Programmed Death Ligand 1 Induction Research Articles

Increased CD8 Tumor Infiltrating Lymphocytes in Colorectal Cancer Microenvironment Supports an Adaptive Immune Resistance Mechanism of PD-L1 Expression.

Background:Tumor cells express programmed death ligand-1 (PD-L1) through several biological processes, thereby having different clinical significance depending on the underlying mechanism of expression. Currently, mechanisms leading to PDL1 gene expression in colorectal cancer (CRC) are not fully understood. Methods:We investigated 98 Indonesia CRC patients to determine PD-L1 protein expressions and their correlations with PD-L1 gene copy number status, tumor infiltrating lymphocytes (TILs), tumor mutational profile, as well as clinicopathologic features. Results:Our investigation demonstrated that 18% of patients positively expressed PD-L1. Further analysis on PD-L1 copy number revealed that all PD-L1+ tumors had normal copy number, indicating that the expression of PD-L1 was not a consequence of genetic amplification of PD-L1. From TILs analysis, there was a significant increase of CD8 in all tumor cells expressing PD-L1 (P=0.0051), indicating that the inducible PD-L1 expression was the prominent mechanism occurred in CRC. Furthermore, the expression of PD-L1 in this CRC population was significantly associated with high frequency of MSI compared to the remainder PD-L1- tumors (P=0.0001), suggesting the natural immunogenicity of tumors via MSI status plays role in attracting immune response. On the other hand, p53 mutations which were frequently observed within Indonesian CRCs (76.5%), they were not associated with PD-L1 expression (p=0.1108), as well as KRAS gene (29.6%; p=0.5772) and BRAF gene mutations (5%; p=0.2171). Conclusion:Our study demonstrated that PD-L1 expressions in CRC were predominantly found as a consequence of infiltrating CD8 T lymphocytes that in part arise in the setting of microsatellite instability. Taken together, our findings further support the role of adaptive immune resistance to drive PD-L1 induction in tumor microenvironment and may provide important rationale for strategy implementation of immunotherapy for CRC cases.

Drug-induced PD-L1 expression and cell stress response in breast cancer cells can be balanced by drug combination

The impact of chemotherapy on tumor-immune system interaction can be either beneficial or harmful, which is represented by the immunogenic cell death (ICD) paradigm or overexpression of the immunosuppressive protein – programmed death ligand 1 (PD-L1). In this study we explore the impact of steroid receptor coactivator inhibitor, other targeted anti-cancer compounds and traditional chemotherapeutic agents on the expression of PD-L1 in four breast cancer (BC) cell lines. Our results show that these agents induce PD-L1 expression, yet the magnitude of this induction varies substantially across the different compounds. In addition, we utilized the E0771 ER + BC cells as a model to examine in greater detail the relationship between pharmacological pressure, cell stress and the induction of PD-L1. Our results imply that drug induced PD-L1 expression occurs in the broader context of cell-stress, without conferring acquired drug-resistance. Furthermore, a balance between BC cytotoxicity, induction of cell-stress and the overexpression of PD-L1 can be achieved through the selection of appropriate combinations of anti-cancer compounds. Therefore, we propose that drug combination can be employed not only for increasing the direct kill of cancer cells, but also as a strategy to minimize the activation of immunosuppressive and cancer cell pro-survival program responses during drug treatment.

Tumor-induced peripheral immunosuppression promotes brain metastasis in patients with non-small cell lung cancer.

Brain metastases are a significant source of morbidity and mortality for patients with lung cancer. Lung cancer can induce local and systemic immunosuppression, promoting tumor growth and dissemination. One mechanism of immunosuppression is tumor-induced expansion of programmed death-ligand 1 (PD-L1) expressing myeloid cells. Here, we investigate peripheral blood immune phenotype in NSCLC patients with or without brain metastasis. Peripheral blood was collected from patients with lung metastatic brain tumors and pre-metastatic lung cancer. Immunosuppressive monocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) were quantified through flow cytometry. T cell reactivity was analyzed via ELISpot. Brain metastasis conditioned media was collected from tumor-derived cell cultures and analyzed for cytokines by ELISA. Naïve monocytes were stimulated with brain metastasis conditioned media to evaluate PD-L1 stimulation. Patients with brain metastatic lung carcinoma demonstrated increased peripheral monocyte PD-L1, MDSC abundance, and Treg percentage compared to early stage pre-metastatic patients and healthy controls. Patients with elevated peripheral monocyte PD-L1 had less reactive T cells and worse survival. Brain metastasis conditioned media stimulation increased monocyte PD-L1, and conditioned media IL-6 levels correlated with PD-L1 induction. Treatment with anti-IL-6 or anti-IL-6 receptor antibodies reduced PD-L1 expression. In summary, patients with lung cancer and brain metastases exhibit multiple markers of peripheral immunosuppression. The frequency of PD-L1+ myeloid cells correlated with the presence of brain metastases.Tumor-derived IL-6 was capable of inducing PD-L1+ myeloid cellsin vitro, suggesting thatmonitoring of immunosuppressive factors in peripheral blood may identify new targets for therapeutic intervention in selected patients.

