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Abstract Colorectal cancer (CRC) is the second common cause of cancer mortality worldwide, and it still lacks effective approaches for relapsed and metastatic CRC. Recently, oncolytic virus has been emerged as a promising immune therapeutic strategy. In this study, we develop a novel oncolytic adenovirus, rAd.mDCN.mCD40L, which drive oncolytic activity by telomerase reverse transcriptase promoter (TERTp). rAd.mDCN.mCD40L expressed both mouse genes of decorin (mDCN) and CD40 ligand (mCD40L), and produced effective cytotoxicity in both human and mouse CRC cells. Moreover, oncolytic adenovirus mediated mDCN over-expression inhibited Met expression in vitro. In CT26 subcutaneous tumor model, intratumorally delivery of oncolytic adenoviruses could inhibit tumor growth and liver metastasis, while mDCN and/or mCD40L armed oncolytic adenoviruses produced much more impressive responses. No obvious toxicity was detected in lung, liver and spleen. Moreover, mDCN and/or mCD40L armed oncolytic adenoviruses altered the immune state to activate anti-tumor responses, including increasing CD8+ T effector cells and CD4+ memory T cells, reducing MDSCs and Tregs in peripheral blood. Furthermore, mDCN and/or mCD40L armed oncolytic adenoviruses mediated mDCN and/or mCD40L expression in tumors, and up-regulated Th1 cytokines and reduced Th2 cytokines in tumors, which will be benefit for remodeling tumor microenvironment. Importantly, rAd.mDCN.mCD40L and rAd.mCD40L prevented tumor liver metastasis much more effectively than rAd.Null and rAd.mDCN. Therefore, rAd.mDCN.mCD40L and rAd.mCD40L are promising approaches for CRC therapy.

Angiotensin II (AngII) receptor subtype 1 (AT1R) is involved in the pathogenesis of preeclampsia (PE). Angiotensin II receptor subtype 2 (AT2R) can antagonize the effects of AT1R, but its effects during pregnancy are not known. We investigated the effect of AT2R on the pathogenesis of PE using a mouse model and recently developed AT2R agonist (compound 21 [C21]). Blastocysts collected from pregnant imprinting control region (ICR) mice were incubated with adenovirus containing the CD40L gene and transferred into the uterine horns of pseudo-pregnant ICR mice to express PE-like features. Osmotic pumps were placed subcutaneously on the dorsal side with C21 or saline. C21 reduced the plasma soluble fms-like tyrosine kinase 1 (sFlt-1) concentration, ameliorating hypertension. The splenic T and B cell profiles in model mice were analyzed by flow cytometry. The gated percentage of IFN-γ-positive Th cells was significantly increased and the percentage of plasma cells in B cells was significantly decreased; however, the percentages were not altered by C21. sFlt-1 and soluble endoglin concentrations in plasma were measured with an enzyme-linked immunosorbent assay, and sFlt-1 was reduced. C21 could become a candidate PE drug as it ameliorated the pathophysiology of PE as a result of decreased production of sFlt-1.

Rheumatoid arthritis (RA) is a systemic autoimmune disease driven by highly active autoantibody‐producing B cells. Activation of B cells is maintained within ectopic germinal centres found in affected joints. Fibroblast‐like synoviocytes (FLS) present in inflamed joints support B‐cell survival, activation, and differentiation. CD27+ memory B cells and naive B cells show very different responses to activation, particularly by CD40 ligand (CD40L). We show that FLS‐dependent activation of human B cells is dependent on interleukin‐6 (IL‐6) and CD40L. FLS have been shown to activate both naive and memory B cells. Whether the activating potential of FLS is different for naive and memory B cells has not been investigated. Our results suggest that FLS‐induced activation of B cells is dependent on IL‐6 and CD40L. While FLS are able to induce plasma cell differentiation, isotype switching, and antibody production in memory B cells, the ability of FLS to activate naive B cells is significantly lower. [ABSTRACT FROM AUTHOR]

The Cd40l –/– mouse is a well-established model of X-linked hyper-immunoglobulin M (IgM) syndrome, an immunodeficiency disorder of human beings characterized by the lack of expression of the CD40 ligand (CD40L) on activated T-cells, predisposing to infections with opportunistic pathogens like Pneumocystis jirovecii. The aim of our study was to describe the pulmonary lesions in Cd40l –/– mice experimentally infected with Pneumocystis murina, in comparison with naturally infected severe combined immunodeficient (SCID) mice. Formalin-fixed paraffin-embedded lungs from 26 Cd40l –/–, 11 SCID, and 5 uninfected Cd40l –/– mice were examined by histology and immunohistochemistry for the presence of the pathogen and for leukocyte populations (CD3, CD4, CD45R/B220, CD8a, Iba-1, Ly-6G, CD206, MHC II, and NKp46/NCR1). Infection was confirmed by immunohistochemistry in 18/26 (69%) Cd40l –/– mice and in 11/11 (100%) SCID mice. Fourteen out of 26 (54%) Cd40l –/– mice had interstitial pneumonia. Twenty-three out of 26 (88%) Cd40l –/– mice had peribronchiolar/perivascular lymphoplasmacytic infiltrates, rich in B-cells and Mott cells. Acidophilic macrophage pneumonia was additionally found in 20/26 (77%) Cd40l –/– mice. Only 4/11 (36%) SCID mice had interstitial pneumonia, but no peribronchiolar/perivascular infiltrates or acidophilic macrophage pneumonia were observed in this strain. This study represents the first description of pulmonary histopathological lesions in Cd40l –/– mice infected with P. murina. We speculate that the singular characteristics of the inflammatory infiltrates observed in Cd40l –/– mice could be explained by the specific immune phenotype of the model. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the second common cause of cancer mortality worldwide, and it still lacks effective approaches for relapsed and metastatic CRC. Recently, oncolytic virus has been emerged as a promising immune therapeutic strategy. In this study, we develop a novel oncolytic adenovirus, rAd.mDCN.mCD40L, which drive oncolytic activity by telomerase reverse transcriptase promoter (TERTp). rAd.mDCN.mCD40L expressed both mouse genes of decorin (mDCN) and CD40 ligand (mCD40L), and produced effective cytotoxicity in both human and mouse CRC cells. Moreover, oncolytic adenovirus mediated mDCN over-expression inhibited Met expression in vitro. In CT26 subcutaneous tumor model, intratumorally delivery of oncolytic adenoviruses could inhibit tumor growth and liver metastasis, while mDCN and/or mCD40L armed oncolytic adenoviruses produced much more impressive responses. No obvious toxicity was detected in lung, liver and spleen. Moreover, mDCN and/or mCD40L armed oncolytic adenoviruses altered the immune state to activate anti-tumor responses, including increasing CD8+ T effector cells and CD4+ memory T cells, reducing MDSCs and Tregs in peripheral blood. Furthermore, mDCN and/or mCD40L armed oncolytic adenoviruses mediated mDCN and/or mCD40L expression in tumors, and up-regulated Th1 cytokines and reduced Th2 cytokines in tumors, which will be benefit for remodeling tumor microenvironment. Importantly, rAd.mDCN.mCD40L and rAd.mCD40L prevented tumor liver metastasis much more effectively than rAd.Null and rAd.mDCN. Therefore, rAd.mDCN.mCD40L and rAd.mCD40L are promising approaches for CRC therapy. [ABSTRACT FROM AUTHOR]

Optical tweezers have profound importance across fields ranging from manufacturing to biotechnology. However, the requirement of refractive index contrast and high laser power results in potential photon and thermal damage to the trapped objects, such as nanoparticles and biological cells. Optothermal tweezers have been developed to trap particles and biological cells via opto-thermophoresis with much lower laser powers. However, the intense laser heating and stringent requirement of the solution environment prevent their use for general biological applications. Here, we propose hypothermal opto-thermophoretic tweezers (HOTTs) to achieve low-power trapping of diverse colloids and biological cells in their native fluids. HOTTs exploit an environmental cooling strategy to simultaneously enhance the thermophoretic trapping force at sub-ambient temperatures and suppress the thermal damage to target objects. We further apply HOTTs to demonstrate the three-dimensional manipulation of functional plasmonic vesicles for controlled cargo delivery. With their noninvasiveness and versatile capabilities, HOTTs present a promising tool for fundamental studies and practical applications in materials science and biotechnology.

