Increased IFN-γ Production Research Articles

An autologous humanized patient-derived-xenograft platform to evaluate immunotherapy in ovarian cancer

ObjectiveThe aim of this study was to “humanize” ovarian cancer patient-derived xenograft (PDX) models by autologous transfer of patient-matched tumor infiltrating lymphocytes (TILs) to evaluate immunotherapies. MethodsOrthotopic high-grade serous ovarian cancer (HGSOC) PDX models were established from three patient donors. Models were molecularly and histologically validated by immunohistochemistry. TILs were expanded from donor tumors using a rapid expansion protocol. Ex vivo TIL and tumor co-cultures were performed to validate TIL reactivity against patient-matched autologous tumor cells. Expression of TIL activation markers and cytokine secretion was quantitated by flow cytometry and ELISA. As proof of concept, the efficacy of anti-PD-1 monotherapy was tested in autologous TIL/tumor HGSOC PDX models. ResultsEvaluation of T-cell activation in autologous TIL/tumor co-cultures resulted in an increase in HLA-dependent IFNγ production and T-cell activation. In response to increased IFNγ production, tumor cell expression of PD-L1 was increased. Addition of anti-PD-1 antibody to TIL/tumor co-cultures increased autologous tumor lysis in a CCNE1 amplified model. Orthotopic HGSOC PDX models from parallel patient-matched tumors maintained their original morphology and molecular marker profile. Autologous tumor-reactive TIL administration in patient-matched PDX models resulted in reduced tumor burden and increased survival, in groups that also received anti-PD-1 therapy. ConclusionsThis study validates a novel, clinically relevant model system for in vivo testing of immunomodulating therapeutic strategies for ovarian cancer, and provides a unique platform for assessing patient-specific T-cell response to immunotherapy.

Abstract B044: Development of a highly potent humanized antibody against CD178: A novel therapeutic target in immuno-oncology

Abstract CD178 (FASLG) is an emerging therapeutic target in immuno-oncology (IO) since CD178/CD95-dependent signalling plays several crucial roles in regulating the immune response to tumours including T cell persistence/survival, T cell exclusion and tumour-dependent T cell killing. Using the mouse hybridoma approach antibodies were raised against the extracellular domain (Pro134-Leu281) of recombinant human CD178. Clone 2B11 was selected for humanisation based on its excellent potency and lack of problem residues in the CDR. Following humanisation by CDR grafting and the introduction of additional mutations to improve affinity and biophysical properties, FBP002 was selected as a potential therapeutic candidate. FB-002 is an IgG4 (S228P) isotype antibody that binds CD178 with high potency in in vitro ELISA assays (IC50 161 pM; 24 ng/mL) and in Surface Plasmon Resonance (SPR) studies (KD of 97 pM). In cellular assays, FBP002 inhibits cell death induced by CD178 in both Jurkat A3 cells (IC50 2.1 ng/mL) and in unstimulated CD4+ human donor T cells (IC50 37 ng/mL). In a model of activation-induced cell death, FBP002 inhibited anti-CD3/anti-CD28 induced cytotoxicity in Jurkat A3 cells (IC50 418 ng/mL). Repeated stimulation of effector T cells can severely limit their function and blunt their anti-tumour response. To investigate the role of CD178 in sustaining T cell activation, we employed a T cell re-stimulation assay by treating human PBMCs with staphylococcal enterotoxin B (SEB) super-antigen for 5 days to activate T cells, followed by IL2 incubation for 14 days, and then a secondary treatment with SEB for 5/6 days. FBP002 significantly (p<0.05) increased cell proliferation following secondary SEB treatment and was also associated with a significant increase in IFNγ production measured by ELISA (p<0.05). FBP002 was more effective than either anti-PD-1 or anti-PD-L1 in this re-stimulation assay and enhanced both PD-1 and PD-L1 activity suggesting a non-overlapping role for CD178 in maintaining the function of activated T cells. To investigate in vivo effects, subcutaneous MC38 murine colon tumours were established in syngeneic immune competent female C57BL/6 mice. Once tumours reached 100 mm3, mice were treated with a surrogate anti-murine CD178 antibody (10 mg/kg i.p. twice weekly), or an isotype control. Anti-CD178 antibody treatment was well tolerated (no body weight loss) and significantly delayed MC38 tumour growth (p<0.05). Immunohistchemistry and image analysis demonstrated that the anti-tumour effect was associated with an increased CD8/Treg ratio in the tumour suggesting that inhibiting CD178 signalling disproportionately protects effector T cells from apoptosis leading to a more effective immune response. In conclusion, we have developed FBP002, a highly potent humanised anti-CD178 antibody that we anticipate will have broad therapeutic potential in the immunological treatment of tumours both as a monotherapy and in combination with established IO agents due to its ability to simultaneously affect multiple aspects of tumour resistance to T cell therapies. Citation Format: Georgia Cerillo, Anderson J Ryan, Peter D Davis. Development of a highly potent humanized antibody against CD178: A novel therapeutic target in immuno-oncology [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B044. doi:10.1158/1535-7163.TARG-19-B044

