-immunosuppressive postnatal BMT and has the potential to significantly expand the applicability of IUHCT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In utero induction of mixed chimerism results in deletion, receptor editing, and functional inactivation of antigen specific alloreactive B cells

Abstract Purpose B cell tolerance is critical for donor cell engraftment across immune barriers after in utero hematopoietic cell transplantation (IUHCT). Using our fetal transplant model we studied how B cells achieve tolerance to allogeneic MHC antigens. Methods 10×106 C57Bl/6-GFP (H2Kb) bone marrow (BM) cells were injected intravenously into gestational day 14 Balb/c (H2Kd) fetuses. Mice were sacrificed at 3 days (P3), 1 and 4 months of age and donor-specific B cell tolerance including deletion, receptor editing and functional inactivation were assessed. Age matched naïve C57Bl/6 and Balb/c mice served as controls. Results The % of alloantigen specific splenic B cells was significantly decreased in allogenic chimeras as compared to controls (p=0.016) consistent with clonal deletion. Alloreactive B cells that escape deletion can undergo receptor editing. There was a significant increase in the % of B cells undergoing editing (lambda light chain expression) at P3 in recipients of IUHCT compared to controls (10.8% vs. 8.4%; p=0.02). Alloreactive B cells can be functionally inactivated such that they no longer make antibodies or express IgM. In contrast to naïve Balb/c mice immunized with C57Bl/6 splenocytes, immunization of recipients of IUHCT failed to elicit an anti-donor antibody response (MFI fold change: IUHCT-0.89 naïve-2.77; p&amp;lt;0.05). Furthermore, IgM expression on alloantigen specific splenocytes in chimeric animals was lower than controls (MFI: chimeric-1206, control-1472 ; p=0.03). Conclusion B cell tolerance after IUHCT is achieved by partial deletion in alloreactive splenic B cells, receptor editing in early mature splenocytes, and the remainder of alloreactive B cells demonstrate a functionally inactive phenotype.

The Intravenous Route of Injection Optimizes Engraftment and Survival in the Murine Model of In Utero Hematopoietic Cell Transplantation

In utero hematopoietic cell transplantation (IUHCT) has the potential to treat a number of congenital hematologic disorders. Clinical application is limited by low levels of donor engraftment. Techniques that optimize donor cell delivery to the fetal liver (FL), the hematopoietic organ at the time of IUHCT, have the potential to enhance engraftment and the clinical success of IUHCT. We compared the 3 clinically applicable routes of injection (intravenous [i.v.], intraperitoneal [i.p.], and intrahepatic [i.h.]) and assessed short- and long-term donor cell engraftment and fetal survival in the murine model of IUHCT. We hypothesized that the i.v. route would promote direct donor cell homing to the FL, resulting in increased engraftment and allowing for larger injectate volumes without increased fetal mortality. We demonstrate that the i.v. route results in (1) rapid diffuse donor cell population of the FL compared with delayed diffuse engraftment after the i.p. and i.h. routes; (2) higher FL and spleen engraftment at early prenatal time points; (3) enhanced stable long-term peripheral blood donor cell engraftment; and (4) improved survival at higher injectate volumes, allowing for higher donor cell doses and increased long-term engraftment. These findings support the use of an i.v. route for clinical protocols of IUHCT.

