Administration Of IL-2 Research Articles

An albumin interleukin 33 fusion protein with enhanced lymph node retention suppresses murine models of multiple sclerosis

Abstract Interleukin-33 (IL-33) is an immunoregulatory cytokine that suppresses pathogenic Th17 T cells. Recombinant IL-33 has been shown to prevent disease onset in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. However, the systemic administration of IL-33 has not been clinically translated, in part because the cytokine’s short half-life makes it difficult to achieve therapeutically effective concentrations in the secondary lymphoid organs. To overcome this challenge, we have recombinantly fused mouse serum albumin (SA) to IL-33. ST2(IL-33R)-expressing mouse group 2 innate lymphoid cells treated with serum albumin fused IL-33 (SA IL-33) increased CD25 expression and IL-13 production in a dose-dependent manner. SA fusion prolonged IL-33 half-life and increased its concentration in the lymph nodes of naïve C57BL/6 mice. Prophylactic administration of SA IL-33 prevented the onset MOG-induced EAE, demonstrating equal efficacy to the clinically approved MS drug fingolimod. In mice with already-established EAE, administration of SA IL-33 reduced disease score and improved weight gain. At the cellular level, SA-IL-33 treatment reduced the frequency of CD45+ cells, including disease causing Rorgt+ CD4+ T cells, in the spinal cord. EAE mice treated with SA-IL-33 displayed an increase in ST2+ FoxP3+ CD25+ Tregs and Th2 T cells in both the spleen and spinal cord draining lymph nodes, suggesting that SA-IL-33 treatment suppresses EAE by expanding the protective type 2 response.

IL-12 alters T-cell metabolism to augment anti-tumor immunotherapy

Abstract To better limit tumor growth, adoptive T cell therapy (ACT), a cutting-edge new strategy for treating cancer, permits patients’ T cells to be modified. Long-lived central memory cells (Tcm), a specific subset of T cells, have been demonstrated through studies conducted in our group and others to be more effective at controlling tumor burden. Powerful inflammatory cytokine IL-12 has been extensively studied for its roles in the interaction of innate and adaptive immunity; however, therapeutic potential has not been optimized. We hypothesized that ex vivo administration of IL-12 may improve metabolic fitness and anti-tumor capabilities of T cells. Using a B16 murine melanoma model and T cells that are reactive to the melanoma epitope gp100, we found ex-vivo conditioning with IL-12 increased stem cell memory (Tscm) and central memory (Tcm) T cell populations. Additionally, we found unique metabolic signatures and proliferation patterns were dependent on IL-12 duration. We also observed enhanced engraftment and tumor control for up to 60 days after adoptively transferring these cells into animals with subcutaneous melanoma tumors. These metabolically conditioned T cells showed improved mitochondrial mass and function, elevated cytokine output, and perforin expression. Aside from switching from glycolysis to oxidative phosphorylation, these cells were also able to continue producing cytokines even when glucose or pyruvate levels dropped. Together, these findings imply that IL-12 modifies T cells’ reliance on glucose for energy while preserving their anti-tumor ability, potentially amplifying their ability to fight tumors in nutrient deprived microenvironments. IL-12 could therefore have a large impact on future ACT strategies. R01 CA250458, R01 CA236379

IL-4 acts through aryl hydrocarbon receptor to antagonize TLR7 induced double negative 2 B cells in lupus

Abstract We recently showed that in systemic lupus erythematosus (SLE), IL-4R signaling is a powerful antagonist that can effectively suppress the development of activated naïve (aNAV) and CD11c +T-bet +IgD −CD27 −double negative 2 (DN2) B cells promoted by both type I and type II IFNs. In the present study, we used the BXD2 mouse model of lupus to determine the mechanism of IL-4 in suppressing the development of DN2 B cells in vivo. Administration of IL-4 significantly inhibited the development of anti-Smith, anti-DNA, and anti-histone autoantibodies induced by TLR7 agonist R848 in BXD2 mice. This was associated with a decreased percentages of CD11c +T-bet +IgD −B cells. Feature-barcoding single cell RNA-sequencing analysis showed that IL-4 modulated B cell development at the transitional stage 2 (T2) and skewed naïve B cells to develop into the CD23 +CD21 −follicular B cells. IL-4 induced the gene encoding interleukin-4-induced1 (IL4i1), an enzyme that metabolizes aromatic amino acids and this was associated with the upregulation of aryl hydrocarbon receptor (AhR) and downstream genes. Metabolomics analysis revealed IL-4 induced AhR agonistic metabolites in B cells including kynurenine (Kyn), indole-3-acetic acid, and indole-3-lactic acid. In the absence of IL-4, Kyn and a potent AhR agonist, formylindolo[3,2-b]carbazole (FICZ), significantly suppressed TLR7 plus IFNβ-induced DN2 B-cell development in vitro. Our results suggest that IL-4 acts through the IL4i1-AhR pathway to inhibit B-cell regulatory response to TLR7 and type I IFN. Identification of small molecular metabolites that act directly in B cells to induce homeostasis may lead to development of orally dosed metabolome modulating therapeutics efficacious in the treatment of SLE. This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311.

