Regulation of NF-κB Nuclear Activity in Peripheral Blood Mononuclear Cells: Role of CD28 Antigen

The NF-κB/rel family of transcription factors regulates the expression of a number of genes, including interleukin 2 (IL-2), IL-2 receptor a chain (Tac), and others, controlling T lymphocyte activation. The CD28 antigen is involved in regulation of T cell activation. To investigate whether CD28 antigen regulates NF-κB factors, we analyzed the effect of an anti-CD28 monoclonal antibody (mAb), CLB-CD28/1, on the nuclear activity of NF-κB complexes in resting and CD3-activated peripheral blood mononuclear cells (PBMC) of 11 donors. Cells were incubated with or without the anti-CD3 mAb OKT3 and/or the mAb CLB- CD28/1. Then nuclear extracts were obtained and analyzed for their binding to a 32p-labeled oligonucleotide, corresponding to the NF-κB 5′-CAAC GGCAGGGGAATCTCCCTCTCCTT-3′ consensus sequence in electrophoretic mobility shift assays. PBMC incubated with control medium did not appear to contain significant levels of NF-κB nuclear activity. The anti-CD28 mAb did not induce any detectable NF-κB nuclear activity in PBMC when used alone, except for two cases. However, cells incubated with the anti-CD3 mAb displayed NF-κB nuclear activity in 7 of the 11 cases. The addition of anti-CD28 to the anti-CD3 mAb-stimulated cells enhanced the levels of NF-κB activity in eight PBMC, while it did not modify PBMC in one sample and partially inhibited the induction of NF-κB in the remaining two samples. The stimulatory effect of anti-CD28 mAb on NF-κB nuclear activity was detected also on CD3-stimulated purified T lymphocytes. By analysis with antisera recognizing the p50 and p65 components of the NF-κB/rel family, NF-κB complexes of CD3+CD28-stimulated PBMC were found to contain both p50 and p65 proteins. An enhanced production of IL-2 was detected in cultures of CD3+CD28-stimulated PBMC. Our results indicate that CD28 triggering can modulate the activity on NF-κB nuclear complexes in T lymphocytes stimulated via CD3. Such an effect appeared not to require the presence of accessory cells (AC) or AC-derived cytokines.