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WO2008026927A2 - Procédé d'affichage des répertoires des récepteurs des lymphocytes t et b - Google Patents

Procédé d'affichage des répertoires des récepteurs des lymphocytes t et b Download PDF

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WO2008026927A2
WO2008026927A2 PCT/NL2007/050429 NL2007050429W WO2008026927A2 WO 2008026927 A2 WO2008026927 A2 WO 2008026927A2 NL 2007050429 W NL2007050429 W NL 2007050429W WO 2008026927 A2 WO2008026927 A2 WO 2008026927A2
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array
tcr
bcr
specific
nucleotides
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WO2008026927A3 (fr
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Nikolaas De Vries
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Academisch Medisch Centrum
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Academic Medical Center
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the invention relates to the fields of medicine and molecular biology, in particular the field of immunology, more in particular the field of T- and B-cell responses and monitors these using advanced DNA and RNA analysis techniques.
  • the recognition molecules of the adaptive immune system cover an enormous diversity of antigens. They are produced by B- and T-lymphocytes, each producing a B cell receptor (BCR) resp. T cell receptor (TCR) of a single specificity. B cells produce the B cell receptor, present on the surface of the B cell. These receptors generally recognize 3 -dimensional structures of antigens, bind the antigen and may lead to B cell activation, and subsequent clonal expansion. B cells may also differentiate to become a plasma cell, which secretes immunoglobulins with the same specificity as the BCR, so- called antibodies. Antibodies may bind to the antigen and may inactivate it, or lead to recruitment of effector functions of the immune system.
  • T cell receptors are present on the membrane surface of T-lymphocytes. These cells can be divided in ⁇ - and ⁇ T cells, expressing an ⁇ - and ⁇ TCR respectively.
  • ⁇ TCRs show a relatively restricted repertoire and probably recognize specific molecular targets in different tissues (1). A recent study suggest they have a key role in presentation of antigen to the immune system (2).
  • ⁇ TCR show a very broad repertoire, ⁇ T cells are the major T-cells in the peripheral blood, and appear to play a key role in adaptive immune responses to specific antigens.
  • the ⁇ TCR recognizes short peptide fragments presented in the antigen binding groove of major histocompatibility (MHC) molecules on the surface of antigen presenting cells (APC).
  • MHC major histocompatibility
  • T-cells may become activated, secrete pro-inflammatory molecules and proliferate to produce daughter cells.
  • the resulting daughter cells share the same specificity of the TCR, forming a so- called T cell clone.
  • activated T cell clones may kill infected or malignant cells expressing the specific MHC/peptide complex (Cytotoxic T-cells).
  • T cell clones activate other immunocompetent cells, e.g. they may trigger B cells to mature to plasma cells and produce antibodies to neutralize the (bacterial) pathogen (Helper T-cells).
  • helper T-cells B cells to mature to plasma cells and produce antibodies to neutralize the (bacterial) pathogen
  • T cells also play a key role in regulation of the adaptive immune response.
  • Adaptive immune responses are important in protecting us from infection and malignancy.
  • the antigen-specific immune response developed in evolution to protect us, recent studies indicate that these responses can also cause substantial damage.
  • autoimmune diseases such as diabetes and rheumatoid arthritis.
  • these responses may also play a role in infectious disease like meningitis (3).
  • the specificity by which the immune system recognizes pathogens, tumor cells and auto-antigens is mainly encoded in the T- and B-cell receptor. A more detailed monitoring and control of specific clonal B- and T-cell responses is therefore important in different areas of health, both in humans and in animals.
  • the TCR beta receptor germline is estimated to encode 40-48 functional V-gene segments (variability), 2 functional D-gene segments (diversity), 13 J-segments (joining) and 2 C-segments (constant) (5;6).
  • Additional diversity is introduced by so-called junctional flexibility between the V, D, and J segments, which causes deletion of a variable number of nucleotides at the coding ends of the joined sequences.
  • random nucleotides may be inserted to link the V-D and D-J gene segments creating the hypervariable NDN region, coding for the complementarity determining region 3 (CDR3) (7;8).
