Interaction between host cell proteins and open reading frames of porcine circovirus type 2.

Postweaning multisystemic wasting syndrome (PMWS) is caused by a systemic inflammation after porcine circovirus type 2 (PCV2) infection. It was one of the most economically important pathogens affecting pig production worldwide before PCV2 vaccine was first introduced in 2006. After the development of a vaccine against PCV2a type, pig farms gradually restored enormous economic losses from PMWS. However, vaccine against PCV2a type could not be fully effective against several different PCV2 genotypes (PCV2b - PCV2h). In addition, PCV2a vaccine itself could generate antigenic drift of PCV2 capsid. Therefore, PCV2 infection still threats pig industry worldwide. PCV2 infection was initially found in local tissues including reproductive, respiratory, and digestive tracks. However, PCV2 infection often leads to a systemic inflammation which can cause severe immunosuppression by depleting peripheral lymphocytes in secondary lymphoid tissues. Subsequently, a secondary infection with other microorganisms can cause PMWS. Eleven putative open reading frames (ORFs) have been predicted to encode PCV2 genome. Among them, gene products of six ORFs from ORF1 to ORF6 have been identified and characterized to estimate its functional role during PCV2 infection. Acquiring knowledge about the specific interaction between each PCV2 ORF protein and host protein might be a key to develop preventive or therapeutic tools to control PCV2 infection. In this article, we reviewed current understanding of how each ORF of PCV2 manipulates host cell signaling related to immune suppression caused by PCV2.

IL-2 delivery by engineered mesenchymal stem cells re-invigorates CD8+ T cells to overcome immunotherapy resistance in cancer.

Immune checkpoint blockade (ICB)-based immunotherapy depends on functional tumour-infiltrating lymphocytes (TILs), but essential cytokines are less understood. Here we uncover an essential role of endogenous IL-2 for ICB responsiveness and the correlation between insufficient IL-2 signalling and T-cell exhaustion as tumours progress. To determine if exogenous IL-2 in the tumour microenvironment can overcome ICB resistance, we engineered mesenchymal stem cells (MSCs) to successfully deliver IL-2 mutein dimer (SIL2-EMSC) to TILs. While MSCs have been used to suppress inflammation, SIL2-EMSCs elicit anti-tumour immunity and overcome ICB resistance without toxicity. Mechanistically, SIL2-EMSCs activate and expand pre-existing CD8+ TILs, sufficient for tumour control and induction of systemic anti-tumour effects. Furthermore, engineered MSCs create synergy of innate and adaptive immunity. The therapeutic benefits of SIL2-EMSCs were also observed in humanized mouse models. Overall, engineered MSCs rejuvenate CD8+ TILs and thus potentiate ICB and chemotherapy.

Exogenous signaling repairs defective T cell signaling inside the tumor microenvironment for better immunity.

It is known that tumor-reactive T cells are initially activated in the draining lymph node, but it is not well known whether and how tumor-infiltrating lymphocytes (TILs) are reactivated in the tumor microenvironment (TME). We hypothesize that defective T cell receptor (TCR) signaling and cosignals in the TME limit T cell reactivation. To address this, we designed a mesenchymal stromal cell-based delivery of local membrane-bound anti-CD3 and/or cosignals to explore their contribution to reactivate T cells inside the TME. Combined anti-CD3 and CD40L rather than CD80 led to superior antitumor efficacy compared with either alone. Mechanistically, TCR activation of preexisting CD8+ T cells synergized with CD40L activation of DCs inside the TME for optimum tumor control. Exogenous TCR signals could better reactivate TILs that then exited to attack distal tumors. This study supplies further evidence that TCR signaling for T cell reactivation in the TME is defective but can be rescued by proper exogenous signals.

Epigenetic Repression of STING by MYC Promotes Immune Evasion and Resistance to Immune Checkpoint Inhibitors in Triple-Negative Breast Cancer.

