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WO1998054223A2 - Procede d'immunologie - Google Patents

Procede d'immunologie Download PDF

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Publication number
WO1998054223A2
WO1998054223A2 PCT/GB1998/001382 GB9801382W WO9854223A2 WO 1998054223 A2 WO1998054223 A2 WO 1998054223A2 GB 9801382 W GB9801382 W GB 9801382W WO 9854223 A2 WO9854223 A2 WO 9854223A2
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specific
cell
cells
antigen
subset
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PCT/GB1998/001382
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WO1998054223A3 (fr
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Richard Andrew Kay
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University Of Dundee
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Priority to AU76631/98A priority Critical patent/AU728909B2/en
Priority to JP50035099A priority patent/JP2001517958A/ja
Priority to CA002291004A priority patent/CA2291004A1/fr
Priority to EP98924427A priority patent/EP1017724A2/fr
Publication of WO1998054223A2 publication Critical patent/WO1998054223A2/fr
Publication of WO1998054223A3 publication Critical patent/WO1998054223A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to an immunological method, in particular it relates to a method of identifying antigen-responsive T cells.
  • T cells are fundamental to the immune process. They play a central role as either regulator or effector in a wide range of immune-driven responses. Their function can be beneficial to the host as in the immune response to infections or tumours but it may also be detrimental such as in autoimmunity, allergy and transplant rejection.
  • T cells recognise peptide antigens presented to them in the context of major-histocompatibility complex-encoded molecules [1,2]. They accomplish this by virtue of a cell-surface, clonally-distributed heterodimer known as the idiotypic T cell receptor (TCR).
  • TCR is usually composed of an ⁇ and ⁇ chain and more rarely a ⁇ and ⁇ chain.
  • Each of these chains has an i-mmunoglobulin-like structure with a variable and a constant domain.
  • the constant domain (as its name suggests) is the same within each chain type (ie all ⁇ chain constant regions are identical) but the variable domain differs between each TCR [1,3-5].
  • variable domain structures arise because the gene which encodes them is formed by the random recombination of smaller gene segments which are imprecisely joined together. These smaller segments are known as variable (N), diversity (D) ( ⁇ and ⁇ chains only) and joining (J) gene segments [6].
  • N variable
  • D diversity
  • J joining
  • Studies of the structure of the chromosomes which encode the TCR chains reveal there are 50-52 functional TCRBV ( ⁇ chain variable gene segments) [7], at least 70 TCRAV (a. chain variable gene segments) [8-14] and 57 TCRAJ (a chain joining gene segments) [15].
  • the molecular biological approach involves enumerating the spectrum of TCR mRNAs expressed at a particular site or in a particular lesion and comparing this to the TCR mRNA repertoire at other sites or in control individuals.
  • a variety of techniques have been employed to accomplish this which are mostly dependent on the polymerase chain reaction (PCR) [32].
  • PCR polymerase chain reaction
  • the most reliable techniques in terms of enumeration have been inverse PCR or anchored PCR.
  • Other semi-quantitative techniques such as family-specific PCR have also been used. This approach is essentially similar to immunohistochemical one but, since all the TCRBV genes are now known, it is more complete than has been achievable to date with a more limited range of anti-V ⁇ monoclonal antibodies.
  • TCR mRNA levels approximate to specific T cell numbers but in order for this to be true two basic assumptions must also be correct: (1) all TCR genes should be transcribed at the same rate; and (2) gene transcription does not vary when T cells are stimulated. As discussed in more detail below, my data suggest that both of these assumptions are incorrect.
  • the TCR can show extraordinarily specificity for peptide antigen presented in the context of MHC molecules, yet the affmity of the TCR for the peptide/MHC complex is low and the off rate for the interaction is high [33-35]. Further complicating this apparent paradox is the fact that as few as 100 peptide/MHC complexes may be required to fully trigger specific T cells, and the fact that sustained TCR to peptide/MHC complex contact is required for full T cell commitment to activation [36- 38].
  • T cell uses its cytoskeleton to move over the surface of the antigen-presenting cell, sequentially making contact with relatively few of the antigen-presenting cell's [APC's] MHC/peptide complexes, with tens of thousands of its own surface TCRs [39]. The summation of each of these signals, over a period of time, leads to the sustained second messenger levels required for a commitment to T cell activation [38-40].
  • TCRBV2 mRNA levels Semi-quantitative measurement of TCRBV2 mRNA levels in patients suffering from toxic-shock syndrome showed that mRNA levels increased sharply during the acute phase of the illness and settled to control levels within approximately three months of this condition being successfully treated.
  • the toxic shock syndrome toxin (TSST-1) is a superantigen specific for rC ⁇ fl V2-encoded TCRs [47,48].
  • the TCR V ⁇ mRNA is not measured on a "per specific T-cell" basis.
  • Kawasaki disease a condition caused by Staphylococci and Streptococci releasing a TSST-1-like superantigen, the rate of production of TCRBV2S1 mRNA by individual T cells increased in the acute phase of the disease and settled to control levels after treatment.
  • TCRBV12 mRNA production rates were shown not to alter after T cells were treated with SEB (a different TCRB V72-specif ⁇ c superantigen) in vitro.
  • SEB a different TCRB V72-specif ⁇ c superantigen
  • the mRNA production rate appeared constant both cell numbers and mRNA levels increased proportionally after this treatment [49,50] .
  • measurement of the increase in specific TCR mRNA production per specific T cell can be used generally to identify antigen-response T cells when the stimulating antigen and responding TCR are not known.
  • Duchmann et al (1993) DNA and Cell Biol. 12, 217-225 describes a purportedly quantitative method for measuring TCR N ⁇ subfamilies by reverse transcriptase (RT)-PCR, but TCR mR ⁇ A is not measured per specific T cell.
  • RT reverse transcriptase
  • One object of the present invention is to provide a method which allows the identification of antigen responsive T cells or the particular TCR involved in an antigen response when the stimulating antigen is not known and when there is no clue or few clues as to what particular T cell or TCR is involved in an antigen response.
  • the method is particularly useful for identifying T cell (and T-cell receptor; TCR) types involved in antigen-mediated diseases.
  • T cell and T-cell receptor; TCR
  • Many human diseases are believed to involve antigen-driven T cells including allergies, autoimmune disease, allograft rejection and acceptance, some infectious diseases such as parasitic diseases, and some cancers. These diseases include, for example, multiple sclerosis, farmer's lung, hay- fever and eczema.
  • TCR gene expression is increased after antigen stimulation in order to replace the receptors which have been lost from the cell surface during antigen triggering.
  • antigen and are therefore phosphorylated and internalised
  • TCR-specific mRNA production rates is a particularly suitable method to discriminate between passively recruited/passively-activated T cells and antigen-specific T cell effectors in any immune process.
  • the rate of mRNA synthesis is measured over a fixed period (ie there is a fixed time between antigen contact with the T cell and the time when the mRNA is measured) and so the rate of mRNA production is equivalent to the amount of mRNA synthesised in a fixed period of time.
  • Chronic diseases involving antigen-mediated processes are believed to involve chronic presentation of antigen to T cells. In these circumstances it can reasonably be assumed that T cells are constantly being triggered by antigen.
  • T cell numbers or mRNA alone In clinical situations, where massive T cell activation occurs (for example in toxic shock syndrome), quantitation of either specific cells or mRNA may be sufficient to make a determination of the T cell responsive to a particular antigen (although it helps to know which T cell receptor subset (such as a particular V ⁇ ) and which superantigen you are looking for).
