Target Immune Cells Research Articles

Adipose Tissue-Resident Immune Cells in Obesity and Type 2 Diabetes.

Inflammation is an important contributor to the pathogenesis of obesity-related type 2 diabetes (T2D). Adipose tissue-resident immune cells have been observed, and the potential contribution of these cells to metabolic dysfunction has been appreciated in recent years. This review focused on adipose tissue-resident immune cells that are dysregulated in the context of obesity and T2D. We comprehensively overviewed emerging knowledge regarding the phenotypic and functional properties of these cells and local factors that control their development. We discussed their function in controlling the immune response cascade and disease progression. We also characterized the metabolic profiles of these cells to explain the functional consequences in obese adipose tissues. Finally, we discussed the potential therapeutic targeting of adipose tissue-resident immune cells with the aim of addressing novel therapeutic approaches for the treatment of this disease.

The Orai Ca2+ channel inhibitor CM4620 targets both parenchymal and immune cells to reduce inflammation in experimental acute pancreatitis.

This work confirms previous reports that CM4620, a small molecule inhibitor of Ca2+ entry via store operated Ca2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells. Key features of acute pancreatitis include excess cellular Ca2+ entry driven by Ca2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology.

Material design for lymph node drug delivery.

A significant fraction of the total immune cells in the body are located in several hundred lymph nodes, in which lymphocyte accumulation, activation and proliferation are organized. Therefore, targeting lymph nodes provides the possibility to directly deliver drugs to lymphocytes and lymph node-resident cells and thus to modify the adaptive immune response. However, owing to the structure and anatomy of lymph nodes, as well as the distinct localization and migration of the different cell types within the lymph node, it is difficult to access specific cell populations by delivering free drugs. Materials can be used as instructive delivery vehicles to achieve accumulation of drugs in the lymph nodes and to target specific lymph node-resident cell subtypes. In this Review, we describe the compartmental architecture of lymph nodes and the cell and fluid transport mechanisms to and from lymph nodes. We discuss the different entry routes into lymph nodes and how they can be explored for drug delivery, including the lymphatics, blood capillaries, high endothelial venules, cell-mediated pathways, homing of circulating lymphocytes and direct lymph node injection. We examine different nanoscale and microscale materials for the targeting of specific immune cells and highlight their potential for the treatment of immune dysfunction and for cancer immunotherapy. Finally, we give an outlook to the field, exploring how lymph node targeting can be improved by the use of materials.

Cancer nanomedicine for combination cancer immunotherapy

Cancer immunotherapy is revolutionizing oncology. However, dose-limiting toxicities and low patient response rates remain major challenges in the clinic. Cancer nanomedicine in combination with immunotherapies offers the possibility to amplify antitumour immune responses and to sensitize tumours to immunotherapies in a safe and effective manner. In this Review, we discuss opportunities for combination immunotherapy based on nanoparticle platforms designed for chemotherapy, photothermal therapy, photodynamic therapy, radiotherapy and gene therapy. We highlight how nanoparticles can be used to reprogramme the immunosuppressive tumour microenvironment and to trigger systemic antitumour immunity, synergizing with immunotherapies against advanced cancer. Finally, we discuss strategies to improve tumour and immune cell targeting while minimizing toxicity and immune-related adverse events, and we explore the potential of theranostic nanoparticles for combination immunotherapy. Cancer nanomedicine in combination with immunotherapies offers the possibility to amplify antitumour immune responses and to sensitize tumours to immunotherapies. In this Review, the authors discuss combination immunotherapy based on nanoparticle platforms designed for chemotherapy, photothermal therapy, photodynamic therapy, radiotherapy and gene therapy.

Harnessing immune checkpoints in myeloid lineage cells for cancer immunotherapy.

Myeloid lineage immune cells, such as macrophages and dendritic cells, play important roles in the induction of antitumor immunity during the initial stage of the cancer-immunity cycle, eliciting antitumor adaptive immunity by phagocytosing cancer cells and processing cancer-specific antigens, and then presenting these antigens to T cells. During this process, cancer cell phagocytosis can be prevented by inhibitory signals, and the signaling cascades that elicit immune responses against cancer antigens can be inhibited by immunosuppressive myeloid cells in the tumor microenvironment. A number of therapeutic strategies for enhancing cancer cell phagocytosis and promoting antitumor immunity by targeting myeloid lineage cells have recently been developed. Here, we discuss recent advances in cancer immunotherapy that involve the targeting of myeloid lineage immune cells to induce effective antitumor immunity.

