The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand.     

Recognition of a specific self-peptide: self-MHC class II complex is critical for positive selection of thymocytes expressing the D10 TCR

We examined the specificity of positive and negative selection by using transgenic mice carrying a variant of the D10 TCR. We demonstrate that a point mutation at position 51 within the CDR2alpha segment significantly reduces the avidity of this TCR for its cognate ligand, but does not impact recognition of nonself MHC class II molecules. Although structural studies have suggested that this TCR site interacts with the MHC class II beta-chain, the avidity of this TCR for its ligand and the function of the T cell can be reconstituted by a point mutation in the bound antigenic peptide. These data demonstrate that the bound peptide can indirectly alter TCR interactions by influencing MHC structure. Remarkably, reducing the avidity of this TCR for a specific antigenic peptide-MHC ligand has a dramatic impact on thymic selection. Positive selection of thymocytes expressing this TCR is nearly completely blocked, whereas negative selection on allogenic MHC class II molecules remains intact. Therefore, the recognition of self that promotes positive selection of the D10 TCR is highly peptide-specific.     

Two MHC surface amino acid differences distinguish foreign peptide recognition from autoantigen specificity

KRN T cells can recognize two self MHC alleles with differing biological consequences. They respond to the foreign peptide RN(42--56) bound to I-A(k) or alternatively initiate autoimmune arthritis by interacting with a self Ag, GPI(282--294), on I-A(g7). Five surface amino acid differences between the two MHC molecules collectively alter which peptide side chains are recognized by the KRN TCR. In this study, it is shown that mutation of only two of these residues, alpha 65 and beta 78, in I-A(k) to their I-A(g7) counterparts is sufficient to allow recognition of the TCR contacts from GPI(282--294). To provide a detailed mechanism for the specificity change, the distinct contributions of each of these two mutations to the global effect on peptide specificity were analyzed. The alpha65 mutation is shown to broaden the spectrum of amino acids permissible at P8 of the peptide. In contrast, the beta 78 mutation alone blocks KRN TCR interaction with I-A(k) and requires the simultaneous presence of the alpha 65 mutation to preserve recognition. In the presence of the alpha 65 mutation, the beta 78 residue broadens peptide recognition at P3 and prevents recognition of the P8 L in RN(42--56), thus producing the observed specificity shift. These results localize the functionally relevant differences between the surfaces of two self-restricted MHC molecules to two residues that have counterbalanced positive and negative contributions to interaction with a single TCR. They highlight how subtle structural distinctions attributable to single amino acids can stand at the interface between foreign Ag responsiveness and pathogenic autoreactivity.     

Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V beta repertoire of the responding CD8+ cytotoxic lymphocyte population.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8-11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR V beta elements. The precise role of the peptide in causing this restricted TCR V beta expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR V beta elements responding to each peptide variant. To focus our analysis solely on the TCR beta-chain, we created a transgenic mouse expressing exclusively the TCR alpha-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR V beta usage consequent to peptide immunization. However, in both C57BL/6 and TCR alpha transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR V beta usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-beta loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR V beta usage, which is consistent with the notion that the TCR beta-chain interacts in vivo preferentially with this region of the MHC/peptide complex.     

Single MHC mutation eliminates enthalpy associated with T cell receptor binding

The keystone of the adaptive immune response is T cell receptor (TCR) recognition of peptide presented by major histocompatibility complex (pMHC) molecules. The crystal structure of AHIII TCR bound to MHC, HLA-A2, showed a large interface with an atypical binding orientation. MHC mutations in the interface of the proteins were tested for changes in TCR recognition. From the range of responses observed, three representative HLA-A2 mutants, T163A, W167A, and K66A, were selected for further study. Binding constants and co-crystal structures of the AHIII TCR and the three mutants were determined. K66 in HLA-A2 makes contacts with both peptide and TCR, and has been identified as a critical residue for recognition by numerous TCR. The K66A mutation resulted in the lowest AHIII T cell response and the lowest binding affinity, which suggests that the T cell response may correlate with affinity. Importantly, the K66A mutation does not affect the conformation of the peptide. The change in affinity appears to be due to a loss in hydrogen bonds in the interface as a result of a conformational change in the TCR complementarity-determining region 3 (CDR3) loop. Isothermal titration calorimetry confirmed the loss of hydrogen bonding by a large loss in enthalpy. Our findings are inconsistent with the notion that the CDR1 and CDR2 loops of the TCR are responsible for MHC restriction, while the CDR3 loops interact solely with the peptide. Instead, we present here an MHC mutation that does not change the conformation of the peptide, yet results in an altered conformation of a CDR3.     

