Ex vivo isolation protocols differentially affect the phenotype of human CD4+ T cells

Leukemic T cell lines have facilitated signal transduction studies but their physiological relevance is restricted. The use of primary T lymphocytes overcomes this limitation but it has long been speculated that methodological aspects of blood collection and the isolation procedure modify the phenotype of the cell. Here we demonstrate that several characteristics of human peripheral T cells are affected by the selection conditions. A significantly higher induction of the chemokine receptor CXCR4 was observed on CD4+ lymphocytes isolated by sheep red blood cell (SRBC) rosetting and CD4 MicroBeads as compared with positively selected CD4+ cells where the antibody/bead complex was immediately detached. These latter cells expressed CXCR4 at levels equivalent to that observed on CD4+ lymphocytes obtained by negative antibody-mediated selection. Furthermore, CD4+ cells isolated by SRBC rosetting and CD4 MicroBeads formed aggregates upon in vitro culture. CD4+ lymphocytes obtained by SRBC rosetting as well as those isolated following positive selection demonstrated basal phosphorylation of the extracellular signal-regulated kinase (ERK)-2. Altogether these data suggest that certain discrepancies concerning signal transduction in primary human T cells can be attributed to the selection conditions. Thus, it is essential to establish the parameters influenced by the isolation protocol in order to fully interpret T cell responses to antigens, chemokines, and cytokines.