Sodium fluoride causes hepatocellular S-phase arrest by activating ATM-p53-p21 and ATR-Chk1-Cdc25A pathways in mice.

In this study, experimental pathology, flow cytometry (FCM), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB) were used to evaluate the effects of sodium fluoride (NaF) on hepatocellular cell cycle progression in mice. A total of 240 ICR mice were divided equally into four groups; the experimental groups received 12, 24, or 48 mg/kg NaF intragastrically for 42 days, while the control group received distilled water. Doses of NaF above 12 mg/kg increased the percentage of cells in S phase (S-phase arrest), reduced percentages of cells in G0/G1 or G2/M phase, and activated the ATM-p53-p21 and ATR-Chk1-Cdc25A pathways. Activation of these pathways was characterized by up-regulation of ATM, p53, p21, ATR, and Chk1 mRNA and protein expression, and down-regulation of Cdc25A, cyclin E, cyclin A, CDK2, CDK4, and proliferating cell nuclear antigen (PCNA) mRNA and protein expression. These results indicate that NaF caused S-phase arrest by activating the ATM-p53-p21 and ATR-Chk1-Cdc25A pathways.

Urine anti-PLA2R antibody is a novel biomarker of idiopathic membranous nephropathy.

Since urine samples more directly reflect kidney alterations and damage than blood samples, we investigated whether urine anti-PLA2R antibody (uPLA2R-Ab) could be utilized similarly to serum anti-PLA2R antibody (sPLA2R-Ab) as a noninvasive biomarker of idiopathic membranous nephropathy (IMN). In this study, we performed a qualitative analysis using an indirect immunofluorescence test (IIFT) and measured uPLA2R-Ab and sPLA2R-Ab concentrations using an enzyme-linked immunosorbent assay (ELISA) in 28 patients with biopsy-proven IMN and 12 patients with secondary membranous nephropathy (SMN). Overall, 64.3% (n=18) of patients with IMN had IIFT-positive sPLA2R-Ab, 67.9% (n=19) of patients with IMN had IIFT-positive uPLA2R-Ab, and none of the SMN patients had IIFT-positive sPLA2R-Ab or uPLA2R-Ab. The titers of the anti-PLA2R antibody from the IMN patients in the urine (10.72±22.24 RU/µmol, presented as uPLA2R-Ab/urine creatinine) and serum (107.36±140.93 RU/ml) were higher than those from the SMN patients (0.51±0.46 RU/µmol, 0.008±0.029 RU/ml, respectively, p<0.05). Statistical analyses indicated that there were positive correlations between uPLA2R-Ab and gPLA2R, sPLA2R-Ab or urinary protein and negative correlations between uPLA2R-Ab and serum albumin in patients with IMN. In conclusion, uPLA2R-Ab is a novel biomarker of IMN. sPLA2R-Ab combined with uPLA2R-Ab might be more helpful for diagnosis and activity in PLA2R associated MN.

Deep sequencing of the T cell receptor ß repertoire reveals signature patterns and clonal drift in atherosclerotic plaques and patients.

The T cell receptor (TCR) ß repertoire directly reflects the status of T cell function. Meanwhile, the immune/inflammatory responses regulated by T cells are the critical determinants of atherosclerosis development. However, due to technical limitations, the composition and molecular characteristics of the TCR repertoire in atherosclerotic patients have not been fully elucidated. In the present study, we use powerful immune repertoire sequencing technology to study this issue. Results show that the utilization of V and/or J genes and the diversity of TCRß repertoire in atherosclerotic plaques are significantly reduced compared to those in the peripheral blood of normal subjects and atherosclerotic patients. The frequencies of the common T cell clones with certain lengths of the complement determining region 3 regions are notably different among all groups. The high-frequency common clones are also increased in the atherosclerotic plaques compared to that in the other two groups. The expansion of several T cell clonotypes (V29-1J2-1, V20-1J1-6, V6-3J2-7 and V11-2J2-2) is validated in atherosclerotic patients. In short, this study reveals that the diversity of TCR ß repertoire significantly decreases in atherosclerotic plaques, probably because of the reduced utilization of VJ genes and marked expansion of some T cell subclones. It provides the basis for understanding the roles of T lymphocytes in the pathogenesis of atherosclerosis.

