Cloning, expression, and functional characterization of TL1A-Ig.

TNF superfamily member 15 (TL1A) is the ligand for TNFR superfamily (TNFRSF)25. We previously reported that TNFRSF25 stimulation with an agonist Ab, 4C12, expands pre-existing CD4(+)Foxp3(+) regulatory T cells (Tregs) in vivo. To determine how the physiological ligand differs from the Ab, we generated a soluble mouse TL1A-Ig fusion protein that forms a dimer of TL1A trimers in solution with an apparent molecular mass of 516 kDa. In vitro, TL1A-Ig mediated rapid proliferation of Foxp3(+) Tregs and a population of CD4(+)Foxp3(-) conventional T cells. TL1A-Ig also blocked de novo biogenesis of inducible Tregs and it attenuated the suppressive function of Tregs. TNFRSF25 stimulation by TL1A-Ig in vivo induced expansion of Tregs such that they increased to 30-35% of all CD4(+) T cells in the peripheral blood within 5 d of treatment. Treg proliferation in vivo was dependent on TCR engagement with MHC class II. Elevated Treg levels can be maintained for at least 20 d with daily injections of TL1A-Ig. TL1A-Ig-expanded Tregs expressed high levels of activation/memory markers KLRG1 and CD103 and were highly suppressive ex vivo. TL1A-Ig-mediated Treg expansion in vivo was protective against allergic lung inflammation, a mouse model for asthma, by reversing the ratio of conventional T cells to Tregs in the lung and blocking eosinophil exudation into the bronchoalveolar fluid. Thus, TL1A-Ig fusion proteins are highly active and tightly controllable agents to stimulate Treg proliferation in vivo, and they are uniquely able to maintain high levels of expanded Tregs by repeated administration.

Purification and crystallization of recombinant human TNF-like ligand TL1A.

TL1A is a recently discovered TNF-like ligand. Because of the interests in the structural basis of the specificity of the bindings of the TNF ligands to the TNF receptors, we sought to crystallize the mature soluble form of human TL1A. To prepare recombinant human TL1A, the coding sequence for mature human soluble TL1A (aa72-aa251) was cloned into Escherichia coli expression vector pDEST14 and the protein was purified in a succession of immobilized metal affinity, hydrophobic interaction, ion exchange and size exclusion chromatography, indicating that mature TL1A may have a metal ligand. The functional activity of recombinant TL1A was confirmed by its ability to bind to DcR3, a soluble decoy receptor of the TNF receptor family that has been previously reported to bind to TL1A. Single crystals of TL1A were obtained in a screen with a crystal screen kit using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. TL1A crystals belong to the tetragonal space group P4(1)2(1)2, with unit cell parameter of a=b=116.734, c=118.927A. The TL1A crystals diffracted to at least 3.2A. Self-rotation functions showed that there are three molecules in the asymmetry unit. Assuming an average partial specific volume of 0.74cm(3)g(-1) for proteins, the water content of the crystal is 62.8%. A preliminary molecular replacement solution was obtained with three TL1A molecules in the asymmetric unit. The three protomers are related by a non-crystallographic 3-fold axis, like those of other TNF ligand family members.