PD-L1 Induction by Cancer-Associated Fibroblast-Derived Factors in Lung Adenocarcinoma Cells.

Cancer-associated fibroblasts (CAFs) exert various effects upon biological behaviours of cancer. In this study, we examined the correlation of CAFs with the intra-tumoural immune system in the lung adenocarcinoma microenvironment. We studied 27 and 113 cases of lung adenocarcinoma tentatively as Cohorts 1 and 2, respectively. The patients in Cohort 1 received epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) for recurrent lung adenocarcinoma. α-smooth muscle actin (α-SMA), a surrogate marker for CAFs, was examined by immunohistochemistry. We then examined the effects of CAFs isolated from lung cancer tissues on programmed death ligand 1 (PD-L1) expression in lung adenocarcinoma cell lines. No significant associations were detected between α-SMA status and the ratios of CD8/CD4 and Foxp3/CD8 in Cohort 1. However, α-SMA status was significantly associated with PD-L1 status in both Cohorts 1 and 2. Conditioned medium of CAFs significantly induced PD-L1 expression in lung adenocarcinoma cell lines, A549, PC-9, and H1975. Among the cytokines examined by antibody array, C-X-C motif chemokine ligand 2 (CXCL2) increased PD-L1 mRNA expression in these cell lines. CXCL2 is therefore considered to have a potential to induce PD-L1 expression in lung adenocarcinoma cells as a result of an interaction between carcinoma cells and CAFs. These findings did firstly demonstrate that CAFs indirectly influenced tumour immunity through increasing PD-L1 expression in lung adenocarcinoma cells.

Abstract 4109: Functional profiling of 17,000 open reading frames identifies regulators of PDL1 expression in HNSCC

Abstract The interaction of Programmed Death Ligand 1 (PD-L1) with the PD-1 receptor serves as an immune checkpoint, dampening anti-tumor immunity. While immunotherapies targeting this pathway (Pembrolizumab and Nivolumab), were approved for use in recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) in 2016, overall response rates are low and few biomarkers have been identified to differentiate responders. We expect a more detailed understanding of PD-L1 checkpoint regulation in HNSCC to provide insight toward addressing these deficits. Thus, we developed a high- throughput screening protocol to discover signals modulating PD-L1 expression. We have employed a lentiviral open reading frame (ORF) library for overexpression of over 14,000 different genes in a pooled format. UM-SCC-49 cells transduced with this library were stained for cell surface PD-L1 and sorted by flow cytometry to select cells with enhanced PD-L1 expression. Sequencing of the selected sub-population was performed to determine ORFs enriched over an unsorted pool. To validate a role for individual target genes in modulating PD-L1 expression, monoclonal populations were expanded from the sorted pool and evaluated for PD-L1 expression. We confirmed increased PD-L1 expression in 7 of these cell lines over wild-type and LacZ overexpressing controls via western blotting and flow cytometry. Furthermore, we selected 5 ORFs of interest overrepresented in the sorted pool for further validation using stably expressed V5 tagged constructs. Target gene expression was confirmed by western blot, and enhanced cell surface PD-L1 expression was validated via flow cytometry for 4/5 cell lines. Thus, we provide evidence for successful identification of signals regulating PD-L1 using a novel high-throughput overexpression screen. Future experiments will aim to clarify the mechanism of PD-L1 induction in response to the signals identified here, and may ultimately provide a more thorough understanding of genetic factors modulating immunosuppression and response to immunotherapy in HNSCC patients. Citation Format: Jacqueline Mann, Samantha Devenport, Aditi Kulkarni, Rebecca Hoesli, Susan Foltin, Nicole Michmerhuizen, Alexey Nesvizhskii, Chad Brenner. Functional profiling of 17,000 open reading frames identifies regulators of PDL1 expression in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4109.

