High Expression Of MHC Class Research Articles

Low-dose decitabine enhances the efficacy of viral cancer vaccines for immunotherapy

Cancer immunotherapy requires a specific antitumor CD8+ Tcell-driven immune response; however, upon genetic and epigenetic alterations of the antigen processing and presenting components, cancer cells escape the CD8+ Tcell recognition. As a result, poorly immunogenic tumors are refractory to conventional immunotherapy. In this context, the use of viral cancer vaccines in combination with hypomethylating agents represents a promising strategy to prevent cancer from escaping immune system recognition. In this study, we evaluated the sensitivity of melanoma (B16-expressing ovalbumin) and metastatic triple-negative breast cancer (4T1) cell lines to FDA-approved low-dose decitabine in combination with PeptiCRAd, an adenoviral anticancer vaccine. The two models showed different sensitivity to decitabine invitro and invivo when combined with PeptiCRAd. In particular, mice bearing syngeneic 4T1 cancer showed higher tumor growth control when receiving the combinatorial treatment compared to single controls in association with a higher expression of MHC class I on cancer cells and reduction in Tregs within the tumor microenvironment. Furthermore, remodeling of the CD8+ Tcell infiltration and cytotoxic activity toward cancer cells confirmed the effect of decitabine in enhancing anticancer vaccines in immunotherapy regimens.

Characterizing hepatocellular carcinoma stem markers and their corresponding susceptibility to NK-cell based immunotherapy.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is the fourth-leading cause of all cancer-related deaths around the world. Liver transplantation, surgery, and local ablation are curative therapies for early-stage HCC. However, post-treatment outcomes can vary based on histopathologic stage. Poorly-differentiated HCC are associated with higher rates of tumor progression and lower overall survival compared to well-differentiated HCC after therapy. In this study, we aimed to characterize the cancer stem cell (CSC) profile of histopathologically-proven well and poorly-differentiated HCCs in an in-vitro environment. We characterized the stem-like profile of each type of HCC based on their surface markers and susceptibility to NK cell-mediated cytotoxicity. Flow cytometry was used to quantify differential expression of MHC-class I, CD54, and CD44 between well- and poorly-differentiated HCCs. Primary untreated NK cells, IL-2 stimulated primary NK cells, and supercharged (sNK) cell-mediated cytotoxicity was assessed against well- and poorly-differentiated HCCs. IFN-γ supernatant from each respective NK cell experimental arm was also used to induce differentiation of HCCs. Finally, we characterized the temporal NK effector cell cytotoxicity using real-time quantitative analysis of imaging and impedance (eSight study). Poorly-differentiated HCCs demonstrated low surface expression of MHC-class I and CD54, and high expression of CD44. Treatment of NK cells secreted IFN-γ or IFN-γ cytokine induced differentiation in HCCs. Poorly-differentiated HCCs in comparison to well-differentiated HCC were more susceptible to NK cell-mediated cytotoxicity in primary NK cells, IL-2 stimulated primary NK cells, and sNK cells. sNK cells induced significantly higher cytotoxicity against well-differentiated HCCs in comparison to untreated or IL-2-stimulated primary NK cells. These findings were recapitulated with real-time quantitative imaging analysis. Poorly-differentiated HCCs were found to have surface marker patterns of CSCs, making them highly susceptible to NK cell-based immunotherapy. NK-cell based therapy can potentially be leveraged as a neoadjuvant or adjuvant therapy in poorly-differentiated HCCs. Supercharged NK cells, which can be rapidly expanded to therapeutic levels, are uniquely capable of lysing both poorly- and well-differentiated HCCs. This finding suggests that sNK cells not only exhibit enhanced features against NK cells' targets but also are capable of activating T cells to induce cytotoxicity against well-differentiated HCCs with high expression of MHC class I.