  • T cell receptor subset such as a particular V ⁇
  • a first aspect of the invention provides a method of identifying an antigen-responsive T cell within a population of T cells, the method comprising the steps of
  • the increase in specific TCR gene expression following antigen stimulation may be determined using any suitable method.
  • mRNA is synthesised and the mRNA is translated into polypeptide. Any protocol for identifying mRNA synthesis may be used and, since mRNA is relatively unstable, measurements of the amount of mRNA over a particular time period is probably a reasonable estimate of the synthesis of new mRNA.
  • Polypeptides are generally more stable than mRNA and so, typically, measuring the amount of specific TCR polypeptide may not distinguish between existing TCR polypeptide and newly synthesised TCR polypeptide.
  • Methods which can distinguish newly synthesised, specific TCR polypeptides and existing, specific TCR polypeptides may, however, be used in the method of the invention but it is preferred if mRNA specific for an individual T cell receptors or for subsets of T cell receptors is measured.
  • the sample containing T cells which have not responded to the antigen may be any suitable control sample as is discussed in more detail below.
  • the control level of expression of a gene encoding a specific T cell receptor or of genes encoding a subset of T cell receptors is measured as for the test sample and, typically, a control level can be set for each specific T cell receptor or subset of T cell receptors.
  • the comparison of the level of specific gene expression in a sample which has responded to the antigen with a level in a sample containing T cells which have not responded to the antigen may be an historic comparison with the levels in a control sample which has been determined separately at an earlier time although, of course, it is particularly preferred if a substantially identical protocol has been used to measure the level of gene expression (eg the amounts of specific T cell receptor mRNA) in the test sample and the control sample.
  • a substantially identical protocol has been used to measure the level of gene expression (eg the amounts of specific T cell receptor mRNA) in the test sample and the control sample.
  • the comparison of the levels of gene expression may be measured in samples taken and, optionally, analysed contemporaneously.
  • a particularly preferred embodiment of the invention provides a method of identifying an antigen-responsive T cell within a population of T cells, the method comprising the steps of
  • T cell receptor mRNA determining individually for each of a plurality of specific T cell receptors, or individually for each of a plurality of subsets of T cell receptors, the amount of T cell receptor mRNA, which mRNA is specific for a T cell receptor or is specific for a subset of T cell receptors, per specific T cell receptor-positive T cell or per specific T cell receptor-positive T cell subset, in the sample obtained in step (1); and (3) determining which T cell receptor mRNA has an increased amount per specific T cell in the samples obtained in step (1) compared to that in a sample containing T cells which have not responded to the antigen.
  • step (1) comprises obtaining (a) a sample containing T cells which have not responded to the antigen and (b) a sample containing T cells which have responded to the antigen and in step (3) it is determined which T cell receptor mRNA has an increased amount per specific T cell in sample (b) compared to sample (a).
  • samples (a) and (b) can be taken and, optionally, the specific TCR mRNA measured, contemporaneously or in some circumstances sample (a) may be an historic test sample.
  • normal ranges of specific TCR gene expression for untriggered T cells can be obtained by reference to normal, healthy individuals.
  • T cells can be obtained by bleeding a suitable number (eg 8 to 20) normal healthy individuals and measuring the numbers of TCR mRNA molecules per T cell for each specific TCR gene or specific subset of TCR genes that one wishes to study.
  • the person-to-person variation in the numbers of TCR- specific mRNA molecules per cell is not great for both TCRBV2S1 and TCRBV3S1 ( Figures 4 and 5). It should be noted that there is little variation in the numbers of specific TCR mRNA molecules per cell between T cells freshly obtained from subjects' peripheral blood (PRE sample) and the same cells cultured for three days in the presence of RPMI 1640 and 10% heat-inactivated foetal calf serum (CON sample) as described in Example 1.
  • the control sample may be from suitable T cells in culture.
  • Ranges for numbers of specific TCR mRNA molecules per cell in antigen-triggered T cells can be derived, in culture, by, for example, triggering the T cells with either superantigens or anti-V ⁇ -specific monoclonal antibodies or anti-CD3 antibodies. The last two methods of triggering are considered to approximate to the situation encountered with conventional antigen in vitro.
  • a suitable number eg 8 to 20
  • a range for triggered TCR mRNA molecules per cell can be obtained.
  • Ranges for triggered TCR mRNA per cell levels may be obtained in vivo if a number of patients suffering with TCRBV-specific diseases are studied during the course of their disease and then again once they had successfully recovered. Examples, as discussed in more detail below, include TCRBV2S1 mRNA levels per T cell in Toxic shock syndrome or Kawasaki disease.
  • the method of the invention may be used to identify a specific antigen- responsive T cell or it may be used to identify a subset of T cells involved in a particular antigen response; for example, the method may be used to identify a T cell receptor subset each of which contain a common V ⁇ segment or a common V ⁇ segment or combinations thereof.
  • T cell receptor subset each of which contain a common V ⁇ segment or a common V ⁇ segment or combinations thereof.
  • smaller and smaller subsets of T cells involved in an antigen response can be identified by, for example, first identifying the specific V ⁇ subset and then the specific V ⁇ -J subset and then the specific V ⁇ subset and then the specific V ⁇ -J subset and then the specific V ⁇ -J/V ⁇ -J subset of TCR.
  • the method is particularly useful in identifying an antigen-responsive T cell, and therefore a specific T cell receptor type, which is associated with a disease state.
  • the population of T cells may be any suitable population of T cells, for example a population of T cells from a mammal. It is preferred if the population of T cells is a population from a human patient; in particular it is preferred if the population of T cells is a population of T cells associated with a disease in a human patient.
  • sample (a) is obtained from a non-diseased site of an individual and the sample containing T cells which have responded to the antigen (“sample (b)”) is obtained from a diseased site of an individual.
  • sample (a) may be obtained from non-diseased synovial samples of a patient whereas sample (b) may be obtained from synovial samples of joints showing signs of rheumatoid arthritis from the same patient.
  • antigen- responsive T cells involved in rheumatoid arthritis may be identified.
  • sample (a) is obtained from a non-diseased individual and the sample containing T cells which have responded to the antigen (“sample (b)") is obtained from a diseased individual.
  • sample (a) may be obtained from a healthy control individual or a convalescent Kawasaki disease patient and sample (b) may be obtained from an acute Kawasaki disease patient.
  • the sample containing T cells which have responded to antigen is a sample obtained from an individual which has been contacted with the antigen (which may be in the form of a disease-causing agent) in vitro.
  • the sample may be a T-cell-containing sample from an individual which is treated with a microorganism in vitro or which is treated in vivo with an antigen or mixture of antigens derived from a microorganism.
  • the population of T cells may be lymphocytes obtained from a peripheral blood sample which are treated with Staphylococcal enter otoxin B (SEB; a superantigen).
  • SEB Staphylococcal enter otoxin B
  • the method may be used to identify an antigen-responsive T cell within a population when the disease-associated antigen is known, it is particularly preferred if the method is used to identify an antigen- responsive T cell within a population of T cells when the antigen or antigens associated with a disease is not known.
  • the method may be used to identify a T cell responsive to a superantigen but it is preferred if it is used to identify a T cell responsive to a conventional antigen.
  • Superantigens are typically the protein products of a number of bacteria and viruses. Their name derives from their ability to stimulate large numbers of T cells compared with that seen with conventional antigens. They differ from conventional antigens in a number of ways: Superantigens function as intact proteins. Conventional antigens are generally small peptides, of between 8 and 12 amino acids long, derived from the internalisation and proteolytic degradation of larger proteins by an antigen-presenting cell.
