Population In Mice Research Articles

1225P - THOR-707, a novel not-alpha IL-2, promotes all key immune system anti-tumoral actions of IL-2 without eliciting vascular leak syndrome (VLS)

BackgroundTHOR-707 is a reprogrammed, site-directed, single pegylated, recombinant human IL-2 (rIL-2) variant that lacks binding affinity for the α chain of the IL-2 receptor. Because THOR-707 has near-native binding affinity for the βγ chain of the IL-2 receptor, it produces an immune-active, CD8+ T effector-driven anti-tumor effect. At the same time because it lacks α chain affinity, it limits the proliferation of immune suppressive CD4+ Tregs and other innate lymphoid cells known to mediate the life-threatening complications of VLS. These pharmacologic properties make THOR-707 an ideal immunooncology drug development candidate. MethodsWe employed in vivo and ex vivo models to study pharmacodynamic responses to THOR-707. ResultsWe demonstrate all critical actions of IL-2 for anti-tumoral responses without inducing VLS, including: (1) maximal extravasation and expansion of key CD8+ T and NK cell populations in mice and NHPs (2) CD8+ T cell infiltration of tumor and lymph nodes in mouse tumor models, critical for antigen presentation by tumor cells and trans-presentation by antigen presenting cells and (3) induction of IFNγ release ex vivo by T cell receptor (TCR)-activated human T cells that is further potentiated by a PD-1 inhibitory antibody, important for upregulation of MHC I and II and enhancement tumor antigen presentation. In mouse syngeneic tumor models, THOR-707 promoted an increase in the number of infiltrating tumor-killing NK and CD8+ T cells without expansion of suppressive CD4+ Treg cells. Expression profiling of tumors revealed promotion of remodeling of cellular populations towards a composition favoring cytolytic phenotypes. Evaluation of TCR clonality revealed that treatment with THOR-707 increased intra-tumoral T cell diversity. ConclusionsTHOR-707 induction of CD8+ T cell tumor infiltration resulted in single agent dose-dependent anti-tumor efficacy, additive efficacy in combination with a PD-1 checkpoint inhibitor, promoted long-term survival in mice and NHPs and rejection of tumor cells on re-challenge. Based on these results, first in human studies are expected to begin this year in solid tumors both as a single agent and in combination with an immune-checkpoint inhibitor. Legal entity responsible for the studySynthorx, Inc. FundingSynthorx, Inc. DisclosureM.E. Milla: Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Full / Part-time employment: Synthorx, Inc.; Licensing / Royalties: Adaptive Biotechnologies, Inc. J.L. Ptacin: Full / Part-time employment: Synthorx, Inc. L. Ma: Full / Part-time employment: Synthorx, Inc. C.E. Caffaro: Full / Part-time employment: Synthorx, Inc. H.R. Aerni: Full / Part-time employment: Synthorx, Inc. K.M. San Jose: Full / Part-time employment: Synthorx, Inc. M.J. Pena: Full / Part-time employment: Synthorx, Inc. R.W. Herman: Full / Part-time employment: Synthorx, Inc. Y. Pavlova: Full / Part-time employment: Synthorx, Inc. D.B. Chen: Full / Part-time employment: Synthorx, Inc. T.K. Ismaili: Full / Part-time employment: Synthorx, Inc. S. Li: Full / Part-time employment: Synthorx, Inc. J. Nguyen: Full / Part-time employment: Synthorx, Inc. N. Singh: Full / Part-time employment: Synthorx, Inc. L.K. Shawver: Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Officer / Board of Directors: Synthorx, Inc.; Shareholder / Stockholder / Stock options: Cleave Biosciences; Shareholder / Stockholder / Stock options: Relay Therapeutics. L.K. Koriazova: Full / Part-time employment: Synthorx, Inc. I.B. Joseph: Full / Part-time employment: Synthorx, Inc.