  • CDR3 complementarity determining region 3
  • BCR antibodies
  • TCR cellular immunity
  • Antibodies are produced in humans or higher animal species by plasma cells as a result of a B cell response, usually in response to exposition to foreign substances, e.g. virus or bacteria, but also in the context of auto-immune disease. In infections these antibodies help the immune system in marking and eliminating pathogens.
  • a second problem with antibody based tests is that the development of each disease-specific test can be very complicated. Problems can arise in identifying and isolating the pathogen, in finding a target within the pathogen against which almost all individuals (human or animal) - irrespective of their genetic background - develop antibodies upon exposure, in acquiring this target in sufficient amounts, and in the further development of a test regarding efficiency, simplicity, reproducibility and validity. Given both problems there is a clear need for a technology that is able to screen the complete repertoire of antibodies for the presence of dominant antibody responses and for changes in these responses. This technology could be used to identify antibody responses accompanying the development of clinical syndromes. In addition it could be used during development of pathogen specific tests or vaccines to study antibody responses in different individuals responding against a specific pathogenic target.
  • T cells In addition to their effector role in the responses against intracellular targets, T cells have an even more important role in the antigen-specific regulation and control of the adaptive immune response, including the regulation of antibody production. Given these important roles of T cells both in the responses against intracellular antigens and in the regulation of adaptive immune responses in general there is a clear need for a technology that rapidly identifies the presence of dominant T cell responses and changes in these responses. Such a tool might be used to study changes in the repertoire of T cell receptors used in different functional subgroups of T cells. Current techniques for the analysis of T cell repertoires (spectratyping, immunoscope II) are very labor- intensive, are not quantitative and lack resolution, as set out below.
  • ⁇ T cells recognize a fragment of a protein (i.e. a peptide), but only then if this peptide is presented on a self-MHC molecule, ⁇ T cells can be identified with labeled complexes of MHC with specific peptide. Since the affinity of the TCR ⁇ for such combinations of MHC and peptide is low, investigators use multimers (e.g. tetramers) of such MHC/peptide combinations (11) or corpuscules (e.g. cells) carrying multiple MHC/peptide complexes (12).
  • multimers e.g. tetramers
  • individual clones can be monitored using ⁇ TCR specific antibodies.
  • DNA sequences of the ⁇ T cell receptor can be acquired after individual cloning and sequencing. All these methods require a priori knowledge of the T-cell clone DNA or the antigen peptide.
  • each of these approaches detects a single TCR ⁇ specificity, rather than screening the whole T ⁇ cell repertoire.
  • a complete screening of the approximately 10 s potential TCR ⁇ specificities (10), specific for each MHC- molecule using large arrays of specific antibodies or tetramers is not feasible due to economical and technical (e.g. weak signal to noise ratios, instability of array) constraints.
  • DNA level For instance tracking of specific clones has been performed using restriction enzymes. Since individual rearrangements differ in nucleotide sequence of the variable region, digests of TCR DNA using the right restriction enzymes may produce restriction patterns specific for different rearrangements. In order to track a clone the restriction pattern of the clone needs to be available and the frequency of the clone needs to be above a certain detection limit. As a screening tool the technique fails since its resolution is too low, and the technique does not produce information that can help in rapid identification and sequencing of the clones involved. Alternatively, when the DNA sequence of the clone is available RT-PCR can be used to track pathogenic T cells (13). As a screening tool this fails since prior knowledge on the DNA sequence is needed (reviewed in (14)).
  • a technique used to screen the TCR repertoire for dominant clones, e.g. after antigen stimulation, is spectratyping, also called immunoscope I (15,23).
  • Individual TCR rearrangements may differ in the length of their CDR3, due to differences in lengths of the gene segments and the process of nucleotide deletion and addition.
  • nucleic acid fragments encoding this region are separated on length.
  • Spectratyping of productive TCR rearrangements produces a series of 7 to 10 peaks, each 3 nucleotides (1 codon) apart, together following a Gaussian curve.
  • the technique can be applied to total TCR mRNA, but also to selected pools of cells, or to products of a specific amplification, e.g.
  • the groups of TCR rearrangements are subsequently separated in a denaturing gel and individual bands are cut out, cloned and sequenced.
