Flow cytometric analysis of protective T-cell response against pulmonary Coccidioides infection

Abstract

The incidence of systemic fungal infections has increased throughout the world, spurring much interest in developing effective vaccines. Coccidioidomycosis, also known as San Joaquin Valley fever, is a potentially life-threatening respiratory mycosis. A vaccine against Coccidioides infection would contribute significantly to the well-being of the approx. 30 million residents in the Southwestern USA as well as the multitude of travelers who annually visit the endemic regions. We have applied a live, attenuated vaccine (ΔT) to explore the nature of vaccine immunity in mice after intranasal challenge with a potentially lethal dose of Coccidioides spores. Coccidioides spores are airborne and highly infectious for mammalian hosts and classified as a bio-safety level 3 agent. T cells are critical in the development of protective immunity against a variety of microorganisms as well as the development of autoimmune disease and allergic responses. Profiles of cytokines detected in lung homogenates of ΔT-vaccinated mice were indicative of a mixed Th1, Th2, and Th17 immune response. We have developed an intracellular cytokine staining and flow cytometric (ICS) technique to measure activated CD4+ and CD8+ T cells and IFN-γ-, IL-4-, IL-5-, and IL-17A-producing T cells in the lungs of mice that are challenged with a potentially lethal dose of Coccidioides spores. The numbers of pulmonary Th1 and Th17 cells during the first 2 weeks post-challenge showed a progressive increase in vaccinated mice and corresponded with reduction of fungal burden. In this protocol, we describe the methodology for culture and isolation of the live, attenuated ΔT spores of Coccidioides used to vaccinate mice, preparation of pulmonary cells, and staining protocol for cell surface markers and intracellular cytokines. This is the most reliable and robust procedure to measure frequencies and numbers of each selected T-cell subsets in lungs of vaccinated versus control mice and can be readily applied to evaluate T-cell response against other microbial infections.