  • Superantigens bind outside the 'peptide binding groove' of the major histocompatibility complex class II molecule on the surface of antigen- presenting cells. Conventional antigenic peptides lie in this groove in the class II molecule.
  • Superantigens generally bind to a specific region of the ⁇ chain of T cell receptor called the fourth hypervariable region. This is encoded by the TCRBV gene: segment alone.
  • the structure of the TCR ⁇ chain and the TCRBJ gene segment may play a minor role in influencing the affinity of superantigen binding.
  • Conventional antigens are thought to be recognised by the complementarity determining regions (especially CDR3 which is formed by the combination of V-(D)-J gene segment recombination and N region additions) of both the ⁇ and ⁇ TCR chains. (See Kay R.A. (1995) Clin. Exp. Immunol. 100, 4-6; and Herman A., et al (1991) Anna. Rev. Immunol. 9, 745-772.)
  • the samples containing T cells may be any suitable samples containing T cells.
  • the sample is a sample of peripheral blood or a sample of bone marrow but it may be any sample from an individual which contains T cells. Samples from an individual which are then cultivated in vitro may also be used.
  • the subset of T cell receptors is a subset wherein each T cell receptor comprises a specific V ⁇ region or segment.
  • the specific V ⁇ region or segment of the TCR mRNA may be recognised using a specific nucleic acid probe which hybridises to the specific V ⁇ segment mRNA.
  • Any convenient method for identifying and quantitating the amount of mRNA containing a specific V ⁇ segment may be used, for example "chip" hybridisation methods of detecting specific mRNA or cDNA may be used.
  • PCR polymerase chain reaction
  • it is particularly preferred in the polymerase chain reaction (PCR) is used; more particularly it is preferred if a quantitative PCR method is used. It will be appreciated that since PCR relies on a DNA template the TCR mRNA should be copied into cDNA prior to or during the PCR process.
  • Methods for synthesising cDNA from mRNA are well known in the art and typically involve hybridising an oligonucleotide primer to the mRNA and synthesising DNA using deoxynucleotides and a reverse transcriptase. Methods for performing polymerase chain reactions are well known in the art. Methods of cDNA synthesis and PCR methods are described in Sambrook et al (1989) Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York, incorporated herein by reference.
  • the subset of T cell receptors is a subset wherein each T cell receptor comprises a specific V ⁇ region or segment.
  • the specific V ⁇ region of the TCR mRNA may be recognised using a specific nucleic acid probe which hybridises to the specific V ⁇ segment mRNA. Similar methods of identifying and quantitating the specific V ⁇ segment-containing TCR mRNA may be used to those for identifying and quantitating the specific V ⁇ segment- containing TCR mRNA.
  • Nucleotide sequence information is available for many of the ⁇ chain variable gene segments (TCRBV) and chain variable gene segments (TCRAV) and ⁇ chain joining gene segments (TCRAJ) as well as for other segments of the TCR genes which are transcribed and spliced into TCR mRNA. Much of this information is available from publicly accessible nucleotide sequence databases such as Gen Bank and EMBL. In particular, the complete 685-kb DNA sequence of the human ⁇ T cell receptor locus is known (Rowen et al (1996) Science 272, 1755-1762). The sequence and its annotations are deposited in the Genome Sequence Data Base with accession numbers L36092, L36190 and U03115, all incorporated herein by reference.
  • Suitable probes and primers for measuring specific V ⁇ , V ⁇ and other TCR gene segments are readily derived from the publicly available sequences for the TCR genes. It is preferred if the amount of T cell receptor mRNA is determined using quantitative PCR; and it is particularly preferred if the quantitative PCR method is reverse-transcription competitive PCR (RT-CPCR). Reverse-transcription competitive PCR (RT-CPCR) is described in detail in Kohsaka et al (1993) NucL Acids Res. 21, 3469-3472 and in Taniguchi et al (1994) J. Immunol. Methods 169, 101-109, both of which are incorporated herein by reference.
  • RT-CPCR Reverse-transcription competitive PCR
  • TCR gene segment-specific oligonucleotide primer For the measurement of any particular TCR gene segment by PCR at least one TCR gene segment-specific oligonucleotide primer is required.
  • TCR gene segment-containing mRNA may be carried out using a pair of PCR primers each of which hybridise within the specific TCR gene segment
  • This approach may substantially prevent amplification of any contaminating genomic DNA; and, therefore, the method is useful for discriminating between TCR cDNA/mRNA and TCR genomic DNA.
  • PCR primers for identifying specific V ⁇ and V ⁇ segments in TCR mRNA are described in Williams et al (1992) J. Clin. Invest. 90, 326-333, incorporated herein by reference, and are shown in Figure 6.
  • identify the combination of specific V ⁇ with specific J segment This may be done, for example, by using a V ⁇ -specific oligonucleotide with an oligonucleotide specific for each J segment in a PCR reaction.
  • the combination with a specific J segment can also be identified using PCR with V ⁇ - and J-specific oligonucleotides.
  • V ⁇ and specific V ⁇ segments may be identified, for example, by using oligonucleotides directed at specific V ⁇ segments in combination with C ⁇ -specific oligonucleotides, or by using oligonucleotides direct at specific V ⁇ segments in combination with C ⁇ -specific oligonucleotides.
  • Combinations of specific V ⁇ and V ⁇ segments with specific J segments can be identified, for example, using substantially the same methods as for V ⁇ -J and V ⁇ -J combinations by using suitable, selective oligonucleotides in a PCR reaction.
  • the number of specific T cell receptor-positive T cells or the number of T cell receptor-positive T cells in a specific subset may be determined using any suitable method. Conveniently, since many antibodies which are specific for specific V ⁇ segments and specific V ⁇ segments of the TCR are available, the determination is made using antibodies which bind to a specific T cell receptor or to a specific subset of T cell receptors (for example to a specific V ⁇ segment of the T cell receptor). Monoclonal antibodies directed at specific V ⁇ and V ⁇ chains are readily available.
  • Immunotech sells the following antibodies (taken over by Coulter Electronics Ltd, Northwell Drive, Luton, Beds LU3 3RH): Antibodies: ⁇ minus V ⁇ l, V ⁇ 2, V ⁇ 3, V ⁇ l, V ⁇ 9, V ⁇ 24, V ⁇ l, V ⁇ 2, V ⁇ 3, V ⁇ 5.1, V ⁇ 5.2, V ⁇ 5.3, V ⁇ 6.1, V ⁇ 8.1 and V ⁇ 8.2, V ⁇ 9, V ⁇ l l, V ⁇ l2, V ⁇ l3.1, V ⁇ l3.6, V ⁇ l4, V ⁇ l6, V ⁇ l7, V ⁇ l8, V ⁇ 20, V ⁇ 21.3, V ⁇ 22.1.
  • monoclonal antibodies against specific T cell receptors may be raised by immunising mice against specific human T cell tumour lines, making mouse B cell hybridomas and then screening the mouse antibodies produced against both the target cell line and different human T cell tumour lines using methods well known in the art. In this way, the hybridoma clones selected are likely to be producing anti-TCR specific monoclonal antibodies.
  • a second approach is to substitute the V region on the ⁇ chain of a mouse TCR with a human V ⁇ region and using the cell line created to immunise mice. In this way, monoclonal antibodies directed at human V ⁇ chains may be generated and much of the background screening is eliminated and a greater range of targets could be generated. Since it is now possible to generate soluble human TCRs, these may be used as targets for raising further monoclonal antibodies against V ⁇ and V ⁇ targets using methods known in the art.
