CD40 Ligand Expression Research Articles

ThPOK represses CXXC5, which induces methylation of histone H3 lysine 9 in Cd40lg promoter by association with SUV39H1: implications in repression of CD40L expression in CD8+ cytotoxic T cells.

CD40 ligand is induced in CD4(+) Th cells upon TCR stimulation and provides an activating signal to B cells, making CD40 ligand an important molecule for Th cell function. However, the detailed molecular mechanisms, whereby CD40 ligand becomes expressed on the cell surface in T cells remain unclear. Here, we showed that CD40 ligand expression in CD8(+) cytotoxic T cells was suppressed by combined epigenetic regulations in the promoter region of the Cd40lg gene, such as the methylation of CpG dinucleotides, histone H3 lysine 9, histone H3 lysine 27, and histone H4 lysine 20. As the transcription factor Th-inducing pox virus and zinc finger/Kruppel-like factor (encoded by the Zbtb7b gene) is critical in Th cell development, we focused on the role of Th-inducing pox virus and zinc finger/Kruppel-like factor in CD40 ligand expression. We found that CD40 ligand expression is moderately induced by retroviral Thpok transduction into CD8(+) cytotoxic T cells, which was accompanied by a reduction of histone H3 lysine 9 methylation and histone H3 lysine 27 methylation in the promoter region of the Cd40lg gene. Th-inducing pox virus and zinc finger/Kruppel-like factor directly inhibited the expression of murine CXXC5, a CXXC-type zinc finger protein that induced histone H3 lysine 9 methylation, in part, through an interaction with the histone-lysine N-methyltransferase SUV39H1. In addition, to inhibit CD40 ligand induction in activated CD4(+) T cells by the CXXC5 transgene, our findings indicate that CXXC5 was one of the key molecules contributing to repressing CD40 ligand expression in CD8(+) cytotoxic T cells.

Specific CD4+T-Cell Reactivity and Cytokine Release in Different Clinical Presentations of Leptospirosis

Clinical manifestations of leptospirosis are highly variable: from asymptomatic to severe and potentially fatal. The outcome of the disease is usually determined in the immunological phase, beginning in the second week of symptoms. The underlying mechanisms, predictive factors, and individual immune responses that contribute to clinical variations are not well understood. The aim of this study was to determine the specifics of CD4(+) T-cell reactivity and cytokine release after stimulation with leptospiral antigens in patients with leptospirosis of different disease severities (patients with mild and severe symptoms) and in control subjects (with and without proven exposure to Leptospira). Whole-blood specimens were stimulated with Leptospira antigens in vitro. Subsequently, intracellular staining of cytokines was performed, and flow cytometry was used to assess the expression of CD40 ligand (CD40L) and the production of gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-2, and tumor necrosis factor alpha (TNF-α) by CD4(+) T cells. The production of inflammatory cytokines such as TNF-α by CD4(+) T cells after stimulation with leptospiral antigens was highest in patients with severe disease. In contrast, the ratio of IL-10 production to TNF-α production was higher in exposed subjects than in patients with mild and severe disease. Levels of proinflammatory cytokines such as TNF-α may be useful markers of the severity of the immunological phase of leptospirosis. IL-10 production by T cells after antigen-specific stimulation may indicate a more successful downregulation of the inflammatory response and may contribute to an asymptomatic course of the disease.

Effects of lipid-lowering treatment on circulating microparticles in patients with diabetes mellitus and chronic kidney disease.

Elevated levels of circulating microparticles (MPs) may contribute to the high cardiovascular risk in diabetes mellitus (DM) and chronic kidney disease (CKD). Therefore, we investigated the effects of lipid-lowering treatment (LLT) with simvastatin alone (S) or with ezetimibe (S+E) on MPs in DM patients with or without CKD. After a placebo run-in period, 18 DM patients with an estimated glomerular filtration rate (eGFR) of 15-59 mL/min (CKD stages 3-4) (DM-CKD) and 21 DM patients with eGFR >75 mL/min (DM-only) were treated with S and S+E in a randomized, double-blind, crossover study. MPs from platelets, monocytes and endothelial cells (PMPs, MMPs and EMPs), and their expression of phosphatidylserine (PS), P-selectin, CD40 ligand (CD40L) and tissue factor (TF) were measured by flow cytometry. At baseline, all types of MPs, except TF-positive MMPs, were elevated in DM-CKD compared with DM-only patients. All MPs, regardless of origin and phenotype, were inversely correlated with eGFR. S reduced the expression of P-selectin, TF and CD40L on PMPs and of TF on MMPs in both patient groups. S+E had no further effect. S also reduced total PS-positive procoagulant MPs, PMPs and MMPs in DM-CKD but not in DM-only patients. DM patients with CKD stages 3-4 had elevated PMPs, EMPs and MMPs compared with DM patients with normal GFR. Simvastatin reduced procoagulant MPs, MMPs and PMPs in DM-CKD patients, suggesting a beneficial reduction of hypercoagulability in this high-risk patient group. Differences between DM-CKD and DM-only patients were counteracted by LLT.

