Genetically engineered IgG1 and nanobody oligomers acquire strong intrinsic CD40 agonism.

Most anti-CD40 antibodies show robust agonism only upon binding to FcγR+ cells, such as B cells, macrophages, or DCs, but a few anti-CD40 antibodies display also strong intrinsic agonism dependent on the recognized epitope and/or isotype. It is worth mentioning, however, that also the anti-CD40 antibodies with intrinsic agonism can show a further increase in agonistic activity when bound by FcγR-expressing cells. Thus, conventional antibodies appear not to be sufficient to trigger the maximum possible CD40 activation independent from FcγR-binding. We proved here the hypothesis that oligomeric and oligovalent anti-CD40 antibody variants generated by genetic engineering display high intrinsic, thus FcγR-independent, agonistic activity. We generated tetra-, hexa- and dodecavalent variants of six anti-CD40 antibodies and a CD40-specific nanobody. All these oligovalent variants, even when derived of bivalent antagonistic anti-CD40 antibodies, showed strongly enhanced CD40 agonism compared to their conventional counterparts. In most cases, the CD40 agonism reached the maximum response induced by FcγR-bound anti-CD40 antibodies or membrane CD40L, the natural engager of CD40. In sum, our data show that increasing the valency of anti-CD40 antibody constructs by genetic engineering regularly results in molecules with high intrinsic agonism and level out the specific limitations of the parental antibodies.

A genetic variant in the CD40 gene is related to HBV infection in the Chinese Han population.

BACKGROUND: CD40 is an important immune costimulatory molecule that has recently been found to be associated with chronic hepatitis B. This study aims to explore the association between CD40 polymorphisms and HBV infection, as well as to investigate the impact of different rs1883832 genotypes on CD40 expression and its effect on the progression of chronic HBV infection. METHODS: We genotyped rs1883832 in 3433 individuals using MassARRAY, and quantified the CD40 expression, including CD40 mRNA, sCD40, and mCD40. The CD40 and HBV infection indicators were assessed to investigate the potential function of rs1883832 in suppressing HBV replication in HepG2.2.15 and HepAD38, CD40L in cytotoxic t lymphocytes (CTLs) and interferon-γ, TNF-α, granzyme B, and perforin were measured to elucidate the mechanism by which CD40 inhibits HBV replication. RESULTS: Our study revealed that the frequencies of CC genotype and C allele of rs1883832 were significantly higher in immune recovery compared to chronic hepatitis B. Individuals with CC genotype exhibited significantly elevated CD40 in serum and B cells compared to TT genotypes in chronic hepatitis B. Additionally, CD40 is capable of inhibiting HBV replication and transcription in hepatocytes by means of interaction with CD40L. A significant negative correlation was found between HBV DNA, HBeAg, and mCD40. Conversely, the expressions of ALT and mCD40 showed a positive correlation, which aligns with the trend of CD40L. CONCLUSIONS: rs1883832 C allele may have a protective role in HBV immune recovery. This protective effect could potentially be attributed to the regulation of CD40 expression. The activation of the anti-HBV immune response, which occurs through binding CD40L on CTL, can suppress HBV DNA replication and potentially facilitate immune recovery in HBV infection.

Clinical and Immunological Features, Genetic Variants, and Outcomes of Patients with CD40 Deficiency.

PURPOSE: Inherited deficiencies of CD40 and CD40 ligand (CD40L) reflect the crucial immunological functions of CD40-CD40L interaction/signaling. Although numerous studies have provided a detailed description of CD40L deficiency, reports of CD40 deficiency are scarce. Herein, we describe the characteristics of all reported patients with CD40 deficiency. METHODS: The PubMed, Embase and Web of Science databases were searched for relevant literature published till 7th August 2023. Study deduplication and identification of relevant reports was performed using the online PICO Portal. The data were extracted using a pre-designed data extraction form and the SPSS software was used for analysis. RESULTS: Systematic literature review revealed 40 unique patients with CD40 deficiency. Respiratory tract and gastrointestinal infections were the predominant clinical manifestations (observed in 93% and 57% patients, respectively). Sclerosing cholangitis has been reported in nearly one-third of patients. Cryptosporidium sp. (29%) and Pneumocystis jirovecii (21%) were the most common microbes identified. Very low to undetectable IgG levels and severely reduced/absent switch memory B cells were observed in all patients tested/reported. Elevated IgM levels were observed in 69% patients. Overall, splice-site and missense variants were the most common (36% and 32%, respectively) molecular defects identified. All patients were managed with immunoglobulin replacement therapy and antimicrobial prophylaxis was utilized in a subset. Hematopoietic stem cell transplantation (HSCT) has been performed in 45% patients (curative outcome observed in 73% of these patients). Overall, a fatal outcome was reported in 21% patients. CONCLUSIONS: We provide a comprehensive description of all important aspects of CD40 deficiency. HSCT is a promising curative treatment option for CD40 deficiency.