Characterizing the role of PIM kinases in the prostate tumor immune microenvironment.

e17080 Background: The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, a family of pro-survival kinases ( PIM1, PIM2, and PIM3), are frequently overexpressed in prostate cancer (PC) and associated with PC aggressiveness and immune evasion. Preclinical data indicate that PIM1 overexpression increases macrophage infiltration and survival using in vivo models of PC. Here, we characterized the association of PIM1, PIM2, and PIM3 with PC immune signatures. Methods: 77 primary prostate and 11 metastatic lymph node samples from treatment-naive metastatic hormone-sensitive PC pts were analyzed by DNA next-generation sequencing (592-gene, NextSeq; WES, NovaSeq) and Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). PC with PIM1/PIM2/PIM3-high(H) and -low(L) expression were classified by top and bottom quartile, respectively. Androgen receptor (AR) signature and Neuroendocrine Prostate Cancer (NEPC) score were calculated based on the expression level of previously defined gene signatures (Hieronymus et al. 2006, Beltran et al. 2016). Pathway enrichment was determined by GSEA (Broad Inst). Immune cell fractions were calculated by deconvolution of WTS using Quantiseq. Statistical significance was determined by chi-square and Mann-Whitney U (p < 0.05) and adjusted for multiple comparisons (q < 0.05). Results: PIM1/PIM2/PIM3-H PC had a higher median MAPK activation score (MPAS) compared to PIM1/PIM2/PIM3-L PC (0.9, 1.3, 0 vs -1.6, -1.4, -1.1 respectively, all p < 0.05). There was no difference in AR signature or NEPC score between PIM-H and PIM-L PC. PIM3-H PC exhibited higher PSA expression (3.15-fold, p < 0.05). PIM1/PIM2-H PC had higher AR expression (1.9 and 3.5-fold, respectively, all p < 0.05). PIM2/PIM3-H PC had enrichment of PI3K/AKT/mTOR, IL2/STAT5, IL6/JAK/STAT3, KRAS, TGFβ, NOTCH, hypoxia signaling, and ROS, P53, OXPHOS pathway and EMT (NES: 1.3-1.5, all FDR < 0.25). PIM1/PIM2-H PC had higher expression of immunostimulatory genes ( IL1β, TNF, and TNFSF13, FC: 1.1-3.1, p < 0.05). PIM1/PIM2/PIM3-H PC had higher expression of MHC class I ( HLA-A, HLA-B, HLA-C, B2M, FC: 1.3-2.5, all p < 0.05) and MHC class II ( HLA-DPA1, HLA-DRB1, HLA-DPB2, TAP1, TAP2, FC: 1.1-3.2, all p < 0.05) genes. PIM3-H PC had increased infiltration of M2 macrophages (8% vs 5.2%) and NK cells (5.2% vs 3.3%), while PIM2-H PC had increased Tregs (1.7% vs 0%) (all p < 0.05). Moreover , PIM1/PIM2/PIM3-H PC had a high T cell inflamed score compared to PIM1/PIM2/PIM3-L PC (66, 80, 56 vs -143, -157, -136, respectively, all p < 0.05). Conclusions: Our data suggest a strong association between PIM expression and increased MAPK activation score, T cell inflamed score, inflammatory, MHC class I and MHC class II gene expression, and differential immune cell infiltration. A better understanding of these differences will provide a rationale for tailored therapeutic approaches for PIM-expressing PC.

High Expression of MHC Class I Overcomes Cancer Immunotherapy Resistance Due to IFNγ Signaling Pathway Defects.

IFNγ signaling pathway defects are well-known mechanisms of resistance to immune checkpoint inhibitors. However, conflicting data have been reported, and the detailed mechanisms remain unclear. In this study, we have demonstrated that resistance to immune checkpoint inhibitors owing to IFNγ signaling pathway defects may be primarily caused by reduced MHC-I expression rather than by the loss of inhibitory effects on cellular proliferation or decreased chemokine production. In particular, we found that chemokines that recruit effector T cells were mainly produced by immune cells rather than cancer cells in the tumor microenvironment of a mouse model, with defects in IFNγ signaling pathways. Furthermore, we found a response to immune checkpoint inhibitors in a patient with JAK-negative head and neck squamous cell carcinoma whose HLA-I expression level was maintained. In addition, CRISPR screening to identify molecules associated with elevated MHC-I expression independent of IFNγ signaling pathways demonstrated that guanine nucleotide-binding protein subunit gamma 4 (GNG4) maintained MHC-I expression via the NF-κB signaling pathway. Our results indicate that patients with IFNγ signaling pathway defects are not always resistant to immune checkpoint inhibitors and highlight the importance of MHC-I expression among the pathways and the possibility of NF-κB-targeted therapies to overcome such resistance.