Lkb1 in dendritic cells restricts CD8+Foxp3+regulatory T cells expansion in vivo

Liver kinase B1 (Lkb1) in dendritic cells (DCs) plays a key role in maintaining immunity homeostasis and adaptive immunity by controlling the CD4+Foxp3+T regulatory cell (CD4+Tregs) pool and T cells activation. However, the function of Lkb1 in DCs for the regulation of CD8+Foxp3+T regulatory cells (CD8+Tregs) has not been addressed. Herein, we found that Lkb1-deficient DCs could lead to excessive CD8+Tregs expansion in multiple organs. We found that OX40 expression was significantly higher in Lkb1-deficient DCs compared with that in wild-type (WT) mice, suggesting a potential pathway of CD8+Treg expansion. Moreover, we found that CD8+Tregs from mice with conditional deletion Lkb1 in DCs (KO) displayed an activated phenotype and expressed higher levels of specific markers, including ICOS and CD103. Interestingly, compared with the WT mice without lipopolysaccharide(LPS) treatment, we found that CD8+Tregs population increased in the WT mice with LPS treatment which can selectively delete Lkb1 protein in DCs. However, there was no significant difference in CD8+Tregs population in the KO mice between LPS treatment group and non-LPS treatment. Collectively, our findings identified Lkb1 in DCs as a crucial regulator of CD8+Treg expansion.

Rats provide a superior model of human stress erythropoiesis

Mouse models are widely used to study human erythropoiesis in vivo. One important caveat using mouse models is that mice often develop significant extramedullary erythropoiesis with anemia, which could mask important phenotypes. To overcome this drawback in mice, here we established in vitro and in vivo rat models for the studies of stress erythropoiesis. Using flow cytometry-based assays, we can monitor terminal erythropoiesis in rats during fetal and adult erythropoiesis under steady state and stress conditions. We used this system to test rat erythropoiesis under phenylhydrazine (PHZ)-induced hemolytic stress. In contrast to mice, rats did not have an increased proportion of early-stage erythroid precursors during terminal differentiation in the spleen or bone marrow. This could be explained by the abundant bone marrow spaces in rats that allow sufficient erythroid proliferation under stress. Consistently, the extent of splenomegaly in rats after PHZ treatment was significantly lower than that in mice. The level of BMP4, which was significantly increased in mouse spleen after PHZ treatment, remained unchanged in rat spleen. We further demonstrated that the bone marrow c-Kit positive progenitor population underwent a phenotype shift and became more CD71 positive and erythroid skewed with the expression of maturing erythroid markers under stress in rats and humans. In contrast, the phenotype shift to an erythroid-skewed progenitor population in mice occurred mainly in the spleen. Our study establishes rat in vitro and in vivo erythropoiesis models that are more appropriate and superior for the study of human stress erythropoiesis than mouse models.

Twist1 Inactivation in Dmp1-Expressing Cells Increases Bone Mass but Does Not Affect the Anabolic Response to Sclerostin Neutralization.

Wnt signaling plays a major role in bone metabolism. Advances in our understanding of secreted regulators of Wnt have yielded several therapeutic targets to stimulate osteoanabolism—the most promising of which is the Wnt inhibitor sclerostin. Sclerostin antibody recently gained approval for clinical use to treat osteoporosis, but the biology surrounding sclerostin antagonism is still incompletely understood. Numerous factors regulate the efficacy of sclerostin inhibition on bone formation, a process known as self-regulation. In previous communications we reported that the basic helix-loop-helix transcription factor Twist1—a gene know to regulate skeletal development—is highly upregulated among the osteocyte cell population in mice treated with sclerostin antibody. In this communication, we tested the hypothesis that preventing Twist1 upregulation by deletion of Twist1 from late-stage osteoblasts and osteocytes would increase the efficacy of sclerostin antibody treatment, since Twist1 is known to restrain osteoblast activity in many models. Twist1-floxed loss-of-function mice were crossed to the Dmp1-Cre driver to delete Twist1 in Dmp1-expressing cells. Conditional Twist1 deletion was associated with a mild but significant increase in bone mass, as assessed by dual energy x-ray absorptiometry (DXA) and microCT (µCT) for many endpoints in both male and female mice. Biomechanical properties of the femur were not affected by conditional mutation of Twist1. Sclerostin antibody improved all bone properties significantly, regardless of Twist1 status, sex, or endpoint examined. No interactions were detected when Twist1 status and antibody treatment were examined together, suggesting that Twist1 upregulation in the osteocyte population is not an endogenous mechanism that restrains the osteoanabolic effect of sclerostin antibody treatment. In summary, Twist1 inhibition in the late-stage osteoblast/osteocyte increases bone mass but does not affect the anabolic response to sclerostin neutralization.

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity.