Potential role of soluble CD40 receptor in chronic inflammatory diseases.

The CD40 receptor and its ligand CD154 are widely expressed in various immune-competent cells. Interaction of CD154 with CD40 is essential for B-cell growth, differentiation, and immunoglobulin class switching. Many other immune-competent cells involved in innate and adaptive immunity communicate through this co-stimulatory ligand-receptor dyad. CD40-CD154 interaction is involved in the pathogenesis of numerous inflammatory and autoimmune diseases. While CD40 and CD154 are membrane-bound proteins, their soluble counterparts are generated by proteolytic cleavage or alternative splicing. This review summarises current knowledge about the impact of single nucleotide polymorphisms in the human CD40 gene and compensatory changes in the plasma level of the soluble CD40 receptor (sCD40) isoform in related pro-inflammatory diseases. It discusses regulation patterns of the disintegrin metalloprotease ADAM17 function leading to ectodomain shedding of transmembrane proteins, such as pro-inflammatory adhesion molecules or CD40. The role of sCD40 as a potential biomarker for chronic inflammatory diseases will also be discussed.

Impact of CD40 gene polymorphisms on the risk of cervical squamous cell carcinoma: a case-control study.

BACKGROUND: Cervical cancer is the fourth most common cancer among women worldwide. Genome-wide association studies have revealed multiple susceptible genes and their polymorphisms for cervical cancer risk. Therefore, we aimed to investigate the correlation between single nucleotide polymorphisms (SNPs) of the CD40 gene and susceptibility to cervical squamous cell carcinoma (CSCC) in a population from the northeastern Han Chinese population. METHODS: The three SNPs (rs1800686, rs3765459, and rs4810485) of the CD40 gene were analyzed by multiplex polymerase chain reaction (PCR) combined with next-generation sequencing methods in 421 patients with CSCC, 594 patients with high-grade squamous intraepithelial lesions (HSIL), and 504 healthy females. Multivariate logistic regression analysis was used to analyze the potential relationship between CD40 gene polymorphisms and CSCC, or HSIL. RESULTS: Our research results showed the AA genotype of rs1800686 had a protective effect on CSCC in comparison to the GG genotype and AG+GG genotypes (AA vs. GG: p = 0.0389 and AA vs. AG+GG: p = 0.0280, respectively). After FDR correction, the results were still statistically significant (p = 0.0389 and p = 0.0389, respectively). Similarly, rs3765459 showed a reduced risk association for CSCC in the codominant and recessive models (AA vs. GG: p = 0.0286 and AA vs. AG+GG: p = 0.0222, respectively). Significant differences remained after FDR correction (p = 0.0286 and p = 0.0286, respectively). However, these differences were no longer significant after the Bonferroni correction. In addition, the genotypes for the rs4810485 polymorphisms were associated with parity of the patients with CSCC. The genotypes for the rs3765459 polymorphisms were significantly correlated with the D-dimer of the patients with CSCC. The 3 SNPs genotypes of the CD40 gene were closely related to the squamous cell carcinoma antigen (SCC) of the patients with HSIL. CONCLUSIONS: The CD40 gene may play a role in the occurrence and development of CSCC.

Inhibition of PI3K p110δ activity reduces IgE production in IL-4 and anti-CD40 stimulated human B cell cultures.

Phosphoinositide 3-kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT-PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease.

CD40 Upregulation in the Retina of Patients With Diabetic Retinopathy: Association With TRAF2/TRAF6 Upregulation and Inflammatory Molecule Expression.

