A chimeric HLA-A2:ß2M:Ig fusion protein for the study of virus-specific CD8+ T-cells.

INTRODUCTION: The response mediated by CD8+ T-cells in the context of infection and vaccination has been thoroughly investigated and represents one of the most important branches that allow for the development of immunity against intracellular pathogens and, thus, the establishment of robust antiviral responses. However, there is a lack of methods to assess antigen-specific CD8+ T-cells. OBJECTIVE: Search for the ideal assays to assess the function of antigen-specific CD8+ T-cells. METHODS: In the present study a chimeric HLA-A2:ß2M:Ig fusion protein was produced, purified, and evaluated in functional CD8+ T-cell response studies using samples from Influenza A patients and humanized mice upon adenoviral vaccination. RESULTS: The HLA-A2:ß2M:Ig molecule, bound to immunodominant viral peptides by passive transfer, was able to induce robust antiviral CD8+ T-cell responses mediated by IFN-γ. The in vitro IFN-γ release assay using the chimeric HLA-A2:ß2M:Ig fusion protein detected bona fide human CD8+ T-cells, demonstrating superior production of IFN-γ by human CD8+ T-cells induced by Influenza A immunodominant GILGFVFTL peptide. Removal of antigen-presenting cells and CD8+ T-cell enrichment improved significantly the IFN-γ production. The chimeric HLA-A2:ß2M:Ig fusion protein also triggered HLA-A2-restricted CD8+ T-cell response in a humanized mouse model upon vaccination with adenovirus encoding HLA-A2-restricted HIV p24 antigen. The results strongly suggest the use of tailor-made assays for detecting HLA-A2-restricted CD8+ T-cell Responses in the Humanized Mouse Model. CONCLUSION: The chimeric HLA-A2:ß2M:Ig fusion protein-based assays provided a sensitive tool that may be paramount to measure virus-specific CD8+ T-cell response in a range of viral infections of clinical relevance.

Functional Human CD141+ Dendritic Cells in Human Immune System Mice.

BACKGROUND: For the purpose of studying functional human dendritic cells (DCs) in a humanized mouse model that mimics the human immune system (HIS), a model referred to as HIS mice was established. METHODS: Human immune system mice were made by engrafting NOD/SCID/IL2Rgammanull (NSG) mice with human hematopoietic stem cells (HSCs) following the transduction of genes encoding human cytokines and human leukocyte antigen (HLA)-A2.1 by adeno-associated virus serotype 9 (AAV9) vectors. RESULTS: Our results indicate that human DC subsets, such as CD141+CD11c+ and CD1c+CD11c+ myeloid DCs, distribute throughout several organs in HIS mice including blood, bone marrow, spleen, and draining lymph nodes. The CD141+CD11c+ and CD1c+CD11c+ human DCs isolated from HIS mice immunized with adenoviruses expressing malaria/human immunodeficiency virus (HIV) epitopes were able to induce the proliferation of malaria/HIV epitopes-specific human CD8+ T cells in vitro. Upregulation of CD1c was also observed in human CD141+ DCs 1 day after immunization with the adenovirus-based vaccines. CONCLUSIONS: Establishment of such a humanized mouse model that mounts functional human DCs enables preclinical assessment of the immunogenicity of human vaccines in vivo.

NLRP3 gain-of-function in CD4+ T lymphocytes ameliorates experimental autoimmune encephalomyelitis.

NLRP3 inflammasome [NLR (nucleotide-binding domain, leucine-rich repeat containing protein) Pyrin-domain-containing 3 ] functions as an innate sensor of several PAMPs and DAMPs (pathogen- and damage-associated molecular patterns). It has been also reported as a transcription factor related to Th2 pattern, although its role in the adaptive immunity has been controversial, mainly because the studies were performed using gene deletion approaches. In the present study, we have investigated the NLRP3 gain-of-function in the context of encephalomyelitis autoimmune disease (EAE), considered to be a Th1- and Th17-mediated disease. We took advantage of an animal model with NLRP3 gain-of-function exclusively to T CD4+ lymphocytes (CD4CreNLRP3fl/fl). These mice presented reduced clinical score, accompanied by less infiltrating T CD4+ cells expressing both IFN-γ and IL-17 at the central nervous system (CNS) during the peak of the disease. However, besides NLRP3 gain-of-function in lymphocytes, these mice lack NLRP3 expression in non-T CD4+ cells. Therefore, in order to circumvent this deficiency, we transferred naive CD4+ T cells from WT, NLRP3-/- or CD4CreNLRP3fl/fl into Rag-1-/- mice and immunized them with MOG35-55 Likewise, the animals repopulated with CD4CreNLRP3fl/fl T CD4+ cells presented reduced clinical score and decreased IFN-γ production at the peak of the disease. Additionally, primary effector CD4+ T cells derived from these mice presented reduced glycolytic profile, a metabolic profile compatible with Th2 cells. Finally, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under a Th2-related cytokine milieu cocktail exhibited in vitro an increased IL-4 and IL-13 production. Conversely, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under Th1 differentiation produced less IFN-γ and T-bet. Altogether, our data evidence that the NLRP3 gain-of-function promotes a Th2-related response, a pathway that could be better explored in the treatment of multiple sclerosis.

