CD4 Knockout Mice Research Articles

CD226 Deletion Reduces Type 1 Diabetes in the NOD Mouse by Impairing Thymocyte Development and Peripheral T Cell Activation.

The costimulatory molecule CD226 is highly expressed on effector/memory T cells and natural killer cells. Costimulatory signals received by T cells can impact both central and peripheral tolerance mechanisms. Genetic polymorphisms in CD226 have been associated with susceptibility to type 1 diabetes and other autoimmune diseases. We hypothesized that genetic deletion of Cd226 in the non-obese diabetic (NOD) mouse would impact type 1 diabetes incidence by altering T cell activation. CD226 knockout (KO) NOD mice displayed decreased disease incidence and insulitis in comparison to wild-type (WT) controls. Although female CD226 KO mice had similar levels of sialoadenitis as WT controls, male CD226 KO mice showed protection from dacryoadenitis. Moreover, CD226 KO T cells were less capable of adoptively transferring disease compared to WT NOD T cells. Of note, CD226 KO mice demonstrated increased CD8+ single positive (SP) thymocytes, leading to increased numbers of CD8+ T cells in the spleen. Decreased percentages of memory CD8+CD44+CD62L– T cells were observed in the pancreatic lymph nodes of CD226 KO mice. Intriguingly, CD8+ T cells in CD226 KO mice showed decreased islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-tetramer and CD5 staining, suggesting reduced T cell receptor affinity for this immunodominant antigen. These data support an important role for CD226 in type 1 diabetes development by modulating thymic T cell selection as well as impacting peripheral memory/effector CD8+ T cell activation and function.

Abstract LB-071: Cross talk between T lymphocytes and macrophages promotes progression of radiation induced lung fibrosis

Abstract Background: Fractionated radiotherapy (RT) is integral part of standard treatment for locally advanced lung cancer. However radiation-induced lung disease limits the application of curative radiation doses and therapy intensification efforts of concurrent chemoradiotherapy, leading to fatal outcomes by local recurrence or metastatic disease even when accepting side effects that decrease the quality of life. So far, no predictive biomarkers exist and no mechanism-based therapies are in clinical use that prevent or treat dose-limiting pneumonitis and lung fibrosis. Own previous work revealed a pathogenic role of the immunomodulatory CD73/adenosine system and of macrophages in radiation-induced pulmonary fibrosis1,2. Moreover, clinical observations associated CD4+ T lymphocytes with adverse effects of RT in the skin and the lung of cancer patients3,4. However, the contribution of RT-induced immune changes to RT-induced lung disease require further definition. Here, we studied the functional role of lymphocytes in radiation-induced lung fibrosis, with a focus on CD4+ T cells. Methods: C57BL/6 wildtype (WT) and RAG-2 knockout (RAG-2-/-) mice received a single dose of whole thorax irradiation with 0 Gy (sham controls) or 15 Gy without or with additional adoptive transfer of CD4+ T cells. We characterized time-dependent immuno-modulation and pathogenesis of lung tissue in flow cytometry, histological and immunohistochemical as well as RNA analyses in comparison to our earlier analyses in or CD73 knockout (CD73-/-) mice. Results: CD4+ T lymphocytes, particulary Treg, accumulated in irradiated lungs of WT mice during the chronic fibrotic phase. While deficiency of B and T cells in RAG2-/- mice led to an earlier onset of fibrosis compared to WT mice, adoptive transfer of CD4+ T cells into RAG2-/- mice led to further aggravation of pulmonary fibrosis. Analysis of immune cell subtypes in adoptively transferred RAG2-/- mice revealed that CD4+ T cells were enhanced, and this was associated with a phenotypic switch in the macrophage population towards M2-like phenotypes that was not observed in CD73-/- mice. Conclusions: We conclude that a reciprocal crosstalk between CD4+ T lymphocytes and macrophages in the irradiated lung environment promotes disease progression, amongst others by regulating macrophage polarization in a microenvironment-dependent manner. 1) Wirsdörfer et al., Cancer Res 2016;76:3045-56. 2) de Leve S et al., FASEB J 2017, 31;2869-2880; Veldwijk MR et al. Clin Cancer Res., 2019, 25:562-572; Nakayama Y et al., Int J Radiat Oncol Biol Phys 1996, 34:459-467. This work was supported by grants of the University Hospital Essen (IFORES) to FW and the DFG (JE275/4-1; GRK1739/2) to VJ. Citation Format: Florian Wirsdörfer, Lena Gockeln, Simone de Leve, Martin Stuschke, Verena Jendrossek. Cross talk between T lymphocytes and macrophages promotes progression of radiation induced lung fibrosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-071.

