Longitudinal Immune Profiling Highlights CD4+ T Cell Exhaustion Correlated with Liver Fibrosis in Schistosoma japonicum Infection.

Schistosomiasis remains an important public health concern. The eggs deposited in livers invoke a Th2-dominant response, which mediates the fibrotic granulomatous response. However, the mechanisms involved in this immunopathological process are still not perfectly clear. Here, we report a single-cell transcriptional landscape of longitudinally collected BALB/c mouse splenocytes at different time points after Schistosoma japonicum infection. We found that exhausted CD4+ T cells were enriched after infection, changing from coproducing multiple cytokines to predominantly producing the Th2 cytokine IL-4. Regulatory B cells had high expression of Fcrl5, Ptpn22, and Lgals1, potentially regulating exhausted CD4+ T cells via direct PD-1-PD-L2 and PD-1-PD-L1 interactions. Within the myeloid compartment, the number of precursor and immature neutrophils sharply increased after infection. Moreover, dendritic cells, macrophages, and basophils showed inhibitory interactions with exhausted CD4+ T cells. Besides, in mouse livers, we found that exhausted CD4+ T cells were distributed around egg granuloma, promoting collagen expression in primary mouse hepatic stellate cells via IL-4 secretion, resulting in liver fibrosis. Our study provides comprehensive characterization of the composition and cellular states of immune cells with disease progression, which will facilitate better understanding of the mechanism underlying liver fibrotic granulomatous response in schistosomiasis.

Upregulation of TRPC5 in hippocampal excitatory synapses improves memory impairment associated with neuroinflammation in microglia knockout IL-10 mice.

BACKGROUND: Members of the transient receptor potential canonical (TRPC) protein family are widely distributed in the hippocampus of mammals and exert respective and cooperative influences on the functions of neurons. The relationship between specific TRPC subtypes and neuroinflammation is receiving increasing attention. METHODS: Using Cx3cr1CreERIL-10-/- transgenic mice and their littermates to study the relationship between TRPC channels and memory impairment. RESULTS: We demonstrated that Cx3cr1CreERIL-10-/- mice displayed spatial memory deficits in object location recognition (OLR) and Morris water maze (MWM) tasks. The decreased levels of TRPC4 and TRPC5 in the hippocampal regions were verified via reverse transcription polymerase chain reaction, western blotting, and immunofluorescence tests. The expression of postsynaptic density protein 95 (PSD95) and synaptophysin in the hippocampus decreased with an imbalance in the local inflammatory environment in the hippocampus. The number of cells positive for ionized calcium-binding adaptor molecule 1 (Iba1), a glial fibrillary acidic protein (GFAP), increased with the high expression of interleukin 6 (IL-6) in Cx3cr1CreERIL-10-/- mice. The nod-like receptor protein 3 (NLRP3) inflammasome was also involved in this process, and the cytokines IL-1ß and IL-18 activated by NLRP3 were also elevated by western blotting. The co-localization of TRPC5 and calmodulin-dependent protein kinase IIα (CaMKIIα) significantly decreased TRPC5 expression in excitatory neurons. AAV9-CaMKIIα-TRPC5 was used to upregulate TRPC5 in excitatory neurons in the hippocampus. CONCLUSIONS: The results showed that the upregulation of TRPC5 improved the memory performance of Cx3cr1CreERIL-10-/- mice related to inhibiting NLRP3 inflammasome-associated neuroinflammation.

Depression-like behavior associated with E/I imbalance of mPFC and amygdala without TRPC channels in mice of knockout IL-10 from microglia.

Depression has a growing impact on public health. Accumulating evidence supports an association between depression and increased immune system activity. IL-10 is a key cytokine that inhibits excessive inflammatory responses and is related to the anti-inflammatory and protective functions of the central nervous system (CNS). Cx3cr1CreERIL-10-/- mice were used in our study. We aimed to identify the role of IL-10 in microglia in depression and anxiety-like behavior. We performed a series of behavioral tests on the mice; the Cx3cr1CreERIL-10-/- male mice showed depression- and anxiety-like behavior compared with the littermates. The expression of transient receptor potential canonical 5 (TRPC5) decreased in both the medial prefrontal cortex (mPFC) and amygdala regions. The cytokines IL-1ß and IL-6 increased, and IL-10 was decreased by western blotting. The knockout mice showed different trends in the effects of synaptic proteins. In the mPFC, IL-10 knockout induced a decrease in NR2B and synaptophysin; in the amygdala region, there was a significant increase in NR2B and PSD95. IL-10 knockout from microglia induced a decrease in GAD67 and parvalbumin (Pv) in the mPFC, but not in the amygdala. Our results showed enhanced depression and anxiety-like behavior in the Cx3cr1CreER IL-10-/- mice, which could be related to an imbalance in local excitatory and inhibitory transmission, as well as neuroinflammation in the mPFC and amygdala. This imbalance was associated with increased local inflammation. Although many studies have demonstrated the role of TRPC channels in emotional responses, our study showed that TRPC was not involved in this process in Cx3cr1CreERIL-10-/- mice.

