CD4 Knockout Research Articles

Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo.

Dysregulated neutrophil (PMN) transmigration across epithelial surfaces (TEpM) significantly contributes to chronic inflammatory diseases, yet mechanisms defining this process remain poorly understood. In the intestine, uncontrolled PMN TEpM is a hallmark of disease flares in ulcerative colitis. Previous in vitro studies directed at identifying molecular determinants that mediate TEpM have shown that plasma membrane proteins including CD47 and CD11b/CD18 play key roles in regulating PMN TEpM across monolayers of intestinal epithelial cells. Here, we show that CD47 modulates PMN TEpM in vivo using an ileal loop assay. Importantly, using novel tissue-specific CD47 knockout mice and in vitro approaches, we report that PMN-expressed, but not epithelial-expressed CD47 plays a major role in regulating PMN TEpM. We show that CD47 associates with CD11b/CD18 in the plasma membrane of PMN, and that loss of CD47 results in impaired CD11b/CD18 activation. In addition, in vitro and in vivo studies using function blocking antibodies support a role of CD47 in regulating CD11b-dependent PMN TEpM and chemotaxis. Taken together, these findings provide new insights for developing approaches to target dysregulated PMN infiltration in the intestine. Moreover, tissue-specific CD47 knockout mice constitute an important new tool to study contributions of cells expressing CD47 to inflammation in vivo.

Impact of CD38 knockout in NK cells on daratumumab-mediated cytotoxicity and cellular metabolism.

e15018 Background: Multiple myeloma (MM) is a neoplasm of plasma cells. These cells express high levels of CD38 and are sensitive to daratumumab (DARA), a CD38-targeting monoclonal antibody. DARA kills MM cells via several mechanisms including antibody dependent cellular cytotoxicity (ADCC). Although DARA has improved the outcome of patients, CR is not achieved in all the cases. Besides, most patients that achieve CR will experience relapse. A potential explanation for blunted efficacy of DARA is downregulation of CD38 levels on MM cells and impaired ADCC due to depletion of CD38high NK cells during therapy. ATRA upregulates CD38 levels on target cells and was proposed to improve the efficacy of DARA. NK cell supplementation was tested in a preclinical model, but that had only a modest effect on DARA efficacy, likely due to their short life via DARA-mediated elimination. Methods: We generated CD38 knockout (CD38KO) NK cells from healthy donors via Cas9 RNPs and investigate if these cells boost DARA activity against MM. RNA-seq and cellular metabolic analysis were performed to examine the effect of CD38 deletion on NK cell function. Results: Knockout efficiency was 81.9 ± 6.9% (mean ± SD, N = 5). Very low off-target effects were seen by whole genome sequencing. When compared to paired CD38 wild type (CD38WT) NK cells, CD38KO NK cells showed lower conjugation (2.57% vs. 11.85%, N = 3, p = 0.04), less fratricide (97.3% vs. 54.2%, mean viability, N = 3, p = 0.01), and superior persistence in mice (18.16% vs. 0.42%, mean frequency in blood, N = 5, p < 0.01) in the presence of DARA. Additionally, CD38KO NK cells exhibited enhanced ADCC against all tested MM cell lines and primary samples including CD38low cell lines and primary MM cells from a patient with disease relapse on DARA. Transcriptomic and cellular metabolic analysis revealed that CD38KO NK cells have favorable metabolic shift with higher mitochondrial respiratory capacity (N = 3, p < 0.01). Although ATRA upregulates CD38 levels on MM cell lines, we observed that ATRA treatment upregulates CD38 levels on circulating NK cells in patients. ATRA upregulated CD38 levels on CD38WT NK cells, but not on CD38KO NK cells, and increased their fratricide. Thus, CD38WT NK cells showed impaired overall DARA-mediated cytotoxicity against MM cells in the presence of ATRA, even though those MM cells were sensitized to DARA. Conclusions: We present proof of concept that adoptive CD38KO NK cell therapy has the potential to maximize the efficacy of DARA against MM. Further investigation into the role of metabolic reprogramming of CD38KO NK cells is warranted.

