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

Abstract

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-αβ (TCRαβ) 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^®, 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)-γ. Multiparametric flow cytometry revealed that CD4⁺ T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ 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.

Govers and colleagues perform direct comparisons between T cell sorting reagents for their ability to enrich T cells. They compare in vitro properties of primary human T cells gene-engineered with gp100280-288 Human Leukocyte Antigen A2-specific T-cell receptorαβ undergoing Magnetic-Activated Cell Sorting either with different peptide-MHC multimers or with an antibody against truncated CD34 (tCD34). Among the multimers, streptamers resulted in the best T cell yield, stability, and expansion ability after sorting, while tCD34-sorted cells resulted in the most significant T cell yield and enrichment.