Cells In KO Mice Research Articles

275-LB: Mitochondrial Fission Factor Can Be a Key Factor in the Regulation of Hepatic Lipid Metabolism

Nonalcoholic fatty liver disease (NAFLD) develops into nonalcoholic steatohepatitis (NASH) when fat accumulates in the liver, leading to hepatocyte apoptosis, inflammation and fibrosis. No effective treatments for NASH have been established. Mitochondria are important organelles that play a central role in de novo lipogenesis and fatty acid β-oxidation. We have shown that mitochondrial fission is enhanced by short-term high-fat diet (HFD) loading, but is impaired by long-term. We speculated that mitochondrial fission inhibition is associated with the development of NASH and generated mice deletion of hepatocyte-specific mitochondrial fission factor (KO) mice. Conditioning HFD, KO mice showed inhibition of hepatocyte mitochondrial fission, increased ER stress and decreased triglyceride secretion from the liver. The hepatic triglyceride contents had significantly increased in KO mice compared to control (CONT), and histological analysis showed a marked increase in large lipid droplets (LDs). In addition to marked fat accumulation, hepatic apoptosis, inflammation and fibrosis were increased in the livers of KO mice, indicating the development of NASH. We noted that large LDs were increased in KO mice and added an additional analysis to determine how mitochondrial fission inhibition is involved in LDs formation in the liver. Oleic acid is a representative monounsaturated fatty acid that promotes LDs deposition in hepatocytes and is mildly cytotoxic by inducing ER stress and apoptosis. Loading primary cultured hepatocytes (PH) with oleic acid increased the number of smaller LDs in PH of CONT mice, whereas LDs in cells of KO mice became larger. Gene expression of Cidec, which acts on the fusion of LDs, was increased in the liver of KO mice, suggesting that inhibition of mitochondrial fission may induce the fusion of LDs and the enlargement of intracellular LDs in the liver. Further analysis is planned, as inhibition of mitochondrial fission may be involved in changes of LDs morphology. Disclosure Y. Takeichi: None. T. Miyazawa: None. Y. Ogawa: None.

QRICH1 is a negative regulator of T cell activation via the CARD11 signalosome

Abstract Controlled lymphocyte activation is crucial for defending against pathogens while preventing diseases such as autoimmunity and lymphoma. In T lymphocytes, the scaffold protein and signaling hub CARD11 mediates NF-κB, JNK, and mTOR pathway activation downstream of T cell receptor (TCR) engagement. We recently identified QRICH1 as a previously unrecognized CARD11 interactor recruited to the CARD11 complex downstream of TCR engagement and discovered that QRICH1 protein levels are upregulated in wild-type activated mouse primary CD8+ T cells. Overexpression of QRICH1 in 293T and Jurkat T cells dose-dependently inhibited NF-κB activation by wild-type CARD11 as well as by constitutively active and oncogenic CARD11 mutants. QRICH1-deficient Jurkat T cells had increased NF-κB activation and IL-2 secretion downstream of TCR stimulation. To study the in vivo role of QRICH1 in immunity, we generated a novel QRICH1 conditional KO mouse model. Compared to control mice, QRICH1 T cell KO mice had decreased splenic CD4+ and CD8+ T cells, despite no differences in thymic CD4+, CD8+, or CD4+CD8+ T cell populations. The CD8+ T cell population in QRICH1 T cell KO mice was skewed towards an effector memory phenotype. QRICH1-deficient primary CD8+ T cells had increased proliferation and production of pro-inflammatory cytokines IFN-γ and TNF-α in response to ex vivo stimulation with anti-CD3/CD28, and QRICH1-deficient OT-1 CD8+ T cells also had increased cytokine production in response to antigen-specific stimulation with Ova peptide. Our findings reveal that QRICH1 negatively regulates T cell activation by modulating CARD11’s signaling output. This work was supported by NIH grants R01AI148143, F31CA254167, and T32GM007445.

