Expression Of Immunomodulatory Molecules Research Articles

Inflammatory disease status and response to TNF blockade are associated with mechanisms of endotoxin tolerance

The mechanisms of endotoxin tolerance (ET), which down-regulate inflammation, are well described in response to exogenous toll-like receptor ligands, but few studies have focused on ET-associated mechanisms in inflammatory disease. As blocking TNF can attenuate the development of ET, the effect of anti-TNF on the expression of key ET-associated molecules in inflammatory auto-immune disease was measured; changes in inflammatory gene expression were confirmed using an ET bioassay. The expression of immunomodulatory molecules was measured in a murine model of arthritis treated with anti-TNF and the expression of ET-associated molecules was measured in whole blood in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients, before and after therapy. The expression of ET-associated genes was also measured in RA patient monocytes before and after therapy, in anti-TNF responders and non-responders. Tnfaip3, Ptpn6 and Irak3 were differentially expressed in affected paws, spleens, lymph nodes and circulating leucocytes in experimental murine arthritis treated with anti-TNF. Prior to therapy, the expression of TNFAIP3, INPP5D, PTPN6, CD38 and SIGIRR in whole blood differed between human healthy controls and RA or AS patients. In blood monocytes from RA patients, the expression of TNFAIP3 was significantly reduced by anti-TNF therapy in non-responders. Prior to therapy, anti-TNF non-responders had higher expression of TNFAIP3 and SLPI, compared to responders. Although the expression of TNFAIP3 was significantly higher in RA non-responders prior to treatment, the post-treatment reduction to a level similar to responders did not coincide with a clinical response to therapy.

Papillary thyroid cancer immune phenotypes via tumor-infiltrating lymphocyte spatial analysis.

Standard-of-care treatment options provide an excellent prognosis for papillary thyroid cancers (PTCs); however, approximately 10% of cases are advanced PTCs, resulting in less than 50% 5-year survival rates. Understanding the tumor microenvironment is essential for understanding cancer progression and investigating potential biomarkers for treatment, such as immunotherapy. Our study focused on tumor-infiltrating lymphocytes (TILs), which are the main effectors of antitumor immunity and related to the mechanism of immunotherapy. Using an artificial intelligence model, we analyzed the density of intratumoral and peritumoral TILs in the pathologic slides of The Cancer Genome Atlas PTC cohort. Tumors were classified into three immune phenotypes (IPs) based on the spatial distribution of TILs: immune-desert (48%), immune-excluded (34%), and inflamed (18%). Immune-desert IP was mostly characterized by RAS mutations, high thyroid differentiation score, and low antitumor immune response. Immune-excluded IP predominantly consisted of BRAF V600E-mutated tumors and had a higher rate of lymph node metastasis. Inflamed IP was characterized by a high antitumor immune response, as demonstrated by a high cytolytic score, immune-related cell infiltrations, expression of immunomodulatory molecules (including immunotherapy target molecules), and enrichment of immune-related pathways. This study is the first to investigate IP classification using TILs in PTC through a tissue-based approach. Each IP had unique immune and genomic profiles. Further studies are warranted to assess the predictive value of IP classification in advanced PTC patients treated with immunotherapy.

Hypothermic Preservation of Adipose-Derived Mesenchymal Stromal Cells as a Viable Solution for the Storage and Distribution of Cell Therapy Products.

Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g., 2-8 °C) could be a feasible alternative. Adipose tissue-derived mesenchymal stromal cells (MSC(AT)) expanded using fetal bovine serum (FBS)- (MSC-FBS) or human platelet lysate (HPL)-supplemented mediums (MSC-HPL) were encapsulated in alginate beads for 30 min, 5 days, and 12 days. After bead release, cell recovery and viability were determined to assess encapsulation performance. MSC identity was verified by flow cytometry, and a set of assays was performed to evaluate functionality. MSC(AT) were able to survive encapsulated for a standard transportation period of 5 days, with recovery values of 56 ± 5% for MSC-FBS and 77 ± 6% for MSC-HPL (which is a negligible drop compared to earlier timepoints). Importantly, MSC function did not suffer from encapsulation, with recovered cells showing robust differentiation potential, expression of immunomodulatory molecules, and hematopoietic support capacity. MSC(AT) encapsulation was proven possible for a remarkable 12 day period. There is currently no solution to completely replace cryopreservation in CGT logistics and supply chain, although encapsulation has shown potential to act as a serious competitor.

Hypoxia preconditioning elicit differential response in tissue-specific MSCs via immunomodulation and exosomal secretion.

