Mary Ann Robinson

A susceptibility locus for multiple sclerosis is linked to the T cell receptor β chain complex

Inheritance of T cell receptor beta chain (TCR beta) genes was analyzed in families of 40 sibling pairs concordant for the relapsing-remitting form of multiple sclerosis (MS). TCR beta haplotypes were determined by segregation analysis of polymorphic markers within the TCR beta complex. The mean proportion of TCR beta haplotypes identical by descent (IBD) inherited by MS sibling pairs was significantly increased compared with expected values (means test, p less than 0.004), whereas the distribution of haplotype sharing was random when MS patients were compared with their unaffected siblings. Furthermore, one allelic form of a TCR beta variable region gene segment was overrepresented on MS chromosomes compared with those parental chromosomes not transmitted to MS offspring both in the MS sibling pair families and in a second group of families containing only one individual affected with MS. These results demonstrate that a gene within the TCR beta complex or a closely linked locus influences susceptibility to MS.

The extent of the human germline T-cell receptor V beta gene segment repertoire

An assessment of the size of the human TCRBV gene segment repertoire based on the identification of TCRBV gene segments in genomic DNA was undertaken. PCR amplification from cloned and uncloned genomic DNA sources, nucleotide sequencing, Southern blot hybridization, and cosmid cloning were used to identify TCRBV gene segments in multiple unrelated individuals. The key advantages to this approach were: (1) TCRBV gene segments which are expressed only at very low levels in cDNA libraries were still detectable, and (2) it was possible to discriminate between alleles at the same locus vs products of different loci. A total of 63 unique TCRBV gene segments were identified and sequenced. Six of these TCRBV gene segments had not been previously described. Thirty-four cosmid clones containing 51 of the 63 identified TCRBV gene segments were isolated and screened for the presence of additional novel TCRBV subfamily members. These results, obtained by a variety of complementary approaches, indicate that the human TCRBV gene segments of which 52 are functional. The availability of the majority of these TCRBV gene segments on cosmid clones should facilitate further investigation of germline TCRBV gene segment polymorphism and putative disease associations.

Identification of a Novel Staphylococcus aureus Two- Component Leukotoxin Using Cell Surface Proteomics

Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.

Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection

Community‐associated methicillin‐resistant Staphylococcus aureus (CA‐MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA‐MRSA pathogenesis are poorly understood and a comprehensive analysis of CA‐MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2‐dimensional gel electrophoresis coupled with automated direct infusion‐tandem mass spectrometry (ADI‐MS/MS) analysis. Eleven known virulence‐related exoproteins differed in abundance between the strains, including alpha‐haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1‐phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host–pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA‐MRSA as a human pathogen.

Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity

The third complementarity-determining region (CDR3) is the only nongermline-encoded hypervariable region of the T cell receptor beta (TCRB) chain, and it is the region that has been predicted to confer fine specificity of the TCR for peptide-MHC complexes. For this reason analysis of TCRB CDR3 heterogeneity may provide insight into immune mechanisms operative in infectious and autoimmune diseases. PBMC stimulated with either mitogen (PHA), superantigen (TSST-1), or nominal antigen (tetanus toxoid) have been compared with unstimulated PBMC using a two-dimensional approach. Analysis of the expressed TCRBV gene repertoire CDR3 length profile coupled with SSCP methodology enabled the discrimination of sequences with the same CDR3 length. For both freshly isolated and PHA stimulated PBMC, a normally distributed spectrum of CDR3 lengths (five or more products) was observed. These products differed by 3 bp (1 amino acid) due to the strict requirement for in-frame rearrangements in the CDR3 region of TCR. By contrast, tetanus toxoid stimulated PBMC had restricted profiles for most TCRBV families after as few as 7 days of incubation. The oligoclonal nature of samples showing CDR3 length restriction was revealed by SSCP analysis and confirmed by sequence determination. Superantigen stimulation resulted in unique patterns of diversity, which included polyclonal expansion of specific TCRBV families as well as oligoclonal expansion of most other TCRBV families. These data reveal complex yet distinct patterns of TCR diversity in response to different T cell activation stimuli.

Spectratype/immunoscope analysis of the expressed TCR repertoire.

Measuring the diversity of TCRs utilized by specific primary and memory T cell responses is critical to the fundamental understanding of regulation of the immune system. This unit describes the Spectratype/Immunoscope technique which permits an in depth analysis of the TCR repertoire present in a variety of biological samples from mice to humans. Spectratype analysis takes advantage of PCR technology to amplify template cDNA corresponding to rearranged transcripts with different CDR3 lengths from specific TCR variable region genes in a competitive manner. The PCR products are then resolved on polyacrylamide sequencing gels to reveal precise sizes in nucleotide base pairs. The unit also includes protocols that have been optimized to process and produce the starting materials required for spectratype analysis.