The Use of Eicosanoids to Enhance Donor Cell Engraftment after in Utero Hematopoietic Cell Transplantation (IUHCT)

IntroductionIn utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive allogeneic transplant approach that has the potential to treat a number of congenital disorders, including hemoglobinopathies and immunodeficiencies. Donor cell engraftment at levels high enough to induce donor specific immune tolerance or to treat a target disease has been elusive and remains a major limitation to the clinical application of IUHCT. One of the most significant barriers to high levels of donor cell engraftment is competition with endogenous fetal hematopoietic stem cells (HSCs) for limited hematopoietic niches following transplant. 16,16-dimethyl-prostaglandin E2 (PGE2) and the epoxyeicosatrienoic acids (EETs) 11,12-EET and 14,15-EET have been shown to enhance donor HSC homing, survival, and cell cycling in postnatal murine and zebrafish models of HSC transplantation. We hypothesized that a single ex vivo treatment of donor cells with these eicosanoids would improve donor cell homing and survival following IUHCT,resulting in higher levels of postnatal donor engraftment.MethodsTen million bone marrow (BM) mononuclear cells from C57Bl/6-GFP mice (H2Kb) were injected intravenously into embryonic day (E14) Balb/c fetuses (H2Kd) via the vitelline vein. Donor cells were treated with vehicle, PGE2, 11,12-EET, or 14,15-EET immediately prior to IUHCT. Early homing to and engraftment of the fetal liver (FL) and spleen (FS) of PGE2 and vehicle treated BM cells at 4, 24 and 72 hours after IUHCT was assessed by flow cytometry. Donor cell survival and apoptosis was also assessed in the FL 96 hours post-IUHCT in these two treatment groups by flow cytometric analysis of intracellular expression of survivin (anti-apoptotic) and anti-caspase 3 (pro-apoptotic). Long-term peripheral blood donor cell engraftment was assessed monthly up to 6 months of age and multilineage engraftment (donor T cells, B cells, granulocytes, and macrophages) was determined at 6 months of age in recipients of all donor cell treatment groups. Statistical analysis was performed using ANOVA with BonferroniÕs multiple comparison test or Kruskal-Wallis with DunnÕs multiple comparison test for normal and non-normal data, respectively. Data reported as mean +/- SEM.ResultsPGE2 pre-treatment produced a significant increase in FL and FS engraftment at 72 hours post-IUHCT compared to vehicle treated donor cells (FL: 37.5 +/- 3.1% vs 21.6 +/- 1.3%; FS: 52.2 +/- 3.7% vs 39.2 +/- 1.8%; p < 0.05). There was no significant increase in donor cell engraftment in the FL at earlier time points associated with PGE2 treatment. PGE2 treatment was also associated with increased survival of donor cells compared to vehicle treated cells as indicated by increased donor cell expression of survivin (19.8±5.6% vs. 4.6±1.3%; p<0.05) and decreased donor cell anti-caspase 3 staining (9.3±1.2% vs. 64.1±10.6%; p<0.05) at 96 hours post-IUHCT. These findings of increased FL/FS engraftment and increased survival at 72 to 96 hours post IUHCT following donor cell treatment with PGE2 translated into significantly increased long-term donor cell engraftment up to 6 months of age. Similar to PGE2, ex vivo treatment of donor cells with 11,12-EET or 14,15-EET resulted in enhanced long-term allogeneic donor cell engraftment (Figure 1). Balanced multilineage engraftment was seen for all treatments suggesting enhanced engraftment at the level of the HSC. No deleterious effects were observed on murine growth or overall health.ConclusionsThe ex vivo treatment of donor cells with eicosanoids including PGE2, 11,12-EET, and 14,15 EET prior to IUHCT results in enhanced long-term multilineage allogeneic donor cell engraftment. Detailed studies of early homing to fetal hematopoietic organs and survival at 96 hours following IUHCT of PGE2 treated donor cells suggest the mechanism of enhanced engraftment is mainly do to a pro-survival effect of PGE2 and related eicosanoids. This work represents a novel application of these eicosanoids in an allogeneic model of IUHCT and highlights their potential to assist in future clinical applications of IUHCT. [Display omitted] DisclosuresZon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

