Development of a novel strategy for engineering high-affinity proteins by yeast display.

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.

Structural and functional consequences of altering a peptide MHC anchor residue.

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-E(k) complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 A resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, approximately 10 A distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.

Eradication of established tumors by CD8+ T cell adoptive immunotherapy.

We generated the DUC18 T cell receptor transgenic mouse expressing an H-2Kd -restricted transgenic T cell receptor specific for the syngeneic CMS5 fibrosarcoma rejection antigen mutated ERK2(136-144). DUC18 mice were capable of specifically eliminating lethal CMS5 tumor challenges, and transfer of DUC18 splenocytes to naive nontransgenic recipients conferred protection from subsequent and established CMS5 tumor burdens. Eradication of established tumor burdens by adoptive transfer of DUC18 splenocytes was dose and time dependent. Transferred tumor-specific T cells remained functional in vivo and capable of rejecting small tumors even in the presence of large, established tumor burdens. These findings highlight the kinetic battle between tumor growth and the production of a tumor-specific response and have critical implications for effective adoptive immunotherapy.

TCR-independent pathways mediate the effects of antigen dose and altered peptide ligands on Th cell polarization.

We examined the role of the peptide/MHC ligand in CD4+ T cell differentiation into Th1 or Th2 cells using a TCR alphabeta transgenic mouse specific for hemoglobin (Hb)(64-76)/I-Ek. We identified two altered peptide ligands of Hb(64-76) that retain strong agonist activity but, at a given dose, induce cytokine patterns distinct from the Hb(64-76) peptide. The ability of these peptides to produce distinct cytokine patterns at identical doses is not due to an intrinsic qualitative property. Each peptide can induce Th2 cytokines at low concentrations and Th1 cytokines at high concentrations and has a unique range of concentrations at which these distinct effects occur. The pattern of cytokines produced from limiting dilution of naive T cells demonstrated that the potential to develop an individual Th1 or Th2 cell is stochastic, independent of Ag dose. We propose that the basis for the observed effects on the Th1/Th2 balance shown by the altered peptide ligands and the amount of Ag dose involves the modification of soluble factors in bulk cultures that are the driving force that polarize the population to either a Th1 or Th2 phenotype.

TCR transgenic mice in which usage of transgenic alpha- and beta-chains is highly dependent on the level of selecting ligand.

We have produced a TCR transgenic mouse that uses a TCR derived from a Th1 clone that is specific for residues 64 to 76 of the d allele of murine hemoglobin presented by I-Ek. Examination of these TCR transgenic mice on an H-2(k/k) background that expressed the nonstimulatory s allele of murine hemoglobin revealed that these mice express many endogenous TCR chains from both alpha and beta loci. We found that this transgenic TCR is also very inefficient at mediating beta selection, thereby showing a direct linkage between beta selection and allelic exclusion of TCR beta. We have also examined these mice on MHC backgrounds that have reduced levels of I-Ek and found that positive selection of cells with high levels of the transgenic TCR depends greatly on the ligand density. Decreasing the selecting ligand density is a means of reducing the number of available selecting niches, and the data reveal that the 3.L2 TCR is used sparingly for positive selection under conditions where the number of niches becomes limiting. The results, therefore, show a way that T cells may get to the periphery with two self-restricted TCRs: one that efficiently mediates positive selection, and another that is inefficient at positive selection with the available niches.

TCR recognition of the Hb(64-76)/I-Ek determinant: single conservative amino acid changes in the complementarity-determining region 3 dramatically alter antigen fine specificity.

