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Immunological Synapses
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RI activation receptor plus a transfected fluorescent-labeled NKG2A lacked actin and lipid rafts at contact sites with anti-NKG2A mAb- or HLA-E-coated beads but had them at contact sites with anti-FcRI
mAb beads. Similar results were seen using primary human NK cells with anti-NKG2A and anti-CD16 mAbs. Immunoprecipitation and immunoblotting showed NKG2A together with SHP-1 in the soluble (nonraft) fraction of lysed cells before and after activation, and contour and channel masking techniques created three-dimensional reconstructed images demonstrating colocalization of phosphorylated NKG2A and SHP-1 at the synapse. Cross-linking of the activating receptor NKG2D increased phosphorylation of Vav1, the actin regulator, and of ERM proteins (ezrin, radixin, and moesin) that link cell surface and cytoskeleton proteins; anti-NKG2A mAb cross-linking inhibited the NKG2D-induced phosphorylation. Cells binding HLA-E-coated beads lacked ezrin at the site of contact. The data show that the NKG2A inhibitory synapse prevents formation of the activating synapse by local actin depolymerization, exclusion of lipid rafts, recruitment of SHP-1, and dephosphorylation of Vav1 and ERM proteins. Targeting Hearing Loss
Patients experiencing autoimmune sensorineural hearing loss (ASNHL), the most common cause of sudden hearing loss in adults, have elevated serum Ab levels to the abundant inner ear protein cochlin. The Tuohy laboratory showed that mice immunized with a mouse cochlin peptide develop hearing loss that can be transferred to naive recipients by peptide-activated CD4+ T cells. However, no direct evidence of T cell reactivity with cochlin exists for humans. In a follow-up to their mouse work, Baek et al. (p. 4203
) used recombinant human cochlin to detect by ELISPOT assays a highly elevated frequency of cochlin-specific IFN-
-secreting T cells and a moderately elevated frequency of cochlin-specific IL-5-secreting T cells in PBMCs from eight ASNHL patients compared with matched normal hearing controls. ASNHL IFN- production was found in purified CD4+ and CD8+ T cells from some patients but was restricted to CD8+ T cells from other patients. Titers of anti-cochlin Ab in sera of ASNHL patients were very high by ELISA compared with normal hearing controls; titers in sera of patients with noise- or age-related hearing loss were higher than in normal hearing controls but lower than in ASNHL patients. The data indicate that cochlin is a T cell target in ASNHL, and perhaps in noise- and/or age-related hearing loss, in humans.
Methylation and Memory
Although effective vaccines rely on induction of memory CD8+ T cells, the signals and factors involved are not completely understood. Kersh (p. 3821
) looked at the role of epigenetic gene methylation in memory CD8+ T cell differentiation in mice lacking methyl-CpG-binding domain protein 2 (MBD2), the transcriptional repressor that binds methylated DNA. Ag-specific CD8+ T cells expanded efficiently, and virus was cleared in wild-type and MBD2–/– mice after lymphocytic choriomeningitis virus (LCMV) infection. Yet, fewer Ag-specific CD8+ T cells were recovered from mutant mouse spleens during the contraction and initial memory phase (days 14–28 postinfection (p.i.)), and only the mutant mice had reduced IL-7R expression on their CD8+ T cells at all time points p.i. as determined by flow cytometry; other surface markers on mutant cells were reduced or enhanced in expression at day 8 p.i. compared with wild-type cells. Mutant cells also had elevated annexin V binding during the contraction phase. The mutant cell marker profile indicated slower acquisition of memory phenotype and longer retention of effector-like properties. Both effector and memory MBD2–/–CD8+ T cells produced less IFN- in response to peptide than wild-type cells. Delayed LCMV clearance, increased viral burden, and severe contraction of the secondary CD8+ T cell response were seen in reinfected mutant mice compared with wild-type controls. SCID mice adoptively transferred with purified MBD2–/–CD8+ T cells had reduced IL-7R expression on their CD8+ T cells, even in the presence of wild-type CD8– T cells. The experiments demonstrate that memory CD8+ T cell induction in LCMV-infected mice requires epigenetic gene silencing by MBD2.
