IFN-γ KO Mice Research Articles

Abstract 715: Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment

Abstract The stroma of solid tumors is populated by myeloid cells, which mostly represent macrophages. Tumor-associated macrophages (TAMs), strongly influenced by cancer cell-derived factors, are key drivers of immunosuppression and support tumor growth and spread to distant sites. Increasing evidence demonstrates their ability to hamper cancer patients' response to most treatments currently applied in the clinic, including immunotherapy. Therefore, strategies to counteract negative effects of TAMs are nowadays gaining momentum at preclinical, translational, and clinical levels. Here, we present the development and evaluation of nanoparticles (NPs) loaded with TLR agonists and/or inhibitors of immunosuppressive pathways to reprogram TAMs and the tumor microenvironment (TME) to unleash an effective immune response to fight against the tumor. Firstly, we have evaluated in vitro the ability of poly(I:C) and/or R848, agonists of TLR3 and TLR7/8 respectively, to reprogram TAMs into antitumor effector cells, and also the efficacy of Stattic and/or Galunisertib, inhibitors of STAT3 and TGF-β pathways respectively, to inhibit immunosuppression by cancer cells and/or TAMs. The best combinations of these drugs were encapsulated in nanoemulsions or polymeric nanocapsules for improved TAM-targeting and pharmacokinetics in vivo. These NPs were characterized for their physicochemical properties and also tested in vitro using primary human macrophages. For in vivo evaluation, subcutaneous and orthotopic murine models of lung cancer (CMT167) were used, showing antitumoral efficacy and TME reprogramming as evaluated by FACS, RNA analysis and multiplex immunofluorescence. Protamine-NCs-loaded with poly(I:C)+R848 and coated with an additional layer of hyaluronic acid functionalized with mannose were used to target the CD206 receptors, showing antitumoral efficacy mediated by TAM reprogramming, evaluated as higher CD86 while lower CD206 and Arg1 expression. Nanoemulsions with a PEGylated surfactant, encapsulating Stattic+Galunisertib+R848 showed faster antitumoral activity versus the free drugs. Experiments performed in IFN-γ KO mice and immune deficient mice (NSG and Balb/c nude) revealed that a fully functional immune system is crucial for the response to the treatment. In conclusion, our work demonstrates the antitumoral efficacy and reprogramming of the tumor microenvironment by combinations of TLR agonists and/or inhibitors of immunosuppression, which can be improved by nanotechnological approaches. Further investigations are ongoing to assess their antitumoral efficacy in other tumor models (i.e. breast and pancreatic cancer), with the final aim of clinical translation. Citation Format: Fernando Torres Andon, Alba Pensado-López, Clement Anfray, Aldo Ummarino, Iago Fernández-Mariño, Lucia Sanjurjo, Jose Crecente-Campo, Eduardo Fernández-Megía, Flavia Castro, Maria Jose Oliveira, Alfonso Calvo, Rosario García Campelo, Alberto Mantovani, Maria Jose Alonso, Paola Allavena. Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 715.

In vitro assay to determine inactivation of Toxoplasma gondii in meat samples

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections. However, most non-heated meat products contain salt and additives, which affect T. gondii viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on T. gondii viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 104 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the T. gondii qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 μL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %–1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values, which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

A Toxoplasma gondii lipoxygenase-like enzyme is necessary for virulence and changes localization associated with the host immune response.

Lipoxygenases (LOXs) are enzymes that catalyze the deoxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid. These modifications create signaling molecules that are best characterized for modulating the immune response. Deletion of the first lipoxygenase-like enzyme characterized for Toxoplasma gondii (TgLOXL1) generated a less virulent strain, and infected mice showed a decreased immune response. This virulence defect was dependent on the mouse cytokine interferon gamma IFNγ. TgLOXL1 changes location from inside the parasite in tissue culture conditions to vesicular structures within the host immune cells during mouse infection. These results suggest that TgLOXL1 plays a role in the modification of the host immune response in mice.

