Biomedical subjects

David M Kranz

Publications and source records attributed to David M Kranz.

At least 19 recordsLinked to original sources

PubMed2007

Crystal structure of a complete ternary complex of TCR, superantigen and peptide-MHC.

'Superantigens' (SAgs) trigger the massive activation of T cells by simultaneous interactions with MHC and TCR receptors, leading to human diseases. Here we present the first crystal structure, at 2.5-A resolution, of a complete ternary complex between a SAg and its two receptors, HLA-DR1/HA and TCR. The most striking finding is that the SAg Mycoplasma arthritidis mitogen, unlike others, has direct contacts not only with TCR Vbeta but with TCR Valpha.

Amino Acid Sequence↗

PubMed2007

Induced sensitization of tumor stroma leads to eradication of established cancer by T cells.

Targeting cancer cells, as well as the nonmalignant stromal cells cross-presenting the tumor antigen (Ag), can lead to the complete destruction of well-established solid tumors by adoptively transferred Ag-specific cytotoxic T lymphocytes (CTLs). If, however, cancer cells express only low levels of the Ag, then stromal cells are not destroyed, and the tumor escapes as Ag loss variants. We show that treating well-established tumors expressing low levels of Ag with local irradiation or a chemotherapeutic drug causes sufficient release of Ag to sensitize stromal cells for destruction by CTLs. This was shown directly using high affinity T cell receptor tetramers for visualizing the transient appearance of tumor-specific peptide-MHC complexes on stromal cells. Maximum loading of tumor stroma with cancer Ag occurred 2 d after treatment and coincided with the optimal time for T cell transfer. Under these conditions, tumor rejection was complete. These findings may set the stage for developing rational clinical protocols for combining irradiation or chemotherapy with CTL therapy.

Adoptive Transfer↗

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

The study of high-affinity TCRs reveals duality in T cell recognition of antigen: specificity and degeneracy.

TCRs exhibit a high degree of Ag specificity, even though their affinity for the peptide/MHC ligand is in the micromolar range. To explore how Ag specificity is achieved, we studied murine T cells expressing high-affinity TCRs engineered by in vitro evolution for binding to hemoglobin peptide/class II complex (Hb/I-Ek). These TCRs were shown previously to maintain Ag specificity, despite having up to 800-fold higher affinity. We compared the response of the high-affinity TCRs and the low-affinity 3.L2 TCR toward a comprehensive set of peptides containing single substitutions at each TCR contact residue. This specificity analysis revealed that the increase in affinity resulted in a dramatic increase in the number of stimulatory peptides. The apparent discrepancy between observed degeneracy in the recognition of single amino acid-substituted Hb peptides and overall Ag specificity of the high-affinity TCRs was examined by generating chimeric peptides between the stimulatory Hb and nonstimulatory moth cytochrome c peptides. These experiments showed that MHC anchor residues significantly affected TCR recognition of peptide. The high-affinity TCRs allowed us to estimate the affinity, in the millimolar range, of immunologically relevant interactions of the TCR with peptide/MHC ligands that were previously unmeasurable because of their weak nature. Thus, through the study of high-affinity TCRs, we demonstrated that a TCR is more tolerant of single TCR contact residue substitutions than other peptide changes, revealing that recognition of Ag by T cells can exhibit both specificity and degeneracy.

Amino Acid Substitution↗

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↗

PubMed2006

Long-range cooperative binding effects in a T cell receptor variable domain.

Although cellular processes depend on protein-protein interactions, our understanding of molecular recognition between proteins remains far from comprehensive. Protein-protein interfaces are structural and energetic mosaics in which a subset of interfacial residues, called hot spots, contributes disproportionately to the affinity of the complex. These hot-spot residues can be further clustered into hot regions. It has been proposed that binding energetics between residues within a hot region are cooperative, whereas those between hot regions are strictly additive. If this idea held true for all protein-protein interactions, then energetically significant long-range conformational effects would be unlikely to occur. In the present study, we show cooperative binding energetics between distinct hot regions that are separated by >20 A. Using combinatorial mutagenesis and surface plasmon resonance binding analysis to dissect additivity and cooperativity in a complex formed between a variable domain of a T cell receptor and a bacterial superantigen, we find that combinations of mutations from each of two hot regions exhibited significant cooperative energetics. Their connecting sequence is composed primarily of a single beta-strand of the T cell receptor variable Ig domain, which has been observed to undergo a strand-switching event and does not form an integral part of the stabilizing core of this Ig domain. We propose that these cooperative effects are propagated through a dynamic structural network. Cooperativity between hot regions has significant implications for the prediction and inhibition of protein-protein interactions.