  • the antibodies may be fluorescently labelled and the cells sorted and counted using a fluorescence-activated cell sorter (FACS) machine.
  • FACS fluorescence-activated cell sorter
  • the antibodies are labelled with any convenient fluorescent compound, for example fluoroscein isothiocyanate (FITC).
  • the number of T cell receptor positive T cells or the number of T cell receptor-positive T cells in a specific subset may be determined by analysing the genomic DNA of the T cell population.
  • Specific T cell DNA which has been somatically rearranged, can be quantified in a similar manner to that of mRNA. Any method which will distinguish unrearranged and rearranged TCR genes may be used in order to determine the number of specific T cells.
  • TCR mRNA which is specific for a T cell receptor or is specific for a subset of T cell receptors in the sample has been enumerated, and once the number of specific T cell receptor-positive T cells or specific T cell receptor-positive T cells of a particular subset has been enumerated, the number of specific TCR mRNA species per specific T cell is computed.
  • control sample is an historic control sample or a sample taken contemporaneously from a separate, healthy individual or from a non-diseased site in the individual form which the test sample has been taken.
  • An increase in the amount of specific T cell receptor mRNA per specific T cell is indicative of that specific T cell (T cell receptor or subset of T cell receptors) being an antigen responsive T cell.
  • the increase in the amount of specific T cell receptor mRNA per specific T cell which is indicative of an antigen response varies depending on the particular antigen and the particular TCR or TCR subset.
  • an increase of greater than about 2 is indicative of a specific T cell response but the increase may be greater than 10 or greater than 100 and it may be greater than 1000.
  • normal ranges of TCR gene expression may be determined for specific T cells or specific subsets of T cells.
  • an increase in the amount of specific T cell receptor mRNA per specific T cell is indicative of an antigen response if the increase is statistically significant by at least one, preferably at least two, and more preferably at least three or more standard deviations above the level of the control (ie the normal range of expression in the unstimulated situation).
  • the method is particularly useful to determine which T cell type is associated with a particular antigen-mediated disease. In some circumstances a predominant T cell type is involved in a disease process and, for example, the same T cell type is involved in the disease in the majority of individuals. Thus, the method is useful if in identifying the
  • T cell type involved in the majority of individuals of a particular disease.
  • the method is also particularly suited for use on individual patients in order to determine the specific T cell type involved in a particular disease in an individual patient. It will be appreciated that treatment of an individual patent may be tailored depending on the T cell type of the patient involved in the disease.
  • a further aspect of the invention provides a method of treating a patient wherein the patient has an antigen-mediated disease the method comprising (a) identifying an antigen-responsive T cell associated with an antigen-mediated disease according to the method of the first aspect of the invention and (b) administering to the patient an effective amount of an agent which ameliorates the disease.
  • the agent which ameliorates the disease is typically an agent which reduces or eliminates the T cell response to the antigen.
  • an agent may be selected which ameliorates the disease.
  • monoclonal antibodies which are directed at a specific V ⁇ segment may be useful, or peptides which are derived from a CDR of a specific V ⁇ segment may be useful.
  • Experimental autoimmune encephalomyelitis (EAE) an animal model of the human condition multiple sclerosis, has provided a prototypic model to test the efficacy of anti-T cell therapy in autoimmune disease.
  • encephalitogenic T cells specific for myelin basic protein were highly restricted, expressing similar TCRs that consisted of V ⁇ 2 and V ⁇ 8.2 (Heber-Katz & Acha-Orbea, 1989) that could be successfully targeted for therapy by V ⁇ 8.2-specific monoclonal antibodies.
  • vaccination with attenuated encephalitogenic T cells also mediated protection against EAE (Lider et al, 1988).
  • TCR peptides derived from the CDR2 or other regions can induce immunoregulation of pathogenic T cells specific for MBP, collagen, heat shock protein, and the P2 protein of peripheral myelin, implicating a potential for therapy in experimental arthritis, neuritis in addition to EAE (Howell et al, 1989; Stevens et al, 1991; Kumar & Sercarz, 1993; Gregorian et al, 1993; Broeren et al, 1994; Matsumoto et al, 1994; Kuhrober et al, 1994; Haqqui er a/, 1995).
  • the invention also includes a method of selecting a treatment for a patient with an antigen-driven disease.
  • Figure 1 shows a comparison of specificity of wild-type- and mutant- specific probes.
  • Figure 1(a) shows a standard curve of amplified BV2S1 wt DNA;
  • Figure 1(b) shows a standard curve of amplified BV2S1 mutant DNA.
  • Figure 2 shows the comparability of optical density (OD 450/630 ) readings obtained with wild-type- and mutant-specific probes. The ratio of ODs from wild-type and mutant probes on an ⁇ construct are shown.
  • Figure 3 describes the measurement of unknown cDNA samples. The quantitation of TCRBV2S1 cDNA is shown.
  • Figure 4 shows a comparison of TCRBV2S1 mRNA production and CD25 expression by V ⁇ 2.1 TCR + T cells in unseparated lymphocyte populations.
  • Figure 5 shows a comparison of TCRBV3S1 mRNA production and CD25 expression by V ⁇ 3.1 TCR + T cells in unseparated lymphocyte populations.
  • Figure 6 shows the sequence of PCR primers suitable for specific amplification of V ⁇ and V ⁇ segments of TCR mRNA (cDNA). See Williams et al (1992) J. Clin. Invest. 90, 326-333 for further details, incorporated herein by reference.
  • Figure 7 shows TCRBV mRNA levels per cell before and after 3 days culture in medium alone or supplemented with the superantigens SEB or TSST-1.
  • Figure 8 is a comparison of CD25 expression and intracellular TCRBV levels in antigen-triggered T cells.
  • Figure 9 shows intracellular TCRBV3S1 mRNA levels obtained from lymphocytes cultured with medium alone (UnRx) or supplemented with anti-CD28 (CD), anti-V ⁇ 3.1 (V ⁇ ) or a combination of the two (V ⁇ + CD).
  • Figure 10 shows the measurement of 15 000 molecules of a cloned TCRBV 17S1 sequence on a single day (grouped) or over a period of 8 weeks (separate).
  • Figure 11 shows the accuracy of measurement of 15 000 molecules of 5 different TCRBV 17S1 wild-type templates with a single mutant BV17S1 clone. Measurements were made using both a B17S1 -specific primer and a TCRBC-specific primer ( ⁇ PCR5')- There was no significant difference between the measurements obtained using either of these primers.
  • Figure 12 shows the measurement of wild-type BV17S1 (clone 7) and mutant BV17S1 (17/2/17 mutant) template using BV17S1 -specific and ⁇ PCR5' primers.
  • Figure 13 shows the measurement of BV17S1 wild-type templates (clone 1 and clone 7) with their respective mutants.
  • Figure 14 shows the measurement of different numbers of molecules of wild-type TCRBV 17S1 template.
  • Figure 15 shows the relative activities different promoter - pXP2 constructs transfected into Jurkat T cells after 40 hours culture in medium alone (•) or supplemented with PMA (O).
  • TCR gene expression is increased after antigen stimulation in order to replace the receptors which have been lost from the cell surface during antigen triggering.
  • T cell activation by cytokines would not involve degradation of cell- surface TCRs
  • I propose that measurement of TCR-specific mRNA production rates is a viable method to discriminate between passively recruited/passively-activated T cells from antigen-specific T cell effectors in any immune process.
