Expansion of quiescent lung adenocarcinoma CD8+ T cells by MUC1-8-mer peptide-T2 cell-β2 microglobulin complexes

Abstract

Adoptive immunotherapy requires the isolation of CD8+ T cells specific for tumor-associated antigens, their expansion in vitro and their transfusion to the patient to mediate a therapeutic effect. MUC1 is an important adenocarcinoma antigen immunogenic for T cells. The MUC1-derived SAPDTRPA (MUC1-8-mer) peptide is a potent epitope recognized by CD8+ T cells in murine models. Likewise, the T2 cell line has been used as an antigen-presenting cell to activate CD8+ T cells, but so far MUC1 has not been assessed in this context. We evaluated whether the MUC1-8-mer peptide can be presented by T2 cells to expand CD25+CD8+ T cells isolated from HLA-A2+ lung adenocarcinoma patients with stage III or IV tumors. The results showed that MUC1-8-mer peptide-loaded T2 cells activated CD8+ T cells from cancer HLA-A2+ patients when anti-CD2, anti-CD28 antibodies and IL-2 were added. The percentage of CD25+CD8+ T cells was 3-fold higher than those in the non-stimulated cells (P=0.018). HLA-A2+ patient cells showed a significant difference (2.3-fold higher) in activation status than HLA-A2+ healthy control cells (P=0.04). Moreover, 77.6% of MUC1-8-mer peptide-specific CD8+ T cells proliferated following a second stimulation with MUC1-8-mer peptide-loaded T2 cells after 10 days of cell culture. There were significant differences in the percentage of basal CD25+CD8+ T cells in relation to the cancer stage; this difference disappeared after MUC1-8-mer peptide stimulation. In conclusion, expansion of CD25+CD8+ T cells by MUC1-8 peptide-loaded T2 cells plus costimulatory signals via CD2, CD28 and IL-2 can be useful in adoptive immunotherapy.

Figure 1

HLA-A2 expression on the T2 cell surface is dose-dependent on the MUC1-8 peptide concentration. T2 cells were incubated with MUC1-8-mer peptide concentrations ranging from 0 to 100 µg/ml in the presence of β₂m (20 µg) for 24 h at 37°C in a 5% CO₂ atmosphere. After peptide loading, the T2 cells were harvested, washed, and stained with FITC-labeled anti-HLA-A2 mAb for flow cytometry. A representative histogram was constructed where the x-axis denotes the HLA-A2 fluorescence intensity of the T2 cells, and the y-axis indicates the cell percentage.

Figure 2

Colocalization of the HLA-A2 molecule with the MUC1-8-mer peptide on the T2 cell surface. T2 cells were loaded with the MUC1-8-mer peptide (100 µg) in the presence of β₂m (20 µg) for 24 h at 37°C in a 5% CO₂ atmosphere. The T2 cells were then harvested, washed, stained with FITC-labeled anti-HLA-A2 and Alexa Fluor 594-labeled goat anti-mouse IgG mAb after anti-CA 27–29 mAb (specific for SAPDTRPA), and prepared for confocal microscopy. (A) Image from one cell with DAPI nuclear staining (blue), (B-D) Same images as (A) where the HLA-A2 molecule is stained green (B), the MUC1-8-mer peptide is stained red (C), and the image merged by triple immunofluorescence (D), revealing colocalization (yellow) between HLA-A2 molecule and MUC1-8-mer peptide. Images were visualized using an LSM-510 zeiss confocal microscope. Data shown are representative of two individual experiments. Scale bar, 10 µm.

Figure 3

MUC1-8-mer peptide-T2 cell complex induces CD25 expression on CD8⁺ T cells from HLA-A2⁺ lung adenocarcinoma patients and healthy individuals. Purified CD8⁺ T cells from patients (A) or healthy individuals (B) were cultured under the different indicated conditions over 6 days. Cells were harvested, stained with FITC-labeled anti-CD8 and PE-labeled anti-CD25 mAbs, and analyzed using flow cytometry (images are shown in panels below each condition). An additional incubation with 7-aminoactinomycin-D (7-AAD) was performed to exclude any dying cells. Each solid circle denotes a sample from one patient, whereas an open circle represents a sample from one healthy control. ^*P<0.05.

Figure 4

Similar increase in CD25⁺CD8⁺ T cells from HLA-A2⁺ patients with stage III or IV tumor disease progression after stimulation by the MUC1-8-mer peptide-T2 complex and costimulatory antibodies. Purified CD8⁺ T cells from patients with stage III or IV were cultured with MUC1-8-mer peptide loaded-T2 cells and costimulatory antibodies to CD28 and CD2 plus IL-2 over 6 days. The cells were harvested, washed and stained with FITC-labeled anti-CD8 and PE-labeled anti-CD25 mAbs, and analyzed using flow cytometry. An additional incubation with 7-aminoactinomycin-D (7-AAD) was performed to exclude dying cells. Each circle denotes a sample from one patient. ^*P<0.03.

Figure 5

Expansion of antigen-specific CD8⁺ T cells after restimulation by the MUC1-8-mer peptide-T2 complex. CFSE-labeled CD8⁺ T cells from HLA-A2⁺ patients were cultured for 10 days under the indicated times and conditions. (A) Histograms show the CFSE intensity of the non-divided cell population in the absence of stimulus (left panels), and cells that have divided numerous times based on sequential fluorescence intensity (middle panels), as well as antigen-specific cells restimulated with the MUC1-8-mer peptide-T2 complex (right panels). (B) Graphic image showing the CD8⁺ T cell proliferation percentage on different days of culture under the conditions indicated.