Wild-type Donor Research Articles

Abstract 226: Novel Athero-protective Role Of Chk1-induced Senp2 S344 Phosphorylation Under Laminar Flow

Atherosclerosis (AthS) tends to form at arterial regions exposed to disturbed (DF) but not laminar (LF) flow. Previously, we reported the significant role of SUMOylation in DF-induced endothelial (EC) activation and subsequent AthS formation via upregulating p53 and ERK5 SUMOylation. LF can inhibit the basal levels of p53 and ERK5 SUMOylation, but how LF inhibits SUMOylation remains unknown. A chemical genetics approach with high-resolution mass spectrometry revealed that the cell-cycle checkpoint kinases CHK1 can phosphorylate SENP2 S344 site, but the functional role remains largely unknown. We aimed to study the functional role of CHK1 in SENP2 function and subsequent AthS. First, we generated phospho-specific SENP2 S344 antibody, and found that LF, but not DF, increases SENP2 S344 phosphorylation. We found the increase of CHK1 S280 phosphorylation (related to nuclear translocation) after LF, suggesting LF-induced nuclear translocation of CHK1. The CHK1 specific inhibitor of GDC0575 and the depletion of CHK1 inhibited LF-induced SENP2 S344 phsophorylation and increased both p53 and ERK5 SUMOylation in ECs. These data suggested the crucial role of CHK1 in LF-induced SENP2 S344 phosphorylation and upregulating de-SUMOylation activity. Next, we generated CRISPR/Cas9-induced Senp2 S344A knock-in (KI) and found that the mutation of SENP2 S344A accelerated p53 and ERK5 SUMOylation. LF-induced reduction of p21, cleaved caspase 3, and ICAM-1 were all reversed by SENP2 S344A mutation. Lastly, we found that the significant acceleration of AthS formation in both ascending aorta/arch (DF area) and descending aorta (LF area) in KI mice compared to wild type (WT) mice after receiving AAV-PCSK9 and high fat diet. We also performed bone marrow transplantation (BMT) after 13 Gy whole body radiation, and larger AthS but only in the aortic arch was observed in BMT mice from WT donor to KI recipient, but not in BMT mice from WT donor to WT recipient, supporting the role of endothelial SENP2 S344 phosphorylation on AthS. The radiation reduced CHK1 expression in ECs, which may explain the different regulatory pattern under BMT or non-BMT. Taken together, these results suggest the critical role of CHK1-mediated SENP2 S344 phosphorylation on LF-induced anti-AthS effects.

Fetal resorption coincides with dysregulated LH secretion in AMH-overexpressing mice

Female anti-Müllerian hormone (AMH) overexpressing (Thy1.2-AMHTg/0) mice experience fetal resorption (miscarriage) by mid-gestation. This study examined whether the ovary, uterine implantation sites and hypothalamus are potential sites of AMH action, as AMH type-2 receptor (AMHR2) expression is reported in each tissue. Pregnancy in Thy1.2-AMHTg/0 mice was compared to wild-type (WT) mice via histological examination of implantation sites, hormone assays, embryo culture and embryo transfer. Uterine AMH and AMHR2 expression was examined by RT-qPCR and immunohistochemistry. The first signs of fetal resorption in the Thy1.2-AMHTg/0 dams occurred at embryonic day 9.5 (E9.5) with 100% of fetuses resorbing by E13.5. Cultured embryos from Thy1.2-AMHTg/0 dams had largely normal developmental rates but a small proportion experienced a minor developmental delay relative to embryos from WT dams. However, embryos transferred from WT donor females always failed to survive to term when transferred into Thy1.2-AMHTg/0 dams. Amh and Amhr2 mRNA was detected in the gravid uterus but at very low levels relative to expression in the ovaries. Progesterone and estradiol levels were not significantly different between WT and Thy1.2-AMHTg/0 dams during pregnancy but luteinizing hormone (LH) levels were significantly elevated in Thy1.2-AMHTg/0 dams at E9.5 and E13.5 relative to WT dams. Collectively, these experiments suggest that AMH overexpression does not cause fetal resorption through an effect on oocytes or preimplantation embryo development. The Thy1.2-AMHTg/0 fetal resorption phenotype is nearly identical to that of transgenic LH overexpression models, suggesting that neuroendocrine mechanisms may be involved in the cause of the miscarriage.