Suppression of Chlamydial Pathogenicity by Nonspecific CD8+ T Lymphocytes.

Chlamydia trachomatis, a leading infectious cause of tubal infertility, induces upper genital tract pathology, such as hydrosalpinx, which can be modeled with Chlamydia muridarum infection in mice. Following C. muridarum inoculation, wild-type mice develop robust hydrosalpinx, but OT1 mice fail to do so because their T cell receptors are engineered to recognize a single ovalbumin epitope (OVA457-462). These observations have demonstrated a critical role of Chlamydia-specific T cells in chlamydial pathogenicity. In the current study, we have also found that OT1 mice can actively inhibit chlamydial pathogenicity. First, depletion of CD8+ T cells from OT1 mice led to the induction of significant hydrosalpinx by Chlamydia, indicating that CD8+ T cells are necessary to inhibit chlamydial pathogenicity. Second, adoptive transfer of CD8+ T cells from OT1 mice to CD8 knockout mice significantly reduced chlamydial induction of hydrosalpinx, demonstrating that OT1 CD8+ T cells are sufficient for attenuating chlamydial pathogenicity in CD8 knockout mice. Finally, CD8+ T cells from OT1 mice also significantly inhibited hydrosalpinx development in wild-type mice following an intravaginal inoculation with Chlamydia Since T cells in OT1 mice are engineered to recognize only the OVA457-462 epitope, the above observations have demonstrated a chlamydial antigen-independent immune mechanism for regulating chlamydial pathogenicity. Further characterization of this mechanism may provide information for developing strategies to reduce infertility-causing pathology induced by infections.

Abstract 452: Role of CD38 in Cardiovascular Performance and Its Use as a Therapeutic Target in Obesity-Induced Diastolic Dysfunction

Background: Cardiac tissue possesses high energetic requirements. During development of heart failure there is a shift from fatty acid oxidation to the utilization of other substrates. This transition leads to the decline in NAD/NADH ratio, which increases susceptibility to stress. CD38 is the main NAD + -consuming enzyme responsible for NAD + levels regulation in tissue and therefore represents a possible therapeutic target in such conditions. Objective: To investigate the role of CD38 in cardiovascular performance and its potential use as a therapeutic target in obesity-induced heart failure. Results: When comparing adult Wild-Type (WT) and CD38 Knock-out (KO) mice we observed that the latter had higher exercise performance, increased heart rate variability (HRV) and decreased heart rate (HR). When comparing WT and CD38 catalytically inactive (CI) mice we observed the same results, showing its relation with CD38 catalytic activity. WT mice treated with an antibody which blocks CD38 activity showed improved exercise capacity and HRV. In both genetically modified or antibody treated groups, NAD+ levels were significantly increased in heart. In order to explore the NAD + role in these findings, we compared WT control and CD38KO treated with FK866 (Nampt inhibitor) and observed that NAD+ levels in heart decreased as well as exercise capacity. We then aimed to demonstrate CD38 role as a target for therapeutic intervention in a model of cardiac strain induced by obesity in aged mice. We compared WT and CD38 CI aged mice, placed in normal diet (ND) or high fat diet (HFD). WT animals fed HFD showed decreased exercise capacity and this effect is reversed in CD38 CI fed HFD. Similarly, WT placed in HFD developed diastolic dysfunction seen as a significant increase in E/e’ ratio, and CD38 CI placed in HFD were protect against the development of this dysfunction. Mechanistically, we observed that blockage of CD38 resulted in decreased protein acetylation and increased SERCA expression. Conclusion: CD38 plays a role in cardiovascular performance by regulation of NAD + homeostasis. In a model of obesity-induced diastolic dysfunction NAD+-boosting by blocking CD38 activity led to protection by up-regulating SERCA expression.

Thrombospondin 1 improves hepatic steatosis in diet-induced insulin-resistant mice and is associated with hepatic fat content in humans.