Cell-autonomous PLCβ1 modulation of neural stem/progenitor cell proliferation during adult hippocampal neurogenesis

PLCβ1 null mouse, a model for epilepsy/schizophrenia, shows enhanced moving activity, seizures, and excessive neurogenesis in the DG of the hippocampus. Since physical or epileptic activity increases neurogenesis, we asked whether the increase of neurogenesis in PLCβ1 null mice was mainly due to the loss of PLCβ1 in stem cells or from in vivo effects of the enhanced movement or seizures of null mice. To avoid in vivo effects, we did neurosphere cultures from the DG of the adult hippocampus and quantified the cell proliferation. We found an increase in the number and size of neurospheres in KO mice cultures, which was similar to the enhancement of in vivo proliferation of DG newborn cells in KO mice. Moreover, the positive effect of high KCl treatment on the proliferation of neurosphere culture was occluded in KO mice. Further DG neurons of PLCβ1 KO mice display increased excitability, consistent with a model for epilepsy. In conclusion, these results suggest cell-autonomous inhibitory roles of PLCβ1 in the proliferation of adult neural stem/progenitor cells in vivo and the excitability of DG granule cells.

P2.07: ASXL1 May Promote the Differentiation and Function of TH17 Cells

Introduction: Kidney transplantation is the most effective method for the treatment of end-stage renal disease. However, immune rejection always restricts the survival rate of the graft. Th17 cells play an important role in renal transplantation rejection while the mechanism regulating Th17 cell differentiation and function is not fully understood. Asxl1 is an epigenetic regulatory factor frequently mutated in myeloid malignancies, but the role of Asxl1 in immune rejection or T cell development and differentiation has not been reported. Method: Lymph nodes were isolated from wild-type mice (CD4Cre-Asxl1F/F, WT) and CD4+ T cell conditional Asxl1 knockout mice (CD4Cre+Asxl1F/F, KO), and CD4+CD25-CD44-CD62L+ naive T lymphocytes were sorted by the method of flow cytometry. The sorted cells mentioned above were stimulated with IL-1β (10 ng/mL), IL-23 (50 ng/mL), IL-6 (50 ng/mL), TGF-β (5 ng/mL), anti-IL-4 (10 μg/mL) and anti-IFN-γ (10 μg/mL) according to the cell density of 1×105/well for 72 h, and then Monensin (2 μmol/L), Brefeldin A (5 μg/mL), Ionomycin (0.5 μg/mL) and PDBU (1 μmol/L) were added for further stimulation for 4 h at 37℃ and 5% CO2. The stimulated cells were collected and the number and proportion of CD4+IL-17+ cells were analyzed by flow cytometry. The differentiation of Th1, Th2 and Treg cells were induced from WT mice by similar methods in vitro, and the expression of Asxl1 in different cell subsets were detected by Real-Time PCR at mRNA level. Results: Compared with the WT mice, the number and proportion of Th17 cells in KO mice were significantly decreased. The mRNA expression level of Asxl1 in Th17 cell subsets from WT mice was the highest. Conclusion: The results suggest that Asxl1 may promote the differentiation and function of Th17 cells as a positive factor. Asxl1 may become a new immune rejection target to regulate the number or function of Th17 cells, which has potential clinical significance in the intervention and prevention of rejection after kidney transplantation.

Single‐Cell Sequencing Reveals Distinct Expression Patterns of the Integrated Stress Response (ISR) Pathway Regulating Cell Survival in Secretory Cells in Colon of Hnrnp I Knockout Mice

ObjectivesHeterogeneous nuclear ribonucleoprotein I (Hnrnp I) is an RNA‐binding protein functions during pre‐mRNA splicing and regulation of alternative splicing events. Previously we found that ablation of Hnrnp I activates NF‐κB signaling in intestinal epithelial cells (IECs), resulting in increased nuclear translocation of P65 (NF‐κB subunit) and upregulation of proinflammatory cytokines and chemokines. Integrated stress response (ISR) signaling was linked to activation of canonical NF‐κB pathway. Therefore, in this study, we aimed to investigate ISR in the colon of Hnrnp I knockout mice. We hypothesize that cellular stress from Hnrnp I knockout triggers ISR, consequently affects the cellular dynamics among secretory cell types on colon epithelium (enteroendocrine, Paneth and goblet cells).MethodsTotal RNA was isolated from total colon cell preparations from WT and KO mice. Single‐cell RNAseq libraries (biological replicates, n=3) were prepared using Chromium Single‐Cell Gene Expression NextGem (v3.1, 10X Genomics). Analyses from raw counts were performed using the Seurat (v3.2.0) R package using default parameters, a computational cell calling algorithm was used to identify cell type using two published annotated mouse cell datasets. Pathway analysis was performed for secretary cell clusters including enteroendocrine cells (EECs), Paneth cells, and goblet cellsResultsComparing to WT, number of EECs in colon was significantly increased in KO mice. Total mRNA level of Atf4, the principal regulator of ISR, was significantly increased in colon secretory cells in KO mice. We also examined mRNA expression of ATF4 target genes in secretory cells. Specifically, gene encoding the regulator of apoptosis and autophagy, members of Bcl2 family, Bnip3, was significantly increased in secretory cells in KO mice, while Mlx and Gabarapl2 (Atg8), both are involved in apoptosis and autophagy signaling pathways, have a trend of increase in secretory cells in KO mice, all comparing to WT.ConclusionsCellular stress from Hnrnp I knockout activated ISR in IECs, specially in those secretory cell types – EECs, Paneth, and goblet cells. The activation of ISR led to both autophagy and apoptosis signaling in these cells through the upregulation of Bnip3, Mlxand Gabarapl2. Taken together, our results suggest that in response to the cellular stresses from the depletion of Hnrnp I in IECs, ISR activation in colonic secretory cells, stimulating apoptosis and autophagy pathway signaling, particularly in EECs, resulting in the proliferation of EECs in KO. The overall manipulation by ISR signaling in the colon secretary cells may present a critical early response to modulations in colon, potentially leading to perturbations to homeostasis among host colon epithelia, colon residential immunity, and gut microbiota.