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by designer drugs (DREADDs) have become the most popular chemogenetic approach used in modern neuroscience. Most studies deliver the ligand clozapine-N-oxide (CNO) using a single intraperitoneal injection, which is suitable for the acute activation/inhibition of the targeted neuronal population. There are, however, only a few examples of strategies for chronic modulation of DREADD-controlled neurons, the majority of which rely on the use of delivery systems that require surgical intervention. Here, we expand on two non-invasive strategies for delivering the ligand CNO to chronically manipulate neural population in mice. CNO was administered either by using repetitive (daily) eye-drops, or chronically through the animal's drinking water. These non-invasive paradigms result in robust activation of the designer receptors that persisted throughout the CNO treatments. The methods described here offer alternatives for the chronic DREADD-mediated control of neuronal activity and may be useful for experiments designed to evaluate behavior in freely moving animals, focusing on less-invasive CNO delivery methods.

Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects.

Targeting the dynamic tumor immune microenvironment (TIME) can provide effective therapeutic strategies for cancer. Neutrophils are the predominant leukocyte population in mice and humans, and mounting evidence implicates these cells during tumor growth and metastasis. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA fibers that are capable of binding tumor cells to support metastatic progression. Here we demonstrate for the first time that circulating NET levels are elevated in advanced esophageal, gastric and lung cancer patients compared to healthy controls. Using pre-clinical murine models of lung and colon cancer in combination with intravital video microscopy, we show that NETs functionally regulate disease progression and that blocking NETosis through multiple strategies significantly inhibits spontaneous metastasis to the lung and liver. Further, we visualize how inhibiting tumor-induced NETs decreases cancer cell adhesion to liver sinusoids following intrasplenic injection - a mechanism previously thought to be driven primarily by exogenous stimuli. Thus, in addition to neutrophil abundance, the functional contribution of NETosis within the TIME has critical translational relevance and represents a promising target to impede metastatic dissemination.

Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice.

Permethrin, a pyrethroid chemical, is widely used as a pesticide because of its rapid insecticidal activity. Although permethrin is considered to exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are unclear. In this study, we investigated the effects of low-level, chronic permethrin exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. We exposed mice to the acceptable daily intake level of permethrin (0.3ppm) in drinking water during the prenatal and postnatal periods. We then examined the effects on the central nervous system in adult male offspring. In the permethrin group, we detected behavior that displayed incomplete adaptation to a novel environment, as well as an impairment in learning and memory. In addition, immunohistochemical analysis revealed an increase in doublecortin- (an immature neuron marker) positive cells in the hippocampal dentate gyrus in the permethrin exposure group compared with the control group. Additionally, in the permethrin exposure group there was a decrease in astrocyte number in the hilus of the dentate gyrus, and remaining astrocytes were often irregularly shaped. These results suggest that exposure to permethrin at low levels in early life affects the formation of the neural circuit base and behavior after maturation. Therefore, in the central nervous system of male mice, low-level, chronic permethrin exposure during the prenatal and postnatal periods has effects that were not expected based on the known effects of permethrin exposure in mature animals.

Gut-associated IgA+ immune cells regulate obesity-related insulin resistance

The intestinal immune system is emerging as an important contributor to obesity-related insulin resistance, but the role of intestinal B cells in this context is unclear. Here, we show that high fat diet (HFD) feeding alters intestinal IgA+ immune cells and that IgA is a critical immune regulator of glucose homeostasis. Obese mice have fewer IgA+ immune cells and less secretory IgA and IgA-promoting immune mediators. HFD-fed IgA-deficient mice have dysfunctional glucose metabolism, a phenotype that can be recapitulated by adoptive transfer of intestinal-associated pan-B cells. Mechanistically, IgA is a crucial link that controls intestinal and adipose tissue inflammation, intestinal permeability, microbial encroachment and the composition of the intestinal microbiome during HFD. Current glucose-lowering therapies, including metformin, affect intestinal-related IgA+ B cell populations in mice, while bariatric surgery regimen alters the level of fecal secretory IgA in humans. These findings identify intestinal IgA+ immune cells as mucosal mediators of whole-body glucose regulation in diet-induced metabolic disease.

Chemogenetic Modulation of Orexin Neurons Reverses Changes in Anxiety and Locomotor Activity in the A53T Mouse Model of Parkinson's Disease.