The MYC oncogene is frequently amplified in triple-negative breast cancer (TNBC). Here, we show that MYC suppression induces immune-related hallmark gene set expression and tumor-infiltrating T cells in MYC-hyperactivated TNBCs. Mechanistically, MYC repressed stimulator of interferon genes (STING) expression via direct binding to the STING1 enhancer region, resulting in downregulation of the T-cell chemokines CCL5, CXCL10, and CXCL11. In primary and metastatic TNBC cohorts, tumors with high MYC expression or activity exhibited low STING expression. Using a CRISPR-mediated enhancer perturbation approach, we demonstrated that MYC-driven immune evasion is mediated by STING repression. STING repression induced resistance to PD-L1 blockade in mouse models of TNBC. Finally, a small-molecule inhibitor of MYC combined with PD-L1 blockade elicited a durable response in immune-cold TNBC with high MYC expression, suggesting a strategy to restore PD-L1 inhibitor sensitivity in MYC-overexpressing TNBC.

Rejuvenation of tumour-specific T cells through bispecific antibodies targeting PD-L1 on dendritic cells.

Bispecific T-cell engagers (BiTEs) preferentially targeting tumour-associated antigens and stimulating CD3-mediated signalling are being used in patients to treat acute B-cell lymphoblastic leukemia. However, the potency of BiTEs in solid tumours is limited by their short half-life and their severe toxicity at relevant therapeutic doses. Here we report the design and in vivo performance of a bispecific antibody that simultaneously targets the murine T-cell co-receptor CD3ε and the murine immune checkpoint programmed-death ligand 1 (PD-L1). In multiple syngeneic tumour models, the bispecific antibody generated higher antitumour immune responses than conventional BiTEs targeting tumour-associated antigens and CD3ε. We found that the durable antigen-specific T-cell responses resulted from the rejuvenation of CD8 T cells, owing to the blockade of PD-L1 on dendritic cells (but not on tumour cells) and co-stimulation by B7-1&2 (a peripheral membrane protein on dendritic cells). Bispecific T-cell engagers targeting dendritic cells rather than tumour cells may represent a general means of T-cell rejuvenation for durable cancer immunotherapy.

A cytokine receptor-masked IL2 prodrug selectively activates tumor-infiltrating lymphocytes for potent antitumor therapy.

As a potent lymphocyte activator, interleukin-2 (IL-2) is an FDA-approved treatment for multiple metastatic cancers. However, its clinical use is limited by short half-life, low potency, and severe in vivo toxicity. Current IL-2 engineering strategies exhibit evidence of peripheral cytotoxicity. Here, we address these issues by engineering an IL-2 prodrug (ProIL2). We mask the activity of a CD8 T cell-preferential IL-2 mutein/Fc fusion protein with IL2 receptor beta linked to a tumor-associated protease substrate. ProIL2 restores activity after cleavage by tumor-associated enzymes, and preferentially activates inside tumors, where it expands antigen-specific CD8 T cells. This significantly reduces IL-2 toxicity and mortality without compromising antitumor efficacy. ProIL2 also overcomes resistance of cancers to immune checkpoint blockade. Lastly, neoadjuvant ProIL2 treatment can eliminate metastatic cancer through an abscopal effect. Taken together, our approach presents an effective tumor targeting therapy with reduced toxicity.

Phc2 controls hematopoietic stem and progenitor cell mobilization from bone marrow by repressing Vcam1 expression.

The timely mobilization of hematopoietic stem and progenitor cells (HSPCs) is essential for maintaining hematopoietic and tissue leukocyte homeostasis. Understanding how HSPCs migrate between bone marrow (BM) and peripheral tissues is of great significance in the clinical setting, where therapeutic strategies for modulating their migration capacity determine the clinical outcome. Here, we identify an epigenetic regulator, Phc2, as a critical modulator of HSPC trafficking. The genetic ablation of Phc2 in mice causes a severe defect in HSPC mobilization through the derepression of Vcam1 in bone marrow stromal cells (BMSCs), ultimately leading to a systemic immunodeficiency. Moreover, the pharmacological inhibition of VCAM-1 in Phc2-deficient mice reverses the symptoms. We further determine that Phc2-dependent Vcam1 repression in BMSCs is mediated by the epigenetic regulation of H3K27me3 and H2AK119ub. Together, our data demonstrate a cell-extrinsic role for Phc2 in controlling the mobilization of HSPCs by finely tuning their bone marrow niche.

