Analysis Of Immune Phenotypes Research Articles

Abstract 4981: Comprehensive analysis of immune phenotypes identifies CD73+ CD163high macrophages as a prognostic biomarker in lung adenocarcinoma

Abstract To better understand the immune profile within the tumor microenvironment (TME) and the potential immune-evasion capacity that may arise from administration of immune checkpoint inhibitors, we investigated immunological phenotypes and their correlations with genomic alterations in order to provide predictive/prognostic implications in lung adenocarcinoma (LAC).Surgical sections with corresponding peripheral blood mononuclear cells were collected from 76 patients with pathologically confirmed Ia-IIIa. The histological contents expressing immune checkpoint molecules in the tumor nest and stromal area were imaged and quantified using a multi-spectral imaging system. Distribution of tumor mutation burden (TMB) and non-synonymous somatic mutations were analyzed in whole exome sequencing (WES) data.Total 76 patients consisted of EGFR-mutant (n=37) and EGFR-wild type (n=39) tumors. As is well known, the number of tumor-infiltrating cytotoxic T cells (174.7 ± 24.4 cells/mm2) and TMB (3.8 ± 0.4) in EGFR-mutant tumors was significantly lower than those of EGFR wild type tumors (394.2 ± 82.3 cells/mm2 and 6.7 ± 1.2) (p=0.0146 and p=0.0254, respectively), elucidating EGFR mutation-mediated immune evasion. The subjects carrying EGFR wild-type were classified into two subgroups by the density of tumor-infiltrating cytotoxic T cells, defined as the high TIL (n=20) and low TIL (n=19). There was great difference of mean density of cytotoxic T cells between low TIL group (129.6 ± 16.8 cells/mm2) and high TIL group (672.8±143.5 cells/mm2). Tumor-infiltrating T cells were granzyme B-positive, suggesting most cells were tumor-specific. Interestingly, no significant difference in TMB was found between the two subgroups (7.5 ± 1.9 in low TIL group vs. 5.8±1.3 in high TIL group; p=0.4642). Tumor proportion score (TPS) of PD-L1 was remarkably higher in high TIL group than low TIL group (29.7 ± 6.5% vs. 13.9 ± 2.8%, p=0.0008, respectively), but no significant differences of tumoral CD73 and IDO between the two groups (p=0.643 and p=0.923, respectively). Even there were no significant differences in counts of tumor-infiltrating macrophages in all types of macrophage, significantly higher counts of PD-L1+ M1-type macrophages (CD68+ CD163low) in the stromal area were discovered in the high TIL group compared to those of the low TIL group (396.2 ± 93.6 vs. 139.8 ± 30.1, p=0.0114, respectively), indicating that most of macrophages resided in the stromal area. Interestingly, the low TIL group exhibited higher counts of CD73+ M2-type macrophages (CD68+ CD163high) in the stromal area than the high TIL group (644.5 ± 110.9 vs. 387.9 ± 73.2, p=0.0639, respectively). Cell count ratio (M1/M2) of macrophages was significantly higher in the high TIL group (p=0.0042), suggesting that the TME with a smaller number of TILs tends to contain more M2-type macrophages. The tumor recurrence was more frequent in the low TIL group compared to the high TIL group with no statistical significance (p=0.127). In surgically resected lung adenocarcinoma patients, the subgroup with a low density of tumor-infiltrating cytotoxic T cells had a large number of CD73+ M2-type macrophages, which may contribute to poor prognosis. Acknowledgement: This study was sponsored by Ono Pharma Co., Ltd Citation Format: Jae-Hwan Kim, Tae-Min Kim, Sung-Gu Her, Kyoung-Ho Pyo, Jeong- Oh Kim, Beung-Chul Ahn, Byoung Chul Cho, Jin Hyoung Kang. Comprehensive analysis of immune phenotypes identifies CD73+ CD163high macrophages as a prognostic biomarker in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4981.

Enhancement of Immune Activities of Mixtures with Sasa quelpaertensis Nakai and Ficus erecta var. sieboldii.

The objective of the present study was to develop a concoction of natural products that could dramatically improve immune function with minimal possible side effects. Sasa quelpaertensis Nakai and Ficus erecta var. sieboldii are plants that are native to Jeju Island, Korea and are known to be rich in physiologically active substances. We prepared a mixture of different proportions and extraction conditions using two natural plants and determined their optimum mixing ratio and extraction method by assessing immune function-related biomarkers in RAW264.7 macrophages. Optimal extract (HR02/04(8:2)-W) was selected from in vitro experiments and its immunity-enhancing efficacy was evaluated in mice. After oral administration of extract to BALB/c mice for 2 weeks, nitric oxide production in the peritoneal exudate cells, natural killer cell cytotoxicity, cytokine expression in splenocytes, and total cell number of immune tissues and phenotype analysis were evaluated. Our results demonstrated that HR02/04(8:2)-W significantly enhanced the immune system by increasing natural killer cell activity, cytokine expression, and total number of cells in immune tissues. In conclusion, our study validates the role of HR02/04(8:2)-W in enhancing immunity and its potential development as a functional food.

