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Single agent immune checkpoint inhibitors have been ineffective for patients with advanced stage and recurrent high grade serous ovarian cancer (HGSOC). Using pre-clinical models of HGSOC, we evaluated the anti-tumor and immune stimulatory effects of an oncolytic adenovirus, MEM-288. This conditionally replicative virus encodes a modified membrane stable CD40L and IFNβ. We demonstrated this virus successfully infects HGSOC cell lines and primary human ascites samples in vitro. We evaluated the anti-tumor and immunostimulatory activity in vivo in immune competent mouse models. Intraperitoneal delivery of MEM-288 decreased ascites and solid tumor burden compared to controls, and treatment generated a systemic anti-tumor immune response. The tumor microenvironment had a higher proportion of anti-tumor macrophages and decreased markers of angiogenesis. MEM-288 is a promising immunotherapy agent in HGSOC, with further pre-clinical studies required to understand the mechanism of action in the peritoneal microenvironment and clinical activity in combination with other therapies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mark Cantwell is an employee, stock owner of Memgen, Inc and has a patent on MEM-288., (Copyright © 2024. Published by Elsevier Inc.)

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

Colorectal cancer (CRC) is the second common cause of cancer mortality worldwide, and it still lacks effective approaches for relapsed and metastatic CRC. Recently, oncolytic virus has been emerged as a promising immune therapeutic strategy. In this study, we develop a novel oncolytic adenovirus, rAd.mDCN.mCD40L, which drive oncolytic activity by telomerase reverse transcriptase promoter (TERTp). rAd.mDCN.mCD40L expressed both mouse genes of decorin (mDCN) and CD40 ligand (mCD40L), and produced effective cytotoxicity in both human and mouse CRC cells. Moreover, oncolytic adenovirus mediated mDCN over-expression inhibited Met expression in vitro. In CT26 subcutaneous tumor model, intratumorally delivery of oncolytic adenoviruses could inhibit tumor growth and liver metastasis, while mDCN and/or mCD40L armed oncolytic adenoviruses produced much more impressive responses. No obvious toxicity was detected in lung, liver and spleen. Moreover, mDCN and/or mCD40L armed oncolytic adenoviruses altered the immune state to activate anti-tumor responses, including increasing CD8 + T effector cells and CD4 + memory T cells, reducing MDSCs and Tregs in peripheral blood. Furthermore, mDCN and/or mCD40L armed oncolytic adenoviruses mediated mDCN and/or mCD40L expression in tumors, and up-regulated Th1 cytokines and reduced Th2 cytokines in tumors, which will be benefit for remodeling tumor microenvironment. Importantly, rAd.mDCN.mCD40L and rAd.mCD40L prevented tumor liver metastasis much more effectively than rAd.Null and rAd.mDCN. Therefore, rAd.mDCN.mCD40L and rAd.mCD40L are promising approaches for CRC therapy., (© 2024. The Author(s).)

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs., (© 2024. The Author(s).)