Nature of T cell epitopes in lupus antigens and HLA-DR determines autoantibody initiation and diversification.

OBJECTIVES: The generation of systemic lupus erythematosus (SLE)-related autoantibodies have been shown to be T cell dependent and antigen driven with HLA-DR restriction. In this study, the initiating antigen(s) and the mechanism of autoantibody diversification were investigated. METHODS: T cell epitopes (T-epitopes) of SmD1 (SmD) were mapped by T-T hybridomas generated from DR3+AE0 mice immunised with SmD and with SmD overlapping peptides. TCRs from the reactive hybridomas were sequenced. The core epitopes were determined. Bacterial mimics were identified by bioinformatics. Sera from DR3+AE0 mice immunised with SmD peptides and their mimics were analysed for their reactivity by ELISA and immunohistochemistry. Samples of blood donors were analysed for HLA-DR and autoantibody specificities. RESULTS: Multiple HLA-DR3 restricted T-epitopes within SmD were identified. Many T-T hybridomas reacted with more than one epitope. Some of them were cross-reactive with other snRNP peptides and with proteins in the Ro60/La/Ro52 complex. The reactive hybridomas used unique TCRs. Multiple T-epitope mimics were identified in commensal and environmental bacteria. Certain bacterial mimics shared both T and B cell epitopes with the related SmD peptide. Bacterial mimics induced autoantibodies to lupus-related antigens and to different tissues. HLA-DR3+ blood donors made significantly more SLE-related autoantibodies. CONCLUSIONS: The unique antigenic structures of the lupus-related autoantigens provide the basis for being targeted and for T and B cell epitope spreading and autoantibody diversification with unique patterns. SLE-related autoantibodies are likely generated from responses to commensal and/or environmental microbes due to incomplete negative selection for autoreactive T cells. The production of SLE-related antibodies is inevitable in normal individuals. The findings in this investigation have significant implications in autoimmunity in general.

Novel T-cell epitopes on Schistosoma japonicum SjP40 protein and their preventive effect on allergic asthma in mice.

Allergic asthma is a chronic inflammatory disease mediated by Th2 cell immune responses. Currently, immunotherapies based on immune deviation are attractive, preventive, and therapeutic strategies for asthma. Many studies have shown that intracellular bacterial infections such as mycobacteria and their components can suppress asthmatic reactions by enhancing Th1 responses, while helminth infections and their proteins can inhibit allergic asthma via immune regulation. However, some helminth proteins such as SmP40, the major egg antigen of Schistosoma mansoni, are found as Th1 type antigens. Using a panel of overlapping peptides, we identified T-cell epitopes on SjP40 protein of Schistosoma japonicum, which can induce Th1 cytokine and inhibit the production of Th2 cytokines and airway inflammation in a mouse model of allergic asthma. These results reveal a novel form of immune protective mechanism, which may play an important role in the modulating effect of helminth infection on allergic asthmatic reactions.

Phospholipase Cε deficiency delays the early stage of cutaneous wound healing and attenuates scar formation in mice.

This study aimed to investigate the role of phospholipase Cε (PLCε) in the skin wound healing process. PLCε, an effect factor of Ras/Rap small G protein, plays a crucial role in skin inflammation by regulating inflammatory cytokines. Inflammatory responses are closely associated with wound healing. Full-thickness skin wounds were made in the PLCε knockout (KO) and wild-type (WT) mice, and the healing process was analyzed. The macroscopic wound closure rate declined in the PLCε KO mice on days 3, 4, and 5 after wounding, following the decreased expression of interleukin (IL)-6, chemokine (C-X-C motif) ligand (Cxcl)-1, Cxcl-2, and chemokine (C-C motif) ligand (Ccl) 20. The proliferation rate of epidermal keratinocytes was not affected by PLCε, but silencing of PLCε resulted in the delayed migration of keratinocytes. Moreover, the scars were found to be much smaller in the PLCε KO mice than in the WT mice. The mRNA expression of Ccl20, collagen (Col) 6a1, and Col17a1 decreased in the PLCε KO mice. These results were in agreement with a previous hypothesis that PLCε might delay the early stage of cutaneous wound healing by inhibiting the migration of keratinocytes, and decrease the expression of Col6a1, Col17a1, and Ccl20 by inhibiting the inflammatory response to reduce scar formation. This study shed light on a novel role of PLCε in wound healing and provided new therapeutic approaches to target PLCε for diminishing scar formation after injury.

