Conceptus interferon gamma is essential for establishment of pregnancy in the pig

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG−/−) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG−/− conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG−/− conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG−/− embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1) and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs.

OP0315 EFFECTOR CD4 T CELLS REQUIRE SURVIVIN FOR REGULATION OF GLUCOSE METABOLISM AND IFNg PRODUCTION

Background:Interferon-gamma (IFNg) producing effector T cells play the leading role in triggering and perpetuation of inflammation in rheumatoid arthritis. Inflammation leads to metabolic reprogramming of T cells and high energy consumption supporting proliferation and IFNg production. Being a part of chromosomal passenger complex, oncoprotein survivin is essential for cell proliferation. It has also been identified as a marker of severe therapy-resistant rheumatoid arthritis. Thus, we aimed to explore the association between survivin and IFNg producing phenotype of CD4 T cells.Objectives:We study if survivin mediates the glucose dependent mechanism of IFNg production in CD4 T cells.Methods:CD4 cells were sorted from the peripheral blood of RA patients and healthy controls, activated with aCD3, cultured in presence of survivin inhibitor YM155 and subjected to RNA sequencing (Illumina, Life Science). IFNg levels in supernatants were measured by ELISA. To study glucose uptake in presence of YM155, CD4 cells were treated with IFNg+aCD3 overnight followed by 2NBD-glucose challenge for 30 min. Uptake of fluorescent 2NBD-glucose probe was measured by flow cytometry. Statistical analysis of RNAseq was performed in R-studio using the Bioconductor package DESeq2.Results:Comparison of the whole-genome transcription profile of CD4 cells different by levels of BIRC5, coding for survivin, demonstrated that the BIRC5hi group expressed significantly higher levels of IFNg (mRNA, p=10-26 and protein, p=10-4). Also, BIRC5hi CD4 cells had higher expression of glucose transporter GLUT1 (SLC2A1, p=0.0064) and of glycolytic enzymes glucose-6-phosphate dehydrogenase (G6PD, p=10-6), pyruvate kinase (PFKP, p=10-6), and lactate dehydrogenase (LDHA, p=10-14). On the contrary, expression of the key regulator of glycolysis 6-phosphofructo-2-kinase (PFKFB3) was significantly lower in the BIRC5hi group (p=4.4x10-5). Notably, expression of glycolytic enzymes G6PD and PFKFB3 correlated strongly to IFNg (r=0.880 and -0.698, respectively), TBX21 (r=0.811 and -0.698) and perforin (r=0.781 and -0.698). To demonstrate functional relevance of the connection between BIRC5 and glucose metabolism, survivin was inhibited in CD4 cell cultures. Survivin inhibition resulted in significant increase of PFKFB3 (p=7x10-6) and LDHA (p=0.0089), leading to inhibition of phosphoglycerate mutase PGAM1 and ATF citrate lyase ACLY (p=0.021 and p=0.0074, respectively), which dignify the restoration of aerobic glycolysis. Importantly, inhibition of survivin decreased 2NBD-glucose uptake by CD4 cells (p=0.031) and reduced expression of GLUT1 (p=0.034). These changes in glucose metabolism were followed by decreased IFNg production in supernatants (p=0.037).Conclusion:The study demonstrates a strong connection between IFNg production and glucose metabolism in CD4 cells. Survivin emerges as an important regulator of glycolysis acting through expression of glycolytic enzymes and glucose transport.Disclosure of Interests:None declared.