Loss of SIRT1 inhibits hematopoietic stem cell aging and age-dependent mixed phenotype acute leukemia

Aging of hematopoietic stem cells (HSCs) is linked to various blood disorders and malignancies. SIRT1 has been implicated in healthy aging, but its role in HSC aging is poorly understood. Surprisingly, we found that Sirt1 knockout improved the maintenance of quiescence of aging HSCs and their functionality as well as mouse survival in serial bone marrow transplantation (BMT) recipients. The majority of secondary and tertiary BMT recipients of aging wild type donor cells developed B/myeloid mixed phenotype acute leukemia (MPAL), which was markedly inhibited by Sirt1 knockout. SIRT1 inhibition also reduced the growth and survival of human B/myeloid MPAL cells. Sirt1 knockout suppressed global gene activation in old HSCs, prominently the genes regulating protein synthesis and oxidative metabolism, which may involve multiple downstream transcriptional factors. Our results demonstrate an unexpected role of SIRT1 in promoting HSC aging and age-dependent MPAL and suggest SIRT1 may be a new therapeutic target for modulating functions of aging HSCs and treatment of MPAL.

Abstract 10691: Single Cell Florescence Imaging in Hypertrophic Cardiomyopathy Reveals a Proarrhythmic Substrate: Implications for Arrhythmogenesis and Antiarrhythmic Therapy

Introduction: Hypertrophic cardiomyopathy (HCM) is associated with high morbidity and mortality and remains the leading cause of sudden death in young people. Despite 5 decades of research, there is no disease modifying therapy for HCM. The cellular mechanisms leading to a proarrhythmic substrate are not yet fully elucidated but may be patient-specific. Here, we present single cell electrophysiology results of adult cardiomyocytes isolated from a cohort of HCM patients who underwent septal reduction therapy with surgical myectomy. Methods: Freshly isolated surgical specimens from HCM patients were enzymatically digested into single ventricular myocytes and compared to discarded wild-type (WT) donor hearts as controls. Myocytes displayed characteristic rod-shaped morphology, clear sarcolemmal ultrastructure, and intact plasma membranes without significant inclusions. After Ca 2+ readaptation and labeling with both a voltage and Ca 2+ sensitive fluorescent dye, cells were field stimulated at various pacing frequencies to obtain voltage and Ca 2+ tracings. APD restitution, response to drug therapy, and baseline data was compared to patient’s clinical data including electrocardiography, pathology, and multimodality imaging. Results: As compared to WT controls, HCM myocytes displayed significantly prolonged APD and Ca 2+ transients, as well as steeper APD restitution at all pacing frequencies tested. These results correlated with clinical QTc prolongation which was evident in HCM patients’ baseline ECGs that was not seen in controls. Despite genetic diversity in HCM samples, the HCM specimens appeared to share a common proarrhythmic phenotype. Finally, pathologic analysis of the myectomy specimens revealed evidence of myocyte hypertrophy, fibrosis, and cellular disarray, consistent with the clinical diagnosis of HCM. Conclusions: The single cell electrophysiology of HCM is markedly different than WT controls and displays a proarrhythmic phenotype. Single cell fluorescence imaging of a patient’s myocytes is a promising approach to elucidate patient specific electrophysiologic signatures and predict precision therapies for these patients.

Cell Competition between Wild-Type and JAK2V617F Mutant Cells Prevents Disease Relapse after Stem Cell Transplantation in a Murine Model of Myeloproliferative Neoplasm