Abstract 6391: Development of a novel strategy for cancer immunotherapy based on tumor immunogenicity improvement by HLA-drug interaction

Abstract [Purpose] Tumors with low T cell infiltrates (poor immunogenicity) display lower probability of clinical response to immune checkpoint inhibitors. To achieve higher immunotherapy efficacy, it is imperative to establish a novel strategy for improving immunogenicity. Abacavir (ABC), an anti-HIV drug, binds to the specific allele of human leukocyte antigen (HLA-B*57:01) and activate CD8+ T cells by presenting altered abnormal peptides. In this study, we considered the immune interaction between ABC and HLA-B*57:01 improving immunogenicity in poorly immunogenic tumors leading to a potent anti-cancer effect. [Methods] Mice were inoculated subcutaneously with tumor cells into the right flanks and interperitoneally treated with either ABC 5 mg or vehicle (water) for consecutive 9 days (B16F10) or 13 days (3LL) after tumor inoculation. Each tumor size was measured using vernier calipers on every 2 days. Activation, proliferation, and IFN-γ secretion of tumor infiltrating CD8+ T cells were analyzed by FACS. [Results and Discussion] ABC treatment showed significant tumor growth inhibition associated with CD8+ T cell infiltration into tumor in mice inoculated with HLA-B*57:01 expressing B16F10 melanoma or Lewis lung carcinoma (3LL) cells, but neither in HLA-B*57:03 (negative allele)-expressing nor control tumors inoculated mice. Moreover, the tumor infiltrating CD8+ T cell were immune-activated and proliferated, with secreting IFN-γ. In contrast, ABC-induced anti-tumor effect was abrogated in CD8+ T cell-depleted mice, IFN-γ KO mice, and CXCR3-blocked mice. The combination therapy with anti-PD-1 antibody treatment induced better inhibition of tumor growth than ABC monotherapy in HLA-B*57:01 expressing B16F10 inoculated mice. These results suggested that ABC/HLA-B*57:01 interaction could improve tumor immunogenicity associated with CD8+ T cell-mediated anti-tumor effect in poorly immunogenic tumors. This finding lays a foundation to develop a novel drug-induced cancer immunotherapy targeting immune resistant tumors. Citation Format: Takeshi Susukida, So-ichiro Sasaki, Tomohiro Shirayanagi, Shigeki Aoki, Kousei Ito, Yoshihiro Hayakawa. Development of a novel strategy for cancer immunotherapy based on tumor immunogenicity improvement by HLA-drug interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6391.

Abstract 021: Activated Cd8 + T Cells Interact With Distal Convoluted Tubules To Promote Salt-sensitive Hypertension Through The Ifnγ-pdl1 Pathway

Background & Hypothesis: The immune system plays a critical role in the development of hypertension. We recently found that CD8 + T cells (CD8Ts) infiltrate the kidneys and stimulate distal convoluted tubular cells (DCTs), upregulating sodium-chloride co-transporter (NCC) activity, leading to excessive sodium retention. However, the precise molecules driving this direct interaction between CD8Ts and DCTs have not yet been identified. Here we hypothesize that activated CD8Ts release gamma interferon (IFNγ) that primes the tubular cell to express PDL1 which functions as a co-stimulatory ligand promoting interactions between activated CD8Ts and DCTs contributing to the development of salt-sensitive hypertension. We predict blocking this molecular pathway will reduce CD8T-homing into the kidney, lowering blood pressure. Results: We found that CD8Ts isolated from DOCA-salt treated hypertensive mice exhibit higher activity compared to those from sham normotensive mice. Pre-activated CD8Ts demonstrated augmented ability to interact with DCTs compared to naïve CD8Ts, and as a consequence, DCTs that had been co-cultured with pre-activated CD8Ts demonstrated increased expression of NCC (7 fold) and increased PDL1 and high sodium retention (47% vs. 0-3%). These effects were abolished by neutralizing IFNγ or knockdown of PDL1 in DCTs. In-vivo results verified the in-vitro studies. We found that IFNγ-KO mice demonstrated reduced blood pressure elevation and T cell infiltration within their kidneys to DOCA-salt compared to WT mice receiving the same treatment (last day systolic blood pressure average 155.2 mmHg compared to 189 mmHg from WT). Blood pressure elevation was blunted in DOCA-salt treated mice with renal tubule-specific knockdown of PDL1 (last day systolic blood pressure average 124 mmHg for mice with siPDL1 nanoparticle and 144 mmHg for mice with scrambled siRNA); NCC elevation was also blunted in this model. Conclusion: Activated CD8Ts demonstrate enhanced ability to interact with DCTs leading to increased expression of NCC and sodium retention through a IFNγ-PDL1 mediated mechanism. Blocking the IFNγ-PDL1 pathway prevents CD8T-DCT interaction both in vitro and in vivo , thereby ameliorating salt-sensitive hypertension.