Parkinson’s disease (PD) is the second most common neurodegenerative disease. PD symptomology is recognized as heterogeneous and in addition to motor function decline includes cognitive, mood, sleep, and metabolic disorders. Previous studies showed early reductions in anxiety and locomotion in the A53T mice model of PD. Since inflammation and astrogliosis are an integral part of PD pathology and impair proper neuronal function, we were keen to investigate if behavioral changes in A53T mice are accompanied by increased inflammation and astrogliosis in the hippocampus (Hipp) and motor cortex (mCtx) brain regions involved in the regulation of anxiety and locomotion, respectively. To test this, we used 3-, 5-, and 7-month-old A53T mice to examine anxiety-like behavior, locomotion, and expression of inflammation and astrogliosis markers in the Hipp and mCtx. Further, we examined the presence of alpha-synuclein accumulation in orexin neurons and orexin neuronal loss. The data show early reductions in anxiety-like behavior as well as increased locomotor activity, which was accompanied by inflammation and astrogliosis in the Hipp and mCtx. Due to the persistence of the orexin neuron population in A53T mice and the involvement of orexin in anxiety and locomotor regulation, we hypothesized that chemogenetic modulation of orexin neurons would reverse the observed reductions in anxiety-like behavior and the increases in locomotor activity in these animals. We showed that chemogenetic activation of orexin neurons in A53T mice restores anxiety-like behavior back to control levels without affecting locomotor activity, whereas the inhibition of orexin neurons reverses the elevated locomotor activity without any effects on anxiety-like behavior. This study exemplifies the complex role of orexin neurons in this model of PD and demonstrates the novel finding that changes in locomotor and anxiety-like behavior are accompanied by inflammation and astrogliosis. Together, these data suggest that the orexin system may play a significant role in early and late stages of PD.

Abstract 1081: Smad3 silences neutrophil anticancer activity in the tumor microenvironment

Abstract Tumor microenvironment (TME) is a new therapeutic target for cancer, however it is highly heterogeneous and still largely unknown. Recently, we revealed that Smad3 is essential for TME-driven cancer progression, better understanding of the underlying mechanism may uncover novel therapeutic targets for cancer. Indeed, Smad3-dependent TME (Smad3-TME) is originated from bone marrow, where neutrophils are the most abundant leukocytes but their role in cancer is still controversial. Here, we are the first study to reveal the regulatory role of Smad3 in tumor-associated neutrophils (TAN). Interestingly, we observed that knockout of Smad3 (Smad3-KO) dramatically increased TAN population in mice bearing syngeneic melanoma B16F10 and lung carcinoma LLC, where the anticancer N1 phenotypes were significantly induced in the SMad3-KO TME-isolated TAN. Unexpectedly, adoptive transfer of Smad3-KO bone marrow-derived neutrophils markedly suppressed the progression of LLC-tumor on Smad3-WT mice in vivo, and their enhanced cancer-killing activity was confirmed by co-culture assay in vitro. Furthermore, microarray analysis identified a total of 4416 differentially expressed genes associated with Smad3-KO neutrophils under cancer condition in vitro, but the presence of Smad3 largely decreased the transcriptome diversity; especially on genes associated with virus defense response including an anticancer cytokine IL-28. Silencing of IL-28 significantly reduced the anticancer efficiency of Smad3-KO TAN on LLC-bearing mice in vivo. Mechanistically, we uncovered that IL-28A serves as a direct Smad3 target gene and specifically down-regulated in neutrophils under TGF-β1 or LLC-secretome stimulations; showing by ChIP and dual-luciferase reporter assays in vitro. Thus, targeting Smad3 precisely on TAN may represent as a novel and effective immunotherapy for cancer. Acknowledgments: This study was supported by Lui Che Woo Institute of Innovative Medicine (CARE program), Research Grants Council of Hong Kong (GRF 14117815, 14121816, 14163317, C7018-16G, TRS T12-402/13N), Health and Medical Research Fund (03140486, 14152321), Innovation and Technology Fund of Hong Kong (ITS/068/18), Direct Grant for Research CUHK (2017.002). Note: This abstract was not presented at the meeting. Citation Format: Patrick Ming-Kuen Tang, Philip Chiu-Tsun Tang, Jeff Yat-Fai Chung, Xiao-Ru Huang, Ka-Fai To, Hui-Yao Lan. Smad3 silences neutrophil anticancer activity in the tumor microenvironment [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 1081.

