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Cytotoxic T-lymphocytes (CTL) are a subset of T-cells that play a critical role in protecting against intracellular infections and cancer, and have the ability to identify and kill infected or transformed cells expressing non-self peptides associated with major histocompatibility (MHC) Class I molecules. Conversely, aberrant CTL activity can contribute to immune-related pathology under conditions of overwhelming infection or autoimmunity. Disease-modifying therapeutics can have unintended effects on CTL, and a growing number of therapeutics are intended to either suppress or enhance CTL or their functions. The susceptibility of CTL to unintended effects from common therapeutic modalities underscores the need for a better understanding of the impact that such therapies have on CTL function and the associated safety implications. While there are reliable ways of quantifying CTL, notably via flow cytometric analysis of specific CTL markers, it has been a greater challenge to implement fit-for-purpose methods measuring CTL function in the context of safety studies of therapeutics. This review focuses on methods for measuring CTL responses in the context of drug safety and pharmacology testing, with the goals of informing the reader about current approaches, evaluating their pros and cons, and providing perspectives on the utility of these approaches for safety evaluation. [ABSTRACT FROM AUTHOR]

In recent years, mRNA-based vaccines have emerged to be a great alternative to DNA-based vaccines due to the safety of not inserting into host genome. However, mRNA molecules are single-stranded nucleic acids that are vulnerable under RNase existing in human skin and tissues. In this study, a self-assembled cationic nanomicelles based on polyethyleneimine-stearic acid (PSA) copolymer were developed to delivery HIV-1 gag encoding mRNA to dendritic cells and BALB/c mice. We evaluated the transfection efficiency and cell uptake efficiency of naked EGFP mRNA, PSA, PEI-2k and PEI-25k nanoparticles format on DC2.4 cell lines. Immune responses after sub-cutaneous administration of gag mRNA to BALB/c mice were notably induced by PSA as compared with naked gag mRNA. We found the PSA/mRNA nanomicelles were potent systems that can effectively deliver mRNA and induce antigen-specific immune response, stimulating various new vaccine strategies using mRNA. [ABSTRACT FROM AUTHOR]

Systemic changes related to cytokine expression levels in women with endometriosis remain a subject of controversy. There are many studies concerning this topic showing differential serum cytokine levels; however, there are limited data presenting cytokine expression at the single-cell level. This study focused on this question by measuring intracellular cytokine staining of activated peripheral CD3+ and CD14+ cells from women with endometriosis (investigative group) compared with those with uterine leiomyoma (control group). Isolated peripheral blood mononuclear cells from women with endometriosis and uterine leiomyoma were stimulated with PMA and ionomycin or with LPS to induce intracellular synthesis of TNF-alpha, IFN-gamma, and IL-8 in subpopulations of CD3+ cells and TNF-alpha, IL-6, IL-10, MCP-1, and IL-8 in CD14+ cells. Comparison of the total groups of patients showed no significant differences in any of the intracellular cytokines investigated in the T cells and monocytes of women with endometriosis compared with controls. When the group of women with endometriosis was divided with regard to severity of disease, a significantly lower percentage of CD3+CD8- lymphocytes stained for IFN-gamma and a significantly higher percentage of CD14+ cells stained for MCP-1 in advanced endometriosis patients compared with the control group were observed. We conclude that peripheral mononuclear cells in women with advanced endometriosis may have differential cytokine synthesis in vitro. These results support the idea that differing immune cell activity measured by intracellular cytokine profiles in women with advanced endometriosis may be more a consequence of the disease than a cause. [ABSTRACT FROM AUTHOR]

BackgroundDevelopment of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials.MethodsAutologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase.ResultsConditions for optimal T-cell expansion using K562-A*0201 APC included input of 2×106 PBMC, a 10 µg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12).DiscussionThis bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients. [ABSTRACT FROM AUTHOR]

Candidate HIV vaccines must show an immune response in order to be considered for further testing and development. What constitutes a “response,” however, is still not clear. While the hunt for a protective vaccine continues, hypotheses are being formed by studying the immune responses across cohorts of people with differing responses to the infection, as well as the immune responses formed by healthy people to other viruses, ones that are generally common and well controlled. Here we examine the functional profile of the immune responses of a group of HIV + long-term non-progressors as measured by intracellular cytokine staining using polychromatic flow cytometry, and compare these responses to those of a larger group of other HIV + people. We describe some of the types of patterns in immune response that are of interest to vaccine researchers, and compare several statistical tests appropriate for this type of data. [ABSTRACT FROM AUTHOR]

The melanoma-associated antigens Melan-A and NY-ESO-1 stimulate different T-cell responses in late-stage melanoma patients. Either CD4+or CD8+T-cell reactivity against NY-ESO-1 was associated with better prognosis, but for Melan-A, only CD8+but not CD4+T-cell responses were associated with longer survival. [ABSTRACT FROM PUBLISHER]