Triggering of T Cell Activation via CD4 Dimers

Moldovan, Maria-Cristina; Sabbagh, Laurent; Breton, Gaëlle; Sékaly, Rafick‐Pierre; Krummel, Matthew F.

doi:10.4049/jimmunol.176.9.5438

Cited by 28 publications

(24 citation statements)

References 56 publications

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“…Silencing of CD4 resulted in the expected impairment of antigen-dependent T-cell activation [13][14][15]. siRNA-mediated knockdown of the Th2 transcription factor GATA-3 abolished the expression of its target genes, the prototypic Th2 cytokines IL-4 and IL-5, as reported for GATA-3 knockout mice [17,18].…”

Section: Discussionmentioning

confidence: 88%

“…Since the CD4 molecule is an important co-stimulator [13][14][15], we analyzed the effect of CD4 knockdown in primary T cells upon antigen-dependent activation in vitro and in vivo. TCR-transgenic (OVA-Tg) CD4 1 T cells were labeled with CFSE and transfected with 1000 pmol of stabilized or conventional CD4 siRNA, stabilized control siRNA (siGFP) or nucleofected but without siRNA.…”

Section: Sirna-transfected T Cells Remain Functional and Maintain Knomentioning

confidence: 99%

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siRNA stabilization prolongs gene knockdown in primary T lymphocytes

et al. 2008

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RNA interference (RNAi)-mediated knockdown of target gene expression represents a powerful approach for functional genomics and therapeutic applications. However, for T lymphocytes, central regulators of immunity and immunopathologies, the application of RNAi has been limited due to the lack of efficient small interfering RNA (siRNA) delivery protocols, and an inherent inefficiency of the RNAi machinery itself. Here, we use nucleofection, an optimized electroporation approach, to deliver siRNA into primary T lymphocytes with high efficiency and negligible impairment of cell function. We identify siRNA stability within the cells as the critical parameter for efficient RNAi in primary T cells. While generally short-lived and immediately lost upon T-cell activation when conventional siRNA is used, target gene knockdown becomes insensitive to cell activation and can persist for up to 2 wk in non-dividing cells with siRNA stabilized by chemical modifications. Targeting CD4 and the transcription factor GATA-3, we show that the use of stabilized siRNA is imperative for functional gene analysis during T lymphocyte activation and differentiation in vitro as well as in vivo.Key words: Gene expression . Immune regulation . T cells Introduction RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing [1][2][3]. Given its ease of application, its high efficiency and remarkable specificity, RNAi holds great promise for broad in vitro and in vivo application in all biomedical areas. Most importantly, RNAi is directly applicable to essentially all somatic cell types including human primary cells, which makes it an invaluable tool to study human gene function and may enable new therapeutic approaches [4,5].Within the immune system T lymphocytes are one major target for siRNA-based gene silencing, since they are key regulators of immune responses and involved in many immune related disorders, like autoimmunity, chronic inflammation or lymphoma.Despite this high potential, the lack of protocols for efficient and sustained siRNA delivery into primary mammalian cells is currently the major obstacle to the use of RNAi. In particular, primary lymphocytes are highly resistant to non-viral transfection using cationic lipids and polymer reagents [6][7][8]. Although lymphocytes can be transfected by electroporation in vitro, this method so far has been rather inefficient, limited to activated cells and was complicated by a severe impairment of cell function and cell viability [9,10]. Recently, a promising approach for the 2616in vivo targeting of lymphocytes has been reported using antibody-protamine fusion proteins to deliver siRNA [11]. However, recent data from small-hairpin RNA transgenic mice indicate that in T lymphocytes the RNAi machinery itself works inefficiently as compared with other cell types [12]. In fact, due to the aforementioned problems with siRNA delivery, the parameters determining RNAi efficien...

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Section: Discussionmentioning

confidence: 88%

Section: Sirna-transfected T Cells Remain Functional and Maintain Knomentioning

confidence: 99%

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

et al. 2008

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“…In human CD4, K 318 and Q 344 were identified as important residues for a non-covalent dimer association between D4 of adjacent CD4 molecules [38], and it has been speculated that this aligning of two CD4 monomers gives the opportunity for efficient forming of a covalently disulfide linked CD4 dimers [37]. has been demonstrated that CD4 dimerization is required for CD4-mediated T-cell activation [38,39], and it has been indicated that the preferred MHC class II co-receptor is disulfide linked dimeric CD4 [37]. Also, the connecting peptide region of the two-domain CD4-like molecules can be important for MHC binding, as a high percentage of prolines (about 21% in halibut CD4-2) may allow an extended structure so that the D1 of CD4-2 can reach the MHC molecule.…”

Section: Discussionmentioning

confidence: 99%

Cloning, characterization, and expression pattern of Atlantic halibut (Hippoglossus hippoglossus L.) CD4-2, Lck, and ZAP-70

Øvergård

Nerland

Patel

2010

Fish & Shellfish Immunology

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Havforskningsinstituttets institusjonelle arkiv Brage IMR - Institutional repository of the Institute of Marine Research b r a g e i m rDette er forfatters siste versjon av den fagfellevurderte artikkelen, vanligvis omtalt som postprint. I Brage IMR er denne artikkelen ikke publisert med forlagets layout fordi forlaget ikke tillater dette. Du finner lenke til forlagets versjon i Brage-posten. Det anbefales at referanser til artikkelen hentes fra forlagets side. Ved lenking til artikkelen skal det lenkes til post i Brage IMR, ikke direkte til pdf-fil.This is the author's last version of the article after peer review and is not the publisher's version, usually referred to as postprint. You will find a link to the publisher's version in Brage IMR. It is recommended that you obtain the references from the publisher's site.Linking to the article should be to the Brage-record, not directly to the pdf-file.

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“…The HIV-1 JRFL Env-eYFP expressor was generated by inserting the codonoptimized sequence of HIV-1 JRFL Env (residues 1 to 711) at the N terminus of enhanced yellow fluorescent protein (eYFP) (from BD Clontech). The CD4-eCFP fusion protein was previously described (38). Transmitted/founder (T/F) envelope expressors from clades C (C1086) (39)(40)(41) and D (190049) were previously described (42), HIV-2 7312A (43, 44) and SIV (45) envelope expressors were previously described.…”

Section: Methodsmentioning

confidence: 99%

Interaction with Cellular CD4 Exposes HIV-1 Envelope Epitopes Targeted by Antibody-Dependent Cell-Mediated Cytotoxicity

Veillette

Désormeaux

Medjahed

et al. 2014

J Virol

244

600

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Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cellmediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins. IMPORTANCEHIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.

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Triggering of T Cell Activation via CD4 Dimers

Cited by 28 publications

References 56 publications

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

Cloning, characterization, and expression pattern of Atlantic halibut (Hippoglossus hippoglossus L.) CD4-2, Lck, and ZAP-70

Interaction with Cellular CD4 Exposes HIV-1 Envelope Epitopes Targeted by Antibody-Dependent Cell-Mediated Cytotoxicity

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