Safety and Tolerability of Immune Checkpoint Inhibitors (PD-1 and PD-L1) in Cancer.

Immunotherapy has emerged in recent years and has revolutionized the treatment of cancer. Immune checkpoint inhibitors, including anti-cytotoxic T lymphocyte antigen-4 (CTLA-4), anti-programmed cell death-1 (PD-1) and anti-programmed cell death ligand-1 (PD-L1) agents, are the first of this new generation of treatments. Anti-PD-1/PD-L1 agents target immune cells by blocking the PD-1/PD-L1 pathway. This blockade leads to enhancement of the immune system and therefore restores the tumour-induced immune deficiency selectively in the tumour microenvironment. However, this shift in the balance of the immune system can also produce adverse effects that involve multiple organs. The pattern of toxicity is different from traditional chemotherapy agents or targeted therapy, and there is still little experience in recognizing and managing it. Thus, toxicity constitutes a real clinical management challenge and any new alteration should be suspected of being treatment-related. The most common toxicities occur in the skin, gastrointestinal tract, lungs, and endocrine, musculoskeletal, renal, nervous, haematologic, cardiovascular and ocular systems. Immune-mediated toxic effects are usually manageable, but toxicities may sometimes lead to treatment withdrawal, and even fulminant and fatal events can occur. Oncologists need to collaborate with internists, clinical immunologists and other specialists to understand, manage and prevent toxicity derived from immunotherapy. This review focuses on the mechanisms of toxicity of anti-PD-1/PD-L1 agents, and its diagnosis and management.

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice.

To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

Characterization of Early Indicators of Cardiac Allograft Vasculopathy Lesions in a Rat Model Using Non-Invasive Cellular MR

Background: Cardiac allograft vasculopathy (CAV) remains a major obstacle to long-term heart allograft survival. A number of studies show that immune mechanisms involved in CAV. Using non-invasive cellular MRI (CMRI) to explore indicators of CAV lesions and characterize its development could provide new insights into the target immune cells that are responsible for the progression of CAV and reveal early markers of the disease before irreversible changes occur. Methods and Results: Rat heart transplant allografts (n = 88) and isografts (n = 22) were employed in this study. CMRI at 4.7-Tesla revealed a few hypointense foci in the graft early on during CAV with more regions becoming involved as the disease progressed. The areas with hypointensity corresponded with infiltrating ED1+ cells labeled with micrometer-sized superparamagnetic iron oxide (MPIO), confirmed by MR microscopy (MRM) at 11.7-Tesla and pathology. MRI abnormalities of the transplanted hearts correlate well with the histopathological findings. MPIO-labeled cells counted by a computer algorithm that analyzed 3D MRM volumes exhibited a strong correlation with those counted by immunohistochemical inspection of ED1+ cell infiltration (R2 = 0.8075). Histology found that in the early phases of CAV, lesions were focal and mostly began in the adventitia. Immunohistochemistry indicated that the infiltrates were mostly ED1+ cells and that their density was significantly correlated with the severity of CAV (p < 0.05). Conclusions: This study illustrates the feasibility of monitoring MPIO-labeled ED1+ cells as an early indicator for CAV before vessel wall changes using non-invasive CMRI, which provides information on the entire heart.

