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: 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: 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: Invariant NKT (iNKT) cells have been shown to be pro-inflammatory or anti-inflammatory in many pathologic conditions including infections, cancer, autoimmune diseases and allergy. Additionally, early expansion of iNKT cells correlates with successful engraftment and low incidence of graft-versus host disease suggesting a role for iNKT cells in tolerance to allogeneic transplantation. iNKT cells recognize glycolipids bound to CD1d via their T cell receptor (TCR), but also express the inhibitory receptors for MHC class I ligands (MHC I.) Little is known regarding the importance of iNKT cell self-recognition of MHC I during development and the occurrence of autoimmunity, allergy or tolerance to in utero hematopoietic cellular transplantation (IUHCT). Given the importance of NK cell self-recognition during development, we hypothesized that, like NK cells, self-engagement of MHC I inhibitory receptors during development alters the phenotype and function of self-responsive iNKT cells. Prenatal allogeneic chimeras were established by in utero transplantation of Balb/c fetal liver light density cells at E14 into age-matched C57BL/6 fetal recipients. A kinetic evaluation of iNKT cell receptor expression and function was performed after birth. iNKT cells were evaluated for expression of activating (Ly49D) and inhibitory (Ly49A, F, G) receptors specific for the donor MHC I (H-2d.) The functional response of iNKT cell subsets was measured following in vivo activation with KRN7000, the ligand for iNKT cells. Intracellular cytokine production (IFN-gamma and IL-4) production was assessed in the alloresponsive Ly49A+ iNKT cell subset and compared to naïve controls and B10.D2 (H2d) mice. We found that a negligible fraction of iNKT cells expressed the activating Ly49D receptor in either chimera or controls (