New strategies for cancer immunotherapy: targeting regulatory T cells

The immunosuppressive action of regulatory T (Treg) cells is one mechanism attributed to the limited success of cancer immunotherapies with checkpoint blockers. Two recent studies report distinct transcriptional profiles of tumor-infiltrating Treg cells and expression of specific molecules, suggesting novel strategies to overcome resistance to cancer immunotherapy.

consequently, increased densities of tumor-infiltrating T reg cells have been associated with poor prognosis in a number of cancers [2,3]. T reg cells can suppress effector T cells through different direct mechanisms, including secretion of inhibitory cytokines, granzyme-mediated cytolysis of effector T cells, and metabolic impairment of effector T cells-for example, by depletion of interleukin-12 (IL-12)-as well as indirect mechanisms such as suppression of dendritic cell maturation and function. However, although widely accepted to be implicated in tumor growth, the molecular phenotypes of tumor-infiltrating T reg cells remain poorly characterized. Identification of molecules expressed specifically in intratumoral T reg cells through genome-wide/transcriptomic analysis has the potential to reveal valuable new targets for therapeutic intervention. Depletion of tumorinfiltrating T reg cells targeting these molecules would shift the balance from immune suppression to immune activation towards tumor cells.

Unique molecular fingerprints of tumorinfiltrating regulatory T cells
So far, most of the knowledge of these immunosuppressive cells has been derived from the study of peripheral blood T reg cells, whereas a complete portrait of tumor-resident T reg cells, including T reg -specific markers, is still lacking. In a recent edition of Immunity, two independent studies, led by Rudensky [4] and Pagani [5], shed light on the features of T reg cells infiltrating common human cancers.
The Rudensky laboratory [4] characterized flowcytometry-sorted T reg cells from human breast carcinomas, peripheral blood, and normal breast parenchyma using RNA sequencing. Tumors with more aggressive phenotypes, such as triple-negative breast cancers (TNBCs), contained higher frequencies of T reg cells, which might imply an active role for T reg cells in cancer progression. RNAsequencing analysis revealed marked transcriptomic differences between tumor-or tissue-resident T reg versus peripheral blood T reg cells, while the gene expression profiles were highly comparable between T reg cells present in breast tumor and normal tissue. This implies that the tissue of residence, rather than the tumor environment, determines the phenotype of infiltrating T reg cells. The gene expression profiles did, however, reveal an enhanced activation of tumor-infiltrating T reg cells compared to T reg cells present in normal breast parenchyma and the upregulation of genes involved in chemokine signaling and immune cell migration. In particular, the marked expression of chemokine receptor 8 (CCR8), a molecule implicated in the modulation of immunogenicity in colorectal cancer (CRC) [6], was identified as a unique feature of intratumoral T reg cells. This was confirmed in T reg cells isolated from other types of cancers, including CRC, lung cancer, melanoma, and angiosarcoma. Moreover, the authors found a strong negative association between the ratio of CCR8/FOXP3-expressing cells and patient survival.
The Pagani laboratory [5] used RNA sequencing to profile human CD4 + T reg cells as well as T helper 1 and T helper 17 cells infiltrating non-small-cell lung cancer (NSCLC), CRC, and the respective matched normal tissues, as well as peripheral blood T reg cells. Their results show a large overlap of the expression profiles from tissue-and tumor-resident T reg cells, yet striking differences with respect to blood T reg cells. Differential expression analysis revealed 309 transcripts consistently upregulated in T reg cells from NSCLC and CRC tumors, but not in other CD4 + T cells subpopulations or in tissue-resident T reg cells. The intratumoral signature includes genes associated with increased suppressor activity, such as the immune checkpoints CTLA4, lymphocyte activation gene 3 (LAG3), and T-cell membrane protein 3 (TIM3). Three of the most highly expressed signature genes, layilin (LAYN), MAGE family member H1 (MAGEH1), and CCR8 (the latter two were also identified as a unique feature of tumor-infiltrating T reg cells by the Rudensky laboratory [4]), were associated with a significant worsening of the 5year survival of both CRC and NSCLC patients. Finally, the authors confirmed the expression signatures shared by CRC-and NSCLC-infiltrating T reg cells via single-cell analysis and demonstrated that they are conserved across different metastatic and non-metastatic cancers.

Potential for new therapeutic strategies for cancer immunotherapy
The presence of T reg cells in the tumor microenvironment represents one of the main obstacles for successful cancer immunotherapy. Since their immunosuppressive action is exerted through different mechanisms, the selective depletion of tumor-infiltrating T reg cells holds promise for unleashing potent antitumor responses. These two resources [4,5] now provide the potential means for targeted depletion of T reg cells. Both studies showed that CCR8 is specifically expressed in tumor-infiltrating T reg cells in multiple tumor types, suggesting that CCR8 represents a promising target to selectively deplete T reg immunosuppressive activity in the tumor microenvironment.
The marked differences between tumor-resident and blood-derived T reg cells reported in these studies highlight the importance of assessing the phenotypes of immune cells at the tumor site. The generation of additional transcriptomic data from other tumor-infiltrating immune cells would tremendously benefit the development and optimization of computational approaches for dissecting complex tumor-immune cell interactions [7], and could contribute to the identification of novel mechanistic insights.
A number of questions remain unanswered and should motivate further experimental and clinical studies. For example, the mechanisms underlying the increased infiltration of T reg cells in aggressive cancers such as TNBC, and the implications for cancer prognosis, remain elusive. Furthermore, heterogeneity of tumor-infiltrating T reg cells has not been addressed. It was shown recently that distinct subpopulations of T reg cells contribute in opposing ways to determining CRC prognosis: CRCs infiltrated with suppression-competent T reg cells had a worse prognosis compared with CRCs infiltrated with non-suppressive T reg cells [8]. Thus, additional studies need to be carried out to validate this observation in other cancer types. Finally, it should be noted that the promising candidate for therapeutic targeting, CCR8, is also expressed in cardiomyocytes and liver [9]; thus, straightforward systemic treatment with CCR8 inhibitors might cause serious immune-related adverse effects. Still, the two studies highlight the importance of assessing the phenotypes of immune cells specifically within the tumor site. Further transcriptomic characterization of tumor-infiltrating T reg cells in other cancers, as well as other immunosuppressive cell populations, will be of the utmost importance for the prevention of resistance to cancer immunotherapies.
Abbreviations CRC: Colorectal cancer; NSCLC: Non-small-cell lung cancer; TNBC: Triple-negative breast cancer; T reg cell: Regulatory T cell