A New Perspective: Mitochondrial Stat3 as a Regulator for Lymphocyte Function.

Stat3 as a transcription factor regulating gene expression in lymphocytes during the immune response is well known. However, since the pioneering studies discovering the presence of Stat3 in mitochondria and its role in regulating mitochondrial metabolism, only a few studies have investigated this non-conventional function of Stat3 in lymphocytes. From this perspective, we review what is known about Stat3 as a transcription factor and what is known and unknown about mitochondrial Stat3 (mitoStat3) in lymphocytes. We also provide a framework to consider how some of the functions previously assigned to Stat3 as regulator of gene transcription could be mediated by mitoStat3 in lymphocytes. The goal of this review is to stimulate interest for future studies investigating mitoStat3 in the immune response that could lead to the generation of alternative pharmacological inhibitors of mitoStat3 for the treatment of chronic inflammatory diseases.

Antitumor activity of kielmeyera coriacea leaf constituents in experimental melanoma, tested in vitro and in vivo in syngeneic mice.

PURPOSE: The antitumor activity of Kielmeyera coriacea (Clusiaceae), a medicinal plant used in the treatment of parasitic, as well as fungal and bacterial infections by the Brazilian Cerrado population, was investigated. METHODS: A chloroform extract (CE) of K. coriacea was tested in the murine melanoma cell line (B16F10-Nex2) and a panel of human tumor cell lines. Tumor cell migration was determined by the wound-healing assay and the in vivo antitumor activity of CE was investigated in a melanoma cell metastatic model. 1H NMR and GC/MS were used to determine CE chemical composition. RESULTS: We found that CE exhibited strong cytotoxic activity against murine melanoma cells and a panel of human tumor cell lines in vitro. CE also inhibited growth of B16F10-Nex2 cells at sub lethal concentrations, inducing cell cycle arrest at S phase, and inhibition of tumor cell migration. Most importantly, administration of CE significantly reduced the number of melanoma metastatic nodules in vivo. Chemical analysis of CE indicated the presence of the long chain fatty compounds, 1-eicosanol, 1-docosanol, and 2-nonadecanone as main constituents. CONCLUSION: These results indicate that K. coriacea is a promising medicinal plant in cancer therapy exhibiting antitumor activity both in vitro and in vivo against different tumor cell lines.

Role of SOCS-1 Gene on Melanoma Cell Growth and Tumor Development.

Melanoma is the most aggressive form of skin cancer, and its incidence has increased dramatically over the years. The murine B16F10 melanoma in syngeneic C57Bl/6 mice has been used as a highly aggressive model to investigate tumor development. Presently, we demonstrate in the B16F10-Nex2 subclone that silencing of SOCS-1, a negative regulator of Jak/Stat pathway, leads to reversal of the tumorigenic phenotype and inhibition of melanoma cell metastasis. SOCS-1 silencing with short hairpin RNA affected tumor growth and cell cycle regulation with arrest at the S phase with large-sized nuclei, reduced cell motility, and decreased melanoma cell invasion through Matrigel. A clonogenic assay showed that SOCS-1 acted as a modulator of resistance to anoikis. In addition, downregulation of SOCS-1 decreased the expression of epidermal growth factor receptor (mainly the phosphorylated-R), Ins-Rα, and fibroblast growth factor receptor. In vivo, silencing of SOCS-1 inhibited subcutaneous tumor growth and metastatic development in the lungs. Because SOCS-1 is expressed in most melanoma cell lines and bears a relation with tumor invasion, thickness, and stage of disease, the present results on the effects of SOCS-1 silencing in melanoma suggest that this regulating protein can be a target of cancer therapy.