Magnetic-Activated Cell Sorting of TCR-engineered T cells using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4 and CD8 T cells

T cell sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100280-288/HLA-A2 specific TCRaß upon Magnetic Activated Cell Sorting (MACS) with different peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that streptamers, when compared to pentamers and tetramers, improved T cell yield as well as level and stability of enrichment of TCR-engineered T cells (> 65% of peptide-MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, streptamers, the only detachable reagent, revealed significant T cell expansion in the 1st week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 mAb resulted in the most significant T cell yield and enrichment of T cells (> 95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared to streptamer, bind about two to three-fold less peptide-MHC but show superior antigen-specific up-regulated expression of CD107a and production of IFN¿. Multi-parameter flow cytometry revealed that CD4 T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN¿ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits towards applications of T cell therapy, especially those that require CD4 T cells.