Mesenchymal stromal cells (MSCs) are emerging as an ideal candidate for regenerative medicine. It is known that the culture conditions impact the cellular properties of MSCs and their therapeutic behavior. Moreover, maintenance of MSCs in low oxygen tension for a short duration has shown to be beneficial for MSCs as it is similar to that of their physiological niche. However, the precise mechanism through which hypoxia pre-conditioning affects MSCs is not clear yet. Thus, in this study, we have investigated the effect of hypoxia exposure (1% O2) on tissue-specific MSCs over a period of time under serum-free culture conditions and evaluated the changes in expression of immuno-modulatory molecules and exosome biogenesis and secretion markers. It was observed that all MSCs responded differentially towards hypoxia exposure as indicated by the expression of HIF-1α. Moreover, this short-term exposure did not induce any changes in MSCs cellular morphology, proliferation rate, and surface marker profiling. In addition, we observed an enhancement in the expression of immunomodulatory factors (HLA-G, PGE-2, and IDO) after hypoxia exposure of 12 to 24h in all tissue-specific MSCs. Interestingly, we have also observed the upregulation in exosome secretion that was further corelated to the upregulation of expression of exosome biogenesis and secretion markers (ALIX, TSG101, RAB27a, RAB27b). Though there was a differential response of MSCs where WJ-MSCs and BM-MSCs showed upregulation of these markers at 6-12h of hypoxia pre-conditioning, while AD-MSCs showed similar changes beyond 24h of hypoxia exposure.

Immunosuppressive properties of human PD-1 + , PDL-1 + and CD80 + dendritic cells from lymph nodes aspirates of lung cancer patients.

Dendritic cells (DCs) play a pivotal role in the homeostasis of the immune system. The tumor microenvironment impairs the proper function of DCs. The immunomodulatory properties of DCs in lung cancer are of interest. In the present study, we analysed DCs subsets and immune cells with the expression of immunomodulatory molecules: PD-1 and PD-L1 and co-stimulatory molecule CD80 in metastatic, non-metastatic lymph nodes (LNs) and peripheral blood (PB). LNs aspirates were obtained during the EBUS/TBNA procedure of 29 patients with primary lung cancer. The cells were analyzed by flow cytometry. We reported a higher percentage of DCs in the metastatic than in the non-metastatic LNs and the PB (0.709% vs. 0.166% vs. 0.043%, p < 0.0001). The proportions of PD-1 + , PD-L1 + and CD80 + DCs were higher in the metastatic LNs than in the non-metastatic ones. A higher proportion of regulatory DCs (DCregs) was found in the metastatic ones than in the non-metastatic LNs (22.5% vs. 3.1%, p = 0.0189). We report that DCs cells show increased expression of PD-1, PD-L1 and CD80 molecules that can interact with T lymphocytes. It can be assumed that mature DCs infiltrating metastatic LNs can develop into DCregs, which are involved in the suppression of anti-tumor response.

Abstract PD3-08: Novel cancer stem cell inhibitor 108600 modulates tumor immunomicroenvironment of triple negative breast cancer (TNBC)

Abstract 108600, a novel CK2/DYRK1/TNIK inhibitor, targets and inhibits cancer stem cells (CSC) in triple negative breast cancer (TNBC), inhibiting tumor growth and metastases in patient-derived xenograft models. CSC’s have been shown to promote immune evasion of several types of cancer. Specifically, over-expression of CK2 has been shown to promote intratumoral recruitment of myeloid derived suppressor cells. We investigated the effects of 108600 treatment on the immune microenvironment of triple negative breast cancer since targeting CSC’s could promote favorable anti-tumor immune responses and mechanistically contribute to tumor growth inhibition. Methods:C57BL/6 mice bearing murine triple negative E0771 tumors were generated by orthotopic injection and treated either with vehicle control or 108600 for 5 days. Tumor growth was assessed by daily caliper measurement All tumors were recovered at endpoint, and processed for RNA sequencing, flow cytometry or Western blot analysis. Results:108600 treatment significantly inhibited growth of E0771 tumors in vivo, demonstrating for the first time efficacy of 108600 against TNBC in immunocompetent models. 108600 treatment decreased intratumoral phosphorylation and expression of 108600 targets AKT1 (Ser 129) and Cyclin D1, which are substrates of CK2α and Dyrk1, respectively. To further explore nature of transcriptional and signaling pathways affected by 108600 treatment in vivo, RNA sequencing was performed. DGE and subsequent annotation analysis showed that various immune (GO:0006955, GO) and inflammatory (GO:0006954, GO) signaling pathways associated with Stat1 (mmu04062, KEGG) and IFNγ (mmu04060, KEGG) were down-regulated in 108600-treated E0771 tumors. GSEA (Gene set enrichment analysis) indicated that the regulatory T cell (Treg) population (GSE42021, GSE40685) was suppressed by 108600 treatment. To validate these findings, tumor infiltrating leukocytes (TIL) were isolated from vehicle or 108600-treated tumors and analyzed by flow cytometry. CD4, CD25, and FOXP3 expression was used to gate the Treg population. The Treg population was significantly suppressed by 108600 treatment, confirming the RNA sequencing results. 108600 also likely regulates expression of immunomodulatory molecules in TNBC tumor cells since 108600 treatment increased PD-L1 surface expression on E0771 cells in vitro. Conclusions:Our study supports 108600, an inhibitor that targets breast cancer stem cells, as a modulator of the immune microenvironment of TNBC. 108600 suppresses the Treg population among the TIL population and increases tumoral expression of PD-L1. Tregs have been associated with disease progression and metastases of TNBC. Further studies are ongoing to clarify the functional consequences of these changes in the setting of tumor growth inhibition. Synergies between 108600 and checkpoint inhibition are also under investigation and could lead to novel combination therapies for TNBC. Citation Format: Katsutoshi Sato, Stacey J. Baker, E. P. Reddy, Hanna Y. Irie. Novel cancer stem cell inhibitor 108600 modulates tumor immunomicroenvironment of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-08.

