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Although personalized neoantigen cancer vaccines have emerged as a promising strategy for cancer treatment, challenges remain to develop immune-stimulatory carriers which allow simultaneous transport of adjuvants and vaccines to lymph nodes (LNs). With inherent immunogenicity, genetic plasticity, and efficiency for large-scale production, M13 phages represent an attractive platform for vaccine delivery as natural bionanomaterials. Here, we report the discovery of an anti-CD40 designed ankyrin repeat protein (DARPin) and propose a bifunctional M13 ph age for neoantigen delivery based on this anti-CD40 DARPin protein (M13 CD40 ). M13 CD40 -based neoantigen vaccines show improved accumulation and prolonged antigen retention in LNs compared with nontargeting phage vaccines due to the abundance of CD40-positive cells in LNs. Besides the intrinsic immunogenicity of phages, M13 CD40 -based neoantigen vaccines also benefit from additional CD40 stimulation due to multiple copies of anti-CD40 DARPins displayed on M13 CD40 phages. Subcutaneous immunization with M13 CD40 -based neoantigen vaccines results in more robust antigen-specific immune responses and superior antitumor efficacy in poorly immunogenic tumor models compared with nontargeting phage vaccines. Combination therapy with PD-1 blockade further enhances T cell cytotoxicity and improves tumor control. To summarize, our findings highlight M13 CD40 as a CD40 nanoagonist as well as an efficient vehicle for LN-targeted delivery of personalized neoantigen vaccines.

In Brief: The CD40 ligand-CD40 signaling system expressed at the maternal-conceptus interface in pigs is involved in regulating maternal immunity by modifying endometrial endothelial cell function during early pregnancy. This paper reveals the role of CD40 ligand-CD40 signaling at the maternal-conceptus interface in pigs., Abstract: To successfully establish and maintain pregnancy in pigs, a variety of factors must work together at the maternal-conceptus interface to form an immune environment appropriate for both the mother and the conceptus. Our transcriptomics study has shown that cluster of differentiation ligand 40 (CD40L) and its receptor CD40, which are known to play important roles in regulating cell- and antibody-mediated immunity, are expressed in the endometrium during early pregnancy. However, the roles of the CD40L and CD40 signaling system are not well understood. Therefore, we determined the expression, regulation and function of CD40L and CD40 at the maternal-conceptus interface in pigs. The endometrium expressed CD40L and CD40 mRNAs at the greatest levels on day 15 of pregnancy. The CD40L protein was localized predominantly to luminal epithelial cells on day 15 of pregnancy, and the CD40 protein was found in luminal epithelial, stromal and vascular endothelial cells in the endometrium during pregnancy. During early pregnancy, the conceptus expressed CD40 but not CD40L and chorioallantoic tissues during mid- to late pregnancy expressed both CD40L and CD40. Interferon-γ increased the expression of CD40L and CD40 in endometrial explants. CD40L increased the migration but not the proliferation of cultured porcine uterine endothelial (pENDO) cells in vitro. In addition, CD40L affected the expression of genes related to angiogenesis, cell adhesion, chemokines and immunity in pENDO cells. These results suggest that the CD40L-CD40 system might play an important role in the establishment of pregnancy in pigs by regulating endometrial endothelial cell function during the implantation period.

B lymphocytes have emerged as an important immune-regulating target. Inoculation with tumor cell membrane-derived vaccines is a promising strategy to activate B cells, yet their efficiency is limited due to lack of costimulatory molecules. To amplify B cell responses against tumor, herein, a spatiotemporally-synchronized antigen-adjuvant integrated nanovaccine, termed as CM-CpG-aCD40, is constructed by conjugating the immune stimulative CpG oligonucleotide and the anti-CD40 antibody (aCD40) onto the membrane vesicles derived from triple negative breast cancer cells. CM-CpG-aCD40 actively accumulates in lymph nodes and is effectively captured by antigen-presenting cells via the recognition of CD40 by aCD40. Tumor antigens on CM-CpG-aCD40 bind to B cell receptors, providing the first stimulation signal for B cells. Meanwhile, the interaction between CpG/Toll like receptor and aCD40/CD40 provides superposed co-stimulation signals, improving the antibody-secreting and antigen-presenting abilities of B cells. The nanovaccine also stimulates dendritic cells to activate CD8 + T cells, and reprograms tumor associated macrophages. CM-CpG-aCD40 activating humoral, cellular, and innate antitumor immunity achieves a tumor inhibition rate of 89.3%, which is further improved to 95.4% when combined with the anti-programmed death ligand 1 (PD-L1) antibody. CM-CpG-aCD40, as a personalized multi-epitope nanovaccine, paves the way for ushering the era of B cell-based immunotherapy., (© 2024 Wiley‐VCH GmbH.)

Statins are widely utilized to reduce cholesterol levels, particularly in cardiovascular diseases. They interface with cholesterol synthesis by inhibiting the 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase enzyme. Besides their primary effect, statins demonstrate anti-inflammatory and immune-modulating properties in various diseases, highlighting the pleiotropic effect of these drugs. The CD40:CD40L signaling pathway is considered a prominent inflammatory pathway in multiple diseases, including autoimmune, inflammatory, and cardiovascular diseases. The findings from clinical trials and in vitro and in vivo studies suggest the potential anti-inflammatory effect of statins in modulating the CD40 signaling pathway and downstream inflammatory mediator. Accordingly, as its classic ligand, statins can suppress immune responses in autoimmune diseases by inhibiting CD40 expression and blocking its interaction with CD40L. Additionally, statins affect intracellular signaling and inhibit inflammatory mediator secretion in chronic inflammatory diseases like asthma and autoimmune disorders such as myasthenia gravis, multiple sclerosis, systemic lupus erymanthus, and cardiovascular diseases like atherosclerosis. However, it is essential to note that the anti-inflammatory effect of statins may vary depending on the specific type of statin used. In this study, we aim to explore the potential anti-inflammatory effects of statins in treating inflammatory diseases by examining their role in regulating immune responses, particularly their impact on the CD40:CD40L signaling pathway, through a comprehensive review of existing literature., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s) under exclusive licence to Maj Institute of Pharmacology Polish Academy of Sciences.)