A cellular automata model of chemotherapy effects on tumour growth: targeting cancer and immune cells

ABSTRACT The effects of therapy on avascular cancer development based on a stochastic cellular automata model are considered. Making the model more compatible with the biology of cancer, the following features are implemented: intrinsic resistance of cancerous cells along with drug-induced resistance, drug-sensitive cells, immune system. Results are reported for no treatment, discontinued treatment after only one cycle of chemotherapy, and periodic drug administration therapy modes. Growth fraction, necrotic fraction, and tumour volume are used as output parameters beside a 2-D graphical growth presentation. Periodic drug administration is more effective to inhibit the growth of tumours. The model has been validated by the verification of the simulation results using in vivo literature data. Considering immune cells makes the model more compatible with the biological realities. Beside targeting cancer cells, the model can also simulate the activation of the immune system to fight against cancer. Abbreviations CA: cellular automata; DSC: drug sensitive cell; DRC: drug resistant cell; GF: growth fraction; NF: necrotic fraction; ODE: ordinary differential equation; PDE: partial differential equation; SCAM: The proposed stochastic cellular automata model

Cytokine responses in pigs after natural infection with classical swine fever virus.

Classical swine fever (CSF) is a highly contagious viral infection that affects domestic and wild pig population. The classical swine fever virus (CSFV) targets immune cells which perturb the immune functions causing immunopathological disorders such as immunosuppression, leukopenia and haemorrhage. In the present study, ELISA and quantitative real-time reverse transcription PCR (qRT-PCR) analysis was employed to determine cytokine profiles in pigs naturally infected with CSFV using whole blood assay (WBA) under field conditions. Significantly higher TNF-α, IL-10, and IL-6 expression levels were found in unvaccinated pigs infected with CSFV (group B) compared to vaccinated pigs that recovered after CSF (group A), the difference being statistically significant (p=0.001). However, the expression of IFN-γ was significantly higher in group A compared to group B (p=0.001). The findings of this field-supported study will help us to understand the immune biology of CSFV infection in infected pigs. The WBA technique can be used as a reliable, fast and feasible in vitro method to assess porcine cellular immune responses as it imitates the porcine blood conditions. Such studies could be of some value in determining the immune status of the ailing animals infected with CSFV. Keywords: classical swine fever virus; immune response; field conditions; interleukin; IFN-γ; TNF-α.

Pattern Recognition Receptors and Liver Failure.

Liver failure is a clinical syndrome with many causes, a complicated pathogenesis, diverse clinical manifestations, and very high mortality. No effective treatment is yet available. Main pathological processes of liver failure include direct damage to parenchymal and nonparenchymal liver cells that might be caused by viruses or drugs, immune-mediated indirect damage, inflammation, and ischemia-hypoxia injury that further strengthen liver damage and lead to endotoxemia. Among these causes, viral or bacterial components (called pathogen-associated and damage-associated molecular patterns) are released during tissue damage and cell death and may be recognized by pattern recognition receptors (PRRs) to induce secretion of inflammatory cytokines and chemokines and activate immune cells. This process is an important mechanism that underlies the progression of liver failure. Research concerning the roles of PRR signaling pathways in liver failure is expected to result in development of immunomodulatory drugs to target specific disease stages, immune cells, and signal transduction molecules. This article briefly introduces the research status of six main PRRs (Toll-like receptors, nucleotide-binding oligomerization domain-like receptors, retinoic-acid-inducible gene I-like receptors, cytosolic DNA sensors, C-type lectin receptors, and inflammasomes) in acute liver failure and acute-on-chronic liver failure and explores further research directions.

MiR-34a is a microRNA safeguard for Citrobacter-induced inflammatory colon oncogenesis.

Inflammation often induces regeneration to repair the tissue damage. However, chronic inflammation can transform temporary hyperplasia into a fertile ground for tumorigenesis. Here, we demonstrate that the microRNA miR-34a acts as a central safeguard to protect the inflammatory stem cell niche and reparative regeneration. Although playing little role in regular homeostasis, miR-34a deficiency leads to colon tumorigenesis after Citrobacter rodentium infection. miR-34a targets both immune and epithelial cells to restrain inflammation-induced stem cell proliferation. miR-34a targets Interleukin six receptor (IL-6R) and Interleukin 23 receptor (IL-23R) to suppress T helper 17 (Th17) cell differentiation and expansion, targets chemokine CCL22 to hinder Th17 cell recruitment to the colon epithelium, and targets an orphan receptor Interleukin 17 receptor D (IL-17RD) to inhibit IL-17-induced stem cell proliferation. Our study highlights the importance of microRNAs in protecting the stem cell niche during inflammation despite their lack of function in regular tissue homeostasis.