The Intravenous Route Optimizes Engraftment and Survival in the Murine Model of in Utero Hematopoietic Cell Transplantation (IUHCT)

IntroductionIUHCT has the potential to treat many congenital hematologic and immune disorders. It is a nonmyeloablative, nonimmunosuppressivetransplant approach that allows engraftment across immune barriers. Obtaining levels of engraftment necessary to treat target diseases remains a significant impediment to the clinical application of IUHCT. One barrier to consistent high-level engraftment is competition from host HSCs for limited niches at the time of transplant. The fetal liver (FL) is the hematopoietic organ in the mouse, large animals, and humans at the proposed time of IUHCT. Techniques that optimize donor cell delivery to the FL may enhance engraftment following IUHCT. In this respect, we sought to identify the route of injection that results in the least degree of mortality while promoting the highest level of engraftment. We compared the intravascular (IV), intraperitoneal (IP), and intrahepatic (IH) delivery of donor hematopoietic cells in a congenic murine model of IUHCT. We hypothesized that the IV route would promote direct homing to the FL resulting in increased levels of postnatal peripheral blood (PB) chimerism, and allow for larger cell doses without deleterious effects on fetal survival.MethodsFive million C57Bl/6-GFP bone marrow mononuclear cells were suspended in a fixed volume of PBS (5 uL) and injected into embryonic day 14 (E14) C57Bl/6 fetuses via the IV, IP, or IH route. Flow cytometry and fluorescence stereomicroscopy (FSM) were used to assess donor cell homing to and engraftment of the FL at 4, 24 and 72 hours and the fetal spleen (FS) at 72 hours post IUHCT. Long-term postnatal PB engraftment was assessed monthly by flow cytometry. Fetal survival to E18 following increasing volumes of PBS injectate at E14 was compared between the different routes of injection. Statistical analysis was performed using ANOVA with Bonferronicorrection and FisherÕs Exact Test.ResultsThe IV route led to significantly higher engraftment of the FL at 24 and 72 hours and the FS at 72 hours post IUHCT (Figure 1). FSM revealed important route-dependent differences in the patterns and kinetics of donor cell homing. IV injected donor cells robustly populated the FL in a homogenous manner within 4 hours of IUHCT. Most IP injected cells remained localized to the peritoneal cavity for over 24 hours post IUHCT with only modest engraftment of the FL. IH injected cells remained within the liver in a focal distribution with notable leaking into the perihepatic space beyond 24 hours post IUHCT. The increased efficiency with which donor cells homed to and engrafted the FL following IV IUHCT translated into superior and stable long-term donor cell engraftment (Figure 2). Fetal mice also demonstrated increased survival following higher injectate volumes administered via the IV route (Figure 3). Tolerance to larger volumes permitted higher donor cell doses to be administered at a given concentration (1x106 cells/mL), which translated into higher long-term donor cell engraftment (6 month chimerism, IV 5x106 donor cells: 4.8 +/- 0.5% vs. IV 20x106 cells: 13.2 +/- 1.0%; p < 0.05).ConclusionsIn a congenic murine model of IUHCT, the IV route of injection results in increased levels of stable long-term donor cell engraftment compared to the IH and IP routes. Mechanistically, this is related to the increased efficiency of donor cell homing to the site of the hematopoietic niche at the time of the transplant. Furthermore, the IV route allows for larger volumes of injectate, and thus potentially higher cell doses, with reduced fetal loss. These observations have important implications in the translation of IUHCT technique to future clinical trials. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Utero Hematopoietic Cell Transplantation Induces Peripheral Tolerance By Upregulating Two Types of Nontraditional Regulatory T Cells

