Ex vivo expanded telomerase-specific T cells are effective in an orthotopic mouse model for pancreatic adenocarcinoma

Abstract

Telomerase activity is over-expressed in nearly all pancreatic carcinomas, but not in chronic pancreatitis. Here, we investigated various protocols for expansion of telomerase-specific T cells for adoptive cell transfer and their use in a syngeneic pancreatic carcinoma mouse model. Telomerase-specific T cells were generated by stimulation of splenocytes from peptide-immunized donor mice with either interleukin (IL)-2, IL-15, artificial antigen-presenting cells, anti-signalling lymphocyte activation molecule (SLAM) microbeads or allogeneic dendritic cells in combination with a limited dilution assay. T cells were tested for antigen specificity in vitro and for anti-tumour activity in syngeneic mice with orthotopically implanted tumours pretreated with cyclophosphamide. The immune cells from recipients were immunophenotyped. During a period of 2 weeks, the expansion approach using IL-2 was very successful in generating a high number of telomerase-specific CD8(+) T cells without losing their function after adoptive cell transfer. Significantly slower tumour growth rate and less metastasis were observed after adoptively transferring telomerase specific CD8(+) T cells, expanded using IL-2. Further investigations showed that anti-tumour efficacy was associated with a significant shift from naive CD8(+) T cells to CD8(+) central memory T cells, as well as recruitment of a high number of dendritic cells. Remarkable amounts of telomerase-specific T cells were detectable in the tumour. Generation of telomerase-specific T cells is feasible, whereat IL-2-based protocols seemed to be most effective and efficient. Antigen-specific T cells showed significant cytotoxic activity in a syngeneic, orthotopic mouse model, whereas central memory T cells but not effector memory T cells appear to be of high importance.

Fig. 1

Telomerase activity in pancreatic cancer. Using a telomerase polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) technique, telomerase activity was investigated (a) in one murine pancreatic carcinoma cell line (Panc02) and 11 human pancreatic carcinoma cell lines (listed in the figure) and (b) in 50 pancreatic carcinoma samples, 25 tissue samples from patients suffering from chronic pancreatitis and 25 samples from healthy pancreas. The dotted line depicts the cut-off.

Fig. 3

Telomerase specificity. In vitro-induced T cells – expanded via interleukin (IL)-2 (either normal or prolonged expansion), IL-15, artificial antigen presenting cells (aAPC) and signalling lymphocyte activation molecule (SLAM) microbeads – were incubated with either murine telomerase peptide-loaded dimer or with human A3 peptide-loaded dimer as control. After being stained with a dimer-specific antibody, cells were processed for specificity analysis using the flow cytometer. The figure depicts representative histograms from at least three independent experiments.

Fig. 2

Expansion approach versus number of cells/ml. T cells were isolated from preimmunized mice and were then plated for 14 days in 96-well plates for limited dilution analysis (LDA), after which T cells were expanded for a further 14 days using artificial antigen presenting cells (aAPC), interleukin (IL)-15, signalling lymphocyte activation molecule (SLAM) microbeads or IL-2 for expansion. A set of T cells was allowed to expand for a prolonged expansion period using IL-2. Shortly before adoptive cell transfer (ACT), the cell count achieved in each expansion approach was determined. Data are shown from at least two independent experiments.

Fig. 4

Anti-tumour activity of adoptively transferred cells. Tumour-bearing mice were treated with adoptively transferred T cells expanded via artificial antigen presenting cells (aAPC), interleukin (IL)-15, signalling lymphocyte activation molecule (SLAM) microbeads and two different IL-2-based protocols. On the day of killing, the tumour volume of all treated mice groups as well as the control was calculated. Data are shown as median and interquartile range (IQR). An asterisk indicates statistical significance. Data are shown from at least three independent experiments.

Fig. 5

(a,b) Immunophenotyping of splenocytes. Tumour-bearing mice were treated as described. Splenocytes from killed animals were investigated by flow cytometry. Data are shown as median and interquartile range (IQR). An asterisk indicates statistical significance. Data are shown from at least three independent experiments.

Fig. 6

(a,b) Comparison of T cell activity generated either by dendritic cell (DC) co-culture or interleukin (IL)-2 expansion. (a) Both cells co-cultured with telomerase-pulsed DCs and those expanded using IL-2 were transferred adoptively into mice. On the day of killing, tumour volume of all treated mice groups as well as the control was calculated. Data are shown as median and interquartile range (IQR). An asterisk indicates statistical significance. (b) On the day of killing, T cells were isolated from the tumours and analysed by dimer stain and subsequent flow cytometric analysis for telomerase-specificity. The figure depicts representative histograms.