Stable, soluble, high-affinity, engineered T cell receptors: novel antibody-like proteins for specific targeting of peptide antigens.

The recent development of T cell receptor phage display opens up the possibility of engineering human T cell receptors with antibody-like binding properties for cell-surface peptide antigens. In this review we briefly discuss recent developments in molecular targeting of peptide antigens. We then discuss potential clinical applications of engineered high-affinity T cell receptors in autoimmunity and cancer.

Recognition of nonpeptide antigens by human V gamma 9V delta 2 T cells requires contact with cells of human origin.

SUMMARY It is becoming apparent that gamma delta T cells form an important part of the adaptive immune response. However, the ligands recognized by gamma delta T cell receptors (TCRs) and the exact biological function of the cells that express this receptor remain unclear. Numerous studies have shown that the dominant human peripheral blood subset of gamma delta T cells, which express a V gamma 9V delta 2 TCR, can activate in response to low molecular weight nonpeptidic molecules. Some of these components have been purified from bacteria or parasites. We examined the activation of polyclonal gamma delta T cell lines, clones with V gamma 9V delta 2 and V gamma 9V delta 1 TCRs, and gamma delta T cells directly ex vivo in response to multiple phosphate, alkylamine and aminobisphosphonate (nBP) antigens and purified protein derivative from Mycobacterium tuberculosis (PPD). V gamma 9V delta 2 T cells were able to respond to multiple small organic molecules of highly variable structure whereas cells expressing a similar V gamma 9 chain paired with a V delta 1 chain failed to recognize these antigens. Thus, the TCR delta chain appears to make an important contribution to the recognition of these antigens. The kinetics of responses to alkylphosphate and alkylamine antigens differ from those of responses to the nBP pamidronate. These different classes of antigen are believed to have differed mechanisms of action. Such differences explain why nBPs can be pulsed onto antigen presenting cells (APCs) and still retain their ability to activate gamma delta T cells while alkylphosphate and alkylamine antigens cannot. We also demonstrate that a substantial proportion of the cells that produce IFN gamma directly ex vivo in response to PPD are gamma delta T cells and that gamma delta T cell activation requires contact with cells of human origin.

The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain.

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.

Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.

Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8alphaalpha.

The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.

Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding.

A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.

Antagonism of cytotoxic T-lymphocyte activation by soluble CD8.

The CD8 co-receptor is important in the differentiation and selection of class I MHC-restricted T cells during thymic development, and in the activation of mature T lymphocytes in response to antigen. Here we show that soluble CD8alphaalpha receptor, despite an extremely low affinity for MHC, inhibits activation of cytotoxic lymphocytes by obstructing CD3 zeta-chain phosphorylation. We propose a model for this effect that involves interference of productive receptor multimerization at the T-cell surface. These results provide new insights into the mechanism of T-cell activation and evidence that CD8 function is exquisitely sensitive to disruption, an effect that might be exploited by molecular therapeutics.

Ultracentrifugation studies on the transmembrane domain of the human erythrocyte anion transporter band 3 in the detergent C12E8.

The dilute solution behaviour of the transmembrane domain (TMD) of the human erythrocyte anion exchanger Band 3 was studied by analytical ultracentrifugation. Sedimentation velocity and equilibrium studies of the TMD solubilized with the detergent C12E8 demonstrate that the protein is a stable dimer in the concentration range 0.1 to 1 mg/ml. There is no evidence of a dissociation at low concentrations or of an association at higher concentrations. Hydrodynamic calculations applying a prolate ellipsoid of revolution and assuming a hydration of w = 0.35 result in an asymmetrical particle with an axial ratio (a/b) of approximately 3.5.

Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, band 3.

Solution studies of the cytoplasmic domain (molecular mass approximately 40kDa) of band 3, the anion exchanger from human erythrocyte membranes, previously suggested a dimeric molecule on the basis of the relative techniques of calibrated gel filtration and calibrated preparative ultracentrifugation. This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography and absolute ultracentrifugation techniques. Sedimentation velocity in the analytical ultracentrifuge combined with calibrated gel chromatography give a molecular mass M of (77 +/- 4) kDa, a value confirmed by low-speed sedimentation equilibrium. Velocity sedimentation in the analytical ultracentrifuge gave a single sedimenting species with an s o 20,w of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation constant Kd, with a value from direct fitting of the concentration distribution curves of (2.8 +/- 0.5) microM, confirmed by a value of approximately 3 microM obtained from fitting a plot of molecular weight Mw,app versus cell loading concentration. Hydrodynamic calculations based on the classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of approximately 10:1, consistent with observations from electron microscopy.

Asymmetric and functional reconstitution of band 3 into pre-formed phosphatidylcholine vesicles.

Human erythrocyte band 3 protein was purified in 0.1% Triton X-100 and reconstituted into pre-formed phosphatidylcholine vesicles by a Triton X-100-mediated procedure [1]. Band 3 (and its transmembrane domain) could be asymmetrically reconstituted into phosphatidylcholine vesicles with retention of sulfate transport activity which showed behaviour characteristic of red cell anion transport in response to pH, H2DIDS and temperature. Successful reconstitution was also possible using high mol ratios of band 3/phosphatidylcholine (1:200), which are not achieved by any other method.

Modification of polyethylene glycol estimation suitable for use with small mammals.

Concentration of polyethylene glycol (PEG) has been measured satisfactorily using only 0.25 ml of intestinal aspirate (containing approximately 2.5 mg/l PEG). Hyden's turbidimetric assay was modified by reading turbidity at 420 mmu. The addition of extra protein was found to facilitate filtration after protein precipitation.