Changes In Immune Cell Populations Research Articles

Determining the mechanism of the immune response to histotripsy ablation of pancreatic cancer

Abstract Histotripsy is a novel, non-thermal, image guided ablation modality that can rapidly kill cells in a targeted region with millimeter precision. Previous work established that there is an immune response to histotripsy ablation, however a mechanism behind this response has not been established. For the current study, we utilized an in vitro model, Pan02 (pancreatic adenocarcinoma), transfected with a known antigen (hemaglutanin/HA), and verified findings in vivo. Cells were ablated at therapeutic and sub-therapeutic doses to determine the release of damage associated molecular pattern (DAMP). For both treatments, gel electrophoresis confirmed the release of damaged DNA, and western blots verified the release of intact HMGB1 and HA proteins. To determine the efficacy of the released DNA to stimulate an immune response, we exposed naïve macrophages to isolated DNA from the treatment supernatant and showed activation by 24 hours. Additionally, to show the potential for a systemic immune response, complete supernatant was given to naïve dendritic cells, that were in turn co-cultured with naïve T cells. CD4+ and CD8+ T cell activation was confirmed with IL-2 and INFg ELISAs as well as flow cytometry. To verify these findings, mice with Pan02 tumors were treated, and demonstrated similar DAMP and antigen release. Further, changes in immune cell populations within the tumor were consistent, with increased macrophages and dendritic cells 24 hours and 7 days and in T cell populations at 7 and 14 days after treatment. These results show the role of specific DAMPs and immune cell populations in responding to histotripsy ablation. Overall, these results indicate a robust immunomodulatory response to histotripsy tumor ablation.

Temporal and spatial modulation of the tumor and systemic immune response in the murine Gl261 glioma model.

Glioblastoma, the most aggressive form of glioma, has a 5-year survival rate of <5%. While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. This study quantifies the temporal and spatial localization of immune cell populations and mediators during glioma development. Eight-week old male C57Bl/6 mice were orthotopically inoculated with 1x106 Gl261 cells and tumor morphology, local and systemic immune cell populations, and plasma cytokines/chemokines assessed at day 0, 1, 3, 7, 14, and 21 post-inoculation by magnetic resonance imaging, chromogenic immunohistochemistry, multiplex immunofluorescent immunohistochemistry, flow cytometry and multiplex immunoassay respectively. From day 3 tumors were distinguishable with >30% Ki67 and increased tissue vascularization (p<0.05). Increasing tumor proliferation/malignancy and vascularization were associated with significant temporal changes in immune cell populations within the tumor (p<0.05) and systemic compartments (p = 0.02 to p<0.0001). Of note, at day 14 16/24 plasma cytokine/chemokines levels decreased coinciding with an increase in tumor cytotoxic T cells, natural killer and natural killer/T cells. Data derived provide baseline characterization of the local and systemic immune response during glioma development. They reveal that type II macrophages and myeloid-derived suppressor cells are more prevalent in tumors than regulatory T cells, highlighting these cell types for further therapeutic exploration.

The enhanced immunopharmacology of VIB4920, a novel Tn3 fusion protein and CD40L antagonist, and assessment of its safety profile in cynomolgus monkeys.

Inhibition of the T- and B-cell interaction through the CD40/CD40 ligand (L) axis is a favourable approach for inflammatory disease treatment. Clinical studies of anti-CD40L molecules in autoimmune diseases have met challenges because of thromboembolic events and adverse haemostasis. VIB4920 (formerly MEDI4920) is a novel CD40L antagonist and Tn3 fusion protein designed to prevent adverse haemostasis and immunopharmacology. We evaluated the pharmacokinetics, activity and toxicity of VIB4920 in monkeys. Cynomolgus monkeys received i.v. or s.c. 5-300 mg·kg-1 VIB4920 or vehicle, once weekly for 1 month (Studies 1 and 2) or 28 weeks (Study 3). VIB4920 exposure and bioavailability were determined using pharmacokinetic analyses, and immune cell population changes via flow cytometry. Pharmacological activity was evaluated by measuring the animals' capacity to elicit an immune response to keyhole limpet haemocyanin (KLH) and tetanus toxoid (TT). VIB4920 demonstrated linear pharmacokinetics at multiple doses. Lymphocyte, monocyte, cytotoxic T-cell and NK cell counts were not significantly different between treatment groups. B-cell counts reduced dose-dependently and the T-cell dependent antibody response to KLH was suppressed by VIB4920 dose-dependently. The recall response to TT was similar across treatment groups. No thromboembolic events or symptoms of immune system dysfunctionality were observed. VIB4920 demonstrated an acceptable safety profile in monkeys. VIB4920 showed favourable pharmacokinetics, dose-dependent inhibition of a neoantigen-specific immune response and no adverse effects on immune function following long-term use. Our data support the use of VIB4920 in clinical trials.

