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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.

Objective: To observe the effects and mechanisms of Maresin2 on the function of DCs(Dendritic cells)., Method: The levels of IL-6, IL-12, TNF-α and IL-1β secreted by BMDCs (Bone marrow-derived Dendritic cells) after Maresin2 treatment were detected by ELISA. At the same time, the expressions of costimulatory molecules CD40 and CD86 on the surface, the ability of phagocytosis of ovalbumin(OVA) antigen, and antigen presentation function in BMDCs were analyzed by flow cytometry. Finally, MAPK and NF-κB pathway signaling phosphorylation in Maresin2-treated BMDCs were detected by western blot., Results: The secretion levels of IL-6, IL-12, TNF-α and IL-1β were significantly decreased in the Maresin2 treatment group after LPS treatment (P < 0.05). The expression levels of CD86 and CD40 were significantly decreased after Maresin2 treatment (P < 0.05). Maresin2 enhanced the phagocytosis ability of ovalbumin(OVA) (P < 0.05), but the ability of antigen presentation of BMDCs with the treatment of Maresin2 changed slightly (P > 0.05). Phosphorylation of p38, JNK, p65, ikka/β and ERK peaked at 15 min in the LPS group, while phosphorylation of p-p38 and p-ERK weakened 30 min and 60 min after treatment with Maresin2., Conclusions: Maresin2 inhibits inflammatory cytokine secretion but enhances phagocytosis via the MAPK/NF-κB pathway in BMDCs, which may contribute to negatively regulating inflammation., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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.)

Hypertension causes platelet activation and adhesion in the brain resulting in glial activation and neuroinflammation. Further, activation of Angiotensin-Converting Enzyme 2/Angiotensin (1-7)/Mas Receptor (ACE2/Ang (1-7)/MasR) axis of central Renin-Angiotensin System (RAS), is known to reduce glial activation and neuroinflammation, thereby exhibiting anti-hypertensive and anti-neuroinflammatory properties. Therefore, in the present study, the role of ACE2/Ang (1-7)/MasR axis was studied on platelet-induced glial activation and neuroinflammation using Diminazene Aceturate (DIZE), an ACE2 activator, in astrocytes and microglial cells as well as in rat model of hypertension. We found that the ACE2 activator DIZE, independently of its BP-lowering properties, efficiently prevented hypertension-induced glial activation, neuroinflammation, and platelet CD40-CD40L signaling via upregulation of ACE2/Ang (1-7)/MasR axis. Further, DIZE decreased platelet deposition in the brain by reducing the expression of adhesion molecules on the brain endothelium. Activation of ACE2 also reduced hypertension-induced endothelial dysfunction by increasing eNOS bioavailability. Interestingly, platelets isolated from hypertensive rats or activated with ADP had significantly increased sCD40L levels and induced significantly more glial activation than platelets from DIZE treated group. Therefore, injection of DIZE pre-treated ADP-activated platelets into normotensive rats strongly reduced glial activation compared to ADP-treated platelets. Moreover, CD40L-induced glial activation, CD40 expression, and NFкB-NLRP3 inflammatory signaling are reversed by DIZE. Furthermore, the beneficial effects of ACE2 activation, DIZE was found to be significantly blocked by MLN4760 (ACE2 inhibitor) as well as A779 (MasR antagonist) treatments. Hence, our study demonstrated that ACE2 activation reduced the platelet CD40-CD40L induced glial activation and neuroinflammation, hence imparted neuroprotection., 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 B.V. All rights reserved.)

CD154, also known as CD40 ligand, is a costimulatory molecule involved in humoral and adaptive immune responses upon pairing with its classical receptor, CD40. The CD154/CD40 dyad is a key participant in the pathogenesis of many autoimmune diseases, including systemic lupus erythematosus (SLE). In SLE, the major cells at play, T and B lymphocytes, are shown to overexpress CD154 and CD40, respectively. Subsequently, these cells and other CD40-positive cells engage in numerous effector functions contributing to SLE development. With the recent identification of additional receptors for CD154, all belonging to the integrin family, the role of CD154 in SLE is more complex and calls for deeper investigation into its biological significance. Many therapeutic strategies directed against the CD154/CD40 couple have been deployed for the treatment of SLE and proved efficient in animal models and human studies. However, the incidence of thromboembolic complications in patients treated with these anti-CD154/CD40 antibodies halted their further clinical assessments and called for another class of therapies targeting these molecules. Second-generation antibodies directed against CD154 or CD40 are showing promising results in the advanced stages of clinical testing. Our review presents a thorough description of CD154 and its receptors, CD40 and the integrin family members in SLE pathogenesis. All these elements of the CD154 system represent important therapeutic targets for the treatment of SLE.