Marek's Disease Virus Infection of Natural Killer Cells.

Natural killer (NK) cells are key players in the innate immune response. They kill virus-infected cells and are crucial for the induction of adaptive immune responses. Marek’s disease virus (MDV) is a highly contagious alphaherpesvirus that causes deadly T cell lymphomas in chickens. Host resistance to MDV is associated with differences in NK cell responses; however, the exact role of NK cells in the control of MDV remains unknown. In this study, we assessed if MDV can infect NK cells and alter their activation. Surprisingly, we could demonstrate that primary chicken NK cells are very efficiently infected with very virulent RB-1B MDV and the live-attenuated CVI988 vaccine. Flow cytometry analysis revealed that both RB-1B and CVI988 enhance NK cell degranulation and increase interferon gamma (IFNγ) production in vitro. In addition, we could show that the MDV Eco Q-encoded oncogene (meq) contributes to the induction of NK cell activation using meq knockout viruses. Taken together, our data revealed for the first time that NK cells are efficiently infectable with MDV and that this oncogenic alphaherpesvirus enhances NK cell degranulation and increased IFNγ production in vitro.

Chimeric Antigen Receptor Modified Memory-like (CAR-ML) NK Cells Exhibit Potent Responses to NK-Resistant Tumors

NK cells are cytotoxic innate lymphoid cells that mediate anti-tumor responses and traffic to most tissues. NK cells activated briefly with IL-12, IL-15, and IL-18 differentiate into memory-like (ML) NK cells with enhanced anti-tumor effects, which has been translated into clinical trials for patients with leukemia. Adoptive cellular therapy with donor ML NK cells were safe and induced CR/CRi in >50% of rel/ref AML patients in a first-in-human phase 1 clinical trial at Washington University (PMID27655849). In preliminary results, donor ML NK cells expand and can persist for months in an immune-compatible recipients during or after allogeneic hematopoietic cell transplantation (HCT), and maintain potent effector function. However, NK cell recognition of many cancer types is limited, since they depend on expression of stress-induced activating receptor ligands. We hypothesized that ML NK cells engineered with chimeric antigen receptors (CAR) would demonstrate improved anti-tumor responses against classically NK-resistant targets. To test this idea, ML NK cells were engineered to express an anti-CD19-CAR, and responses against NK-resistant B-cell malignancies evaluated in vitro and in vivo. CAR-modified primary human ML NK cells (CAR-ML) were transduced with an anti-CD19-CD8a-41BB-CD3z-GFP (CD19-CAR-ML) lentivirus. This differentiation and transfection approach resulted in approximately 15-25% of ML NK cells transduced with the CAR construct, as determined flow cytometry staining for soluble CD19 and GFP. As an additional control, αCD33-CD8a-41BB-CD3z-GFP CAR-transduced ML NK cells and GFP- internal control ML NK cells were used. Here, in vitro functional assays were used to determine if CD19-CAR enhances ML NK cells in an antigen (CD19)-specific manner. CD19-CAR-ML (GFP+) and control ML NK (GFP-) cells were evaluated for functional responses to CD19-positive or CD19-negative tumor targets in vitro. CD19-CAR-ML NK cells demonstrated significantly increased IFN-γ production (44±4% vs. 15±3%, p<0.001); mean ± SEM) and degranulation (31±4% vs. 5±1%, p<0.001) against NK-resistant CD19+ Raji targets, compared to control GFP- and CD33-CAR ML NK cells. To understand the contributions of ML differentiation on the enhanced functionality of CD19-CAR modified NK cells, we compared CD19-CAR-ML NK cells to control CD19-CAR NK cells that were treated with IL-15 only. CD19-CAR-ML NK cells also exhibited significantly increased effector responses compared to control CAR NK cells against CD19+ targets (p<0.01). CD19-CAR-ML NK cells responded similarly to GFP- ML NK cells against CD19-negative Kasumi leukemia targets. Finally, CD19-CAR-ML NK cells also exhibited significantly enhanced killing, degranulation, and IFN-γ production against primary CD19+ follicular lymphoma targets from patient lymph nodes (p<0.01). To establish the translational utility of this approach, autologous CD19-CAR-ML NK cells generated from lymphoma patients demonstrated significantly increased IFN-γ production (p<0.05) and degranulation (p<0.01) against their own CD19+ lymphoma targets, compared to control ML NK cells (GFP-). These data confirm contributions of both ML differentiation and CAR expression in the enhanced antigen-specific, anti-tumor responses observed in CAR-ML NK cells. To test the expansion and persistence of ML NK cells transduced with CD19 or CD33-CAR were transferred into CD19+ Raji-bearing NSG mice and supported in vivo with IL-15. After three weeks, mice were sacrificed and NK (CD56) cell persistence and tumor (CD19) burden assessed by flow cytometry. CD19-CAR ML recipient mice had reduced tumor burden in the BM, spleen, and blood compared to CD33-CAR ML treated mice. Notably, CD19-CAR-ML (GFP+) were increased to >70% of human NK cells from 20% in the CD33-CAR-ML recipient mice, suggesting antigen-specific CAR-expressing NK cells expand or survive better in vivo than non-transduced ML or non-specific CAR-ML NK cells in vivo. Thus, combining CAR with ML differentiation results in NK cells with enhanced responses to NK resistant tumors. These studies warrant continued CAR-ML development and provide the pre-clinical rationale for translating this combination NK cell therapy approach to the clinic. Figure Disclosures Fehniger: Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).

