Induced IFN-γ Production Research Articles

Abstract 2072: pH-activatable IL-12-loaded polymeric micelles safely enhance antitumor efficacy as monotherapy and in combination with immune checkpoint inhibitors

Abstract Background: IL-12 is a pro-inflammatory cytokine with antitumor potential that activates both innate and adaptive immunity. However, IL-12 shows poor pharmacokinetics and intolerable toxicities due to unspecific distribution. Herein, we present pH-sensitive polymeric micelles loading IL-12 (IL-12/m) that release fully active IL-12 after sensing intratumoral pH to safely potentiate immunotherapy. Methods: IL-12/m with 40 nm in diameter were prepared by self-assembly in phosphate buffer. The IL-12 release was tested at different pH. The stimulation of IFN-γ release from IL-12 and IL-12/m was studied in mouse splenocytes. Pharmacokinetic, pharmacodynamic, and antitumor activity were studied in immunosuppressed (B16F10 and 4T1) mouse tumor models (staged ≥ 200 mm3) and in a lung metastasis model (4T1). The safety of IL-12 was evaluated by tracing levels of cytokines and blood markers of toxicity. Results: IL-12/m selectively activated at intratumoral pH 6.5, releasing more than 80% of IL-12 in 24 h. While IL-12/m did not induce IFN-γ production in splenocytes, the released IL-12 showed similar activity to native IL-12. In mice, IL-12/m prolonged blood circulation with minimal leakage, yielding a 3-fold longer half-life than IL-12. Moreover, the accumulation in tumor for IL-12/m (7.5%ID/g of tumor) was 3-fold higher compared that of free IL-12, with more than 90% of IL-12/m being activated. The tumors treated with IL-12/m showed 2-3-fold higher levels of IFN-γ, IL-6 and TNF-α than those treated with IL-12, whereas anti-inflammatory IL-10 was 2-fold lower for IL-12/m. In blood and organs, IL-12/m lowered the cytokine levels compared to IL-12. Also, IL-12/m avoided the tachyphylaxis of IL-12 without peak effects of IFN-γ, and the blood markers of toxicity, i.e., BUN, ALT and AST, remained close to control levels, supporting the tumor selective activation of IL-12/m. IL-12/m was efficacious in the B16F10 tumor model (10 µg iv 3 injections every 4 days) as monotherapy, and in combination with anti-PD-1 (10 mg/kg IP 3 injections every 4 days), which led to 6 of 10 complete regressions (CR). IL-12/m was also effective at 1 µg (5 injections every 2 days) against 4T1 tumors. IL-12/m increase the number and activation of CD8+ T cells and NK cells in tumors. The combination of IL-12/m (10 µg iv 3 injections every 4 days) with anti-PD1 and anti-CTLA-4 (both 10 mg/kg IP 3 injections every 4 days) achieve complete CR in a spontaneous model of lung metastasis. Responders showed immunological memory after being rechallenged with fresh tumor cells. Conclusions: IL-12/m, a pH-activated polymeric micelle loading IL-12, masked the activity of IL-12 in healthy tissues, while it unleashed full potency of IL-12 in tumors, improving tolerability and efficacy. IL-12/m presented greater antitumor activity than IL-12 as monotherapy and in combination with immune checkpoint inhibitors. Citation Format: Horacio Cabral, Pengwen Chen, Kazuhiko Kakimi, Kazunori Kataoka, Takuya Miyazaki, Koji Nagaoka. pH-activatable IL-12-loaded polymeric micelles safely enhance antitumor efficacy as monotherapy and in combination with immune checkpoint inhibitors [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 2072.

Type I interferon co‐stimulation enhances γδ intraepithelial lymphocyte IFNγ production

Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) constantly survey the intestinal epithelium, facilitating a localized effector response to limit pathogen invasion. Despite constant γδ TCR triggering in vivo, these IELs remain immunologically quiescent, suggesting that a co‐stimulatory signal is required to overcome their activation threshold. Type I interferon (IFN) is a family of pro‐inflammatory cytokines that are critical for host defense against bacterial and viral infection. Activated γδ IELs limit viral replication in intestinal epithelial cells by producing type I IFNs, such as IFNα; however, the role of IFNα in mediating γδ IEL antiviral effector function remains unknown. To determine whether IFNα directly activates these sentinel lymphocytes, murine small intestinal γδ IELs were isolated, sorted, and stimulated ex vivo with 1μg/mL αCD3, 10 ng/mL IFNα, or both for 5 h. Following treatment with αCD3 and IFNα, we observed a 27%±9.3 increase in IFNγ+ γδ IELs (p<0.0001) and a 2.5‐fold increase in IFNγ mean fluorescence intensity (MFI)(p<0.0001) compared to αCD3 stimulation. Interestingly, IFNα treatment alone was not sufficient to induce IFNγ production. To determine the threshold of TCR signaling necessary for this enhanced IFNγ production, we treated γδ IELs with suboptimal concentrations of αCD3 (0.01 or 0.1 μg/mL), to mimic constant TCR triggering in vitro.At 0.1 μg/mL αCD3, IFNα induced a 27%±7.2 increase in frequency of IFNγ+ γδ IELs (p<0.01) and a significant increase in MFI of γδ IEL IFNγ production over αCD3 treatment alone (p<0.001). With a low level of TCR priming (0.1 μg/mL αCD3), a low dose of IFNα (0.1 ng/mL) was able to stimulate a 22%±6.8 increase in IFNγ+ γδ IELs compared to αCD3 treatment alone. However, higher concentrations of IFNα failed to further enhance γδ IEL IFNγ production. Stimulation of γδ IELs with IFNα ex vivo was sufficient to induce STAT1 and STAT4 phosphorylation; therefore, we asked whether STAT1 or STAT4 is required for IFNα‐induced IFNγ production. To this end, we treated WT, STAT1 KO, or STAT4 KO γδ IELs with 1 μg/mL αCD3, 10 ng/mL IFNα, or both and found that the IFNα‐induced increase in IFNγ production was abrogated in STAT4‐, but not STAT1‐deficient γδ IELs (p<0.0001). To assess changes in STAT1 and STAT4 expression in γδ IELs during acute viral infection, WT mice were infected orally with 1.5x106 PFU murine gamma herpesvirus (MHV). We observed a 16%±3 increase in the frequency of γδ IELs 3 days post‐infection compared to controls, which corresponded with a decrease in total STAT1 (MFI: 905±95 vs 638±102, p<0.01). Taken together, our data demonstrate that γδTCR signaling is required for IFNα‐induced IFNγ production in a STAT4‐dependent manner. These findings suggest that IFNα signaling in conjunction with constant TCR triggering promotes the rapid activation and expansion of γδ IELs, which may enhance their effector response to microbial pathogens.