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism across immune barriers without myeloablation/immunosuppression. It has the potential to induce donor specific tolerance (DST) for postnatal nonmyeloablative cellular transplants to treat target diseases such as Sickle cell disease. Partial deletion of donor reactive host T cells and host reactive donor T cells is responsible for tolerance and the absence of GVHD following IUHCT. The remaining nondeleted donor and host specific T cells are thought to undergo anergy. The mechanisms by which anergy and peripheral tolerance are achieved following IUHCT are not completely understood. Studies in both mice and humans have demonstrated the importance of regulatory T cells in maintaining tolerance following postnatal allogeneic transplantation. Subsets of regulatory T cells include traditional regulatory T cells (Tregs; CD4+ CD25+ FoxP3+), Type 1 regulatory T cells (Tr1 cells; CD4+ CD49b+ CD223+), and LAP+ regulatory T cells (LAP+ cells; CD4+ CD25- LAP+). These T regulatory populations inhibit T cell activation and proliferation through, among other mechanisms, the secretion of immunosuppressive cytokines including IL-10. During pregnancy, there is upregulation of circulating IL-10 levels in maternal and fetal blood, and maternal antigen specific fetal Tregs are induced in peripheral lymphoid tissue. We propose that IUHCT takes advantage of these tolerogenic features of the fetus to promote the peripheral induction of regulatory T cells capable of suppressing reactive T cell clones that escape thymic deletion.Methods: 10x106 bone marrow cells from 6-8 week old C57Bl/6 (B6) FoxP3GFP (H2Kb) mice were injected intravenously into allogeneic gestational day 14 Balb/c FoxP3GFP fetal recipients (H2Kd). Uninjected B6 FoxP3GFP and Balb/c FoxP3GFP mice served as controls. Mice were sacrificed at 2 and 4 weeks of age. Peripheral blood donor chimerism was determined by flow cytometry at the time of sacrifice. Splenocytes were analyzed for expression of CD4, H2Kb, H2Kd, CD49b, CD223, CD25, and LAP to determine the percentage of donor and host Tregs, Tr1 cells, and LAP+ cells within the CD4+ population. CD4+ splenocytes were also assessed for intracellular IL-10 expression. Statistically significant differences between chimeric and uninjected control animals were determined using a two-tailed student's t-test.Results: Chimeric animals demonstrated a 4- and 5-fold increase in donor Tr1 cells compared to uninjected B6 FoxP3GFP controls at 2 (2.4% vs. 0.6%, p=0.03) and 4 weeks of age (2.4% vs. 0.5%, p=0.007). There was no significant difference in host Tr1 cell levels of chimeric mice compared to uninjected Balb/c FoxP3GFP controls at either 2 (0.73% vs. 0.61%, p=0.4) or 4 weeks of age (1.0% vs. 0.53%, p=0.09). Similar to Tr1 cells, LAP+ cells of donor origin were increased compared to B6 FoxP3GFP controls at 2 (2.2% vs. 0.8%, p=0.008) and 4 weeks of age (3.2% vs. 0.6%, p=0.03). Additionally, host LAP+ cells were increased compared to Balb/c FoxP3GFP controls at 2 (2.6% vs. 1.5%, p=0.0004) and 4 weeks of age (3.0% vs. 1.1%, p=0.0005). The increase in Tr1 and LAP+ cells was associated with a significant decrease in traditional Tregs in both the host and donor population at 2 and 4 weeks of age (Figure 1). Elevated levels of Tr1 and LAP+ cells did not correlate with the absolute levels of donor cell engraftment but was dependent on achieving a threshold level of chimerism known to be associated with DST. Furthermore, the MFI of intracellular IL-10 among donor CD4+ splenocytes was significantly increased compared to those from B6 FoxP3 controls at 2 weeks of age (Figure 2), demonstrating immunosuppressive activity consistent with elevated Tr1 and LAP+ levels.Conclusion: The induction of donor and host tolerance following IUHCT is important for maintaining successful long-term engraftment and preventing GVHD. DST also allows for postnatal nonmyeloablative cellular transplants to increase engraftment to therapeutically relevant levels. We demonstrate a potentially important role that nontraditional Tr1 and LAP+ regulatory T cells play in maintaining donor cell engraftment and peripheral tolerance in the setting of IUHCT. Future investigations into the ability of these cells to augment tolerance induction after inefficient IUHCT have the potential to expand the clinical promise of IUHCT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mobilization of Host Hematopoietic Cells Increases Engraftment after in Utero Hematopoietic Cell Transplantation (IUHCT)

