OVA-specific memory CD4 T cells were generated as described ( 10, 11) by in vitro priming of DO11.10 CD4 T cells with 1.0 μg/ml OVA peptide and APC and adoptive transfer of the resultant primed/effector cells into RAG2 −/− adoptive hosts, with persisting memory CD4 T cells recovered 2–5 mo posttransfer. OVA-specific naive CD4 T cells were isolated from the spleens of DO11.10 or DO11.10×RAG2 −/− mice as described ( 10). ![]() Generation and isolation of naive and memory CD4 T cells Louis, MO), and cell-permeable p50 inhibitor and inactive control peptides were purchased from CalBiochem/EMD Biosciences (San Diego, CA). ![]() HPLC-purified OVA peptide (323–339 IASQAVHAAHAEINEAGA) was synthesized by the Biopolymer Laboratory at the University of Maryland. For Western blotting, rabbit anti- p105/50 was purchased from Abcam, anti-actin (sc47778) from Santa Cruz Biotechnology, and mouse anti–T-bet (4B10) was purchased from BD Pharmingen. ChIP-grade Abs specific for NF-κB (p50) and T-bet were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-histone H3 and anti-p50/105 Abs were from Abcam (Cambridge, MA). Materials and Methodsįluorescently conjugated anti–IFN-γ, anti–IL-2, anti-CD25, anti-CD44, anti-CD62L, and anti-CD4 Abs were purchased from BD Pharmingen (San Diego, CA) anti–T-bet was from eBioscience (San Diego, CA), and KJ1-26 specific for DO11.10 TCR was from Caltag Laboratories (Burlingame, CA). The identification of a transcriptional regulator for memory immune responses is important for memory modulation in vaccines, autoimmunity, and transplantation. Our results indicate differential control of IFN-γ transcription in naive and memory CD4 T cells and a molecular mechanism for rapid memory recall responses through NF-κB p50 activity and promoter engagement. Moreover, inhibition of NF-κB activity by ammonium pyrrolidine dithiocarbamate (PDTC a pharmacological inhibitor) or by a cell-permeable peptide inhibitor of NF-κB p50 translocation abrogated early recall responses by memory CD4 T cells. Chromatin immunoprecipitation (ChIP) analyses further revealed that NF-κB but not T-bet was engaged on the IFN-γ promoter in memory CD4 T cells at early times after Ag stimulation. We demonstrate that rapid IFN-γ production by Ag-specific memory CD4 T cells is regulated on the transcriptional level and occurred from a population that did not upregulate T-bet expression but was associated with NF-κB p50 nuclear translocation and increased nuclear p50 expression. We focused on the role of T-bet and NF-κB in memory recall because of their known role in regulating IFN-γ production during primary responses. ![]() In this study, we hypothesized that rapid recall production of the effector cytokine IFN-γ by memory CD4 T cells was regulated by the enhanced activity of transcription factors. Our results reveal a molecular mechanism for memory T cell recall through enhanced NF-κB p50 activation and promoter engagement, with important implications for memory T cell modulation in vaccines, autoimmunity, and transplantation. Moreover, pharmacologic inhibition of NF-κB activity or peptide-mediated inhibition of NF-κB p50 translocation abrogated early memory T cell signaling and TCR-mediated effector function. We identified rapid induction of NF-κB transcriptional activity and increased engagement of NF-κB on the IFN-γ promoter at rapid times after TCR stimulation of memory compared with naive CD4 T cells. ![]() By contrast, immediate IFN-γ production by Ag-stimulated memory CD4 T cells occurred in the absence of significant nuclear T-bet expression or T-bet engagement on the IFN-γ promoter. In naive CD4 T cells, IFN-γ production only occurred after sustained Ag activation and was associated with high expression of the T-bet transcription factor required for Th1 differentiation and with T-bet binding to the IFN-γ promoter as assessed by chromatin immunoprecipitation analysis. In this study, we investigated transcriptional mechanisms for rapid IFN-γ production by Ag-specific memory CD4 T cells. Memory T cells are distinguished from naive T cells by their rapid production of effector cytokines, although mechanisms for this recall response remain undefined.
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