Expression of IFN-γ Upon Triggering of Activating Ly49D NK Receptors In Vitro and In Vivo: Costimulation with IL-12 or IL-18 Overrides Inhibitory Receptors

AbstractInterleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

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Stimulation of Interleukin-10 Production by Acidic β-Lactoglobulin-Derived Peptides Hydrolyzed with Lactobacillus paracasei NCC2461 Peptidases

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.2.266-271.2004 ◽

2004 ◽

Vol 11 (2) ◽

pp. 266-271 ◽

Cited By ~ 48

Author(s):

Guénolée Prioult ◽

Sophie Pecquet ◽

Ismail Fliss

Keyword(s):

Oral Tolerance ◽

Immunosuppressive Agents ◽

Interleukin 10 ◽

Lactobacillus Paracasei ◽

In Vitro Hydrolysis ◽

Ifn Γ ◽

Immunomodulatory Peptides ◽

Β Lactoglobulin

ABSTRACT We have previously demonstrated that Lactobacillus paracasei NCC2461 may help to prevent cow's milk allergy in mice by inducing oral tolerance to β-lactoglobulin (BLG). To investigate the mechanisms involved in this beneficial effect, we examined the possibility that L. paracasei induces tolerance by hydrolyzing BLG-derived peptides and liberating peptides that stimulate interleukin-10 (IL-10) production. L. paracasei peptidases have been shown to hydrolyze tryptic-chymotryptic peptides from BLG, releasing numerous small peptides with immunomodulating properties. We have now shown that acidic tryptic-chymotryptic peptides stimulate splenocyte proliferation and gamma interferon (IFN-γ) production in vitro. Hydrolysis of these peptides with L. paracasei peptidases repressed the lymphocyte stimulation, up-regulated IL-10 production, and down-regulated IFN-γ and IL-4 secretion. L. paracasei NCC2461 may therefore induce oral tolerance to BLG in vivo by degrading acidic peptides and releasing immunomodulatory peptides stimulating regulatory T cells, which function as major immunosuppressive agents by secreting IL-10.

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MyD88-Dependent Glucose Restriction and Itaconate Production Control Brucella Infection

Infection and Immunity ◽

10.1128/iai.00156-21 ◽

2021 ◽

Author(s):

Carolyn A. Lacey ◽

Bárbara Ponzilacqua-Silva ◽

Catherine A. Chambers ◽

Alexis S. Dadelahi ◽

Jerod A. Skyberg

Keyword(s):

Glucose Transporter ◽

Production Control ◽

Brucella Melitensis ◽

Underlying Mechanism ◽

Protective Role ◽

Ifn Γ ◽

Glucose Restriction ◽

Myd88 Signaling

Brucellosis is one of the most common global zoonoses and is caused by facultative intracellular bacteria of the genus Brucella . Numerous studies have found that MyD88 signaling contributes to protection against Brucella , however the underlying mechanism has not been entirely defined. Here we show that MyD88 signaling in hematopoietic cells contributes both to inflammation and to control of Brucella melitensis infection in vivo . While the protective role of MyD88 in Brucella infection has often been attributed to promotion of IFN-γ production, we found that MyD88 signaling restricts host colonization by B. melitensis even in the absence of IFN-γ. In vitro , we show that MyD88 promotes macrophage glycolysis in response to B. melitensis . Interestingly, a B. melitensis mutant lacking the glucose transporter, GluP, was more highly attenuated in MyD88 -/- than in WT mice, suggesting MyD88 deficiency results in an increased availability of glucose in vivo which Brucella can exploit via GluP. Metabolite profiling of macrophages identified several metabolites regulated by MyD88 in response to B. melitensis , including itaconate. Subsequently, we found that itaconate has antibacterial effects against Brucella and also regulates the production of pro-inflammatory cytokines in B. melitensis -infected macrophages. Mice lacking the ability to produce itaconate were also more susceptible to B. melitensis in vivo . Collectively, our findings indicate that MyD88-dependent changes in host metabolism contribute to control of Brucella infection.

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A Signal Transducer and Activator of Transcription (Stat)4-independent Pathway for the Development of T Helper Type 1 Cells

Journal of Experimental Medicine ◽

10.1084/jem.188.6.1191 ◽

1998 ◽

Vol 188 (6) ◽

pp. 1191-1196 ◽

Cited By ~ 127

Author(s):

Mark H. Kaplan ◽

Andrea L. Wurster ◽

Michael J. Grusby

Keyword(s):