Bioactive Polysaccharide Nanoparticles Improve Radiation-Induced Abscopal Effect through Manipulation of Dendritic Cells.

Radiotherapy was considered to induce an abscopal effect initiated through antigen release and presented by dendritic cells (DC), while the immunosuppressive tumor microenvironment (TEM) attenuated the effects. Herein, we utilized bioactive polysaccharides extracted from the natural herb Astragalus membranaceus and developed polysaccharide nanoparticles (ANPs) that can reverse TEM and, accordingly, enhance the radiation-induced abscopal effect. ANP showed ability to prolong the survival rate of tumor-bearing mice. In addition, ANP dramatically inhibited the growth of the primary tumor subjected to radiation as well as the secondary tumor distant from the primary lesion. Mechanistic study demonstrated that an ANP-induced immune response was mainly reflected by DC activation, represented by phenotypic maturation and enhanced antigen presentation through the TLR4 signaling pathway. Mature DC induced by ANP migrated to the tumor-draining lymph node and initiated T-cell expansion. Specifically, DC activation was successfully translated into an increase in CD4+ T/Treg and CD8+ T/Treg ratios within both primary (irradiated) and secondary (unirradiated) tumors. Our results also indicated that the systemic antitumor immune response and immune memory were enhanced with the increase in IFN-γ production and effector memory T-cell population. Our work provided a novel strategy to facilitate the incorporation of immunoactive macromolecules purified from natural herbs into modern nanotechnology in the era of immunotherapy.

IL-10 Has Differential Effects on the Innate and Adaptive Immune Systems of Septic Patients.