Conceptus interferon gamma is essential for pregnancy maintenance in the pig

Establishment and maintenance of pregnancy in the pig is a complex process that relies on adequate communication between the conceptus and maternal uterine endometrium. During the peri-implantation period, the conceptuses produce and secrete estrogens, interleukin 1 beta 2, prostaglandins and other biological factors into the uterine lumen that change the uterine epithelium to become receptive to the attaching conceptuses as well as promote proper conceptus development. Following elongation, beginning on day 12 of pregnancy, the conceptus is known to secrete two different types of interferons. The pig conceptus secretes both type I (interferon delta, IFND) and type II (interferon gamma, IFNG) interferons during this time. CRISPR/Cas9 gene editing and somatic cell nuclear transfer (SCNT) technologies were used to create an IFNG loss-of-function study in pigs. Blastocyst stage embryos that were IFNG[superscript +/+] or IFNG[superscript -/-] were transferred into recipient gilts and their reproductive tracts were collected on days 15 and 17 of pregnancy. Elongated conceptuses were flushed from recipient gilts on day 15 IFNG[superscript +/+] (4/4) and IFNG[superscript -/-] (4/4) recipient gilts. On day 17 of pregnancy, all IFNG[superscript +/+] recipient gilts (4/4) contained elongated viable conceptuses; however, conceptuses were only recovered from 2 of 8 IFNG[superscript -/-] embryo recipient gilts. In all IFNG[superscript -/-] pregnancies, the conceptuses were thin and fragmented compared to IFNG+/+ conceptuses. Additionally, the reproductive tracts that received IFNG[superscript -/-] conceptuses which were not pregnant on day 17 appeared hyperemic, inflamed and edematous. IFNG was localized to the trophectoderm of IFNG[superscript +/+] conceptuses on both day 15 and 17 of pregnancy. However, IFNG expression was not detected in IFNG[superscript -/-] conceptuses on either day 15 or day 17 of pregnancy. Conceptus IFNG mRNA expression was significantly affected by genotype (P [equals] 0.0006) and day (P [less than] 0.0001). Total IFNG was significantly lower (P [equals] 0.0018) in ULF of IFNG[superscript -/-] embryo recipient gilts compared to IFNG[superscript +/+] embryo recipient gilts. IFNG[superscript +/+] conceptuses induced endometrial folding and recruited large numbers of immune cells to the endometrial stroma beneath the site of conceptus attachment compared to less endometrial folding and presence of immune cells in IFNG[superscript -/-] pregnancies on day 15. An additional group of recipient gilts received either IFNG[superscript +/+] embryos (n [equals] 6) or both IFNG[superscript -/-] and IFNG[superscript +/+] embryos (n [equals] 5) to determine if the presence of IFNG producing conceptuses could rescue the IFNG-/- embryos beyond day 17 of pregnancy. On day 30 of pregnancy, 3/6 of IFNG[superscript +/+] embryo recipient gilts contained 3-4 viable embryos, however, only 1 of 5 IFNG[superscript -/-] and IFNG[superscript +/+] cotransferred recipient gilts maintained pregnancy to day 30. Genotyping indicated that all five (1 healthy, 4 degenerating) embryos were IFNG[superscript +/+]. These results indicate conceptus IFNG production is not essential for early conceptus development, rapid elongation or establishment of pregnancy. However, conceptus IFNG production does appear to be necessary for survival during the period of placental attachment beyond day 15.