  • the denaturing gel alone still has insufficient resolution to perform direct sequencing.
  • the technique is laborious, time-costly, and does not permit good quantitative comparisons between samples.
  • T cell receptor mRNA analysis is anchored RT-PCR (16), which allows amplification of T cell clones unbiased with respect to V family usage.
  • This method allows PCR amplification of all T-cell clones simultaneously, it requires subsequent cloning and sequencing of the individual TCR sequences. The technique is therefore also laborious and time-costly, and the sensitivity is limited and dependent on the number of clones analyzed.
  • Ogle et al (24) describe a technique where they hybridize TCR and BCR nucleotide sequences to a random array. Although this may be used to estimate lymphocyte diversity, it cannot be used to identify dominant clones or clones that increase or decrease in size.
  • Baner et al (25) describe a method in which they analyze V-beta usage. This analysis does not yield information on CDR3 region and has low resolution.
  • Lebed et al (26) describe how a PCR product acquired using a TCR V beta and C beta primer, is hybridized to a universal microarray. This method can be used to assess the complexity of mixtures, but cannot be used to identify dominant clones or clones that increase or decrease in size.
  • WO 03/044225 teaches a technique for hybridization of BCR or TCR PCR products to an array.
  • the description does not provide for hybridization to selected oligonucleotides consisting of a combination of a constant part (e.g. J-, D- or V- sequence) coupled to a random nucleotide sequence.
  • the technique does not make use of the deletion of terminal nucleotides of V-, D- and J-genes (or NDN junctional flexibility and hypermutation) to create resolution for a display of the full repertoire.
  • the technique does not use ligation technology, but rather detects the hybridization complexes themselves. This technology is far less sequence specific than the method disclosed in this specification.
  • WO2005084134 describes a technique for sequencing antibody and T cell receptor genes.
  • This patent application describes a method wherein TCR or BCR DNA or RNA is hybridized to oligonucleotides.
  • the technique also encompasses the use of oligonucleotides on the stretches where germline-sequences end and where the random NDN sequences resulting from VDJ recombination, junctional flexibility (deletion and insertion of nucleotides at the N-terminals of V-, D- or J segments) and hypermutation start.
  • this method is designed for sequencing of the complete TCR or BCR sequences and does not create the resolution to follow all individual clones.
  • the current invention provides a solution for the problems described above and the specification provides a process, comprising a high-throughput method based on micro-array technology, that can be applied to screen a B cell / plasma cell and T cell receptor repertoire of all BCR and/or TCR gene rearrangements, for dominant clones and for changes in these repertoires over time or interindividually.
  • the method provides a readout on the molecular level for rearranged and/or hypermutated B- and/or T-cell receptor repertoires, and changes in the repertoire after events such as infections.
  • the size of the TCR or BCR repertoire is too large to test on a single microarray carrying oligonucleotides.
  • the invention provides a method that for each rearranged clone creates an identifier based on the V-, D- or J- gene segment used in the rearrangement, the number of N-terminal nucleotides deleted from this V-, D- or J-gene segment and the sequence of the first few nucleotides that follow this rearranged gene segment Depending on the choice of cell nucleic acid material, amplification system, primers, annealer sequences and oligonucleotide array, this identifier in most instances will be unique for a given clone.
  • the identifier produced is specific for the clone, even on re-evalution, and allows direct sequencing of the full receptor sequence. To increase specificity, the sequence of the first few nucleotides following the rearranged V-, D- or J-gene segment is tested using ligation technology rather than hybridization technology.
  • Application of this tool facilitates (a) identification of substances (e.g. pathogens, allergens or toxins) that induce an immune response, e.g. in infectious or autoimmune diseases, already in early stages of initial contact with the substance, (b) advances in diagnostics, (c) tools for monitoring the immune response in disease with applications in prognostics, and (d) identification of targets for new methods and means for treatment of diseases.
  • substances e.g. pathogens, allergens or toxins
  • the current invention provides a process for screening and monitoring a T- or B- cell repertoire.
  • the method according to the invention may be used to detect the repertoire of the ⁇ / ⁇ TCR, the ⁇ / ⁇ TCR, or BCR receptors of all isotypes (IgA, IgE, IgG and/or IgM), and both heavy and/or light chains ( ⁇ and/or K).