Characterization of stable hypoxia-preconditioned dental pulp stem cells compared with mobilized dental pulp stem cells for application for pulp regenerative therapy

BackgroundDental pulp stem cells (DPSCs) have been developed as a potential source of mesenchymal stem cells (MSCs) for regeneration of dental pulp and other tissues. However, further strategies to isolate highly functional DPSCs beyond the colony-forming methods are required. We have demonstrated the safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. The device for isolation of MDPSCs, however, is not cost-effective and requires a prolonged cell culture period. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness. Therefore, in this investigation, we attempted to improve the clinical utility of DPSCs by hypoxia-preconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry.MethodsColony-forming DPSCs were isolated and preconditioned with hypoxia in a stable closed cultured system and compared with MDPSCs isolated from the individual dog teeth. We examined the proliferation rate, migration potential, anti-apoptotic activity, and gene expression of the stem cell markers and angiogenic/neurotrophic factors. Trophic effects of the conditioned medium (CM) were also evaluated. In addition, the expression of immunomodulatory molecules upon stimulation with IFN-γ was investigated. The pulp regenerative potential and transplantation safety of hpDPSCs were further assessed in pulpectomized teeth in dogs by histological and immunohistochemical analyses and by chemistry of the blood and urine tests.ResultshpDPSCs demonstrated higher proliferation rate and expression of a major regulator of oxygen homeostasis, HIF-1α, and a stem cell marker, CXCR-4. The direct migratory activity of hpDPSCs in response to G-CSF was significantly higher than MDPSCs. The CM of hpDPSCs stimulated neurite extension. However, there were no changes in angiogenic, migration, and anti-apoptotic activities compared with the CM of MDPSCs. The expression of immunomodulatory gene, PTGE was significantly upregulated by IFN gamma in hpDPSCs compared with MDPSCs. However, no difference in nitric oxide was observed. The regenerated pulp tissue was quantitatively and qualitatively similar in hpDPSC transplants compared with MDPSC transplants in dog teeth. There was no evidence of toxicity or adverse events of the hpDPSC transplantation.ConclusionsThese results demonstrated that the efficacy of hpDPSCs for pulp regeneration was identical, although hpDPSCs improved stem cell properties compared to MDPSCs, suggesting their potential clinical utility for pulp regeneration.

PD-L1+ dendritic cells in the tumor microenvironment correlate with good prognosis and CD8+ T cell infiltration in colon cancer.

BackgroundThe prognostic value of tumor‐associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c+ DC co–expressing the immunoinhibitory molecule PD‐L1 and their spatial relationship with CD8+ T‐cells in patients treated for stage III colon cancer.MethodsTissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD‐L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan‐Meier estimates and Cox regression were used to assess survival.ResultsIntratumoral CD8+ cell density (HR = .52, 95% confidence interval [CI] .33‐.83, P = .007), stromal CD11c+ cell density (HR = .52, 95% CI .33‐.83, P = .006), intratumoral CD11c+PD‐L1+ cell density (HR = .57, 95% CI .35‐.92, P = .021), and stromal CD11c+PD‐L1+ cell density (HR = .48, 95% CI .30‐.77, P = .003) on leading‐edge cores were all significantly associated with good survival. CD8+ cell density was positively correlated with both CD11c+ cell density and CD11c+PD‐L1+ cell density in tumor epithelium and stromal compartments.ConclusionHere we showed that PD‐L1‐expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically “hot” tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor‐associated DC may help to further elucidate their prognostic value.

Interacting Genetic Lesions of Melanoma in the Tumor Microenvironment: Defining a Viable Therapy.

