Important Mechanism Of Immune Evasion Research Articles

The biological function of Serpinb9 and Serpinb9-based therapy.

Recent breakthroughs in discovering novel immune signaling pathways have revolutionized different disease treatments. SERPINB9 (Sb9), also known as Proteinase Inhibitor 9 (PI-9), is a well-known endogenous inhibitor of Granzyme B (GzmB). GzmB is a potent cytotoxic molecule secreted by cytotoxic T lymphocytes and natural killer cells, which plays a crucial role in inducing apoptosis in target cells during immune responses. Sb9 acts as a protective mechanism against the potentially harmful effects of GzmB within the cells of the immune system itself. On the other hand, overexpression of Sb9 is an important mechanism of immune evasion in diseases like cancers and viral infections. The intricate functions of Sb9 in different cell types represent a fine-tuned regulatory mechanism for preventing immunopathology, protection against autoimmune diseases, and the regulation of cell death, all of which are essential for maintaining health and responding effectively to disease challenges. Dysregulation of the Sb9 will disrupt human normal physiological condition, potentially leading to a range of diseases, including cancers, inflammatory conditions, viral infections or other pathological disorders. Deepening our understanding of the role of Sb9 will aid in the discovery of innovative and effective treatments for various medical conditions. Therefore, the objective of this review is to consolidate current knowledge regarding the biological role of Sb9. It aims to offer insights into its discovery, structure, functions, distribution, its association with various diseases, and the potential of nanoparticle-based therapies targeting Sb9.

Deep mutagenesis scanning using whole trimeric SARS-CoV-2 spike highlights the importance of NTD-RBD interactions in determining spike phenotype.

New variants of SARS-CoV-2 are continually emerging with mutations in spike associated with increased transmissibility and immune escape. Phenotypic maps can inform the prediction of concerning mutations from genomic surveillance, however most of these maps currently derive from studies using monomeric RBD, while spike is trimeric, and contains additional domains. These maps may fail to reflect interdomain interactions in the prediction of phenotypes. To try to improve on this, we developed a platform for deep mutational scanning using whole trimeric spike. We confirmed a previously reported epistatic effect within the RBD affecting ACE2 binding, that highlights the importance of updating the base spike sequence for future mutational scanning studies. Using post vaccine sera, we found that the immune response of vaccinated individuals was highly focused on one or two epitopes in the RBD and that single point mutations at these positions can account for most of the immune escape mediated by the Omicron BA.1 RBD. However, unexpectedly we found that the BA.1 RBD alone does not account for the high level of antigenic escape by BA.1 spike. We show that the BA.1 NTD amplifies the immune evasion of its associated RBD. BA.1 NTD reduces neutralistion by RBD directed monoclonal antibodies, and impacts ACE2 interaction. NTD variation is thus an important mechanism of immune evasion by SARS-CoV-2. Such effects are not seen when pre-stabilized spike proteins are used, suggesting the interdomain effects require protein mobility to express their phenotype.

EP08.01-013 Ramucirumab plus Atezolizumab in Patients with Stage IV NSCLC Previously Treated with Immune Checkpoint Blockade

Angiogenesis is an important mechanism of immune evasion and there are emerging data suggesting a benefit from combining immune checkpoint inhibitors (ICIs) with anti-angiogenic therapies. We evaluated the combination of atezolizumab and ramucirumab in patients (pts) with previously treated non-small cell lung cancer (NSCLC).

T Cell Versus T Cell; A Study of the Immune Checkpoint Landscape in Cutaneous T Cell Lymphoma

