Search

Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig­naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC. Significance: HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease. This article is highlighted in the In This Issue feature, p. 1825

Supplementary Data from IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma

Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig­naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC.Significance:HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease.This article is highlighted in the In This Issue feature, p. 1825

Natural killer (NK) cells have crucial roles in the innate immunosurveillance of cancer and viral infections. They are 'first responders' that can spontaneously recognize abnormal cells in the body, rapidly eliminate them through focused cytotoxicity mechanisms and potently produce pro-inflammatory cytokines and chemokines that recruit and activate other immune cells to initiate an adaptive response. From the initial discovery of the diverse cell surface receptors on NK cells to the characterization of regulatory events that control their function, our understanding of the basic biology of NK cells has improved dramatically in the past three decades. This advanced knowledge has revealed increased mechanistic complexity, which has opened the doors to the development of a plethora of exciting new therapeutics that can effectively manipulate and target NK cell functional responses, particularly in cancer patients. Here, we summarize the basic mechanisms that regulate NK cell biology, review a wide variety of drugs, cytokines and antibodies currently being developed and used to stimulate NK cell responses, and outline evolving NK cell adoptive transfer approaches to treat cancer.

Pancreatic ductal adenocarcinoma (PDAC) is soon to be the second deadliest types of cancer, with a 5-year survival of only 10%. The unique features of PDAC are the expansion of cancer-associated fibroblasts (CAFs), presence of a dense fibrous stroma with high bundled collagen I and immunosuppression, representing a challenge for therapies. Ways to overcome these unique pro-tumor features will be key for the development of better therapeutic strategies for PDAC. Herein we report that, in addition to the identified ectopic expression of NGL-1 in PDAC cells, it is also expressed in the stroma (CAFs and immune cells) of PDAC patients, inversely correlating with overall survival. Stromal NGL-1 was important for tumorigenesis in vivo, as NGL-1 knockout mice (KO) orthotopically allografted with pancreatic cancer cells presented less tumor burden. Further analysis showed that the tumors from KO mice presented more CD8+ T cells and less immunosuppressive cytokines, such as TGFβ. Single cell RNAseq analysis of these tumors showed decreased expression of pro-tumor factors, such as immune checkpoint molecules in T cells and TGFβ related genes across different cellular compartments (epithelial, immune and fibroblasts). In accordance with these results, these tumors presented a limited amount of desmoplastic bundled collagen, suggestive of a TGFβ-deficient environment. In order to further dissect between the NGL-1-dependent contributions of immune vs. local stroma (e.g., CAFs) cells, we generated bone marrow chimeras and performed orthotopic injections to generate tumors. The loss of NGL-1 in each cellular compartment alone failed to phenocopy the full body loss of NGL-1, suggesting that NGL-1 in both immune cells and local stromal are important for tumorigenesis. Functionally, NGL-1 deficient CAFs failed to support the survival of starved PDAC cells in vitro, downregulated important myofibroblastic molecules such as p-smad, and produced less immunosuppressive cytokines, suggesting a role for NGL-1 in key pro-tumor features of CAFs. In fact, the fibroblastic NGL-1 dependent immunomodulatory effects were confirmed with human CD8+ T cells from healthy donors, which lost their cytotoxic profile in the presence of conditioned media (CM) from NGL-1+ CAFs, but were able to keep this profile when exposed to CM from NGL-1 deficient CAFs. Bone marrow derived macrophages from KO mice produced less pro-tumor cytokines and CD8+ T cells lacking NGL-1 proliferated more than those from wild type animals, when stimulated in vitro. Mechanistically, while immune cells and CAFs deficient in NGL-1 are both tumor suppressive, the latter can regain pro-tumor functions in response to TGFβ, explaining the need for a global modulation of NGL-1 expression for an anti-tumor effect. Finally, NGL-1 KO mice orthotopically allografted with PDAC cells responded better (smaller tumors) to chemotherapeutical regimen (FOLFIRINOX) compared to WT animals. All these results point to NGL-1 as a potential new target to modulate immunosuppression and tumorigenesis in pancreatic cancer. Citation Format: Debora Barbosa Vendramini Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Esteban Martinez, Stephen Sykes, Nina Steele, Marina Pasca di Magliano, Dmitry Zhigarev, Charline Ogier, Huamin Wang, Igor Astsaturov, Kerry Campbell, Edna Cukierman. Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR017.