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.)

Background: Anti-Cluster of differentiation (CD)-40-induced colitis, driven by innate inflammatory responses in the intestine, is a potent animal model exhibiting IBD pathophysiology including diarrhea. However, the ion transport basis of diarrhea and some key mucosal pathways (Paneth cells, stem cell niche, and mechanosensory) in this model have not been investigated., Methods: Mucosal scrapings and intestinal tissue from control and CD40 antibody (150 µg) treated Rag2-/- mice were examined for gut inflammation, Paneth cell numbers, expression of key transporters, tight/adherens junction proteins, stem cell niche, and mechanosensory pathway via hematoxylin and eosin staining, quantitative polymerase chain reaction, and western blotting., Results: Compared with control, anti-CD40 antibody treatment resulted in a significant loss of body weight (P < .05) and diarrhea at day 3 postinjection. Distal colonic tissues of anti-CD40 mice exhibited increased inflammatory infiltrates, higher claudin-2 expression, and appearance of Paneth cell-like structures indicative of Paneth cell metaplasia. Significantly reduced expression (P < .005) of downregulated in adenoma (key Cl- transporter), P-glycoprotein/multidrug resistantance-1 (MDR1, xenobiotic transporter), and adherens junction protein E-cadherin (~2-fold P < .05) was also observed in the colon of anti-CD40 colitis mice. Interestingly, there were also marked alterations in the stem cell markers and upregulation of the mechanosensory YAP-TAZ pathway, suggesting the activation of alternate regeneration pathway post-tissue injury in this model., Conclusion: Our data demonstrate that the anti-CD40 colitis model shows key features of IBD observed in the human disease, hence making it a suitable model to investigate the pathophysiology of ulcerative colitis (UC)., (Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation 2024.)

The involvement of Group 3 innate lymphoid cells (ILC3s) and dendritic cells (DCs) in chronic lung inflammation has been increasingly regarded as the key to understand the inflammatory mechanisms of smoke-related chronic obstructive pulmonary disease (COPD). However, the mechanism underlying the engagement of both remains unclear. Our study aimed to explore NCR - ILC3 differentiation in the lungs of mice exposed to cigarette smoke (CS) and to further investigate whether DCs activated by CS exposure contribute to the differentiation of ILCs into NCR - ILC3s. The study involved both in vivo and in vitro experiments. In the former, the frequencies of lung NCR - ILC3s and NKp46 - IL-17A + ILCs and the expression of DCs, CD40, CD86, IL-23, and IL-1β quantified by flow cytometry were compared between CS-exposed mice and air-exposed mice. In the latter, NKp46 - IL-17A + ILC frequencies quantified by flow cytometry were compared after two cocultures, one involving lung CD45 + Lin - CD127 + ILCs sorted from air-exposed mice and DCs sifted by CD11c magnetic beads from CS-exposed mice and another including identical CD45 + Lin - CD127 + ILCs and DCs from air-exposed mice. The results indicated significant increases in the frequencies of NCR - ILC3s and NKp46 - IL-17A + ILCs; in the expression of DCs, CD40, CD86, IL-23, and IL-1β in CS-exposed mice; and in the frequency of NKp46 - IL-17A + ILCs after the coculture with DCs from CS-exposed mice. In conclusion, CS exposure increases the frequency of lung ILCs and NCR - ILC3s. CS-induced DC activation enhances the differentiation of ILCs into NCR - ILC3s, which likely acts as a mediating step in the involvement of NCR-ILC3s in chronic lung inflammation.

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.)

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).)