Differential activation of chicken gamma delta T cells from different tissues by Toll-like receptor 3 or 21 ligands

Gamma delta (γδ) T cells are highly enriched in mucosal barrier sites including intestinal tissues where microbial infections and tumors often originate in mammals. Human γδ T cells recognize stress antigens and microbial signals via their T cell receptor (TCR), natural killer (NK) receptors, and pattern recognition receptors. However, little is known about antigens or ligands capable of stimulating chicken γδ T cells. The results of the present study demonstrated that polyinosinic-polycytidylic acid (poly(I:C)), a Toll-like receptor (TLR)3 ligand, significantly induced upregulation of CD8α molecules on circulating and lung γδ T cells. Moreover, poly(I:C) stimulation induced interferon (IFN)-γ production from splenic and lung CD8α+ γδ T cells while Cytosine-phosphate-Guanine oligodeoxynucleotides (CpG-ODN) 2007, a TLR21 ligand, stimulation induced IFN-γ production by circulating γδ T cells. Neither poly(I:C) nor CpG-ODN 2007 stimulation elicited degranulation of γδ T cells. Additionally, the results revealed that CpG-ODN 2007 induced IFN-γ production from TCR-stimulated γδ T cells sorted from spleen. In our experiments, isopentenyl pyrophosphate (IPP), 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), or zoledronate (Zol) stimulation did not induce IFN-γ production or degranulation in γδ T cells. Taken together, a combination of CpG-ODN 2007 and anti-CD3ε monoclonal antibodies (mAbs) can stimulate chicken γδ T cells and induce production of IFN-γ by these cells while IFN-γ production by γδ T cells induced by stimulation of poly(I:C) needs signals from other cells. These results suggest that chicken γδ T cells can sense invading pathogens via TLRs and produce IFN-γ as a first line of defense.

Blocking EP4 down-regulates tumor metabolism and synergizes with anti-PD-1 therapy to activate natural killer cells in a lung adenocarcinoma model.

Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) pathway, is produced by tumors and surrounding stromal cells. It stimulates tumor progression, promotes angiogenesis and suppresses the anti-tumor response. Pharmacological inhibition of PGE2 synthesis has been shown to suppress tumor initiation and growth in vivo. In the current study, we demonstrated that the growth of the Ptgs2-deficient 3LL lung adenocarcinoma cell line was down-regulated in vivo through natural killer (NK) cell activation and a reduction in the population of polymorphonuclear leukocyte-myeloid-derived suppressor cells (PMN-MDSCs) and tumor-associated macrophages (TAMs). On the basis of these results, the therapeutic effect of ONO-AE3-208 (EP4i), an inhibitor of EP4 (a PGE2 receptor), combined with anti-PD-1 antibody was evaluated. EP4i, but not anti-PD-1 antibody, decreased tumor metabolism including glycolysis, fatty acid oxidation and oxidative phosphorylation. EP4i induced IFNγ production from only NK cells (not from T cells) and a shift from M2-like to M1-like macrophages in TAMs. These effects were further enhanced by anti-PD-1 antibody treatment. Although CD8 T-cell infiltration was increased, IFNγ production was not significantly altered, even with combination therapy. Tumor hypoxia was ameliorated by either EP4i or anti-PD-1 antibody treatment, which was further affected by the combination. Normalization of tumor vessels was significant only for the combination therapy. The results indicated a novel effect of EP4i for the metabolic reprogramming of tumors and revealed unique features of EP4i that can synergize with anti-PD-1 antibody to promote IFNγ production by NK cells, polarize TAMs into the M1 phenotype, and reduce hypoxia through normalization of the tumor vasculature.