Deep learning-based immune phenotype analysis reveals distinct resistance pattern of immune checkpoint inhibitor in non-small cell lung cancer.

3119 Background: Resistance pattern and biological mechanism of immune checkpoint inhibitor (ICI) has been poorly understood. Sine suggested resistance mechanisms would be either innate resistance caused by lack of immune recruitment or acquired immune evasion after durable response of ICI treatment, we hypothesized that resistance pattern of tumor microenvironment would be distinct according to duration of ICI response in non-small cell lung carcinoma (NSCLC). In the current study, we applied deep-learning-based classification of three immune phenotypes (3IP): inflamed, excluded, and desert, to objectively assess the immunologic status of tumor microenvironment. Methods: Deep-learning algorithm of H&E Whole-Slide Images (WSI), called Lunit-SCOPE, was trained with 1,824 H&E WSI of NSCLC from Samsung Medical Center (SMC). WSI was divided into patches and each patch (~10 high-power fields) was classified as inflamed, excluded and desert, based on both quantity and localization of immune cells. Among NSCLC patients treated with ICI in SMC, 87 paired treatment-naïve (Pre, patch N = 15,415) and post-progression (Post, patch N = 18,197) tumor tissues were analyzed for Lunit-SCOPE. Results: In 87-paired samples, proportions of excluded and desert phenotypes were increased in post-progression tumor tissues (excluded; Pre 26.8% versus Post 32.5%, desert; Pre 19.5% versus Post 25.3%). Focused on 29 patients classified as inflamed in treatment-naïve, proportion of immune phenotypes of post-progression were clearly different according to duration of response, divided by median progression-free survival (PFS) of 3.7 m. Patients with rapid progression without ICI response (PFS < 3.7 m) turned into desert type (46.2%), whereas durable responder (PFS ≥ 3.7 m) either still remained on inflamed phenotype (42.9%) or turned into excluded phenotype (21.4%). Patients who remained on inflamed phenotype had favorable overall survival after progression on ICI, compared to turned into desert type (median survival not reached versus 6.6 m, P= 0.0296). Conclusions: Resistance patterns of ICI are distinct according to duration of response in patients with inflamed phenotype. Rapid progressor turns off immune into desert phenotype whereas most durable responder keeps immune recruitment into tumor microenvironment, which needs tailored strategy to overcome ICI resistance.

Immune phenotypic analysis using pre-designed BD® AbSeq pan immune-response panel

Abstract High-throughput single cell RNA sequencing (scRNA-seq) has recently emerged as a powerful tool for profiling complex cell populations. While traditional scRNA-seq captures information at transcript level, recent technology using DNA-barcoded antibodies enable multiomic analysis, examining protein alongside mRNA. Although this approach allows high parameter antibody panels, the sequencing cost can escalate quickly for high plexy panel with high antigen density markers. Therefore, thought should be put on panel design in order to gain meaningful results efficiently. Here, we have designed a panel for analyzing multiple immune responses, BD® AbSeq pan immune-response panel. Our immune response panel excludes abundant cell lineage markers which take up a huge amount of reads but provide only cell lineage information that can be easily resolved by RNA profiles only. This panel is focused on analyzing the various immune responses in multiple cell lineages. Therefore, it has a potential to be used to uncover phenotypic changes upon disease status with various immune stimuli. To test the capability of this AbSeq panel in discovery of phenotypic changes in multiple immune response, we have tested it in the various scenarios. We propose that the BD® AbSeq pan immune-response panel can be used to evaluate unpredictable disease phenotypes to identify the cell types affected from the disease as well as the responsible molecular pathways of the disease at a single cell level. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Mucosal Profiling of Pediatric-Onset Colitis and IBD Reveals Common Pathogenics and Therapeutic Pathways

Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth ofinfants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial Tcells, and platelet aggregation andrelease of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.

Inhibition of Δ24-dehydrocholesterol reductase activates pro-resolving lipid mediator biosynthesis and inflammation resolution

Targeting metabolism through bioactive key metabolites is an upcoming future therapeutic strategy. We questioned how modifying intracellular lipid metabolism could be a possible means for alleviating inflammation. Using a recently developed chemical probe (SH42), we inhibited distal cholesterol biosynthesis through selective inhibition of Δ24-dehydrocholesterol reductase (DHCR24). Inhibition of DHCR24 led to an antiinflammatory/proresolving phenotype in a murine peritonitis model. Subsequently, we investigated several omics layers in order to link our phenotypic observations with key metabolic alterations. Lipidomic analysis revealed a significant increase in endogenous polyunsaturated fatty acid (PUFA) biosynthesis. These data integrated with gene expression analysis, revealing increased expression of the desaturase Fads6 and the key proresolving enzyme Alox-12/15 Protein array analysis, as well as immune cell phenotype and functional analysis, substantiated these results confirming the antiinflammatory/proresolving phenotype. Ultimately, lipid mediator (LM) analysis revealed the increased production of bioactive lipids, channeling the observed metabolic alterations into a key class of metabolites known for their capacity to change the inflammatory phenotype.