Melanoma is the most aggressive form of skin cancer with an estimated 106,110 newly diagnosed cases in the United States of America in 2021 leading to an approximated 7180 melanoma-induced deaths. Cancer typically arises from an accumulation of somatic mutations and can be associated with mutagenic or carcinogenic exposure. A key characteristic of melanoma is the extensive somatic mutation rate of 16.8 mutations/Mb, which is largely attributed to UV exposure. Bearing the highest mutational load, many of them occur in key driver pathways, most commonly the BRAFV600E in the mitogen-activated protein kinase (MAPK) pathway. This driver mutation is targeted clinically with FDA-approved therapies using small molecule inhibitors of oncogenic BRAFV600E and MEK, which has greatly expanded therapeutic intervention following a melanoma diagnosis. Up until 2011, therapeutic options for metastatic melanoma were limited, and treatment typically fell under the spectrum of surgery, radiotherapy, and chemotherapy.Attributed to the extensive mutation rate, as well as having the highest number of neoepitopes, melanoma is deemed to be extremely immunogenic. However, despite this highly immunogenic nature, melanoma is notorious for inducing an immunosuppressive microenvironment which can be relieved by checkpoint inhibitor therapy. The two molecules currently approved clinically are ipilimumab and nivolumab, which target the molecules CTLA-4 and PD-1, respectively.A plethora of immunomodulatory molecules exist, many with redundant functions. Additionally, these molecules are expressed not only by immune cells but also by tumor cells within the tumor microenvironment. Tumor profiling of these cell surface checkpoint molecules is necessary to optimize a clinical response. The presence of immunomodulatory molecules in melanoma, using data from The Cancer Genome Atlas and validation of expression in two model systems, human melanoma tissues and patient-derived melanoma cells, revealed that the expression levels of B and T lymphocyte attenuator (BTLA), TIM1, and CD226, concurrently with the BRAFV600E mutation status, significantly dictated overall survival in melanoma patients. These molecules, along with herpesvirus entry mediator (HVEM) and CD160, two molecules that are a part of the HVEM/BTLA/CD160 axis, had a higher expression in human melanoma tissues when compared to normal skin melanocytes and have unique roles to play in T cell activation. New links are being uncovered between the expression of immunomodulatory molecules and the BRAFV600E genetic lesion in melanoma. Small molecule inhibitors of the MAPK pathway regulate the surface expression of this multifaceted molecule, making BTLA a promising target for immuno-oncology to be targeted in combination with small molecule inhibitors, potentially alleviating T regulatory cell activation and improving patient prognosis.

PIM Kinases Promote Survival and Immune Escape in Primary Mediastinal Large B-Cell Lymphoma through Modulation of JAK-STAT and NF-κB Activity

Primary mediastinal large B-cell lymphoma (PMBL) cells depend on the constitutive activity of NF-κB and STAT transcription factors, which drive expression of multiple molecules essential for their survival. In a molecularly related B-cell malignant tumor (classic Hodgkin lymphoma), tumor Reed-Sternberg cells overexpress oncogenic (proviral integration site for Moloney murine leukemia virus (PIM) 1, 2, and 3 kinases in a NF-κB- and STAT-dependent manner and PIMs enhance survival and expression of immunomodulatory molecules. Given the multiple overlapping characteristics of Reed-Sternberg and PMBL cells, we hypothesized that PIM kinases may be overexpressed in PMBL and involved in PMBL pathogenesis. The expression of PIM kinases in PMBL diagnostic biopsy specimens was assessed and their role in survival and immune escape of the tumor cells was determined. PIMs were abundantly expressed in primary tumors and PMBL cell lines. Inhibition of PIM kinases was toxic to PMBL cells, attenuated protein translation, and down-regulated NF-κB- and STAT-dependent transcription of prosurvival factors BCL2A1, BCL2L1, and FCER2. Furthermore, PIM inhibition decreased expression of molecules engaged in shaping the immunosuppressive microenvironment, including programmed death ligand 1/2 and chemokine (C-C motif) ligand 17. Taken together, our data indicate that PIMs support PMBL cell survival and immune escape and identify PIMs as promising therapeutic targets for PMBL.