Fasciola hepatica fatty acid binding protein (Fh12) induces apoptosis and tolerogenic properties in murine bone marrow derived dendritic cells

In a previous study we demonstrated that Fasciola hepatica fatty acid binding protein (Fh12) significantly suppress macrophage function by inhibiting IL-6, IL-1β, tumor necrosis factor (TNF)-α and IL-12 production in TLR4-stimulated murine macrophages, an effect mediated through the signaling of CD14 co-receptor without affecting the viability of these cells. Given that dendritic cells (DCs) are immune cells that play a central role in the initiation of primary immune responses and that are the only antigen-presenting cells capable of stimulating naïve T-cells, in the present study we investigated the effect of Fh12 on DCs. We found that Fh12 exerts a strong suppressive effect on activation and function of DCs. However, in contrast to the effect observed on macrophages, Fh12 induces early and late apoptosis of DCs being this phenomenon dose-dependent and CD14-coreceptor independent. At low concentration Fh12 modulates the LPS-induced DCs maturation status by suppressing the MHC-II, and co-stimulatory molecules CD40 and CD80 surface expression together with the pro-inflammatory cytokines IL-12p70 and IL-6 production whereas increase the IL-10 levels. Besides, Fh12 decreased the ability of LPS-activated DCs to induce IFN-γ production against allogeneic splenocytes, while increasing IL-4 production. We have described for the first time the ability of Fh12 to modify selectively the viability of DCs by apoptosis induction. The selective diminution in DCs survival could be a F. hepatica strategy in order to prevent a host immune response during the earliest phases of infection.

Precision Co-Targeting of the Thymic Stromal Lymphopoietin Receptor in Childhood CRLF2-Rearranged Acute Lymphoblastic Leukemia

Introduction : Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with high rates of chemoresistance and relapse. CRLF2 (cytokine receptor-like factor 2) rearrangements occur in 50% of Ph-like and 60% of Down Syndrome (DS)-associated ALL and induce constitutive JAK/STAT and other kinase signaling. Current clinical trials are studying chemotherapy with the JAK inhibitor ruxolitinib in patients with CRLF2-rearranged Ph-like ALL, but results are not yet known. While chimeric antigen receptor T-cell (CART) immunotherapies have induced remarkable remissions in children with relapsed/refractory B-ALL, approximately 50% of CD19CART-treated patients will relapse again, many with CD19 antigen loss. New therapies are needed to prevent relapse and overcome immunotherapeutic resistance.Methods : We previously developed CAR T cells targeting the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) and demonstrated potent preclinical activity in Ph-like ALL models (Qin Blood 2015), which has led to a soon-to-open phase 1 clinical trial for patients with relapsed/refractory CRLF2-overexpressing ALL. In the current preclinical studies, we hypothesized that combinatorial targeting with bispecific TSLPRxCD19CART or TSLPRxCD22CART (Ross Cancer Res 2020) or with TSLPRCART + ruxolitinib will have superior activity against CRLF2-rearranged Ph-like and DS-ALL.Results : TSLPRCART treatment of CRLF2-rearranged ALL cell line (n=1) and patient-derived xenograft (PDX) models potently inhibited leukemia proliferation in vitro and in vivo and induced long-term ‘cure’ of xenograft mice. However, co-administration of TSLPRCART + ruxolitinib markedly diminished in vivo T cell numbers, blunted cytokine production, and facilitated leukemia relapse, which could be abrogated by delaying ruxolitinib. Importantly, ruxolitinib co-treatment prevented severe TSLPRCART-induced cytokine release syndrome (CRS) and animal death. Interestingly, ruxolitinib withdrawal led to return of T-cell functionality with re-detection of TSLPRCART in peripheral blood, induction of IFN-γ production, and leukemia clearance upon CRLF2+ ALL rechallenge (Figure 1).Conclusions: In these preclinical studies, we report potent activity of TSLPRCART in cell line (n=1) and PDX models of childhood CRLF2-rearranged Ph-like ALL (n=2) and DS-ALL (n=2) and, interestingly, deleterious effects of concomitant JAK inhibition upon CAR T cell functionality. We demonstrated that ruxolitinib co-administration impaired in vivo TSLPRCART-induced ALL cell killing but was also beneficial in protection against life-threatening cytokine release syndrome in co-treated animals. Importantly, TSLPRCART was not eliminated, only suppressed, by JAKi co-treatment with restoration of T cell functionality upon ruxolitinib removal and/or leukemia relapse/rechallenge studies. Ongoing studies are defining optimal TSLPRCART + ruxolitinib sequence(s) to maximize both anti-leukemia efficacy and potential CRS mitigation, as well as assessing in vivo efficacy of bispecific TSLPRCARTs in CRLF2-R Ph-like ALL and DS-ALL PDX models for future translation and clinical evaluation in next-generation trials. [Display omitted] DisclosuresFry: ElevateBio: Research Funding; Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tasian: Aleta Biotherapeutics: Consultancy; Kura Oncology: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

TLR-4 Agonist Induces IFN-γ Production Selectively in Proinflammatory Human M1 Macrophages through the PI3K-mTOR- and JNK-MAPK-Activated p70S6K Pathway.

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.

