Dual conformations of a T cell receptor V alpha homodimer: implications for variability in V alpha V beta domain association

The crystal structure of a mutant T cell receptor (TCR) V alpha domain containing a grafted third complementarity-determining region (CDR3) from a different V alpha was determined at 2.3 A resolution by molecular replacement using the wild-type V alpha structure as a search model. Like the wild-type V alpha domain, the mutant crystallized as a homodimer very similar to TCR V alpha V beta and antibody V(L)V(H) heterodimers, with the CDR loops disposed to form part of the antigen-binding site. However, the relative orientation of the two chains in the mutant V alpha homodimer differs from that in the wild-type by a rotation of 14 degrees such that the buried surface area in the dimer interface of the mutant is 140 A2 less than in the wild-type. While the residues forming the interface are essentially the same in the two structures, there are only four pairs of interface hydrogen bonds in the case of the mutant compared with eight for the wild-type. These results suggest that multiple relative orientations of the V alpha and V beta domains of TCRs may be possible, providing a significant contribution to TCR combining site diversity.     

A functionally significant allelic polymorphism in a T cell receptor V beta gene segment

The effect of an allelic polymorphism in the BV1S1 gene segment on recognition of major histocompatibility complex (MHC)-peptide complexes by a specific T cell receptor (TCR) was studied using RBL 2H3 cells transfected with TCR-CD3 zeta chimeric receptors. An HLA-A2-restricted human immunodeficiency virus (HIV) pol-specific cytotoxic T lymphocyte (CTL) clone utilizing the BV1S1A2 gene in combination with AV2S1A2 was identified and the extracellular domains of the TCR were fused to CD3 zeta. In degranulation assays RBL 2H3 transfectants expressing this receptor maintained the specificity of the parental CTL clone. The allelic variant BV1S1A1N1 containing a glutamine for histidine substitution at position 48 in the loop of the second complementarity-determining region was generated by site-directed mutagenesis. Transfection of this molecule as a CD3 zeta chimera together with the original AV2S1A2 CD3 zeta molecule resulted in cell surface expression of both chains but a loss of recognition of HLA-A2 HIV pol peptide-pulsed targets. The effect of this polymorphism on MHC-peptide recognition supports current models of TCR MHC-peptide interaction and provides evidence for a functional role for polymorphism in the TCRV genes.     

A T cell receptor beta chain variable region polymorphism associated with radiographic progression in rheumatoid arthritis

OBJECTIVE: In rheumatoid arthritis (RA) genetic factors influence susceptibility to disease and progression. Identifying these genetic factors may give more insight into the aetiology and pathogenesis of this disease. Furthermore, if these genetic markers can predict progression in an early stage of disease, timely institution of more aggressive treatment in patients with a bad prognosis may help to prevent joint damage. Several studies have shown that HLA-DRB1 alleles are associated with RA, whereas others have indicated that genes not linked to the HLA complex are also involved. Candidates for such genes are the T cell receptor (TCR) alpha/beta genes. METHODS: The association of a polymorphism in a TCR beta chain variable region gene (TCR-V beta 8) with both risk for RA and radiographic progression of joint disease was analysed after a three year follow up. A cohort of 118 white patients with a duration of disease shorter than one year at entry, and 110 white controls were typed for this (BamHI) TCR-V beta 8 polymorphism. RESULTS: The distribution of the two alleles, 2.0 and 23.0 kb, was identical in patients and controls. Radiographic progression (modified Sharp method) after a three year follow up, studied in 111 patients, was significantly less in the group possessing the 2.0 kb allele (p = 0.03). CONCLUSION: This does not confirm the reported association of the (BamHI) TCR-V beta 8 2.0 kb allele with RA. By contrast with previous findings in smaller studies, in the present study this 2.0 kb allele was protective against radiographic progression. Because well known prognostic variables in RA were corrected for, the findings indicate that the TCR-V beta 8 polymorphism studied is a new prognostic marker for this disease.     

T cell receptor V beta repertoire in HIV-infection individuals: lack of evidence for selective V beta deletion

The gradual decline of CD4+ T lymphocytes in HIV-infected individuals culminates in the lethal immunosuppression of AIDS. The mechanism of CD4+ T cell loss is currently unknown, but has recently been suggested to occur as a result of an HIV-encoded superantigen which facilitates a selective deletion of T cells expressing specific V beta genes. To verify and extend such observations, peripheral blood leucocytes (PBL) from 15 HIV+ individuals, 10 of which had very low CD4 T cell counts (< 200/mm3), were analysed for T cell receptor (TCR) V beta gene expression. In contrast to a recent study, the results presented here fail to provide evidence that selective loss of V beta-bearing T cells occurs in HIV+ individuals. Furthermore, when PBL from HIV+ individuals were stimulated with Staphylococcal enterotoxin B (SEB), T cells expressing V beta subfamilies known to engage this superantigen were expanded, indicating that such cells were not deleted and were responsive to stimulation by a bacterial superantigen. Collectively, these data suggest that CD4 loss in HIV patients does not occur in a V beta-selective, superantigen-mediated fashion.     