  • T cell receptor (TCR) messenger RNA (mRNA) is measured by a reverse transcription competitive polymerase chain reaction (RT-CPCR) (see, for example, Kohsaka et al (1993) NucL Acids Res. 21, 3469-3472; and Taniguchi et al (1994) J. Immunol. Methods 169, 101-109.
  • RT-CPCR reverse transcription competitive polymerase chain reaction
  • a mutant template is added at different concentrations to aliquots of wild-type cDNA template and the ratio of the two is determined by mutant and wild-type-specific oligonucleotide probes after PCR. Quantification is expressed as the number of molecules of specific TCR mRNA expressed per specific TCR + ve T cell.
  • TCRBV gene mutants are manufactured by cloning amplified, wild-type PCR products into a vector such as PCRscript according to manufacturer's instructions and mutating this cloned template.
  • the mutation is performed using a PCR-based method known as gene SOEing (sequence overlap extension) according to the methods of Higuchi et al (1988) and Ho et al (1989). Briefly, the wild-type sequence is amplified in two halves, in separate reactions. The first reaction amplifies the upstream half of the template using the upstream TCRB V-specific primer and a mutational downstream primer.
  • the mutational primer anneals to the template just upstream of the site to be mutated and carries 12 to 15 bp of the new mutation sequence at its 5' end.
  • the second reaction which is carried out separately, amplifies the lower half of the template. It utilises the downstream -TC7?-5C-specif ⁇ c primer and an upstream mutational primer.
  • the upstream mutational primer anneals to the template just downstream of the site to be mutated and carries 12 to 15 bp of the new mutation sequence at its 5' end. After amplification, each half is purified free of wild-type template, primers and Taq polymerase and diluted to be at equal concentration.
  • the two halves are then placed together in a PCR mix along with the TCRBV- and rC& ⁇ C-specific primers only and amplified in a 'hot- started' PCR reaction.
  • the 2 halves are annealed together by virtue of their overlapping mutation sequence and a new mutant template is created by the PCR process.
  • the new PCR product can then be re-cloned back into a vector such as PCRscript and sequenced until an error-free clone is identified.
  • Using this process we have replaced the CATCAGAAGCAGAGATCTCC sequence in the wild-type TCRBC region with the GATGTCAAGCTGGTCGAGAA sequence from the corresponding region of the TCRAC gene.
  • This mutation was designed not to affect the overall size, dG/dC:dA/dT content or the primer annealing sequences of the original wild-type template.
  • the mutated template amplifies with equal efficiency as the wild type template in a number of TCRBV- specific PCR reactions. See Higuchi R., et al (1988) Nucleic Acids Res. 15, 7351-7367 and Ho S.N., et al (1989) Gene 77, 51-9, for further details on gene SOEing.
  • Lymphocytes were obtained from a peripheral blood sample from a normal donor and cultured for three days at 1 x 10 6 cells/ml in vitro in RPMI 1640 with penicillin, streptomycin, glutamine and 10% (v/v) heat- inactivated foetal calf serum. Cells were incubated in this medium alone or medium supplemented with either 100 ng/ml Staphylococcal enterotoxin B (SEB a superantigen which binds V ⁇ 3+ cells amongst others but not V ⁇ 2+ T cells) or 10 ng/ml TSST-1 (which binds V ⁇ 2 + T cells but not V ⁇ 3+ ones) [511. 31
  • SEB Staphylococcal enterotoxin B
  • TSST-1 which binds V ⁇ 2 + T cells but not V ⁇ 3+ ones
  • Total RNA is extracted from T cells either in vivo or in vitro using the RNeasyTM extraction kit according to manufacturer's instructions (Qiagen) following cell lysis and DNA shearing by the QiashredderTM (Qiagen). DNA contamination may be removed by treatment with RNase-deficient DNase for 1 hr at 37 °C followed by heat inactivation of the enzyme by incubation at 75 °C for 5 mins. Total RNA concentrations may be sometimes measured at this point but are often less than can be quantified by spectrometry.
  • RNA is then reverse transcribed with 100 U M-MLV reverse transcriptase (Superscript 11 ; Life Technologies) in a final volume of 20 ⁇ l for 90 min following a 'hot start' according to manufacturer's instructions.
  • M-MLV reverse transcriptase Superscript 11 ; Life Technologies
  • One ⁇ l of cDNA is amplified with O.lx, lx and lOx molecules of mutant plasmid DNA.
  • Estimations of cDNA concentration can be made by amplification of 1 ⁇ l of cDNA against a standard curve of cloned specific-TCRBV gene product of known concentration.
  • the PCR reaction is in 50 ⁇ l final volume using 1 U Red HotTM Taq polymerase (Advanced Biotechnologies) with between 10 and 25 pMol each of a TCRBV-specific and an aminated-TCRBC-specific oligonucleotide primer, 200 ⁇ M dNTPs and 2.0 mM MgCl 2 .
  • the reaction is 'hot started' at 95 °C for 10 min, and cooled to 25 °C at a rate of 3°C per min on a PHC-3 thermal cycler (Techne).
  • primers are designed which anneal to the CDR1 or CDR2 regions of the gene as these are the areas most likely to differ from other TCRBV sequences.
  • the amplified PCR products are separated from the unincorporated primers using the 'clean up' kit (Advanced Biotechnologies Ltd) according to the manufacturer's instructions.
  • the purified PCR product is eluted in 90 ⁇ l H 2 0 and mixed with an equal volume of MES/EDTA buffer (50 mM (2-[N- morpholino]ethanesulfonic acid), 1 mM EDTA).
  • the single-stranded DNA bound to the plate is probed either with a Biotin-conjugated, wild-type-specific (2 wells) or a Biotin-conjugated, 33 mutant-specific (2 wells) oligonucleotide dissolved in HW for 90 min at 42°C.
  • the plates are then washed three times with HW2 buffer (2XSSC + 0.1 % (v/v) n-lauroylsarcosine) and once with buffer B (100 mM Tris- HC1 pH 7.5 + 800 mM NaCl + 0.5% (w/v) Blocking reagent (Boehringer Mannheim)).
  • the bound probes are then detected by incubating the wells for 1 hr at 37 °C with 100 ⁇ l ABC (Avidin Biotin Complex) streptavidin peroxidase (Dako Ltd) made up according to the manufacturer's instructions and diluted 1:10000 in Buffer B.
  • the plate is then washed IX with Buffer B and 5X with Buffer A (100 mM Tris- HCl pH 7.5 and 800 mM NaCl).
  • Peroxidase activity was detected by adding a 100 ⁇ l of TMB substrate (1 mg/ml) in a phosphate-citrate buffer with 1 ⁇ l H 2 0 2 .
  • the reaction is stopped with 100 ⁇ l of a IM H S0 4 solution and read on a dual wavelength ELISA reader at 450 nM with correction at 630 nM.
  • the amount of mutant and wild-type product in each reaction is directly in proportion to their optical densities. Plotting log 10 optical density (450/630) against log 10 initial mutant reaction concentration allows derivation of initial wild-type cDNA concentration. The point where the l°g l o optical density ratio is 0 is the point where the two templates were at the same initial concentration.
  • the comparability of the optical densities derived from mutant- and wild-type-specific oligonucleotide probes was determined by constructing a template which contained one copy of each of these sequences contained within a genetic region spanned by the TCRBV2SI and TCRBC PCR primer annealing sequences. This construct was 34 manufactured by restricting wild-type and mutant clones with Hpal and Bali. The appropriate fragments were ligated together, and re-amplified using the TCRBV2S1 and -TCtf ⁇ C-specific primers. The new PCR product was cloned into the PCRscript vector.