Abstract PR17: Immunogenomic landscape of pediatric solid malignancies

Abstract Malignancy remains the leading cause of disease-related death in children. To identify potential tumor-driving molecular targets and immunogenomic profiles in pediatric cancers, we performed RNA-seq analysis on a cohort of 788 pediatric solid malignant tumors across 14 different diagnoses in conjunction with additional 147 normal tissues for comparison. Sequencing data were analyzed for expressed mutations, fusion events, and expressional patterns, providing therapeutic targets and rich cancer biology for these childhood cancers. Furthermore, we describe immunogenomic features of these solid tumors including immune cell infiltrate, neoantigen expression, expression of immunomodulatory molecules, and T-cell receptor repertoire. Across the cohort, we observed a striking correlation between the expressed neoantigen burden in tumors and enrichment of the effector immune signatures. Histology-specific immunogenomic patterns were also apparent. Several of the pediatric cancers such as alveolar soft part sarcoma and osteosarcoma exhibit rich immune cell infiltration and evidence for activated T-cell activities, whereas others such as Wilms’ tumors and synovial sarcoma generally have a very low T-cell infiltration. We demonstrate that RNA-seq is a powerful tool to identify clinically relevant and histology-specific recurrent mutations, novel oncogenic fusions, and translationally relevant immunogenomic patterns for pediatric cancers. This study also represents one of the largest of its type to date and provides a framework for future translational efforts in pediatric cancer. This abstract is also being presented as Poster A69. Citation Format: Jun S. Wei, Andrew S. Brohl, Sivasish Sindiri, Young K. Song, Sushma Najaraj, Vineela Gangalapudi, Xinyu Wen, Marc Ladanyi, Javed Khan. Immunogenomic landscape of pediatric solid malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR17.

Abstract A23: Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline

Abstract Sarcomas are rare malignancies of mesenchymal origin for which current treatment options are very limited. Due to the vast heterogeneity of the various subtypes, the development of new targeted therapies requires a customized approach. Checkpoint blockade therapies are a promising alternative that still have not been systematically investigated for sarcoma patients. Thus, utilizing the infrastructure of the Cell Therapy Core Facility (CTC) at Nova Southeastern University (NSU), we developed a clinic-to-bench pipeline that enables the processing of fresh tumor material after surgical excision to generate primary sarcoma cell lines for biobanking and research purposes. Using this pipeline, we have extensively characterized mRNA, miRNA and cell surface expression profiles from more than 40 sarcoma cell lines to date, as well as validated their tumorigenicity by assessing expression of sarcoma/cancer associated genes such as Birc5, Birc2, Bcl2, Ewsr1-fli1, Syt/Ssx and Pcna. Moreover, we assessed the expression of immunomodulatory molecules that may suppress antitumor responses, such as PD-L1, PD-L2, OX40L and CD40L, using multicolor flow cytometry. Detailed characterization of the immune profile of 7 bone-related and 7 soft tissue sarcoma cell lines, assessed after 12 weeks of in vitro serial passaging, revealed a common signature in the cell surface expression of proliferative marker PCNA, and lymphocyte ligands CD112 and CD115. Moreover, immunoprofiling of the PCNA/CD112/CD155+ sarcoma cell lines (n=14) demonstrated persistent expression of PD-L2 in all cell lines and the potential applications of currently clinically approved anti-PD-1 checkpoint inhibitors for sarcoma treatment. Importantly, the conserved expression of these proteins enabled the distinction of sarcoma tumor cells from other cells in the fresh primary tumor mix directly after isolation. Thus, we next performed a comparative analysis of the immune profile of 6 freshly isolated sarcoma tumors and their respective cell lines. Comparison of the surface expression profiles of the generated cell lines and the corresponding primary tumor cells showed that the initial phenotype was preserved after more than 12 weeks of expansion and after cryopreservation. This proves that the generated cell lines are an accurate tool that can be used for the development of novel personalized immunotherapies. Most importantly, our results enabled the design of a translational research pipeline that allows the systematic characterization of the immune profile of understudied sarcomas for the first time in order to gain insight into which patients may directly benefit from available immunotherapies and which may be eligible to enroll in clinical trials assessing the efficacy of checkpoint inhibitors. In summary, the generated sarcoma biobank serves as a unique and highly representative primary resource that can be utilized to identify potential candidates for novel targeted and personalized immunotherapies for the treatment of sarcomas. Citation Format: Anna-Maria Georgoudaki, Robin Krueger, Michelle Hartman, Tamara Chinn, Dustin Tran, Reneé Potens, Wendy Weston, H. Thomas Temple, Adil D. Duru. Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A23.

Abstract 3653: Immunogenomic landscape of pediatric solid malignancies

Abstract Malignancy remains the leading cause of disease-related death in children. To identify potential tumor-driving molecular targets and immunogenomic profiles in pediatric cancers, we performed RNA-seq analysis on a cohort of 792 pediatric solid malignant tumors across 14 different diagnoses in conjunction with additional 147 normal tissues for comparison. Sequencing data was analyzed for expressed mutations, fusion events, and expressional patterns, providing therapeutic targets and rich cancer biology for these childhood cancers. Furthermore, we describe immunogenomic features of the tumors including immune cell infiltrate, neoantigen expression, expression of immunomodulatory molecules, and T cell receptor repertoire. Across the cohort, we observed a striking correlation between the expressed neoantigen burden in tumors and enrichment of the effector immune signatures. Histology-specific immunogenomic patterns were also apparent. Several of the pediatric cancers such as alveolar soft part sarcoma and osteosarcoma exhibit rich immune cell infiltration and evidence for activated T cell activities, whereas others such as Ewing’s sarcoma and yolk sac tumors generally have a very low T cell infiltration. We demonstrate that RNA-seq is a powerful tool to identify clinically relevant and histology-specific recurrent mutations, novel oncogenic fusions, and translationally relevant immunogenomic patterns for pediatric cancers. This study also represents one of the largest of its type to date and provides a framework for future translational efforts in pediatric cancer. Citation Format: Jun S. Wei, Andrew S. Brohl, Young K. Song, Sushma Najaraj, Vineela Gangalapudi, Ashley Walton, Xinyu Wen, Marc Ladanyi, Javed Khan. Immunogenomic landscape of pediatric solid malignancies [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 3653.