Bacterial Toxins↗

PubMed2005

Class II-restricted T cell receptor engineered in vitro for higher affinity retains peptide specificity and function.

The T cell receptor (TCR) alphabeta heterodimer determines the peptide and MHC specificity of a T cell. It has been proposed that in vivo selection processes maintain low TCR affinities because T cells with higher-affinity TCRs would (i) have reduced functional capacity or (ii) cross-react with self-peptides resulting in clonal deletion. We used the class II-restricted T cell clone 3.L2, specific for murine hemoglobin (Hb/I-E(k)), to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR (Vbeta-linker-Valpha) was mutagenized and selected for thermal stability and surface expression in a yeast display system. Stabilized mutants were used to generate a library with CDR3 mutations that were selected with Hb/I-E(k) to isolate a panel of affinity mutants with K(D) values as low as 25 nM. Kinetic analysis of soluble single-chain TCRs showed that increased affinities were the result of both faster on-rates and slower off-rates. T cells transfected with the mutant TCRs and wild-type TCR responded to similar concentrations of peptide, indicating that the increased affinity was not detrimental to T cell activation. T cell transfectants maintained exquisite hemoglobin peptide specificity, but an altered peptide ligand that acted as an antagonist for the wild-type TCR was converted to a strong agonist with higher-affinity TCRs. These results show that T cells with high-affinity class II reactive TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of T cell activation.

Amino Acid Sequence↗

PubMed2005

Estrogen induction of the granzyme B inhibitor, proteinase inhibitor 9, protects cells against apoptosis mediated by cytotoxic T lymphocytes and natural killer cells.

Exposure to estrogens is associated with an increased risk of developing breast, cervical, and liver cancer. Estrogens strongly induce the human granzyme B inhibitor, proteinase inhibitor 9 (PI-9). Because cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the granzyme pathway to induce apoptosis of target cells, we tested the ability of activated CTLs and the human NK cell line, YT cells, to lyse human liver cells. Estrogen induction of PI-9 protected the liver cells against CTL and NK cell-mediated, granzyme-dependent, apoptosis. Knockdown of PI-9 by RNA interference blocked the protective effect of estrogen. This work demonstrates that estrogens can act on target cells to control their destruction by immune system cells and shows that induction of PI-9 expression can inhibit both CTL and NK cell-mediated apoptosis. Estrogen induction of PI-9 may reduce the ability of cytolytic lymphocytes-mediated immune surveillance to destroy newly transformed cells, possibly providing a novel mechanism for an estrogen-mediated increase in tumor incidence.

Animals↗

PubMed2005

Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1.

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.

Alanine↗

PubMed2005

Neuronal localization of indoleamine 2,3-dioxygenase in mice.

Indoleamine 2,3-dioxygenase (IDO) catabolizes tryptophan to kynurenine. In the immune system, the reduction in tryptophan and increase in kynurenine act to suppress T-cell function. In the nervous system, kynurenine can be further metabolized to quinolinic acid, which can be neurotoxic. IDO is known to be expressed by microglia and its levels are upregulated by interferon-gamma (IFNgamma). We report here that IDO immunoreactivity is also localized in neurons, and that IDO is upregulated by IFNgamma in neurons of the hippocampus. Thus, neuronal IDO could contribute to the vulnerability of neurons to inflammatory conditions.

Animals↗

PubMed2005

Structural basis of affinity maturation and intramolecular cooperativity in a protein-protein interaction.

Although protein-protein interactions are involved in nearly all cellular processes, general rules for describing affinity and selectivity in protein-protein complexes are lacking, primarily because correlations between changes in protein structure and binding energetics have not been well determined. Here, we establish the structural basis of affinity maturation for a protein-protein interaction system that we had previously characterized energetically. This model system exhibits a 1500-fold affinity increase. Also, its affinity maturation is restricted by negative intramolecular cooperativity. With three complex and six unliganded variant X-ray crystal structures, we provide molecular snapshots of protein interface remodeling events that span the breadth of the affinity maturation process and present a comprehensive structural view of affinity maturation. Correlating crystallographically observed structural changes with measured energetic changes reveals molecular bases for affinity maturation, intramolecular cooperativity, and context-dependent binding.

Amino Acid Substitution↗

PubMed2005

T cell receptor CDRs: starring versus supporting roles.

