Expression Of Immune Checkpoint Ligands Research Articles

Abstract 1407: Investigating the effect of an aged liver microenvironment on immune surveillance and predisposition to cancer​

Abstract The incidence of liver cancer is increasing and there is an urgent need for new therapies and preventative strategies. Age is a major risk factor for liver cancer, the reasons for which are not well defined. One of the hallmarks of aging is immune dysfunction which affects liver homeostasis potentially making it prone to cancer. To understand the role of immune dysfunction in aging liver, we analyzed immune cells from young and old livers. As is reported before, in aged livers we observed an increase in CD101+PD1+CD8+ T cell population as well as an increase in an IFNγ+TNFα+ population suggesting an increase in T cell response in an aging liver. We next sought to understand the cause of T cell exhaustion in aged livers. Cell cluster analysis of the young (4-month-old) and old (22 month old) livers by scRNA seq revealed a cell population unique to the aged livers, showing increased expression of immune checkpoint ligands as well as tumor-initiating cell markers. We validated this data by flow cytometry. We next functionally probed the immune environment of aged liver, by testing the effect of cognate antigen expression in young and old hepatocytes on adoptively transferred activated P14 CD8+ T cells isolated from a young P14 mouse. Analysis of the ex vivo activated P14 CD8+ T cells from young and old antigen-expressing livers by flow cytometry and scRNA sequencing revealed increased exhaustion and cytokine-deficiency in old mice as compared to the young mice. This study explores the age-associated alterations in the immune cells in the liver, aiming to alter liver-resident immune cells and their metabolic states in a way that promotes anti-tumor immunity in an aging liver. Citation Format: Armin Aabish Gandhi, Filipe Araujo Hoffmann, Michael LaPorte II, Aaron Havas, Adarsh Rajesh, Andrew Davis, Marcos G. Teneche, Jessica Proulx, Susan Kaech, Peter D. Adams. Investigating the effect of an aged liver microenvironment on immune surveillance and predisposition to cancer​ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1407.

Abstract 6212: A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden that leads to the generation of few neo-epitopes derived from mutated genes that can be recognized by cytotoxic T cells. The immunosuppressive microenvironment of PDAC comprises suppressive cell populations that include myeloid cells, cancer associated fibroblast subpopulations, and regulatory T cells that reduce T cell infiltration and cytotoxic function. We have developed a murine personalized cancer vaccine to enhance cytotoxic T cell function against PDAC, PancVAX (Kinkead et al, JCI Insight 2018). Survival benefit with PancVAX was dependent on the addition of immune checkpoint inhibitor (ICI) therapy to increase the number of cytotoxic CD8 T cells infiltrating tumors and maintain their cytotoxic function (Huff et al, JCI Insight 2023). Yet, the impact of cell-cell communication on effector T cell function arising from the immunosuppressive microenvironment of PDAC remains unknown. We previously developed a ligand-receptor inference method called Domino that infers inter- and intra-cellular signaling networks in single-cell RNA-seq data (Cherry et al, Nat Biomed Eng 2021). However, this technique is limited to data from single conditions. Understanding how this combination immunotherapy alters cellular signaling networks requires new computational tools that identify changes in regulatory networks between treatment conditions. We developed capabilities for assessment of differential signaling with Domino founded on a basis of differential signal receipt. Active receptors are identified by receptor expression in recipient cells that is correlated with downstream transcription factors in the intracellular signaling cascade. Identification of causative ligands for receptor activation is assessed by a probability of interaction for ligand-receptor interactions between cell types. This probability metric considers mean expression of ligand and receptor genes as well as the number of sender cells for comparison across treatment groups. Comparing communication networks in PancVAX alone to PancVAX with ICI, PancVAX with ICI enhances cell-cell adhesion and interferon gamma signaling in cytotoxic CD8 T cells towards exhausted CD8 T cells. Macrophages in PancVAX with ICI enhance expression of immune checkpoint ligands towards CD8 T cells, despite the presence of blocking ICI antibodies. Intracellular signaling downstream of PD-1 in exhausted T cells shows diminished numbers of transcription factors linked to PD-1 signaling. This software informs prioritization of inhibitory signals for modulation in future immunotherapy regimens and can be applied generally to other tumor subtypes and treatment contexts. Citation Format: Jacob T. Mitchell, Atul Deshpande, Amanda L. Huff, Chris Cherry, Sushma Nagaraj, Kavita Krishnan, Dmitrijs Lvovs, Jennifer H. Elisseeff, Elizabeth M. Jaffee, Neeha Zaidi, Elana J. Fertig. A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6212.