Nitric oxide synthase inhibition abolishes exercise-mediated protection against isoproterenol-induced cardiac hypertrophy in female mice.

OBJECTIVE: Exercise training (ET) provides a cardioprotective effect against pathological cardiac hypertrophy. Nitric oxide (NO) plays an important role in modulating cardiac hypertrophy. However, few studies explore the relationship between NO signaling and the inhibitory effect of ET on pathological cardiac remodeling. METHODS: In this study, we evaluated ET effects on isoproterenol (ISO)-induced cardiac hypertrophy in female mice. Moreover, L-NAME (Nω-nitro-L-arginine methyl ester), a nonselective NO synthase (NOS) inhibitor, was used to assess the involvement of NO signaling in cardiac hypertrophy. Morphological and echocardiographic variables were assessed. Cardiac hypertrophy-related gene expression was detected by real-time PCR and the protein levels of NOS signaling molecules were determined by Western blot. RESULTS: L-NAME treatment prevented the beneficial effects of ET against the increase in heart weight (HW)/body weight (BW), HW/tibia length and lung weight/BW and echocardiographic variables following ISO injection. Also, L-NAME co-administration reversed ET-induced inhibition of myocardial fibrosis and fetal gene reactivation in ISO-treated mice. Furthermore, L-NAME treatment prevented ET-mediated up-regulation of phosphorylated endothelial NOS and plasma NO in ISO-treated mice. CONCLUSIONS: Our findings demonstrate that L-NAME treatment could abolish ET-induced cardioprotection against pathological cardiac hypertrophy and that NOS modulation may be involved in the antihypertrophic effects induced by ET.

6-Valent Virus-Like Particle-Based Vaccine Induced Potent and Sustained Immunity Against Noroviruses in Mice.

Norovirus is a major cause of acute gastroenteritis worldwide, and no vaccine is currently available. The genetic and antigenic diversity of Norovirus presents challenges for providing broad immune protection, which calls for a multivalent vaccine application. In this study, we investigated the possibility of developing a virus-like particle (VLP)-based 6-valent Norovirus vaccine candidate (Hexa-VLPs) that covers GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 genotypes. Hexa-VLPs (30 µg) adjuvanted with 500 µg of aluminum hydroxide (alum) were selected as the optimal immunization dose after a dose-escalation study. Potent and long-lasting blockade antibody responses were induced by 2-or 3-shot Hexa-VLPs, especially for the emerging GII.P16-GII.2 and GII.17 (Kawasaki 2014) genotypes. Hexa-VLPs plus alum elicited Th1/Th2 mixed yet Th2-skewed immune responses, characterized by an IgG1-biased subclass profile and significant IL-4+ T-cell activation. Notably, simultaneous immunization with a mixture of six VLPs revealed no immunological interference among the component antigens. These results demonstrate that Hexa-VLPs are promising broad-spectrum vaccines to provide immunoprotection against major GI/GII epidemic strains in the future.

Exercise Training Protects Against Heart Failure Via Expansion of Myeloid-Derived Suppressor Cells Through Regulating IL-10/STAT3/S100A9 Pathway.

