Biomedical subjects

Frances Crawford

Publications and source records attributed to Frances Crawford.

10 recordsLinked to original sources

PubMed2024

Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

Animals↗

PubMed2006

Interface-disrupting amino acids establish specificity between T cell receptors and complexes of major histocompatibility complex and peptide.

T cell receptors (TCRs) bind complexes of cognate major histocompatibility complex (MHC) and peptide at relatively low affinities (1-200 microM). Nevertheless, TCR-MHC-peptide interactions are usually specific for the peptide and the allele encoding the MHC. Here we show that to escape thymocyte negative selection, TCRs must interact with many of the side chains of MHC-peptide complexes as 'hot spots' for TCR binding. Moreover, even when the 'parental' side chain did not contribute binding affinity, some MHC-peptide residues contributed to TCR specificity, as amino acid substitutions substantially reduced binding affinity. The presence of such 'interface-disruptive' side chains helps to explain how TCRs generate specificity at low-affinity interfaces and why TCRs often 'accommodate' a subset of amino acids at a given MHC-peptide position.

Amino Acids↗

PubMed2006

Use of baculovirus MHC/peptide display libraries to characterize T-cell receptor ligands.

Peptide/protein display libraries are powerful tools for identifying and manipulating receptor/ligand pairs. While the large size of bacterial phage display libraries has made them the platform of choice in many applications, often considerable engineering has been required to achieve display of properly folded and active eukaryotic proteins, such as antibodies. This problem has been partially solved in several eukaryotic display systems, e.g. using yeast or retroviruses, but these systems have their own limitations. Recently, baculovirus has been developed as a display system using the virus itself or infected insect cells as the display platform. Here, we review the development and use of baculovirus-infected cells as a platform for display libraries of peptides bound to major histocompatibility complex (MHC) class I (MHCI) or class II (MHCII). We have used fluorescent multimeric soluble T-cell receptors (TCRs) to screen these libraries and to identify peptide antigen mimotopes. We also present some improvements to this system that allow very large libraries to be constructed and screened. We have used these libraries to examine the role of MHCII-bound peptides in the presentation of the staphylococcal enterotoxin A (SEA) and to manipulate an MHCI tumor-associated antigen.

Amino Acid Sequence↗

PubMed2005

How the T cell repertoire becomes peptide and MHC specific.

T cells bearing alphabeta T cell receptors (TCRs) recognize antigens in the form of peptides bound to class I or class II major histocompatibility proteins (MHC). TCRs on mature T cells are usually very specific for both peptide and MHC class and allele. They are picked out from a precursor population in the thymus by MHC-driven positive and negative selection. Here we show that the pool of T cells initially positively selected in the thymus contains many T cells that are very crossreactive for peptide and MHC and that subsequent negative selection establishes the MHC-restriction and peptide specificity of peripheral T cells. Our results also suggest that germline-encoded TCR variable elements have an inherent predisposition to react with features shared by all MHC proteins.

Alleles↗

PubMed2005

Using a baculovirus display library to identify MHC class I mimotopes.

We have developed a baculovirus-based display system for identifying antigen mimotopes for MHC class I-specific T cells. The mouse MHC class I molecule, Dd, was displayed on baculovirus-infected insect cells with a library of 9- and 10-mer peptides tethered via a flexible linker to the N terminus of beta2 microglobulin. As a test case, the library was screened by flow cytometry by using a multimeric fluorescent alphabetaTCR from a mouse T cell specific for Dd plus an unknown self peptide. A mimotope was identified that, when bound to Dd, stimulated the T cell to secret IL-2. The sequence of the mimotope was used to identify a self peptide present in a mouse protein, Spin. The Spin peptide, when complexed with Dd, also activated the T cell. This technique should be generally useful in identifying and manipulating MHC class I peptide mimotopes and epitopes.

Amino Acid Sequence↗

PubMed2005

Class II major histocompatibility complex-peptide tetramer staining in relation to functional avidity and T cell receptor diversity in the mouse CD4(+) T cell response to a rheumatoid arthritis-associated antigen.

