Clinical-Scale Combined CD4/CD8 Depletion for Donor Innate Lymphocyte Infusion (DILI).

The ability to selectively deplete or enrich cells of specific phenotype by immunomagnetic selection procedures holds significant promise for application in adoptive immunotherapy protocols. In vitro and in vivo studies demonstrated that MHC-independent effector cells of the innate immune system such as natural killer (NK) cells and γδ T cells have potent anti-tumor activity without inducing Graft-versus-Host disease (GvHD). However, current clinical-scale approaches to reduce the risk of GvHD usually use T-cell depletion (such as CD34⁺ selection or CD3⁺ depletion). However, both procedures also deplete γδ T cells which may be advantageous in mediating Graft-versus tumor effects and augmenting innate immune response against infections. Here we present a new method for depletion of T cells with potential GvHD reactivity and simultaneously enrichment of innate lymphocytes (NK cells and γδ T cells) from standard leukapheresis products by using a single-step immunomagnetic protocol which efficiently depletes CD4⁺ and CD8⁺ αβ T cells under good manufacturing conditions (GMP). Efficiency of CD4⁺ and CD8⁺ T cell depletion from (unstimulated) leukapheresis products (n=6) containing up to 2.0 × 10¹⁰ white blood cells, was demonstrated by 4-color flow cytometric analysis (mean log depletion of CD4⁺ cells: 4.12 (3.17–4.9); mean log depletion of CD8⁺ cells: 3.77 (2.97–4.54)). In addition to efficient depletion of CD4⁺ and CD8⁺ cells, immunomagnetic CD4/CD8 depletion resulted in enrichment of NK cells and γδ T cells (mean NK cell recovery: 38%(19–72), mean γδ T cell cell recovery: 50%(34–79)). In vitro assays of the final product demonstrated that NK cells and γδ T cells preserved their proliferative and cytotoxic capacity. We conclude that simultaneous depletion of CD4⁺ and CD8⁺ cells is feasible and can be performed in large scale under GMP conditions with sufficient depletion efficacy of αβ T cells and recovery of functionally intact innate effector lymphocytes (NK cells and γδ T cells) for potential use in adoptive immunotherapy studies.