CIAP1/2 Antagonism Induces Antigen-Specific T Cell-Dependent Immunity.

Checkpoint blockade immunotherapy has failed in pancreatic cancer and other poorly responsive tumor types in part due to inadequate T cell priming. Naive T cells can receive costimulation not only via CD28 but also through TNF superfamily receptors that signal via NF-κB. Antagonists of the ubiquitin ligases cellular inhibitor of apoptosis protein (cIAP)1/2, also called second mitochondria-derived activator of caspases (SMAC) mimetics, induce degradation of cIAP1/2 proteins, allowing for the accumulation of NIK and constitutive, ligand-independent activation of alternate NF-κB signaling that mimics costimulation in T cells. In tumor cells, cIAP1/2 antagonists can increase TNF production and TNF-mediated apoptosis; however, pancreatic cancer cells are resistant to cytokine-mediated apoptosis, even in the presence of cIAP1/2 antagonism. Dendritic cell activation is enhanced by cIAP1/2 antagonism invitro, and intratumoral dendritic cells show higher expression of MHC class II in tumors from cIAP1/2 antagonism-treated mice. In this study, we use invivo mouse models of syngeneic pancreatic cancer that generate endogenous T cell responses ranging from moderate to poor. Across multiple models, cIAP1/2 antagonism has pleiotropic beneficial effects on antitumor immunity, including direct effects on tumor-specific T cells leading to overall increased activation, increased control of tumor growth invivo, synergy with multiple immunotherapy modalities, and immunologic memory. In contrast to checkpoint blockade, cIAP1/2 antagonism does not increase intratumoral T cell frequencies. Furthermore, we confirm our previous findings that even poorly immunogenic tumors with a paucity of T cells can experience T cell-dependent antitumor immunity, and we provide transcriptional clues into how these rare T cells coordinate downstream immune responses.

Stemness Correlates Inversely with MHC Class I Expression in Pediatric Small Round Blue Cell Tumors.

Simple SummaryTumors occurring at a young age are distinct from tumors in older individuals, clinically and pathologically. As small round blue cell tumors (SRBCTs), they often show a resemblance to stem cells and immature precursor cells during embryonal development. Recently, immunotherapy has become an option for a subset of patients with limited success. We observed that in almost all the pediatric SRBCT types investigated (n = 1134) there was an inverse relationship, when comparing genes highly expressed in stem cells with genes encoding MHC class I molecules. MHC class I molecules are important in tumor cell recognition by cytotoxic T cells. We suspect that these tumors are derived from multipotent precursor cells that naturally show a low MHC class I expression and a lack of immune recognition necessary for prenatal proliferation and development.Recently, immunotherapeutic approaches have become a feasible option for a subset of pediatric cancer patients. Low MHC class I expression hampers the use of immunotherapies relying on antigen presentation. A well-established stemness score (mRNAsi) was determined using the bulk transcriptomes of 1134 pediatric small round blue cell tumors. Interestingly, MHC class I gene expression (HLA-A/-B/-C) was correlated negatively with mRNAsi throughout all diagnostic entities: neuroblastomas (NB) (n = 88, r = −0.41, p < 0.001), the Ewing’s sarcoma family of tumors (ESFT) (n = 117, r = −0.46, p < 0.001), rhabdomyosarcomas (RMS) (n = 158, r = −0.5, p < 0.001), Wilms tumors (WT) (n = 224, r = −0.39, p < 0.001), and central nervous system-primitive neuroectodermal tumors CNS-PNET (r = −0.49, p < 0.001), with the exception of medulloblastoma (MB) (n = 76, r = −0.24, p = 0.06). The negative correlation of MHC class I and mRNAsi was independent of clinical features in NB, RMS, and WT. In NB and WT, increased MHC class I was correlated negatively with tumor stage. RMS patients with a high expression of MHC class I and abundant CD8 T cells showed a prolonged overall survival (n = 148, p = 0.004). Possibly, low MHC class I expression and stemness in pediatric tumors are remnants of prenatal tumorigenesis from multipotent precursor cells. Further studies are needed to assess the usefulness of stemness and MHC class I as predictive markers.

