Intracellular IFN-γ Levels Research Articles

Unconventional T Cells Influence Clinical Outcome After Allogeneic Hematopoietic Cell Transplantation.

We evaluated the impact of early recovery of mucosal-associated invariant T cells (MAIT) and gamma-delta (γδ) T cells, especially Vδ2+ T cells, on the clinical outcomes of 76 patients who underwent allogeneic hematopoietic cell transplantation (allo-HCT). MAIT cells were identified at day 20-30 post-transplant using flow cytometry and defined as CD3+ TCRVα7.2+CD161+. Two subsets of Vδ2+ T cells were analyzed according to the expression of CD26. The cytotoxicity profile of MAIT and Vδ2+ T cells was analyzed according to the intracellular expression of perforin and granzyme B, and intracellular IFN-γ was evaluated after in vitro activation. CD26+Vδ2+ T cells displayed higher intracellular levels of IFN-γ, whereas CD26- Vδ2+ T were found to be more cytotoxic. Moreover, MAIT cell frequency was correlated with the frequency of Vδ2+ T cells with a better correlation observed with Vδ2+CD26+ than with the Vδ2+CD26- T cell subset. By using the composite endpoint graft-versus-host disease (GvHD)-free,relapse-free survival(GRFS) as the primary endpoint, we found that patients with a higher MAIT cell frequency at day 20-30 after allo-HCT had a significantly increased GRFS and a better overall survival (OS) and disease-free survival (DFS). Moreover, patients with a low CD69 expression by MAIT cells had an increased cumulative incidence of grade 2-4 acute GvHD (aGvHD). These results suggest that MAIT cell reconstitution may provide mitigating effects early after allo-HCT depending on their activation markers and functional status. Patients with a high frequency of Vδ2+CD26+ T cells had a significantly higher GRFS, OS and DFS, but there was no impact on cumulative incidence of grade 2-4 aGVHD, non-relapse mortality and relapse. These results revealed that the impact of Vδ2+ T cells on the success of allo-HCT may vary according to the frequency of the CD26+ subset.

Human Immunodeficiency Virus-Human Pegivirus Coinfected Individuals Display Functional Mucosal-Associated Invariant T Cells and Follicular T Cells Irrespective of PD-1 Expression.

Human pegivirus (HPgV) appears to alter the prognosis of HIV disease by modulating T cell homeostasis, chemokine/cytokine production, and T cell activation. In this study, we evaluated if HPgV had any 'favorable' impact on the quantity and quality of T cells in HIV-infected individuals. T cell subsets such as CD4lo, CD4hi, and CD8+ T cells, CD4+ MAIT cells, CD8+ MAIT cells, follicular helper T (TFH) cells, and follicular cytotoxic T (TFC) cells were characterized based on the expression of markers associated with immune activation (CD69, ICOS), proliferation (ki67), cytokine production (TNF-α, IFN-γ), and exhaustion (PD-1). HIV+HPgV+ individuals had lower transaminase SGOT (liver) and GGT (biliary) in the plasma than those who were HPgV-. HIV/HPgV coinfection was significantly associated with increased absolute CD4+ T cell counts. HIV+HPgV+ and HIV+HPgV- individuals had highly activated T cell subsets with high expression of CD69 and ICOS on bulk CD4+ and CD8+ T cells, CD4+ MAIT cells, CD8+ MAIT cells, and CXCR5+CD4+ T cells and CXCR5+CD8+ T cells compared with healthy controls. Irrespective of immune activation markers, these cells also displayed higher levels of PD-1 on CD4+ T and CD8+ T cells . Exploring effector functionality based on mitogen stimulation demonstrated increased cytokine production by CD4+ MAIT and CD8+ MAIT cells. Decrease in absolute CD4+ T cell counts correlated positively with intracellular IFN-γ levels by CD4lo T cells, whereas increase of the same correlated negatively with TNF-α in the CD4lo T cells of HIV+HPgV+ individuals. HIV/HPgV coinfected individuals display functional CD4+ and CD8+ MAIT, TFH, and TFC cells irrespective of PD-1 expression.

Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice

ObjectiveRecurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models.MethodsExosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR.ResultsIFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3′ UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity.ConclusionOur study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal–fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.Graphical

The role of cytokines and T-bet, GATA3, ROR-γt, and FOXP3 transcription factors of T cell subsets in the natural clinical progression of Type 1 Diabetes.