Abstract 1378: CD8 T cell mediated killing of MHC class 1 negative tumors requires antigen presenting myeloid cells and interferon gamma

Abstract Major Histocompatibility Complex (MHC) Class I downregulation is a well described mechanism of tumor immune escape, posing a challenge for T cell based immunotherapies including immune checkpoint blockade (ICB). Recent studies, however, have demonstrated mixed roles of MHC Class 1 and the critical component beta-2-microglobulin (β2m) expression in cancer progression and ICB response, with some studies showing inactivation of antigen presentation to be associated with resistance to ICB and others showing low β2m expression to be associated with favorable prognosis. Glioblastoma (GBM) in particular expresses little or no MHC Class 1 and patients remain unresponsive to ICB. We thus sought to evaluate the role of MHC Class 1 in ICB, given that we have previously demonstrated that combination ICB with anti-PD-1 and co-stimulation with 4-1BB agonism has marked efficacy against intracranial murine glioma tumors in a CD8 T cell dependent manner. Surprisingly, in a CT2A murine glioma tumor line expressing the antigen TRP2 and lacking cell surface MHC I (CT2A-TRP2-β2mKO), the efficacy of combination 4-1BB and PD-1 therapy (ICB) was re-demonstrated in a CD8 dependent manner, independent of NK cells, CD4 T cells, and B cells. Furthermore, the efficacy of immunotherapy against intracranial CT2A-TRP2-β2mKO was demonstrated to be antigen dependent, with an adoptive lymphocyte transfer (ALT) of TRP2 TCR transduced T cells (TRP2 T cells) into a CD8KO mouse sufficient to eliminate CT2A-TRP2-β2mKO in the setting of ICB. Additionally, an ALT of TRP2 T cells did not kill CT2A-β2mKO tumors in the setting of ICB, while OT-1 mice whose CD8+ T cells primarily recognize OVA peptide with CT2A-TRP2-β2mKO tumors did not respond to ICB. In vitro studies revealed that TRP2 T cells demonstrated anti-tumor cytotoxicity against MHC Class I negative CT2A-TRP2-β2mKO tumor cells in the presence of TRP2 loaded bone marrow derived macrophages (TRP2 Mφ), but neither cell type was individually sufficient to induce tumor cell death, while the combination of TRP2 T cells and TRP2 Mφ demonstrated no cytotoxicity against CT2A-β2mKO tumors. Transwell experiments in which TRP2 Mφ and CT2A-TRP2-β2mKO tumor cells were separated by a 0.5µm membrane permeable to T cells but not Mφ or tumor cells revealed that contact between TRP2 Mφ and tumor cells was not necessary to induce T cell dependent killing. Indeed, tumor-bearing β2mKO bone marrow chimeras lacking MHC class 1 on hematopoeitically derived cells did not respond to ICB, highlighting the importance of antigen presentation from myeloid cells. The mechanism of killing was found to be dependent on interferon gamma (IFNγ), as IFNγKO mice did not respond to ICB. These findings demonstrate that tumors with low MHC Class 1 expression may still be targeted by T cell dependent immunotherapies such as ICB when antigen presentation can occur from myeloid cells. Citation Format: Emily Lerner, Vincent D'Anniballe, William Tomaszewski, Jonathan Perera, Xiuyu Cui, Jessica Waibl-Polania, Daniel Wilkinson, Michael D. Gunn, Peter E. Fecci, Karolina Woroniecka. CD8 T cell mediated killing of MHC class 1 negative tumors requires antigen presenting myeloid cells and interferon gamma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1378.

TNFα Controls the Delicate Balance between Erythropoiesis and Stem Cell Exhaustion during Inflammatory Stress

