Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry.

Abstract

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

• Authors: Jocelyn H Wright, Li-Ya Huang, Stephanie Weaver, L Diego Archila, Megan S McAfee, Alexandre V Hirayama, Aude G Chapuis, Marie Bleakley, Anthony Rongvaux, Cameron J Turtle, R Savanh Chanthaphavong, Jean S Campbell, Robert H Pierce
• Journal: Journal of immunological methods (J Immunol Methods) Vol. 492 Pg. 112955 (05 2021) ISSN: 1872-7905 [Electronic] Netherlands
• PMID: 33383062 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2020 Elsevier B.V. All rights reserved.

Chemical References

• Receptors, Chimeric Antigen

Topics

• Animals
• Biopsy
• Cell Engineering
• Coculture Techniques
• Genetic Vectors (genetics)
• Hepatitis B Virus, Woodchuck (genetics)
• Humans
• Immunohistochemistry (methods)
• Immunophenotyping (methods)
• In Situ Hybridization, Fluorescence
• Lentivirus (genetics)
• Lymph Nodes (pathology)
• Male
• Mice
• Mice, Transgenic
• Models, Animal
• Paraffin Embedding
• Receptors, Chimeric Antigen (analysis, genetics, immunology)
• Skin (cytology, immunology, pathology)
• T-Lymphocytes (immunology, metabolism, transplantation)
• Tissue Fixation
• Transduction, Genetic
• Transplantation Chimera