Tumor-released survivin induces a type-2 t cell response and decreases cytotoxic T cell function, in vitro

Abstract

Clinical studies of T cell profiles from cancer patients have shown a skewing toward a type-2 T cell response with decreased cytotoxic T cell function. However, the primary cause of this shift remains unknown. Here we show that tumor-released Survivin, an inhibitor of apoptosis (IAP) protein and tumor-specific antigen, is taken up by T cells and alters their response. The addition of Survivin to T cell cultures resulted in decreased T cell proliferation and reduced cytotoxic CD8(+) T cell function. Additionally, type 1 cell numbers and IFN-γ and IL-2 production were significantly reduced, while IL-4 release and type 2 T cell numbers increased. In contrast, the function and numbers of Th17 and T regulatory cells were not affected. These studies show that tumor-released Survivin modulates T cells resulting in a phenotype similar to that observed in cancer patients with a polarity shift from a type 1 to a type 2 response.

Fig. 1

Normal T cells take up tumor-released Survivin. a Normal T cells were isolated by magnetic separation and cultured with FLAG-HA-Survivin or co-cultured with POZn-WT-Survivin cells for 24 h. Confocal images show uptake and surface binding of Survivin using a 60x oil immersion objective lens (Red – Actin, Green – FLAG-HA-Survivin, blue – DAPI). Representative of 2 independent experiments. b Surface and intracellular mean fluorescence intensity of HA in CD4⁺ (top) and CD8⁺ (bottom) T cells over time. Representative of 3 independent experiments. (Mean ± SD for 3 independent experiments; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 Intracellular vs. Surface). c Western blotting of T cells shows the presence of intracellular Survivin and FLAG tagged Survivin over time. Blots are representative of 3 independent experiments. d Densitometry shows Mean ± SD for 3 independent experiments, 2 blots each, n = 6

Fig. 2

T cell proliferation is inhibited by culture with tumor-released Survivin. a Representative Ki-67 staining at 72 h post-stimulation with anti-CD3/anti-CD28 microbeads. b Ki-67 expression was evaluated over time in T cells cultured with FLAG-HA-Survivin. CD4⁺Ki-67⁺ and CD8⁺Ki-67⁺ cell numbers are decreased after culture with FLAG-HA-Survivin compared to control. c and d CFSE dilution post-activation is significantly reduced by Survivin in CD8⁺ and CD4⁺ T cells compared to controls at 72, 96 and 120 h. e and f BrdU incorporation is diminished in CD8⁺ and CD4⁺ T cell cultures containing Survivin. Results in a, c and e are representative of 3 independent experiments. Data in b, d, and f are Means ± SD of 3 independent experiments, n = 9. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Survivin does not affect T cell activation. a CD25 and CD69 staining of CD4⁺ and CD8⁺ T cells cultured with anti-CD3/anti-CD28 microbeads for 24 h show no change in CD25⁺CD69⁺ cell numbers. Results representative of 3 independent experiments. b CD25 and CD69 MFI are and unchanged in CD8⁺ and CD4⁺ Survivin-cultured cells. Results show Mean ± SD, n = 9

Fig. 4

Survivin decreases cytotoxicity of CD8⁺ T cells. CD8⁺ cells from a single donor were cultured with Survivin for 24 h and their cytotoxic function assessed by the CytoTox 96 Non-Radioactive Cytotoxicity Assay. Results show Mean ± SD of 3 independent experiments, n = 3. *p < 0.05

Fig. 5

Polarization to a type 2 phenotype by Survivin. Survivin significantly decreases CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ and increases CD4⁺Il-5⁺, CD8⁺Il-5⁺, CD4⁺IL-13⁺, CD8⁺IL-13⁺, and CD4⁺IL-4⁺ cell numbers. No change was seen in IL-17⁺ cell numbers. Results show Mean ± SD from 3 independent experiments, n = 9. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 6

Type 1 cytokines are decreased and Type 2 cytokines increased by Survivin. Cytokine ELISA shows decreased production of inflammatory cytokines IFN-γ and IL-2 by CD4⁺ and CD8⁺ T cells and increased IL-4 and IL-13 production by CD4⁺ cells in the presence of Survivin. No change was observed in IL-5 or IL-10 release. Results show Mean ± SD from 2 independent experiments, n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001