Striated Preferentially Expressed Protein Kinase (SPEG)-Deficient Skeletal Muscles Display Fewer Satellite Cells with Reduced Proliferation and Delayed Differentiation

Centronuclear myopathies (CNMs) are a subtype of congenital myopathies characterized by skeletal muscle weakness and an increase in the number of central myonuclei. SPEG (striated preferentially expressed protein kinase) has been identified as the sixth gene associated with CNM, and it has been shown that striated muscle-specific Speg-knockout (KO) mice have defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling. The impact of SPEG deficiency on the survival and function of myogenic cells remains to be deciphered. In this study, the authors examined the overall population, proliferation, and differentiation of myogenic cells obtained from striated muscle-specific Speg-KO mice and compared them with wild-type (WT) controls. SPEG-deficient skeletal muscles contained fewer myogenic cells, which on further study demonstrated reduced proliferation and delayed differentiation compared with those from WT muscles. Regenerative response to skeletal muscle injury in Speg-KO mice was compared with that of WT mice, leading to the identification of similar abnormalities including fewer satellite cells, fewer dividing cells, and an increase in apoptotic cells in KO mice. Overall, these results reveal specific abnormalities in myogenic cell number and behavior associated with SPEG deficiency. Similar satellite cell defects have been reported in mouse models of MTM1- and DNM2-associated CNM, suggestive of shared underlying pathways.

Abstract 941: B cells are required to generate optimal antitumor immunity in response to PD-1 blockade treatment

Abstract Background: Melanoma is the most deadly form of skin cancer. Although, the immunogenic nature of melanoma makes this disease susceptible to immunotherapy, metastatic melanoma remains highly resistant to established immunotherapies like cancer vaccines. Anti-PD-1 antibodies alone showed great clinical activity but were still ineffective in many melanoma patients. These reports highlight the need to understand the molecular mechanism of immunotherapy induced anti-melanoma immune responses to develop new approaches for effective therapy against melanoma. B cells augment CD8+ T cell-mediated immunity during autoimmunity and allograft rejection, but the role of B cells in anti-tumor CD8+ T cell immunity and in cancer patient survival is controversial. Recently, we reported that intratumoral stimulation of TLR-7/8 in a mouse model of melanoma induced tumor regression and activation of B cells. B cells were required for tumor suppression in an antibody-independent manner. Also, we found that melanoma patients who responded to anti-PD-1 blockade therapy had significantly more B cells and CD8+ T cells in tumors than non-responders. Based on these evidences, we hypothesized that activated B cells induce and/or enhance anti-melanoma CD8+ T cell immunity in response to immunotherapy. Methods: B16 melanoma mouse model was used. Wild type and B cell Knockout mice were used to determine treatment efficacy and generation of tumor-specific CD8+T cells in response to PD-L1 blockade and/or TLR-7/8 activation. TLR-7/8 agonist activated B cells were isolated and adoptively transferred into tumor-bearing B cell KO mice, which were treated with anti-PD-L1.Therapeutic efficacy and melanoma-specific CD8+ T cell immunity were determined. Results: We found that absence of B cells not only decreases the efficacy of TLR-7/8 agonist or anti-PDL-1 abs monotherapy but also TLR-7/8 agonist and anti-PD-L1 abs combination therapy. In addition, mice were unable to develop potent anti- melanoma CD8+T cell immunity in the absence of B cells in response to either mono or combination therapy. Interestingly, we reported prolonged survival of tumor bearing B cell KO mice which received TLR-7/8 agonist activated B cells compared to inactivated B cells recipients in response to anti-PD-L1 antibody therapy. Conclusions: These results suggest that activated B cells enhance anti-melanoma CD8+ T cell immunity in response to immunotherapy. We expect our findings to lead the field closer to development of biomarkers based on B cell activation that predict response to immunotherapy. Our findings may also facilitate design of more effective anti-melanoma immunotherapy. Citation Format: Shubhra Singh, Karishma Bavisi, Jason Roszik, Adi Diab, Richard E. Davis, Patrick Hwu, Willem W. Overwijk, Manisha Singh. B cells are required to generate optimal antitumor immunity in response to PD-1 blockade treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 941.

