Direct Transfer of p65 into T Lymphocytes from Systemic Lupus Erythematosus Patients Leads to Increased Levels of Interleukin-2 Promoter Activity

Herndon, Thomas M.; Juang, Yuang-Taung; Solomou, Elena E.; Rothwell, Stephen W.; Gourley, Mark F.; Tsokos, George C.

doi:10.1006/clim.2002.5192

Cited by 43 publications

(33 citation statements)

References 38 publications

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“…Investigators at our laboratory have demonstrated that reconstitution of p65 NF-B restores CD3-mediated IL-2 production in SLE T cells (11), as does the elimination of the transcription repressor CREM using an antisense CREM plasmid (18). In those approaches, though, a suboptimal dose of phytohemagglutinin A was used to condition the cells for gene transfer, and this process may have altered cell physiology.…”

Section: Discussionmentioning

confidence: 99%

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Reconstitution of deficient T cell receptor ζ chain restores T cell signaling and augments T cell receptor/CD3–induced interleukin‐2 production in patients with systemic lupus erythematosus

Nambiar

Fisher

Warke

et al. 2003

Arthritis & Rheumatism

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101

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Objective. T cells from a majority of patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) chain, a subunit of the TCR/CD3 complex. This study was undertaken to explore the possibility that forced expression of TCR chain may reverse the known signaling abnormalities and defective interleukin-2 (IL-2) production in SLE T cells.Methods. Freshly isolated SLE T cells were transfected with TCR chain construct in a eukaryotic expression vector at high efficiency, by a recently developed nucleoporation technique. Restoration of TCR/ CD3-mediated signaling was studied in the chaintransfected cells.Results. In SLE T cells transfected with TCR chain, surface expression of TCR chain was increased and the TCR/CD3-induced increased free intracytoplasmic calcium concentration response was normalized, as was hyperphosphorylation of cellular substrates. Simultaneously, the previously noted increased expression of the Fc receptor ␥ chain was diminished in SLE T cells transfected with the chain expression vector, and the surface membrane clusters of cell signaling molecules were redistributed to a more continuous pattern. TCR chain replacement also augmented the expression of diminished TCR/CD3-mediated IL-2 production in SLE T cells, associated with increased expression of the p65 subunit of nuclear factor B in the nuclear fractions of these T cells. Conclusion. These results suggest that reconstitution of deficient TCR chain can reverse the TCR/ CD3-mediated signaling abnormalities as well as the defective IL-2 production in T cells of patients with SLE.It is well recognized that T cells from patients with systemic lupus erythematosus (SLE) display a number of signaling abnormalities (1). Many of the identified molecular aberrations explain certain established cell and cytokine defects, whereas the mechanisms of other defects have not yet been elucidated. Our group and others have demonstrated that the expression of the subunit of the T cell receptor (TCR) is decreased in a majority of SLE patients (2-4) and that this defect persists over time and is independent of disease activity (5).Despite the decreased expression of the TCR chain in SLE T cells, crosslinking of the TCR/CD3 complex leads to increased free intracytoplasmic calcium concentration ([Ca 2ϩ ] i ) response (6) and protein tyrosine phosphorylation (2,4). These events appear to occur because the Fc receptor (FcR) ␥ chain becomes a functional part of the TCR/CD3 complex (7). In support

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Section: Discussionmentioning

confidence: 99%

“…Our group recently demonstrated that substitution of the defective p65 subunit of NF-B enhanced TCR/CD3-mediated IL-2 production in SLE T cells (11).…”

mentioning

confidence: 99%

Reconstitution of deficient T cell receptor ζ chain restores T cell signaling and augments T cell receptor/CD3–induced interleukin‐2 production in patients with systemic lupus erythematosus

Nambiar

Fisher

Warke

et al. 2003

Arthritis & Rheumatism

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101

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“…We capitalized on a recently developed efficient electroporation-based fresh T cell transfection protocol (22) and a transfection technique by nucleoporation, which enabled increased expression of Fc⑀RI␥ chain encoded in an expression vector. We report that the Fc⑀RI␥ chain containing CD3 complex leads to increased T cell responsiveness.…”

Section: Forced Expression Of the Fc Receptor ␥-Chain Renders Human Tmentioning

confidence: 99%

“…After several modifications of the electroporation method, we established a method to transfect primary T cells routinely with 25-30% efficiency (22). Briefly, the PBMCs were incubated overnight at a density of 3 ϫ 10 6 /ml in RPMI 1640 containing penicillin/streptomycin and 10% fetal bovine serum or autologous serum and 1 g/ml PHA for 18 -20 h. T cells were isolated by negative selection of non-T cells by MACS separation as described above.…”