P143 EPITHELIAL-DERIVED SELENOPROTEIN P PROTECTS FROM COLITIS-ASSOCIATED CARCINOGENESIS

Abstract Patients with inflammatory bowel disease demonstrate selenium nutritional deficiencies and are at increased risk for colon cancer. Previously, we determined that global loss of the selenium-containing antioxidant protein, Selenoprotein P (SELENOP), exacerbates experimental colitis-associated cancer (CAC). Using a variety of tissue-specific knockout mouse lines, we further determined that epithelial-derived SELENOP served as the primary mediator of disease severity, and Villin-Cre; Selenop f/f (Selenop ΔIEC) mice have increased tumor incidence, number, size, and dysplasia as compared to WT cohorts. We next aimed to establish the mechanism by which epithelial-derived SELENOP modifies inflammatory tumorigenesis. We first investigated SELENOP’s effect on tumor promotion, and although Selenop ΔIEC tumors were larger, tumor cell proliferation was unchanged. Because global loss of SELENOP modified immune cell recruitment, we next characterized tumoral immunoinfiltrates by immunohistochemistry and seq-ImmuCC analysis of RNA-sequencing results. Neither metric identified significant changes in immune cell populations in either tumor or adjacent normal tissues. Further, histological endpoint analysis noted no change in degree of inflammation in Selenop ΔIEC colons at the end of the azoxymethane/dextran sodium sulfate protocol. Thus, epithelial-derived SELENOP likely does not augment inflammatory tumorigenesis via tumor promotion or inflammatory means by the measured parameters. We next hypothesized that loss of epithelial SELENOP modifies tumor initiation, and in support of this, analysis of endoscopy videos identified increased tumor numbers at early stages of experimental carcinogenesis. Tumors from Selenop ΔIEC mice also showed increased staining for markers of apoptosis and DNA damage, suggesting earlier tumor initiation may be due to increased oxidative stress. Further, myeloid-specific Selenop knockout, which did not alter inflammatory tumorigenesis, upregulated other antioxidant selenoproteins which may compensate for SELENOP loss, but this increase was not observed in the Selenop ΔIEC model. Interestingly, query of the Predicting Response to Standardized Pediatric Colitis Therapy (PROTECT) cohort indicated that SELENOP is downregulated in UC tissues, with greater downregulation in severe disease, but levels of other key antioxidant selenoproteins were again unchanged. Finally, to more clearly investigate SELENOP loss in epithelial cells, SELENOP was knocked down in human adult ulcerative colitis organoids. Here, knockdown induced a pronounced growth defect, increased apoptosis, and increased oxidative stress as measured by CellRox staining intensity. Taken together, these results suggest a model in which epithelial SELENOP protects from CAC by buffering oxidative stress in colonic epithelial cells in the setting of an inflammatory microenvironment.

Increased oxidative phosphorylation in lymphocytes does not atone for decreased cell numbers after burn injury.

The acute systemic inflammatory response syndrome (SIRS) and multiorgan dysfunction (MOD) that occur in large burn injuries may be attributed, in part, to immunosuppressive responses such as decreased lymphocytes. However, the mitochondrial bioenergetics of lymphocytes after severe burn injury are poorly understood. The purpose of this study was to examine mitochondrial function of lymphocytes following severe burns in a swine model. Anesthetized Yorkshire swine (n = 17) sustained 40% total body surface area full-thickness contact burns. Blood was collected at pre-injury (Baseline; BL) and at 24 and 48 h after injury for complete blood cell analysis, flow cytometry, cytokine analysis, and ficoll separation of intact lymphocytes for high-resolution mitochondrial respirometry analysis. While neutrophil numbers increased, a concomitant decrease was found in lymphocytes (P < 0.001) after burn injury, which was not specific to CD4+ or CD8+ lymphocytes. No changes in immune cell population were observed from 24 h to 48 h post-injury. IL 12-23 decreased while a transient increase in IL 4 was found from BL to 24h (P < 0.05). CRP progressively increased from BL to 24h (P < 0.05) and 48h (P < 0.001) post-injury. Routine and maximal mitochondrial respiration progressively increased from BL to 24h (P < 0.05) and 48 h post-injury (P < 0.001). No changes were found in leak respiration or residual oxygen consumption. When considering the reduction in lymphocyte number, the total peripheral lymphocyte bioenergetics per volume of blood significantly decreased from BL to 24h and 48h (P < 0.05). For the first time, we were able to measure mitochondrial activity in intact lymphocyte mitochondria through high-resolution respirometry in a severely burned swine model. Our data showed that the non-specific reduction in peripheral T cells after injury was larger than the increased mitochondrial activity in those cells, which may be a compensatory mechanism for the total reduction in lymphocytes. Additional studies in the metabolic activation of T cell subpopulations may provide diagnostic or therapeutic targets after severe burn injury.

