Invariant natural killer T cells regulate breast cancer response to radiation and CTLA-4 blockade

Abstract

Purpose: Immunoregulatory and suppressive mechanisms represent major obstacles to the success of immunotherapy in cancer patients. We have shown that the combination of radiotherapy to the primary tumor and CTL-associated protein 4 (CTLA-4) blockade induces antitumor immunity, inhibiting metastases and extending the survival of mice bearing the poorly immunogenic and highly metastatic 4T1 mammary carcinoma. Similarly to patients with metastatic cancer, however, mice were seldom cured. Here we tested the hypothesis that invariant natural killer T (iNKT) cells, a subset with unique regulatory functions, can regulate the response to radiotherapy and CTLA-4 blockade.

Experimental design: The growth of 4T1 primary tumors and lung metastases was compared in wild-type and iNKT cell-deficient (iNKT-/-) mice. Treatment was started on day 13 when the primary tumors were palpable. Mice received radiotherapy to the primary tumor in two doses of 12 Gy in combination or not with 9H10 monoclonal antibody against CTLA-4. Response to treatment was assessed by measuring primary tumor growth delay/regression, survival, and number of lung metastases.

Results: The response to radiotherapy plus 9H10 was markedly enhanced in the absence of iNKT cells, with 50% of iNKT-/- versus 0% of wild-type mice showing complete tumor regression, long-term survival, and resistance to a challenge with 4T1 cells. Administration of the iNKT cell activator alpha-galactosylceramide did not enhance the response of wild-type mice to radiotherapy plus 9H10. Tumor-infiltrating iNKT cells were markedly reduced in wild-type mice treated with radiotherapy plus 9H10.

Conclusions: iNKT cells play a major role in regulating the response to treatment with local radiotherapy and CTLA-4 blockade.

Figures

Figure 1. CD8 T cell-mediated inhibition of metastases in untreated tumor-bearing mice lacking iNKT cells

(A) Mice received 5×104 4T1 cells s.c. on Day 0. The number of metastatic cells in the lungs at Day 34 was significantly lower for the iNKT−/− than for the WT mice (p=0.0009). Each symbol represents one animal. (B, C) mAb-mediated CD4 and CD8 T-cell depletion was started on Day −1, and maintained up to the day of sacrifice. Mice were sacrificed on Day 33 to determine the tumor weigh (B) and number of metastatic cells in the lungs (C). Bars represent the mean ± SE of 8 to 10 animals/group. (B) There was no significant tumor weight difference (p>0.6) between WT and iNKT−/− mice non-depleted or depleted of CD8 or CD4 T cells. However, tumor weight was significantly increased in mice depleted of both T cell subsets (p<0.01 when compared to all other groups). (C) The number of metastases in iNKT−/− mice depleted of CD8 T cells (CD8) or CD4 and CD8 T cells (CD4+CD8) was significantly higher than in non-depleted iNKT−/− mice (p=0.004 and 0.001, respectively). In contrast, CD4 depletion did not significantly increase the number of metastases (p=0.94). Whereas there was a significant difference between WT and non-depleted iNKT−/− mice in the number of metastases (p=0.043), no significant difference was seen between WT and iNKT−/− mice after CD8 T cell depletion by itself or in combination with CD4 T cell depletion. (D) 4T1 cells are not intrinsically immunogenic in iNKT−/− mice. WT and iNKT−/− mice (N=5/group) were vaccinated weekly for 3 weeks with 106 irradiated (100 Gy) 4T1 cells s.c. in the left flank (+) or mock vaccinated with DMEM (−). Seven days after the last vaccination mice were challenged with 5×104 viable 4T1 cells and followed for tumor development. All mice developed tumors and there was significant difference in tumor weight in vaccinated WT (p=0.94) or iNKT (p=0.62) mice on Day 26.

Figure 2. Therapeutic response of iNKT−/− mice with established 4T1 carcinoma to treatment with local RT and CTLA-4 blockade

Treatment was started on Day 13 post-s.c. inoculation with 4T1 cells. RT was delivered in two fractions of 12 Gy to the s.c. tumors on Day 13 and 14. Ab were given i.p. 1, 4 and 7 days post-RT. (A) Tumor growth delay in iNKT−/− mice treated with control hamster IgG (IgG) (open traingles, N=8), 9H10 (closed triangles, N=9), RT + IgG (open circles, N=9), or RT + 9H10 (closed circles, N=9). Tumor volume is shown as the mean ± SE for animals with tumors in each treatment group up to Day 32 when all animals were alive. The number of mice with complete tumor regression over the total number of mice per group is indicated. Tumor volume differences between treated and control (IgG) mice were statistically significant (p

Figure 3. Comparison between WT and iNKT−/− mice with established 4T1 carcinoma in the response to treatment with local RT and CTLA-4 blockade

