Characterization of T cell responses in peanut-allergic patients by polychromatic flow cytometry

Abstract

Rationale Antigen-specific effector T cells from allergic individuals are rare. Others have reported the use of CFSE to identify antigen-specific lymphocytes. We sought to characterize the phenotype of antigen-responsive CD4 T cells in peanut-allergic individuals. Methods Challenge-proven peanut allergy patients were recruited. PBMC were CFSE-labeled at a range of concentrations. Following culture, cells were stimulated with low (5 ng/250ng) or high (50 ng/1ug) concentrations of PMA and calcium ionophore. Cells were stained with ethidium monazide bromide, then fixed, permeabilized and stained for flow cytometry. Results CFSE titration experiments revealed substantial suppression of lymphocyte responses to both antigen and mitogen in a dose-dependent manner. Low PMA/calcium ionophore stimulation was optimum for the detection of cytokine with minimum cell death. The ability to incorporate the live/dead cell discriminator, EMA, significantly improved the measurement of proliferation and intracellular cytokine (particularly IL-4). There was a consistent two-to-four fold increase in percent of IL-4 +CD3 +CD4 + over spontaneous levels in response to peanut antigen. In contrast, non-peanut allergic individuals had no appreciable IL-4. IL-4 percentage was higher to peanut extract than to SEB, suggesting the phenotype is antigen-specific. Unexpectedly, there was a higher percent IL-4 + lymphocytes among the non-proliferating population. For many patients, the majority of IL-4 + CD3 +CD4 + lymphocytes were undivided and CD25 −. In contrast, the percent of ifn-γ + cells in the zproliferating subset was substantially higher than in the nondividing population. Conclusions Evaluation of allergen-specific lymphocytes is sensitive to methodology. under the conditions of this study, there is an important th2 effector population with low proliferative capacity.