Functional characterization of a short peptidoglycan recognition protein from Chinese giant salamander (Andrias davidianus).

Peptidoglycan (PGN) recognition proteins (PGRPs) are important pattern recognition receptors (PRRs) involved in immune defense against bacterial infections. In this study, a short PGRP (termed AdPGRP-S1) was cloned and functionally characterized from Chinese giant salamander (Andrias davidianus), the largest extant urodela amphibian species. AdPGRP-S1 was 184 aa in length and shared 38.7%-54.9% sequence identities with other vertebrates' short PGRPs. It contained one typical PGRP domain at the C-terminal region and several conserved amino acid (aa) residues involved in amidase and PGN binding. AdPGRP-S1 was constitutively expressed in all tissues examined, with the highest expression level seen in spleen and intestine. It has been shown that AdPGRP-S1 could bind and degrade Lys-PGN and Dap-PGN. Further, AdPGRP-S1 had antibacterial activity against the Gram-negative bacteria, Edwardsiella tarda, and was able to trigger the activation of NF-κB signaling. These results demonstrated that AdPGRP-S1 possesses multiple functions in pathogen recognition, mediating ceullular signaling, and initiating antibacterial response. This is the first functional study of a salamander PGRP, providing insight to further understand the functional evolution of verterbates' PGRPs.

Peripheral blood microRNAs expression is associated with infant respiratory syncytial virus infection.

MicroRNAs respond to the inflammatory responses induced by RNA virus infection. In this study, we investigated the specific microRNA profile in the peripheral blood of infants infected with respiratory syncytial virus (RSV). Blood specimens were analyzed using microRNA microarrays, followed by quantitative RT-PCR. A specific microRNA profile in the peripheral blood of RSV-infected infants was identified for the first time. MiR-106b-5p, miR-20b-5p, and miR-342-3p were upregulated, while miR-320e, miR-320d, miR-877-5p, miR-122-5p, and miR-92b-5p were downregulated. Pathway analysis indicated that the dysregulated microRNAs were involved in inflammatory and immune responses, including Wnt, TGF-ß, insulin, and T and B cell receptor signaling. These results demonstrate that RSV infection associates with a distinct microRNA fingerprint and suggest that RSV induces inflammatory responses in infants.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol ameliorates arthritic joints through reducing neutrophil infiltration mediated by IL-6/STAT3 and MIP-2 activation.

The pathogenesis of rheumatoid arthritis (RA) has been implicated neutrophil extracellular traps (NETs) formation which could generate autoantigen. Neutrophil contributes to initiate and maintain the inflammatory process in the joint. In this study, we show that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) decreases neutrophil migration by regulating the activity of STAT3, a regulator of IL-6 and MIP-2 expression. PLAG caused a decrease in IL-6 production in the RAW264.7 macrophage cell line and in rheumatoid arthritis-fibroblast-like synoviocytes via the regulation of STAT3 signaling without affecting NF-κB signaling. In a mouse model of collagen-induced arthritis (CIA), arthritic symptoms were recapitulated, with increased IL-6 level in the synovium, and PLAG treatment restored IL-6 to a level comparable to that achieved with commercial therapeutics (such as Remicade or methotrexate). Staining of joint tissue with neutrophil-specific antibody showed that PLAG significantly reduced the infiltration of neutrophils into the joint synovium of CIA mice. The inhibitory effect of PLAG on IL-6/STAT3 or MIP-2 signaling also reduced the migration of differentiated neutrophils in vitro. Therefore, PLAG inhibits the infiltration of destructive neutrophils into inflammatory sites, and can be utilized as a potent therapeutic agent for the treatment of sustained inflammation and joint destruction.

