Abstract
CD26 is a widely distributed 110-kDa cell surface glycoprotein with an important role in T-cell costimulation. We demonstrated previously that CD26 binds to caveolin-1 in antigen-presenting cells, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1 in antigen-presenting cells. IRAK-1 is then subsequently phosphorylated to up-regulate CD86 expression, resulting in subsequent T-cell proliferation. However, it is unclear whether caveolin-1 is a costimulatory ligand for CD26 in T-cells. Using soluble caveolin-1-Fc fusion protein, we now show that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-kappaB activation in a T-cell receptor/CD3-dependent manner. We also demonstrated that the cytoplasmic tail of CD26 interacts with CARMA1 in T-cells, resulting in signaling events that lead to NF-kappaB activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IkappaB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule.
Highlights
CD26 is a 110-kDa cell surface glycoprotein with known dipeptidyl peptidase IV (DPPIV,4 EC 3.4.14.5) activity in its extracellular domain (1–3) and is capable of cleaving N-terminal dipeptides with either L-proline or L-alanine at the penultimate position (2)
In our previous study (14), we identified caveolin-1 in antigen-presenting cells (APC) as a binding protein for CD26, and we demonstrated that CD26 on activated memory T-cells directly faces caveolin-1 on tetanus toxoid-loaded monocytes in the contact area, which was revealed as the immunological synapse for T-cell-APC interaction
(1 ϫ 108) that were stimulated for 10 min with anti-CD3 We examined whether the generated N-terminal domain (NT)-Fc fusion protein alone or with anti-CD3 plus N-terminal region of human caveolin-1 (NT-Fc) were lysed with 1 ml of binds to CD26
Summary
Expression and Purification of Fc Proteins—For initial attempts at expression of soluble forms of Fc fusion proteins, human IgG1 Fc cassette vector was made using pCAG-EB6MCS vector (28, 29). The Fc fusion protein containing amino acids 1–10 of human CD26 cytoplasmic tail (CD26 aa1–10) was constructed in identical fashion, using the primers described in the Supplemental Material (pCAG-EB6huECDSP-CD26 aa1–10-Fc␥1). After constructs were confirmed by DNA sequencing, plasmids were transfected to JPM50.6 cells using the Nucleofector II device according to the manufacturer’s instruction. For stimulation experiments using the expression system, CHO-K1 cells were transfected with GFP-fused full-length caveolin-1 or SCD-deleted caveolin-1 expression plasmids, with the constructs being described previously (14), using Lipofectamine2000 reagent (Invitrogen). For IL-2 production assay using Jurkat T-cell lines, JPM50.6, or their transfectants, 5 ϫ 105 cells/well in 200 l of culture media were incubated at 37 °C in the presence of the indicated plate-bound antibodies and/or NT-Fc proteins. Preparation of Lysates or Lipid Raft Fractionation, Immunoprecipitation, and Western Blotting—Stimulated or unstimulated cells were pelleted and lysed with TBSD buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.1% digitonin, Statistics—Student’s t test was used to determine whether the difference between control and sample was significant (p Ͻ 0.05 being significant)
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