Antigenic peptide binding to MHC class II molecules at increased peptide concentrations

Localization of soluble major histocompatibility class II-peptide complexes on T cell surface

“…Purification of HLA-DR2 was carried out by immunoaffinity chromatography in the presence of 0.05 % n-dodecyl maltoside (DM) detergent as described recently . Pooled DR2 fractions were complexed with a MBP peptide analog MBP(83-102)Y 83 [Ac-YDENPVVHFFKNIVTPRTPP-NH2] under optimized binding conditions Nag et al, 1994a). Final complex preparation was passed through Sephacryl S-200 gel filtration column to remove the unbound free peptide.…”

“…Purified MHC class II molecules isolated from cell surfaces are known to bind antigenic peptides in vitro (Jardetzky et al, 1990;Nag et al, 1994a;Sette et al, 1992) and MHC class II-peptide complexes can recognize TCRs on cloned T cells (Allen et al, 1987;Babbit et al, 1985;Buus et al, 1987a,b;Nag et al, 1992aNag et al, ,b, 1993Schwartz, 1990;McConnell, 1986, 1987). Typically such TCR engagement by MHC-peptide complexes can be measured by changes in cytokine levels of T cells (Le Gros et al, 1990;Swain et al, 1988;Tanaka et al, 1993).…”

“…Due to fractional occupancy of MHC molecules with exogenous antigenic peptides (Buus et al, 1986(Buus et al, , 1987Luescher et al, 1988;Nag et al, 1994a) and low affinity of soluble MHC-peptide complexes to TCRs (Corr et al, 1994;Jorgensen et al, 1992;Matsui et al, 1991Matsui et al, , 1994Nag et al, 1994b;Sykulev et al, 1994), evidence for direct visualization of bound MHC-peptide complexes on the surface of T cell has not yet been demonstrated.…”

Peptide Binding Inhibits Aggregation of Soluble MHC Class II in Solution

“…Af nity-puri ed MHC class II molecules when incubated with antigenic peptides show a speci c binding ef ciency at the optimized conditions in vitro (13,21). A method developed in our laboratory for the preparation of native HLA-DR2-peptide complexes with maximum peptide occupancy was used for af nity-puri ed HLA-DR2 containing both DRB1*1501 and DRB5*0101 class II molecules and an immunodominant pep- peptide, followed by preparative gel-ltration chromatography of the product to remove excess peptide as well as to purify functionally active complex from its aggregates.…”

“…Prior to reloading LMW and HMW aggregate complex, the percent bound MBP(83-102)Y 83 associated with DR2 was quantitated by acid extraction followed by reverse-phase HPLC with a C 18 (0.21 £ 15 cm) Vydac 218TP5215 narrow-bore column (20,21). Class II complexes (LMW and HMW) were incubated with biotinylated MBP(83-102)Y 83 at various binding conditions such as (a) excess of biotinylated MBP peptide, (b) changing the pH of the binding buffer, (c) increasing temperature, and (d) exposure to reducing agent for 3 days at 37 ± C. The resulting complexes were neutralized and analyzed by the antibody-capture plate assay (13) ± C, with and without added 2 mM DTT).…”