Large-scale depletion of CD25⁺ regulatory T cells from patient leukapheresis samples

The ability to selectively enrich or deplete T lymphocytes of specific phenotype and function holds significant promise for application in adoptive immunotherapy protocols. Although CD4⁺ T cells can have an impact on CD8⁺ T-cell effector function, memory, and maintenance, a subset of CD4⁺ T cells, CD25⁺ regulatory T cells (T_reg), can regulate peripheral self tolerance and possess the ability to suppress antitumor responses. The authors report the ability to selectively deplete CD25⁺ T_reg cells from patient leukapheresis samples using a clinical-grade, large-scale immunomagnetic system. Using leukapheresis samples containing up to 1.3 × 10¹⁰ white blood cells, efficient depletion of T_reg cells was measured by flow cytometric analysis of CD25 expression and FOXP3 expression on post-depletion products. Remnant CD25⁺ cells could not be detected in CD25-depleted products after short-term culture in IL-2 or enriched following secondary immunomagnetic selection for CD25⁺ cells, confirming that efficient depletion had occurred. In parallel to efficient enrichment of CD25⁺ cells, immunomagnetic selection resulted in the recovery of T_reg cells, since CD25⁺ lymphocytes removed during depletion were primarily composed of CD4⁺ T cells that expressed high levels of FOXP3 and possessed suppressive activity against autologous TCR-stimulated CD4⁺ CD25⁺ T cells in vitro. These results show that selective separation of functional CD25⁺ T_reg cells from large-scale samples can be performed in large scale under clinical-grade conditions with sufficient selection, recovery, viability, ability to expand, and function for potential use in adoptive immunotherapy.