  • This template was sequenced to ensure it contained error-free annealing sites for the TCRBV2SI, TCRBC, wild-type and mutant sequences.
  • This template was then amplified using a biotinylated rC ⁇ _3C-specific primer, and bound at various dilutions to a streptavidin-coated plate.
  • a specific TCR ⁇ chain sequence is to be quantified this is performed using the above method using either a specific TCRBV-TCRBJ combination of oligonucleotide primers or a specific TCRBV-N region combination of oligonucleotide primers.
  • the mutation is best placed within the TCRBV sequence. It can be performed by gene SOEing as before and the most convenient mutation is to replace a stretch of sense sequence with antisense sequence. This leaves the G/C:A/T content the same and it is certain that both mutant and wild- type probes will have equal affinity.
  • the PCR annealing site which is furthest away from the probe target sequence should be used as the end whose PCR oligomer is biotinylated.
  • V ⁇ + T cell numbers and the amounts of TCRBV-specific mRNA molecules are enumerated.
  • Specific T cell numbers is measured by using a combination of V ⁇ -specific monoclonal antibodies and cell counting.
  • TCR and CD25 Immunohistochemistry Aliquots of 5 x 10 5 cells were pelleted and resuspended in 50 ml PBS pH 7.6 with 0.01 % (w/v) sodium azide and 10% (v/v) heat inactivated normal human serum. These were incubated with 20 ml FITC-conjugated anti-V ⁇ 2, anti-V ⁇ 3 or anti-CD3 and counter stained with 10 ml RPE-conjugated anti-CD25 for 40 min on ice. Cells were then washed x 3 and fixed in 500 ml of PBS with 0.5% (v/v) paraformaldehyde buffered to pH 7.4. Samples were processed on a FACScan analyser (Beckton Dickinson).
  • TCRBV-containing DNA can be quantified.
  • Specific T cell DNA which has been somatically re-arranged, can be quantified as for specific ⁇ chain mRNA as CPCR.
  • a TCRBV-specific oligomer is used in combination with TCRBJ-specific primers covering all the thirteen TCRBJ sequences known to occur. The mutation, as above, should be placed in the TCRBV sequence. Once numbers of DNA molecules are known for each TCRBJ combination the numbers of T cells can be calculated as each T cell should contain only one BV-BJ combination.
  • TCRA mRNA total and specific, may also be achieved using the RT-CPCR method. In the first instance, TCRA mutants have to be manufactured.
  • mutants In the reverse of the TCRB method, mutants have the GATGTCAAGCTGGTCGAGAA wild-type TCRAC sequence replaced with the equivalent sequence from the TCRBC chain, CATCAGAAGCAGAGATCTCC by gene SOEing. This may be performed to sequences derived using TCRAV- and rC_R- C-specific primers (for quantification of specific ⁇ chain message) or in sequences amplified using two TC C-specific primers (for quantitation of total ⁇ mRNA).
  • TCRBV cDNA measurement Measurement of total ⁇ mRNA levels using TCRAC- specific primers is by far superior to quantifying the levels of TCRBC mRNA. This is because TCR ⁇ chain mRNA is also made by ⁇ T cells whereas TCR ⁇ chain mRNA is not.
  • TCRAV-TCRAJ TCRBV-TRCBJ quantitation. Namely, the mutation is placed in the TCRAV region and is conveniently the antisense of the existing sequence. Either, TCRAV- or TCRAJ-specific primers may be biotinylated depending on which is further from the oligomer probe annealing site. 37 Results are shown in Figures 1 to 5.
  • Figure 1 shows a comparison of specificity of wild-type- and mutant- specific probes.
  • a TCRBV2S1 mRNA mRNA transcript was reverse transcribed, amplified by PCR and cloned into the pBluescript vector. It was mutated by gene SOEing so that a 20 bp sequence in the TCRBC region was replaced with a 20 bp sequence from the corresponding region of the TCRAC gene. The mutant was also cloned into pBluescript. Varying numbers of molecules of wild-type (wt) and mutant (mut) sequence (from 10 to 10 molecules) were amplified by PCR using TCRBV2S1- and -TCit ⁇ C-specific primers.
  • the amplified templates were attached to an amine-binding plate by virtue of the aminated-group added to the 5' end of the TCRBC-specific primer according to the method described earlier.
  • Figure 1 shows the results of probing wt and mut amplicons with both wt- and mut-specific probes.
  • Figure 2 shows the comparability of optical density (OD 450/630 ) readings obtained with wild-type- and mutant-specific probes.
  • a construct containing both wild-type and mutant sequences was manufactured (see above) and cloned.
  • Different numbers of constructs (0 to 10 6 molecules) were amplified with TCRBV2S1- and rCftBC-specific primers, attached to an amine binding ELISA plate and then probed with wild-type- and mutant-specific probes (as described above).
  • the ODs obtained from each probe against the construct target were precisely comparable over the range 0 to 10 6 molecules.
  • Figure 3 describes the measurement of unknown cDNA samples. Aliquots of an unknown quantity of TCRBV2S1 cDNA are mixed with varying known amounts of mut TCRBV2SI and the two are co-amplified in a series of PCR reactions. After having been assayed using the DNA capture ELISA, the ratios of mutant to wild-type amplicons are plotted against the starting mutant template concentration. The point where the log of the ratio is 0 (ie the ratio of mutant to wild-type amplicon is 1) is the point where wild-type and mutant templates were present at the same initial concentration.
  • FIG. 4 shows a comparison of TCRBV2SI mRNA production and CD25 expression by V ⁇ 2.1 TCR + T cells in unseparated lymphocyte populations.
  • the V ⁇ 2.1 T cells within a population of unseparated peripheral blood lymphocytes, were analysed for TCRBV2S1 mRNA production (molecules per cell) and CD25 expression (% positivity). The analyses took place prior to culture (PRE) and three days after being cultured in the presence of medium alone (CON), or medium supplemented with either 100 ng/ml staphylococcal enterotoxin B (SEB) or 10 ng/ml toxic shock syndrome toxin- 1 (TSST-1). Four normal individuals were examined.
  • CON medium alone
  • SEB staphylococcal enterotoxin B
  • TSST-1 toxic shock syndrome toxin- 1
  • TCRBV2S1 production was much higher in the appropriately stimulated (TSST-1) cell cultures than with any of the controls.
  • the magnitude of increase in mRNA production was much larger than that seen in CD25 positivity suggesting that analysis of specific TCR mRNA production 39 rates gives the clearest indication of the antigen-driven T cells within an unseparated lymphocyte population.
  • FIG. 5 is a comparison of TCRBV3S1 mRNA production and CD25 expression by V ⁇ 3.1 TCR + T cells in unseparated lymphocyte populations.
  • the V ⁇ 3.1 T cells within a population of unseparated peripheral blood lymphocytes, were analysed for TCRBV3S1 mRNA production (molecules per cell) and CD25 expression (% positivity).
  • the analyses took place prior to culture (PRE) and three days after being cultured in the presence of medium alone (CON), or medium supplemented with either 100 ng/ml staphylococcal enterotoxin B (SEB) or 10 ng/ml toxic shock syndrome toxin- 1 (TSST-1).
  • CON medium alone
  • SEB staphylococcal enterotoxin B
  • TSST-1 toxic shock syndrome toxin- 1
  • TCRBV3S1 mRNA production were highest in the appropriately stimulated lymphocytes (SEB). An increased amount of TCRBV3S1 was seen in TSST-1 stimulated cultures compared with PRE and CON controls. This was still significantly less than that observed in SEB-stimulated cultures. There is no significant difference in the CD25 expression by the V ⁇ 3.1 T cells in the TSST-1 and SEB cultures. This confirms that analysis of specific mRNA production per specific T cell provides the clearest indication of identity of the antigen-driven T cells within unseparated lymphocyte populations.