Section: Transfection Of T Cellsmentioning

confidence: 99%

“…Human T cells were transfected using the electroporation method, and the transfection efficiency was determined by FACS analysis of the expressed GFP (22). Plasmid pIRES containing enhanced GFP was used as a positive control, and a vector containing ␤-galactosidase gene was used as a negative control.…”

Section: Expression Of Fc⑀ri␥ Chain In Transfected T Cellsmentioning

confidence: 99%

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Forced Expression of the Fc Receptor γ-Chain Renders Human T Cells Hyperresponsive to TCR/CD3 Stimulation

Nambiar

Fisher

Kumar

et al. 2003

The Journal of Immunology

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High level expression of FcεRIγ chain replaces the deficient TCR ζ-chain and contributes to altered TCR/CD3-mediated signaling abnormalities in T cells of patients with systemic lupus erythematosus. Increased responsiveness to Ag has been considered to lead to autoimmunity. To test this concept, we studied early signaling events and IL-2 production in fresh cells transfected with a eukaryotic expression vector encoding the FcεRIγ gene. We found that the overexpressed FcεRIγ chain colocalizes with the CD3ε chain on the surface membrane of T cells and that cross-linking of the new TCR/CD3 complex leads to a dramatic increase of intracytoplasmic calcium concentration, protein tyrosine phosphorylation, and IL-2 production. We observed that overexpression of FcεRIγ is associated with increased phosphorylation of Syk kinase, while the endogenous TCR ζ-chain is down-regulated. We propose that altered composition of the CD3 complex leads to increased T cell responsiveness to TCR/CD3 stimulation and sets the biochemical grounds for the development of autoimmunity.

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Identification of microRNA‐31 as a novel regulator contributing to impaired interleukin‐2 production in T cells from patients with systemic lupus erythematosus

Fan¹,

Liang²,

Tang³

et al. 2012

Arthritis & Rheumatism

100

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Objective. MicroRNAs (miRNAs) function to finetune the control of immune cell signaling. It is well established that there are abnormalities in the interleukin-2 (IL-2)-related signaling pathways in systemic lupus erythematosus (SLE). The miR-31 micro-RNA has been found to be markedly underexpressed in patients with SLE, and thus the present study was undertaken to investigate the role of miR-31 in IL-2 defects in lupus T cells.Methods. Expression levels of miR-31 were quantitated using TaqMan miRNA assays. Transfection and stimulation of cultured cells followed by TaqMan quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and reporter gene assays were conducted to determine the biologic function of miR-31. NF-AT nuclear translocation and expression were quantitatively measured using an ImageStream cytometer. Bioinformatics analysis, small interfering RNA (siRNA) knockdown, and Western blotting were performed to validate miR-31 targets and effects.Results. The expression of miR-31 was significantly decreased in lupus T cells, and this was positively correlated with the expression of IL-2. Overexpression of miR-31 in T cells increased the production of IL-2 by altering NF-AT nuclear expression and IL2 promoter activity, while knockdown of endogenous miR-31 reduced IL-2 production. RhoA expression was directly repressed by miR-31 in T cells. Of note, siRNA-mediated knockdown of RhoA enhanced IL2 promoter activity and, consequently, up-regulated IL-2 production. RhoA expression was consistently up-regulated and negatively correlated with the levels of miR-31 in lupus T cells. Manipulation of miR-31 expression in lupus T cells restored the expression of IL-2 at both the messenger RNA and protein levels.Conclusion. MicroRNA-31 is a novel enhancer of IL-2 production during T cell activation. Dysregulation of miR-31 and its target, RhoA, could be a novel molecular mechanism underlying the IL-2 deficiency in patients with SLE.Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disorder characterized by chronic

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Direct Transfer of p65 into T Lymphocytes from Systemic Lupus Erythematosus Patients Leads to Increased Levels of Interleukin-2 Promoter Activity

Cited by 43 publications

References 38 publications

Reconstitution of deficient T cell receptor ζ chain restores T cell signaling and augments T cell receptor/CD3–induced interleukin‐2 production in patients with systemic lupus erythematosus

Reconstitution of deficient T cell receptor ζ chain restores T cell signaling and augments T cell receptor/CD3–induced interleukin‐2 production in patients with systemic lupus erythematosus

Forced Expression of the Fc Receptor γ-Chain Renders Human T Cells Hyperresponsive to TCR/CD3 Stimulation

Identification of microRNA‐31 as a novel regulator contributing to impaired interleukin‐2 production in T cells from patients with systemic lupus erythematosus

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