Striking effect of low-dose radiotherapy combined with PD-1 blockade on small cell lung cancer in mice and refractory patients (Achilles Study).

e20608 Background: Immune checkpoint inhibitors (ICIs) provide a durable and long-term benefit but still unsatisfactory clinical efficacy for extensive-stage small cell lung cancer (ES-SCLC). An optimized dose and schedule of radiotherapy for ES-SCLC remain to be studied. We hypothesized that the addition of low-dose radiotherapy (LDRT) to ICI could improve the efficacy of ES-SCLC. Methods: In SCLC bearing mice, the tumor was irradiated with LDRT in dose-escalation starting at 3Gy×1 fractions (f) up to 3Gy×7f, and combined with PD-1 antibody injection (0.2mg/mouse, i.p, every 3 days). Mice were followed for tumor growth and survival. The tumor microenvironment was dynamically analyzed every 3 days till day 18 by flow cytometry and immunofluorescence. 15 patients (pts) with relapsed ES-SCLC who received the treatment with LDRT and PD-1 blockade were retrospectively reviewed. Results: LDRT with 3Gy×5f delivered over 5 days (LDRT15Gy/5f) was determined to be the optimized dose when combined with PD-1 blockade. Combined group exhibited obvious growth retardation and prolonged survival compared to either monotherapy.The most robust infiltration of T cells in combined group was observed on day 9 after start of treatment. Bulk and single-cell RNA-seq showed significant immune cells infiltration, predominately CD8+ T cells and M1 macrophage. The activation and degranulation of CD8+ T cells in combination group were enhanced. Intra-tumoral CD8+ T cells were mainly assigned to effector memory (Tem) cells, then exhausted precursor (Texp) cells and exhausted (Tex) cells. Pseudo-time analysis supported a state evolution model that CD8+ Tem cells differentiate into intra-tumoral CD8+ Tex cells through an intermediate CD8+ Texp state. Tumor cells were divided into clusters 0, 1, and 2. Cluster 0 nearly disappeared while the proportion of cluster 1 increased in combination therapy. Cluster 1 enriched inflammatory response pathways and higher expression of MHC class I versus clusters 0 and 2. Additionally, the density of intra-tumoral microvessel was decreased, while vascular perfusion and the number of pericyte-covered vessels increased in combined group. Correspondingly, 15 recurrent ES-SCLC pts who treated with up-front LDRT15Gy/5f plus PD-1 blockade showed an ORR of 80%. Median PFS and OS were 4.3 months and 10.9 months. PFS rates at 6, 12 and 24 months were 40%, 20% and 6.7%, and OS rates were 60%, 40% and 24%, respectively. No patient experienced above grade 4 radiation- and immunotherapy-related toxicity. Conclusions: Our study is the first report of the synergistic effect of LDRT15Gy/5f and PD-1 blockade in SCLC mice model and recurrent ES-SCLC pts. The LDRT15Gy/5f demonstrates both immunologic adjuvant and cytotoxic effect on SCLC, and is safe and feasible in clinical pts. Further translational research, Match trial (NCT04622228), is ongoing.

OMIP-051 - 28-color flow cytometry panel to characterize B cells and myeloid cells.

OMIP-051 - 28-color flow cytometry panel to characterize B cells and myeloid cells.

Fecal microbiome from patients with ulcerative colitis is potent to induce inflammatory responses

Ulcerative colitis (UC) is a chronic idiopathic disease affecting the colon. Patients with UC display a number of alterations in immune-related molecules and cells, as well as dysbiosis in the intestinal microbiota. It remains unclear whether the alterations in the patients' immune systems are initiating factors of UC or a result from external insults. Also, precisely how these intestinal microorganisms affect UC development is not completely understood. To answer these questions, fecal bacteria were collected from UC patients during the active phase (UC-active), UC patients during the remission phase (UC-remission), and healthy controls. The fecal bacteria were then used to stimulate monocytes from three additional healthy subjects. We found that fecal bacteria from both UC-active and UC-remission patients presented higher capacity than fecal bacteria from healthy controls, resulting higher expression of MHC class I and MHC class II molecules, as well as higher expression of costimulatory molecules CD80 and CD86. The production of multiple cytokines, including IL-6, TNF-α, IL-10, and IL-12, were higher following stimulation with fecal bacteria from UC-active and UC-remission patients. Notably, when fecal bacteria were diluted to lower concentration, the bacteria from UC-active patients was clearly more effective at activating monocytes than those from UC-remission patients and controls. Collectively, our results revealed that the fecal bacteria from UC patients could cause stronger inflammatory responses than fecal bacteria from healthy controls.

