The Journal of Immunology, 2007, 178: 5409-5410.
Copyright © 2007 by The American Association of Immunologists, Inc.
IN THIS ISSUE
T Cells versus Vaccine Vectors
One strategy to achieve long-term protection against pathogens causing AIDS, tuberculosis, or malaria is to develop vectors that express a DNA vaccine. To date, this effect has been hampered by host elimination of plasmid DNA immunogens. To understand the basis of the damping of these vaccines, Greenland et al. (p. 5652
) used an In vivo Imaging System (IVIS) to follow expression of nonreplicating vaccine vectors expressing luciferase in immunized mice injected with luciferin. Wild-type mice expressed luciferase maximally at 48 h or 14 days after injection with an adenovirus construct or a plasmid construct, respectively; luciferase expression declined rapidly after the peaks. Luciferase expression in nude mice peaked at day 2 or 4 following immunization with the adenovirus or plasmid construct, respectively, and did not decline. No luciferase-specific Ab responses were detected with either vector. Appearance of Ag-specific CD8+ T cells coincided with the damping of vaccine Ag expression in mice immunized with a plasmid expressing a luciferase-viral peptide fusion protein. A vaccine that elicited a low-level T cell response persisted unless administered with adjuvant. Histology and immunostaining showed an inflammatory CD3+ T cell infiltrate and apoptotic myocytes and T cells at the injection site in wild-type mice. Immunized Rag1–/– or Fas–/– mice had persistent, high-level luciferase and Ag expression as measured by IVIS. Vaccine Ag expression was more persistent, and immune responses were higher after a boosting inoculation in Fas–/– vs wild-type mice. This approach incorporating IVIS shows that vaccine-elicited T cells may induce Fas-mediated apoptosis of vector-containing cells.
IL-21 and H. pylori Gastritis
Inflammation of gastric mucosa is associated with a T cell-mediated response to infection by Helicobacter pylori. IL-21 produced by activated CD4+ T cells enhances influx of immune cells into inflamed tissues. In looking at a possible link between IL-21 and H. pylori gastritis, Caruso et al. (p. 5957
) found higher IL-21 protein and mRNA expression in gastric mucosa from H. pylori-infected patients with gastritis compared with samples from uninfected patients with gastritis or normal controls. Expression of p21 was reduced in patients successfully treated for H. pylori. IL-21R was detected in gastric epithelial cell extracts and in epithelial cell-depleted mucosal samples from H. pylori-infected patients and controls and on two gastric cancer epithelial cell lines. IL-21R protein expression on cells was enhanced by incubation with any of three different H. pylori strains or by treatment with IL-1
, TNF-
, or IFN-
. IL-21 increased protein and mRNA synthesis of matrix metalloproteinase (MMP)-2 and MMP-9 in the gastric cell lines; I
B- protein was reduced, and NF-B binding complex formation was increased as determined by EMSA. A specific inhibitor of NF-B activation abrogated its DNA binding activity in IL-21-treated cell cultures. Reduced MMP-2 and MMP-9 protein levels were measured in the inhibitor-treated cultures and in ex vivo organ cultures of gastric biopsies from infected patients treated with an anti-IL-21 Ab. The authors suggest that an H. pylori-driven inflammatory response stimulates gastric tissue production of IL-21, NF-B-induced synthesis of MMP-2 and MMP-9, and expression of IL-21R.
BAFFled B Cells
The progression of B cell development beyond the transitional type 2 stage and B cell homeostasis is dependent on B cell activating factor (BAFF). BAFF binds to B cells, but its precise role in B cell differentiation is not known. Darce et al. (p. 5612
) showed that only human CD19+ PBMCs reacted with an anti-BAFF Ab. Immunoblotting and a soluble decoy receptor approach revealed that the Ab-reactive species was the processed, soluble form of BAFF. BAFF was detected on naive and memory, but not on germinal center (GC), B cells from human tonsil tissue. All three cell populations expressed one to three BAFF receptors, and GC B cells bound added soluble BAFF. Peripheral blood B cells lost prebound BAFF after in vitro activation by CD40L plus IL-2 and IL-10 (T cell dependent or TD) or by CpG (T cell independent or TI) but retained the ability to bind exogenous BAFF. BAFF promoted proliferation and increased Ig secretion of TD-activated naive B cells, and it improved survival of TI-activated memory B cells but inhibited their differentiation into Ig-secreting cells. TI or TD activation of memory B cells induced markers of Ig-secreting cells, and BAFF increased TD-induced, but decreased TI-induced, differentiation. The authors propose that limited exposure of B cells to BAFF within the GC maintains the B cell differentiation program, whereas BAFF controls the balance between Ig-secreting cells and memory B cell development outside the GC.
Susceptibility to HIV-1/AIDS
Since the number of CCR5 receptors on CD4+ T cells determines susceptibility to HIV-1, it is important to understand the molecular mechanisms regulating CCR5 gene expression. Mummidi et al. (p. 5668
) used two PCR-based assays to determine that CCR5 mRNA isoforms originating within exon 1 were poorly expressed in quiescent human PBMCs and purified CD4+ T cells compared with truncated exon 2 transcripts. TCR activation of the cells up-regulated expression only of exon 1-containing transcripts. A reporter plasmid carrying the CCR5 exon 1 promoter in transformed T cells was weakly expressed compared with one carrying the CCR5 exon 2 promoter. In contrast, equivalent expression of either transfected plasmid was seen in unstimulated PBMCs or purified CD4+ T cells from normal donors; TCR activation increased expression of only exon 1. An open chromatin configuration was detected at four of nine conserved noncoding sequences upstream of the CCR5 locus in memory, but not naive, CD4+ T cells; acetylated histones were enriched at the exon 1 promoter. Surface expression and mRNA levels of CCR5 were higher in activated T cells overexpressing the Oct-2 (octamer-2) transcription factor but lower in cells overexpressing Oct-1. Engineered mutations within the Oct-2 binding site in the exon 1 promoter reduced CCR5 plasmid expression in activated PBMCs, and an Oct-2 short interfering RNA blocked exon 1 transcription in primary T cells and in activated PBMCs. The exon 1 promoter region from chimpanzee or African green monkey had a single nucleotide difference in the consensus octamer binding site compared with humans and did not bind Oct-1 and Oct-2. The authors propose that CCR5 expression via Oct-2 transactivation of the CCR5 exon 1 promoter occurs in activated CD4+ T cells and that the nonhuman primate hosts are protected from viral infection by mutated octamer binding sites.