Abstract 3962: Chemotherapy induces alteration of programmed death ligand-1 expression in gastric cancer

Abstract Background Cancer immunotherapy targeting the programmed cell death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway have been demonstrated favorable therapeutic effect in gastric cancer (GC). However, it remains unclear that PD-L1 expression in GC tissue can be a biomarker to predict the therapeutic effect of immune checkpoint inhibitors. We hypothesize that prior therapy, including chemotherapy and radiation therapy can lead to the alteration of PD-L1 status, resulting in imprecise evaluation of PD-L1 expression. The aim of this study is to clarify the effect of cytotoxic agents on PD-L1 expression in GC cells in time and dose depending manner. Methods PD-L1 expression of GC cell lines were confirmed by quantitative PCR and western blotting. Two gastric cancer cell lines, which have low PD-L1 expression, are selected and used in subsequent in vitro experiment. We evaluated the expression of PD-L1 in these cell lines by quantitative PCR and western blotting after exposure to cytotoxic agents at various concentrations and exposure times. Furthermore, we examined whether the expression of PD-L1 induced by cytotoxic agents was maintained after ceasing the 5-FU exposure. The gene pathway regulating PD-L1 induction was also examined. Results The PD-L1 expression of the selected GC cell lines were upregulated, depending on the concentration and exposure time of cytotoxic agents. Furthermore, the PD-L1 expression induced by cytotoxic agents was maintained even after cessation of exposure. We clarify that the upregulation of PD-L1 expression was induced via p38 MAPK activation. Conclusion We demonstrated that cytotoxic agent can alter the expression of PD-L1 in GC cells through the activation of p38 MAPK pathway in dose and time depending manner. Our findings suggested that the modified administration of immune checkpoint inhibitors can improve the antitumor response, leading to promising new treatment strategy against GC. Citation Format: Kohei Yamashita, Masaaki Iwatsuki, Shiro Iwagami, Kojiro Eto, Yuki Koga, Yuki Kiyozumi, Yoshifumi Baba, Naoya Yoshida, Hideo Baba. Chemotherapy induces alteration of programmed death ligand-1 expression in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3962.

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

Upregulation of programmed death-ligand 1 (PD-L1) on circulating and tumor-infiltrating myeloid cells is a critical component of GBM-mediated immunosuppression that has been associated with diminished response to vaccine immunotherapy and poor survival. Although GBM-derived soluble factors have been implicated in myeloid PD-L1 expression, the identity of such factors has remained unknown. This study aimed to identify factors responsible for myeloid PD-L1 upregulation as potential targets for immune modulation. Conditioned media from patient-derived GBM explant cell cultures was assessed for cytokine expression and utilized to stimulate naïve myeloid cells. Myeloid PD-L1 induction was quantified by flow cytometry. Candidate cytokines correlated with PD-L1 induction were evaluated in tumor sections and plasma for relationships with survival and myeloid PD-L1 expression. The role of identified cytokines on immunosuppression and survival was investigated in vivo utilizing immunocompetent C57BL/6 mice bearing syngeneic GL261 and CT-2A tumors. GBM-derived IL6 was identified as a cytokine that is necessary and sufficient for myeloid PD-L1 induction in GBM through a STAT3-dependent mechanism. Inhibition of IL6 signaling in orthotopic murine glioma models was associated with reduced myeloid PD-L1 expression, diminished tumor growth, and increased survival. The therapeutic benefit of anti-IL6 therapy proved to be CD8+ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy. Our findings suggest that disruption of IL6 signaling in GBM reduces local and systemic myeloid-driven immunosuppression and enhances immune-mediated antitumor responses against GBM.

Neddylation inhibition upregulates PD-L1 expression and enhances the efficacy of immune checkpoint blockade in glioblastoma.