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Complementarity Determining Regions↗

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

Folate-mediated targeting of T cells to tumors.

Recently various strategies have been developed to exploit in a clinical setting the well established finding that T cells can specifically recognize and destroy tumor cells. Several independent approaches to the targeting of T cells against cancer have been explored, including the use of bispecific antibodies (anti-T cell/anti-tumor cell) to redirect T cells, vaccines to induce tumor-reactive T cells, and adoptive transfer of ex vivo activated, tumor-reactive T cells. In this review, we focus on studies in which high-affinity folate receptors (FRs) on tumor cells have served as targets for redirecting or enhancing the effectiveness of activated T cells. Bispecific antibody conjugates of folate and antibodies to the T cell receptor (TCR) complex can generate tumor-reactive T cell responses. The development of folate/antibody conjugates specific for the T cell co-stimulatory molecule CD28 could yield activated T cells that recognize endogenous peptide-major histocompatibility complex (MHC) antigens on tumor cells. Finally, we discuss a less investigated area in which high-affinity FRs on macrophages, or other antigen presenting cells (APCs), may provide opportunities in the design of tumor-antigen-specific vaccines.

Adoptive Transfer↗

PubMed2004

Expression profiling using a hexamer-based universal microarray.

We describe a transcriptional analysis platform consisting of a universal micro-array system (UMAS) combined with an enzymatic manipulation step that is capable of generating expression profiles from any organism without requiring a priori species-specific knowledge of transcript sequences. The transcriptome is converted to cDNA and processed with restriction endonucleases to generate low-complexity pools (approximately 80-120) of equal length DNA fragments. The resulting material is amplified and detected with the UMAS system, comprising all possible 4,096 (4(6)) DNA hexamers. Ligation to the arrays yields thousands of 14-mer sequence tags. The compendium of signals from all pools in the array-of-universal arrays comprises a full-transcriptome expression profile. The technology was validated by analysis of the galactose response of Saccharomyces cerevisiae, and the resulting profiles showed excellent agreement with the literature and real-time PCR assays. The technology was also used to demonstrate expression profiling from a hybrid organism in a proof-of-concept experiment where a T-cell receptor gene was expressed in yeast.

3' Untranslated Regions↗

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).

Animals↗

PubMed2003

Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b).

The Ly49 family of natural killer (NK) receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I. Ly49 receptors show complex patterns of MHC class I cross-reactivity and, in certain cases, peptide selectivity. To investigate whether specificity differences result from topological differences in MHC class I engagement, we determined the structure of the peptide-selective receptor Ly49C in complex with H-2K(b). The Ly49C homodimer binds two MHC class I molecules in symmetrical way, a mode distinct from that of Ly49A, which binds MHC class I asymmetrically. Ly49C does not directly contact the MHC-bound peptide. In addition, MHC crosslinking by Ly49C was demonstrated in solution. We propose a dynamic model for Ly49-MHC class I interactions involving conformational changes in the receptor, whereby variations in Ly49 dimerization mediate different MHC-binding modes.

Animals↗

PubMed2003

Dissecting cooperative and additive binding energetics in the affinity maturation pathway of a protein-protein interface.

When two proteins associate they form a molecular interface that is a structural and energetic mosaic. Within such interfaces, individual amino acid residues contribute distinct binding energies to the complex. In combination, these energies are not necessarily additive, and significant positive or negative cooperative effects often exist. The basis of reliable algorithms to predict the specificities and energies of protein-protein interactions depends critically on a quantitative understanding of this cooperativity. We have used a model protein-protein system defined by an affinity maturation pathway, comprising variants of a T cell receptor Vbeta domain that exhibit an overall affinity range of approximately 1500-fold for binding to the superantigen staphylococcal enterotoxin C3, in order to dissect the cooperative and additive energetic contributions of residues within an interface. This molecular interaction has been well characterized previously both structurally, by x-ray crystallographic analysis, and energetically, by scanning alanine mutagenesis. Through analysis of group and individual maturation and reversion mutations using surface plasmon resonance spectroscopy, we have identified energetically important interfacial residues, determined their cooperative and additive energetic properties, and elucidated the kinetic and thermodynamic bases for molecular evolution in this system. The summation of the binding free energy changes associated with the individual mutations that define this affinity maturation pathway is greater than that of the fully matured variant, even though the affinity gap between the end point variants is relatively large. Two mutations in particular, both located in the complementarity determining region 2 loop of the Vbeta domain, exhibit negative cooperativity.

Algorithms↗