146P - Expression of immunomodulatory molecule, programmed death ligand-1 in triple negative invasive breast cancer in the Indian population

146P - Expression of immunomodulatory molecule, programmed death ligand-1 in triple negative invasive breast cancer in the Indian population

Gut epithelial cell-derived exosomes trigger posttrauma immune dysfunction.

Exosomes are extracellular vesicles that act as endogenous mediators of the immune response. We have previously shown that exosomes released into mesenteric lymph (ML) following trauma (T)/hemorrhagic shock (HS) induce proinflammatory cytokine production in macrophages and are involved in the pathogenesis of postshock acute lung injury. However, the cellular origin of ML exosomes and their role in the posttrauma immune response remains unclear. We hypothesized that exosomes released from damaged-intestinal epithelial cells contribute to posttrauma immune dysfunction by altering the function of dendritic cells (DCs), key regulators of the adaptive immunity. Male rats underwent cannulation of the femoral artery, jugular vein and ML duct. T/HS was induced by laparotomy and 60 minutes of hemorrhagic shock followed by resuscitation. The ML was collected before (preshock) and after T/HS (post-T/HS) for isolation of exosomes. Surface epitopes of exosomes isolated from ML were assessed by flow cytometry to determine their cellular origin and phenotypic changes. The immunomodulatory effects of ML exosomes on DCs were assessed by Annexin V apoptosis assay, expression of costimulatory molecules, and antigen-presenting capacity to lymphocytes. Exosomes isolated from ML highly expressed CD63 (exosome marker) and epithelial cell-specific marker, suggesting their derivation from intestinal epithelial cells. The expression of immunomodulatory molecules, such as major histocompatibility complex class II and Fas ligand on ML exosomes, was significantly increased after T/HS. Coincubation of DCs with exosomes isolated from ML after T/HS increased DC apoptosis twofold compared with preshock ML exosomes. Furthermore, post-T/HS ML exosomes significantly suppressed lipopolysaccharide-mediated expression of CD80 and CD86 on DCs as well as decreased their antigen-presenting capacity to induce lymphocytes proliferation. Gut epithelial cells release immunomodulatory exosomes into the ML after T/HS and resuscitation. Mesenteric lymph exosomes may be critical mediators of posttraumatic immunosuppression causing depletion and dysfunction of DCs.

Abstract 3656: Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells

Abstract Melanoma is one of the deadliest forms of skin cancer, with a dim prognosis when metastasized, leading to the highest number of skin cancer related deaths. In recent years there has been a focus on the use of checkpoint inhibitor therapies like anti-CTLA-4, anti-PD-1 and anti-PD-L1 to treat melanoma. PD-L1, HVEM and VISTA expression on cancer cells has been shown to promote immune evasion and tumor survival. CTLA-4, when engaged on tumor cells leads to their apoptosis and LIGHT signaling leads to recruitment of T cells and effective tumor clearance. Thus, since these molecules do not function in isolation, we need to consider tissue pleiotropy and the expression of co-stimulatory and co-inhibitory molecules on tumor tissue. Our laboratory has characterized and established five primary patient derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM and GLM-2. In an effort to understand tumor tissue pleiotropy, we conducted a comprehensive expression pattern screening of eight co-inhibitory and ten co-stimulatory molecules by RT-PCR. Among others, VISTA, a CD4+ T cell suppressor, HVEM, an immune evasion regulator, LAG3 and TIM3, potential immune checkpoint targets, were seen to be differentially expressed in these primary cell lines. Current therapies target CTLA-4, PD-1 and PD-L1, which also exhibit varying expression in our primary cell lines, pointing to the importance of considering tissue expression of these molecules when administering these novel immunotherapies. Additionally, 80% of melanoma patients are positive for the BRAFV600E genetic lesion and are administered vemurafenib (PLX4032), an inhibitor of the overactive mutated BRAF. Our in vitro studies show that treatment with PLX4032, changed the expression of CTLA-4 and PD-L1 in these primary cells. We observed a decrease in PD-L1 protein expression in MEL-V and GLM-2 and an increase in PD-L1 protein expression in MEL-2 and KFM, the four BRAFV600E positive cell lines. CTLA-4 protein expression demonstrated an upward trend on treatment with PLX4032. This synergy observed between treatment with drugs targeting genetic lesions and the expression of immunomodulatory molecules warrants characterization of tumor biopsies prior to designing an effective combinatorial therapy regime. Our long term goal is to optimize combinatorial immune and drug therapies directed against both co-stimulators and checkpoint inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Tuli, Ghada Ben Rahoma, Sarnath Singh, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2017-3656