Th cells play an important role in pathogenesis of type 1 diabetes (T1D). Peripheral blood mononuclear cells were isolated from peripheral blood samples from newly diagnosed (ND), 1-year (1YD), and 5-year T1D (5YD) patients (n:8 of each group), 8 healthy controls (HC), and cultured for 24h under unstimulated (US) and stimulated conditions. Cell ratios of Th1, Th2, Th17, Treg, and intracellular levels of IFN-γ, TNF-α, IL-10, TGF-β, IL-5, IL-13, IL-17, and IL-21 cytokines were evaluated using the flow cytometry. mRNA expressions of transcription factors T-bet, GATA3, ROR-γt, and FOXP3 of these cells were determined by real-time PCR. Reduced CD4+CD25high cell ratios were detected in ND. CD4+CD25high cells were found to be reduced in ND and 1YD compared to HC under IL-2-stimulated conditions. Intracellular IFN-γ and TNF-α levels were low in all patients under US and IL-12-stimulated conditions. IL-17A and IL-21 were found to be high in patients with IL-6-stimulated conditions. Expressions of IL-10 and TGF-β have been observed to be reduced in patients. Th1/Th2, Th17/Treg, and Th1/Treg ratios were higher in patient groups. FOXP3 and GATA3 mRNA expressions were found to be low in patients, while RORγt and T-bet mRNA levels were higher than HC. Th1, Th17, and Treg cells and their cytokines have been shown to be associated with type 1 diabetes.

Immunophenotype and function of circulating myeloid derived suppressor cells in COVID-19 patients

The pathogenesis of coronavirus disease 2019 (COVID-19) is not fully elucidated. COVID-19 is due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes severe illness and death in some people by causing immune dysregulation and blood T cell depletion. Increased numbers of myeloid-derived suppressor cells (MDSCs) play a diverse role in the pathogenesis of many infections and cancers but their function in COVID-19 remains unclear. To evaluate the function of MDSCs in relation with the severity of COVID-19. 26 PCR-confirmed COVID-19 patients including 12 moderate and 14 severe patients along with 11 healthy age- and sex-matched controls were enrolled. 10 ml whole blood was harvested for cell isolation, immunophenotyping and stimulation. The immunophenotype of MDSCs by flow cytometry and T cells proliferation in the presence of MDSCs was evaluated. Serum TGF-β was assessed by ELISA. High percentages of M-MDSCs in males and of P-MDSCs in female patients were found in severe and moderate affected patients. Isolated MDSCs of COVID-19 patients suppressed the proliferation and intracellular levels of IFN-γ in T cells despite significant suppression of T regulatory cells but up-regulation of precursor regulatory T cells. Serum analysis shows increased levels of TGF-β in severe patients compared to moderate and control subjects (HC) (P = 0.003, P < 0.0001, respectively). The frequency of MDSCs in blood shows higher frequency among both moderate and severe patients and may be considered as a predictive factor for disease severity. MDSCs may suppress T cell proliferation by releasing TGF-β.

Triptolide leads to hepatic intolerance to exogenous lipopolysaccharide and natural-killer-cell mediated hepatocellular damage by inhibiting MHC class I molecules

BackgroundTripterygium wilfordii Hook. F (TWHF) is used as a traditional Chinese medicine, called thunder god vine, based on its efficacy for treating inflammatory diseases. However, its hepatotoxicity has limited its clinical application. Triptolide (TP) is the major active and toxic component of TWHF. Previous studies reported that a toxic pretreatment dose of TP leads to hepatic intolerance to exogenous lipopolysaccharide (LPS) stimulation, and to acute liver failure, in mice, but the immune mechanisms of TP-sensitised hepatocytes and the TP-induced excessive immune response to LPS stimulation are unknown. PurposeTo identify both the key immune cell population and mechanism involved in TP-induced hepatic intolerance of exogenous LPS. Study DesignIn vitro and in vivo experiments were conducted to investigate the inhibitory signal of natural killer (NK) cells maintained in hepatocytes, and the ability of TP to impair that signal. MethodsFlow cytometry was performed to determine NK cell activity and hepatocyte histocompatibility complex (MHC) class I molecules expression; the severity of liver injury was determined based on blood chemistry values, and drug- or cell-mediated hepatocellular damage, by measuring lactate dehydrogenase (LDH) release. In vivo H-2Kb transduction was carried out using an adeno-associated viral vector. ResultsInterferon (IFN)-γ-mediated necroptosis occurred in C57BL/6N mice treated with 500 μg TP/kg and 0.1 mg LPS/kg to induce fulminant hepatitis. Primary hepatocytes pretreated with TP were more prone to necroptosis when exposed to recombinant murine IFN-γ. In mice administered TP and LPS, the intracellular IFN-γ levels of NK cells increased significantly. Subsequent study confirmed that NK cells were activated and resulted in potent hepatocellular toxicity. In vivo and in vitro TP administration significantly inhibited MHC class I molecules in murine hepatocytes. An in vitro analysis demonstrated the susceptibility of TP-pretreated hepatocytes to NK-cell-mediated cytotoxicity, an effect that was significantly attenuated by the induction of hepatocyte MHC-I molecules by IFN-α. In vivo induction or overexpression of hepatocyte MHC-I also protected mouse liver against TP and LPS-induced injury. ConclusionThe TP-induced inhibition of hepatocyte MHC-I molecules expression leads to hepatic intolerance to exogenous LPS and NK-cell mediated cytotoxicity against self-hepatocytes. These findings shed light on the toxicity of traditional Chinese medicines administered for their immunomodulatory effects