Introduction.Tumor infiltrating T cells (TILs) are capable of recognising tumor-specific mutations presented as peptides on MHC class I. Overexpression of immune checkpoint ligands on cancer cells, and the corresponding expression of the receptors on T cells, appears to be an important mechanism of immune evasion, exploited with success in immune checkpoint therapy. However, when the cancer cell is itself a T cell, the microenvironment interaction is likely to be more complex. To investigate this further we utilized the clonal nature of mycosis fungoides (MF), a primarily CD4 T cell cancer, by targeting the TCR receptor, differentiating between TIL and tumor in the microenvironment.Methods.43 patients with mycosis fungoides (MF) were prospectively consented to the study, and a skin biopsy and blood sample were obtained. The skin biopsy was macerated and digested with collagenase D, and a TCR V-beta clonogram was used to identify the tumor population. Flow cytometry was used to isolate five cell populations: peripheral blood CD4 and CD8, TIL CD4 and CD8, and tumor cells. The expression of a panel of immune checkpoint receptors (PD-1, TIM-3, LAG-3, Fas & TIGIT) and ligands (PD-L1, PD-L2, Galectin 9, Fas ligand) were measured using conjugated antibodies.Results.46 biopsies from MF patients with stage IA(n=6), IB(n=18), IIA(n=1), IIB(N=14), IIA(n=1) & IVA(n=3) were analysed with paired blood. The technique was effective in distinguishing tumour from TIL in 20 cases, isolating a population with CD7 downregulation, high forward and side scatter (indicative of larger and more complex cells) and a unique immune checkpoint profile (figure 1a & 1b). Tumour infiltrating lymphocytes of both CD4 and CD8 phenotype demonstrated significant overexpression of immune checkpoint receptors PD-1 (p < 0.0001), TIGIT (p < 0.0001) & fas (p < 0.0001), with an increased population of T regulatory cells in the CD4 cells (p < 0.001) and CD8 expressing high TIM-3 (p < 0.0001) compared to peripheral blood. While the TIL population was similar across the demographic, the tumour population demonstrated a significantly varied phenotype, with up and down-regulation of immune checkpoints and HLA-DR (figure 1c). Principal component analysis (figure 1d) revealed three tumour subtypes:1. High PD-L1, Fas, HLA-DR (n=4)2. High PD-1. Low PD-L1 (n=12)3. Low PD-1, PD-L1, Fas (n=3)These subtypes did not relate to disease type, stage, presence of large cell transformation or folliculotropic disease, though those with advanced disease had tumor phenotypes which differed more from the TILs. Tumor cells not only expressed MHC class I and HLA-DR, but upregulated these compared to peripheral blood (p < 0.001).Discussion.The TIL phenotype in MF is surprisingly homogeneous across the demographic, demonstrating upregulation of immune checkpoint receptors and an exhaustion phenotype seen in other cancers. However, the tumor cells did not show consistent upregulation of immune checkpoint ligands, instead their phenotype across patients was heterogeneous with both up and down-regulation. Expression of immune checkpoint ligands by the tumor cells may have negative growth consequences for both tumor cells as well as the immune system, particular since tumor T cells often express immune checkpoint receptors. We did not see concurrent high expression of PD-L1 and PD-1 on tumor cells, which would be expected to be detrimental to tumor survival. Immune checkpoint ligand over-expression is unlikely to be the main method of immune evasion in mycosis fungoides, and as a result, immune checkpoint inhibition may have unexpected responses in this disease.Legends.Figure 1a. TCR V-beta antibody used to separate three CD4 populations (red: peripheral blood CD4, green: CD4 TIL, blue: tumor) and compare HLA-DR expression.Figure 1b. tSNE clustering of cells in the tumour microenvironment, demonstrating clear separation of three populations - tumor, CD4 TIL and CD8 TIL. Coloring by expression of PD-L1 (red: high, blue: low).Figure 1c. Heatmap of immune checkpoint markers (red: high, blue: low) in the tumor cell population only. This demonstrates the heterogeneity, particularly in PD-1, Fas, PD-L1 and HLA-DR expression.Figure 1d. Principal component analysis demonstrating the observation that TIL expression of immune checkpoints are similar between patients (green and blue), whereas tumor expression of these markers (red) can vary markedly and cluster. [Display omitted] DisclosuresScarisbrick:NHS: Employment; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Mallinckrodt: Consultancy, Honoraria; Innate Pharma: Consultancy; Actelion: Consultancy.

Targeting tumor-associated macrophages in the tumor microenvironment.