385 Ruxolitinib cream ameliorates a preclinical model of skin dermatitis via modulation of inflammatory T-cell subsets

Allergic contact dermatitis, is driven by an overreaction of the body’s immune system to a normally harmless substance resulting in skin inflammation. Preclinically, topical administration of the T helper 2 (Th2) hapten fluorescein isothiocyanate (FITC) drives an immunogenic sensitization in mice and recall challenge on the ear triggers a localized inflammatory contact hypersensitivity. Pharmacological inhibition of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway has previously demonstrated efficacy in a Phase 2 clinical trial in patients with atopic dermatitis (AD) (NCT03011892). Ruxolitinib cream, a topical JAK1/2 inhibitor, is currently being evaluated in clinical trials for the treatment of mild-to-moderate AD (NCT03745638, NCT03538041 and NCT03745651). The experimental goal was to evaluate the dose-dependent efficacy of ruxolitinib cream as a topical monotherapy for the modulation of experimentally induced contact dermatitis in mice. Acute and chronic dermatitis was induced in Balb/C mice via FITC sensitization and subsequent recall response. Topical administration of ruxolitinib cream was compared to steroid (Betamethasone cream, 0.05%). Efficacy was quantified via inflammatory edema, immune phenotyping, tissue histology and immune pathway analysis. Topical administration of 1.5% ruxolitinib cream (q.d. and b.i.d) dose-dependently reduced inflammatory swelling and lichenification following recall FITC challenge in both acute (p<0.05) and chronic (p<0.05) contact dermatitis. Ruxolitinib efficacy was associated with a significant (p<0.01), dose-dependent, reduction in Th2 lymphocytes within the auricular (draining) lymph node). Pathway analysis revealed downregulation of inflammatory mechanisms consistent with JAK1/2 inhibition. Ruxolitinib cream was highly effective at inhibiting contact dermatitis in mice, ameliorated pro-inflammatory mechanisms and reduced skin thickening. Together, the data suggest that ruxolitinib cream may have potential as a therapeutic agent for the treatment of contact dermatitis.

Genetic associations of T cell cancer immune response with tumor aggressiveness in localized prostate cancer patients and disease reclassification in an active surveillance cohort

ABSTRACT Determining prostate cancer (PCa) aggressiveness and reclassification are critical events during the treatment of localized disease and for patients undergoing active surveillance (AS). Since T cells play major roles in cancer surveillance and elimination, we aimed to identify genetic biomarkers related to T cell cancer immune response which are predictive of aggressiveness and reclassification risks in localized PCa. The genotypes of 3,586 single nucleotide polymorphisms (SNPs) from T cell cancer immune response pathways were analyzed in 1762 patients with localized disease and 393 who elected AS. The aggressiveness of PCa was defined according to pathological Gleason score (GS) and D’Amico criteria. PCa reclassification was defined according to changes in GS or tumor characteristics during subsequent surveillance biopsies. Functional characterization and analysis of immune phenotypes were also performed. In the localized PCa cohort, seven SNPs were significantly associated with the risk of aggressive disease. In the AS cohort, another eight SNPs were identified as predictors for aggressiveness and reclassification. Rs1687016 of PSMB8 was the most significant predictor of reclassification. Cumulative analysis showed that a genetic score based on the identified SNPs could significantly predict risk of D’Amico high risk disease (P-trend = 2.4E-09), GS4 + 3 disease (P-trend = 1.3E-04), biochemical recurrence (P-trend = 0.01) and reclassification (P-trend = 0.01). In addition, the rs34309 variant was associated with functional somatic mutations in the PI3K/PTEN/AKT/MTOR pathway and tumor lymphocyte infiltration. Our study provides plausible evidence that genetic variations in T cell cancer immune response can influence risks of aggressiveness and reclassification in localized PCa, which may lead to additional biological insight into these outcomes. Abbreviations: PCa, prostate cancer; AS, active surveillance; GS, Gleason score; PSA, prostate specific antigen; TCGA, The Cancer Genome Atlas; SNP, single nucleotide polymorphisms; UFG, unfavorable genotype.