Glia-immune interactions post-ischemic stroke and potential therapies.

This article reviews glial cell interactions with the immune system post-ischemic stroke. Research has shown that glial cells in the brain play a role in altering phenotypes of other glial cells and have downstream immune cell targets ultimately regulating a neuroinflammatory response. These interactions may play a deleterious as well as beneficial role in stroke recovery. Furthermore, they may provide a novel way to approach potential therapies, since current stroke drug therapy is limited to only one Food and Drug Administration-approved drug complicated by a narrow therapeutic window. Until this point, most research has emphasized neuroimmune interactions, but little focus has been on bidirectional communication of glial-immune interactions in the ischemic brain. By expanding our understanding of these interactions through a compilation of glial cell effects, we may be able to pinpoint major modulating factors in brain homeostasis to maintain or discover ways to suppress irreversible ischemic damage and improve brain repair.

Knockout Tumor Microenvironment HO-1 Neutralizes Myeloid-Derived Suppressor Cells and Enhances the Antitumor Effect of PD-1 Inhibition in Murine Models of Acute Myeloid Leukemia

Background/Aims: Myeloid-derived suppressor cells (MDSCs) has been shown to be involved in tumor immune escape mechanisms and B cell-specific immunity in Acute myeloid leukemia (AML) patients. In our previous study shown that HO-1 protein have immunomodulatory activity including targeting of immune suppressor cells in the tumor microenvironment. Thus, we decided to assess whether HO-1 could enhance anti-PD-1 treatment and investigate those alterations in the immunosuppressive tumor microenvironment that contribute to the combined antitumor activity.Method: We utilized C57/BL6 (HO-1 knockout) mouse were radiation with 6.5GY once in a row to repress residual immunity. Malignant tumor HL-60 cells respectively (1×107 cells) per animal were injected subcutaneously into the right abdomen. The xenograft mouse models of AML were euthanised on the 14th day after treatment with a PD-1 inhibitor once a day (20 mg/kg). The tumor volumes were measured and calculated by ruler. Survival curve of individual groups was evaluated from the first day of treatment until death using Kaplan-Meier curves.Results: HO-1 gene knockout enhanced the antitumor effect of PD-1 inhibition in xenograft mouse models of AML by reducing tumor growth and increasing survival. HO-1 gene knockout inhibited the immunosuppressive function of both polymorphonuclear (PMN)- and monocytic-myeloid derived suppressor cell (M-MDSC) populations. Analysis of MDSC response to HO-1 gene knockout revealed significantly reduced arginase-1, iNOS, and COX-2 levels, suggesting potential mechanisms for the altered function. We also observed significant alterations in cytokine/chemokine release in vivo with a shift toward a tumor-suppressive microenvironment.Conclusions: Our results demonstrate that HO-1 gene knockout enhances the antitumor effect of PD-1 targeting through functional inhibition of MDSCs and a transition away from an immune-suppressive tumor microenvironment.[Key words] HO-1 gene knockout; PD-1 inhibitor; tumor microenvironment; MDSCs; AML DisclosuresNo relevant conflicts of interest to declare.

Pre-clinical development of Listeria-based nanovaccines as immunotherapies for solid tumours: insights from melanoma

ABSTRACTGold glyconanoparticles loaded with the listeriolysin O peptide 91–99 (GNP-LLO91-99), a bacterial peptide with anti-metastatic properties, are vaccine delivery platforms facilitating immune cell targeting and increasing antigen loading. Here, we present proof of concept analyses for the consideration of GNP-LLO91-99 nanovaccines as a novel immunotherapy for cutaneous melanoma. Studies using mouse models of subcutaneous melanoma indicated that GNP-LLO91-99 nanovaccines recruite and modulate dendritic cell (DC) function within the tumour, alter tumour immunotolerance inducing melanoma-specific cytotoxic T cells, cause complete remission and improve survival. GNP-LLO91-99 nanovaccines showed superior tumour regression and survival benefits, when combined with anti-PD-1 or anti-CTLA-4 checkpoint inhibitors, resulting in an improvement in the efficacy of these immunotherapies. Studies on monocyte-derived DCs from patients with stage IA, IB or IIIB melanoma confirmed the ability of GNP-LLO91-99 nanovaccines to complement the action of checkpoint inhibitors, by not only reducing the expression of cell-death markers on DCs, but also potentiating DC antigen-presentation. We propose that GNP-LLO91-99 nanovaccines function as immune stimulators and immune effectors and serve as safe cancer therapies, alone or in combination with other immunotherapies.