Immune checkpoint ligands expressed on mature high endothelial venules predict poor prognosis of NSCLC: have a relationship with CD8+ T lymphocytes infiltration.

An insufficient number of intratumoral CD8+ T lymphocytes is a major barrier to antitumor immunity and immunotherapy. High endothelial venules (HEVs) are the major sites through which lymphocytes enter tumors; however, the molecular mechanism through which HEVs mediate CD8+ T lymphocyte infiltration remains poorly understood. Forty-two patients with stage IIIA lung adenocarcinoma, who underwent surgery, were recruited. Multiplex immunohistochemical staining was conducted on tumor tissues to detect the immune checkpoint ligands (ICLs) expressed in the HEVs, blood vessels, and lymphatics. A new ICL score model was constructed to evaluate ligand expression. The relationship between ICL score, tumor-infiltrating CD8+ T cell frequency, and survival of patients was investigated. Mature HEVs, but not blood vessels or lymphatics, mediated CD8+ T cell infiltration. However, the ICLs expressed on mature HEVs could negatively regulate CD8+ T cell entry into tertiary lymphoid structures (TLSs). In addition, according to the results obtained using our ICLtotal score model, the expression of ICLs on HEVs was observed to be a predictor of both CD8+ T cell infiltration and survival, in which a high ICLtotal score > 1 represent a weak CD8+ T cell infiltration and a high ICLtotal score > 2 predicts poor survival. Using the ICL score model, we discovered that ICLs expressed on HEVs are indicative of CD8+ T cell subset infiltration in TLSs, as well as of patient survival with lung cancer.

Cancer-derived non-coding RNAs endow tumor microenvironment with immunosuppressive properties.

Non-coding RNAs (ncRNAs) have attracted extensive attention due to their vital roles in tumorigenesis and progression, especially in the immunotherapy resistance. Tumor immunotherapy resistance is a crucial factor hindering the efficacy of tumor treatments, which can be largely attributed to the immunosuppressive properties of tumor microenvironment. Current studies have revealed that cancer-derived ncRNAs are involved in the formation of tumor immunosuppressive microenvironment (TIME) through multiple ways. They not only promote the expression of immune checkpoint ligands (e.g., PD-L1, CD47, Gal-9, and CD276) on cancer cell surfaces, but also enhance the secretion of immunosuppressive cytokines (e.g., TGF-β, IL-6, IL-10, VEGF, and chemokines). Cancer-derived ncRNAs could also be transferred into surrounding immune-related cells through extracellular vesicles, thereby inhibiting the cytotoxicity of CD8+ T cells and NK cells, restraining the DC-mediated antigen presentation, inducing the immunosuppressive phenotype transformation of TAMs and CAFs, and enhancing the immunosuppressive functions of Tregs and MDSCs. Herein, we summarize the roles of cancer-derived ncRNAs in regulating TIME formation and further explore their potential applications as prognostic biomarkers and immunotherapeutic targets, which will help us to address the TIME-mediated immunotherapy resistance in the future. This article is categorized under: RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Effect of Chemotherapeutics on In Vitro Immune Checkpoint Expression in Non-Small Cell Lung Cancer.

Objectives: Immune checkpoint (ICP) expression in tumor cells could directly or indirectly affect the results of immunotherapy. ICP ligands on tumor cells usually bind their immune cell receptors to inhibit the activity, resulting in tumor immune escape. Thus, the purpose of this study was to ascertain the impact of various chemotherapeutic drugs on ICP expression in non-small cell lung cancer (NSCLC) cell lines with different pathological subtypes to provide a basis for the development of a superior regimen of chemotherapy combined with ICP blockade. Methods: Several first-line chemotherapy agents (cisplatin, carboplatin, paclitaxel, gemcitabine, vinorelbine, and pemetrexed) were selected to treat different NSCLC cell lines (squamous carcinoma H1703, adenocarcinoma A549, and large cell cancer H460) for 72 hours, and then the changes in ICP expression in the tumor cells were observed through flow cytometry. Results: Cisplatin, carboplatin, and paclitaxel upregulated the expressions of programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2) in A549 and H460 cell lines. Meanwhile, vinorelbine and pemetrexed upregulated PD-L1 and PD-L2 in H1703, A549, and H460 cell lines. Paclitaxel, gemcitabine, vinorelbine, and pemetrexed significantly upregulated the expressions of both galectin-9 and high-mobility group box protein 1 (HMGB1) in the A549 cell line. Cisplatin and paclitaxel significantly upregulated the expressions of major histocompatibility complex-II (MHC-II), galectin-3, α-synuclein, and fibrinogen-like protein 1 (FGL1) in A549 and H460 cell lines. In addition, cisplatin and vinorelbine significantly upregulated the expressions of both CD155 and CD112 in the H460 cell line. Vinorelbine upregulated MHC-I in all three cell lines. Conclusion: Chemotherapy agents have different effects on the expression of ICP ligands in tumor cells with different pathological types, and this may affect the efficacy of combined immunotherapy. These results provide a theoretical basis for further selection and optimization of the combination of chemotherapy and immunotherapy.