CD8 regulatory T cells during Zika virus infection

Abstract Introduction Flavivirus infections are cause of great concern worldwide due to recurrent epidemics and high mortality rates, being a major public health problem. The relevance to study Zika virus infection was highlighted after the discovery that ZIKV infection during pregnancy could cause microcephaly, besides others abnormalities. Knowing that ZIKV, like other flaviviruses, can modulate host innate and adaptive immune response, we aim to understand the role of CD8+ regulatory T cells in the control of viral replication in an experimental infection. Methods C57BL/6 Foxp3GFP and C57BL/6 CD8−/− mice were infected with 106 PFU of ZIKV (Brazilian strain), followed by viral quantification, gene expression, and flow cytometry analysis. Results Infection of CD8 knockout mice presented viral load with an oscillatory profile compared to WT mice, and analysis of CD8 T cells populations in WT mice showed that CD8Foxp3 cells are induced on the first day post infection and it pool of cells are maintained during infection, as could be noted on day 30 post infection. These cells are related to increase of viral replication as shown in cell transfer experiment. However, naïve CD8Foxp3 cells are responsible to increase virus replication, while CD8Foxp3 cells from infected mice seems to be not capable to respond to antigen. Conclusion This finding leads us to believe that there is an attempt by the pathogen to modulate the adaptive immune response, in order to facilitate its replication and permanence in the host. In short, our work seeks to elucidate the immunobiology of the ZIKV infection and to correlate this with the susceptibility to the Congenital Syndrome caused by Zika virus.

Nexus between CNS resident microglia and migrating peripheral T cells pave the way for host immunity against neurotropic virus infection

Abstract Neurotropic virus induced neuroinflammation initiates with the activation of brain resident immune cells which causes a heightened release of chemokines/cytokines leading to a compromise in the blood brain barrier integrity. This accentuates the upregulation of regulatory molecules like antiviral Interferon stimulating gene induced tetratricopeptide repeats protein, Ifit2, and augments infiltration of peripheral immune cells. Among the early infiltrating immune cells, a large population is composed of neutrophils and monocyte/macrophages which, along with brain resident microglia, orchestrate the full panoply of innate immune responses. Ifit2 has been shown to be beneficial in protecting mice from lethal neurotropic virus infection by restricting viral replication and amplifying antiviral responses. Using a murine neurotropic coronavirus infection in Ifit2−/− mice, we report that in the absence of Ifit2, viral replication is significantly increased and mice develop severe neuropathy. Despite the enormous viral load, Ifit2-deficient mice are impaired in microglial activation and recruitment of peripheral T cells into the CNS. To further confirm the role of migratory T cells in viral-induced neuroinflammation and neurodegeneration, studies were conducted in CD4−/− mice. Results showed no significant change in acute stage pathogenesis, whereas CD4−/− mice showed accelerated loss of axon-myelin coherence during the chronic stage. Together, studies in Ifit2 and CD4 knockout mice reveal that the interplay between brain resident microglia and migratory helper T cells is important for strengthening host immunity against neurotropic virus infection.

CD38 Causes Autophagic Flux Inhibition and Cardiac Dysfunction Through a Transcriptional Inhibition Pathway Under Hypoxia/Ischemia Conditions.

Induced autophagy is protective against myocardial hypoxia/ischemia (H/I) injury, but evidence regarding the extent of autophagic clearance under H/I and the molecular mechanisms that influence autophagic flux has scarcely been presented. Here, we report that CD38 knockout improved cardiac function and autophagic flux in CD38–/– mice and CD38–/– neonatal cardiomyocytes (CMs) under H/I conditions. Mechanistic studies demonstrated that overexpression of CD38 specifically downregulated the expression of Rab7 and its adaptor protein pleckstrin homology domain-containing protein family member 1 (PLEKHM1) through nicotinamide adenine dinucleotide (NAD)-dependent and non-NAD-dependent pathways, respectively. Loss of Rab7/PLEKHM1 impaired the fusion of autophagosomes and lysosomes, resulting in autophagosome accumulation in the myocardium and consequent cardiac dysfunction under H/I conditions. Thus, CD38 mediated autophagic flux blockade and cardiac dysfunction in a Rab7/PLEKHM1-dependent manner. These findings suggest a potential therapeutic strategy involving targeted suppression of CD38 expression.

