Partial T cell activation with an altered superantigenic ligand

T cells have the capacity to respond to ligands as full, weak, partial or null agonists, or indeed as antagonists. In the present paper, it is reported that staphylococcal enterotoxin B (SEB) mutated in a T cell receptor (TCR) contact site (SEBDelta61Y) behaves as an altered ligand for a T cell clone (AC20) that expresses the Vbeta17 TCR. The T cells were partially activated by SEBDelta61Y, as shown by TCR down-modulation and up-regulation of the IL-2 receptor. However, these cells did not secrete IL-2, IL-3, IL-4 or IFN-gamma, nor did they proliferate. Analysis of intracellular protein tyrosine phosphorylation after cellular activation provided further evidence that SEBDelta61Y could transduce a signal via the Vbeta17 TCR. The events following receptor ligation were clearly different when the T cells were stimulated with SEB or SEBDelta61Y, manifested as both quantitatively and qualitatively different patterns of phosphorylation of intracellular substrates. In contrast, only quantitative differences were apparent when a transfectant expressing the same alpha/beta TCR was stimulated with the different superantigens. Together, these results provide the first demonstration that altered TCR ligands are not restricted to peptides substituted at secondary TCR contact residues. Rather, an altered superantigenic ligand mutated in the TCR binding site can behave as a partial agonist.     

Molecular basis of TCR selectivity, cross-reactivity, and allelic discrimination by a bacterial superantigen: integrative functional and energetic mapping of the SpeC-Vbeta2.1 molecular interface

Superantigens activate large fractions of T cells through unconventional interactions with both TCR beta-chain V domains (Vbetas) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vbeta2(+) T cells. Herein, we have analyzed the SpeC-Vbeta2.1 interaction by mutating all SpeC residues that make contact with Vbeta2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vbeta2.1(+) Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC "functional epitope" for TCR engagement. Although only two SpeC residues (Tyr(15) and Arg(181)) are critical for activation of virtually all human CD3(+) T cells, a larger cluster of four hot spot residues are required for interaction with Vbeta2.1. Three of these residues (Tyr(15), Phe(75), and Arg(181)) concentrate their binding energy on the CDR2 loop residue Ser(52a), a noncanonical residue insertion found only in Vbeta2 and Vbeta4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vbeta2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vbeta2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vbeta domain subfamily.     

Continuous exposure of mice to superantigenic toxins induces a high-level protracted expansion and an immunological memory in the toxin-reactive CD4+ T cells

We analyzed the responses of several T cell fractions reactive with superantigenic toxins (SAGTs), staphylococcal enterotoxin A (SEA), or Yersinia pseudotuberculosis-derived mitogen (YPM) in mice implanted with mini-osmotic pumps filled with SEA or YPM. In mice implanted with the SEA pump, SEA-reactive Vbeta3(+)CD4(+) T cells exhibited a high-level protracted expansion for 30 days, and SEA-reactive Vbeta11(+)CD4(+) T cells exhibited a low-level protracted expansion. SEA-reactive CD8(+) counterparts exhibited only a transient expansion. A similar difference in T cell expansion was also observed in YPM-reactive T cell fractions in mice implanted with the YPM pump. Vbeta3(+)CD4(+) and Vbeta11(+)CD4(+) T cells from mice implanted with the SEA pump exhibited cell divisions upon in vitro restimulation with SEA and expressed surface phenotypes as memory T cells. CD4(+) T cells from mice implanted with the SEA pump exhibited high IL-4 production upon in vitro restimulation with SEA, which was due to the enhanced capacity of the SEA-reactive CD4(+) T cells to produce IL-4. The findings in the present study indicate that, in mice implanted with a specific SAGT, the level of expansion of the SAGT-reactive CD4(+) T cell fractions varies widely depending on the TCR Vbeta elements expressed and that the reactive CD4(+) T cells acquire a capacity to raise a memory response. CD8(+) T cells are low responders to SAGTs.     