  • TCR and BCR polynucleotides are extracted as genomic DNA or total RNA or messenger RNA from cellular nucleic acid material.
  • the cellular material may be obtained from any subject, preferably a vertebrate, more preferably a mammal and most preferably a human. It may be obtained from peripheral blood, body fluids or tissue. It may contain all T- and/or B-cells, or a subgroup of T- and/or B-cells selected on surface markers, physical characteristics, and proliferative and/or functional responses to different stimuli.
  • mRNA gene transcripts may be used to synthesize a first strand of cDNA molecules, optionally followed by synthesis of the complementary DNA strands.
  • the polynucleotides encoding the hypervariable CDR domains (complementarity determining regions), comprising part of the T- or B-cell repertoire diversity are amplified such that in case of CDR3 the amplification product comprises at least the sequence of the NDN hypervariable region, completely or partially, linked to a nucleotide sequence derived from the V (variability), D (diversity) and/or J (junctional) gene segments.
  • the product from the amplification reaction includes at least one of the junctional flexibility boundaries separating the V, D and J gene segments, in order to fully exploit detection of diversity created by N-terminal deletions from the V and J gene segments at the boundaries and optionally from the diversity (D) elements that may be located between the V and J gene segments.
  • Amplification of polynucleotides can be performed using several different methodologies, e.g. polymerase chain reaction (PCR), NASBA or in vitro transcription.
  • the preferred amplification method used by the invention is the polymerase chain reaction (PCR).
  • the CDR3 region which contains most of the specificity and variability, of heavy or light chains of immunoglobulins or one of the T cell receptor chains (alpha, beta, gamma or delta) is amplified using primers located on both sides of the CDR3.
  • Amplification of rearranged human TCR beta genes can thus be performed as described for TCR-beta by Pannetier C et al. Proc. Natl. Acad. Sci. USA 1993;90: 4319-4323.
  • Human immunglobulin genes can thus be amplified as described by Wang & Stollar, J Immunol Methods, 244(1-2): 217-25, 2000.
  • the V-primer used for amplification may be any primer capable of hybridizing to any location on the V-segment or the complement thereof.
  • TCR T cell receptor
  • Primers may be selected such that the primer is highly specific, distinguishing only one of the 40 - 48 functional human V ⁇ gene segments identified so far, or one of the 43 - 45 functional Va gene segments.
  • the primer may also be chosen to be generic, i.e. capable of hybridizing to and facilitating amplification of a subset of more than one V ⁇ or Va gene segments.
  • V-specific primers may be selected to amplify part or all of the ⁇ / ⁇ TCR repertoire.
  • the C or J gene segment specific primer capable of hybridizing on the opposite part of the NDN region with reference to the V-primer, may be a primer specific for one of the two C or 13 J gene segments. It may also be a generic primer that is capable of amplifying all or a specific subset of the TCR C or J segments. Likewise, C- or J-specific primers may be selected to amplify part or all of the ⁇ , ⁇ or ⁇ TCR repertoire. In a preferred embodiment, the entire BCR and/or TCR spectrum of an individual or sample is amplified by using all available primers combinations between V and C/J gene segment specific primers.
  • the template DNA or cDNA may be conveniently split in portions, preferably in a microtiter / microwell format to handle all primer combinations and allowing screening of the entire repertoire of gene rearrangements.
  • primers can be designed to amplify the K and/or ⁇ light chain CDRs and more preferably the heavy chain CDRs, most preferably CDR3, using primer pairs that are specific for at least one and optionally more gene segments from the at least 30 - 35 functional V kappa and 5 functional J kappa gene segments, 29 - 33 functional V lambda and 5 functional J lambda gene segments, and 38 - 46 functional VH gene segments combined with 23 functional DH or 6 functional JH gene segments.
  • the genomic DNA or cDNA template is divided over a number of amplification reactions, each with different primer pairs / combinations. For instance, for visualizing the repertoire of the TCR ⁇ , primers specific for each of the 40 - 48 functional V ⁇ segments may be combined with primers for all of the 13 functional J segments and/or all 2 functional C segments, yielding the full repertoire for analysis on the array.
