Effects of recombinant human IL-21 on the cytokine profiles of tree shrew spleen lymphocytes.

<p>*represents a statistically significant difference between two groups (<i>P</i><0.05).</p

The IL-21 expression in tree shrew samples.

<p>(A) Concentrations of IL-21 in supernatant from tree shrew spleen lymphocytes induced with ConA by ELISA. (B) Quantification of IL-21 mRNA expression in tree shrew spleen lymphocytes induced by ConA. *represents a significant difference compared to stimulation with ConA and the unstimulated negative control (<i>P</i><0.05). (C) The IL-21 expression of tree shrews spleen lymphocytes and human peripheral blood mononuclear cells were detected by flow cytometry staining.</p

The reactivity of anti-human IL-21 antibody to tree shrew IL-21 expressed by transfected Huh7 cells.

<p>(A) FCM represents flow cytometry staining; (B) ELISA represents enzyme-linked immunosorbent assay; (C) WB represents western blotting. CG represents the control group with pEGFP-N3 plasmid transfection; EG represents the experimental group with pEGFP-N3/tsIL-21 plasmid transfection.</p

Predicted three-dimensional structures of the tree shrew and human IL-21 protein.

<p>(A) Secondary structure of tree shrew IL-21 (right) compared with human IL-21 (left). Red represents α helices, cyan represents β sheets, green represents β turns, white represents random coils, and yellow represents N-glycosylation sites. (B) Hydrophobicity of tree shrew IL-21 (right) compared with human IL-21 (left). Blue represents hydrophilicity, brown represents hydrophobicity, white represents transition. (C) Surface charge of tree shrew IL-21 (right) compared with human IL-21 (left). Blue represents negative charge, red represents positive charge, white represents no electrical charge.</p

Homology analysis of the IL-21 gene.

<p>(A) Phylogenetic tree of the IL-21 gene based on the amino acid sequences among eighteen species. Alignment of tree shrew and human IL-21 coding sequences (B) and amino acid sequences (C).</p

Primers used in the study.

<p>Primers used in the study.</p

Assessment of sequence homology and immunologic cross-reactivity between tree shrew (<i>Tupaia belangeri</i>) and human IL-21

<div><p>Many studies have indicated that the expression of interleukin-21 (IL-21) is associated with the pathogenesis of certain liver diseases. However, in alternative animal models of liver diseases, it remains unknown whether the tree shrew could be utilized to analyze the relationship between IL-21 and liver diseases. Here, the phylogenetic tree, sequence alignment and protein structure model of tree shrew and human IL-21 were analyzed using bioinformatics software. A pEGFP-N3/tsIL-21 eukaryotic expression vector of tree shrew IL-21 (tsIL-21) was constructed, and IL-21 expression by the vector-transfected Huh7 cells was evaluated using the newly established quantitative real-time PCR and immunologic protocols for assessing human IL-21. The cytokine profiles were also evaluated in tree shrew spleen lymphocytes induced by recombinant human IL-21 or concanavalin A. It was found that the coding sequence (CDS) of tsIL-21 amplified from spleen lymphocytes belonged to the predicted sequence. The tsIL-21 was closely clustered with primate IL-21 rather than rodent IL-21, and it had an alignment of 83.33% with the human IL-21 nucleotide sequence and 69.93% with the amino acid sequence. The profiles of secondary structure, hydrophobicity and surface charge of tsIL-21 were also similar with those of human IL-21. The tsIL-21 expressed by the vector-transfected Huh7 cells could be identified by their different sources of antibodies against human IL-21, which were all dose-dependent. Recombinant human IL-21 could induce the change of the cytokine profiles of tree shrew spleen lymphocytes, which showed a higher expression of IL-10 and IFN-γ rather than IL-2, IL-4, IL-17, TNF-a and IL-21 during the five-day stimulation. These results indicate that tsIL-21 has a high degree of homology, structural similarity and immunological cross-reactivity with human IL-21 and also confirm the accuracy of this predicted tsIL-21CDS. The protocols utilized in this study will lead to the experimental feasibility of further IL-21-related studies in vivo.</p></div

TCRγδ⁺CD4⁻CD8⁻ T Cells Suppress the CD8⁺ T-Cell Response to Hepatitis B Virus Peptides, and Are Associated with Viral Control in Chronic Hepatitis B

<div><p>The immune mechanisms underlying failure to achieve hepatitis B e antigen (HBeAg) seroconversion associated with viral control in chronic hepatitis B (CHB) remain unclear. Here we investigated the role of CD4⁻CD8⁻ T (double-negative T; DNT) cells including TCRαβ⁺ DNT (αβ DNT) and TCRγδ⁺ DNT (γδ DNT) cells. Frequencies of circulating DNT cell subsets were measured by flow cytometry in a retrospective cohort of 51 telbivudine-treated HBeAg-positive CHB patients, 25 immune tolerant carriers (IT), 33 inactive carriers (IC), and 37 healthy controls (HC). We found that γδ DNT cell frequencies did not significantly change during treatment, being lower at baseline (<i>P</i> = 0.019) in patients with HBeAg seroconversion after 52 weeks of antiviral therapy (n = 20) than in those without (n = 31), and higher in the total CHB and IT than IC and HC groups (<i>P</i><0.001). αβ DNT cell frequencies were similar for all groups. In vitro, γδ DNT cells suppressed HBV core peptide-stimulated interferon-γ and tumor necrosis factor-α production in TCRαβ⁺CD8⁺ T cells, which may require cell–cell contact, and could be partially reversed by anti-NKG2A. These findings suggest that γδ DNT cells limit CD8⁺ T cell response to HBV, and may impede HBeAg seroconversion in CHB.</p></div

Clinical characteristics of subjects in cross-sectional study.

<p>ALT, alanine aminotransferase; AST, Aspartate aminotransferase; HBeAg, HBV e antigen; HC, healthy controls; IC, inactive carriers; IT, immune tolerant carriers; M/F: Male/female; NA, not applicable.</p>a)<p>Data are median (interquartile range).</p

Mechanisms associated with the suppression by γδ DNT cells.

<p>(A) The suppression of HBV core peptide-induced production of TNF-α or IFN-γ in CD8⁺αβ T cells could be partly abrogated by inserting a membrane between the CD8⁺ αβ T cells and the γδ DNT cells in a transwell assay of cells from four patients with CHB. Lines link the data from the same patients. (B) γδ DNT cells were isolated and pre-incubated with either anti-NKG2A, anti-HLA-E, or mouse IgG1 isotype before being co-cultured with γδ T(−)PBMC. After 10 days of stimulation with peptides, production of TNF-α (black bars) and IFN-γ (blank bars) in CD8⁺αβ T cells was detected. Data from three patients are shown (core, HBV core peptides pool; gag, HIV gag peptide). DNT cells, double-negative T cells; IFN, interferon; PBMC, peripheral blood mononuclear cells; TNF, tumor necrosis factor.</p