Pig tissue factor pathway inhibitor α fusion immunoglobulin inhibits pig tissue factor activity in human plasma moderately more efficiently than the human counterpart.

OBJECTIVE: To determine the efficacy of soluble pig tissue factor pathway inhibitor fusion immunoglobulin (TFPI-Ig) in blocking pig to human xenogeneic blood coagulation. RESULTS: To generate pig TFPI-Ig or human TFPI-Ig, expression vector containing cDNA encoding pig TFPIα or human TFPIα combined with human constant Ig heavy chain region was cloned and introduced into CHO cells. After purification of pig TFPI-Ig and human TFPI-Ig, the inhibition of each recombinant protein on pig tissue factor (TF)-mediated blood coagulation was examined in human plasma. Compared to human TFPI-Ig, pig TFPI-Ig inhibited pig TF activity and thrombin generation in human plasma more efficiently at certain concentrations. CONCLUSIONS: Pig TFPI-Ig will be be useful as a therapeutic protein to treat pig to human xenogeneic blood coagulation.

Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice.

Severe hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, ß) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl-/- mice show more severe symptoms than do wild-type (Axl+/+) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.

Molecular Cloning and Expression Analysis of Pig Cd90.

The CD90 (Thy-1) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that transfers signals involved in many biological events including cell activation, cell migration, cell adhesion, and tumor suppression. In this study, we cloned pig CD90 cDNA and determined its complete cDNA sequence. Pig CD90 cDNA contained an open reading frame (486 bp) encoding 161 amino acids with three putative N-glycosylation sites and four well-conserved cysteine residues, which form a possible disulfide bond within the extracellular domain among mammalian species. Pig CD90 mRNA was detected in various tissues, indicating the multicellular functions of CD90 in pigs. Flow cytometry analyses demonstrated that anti-human CD90 antibody recognizes a pig CD90 on the cell surface. Moreover, immunohistochemistry analysis revealed that CD90 expression is widely diffused in several pig tissues. Further studies will be necessary to define the functional contribution of CD90 during specific infectious diseases in pigs.

The ORF3 protein of porcine circovirus type 2 promotes secretion of IL-6 and IL-8 in porcine epithelial cells by facilitating proteasomal degradation of regulator of G protein signalling 16 through physical interaction.

Porcine circovirus type 2 (PCV2) is the main aetiological agent of postweaning multisystemic wasting syndrome. The mechanism of pathogenicity associated with PCV2 infection is still not fully understood. Nevertheless, the fact that large amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection may induce chronic inflammatory responses followed by the destruction of lymphoid tissues. However, how PCV2 infection causes an excessive inflammatory response in the host immune system during the early stage of PCV2 infection has still not been elucidated. In this study, we show that direct interaction between the PCV2 ORF3 and regulator of G protein signalling 16 (RGS16) within the cytoplasm of host cells leads to ubiquitin-mediated proteasomal degradation of RGS16. Facilitated degradation of the RGS16 by PCV2 ORF3 further enhances NFκB translocation into the nucleus through the ERK1/2 signalling pathway and increased IL-6 and IL-8 mRNA transcripts. Consequently, more severe inflammatory responses and leukocyte infiltration occur around host cells. This evidence may be the first clue explaining the molecular basis of how excessive amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection.

Trafficking of LAG-3 to the surface on activated T cells via its cytoplasmic domain and protein kinase C signaling.

