CD40-CD40L Interaction Research Articles

Mechanotransduction governs CD40 function and underlies X-linked hyper-IgM syndrome.

B cell maturation depends on cognate interactions between the T and B cells. Upon interaction with CD40 ligand (CD40L) on T cells, CD40 delivers costimulatory signals alongside B cell antigen receptor (BCR) signaling to regulate affinity maturation and antibody class switch. Mutations affecting CD40-CD40L interactions cause abnormal antibody responses in immunodeficiencies known as X-linked hyper-IgM syndrome (X-HIgM). Here, we study the CD40-mediated mechanotransduction in B cells, which likely occurs during their physical contacts with T cells. We found that CD40 forms catch bond with CD40L that lasts longer at larger forces, both B and T cells exert tension on CD40-CD40L bonds, and force enhances CD40 signaling and antibody class switch. X-HIgM CD40L mutations impair catch bond formation, suppress endogenous tension, and reduce force-enhanced CD40 signaling, leading to deficiencies in antibody class switch. Our findings highlight the role of mechanotransduction in CD40 function and provide insights into the mechanisms underlying X-HIgM syndrome.

CD4+ T cell help during early acute hepacivirus infection is critical for viral clearance and the generation of a liver-homing CD103+CD49a+ effector CD8+ T cell subset.

In hepatitis C virus (HCV) infection, CD4+ and CD8+ T cells are crucial for viral control. However, a detailed understanding of the kinetic of CD4+ T cell help and its role in the generation of different CD8+ T cell subsets during acute infection is lacking. The absence of a small HCV animal model has impeded mechanistic studies of hepatic antiviral T cell immunity and HCV vaccine development. In this study, we used a recently developed HCV-related rodent hepacivirus infection mouse model to investigate the impact of CD4+ T cell help on the hepatic CD8+ T cell response and viral clearance during hepacivirus infection in vivo. Our results revealed a specific kinetic of CD4+ T cell dependency during acute infection. Early CD4+ T cell help was essential for CD8+ T cell priming and viral clearance, while CD4+ T cells became dispensable during later stages of acute infection. Effector CD8+ T cells directly mediated timely hepacivirus clearance. An analysis of hepatic CD8+ T cells specific for two different viral epitopes revealed the induction of subsets of liver-homing CD103+CD49a+ and CD103-CD49a+ effector CD8+ T cells with elevated IFN-γ and TNF-α production. CD103+CD49a+ T cells further persisted as tissue-resident memory subsets. A lack of CD4+ T cell help and CD40L-CD40 interactions resulted in reduced effector functions and phenotypical changes in effector CD8+ T cells and a specific loss of the CD103+CD49a+ subset. In summary, our study shows that early CD4+ T cell help through CD40L signaling is essential for priming functional effector CD8+ T cell subsets, including unique liver-homing subsets, and hepacivirus clearance.

Failed Downregulation of PI3K Signaling Makes Autoreactive B Cells Receptive to Bystander T Cell Help.

The role of T cell help in autoantibody responses is not well understood. Because tolerance mechanisms govern both T and B cell responses, one might predict that both T cell tolerance and B cell tolerance must be defeated in autoantibody responses requiring T cell help. To define whether autoreactive B cells depend on T cells to generate autoantibody responses, we studied the role of T cells in murine autoantibody responses resulting from acute B cell-specific deletion of regulatory phosphatases. Ars/A1 B cells are DNA reactive and require continuous inhibitory signaling by the tyrosine phosphatase SHP-1 and the inositol phosphatases SHIP-1 and PTEN to maintain unresponsiveness. Acute B cell-restricted deletion of any of these phosphatases results in an autoantibody response. In this study, we show that CD40-CD40L interactions are required to support autoantibody responses of B cells whose anergy has been compromised. If the B cell-intrinsic driver of loss of tolerance is failed negative regulation of PI3K signaling, bystander T cells provide sufficient CD40-mediated signal 2 to support an autoantibody response. However, although autoantibody responses driven by acute B cell-targeted deletion of SHP-1 also require T cells, bystander T cell help does not suffice. These results demonstrate that upregulation of PI3K signaling in autoreactive B cells, recapitulating the effect of multiple autoimmunity risk alleles, promotes autoantibody responses both by increasing B cells' cooperation with noncognate T cell help and by altering BCR signaling. Receptiveness to bystander T cell help enables autoreactive B cells to circumvent the fail-safe of T cell tolerance.