Background: Ideally, vaccination should induce protective long-lived humoral and cellular immunity. Current licensed COVID-19 mRNA vaccines focused on the spike (S) region induce neutralizing antibodies that rapidly wane., Methods: Herein, we show that a subunit vaccine (CD40.CoV2) targeting spike and nucleocapsid antigens to CD40-expressing cells elicits broad specific human (hu)Th1 CD4 + and CD8 + T cells in humanized mice., Findings: CD40.CoV2 vaccination selectively enriched long-lived spike- and nucleocapsid-specific CD8 + progenitors with stem-cell-like memory (Tscm) properties, whereas mRNA BNT162b2 induced effector memory CD8 + T cells. CD8 + Tscm cells produced IFNγ and TNF upon antigenic restimulation and showed a high proliferation rate. We demonstrate that CD40 activation is specifically required for the generation of huCD8 + Tscm cells., Interpretation: These results support the development of a CD40-vaccine platform capable of eliciting long-lasting T-cell immunity., Funding: This work was supported by Inserm, Université Paris-Est Créteil, and the Investissements d'Avenir program, Vaccine Research Institute (VRI), managed by the ANR., Competing Interests: Declaration of interests The authors SZ, GZ, MC, SC, and YL are named inventors on patent applications based on this work held by Inserm Transfert. The remaining authors declare no competing interests. Inserm Transfert provided a license for CD40-targeting vaccines to the biotech company LinKinVax., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

CD40-CD40-ligand (CD40L) interaction plays crucial immunoregulatory roles, as CD40 signals through different signaling intermediates to convert the messages from CD40L to effector functions. Being a TNFα receptor family member, CD40 binds TNFα receptor-associated factors, assembles signalosome complexes and decrypts the messages from CD40L through different signaling modules to result in residue-specific effector functions. The evidence for such a residue-specific message encryption first came from the CD40L mutations resulting in X-linked hyper-IgM syndrome, as the extent of effects varied with the residue mutated. The structural studies on the CD40-CD40L interaction implied differential involvement of the interacting residues on CD40L in influencing the effector functions. Three lines of evidence indicate the previously implied residue-specific message encryption in CD40L: screening of a dodecameric peptide library for CD40 binders identified two peptides with different sequences resulting in counteractive effector functions in macrophages; a series of CD40L mutants identified that the mutations in these residues selectively affected CD40 signaling and macrophage effector functions; and, a panel of 40-mer peptides, representing the CD40-interacting domain of mouse CD40L, with single substitutions resulted in altered CD40 signaling through various signaling intermediates and effector functions in mouse macrophages. We therefore construct the first-ever message encryption-decryption in a biological receptor-ligand system wherein the CD40L residues that interact with CD40 residues have encrypted messages, which are decoded by CD40 signaling to result in residue-specific effector functions. This review presents a novel perspective of receptor-ligand interaction as a system of message transmission, message decoding by signaling, and its transcription to various read-outs. [250 words]., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

CD40 agonist antibodies (αCD40) have shown promising anti-tumor response in both preclinical and early clinical studies. However, its systemic administration is associated with immune- and hepato-toxicities which hampers its clinical usage. In addition, αCD40 showed low tumor retention and induced PD-L1 expression which makes tumor microenvironment (TME) immunosuppressive. To overcome these issues, in this study, we have developed a multifunctional Immunosome where αCD40 is conjugated on the surface and RRX-001, a small molecule immunomodulator was encapsulated inside it. Immunosomes showed higher tumor accumulation till 96 h of administration and displayed sustained release of αCD40 in vivo. Immunosomes significantly delayed tumor growth and showed tumor free survival in mice bearing GL-261 glioblastoma by increasing the population of CD45 + CD8 + T cells, CD45 + CD20 + B cells, CD45 + CD11c + DCs and F4/80 + CD86 + cells in TME. Immunosome significantly reduced the population of T-regulatory cells, M2 macrophage, and MDSCs and lowered the PD-L1 expression. Moreover, Immunosomes significantly enhanced the levels of Th1 cytokines (IFN-γ, IL-6, IL-2) over Th2 cytokines (IL-4 and IL-10) which supported anti-tumor response. Most interestingly, Immunosomes averted the in vivo toxicities associated with free αCD40 by lowering the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-6, IL-1α and reduced the degree of liver damage. In addition, Immunosomes treated long-term surviving mice showed tumor specific immune memory response which prevented tumor growth upon rechallenge. Our results suggested that this novel formulation can be further explored in clinics to improve in vivo anti-tumor efficacy of αCD40 with long-lasting tumor specific immunity while reducing the associated toxicities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Medullary thymic epithelial cells (mTECs) play a crucial role in suppressing the onset of autoimmunity by eliminating autoreactive T cells and promoting the development of regulatory T cells in the thymus. Although mTECs undergo turnover in adults, the molecular mechanisms behind this process remain unclear. This study describes the direct and indirect roles of receptor activator of NF-κB (RANK) and CD40 signaling in TECs in the adult thymus. Flow cytometric and single-cell RNA-seq (scRNA-seq) analyses suggest that the depletion of both RANK and CD40 signaling inhibits mTEC differentiation from CCL21 + mTEC progenitors to transit-amplifying TECs in the adult thymus. Unexpectedly, this depletion also exerts indirect effects on the gene expression of TEC progenitors and cortical TECs. Additionally, the expression levels of AP-1 genes, which enable the further subdivision of TEC progenitors, are up-regulated following the depletion of RANK and CD40 signaling. Overall, our data propose that RANK and CD40 signaling cooperatively maintain mature mTEC frequency in the adult thymus and sustain the characteristics of TEC progenitors through an indirect mechanism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hayama, Ishii, Miyauchi, Yoshida, Hagiwara, Muramtatu, Namiki, Endo, Miyao, Akiyama and Akiyama.)

Chimeric antigen receptor T (CAR-T) cells show remarkable efficacy for some hematological malignancies. However, CAR targets that are expressed at high level and selective to tumors are scarce. Several strategies have been proposed to tackle the on-target off-tumor toxicity of CAR-T cells that arise from suboptimal selectivity, but these are complicated, with many involving dual gene expression for specificity. In this study, we show that switchable CAR-T cells with a tumor targeting adaptor can mitigate on-target off-tumor toxicity against a low selectivity tumor antigen that cannot be targeted by conventional CAR-T cells, such as CD40. Our system is composed of anti-cotinine murine CAR-T cells and cotinine-labeled anti-CD40 single chain variable fragments (scFv), with which we show selective tumor killing while sparing CD40-expressing normal cells including macrophages in a mouse model of lymphoma. Simple replacement of the tumor-targeting adaptor with a suicidal drug-conjugated tag may further enhance safety by enabling permanent in vivo depletion of the switchable CAR-T cells when necessary. In summary, our switchable CAR system can control CAR-T cell toxicity while maintaining therapeutic efficacy, thereby expanding the range of CAR targets., Competing Interests: Competing interests K.C. and E.Y.C. are founders and shareholders of Ticaros Inc. H.B.P., J.E.L., and G.R.J. are currently employees of Ticaros. K.C., J.C., H.B.P., K.H.K., S.I.K., and G.R.J. are co-inventors on the pending patent for anti-CD40 switchable CAR T cells. The other authors declare no competing interests., (© 2024. The Author(s).)

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.