The molecular basis of class II MHC allelic control of T cell responses.

To identify the molecular basis for the effects of MHC molecule polymorphism on T cell responses, we have combined functional T cell response testing with measurements of peptide binding to the class II MHC molecules on transfected cells. Our studies identify a small subset of spatially localized polymorphic residues of the E alpha E beta dimer (strand residue beta 29, and helix residues beta 72 and beta 75) regulating cytochrome c peptide presentation by two distinct mechanisms. The first effect is on quantitative control of net peptide binding. The replacement of the valine found at position beta 29 in E beta k with the glutamic acid found in E beta b results in a selective loss of pigeon cytochrome peptide but not moth cytochrome peptide binding to the resultant mutant E alpha E beta k molecule. Reciprocally, the replacement of glutamic acid at beta 29 in E beta b with valine results in a gain of pigeon peptide binding. These changes in binding parallel changes in T cell responses in vitro to these peptide-E alpha E beta combinations and mirror the in vivo immune response gene phenotypes of mice expressing E alpha E beta k and E alpha E beta b. E alpha E beta s molecules, which have a beta 29 glutamic acid, are nevertheless able to bind and present pigeon cytochrome peptides, and this is due to changes in helix residues beta 72 and beta 75 that compensate for the negative effect of the beta 29 glutamic acid. The second activity is a critical change in the conformation of the peptide bound to the same extent by distinct MHC molecules, as revealed by changes in T cell responses to moth cytochrome peptides presented by two E alpha E beta molecules differing only at position beta 29. Both of these effects can be ascribed to a single polymorphic residue modeled to be inaccessible to TCR contact (beta 29), providing a striking demonstration of how MHC molecule polymorphism can modify T cell-dependent immune responses without direct physical participation in the receptor recognition event.     

Diverse fine specificity and receptor repertoire of T cells reactive to the major VP1 epitope (VP1230-250) of Theiler's virus: V beta restriction correlates with T cell recognition of the c-terminal residue.

Theiler's murine encephalomyelitis virus induces chronic demyelinating disease in genetically susceptible mice. The histopathological and immunological manifestation of the disease closely resembles human multiple sclerosis, and, thus, this system serves as a relevant infectious model for multiple sclerosis. The pathogenesis of demyelination appears to be mediated by the inflammatory Th1 response to viral epitopes. In this study, T cell repertoire reactive to the major pathogenic VP1 epitope region (VP1233-250) was analyzed. Diverse minimal T cell epitopes were found within this region, and yet close to 50% of the VP1-reactive T cell hybridomas used V beta 16. The majority (8/11) of the V beta 16+ T cells required the C-terminal amino acid residue on the epitope, valine at position 245, and every T cell hybridoma recognizing this C-terminal residue expressed V beta 16. However, the complementarity-determining region 3 sequences of the V beta 16+ T cell hybridomas were markedly heterogeneous. In contrast, such a restriction was not found in the V alpha usage. Only restricted residues at this C-terminal position allowed for T cell activation, suggesting that V beta 16 may recognize this terminal residue. Further functional competition analysis for TCR and MHC class II-contacting residues indicate that many different residues can be involved in the class II and/or TCR binding depending on the T cell population, even if they recognize the identical minimal epitope region. Thus, recognition of the C-terminal residue of a minimal T cell epitope may associate with a particular V beta (but not V alpha) subfamily-specific sequence, resulting in a highly restricted V beta repertoire of the epitope-specific T cells.     

Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition.

The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.     