To identify the structural basis of Ag fine specificity, TCR sequences from a panel of Hb(64-76)/I-Ek-specific T cells were compared and found to be restricted in variable (V) gene usage, predominantly using BV1 or BV15 and AV4 or AV10 genes. TCRA and TCRB junctional sequences were extremely diverse. No conservation of length or position was found, which distinguishes this response from others, but correlates with the range of fine specificities that these T cells display. A remarkable subtlety in the recognition of Hb(64-76) was revealed from the study of the response to the D73 variant of Hb(64-76), which contains a conservative change in an MHC anchor residue not affecting the binding affinity to I-Ek. To one group of T cells this determinant was non-cross-reacting with Hb(64-76), whereas another recognized both Ags. Interesting, they all used a different constellation of TCRBV genes than that found in Hb(64-76) recognition. To limit the variability in the anti-Hb(64-76) TCR repertoire, transgenic mice expressing a fixed TCRB rearrangement from a Hb(64-76)-specific T cell were used. In Hb(64-76)-specific TCR from these mice, the endogenous alpha-chains pairing with the transgenic beta-chain were highly restricted in their AV gene usage. A comparison of two pairs of closely related T cells of these endogenous TCR variants, one differing by a single, conservative substitution in the complementarity-determining region 3 and the other containing a positional switch of two amino acids, revealed dramatically different fine specificities. Overall, these findings highlight the exquisite sensitivity of the TCR- peptide/MHC interaction to subtle alterations in any of the components.

Myocarditis-inducing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart.

Immune interactions in the heart were studied using a murine model of myosin-induced autoimmune myocarditis. A T cell hybridoma specific for mouse cardiac myosin was generated from A/J mice and used to demonstrate that endogenous myosin/I-Ak complexes are constitutively expressed on antigen-presenting cells in the heart. This T cell hybridoma, Seu.5, was used as a functional probe to identify a myocarditis-inducing epitope of cardiac myosin. Overlapping peptides based on the cardiac myosin heavy chain alpha (myhc alpha) sequences were synthesized and tested for their ability to stimulate Seu.5 T cells. One peptide, myhc alpha (325-357) strongly stimulated the Seu.5 T cells, localizing the epitope to this region of the myhc alpha molecule. Using truncated peptides, the epitope was further localized to residues 334-352. The myhc alpha (334-352) peptide strongly induced myocarditis when administered to A/J mice, which was histologically indistinguishable from that induced by myosin. The myhc alpha (334-352) epitope was present in cardiac myosin and not skeletal muscle myosins, providing a biochemical basis for the cardiac specificity of this autoimmune disease. Induction of myocarditis by this epitope was restricted to the myhc alpha isoform and not the myhc beta isoform, suggesting there may be a difference in the efficiency of generating tolerance to these isoforms of cardiac myosin, which are differentially developmentally regulated. The myhc alpha (334-352) epitope bound to purified I-Ak molecules in a similar manner to other I-Ak-restricted immunogenic epitopes, HEL(48-61) and RNase(43-56). Importantly, the myhc alpha (334-352) epitope was able to bind to I-Ak molecules on the surface of antigen-presenting cells in a stable manner. These findings demonstrate that autoantigenic epitopes can behave in a dominant manner and constitutively bind to class II molecules in the target organ in a similar manner to foreign immunogenic epitopes.

Binding to Ia protects an immunogenic peptide from proteolytic degradation.

A 34 amino acid hen egg-white lysozyme (HEL) peptide was designed and synthesized to investigate if an immunogenic peptide once bound to an Ia molecule becomes proteolytically inaccessible. The determinant recognized by T cells, HEL(52-61) was composed of L-amino acids whereas the 12 amino acid extension on each side of this core were composed of D-epimers. This peptide, HEL(40-73) was resistant to proteolysis, except in the core region, where any cleavage would destroy the determinant. Initially HEL(40-73) was shown to be able to stimulate the HEL specific T cell, 3A9, indicating that an I-Ak molecule can bind and present large peptides that extend beyond the theoretical binding groove. HEL(40-73) was then used to examine the proteolytic sensitivity of determinants recognized by T cells. If HEL(40-73) was treated with chymotrypsin before binding to I-Ak, the determinant was totally destroyed; however, if HEL(40-73) was allowed to first bind to I-Ak, then the determinant became resistant to chymotrypsin cleavage. Thus an Ia molecule can protect a determinant from proteolytic degradation, a finding that has important implications for proposed pathways of Ag processing.