The mTORC1 component RPTOR is required for maintenance of the foundational spermatogonial stem cell pool in mice†.

The self-renewal, proliferation, and differentiation of the spermatogonial populations must be finely coordinated in the mammalian testis, as dysregulation of these processes can lead to subfertility, infertility, or the formation of tumors. There are wide gaps in our understanding of how these spermatogonial populations are formed and maintained, and our laboratory has focused on identifying the molecular and cellular pathways that direct their development. Others and we have shown, using a combination of pharmacologic inhibitors and genetic models, that activation of mTOR complex 1 (mTORC1) is important for spermatogonial differentiation in vivo. Here, we extend those studies to directly test the germ cell-autonomous requirement for mTORC1 in spermatogonial differentiation. We created germ cell conditional knockout mice for "regulatory associated protein of MTOR, complex 1" (Rptor), which encodes an essential component of mTORC1. While germ cell KO mice were viable and healthy, they had smaller testes than littermate controls, and no sperm were present in their cauda epididymides. We found that an initial cohort of Rptor KO spermatogonia proliferated, differentiated, and entered meiosis (which they were unable to complete). However, no self-renewing spermatogonia were formed, and thus the entire germline was lost by adulthood, resulting in Sertoli cell-only testes. These results reveal the cell autonomous requirement for RPTOR in the formation or maintenance of the foundational self-renewing spermatogonial stem cell pool in the mouse testis and underscore complex roles for mTORC1 and its constituent proteins in male germ cell development.

Sostdc1 regulates natural killer cell maturation and cytotoxicity

Abstract Natural killer (NK) cells are specialized lymphocytes with innate ability to eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified novel roles for sclerostin domain containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (KO) mice display a progressive accumulation of transitional NK cells (CD27+CD11b+, tNK) with age, indicating a partial developmental block. The Ly49 repertoire on NK cells in KO mice is also changed. Lower frequencies (%) of KO splenic tNKs express inhibitory Ly49G2, but higher % of activating Ly49H+ and D+ cells. However, the % of Ly49I+, G2+, H+ and D+ populations were universally decreased at the most mature (CD27− CD11b+, mNK) stage. We hypothesized that the Ly49 repertoire in Sostdc1-KO mice would correlate with NK killing ability. We observed that KO NK cells are hyporesponsive against MHC-I-deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-gamma expression to controls. Further investigation is required to determine Sostdc1’s role in NK cell cytotoxicity and self/non-self recognition. Sostdc1 is expressed in the bone periosteum, and we discovered Sostdc1 expression in NK cells. Consistent with this, reciprocal BM transplant experiments showed that Sostdc1 regulates NK cells through cell-extrinsic and cell-intrinsic manners. Transcriptomic profiling should identify if Sostdc1’s known roles in Wnt and BMP signaling are active, and reveal novel effects of Sostdc1 on NK cells. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation, and NK cell cytotoxicity, and identify potential NK cell niches.

Abstract 1787: miR-150 as a predictor of hematopoietic reconstitution after myeloablation