Effect of Danhong Injection Combined with Naoxintong Tablets on Prognosis and Inflammatory Factor Expression in Acute Coronary Syndrome Patients Undergoing Percutaneous Coronary Intervention.

Danhong is a Chinese medical component that has been broadly used to treat various cerebrovascular diseases. This work aimed to investigate the effect of Danhong injection combined with Naoxintong tablets on the short-term prognosis and expression of inflammatory factor-soluble CD40 ligand (sCD40L) in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). A total of 100 ACS patients with PCI were randomly divided equally into treatment and control groups. The control group was treated with conventional secondary prevention of coronary heart disease. Based on secondary prevention, Danhong injection combined with Naoxintong tablets was administered in the treatment group. The incidences of major adverse cardiovascular events and cardiac functions, including ejection fraction (EF) and six-minute walk test distance, during hospital discharge and at the third postoperative month were observed. The serum sCD40 levels at different times were also noted. There were 2 patients in the treatment group and 7 in the control group that were lost during follow-up, so the collected data were from only 48 patients in the treatment and 43 in the control group. During hospital discharge and at the third postoperative month, no significant difference in death, myocardial infarction, stroke, angina pectoris and readmission were observed between the two groups (p > 0.05). Upon hospital discharge, EF, six-minute walk test distance and serum sCD40L level in the two groups were not significantly different (p > 0.05). At the third postoperative month, EF and six-minute walk test distance in treatment group were significantly higher than those in the control group (p < 0.05), and the serum sCD40L level in the treatment group was significantly lower than that in the control group (p < 0.01). In addition, serum sCD40L levels in the two groups at the third postoperative month were significantly lower than those during hospital discharge (p < 0.01). Danhong injection combined with Naoxintong tablets decreased serum sCD40L level and improved cardiac functions in ACS patients undergoing PCI at 3 months following discharge, but not at discharge. Acute coronary syndrome; Danhong injection; Naoxintong tablets; sCD40.

Abstract A115: The CD95 ligand inhibitor APG 101 reduces tumor recurrence and metastasis in an adjuvant orthotopic mouse model of pancreatic cancer as well as primary tumor load in a palliative setting