Molecular Mechanisms of Stem Cell Reprogramming by CD47 Antagonists in Primary Human Cells

CD47 is a ubiquitously expressed transmembrane receptor whose interaction with signal regulatory protein alpha on macrophages initiates anti‐phagocytic signaling. Additionally, thrombospondin‐1 (THBS1) binding to CD47 induces cell‐autonomous signaling that regulates cell proliferation, differentiation, and recovery from genotoxic stress. Previous studies showed that CD47‐ or THBS1‐null primary lung endothelial cells exposed to ionizing radiation have higher viability than primary wildtype cells. The mechanisms that enhance the viability of CD47‐null primary cells include enhanced autophagy, anabolic metabolism, and increased expression of c‐Myc and other stem cell transcription factors. The latter enable null cells to spontaneously form embryoid bodies. These results suggest that loss of CD47 may generally facilitate cell reprogramming and self‐renewal. Induced pluripotent stem cells (iPS) can be induced by introducing Yamanaka stem cell factors (MYC, KLF4, SOX2, and OCT4) into primary somatic cells, but iPS cells can form teratomas and thus pose risk of cancer when used for regenerative medicine. Therefore, alternative approaches are needed to overcome these limitations. We propose that CD47 gene knock out (KO) via CRISPR or treating with CD47 antagonists from primary human cells could be a new, simpler, and more effective way to develop multipotent stem cells. To investigate this, we have studied the effects of CD47 knockout and antagonists on embryoid body formation and mRNA expression of stem cell factors using fibroblasts, adipocytes, and human umbilical vein cells (HUVEC). Reprogramming was measured by quantifying embryoid body formation, cell viability, and mRNA expression using real‐time PCR. Preliminary results with HUVEC, fibroblast, and adipocyte primary cells treated with CD47 knockout or antagonists have shown enhanced cell proliferation and expression of stem cell factors similar to murine cd47‐null lung endothelial cells. These results suggest that CD47 plays a key regulatory role in stem cell reprogramming of human primary cells.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIH/National Cancer Institute.

Abstract 103: Cd26 Knockout And Inhibition Promotes Dorsal Wound Healing Via Modulation Of Engrailed-1 Positive Fibroblasts

Purpose: Engrailed-1 (En1) positive fibroblasts are responsible for scar formation in the dorsal skin of adult mice during the postnatal period. The cell surface marker CD26, also known as dipeptidyl-peptidase-4 (DPP4), is expressed by the vast majority of En1 positive fibroblasts and can be used to isolate this lineage of scar-forming fibroblasts. We have previously shown that inhibition of CD26 with Diprotin results in decreased cutaneous scarring during wound healing. To further interrogate this biology, we hypothesized that inhibition of CD26 with a small molecule CD26/DPP4 inhibitor (MK0626) or CD26 knockout mice, might improve the rate of wound healing, decrease scar fibrosis, and achieve a more regenerative phenotype in the context of wound healing. Methods: The effects of MK0626 were initially evaluated on NIH 3T3 fibroblasts in vitro. Migration, measured by scratch assay, and proliferation were assessed on treatment and control specimens at 12 and 24 hours. Results were analyzed using ImageJ. To test the effects of MK0262 in vivo, bilateral full thickness wounds were created in the dorsal dermis of 10-week old C57Bl/6 (wild-type) mice. Equivalent wounds were also made in the dorsal dermis of En1mTmG CD26 knockout mice (CD26-/+;En1Cre;R26mTmG). A stented-wound healing model developed in our laboratory, which better mimics human wound healing kinetics, was used. The wild-type mice received oral MK0626 at a high dose (30 mg/kg), low dose (3 mg/kg), or saline control every other day over the course of wound healing. Wounds were harvested at post-operative day 14. Results:In vitro scratch assay showed significantly increased fibroblast migration and increased proliferation with treatment of MK0626 compared with vehicle control. In vivo, orally-administered MK0626 significantly improved the rate of wound closure in wild-type mice. Dermal scar thickness (as well as En-1 expression) was decreased in En1mTmG CD26 knockout mice when compared with En1mTmG control mice. Conclusions: Modulation of CD26 expression shows therapeutic potential to encourage regenerative wound healing. Specifically, our in vivo experiments show that with oral CD26 inhibition or CD26 knockout, the wound closure rate is increased and the dermal scar is thinner, the latter of which correlates with decreased presence of the En1-lineage-positive fibroblasts. Accelerated wound closure with decreased scarring is immensely beneficial from a clinical perspective.

