title: Lysosome-associated membrane glycoprotein 1 predicts fratricide amongst T cell receptor transgenic CD8+ T cells directed against tumor-associated antigens.
abstract: OBJECTIVE
  Autologous as well as allogeneic CD8+ T cells transduced with tumor antigen specific T cell receptors (TCR) may cause significant tumor lysis upon adoptive transfer. Besides unpredictable life-threatening off-target effects, these TCRs may unexpectedly commit fratricide. We hypothesized lysosome-associated membrane glycoprotein 1 (LAMP1, CD107a) to be a marker for fratricide in TCR transgenic CD8+ T cells.
  
  
  METHODS
  We identified HLA-A*02:01/peptide-restricted T cells directed against ADRB3295. After TCR identification, we generated HLA-A*02:01/peptide restricted TCR transgenic T cells by retroviral transduction and tested T cell expansion rates as well as A*02:01/peptide recognition and ES killing in ELISpot and xCELLigence assays. Expansion arrest was analyzed via Annexin and CD107a staining. Results were compared to CHM1319-TCR transgenic T cells.
  
  
  RESULTS
  Beta-3-adrenergic receptor (ADRB3) as well as chondromodulin-1 (CHM1) are over-expressed in Ewing Sarcoma (ES) but not on T cells. TCR transgenic T cells demonstrated HLA-A*02:01/ADRB3295 mediated ES recognition and killing in ELISpot and xCELLigence assays. 24h after TCR transduction, CD107a expression correlated with low expansion rates due to apoptosis of ADRB3 specific T cells in contrast to CHM1 specific transgenic T cells. Amino-acid exchange scans clearly indicated the cross-reactive potential of HLA-A*02:01/ADRB3295- and HLA-A*02:01/CHM1319-TCR transgenic T cells. Comparison of peptide motive binding affinities revealed extended fratricide among ADRB3295 specific TCR transgenic T cells in contrast to CHM1319.
  
  
  CONCLUSIONS
  Amino-acid exchange scans alone predict TCR cross-reactivity with little specificity and thus require additional assessment of potentially cross-reactive HLA-A*02:01 binding candidates. CD107a positivity is a marker for fratricide of CD8+ TCR transgenic T cells.
authors.affiliation: Laboratory for Functional Genomics and Transplantation Biology, Departments of Pediatrics and Children's Cancer Research Center, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kirschner
authors.affiliation: Laboratory for Functional Genomics and Transplantation Biology, Departments of Pediatrics and Children's Cancer Research Center, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Thiede
authors.affiliation: Laboratory for Functional Genomics and Transplantation Biology, Departments of Pediatrics and Children's Cancer Research Center, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.|Laboratory for Immunotherapy, Dr. von Hauner Children's Hospital, Medical center of the LMU Munich, Munich, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Blaeschke
authors.affiliation: Laboratory for Functional Genomics and Transplantation Biology, Departments of Pediatrics and Children's Cancer Research Center, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Günther H S
authors.initials: GH
authors.lastname: Richter
authors.affiliation: Laboratory for Pediatric Sarcoma Biology, Institute of Pathology of the LMU Munich, Munich, Germany.
authors.forename: Julia S
authors.initials: JS
authors.lastname: Gerke
authors.affiliation: Laboratory for Pediatric Sarcoma Biology, Institute of Pathology of the LMU Munich, Munich, Germany.
authors.forename: Michaela C
authors.initials: MC
authors.lastname: Baldauf
authors.affiliation: Laboratory for Pediatric Sarcoma Biology, Institute of Pathology of the LMU Munich, Munich, Germany.|German Cancer Consortium (DKTK), Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Thomas G P
authors.initials: TG
authors.lastname: Grünewald
authors.affiliation: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Busch
authors.affiliation: Laboratory for Functional Genomics and Transplantation Biology, Departments of Pediatrics and Children's Cancer Research Center, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Burdach
authors.affiliation: Laboratory for Functional Genomics and Transplantation Biology, Departments of Pediatrics and Children's Cancer Research Center, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Thiel
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D019264:Adoptive Transfer; D017302:Annexins / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology*; D002469:Cell Separation; D003106:Colon / Q000378:metabolism; D005434:Flow Cytometry; D005838:Genotype; D015789:HLA-A2 Antigen / Q000378:metabolism; D006801:Humans; D016219:Immunotherapy, Adoptive; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000166:cytology; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D022702:Receptors, Adrenergic, beta-3 / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D012160:Retina / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D019076:Transgenes
chemical_list: C568534:ADRB3 protein, human; D048868:Adaptor Proteins, Signal Transducing; D017302:Annexins; D000951:Antigens, Neoplasm; C081787:CHM protein, human; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D022702:Receptors, Adrenergic, beta-3; D011948:Receptors, Antigen, T-Cell
keywords: CD107a; TCR transgenic T cells; amino-acid exchange scan; cross reactivity; fratricide