Limiting Crohn’s Inflammation
Although the inflamed mucosa of patients with Crohn’s disease (CD) has high levels of IL-22 produced by Th1 cells, the role of IL-22 in CD is not known. On p. 5973
, Wolk et al. found elevated levels of IL-22 in the blood of CD patients compared with healthy controls. In the experimental colitis mouse model for CD, elevated IL-22 mRNA was detected in inflamed gut and in mesenteric lymph nodes of affected animals. Binding studies demonstrated higher affinity of IL-22 with its soluble receptor, IL-22 binding protein (IL-22BP), than with its membrane receptor IL-22R. IL-22BP mRNA levels were lower in the inflamed mouse colon but not in mesenteric lymph nodes. Healthy mice injected with IL-22 had plasma levels of LPS binding protein (LBP) 48 h later that were elevated compared with mice injected with PBS; liver, but not lung or kidney, tissues had increased LBP mRNA expression. Liver cells stimulated with IL-22 in vitro had increased STAT1 and STAT3 phosphorylation and released more LBP into culture supernatants than control cultures. IL-22 increased LBP production stimulated by IL-1, TNF-, or IL-6; IL-22BP or an Ab against one chain of the IL-22R dose dependently inhibited the effect. Blood plasma LBP levels were increased in both mouse colitis and CD as measured by ELISA, but free LPS levels were only slightly higher in CD patients than in healthy controls. The authors suggest that increased liver production of LBP by IL-22 sequesters LPS within the intestine and prevents systemic inflammation in CD.
Regulating Bone Turnover
Protein inhibitor of activated STAT3 (PIAS3) is a binding partner for microphthalmia transcription factor (Mitf), important in osteoclast development. However, a direct role for PIAS3 in osteoclastogenesis has not been shown. On p. 5588
, Kim et al. found by real-time PCR that PIAS3 mRNA expression was down-regulated in murine bone marrow-derived monocyte/macrophage cells (BMMs) treated with stimulators of osteoclast formation (TNF-, IL-1, and IL-6) but was up-regulated by treatment with suppressors (IL-4, IFN-, -, and -) of osteoclast differentiation induced by TNF-related activation-induced cytokine (RANKL). The number and size of multinuclear osteoclasts and expression of NFATc1 and osteoclast-associated receptor (OSCAR) were reduced in RANKL-treated BMMs transduced with a retrovirus overexpressing PIAS3 protein compared with controls; in contrast, a PIAS3 short interfering RNA increased osteoclastogenesis and expression of NFATc1 and OSCAR. PIAS3 overexpression also reduced induction of OSCAR by transcription factors NFATc1 or Mitf and reduced c-fos transactivation of the NFATc1 promoter. Anti-PIAS3 Ab coimmunoprecipitated Mitf, c-fos, or NFATc1 from lysates of mouse cells cotransfected with vectors expressing one of those proteins plus PIAS3. Chromatin immunoprecipitation assay using anti-histone deacetylase 1 (HDAC1) Ab demonstrated decreased binding of HDAC1 to the NFATc1 promoter in bone marrow-derived osteoclasts vs BMMs; overexpression of PIAS3 increased HDAC1 binding to the NFATc1 promoter in both cell types and increased HDAC1 binding to the OSCAR promoter in osteoclasts. The data show that PIAS3 down-regulation of RANKL-mediated osteoclastogenesis occurs by HDAC1-mediated corepression of NFATc1 and OSCAR.
Autoantibody Repertoire in Pemphigus
Autoantibodies against desmosomal glycoprotein desmoglein (Dsg)1 or Dsg1 plus Dsg3 are responsible for the skin blistering and lesions in mucosal pemphigus vulgaris (PV) and mucosal and skin PV, respectively. However, few details about the autoantibody repertoires in patients suffering from PV are available. Qian et al. (p. 5982
) generated 73 heterohybridomas by fusing peripheral blood B cells from 6 PV patients with mouse myeloma cells and found by Dsg-specific ELISA that two-thirds of them were IgM and one-third were IgG. Nearly all (67) of the hybridomas reacted with both Dsg1 and Dsg3, consistent with the extensive ectodomain homology of the two proteins. Sequence analyses of RT-PCR amplified VH and VL sequences revealed six sets of clonally related hybridomas from four of the patients. A bias for two VH gene segments in IgG, but not in IgM, hybridomas was noted; VL usage was more diverse among the IgG hybridomas. Divergence from germline sequences indicated somatic hypermutation, especially among the IgG hybridomas. More than 90% of the IgG H chains had six or more mutations, predominantly amino acid replacements within the CDR regions. Shared mutations among independent IgG and IgM hybridomas also were located in H chain CDRs. Both shared and unique somatic mutations were detected among members of the same clone. Most early replacement mutations were nonconservative, whereas those that occurred later were conservative. The results provide evidence for Ag selection of the anti-Dsg response at the IgM and IgG stages in PV patients.
Summaries written by Dorothy L. Buchhagen, Ph.D.
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