Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Mar 1;146(5):1409-1420.
doi: 10.1002/ijc.32777. Epub 2019 Dec 4.

A TLR7 agonist strengthens T and NK cell function during BRAF-targeted therapy in a preclinical melanoma model

Lydia Bellmann  1 , Giuseppe Cappellano  1 , Johanna F Schachtl-Riess  1 , Anastasia Prokopi  1 , Athanasios Seretis  1 , Daniela Ortner  1 , Christoph H Tripp  1 , Constance E Brinckerhoff  2 , David W Mullins  3 , Patrizia Stoitzner  1
Affiliations

A TLR7 agonist strengthens T and NK cell function during BRAF-targeted therapy in a preclinical melanoma model

Lydia Bellmann et al. Int J Cancer. .

Erratum in

  • Erratum.
    [No authors listed] [No authors listed] Int J Cancer. 2020 Jul 1;147(1):E9. doi: 10.1002/ijc.32940. Epub 2020 Mar 6. Int J Cancer. 2020. PMID: 32359118 Free PMC article. No abstract available.

Abstract

Therapeutic success of targeted therapy with BRAF inhibitors (BRAFi) for melanoma is limited by resistance development. Observations from preclinical mouse models and recent insights into the immunological effects caused by BRAFi give promise for future development of combination therapy for human melanoma. In our study, we used the transplantable D4M melanoma mouse model with the BRAFV600E mutation and concomitant PTEN loss in order to characterize alterations in tumor-infiltrating effector immune cells when tumors become resistant to BRAFi. We found that BRAFi-sensitive tumors displayed a pronounced inflammatory milieu characterized by high levels of cytokines and chemokines accompanied by an infiltration of T and NK cells. The tumor-infiltrating effector cells were activated and produced high levels of IFN-γ, TNF-α and granzyme B. When tumors became resistant and progressively grew, they reverted to a low immunogenic state similar to untreated tumors as reflected by low mRNA levels of proinflammatory cytokines and chemokines and fewer tumor-infiltrating T and NK cells. Moreover, these T and NK cells were functionally impaired in comparison to their counterparts in BRAFi-sensitive tumors. Their effector cell function could be restored by additional peritumoral treatment with the TLR7 agonist imiquimod, a clinically approved agent for nonmelanoma skin cancer. Indeed, resistance to BRAFi therapy was delayed and accompanied by high numbers of activated T and NK cells in tumors. Thus, combining BRAFi with an immune stimulating agent such as a TLR ligand could be a promising alternative approach for the treatment of melanoma.

Keywords: BRAF inhibitor resistance; T cell and NK cell immunity; melanoma; targeted therapy.