Initial experience of anti-PD1 therapy with nivolumab in advanced hepatocellular carcinoma.

PURPOSE: To evaluate efficacy and safety of anti-PD1 therapy with nivolumab for treatment of advanced hepatocellular carcinoma (HCC). METHODS: From Jan 2016 to Jan 2017, eleven cases of HCC (average age of 51.8-year), 4 at stage B and 7 at stage C, according to Barcelona Clinic Liver Cancer staging, were treated with nivolumab. There were 4 patients with lung metastasis, 1 with portal vein tumor thrombus, 1 with abdominal metastasis and 1 with bone metastasis. The protocol was nivolumab, 3 mg/kg, on day 1, q3w. All patients were treated for more than 4 cycles. During anti-PD1 treatment period, 6 patients also received sorafenib and 1 patient received cytokine-induced killer cell therapy. Objective response and clinical adverse events were evaluated retrospectively. RESULTS: Patients underwent a total of 80 cycles of nivolumab therapy, ranging between 4 and 18 cycles per patient. Nivolumab was associated with a disease control rate of 81.8%, with an objective response of 63.6% (Modified Response Evaluation Criteria in Solid Tumors). No adverse effects related to nivolumab were noted. CONCLUSION: Our experience shows that nivolumab could achieve acceptable outcome in HCC patients and may serve as an optional treatment, especially for patients who failed to gain a benefit from routine treatments.

Hepatic stroma-educated regulatory DCs suppress CD8+ T cell proliferation in mice.

Liver dendritic cells (DCs) display immunosuppressive activities and inhibit the CD4+ T cell response. The present study assessed whether and how liver DCs suppress CD8+ T cells. We found that bone marrow-derived mature DCs incubated with liver stromal cells were characterized by a longer life span, reduced CD11c, IA/IE, CD80, CD86, and CD40 expression, and increased CD11b expression. These unique liver stromal cell-educated mature DCs (LSed-DCs) stimulated CD8+ T cells to express CD25 and CD69, but inhibited their proliferation. CD8+ T cell suppression depended on soluble factors released by LSed-DCs, but not cell-cell contact. Compared with mature DCs, LSed-DCs produced more nitric oxide and IL-10. Addition of a nitric oxide synthase inhibitor, PBIT, but not an IL-10-blocking mAb, reversed LSed-DC inhibition of CD8+ T cell proliferation. We also found that LSed-DCs reduced CD8+ T cell-mediated liver damage in a mouse model of autoimmune hepatitis. These results demonstrate that the liver stroma induces mature DCs to differentiate into regulatory DCs that suppress CD8+ T cell proliferation, and thus contribute to liver tolerance.

Colonization, mortality, and host cytokines response to enterohemorrhagic Escherichia coli in rabbits.

The major virulence factor of enterohemorrhagic Escherichia coli in infections is its ability to cause attaching and effacing lesions in enterocytes, as well as to produce Shiga toxins. To clarify the pathogenic mechanism and host innate immune responses of enterohemorrhagic Escherichia coli in rabbits, experimental infections with TS and MY strains were conducted. Among the results, although the MY strain's pathogenicity was stronger than the TS, typical symptoms were observed in both groups of bacterial-infected rabbits. Pathological changes in the heart, liver, and spleen of rabbits infected with the MY strain were more severe than those infected with the TS strain, pro-inflammatory cytokines IL-1ß, IL-6, IL-8, IFN-γ, and TNF-α were induced by both strains, and α- and ß-defensin were significantly upregulated at 3 d postinfection. Moreover, in the spleen, the MY strain induced greater expressions of α- and ß-defensins than did the TS strain. However, in the liver, the TS strain induced greater expressions of α- and ß-defensins than did the MY strain. Most likely, different replications of the MY and TS strains in the liver and spleen induced different host immune responses. Altogether, the findings provide new insights into the occurrence and development of enterohemorrhagic Escherichia coli-mediated diseases in rabbits.