Anti-PDL1/IL-15 fusion protein increases rare effector cells in cynomolgus monkeys and mice

Abstract Background Kadmon has generated an anti-PDL1/IL-15 fusion protein (KD033) by combining a proprietary, fully human, high affinity anti-human/mouse PD-L1 antibody with human IL-15. Initial assessment of KD033 showed enhanced tolerability relative to a non-targeted IL-15 fusion protein, in addition to its potent anti-tumor activity and immune memory generation. KD033 increased CD8+ T and NK cells in peripheral blood of humans, cynomolgus monkeys and mice as expected from on target engagement of IL-15. In this study, we evaluated additional immune cell population changes in mice and cynomolgus monkeys treated with KD033. Methods Tumor size, measured at day 7 in syngeneic subcutaneous CT26 colorectal cancer mouse model after a single KD033 surrogate treatment, was used to define mice as either KD033 responders (decreasing tumor volumes) or non-responders (no change or increasing tumor volumes). Peripheral blood from KD033 responders and non-responders as well as from cynomolgus monkeys treated with KD033 at predose, 24 and 168 hours after dosing was analyzed by flow cytometry for various immune cell populations. Results Granzyme B+ CD8+ T and gamma delta T cells in peripheral blood were specifically increased in KD033 surrogate responder mice. These increases were not observed in non-responder mice. Greater increases in NKT-like cells were also observed in KD033 surrogate best responders. This is in contrast to other immune populations, such as CD8+ T and B cells, which were similarly impacted, in both KD033 surrogate responder and non-responder mice. In cynomolgus monkeys, CD3+CD4-CD8- cells, which includes gamma delta T cells, were one of the immune populations with the highest increases at 168 hours post dose. Conclusion KD033 administration increases CD8+ T and NK cells, consistent with IL-15 pharmacodynamics. In addition, analysis of peripheral blood from KD033 surrogate best and non-responder mice demonstrated increases in different immune populations, which can facilitate their use as in-treatment biomarkers of KD033 activity in tumors. Importantly, KD033 administration resulted in increases in rare cytotoxic cells such as NKT and gamma delta T cells in peripheral blood which may contribute to its anti-tumor activity and immunity. Legal entity responsible for the study Kadmon Corporation LLC. Funding Kadmon Corporation LLC. Disclosure S. Martomo: Full / Part-time employment: Kadmon Corporation. D. Lu: Full / Part-time employment: Kadmon Corporation. Z. Polonskaya: Full / Part-time employment: Kadmon Corporation. X. Luna: Full / Part-time employment: Kadmon Corporation. F. Miyara: Full / Part-time employment: Kadmon Corporation. J. Patel: Full / Part-time employment: Kadmon Corporation.

Longitudinal immune characterization of syngeneic tumor models to enable model selection for immune oncology drug discovery

BackgroundThe ability to modulate immune-inhibitory pathways using checkpoint blockade antibodies such as αPD-1, αPD-L1, and αCTLA-4 represents a significant breakthrough in cancer therapy in recent years. This has driven interest in identifying small-molecule-immunotherapy combinations to increase the proportion of responses. Murine syngeneic models, which have a functional immune system, represent an essential tool for pre-clinical evaluation of new immunotherapies. However, immune response varies widely between models and the translational relevance of each model is not fully understood, making selection of an appropriate pre-clinical model for drug target validation challenging.MethodsUsing flow cytometry, O-link protein analysis, RT-PCR, and RNAseq we have characterized kinetic changes in immune-cell populations over the course of tumor development in commonly used syngeneic models.ResultsThis longitudinal profiling of syngeneic models enables pharmacodynamic time point selection within each model, dependent on the immune population of interest. Additionally, we have characterized the changes in immune populations in each of these models after treatment with the combination of α-PD-L1 and α-CTLA-4 antibodies, enabling benchmarking to known immune modulating treatments within each model.ConclusionsTaken together, this dataset will provide a framework for characterization and enable the selection of the optimal models for immunotherapy combinations and generate potential biomarkers for clinical evaluation in identifying responders and non-responders to immunotherapy combinations.

IMMU-04. TEMPORAL AND SPATIAL MODULATION OF THE IMMUNE RESPONSE OF THE MURINE GL261 GLIOMA TUMOUR MICROENVIRONMENT

Abstract INTRODUCTION Glioma is a debilitating and early fatal cancer arising in the glial cells of brain. Glioblastoma, the most aggressive form of gliomas, has a 5-year survival rate of 5% with treatment options limited to surgery, radiotherapy or chemotherapy with temozolomide. OBJECTIVE While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. In this study we quantified the temporal and spatial localisation of immune cell populations and mediators during glioma development. METHODS Mice were inoculated with 1x106 Gl261 cells at AP0.1mm, ML1.0mm, DV2.6mm Bregma. Tumour morphology, local and systemic immune cell populations were assessed at Day-0, 1, 3, 7, 14, and 21 post-inoculation by MRI, immunohistochemistry for Ki67+ proliferation and CD31+ vessel density; local immune infiltrate by multiplex immunofluorescence (VECTRA®;PerkinElmer); systemic immunity in spleen, bone marrow and peripheral blood by 16-parameter flow cytometry (LSRFortessa™;BD Biosciences); and multiplex immunoassay (Bio-Plex®;Bio-Rad) for plasma cytokines/chemokines. RESULTS From Day-3 tumours were distinguishable with high Ki67+ (&gt; 30%) and increased tissue vascularisation (26.38±1.45 vessels/field; p&lt; 0.05). Ki67 remained high until Day-21 with visible necrotic regions, increased vessel density and lumen area within tumour region. Increasing tumour proliferation/malignancy and vascularisation were associated with significant temporal changes in immune cell populations within the tumour (p&lt; 0.05) and systemic compartments (p=0.02- p&lt; 0.0001). Of note, at Day-14 NK, M1, PMN-MDSCs and M-MDSCs in the tumour infiltrate declined, coinciding with a decrease in 16/24 plasma cytokine/chemokines levels. Tumour infiltrating immune cell populations did not correlate with peripheral blood populations. However, a decrease in plasma cytokine/chemokine levels may indicate ‘immune exhaustion’. CONCLUSIONS The data derived provide baseline characteristics to study changes associated with multi-modal treatment strategies and to sequence therapies to maintain immune modulation. This information will contribute to the identification of novel combination therapies.