Expression Modulation of Immune Checkpoint Molecules by Ibrutinib and Everolimus Through STAT3 in MCF-7 Breast Cancer Cells

Tumor-targeted therapy with small-molecule inhibitors (SMIs) has been demonstrated to be a highly effective therapeutic strategy for various cancers. However, their possible associations with immune evasion mechanisms remain unknown. This study examined the association of inhibitors of the protein kinase B (AKT), mammalian target of rapamycin (mTOR), and Bruton’s tyrosine kinase (BTK) signaling pathways with the expression of immune checkpoint ligands programmed death-ligand 1 (PD-L1), CD155, and galectin-9 (Gal-9) in a breast cancer cell line. MCF-7 cells were treated with everolimus, MK-2206, and ibrutinib. An MTT assay was used to determine the optimal dose for all drugs. A real-time polymerase chain reaction was utilized to measure the mRNA expression of PD-L1, CD155, and Gal-9. The western blot technique was also employed to evaluate the protein expression of the phosphorylated signal transducer and activator of transcription 3 (STAT3). The optimal doses of everolimus, MK-2206, and ibrutinib were observed to be 200, 320, and 2000 nM, respectively. The PD-L1 and CD155 mRNA expression was significantly decreased following the treatment with everolimus and ibrutinib, but not with MK-2206. There were no differences in Gal-9 expression between the single-treated and control groups; however, combined treatment with everolimus and ibrutinib increased its mRNA expression. Everolimus and ibrutinib both inhibited constitutive STAT3 phosphorylation in MCF-7, which was more pronounced in combination treatment. The findings regarding the modulation of PD-L1, CD155, and Gal-9 molecules by SMIs emphasize the crosstalk between the expression of these immune checkpoint molecules and AKT/mTOR/BTK signaling pathways through STAT3 as a critical transcription factor.

The Effects of PI3K/Akt/mTOR Signaling Pathway Inhibitors on the Expression of Immune Checkpoint Ligands in Acute Myeloid Leukemia Cell Line.

Up-regulation of immune checkpoint ligands is considered as one of the most important immune escape mechanisms in acute myeloid leukemia (AML). Herein, we investigate a relationship between the inhibition of PI3K/Akt/mTOR signaling pathways and the regulation of immune checkpoint ligands in AML cells. The HL-60 cell line was treated with idelalisib as PI3K inhibitor, MK-2206 as Akt inhibitor, and everolimus as mTOR inhibitor either in a single or combined format. Cell viability and apoptosis were evaluated using MTT and flow cytometry assays, respectively. The relative expression of PD-L1, galectin-9, and CD155 was determined by real-time PCR. Our findings demonstrated decreased proliferation and increased apoptosis of HL-60 cells after treatment with idelalisib, MK-2206, and everolimus. As expected, the combined treatment showed a more inhibiting effect than the single treatment. Interestingly, our results elucidated that the expression of PD-L1 and Gal-9 but not MK-2206 decreased after treatment with idelalisib and everolimus. Regarding CD155, the expression of this molecule was downregulated after treatment with everolimus, but not idelalisib and MK-2206. However, combined treatment of HL-60 cells with two or three inhibitors decreased the expression levels of PD-L1, Gal-9, and CD155 checkpoint ligands. We showed that PI3K/Akt/mTOR pathway inhibitors not only serve as cytotoxic drugs but also regulate the expression of immune checkpoint ligands and interfere with the immune evasion mechanisms of AML leukemic cells. Combinational treatment approaches to block these pathways might be a promising and novel therapeutic strategy for AML patients via interfering in immune escape mechanisms.

