Flow cytometric methods for assessment of cell-mediated immune responses

Abstract

The aim of this thesis was to develop and improve flow cytometry-based methods for assessment of cellmediated immune responses (CMI) induced by natural infection or vaccination. Traditionally, the measurement of anti genspecific cellular immune responses has been based on the use of complex, labourintensive, and less informative bulkresults assays (51Cr-release, 3 3H-thymidin uptake and Limiting dilution assay, LDA). Alternative techniques for assessment have been developed (tetramer-staining, ELISspot, intracellular cytokine staining, ICS), with pros and cons for each system. We have focused on the assessment of cytokine production and cytolytic capacity, two activities of importance for evaluation of CMI to viral infections, especially HIV. Flow cytometry technology offers the possibility for simultaneous analysis of multiple characteristics and functions of single cells at high rate.

We have developed two lytic assays for the assessment of natural killer (NK) cell mediated and HIV-antigen-specific Tcell mediated cytotoxicity (FC-CTL). In the NK cell assay, the cell populations are separated by post-culture immunophenotyping and cell death is detected by uptake of propidium iodide (PI), which permits the combined assessment of NK cell lysis and conjugate formation between target and effector cells. The assay is sensitive, very easy to perform and more informative than other lytic assays. The FC-CTL is based on dual staining with 5-(and -6)carboxyfluorescein diacetate succinimidyl ester, CFSE (for tracing target cells) and PI (for cell-death), which permits identification of several cell populations including viable and dead target cells at different stages of cell-death; live and dead effector cells; and also enumeration of lysed 'not-detectable' target cells. Determination of HIV-specific cytolysis is based on the enumeration of viable target cells rather than percentage of dead cells as commonly used for other assays. The FC-CTL is well correlated to, but more sensitive than the conventional 51Cr-release assay.

The standard procedure for ICS is based on short-term (6h) activation of PBMCs or undiluted whole blood. We performed kinetic studies of cytokine production with regard to incubation time and dilution of whole blood samples for specific and non-specific activation. We consistently recorded higher frequencies of responding T-cells in samples of diluted blood (115-1 A 0) cultured for several days (long-term (LT-ICS), as compared to short-term incubation. The increased levels of cytokines, in particular IFN-gamma with peak detection at 72h, are due to expansion of the antigen specific cytokine-producing cells.

The new assays (LT-ICS and FC-CTL) and conventional methods (ELIspot and Cr-release) were subsequently applied for a study of HIV-1 infected (>5 years) treatment-naïve individuals with CD4+-cell counts >400 and with low or high viral load. The levels of IFN-gamma responses and cytolytic activity were consistently higher in subjects with low viral load, against all HIV-antigen tested (Gag, Nef and avipox-based antigens), as assessed by all methods employed. By the LT-ICS (48h), the difference between the two patient-groups was statistically significant for CD4+ T-cell responses against all HIV-antigens tested. The strong HIV-specific cell-mediated immune responses found in subjects with stable CD4+ T-cell counts and low viral load, support the idea that strong HIV-1 -specific cellular immunity is protective.

Both cytolytic assays and the LT-ICS can be further expanded for simultaneous assessment of several different molecules expressed on cell-surface or present intracellular, especially if the 3-4-laser flow cytometers with ability to detect up to 18 colours are utilized. The new assays presented in this thesis will be valuable tools for research regarding evaluation of CMI against viral infections such as HIV and for monitoring in vaccine studies, in particular if immune responses may appear at low levels. The NK cell cytotoxicity assay has been further developed for use of whole blood and is now employed for a study of the role of innate immunity for control of herpes simplex infection, and the LT-ICS will be used as one of the assays for evaluation of CMI in a phase 1 HIV- 1 vaccine trial in Stockholm.