  • TCR production rates increase greatly when T cells are stimulated by their specific antigen but the magnitude of this increase varies for different TCR genes.
  • TCR mRNA molecules per cell are increased in passive cultures with activated lymphocytes but not to the same degree as when the T cell is directly stimulated with antigen.
  • CD25 + expression appears to be independently regulated from TCR gene transcription.
  • TCR mRNA molecules per T cell This is an example of measuring TCR mRNA molecules per T cell as an index of antigen-mediated TCR triggering in vivo.
  • TCRBJ2S1 gene segment and bearing a distinct N region sequence almost exclusively (Tavakol Afshari et al (1997) Transpl. Immunol. , in press).
  • a number of DA and DA X Lewis F t rats are obtained and injected with unseparated DA lymphocytes into their hind footpads. Over a period of 14 days draining lymph nodes are obtained from injected rats, the DA lymphocytes are purified by eliminating RT1 1 cell surface marker-positive lymphocytes (F, cells) and the numbers of TCRBV6SI-TCRBJ2SI mRNA and genomic DNA molecules are measured essentially as described in Example 1.
  • the DA lymphocytes obtained from DA X Lewis have higher TCRBV6S1-TCRBJ2S1 mRNA to DNA ratios (indicating higher levels of specific TCR mRNA per specific T cell) than those obtained from control (DA) animals.
  • Example 3 Measurement of levels of TCRBV13S2 mRNA per V( 13.2 TCR-positive T cell in Hypergammaglobulinaemic Primary Sj ⁇ gren's Syndrome (HGPSS)
  • HGPSS is an autoimmune disorder characterised by T cell infiltration with and immune destruction of lacrimal and salivary glands.
  • TCRBV13S2 a genetic susceptibility to this condition encoded by the TCRBV13S2 gene and elevated levels of TCRBV13 mRNA in the salivary glands of patients with this condition (Kay et al, 1995; Sumida et al, 1992).
  • Biopsies of minor salivary glands and peripheral blood samples are obtained from patients with this condition.
  • Minor salivary gland biopsies from patients undergoing dental surgery for unrelated conditions (impacted wisdom teeth removal) are also obtained.
  • the biopsies are collagenase digested.
  • the lymphocytes from digested biopsies and the peripheral blood samples are purified by separation 42 through a Ficoll gradient and the V ⁇ l3.2+ T cell numbers are quantified by a combination of white cell counts and FACS analysis following immunohistochemical staining using an anti-V ⁇ l3.2, FITC- conjugated monoclonal antibody.
  • the TCRBV13S2 mRNA levels are measured as described in Example 1.
  • the levels of TCRBV13S2 mRNA per specific V ⁇ l3.2+ T cell are higher in the HGPSS patients' salivary glands than in the HGPSS patients' peripheral blood (selective homing to and triggering within diseased tissue) or in the salivary glands of patients with irrelevant dental disorders. (See Kay R.A.
  • Lymphocytes were separated from the peripheral blood samples from 4 normal donors and cultured for 3 days at 1.5 x 10 6 cells/ml in RPMI 1640 with penicillin, streptomycin, glutamine and 10% (v/v) heat- inactivated foetal calf serum. Cells were incubated in medium alone or medium supplemented with either 100 ng/ml SEB or 10 ng/ml TSST-1. Messenger RNA levels were measured as using the contaminant reverse- transcription PCR as described previously in Example 1. The numbers of V ⁇ 2.1 + and V ⁇ 3.1 + cells along with their CD25 status was performed by FACS analysis as described earlier in Example 1. Measurements were made prior to (PRE) as well as after 3 days culture.
  • FIG. 7 shows TCRBV mRNA levels per cell before and after 3 days culture in medium alone or supplemented with the superantigens SEB or TSST-1.
  • Figure 8 is a comparison of CD25 expression and intracellular TCRBV levels in antigen-triggered T cells.
  • Example 5 T cell response to stimulation with conventional antigen
  • Lymphocytes were separated from a peripheral blood sample and cultured as before for 3 days in medium alone or medium supplemented with an anti-V ⁇ 3.1 -specific monoclonal antibody, an anti-CD28-specific monoclonal antibody or both antibodies in combination.
  • Figure 9 shows the intracellular TCRBV3SI mRNA levels obtained from lymphocytes cultured with medium alone (UnRx) or supplemented with anti-CD28 (CD), anti- V ⁇ 3.1 (V ⁇ ) or a combination of the two (V ⁇ + CD).
  • TCRBVI7S 1 -specific mRNA was reverse transcribed, amplified, cloned and sequenced.
  • the clone was mutated, as previously described, in the TCRBC region so that it contained a short TCRAC sequence and could be used as a measurement contaminant for the wild-type template.
  • Figure 10 shows the measurement of 15,000 molecules of a cloned TCRBVI7S1 sequence on a single day (grouped) or over a period of 8 weeks (separate).
  • variable domains of the ⁇ chain of the TCR are encoded by a combination of 3 smaller gene segments. Therefore in each different TCRBV17S1 -encoded TCR, the TCRBV17S1 gene segment will be 45 combined with a different BD and BJ gene segment. In order to see if this physiological variation affects the accuracy of measurement, a mutant BV17S1 clone (using BD1 and BJISI) was used to measure 5 different BV17S1 wild-type templates. One of these templates had the same BD and BJ combination as the mutant; the other 4 did not.
  • Figure 11 shows the results of measuring these 5 different BV17S1 clones, with a single mutant contaminant using both a BV17S1- specific primer (which includes the regions of genetic variability) and a ⁇ PCR5' primer (which excludes the regions of genetic variability).
  • results show that the genetic variation that naturally occurs in TCR gene rearrangement does not affect the ability of a single mutant clone to provide accurate data. Furthermore, a single mutant clone can measure a polyclonal population of TCR sequences or a single TCR template with equal efficiency (data not shown).
  • Figure 11 shows the accuracy of measurement of 15,000 molecules of 5 different TCRBVI7SI wild-type templates with a single mutant BVI7SI clone. Measurements were made using both a _9V7ZS7-specif ⁇ c primer and a _rC--_3C-specific primer ( ⁇ PCR5') were used. There was no significant difference between the measurements obtained using either of these primers.
  • BV17S1 wild-type templates This mutant was identical to the wild-type in all but one respect.
  • a short sequence approximately 60 bp long, was substituted from the middle of the BV2S1 variable gene into the middle of the BV17SI sequence (17/2/17 mutant).
  • Measurements were made of the two templates using both the BVI7S1- specific primer (which anneals 5' to the mutation) and ⁇ PCR5' primer (which anneals downstream to the mutation). The results are shown in Figure 12.
  • Figure 12 shows the measurement of wild-type BV17S1 (clone 7) and mutant BV17S1 ( ⁇ 1I2I ⁇ 1 mutant) template using BV17S1- specific and ⁇ PCR5' primers.
  • the assay was assessed in its ability to measure a range of wild- type molecule numbers.
  • One hundred thousand, 10,000 and 1,000 molecules of TCRBV17S1 template were measured ( Figure 14).
  • the inter-assay coefficient of variation was comparable over this range of molecule numbers and was approximately 23% .
  • the test easily distinguished a ten-fold difference in template numbers.
  • the measurements were designed to cover a range that exceeded, in both directions, any molecule numbers which have so far been encountered in the experiments in vitro.