Abstract The identification of biomarkers that allow evaluation of myeloablation and hematopoietic reconstitution time frame is important for tailoring the radiation or chemo therapy in cancer patients. The fate of hematopoietic cells is regulated at multiple stages by various genes and regulatory microRNAs. In this context, miR-150 has been demonstrated as a major regulator of lymphoid lineage differentiation and development. Our mouse model studies shows a novel role of miR-150 in imparting radiation resistance. We observed a prolonged survival benefit in mice where both the copies of miR-150 genes are deleted in comparison to that of wildtype mice in the same C57BL/6 background (p<0.05). The survival benefit after total body irradiation was found to be associated with lower apoptosis and early reconstitution in hematopoietic stem cells (lin-ckit+sca1+/-) in bone marrow. Furthermore, the recovery kinetics at sub lethal dose (6Gy) showed that HSCs in miR-150-/- mice recovers faster. Myeloid (Mac1+Gr1+) and lymphoid (CD45+CD19+) lineage cells reconstitutes within 14 days after radiation in KO mice compared to 21 days or more in WT mice. We observed a statistically significant upregulation in myeloid cells in KO mice which implicates an early recovery from radiation induced neutropenia and hence more survivability. In addition, the enhanced bone marrow reconstitution was reflected in peripheral blood counts where a noticeable difference (p<0.05) in blood RBCs lymphocytes, monocytes, neutrophils, and basophiles were noted when compared sub lethally irradiated KO with WT mice. Altogether we demonstrate that the level of miR-150 regulates the bone marrow cells distribution and hence could be targeted for achieving accelerated hematopoietic recovery after myeloablation. These findings may be translated to human cancer patients for therapy guiding based on miR-150 expression level. Besides its use in prediction of reconstitution, miR-150 in the circulating system could provide a direct readout of functional marrow and hence hematological acute radiation syndrome (H-ARS). Citation Format: Marshleen Yadav, Arnab Chakravarti, Naduparambil K. Jacob. miR-150 as a predictor of hematopoietic reconstitution after myeloablation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1787. doi:10.1158/1538-7445.AM2017-1787

Cell-autonomous requirement for mammalian target of rapamycin (Mtor) in spermatogonial proliferation and differentiation in the mouse†.

Spermatogonial stem cells must balance self-renewal with production of transit-amplifying progenitors that differentiate in response to retinoic acid (RA) before entering meiosis. This self-renewal vs. differentiation fate decision is critical for maintaining tissue homeostasis, as imbalances cause defects that can lead to human testicular cancer or infertility. Little is currently known about the program of differentiation initiated by RA, and the pathways and proteins involved are poorly defined. We recently found that RA stimulation of the Phosphatidylinositol 3-kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) kinase signaling pathway is required for differentiation, and that short-term inhibition of mTOR complex 1 (mTORC1) by rapamycin blocked spermatogonial differentiation in vivo and prevented RA-induced translational activation. Since this phenotype resulted from global inhibition of mTORC1, we created conditional germ cell knockout mice to investigate the germ cell-autonomous role of MTOR in spermatogonial differentiation. MTOR germ cell KO mice were viable and healthy, but testes from neonatal (postnatal day (P)8), juvenile (P18), and adult (P > 60) KO mice were smaller than littermate controls, and no sperm were produced in adult testes. Histological and immunostaining analyses revealed that spermatogonial differentiation was blocked, and no spermatocytes were formed at any of the ages examined. Although spermatogonial proliferation was reduced in the neonatal testis, it was blocked altogether in the juvenile and adult testis. Importantly, a small population of self-renewing undifferentiated spermatogonia remained in adult testes. Taken together, these results reveal that MTOR is dispensable for the maintenance of undifferentiated spermatogonia, but is cell autonomously required for their proliferation and differentiation.

Possible pathogenic engagement of soluble Semaphorin 4D produced by γδT cells in medication-related osteonecrosis of the jaw (MRONJ)

Prior consensus held that medication-related osteonecrosis of the jaw (MRONJ) lesion was composed of necrotic bone; however, more recent studies have identified inflammatory infiltrates in the lesion. Herein, we report that remarkably elevated infiltrating γδT cells (90% of lymphocytes) express Semaphorin 4D (Sema4D) in human patient with MRONJ lesion, whereas γδT cells only account for 2–5% of lymphocytes in blood. Importantly, Sema4D is implicated in the pathogenesis of T cell-mediated inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Indeed, in a mouse model of MRONJ, an elevated number of γδT, but not αβT, cells infiltrating in the MRONJ-like lesion was observed. Both elevated soluble Sema4D (sSema4D) production accompanied by pro-inflammatory cytokines, including TNF-α IFN-γ and IL-1β, and Sema4D-expressing γδT cells were detected in mouse MRONJ-like lesion. Activated γδT cells produced sSema4D in vitro, which could promote TNF-α production from macrophages. Meanwhile, γδT cell-KO mice were resistant to the induction of MRONJ and, hence, showed no elevation of local productions of Sema4D and TNF-α. Finally, systemic administration of anti-Sema4D neutralizing mAb suppressed the onset of MRONJ in wild-type mice in conjunction with diminished level of TNF-α. These results suggested a critical pathogenic engagement of Sema4D produced by γδT cells in the development of MRONJ.