Interleukin-1β in peripheral monocytes is associated with seizure frequency in pediatric drug-resistant epilepsy

In this study, we assessed circulating immune cells and plasma cytokine levels in 15 pediatric patients with drug-resistant epilepsy (DRE). DRE patients had a significantly higher percentage of CD14+ monocytes positive for IL-1β, IL-1 receptor antagonist, IL-6, and TNF-α than controls. Significantly higher intracellular levels of IFN-γ in CD4+ T cells and NK cells were also found in DRE patients. The level of IL-1β+ CD14+ monocytes correlated with seizure frequency, and intracellular levels of IFN-γ in NKT-like cells were negatively correlated with the duration of epilepsy. Peripheral immune cells might be involved in the pathogenesis of DRE.

Impaired NK cell activation during acute dengue virus infection: A contributing factor to disease severity

Dengue viral (DENV) infection has a broad clinical spectrum ranging from classical febrile illness to life-threatening disease. Literature suggests that spectrum of illness could be due to differences in innate immune-responses; however, the knowledge is still at infancy. Amongst the various cells involved in innate immune responses, NK cells play a central role, particularly in anti-viral immunity. Thus in this study we have evaluated the role of NK-cells during acute-DENV infection and its influence on severity of disease, by analyzing activation, cytotoxic receptors, cytolytic granule contents and degranulation markers on NK-cells during different stages of infection.Based on the clinical manifestations and severity of the disease, DENV patients were classified into patients with dengue without warning signs (DF), dengue with warning signs (DFWS) and severe dengue (SD) patients. During acute-DENV infection, though there was no alteration in frequency of NK-cells, significant increase in frequency of CD56bright subset in DF patients (p < 0.05) was observed, while it remained unaltered in SD patients. We also found that, CD56dim NK-cell subset of DF patients had elevated CD69 expression, granzyme B and intracellular IFN-γ levels compared to SD patients (p < 0.05). Amongst the NK-cell cytotoxicity receptor (NCR), NKp30 receptor was significantly elevated in DF patients (p < 0.05), however in SD patients it was comparable to healthy controls. This receptor is essential for dendritic cells-NK-cells crosstalk for initiating adaptive immune response. IL-15 is known to induce NKp30 expression, which was also seen to be elevated in DF patients (p < 0.05) but unaltered in SD patients. In SD patients, even post-6 days of infection i.e. during recovery phase, CD69 and NKp30 expression did not raise, suggesting impaired NK-cell response in these patients.To summarize, our study reports, that efficient NK cell response during acute phase of DENV infection is crucial for preventing severity of the disease. This study helps in understanding the dynamics of NK cell response in immunopathogenesis of DENV infection; which is crucial for development of efficacious therapeutics as well as vaccine.

Abstract 4146: Calcineurin represses IL-12 secretion in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) remains a leading cause of disease-related deaths in children. Although immuno-therapeutics have become a powerful strategy for the treatment of ALL, the mechanisms by which the leukemia cells avoid recognition and clearance by the immune system have not been clearly defined. Previous studies by our group defined leukemia-cell calcineurin (Cn) as a critical mediator of immune evasion in an aggressive, clinically relevant, murine model of BCR-ABL1+ B-ALL, as knocking it down in leukemia cells extended survival in immunocompetent, but not B and/or T cell-deficient mice (P=0.001). To identify downstream mediators of Cn-dependent immune evasion, we performed a cytokine array that demonstrated increased levels in chemokines and cytokines in the supernatant of Cn-deficient leukemia compared to the control. To validate this finding, ELISA was performed to quantify cytokine production and secretion. IL-12 secretion in the supernatant of Cn-deficient leukemia was significantly increased compared to the control. Ex vivo analyses demonstrated that T cell stimulation with the supernatant of Cn-deficient leukemia cells led to CD8+ T cell activation and CD4+ T cell differentiation into Th1 cells, as determined by measuring intracellular levels of IFN-γ and TNF-α. Furthermore, we found that the enhanced T cell activation by factors secreted from Cn-deficient leukemia was ablated by IL-12 neutralizing antibodies. Notably, analyses of publicly available gene expression profiles demonstrate that children with ALL in which either IL-12A or IL-12B is expressed at high levels have better outcomes, suggesting that IL-12 has clinical relevance. Importantly, treatment of mice with recombinant IL-12 (rIL-12) dramatically reduced the leukemia burden and prolonged survival in WT mice with parental leukemia as compared to vehicle treated WT controls (P=0.002). Successfully treated mice survived a second challenge with leukemia, suggesting the establishment of immunologic memory in these mice. In addition, rIL-12 treated Rag1-/- mice with leukemia had a similar reduction in leukemia burden, and transient prolongation of survival as compared to vehicle treated Rag1-/- mice (P=0.003). Given that Rag1-/- recipients lack mature B and T-cells, these results suggest that IL-12 responsive innate immune cells, such as NK cells, also play a major role in the effective suppression of leukemia cells. To begin to address this hypothesis, WT, Rag1-/-, IL2rg-/-, and double knockout (DKO) mice lacking B, T and NK cells (Rag1-/-/IL2rg-/-) were engrafted with calcineurin-deficient leukemia. A trend was observed in which DKO mice showed the highest rate of leukemia progression compared to the other groups. Overall, these data suggest that Cn-mediated inhibition of IL-12 secretion contributes to evasion of both innate and adaptive immunity by leukemia cells, and that novel strategies for therapeutic IL-12 delivery are needed. Citation Format: Rae Hunter, Jairo Fonseca, Jodi Dougan, Lori A. Gardner, Jennifer Rabe, Manali Rupji, Bhakti Dwivedi, Curtis J. Henry, Christopher C. Porter. Calcineurin represses IL-12 secretion in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4146.