Novel in vitro invariant natural killer T cell functional assays

BackgroundInvariant Natural Killer T (iNKT) cells are innate lymphocytes bridging the innate and adaptive immune systems and are critical first responders against cancer and infectious diseases. iNKT cell phenotype and functionality are studied using in vitro stimulation assays assessing cytokine response and proliferation capabilities. The most common stimulant is the glycolipid α-Galactosyl Ceramide (α-GalCer), which stimulates iNKT cells when presented by CD1d, an MHC class I-like molecule expressed by antigen-presenting cells (APC). Another stimulant used is α-GalCer-loaded DimerX, a CD1d-Ig fusion protein which stimulates iNKT cells in an APC-independent fashion. Here, we demonstrate use of the PBS-57-loaded CD1d-tetramer as an APC-independent stimulant, where PBS-57 is an α-GalCer analogue. MethodsUsing healthy fresh (n = 4) and frozen (n = 7) peripheral blood mononuclear cells (PBMCs), 10-h cytokine response (measuring IFN-γ production) and 10-day proliferation assays were performed assessing iNKT functionality using α-GalCer, CD1d-tetramer and DimerX stimulants. ResultsAll stimulants effectively induced IFN-γ production in both fresh and frozen PBMC. After the 10-h activation, CD1d-tetramer was significantly more effective than α-GalCer (p = 0.032) in inducing IFN-γ production in fresh PBMC and significantly more effective than both α-GalCer (p = 0.004) and DimerX (p = 0.021) in frozen PBMC. Similarly, all stimulants induced strong proliferation responses in all samples, although this was only significant in the frozen PBMC. No significant differences in proliferation were observed between stimulants. SignificanceThis study supports PBS-57-loaded CD1d-tetramer as an effective in vitro APC-independent iNKT cell stimulant, which is comparable to or even more effective than α-GalCer and DimerX. As CD1d is downregulated during infectious disease and cancer as evasion strategies, in vitro assays which are APC-independent can assist in providing objective insight to iNKT activation by not relying on CD1d expression by APCs. Overall, the novel CD1d-tetramer stimulation equips researchers with an expanded “toolkit” to successfully assess iNKT cell function.

Interleukin-10 induces interferon-γ-dependent emergency myelopoiesis.

In emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production are observed and often mediated by the pro-inflammatory cytokine interferon gamma (IFN-γ). Interleukin-10 (IL-10) inhibits IFN-γ secretion, but paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work, we use different invivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also find that IL-10, unexpectedly, reprograms CD4 and CD8 Tcells toward an activation state that includes IFN-γ production by these Tcell subsets invivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening additional perspectives for the design of IL-10-based immunotherapies.

The Flagellin:Allergen Fusion Protein rFlaA:Betv1 Induces a MyD88- and MAPK-Dependent Activation of Glucose Metabolism in Macrophages.

TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs). BMDMs differentiated from BALB/c, C57BL/6, TLR5−/−, or MyD88−/− mice were pre-treated with inhibitors, stimulated with rFlaA:Betv1 or respective controls, and analyzed for activation, cytokine secretion, metabolic state, RNA transcriptome, and modulation of allergen-specific Th2 responses. Stimulation of BMDMs with rFlaA:Betv1 resulted in MyD88-dependent production of IL-1β, IL-6, TNF-α, IL-10, CD69 upregulation, and a pronounced shift towards glycolysis paralleled by activation of MAPK, NFκB, and mTOR signaling. Inhibition of either mTOR (rapamycin) or SAP/JNK-MAPK signaling (SP600125) resulted in dose-dependent metabolic suppression. In BMDM and T cell co-cultures, rFlaA:Betv1 stimulation suppressed rBet v 1-induced IL-5 and IL-13 secretion while inducing IFN-γ production. mRNA-Seq analyses showed HIF-1a, JAK, STAT, phagosome, NLR, NFκB, TNF, TLR, and chemokine signaling to participate in the interplay of cell activation, glycolysis, and immune response. rFlaA:Betv1 strongly activated BMDMs, resulting in MyD88−, MAPK−, and mTOR-dependent enhancement of glucose metabolism. Our results suggest macrophages are important target cells to consider during restauration of allergen tolerance during AIT.

The effect of carvacrol on respiratory syncytial virus infection in mice model: caution in the use of herbal medicines

Background and Objectives:Respiratory syncytial virus (RSV) is one of the most common viruses associated with acute lower respiratory tract infections in infants, young children, and the elderly. Due to a lack of effective anti-viral drugs or vaccines, using an immunomodulatory strategy is probably the best option to decrease the burden of RSV disease. Here, we studied carvacrol as a known immunomodulator on RSV infection outcome in a mice model.Materials and Methods:Balb/c mice were infected by intranasal inoculation of RSV-A2, and treatment started daily 24 h after infection. Mice were sacrificed on day five after infection and experimental analyses were performed to study airway immune cell influx, CD4 and CD8 subtypes, cytokine/chemokine secretion, lung histopathology, and viral load.Results:Results showed that using carvacrol enhanced immune cell influx, cytokine/chemokine production, and virus titer, and aggravated lung pathology. Our result showed that carvacrol administration increased viral titer compared to the RSVPBS group. Also, carvacrol significantly induced IFN-γ production and did not induce IL-10 production. Besides, carvacrol non-significantly increased lymphocytes and monocytes count but did not affect the neutrophil count.Conclusion:Carvacrol at the concentration of 80 (mg/kg) did not show immunomodulatory activity to alleviate the RSV infection outcome. Further research is needed to uncover the effects of the carvacrol intervention on virus replication and immune responses following RSV infection. Many herbal remedies in use contain carvacrol. However, the use of herbal remedies to treat viral respiratory infections such as RSV has to be performed with caution.

