Mucosa-Specific Targets for Regulation of IFN-γ Expression: Lamina Propria T Cells Use Differentcis-Elements than Peripheral Blood T Cells to Regulate Transactivation of IFN-γ Expression

Abstract

Activation of lamina propria (LP) T cells via the CD2 pathway enhances IFN-γ (IFN-γ) secretion with further enhancement after CD28 coligation. The molecular mechanisms regulating IFN-γ expression in LP T cells remain unknown. Previous studies in PBL and T cell lines identified cis- and trans-regulatory elements in TCR-mediated expression of IFN-γ. This study examines CD2 and PMA/ionophore-responsive IFN-γ promoter elements. Activation of LPMC via CD2-induced IFN-γ secretion and a parallel up-regulation of mRNA expression. CD28 coligation enhanced mRNA stability without up-regulating transcription as measured by nuclear run-on. Transfection of a −2.7-kb IFN-γ promoter-reporter construct into PBL and LP mononuclear cells (LPMC) revealed significant promoter activity after CD2 activation, with additional transactivation after CD2/CD28 costimulation in PBL, but not in LPMC. Functional analysis using truncated promoter fragments identified distinct cis-regulatory regions selectively transactivating IFN-γ expression in PBL compared with LPMC. In PBL, CD2 activation elements reside within the −108- to +64-bp region. However, in LPMC the upstream region between −204 and −108 bp was essential. Transfection of the proximal and distal AP-1-binding elements, as well as TRE/AP-1 constructs, revealed functional activation of AP-1 subsequent to CD2 signaling, with activation critical in PBL but diminished in LPMC. Electromobility shift analysis using oligonucleotides encompassing the proximal, distal, and BED/AP-1-binding regions failed to demonstrate selective transactivation after CD2 signaling of LPMC. This report provides evidence that activation of LPMC results in transactivation of multiple promoter elements regulating IFN-γ expression distinct from those in PBL.

An IFNG SNP with an estrogen-like response element selectively enhances promoter expression in peripheral but not lamina propria T cells

“…Mucosa-specific IFNG response elements have previously been identified within the À204 and À108 bp region. 14 In the present study, we show that an SNP (À179G/T) recently detected within this region 17 exhibits selective allele-specific enhanced expression compared to the common allele in PBMC, but not in LPMC. In PBMC transfected with reporter constructs containing the polymorphic À179T, there is a CD2-mediated doubling of promoter activity compared to the common À179G allele.…”

“…[6][7][8] We have previously demonstrated that there are mucosa-specific mechanisms for T-cell cytokine gene regulation of IFN-g expression, which differ from those seen in PBL. 11,[13][14][15]22 Studies by other groups have indicated that regulation of IFN-g gene expression in primary T cells differs from that observed in tumor T-cell lines, and likewise, differs in naïve compared to memory T-cell subsets. [23][24][25] Differential cytokine gene regulation may be controlled, in part, Novel ERE-like cis-element in IFNG promoter R Gonsky et al by DNA methylation patterns.…”

“…[13][14][15]22 Our studies defined mucosal-specific functional divergence within the enhancer and repressor regions of the IFNG promoter. The data presented here expand on these findings.…”

“…14 In contrast, in lamina propria mononuclear cells (LPMC), a significant CD2 response element resides in the À204 and À108 bp region, a region previously reported in PBL and T-cell lines to possess an essential AP-1 binding site, but CD2-mediated activation of IFNG expression is not altered by deletion of this AP-1 site. 14 More recently, we have shown that CD2 stimulation activates the Janus protein tyrosine kinase/ signal transducers and activators of transcription (STAT) pathway in PBMC differently than in LPMC. In LPMC, CD2 signaling results in enhanced phosphotyrosine STAT1 and STAT4 and phosphoserine STAT1.…”

“…[13][14][15] In PBL, CD2 activation elements have been mapped mainly to the À108 to þ 64 bp region, encompassing the proximal and distal activator protein 1 (AP-1) binding sites. 14 In contrast, in lamina propria mononuclear cells (LPMC), a significant CD2 response element resides in the À204 and À108 bp region, a region previously reported in PBL and T-cell lines to possess an essential AP-1 binding site, but CD2-mediated activation of IFNG expression is not altered by deletion of this AP-1 site. 14 More recently, we have shown that CD2 stimulation activates the Janus protein tyrosine kinase/ signal transducers and activators of transcription (STAT) pathway in PBMC differently than in LPMC.…”