166. TGF-β Restricts T-cell IFNg Production in Pulmonary Tuberculous Granulomas

Background IFNg production by CD4 T cells has been thought to be critical for immunity against Mycobacterium tuberculosis (Mtb); however, recent studies show that IFNg-producing CD4 T cells are more effective at preventing dissemination than controlling Mtb in the lung. Because optimal control of Mtb infection requires direct interactions between CD4 T cells and Mtb-infected cells presenting cognate antigen on MHCII, we sought to determine the location of CD4 T-cell antigen recognition and IFNg production in the Mtb-infected lung. Methods We infected mice with an ultra-low dose (ULD) of Mtb (1–3 CFU), a model developed in our laboratory which results in well-circumscribed granulomas that recapitulate many features of human Mtb granulomas. Using immunohistochemistry and quantitative imaging, we examined their lungs 35 days later for phenotypic and spatial analysis of T-cell receptor (TCR) signaling (using IRF4) and IFNγ production. We tested the antigen specificity of these responses with an adoptive transfer of both Mtb-specific and OVA-specific control CD4 T cells into ULD Mtb-infected mice. To assess the role of TGFβ signaling on T-cell localization and function, we performed the same analysis in mice lacking the TGFβ receptor (TGFβR) on T cells. Results Within Mtb-infected lungs, many T cells localize near Mtb cells and undergo TCR signaling. Despite this, we found very few cells producing IFNγ within the granuloma (Figure 1). In our adoptive transfer experiment, both cell types infiltrated the granuloma. The Mtb-specific, but not OVA-specific, T cells had active TCR engagement though only a small fraction of these cells produced IFNγ, and this IFNγ was diminished near Mtb (Figure 2). Conversely, in the TGFβR conditional knockout, we found increased IFNγ production that was highest within the granuloma (Figure 3). Conclusion Despite ongoing TCR stimulation in T cells, IFNγ production is restricted in areas where cognate interactions are most likely to occur. TGFβ plays a critical role in mediating this effect, as T cells lacking the receptor can produce more IFNγ near infected cells. These findings help explain why IFNγ-producing T cells have limited capacity to control pulmonary Mtb infection and could guide new strategies for vaccine and immunotherapeutic development. Disclosures All authors: No reported disclosures.

Evaluation of Immune Recovery Following Autologous Hematopoietic Cell Transplantation in HIV-Related Lymphoma: Results of the BMT CTN 0803/AMC 071 Trial