Abstract 5658: From staining to analysis: End-to-end application of InSituPlexTM technology for multiplexed immuno-profiling in FFPE tumor samples

Abstract Introduction: The development of new and improved cancer diagnostic and therapeutic methods relies on a deep understanding of the protein expression profile in the tumor microenvironment. Conventional immunohistochemistry (IHC) methods only provide insights into a limited number of markers of interest, prompting the need for the development of multiplexed methods to assess and characterize multiple cell phenotypes and their spatial context in a single sample. We have developed an approach to enable high-multiplexing detection of protein markers in FFPE tumor samples using an integrated workflow with the Leica Bond RX autostainer and Leica Versa whole slide scanner. Methods: A cocktail of primary antibodies of interest, modified with unique, addressable DNA barcodes, is applied to tissue samples in a single staining step. The barcodes are then amplified simultaneously to improve sensitivity. Finally, a cocktail of fluorescently labeled oligonucleotides, complementary to the barcodes on the antibodies, is used to tag the different targets of interest for multiplexed imaging. Formalin-fixed, paraffin-embedded samples from both human tonsil and primary tumor biopsies were stained manually and automatically on the BondRX, using a panel of antibodies specific to cancer immuno-profiling markers. Images were acquired using the Leica Versa whole slide scanner, and analyzed with Leica Aperio Image Analysis and Indica Labs Halo software. Results: Compared to typical multiplex IHC methods, the InSituPlex single staining step offers multiple advantages. First, the workflow preserves the integrity and antigenicity of the samples by avoiding any antibody stripping steps and streamlining the assay development process by eliminating the need to optimize staining order. In addition, the total assay time stays constant regardless of the multiplexing level. Adding targets to the panel does not increase staining time, facilitating the optimization of higher multiplex assays, as well as the development of streamlined automated assays. A fully automated staining protocol was developed on the BondRX by modifying assay parameters such as reagent concentrations and volumes, optimizing the timing of each step, and evaluating the impact on the resulting images in terms of specificity, sensitivity and overall signal to background ratio. After optimization, the automated staining protocol delivered similar or better results compared to the manual assay protocol, while decreasing the total assay time by approximately 40%. The optimized protocol was subsequently used to carry out a multiplexed analysis of immune cell phenotypes in tumor samples. Conclusions: the InSituPlex technology enables multiplexed immune-profiling in tumor FFPE sections, and provides a streamlined, high-throughput workflow with existing automated staining and imaging instrumentation. Citation Format: Mael Manesse, Amanda Bares, Heike Boisvert, Stephanie Walter. From staining to analysis: End-to-end application of InSituPlexTM technology for multiplexed immuno-profiling in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5658.

Abstract 1747: Hyaluronan (HA) accumulation restricts CD8+ T cell numbers and skews tumor-associated macrophage (TAM) phenotype in mouse syngeneic pancreatic tumors

Abstract The glycosaminoglycan hyaluronan (HA) is abundant in the tumor microenvironment (TME) of diverse solid tumor types, and tumor accumulation of HA is associated with poor patient outcome. We previously demonstrated, in multiple HA-rich mouse syngeneic tumor models, that enzymatic degradation of HA using PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20) enhanced tumor growth inhibition and increased tumor accumulation of T and NK cells when combined with a checkpoint inhibitor. However, the ability of HA accumulation in the TME to alter the composition of tumor-infiltrating immune cells has not been well characterized. Here, we addressed this question by performing a detailed immune phenotypic analysis of HA-poor and HA-rich tumors using a mouse syngeneic pancreatic tumor model. To achieve this, we transduced Pan02 cells with a lentivirus encoding hyaluronan synthase 3 (HAS3, Pan02-HAS3) or empty vector (EV, Pan02-EV). After peritibial implantation, Pan02-HAS3 tumors contained 13.4-fold higher levels of HA than Pan02-EV tumors, as determined by HA ELISA. Both variants grew with similar kinetics to the Pan02 parental line. Flow cytometric analysis of tumor-infiltrating immune cells in four independent and identical studies revealed an average 6.2-fold reduction in the number of CD8+ T cells in Pan02-HAS3 tumors compared with Pan02-EV. Likewise, numbers of CD4+ T cells and NK cells were reduced by an average of 4.8- and 2.9-fold, respectively, in Pan02-HAS3 tumors compared with Pan02-EV. In contrast, we did not observe consistent differences in the numbers of tumor-associated macrophages (TAMs), neutrophils/granulocytic myeloid-derived suppressor cells (G-MDSC), monocytes/monocytic (M)-MDSC, or dendritic cells (DCs) between the two tumor variants. However, within the TAM population, cells displaying an “M2-like” phenotype (CD206high MHCIIlow) were present at an average 10.5-fold higher frequency in Pan02-HAS3 tumors compared with Pan02-EV. Similarly, the percentage of DCs that were CD103+ CD11b- was reduced by 1.4-fold in Pan02-HAS3 tumors compared with Pan02-EV. Together, our data indicate that HAS3-driven HA accumulation in Pan02 mouse syngeneic tumors induces several aspects of immunosuppression: it restricts the numbers of tumor-infiltrating lymphocytes, and skews TAMs and DCs toward immunosuppressive phenotypes. These findings are consistent with our previous work, which demonstrated that combining PEGPH20 with anti-PD-L1 increased tumor-infiltrating lymphocyte numbers and reduced the frequency of CD206high MHCIIlow TAMs in Pan02-HAS3 tumors. Our results support the ongoing clinical evaluation of PEGPH20 in combination with immunotherapy in HA-rich tumor indications (NCT02563548 and NCT03193190). Citation Format: Benjamin Thompson, Trevor Kimbler, Jisook Lee, Chunmei Zhao, Kelly Chen, Renee Clift, Curtis B. Thompson, Sanna Rosengren, Daniel C. Maneval. Hyaluronan (HA) accumulation restricts CD8+ T cell numbers and skews tumor-associated macrophage (TAM) phenotype in mouse syngeneic pancreatic tumors [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 1747.

