iNKT cells control mouse spontaneous carcinoma independently of tumor-specific cytotoxic T cells

Abstract

Background: CD1d-restricted invariant NKT (iNKT) cells are a subset of T lymphocytes endowed with innate effector functions that aid in the establishment of adaptive T and B cell immune responses. iNKT cells have been shown to play a spontaneous protective role against experimental tumors. Yet, the interplay between iNKT and tumor-specific T cells in cancer immune surveillance/editing has never been addressed. The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a realistic model of spontaneous oncogenesis, in which the tumor-specific cytotoxic T cell (CTL) response undergoes full tolerance upon disease progression.

Principal findings: We report here that lack of iNKT cells in TRAMP mice resulted in the appearance of more precocious and aggressive tumors that significantly reduced animal survival. TRAMP mice bearing or lacking iNKT cells responded similarly to a tumor-specific vaccination and developed tolerance to a tumor-associated antigen at comparable rate.

Conclusions: Hence, our data argue for a critical role of iNKT cells in the immune surveillance of carcinoma that is independent of tumor-specific CTL.

Figure 1. Survival of TRAMP mice lacking iNKT cells is reduced.

Kaplan-Maier plot reporting the survival curves of groups of male TRAMP (black squares; n = 21) and TRAMPJα18^−/− mice (black circles; n = 21). Animals were examined twice a week and euthanized when signs of bulky prostate tumor and/or distress were manifest. At necropsy, the anatomy and histology of the UGA was analyzed as indicated in the Material and Methods section. Animals were attributed a disease score≥4. Statistical comparison (Log-Rank test) between the survival curves: p<0.0001.

Figure 2. Tumor onset is accelerated in TRAMP mice lacking iNKT cells.

Age- and sex-matched male TRAMP (white bars; n = 97), TRAMPJα18^−/− (black bars; n = 51) and WT mice (gray bars; n = 97) were killed at the indicated week after birth and the anatomy and histology of the UGA was analyzed as indicated in the Material and Methods section. Animals affected by neuroendocrine tumors were excluded. A, UGA weight (g), expressed as average±SEM, of TRAMP, TRAMPJα18^−/− and WT mice killed at weeks 6–8 (n = 5, 8 and 19, respectively), 12–16 (n = 16, 17 and 26), 17–24 (n = 30, 15 and 26) and 25–28 (n = 46, 11 and 26), respectively. B, ratio of the weight of UGA and body weight subtracted of the UGA in the groups of animals reported above. C, Disease score, expressed as average±SEM, of the groups of animals reported above. Statistical analysis of collected data was performed using the Student's t-test (A and B) and the Mann-Whitney test (C); ***p<0.001, **0.001<p<0.05, *0.01<p<0.05.

Figure 3. The immune response against Tag is comparable in TRAMP and TRAMPJα18^−/− mice.

Tag-IV-pulsed DC were injected once i.d. into 6- (panels A–C) and 16-week (D–F) old male TRAMP (A and D), TRAMPJα18^−/− (B and E) and WT age- and sex-matched littermates (C and F). After 7 days, animals were killed and their splenocytes were stimulated in vitro with irradiated B6/K-0 cells, and tested 5 days later for cytotoxic activity (measured as ⁵¹Cr release); un-pulsed (black squares) or Tag-IV-pulsed (black diamonds) RMA and B6/K-0 (black circles) cells were used as targets. Data correspond to one out of at least three independent experiments, which gave similar results.

Figure 4. Tolerance in tumor-bearing mice is specific for the PC-related Ag Tag.

TRP-2-pulsed DC were injected once i.d. into 16-week old male TRAMP (left panel), TRAMPJα18^−/− (middle panel) and WT age- and sex-matched littermates (right panel). After 7 days, animals were killed and their splenocytes were stimulated in vitro with the TRP-2 peptide, and tested 5 days later for cytotoxic activity (measured as ⁵¹Cr release); un-pulsed (black diamonds) or TRP-2-pulsed (black triangles) RMA cells were used as targets. Each panel is representative of at least two independent experiments.

Figure 5. αGal-Cer-mediated release of iNKT-associated cytokines.

αGalCer (2 µg/mouse) was administered i.v. to TRAMP (white circles) and WT (black circles) littermates, and blood samples were collected from the tail vein 2, 6 and 24 hours later, and the serum content of IL-4 and IFN-γ was determined by standard ELISA. Values are reported as concentration of the cytokine (pg/ml) in the sera of each animal (3–4/experimental group) analyzed at the indicated time points. Reported data are representative of at least two independent experiments.