CD40 Ligand Research Articles

Alterations in the Serum Proteome Following Electroconvulsive Therapy for a Major Depressive Episode: A Longitudinal Multicenter Study

BackgroundElectroconvulsive therapy (ECT) is the most effective treatment for severe depression, but the biological changes induced by ECT remain poorly understood. MethodsThis study investigated alterations in blood serum proteins in 309 patients receiving ECT for a major depressive episode. We analyzed 201 proteins in samples collected at 3 time points (T): just before the first ECT treatment session (T0), within 30 minutes after the first ECT session (T1), and just before the sixth ECT session (T2). ResultsUsing statistical models to account for repeated sampling, we identified 152 and 70 significantly (<5% false discovery rate) altered proteins at T1 and T2, respectively. The most pronounced alterations at T1 were transiently increased levels of prolactin, myoglobin, and kallikrein-6. However, most proteins had decreased levels at T1, with the largest effects observed for pro-epidermal growth factor, proto-oncogene tyrosine-protein kinase Src, tumor necrosis factor ligand superfamily member 14, sulfotransferase 1A1, early activation antigen CD69, and CD40 ligand. The change of several acutely altered proteins correlated with electric current and pulse frequency in a dose-response–like manner. Over a 5-session course of ECT, some acutely altered levels were sustained while others increased, e.g., serine protease 8 and chitinase-3-like protein 1. None of the studied protein biomarkers were associated with clinical response to ECT. ConclusionsWe report experimental data on alterations in the circulating proteome triggered by ECT in a clinical setting. The findings implicate hormonal signaling, immune response, apoptotic processes, and more. None of the findings were associated with clinical response to ECT.

Evaluation of the CD40 receptor-ligand system in the patients with atrial fibrillation of non-valvular genesis

Thromboembolic syndrome is the most dangerous complication of atrial fibrillation which develops in about 8-15% of cases, thus presuming the role of persisting left-heart thrombosis in presence of anticoagulant therapy in some patients. When activated, the blood platelets express multiple copies of CD40L on their membrane. Hence, the soluble form of CD40 ligand is considered a marker of platelet activation and pathogenic processes associated with increased activity of the thrombotic system. Our aim was to study the content of CD40, soluble CD40 ligand and thrombomodulin in the patients with atrial fibrillation of non-valvular genesis receiving anticoagulant therapy, discerning those with a history of thrombotic complications, and the cases with atrial fibrillation, however, free of thrombotic complications. The study group included 22 healthy volunteers and 60 patients diagnosed with atrial fibrillation who received anticoagulant therapy, of whom 21 patients have developed thrombotic complications in the course of adequate anticoagulant therapy. Quantitative assays of CD40, soluble CD40 ligand and soluble thrombomodulin were performed by enzyme immunoassay using Core Facility “Medical Genomics”, Tomsk National Research Medical Center. Concentration of soluble CD40 ligand in both groups of the patients with atrial fibrillation significantly exceeded appropriate values in the group of healthy volunteers. CD40L content was increased in the group of patients with thrombotic complications against the group of patients without thrombotic complications. Thrombomodulin content in blood serum was decreased in the patients with thrombotic complications, as compared to both thrombosis-free patients, and to practically healthy volunteers. The study of CD40/CD40L system and thrombomodulin showed that the patients with thrombotic complications exhibited higher serum level of soluble CD40L, with a simultaneous decrease of thrombomodulin, a physiological anticoagulant. A comparative analysis of the CD40/sCD40L system showed increased concentrations of the biomarkers in females, when compared to males.

Platelet Reactivity and Inflammatory Phenotype Induced by Full-Length Spike SARS-CoV-2 Protein and Its RBD Domain.

A state of immunothrombosis has been reported in COVID-19. Platelets actively participate in this process. However, little is known about the ability of SARS-CoV-2 virus proteins to induce platelet activity. Platelet-rich plasma (PRP) was incubated with spike full-length protein and the RBD domain in independent assays. We evaluated platelet activation through the expression of P-selectin and activation of glicoprotein IIbIIIa (GP IIbIIIa), determined by flow cytometry and the ability of the proteins to induce platelet aggregation. We determined concentrations of immunothrombotic biomarkers in PRP supernatant treated with the proteins. We determined that the spike full-length proteins and the RBD domain induced an increase in P-selectin expression and GP IIbIIIa activation (p < 0.0001). We observed that the proteins did not induce platelet aggregation, but favored a pro-aggregating state that, in response to minimal doses of collagen, could re-establish the process (p < 0.0001). On the other hand, the viral proteins stimulated the release of interleukin 6, interleukin 8, P-selectin and the soluble fraction of CD40 ligand (sCD40L), molecules that favor an inflammatory state p < 0.05. These results indicate that the spike full-length protein and its RBD domain can induce platelet activation favoring an inflammatory phenotype that might contribute to the development of an immunothrombotic state.