Purpose: CD40 is upregulated in the retinas of diabetic mice, drives pro-inflammatory molecule expression, and promotes diabetic retinopathy. The role of CD40 in diabetic retinopathy in humans is unknown. Upregulation of CD40 and its downstream signaling molecules TNF receptor associated factors (TRAFs) is a key feature of CD40-driven inflammatory disorders. We examined the expression of CD40, TRAF2, and TRAF6 as well as pro-inflammatory molecules in retinas from patients with diabetic retinopathy. Methods: Posterior poles from patients with diabetic retinopathy and non-diabetic controls were stained with antibodies against von Willebrand factor (labels endothelial cells), cellular retinaldehyde-binding protein (CRALBP), or vimentin (both label Müller cells) plus antibodies against CD40, TRAF2, TRAF6, ICAM-1, CCL2, TNF-α, and/or phospho-Tyr783 phospholipase Cγ1 (PLCγ1). Sections were analyzed by confocal microscopy. Results: CD40 expression was increased in endothelial and Müller cells from patients with diabetic retinopathy. CD40 was co-expressed with ICAM-1 in endothelial cells and with CCL2 in Müller cells. TNF-α was detected in retinal cells from these patients, but these cells lacked endothelial/Müller cell markers. CD40 in Müller cells from patients with diabetic retinopathy co-expressed activated phospholipase Cγ1, a molecule that induces TNF-α expression in myeloid cells in mice. CD40 upregulation in endothelial cells and Müller cells from patients with diabetic retinopathy was accompanied by TRAF2 and TRAF6 upregulation. Conclusions: CD40, TRAF2, and TRAF6 are upregulated in patients with diabetic retinopathy. CD40 associates with expression of pro-inflammatory molecules. These findings suggest that CD40-TRAF signaling may promote pro-inflammatory responses in the retinas of patients with diabetic retinopathy.

Upregulation of Inflammatory Mediators in Peripheral Blood CD40+ Cells in Children with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a common and severe neurodevelopmental disorder in early childhood, defined as social and communication deficits and repetitive and stereotypic behaviours. The aetiology is unknown in most cases. However, several studies have identified immune dysregulation as potentially promoting ASD. Among the numerous immunological findings in ASD, reports of increased pro-inflammatory markers remain the most consistently observed. C-C chemokine receptor type 1 (CCR1) activation is pro-inflammatory in several neurological disorders. Previous evidence has implied that the expression of chemokine receptors, inflammatory mediators, and transcription factors play a pivotal role in several neuroinflammatory disorders. There have also been reports on the association between increased levels of proinflammatory cytokines and ASD. In this study, we aimed to investigate the possible involvement of CCR1, inflammatory mediators, and transcription factor expression in CD40+ cells in ASD compared to typically developing controls (TDC). Flow cytometry analysis was used to determine the levels of CCR1-, IFN-γ-, T-box transcription factor (T-bet-), IL-17A-, retinoid-related orphan receptor gamma t (RORγt-), IL-22- and TNF-α-expressing CD40 cells in PBMCs in children with ASD and the TDC group. We further examined the mRNA and protein expression levels of CCR1 using real-time PCR and western blot analysis. Our results revealed that children with ASD had significantly increased numbers of CD40+CCR1+, CD40+IFN-γ+, CD40+T-bet+, CD40+IL-17A+, CD40+RORγt+, CD4+IL-22+, and CD40+TNF-α+ cells compared with the TDC group. Furthermore, children with ASD had higher CCR1 mRNA and protein expression levels than those in the TDC group. These results indicate that CCR1, inflammatory mediators, and transcription factors expressed in CD40 cells play vital roles in disease progression.

Tumor Cell Nanovaccines Based on Genetically Engineered Antibody-Anchored Membrane.