Rituximab treatment in adults with refractory minimal change disease or focal segmental glomerulosclerosis.

Rituximab (RTX) may benefit patients with glomerular disease who suffer from focal segmental glomerular sclerosis (FSGS) or minimal change disease (MCD). Here, we have described our experience treating 6 FSGS and 9 MCD patients with steroid-dependent/refractory nephrotic syndrome (NS) with RTX. Patients received RTX (375 mg/m2) intravenously on days 1, 8, 23, and 29. During a median follow-up of 8 months (range, 3-36 months) after RTX administration, all patients achieved complete or partial remission. Relapses decreased by approximately 30-fold compared with the year preceding RTX treatment, and an 89.27% reduction in proteinuria was observed. Furthermore, RTX treatment could decrease medical costs by 76.52% compared with the costs associated with the long-term use (for 12-13 months) of steroids and immunosuppressive drugs. In conclusion, RTX treatment was safe and effective for patients with refractory FSGS or MCD.

Dissecting the mechanisms involved in anti-human T-lymphocyte immunoglobulin (ATG)-induced tolerance in the setting of allogeneic stem cell transplantation - potential implications for graft versus host disease.

Polyclonal anti-human thymocyte globulins (ATG) have been recently shown to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic stem cell transplantation (HSCT) from both sibling and unrelated donors. Induction of regulatory T cells has been suggested as one of the possible mechanisms. The aim of current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance. Phenotypic characterization revealed a significant increase in the expression of FoxP3, GITR, CD95, PD-1 and ICOS as well as the complement inhibitory molecules CD55, CD58 and CD59 on CD4+CD25+ T cells upon ATG treatment. Addition of ATG-treated cells to autologous and allogeneic peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/anti-CD28 antibodies resulted in significant inhibition of proliferation. Moreover, T-cell activation and IFNγ secretion were reduced in the presence of ATG-induced Treg cells. The CD4+CD25+CD127-low Treg fraction sorted from ATG-treated culture demonstrated greater suppressive potency than negative fraction. Conditioned medium produced by ATG-treated but not IgG-treated cells contained TGFß and suppressed T cell proliferation and activation in a TGFß receptor-dependent manner. TGFß receptor kinase inhibitor SB431542 interfered with the suppressive activity of ATG-primed cells, enabling partial rescue of proliferation and IFNγ secretion. Moreover, SB431542 prevented Treg phenotype induction upon ATG treatment. Altogether, our data reveal the role of TGFß signaling in ATG-mediated immunosuppression and further support the use of ATG, a potent inducer of regulatory T cells, for prevention of GVHD post HSCT and potentially other therapeutic applications.

The TRPV1 ion channel regulates thymocyte differentiation by modulating autophagy and proteasome activity.

Autophagy and the ubiquitin-proteasome system (UPS) control thymus cell homeostasis under resting and endoplasmic reticulum (ER) stress conditions. Several evidence support a cross-talk between UPS and autophagy; abrogation of UPS responses stimulates autophagy, and vice versa the inhibition of autophagy alters the UPS functions. Herein, we found that TRPV1 activation induces ER stress, proteasome dysfunction and autophagy in thymocytes by modulating the expression of UPR-related genes. The TRPV1-mediated autophagy prevents the UPR activation by inhibiting BiP, Grp94 and ERp57 chaperone protein expression. Thymocytes from TRPV1 KO mice display both autophagy and proteasome dysfunctions, resulting in increased apoptotic cells and reduced total DP thymocyte number. In addition, positive selection of thymocytes triggered by anti-TCRß/CD2 Ab-mediated costimulation induces apoptosis in thymocytes from TRPV1 KO as compared with WT mice. Stimulation of TRPV1 KO thymocytes with anti-TCRß/CD2 mAbs modulates the expression of CD4 antigen on purified DP thymocytes, with reduced number of mature, single positive (SP) CD4 and increased number of immature SP CD4low and DP CD4lowCD8+ thymocytes, further supporting the intrinsic role of TRPV1 in T cell maturation. Finally, a reduction in CD8+ and CD4+ T cells is evidenced in the peripheral blood and spleen of TRPV1 KO, as compared with WT mice. Therapeutic strategy by restraining or stimulating the TRPV1 expression and functions in thymocytes might represent a new pharmacological tool in the regulation of different inflammatory T cell responses.