Abstract Since the first description of CD95/Fas this member of the death receptor superfamily has been regarded as paradigmatic for death receptors, capable of induction of caspase-driven apoptosis. More recently, CD95 has also emerged as an inducer of other signaling pathways including pro-inflammatory and migratory mechanisms (for an overview see Roeder C. et al., Eur J Cell Biol. 2011). Previously, we demonstrated a strong induction of NFkB activity after CD95 stimulation of pancreatic cancer cells resulting in increased invasiveness under in vitro conditions. Inhibition of CD95 mediated mechanisms in tumor gains importance since malignant cells may misuse this system for progression as demonstrated by others eg. for ovarian cancer and glioblastoma. From a clinical point of view, several observations also point into the same direction. Studies on colorectal cancer, head and neck, hepatocellular carcinoma for example suggest CD95 ligand expression to correlate negatively with patients prognosis. APG101 is an inhibitor of CD95 ligand consisting of the CD95 receptor extracellular domain fused to the Fc domain of IgG. A randomized controlled phase II ‘proof-of-concept’ study in patients with recurrent glioblastoma was successfully completed recently (NCT01071837). Up to date there exist no in vivo studies concerning the influence of blocking the interaction between CD95 and its ligand using this recombinant inhibitor in pancreatic cancer. Thus we tested this novel tool in a palliative and adjuvant orthotopic xenotransplantation model of human pancreatic cancer. In this study PancTuI Luc cells were used, which establish a highly infiltrating primary tumor with very low/no metastatic capacity in the liver. All mice (n=8/group) survived the operative procedures for the palliative treatment and were randomly assigned into five groups: control group; treatment with different concentrations of APG101: 12.5, 25, 50, and 100 µg/g bw. (i.p. injection, twice per week). Upon tumor resection (2/3rd pancreatectomy 10 days after tumor cell inoculation) local as well as distant recurrent disease (primarily in the liver) is regularly observed. All mice surviving the operative procedures for the adjuvant treatment (n=9/group) were randomly assigned into two groups: control group with 200 µl saline 0.9 % i.p. and the APG 101 group with a dose of 25 µg/g bw i.p (twice a week). Treatment was started three days after subtotal pancreatectomy and lasted for 28 days until sacrifice of the animals. Palliative treatment: mean tumor weight was 353 ± 179 mg for animals in the control group compared to 210.4 ± 86.5 mg to the mice treated with 25 µg APG101/g bw (p = 0.09) and 221.4 ± 94 mg (p = 0.021) in the group treated with 50 µg APG101/g bw Tumor volume was significantly reduced in all treatment groups, again with 25 µg APG101/g bw achieving maximal growth inhibition. Within the 4 weeks no metastases occurred in any group as previously observed within this model. Adjuvant treatment: All mice developed recurrent tumors. The mean weight of locally recurrent tumor was 887.5 ± 467.3 mg in the control group (treated with 0.9% saline). The other group treated with APG101 (25 µg/g bw) showed a local tumor recurrence with a significant lower tumor weight (p=0.027). The average number of metastases per mouse in the therapy group was 2.3 versus 4 metastases / mouse in the control group. Reduced tumor growth in both therapeutic settings was reflected by a reduced Ki67 proliferation index. Our data underline the importance of the CD95 antagonist APG101 as a new option in pancreatic cancer therapy that might be further evaluated also in combination with chemotherapies. Note: These results were published in the International Journal of Cancer as a full-length article before the online publication of this abstract. Citation information: Inhibition of IL-6 signaling significantly reduces primary tumor growth and recurrencies in orthotopic xenograft models of pancreatic cancer. Goumas FA, Holmer R, Egberts JH, Gontarewicz A, Heneweer C, Geisen U, Hauser C, Mende MM, Legler K, Röcken C, Becker T, Waetzig GH, Rose-John S, Kalthoff H. Int J Cancer. 2015 Jan 21. doi: 10.1002/ijc.29445. [Epub ahead of print] PMID: 25604508. Citation Format: Freya Goumas, Khalid Rhashid, Anna Trauzold, Harald Fricke, Michael Kluge, Thomas Becker, Jan Egberts, Holger Kalthoff. The CD95 ligand inhibitor APG 101 reduces tumor recurrence and metastasis in an adjuvant orthotopic mouse model of pancreatic cancer as well as primary tumor load in a palliative setting. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A115.

A CB2-Selective Cannabinoid Suppresses T-Cell Activities and Increases Tregs and IL-10.

We have previously shown that agonists selective for the cannabinoid receptor 2 (CB2), including O-1966, inhibit the Mixed Lymphocyte Reaction (MLR), an in vitro correlate of organ graft rejection, predominantly through effects on T-cells. Current studies explored the mechanism of this immunosuppression by O-1966 using mouse spleen cells. Treatment with O-1966 dose-relatedly decreased levels of the active nuclear forms of the transcription factors NF-κB and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures generated using a PCR T-cell activation array showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. Further, an increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures. Pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-related decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. These data support the potential of CB2-selective agonists as useful therapeutic agents to prolong graft survival in transplant patients, and strengthens their potential as a new class of immunosuppressive agents with broader applicability.