As part of our work to develop small-molecule inhibitors (SMIs) of the CD40-CD40L(CD154) costimulatory protein-protein interaction, here, we describe the ability of two of our most promising SMIs, DRI-C21041 and DRI-C21095, to prolong the survival and function of islet allografts in two murine models of islet transplantation (under the kidney capsule and in the anterior chamber of the eye) and to prevent autoimmune type 1 diabetes (T1D) onset in NOD mice. In both transplant models, a significant portion of islet allografts (50%-80%) remained intact and functional long after terminating treatment, suggesting the possibility of inducing operational immune tolerance via inhibition of the CD40-CD40L axis. SMI-treated mice maintained the structural integrity and function of their islet allografts with concomitant reduction in immune cell infiltration as evidenced by direct longitudinal imaging in situ . Furthermore, in female NODs, three-month SMI treatment reduced the incidence of diabetes from 80% to 60% (DRI-C21041) and 25% (DRI-C21095). These results ( i ) demonstrate the susceptibility of this TNF superfamily protein-protein interaction to small-molecule inhibition, ( ii ) confirm the in vivo therapeutic potential of these SMIs of a critical immune checkpoint, and ( iii ) reaffirm the therapeutic promise of CD40-CD40L blockade in islet transplantation and T1D prevention. Thus, CD40L-targeting SMIs could ultimately lead to alternative immunomodulatory therapeutics for transplant recipients and prevention of autoimmune diseases that are safer, less immunogenic, more controllable (shorter half-lives), and more patient-friendly (i.e., suitable for oral administration, which makes them easier to administer) than corresponding antibody-based interventions., Competing Interests: The authors declare the following competing financial interest: The University of Miami has filed a patent on the DRI-C compounds with PB as inventor. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer VV declared a shared affiliation, with no collaboration, with the authors to the handling editor at the time of the review., (Copyright © 2024 Chuang, Alcazar, Watts, Abdulreda and Buchwald.)

CD40-CD40 ligand interactions are critical for controlling Pneumocystis infection. However, which CD40-expressing cell populations are important for this interaction have not been well defined. We used a cohousing mouse model of Pneumocystis infection, combined with flow cytometry and quantitative polymerase chain reaction, to examine the ability of different populations of cells from C57BL/6 mice to reconstitute immunity in CD40 knockout mice. Unfractionated splenocytes, as well as purified B cells, were able to control Pneumocystis infection, while B cell-depleted splenocytes and unstimulated bone marrow-derived dendritic cells were unable to control infection in CD40 knockout mice. Pneumocystis antigen-pulsed bone marrow-derived dendritic cells showed early but limited control of infection. Additional findings were consistent with recent studies that suggested a role for antigen presentation by B cells; specifically, by using cells from immunized animals, B cells were able to present Pneumocystis antigens to induce proliferation of T cells. Thus, CD40 expression by B cells appears necessary for robust immunity to Pneumocystis., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

B cells play a key role in humoral immune responses by producing antibodies. Although there are numerous research on memory B cells definition markers and cytokines on B cell development, different studies have yielded contradictory conclusions due to species studied, the different cells and stimulating agents used. In the current study, we conducted a detailed characterization of B cells in human CBMCs, PBMCs and tonsil, including expression of Igs, activation and memory markers. Furthermore, we found that considerable amounts of IgA and IgG were expressed by CD27 - B cells. These "Atypical" memory B cells corresponded to approximately 50% of IgG + and IgA + B cells in blood, this proportion even reached 90% in tonsil. In addition, we investigated the effect of IL-21 and TGF-β1 on the membrane-bound form and secreted form of Igs using PBMCs and purified blood B cells. There were actual differences between the effect of cytokines on Igs secretion and surface expression. Our study will be helpful to advance the knowledge and understanding of humoral memory., (© 2024. The Author(s).)

Dendritic cells (DCs) are critical mediators of antigen priming and T-cell activation. Zymogen granule protein 16 (ZG16) is demonstrated as an anti-oncogene in T-cell mediated antitumor immunity, but its effect on DCs is largely unknown. Herein, we wonder whether ZG16 affects the activation of DCs in pancreatic cancer. Firstly, the increased ZG16 expression was observed during the maturation of DCs derived from mouse bone marrow or human peripheral blood. Then, overexpression of ZG16 or exogenous introduction of recombinant ZG16 protein induced the expression of MHC II, CD86, CD84, and CCR7 on the surface of DCs, thereby facilitating the secretion of proinflammatory mediators IL-1β, IL-6, TNF-α, and IL-12/p70, supporting the promoting effect of ZG16 on DC maturation. By establishing the subcutaneous and orthotopic mouse models of pancreatic cancer, we confirmed that intraperitoneal injection of recombinant ZG16 protein (Re-mZG16) could induce tumor regression by stimulating DC maturation and enhancing antitumor responses of CD4 + , CD8 + , PD-1 + , and Ctla4+ cells. Besides, Re-mZG16 in combination with gemcitabine showed a synergistic effect in the treatment of pancreatic cancer. Mechanistically, we demonstrated that ZG16 inhibited the ubiquitination and degradation of CD40, which depended on the lectin domain of ZG16. In conclusion, this study provided a novel insight into the role of ZG16-CD40 axis in DC-based immunotherapy for pancreatic cancer., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Trained immunity is characterized by histone modifications and metabolic changes in innate immune cells following exposure to inflammatory signals, leading to heightened responsiveness to secondary stimuli. Although our understanding of the molecular regulation of trained immunity has increased, the role of adaptive immune cells herein remains largely unknown. Here, we show that T cells modulate trained immunity via cluster of differentiation 40-tissue necrosis factor receptor-associated factor 6 (CD40-TRAF6) signaling. CD40-TRAF6 inhibition modulates functional, transcriptomic, and metabolic reprogramming and modifies histone 3 lysine 4 trimethylation associated with trained immunity. Besides in vitro studies, we reveal that single-nucleotide polymorphisms in the proximity of CD40 are linked to trained immunity responses in vivo and that combining CD40-TRAF6 inhibition with cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4-Ig)-mediated co-stimulatory blockade induces long-term graft acceptance in a murine heart transplantation model. Combined, our results reveal that trained immunity is modulated by CD40-TRAF6 signaling between myeloid and adaptive immune cells and that this can be leveraged for therapeutic purposes., Competing Interests: Declaration of interests J.O., L.A.B.J., M.G.N., and W.J.M.M. declare that they are scientific founders of Trained Therapeutics Discovery., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glioblastoma multiforme (GBM) is one of the most aggressive and deadly forms of brain cancer, which has a very complex tumor microenvironment (TME) promoting tumor growth, immune evasion, and resistance to therapy. The main players within this environment are represented by cytokines such as Interleukin-4, Interleukin-6, and Interleukin-13, along with the costimulatory molecule CD40. The paper draws back the curtain on the complex interactions played out by these molecules in contributing to the formation of a TME within GBM. IL-4 and IL-13 induce an immunosuppressive environment through the polarization of tumor-associated macrophages (TAMs) into a pro-tumoral M2 phenotype. In contrast, IL-6 takes part in the activation of the JAK-STAT3 pathway, enhancing survival and proliferation of tumor cells. In this context, CD40 either induces anti-tumor immunity through APC activation or facilitates tumors by angiogenesis and survival pathways. The synergistic actions of these molecules create feedback loops that keep up the malignancy of GBM and present a big problem for therapy. Knowledge of these interactions opens new ways for the development of multi-targeted therapeutic strategies at the other end. This may result in the interruption of the tumor-supportive environment in GBM, reducing tumor growth and improving patient outcomes by targeting IL-4, IL-6, IL-13, and CD40 simultaneously., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Visceral leishmaniasis (VL) is a neglected tropical disease caused by parasites from the Leishmania (L.) donovani complex. VL is characterised by uncontrolled parasite replication in spleen, liver and bone marrow, and by an impaired immune response and high systemic levels of inflammation. Monocytes have been poorly characterised in VL patients. The aim of this study was to evaluate the expression levels of markers involved in the regulation of T cell responses on different subsets of monocytes from the blood of VL patients and healthy non-endemic controls (HNEC). Monocytes can broadly be divided into three subsets: classical, intermediate and non-classical monocytes. Our results show that the percentages of all three subsets stayed similar at the time of VL diagnosis (ToD) and at the end of anti-leishmanial treatment (EoT). We first looked at co-stimulatory receptors: the expression levels of CD40 were significantly increased on classical and intermediate, but not non-classical monocytes, at ToD as compared to EoT and HNEC. CD80 expression levels were also increased on intermediate monocytes at ToD as compared to EoT and HNEC, and on classical monocytes only as compared to HNEC. The levels of CD86 were similar at EoT and ToD and in HNEC on classical and intermediate monocytes, but significantly higher at EoT on non-classical monocytes. We also looked at an inhibitory molecule, PD-L1. Our results show that the expression levels of PD-L1 were significantly higher on all three monocyte subsets at ToD as compared to HNEC, and to EoT on classical and intermediate monocytes. These results show that monocytes from the blood of VL patients upregulate both co-stimulatory and inhibitory receptors and that their expression levels are restored at EoT., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Adem et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ligation of the B cell antigen receptor (BCR) initiates humoral immunity. However, BCR signaling without appropriate co-stimulation commits B cells to death rather than to differentiation into immune effector cells. How BCR activation depletes potentially autoreactive B cells while simultaneously primes for receiving rescue and differentiation signals from cognate T lymphocytes remains unknown. Here, we use a mass spectrometry-based proteomic approach to identify cytosolic/nuclear shuttling elements and uncover transcription factor EB (TFEB) as a central BCR-controlled rheostat that drives activation-induced apoptosis, and concurrently promotes the reception of co-stimulatory rescue signals by supporting B cell migration and antigen presentation. CD40 co-stimulation prevents TFEB-driven cell death, while enhancing and prolonging TFEB's nuclear residency, which hallmarks antigenic experience also of memory B cells. In mice, TFEB shapes the transcriptional landscape of germinal center B cells. Within the germinal center, TFEB facilitates the dark zone entry of light-zone-residing centrocytes through regulation of chemokine receptors and, by balancing the expression of Bcl-2/BH3-only family members, integrates antigen-induced apoptosis with T cell-provided CD40 survival signals. Thus, TFEB reprograms antigen-primed germinal center B cells for cell fate decisions., (© 2024. The Author(s).)