Reconstitution of CD8+ T cells by retroviral transfer of the TCR alpha beta-chain genes isolated from a clonally expanded P815-infiltrating lymphocyte

Gene transfer of TCR alphabeta-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vbeta subfamily usage of CD8(+) TIL in the tumor sites demonstrated that T cells using Vbeta10 efficiently infiltrated and expanded clonally. The TCR alpha- and beta-chain sequences derived from a tumor-infiltrating CD8(+)/Vbeta10(+) single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR alphabeta-chain genes were retrovirally introduced into CD8(+) T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2L(d) mAb. Furthermore, T cells containing both TCR alpha- and beta-chains, but not TCR beta-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8(+) T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.     

TCR V beta 8+ T cells prevent development of toxoplasmic encephalitis in BALB/c mice genetically resistant to the disease

BALB/c are genetically resistant to development of toxoplasmic encephalitis (TE) when infected with Toxoplasma gondii, whereas CBA/Ca mice are susceptible. We compared TCR Vbeta chain usage in lymphocytes infiltrated into brains between these animals following infection. TCR Vbeta8(+) cells were the most frequent T cell population in brains of infected, resistant BALB/c mice, whereas TCR Vbeta6(+) T cells were more prevalent than Vbeta8(+) T cells in brains of infected, susceptible CBA/Ca mice. Adoptive transfer of Vbeta8(+) immune T cells, obtained from infected BALB/c mice, prevented development of TE and mortality in infected athymic nude mice that lack T cells. In contrast, adoptive transfer of Vbeta6(+) immune T cells did not prevent development of TE or mortality in the nude mice. The protective activity of Vbeta8(+) immune T cells was greater than that of the total Vbeta8(-) population. In addition, Vbeta8(+) immune T cells produced markedly greater amounts of IFN-gamma than did the Vbeta8(-) population after stimulation with tachyzoite lysate Ags in vitro. Thus, Vbeta8(+) T cells appear to play a crucial role in the genetic resistance of BALB/c mice against development of TE.     

Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II receptor HLA-DR1

Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.     

CTL recognition of a protective immunodominant influenza A virus nucleoprotein epitope utilizes a highly restricted Vbeta but diverse Valpha repertoire: functional and structural implications

To investigate protective immunity conferred by CTL against viral pathogens, we have analyzed CD8(+) T cell responses to the immunodominant nucleoprotein epitope (NP(366-374)) of influenza A virus in B6 mice during primary and secondary infections in vivo. Unlike the highly biased TCR Vbeta repertoire, the associated Valpha repertoire specific for the NP(366-374)/D(b) ligand is quite diverse. Nonetheless, certain public and conserved CDR3alpha clonotypes with distinct molecular signatures were identified. Pairing of public Valpha and Vbeta domains creates an alphabeta TCR heterodimer that binds efficiently to the NP(366-374)/D(b) ligand and stimulates T cell activation. In contrast, private TCRs, each comprising a distinct alpha chain paired with the same public beta chain, interact very differently. Molecular dynamics simulation reveals that the conformation and mobility of the shared Vbeta CDR loops are governed largely by the associated Valpha domains. These results provide insight into molecular principles regarding public versus private TCRs linked to immune surveillance after infection with influenza A virus.     

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.     

Altered peptide ligand-mediated TCR antagonism can be modulated by a change in a single amino acid residue within the CDR3 beta of an MHC class I-restricted TCR

The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8-10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the beta-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3 beta loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.     

Distinct footprints of TCR engagement with highly homologous ligands

T cell receptor engagement promotes proliferation, differentiation, survival, or death of T lymphocytes. The affinity/avidity of the TCR ligand and the maturational stage of the T cell are thought to be principal determinants of the outcome of TCR engagement. We demonstrate in this study that the same mouse TCR preferentially uses distinct residues of homologous peptides presented by the MHC molecules to promote specific cellular responses. The preference for distinct TCR contacts depends on neither the affinity/avidity of TCR engagement (except in the most extreme ranges), nor the maturity of engaged T cells. Thus, different portions of the TCR ligand appear capable of biasing T cells toward specific biological responses. These findings explain differences in functional versatility of TCR ligands, as well as anomalies in the relationship between affinity/avidity of the TCR for the peptide/MHC and cellular responses of T cells.     