PURPOSE: Graves orbitopathy (GO) is an orbital manifestation of autoimmune Graves disease, and orbital fibroblast is considered a target cell, producing pro-inflammatory cytokines and/or differentiating into adipocytes. Adipose tissue has been focused on as an endocrine and inflammatory organ secreting adipokines. We investigated the pathogenic role of a specific adipokine, adipsin, known as complement factor D in Graves orbital fibroblasts. METHODS: The messenger RNA (mRNA) expression of multiple adipokines was investigated in adipose tissues harvested from GO and healthy subjects. Adipsin protein production was analyzed in primary cultured orbital fibroblasts under insulin growth factor (IGF)-1, CD40 ligand (CD40L) stimulation, and adipogenesis. The effect of blocking adipsin with small interfering RNA (siRNA) on pro-inflammatory cytokine production and adipogenesis was evaluated using quantitative real-time PCR, Western blot, and ELISA. Adipogenic differentiation was identified using Oil Red O staining. RESULTS: Adipsin gene expression was significantly elevated in GO tissue and increased after the stimulation of IGF-1 and CD40L, as well as adipocyte differentiation in GO cells. Silencing of adipsin suppressed IGF-1-induced IL-6, IL-8, COX2, ICAM-1, CCL2 gene expression, and IL-6 protein secretion. Adipsin suppression also attenuated adipocyte differentiation. Exogenous treatment of recombinant adipsin resulted in the activation of the Akt, ERK, p-38, and JNK signaling pathways. CONCLUSIONS: Adipsin, secreted by orbital fibroblasts, may play a distinct role in the pathogenesis of GO. Inhibition of adipsin ameliorated the production of pro-inflammatory cytokines and adipogenesis in orbital fibroblasts. Our study provides an in vitro basis suggesting adipsin as a potential therapeutic target for GO treatment.

Cancer remains a significant challenge in medicine due to its complexity and heterogeneity. Biomarkers have emerged as vital tools for cancer research and clinical practice, facilitating early detection, prognosis assessment, and treatment monitoring. Among these, CD40 ligand (CD40L) has gained attention for its role in immune response modulation. Soluble CD40 ligand (sCD40L) has shown promise as a potential biomarker in cancer diagnosis and progression, reflecting interactions between immune cells and the tumor microenvironment. This review explores the intricate relationship between sCD40L and cancer, highlighting its diagnostic and prognostic potential. It discusses biomarker discovery, emphasizing the need for reliable markers in oncology, and elucidates the roles of CD40L in inflammatory responses and interactions with tumor cells. Additionally, it examines sCD40L as a biomarker, detailing its significance across various cancer types and clinical applications. Moreover, the review focuses on therapeutic interventions targeting CD40L in malignancies, providing insights into cellular and gene therapy approaches and recombinant protein-based strategies. The clinical effectiveness of CD40L-targeted therapy is evaluated, underscoring the need for further research to unlock the full potential of this signaling pathway in cancer management. [ABSTRACT FROM AUTHOR]

During coronary artery bypass grafting (CABG), the surgical procedure, particularly the manipulation of the major arteries of the heart, induces a significant inflammatory state that may compromise platelet function to the extent that platelet transfusion is required. Given stored platelets as a major source of biological mediators, this study investigates the effects of platelet transfusion on the major pro‐aggregatory, pro‐inflammatory and immunomodulatory markers of platelets. Platelets from 20 patients, 10 who received platelet transfusion and 10 without, were subjected to flow cytometery where P‐selectin and CD40 ligand (CD40L) expressions and PAC‐1 binding (activation‐specific anti GPIIb/GPIIIa antibody) analysed at five‐time points of 24 h before surgery, immediately, 2 h, 24 h and 1 week after surgery. Analysis of intra‐platelet transforming growth factor‐beta‐1 (TGF‐β1) was also conducted using western blotting. Patients with platelet transfusion showed increased levels of P‐selectin, CD40L and intra‐platelet TGF‐β1 2‐h after surgery compared to those without transfusion (p < 0.05). PAC‐1 binding was increased 24 h after surgery in transfused patients (p < 0.05). Given the significant post‐transfusion elevation of platelet TGF‐β1, P‐sel/CD40L reduction in transfused patients a week after was of much interest. This study showed for the first time the significant effects of platelet transfusion on the pro‐inflammatory, pro‐aggeregatory and immunomodulatory state of platelets in CABG patients, which manifested with immediate, midterm and delayed consequences. While the increased pro‐inflammatory conditions manifested as an immediate effect of platelet transfusion, the pro‐aggregatory circumstances emerged 24 h post‐transfusion. A week after surgery, attenuations of pro‐inflammatory markers of platelets in transfused patients were shown, which might be due to the immunomodulatory effects of TGF‐β1. [ABSTRACT FROM AUTHOR]

Follicular CD8+CXCR5+ T cells are a specialized CD8+ T cell subset with unique follicular-homing capabilities that have been reported to display effector functions in viral immunity, tumor immunity, and autoimmunity. CD8+CXCR5+ T cells exhibit B cell helper functions and express CD40L, ICOS, programmed cell death protein 1 (PD-1), and BCL-6, the transcriptional regulator of CD4+CXCR5+ T follicular helper cells and of germinal center B cells. HIV is known to be sequestered in lymphoid follicles, and CD8+CXCR5+ T cell frequency is a marker for disease severity, given that HIV-infected patients with lower numbers of circulating CD8+CXCR5+ T cells display lower CD4+ T cell counts. Likewise, several groups have reported a direct correlation between the quantity of CD8+CXCR5+ T cells and suppression of HIV viral load. In this study, we observed elevated absolute numbers of CD8+CXCR5+ and CD8+CXCR5+BCL-6+PD-1+ T cells in the blood of HIV-infected participants of the Multicenter AIDS Cohort Study. We further demonstrated in vitro that activated human CD8+CXCR5+ T cells isolated from peripheral blood and tonsil from healthy donors show increased CD40L expression and induce the production of PD ligand 1 (PD-L1)+IgG+ B cells. Moreover, absolute numbers of CD8+CXCR5+ T cells significantly and positively correlated with numbers of PD-L1+ B cells found in blood of HIV-infected individuals. Altogether, these results show that activated CD8+CXCR5+ T cells have the ability to activate B cells and increase the percentage of PD-L1+ and PD-L1+IgG+ B cells, which provides insights into the early events of B cell activation and differentiation and may play a role in disease progression and lymphomagenesis in HIV-infected individuals.