CD40L-CD40-TRAF signaling plays a role in atherosclerosis progression and affects the pathogenesis of coronary heart disease (CHD). We tested the hypothesis that CD40L-CD40-TRAF signaling is a potential therapeutic target in hyperlipidemia, diabetes, and hypertension. In mouse models of hyperlipidemia plus diabetes (db/db mice) or hypertension (1 mg/kg/d angiotensin-II for 7 days), TRAF6 inhibitor treatment (2.5 mg/kg/d for 7 or 14 days) normalized markers of oxidative stress and inflammation. As diabetes and hypertension are important comorbidities aggravating CHD, we explored whether the CD40L-CD40-TRAF signaling cascade and their associated inflammatory pathways are expressed in CHD patients suffering from comorbidities. Therefore, we analyzed vascular bypass material (aorta or internal mammary artery) and plasma from patients with CHD with diabetes and/or hypertension. Our Olink targeted plasma proteomic analysis using the IMMUNO-ONCOLOGY panel revealed a pattern of step-wise increase for 13/92 markers of low-grade inflammation with significant changes. CD40L or CD40 significantly correlated with 38 or 56 other inflammatory targets. In addition, specific gene clusters that correlate with the comorbidities were identified in isolated aortic mRNA of CHD patients through RNA-sequencing. These signaling clusters comprised CD40L-CD40-TRAF, immune system, hemostasis, muscle contraction, metabolism of lipids, developmental biology, and apoptosis. Finally, immunological analysis revealed key markers correlated with comorbidities in CHD patients, such as CD40L, NOX2, CD68, and 3-nitrotyrosine. These data indicate that comorbidities increase inflammatory pathways in CHD, and targeting these pathways will be beneficial in reducing cardiovascular events in CHD patients with comorbidities., (© 2024. The Author(s).)

Anti-CD40 antibodies (Abs) have been shown to induce antitumor T-cell responses. We reported that the engineered agonistic anti-CD40 Ab (5C11, IgG4 isotype) recognized human CD40 antigen expressed on a human B lymphoblastoid cell line as well as on splenic cells isolated from humanized CD40 mice. Of note, a single high dosage of 5C11 was able to prohibit tumor growth in parallel with an increase in the population of infiltrated CD8 + T cells. Furthermore, the antitumor effects of 5C11 were enhanced in the presence of β-glucan along with an increase in the population of infiltrated CD8 + T cells. In addition, the numbers of CD86 + TAMs and neutrophils were elevated in the combination of 5C11 and β-glucan compared with either 5C11 or β-glucan alone. Furthermore, the abundance of Faecalibaculum, one of the probiotics critical for tumor suppression, was obviously increased in the combination of 5C11 and β-glucan-treated mice. These data reveal a novel mechanism of tumor suppression upon the combination treatment of 5C11 and β-glucan and propose that the combination treatment of agonistic anti-human CD40 antibody 5C11 and β-glucan could be a promising therapeutic strategy for cancer patients., Competing Interests: Declaration of competing interest None., (Copyright © 2024 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Objectives: Chronic rejection remains the leading cause of progressive decline in graft function. Accumulating evidence indicates that macrophages participate in chronic rejection dependent on CD40-CD40L. The FOS family members are critical in inflammatory and immune responses. However, the mechanisms underlying the role of FOS family members in chronic rejection remain unclear. In this study, we aimed to elucidate the role and underlying mechanisms of FOS-positive macrophages regulated by CD40 that mediate chronic allograft rejection., Materials and Methods: We downloaded publicly accessible chronic rejection kidney transplant single-cell sequencing datasets from the gene expression omnibus database. Differentially expressed genes between the CD40hi and CD40low macrophage chronic rejection groups were analyzed. We established a chronic rejection mouse model by using CTLA-4-Ig. We treated bone marrow-derived macrophages with an anti-CD40 antibody. We assessed expression of the FOS family by flow cytometry, real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. We identified altered signaling pathways by using RNA sequencing analysis. We detected DNA specifically bound to transcription factors by using ChIP-sequencing, with detection of the degree of graft fibrosis and survival., Results: FOS was highly expressed on CD40hi macrophages in patients with chronic transplantrejection. Mechanistically, we showed that CD40 activated NF-κB2 translocation into the nucleus to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant.We showed thatNF-κB2 regulated c-Fos and FosB expression by binding to the c-fos and fosb promoter regions. Inhibition of c-Fos/activator protein-1 decreased graft fibrosis and prolonged graft survival., Conclusions: CD40 may activate transcription factor NF-κB2 translocation into the nucleus of macrophages to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant. Inhibition of c-Fos/activator protein-1 decreased grafts fibrosis and prolonged graft survival.