Costimulatory Molecules CD80/86 Trigger Non-Specific Cytotoxic Cell of Nile tilapia (Oreochromis niloticus) to Kill CIK Cells

The teleost non-specific cytotoxic cell (NCC), as the evolutionary precursors of NK cells, is an important cytotoxic cell population in the innate immune system of teleost. We have recently realized that costimulatory CD80/86 have conservation in structural and interactional features with its ligand CD28 in Nile tilapia (Oreochromis niloticus). However, the ability of CD80/86 to regulate NCC activity has not been fully investigated. In the present study, we first obtained the recombinant fusion CD80/86 protein from O. niloticus (rOn-CD80/86). Then, NCC incubation with rOn-CD80/86 resulted in a significant production of NCC effector cytokines, including tumor necrosis factor-alpha, cellular apoptosis susceptibility and NK-lysin. Furthermore, NCC treatment with rOn-CD80/86 could significantly improve the ability to kill kidney cells of Grass carp (CIK) and up-regulate the activities of caspase-1 and caspase-3 in CIKs. The yeast, two-hybrid assay showed that On-CD80/86 cannot directly interact with non-specific cytotoxic cell receptor protein-1 of O. niloticus (On-NCCRP-1). The single-cell RNA-Seq data of Nile tilapia head kidney lymphocytes analysis found On-CD28 did not exhibit expression on NCCs subsets. The above results suggest that costimulatory molecules On-CD80/86 is independent of On-NCCRP-1 and On-CD28 receptor in modulating NCC killing activity in vitro of Nile tilapia. The results also provide more insights into the mechanism of NCC activity regulation.

Allogeneic cord blood regulatory T cells can resolve lung inflammation

Background aimsCD4+CD25+CD127lo regulatory T cells (Tregs) are responsible for maintaining immune homeostasis. Tregs can be rendered defective and deficient as a result of the immune imbalance seen in lung injury, and such dysfunction can play a major role in continued tissue inflammation. The authors hypothesized that adoptive therapy with healthy allogeneic umbilical cord blood (UCB)-derived Tregs may be able to resolve inflammation. ResultsEx vivo-expanded UCB Tregs exhibited a unique phenotype with co-expression of CD45RA+CD45RO+ >80% and lung homing markers, including CD49d. UCB Tregs did not turn pathogenic when exposed to IL-6. Co-culture with increasing doses of dexamethasone led to a synergistic increase in UCB Treg-induced apoptosis of conventional T cells (Tcons), which translated into significantly higher suppression of proliferating Tcons, especially at a lower Treg:Tcon ratio. Multiple injections of UCB Tregs led to their preferential accumulation in lung tissue in an immune injury xenogenic model. A significant decrease in lung resident cytotoxic CD8+ T cells (P = 0.0218) correlated with a sustained decrease in their systemic distribution compared with controls (P < 0.0001) (n = 7 per arm) as well as a decrease in circulating human soluble CD40 ligand level (P = 0.031). Tissue architecture was preserved in the treatment arm, and a significant decrease in CD3+ and CD8+ burden was evident in immunohistochemistry analysis. ConclusionsUCB Treg adoptive therapy is a promising therapeutic strategy for treatment of lung injury.

CD47-SIRPα axis blockade in NASH promotes necroptotic hepatocyte clearance by liver macrophages and decreases hepatic fibrosis.

Necroptosis contributes to hepatocyte death in nonalcoholic steatohepatitis (NASH), but the fate and roles of necroptotic hepatocytes (necHCs) in NASH remain unknown. We show here that the accumulation of necHCs in human and mouse NASH liver is associated with an up-regulation of the "don't-eat-me" ligand CD47 on necHCs, but not on apoptotic hepatocytes, and an increase in the CD47 receptor SIRPα on liver macrophages, consistent with impaired macrophage-mediated clearance of necHCs. In vitro, necHC clearance by primary liver macrophages was enhanced by treatment with either anti-CD47 or anti-SIRPα. In a proof-of-concept mouse model of inducible hepatocyte necroptosis, anti-CD47 antibody treatment increased necHC uptake by liver macrophages and inhibited markers of hepatic stellate cell (HSC) activation, which is responsible for liver fibrogenesis. Treatment of two mouse models of diet-induced NASH with anti-CD47, anti-SIRPα, or AAV8-H1-shCD47 to silence CD47 in hepatocytes increased the uptake of necHC by liver macrophages and decreased markers of HSC activation and liver fibrosis. Anti-SIRPα treatment avoided the adverse effect of anemia found in anti-CD47-treated mice. These findings provide evidence that impaired clearance of necHCs by liver macrophages due to CD47-SIRPα up-regulation contributes to fibrotic NASH, and suggest therapeutic blockade of the CD47-SIRPα axis as a strategy to decrease the accumulation of necHCs in NASH liver and dampen the progression of hepatic fibrosis.

