Fig. S3. CD103+CD56+Lin- ILCs exhibit features that are associated with Tregs.

Fig. S5. Identifying cellular clusters from scRNA-seq of ovarian tumours.

Fig. S8. CD56+ ILCs cluster 0 from CITE-seq are composed of predominately CD103+CD49a+ expressing cells.

Fig. S2. CD103+CD56+Lin- ILCs express distinct gene expression compared to CD103-CD56+Lin-ILCs.

Fig. S6. CD103+CD56+ ILCs have distinct transcriptional networks revealed by SCENIC analysis.

Fig. S9. CD103+CD56+Lin- ILCs express distinct signatures compared to other ILC subsets.

Fig. S7. Intratumoural CD103+CD56+Lin- ILCs displayed unique profile of transcription factors.

Fig S4. CD101 and GITR expression is expressed on CD103+CD49a+CD56+Lin- ILCs but not other NK cell subsets.

Proportion of cells per clusters for each patient sample from scRNA-seq and CITE-seq datasets.

Innate lymphoid cells (ILCs) with inhibitory properties have recently been identified in cancer, but the specific surface markers or transcription factors distinguishing these populations from conventional ILCs are not well-defined. In this study, we found that CD103 identifies CD56+ ILCs that are associated with poor proliferative capacity in tumor-infiltrating lymphocyte (TIL) cultures from epithelial ovarian carcinoma (EOC). These CD103+CD56+ ILCs expressed distinct surface markers (e.g., CD101, CD49a, GITR) and exhibited a unique transcriptomic profile compared to other intratumoral ILC subsets (ILC1s, ILC2s, ILC3s, NK cells). Using scRNA-seq, we discovered that tumors with a high abundance of CD103+CD56+ ILCs were linked to CD8+ T cells with low expression of genes related to T cell activation and TCR signaling. Mass cytometry imaging further revealed that CD8+ T cells in direct contact with CD103+NKp46+ ILCs had lower expression of the cytotoxic molecule GZMB and the proliferation marker Ki67 compared to CD8+ T cells not in contact with these ILCs. Additionally, CD103+CD56+ ILCs inhibited autologous CD8+ T cells in vitro. Overall, our study identifies CD103+CD56+ inhibitory ILCs as being associated with dysfunctional CD8+ T cells within the tumor microenvironment of EOC. Further characterization of these inhibitory ILCs may provide novel therapeutic targets to enhance anti-tumor immune responses and improve clinical outcomes for patients with EOC. This work was supported by CIHR foundation award (CIHR FDN #143220), a TRANSCAN CIHR grant (CIHR – TRN #184713), a CHRYSALIDS TRANSCAN3 CCS grant, and a CCS grant (CCSRI # 706152) that was generously funded in memory Mr. Jim Jo Tak. This work was also supported by the Wolfund Immunotherapy Fund at the Princess Margaret Cancer Foundation, which was generously donated by the Wolfond Family. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Immunotherapies have had unprecedented success in the treatment of multiple cancer types, albeit with variable response rates. Unraveling the complex network of immune cells within the tumor microenvironment (TME) may provide additional insights to enhance antitumor immunity and improve clinical response. Many studies have shown that NK cells or innate lymphoid cells (ILC) have regulatory capacity. Here, we identified CD103 as a marker that was found on CD56+ cells that were associated with a poor proliferative capacity of tumor-infiltrating lymphocytes in culture. We further demonstrated that CD103+CD56+ ILCs isolated directly from tumors represented a distinct ILC population that expressed unique surface markers (such as CD49a and CD101), transcription factor networks, and transcriptomic profiles compared with CD103-CD56+ NK cells. Using single-cell multiomic and spatial approaches, we found that these CD103+CD56+ ILCs were associated with CD8+ T cells with reduced expression of granzyme B. Thus, this study identifies a population of CD103+CD56+ ILCs with potentially inhibitory functions that are associated with a TME that includes CD8+ T cells with poor antitumor activity. Further studies focusing on these cells may provide additional insights into the biology of an inhibitory TME.