Tu1865 - High Dimensional Immune Phenotyping and Transcriptional Analysis Reveal Robust Recovery of Viable Human Immune and Epithelial Cells from Frozen Gastrointestinal Tissue

Tu1865 - High Dimensional Immune Phenotyping and Transcriptional Analysis Reveal Robust Recovery of Viable Human Immune and Epithelial Cells from Frozen Gastrointestinal Tissue

Terminal differentiation of T cells is strongly associated with CMV infection and increased in HIV-positive individuals on ART and lifestyle matched controls.

HIV-1-positive individuals on successful antiretroviral therapy (ART) are reported to have higher rates of age-associated non-communicable comorbidities (AANCCs). HIV-associated immune dysfunction has been suggested to contribute to increased AANCC risk. Here we performed a cross-sectional immune phenotype analysis of T cells in ART-treated HIV-1-positive individuals with undetectable vireamia (HIV-positives) and HIV-1-negative individuals (HIV-negatives) over 45 years of age. In addition, two control groups were studied: HIV negative adults selected based on lifestyle and demographic factors (Co-morBidity in Relation to AIDS, or COBRA) and unselected age-matched donors from a blood bank. Despite long-term ART (median of 12.2 years), HIV-infected adults had lower CD4+ T-cell counts and higher CD8+ T-cell counts compared to well-matched HIV-negative COBRA participants. The proportion of CD38+HLA-DR+ and PD-1+ CD4+ T-cells was higher in HIV-positive cohort compared to the two HIV-negative cohorts. The proportion CD57+ and CD27−CD28− cells of both CD4+ and CD8+ T-cells in HIV-positives was higher compared to unselected adults (blood bank) as reported before but this difference was not apparent in comparison with well-matched HIV-negative COBRA participants. Multiple regression analysis showed that the presence of an increased proportion of terminally differentiated T cells was strongly associated with CMV infection. Compared to appropriately selected HIV-negative controls, HIV-positive individuals on ART with long-term suppressed viraemia exhibited incomplete immune recovery and increased immune activation/exhaustion. CMV infection rather than treated HIV infection appears to have more consistent effects on measures of terminal differentiation of T cells.

A clinical research on imaging and pathological features of primary central nervous system lymphoma

Objective To investigate the clinical features, imaging and pathological characteristics of primary central nervous system lymphoma (PCNSL) in order to increase the diagnostic accuracy of PCNSL in clinic. Methods A retrospective analysis of 6 cases with PCNSL confirmed by pathological and immunohistochemical examination, treated in our hospital from December 2013 to August 2014 was performed. The clinical presentations, lesion locations, imaging manifestations and histopathological features were analysed. Results The symptoms were various, among which headache and dizziness were frequently presented. Five cases were identified to have solitary tumor foci and the other one had multiple lesions. Among 7 lesions, 4 were located in cerebral hemisphere, one was located in cerebellum, one in lateral ventricle and one in thalamus. Moreover, 2 lesions were involved in corpus callosum. The lesions presented in roundish shape or irregular shape with clear boundary, minimal edema and mass effect. CT showed high-density of the lesions. All lesions showed iso- or slightly hypo-intense signal on T 1 WI and iso- or mild hyper-intense on T 2 WI, and homogeneous enhancement after contrast. All cases underwent operations. The tumors appeared flesh red or grey-white, soft and without capsules. Under microscopic examination, the tumor cells were distributed around the blood vessels as sleeve-like infiltration. Immune phenotype analysis showed all tumors were B-cell lymphoma. Conclusions PCNSL has certain imaging and pathological features and most of them are diffuse large B-cell lymphoma (DLBCL). Pathological examination and immune phenotype analysis are gold standard in making confirmed diagnosis. DOI: 10.3969/j.issn.1672-6731.2016.11.014

Establishment of specific cytotoxic T lymphocyte culture system and its inhibitory effect on ovarian cancer.