Anemia of Inflammation (AI) is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, or cancer. Induced expression of hepcidin by pro-inflammatory cytokines results in iron-restricted anemia. In particular, abnormally elevated levels of the cytokine Tumor Necrosis Factor-α (TNFα) is a hallmark of AI, however its contribution to the pathophysiology of AI is not well understood.In this study, we investigated the role of TNFα in the development of anemia in a TNFα knockout (TNFαKO) mouse model of AI, which is induced with a single intraperitoneal (i.p.) injection of heat-killed Brucella Abortus (BA) (Kim et al. Blood). We hypothesized that TNFαKO mice would show a less severe form of AI compared to WT animals when challenged with BA.Our results showed that WT-BA mice developed severe anemia within 2 weeks, which was resolved by 8 weeks, whereas the TNFαKO mice developed leukocytosis and an irreversible macrocytic, hyperchromic anemia. Serum analysis at 8 weeks showed that erythropoietin (EPO) and iron parameters were elevated in TNFαKO compared to WT mice, which ruled out iron-restriction as the cause for the persistent anemia. However, serum cytokine measurements of TNFαKO mice at 4 weeks showed continual elevation of interleukin (IL)-12p40 and Interferon-γ (IFNγ) compared to WT-BA controls. We hypothesized that TNFα served an anti-inflammatory role that restrained prolonged inflammation after BA, and in its absence, pro-inflammatory macrophages continuously secreted IL-12p40 levels and induced the proliferation of IFNγ secreting cells.To test if concurrent loss of IFNγ would correct the inflammatory phenotype, we crossed TNFαKO with IFNγKO mice (DKO). Indeed, IFNγKO and DKO mice challenged with BA showed complete reversal of the anemic phenotype present in WT-BA and TNFαKO-BA at 2 weeks. Additionally, serum levels of IL-12p40 were normalized by 4 weeks in IFNγKO-BA and DKO-BA compared to TNFαKO-BA mice. Flow cytometry analysis of the bone marrow (BM) and spleen (SPL) at 8 weeks showed T-lymphocytes and macrophages were markedly expanded, whereas erythrocytes and B-lymphocytes were reduced in TNFαKO-BA mice. However, only modest differences in erythrocytes, macrophage, T- and B-lymphocyte in WT-BA, IFNγKO-BA and DKO-BA in the BM and SPL were detected. Additionally, we performed immunohistochemistry using an anti-CD3 antibody in SPL and livers of BA treated mice sacrificed at 8 weeks. We found complete disorganization of the white pulp in the SPL and infiltration of T-Lymphocytes in livers of TNFαKO-BA but not in WT-BA, IFNγKO-BA or DKO-BA animals.These results led us to question if lack of TNFα skewed the BM towards T-Lymphocytes. We investigated the hematopoietic stem cell (HSC) LSK, multipotent myeloid progenitors (MPP) LK, and common lymphoid progenitor (CLP) compartments by flow cytometry. Shockingly, the TNFαKO had over a 30% increase of Lin-cKit+Sca1+ LSKs, compared to TNFαKO controls, while the Lin-cKit+Sca1- LK population was drastically reduced. By contrast, the Lin-CD217-cKit+Sca1+ CLP population was expanded by more than 40% in the TNFαKO-BA compared to TNFαKO controls.Considering this data, it is difficult to disentangle the effects of TNFα in AI in vivo from its role in regulating upstream stem and progenitor cell differentiation using a germline KO strategy. However, our observations reinforce the link between iron homeostasis and HSC self-renewal and provide a new model to study inflammation associated bone marrow failure. The TNFα-BA mice displayed severe anemia, which seems to result from persistent IFNγ elevation. A recent study identified TNFα as a major pro-survival and pro-regeneration factor for HSCs (Yamashita & Passegue, Cell Stem Cell). Other studies have shown that IFNγ restricts HSCs self-renewal (Chen et al. Blood). Our results in the TNFαKO-BA treated mice suggest that TNFα preserves balanced progenitor output by countering the action of IFNγ at the HSC level. Ongoing work aims to understand the relationship between TNFα and IFNγ in regulating HSC quiescence, self-renewal, and overall pool size. DisclosuresPaulson: Forma Therapeutics: Consultancy. Rivella: Meira GTx: Consultancy; Ionis Pharmaceuticals: Consultancy.

The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides.

CD47 is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. We utilized CRISPR/Cas-9 CD47 knock-out, depletion of NK cells, and mice genetically deficient in IFN-γ to elucidate the mechanism of anti-CD47 therapy in a murine model of cutaneous T cell lymphoma (CTCL). CD47 was found to be a crucial factor for tumor progression since CD47 KO CTCL exhibited a delay in tumor growth. The treatment of CD47 WT murine CTCL with anti-CD47 antibodies led to a significant reduction in tumor burden as early as four days after the first treatment and accompanied by an increased percentage of cytotoxic NK cells at the tumor site. The depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti-CD47. Notably, the treatment of CD47 WT tumors in IFN-γ KO mice with anti-CD47 antibodies was efficient, demonstrating that IFN-γ was not required to mediate anti-CD47 therapy. We were able to potentiate the therapeutic effect of anti-CD47 therapy by IFN-α. That combination resulted in an increased number of cytotoxic CD107a + IFN-γ-NK1.1 cells and intermediate CD62L + NKG2a-NK1.1. Correlative data from a clinical trial (clinicaltrials.gov, NCT02890368) in patients with CTCL utilizing SIRPαFc to block CD47 confirmed our in vivo observations.

Contributions of IFN-γand granulysin to the clearance of Plasmodium yoelii blood stage.