Despite the promise in whole-tumor cell vaccines, a key challenge is to overcome the lack of costimulatory signals. Here, agonistic-antibody-boosted tumor cell nanovaccines are reported by genetically engineered antibody-anchored membrane (AAM) technology, capable of effectively activating costimulatory pathways. Specifically, the AAM can be stably constructed following genetic engineering of tumor cell membranes with anti-CD40 single chain variable fragment (scFv), an agonistic antibody to induce costimulatory signals. The nanovaccines are versatilely designed and obtained based on the anti-CD40 scFv-anchored membrane and nanotechnology. Following vaccination, the anti-CD40 scFv-anchored membrane nanovaccine (Nano-AAM/CD40) significantly facilitates dendritic cell maturation in CD40-humanized transgenic mice and subsequent adaptive immune responses. Compared to membrane-based nanovaccines alone, the enhanced antitumor efficacy in both "hot" and "cold" tumor models of the Nano-AAM/CD40 demonstrates the importance of agonistic antibodies in development of tumor-cell-based vaccines. To expand the design of nanovaccines, further incorporation of cell lysates into the Nano-AAM/CD40 to conceptually construct tumor cell-like nanovaccines results in boosted immune responses and improved antitumor efficacy against malignant tumors inoculated into CD40-humanized transgenic mice. Overall, this genetically engineered AAM technology provides a versatile design of nanovaccines by incorporation of tumor-cell-based components and agonistic antibodies of costimulatory immune checkpoints.

Myeloid CD40 deficiency reduces atherosclerosis by impairing macrophages' transition into a pro-inflammatory state.

AIMS: CD40 and its ligand, CD40L, play a critical role in driving atherosclerotic plaque development. Disrupted CD40-signalling reduces experimental atherosclerosis and induces a favourable stable plaque phenotype. We recently showed that small molecule-based inhibition of CD40-tumour necrosis factor receptor associated factor-6 interactions attenuates atherosclerosis in hyperlipidaemic mice via macrophage-driven mechanisms. The present study aims to detail the function of myeloid CD40 in atherosclerosis using myeloid-specific CD40-deficient mice. METHOD AND RESULTS: Cd40flox/flox and LysM-cre Cd40flox/flox mice on an Apoe-/- background were generated (CD40wt and CD40mac-/-, respectively). Atherosclerotic lesion size, as well as plaque macrophage content, was reduced in CD40mac-/- compared to CD40wt mice, and their plaques displayed a reduction in necrotic core size. Transcriptomics analysis of the CD40mac-/- atherosclerotic aorta revealed downregulated pathways of immune pathways and inflammatory responses. Loss of CD40 in macrophages changed the representation of aortic macrophage subsets. Mass cytometry analysis revealed a higher content of a subset of alternative or resident-like CD206+CD209b- macrophages in the atherosclerotic aorta of CD40mac-/- compared to CD40wt mice. RNA-sequencing of bone marrow-derived macrophages of CD40mac-/- mice demonstrated upregulation of genes associated with alternatively activated macrophages (including Folr2, Thbs1, Sdc1, and Tns1). CONCLUSIONS: We here show that absence of CD40 signalling in myeloid cells reduces atherosclerosis and limits systemic inflammation by preventing a shift in macrophage polarization towards pro-inflammatory states. Our study confirms the merit of macrophage-targeted inhibition of CD40 as a valuable therapeutic strategy to combat atherosclerosis.

Adipocytes control hematopoiesis and inflammation through CD40 signaling.

The co-stimulatory CD40-CD40L dyad plays an important role in chronic inflammatory diseases associated with aging. Although CD40 is mainly expressed by immune cells, CD40 is also present on adipocytes. We aimed to delineate the role of adipocyte CD40 in the aging hematopoietic system and evaluated the effects of adipocyte CD40 deficiency on cardiometabolic diseases. Adult adipocyte CD40-deficient mice (AdiCD40KO) mice had a decrease in bone marrow hematopoietic stem cells (Lin-Sca+cKit+, LSK) and common lymphoid progenitors, which was associated with increased bone marrow adiposity and T-cell activation, along with elevated plasma corticosterone levels, a phenotype that became more pronounced with age. Atherosclerotic AdiCD40koApoE-/- (CD40AKO) mice also displayed changes in the LSK population, showing increased myeloid and lymphoid multipotent progenitors, and augmented corticosterone levels. Increased T-cell activation could be observed in bone marrow, spleen, and adipose tissue, while the numbers of B cells were decreased. Although atherosclerosis was reduced in CD40AKO mice, plaques contained more activated T cells and larger necrotic cores. Analysis of peripheral adipose tissue in a diet-induced model of obesity revealed that obese AdiCD40KO mice had increased T-cell activation in adipose tissue and lymphoid organs, but decreased weight gain and improved insulin sensitivity, along with increased fat oxidation. In conclusion, adipocyte CD40 plays an important role in maintaining immune cell homeostasis in bone marrow during aging and chronic inflammatory diseases, particularly of the lymphoid populations. Although adipocyte CD40 deficiency reduces atherosclerosis burden and ameliorates diet-induced obesity, the accompanying T-cell activation may eventually aggravate cardiometabolic diseases.