Hepatocellular carcinoma (HCC) is associated with increased soluble CD40 levels. This study aimed to investigate CD40's role in liver tumor progression. CD40 levels were examined in HCC patient tissues and various HCC cell lines, and their interaction with CD4 + T cells was studied. RNA sequencing analysis was performed to explore the mechanisms of CD40 induction. Poorly differentiated HCC tumor tissues exhibited high membrane-bound CD40 expression, in contrast to nontumor areas. Poorly differentiated HCC cell lines showed high expression of membrane-bound CD40 with low CD40 promoter methylation, which was the opposite of that observed in the well-differentiated HCC cell lines. Solely modulating CD40 expression in HCC cells exerted no direct consequences on cell growth or appearance. Interestingly, the human hepatoma cell line HLF co-cultured with activated (CD40 ligand + ) CD4 + T cells had increased CD40 levels and a modest 3.2% dead cells. The percentage of dead cells increased to 10.9% and underwent preneutralizing CD40 condition, whereas preblocking both CD40 and integrin α5β1 concomitantly caused only 1.9% cell death. RNA sequencing of co-cultured HLFs with activated CD4 + T cells revealed the up-regulation of interferon and immune-response pathways. Increased interferon-γ levels in the activated T-cell media stimulated the Janus kinase/signal transducer and activator of transcription 3 pathway, resulting in increased CD40 expression in HLF. Collectively, CD40 expression in poorly differentiated HCC cells prevented cell death by interacting with CD40 ligand in activated T cells. Targeting CD40 may represent a promising anticancer therapy., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Hyperglycemia reinforces pro-inflammatory conditions that enhance CD40 expression in endothelial cells (EC). Thymine to cytosine transition (-1T > C) in the promoter of the CD40 gene (rs1883832) further increases the abundance of CD40 protein on the EC surface. This study examines potential associations of the -1T > C SNP of the CD40 gene with type 1 (T1D) or type 2 (T2D) diabetes. Moreover, it investigates the impact of a pro-inflammatory diabetic microenvironment on gene expression in human cultured umbilical vein EC (HUVEC) derived from CC- vs. TT-genotype donors., Methods: Tetra-ARMS-PCR was used to compare genotype distribution in 252 patients with diabetes. Soluble CD40 ligand (sCD40L) and soluble CD40 receptor (sCD40) plasma levels were monitored using ELISA. RNA-sequencing was performed with sCD40L-stimulated CC- and TT-genotype HUVEC. Quantitative PCR, Western blot, multiplex-sandwich ELISA array, and immunocytochemistry were used to analyse changes in gene expression in these cells., Results: Homozygosity for the C-allele was associated with a significant 4.3-fold higher odds of developing T2D as compared to individuals homozygous for the T-allele. Inflammation and endothelial-to-mesenchymal transition (EndMT) driving genes were upregulated in CC-genotype but downregulated in TT-genotype HUVEC when exposed to sCD40L. Expression of EndMT markers significantly increased while that of endothelial markers decreased in HUVEC following exposure to hyperglycemia, tumour necrosis factor-α and sCD40L., Conclusions: The -1T > C SNP of the CD40 gene is a risk factor for T2D. Depending on the genotype, it differentially affects gene expression in human cultured EC. CC-genotype HUVEC adopt a pro-inflammatory and intermediate EndMT-like phenotype in a pro-diabetic microenvironment., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

CD40 is a member of the tumor necrosis factor receptor superfamily, and it is widely expressed on immune and non-immune cell types. The interaction between CD40 and the CD40 ligand (CD40L) plays an essential function in signaling, and the CD40/CD40L complex works as an immune checkpoint molecule. CD40 has become a therapeutic target, and a variety of agonistic/antagonistic anti-CD40 monoclonal antibodies (mAbs) have been developed. To better understand the mode of action of anti-CD40 mAbs, we determined the X-ray crystal structures of dacetuzumab (agonist) and bleselumab (antagonist) in complex with the extracellular domain of human CD40, respectively. The structure reveals that dacetuzumab binds to CD40 on the top of cysteine-rich domain 1 (CRD1), which is the domain most distant from the cell surface, and it does not compete with CD40L binding. The binding interface of bleselumab spread between CRD2 and CRD1, overlapping with the binding surface of the ligand. Our results offer important insights for future structural and functional studies of CD40 and provide clues to understanding the mechanism of biological response. These data can be applied to developing new strategies for designing antibodies with more therapeutic efficacy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets., (Copyright © 2024 Elsevier B.V. All rights reserved.)

B cells, despite their several unique functionalities, remain largely untapped for use as an adoptive cell therapy and are limited to in vitro use for antibody production. B cells can be easily sourced, they possess excellent lymphoid-homing capabilities, and they can act as antigen-presenting cells (APCs), offering an alternative to dendritic cells (DCs), which have shown limited efficacy in the clinical setting. Soluble factors such as IL-4 and anti-CD40 antibody can enhance the activation, survival, and antigen-presenting capabilities of B cells; however, it is difficult to attain sufficiently high concentrations of these biologics to stimulate B cells in vivo . Micropatches as Cell Engagers (MACE) are polymeric microparticles, surface functionalized with anti-CD40 and anti-IgM, which can attach to B cells and simultaneously engage multiple B-cell receptors (BCR) and CD40 receptors. Stimulation of these receptors through MACE, unlike free antibodies, enhanced the display of costimulatory molecules on the B-cell surface, increased B-cell viability, and improved antigen presentation by B cells to T cells in vitro . B-cell activation by MACE further synergized with soluble IL-4 and anti-CD40. MACE also elicited T-cell chemokine secretion by B cells. Upon intravenous adoptive transfer, MACE-bound B cells homed to the spleen and lymph nodes, key sites for antigen presentation to T cells. Adoptive transfer of MACE-B cells pulsed with the CD4+ and CD8+ epitopes of ovalbumin significantly delayed tumor progression in a murine subcutaneous EG7-OVA tumor model, demonstrating the functional benefit conferred to B cells by MACE.

Although Multiple Sclerosis (MS) is primarily thought to be an autoimmune condition, its possible viral etiology must be taken into consideration. When mice are administered neurotropic viruses like mouse hepatitis virus MHV-A59, a murine coronavirus, or its isogenic recombinant strain RSA59, neuroinflammation along with demyelination are observed, which are some of the significant manifestations of MS. MHV-A59/RSA59 induced neuroinflammation is one of the best-studied experimental animal models to understand the viral-induced demyelination concurrent with axonal loss. In this experimental animal model, one of the major immune checkpoint regulators is the CD40-CD40L dyad, which helps in mediating both acute-innate, innate-adaptive, and chronic-adaptive immune responses. Hence, they are essential in reducing acute neuroinflammation and chronic progressive adaptive demyelination. While CD40 is expressed on antigen-presenting cells and endothelial cells, CD40L is expressed primarily on activated T cells and during severe inflammation on NK cells and mast cells. Experimental evidences revealed that genetic deficiency of both these proteins can lead to deleterious effects in an individual. On the other hand, interferon-stimulated genes (ISGs) possess potent antiviral properties and directly or indirectly alter acute neuroinflammation. In this review, we will discuss the role of an ISG, ISG54, and its tetratricopeptide repeat protein Ifit2; the genetic and experimental studies on the role of CD40 and CD40L in a virus-induced neuroinflammatory demyelination model., (© 2023 International Union of Biochemistry and Molecular Biology.)