The innate lymphoid cell (ILC) family is composed of heterogeneous innate effector and helper immune cells that preferentially reside in tissues where they promote tissue homeostasis. In cancer, they have been implicated in driving both pro- and anti-tumor responses. This apparent dichotomy highlights the need to better understand differences in the ILC composition and phenotype within different tumor types that could drive seemingly opposite anti-tumor responses. Here, we characterized the frequency and phenotype of various ILC subsets in melanoma metastases and primary epithelial ovarian tumors. We observed high PD-1 expression on ILC subsets isolated from epithelial ovarian tumor samples, while ILC populations in melanoma samples express higher levels of LAG-3. In addition, we found that the frequency of cytotoxic ILCs and NKp46+ILC3 in tumors positively correlates with monocytic cells and conventional type 2 dendritic cells, revealing potentially new interconnected immune cell subsets in the tumor microenvironment. Consequently, these observations may have direct relevance to tumor microenvironment composition and how ILC subset may influence anti-tumor immunity.

Pancreatic cancer remains an unmet medical need. Late diagnosis and the lack of efficient treatment significantly impact the prognosis of patients suffering from pancreatic cancer. Improving patient outcomes requires a deeper comprehension of the tumor ecosystem. To achieve this, a thorough exploration of the tumor microenvironment using pre-clinical models that accurately replicate human disease is imperative, particularly in understanding the dynamics of immune cell subsets. Surprisingly, the impact of model variations on the composition of the tumor microenvironment has been largely neglected. In this study, we introduce an orthotopic model of pancreatic ductal adenocarcinoma and a spontaneous model of insulinoma. Our findings reveal striking differences in the innate lymphoid cell infiltrate, highlighting the importance of considering model-specific influences when investigating the tumor microenvironment.

The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated T cells synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance T cell metabolism and anti-tumor activity.

NK cells have been shown to exhibit inflammatory and immunoregulatory functions in a variety of healthy and diseased settings. In the context of chronic viral infection and cancer, distinct NK cell populations that inhibit adaptive immune responses have been observed. To understand how these cells arise and further characterize their immunosuppressive role, we examined in vitro conditions that could polarize human NK cells into an inhibitory subset. TGF-β1 has been shown to induce regulatory T cells in vitro and in vivo; we therefore investigated if TGF-β1 could also induce immunosuppressive NK-like cells. First, we found that TGF-β1/IL-15, but not IL-15 alone, induced CD103+CD49a+ NK-like cells from peripheral blood NK cells, which expressed markers previously associated with inhibitory CD56+ innate lymphoid cells, including high expression of GITR and CD101. Moreover, supernatant from ascites collected from patients with ovarian carcinoma also induced CD103+CD49a+ NK-like cells in vitro in a TGF-β-dependent manner. Interestingly, TGF-β1/IL-15-induced CD103+CD56+ NK-like cells suppressed autologous CD4+ T cells in vitro by reducing absolute number, proliferation, and expression of activation marker CD25. Collectively, these findings provide new insight into how NK cells may acquire an inhibitory phenotype in TGF-β1-rich environments.

Natural killer (NK) cell activity is influenced by cytokines and microenvironment factors, resulting in remarkably diverse functions, by contributing to inflammatory responses or serving as rheostats of adaptive immunity. Using single cell RNA sequencing (scRNA-seq), we identified a TGFb1highCD56brightNK cell population associated with hematopoietic stem cell transplant recipients protected from acute graft-versus-host disease (GVHD). We further define a role for the combination of interleukin-2 (IL-2) and transforming growth factor R1 (TGF-R1) in promoting a regulatory phenotype in NK cells. "Induced"regulatory NK cells produce high amounts of TGF-R1, inhibited T cells, could promote naive T cells differentiation into regulatory T cells, and exhibited a unique transcriptional program that includes expression of IKZF2 (HELIOS) and ZNF683 (HOBIT). This phenotype was not stable, and "induced"regulatory NK cells lost the ability to secrete TGF-R1 upon exposure to different cytokines. These findings define protective CD56brightNK cells post-hematopoietic stem cell transplantation, and demonstrate the combination of IL-2 and TGF-R1 promotes regulatory activity in NK cells.