Pevonedistat (MLN4924), a specific NEDD8-activating enzyme inhibitor, has been considered as a promising treatment for glioblastoma, which is currently in Phase I/II clinical trials. On the other hand, inhibition of neddylation pathway substantially upregulates the expression of T cell negative regulator programmed death-ligand 1 (PD-L1), which might account for the potential resistance via evasion of immune surveillance checkpoints. Whether administration of anti-PD-L1 enhances the efficacy of pevonedistat through a cytotoxic T cell-dependent mechanism in glioblastoma needs to be investigated. Here, we report that depletion of neddylation pathway key enzymes markedly elevates PD-L1 expression in glioblastoma cancer cells. Consistently, neddylation inhibitor pevonedistat significantly enhances PD-L1 expression in both glioblastoma cancer cell lines and animal models. Mechanistically, pevonedistat increases PD-L1 mRNA levels mainly through inhibiting Cullin1-F-box and WD repeat domain-containing 7 E3 ligase activity and accumulating c-MYC proteins, a direct transcriptional activator of PD-L1 gene expression. In addition, inhibition of Cullin3 activity by pevonedistat also blocks PD-L1 protein degradation. Importantly, pevonedistat attenuates T cell killing through PD-L1 induction, and blockade of PD-L1 restores the sensitivity of pevonedistat-treated glioblastoma cancer cells to T cell killing. The combination of pevonedistat and anti-PD-L1 therapy compared to each agent alone significantly increased the therapeutic efficacy in vivo. Our study demonstrates inhibition of neddylation pathway suppresses cancer-associated immunity and provides solid evidence to support the combination of pevonedistat and PD-L1/programmed cell death protein 1 immune checkpoint blockade as a potential therapeutic strategy to treat glioblastoma.

PC-1 Genetic and pharmaceutical inactivation of hypoxia-inducible factor 1α reveals a molecular switch from graft vs host diseases to graft vs leukemia effect

PC-1 Genetic and pharmaceutical inactivation of hypoxia-inducible factor 1α reveals a molecular switch from graft vs host diseases to graft vs leukemia effect

Modulation of immune checkpoint molecule expression in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy for which novel therapeutics with improved efficacy are greatly needed. To provide support for clinical immune checkpoint blockade, we comprehensively evaluated the expression of therapeutically targetable immune checkpoint molecules on primary MCL cells. MCL cells showed constitutive expression of Programmed Death 1 (PD-1) and Programmed Death Ligand 1 (PD-L1), variable CD200, absent PD-L2, Lymphocyte Activation Gene 3 (LAG-3), and Cytotoxic T-cell Associated Protein 4 (CTLA-4). Effector cells from MCL patients expressed PD-1. Co-culture of MCL cells with T-cells induced PD-L1 surface expression, a phenomenon regulated by IFNγ and CD40:CD40L interaction. Induction of PD-L1 was attenuated by concurrent treatment with ibrutinib or duvelisib, suggesting BTK and PI3K are important mediators of PD-L1 expression. Overall, our data provide further insight into the expression of checkpoint molecules in MCL and support the use of PD-L1 blocking antibodies in MCL patients.

Upregulation of PD-L1 on Myeloma Cells By Immunomodulatory Agents Potentiates the Effect of Durvalumab