"Double-duty" conventional dendritic cells in the amphibian Xenopus as the prototype for antigen presentation to B cells.

Two populations of dendritic cells (DCs) are found in mammals, one derived from hematopoietic precursors (conventional/cDC), and another derived from mesenchymal precursors, the follicular DC (FDC); the latter is specialized for antigen presentation to B cells, and has only been definitively demonstrated in mammals. Both cDC and FDC are necessary for induction of germinal centers (GC) and GC-dependent class switch recombination (CSR) and somatic hypermutation (SHM). We demonstrate that inXenopus, an amphibian in which immunoglobulin CSR and SHM occur without GC formation, a single type of DC has properties of both cDC and FDC, including high expression of MHC class II for the former and display of native antigen at the cell surface for the latter. Our data confirm that the advent of FDC functionality preceded emergence ofbona fideFDC, which was in turn crucial for the development of GC formation and efficient affinity maturation in mammals.

Primary Tumors Limit Metastasis Formation through Induction of IL15-Mediated Cross-Talk between Patrolling Monocytes and NK Cells.

Metastases are responsible for the vast majority of cancer-related deaths. Although tumor cells can become invasive early during cancer progression, metastases formation typically occurs as a late event. How the immune response to primary tumors may dictate this outcome remains poorly understood, which hampers our capacity to manipulate it therapeutically. Here, we used a two-step experimental model, based on the highly aggressive B16F10 melanoma, that temporally segregates the establishment of primary tumors (subcutaneously) and the formation of lung metastases (from intravenous injection). This allowed us to identify a protective innate immune response induced by primary tumors that inhibits experimental metastasis. We found that in the presence of primary tumors, increased numbers of natural killer (NK) cells with enhanced IFNγ, granzyme B, and perforin production were recruited to the lung upon metastasis induction. These changes were mirrored by a local accumulation of patrolling monocytes and macrophages with high expression of MHC class II and NOS2. Critically, the protective effect on metastasis was lost upon patrolling monocyte or NK cell depletion, IL15 neutralization, or IFNγ ablation. The combined analysis of these approaches allowed us to establish a hierarchy in which patrolling monocytes, making IL15 in response to primary tumors, activate NK cells and IFNγ production that then inhibit lung metastasis formation. This work identifies an innate cell network and the molecular determinants responsible for "metastasis immunosurveillance," providing support for using the key molecular mediator, IL15, to improve immunotherapeutic outcomes. Cancer Immunol Res; 5(9); 812-20. ©2017 AACR.

Vaccination efficacy with marrow mesenchymal stem cell against cancer was enhanced under simulated microgravity

Stem cell vaccination can induce consistent and strong anti-tumor immunity against cancer in mice model. The antigenic similarity between tumors and embryos has been appreciated for many years and reflects the expression of embryonic gene products by cancer cells and/or cancer-initiating stem cells. Taking advantage of this similarity, we have tested a prophylactic lung cancer vaccine composed of allogeneic murine MSCs. Based on this conception, we first compared their tumor vaccines intervention effects of adult MSCs and MSCs under simulated microgravity (MSC/SMG). In this study, BALB/c mice were vaccinated with MSCs or MSC/SMG, compared with mice vaccinated with phosphate buffered saline (PBS) as negative controls. We then subcutaneously implanted the A549 human lung cancer cell line into vaccinated mice and monitored tumor growth potential in vivo. The smaller tumor size and less tumor weight were observed in mice vaccinated with MSCs or MSC/SMG, compared with that of the Control group. Particularly, it was much more significant in the group of MSC/SMG than that group of the MSCs. Vaccination with SMG treated MSCs inhibited proliferation and promoted apoptosis of tumor tissue. SMG/MSC vaccination induced bothTh1-mediated cytokine response; CD8-dependent cytotoxic response which reduced the proportion of Treg cells. Furthermore, SMG/MSC vaccination significantly increased MHC1 and HSPs proteins expression. In conclusion, we demonstrated the SMG could improve tumor-suppressive activity of MSC. The enhanced anti-tumor immune response of MSCs/SMG was strongly associated with the higher expression of MHC class I molecule on DCs, and the abundance of HSPs in the SMG treated MSCs may make antigens in the MSC more cross-presentable to the host DCs for generating protective antitumor activity. This study gains an insight into the mechanism of MSCs anti-tumor efficacy and gives a new strategy for cancer therapies in the future.