Respiratory infections in X-linked hyper-IgM syndrome with CD40LG mutation: a case series of seven children in China

BackgroundX-linked hyper-immunoglobulin M (XHIGM), a primary immunodeficiency syndrome caused by mutations in the CD40 ligand gene(CD40LG), presents with recurrent respiratory infections in pediatric patients. We aimed to evaluate the spectrum of clinical features and respiratory pathogens in pediatric patients with XHIGM in China.MethodsWe retrospectively reviewed seven pediatric patients who were diagnosed with XHIGM and received follow-up treatment at the Guangzhou Women and Children’s Medical Center between January 2010 and January 2021. We determined their clinical characteristics, causative pathogens, and prognosis by performing peripheral immunological and genetic tests.ResultsThere were seven boys with age ranging from 4–20 months (median age, 13 months). Four of the seven respiratory infections were caused by Talaromyces marneffei(T. marneffei). Two patients had viral infections caused by cytomegalovirus (CMV) and human adenovirus respectively. One patient had a mixed infection caused by Pneumocystis carinii and CMV. Except for one child who died of respiratory failure, one patient received hematopoietic stem cell transplantation (HSCT) and recovered well, the other five patients survived with regular infusions of intravenous immunoglobulin (IVIg) during the follow-up period. Six patients had reduced antibody levels, especially IgG, IgA, and IgE levels. Increased serum IgM levels were detected in four cases, and three cases presented normal IgM levels at onset. All children were diagnosed with XHIGM with CD40LG variation. Three novel mutations were identified in the present study.ConclusionsOur study suggests that respiratory infections usually begin within 2 years old, fungi and viruses are important pathogens causing respiratory infections in children with XHIGM. In endemic areas, T. marneffei is the common pathogen of respiratory tract infection in children with the disease.

Relevance of Monitoring Thymic Function and Recovery in Interpreting Immune Reconstitution Post Hematopoietic Cell Transplantation in Inborn Errors of Immunity

Introduction Allogeneic hematopoietic cell transplantation (HCT) is the treatment of choice for many inborn errors of immunity (IEI). Impaired recovery of thymic function predicts poor clinical outcomes after HCT, especially in pediatric patients with IEI. CD31+CD4+CD45RA+ T cells are recognized as recent thymic emigrants (RTE) or true naïve T cells, and indicators of thymic output in HCT recipients. Reduced intensity-conditioning (RIC) and reduced toxicity-conditioning (RTC) regimens are increasingly used to limit organ toxicity and late effects of HCT. However, it is not known if they facilitate thymic recovery. Furthermore, graft-versus-host disease (GVHD) and its treatment can lead to prolonged thymic dysfunction. Methods Pediatric normal values and percentiles for a flow cytometry based RTE assay were developed. In this prospective study, we used RIC with fludarabine, melphalan, alemtuzumab, RTC with busulfan, fludarabine, alemtuzumab or myeloablative conditioning regimen (MAC) with busulfan, cyclophosphamide, alemtuzumab in patients with IEI. For GVHD prophylaxis cyclosporine A or tacrolimus together with mycophenolate mofetil were used. We assessed RTE immune reconstitution at 100 days, 6 months, 1 year and 2 years after HCT. Institutional review board of the University of Minnesota approved the study protocol. We hypothesized that thymic-derived immune reconstitution is negatively affected by occurrence of acute GvHD, age at transplantation (negatively with higher age) and type of conditioning (negatively with more intensive conditioning). Linear mixed model was performed for the repeated measures of RTE (after square root transformation) with covariates time (discrete), age at transplant (continuous), conditioning regimen, acute GHVD and all the second-order interactions with time. Results Between January 1st 2012 and December 31st 2021 45 patients with IEI were transplanted. Two patients did not receive the conditioning regimen per protocol, one patient received alemtuzumab only and two patients received no conditioning. These five patients were excluded from the final analysis. Thirteen (32%) of 40 patients were female. Median age at transplantation was 2.64 years (IQR 0.89 to 10.09). Median time to neutrophil engraftment was 12 days (IQR 10 to 14). Median follow-up time was 46.5 months (IQR 22.8 to 62.7). The overall survival was 82.5% (95%CI 66.8% to 91.2%). 13 (32%) received RIC, 23 (57%) RTC and 4 (10%) MAC. 10 (25%) patients had a matched related donor, 24 (60%) had a matched unrelated donor and 6 (15%) received an umbilical cord blood transplant. 7 (17.5%) patients had Chronic Granulomatous Disease, 5 (12.5%) Hemophagocytic lymphohistiocytosis, 5 (12.5%) Severe congenital neutropenia, 4 (10.0%) CD40 Ligand deficiency, 4 (10.0%) Severe combined immunodeficiency, 4 (10.0%) immunodeficiency not otherwise specified, 3 (7.5%) Wiskott-Aldrich syndrome, and 8 (20%) other primary immunodeficiency as underlying diagnosis. Patients with more intensive conditioning had lower RTE absolute values at one- and two-years post-transplant than patients with less intensive conditioning regimen (Figure 1A). Patients with acute GHVD occurring in the first 100 days after transplantation had lower RTE absolute values at one- and two-years post-transplant compared to patients without GVHD (Figure 1B). In the linear mixed model for the repeated measures of RTE (after square root transformation), we found at one-year post-transplantation a significant difference with increasing age (p<0.0001), between any grade acute GVHD and no acute GVHD (p=0.03) and between MAC and RIC (p=0.01) and RTC and RIC (p<0.01) (Table 1). These results remained robust at two-year follow-up as well. Conclusions Serial measurement of RTE is an useful immune reconstitution marker for assessment of thymic function after HCT. In patients with IEI, age at transplantation, occurrence of acute GHVD and intensity of conditioning regimen were strongly associated with slower thymic-derived immune reconstitution. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract IA005: Generating T cell responses against pancreatic cancer

