CD4+ CD8+ cells are rare among in vitro activated mouse or human T lymphocytes

The predominant cell type in the thymus expresses both of the function-associated T cell surface markers, CD4 and CD8, but CD4+ CD8+ cells are rare or absent outside the thymus. Double expression has therefore been assumed to be an indication of immaturity. However, recent reports have suggested that CD4+ CD8+ cells can appear in cultures of activated mature cells. We have therefore activated human peripheral blood lymphocytes and mouse spleen cells, lymph node cells, and cortisone-resistant thymocytes using a number of different stimulation regimes, and we have analyzed them at various times for CD4 and CD8 expression. In all cases, upon analysis of cultured cells by flow cytometry, CD4+ CD8+ cells were rare. A combination of microscopic analysis, cell sorting followed by microscopic analysis, and careful staining controls demonstrated that even when flow cytometry showed some CD4+ CD8+ cells, most of these were artifacts in the form of doublets or clumps of single positive cells or dead cells. Taking this into account, CD4+ CD8+ cells made up less than 1% in the mouse and less than 3% in the human T cell cultures at any time periods. We therefore found no evidence for the generation of large numbers of CD4+ CD8+ cells in cultures of mouse or human T cells.