BACKGROUND: Exercise training (ET) has a protective effect on the progression of heart failure, however, the specific mechanism has not been fully explored. Myeloid-derived suppressor cells (MDSCs) are a group of myeloid-derived immunosuppressive cells, which showed a protective effect in the progression of heart failure. Thus, we hypothesized that the protective effect of ET on heart failure may be related to the infiltration of MDSCs. METHODS: C57BL/6 mice were made to run on a treadmill 6× a week for 4 weeks followed by isoproterenol injection from third week. Heart function was evaluated by echocardiography and the proportion of MDSCs was detected by flow cytometry. Hypertrophic markers, cardiac fibrosis, and inflammatory factors were detected by real-time PCR, ELISA, histological staining, and Western blot. RESULTS: ET treatment in isoproterenol-induced heart failure mice (n=7) enhanced cardiac function (57% increase in FS%, P=0.002) and improved morphological changes compared with isoproterenol mice (n=17). ET further caused 79% increasing in cardiac MDSCs in isoproterenol mice (P<0.001). In addition, depletion of MDSCs by 5-Fluorouracil blunted the cardio-protective effect of ET. T-cell proliferation assay showed that ET did not affect the suppressive activity of MDSCs. Furthermore, we found that ET activated the secretion of IL (interleukin)-10 by macrophages in isoproterenol mice. MDSCs expansion and cardio protection was not present in tamoxifen-inducible macrophage-specific IL-10 knockout mice. Western blot results confirmed that IL-10 regulated the differentiation of MDSCs through the translocation of p-STAT3 (signal transducer and activator of transcription 3)/S100A9 (S100 calcium-binding protein A9) to the nucleus. CONCLUSIONS: ET could increase MDSCs by stimulating the secretion of IL-10 from macrophage, which was through IL-10/STAT3/S100A9 signaling pathway, thereby achieving the role of heart protection.

Intranasal delivery of MSC-derived exosomes attenuates allergic asthma via expanding IL-10 producing lung interstitial macrophages in mice.

Mesenchymal stem cells (MSCs) have been investigated in preventing and treating allergic asthma in many reports. Recently, MSC-derived exosomes (MSC-Exo) were showed a promising alternative to stem cell-based therapy in many kinds of diseases. However, the effect of MSC-Exo on allergic asthma has not been investigated thoroughly thus far. Here, we aimed to investigate the immunomodulation effect of MSC-Exo in a murine model of asthma and explore the underlying mechanisms. BALB/c mice were sensitized and challenged by OVA to establish asthma model. MSC-Exo were intranasally delivered before or during challenge and the protective effect were assessed after the last OVA challenge. Allergic airway inflammation elicited by OVA were significantly attenuated by intranasal delivery of MSC-Exo. To explore the protective mechanism of MSC-Exo, lung interstitial macrophages (IMs) and alveolar macrophages (AMs) were analyzed by flow cytometry and the origin of IMs were traced. Lung IMs ratios were significantly enhanced and high level of IL-10 was produced after MSC-Exo intranasal delivery. IMs ratios were not obviously affected by CCR2 inhibitor or Clodronate liposome administration, whereas significantly decreased in splenectomized mice. Cx3cr1+ cell specific IL-10 conditionally deficient mice were used to further examine the role of IL-10 producing IMs in allergic asthma. IMs-mediated protection was dependent on IL-10, given that the protection disappeared in Cx3cr1-IL-10-/-mice. In conclusion, intranasal delivery of MSC-Exo could substantially expand lung IL-10-producing IMs, which may originate from spleen, thus contribute to protection against allergic asthma in mice.

Chronic stress promotes breast carcinoma metastasis by accumulating myeloid-derived suppressor cells through activating ß-adrenergic signaling.

Numerous studies have found that chronic stress could promote tumor progression and this may be related to inhibtion of immune system. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells with immunosuppressive activity. MDSCs may represent a key link between chronic stress and tumor progression. However, the role of stress-induced MDSCs in breast cancer progression is unclear. The present study showed that pre-exposure of chronic stress could lead to MDSCs elevation and facilitated breast cancer metastasis in tumor-bearing mice. Adoptive transfer of MDSCs could significantly increase lung metastatic foci. In contrast, lung metastasis could be alleviated by depleting endogenous MDSCs with Gr-1 antibody. The concentration of norepinephrine in serum and the expression of tyrosine hydroxylase in bone marrow could be significantly elevated by chronic stress. Moreover, propranolol, an inhibitor of ß-adrenergic signaling, could inhibit breast carcinoma metastasis and prevent the expansion of chronic stress-induced MDSCs. Further study revealed that the expressions of IL-6 and JAK/STAT3 signaling pathways were upregulated by chronic stress in mice, and this upregulation could be inhibited by propranolol. Blocking the IL-6 signal or inhibiting the activation of the JAK/STAT3 signaling pathway could reduce tumor growth and metastasis by attenuating the accumulation of MDSCs in vivo. Besides, propranolol inhibited the expression of IL-6 in supernatant of 4T1 cells induced by isoproterenol and reduced the proportion of inducible MDSCs in vitro. Taken together, these data indicated that chronic stress may accumulate MDSCs via activation of ß-adrenergic signaling and IL-6/STAT3 pathway, thereby promoting breast carcinoma metastasis.