Alteration of the mare's immune system by the synthetic progestin, altrenogest.

Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown. Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7days. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines. In vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1β in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1β expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares. P4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have.

255-LB: The Role of Th1, Th2, Th17, and Treg Cells in Various Clinical Phases of Type 1 Diabetes

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycaemia due to insulin production/secretion disorder or insulin resistance caused by various pathogenetic mechanisms. T helper cells (Th) play an important role in the pathogenesis of the disease. In our study, peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood samples from newly diagnosed, 1 year and 5 year type 1 diabetes (T1D) patients (8 patients) and 8 healthy controls, and cultured for 24 hours under unstimulated and stimulated conditions. After culture, the cell ratios of Th1, Th2, Th17, and regulator T (Treg) and the intracellular levels of IFN-γ, TNF-α, IL-10, TGF-β, IL-5, IL-13, IL-17 and IL-21 cytokines were evaluated using Flow Cytometry; whereas mRNA expressions of the transcription factors T-bet, GATA3, ROR-γt and FOXP3 of these cells were determined by Real-Time PCR. Reduced CD4+CD25high cell ratios were detected in newly diagnosed T1D patients under unstimulated conditions. CD4+CD25high cells were found to be reduced in the newly diagnosed and 1 year group of patients compared to healthy controls under IL-2 stimulated conditions. Intracellular IFN-γ and TNF-α levels were low in all patients under unstimulated and IL-12 stimulated conditions. IL-17A and IL-21 were found to be high in patients with unstimulated and IL-6 stimulated conditions. Expressions of IL-10 and TGF-β have been observed to be reduced in patients. Th1/Th2, Th17/Treg and Th1/Treg ratios were higher in the patient groups. FOXP3 and GATA3 mRNA expressions were found to be low in patients, while RORγt and T-bet mRNA levels were higher than healthy controls under stimulated and unstimulated conditions. Our results suggest that Th cell subgroups play an important role in the pathogenesis of T1D. Disclosure B. Aydin Ozgur: None. S. Cinar: None. D. Ozturk: None. E. Coskunpinar: None. A.O. Gurol: None. M. Yilmaz: None.

