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Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity

Abstract : The interaction between 4-1BB ligand (CD137L), a member of the tumor necrosis factor superfamily, and its receptor 4-1BB provides a co-stimulatory signal for T lymphocyte proliferation and survival. However, the structure of 4-1BBL has not been thoroughly investigated, and none of the human recombinant 4-1BBL molecules available have been described as capable of co-stimulating T cells. The present work provides a model of the three-dimensional structure of the tumor necrosis factor homology domain of 4-1BBL and describes the production of a recombinant human soluble 4-1BBL whose originality lies in that it contains the whole extracel-lular tail preceding the tumor necrosis factor homology domain and an AviTag peptide (AviTag-4-1BBL) allowing enzymatic biotinyla-tion and multimerization via streptavidin. We provide evidence that this chimeric protein exists as a homotrimer, whereas commercial FLAG-tagged 4-1BBL does not. This resulted in a much higher affinity for 4-1BB (1.2 nM) as compared with FLAG-4-1BBL (55.2 nM). We demonstrate that the single extracellular cysteine residue in the tail (Cys-51) could form a disulfide bond, both in our recom-binant protein and in physiologically expressed 4-1BBL. The mutation of this cysteine residue exerted no effect on trimerization but increased the dissociation rate of AviTag-4-1BBL from 4-1BB. In its soluble form, AviTag-4-1BBL did not stimulate purified T cells but dramatically inhibited proliferation of peripheral blood mononu-clear cells stimulated with anti-CD3 mAb. In contrast, a very significant co-stimulatory effect was observed on purified T cells once AviTag-4-1BBL was immobilized onto streptavidin beads. In addition , we show that the cross-linking of two trimeric AviTag-4-1BBL molecules was the minimum step required to elicit significant costimulatory activity.
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Contributor : Agnès Quemener <>
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Catherine Rabu, Agnès Quéméner, Yannick Jacques, Klara Echasserieau, Patricia Vusio, et al.. Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2005, 280, pp.41472 - 41481. ⟨10.1074/jbc.M506881200⟩. ⟨inserm-02442588⟩

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