BackgroundNonalcoholic fatty liver disease (NAFLD) is associated with altered production of secreted proteins. Increased understanding of secreted proteins could lead to improved prediction and treatment of NAFLD. Here, we aimed to discover novel secreted proteins in humans that are associated with hepatic fat content using unbiased proteomic profiling strategy, and how the identified Thbs1 modulates lipid metabolism and hepatic steatosis.MethodNAFLD patients were enrolled and treated with lifestyle intervention. Patients who underwent liver biopsy were enrolled for analyzing the correlation between circulating Thbs1 and liver steatosis. Mice were fed on high-fat, high-sucrose diet and treated with recombinant Thbs1. Primary hepatocytes isolated from CD36 knockout (CD36-/-) mice and their wild-type littermates (controls) were treated with glucose plus insulin for 24 h together with or without recombinant Thbs1.FindingSerum Thbs1 levels are increased in participants with NAFLD and positively associated with liver steatosis grades. Improvement of liver steatosis after lifestyle intervention was accompanied with significant reduction of serum Thbs1 levels. Pharmacological administration of recombinant human Thbs1 attenuates hepatic steatosis in diet-induced obese mice. Treatment with Thbs1 protein or stably overexpression of Thbs1 causes a significant reduction of lipid accumulation in primary hepatocytes or HepG2 cells exposed to high glucose plus insulin, suggesting that Thbs1 regulates lipid metabolism in a hepatocyte-autonomous manner. Mechanistically, Thbs1 inhibits cleavage and processing of SREBP-1, leading to a reduction of target lipogenic gene expression and hepatic steatosis. Inhibitory effects of Thbs1 on lipogenesis and triglyceride accumulation are abrogated in CD36 deficient primary hepatocytes exposed to high glucose plus insulin. Interestingly, beneficial effects of Thbs1 on lipid accumulation are observed in primary hepatocytes treated with a Thbs1 nonapeptide mimetic ABT-526.InterpretationThbs1 is a biomarker for NAFLD in humans, and pharmacological and genetic approaches for the modulation of Thbs1 activity may have the therapeutic potential for treating hepatic steatosis.FundA full list of funding bodies that contributed to this study can be found in the Funding Sources section.

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.

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.

Deficiency of platelet adhesion molecule CD226 causes megakaryocyte development and platelet hyperactivity.

This study used constitutive CD226 gene knockout (KO) mice as a model to investigate the functions and mechanisms of CD226 in megakaryocyte (MK) maturation and platelet activation. Although CD226 deficiency did not cause MK polyploidization or platelet granule abnormalities, increased MK counts were detected in the femora bone marrow (BM) and spleen of CD226 KO mice. Particularly, CD226 KO mice have a more extensive membrane system in MKs and platelets than wild-type (WT) mice. We also demonstrated that CD226 KO mice displayed increased platelet counts, shortened bleeding time, and enhanced platelet aggregation. CD226 KO platelets had an increased mature platelet ratio compared to the control platelets. In addition, the observed reduction in bleeding time may be due to decreased nitric oxide (NO) production in the platelets. Platelet-specific CD226-deficient mice showed similar increased MK counts, shortened bleeding time, enhanced platelet aggregation, and decreased NO production in platelets. Furthermore, we performed middle cerebral artery occlusion-reperfusion surgery on WT and CD226 KO mice to explore the potential effect of CD226 on acute ischemia-reperfusion injury; the results revealed that CD226 deficiency led to significantly increased infarct area. Thus, CD226 is a promising candidate for the treatment of thrombotic disorders.

Abstract A36: CXCL14-mediated antigen-specific CD8+ T-cell responses suppress HPV-positive head and neck cancer

Abstract Human papillomaviruses (HPVs) cause over 5% of all human cancer incidences, including a subset of head and neck squamous cell carcinoma (HNSCC), resulting in about half a million deaths every year. Persistent infection with high-risk HPV is necessary for development of HPV-associated malignancies. To establish persistence in the host, HPV must evade host antiviral defenses, including the innate and adaptive immune responses. As antiviral and antitumor immune responses share similar mechanisms, it is likely that HPV-induced immune suppression leads to immune evasion and survival of HPV-infected cancer cells. We have previously shown that expression of the chemokine CXCL14 is significantly downregulated by promoter methylation mediated by the HPV oncoprotein E7 during cancer progression. Restoration of CXCL14 expression in HPV-positive HNSCC cells dramatically suppresses tumor growth through an immune-dependent mechanism in mice. While CXCL14 recruits NK and CD8+ T cells to the tumor microenvironment, the roles of NK and T cells to enact the CXCL14-mediated tumor suppression remained undefined. To determine the roles of NK and CD8+ T cells, tumor growth was assessed in wild-type C57BL/6 mice depleted of NK or CD8+ T cells and CD8-knockout mice injected with the HPV-positive HNSCC cells re-expressing CXCL14. The results show that that CD8+ T cells are required for CXCL14-mediated tumor suppression. The antitumor CD8+ T-cell responses require antigen specificity as a transgenic model of mice with a restricted CD8+ T-cell receptor failed to control tumor growth. Counteracting the HPV-mediated downregulation of major histocompatibility complex class I (MHC-I), CXCL14 expression restores MHC-I expression on HPV-positive HNSCC cells. Furthermore, knockdown of MHC-I expression in CXCL14 expressing HNSCC cells results in loss of tumor suppression, defining a critical role for antigen presentation. These results suggest that CXCL14 expression drives antigen-specific CD8+ T-cell responses and suppresses tumor growth through the restoration of MHC-I expression in HPV-positive HNSCC. Our findings provide useful insight into the CXCL14-mediated antitumor mechanism that increases CD8+ T-cell recognition of tumor cells and could boost the efficacy of current immunotherapies. Citation Format: Joseph A. Westrich, Daniel W. Vermeer, Stephanie Bonney, Alexa Silva, Jennifer N. Berger, Marianna Madeo, Louis Cicchini, John H. Lee, William C. Spanos, Dohun Pyeon. CXCL14-mediated antigen-specific CD8+ T-cell responses suppress HPV-positive head and neck cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A36.