ATIM-40. CIRCULATING MYELOID-DERIVED SUPPRESSOR CELLS PREDICT FAVORABLE RESPONSE TO IMMUNE CHECKPOINT THERAPY IN A RANDOMIZED TRIAL OF NIVOLUMAB AND BEVACIZUMAB IN RECURRENT GBM

Abstract Glioblastoma (GBM) creates an immunosuppressive environment that presents a challenge to efficacy of immunotherapeutic approaches. Results from the CheckMate-143 trial demonstrated responses in 8% of patients with nivolumab, underscoring the need for further insight into the mechanisms and markers of immune suppression and response. Given a limited set of biomarkers predictive of immunotherapy response in GBM, we explored the changes in immune cell populations in nivolumab and bevacizumab-treated GBM patients pre and post-treatment in order to help predict response. In these studies, we utilized traditional and newly developed approaches, including mass cytometry time-of-flight (CyTOF), single-cell RNA sequencing, and 10X Genomics simultaneous cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). We analyzed patients’ samples in a randomized, phase 2 study of nivolumab and bevacizumab at GBM first recurrence (NCT03452579). Nine patients were identified as responders or non-responders at 8 weeks after therapy initiation. Utilizing peripheral blood samples, we observed a 6.4-fold decrease in immunosuppressive myeloid-derived suppressor cells (MDSCs) between baseline and first imaging follow-up in responders compared to non-responders, with a 4.9-fold decrease in the granulocytic MDSC (G-MDSC) subtype in responders over non-responders. While no significant changes in overall T-cell numbers were noted, expression of PD-1 on CD4+ T cells was significantly elevated at baseline and follow-up in responders as compared to non-responders – signatures which were confirmed by CyTOF. Given these immunophenotypic changes, preliminary results of a detailed investigation of this cohort by CITE-seq indicate that responders had increased IL7R-positive T cells post-treatment, which was not observed in non-responders. These results are currently being validated in an additional 40 patients that have been enrolled. Altogether, differences in immunophenotypes that were specific to responders and non-responders were observed, and characterization of these immune populations may be helpful in identifying GBM patients likely to benefit from immunotherapy.

Modulatory Effect of the Euro-Lupus Low-Dose Intravenous Cyclophosphamide Regimen on Circulating Immune Cells in Systemic Lupus Erythematosus.

A Euro-Lupus regimen of low-dose intravenous cyclophosphamide (CFA) is commonly used to treat severe organ manifestations of systemic lupus erythematosus (SLE), particularly lupus nephritis (LN). There are no data on the distributions and dynamics of immune cell populations in patients with various treatment outcomes. The circulating immune cells of 11 female SLE patients were assessed before and after Euro-Lupus regimen (cumulative dose of 3000mg CFA) by flow cytometry together with those of 16 healthy women. A subanalysis was performed in LN patients who achieved complete remission (CR; n = 3), partial remission (PR; n = 4), and no response (NR; n = 2). In SLE, the Euro-Lupus regimen decreased the percentage and absolute count of B cells; increased the percentage of CD8+ T cells, T regulatory cells, neutrophils, and monocyte subsets; and activated T and NK cells compared to healthy controls (P < 0.050). Patients with LN achieving CR had significantly lower proportions of CD27+ B memory cells compared to poor responders (PR/NR, P = 0.035). The post-treatment percentages and absolute numbers of B cells, T cells, NK cells, monocytes, and neutrophils showed high inter-individual variability with no association with treatment outcome. Our pilot study revealed the dynamics of changes in immune cell populations in SLE patients during a Euro-Lupus regimen, mainly the lowering of B cells. In LN patients who achieved CR, a lower proportion of CD27+ B memory cells was evident compared to poor responders (PR/NR). Further studies on usefulness of monitoring immune cells for treatment response prediction on larger cohorts are needed.

Peripheral changes in immune cell populations and soluble mediators after anti-PD-1 therapy in non-small cell lung cancer and renal cell carcinoma patients.