Nanoparticle Ligand-Decoration Procedures Affect in Vivo Interactions with Immune Cells.

Ligand-decorated nanoparticles are extensively studied and applied for in vivo drug delivery and molecular imaging. Generally, two different ligand-decoration procedures are utilized; ligands are either conjugated with nanoparticle ingredients and incorporated during nanoparticle preparation, or they are attached to preformed nanoparticles by utilizing functionalized reactive surface groups (e.g., maleimide). Although the two procedures result in nanoparticles with very similar physicochemical properties, formulations obtained through the latter manufacturing process typically contain nonconjugated reactive surface groups. In the current study, we hypothesized that the different ligand-decoration procedures might affect the extent of interaction between nanoparticles and immune cells (especially phagocytes). In order to investigate our hypothesis, we decorated lipidic nanoparticles with a widely used cyclic Arg-Gly-Asp (cRGD) peptide using the two different procedures. As proven from in vivo experiments in mice, the presence of nonconjugated surface moieties results in increased recognition by the immune system. This is important knowledge considering the emerging focus on understanding and optimizing ways to target and track immune cells and the development of nanomedicine-based strategies in the field of immunotherapy.

Nanoimmunotherapy to treat ischaemic heart disease.

Atherosclerosis is a chronic disease of the large arteries and the underlying cause of myocardial infarction and stroke. Atherosclerosis is driven by cholesterol accumulation and subsequent inflammation in the vessel wall. Despite the clinical successes of lipid-lowering treatments, atherosclerosis remains one of the major threats to human health worldwide. Over the past 20 years, insights into cardiovascular immunopathology have provided a plethora of new potential therapeutic targets to reduce the risk of atherosclerosis and have shifted the therapeutic focus from lipids to inflammation. In 2017, the CANTOS trial demonstrated for the first time the beneficial effects of targeting inflammation to treat cardiovascular disease by showing that IL-1β inhibition can reduce the recurrence rate of cardiovascular events in a large cohort of patients. At the same time, preclinical studies have highlighted nanotechnology approaches that facilitate the specific targeting of innate immune cells, which could potentially generate more effective immunomodulatory treatments to induce disease regression and prevent the recurrence of cardiovascular events. The clinical translation of such nanoimmunotherapies and their application to treat patients with ischaemic heart disease are challenges that lie ahead.

Targeting immune cells to ease pain

When looking for new ways to treat chronic pain, researchers and doctors want drugs that have less addiction potential than opioids do. Novartis is currently testing a compound called EMA401 in Pha...

Abstract 3775: Use of RLI-15 a clinical grade fusion protein with IL-15 superagonistic activity for the activation of anti-tumor immune response