Abstract The ultimate promise of tumor immunotherapy is dependent on the generation of an effective immune response against cancer, including CD4 and CD8 T cells. However, driving T cell responses in tumors such as pancreatic ductal adenocarcinoma (PDAC) requires overcoming significant hurdles in the tumor microenvironment (TME). Local and systemic suppression of dendritic cell function depresses endogenous T cell immunity against PDAC, which is further exacerbated by tumor cell intrinsic recruitment of suppressive myeloid cells and macrophages to the tumor site. To bypass these barriers, we’ve utilized agonistic anti-CD40 antibody, which mimics the endogenous CD40 ligand, to license and mature dendritic cells and re-educate tumor-associated myeloid cells towards a Th1/M1 phenotype in the PDAC TME. Using the KrasLSL-G12D/+,Trp53LSL-R172H/+,Pdx1-Cre (KPC) genetically engineered mouse model of pancreatic cancer, we show that combining standard-of-care chemotherapy with CD40 agonism promotes clonal T cell activation and expansion locally in the tumor site, resulting in regressions and cures of established tumors. This CD40-induced T cell response was further enhanced by the addition of dual immune checkpoint blockade (anti-PD-1 and anti-CTLA-4), but was independent of classical innate immune sensors such as Type I interferon receptor (IFNAR) and toll-like receptors (TLRs) previously thought to be critical for the generation of T cell immunity against tumors. These studies formed the preclinical rationale for trials investigating the use of agonistic CD40 therapy for patients with resectable or metastatic PDAC, and provided insight for immunological correlates of response in the clinical setting. More recently, we have reported on the negative impact of chemotherapy on the developing anti-tumor T cell response, emphasizing the need for additional investigations into the combination and sequencing of treatment modalities for enhanced outcomes in patients with PDAC. These studies reveal the potency of using an immunologically relevant mouse model to interrogate immune interventions in PDAC for patients with the greatest unmet clinical need. Citation Format: Katelyn T. Byrne. Generating T cell responses against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA005.

First-in-Human (FIH) Study of the Fully-Human Kappa-Lambda CD19/CD47 Bispecific Antibody TG-1801 in Patients (pts) with B-Cell Lymphoma

