Antitumor activity of T cells generated from lymph nodes draining the SEA-expressing murine B16 melanoma and secondarily activated with dendritic cells

Abstract

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFN gamma and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.

Keywords: Adoptive therapy; B16 melanoma; Staphylococcal enterotoxin A (SEA); T lymphocyte; dendritic cells (DC).

Figure 1

A: Scheme for construction of recombinant expression vector pcDNA3.1 (+)/SEA. B: Restriction enzyme digestion of the recombinant expression vector pcDNA3.1-SEA. 1. λDNA/HindIII ladder; 2. pcDNA3.1-SEA/NheI + EcoRI; 3. pcDNA3.1-SEA/EcoRI; M: DL2000 marker. C: Transfected SEA fragment confirmed by RT-PCR product on agarose gel electrophoresis. Lanes 1-3: Three clones of SEA-B16 cells; M: DNA marker (DL2000).

Figure 1

A: Scheme for construction of recombinant expression vector pcDNA3.1 (+)/SEA. B: Restriction enzyme digestion of the recombinant expression vector pcDNA3.1-SEA. 1. λDNA/HindIII ladder; 2. pcDNA3.1-SEA/NheI + EcoRI; 3. pcDNA3.1-SEA/EcoRI; M: DL2000 marker. C: Transfected SEA fragment confirmed by RT-PCR product on agarose gel electrophoresis. Lanes 1-3: Three clones of SEA-B16 cells; M: DNA marker (DL2000).

Figure 1

A: Scheme for construction of recombinant expression vector pcDNA3.1 (+)/SEA. B: Restriction enzyme digestion of the recombinant expression vector pcDNA3.1-SEA. 1. λDNA/HindIII ladder; 2. pcDNA3.1-SEA/NheI + EcoRI; 3. pcDNA3.1-SEA/EcoRI; M: DL2000 marker. C: Transfected SEA fragment confirmed by RT-PCR product on agarose gel electrophoresis. Lanes 1-3: Three clones of SEA-B16 cells; M: DNA marker (DL2000).

Figure 2

Morphological observation of DCs under phase contrast microscope, scanning electron microscope, and transmission electron microscope after cell culture for 7 days.

Figure 3

Proliferation of TDLN cells stimulated with different conditions. Multiple inguinal TDLNs were pooled from groups of mice and were processed for lymphoid cell suspension. T cells were purified using the nylon wool column. Purified T cells were stimulated under the following conditions: Group A, D: Anti-CD3 + anti-CD28 + IL-2 plus DC and tumor lysates; Group B, E: Anti-CD3 + anti-CD28 + IL-2; Group C, F: Un-stimulated (medium only). 100μl of cell suspension was added to each well of 96-well culture plate in triplicate. Stimuli described above were added at 100μl/well. After culture for 48 hours, cell proliferation was measured with MTT analysis. P<0.01: A vs. B; A vs. C; A vs. D. B vs. E; C vs. F. D vs. E; D vs. F.

Figure 4

SEA TDLN cells activated with anti-CD3/anti-CD28/IL-2 plus DC and tumor antigen demonstrated the most potent therapeutic efficacy. In these adoptive transfer experiments, pulmonary metastases were induced by tail vein injection of 1 x 10⁶ of wt B16 cells in 0.2 ml PBS in B6 mice. Three days later, the tumor-bearing mice were treated with variously activated TDLN cells (2 x 10⁶ cells/mouse) by tail vein injection. Approximately 10 days after T cell transfer, all mice were randomized and sacrificed, and lungs were harvested for enumeration of pulmonary metastatic nodules. TDLN cells were activated with anti-CD3/anti-CD28/IL-2 plus DC and tumor antigen (Group A, D); anti-CD3/anti-CD28/IL-2 (Group B, E), or un-stimulated (Group C, F). P<0.01: A vs. B; A vs. C; A vs. D; B vs. E; C vs. F; D vs. E; D vs. F.

Figure 5

In vitro CTL activity of TDLN cells stimulated with different conditions. SEA TDLN and wt TDLN T cells were stimulated using anti-CD3 + anti-CD28 + IL-2 with (group A, D) or without (group B, E) DC plus tumor lysate, or left un-stimulated (group C, F), as in Figure 3. Stimulated T cells were washed, re-suspended at 4×10⁶/ml and mixed with ³H-TdR-labeled B16 cells at 40: 1, 20: 1, and 10: 1 in U-bottom 96-well plate. After culture at 37^oC with 5% CO₂ for 18 hours, cpm was measured and the killing index was calculated.

Figure 6

IFNγ and IL-10 expression on activated TDLN cells. SEA TDLN and wt TDLN T cells were stimulated using anti-CD3 + anti-CD28 + IL-2 with (groups A, D) or without (groups B, E) DC plus tumor lysate, or left un-stimulated (groups C, F), as in Figure 3. After activation for 48 hours, TDLN T cells were permeablized and stained with FITC-conjugated anti-IFN-γ or PE-conjugated anti-IL-10. FACS analysis was then performed.