Bovine gamma/delta T cells are stimulated by bovine coronavirus antigens identified by a soluble T cells receptor

Gamma/delta (γδ) T cells in cattle account for an abundant T cell population. However, little is known regarding the function of γδ T cells as immune cells compared to αβ T cells. Not many pathogen-related antigens have been defined and known to stimulate γδ T cells. To address this information gap, we constructed a soluble receptor for bovine γδ T cells (sγδTCR) that was later used to identify two proteins (156 kDa and 102 kDa) or protein fragments expressed by bovine coronavirus (BCov). The molecular weight of the larger protein suggests it could be the spike glycoprotein of BCov. Subsequently, we used the identified viral proteins to study the reactivity of bovine γδ T cells. In vitro assays showed that purified preparations of the two proteins stimulated WC1+ γδ T cells isolated from cattle. A 4-fold increase in IFN production and a significantly higher expression of MHC class II was observed. However, these viral ligands could not stimulate γδ T cells to synthesize IL-8 or GM-CSF, known to be produced by γδ T cells when stimulated with bacterial antigens. Although the γδ T cells assessed here appeared activated by way of IFN and MHC II expression, surface markers such as CD2, CD25, CD44, CD62L and CD335 were not expressed at significant levels. Further, the activation elicited by viral ligands was not sufficient to induce cytotoxic capability in γδ T cells in vitro as measured by a flow cytometry-based cytotoxicity assay. This in vitro study shows that WC1+ γδ T cells can directly recognize viral antigen

Abstract A127: Innate crosstalk between ILC and interstitial macrophages promotes lung cancer regression in response to IL-12 therapy

Abstract Malignant tumors maintain a dominant immunosuppressive microenvironment that facilitates their progression and dissemination, mediated by the secretion of soluble factors, recruitment of different subsets of immune cells and expression of immune-regulatory receptors. The immune-modulatory cytokine interleukin-12 (IL-12) is a promising alternative to overcome this suppressive microenvironment because of its direct role in the activation of innate and adaptive immunity and potent anti-angiogenic effect. Despite its potent activity in experimental models, the clinical use of IL-12 for the treatment of human cancers has proven difficult because of its toxicity. The lung is a preferred site of metastasis for several types of tumor. Therefore, we hypothesized that local delivery of IL-12 could improve the immune response against lung metastases via modification of the tumor microenvironment and avoiding systemic toxicity. Therefore, we evaluated the anti-tumor activity of IL-12 in a murine model of metastatic B16 melanoma by intranasal delivery lentiviral vector encoding IL-12 (IL-12 LV). This delivery route resulted in transduction of alveolar macrophages and a drastic reduction of lung metastases. Whilst treated mice survived longer than controls, they eventually succumbed to distant tumor lesions while their lungs remained tumor-free. IL-12 LV treatment was associated with local infiltration and proliferation of effector subsets (NK, CD8+, CD4+ T cells) that expressed high levels of granzyme B and pro-inflammatory cytokines. These cells, however, are dispensable as IL12 LV was equally effective in models of NK and T cell ablation. Interferon gamma (IFNγ) was identified as an essential cytokine in the mechanism of IL-12 LV mediated tumoricidal activity. IFNg expression under IL-12 LV treatment was observed in NK, T cell and Innate Like Cell (ILC) populations, the latter of which sufficiently sustains IFNγ dependant activity in the absence of NK and T cells. The myeloid populations of the IL-12 LV treated lung were phenotyped and expansion of Interstitial Macrophages with high expression of MHC class II and inducible nitric oxide synthase (iNOS) was observed in an IFNγ dependant manner, therefore transgenic models of macrophage associated IFNγ insensitivity are currently being employed in order to elucidate the essential cytotoxic status of Interstitial Macrophages in models of IL12-LV mediated tumor rejection. We conclude that activation of innate and adaptive immunity by local genetic delivery of IL-12 is effective for the treatment of metastatic melanoma in the lung. Despite its local effectiveness, systemic protection might require further combination therapy, such as vaccination or checkpoint inhibition. Citation Format: Jake Y. Henry, Frederick Arce, Burkhard Becher, Karl S. Peggs, Sergio A. Quezada. Innate crosstalk between ILC and interstitial macrophages promotes lung cancer regression in response to IL-12 therapy. [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 A127.