Expression of the transcriptional regulator ZFP318 is induced in germinal center (GC)-exiting memory B cell precursors and memory B cells (MBCs). Using a conditional ZFP318 fluorescence reporter that also enables ablation of ZFP318-expressing cells, we found that ZFP318-expressing MBCs were highly enriched with GC-derived cells. Although ZFP318-expressing MBCs constituted only a minority of the antigen-specific MBC compartment, their ablation severely impaired recall responses. Deletion of Zfp318 did not alter the magnitude of primary responses but markedly reduced MBC participation in recall. CD40 ligation promoted Zfp318 expression, whereas B cell receptor (BCR) signaling was inhibitory. Enforced ZFP318 expression enhanced recall performance of MBCs that otherwise responded poorly. ZFP318-deficient MBCs expressed less mitochondrial genes, had structurally compromised mitochondria, and were susceptible to reactivation-induced cell death. The abundance of ZFP318-expressing MBCs, instead of the number of antigen-specific MBCs, correlated with the potency of prime-boost vaccination. Therefore, ZFP318 controls the MBC recallability and represents a quality checkpoint of humoral immune memory., Competing Interests: Declaration of interests Y.W. and H.Q. are co-founders of Emergent Biomed Solutions, Ltd., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The combination of radiotherapy/chemoradiotherapy and immune checkpoint blockade can result in poor outcomes in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Here, we show that combining ATR inhibition (ATRi) with radiotherapy (RT) increases the frequency of activated NKG2A + PD-1 + T cells in animal models of HNSCC. Compared with the ATRi/RT treatment regimen alone, the addition of simultaneous NKG2A and PD-L1 blockade to ATRi/RT, in the adjuvant, post-radiotherapy setting induces a robust antitumour response driven by higher infiltration and activation of cytotoxic T cells in the tumour microenvironment. The efficacy of this combination relies on CD40/CD40L costimulation and infiltration of activated, proliferating memory CD8 + and CD4 + T cells with persistent or new T cell receptor (TCR) signalling, respectively. We also observe increased richness in the TCR repertoire and emergence of numerous and large TCR clonotypes that cluster based on antigen specificity in response to NKG2A/PD-L1/ATRi/RT. Collectively, our data point towards potential combination approaches for the treatment of HNSCC., (© 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.)

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).)

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.

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.)

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.)

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.)

In situ vaccines (ISVs) utilize the localized delivery of chemotherapeutic agents or radiotherapy to stimulate the release of endogenous antigens from tumors, thereby eliciting systemic and persistent immune activation. Recently, a bioinspired ISV strategy has attracted tremendous attention due to its features such as an immune adjuvant effect and genetic plasticity. M13 bacteriophages are natural nanomaterials with intrinsic immunogenicity, genetic flexibility, and cost-effectiveness for large-scale production, demonstrating the potential for application in cancer vaccines. In this study, we propose an ISV based on the engineered M13 bacteriophage targeting CD40 (M13 CD40 ) for dendritic cell (DC)-targeted immune stimulation, named H-GM-M13 CD40 . We induce immunogenic cell death and release tumor antigens through local delivery of ( S )-10-hydroxycamptothecin (HCPT), followed by intratumoral injection of granulocyte-macrophage colony stimulating factor (GM-CSF) and M13 CD40 to enhance DC recruitment and activation. We demonstrate that this ISV strategy can result in significant accumulation and activation of DCs at the tumor site, reversing the immunosuppressive tumor microenvironment. In addition, H-GM-M13 CD40 can synergize with the PD-1 blockade and induce abscopal effects in cold tumor models. Overall, our study verifies the immunogenicity of the engineered M13 CD40 bacteriophage and provides a proof of concept that the engineered M13 CD40 phage can function as an adjuvant for ISVs.

Tumor molecular data sets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning (ML) to analyze a single-cell, spatial, and highly multiplexed proteomic data set from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcomes. We designed a multiplex immunohistochemistry antibody panel to compare T-cell functionality and spatial localization in resected tumors from treatment-naïve patients with localized pancreatic ductal adenocarcinoma (PDAC) with resected tumors from a second cohort of patients treated with neoadjuvant agonistic CD40 (anti-CD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both cohorts were assayed, and over 1,000 tumor microenvironment (TME) features were quantified. We then trained ML models to accurately predict anti-CD40 treatment status and disease-free survival (DFS) following anti-CD40 therapy based on TME features. Through downstream interpretation of the ML models' predictions, we found anti-CD40 therapy reduced canonical aspects of T-cell exhaustion within the TME, as compared with treatment-naïve TMEs. Using automated clustering approaches, we found improved DFS following anti-CD40 therapy correlated with an increased presence of CD44+CD4+ Th1 cells located specifically within cellular neighborhoods characterized by increased T-cell proliferation, antigen experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of ML in molecular cancer immunology applications, highlight the impact of anti-CD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for anti-CD40-treated patients with PDAC., (©2024 American Association for Cancer Research.)