Lack of estrogen receptor α in astrocytes of progranulin-deficient mice.

Progranulin (PGRN) is a multifunctional growth factor with functions in neuroprotection, anti-inflammation, and neural progenitor cell proliferation. These functions largely overlap with the actions of estrogen in the brain. Indeed, we have previously shown that PGRN mediates the functions of estrogen, such as masculinizing the rodent brain and promoting adult neurogenesis. To evaluate the underlying mechanism of PGRN in mediating the actions of estrogen, the localization of estrogen receptor α (ERα) in the brains of wild-type (WT) and PGRN-deficient (KO) mice was investigated. First, double-labeling immunofluorescence was performed for ERα with neuronal nuclei (NeuN), ionized calcium-binding adaptor molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP), as markers for neurons, microglia, and astrocytes, respectively, in female mice in diestrous and estrous stages. ERα-immunoreactive (IR) cells were widespread and co-localized with NeuN in brain sections analyzed (bregma –1.06 to –3.16 mm) of both WT and KO mice. In contrast, expression of ERα was not observed in Iba1-IR cells from both genotypes. Interestingly, although ERα was co-localized with GFAP in WT mice, virtually no ERα expression was discernible in GFAP-IR cells in KO mice. Next, the brains of ovariectomized adult female, adult male, and immature female mice were subjected to immunostaining for ERα and GFAP. Again, co-localization of ERα with GFAP was observed in WT mice, whereas this co-localization was not detected in KO mice. These results suggest that PGRN plays a crucial role in the expression of ERα in astrocytes regardless of the estrous cycle stage, sex, and maturity.

Absence of Vasoactive Intestinal Peptide Signaling Significantly Enhances T Cell Survival Following MCMV Infection Via Increased Expression of Bcl-2 and Bcl-XL

BackgroundThe nervous system's influence on immune responses has become increasingly appreciated following the identification of a variety of immunomodulatory products secreted from neurons. One such product, vasoactive intestinal peptide (VIP), has been identified as an anti-inflammatory mediator exerting effects on both the innate and adaptive arms of the immune system. Interference with this pathway through blocking the VIP receptors VPAC1 and VPAC2 has been demonstrated to enhance T cell responses to viral infection as well as cancer. VIP antagonism results in increased numbers of antigen-specific cells in the peripheral blood and a shift in the balance of co-stimulatory and co-inhibitory molecules towards a pro-inflammatory phenotype. The consequences of such effects are a reduction in viral load in a model of MCMV infection and a reduced tumor burden and increased survival in a model of lymphoma.MethodsIn order to better understand the molecular mechanisms underlying the effects of VIP absence or antagonism, we used the MCMV infection model. Male VIP WT and VIP KO mice were infected with 105 pfu MCMV ip and sacrificed at various time points. Spleens were harvested and T cells were FACS sorted based on the expression of activation markers. Expression of pro and anti-apoptotic molecules was assessed via Western blot. Expression of VIP and its receptor VPAC1 was determined using qRT-PCR of extracted mRNA. In vitro T cell proliferation was assessed using CFSE dilution of splenocytes stimulated with CD3/CD28 beads and IL-2.ResultsWe first examined levels of VIP and VPAC1 over the course of MCMV infection via qRT-PCR of extracted mRNA. Expression of VIP and VPAC1 was found to change in response to infection with an initial decrease in the levels of VPAC1 followed by an increase back to baseline levels. The pattern of VIP expression was opposite with an initial increase followed by a decrease. In order to gain more mechanistic insight into the effects of VIP signaling in T cells during MCMV infection, activated splenic CD8 T cells from WT mice and mice lacking VIP were FACS sorted at various time points. Activated CD69+CD8+ T cells from KO mice expressed higher levels of the anti-apoptotic protein Bcl-XL both during the initial response and at the peak by Western blot (Figure 1). Intracellular staining revealed an increased frequency of Bcl-2-expressing CD8 T cells in KO mice at the peak of the response (Figure 1). Proliferation of CFSE-labeled CD8 T cells in response to in vitro CD3/CD28 stimulation was found to be identical in wild type and VIP knockout cells suggesting modified T cell survival rather than proliferation. The negative influence of VIP on T cell survival resulted in significantly higher T cell frequency and a significant increase in the absolute numbers of antigen-specific splenic CD8 T cells in knockout mice (Figure 2). These results provide further support for VIP antagonism as a viable option for th e enhancement of CD8 T cell-mediated immunity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proteomic Alterations in B Lymphocytes of Sensitized Mice in a Model of Chemical-Induced Asthma.