Lymphocyte activation gene-3 (LAG-3; CD223), a structural homolog of CD4, binds to MHC class II molecules. Recent research indicated that signaling mediated by LAG-3 inhibits T cell proliferation, and LAG-3 serves as a key surface molecule for the function of regulatory T cells. Previous reports demonstrated that the majority of LAG-3 is retained in the intracellular compartments and is rapidly translocated to the cell surface upon stimulation. However, the mechanism by which LAG-3 translocates to the cell surface was unclear. In this study, we examined the trafficking of human LAG-3 under unstimulated as well as stimulated conditions of T cells. Under the unstimulated condition, the majority of LAG-3 did not reach the cell surface, but rather degraded within the lysosomal compartments. After stimulation, the majority of LAG-3 translocated to the cell surface without degradation in the lysosomal compartments. Results indicated that the cytoplasmic domain without Glu-Pro repetitive sequence is critical for the translocation of LAG-3 from lysosomal compartments to the cell surface. Moreover, protein kinase C signaling leads to the translocation of LAG-3 to the cell surface. However, two potential serine phosphorylation sites from the LAG-3 cytoplasmic domain are not involved in the translocation of LAG-3. These results clearly indicate that LAG-3 trafficking from lysosomal compartments to the cell surface is dependent on the cytoplasmic domain through protein kinase C signaling in activated T cells.

Molecular cloning and expression analysis of pig CD7.

CD7 is an integral membrane protein which mediates an important signal to mediate the differentiation, activation, and regulation of some T cells and NK cells. However, only human and mouse CD7 have been identified and studied among mammalian species. In this study, we cloned pig CD7 cDNA and determined its complete cDNA sequence. Pig CD7 cDNA contained an open reading frame (627 bp) encoding 208 amino acids with well conserved motifs involved in signal transduction within cytoplasmic tail among mammalian species. Pig CD7 mRNA was detected by RT-PCR in mainly lymphoid tissues, indicating the conserved functions of CD7 in pigs. Moreover, we generated soluble pig CD7 fusion immunoglobulin (pig CD7Ig) containing extracellular domain of pig CD7 to test whether pig CD7 binds to pig galectin-3. Flow cytometry and immunohistochemistry analyses indicated that soluble pig CD7Ig can bind to galectin-3 expressed in macrophages and epithelial cells of small intestine. These results help to analyze the structural relationship between CD7 and its ligand transferring signal transduction among mammalian species.

Human cytomegalovirus (HCMV) US2 protein interacts with human CD1d (hCD1d) and down-regulates invariant NKT (iNKT) cell activity.

To avoid host immune surveillance, human cytomegalovirus (HCMV) encoded endoplasmic reticulum (ER)-membrane glycoprotein US2, which interferes with antigen presenting mechanism of Major histocompatibility complex (MHC) class Ia and class II molecules. However, not many attempts have been made to study the effect of HCMV US2 on the expression of MHC class Ib molecules. In this study, we examined the effect of HCMV US2 on the expression and function of human CD1d (hCD1d), which presents glycolipid antigens to invariant NKT (iNKT) cells. Our results clearly showed that the physiological interaction between ER lumenal domain of HCMV US2 and α3 domain of hCD1d was observed within ER. Compared with mature form of hCD1d, immature form of hCD1d is more susceptible to ubiquitin-dependent proteasomal degradation mediated by HCMV US2. Moreover, the ectopic expression of HCMV US2 leads to the down-modulation of iNKT cell activity without significant change of hCD1d expression. These results will advance our understanding of the function of HCMV US2 in immune evasive mechanisms against anti-viral immunity of iNKT cells.

Molecular cloning and expression analysis of pig CD138.

CD138 (syndecan-1) interacts with various components of the extracellular matrix and associates with the actin cytoskeleton. In this study, we cloned pig CD138 cDNA and determined its complete cDNA sequence. Pig CD138 cDNA contained an open reading frame (930 bp) encoding 309 amino acids with five well conserved putative glycosaminoglycan attachment sites, a putative cleavage site for matrix metalloproteinases, and conserved motifs involved in signal transduction among mammalian species. Pig CD138 mRNA was detected in various tissues, including lymphoid and non-lymphoid organs, indicating the multicellular functions of CD138 in pigs. Western blot and flow cytometry analyses detected an approximate 35 kDa pig CD138 protein expressed on the cell surface. Further immunohistochemistry analysis revealed that CD138 expression was mainly observed in submucosa and lamina propria of the pig small intestine. Further study will be necessary to define the functional importance of CD138 during specific infectious diseases in pigs.