Evaluation of Helicobacter pylori OipA protein as a vaccine candidate and propolis as an adjuvant in C57BL/6 mice.

Objective(s):Outer inflammatory protein A (OipA) is an essential adhesin of Helicobacter pylori. We aimed to evaluate the effects of a recombinant OipA in the induction of crucial cytokines as a vaccine candidate and propolis as an adjuvant in C57BL/6 mice.Materials and Methods:C57BL/6 mice were divided into nine groups according to the disposition of antigen and adjuvant and route of administration: subcutaneous (sc) or gavage. The administrated recombinant purified OipA and propolis concentrations were 10 μg/ml and 40 μg/ml, respectively. After vaccination, we measured expression levels of IFN-γ and IL-4 cytokine genes in the spleen cells of mice by real-time PCR. Results:All results were contrasted with the negative sample. By sc injection, the expression of INF-γ was increased 3.5 and 2.9-fold for OipA and OipA plus propolis, respectively. By gavage 4.4 and 11-fold increase was found for OipA and OipA plus propolis, respectively. The administration of propolis by gavage showed more increase than Sc injection concerning the production of INF-γ. The 11-fold increase for injection of OipA plus propolis by gavage was comparable OipA plus Freund’s adjuvant injected subcutaneously. This result suggested an excellent immunological response toward OipA concerning the production of INF-γ in mice. In all cases there were no notable IL-4 production increases.Conclusion:The results confirm the efficiency of OipA in induction of IFN-γ production, and thereby the cellular immune response. Propolis could be a suitable adjuvant.

A Novel CD73 Inhibitor SHR170008 Suppresses Adenosine in Tumor and Enhances Anti-Tumor Activity with PD-1 Blockade in a Mouse Model of Breast Cancer.

IntroductionCD73 and adenosine support growth-promoting neovascularization, metastasis, and survival in cells, and promote anti-PD-1 mAb therapy-induced immune escape. Consequently, developing a CD73 inhibitor as monotherapy and a potential beneficial combination partner with immune-checkpoint inhibitors needs investigation.MethodsCD73 inhibitors were evaluated in vitro with soluble and membrane-bound CD73 enzymes, as well as its PD biomarker responses in human peripheral blood mononuclear cells (PBMC) by flow cytometry and ELISA. The binding modes of the molecules were analyzed via molecular modeling. The anti-tumor activity and synergistic effect of SHR170008 in combination with anti-PD-1 mAb were evaluated in a syngeneic mouse breast cancer model.ResultsSHR170008 was discovered during the initial structural modifications on the link between the ribose and the α-phosphate of AMPCP, which significantly improved the stability of the compound confirmed by the metabolite identification study. Further modifications on the adenine base of AMPCP improved the potency due to forming stronger interactions with CD73 protein. It exhibited potent inhibitory activities on soluble and endogenous membrane-bound CD73 enzymes, and induced IFNγ production, reversed AMP-suppressed CD25+ and CD8+/CD25+ expression, and enhanced granzyme B production on CD8+ T cells in human PBMC. SHR170008 showed dose-dependent anti-tumor efficacy with suppression of adenosine in the tumors in EMT6 mouse breast tumor model. The increase of adenosine in tumor tissue by anti-PD-1 mAb alone was suppressed by SHR170008 in the combination groups. Simultaneous inhibition of CD73 and PD-1 neutralization synergistically enhanced antitumor immunity and biomarkers in response, and exposures of SHR170008 were correlated with the efficacy readouts.ConclusionOur findings suggest that CD73 may serve as an immune checkpoint by generating adenosine, which suppresses the antitumor activity of anti-PD-1 mAb, and inhibition of CD73 may be a potential beneficial combination partner with immune-checkpoint inhibitors to improve their therapeutic outcomes in general.

Curcuma longa enhances IFN-γ secretion by natural killer cells through cytokines secreted from macrophages.

Interferon-γ (IFN-γ) regulates the human immune system. To study the interaction between macrophages and natural killer (NK) cells, we established a THP-1 macrophage-conditioned media. Among the 58 natural plant extracts tested, Curcuma longa exerted the strongest IFN-γ-enhancing effect in NK-92 cells through THP-1 macrophages. C. longa extract (CLE) enhanced IFN-γ secretion 2.3- and 4.2-fold at 50 and 100µg/ml, respectively. Therefore, we evaluated its IFN-γ-enhancing effect in vitro. Although NK-92 cells did not produce IFN-γ following treatment with C. longa, enhanced IFN-γ secretion was observed after treatment with THP-1 macrophage-conditioned media. We hypothesized that the cytokines secreted by the CLE-treated THP-1 macrophages are responsible for stimulating NK-92 cells. Cytokine array results show upregulation of cytokines, including MIP-1α, CXCL-1, IL-1β, PAI-1, and TNF-α, in CLE-treated THP-1 macrophages. To determine the cytokines responsible for augmenting IFN-γ secretion, NK-92 cells were stimulated with MIP-1α, CXCL-1, IL-1β, or PAI-1. Enzyme-linked immunosorbent assay results show that all cytokines induced IFN-γ production, although the dose response was somewhat varied. High-performance liquid chromatography analysis of CLE revealed the concentrations of three active curcuminoids, curcumin, demethoxycurcumin, and bisdemethoxycurcumin, as 6.70%, 1.00%, and 0.95%, respectively. Their mixture (with concentrations comparable to their occurrence in CLE) exerted an effect similar to that of the whole CLE. Our findings reveal that CLE indirectly stimulated NK-92 cells to secrete IFN-γ, which is mediated by cytokines produced from THP-1 macrophages. Further, we identified three curcuminoids partly responsible for this IFN-γ-enhancing effect. Therefore, C. longa can be used as a functional food ingredient owing to its immune-boosting ability. PRACTICAL APPLICATION: This study demonstrates that CLE stimulates THP-1 macrophages to secrete cytokines, which can in turn stimulate IFN-γ production by NK-92 cells. A mixture of three curcuminoids present in the extract exerted effects similar to whole CLE, demonstrating that the curcuminoids are partly responsible for the IFN-γ-enhancing effect of C. longa. Since IFN-γ is a key regulator of human immune system, these results suggest the potential use of C. longa as an immune-boosting functional food ingredient.