Complementarity-determining region 1 sequence requirements drive limited V alpha usage in response to influenza hemagglutinin 307-319 peptide

We have developed a T cell activation-based system that allows for the selection of TCRs with defined peptide/MHC specificities from libraries in which complementarity-determining region (CDR) sequences have been randomized by in vitro mutagenesis. Using this system, we have explored the sequence requirements for CDR1 and CDR2 of the TCR alpha-chain in a human T cell response characterized by restricted Valpha and Vbeta usage. Libraries of T cells expressing receptors built on the framework of a TCR specific for the influenza virus peptide hemagglutinin 307-319 presented by HLA-DR4, but with random sequences inserted at CDR1alpha or CDR2alpha, were selected for response to the same peptide/MHC ligand. A wide variety of CDR2alpha sequences were found to be permissive for recognition. Indeed, >25% of T cell clones chosen at random displayed a significant response. In contrast, a similar challenge of a randomized CDR1alpha library yielded only the parental sequence, and then only after multiple rounds of selection. T cell clones cross-reactive on closely related HLA alleles (subtypes of DR4) could be isolated from randomized libraries, but not clones restricted by more distantly related alleles such as HLA-DR1. These results indicate that, in the context of this T cell response, the structural requirements for recognition at CDR1alpha are significantly more restricted than at CDR2alpha. This system for mutation and selection of TCRs in vitro may be of use in engineering T cells with defined specificities for therapeutic applications.     

Functional mapping of MHC class II polymorphic residues. The alpha-chain controls the specificity for binding an Ad-versus an A k-restricted peptide and the beta-chain region 65-67 controls T cell recognition but not peptide binding.

MHC proteins are polymorphic cell surface glycoproteins involved in the binding of peptide Ag and their presentation to T lymphocytes. The polymorphic amino acids of MHC proteins are primarily located in the N-terminal domains and are thought to influence T cell recognition both by influencing the binding of peptide Ag and by direct contact with the T cell receptor. In order to determine the relative importance of individual polymorphic amino acids in Ag presentation, a number of groups have taken the approach of interchanging polymorphic amino acids between different alleles of MHC protein in an attempt to define which of the polymorphisms influence peptide binding and which influence T cell recognition by direct contact with the TCR. The peptide OVA323-339 has been previously shown to bind to the MHC class II protein Ad and to have a much lower affinity for Ak, whereas the peptide hen egg lysozyme 46-61 binds well to Ak and poorly to Ad. In the present report, we have analyzed the ability of purified wild-type MHC class II proteins as well as the ability of three different hybrid molecules between Ad and Ak to bind and present these peptides. We find that the alpha-chain of the MHC class II protein plays a critical role in the binding of HEL46-61 and confers the specificity for binding OVA323-339, regardless of which beta-chain is present. We also find that the beta-chain region 65-67 does not control the specificity of peptide binding to the MHC protein, but is important in T cell responses to preformed MHC-peptide complexes, suggesting a role for this region in contacting the TCR.     

Crystal structure of a T cell receptor Valpha11 (AV11S5) domain: new canonical forms for the first and second complementarity determining regions.

We describe the X-ray crystallographic structure of a murine T cell receptor (TCR) Valpha domain ("Valpha85.33"; AV11S5-AJ17) to 1.85 A resolution. The Valpha85.33 domain is derived from a TCR that recognizes a type II collagen peptide associated with the murine major histocompatibility complex (MHC) class II molecule, I-A(q). Valpha85.33 packs as a Valpha-Valpha homodimer with a highly symmetric monomer-monomer interface. The first and second complementarity determining regions (CDR1 and CDR2) of this Valpha are shorter than the CDRs corresponding to the majority of other Valpha gene families, and three-dimensional structures of CDRs of these lengths have not been described previously. The CDR1 and CDR2 therefore represent new canonical forms that could serve as templates for AV11 family members. CDR3 of the Valpha85.33 domain is highly flexible and this is consistent with plasticity of this region of the TCR. The fourth hypervariable loop (HV4alpha) of AV11 and AV10 family members is one residue longer than that of other HV4alpha regions and shows a high degree of flexibility. The increase in length results in a distinct disposition of the conserved residue Lys68, which has been shown in other studies to play a role in antigen recognition. The X-ray structure of Valpha85.33 extends the database of canonical forms for CDR1 and CDR2, and has implications for antigen recognition by TCRs that contain related Valpha domains.     

