MHC class III lymphocyte antigens 6 as endogenous immunotoxins: Unlocking immunotherapy in proficient mismatch repair colorectal cancer.

A majority of cancers, including colorectal cancer (CRC) with intact DNA mismatch repair, exhibit a paralyzed antitumor immune response and resistance to immune checkpoint inhibitors. Members of MHC class III lymphocyte antigen 6G (LY6G) encode glycosylphosphatidylinositol (GPI) proteins anchored to the membrane. Snake venom neurotoxins and LY6G proteins share a three-finger (3F) folding domain. LY6 proteins such as LY6G6D are gaining a reputation as excellent tumor-associated antigens that can potently inhibit anti-tumor immunity in cancers with proficient mismatch repair. Thus, we called MHC class III LY6G endogenous immunotoxins. Since the discovery of LY6G6D as a tumor-associated antigen, T-cell engagers (TcEs) have been developed to simultaneously bind LY6G6D on cancer cells and CD3 on T cells, improving the treatment of metastatic solid tumors that are resistant to ICIs. We present a current understanding of how alterations in MHC class III genes inhibit antitumor immunity, and how these understandings can be turned into effective treatments for patients who are refractory to standard immunotherapy. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics Cancer > Molecular and Cellular Physiology.

Cinobufagin induces FOXO1-regulated apoptosis, proliferation, migration, and invasion by inhibiting G9a in non-small-cell lung cancer A549 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Bufonis (VB), an animal drug called Chansu in China, is the product of the secretion of Bufo gargarizans Cantor or B. melanostictus Schneider. As a traditional Chinese medicine (TCM) for a long time, it has been widely used in the treatment of heart failure, ulcer, pain, and various cancers. Cinobufaginn (CNB), the cardiotonic steroid or bufalene lactone extracted from VB, has the effects of detoxification, detumescence, and analgesia. AIM OF THE STUDY: The present study aimed to define the effects of CNB on non-small-cell lung cancer (NSCLC) and identify the potential molecular mechanisms. MATERIALS AND METHODS: A549 cells were treated with cinobufagin and cell viability, apoptosis, migration, and invasion were then evaluated using Cell Counting Kit-8 (CCK8) assays, flow cytometry, and Transwell assays, respectively. Moreover, the levels of proliferating cell nuclear antigen (PCNA), cytokeratin8 (CK8), poly ADP-ribose polymerase (PARP), Caspase3, Caspase8, B-cell lymphoma/lewkmia-2(Bcl-2), Bcl2-Associated X(Bax), forkhead box O1 (FOXO1), and euchromatic histone-lysine N-methyltransferase2 (G9a, EHMT2) in A549 cells were evaluated using qRT-PCR and/or Western blot analysis (WB), Co-IP, immunofluorescence, and immunohistochemistry. An in vivo imaging system, TUNEL, Immunofluorescence, and immunohistochemistry were also used to detect proliferating cell nuclear antigen(PCNA), Ki67, E-Cadherin(E-Cad), FOXO1, and G9a in mouse xenograft model experiments. RESULTS: CNB suppressed cell proliferation, migration, and invasion but promoted apoptosis in A549 cells in a dose- and time-dependent manner, while cinobufagin had no cytotoxic effect on BEAS-2B cells. In vivo, cinobufagin inhibited the proliferation, migration, and invasion of A549 cells and promoted their apoptosis. The occurrence of the above phenomena was accompanied by an increase in FOXO1 expression and a decrease in G9a expression. In A549 cells, CNB did not reverse the changes in the proliferation, migration, invasion, and apoptosis of A549 cells after FOXO1 was successfully silenced. CONCLUSION: Our study provides the first evidence that cinobufagin suppresses the malignant biological behaviours of NSCLC cells in vivo and in vitro and suggests that mechanistically, this effect may be achieved by inhibiting the expression of the histone methyltransferase G9a and activating the tumour suppressor gene FOXO1. Taken together, our findings provide important insights into the molecular mechanism underlying cinobufagin's anticancer activity, and suggest that cinobufagin could be a candidate for targeted cancer therapy.

Mesenchymal stem cells from human umbilical cord regulate the expression of major histocompatibility complex in human neural stem cells and their lineages.

hNSCs (human neural stem cells) derived from embryonic tissue and aborted fetal brains are considered to be the most promising candidates for neurodegenerative and other CNS(central nervous system) diseases. However, the most common problem, which limited successful use of these allogeneic hNSC therapy, is immune rejection. Mesenchymal stem cells (MSCs) from human umbilical cord (hUC-MSCs) are receiving increasing attention for their immune-modulatory properties. In the current studies, we firstly investigated the immunogenecity of hNSCs as well as their lineages in cultures with the presence or absence of interferon gamma (IFNγ), a pro-inflammatory factors. Our data revealed that the majority of hNSCs and astrocytes expressed MHCI (major histocompatibility complex class I) while neurons hardly expressed MHCI (<5%) in the absence of IFNγ. In addition, neither hNSCs nor neurons expressed MHCII while a subpopulation (about 18 %) of astrocytes expressed MHCII without IFNγ stimulation. However, the addition of IFNγ in cultures significantly increased the expressions of MHCII on hNSCs and astrocytes. However, IFNγ did not affect the expression of MHCI on hNSCs and astrocytes. We then investigated whether hUC-MSCs had the capacity of regulating the immunogenecity of hNSCs as well as their lineages in a co-culture system. We found that hUC-MSCs did not affect the expression of MHCI on hNSCs and their lineages, however, these cells were able to significantly inhibit the IFNγ-induced up-regulation of MHCII on hNSCs and astrocytes (p < 0.001). Thus, our results suggest that hUC-MSCs may serve as potentially useful modulators to reduce the immunogenicity of allogeneic hNSCs in clinical application.

