Constant Region Primer Research Articles

Detection of T cell receptors in early rheumatoid arthritis synovial tissue.

Synovial tissue is rarely available from patients with early synovitis, with the exception of synovial biopsies. However, T cell populations early in the development of synovitis may be enriched in antigen-specific cells and critical to disease pathogenesis. To investigate the T cell repertoire in early synovitis, we utilized a PCR protocol for detection of T cell receptor (TCR) transcripts present in small amounts of synovial tissue. To expand the substrate for PCR, preamplification of cDNA was performed with a 3' constant region primer plus either a mixture of variable region primers or random hexanucleotides. Utilizing this method improved the sensitivity of detection. This technique is applied here to the analysis of TCR transcripts in synovial biopsies from individuals with early rheumatoid arthritis (RA) and non-RA synovitis. TCR alpha-chain transcripts were detectable in 5/5 RA and 4/4 non-RA specimens evaluated, with beta-chain transcripts detected in 4/5 early RA and 4/4 non-RA specimens evaluated. Confirmation of transcripts by sequencing of cloned PCR products verified the specificity of amplification. The most frequently expressed TCR V region families in early RA synovitis were V alpha 11, V alpha 14, V alpha 28, V beta 7, V beta 9 and V beta 17. Several of these V regions have previously been implicated in studies of chronic RA synovitis. J alpha and J beta region usage was similar to that seen in chronic RA, and conserved N region motifs were apparent. We conclude that it is possible to detect TCR transcripts in small synovial biopsies from individuals with early arthritis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ex vivo clonotype primer-directed gene amplification to identify malignant T cell repertoires.

A novel strategy that utilizes input genomic DNA and overcomes limitations encountered with traditional RNA reverse transcription-polymerase chain reaction (PCR) amplification methodology is described to screen for T cell receptor (TCR) repertoires. The methodology has been developed to identify individual T cell clonotypes with regard to their unique receptor beta chain variable/diversity/joining (VDJ) region gene rearrangement. The technique avoids preselection for a given antigen specificity and is therefore independent of artificial bias introduced by in vitro cell population expansion. This technique was used to detect and identify genetically of malignant clones from heterogeneous mononuclear cell populations from an array of hemato-oncological disorders, including mycosis fungoides/Sézary Syndrome, adult T cell leukemia, and large granular lymphoproliferative disease. An initial primary PCR, directed by a TCR-J beta generic primer and a complement of family-specific TCR-V beta primers, defines predominant T cell receptor variable gene usage. Use of a TCR-J beta generic primer supplants the use of a constant region primer anchor and thus eliminates the need to target mRNA. The process of variable gene screening also expedites gene sequencing. By sequencing through the VDJ juxtaposed region, i.e., the third complementarity determinant region, clonotype-specific primers are developed and used in a secondary clonotype primer-directed PCR (CPD-PCR) to detect, with extreme sensitivity and specificity, unique T cell clonal repertoires. Analysis of the products of the CPD-PCR permits the detection of a single malignant cell among one million polyclonal cells and supercedes the constraints of prior studies that provide a limited evaluation of family variable gene repertoire usage. This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease-free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones.

Development of the non-palindromic adaptor polymerase chain reaction (NPA-PCR) for the amplification of alpha- and beta-chain T-cell receptor cDNAs.

We have developed a highly efficient new method for the amplification of alpha- and beta-chain human T-cell receptor (TCR) cDNAs. This method is designated non-palindromic adaptor polymerase chain reaction (NPA-PCR). cDNA was synthesized from total RNA isolated from mononuclear leucocytes, using either an oligo (dT)15-NotI or a C alpha-NotI or a C beta-NotI primer and RNase H-negative reverse transcriptase. The double-stranded cDNA was ligated with the non-palindromic adaptors EcoRI-XmnI [d(ATTCGAACCCCTTCG)] and XmnI G strand [d(pCGAAGGGGTTCG)] (phosphorylated), which resulted in the addition of the EcoRI-XmnI site in both 5' and 3' ends. These two non-palindromic adaptors, EcoRI-XmnI and XmnI G strand, are complementary to each other and both are required for ligation. The EcoRI-XmnI adaptor was removed from the 3' end by treatment with NotI restriction nuclease, whereas it was retained at the 5' end. The non-palindromic adaptor EcoRI-XmnI was used as the 5' amplification primer. C alpha or C beta constant region primers were used as 3' amplification primers. The amplified cDNAs were cloned and the plasmids were used to transform DH5 alpha competent cells. Over 1000 white colonies per 0.1-0.25 micrograms of total RNA or per 10,000 to 50,000 human peripheral blood mononuclear cells were obtained after amplification of either the alpha- or the beta-chain TCR cDNAs. Between 40 and 62% of the colonies (range from five donors) were positive after screening with either a C alpha or a C beta probe, located 5' to the C alpha and C beta amplification primers. A total of 50 amplified alpha- or beta-chain cDNA positive clones from two normal donors were randomly chosen and sequenced, and the sequences obtained were typical of alpha beta TCR. Two new J alpha gene segments were identified. Approximately 30% of the alpha-chain positive clones have 5' untranslated region, and most of the remaining alpha- or beta-chain TCR clones started from the initiation codon or near the 5' end. NPA-PCR has several advantages over existing PCR methods for the amplification of cDNAs with unknown or variable 5' end, such as the T-cell antigen receptors and the immunoglobulins. Among these advantages is that only one 5' end extension primer is required. Because of the large number of TCR V alpha and V beta families, a large number of different 5' end primers are required for amplification of alpha beta TCR cDNAs by conventional PCR.