Unraveling a hotspot for TCR recognition on HLA-A2: evidence against the existence of peptide-independent TCR binding determinants

T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.     

The X-ray crystal structure of a Valpha2.6Jalpha38 mouse T cell receptor domain at 2.5 A resolution: alternate modes of dimerization and crystal packing.

We describe here the structure of a murine T cell receptor (TCR) Valpha2.6Jalpha38 (TCRAV2S6J38) domain, derived from a T cell hybridoma with specificity for the H-2Ddmajor histocompatibility complex class I molecule bound to a decamer peptide, P18-I10, from the HIV envelope glycoprotein gp120, determined by X-ray crystallography at 2.5 A resolution. Unlike other TCR Valpha domains that have been studied in isolation, this one does not dimerize in solution at concentrations below 1 mM, and the crystal fails to show dimer contacts that are likely to be physiological. In comparison to other Valpha domains, this Valpha2.6 shows great similarity in the packing of its core residues, and exhibits the same immunoglobulin-like fold characteristic of other TCR Valpha domains. There is good electron density in all three complementarity-determining regions (CDRs), where the differences between this Valpha domain and others are most pronounced, in particular in CDR3. Examination of crystal contacts reveals an association of Valpha domains distinct from those previously seen. Comparison with other Valpha domain structures reveals variability in all loop regions, as well as in the first beta strand where placement and configuration of a proline residue at position 6, 7, 8, or 9 affects the backbone structure. The great variation in CDR3 conformations among TCR structures is consistent with an evolving view that CDR3 of TCR plays a plastic role in the interaction of the TCR with the MHC/peptide complex as well as with CDR3 of the paired TCR chain.     

Immunobiological analysis of TCR single-chain transgenic mice reveals new possibilities for interaction between CDR3alpha and an antigenic peptide bound to MHC class I

The interaction between TCRs and peptides presented by MHC molecules determines the specificity of the T cell-mediated immune response. To elucidate the biologically important structural features of this interaction, we generated TCR beta-chain transgenic mice using a TCR derived from a T cell clone specific for the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2K(b). We immunized these mice with VSV8 or analogs substituted at TCR contact residues (positions 1, 4, and 6) and analyzed the CDR3alpha sequences of the elicited T cells. In VSV8-specific CTLs, we observed a highly conserved residue at position 93 of CDR3alpha and preferred Jalpha usage, indicating that multiple residues of CDR3alpha are critical for recognition of the peptide. Certain substitutions at peptide position 4 induced changes at position 93 and in Jalpha usage, suggesting a potential interaction between CDR3alpha and position 4. Cross-reactivity data revealed the foremost importance of the Jalpha region in determining Ag specificity. Surprisingly, substitution at position 6 of VSV8 to a negatively charged residue induced a change at position 93 of CDR3alpha to a positively charged residue, suggesting that CDR3alpha may interact with position 6 in certain circumstances. Analogous interactions between the TCR alpha-chain and residues in the C-terminal half of the peptide have not yet been revealed by the limited number of TCR/peptide-MHC crystal structures reported to date. The transgenic mouse approach allows hundreds of TCR/peptide-MHC interactions to be examined comparatively easily, thus permitting a wide-ranging analysis of the possibilities for Ag recognition in vivo.     

Vdelta1 T cell receptor binds specifically to MHC I chain related A: molecular and biochemical evidences

Human MHC class I chain-related A (MICA) is a tumor-associated antigen that can be recognized by Vdelta1 subset of tumor-infiltrating gammadelta T cells. We previously reported that immobilized recombinant MICA protein could induce the proliferation of tumor-infiltrating Vdelta1 gammadelta T cells in vitro. But there has been no direct evidence showing the engagement of gammadelta T cell receptors (TCR) of the induced cells with MICA. In the current investigation, we show that MICA induces specific cytolytic activity of the expanded gammadelta T cells. We expressed the coupled V domains from the MICA-induced T cells as a single polypeptide chain Vdelta Vgamma TCR (gammadelta scTCR). Such scTCR can specifically bind MICA of HeLa cells. Direct interaction of gammadelta scTCRs with in vitro expressed MICA was monitored using an IAsys biosensor. We found that the Vdelta1 scTCR can specifically bind to immobilized MICA molecule and MICA alpha1alpha2 domains are responsible for the binding reaction.     