  • Figure 14 shows the measurement of different numbers of molecules of wild-type TCRBV17SI template.
  • the contaminant, quantitative PCR method is capable of measuring the numbers of TCR mRNA molecules likely to be encountered in vitro (and probably in vivo) with an accuracy of about 23% .
  • this test should (and does) easily detect T cell triggering.
  • a single mutant template for each TCRBV gene is all that is necessary to measure monoclonal, oligoclonal and polyclonal TCR mRNA populations and the accuracy of the assay does not appear to drift with time.
  • Example 1 Data from Example 1 shows that intracellular mRNA levels varied between different TCRBV gene both at rest and after activation. Given the sequence differences that occur between individual TCRBV gene promoters it seems likely that this represents differences in gene transcription.
  • rat TCRBV promoters were isolated from genomic DNA by PCR cloned into the pGEM-T Easy vector (Promega), and sequenced. These promoters from different TCRBV genes were subcloned into the luciferase reporter gene vector, pXP2. These constructs were transfected by electroporation along with a co- transfectant control (a thymidine kinase promoter controlling renilla luciferase expression) into the human T cell line, Jurkat. The cells were cultured for 40 hours after transfection either in medium alone or medium supplemented with PMA at 10 ng/ml. The PMA pharmacologically mimics TCR triggering.
  • a co- transfectant control a thymidine kinase promoter controlling renilla luciferase expression
  • TCRBV genes (even ones in the same family such as BV5S1 and BV5S2) are transcribed at different rates from each other both at rest and after activation. These data appear to confirm the earlier suggestions that differences in intracellular TCR mRNA levels might reflect differences in -TC- V-specific mRNA 49 production. It should be noted that the magnitude of the differences seen in intracellular mRNA levels is greater than those observed with the reporter constructs. This is probably due to not including the TCRB enhancer in the reporter constructs. The TCRB enhancer is relatively very strong compared to the promoter and increases luciferase production markedly in transfection experiments. Figure 15 shows the relative activities different promoter-pXP2 constructs transfected into Jurkat T cells after 40 hours culture in medium alone (•) or supplemented with PMA (O).
  • (5) shows luciferase reporter gene assay data using rat TCR gene promoters transfected in human Jurkat T cell lines which demonstrate that TCR gene transcription is increased after T cell stimulation and does vary, both at rest and after stimulation, with different TCRBV genes.

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Abstract

On décrit un procédé d'identification d'une cellule T sensibilisée par l'antigène parmi une population de cellules T. Le procédé consiste à: 1) se procurer un échantillon contenant des cellules T sensibles à l'antigène; 2) déterminer séparément, pour chaque récepteur d'un ensemble de récepteurs spécifiques de cellules T, ou séparément pour chaque sous-population d'un ensemble de sous-populations de récepteurs de cellules T, si l'expression d'un gène codant un récepteur spécifique de cellules T, ou si l'expression de gènes codant une sous-population de récepteurs de cellules T a augmenté par cellule T sensible au récepteur de cellules T spécifiques ou si elle a augmenté par sous-population de cellule T sensible au récepteur de cellules T spécifiques, par comparaison avec l'expression dudit gène ou desdits gènes dans un échantillon contenant des cellules T insensibles à l'antigène. Le procédé permet d'identifier efficacement des cellules T sensibilisées par l'antigène associées à un état pathologique tel que la polyarthrite rhumatoïde.
PCT/GB1998/001382 1997-05-27 1998-05-27 Procede d'immunologie WO1998054223A2 (fr)

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AU76631/98A AU728909B2 (en) 1997-05-27 1998-05-27 Immunological method
JP50035099A JP2001517958A (ja) 1997-05-27 1998-05-27 免疫学的方法
CA002291004A CA2291004A1 (fr) 1997-05-27 1998-05-27 Procede d'immunologie
EP98924427A EP1017724A2 (fr) 1997-05-27 1998-05-27 Procede d'immunologie

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GBGB9710820.3A GB9710820D0 (en) 1997-05-27 1997-05-27 Immunological method

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
EP1243327A1 (fr) * 2001-03-19 2002-09-25 BioChip Technologies GmbH Surface avec un motif de cellules et méthode pour sa production
WO2003044225A3 (fr) * 2001-11-23 2003-12-04 Bayer Ag Etablissement du profil du repertoire des genes immunitaires
US9803246B2 (en) 2011-06-28 2017-10-31 International Institute Of Cancer Immunology, Inc. Receptor gene for peptide cancer antigen-specific T cell
US10093977B2 (en) 2007-03-05 2018-10-09 International Institute Of Cancer Immunology, Inc. Cancer antigen-specific T-cell receptor gene, peptide encoded by the gene, and use of them
US10500257B2 (en) 2003-06-27 2019-12-10 International Institute Of Cancer Immunology, Inc. Method of selecting WT1 vaccine adaptive patient

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FR2671356B1 (fr) * 1991-01-09 1993-04-30 Inst Nat Sante Rech Med Procede de description des repertoires d'anticorps (ab) et des recepteurs des cellules t (tcr) du systeme immunitaire d'un individu.
EP0602178A4 (en) * 1991-08-28 1995-10-25 Wistar Inst T cell receptor-based therapy for rheumatoid arthritis.
WO1994014067A1 (fr) * 1992-12-14 1994-06-23 T Cell Sciences, Inc. Diagnostic et traitement de la sarcoïdose
AU3878697A (en) * 1996-06-20 1998-02-02 Cornell Research Foundation Inc. Identification of abnormalities in the expression of t and cell antigen receptors as indicators of disease diagnosis, prognosis and therapeutic predictors

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1243327A1 (fr) * 2001-03-19 2002-09-25 BioChip Technologies GmbH Surface avec un motif de cellules et méthode pour sa production
WO2002074434A3 (fr) * 2001-03-19 2003-10-30 Genovac Ag Surface comportant un motif de cellules et procede de fabrication de cette surface
WO2003044225A3 (fr) * 2001-11-23 2003-12-04 Bayer Ag Etablissement du profil du repertoire des genes immunitaires
US10500257B2 (en) 2003-06-27 2019-12-10 International Institute Of Cancer Immunology, Inc. Method of selecting WT1 vaccine adaptive patient
US10093977B2 (en) 2007-03-05 2018-10-09 International Institute Of Cancer Immunology, Inc. Cancer antigen-specific T-cell receptor gene, peptide encoded by the gene, and use of them
EP3492590A3 (fr) * 2007-03-05 2019-06-19 International Institute of Cancer Immunology, Inc. Gène du récepteur des lymphocytes t spécifiques de l'antigène du cancer, peptide codé par le gène et leur utilisation
US10669584B2 (en) 2007-03-05 2020-06-02 International Institute Of Cancer Immunology, Inc. Cancer antigen-specific T-cell receptor gene, peptide encoded by the gene, and use of them
US9803246B2 (en) 2011-06-28 2017-10-31 International Institute Of Cancer Immunology, Inc. Receptor gene for peptide cancer antigen-specific T cell
US10648036B2 (en) 2011-06-28 2020-05-12 International Institute Of Cancer Immunology, Inc. Receptor gene for peptide cancer antigen-specific T cell

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GB9710820D0 (en) 1997-07-23
AU728909B2 (en) 2001-01-18
AU7663198A (en) 1998-12-30
EP1017724A2 (fr) 2000-07-12
JP2001517958A (ja) 2001-10-09
WO1998054223A3 (fr) 1999-03-04
CA2291004A1 (fr) 1998-12-03

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