Biomedical subjects

Susan E Brophy

Publications and source records attributed to Susan E Brophy.

3 recordsLinked to original sources

PubMed2006

Cellular uptake followed by class I MHC presentation of some exogenous peptides contributes to T cell stimulatory capacity.

The T cell stimulatory activity of peptides is known to be associated with the cell surface stability and lifetime of the peptide-MHC (pepMHC) complex. In this report, soluble high-affinity T cell receptors (TCRs) that are specific for pepMHC complexes recognized by the mouse CD8+ clone 2C were used to monitor the cell surface lifetimes of synthetic agonist peptides. In the 2C system, L(d)-binding peptide p2Ca (LSPFPFDL) has up to 10,000-fold lower activity than peptide QL9 (QLSPFPFDL) even though the 2C TCR binds to p2Ca-L(d) and QL9-L(d) complexes with similar affinities. Unexpectedly, p2Ca-L(d) complexes were found to have a longer cell surface lifetime than QL9-L(d) complexes. However, the strong agonist activity of QL9 correlated with its ability to participate in efficient intracellular delivery followed by cell surface expression of the peptide, resulting in high and persistent surface levels of QL9-L(d). The ability of target cells to take up and present QL9 was observed with TAP-deficient cells and TAP-positive cells, including dendritic cells. The process was brefeldin A-sensitive, indicating a requirement for transport of the pepMHC through the ER and/or golgi. Thus, strong T cell stimulatory activity of some pepMHC complexes can be accomplished not only through long cell surface lifetimes of the ligand, but through a mechanism that leads to delayed presentation of the exogenous antigen after intracellular uptake.

Amino Acid Sequence↗

PubMed2006

Engineering and characterization of a stabilized alpha1/alpha2 module of the class I major histocompatibility complex product Ld.

The major histocompatibility complex (MHC) is the most polymorphic locus known, with thousands of allelic variants. There is considerable interest in understanding the diversity of structures and peptide-binding features represented by this class of proteins. Although many MHC proteins have been crystallized, others have not been amenable to structural or biochemical studies due to problems with expression or stability. In the present study, yeast display was used to engineer stabilizing mutations into the class I MHC molecule, Ld. The approach was based on previous studies that showed surface levels of yeast-displayed fusion proteins are directly correlated with protein stability. To engineer a more stable Ld, we selected Ld mutants with increased surface expression from randomly mutated yeast display libraries using anti-Ld antibodies or high affinity, soluble T-cell receptors (TCRs). The most stable Ld mutant, Ld-m31, consisted of a single-chain MHC module containing only the alpha1 and alpha2 domains. The enhanced stability was in part due to a single mutation (Trp-97 --> Arg), shown previously to be present in the allele Lq. Mutant Ld-m31 could bind to Ld peptides, and the specific peptide.Ld-m31 complex (QL9.Ld-m31) was recognized by alloreactive TCR 2C. A soluble form of the Ld-m31 protein was expressed in Escherichia coli and refolded from inclusion bodies at high yields. Surface plasmon resonance showed that TCRs bound to peptide.Ld-m31 complexes with affinities similar to those of native full-length Ld. The TCR and QL9.Ld-m31 formed complexes that could be resolved by native gel electrophoresis, suggesting that stabilized alpha1/alpha2 class I platforms may enable various structural studies.

Alleles↗

PubMed2003

A yeast display system for engineering functional peptide-MHC complexes.

In a cellular immune response, antigenic peptides derived by intracellular processing of foreign pathogens are bound to the class I major histocompatability complex (MHC I) and presented to CD8(+) cytotoxic T cells. Although the crystal structures of several different MHC products have been solved, many MHC molecules, including some associated with diseases, have not been amenable to biochemical and structural studies. The variability in this success is based largely on the fact that peptide-MHC complexes vary extensively in their stability. These properties also are intimately tied to the biological activity of the complexes. The ability to apply the techniques of directed evolution to this system in order to engineer stable complexes has been complicated by the trimeric structure of peptide-MHC complexes, requiring association of three polypeptides: the heavy chain, beta2-microglubulin (beta2m), and a short peptide. We show here that single-chain forms of peptide-MHC complexes can be expressed as Aga-2 fusions on the surface of yeast. Three different complexes, SIYRYYGL-K(b)-beta2m (SIYR-K(b)), EQYKFYSV-K(b)-beta2m (dEV8-K(b)), and SIINFEKL-K(b)-beta2m (OVA-K(b)), were expressed on yeast and detected by flow cytometry with a conformation-specific anti-K(b) antibody (B.8.24.3). In addition, yeast displaying K(b) loaded with exogenous SIYR and OVA peptides were recognized by a high-affinity T cell receptor that is specific for SIYR-K(b) and by an antibody (25.D1-16) that is specific for OVA-K(b), respectively. Finally, yeast that display the SIYRYYGL-K(b) also directly stimulated CD69 up-regulation on naive 2C T cells. Hence, yeast display represents a technology that can be used for directed evolution of any of the components of the trimeric pep-MHC complex.

Animals↗