A particular TCR beta variable region used by T cells infiltrating kidney transplants

Immune tolerance to MHC class II identical renal grafts is achievable in miniature swine following a short immunosuppressive treatment. Like in clinical transplants, swine-accepted allografts are primarily infiltrated by CD8(+) T cells, which are noncytotoxic to the renal tissue. However, the actual specificity and function of these intragraft-infiltrating lymphocytes remain poorly understood. To develop the molecular tools to study TCR-associated functions of graft-infiltrating cells in a preclinical transplantation model, we have determined the nucleotide sequence of 19 pig Vbeta, 12 Jbeta, and two Dbeta. Sequence comparisons identified 17 different Vbeta families and two Jbeta clusters homologous to the human Jbeta1 and Jbeta2. The fact that the pig Jbeta1 segments were always found joined to the Dbeta1-like sequence in numerous rearranged TCR beta cDNA suggests the existence of two D-J clusters in swine. These results also imply that the polymorphism of the porcine TCR beta segments is similar to that found in human. Finally, we report the discovery of a new and functional Vbeta subfamily named Vbeta100, which exhibited similarity to the murine Vbeta2 sequence but had no described Vbeta homolog in humans. Pilot spectratyping studies on Vbeta usage revealed a clonal dominance of Vbeta100(+) T cell subsets among infiltrating cells in two accepted grafts.     

Requirement for CD40 Ligand, CD4+ T Cells, and B Cells in an Infectious Mononucleosis-Like Syndrome

Respiratory challenge with the murine gammaherpesvirus 68 (gammaHV-68) results in productive infection of the lung, the establishment of latency in B lymphocytes and other cell types, transient splenomegaly, and prolonged clonal expansion of activated CD8(+) CD62L(lo) T cells, particularly a Vbeta4(+) CD8(+) population that is found in mice with different major histocompatibility complex (MHC) haplotypes. Aspects of the CD8(+)-T-cell response are substantially modified in mice that lack B cells, CD4(+) T cells, or the CD40 ligand (CD40L). The B-cell-deficient mice show no increase in Vbeta4(+) CD8(+) T cells. Similar abrogation of the Vbeta4(+) CD8(+) response is seen following antibody-mediated depletion of the CD4(+) subset, through the numbers of CD8(+) CD62L(lo) cells are still significantly elevated. Virus-specific CD4(+)-T-cell frequencies are minimal in the CD40L(-/-) mice, and the Vbeta4(+) CD8(+) population remains unexpanded. Apparently B-cell-CD4(+)-T-cell interactions play a part in the gammaHV-68 induction of both splenomegaly and non-MHC-restricted Vbeta4(+) CD8(+)-T-cell expansion.     

Valpha24-JalphaQ-independent,CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes

Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid alpha-galactosylceramide (alphaGC), to a highly conserved NKT cell subset expressing an invariant TCR Valpha24-JalphaQ paired with Vbeta11 chain (Valpha24(+)Vbeta11(+) invariant NK T cell (NKT(inv))). The developmental pathway of Valpha24(+)Vbeta11(+)NKT(inv) is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-alphaGC-tetramers, we demonstrate that in humans, TCR variable domains other than Valpha24 and Vbeta11 can mediate specific recognition of CD1d-alphaGC. In contrast to Valpha24(+)Vbeta11(+)NKT(inv) cells, Valpha24(-)/CD1d-alphaGC-specific T cells express either CD8alphabeta or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8alphabeta(+)Valpha24(-)/CD1d-alphaGC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than Valpha24(+)/CD1d-alphaGC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-alphaGC complex in humans is not uniformly restricted to the Valpha24-JalphaQ/Vbeta11 NKT cell subset, but can be mediated by a diverse range of Valpha and Vbeta domains. The existence of a diverse repertoire of CD1d-alphaGC-specific T cells in humans strongly supports their Ag-driven selection.     

Cutting edge: influence of the TCR Vbeta domain on the selection of semi-invariant NKT cells by endogenous ligands

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.     

MHC class I allele dosage alters CD8 expression by intestinal intraepithelial lymphocytes

The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.     

