Molecular Recognition of Human CD1b Antigen Complexes: Evidence for a Common Pattern of Interaction with αβ TCRs

Abstract

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the α1 and α2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the α helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both α helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.

Direct Measurement of Antigen Binding Properties of CD1 Proteins Using Fluorescent Lipid Probes

“…Proteins-A substantial number of site-directed mutations in the ␣1 and ␣2 domains of human CD1b have been reported to affect specific lipid antigen presentation to CD1b-restricted T cells (17,32). Thus far, it has not been determined directly whether the effects of these mutations are due to a reduction in binding of lipid antigens by CD1b or to interference with the stable docking of the TCR on the surface of CD1b.…”

“…CD1-transfected HeLa cells and 293 cells have been described previously (17,23). JRT3.T1.5, a T cell receptor (TCR) ␤ chain-deficient mutant of the Jurkat T leukemia line, was also obtained from ATCC.…”

“…The resulting constructs were verified by sequencing and subsequently used to stably transfect HeLa cells or transiently transfect 293T cells using LipofectAMINE. HeLa and 293T transfectants expressing the native two-chain forms of CD1b, CD1c, and CD1d have been described previously (17,23,33). The surface expression of membrane-bound forms of scCD1 or native CD1 was analyzed by FACS as follows.…”

“…Single chain CD1 mutant constructs were generated by replacing the wild type CD1b sequence between the BamHI and NotI sites of the scCD1b/pcDNA3 construct with analogous fragments containing the desired alanine substitution mutations. The mutant CD1b fragments were amplified by PCR from the previously reported CD1b mutant constructs (17,32) by using the following primers: 5Ј-ggatccggttctggaggtggaggttcagaacatgcctcccag and 5Ј-cccgcggccgcccactaatggtgatggtgatggtggcccccaattgagccaatggaggt. The resulting scCD1b mutant constructs were fully sequenced to confirm the specific mutations and were used to produce stable CHO transfectants as described above.…”

“…This mechanism of binding positions the polar head group or hydrophilic cap of the bound lipid at or near the opening of the groove where it can make direct contacts with T cell antigen receptors (17)(18)(19)(20).…”