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Inactivating Treg Cells
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Prion Protein and T Cells
Cellular prion protein (PrPC), expressed on a variety of cells including thymocytes, binds copper. Although copper deficiency impairs the immune response, the effect of PrPC on T cell function has not been explored. Jouvin-Marche et al. (p. 3490
) noted thymic atrophy in 10-wk-old mice expressing high levels of the PrPC transgene. Double-positive (DP) thymocytes and single-positive (SP) CD4+ T cells were reduced compared with wild-type controls. Double-negative (DN) thymocytes, especially in the DN3 compartment, were increased greatly in transgenic mice. PrPC overexpression promoted differentiation of 
T cells while decreasing that of 
T cells. CD25 levels were higher and CD5 levels were lower on DP and CD4+ SP cells from transgenic vs wild-type animals. Transgenic CD4+ SP cells also had lower levels of CD69 expression. PrPC expression was high on DP and DN3 cells and either high on SP thymocytes or intermediate (on CD4+ T cells) or low (on CD8+ T cells). Expression of TCR on SP cells was inversely proportional to PrPC expression. Hemopoietic cell reconstitution experiments in lethally irradiated hosts demonstrated that retardation in T cell development and accumulation of T cells occurred only in wild-type animals that received PrPC transgenic bone marrow but not the converse. Transgenic animals had increased levels of copper in brain and thymus but not spleen. Increased dietary copper resulted in increased DP and decreased DN cell percentages compared with transgenic mice not given copper. The authors suggest that an antioxidant environment resulting from overexpression of PrPC favors T cell maturation and/or survival.
CD8+ T Cell Response to CMV
Both human CMV (HCMV) and mouse CMV (MCMV) establish lifelong latent infections in their hosts with extremely large CD8+ T cell responses. However, the cause of the dominance of CMV-specific T cells is not understood. Munks et al. (p. 3760
)screened an H-2b fibroblast library expressing 170 cloned open reading frames (ORFs) of MCMV with splenocytes from MCMV-infected C57BL/6 (B6) mice. Intracellular cytokine staining for IFN- identified 27 ORFs, predominantly from early viral genes, that elicited an enhanced CD8+ T cell response. A combination of subcloning, computer-based epitope prediction and peptide synthesis identified major epitopes. Eighteen of the identified epitopes accounted for 51% of all responses of CD8+ T cells from spleens of acutely infected mice, and between 49 and 59% of all activated CD8+ T cells were specific for MCMV. An intracellular cytokine staining assay showed no obvious correlation between T cell functional avidity for different peptides and immunodominance of an epitope. Whereas other mouse strains sharing the H-2b haplotype with B6 responded to all epitopes tested, the total CD8+ T cell response was highest in B6. BALB/c H-2d-restricted epitopes dominated the response in F1 mice expressing both H-2b and H-2d haplotypes on a BALB/c background, whereas H-2b and H-2d-restricted epitopes were codominant in responses of F1 mice with a B6 background. The study demonstrates great diversity in the CD8+ T cell response to MCMV in mice and emphasizes the importance of genetic background on the immunodominance hierarchy.
CR1/2 and Viral-Induced Myocarditis
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and increased IL-1 levels in their hearts during acute myocarditis. Although immune complex deposition occurred in hearts of both infected wild-type and CR1/2-deficient mice, IgG deposition was greater in CR1/2-deficient hearts. The experiments indicate that CR1/2 expression protects mice against CVB3-induced myocarditis by reducing immune complex deposition, inflammation, and fibrosis. Leukocyte Migration
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-parvin, through its interaction with ILK in yeast two-hybrid screens against human cDNA libraries and raised a rabbit polyclonal Ab against it. -Parvin was expressed in monocyte-derived cell lines and in one established T cell line. Only deletion mutants containing the C-terminal half of -parvin interacted with full-length ILK. Coprecipitation of ILK and -parvin was seen from lysates of cells cotransfected with the two molecules and from lysates of unstimulated cells. GFP--parvin or GFP-C-terminal fragment localized with ILK at vinculin-positive focal adhesions and on surface blebs during early stages of PMA-stimulated cell spreading on fibronectin (FN). ILK, -parvin, and F-actin concentrated at the leading edge of polarized PMA-stimulated cells. Overexpression of the C-terminal half of -parvin abolished MCP-1-induced adhesion of cells to FN and blocked cell spreading and polarization of PMA-stimulated cells. Overexpression of the N-terminal half of -parvin enhanced cell adhesion in a process requiring a key cytoskeletal protein; anti-1 integrin Ab blocked the effect. Introduction of a -parvin short hairpin RNA suppressed expression of ILK and an ILK-interacting protein, reduced MCP-1-induced cell adhesion, inhibited formation of focal adhesions and surface blebs, blocked polarization, and decreased PMA-stimulated cell spreading on FN and cell growth and survival. Overexpressed -parvin coprecipitated with several other cytoskeletal components. The data indicate that -parvin-ILK complex formation during cell-substrate interactions is essential for leukocyte migration. Modifying mRNA Splicing
Antisense oligonucleotides (ASOs) are used to target destruction of mRNA transcripts, but their use in regulation or redirection of mRNA splicing had not been established. Vickers et al. (p. 3652
) designed an ASO directed against the donor splice site of MyD88 RNA exon II. A human kidney cell line treated in vitro with the chemically modified splicing ASO exhibited a shift of MyD88 mRNA from a long form to a short form lacking exon II as determined by quantitative RT-PCR and Northern blots. (Short-form MyD88 does not activate NF-
B in cells exposed to IL-1.) MyD88 short- and long-form protein ratios on immunoblots correlated with the mRNA results. A control ASO had no effect. IL-1-mediated up-regulation of ICAM-1 and IL-8 mRNAs and expression of a pNFB reporter plasmid were reduced in cells pretreated with the splicing ASO. Whereas a small-interfering RNA or an active RNase H-dependent ASO targeting MyD88 mRNA resulted in loss of MyD88 mRNA and protein, only long-form MyD88 mRNA and protein were reduced by treatment with the splicing ASO. A different splicing ASO was shown to enhance short-form MyD88 splicing in a mouse macrophage cell line. The majority of liver, adipose tissue, and intestine MyD88 mRNA and protein was of short-form in mice injected with the splicing ASO twice weekly for 2 wk compared with controls. No short-form was detected on immunoblots of the tissue proteins. IL-1 challenge of control, but not splicing ASO-treated, mice resulted in up-regulation of serum amyloid A RNA. The authors demonstrate effective redirection of MyD88 RNA splicing and inhibition of NF-B activation by IL-1 using ASOs in human and mouse cells in culture and in mice.
Poxviral Virulence Factors
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Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
, and Immune Complex Deposition in the Heart
-Parvin-Integrin-Linked Kinase Complex Is Critically Involved in Leukocyte-Substrate Interaction
Signaling in Cell Culture and in Mice with a 2'-O-Methoxyethyl-Modified Oligonucleotide
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