Zinc oxide nanoparticles provide an adjuvant effect to ovalbumin via a Th2 response in Balb/c mice

Zinc oxide nanoparticles (ZNPs) have been used in dietary supplements and may cause an immunomodulatory effect. The present study investigated the effect of ZNPs on antigen-specific immune responses in mice sensitized with the T-cell-dependent antigen ovalbumin (OVA). BALB/c mice were intraperitoneally administered ZNPs (0.25, 0.5, 1 and 3mg) once, in combination with OVA, and the serum antibodies, splenocyte reactivity and activation of antigen-presenting cells were examined. The serum levels of OVA-specific IgG1 and IgE were found significantly enhanced by treatment with ZNPs over control. An increased level of IL-2, IL-4, IL-6, IL-17 and decreased level of IL-10 and TNF-α in splenocytes administered with ZNPs were observed in comparison with control. The ZNPs and OVA-stimulated T lymphocytes showed enhanced proliferation compared with control. Macrophages and B cells showed high expression of MHC class II, whereas higher expression of CD11b in macrophages of the ZNPs and ZNPs/OVA treated groups was observed. The lungs and spleen had increased eosinophils and mast cell numbers. Also, myeloperoxidase activity in lungs was found to be increased by 2.5-fold in the case of ZNPs and 3.75-fold increase in ZNPs/OVA, whereas in intestine, there was significant increase in both the groups. Increased expression of the genes for GATA-3, SOCS-3, TLR-4, IL-13 and IL-5 in the intestine was observed. Collectively, these data indicate that systemic exposure to a single administration of ZNPs could enhance subsequent antigen-specific immune reactions, including the serum production of antigen-specific antibodies, and the functionality of T cells.

Multidisciplinary approaches to enhance BRAF targeted therapy in melanoma (P2092)

Abstract 40-50% of patients with melanoma are identified with BRAF mutations. About 80% of the BRAF mutations carry the V600E mutation. These patients are treated with BRAF inhibitors alone or in combination with other cell signaling pathway inhibitors. However, a relapse of the disease is observed within 6-8 months after the commencement of the therapy. Some studies hint that NK cell based therapy can be effective in controlling melanoma. In this study multidisciplinary approaches were tested to understand the mechanism of Vemurafenib resistance by a BRAFV600E melanoma. Vemurafenib is a BRAFV600E inhibitor. Vemurafenib resistant melanoma cell lines were raised from the sensitive cell lines by drug treatment till 80-90% of cells showed resistance. Both the resistant and sensitive cell lines were tested in a NK cell cytotoxicity assay in vitro by standard 51Cr-release assay. The Vemurafenib resistant cell lines were found to have a reduced sensitivity to NK cell killing. Though there was no significant difference in expression of NK cell activating ligands on the surface between the resistant and sensitive cell lines, there was a tendency of higher expression of MHC class I on Vemurafenib resistant cell lines. BRAF exon15 sequencing shows no difference between Vemurafenib resistant and sensitive cell types. Experiments are in progress to understand the mechanism(s) of the reduced sensitivity of the Vemurafenib resistant cell lines to NK cell killing.

