Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Aug 8;7:12335.
doi: 10.1038/ncomms12335.

Interdependent IL-7 and IFN-γ Signalling in T-cell Controls Tumour Eradication by Combined α-CTLA-4+α-PD-1 Therapy

Affiliations
Free PMC article

Interdependent IL-7 and IFN-γ Signalling in T-cell Controls Tumour Eradication by Combined α-CTLA-4+α-PD-1 Therapy

Lewis Zhichang Shi et al. Nat Commun. .
Free PMC article

Abstract

Combination therapy with α-CTLA-4 and α-PD-1 has shown significant clinical responses in different types of cancer. However, the underlying mechanisms remain elusive. Here, combining detailed analysis of human tumour samples with preclinical tumour models, we report that concomitant blockade of CTLA-4 and PD-1 improves anti-tumour immune responses and synergistically eradicates tumour. Mechanistically, combination therapy relies on the interdependence between IL-7 and IFN-γ signalling in T cells, as lack of either pathway abrogates the immune-boosting and therapeutic effects of combination therapy. Combination treatment increases IL-7Rα expression on tumour-infiltrating T cells in an IFN-γ/IFN-γR signalling-dependent manner, which may serve as a potential biomarker for clinical trials with immune checkpoint blockade. Our data suggest that combining immune checkpoint blockade with IL-7 signalling could be an effective modality to improve immunotherapeutic efficacy. Taken together, we conclude that combination therapy potently reverses immunosuppression and eradicates tumours via an intricate interplay between IFN-γ/IFN-γR and IL-7/IL-7R pathways.

Conflict of interest statement

Drs Sharma and Allison are founders of Jounce Therapeutics. Dr Sharma also serves as a consultant for Bristol-Myers Squibb (BMS), Amgen and Glaxo SmithKline. Dr Allison is an inventor of intellectual property owned by the UC-Berkeley, and licensed to BMS and has received royalties from BMS. Dr Allison is also inventor of intellectual property owned by Memorial-Sloan Kettering Cancer Center and licensed to Merck. All other authors have no conflict of interest to disclose.