The present study aimed to establish a novel method for efficiently inducing cytotoxic T lymphocytes (CTLs) in vitro, in order to develop an immune-based therapy for suppressing and killing ovarian cancer cells with a high safety and efficacy. Peripheral blood mononuclear cells (PBMCs) were stimulated with CpG oligodeoxynucleotide (CpGODN) and ginsenoside Rg1, which were united as an immune adjuvant, and human epidermal growth factor receptor 2 (HER2/neu) antigen peptide, in order to establish a specific CTL culture system in vitro. Chromosome karyotype analysis, growth curve construction and flow cytometric analysis of immune phenotypes, including cluster of differentiation (CD)3, CD4 and CD8, were performed to characterize the stimulated PBMCs in vitro. Subsequently, SKOV3 ovarian cancer cells were treated with the specific CTL culture system in vitro, and MTT assays were performed to test the inhibitory and lethal effects of the CTLs on SKOV3 cells. The number of CTLs was significantly increased from day 7 of stimulation with the specific mixture (CpGODN, ginsenoside Rg1 and HER2/neu) (P<0.01), and plateaued on day 19. Following activation, the number of CD3+, CD3+CD4+ and CD3+CD8+ cells was significantly increased (P<0.01). The lymphocyte karyotype did not change following exposure to antigen. After treatment with the specific CTL system, the number of SKOV3 cells in the experimental group was significantly reduced compared with that in the control group (P<0.01). The results of the present study suggested that two novel immune adjuvants, CpGODN and ginsenoside Rg1, could be combined with the HER2/neu antigen peptide to establish a specific CTL culture system in vitro. This system demonstrated a high antigen specificity, safety and proliferative ability, and exerted significant lethal and inhibitory effects on SKOV3 cells in vitro.

XLF deficiency results in reduced N-nucleotide addition during V(D)J recombination

AbstractRepair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining pathway (NHEJ) is important not only for repair of spontaneous breaks but also for breaks induced in developing lymphocytes during V(D)J (variable [V], diversity [D], and joining [J] genes) recombination of their antigen receptor loci to create a diverse repertoire. Mutations in the NHEJ factor XLF result in extreme sensitivity for ionizing radiation, microcephaly, and growth retardation comparable to mutations in LIG4 and XRCC4, which together form the NHEJ ligation complex. However, the effect on the immune system is variable (mild to severe immunodeficiency) and less prominent than that seen in deficiencies of NHEJ factors ARTEMIS and DNA-dependent protein kinase catalytic subunit, with defects in the hairpin opening step, which is crucial and unique for V(D)J recombination. Therefore, we aimed to study the role of XLF during V(D)J recombination. We obtained clinical data from 9 XLF-deficient patients and performed immune phenotyping and antigen receptor repertoire analysis of immunoglobulin (Ig) and T-cell receptor (TR) rearrangements, using next-generation sequencing in 6 patients. The results were compared with XRCC4 and LIG4 deficiency. Both Ig and TR rearrangements showed a significant decrease in the number of nontemplated (N) nucleotides inserted by terminal deoxynucleotidyl transferase, which resulted in a decrease of 2 to 3 amino acids in the CDR3. Such a reduction in the number of N-nucleotides has a great effect on the junctional diversity, and thereby on the total diversity of the Ig and TR repertoire. This shows that XLF has an important role during V(D)J recombination in creating diversity of the repertoire by stimulating N-nucleotide insertion.

Abstract 4885: Intratumoral injection of G100 (TLR4 agonist glycopyranosyl lipid A) modulates tumor microenvironment and induces CD8 T cell-dependent, systemic anti-tumor immunity

Abstract The tumor microenvironment (TME) plays a critical role in controlling the balance between tumor progression and immune surveillance. Increased infiltration of T cells, especially CD8 T cells has been associated with a good prognosis. We hypothesized that G100, a novel synthetic TLR4 agonist, can modulate TME when directly injected into the tumor and trigger both a local and systemic effective immune response. Balb/c mice with implanted syngeneic A20 lymphoma received intratumoral (IT) injection of G100 (10 ìg) or control PBS three times a week. This treatment significantly inhibited tumor growth and resulted in complete tumor regression in approximately 60% of treated mice. To investigate the effects of G100 on TME, tumors were collected after three IT G100 injections for gene expression analysis by Nanostring and immune phenotyping analysis by FACS. Out of the 770 immune response genes included in the mouse PanCancer Immune Profiling panel, 295 genes were significantly upregulated in G100 treated tumors. The upregulated genes include DC function-related genes (CD40, CD83, CD86, and Ly96) and T cell and NK cell function genes and multiple chemokines and cytokines (IL1b, IL12A, IL18, IL6, IFNã, FcãR4, ICOS, GZMB, CCL3, CCL5, CCL7, CXCL1, CXCL2, CXCL11, CCR5, CCR6, and CCR7). T cell exhaustion markers (CTLA4 and LAG3) and CD274 (PD-L1) were also induced. G100-induced inflammation is also reflected at the cellular level as shown by increased T cells and NK cells in tumor via FACS analysis. To determine the immune cells that mediated tumor rejection, mice were selectively depleted of CD4 or CD8 T cells during G100 treatment. Results showed that the anti-tumor effect of G100 is dependent on CD8 T cells. G100-induced tumor protection was durable as mice surviving the first tumor challenge rejected a secondary tumor challenge without additional G100 treatment. Altogether, our results showed that IT G100 induces a proinflammatory cytokine and chemokine milieu that changes a “cold” tumor to a “hot” tumor, which facilitates the development of a CD8 T cell-dependent potent and durable anti-tumor effect. The induction of PD-L1 also suggests the potential synergy between G100 and checkpoint blockade therapy. These preclinical data support an on-going clinical trial of IT G100 in patients with follicular non-Hodgkin's lymphoma (NCT02501473), alone and in combination with anti-PD-1 therapy (pembrolizumab). Citation Format: Hailing Lu, Jessica Hewitt, Jan ter Meulen. Intratumoral injection of G100 (TLR4 agonist glycopyranosyl lipid A) modulates tumor microenvironment and induces CD8 T cell-dependent, systemic anti-tumor immunity. [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 4885.