T cell receptor beta chain dimers on immature thymocytes from normal mice

During T cell development the T cell receptor (TCR) beta chain is expressed before the TCR alpha chain. Experiments in TCR beta transgenic severe combined immune deficiency (SCID) mice have shown that the TCR beta protein can be expressed on the cell surface of immature thymocytes in the absence of the TCR alpha chain and that the TCR beta protein controls T cell development with regard to cell number, CD4/CD8 expression and allelic exclusion of the TCR beta chain. Subsequent experiments have shown that on the surface of thymocytes from TCR beta transgenic SCID mice the TCR beta protein can be expressed in a monomeric and dimeric form whereas only the dimeric form was found on the surface of a TCR beta-transfected, immature T cell line. The results presented here show that normal thymocytes from 16-day-old fetuses likewise express only the dimeric form and that the monomeric form on the surface of thymocytes from transgenic mice results from glycosyl phosphatidylinositol linkage. Our results show for the first time that under physiological conditions a TCR beta dimer can be expressed on the cell surface without the TCR alpha chain.     

Molecular modeling of interleukin-8 receptor beta and analysis of the receptor-ligand interaction

A structural model of interleukin-8 receptor type beta (IL-8R-beta) was constructed based on the structure of bacteriorhodopsin. High temperature molecular dynamics simulations were performed to search the possible conformations of loop regions in IL-8R-beta which recognize the ligand. The crystal structure of interleukin 8 (IL-8) was used as a geometric constraint of the extracellular loop regions of IL-8R-beta in the conformational search. 500 complex structures were extracted from the dynamics trajectory and five plausible models were selected based on the binding energy and known experimental data. To study further the interaction between IL-8R-beta and its ligands, the complex of IL-8R-beta and platelet factor 4 (PF4) C-terminal peptide was also modeled by molecular dynamics simulations. From these models, the N-terminus, extracellular domain 3 and extracellular domain 4 of IL-8R-beta were found to be important for ligand binding. Key residues of these regions involved in ligand binding were characterized. These models provide insight into the structural basis of biological activity of IL-8 and PF4 and may guide the design of potential therapeutic agents targeting IL-8 receptors. Furthermore, the approach developed from this study may have implications for the understanding of other chemokine receptor-ligand interactions that have been recently suggested to be involved in HIV infection.     

Restrictions of T cell receptor beta chain repertoire in the peripheral blood of patients with systemic lupus erythematosus

OBJECTIVE: To investigate whether oligoclonal T cell populations occur in peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE). METHODS: RNA was extracted from the lymphocytes isolated from whole peripheral blood of five female patients fulfilling ARA criteria for SLE and two healthy female controls, and synthesised into cDNA. CDR3 length spectratyping was performed using a polymerase chain reaction (PCR) run to saturation followed by a primer extension with a radioactively labelled primer. The resulting samples, one for each of the 23 V beta families, were then run on a polyacrylamide sequencing gel to examine the T cell receptor beta chain repertoire at the level of VDJ length heterogeneity. RESULTS: The two healthy female controls showed faint oligoclonal bands in only two and three V beta families respectively. Three of the patients showed a similar degree of oligoclonality to the controls, while the other two, who had active disease as shown by SLAM scores of 17 and 19 and in one case low C4 and raised C3dg levels, showed marked oligoclonality of their T cell beta chain repertoire affecting more than 17 of the 23 V beta families analysed. CONCLUSIONS: Using the technique of CDR3 length spectratyping, restriction of T cell receptor beta chain usage by peripheral blood T cells in patients with SLE has been demonstrated for the first time.     

An unusual skin lesion in a pediatric patient with Wegener's granulomatosis

Cutaneous manifestations occur in a significant number of patients with Wegener's granulomatosis (WG); however, the presentation and histopathology of these lesions are highly variable and may present problems in diagnosis. We report the presentation of a single large skin lesion in a pediatric patient with a history of WG and the characterization of this lesion by magnetic resonance imaging (MRI) and histopathology. MRI was helpful in delineating the extent of the lesion, although a skin biopsy was necessary to confirm the diagnosis of the vasculitic nature of the lesion.     

