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.

Tumor-infiltrating lymphocytes (TILs) from patients with glioma

“…TILs and PBMCs were stained using the same Ab cocktail as described previously 47 and in detail in the supplementary data. Analysis was performed using a FACS Aria flow cytometer (BD Biosciences, Stockholm, Sweden) and FlowJo software.…”

“…TILs were also exposed to autologous tumor cells for 12 h and tested for cytokine production. Stimulation was stopped by incubating cells at 4 C. At day 2, TILs were washed and stained using the same Ab cocktail and protocol as described 47 (in detail in the supplementary data).…”

T Cell Genesis: In Vitro Veritas Est ?

“…TILs can be found in several solid tumors, including in gastrointestinal [57] and pancreatic cancers [58] and their presence correlates with a more positive clinical outcome. Therefore, the methodology used for the isolation, ex vivo expansion and reintroduction of TILs to the patient provides a compelling option for cancer treatment.…”

Adoptive immunotherapy via CD4+ versus CD8+ T cells

“…These include tumor-infiltrating T cells (TILs) that have been expanded and activated ex vivo after isolation from tumor (Dudley et al, 2005;Meng et al, 2016a), T cell receptor (TCR)-modified T cells that are modified ex vivo to express a particular TCR for more specific tumor recognition (Spear et al, 2016), and chimeric antigen receptor (CAR)-engineered T cells (where the receptor is engineered by fusing an antibody (Ab) with the TCR to improve tumor recognition and interaction) (Brown et al, 2015). The above manipulations are part of an effort to improve the clinical efficacy of ACT.…”

“…Thus, cytokine growth factors used in ex vivo expansion of vaccine-primed CD4+ T cells can significantly improve antitumor immune responses. The choice of cytokine growth factor, therefore, is critical (Meng et al, 2016a;Phan-Lai et al, 2015). Specifically, IL2/IL21 (perhaps also in combination with other cytokines) can give rise to Th1/Th17 polyfunctional T cells and induce durable responses in mice and patients (Phan-Lai et al, 2015).…”