IntroductionDisease relapse after allogeneic stem cell transplantation is a major cause of treatment-related morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). The cellular and molecular mechanisms for MPN relapse are not well understood. In this study, we investigated the role of cell competition between wild-type and JAK2V617F mutant cells in MPN disease relapse after stem cell transplantation.MethodsJAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) were crossed with Tie2-cre mice to express JAK2V617F specifically in all hematopoietic cells and vascular endothelial cells (Tie2FF1), so as to model the human diseases in which both the hematopoietic stem cells and endothelial cells harbor the mutation.ResultsTo investigate the underlying mechanisms for MPN disease relapse, we transplanted wild-type CD45.1 marrow directly into lethally irradiated Tie2FF1 mice or age-matched control mice(CD45.2). During a 6-7mo follow up, while all wild-type control recipients displayed full donor engraftment, ~60% Tie2FF1 recipient mice displayed recovery of the JAK2V617Fmutant hematopoiesis (mixed donor/recipient chimerism) 10 weeks after transplantation and developed a MPN phenotype with neutrophilia and thrombocytosis, results consistent with our previous report. Using CD45.1 as a marker for wild-type donor and CD45.2 for JAK2V617F mutant recipient cells, we found that the wild-type HSCs (Lin -cKit +Sca1 +CD150 +CD48 -) were severely suppressed and the JAK2V617F mutant HSCs were significantly expanded in the relapsed mice; in contrast, there was no significant difference between the wild-type and mutant HSC numbers in the remission mice. (Figure 1)Cell competition is an evolutionarily conserved mechanism in which “fitter” cells out-compete their “less-fit” neighbors. We hypothesize that competition between the wild-type donor cells and JAK2V617F mutant recipient cells dictates the outcome of disease relapse versus remission after stem cell transplantation. To support this hypothesis, we found that there was no significant difference in cell proliferation, apoptosis, or senescence between wild-type and JAK2V617F mutant HSPCs in recipient mice who achieved disease remission; in contrast, in recipient mice who relapsed after the transplantation, wild-type HSPC functions were significantly impaired (i.e., decreased proliferation, increased apoptosis, and increased senescence), which could alter the competition between co-existing wild-type and mutant cells and lead to the outgrowth of the JAK2V617F mutant HSPCs and disease relapse. (Figure 2)To understand how wild-type cells prevent the expansion of JAK2V617F mutant HSPCs, we established a murine model of wild-type and JAK2V617F mutant cell competition. In this model, when 100% JAK2V617F mutant marrow cells (from the Tie2FF1 mice) are transplanted alone into lethally irradiated wild-type recipients, the recipient mice develop a MPN phenotype ~4wks after transplantation; in contrast, when a 50-50 mix of mutant and wild-type marrow cells are transplanted together into the wild-type recipient mice, the JAK2V617F mutant donor cells engraft to a similar level as the wild-type donor cells and the recipient mice displayed normal blood counts during more than 4-months of follow up. In this model, compared to wild-type HSPCs, JAK2V617F mutant HSPCs generated significantly more T cells and less B cells in the spleen, and more myeloid-derived suppressor cells (MDSCs) in the marrow; in contrast, there was no difference in T, B, or MDSC numbers between recipients of wild-type HSPCs and recipients of mixed wild-type and JAK2V617F mutant HSPCs. We also found that program death ligand 1 (PD-L1) expression was significantly upregulated on JAK2V617F mutant HSPCs compared to wild-type cells, while PD-L1 expression on mutant HSPCs was significantly decreased when there was co-existing wild-type cell competition. These results indicate that competition between wild-type and JAK2V617F mutant cells can modulate the immune cell composition and PD-L1 expression induced by the JAK2V617F oncogene. (Figure 3)ConclusionOur study provides the important observations and mechanistic insights that cell competition between wild-type donor cells and JAK2V617F mutant recipient cells can prevent MPN disease relapse after stem cell transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

JMML tumor cells disrupt normal hematopoietic stem cells by imposing inflammatory stress through overproduction of IL-1β

AbstractDevelopment of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1β, but not IL-6, T NF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1β receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R−/− double mutant mice. These findings suggest IL-1β signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML.

Inducible SBDS Deficiency and Germline FANCC/FANCG Deficiency Differentially Impact the Capacity of Bone Marrow Niches to Engraft Hematopoietic Stem Cells after Transplantation