Patients with non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) have shown benefit from anti-PD-1 therapies. However, not all patients experience tumor shrinkage, durable responses or prolonged survival, demonstrating the need to find response markers. In blood samples from NSCLC and RCC patients obtained before and after anti-PD-1 treatment, we studied leukocytes by complete blood cell count, lymphocyte subsets using flow cytometry and plasma concentration of nine soluble mediators, in order to find predictive biomarkers of response and to study changes produced after anti-PD-1 therapy. In baseline samples, discriminant analysis revealed a combination of four variables that helped differentiate stable disease-response (SD-R) from progressive disease (PD) patients: augmented frequency of central memory CD4+ T cells and leukocyte count was associated with response while increased percentage of PD-L1+ natural killer cells and naïve CD4+ T cells was associated with lack of response. After therapy, differential changes between responders and non-responders were found in leukocytes, T cells and TIM-3+ T cells. Patients with progressive disease showed an increase in the frequency of TIM-3 expressing CD4+ and CD8+ T cells, whereas SD-R patients showed a decrease in these subsets. Our findings indicate that a combination of immune variables from peripheral blood(PB) could be useful to distinguish response groups in NSCLC and RCC patients treated with anti-PD-1 therapy. Frequency of TIM-3+ T cells showed differential changes after treatment in PD vs SD-R patients, suggesting that it may be an interesting marker for monitoring progression during therapy.

Leveraging cell-specific differentially methylated regions to identify leukocyte infiltration in adipose tissue.

Obesity is understood to be an inflammatory condition characterized in part by changes in resident immune cell populations in adipose tissue. However, much of this knowledge has been obtained through experimental animal models. Epigenetic mechanisms, such as DNA methylation may be useful tools for characterizing the changes in immune cell populations in human subjects. In this study, we introduce a simple and intuitive method for assessing cellular infiltration by blood into other heterogeneous, admixed tissues such as adipose tissue, and apply this approach in a large human cohort study. Associations between higher leukocyte infiltration, measured by evaluating a distance measure between the methylation signatures of leukocytes and adipose tissue, and increasing body mass index (BMI) or android fat mass (AFM) were identified and validated in independent replication samples for CD4 (pBMI = 0.009, pAFM = 0.020), monocytes (pBMI = 0.001, pAFM = 4.3 × 10-4 ), and dendritic cells (pBMI = 0.571, pAFM = 0.012). Patterns of depletion with increasing adiposity were observed for plasma B (pBMI = 0.430, pAFM = 0.004) and immature B (pBMI = 0.022, pAFM = 0.042) cells. CD4, dendritic, monocytes, immature B, and plasma B cells may be important agents in the inflammatory process. Finally, the method used to assess leukocyte infiltration in this study is straightforwardly extended to other cell types and tissues in which infiltration might be of interest.

Symposium review: Immunological detection of the bovine conceptus during early pregnancy

Infertility and subfertility reduce the economic viability of dairy production. Inflammation reduces conception rates in dairy cattle, but surprisingly little information exists about the populations and the functions of immune cells at the conceptus-maternal interface during the periattachment period in dairy cattle. Early pregnancy is accompanied by immune stimulation at insemination and conceptus secretion of IFN-τ, pregnancy-associated glycoproteins, prostaglandins, and other molecules whose effects on immune function during early pregnancy have not been determined. Our working hypothesis is that pregnancy induces changes in immune cell populations and functions that are biased toward immunological tolerance, tissue remodeling, and angiogenesis. This review summarizes current knowledge, starting with insemination and proceeding through early pregnancy, as this is the period of maximal embryo loss. Results indicated that early pregnancy is accompanied by a marked increase in the proportion of endometrial immune cells expressing markers for natural killer (CD335) cells and cytotoxic T cells (CD8) along with an increase in cells expressing major histocompatibility class II antigens (macrophages and dendritic cells). This is accompanied by increased abundance of mRNA for IL-15, a natural killer growth factor, and IL-10 in the endometrium during early pregnancy. Furthermore, expression of indoleamine 2,3 dioxygenase was 15-fold greater in pregnant compared with cyclic heifers at d 17, but then declined by d 20. This enzyme converts tryptophan to kynurenine, which alters immune function by creating a localized tryptophan deficiency and by activation of the aryl hydrocarbon receptor and induction of downstream tolerogenic mediators. Expression of the aryl hydrocarbon receptor is abundant in the bovine uterus, but its temporal and spatial regulation during early pregnancy have not been characterized. Pregnancy is also associated with increased expression of proteins known to inhibit immune activation, including programed cell death ligand-1 (CD274), lymphocyte activation gene-3 (CD223), and cytotoxic T-lymphocyte associated protein-4 (CD152). These molecules interact with receptors on antigen-presenting cells and induce lymphocyte tolerance. Current results support the hypothesis that early pregnancy signaling in dairy heifers involves changes in the proportions of immune cells in the endometrium as well as induction of molecules known to mediate tolerance. These changes are likely essential for uterine wall remodeling, placentation, and successful pregnancy.