OBJECTIVE: Although studies have suggested that human cartilage (HC) gp-39 may be an antigen recognized by autoreactive CD4(+) T cells in rheumatoid arthritis, we previously failed to identify specific CD4(+) T cells in patients' synovial fluid or blood using a class II major histocompatibility complex-peptide tetramer composed of the immunodominant HC gp-39(263-275) epitope covalently linked to DR4. We undertook this study to better understand the parameters for specific binding of this tetramer. METHODS: DR4-transgenic mice were immunized with the HC gp-39 peptide, and a set of peptide-responsive hybridomas was derived. Hybridomas were stained with the DR4-gp-39 tetramer and cultured with increasing amounts of peptide in the presence of DR4-expressing antigen-presenting cells to determine functional avidity. RESULTS: Great variability was apparent in the ability of the tetramer to stain the hybridomas, and there was a strong correlation between the intensity of tetramer staining and functional avidity. Importantly, nearly 30% of the hybridomas did not stain with tetramer, and these cells exhibited relatively low functional avidity. Although the addition of an anti-T cell receptor (anti-TCR) monoclonal antibody during the staining procedure enhanced binding of the tetramer to a number of the hybridomas, a significant percentage remained unstainable. Analysis of TCR expression showed that >90% of the hybridomas expressed the same TCR beta-chain variable region (V(beta)10), and sequencing of the TCR junctional regions showed diversity in the third complementarity-determining region. CONCLUSION: These results suggest that immune responses dominated by relatively low-affinity TCR interactions, such as those that may occur in autoimmune disease, will be difficult to detect using standard tetramer techniques.

Animals↗

PubMed2004

Mimotopes for alloreactive and conventional T cells in a peptide-MHC display library.

The use of peptide libraries for the identification and characterization of T cell antigen peptide epitopes and mimotopes has been hampered by the need to form complexes between the peptides and an appropriate MHC molecule in order to construct a complete T cell ligand. We have developed a baculovirus-based peptide library method in which the sequence encoding the peptide is embedded within the genes for the MHC molecule in the viral DNA, such that insect cells infected with virus encoding a library of different peptides each displays a unique peptide-MHC complex on its surface. We have fished in such a library with two different fluorescent soluble T cell receptors (TCRs), one highly peptide specific and the other broadly allo-MHC specific and hypothesized to be much less focused on the peptide portion of the ligand. A single peptide sequence was selected by the former alphabetaTCR that, not unexpectedly, was highly related to the immunizing peptide. As hypothesized, the other alphabetaTCR selected a large family of peptides, related only by a similarity to the immunizing peptide at the p5 position. These findings have implications for the relative importance of peptide and MHC in TCR ligand recognition. This display method has broad applications in T cell epitope identification and manipulation and should be useful in general in studying interactions between complex proteins.

Amino Acid Sequence↗

PubMed2003

Negative selection imparts peptide specificity to the mature T cell repertoire.

The T cell alphabeta receptor (TCR) recognizes foreign peptide antigens bound to proteins encoded in the MHC. The MHC portion of this complex contributes much to the footprint of the TCR on the ligand, yet T cells are usually very specific for individual foreign peptides. Here, we show that the development of peptide-specific T cells is not intrinsic to thymocytes that undergo thymic-positive selection but is an outcome of eliminating, through negative selection, thymocytes bearing TCRs with extensive peptide cross-reactivity. Hence, thymic-negative selection imposes peptide specificity on the mature T cell repertoire.

Amino Acid Sequence↗

PubMed2002

Alternate interactions define the binding of peptides to the MHC molecule IA(b).

We have solved the crystal structure of the MHCII molecule, IA(b), containing an antigenic variant of the major IA(b)-binding peptide derived from the MHCII IEalpha chain. The four MHC pockets at p1, p4, p6, and p9 that usually bind peptide side chains are largely empty because of alanines in the peptide at these positions. The complex is nevertheless very stable, apparently because of unique alternate interactions between the IA(b) and peptide. In particular, there are multiple additional hydrogen bonds between the N-terminal end of the peptide and the IA(b) alpha chain and an extensive hydrogen bond network involving an asparagine at p7 position of the peptide and the IA(b) beta chain. By using knowledge of the shape and size of the traditional side chain binding pockets and the additional possible interactions, an IA(b) peptide-binding motif can be deduced that agrees well with the sequences of known IA(b)-binding peptides.

Amino Acid Motifs↗

PubMed2002

Structural basis of cytochrome c presentation by IE(k).

The COOH-terminal peptides of pigeon and moth cytochrome c, bound to mouse IE(k), are two of the most thoroughly studied T cell antigens. We have solved the crystal structures of the moth peptide and a weak agonist-antagonist variant of the pigeon peptide bound to IE(k). The moth peptide and all other peptides whose structures have been solved bound to IE(k), have a lysine filling the p9 pocket of IE(k). However, the pigeon peptide has an alanine at p9 shifting the lysine to p10. Rather than kinking to place the lysine in the anchor pocket, the pigeon peptide takes the extended course through the binding groove, which is characteristic of all other peptides bound to major histocompatibility complex (MHC) class II. Thus, unlike MHC class I, in which peptides often kink to place optimally anchoring side chains, MHC class II imposes an extended peptide conformation even at the cost of a highly conserved anchor residue. The substitution of Ser for Thr at p8 in the variant pigeon peptide induces no detectable surface change other than the loss of the side chain methyl group, despite the dramatic change in recognition by T cells. Finally, these structures can be used to interpret the many published mutational studies of these ligands and the T cell receptors that recognize them.

Amino Acid Motifs↗