A Novel Dendritic Cell-Derived Vaccine in Multiple Myeloma

Introduction: Despite recent advances in the treatment of multiple myeloma (MM), curative outcomes remain elusive with standard therapies. MM is characterized by immune dysregulation and a loss of tumor-associated T cell surveillance contributing to disease evolution. Cancer vaccines have emerged as a promising immunotherapeutic strategy that seeks to reestablish anti-tumor immunity through the effective presentation of tumor associated antigens in the context of positive costimulation and activation signals. DCOne is a cell based tumor vaccine derived from an acute myelogenous leukemia cell line, differentiated toward a dendritic cell (DC) phenotype (DCPrime, Netherlands). DCOne strongly expresses WT-1, PRAME, RHAMM, survivin, and MUC-1. Since MM cells commonly express these tumor-associated antigens as well, we explored the efficacy of DCOne in inducing myeloma-specific immunity.Methods/Results: We first investigated the capacity of DCOne to polarize T cells toward an activated phenotype. Peripheral blood mononuclear cells (PBMCs) derived from MM patients were cultured in the presence or absence of DCOne and pulsed with whole cell DCOne lysate 24 hours prior to analysis. Intracellular levels of IFN-γ and perforin in CD8+ T cells and intracellular levels of IL-10 in CD4+ T cells were assessed via FACS analysis. Following 10 days of co-culture, we observed an increased percentage of CD8+ IFN-γ+ T cells and an increased percentage of CD8+ perforin+ T cells in PBMCs co-cultured with DCOne versus those not co-cultured with DCOne (10.8% versus 3.3%, p=0.02, n=11 and 6.6% versus 1.7%, p=0.045, n=10, respectively). In contrast, exposure of PBMCs to DCOne did not alter the percentage of CD4+ IL-10+ T cells (p=0.53, n=8). Given the observed increase in IFN-γ- and perforin-positive CD8+ T cells after co-culture of MM PBMCs with DCOne, we investigated whether CD8+ T cells co-cultured with DCOne exhibited enhanced killing of autologous tumor cells in a standard cytotoxic T lymphocyte (CTL) assay measuring Granzyme B activity by FACS analysis. DCOne potently induced CTL-mediated killing of autologous MM cells as determined by an increased percentage of CD8+ Granzyme B+ cells in stimulated versus control cells (27.4% versus 12.5%, p=0.05, n=3).We next investigated the mechanism of action by which DCOne elicited a MM-specific response. We postulated that the allogeneic cell line would induce immune activation in part through the transfer of antigen to PBMC-derived antigen presenting cells; these antigen presenting cells would in turn be capable of eliciting response via MHC restricted presentation of antigen to autologous T cell populations. Consistent with this hypothesis, we demonstrated that 32% of patient-derived PBMCs exhibited uptake of DCOne RNA following co-culture (n=3). Uptake of DCOne RNA by patient-derived PBMCs was completely abrogated with Transwell separation. Next, we examined how patient-derived PBMCs take up DCOne antigens. Using Western Blot analysis, we found that MUC-1 and survivin are expressed in extracellular vesicles (EVs) derived from DCOne lysate. Thus, we conclude that DCOne antigens are trafficked via EVs secreted by the DCOne parent cell, whereupon they are taken up by MM patient-derived PBMCs.Conclusion: In conclusion, when cultured in vitro with MM patient-derived PBMCs, DCOne results in increased IFN-γ- and perforin-positive CD8+ T cells, as well as induction of autologous tumor killing by these cells. DCOne RNA is taken up by MM patient-derived PBMCs and trafficked to the extracellular environment via EVs. DCOne demonstrates in vitro efficacy as an "off-the-shelf" strategy for stimulating MM-specific immunity. A clinical trial is being planned. DisclosuresKruisbeek:DCPrime: Employment, Other: Founder, CSO, and CEO of DCPrime. Van Wetering:DCPrime: Employment. Arnason:Gilead: Consultancy. Rosenblatt:DCPrime: Research Funding; BMS: Research Funding; Astex: Research Funding. Avigan:Astex: Research Funding; DCPrime: Research Funding.

Defining Fundamental B Cell-Subset Dysfunction in Myeloma

One of the most prominent features of multiple myeloma (MM) has been immune deficiency which predisposes patients to infectious complications and suppresses development of anti-MM immune responses. We and others have previously described the T cell dysfunction in Th1, Treg and Th17 cells, plasmacytoid dendritic cells and myeloid-derived suppressor cells (MDSC). However, the most fundamental and long identified deficiency is in the humoral immune response. Suppression of uninvolved immunoglobulins (UIgs) have been well described (i.e. suppression of serum IgA and IgM in IgG myeloma); and antibody responses to vaccination have been inadequate. However, very limited information is available regarding B cell function and how UIgs are suppressed in myeloma. We have now evaluated six different B cell subsets (B1a, B1b, B2, Breg, IRA-B, and MZ) in peripheral blood (PBMC) and bone marrow (BM) to understand alterations in B cell immune function in MM. We have observed significantly lower ratio of B2 (normal B cell-subset) and B1a (natural antibody-producing cells) subsets (10±4 vs 57±17; p < 0.05) and B2 and Breg (regulatory B cell-subset) subsets (14±4 vs 45±13; p< 0.05) in PBMC from MM patients (N=19) compared with healthy donor (N=33) respectively. Similar results were observed in BM samples from MM patients (N=18) compared with healthy donors (N=12); B2/B1a subset (2.4±0.6 vs 8±1.3; p < 0.05) and B2/Breg subset (8±1.4 vs 43.7±8.4; p< 0.05) respectively. To understand whether MM cells directly or indirectly alter B cell-subsets, we incubated myeloma cells (N=4) with healthy donor PBMCs, and analyzed B cell subsets after 3 days. We observed significant elevation in B1 subset (2.5 fold of control) and reduced B2 subset (89±3% of control). When we incubated PBMCs with IL-17A over-expressing MM cells (N=3), we observed further significant reduction in B2 subset (74% of control). When normal PBMCs are cultured in IL-17A (N=4) we observed significantly increased IL-10-producing Breg subset (28% of control). Similarly, co-culture of healthy B cells with MDSC led to significant increase (3.8 times) in Breg cell- population (N=3) compared with control group. To study the impact of B cell dysfunction on T cell function in MM, we activated normal PBMC via anti-CD3 antibody, in the presence or absence of B cells, and measured intra-cellular IFN-γ levels in CD69+ cells. We observed that the absence of B cells significantly inhibited interferon-producing T cells compared to control (by 43%; p<0.05). Importantly, following removal of CD25+ cells (Tregs and activated memory T cells), with or without B cells, we did not observe any difference in the inhibition of IFN-γ, indicating that B cells influence memory T cells rather than naïve T cells for the production of IFN-γ. To evaluate impact of lenalidomide on this interaction, we stimulated purified normal donor CD45RO memory T cells with Th1 polarizing cocktail in the presence or absence of purified normal B cells or B cells from MM patient (MM-B) in presence of lenalidomide and observed thatlenalidomide significantly improved MM-B cell-mediated IFN-γ-producing Th1 responses (by 32%, p<0.05) compared to normal B cell-mediated Th1 responses. In an effort to evaluate whether any therapy may improve the B cell function, we cultured normal PBMCs in the presence of lenalidomide (N=9) and observed reduction in Breg subset by 40% of control. To evaluate the effect of therapy on B cell-subsets in MM, we analyzed B cell subsets in PBMC from newly-diagnosed and lenalidomide-treated MM patients and observed that lenalidomide-treated group showed significant (p<0.05) improvement in B cell subsets (increased B2 and lower B1 cells) even before clinical response. These results suggest that immunomodulatory agents may be able to re-program humoral immunity in these patients. In summary, we report that the myeloma cell driven skewed B cell subset distribution with consequent B cell dysfunction drives the observed abnormalities in humoral/cell mediated immunity. The current therapeutic interventions, besides providing deep clinical responses, may also improve B cell function with impact on long term outcome. DisclosuresNo relevant conflicts of interest to declare.