Introduction and AimThe role of B-lymphocytes in chemical-induced asthma is largely unknown. Recent work demonstrated that transferring B lymphocytes from toluene diisocyanate (TDI)-sensitized mice into naïve mice, B cell KO mice and SCID mice, triggered an asthma-like response in these mice after a subsequent TDI-challenge. We applied two-dimensional difference gel electrophoresis (2D-DIGE) to describe the “sensitized signature” of B lymphocytes comparing TDI-sensitized mice with control mice.ResultsSixteen proteins were identified that were significantly up- or down-regulated in B lymphocytes of sensitized mice. Particularly differences in the expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 could be correlated to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses.ConclusionThis study revealed important alterations in the proteome of sensitized B cells in a mouse model of chemical-induced asthma, which will have an important impact on the B cell function.

Abstract P178: Depletion of Gamma-Delta T Cells Protects Against Toll-Like Receptor-Induced Preeclampsia in Mice

Preeclampsia (PE), a hypertensive disorder of pregnancy, is associated with vascular endothelial dysfunction and excessive immunity and inflammation. However, it is unclear which innate immune cells propagate the pro-inflammatory state. Gamma-delta T (gdT) cells can secrete tolerogenic anti-inflammatory cytokines or cytotoxic pro-inflammatory cytokines depending on their activation status. gdT cells from women with PE produce significantly more IFNg and perforin and are less susceptible to apoptosis than gdT cells from normal pregnant women. We hypothesized that Toll-like receptor (TLR) activation in gdT cells induces inflammation and causes PE-like features in mice and that gdT cell KO mice would be resistant to developing TLR-induced PE-like features. Activation of splenocytes isolated from day 14 normal pregnant mice with the TLR3 agonist poly I:C or the TLR7 agonist R837 for 24 hours significantly increased gdT cells as well as IFNg and TNFa production. We have reported that poly I:C or R837 treatment of normal pregnant mice elicits a pregnancy-dependent PE-like syndrome by inducing a pro-inflammatory immune response. Pregnant poly I:C-treated and R837-treated mice had significantly increased splenic levels of gdT cells and plasma levels of IFNg and TNFa compared to pregnant vehicle-treated mice. Pregnant gdT cell KO mice treated with poly I:C or R837 did not develop hypertension or endothelial dysfunction. These data demonstrate that gdT cells mediate the TLR-induced PE-like features in mice and depletion of gdT cells may reduce the severity of PE in women.

The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution.

Air pollution is known to exacerbate chronic inflammatory conditions of the lungs including pulmonary hypertension, cardiovascular diseases and autoimmune diseases. Directly pathogenic antibodies bind pro-inflammatory cell receptors and cause or exacerbate inflammation. In contrast, anti-inflammatory antibody isotypes (e.g. mouse immunoglobulin G1, IgG1) bind inhibitory cell receptors and can inhibit inflammation. Our previous studies showed that co-exposure to antigen and urban ambient particulate matter (PM2.5) induced severe pulmonary arterial thickening and increased right ventricular systolic pressures in mice via T-cell produced cytokines, Interleukin (IL)-13 and IL-17A. The aim of the current study was to understand how B cell and antibody responses integrate into this T cell cytokine network for the pulmonary hypertension phenotype. Special focus was on antigen-specific IgG1 that is the predominant antibody in the experimental response to antigen and urban ambient PM2.5. Wild type and B cell-deficient mice were primed with antigen and then challenged with antigen and urban particulate matter and injected with antibodies as appropriate. Our data surprisingly showed that B cells were necessary for the development of increased right ventricular pressures and molecular changes in the right heart in response to sensitization and intranasal challenge with antigen and PM2.5. Further, our studies showed that both, the increase in right ventricular systolic pressure and right ventricular molecular changes were restored by reconstituting the B cell KO mice with antigen specific IgG1. In addition, our studies identified a critical, non-redundant role of B cells for the IL-17A-directed inflammation in response to exposure with antigen and PM2.5, which was not corrected with antigen-specific IgG1. In contrast, IL-13-directed inflammatory markers, as well as severe pulmonary arterial remodeling induced by challenge with antigen and PM2.5 were similar in B cell-deficient and wild type mice. Our studies have identified B cells and antigen specific IgG1 as potential therapeutic targets for pulmonary hypertension associated with immune dysfunction and environmental exposures.