Strategies to optimize interactions between the recipient bone marrow (BM) microenvironment and donor hematopoietic stem cells (HSC) could improve hematopoietic stem cell transplantation (HSCT) outcomes by reducing graft failure rates, improving blood and immune reconstitution efficiency, and reducing intensive cytotoxic conditioning requirements. Such methods are particularly relevant for patients with inherited bone marrow failure syndromes (iBMFs), rare genetic disorders for which HSCT is required to cure bone marrow failure (BMF), but for which HSCT is also associated with a high incidence of delayed donor engraftment, graft failure and morbidity due to chemotherapy and radiation sensitivity.In previous studies of the MPL-/- model of congenital amegakaryocytic thrombocytopenia, we discovered that decreased numbers of megakaryocytes and/or long-term (LT)-HSC caused by absent thrombopoietin/MPL signaling resulted in diminished capacity of the MPL-/- BM niche microenvironment to efficiently engraft wildtype (WT) donor HSC after HSCT. This observation led us to hypothesize that pre-existing BMF, regardless of cause, may generally alter HSC niche elements critical for engraftment after HSCT. We now present data testing this hypothesis in mouse models of Fanconi Anemia (FA) and Shwachman-Diamond Syndrome (SDS).To model FA, we used double mutant FANCC-/-;FANCG-/- mice, which have previously been demonstrated to develop manifestations of BMF. In our facility, FANCC-/-;FANCG-/- mice did not develop evidence of BMF, as measured by blood counts, BM cellularity, and HSC quantitation out to 1 year of age even with induction of HSC cycling by polyinosinic-polycytidilic acid (pIpC). Using SCT assays in which GFP+ WT donor BM was transplanted into FA recipients (FANCC-/-, FANCG-/-, FANCC-/-;FANCG-/-) or WT controls after 800-1100 cGy total body irradiation (TBI), efficiency and durability of donor engraftment was assessed at 3 weeks and 5 months, respectively, after primary SCT by competitive secondary HSCT. Unexpectedly, all FA recipients demonstrated more rapid BM reconstitution and enhanced early hematopoietic progenitor engraftment of WT donor BM compared with WT controls. However, there was no significant difference in LT-HSC engraftment efficiency or durability in the FA recipient groups versus WT controls.To model SDS, we first attempted to generate a model of osteoblast-specific SBDS deletion by crossing Col1A1Cre mice with mice carrying floxed SBDS alleles (SBDSfl/fl). However, this Col1A1CreSBDSExc model proved to be embryonic lethal at E12-E15 due to profound developmental abnormalities. We thus generated an inducible model by crossing SBDSfl/fl mice with Mx1Cre+ mice, and inducing SBDS deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by pIpC administration. SBDS deletion induced by pIpC was confirmed with PCR methods.Within 4 weeks of pIpC induction, Mx1CreSBDSExc mice show evidence of BMF, including a 40% reduction in platelet counts, inverted myeloid/lymphoid cell ratio in blood, and up to 80% reduction in immunophenotypic BM LT-HSC. When we attempted HSCT with GFP+ WT donor BM in Mx1CreSBDSExc and littermate control recipients following 1100 cGy TBI, 90% of Mx1CreSBDSExc recipients died by day 9 after HSCT, whereas all littermate controls survived. BM sections from Mx1CreSBDSExc recipients demonstrated persistent aplasia out to 1 week post-HSCT, suggesting that Mx1CreSBDSExc died from primary graft failure. Competitive secondary HSCT performed from BM harvested 1 week after primary HSCT demonstrated severe deficits in GFP+ WT donor HSC engraftment in Mx1CreSBDSExc recipients. Our initial studies as to why donor engraftment fails in Mx1CreSBDSExc recipients have identified that compared to WT, Mx1CreSBDSExc mice show significantly reduced expansion of osteolineage niche cells after TBI, a response that we have previously shown to be critical for efficient donor engraftment after HSCT.Taken together, our data demonstrate that pre-existing BMF and SBDS deficiency in BM osteolineage cells, but not FANCC or FANCG deficiency without co-existent BMF, significantly reduce the capacity of the BM niche to engraft donor HSC. Future studies defining specific niche deficits in our inducible SBDS deficiency model will provide critical insights into cellular and molecular pathways required for efficient engraftment after HSCT. DisclosuresNo relevant conflicts of interest to declare.

Graft Versus Host Disease Activity of Donor T Cells Is Regulated By Vasoactive Intestinal Polypeptide Synthesized By Donor Plasmacytoid Dendritic Cells

Introduction: Increased numbers of plasmacytoid dendritic cells (pDC) in the bone marrow graft improve clinical outcomes with decreased graft-versus-host disease (GvHD) and improved survival in previous pre-clinical murine experiments (Lu Y, Blood. 2012;119:1075-1085) and in clinic data from BMT CTN 201 (Waller JCO 2014; 32:2365-72). The mechanism by which donor pDC limit severe GvHD and improve survival after allo-BMT is unknown. We have previously described higher levels of donor T cell activation and expansion (measured as bio-luminescent radiance) among recipients of VIP-KO pDC (Li, J.X Blood 2016 128:2154). We hypothesize that blocking VIP signaling augment the magnitude of T cell activation and expansion, and that modulating the VIP pathway may be a potential therapeutic target for regulating GvHD in allo-HSCT.Methods:We used a MHC mismatched C57BL/6 àB10BR allo-BMT model, transplanting purified populations of FACS sorted Lin-Sca-1+C-kit+ (LSK) stem cells combined with FACS purified BM pDC and MACS purified splenic T cells. Lethally irradiated B10BR recipients received 5,000 HSC, 50,000 pDC from wild type (WT) or VIP-KO donor and 1×106 T cells from WT C57BL6 donor mice. Survival and GvHD were measured based on fur texture, posture, activity, skin integrity and weight body loss. To test VIP production by pDC cells, we purified donor pDC from VIP-GFP C57 BL/6 mice, in which GFP transcription is under the control of the VIP gene promotor, and transplanted them into lethally irradiated MHC mis-matched B10.BR recipients. VIP expression in mice receiving pDC cells from a VIP-GFP transgenic donor was detected by fluorescence confocal microscopy. Immune polarization of donor T cells was accessed by intracellular cytokine staining and flow cytometry. Mice transplanted with VIP-GFP pDC cells were sacrificed on D10 and spleens were collected in OCT.Results: Survival of recipients receiving HSC, T cells and WT pDC was 71% on day100 post-transplant while survival of those receiving VIP-KO pDC was 31% (Fig 1A). Splenocytes were obtained from recipients on day 3 post-transplant. IFN-γexpression was higher and IL-10 epression was significantly lower in donor CD8+ T cells in mice receiving VIP-KO pDC compared with WT pDC (Fig 1B). Confocal microscopy showed VIP expression in donor pDC among recipients of VIP-GFP pDC (Fig 2)Conclusion:These data indicated VIP signaling occurs via interactions between donor T cells and donor pDC. Local production of VIP inhibits activation and Th1 immune polarization of donor T cells. In murine allo-BMT, absence of VIP expression in pDC leads to increased T cell activation, expansion, allo-reactivity and Th1 polarization resulting in worse survival. Thus VIP is a critical pathway in regulating GvHD and GvL action of donor T cells and an attractive pharmacological target for GvHD prevention. [Display omitted] DisclosuresWaller:AMGEN: Consultancy; PRA: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Celldex: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Helocyte: Consultancy; Cerus: Equity Ownership; National Institutes of Health: Research Funding; Chimerix: Equity Ownership; Coulter Foundation: Research Funding; Katz Foundation: Research Funding.