Abstract 1714: Preclinical characterization of DF6002/BMS-986415, a novel differentiated IL-12 Fc-fusion protein with robust antitumor activity as monotherapy or in combination with anti-PD-1

Abstract Background: Interleukin 12 (IL-12) is a proinflammatory cytokine that plays a central role in regulating both innate and adaptive antitumor immune responses. Clinically, administration of recombinant human IL-12 (rhIL-12) has been limited by a short half-life (t1/2) requiring frequent dosing, causing tachyphylaxis as well as toxic side effects. DF6002/BMS-986415 is an IL-12 Fc-fusion protein designed to have a prolonged t1/2 while retaining equivalent potency to rhIL-12 and promoting sustained IFN-γ response with reduced toxicity. Here we present the preclinical evaluation of DF6002/BMS-986415 ± anti–PD-1. Methods: DF6002/BMS-986415 and rhIL-12 stimulation of IFN-γ release from human and cynomolgus macaque (Cyno) peripheral blood mononuclear cells (PBMCs) and in vivo exposure in Cynos was assessed. As rhIL-12 is inactive in mice, pharmacokinetic, pharmacodynamic, and antitumor effects were evaluated with mDF6006, a mouse surrogate of DF6002/BMS-986415, and recombinant mouse IL-12 (rmIL-12). Tumor growth was evaluated in inflamed (CT26 and MC38) and immunosuppressed (4T1 and B16F10) mouse tumor models (staged ≥ 200 mm3). Results: DF6002/BMS-986415 induced IFN-γ production in human (EC50, 3.35 pM) and Cyno (EC50, 3.09 pM) PBMCs comparable to rhIL-12. DF6002/BMS-986415 was well tolerated at doses yielding greater exposure vs equimolar rhIL-12 in Cynos. In mice, the mouse surrogate mDF6006 vs rmIL-12 yielded a 5-fold increase in t1/2 and a 40-fold greater serum IFN-γ exposure. In contrast to rmIL-12, mDF6006 achieved protracted IFN-γ levels without peak effects associated with increased toxicity; serum IFN-γ Cmax occurred after day 6 and subsided by day 8. mDF6006 was efficacious across mouse tumor models tested. In the CT26 tumor model, mDF6006 (1 µg IP QW) monotherapy showed greater antitumor activity vs equimolar doses of rmIL-12 and led to complete regression (CR) in mice with large tumors (> 800 mm3); immunological memory response was observed in responders rechallenged with fresh tumor cells. In the B16F10 tumor model, mDF6006 (0.5 µg equivalent rmIL-12 SC QW) + anti–PD-1 (10 mg/kg IP Q2W) yielded a higher CR rate (mDF6006 + anti-PD-1, 4 of 10 CR; mDF6006, 1 of 10 CR) and increased survival vs either agent alone. An increase in the number and activation of CD4+ and CD8+ T cells as well as NK cells was observed 4 days after administration of mDF6006 (0.5 µg) in the B16F10 model. Conclusions: DF6002/BMS-986415, a novel IL-12 Fc-fusion protein, retained the full potency of rhIL-12 and showed improved exposure and tolerability; mDF6006 exhibited greater antitumor activity than rmIL-12. These data support the ongoing first-in-human phase 1/2 study evaluating DF6002/BMS-986415 as a novel therapeutic for the treatment of solid tumors in combination with nivolumab (NCT04423029). Citation Format: Eva Gutierrez, Mitchell Bigelow, Colin LaCroix, Patrick Kirby, Lynn Markowitz, Michael Naill, Steve O'Neil, Philip Ansumana Hull, John Engelhardt, Jean-Marie Cuillerott, Ann Cheung, Asya Grinberg, Nicolai Wagtmann. Preclinical characterization of DF6002/BMS-986415, a novel differentiated IL-12 Fc-fusion protein with robust antitumor activity as monotherapy or in combination with anti-PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1714.