Background: Clinical outcomes for patients with HIV-related lymphomas who have undergone autologous hematopoietic cell transplantation (AHCT) are similar to HIV-negative patients (Alvarnas et al., Blood 2016). Here we report a detailed, longitudinal immunophenotypic and functional evaluation of immune recovery of patients enrolled on the BMT CTN 0803/AMC 071 multicenter phase II study (clinicaltrials.gov NCT01141712). Methods: Comprehensive analysis of cellular immunome was performed using 5 color flow cytometry. Acquisition and analysis was performed via FC500 cytometry analyzers with CXP software and prism plot. Comparisons were made between HIV+ and HIV- cohorts of peripheral blood mononuclear cell (PBMC) subsets at 56, 180, and 360 days post AHCT. The HIV- cohort was collected from 30 multiple myeloma patients enrolled in a longitudinal immune recovery study after AHCT (median age 52.5 years (18-71); 57% male, no post AHCT exposure to IMID or other treatment). Control samples were collected from 72 healthy volunteers (median age of 49 (21-68); 53%, M). A Wilcoxon rank sum test was utilized to compare the HIV+ and HIV- groups to controls and to each other at each time point for 18 immune cell subsets common to all three panels. An unsupervised analysis was performed utilizing a principal component analysis (PCA) to look for overall differences in the cohorts. Similar methodologies were used to compare HIV+ to controls that analyzed 100 PBMC subsets. Functional immune recovery was evaluated by IFNg Elispot assay where 2x105 PBMC collected at each time point were pulsed with control, EBV (BZLF1) or HIV (GAG) pepmix preparations. As a control for TCR responsiveness, anti CD3/CD28 antibody-beads were used to immobilize TCR in ELISPOT assay. T cell responses from PBMCs of each of the three time points of HIV+ patients on trial were compared to PBMCs from HIV- donors (n=6). Results: Wilcoxon Rank sum tests show significant differences between transplant patients and controls and between HIV+ and HIV- patients at all visits. There are fewer cell subsets significantly different at day 365 compared to day 56 or 120 in all comparisons. The PCA showed group differences between HIV+, HIV- and control subjects. CD3+/HLA-DR+ (late activation), CD8+/CD25- (cytotoxic T cells) and CD3+/CD314+ (T cells with activating NKG2D) were found to be more prevalent in HIV+ transplant patients. These findings may be consistent with expanded populations of chronically activated cytotoxic T lymphocytes in HIV+ transplant patients. Subsets of NK, Th1 and Th2 cells showed statistically significant differences between HIV+ (low), HIV- (higher) and controls (higher). When the principal components are plotted by visit there is a pattern of both HIV+ and HIV- transplant patients clustering closer to controls as patients recover from AHCT. The PCA was also utilized to compare the HIV+ cohort to controls which had the same panel of cell subsets tested and allowed for the use of 100 cell subsets in the analysis. This analysis showed a similar group separation and pattern of clustering closer to controls in later visits. These findings demonstrate complex interactions between T and NK cell subsets. Functional assessment of antigen-specific T cell responsiveness was evaluated in Elispot assays with EBV (BZLF1) and HIV (GAG) recall antigens and anti-CD3/CD28 controls. Of 30 evaluable patients, 28 HIV+ patients demonstrated measurable IFNg production in response to GAG (spots/2x105 PBMC, range: 8-615), 21 showed measurable response to BZLF1 pepmix (range 12-450); and all patients demonstrated responsiveness to anti CD3/CD28 stimulation. Magnitude of IFNg production from HIV+ samples was generally higher than that observed healthy, HIV- controls. Assessment of NK cell responsiveness is currently underway. Conclusions: While clinical outcomes following AHCT between HIV+ and HIV- patients is comparable, clear distinctions were observed with immune recovery of specific PBMC subsets during the first year following AHCT with differences diminishing as patients recover post transplant. Longitudinal immune responsiveness of PBMC from HIV+ patients to EBV and HIV recall antigens and TCR stimulation generally showed more robust IFNg production compared to PBMCs from HIV- volunteer controls. These data provide further justification supporting AHCT as an option for HIV+ patients provided they meet standard transplant criteria. Disclosures Little: This study was coordinated by the ECOG-ACRIN Cancer Research Group (Robert L. Comis, MD and Mitchell D. Schnall, MD, PhD, Group Co-Chairs) and supported by the National Cancer Institute of the National Institutes of Health under the following award number: Employment. Noy:Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding. Krishnan:celgene: Consultancy, Speakers Bureau; takeda: Consultancy, Speakers Bureau; janssen: Consultancy, Speakers Bureau; onyx: Speakers Bureau. Hofmeister:Signal Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Arno Therapeutics, Inc.: Research Funding; Incyte, Corp: Membership on an entity's Board of Directors or advisory committees; Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Karyopharm Therapeutics: Research Funding; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees. Forman:Mustang Therpapeutics: Other: Construct licensed by City of Hope. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentech: Research Funding. Baiocchi:Essanex: Research Funding.

pVAX14 DNA, An Immunoadjuvant, Extends Life-Span As Add-On Treatment To Azacitidine (AZA) In A Mouse Model Of High Risk Myelodysplastic Syndrome (HR-MDS)