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is a leading cause of preventable blindness. We have developed in vivo mouse models of acute and chronic C. trachomatis genital infection to explore the significance of macrophage-directed response in mediating immune activation/suppression. Our findings reveal that during chronic and repeated C. trachomatis infections, Th 1 response is abated while T reg response is enhanced. Additionally, an increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is observed during chronic infection. We have also observed that M 2 macrophages with low CD40 expression promote Th 2 and T reg differentiation leading to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to an M 2 phenotype. C. trachomatis infection prevents the increase in CD40 expression as observed in western blots and flow cytometric analysis. Insufficient IFNγ, as observed during chronic infection, leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFNγ responsiveness in macrophages via hampering IFNγRI and IFNγRII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFNγ supplementation. M 2 macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th 2 and T reg differentiation which could not be reverted even by IFNγ supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFNγ, thus promoting immunosuppression. In summary, C. trachomatis modulates the innate immune cells, attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages., (Copyright © 2024 Elsevier B.V. All rights reserved.)

CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily of receptors expressed on a variety of cell types. The CD40-CD40L interaction gives rise to many immune events, including the licensing of dendritic cells to activate CD8 + effector T cells, as well as the facilitation of B cell activation, proliferation, and differentiation. In malignant cells, the expression of CD40 varies among cancer types, mediating cellular proliferation, apoptosis, survival and the secretion of cytokines and chemokines. Agonistic human anti-CD40 antibodies are emerging as an option for cancer treatment, and early-phase clinical trials explored its monotherapy or combination with radiotherapy, chemotherapy, immune checkpoint blockade, and other immunomodulatory approaches. In this review, we present the current understanding of the mechanism of action for CD40, along with results from the clinical development of agonistic human CD40 antibodies in cancer treatment (selicrelumab, CDX-1140, APX005M, mitazalimab, 2141-V11, SEA-CD40, LVGN7409, and bispecific antibodies). This review also examines the safety profile of CD40 agonists in both preclinical and clinical settings, highlighting optimized dosage levels, potential adverse effects, and strategies to mitigate them., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: 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., Background: 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., Methods: 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., Results: 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., Conclusion: 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., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Previously, we established that as a function of its mode of interaction with its ligand or cellular conditions such as membrane lipids, preexisting signaling intermediates activation status, a transmembrane receptor, as represented here with CD40, can induce counteractive cellular responses. Using CD40-binding peptides, recombinant mutated CD40-ligands, and an agonistic antibody, we have established the functional duality of CD40. CD40 builds up two constitutionally different signalosomes on lipid raft and non-raft membrane domains initiating two different signaling pathways. Although this initial signaling may be modified by the pre-existing signaling conditions downstream and may be subjected to feed-forward or negative signaling effects, the initial CD40-CD40L interaction plays a crucial role in the functional outcome of CD40. Herein, we have reviewed the influence of interaction between the CD40-CD40L evoking the functional duality of CD40 contingent upon different physiological states of the cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Homozygosity for the C allele of the -1T>C single nucleotide polymorphism (SNP) of the CD40 gene (rs1883832) is associated with susceptibility to coronary heart disease (CHD), enhanced CD40 expression, and shedding. The disintegrin metalloprotease ADAM17 can cleave various cell surface proteins. This study investigates an association between ADAM17-mediated CD40 shedding and inflammation in CC genotype human endothelial cells., Methods: Human umbilical vein endothelial cells (HUVEC) carrying the CC genotype were stimulated with soluble CD40 ligand (sCD40L) or tumor necrosis factor-α (TNFα). Messenger RNA and protein expression were determined with standard methods. Levels of high sensitive c-reactive protein (hs-CRP), interleukin-6 (IL-6), and sCD40 in plasma samples from patients with CHD were assessed using ELISA., Results: ADAM17 surface abundance was elevated following stimulation with CD40L and TNFα just as its regulator iRhom2. Inhibition of ADAM17 prevented TNFα-induced sCD40 and soluble vascular cell adhesion molecule-1 release into the conditioned medium and reinforced CD40 surface abundance. Secondary to inhibition of ADAM17, stimulation with CD40L or TNFα upregulated monocyte chemoattractant protein-1 mRNA and protein. Levels of sCD40 and the inflammatory biomarkers hs-CRP and IL-6 were positively correlated in the plasma of patients with CHD., Conclusions: We provide a mechanism by which membrane-bound CD40 is shed from the endothelial cell surface by ADAM17, boosting sCD40 formation and limiting downstream CD40 signaling. Soluble CD40 may represent a robust biomarker for CHD, especially in conjunction with homozygosity for the C allele of the -1T>C SNP of the CD40 gene.

Macrophages are critical in the pathogenesis of a diverse group of viral pathogens, both as targets of infection and for eliciting primary defense mechanisms. Our prior in vitro work identified that CD40 signaling in murine peritoneal macrophages protects against several RNA viruses by eliciting IL-12, which stimulates the production of interferon gamma (IFN-γ). Here, we examine the role of CD40 signaling in vivo. We show that CD40 signaling is a critical, but currently poorly appreciated, component of the innate immune response using two distinct infectious agents: mouse-adapted influenza A virus (IAV, PR8) and recombinant VSV encoding the Ebola virus glycoprotein (rVSV-EBOV GP). We find that stimulation of CD40 signaling decreases early IAV titers, whereas loss of CD40 elevated early titers and compromised lung function by day 3 of infection. Protection conferred by CD40 signaling against IAV is dependent on IFN-γ production, consistent with our in vitro studies. Using rVSV-EBOV GP that serves as a low-biocontainment model of filovirus infection, we demonstrate that macrophages are a CD40-expressing population critical for protection within the peritoneum and T-cells are the key source of CD40L (CD154). These experiments reveal the in vivo mechanisms by which CD40 signaling in macrophages regulates the early host responses to RNA virus infection and highlight how CD40 agonists currently under investigation for clinical use may function as a novel class of broad antiviral treatments.