P. vivax-infected Retics (iRetics) express human leukocyte antigen class I (HLA-I), are recognized by CD8+ T cells and killed by granulysin (GNLY) and granzymes. However, how Plasmodium infection induces MHC-I expression on Retics is unknown. In addition, whether GNLY helps control Plasmodium infection in vivo has not been studied. Here, we examine these questions using rodent infection with the P. yoelii 17XNL strain, which has tropism for Retics. Infection with P. yoelii caused extramedullary erythropoiesis, reticulocytosis and expansion of CD8+CD44+CD62L- IFN-γ-producing T cells that form immune synapses with iRetics. We now provide evidence that MHC-I expression by iRetic is dependent on IFN-γ-induced transcription of IRF-1, MHC-I and β2-microglobulin (β2-m) in erythroblasts. Consistently, CTLs from infected wild type (WT) mice formed immune synapses with iRetics in an IFN-γ- and MHC-I-dependent manner. When challenged with P. yoelii 17XNL, WT mice cleared parasitemia and survived, while IFN-γ KO mice remained parasitemic and all died. β2-m KO mice that do not express MHC-I and have virtually no CD8+ T cells had prolonged parasitemia, and 80% survived. Because mice do not express GNLY, GNLY-transgenic mice can be used to assess the in vivo importance of GNLY. Parasite clearance was accelerated in GNLY-transgenic mice and depletion of CD8+ T cells ablated the GNLY-mediated resistance to P. yoelii. Altogether, our results indicate that in addition to previously described mechanisms, IFN-γ promotes host resistance to the Retic-tropic P. yoelii 17XNL strain by promoting MHC-I expression on iRetics that become targets for CD8+ cytotoxic T lymphocytes and GNLY.

GRIM-19 Ameliorates Multiple Sclerosis in a Mouse Model of Experimental Autoimmune Encephalomyelitis with Reciprocal Regulation of IFNγ/Th1 and IL-17A/Th17 Cells.

The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain. Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord. Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM19 OVN. GRIM-19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

Regulatory Role for NK Cells in a Mouse Model of Systemic Juvenile Idiopathic Arthritis.

Mice deficient in IFN-γ (IFN-γ knockout [KO] mice) develop a systemic inflammatory syndrome in response to CFA, in contrast to CFA-challenged wild-type (WT) mice who only develop a mild inflammation. Symptoms in CFA-challenged IFN-γ KO resemble systemic juvenile idiopathic arthritis (sJIA), a childhood immune disorder of unknown cause. Dysregulation of innate immune cells is considered to be important in the disease pathogenesis. In this study, we used this murine model to investigate the role of NK cells in the pathogenesis of sJIA. NK cells of CFA-challenged IFN-γ KO mice displayed an aberrant balance of activating and inhibitory NK cell receptors, lower expression of cytotoxic proteins, and a defective NK cell cytotoxicity. Depletion of NK cells (via anti-IL-2Rβ and anti-Asialo-GM1 Abs) or blockade of the NK cell activating receptor NKG2D in CFA-challenged WT mice resulted in increased severity of systemic inflammation and appearance of sJIA-like symptoms. NK cells of CFA-challenged IFN-γ KO mice and from anti-NKG2D-treated mice showed defective degranulation capacities toward autologous activated immune cells, predominantly monocytes. This is in line with the increased numbers of activated inflammatory monocytes in these mice which was particularly reflected in the expression of CCR2, a chemokine receptor, and in the expression of Rae-1, a ligand for NKG2D. In conclusion, NK cells are defective in a mouse model of sJIA and impede disease development in CFA-challenged WT mice. Our findings point toward a regulatory role for NK cells in CFA-induced systemic inflammation via a NKG2D-dependent control of activated immune cells.

Defect of Interferon γ Leads to Impaired Wound Healing through Prolonged Neutrophilic Inflammatory Response and Enhanced MMP-2 Activation.

Interferon (IFN)-γ is mainly secreted by CD4+ T helper 1 (Th1), natural killer (NK) and NKT cells after skin injury. Although IFN-γ is well known regarding its inhibitory effects on collagen synthesis by fibroblasts in vitro, information is limited regarding its role in wound healing in vivo. In the present study, we analyzed how the defect of IFN-γ affects wound healing. Full-thickness wounds were created on the backs of wild type (WT) C57BL/6 and IFN-γ-deficient (KO) mice. We analyzed the percent wound closure, wound breaking strength, accumulation of leukocytes, and expression levels of COL1A1, COL3A1, and matrix metalloproteinases (MMPs). IFN-γKO mice exhibited significant attenuation in wound closure on Day 10 and wound breaking strength on Day 14 after wound creation, characteristics that are associated with prolonged neutrophil accumulation. Expression levels of COL1A1 and COL3A1 mRNA were lower in IFN-γKO than in WT mice, whereas expression levels of MMP-2 (gelatinase) mRNA were significantly greater in IFN-γKO than in WT mice. Moreover, under neutropenic conditions created with anti-Gr-1 monoclonal antibodies, wound closure in IFN-γKO mice was recovered through low MMP-2 expression levels. These results suggest that IFN-γ may be involved in the proliferation and maturation stages of wound healing through the regulation of neutrophilic inflammatory responses.