482. Editing of the CD40L Gene Restores Regulated CD40L Expression in X-HIGM Patient T Cells

X-linked hyper-IgM syndrome (X-HIGM) is a primary immunodeficiency syndrome characterized by insufficient CD40 Ligand (CD40L) expression, resulting in defective T-cell help, impaired immunoglobulin class-switching and recurrent opportunistic infections. Previously, constitutive CD40L expression in murine bone marrow cells and thymocytes was achieved by retroviral gene transfer and successfully corrected immune function. However, treated animals developed severe lymphoproliferative disease, likely due to constitutive CD40L surface expression. This demonstrated the importance of preserving endogenous gene regulation in this and other gene therapy endeavors. Here, we report efficient, on-target Homology Directed Repair (HDR) editing of the CD40L locus in primary human T cells using a combination of TALEN gene targeting and a donor template delivered transiently by recombinant Adeno-Associated Virus (rAAV). Our TALEN and donor template reagents were designed to insert a coding sequence (GFP or CD40L) upstream of the endogenous translation start site within Exon 1, thus allowing transgene expression to be regulated by the endogenous CD40L promoter, and included the CD40L 3’UTR to preserve known post-transcriptional regulation features. The kinetics of GFP and edited CD40L surface expression after PMA-Ionomycin stimulation paralleled that of endogenous CD40L in unedited T-cells. Additionally, activated T-cells expressing edited CD40L were able to bind CD40-Ig similar to unedited cells. When T-cells from X-HIGM patients were edited in this fashion, CD40L expression and CD40-Ig binding was restored, and repaired activated X-HIGM T-cells induced the expression of early activation markers and class switching of naive B-cells in vitro. Finally, we found no differences in TCR repertoire or engraftment and stability in NSG mice between T cells that underwent gene editing vs. unedited cells. These results demonstrate the feasibility of site-directed gene repair to restore normally regulated CD40L protein expression and functional T-cell help.

Selective inhibition of Kv1.3 channels in memory T cells by targeted delivery with nanoparticles suppresses CD40 ligand expression in SLE (THER5P.908)

Abstract The pathogenesis of Systemic Lupus Erythematosus (SLE) is characterized by hyperactive memory T cells (TM). In SLE, the TM cells show a Ca2+-dependent increase in the costimulatory CD40 ligand (CD40L) which binds CD40 on B cells, resulting in B cell activation and autoantibody production. CD40L is widely recognized as a potential target for developing new therapies for SLE. Cytosolic Ca2+ levels, which increase during T cell activation and mediate CD40L expression, are regulated by Kv1.3 channels. We have developed lipid nanoparticles that can deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to TM cells and decrease the activation-induced Ca2+ influx (Hajdu et al., Biomaterials 2013). We studied whether these Kv1.3-NPs decrease the Ca2+-dependent overexpression of CD40L in SLE peripheral blood TM cells. We observed that nuclear translocation of NFAT, a transcription factor upstream to CD40L, in activated TEM cells was reduced by ~50% after treatment with Kv1.3-NPs. Furthermore, Kv1.3-NP treatment selectively reduced CD40L expression in activated TEM cells by ~40% in healthy donors and ~60% in SLE patients, while CD40L levels remained unchanged in cells that did not incorporate the nanoparticles. Interestingly, we observed that treatment with Kv1.3-NPs in SLE patients resulted in T cell phenotype switching from a predominantly TM phenotype to a naïve phenotype. Our findings open the possibility of using Kv1.3-NPs as potential targeted immune suppressive therapeutic agents in SLE.

Glia Maturation Factor Stimulates Release of Proinflammatory Mediators from Mast Cells

We have investigated if brain protein glia maturation factor (GMF) and Parkinson's disease (PD)‐relevant toxin 1‐methyl‐4‐phenylpyridinium (MPP+) and α‐synuclein can activate mast cells to release inflammatory mediators. Mast cells are implicated and suggested as a therapeutic target in neuroinflammation but its role in PD is not yet understood. We have analyzed the effect of recombinant GMF, MPP+, α‐synuclein and recombinant IL‐33 on mouse bone marrow‐derived cultured mast cells (BMMCs), human umbilical cord blood‐derived cultured mast cells (hCBMCs), and mouse embryonic brain‐derived cultured astrocytes. The release of cytokines/chemokines was determined by ELISA and the expression of co‐stimulatory molecules CD40 and CD40Ligand by flow cytometry. GMF significantly released chemokine (C‐C motif) ligand 2 (CCL2) from BMMCs when compared to un‐treated control cells. GMF, α‐synuclein and MPP+ activated BMMCs to release interleukin‐1β (IL‐1β), and IL‐8 release from hCBMCs. IL‐33, an IL‐1 family member induced the expression of GMF in human mast cells. GMF induced more tumor necrosis factor‐alpha (TNF‐α) release from astrocyte‐ mast cell co‐culture. Flow cytometry showed increased IL‐33 expression by GMF and MPP+in astrocytes. GMF also induced the expression of CD40 in astrocytes. In conclusion, proinflammatory mediator release from mast cells following GMF, MPP+ or α‐synuclein treatment, and significant release of GMF by mast cells indicate a novel drug target for neurodegenerative diseases including PD.

Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40(+) tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40(+) tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19(+) systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy.

Memory T follicular helper CD4 T cells.

T follicular helper (Tfh) cells are the subset of CD4 T helper cells that are required for generation and maintenance of germinal center reactions and the generation of long-lived humoral immunity. This specialized T helper subset provides help to cognate B cells via their expression of CD40 ligand, IL-21, IL-4, and other molecules. Tfh cells are characterized by their expression of the chemokine receptor CXCR5, expression of the transcriptional repressor Bcl6, and their capacity to migrate to the follicle and promote germinal center B cell responses. Until recently, it remained unclear whether Tfh cells differentiated into memory cells and whether they maintain Tfh commitment at the memory phase. This review will highlight several recent studies that support the idea of Tfh-committed CD4 T cells at the memory stage of the immune response. The implication of these findings is that memory Tfh cells retain their capacity to recall their Tfh-specific effector functions upon reactivation to provide help for B cell responses and play an important role in prime and boost vaccination or during recall responses to infection. The markers that are useful for distinguishing Tfh effector and memory cells, as well as the limitations of using these markers will be discussed. Tfh effector and memory generation, lineage maintenance, and plasticity relative to other T helper lineages (Th1, Th2, Th17, etc.) will also be discussed. Ongoing discoveries regarding the maintenance and lineage stability versus plasticity of memory Tfh cells will improve strategies that utilize CD4 T cell memory to modulate antibody responses during prime and boost vaccination.

Splenic TFH expansion participates in B-cell differentiation and antiplatelet-antibody production during immune thrombocytopenia

AbstractAntiplatelet-antibody-producing B cells play a key role in immune thrombocytopenia (ITP) pathogenesis; however, little is known about T-cell dysregulations that support B-cell differentiation. During the past decade, T follicular helper cells (TFHs) have been characterized as the main T-cell subset within secondary lymphoid organs that promotes B-cell differentiation leading to antibody class-switch recombination and secretion. Herein, we characterized TFHs within the spleen of 8 controls and 13 ITP patients. We show that human splenic TFHs are the main producers of interleukin (IL)-21, express CD40 ligand (CD154), and are located within the germinal center of secondary follicles. Compared with controls, splenic TFH frequency is higher in ITP patients and correlates with germinal center and plasma cell percentages that are also increased. In vitro, IL-21 stimulation combined with an anti-CD40 agonist antibody led to the differentiation of splenic B cells into plasma cells and to the secretion of antiplatelet antibodies in ITP patients. Overall, these results point out the involvement of TFH in ITP pathophysiology and the potential interest of IL-21 and CD40 as therapeutic targets in ITP.

Self-complementary adeno-associated virus 5-mediated gene transduction of a novel CD40L mutant confers direct antitumor effects in lung carcinoma.

CD40 ligand (CD40L) gene therapy offers a potentially useful option for lung cancer due to its multiple antitumor activities. However, membrane-bound CD40L may be proteolytically cleaved to form soluble CD40L (sCD40L), which results in adverse effects. In a previous study by our group, it was demonstrated that recombinant self-complementary adeno-associated virus 5 (scAAV5) efficiently delivered genes to lung cancer cells. In the present study, an scAAV5 expressing a non-cleavable human CD40L mutant (scAAV5-CD40L-M) was generated and its direct antitumor effects in lung cancer were evaluated. Transduction with scAAV5-CD40L-M resulted in effective expression of CD40L on the cell surface with low levels of cleaved sCD40L, which significantly reduced the percentage of viable cells and promoted caspase-3-dependent apoptosis of CD40-positive lung carcinoma A549 cells, compared with scAAV5-CD40L transduction (P<0.05). Furthermore, treatment with scAAV5-CD40L-M exerted a significant antitumor effect against CD40-positive A549 xenografts by inducing apoptosis (P<0.05) with few side effects. Gene therapy using an scAAV5 vector expressing non-cleavable human CD40L mutant may therefore have direct antitumor effects against CD40-positive lung cancers. These tumoricidal effects of scAAV5-CD40L-M treatment make it a promising therapeutic technique for the treatment of lung cancer.