NK cell activation by the heat shock protein gp96 (VAC12P.1018)

Abstract Heat Shock Proteins (HSPs) are key mediators in the development of immune responses. Following immunization with HSPs chaperoning tumor antigens, anti-tumor immune responses are elicited. The current model of HSP mediated anti-tumor immunity suggests HSPs bind to and are internalized by antigen presenting cells (APCs), which results in the priming of effector T cells that subsequently inhibit tumor growth. In addition to APCs and T cells, NK cells have also been demonstrated to be essential for HSP mediated anti-tumor immunity. However what role NK cells play in eliciting immunity is poorly understood. In these studies, we aim to characterize the activation of NK cells following stimulation with the prototypical HSP, gp96. Our results show that, following incubation with gp96 in vitro, we observe an increase in the frequency of NK cells containing the intracellular effector proteins granzyme B and IFNγ. Although intracellular levels of IFNγ are increased, we do not detect IFNγ secretion and the intracellular levels are less than what is observed following cytokine mediated activation. Due to these responses, we are currently investigating NK cells for the presence of the HSP receptor CD91. Collectively, these data indicate that directly, HSP stimulation results in partial NK cell activation, and other mechanisms, such as indirect activation via HSP activated APCs, may be necessary to observe optimal NK cell effector functions.

Interleukin-7 Enhances the in Vivo Anti-tumor Activity of Tumor-reactive CD8+T cells with Induction of IFN-gamma in a Murine Breast Cancer Model

Interleukin-7 (IL-7) is a potent anti-apoptotic cytokine that enhances immune effector cell functions and is essential for lymphocyte survival. While it known to induce differentiation and proliferation in some haematological malignancies, including certain types of leukaemias and lymphomas, little is known about its role in solid tumours, including breast cancer. In the current study, we investigated whether IL-7 could enhance the in vivo antitumor activity of tumor-reactive CD8+ T cells with induction of IFN-γ in a murine breast cancer model. Human IL-7 cDNA was constructed into the eukaryotic expression plasmid pcDNA3.1, and then the recombinational pcDNA3.1-IL-7 was intratumorally injected in the TM40D BALB/C mouse graft model. Serum and intracellular IFN-γ levels were measured by ELISA and flow cytometry, respectively. CD8+ T cell-mediated cytotoxicity was analyzed using the MTT method. Our results showed that IL-7 administration significantly inhibited tumor growth from day 15 after direct intratumoral injection of pcDNA3.1-IL-7. The anti-tumor effect correlated with a marked increase in the level of IFN-γ and breast cancer cells-specific CTL cytotoxicity. In vitro cytotoxicity assays showed that IL-7-treatment could augment cytolytic activity of CD8+ T cells from tumor bearing mice, while anti-IFN-γ blocked the function of CD8+ T cells, suggesting that IFN-γ mediated the cytolytic activity of CD8+ T cells. Furthermore, in vivo neutralization of CD8+ T lymphocytes by CD8 antibodies reversed the antitumor benefit of IL-7. Thus, we demonstrated that IL-7 exerts anti-tumor activity mainly through activating CD8+ T cells and stimulating them to secrete IFN-γ in a murine breast tumor model. Based on these results, our study points to a potential novel way to treat breast cancer and may have important implications for clinical immunotherapy.