CD4 + T cells reverse surface antigen persistence in a mouse model of HBV replication

ABSTRACT Hepatitis B virus (HBV) is responsible for over 800,000 deaths yearly worldwide. While chronic HBV infection currently has no highly effective cure, immunotherapy has generated interest as a potential therapeutic solution due to the connection between viral chronicity and the magnitude of HBV-specific immune responses. Suitable animal models for studying successful immune responses to HBV are limited due to the lack of natural viral tropism for rodents. Using a mouse model of HBV replication, we found that CD4 + T cells are essential for the antibody-mediated clearance of hepatitis B surface antigen (HBsAg) and that early transient inhibition of the CD4 + T cell-B cell axis imparts HBsAg persistence through CD4 + T cell functional impairment. HBsAg resolution in BALB/c mice transduced with an adeno-associated virus encoding the HBV genome (AAV-HBV) depended on B cells, MHCII, and CD4 + T cells, but not CD8 + T cells, indicative of antibody-mediated control. Furthermore, disrupting the CD40-CD40L interaction blunted HBsAg clearance and the generation of HBsAg-specific B cells. Finally, HBsAg persistence induced by transient CD4 depletion was reversible by the transfer of total splenocytes or purified CD4 + T cells from immunized mice. These findings demonstrate the T cell-dependent nature of HBsAg-resolving antibody responses as well as the ability of CD4 + T cells to reverse HBsAg persistence. IMPORTANCE Hepatitis B virus (HBV) is a leading causative agent of viral hepatitis. A preventative vaccine has existed for decades, but only limited treatment options are available for people living with chronic HBV. Animal models for studying HBV are constrained due to narrow viral tropism, impeding understanding of the natural immune response to the virus. Here, using a vector to overcome the narrow host range and establish HBV replication in mice, we identified the role of helper T cells in controlling HBV. We show that helper T cells promote the B cell’s ability to generate antibodies that remove HBV and its associated surface antigen from the blood and that transfer of purified helper T cells from HBV-immunized mice can reverse the accumulation of virus and antigen, furthering our understanding of the immune response to HBV.

CD40-CD40 ligand interaction between periodontal ligament cells and cementoblasts enhances periodontal tissue remodeling in response to mechanical stress.

We analyzed the localization and expression of Cluster of differentiation 40 ligand (CD40L) in murine periodontal tissue applied with the orthodontic force to determine the CD40L-expressing cells under mechanical stress. Furthermore, we investigated whether CD40-CD40L interaction played an important role in transducing mechanical stress between periodontal ligament (PDL) cells and cementoblasts and remodeling the periodontal tissue for its homeostasis. PDL is a complex tissue that contains heterogeneous cell populations and is constantly exposed to mechanical stress, such as occlusal force. CD40 is expressed on PDL cells and upregulated under mechanical stress. However, whether its ligand, CD40L, is upregulated in periodontal tissue in response to mechanical stress, and which functions the CD40-CD40L interaction induces by converting the force to biological functions between the cement-PDL complex, are not fully understood. The orthodontic treatment was applied to the first molars at the left side of the upper maxillae of mice using a nickel-titanium closed-coil spring. Immunohistochemistry was performed to analyze the localization of CD40L in the periodontal tissue under the orthodontic force. Human cementoblasts (HCEM) and human PDL cells were stretched in vitro and analyzed CD40L and CD40 protein expression using flow cytometry. A GFP-expressing CD40L plasmid vector was transfected into HCEM (CD40L-HCEM). CD40L-HCEM was co-cultured with human PDL cells with higher alkaline phosphatase (ALP) activity (hPDS) or lower ALP (hPDF). After co-culturing, cell viability and proliferation were analyzed by propidium iodide (PI) staining and bromodeoxyuridine (BrdU) assay. Furthermore, the mRNA expression of cytodifferentiation- and extracellular matrix (ECM)-related genes was analyzed by real-time PCR. Immunohistochemistry demonstrated that CD40L was induced on the cells present at the cementum surface in periodontal tissue at the tension side under the orthodontic treatment in mice. The flow cytometry showed that the in vitro-stretching force upregulated CD40L protein expression on HCEM and CD40 protein expression on human PDL cells. Co-culturing CD40L-HCEM with hPDF enhanced cell viability and proliferation but did not alter the gene expression related to cytodifferentiation and ECM. In contrast, co-culturing CD40L-HCEM with hPDS upregulated cytodifferentiation- and ECM-related genes but did not affect cell viability and proliferation. We revealed that in response to a stretching force, CD40L expression was induced on cementoblasts. CD40L on cementoblasts may interact with CD40 on heterogeneous PDL cells at the necessary time and location, inducing cell viability, proliferation, and cytodifferentiation, maintaining periodontal tissue remodeling and homeostasis.