Boosting of tau protein aggregation by CD40 and CD48 gene expression in Alzheimer's disease.

Neurodegenerative diseases result from the interplay of abnormal gene expression and various pathological factors. Therefore, a disease-specific integrative genetic approach is required to understand the complexities and causes of target diseases. Recent studies have identified the correlation between genes encoding several transmembrane proteins, such as the cluster of differentiation (CD) and Alzheimer's disease (AD) pathogenesis. In this study, CD48 and CD40 gene expression in AD, a neurodegenerative disease, was analyzed to infer this link. Total RNA sequencing was performed using an Alzheimer's disease mouse model brain and blood, and gene expression was determined using a genome-wide association study (GWAS). We observed a marked elevation of CD48 and CD40 genes in Alzheimer's disease. Indeed, the upregulation of both CD48 and CD40 genes was significantly increased in the severe Alzheimer's disease group. With the elevation of CD48 and CD40 genes in Alzheimer's disease, associations of protein levels were also markedly increased in tissues. In addition, overexpression of CD48 and CD40 genes triggered tau aggregation, and co-expression of these genes accelerated aggregation. The nuclear factor kappa B (NF-ĸB) signaling pathway was enriched by CD48 and CD40 gene expression: it was also associated with tau pathology. Our data suggested that the CD48 and CD40 genes are novel AD-related genes, and this approach may be useful as a diagnostic or therapeutic target for the disease.

The rs1883832 Polymorphism (CD40-1C>T) Affects the Intensity of IgA Responses after BNT162b2 Vaccination

The effectiveness of coronavirus disease 2019 (COVID-19) vaccination strategies is affected by several factors, including the genetic background of the host. In our study, we evaluated the contribution of the functional polymorphism rs1883832 affecting the Kozak sequence of the TNFSF5 gene (c.-1C>T), encoding CD40, to humoral immune responses after vaccination with the spike protein of SARS-CoV-2. The rs1883832 polymorphism was analyzed by PCR-RFLP in 476 individuals (male/female: 216/260, median age: 55.0 years, range: 20−105) of whom 342 received the BNT162b2 mRNA vaccine and 134 received the adenovirus-based vector vaccines (67 on ChAdOx1-nCoV-19 vaccine, 67 on Ad.26.COV2.S vaccine). The IgG and IgA responses were evaluated with chemiluminescent microparticle and ELISA assays on days 21, 42, and 90 after the first dose. The T allele of the rs1883832 polymorphism (allele frequency: 32.8%) was significantly associated with lower IgA levels and represented, as revealed by multivariable analysis, an independent risk factor for reduced anti-spike protein IgA levels on days 42 and 90 following BNT162b2 mRNA vaccination. Similar to serum anti-spike IgA levels, a trend of lower anti-spike IgA concentrations in saliva was found in individuals with the T allele of rs1883832. Finally, the intensity of IgA and IgG responses on day 42 significantly affected the prevalence of COVID-19 after vaccination. The rs1883832 polymorphism may be used as a molecular predictor of the intensity of anti-spike IgA responses after BNT162b2 mRNA vaccination.

Mechanisms of CD40-dependent cDC1 licensing beyond costimulation.

CD40 signaling in classical type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1s, including Cd70, Tnfsf9, Ptgs2 and Bcl2l1, and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T cell expansion. Loss of 4-1BB, alone or in Cd27-/- mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased caspase activation in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation by Cd40-/- cDC1s to CD8+ T cells, which were reversed by re-expression of Bcl2l1. Thus, CD40 signaling in cDC1s not only induces costimulatory ligands for CD8+ T cells but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.

RelB contributes to the survival, migration and lymphomagenesis of B cells with constitutively active CD40 signaling.