Sepsis, a disease of divergent pro- and anti-inflammatory-mediated pathways, has a high prevalence of morbidity and mortality, yet an understanding of potential unifying mediators between these pathways that may improve clinical outcomes is largely unclear. IL-10 has classically been designated an immunosuppressive cytokine, although recent data suggest that under certain conditions IL-10 can be immune stimulatory. We sought to further investigate the effect of IL-10 on innate and adaptive immunity in an in vitro human observational cohort study in patients with sepsis via modulation of IL-10 on IFN-γ production by T cells and TNF-α production and HLA-DR expression by monocytes. These results were compared with critically ill nonseptic patients and healthy volunteers. ELISpot analysis was performed using PBMC fraction from patient whole-blood samples. Finally, to provide additional potential clinical relevance, we examined the effect of IL-10 on T cell IFN-γ production in an in vivo cecal ligation and puncture model of sepsis using C57 black/J6 female mice. We found that inhibition of IL-10 significantly increased both production of T cell IFN-γ and monocyte TNF-α, whereas addition of IL-10 increased T cell IFN-γ production but decreased monocyte production of TNF-α and HLA-DR expression. There was no significant effect of IL-10 on control cohorts. IL-10-treated septic mice demonstrated increased IFN-γ production in splenocytes. Thus, IL-10 demonstrates both pro- and anti-inflammatory effects in the septic microenvironment, which is likely cell and context dependent. Further elucidation of relevant signaling pathways may direct future therapeutic targets.

Variation in interferon-γ production between QFT-Plus and QFT-GIT assays in TB contact investigation.

Individuals with recent contact with active tuberculosis (TB) patients were screened for latent tuberculosis infection (LTBI) considering their greater relative risk for developing active TB. QuantiFERON®-TB Gold Plus (QFT-Plus) assay offers two TB-specific antigen tubes (TB1 and TB2). TB2 is designed to elicit both CD4 and CD8 T-cell responses. This mechanism could lead to increased sensitivity as compared to the QuantiFERON®-TB Gold In-Tube (QFT-GIT) assay. Our objective was to compare the LTBI diagnostic capability of QFT-Plus with that of QFT-GIT. A total of 412TB contacts (median age 44 years) were prospectively enrolled. We conducted both QFT-Plus and QFT-GIT assays concurrently. We also analyzed production of interferon-γ (IFN-γ) in response to TB-specific antigens. The positivity rates in QFT-Plus and QFT-GIT were 7.5% (95% confidence interval (CI), 5.4-10.5) and 5.8% (CI, 3.9-8.5), respectively, showing a significant difference (P<0.05). The median (inter-quartile range [IQR]) of IFN-γ[QFT-Plus]/IFN-γ[QFT-GIT] was 1.41 (1.00-2.00). QFT-Plus produced 1.4 times IFN-γ. QFT-Plus revealed significantly higher positivity rate compared with QFT-GIT, which might be attributed to increased IFN-γ production. The cut-off in both QFT-Plus and QFT-GIT was 0.35 IU/mL. These effects might resulted in the higher positivity rate in QFT-Plus.

The E3 Ubiquitin Ligase Asb2α in T Helper 2 Cells Negatively Regulates Antitumor Immunity in Colorectal Cancer.

The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in Th1 and Th2 cells. A Th1-dominated immune response predicts positive outcomes in colorectal cancer. The E3 ubiquitin ligase, Asb2α, is expressed in Th2 cells, but its roles in T-cell maturation and cancer are unclear. We provide evidence that the Th2 master regulator, Gata3, induces Asb2 Loss of Asb2 did not affect Th differentiation ex vivo, but reduced IL4 production from Th2 cells. We found that high ASB2 expression was associated with poor outcome in colorectal cancer. Loss of Asb2 from hematopoietic cells promoted a Th1 response and attenuated colitis-associated tumorigenesis in mice. Diminished Th2 function correlated with increased IFNγ production and an enhanced type 1 antitumor immune response in Asb2-deficient mice. Our work suggests that Asb2α promotes a Th2 phenotype in vivo, which in turn is associated with tumor progression in a mouse model of colitis.

Abstract 3187: Engineering a new generation of cell therapies for solid tumor oncology using the SQZ platform