Introduction: Cancer cells elude anti-tumor immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors. Blockade of signal regulatory protein (SIRP)-α on macrophages, or of its ligand CD47 expressed on tumor cells, improves tumor cell elimination however CD47 is a challenging target given its ubiquitous distribution in healthy tissues. Combination of CD47 blockade with the anti-CD20 antibody rituximab has resulted in encouraging clinical activity (Advani 2018), and CD19 is also an established target of multiple B-NHL therapies. TG-1801 is a bispecific agent designed with a CD19 high-affinity arm and a blocking CD47 arm for concurrent target engagement on the tumor cells surface. Herein we report results of the FIH study for the first time. Methods: The primary objectives were to characterize the safety profile and to determine the recommended phase 2 dose (RP2D) of TG-1801. Other objectives included assessment of pharmacokinetics (PK), preliminary antitumor activity (Lugano 2014), and pharmacodynamics (PD: B-cell depletion). Eligible patients must have had relapsed/refractory (R/R) B-cell lymphoma after at least one prior standard therapy. A 3+3 design was utilized to evaluate sequentially higher doses of TG-1801 at the following dose levels: 20 mg, 60 mg, 180 mg, 360 mg, and 500 mg. In the monotherapy arm, treatment consisted of weekly fixed dosing 1h-infusions of TG-1801 in a 4-week cycle: day (D)1, D8, D15, and D22 of cycle (C) 1 and C2. Patients who had at least stable disease after 2 cycles were given the option of receiving TG-1801 once every 4 weeks for an additional 4 infusions. Intra-patient dose escalations were permitted. An additional arm was enrolled with the combination therapy of ublituximab (anti-CD20 mAb) plus TG-1801. Patients received TG-1801 and ublituximab infusions in a 4 week-cycle: D1, D8, and D15 of C1; D1 of C2 thru C6; and D1 of C9 and every 3 cycles thereafter, up to 24 cycles. A 3+3 design was also utilized to evaluate two dose levels of TG-1801 (300 mg and 400 mg) with a standard dose of ublituximab (900 mg). Results: As of May 2022, 14 pts (MZL = 5, DLBCL including Richter's transformation = 5, FL = 4) received monotherapy and 16 pts received combination therapy (DLBCL = 9, FL = 4, MZL = 2, MCL = 1). Patients were heavily pretreated: median 3 prior systemic therapies (range, 1 - 8), all had received ≥1 prior anti-CD20; 14 pts (46%) were refractory to their last prior therapy. Twenty-one (70%) had extranodal disease, and 9 (30%) had bulky disease. One DLT of grade (G) 4 thrombocytopenia occurred at the 500 mg dose level. The most common (>20%) treatment emergent adverse events (TEAEs) at monotherapy doses of 20 mg to 360 mg (n=10) included fatigue, thrombocytopenia, and infusion-related reaction (3 pts each), with no G3/4 TEAE occurring in >20% of pts. Combination therapy was also well tolerated (TG-1801 at 300 mg N=3, 400 mg N=13) without DLT. The most common TEAE (≥20%) for combination therapy were: anemia (31%), headache, abdominal pain, fatigue, and thrombocytopenia (25% each), with no G3/4 TEAE occurring in >20% of pts. There were 2 permanent treatment discontinuations due to TEAE (1 infusion reaction [500 mg monotherapy], 1 rash [360 mg monotherapy]). Three partial responses (PR) were observed on monotherapy (n=14), all at 360 mg TG-1801, including 1 MZL pt with 7 prior lines that included PI3Ki, BTKi, auto transplant, and multiple prior anti-CD20s. All 3 pts completed 6 cycles of treatment and continued to further cycles of re-treatment. In the combination arm (n=16), a 44% response rate was observed with 1 complete response (CR) in a pt with FL and 6 PR (5 pts with DLBCL and 1 pt with FL) for a 56% ORR for DLBCL patients and 50% ORR for FL patients. The median duration of combination therapy exposure was 8.7 mo (range of 1-20 mo). Responses appear durable, with ten pts remaining on study, with 1 pt receiving 360 mg monotherapy and 9 pts receiving combination therapy. Dose dependent increases in exposure were observed and pharmacodynamic analysis demonstrated a >50% depletion of circulating lymphocytes within 7 days of the first infusion in all evaluable pts. Conclusions: TG-1801 monotherapy and combination therapy demonstrates clinical activity, particularly in relapsed and refractory DLBCL, with an acceptable preliminary safety profile. This study (NCT03804996) has completed enrollment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Do Levels of the Platelet Activation Markers sCD40 L and SCUBE 1 Differ between Laboratory-Confirmed and Clinically Diagnosed COVID-19 Patients?