Phenotypic changes of lymphocyte populations in psoriasiform dermatitis animal model.

Psoriasis is a T cell mediated, chronic inflammatory autoimmune skin disease that affects up to 2–3% of the global population and leads to a decrease in quality of life. Experimental data accumulated in recent years highlighted the important role played by the immune system in the pathogenesis of this disease. Non-human psoriasis models are an important research tool that attempts to reproduce the clinical features of the disease in order to explain the pathogenesis of psoriasis and to identify possible therapeutic targets. Imiquimod-based murine model of psoriatic dermatitis is an alternative to traditional models of experimental psoriasis in mice and the induced dermatitis closely mimics the pathologic changes in human psoriasis. In order to emphasize changes in immune cell populations involved in lesion pathogenesis, we performed a murine model of psoriasiform dermatitis model by topical IMQ application. The progress and the severity of IMQ-induced skin inflammation were clinically (PASI score) and histopathologically evaluated. The immunological changes induced by IMQ treatment in lymphocyte populations from peripheral blood and spleen were evaluated by flow cytometry. The main changes observed in peripheral blood were the significantly increased T-CD8a+ lymphocyte and NK1.1+ cell percentages and the decreased T-CD4+ and B lymphocyte percentages in IMQ-treated mice. In spleen samples, lymphocytes showed the same tendency of variation as in peripheral blood, but without statistical significance. A significant decrease of B cells percentages was observed in spleen suspensions. Data obtained in skin samples may suggest the involvement of CD3ε+, CD4+ and CD8a+ cells in the lesional process. This murine model was analyzed by performing a basic cellular profile at three levels: peripheral blood, spleen and skin. The evaluation aimed to establish the immune framework of this experimental model that could further be used for etipathogenic mechanism identification and/or for studies regarding targeted therapies.

Abstract A76: Immunologic changes following intraperitoneal administration of a formulated IL-12 plasmid in combination with standard neoadjuvant chemo in newly diagnosed advanced stage ovarian cancer patients

Abstract Background: This abstract describes the translational component of a phase I study of weekly intraperitoneal (IP) GEN-1, an IL-12 plasmid formulated with polyethyleneglycol-polyethyleneimine cholesterol lipopolymer, in combination with standard intravenous (IV) weekly taxane (T) and carboplatinum (C) every 3 weeks in epithelial ovarian, fallopian tube, or primary peritoneal cancer (EOC) patients undergoing neoadjuvant chemotherapy (NAC). Local administration of GEN-1 could provide persistent IL-12 secretion to induce type-1 immune responses in the immunosuppressive tumor microenvironment. Our hypothesis is that the GEN-1 plus NAC treatment will reprogram the tumor immune microenvironment towards a potent antitumor immune response. We have previously reported the clinical finding that GEN-1 is safe up to 79 mg/m2 IP weekly up to eight treatments. The most common related toxicities were Grade 1 nausea, vomiting, abdominal pain, and fatigue. Patients continue to be followed for progression and any drug-related safety events. Methods: Newly diagnosed advanced-stage EOC patients being treated with NAC were eligible. The trial utilized a 3+3 design with GEN-1 IP dose levels ranging from 36 mg/m2 to 79 mg/m2 weekly for 8 treatments with concurrent IV T/C. Tumor samples were collected at time of diagnostic laparoscopy and interval debulking (IDB). Blood and peritoneal ascites were collected before and after treatment. The immunologic analysis included frequency of immune cell populations and cytokine levels in tumor, blood, or peritoneal ascites. The levels of cytokines IL-12, IFN-γ, TNF-α, TGF-β, IL-10, and VEGF were quantified in blood plasma and ascites samples with commercially available cytokine-specific ELISA kits. The tumor tissue specimens were paraffin embedded and serial sections were stained with specific antibodies for various T-cell markers using immunohistochemistry. The density of various T-cells, dendritic cells, and myeloid-derived suppressor cells in blood or ascites was determined by flow cytometry. Results: The interim results obtained from Cohorts 1–4 (stage 1) show post-treatment changes in immune cell populations, including an increase in cytotoxic CD8+ T-cells and decrease in several immunosuppressive markers including FoxP3+, PD-1, PDL-1, and IDO-1. The cell ratio of CD8+ to all immunosuppressive markers appeared to be skewing beneficially towards more cytotoxic T lymphocyte and away from suppressive cells in most patients. There were no significant changes in the frequency of circulating T cells (or cytokine response). Analysis of ascites before and 24 hours after IP administration of GEN-1+NAC showed evidence of IL-12 gene transfer and activation of downstream signaling pathways, including increases in IFN-γ levels and inhibition of peritoneal VEGF levels. The cytokine response was predominantly in ascites while comparatively little changes were observed in blood plasma. Thus, the immunomodulatory and therapeutic effect of GEN-1 treatment is primarily localized within the tumor microenvironment and does not appear to have any untoward effect on systemic immune responses. The changes in cytokine levels appeared to be GEN-1 dose dependent. Additional analyses from this study are in progress. Conclusions: Adding GEN-1 to neoadjuvant T/C is safe and biologically active in EOC patients. Based on preliminary analyses of Cohorts 1–4 (stage 1), the treatment of EOC patients with GEN-1 and NAC has resulted in favorable immunologic responses. Final translational results will be presented at the meeting. Clinical trial information: NCT02480374. Citation Format: Khursheed Anwer, Junko Matsuzaki, Wiam Bshara, Amit Lugade, Angela Omilian, Premal H. Thaker, William H. Bradley, Rebecca Arend, Camille Gunderson, Jason Fewell, Nicholas Borys, Lauren Musso, Adekunle Odunsi. Immunologic changes following intraperitoneal administration of a formulated IL-12 plasmid in combination with standard neoadjuvant chemo in newly diagnosed advanced stage ovarian cancer patients. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A76.