PubMed Disclaimer

Figures

Figure 1
Figure 1
BRAFi treatment causes a transient infiltration of T and NK cells. (a) Tumor growth curve upon s.c. injection with 3 × 105 D4M cells. On Day 8 (dotted vertical line), mice received either control chow (dashed lines = untreated) or BRAFi‐containing chow (blue and black lines). One representative graph is shown (n ≥ 12/group). (b–d) Tumors from mice shown in (a) were analyzed, and (b) tumor weight was measured. (c) Flow cytometry analysis of tumor‐infiltrating CD45+ immune cells, (d) tumor‐infiltrating NK cells (NK1.1+) and T cells (CD3+) from untreated, BRAFi‐sensitive and BRAFi‐resistant tumors. Percentages and cell numbers/g tumor are shown as summary graphs for at least two independent experiments (n ≥ 16/group). [Color figure can be viewed at http://wileyonlinelibrary.com]
Figure 2
Figure 2
BRAFi‐sensitive and resistant tumors display distinct chemokine, cytokine and tumor antigen expression patterns. (a–d) RT‐qPCR was performed with untreated, BRAFi‐sensitive and BRAFi‐resistant tumors to determine mRNA levels for (a) chemokines CCL2, CCL3, CCL4, CXCL9, CXCL10, (b) proinflammatory cytokines IL‐12a, IL‐12b, IL‐15, IL‐18, (c) immunosuppressive cytokines IL‐10 and TGF‐β1 and (d) melanoma‐associated antigens gp100 and trp‐2 relative to TATA binding protein (Tbp; n = 6/group). [Color figure can be viewed at http://wileyonlinelibrary.com]
Figure 3
Figure 3
Tumor‐infiltrating NK and T cells exhibit an activated phenotype during BRAFi treatment. (a and b) Percentages of (a) CD69+, (b) PD‐1+ and TIM‐3+ cells of NK and T cells infiltrating untreated, BRAFi‐sensitive and BRAFi‐resistant tumors were determined by flow cytometry. Summary graphs for at least two independent experiments are shown (n ≥ 7/group). (c) RT‐qPCR was performed with untreated, BRAFi‐sensitive and BRAFi‐resistant tumors to determine mRNA levels for the inhibitory ligands PD‐L1, PD‐L2 and galectin‐9 relative to TATA binding protein (Tbp; n = 6/group). [Color figure can be viewed at http://wileyonlinelibrary.com]
Figure 4
Figure 4
NK and T cells in BRAFi‐sensitive tumors produce cytokines and possess the ability to kill tumors cells. (a and b) Flow cytometry analysis showing the percentages of (a) IFN‐γ+ and (b) TNF‐α+ cells of CD4+ and CD8+ T cells as well as NK cells infiltrating untreated, BRAFi‐sensitive and BRAFi‐resistant tumors evaluated after in vitro restimulation. Summary graphs for two independent experiments are shown (n ≥ 6/group). (c) Flow cytometry analysis showing the percentages of granzyme B+ cells of NK and CD8+ T cells infiltrating untreated, BRAFi‐sensitive and BRAFi‐resistant after in vitro restimulation. Summary graphs for two independent experiments are shown (n ≥ 8/group). (d) Percentages of NKG2D+ cells of NK and T cells infiltrating untreated, BRAFi‐sensitive and BRAFi‐resistant tumors were determined by flow cytometry. Summary graphs for two independent experiments are shown (n ≥ 8/group). (e) RT‐qPCR was performed with untreated, BRAFi‐sensitive and BRAFi‐resistant tumors to determine mRNA levels for the NKGD2 ligands Rae1, Ulbp1 and H60b relative to TATA binding protein (Tbp) (n ≥ 5/group). (f) Percentages of MHC‐class I+ cells of viable CD45− tumor cells infiltrating untreated, BRAFi‐sensitive and BRAFi‐resistant tumors were determined by flow cytometry analysis. Summary graph for one experiment is shown (n ≥ 4/group). [Color figure can be viewed at http://wileyonlinelibrary.com]
Figure 5
Figure 5
Additional application of the TLR7A during BRAFi treatment prolongs tumor growth control in D4M melanoma. (a) Tumor growth curve after s.c. injection with 3 × 105 murine melanoma D4M cells. On Day 8 (first dotted vertical line), mice were switched to BRAFi‐containing chow and on Day 19 (second dotted vertical line) mice were given s.c. peritumorally 2 mg/kg of the TLR7A imiquimod (blue lines) twice per week or PBS (black lines). Summary graph for two independent experiments is shown (n = 10–11/group). (b) Size and weight of tumors from (a) were assessed at Days 41–47 or when tumors reached a size over 75 mm2. (c) Percentages of tumor‐infiltrating NK and T cells from PBS‐treated or TLR7A‐treated tumors were determined by flow cytometry. Summary graphs for two independent experiments are shown (n = 9/group). (d) Percentages of CD69+, PD‐1+ and NKG2D+ cells of NK and T cells infiltrating PBS‐treated or TLR7A‐treated tumors were determined by flow cytometry. Summary graphs for 1–2 experiments are shown (n ≥ 4/group). (e) Percentages of IFN‐γ+ and TNF‐α+ cells of CD4+ and CD8+ T cells as well as NK cells infiltrating PBS‐treated or TLR7A‐treated tumors were determined upon in vitro restimulation. Summary graph for one experiment is shown (n ≥ 3/group). [Color figure can be viewed at http://wileyonlinelibrary.com]
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018;68:7–30. - PubMed
    1. Flaherty KT, Hodi FS, Fisher DE. From genes to drugs: targeted strategies for melanoma. Nat Rev Cancer 2012;12:349–61. - PubMed
    1. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949–54. - PubMed
    1. Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011;364:2507–16. - PMC - PubMed
    1. Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAF‐mutated metastatic melanoma: a multicentre, open‐label, phase 3 randomised controlled trial. Lancet 2012;380:358–65. - PubMed

Publication types

MeSH terms