Tumor-associated macrophages (TAMs) are the most abundant population type of tumor-infiltrating immune cells found in the tumor microenvironment (TME), and are evolutionarily associated with microvessel density in tumor tissues. TAMs can be broadly divided into M1-like and M2-like TAMs, which demonstrate antitumor and pro-tumor activity in the TME, respectively. Studies have indicated that: i) The predominate presence of M2-like TAMs in the TME can result in tumor immunosuppression and chemoresistance; ii) the ratio of M1-like to M2-like TAMs in the TME is positively correlated with better long-term prognosis of patients with cancer; iii) epigenetic silencing, preventing the secretion of M1-like TAM-associated molecules, is an important immune evasion mechanism during tumor progression; and iv) the transformation from M2-like to M1-like TAMs following exposure to specific conditions can result in tumor regression. The present study discusses the molecular events underlying the recruitment of macrophages and their polarization into M1-like or M2-like TAMs, and their differential roles in angiogenesis, angiostasis, invasion, metastasis and immune activity in the TME. This insight may inform the improved design of TAM-targeted cancer immunotherapy. Some of these therapeutic strategies show promising effects; however, challenges remain.

Inhibition of MICA and MICB Shedding Elicits NK-Cell-Mediated Immunity against Tumors Resistant to Cytotoxic T Cells.

Resistance to cytotoxic T cells is frequently mediated by loss of MHC class I expression or IFNγ signaling in tumor cells, such as mutations of B2M or JAK1 genes. Natural killer (NK) cells could potentially target such resistant tumors, but suitable NK-cell-based strategies remain to be developed. We hypothesized that such tumors could be targeted by NK cells if sufficient activating signals were provided. Human tumors frequently express the MICA and MICB ligands of the activating NKG2D receptor, but proteolytic shedding of MICA/B represents an important immune evasion mechanism in many human cancers. We showed that B2M- and JAK1-deficient metastases were targeted by NK cells following treatment with a mAb that blocks MICA/B shedding. We also demonstrated that the FDA-approved HDAC inhibitor panobinostat and a MICA/B antibody acted synergistically to enhance MICA/B surface expression on tumor cells. The HDAC inhibitor enhanced MICA/B gene expression, whereas the MICA/B antibody stabilized the synthesized protein on the cell surface. The combination of panobinostat and the MICA/B antibody reduced the number of pulmonary metastases formed by a human melanoma cell line in NOD/SCID gamma mice reconstituted with human NK cells. NK-cell-mediated immunity induced by a mAb specific for MICA/B, therefore, provides an opportunity to target tumors with mutations that render them resistant to cytotoxic T cells.

Staphylococci evade the innate immune response by disarming neutrophils and forming biofilms.

Staphylococcusaureus and Staphylococcusepidermidis can cause many types of infections, ranging from skin infections to implant-associated infections. The primary innate immune response against bacterial infections involves complement activation, recruitment of phagocytes (most importantly neutrophils), and subsequent killing of the pathogen. However, staphylococci are not innocent bystanders; they actively obstruct this immune attack. To do that, S.aureus secretes several immune-evasion proteins to resist attack by the innate immune system. Furthermore, S.aureus and S.epidermidis are known for their ability to form biofilms on implanted medical devices and host tissues, which provides another important immune-evasion mechanism. Understanding these different strategies to resist immune attack will help to develop novel therapies against staphylococcal infections.

Suppression of tumor antigen presentation during aneuploid tumor evolution contributes to immune evasion

ABSTRACTAnti-tumor immune responses impede tumor formation, and cancers have evolved many mechanisms of immune evasion. Confirming earlier findings, we show that human tumors with high chromosomal instability (CIN+) are significantly less immunogenic, as judged by tumor lymphocyte infiltration, compared to those with more stable genomes (CIN-). This finding is paradoxical, as genomic instability is expected to evoke an innate immune response. Importantly, CIN+ tumors and cell lines exhibited suppressed expression of proteins involved in MHC class I antigen presentation at least partly due to DNA hypermethylation of the corresponding genes. Using a mouse model of the in vivo evolution of aneuploid tumors, we found that the induction of chromosomal instability in tumor cells is highly immunogenic due to the activation of the STING/TBK1 pathway and consequent increased interferon signaling and antigen presentation. However, tumors evolving under immune pressure suppress the STING/TBK1 and antigen presentation pathways and evade anti-tumor immune responses. In contrast, CIN+ tumors that develop under low immune pressure in both humans and mice retain efficient MHC class I antigen presentation and immunogenicity. Altogether, this study identifies an important mechanism of immune evasion in chromosomally unstable tumors.

Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription.

Innate immunity is the first line of host defense against viral invasion. The induction of type I interferons (IFNs) and inflammatory cytokines is essential to host antiviral immune responses, which are also key targets of viral immune evasion. Human cytomegalovirus (HCMV) can establish long-term latent infections, in which immune evasion is a pivotal step. In this study, we identified HCMV protein UL44, a DNA polymerase processivity factor, as an inhibitor of the interferon regulatory factor 3 (IRF3)- and NF-κB-dependent antiviral response. Ectopic expression of UL44 inhibited HCMV-triggered induction of downstream effector genes and enhanced viral replication. Conversely, knockdown of UL44 potentiated HCMV-triggered induction of downstream antiviral genes. UL44 interacted with IRF3 and p65, and it inhibited the binding of IRF3 and NF-κB to the promoters of their downstream antiviral genes. These findings reveal an important mechanism of immune evasion by HCMV at the transcriptional level.IMPORTANCE Induction of type I IFNs and inflammatory cytokines plays pivotal roles in host antiviral innate immune responses. Viruses have evolved various mechanisms to interfere with these processes. HCMV causes severe ailments in immunodeficient populations and is a major cause of birth defects. It has been shown that HCMV antagonizes host innate immune defenses, which is important for establishing immune evasion and latent infection. In this study, we identified the HCMV DNA polymerase subunit UL44 as a suppressor of antiviral innate immune responses. Overexpression of UL44 impaired HCMV-triggered induction of type I IFNs and other antiviral genes and thus potentiated viral replication, whereas UL44 deficiency showed opposite effects. Mechanistic studies indicated that UL44 acts by inhibiting the binding of IRF3 and NF-κB to the promoters of downstream antiviral genes. These findings defined an important mechanism of HCMV immune evasion at the transcriptional level, which may provide a therapeutic target for the treatment of HCMV infection.

Programmed death ligand-1, tumor infiltrating lymphocytes and HLA expression in Chinese extrahepatic cholangiocarcinoma patients: Possible immunotherapy implications.

Immunotherapy might be an effective treatment in extrahepatic cholangiocarcinoma (eCCA), a tumor with extremely limited therapeutic options. Our study is to characterize the programmed death ligand-1 (PD-L1) protein expression and cancer microenvironment profiles in surgically resected eCCA samples. PD-L1 positivity was observed on tumor cells (32.3%) as well as on tumor-associated macrophages (74.2%). PD-L1 expression by eCCA correlated significantly with immune parameters such as intra-tumoral CD3+ tumor infiltrating lymphocytes (TILs) density (P = 0.002), intra-tumoral CD8+ TILs density (P < 0.001), and the expression pattern of human leukocyte antigen (HLA) class I (P < 0.001). Immunofluorescence showed that PD-L1 positive tumor cells were adjacent to PD-1 positive cells and the stroma covered with interferon-γ. Correlation with clinicopathological parameters and survival analyses revealed that PD-L1 positivity in eCCA was related to the absence of venous invasion (P = 0.030), improved overall survival (P = 0.020) and progressionfree survival (P = 0.011). HLA class I molecules defect, which is an important mechanism of immune evasion, was frequently observed in eCCA (50.0%) and was associated with a decreased number of intra-tumoral CD8+ TIL density (P = 0.028). Additionally, the presence of unusually high numbers of tumor-associated macrophages (TAMs) subsets M2 in most of eCCA (74.2%) was noted. Our study indicated that PD-L1 expression in association with intra-tumoral TILs infiltration and HLA class I expression in 32.3% of the eCCA reflects an active immune microenvironment potentially responsive to PD-1/PD-L1 inhibitors. In addition, the combination of macrophage-targeting agents may provide therapeutic synergy for future immunotherapy.