Ex vivo fucosylation promotes the GVL effect of NK cells after Allo-HSCT

Abstract Recent studies demonstrated that ex vivo fucosylation can increase the persistence and anti-GVHD potency of Treg cells. Fucosylation also improves human cord blood engraftment. However, the role of fucosylation in graftversus- leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) remains unknown. Here, we established a murine GVL model by intravenously injecting luciferase (lus+)/yfp-expressing B-cell lymphoma (A20) cells and founded that adoptive transfer of fucosylated NK cells markedly prolonged the survival than control NK cells. Ex vivo fucosylation significantly upregulated IFN-γ production in NK cells and enhanced their cytotoxic activity. Flow cytometry analysis revealed that fucosylation promoted the infiltration of NK cells, which may be due to the elevated binding to CD62E. Further studies are needed to confirm the contribution of IFN-γ by using IFN-γ KO mice and investigate the detailed mechanisms of fucosylated NK cells in the regulation of GVL responses.

Reconstitution of iNKT cells enhances antitumor immunity through liver education of iNKT cells and modulation of tumor microenvironment

Abstract Invariant natural killer (iNKT) cells are a specific population of T cells that functionally are more like innateimmune regulatory cells and play important roles in antitumor immunity. It is reported that reconstitution of iNKT cells enhances antitumor immunity in human and mice, however, the underlying mechanism is not known. Using thymocytes and splenocytes to reconstitute iNKT cells in iNKT KO mice, we found that reconstitution of iNKT cells dramatically inhibits tumor development, progression and metastasis, and increases the survival of tumor-bearing mice. Most of the transferred cells accumulated in the liver. After two weeks of transfer around 70% of the transferred thymocytes in the liver differentiated into iNKT cells. Transferring CXCR6− thymocytes enhanced antitumor immunity, transferring CXCR6− splenocytes inhibited antitumor immunity. The enhanced antitumor immunity was abolished in IFN-γ and iNKT double KO mice, which suggests that this antitumor immunity is IFN-γ-dependent. Depletion of NK cells, but not CD8+ T cells or CD4+ T cells, diminishes iNKT cell reconstitution-induced antitumor immunity. Reconstitution of iNKT cells increased tumor-infiltrated lymphocytes (TIL), specifically IFN-γ-producing CD8+ T cells and NK cells, but decreased MDSC and M2 macrophages and down regulated the expression of TGFβ, Arginase and iNOS in the tumor. Taken together, liver education of iNKT cells plays a key role in iNKT cell reconstitution-induced antitumor immunity. This enhanced antitumor immunity depends on IFN-γ and NK cells of the host through modulating tumor microenvironment.

Interferon-gamma deficiency protects against aging-related goblet cell loss.

Aging is a well-recognized risk factor for dry eye. Interferon-gamma (IFN-γ) has been implicated in conjunctival keratinization and goblet cell loss in dry eye. We investigated the role of IFN-γ in age-related dry eye by evaluating young (8 weeks) and aged (15 months; 15M) C57BL/6 (B6) and IFN-γKO mice. Age effects on the conjunctiva and cornea epithelium were assessed with PAS staining and corneal staining, respectively. Expression of T cell-related cytokines (IL-17A, IFN-γ), chemokines (CXCL10 and CCL20), in the ocular surface epithelium was evaluated by real time PCR. A significant decrease in filled goblet cells was noted in 15M B6 mice and this was significantly lower than age and sex-matched IFN-γKO mice. Aged male B6 had significantly higher IFN-γ, and CXCL10 mRNA in their conjunctiva than female B6 mice. Aged IFN-γKO females had significantly higher IL-17A mRNA in conjunctiva than IFN-γKO males and B6 mice. Corneal barrier dysfunction was observed in 15M female B6 and aged IFN-γKO mice of both sexes; however it was significantly higher in IFN-γKO compared to B6 mice. While there was a significant increase in IL 17A, and CCL20 in corneas of aged female B6 and IFN-γKO mice compared to males, these changes were more evident in aged female IFN-γKO group.Partial resistance of IFN-γKO mice to aging-induced goblet cell loss indicates IFN-γ is involved in the age-related decline in conjunctival goblet cells. Increased corneal IL-17A expression paralleled corneal barrier disruption in aging female of both strains. IFN-γ appears to suppress IL-17A on the ocular surface.

