Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Apr 20;101(16):6098-103.
doi: 10.1073/pnas.0308688101. Epub 2004 Apr 12.

CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappaB subunits on IL-8 and Bcl-xL gene promoters

Barbara Marinari  1 , Antonio Costanzo, Valeria Marzano, Enza Piccolella, Loretta Tuosto
Affiliations

CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappaB subunits on IL-8 and Bcl-xL gene promoters

Barbara Marinari et al. Proc Natl Acad Sci U S A. .

Abstract

CD28 is one of the most important costimulatory receptors necessary for full T lymphocyte activation. The CD28 receptor can enhance T cell antigen receptor (TCR) signals, as well as deliver independent signals. Indeed, CD28 engagement by B7 can generate TCR-independent signals leading to IkappaB kinase and NF-kappaB activation. Here we demonstrate that the TCR-independent CD28 signal leads to the selective transcription of survival (Bcl-xL) and inflammatory (IL-8 and B cell activation factor, but not proliferative (IL-2), genes, in a NF-kappaB-dependent manner. CD28-stimulated T cells actively secrete IL-8, and Bcl-xL up-regulation protects T cells from radiation-induced apoptosis. The transcription of CD28-induced genes is mediated by the specific recruitment of RelA and p52 NF-kappaB subunits to target promoters. In contrast, p50 and c-Rel, which preferentially bind NF-kappaB sites on the IL-2 gene promoter after anti-CD3 stimulation, are not involved. Thus, we identify CD28 as a key regulator of genes important for both survival and inflammation.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
CD28 engagement by B7 induces IL-8, Bcl-xL, and BAFF mRNA expression in primary human T cells. (A) RT-PCR analysis of IL-8-, Bcl-xL-, BAFF-, and CCR7-specific mRNA expressions in human primary T cells stimulated for different times with adherent Dap3/B7 cells. (B) Peripheral and cord blood CD4+ T cells were stimulated for 8 h with adherent Dap3/B7 cells (lanes 1–4). Cord blood CD4+ T cells were also stimulated with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) plus ionomycin (0.5 μg/ml) as positive control (P/I, lane 5). IL-8, Bcl-xL, and BAFF mRNA expressions were analyzed by RT-PCR. Data represent three independent experiments.
Fig. 2.
Fig. 2.
Inhibitors of the NF-κB pathway interfere with CD28-induced gene expression. Human T cells were pretreated for 2 h with 10 mM aspirin, 25 μM l-1-tosylamido-2-phenylethyl chloromethyl (TPCK), 10 μM N-acetyl-leucylleucyl-norleucinal (ALLnL), or 30 μM PGA1 and then stimulated for 6 h with adherent Dap3/B7 cells. IL-8, Bcl-xL, and BAFF mRNA expressions were analyzed by RT-PCR. Data represent three independent experiments.
Fig. 3.
Fig. 3.
CD28 induces the early degradation of IκBα without affecting p100 processing. Cytoplasmic extracts from human primary T cells stimulated with adherent Dap3/B7 cells were analyzed by Western blotting with anti-IκBα- and anti-p52 antibodies. (A Middle) An early and partial degradation of IκBα was observed after CD28 engagement. (A and B Top) In contrast, no detectable p100 processing was found at any time. The blots were reprobed with anti-RelA antibodies to verify equal loading of proteins. Data represent three independent experiments.
Fig. 4.
Fig. 4.
CD28 ligation induces nuclear translocation of RelA and p52. Nuclear fractions from human T cells stimulated with adherent Dap3/B7 cells for different times were analyzed by Western blotting for the presence of RelA, p52, p50, RelB, and c-Rel. Expression of IKKα was analyzed to verify the purity of the nuclear extracts. Data represent three independent experiments.
Fig. 5.
Fig. 5.
CD28 engagement by B7 induces the nuclear translocation of preexisting RelA/p52 heterodimers. (A) Cytoplasmic extracts from human primary T cells were subjected to immunoprecipitation (IP) with unrelated antibody (C) or anti-p50- or anti-p52-specific antibodies. Immunoprecipitations and total lysates (TL) were analyzed for the presence of RelA, p52, and p50 by Western blotting. (B) Cytoplasmic extracts from human T cells, stimulated for 30 and 60 min with adherent Dap3/B7 cells, were immunoprecipitated with anti-p52 antibody. Anti-IκBα, -RelA, and -p52 Western blotting was performed. (C) Human T cells were stimulated for 30 min with adherent Dap3/B7 cells, and both RelA and RelB Western blotting was performed on nuclear anti-p50 and anti-p52 immunoprecipitates. Each immunoprecipitate was analyzed for p50 and p52 content. Data represent three independent experiments.
Fig. 6.
Fig. 6.
CD28 stimulation of human T cells induces the selective recruitment of RelA and p52 to the IL-8 and Bcl-xL promoters. Human primary T cells were stimulated with adherent Dap3/B7 cells for different times. ChIP assays were performed by using anti-RelA, -p52, -RelB, -p50 and -RNA polymerase II (pol II) antibodies or no antibody (NA) as control. (A and B) Immunoprecipitated DNA was analyzed by PCR with IL-8 (A) and Bcl-xL (B) promoter-specific primers. (C) RelA and p52 recruitment to IL-2 promoter in human T cells stimulated with Dap3/B7 for different times. (Right) The kinetics of the induction of IL-2 mRNA (evaluated by RT-PCR) is shown. (D) RelA, p50, c-Rel, or p52 recruitment to the IL-2 promoter in human T cells stimulated with anti-CD3 (UCHT1) antibody. (Right) The kinetic of induction of IL-2, IL-8, and Bcl-xL mRNAs (evaluated by RT-PCR) is shown. Data represent three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lanzavecchia, A., Iezzi, G. & Viola, A. (1999) Cell 96, 1-4. - PubMed
    1. Wulfing, C. & Davis, M. M. (1998) Science 282, 2266-2269. - PubMed
    1. Michel, F., Attal-Bonnefoy, G., Mangino, G., Mise-Omata, S. & Acuto, O. (2001) Immunity 15, 935-945. - PubMed
    1. Tuosto, L. & Acuto, O. (1998) Eur. J. Immunol. 28, 2131-2142. - PubMed
    1. Kaga, S., Ragg, S., Rogers, K. A. & Ochi, A. (1998) J. Immunol. 160, 24-27. - PubMed

Publication types