Background:IUHCT is a non-myeloablative non-immunosuppressive transplant approach that allows for donor cell engraftment across allogeneic barriers. It has the potential to treat a large number of congenital hematologic, genetic, and immunologic disorders. A significant barrier to the clinical application of IUHCT is the low level of donor cell engraftment secondary to competition for limited space in the developing hematopoietic niche. AMD3100, an inhibitor of the chemokine receptor CXCR4, and Firategrast, a small molecule inhibitor of the α4β1 integrin, have been shown to disrupt the retention of hematopoietic stem cells (HSCs) in postnatal bone marrow (BM) transplant models. We hypothesized that administration of these agents to a pregnant dam prior to IUHCT would mobilize endogenous fetal HSCs from the fetal liver (FL) hematopoietic niche resulting in preferential homing of donor HSCs and enhanced long-term donor cell engraftment.Methods:AMD3100 (5 mg/kg) and Firategrast (100 mg/kg) were maternally administered alone or in combination to time-dated Balb/c dams at either embryonic day 17 (E17) (pharmacokinetic studies) or E14 (endogenous HSC mobilization, donor cell homing, and long-term engraftment studies). Maternal and fetal sera were analyzed by mass spectrometry at 15, 45, 90, and 180 minutes following AMD3100 or Firategrast administration to determine serum drug concentrations. Based on these pharmacokinetic studies, FLs were harvested 60 minutes following AMD3100 and/or Firategrast administration and assessed by flow cytometry for lineage- sca-1+ c-kit+ (LSK) cells to determine endogenous HSC mobilization. Short-term homing and long-term engraftment were subsequently assessed in Balb/c fetal mice (H2Kd) intravenously injected with 10x106 B6 GFP (H2Kb) BM cells following maternal administration of AMD3100, Firategrast, or AMD3100+Firategrast (Combination). For homing analysis, the proportion of donor LSK within the donor lymphocyte compartment of the FL was measured 24 hours after IUHCT. Long-term engraftment of donor cells was assessed monthly by flow cytometry of peripheral blood (PB) through 6 months of age. Multilineage PB engraftment was assessed at 9 months of age. Statistical analyses were performed using one-way or two-way ANOVA with Bonferroni post-hoc tests.Results:Maternal administration of either agent resulted in transplancental passage with peak fetal serum concentrations occurring between 45 and 90 minutes for AMD3100, and between 15 and 90 minutes for Firategrast. Treatment with Firategrast or Combination (AMD3100+Firategrast) resulted in significant mobilization of endogenous HSCs from the FL, with Firategrast eliciting the greatest mobilization vs. control (no treatment) (2.8x103 vs. 9.6x103 HSCs/FL, p<0.05) (Figure 1). Maternal administration of Firategrast or Combination prior to IUHCT translated into 1) significantly increased short-term homing of allogeneic donor LSK in the fetal liver 24 hours after IUHCT (Control: 1.5% ± 0.10 donor LSK in donor lymphocyte population; Firategrast: 2.4% ± 0.3, p = 0.0135; Combination: 2.4% ± 0.1, p = 0.0136), and 2) sustained enhancement of long-term allogeneic engraftment through 6 months of age (Figure 2). There was no apparent benefit to Combination treatment over Firategrast treatment alone. All chimeric mice demonstrated multilineage engraftment at 9 months of age supporting engraftment at the level of the HSC or early progenitor cell following IUHCT.Conclusion:Maternal administration of antagonists of the CXCR4/SDF-1a and a4b1/VCAM-1 pathways results in mobilization of endogenous fetal HSCs from the FL hematopoietic niche and preferential homing of donor HSCs to the FL when administered prior to IUHCT. The combination of agents targeting both the CXCR4/SDF-1a and a4b1/VCAM-1 pathways in the current study did not yield a synergistic effect on mobilization and engraftment. This work demonstrates the potential of mobilizing endogenous fetal HSCs as a strategy to overcome barriers to successful engraftment in utero bringing IUHCT closer to clinical application. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of B Cell Tolerance after in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive transplant approach that results in donor cell engraftment across immune barriers. Although a significant amount of work has investigated the fate of T cells following IUHCT, little attention has been paid to B cell tolerance and the fate of donor derived host reactive or host derived donor reactive B cells following IUHCT. B cell tolerance is broadly believed to occur by a combination of 3 mechanisms: deletion, receptor editing, and functional inactivation (anergy). In the current study we attempt to elucidate the mechanism(s) by which B cell tolerance occurs following IUHCT.Methods: 10x106 donor bone marrow (BM) cells were injected intravenously via the vitelline vein into gestational day 14 murine fetuses. IUHCT was performed in the congenic (C57Bl/6-GFP [H2Kb ] into C57Bl/6 [H2Kb ]) and allogeneic (C57Bl/6-GFP into Balb/c [H2Kd ]) strain combinations. Naive Balb/c and C57Bl/6 mice served as controls. Mice were sacrificed at day of life 3 (P3), 1 month and 4 months of age at which time their BM, spleen, and serum were harvested. To assess B cell deletion, flow cytometry was used to determine the absolute # and % of host and donor immature and pre B cells in the BM. Additionally, apoptosis of host and donor BM derived B cells was determined by annexin staining. Central receptor editing was evaluated using RT qPCR to measure the amount of Vκ-RS rearrangments in BM pre-B cells. Peripheral receptor editing was studied by calculating the % of λ light chains in mature splenocytes identified by flow cytometry. Finally, functional inactivation of donor reactive host B cells was assessed by measuring anti-H2Kb serum antibodies (ab) of allogeneic chimeras, naive, and immunized mice at 1 month of age.Results: The absolute number of BM immature B cells was decreased in allogeneic recipients of IUHCT compared to noninjected Balb/c controls at 1 month of age (fig 1). This effect was lost by 4 months of age. The decrease in B cells resulted primarily from a decrease in immature donor as opposed to host B cells compared to controls (% immature donor B cells in allogeneic recipients vs. controls: 16.2% vs. 39.9%; p<0.0005). Donor B cells in allogeneic chimeras also demonstrated a trend toward increased apoptosis compared to controls (24.8 vs. 18.7%; p=0.2) which was not seen in immature host B cells (18.3 vs. 18.6%; p=0.9). There was no significant decrease in the absolute number of immature B cells or increased apoptosis in congenic recipients compared to uninjected controls. These findings suggest deletion of autoreactivedonor B cells.Light chain receptor editing involves rearrangements within the κ and λ gene loci and may occur in BM pre-B cells or mature B cells in the spleen. We found no difference in the Vκ-RS rearrangements of pre B cells in allogeneic chimeras and controls at 1 month. In contrast, the quantity of total λ+ mature splenic B cells was increased in allogeneic chimeras at P3 (10.8 vs. 8.4%; p=0.02) and resulted from an increased host λ+ % compared to controls (10.8 vs. 8.4% p=0.03) suggesting peripheral receptor editing of host cells (fig.2). The λ+ % increase in allogeneic chimeras was lost by 1 month.Autoreactive B cells that escape deletion and receptor editing can be functionally inactivated. Neither allogeneic nor naive mice developed ab to H2Kb splenocytes, however, Balb/c mice immunized to H2kb antigen showed high ablevels (MFI fold change: allo-0.89 naive-1.37 imm-2.77; p<0.05).Conclusion:B cell tolerance after IUHCT is achieved by distinct mechanisms for host and donor cell populations. Donor derived host reactive B cells undergo deletion and apoptosis while receptor editing and functional inactivation are the primary mechanisms of B cell tolerance of host derived donor reactive B cells. We hope use this and future studies of antigen specific B cell tolerance to harness the immunologic potential of IUHCT for many hematopoietic and immunologic congenital diseases. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Consensus statement from the first international conference for in utero stem cell transplantation and gene therapy.

