Biochemical and functional characterization of soluble multivalent MHC L(d)/Fc gamma 1 and L(d)/Fc mu chimeric proteins loaded with specific peptides.
Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)-peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen-specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H-2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes mu or gamma1. Stable transfectants of these L(d)/Fc gamma1 and L(d)/Fc mu genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/ Fc gamma1 and L(d)/Fc mu monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fc gamma1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fc gamma1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.
Lepley
D M
DM
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-3135, USA.
Gillanders
W E
WE
Myers
N B
NB
Robinson
R A
RA
Beisel
K W
KW
Wisecarver
J L
JL
Pirruccello
S J
SJ
Lee
D R
DR
Hansen
T H
TH
Rubocki
R J
RJ
D000818:Animals; D000939:Epitopes / Q000737:chemistry; D006031:Glycosylation; D006649:Histocompatibility Antigens / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D010449:Peptide Mapping; D011487:Protein Conformation; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry; D011961:Receptors, Fc / Q000737:chemistry* / Q000235:genetics; D017452:Receptors, IgG / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D000939:Epitopes; D006649:Histocompatibility Antigens; D012333:RNA, Messenger; D011961:Receptors, Fc; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; C035311:immunoglobulin M receptor