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.

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.

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.

Antibody-suppressor CD8+ T Cells Require CXCR5.

We previously reported the novel activity of alloprimed CD8 T cells that suppress posttransplant alloantibody production. The purpose of the study is to investigate the expression and role of CXCR5 on antibody-suppressor CD8 T-cell function. C57BL/6 mice were transplanted with FVB/N hepatocytes. Alloprimed CD8 T cells were retrieved on day 7 from hepatocyte transplant recipients. Unsorted or flow-sorted (CXCR5CXCR3 and CXCR3CXCR5) alloprimed CD8 T-cell subsets were analyzed for in vitro cytotoxicity and capacity to inhibit in vivo alloantibody production following adoptive transfer into C57BL/6 or high alloantibody-producing CD8 knock out (KO) hepatocyte transplant recipients. Alloantibody titer was assessed in CD8 KO mice reconstituted with naive CD8 T cells retrieved from C57BL/6, CXCR5 KO, or CXCR3 KO mice. Antibody suppression by ovalbumin (OVA)-primed monoclonal OVA-specific t-cell receptor transgenic CD8+ T cells (OT-I) CXCR5 or CXCR3 CD8 T-cell subsets was also investigated. Alloprimed CXCR5CXCR3CD8 T cells mediated in vitro cytotoxicity of alloprimed "self" B cells, while CXCR3CXCR5CD8 T cells did not. Only flow-sorted alloprimed CXCR5CXCR3CD8 T cells (not flow-sorted alloprimed CXCR3CXCR5CD8 T cells) suppressed alloantibody production and enhanced graft survival when transferred into transplant recipients. Unlike CD8 T cells from wild-type or CXCR3 KO mice, CD8 T cells from CXCR5 KO mice do not develop alloantibody-suppressor function. Similarly, only flow-sorted CXCR5CXCR3 (and not CXCR3CXCR5) OVA-primed OT-I CD8 T cells mediated in vivo suppression of anti-OVA antibody production. These data support the conclusion that expression of CXCR5 by antigen-primed CD8 T cells is critical for the function of antibody-suppressor CD8 T cells.

A role for the CD38 rs3796863 polymorphism in alcohol and monetary reward: evidence from CD38 knockout mice and alcohol self-administration, [11C]-raclopride binding, and functional MRI in humans

ABSTRACTBackground: Cluster of differentiation 38 (CD38) is a transmembrane protein expressed in dopaminergic reward pathways in the brain, including the nucleus accumbens (NAc). The GG genotype of a common single nucleotide polymorphism (SNP) within CD38, rs3796863, is associated with increased social reward.Objective: Examine whether CD38 rs3796863 and Cd38 knockout (KO) are associated with reward-related neural and behavioral phenotypes.Methods: Data from four independent human studies were used to test whether rs3796863 genotype is associated with: (1) intravenous alcohol self-administration (n = 64, 30 females), (2) alcohol-stimulated dopamine (DA) release measured using 11C-raclopride positron emission tomography (n = 22 men), (3) ventral striatum (VS) response to positive feedback measured using a card guessing functional magnetic resonance imaging (fMRI) paradigm (n = 531, 276 females), and (4) resting state functional connectivity (rsfc) of the VS (n = 51, 26 females). In a fifth study, we used a mouse model to examine whether cd38 knockout influences stimulated DA release in the NAc core and dorsal striatum using fast-scanning cyclic voltammetry.Results: Relative to T allele carriers, G homozygotes at rs3796863 within CD38 were characterized by greater alcohol self-administration, alcohol-stimulated dopamine release, VS response to positive feedback, and rsfc between the VS and anterior cingulate cortex. High-frequency stimulation reduced DA release among Cd38 KO mice had reduced dopamine release in the NAc.Conclusion: Converging evidence suggests that CD38 rs3796863 genotype may increase DA-related reward response and alcohol consumption.