CD40 ligand stimulation affects the number and memory phenotypes of human peripheral CD8+ T cells

BackgroundCD40L is primarily expressed on activated CD4+ T cells and binds to CD40 which is expressed by various cells including dendritic cells, macrophages and B lymphocytes. While CD40-CD40L interaction is known to be direct between B cells and CD4+ T cells which results in proliferation and immunoglobulin isotype switching, antigen presenting cells (APCs) were thought to be involved in the delivery of CD4+ help to CD8+ T cells by cross-talk between CD4+ and CD8+ T cells and APCs. However, subsequent study demonstrated that CD40L signal can be directly delivered to CD8+ T cells by CD40 expression on CD8+ T cells. Since most studies have been carried out in murine models, we aimed to investigate the direct effect of CD40L on human peripheral CD8+ T cells.ResultsHuman peripheral CD8+ T cells were isolated to exclude the indirect effect of B cells or dendritic cells. Upon activation, CD40 expression on CD8+ T cells was transiently induced and stimulation with artificial APCs expressing CD40L (aAPC-CD40L) increased the number of total and central memory CD8+ T cells and also pp65 specific CD8+ T cells. Stimulation with aAPC-CD40L also resulted in higher proportion of central memory CD8+ T cells.ConclusionsOur study suggests that CD40L has an effect on the increased number of CD8+ T cells through CD40 expressed on activated CD8+ T cells and has influence on memory CD8+ T cell generation. Our results may provide a new perspective of the effect of CD40L on human peripheral CD8+ T cells, which differ according to the memory differentiation status of CD8+ T cells.

Involvement of CD40-CD40L and ICOS-ICOSL in the development of chronic rhinosinusitis by targeting eosinophils.

Chronic rhinosinusitis (CRS), whose prevalence and pathogenesis are age-related, is characterized by nasal tissue eosinophil infiltration. CD40-CD40 ligand (CD40L) pathway involves in the eosinophil-mediated inflammation, and inducible co-stimulator (ICOS)-ICOS ligand (ICOSL) signal can strengthen CD40-CD40L interaction. Whether CD40-CD40L and ICOS-ICOSL have a role in the development of CRS remains unknown. The aim of this study is to investigate the association of CD40-CD40L and ICOS-ICOSL expression with CRS and underlying mechanisms. Immunohistology detected the expression of CD40, CD40L, ICOS, and ICOSL. Immunofluorescence was performed to evaluate the co-localizations of CD40 or ICOSL with eosinophils. Correlations between CD40-CD40L and ICOS-ICOSL as well as clinical parameters were analyzed. Flow cytometry was used to explore the activation of eosinophils by CD69 expression and the CD40 and ICOSL expression on eosinophils. Compared with the non-eCRS subset, ECRS (eosinophilic CRS) subset showed significantly increased CD40, ICOS, and ICOSL expression. The CD40, CD40L, ICOS, and ICOSL expressions were all positively correlated with eosinophil infiltration in nasal tissues. CD40 and ICOSL were mainly expressed on eosinophils. ICOS expression was significantly correlated with the expression of CD40-CD40L, whereas ICOSL expression was correlated with CD40 expression. ICOS-ICOSL expression positively correlated with blood eosinophils count and disease severity. rhCD40L and rhICOS significantly enhanced the activation of eosinophils from patients with ECRS. Tumor necrosis factor-α (TNF-α) and interleukin-5 (IL-5) obviously upregulated CD40 expression on eosinophils, which was significantly inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor. Increased CD40-CD40L and ICOS-ICOSL expressions in nasal tissues are linked to eosinophils infiltration and disease severity of CRS. CD40-CD40L and ICOS-ICOSL signals enhance eosinophils activation of ECRS. TNF-α and IL-5 regulate eosinophils function by increasing CD40 expression partly via p38 MAPK activation in patients with CRS.