A method for in vitropolarization of pathogenic ST2-expressing memory Th2 cells

Abstract IL-33 and its receptor subunit ST2 (IL1RL1) have been linked to type 2 eosinophilic asthma in multiple human and mouse studies. In particular, recent clinical trials demonstrate that monoclonal antibodies targeting IL-33/ST2 are beneficial to subsets of asthma patients. ST2 +memory Th2 cells (mTh2) are important effector cells in IL-33-mediated type 2 eosinophilic asthma. Herein we describe a unique method for polarizing a subset of pathogenic ST2 +mTh2 (ST2 +mTh2p) cells from naïve mouse CD4 T cells in vitro, which does not require multi-round activation/resting or co-culturing with antigen-presenting cells as reported in prior studies. The ST2 +mTh2p cells proliferate and produce large amounts of type 2 cytokines upon IL-33 treatment [without involving T-cell receptor (TCR) ligation]. IL-33 stimulation induces metabolic reprogramming of ST2 +mTh2p, as indicated by the increased mitochondrial respiration and glycolytic rate in Seahorse assays as well as upregulated key metabolites and metabolic genes involved in these two bioenergetic pathways. Interestingly, IL-33 stimulation also increases the metabolites and the expression of key enzymes for the production of polyamines (the important regulators of cell proliferation). Moreover, ST2 +mTh2p cells are highly pathogenic, as they are capable of propagating and producing type 2 cytokines in the lung, resulting in strong eosinophilic inflammation induced by intranasal IL-33 administration alone after adoptive transfer to Rag2−/−IL2r−/−mice (lacking both innate and adaptive lymphocytes). In summary, the current study provides useful tools to investigate IL-33/ST2-regulated molecular mechanisms and immunological functions of ST2 +mTh2p cells both in vitroand in vivo. R0 1HL144497

Monocyte-derived IL-6 Programs Microglia to Rebuild Damaged Brain Vasculature

Abstract Cerebrovascular injury (CVI) is a common pathology caused by infections, injury, stroke, neurodegeneration, and autoimmune disease. Rapid resolution of a CVI requires a coordinated innate immune response. In this study, we sought mechanistic insights into how CNS infiltrating monocytes program resident microglia to mediate angiogenesis and cerebrovascular repair following intracerebral hemorrhage. In the penumbrae of human stroke brain lesions, we identified a subpopulation of microglia that express VEGFA. These cells, termed repair associated microglia (RAM), were also observed in a rodent model of CVI and co-expressed IL-6Ra. Cerebrovascular repair did not occur in IL-6 knockouts or in mice lacking microglial IL-6Ra expression, and single cell transcriptomic analyses revealed faulty RAM programming in the absence of IL-6 signaling. Infiltrating CCR2+ monocytes were the primary source of IL-6 following CVI and were required to endow microglia with proliferative and pro-angiogenic properties. Faulty RAM programming in the absence of IL-6 or inflammatory monocytes resulted in poor cerebrovascular repair, neuronal destruction, and sustained neurological deficits that were all restored via exogenous IL-6 administration. These data provide a molecular and cellular basis for how monocytes instruct microglia to repair damaged brain vasculature and promote functional recovery after injury.

Plasticity between type 2 innate lymphoid cell subsets and amphiregulin expression regulates epithelial repair in biliary atresia.

