Distinct regulatory elements are required for faithful expression of human CD4 in T cells, macrophages, and dendritic cells of transgenic mice

Abstract

To identify the regulatory elements controlling expression of the human CD4 (hCD4) gene in different cell types of the immune system, deletion and chimeric (human/murine) reporter genes were constructed and tested in transgenic (Tg) mice. Regulatory elements required for the proper hCD4 expression in the immature double-positive thymic T cells were identified in the enhancer and in the 3 end of intron 1. Expression of hCD4 in macrophages is controlled by at least 2 sets of regulatory elements: one present in front of exon 1 and the second at the 5 end of intron 1. The hCD4 elements required for expression on both myeloid and lymphoid CD8␣ ؉ dendritic cells (DCs) from lymph node and thymus were found IntroductionThe CD4 cell surface receptor is expressed on many mouse or human cell populations, namely, on very early T-cell precursors (CD4 Low CD44 ϩ CD25 Ϫ ), on immature double-positive (DP) CD4 ϩ CD8 ϩ T cells, on mature single-positive (SP) CD4 ϩ CD8 Ϫ T cells, 1,2 and on a subpopulation of CD8␣ Ϫ dendritic cells (DCs). 3,4 In humans, CD4 is also expressed in monocytes, macrophages, microglial cells, and some DCs. 5,6 Several regulatory elements of the murine and human CD4 genes (mCD4, hCD4) have been identified and tested in transgenic (Tg) animal studies (reviewed by Ellmeier et al 1 and Killeen and Littman 2 ). We have exploited the structural homology, but the different patterns of expression, of the mCD4 and hCD4 genes and made chimeric mCD4/hCD4 transgenes, to map more precisely the regulatory regions of the hCD4 gene controlling its expression in macrophages and in DCs. Study design Tg miceConstructs CD4A, CD4B, and CD4C were described previously. 7 Other constructs were generated from the CD4C DNA by replacing the 2.6-kilobase pair (kbp) SacI fragment with the mouse promoter (CD4E), or the 9-kbp EcoRV-XbaI fragment with the murine silencer (0.5 kbp) (CD4F), or by deleting the 6-kbp EcoRV fragment (CD4H). Tg mice were generated as described elsewhere. 7 Flow cytometryCell suspensions were prepared from lymphoid organs and stained with antibodies, as previously described. 7 Peritoneal macrophages were plated overnight. Between 80% and 95% of them stained positive for Mac-1 ( Figure 2B), Mac3, and F4/80 (data not shown). DCs were isolated from lymph node (LN) 8 and thymus. 9 Results and discussionThe Tg mice harboring 6 different DNAs were generated ( Figure 1). Northern blot analysis revealed that expression was highest in the thymus but was also detectable in the spleen and LNs, and was negative in all other organs tested (liver, heart, kidney, lung, intestine, muscle, brain; data not shown). This result is consistent with previous studies indicating that the human and mouse CD4 promoter/enhancer alone or in combination is sufficient to restrict expression to hematopoietic tissues. 7,10-13 As expected, the level of expression varied among different founders carrying the same construct, an effect most likely related to the site of integration of the inoculated DNA.Expression of hCD4 in DP CD4 ؉ CD8 ؉ T ...

The Nef-Mediated AIDS-Like Disease of CD4C/Human Immunodeficiency Virus Transgenic Mice Is Associated with Increased Fas/FasL Expression on T Cells and T-Cell Death but Is Not Prevented in Fas-, FasL-, Tumor Necrosis Factor Receptor 1-, or Interleukin-1β-Converting Enzyme-Deficient or Bcl2-Expressing Transgenic Mice

“…In addition, the development of the severe lymphadenopathy (mainly caused by the accumulation of DN B220 ϩ T cells), associated with the loss of Fas or FasL (84), was almost completely abrogated in lpr/lpr and gld/gld CD4C/HIV Tg mice. Since expression of HIV-1 could be documented in these DN B220 ϩ T cells, this suggests that these cells may be derived either from thymic CD4 ϩ precursors or from more mature CD4 ϩ T cells, but possibly not from CD8 ϩ T cells in which the CD4C promoter (driving expression of the HIV-1 transgene) is poorly active (26,27). This would be consistent with the observation that their accumulation appears to be thymus dependent, since neonatal thymectomy has been reported to abrogate their accumulation (85).…”

Cardiac Disease in Transgenic Mice Expressing Human Immunodeficiency Virus-1 Nef in Cells of the Immune System

“…31,32,47,48 No evidence of expression could be documented in smooth, skeletal muscle, endothelial, or in various epithelial cells. To further confirm that Tg expression was restricted in the heart to cells of hematopoietic origin, Tg expression was assessed by in situ hybridization using HIV-1-specific riboprobes, 31 both in CD4C/HIV MutA and CD4C/ HIV MutG Tg hearts.…”

Expression of Simian Immunodeficiency Virusnefin Immune Cells of Transgenic Mice Leads to a Severe AIDS-Like Disease

“…The cell type specificity of SIV nef transgene expression determined by the CD4C promoter in Tg mice is likely to contribute to the resemblance of the phenotype to human and simian AIDS. Indeed, other Tg mice expressing only HIV Nef under the regulation of the long terminal repeat (LTR) or of other T-cell-specific promoter/ enhancer elements (11,19,20,48,62) of the macrophage/dendritic lineages of Tg mice (29)(30)(31)(32). The expression of the transgene in mice seems to be following the tissue specificity of this promoter, thus targeting the same cell populations affected by the virus in natural infections.…”