Delayed Type Hypersensitivity ◽

Th2 Cells ◽

Signal Transducer ◽

T Helper ◽

Th1 Cell ◽

Th Cells ◽

Th Cell ◽

Ifn Γ

The differentiation of T helper (Th) cells is regulated by members of the signal transducer and activator of transcription (STAT) family of signaling molecules. We have generated mice lacking both Stat4 and Stat6 to examine the ability of Th cells to develop in the absence of these two transcription factors. Stat4, Stat6−/− lymphocytes fail to differentiate into interleukin (IL)-4–secreting Th2 cells. However, in contrast to Stat4−/− lymphocytes, T cells from Stat4, Stat6−/− mice produce significant amounts of interferon (IFN)-γ when activated in vitro. Although Stat4, Stat6−/− lymphocytes produce less IFN-γ than IL-12–stimulated control lymphocytes, equivalent numbers of IFN-γ–secreting cells can be generated from cultures of Stat4, Stat6−/− lymphocytes activated under neutral conditions and control lymphocytes activated under Th1 cell–promoting conditions. Moreover, Stat4, Stat6−/− mice are able to mount an in vivo Th1 cell–mediated delayed-type hypersensitivity response. These results support a model of Th cell differentiation in which the generation of Th2 cells requires Stat6, whereas a Stat4-independent pathway exists for the development of Th1 cells.

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Stable Transfection of the BovineNRAMP1 Gene into Murine RAW264.7 Cells: Effect onBrucella abortus Survival

Infection and Immunity ◽

10.1128/iai.69.5.3110-3119.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3110-3119 ◽

Cited By ~ 62

Author(s):

Robert Barthel ◽

Jianwei Feng ◽

Jorge A. Piedrahita ◽

David N. McMurray ◽

Joe W. Templeton ◽

...

Keyword(s):

Cell Lines ◽

In Vitro Model ◽

Regulatory Control ◽

Natural Resistance ◽

Ifn Γ ◽

Flanking Region ◽

Vitro Model ◽

Raw264.7 Macrophage

ABSTRACT Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. BovineNRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovine NRAMP1 as stable transgenes under the regulatory control of the bovineNRAMP1 promoter in the murine RAW264.7 macrophage cell line (Bcg s ) to analyze the regulation of the NRAMP1 gene and its role in macrophage function. We demonstrated that the 5′-flanking region of bovineNRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3′ untranslated region critically affects the expression of bovineNRAMP1 and the control of in vitro replication ofBrucella abortus but not Salmonella enterica serovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-γ)- and IFN-γ–lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h.

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323 Immunogenic syngeneic model MC38-OVA for the preclinical evaluation of immune evasion and checkpoint blockade

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.323 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A348-A348

Author(s):

Jessie Wang ◽

Kaixia Lian ◽

Jia Zheng ◽

Chenpan Nie ◽

Annie An ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Immunity ◽

Immune Evasion ◽

Syngeneic Mouse ◽

Ifn Γ ◽

Draining Lymph Nodes ◽

Syngeneic Tumors

BackgroundThe development of immuno-oncology (I/O) therapeutics has revolutionized the cancer treatment landscape. Despite this achievement, the mechanism behind limited responses is poorly understood. Tumor immune evasion has been reported to arise through the loss of tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways, which are crucial to CD8+ T cell-mediated killing. Syngeneic mouse models have been widely used as they have an intact immune system, are easily accessible, and have a vast array of historical data for comparison. However, limited syngeneic models respond to immune checkpoint inhibitors, possibly due to low intrinsic immunogenicity. The expression of ovalbumin (OVA) has previously shown to sufficiently alter the susceptibility of syngeneic tumors to host T cell-mediated responses. In this study, the newly developed OVA-expressing MC38 syngeneic line was characterized for tumor immunity, checkpoint blockade response and response durability.MethodsMurine colon cancer MC38 cells were transduced by lentiviral vector with chicken OVA coding cDNA. A single clone was selected, and OVA expression was confirmed by western blot. The MC38-OVA cells were subcutaneously implanted into immunocompetent mice to evaluate the tumorigenicity and in vivo response to anti-PD-1 antibody treatment. Blood was collected 2 days post final dose of anti-PD-1 treatment for phenotypic analysis by FACS. Spleen and tumor draining lymph nodes were collected at termination for FACS analysis of IFN-γ+ T cells and OVA specific CD8+ T cells. Adoptive transfer was evaluated by challenge studies in both MC38-OVA and MC38 tumor-bearing mice with T cells derived from MC38-OVA mice, anti-PD-1 cured mice and OT-I mice. In vitro killing assays were performed to evaluate the function of adoptive CD3+ T cells transfer.ResultsOVA-expressing MC38 presented complete regression under anti-PD-1 treatment in vivo. T cell expansion was observed after anti-PD-1 treatment in peripheral blood with increased IFN-γ+ T cells in both tumor-draining lymph nodes and spleen. Additionally, anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited MC38-OVA and MC38 tumor growth following adoptive transfer. CD3+ T cells from MC38-OVA-bearing mice and OT-I mice showed anti-tumor immunity in vivo. In vitro killing assay demonstrated increased immunity.ConclusionsSyngeneic mouse tumor models are preferred preclinical models for I/O research, despite limited intrinsic immunogenicity. OVA expression in syngeneic tumors largely increased T cell-mediated immunity to enhance antigen-specific T cell responses during tumorigenesis, providing novel immunogenic models for preclinical immunotherapy evaluation.