Ectonucleotidase CD39 is highly expressed on ATLL cells and is responsible for their immunosuppressive function

Adult T-cell leukemia/lymphoma (ATLL) patients have an extremely poor prognosis, partly due to their immunosuppressive state. The majority of ATLL patients have leukemic cells with phenotype similar to Tregs, prompting suggestions that ATLL cells themselves have immunosuppressive functions. In this study, we detected CD39 expression on ATLL cells, particularly frequent on aggressive subtypes. CD39 and CD73 convert extracellular adenosine triphosphate (ATP) into adenosine, a key player in Tregs’ immunosuppression. In vitro culture, both CD39+ ATLL cells and normal Tregs converted rapidly extracellular ATP to AMP, which was disturbed by CD39 inhibitors, and was negated in the CD39 knockout MJ cell line. The proliferation of cocultured CD4+/CD8+ normal T cells was suppressed by CD39+ MJ cells, but not by CD39 knockout MJ cells. Supplemented ATP was exhausted by an EG7-OVA T-cell line with stable CD39 induction, but not by mock. When these cell lines were subcutaneously transplanted into murine flanks, Poly(I:C) peritoneal administration reduced tumor size to 1/3 in mock-transplanted tumors, but not in CD39 induced tumors. Overall, we found that ATLL cells express CD39 at a high rate, and our results suggest that this helps ATLL cells escape antitumor immunity through the extracellular ATPDase-Adenosine cascade. These findings will guide future clinical strategies for ATLL treatment.

Cd9 Protects Photoreceptors from Injury and Potentiates Edn2 Expression.

PurposeCd9 is a tetraspanin membrane protein that plays various roles in tissue development and disease pathogenesis, especially in cancer, but the expression patterns and function of Cd9 in retinal development and disease are not well understood. We asked its roles during retinal photoreceptor degeneration by using CD9-knockout mice.MethodsCd9 knockout mice and rd1 mice were used to examine roles of Cd9 for progression of photoreceptor degeneration. Reverse transcription-polymerase chain reaction and immunohistochemistry were mainly used as analytical methods.ResultsCd9 transcripts were only weakly expressed in retina at embryonic day 14, but its expression level subsequently increased and peaked at around postnatal day 12. In 6-week-old female mice derived retina, mRNA expression decreased slightly but was maintained at a significant level. Published RNA-sequencing data and immunohistochemistry indicated that Cd9 was expressed abundantly in Müller glia and weakly in other retinal neurons. Notably, when photoreceptors were damaged, Cd9 expression was increased in rod photoreceptors and decreased in Müller glia. Cd9 knockout mice retinas developed normally; however, once the retina suffered damage, degeneration of photoreceptors was more severe in Cd9 knockout retinas than control retinas. Induction of Edn2, which is known to protect against photoreceptor damage, was severely hampered. In addition, induction of Socs3, which is downstream of gp130 (Il6st), was weaker in Cd9 knockout retinas.ConclusionsTaken together, these findings indicate that, although Cd9 was dispensable for normal gross morphological development, it protected rod photoreceptors and enhanced Edn2 expression, possibly through modulation of gp130 signaling.

Bone marrow dendritic cells deficient for CD40 and IL-23p19 are tolerogenic in vitro.