HDAC is indispensable for IFN-\u03b3-induced B7-H1 expression in gastric cancer

BackgroundB7 homolog 1 (B7-H1) overexpression on tumor cells is an important mechanism of immune evasion in gastric cancer (GC). Elucidation of the regulation of B7-H1 expression is urgently required to guide B7-H1-targeted cancer therapy. Interferon gamma (IFN-γ) is thought to be the main driving force behind B7-H1 expression, and epigenetic factors including histone acetylation are recently linked to the process. Here, we investigated the potential role of histone deacetylase (HDAC) in IFN-γ-induced B7-H1 expression in GC. The effect of Vorinostat (SAHA), a small molecular inhibitor of HDAC, on tumor growth and B7-H1 expression in a mouse GC model was also evaluated.ResultsRNA-seq data from The Cancer Genome Atlas revealed that expression of B7-H1, HDAC1–3, 6–8, and 10 and SIRT1, 3, 5, and 6 was higher, and expression of HDAC5 and SIRT4 was lower in GC compared to that in normal gastric tissues; that HDAC3 and HDAC1 expression level significantly correlated with B7-H1 in GC with a respective r value of 0.42 (p < 0.001) and 0.21 (p < 0.001). HDAC inhibitor (Trichostatin A, SAHA, and sodium butyrate) pretreatment suppressed IFN-γ-induced B7-H1 expression on HGC-27 cells. HDAC1 and HDAC3 gene knockdown had the same effect. SAHA pretreatment or HDAC knockdown resulted in impaired IFN-γ signaling, demonstrated by the reduction of JAK2, p-JAK1, p-JAK2, and p-STAT1 expression and inefficient STAT1 nuclear translocation. Furthermore, SAHA pretreatment compromised IFN-γ-induced upregulation of histone H3 lysine 9 acetylation level in B7-H1 gene promoter. In the grafted mouse GC model, SAHA treatment suppressed tumor growth, inhibited B7-H1 expression, and elevated the percentage of tumor-infiltrating CD8+ T cells.ConclusionHDAC is indispensable for IFN-γ-induced B7-H1 in GC. The study suggests the possibility of targeting B7-H1 using small molecular HDAC inhibitors for cancer treatment.

Human NK Cells Develop an Exhaustion Phenotype During Polar Degranulation at the Aspergillus fumigatus Hyphal Synapse.

Pulmonary aspergillosis is an opportunistic fungal infection affecting immunocompromised individuals. Increasing understanding of natural killer (NK) cell immunobiology has aroused considerable interest around the role of NK cells in pulmonary aspergillosis in the immunocompromised host. Murine studies indicate that NK cells play a critical role in pulmonary clearance of A. fumigatus. We show that the in vitro interaction between NK cells and A. fumigatus induces partial activation of NK cell immune response, characterised by low-level production of IFN-γ, TNF-α, MIP-1α, MIP-1β, and RANTES, polarisation of lytic granules and release of fungal DNA. We observed a contact-dependent down-regulation of activatory receptors NKG2D and NKp46 on the NK cell surface, and a failure of full granule release. Furthermore, the NK cell cytokine-mediated response to leukaemic cells was impaired in the presence of A. fumigatus. These observations suggest that A. fumigatus-mediated NK cell immunoparesis may represent an important mechanism of immune evasion during pulmonary aspergillosis.

Abstract 4550: CD80 vIgD-Fc proteins combine checkpoint antagonism and costimulatory signaling for potent antitumor immunity