Abstract Effective T cell priming is crucial to induce anti-tumor CD8+ T cell responses and requires the efficient presentation of antigen on major histocompatibility complex class I (MHC-I) by antigen presenting cells (APCs). Previous efforts using dendritic cells to prime CD8+ T cell responses have proven difficult due to limited cell availability in the blood and challenges delivering antigen to the APC cytosol, a necessary step for MHC-I presentation and CD8+ T cell activation. To overcome this limitation, we deliver antigen directly to the cytosol of target APCs using the microfluidics-based SQZ platform. SQZ uniquely facilitates antigen loading into both professional and unconventional APCs, including B cells, T cells, and heterogenous populations of cells, which can be easily obtained directly from the blood. Protein and peptide antigens are delivered using SQZ to each of these APCs effectively, leading to efficient presentation of immunogenic epitopes on MHC-I. Here, we demonstrate that murine SQZ-APCs can stimulate antigen-specific CD8+ T cell responses in vitro and in vivo as measured by expansion of antigen-specific T cells and production of IFNγ. In the TC-1 tumor model for HPV-associated cancers, antigen-loaded SQZ-APCs have strong anti-tumor effects both prophylactically and therapeutically. Following therapeutic immunization, the anti-tumor responses correlate with an increase in antigen-specific CD8+ tumor infiltrating lymphocytes compared to untreated mice. In addition, compared to a traditional subcutaneous peptide vaccine, SQZ-APCs elicit a five-fold greater intratumoral CD8+ T cell response and drive significantly more tumor growth inhibition. Importantly, this SQZ-enabled cancer cell therapy translates to human B cells, T cells, and heterogenous populations of cells engineered to function as APCs. When a peptide is delivered to the cytosol using SQZ, all of these primary human cells activate antigen-specific CD8+ T cell responses in vitro by stimulation of IFNγ from antigen-specific CD8+ T cell responders. In comparison to cells incubated in the presence of peptide antigen, SQZ-APCs stimulate a 10-fold increase in IFNγ production from antigen-specific CD8+ responder T cells (n=13 donors). Finally, the SQZ process has been scaled to engineer human SQZ-APCs in preparation for clinical trials with a throughput of greater than 4 billion cells SQZ’d per minute. Collectively, these findings highlight the significant clinical potential of the SQZ platform to engineer potent APCs for a new generation of cancer cell therapies. Citation Format: Kelan A. Hlavaty, Matthew G. Booty, Scott Loughhead, Katarina Blagovic, Alfonso Vicente-Suarez, Defne Yarar, Howard Bernstein, Armon Sharei. Engineering a new generation of cell therapies for solid tumor oncology using the SQZ platform [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 3187.

Poliglusam Nanoparticles Activate T Cell Response in Breast Cancer Cell: an In Vivo and In Vitro Study.

Poliglusam nanoparticles are potential therapeutic agents for the treatment of cancer. In particular, their efficacy has been reported as delivery systems in breast cancer. The aim of this study is to propose a new immunotherapeutic strategy, using Poliglusam nanoparticles as activators of the human immune response. Poliglusam nanoparticles were synthesized and characterized using both dynamic light scattering and electron microscopy. Whilst, their effectiveness in immune stimulation and detection of apoptosis was evaluated by cytokine and TUNEL assays. Finally, the cytokines pattern in splenocytes revealed an increase in IFN-γ production. The results of cytotoxicity on 4T1 cells show an increase in the mortality rate with respect to the control cell line. The rate of apoptosis induced by Poliglusam nanoparticles on 4T1 mouse breast cancer cell line is about 45% higher compared to MCF-7 human cells line, revealing the natural tendency of Poliglusam in increasing the production of IFN-γ in cancer cells. At the state-of-art of the knowledge, very few information have been achieved on the immunological effects of Poliglusam. This work is one of the first studies for the identification of non-functionalized Poliglusam nanoparticles impact on breast cancer. Thus, their immunotherapeutic effect, combined with an anticancer drug, can be employed as potential effective drug for eliminating breast cancer cells in the future.