Although a dysregulated type 1 immune response is integral to the pathogenesis of biliary atresia, studies in both humans and mice have uncovered a type 2 response, primarily driven by type 2 innate lymphoid cells. In nonhepatic tissues, natural type 2 innate lymphoid cell (nILC2s) regulate epithelial proliferation and tissue repair, whereas inflammatory ILC2s (iIlC2s) drive tissue inflammation and injury. The aim of this study is to determine the mechanisms used by type 2 innate lymphoid cell (ILC2) subpopulations to regulate biliary epithelial response to an injury. Using Spearman correlation analysis, nILC2 transcripts, but not those of iILC2s, are positively associated with cholangiocyte abundance in biliary atresia patients at the time of diagnosis. nILC2s are identified in the mouse liver through flow cytometry. They undergo expansion and increase amphiregulin production after IL-33 administration. This drives epithelial proliferation dependent on the IL-13/IL-4Rα/STAT6 pathway as determined by decreased nILC2s and reduced epithelial proliferation in knockout strains. The addition of IL-2 promotes inter-lineage plasticity towards a nILC2 phenotype. In experimental biliary atresia induced by rotavirus, this pathway promotes epithelial repair and tissue regeneration. The genetic loss or molecular inhibition of any part of this circuit switches nILC2s to inflammatory type 2 innate lymphoid cell-like, resulting in decreased amphiregulin production, decreased epithelial proliferation, and the full phenotype of experimental biliary atresia. These findings identify a key function of the IL-13/IL-4Rα/STAT6 pathway in ILC2 plasticity and an alternate circuit driven by IL-2 to promote nILC2 stability and amphiregulin expression. This pathway induces epithelial homeostasis and repair in experimental biliary atresia.

MWTX-330, an IL 12 INDUKINE Molecule, Activates and Reshapes Tumor-infiltrating CD8+ T and NK Cells to Generate Antitumor Immunity.

IL-12 is a pleotropic inflammatory cytokine that has broad stimulatory effects on various immune cell populations, making it an attractive target for cancer immunotherapy. However, despite generating robust antitumor activity in syngeneic murine tumor models, clinical administration of IL-12 has been limited by severe toxicity. mWTX-330 is a selectively inducible INDUKINE molecule comprised of a half-life extension domain and an inactivation domain linked to chimeric IL-12 by tumor protease-sensitive linkers. Systemic administration of mWTX-330 in mice was well tolerated, resulted in robust antitumor immunity in multiple tumor models, and preferentially activated tumor-infiltrating immune cells rather than immune cells present in peripheral tissues. Antitumor activity was dependent on in vivo processing of the protease cleavable linkers and required CD8+ T cells for full efficacy. Within the tumor, mWTX-330 increased the frequency of cross-presenting dendritic cells (DCs), activated natural killer (NK) cells, skewed conventional CD4+ T cells toward a T helper 1 (TH1) phenotype, drove regulatory T cells (Treg) fragility, and increased the frequency of polyfunctional CD8+ T cells. mWTX-330 treatment also increased the clonality of tumor-infiltrating T cells by expanding underrepresented T-cell receptor (TCR) clones, drove CD8+ T and NK cells towards increased mitochondrial respiration and fitness, and decreased the frequency of TOX+ exhausted CD8+ T cells within the tumor. A fully human version of this INDUKINE molecule was stable in human serum, was reliably and selectively processed by human tumor samples, and is currently in clinical development. .

Interleukin-33 facilitates liver regeneration through serotonin-involved gut-liver axis.

Insufficient liver regeneration causes post-hepatectomy liver failure and small-for-size syndrome. Identifying therapeutic targets to enhance hepatic regenerative capacity remains urgent. Recently, increased IL-33 was observed in patients undergoing liver resection and in mice after partial hepatectomy (PHx). The present study aims to investigate the role of IL-33 in liver regeneration after PHx and to elucidate its underlying mechanisms. We performed PHx in IL-33 -/- , suppression of tumorigenicity 2 (ST2) -/- , and wild-type control mice, and found deficiency of IL-33 or its receptor ST2 delayed liver regeneration. The insufficient liver regeneration could be normalized in IL-33 -/- but not ST2 -/- mice by recombinant murine IL-33 administration. Furthermore, we observed an increased level of serotonin in portal blood from wild-type mice, but not IL-33 -/- or ST2 -/- mice, after PHx. ST2 deficiency specifically in enterochromaffin cells recapitulated the phenotype of delayed liver regeneration observed in ST2 -/- mice. Moreover, the impeded liver regeneration in IL-33 -/- and ST2 -/- mice was restored to normal levels by the treatment with (±)-2,5-dimethoxy-4-iodoamphetamine, which is an agonist of the 5-hydroxytrytamine receptor (HTR)2A. Notably, in vitro experiments demonstrated that serotonin/HTR2A-induced hepatocyte proliferation is dependent on p70S6K activation. Our study identified that IL-33 is pro-regenerative in a noninjurious model of liver resection. The underlying mechanism involved IL-33/ST2-induced increase of serotonin release from enterochromaffin cells to portal blood and subsequent HTR2A/p70S6K activation in hepatocytes by serotonin. The findings implicate the potential of targeting the IL-33/ST2/serotonin pathway to reduce the risk of post-hepatectomy liver failure and small-for-size syndrome.