Abstract 1050: Characterization of human cancer cell line xenografts in humanized mice

Abstract Studying immune-oncology (IO) agents that target the human immune system in mice have posed challenges for oncology researchers. Since the commercial introduction of humanized mice, antitumor efficacy studies can now be performed with human cancer cells within mice bearing the components of a human immune system. However, development and characterization of these models is necessary to understand which model may be best for different agents. To this end, we characterized A375, A549, Caki-1, H1299, H1975, HCC827, HCT116, KU19-19, MDA-MB-231, and RKO human cancer cell lines in CD34+ humanized NSG mice for tumor growth rate and immune cell profiling in tumor and spleen compartments. We found that CD4+ T cell composition in both the spleen and tumor varied among the models, with A375, Caki-1, and RKO expressing higher levels of CD4+ T cells compared to the other models. Many of the models had moderate levels of CD8+ T cells in the tumor. Spleen analysis revealed higher NK cell populations in mice bearing Caki-1 tumors, while A549 tumor samples had a higher percentage of NK cells relative to the other models. Surprisingly, many of the tumor models showed an abundance of myeloid cells in the spleen and tumor compartments, although these were mostly comprised of B cells and dendritic cells and lower levels of macrophages and MDSCs. The expression level and percentage of cells expressing PD-1, and PD-L1 in the models were also determined. Furthermore, the antitumor response of select models to PD-1 and PD-L1 antibodies will be presented. In summary, these data demonstrate that there are tumor-intrinsic factors that influence the immune cell repertoire within tumors and spleen. These data may also aid in selection of tumor models to test antitumor activity of novel IO agents. Citation Format: Christina Stevens, Christopher Maddage, Kerri Lasky, Jonathan Rios-Doria, Holly Koblish. Characterization of human cancer cell line xenografts in humanized mice [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 1050.

Abstract LB-139: The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer

Abstract Background: Regulatory T cells (Tregs) are potent immunosuppressive cells of the immune system. They facilitate the survival and progression of tumor through their ability to suppress the antitumor immunity. The expression of tumor necrosis factor receptor type II (TNFR2) on Tregs is reported to identify the maximally suppressive Treg population in both mice and human. According to our previous research, expression of TNFR2 appeared to correlate better than CD25+ and CD127− with FoxP3 expression. Aim: The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer are needed to be studied. Methods: The expression and phenotype of TNFR2 + Tregs in peripheral blood and tumor tissues of 33 cases of gastric cancer patients and in peripheral blood of 15 normal controls were detected by flow cytometry. The ratio of Foxp3+Tregs and TNFR2+Tregs of 145 cases of gastric cancer patients was analyzed by multicolor immunofluorescence assay. We analyzed their relationship with gastric cancer stage, metastasis and other clinical indicators by clinical and pathological data. Results: Flow cytometry showed that the expression of TNFR2 in human peripheral blood Tregs was significantly higher than that in conventional T cells (Tconvs) in peripheral blood CD4 + T cell subsets of gastric cancer patients and healthy individuals (P<0.05). The proportion of TNFR2 + cells in peripheral blood Tregs cells was significantly higher in patients with gastric cancer than in normal subjects (P <0.05). Tregs and Tconvs cells expressed very low level of CTLA-4 and TIM-3 in both normal controls and gastric cancer patients. Multicolour immunofluorescence showed that the ratio of Foxp3 + Tregs and TNFR2 + Tregs was increased in the tumor tissue, and TNFR2 + Tregs cells showed a tendency to increase with tumor progression and lymphatic metastasis. Higher levels of infiltration of TNFR2+ Tregs cells are significantly associated with patients’ OS (P<0.05) and DFS (P<0.05). Conclusion: Our findings revealed that TNFR2 could be a promising molecular marker associated with poor prognosis. The mechanism by which TNFR2+Tregs promote tumor survival and progression in gastric cancer is still needed further research. Citation Format: Yang Qu, Gang Zhao, Hui Li. The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer [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 LB-139.

γδ T cells shape memory-phenotype αβ T cell populations in non-immunized mice.