Figures

Figure 1
Figure 1. α-CTLA-4 upregulates the PD-1/PD-L1 inhibitory pathway in human and murine bladder tumours.
(a,b) Surface expression of CTLA-4 and PD-1 in Foxp3-CD4+ TILs isolated from patients with bladder cancer (a) and from murine MB49 bladder tumours (b). (c,d) PD-L1 expression on human (c) and mouse (d) bladder tumour cells analyzed by flow cytometry. (e) PD-1 expression on immune cells from pre- and post-α-CTLA-4-treated human bladder tumour by IHC. Representative IHC photos (arrows indicate positively-stained cells) and pooled results from all the patients are shown. (f) PD-1 expression on Foxp3-CD4+ TILs isolated from MB49 tumour-bearing mice treated with (α-CTLA-4) or without α-CTLA-4 (UnTx) were analyzed by flow cytometry. Histograms of PD-1 (left) and pooled results (right) from five mice in one representative experiment are shown. (g,h) PD-L1 expression on tumour cells (g) and immune cells (H) of pre- and post-α-CTLA-4-treated human bladder samples by IHC. Representative IHC photos (arrows indicate positively-stained cells) and pooled results from all the matched cases are shown. Data in scatter plots and the bar graph are means±s.e.m. Representative results (b, d, and f) from three independent experiments are shown. *P<0.05; **P<0.01; ***P<0.001 by two-tailed unpaired Student's t-test.
Figure 2
Figure 2. Combined PD-1 and CTLA-4 blockade eradicates MB49 bladder tumours in mice.
Mice bearing 6 days palpable MB49 tumour were left untreated (UnTx) or treated with α-CTLA-4, α-PD-1 or combination of both (Combo) or their corresponding isotype controls (α-CTLA-4 iso, α-PD-1 iso or Combo iso). (a) Individual tumour growth. Tumour size is presented as length × width (mm2) and ratios of tumour-growing mice in each groups are shown as insets on each panel. (b) Survival curves from one representative experiment are shown. Data are representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.001 by log-rank (Mantel–Cox) test.
Figure 3
Figure 3. Combination therapy-mediated MB49 tumour rejection is dependent on CD4+ and CD8+ T cells.
Isolated TILs from 13–15 days tumours, 1–3 days after the last treatment with α-CTLA-4, α-PD-1, combination of both (Combo) were analyzed by flow cytometry for (a) Total CD4+ T cells and CD8+ T cells. Representative flow panels are shown on the left and pooled results from five mice are depicted in bar graphs on the right. (b) Foxp3+ Treg in CD4+ T cells. Pooled results from five mice are shown in the right bar graph. (c) Ratios of Foxp3 to Foxp3+ in CD4+ T cells. (d) Ratios of CD8+ T cells to Foxp3+ CD4+ T cells. Mice depleted of CD4+ (GK1.5), CD8+ (2.43) or both (GK1.5+2.43) were treated with the combo and mouse survivals are presented (e). Data in the bar graphs are means±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by one-way ANOVA with Bonferroni's post hoc test. Data are representative of three independent experiments.
Figure 4
Figure 4. Combination therapy enhances polyfunctionality of CD4+ and CD8+ TILs.
(a) Purified CD4+ from TILs described as in Fig. 3 co-cultured with irradiated MB49 cells and splenic DCs in vitro were analyzed to determine the percentages of cells positive for IL-2 and IFN-γ. (b) Summarized results of dual IFN-γ+IL-2+ producers from five mice. (cf) Isolated TILs without further purification were stimulated briefly with PMA and ionomycin in vitro, and then analyzed for IFN-γ and TNF-α production in CD4+ TILs (c) or in CD8+ TILs (e). Percentages of dual IFN-γ+TNF-α+ producers in CD4+ or CD8+ TILs from fiev mice are depicted in (d) and (f), respectively. Data in the bar graphs are means±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by one-way ANOVA with Bonferroni's post hoc test. Data are representative of three independent experiments.
Figure 5
Figure 5. Combination therapy induces memory response and therapeutic benefits by engaging IL-7 signalling.
(a,b) Isolated splenocytes from tumour-bearing WT mice with regressing tumours treated with mono- or combo therapy were analyzed for: (a) Frequencies of CD4+CD44hiCD122+ (TCM) and (b) Frequencies of CD8+CD44hiCD122+ (TCM) cells. Pooled results of percentage and absolute cell number of TCM from five mice are depicted as bar graphs. (c) Combo-treated WT mice that previously rejected MB49 tumours were re-challenged with MB49 tumour cells or unrelated B16-BL6 melanoma cells. Tumour growth (mm2) is shown. (d) Surface expression of IL-7Rα on CD4+ and CD8+ TILs as described in Fig. 3 and MFIs of IL-7Rα from five mice are presented (right). (e) mRNA expression of IL-7Rα in sorted CD4+ and CD8+ TILs from untreated and combo-treated mice by real-time RT-PCR. Results were normalized to the expression of housekeeping gene (β-actin). (f) Survival of WT and Il7r−/− tumour-bearing mice treated with combination therapy. (g) Survival of tumour-bearing mice pretreated with blocking antibody against IL-7 (M25) upon combination therapy. (h) Abundance of CD4+ and CD8+ TILs isolated from Il7r−/− tumour-bearing mice as described in f. Data are means±s.e.m. of five mice in each group. NS, no statistical significance; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by one-way ANOVA with Bonferroni's post hoc test (a,b), two-tailed unpaired Student's t-test (d,e) or log-rank (Mantel–Cox) test (g). Data are representative of two to three independent experiments.
Figure 6
Figure 6. Combination therapy requires an intact loop of IL-7 and IFN-γ signalling pathways to reject MB49 tumours.
(a) Survival of WT and Ifngr1−/− MB49 tumour-bearing mice untreated (UnTx) or treated with combo. (b) Survival of tumour-bearing mice pretreated with blocking antibody against IFN-γ (XMG1.2) and then treated with combination therapy. (c) Sublethally irradiated CD45.1 mice were adoptively transferred with WT, Ifngr1−/− or Il7r−/− total T cells, followed by MB49 tumour inoculation and combination therapy. Tumour growth kinetics is shown. (d) Rag-1−/− mice were adoptively transferred with WT, Ifngr1−/− or Il7r−/− total T cells, followed by MB49 tumour inoculation and combination therapy. Tumour sizes on day 12 post-tumour inoculation are shown. (e) CD45.1 mice were treated as described above in c and mouse survivals are illustrated. (f,g) Isolated splenocytes from Il7r−/− (f) and Ifngr1−/− (g) tumour-bearing mice as described in Fig. 3 were analyzed for frequencies of CD4+CD44hiCD122+ (TCM) cells. Pooled results from five mice are depicted as bar graphs. (h,i) Isolated TILs from Ifngr1−/− tumour-bearing mice treated with combination therapy were examined for IL-7Rα expression in CD4+ (H) or CD8+ (i). Data are means±s.e.m. of five mice in each group. NS, no statistical significance; *P<0.01; **P<0.01 by one-way ANOVA with Bonferroni's post hoc test (c,d), log-rank (Mantel–Cox) test (e) or two-tailed unpaired Student's t-test (fi). Data are representative of two to three independent experiments.

Similar articles

See all similar articles

Cited by 24 articles

See all "Cited by" articles

References

    1. Ishida Y., Agata Y., Shibahara K. & Honjo T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 11, 3887–3895 (1992). - PMC - PubMed
    1. Sharma P. & Allison J. P. The future of immune checkpoint therapy. Science 348, 56–61 (2015). - PubMed
    1. Tivol E. A. et al. . Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3, 541–547 (1995). - PubMed
    1. Leach D. R., Krummel M. F. & Allison J. P. Enhancement of antitumor immunity by CTLA-4 blockade. Science 271, 1734–1736 (1996). - PubMed
    1. Hodi F. S. et al. . Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 363, 711–723 (2010). - PMC - PubMed

Publication types

MeSH terms

Feedback