Detection of Antibodies in Serum and Lymphocytes of Patients with Hemorraghic Fever with Renal Syndrome

Hemorrhagic Fever with Renal Syndrome (HFRS) is zoonotic disease, endemic in the republic of Tatarstan. Increased endothelial barrier permeability, resulting in tissue edema, leukocyte migration and hemorrhages, is characteristic pathological finding in HFRS. Cytokine storm hypothesis was suggested to explain hantavirus pathogenesis, where over activation of pro-inflammatory cytokines was suggested to cause endothelial cell damage and increased leukocyte infiltration of target tissue. Hantavirus nucleocapsid (N) protein and glycoprotein proteins (G1 and G2) were suggested to be the most immunogenic viral proteins. However, information on immunogenic epitopes triggering humoral and cellular immune response is limited.Materials and methods. Blood and serum samples were collected from HFRS patients admitted to Republican Clinical Hospital for Infectious Disease named after Agafonov, Republic of Tatarstan along with controls matched for the region. The Institutional Review Board of the Kazan Federal University approved this study and informed consent was obtained from each study subject according to the guidelines approved under this protocol (article 20, Federal Law “Protection of Health Rights of Citizens of Russian Federation” N323- FZ, 11.21.2011).Peptide library consisting of overlapping peptides (15 aa; 5 aa overlap) of N, G1 and G2 of Puumala virus (PPUV) were generated. Total of 150 peptides were tested. PUUV was selected because it is endemic in Republic of Tatarstan causing HFRS.Immunogenic epitopes inducing antibody response in HFRS were analyzed using direct ELISA. Peptides were used to coat the plate (1 µg/well), followed by incubation with HFRS serum. Control serum samples were used to determine peptide non-specific reactivity. Statistical analysis was conducted using Minitab software; differences between medians of HFRS and control groups were analyzed using the Mann-Whitney test for non-parametric data. Data was considered significant at p ≤ 0.05.T-lymphocyte epitopes were analyzed using ELISpot assay. Blood from HFRS cases was collected in vacuum tubes containing sodium citrate (Vacuutest, Apexmed). Confirmation of PUUV infection was done by PCR (n=10). Peripheral blood mononuclear cells (PBMC) were isolated using ficoll density gradient separation (p=1,077 g/l). Isolated mononuclear leukocytes were cultured in 96-well plates coated with anti-IFNγ antibody. Leukocytes were incubated with each peptide for 48 hours. PBMC treated with phytohaemoagglutinine (PHA) served as a positive control. Spots were visualized using AEC substrate and counted by direct microscopy.Results. PUUV RNA was detected in all blood samples. Serum samples from 10 HFRS cases and 10 controls were analyzed on reactivity to the overlapping peptides coding nucleocapsid and G1 and G2 glycoproteins. Reactivity of eleven peptides (150 total) was statistically significant in HFRS serum as compared to controls (Figures 1a, 1b). Interestingly, the intensity of reactivity between HFRS serum and glycoprotein peptides was 1.5±0.2 times higher than that of N protein.Several G and N protein peptides were identified as inducing IFN-γ production by leukocytes. These peptides are M82 (YTRKACIQLGTEQTC), M69 (TDLELDFSLPSSASY), N3 (ARQKLKDAERAVEVY), N2 (ITRHEQQLVVARQKL), N1 (MSDLTDIQEEITRHE) and M104 (NLVRGQNTVHIVGKG) (Figure 1c).Collected data revealed that the most immunogenic peptides were M104 (NLVRGQNTVHIVGKG) and N3 (ARQKLKDAERAVEVY) from hantavirus G and N proteins respectively. These peptides revealed strong reactivity in ELISA and ELISpot assays suggesting that they play role in activation of humoral and immune responses.Conclusion. Data confirms that both N and G proteins of PUUV are immunogenic. New epitopes were identified on N and G proteins which can stimulate humoral and cellular immunity.This work was supported by Russian Science Foundation grant №15-14-00016. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel TGF-β2/Interleukin Receptor Signal Conversion Platform That Protects CAR/TCR T Cells from TGF-β2-Mediated Immune Suppression and Induces T Cell Supportive Signaling Networks

Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder the efficacy of adoptively transferred T cells. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces hetero-dimerization of these receptors and phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. In several previous reports, a dominant negative receptor (DNR) version of TGFBR2, that does not contain signaling domains, was shown to protect T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs.Herein, we report the development of a novel TGF-β signal conversion platform that provides a T cell supportive signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of a T cell stimulating interleukin receptor. The signaling domains of interleukin receptors that were chosen for this platform are naturally activated via cytokine-induced hetero-dimerization and induce STAT transcription factor signaling pathways beneficial to effector T cell function and persistence. Based on the ability of lentiviral vectors to express large and complex gene expression cassettes, we constructed single vectors encoding both chimeric TGF-β receptors (CTBR) in the context of either a CAR or transgenic TCR. This required expression of four unique proteins from a single lentiviral vector when CTBR was combined with a transgenic TCR consisting of unique alpha and beta chains.To demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on multiple functional attributes of CTBR expressing T cells, including gene expression, cytokine production, cytotoxicity, and metabolic function. As anticipated, the STAT signaling signature induced by a particular CTBR was predictive of the functional consequence of exposure to TGF-β. In the case of a CTBR based on IL-12 signaling domains (CTBR12) that strongly induced STAT4 phosphorylation, CTBR12 expressing T cells secreted significantly greater amounts of IFNγ than control T cells following activation in the presence of TGF-β. This result was consistent with IL-12-mediated STAT4 signaling, which induces IFNγ production in effector T cells. We next evaluated CTBR expressing T cells in a stringent serial re-stimulation assay in the presence of TGF-β but absence of exogenous IL-2 cytokine support. Antigen driven T cell expansion was severely limited by exposure to TGF-β in this assay, and either CAR or transgenic TCR expressing cells ultimately lost cytotoxic activity over time. T cells overexpressing the dominant negative TGF-β receptor failed to expand and clear tumor cells following multiple rounds of antigen-driven stimulation in the presence of TGF-β, recapitulating the impact of tumor-derived TGF-β on infiltrating lymphocyte function. On the other hand, CTBR expressing cells maintained their ability to expand and kill tumor targets in the presence of TGF-β.Together, these data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into a STAT signal that supports T cell effector function. The specific impact on T cell function is dependent on the nature of the signaling domains utilized. This platform is relevant in the context of both CAR T cells and transgenic TCR T cells, and has the potential to produce superior T cell responses in the immunosuppressive tumor microenvironment. DisclosuresBoyerinas:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Murray:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio: Employment, Equity Ownership. Parsons:bluebird bio: Employment, Equity Ownership. Seidl:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership, Patents & Royalties.