Abstract Introduction: PD-1 pathway antagonists have revealed the importance of checkpoint pathways in regulating antitumor immunity, but an existing immune response is generally required for clinical efficacy. Specific T cell costimulation through CD28 is central to this process, but the CD28 ligands CD80 and CD86 are often poorly expressed in the tumor microenvironment, accounting for a second important mechanism of immune evasion by tumors. In contrast, PD-L1 expression has been found extensively in multiple tumor cell types. Therapeutics that combine PD-L1/PD-1 antagonism coupled with PD-L1 dependent CD28 agonism may therefore provide a more potent, yet safe immunotherapeutic approach. Experimental Procedures: The variant Ig Domain (vIgD)TM platform has generated a diversity of human CD80 variants using yeast display affinity maturation and selections against all three CD80 counterstructures CD28, CTLA-4, and PD-L1. CD80 vIgDs were produced in a mammalian expression system as recombinant Fc fusion proteins (CD80 vIgD-Fc proteins) and their binding properties were quantified by flow cytometry. Functional activity was determined in vitro by assessing responses from human primary T cells or an IL-2-luciferase Jurkat T cell reporter line stimulated with PD-L1-expressing artificial antigen presenting cells (aAPCs). In vitro human T cell cytotoxicity assays with a human PD-L1-expressing tumor line were also performed. Antitumor activity was assessed in vivo with mice implanted with human PD-L1 transduced MC38 tumors. Data Summary: The human CD80 IgV fragment was found to be optimal for high affinity PD-L1 and CD28 binding. A large panel of CD80 vIgD-Fc proteins demonstrated a range of binding towards CD28, PD-L1, and/or CTLA-4, and CD80 vIgD-Fc proteins with high affinity for PD-L1 antagonized the PD-L1/PD-1 interaction. Some CD80 vIgD-Fc proteins agonized CD28 in a PD-L1 dependent fashion with increased luciferase activity in the Jurkat reporter assay as well as increased cytokine production by primary human T cells when stimulated with a PD-L1 expressing aAPC in vitro. The same candidates also showed specific killing of human PD-L1 expressing tumor cells in vitro compared to the parental tumor line lacking PD-L1 expression. Importantly, selected CD80 vIgD-Fc proteins caused significant tumor reduction in the MC38 in vivo tumor model. Conclusion: Engineered CD80 vIgD-Fc proteins that deliver a localized CD28 costimulatory signal to T cells while simultaneously antagonizing the inhibitory PD-L1/PD-1 pathway may provide a transformative mechanism of action to drive potent, tolerable antitumor immunity. Preclinical development of therapeutic candidates is under way. Citation Format: Ryan Swanson, Mark F. Maurer, Chris L. Navas, Chelsea J. Gudgeon, Joseph L. Kuijper, Martin Wolfson, Katherine E. Lewis, Stacey R. Dillon, Steve D. Levin, Michael G. Kornacker. CD80 vIgD-Fc proteins combine checkpoint antagonism and costimulatory signaling for potent antitumor immunity [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 4550.

Human Cytomegalovirus Tegument Protein UL82 Inhibits STING-Mediated Signaling to Evade Antiviral Immunity

Recognition of human cytomegalovirus (HCMV) DNA by the cytosolic sensor cGAS initiates STING-dependent innate antiviral responses. HCMV can antagonize host immune responses to promote latency infection. However, it is unknown whether and how HCMV targets the cGAS-STING axis for immune evasion. Here we identified the HCMV tegument protein UL82 as a negative regulator of STING-dependent antiviral responses. UL82 interacted with STING and impaired STING-mediated signaling via two mechanisms. UL82 inhibited the translocation of STING from the ER to perinuclear microsomes by disrupting the STING-iRhom2-TRAPβ translocation complex. UL82 also impaired the recruitment ofTBK1 and IRF3 to the STING complex. The levels of downstream antiviral genes induced by UL82-deficientHCMV were higher than those induced bywild-type HCMV. Conversely, wild-type HCMV replicated more efficiently than the UL82-deficient mutant. These findings reveal an important mechanism of immune evasion by HCMV.

Abstract 4024: Epigenetic priming potentiates immune checkpoints inhibitors in ovarian cancer