Abstract 4094: The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo

Abstract Background: G100, which contains the synthetic TLR4 agonist Glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water emulsion (SE) for intratumoral therapy, is currently in clinical development in combination with pembrolizumab for the treatment of B-cell follicular lymphoma (FL). The current study was undertaken to investigate the interaction of G100 with rituximab, the anti-CD20 mAb that is standard-of-care treatment for FL. Methods: In vitro studies were performed using peripheral blood mononuclear cells (PBMC) from healthy human donors to investigate the effect of GLA-AF (an aqueous formulation of GLA) on natural killer (NK) cell activation. IFNγ production in NK cells after activation with plate-bound anti-CD16 or target K562 leukemia cells was measured by intracellular cytokine staining (ICS). Rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) was measured using pretreated or untreated PBMC and rituximab-coated Raji Burkitt’s lymphoma cells as target cells. In vivo studies were carried out in Balb/c mice with bilateral A20 lymphoma. Mice received G100 alone (10μg, intratumoral, 3 injections/week), anti-CD20 alone (clone 5D2, provided by Genentech, 200μg, intraperitoneal, 1 injection/week), or G100+anti-CD20 for a total of 3 weeks. Results: Incubation of PBMC from 3 different donors with GLA-AF (1 μg/mL) resulted in NK cell activation as demonstrated by increased IFNγ production, as well as enhanced response to the K562 target cells and anti-CD16. When PBMC from 5 normal donors were used in rituximab-mediated ADCC assay, pretreated PBMCs had significantly enhanced lysis of target Raji cells compared to untreated PBMC (p&amp;lt;0.05). In mice with bilateral A20 tumors, the combination of G100 with anti-CD20 resulted in significantly better tumor control and prolonged survival (n=5 per group, p&amp;lt;0.05). Conclusions: G100 enhances NK cell activity and rituximab-mediated ADCC in human PBMC. Combination of G100 and anti-CD20 mAb has synergistic anti-tumor effects in a mouse lymphoma model. These data support clinical testing of G100 with rituximab in patients with FL. Citation Format: Hailing Lu, Alec Betancur, Jan ter Meulen. The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo [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 4094.

Armored Inducible Expression of IL-12 Enhances Antitumor Activity of Glypican-3-Targeted Chimeric Antigen Receptor-Engineered T Cells in Hepatocellular Carcinoma.

Adoptive immunotherapy based on chimeric antigen receptor-modified T (CAR-T) cells has been demonstrated as one of the most promising therapeutic strategies in the treatment of malignancies. However, CAR-T cell therapy has shown limited efficacy for the treatment of solid tumors. This is, in part, because of tumor heterogeneity and a hostile tumor microenvironment, which could suppress adoptively transferred T cell activity. In this study, we, respectively, engineered human- or murine-derived-armored glypican-3 (GPC3)-specific CAR-T cells capable of inducibly expressing IL-12 (GPC3-28Z-NFAT-IL-12) T cells. The results showed that GPC3-28Z-NFAT-IL-12 T cells could lyse GPC3+ tumor cells specifically and increase cytokine secretion compared with GPC3-28Z T cells in vitro. In vivo, GPC3-28Z-NFAT-IL-12 T cells augmented the antitumor effect when encountering GPC3+ large tumor burdens, which could be attributed to IL-12 increasing IFN-γ production, favoring T cells infiltration and persistence. Furthermore, in immunocompetent hosts, low doses of GPC3-m28Z-mNFAT-mIL-12 T cells exerted superior antitumor efficacy without prior conditioning in comparison with GPC3-m28Z T cells. Also, mIL-12 secretion decreased regulatory T cell infiltration in established tumors. In conclusion, these findings demonstrated that the inducible expression of IL-12 could boost CAR-T function with less potential side effects, both in immunodeficient and immunocompetent hosts. The inducibly expressed IL-12-armored GPC3-CAR-T cells could broaden the application of CAR-T-based immunotherapy to patients intolerant of lymphodepletion chemotherapy and might provide an alternative therapeutic strategy for patients with GPC3+ cancers.

The Role of O-Antigen in LPS-Induced Activation of Human NK Cells.

NK cells can be stimulated by bacterial lipopolysaccharides (LPS). Unlike macrophages, human NK cells do not express or express very low level of surface TLR4 receptor normally required for the LPS stimulation. This has led to the assumption that the mechanisms of stimulating action of LPS on macrophages and NK cells differs. In this work, we investigated the effects of different forms of E. coli LPS, including mutants lacking O-antigen structures, and deacylated LPS on IFNγ production by purified human NK cells. The main findings were the following: (1) NK cells were more sensitive to the S-forms of LPS than the R-forms (LPS lacking O-antigen); (2) LPS triggered a significant increase in IFNγ production by NK cells in concentrations about 1000 times higher than those that can induce cytokine production by macrophages; (3) the composition and structure of saccharide part of LPS have a strong influence on its observed effects on NK cells; and (4) LPS fully retained the ability to trigger cytokine production in NK cells in serum-free media. The acquired data demonstrated that the presence and structure of O-antigen affects the LPS-induced activation of human NK cells.