Limited T cell receptor beta-chain usage in the sperm whale myoglobin 110-121/E alpha dA beta d response by H-2d congenic mouse strains.

The specificity and TCR gene usage of a panel of sperm whale myoglobin (SpWMb)-reactive T cell clones from DBA/2 mice have previously been characterized, to study structure-function relationships between components of the ternary complex consisting of Ag, TCR, and MHC class II molecules, whose interaction leads to Th cell activation. These DBA/2 clones were specific for epitopes within the residue 110 to 121 region of SpWMb, in the context of the mixed isotype molecule E alpha dA beta d, and expressed the TCR V beta 8.2 gene element. SpWMb-specific T cell hybridomas from the H-2d-congenic B10.D2 mouse strain, which differs from the DBA/2 strain only in the non-MHC background, were generated and compared with the T cell hybridomas from DBA/2 mice, in order to investigate the influence of non-MHC genes on the specificity of the T cell response to the 110-121 epitope. V beta usage by these hybridomas was very homogeneous; three of three DBA/2 and eight of nine B10.D2 hybridomas specific for the 110-121 epitope, in the context of the mixed isotype molecule E alpha dA beta d, expressed the V beta 8.2 gene product. Nucleotide and amino acid sequences of D beta, J beta, and N regions were also similar. One 110-121/E alpha dA beta d-specific B10.D2 hybridoma used V beta 7, a V beta that is clonally deleted in DBA/2 mice. These experiments suggest that a similar set of TCR beta genes are used to respond to a given epitope, regardless of non-MHC background, and they support the hypothesis that, despite great variability between individuals in their non-MHC background genes, human HLA-associated diseases might result from the formation of a particular ternary complex consisting of a shared MHC molecule, a common "disease-associated" epitope, and a shared TCR.     

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.     

Amino acid residues in the T cell receptor CDR3 determine the antigenic reactivity patterns of insulin-reactive hybridomas

We examined TCR gene usage in a panel of beef insulin/I-Ad-restricted T cell hybrids obtained from BALB/c mice. These hybrids demonstrated several distinct patterns of reactivity defined by their ability to respond to species variants of insulin. Correlation of TCR-alpha and -beta-gene usage with these patterns of reactivity demonstrated that TCR gene usage was restricted within Ag reactivity groups. In particular, V-J junctional regions (CDR3 equivalent) were restricted with conserved junctional amino acid motifs present in both TCR-alpha- and -beta-chains. Comparison of TCR gene usage in hybrids expressing identical V alpha and V beta gene segments but demonstrating different patterns of reactivity revealed that changes in either J alpha and/or J beta gene segment usage could alter antigenic reactivity. Indeed, single or limited amino acid differences within the CDR3 region were sufficient to markedly alter fine specificity. These data demonstrate the critical role for CDR3 in determining antigenic reactivity in beef insulin-reactive hybrids and are compatible with the current model of TCR/peptide/MHC interaction.     

A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: (1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide—MHC, or (2) introduction of a chimeric antigen receptor, including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vα-linker-Vβ) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen-loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins.     

Superantigens interact with MHC class II molecules outside of the antigen groove

Superantigens, including the staphylococcal enterotoxins and the minor lymphocyte stimulatory antigens, are highly potent immunostimulatory molecules, capable of activating virtually all T cells that express particular T cell receptor (TCR) variable regions. Superantigen stimulation of T lymphocytes depends on major histocompatibility complex (MHC) class II molecules, so there has been some debate as to whether superantigens interact with the antigen binding "groove" on class II complexes, just like conventional peptide antigens, or whether they bind elsewhere and serve as TCR coligands. We compared the presentation of peptide antigens and superantigens by a panel of mutant-presenting cell lines, each displaying an A kappa alpha chain with a single alanine replacement along the alpha helix proposed to form one face of the groove. The negligible effect of these 30 mutations on superantigen presentation, versus their drastic consequences for peptide presentation, prompts us to conclude that superantigens interact with MHC class II molecules outside the groove.