CD40LG genotype does not correlate with clinical phenotype in X-linked hyper-IgM syndrome

IntroductionCD40 ligand is expressed on follicular helper T cells and binds to CD40 on B cells and antigen presenting cells (APC). The CD40L-CD40 interaction is integral to germinal center formation, immunoglobulin class switching and affinity maturation, as well as APC mediated cytokine secretion required for an effective T cell response. Patients with mutations in the CD40LG gene develop X-linked Hyper-IgM (XHIGM) syndrome characterized by recurrent sinopulmonary, opportunistic infections, liver disease and malignancy. Early studies identified no clear genotype/phenotype correlation; but a milder phenotype has been reported associated with certain variants such as T254M. PurposeTo understand if specific genetic mutations in CD40LG correlate with disease severity we developed a clinical severity scoring system and retrospectively analyzed the relationship between severity and genotype in 109 individuals with XHIGM. ResultsThe majority of reported mutations were missense (41%), splice site (27%), nonsense (13%) or deletion (13%) mutations. There was not a statistically significant difference in severity or survival among mutation type groups. We identified 10 unrelated individuals with amino acid substitution T254M and found a range of clinical phenotypes from mild disease with late onset to early manifestations, liver disease and death due to cholangiocarcinoma. ConclusionOur data confirms the lack of genotype/phenotype correlation in XHIGM and suggest the need to develop a clinical scoring system that would allow for better understanding of an individual prognostic outcome.

Novel peptide inhibitors targeting CD40 and CD40L interaction: A potential for atherosclerosis therapy

Atherosclerosis is a chronic inflammatory disease characterized by plaque build-up in the arteries, leading to the obstruction of blood flow. Macrophages are the primary immune cells found in the atherosclerotic lesions and are directly involved in atherosclerosis progression. Macrophages are derived from extravasating blood monocytes. The monocytic CD40 receptor is important for monocyte recruitment on the endothelium expressing the CD40 ligand (CD40L). Thus, targeting monocyte/macrophage interaction with the endothelium by inhibiting CD40-CD40L interaction may be a promising strategy for attenuating atherosclerosis. Monoclonal antibodies have been used against this target but shows various complications. We used an array of computer-aided drug discovery tools and molecular docking approaches to design a therapeutic inhibitory peptide that could efficiently bind to the critical residues (82Y, 84D, and 86N) on the CD40 receptor essential for the receptor's binding to CD40L. The initial screen identified a parent peptide with a high binding affinity to CD40, but the peptide exhibited a positive hepatotoxicity score. We then designed several novel peptidomimetic derivatives with higher binding affinities to CD40, good physicochemical properties, and negative hepatotoxicity as compared to the parent peptide. Furthermore, we conducted molecular dynamics simulations for both the apo and complexed forms of the receptor with ligand, and screened peptides to evaluate their stability. The designed peptidomimetic derivatives are promising therapeutics targeting the CD40-CD40L interaction and may potentially be used to attenuate atherosclerosis.

Monitoring the Interaction Between Dendritic Cells and T Cells In Vivo with LIPSTIC.