Acute myeloid leukemia (AML) is an aggressive malignancy that requires rapid treatment with chemotherapies to reduce tumor burden. However, these chemotherapies can compromise lymphocyte function, thereby hindering normal anti-tumor immune responses and likely limiting the efficacy of subsequent immunotherapy. To better understand these negative impacts, we assessed the immunological effects of standard-of-care AML therapies on lymphocyte phenotype and function over time. When compared to healthy donors, untreated AML patients showed evidence of lymphocyte activation and exhaustion and had more prevalent CD57+NKG2C+ adaptive NK cells, which was independent of human cytomegalovirus (HCMV) status. HMA/venetoclax treatment resulted in a greater fraction of T cells with effector memory phenotype, inhibited IFN-γ secretion by CD8+ T cells, upregulated perforin expression in NK cells, downregulated PD-1 and 2B4 expression on CD4+ T cells, and stimulated Treg proliferation and CTLA-4 expression. Additionally, we showed increased expression of perforin and CD39 and enhanced IFN-γ production by T cells from pre-treatment blood samples of venetoclax-resistant AML patients. Our results provide insight into the lymphocyte status in previously untreated AML patients and the effects of standard-of-care treatments on their biology and functions. We also found novel pre-treatment characteristics of T cells that could potentially predict venetoclax resistance.

Acute myeloid leukemia (AML) accounts for about one third of all leukemias, with half of all leukemia deaths. The low 5-year survival rate and manifestation of this deadly disease in old age reinforce the need for new safe and effective AML therapies. Considering the exceptional role of immune cells in the recognition and elimination of tumor cells, one of these methods is immunotherapy. However, for the development of immunotherapeutic approaches, it is necessary to clearly understand the role of certain effector cells in the pathogenesis of the disease, as well as to have the knowledge about the phenotypic characteristics of these cells. Natural killer (NK) cells play important roles in innate cancer immunosurveillance, and some published data indicate that the antitumor function of NK cells is reduced in AML patients. To expand upon previous work, we performed a comprehensive analysis of the phenotypic and functional characteristics of NK cells in previously untreated AML patients that took into account a wide variety of biomarkers. The goals of this study were to define the phenotypic and functional differences in NK cells from AML patients and healthy donors and determine how these parameters affect outcome of the disease. We used 14-color flow cytometry to assess more than 30 measurable markers on NK cells from the peripheral blood of 33 untreated AML patients and age-matched healthy controls. In addition, NK cells were tested for interferon-γ responses under antibody-dependent cellular cytotoxicity conditions. Results in AML patients were then compared to healthy donors. We found that the NK cells of patients with AML have a significantly lower capacity to secrete IFN-γ and showed numerous signs of an exhausted phenotype, as compared to healthy controls. These included increased surface expression of CD39, PD-1, and LILRB1 on NK cells from AML patients. Interestingly, surface expression of the TIGIT checkpoint receptor did not differ between AML patients and healthy donors, but surface expression of the activating receptor DNAM-1, which shares the same ligands on tumor cells, was decreased. All of these data confirm that the NK cells of AML patients are functionally impaired. We also noted that the frequency of CD57 + NKG2C + KIR + memory-like "adaptive" NK cells was greater in the blood of AML patients. The proportion of adaptive NK cells did not correlate with the age of donors. Phenotypic features of this cell subpopulation from the AML patients included significantly increased expression levels of CD56 and significantly lower expression of the activating receptor DNAM-1. Disclosures Zhigarev: Janssen R&D: Research Funding; Immunitas Therapeutics: Consultancy; Tavotek Biotherapeutics: Consultancy. Drenberg: Janssen R&D: Current Employment. Campbell: Janssen R&D: Research Funding.

BackgroundAcute myeloid leukemia (AML) is a heterogeneous group of malignant bone marrow diseases, characterized by massive and uncontrolled proliferation of myeloid precursor cells, which alters normal blood cell ratios. This disease is common to older adults and collectively displays one of the lowest 5-year overall survival rates (MethodsMore than 100 blood samples were obtained from 33 previously untreated AML patients. More than 50 measurable biomarkers were analyzed using 14-color flow cytometry to assess immune phenotypes of T and NK cells in peripheral blood of AML patients prior to treatment and at up to four timepoints after initiation of treatment with HMA or chemotherapy.ResultsWe found several significant changes in immune cell phenotype and function that occur in response to these therapies. Treatment with HMAs was strikingly less impactful on immune cells in patients compared to previously published in vitro studies. Nevertheless, HMA treatment increased perforin levels in T and NK cells, inhibited IFN-gamma secretion by CD8+ T cells, and changed expression of several checkpoint molecules. While chemotherapy caused fewer phenotypic changes it dramatically decreased the total number of immune cells. We also determined viable, functional and phenotypical recovery periods for immune effector cells after the treatments.ConclusionsOur results are important for introducing new second line immunotherapies to these chemotherapeutic regimens for treating AML and to improve overall understanding of immune cell behavior under conditions of anti-tumor treatment.AcknowledgementsSupported by grants from Janssen and the U.S./Israel Binational Science Foundation.Ethics ApprovalThe study was approved by the Fox Chase Cancer Center Institutional Review Board, approval number 17-8010, and all patients provided informed consent before taking part in the study.