Abstract P3-16-01: Immune characterization of inflammatory breast cancer and correlation to pathological complete response

Abstract Background: Treatment of inflammatory breast cancer (IBC) includes neoadjuvant chemotherapy (NAC) followed by mastectomy and radiation. Responses are limited however with low pathological complete response (pCR) rates and poor survival. Recent RNA expression studies suggest that activated T cell signaling pathways and immunomodulatory markers such as PD-L1 are associated with a higher pCR rate in IBC; however comprehensive studies of tumor infiltrating lymphocytes (TIL) and protein expression of immunomodulatory molecules are lacking. There is a critical need therefore to study molecular and immune determinants of therapeutic response in IBC, with the goal of identifying biomarkers and actionable strategies to improve treatment outcomes. Methods: Baseline core biopsies from 36 IBC patients, 22 with stage III and 14 with stage IV disease were evaluated. Of these, 21 stage III and 10 stage IV patients underwent mastectomy following NAC, the latter for palliative purposes. Tumor subtype distribution was 14 patients with HER2-/HR-, 6 with HER2+/HR-, 11 with HER2-/HR+, and 5 with HER2+/HR+ disease. TIL infiltration in the tumor stroma was quantified on H&amp;E slides based on consensus guidelines as well as by immunohistochemistry (IHC) staining for CD8. PD-L1 expression in the TIL and invasive tumor was evaluated by IHC in tumors with &amp;gt;1%TIL. Results: Stromal TIL were found in the invasive tumor on pretreatment biopsies in 26 (72%) patients, with TIL percentages ranging from 1% to 60% (mean=11.6; sd=13.8); of note, 1 patient biopsy sample only had tumor emboli on the tissue block and therefore was not evaluable. Higher TIL infiltrate was noted in stage III versus stage IV disease (mean TIL 11.6% versus 3.5%, p=0.028). Mean TIL infiltrate was 11.5% in HER2-/HR-, 10.0% in HER2+/HR-, 10.4% in HER2+/HR+, and 3.6% in HER2-/HR+ tumors (p=NS). At mastectomy, 7/21 stage III patients and 1/10 stage IV patients achieved a pCR. Mean TIL was 13.4% in the pCR group versus 8.2% in the non-pCR group (p=0.37) CD8 and PD-L1 staining was performed on samples with &amp;gt;1%TIL (n=15, of which 14 samples were available for additional staining). An average of 42% of TIL stained positive for CD8 (range 10-80%). There was no significant relationship between %CD8 and pCR, stage, or receptor status. None of these 14 patients demonstrated membranous PD-L1 positivity but all had focal weak cytoplasmic staining in the lymphocytes. Conclusions: Differences exist in the presence of stromal TIL in distinct groups within IBC (stage III versus stage IV disease and across histologic subtypes) and may contribute to differential responses to therapy. When comparing these results to published non-IBC literature (FinHER trial), our IBC patient cohort had lower TIL infiltrate in several histologic subtypes (HER2-/HR- 11.5% vs 25%, p=0.015), HER2+/HR-(10% vs 20%, p=0.10), and HER-/HR+ disease (3.6 vs 7.5%, p=0.01); TIL was comparable for HER2+/HR+ disease. Additional studies are underway (including multiplex analysis of myeloid and lymphoid markers, T cell receptor sequencing, and molecular profiling) in pre-treatment and surgical samples to better understand mechanisms of treatment response and resistance. Citation Format: Reddy SM, Wargo JA, Reuben A, Reuben J, Woodward W, Ueno N, Mittendorf EA, Krishnamurthy S. Immune characterization of inflammatory breast cancer and correlation to pathological complete response [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-16-01.

Abstract 4485: Regulation of chronic lymphocytic leukemia (CLL) immunobiology by histone deacetylase 6 (HDAC6)