Background and Aims: Hyperglycemia reinforces pro-inflammatory conditions that enhance CD40 expression in endothelial cells (EC). Thymine to cytosine transition (-1T > C) in the promoter of the CD40 gene (rs1883832) further increases the abundance of CD40 protein on the EC surface. This study examines potential associations of the -1T > C SNP of the CD40 gene with type 1 (T1D) or type 2 (T2D) diabetes. Moreover, it investigates the impact of a pro-inflammatory diabetic microenvironment on gene expression in human cultured umbilical vein EC (HUVEC) derived from CC- vs. TT-genotype donors., Methods: Tetra-ARMS-PCR was used to compare genotype distribution in 252 patients with diabetes. Soluble CD40 ligand (sCD40L) and soluble CD40 receptor (sCD40) plasma levels were monitored using ELISA. RNA-sequencing was performed with sCD40L-stimulated CC- and TT-genotype HUVEC. Quantitative PCR, Western blot, multiplex-sandwich ELISA array, and immunocytochemistry were used to analyse changes in gene expression in these cells., Results: Homozygosity for the C-allele was associated with a significant 4.3-fold higher odds of developing T2D as compared to individuals homozygous for the T-allele. Inflammation and endothelial-to-mesenchymal transition (EndMT) driving genes were upregulated in CC-genotype but downregulated in TT-genotype HUVEC when exposed to sCD40L. Expression of EndMT markers significantly increased while that of endothelial markers decreased in HUVEC following exposure to hyperglycemia, tumour necrosis factor-α and sCD40L., Conclusions: The -1T > C SNP of the CD40 gene is a risk factor for T2D. Depending on the genotype, it differentially affects gene expression in human cultured EC. CC-genotype HUVEC adopt a pro-inflammatory and intermediate EndMT-like phenotype in a pro-diabetic microenvironment., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Although Multiple Sclerosis (MS) is primarily thought to be an autoimmune condition, its possible viral etiology must be taken into consideration. When mice are administered neurotropic viruses like mouse hepatitis virus MHV-A59, a murine coronavirus, or its isogenic recombinant strain RSA59, neuroinflammation along with demyelination are observed, which are some of the significant manifestations of MS. MHV-A59/RSA59 induced neuroinflammation is one of the best-studied experimental animal models to understand the viral-induced demyelination concurrent with axonal loss. In this experimental animal model, one of the major immune checkpoint regulators is the CD40-CD40L dyad, which helps in mediating both acute-innate, innate-adaptive, and chronic-adaptive immune responses. Hence, they are essential in reducing acute neuroinflammation and chronic progressive adaptive demyelination. While CD40 is expressed on antigen-presenting cells and endothelial cells, CD40L is expressed primarily on activated T cells and during severe inflammation on NK cells and mast cells. Experimental evidences revealed that genetic deficiency of both these proteins can lead to deleterious effects in an individual. On the other hand, interferon-stimulated genes (ISGs) possess potent antiviral properties and directly or indirectly alter acute neuroinflammation. In this review, we will discuss the role of an ISG, ISG54, and its tetratricopeptide repeat protein Ifit2; the genetic and experimental studies on the role of CD40 and CD40L in a virus-induced neuroinflammatory demyelination model., (© 2023 International Union of Biochemistry and Molecular Biology.)

X-linked hyper-immunoglobulin M (X-HIGM) syndrome and autosomal recessive hyper-immunoglobulin E syndrome (HIES) are rare inborn errors of immunity characterized by recurrent infections due to immune system impairment. In this study, we identified a novel hemizygous CD40 ligand (CD40L) mutation and compound heterozygous dedicator of cytokinesis-8 (DOCK8) mutations in two Han Chinese families with X-HIGM and HIES, respectively. We aimed to investigate the association between their genotypes and phenotypes. Genomic DNA was extracted from peripheral blood samples obtained from the families. Whole exome sequencing and Sanger sequencing were performed to identify and verify pathogenic variants in the two families. Clinical analyses of the probands were also performed. A novel hemizygous mutation of CD40L in exon 2 (c.257delA) was identified in the first proband, resulting in the substitution of glycine with glutamic acid at codon 86 of the protein. This leads to premature termination of translation at downstream codon 9 (p.E86Gfs*9). Sanger sequencing confirmed that the variant was inherited from the mother. The second proband carried two novel compound heterozygous mutations in DOCK8: one at exon 14 (c.1546C > G) inherited from the father, and the other at intron 41 (c.5355 + 6C > T; splicing) inherited from the mother. This study enhances our understanding of the pathogenetic mutation spectrum of CD40L and DOCK8 genes, facilitating the prenatal diagnosis of X-HIGM and HIES and enabling timely treatment of patients., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Immunostimulatory gene therapy using oncolytic viruses is currently evaluated as a promising therapy for cancer aiming to induce anti-tumour immunity. Here, we investigate the capacity of oncolytic adenoviruses (LOAd) and their transgenes to induce immunogenicity in the infected tumour cells. Oncolysis and death-related markers were assessed after infection of eight human solid cancer cell lines with different LOAd viruses expressing a trimerized, membrane-bound (TMZ)-CD40L, TMZ-CD40L and 41BBL, or no transgenes. The viruses induced transgene expression post infection before they were killed by oncolysis. Death receptors TRAIL-R1, TRAIL-R2 and Fas as well as immunogenic cell death marker calreticulin were upregulated in cell lines post infection. Similarly, caspase 3/7 activity was increased in most cell lines. Interestingly, in CD40 + cell lines there was a significant effect of the TMZ-CD40L-encoding viruses indicating activation of the CD40-mediated apoptosis pathway. Further, these cell lines showed a significant increase of calreticulin, and TRAIL receptor 1 and 2 post infection. However, LOAd viruses induced PD-L1 upregulation which may hamper anti-tumour immune responses. In conclusion, LOAd infection increased the immunogenicity of infected tumour cells and this was potentiated by CD40 stimulation. Due to the simultaneous PD-L1 increase, LOAd viruses may benefit from combination with antibodies blocking PD1/PD-L1., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)