Effects of baicalein on IL-1ß-induced inflammation and apoptosis in rat articular chondrocytes.

In osteoarthritis (OA), activated synoviocytes and articular chondrocytes produce pro-inflammatory cytokines, such as IL-1ß, that promote chondrocyte apoptosis and activate the NF-κB signaling pathway to induce catabolic factors. In this study, we examined the anti-inflammatory and anti-apoptotic effect of baicalein on IL-1ß signaling and NF-κB-regulated gene products in rat chondrocytes. Rat chondrocytes were pretreated with 10 ng/ml IL-1ß for 24 h and then co-treated with 10 ng/ml IL-1ß and 50 µM baicalein for 0, 12, 24, 36 and 48h. The expression levels of poly(ADP-ribose) polymerase (PARP), Bcl-2, caspase-3, matrix metalloproteinase (MMP)-9, MMP-3, cyclooxygenase (COX)-2 and SOX-9 were detected by Western blot and quantitative reverse transcription-PCR (qPCR). The effects of baicalein on the translocation and phosphorylation of the NF-κB system were studied by Western blotting and immunofluorescence. Baicalein stimulated the expression of anti-apoptotic genes and reduced the pro-apoptotic and pro-inflammatory gene products in chondrocytes. Baicalein promoted SOX-9 expression in a time-dependent manner in chondrocytes. Baicalein inhibited the NF-κB activation that was induced by IL-1ß in a time-dependent manner in chondrocytes. Our results suggest that the anti-inflammatory and anti-apoptotic effects of baicalein are mediated through the inhibition of the translocation of phosphorylated p65 to the nucleus.

Canis familiaris allergen Can f 6: expression, purification and analysis of B-cell epitopes in Chinese dog allergic children.

Dog allergy is common worldwide. However, the allergenicity of dog allergy is still unclear in China as well as in special group, such as children. In this study, we chose Can f 6, a major dog allergen which belongs to the lipocalin to study its allergenicity in Chinese dog allergic children. Can f 6 gene was subcloned into pET-28a vector and transformed into E. coli BL21 (DE3) cells for expression. The recombinant Can f 6 was purified by nickel affinity chromatography, identified by SDS-PAGE, and tested for its allergenicity by Western blot with sera and basophil activation test. Secondary structures, B cell epitopes and homology modeling of Can f 6 were predicted by using a series of bioinformatical approaches. And the verification of B cell epitopes was detected by ELISA. The recombinant allergen showed an explicit band with the molecular weight of 20 kDa by SDS-PAGE. Sera from 56.3 % (18/32) of dog-allergic children patients reacted with Can f 6. The induction of the expression of CD63 and CCR3 of dog allergic children in passively sensitized basophils was up to approximately 5.0 times higher than healthy subjects. The secondary structure of Can f 6 contains 3 α-helices, 9 ß-sheets and random coils. Five B cell epitopes of Can f 6 were predicted and were confirmed successfully by ELISA. The results showed Can f 6 is a major allergen in Chinese children, which provides a basis for further study of Can f 6 in diagnosis and treatment of symptoms in children in China. The structural information of Can f 6 will help to form a foundation for the future design of vaccines and therapies for Can f 6 related allergies.

Inhibition of autoimmune Th17 cell responses by pain killer ketamine.