Size and composition of γδ T cell populations change dramatically with tissue location, during development, and in disease. Given the functional differentiation of γδ T cell subsets, such shifts might alter the impact of γδ T cells on the immune system. To test this concept, and to determine if γδ T cells can affect other immune cells prior to an immune response, we examined non-immunized mice derived from strains with different genetically induced deficiencies in γδ T cells, for secondary changes in their immune system. We previously saw extensive changes in pre-immune antibodies and B cell populations. Here, we report effects on αβ T cells. Similarly to the B cells, αβ T cells evidently experience the influence of γδ T cells at late stages of their pre-immune differentiation, as single-positive heat stable antigen-low thymocytes. Changes in these and in mature αβ T cells were most prominent with memory-phenotype cells, including both CD8+ and CD4+ populations. As previously observed with B cells, most of the effects on αβ T cells were dependent on IL-4. Unexpectedly, IL-4 seemed to be produced mainly by αβ T cells in the non-immunized mice, albeit strongly regulated by γδ T cells. Similarly to our findings with B cells, changes of αβ T cells were less pronounced in mice lacking all γδ T cells than in mice lacking only some, suggesting that the composition of the γδ T cell population determines the nature of the γδ-influence on the other pre-immune lymphocytes.

PS1567 UNCOUPLING INK PROTEINS FROM CDK4/CDK6 IN HSCS – UNCOUPLING PROLIFERATION FROM SENESCENCE

Background:Cyclin‐dependent kinase (CDK) 4 and CDK6 are highly related cell‐cycle kinases that regulate progression from G1 to S‐phase. CDK4/6 are activated upon binding of D‐type cyclins and subsequently phosphorylate the retinoblastoma protein (Rb). CDK4/6 inhibitor proteins, the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) as major player, adds another layer of complexity. Hyperactivity of CDK4 or CDK6 is frequently observed in hematopoietic malignancies due to loss of p16INK4a. Besides, CDK6 has also been identified as transcriptional regulator driving complex transcriptional programs. CDK4/6 inhibitors are approved in breast cancer treatment and are currently tested in hematopoietic malignancies.Aims:We aimed to unravel the consequences of the loss of these inhibitory INK‐dependent mechanisms for hematopoiesis.Methods:We used knock‐in mice expressing a CDK6 (CDK6R31C, Cdk6 R/R ) and/or a CDK4 (CDK4R24C, Cdk4 R/R ) mutant insensitive to INK4‐mediated inhibition and analyzed stem cell populations. Further we tested the self‐renewal capacity of mutant hematopoietic progenitors under conditions of hematopoietic stress.Results:Despite comparable bone marrow cellularity, we found a significant reduction in LSK cells and HSC populations in mice harboring Cdk4 R/R or Cdk6 R/R mutation. The combination of Cdk4 R/R /Cdk6 R/R in double‐knock‐in mice rescued these defects in all progenitor stages. Serially transplanted Cdk4 R/R /Cdk6 R/R double‐knock‐in HSCs display enhanced stem cell proliferation. They maintain the ability to give rise to both myeloid and lymphoid lineages. In response to hematopoietic stress (5‐FU, serial and competitive total bone marrow transplantation), CDK4R24C/CDK6R31C prevents stem cell exhaustion and show robust recovery in stem cell numbers. Low‐input RNAseq of long‐term stress‐triggered HSCs will unravel mechanisms uncoupling proliferation from senescence.Summary/Conclusion:HSCs with disrupted CDK4/6 – INK4 binding show enhanced proliferation, reduced exhaustion and improved transplant ability. Analysis of the underlying molecular alterations may open novel therapeutic options for CDK4/6 hyperactive hematopoietic malignancies.

Timed Feeding Protocols While Consuming a High Fat Diet Do Not Alter Gut Fungal Populations (OR23-06-19)

ObjectivesThe gut microbiome participates in host metabolic regulation. While the vast majority of microbiome research has focused on bacterial populations, other microorganisms also colonize the mammalian intestine and likely play functionally important roles in host metabolism. The objective of current study was to characterize the role of dietary composition and timing strategies upon gut fungal populations. MethodsC57BL/6 male mice were randomized to a Chow diet or a high-fat diet (HFD) for 6 weeks, followed by a switch from HFD to 1) Chow (sChow), 2) Purified High Fiber – Daniel Fast (DF), 3) HFD ad lib, 4) HFD time restricted (TRF), 5) HFD alternative day fasting (ADF), or 6) HFD 60% caloric restriction (CR) for an additional 8 weeks. Ileal, cecal and serial fecal samples were collected for next generation sequencing of ITS2 rRNA to examine the gut mycobiome. ResultsWe observed dramatic reductions in alpha diversity in fecal fungal populations when animals consumed the HFD compared with Chow. HFD resulted in dramatic reduction in the relative abundance of the fungal order Saccharomyces, with a concomitant increases in the genus Candida and Hanseniaspora. In response to dietary switch from HFD to Chow, fungal taxonomic composition, alpha, and beta diversity transitioned to a population clustering more similarity with Chow by weeks 4 and 8 of intervention. After 8 weeks on the respective dietary interventions, alpha diversity of the ileal, cecal and fecal fungal population in mice consuming DF or various HFD fasting protocols remained similar to the HFD controls. Saccharomycetales remained the dominant genus present in HFD and DF groups. However, amongst these groups, the DF group (fecal sample) showed the greatest increase in Saccharomyces, but time-restricted feeding protocols also showed increased levels of Saccharomyces. ConclusionsWhile fasting protocols on HFD are associated with improved metabolic outcomes, these data demonstrate that – similar to microbial populations within the microbiome – diet remains the largest driver of microorganism community composition. To our knowledge, this is the first investigation into the role of dietary timing strategies upon the gut fungal communities ever reported. Funding SourcesUniversity of Memphis. Children’s Foundation Research Institute, Memphis.