Abstract LB093: Engineering tumor infiltrating lymphocytes from sarcoma and colorectal tumors with membrane bound IL-15 for IL-2 independent expansion and enhanced cytotoxicity against autologous tumor cell lines

Abstract Tumor infiltrating lymphocytes (TIL) have shown promising efficacy in immunologically “hot” solid tumors that have a high level of T cell infiltration, such as melanoma. However, current treatment regimens require high dose IL-2 administration to support TIL survival, which limits their clinical applications due to IL-2 related toxicity. Obsidian Therapeutics is engineering TIL with membrane bound IL-15 (mbIL15) to eliminate the dependence of TIL on exogenous IL-2, potentially enhancing the tolerability of TIL therapies. Because there is a high unmet medical need in patients with tumors colder than melanoma, we evaluated mbIL15-engineered TIL expansion and functionality from colorectal cancer (CRC) and sarcoma biopsies. Using an IL2-independent, proprietary rapid expansion process (REP) we successfully expanded mbIL15-engineered TIL from both CRC and sarcoma, despite significantly lower T-cell numbers in the tumor tissues (average of 12-15% CD45+ TIL versus 64% in melanoma). mbIL15-engineered TIL showed an enrichment for CD8+ T cells throughout the REP and high T cell receptor variable beta chain (TCR Vbeta) diversity. mbIL15-engineered TIL also exhibited lower immune checkpoint expression (LAG3/PD-1) and higher activation marker expression (CD25/CD27/CD28) when compared with unengineered TIL expanded from the same tumors using a conventional REP with IL-2. mbIL15-engineered TIL were polyfunctional, as defined by expression of more than one effector molecules (CD107a, perforin, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and granzyme b) in response to CD3/CD28 stimulation. To examine the cytotoxic function of mbIL15-engineered TIL, we developed autologous cell lines from the same tumors that were used to generate the TIL. Using whole exome and RNA sequencing we found that the autologous tumor cell lines maintained expression of conserved tumor antigens and HLA-expression when compared with the primary tumor. When co-cultured with the autologous tumor cell lines, mbIL15-engineered TIL secreted higher levels of IFN-γ and induced higher cytotoxicity as compared to unengineered TIL cultured with IL-2. Taken together, these data demonstrate that mbIL15-engineered TIL can successfully be expanded from comparatively “cold” tumors with low T-cell infiltration, such as CRC and sarcoma, while maintaining high TCR diversity and polyfunctionality and demonstrating higher cytokine production and cytotoxic activity against autologous tumor lines, compared to conventional TIL with IL2. Citation Format: Zheng Ao, Carmela Passaro, Bulent A. Aksoy, Balazs Koscso, Rachel Burga, Kyle Pedro, Natasha Ly, Nirzari Shah, Alonso V. Ocando, Gauri Kulkarni, Trisha Timpug, Seth M. Pollack, Jan ter Meulen, Michelle L. Ols. Engineering tumor infiltrating lymphocytes from sarcoma and colorectal tumors with membrane bound IL-15 for IL-2 independent expansion and enhanced cytotoxicity against autologous tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB093.