Activation of CD40-signaling contributes to the initiation, progression and drug resistance of B cell lymphomas. We contributed to this knowledge by showing that constitutive CD40-signaling in B cells induces B cell hyperplasia and finally B cell lymphoma development in transgenic mice. CD40 activates, among others, the non-canonical NF-ĸB signaling, which is constitutively activated in several human B cell lymphomas and is therefore presumed to contribute to lymphopathogenesis. This prompted us to study the regulatory role of the non-canonical NF-ĸB transcription factor RelB in lymphomagenesis. To this end, we crossed mice expressing a constitutively active CD40 receptor in B cells with conditional RelB-KO mice. Ablation of RelB attenuated pre-malignant B cell expansion, and resulted in an impaired survival and activation of long-term CD40-stimulated B cells. Furthermore, we found that hyperactivation of non-canonical NF-кB signaling enhances the retention of B cells in the follicles of secondary lymphoid organs. RNA-Seq-analysis revealed that several genes involved in B-cell migration, survival, proliferation and cytokine signaling govern the transcriptional differences modulated by the ablation of RelB in long-term CD40-stimulated B cells. Inactivation of RelB did not abrogate lymphoma development. However, lymphomas occurred with a lower incidence and had a longer latency period. In summary, our data suggest that RelB, although it is not strictly required for malignant transformation, accelerates the lymphomagenesis of long-term CD40-stimulated B cells by regulating genes involved in migration, survival and cytokine signaling.

LTBP4, SPP1, and CD40 Variants: Genetic Modifiers of Duchenne Muscular Dystrophy Analyzed in Serbian Patients.

BACKGROUND: Clinical course variability in Duchenne muscular dystrophy (DMD) is partially explained by the mutation location in the DMD gene and variants in modifier genes. We assessed the effect of the SPP1, CD40, and LTBP4 genes and DMD mutation location on loss of ambulation (LoA). METHODS: SNPs in SPP1-rs28357094, LTBP4-rs2303729, rs1131620, rs1051303, rs10880, and CD40-rs1883832 were genotyped, and their effect was assessed by survival and hierarchical cluster analysis. RESULTS: Patients on glucocorticoid corticosteroid (GC) therapy experienced LoA one year later (p = 0.04). The modifying effect of SPP1 and CD40 variants, as well as LTBP4 haplotypes, was not observed using a log-rank test and multivariant Cox regression analysis. Cluster analysis revealed two subgroups with statistical trends in differences in age at LoA. Almost all patients in the cluster with later LoA had the protective IAAM LTBP4 haplotype and statistically significantly fewer CD40 genotypes with harmful T allele and "distal" DMD mutations. CONCLUSIONS: The modifying effect of SPP1, CD40, and LTBP4 was not replicated in Serbian patients, although our cohort was comparable in terms of its DMD mutation type distribution, SNP allele frequencies, and GC-positive effect with other European cohorts. Cluster analysis may be able to identify patient subgroups carrying a combination of the genetic variants that modify LoA.

Extensive involvement of CD40 and CD154 costimulators in multiple T cell-mediated responses in a perciform fish Larimichthys crocea.

CD40 and CD154 are well-characterized costimulatory molecules involved in adaptive humoral immunity in humans and other mammals. These two costimulatory molecules were found to be originated from teleost fish during vertebrate evolution. However, the functionality of fish CD40 and CD154 remains to be explored. In this study, we identified the CD40 and CD154 homologs (LcCD40 and LcCD154) from large yellow croaker (Larimichthys crocea), a marine species of the perciform fish family. The LcCD40 and LcCD154 share conserved structural features to their mammalian counterparts, and are widely expressed in immune-relevant tissues and leukocytes at different transcriptional levels. Immunofluorescence staining and FCM analysis showed that LcCD40 and LcCD154 proteins are distributed on MHC-II+ APCs and CD4-2+ T cells, and are significantly upregulated in response to antigen stimulation. Co-IP assay exhibited strong association between LcCD40 and LcCD154 proteins. Blockade of LcCD154 with anti-LcCD154 antibody (Ab) or recombinant soluble LcCD40-Ig fusion protein remarkably decreased the MHC-II+ APC-initiated CD4+ T cell response upon Aeromonas hydrophila stimulation, and alloreactive T cell activation as examined by mixed lymphocyte reaction (MLR). These findings highlight the costimulatory role of LcCD40 and LcCD154 in T cell activities in Larimichthys crocea. Thus, the CD40 and CD154 costimulators may extensively participate in the regulation of multiple T cell-mediated immune responses in teleost fish. It is anticipated that this study would provide a cross-species understanding of the evolutionary history of CD40 and CD154 costimulatory signals from fish to mammals.