Gastrointestinal dysfunction in patients and mice expressing the autism-associated R451C mutation in neuroligin-3.

Gastrointestinal (GI) problems constitute an important comorbidity in many patients with autism. Multiple mutations in the neuroligin family of synaptic adhesion molecules are implicated in autism, however whether they are expressed and impact GI function via changes in the enteric nervous system is unknown. We report the GI symptoms of two brothers with autism and an R451C mutation in Nlgn3 encoding the synaptic adhesion protein, neuroligin‐3. We confirm the presence of an array of synaptic genes in the murine GI tract and investigate the impact of impaired synaptic protein expression in mice carrying the human neuroligin‐3 R451C missense mutation (NL3R451C). Assessing in vivo gut dysfunction, we report faster small intestinal transit in NL3R451C compared to wild‐type mice. Using an ex vivo colonic motility assay, we show increased sensitivity to GABAA receptor modulation in NL3R451C mice, a well‐established Central Nervous System (CNS) feature associated with this mutation. We further show increased numbers of small intestine myenteric neurons in NL3R451C mice. Although we observed altered sensitivity to GABAA receptor modulators in the colon, there was no change in colonic neuronal numbers including the number of GABA‐immunoreactive myenteric neurons. We further identified altered fecal microbial communities in NL3R451C mice. These results suggest that the R451C mutation affects small intestinal and colonic function and alter neuronal numbers in the small intestine as well as impact fecal microbes. Our findings identify a novel GI phenotype associated with the R451C mutation and highlight NL3R451C mice as a useful preclinical model of GI dysfunction in autism. Autism Res 2019, 12: 1043–1056. © 2019 International Society for Autism Research, Wiley Periodicals, Inc.Lay SummaryPeople with autism commonly experience gastrointestinal problems, however the cause is unknown. We report gut symptoms in patients with the autism‐associated R451C mutation encoding the neuroligin‐3 protein. We show that many of the genes implicated in autism are expressed in mouse gut. The neuroligin‐3 R451C mutation alters the enteric nervous system, causes gastrointestinal dysfunction, and disrupts gut microbe populations in mice. Gut dysfunction in autism could be due to mutations that affect neuronal communication.

TRPV1 Contributes to Cerebral Malaria Severity and Mortality by Regulating Brain Inflammation

Transient receptor potential vanilloid 1 (TRPV1) is a Ca+2-permeable channel expressed on neuronal and nonneuronal cells, known as an oxidative stress sensor. It plays a protective role in bacterial infection, and recent findings indicate that this receptor modulates monocyte populations in mice with malaria; however, its role in cerebral malaria progression and outcome is unclear. By using TRPV1 wild-type (WT) and knockout (KO) mice, the importance of TRPV1 to this cerebral syndrome was investigated. Infection with Plasmodium berghei ANKA decreased TRPV1 expression in the brain. Mice lacking TRPV1 were protected against Plasmodium-induced mortality and morbidity, a response that was associated with less cerebral swelling, modulation of the brain expression of endothelial tight-junction markers (junctional adhesion molecule A and claudin-5), increased oxidative stress (via inhibition of catalase activity and increased levels of H2O2, nitrotyrosine, and carbonyl residues), and diminished production of cytokines. Plasmodium load was not significantly affected by TRPV1 ablation. Repeated subcutaneous administration of the selective TRPV1 antagonist SB366791 after malaria induction increased TRPV1 expression in the brain tissue and enhanced mouse survival. These data indicate that TRPV1 channels contribute to the development and outcome of cerebral malaria.

