title: Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer.
abstract: Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-[gamma] production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR V[beta] was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8(+) T cells. CD3(+)CD8(+), CD3(+)CD4(+), and CD3(+)CD4(-)CD8(-)[double-negative (DN) T cells] resided predominantly in central (CD45RA(-)CCR7(+)) and effector (CD45RA-CCR7-) memory subsets. CD8(+) TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4(+) TILs showed only up to 12% LAG-3(+) staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8(+)) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-[alpha] and IFN-[gamma] production. Twelve of 17 of CD8(+) TILs showed preferential expansion of certain TCR V[beta] families (eg, 99.2% V[beta]13.2 in CD8(+) TILs, 77% in the V[beta]1, 65.9% in the V[beta]22, and 63.3% in the V[beta]14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8(+) V[beta]13.2) reacting strongly against autologous tumor defined by INF-[gamma] production or by cytotoxicity. We have optimized methods for generating pancreatic cancer–specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.
authors: Departments of *Laboratory Medicine, Division of Therapeutic Immunology ‡Microbiology, Tumor and Cell Biology, Karolinska Institute Departments of †Clinical Science, Intervention and Technology §Laboratory Medicine, Division of Pathology ∥Neurosurgery ¶CAST, Center for Allogeneic Stem Cell Transplantation, Karolinska Hospital, Stockholm, Sweden.
authors: Qingda
authors: Q
authors: Meng
authors: Zhenjiang
authors: Z
authors: Liu
authors: Elena
authors: E
authors: Rangelova
authors: Thomas
authors: T
authors: Poiret
authors: Aditya
authors: A
authors: Ambati
authors: Lalit
authors: L
authors: Rane
authors: Shanshan
authors: S
authors: Xie
authors: Caroline
authors: C
authors: Verbeke
authors: Ernest
authors: E
authors: Dodoo
authors: Marco
authors: M
authors: Del Chiaro
authors: Matthias
authors: M
authors: Löhr
authors: Ralf
authors: R
authors: Segersvärd
authors: Markus J
authors: MJ
authors: Maeurer
mesh_terms: D000230:Adenocarcinoma / Q000276:immunology / Q000628:therapy*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000951:Antigens, Neoplasm / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D005260:Female; D006801:Humans; D007156:Immunologic Memory; D016219:Immunotherapy, Adoptive / Q000379:methods*; D008213:Lymphocyte Activation; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D010190:Pancreatic Neoplasms / Q000276:immunology / Q000628:therapy*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D016207:Cytokines; D011948:Receptors, Antigen, T-Cell