A Combo offer to deal with Melanoma: Latest Development with Immune Checkpoint Inhibitors

Modern Science has experienced huge breakthroughs over the last decades, one of the major ones being the discovery of immune checkpoints and their inhibitors. The checkpoint inhibitors revolutionized the stagnant field of cancer biology and were responsible for the rise of cancer immunotherapies. Immunotherapies command the immune system to destroy tumors by using antibodies against specific immunosuppressive surface receptors.  Programmed cell death protein-1 (PD-1) is one such receptor expressed on T cells that interacts with programmed cell death ligand-1 (PD-L1) present on tumor cells, which leads to inactivation and subsequent anergy (absence of normal immune response) of T cells. Cytotoxic T lymphocyte-associated protein-4 (CTLA-4) is another receptor that is constitutively expressed on regulatory T cells and enhances its role by inhibiting the functions of other T cells and antigen-presenting cells, hence helping tumor growth. Monoclonal antibody treatments targeting these protein receptors are now widely used in clinics and have helped in saving countless lives already. The success has increased interest in not only finding newer targets to block exhausting signals but also improving existing immunotherapies.

Another technology that has greatly helped with finding targets for cancer immunotherapies is single-cell RNA (scRNA) sequencing. With the advent of scRNA seq technology, understanding the complex tumor microenvironments – rich in a wide range of immune cells like T cells, NK cells, innate lymphoid cells, eosinophils, dendritic cells, and macrophages – has become much easier. Populations of these cells vary depending on the type of tumor. Using insights from the single-cell mRNA sequencing, it is now possible to understand and map the underlying transcriptional profiles of these varieties of cells to further elucidate the ‘what’ and ‘how’ of their individual role amongst tumor cells.

Two studies published in Nature Immunology by huge collaborative groups from across the world use scRNA sequencing amongst other methods to study the role of Innate Lymphoid Cells (ILCs) and T follicular regulatory cells in tumors. Both studies converge at anti-PD-1 treatment and provide better combinatorial therapy options.

Innate Lymphoid Cells & Eosinophils

Jacquelot and colleagues began with investigating the role of ILCs in melanoma. They analyzed tumor infiltrates by scRNA sequencing and found that all types of ILCs (ILC1, ILC2, ILC3) and NK cells were present in the tumors. Out of all these cells, the absence of ILC2s had the most adverse effect on tumor progression, pointing to their importance in regulating tumor growth. ILC2s play a controversial role across different types of tumors. Some studies have shown ILC2s to be anti-inflammatory and suppress immune responses while others have shown ILC2s to elicit antitumor responses and have anti-metastatic tendencies.

In this study, ILC2s were playing an antitumor role, so the next step was to figure out how. One of the genes that was upregulated in these ILC2s was Csf2 which codes for granulocyte-macrophage colony-stimulating factor (GM-CSF) that is an essential cytokine for early development of the myeloid lineage of cells. ILC2s were the only cells producing high levels of GM-CSF in the milieu. When eosinophils were treated in vitro with GM-CSF, their survival was enhanced, and major eosinophil cytotoxicity genes were also upregulated. On further experimentation, they found that knocking out Csf2 had a direct effect on eosinophil circulation and infiltration into the tumor. It was inferred that activated ILC2s recruited and nourished eosinophils that aided in fighting the tumor.

The tumor-infiltrating ILC2 cells also expressed higher PD-1. Hence, using anti-PD-1 therapy did not only stop T cells from becoming inactive but also sustained the activated ILC2s. Previous studies state that IL-33 mediated activation of ILC2s can impact tumor progression both positively and negatively. Here, the researchers found that IL-33 administration with anti-PD-1 had a significant reduction of tumor load compared to using only PD-1 or only IL-33.

Regulatory T Cells & T Follicular Regulatory Cells

Regulatory T cells (Treg) and T follicular regulatory cells (T_FR) are two of the many T cell populations present in any tumor microenvironment. As expected, a large number of these regulatory cells can worsen tumor progression because of their immune-suppressive nature. Eschweiler et al. provided invaluable insights into Treg and T_FR dynamics and showed how sequential immune checkpoint blockade can go a long way in treating melanoma.

Using a mix of clinical and laboratory single-cell RNA seq data, they established that Tregs and T_FR cells make up a substantial amount of tumor-infiltrating CD4+ T cells. They also showed that Tregs that were specific for tumor-associated antigens could clonally expand intratumorally to produce large populations of T_FR cells that are better at suppressing effector functions than Tregs. For example, T_FR cells reduced CD8+ cell-mediated IFNγ secretion by four-fold and TNFα and IL2 by two-fold, when compared to Tregs. All this data pointed to the importance of curbing T_FR populations to improve the immune-mediated response against tumors.

T_FR cells also expressed the highest amount of PD-1 and CTLA-4 amongst all other CD4+ cells. When mice were treated with anti-PD-1, the number of Tregs and T_FR cells were drastically increased, signifying that Tregs were responding to PD-1 blockade by expanding their numbers and converting to T_FR cells, ultimately increasing their net immune suppression. In absence of T_FR cells from the mice, anti-PD1 treatment significantly reduced the tumor load.

Two is better than one

Jacquelot et al concluded their studies by suggesting a combinatorial therapy using IL-33 with anti-PD-1, to activate ILC2s and increase the subsequent eosinophil-associated tumor cytotoxicity. On the other hand, Eschweiler et al concluded that using anti-CTLA-4 therapy before administering anti-PD-1 led to a drastic reduction in Tregs and TFR cells and significantly increased its immunotherapeutic potential. Both the studies provide improved mechanistic understanding of the tumor microenvironment and show how small changes to treatment procedures can largely improve clinical outcomes. We hope that these combinatorial therapies will be effective in clinical trials and save cancer patients all over the world.

Sources:

• Jacquelot N, Seillet C, Wang M, Pizzolla A, Liao Y, Hediyeh-Zadeh S, Grisaru-Tal S, Louis C, Huang Q, Schreuder J, Souza-Fonseca-Guimaraes F, de Graaf CA, Thia K, Macdonald S, Camilleri M, Luong K, Zhang S, Chopin M, Molden-Hauer T, Nutt SL, Umansky V, Ciric B, Groom JR, Foster PS, Hansbro PM, McKenzie ANJ, Gray DHD, Behren A, Cebon J, Vivier E, Wicks IP, Trapani JA, Munitz A, Davis MJ, Shi W, Neeson PJ, Belz GT. Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma. Nat Immunol. 2021 Jul;22(7):851-864. doi: 10.1038/s41590-021-00943-z.
• Eschweiler S, Clarke J, Ramírez-Suástegui C, Panwar B, Madrigal A, Chee SJ, Karydis I, Woo E, Alzetani A, Elsheikh S, Hanley CJ, Thomas GJ, Friedmann PS, Sanchez-Elsner T, Ay F, Ottensmeier CH, Vijayanand P. Intratumoral follicular regulatory T cells curtail anti-PD-1 treatment efficacy. Nat Immunol. 2021 Jun 24. doi: 10.1038/s41590-021-00958-6. Epub ahead of print. PMID: 34168370.

Check out Antibuddies’ blog post “A Combo offer to deal with Melanoma: Latest Development with Immune Checkpoint Inhibitors”.

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