T Cell Versus T Cell; A Study of the Immune Checkpoint Landscape in Cutaneous T Cell Lymphoma

Introduction.Tumor infiltrating T cells (TILs) are capable of recognising tumor-specific mutations presented as peptides on MHC class I. Overexpression of immune checkpoint ligands on cancer cells, and the corresponding expression of the receptors on T cells, appears to be an important mechanism of immune evasion, exploited with success in immune checkpoint therapy. However, when the cancer cell is itself a T cell, the microenvironment interaction is likely to be more complex. To investigate this further we utilized the clonal nature of mycosis fungoides (MF), a primarily CD4 T cell cancer, by targeting the TCR receptor, differentiating between TIL and tumor in the microenvironment.Methods.43 patients with mycosis fungoides (MF) were prospectively consented to the study, and a skin biopsy and blood sample were obtained. The skin biopsy was macerated and digested with collagenase D, and a TCR V-beta clonogram was used to identify the tumor population. Flow cytometry was used to isolate five cell populations: peripheral blood CD4 and CD8, TIL CD4 and CD8, and tumor cells. The expression of a panel of immune checkpoint receptors (PD-1, TIM-3, LAG-3, Fas & TIGIT) and ligands (PD-L1, PD-L2, Galectin 9, Fas ligand) were measured using conjugated antibodies.Results.46 biopsies from MF patients with stage IA(n=6), IB(n=18), IIA(n=1), IIB(N=14), IIA(n=1) & IVA(n=3) were analysed with paired blood. The technique was effective in distinguishing tumour from TIL in 20 cases, isolating a population with CD7 downregulation, high forward and side scatter (indicative of larger and more complex cells) and a unique immune checkpoint profile (figure 1a & 1b). Tumour infiltrating lymphocytes of both CD4 and CD8 phenotype demonstrated significant overexpression of immune checkpoint receptors PD-1 (p < 0.0001), TIGIT (p < 0.0001) & fas (p < 0.0001), with an increased population of T regulatory cells in the CD4 cells (p < 0.001) and CD8 expressing high TIM-3 (p < 0.0001) compared to peripheral blood. While the TIL population was similar across the demographic, the tumour population demonstrated a significantly varied phenotype, with up and down-regulation of immune checkpoints and HLA-DR (figure 1c). Principal component analysis (figure 1d) revealed three tumour subtypes:1. High PD-L1, Fas, HLA-DR (n=4)2. High PD-1. Low PD-L1 (n=12)3. Low PD-1, PD-L1, Fas (n=3)These subtypes did not relate to disease type, stage, presence of large cell transformation or folliculotropic disease, though those with advanced disease had tumor phenotypes which differed more from the TILs. Tumor cells not only expressed MHC class I and HLA-DR, but upregulated these compared to peripheral blood (p < 0.001).Discussion.The TIL phenotype in MF is surprisingly homogeneous across the demographic, demonstrating upregulation of immune checkpoint receptors and an exhaustion phenotype seen in other cancers. However, the tumor cells did not show consistent upregulation of immune checkpoint ligands, instead their phenotype across patients was heterogeneous with both up and down-regulation. Expression of immune checkpoint ligands by the tumor cells may have negative growth consequences for both tumor cells as well as the immune system, particular since tumor T cells often express immune checkpoint receptors. We did not see concurrent high expression of PD-L1 and PD-1 on tumor cells, which would be expected to be detrimental to tumor survival. Immune checkpoint ligand over-expression is unlikely to be the main method of immune evasion in mycosis fungoides, and as a result, immune checkpoint inhibition may have unexpected responses in this disease.Legends.Figure 1a. TCR V-beta antibody used to separate three CD4 populations (red: peripheral blood CD4, green: CD4 TIL, blue: tumor) and compare HLA-DR expression.Figure 1b. tSNE clustering of cells in the tumour microenvironment, demonstrating clear separation of three populations - tumor, CD4 TIL and CD8 TIL. Coloring by expression of PD-L1 (red: high, blue: low).Figure 1c. Heatmap of immune checkpoint markers (red: high, blue: low) in the tumor cell population only. This demonstrates the heterogeneity, particularly in PD-1, Fas, PD-L1 and HLA-DR expression.Figure 1d. Principal component analysis demonstrating the observation that TIL expression of immune checkpoints are similar between patients (green and blue), whereas tumor expression of these markers (red) can vary markedly and cluster. [Display omitted] DisclosuresScarisbrick:NHS: Employment; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Mallinckrodt: Consultancy, Honoraria; Innate Pharma: Consultancy; Actelion: Consultancy.