Juglone eliminates MDSCs accumulation and enhances antitumor immunity

Myeloid-derived suppressor cells (MDSCs) contribute to immune activity suppression and promote the tumor progression. Elimination of MDSCs is a promising cancer therapeutic strategy, and some chemotherapeutic agents have been reported to hamper tumor progression by suppressing MDSCs. Juglone has been showed to exert a direct cytotoxic effect on tumor cells. However, the effect of juglone on MDSCs and anti-tumor immune statue has remained unexplored. In our study, we observed that juglone suppressed tumor growth and metastasis markedly, and the tumor growth suppression in immunocompetent mice was more drastic than that in immunodeficient mice. Juglone reduced the accumulation of MDSCs and increased IFN-γ production by CD8+ T cells. Consistently, juglone affected myeloid cells differentiation and maturation, impairing the immunosuppressive functions of MDSCs. Moreover, juglone down-regulated the level of IL-1β which was mediating accumulation of MDSCs. In addition, juglone inhibited 5FU-induced liver injury in a colorectal carcinoma-bearing mice model. Thus, our work suggests that the anti-tumor effect of juglone is mediated, at least in part, by eliminating accumulation of MDSCs.

CD4+CD25+Foxp3+ regulatory T cells suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood.

Studies have shown that CD4CD25Foxp3Treg cells suppress NKG2D expression on NK cells via a cell contact-dependent mechanism and increased TGF-β and IL-10 production in some cancer models. We herein aimed to explore whether CD4CD25Foxp3Tregs suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood and elucidate the exact mechanism underlying this phenomenon. To explore the function of NKG2D, NK cell cultures were treated with an NKG2D-blocking antibody to block these receptors. Additionally, TGF-β- and IL-10-blocking antibodies were added to NK and CD4CD25Foxp3Treg cell cocultures to evaluate whether the latter cells suppress NKG2D expression of NK cells via increasing the production of TGF-β and IL-10. The expression of NKG2D on NK cells was detected by 3-color flow cytometry, and NK cell activity was assessed by 3 assays: a nonradioactive cytotoxicity assay, an ELISA measuring IFN-γ production and a flow cytometry assay to evaluate CD107a expression. Blocking NKG2D decreased NK cell cytotoxicity, IFN-γ production and CD107a expression. Moreover, blocking TGF-β and IL-10 substantially increased the NKG2D expression in NK and CD4CD25Foxp3Treg cell cocultures. Similarly, blocking TGF-β and IL-10 enhanced NK cell cytotoxicity, IFN-γ production and CD107a expression; Transwell insert assays also revealed increased IFN-γ production and CD107a and NKG2D expression. CD4CD25Foxp3Tregs suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood via a cell contact-dependent mechanism and increased TGF-β and IL-10 production.

Acetate Promotes T Cell Effector Function during Glucose Restriction

Competition for nutrients like glucose can metabolically restrict Tcells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8+ Tcells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Exvivo acetate treatment increases IFN-γ production by exhausted Tcells, whereas reducing ACSS expression in Tcells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive Tcells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote Tcell function during cancer.

Association of glycemic status and interferon-γ production with leukocytes and platelet indices alterations in type2 diabetes

AimsThe present study aimed to evaluate the correlation between glycemic status and the inflammation biomarkers; leukocytes, platelets indices and interferon gamma (IFN-γ) production in type 2 diabetes mellitus (T2DM) patients regarding diabetic complications. MethodsStudy was conducted on 158 patients allocated as normal healthy subjects (50) and 108 patients diagnosed as T2DM. The diabetic patients were subdivided into six groups according to metformin administration as mono-or dual therapies. ResultsThe current results exhibited a significant elevation in systolic blood pressure, total and LDL-cholesterol levels and IFN-γ as well as a noticeable decrease in HDL-cholesterol and anti-atherogenic factor values compared to the healthy patients. Leukocytes and neutrophils count, main platelets volume (MPV) and platelet distribution width (PDW) values revealed noticeable elevations in most treated T2DM groups, while a marked depletion was recorded in platelets count compared to healthy subjects. Glycemic control, most treated diabetic patients with metformin mono- and dual therapies showed an ameliorative effect in HbA1c, IFN-γ, MPV, and PDW values compared to recent diabetic ones. ConclusionDiabetes was correlated significantly with dyslipidemia and atherogenic risk in parallel with an increase in IFN-γ production and hematological inflammatory biomarkers; leukocytes, neutrophil/lymphocyte and platelet/lymphocyte ratios, MPV and PDW values. The amelioration in inflammatory biomarkers was associated with improvement in glycemic control.