On April 17–18, 2014, basic and translational scientists and clinicians convened in San Francisco, CA for a conference in fetal stem cell transplantation, stem cell biology, tolerance, and gene therapy. The purpose of the meeting (http://pedsurglab.surgery.ucsf.edu/news–events/fetal-symposium-2014.aspx) was to outline the goals of in utero transplantation, review the barriers that have been encountered, and learn about new developments that can be applied to the field. Information discussed at this conference will help pave the way for developing novel strategies to achieve therapeutic engraftment levels in the fetus, and identify ways to safely translate these strategies to a wide range of clinical applications. We held a final consensus session to achieve an international agreement for future pre-clinical and clinical studies of in utero hematopoietic cell transplantation (IUHCT). We agreed on the following items: In utero transplantation is a viable strategy to treat fetuses with selective congenital disorders. Given recent publications that the maternal immune response can limit engraftment, the clinical strategy for IUHCT should involve transplantation of autologous or maternal-derived cells. The host immune response may be a limiting factor that might be circumvented with early cell delivery. The fetal microenvironment plays a primary role in supporting the engraftment and expansion of transplanted cells and requires further investigation. Recent data from large animal studies suggests that intravascular injection may be the delivery route of choice to achieve engraftment of hematopoietic stem cells in the fetus. Currently, there is no proven safe method of host conditioning in the fetus. Until specific, non-toxic conditioning methods (such as antibody-mediated depletion of host HSC) are optimized in pre-clinical models, large cell doses should be used to overcome host competitive barriers. Experimental model data are sufficient to warrant a phase 1 clinical trial of IUHCT for select fetuses. The most suitable hematological diseases are hemoglobinopathies such as sickle cell disease and thalassemia, given their high morbidity/mortality, the availability of reliable prenatal screening programs, and the paucity of optimum postnatal care options. The value of alternative cells, such as mesenchymal stromal cells (MSC) and amniotic fluid-derived cells, for other appropriate congenital pathologies warrants investigation. Reports of using MSC in utero to treat osteogenesis imperfecta (OI) in a limited number of patients are promising and suggest that, after optimization, MSC could be used to improve/treat OI. Treatment of the fetal patient using gene therapy and gene-modified cells have great future potential and should be fields of active investigation. A new society focused on fetal stem cell transplantation and gene therapy will be formed (FeTIS: Fetal Transplantation and Immunology Society), with the mission of accelerating clinical applications of stem cell transplantation and gene therapy approaches to treat fetuses with congenital anomalies. The society should develop and maintain an international registry of treated patients and their outcomes to facilitate reporting and sharing of results. This database will not be publicly accessible and data will be anonymized. The society will provide a forum for members to share best practice and clinical governance for in utero stem cell and gene therapy cases.

Partial rescue of mucopolysaccharidosis type VII mice with a lifelong engraftment of allogeneic stem cells in utero.

In utero hematopoietic cell transplantation (IUHCT) has been performed in Mucopolysaccharidosis Type VII (MPSVII) mice, but a lifelong engraftment of allogeneic donor cells has not been achieved. In this study, we sought to confirm a lifelong engraftment of allogeneic donor cells immunologically matched to the mother and to achieve partial rescue of phenotypes in the original MPSVII strain through IUHCT by intravenous injection. We performed in vitro fertilization in a MPSVII murine model and transferred affected embryos to ICR/B6-GFP surrogate mothers in cases where fetuses receiving IUHCT were all homozygous. Lineage-depleted cells from ICR/B6-GFP mice were injected intravenously at E14.5. Chimerism was confirmed by flow cytometry at 4 weeks after birth, and β-glucuronidase activity in serum and several phenotypes were assessed at 8 weeks of age or later. Donor cells in chimeric mice from ICR/B6-GFP mothers were detected at death, and were confirmed in several tissues including the brains of sacrificed chimeric mice. Although the serum enzyme activity of chimeric mice was extremely low, the engraftment rate of donor cells correlated with enzyme activity. Furthermore, improvement of bone structure and rescue of reproductive ability were confirmed in our limited preclinical study. We confirmed the lifelong engraftment of donor cells in an original immunocompetent MPSVII murine model using intravenous IUHCT with cells immunologically matched to the mother without myeloablation, and the improvement of several phenotypes.