Low-dose IL-2 enhances the generation of IL-10-producing immunoregulatory B cells

Dysfunction of interleukin-10 producing regulatory B cells has been associated with the pathogenesis of autoimmune diseases, but whether regulatory B cells can be therapeutically induced in humans is currently unknown. Here we demonstrate that a subset of activated B cells expresses CD25, and the addition of low-dose recombinant IL-2 to in vitro stimulated peripheral blood and splenic human B cells augments IL-10 secretion. Administration of low dose IL-2, aldesleukin, to patients increases IL-10-producing B cells. Single-cell RNA sequencing of circulating immune cells isolated from low dose IL2-treated patients reveals an increase in plasmablast and plasma cell populations that are enriched for a regulatory B cell gene signature. The transcriptional repressor BACH2 is significantly down-regulated in plasma cells from IL-2-treated patients, BACH2 binds to the IL-10 gene promoter, and Bach2 depletion or genetic deficiency increases B cell IL-10, implicating BACH2 suppression as an important mechanism by which IL-2 may promote an immunoregulatory phenotype in B cells.

Abstract 1824: Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity

Abstract Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Although recombinant mIL18 has demonstrated efficacy in mouse tumor models, recombinant human IL18 has not been efficacious in clinical studies, likely due to upregulation of IL18 binding protein (IL18BP), a negative regulator of the IL18 signaling axis. IL18BP is induced 10 - 100 fold after IL18 administration, binds to IL18 with high affinity, and prevents IL18 binding to the IL18 receptor. Analysis of expression data from the TCGA database as well as archived patient samples from select indications revealed that both IL18 and IL18BP are co-expressed in a number of tumor types, suggesting there may be a preexisting pool of IL18/IL18BP complex at the site of the tumor in some patients. Inhibition of IL18/IL18BP binding using a monoclonal antibody may therefore allow release of IL18 in situ, leading to IL18-mediated proinflammatory effects and enhanced anti-tumor immunity. Although the interaction of IL18 with IL18BP is a high affinity interaction, we have been able to identify very high affinity antibodies which can both inhibit complex formation and disrupt existing mouse IL18/IL18BP complexes. These antibodies are active in a number of cell-based functional assays, resulting in potent stimulation of interferon gamma production from cell lines or splenocytes. In vivo, we observed efficacy in a mouse syngeneic tumor model using an anti-mIL18BP antibody. RNA and protein analysis demonstrated expression of both IL18BP and IL18 within the tumors. Combined, these data suggest that inhibition of IL18/IL18BP binding may prove efficacious in the treatment of cancers where existing pools of IL18/IL18BP complexes prevent an IL18-mediated proinflammatory response. Experiments exploring anti-IL18BP therapy in combination with other checkpoint inhibitors such as anti-PD-1 are under current investigation. Citation Format: H. Toni Jun, Deborah A. Witherden, Eric K. Elliot, Christine M. Chidester, Maggie Willen, Bryan R. Peguero, Merrick Chai, Robert A. Horlick, David J. King. Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity [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 1824.

Abstract 1847: Engineered cell surface tag-targeted IL-2 and IL-21 selectively and safely enhance CAR-T anti-tumor activity via different mechanisms

Abstract The treatment of some patients with hematological malignancies has been transformed by chimeric antigen receptor T (CAR-T) cells. However, a large clinical need remains for more effective CAR-T therapies and to expand their use to broader patient groups including those with solid tumors. The exogenous administration of IL-2 and IL-21 were shown to enhance the engraftment, persistence, and functionality of CAR-Ts in preclinical models. However, clinical use of such cytokines is limited due to their pleiotropic nature that can result in toxicity and undesired effects on endogenous immune cells. To overcome this, we applied our cis-targeting technology to develop CAR-T specific IL-2 and IL-21 molecules that selectively activate CAR-Ts and exhibit minimal activity on non-CAR cells. Cis-targeted IL-2 and IL-21 molecules are comprised of a targeting antibody fused to an affinity-attenuated cytokine mutein, the activity of which is rescued due to the avidity provided by the targeting arm. This enables the highly selective delivery of cytokine support to human CAR-T cells. We engineered two types of fusions targeting either 1) the CAR directly without blocking CAR antigen recognition (CAR-IL2) or 2) an exogenous cell surface tag (non-signaling truncated epidermal growth factor receptor [EGFRt]) co-expressed with the CAR (EGFRt-IL2 and EGFRt-IL21). We characterized these molecules in vitro and in vivo. Both CAR- and EGFRt-targeted molecules demonstrated >100-fold preferential STAT activation of CAR-T cells over non-CAR cells. Unexpectedly, CAR-targeted fusions induced substantial antigen independent cytokine release, whereas fusions targeting the EGFRt tag did not. We found that antigen independent cytokine release was more suppressed by a LCK inhibitor rather than a JAK inhibitor, suggesting that downstream CAR signaling was the major contributor, compared to cytokine receptor signaling. In vivo studies examined the anti-tumor activity of the tag-targeted cytokine fusions given their desired safety profile. A single dose of either EGFRt-IL2 or EGFRt-IL21 on day 1 or day 7 following a sub-optimal infusion of CD19 CAR-T cells (0.1 x106) induced strong tumor regression and relapse free survival for >70 days in an aggressive NALM-6 leukemia model. EGFRt-IL21 induced a sustained 10-fold expansion of CAR-T numbers over PBS in the blood by day 7. In contrast, EGFRt-IL2 induced a delayed but greater expansion (1000-fold over PBS) by day 14 suggestive of a mechanistic difference of the two cis-targeted molecules. Analysis of EGFRt-IL2 and EGFRt-IL21 in a solid tumor model using CAR-T cells targeting endogenous tumor antigen showed similar anti-tumor effects. Cis-targeted IL-2 and IL-21 fusion molecules directed by anti-tag EGFRt antibodies confer selective enhancement of CAR-T cells with a minimal safety risk, representing a promising approach as an adjuvant CAR-T therapy. Citation Format: Nathan D. Mathewson, Wei Chen, Paul Bessette, Sara Sleiman, Meghana Sukthankar, Kelly D. Moynihan, Chris Kimberlin, Terrence Park, Audrey Hollingsworth, Saar Gill, Andy Yeung, Ivana Djuretic. Engineered cell surface tag-targeted IL-2 and IL-21 selectively and safely enhance CAR-T anti-tumor activity via different mechanisms [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 1847.