Abstract A195: Tremelimimab activates CD4 and CD8+ T cells in patients with hepatocellular carcinoma

Abstract Introduction: Tremelimumab is a fully human monoclonal antibody that binds to CTLA-4 expressed on the surface of activated T lymphocytes and results in inhibition of B7-CTLA-4-mediated downregulation of T-cell activation. Patients with advanced hepatocellular carcinoma, who progressed on standard of care, were treated with tremelimumab and tumor ablation (radiofrequency ablation and transcatheter arterial chemoembolization) to augment anti-tumor immunity. Treatment was safe and feasible. Encouraging clinical activity has been seen with objective confirmed partial responses in 4/12 (33%) evaluable patients and a time to progression of 7.4 months. Here we report first results from immunoanalysis evaluating tumor biopsies, immune cell subsets in peripheral blood and serum viral loads in patients with chronic HBV or HCV infection. Methods: 20 Patients with HCC were treated with tremelimumab and tumor ablation on clinical trial NCT01853618. Tumor biopsies were taken at the time of ablation and peripheral blood mononuclear cells (PBMC) were collected before and during treatment in a subset of patients. Serum samples were tested for viral load (quantitative HCV RNA). Serum HBsAg titers were measured by chemiluminescent microparticle immunoassay (CMIA) using the ARCHITECT platform (Abbott Laboratories, Chicago, IL), as per the manufacturer's instructions. Tumor biopsies with analyzed by immunohistochemistry for CD3, CD4, CD8, CD20 and Granzyme B. Eleven-color flow cytometry was performed to study PBMC using the following antibodies: CD4, CD3, CD4, CD8, CD11c, CD14, CD19, CD20, CD25, CD38, CD45RA, CD56, CD123, CD127, CCR7, CCR4, CXCR3, PD-1, 4-1BB, TIM3, CTLA4, PD-L1, ICOS, HLA-DR. Statistical analysis was done using the Wilcoxon signed rank test. Results presented are preliminary and update data will be presented at meeting. Results: Immunohistochemical analysis of tumor biopsies demonstrated an increase in the CD3+CD8+ T cell population in tumors. CD8+ T cells stained positive for Granzyme A. No changes in the number of CD68+ macrophages were seen. Multi-color flow cytotometry PBMC revealed statistically significant changes after the 1st cycle of activated CD4+ and CD8+ T cells. There was a trend towards a reduction in CD4+ Tregs. Activated CD8+ T cells remained elevated for more than three months. Eight of 9 patients with quantifiable HCV experienced a marked reduction in viral load. Four of 4 HBV patients experienced a reduction in quantitative HepBsAg. Analysis of immune cell phenotype and more in depth analysis of tumor samples is ongoing and will be reported. Conclusions: Tremelimumab in combination with subtotal TACE or RFA leads to an accumulation of intratumoral CD8+ T cells, an activation of CD4+ and CD8+ T cells in peripheral blood and reductions in HCV viral load and HBsAg. Citation Format: Firouzeh Korangy, Mei ElGindi, Drew Pratt, David Venzon, Austin Duffy, Oxana Makarova-Rusher, Sid Kerkar, David Kleiner, Bradford Wood, Tim Greten. Tremelimimab activates CD4 and CD8+ T cells in patients with hepatocellular carcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A195.

Rapamycin-conditioned dendritic cells induced immune tolerance through the regulation of Treg/Th17 cells in mice

To investigate the effect of tolerogenic dendritic cells (Tol-DC) generated by Rapamycin (Rapa) on the differentiation of Treg/Th17 cells and explore the possible mechanism of tolerance induction. DC progenitors from mouse bone marrow were propagated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin (IL)-4 stimulation for 6 days in the presence or absence of Rapa (20 ng/ml). During DC culture, morphology of cell was observed under electron microscope. Cell surface expression of CD11c, CD40 and CD80 was analyzed by flow cytometry. The antigen-presenting function of DC was determined by one-way mixed leukocyte reactions. In vivo, the recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control, Rapa, immature DC (imDC) and Tol-DC group. The survival time of the skin allograft was observed and Treg/Th17 cells were analyzed by flow cytometry in each group. The immunephenotypic analysis showed that in comparison with those in the control group and the LPS group the expression of the co-stimulatory molecules CD40 and CD80 were significantly lower in the Rapa-group and Rapa+LPS group. The ability to stimulate proliferation of T cells of the same genotype in the Rapa-group was significantly inhibited (P<0.01). In the in vivo experiment, the mice's survival time remarkably prolonged, the percentage of Treg cells was enhanced and Th17 cells was reduced in the mice's spleen in Tol-DC group. Tol-DC generated by Rapamycin can significantly induce immune tolerance through up-regulate Tregs and down-regulate Th17 cells. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs, which can be loaded with donor antigen, and potentially used to promote organ transplant tolerance.

