TgMIC6 inhibition of autophagy is partially responsible for the phenotypic differences between Chinese 1 Toxoplasma gondii strains.

Chinese1 is the predominant Toxoplasma gondii lineage in China, and significant phenotypic differences are observed within the lineage. WH3 and WH6 are two representative strains of Chinese 1, which exhibit divergent virulence and pathogenicity in mice. However, virulence determinants and their modulating mechanisms remain elusive. A global genome expression analysis of the WH3 and WH6 transcriptional profiles identified microneme secretory protein 6 (MIC6), which may be associated with the phenotypic difference observed in WH3. In the present study, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technique was used to generate a T. gondii microneme secretory protein (TgMIC6) knockout in WH3. Wild-type mice and different mouse and human cell lines were infected with the WH3, WH3-Δmic6, and WH6 strains. The survival rate of mice, related cytokine levels in serum, and the proliferation of parasites were observed. These results suggested that TgMIC6 is an important effector molecule that determines the differential virulence of WH3 in vivo and in vitro. Furthermore, MIC6 may enhance WH3 virulence via inhibition of host cell autophagy and activation of key molecules in the epidermal growth factor receptor (EGFR)-Akt-mammalian target of rapamycin (mTOR) classical autophagy pathway. CD40L was cleared in vivo by i.p injection of CD40L monoclonal antibody, and it was found that the virulence of WH3-Δmic6 to mice was restored to a certain extent in the absence of CD40L. This study elucidates the virulence determinants and immune escape strategies of Toxoplasma gondii in China. Moreover, these data will aid the development of effective strategies for the prevention and control of toxoplasmosis.

Platelet transfusion enhances pro-aggregatory status shortly after coronary 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-ß1) was also conducted using western blotting. Patients with platelet transfusion showed increased levels of P-selectin, CD40L and intra-platelet TGF-ß1 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-ß1, 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-ß1.

Synergy and antagonism in the integration of BCR and CD40 signals that control B-cell proliferation.

In response to infection or vaccination, a successful antibody response must enrich high-affinity antigen-reactive B-cells through positive selection, but eliminate auto-reactive B-cells through negative selection. B-cells receive signals from the B-cell receptor (BCR) which binds the antigen, and the CD40 receptor which is stimulated by neighboring T-cells that also recognize the antigen. How BCR and CD40 signaling are integrated quantitatively to jointly determine B-cell fate decision and proliferation remains unclear. To investigate this, we developed a differential-equations-based model of the BCR and CD40 signaling networks activating NFκB. Our model accurately recapitulates the NFκB dynamics of B-cells stimulated through their BCR and CD40 receptors, correctly predicting that costimulation induces more NFκB activity. However, when linking it to established cell fate decision models of cell survival and cell cycle control, it predicted potentiated population expansion that was not observed experimentally. We found that this discrepancy was due to a time-dependent functional antagonism exacerbated by BCR-induced caspase activity that can trigger apoptosis in founder cells, unless NFκB-induced survival gene expression protects B-cells in time. Guided by model predictions, sequential co-stimulation experiments revealed how the temporal dynamics of BCR and CD40 signaling control the fate decision between negative and positive selection of B-cell clonal expansion. Our quantitative findings highlight a complex non-monotonic integration of BCR and CD40 signals that is controlled by a balance between NFκB and cell-death pathways, and suggest a mechanism for regulating the stringency of B-cell selection during an antibody response.

Analysis of the expression of cytokines and chemokines, platelet-leukocyte aggregates, sCD40L and sCD62P in cutaneous melanoma.

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.

Iscalimab Combined With Transient Tesidolumab Prolongs Survival in Pig-to-Rhesus Monkey Renal Xenografts.

