Search

Introduction In 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. Methods Ten 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. Results PGE2 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 Conclusions The 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. Disclosures Zon: 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.

Abstract 4668 Introduction In Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been analyzed. In this study, we utilized the murine model of allogeneic IUHCT and analyzed donor and host thymocyte development. Methods Bone marrow (BM) cells (10×106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal time points the thymocytes were analyzed by multi-color flow cytometry. Results The results demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocytes and from thymocyte development in normal B6 and Balb/c control mice. Though each subpopulation of the host's thymocytes showed comparable levels to the normal untransplanted Balb/c mice, the donor BM-derived thymocytes demonstrated significantly higher proportions of CD4+CD8- and CD4-CD8+ single positive cells, and a dramatically lower proportion of CD4+CD8+ double positive cells compared to their donor-derived counterparts, respectively. These discrepancies increased with each analysis time point up to 12 weeks. Immature single positive cells, including both TCRb-CD4+CD8- and TCRb-CD4-CD8+ cells, were significantly higher in the donor-derived thymocytes than the host, indicating the development of the donor BM-derived thymocytes were impeded at the DN-DP stage in this specific allogeneic IUHCT setting. In addition, we also found higher frequencies and more potent suppressive capacity of regulatory T cells in the donor-derived T cell compartment than in the host T cell compartment. In the reversed allogeneic strain combination, i.e. IUHCT of Foxp3GFP Balb/c BM MNCs into the E14 Foxp3GFP B6 recipients, thymocytes of both origins showed imbalanced intra-thymic development that differed from the normal mouse. Conclusion Our data in the current study suggests that immune reconstitution of the donor bone marrow derived thymocytes differs from that of the host cells and that of normal mice in an allogeneic IUHCT system. The mechanism of the altered thymic development in allogeneic IUHCT, and the potential importance of these observations for clinical IUHCT remains to be determined. Bone marrow (BM) cells (10×106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal time points the thymocytes were analyzed by multicolor flow cytometry (Fig. A), and the expression of TCR¦Â in each individual subpopulation in 4-week old mice was measured (Fig. B). Disclosures: No relevant conflicts of interest to declare.

Abstract 1904 Introduction: In Utero Hematopoietic Cell Transplantation (IUHCT) is a potential treatment for congenital hematologic disorders. The rationale is to utilize normal events during hematopoietic and immunologic development to facilitate hematopoietic engraftment. While the risk of graft versus host disease (GVHD) after IUHCT would be anticipated to be high, based on Medawar's tenets, surprisingly little GVHD has been observed after experimental IUHCT and IUHCT induced GVHD has not been previously characterized. To better understand the potential risk of IUHCT induced GVHD prior to clinical application, we attempted to intentionally induce GVHD pathophysiology in the allogeneic murine model of IUHCT. Methods: Bone marrow (BM) cells (10×106) from C57/BL6 (B6, H2kb, GFP−) mice were co-injected with increasing doses of splenocytes from B6-GFP (H2kb, GFP+) mice intravenously into Balb/c (H2kd) fetuses at embryonic day 14 (E14). Control groups included: 1) 10×106 B6 BM cells alone; 2) same doses of B6-GFP splenocytes alone; and 3) IUHCT of congenic donor cell populations into B6 recipients. Surviving pups were assessed using a clinical GVHD scale and separate cohorts were harvested at pre- and post-natal time-points for analysis by fluorescence stereoscopic microscopy (FSM), flow cytometry for assessment of donor and host cell populations in hematopoietic tissues, and histologic assessment of target tissues for GVHD. Results: Prenatal Analysis - In the allogenic groups that received 10×106 BM cells plus increasing doses of splenocytes (0.1×106 to 10×106 splenocytes/fetus) a consistent distribution of GFP positive cells could be seen by whole body FSM in lymphohematopoietic sites (spleen, lymph nodes, thymus and fetal liver) at E16 that did not differ significantly from congenic controls. By E19 – E20 however, marked proliferation of the cells occurred with clear infiltration of GVHD target organs (skin, lungs, liver, intestine). This expansion was proportionate to the dose of splenocytes and was not seen in congenic or BM only controls. Increased mortality was not observed until doses greater than 5 × 106 splenocytes were given (approx. 5 × 1011 splenocytes/kg est. fetal wt.) with perinatal death likely attributable to respiratory insufficiency. Postnatal Analysis - In the allogeneic groups there was minimal clinical evidence of GVHD up to and inclusive of the 5×106 splenocyte dose. A few pups showed poor growth relative to the normal growth curve and mild fur ruffling, but these resolved by P15, and there was no further clinical evidence of GVHD and no postnatal deaths up to P60. In harvested tissues many GFP+ cells persisted in GVHD target organs, however, no GVHD histopathology was observed at any time. Flow cytometric analysis confirmed a proliferation of donor splenocytes that peaked around P5-P10. Lymphocyte subset analysis revealed an expansion of predominantly CD8+ T-cells. Alkaline phosphatase in the host liver, as a marker of GVHD was increased during the same time course and returned to normal by P15. An increase of host and donor BM derived CD4+ cells was also observed that peaked around P15-17. Further analysis confirmed an increase in both T-regulatory cells and activated CD4+ T-cells compared to normal or BM only controls. The most remarkable observation was a dramatic graft versus hematopoietic (GVH) effect with an increase in donor BM derived chimerism at P60 to > 80% with no clinical GVHD. These findings were seen only in the allogenic group that received both splenocytes and bone marrow cells, not in the other groups. In the group that received only bone marrow cells, the chimerism remained at low levels (Figure 1). Conclusion: Our data demonstrates that the fetus is in fact resistant to GVHD relative to myeloablated postnatal BMT recipients. While there is an aggressive alloimmune activation and expansion of donor T-cells, clinical manifestations are mild and are reversed by what appears to be a T-regulatory cell expansion. In our model, the alloimmune activation phase is accompanied by a potent GVH response providing a competitive advantage for donor cells and resulting in marked enhancement of donor cell engraftment without clinical evidence of GVHD. Further work in this model is directed toward defining regulatory and effector mechanisms involved in controlling GVHD and the GVH effect. Disclosures: No relevant conflicts of interest to declare.