Aim: Thromboembolic complications are an important cause of mortality and morbidity in coronavirus disease 2019 (COVID-19) patients. The purpose of this study was to compare levels of the platelet activation markers soluble CD40 ligand (sCD 40L) and signal peptide-CUB-EGF domain-containing protein 1 (SCUBE 1) and hematological parameters between laboratory-confirmed and clinically diagnosed COVID-19 patients. No previous studies have investigated levels of these markers in laboratory-confirmed and clinically diagnosed COVID-19 patients. Material and Method: Fifty-one laboratory-confirmed and clinically diagnosed COVID-19 patients with no exclusion criteria were enrolled in the study. Blood specimens were collected for SCUBE1, sCD40 L, and hematological and biochemical parameter measurement. These parameters from laboratoryconfirmed and clinically diagnosed COVID-19 patients were then compared. Results: SCUBE1 and sCD40L levels were significantly higher in the laboratory-confirmed group compared to the clinically diagnosed group (p&lt;0.05 and p =0.005, respectively). Time elapsing between onset of symptoms and presentation to hospital was significantly shorter in the laboratory-confirmed group, while rates of contact with COVID-19 patients were significantly higher (p &lt;0.001 and p &lt;0.005, respectively). SCUBE 1 levels were significantly negatively correlated with ferritin and C-reactive protein (CRP) (p&lt; 0.05, r= - 0.322 and p&lt; 0.05, r= - 0.351, respectively). Conclusion: This study shows, for the first time in the literature, that levels of the platelet activation markers SCUBE1 and sCD40L are significantly higher in laboratory-confirmed COVID-19 patients compared to clinically diagnosed individuals.

UNDETECTABLE IGE AS A SENTINEL BIOMARKER FOR HUMORAL IMMUNODEFICIENCY

<h3>Introduction</h3> The clinical significance of undetectable IgE (<2 IU/mL) in patients with otherwise normal immunoglobulins is unclear. We hypothesize that undetectable IgE reflects an impairment in isotype switching that could portend future development of a broader defect in humoral immunity. <h3>Methods</h3> We recruited 17 healthy adults with IgE <2 IU/mL. We compared their response to immunization with a Salmonella typhi polysaccharide vaccine relative to 16 randomly selected adults with IgE >2 IU/mL. We assessed pre-and post-vaccine IgG titers 4-6 weeks after vaccination, with a normal Salmonella typhi IgG index (STIGG index) defined as a >2-fold increase in titers. We isolated peripheral blood lymphocytes from 14 patients with undetectable IgE and induced IgE production <i>in vitro</i> via stimulation with CD40 ligand (CD40L) and IL-4. IgE production was assessed through semi-quantitative PCR of ε-germline and IgE heavy chain transcripts. <h3>Results</h3> 4/17 patients with undetectable IgE had a STIGG index ≤2, compared with 0/16 patients in the control group (p-value = 0.0582, Fisher's exact test). ε-germline and IgE heavy chain mRNA were detected in peripheral lymphocytes from all of the assessed patients with undetectable IgE. <h3>Conclusion</h3> An impaired polysaccharide vaccine response occurred more frequently in patients with undetectable IgE than in patients with an IgE >2 IU/mL, but the difference in this small pilot study was nonsignificant. A larger sample size is needed to clarify these findings. Peripheral blood lymphocytes isolated from patients with undetectable IgE are capable of producing IgE transcripts in response to CD40L/IL-4 stimulation.

GM-CSF-activated human dendritic cells promote type 1 T follicular helper cell polarization in a CD40-dependent manner.

T follicular helper (Tfh) cells regulate humoral responses and present a marked phenotypic and functional diversity. Type 1 Tfh (Tfh1) cells were recently identified and associated with disease severity in infection and autoimmune diseases. The cellular and molecular requirements to induce human Tfh1 differentiation are not known. Here, using single-cell RNA sequencing (scRNAseq) and protein validation, we report that human blood CD1c+ dendritic cells (DCs) activated by GM-CSF (also known as CSF2) drive the differentiation of naive CD4+ T cells into Tfh1 cells. These Tfh1 cells displayed typical Tfh molecular features, including high levels of PD-1 (encoded by PDCD1), CXCR5 and ICOS. They co-expressed BCL6 and TBET (encoded by TBX21), and secreted large amounts of IL-21 and IFN-γ (encoded by IFNG). Mechanistically, GM-CSF triggered the emergence of two DC sub-populations defined by their expression of CD40 and ICOS ligand (ICOS-L), presenting distinct phenotypes, morphologies, transcriptomic signatures and functions. CD40High ICOS-LLow DCs efficiently induced Tfh1 differentiation in a CD40-dependent manner. In patients with mild COVID-19 or latent Mycobacterium tuberculosis infection, Tfh1 cells were positively correlated with a CD40High ICOS-LLow DC signature in scRNAseq of peripheral blood mononuclear cells or blood transcriptomics, respectively. Our study uncovered a novel CD40-dependent Tfh1 axis with potential physiopathological relevance to infection. This article has an associated First Person interview with the first author of the paper.

Selected Factors of Vascular Changes: The Potential Pathological Processes Underlying Primary Headaches in Children.