Introduction: Programmed death-ligand 1 (PD-L1) shows increased expression on plasma cells from multiple myeloma (MM) patients as compared with those from monoclonal gammopathy of undetermined significance patients and healthy volunteers. PD-L1 inhibits tumor-specific cytotoxic T lymphocyte-mediated responses via interaction with PD-1. Our previous study showed that PD-L1 on MM cells is associated with aggressive myeloma behaviors including cell proliferation and drug resistance, and furthermore the interaction of PD-L1 with PD-1 molecules induced drug resistance in MM cells by antiapoptotic responses through the Akt signaling pathway (Tamura et al. Leukemia 2013, Cancer Immunol Res 2016). We next investigated the regulation of PD-L1 expression by immunomodulatory drugs (IMDs) and antimyeloma effects of the anti-PD-L1 antibody durvalumab (DUR) in combination with IMDs.Methods: 1) The expression of mRNA and protein in MM cell lines (MOSTI-1, U266, KMS18, OPM2, MM.1S) and plasma cells from MM patients was analyzed using real-time PCR/DNA microarray and flow cytometry/Western blotting, respectively. 2) PD-L1 induction by IMDs was evaluated based on promoter activity in the luciferase assay. 3) Cereblon (CBRN)-knockdown and -knockout MM cell lines were established using the lentiviral shRNA and CRISPR/Cas9 system, respectively. 4) MM.1S cells were inoculated subcutaneously into immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice and then MM1.S-engrafted mice were administered lenalidomide (LEN; 25 mg/kg per day) orally for 12 days. 5) CD3+ T cells were isolated from peripheral blood mononuclear cells of healthy volunteers, and then T cells activated with anti-CD3/CD28 antibodies were co-cultured with X-irradiated MM cells for 4 days. T-cell proliferation and activation were assessed using the CFSE cell division assay and expression levels of activation markers CD25 and CD45RO, respectively.Results: 1) PD-L1 expression was upregulated in MM cell lines by LEN and pomalidomide (POM) in a concentration-dependent manner, but not by thalidomide. Its expression on plasma cells, but not on lymphocytes and monocytes, obtained from relapsed/refractory MM patients was enhanced by LEN and POM. This PD-L1 induction was not seen in CRBN-knockdown and -knockout MM cell lines. To clarify this mechanism, gene expression profiling analysis was performed in MM cell lines with and without POM treatment, demonstrating that not only PD-L1 but also a proliferation-inducing ligand (APRIL) was enhanced by POM. APRIL expression was not induced by POM in CRBN-knockdown and -knockout MM cell lines. PD-L1 upregulation on MM cells by POM was inhibited by either recombinant B cell maturation antigen (BCMA)-Ig as an APRIL inhibitor, the antibody against BCMA, one of the receptors for APRIL, or MEK/ERK inhibitor U0126. Furthermore, PD-L1 and APRIL expression in MM.1S cells engrafted in NOG mice were upregulated by LEN treatment. On the other hand, LEN/POM also increased PD-L1 promoter activity in BCMA- and APRIL-negative HeLa cells. 2) When T cells were co-cultured with LEN- or POM-treated U266 cells (PD-L1-upregulated cells), the proliferation of CD4+ and CD8+ T cells was inhibited in comparison with that in untreated cells. This T-cell suppression did not occur when DUR and LEN/POM were added during co-cultivation of LEN/POM-treated U266 cells with T cells. Moreover, the population of both CD25+CD45RO+ double-positive CD4+ and CD8+ T cells co-cultured with POM-treated U266 cells was increased by combined POM and DUR treatment compared with DUR alone.Conclusion: Our study revealed that IMDs enhanced PD-L1 expression on MM cells as well as APRIL production through CRBN binding, and one mechanism may involve the APRIL-BCMA interaction. Another mechanism may be directly involved in PD-L1 downregulation by IKZF1 degradation through CRBN. Furthermore, T-cell inhibition induced by PD-L1-upregulated MM cells was effectively recovered after combination treatment with DUR and LEN or POM. These results suggest that DUR combined with IMDs may be a reasonable treatment for relapsed/refractory MM patients. DisclosuresIto:Celgene: Honoraria. Handa:JVC KENWOOD Corporation: Other: Leadership Position/Advisory Role; SYSMEX CORPORATION: Other: Leadership Position/Advisory Role; TAMAGAWA SEIKI Co.,Ltd.: Other: Leadership Position/Advisory Role; Celgene: Honoraria, Other: Supplied with reagents and drugs., Research Funding. Tamura:Takeda Pharmaceutical: Honoraria; Ono Pharmaceutical: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria, Research Funding.

Combined Blockade of IL6 and PD-1/PD-L1 Signaling Abrogates Mutual Regulation of Their Immunosuppressive Effects in the Tumor Microenvironment.

