ALBERT

Description: Exencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord.

Description: Clinical application of allogeneic bone marrow transplantation (BMT) has been limited by toxicity related to cytoreductive conditioning and immune response. In utero hematopoietic stem cell transplantation (IUHSCT) is a nonablative approach that achieves mixed chimerism and donor-specific tolerance but has been limited by minimal engraftment. We hypothesized that mixed chimerism achieved by IUHSCT could be enhanced after birth by nonmyeloablative total body irradiation (TBI) followed by same-donor BMT. To test this hypothesis, mixed chimerism was created by IUHSCT in a major histocompatibility complex-mismatched strain combination. After birth, chimeric animals received nonmyeloablative TBI followed by transplantation of donor congenic bone marrow cells. Our results show that: (1) low-level chimerism after IUHSCT can be enhanced to high-level chimerism by this strategy; (2) enhancement of chimerism is dependent on dose of TBI; (3) the mechanism of TBI enhancement is via a transient competitive advantage for nonirradiated hematopoietic stem cells; (4) engraftment observed in the tolerant, fully allogeneic IUHSC transplant recipient is equivalent to a congenic recipient; and (5) host-reactive donor lymphocytes are deleted with no evidence of graft-versus-host disease. This study supports the concept of prenatal tolerance induction to facilitate nonmyeloablative postnatal strategies for cellular therapy. If clinically applicable, such an approach could dramatically expand the application of IUHSCT.

Description: Despite clinical success with in utero hematopoietic stem cell transplantation in immunodeficient recipients, only microchimerism (

Description: Invariant NKT (iNKT) cells are glycolipid-reactive alpha/beta T cells which have an important role in the regulation of GVHD after allogeneic bone marrow transplantation. During thymic development, murine iNKT cells divide into three transcriptionally distinct lineages-NKT1, NKT2, and NKT17 that differ in their cytokine expression profile both at rest and upon antigen recognition via their TCR. Given that the lineage profile of iNKT cells varies dramatically between inbred strains of mice, it has been postulated that recognition of allospecific glycolipids determines iNKT cell lineage-fate decisions. Therefore, we challenged this hypothesis in a murine model of prenatal allogeneic transplantation to determine if the lineage commitment of immature iNKT cells was intrinsically programmed or extrinsically regulated by the allospecific environment during development. Prenatal allogeneic chimeras were established by in utero transplantation of E14 fetal liver light density cells into age-matched allogeneic fetal recipients (Balb/c to B6 or B6 to Balb/c). In this model, immature iNKT cells of both donor and host origin have the capacity to participate in education as CD1d on bone marrow-derived cells regulate the maturation of developing iNKT cells. This permitted an analysis of the impact of either host-to-donor or donor-to-host environmental cues in directing iNKT cell lineage-fate decisions. iNKT cell populations were identified using flow cytometric analysis of the transcription factors PLZF and T-bet. The lineage profile for donor and host thymic iNKT cells from chimeric mice were compared to the thymic iNKT cell population in naïve controls. As shown, B6 iNKT cells in prenatal chimeras exhibited a predominance of the NKT1 lineage in either the donor or the host situation similar to their frequency in naïve B6 controls (figure A). Conversely, Balb/c iNKT cells in both the donor and the host situation exhibited skewing toward an NKT1 lineage profile and away from the NKT2 lineage bias seen in naïve Balb/c controls (figure B). Furthermore, the expression of the H-2d MHC class I-reactive Ly49A receptor by Balb/c iNKT cells strongly correlates with the NKT1 lineage fate in control animals. However, both donor and host Balb/c cells demonstrated reduced correlation between Ly49A expression and NKT1 lineage fate indicating that the presence of H-2b expressing B6 cells diminished the ability of H-2d -reactive Ly49A to dictate lineage fate decisions. This study uniquely demonstrates the potential for cell-extrinsic signals in guiding iNKT cell lineage fate in an asymmetric fashion. Specifically, we find that: 1) the B6 iNKT lineage profile is intrinsically determined and unaffected by exposure to allogeneic Balb/c cells during development; 2) the Balb/c iNKT cell lineage profile is extrinsically determined and dominantly skewed toward an NKT1 lineage by exposure to even small numbers of B6 cells during development; and 3) the exposure to B6 cells overrides the contribution of Ly49A to developmental decisions made by Balb/c iNKT cells. Future studies will explore the regulatory interactions that govern allospecific iNKT cell lineage fate decisions and the resulting impact on the pro-inflammatory or immunoregulatory function of iNKT cells in clinically-relevant models. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (106 LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.