Single-trial properties of place cells in control and CA1 NMDA receptor subunit 1-KO mice.

The NMDA receptor plays a key role in synaptic plasticity and its disruption leads to impaired spatial representation in the CA1 area of the hippocampus, with place cells exhibiting larger place fields (McHugh et al., 1996). Place fields are defined by the spatial and nonspatial inputs of a given place and context, by intrinsic network processes, such as phase precession, but also by the matching of these inputs to a pre-existing spatial representation. Larger place fields may be a consequence of spatially widened firing upon a single crossing of a place field, or of increased variability in place field positions across traversals. We addressed this question by monitoring CA1 place cell activity, with tetrodes, in control and KO mice lacking the NMDA receptor in this region. In individual crossings of the field, we found no difference between genotypes in place field size; the larger, overall place field size turns out to be a consequence of jitter across trials. We suggest that this jitter reflects a deficit in the matching of current spatial inputs to the stored spatial representation of the track. This is supported by the finding that deficits in place field size and spatial information are rescued by extensive exposure of the mouse to the track, which may echo an increased influence of memory retrieval processes in CA3 on firing in CA1.

CD275/ICOSL-independent germinal centers and autoantibody in autoimmune-prone RasGRP1-deficient mice (BA13P.125)

Abstract Lymphopenia results in favorable microenvironments for the expansion and activation of autoreactive lymphocytes. Mice deficient in guanine nucleotide exchange factor Rasgrp1 (KO) are T lymphopenic early in life and develop a lymphoproliferative disorder with features of human SLE. Our previous work revealed that autoreactive B cells lacking RasGRP1 break tolerance early during development as well as during germinal center responses, suggesting both T cell-independent and -dependent mechanisms are responsible. To understand whether T cells are involved in the breach of tolerance in germinal centers, we examined T effector cells in KO mice. In addition to the expected increase in Th1 cells, the frequency and number of Th17 cells were also elevated in KO mice. Th17 cells were positive for CXCR5 and Bcl-6, localized to germinal centers and upregulated levels of CD278/ICOS coordinately with the appearance of germinal centers, all attributes of T follicular helper cells (Tfh17). To determine whether CD278-CD275 interactions were required for the development of Tfh17 cells and for autoantibody, we crossed KO and CD275-deficient mice. Interestingly, KO mice lacking CD275 formed germinal centers with an elevated frequency of Th17 cells and similar titers of autoantibodies. From these data, we suggest that CD278-CD275 interactions are not required for germinal centers and autoantibody production in mice lacking RasGRP1.

B7-H1 blockade improves efficacy of regulatory T cell depletion as cancer immunotherapy by reducing Treg regeneration, possibly through tumor B7-H1 effects (VAC3P.950)

Abstract Immune co-signaling blockade is effective cancer immunotherapy but is not curative alone. Treg depletion has shown potential human anti-cancer efficacy, but is also not curative alone. As B7-H1 immune co-signaling facilitates Treg generation, we hypothesized that adding αB7-H1 to Treg depletion would improve treatment by hindering Treg regeneration. We show here that αB7-H1 plus denileukin diftitox (DD) to deplete Tregs is a synergistic combo that effects complete and durable (>7 mos) tumor regressions in bulky B16 melanoma, whereas neither agent alone does. αB7-H1 efficacy alone or with DD is lost in IFN-γ KO or T cell KO mice, and is mediated in part by tumor-specific CD8+ (but not CD4+) T cells. αB7-H1 is highly effective in B7-H1 KO mice, demonstrating a major role for tumor B7-H1 in treatment efficacy. Reducing tumor B7-H1-mediated Treg generation appears to be a mechanism as predicted. The αB7-H1/DD combo was also effective in ID8 ovarian cancer showing broad application. We engineered B7-H1hi/lo B16 tumors for further immune studies. Surprisingly, B7-H1hi B16 grew faster than B7-H1lo in RAG KO, suggesting B7-H1 as an adaptive immunity-independent tumor growth/survival factor. IFNγ upregulated tumor B7-H1, but αB7-H1 did not prevent IFN-γ-mediated tumor inhibition in vitro. B7-H1hi/lo tumors in B7-H1 KO are being used to define tumor vs host effects of αB7-H1 ± Treg depletion. As αB7-H1 and DD have been tested clinically, this combo is rapidly clinically translated.