Lineage-Specific Chimerism and Outcome After Hematopoietic Stem Cell Transplantation for DOCK8 Deficiency

Bi-allelic variants in the dedicator of cytokinesis 8 (DOCK8) gene cause a combined immunodeficiency, characterized by recurrent sinopulmonary and skin infections, food allergies, eczema, eosinophilia, and elevated IgE. Long-term outcome is poor given susceptibility to infections, malignancy, and vascular complications. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option and has shown promising outcome. The impact of mixed chimerism on long-term outcome is unclear. We reasoned that reversal of disease phenotype would depend on cell lineage-specific chimerism. DOCK8 variants were confirmed by Sanger and/or exome sequencing and immunoblot and/or intracellular flow cytometry. Donor chimerism was analyzed by XY-fluorescence in situ hybridization or quantitative short tandem repeat PCR. Outcome was assessed by laboratory tests, lymphocyte subsets, intracellular DOCK8 protein flow cytometry, T-cell proliferation analysis, and multiparameter immunoblot allergy screening. We report on nine patients, four of whom with mixed chimerism, with a median follow-up of 78 months after transplantation. Overall, we report successful transplantation with improvement of susceptibility to infections and allergies, and resolution of eczema in all patients. Immunological outcome in patients with mixed chimerism suggests a selective advantage for wild-type donor T-cells but lower donor B-cell chimerism possibly results in a tendency to hypogammaglobulinemia. No increased infectious and allergic complications were associated with mixed chimerism. Aware of the relatively small cohort size, we could not demonstrate a consistent detrimental effect of mixed chimerism on clinical outcomes. We nevertheless advocate aiming for complete donor chimerism in treating DOCK8 deficiency, but recommend reduced toxicity conditioning.

Pertussis toxin inhibits encephalitogenic T cell infiltration and promotes a B cell driven disease during TH17-EAE

Abstract Pertussis toxin (PTX) is required as a co-adjuvant to induce disease in active Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The objective of this study was to investigate the role of PTX during adoptive transfer TH17-EAE and observe alterations in the disease phenotype. TH17-EAE was induced by transferring myelin-specific TH17 cells, harvested from MOG35-55 immunized wild type (WT) donor mice, into WT or B cell deficient (μMT) recipient mice. The recipient mice also received PTX or PBS on days 0 and 2 and monitored for paralysis. In WT mice, PTX significantly reduced disease severity and delayed onset of paralysis. PTX reduced accumulation of pathogenic T helper cells and myeloid cells in the CNS, which we found to be associated with PTX inhibition of chemokine receptor dependent trafficking. Strikingly, our data shows that PTX promotes the accumulation of B cells in the CNS and elevates the capacity of B cells to present antigen to T cells in the periphery, suggesting an altered disease toward a B cell-dependent pathology. We found that without PTX treatment, the severity of disease was equivalent between WT and μMT mice. In contrast, with PTX treatment, the μMT mice had significantly less disease compared to the WT mice. In conclusion, our data suggests that during TH17-EAE, PTX restricts the trafficking of pathogenic T helper cells and myeloid cells into the CNS and thereby delays the disease onset. However, not only PTX does not alter trafficking of B cells into the CNS it also stimulates a more inflammatory B cell phenotype. This study provides novel insights to interactions between TH17 and B cells in mice and has defined a new model to study the pathologic functions of B cells during multiple sclerosis.