A CD40 variant is associated with systemic bone loss among patients with rheumatoid arthritis.

OBJECTIVES: Little is known about genes predisposing to systemic bone loss (SBL) in rheumatoid arthritis (RA). Therefore, we examined the association between SBL and variants of genes playing a critical role in both immune response and bone homeostasis among patients with RA. METHODS: IRAK-1 rs3027898, IRAK-2 rs3844283, IRAK-2 rs708035, IFIH1 rs1990760, CD40 rs48104850, TNFAIP3 rs2230926, and miR146-a rs2910164 were genotyped in 176 adult RA patients. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DXA). RESULTS: Low BMD was observed in 116 (65.9%) patients. Among them, 60 (34.1%) had low femoral neck (FN) Z score, 72 (40.9%) had low total femur (TF) Z score, and 105 (59.6%) had low lumbar spine (LS) Z score. Among all the SNPs assessed, only CD40 rs4810485 was found to be associated with reduced TF Z score with the CD40 rs4810485 T allele protecting against reduced TF Z score (OR = 0.40, 95% CI = 0.23-0.68, p = 0.0005). This association was confirmed in the multivariate logistic regression analysis (OR = 0.31, 95% CI = 0.16-0.59, p = 3.84 × 10-4). Moreover, median FN BMD was reduced among RA patients with CD40 rs4810485 GG genotype compared to RA patients harbouring CD40 rs4810485 TT and GT genotypes (0.788 ± 0.136 versus 0.826 ± 0.146 g/cm2, p = 0.001). IRAK-1 rs3027898, IRAK-2 rs3844283, rs708035, IFIH rs1990760, TNFAIP3 rs2230926, and miR146-a rs2910164 were not found to be associated with SBL. CONCLUSION: This study for the first time ever demonstrated an association between a CD40 genetic variant and SBL among patients with RA. KEY POINTS: • CD40 rs4810485 GG genotype is associated with decreased BMD among patients with RA. • CD40 rs4810485 might serve as a genetic marker for SBL in RA. • CD40 genetic variations might be integrated in future development of more effective therapeutic interventions for prevention of SBL in RA.

Genetic polymorphisms of FCGR2A, ORAI1 and CD40 are associated with risk of lung cancer.

FCGR2A, ORAI1 and CD40 are all involved in the immune and inflammatory responses in the human body, whereas its association with lung cancer is still unclear. This study aimed to investigate the effects of polymorphisms in these genes on the susceptibility to lung cancer. Six candidate single nucleotide polymorphisms (SNPs) were genotyped using a MassARRAY platform in a discovery cohort, including 400 lung cancer patients and 400 healthy controls, and validated in a replication cohort, including 529 lung cancer cases and 532 controls. Comparing the allele frequency distributions, we found that the rs1801274-G, rs511278-T and rs1883832-T were risk alleles for lung cancer (P < 0.05), whereas the minor allele of rs12320939-T was a protective allele for the disease (P = 0.037). Comparing the genotype frequency distributions, we found that rs1801274-GG, rs511278-CT and of rs1883832-TT were risk genotype for lung cancer (P < 0.05). Genetic model analysis showed that the rs1801274 A>G was correlated with an elevated risk of lung cancer in recessive and log-additive models (P < 0.05); rs511278 C>T exhibited an increased risk of disease in dominant and log-additive models (P < 0.05); rs1883832 C>T had a strong relationship with risk of disease in all three models (P < 0.001), whereas rs12320939 G>T was correlated to a reduced risk of disease in recessive and log-additive models (P < 0.05). Finally, the association between the above SNPs and lung cancer risk was validated in a replication cohort (P < 0.05). These results shed new light on the association between immune-related genes and risk of lung cancer, and might be useful for the identification of high-risk individuals.