Immunologic Characterization of Tumor Antigen-Specific T Cells in Multiple Myeloma: Implication in Immune Checkpoint Blockade

Various immune checkpoint inhibitory molecules expressed on tumor-recognizing T cells are a critical component of the immunosuppressive tumor microenvironment. Trials of monoclonal antibody drugs targeting immune checkpoint have achieved noteworthy clinical benefit in many types of solid tumors, including melanoma and non-small cell lung carcinoma, by blocking inhibitory signals and restoring anti-tumor immune response. In contrast, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant single-agent anti-tumor efficacy. To predict and improve the clinical responsiveness to immune checkpoint blocking treatment in multiple myeloma, explicit functional characterization of tumor antigen-specific T cells is an essential prerequisite. In the present study, we investigated the immunologic characteristics of marrow-infiltrating myeloma antigen-specific T cells, focusing on the expression of T cell inhibitory molecules.First, we examined the expression of immune checkpoint ligands by malignant plasma cells in bone marrow of newly diagnosed multiple myeloma patients, using multicolor flow cytometry. As a result, we found that malignant plasma cells, defined as CD14-CD19-CD138+CS1+CD56hi, expressed PD-L1 abundantly. Interestingly, frequency of PD-L1-expressing plasma cells in bone marrow correlated positively with tumor burden and clinical stage. Several immune checkpoint ligands, including PD-L1 and galectin-9, were also expressed in various cellular components of bone marrow other than tumor cells.Next, we investigated the immunophenotypes of marrow-infiltrating CD8+ T cells. Marrow-infiltrating CD8+ T cells showed significantly higher PD-1 expression in multiple myeloma patients, compared to control group. In myeloma patients, PD-1+ CD8+ T cells were more enriched in bone marrow compartment, than in peripheral circulation. PD-1+ marrow-infiltrating CD8+ T cells co-expressed other types of T cell inhibitory receptors including Tim-3, LAG3 and TIGIT.In selected patients, we could successfully define CD8+ T cell population specifically recognizing the HLA-A2-restricted epitope “LLLGIGILV”, included in myeloma tumor antigen HM1.24, using MHC multimer technique. We further characterized the immunophenotypes of myeloma antigen-specific CD8+ T cells. Myeloma antigen-specific CD8+ T cells were exclusively enriched in effector-memory T cell population and vast majority of them expressed high level of PD-1. They also express other types of T cell inhibitory receptors simultaneously. Strikingly, myeloma antigen-specific CD8+ T cells expressed high level of Eomes, indicating that they were profoundly exhausted functionally and that a simple blockade of PD-1/PD-L1 axis might not be sufficient to reinvigorate their anti-myeloma activity. When marrow-infiltrating T cells of myeloma patients were ex vivo cultured and stimulated with myeloma antigens, however, their antigen-specific proliferation was significantly restored in the presence of PD-1 blocking in some cases.Collectively, although PD-1/PD-L1 axis acts as a major component of immunosuppressive microenvironment in multiple myeloma, the clinical efficacy of PD-1 blockades in multiple myeloma might be hampered by (1) heterogeneity and multiplicity in expression patterns of T cell inhibitor receptors and (2) molecular depth of T cell exhaustion. Therefore, for successful rejuvenation of anti-myeloma T cell responses, combination immunotherapeutic approaches are required. Our results provides a basis for utilizing multiple immune checkpoint inhibitors targeting different molecules and incorporating immunomodulatory agents into the immune checkpoint inhibitor in treatment of multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Melphalan flufenamide inhibits osteoclastogenesis by suppressing proliferation of monocytes

Myeloma bone disease is a major complication in multiple myeloma affecting quality of life and survival. It is characterized by increased activity of osteoclasts, bone resorbing cells. Myeloma microenvironment promotes excessive osteoclastogenesis, a process of production of osteoclasts from their precursors, monocytes. The effects of two anti-myeloma drugs, melphalan flufenamide (melflufen) and melphalan, on the activity and proliferation of osteoclasts and their progenitors, monocytes, were assessed in this study. In line with previous research, differentiation of monocytes was associated with increased expression of genes encoding DNA damage repair proteins. Hence monocytes were more sensitive to DNA damage-causing alkylating agents than their differentiated progeny, osteoclasts. In addition, differentiated progeny of monocytes showed increased gene expression of immune checkpoint ligands which may potentially create an immunosuppressive microenvironment. Melflufen was ten-fold more active than melphalan in inhibiting proliferation of osteoclast progenitors. Furthermore, melflufen was also superior to melphalan in inhibition of osteoclastogenesis and bone resorption. These results demonstrate that melflufen may exert beneficial effects in patients with multiple myeloma such as reducing bone resorption and immunosuppressive milieu by inhibiting osteoclastogenesis.