Expression pattern and functional role of Phc2 during activation of helper T cells after antigenic stimulation.

Polycomb group (PcG) proteins, which are conserved from invertebrates to mammals, are associated with epigenetic regulation of many cell fates. The activities of PcG proteins are largely associated with modulation of specific immune reactions. However, no study has attempted to explore the role of Phc2, a subunit of polycomb repressive complex 1, on helper T (Th) cell activation. Presently, Phc2 expression was down-regulated in activated Th cells. The ectopic expression of Phc2 in Th cells inhibited Th cell proliferation and secretion of interleukin-2 from Th cells upon antigen-specific activation. Phc2 may act as a negative regulator that inhibits the activity of Th cells.

Saponins from soy bean and mung bean inhibit the antigen specific activation of helper T cells by blocking cell cycle progression.

Treatment of helper T (Th) cells with saponins from soy bean and mung bean prevented their activation by inhibiting cell proliferation and cytokine secretion. However, the saponins did not affect the expression of major histocompatibility complex class II (A(b)) and co-stimulatory molecule (CD86) on professional antigen-presenting cells. Instead, the saponins directly inhibited Th cell proliferation by blocking the G(1) to S phase cell cycle transition. Moreover, blocking of the cell cycle by the saponins was achieved by decreased expression of cyclin D1 and cyclin E, and constitutive expression of p27(KIP1). Saponins also increased stability of p27(KIP1) in Th cells after antigenic stimulation.

Generation and evaluation of the efficacy of rhesus monkey soluble cytotoxic T lymphocyte-associated antigen-4 in the allogeneic mixed lymphocyte reaction.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is a transmembrane protein that is structurally similar to CD28. As CTLA-4 has a much higher binding affinity to B7 than CD28, several approaches using soluble CTLA-4 have been tried to down-regulate T cell activity by blocking the interaction between CD28 and B7. We constructed soluble rhesus monkey CTLA-4 immunoglobulin (CTLA-4Ig) containing a critical binding site to B7 combined with a constant Ig heavy chain region in a mammalian system. Flow cytometry analyses indicated that soluble rhesus monkey CTLA-4Ig bound to rhesus monkey CD86 (B7.2). Moreover, soluble rhesus monkey CTLA-4Ig more effectively blocked the rhesus monkey-rhesus monkey allogeneic mixed lymphocyte reaction compared with that of humans. These results indicate that soluble rhesus monkey CTLA-4Ig may be useful in preclinical trials in a rhesus monkey model.

The mouse small ubiquitin-like modifier-2 (SUMO-2) inhibits interleukin-12 (IL-12) production in mature dendritic cells by blocking the translocation of the p65 subunit of NFκB into the nucleus.

Post-translational modification by small ubiquitin-like modifier (SUMO) is involved in several significant cellular events. In particular, SUMO-1 and SUMO-4 modifications of IκBα have been shown to be actively involved in NFκB regulation. However, among the SUMO family, the specific function of SUMO-2/3 remains relatively unknown. In addition, it is not clear whether SUMO-2/3 follows the same functional role as SUMO-1 and SUMO-4 during the activation of NFκB. In this study, we examined the influence of mouse SUMO-2 during the maturation of dendritic cells (DCs). Our results showed that the ectopic expression of SUMO-2 does not affect the cell surface expression of MHC class II molecule (A(b)) and co-stimulatory molecules (CD80 and CD86), and the efficiency of antigen uptake. However, the ectopic expression of mouse SUMO-2 inhibited IL-12 secretion by blocking the translocation of the p65 subunit of NFκB into the nucleus, which led to the polarization of naïve CD4(+) T cells to T helper 2 (Th2) shift in vitro. Further analyses showed that SUMO-2 directly modified IκBα. These results indicate that the functional role of SUMO-2/3 in the regulation of NFκB activity was conserved during evolution.