Thymic microbiota-specific T cells role in intestinal inflammation

Abstract Inflammatory bowel disease (IBD) patients, including Crohn’s disease and ulcerative colitis, exhibit dysregulated immune responses. Although IBD etiology remains uncertain, microbiota-reactive T cells are thought to play a role in pathogenesis. It has been shown that gut microbiota and their metabolites play a role in modulating mucosal immune responses and thus, have the potential to influence the development of IBD. Although host-microbe interactions are required to balance effector and regulatory T cells responses, it remains unknown how microbiota-specific T cells are generated. Here we studied how microbiota-specific T cells develop and how they can drive intestinal inflammation. Colonization of mice at weaning induced the expansion of naive microbiota-specific T cells in the thymus before peripheral distribution. Transfer of naive thymic microbiota-specific T cells into immunodeficient mice induced intestinal inflammation by differentiating into Th1 or Th17 cells. We identified that CX3CR1+ dendritic cells (CX3+DCs) were required for this process, as depleting these cells decreased the expansion of thymic microbiota-specific T cells. Transfer of thymic T cells from mice lacking CX3+ DCs induced less colitis in immunodeficient mice compared to wildtype donors. Moreover, inhibiting CX3+ DCs antigen-presentation suppressed induction of microbiota-specific T cells and transfer of these cells promoted less intestinal inflammation. Together, our data suggests CX3+ DCs drive T cell selection of microbiota-specific T cells in the thymus, which can differentiate into intestinal effector T cells mediating pathology in immunodeficient hosts.

STAT1 protects CD4 T cells from deletion by NK cells during influenza infection and promotes Th1 identity that can be enhanced through STAT4 to improve their anti-viral impact

Abstract Most CD4 T cells responding to influenza A virus (IAV) fit prototypical Th1 criteria including high expression of the transcription factor T-bet, the Th1 ‘master regulator’, and strong IFNg production. Optimal Th1 development in vitro also requires cytokine-dependent activation of the transcription factors STAT4 and STAT1, but how these pathways impact anti-viral responses is not clear. Here, to focus on the roles of CD4 T cell-intrinsic STAT4 and STAT1, we transferred STAT-deficient (STAT−/−) or wildtype CD4 T cells recognizing virus to congenic wildtype hosts, challenged with IAV, and analyzed donor effector responses in the infected lungs. STAT4−/− and wildtype cells reached similar numbers with no differences seen in their Th1 identity. In contrast, STAT1−/− donor cells were barely detected in host mice but reached similar numbers to wildtype donors when NK cells were depleted. Analysis of STAT1−/− cells in NK cell-depleted hosts revealed severely compromised Th1 identity that mirrored T-bet−/− donor cell responses. While these findings suggest that full Th1-polarization during IAV infection does not require STAT4, treatment of mice with IL-12 dramatically increases Th1 attributes in wildtype but not STAT4−/−CD4 T cells. This indicates that the wildtype IAV-primed CD4 T cell response is not strongly Th1-polarized. Finally, priming CD4 T cells in vitro with STAT4- and STAT1-activating cytokines promotes effector cells better able to combat IAV when transferred to unprimed mice than when only STAT1 activating signals are present. Enhancing Th1 identity through harnessing CD4 T cell-intrinsic STAT4 activation can thus improve their anti-viral impact during infection, with important implications for vaccine design.

Preclinical Application of Reduced Manipulated Processing Strategy to Collect Transplantable Hepatocytes: A Pilot and Feasibility Study.

Background: The complex isolation and purification process of hepatocytes for transplantation is labor intensive and with great contamination risk. Here, as a pilot and feasibility study, we examined in vitro and in vivo hepatocyte isolation feasibility and cell function of Cell Saver® Elite®, an intraoperative blood-cell-recovery system. Methods: Rat and pig liver cells were collected using this system and then cultured in vitro, and their hepatocyte-specific enzymes were characterized. We then transplanted the hepatocytes in an established acute liver–injured (retrorsine+D-galactosamine-treated) rat model for engraftment. Recipient rats were sacrificed 1, 2, and 4 weeks after transplantation, followed by donor-cell identification and histological, serologic, and immunohistopathological examination. To demonstrate this Cell Saver® strategy is workable in the first place, traditional (classical) strategy, in our study, behaved as certainty during the cell manufacturing process for monitoring quality assurance throughout the course, from the start of cell isolation to post-transplantation. Results: We noted that in situ collagenase perfusion was followed by filtration, centrifugation, and collection in the Cell Saver® until the process ended. Most (>85%) isolated cells were hepatocytes (>80% viability) freshly demonstrating hepatocyte nuclear factor 4α and carbamoyl-phosphate synthase 1 (a key enzyme in the urea cycle), and proliferating through intercellular contact in culture, with expression of albumin and CYP3A4. After hepatocyte transplantation in dipeptidyl peptidase IV (−/−) rat liver, wild-type donor hepatocytes engrafted and repopulated progressively in 4 weeks with liver functional improvement. Proliferating donor hepatocyte–native biliary ductular cell interaction was identified. Post-transplantation global liver functional recovery after Cell Saver and traditional methods was comparable. Conclusions: Cell Saver® requires reduced manual manipulation for isolating transplantable hepatocytes.