Recently emerging cancer immunotherapies combine the applications of therapeutics to disrupt the immunosuppressive conditions in tumor-bearing hosts. In this study, we found that targeting the proinflammatory cytokine IL6 enhances tumor-specific Th1 responses and subsequent antitumor effects in tumor-bearing mice. IL6 blockade upregulated expression of the immune checkpoint molecule programmed death-ligand 1 (PD-L1) on melanoma cells. This PD-L1 induction was canceled in IFNγ-deficient mice or CD4+ T cell-depleted mice, suggesting that CD4+ T cell-derived IFNγ is important for PD-L1 induction in tumor-bearing hosts. In some patients with melanoma, however, treatment with the anti-PD-1 antibody nivolumab increased systemic levels of IL6, which was associated with poor clinical responses. This PD-L1 blockade-evoked induction of IL6 was reproducible in melanoma-bearing mice. We found that PD-1/PD-L1 blockade prompted PD-1+ macrophages to produce IL6 in the tumor microenvironment. Depletion of macrophages in melanoma-bearing mice reduced the levels of IL6 during PD-L1 blockade, suggesting macrophages are responsible for the IL6-mediated defective CD4+ Th1 response. Combined blockade of the mutually regulated immunosuppressive activities of IL6 and PD-1/PD-L1 signals enhanced expression of T cell-attracting chemokines and promoted infiltration of IFNγ-producing CD4+ T cells in tumor tissues, exerting a synergistic antitumor effect, whereas PD-L1 blockade alone did not promote Th1 response. Collectively, these findings suggest that IL6 is a rational immunosuppressive target for overcoming the narrow therapeutic window of anti-PD-1/PD-L1 therapy.Significance: These findings advance our understanding of IL6-PD1/PD-L1 cross-talk in the tumor microenvironment and provide clues for targeted interventional therapy that may prove more effective against cancer. Cancer Res; 78(17); 5011-22. ©2018 AACR.

The Interplay of Notch Signaling and STAT3 in TLR-Activated Human Primary Monocytes.

The highly conserved Notch signaling pathway essentially participates in immunity through regulation of developmental processes and immune cell activity. In the adaptive immune system, the impact of the Notch cascade in T and B differentiation is well studied. In contrast, the function, and regulation of Notch signaling in the myeloid lineage during infection is poorly understood. Here we show that TLR signaling, triggered through LPS stimulation or in vitro infection with various Gram-negative and -positive bacteria, stimulates Notch receptor ligand Delta-like 1 (DLL1) expression and Notch signaling in human blood-derived monocytes. TLR activation induces DLL1 indirectly, through stimulated cytokine expression and autocrine cytokine receptor-mediated signal transducer and activator of transcription 3 (STAT3). Furthermore, we reveal a positive feedback loop between Notch signaling and Janus kinase (JAK)/STAT3 pathway during in vitro infection that involves Notch-boosted IL-6. Inhibition of Notch signaling by γ-secretase inhibitor DAPT impairs TLR4-stimulated accumulation of NF-κB subunits p65 in the nucleus and subsequently reduces LPS- and infection-mediated IL-6 production. The reduced IL-6 release correlates with a diminished STAT3 phosphorylation and reduced expression of STAT3-dependent target gene programmed death-ligand 1 (PD-L1). Corroborating recombinant soluble DLL1 and Notch activator oxaliplatin stimulate STAT3 phosphorylation and expression of immune-suppressive PD-L1. Therefore we propose a bidirectional interaction between Notch signaling and STAT3 that stabilizes activation of the transcription factor and supports STAT3-dependent remodeling of myeloid cells toward an immuno-suppressive phenotype. In summary, the study provides new insights into the complex network of Notch regulation in myeloid cells during in vitro infection. Moreover, it points to a participation of Notch in stabilizing TLR-mediated STAT3 activation and STAT3-mediated modulation of myeloid functional phenotype through induction of immune-suppressive PD-L1.

Abstract 1024: Tumor intrinsic properties associate with differential effects on CD8+ tumor-infiltrating lymphocyte density and immune gene expression in non-small cell lung cancer (NSCLC) samples

