title: Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors.
abstract: BACKGROUND
  T cells expressing chimeric antigen receptors (CARs) have shown exciting promise in cancer therapy, particularly in the treatment of B-cell malignancies. However, optimization of CAR-T cell production remains a trial-and-error exercise due to a lack of phenotypic benchmarks that are clearly predictive of anti-tumor functionality. A close examination of the dynamic changes experienced by CAR-T cells upon stimulation can improve understanding of CAR-T-cell biology and identify potential points for optimization in the production of highly functional T cells.
  
  
  METHODS
  Primary human T cells expressing a second-generation, anti-CD19 CAR were systematically examined for changes in phenotypic and functional responses to antigen exposure over time. Multi-color flow cytometry was performed to quantify dynamic changes in CAR-T cell viability, proliferation, as well as expression of various activation and exhaustion markers in response to varied antigen stimulation conditions.
  
  
  RESULTS
  Stimulated CAR-T cells consistently bifurcate into two distinct subpopulations, only one of which (CAR(hi)/CD25(+)) exhibit anti-tumor functions. The use of central memory T cells as the starting population and the resilience-but not antigen density-of antigen-presenting cells used to expand CAR-T cells were identified as critical parameters that augment the production of functionally superior T cells. We further demonstrate that the CAR(hi)/CD25(+) subpopulation upregulates PD-1 but is resistant to PD-L1-induced dysfunction.
  
  
  CONCLUSIONS
  CAR-T cells expanded ex vivo for adoptive T-cell therapy undergo dynamic phenotypic changes during the expansion process and result in two distinct populations with dramatically different functional capacities. Significant and sustained CD25 and CAR expression upregulation is predictive of robust anti-tumor functionality in antigen-stimulated T cells, despite their correlation with persistent PD-1 upregulation. The functionally superior subpopulation can be selectively augmented by careful calibration of antigen stimulation and the enrichment of central memory T-cell type.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, 420 Westwood Plaza, Boelter Hall 5531, Los Angeles, CA, 90095, USA. zenan.chang@ucla.edu.|Molecular Biology Institute, University of California-Los Angeles, 611 Charles E. Young Drive East, Boyer Hall 168B, Los Angeles, CA, 90095, USA. zenan.chang@ucla.edu.
authors.forename: ZeNan L
authors.initials: ZL
authors.lastname: Chang
authors.affiliation: Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, WAB 563, Boston, MA, 02115, USA. pamela_silver@hms.harvard.edu.
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Silver
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, 420 Westwood Plaza, Boelter Hall 5531, Los Angeles, CA, 90095, USA. yvonne.chen@ucla.edu.
authors.forename: Yvonne Y
authors.initials: YY
authors.lastname: Chen
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D060890:B7-H1 Antigen / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018628:Gene Dosage; D006801:Humans; D007156:Immunologic Memory; D008213:Lymphocyte Activation / Q000276:immunology; D016131:Lymphocyte Subsets / Q000276:immunology; D010641:Phenotype; D061026:Programmed Cell Death 1 Receptor / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D019076:Transgenes; D015854:Up-Regulation / Q000235:genetics
chemical_list: D015703:Antigens, CD; D060890:B7-H1 Antigen; C423236:CD274 protein, human; C105992:PDCD1 protein, human; D061026:Programmed Cell Death 1 Receptor; D011948:Receptors, Antigen, T-Cell