Abstract 5075: B-cell expression of AICDA and MME [CALLA, CD10] is predictive of a subsequent non-Hodgkin lymphoma diagnosis

Abstract Background: Mounting evidence from epidemiologic studies of serum biomarkers highlights the importance of inflammation and immune activation preceding the diagnosis of B-cell non-Hodgkin lymphoma (NHL). Although chronic B-cell hyperactivation is likely involved in the pathogenesis of NHL, an important gap in knowledge remains present as the source and targets of these inflammatory and immune activation associated factors are mainly undefined. Targeted studies of B-cell gene expression can help fill this gap, further define the immune environment from which these tumors arise, and assist in identifying biomarkers for early detection of B-cell NHLs. Methods: This study was based in a large national cohort of HIV+ men, the Multicenter AIDS Cohort Study, who are at increased risk for developing NHL. 144 HIV+ B-cell NHL cases were matched to 144 HIV+ controls on follow-up time and sample availability. B-cells isolated from viably frozen peripheral blood mononuclear cells, collected 2 to 13 years (median 4.7 years) prior to NHL diagnosis or control match date, were used to measure messenger RNA (mRNA) levels of 16 molecules that are associated with B-cell activation, using the Quantigene® Plex 2.0 Assay Kit (Affymetrix) on the Luminex platform. Expression levels were normalized by the geomean of five housekeeping genes. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by conditional logistic regression for gene expression categorized as detectable versus undetectable. Results: Activation-induced cytidine deaminase (AICDA) was detected in 8.3% of cases and 2.8% of controls, OR=3.7, 95% CI=1.0-13.1. Membrane metallo-endopeptidase (MME) was detected in 59.7% of cases and 29.9% of controls, OR=3.9, 95% CI=2.2-6.8. The AICDA and MME associations with NHL were similar across subgroups of site, EBV tumor status, and lag time between sample collection and NHL diagnosis date. AICDA, but not MME, was more strongly associated with NHL among those who were the least immunosuppressed (higher CD4+ T cell count and low HIV RNA levels). In subgroup analyses, CD40 ligand (CD40LG) expression was associated with NHL with a time lag time of &amp;lt;6 years, OR=3.0, 95% CI=1.0-9.3, and Interleukin-6 (IL6) expression was associated with NHL among those with a CD4+ T cell count &amp;lt;300, OR=3.9, 95% CI=0.9-17. Interpretation &amp; Conclusions: AICDA expression is essential for antibody specificity and diversity but its aberrant expression has been implicated in lymphomagenesis. MME is a signature marker of B-cell NHLs of germinal center origin and has previously been associated with NHL survival. We found that AICDA and MME expression precedes the clinical diagnosis of NHL in an HIV+ cohort. Additionally, late CD40LG expression and IL6 expression among the more severely immunosuppressed individuals were associated with NHL risk in this study. These molecules should be studied further for possible early detection and clinical utility. Citation Format: Shehnaz K. Hussain, Marta Epeldegui, Larry I. Magpantay, Elizabeth Crabb Breen, Emilee Knowlton, Steven Wolinksy, Lisa P. Jacobson, Jay H. Bream, Roger Detels, Zuo-Feng Zhang, Otoniel Martinez-Maza. B-cell expression of AICDA and MME [CALLA, CD10] is predictive of a subsequent non-Hodgkin lymphoma diagnosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5075. doi:10.1158/1538-7445.AM2014-5075

Ginkgolide B inhibits platelet release by blocking Syk and p38 MAPK phosphorylation in thrombin-stimulated platelets

IntroductionAtherosclerosis is a chronic vascular inflammatory disease. Platelets play a critic role in the initiation of vascular inflammation in atherosclerosis. In the present study, we investigated the effects of ginkgolide B on the inhibition of platelet release and the potential mechanisms. MethodsExperiments were performed in freshly human platelets. Platelet aggregation and ATP release were measured with a Lumi-aggregometer. Thrombin (0.5 U/ml) was used to induce platelet activation. Protein expression and phosphorylation was examined by Western blotting. ResultsThe results showed that ginkgolide B significantly suppressed ATP release by 50.8% in thrombin-activated platelets. Ginkgolide B completely abolished the expression of platelet factor 4 (PF4) and CD40 Ligand (CD40L). Moreover, ginkgolide B fully attenuated the phosphorylation of Syk and p38MAPK. Similarly, R788 (a syk inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited the expression PF4 and CD40L, respectively. Furthermore, the combination of low concentrations of ginkgolide B and R788 or SB203580 has synergistic inhibition on the expression of PF4 and CD40L. Ginkgolide B partially reduced calcium efflux by 52.7% in thrombin-stimulated platelets. ConclusionGinkgolide B potently inhibited the expression of PF4 and CD40L in thrombin-activated platelets. Ginkgolide B partially decreased ATP release and Ca2+ efflux. The mechanism might be associated with the inhibition of Syk and p38 MAPK phosphorylation. These results demonstrated that ginkgolide B might be a promising drug on inhibiting platelet function and reducing inflammation in atherosclerosis.