Resveratrol prevents ISO-induced myocardial remodeling associated with regulating polarization of macrophages through VEGF-B/AMPK/NF-kB pathway.

Macrophage expansion and inflammatory responses are involved in induction of cardiac remodeling. Resveratrol has strong anti-inflammatory effects, however its effect on macrophage infiltration and polarization is unknown. This study aimed to investigate the anti-inflammatory effects of RSV on ISO-induced myocardial remodeling in mice and its regulatory role in macrophage polarization. BALB/c mice were orally administered with RSV (100 mg/kg) daily for one week, then were subcutaneously injected with ISO (50 mg/kg) daily for another week. ISO injections to mouse caused cardiac dysfunction evidenced by cardiac hypertrophy and cardiomyocyte fibrosis. Meanwhile, macrophage M1 polarization was found in ISO treated mice, which was evidenced by increased percentage of Ly6Clow macrophages in the heart, levels of M1 cytokines and expression of CD68, and decreased percentage of Ly6Chigh macrophage, levels of M2 cytokines and expression of CD206. All these changes in cardiac and macrophage M1 polarization were ameliorated when mice were pretreated with RSV. The effect of RSV on macrophage polarization was also tested in RAW264.7 cells. It was found that pre-treatment with RSV decreased the levels of M1 marker or proinflammatory cytokines, while increased the levels of M2 markers in ISO treated cells. In addition, it was found that RSV could upregulate the expression of VEGF-B and the activity of AMPK, while it downregulated the expression of phosphorylated NF-κB p65 both in RAW264.7 cells and in mice. Furthermore, pretreatment with VEGF-B siRNA greatly reversed changes in almost all above parameters evoked by RSV in RAW264.7 cells. Therefore, our findings suggest RSV has potential therapeutic effects in ISO-induced myocardial injury, which may be by inhibiting the M1 polarization of macrophages through VEGFB/AMPK/NF-кB pathway.

Resveratrol protects against isoproterenol induced myocardial infarction in rats through VEGF-B/AMPK/eNOS/NO signalling pathway.

According to our previous results, resveratrol (RSV, 3, 5, 4-trihydroxystilbene), a naturally polyphenolic phytoalexin, could attenuate myocardial ischemia/reperfusion injury through up-regulation of vascular endothelial growth factor B (VEGF-B) in isolated rat heart or H9c2 cells. However, the molecular mechanism remains unclear. In this study, we investigated the protective effect of RSV on myocardial infarction (MI) in rats and further explored the underlying signal pathway after VEGF-B. Rats received RSV or normal saline by intragastric administration for 7 consecutive days and followed by subcutaneously isoproterenol (ISO) or normal saline injections for another 2 days. We found that RSV pretreatment prevented the unfavourable changes in HW/BW, HW/TL, infarct size, and cell apoptosis in ISO-treated rats. Moreover, superoxide and malondialdehyde (MDA) production were significantly reduced and superoxide dismutase (SOD) was increased by RSV in ISO-treated rats. Furthermore, it showed that RSV pretreatment increased VEGF-B, p-eNOS and p-AMPK expression, and NO production in ISO-treated rats. Using Neonatal Rat Ventricular Myocytes (NRVM), we found that VEGF-B siRNA could abolish the cardio-protective effect of RSV. The enhanced ratios of eNOS phosphorylation to eNOS expression induced by RSV were markedly reversed by VEGF-B siRNA in NRVM also. Meantime, we found that the effect of VEGF-B knock-down on eNOS activation was rescued by AMPK activator AICAR. L-NAME, a NOS inhibitor, could inhibit RSV enhanced eNOS phosphorylation but had no effect on VEGF-B expression in NRVM or in rats. Collectively, our results indicate that RSV exerts cardio-protection from ISO-induced myocardial infarction through VEGF-B/AMPK/eNOS/NO signalling pathway.

CpG oligodeoxynucleotide and montanide ISA 206 adjuvant combination augments the immune responses of a recombinant FMDV vaccine in cattle.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. To prevent the spread of FMDV, inactivated virus vaccines are used to immunize animals in developing countries. However, there are safety concerns. In addition, it is difficult to distinguish the vaccinated animals from those naturally infected ones. In our lab, we have developed a recombinant FMDV vaccine named A7. A7 contained multiple B cell and T cell epitopes, which reside in a capsid protein (VP1) of FMDV. To enhance its immunogenicity, A7 was formulated with CpG ODN RW03 in combination with Montanide ISA 206 (ISA), and the resultant vaccine (A7+ISA+CpG ODN RW03) was used to immunize mice and cattle. It was found that CpG ODN RW03 and ISA combination could facilitate A7 to induce a vigorous and long-lasting specific antibody response in mice and cattle. After FMDV challenge, 80% (4/5) of the calves immunized with A7+ISA+CpG ODN RW03 were protected, which was superior to those immunized with A7+ISA (25%, 1/4) or inactivated FMDV vaccine (50%, 2/4). These findings suggest that CpG ODN RW03 could be used with Montanide ISA 206 as a potent adjuvant for recombinant FMDV in cattle.