Anti-PD-1 Antibody Administration following Hip Fracture Surgery Reverses Immune Dysfunction and Decreases Susceptibility to Infection

The aim of this investigation was to assess expression of programmed death-1 (PD-1) and inflammatory status after hip fracture surgery in aged mice and to evaluate the effect of anti-PD-1 antibody intervention. Male C57BL/6 mice aged 22-28 months underwent hip fracture and femoral intramedullary pinning or a sham procedure. Expression of PD-1 was measured on CD4+ and CD8+ T cells. Additionally, the effects of anti-PD-1 antibody on lymphocyte apoptosis, cytokine production, bacterial clearance, and survival were determined. Expression of PD-1 on T cells was upregulated in mice after hip fracture and surgery compared to sham controls. Administration of anti-PD-1 antibody prevented T lymphocyte apoptosis, increased IFN-γ production in splenocytes, and decreased systemic inflammation. Antibody blockade of PD-1 significantly decreased susceptibility to bacteria and improved survival rates of aged mice after hip fracture and surgery followed by the induction of Pseudomonas aeruginosa pneumonia. This study showed that hip fracture and surgical trauma cause significant increases in PD-1 expression in aged mice. Antibody blockade of PD-1 partially reverses T cell apoptosis, decreases the systemic inflammatory response and susceptibility to bacteria, and reduces mortality.

Association of Cytomegalovirus DNA and Immunologic Markers of Cardiovascular Disease.

BackgroundPersons living with human immunodeficiency virus (HIV) (PLWH) with high cytomegalovirus (CMV)–specific interferon (IFN) γ response have increased numbers of endothelium homing receptor (CX3CR1)+–expressing cells that are associated with cardiovascular disease. The current study was performed to investigate the effect of cellular levels of CMV DNA on these markers.MethodsEighty paired peripheral blood mononuclear cell samples were collected ≥12 months apart from 40 CMV-seropositive PLWH with suppressed HIV RNA, who started antiretroviral therapy at median of 3-months of infection. The samples were assessed for CMV-specific IFN-γ response by means of enzyme-linked immunospot assay, and participants were classified as low responders (LRs) or high responders (HRs) based on IFN-γ production (≤100 or >100 spot-forming units [SFUs]/105 cells).ResultsOf the 40 participants, 26 (65%) were HRs and 14 (35%) LRs at baseline, which did not change over time or by CMV levels (median at first/second time points, 383/308 SFUs/106 cells for HRs vs 21/41 SFUs/106 for LRs). A decrease in IFN-γ over time was associated with higher CMV DNA levels (P < .01). High CMV response was also associated with increased CD28+CD27−CD4+ T cells expressing CX3CR1 (P < .001). Similarly, increased IFN-γ production was associated with increased CMV-specific CX3CR1+CD28+CD27−CD4+ and CD8+ T cells (P < .001).ConclusionsThese findings demonstrate that levels of CMV-specific IFN-γ response in PLWH are stable over time, and that HRs have increased circulating T cells expressing CX3CR1 that may put them at increased risk of cardiovascular disease and other inflammatory diseases.

Combination of Synthetic Long Peptides and XCL1 Fusion Proteins Results in Superior Tumor Control

Cross-presenting Xcr1+CD8α DCs are attractive APCs to target for therapeutic cancer vaccines, as they are able to take up and process antigen from dying tumor cells for their MHCI-restricted presentation to CD8 T cells. To this aim, we developed fusion proteins made of the Xcr1 ligand Xcl1 fused to an OVA synthetic long peptide (SLP) and IgG1 Fc fragment. We demonstrated the specific binding and uptake of the Xcl1 fusion proteins by Xcr1+ DCs. Most importantly, their potent adjuvant effect on the H-2Kb/OVA specific T cell response was associated with a sustained tumor control even against the poorly immunogenic B16-OVA melanoma tumor. The increased tumor protection correlated with higher tumor infiltration of antigen-specific CD8+ T cells, increased IFNγ production and degranulation potential. Altogether, these results demonstrate that therapeutic cancer vaccines may be greatly improved by the combination of SLP antigen and Xcl1 fusion proteins.