The T-cell-receptor repertoire in the synovial fluid of a patient with rheumatoid arthritis is polyclonal.

We have analyzed the T-cell-receptor repertoire expressed in the synovial fluid of a patient with rheumatoid arthritis by using an inverse polymerase chain reaction. Total RNA was isolated from Ficoll-purified mononuclear cells and converted into circularized double-stranded cDNA. Specific amplification of alpha- and beta-chain variable regions (V alpha and V beta) was achieved with inverted alpha- and beta-chain constant region (C alpha and C beta) primer pairs, and the amplification products were cloned into phage vectors. A total of 78 alpha and 76 beta clones were sequenced, and 67 and 72 productively rearranged alpha and beta genes were identified, respectively. Thirty-one V alpha, 33 alpha-chain joining region (J alpha), 29 V beta, and 12 beta-chain joining region (J beta) gene segments were found in the productively rearranged clones, indicating that the T-cell repertoire expressed in the synovial fluid of this RA patient is highly heterogenous and polyclonal. Comparison of peripheral blood and synovial fluid repertoires showed that the most abundant V beta sequences, V beta 2.1 and V beta 3.1, were enriched in the inflamed joint by a factor of 2 to 3. It is possible that T cells expressing these V beta gene segments, which recognize bacterial superantigens, play a role in the disease.

Construction of representative immunoglobulin variable region cDNA libraries from human peripheral blood lymphocytes without in vitro stimulation

We have developed a sensitive and specific method for preparation of representative IgM and IgG cDNA libraries of peripheral blood lymphocytes without in vitro stimulation of B cells and without primer-based bias. The procedure involves preparation of double-stranded cDNA from initial Cμ and Cγ constant region primers. Linkers are attached to the cDNA and nonselective polymerase chain reaction (PCR) amplification is performed with the linkers as primers. This product is ligated to M13 RF DNA, and selective PCR amplification is achieved with a nested constant region primer and a vector primer. The product is cloned into M13 phage. More than 85% of the white plaques are positive with a J H probe. Diversity of the library is confirmed by hybridization with V H gene probes and by DNA sequencing. A sample of 7–30 ml of blood from a normal human adult can yield both μ and γ cDNA libraries. Analysis of the libraries yields a picture of Ig variable gene usage at the time of sampling.

PCR amplification of antibody genes

A general method for directly obtaining the DNA sequence of the variable regions of any immunoglobulin chain by using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5′ signal peptide or framework 1 and a conserved 3′ constant region primer were used for enzymatic amplification of the heavy and light chain variable regions from human hybridomas producing monoclonal antibodies that recognize epitopes on HIV-1 (gp120, gp42), digoxin, tetanus, immunogibulins (rheumatoid factor), and MHC class I and II proteins. Both γ and μ heavy chains and κ and λ light chains were successfully amplified and subsequently cloned. A similar strategy was used to amplify variable regions of mouse heavy and light chain variable regions from hydridomas recognizing human T cell antigens (CD3, CD6), an outer membrane protein of Neisseria meningiditis , carcinoembryonic antigen, fibrin, and PRP, the major carbohydrate antigen of Haemophilus influenza . Variable regions can be amplified from single hybridoma cells isolated by micromanipulation or fluorescence-activated cell sorter. The method makes it possible to construct antibody libraries in phage without resort to hybridoma technology. Sensitive studies of B-cell differentiation and ontogeny, B-lymphoma oncogenesis and heterogeneity, and antibody diversity, as well as immortalization and manipulation of the antibody repertoire for diagnostic and therapeutic purposes, are now possible.

Cloning and sequencing of imniunoglobulin variable-region genes using degenerate oligodeoxy-ribonucleotides and polymerase chain reaction

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5′-oligodeoxy-ribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from γ and μ heavy chains and from κ light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5′-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from γ and μ isotypes and the universal light-chain primer was used to amplify three separate κ light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG 1/κ, IgG 2b/κ and IgM/κ isotypes.

Early onset of somatic mutation in immunoglobulin VH genes during the primary immune response.

The dynamics of somatic mutation in Ig variable genes was investigated in order to define a population of B cells undergoing mutation. BALB/cJ mice were injected with PC-KLH, and splenic RNA was prepared 5, 7, and 13 d later. The mRNA was annealed to gamma constant region primers to make cDNA transcripts encoding VH genes. 103 cDNA clones corresponding to 18 different genes from the VH7183, VH3660, and VHS107 subfamilies were sequenced to identify mutation. VH genes had a low level of mutation on day 5 after immunization and accumulated more mutation by day 7 at a rate of 10(-3) mutations per nucleotide per generation. However, by day 13, the number of mutations per gene did not increase, and most of the substitutions encoded replacement amino acid changes that were clustered in the hypervariable regions, indicating that the mutational process was less active during the second week and that antigen selection had occurred. The data are consistent with a developmentally regulated mechanism in which mutation is activated during the first week of the primary immune response for a limited time period, after which selection acts to preserve the beneficial mutants.

Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction

A general method to directly obtain the DNA sequence of the variable regions of any immunoglobulin chain using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5' signal peptide and a conserved 3' constant region primer were used for enzymatic amplification of each of the heavy and light chain variable regions of a human hybridoma producing a monoclonal antibody recognizing an epitope of gp120 of the human immunodeficiency virus 1. The amplified DNA segments were cloned and the sequence was determined for the heavy chain variable region. This method will greatly facilitate structural and functional studies of immunoglobulins by reducing the effort to clone and sequence the members of the immunoglobulin as well as other multigene families.