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.)

The interaction between CD40 and CD40 ligand (CD40L) a crucial co-stimulatory signal for activating adaptive immune cells, has a noteworthy role in atherosclerosis. It is well-known that atherosclerosis is linked to immune inflammation in blood vessels. In atherosclerotic lesions, there is a multitude of proinflammatory cytokines, adhesion molecules, and collagen, as well as smooth muscle cells, macrophages, and T lymphocytes, particularly the binding of CD40 and CD40L. Therefore, research on inhibiting the CD40-CD40L system to prevent atherosclerosis has been ongoing for more than 30 years. However, it's essential to note that long-term direct suppression of CD40 or CD40L could potentially result in immunosuppression, emphasizing the critical role of the CD40-CD40L system in atherosclerosis. Thus, specifically targeting the CD40-CD40L interaction on particular cell types or their downstream signaling pathways may be a robust strategy for mitigating atherosclerosis, reducing potential side effects. This review aims to summarize the potential utility of the CD40-CD40L system as a viable therapeutic target for atherosclerosis., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

Subunit vaccines hold substantial promise in controlling infectious diseases, due to their superior safety profile, specific immunogenicity, simplified manufacturing processes, and well-defined chemical compositions. One of the most important end-targets of vaccines is a subset of lymphocytes originating from the thymus, known as T cells, which possess the ability to mount an antigen-specific immune response. Furthermore, vaccines confer long-term immunity through the generation of memory T cell pools. Dendritic cells are essential for the activation of T cells and the induction of adaptive immunity, making them key for the in vitro evaluation of vaccine efficacy. Upon internalization by dendritic cells, vaccine-bearing antigens are processed, and suitable fragments are presented to T cells by major histocompatibility complex (MHC) molecules. In addition, DCs can secrete various cytokines to crosstalk with T cells to coordinate subsequent immune responses. Here, we generated an in vitro model using the immortalized murine dendritic cell line, DC2.4, to recapitulate the process of antigen uptake and DC maturation, measured as the elevation of CD40, MHC-II, CD80 and CD86 on the cell surface. The levels of key DC cytokines, tumor necrosis alpha (TNF-α) and interleukin-10 (IL-10) were measured to better define DC activation. This information served as a cost-effective and rapid proxy for assessing the antigen presentation efficacy of various vaccine formulations, demonstrating a strong correlation with previously published in vivo study outcomes. Hence, our assay enables the selection of the lead vaccine candidates based on DC activation capacity prior to in vivo animal studies., 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 Lu, Kong, Zhang, Firdaus, Wells, Stephenson, Toth, Skwarczynski and Cruz.)

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.)

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-β (TGF-β) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-β signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-β-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-β-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-β-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

CD40 signaling upregulates BCL-XL and MCL-1 expression in the chronic lymphocytic leukemia (CLL) lymph node microenvironment, affording resistance to the BCL-2 inhibitor, venetoclax. Venetoclax resistance in the therapeutic setting and after long-term laboratory selection has been linked to metabolic alterations, but the underlying mechanism(s) are unknown. We aimed here to discover how CD40 stimulation as a model for tumor microenvironment-mediated metabolic changes, affects venetoclax sensitivity/resistance. CD40 stimulation increased oxidative phosphorylation and glycolysis, but only inhibition of oxidative phosphorylation countered venetoclax resistance. Furthermore, blocking mitochondrial import of pyruvate, glutamine or fatty acids affected CLL metabolism, but did not prevent CD40-mediated resistance to venetoclax. In contrast, inhibition of the electron transport chain (ETC) at complex I, III or V attenuated CLL activation and ATP production, and downregulated MCL-1 and BCL-XL, correlating with reduced CD40 surface expression. Moreover, ETC inhibition equaled mTOR1/2 but not mTOR1 inhibition alone for venetoclax resistance, and all three pathways were linked to control of general protein translation. In line with this, ETC plus mTOR inhibition synergistically counteracted venetoclax resistance. These findings link oxidative CLL metabolism to CD40 expression and cellular signaling, and may hold clinical potential.