Tumor interferon signaling and suppressive myeloid cells are associated with CAR T-cell failure in large B-cell lymphoma

AbstractAxicabtagene ciloleucel (axi-cel) is a chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory large B-cell lymphoma (LBCL). This study evaluated whether immune dysregulation, present before CAR T-cell therapy, was associated with treatment failure. Tumor expression of interferon (IFN) signaling, high blood levels of monocytic myeloid-derived suppressor cells (M-MDSCs), and high blood interleukin-6 and ferritin levels were each associated with a lack of durable response. Similar to other cancers, we found that in LBCL tumors, IFN signaling is associated with the expression of multiple checkpoint ligands, including programmed cell death–ligand 1, and these were higher in patients who lacked durable responses to CAR-T therapy. Moreover, tumor IFN signaling and blood M-MDSCs associated with decreased axi-cel expansion. Finally, patients with high tumor burden had higher immune dysregulation with increased serum inflammatory markers and tumor IFN signaling. These data support that immune dysregulation in LBCL promotes axi-cel resistance via multiple mechanistic programs: insufficient axi-cel expansion associated with both circulating M-MDSC and tumor IFN signaling, which also gives rise to expression of immune checkpoint ligands.

85P MiRNAome deregulation can contribute to overexpression of immune checkpoints: An epigenetic mechanism of checkpoint inhibitor therapy failure

One of the main immune escape mechanisms used by tumors is the exploitation of immune checkpoints – receptors on surface of immune cells, which, after binding their ligands, can suppress immune response. This research aims to identify in what way the shifts in miRNA signature can contribute to the expression of immune checkpoint ligands on cancer cells. MiRNA targets within gene transcripts were predicted in silico using the TargetScan software. Targets of miRNAs miR-15/16, miR-34, miR-140, miR-141/200, miR-145, miR-148/152, miR-199, miR-200, miR-202, miR-214, miR-302, miR-320 and miR-520 were found in PDL1 gene transcript. Down-regulation of these miRNAs is characteristic to the cancer cells and, therefore, allows reactivation and hyperexpression of gene encoding PD-1 ligand. Moreover, multiple targets of down-regulated miRNAs let-7, miR-1/206, miR-15/16, miR-22, miR-26, miR-29, miR-31, miR-34, miR-124, miR-125, miR-128, miR-129, miR-133a/b, miR-138, miR-140, miR-141/200, miR-143, miR-145, miR-148/152, miR-149, miR-194, miR-199, miR-204, miR-205, miR-214, miR-218, miR-302, miR-326, miR-449, miR-506, miR-520 and miR-655 were revealed in transcripts of genes encoding other immune checkpoint ligands – PDL2 (PDCD1LG2), B7-H3 (CD276), B7-H4 (VTCN1), HHLA2, galectin 9 (LGALS9), HVEM, PVR (Necl-5), PVRL2 (Nectin 2) and CD200. In addition, transcript of FASL gene encoding the Fas ligand carries targets of down-regulated miRNAs let-7, miR-22, miR-141/200, miR-149, miR-194, miR-199, miR-214 and miR-302/520. Down-regulation of tumor-suppressive miRNAs can be responsible for overexpression of genes encoding the key immune checkpoint ligands. As a result, cancer cells can ensure immune privilege. This resistance is multiple, because the shifts in miRNAome can cause hyperexpression of many immune checkpoint ligands at one time. Therefore, inhibition of one of the immune checkpoint axes can be easily overcome by overexpression of others due to the most profound down-regulation of the miRNA expression that is well-known phenomenon during the tumor progression and drug administration. Probably, this explains the long-term failure of immunotherapy.

Combination of PD-L1 and PVR determines sensitivity to PD-1 blockade.

Expression of immune checkpoint ligands (ICLs) is necessary to trigger the inhibitory signal via immune checkpoint receptors (ICRs) in exhausted T cells under tumor immune microenvironment. Nevertheless,to our knowledge, ICL expression profile in cancer patients has not been investigated. Using previously reported RNA-seq data sets, we found that expression of ICLs was patient specific but their coexpression can be patterned in non-small-cell lung cancers (NSCLCs). Since the expression of PD-L1 and poliovirus receptor (PVR) among various ICLs was independently regulated, we could stratify the patients who were treated with anti-PD-1 later into 4 groups according to the expression level of PD-L1 and PVR. Of interest, high PVR and low PVR expressions in PD-L1-expressing patients enriched nonresponders and responders to PD-1 blockade, respectively, helping in further selection of responders. Using a genetically engineered cancer model, we also found that PVR-deficient and PD-L1-sufficient tumor-bearing mice were highly sensitive to anti-PD-1 therapy, whereas PVR-sufficient and PD-L1-deficient tumor-bearing mice were resistant to anti-PD-1 therapy. Taken together, our study provides a concept that combinatorial expression patterns of PVR and PD-L1 are key determinants for PD-1 blockade and furthermore suggest a better therapeutic usage of immune checkpoint blockades (ICBs).