Interferon-γ: teammate or opponent in the tumour microenvironment?

Cancer immunotherapy offers substantive benefit to patients with various tumour types, in some cases leading to complete tumour clearance. However, many patients do not respond to immunotherapy, galvanizing the field to define the mechanisms of pre-existing and acquired resistance. Interferon-γ (IFNγ) is a cytokine that has both protumour and antitumour activities, suggesting that it may serve as a nexus for responsiveness to immunotherapy. Many cancer immunotherapies and chemotherapies induce IFNγ production by various cell types, including activated T cells and natural killer cells. Patients resistant to these therapies commonly have molecular aberrations in the IFNγ signalling pathway or express resistance molecules driven by IFNγ. Given that all nucleated cells can respond to IFNγ, the functional consequences of IFNγ production need to be carefully dissected on a cell-by-cell basis. Here, we review the cells that produce IFNγ and the different effects of IFNγ in the tumour microenvironment, highlighting the pleiotropic nature of this multifunctional and abundant cytokine.

Staphylococcal Enterotoxin Superantigens Induce Prophylactic Antiviral Activity Against Encephalomyocarditis Virus In Vivo and In Vitro.

The staphylococcal enterotoxins (SEs) are classified as superantigens due to their potent stimulation of the immune system resulting in T cell activation and prodigious cytokine production and toxicity. This study examined the ability of superantigens to induce prophylactic antiviral activity in vivo and in vitro and evaluated potential superantigen mimetic peptides. Prophylactic treatment of mice in vivo with intraperitoneal injections of SE superantigens SEA and SEB (both at 20 μg/day for 3 days) prevented encephalomyocarditis virus (EMCV)-induced lethality in 100% and 80% of mice, respectively, as compared with control saline-treated groups in which EMCV was lethal to all mice. Furthermore, SEA (2 μg/mL) and SEB (1 μg/mL) induced antiviral activity in mouse splenocytes to produce an antiviral factor since their supernatant prevented EMCV lysis of L929 cells in tissue culture. It was found that superantigens do not directly prevent EMCV infection, but rather indirectly through inducing interferon gamma (IFNγ) production in cells as the antiviral factor. Evaluation of various superantigen mimetic peptides showed that one peptide (SEA3) had superantigen-like activity by inducing IFNγ production in cells but without the cellular proliferation, as associated with superantigens. However, the induction of IFNγ activation by the SEA3 peptide was not as pronounced, and took a much higher peptide concentration, when compared with the parent superantigen. If the negative side effects of superantigens can be eliminated, their beneficial properties can be harnessed for prophylactic treatment of viral infections and other pathologies requiring a robust immune response.

Effect of antiplague vaccination on phagocytic activity of human blood granulocytes

Anti-plague vaccination stimulates phagocytosis of Yersinia pestis cells by blood leukocytes of laboratory animals. However its effect on the phagocytic activity of human white blood cells towards plague microbes has not been studied. In this work, flow cytometry was used to study phagocytic activity of blood granulocytes towards Y. pestis, Escherichia coli and Staphylococcus aureus in the people vaccinated, or not vaccinated against plague. These persons inhabited the territories of natural plague foci in Russian Federation. The results of phagocytic activity were evaluated in microvolumes of whole blood. The induced IFNγ production was evaluated in blood by enzyme immunoassay technique, and the titers of specific antibodies to plague-specific F1 antigen were determined in blood serum samples. It was found, that the subjects who have never been vaccinated against plague had 3-fold lower granulocyte phagocytic activity towards Y. pestis than the persons repeatedly vaccinated with live plague vaccine, and 5-fold lower than phagocytosis of E. coli and S. aureus. The next annual revaccination caused additional stimulation of in vitro phagocytic capacity of blood leukocytes with respect to plague microbes. Individual values of phagocytic indices correlated with serum antibody titers (r = 0.65, p = 0.0004), and with levels of IFNγ production (r = 0.73, p = 0.0004). Thus, the obtained data confirmed dependence between phagocytic activity of human blood leukocytes and the pathogen type, thus allowing to demonstrate a connection between activated phagocytic response to Y. pestis cells in live plague-vaccinated individuals with other immune indexes of vaccine efficiency (specific antibodies to F1, induced IFNγ production). Further research in this direction will bring us closer to development of an informative test for assessing the intensity of antiplague immunity.