Intraperitoneal administration of poly(I:C) with polyethylenimine leads to significant antitumor immunity against murine ovarian tumors

Ovarian cancer is currently the most lethal gynecologic cancer in the United States. There is an urgent need for the development of innovative therapies against ovarian cancer, such as immunotherapy. The toll-like receptor 3 ligand, polyriboinosinic:polyribocytidylic acid (poly(I:C), has emerged as a promising adjuvant for activating the host immune responses for the control of tumors. We reasoned that a strategy to enhance the intracellular uptake of poly(I:C) will likely improve the poly(I:C) adjuvant effect. Since polyethylenimine (PEI) has been shown to increase the transfection efficiency of nucleic acids, we characterized the antitumor effects in mouse ovarian surface epithelial cells (MOSEC) tumor-bearing mice treated intraperitoneally with poly(I:C) and PEI. We observed that tumor-bearing mice treated with poly(I:C) and PEI generated significantly better therapeutic antitumor effects against MOSEC tumors compared with treatment with poly(I:C) alone. Furthermore, we found that NK cells play a significant role in the antitumor effects generated by treatment with poly(I:C) in combination with PEI. Intraperitoneal administration of poly(I:C) with PEI led to the uptake of poly(I:C) mainly by CD11b+ macrophages, resulting in the high expression of MHC class II and IL-12 (M1 phenotype). In addition, adoptive transfer of CD11b+ macrophages from mice treated with poly(I:C) and PEI was found to lead to increased number of activated NK cells in the recipient mice. Taken together, our data indicate that PEI can potentially be used to improve the uptake of poly(I:C) by CD11b+ macrophages, leading to the activation of NK cells and the control of murine ovarian tumors.

Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis

BackgroundSarcoidosis is a systemic disease of unknown aetiology characterised by granuloma formation and the presence of interferon γ (IFNγ)-producing T cells that cause inflammation and tissue damage in multiple organs,...

Ikaros null mice demonstrate defects in dendritic cell development. (36.17)

Abstract Ikaros is a transcription factor that is expressed almost exclusively in cells of the hematopoietic lineage. Ikaros null mice lack several hematopoietic cell lineages including some dendritic cell (DC) subsets. Additionally, several Ikaros null bone marrow (BM) progenitor populations have a decreased expression of the receptor tyrosine kinase, Flt3. DCs can be generated in vitro by culture of BM with either GM-CSF or Flt3L (the ligand for Flt3). Our studies find that loss of Ikaros leads to a specific defect in the ability of BM precursors to generate DCs in response to culture with Flt3L but not in response to culture with GM-CSF. Those DCs that do develop from the culture of Ikaros null BM with Flt3L have an activated phenotype with higher expression of MHC class II and the costimulatory molecules CD40, CD80 and CD86 than their wild type counterparts. An examination of gene expression in Ikaros null BM progenitors cultured with Flt3L shows an inappropriately low expression of Irf4, Irf8 and Spib, genes encoding transcription factors known to be important for DC development. Studies are currently underway to determine whether enforced expression of Ikaros, Flt3 or one of the above-mentioned transcription factors can rescue DC development upon culture of Ikaros null BM with Flt3L. These studies will provide a potential molecular mechanism for the impaired development of DCs in Ikaros null mice.

Injection of bleomycin in newborn mice induces autoimmune sialitis that is transferred by CD4 T cells

Bleomycin (BLM) induces cellular apoptosis or necrosis by producing reactive oxygen species, and has been used to induce scleroderma in adult mice. We wondered whether BLM induces the same pathological phenotype in newborn mice as in adult mice. BLM was subcutaneously administrated to newborn BALB/c mice. At 1 month of age, BLM-treated mice showed severe destruction of salivary glands with enlargement of nearby lymph nodes. These nodes contained CD4(+) T cells and B220(+)cells with high expression of MHC class II molecules. In addition, autoantibodies were detected by HEp-2 staining and western blotting. The cell transfer experiments were performed to evaluate the role of autoimmune phenomena in these pathological changes. Following the transfer of enriched CD4(+) T cells to 1-month-old BALB/c nude mice, the salivary glands were severely damaged with CD4(+) T cell and B220(+) cells infiltrations. The number of T-cell antigen receptor Vbeta 8.3(+) CD4(+) T cells was significantly increased in BLM-treated murine spleen. These findings will provide new insights into the causal factors of environment in autoimmunity and the relationship between autoreactive CD4(+) T cells and autoantibodies.