Objective(s):In addition to pro-inflammatory role, dendritic cells (DCs) can also be anti-inflammatory when they acquire tolerogenic phenotype. In this study we tested the role of CD40 and IL-23p19 in antigen presenting function of bone marrow-derived DCs (BMDCs) by comparing BMDCs derived from CD40 knockout (CD40KO-DCs) and IL-23p19 (IL-23p19KO-DCs) knockout mice with those from C57BL/6 mice (Cont-DCs). We have focused on CD40 and IL-23, as these molecules have well established pro-inflammatory roles in a number of autoimmune and inflammatory diseases. Materials and Methods:The expression of maturation markers MHC II and co-stimulatory molecules CD40, CD80, and CD86 was analyzed by flow cytometry, while the expression of CD40 and IL-23p19 was measured by RT-PCR. The capacity of BMDCs to activate CD4+ T cells was evaluated by 3H-thymidine incorporation, and the capacity of BMDCs to uptake antigen was evaluated using fluorescent OVA and flow cytometry. Results:The lack of CD40 or IL-23p19 had no effect on uptake of FITC-OVA by the DCs, confirming their immature phenotype. Moreover, CD40KO-DCs had significantly reduced capacity to stimulate proliferation of CD4+ T cells. CD4+ T cells activated by CD40KO-DCs and IL-23p19KO-DCs produced significantly less IFN-γ (P-value ≤0.05), while CD4+ T cells stimulated by IL-23p19KO-DCs produced less GM-CSF and more IL-10 than Cont-DCs. Conclusion:This study shows that CD40KO-DCs and IL-23p19KO-DCs have a marked tolerogenic potency in vitro. Future in vivo studies should determine if and to what extent DCs lacking CD40 or IL-23 have a potential to be useful in therapy of autoimmune inflammation.

CD73 or CD39 Deletion Reveals Different Mechanisms of Formation for Spontaneous and Mechanically Stimulated Adenosine and Sex Specific Compensations in ATP Degradation.

Adenosine is important for local neuromodulation, and rapid adenosine signaling can occur spontaneously or after mechanical stimulation, but little is known about how adenosine is formed in the extracellular space for those stimulations. Here, we studied mechanically stimulated and spontaneous adenosine to determine if rapid adenosine is formed by extracellular breakdown of adenosine triphosphate (ATP) using mice globally deficient in extracellular breakdown enzymes, either CD39 (nucleoside triphosphate diphosphohydrolase 1, NTPDase1) or CD73 (ecto-5'-nucleotidase). CD39 knockout (KO) mice have a lower frequency of spontaneous adenosine events than wild-type (WT, C57BL/6). Surprisingly, CD73KO mice demonstrate sex differences in spontaneous adenosine; males maintain similar event frequencies as WT, but females have significantly fewer events and lower concentrations. Examining the mRNA expression of other enzymes that metabolize ATP revealed tissue nonspecific alkaline phosphatase (TNAP) was upregulated in male CD73KO mice, but not secreted prostatic acid phosphatase (PAP) or transmembrane PAP. Thus, TNAP upregulation compensates for CD73 loss in males but not in females. These sex differences highlight that spontaneous adenosine is formed by metabolism of extracellular ATP by many enzymes. For mechanically stimulated adenosine, CD39KO or CD73KO did not change stimulation frequency, concentration, or t1/2. Thus, the mechanism of formation for mechanically stimulated adenosine is likely direct release of adenosine, different than spontaneous adenosine. Understanding these different mechanisms of rapid adenosine formation will help to develop pharmacological treatments that differentially target modes of rapid adenosine signaling, and all treatments should be studied in both sexes, given possible differences in extracellular ATP degradation.

CD38 in Neurodegeneration and Neuroinflammation.

Neurodegenerative diseases are characterized by neuronal degeneration as well as neuroinflammation. While CD38 is strongly expressed in brain cells including neurons, astrocytes as well as microglial cells, the role played by CD38 in neurodegeneration and neuroinflammation remains elusive. Yet, CD38 expression increases as a consequence of aging which is otherwise the primary risk associated with neurodegenerative diseases, and several experimental data demonstrated that CD38 knockout mice are protected from neurodegenerative and neuroinflammatory insults. Moreover, nicotinamide adenine dinucleotide, whose levels are tightly controlled by CD38, is a recognized and potent neuroprotective agent, and NAD supplementation was found to be beneficial against neurodegenerative diseases. The aims of this review are to summarize the physiological role played by CD38 in the brain, present the arguments indicating the involvement of CD38 in neurodegeneration and neuroinflammation, and to discuss these observations in light of CD38 complex biology.

