Immunology

Natural killer (NK) cells are the major effectors of the innate immune system when activated resulting in modulation of immune response of the host defense through target cell lysis and secretion of cytokines. Precise functions of NK cells are essential for the treatment outcome of different virus infections and malignant diseases. NK cells impart cytotoxic effect to the target cells lacking MHC class I molecules and thus the final readout of the activity is death of target cells. The NK cell function is evaluated by the ⁵¹Cr-release and/or flow cytometry-based assays. In the present protocol, we have determined the activation of NK cells by the liberation of IL-10 and IFNγ, and subsequently its function by enumerating the number of dead tumor cells originally isolated from the ascitic fluid of ovarian cancer patients. The entire assay is based on cells of the healthy donors and patients. Besides determining function, this method is able to demarcate between NK-cell sensitive and insensitive tumor cells. This technique enables researchers to study NK cell functions in healthy donors or in patients to reveal their impact on different malignancies and to further discover new therapeutic strategies.

Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and -10 are fully activated by several proteolytic cleavage steps and induce the caspase cascade leading to apoptotic cell death. Analysing the processing of procaspases-8 and -10 by Western blot is a commonly used method to study the induction of apoptosis by death receptor stimulation. To analyse procaspase-8 and -10 cleavage, cells are stimulated with a death ligand for different time intervals, lysed and subjected to Western blot analysis using anti-caspase-8 and anti-caspase-10 antibodies. This allows monitoring the caspase cleavage products and thereby induction of apoptosis.

Cytotoxic CD8⁺ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of antigen-specific cytotoxic T cells can be assessed functionally in the so-called chromium 51 (⁵¹Cr) release assay, which was developed almost 50 years ago in our institution (Brunner et al., 1968). Radioactively-labelled cells deficient for endogenous antigen presentation [e.g., transporter for antigen presentation (TAP)-deficient T2 cells] and stably transfected with the MHC of interest (e.g., HLA-A2⁺) are typically used as targets during this 4h assay. Alternatively, ⁵¹Cr-labelled virus-infected or tumor cell lines presenting immunogenic antigens endogenously can serve as target cells (e.g., for the assessment of tumor recognition).

In a peptide titration assay (section A), radioactively labelled target cells are pulsed with a serial dilution of the antigenic peptide and incubated at an effector (e.g., a CD8⁺ T cell clone) to target (⁵¹Cr -T2 cells) ratio (E:T) of 10:1 in a 96-well V-bottom plate for 4 h at 37 °C. In a tumor killing assay (section B), cytotoxic CD8⁺ effector cells are incubated at different ratios with the ⁵¹Cr-labelled target cell line (typically at E:T ratios of 30:1, 10:1, 3:1 and 1:1) in the presence or absence of the specific antigenic peptide (1 μM) and incubated for 4 h at 37 °C. At the end of the test, the amount of radioactivity release from the lysed target cells is determined in the supernatant using a liquid scintillation counter. The percentage of specific lysis, as well as the EC50 (i.e., 50% of maximal killing) and EMax values are then calculated, providing quantitative information about the antigen-specific functional avidity (i.e., the relative efficiency of T cell function based on antigen recognition via a defined TCR and maximal killing capacity of the analyzed T cells).

Cytotoxic CD8⁺ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8⁺ T cell killing activity, we use the in vivo killing assay (IVK). Here we describe the protocol for the lysis of cells expressing a CD8⁺ T cell epitope of the OVA protein (SIINFEKL). Mice are previously immunized with the OVA protein and 7 days after immunization, they receive a mix of target cells, prepared from naive C57BL/6 spleen cells pulsed with the SIINFEKL peptide and labeled with high level of CFSE and of non-pulsed control cells labeled with low level of CFSE. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE^high cells and CFSE^low cells. The percentage of lysis is calculated by the difference between CFSE high versus low in immunized vs non-immunized mice.

Measuring the ability of antigen-specific CD8⁺ T cell to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.

Natural killer (NK) cells comprise 5–20% of peripheral blood mononuclear cells (PBMC) in humans. In addition to their fundamental roles in the defense against viral infections and tumor surveillance, NK cells help shape adaptive immune responses through their production of cytokines. NK cells are traditionally identified as CD3^neg, CD14^neg, CD19^neg lymphocytes expressing CD56. Using a combination of markers that includes CD56 and CD7 greatly increases the ability to define the phenotype and function of NK cell subsets. Two key markers of NK cell function are the production of IFNγ and the release of cytotoxic granules measured by the expression of CD107a. Here we describe a method to assess IFNγ and CD107a expression in NK cells following stimulation with target cells or cytokines. This method can be used to assess the general functional capacity of NK cells in peripheral blood mononuclear cells from a wide range of study participants.

Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections and cancers in vivo. Here we developed a flow cytometry based protocol for ADCC assay using human peripheral blood mononuclear cells (PBMCs) as effector cells. Using this protocol, we determined the ADCC activity of convalescent plasma IgGs from six H1N1-infected human subjects in China, and identified two dominant ADCC epitopes, designated E1 [amino acid (AA) 92-117] and E2 (AA 124-159), on haemagglutinin of pandemic H1N1 influenza virus by epitope mapping of the convalescent plasma IgGs with different levels of ADCC activity. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer a comprehensive immune protection against viral infections.

Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the “gold standard” chromium release assay for measuring the target cell killing capacity of NK cells. Key features of this cytotoxicity assay are that it is performed with sorted NK cells as the effectors and any Major Histocompatibility Class I (MHC-I)-low or deficient tumor cell line can be used as the target cells.

⁵¹Cr-release assays are commonly used for the precise and accurate quantification of cytotoxicity, particularly in the study of tumor cytolysis. This test has the advantage of requiring only very few cells.