Interactions between different cell types are key for immune function. Traditionally, interactions have been investigated in vivo by intravital two-photon microscopy, but the molecular characterization of the cells participating in a specific interaction is limited by the inability to retrieve the cells for downstream analysis. We recently developed an approach to label cells undergoing specific interactions in vivo, which we called LIPSTIC (Labeling Immune Partnership by Sortagging Intercellular Contacts). Here, we provide detailed instructions on how to track CD40-CD40L interactions between dendritic cells (DCs) and CD4+ T cells using genetically engineered LIPSTIC mice. This protocol requires expertise in animal experimentation and multicolor flow cytometry. Once mouse crossing has been achieved, it takes 3days or more to complete, depending on the kinetics of the interactions that the researcher wishes to investigate.

Immunity to Cryptosporidium: Lessons from Acquired and Primary Immunodeficiencies.

Cryptosporidium is a ubiquitous protozoan parasite that infects gut epithelial cells and causes self-limited diarrhea in immunocompetent individuals. However, in immunocompromised hosts with global defects in T cell function, this infection can result in chronic, life-threatening disease. In addition, there is a subset of individuals with primary immunodeficiencies associated with increased risk for life-threatening cryptosporidiosis. These patients highlight MHC class II expression, CD40-CD40L interactions, NF-κB signaling, and IL-21 as key host factors required for resistance to this enteric pathogen. Understanding which immune deficiencies do (or do not) lead to increased risk for severe Cryptosporidium may reveal mechanisms of parasite restriction and aid in the identification of novel strategies to manage this common pathogen in immunocompetent and deficient hosts.

Tumor resident memory CD8 T cell formation and concomitant tumor immunity is CD40L dependent and CD4 independent.

Abstract The implantation of cancer cell lines into immunocompetent mice acts as a vaccination event, resulting in T cell immunity that impacts both tumor progression and the response to subsequent therapy. We have previously shown that blocking immunity at tumor implantation using anti-CD40L antibodies blocks development of an intratumoral antigen-specific CD8 T cell population with tissue resident memory (Trm) phenotypes, and also limits responsiveness to radiation therapy and immunotherapy combinations. Using pancreatic tumors derived from Pdx1-Cre and Pdx1-Cre /R26LSL-LSIY mice crossed with KrasLSL-G12D/+Tp53LSL-R172H/+ mice, we explored the role of CD4 T cells in providing CD40-CD40L help to generate Trm and conventional T central memory CD8 T cells (Tcm) cells in the tumor and systemically. Using depleting antibodies, we identified that Trm cells in the tumor could form independently of CD4 T cells but were lost following CD40L blockade. By contrast, systemically circulating Tcm cells that could re-expand after antigen-specific challenge were dependent on both CD4 T cells and CD40-CD40L interactions. Notably, concomitant immunity generated by tumor implantation that can prevent tumor growth on the opposite flank could also occur independently of CD4 T cells, but was lost on CD40L blockade. Systemic vaccination with Listeria monocytogenes expressing SIY efficiently induced the formation of SIY-specific Tcm cells but did not impact tumor implantation. These data suggest that tumor implantation generates Trm cells independently of CD4 T cells, and that these cells, not Tcm cells, sustain concomitant immunity. These data may impact immune therapies designed to control metastatic seeding in patients. Supported by grants from NIH (R01CA182311, R01CA244142, R01CA208644).

The CD40/CD40 ligand system in linking acute neuroinflammation with chronic progressive demyelination