Abstract WP323: Genetic Knockout of CD38 in Mice Protects Against Ischemic Stroke With Smaller Infarct Volumes and Decreased Motor Impairment

Introduction: Endothelial dysfunction is an important mediator of post ischemic injury of the heart and brain following ischemia/reperfusion (I/R). We have reported that CD38 activation in heart I/R models leads to NADPH depletion with endothelial nitric oxide synthase (eNOS) impairment, loss of endothelial-mediated coronary dilatation and increased myocardial infarction. While CD38 knockout or inhibition prevents this dysfunction and decreases myocardial infarction, the role of CD38 in ischemic stroke remains uncertain. Hypothesis: We hypothesize that loss of CD38 expression and activity through gene knockout is protective with smaller infarct volume. Methods: 15 week-old male CD38 knock out (KO) n=12 and wild type (WT) mice n=12 underwent middle cerebral artery occlusion (MCAO) for 60 minutes. Stroke volume was calculated using T2 MRI sequences on a 9.4 T MRI system acquired 48 hours post stroke with images analyzed using Osirix software. The ratios of the stroke volume to the affected hemisphere volume and the compensated swelling infarction volume percentage of normal hemisphere were calculated using established methods. Open field test to measure motor impairment was performed at baseline and 48 hours post stroke (KO, n=12, WT n=6). Statistical analysis was completed in STATA using Man-Whitney U test and T-test to compare infarct volumes and cognitive parameters. Values are shown as mean ± SD. P value < 0.05 was considered significant. Results: At 48 hours, brain MRI showed a smaller percentage of cerebral hemisphere affected by stroke in CD38 KO compared to WT (25.9±3.7 vs 41.1±9.4, respectively P=0.0001) and a smaller percentage of compensated swelling infarction volume of normal hemisphere in KO mice compared to WT (19.6±3 vs 33.5±9, respectively P=0.0001). Open field test showed significant post stroke motor impairment in WT compared to CD38KO mice (distance travelled 1.8±1 m vs 4.7±3 m, P=0.04, respectively and average speed 0.006±0.003 m/s vs 0.016±0.01m/s, P=0.04, respectively). Conclusions: Infarct volumes are smaller and motor impairment is decreased in CD38 KO mice compared to WT demonstrating that gene knockout of CD38 confers neuroprotection against acute ischemic brain injury.

CD4+ T Cell-Derived NGAL Modifies the Outcome of Ischemic Acute Kidney Injury.

CD4+ T cells mediate the pathogenesis of ischemic and nephrotoxic acute kidney injury (AKI). However, the underlying mechanisms of CD4+ T cell-mediated pathogenesis are largely unknown. We therefore conducted unbiased RNA-sequencing to discover novel mechanistic pathways of kidney CD4+ T cells after ischemia compared with normal mouse kidney. Unexpectedly, the lipocalin-2 (Lcn2) gene, which encodes neutrophil gelatinase-associated lipocalin (NGAL) had the highest fold increase (∼60). The NGAL increase in CD4+ T cells during AKI was confirmed at the mRNA level with quantitative real-time PCR and at the protein level with ELISA. NGAL is a potential biomarker for the early detection of AKI and has multiple potential biological functions. However, the role of NGAL produced by CD4+ T cells is not known. We found that ischemic AKI in NGAL knockout (KO) mice had worse renal outcomes compared with wild-type (WT) mice. Adoptive transfer of NGAL-deficient CD4+ T cells from NGAL KO mice into CD4 KO or WT mice led to worse renal function than transfer of WT CD4+ T cells. In vitro-simulated ischemia/reperfusion showed that NGAL-deficient CD4+ T cells express higher levels of IFN-γ mRNA compared with WT CD4+ T cells. In vitro differentiation of naive CD4+ T cells to Th17, Th1, and Th2 cells led to significant increase in Lcn2 expression. Human kidney CD4+ T cell NGAL also increased significantly after ischemia. These results demonstrate an important role for CD4+ T cell NGAL as a mechanism by which CD4+ T cells mediate AKI and extend the importance of NGAL in AKI beyond diagnostics.

Extracellular NAD+ enhances PARP-dependent DNA repair capacity independently of CD73 activity