Fibroblast Expression of Thy-1 Protects Grafts from Chronic Lung Allograft Dysfunction

<h3>Purpose</h3> Multiple immunologic and physiologic insults can contribute to chronic lung allograft dysfunction (CLAD). Thy-1 is a surface glycoprotein that can downregulate fibroblast differentiation to myofibroblasts and fibroblast activation. The role of Thy-1 in CLAD has not been explored. <h3>Methods</h3> We utilized the minor antigen mismatched C57BL/6 (B6)→C57BL/10 (B10) model of murine lung transplantation. To mimic bacterial infection some mice received LPS (5μg in 50μl PBS/dose) intratracheally every 4 days until sacrifice on postoperative day 30 while some mice were treated with saline. We transplanted either wild type B6 or Thy-1 deficient grafts to B10 recipient mice (B6^wt→B10 vs. B6^Thy1-/−→B10). Grafts were assessed based on histological grading for obliterative bronchiolitis (OB) and parenchymal fibrosis. To assess the role of LPS on fibroblast-specific Thy-1 expression, we utilized primary cell cultures of murine lung fibroblasts isolated from B6 mice, using media containing 1μg/ml of LPS. <h3>Results</h3> LPS treatment resulted in CLAD which was more severe in B6^Thy1-/- donors(Figure A).More importantly immunohistochemical evaluation demonstrated that that all OB and fibrotic lesion, even in wild-type donors consisted of Thy-1 negative fibroblast. In vitro administration of LPS significantly downregulated Thy-1 in wild-type fibroblast through the downregulation of protein-specific mRNA (<i>P</i><0.05) (Figure B). <h3>Conclusion</h3> We for the first time demonstrate the link between the downregulation of Thy-1 and CLAD. We also demonstrate that LPS results in loss of Thy-1 on stromal cells thus contributing to proliferative airway lesions and parenchymal fibrosis. Our findings suggest that Thy-1 replacement may serve as a therapeutic option to prevent chronic rejection.

Interpretation of BRCA2 Splicing Variants: A Case Series of Challenging Variant Interpretations and the Importance of Functional RNA Analysis

A substantial proportion of pathogenic variants associated with an increased risk of hereditary cancer are sequence variants affecting RNA splicing. The classification of these variants can be complex when both non-functional and functional transcripts are produced from the variant allele. We present four BRCA2 splice site variants with complex variant interpretations (BRCA2 c.68-3T>G, c.68-2A>G, c.425G>T, c.8331+2T>C). Evidence supporting a pathogenic classification is available for each variant, including in silico models, absence in population databases, and published functional data. However, comprehensive RNA analysis showed that some functional transcript may be produced by each variant. BRCA2 c.68-3T>G results in a partial splice defect. For BRCA2 c.68-2A>G and c.425G>T, aberrant splicing was shown to produce a potentially functional, in-frame transcript. BRCA2 c.8331+2T>C may utilize a functional GC donor in place of the wild-type GT donor. The severity of cancer history for carriers of these variants was also assessed using a history weighting algorithm and was not consistent with pathogenic controls (carriers of known pathogenic variants in BRCA2). Due to the conflicting evidence, our laboratory classifies these BRCA2 variants as variants of uncertain significance. This highlights the importance of evaluating new and existing evidence to ensure accurate variant classification and appropriate patient care.

Epilepsy, status epilepticus, and hemiplegic migraine coexisting with a novel SLC4A4 mutation.

Recessive mutations in the SLC4A4 gene cause a syndrome characterised by proximal renal tubular acidosis (pRTA), mental retardation, dental and ocular abnormalities, and hemiplegic migraine. Rare cases involving the development of epilepsy or its severe complication-status epilepticus-have been described. The clinical and genetic status of four affected members in a Spanish family was studied. The SLC4A4 gene mutation was detected with a next-generation sequencing (NGS) panel in the proband, and Sanger confirmed the putative mutations in affected relatives. In silico analysis was performed to elucidate the putative effect of mutation on the splicing process. A novel mutation, c.2562+2T>G, was identified in the homozygous state in all diseased members of the family. This mutation affected a canonical splice site and is predicted to abolish the wild-type donor site, which predicts a premature truncated NBCe1 protein with cotransport activity. The resulting protein lacks the 190 amino acids of the carboxyl-terminus, and the effect is likely to be a loss of function. All patients suffered from severe pRTA and ocular abnormalities, and the adults also suffered from neurological complications, such as hemiplegic migraine and/or epilepsy. Two developed life-threatening status epilepticus, although they fully recovered and remained free of seizures with valproate. These results expand the clinical and mutational spectra of SLC4A4-related disease and have implications for understanding the potential role of NBCe1 in the pathophysiologic processes of hemiplegic migraine and epilepsy/status epilepticus associated with the mutation.