Sea Squirt C3a Receptor
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MHC Class I and AIDS-Free Survival
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only in response to the variant, but not KF11, peptide. Tetramer decay analysis indicated that the KF11 tetramer had weaker binding kinetics to a variant-specific CTL clone. Crystal structures of either peptide bound with HLA-B*5703 revealed a central peptide "bulge." TCR V region usage and CDR3 sequence analysis showed use of a V
17/V15 TCR in CTLs from patients infected with KF11 with almost complete conservation of aa usage in V and V CDR3 regions; viral variant CTLs used a V15/V15 TCR combination with a V15 CDR3 loop of different amino acid composition than the conserved KF11 TCR. This combination of structural and cellular studies indicates that HIV-1-specific CTLs cross-recognize peptides of an immunodominant HIV-1 and its most common mutant but respond only to mutant peptide. Silencing CIITA in Plasma Cells
Transcriptional loss of MHC class II and MHC CIITA genes accompanies maturation of B cells to plasma cells. Silencing of these genes could occur by loss in accessibility of factors to the CIITA pIII promoter, which controls CIITA expression in B cells. Green et al. (p. 3865 ) found by EMSA and chromatin immunoprecipitation assays that the Sp1 transcription factor bound to the activation response element 1 in pIII. In addition to Sp1, three other transcription factors associated with pIII in a human B cell line but had reduced association in a human plasma cell line. Human and mouse B cell lines had high levels of histone H3 acetylation at three lysine residues and modest levels at H4, all of which were greatly reduced or absent in human and mouse plasma cell lines. Levels of dimethylation and trimethylation of H3 at a fourth lysine residue were high in the B cells but reduced and absent, respectively, in the plasma cell lines. Acetylation of H3 at lysine 9 was high in the B cell lines, indicating transcriptional activation, whereas dimethylation at the site was seen only in the plasma cell lines, indicating active transcriptional silencing. Similar patterns of nucleosome modifications were detected at the other three CIITA promoters in the human and mouse cell lines. Factor binding and histone acetylation in primary murine splenic B cells during in vitro differentiation into plasma cells showed changes at pIII that were similar to those seen for the cell lines. The data indicate that loss of CIITA expression during differentiation of B cells to plasma cells is due to a loss of accessibility to transcription by progressive stages of deacetylation and methylation during chromatin remodeling at its promoter.
RANK(L)ing Endotoxin Shock
Osteoclast differentiation factor, RANKL, and the decoy receptor osteoprotegerin (OPG or osteoclast inhibitory factor) primarily function in bone biology. However, published observations suggest a role for these molecules in the innate immune system. Maruyama et al. (p. 3799
) found that LPS-induced production of TNF-, IL-6, and IL-12 by wild-type or Fos–/– M-CSF-dependent bone marrow-derived macrophages and of TNF- by wild-type spleen-derived and peritoneal macrophages was prevented by pretreatment of cells with RANKL. The induced tolerance was reversible by removal of RANKL or treatment of cells with GM-CSF before LPS exposure. MyD88 mRNA levels decreased in RANKL-treated cells, but TLR4 mRNA levels did not change. RANKL also suppressed the cytokine response to other bacterial components. Reduced RANKL and increased OPG serum levels were measured in mice after LPS administration or after oral infection with Salmonella. Mice deficient in RANKL had higher serum levels of TNF- and IL-6 after LPS injection, whereas mice deficient in OPG had high levels of RANKL and produced low amounts of IL-6 in response to LPS injection compared with wild-type controls. All RANKL-deficient mice died, but 90% of wild-type mice were alive, by 24 h after receiving LPS i.p. A lethal i.p. dose of LPS killed 90% of wild-type mice, but 60% of animals given a mixture of RANKL and M-CSF survived. The authors demonstrate that RANKL-induced tolerance in macrophages, mediated by down-regulation of MyD88, protects mice against endotoxin shock and suggest that the RANKL/OPG ratio is a good indicator of endotoxemia and bacterial infection.
Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
B Ligand and Osteoprotegerin Regulate Proinflammatory Cytokine Production in Mice
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