CD40 Induces Antigen Transporter and Immunoproteasome Gene Expression in Carcinomas via the Coordinated Action of NF-κB and of NF-κB-Mediated De Novo Synthesis of IRF-1

Abstract

Cancer cells may evade immune surveillance as a result of defective antigen processing and presentation. In this study, we demonstrate that CD40 ligation overcomes this defect through the coordinated action of the transcription factors NF-B and interferon regulatory factor 1 (IRF-1). We show that unlike interferon signaling, which triggers the STAT1-mediated transcriptional activation of IRF-1, the ligation of CD40 in carcinomas induces the rapid upregulation of IRF-1 in a STAT1-independent but NF-B-dependent manner. The transcriptional activation of IRF-1 is controlled largely by the recruitment of p65 (RelA) NF-B to the IRF-1 promoter following the engagement of a TAK1/IB kinase ␤/IB␣ signaling pathway downstream of CD40. NF-B and de novo-synthesized IRF-1 converge to regulate the expression of genes involved in antigen processing and transport, as evident from the sequential recruitment of NF-B and IRF-1 to the promoters of the genes encoding transporter for antigen processing 1 (TAP1), TAP2, tapasin, and low-molecular-mass polypeptides LMP2 and LMP10. Moreover, the RNA interference-mediated knockdown of IRF-1 reduced, whereas the inhibition of NF-B abolished, the effects of CD40 on TAP1 and LMP2 upregulation in carcinoma cells. Collectively, these data reveal a novel "feed-forward" mechanism induced by NF-B which ensures that acutely synthesized IRF-1 operates in concert with NF-B to amplify the immunoproteasome and antigenprocessing functions of CD40.Among the nine known members of the interferon regulatory factor (IRF) family of transcription factors, IRF-1 has attracted significant attention as a master regulator of genes involved in the development of innate and adaptive immunity (reviewed in reference 59). Indeed, studies of mice with a null mutation in the irf-1 alleles (irf-1 Ϫ/Ϫ ) have revealed that IRF-1 plays a crucial role in interferon (IFN)-induced antiviral and antibacterial responses. When challenged with pathogens, irf-1 Ϫ/Ϫ mice display compromised Th1 cell differentiation associated with defects in interleukin-12 p35 subunit (IL-12p35) and inducible nitric oxide synthase (iNOS) gene expression in macrophages and concomitant hyporesponsiveness of CD4 ϩ T lymphocytes and natural killer cells to 37). In line with this observation, the promoters of the IL-12p35 and iNOS genes possess functional IRF-1 binding motifs (42). IRF-1 also plays an important role in the transcriptional control of the transporter for antigen processing TAP1 and the immunoproteasome component LMP2 in response to IFN-␥ (41, 68), thereby influencing the presentation of viral and tumor antigens to CD8 ϩ T cells. Moreover, IRF-1 participates in an autoregulatory loop in the context of type I IFN signaling in which IRF-1 is both a target and a transcriptional activator of 20,72).In addition to its role in regulating the immune response to pathogens, IRF-1 has been proposed to function as a tumor suppressor (reviewed in reference 59) and to influence p53 activity (10). Thus, the loss of IRF-1 dramatically exacerbates susceptib...

CD40 Stimulates a “Feed-Forward” NF-κB–Driven Molecular Pathway That Regulates IFN-β Expression in Carcinoma Cells

“…HEK 293 cells (1.5 3 10 5 cells/well in a 12-well plate) were transfected with 50 ng pIFN-b/Luc and 50 ng of a Renilla plasmid and plasmids expressing CD40WT, CD40mT2/T3, CD40 mT2/T3/T6, or HA-p52 by using Lipofectamine (Invitrogen) in Opti-MEM medium (Invitrogen), according to the manufacturer's instructions. Following a 10-h incubation with the transfection mixture, the medium was changed to DMEM supplemented with 10% FCS, and Luciferase and Renilla values were measured 24 h later as described previously (15).…”

“…The p52 Ab was purchased from Cell Signaling Technology, the IRF7 from Acris and the actin Ab from Sigma-Aldrich. Immunoblotting was performed as described previously (15).…”

“…The antiviral activity of CD40L has also been documented in the mouse (11), and increasing experimental and clinical evidence suggests that activation of the CD40 pathway could be exploited for cancer therapy (12,13). Indeed, the stimulation of tumor cells with CD40L induces the expression of components of the Ag presentation machinery required for the engagement of antitumor immune responses (14) through the sequential activation of NF-kB and its target, IRF1 (15). Interestingly, IRF1 participates in an autoregulatory loop in the context of type I IFN signaling in which IRF1 is both a target and a transcriptional activator of IFN-b (16,17).…”

Viral IL-10 down-regulates the “MHC-I antigen processing operon” through the NF-κB signaling pathway in nasopharyngeal carcinoma cells

“…Therefore, they concluded that LMP-1 could increase the antigen presentation of both TAP-1 and TAP-2 through NFjB. Moschonas et al (2003) discovered that the CD40 ligand could induce antigen presentation and related gene expression (TAP-1, TAP-2, LMP-2, LMP-10 and tapasin). These gene promoters all contained NF-jB binding regions.…”

“…In vitro, vIL-10 can down-regulate the expression of TAP-1, TAP-2, LMP-2, LMP-7 and HLA-I in CNE-2 cells (data is not shown). The expression of TAP-1, TAP-2, LMP-2, LMP-7 and HLA-I involves the activation of NF-jB and its recruitment to the promoter elements proximal to the gene transcription start sites (Moschonas et al 2003;Molinero et al 2004).…”