Recognition of the influenza hemagglutinin by class II MHC-restricted T lymphocytes and antibodies. I. Site definition and implications for antigen presentation and T lymphocyte recognition.

We have identified the site encompassing residues 126-145 on the A/Japan/57 influenza hemagglutinin molecule that is recognized in association with HLA-DRw11 by a clonal population of human, influenza specific, CD4+ cytolytic T lymphocytes. The critical core sequence of the T cell determinant spans hemagglutinin residues 129-140 and overlaps a putative antibody binding site. Hemagglutinins of influenza field strains that are not recognized by the T cell clones contain sequence alterations within the 129-140 target site of the CD4+ T cells. Functional analyses, with synthetic peptides, of the contribution of each of the residues within the sequence toward the capacity of the antigenic fragment to associate with both the restriction element and the TCR revealed a continuous linear array of residues necessary for MHC binding and/or Ag receptor engagement. At least one residue, the lysine at position 134, was shown to be critical for both DRw11 association and TCR recognition. The significance of these findings for recognition of glycoproteins by human CD4+ T cells is discussed.     

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.     

Enzymatically mediated engineering of multivalent MHC class II-peptide chimeras

We previously reported the genetic engineering of the first soluble, bivalent major histocompatibility complex (MHC) class II-peptide ligand for T-cell receptor (TCR). This ligand binds stably and specifically to cognate T-cells and exhibits immunomodulatory effects in vitro and in vivo. The increase in valence of MHC class II-peptide ligands was shown to parallel their avidity for cognate TCRs and potency in stimulating cognate T-cells. We describe a new enzymatic method to increase the valence of MHC-peptide ligands by cross-linking the N-glycan moieties of dimeric MHC II-peptide units through a flexible, bifunctional polyethylene glycol linker. Using this method, we generated covalently stabilized tetravalent and octavalent MHC II-peptide ligands which bound stably and specifically to cognate TCR and preserved their structural integrity in blood and lymphoid organs for 72 h. Depending on the TCR/CD4 occupancy and degree of TCR/CD4 co-clustering, the multivalent MHC II-peptide ligands polarized efficiently the antigen-specific CD4(+) T-cells toward type 2 cell differentiation or induced T-cell anergy and apoptosis. The enzymatically mediated engineering of multivalent MHC-peptide ligands for cognate TCRs may provide rational grounds for the development of new therapeutic agents endowed with strong modulatory effects on antigen-specific T-cells.     

High affinity T cell receptors from yeast display libraries block T cell activation by superantigens

The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.     

Homology between an alloantigen and a self MHC allele calibrates the avidity of the alloreactive T cell repertoire independent of TCR affinity

The self-restricted T cell repertoire exhibits a high frequency of alloreactivity. Because these alloreactive T cells are derived from the pool of cells selected on several different self MHC alleles, it is unknown how development of the alloantigenic repertoire is influenced by homology between a self MHC allele and an alloantigen. To address this, we used the 2C transgenic TCR that is selected by K(b), is alloreactive for L(d), and cross-reacts with L(q). L(q) is highly homologous to L(d) and binds several of the same peptide ligands, including p2Ca, the peptide recognized by 2C. We find that L(d)/p2Ca is a high avidity agonist ligand, whereas L(q)/p2Ca is a low avidity agonist ligand for 2C T cells. When mice transgenic for the 2C TCR are bred to L(q)-expressing mice, 2C(+) T cells develop; however, they express lower levels of either the 2C TCR or CD8 and require a higher L(d)/p2Ca ligand density to be activated than 2C(+) T cells selected by K(b). Furthermore, the 2C T cells selected in the presence of L(q) fail to detect L(q)/p2Ca complexes even at high ligand density. Thus, despite possessing the identical TCR, there is a functional avidity difference between 2C(+) T cells selected in the presence of L(q) vs K(b). These data provide evidence that homology between the selecting ligand and an alloantigen can influence the avidity of the T cell repertoire for the alloantigen, and suggest that thymic selection can fine tune T cell avidity independent of intrinsic TCR affinity.