Abstract Introduction: Anti-tumor immune response is controlled by a complex interaction between the immune system, tumor cells, and associated stroma. Expression of major histocompatibility complex class I (MHC I) and MHC class II (MHC II) antigens may be dysregulated in cancer, leading to alterations in the tumor antigen presentation profile. Stage I–III resected NSCLC tumor samples were profiled to study the relationship between MHC I, MHC II, and programmed death ligand 1 (PD-L1) tumor cell (TC) expression with tumor-associated inflammation. Here we report the potential impact of respective profiles on tumor immune response. Methods: 53 adenocarcinoma (NSCLC-AD) and 51 squamous cell carcinoma (NSCLC-SQ) stage I–III resected formalin-fixed, paraffin-embedded tumor specimens from commercial sources were stained by immunohistochemistry for MHC I (HLA-A,B,C), MHC II (HLA-DP,DQ,DR), and PD-L1 and TC expression was assessed by manual pathologist review. CD8+ cell density was quantified using Definiens image analysis algorithms for the intraepithelial tumor region. RNA extracted from the samples was analyzed by RNAseq, with data available for 48 NSCLC-AD and 46 NSCLC-SQ specimens. A 25-gene IFN-gamma gene signature (IFNG score) was used to study the association of T-cell inflammation with other biomarkers. Results: Of all NSCLC-AD and NSCLC-SQ specimens, 85% showed either complete (<20% TC) or partial (20–80% TC) loss of TC MHC I, while TC MHC II was increased (>1%; MHCIIhi) in 60% of NSCLC-AD and 18% of NSCLC-SQ. Quantitative analysis of CD8 in the tumor microenvironment (TME) revealed a significantly reduced intraepithelial density of CD8+ tumor-infiltrating lymphocytes (TILs) in NSCLC-AD (P=0.009) and NSCLC-SQ (P=0.01) with complete MHC I loss. In contrast, intraepithelial CD8+ TILs were significantly increased in MHCIIhi tumors for both NSCLC-AD (P=0.004) and NSCLC-SQ (P=0.006). Tumors displaying both MHCIIhi and retained MHC I demonstrated the highest TIL density. Gene expression associated with IFN-gamma response was increased in tumors with retained MHC I (P=0.0036) and MHCIIhi samples (P<0.001). In NSCLC-AD and NSCLC-SQ, 38% and 59% had PD-L1 expression ≥1%, respectively. PD-L1 expression correlated with CD8 density and IFNG score in NSCLC-AD, but not in NSCLC-SQ. Conclusions: Understanding the role of the antigen presentation machinery in immune activation and evasion in the TME is important to predict responses to immunotherapy. These data suggest that the expression of MHC I, MHC II, and PD-L1 by tumor cells may play a role in guiding the localization of CD8+ T cells in the TME with differential effects dependent on histologic subtype. The lack of correlation between inflammation and PD-L1 expression in stage I–III NSCLC-SQ suggests an alternate PD-L1 induction mechanism in a subset of those tumors. Citation Format: Cyrus Hedvat, Keyur Desai, Dimple Pandya, Peter Szabo, Johannes Zimmermann, Jan Lesniak, Scott Ely, Sujaya Srinivasan, Xi-Tao Wang, Michele French, Robin Edwards. Tumor intrinsic properties associate with differential effects on CD8+ tumor-infiltrating lymphocyte density and immune gene expression in non-small cell lung cancer (NSCLC) samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1024.

Egr2-independent, Klf1-mediated induction of PD-L1 in CD4+ T cells

Programmed death ligand 1 (PD-L1)-mediated induction of immune tolerance has been vigorously investigated in autoimmunity and anti-tumor immunity. However, details of the mechanism by which PD-L1 is induced in CD4+ T cells are unknown. Here, we revealed the potential function of Klf1 and Egr2-mediated induction of PD-L1 in CD4+ T cells. We focused on the molecules specifically expressed in CD4+CD25−LAG3+ regulatory T cells (LAG3+ Tregs) highly express of PD-L1 and transcription factor Egr2. Although ectopic expression of Egr2 induced PD-L1, a deficiency of Egr2 did not affect its expression, indicating the involvement of another PD-L1 induction mechanism. Comprehensive gene expression analysis of LAG3+ Tregs and in silico binding predictions revealed that Krüppel-like factor 1 (Klf1) is a candidate inducer of the PD-L1 gene (Cd274). Klf1 is a transcription factor that promotes β-globin synthesis in erythroid progenitors, and its role in immunological homeostasis is unknown. Ectopic expression of Klf1 induced PD-L1 in CD4+ T cells through activation of the PI3K-mTOR signaling pathway, independent of STATs signaling and Egr2 expression. Our findings indicate that Klf1 and Egr2 are modulators of PD-L1-mediated immune suppression in CD4+ T cells and might provide new insights into therapeutic targets for autoimmune diseases and malignancies.

Salmonella enterica Serovar Typhimurium Increases Functional PD-L1 Synergistically with Gamma Interferon in Intestinal Epithelial Cells via Salmonella Pathogenicity Island 2.