Interferon gamma protects neonatal neural stem/progenitor cells during measles virus infection of the brain.

BackgroundIn the developing brain, self-renewing neural stem/progenitor cells (NSPC) give rise to neuronal and glial lineages. NSPC survival and differentiation can be altered by neurotropic viruses and by the anti-viral immune response. Several neurotropic viruses specifically target and infect NSPCs, in addition to inducing neuronal loss, which makes it difficult to distinguish between effects on NSPCs that are due to direct viral infection or due to the anti-viral immune response.MethodsWe have investigated the impact of anti-viral immunity on NSPCs in measles virus (MV)-infected neonates. A neuron-restricted viral infection model was used, where NSPCs remain uninfected. Thus, an anti-viral immune response was induced without the confounding issue of NSPC infection. Two-transgenic mouse lines were used: CD46+ mice express the human isoform of CD46, the MV entry receptor, under the control of the neuron-specific enolase promoter; CD46+/IFNγ-KO mice lack the key anti-viral cytokine IFNγ. Multi-color flow cytometry and Western Blot analysis were used to quantify effects on NSPC, neuronal, and glial cell number, and quantify effects on IFNγ-mediated signaling and cell markers, respectively.ResultsFlow cytometric analysis revealed that NSPCs were reduced in CD46+/IFNγ-KO mice at 3, 7, and 10 days post-infection (dpi), but were unaffected in CD46+ mice. Early neurons showed the greatest cell loss at 7 dpi in both genotypes, with no effect on mature neurons and glial cells. Thus, IFNγ protected against NSPC loss, but did not protect young neurons. Western Blot analyses on hippocampal explants showed reduced nestin expression in the absence of IFNγ, and reduced doublecortin and βIII-tubulin in both genotypes. Phosphorylation of STAT1 and STAT2 occurred independently of IFNγ in the hippocampus, albeit with distinct regulation of activation.ConclusionsThis is the first study to demonstrate bystander effects of anti-viral immunity on NSPC function. Our results show IFNγ protects the NSPC population during a neonatal viral CNS infection. Significant loss of NSPCs in CD46+/IFNγ-KO neonates suggests that the adaptive immune response is detrimental to NSPCs in the absence of IFNγ. These results reveal the importance and contribution of the anti-viral immune response to neuropathology and may be relevant to other neuroinflammatory conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0571-1) contains supplementary material, which is available to authorized users.

Microencapulated rapamycin prevents carcinogen and inflammation driven colon cancer through immune mechanisms

Abstract We tested microencapsulated rapamycin (eRapa, ~2.2 mg/kg rapamycin/mouse/day) in carcinogen (azoxymethane, AOM) + inflammatory agent (dextran sodium sulfate, DSS) colon cancer. WT BL6 mice fed eRapa before, and during AOM/DSS had significantly fewer colon tumors and tumor burden than control fed mice (empty microcapsules). eRapa prevented colon cancer in δ TCR KO mice lacking γδ T cells but not in IFN-γ KO mice. In carcinogen (DMBA) + inflammatory agent (TPA) skin cancer, IFN-γ and γδ T cells were both needed for eRapa cancer prevention, showing tumor-specific and common immune requirements for eRapa-mediated cancer prevention. In βδ TCR KO mice lacking all T cells, AOM/DSS induced no cancer or only few tumors, suggesting αβ T cells are required for colon neoplasia and cancer in the AOM/DSS model. Protection from acute colitis in this model usually predicts colon cancer protection. Strikingly, however, eRapa did not prevent acute clinical or histologic colitis induced by DSS, despite cancer protection, suggesting effects on chronic colitis, or induction of cancer-protective but not acute colitis-protective mechanisms. In acute colitis, eRapa significantly decreased spleen and colon weights and CD3+CD4+ T cells, and increased mesenteric lymph node γδ T cells (with decreased Vγ1.1+ and increased Vγ2+ subsets) consistent with altered inflammation and reduced CD4-CXCR3+ and CD4-α4β7 T cells (likely γδ T cells) consistent with altered trafficking but did not affect CCR9+ T cells. In chronic colitis, eRapa significantly increased γδ T cells (with no changes in Vγ1.1+ or Vγ2+ subsets) that could mediate cancer protection. These novel immune effects of rapamycin help define its cancer prevention mechanisms and define novel clinical uses.