Activation of human natural killer cells by the novel innate immune modulator recombinant Eimeria antigen

The safe and effective activation of the innate and adaptive immune systems are crucial in the implementation of immunotherapeutic modalities for the prevention and treatment of human diseases. Eimeria antigen (EA) and its recombinantly expressed analog (rEA) are extremely effective activators of innate immunity in mice. The effects of rEA in the mouse are primarily mediated through the TLR11/12 MyD88 signaling system. Human cells lack functional TLR11 and TLR12, suggesting that rEA would not be effective in providing beneficial immune activation in humans. In the current report we provide definitive evidence that the treatment of human peripheral blood mononuclear cell (PBMC) cultures with rEA significantly up regulates CD69, CD107, NKG2D levels on NK cells. Furthermore, rEA stimulates human NK cell effector functions including increasing intracellular levels of IFNγ and Granzyme B. These responses are positively correlated with an improved capacity of rEA stimulated human PBMCs to kill NK cell-sensitive human K562 tumor cells. Importantly, rEA-triggered innate immune responses was not associated with increased pro-inflammatory cytokines and chemokines production. These data confirm a previously unidentified role for rEA in human immune cell activation, and suggests the utilization of rEA in immunotherapies against a variety of infectious diseases and cancers.

Reprogramming Aberrant B Cell-Subsets to Improve Immune Function in Multiple Myeloma

AbstractAbstract 3986B cell-malignancies exhibit considerable immune dysfunction particularly in multiple myeloma (MM). We have previously demonstrated that in T cell-compartment, regulatory T helper cells are dysfunctional in multiple myeloma (MM) while Th17 cells are significantly elevated and IL-17 produced by them is associated with MM cell growth and survival as well as suppressed immune responses and bone disease. We have here investigated the B cell-subsets and their ability to re-program anti-tumor immunity in MM. We have first characterised four different B cell-subsets (B1a, B1b, B2 and regulatory B cells) using 10-color flow cytometric analysis in both peripheral blood and bone-marrow (BM) samples from MM patients compared with normal healthy donors. We observe that CD5+ B1a-B cells are significantly elevated in peripheral blood of MM patients (N=7) compared to healthy donor (N=15) (42±8% vs 13±3%, respectively, p<0.05); while normal B cells (B2 cells) are significantly reduced in peripheral blood (29.8±6.5, p<0.05) and in the BM samples (11±4.8, N=4, p<0.05) of MM patients compared to healthy donors (59±3, and 60.2±2, N=10, respectively). We also observed that both B1b (47.9±18 vs. 22.8±4) and regulatory B cells (7.1±4.5 vs. 1.54±0.3) are elevated in BM samples of MM compared to healthy donors, however there were no differences in B1b and regulatory B cells in the peripheral blood of MM compared to healthy donor samples. Interestingly, in myeloma we observe higher levels of activated B cell subsets but lower levels of memory B cell subsets compared to healthy donors. These results, particularly very low levels of normal B cells in MM patients, may explain the decreased levels of uninvolved immunoglobulin in MM.As removal of B cell population has been shown to re-program T helper cell populations, we next investigated impact of B cell population on T cell activation. We activated normal PBMC via the anti-CD3 antibody, in the presence or absence of B or CD25+ cells and measured intra-cellular IFN-γ levels in CD69+ cells. We found that the absence of B cells significantly inhibited interferon-producing T cells compared to PBMC (by 43%; p<0.05). Importantly, following removal of CD25+ cells, which consists of both Tregs and activated memory T cells, with or without B cells, we did not observe any difference in the inhibition of IFN-γ, indicating that B cells are influencing memory T cells rather than naïve T cells for the production of IFN-γ. This prompted us to identify the phenotypic signature of regulatory T cell populations when purified memory T cells are polarized with the regulatory T cell cocktail in presence or absence of B cells. We observed that B cells reduce FOXP3 expression by 18 %(N=5) and establish cognitive interactions with T cells. This occurred by increasing the expression of GITR (154%) and CTLA4 (54%); while reducing PD1 (−24%) and OX40 (−21%) expression on T cells without affecting HLA expression. We have also observed these improvements by B cell modulation on T cells in MM. Our results indicate that targeting these re-programmable capabilities of B cells to modulate T helper cell populations may enable us to improve T cell function in MM; and may improve immune function in MM and also allow us to enhance responses to vaccinations. Disclosures:Ghobrial:Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other. Richardson:Novartis: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees. Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity’s Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

Consequences of TCDD treatment on intra-hepatic lymphocytes during liver regeneration