Abstract RLI-15, a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain represents a promising candidate for the induction of anti-tumor immunity. RLI-15 was designed to bypass the need of endogenous IL-15Rα, thereby leveraging the activity of IL-15 in vivo on target immune cells. RLI-15 stimulates the proliferation and the cytotoxic activity of natural killer (NK) cells and memory CD8+ T cells with no significant expansion and activation of regulatory T cell compartment. RLI-15 was previously shown to exhibit a potent anti-metastatic activity in B16F10 melanoma and Renca renal cell carcinoma mouse models. RLI-15 also significantly delayed tumor growth and prolonged survival when combined with anti-PD1 therapy in CT26 and MC38 colon carcinoma models. Here, we report that the combination treatment with clinical-grade RLI-15 and an anti-PD1 antibody leads to a significant anti-tumor efficacy in a TRAMP-C2 prostate cancer mouse model with 70 % of mice remaining tumor free after the treatment. We evaluated the optimal schedule of such combination therapy to set the basis for the design of upcoming clinical trials. We further tested how the administration schedule affects the pharmacodynamics properties of clinical-grade RLI-15 and translates into the anti-tumor efficacy in metastatic Renca and CT26 mouse models. In cynomolgous monkeys, various schedules of administration of RLI-15 showed a dose-dependent expansion of peripheral blood lymphocytes, predominantly of NK cell and memory CD8+ T cell compartments. The toxicity in mice and cynomolgous monkeys was evaluated to determine the maximal tolerated dose of RLI-15. Furthermore, the activity of clinical-grade RLI-15 was tested in vitro on human PBMCs and the superiority over IL-2 and IL-15 stimulatory capacity has been confirmed. The complex analysis of RLI-15 behavior and of the induced anti-tumor immune response will be explored in the design of a planned Phase I clinical study in patients with both solid tumors and hematological malignancies. Citation Format: Irena Adkins, Lenka Sadilkova, Nada Hradilova, Jakub Tomala, Barbora Tomalova, Marek Kovar, Romana Mikyskova, Milan Reinis, Guy de Martynoff, David Bechard, Ulrich Moebius, Radek Spisek. Use of RLI-15 a clinical grade fusion protein with IL-15 superagonistic activity for the activation of anti-tumor immune response [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 3775.

Abstract 1600: Expanded multiplexing capability in brightfield immunohistochemistry utilizing multiple chromogen chemistries and detection enzymes

Abstract Introduction Immunohistochemistry (IHC) is a valuable means to examine the distribution of protein expression at the subcellular level throughout human tissues while retaining morphology of the cells and tissue. Given the large and growing number of established protein biomarkers and the often very limited amount of tissue provided by tumor biopsy, the ability to evaluate multiple biomarkers on a single-slide mounted specimen via multiplexed IHC is of growing importance. However, due to the limited number of conventional chromogens, their broad absorbance spectra, and deposition chemistry based on only two enzymes, brightfield multiplexing has been typically limited to only two protein targets. Methods To expand multiplexing capability, our project objective was to reduce the antibody incubation, detection, and enzyme deactivation steps to produce high-level multiplexed chromogenic assays with short run times and minimal exposure to potentially destructive enzyme deactivation reagents. We utilized unmodified and tagged primary antibodies with secondary anti-primary and anti-tag antibodies conjugated to several enzymes employing orthogonal enzymatic detection systems. Peroxidase-catalyzed tyramide signal amplification (TSA) and quinone methide signal amplification (QMSA) chemistry adapted to two different hydrolase enzymes provided several compatible chromogens with relatively narrow absorbance bands and good spectral separation. All assays were fully automated and performed on the Benchmark XT platform. Results Multiplex IHC staining was demonstrated using combinations of TSA and QMSA, with the former catalyzed by horseradish peroxidase (HRP) and the latter established to work successfully with two different hydrolase enzymes: alkaline phosphatase (AP) and neuraminidase. Three- and four-chromogen multiplex assays were demonstrated on a variety of targets including breast cancer (Her2, ER, PR, Ki-67) and immune cell targets (CD3, CD8, cytokeratin, FOXP3, PD-L1) on a variety of tumor and tonsil tissues. Use of both tagged and untagged primary antibodies and up to three enzymes allow reduction of antibody removal and enzyme deactivation steps, significantly reducing assay time. Quantitative comparisons of multiplex staining to ‘gold standard' DAB staining on serial sections verified the multiplex assay performance. Conclusion We have expanded on the current HRP and AP brightfield IHC detection technologies by utilizing TSA- and QMSA-based chromogens and introducing the new neuraminidase detection chemistry. These detection chemistries combined with tagged and untagged primary antibodies provided up to four-chromogen brightfield multiplex IHC with verified assay performance. Citation Format: Esteban Roberts, William Day, Brian D. Kelly, Nathan W. Polaske, Julia Ashworth-Sharpe, Donald Johnson, Mark Lefever, Jerry Kosmeder, Hongjun Zhang, Jian Zhang, Tsu-Shuen Tsao, Mike Farrell, Joerg Bredno, Robert Ochs, Larry Morrison. Expanded multiplexing capability in brightfield immunohistochemistry utilizing multiple chromogen chemistries and detection enzymes [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 1600.