Changes in nicotinamide adenine dinucleotide (NAD+) levels that compromise mitochondrial function trigger release of DNA damaging reactive oxygen species. NAD+ levels also affect DNA repair capacity as NAD+ is a substrate for PARP-enzymes (mono/poly-ADP-ribosylation) and sirtuins (deacetylation). The ecto-5′-nucleotidase CD73, an ectoenzyme highly expressed in cancer, is suggested to regulate intracellular NAD+ levels by processing NAD+ and its bio-precursor, nicotinamide mononucleotide (NMN), from tumor microenvironments, thereby enhancing tumor DNA repair capacity and chemotherapy resistance. We therefore investigated whether expression of CD73 impacts intracellular NAD+ content and NAD+-dependent DNA repair capacity. Reduced intracellular NAD+ levels suppressed recruitment of the DNA repair protein XRCC1 to sites of genomic DNA damage and impacted the amount of accumulated DNA damage. Further, decreased NAD+ reduced the capacity to repair DNA damage induced by DNA alkylating agents. Overall, reversal of these outcomes through NAD+ or NMN supplementation was independent of CD73. In opposition to its proposed role in extracellular NAD+ bioprocessing, we found that recombinant human CD73 only poorly processes NMN but not NAD+. A positive correlation between CD73 expression and intracellular NAD+ content could not be made as CD73 knockout human cells were efficient in generating intracellular NAD+ when supplemented with NAD+ or NMN.

Natural Killer and CD8 T Cells Contribute to Protection by Formalin Inactivated Respiratory Syncytial Virus Vaccination under a CD4-Deficient Condition.

Respiratory syncytial virus (RSV) causes severe pulmonary disease in infants, young children, and the elderly. Formalin inactivated RSV (FI-RSV) vaccine trials failed due to vaccine enhanced respiratory disease, but the underlying immune mechanisms remain not fully understood. In this study, we have used wild type C57BL/6 and CD4 knockout (CD4KO) mouse models to better understand the roles of the CD4 T cells and cellular mechanisms responsible for enhanced respiratory disease after FI-RSV vaccination and RSV infection. Less eosinophil infiltration and lower pro-inflammatory cytokine production were observed in FI-RSV vaccinated CD4KO mice after RSV infection compared to FI-RSV vaccinated C57BL/6 mice. NK cells and cytokine-producing CD8 T cells were recruited at high levels in the airways of CD4KO mice, correlating with reduced respiratory disease. Depletion studies provided evidence that virus control was primarily mediated by NK cells whereas CD8 T cells contributed to IFN-γ production and less eosinophilic lung inflammation. This study demonstrated the differential roles of effector CD4 and CD8 T cells as well as NK cells, in networking with other inflammatory infiltrates in RSV disease in immune competent and CD4-deficient condition.

The Effect of in vivo Macrophage Depletion on Skin Allograft Rejection in Wild-type and CD8 Knockout Mice

The Effect of in vivo Macrophage Depletion on Skin Allograft Rejection in Wild-type and CD8 Knockout Mice

CD38, CD157, and RAGE as Molecular Determinants for Social Behavior.

Recent studies provide evidence to support that cluster of differentiation 38 (CD38) and CD157 meaningfully act in the brain as neuroregulators. They primarily affect social behaviors. Social behaviors are impaired in Cd38 and Cd157 knockout mice. Single-nucleotide polymorphisms of the CD38 and CD157/BST1 genes are associated with multiple neurological and psychiatric conditions, including autism spectrum disorder, Parkinson’s disease, and schizophrenia. In addition, both antigens are related to infectious and immunoregulational processes. The most important clues to demonstrate how these molecules play a role in the brain are oxytocin (OT) and the OT system. OT is axo-dendritically secreted into the brain from OT-containing neurons and causes activation of OT receptors mainly on hypothalamic neurons. Here, we overview the CD38/CD157-dependent OT release mechanism as the initiation step for social behavior. The receptor for advanced glycation end-products (RAGE) is a newly identified molecule as an OT binding protein and serves as a transporter of OT to the brain, crossing over the blood–brain barrier, resulting in the regulation of brain OT levels. We point out new roles of CD38 and CD157 during neuronal development and aging in relation to nicotinamide adenine dinucleotide+ levels in embryonic and adult nervous systems. Finally, we discuss how CD38, CD157, and RAGE are crucial for social recognition and behavior in daily life.