Usage of human T-cell receptor Vβ, Jβ, Cβ, and Vα gene segments is not proportional to gene number

Abstract Certain T-cell receptor (TCT) β-chain variable (V), joining (J), and constant (C) gene segments, as well as TCRα-chain V gene segments, are disproportionally represented in TCR α and β cDNA libraries derived from PHA-stimulated peripheral blood lymphocytes. Sequences of 138 TCRα clones and 96 TCRβ clones were determined and of these 128 TCRα clones and 88 TCRβ clones were found to contain unique combinations of V, J, and C gene segments or to display diversity in N region nucleotides. The frequency of the V, J, and C genes used in the assembly of unique transcripts was ascertained. Of the 24 reported Vβ genes, families, 21 were observed among the 88 TCRβ clones including four Vβ families (Vβ1, Vβ2, Vβ3, and Vβ4) that were represented in the sample 2 1 2 −5 times more frequently than would be expected on the basis of copy number within the gene complex. Seventy-eight percent of the clones were positive for Cβ2 and more than half of the clones (53%) used one of two Jβ2 genes: Jβ2.1 was present in 27 clones and Jβ2.7 in 20 clones. TCR Vα families were also disproportionately represented in this sample. Twenty-five of 30 Vα families were observed in the sample of 128 clones including six recently reported Vα families. Three Vα families, Vα2, Vα8, and Vα23, accounted for ≈40% of the TCRα clones and were represented at 18%, 9.4%, and 13.3%, respectively. Both Vα2 and Vα8 gene families contain more than one gene; thus the number of clones observed in these families may, in part, be related to gene number. However, Vα23, which appears to be a single-copy gene family, is significantly overrepresented in this sample. Although disproportional usage of Vβ genes may be accounted for by superantigen exposure, reasons for disproportional usage of Jβ, Cβ, and Vα genes are presently unknown.

Contributions of CD4+, CD8+, and CD4+CD8+ T cells to skewing within the peripheral T cell receptor β chain repertoire of healthy macaques

Diversity in the peripheral T cell receptor repertoire of rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) has been studied by examining the profile of CDR3 lengths in TCR beta chains. Expressed CDR3 length distribution profiles for individual TCRBV families were obtained from total peripheral blood mononuclear cells (PBMC) and T cell subsets isolated from PBMC. These studies reveal that the T cell receptor repertoire of PBMC from healthy macaques often exhibits skewing in TCRBV family CDR3 profiles. The skewing of TCRBV family CDR3 profiles was evident as discrete expanded length(s) and was detected in up to 50% of the PBMC profiles. Analyses of separated T cell populations demonstrated that the CD8+ T cell subset was responsible for the majority of observed skewing in CDR3 length profiles. However, CD4+ T cells were also shown to contribute to the skewed peripheral PBMC repertoire in these animals. While certain TCRBV families frequently displayed skewed profiles, there was no concordance in the particular CDR3 lengths expanded among the different animals. Furthermore, an additional feature of the peripheral blood of the animals studied was the presence of an unusual population of extrathymic CD4 and CD8+ (double-positive) T cells (up to 9.6% in the PBMC of rhesus macaques). The double-positive T cells could be differentiated from CD4 single-positive and CD8 single-positive T cells by their increased surface expression of LFA-1 and decreased CD62L expression. The percentage of the double-positive T cells was higher in rhesus than pig-tailed macaques and contributed substantially to the peripheral T cell repertoire.

The human T-cell receptor variable gene segment TCRBV6S1 has two null alleles

The extent of polymorphism in TCRBV6S1 was examined by screening 203 individuals of diverse ethnic backgrounds by using SSCP. Three alleles were detected, including two that were described previously (TCRBV6S1*1 and *2P). The third allele (TCRBV6S1*3P), identified in these studies, is a pseudogene because, similar to allele *2P, it contains a substitution of a highly conserved cysteine residue near CDR3. Among a panel of 126 Caucasian donors, alleles *1, *2P, and *3P were observed to have frequencies of 0.72, 0.12, and 0.16, respectively. The extent of this survey suggests that it is unlikely for there to be additional common variants of TCRBV6S1. The approach used here enables rapid typing for polymorphism in a TCRBV gene that results in an allelically determined hole in the TCR repertoire.

Molecular determination of T-cell receptor α and β chain genotypes in human families

Abstract Polymorphism in genes encoding the α and β chain of the human T cell receptor has been detected by Southern blot analysis. Genomic DNA samples were isolated from B lymphoblastoid cell lines derived from members of families, each family including at least one individual with a recombinant HLA haplotype. T cell receptor α and β chain haplotypes could be assigned in the families on the basis of observed restriction fragment length polymorphism (RFLP). Polymorphism in the α chain gene was detected in Bg1II digests using an α chain probe that included the V, J, C, and 3′ untranslated sequences. A probe consisting of only the constant region (Cα) revealed no polymorphism indicating that the polymorphic fragment hybridized to V, J, or 3′ untranslated sequences of the α chain. Polymorphism in β chain genes was observed in Bg1II digested DNA samples using a probe that corresponds to the constant region (Cβ). Polymorphic Cβ restriction fragments of 10.0 and 9.2 kilobase segregated in six of the eight families studied. Recent structural data for the Cβ region suggest that the polymorphic Bg1ii site lies in the region 5′ to the Cβ2 gene. These polymorphisms should serve as markers for α and β chain complexes allowing genetic studies of these immunologically important gene families.