The prevalence, social consequences and complicated pathogenesis make headaches in children a significant clinical issue. Studies in adults suggest that primary headaches could be the first sign of atherosclerosis and platelet aggregation. To analyze the blood levels of selected biomarkers of vascular changes potentially associated with a higher risk of atherosclerosis in children with primary headaches. The medical family history, brain-derived neurotrophic factor (BDNF), soluble CD40 ligands (sCD40L), endothelial plasminogen activator inhibitor (PAI I), vascular endothelial growth factor (VEGF) and intima-media thickness (IMT) measurements were performed in the 83 children (52 with primary headaches, 31 controls). Selected factors were compared with basic laboratory parameters that are potentially related to atherosclerosis: C-reactive protein (CRP) and lipid concentration. There were no significant differences in biomarkers of vascular changes in the study group and controls in general. In the study group, boys had a higher BDNF level than girls (p = 0.046). Normal-weight migraine patients had significantly higher PAI-I levels than controls (p = 0.034). A positive correlation between PAI-1 and triglycerides (TG) was observed. IMT did not differ between children with primary headaches and controls; however, IMT showed a positive correlation with BMI z-score and TG. Children with headaches had, more often, a positive family history of cardiovascular disease (p = 0.049). There were no clear clinical changes indicative of atherosclerosis in the study population. However, some trends are visible. Primary headaches are more often related to a family history of cardiovascular diseases. IMT is associated with TG levels and BMI z-score. The measured biomarkers of vascular changes show mutual relations.

A phase I study of an adenoviral vector delivering a MUC1/CD40-ligand fusion protein in patients with advanced adenocarcinoma

Cancer vaccines as immunotherapy for solid tumours are currently in development with promising results. We report a phase 1 study of Ad-sig-hMUC1/ecdCD40L (NCT02140996), an adenoviral-vector vaccine encoding the tumour-associated antigen MUC1 linked to CD40 ligand, in patients with advanced adenocarcinoma. The primary objective of this study is safety and tolerability. We also study the immunome in vaccinated patients as a secondary outcome. This trial, while not designed to determine clinical efficacy, reports an exploratory endpoint of overall response rate. The study meets its pre-specified primary endpoint demonstrating safety and tolerability in a cohort of 21 patients with advanced adenocarcinomas (breast, lung and ovary). The maximal dose of the vaccine is 1 ×1011 viral particles, with no dose limiting toxicities. All drug related adverse events are of low grades, most commonly injection site reactions in 15 (71%) patients. Using exploratory high-dimensional analyses, we find both quantitative and relational changes in the cancer immunome after vaccination. Our data highlights the utility of high-dimensional analyses in understanding and predicting effective immunotherapy, underscoring the importance of immune competency in cancer prognosis.

CD44 and CD221 directed magnetic cubosomes for the targeted delivery of helenalin to rhabdomyosarcoma cells

Confining chemotherapy to tumour sites by means of active targeting nanoparticles (NPs) may increase the treatment effectuality while reducing potential side effects. Cubosomes are one of the next-generation drug delivery nanocarriers by virtue of their biocompatibility and bioadhesion, sizeable payload encapsulation and high thermostability. Herein, an active tumour targeting system towards rhabdomyosarcoma (RMS) cells was evaluated. Cubosomes were loaded with helenalin (a secondary metabolite from Arnica plants), which we have previously shown to induce apoptosis in RMS cells. The functionalization of the cubosomes was accomplished to enable binding to membrane receptors and translocation under a magnetic field. RMS cells overexpress CD44 and CD221 on their membrane surface and, therefore, hyaluronic acid (HA, a ligand for CD44) and antibodies (Abs) against CD221 were coupled to cubosomes via electrostatic attraction and the thiol-Michael reaction, respectively. Magnetization of the cubic phase NPs was achieved by embedding superparamagnetic iron oxide NPs (SPIONPs) into the cubic matrix. Single-function and multi-function cubosomes had Im3m cubic phase structures with well-organized lattice patterns. Conjugation with 2% HA or anti-CD221 half Abs and/or 1% SPIONPs showed significantly higher uptake into RMS cells compared to unfunctionalized cubosomes. CD44 and CD221 directed magnetic (triple-function) cubosomes were capable of internalizing into RMS cells in an energy-independent mechanism. Helenalin-laden triple functionalized cubosomes showed limited impact on the viability of control fibroblast cells, while they induced a high degree cytotoxicity against RMS cells. Profound tumour cell death was observed in both two-dimensional (2D) culture and three-dimensional (3D) tumour spheroids.

Mechanisms of CD40-dependent cDC1 licensing beyond costimulation.

