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 the 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 gp100(280-288)/HLA-A2-specific T cell receptor-alpha beta (TCR alpha beta) on magnetic-activated cell sorting (MACS) with various peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that Streptamer (R), when compared with 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, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (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 with Streptamer, bound about 2- to 3-fold less peptide-MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-gamma. Multiparametric flow cytometry revealed that CD4(+) T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-gamma production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4(+) T cells.