Background We have established animal models of MDS and acute myelogenous leukemia (AML) using NRASD12 and overexpression of BCL-2 (Omidvar Cancer Res 2007). These models have identified a novel MDS biomarker, the RAS:BCL-2 complex (Le Pogam, Leuk Res 2013) and a BH3 mimetic inhibitor, ABT-737, was shown to target leukemic cells and increase life span in both models (Beurlet, Blood 2013, Gorombei, EHA 2013). Here we have studied the benefit of an immunotherapeutic approach in HR-MDS by taking advantage of the reported immunomodulating effect of azacitidine (AZA) (Goodyear, Blood 2010) and of a non-specific DNA vaccine (pVAX14) we have designed. Methods pVAX14 is a non-specific DNA plasmid which, in an APL mouse model , gives similar survival to the specific PML-RARA DNA we previously designed (Padua, Nat Med 2003) (results submitted to ASH 2013). pVAX14 is a novel construct containing GC-rich sequences and coding for unique peptides, 3 of which we have shown to be immunogenic; ATRA was combined for its immunomodulatory properties. Survival efficacy was measured in mice treated with AZA alone (1mg/kg intraperitoneally 3 times per week until death), pVAX14 (6 weekly injections of 50 mg DNA injected intradermally, ATRA (10 mg 21-day release pellets) or their combinations, and untreated controls. MDS was monitored with biomarkers previously validated for this model, (Beurlet Blood 2013): peripheral blood (PB) counts, PB blasts (Mac1hi/Gr1lo) and spleen cell AKT, MEK1 and ERK1/2 levels by the Nanoimmunoassay (NIA) (Fan, Nat Med 2009). Immunomonitoring was based on lymphocyte subpopulations including Memory T-cells (CD4+/CD44hi/CD62Llo), IFNg production (ELISPOT) and Toll-like receptor-9 (TLR-9) activation measured on MYD88 transcripts by RQ-PCR. Results 1) Survival benefit: in a first cohort of HR-MDS mice, pVAX14 treatment significantly prolonged survival (median survival 100 days in treated versus 10 days in untreated mice) (Kaplan Meier p<0.0001, Fig. 1). In a second pilot cohort, AZA+pVAX14+ATRA appeared superior (4/4 mice alive at 70 days) to AZA+pVAX14 or AZA+ATRA (2/4 alive at 70 days in both groups) and AZA alone (1/5 alive at 70 days). In a third larger confirmatory cohort, median survival was 65 days with the AZA+pVAX14+ATRA combination, 40 days with AZA alone (Kaplan Meier p= 0.044 compared with AZApVAX14+ATRA) and 10 days in untreated mice (p<0.0001 compared with AZA alone) (Fig. 2). 2) Hematological parameters: survival advantage of mice treated with pVAX14 alone, AZA alone and AZA+pVAX14+ATRA was corroborated with lack of leukemic progression assessed on days 13, 32 and 55, as shown by stable platelet counts and peripheral blasts (Mac-1hi/GR-1lo population) and downregulation of RAS signaling proteins with dephosphorylation of AKT, MEK1 and ERK1/2. 3) Enhanced immune responses: survival advantage and absence of leukemic progression were correlated with enhanced immune responses: increased IFNg production (p<0.03) and expression of MYD88 (p<0.05) were seen in mice treated with pVAX14 alone compared to untreated mice); Tmem were significantly increased in treated mice, with values highest for AZA+pVAX14+ATRA (p<0.005 compared to untreated HR-MDS mice). Conclusions 1) AZA increases survival in this HR-MDS model. Immune mechanisms seem to be implicated but we are currently analyzing other potential mechanisms of action, including DNA methylation 2) pVAX14+ATRA as add-on therapy to AZA further improves survival, and potentiates the immune responses initiated by AZA. This adjuvant DNA immunotherapy may thus be a promising approach for MDS treatment. PG and CLP contributed equally to this work. Disclosures: Pierre: Celgene: Honoraria, Research Funding. Christine:VivaVacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study., I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees. Padua:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study., I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees.

Activation Of Natural Killer Cells By Soypeptide Lunasin and Cytokine: Implication In Cancer Immunotherapy For Lymphoma

Introduction Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that chemotherapy-treated lymphoma patients have acquired deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNg production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer (NK) cells. Experimental Design PBMCs of healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Results Adding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The expression level of CD16 and granzyme B was increased in CD56-bright population of NK cells following stimulation with lunasin and cytokine. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNg production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines had higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. Moreover, lunasin augmented antibody-dependent cellular cytotoxicity (ADCC) of NK cells against anti-CD20 coated human B-lymphoma cell line. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Conclusion We have discovered a novel use of lunasin that exerts synergistic effects together with IL-12 or IL-2. This synergism leads to stronger NK-mediated anti-tumor activity, suggesting the potential clinical use of lunasin to augment the therapeutic responses in cytokine-based immunotherapy. Disclosures: No relevant conflicts of interest to declare.