OBJECTIVE: To evaluate the clinically relevant anti-CD40 antibody iscalimab for baseline immunosuppression in a preclinical pig-to-rhesus renal xenograft model. SUMMARY BACKGROUND DATA: CD40/CD40L co-stimulation blockade-based immunosuppression has been more successful than calcineurin-based protocols in prolonging xenograft survival in preclinical models. METHODS: GGTA1 knockout/CD55 transgenic pig kidneys were transplanted into rhesus monkeys (n = 6) receiving an iscalimab-based immunosuppressive regimen. RESULTS: Two grafts were lost early (22 and 26 days) because of ectatic donor ureters with otherwise normal histology. The other recipients survived 171, 315, 422, and 439 days with good renal function throughout the posttransplant course. None of the recipients experienced serious infectious morbidity. CONCLUSIONS: It may be reasonable to evaluate an iscalimab-based immunosuppressive regimen in clinical renal xenotransplantation.

Trained immunity is regulated by T cell-induced CD40-TRAF6 signaling.

Trained immunity is characterized by histone modifications and metabolic changes in innate immune cells following exposure to inflammatory signals, leading to heightened responsiveness to secondary stimuli. Although our understanding of the molecular regulation of trained immunity has increased, the role of adaptive immune cells herein remains largely unknown. Here, we show that T cells modulate trained immunity via cluster of differentiation 40-tissue necrosis factor receptor-associated factor 6 (CD40-TRAF6) signaling. CD40-TRAF6 inhibition modulates functional, transcriptomic, and metabolic reprogramming and modifies histone 3 lysine 4 trimethylation associated with trained immunity. Besides in vitro studies, we reveal that single-nucleotide polymorphisms in the proximity of CD40 are linked to trained immunity responses in vivo and that combining CD40-TRAF6 inhibition with cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4-Ig)-mediated co-stimulatory blockade induces long-term graft acceptance in a murine heart transplantation model. Combined, our results reveal that trained immunity is modulated by CD40-TRAF6 signaling between myeloid and adaptive immune cells and that this can be leveraged for therapeutic purposes.

Combination of Anti-CD40 and Anti-CD40L Antibodies as Co-Stimulation Blockade in Preclinical Cardiac Xenotransplantation.

The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac xenotransplantation. However, it is still unclear which single antibody directed against CD40 or CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For example, the high doses of antibody administered in previous experiments might not be feasible for the treatment of humans, while thrombotic side effects were described for first-generation anti-CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy. Importantly, no incidence of thromboembolic events associated with the administration of the anti-CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two were terminated deliberately after 90 days with the baboons in excellent condition and two were extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical signs, histopathology revealed fungal infections in all four recipients. This study provides, for the first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this immune checkpoint.

The role of immune cells and immune related genes in the tumor microenvironment of papillary thyroid cancer and their significance for immunotherapy.

Papillary thyroid carcinoma (PTC) is the most common pathological type of thyroid cancer (THCA) and shows a better prognosis than other types. However, further research is needed to determine the risk of PTC. We herein used the CIBERSORT algorithm to analyze the gene-expression profile obtained from TCGA, estimated the infiltration ratio of 22 immune cell types in tumor tissues and normal tissues, analyzed the differential expression of immune-related genes, and identified immune cells and immune-related genes related to clinical progress and prognosis. We uncovered 12 immune cell types and nine immune-related genes that were closely correlated with TNM staging, and two immune cell types (activated NK cells and γδT cells) and one immune-related gene (CD40LG) that were associated with prognosis. After evaluation, four immune cell types could be used to determine low-risk PTC, with six immune cell types and six immune-related genes closely associated with high-risk PTC. The type and quantity of infiltrating immune cells in the microenvironment of PTC, as well as immune-related genes, appear to be closely related to tumor progression and can therefore be used as important indicators for the evaluation of patient prognosis. We posit that the study of immune cells and immune-related genes in the tumor microenvironment will facilitate the determination of low-risk PTC more accurately, and that this will greatly promote the development of high-risk PTC immunotherapy.

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.

Effect of Diminazene Aceturate, an ACE2 activator, on platelet CD40L signaling induced glial activation in rat model of hypertension.

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

Differential response of IgM and IgG memory B cell populations to CD40L: insights of T cell - memory B cell interactions.