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

Impaired TIGIT expression on B cells drives circulating follicular helper T cell expansion in multiple sclerosis.

B cell depletion in patients with relapsing-remitting multiple sclerosis (RRMS) markedly prevents new MRI-detected lesions and disease activity, suggesting the hypothesis that altered B cell function leads to the activation of T cells driving disease pathogenesis. Here, we performed comprehensive analyses of CD40 ligand– (CD40L-) and IL-21–stimulated memory B cells from patients with MS and healthy age-matched controls, modeling the help of follicular helper T cells (Tfh cells), and found a differential gene expression signature in multiple B cell pathways. Most striking was the impaired TIGIT expression on MS-derived B cells mediated by dysregulation of the transcription factor TCF4. Activated circulating Tfh cells (cTfh cells) expressed CD155, the ligand of TIGIT, and TIGIT on B cells revealed their capacity to suppress the proliferation of IL-17–producing cTfh cells via the TIGIT/CD155 axis. Finally, CCR6+ cTfh cells were significantly increased in patients with MS, and their frequency was inversely correlated with that of TIGIT+ B cells. Together, these data suggest that the dysregulation of negative feedback loops between TIGIT+ memory B cells and cTfh cells in MS drives the activated immune system in this disease.

Role of CD40 ligand-mediated endothelial cell-monocyte interaction at atherosclerosis predilection sites

BackgroundMonocyte recruitment into the vessel wall at atherosclerosis predilection sites is essential for lesion development in the early phase of atherosclerosis. Platelets interacting with ultra-large von Willebrand Factor (ULVWF) multimers deposited after CD40 receptor ligation on the endothelial surface form adhesive bridges and facilitate monocyte diapedesis. We hypothesise that enhanced endothelial CD40 expression at arterial bifurcations is responsible for monocyte recruitment and that its absence reduces susceptibility to atherosclerosis. MethodsY-shaped channel slides covered with endothelial cells (HUVEC) and isolated perfused carotid artery bifurcations from different mouse lines were used for adhesion studies with isolated fluorescent dye-labelled platelets and monocytes. Monocyte adherence was quantified via fluorescence imaging. Oil Red O staining visualised aortic atherosclerotic plaques, and mRNA expression was determined by qRT-PCR. ResultsIn response to soluble CD40 ligand (sCD40L) stimulated ULVWF release, the number of monocytes bound distal to the bifurcation of the Y-slide was 1.8-fold greater than without stimulation. The number of adherent monocytes in sCD40L-treated carotid artery bifurcations was 6 to 12.3-fold greater in ApoE knockout mice as compared to bifurcations derived from CD40/ApoE-deficient or control mice. CD40 mRNA expression was 2-fold higher in carotid artery bifurcations of ApoE knockout mice as compared to the proximal unbranched segment. Introduction of the CD40 knockout into the ApoE-/- background reduced the atherosclerosis burden along the entire aorta of these mice by 60 %. ConclusionsOur data demonstrate the importance of endothelial CD40 expression at atherosclerosis predilection sites for endothelial cell-platelet-monocyte interaction in the early phase of atherosclerosis.

Inflammatory and Prothrombotic Biomarkers, DNA Polymorphisms, MicroRNAs and Personalized Medicine for Patients with Peripheral Arterial Disease.

Classical risk factors play a major role in the initiation and development of atherosclerosis. However, the estimation of risk for cardiovascular events based only on risk factors is often insufficient. Efforts have been made to identify biomarkers that indicate ongoing atherosclerosis. Among important circulating biomarkers associated with peripheral arterial disease (PAD) are inflammatory markers which are determined by the expression of different genes and epigenetic processes. Among these proinflammatory molecules, interleukin-6, C-reactive protein, several adhesion molecules, CD40 ligand, osteoprotegerin and others are associated with the presence and progression of PAD. Additionally, several circulating prothrombotic markers have a predictive value in PAD. Genetic polymorphisms significantly, albeit moderately, affect risk factors for PAD via altered lipoprotein metabolism, diabetes, arterial hypertension, smoking, inflammation and thrombosis. However, most of the risk variants for PAD are located in noncoding regions of the genome and their influence on gene expression remains to be explored. MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that modulate gene expression at the post-transcriptional level. Patterns of miRNA expression, to some extent, vary in different atherosclerotic cardiovascular diseases. miRNAs appear to be useful in the detection of PAD and the prediction of progression and revascularization outcomes. In conclusion, taking into account one’s predisposition to PAD, i.e., DNA polymorphisms and miRNAs, together with circulating inflammatory and coagulation markers, holds promise for more accurate prediction models and personalized therapeutic options.