Abstract B03: Targeting tumor-intrinsic metabolic node in pancreatic cancer causes tumor regression, remodels extracellular matrix, and sensitizes to anti-PD1 therapy

Abstract Background: Pancreatic adenocarcinoma (PDAC) is an extremely immunosuppressive and heterogenous neoplasm for which immune checkpoint inhibitor therapy has not been very successful. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on one hand and also influence the extracellular matrix in the tumor microenvironment. Our study aims to sensitize pancreatic cancer to anti PD-1 therapy by targeting the HBP pathway. Methods: 6-Diazo-5-oxo L-norleucine or DON was used as a GFAT inhibitor. Tetracycline-inducible GFAT knockout cell lines were made using S2-VP10 as the parent cell line. Results: High-dose monotherapy of DON (1mg/kg/3days a week) caused significant decrease in tumor volume in immunocompetent KPC mice along with significant reduction in metastasis and circulating tumor cells. Circulating tumor cells (CTCs) were also found to be significantly reduced in DON-treated mice as compared to control, leading to reduced metastasis. Combination of low-dose DON (0.5mg/kg/3 days a week) with low-dose anti PD-1 (100 μgX3 times) caused an impressive reduction in tumor volume along with an increase in survival in KPC tumor-bearing mice. Flow-based analysis of tumor cells show a significant influx of CD8+ T cells along with an increase in M1 polarization of macrophages. Survival study of CD8 knockout mice (CD8KO), orthotopically implanted with KPC and fibroblast cells, show that at low doses DON loses its anticancer effect in absence of CD8 as demonstrated by decrease in survival in CD8KO mice as compared to tumor-bearing wild-type mice. Conclusion: Novel therapeutic strategies that can make PDAC more amenable to immune therapy are urgently needed. In this context, our study is extremely timely as it targets specific metabolic pathways in cancer cells to overcome immune resistance and sensitize the tumor to anti-PD1 therapy. Citation Format: Nikita S. Sharma, Vineet K. Gupta, Vanessa Garrido, Roey Hadad, Brittany Durden, Kousik Kesh, Anthony Ferrantella, Bhuwan Giri, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. Targeting tumor-intrinsic metabolic node in pancreatic cancer causes tumor regression, remodels extracellular matrix, and sensitizes to anti-PD1 therapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B03.

CD28 Deficiency Ameliorates Thoracic Blast Exposure-Induced Oxidative Stress and Apoptosis in the Brain through the PI3K/Nrf2/Keap1 Signaling Pathway.

Blast exposure is a worldwide public health concern, but most related research has been focused on direct injury. Thoracic blast exposure-induced neurotrauma is a type of indirect injuries where research is lacking. As CD28 stimulates T cell activation and survival and contributes to inflammation initiation, it may play a role in thoracic blast exposure-induced neurotrauma. However, it has not been investigated. To explore the effects of CD28 on thoracic blast exposure-induced brain injury and its potential molecular mechanisms, a mouse model of thoracic blast exposure-induced brain injury was established. Fifty C57BL/6 wild-type (WT) and fifty CD28 knockout (CD28−/−) mice were randomly divided into five groups (one control group and four model groups), with ten mice (from each of the two models) for each group. Lung and brain tissue and serum samples were collected at 12 h, 24 h, 48 h, and 1 week after thoracic blast exposure. Histopathological changes were detected by hematoxylin-eosin staining. The expressions of inflammatory-related factors were detected by ELISA. Oxidative stress in the brain tissue was evaluated by determining the generation of reactive oxygen species (ROS) and the expressions of thioredoxin (TRX), malondialdehyde (MDA), SOD-1, and SOD-2. Apoptosis in the brain tissue was evaluated by TUNEL staining and the levels of Bax, Bcl-xL, Bad, Cytochrome C, and caspase-3. In addition, proteins of related pathways were also studied by western blotting and immunofluorescence. We found that CD28 deficiency significantly reduced thoracic blast exposure-induced histopathological changes and decreased the levels of inflammatory-related factors, including IL-1β, TNF-α, and S100β. In the brain tissue, CD28 deficiency also significantly attenuated thoracic blast exposure-induced generation of ROS and expressions of MDA, TRX, SOD-1, and SOD-2; lowered the number of apoptotic cells and the expression of Bax, cleaved caspase-3, Cytochrome C, and Bad; and maintained Bcl-xL expression. Additionally, CD28 deficiency significantly ameliorated thoracic blast exposure-induced increases of p-PI3K and Keap1 and the decrease of Nrf2 expression in the brain. Our results indicate that CD28 deficiency has a protective effect on thoracic blast exposure-induced brain injury that might be associated with the PI3K/Nrf2/Keap1 signaling pathway.