Expression of the costimulatory molecule CD40 on both B cells and dendritic cells (DCs) is required for induction of experimental autoimmune encephalomyelitis (EAE), and cell-autonomous CD40 expression on B cells is required for primary T-dependent (TD) Ab responses. We now ask whether the function of CD40 expressed by different cell types in these responses is mediated by the same or different cytoplasmic domains. CD40 has been reported to possess multiple cytoplasmic domains, including distinct TRAF6 and TRAF2/3 binding motifs. To elucidate the in vivo function of these motifs in B cells and DCs involved in EAE and TD germinal center responses, we have generated knock-in mice containing distinct CD40 cytoplasmic domain TRAF-binding site mutations and have used these animals, together with bone marrow chimeric mice, to assess the roles that these motifs play in CD40 function. We found that both TRAF2/3 and TRAF6 motifs of CD40 are critically involved in EAE induction and demonstrated that this is mediated by a role of both motifs for priming of pathogenic T cells by DCs. In contrast, the TRAF2/3 binding motif, but not the TRAF6 binding motif, is required for B cell CD40 function in TD high-affinity Ab responses. These data demonstrate that the requirements for expression of specific TRAF-binding CD40 motifs differ for B cells or DCs that function in specific immune responses and thus identify targets for intervention to modulate these responses., (Copyright © 2023 by The American Association of Immunologists, Inc.)

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., (© 2023 The Authors. Immunology published by John Wiley & Sons Ltd.)

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.)

CD40, a member of the tumor necrosis factor receptor (TNFR) family, is known to be involved in immune system regulation, acting as a costimulatory molecule, and in antitumor responses against cancer cells. It is a protein that is expressed in different types of cells, including immune cells and cancer cells (e.g., cervical cancer, breast cancer, melanoma). In this study, we investigated CD40/CD40L transcriptional and protein levels in cervical cancer cell lines and tumors. Higher CD40 expression was observed in cervical cancer cell lines derived from squamous cell carcinomas than from adenocarcinomas. Search of CD40/CD40L expression in cervical cancer tissues in public data sets revealed that about 83% of squamous cell carcinomas express CD40 compared to other cervical tumor subtypes. Moreover, expression of CD40 and CD40L in squamous cervical carcinomas is associated with better overall survival. Therefore, these proteins could be explored as prognostic markers in cervical cancers.

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.)

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.)

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.

Thyroid-associated ophthalmopathy (TAO) is the most common autoimmune inflammatory diseases of the orbit. The CD40-CD40L pathway has been regarded as a potential molecular mechanism contributing to the development and progression of TAO, and RNA aptamers with specific binding affinity to CD40 (CD40Apt) represents a promising inhibitor of the CD40-CD40L signaling in TAO treatment. In this study, CD40Apt was confirmed to specifically recognize mouse CD40-positive ortibtal fibroblast. Mouse orbital fibroblasts were isolated from TAO mice model orbital tissues and validated. In TGF-β-induced orbital fibroblast activation model in vitro, CD40Apt administration inhibited TGF-β-induced cell viability, decreased TGF-β-induced α-SMA, Collagen I, Timp-1, and vimentin levels, and suppressed TGF-β-induced phosphorylation of Erk, p38, JNK, and NF-κB. In TAO mice model in vivo, CD40Apt caused no significant differences to the body weight of mice; furthermore, CD40Apt improved the eyelid broadening, ameliorated inflammatory infiltration and the hyperplasia in orbital muscle and adipose tissues in model mice. Concerning orbital fibroblast activation, CD40Apt reduced the levels of CD40, collagen I, TGF-β, and α-SMA in orbital muscle and adipose tissues of model mice. Finally, CD40Apt administration significantly suppressed Erk, p38, JNK, and NF-κB phosphorylation. In conclusion, CD40Apt, specifically binds to CD40 proteins in their natural state on the cell surface with high affinity, could suppress mouse orbital fibroblast activation, therefore improving TAO in mice model through the CD40 and downstream signaling pathways. CD40Apt represents a promising antagonist of the CD40-CD40L signaling for TAO treatment., (© 2023. The Author(s).)

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.