Prolongation of heart allograft survival after long-term expression of soluble MHC class I antigens and vIL-10 in the liver by AAV-plasmid-mediated gene transfer.

INTRODUCTION: The essential prerequisite for successful gene therapy in vivo is an effective and long-lasting transfer of the desired gene into the respective cell type or tissue. Over the last decades, many different methods have been developed for this purpose. The use of plasmid DNA seems to be a good alternative to the commonly used viral vectors because its large-scale production is simple, and side effects are low. Unfortunately, most reports describe only short-term expression in vivo, probably due to the lack of genomic integration in the target cell. This problem can possibly be addressed by the use of adeno-associated virus plasmids (AAV plasmids), where the coding sequences are cloned between the AAV-specific inverted terminal repeats. Here, we report our results after allogeneic heart transplantation, which followed AAV-plasmid-mediated gene transfer of the rat soluble major histocompatibility complex class I antigen RT1.A(a) and viral interleukin (vIL)-10 in the "high"-responder Dark Agouti to Lewis rat strain combination. RESULTS: A high and stable long-term expression was achieved by in vivo transfection of the liver using AAV plasmids. Serum levels over 1,000 ng/ml of soluble RT1.A(a) and over 300 pg of vIL-10, respectively, were achieved. Expression levels remained high for up to several months. A mean prolongation of heart allograft survival of 1 to 2 days was demonstrated after transfection of either RT1.A(a) or vIL-10.

[Inhibition of tumor growth in rat liver induced by tumor-specific transfer factor].

A transfer factor (TF) specific to antigens of Geren's carcinoma in rat was developed using an original model of intraorganic growth. Intravenous injection (1pg/g body) inhibited primary tumor node growth in the liver by 78% and blocked dissemination to peritoneal viscera. Its effect was due to an immunospecific component promoting antitumor immunological response. The latter presented as generation of cytotoxic effector cells, vascular disorders in tumor parenchyma thus blocking tumor cell proliferation. Possible applications of tumor-specific TF for biotherapy of cancer patients are discussed.

Peptidic complex mixtures as therapeutic agents in CNS autoimmunity.

Limited success with antigen-specific immunotherapies has led to the identification of novel approaches which consider the degeneracy of the T cell response, i.e. their ability to respond to multiple antigenic peptides. Random complex mixtures of polypeptides such as glatiramer acetate (GA) were among the first to be applied as immunodulators that take into account T cell degeneracy. While the mechanisms of action are not completely understood, the immunogenicity of GA, its strong major histocompatability complex (MHC) binding, immune deviation and bystander suppression all appear to be important. In the present study we have designed peptidic complex mixtures (CM) of varied lengths and compositions to test their potential as immunomodulating agents. CM were synthesized that had defined lengths and contained aa corresponding to binding motifs of MHC class II molecules relevant in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), specifically HLA-DRB1*1501 and HLA-DRB5*0101, which are related to MS, and H2-IA(s) associated with EAE in SJL mice. Additional CM were designed based on specificity profiles derived from positional scanning synthetic combinatorial library (PS-SCL) testing of a GA-specific T cell clone (TCC). Several mixtures were strongly stimulatory for peripheral blood mononuclear cells (PBMC) from MS patients and healthy donors suggesting a high degree of cross-reactivity with other peptide antigens. A subset of these mixtures exhibited cross-reactivity to myelin antigens and prophylactic efficacy in reducing the severity of EAE. Based on these observations we envision mixture-based peptidic compounds can be developed not only for immunotherapeutic purposes in autoimmune diseases and cancer, but also in vaccine development.

[Significance of allopeptide-induced expression of Fas ligand (FasL) in parenchyma of allogenic heart transplants as the cause of donor-specific tolerance induction]. / Die Bedeutung der Allopeptid-induzierten Expression von Fas-Ligand (FasL) im Parenchym allogener Herztransplantate als Ursache der spenderspezifischen Toleranzinduktion.

This study proves the tolerogenicity of polymorphic allopeptides. Combined administration of peptides derived from the alpha 1 (intrathymal) and the alpha 2 (intraperitoneal) helical region of the donor RT1.A(a) molecule induced specific tolerance in a rat model of cardiac allotransplantation. The underlying tolerance mechanism was mediated by selective depletion of alloreactive T cells within the thymus and Fas-L-induced apoptosis within the graft.

Interactions of major and minor histocompatibility antigens in the graft-versus-host reaction.

The greater severity of the graft-versus-host (GVH) reaction induced by grafting C57BL/6(B6) lymphoid or bone marrow cells to irradiated (B6 x DBA/2)F1 recipients, as compared to that induced in H-2-identical (B6 x BALB/c)F1 recipients, has been shown to arise from a synergistic effect of H-2d and DBA/2 minor histocompatibility antigens (MiHAs), and not from a difference in the intensity of genetic resistance to B6 cell grafts exhibited by the two F1s. Furthermore, detection of such synergistic effects depends upon choice of an appropriate assay system; GVH splenomegaly results cannot be used to predict the outcome of a systemic GVH reaction.