Alex So

The clinical and immunogenetic features of patients with autoantibodies to the nucleolar antigen PM-Scl.

The clinical and laboratory features of 32 patients with anti-PM-Scl were studied. Patients with this rare autoantibody suffered from a homogenous overlap connective tissue disease defined by Raynaud phenomenon (32/32), features of scleroderma (31/32), arthritis (31/32, erosive in 9/32), myositis (28/32), lung restriction (25/32), calcinosis (15/32), and sicca (11/32). Significant renal and neurologic involvement was uncommon. All patients examined (22/22) had HLA-DR3, and 50% of these patients were homozygous. Our patients responded favorably to moderate immunosuppression and, with therapy, the disease generally has a good prognosis; over 50% of our series (17/32) remained well on minimal or no immunosuppression after a median follow-up of 8 years.

PCR-based analysis of the TCR repertoire in human autoimmune diseases

Characterization of T cells at sites of autoimmune damage has been difficult. Now, however, polymerase chain reaction (PCR)-based methods are being used to analyse T-cell receptor (TCR) gene expression in such lesions. Here, Christopher Marguerie, Claudio Lunardi and Alex So summarize and interpret the most recent findings and describe the current understanding of TCR usage in autoimmune diseases.

An altered repertoire of T cell receptor V gene expression by rheumatoid synovial fluid T lymphocytes

The pattern of T cell receptor V gene expression by lymphocytes from rheumatoid synovial fluid and paired peripheral blood samples was compared using a polymerasc chain reaction (PCR)‐based assay. Eight rheumatoid arthritis (RA) patients who had varying durations of disease (from 2 to 20 years) were studied. In all patients there was evidence of a different pattern of V gene expression between the two compartments. Significantly increased expression of at least one Vα or Vβ gene family by synovial fluid T cells was observed in all the patients studied. Three different Vα (Vα 10, 15 and 18) and three Vβ (Vβ 4, 5 and 13) families were commonly elevated. Sequencing of synovial Vβ transcripts demonstrated that the basis of increased expression of selected V gene families in the synovial fluid was due to the presence of dominant clonotypes within those families, which constituted up to 53% of the sequences isolated from one particular synovial V gene family. There were considerable differences in the NDJ sequences found in synovial and peripheral blood T cell receptor (TCR) transcripts of the same Vα gene family. These data suggest that the TCR repertoire in the two compartments differs, and that antigen‐driven expansion of particular synovial T cell populations is a component of rheumatoid synovitis, and is present in all stages of the disease.

A new polymorphic marker of the T-cell antigen receptor α chain genes in man

The restriction fragment length polymorphism of the unrearranged T-cell antigen receptor (Tcr) α chain gene was investigated. Taq I digests, when probed with a Tcr α chain cDNA probe, revealed polymorphic bands of 7.0, 2.0, and 1.4 kb, due to variations around the Cα gene, and the V gene cluster. Family studies confirmed the segregation of these polymorphic bands as allelic markers. These polymorphisms provide a new marker for the analysis of genetic variation of the Tcr a chain, and the influence of variation of the Tcr genes on the immune response.

Reduction in T gamma delta cell numbers and alteration in subset distribution in systemic lupus erythematosus.

We have studied the distribution of Tγδ cells in the peripheral blood of 35 patients with systemic lupus erythematosus (SLE) and 36 age‐matched controls. The monoclonal antibodies A13, BB3 and TiγA, which are specific for the Vδ1, Vδ2 and Vδ9 gene products respectively, were used to define Tγδ cell subsets. A significantly lower frequency of Tγδ cells was found in peripheral blood lymphocytes of SLE patients compared with normal subjects (3.2%versus 5.9%). There was a marked reduction in the Vδ2+ subset of Tγδ cells, which resulted in a reversal of the ratio of Vδ2+/Vδ1+ cells from 4.34 to 0.56. No correlation was found with either clinical or laboratory measures of disease activity. These results suggest that the observed changed in Tγδ subset distribution are related to the SLE itself, and not secondary to changes in disease activity.

HLA class I expression on erythrocytes and platelets from patients with systemic lupus erythematosus, rheumatoid arthritis and from normal subjects

It has previously been shown, by a haemagglutination assay, that patients with systemic lupus erythematosus (SLE) express increased levels of HLA class I on erythrocytes compared with normal subjects and patients with rheumatoid arthritis (RA). A radioligand‐binding assay, using monoclonal antibody W6/32, was devised to quantify HLA class I expression on erythrocytes and platelets. An increased number of class I molecules was expressed on erythrocytes from 45 patients with SLE (mean = 354 molecules per cell, median = 255 molecules, range = 30–1270 molecules per cell), compared with cells from 46 normal subjects (mean = 132, median = 78, range = 40–550) and 31 RA patients (mean = 132, median = 89, range = 26–497). The presence of HLA‐B7 correlated with increased class I expression on erythrocytes from both normal subjects and patients with SLE. Levels of HLA class I in serum were measured. All subjects with HLA‐A9 (A23, 24) showed higher levels of serum class I than their A9‐negative counterparts, and there was no difference in levels between SLE patients and normal subjects. There were no correlations between class I levels in serum and on erythrocytes amongst SLE patients or normal subjects. Red cells were fractionated, according to their age in vivo, on Percoll gradients. Class I levels fell with increasing erythrocyte age in all individuals, but were higher in all fractions from SLE patients compared with age‐matched fractions from normal subjects. HLA‐B7‐positive erythrocytes also expressed higher class I levels in each Percoll fraction, compared with their HLA‐B7‐negative counterparts, suggesting that enhanced B7 expression is not due to greater structural stability of this class I allotype. These data are compatible with the hypothesis that class I is expressed as an intrinsic protein of erythrocyte membranes and that expression is increased amongst patients with SLE.