CD40 signaling has long been a target in autoimmunity. Attempts to block signaling between CD40 and CD154 during clinical trials using monoclonal antibodies suffered severe adverse events. Previously, we developed a peptide, KGYY 15 , that targets CD40 and, in preclinical trials, prevents type 1 diabetes in >90% of cases and reverses new-onset hyperglycemia in 56% of cases. It did so by establishing normal effector T-cell levels rather than ablating the cells and causing immunosuppression. However, the relationship between KGYY 15 and other elements of the complex signaling network of CD40 is not clear. Studying interactions between proteins from autoimmune and nonautoimmune mice, we demonstrate interactions between CD40 and integrin CD11a/CD18, which complicates the understanding of the inflammatory nexus and how to prevent autoinflammation. In addition to interacting with CD40, KGYY 15 interacts with the integrins CD11a/CD18 and CD11b/CD18. We argue that modulation of CD40-CD154 signaling may be more advantageous than complete inhibition because it may preserve normal immunity to pathogens., Competing Interests: Conflict of interest D. H. W. Jr is the founder and chief scientific officer of Op-T LLC., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The capacity to induce tumour-cell specific apoptosis represents the most unique feature of the TNF receptor (TNFR) family member CD40. Recent studies on the signalling events triggered by its membrane-presented ligand CD40L (mCD40L) in normal and malignant epithelial cells have started to unravel an exquisite context and cell type specificity for the functional effects of CD40. Here, we demonstrate that, in comparison to other carcinomas, mCD40L triggered strikingly more rapid apoptosis in colorectal carcinoma (CRC) cells, underpinned by its ability to entrain two concurrently operating signalling axes. CD40 ligation initially activates TNFR-associated factor 3 (TRAF3) and subsequently NADPH oxidase (NOX)/Apoptosis signal-regulating kinase 1 (ASK1)-signalling and induction of reactive oxygen species (ROS) to mediate p38/JNK- and ROS-dependent cell death. At that point, p38/JNK signalling directly activates the mitochondrial pathway, and triggers rapid induction of intracellular TNF-related apoptosis-inducing ligand (TRAIL) that signals from internal compartments to initiate extrinsic caspase-10-asscociated apoptosis, leading to truncated Bid (tBid)-activated mitochondrial signalling. p38 and JNK are essential both for direct mitochondrial apoptosis induction and the TRAIL/caspase-10/tBid pathway, but their involvement follows functional hierarchy and temporally controlled interplay, as p38 function is required for JNK phosphorylation. By engaging both intrinsic and extrinsic pathways to activate apoptosis via two signals simultaneously, CD40 can accelerate CRC cell death. Our findings further unravel the multi-faceted properties of the CD40/mCD40L dyad, highlighted by the novel TNFR crosstalk that accelerates tumour cell-specific death, and may have implications for the use of CD40 as a therapeutic target.

Introduction: A genome-wide association analysis revealed a rheumatoid arthritis (RA)-risk-associated genetic locus on chromosome 9, which contained the tumor necrosis factor receptor-associated factor 1 (TRAF1). However, the detail mechanism by TRAF1 signaled to fibroblast-like synoviocytes (FLSs) apoptosis remains to be fully understood., Materials and Methods: Synovial tissue of 10 RA patients and osteoarthritis patients were obtained during joint replacement surgery. We investigated TRAF1 level and FLSs apoptosis percentage in vivo and elucidated the mechanism involved in the regulation of apoptotic process in vitro., Results: We proved the significant increase of TRAF1 level in FLSs of RA patients and demonstrated that TRAF1 level correlated positively with DAS28 score and negatively with FLSs apoptosis. Treatment with siTRAF1 was able to decrease MMPs levels and the phosphorylated forms of JNK/NF-κB in vitro. Moreover, JNK inhibitor could attenuate expression of MMPs and increase percentage of apoptosis in RA-FLSs, while siTRAF1 could not promote apoptosis when RA-FLSs were pretreated with JNK activator., Conclusions: High levels of TRAF1 in RA synovium play an important role in the synovial hyperplasia of RA by suppressing apoptosis through activating JNK/NF-kB-dependent signaling pathways in response to the engagement of CD40., (© 2022. The Japanese Society Bone and Mineral Research.)

Objective: The aim of the study was to explore the relevance of CD40, CD86, and GSTO1 with the pathogenesis of COPD., Methods: Patients with acute exacerbation of COPD were contrasted with the healthy and nonsmoking ones and smoking but without COPD ones. The changes of CD40, CD86, and GSTO1 in the peripheral blood, collected from different groups, were detected by flow cytometry and western blotting, respectively., Results: Compared with the nonsmoking group and smoking but without the COPD group, the expression of CD40 and CD86 of the patients with COPD increased significantly, but the expression of GSTO1 decreased. CD40 and CD86 were negatively correlated with FEV1%, while GSTO1 was positively correlated with FEV1% and negatively correlated with CD40 and CD86., Conclusion: CD40, CD86, and GSTO1 may play a role in the pathogenesis of COPD, and they are related to the severity of COPD and the degree of changes in the lung function., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Desheng Sun et al.)

CD154, an inflammatory mediator also known as CD40 ligand, has been identified as a novel binding partner for some members of the integrin family. The αIIbβ3, specifically expressed on platelets, was the first integrin to be described as a receptor for CD154 after CD40. Its interaction with soluble CD154 (sCD154) highly contributes to thrombus formation and stability. Identifying αIIbβ3 opened the door for investigating other integrins as partners of CD154. The αMβ2 expressed on myeloid cells was shown capable of binding CD154 and contributing as such to cell activation, adhesion, and release of proinflammatory mediators. In parallel, α5β1 communicates with sCD154, inducing pro-inflammatory responses. Additional pathogenic effects involving apoptosis-preventing functions were exhibited by the CD154-α5β1 dyad in T cells, conferring a role for such interaction in the survival of malignant cells, as well as the persistence of autoreactive T cells. More recently, CD154 receptors integrated two new integrin members, αvβ3 and α4β1, with little known as to their biological significance in this context. This article provides an overview of the novel role of integrins as receptors of CD154 and as critical players in pro-inflammatory and apoptotic responses.

Abstract: One in 5 patients with diabetes suffers from chronic pain with neuropathic characteristics, but the pathophysiological mechanisms underlying the development of neuropathic pain in patients with diabetic distal symmetrical polyneuropathy (DSP) are poorly understood. Systemic low-grade inflammation has been implicated, but there is still a considerable knowledge gap concerning its scope and meaning in this context. The aim of the study was to establish the broad inflammatory signature of painful diabetic DSP in serum samples from the Pain in Neuropathy Study, an observational cross-sectional multicentre study in which participants underwent deep phenotyping. In the present two cohorts exploration-replication study (180 participants in each cohort), serum samples from Pain in Neuropathy Study participants were analyzed with the Olink INFLAMMATION panel (Olink Bioscience, Uppsala, Sweden) that enables the simultaneous measurement of 92 inflammation-related proteins (mainly cytokines, chemokines, and growth factors). In both the exploration and the replication cohort, we identified a high-inflammation subgroup where 14 inflammation-related proteins in particular were associated with more neuropathy and higher pain intensity. The top 3 proteins were hepatocyte growth factor, colony-stimulating factor 1, and CD40 in both cohorts. In the exploratory cohort, additional clinical data were available, showing an association of inflammation with insomnia and self-reported psychological distress. Hence, this cross-sectional exploration-replication study seems to confirm that low-grade systemic inflammation is related to the severity of neuropathy and neuropathic pain in a subgroup of patients with diabetic DSP. The pathophysiological relevance of these proteins for the development of neuropathic pain in patients with diabetic DSP must be explored in more depth in future studies., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.)