Abstract Background: Ovarian cancer (OC) progression is accompanied by the establishment of stable and transcriptionally repressive epigenetic modifications. An important mechanism of immune evasion is represented by epigenetic silencing of tumor antigens (NY-ESO-1, Muc16 and MAGE). We hypothesize that by reversing DNA methylation, DNA methyl transferase inhibitors (DNMTIs) restore the expression of such antigens, potentiating anti-tumor immune response. The targeting of immune checkpoints regulated by programmed cell death protein-1(PD-1) signaling represents a novel therapeutic strategy in cancer, including in OC. Here we set out to measure the anti-tumor effects of epigenetic priming in combination with PD-1/PDL-1 blockade in OC preclinical models. Methods: The ID8 intraperitoneal (ip) immunocompetent syngeneic mouse model was used to measure the effects of the novel DNMTI guadecitabine (SGI-110, Astex Pharmaceuticals Inc) and PDL1 blockade. The experimental groups consisted of non-specific IgG (control), guadecitabine 2mg/m2 sq bi-weekly, murine anti-PDL1 inhibitory antibody (10mg/kg) bi-weekly and combination of guadecitabine with anti-PDL1 antibody (n = 6 mice/group, 3 week treatment). Immune cells collected from the ascites and spleens of tumor bearing mice were either directly processed or co-cultured with ID8 cells for 48 hours. Cells were immuno-phenotyped by flow cytometry. In OC cells treated with guadecitabine, changes in gene expression were analyzed by real time-PCR. Results: In ID8 tumors bearing mice, the combination of PDL1 blockade with guadecitabine significantly decreased primary tumor formation (P&amp;lt;0.05) and malignant ascites accumulation (P&amp;lt;0.0001) compared with the control group. Treatments were well tolerated without detectable weight changes attributable to toxicity. CD8+ cell fractions expressing the exhaustion markers (PD1+ or CTL4+) isolated from the ascites and spleens of control mice were diminished by guadecitabine, PD-L1 inhibitory antibody, and the combination treatment. Guadecitabine and the combination regimen increased CD8+/MHC1+ and CD8+/CD40+ cell populations. Treatment with DNMTIs significantly increased the expression of tumor antigens Muc16, Mage A2, A11, and NY-ESO 1 (p&amp;lt;0.01) in several OC cell lines. Conclusions: Guadecitabine in combination with anti-PDL1 antibody induced striking anti-tumor effects in an immunocompetent OC syngeneic model by activating cytotoxic T-cells. These data support clinical strategies utilizing epigenetic priming using DNMTI in combination with immune checkpoints inhibitors. Citation Format: Yu Qing, Salvatore Condello, Katie J. Meyer, Andrea Caperell-Grant, Kenneth P. Nephew, Daniela Matei. Epigenetic priming potentiates immune checkpoints inhibitors in ovarian cancer. [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 4024.

Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection

Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors.

PD-1 Blockade with Pembrolizumab in Patients with Classical Hodgkin Lymphoma after Brentuximab Vedotin Failure: Safety, Efficacy, and Biomarker Assessment

PD-1 Blockade with Pembrolizumab in Patients with Classical Hodgkin Lymphoma after Brentuximab Vedotin Failure: Safety, Efficacy, and Biomarker Assessment

Pembrolizumab in Combination with Lenalidomide and Low-Dose Dexamethasone for Relapsed/Refractory Multiple Myeloma (RRMM): Keynote-023

Pembrolizumab in Combination with Lenalidomide and Low-Dose Dexamethasone for Relapsed/Refractory Multiple Myeloma (RRMM): Keynote-023

Phase 1b Study of PD-1 Blockade with Pembrolizumab in Patients with Relapsed/Refractory Primary Mediastinal Large B-Cell Lymphoma (PMBCL)