Abstract In CLL, an immunosuppressive phenotype enables the malignant B cell to evade immune detection leading to immune suppression. In recent years, epigenetic changes prompted by proteins known as histone deacetylases (HDACs) have gained special attention predominantly because of their active role in the regulation of pathogenesis and immune-related pathways in CLL; though the precise mechanism by which these regulatory events take place has yet to be elucidated. In addition to the well-recognized role of histone deacetylase inhibitors (HDACi) in the control of cell cycle and apoptosis, HDACi have a potential role in modulating the immunobiology of CLL. Remarkably, HDACi alter the inflammatory status of immune cells and tumor cells themselves. Among the family of HDACs, we have recently demonstrated that HDAC6 has a regulatory role in transcriptional regulation of IL-10, and is responsible for T cell anergy in murine and human antigen presenting cells. Activation of T-cells is predominantly dependent on both co-stimulatory and co-inhibitory molecules, including PD-1/PD-L1 as well as CTLA4-B7, OX40-OX40L, CD40-CD154, GAL9-TIM3, and 41BBL/41BB. Furthermore, most of these molecules have been identified to have epigenetically induced changes in expression. Previously we had reported that expression of HDAC6 is increased in CLL patient samples. Recent studies from our lab show that selective HDAC6 inhibitors modify the expression of immunomodulatory molecules, which may ultimately lead to increases in the immunogenicity of CLL. Thus far, findings from our lab reveal that CLL cells treated with HDAC6i show 1) a dose dependent cell kill, 2) a reduction of IL-10—an important cytokine in the regulation of cell proliferation in CLL, and 3) synergistic reduction of viability when combined with the BTK inhibitor ibrutinib. Subsequently, we have also demonstrated that MEC2-HDAC6KD cells exhibit an increase in MHCII and a decrease in PD-L1 expression. Interestingly, we also observed a decrease in the expression of PD-L1 and other immune checkpoint markers in CLL cell lines treated with low-doses of HDAC6i. Additionally, malignant B cells isolated from euTCL1 mice and treated ex vivo with HDAC6i become more immunogenic and elicit greater type I allogeneic T cell immune response. Lastly, utilizing our in vivo CLL murine models (euTCL1, and euTCL1-HDAC6KO), we have been able to demonstrate a reduction in circulating lymphocytes in euTCL1-HDAC6KO, as well as euTCL1 mice receiving systemic administration of HDAC6i. In conclusion, selective inhibition of HDAC6 in CLL results in the reduction of co-inhibitory molecules, dose dependent increases in cell killing as single treatment as well as strong synergy when combined with ibrutinib. These findings could provide a successful combination immunotherapeutic strategy for the treatment of CLL. Citation Format: Eva Sahakian, Kamira Maharaj, John Powers, Renee M. Fonesca, Susan Deng, Javier Pinilla-Ibraz, Steven N. Quayle, Simon S. Jones. Regulation of chronic lymphocytic leukemia (CLL) immunobiology by histone deacetylase 6 (HDAC6). [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 4485.

Abstract SY26-03: Immune effects of targeted therapy and implications for combination strategies

Abstract SY26-03: Immune effects of targeted therapy and implications for combination strategies

Abstract IA31: Combining targeted therapy and immune checkpoint blockade to improve responses

Abstract There have been significant advances in treatment of metastatic melanoma over the past several years through the use of targeted therapy and immune checkpoint blockade. Oncogenic mutations in the BRAF gene occur in over half of melanoma tumors, and treatment with agents targeting this mutation results in objective responses in the majority of patients. However significant limitations exist as responses are not durable, with the majority of patients progressing on therapy within 6-10 months. Another area of significant progress involves the use of immune checkpoint blockade, such as with monoclonal antibodies blocking CTLA-4 and the PD-1 pathway. In contrast, treatment of patients with immune checkpoint inhibitors results in a lower response rate than those observed with targeted therapy, but ones that tend to be more durable. With these advances, we are posed with therapeutic dilemmas with regard to timing and sequence of therapy. Namely, there is significant debate as to whether to begin treatment with targeted therapy versus immune checkpoint blockade upfront, and at which point to change treatment strategy. There is growing evidence of potential synergy in combining treatment with BRAF-targeted therapy and immune checkpoint blockade, and this concept is being empirically tested in clinical trials. There a strong scientific rationale for combining these therapies, as treatment with BRAF inhibitors is associated with increased immunogenicity of melanoma tumors within 2 weeks of therapy - with increased melanoma antigens and CD8+ T cell infiltrate, and decreased immunosuppressive cytokines and VEGF. BRAF inhibitors may also directly activate T cells through paradoxical activation of the MAPK pathway. However there is a concurrent increase in expression of immunomodulatory molecules (such as PD-L1) within 2 weeks of therapy which is likely related to T cell infiltrate, though also suggest a possible immune mechanism of resistance. Clinical trials and studies in murine models are currently underway to explore synergy between these therapies, and insights gained will be discussed herein. It is becoming increasingly apparent that complexities exist regarding appropriate timing and sequence in these combination strategies to optimize responses and minimize toxicity. Ultimately, ideal approaches using combined BRAF-targeted therapy and immune checkpoint blockade will be built on a deep understanding of the molecular and immune effects of each of these therapies in isolation, as well as in combination. Citation Format: Jennifer A. Wargo. Combining targeted therapy and immune checkpoint blockade to improve responses. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA31.