2385 Role of the antioxidant enzyme catalase in respiratory syncytial virus infection

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to further evaluate underlying disease parameters in respiratory syncytial virus (RSV) infection, that is reduction in antioxidant potential, and determining if supplementation of the antioxidant enzyme catalase could be employed as a potential therapeutic. METHODS/STUDY POPULATION: Nasopharyngeal secretions were obtained from patients (&lt;2 years old) verified for RSV infection, and assessed for catalase activity and correlated with disease parameters. In addition, the BALB/c animal model of RSV infection was utilized to directly study the effect of supplemental catalase on RSV-related disease parameters in vivo. The catalase formulation used in these studies is pegylated, and has been tested to provide long-term increased catalase activity in vivo. We are also currently working on designing an in vitro model of catalase supplementation in A549 bronchial epithelial cells. RESULTS/ANTICIPATED RESULTS: Our preliminary data shows that patients with more severe disease (based on hospitalization, oxygen supplementation) have significantly lower levels of catalase activity (p&lt;0.02). Additionally, when pegylated-Catalase (PG-CAT) treatment is utilized in RSV infection of mice, there is significant improvement in several disease parameters. PG-CAT-treated mice show an attenuated body weight loss (p&lt;0.001) and clinical disease (p&lt;0.02), and also have lower levels of key pro-inflammatory cytokines including CXCL1 and TNF-α. PG-CAT treatment also resulted in a minor decrease in viral titer, which is being further evaluated. In addition, PG-CAT treatment resulted in an improvement in airway hyperresponsiveness observed at baseline, we are further characterizing this improvement and also conducting methacholine challenges. Currently, we are working to determine the underlying mechanism through which PG-CAT results in these improvements, and whether it is through changes in immune cell populations, cellular signaling or apoptosis signaling pathways (i.e., caspases). DISCUSSION/SIGNIFICANCE OF IMPACT: RSV is the leading cause of viral pneumonia and bronchiolitis in infants, with no vaccines or effective therapeutics available currently. Our study indicates that catalase activity could be used as a potential correlate for disease severity and be used as an indicator of disease during patient treatment. Additionally, and more importantly supplementation of catalase could be used as a potential therapeutic for treatment of RSV.

The Effect of Helminth-Induced Immunomodulatory Therapy (HINT) in Relapsing Remitting Multiple Sclerosis

Abstract This study examines the immune response stimulated by Trichuris suis ova when used as a treatment for Multiple Sclerosis. The hygiene hypothesis posits that insufficient exposure to microorganisms due to modern sanitation can predispose susceptible individuals to immune dysregulation. A pilot HINT 1 study indicated that TSO could effectively regulate the immune system of relapsing-remitting MS patients. Here, we investigate this hypothesis further with a longer treatment period. The HINT phase 2 clinical trial consisted of 16 treatment-naïve patients with newly diagnosed RRMS. Patients were given TSO buffered with 15mL of Phosphate buffered saline every two weeks. Anti-TSO immune response was measured by IgG1, IgA and IgE ELISAs of patient serum and eosinophilia count. Changes in immune cell populations were monitored by flow cytometry of peripheral blood mononuclear cells. CD4+ T cells were further analyzed as Th1, Th2, Th17, TREG, and TFH cells. During TSO treatment, the average number of new Gd+ lesions in the HINT patients was reduced. In addition, there was a significant increase in the anti-TSO immune responses as measured by IgG1 and IgE ELISAs, and increased eosinophilia during the second month of treatment. Also, TSO administration led to a decrease in the percentage of activated CD4+ and CD8+ T cells during therapy accompanied by an increase in the presence of TREG cells during treatment, a reduction in the presence on TFH cells in circulation, and an increase in IL-4-expressing T cells. This data implies a shift from a Th1 inflammatory state to a Th2 anti-inflammatory state during HINT II TSO treatment and suggests that immunoregulatory therapy through probiotic helminth treatment could be a viable option as a treatment for MS.

Canonical NF-κB signaling in myeloid cells promotes lung metastasis in a mouse breast cancer model.