Introduction: Like classical Hodgkin lymphoma (cHL), PMBCL frequently harbors genetic alterations of the 9p24.1 locus, leading to overexpression of the PD-1 ligands, PD-L1 and PD-L2. This provides a possible mechanism of immune evasion and suggests that PMBCL could have a genetically determined vulnerability to PD-1 blockade. Pembrolizumab is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands. Pembrolizumab has already demonstrated robust antitumor activity in advanced solid tumors and in cHL. KEYNOTE-013 (NCT01953692) is a multicenter, multicohort phase 1b trial testing the safety and preliminary efficacy of pembrolizumab in patients with hematologic malignancies. Based on its genetics, PMBCL was included as an independent cohort in this trial. Here we report the preliminary results in this patient population.Methods: The PMBCL cohort of KEYNOTE-013 is enrolling patients with relapsed/refractory (R/R) disease who have relapsed after or are ineligible for autologous stem cell transplant (ASCT). Pembrolizumab is administered intravenously at a dose of 10 mg/kg every 2 weeks for up to 2 years or until confirmed disease progression or unacceptable toxicity. The primary end points are safety and antitumor activity. Response is being evaluated using computed tomography (CT) and positron emission tomography (PET) at week 12 and every 8 weeks thereafter, using IHP 2007 criteria. Other end points include complete remission (CR) rate, duration of response (DOR), and exploratory biomarker analyses.Results: As of July 23, 2015, 10 patients with R/R PMBCL with a median age of 28 (23-62) years have been enrolled in this cohort. Patients were heavily pretreated: 40% had ≥4 prior lines of therapy, and 60% had prior radiation. Six patients (60%) experienced at least 1 adverse event (AE) of any grade related to study treatment. These treatment-related AEs, all grade 1/2, were: hypothyroidism and decreased appetite (2 patients each), and diarrhea, nausea, vomiting, fatigue, edema, weight loss, and arthralgia (1 patient each). There were no grade 3-5 treatment-related AEs. Two patients experienced a serious AE (grade 3 infectious pneumonia) unrelated to study drug. No patient discontinued for toxicity.Nine patients were evaluable for response (1 discontinued treatment based on clinical progression before week 12). The objective response rate (ORR) was 44% (4/9), with 1 patient achieving a CR and 3 patients achieving a partial response. The intent-to-treat ORR was 40%. With a median follow-up of 144 days, the median DOR has not been reached (1+ to 291+) days, with all 4 responses ongoing at the time of data cutoff. Six of 10 patients have discontinued study treatment because of disease progression, and 4 patients remain on study.Conclusion: The preliminary results of KEYNOTE-013 indicate that PD-1 blockade with pembrolizumab is associated with a tolerable safety profile and a promising response rate in heavily pretreated patients with R/R PMBCL. Those patients often have a very poor outcome with conventional therapy, justifying further studies of pembrolizumab in this population. DisclosuresZinzani:Gilead: Membership on an entity's Board of Directors or advisory committees; J&J: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Off Label Use: The PD-1 pathway is an important mechanism of immune evasion for many tumors. Pembrolizumab is a humanized monoclonal antibody that blocks the interaction of PD-1 with its ligands PD-L1 and PD-L2 on the tumor cell surface and, based upon pembrolizumab's antitumor immune activity in several solid tumors, it may be an effective option for treating hematological malignancies.. Ribrag:Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Esai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Moskowitz:Genentech: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Honoraria, Research Funding; Pharmacyclics: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Kuruvilla:Merck: Honoraria; Bristol-Myers Squibb: Honoraria; Hoffmann LaRoche: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Lundbeck: Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy; Karyopharm: Honoraria. Balakumaran:Merck: Employment, Equity Ownership; Amgen: Equity Ownership. Snyder:Merck: Employment, Equity Ownership. Marinello:Merck: Employment, Equity Ownership. Shipp:BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees. Armand:Infinity: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Sequenta, Inc.: Research Funding; BMS: Research Funding.

MDSCs enhance tumor cell proliferation in a caspase-1 related inflammasome cytokines dependent way

Myeloid-derived-suppression cells (MDSCs) are believed to be an important immune evasion mechanism by suppressing T cells. We investigated whether MDSCs have direct T cell independent pro-carcinogenic effect on tumor cells. We sorted the monocytic CD14+/CD11b+/HLA-DRlow MDSCs from head and neck squamous cell carcinoma (HNSCC) patients and found that MDSCs increased the proliferation index of HNSCC cells. Similar results were seen from co-culturing murine MDSCs and CT26 and B16 cells. Supernatant from the MDSCs were found to secrete inflammasome cytokines IL-1b and IL-18. MDSC's enhancement of proliferative index of the tumor was found to be caspase-1 dependent using FLICA. To test this in vivo, T cell depleted caspase-1 null mice showed significant decrease in tumor growth rate. To confirm the importance of myeloid inflammasome signaling in carcinogenesis, we suppressed MyD88 gene in tumor cell line Cal27 and found that the ability of MDSCs to promoting tumor proliferation is diminished. Taken together, our findings demonstrate that MDSCs can promote tumor cells proliferation on a inflammasome cytokines dependent manner.