CD40L is expressed in activated T cells, and it plays a major role in immune response and is a major therapeutic target for inflammation. High IgM syndrome type 1 (HIGM1) is a congenital functional defect in CD40L/CD40 signaling due to defective CD40L. CD40L is also stored in platelet granules and transported to the surface upon platelet activation. Platelet integrin αIIbβ3 is known to bind to fibrinogen and activation of αIIbβ3 is a key event that triggers platelet aggregation. Also, the KGD motif is critical for αIIbβ3 binding and the interaction stabilizes thrombus. Previous studies showed that CD40L binds to and activates integrins αvβ3 and α5β1 and that HIGM1 mutations are clustered in the integrin-binding sites. However, the specifics of CD40L binding to αIIbβ3 were unclear. Here, we show that CD40L binds to αIIbβ3 in a KGD-independent manner using CD40L that lacks the KGD motif. Two HIGM1 mutants, S128E/E129G and L155P, reduced the binding of CD40L to the classical ligand-binding site (site 1) of αIIbβ3, indicating that αIIbβ3 binds to the outer surface of CD40L trimer. Also, CD40L bound to the allosteric site (site 2) of αIIbβ3 and allosterically activated αIIbβ3 without inside-out signaling. Two HIMG1 mutants, K143T and G144E, on the surface of trimeric CD40L suppressed CD40L-induced αIIbβ3 activation. These findings suggest that CD40L binds to αIIbβ3 in a manner different from that of αvβ3 and α5β1 and induces αIIbβ3 activation. HIGM1 mutations are clustered in αIIbβ3 binding sites in CD40L and are predicted to suppress thrombus formation and immune responses through αIIbβ3.

Platelets have a wide range of functions including critical roles in hemostasis, thrombosis, and immunity. We hypothesized that during acute inflammation, such as in life-threatening sepsis, there are fundamental changes in the sites of platelet production and phenotypes of resultant platelets. Here, we showed during sepsis that the spleen was a major site of megakaryopoiesis and platelet production. Sepsis provoked an adrenergic-dependent mobilization of megakaryocyte-erythrocyte progenitors (MEPs) from the bone marrow to the spleen, where IL-3 induced their differentiation into megakaryocytes (MKs). In the spleen, immune-skewed MKs produced a CD40 ligandhi platelet population with potent immunomodulatory functions. Transfusions of post-sepsis platelets enriched from splenic production enhanced immune responses and reduced overall mortality in sepsis-challenged animals. These findings identify a spleen-derived protective platelet population that may be broadly immunomodulatory in acute inflammatory states such as sepsis.

Emerging evidence shows that tumor cells secrete extracellular vesicles (EVs) that carry bioactive cell surface markers, such as programmed death-ligand 1 (PD-L1), which can modulate immune responses and inhibit anti-tumor responses, potentially playing a role in lymphomagenesis and in promoting the growth of these cancers. In this study, we investigated the role of EVs expressing cell surface molecules associated with B cell activation and immune regulation. We measured levels of EVs derived from plasma from 57 subjects with AIDS-related non-Hodgkin lymphoma (AIDS-NHL) enrolled in the AIDS Malignancies Consortium (AMC) 034 clinical trial at baseline and post-treatment with rituximab plus concurrent infusional EPOCH chemotherapy. We found that plasma levels of EVs expressing PD-L1, CD40, CD40L or TNF-RII were significantly reduced after cancer treatment. AIDS-NHL patients with the diffuse large B cell lymphoma (DLBCL) tumor subtype had decreased plasma levels of EVs bearing PD-L1, compared to those with Burkitt's lymphoma. CD40, CD40L and TNF-RII-expressing EVs showed a significant positive correlation with plasma levels of IL-10, CXCL13, sCD25, sTNF-RII and IL-18. Our results suggest that patients with AIDS-NHL have higher levels of EVs expressing PD-L1, CD40, CD40L or TNF-RII in circulation before cancer treatment and that levels of these EVs are associated with levels of biomarkers of microbial translocation and inflammation.

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

Objective：To discuss the effect of CD40 ligand (CD40L) on the biological behavior of the human monocytic leukemia THP-1 cells through long non-coding RNA（lncRNA) linc00239, and to clarify its potential mechanism. Methods：The linc00239 over-expression vector (pcDNA-linc00239) and interference vector (sh-linc00239) were constructed and transfected into the THP-1 cells. Real-time fluorescence quantitative PCR (RT-qPCR) method was used to detect the transfection efficiency. The THP-1 cells were divided into control group, vector group, pcDNA-linc00239 group, sh-linc00239 group, vector+CD40L group, pcDNA-linc00239+CD40L group, and sh-linc00239+CD40L group. RT-qPCR method was used to detect the expression levels of linc00239 in the cells in various groups； CCK-8 assay was used to detect the proliferation activities of the cells in various groups；flow cytometry was used to detect the percentages of the cells at different cell cycles and the apoptotic rates of the cells in various groups；RT-qPCR and Western blotting methods were used to to detect the expression levels of B-cell lymphoma-2（Bcl-2) and Bcl-2-associated X protein (Bax) mRNA and proteins in the cells in various groups； Western blotting method was used to detect the expression levels of protein kinase B (AKT) and phosphorylated AKT (p-AKT) proteins in the cells in various groups, and the ratio of p-AKT/AKT was calculated. Results：Compared with vector group, the proliferation activity of the cells and the percentage of the cells at G2 phase in pcDNA-linc00239 group were significantly increased (P<0. 05 or P<0. 01), the expression levels of linc00239, Bcl-2 mRNA and protein, and the ratio of p-AKT/AKT were significantly increased (P<0. 05 or P<0. 01), the percentage of the cells at G1 phase, apoptotic rate, and expression levels of Bax mRNA and protein in the cells were significantly decreased (P<0. 05) ； compared with vector group, the proliferation activity of the cells and percentage of the cells at G2 phase, expression levels of linc00239, Bcl-2 mRNA and protein, and ratio of p-AKT/AKT in the cells in sh-linc00239 group and vector+CD40L group were significantly decreased (P<0. 05 or P<0. 01), while the percentage of the cells at G1 phase, apoptotic rate, and the expression levels of Bax mRNA and protein in the cells were significantly increased (P<0. 05 or P<0. 01) ；compared with pcDNA-linc00239 group, the proliferation activity of the cells and percentage of cells at G2 phase in pcDNA-linc00239+CD40L group were significantly decreased (P<0. 05 or P<0. 01), the expression levels of linc00239, Bcl-2 mRNA and protein, and ratio of p-AKT/AKT were significantly decreased (P<0. 05 or P<0. 01), while the percentage of cells at G1 phase, apoptotic rate, and the expression levels of Bax mRNA and protein were significantly increased (P<0. 05 or P<0. 01) ；compared with sh-linc00239 group, the proliferation activity of the cells and percentage of cells at G2 phase in sh-linc00239+CD40L group were significantly decreased (P<0. 05 or P<0. 01), the expression levels of linc00239, Bcl-2 mRNA and protein, and ratio of p-AKT/ AKT were significantly decreased (P<0. 05 or P<0. 01), and the percentage of the cells at G1 phase, apoptotic rate, and expression levels of Bax mRNA and protein were significantly increased (P<0. 05 or P<0. 01) . Conclusion：CD40L can inhibit the proliferation and cell cycle progression of the THP-1 cells through linc00239 and induce the apoptosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvβ3 and α5β1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4β1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvβ3, α5β1, and α4β1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.

