title: 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.
authors.affiliation: Laboratory of Experimental Tumor Immunology, Department of Medical Oncology, Erasmus University Medical Center-Daniel den Hoed Cancer Center, 3015 GE Rotterdam, The Netherlands.
authors.forename: Coen
authors.initials: C
authors.lastname: Govers
authors.forename: Cor
authors.initials: C
authors.lastname: Berrevoets
authors.forename: Elike
authors.initials: E
authors.lastname: Treffers-Westerlaken
authors.forename: Marieke
authors.initials: M
authors.lastname: Broertjes
authors.forename: Reno
authors.initials: R
authors.lastname: Debets
mesh_terms: D000911:Antibodies, Monoclonal; D018952:Antigens, CD34 / Q000235:genetics / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D015789:HLA-A2 Antigen / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008280:Magnetics / Q000379:methods*; D010446:Peptide Fragments; D010455:Peptides / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D058951:gp100 Melanoma Antigen / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D005819:Genetic Markers; D015789:HLA-A2 Antigen; D052118:Lysosomal-Associated Membrane Protein 1; D010446:Peptide Fragments; D010455:Peptides; D016693:Receptors, Antigen, T-Cell, alpha-beta; D058951:gp100 Melanoma Antigen; C548641:gp100(280-288) melanoma antigen peptide; D007371:Interferon-gamma