479 (PB-112) - Abnormalities in miRNAome contribute to aberrant expression of immune checkpoint ligands on breast cancer cells: An epigenetic mechanism of tumor immune privilege

479 (PB-112) - Abnormalities in miRNAome contribute to aberrant expression of immune checkpoint ligands on breast cancer cells: An epigenetic mechanism of tumor immune privilege

Immunologic Milieu of Mature T-Cell and NK-Cell Lymphomas—Implications for Therapy

T-cells and natural killer (NK) cells share the same ontogeny, and lymphomas derived from them are clinically diverse, occurring in nodal and extranodal sites. In addition to inherent properties of these lymphomas, their microenvironment also impacts on pathogenesis and response to therapy. An understanding of the milieu of T-cell and NK cell lymphomas has important implications on treatment. Components of the microenvironment include tumour-associated macrophages (TAM), non-neoplastic T-cells and B-cells, eosinophils, dendritic cells, endothelial cells and blood vessels. TAM typically undergoes M2 polarization, promoting angiogenesis and inhibiting anti-tumour cellular immunity. In lymphomas of follicular helper T-cell derivation, increased B-cell proliferation occurs, which may in turn enhance neoplastic T-cell growth. The expression of immune checkpoint ligands on TAM, dendritic cells or lymphoma cells induces an immunosuppressive environment conducive to neoplastic proliferation. Strategies against this complex cellular and immunologic microenvironment have shown promises. These include the use of signal transduction inhibitors, monoclonal antibodies against chemokines or non-neoplastic microenvironmental cells, immunomodulatory drugs and immune checkpoint blockade. As T-cell and NK cell lymphomas are highly heterogeneous, clinical trials to demonstrate efficacy of a given therapeutic approach requires careful design aiming at enriching patient populations who will best respond. Targeting of the immunologic milieu in T-cell and NK-cell lymphomas offers exciting challenges and opportunities.

MYC Inhibition and TP53 Activation Synergistically Suppress Immune Checkpoint Ligand PD-L1 Expression in AML Cells

Expression of immune checkpoint ligands is a mechanism that many tumors use to escape attack by host immune cells. PD-L1, the ligand for checkpoint receptor PD-1 on T cells, is often expressed on tumor cells. Engagement of PD-1 on T cells by PD-L1 on tumor cells attenuates T-cell receptor signaling and suppresses anti-tumor response. PD-1 and PD-L1 blocking antibodies have been implemented clinically as treatment for many cancers, but the pattern of PD-L1 expression on AML is not well characterized. To answer this question, we studied how PD-L1 expression on AML is regulated under in vitro conditions that simulate the leukemia-host microenvironment.We examined surface expression of PD-L1 by flow cytometry on 4 AML lines, THP-1, KG1, KG1a, HL60, and a CML line, K562. Under basal conditions, these lines expressed no or low levels of PDL1. The AML cells were then subjected to conditions that mimic the leukemia-host microenvironment. AML cells were stained with the green fluorescent dye CFSE and co-cultured with Ficoll-separated PMNCs from healthy donors. After a day of co-culture, expression of PD-L1 was analyzed on AML cells and PD-1, CD25 and CD69 activation markers on PMNCs. Only a small increase of PD-L1, up to 2-4 fold, was seen on AML cells under this condition. To simulate the pro-inflammatory milieu in the tumor microenvironment, anti-CD3/CD28 microbeads were then added in culture to activate T cells. We observed a marked up-regulation of PD-L1 on AML cells, up to 5-60 fold, plus prominent expression of PD-1, CD25 and CD69 on T cells. These findings were confirmed by an alternative method of T cell activation in which AML cells were first coated with an anti-CD123 antibody, linked to anti-CD3/CD28 antibodies via a biotin-streptavidin bridge, and then cultured with PMNCs. To test whether pro-inflammatory cytokines were the sole inducers of PD-L1 expression, AML cells were treated with IFN-γ or TNF-α alone. IFN-γ treatment enhanced PD-L1 expression by 2-10 fold, while TNF-α showed a <2-fold increase. These results show that expression of PD-L1 on AML is dynamically regulated through interaction with activated T cells, by multiple mechanisms including cytokine production and cell-cell interaction.MYC has been shown to regulate PD-L1 expression on T-ALL and solid tumors (Science 2016; 352:227). We asked whether MYC inhibition would suppress PD-L1 on AML. AML and PMNCs were co-cultured in the presence of anti-CD3/CD28 beads, with JQ1, a BET bromodomain inhibitor that blocks MYC expression. JQ1 inhibited PD-L1 expression by >90%. Dose-effect titration showed sigmoidal curves with ED50 of 0.03 to 0.1 μM for the 5 AML lines. Treatment with another MYC inhibitor, CPI-203, yielded similar results. These observations indicate that MYC inhibition can suppress PD-L1 expression on AML induced by activated T cells. TP53 has been shown to regulate PD-L1 expression on non-small cell lung cancer (JNCI 2016; 108:djv303). We asked whether TP53 activation in AML would also affect PD-L1 expression. Since the AML lines we used did not express wild-type TP53, we overexpressed TP53 in these cells by transfecting with a TP53-GFP plasmid. Expression of TP53 in the cells decreased PD-L1 levels by >80%. Treatment of the cells with pifithrin, an inhibitor that blocks trans-activating function of TP53, did not rescue PD-L1 expression, suggesting that the effect of TP53 on PD-L1 expression is independent of its canonical trans-activating pathway. We asked if MYC and TP53 would synergistically affect PD-L1 expression on AML. We transfected AML cells with the TP53-GFP plasmid and co-cultured the cells with PMNCs and anti-CD3-/CD28 beads, in the presence or absence of JQ1. We found that JQ1 treatment of TP53-transfected cells further decreased PD-L1 expression by another 15%, indicating that MYC and TP53 independently and synergistically affect PD-L1 expression on AML.In summary, PD-L1 expression on AML cells is dynamically up-regulated upon interaction with activated T cells and suppressed by perturbation of the MYC and TP53 pathways. These findings have implications in the use of immune effector cell therapy against AML, since the activated effector cells could up-regulate PD-L1 expression on target cells and attenuate anti-leukemia effects. MYC inhibitors and TP53 activators could potentially be used in combination to suppress PD-L1 up-regulation and abrogate the ability of AML cells to escape host immune elimination. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2320: CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer

Abstract Non-small-cell lung cancers (NSCLC) account for 85% of lung cancers and are mostly diagnosed at advanced stage. Drugs interrupting immune checkpoints, such as anti-CTLA-4, anti-PD-1, anti-PD-L1, have proven to unleash anti-tumor immunity and mediate durable cancer regressions in a portion of patients with NSCLC. Epithelial-to-mesenchymal transition (EMT) is a developmental process that enables reprogramming of polarized epithelial cells towards a mesenchymal phenotype with migratory and invasive properties. EMT has been involved in escape from oncogenic-induced senescence, resistance to chemotherapy and development of metastatic disease. Moreover, EMT promotes cancer cell plasticity and favors their adaptability to selective pressures. Here we made use of in silico approaches and further in vivo validation to evaluate the potential role of EMT as a major escape mechanism to tumor immunosurveillance in NSCLC. We performed an initial in silico analysis to assess the expression of immune checkpoint ligands (ICPLs) in 129 NSCLC cell lines (CCLE), in relation to their EMT status defined by a 72-gene EMT signature that was previously reported in NSCLC. Unsupervised hierarchical clustering analysis revealed that the expression of selected ICPLs was associated with the mesenchymal or epithelial phenotype. In particular, CD70, a member of the tumor necrosis factor superfamily, was significantly associated with the mesenchymal status (p &amp;lt; 0.001). We identified an E-box sequence (CANNTG) in CD70 gene promoter thus suggesting the possible regulation of CD70 by ZEB1 and/or SNAI. We extended the in silico analysis to a set of 488 adenocarcinomas (ADC) and 501 squamous cell carcinomas (SCC) from the TCGA and confirmed that CD70 expression was associated with a mesenchymal status (q-value &amp;lt;0.001). In a set of E-like (H441 and H1650) and M-like (H23 and HCC44) NSCLC cell lines we validated the association between an increased level of CD70 by flow cytometry and the mesenchymal status. Expression of CD70 by immunohistochemistry was observed in 5/51 lung ADC (10%), 6/45 (13%) lung SCC, 16/26 (63%) pulmonary sarcomatoid carcinomas and 2/10 (20%) small cell lung carcinomas. Interestingly, in pulmonary sarcomatoid carcinomas, CD70 expression was closely associated with the mesenchymal component. Our results suggest an association between the expression of CD70 and the mesenchymal phenotype, thus shedding light on the potential role of CD70 in immune escape of a subset of NSCLC. Citation Format: Sandra Ortiz-Cuaran, Aurélie Swalduz, Maria A. Albaret, Christine Menetrier-Caux, Véronique Haddad, Arnaud Paré, Geneviève De Souza, Anne P. Morel, Maurice Pérol, Christophe Caux, Sylvie Lantuejoul, Alain Puisieux, Pierre Saintigny. CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2320.