Nontyphoidal serovars of Salmonella enterica are pathogenic bacteria that are common causes of food poisoning. Whereas Salmonella mechanisms of host cell invasion, inflammation, and pathogenesis are mostly well established, a new possible mechanism of immune evasion is being uncovered. Programmed death ligand 1 (PD-L1) is an immunosuppressive membrane protein that binds to activated T cells via their PD-1 receptor and thereby halts their activation. PD-L1 expression plays an essential role in the immunological tolerance of self-antigens but is also exploited for immune evasion by pathogen-infected cells and cancer cells. Here, we show for the first time that Salmonella infection of intestinal epithelial cells causes the induction of PD-L1. The increased expression of PD-L1 through Salmonella infection was seen in both human and rat intestinal epithelial cell lines. We determined that cellular invasion by the bacteria is necessary for PD-L1 induction, potentially indicating that Salmonella strains are delivering mediators from inside the host cell that trigger the increased PD-L1 expression. Using knockout mutants, we determined that this effect largely originates from the Salmonella pathogenicity island 2. We also show for the first time in any cell type that Salmonella combined with gamma interferon (IFN-γ) causes a synergistic induction of PD-L1. Finally, we show that Salmonella plus IFN-γ induction of PD-L1 decreased the cytokine production of activated T cells. Understanding Salmonella immune evasion strategies could generate new therapeutic targets and help to manipulate PD-L1 expression in other diseases.

MicroRNA-155 induction via TNF-α and IFN-γ suppresses expression of programmed death ligand-1 (PD-L1) in human primary cells

Programmed death ligand-1 (PD-L1) is a critical regulator of T cell function contributing to peripheral immune tolerance. Although it has been shown that posttranscriptional regulatory mechanisms control PD-L1 expression in cancer, it remains unknown whether such regulatory loops operate also in non-transformed cells. Here we studied PD-L1 expression in human dermal lymphatic endothelial cells (HDLECs), which play key roles in immunity and cancer. Treatment of HDLECs with the pro-inflammatory cytokines IFN-γ and TNF-α synergistically up-regulated PD-L1 expression. IFN-γ and TNF-α also affected expression of several microRNAs (miRNAs) that have the potential to suppress PD-L1 expression. The most highly up-regulated miRNA following IFN-γ and TNF-α treatment in HDLECs was miR-155, which has a central role in the immune system and cancer. Induction of miR-155 was driven by TNF-α, the effect of which was significantly enhanced by IFN-γ. The PD-L1 3′-UTR contains two functional miR-155-binding sites. Endogenous miR-155 controlled the kinetics and maximal levels of PD-L1 induction upon IFN-γ and TNF-α treatments. We obtained similar findings in dermal fibroblasts, demonstrating that the IFN-γ/TNF-α/miR-155/PD-L1 pathway is not restricted to HDLECs. These results reveal miR-155 as a critical component of an inflammation-induced regulatory loop controlling PD-L1 expression in primary cells.

Defective programmed death ligand-1 (PD-L1) expression on monocytes from systemic lupus erythematosus (SLE) patients can be corrected by a soluble T lymphocyte factor (99.15)

Abstract PD-L1 is an inhibitory protein expressed on monocytes (Mo) to maintain peripheral T cell tolerance. We previously reported deficient expression of PD-L1 on Mo from patients with active SLE. To investigate the mechanisms for dysregulated PD-L1 expression in SLE we isolated peripheral blood mononuclear cells (PBMC) from juvenile SLE patients and age-matched controls. PD-L1 expression was measured by flow cytometry and realtime quantitative RT-PCR. Cytokines were analyzed by Luminex. We found that PD-L1 expression on SLE Mo was significantly induced by PBMC or CD4+ T lymphocytes from healthy subjects, and also by supernatant from healthy PBMC. Conversely, PD-L1 expression was decreased in healthy donor Mo by addition of SLE PBMC or supernatant. SLE cells had an increased rate of apoptosis, and inhibition of caspase activity led to decreased apoptosis and upregulation of PD-L1. PD-L1 mRNA expression did not correlate with protein expression. Among 15 cytokines tested, only IL-2 and IL-10 were elevated in plasma of SLE patients with active disease. These results suggest that expression of PD-L1 in juvenile SLE is controlled by caspases at the protein level via soluble factors normally produced by T lymphocytes. Abnormal T lymphocyte activity in SLE may prevent induction of PD-L1, leading to loss of peripheral tolerance. Supported by the Lupus Foundation and the Lupus Research Institute