Abstract CD4+ T cells play critical roles in mediating adaptive immunity and developing protective host immunity by eliminating infectious viruses. CD40 Ligand expressed on CD4+ T cells known to interact with its cognate receptor CD40 expressed on monocyte/macrophage/microglia to induce several immune functions during inflammation and host immunity. Our study aimed to delineate a potential CD4+ T cell-microglia nexus at the cellular and molecular level using CD4−/− and CD40L−/− mice. Results showed that the absence of CD40L renders mice highly susceptible to murine-coronavirus-MHV-A59/RSA59 infection due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS at the acute-adaptive bridging phase of neuroinflammation. CD40L deficiency significantly impaired the priming, expansion, and activation of lymphoid cells in the deep cervical lymph nodes. Additionally, both CD4−/− and CD40L−/− mice presented with severe chronic phase demyelination and axonal pathology in the brain and spinal cord which was correlated with prolonged virus persistence. Results from this study provide exciting information about a critical role of CD40L in mediating RSA59 induced activation of CD4+ T cells in the CLN and their consequent infiltration into CNS in addition to affecting the numbers and activation of monocyte/macrophage/microglia during the acute-adaptive transition phase. CD40-CD40L interaction in linking the innate-acute and chronic-adaptive immune responses may suggest a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to autoimmune suppression, which holds only for autoimmune mechanisms of inflammatory demyelination. This work was supported by a Department of Biotechnology, India, research grant (BT/PR 20922/MED/122/37/2016). We thank the Council of Scientific and Industrial Research (CSIR) India for providing fellowships to F.S. the Ministry of Human Resource Development (MHRD), India for providing fellowship to D.C; and the University Grants Commission (UGC), India, for providing fellowship to M.K.

IRF5 regulation of CD4+ T cell metabolism controls CD40L expression

Abstract Systemic Lupus Erythematosus (SLE) is a devastating autoimmune disease that results from failure of the immune system to distinguish “self” from “non-self”. Studies in our lab and others demonstrated that human SLE CD4+ T cells have elevated levels of IRF5 and increased metabolism, while Irf5 knockout murine CD4+ T cells show diminished utilization of oxidative phosphorylation and glycolysis, respectively. However, how IRF5 contributes to CD4+ T cell support of B cell function and dysfunction has not been fully elucidated. Here, using IRF5 KO C57BL/6J mice, we show that loss of IRF5 in CD4+ T cells directly contributes to defects in plasmablast generation. Examination of the CD40-CD40L interaction shows significant decreases in CD40L expression in Irf5 KO CD4+ T cells. Examination of Cd40lg transcript expression shows no difference in mRNA expression in Irf5 KO murine CD4+ T cells when compared to WT. This finding indicated a novel post-transcriptional regulatory role for IRF5. Examination of the canonical mTOR pathway, a key translational regulator, shows decreased phosphorylation of the S6 ribosomal protein upon loss of Irf5, indicating diminished activity of the mTOR pathway. mTOR inhibition with rapamycin in turn results in decreased CD40L expression, supporting a role for mTOR in CD40L regulation. As mTOR signaling is known to be regulated by cellular metabolism, we examined if loss of IRF5 results in altered T cell metabolism. We found that Irf5 KO T cells have decreased glucose metabolism and increased glutamine metabolism. This data has increased our understanding of how metabolism influences CD4+ T cell function and provided insight into novel post-translational regulatory roles for IRF5 via T cell metabolic regulation.

CD40-CD40L in Neurological Disease.

Immune-inflammatory conditions in the central nervous system (CNS) rely on molecular and cellular interactions which are homeostatically maintained to protect neural tissue from harm. The CD40–CD40L interaction upregulates key proinflammatory molecules, a function best understood in the context of infection, during which B-cells are activated via CD40 signaling to produce antibodies. However, the role of CD40 in neurological disease of non-infectious etiology is unclear. We review the role of CD40–CD40L in traumatic brain injury, Alzheimer’s Disease, Parkinson’s Disease, stroke, epilepsy, nerve injury, multiple sclerosis, ALS, myasthenia gravis and brain tumors. We also highlight therapeutic advancements targeting the CD40 system to either attenuate the neuroinflammatory response or leverage the downstream effects of CD40 signaling for direct tumor cell lysis.

CD40 Cross-Linking Induces Migration of Renal Tumor Cell through Nuclear Factor of Activated T Cells (NFAT) Activation.