Molecular characterization of the di-leucine-based internalization motif of the T cell receptor

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinases. We have recently shown that both phosphorylation of Ser-126 and the presence of the di-leucine sequence Leu-131 and Leu-132 in CD3 gamma are required for protein kinase C (PKC)-mediated TCR down-regulation. To identify additional residues required for PKC-mediated phosphorylation of CD3 gamma and for TCR down-regulation, an alanine scanning of CD3 gamma was done. Mutations of Arg-124, Ser-126, Lys-128, and Gln-129 inhibited both phosphorylation and TCR down-regulation, whereas mutation of Asp-127 only inhibited down-regulation. Further analyses demonstrated a discrepancy between the ability to be phosphorylated on CD3 gamma and to down-regulate the TCR in several transfectants. Phosphorylation was not as strictly dependent on the nature and position of the phosphoacceptor group and basic residues as were the subsequent steps involved in TCR down-regulation. Our results suggest that PKC-mediated TCR down-regulation may be regarded as a two-step process. 1) Recognition and phosphorylation of CD3 gamma by PKC. In this process Arg-124, Ser-126, Lys-128, and Gln-129 are important. 2) Recognition of phosphorylated CD3 gamma by molecules involved in receptor internalization. In this process Ser(P)-126, Asp-127, Leu-131, and Leu-132 are important.     

Allelic polymorphism in the coding region of human T cell receptor V beta 12 gene

Many bilateral distal extension removable partial dentures possess insufficient guidance to control their dislodgement. When such a situation is observed, the tips of retentive clasps must be located in both an occlusogingival and mesiodistal (away from the denture base area) undercut. The specific location can be identified with a dental surveyor that has been adapted to function in two planes. Details of the adaptation process and use of the surveyor are described.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

On defining the rules for interactions between the T cell receptor and its ligand: a critical role for a specific amino acid residue of the T cell receptor beta chain

The specificity of T cell-mediated immune responses is primarily determined by the interaction between the T cell receptor (TCR) and the antigenic peptide presented by the major histocompatibility complex (MHC) molecules. To refine our understanding of interactions between the TCR and the antigenic peptide of vesicular stomatitis virus (VSV) presented by the class I MHC molecule H-2Kb, we constructed a TCR alpha chain transgenic mouse in a TCR alpha-deficient background to define specific structural features in the TCR beta chain that are important for the recognition of the VSV/H-2Kb complex. We found that for a given peptide, a peptide-specific, highly conserved amino acid could always be identified at position 98 of the complementarity-determining region 3 (CDR3) loop of TCR beta chains. Further, we demonstrated that substitutions at position 6, but not position 1, of the VSV peptide induced compensatory changes in the TCR in both the amino acid residue at position 98 and the length of the CDR3beta loop. We conclude that the amino acid residue at position 98 of the CDR3beta loop is a key residue that plays a critical role in determining the specificity of TCR-VSV/H-2Kb interactions and that a specific length of the CDR3beta loop is required to facilitate such interactions. Further, these findings suggest that the alpha and beta chains of TCRs interact with amino acid residue(s) toward the N and C termini of the VSV peptide, respectively, providing functional evidence for the orientation of a TCR with its peptide/MHC ligand as observed in the crystal structures of TCR/peptide/MHC complexes.     

Clinical quiz. Differential diagnosis of a patient with Wegener''s granulomatosis

STUDY OBJECTIVE: To describe a case series of emergency medical technician-basic (EMT-B)-administered epinephrine for anaphylaxis in a wilderness setting. DESIGN: Case series of patients in anaphylaxis who received epinephrine subcutaneously from EMT-Bs. SETTING: National park rural/wilderness emergency medical service system covering 863,000 acres and serving approximately 2 million annual visitors. PARTICIPANTS: Prehospital care providers were National Park Service rangers with EMT-B training. Patients in the series were visitors to Sequoia and Kings Canyon National Parks with anaphylaxis resulting from insect stings. INTERVENTION: Subcutaneous administration of epinephrine by EMT-Bs trained in recognition, understanding, and treatment of anaphylaxis. RESULTS: Eight patients with anaphylaxis resulting from Hymenoptera stings, from June 1992 through September 1993, received EMT-B-administered epinephrine. All patients improved clinically after treatment with epinephrine within 25 minutes. No major side effects occurred. CONCLUSION: Our data suggest that EMT-B-administered epinephrine is safe when used by EMT-Bs in the rural/wilderness setting, with appropriate physician supervision. Further study in large trials will be required to demonstrate safety and efficacy.