The tumor microenvironment (TME) strongly affects the clinical outcomes of immunotherapy. This study aimed to activate the antitumor immune response by manipulating the TME by transfecting genes encoding relevant cytokines into tumor cells using a synthetic vehicle, which is designed to target tumor cells and promote the expression of transfected genes. Lung tumors were formed by injecting CT26.WT intravenously into BALB/c mice. Upon intravenous injection of the green fluorescent protein‐coding plasmid encapsulated in the vehicle, 14.2% tumor‐specific expression was observed. Transfection of the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and CD40 ligand (L)‐plasmid combination and interferon gamma (IFNγ) and CD40L‐plasmid combination showed 45.5% and 54.5% complete remission (CR), respectively, on day 60; alternate treatments with both the plasmid combinations elicited 66.7% CR, while the control animals died within 48 days. Immune status analysis revealed that the density of dendritic cells significantly increased in tumors, particularly after GM‐CSF‐ and CD40L‐gene transfection, while that of regulatory T cells significantly decreased. The proportion of activated killer cells and antitumoral macrophages significantly increased, specifically after IFNγ and CD40L transfection. Furthermore, the level of the immune escape molecule programmed death ligand‐1 decreased in tumors after transfecting these cytokine genes. As a result, tumor cell‐specific transfection of these cytokine genes by the synthetic vehicle significantly promotes antitumor immune responses in the TME, a key aim for visceral tumor therapy. [ABSTRACT FROM AUTHOR]

We aimed to investigate the role of the CD40-CD40 ligand (CD40L) pathway in inflammation-mediated angiogenesis in proliferative diabetic retinopathy (PDR). We analyzed vitreous fluids and epiretinal fibrovascular membranes from PDR and nondiabetic patients, cultures of human retinal microvascular endothelial cells (HRMECs) and Müller glial cells and rat retinas with ELISA, immunohistochemistry, flow cytometry and Western blot analysis. Functional tests included measurement of blood–retinal barrier breakdown, in vitro angiogenesis and assessment of monocyte-HRMEC adherence. CD40L and CD40 levels were significantly increased in PDR vitreous samples. We demonstrated CD40L and CD40 expression in vascular endothelial cells, leukocytes and myofibroblasts in epiretinal membranes. Intravitreal administration of soluble (s)CD40L in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, VEGF, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). sCD40L induced upregulation of VEGF, MMP-9, MCP-1 and HMGB1 in cultured Müller cells and phospo-ERK1/2, p65 subunit of NF-ĸB, VCAM-1 and VEGF in cultured HRMECS. TNF-α induced significant upregulation of CD40 in HRMECs and Müller cells and VEGF induced significant upregulation of CD40 in HRMECs. sCD40L induced proliferation and migration of HRMECs. We provide experimental evidence supporting the involvement of the CD40L-CD40 pathway and how it regulates inflammatory angiogenesis in PDR. [ABSTRACT FROM AUTHOR]

Although elevated serum levels of soluble CD40 ligand (sCD40L) were reported in patients with cancer, the importance of high sCD40L levels in clinical oncology remains unknown. We conducted a post hoc analysis of the AMATERASU randomized clinical trial of vitamin D3 supplementation (2000 IU/day) in patients with digestive tract cancer to assess its significance. Serum sCD40L levels were measured by ELISA in 294 residual samples, and were divided into tertiles. In patients with colorectal cancer (CRC), 5-year relapse-free survival (RFS) rates in the middle and highest tertiles were 61.6% and 61.2%, respectively, which was significantly lower than 83.8% in the lowest tertile. A Cox proportional hazard analysis showed that the lowest tertile had a significantly lower risk of relapse or death than the highest tertile even with multivariate adjustment (hazard ratio (HR), 0.30; 95% confidence interval (CI), 0.11–0.80; p = 0.016). In the subgroup of CRC patients with the highest tertile of sCD40L, the 5-year RFS rate in the vitamin D group was 77.9%, which was significantly higher than 33.2% in the placebo group (HR, 0.30; 95% CI, 0.11–0.81; p = 0.018 [Pinteraction = 0.04]). In conclusion, elevated sCD40L might be a biomarker of poor prognosis in patients with CRC, but vitamin D supplementation might improve RFS in patients with high sCD40L. [ABSTRACT FROM AUTHOR]

Summary: Mucosa-associated invariant T (MAIT) cells are evolutionarily conserved, innate-like T lymphocytes with enormous immunomodulatory potentials. Due to their strategic localization, their invariant T cell receptor (iTCR) specificity for major histocompatibility complex-related protein 1 (MR1) ligands of commensal and pathogenic bacterial origin, and their sensitivity to infection-elicited cytokines, MAIT cells are best known for their antimicrobial characteristics. However, they are thought to also play important parts in the contexts of cancer, autoimmunity, vaccine-induced immunity, and tissue repair. While cognate MR1 ligands and cytokine cues govern MAIT cell maturation, polarization, and peripheral activation, other signal transduction pathways, including those mediated by costimulatory interactions, regulate MAIT cell responses. Activated MAIT cells exhibit cytolytic activities and secrete potent inflammatory cytokines of their own, thus transregulating the biological behaviors of several other cell types, including dendritic cells, macrophages, natural killer cells, conventional T cells, and B cells, with significant implications in health and disease. Therefore, an in-depth understanding of how costimulatory pathways control MAIT cell responses may introduce new targets for optimized MR1/MAIT cell-based interventions. Herein, we compare and contrast MAIT cells and mainstream T cells for their expression of classic costimulatory molecules belonging to the immunoglobulin superfamily and the tumor necrosis factor (TNF)/TNF receptor superfamily, based not only on the available literature but also on our transcriptomic analyses. We discuss how these molecules participate in MAIT cells' development and activities. Finally, we introduce several pressing questions vis-à-vis MAIT cell costimulation and offer new directions for future research in this area. The CD40L-CD40 costimulatory axis modulates mucosa-associated invariant T (MAIT) cells' functions and mediates their bidirectional crosstalk with other important immunocytes. Activated MAIT cells rely on this pathway to promote dendritic cell maturation, resulting in type-I cytokine production as well as follicular helper T cell expansion in lymph nodes. In addition, CD40L signaling in MAIT cells enhances B cell activation and antibody responses. [ABSTRACT FROM AUTHOR]

Simple Summary: The tight regulation of the expression of cytokines, surface receptors and co-stimulatory and co-inhibitory molecules is necessary for a well-functioning immune system. There are various cellular processes that regulate the expression of these immune regulatory proteins, for instance at the RNA transcript level. Epitranscriptomic regulation, involving RNA binding proteins and modifications, can determine the turnover and translation of mRNA transcripts. N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic cells and it was demonstrated that m6A is involved in the early differentiation of CD4+ T lymphocytes. However, the function of m6A in CD4+ T cell activation and function is still incompletely understood. Here, we demonstrate that m6A regulates the activation of CD4+ T lymphocytes via the regulation of CD40 ligand expression, a key co-stimulatory molecule expressed on the cell surface of CD4+ T cells. The discovery of this novel function of m6A and its regulatory proteins contributes to our general understanding of CD4+ T cell activation, gene expression regulation and autoimmune disease pathogenesis. T cell activation is a highly regulated process, modulated via the expression of various immune regulatory proteins including cytokines, surface receptors and co-stimulatory proteins. N6-methyladenosine (m6A) is an RNA modification that can directly regulate RNA expression levels and it is associated with various biological processes. However, the function of m6A in T cell activation remains incompletely understood. We identify m6A as a novel regulator of the expression of the CD40 ligand (CD40L) in human CD4+ lymphocytes. Manipulation of the m6A 'eraser' fat mass and obesity-associated protein (FTO) and m6A 'writer' protein methyltransferase-like 3 (METTL3) directly affects the expression of CD40L. The m6A 'reader' protein YT521-B homology domain family-2 (YTHDF2) is hypothesized to be able to recognize and bind m6A specific sequences on the CD40L mRNA and promotes its degradation. This study demonstrates that CD40L expression in human primary CD4+ T lymphocytes is regulated via m6A modifications, elucidating a new regulatory mechanism in CD4+ T cell activation that could possibly be leveraged in the future to modulate T cell responses in patients with immune-related diseases. [ABSTRACT FROM AUTHOR]

Immunization with recombinant ALVAC/gp120 alum vaccine provided modest protection from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) acquisition in humans and macaques. Vaccine-mediated protection was associated with the elicitation of IgG against the envelope V2 loop and of envelope-specific CD4+ T cell responses. We hypothesized that the simultaneous expression of the costimulatory molecule CD40L (CD154) by the ALVAC-HIV vector could increase both protective humoral and cellular responses. We engineered an ALVAC-SIV coexpressing CD40L with SIVmac251 (ALVAC-SIV/CD40L) gag, pol, and env genes. We compared its immunogenicity in macaques with that of a canonical ALVAC-SIV, with both given as a vector-prime/gp120 in alum boost strategy. The ALVAC-SIV/CD40L was superior to the ALVAC-SIV regimen in inducing binding and tier 1 neutralizing antibodies against the gp120. The increase in humoral responses was associated with the expression of the membrane-bound form of the CD40L by CD4+ T cells in lymph nodes. Unexpectedly, the ALVAC-SIV/CD40L vector had a blunting effect on CD4+ Th1 helper responses and instead favored the induction of myeloid-derived suppressor cells, the immune-suppressive interleukin-10 (IL-10) cytokine, and the down-modulatory tryptophan catabolism. Ultimately, this strategy failed to protect macaques from SIV acquisition. Taken together, these results underlie the importance of balanced vaccine-induced activating versus suppressive immune responses in affording protection from HIV.IMPORTANCE CD40-CD40 ligand (CD40L) interaction is crucial for inducing effective cytotoxic and humoral responses against pathogens. Because of its immunomodulatory function, CD40L has been used to enhance immune responses to vaccines, including candidate vaccines for HIV. The only successful vaccine ever tested in humans utilized a strategy combining canarypox virus-based vector (ALVAC) together with an envelope protein (gp120) adjuvanted in alum. This strategy showed limited efficacy in preventing HIV-1/SIV acquisition in humans and macaques. In both species, protection was associated with vaccine-induced antibodies against the HIV envelope and CD4+ T cell responses, including type 1 antiviral responses. In this study, we tested whether augmenting CD40L expression by coexpressing it with the ALVAC vector could increase the protective immune responses. Although coexpression of CD40L did increase humoral responses, it blunted type 1 CD4+ T cell responses against the SIV envelope protein and failed to protect macaques from viral infection.

While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3-/- mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b-CD103- double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses.

HIV-1-infected individuals are treated with lifelong antiretroviral drugs to control the infection. A means to strengthen the antiviral T cell response might allow them to control viral loads without antiretroviral drugs. We report the development of a lentiviral vector-based dendritic cell (DC) vaccine in which HIV-1 antigen is co-expressed with CD40 ligand (CD40L) and a soluble, high-affinity programmed cell death 1 (PD-1) dimer. CD40L activates the DCs, whereas PD-1 binds programmed death ligand 1 (PD-L1) to prevent checkpoint activation and strengthen the cytotoxic T lymphocyte (CTL) response. The injection of humanized mice with DCs transduced with vector expressing CD40L and the HIV-1 SL9 epitope induced antigen-specific T cell proliferation and memory differentiation. Upon HIV-1 challenge of vaccinated mice, viral load was suppressed by 2 logs for 6 weeks. Introduction of the soluble PD-1 dimer into a vector that expressed full-length HIV-1 proteins accelerated the antiviral response. The results support development of this approach as a therapeutic vaccine that might allow HIV-1-infected individuals to control virus replication without antiretroviral therapy.

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Systemic lupus erythematosus (SLE) is a systemic, autoimmune disease characterized by chronic inflammation and organ damage. Dapirolizumab pegol inhibits CD40 ligand (CD40L) and is currently undergoing phase 3 trials for the treatment of SLE. To describe the pharmacokinetic characteristics of dapirolizumab pegol and the relationship between exposure and probability of achieving a British Isles Lupus Assessment Group‐based Composite Lupus Assessment (BICLA) response, a population pharmacokinetic (popPK) model and an exposure‐response model were developed, based on results of the phase 2b trial (RISE; NCT02804763) of dapirolizumab pegol in SLE. Dapirolizumab pegol pharmacokinetics were found to be dose proportional and well described by a 2‐compartment model with first‐order elimination from the central compartment. In the popPK model, body weight was the only significant covariate. The average concentration of dapirolizumab pegol, derived from the popPK model, was incorporated into the exposure‐response model. Overall, the exposure‐response model showed that treatment with dapirolizumab pegol increased the probability of transitioning from BICLA "Nonresponder" to "Responder." No significant covariates on BICLA responder status were identified. Notably, the half maximal effective concentration was greater for the transition from "Responder" to "Nonresponder" (150 µg/mL) than the transition from "Nonresponder" to "Responder" (12 µg/mL), indicating that sustained dapirolizumab pegol concentrations may be required to maintain BICLA response. In conclusion, dapirolizumab pegol pharmacokinetics were as expected for a PEGylated molecule and results from the exposure‐response model indicate that a favorable dapirolizumab pegol effect was identified for both BICLA "Nonresponder" to "Responder" and "Responder" to "Nonresponder" transition probabilities. [ABSTRACT FROM AUTHOR]

Mucosa‐associated lymphoid tissue (MALT) lymphoma is a B‐cell tumour that develops over many decades in the stomachs of individuals with chronic Helicobacter pylori infection. We developed a new mouse model of human gastric MALT lymphoma in which mice with a myeloid‐specific deletion of the innate immune molecule, Nlrc5, develop precursor B‐cell lesions to MALT lymphoma at only 3 months post‐Helicobacter infection versus 9–24 months in existing models. The gastric B‐cell lesions in the Nlrc5 knockout mice had the histopathological features of the human disease, notably lymphoepithelial‐like lesions, centrocyte‐like cells, and were infiltrated by dendritic cells (DCs), macrophages, and T‐cells (CD4+, CD8+ and Foxp3+). Mouse and human gastric tissues contained immune cells expressing immune checkpoint receptor programmed death 1 (PD‐1) and its ligand PD‐L1, indicating an immunosuppressive tissue microenvironment. We next determined whether CD40L, overexpressed in a range of B‐cell malignancies, may be a potential drug target for the treatment of gastric MALT lymphoma. Importantly, we showed that the administration of anti‐CD40L antibody either coincident with or after establishment of Helicobacter infection prevented gastric B‐cell lesions in mice, when compared with the control antibody treatment. Mice administered the CD40L antibody also had significantly reduced numbers of gastric DCs, CD8+ and Foxp3+ T‐cells, as well as decreased gastric expression of B‐cell lymphoma genes. These findings validate the potential of CD40L as a therapeutic target in the treatment of human gastric B‐cell MALT lymphoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Talaromyces (Penicillium) marneffei is an AIDS-defining infection in Southeast Asia and is associated with high mortality. It is rare in non-immunosuppressed individuals, especially children. Little is known about host immune response and genetic susceptibility to this endemic fungus. Genetic defects in the interferon-gamma (IFN-γ)/STAT1 signaling pathway, CD40/CD40 ligand- and IL12/IL12-receptor-mediated crosstalk between phagocytes and T-cells, and STAT3-mediated Th17 differentiation have been reported in HIV-negative children with talaromycosis and other endemic mycoses such as histoplasmosis, coccidioidomycosis, and paracoccidioidomycosis. There is a need to design a diagnostic algorithm to evaluate such patients. In this article, we review a cohort of pediatric patients with disseminated talaromycosis referred to the Asian Primary Immunodeficiency Network for genetic diagnosis of PID. Using these illustrative cases, we propose a diagnostics pipeline that begins with immunoglobulin pattern (IgG, IgA, IgM, and IgE) and enumeration of lymphocyte subpopulations (T-, B-, and NK-cells). The former could provide clues for hyper-IgM syndrome and hyper-IgE syndrome. Flow cytometric evaluation of CD40L expression should be performed for patients suspected to have X-linked hyper-IgM syndrome. Defects in interferon-mediated JAK-STAT signaling are evaluated by STAT1 phosphorylation studies by flow cytometry. STAT1 hyperphosphorylation in response to IFN-α or IFN-γ and delayed dephosphorylation is diagnostic for gain-of-function STAT1 disorder, while absent STAT1 phosphorylation in response to IFN-γ but normal response to IFN-α is suggestive of IFN-γ receptor deficiency. This simple and rapid diagnostic algorithm will be useful in guiding genetic studies for patients with disseminated talaromycosis requiring immunological investigations.

BACKGROUND:Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. OBJECTIVE:To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. METHODS:Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. RESULTS:CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. CONCLUSION:Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity.

X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

Activated platelets are involved in the atherogenic stage of atherosclerosis, while they can also progress it to atherothrombosis which may cause an ischemic state and organ failure. In general, coronary artery disease (CAD) is considered as common and severe clinical consequence of atherosclerosis, manifesting as a chronic inflammatory condition with the release of platelet mediators, among which the importance of platelet-borne TGF-β1 is not yet well understood. Hence, for the first time, this study aimed to examine platelet level of TGF-β1 (latent/mature) in CAD-patients and its association with the expression of platelet pro-inflammatory molecules. Platelet from stable CAD-patients candidate for CABG and healthy controls were subjected to flowcytometry analysis to evaluate P-selectin and CD40L expressions and PAC-1 binding. Platelet-borne and soluble TGF-β1, both mature/active and latent forms were also examined with western blotting. Higher expression levels of P-selectin and CD40L in patients with CAD than in controls were associated with comparable levels of PAC-1 binding in both groups. Platelet TGF-β1 levels were also significantly higher in patients, while their platelets showed clear bands of mature TGF-β1 that were barely visible in healthy individuals. Soluble TGF-β1 was also higher in patients. Significant correlations between mature/active TGF-β1 and platelet pro-inflammatory markers (P-selectin and CD40L) as well as common indicators of inflammation (CRP and ESR) were observed in CAD patients. In this study, given the insignificant changes in pro-aggregatory potentials in stable CAD, the pro-inflammatory state of platelets may be more involved in disease development and progression. Direct correlations between active platelet-borne TGF-β1 and pro-inflammatory markers with its presence in CAD-patients, which was almost absent in the platelets of healthy individuals, may also underscore the significant contribution of platelet-borne TGF-β1 to the pathogenesis of the disease. [ABSTRACT FROM AUTHOR]

Background: Xenotransplantation has made significant advances recently using pigs genetically engineered to remove carbohydrate antigens, either alone or with addition of various human complement, coagulation, and anti-inflammatory ''transgenes''. Here we evaluated results associated with gene-edited (GE) pig hearts transplanted in baboons using an established costimulation-based immunosuppressive regimen and a cold-perfused graft preservation technique., Methods: Eight baboons received heterotopic abdominal heart transplants from 3-GE (GalKO.β4GalNT2KO.hCD55, n = 3), 9-GE (GalKO.β4GalNT2KO.GHRKO.hCD46.hCD55. TBM.EPCR.hCD47. HO-1, n = 3) or 10-G (9-GE+CMAHKO, n = 2) pigs using Steen's cold continuous perfusion for ischemia minimization. Immunosuppression (IS) included induction with anti-thymocyte globulin and αCD20, ongoing αCD154, MMF, and tapered corticosteroid., Results: All three 3-GE grafts functioned well initially, but failed within 5 days. One 9-GE graft was lost intraoperatively due to a technical issue and another was lost at POD 13 due to antibody mediated rejection (AMR) in a baboon with a strongly positive pre-operative cross-match. One 10-GE heart failed at POD113 with combined cellular and antibody mediated rejection. One 9-GE and one 10-GE hearts had preserved graft function with normal myocardium on protocol biopsies, but exhibited slowly progressive graft hypertrophy until elective necropsy at POD393 and 243 respectively. Elevated levels of IL-6, MCP-1, C-reactive protein, and human thrombomodulin were variably associated with conditioning, the transplant procedure, and clinically significant postoperative events., Conclusion: Relative to reference genetics without thrombo-regulatory and anti-inflammatory gene expression, 9- or 10-GE pig hearts exhibit promising performance in the context of a clinically applicable regimen including ischemia minimization and αCD154-based IS, justifying further evaluation in an orthotopic model., (Copyright © 2024 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients.

Both platelet count and function change after treatment of immune thrombocytopenia. Platelet function can be measured by plasma markers, including platelet activity [e.g., soluble P-selectin (sP-selectin) and soluble CD40 ligand (sCD40L)] and platelet turnover markers [e.g., glycocalicin (GC)]. Patients were classified into no response (NR, including new diagnosis), partial response (PR) and complete response (CR). One hundred and sixteen samples (29 CR, 32 PR, 55 NR) from 79 patients were collected. Plasma markers (sP-selectin, sCD40L and GC) were measured by ELISA. Platelet counts and mean platelet volume (MPV) were obtained in the clinical laboratory using GenS System-2. The results showed that responsive patients (PR + CR) had higher levels of sP-selectin (P = 0.026) and sCD40L (P = 0.001). Although there was no difference in MPV (P = 0.077) or GC (P = 0.078), there was a marked decrease of GC index (P < 0.001) in responsive patients. Paired sample analysis showed no difference in sP-selectin, sCD40L, MPV or GC but significant difference in GC index (P = 0.017) between NR and PR. Another paired sample analysis showed no difference in sP-selectin, sCD40L, MPV or GC but significant difference in GC index (P = 0.029) between PR and CR. Patients with refractory and newly diagnosed disease had a significant difference in GC (P = 0.020) and sCD40L (P = 0.001), despite similarly low platelet counts. In conclusion, platelet activity markers (sP-selectin and sCD40L) and GC indices change in parallel with treatment response. Plasma levels of GC and sCD40L may be predictors of treatment response. [ABSTRACT FROM AUTHOR]

Background and Purpose CD40 and CD40 ligand (CD40L) are costimulatory molecules of the tumor necrosis factor receptor superfamily and well known for their involvement in inflammatory diseases: atherosclerotic mouse models with disrupted CD40 signalling develop lesions of reduced size with a more stable plaque profile. This study investigated the potential of plasma and intraplaque levels of CD40 and CD40L as markers for cardiovascular disease (CVD) in humans and their association with plaque stability. Methods Soluble CD40 and CD40L (sCD40L) were measured in plasma in 1,437 subjects from The SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort. Intra-plaque levels of sCD40 and sCD40L were measured in atherosclerotic plaque homogenates from 199 subjects of the Carotid Plaque Imaging Project (CPIP) cohort. Results Both plasma sCD40 and sCD40L levels were elevated in individuals with prevalent stroke, while sCD40 levels also were higher in individuals with a prior acute myocardial infarction. Plasma levels of sCD40 correlated with carotid intima-media thickness and total carotid plaque area and were associated with risk of cardiovascular events over a 3-year follow-up period. Intra-plaque levels of sCD40 and sCD40L were associated with plaque components characteristic for plaque vulnerability and extracellular matrix remodelling. Conclusions Higher plasma sCD40 and sCD40L levels are associated with prevalent CVD. Plasma sCD40 levels also correlate with the severity of carotid atherosclerosis and predict future cardiovascular events, while intra-plaque levels correlate with a vulnerable plaque phenotype. Our findings thus demonstrate that elevated levels of sCD40 and sCD40L are markers of CVD.

BackgroundX-linked hyper-IgM syndrome (XHIGM) is a primary immunodeficiency with high morbidity and mortality compared with those seen in healthy subjects. Hematopoietic cell transplantation (HCT) has been considered a curative therapy, but the procedure has inherent complications and might not be available for all patients.ObjectivesWe sought to collect data on the clinical presentation, treatment, and follow-up of a large sample of patients with XHIGM to (1) compare long-term overall survival and general well-being of patients treated with or without HCT along with clinical factors associated with mortality and (2) summarize clinical practice and risk factors in the subgroup of patients treated with HCT.MethodsPhysicians caring for patients with primary immunodeficiency diseases were identified through the Jeffrey Modell Foundation, United States Immunodeficiency Network, Latin American Society for Immunodeficiency, and Primary Immune Deficiency Treatment Consortium. Data were collected with a Research Electronic Data Capture Web application. Survival from time of diagnosis or transplantation was estimated by using the Kaplan-Meier method compared with log-rank tests and modeled by using proportional hazards regression.ResultsTwenty-eight clinical sites provided data on 189 patients given a diagnosis of XHIGM between 1964 and 2013; 176 had valid follow-up and vital status information. Sixty-seven (38%) patients received HCT. The average follow-up time was 8.5 ± 7.2 years (range, 0.1-36.2 years). No difference in overall survival was observed between patients treated with or without HCT (P = .671). However, risk associated with HCT decreased for diagnosis years 1987-1995; the hazard ratio was significantly less than 1 for diagnosis years 1995-1999. Liver disease was a significant predictor of overall survival (hazard ratio, 4.9; 95% confidence limits, 2.2-10.8; P

PurposeCluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox.MethodsHuman RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry.ResultsLigation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7-mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner.ConclusionsCluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration.

Chronic HIV infection is associated with accumulation of germinal center (GC) T follicular helper (Tfh) cells in the lymphoid tissue. The GC Tfh cells can be heterogeneous based on the expression of chemokine receptors associated with T helper lineages, such as CXCR3 (Th1), CCR4 (Th2), and CCR6 (Th17). However, the heterogeneous nature of GC Tfh cells in the lymphoid tissue and its association with viral persistence and Ab production during chronic SIV/HIV infection are not known. To address this, we characterized the expression of CXCR3, CCR4, and CCR6 on GC Tfh cells in lymph nodes following SIVmac251 infection in rhesus macaques. In SIV-naive rhesus macaques, only a small fraction of GC Tfh cells expressed CXCR3, CCR4, and CCR6. However, during chronic SIV infection, the majority of GC Tfh cells expressed CXCR3, whereas the proportion of CCR4(+) cells did not change, and CCR6(+) cells decreased. CXCR3(+), but not CXCR3(-), GC Tfh cells produced IFN-γ (Th1 cytokine) and IL-21 (Tfh cytokine), whereas both subsets expressed CD40L following stimulation. Immunohistochemistry analysis demonstrated an accumulation of CD4(+)IFN-γ(+) T cells within the hyperplastic follicles during chronic SIV infection. CXCR3(+) GC Tfh cells also expressed higher levels of ICOS, CCR5, and α4β7 and contained more copies of SIV DNA compared with CXCR3(-) GC Tfh cells. However, CXCR3(+) and CXCR3(-) GC Tfh cells delivered help to B cells in vitro for production of IgG. These data demonstrate that chronic SIV infection promotes expansion of Th1-biased GC Tfh cells, which are phenotypically and functionally distinct from conventional GC Tfh cells and contribute to hypergammaglobulinemia and viral reservoirs.

MicroRNA (miRNA) deficiency impairs the generation of T follicular helper (Tfh) cells, but the contribution of individual miRNAs to this phenotype remains poorly understood. In this study, we performed deep sequencing analysis of miRNAs expressed in Tfh cells and identified a five-miRNA signature. Analyses of mutant mice deficient of these miRNAs revealed that miR-22 and miR-183/96/182 are dispensable, but miR-155 is essential for the generation and function of Tfh cells. miR-155 deficiency led to decreased proliferation specifically at the late stage of Tfh cell differentiation and reduced CD40 ligand (CD40L) expression on antigen-specific CD4(+) T cells. Mechanistically, miR-155 repressed the expression of Peli1, a ubiquitin ligase that promotes the degradation of the NF-κB family transcription factor c-Rel, which controls cellular proliferation and CD40L expression. Therefore, our study identifies a novel miR-155-Peli1-c-Rel pathway that specifically regulates Tfh cell generation and function.

Whether biomarkers associated with cardiovascular disease risk also predict incident diabetes mellitus (DM) is unknown. Our objective was to determine if a panel of 18 biomarkers previously associated with risk of cardiovascular disease also predicts incident DM in statin-treated patients with coronary artery disease (CAD). The Treating to New Targets (TNT) study is a randomized trial that compared the efficacy of high (80 mg) versus low (10 mg) dose atorvastatin for the secondary prevention of coronary heart disease events. Fasting plasma levels of standard lipids and of 18 emerging CAD risk biomarkers were obtained after an 8-week run-in period on atorvastatin 10 mg in a random sample of 1,424 TNT patients. After exclusion of patients with DM at baseline (n = 253), 101 patients developed DM during the median follow-up of 4.9 years. Patients with incident DM had lower levels of total and high-molecular weight adiponectin, lipoprotein-associated phospholipase A2 (Lp-PLA2), soluble receptor of advanced glycation end products, and vitamin D compared with patients without incident DM. In contrast, insulin, soluble CD40 ligand, and soluble intercellular adhesion molecule-1 levels were higher in patients with incident DM compared with those without. Plasma levels of C-reactive protein, cystatin C, lipoprotein(a), monocyte chemotactic protein-1, matrix metalloproteinase-9, myeloperoxidase, neopterin, N-terminal fragment of pro-B-type natriuretic peptide, osteopontin, and soluble vascular cell adhesion molecule-1 were comparable in patients with and without incident DM. After multivariate adjustment, total and high-molecular weight adiponectin as well as Lp-PLA2 were negatively associated with incident DM. Results of this study suggest that plasma lipids and some emerging CAD risk biomarkers, such as adiponectin and Lp-PLA2, may be useful for predicting incident DM in statin-treated patients with stable CAD.

PURPOSE: The United States Immunodeficiency Network (USIDNET) patient registry was used to characterize the presentation, genetics, phenotypes, and treatment of patients with Hyper IgM Syndrome (HIGM). METHODS: The USIDNET Registry was queried for HIGM patient data collected from October 1992 to July 2015. Data fields included demographics, criteria for diagnosis, pedigree analysis, mutations, clinical features, treatment and transplant records, laboratory findings, and mortality. RESULTS: Fifty-two physicians entered data from 145 patients of ages 2 months to 62 years (median 12 years); 131 were males. Using patients age at last entry, data from 2072 patient years are included. Mutations were recorded for 85 subjects; 82 were in CD40LG. Eighteen subjects had non-X-linked HIGM. 40 % had a normal serum IgM and 15 %, normal IgA. Infections were reported for 91 %, with pulmonary, ear, and sinus infections being the most common. 42 % had Pneumocystis jirovecii pneumonia; 6 % had Cryptosporidium. 41 % had neutropenia. 78 % experienced non-infectious complications: chronic diarrhea (n = 22), aphthous ulcers (n = 28), and neoplasms (n = 8) including colon cancer, adrenal adenoma, liver adenocarcinoma, pancreatic carcinoid, acute myeloid leukemia, hepatoma, and, in a female with an autosomal dominant gain of function mutation in PIK3CD, an ovarian dysgerminoma. Thirteen patients had a hematopoietic marrow or stem cell transplant; three had solid organ transplants. Thirteen were known to have died (median age = 14 years). CONCLUSIONS: Analysis of the USIDNET Registry provides data on the common clinical features of this rare syndrome, and in contrast with previously published data, demonstrates longer survival times and reduced gastrointestinal manifestations.

Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. T cells were analyzed by multicolor flow cytometry of PBMC after re-stimulation with TT in vitro. Reactive T helper (Th) cells were characterized by increased frequencies of CD4+CD154+ lymphocytes in in vitro TT-re-stimulated PBMC on day 14 after vaccination of the horses compared to pre-vaccination. The majority of all CD154+ cells after TT re-stimulation were CD4+ Th cells, but CD154 was also induced on CD4- cells albeit in lower frequencies. CD154+CD4+ Th cells were enriched in cytokine-expressing cells compared to CD154-CD4+ Th cells. Similar to the CD4+CD154+ frequencies, CD4+IL-4+, CD4+IFN-γ+ and CD4+TNF-α+ were increased after vaccination, but IL-4+ increased later than IFN-γ+ and CD4+TNF-α+, which already exceeded pre-vaccination frequencies on day 7. CD4+CD154+ frequencies correlated positively with those of CD4+IL-4+ (Th2) on day 14, and negatively with CD4+IFN-γ+ induction on day 7, but did not correlate with CD4+TNF-α+ frequencies or TT-specific antibody concentrations. CD154 appears to be a useful marker of antigen-reactive equine Th cells in combination with cytokine expression. The T cell analyses established here with TT can be applied to other antigens relevant for infections or allergies of horses and in horse models for translational research. [ABSTRACT FROM AUTHOR]

CD40 ligand (CD40L) deficiency is a rare inborn error of immunity presenting with heterogeneous clinical manifestations. While a detailed characterization of patients affected by CD40L deficiency is essential to an accurate diagnosis and management, information about this disorder in Latin American patients is limited. We retrospectively analyzed data from 50 patients collected by the Latin American Society for Immunodeficiencies registry or provided by affiliated physicians to characterize the clinical, laboratory, and molecular features of Latin American patients with CD40L deficiency. The median age at disease onset and diagnosis was 7 months and 17 months, respectively, with a median diagnosis delay of 1 year. Forty-seven patients were genetically characterized revealing 6 novel mutations in the CD40LG gene. Pneumonia was the most common first symptom reported (66%). Initial immunoglobulin levels were variable among patients. Pneumonia (86%), upper respiratory tract infections (70%), neutropenia (70%), and gastrointestinal manifestations (60%) were the most prevalent clinical symptoms throughout life. Thirty-five infectious agents were reported, five of which were not previously described in CD40L deficient patients, representing the largest number of pathogens reported to date in a cohort of CD40L deficient patients. The characterization of the largest cohort of Latin American patients with CD40L deficiency adds novel insights to the recognition of this disorder, helping to fulfill unmet needs and gaps in the diagnosis and management of patients with CD40L deficiency. [ABSTRACT FROM AUTHOR]

Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. T cells were analyzed by multicolor flow cytometry of PBMC after re-stimulation with TT in vitro. Reactive T helper (Th) cells were characterized by increased frequencies of CD4+CD154+ lymphocytes in in vitro TT-re-stimulated PBMC on day 14 after vaccination of the horses compared to pre-vaccination. The majority of all CD154+ cells after TT re-stimulation were CD4+ Th cells, but CD154 was also induced on CD4- cells albeit in lower frequencies. CD154+CD4+ Th cells were enriched in cytokine-expressing cells compared to CD154-CD4+ Th cells. Similar to the CD4+CD154+ frequencies, CD4+IL-4+, CD4+IFN-γ+ and CD4+TNF-α+ were increased after vaccination, but IL-4+ increased later than IFN-γ+ and CD4+TNF-α+, which already exceeded pre-vaccination frequencies on day 7. CD4+CD154+ frequencies correlated positively with those of CD4+IL-4+ (Th2) on day 14, and negatively with CD4+IFN-γ+ induction on day 7, but did not correlate with CD4+TNF-α+ frequencies or TT-specific antibody concentrations. CD154 appears to be a useful marker of antigen-reactive equine Th cells in combination with cytokine expression. The T cell analyses established here with TT can be applied to other antigens relevant for infections or allergies of horses and in horse models for translational research.

Background: Cutaneous melanoma (CM) is a malignancy with a variable incidence worldwide and a poor advanced-stage prognosis. Melanoma growth is closely associated with the immune system., Methods: A cross-sectional study was performed on CM patients admitted at the Hospital de Cancer de Pernambuco (HCP) between 2015 and 2018. Fifty-one CM patients were included, and 30 healthy individuals. The study aimed to evaluate the association of platelet activation mechanisms and inflammatory response in patients with cutaneous melanoma., Results: Elevated serum IL10 and low serum TNF levels in CM patients compared to controls (p < 0.05). High IL6 levels in patients with negative lymph nodes LN (-) compared to positive lymph nodes group (LN +, p = 0.0005). Low RANTES levels in patients compared to controls (p < 0.05). Elevated levels of platelet-lymphocyte (PLA), platelet-monocytes (PMA), and platelet-neutrophils (PNA) aggregates were observed in patients compared to controls (p < 0.05). CM patients with stage II had lower PMA levels than stages I and III (p < 0.05). High PMA levels were observed in patients with LN (+) compared to the LN (-) group (p < 0.0001). Patients with SSM had high levels of sCD40L and sCD62P compared to controls (p < 0.05)). High sCD40L levels in stage II compared to the stage III group, and sCD62P in stages I and II compared to the stage III group (p < 0.05). High sCD62P levels in patients with LN (-) compared to the group LN (+) (p < 0.05)., Conclusion: It was observed the immunosuppressive profile in CM may favor tumor progression. High levels of platelet-leukocyte aggregates, sCD40L, and sCD62P may be associated with the worst prognosis., (© 2024 Wiley Periodicals LLC.)

Tuberculosis (TB) pathologic lesions in rhesus macaques resemble those in humans. The expression levels of several host TB candidate genes in the peripheral blood mononuclear cells (PBMCs) of six rhesus macaques experimentally infected with Mycobacterium tuberculosis were quantified pre-infection and at several dates post-infection. Quantitative measures of TB histopathology in the lungs including: granuloma count, granuloma size, volume of granulomatous and non-granulomatous lesions, and direct bacterial load, were used as the outcomes of a multi-level Bayesian regression model in which expression levels of host genes at various dates were used as predictors. The results indicate that the expression levels of TR4, CD40, CD40L, FAS (CD95) and TNF in PBMC were associated with quantitative measures of the severity of TB histopathologic lesions in the lungs of the study animals. Moreover, no reliable association between the expression levels of IFNE in PBMCs and the severity of TB lesions in the lungs of the study animals was found. In conclusion, PBMC expression profiles derived from the above-listed host genes might be appropriate biomarkers for probabilistic diagnosis and/or prognosis of TB severity in rhesus macaques.