An inflammatory tumor microenvironment is a common characteristic of solid tumors. It is the result of a complex interplay between tumor cells, tumor infiltrating immune cells and other stromal cells. Myeloid cells in the tumor microenvironment are considered major drivers of tumor progression and metastasis and increased numbers of these cells are associated with poor prognosis in various cancer patients. The transcription factor NF-κB is considered the master regulator of inflammatory gene expression and immune cell function. Its activation in various cells of the tumor microenvironment contributes essentially to tumorigenesis. In the present study, the role of canonical NF-κB signaling in myeloid cells in metastatic breast cancer was addressed by myeloid-specific deletion of Ikkβ in the MMTV polyoma middle T (PyMT) mouse model. Ikkβ deletion in myeloid cells did not affect primary mammary tumor growth but significantly reduced lung metastasis. While dissemination from the primary tumor was unaltered, myeloid-specific Ikkβ loss resulted in a strong up-regulation of pro-inflammatory genes and changes in immune cell populations in the lung, creating a tumor-suppressive microenvironment at the distant site. Thus, canonical NF-κB signaling in myeloid cells creates a permissive lung microenvironment that supports breast to lung metastasis.

Head and neck squamous cell carcinoma: Genomics and emerging biomarkers for immunomodulatory cancer treatments.

Head and neck squamous cell carcinoma (HNSCC) comprises a heterogeneous group of tumors that arise from the squamous epithelium of the oral cavity, oropharynx, larynx and hypopharynx. While many HNSCCs are related to classical etiologic factors of smoking and alcohol, a clinically, genomically, and immunologically distinct subgroup of tumors arise from the epithelium of the tonsil and the base of tongue as a result of infection with Human Papilloma Virus (HPV). In this review we describe the genomic and immunologic landscape of HNSCC, highlighting differences between HPV-positive and HPV-negative HNSCC. While HPV-negative tumors are characterized by tobacco-associated mutations in genes including TP53 and CDKN2A, in HPV-positive HNSCC integration of viral genome from HPV into the host cellular genome results in expression of the E6 and E7 viral oncoproteins, with consequent degradation of p53 and functional inactivation of Rb. The immune microenvironment of HNSCC is characterized by changes in immune cell populations, immune checkpoints, as well as tumor or microenvironmental factors that alter the balance of the immune milieu in favor of immunosuppression, allowing tumor evasion and escape from immune surveillance. Immune therapies, in particular those targeting the PD1 receptor or its ligand PD-L1, including nivolumab, pembrolizumab, durvalumab, and atezolizumab have shown significant efficacy in subsets of patients with HNSCC. Current trials are evaluating the efficacy of these agents in combination with chemotherapy, radiotherapy and other immune therapies including CTLA-4 and IDO-1 inhibitors. While biomarkers including PD-L1 expression, PD-L2 expression and the interferon-gamma gene signature show potential to predict benefit from checkpoint inhibitor therapy - it is hoped that improved understanding of the genomic and immune landscape will lead to ways to improved strategies to stratify patients and to select which HNSCC are most likely to benefit from these therapies.

Aged garlic extract supplementation modifies inflammation and immunity of adults with obesity: A randomized, double-blind, placebo-controlled clinical trial

Obesity is a serious global health issue and often results in low-grade systemic inflammation, increasing the risk for several chronic diseases. If obesity-induced inflammation could be reduced, fewer complications and co-morbidities might occur. To investigate whether daily supplementation with aged garlic extract (AGE) could reduce chronic inflammation and improve immune function in adults with obesity. Fifty-one healthy adults with obesity (mean age 45.6±1.6 years, mean BMI 36.1±0.9kg/m2) were recruited to participate in a parallel, double-blind, placebo-controlled, randomized study. After being matched by BMI, participants were randomized into the AGE supplementation or placebo group. Participants were asked to take a divided daily dose of 3.6g AGE or placebo, with food for 6 weeks. Blood lipid and inflammatory markers were assessed at baseline and after 6 weeks of supplementation. Additionally, peripheral blood mononuclear cells (PBMC) were isolated from whole blood and used to detect changes in immune cell populations and levels of cytokine secretion. A one-way ANCOVA was performed to evaluate differences between the two groups, controlling for respective baseline values. At the end of study, serum IL-6 (p=0.04) and TNF-α (p=0.05) of participants consuming AGE were significantly lower than those consuming the placebo capsules. PBMC flow cytometry results showed that participants from the AGE group had a higher proportion of γδ-T cells (p=0.03) and a lower proportion of NKT cells (p=0.02) in the total population of lymphocytes. There was no difference in percentage of NK cells between the two groups. A significant difference in blood LDL concentration was also observed (p=0.05). Total cholesterol and non-HDL cholesterol tended to differ between participants from the AGE group and those from the placebo group, although values did not achieve statistical significance. Six weeks of AGE consumption modulated immune cell distribution, prevented the increase of serum TNF-α and IL-6 concentrations and reduced blood LDL concentration in adults with obesity. AGE, taken consistently, may be beneficial in preventing the development of chronic diseases associated with low-grade inflammation in adults with obesity. Registered under ClinicalTrials.gov with the identifier code NCT01959646.