Comparison of response to treatment with RT and 9H10 (solid lines) or IgG (broken lines) in WT (black) and iNKT−/− (red) 4T1-tumor bearing mice. Treatment was started on Day 13 post-s.c. inoculation in the flank. RT was delivered in two fractions of 12 Gy to the s.c. tumors on Day 13 and 14. Ab were given i.p. 1, 4 and 7 days post-RT. (A) Tumor volume is shown as the mean ± SE for animals with tumors in each treatment group up to Day 29 when all animals were alive. The number of mice with complete tumor regression by Day 29 over the total number of mice per group is indicated. Two additional iNKT−/− mice had complete tumor regression after Day 29. Primary tumor growth was not significantly different in WT and iNKT−/− mice receiving the control IgG (p>0.05) at all time points. In contrast, iNKT−/− mice receiving RT+9H10 had a significantly lower tumor volume than WT mice receiving RT+9H10 (p

Figure 4. Administration of αGC does not enhance the response of 4T1 tumor-bearing WT mice to local RT and CTLA-4 blockade

Treatment was started on Day 13 post-s.c. inoculation in the flank. RT was delivered in two fractions of 12 Gy to the s.c. tumors on Day 13 and 14. 9H10 was given i.p. 1, 4 and 7 days post-RT. αGC (100 ng) was given i.p. twice per week starting on Day 1 post-RT. WT mice (N=5/group) were treated with (A) vehicle (closed circles), 9H10 + vehicle (closed diamonds), RT + vehicle (closed squares), RT + 9H10 + vehicle (closed triangles), (B) αGC (open circles), 9H10 + αGC (open diamonds), RT + αGC (open squares), or RT + 9H10 + αGC (open triangles). Tumor volume is shown as the mean ± SEM for animals with tumors in each treatment group up to Day 27 when all animals were alive. RT caused a significant (p

Figure 5. iNKT cells infiltrating 4T1 tumors are reduced by treatment with local RT and CTLA-4 blockade

WT mice were injected with 4T1 cells on Day 0 and left untreated (None) or treated with local RT and 9H10 (RT+9H10) as described in the legend of Figure 3. Tumors and lungs were harvested on Day 29, and obtained single cell suspensions stained FITC-anti-CD3 mAb and CD1d-PBS-57 PE or control CD1d-Vehicle PE tetramers to identify iNKT cells, followed by flow cytometry analysis. The lymphocyte gate was set based on the scattered plots in the spleen. (A) Histograms showing tumor-derived cells from untreated mice stained with CD3 and CD1d-PBS-57 or control tetramers, as indicated, and gated on CD3+ cells. Numbers indicate the percentage of cells in the square gate. (B) The percentage of cells in the lymphocyte gate positive for CD3 and CD1d-PBS-57 was multiplied for the percentage of cells in the lymphocyte gate and for the total number of viable cells isolated from the tumors, and divided for the tumor weight to obtain the number of cells per mg of tumor in treated (RT+9H10, white bar) and untreated (None, black bar) mice. (C) Percentage of CD3+ T cells that bind the CD1d-PBS-57 tetramers in tumor and lungs of treated (RT+9H10, white bars) and untreated (None, black bars) mice, as indicated. All data are from 4 to 5 mice of each group. Errors bars are absent because pooling of tumors and lungs within each group was necessary to obtain sufficient cells for analysis.

Figure 6. Accumulation of MDSC and TGFβ production in WT and iNKT−/− mice bearing 4T1 tumors

WT and NKT−/− mice were inoculated s.c. with 5×104 4T1 cells on Day 0 (N=5/group). 3 non-tumor-bearing mice of each genotype served as controls. On Day 14 primary tumors and spleens were harvested and obtained cell suspensions were stained with PE-anti-CD11b and Cy-anti-Gr-1 mAbs to detect MDSC. (A) Representative histograms of spleen from one healthy (top row) and one tumor-bearing (middle row) mouse showing the CD11b+Gr-1+ MDSC. Histograms from pooled tumors isolated from 5 WT and 5 iNKT−/− mice showing the CD11b+Gr-1+ MDSC (bottom row). Numbers indicate the percentage of MDSC. (B) Percentage of spleen MDSC in relation to the tumor weight in WT (closed triangles) and iNKT−/− (open circles) mice. Each symbol represents an individual mouse. For control healthy mice, one symbol representing the mean of 3 animals is shown. (C) Splenocytes isolated from tumor-free (−) (N=3) and tumor-bearing (+) (N=5) mice were cultured o.n. and the concentration of TGFβ in the culture supernatants was determined by ELISA. Data are the mean ± SE. In both WT and iNKT−/− mice spleen cells derived from tumor-bearing mice produced significantly more TGFβ than spleen cells derived from healthy mice (p<0.05). Although the baseline production of TGFβ did not differ significantly between WT and iNKT−/− healthy mice (p=0.35), tumor-bearing iNKT−/− mice produced more TGFβ than tumor-bearing WT mice (p<0.01).