  • the expressed immune genes comprising the TCR and BCR (heavy and light chain) receptor coding gene transcripts (mRNA molecules) are reverse transcribed using an oligo dT primer or alternatively an immune gene specific primer complementary at least in part to the sequence in the immune gene constant region.
  • a V-gene specific PCR can subsequently be performed using a primer specifically encoding part of this V-gene and a primer that encodes a C- or J-gene segment, partially or completely.
  • a generic amplification of all V-region genes can subsequently be accomplished by (1) using mixes of V-specific primers, (2) adding a novel primer recognition site to the V-region side by transcription and/or amplification with V-specific primers containing the novel primer sequence in the 5' tail, (3) adding a primer recognition site to the V-region side by ligation, (4) by tailing of the cDNA with nucleotide residues using the enzyme terminal deoxynucleotidyl transferase or another enzyme encoding terminal transferase activity, subsequently using a template switching strategy (Zhu YY et al. Biotechniques. 2001 Apr;30(4):892- 7; Douek DC et al. J Immunol. 2002 Mar 15;168(6):3099-104).
  • the primers used in the transcription or amplification reaction may be physically linked to a molecular tag allowing separate detection and/or isolation of the (+) and (-) strands, such as an immunological hapten such as biotin or digoxigenin, or a fluorochrome (such as Cy3, Cy5, GFP, Fitc, Trite, etc.), a radiochemical ( 32 P, 33 P, 35 S, 125 I, 3 H) or enzyme (alkaline phosphatase, horseradish peroxidase and the like).
  • an immunological hapten such as biotin or digoxigenin
  • a fluorochrome such as Cy3, Cy5, GFP, Fitc, Trite, etc.
  • a radiochemical 32 P, 33 P, 35 S, 125 I, 3 H
  • enzyme alkaline phosphatase, horseradish peroxidase and the like.
  • Primer and annealer oligonucleotides used in the method of the invention may comprise DNA nucleotides, RNA nucleotides, 2'-0 substituted ribonucleotides, including alkyl and methoxy ethyl substitutions, peptide nucleic acid (PNA), locked nucleic acid (LNA) and morpholino antisense oligonucleotides and ethylene-bridged nucleotides (ENA) and combinations thereof.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • ENA ethylene-bridged nucleotides
  • CDR3 containing amplification products from rearranged TCR or BCR encoding genes or gene-transcripts are hybridized to a labeled complementary annealer oligonucleotide that is capable of hybridizing to the CDR3- oriented end of the rearranged gene segments, preferably adjacent within 5 to 15, more preferably within 13 nucleotides to the junction of the V or J segment or to either end of the rearranged D segment.
  • the hybridization of the annealer oligonucleotide and subsequent ligation is performed before addition to the array or during hybridization of the polynucleotides to the array that comprises oligonucleotides on a solid carrier.
  • the annealer may be physically linked to a molecular tag allowing separate detection and optionally isolation of the (+) and (-) strands, such as an immunological haptens, fluorochromes, radiochemicals or enzymes as mentioned above.
  • a molecular tag such as an immunological haptens, fluorochromes, radiochemicals or enzymes as mentioned above.
  • DNA originating from a pool of T- or B-cells the amplified TCR and/or BCR encoding polynucleotides or the complex of a polynucleotide with an annealer are hybridized to oligonucleotides on one or more solid carriers.
  • the design of the solid carrier is such, that it links oligonucleotide sequence information to specific locations, time-points or tags (e.g. color).
  • the solid carrier is a DNA micro-array or DNA chip, a technique well described in the art (Ewis et al, Expert Rev MoI Diagn 2005;5:315; Schena et al Science 1995;270:467; Lockhart DJ et al., Nature Biotechnol 1996;14:1675).
  • the solid carriers may also be in the form of beads with one or more oligonucleotides attached to their surface, such as applied in the Luminex or Illumina systems (Jian-Bing Fan, Sean X. Hu, William C. Craumer, and David L. Barker, BioTechniques 39 Supplement, S583-S588, 2005., Smith PL, WalkerPeach CR, Fulton RJ, DuBois DB, 1998, Clinical Chemistry, 44: 2054-2060 ).