CD40 crosslinking plays an important role in regulating cell migration, adhesion and proliferation in renal cell carcinoma (RCC). CD40/CD40L interaction on RCC cells activates different intracellular pathways but the molecular mechanisms leading to cell scattering are not yet clearly defined. Aim of our study was to investigate the main intracellular pathways activated by CD40 ligation and their specific involvement in RCC cell migration. CD40 ligation increased the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH (2)-terminal kinase (JNK) and p38 MAPK. Furthermore, CD40 crosslinking activated different transcriptional factors on RCC cell lines: AP-1, NFkB and some members of the Nuclear Factor of Activated T cells (NFAT) family. Interestingly, the specific inhibition of NFAT factors by cyclosporine A, completely blocked RCC cell motility induced by CD40 ligation. In tumor tissue, we observed a higher expression of NFAT factors and in particular an increased activation and nuclear migration of NFATc4 on RCC tumor tissues belonging to patients that developed metastases when compared to those who did not. Moreover, CD40-CD40L interaction induced a cytoskeleton reorganization and increased the expression of integrin β1 on RCC cell lines, and this effect was reversed by cyclosporine A and NFAT inhibition. These data suggest that CD40 ligation induces the activation of different intracellular signaling pathways, in particular the NFATs factors, that could represent a potential therapeutic target in the setting of patients with metastatic RCC.

CD40L-CD40 interaction avidity as coronary artery disease predictive factor

Background: Coronary artery disease (CAD) can occur due to atherosclerosis in coronary arteries. Platelet agreement plays an important role in the pathophysiology of CAD. CD40L is a surface antigen on activated platelets. CD40L will bind to CD40 which is expressed by macrophages and endothelials then this activation results in reduced thickness and stability of atherosclerotic plaque and then thrombus appears. Thrombus ultimately inhibits blood flow to the coronary arteries. Proper measurement of these activities can describe the occurrence of CADs driven by platelets.
 Objectives: Measuring the avidity of CD40L-CD40 interactions so that the relationship between CD40L activity and CAD events can be seen.
 Materials and Methods: This research is non-experimental in nature and uses Case control designs. The subjects of the study were CAD patients at RSUD Dr. Soedirman Kebumen. Patients who met the inclusion and exclusion criteria were then examined and healthy patients were matched and CAD patients matched. The subject’s blood is then drawn and sent to Clinical Pathology Laboratory, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada. Blood samples were then examined using flow cytometry to determine CD40L levels. The results obtained were then analyzed by logistic regression test with Medcalc software.
 Results and discussion: Twenty-six subjects participated in this study, with 13 subjects were CAD patients and 13 healthy control subjects.The percentage of platelets expressing CD62P+ CD40L+ in the CAD group was higher than in the control group (p = 0.0015). Statistical analysis with T test showed that there were significant differences in CD40-expressing platelets between the CAD group and the control group (p = 0.0029).The study conclude that the avidity of CD40L-CD40 interactions as indicated by CD40 expression is related to CAD events. CD40 expression was higher in subjects with CAD compared to controls.
 Bangladesh Journal of Medical Science Vol.20(4) 2021 p.796-800

Evaluation of Pre-miRNA-146a rs2910164 Variations and sCD40 Ligand in Egyptian Patients with Rheumatoid Arthritis

Abstract Background: Patients with Rheumatoid Arthritis (RA) are at greater risk of developing Cardiovascular (CV) events with higher rate of subsequent mortality. MicroRNAs (miR-NAs) such as MiR-146a are thought to have an important role in the pathogenesis of RA. CD40-CD40L interaction is poten-tially involved in atherogenesis and plaque rupture in RA, it can be a risk factor in development of accelerated vascular complications in RA patients. Aim of Work: Our aim is to evaluate the level of sCD40L, investigate the genetic variations (polymorphisms) in pre-miRNA-146a rs2910164 in a cohort of Egyptian RA patients and detect their possible relation to CV risk in RA. Methods: 108 RA patients were enrolled in our study and were divided into 2 groups according to presence and absence of CV risk along with 49 controls. Genotyping of pre-microRNA-146a rs2910164 polymorphism was done by Taq-Man real-time PCR and serum sCD40L levels were measured using human soluble CD40L ELISA assay. Results: sCD40L concentration was significantly lower in RA patients without CV risk and RA patients with CV risk when compared to control group (p < 0.001), but there was no statistically significant difference when comparing both patients' groups (p=0.5). The genotype and allele frequencies of pre-microRNA-146a rs2910164 did not show a statistically significant difference between the studied groups. When comparing RA group without CV risk, RA group with CV risk and control group, GG genotype was 41.8%, 39.6% and 32.7%, GC was 40%, 49.1% and 51% and CC was 18.2%, 11.3% and 16.3%, respectively. G allele frequency in RA without CV risk group, RA with CV risk group and control group was 35.2%, 35.2% and 29.5% and C allele frequency was 34.7%, 31.4% and 33.9%, respectively. Conclusion: Pre-miRNA-146a rs2910164 gene variations were not associated with RA and does not increase the sus-ceptibility to cardiovascular disease. Low serum levels of sCD40L were detected in RA patients irrespective of the presence of cardiovascular risk. Evaluating sCD40L in patients not receiving any treatment and comparing them with post-treatment levels is recommended.