Oral administration of chicken NK-lysin or recombinant chicken IL-7 improves vaccine efficacy of Eimeria tenella elongation factor-1α (EF-1α) against coccidiosis in commercial broiler chickens

The synergistic effects of orally-delivered chicken NK-lysin peptide 2 (cNK-2) or recombinant chicken IL-7 (rchIL-7) on vaccination with recombinant Eimeria elongation factor-1α (rEF-1α) against Eimeria maxima (E. maxima) infection was investigated in broiler chickens. Chickens were divided into six groups: control (CON, no Eimeria infection), non-immunized control (NC, PBS), Vaccination 1 (VAC 1, rEF-1α plus cNK-2), Vaccination 2 (VAC 2, rchIL-7 plus cNK-2), Vaccination 3 (VAC 3, rEF-1α/rchIL-7 plus cNK-2), and Vaccination 4 (VAC 4, rEF-1α/rchIL-7 plus cNK-2). All groups, except the CON and NC, were orally treated with cNK-2 for 5 days. The first immunization, except for the VAC 4 group, was performed intramuscularly on day 4, and the second immunization was given with the same concentration of components as the primary immunization one week later. The immunization of the VAC 4 group was carried out by an oral inoculation on the same days. On day 19, all chickens except the CON group, were orally challenged with E. maxima (1.0 × 104 oocysts/chicken). The in vivo vaccination results showed that the VAC 1 and VAC 3 groups produced high (p < 0.05) levels of serum antibody titers to rEF-1α, and the VAC 3 showed enhanced (p < 0.05) levels of serum IL-7. Furthermore, the VAC 3 group showed significantly (p < 0.01) greater body weight gains at 6- and 9-days post-E. maxima infection (dpi) with reduced oocyst shedding at 6 dpi. The average jejunal lesion score of the NC group was 2.5 whereas the VAC 1 group showed a significantly (p < 0.05) lower lesion scores at 6 dpi. E. maxima infection significantly (P < 0.05) up-regulated the expression levels of cytokines (IL-6, IL-10 and IFN-γ) in the jejunum at 4 dpi, but those expressions were down-regulated in VAC 1 or VAC 3 groups. Moreover, the gene expression levels of Jam 2 and Occludin, were significantly (P < 0.05) decreased following E. maxima infection in jejunum at 4 dpi (NC), but their expressions were increased in the VAC 3 group. Collectively, these results showed that the efficacy of rEF-1α vaccination was significantly enhanced when rEF-1α vaccine co-immunized with chIL-7 or cNK-2.

IL-10 Negatively Controls the Primary T Cell Response of Tilapia by Triggering the JAK1/STAT3/SOCS3 Axis That Suppresses NF-κB and MAPK/ERK Signaling.