Analysis of T cell receptor V alpha polymorphisms in rheumatoid arthritis

OBJECTIVE To test for association of T cell receptor (TCR) V alpha polymorphisms and rheumatoid arthritis (RA) in British and Swiss white populations. METHODS TCRAV polymorphisms were analysed in RA patients and controls by single strand conformational polymorphism (SSCP) analysis. Associations were sought between defined genotypes and RA, and the effect of HLA-DR4 status analysed. Putative associations were then retested further in new groups of patients and controls. Overall, 360 RA patients and 197 controls were studied. RESULTS No association between TCRAV5S1, V6S1, V8S1, V17S1 or V21S1 polymorphisms and RA were observed in the initial population screened. Stratification for DR4 status showed an increase of V5S1*01/*01 in DR4 positive versus DR4 negative patients (χ2 = 7.19, p=0.028 (2df), p=0.14 after correction for multiple comparisons). This putative association was tested in three further patient groups, none of which showed significant increase of V5S1*01/*01 in DR4 positive patients, although an overall trend towards an increase in V5S1*01/*01 was observed. CONCLUSION No evidence was found for a strong association of TCRAV genes and RA in a white population. However, these results suggest a weak association of V5S1*01/*01 with DR4 positive RA, although this requires confirmation using larger groups of patients and controls.

Identification of novel human T-cell receptor Vβ gene segments by the anchored-polymerase chain reaction

We have studied the diversity of the expressed human T-cell receptor (TCR) β-chain repertoire by analysis of mRNA from unstimulated peripheral blood T-cells. The anchored-polymerase chain reaction (PCR) was used to isolate TCRB transcripts. Of 20 full or near full-length functional transcripts sequenced, two were novel TCRVB gene segments. They have strong sequence similarities to the known TCRBV5, and 8 subfamilies. Southern blot analysis and sequence-specific oligonucleotide hybridization confirmed: a) that these sequences are present in genomic DNA; b) their relationship to the known TCRBV families. A TCRBV sequence similar to a recently identified novel TCRBV24 subfamily was also found. We show by southern blotting that this sequence forms a single member subfamily, and by deletion analysis of T-cell lines, we have mapped this sequence to lie between the genes which encode the TCRBV8.1 and TCRBV5.3 gene segments. The results show that the anchored PCR is a powerful tool in the analysis of the TCR repertoire, which may contain more V gene segments than previously defined.

T-cell receptor variable alpha (TCRAV) polymorphisms in European, Chinese, South American, AfroCaribbean, and Gambian populations.

Abstract Interactions involving the T-cell receptor (TCR) and major histocompatibility complex (MHC) are fundamental to the generation of a specific immune response. The study of interpopulation differences in TCR genes may identify those genes which are subject to selection, and also provides useful information for future genetic studies in these populations. In this study we present analysis of five TCRAV polymorphisms, for V5S1, V6S1, V8S1, V17S1, and V21S1 loci in five human populations by single-strand conformational polymorphism (SSCP) analysis. Caucasian, Chinese, Gambian, AfroCaribbean, and South American Indians (Mapuches) showed marked interpopulation variation for both the silent (V5S1, V17S1, and V21S1) and coding (V6S1 and V8S1) polymorphisms. In general the alleles were conserved in the different populations, but new, additional variants were found for V5S1 and V17S1 in Gambians and Caucasians. V6S1 overall showed the highest nucleotide diversity, and V6S1 genotype distributions were skewed away from expected values in Chinese and Mapuches. Analysis of allelic associations showed a general lack of linkage disequilibrium between the loci, which was reflected by the absence of strong population-specific haplotypes.

Physical mapping of the human T-cell receptor beta gene complex, using yeast artificial chromosomes

Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell β chain gene complex (TCRB). Variable region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identified, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes (BC) when analyzed by pulse field gel electrophoresis. Restriction enzyme mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragments of 280 110, 90, and 125 kbp and was in accordance with published data. In addition comparison of hybridization results of Sfi I-restricted B3 and genomic DNA from the parental cell line GM1416B revealed identical banding patterns. The data thus showed YAC B3 encoded a complete and unrearranged TCRB gene locus of some 600–620 kbp. The map was further resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene families. Flourescent in situ hybridization of B3 to spreads of human metaphase chromosomes localized B3 to 7q35. However, two additional signals were obtained; one attributable to the TCRBV orphon cluster on 9p21, the second to the long arm of chromosome 2. Polymerase chain reaction amplification of a chromosome 2 somatic cell hybrid, using primers for all 25 TCRBV gene families, revealed that the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCRB locus flanked by chromosome 2 sequences.