Description: Prenatal transplantation capitalizes on the unique fetal environment, allowing for life-long engraftment of allogeneic stem cells without the need for harsh conditioning regimens. A prerequisite for stable engraftment of allogeneic cells likely requires the negative selection of donor-specific host effector T cells (Teff) and the support of donor-specific regulatory T cells (Tregs). However, little is known about the interplay between these cell types during development. The purpose of this study was to characterize the dynamic relationship between donor-specific Teff and Tregs as they emerge during development. Prenatal allogeneic chimeras were established by in utero transplantation of E14 fetal liver light density cells into age-matched allogeneic fetal recipients (Balb/c to B6 or B6 to Balb/c). In this model, immature T cells from B6 mice expressing TCRv-beta-5, 11, and 12 are negatively selected by mtv-8 superantigen complexed with I-E class MHC II on Balb/c cells. As alpha/beta TCR rearrangement does not occur until E16, this established transplantation model allows for alloantigen to be present from the earliest stages of thymic selection. Kinetic analysis of donor-specific T cell populations was performed in peripheral blood paired with in depth analysis in thymus and spleen in control and chimeric mice. Negative selection of donor-specific Teff cells occurs at an unexpectedly slow pace in Balb/c to B6 prenatal chimeras. Donor-specific CD4 and CD8 Teff are significantly decreased in frequency at 4 weeks of age but do not reach maximal deletion until 12 weeks of age (TCRv-beta-5 data shown in Figure A). Further analysis demonstrated that this slow elimination of donor-specific Teff was paired with an early increase in the frequency of donor-specific Tregs at 4 weeks of age (TCRv-beta-5 data shown in Figure B.) This increase in donor-specific Tregs likely occurred as a result of peripheral expansion as there was no change in the frequency of donor-specific Tregs in the thymus (Figure B) and no change in the frequency of these cells that expressed the markers of thymically derived natural Tregs neuropilin-1 or helios (data not shown). In agreement with this hypothesis, donor-specific splenic Tregs incorporated BrdU at a higher rate than other Tregs in young mice indicating a potential expansion of donor-specific Tregs in the periphery (TCRv-beta-5 data shown in Figure C.) Collectively, these data demonstrate that prenatal transplantation is characterized by: 1) a surprisingly slow reduction of donor-specific Teff subsets; 2) an early expansion of donor-specific Tregs. Further studies will explore the role of donor-specific Tregs in controlling early immunity to prenatally encountered antigens. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: The failure to achieve durable engraftment following prenatal transplantation in immunologically normal human fetal recipients calls for a closer examination of the fetal immune response to allotransplantation. Previous studies in mice suggest that the fetal innate immune system functions as a critical barrier to allogeneic engraftment mediated by recognition of MHC class Ib antigens. We hypothesized that Qa-2 (the putative murine homolog for HLA-G) might play an essential role in the modulation of fetal immune response to prenatally transplanted allogeneic cells. To address this hypothesis, we utilized B6.K1 mice as a donor strain. B6.K1 mice are Qa-2 deficient and are congenic with wild-type B6.Ly5.2 mice. Light density mononuclear cells (LDMCs) were harvested from the livers of 14 dpc fetal B6.K1 or B6.Ly5.2 mice and transplanted into age-matched allogeneic Balb/c fetal recipients at a dose of 105 cells per fetus. Following delivery, peripheral blood chimerism was assessed serially in the recipients. Survival to weaning was similar between the groups without evidence of GVHD. At 3 weeks of age, recipients of B6.K1 cells demonstrated significantly lower peripheral blood chimerism levels than recipients of B6.Ly5.2 control cells. By 6 months of age, nearly all of the recipients of B6.K1 cells had lost their chimerism. Conversely, the chimerism levels in recipients of B6.Ly5.2 control cells remained stable suggesting that donor Qa-2 expression was essential for allograft survival. To assess the competitive capacity of the B6.K1 donor cells in the absence of immunologic disparity, B6.K1 or B6.Ly5.2 fetal liver LDMCs were transplanted into congenic B6.Ly5.1 hosts at the same cell dose per fetus. This resulted in stable long-term engraftment of the B6.K1 cells in all recipients. Chimerism levels were identical to those recipients who received B6.Ly5.2 control cells, confirming that the engraftment disparities observed in the allogeneic recipients resulted from immunologic rejection. To assess the resilience of this apparent Qa-2-dependent innate immune barrier, the allogeneic transplantation experiments were then repeated at a ten-fold higher donor cell dose (106 cells/fetus). Early chimerism levels remained significantly lower in allogeneic recipients of Qa-2 deficient cells compared to controls. However, recipients of B6.K1 cells maintained their engraftment for more than 6 months indicating that the Qa-2-dependent fetal immune barrier may be overcome with higher levels of circulating antigen. From these experiments we conclude: Host allorecognition of the class Ib antigen Qa-2 is crucial for durable engraftment following in utero transplantation; The failed engraftment of Qa-2 deficient hematopoietic cells does not result from a defective competitive engraftment capacity; Qa-2 dependent fetal immune rejection may be diminished by higher levels of early chimerism. These experiments provide direct evidence for the critical role of MHC class Ib antigens in regulation of the fetal immune response to allotransplantation. Additionally, the demonstration of reliable engraftment following transplantation of higher cell doses provides a translationally relevant approach to enhance the clinical success of prenatal transplantation in immunologically normal hosts.

Description: Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (106 LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.