The effect of prasugrel on ADP-stimulated markers of platelet activation in patients with sickle cell disease

Platelets of patients with sickle cell disease (SCD) show evidence of mild activation in the non-crisis steady state and greater activation during vaso-occlusive crises (VOC). Prasugrel, a potent inhibitor of ADP-mediated platelet activation and aggregation, may be useful in attenuating VOC. We compared platelet responses to ADP stimulation in patients with SCD and healthy subjects before and after treatment with prasugrel. In a phase 1 study, platelet biomarker levels were assessed in 12 adult patients with SCD and 13 healthy subjects before and after 12 ± 2 days of 5.0 or 7.5 mg/day prasugrel. The following were determined in whole blood samples stimulated with 20 µM ADP: (i) percentages of monocytes and neutrophils with adherent platelets (cell–platelet aggregates); (ii) the relative number (mass) of platelets associated with each monocyte and neutrophil as reported by CD61 mean fluorescence intensity (MFI) of the monocyte–platelet and neutrophil–platelet aggregates; (iii) the percentages of platelets positive for surface expression of CD40 ligand (CD40L), P-selectin (CD62p) and activated glycoprotein IIb-IIIa (GPIIb-IIIa); and (iv) the percentages of platelets and monocyte–platelet aggregates positive for surface tissue factor (TF) expression. At baseline, there were no significant differences between cohorts in the percentages of platelets expressing activation biomarkers. Following 12 days of prasugrel administration, the percentages of platelets expressing activation biomarkers following ADP stimulation were reduced in both cohorts, and there were no significant differences between groups. Both patients with SCD and healthy subjects had significant reductions in the monocyte–platelet and neutrophil–platelet aggregate MFI and the percentage of platelets expressing P-selectin and activated GPIIb-IIIa (all p < 0.05). Healthy subjects also had significant reductions in monocyte–platelet aggregate percentages (p = 0.004), neutrophil–platelet aggregate percentages (p = 0.011) and the percentage of CD40L-positive platelets (p = 0.044) that were not observed in patients with SCD. Prasugrel administration to SCD patients attenuates ex vivo ADP-stimulated platelet activation as measured by the percentage of platelets positive for P-selectin and GPIIb–IIIa, thus reducing the proportion of platelets that may participate in aggregates. Furthermore, prasugrel decreases ex vivo ADP-stimulated platelet aggregation with monocytes and neutrophils as measured by the monocyte–platelet and neutrophil–platelet aggregate MFI. This implies that in the presence of prasugrel, fewer platelets adhere to monocytes and neutrophils, which may result in reducing cell–platelet aggregate size. Therefore, reduced platelet reactivity and decreased size of leukocyte–platelet aggregates suggest additional mechanisms by which prasugrel may provide benefit to patients with SCD and support further investigation of possible therapeutic benefits of prasugrel in this population.

Critical role for bone marrow stromal antigen 2 in acute Chikungunya virus infection.

Bone marrow stromal antigen 2 (BST-2; also known as tetherin or CD317) is an IFN-inducible gene that functions to block the release of a range of nascent enveloped virions from infected host cells. However, the role of BST-2 in viral pathogenesis remains poorly understood. BST-2 plays a multifaceted role in innate immunity, as it hinders retroviral infection and possibly promotes infection with some rhabdo- and orthomyxoviruses. This paradoxical role has probably hindered exploration of BST-2 antiviral function in vivo. We reported previously that BST-2 tethers Chikungunya virus (CHIKV)-like particles on the cell plasma membrane. To explore the role of BST-2 in CHIKV replication and host protection, we utilized CHIKV strain 181/25 to examine early events during CHIKV infection in a BST-2(-/-) mouse model. We observed an interesting dichotomy between WT and BST-2(-/-) mice. BST-2 deficiency increased inoculation site viral load, culminating in higher systemic viraemia and increased lymphoid tissues tropism. A suppressed inflammatory innate response demonstrated by impaired expression of IFN-α, IFN-γ and CD40 ligand was observed in BST-2(-/-) mice compared with the WT controls. These findings suggested that, in part, BST-2 protects lymphoid tissues from CHIKV infection and regulates CHIKV-induced inflammatory response by the host.