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Interleukin-15 May Be Responsible for Early Activation of Intestinal Intraepithelial Lymphocytes after Oral Infection with Listeria monocytogenes in Rats

Infection and Immunity ◽

10.1128/iai.66.12.5677-5683.1998 ◽

1998 ◽

Vol 66 (12) ◽

pp. 5677-5683 ◽

Cited By ~ 53

Author(s):

Kenji Hirose ◽

Hirohiko Suzuki ◽

Hitoshi Nishimura ◽

Akio Mitani ◽

Junji Washizu ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Cell Receptor ◽

Intraepithelial Lymphocytes ◽

Oral Infection ◽

Epithelial Cell Line ◽

Intestinal Intraepithelial Lymphocytes ◽

Interleukin 15 ◽

Ifn Γ

ABSTRACT Exogenous interleukin-15 (IL-15) stimulates intestinal intraepithelial lymphocytes (i-IEL) from mice to proliferate and produce gamma interferon (IFN-γ) in vitro. To determine whether endogenous IL-15 is involved in activation of i-IEL during intestinal infection, we examined IL-15 synthesis by intestinal epithelial cells (i-EC) after infection with Listeria monocytogenes in rats. In in vitro experiments, invasion of L. monocytogenes into IEC-6 cells, a rat small intestine epithelial cell line, evidently induced IL-15 mRNA expression coincident with nuclear factor κB (NF-κB) activation, which is essential for IL-15 gene expression. IL-15 synthesis was detected in rat i-EC on day 1 after an oral inoculation of L. monocytogenes in vivo. The numbers of T-cell receptor (TCR) γδ+ T cells, NKR.P1+cells, and CD3+ CD8+ αα cells in i-IEL were significantly increased on day 1 after oral infection. The i-IEL from infected rats produced larger amounts of IFN-γ upon stimulation with immobilized anti-TCR γδ or anti-NKR.P1 monoclonal antibodies. These results suggest that IL-15 produced by i-EC may stimulate significant fractions of i-IEL to produce IFN-γ at an early phase of oral infection with L. monocytogenes.

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Local Immune Responses to Chlamydia pneumoniae in the Lungs of BALB/c Mice during Primary Infection and Reinfection

Infection and Immunity ◽

10.1128/iai.66.11.5113-5118.1998 ◽

1998 ◽

Vol 66 (11) ◽

pp. 5113-5118 ◽

Cited By ~ 46

Author(s):

Jenni M. Penttilä ◽

Marjukka Anttila ◽

Mirja Puolakkainen ◽

Aino Laurila ◽

Kari Varkila ◽

...

Keyword(s):

Primary Infection ◽

Bacterial Infections ◽

Chlamydia Pneumoniae ◽

Mononuclear Cells ◽

Relative Increase ◽

Culturable Bacteria ◽

Ifn Γ ◽

Tissue Reactions

ABSTRACT Cell-mediated immune (CMI) responses play a major role in protection as well as pathogenesis of many intracellular bacterial infections. In this study, we evaluated the infection kinetics and assessed histologically the lymphoid reactions and local, in vitro-restimulated CMI responses in lungs of BALB/c mice, during both primary infection and reinfection with Chlamydia pneumoniae. The primary challenge resulted in a self-restricted infection with elimination of culturable bacteria by day 27 after challenge. A mild lymphoid reaction characterized the pathology in the lungs. In vitro CMI responses consisted of a weak proliferative response and no secretion of gamma interferon (IFN-γ). The number of lung-derived mononuclear cells increased substantially during the primary infection; the largest relative increase was observed in B cells (B220+). After reinfection, the number of lung-derived mononuclear cells increased further, and the response consisted mainly of T cells. The reinfection was characterized in vivo by significant protection from infection (fewer cultivable bacteria in the lungs for a shorter period of time) but increased local lymphoid reaction at the infection site. In vitro, as opposed to the response in naive mice, acquired immunity was characterized by a strongly Th1-biased (IFN-γ) CMI response. These results suggest that repeated infections with C. pneumoniae may induce Th1-type responses with similar associated tissue reactions, as shown in C. trachomatis infection models.

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A 70-Kilodalton Recombinant Heat Shock Protein ofCandida albicans Is Highly Immunogenic and Enhances Systemic Murine Candidiasis

Infection and Immunity ◽

10.1128/iai.66.5.2154-2162.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 2154-2162 ◽

Cited By ~ 52

Author(s):

Carla Bromuro ◽

Roberto La Valle ◽

Silvia Sandini ◽

Francesca Urbani ◽

Clara M. Ausiello ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Mononuclear Cells ◽

Cell Mediated Immunity ◽

Healthy Human ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha ◽

Ifn Γ

ABSTRACT The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-γ), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-γ was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-γ upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.

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