Ketamine is widely used in animals and humans as a systemic anesthetic. Although several immune-modulatory functions of ketamine have been reported, the effects of ketamine on the differentiation of Th17 cell are unknown. We found that ketamine significantly diminished the frequency of IL-17-producers among CD4+ T cells stimulated under Th17-skewing conditions. Mechanistic studies showed that ketamine had little effect on the production of Th17-inducing cytokines by dendritic cells and the proliferation of T cells in response to anti-CD3; however it significantly hampered IL-21 expression as well as STAT3 phosphorylation in T cells upon IL-6 stimulation. Moreover, MOG-reactive CD4+ T cells expanded in the presence of ketamine produced reduced amounts of Th17 cytokines, leading to diminished EAE severity when transferred into TCRß-deficient mice in comparison to those treated with vehicle. These findings demonstrate that ketamine suppresses autoimmune Th17 cell responses by inhibiting the differentiation as well as the reactivation of Th17 cells.

B cells regulate thymic CD8+T cell differentiation in lupus-prone mice.

Previous studies have shown that under normal physiological conditions thymic B cells play a critical function in T cell negative selection. We tested the effect of thymic B cells on thymic T-cell differentiation in autoimmune diseases including systemic lupus erythematosus (SLE). We found that thymic B cells and CD8- CD4+ and CD4-CD8+T cells increased, whereas CD4+CD8+T cells decreased in lupus-prone mice. Once B cells were reduced, the change was reversed. Furthermore, we found that B cells blocked thymic immature single positive (ISP) CD4-CD8+CD3lo/-RORγt- T cells progression into CD4+CD8+T cells. Interestingly, we found a novel population of thymic immature T cells (CD4-CD8+CD3loRORγt+) that were induced into mature CD4-CD8+CD3+RORγt+T cells by B cells in lupus-prone mice. Importantly, we found that IgG, produced by thymic B cells, played a critical role in the differentiation of thymic CD8+ISP and mature RORγt+CD8+ T cells in lupus-prone mice. In conclusion, B cells blocked the differentiation from thymic CD8+ISP and induced the differentiation of a novel immature CD4-CD8+CD3loRORγt+T cells into mature RORγt+CD8+ T cells by secreting IgG antibody in lupus-prone mice.

Epigallocatechin-3-gallate (EGCG) up-regulates miR-15b expression thus attenuating store operated calcium entry (SOCE) into murine CD4+ T cells and human leukaemic T cell lymphoblasts.

CD4+ T cells are key elements in immune responses and inflammation. Activation of T cell receptors in CD4+ T cells triggers cytosolic Ca2+ release with subsequent store operated Ca2+ entry (SOCE), which is accomplished by the pore forming Ca2+ release activated Ca2+ (CRAC) channel Orai1 and its regulator stromal cell-interaction molecule 2 (STIM2). Green tea polyphenol epigallocatechin-3-gallate (EGCG) acts as a potent anti-inflammatory and anti-oxidant agent for various types of cells including immune cells. However, how post-transcriptional gene regulators such as miRNAs are involved in the regulation of Ca2+ influx into murine CD4+ T cells and human Jurkat T cells through EGCG is not defined. EGCG treatment of murine CD4+ T cells significantly down-regulated the expression of STIM2 and Orai1 both at mRNA and protein levels. Furthermore, EGCG significantly decreased SOCE in both murine and human T cells. EGCG treatment increased miRNA-15b (miR-15b) abundance in both murine and human T cells. Bioinformatics analysis reveals that miR-15b, which has a STIM2 binding site, is involved in the down-regulation of SOCE. Overexpression of miR-15b significantly decreased the mRNA and protein expression of STIM2 and Orai1 in murine T cells. Treatment of Jurkat T cells with 10 µM EGCG further decreased mTOR and PTEN protein levels. EGCG decreased mitochondrial membrane potential (MMP) in both human and murine T cells. In conclusion, the observations suggest that EGCG inhibits the Ca2+ entry into murine and human T cells, an effect accomplished at least in part by up-regulation of miR-15b.

Immunology repertoire study of pulmonary sarcoidosis T cells in CD4+, CD8+ PBMC and tissue.