Memory B cells (mBCs) are characterized by their long-term stability, fast reactivation, and capability to rapidly differentiate into antibody-secreting cells (ASCs). However, the role of T cells in the differentiation of mBCs, in contrast to naive B cells, remains to be delineated. We study the role of T cells in mBC responses, using CD40L stimulation and autologous T-B co-cultures. Our results showed that increased CD40L levels led to a selective increased proliferation of IgM+ mBC, which did not class-switched, resulting in higher frequencies of IgM+ ASCs and a lower frequency of IgG+ ASCs. The IgG+/IgA+ mBCs were unaffected. We further compared the transcription of immune-related genes in IgM+ and IgG+ pre-plasmablasts cultured at high (500 ng/mL) and low (50 ng/mL) CD40L levels. In response to increased CD40L levels, both populations exhibited a core response to genes related to activation (TRAF1, AKT3, CD69, and CD80). However, they differed in genes related to cytokine/chemokine/homing interactions (CCL3/4/17, LTA, NKX2-3, BCL2 and IL21R) and cell-cell interactions (HLADR, CD40, and ICOSL), which were largely confined to IgG+ cells. Our findings revealed that in co-cultures with a high T-ratio, the response was similar to that found in cultures with high CD40L levels. These results suggest that IgG+ mBCs have a greater capacity for proliferation and T cell interaction, and weaker migration capabilities, leading to a preference for an IgG response over IgM in the short term. This adaptable response could fine-tune the memory repertoire with different functions of IgG versus IgM mBCs.

JAK inhibitors attenuate hyperactivation of nonswitched memory B cells in rheumatoid arthritis patients in remission.

OBJECTIVE: To investigate the distribution and activation of B-cell subpopulations in rheumatoid arthritis (RA) patients treated with Janus kinase inhibitors (JAKis) and to analyze their correlation with disease remission. METHODS: Peripheral blood samples were collected from 23 adult healthy controls and 58 RA patients, 31 of whom were treated with JAKis and assessed during a 24-month follow-up. The number of peripheral B-cell subpopulations (including naive B cells, nonswitched memory B (NSMB) cells, switched memory B cells, and double-negative B cells), their activation, and phosphorylation of SYK and AKT upon B-cell receptor (BCR) stimulation in each population were analyzed by flow cytometry. RESULTS: Compared with that in healthy controls, the frequency of NSMB cells was significantly lower in new-onset untreated RA patients. However, expression of CD40, CD80, CD95, CD21low and pAKT significantly increased in these NSMB cells. Additionally, the number of NSMB cells correlated negatively with DAS28-ESR and IgG and IgA levels in these patients; expression of CD80, CD95 and CD21low on NSMB cells correlated positively with DAS28-ESR and IgG and IgA levels. After treatment with JAKis, the serum IgG concentration significantly decreased in RA patients in remission, but CD40, CD95 and pAKT levels in NSMB cells significantly decreased. CONCLUSION: RA patients present different B-cell subpopulations, in which the frequency of NSMB cells is negatively associated with disease activity. However, treatment with JAKis can inhibit activation of NSMB cells, restore the balance of kinase phosphorylation, and facilitate disease remission in RA patients.

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.

Intratumoral NKT cell accumulation promotes antitumor immunity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a potentially lethal disease lacking effective treatments. Its immunosuppressive tumor microenvironment (TME) allows it to evade host immunosurveillance and limits response to immunotherapy. Here, using the mouse KRT19-deficient (sgKRT19-edited) PDA model, we find that intratumoral accumulation of natural killer T (NKT) cells is required to establish an immunologically active TME. Mechanistically, intratumoral NKT cells facilitate type I interferon (IFN) production to initiate an antitumor adaptive immune response, and orchestrate the intratumoral infiltration of T cells, dendritic cells, natural killer cells, and myeloid-derived suppressor cells. At the molecular level, NKT cells promote the production of type I IFN through the interaction of their CD40L with CD40 on myeloid cells. To evaluate the therapeutic potential of these observations, we find that administration of folinic acid to mice bearing PDA increases NKT cells in the TME and improves their response to anti-PD-1 antibody treatment. In conclusion, NKT cells have an essential role in the immune response to mouse PDA and are potential targets for immunotherapy.

Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness.

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation, which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared with conventional CARs and demonstrated the up-regulated genes associated with T cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B cell lymphoblastic leukemia and B cell non-Hodgkin lymphoma xenograft models while demonstrating persistent T cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

Assembly and activation of EBV latent membrane protein 1.

Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.

The expression of immune co-stimulators as a prognostic predictor of head and neck squamous cell carcinomas and oral squamous cell carcinomas.

Background/purpose: T cells require second immune checkpoint molecules for activation and immune memory after antigen presentation. We found that inducible co-stimulator (ICOS) has been a favorable prognostic factor amongst B7 immune checkpoint co-stimulators (ICSs) families in head and neck squamous cell carcinoma (HNSCC) and oral SCC (OSCC). Materials and methods: This study analyzed the expression of non-B7 tumor necrosis factor (TNF) superfamily ICSs in the Cancer Genome Atlas (TCGA) HNSCC cohort, our OSCC cohort, and TCGA pan-cancer datasets. The correlation in expression, prognosis, and immune status was assessed. Results: The higher expression of CD27, CD30, CD40L, death domain 3 (DR3), and OX40, presumably on the T cell surface, defined better overall survival of HNSCC patients. Besides, CD27, CD30, CD40L, and OX40 were highly correlated with ICOS expression in tumors. CD27, CD40L, and DR3 expression are higher in HPV+ HNSCC tumors than in HPV- tumors. The combined expression level of CD27/OX40 or CD27/CD40L/OX40 enables the potent survival prediction of small, less nodal involvement, early stage, and HPV + tumor subsets. Tumors expressing high CD27, CD30, CD40L, ICOS, and OX40 exhibited enhanced immune cell infiltration. The high correlation in the expression of these ICSs was also noted in the vast majority of tumor types in TCGA datasets. Conclusion: The findings of this study not only confirm the potential of the concordant stimulation of CD27, CD30, CD40L, ICOS, and OX40 as a crucial strategy in cancer immunotherapy but also inspire further exploration into the field, highlighting the promising future of cancer treatment.

Investigation of activation-induced markers (AIM) in porcine T cells by flow cytometry.

Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25+CD69+CD40L+TNF-α- T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25+CD69+TNF-α- T cells were identified. Within CD8α+ CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs.

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.

Efficacy of IFN-γ, sCD40L, and Poly(I:C) Treated Bone Marrow-Derived Macrophages in Murine Mammary Carcinoma.

INTRODUCTION: Here, we explored methods to generate anti-tumor bone marrow-derived macrophages (BMDM) and how delivery of the BMDM at early tumor sites could impact disease progression. METHODS: BMDM treated with IFN-γ, sCD40L, poly(I:C), and a combination of the three were assessed. RESULTS: Treatment with sCD40L had no significant impact on the BMDM. Treating BMDM with IFN-γ impacted IL-1ß, MHC Class II, and CD80 expression. While poly(I:C) treatment had a greater impact on the BMDM than IFN-γ when assessed by the in vitro assays, the BMDM treated with poly (I:C) had mixed results in vivo where they decreased growth of the EMT6 tumor, did not impact growth of the 168 tumor, and enhanced growth of the 4T1 tumor. The combination of poly(I:C), IFN-γ, and sCD40L had the greatest impact on the BMDM in vitro and in vivo. Treatment with all three agonists resulted in increased IL-1ß, TNF-α, and IL-12 expression, decreased expression of arginase and mrc, increased phagocytic activity, nitrite production, and MHC Class II and CD80 expression, and significantly impacted growth of the EMT6 and 168 murine mammary carcinoma models. DISCUSSION: Collectively, these data show that treating BMDM with poly(I:C), IFN-γ, and sCD40L generates BMDM with more consistent anti-tumor activity than BMDM generated with the individual agonists.