The Fas Ligand Inhibitor APG101 in Transfusion Dependent Patients with Low Risk MDS: Interim Results from a Phase I Study

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous diseases characterized by clonal, ineffective hematopoiesis resulting in peripheral cytopenia and an increased risk of progression to acute myeloid leukemia. Particularly in low risk MDS a pro-apoptotic milieu has been found with increased levels of apoptosis-promoting factors such as tumor necrosis factor and fas ligand (CD95 ligand). This is thought to be a major pathomechanism in low risk MDS resulting in increased apoptosis of medullary erythroid progenitors leading to peripheral cytopenia. APG101 is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1. APG101 binds to CD95 ligand expressed on effector cells as well as to functionally active ligand in solution, thus blocking the interaction between CD95 and its ligand. Here, we report on interim results of a phase I study in transfusion dependent low risk MDS patients treated with APG101.Methods: A total of 15 patients have been enrolled by July 30th 2014. All patients gave their written informed consent. Patients were treated for 12 weeks with weekly intravenous infusions of 100 mg or 400 mg APG101, respectively. Thereafter the treatment was discontinued and the patients were followed for another 6 months. Bone marrow biopsies were taken prior to the first APG101 dosing (baseline), at the end of treatment (EOT, week 12), at week 25 (follow up) and at week 37 (follow up). Methylcellulose assays on isolated CD34+ cells from the bone marrow aspirates at the different time points were performed. In addition, bone marrow samples at each time point were subjected to amplicon deep sequencing to detect gene mutations prior to APG101 dosing and to follow the mutational allele burden during and after treatment. This was done by sequencing of genomic DNA from ficollized mononuclear bone marrow cells on a Roche 454 GS Junior system.Results: At data cut-off, 5 patients (3 male) had completed the study. Median age was 77 (range 65-78). WPSS categories were low in 2 patients and intermediate in 3 patients, respectively. According to the exclusion criteria none of the patients showed a medullary blast count ≥ 5%. Two patients received 400 mg weekly while 3 patients received 100 mg APG101 weekly, respectively. All patients stayed on treatment as scheduled and no adverse events were reported so far. Careful evaluation of bone marrow smears as well as immune phenotypic analysis of bone marrow aspirates showed no increase in bone marrow blasts at any time point. Moreover, quantification of the allele burden of pre-existing mutations (mutations of ASXL1 and SF3B1 in one patient and DNMT3A in the other patient) showed no significant expansion of the mutated clones. Ex vivo differentiation analyses revealed an increase in granulocytic colony forming as well as CFU-E/BFU-E forming capacity. With regard to efficacy, 4 of 5 patients showed a decrease in transfusion frequencies, which so far did not fulfill the IWG 2006 criteria for hematologic improvement (HI).Conclusions: APG101 is a well tolerable compound in transfusion-dependent low risk MDS patients, particularly in elderly patients. None of the patients discontinued treatment due to toxicity. Monitoring did not reveal any signs of progress i.e. increase in medullary blasts in any of the patients. Deep amplicon sequencing did not show any signs of expansion of preexisting clones during therapy, i.e. APG101 did not provide malignant clones with a survival benefit. Although no major or minor HI (IWG 2006) was seen in 5 analyzed patients, the majority of patients showed a slight reduction in transfusion need which makes APG101 an interesting target for further investigation. DisclosuresKunz:Apogenix GmbH: Employment. Fricke:Apogenix GmbH: Employment. Nolte:Apogenix GmbH: Consultancy, Research Funding.

Comparative Immune Phenotypic Analysis of Cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in Immune-Competent Individuals: Proportional Representation of CD8+ T-Cells but Not FoxP3+ Regulatory T-Cells Is Associated with Disease Stage

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3+ T-cells than photo-damaged skin, however, the abundance of CD3−CD56+ Natural Killer (NK) cells, CD11c+HLA-DR+ conventional Dendritic Cells (cDC), BDCA-2+HLA-DR+ plasmacytoid DC (pDC), FoxP3+ Regulatory T-cells (T-reg), Vα24+Vβ11+ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4+ T-cells were associated with SCC, yet CD8+ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8+ T-cells than IEC. We propose that differences in CD8+ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4+ T-cells to CD8+ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.