Sarcoidosis is a systemic granulomatous disorder highly related with immune response. The diversity and stability of the immune system could be measured by hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). Here we used a combination of multiplex PCR and next-generation sequencing to conduct a good quality analysis of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from 7 sarcoidosis patients and lung sarcoidosis tissue from 6 patients. The length distribution of CDR3 sequences identified a significant difference among CD4+, CD8+ and tissue samples. The analysis of Gini coefficient, Shannon entropy and HEC number showed that they all presents in sarcoidosis tissue group clones in a more skewed manner than that of in PMBCs groups. 2 nucleotide sequences and 2 amino acid sequences were shared by all samples. The comparison of TRBV, TRBJ usage and VJ combination frequency identified 2 TRBV genes, 2 TRBJ genes differentially expressed among different groups and different higher usage and lower usage of V-J combinations between each group.

Toxoplasma gondii excretory/secretory antigens (TgESAs) suppress pro-inflammatory cytokine secretion by inhibiting TLR-induced NF-κB activation in LPS-stimulated murine macrophages.

Excretory/secretory antigens (ESAs) produced by Toxoplasma gondii enable this parasite to invade and survive within the host cells through immunomodulation. In this study, the modulating effects of T. gondii excretory/secretory antigens (TgESAs) on the Ana-1 murine macrophage cell line were evaluated. Ana-1 cells were incubated with several concentrations of TgESAs, and the resulting effects on cellular viability, phagocytotic ability, and apoptosis induction were determined. Pro-inflammatory and anti-inflammatory cytokine secretion, nitric oxide production, toll-like receptor expression, and nuclear translocation of NF-κB were all measured after incubation with TgESAs. After TgESAs treatment, the proliferation and phagocytosis ability of Ana-1 cells decreased, and apoptosis was induced in a dose dependent manner. Analysis of Ana-1 cell culture supernatants showed that TgESAs not only upregulated secretion of anti-inflammatory cytokines (interleukin-10 and transforming growth factor-ß1), they also inhibited secretion of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1ß). Additionally, TgESAs inhibited nitric oxide production, toll-like receptor (TLR) 2 and 4 activation, and the nuclear translocation of NF-κB in lipopolysaccharide-stimulated Ana-1 macrophages. These results suggest TgESAs inhibit the functional activity of Ana-1 murine macrophages by inhibiting TLR-induced NF-κB activation, which suppresses pro-inflammatory cytokine secretion.

High-affinity human PD-L1 variants attenuate the suppression of T cell activation.

The activated T cells can be suppressed by programed death-1 (PD-1) axis through low affinity interaction between PD-1 and PD-ligand 1 (PD-L1) in solution or on antigen presenting cells. In clinic, the concentration of soluble PD-L1 in peripheral blood negatively correlates with cancer prognosis. However, there is little information about the relation between the affinity of PD-1/PD-L1 interaction and the suppressive capacity of PD-1 axis. In this study, we analyzed inhibitory roles of high affinity soluble human PD-L1 (hPD-L1) variants, which were generated with directed molecular evolution. Resultant two clones L3C7-hPD-L1 and L3B3-hPD-L1 showed over 20 folds greater affinity than that of native hPD-L1. We found that L3B3-hPD-L1 and L3C7-hPD-L1 could compete with an anti-PD-1 antibody (EH12.1) for binding to hPD-1. More importantly, although native soluble hPD-L1 can induce suppressive effects on activated T cells, we found L3B3-hPD-L1 and L3C7-hPD-L1 attenuated the strength of PD-1 axis for suppressing the proliferation and interferon γ (IFN-γ) secretion of PBMC. In conclusion, our data provide direct evidence in which immune checkpoint receptor-ligand interactive strength can alter the the suppressive function, in particular, the suppressive capacity of PD-1 axis could be decreased with enhanced affinity of soluble PD-L1 and PD-1 interaction. Our study might provide a new direction for manipulating immune checkpoints.