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1β-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The use of tumor mutation-derived neoantigen represents a promising approach for cancer vaccines. Preclinical and early phase human clinical studies have shown the successful induction of tumor neoepitope-directed responses; however, overall clinical efficacy of neoantigen vaccines has been limited. One major obstacle of this strategy is the prevailing lack of sufficient understanding of the mechanism underlying the generation of neoantigen-specific CD8 + T cells. Here, we report a correlation between antitumor efficacy of neoantigen/toll-like receptor 3 (TLR3)/CD40 agonists vaccination and an increased frequency of circulating antigen-specific CD8 + T cells expressing CX3C chemokine receptor 1 (CX3CR1) in a preclinical model. Mechanistic studies using mixed bone marrow chimeras identified that CD40 and CD80/86, but not CD70 signaling in Batf3-dependent conventional type 1 dendritic cells (cDC1s) is required for the antitumor efficacy of neoantigen vaccine and generation of neoantigen-specific CX3CR1 + CD8 + T cells. Although CX3CR1 + CD8 + T cells exhibited robust in vitro effector function, in vivo depletion of this subset did not alter the antitumor efficacy of neoantigen/TLR3/CD40 agonists vaccination. These findings indicate that the vaccine-primed CX3CR1 + subset is dispensable for antitumor CD8 + T cell responses, but can be used as a blood-based T-cell biomarker for effective priming of CD8 + T cells as post-differentiated T cells. Taken together, our results reveal a critical role of CD40 and CD80/86 signaling in cDC1s in antitumor efficacy of neoantigen-based therapeutic vaccines, and implicate the potential utility of CX3CR1 as a circulating predictive T-cell biomarker in vaccine therapy., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Signalling by IFN-y and CD40 is known to trigger anti-microbial activity in macrophages infected with Toxoplasma gondii, but their effects on infected neurons are less well known. Here, we compared how stimulation with IFN-y and an agonistic anti-CD40 mAb impacts infection and cyst formation in the mouse neuroblastoma cell line Neuro-2a relative to bone marrow-derived macrophages. Both IFN-y and CD40 mAb decreased cyst emergence in Neuro-2a cells. In macrophages, these stimuli decreased infection, but had no impact on infection in the neuroblastoma cell line. Resistance to killing in Neuro-2a cells may explain why neurons preferentially harbour parasites during chronic infection in the brain., (© 2021 John Wiley & Sons Ltd.)

The relationship between cancer and autoimmunity is complex. However, the incidence of solid tumors such as melanoma has increased significantly among patients with previous or newly diagnosed systemic autoimmune disease (AID). At the same time, immune checkpoint blockade (ICB) therapy of cancer induces de novo autoinflammation and exacerbates underlying AID, even without evident antitumor responses. Recently, systemic lupus erythematosus (SLE) activity was found to drive myeloid-derived suppressor cell (MDSC) formation in patients, a known barrier to healthy immune surveillance and successful cancer immunotherapy. Cross-talk between MDSCs and macrophages generally drives immune suppressive activity in the tumor microenvironment. However, it remains unclear how peripheral pregenerated MDSC under chronic inflammatory conditions modulates global macrophage immune functions and the impact it could have on existing tumors and underlying lupus nephritis. Here we show that pathogenic expansion of SLE-generated MDSCs by melanoma drives global macrophage polarization and simultaneously impacts the severity of lupus nephritis and tumor progression in SLE-prone mice. Molecular and functional data showed that MDSCs interact with autoimmune macrophages and inhibit cell surface expression of CD40 and the production of IL27. Moreover, low CD40/IL27 signaling in tumors correlated with high tumor-associated macrophage infiltration and ICB therapy resistance both in murine and human melanoma exhibiting active IFNγ signatures. These results suggest that preventing global macrophage reprogramming induced by MDSC-mediated inhibition of CD40/IL27 signaling provides a precision melanoma immunotherapy strategy, supporting an original and advantageous approach to treat solid tumors within established autoimmune landscapes. SIGNIFICANCE: Myeloid-derived suppressor cells induce macrophage reprogramming by suppressing CD40/IL27 signaling to drive melanoma progression, simultaneously affecting underlying autoimmune disease and facilitating resistance to immunotherapy within preexisting autoimmune landscapes., (©2021 American Association for Cancer Research.)

The NF-κB family of transcriptional activators is responsible for the expression of numerous genes that control key functions such as cell development and survival. Subunit p50 has been studied extensively and is known to include 13 tyrosines, but the extent and pattern of tyrosine phosphorylation that accompanies p50 function has not been defined in the literature, especially at the level of selectivity of gene expression. In this study, phosphorylated tyrosine (pTyr) was site-selectively incorporated into the p50 subunit using an E. coli in vitro expression system containing a modified ribosome. In human T cells, the NF-κBs containing a pTyr at position 60 or 82 of p50 strongly increased the expression of CD40, which is a potential target for cancer or viral immunotherapy. Promoter DNA binding was studied for CD40 promoters, and verified two pTyr residues in NF-κB p50/p65 heterodimers that facilitated this process, and that support the possible importance of phosphorylation stoichiometry. This study defines a new approach for studying tyrosine residues whose phosphorylation alters protein binding to DNA promoters, and contributes to the facility of DNA expression.

Highlights: CD40 expression correlates with the type I anti-tumor response and better survival. Pan-cancer bioinformatics characterization reveals reduced CD40 expression in 11 cancer types, including RAS mut melanoma compared to nevi. RAS mutation correlates with reduced CD40 expression in malignant melanoma. CD40 expression is associated with better response to immune checkpoint blockade therapy in melanoma., (© 2021. The Author(s).)

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19 + target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19 + target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model., Competing Interests: Declaration of interests H.K., research funding from Chugai Pharmaceutical, Kyowa Hakko Kirin, Zenyaku Kogyo, FUJIFILM Corporation, Daiichi Sankyo, Astellas Pharma, Otsuka Pharmaceutical, Nippon Shinyaku, Eisai, Pfizer Japan, Takeda Pharmaceutical, Novartis Pharma K.K., Sumitomo Dainippon Pharma, Sanofi K.K., and Celgene Corporation; consulting fees from Astellas Pharma, Amgen Astellas BioPharma K.K., and Daiichi Sankyo; honoraria from Bristol-Myers Squibb, Astellas Pharma, and Novartis Pharma K.K. M.H., an inventor on patent applications related to CAR-T technologies that have been filed by the Fred Hutchinson Cancer Research Center, Seattle, WA, USA. The other authors declare no competing interests., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Unregulated neuro-inflammation mediates seizures in temporal lobe epilepsy (TLE). Our aim was to determine the effect of CD40-CD40L activation in experimental seizures. CD40 deficient mice (CD40KO) and control mice (wild type, WT) received pentenyltetrazole (PTZ) or pilocarpine to evaluate seizures and status epilepticus (SE) respectively. In mice, anti-CD40L antibody was administered intranasally before PTZ. Brain samples from human TLE and post-seizure mice were processed to determine CD40-CD40L expression using histological and molecular techniques. CD40 expression was higher in hippocampus from human TLE and in cortical neurons and hippocampal neural terminals after experimental seizures. CD40-CD40L levels increased after seizures in the hippocampus and in the cortex. After SE, CD40L/CD40 levels increased in cortex and showed an upward trend in the hippocampus. CD40KO mice demonstrated reduction in seizure severity and in latency compared to WT mice. Anti-CD40L antibody limited seizure susceptibility and seizure severity. CD40L-CD40 interaction can serve as a target for an immuno-therapy for TLE., (© 2021. The Author(s).)