Biomedical subjects

Phillip D Holler

Publications and source records attributed to Phillip D Holler.

7 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

Development of CD4+ T cells expressing a nominally MHC class I-restricted T cell receptor by two different mechanisms.

Differences in T cell receptor (TCR) signaling initiated by interactions among TCRs, coreceptors, and self-peptide-MHC complexes determine the outcome of CD4 versus CD8 lineage of T cell differentiation. The H-2Ld and Kbm3 alloreactive 2C TCR is positively selected by MHC class I Kb and a yet-to-be identified nonclassical class I molecule to differentiate into CD8+ T cells. Here we describe two mechanisms by which CD4+ 2C T cells can be generated in 2C TCR-transgenic mice. In the RAG-/- background, development of CD4+ 2C T cells requires the expression of both I-Ab and the TAP genes, indicating that both MHC class I and II molecules are required for positive selection of these T cells. Notably, only some of the 2C+ RAG-/- mice (approximately 30%) develop CD4+ 2C T cells, with frequencies in individual mice varying from 0.5% to as high as approximately 50%. In the RAG+ background, where endogenous TCRalpha genes are rearranged and expressed, CD4+ 2C T cells are generated because these cells express the 2C TCR as well as additional TCRs, consisting of the 2C TCRbeta and endogenous TCRalpha chains. Similarly, T cells expressing the OT-1 TCR, which is nominally MHC class I-restricted, can also develop into CD4+ T cells through the same two mechanisms. Thus, expression of two TCRs by a single thymocyte, TCR recognition of multiple MHC molecules, and heterogeneity of TCR, coreceptors, and peptide-MHC interactions in the thymus all contribute to the outcome of CD4 versus CD8 lineage development.

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PubMed2004

High-affinity, peptide-specific T cell receptors can be generated by mutations in CDR1, CDR2 or CDR3.

The third complementarity-determining regions (CDR3s) of antibodies and T cell receptors (TCRs) have been shown to play a major role in antigen binding and specificity. Consistent with this notion, we demonstrated previously that high-affinity, peptide-specific TCRs could be generated in vitro by mutations in the CDR3alpha region of the 2C TCR. In contrast, it has been argued that CDR1 and CDR2 are involved to a greater extent than CDR3s in the process of MHC restriction, due to their engagement of MHC helices. Based on this premise, we initiated the present study to explore whether higher affinity TCRs generated through mutations in these CDRs or other regions would lead to significant reductions in peptide specificity (i.e. the result of greater binding energy gained through interactions with major histocompatibility complex (MHC) helices). Yeast-display technology and flow sorting were used to select high-affinity TCRs from libraries of CDR mutants or random mutants. High-affinity TCRs with mutations in the first residue of the Valpha, CDR1, CDR2, or CDR3 were isolated. Unexpectedly, every TCR mutant, including those in CDR1 and CDR2, retained remarkable peptide specificity. Molecular modeling of various mutants suggested that such exquisite specificity may be due to: (1) enhanced electrostatic interactions with key peptide or MHC residues; or (2) stabilization of CDRs in specific conformations. The results indicate that the TCR is positioned so that virtually every CDR can contribute to the antigen-specificity of a T cell. The conserved diagonal docking of TCRs could thus orient each CDR loop to sense the peptide directly or indirectly through peptide-induced effects on the MHC.

Amino Acid Sequence↗

PubMed2004

T cell receptors: affinities, cross-reactivities, and a conformer model.

Based on findings with the T cell receptor from mouse CTL clone 2C, and other TCRs, we propose a model that could account for degeneracy of T cell recognition. This "conformer model" holds that a single TCR exists in multiple conformations that are in equilibrium. The model is consistent with (1) the characterization of multiple ligands that bind to the 2C TCR, and other TCRs, (2) the binding properties and predicted structural features of various 2C TCR mutants, including higher affinity variants isolated by directed evolution, and (3) the three-dimensional structures of TCRs and antibodies, with emphasis on the conformational diversity exhibited by proteins with the same primary amino acid sequence. We propose that the advantages of multiple conformers (e.g. ability of a single T cell to recognize many different ligands) outweigh the disadvantages (e.g. lower TCR affinity and possibly T cell sensitivity; detrimental cross-reactivity with structurally unrelated self-ligands).

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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↗

PubMed2003

Quantitative analysis of the contribution of TCR/pepMHC affinity and CD8 to T cell activation.

The relative roles of CD8, TCR:pepMHC affinity, and TCR:pepMHC dissociation rate in T cell activation have remained controversial. To determine the relationships among these factors, we used T cells transfected with normal and in vitro engineered alphabeta TCRs, in the presence or absence of CD8. The TCRs exhibited a wide range of affinities (K(D) values of 80 microM to 5 nM). T cells with the highest affinity TCRs were efficiently stimulated by peptide, with or without CD8. In contrast, CD8 was required for T cells that expressed TCRs with affinities typical of syngeneic reactions (K(D) values above approximately 3 microM). The results suggest that virtually all normal syngeneic interactions require CD8, which enhances peptide sensitivity by one million-fold or more.

Animals↗

PubMed2002

TCRs with high affinity for foreign pMHC show self-reactivity.

T cells with high-affinity T cell receptors (TCRs) for a foreign peptide-major histocompatibility complex (pMHC) appear to be negatively selected, even though they have never seen the foreign antigen. To examine how this process operates, we used in vitro yeast display to isolate high-affinity TCRs from the T cell clone 2C. The TCRs showed fast on-rates, which were consistent with reduced CDR (complementarity determining region) flexibility, and cross-reactivity with other cognate pMHCs. T cell hybridomas transfected with a high-affinity TCR were stimulated by endogenous self-pMHC, which suggested that T cells bearing the TCR would be negatively selected. The immune system appears to maintain a repertoire of flexible, low-affinity TCRs at the expense of more effective high-affinity TCRs.

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