IFN-γ production by CD4 CD8 double positive cells is essential for expansion of Proteobacteria in the neonatal murine intestine.

Abstract Background Expansion of Proteobacteria is seen in the neonatal intestine in both humans and mice. Proteobacteria are also significantly over-represented in preterm infants that develop Necrotizing Enterocolitis (NEC). Neonatal mice are more susceptible to models of NEC. The mechanism of expansion of Proteobacteria in neonatal mice and susceptibility to NEC remain undefined. Objective To identify immune modulators that favor expansion of Proteobacteria in the neonatal murine intestine. Methods qRT-PCR of bacterial 16S rRNA gene sequence tags of colonic fecal contents from 1–3 week (wk) old wild type (WT), IFN-γ knock out and Rag deficient mice was performed to identify major bacterial groups. qRT-PCR and ELISA of whole colon were used to profile cytokine expression. Flow-cytometry of lamina propria cells was used to characterize cells in 1-,2- and 3-wk old mice. IFN-γ neutralizing Ab was given IP to 3 day old mice and then similarly examined at 2 wks. Results 2-wk-old WT mice showed increased expansion of Proteobacteria and increased production of IFN-γ. IFN-γ KO mice had minimal expansion of Proteobacteria in the colon, this was also seen in WT mice blocked with IFN-γ Ab. Flow-cytometry revealed a unique subset of CD4 CD8 double positive cells, only present in neonatal mice, that produce IFN-γ in WT mice. Rag deficient mice did not have this subset of cells and also had minimal colonization by Proteobacteria at 2-wks. Conclusions IFN-γ promotes the expansion of Proteobacteria in the neonatal intestine. A unique and transient subset of CD4 CD8 cells, that are absent in Rag deficient mice, appear to be responsible for the increased IFN-γ in neonatal mice. This is a potential target for intervention in the prevention of NEC in preterm infants.

Leishmania (Viannia) braziliensis amastigotes from patients with mucosal leishmaniasis have increased ability to disseminate and are controlled by nitric oxide at the early stage of murine infection.

Mucosal leishmaniasis (ML) caused by Leishmania (Vianna) braziliensis usually appears after the healing of the primary lesion when amastigotes disseminate from the infection site to the mucosal area. Here, we investigated murine infection with amastigotes obtained from patients with ML or localized cutaneous leishmaniasis (LCL). Amastigotes were used to infect wild type, IFN-γ KO and inducible nitric oxide synthase (iNOS) KO mice. Amastigotes from patients with LCL induced lesions that appeared earlier in IFN-γ KO than parasites from ML. The lesion after infection with ML appeared early in iNOS KO than in IFN-γ KO mice and in iNOS KO mice parasites from ML and LCL cause similar lesions at the initial phase of infection, while parasites from ML induced greater lesions than the ones from LCL at the late phase. A greater number of parasites were observed in spleen of IFN-γ KO and iNOS KO mice infected with amastigotes from patients with ML than those with LCL. Parasites from ML infect a lower percentage of macrophages and are killed independent on IFN-γ and dependent on NO. The data suggest that amastigotes responsible for mucosal lesion in humans develop slowly on the initial phase of infection due to high susceptibility to NO and they have an increased ability to disseminate.

Contribution of Invariant Natural Killer T Cells to Skin Wound Healing

In the present study, we determined the contribution of invariant natural killer T (iNKT) cells to the skin wound healing process. In iNKT cell-deficient (Jα18KO) mice lacking iNKT cells, wound closure was significantly delayed compared with wild-type mice. Collagen deposition, expression of α-smooth muscle actin and CD31, and wound breaking strength were significantly attenuated in Jα18KO mice. The adoptive transfer of liver mononuclear cells from wild-type but not from Jα18KO or interferon (IFN)-γ gene-disrupted (IFN-γKO) mice resulted in the reversal of this impaired wound healing in Jα18KO mice. IFN-γ expression was induced in the wounded tissues, which was significantly decreased at 6, 12, and 24 hours, but increased on day 3 after wounding in Jα18KO mice. The main source of the late-phase IFN-γ production in Jα18KO mice were neutrophils rather than NK cells and T cells. Administration of α-galactosylceramide, an activator of iNKT cells, resulted in the acceleration of wound healing on day 3 in wild-type mice. This effect was not observed in IFN-γKO mice. These results indicate that iNKT cells play important roles in wound healing. The iNKT cell-induced IFN-γ production may regulate the wound healing process in the early phase.