CD40L expression in plasma of volunteers following LPS administration: A comparison between assay of CD40L on platelet microvesicles and soluble CD40L

CD40 ligand (CD40L) is a transmembrane protein that is mainly expressed on activated T cells and platelets. This protein, however, may also be shed from cells and circulate in the blood in a soluble form. “Soluble CD40L” has attracted interest as a biomarker as it can interact with CD40 and elicit cellular responses involved in the pathophysiology of various thrombotic and inflammatory conditions. As platelets can release microvesicles following activation, we investigated the expression of CD40L on circulating microvesicles as well as CD40L in plasma, in an experimental model of inflammation in healthy volunteers (i.e., intravenous lipopolysaccharide administration). We studied CD40L quantified as CD40L-positive platelet microvesicles by flow cytometry, and as CD40L in plasma (“soluble CD40L”) by an ELISA. Results of these studies showed that levels of CD40L exposed on platelet microvesicles were significantly increased after lipopolysaccharide administration. ELISA measurements of CD40L in plasma (“soluble CD40L”) did not show any significant increase in plasma levels over time. Separation of soluble and vesicle-bound CD40L by high-speed centrifugation indicated that the ELISA can also detect CD40L on microvesicles, as a trend toward increased concentrations were observed in the pellet of high-speed centrifuged samples (i.e., in samples in which microvesicles are enriched). Together, these findings suggest that platelet microvesicles are a source of CD40L in the circulation and that CD40L exposure on platelet microvesicles increases following experimentally induced inflammation. Our data also suggest that determining levels of CD40L on microvesicles in plasma samples may provide a more sensitive detection of changes in CD40L expression than measurement of “soluble CD40L” in plasma with an ELISA. In addition, information regarding the cellular source of CD40L can be obtained with a flow cytometry-based microvesicle assay in a way not possible with an ordinary ELISA.

X-linked hyper IgM syndrome: clinical, immunological and molecular features in patients from India.

X-linked hyper-IgM (XHIM) is a primary immunodeficiency disorder characterized by recurrent infections, low serum IgG and IgA and normal or elevated IgM. It results from mutations in the CD40 ligand (CD40L) gene. Confirmation of diagnosis with identification of underlying molecular defect is important for the initiation of appropriate therapeutic interventions, including immunoglobulin replacement, antibiotics and bone marrow transplantation. To investigate the molecular basis of XHIM, we evaluated 7 patients with suspected XHIM and abnormal CD40L expression on activated CD4(+) T lymphocytes. The entire coding region and intronic splice sites of the CD40L gene were sequenced from the genomic DNA of the patients. 7 mutations; 3 nonsense (c.172delA, c.A229T, c.C478T), 1 missense (c.A506G) and 3 splice sites [c.346+2(T→C), c.289-1(G→C), c.346+1(G→T)] were identified, out of which 5 were novel. A wide heterogeneity in the nature of mutations has been observed in Indian XHIM patients in the present study. Identification of mutations in this rare disorder will help in genetic diagnosis in affected families which could be further useful in prenatal diagnosis.

A novel disease-causing CD40L mutation reduces expression of CD40 ligand, but preserves CD40 binding capacity

Mutations in CD40 ligand (CD40L) that permit residual CD40L expression typically impair binding of CD40. We report a male patient who presented with recurrent bacterial respiratory tract infections, normal IgM, decreased IgG, absent IgA levels, and CD40L expression at ~50% of the level observed in the normal control. He subsequently developed autoimmunity, inflammatory bowel disease, severe opportunistic infections suggestive of a combined immunodeficiency, and a cervical spine schwannoma. Whole exome sequencing of the patient's genomic DNA revealed a novel missense mutation (p.H47Y) in CD40L. Although this mutation was predicted to be benign in silico, flow cytometry at 13 years of age demonstrated markedly decreased CD40L expression (~32% of normal control) that retained the capacity to bind soluble CD40-Ig, suggesting that the mutation impairs CD40L surface expression without affecting its affinity for CD40. This case highlights the variability in the clinical evolution and phenotype of CD40L deficiency.