Breast cancer is the most common cancer in women and continues to have a significant impact in cancer-associated deaths worldwide. Investigating the complex roles of infiltrating immune subsets within the tumor microenvironment (TME) will enable a better understanding of disease progression and reveal novel therapeutic strategies for patients with breast cancer. The mammary-specific expression of polyomavirus middle T oncoprotein (MMTV-PyMT) was first established in 1992 by William Muller and is the most commonly used genetically engineered mouse model (GEMM) for breast cancer research. Innate lymphoid cells (ILCs) are composed of a diverse family of effector cells known to play important roles in defense against pathogens, tissue homeostasis, and tumor immunity. In mice, group 1 ILCs are composed of NK cells and ILC1s, which have been shown to have differential roles within the TME. Here, we provide a detailed methodology in characterizing tumor-infiltrating NK cells and ILC1s in MMTV-PyMT breast tumor model.

Supplementary Figure from Overproduction of IFNγ by Cbl-b–Deficient CD8+ T Cells Provides Resistance against Regulatory T Cells and Induces Potent Antitumor Immunity

Background: Regulatory innate lymphoid cells (ILCregs) are an emerging heterogenous subset of innate lymphoid cells that suppress immunity in several healthy and diseased contexts, including cancer. Previously, we identified CD56+ ILCregs from in vitro expanded tumour-infiltrating lymphocyte (TIL) cultures in patients with epithelial ovarian carcinoma (EOC). However, lineage-defining surface markers or transcription factors that distinguishes these regulatory populations from other conventional NK cells have not been identified. The objective of this study is to characterize markers that define ILCregs directly from primary tumours and investigate its role within the tumour microenvironment (TME). Methods: Women with suspected abdominal mass were recruited and consented at Gynecology Cancer Clinic at Princess Margaret Hospital. Surgically resected EOC tumours were processed and analyzed by flow cytometry and single-cell RNA-sequencing. Suppression assays were performed by co-culture of tumour-infiltrating CD103+CD56+ ILCregs with autologous TILs for 4 days in vitro with soluble αCD3. Results: We found that CD103 expressing CD56+ ILCregs were associated with poor clinical prognosis in patients with ovarian carcinoma. CD103+CD56+ ILCregs displayed reduced expression of cytolytic related markers (i.e., GZMB, CD107a, CD16) compared to CD103-CD56+ NK cells and had a distinct transcriptomic profile from NK cells. Interestingly, CD103+CD56+ ILCregs displayed distinct surface markers (i.e., CD49a, CD69, GITR) and transcription factor networks that were associated with Treg development, differentiation, and suppressive function. Intratumoural CD103+CD56+ ILCregs suppressed autologous CD8+ T cells by downregulation of granzyme B in vitro. Concordantly, the abundance of CD103+CD56+ ILCregs in patient tumours were associated with reduced activated CD8+ T cells within the TME. Conclusion: This study identified CD103+CD56+ ILCregs in the TME of patients with EOC that are associated with poor clinical prognosis and plays a key role in regulating T cells. Further investigations into underlying immunoregulatory networks, including ILCregs, within the TME may provide novel targets to improve prognosis for patients with EOC. Citation Format: Douglas C Chung, Jehan Vakharia, Kathrin Warner, Nicolas Jacquelot, Azin Sayad, SeongJun Han, Maryam Ghaedi, Carlos R Garcia-Batres, Alisha Elford, Ben X Wang, Linh T Nguyen, Patricia A Shaw, Blaise A Clarke, Marcus Q Bernardini, Sarah E Ferguson, Pamela S Ohashi. CD103+CD56+ innate lymphoid cells are associated with an immunosuppressive microenvironment in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A009.