Toxoplasma gondii is an intracellular protozoan parasite which can infect most warm-blooded animals and humans. Among the different mouse models, C57BL/6 mice are more susceptible to T. gondii infection compared to BALB/c mice, and this increased susceptibility has been attributed to various factors, including T-cell responses. Dendritic cells (DCs) are the most prominent type of antigen-presenting cells and regulate the host immune response, including the response of T-cells. However, differences in the DC responses of these mouse strains to T. gondii infection have yet to be characterized. In this study, we cultured bone marrow-derived DCs (BMDCs) from BALB/c and C57BL/6 mice. These cells were infected with T. gondii. The activation of the BMDCs was assessed based on the expression of cell surface markers and cytokines. In the BMDCs of both mouse strains, we detected significant increases in the expression of cell surface T-cell co-stimulatory molecules (major histocompatibility complex (MHC) II, CD40, CD80, and CD86) and cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-12p40, IL-1β, and IL-10) from 3 h post-T. gondii infection. The expression of MHC II, CD40, CD80, CD86, IFN-γ, IL-12p40, and IL-1β was significantly higher in the T. gondii-infected BMDCs obtained from the C57BL/6 mice than in those from the BALB/c mice. These findings indicate that differences in the activation status of the BMDCs in the BALB/c and C57BL/6 mice may account for their differential susceptibility to T. gondii.

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.)

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.

The utility of spatial immunobiomarker quantitation in prognostication and therapeutic prediction is actively being investigated in triple-negative breast cancer (TNBC). Here, with high-plex quantitative digital spatial profiling, we map and quantitate intraepithelial and adjacent stromal tumor immune protein microenvironments in systemic treatment-naïve (female only) TNBC to assess the spatial context in immunobiomarker-based prediction of outcome. Immune protein profiles of CD45-rich and CD68-rich stromal microenvironments differ significantly. While they typically mirror adjacent, intraepithelial microenvironments, this is not uniformly true. In two TNBC cohorts, intraepithelial CD40 or HLA-DR enrichment associates with better outcomes, independently of stromal immune protein profiles or stromal TILs and other established prognostic variables. In contrast, intraepithelial or stromal microenvironment enrichment with IDO1 associates with improved survival irrespective of its spatial location. Antigen-presenting and T-cell activation states are inferred from eigenprotein scores. Such scores within the intraepithelial compartment interact with PD-L1 and IDO1 in ways that suggest prognostic and/or therapeutic potential. This characterization of the intrinsic spatial immunobiology of treatment-naïve TNBC highlights the importance of spatial microenvironments for biomarker quantitation to resolve intrinsic prognostic and predictive immune features and ultimately inform therapeutic strategies for clinically actionable immune biomarkers., (© 2023. The Author(s).)

The outcome of patients with acute myeloid leukemia remains poor, and immunotherapy has the potential to improve this. T cells expressing chimeric antigen receptors or bispecific T-cell engagers targeting CD123 are actively being explored in preclinical and/or early phase clinical studies. We have shown that T cells expressing CD123-specific bispecific T-cell engagers (CD123.ENG T cells) have anti-acute myeloid leukemia activity. However, like chimeric antigen receptor T cells, their effector function diminishes rapidly once they are repeatedly exposed to antigen-positive target cells. Here we sought to improve the effector function of CD123.ENG T cells by expressing inducible co-stimulatory molecules consisting of MyD88 and CD40 (iMC), MyD88 (iM), or CD40 (iC), which are activated by a chemical inducer of dimerization. CD123.ENG T cells expressing iMC, iM, or iC maintained their antigen specificity in the presence of a chemical inducer of dimerization, as judged by cytokine production (interferon-γ, interleukin-2) and their cytolytic activity. In repeat stimulation assays, activating iMC and iM, in contrast to iC, enabled CD123.ENG T cells to secrete cytokines, expand, and kill CD123-positive target cells repeatedly. Activating iMC in CD123.ENG T cells consistently improved antitumor activity in an acute myeloid leukemia xenograft model. This translated into a significant survival advantage in comparison to that of mice that received CD123.ENG or CD123.ENG.iC T cells. In contrast, activation of only iM in CD123.ENG T cells resulted in donor-dependent antitumor activity. Our work highlights the need for both toll-like receptor pathway activation via MyD88 and provision of co-stimulation via CD40 to consistently enhance the antitumor activity of CD123.ENG T cells.