CD40-CD40L interaction is critical in mounting host immunity against m-CoV Mouse Hepatitis Virus induced neuroinflammatory demyelination

Abstract Chronic progressive neuroinflammatory disease multiple sclerosis (MS) is characterized by loss of neuronal functions resulting from demyelination with or without axonal degeneration. Infiltration of T lymphocytes and activation of microglia and their interplay are the major pathophysiological events leading to neurodegeneration in MS. Our studies in Mouse Hepatitis Virus (MHV) induced neuroinflammatory model demonstrated a protective role of CNS infiltrating CD4+ T cells. In the absence of CD4+ T cells, microglial activation fails to resolve, and mice are more susceptible to acute poliomyelitis and chronic demyelination with axonal bulbar vacuolation. Our studies also revealed that CD40L (expressed on CD4+ T cells) upregulates upon MHV infection but is downregulated in CD4−/− mice CNS. This led to a further delineation of the CD4-microglia nexus at the molecular level using CD40L−/− mice. Results showed that the absence of CD40L renders mice highly susceptible to MHV infection due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS at the acute-adaptive bridging phase (day 7–10 p.i.) of inflammation. Moreover, CD40L−/− mice exhibited severe demyelination, axonal loss, and persistent poliomyelitis at the chronic phase of infection, highlighting the protective role of CD40-CD40L in MHV induced neuroinflammatory demyelination. Together, these studies highlight that migration of peripheral T cells and their interaction with microglia via CD40-CD40L is essential to eliminate the virus and provide long-term neuroprotection. These findings can lead to designing potential therapeutic interventions against MS, targeting CD40-CD40L interaction.

Effect of age and sex on immune checkpoint expression and kinetics in human T cells.

BackgroundImmune checkpoints are crucial molecules in maintaining a proper immune balance. Even though age and sex are known to have effects on the immune system, the interplay between age, sex and immune checkpoint expression by T cells is not known. The aim of this study was to determine whether age and sex affect immune checkpoint expression by T cells and if age and sex affect the kinetics of immune checkpoint expression following ex vivo stimulation. In this study, whole blood samples of 20 healthy young adults (YA, 9 males and 11 females) and 20 healthy older adults (OA, 9 males and 11 females) were stained for lymphocyte lineage markers and immune checkpoints and frequencies of CD28+, PD-1+, VISTA+ and CD40L+ T cells were determined. Immune checkpoint expression kinetics were studied following ex vivo anti-CD3/anti-CD28 stimulation of T cells from young and older healthy adults.ResultsWe report an age-associated increase of CD40L + CD4+ and CD40L + CD8+ T-cell frequencies, whereas CD40+ B-cell frequencies were decreased in older adults, suggesting modulation of the CD40L-CD40 interaction with age. Immune checkpoint expression kinetics revealed differences in magnitude between CD4+ and CD8+ T cells independent of age and sex. Further analysis of CD4+ T-cell subsets revealed an age-associated decrease of especially PD-1 + CD4+ memory T cells which tracked with the female sex.ConclusionCollectively, our results demonstrate that both age and sex modulate expression of immune checkpoints by human T cells. These findings may have implications for optimising vaccination and immune checkpoint immunotherapy and move the field towards precision medicine in the management of older patient groups.