Phenotypic analysis of alveolar macrophage populations in old mice and their response to Mycobacterium tuberculosis infection

Abstract The elderly are more susceptible to pulmonary infections including tuberculosis. Here, we examined the impact of aging on the phenotype and response of mouse alveolar macrophages (AMs) to Mycobacterium tuberculosis (M.tb). AMs were isolated from bronchoalveolar lavage fluid (BALF) of young (3 month) and old (18 month) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNFα and MIF than young mice and old mice contained higher levels of CCL2, IFN-β, IL-1β and MIF in their BALF. Flow cytometry analysis of AMs showed two distinct AM populations, a major CD11c+ CD11b− and a minor CD11c+CD11b+ population, the latter was significantly increased in old mice. Expression of CD206, TLR2, CD16/CD32, MHC class II and CD86 was higher in the CD11c+CD11b+ AMs and these AMs expressed monocytic markers Ly6C, C3CR1, and CD115, suggesting they are of monocytic origin. Sorted CD11c+CD11b+ AMs expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c+ CD11b− AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c+CD11b+ AMs phagocytosed more M.tb, and M.tb in these AMs expressed higher RNA levels of genes required its survival. Our studies identify two distinct AM populations in old mice, specifically a novel CD11c+CD11b+ AM population which is hyper-inflammatory and phagocytoses M.tb more efficiently, yet is more permissive for M.tb survival than the CD11c+CD11b− population which is more immuno-regulatory in nature. Our results also indicate that the lung cytokine environment of old mice contains an unusual array of both pro-inflammatory and immune-regulatory cytokines.

Characterization of mouse peritoneal macrophages using novel flow cytometry antibodies

Abstract Peritoneal resident macrophages are one of the most extensively studied macrophage populations in mouse. These cells can be divided into at least two discrete populations: small peritoneal macrophages (SPM) and large peritoneal macrophages (LPM). Under steady state conditions the monocyte-derived SPM constitute a minor population. They have a F4/80 (low) MHC-II (high) phenotype and constitutively produce cytokine RELM-alpha, known to be involved in anti-helminth response. LPM originate from embryonic progenitors and are normally more abundant. They express high levels of F4/80 and low levels of MHCII, and constitutively produce the B-cell attracting chemokine CXCL13. Here, we examined the homogeneity of each of the populations in C57Bl/6 and Balb/c mice. Our flow cytometric analysis with an array of novel antibodies, including anti-VSIG4 (CRIg), arginase 1, CD206, and CD163, revealed diversity within the LPM population. This phenotypic diversity most likely reflects differences in function and activation status, and will need further investigation.

The inoculation of α-Galactosylceramide and HPV18-E1 protein promote a cytokines profile involved in the generation of central memory CD8 + T cells

Abstract CD8+ mediated immune response plays a major role in the clearance of Human Papillomavirus (HPV) infected cells. In the natural history of an HPV infection, the expression of HPV-E1 helicase during viral replication makes it a potential immunological target. Currently, there are no therapeutic vaccines that eliminate the lesions caused by HPV. Nevertheless, there are many studies on developing therapeutic vaccines against HPV. Vaccines and immunological therapies mostly use adjuvants, particularly α-galactocilceramide (α-GalCer) is an adjuvant that promotes a CD8+ T lymphocyte response mainly. We demonstrate in previous work, that immunization of HPV type 18 E1 protein along with α-GalCer adjuvant elicited CD8 T cell-immune response, able to eliminate tumor cells expressing HPV18 E1 protein. In this study, we analyzed the effect of α-GalCer in the generation of memory T CD8+ populations. We evaluated the serum levels of IL-15, IL-7, IL-4, IL-31, IL-2, TNF-α and the number of memory CD8+ T cells. The serum levels of IL-15, IL-7, IL-4 and TNF- α are increase in mice inoculated with α-GalCer plus HPV18-E1 with respect to mice inoculated with α-GalCer or HPV18-E1 protein. These data correlate with the increase of central memory T CD8+ population in mice inoculated with α-GalCer plus HPV18-E1. These data indicate that the administration of α-GalCer along with HPV18-E1 protein helps the generation of central memory CD8 + T lymphocytes. This adjuvant could be useful in the development of new therapeutic strategies to clearance HPV established infections. INCAN: (017/076/17) (CEI/1076/17)