[Computer-aided-designing and eukaryotic expression of recombinant HPV 16 vcaccine].

AIM: To develop a recombinant protein vaccine with epitopes on L1 protein for prevention of HPV16 infection. METHODS: One B cell epitope and one T cell epitope from HPV16 L1 protein were chosen to construct genes encoding different recombinant proteins. Then all the 3D structures of the proteins were predicted by Geno3D in internet. An optimal protein named RH was chosen in which three B epitopes' location simulates that in the real HPV16 L1. Gene X (consisting of B+T+T) and gene Y (consisting of B+T) were constructed by multiple PCR. RH gene (consisting of X+X+X+X+Y+Y+Y) was constructed, sub-cloned into pFastbac1, and expressed in Sf9. RESULTS: RH protein with epitopes on HPV16 L1 was designed and predicted by Geno3D. RH gene was constructed and sub cloned into pFastbac1. Finally, it was successfully expressed in baculovirus expression system. CONCLUSION: A HPV16 recombinant vaccine is successfully constructed and expressed.

A novel canine favored CpG oligodeoxynucleotide capable of enhancing the efficacy of an inactivated aluminum-adjuvanted rabies vaccine of dog use.

In order to develop novel canine CpG ODNs as adjuvant for rabies vaccine of dog use, a panel of CpG ODNs containing different CpG motifs was designed and screened for their ability to induce the proliferation of canine splenocytes. Three AACGTT motif-containing CpG ODNs, designated as YW07, YW08 and YW09, respectively, were outshined with stronger ability to activate canine immune cells. The CpG ODNs were tested for their adjuvant activity for rabies vaccine in mice and dogs. It was found that YW07 could facilitate the rabies vaccine to induce more vigorous and long-lasting specific antibody response in mice and dogs, respectively. These findings suggest that YW07, a canine favored CpG ODN, could be used as a novel adjuvant for developing more efficient rabies vaccine of dog use.

Human microsatellite DNA mimicking oligodeoxynucleotides down-regulate TLR9-dependent and -independent activation of human immune cells.

To develop novel immunoregulatory oligodeoxynucleotides (ODNs), we have designed a series of ODNs based on the sequences in human microsatellite (MS) DNA. The ODNs, designated as human MS DNA mimicking ODNs (MS ODNs), have been studied for their inhibitory effects on human immune cells activated by TLR9-dependent and -independent stimulations. We find for the first time that MS08, a MS ODN composed entirely of TC dinucleotide (TC) repeats, inhibits CpG ODN (TLR9 ligand)-induced human PBMCs proliferation, CD80 and CD86 expression and production of interferon. In addition, MS08 also inhibits the proliferation of human PBMCs stimulated by PHA, PMA and alloantigens in a TLR9-independent manner. The inhibition correlates with competition of binding and uptake between MS08 and CpG ODN in human PBMCs. Structurally, TC, CT or CCT are revealed as essential suppressive motifs required for the inhibition. These findings suggest that TC repeat containing MS ODN could be of therapeutic use in pathologic situations due to excessive activation of immune cells.

[Establishment, optimization and application of a screening method for immunoregulatory oligodeoxynucleotides].

AIM: To establish a set of reasonable and convenient experimental system to provide a screening method for the development of novel immunoregulatory oligodeoxynucleotides. METHODS: The human PBMCs were stimulated by CpG ODN and/or immunoregulatory ODN. The cell proliferation and anti-viral activity of the supernatant induced by CpG ODN were examined by thymidine incorporation and anti-viral bioassay to evaluate the immunoregulatory activity of candidate ODN. The experimental conditions were also optimized. RESULTS: A screening method on which A151, a positive immunoregulatory ODN, inhibited the proliferation and anti-viral activity of the CpG ODN-induced human PBMCs was successfully established. CONCLUSION: The successful establishment of CpG ODN based screening method lays the foundations for further development of novel immunoregulatory oligodeoxynucleotides.