Bone marrow transplantation into Abcd1-deficient mice: Distribution of donor derived-cells and biological characterization of the brain of the recipient mice.

X-linked adrenoleukodystrophy (X-ALD) is a severe inherited metabolic disease with cerebral inflammatory demyelination and abnormal accumulation of very long chain fatty acid (VLCFA) in tissues, especially the brain. At present, bone marrow transplantation (BMT) at an early stage of the disease is the only effective treatment for halting disease progression, but the underlying mechanism of the treatment has remained unclear. Here, we transplanted GFP-expressing wild-type (WT) or Abcd1-deficient (KO) bone marrow cells into recipient KO mice, which enabled tracking of the donor GFP+ cells in the recipient mice. Both the WT and KO donor cells were equally distributed throughout the brain parenchyma, and displayed an Iba1-positive, GFAP- and Olig2-negative phenotype, indicating that most of the donor cells were engrafted as microglia-like cells. They constituted approximately 40% of the Iba1-positive cells. Unexpectedly, no decrease of VLCFA in the cerebrum was observed when WT bone marrow cells were transplanted into KO mice. Taken together, murine study suggests that bone marrow-derived microglia-like cells engrafted in the cerebrum of X-ALD patients suppress disease progression without evidently reducing the amount of VLCFA in the cerebrum.

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

Novel compound heterozygous mutation in WEE2 is associated with fertilization failure: case report of an infertile woman and literature review

BackgroundFertilization failure after intracytoplasmic sperm injection continues to affect couples and the etiology is not well-understood.Case presentationWe characterized a couple with 2-year history of primary unexplained infertility. Three different assisted reproduction attempts (IVF + rescue ICSI, ICSI and ICSI-AOA) showed repeated fertilization failure for MII oocyte retrieval after controlled ovarian hyperstimulation. After whole-exome sequencing and sanger sequencing of the couple and their family members, variant pathogenicity was assessed using SIFT, PolyPhen2, Mutation Taster, and Human Splicing Finder software. We identified novel compound heterozygous mutations, c.1535 + 3A > G and c.946C > T (p. Leu316Phe), in WEE2 in the female proband. Trios analysis of the variations revealed an autosomal recessive pattern. c.1535 + 3A > G in WEE2 was predicted to break the wild-type donor site and affect splicing, and the missense mutation c.946C > T (p. Leu316Phe) of WEE2 was predicted to be pathogenic.ConclusionA novel compound heterozygous mutation in WEE2 was identified in an infertile female who experienced repeated fertilization failure even after ICSI-AOA. These novel mutations in WEE2 provided genetic evidence for fertilization failure.

Tristetraprolin Overexpression in Non-hematopoietic Cells Protects Against Acute Lung Injury in Mice.

Tristetraprolin (TTP) is a mRNA binding protein that binds to adenylate-uridylate-rich elements within the 3′ untranslated regions of certain transcripts, such as tumor necrosis factor (Tnf) mRNA, and increases their rate of decay. Modulation of TTP expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. Accordingly, we tested the role of TTP in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. LPS-challenged TTP-knockout (TTPKO) mice, as well as myeloid cell-specific TTP-deficient (TTPmyeKO) mice, exhibited significant increases in lung injury, although these responses were more robust in the TTPKO. Mice with systemic overexpression of TTP (TTPΔARE) were protected from ALI, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ALI. Interestingly, while irradiated wild-type (WT) mice reconstituted with TTPKO hematopoietic progenitor cells (HPCs) showed exaggerated ALI, their reconstitution with the TTPΔARE HPCs mitigated ALI. The reconstitution of irradiated TTPΔARE mice with HPCs from either WT or TTPΔARE donors conferred significant protection against ALI. In contrast, irradiated TTPΔARE mice reconstituted with TTPKO HPCs had exaggerated ALI, but the response was milder as compared to WT recipients that received TTPKO HPCs. Finally, the reconstitution of irradiated TTPKO recipient mice with TTPΔARE HPCs did not confer any protection to the TTPKO mice. These data together suggest that non-HPCs-specific overexpression of TTP within the lungs protects against ALI via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration.