CD40 Ligand Research Articles

Lipid nanoparticle encapsulation of a Delta spike-CD40L DNA vaccine improves effectiveness against Omicron challenge in Syrian hamsters

The effectiveness of mRNA vaccines largely depends on their lipid nanoparticle (LNP) component. Herein, we investigate the effectiveness of DLin-KC2-DMA (KC2) and SM-102-based LNPs for the intramuscular delivery of a plasmid encoding B.1.617.2 (Delta) spike fused with CD40 ligand. LNP encapsulation of this CD40L-adjuvanted DNA vaccine with either LNP formulation drastically enhanced antibody responses, enabling neutralization of heterologous Omicron variants. The DNA-LNP formulations provided excellent protection from homologous challenge, reducing viral replication, and preventing histopathological changes in the pulmonary tissues. Moreover, theDNA-LNP vaccines maintained a high level of protection against heterologous Omicron BA.5 challenge despite a reduced neutralizing response. In addition, we observed that DNA-LNP vaccination led to the pulmonary downregulation of interferon signaling, interleukin-12 signaling, and macrophage response pathways following SARS-CoV-2 challenge, shedding some light on the mechanisms underlying the prevention of pulmonary injury. These results highlight the potential combination of molecular adjuvants with LNP-based vaccine delivery to induce greater and broader immune responses capable of preventing inflammatory damage and protecting against emerging variants. These findings could be informative for the future design of both DNA and mRNA vaccines.

High-volume hemofiltration does not protect human kidney endothelial and tubular epithelial cells from septic plasma-induced injury

High volume hemofiltration (HVHF) could remove from plasma inflammatory mediators involved in sepsis-associated acute kidney injury (SA-AKI). The IVOIRE trial did not show improvements of outcome and organ dysfunction using HVHF. The aim of this study was to evaluate in vitro the biological effects of plasma of patients treated by HVHF or standard volume hemofiltration (SVHF). We evaluated leukocyte adhesion, apoptosis and functional alterations of endothelial cells (EC) and tubular epithelial cells (TEC). In vitro data were correlated with plasma levels of TNF-α, Fas-Ligand (FasL), CD40-Ligand (CD40L), von Willebrand Factor (vWF) and endothelial-derived microparticles. An experimental model of in vitro hemofiltration using LPS-activated blood was established to assess cytokine mass adsorption during HVHF or SVHF. Plasma concentrations of TNF-ɑ, FasL, CD40L and von Willebrand Factor (vWF) were elevated at the start (d1h0) of both HVHF and SVHF, significantly decreased after 6 h (d1h6), remained stable after 12 h (d1h12) and then newly increased at 48 h (d3h0). Plasma levels of all these molecules were similar between HVHF- and SVHF-treated patients at all time points considered. In addition, the levels of endothelial microparticles remained always elevated, suggesting the presence of a persistent microvascular injury. Plasma from septic patients induced leukocyte adhesion on EC and TEC through up-regulation of adhesion receptors. Moreover, on EC, septic plasma induced a cytotoxic and anti-angiogenic effect. On TEC, septic plasma exerted a direct pro-apoptotic effect via Fas up-regulation and caspase activation, loss of polarity, altered expression of megalin and tight junction molecules with an impaired ability to internalize albumin. The inhibition of plasma-induced cell injury was concomitant to the decrease of TNF-α, Fas-Ligand and CD40-Ligand levels. The protective effect of both HVHF and SVHF was time-limited, since a further increase of circulating mediators and plasma-induced cell injury was observed after 48 h (d3h0). No significant difference of EC/TEC damage were observed using HVHF- or SVHF-treated plasma. The in vitro hemofiltration model confirmed the absence of a significant modulation of cytokine adsorption between HVHF and SVHF. In comparison to SVHF, HVHF did not increase inflammatory cytokine clearance and did not reverse the detrimental effects of septic plasma-induced EC and TEC injury. Further studies using adsorptive membranes are needed to evaluate the potential role of high dose convective therapies in the limitation of the harmful activity of plasma soluble factors involved in SA-AKI.Trial registration IVOIRE randomized clinical trial; ClinicalTrials.gov (NCT00241228) (18/10/2005).

Neu5Gc-mediated high-affinity interaction is dispensable for CD22 cis-ligands to regulate B cell signaling

CD22 (also known as Siglec-2) is an inhibitory receptor expressed in B cells. CD22 specifically recognizes α2,6 sialic acid and interacts with α2,6 sialylated membrane proteins expressed on the same cell (cis-ligands) and those derived from outside of the cell (trans-ligands). Previously, CD22 cis-ligands were shown to regulate the activity of CD22, thereby regulating both BCR ligation-induced signaling and low-level "tonic" signaling in the absence of BCR ligation that regulates the survival and differentiation of B cells. Mouse CD22 prefers Neu5Gc to Neu5Ac thereby binding to α2,6-linked Neu5Gc with high affinity. Although human CD22 binds to a distinct α2,6 sialylated glycan with high affinity, expression of high-affinity ligands is regulated in a conserved and stringent manner. However, how high- versus low-affinity CD22 ligands regulate B cells is poorly understood. Here we demonstrate that the interaction of CD22 with the endogenous ligands enhances BCR ligation-induced signaling but reduces tonic signaling in Cmah-/- mouse B cells deficient in Neu5Gc as well as wild-type B cells. Moreover, Cmah-/- B cells do not show alterations in the phenotypes correlated to tonic signaling. These results indicate that low-affinity interaction of the CD22 cis-ligands with CD22 is sufficient for the regulation of B cell signaling, and suggest that expression of high-affinity CD22 ligands might be involved in the regulation of B cells by competing for the binding of CD22 with exogenous trans-ligands of CD22.

Platelet transfusion enhances pro-aggregatory status shortly aftercoronary artery bypass grafting (CABG while modulating platelet pro-inflammatory state 1-week post-surgery.

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 24h 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.

Soluble CD40 Ligand as a Promising Biomarker in Cancer Diagnosis.

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.

CD44-hyaluronan mediating endocytosis of iron-platinum alloy nanoparticles induces ferroptotic cell death in mesenchymal-state lung cancer cells with tyrosine kinase inhibitor resistance

While tyrosine kinase inhibitor resistance in cancer is a critical issue in the medical field, it is important for clinical testing as well, since it affects the ultimate outcome of cancer therapy. Yet, no effective solutions have been implemented till date. Clinical observations after tyrosine kinase inhibitor treatment reveal that acquired resistance inevitably limits the curative effects of non-small cell lung cancer treatment because of mutations in the epidermal growth factor receptor gene, which are accompanied by epithelial-mesenchymal transition. Here, for the first time, we report that the transmembrane glycoprotein CD44, which is associated with epithelial-mesenchymal transition, chemoresistance, and cancer progression, mediates enhanced endocytosis of iron-platinum alloy nanoparticles (FePt NPs) in the mesenchymal-state gefitinib-resistant (GR+ and M6) cells, via the binding of the CD44 ligand, hyaluronan, to the surface-absorbed hyaluronan-binding protein 2. Upon treatment with FePt NPs, there was higher cellular uptake in mesenchymal-state GR+ and M6 cells, resulting from cell death through ferroptosis and mitochondrial dysfunction, as compared to that observed in the epithelial-state cells. Mechanistically, inactivation of dihydroorotate dehydrogenase elevated the production of mitochondrial lipid peroxidation, and enhanced the cell death in the epithelial-state HCC827 cells, thereby indicating its role in defense against FePt NPs-induced ferroptosis. Furthermore, induction of ferroptosis has been shown to specifically promote the cell death of drug-tolerant “persister” cells and reverse their resistance as well. Therefore, we concluded that FePt NPs preferentially target mesenchymal drug-tolerant “persister” cells and promote ferroptosis, to overcome their resistance. Statement of significanceIn the present study, we identified FePt NPs as an innovative agent for cancer treatment, particularly in mesenchymal-state cells that exhibit TKI resistance. Mesenchymal-state cancer cells showed enhanced uptake of FePt NPs via CD44-HA-mediated endocytosis, accompanied by severe cell death and mitochondrial morphology alterations, in comparison to epithelial-state cells. We further elucidated the mechanism underlying FePt NPs-induced ferroptotic cell death as via a burst of mitochondrial LPO and DHODH protein inactivation. In addition, we found that FePt NPs inhibit tumor growth in TKI-resistant mesenchymal GR+ cell-bearing mice with better efficacy than the ferroptotic inducer RSL3. Our current findings on using FePt NPs to overcome TKI resistance through ferroptosis activation may offer a alternative strategy for improved cancer treatment.

Fibroblast-like synoviocytes preferentially induce terminal differentiation of IgD+ memory B cells instead of naïve B cells.

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.

Targeting exhausted cytotoxic T cells through CTLA-4 inhibition promotes elimination of neoplastic cells in human myelofibrosis xenografts.

Myeloproliferative neoplasms represent a group of clonal hematopoietic disorders of which myelofibrosis (MF) is the most aggressive. In the context of myeloid neoplasms, there is a growing recognition of the dysregulation of immune response and T-cell function as significant contributors to disease progression and immune evasion. We investigated cytotoxic T-cell exhaustion in MF to restore immune response against malignant cells. Increased expression of inhibitory receptors like CTLA-4 was observed on cytotoxic T cells from MF patients together with a reduced secretion of IFNɣ and TNFɑ. CTLA-4 ligands CD80 and CD86 were increased on MF granulocytes and monocytes highlighting a possible role for myeloid cells in suppressing T-cell activation in MF patients. Unlike healthy donors, the activation of cytotoxic T cells from MF patients was attenuated in the presence of myeloid cells and restored when T cells were cultured alone or treated with anti-CTLA-4. Moreover, anti-CTLA-4 treatment promoted elimination of neoplastic monocytes and granulocytes in a co-culture system with cytotoxic T cells. To test CTLA-4 inhibition in vivo, patient-derived xenografts were generated by transplanting MF CD34+ cells and by infusing homologous T cells in NSGS mice. CTLA-4 blockade reduced human myeloid chimerism and led to T-cell expansion in spleen and bone marrow. Overall, these findings shed light on T-cell dysfunction in MF and suggest that CTLA-4 blockade can boost the cytotoxic T cell-mediated immune response against tumor cells.

Crizotinib resistance reversal in ALK-positive lung cancer through zeolitic imidazolate framework-based mitochondrial damage

Crizotinib (CRZ), one of anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs), has emerged as a frontline treatment for ALK-positive (ALK+) lung adenocarcinoma. However, the overexpression of P-glycoprotein (P-gp, a mitochondrial adenosine triphosphate (ATP)-dependent protein) in lung adenocarcinoma lesions causes multidrug resistance (MDR) and limits the efficacy of CRZ treatment. Herein, a mitochondria-targeting nanosystem, zeolitic imidazolate framework-90@indocyanine green (ZIF-90@ICG), was fabricated to intervene in mitochondria and overcome drug resistance. Due to the zinc ion (Zn2+) interference of ZIF-90 and the photodynamic therapy (PDT) of ICG, this nanosystem is well suited for damaging mitochondrial functions, thus downregulating the intracellular ATP level and inhibiting P-gp expression. In addition, systematic bioinformatics analysis revealed the upregulation of CD44 in CRZ-resistant cells. Therefore, hyaluronic acid (HA, a critical target ligand of CD44) was further modified on the surface of ZIF-90@ICG for active targeting. Overall, this ZIF-90@ICG nanosystem synergistically increased the intracellular accumulation of CRZ and reversed CRZ resistance to enhance its anticancer effect, which provides guidance for nanomedicine design to accurately target tumours and induce mitochondrial damage and represents a viable regimen for improving the prognosis of patients with ALK-TKIs resistance. Statement of significanceThe original aim of our research was to combat multidrug resistance (MDR) in highly aggressive and lethal lymphoma kinase-positive (ALK+) lung adenocarcinoma. For this purpose, a cascade-targeted system was designed to overcome MDR, integrating lung adenocarcinoma-targeted hyaluronic acid (HA), mitochondrion-targeted zeolitic imidazolate framework-90 (ZIF-90), the clinically approved drug crizotinib (CRZ), and the fluorescence imaging agent/photosensitizer indocyanine green (ICG). Moreover, using a “two birds with one stone” strategy, ion interference and oxidative stress induced by ZIF-90 and photodynamic therapy (PDT), respectively, disrupt mitochondrial homeostasis, thus downregulating adenosine triphosphate (ATP) levels, inhibiting MDR-relevant P-glycoprotein (P-gp) expression and suppressing tumour metastasis. Overall, this research represents an attempt to implement the concept of MDR reversal and realize the trade-offs between MDR and therapeutic effectiveness.

Development of an immunoassay for quantification of soluble human CD40L (CD154) in plasma and serum samples

When the membrane protein CD40 ligand (CD40L) on activated T cells binds the receptor CD40 on B-cells, it provides a co-stimulatory signal for B cell activation. Dysregulation of the CD40L:CD40 axis is associated with inflammatory and autoimmune diseases. The presence of soluble CD40L (sCD40L) in plasma is implicated in several diseases, from cardiovascular and autoimmune diseases to different types of cancer, and sCD40L has been suggested as a valuable marker of disease. If sCD40L is to be used as a biomarker, being able to precisely measure and quantify the levels of sCD40L in human blood samples is of utmost importance. We demonstrate the development of a sandwich-type time-resolved immunofluorometric assay for quantification of sCD40L in plasma or serum samples. For this, we generate 29 monoclonal anti-CD40L antibodies, and from these, we select the optimal combination of capture antibody and detection antibody. A number of variables were tested: the influence of the type of sample (comparing 3 different blood collection tubes for serum sampling and 4 different types of tubes for plasma sampling), the influence of freeze-thaw cycles, the influence of sampling time during night and day, and the influence of centrifugation of the samples. We found a very similar level of sCD40L in paired EDTA plasma and serum samples. Out of 100 healthy blood donor samples 61 had a level of sCD40L below the detection level of the assay, whereas the remaining 39 samples had ranging levels of sCD40L from 1.14 to 33.14 ng/mL. In summary, we present a time-resolved immunofluorometric assay based on paired monoclonal antibodies, ensuring high specificity, sensitivity, and homogeneity. The Eu3+-based assay additionally provides consistent assay readouts due to the extended decay time not seen in standard enzyme-linked immunosorbent assays. The assay paves the way for specific and consistent quantification of sCD40L in human plasma and serum samples.

Cardiometabolic Co-morbidity Burden and Circulating Biomarkers in Patients With Chronic Coronary Disease in the ISCHEMIA Trials

Cardiometabolic co-morbidities, diabetes (DM), hypertension (HTN), and obesity contribute to cardiovascular disease. Circulating biomarkers facilitate prognostication for patients with cardiovascular disease. We explored the relation between cardiometabolic co-morbidity burden in patients with chronic coronary disease and biomarkers of myocardial stretch, injury, inflammation, and platelet activity. We analyzed participants from the International Study of Comparative Health Effectiveness with Medical and Invasive Approaches (ISCHEMIA) trials biorepository with plasma biomarkers (N-terminal probrain natriuretic peptide, high-sensitivity cardiac troponin T, high-sensitivity C-reactive protein, interleukin-6, soluble CD40 ligand, and growth differentiation factor-15) and clinical risk factors (hemoglobin A1c [HbA1c], systolic blood pressure [SBP], and body mass index [BMI]) at baseline. We defined cardiometabolic co-morbidities as DM, HTN, and obesity at baseline. Co-morbidity burden is characterized by the number and severity of co-morbidities. Controlled co-morbidities were defined as HbA1c <7% for those with DM, SBP <130 mm Hg for those with HTN, and BMI <30 kg/m2. Severely uncontrolled was defined as HbA1c ≥8%, SBP ≥160 mm Hg, and BMI ≥35 kg/m2. We performed linear regression analyses to examine the association between co-morbidity burden and log-transformed biomarker levels, adjusting for age, gender, estimated glomerular filtration rate controlled for hemodialysis, and left ventricular ejection fraction. A total of 752 participants (mean age 66 years, 19% women, 84% White) were included in this analysis. Self-reported Black race, current smokers, history of myocardial infarction, and heart failure had a greater cardiometabolic co-morbidity burden. The presence of ≥1 severely uncontrolled co-morbidity was associated with significantly higher baseline levels of high-sensitivity cardiac troponin T, high-sensitivity C-reactive protein, interleukin-6, and growth differentiation factor-15 than participants with no co-morbidities. In conclusion, increasing cardiometabolic co-morbidity burden in patients with chronic coronary disease is associated with higher levels of circulating biomarkers of myocardial injury and inflammation.

CD40 ligand antagonist dazodalibep in Sjögren’s disease: a randomized, double-blinded, placebo-controlled, phase 2 trial

Sjögren’s disease (SjD) is a chronic, systemic autoimmune disease with no approved disease-modifying therapies. Dazodalibep (DAZ), a novel nonantibody fusion protein, is a CD40 ligand antagonist that blocks costimulatory signals between T and B cells and antigen-presenting cells, and therefore may suppress the wide spectrum of cellular and humoral responses that drive autoimmunity in SjD. This study was a phase 2, randomized, double-blinded, placebo (PBO)-controlled trial of DAZ with a crossover stage in two distinct populations of participants with SjD. Population 1 had moderate-to-severe systemic disease activity and population 2 had an unacceptable symptom burden and limited systemic organ involvement. All participants had a diagnosis of SjD, with 21.6% and 10.1% having an associated connective tissue disease (rheumatoid arthritis or systemic lupus erythematosus) in populations 1 and 2, respectively. The remaining participants would be considered as having primary Sjögren’s syndrome. The primary endpoint for population 1 (n = 74) was the change from baseline in the European League Against Rheumatism Sjögren’s Syndrome Disease Activity Index at day 169. The primary endpoint for population 2 (n = 109) was the change from baseline in the European League Against Rheumatism Sjögren’s Syndrome Patient Reported Index at day 169. The primary endpoints (least squares mean ± standard error) were achieved with statistical significance for both population 1 (DAZ, −6.3 ± 0.6; PBO, −4.1 ± 0.6; P = 0.0167) and population 2 (DAZ, −1.8 ± 0.2; PBO, −0.5 ± 0.2; P = 0.0002). DAZ was generally safe and well tolerated. Among the most frequently reported adverse events were COVID-19, diarrhea, headache, nasopharyngitis, upper respiratory tract infection, arthralgia, constipation and urinary tract infection. In summary, DAZ appears to be a potential new therapy for SjD and its efficacy implies an important role for the CD40/CD40 ligand pathway in its pathogenesis. ClinicalTrials.gov identifier: NCT04129164.

Multi-omic Analysis of Human B-cell Activation Reveals a Key Lysosomal BCAT1 Role in mTOR Hyperactivation by B-cell receptor and TLR9.

B-lymphocytes play major adaptive immune roles, producing antibody and driving T-cell responses. However, how immunometabolism networks support B-cell activation and differentiation in response to distinct receptor stimuli remains incompletely understood. To gain insights, we systematically investigated acute primary human B-cell transcriptional, translational and metabolomic responses to B-cell receptor (BCR), Toll-like receptor 9 (TLR9), CD40-ligand (CD40L), interleukin-4 (IL4) or combinations thereof. T-independent BCR/TLR9 co-stimulation, which drives malignant and autoimmune B-cell states, jointly induced PD-L1 plasma membrane expression, supported by NAD metabolism and oxidative phosphorylation. BCR/TLR9 also highly induced the transaminase BCAT1, which localized to lysosomal membranes to support branched chain amino acid synthesis and mTORC1 hyperactivation. BCAT1 inhibition blunted BCR/TLR9, but not CD40L/IL4-triggered B-cell proliferation, IL10 expression and BCR/TLR pathway-driven lymphoma xenograft outgrowth. These results provide a valuable resource, reveal receptor-mediated immunometabolism remodeling to support key B-cell phenotypes including PD-L1 checkpoint signaling, and identify BCAT1 as a novel B-cell therapeutic target.

Checkpoint Inhibitors in Dogs: Are We There Yet?

Immune checkpoint inhibitors (ICI) have revolutionised cancer treatment in people. Immune checkpoints are important regulators of the body's reaction to immunological stimuli. The most studied immune checkpoint molecules are programmed death (PD-1) with its ligand (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) with its ligands CD80 (B7-1) and CD86 (B7-2). Certain tumours can evade immunosurveillance by activating these immunological checkpoint targets. These proteins are often upregulated in cancer cells and tumour-infiltrating lymphocytes, allowing cancer cells to evade immune surveillance and promote tumour growth. By blocking inhibitory checkpoints, ICI can help restore the immune system to effectively fight cancer. Several studies have investigated the expression of these and other immune checkpoints in human cancers and have shown their potential as therapeutic targets. In recent years, there has been growing interest in studying the expression of immune checkpoints in dogs with cancer, and a few small clinical trials with ICI have already been performed on these species. Emerging studies in veterinary oncology are centred around developing and validating canine-targeted antibodies. Among ICIs, anti-PD-1 and anti-PD-L1 treatments stand out as the most promising, mirroring the success in human medicine over the past decade. Nevertheless, the efficacy of caninized antibodies remains suboptimal, especially for canine oral melanoma. To enhance the utilisation of ICIs, the identification of predictive biomarkers for treatment response and the thorough screening of individual tumours are crucial. Such endeavours hold promise for advancing personalised medicine within veterinary practice, thereby improving treatment outcomes. This article aims to review the current research literature about the expression of immune checkpoints in canine cancer and the current results of ICI treatment in dogs.

Pneumocystis murina lesions in lungs of experimentally infected Cd40l-/- mice.

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.

Intranasal and Intramuscular Immunization with a Universal Influenza A Virus Vaccine Induces Distinct and Protective Immune Responses in Mice

Abstract Influenza infections cause more than half a million deaths annually. Despite the prevalence of influenza vaccines, the vaccine efficacy varies widely (19-54%) due frequent mutations of the viral genome, a problem exacerbated by cross-species transmission. Although intranasal (IN) administration could enhance effectiveness, most current vaccines are intramuscular (IM) due to limited understanding of the IN mechanism. This study aims to compare the mechanism of protection conferred by an IN or IM delivered universal influenza vaccine. We designed a vector expressing a conserved antigen, the nucleoprotein (NP), fused with CD40 ligand, acting as both a targeting ligand and a molecular adjuvant. Only IN vaccinated mice were protected against H1N1, H3N2, and a highly pathogenic and transmissive H5N1 avian strain, showing high antibody levels and distinct cytokines along the respiratory tract. To further characterize the cell-mediated response, IN or IM vaccinated animals were treated with an agonist for sphingosine-1-phosphate receptors, inhibiting lymphocyte egress. After the treatment, only IN was protective against the viral challenge, indicating that IN induced a potent local cell-mediated defense without circulating lymphocytes. Further flow cytometric analysis revealed that IN induced proliferative tissue-resident T cells in the lungs. These findings suggest that IN vaccination induces superior cross-subtype protection by stimulating robust mucosal immunity.

Moving from Insulin Substitution to the Treatment of the Underlying Autoimmune Disease in Type 1 Diabetes.

Currently, a paradigm change occurs in type 1 diabetes from insulin substitution to the treatment of the underlying autoimmune disease. Teplizumab, a humanized monoclonal anti-CD3 antibody, is the first FDA-approved disease-modifying treatment of preclinical stage 2 diabetes. Research of drugs like golimumab, a monoclonal antibody specific for TNF alpha, baricitinib, a tyrosine kinase inhibitor, or frexalimab, a monoclonal antibody against the CD40 ligand, is still ongoing. Repurposing drugs that have been used in other indications like the calcium channel blocker verapamil, antithymocyte globulin (ATG), an antibody preparation used in solid organ transplantation, glucagon-like peptide-1 agonists utilized in type 2 diabetes and obesity, or the antiviral drugs pleconaril and ribavirin have shown positive effects in preserving beta-cell function. While new therapies to halt autoimmunity and restore beta cells in stages one to three are being developed, replacing beta-cell function via inducible pluripotent stem cells have shown glucose control and insulin independence in long-standing type 1 diabetes, albeit with concomitant immunosuppression. Multicenter multinational initiatives developing a clinical trial network like INNODIA or a research platform with the goal of stopping type 1 diabetes in its early stages like EDENT1FI will be instrumental to study these new strategies.

Immunological Characteristics of Hepatic Dendritic Cells in Patients and Mouse Model with Liver Echinococcus multilocularis Infection.

The cestode Echinococcus multilocularis, which mainly dwells in the liver, leads to a serious parasitic liver disease called alveolar echinococcosis (AE). Despite the increased attention drawn to the immunosuppressive microenvironment formed by hepatic AE tissue, the immunological characteristics of hepatic dendritic cells (DCs) in the AE liver microenvironment have not been fully elucidated. Here, we profiled the immunophenotypic characteristics of hepatic DC subsets in both clinical AE patients and a mouse model. Single-cell RNA sequencing (scRNA-Seq) analysis of four AE patient specimens revealed that greater DC numbers were present within perilesional liver tissues and that the distributions of cDC and pDC subsets in the liver and periphery were different. cDCs highly expressed the costimulatory molecule CD86, the immune checkpoint molecule CD244, LAG3, CTLA4, and the checkpoint ligand CD48, while pDCs expressed these genes at low frequencies. Flow cytometric analysis of hepatic DC subsets in an E. multilocularis infection mouse model demonstrated that the number of cDCs significantly increased after parasite infection, and a tolerogenic phenotype characterized by a decrease in CD40 and CD80 expression levels was observed at an early stage, whereas an activated phenotype characterized by an increase in CD86 expression levels was observed at a late stage. Moreover, the expression profiles of major immune checkpoint molecules (CD244 and LAG3) and ligands (CD48) on hepatic DC subsets in a mouse model exhibited the same pattern as those in AE patients. Notably, the cDC and pDC subsets in the E. multilocularis infection group exhibited higher expression levels of PD-L1 and CD155 than those in the control group, suggesting the potential of these subsets to impair T cell function. These findings may provide valuable information for investigating the role of hepatic DC subsets in the AE microenvironment and guiding DC targeting treatments for AE.

Platelet activity, coagulation, and fibrinolysis in long-term users of anabolic-androgenic steroids compared to strength-trained athletes

IntroductionUse of anabolic-androgenic steroids (AAS) is associated with adverse cardiovascular (CV) effects, including potential prothrombotic effects. This study aimed to assess platelet activation and aggregation, coagulation, and fibrinolysis, in long-term AAS users compared to non-using strength-trained athletes. Materials and methodsThirty-seven strength-trained men using AAS were compared to seventeen non-using professional strength-trained athletes at similar age (median 33 years). AAS use was verified by blood and urine analyses. Platelet Function Analyzer 100 (PFA-100) and whole blood impedance aggregometry with thrombin, arachidonic acid, and ADP as agonists, were performed to evaluate platelet aggregation. ELISA methods were used for markers of platelet activation. Fibrinogen, D-dimer, the coagulation inhibitors protein S and C activity, and antithrombin were measured by routine. Fibrinolysis was evaluated by Plasminogen Activator Inhibitor-1 (PAI-1) activity. ResultsThere were no significant differences in platelet aggregation between the two groups. Von Willebrand factor was lower among the AAS users (p < 0.01), and P-Selectin was slightly higher (p = 0.05), whereas CD40 Ligand, β-thromboglobulin, and thrombospondin did not differ significantly. No differences were found in the assessed coagulation inhibitors. Higher D-dimer levels (p < 0.01) and lower PAI-1 activity (p < 0.01) were found among the AAS users. ConclusionsThe investigated long-term users of AAS did not exhibit elevated platelet activity compared to strength-trained non-using athletes. However, AAS use was associated with higher D-dimer levels and lower PAI-1 activity. These findings suggest that any prothrombotic effect of long-term AAS use may predominantly involve other aspects of the hemostatic system than blood platelets.

B cells in the pneumococcus-infected lung are heterogeneous and require CD4+ T cell help including CD40L to become resident memory B cells.

Recovery from respiratory pneumococcal infections generates lung-localized protection against heterotypic bacteria, mediated by resident memory lymphocytes. Optimal protection in mice requires re-exposure to pneumococcus within days of initial infection. Serial surface marker phenotyping of B cell populations in a model of pneumococcal heterotypic immunity revealed that bacterial re-exposure stimulates the immediate accumulation of dynamic and heterogeneous populations of B cells in the lung, and is essential for the establishment of lung resident memory B (BRM) cells. The B cells in the early wave were activated, proliferating locally, and associated with both CD4+ T cells and CXCL13. Antagonist- and antibody-mediated interventions were implemented during this early timeframe to demonstrate that lymphocyte recirculation, CD4+ cells, and CD40 ligand (CD40L) signaling were all needed for lung BRM cell establishment, whereas CXCL13 signaling was not. While most prominent as aggregates in the loose connective tissue of bronchovascular bundles, morphometry and live lung imaging analyses showed that lung BRM cells were equally numerous as single cells dispersed throughout the alveolar septae. We propose that CD40L signaling from antigen-stimulated CD4+ T cells in the infected lung is critical to establishment of local BRM cells, which subsequently protect the airways and parenchyma against future potential infections.