The braking mechanisms to protect the host from tissue damage and inflammatory disease caused by an overexuberant immune response are common in many T cell subsets. However, the negative regulation of T cell responses and detailed mechanisms are not well understood in early vertebrates. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell immunity by IL-10. Tilapia encodes an evolutionarily conserved IL-10, whose expression in lymphocytes is markedly induced during the primary adaptive immune response against Aeromonas hydrophila infection. Activated T cells of tilapia produce IL-10, which in turn inhibits proinflammatory cytokine expression and suppresses PHA-induced T cell activation. Moreover, administration of IL-10 impairs the proliferation of tilapia T cells, reduces their potential to differentiate into Th subsets, and cripples the cytotoxic function, rendering the animals more vulnerable to pathogen attack. After binding to its receptor IL-10Ra, IL-10 activates the JAK1/STAT3 axis by phosphorylation and enhances the expression of the suppressor of cytokine signaling 3 (SOCS3), which in turn attenuates the activation of the NF-κB and MAPK/ERK signaling pathways, thus suppressing the T cell response of tilapia. Our findings elucidate a negative regulatory mechanism of T cell immunity in a fish species and support the notion that the braking mechanism of T cells executed through IL-10 existed prior to the divergence of the tetrapod lineage from teleosts. Therefore, this study, to our knowledge, provides a novel perspective on the evolution of the adaptive immune system.

Anti-Vascular Endothelial Growth Factor Therapy Abolishes Glioma-Associated Endothelial Cell-Induced Tumor Invasion.

Tumor-remodeled endothelial cells not only facilitate the formation of tumor angiogenesis but also promote tumorigenesis. In this study, we aimed to explore the interaction between glioma-associated endothelial cells (GAEs) and glioma cells. We found that different subtypes of glioma owned distinct GAE abundance. Glioma patients with high GAE abundance exhibited poor prognosis. Both the results of the bioinformatics analysis and the in vitro co-culture system assay revealed that GAE promoted glioma cell invasion. Besides, anti-vascular endothelial growth factor (VEGF) therapy partially abolished the effects of GAE on gliomas. Moreover, anti-VEGF therapy upregulated IL-2 expression in GAE, and exogenous IL-2 administration inhibits GAE-induced glioma cell invasion. Collectively, our present study provides a novel outstanding of the interaction between GAE and glioma cells.

IL-27 deficiency inhibits proliferation and invasion of trophoblasts via the SFRP2/Wnt/β-catenin pathway in fetal growth restriction.

Background: Fetal growth restriction (FGR) is characterized by restricted fetal growth and dysregulated placental development. The etiology and pathogenesis still remain elusive. IL-27 shows multiple roles in regulating various biological processes, however, how IL-27 involves in placentation in FGR pregnancy hasn't been demonstrated. Methods: The levels of IL-27 and IL-27RA in FGR and normal placentae were determined by immunohistochemistry, western blot and RT-PCR. HTR-8/SVneo cells and Il27ra-/- murine models have been adopted to evaluate the effects of IL-27 on the bio-functions of trophoblast cells. GO enrichment and GSEA analysis were performed to explore the underlying mechanism. Findings: IL-27 and IL-27RA was lowly expressed in FGR placentae and administration of IL-27 on HTR-8/SVneo could promote its proliferation, migration and invasion. Comparing with wildtypes, Il27ra-/- embryos were smaller and lighter, and the placentae from which were poorly developed. In mechanism, the molecules of canonical Wnt/β-catenin pathway (CCND1, CMYC, SOX9) were downregulated in Il27ra-/- placentae. In contrast, the expression of SFRP2 (negative regulator of Wnt) was increased. Overexpression of SFRP2 in vitro could impair trophoblast migration and invasion capacity. Interpretation: IL-27/IL-27RA negatively regulates SFRP2 to activate Wnt/β-catenin, and thus promotes migration and invasion of trophoblasts during pregnancy. However, IL-27 deficiency may contribute to the development of FGR by restricting the Wnt activity.

Treg-based immunotherapy for antigen-specific immune suppression and stable tolerance induction: a perspective.