Increasing evidence demonstrates a physiological role for the aryl hydrocarbon receptor (AhR) in regulating hepatocyte cell cycle progression. Previous studies have used a murine model of liver regeneration to show that exposure to the potent exogenous AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suppresses hepatocyte proliferation in vivo. Based on recent reports that natural killer (NK) cells negatively regulate liver regeneration, coupled with the well-established immunomodulatory effects of TCDD, it was hypothesized that alterations in lymphocyte activation contribute to the suppression of liver regeneration in TCDD-treated mice. To test this, mice were treated with TCDD (20 μg/kg) 1 day prior to 70% partial hepatectomy (PH), in which two-thirds of the liver was surgically resected. Lymphocytes were collected from the remnant liver and analyzed by flow cytometry. Whereas exposure to TCDD did not alter the number of NK cells or CD3+ T-cells recovered from the regenerating liver, it reduced the percentage and number of intra-hepatic NKT cells 42 h after PH. With regard to lymphocyte activation, TCDD treatment transiently increased CD69 expression on NK and NKT cells 12 h after PH, but had no effect on intracellular levels of IFNγ in NK, NKT, or CD3+ T-cells. To determine the relevance of NK cells to the suppression of liver regeneration by TCDD, mice were treated with anti-Asialo GM-1 (ASGM-1) antibody to deplete NK cells prior to TCDD treatment and PH, and hepatocyte proliferation was measured using bromodeoxyuridine incorporation. Exposure to TCDD was found to inhibit hepatocyte proliferation in the regenerating liver of NK cell-depleted mice and control mice to the same extent. Hence, it is unlikely that enhanced numbers or increased activation of NK cells contribute to the suppression of liver regeneration in TCDD-treated mice.

Maturation of Monocyte-Derived Dendritic Cells Induced by Dual Targeting of Endocytosis Receptor and CD40

AbstractAbstract 4293Dendritic cells (DC) are professional antigen presenting cells, which can induce and regulate adaptive immune responses. Hence, DC are attractive candidates for anti-tumor vaccination strategies. The aim of this study was to create recombinant bispecific Fab to target and activate DC, by using derivates of monovalent agonistic CD40 scFv and endocytosis receptors FcγRI (CD64), FcαR (CD89), DEC205 (CD205), as well as DC-SIGN (CD209), respectively. The recombinant bispecific molecules were expressed in the eukaryotic cell line Hek 293T, enriched to homogeneity by affinity chromatography and analyzed for specific binding to the targeted receptors. Functional analysis of these molecules indicated, that concurrent engagement of CD40 and endocytosis receptors expressed by immature IL-4/GM-CSF cultured monocytes-derived DC induced maturation of these DC. This was suggested by changes of DC phenotype, especially enhanced surface expression of co-stimulatory molecules like CD80 and CD86, and gain of CD83 expression as measured by flow cytometry. To particularly analyze the T cell activation properties of these DC, MACS enriched T cells of autologous donors were co-cultivated with antigen-loaded and subsequently maturated DC for seven days. After restimulation of T cells, flow cytometric detection of intracellular IFN-γ levels and surface expression of CD69 used as markers for T cell activation implies, that these DC exhibit elevated properties to activate antigen specific T cells. In contrast, a bispecific [CD64scFv × CD40scFv] Fab used as control failed to induce maturation of DC lacking CD64 expression, which may be due to abrogation of bivalent binding of this molecule and therefore CD40 cross-linking. Moreover, dual targeting with these novel immunoconstructs when linked to peptides of a tumor-associated antigen may allow direct antigen delivery to DC in combination with DC activation, and thus anti-tumor vaccination in vivo. Disclosures:No relevant conflicts of interest to declare.

Lipopolysaccharide-Mediated Mast Cell Activation Induces IFN-γ Secretion by NK Cells

Mast cells (MCs) that are well known for their important effector function in IgE-associated immune responses play a key role in innate immune defenses. In this study, we investigate the interaction between MCs and NK cells in vitro and in vivo. We show that mouse bone marrow-derived cultured MCs activated with LPS, polyinosinic-polycytidylic acid, or CpG can stimulate NK cells to secrete increasing concentrations of IFN-gamma. MCs induce a 20-fold increase in IFN-gamma release from NK cells after LPS stimulation. This enhancement of IFN-gamma secretion is cell contact dependent and TNF-alpha independent. Furthermore, we show that this interaction is in part mediated by OX40 ligand on MCs. NK cell-mediated cytotoxicity was not affected by the presence of MCs. Intracellular IFN-gamma levels in splenic NK cells are significantly decreased after i.p. injection of LPS in mast cell-deficient (C57BL/6 Kit(wsh/wsh)) mice in comparison with wild-type mice. In conclusion, our data show for the first time a direct mast cell-dependent NK cell activation. This interaction might play an important role in innate immune defense, as it is dependent on the presence of stimulators relevant in innate immune responses.