FoxP3-expressing regulatory T cells (Tregs), whether naturally generated in the immune system or unnaturally induced from conventional T cells (Tconvs) in the laboratory, have much therapeutic value in treating immunological diseases and establishing transplantation tolerance. Natural Tregs (nTregs) can be selectively expanded in vivo by administration of low-dose IL-2 or IL-2 muteins for immune suppression. For adoptive Treg cell therapy, nTregs can be expanded in vitro by strong antigenic stimulation in the presence of IL-2. Synthetic receptors such as CAR can be expressed in nTregs to equip them with a particular target specificity for suppression. In addition, antigen-specific Tconvs can be converted in vitro to functionally stable Treg-like cells by a combination of antigenic stimulation, FoxP3 induction, and establishment of the Treg-type epigenome. This review discusses current and prospective strategies for Treg-based immune suppression and the issues to be resolved for achieving stable antigen-specific immune suppression and tolerance induction in the clinic by targeting Tregs.

CD56bright natural killer cells preferentially kill proliferating CD4+ T cells.

Human CD56br natural killer (NK) cells represent a small subset of CD56+ NK cells in circulation and are largely tissue-resident. The frequency and number of CD56br NK cells in blood has been shown to increase following administration of low-dose IL-2 (LD-IL2), a therapy aimed to specifically expand CD4+ regulatory T cells (Tregs). Given the potential clinical application of LD-IL-2 immunotherapy across several immune diseases, including the autoimmune disease type 1 diabetes, a better understanding of the functional consequences of this expansion is urgently needed. In this study, we developed an in vitro co-culture assay with activated CD4+ T cells to measure NK cell killing efficiency. We show that CD56br and CD56dim NK cells show similar efficiency at killing activated CD4+ conventional T (Tconv) and Treg cell subsets. However, in contrast to CD56dim cells, CD56br NK cells preferentially target highly proliferative cells. We hypothesize that CD56br NK cells have an immunoregulatory role through the elimination of proliferating autoreactive CD4+ Tconv cells that have escaped Treg suppression. These results have implications for the interpretation of current and future trials of LD-IL-2 by providing evidence for a new, possibly beneficial immunomodulatory mechanism of LD-IL-2-expanded CD56br NK cells.

IL-33/ST2 axis promotes remodeling of the extracellular matrix and drives protective microglial responses in the mouse model of perioperative neurocognitive disorders.

Anesthesia and surgery induce cognitive impairment via uncertain mechanisms. Increasing evidence has suggested that microglial activity mediated by IL-33 /ST2 plays a critical role in immune regulation and inflammatory responses. Yet, the implications for microglia activity mediated by IL-33 in perioperative neurocognitive disorders (PND) are not well established. We showed that IL-33 and ST2 were downregulated in the hippocampus after anesthesia and surgery, and the expression of aggrecan, remodeling by microglia, was upregulated. Meanwhile, the expression of pro-inflammatory cytokines (IL-6 and IL-1β) and M1-like microglia marker (iNOS) increased, and the expression of M2-like microglia marker (CD206) decreased. Notably, the administration of IL-33 attenuated neuroinflammation and shifted the polarization of microglia in the hippocampus after anesthesia and surgery. Furthermore, IL-33 treatment rescued the increase of aggrecan, loss of dendritic spines, and impairment of LTP, improving cognitive performance. In conclusion, our study suggests that microglia activity mediated by IL-33/ST2 plays a vital role in cognitive impairments after anesthesia and surgery, which may serve as a therapeutic target for PND.

Dectin-1/IL-15 Pathway Affords Protection against Extrapulmonary Aspergillus fumigatus Infection by Regulating Natural Killer Cell Survival

Aspergillus fumigatus is a ubiquitous, yet potentially pathogenic, mold. The immune system employs innate receptors, such as dectin-1, to recognize fungal pathogens, but the immunological networks that afford protection are poorly explored. Here, we investigated the role of dectin-1 in anti-A. fumigatus response in an experimental model of acute invasive aspergillosis. Mice lacking dectin-1 presented enhanced signs of inflammation, with increased production of inflammatory cytokines and neutrophil infiltration, quickly succumbing to the infection. Curiously, resistance did not require T/B lymphocytes or IL-17. Instead, the main effector function of dectin-1 was the preservation of the NK cell population in the kidneys by the provision of the cytokine IL-15. While the depletion of NK cells impaired host defense in wild-type mice, IL-15 administration restored antifungal responses in dectin-1-deficient mice. Our results uncover a new effector mechanism for dectin-1 in anti-Aspergillus defense, adding an alternative approach to understand the pathophysiology of this infection.