  • the amplified CDR preferably CDR3, sequences are capable of annealing under conditions conducive to hybridization to the oligonucleotides fixed to the solid carrier.
  • Arrays in particular DNA microarrays, comprising predetermined random hexamers, preferably all 4096 available permutations thereof (4 6 ), are a preferred embodiment in the method of the invention but alternatively array formats consisting of random trimers, tetramers, pentamers, heptamers, octamers, nonamers, decamers, etc. optionally combined with BCR and TCR specific sequences, can also be used in other embodiments of the invention as explained below and in figures 1, 5 and 6.
  • Predetermined random oligonucleotide sequences are herein defined as random permutations of nucleotides and nucleotide analogs, preferably the four DNA nucleotides A, T, C, or G.
  • the position on the array of each random permutation is known or can be traced.
  • the oligonucleotides on the array may be attached by a spacer molecule, the spacer molecule may also comprise or consist of an irrelevant nucleotide sequence or a nucleotide sequence wherein the nucleotides are chemically modified to facilitate attachment to a solid carrier surface.
  • Hybridization can be carried out under standard conditions known to the skilled person and/or provided by the manufacturer of commercially available DNA arrays.
  • Detection of the hybridized / annealed CDR3 sequences may take place by several methods known in the art, most preferably using fluorochromes, such as, but not limited to Cy3 and/or Cy5.
  • Fluorochromes such as, but not limited to Cy3 and/or Cy5.
  • Signal amplification systems are described in the art (Stears RL, Getts RC, Gullans SR, Physiol. Genomics 3(2). 93-99 (2000); Karsten SL, Van Deerlin VM, Sabatti C et al. Nucleic Acids Res. 30(2), e4 (2002)) and may be applied by a skilled artisan to enhance signals generated from DNA microarray analysis to increase the sensitivity of the method of the invention .
  • the CDR3 containing amplification products are hybridized to oligonucleotides on one or more solid carriers / arrays in the presence of a DNA ligase. After ligation the complexes may be denatured, allowing removal of unligated DNA and detection of the ligated labeled annealer oligonucleotides on the array.
  • the array consists of random oligonucleotides, e.g. hexamer sequences, optionally preceded by TCR or BCR specific stretches, such as but not limited to a stretch of 10 to 30 nucleotides complementary to a V-, D- or J segment of the TCR or
  • this array contains all permutations of these oligonucleotide sequences.
  • the array contains all permutations except those permutations in which the oligonucleotide sequence on the array encodes that part of the germline sequence of the V or J segment analyzed or the complement thereof which immediately follows the annealer sequence or the complement thereof.
  • BCR B-cell receptor
  • TCR or BCR chain from the DNA or cDNA obtained in a), using a primer pair complementary to a V, D, J or C gene segment of a TCR or BCR chain, wherein optionally one of the primers is provided with a specific tag or hapten, to isolate a (+) or (-) DNA strand, followed by, in any order, step c) and step d); c) hybridizing the product of step b) or step d) with a labeled annealer oligonucleotide that comprises nucleotides complementary to or identical to part or all of a V, D or J gene segment and that is capable of hybridizing in close proximity to a junction between two of a V, D and J gene segment; d) hybridizing the product of step b) or step c) to a solid carrier comprising an array of predetermined random oligonucleotide sequences, in the presence of a DNA ligase; and, e) detecting the ligated labeled annea
  • a specifically labeled annealer oligonucleotide having a sequence capable of hybridizing to the CDR3-oriented end of a rearranged V or J segment or to either end of a rearranged D segment, i.e. at the hypervariable junctions between D and V gene segments or the D and J gene segments.
  • the annealer is added to a suitable nucleic acid sample of amplified CDR sequences.
  • the annealer oligonucleotide is added before or during hybridization of amplified CDR fragments to the array, in the presence of a DNA ligase.
  • the labeled annealer will only be ligated by a DNA ligase if the annealer sequence is hybridized directly adjacent to a predetermined random sequence on the array, preferably a random hexamer.
  • the labeled annealer oligonucleotide comprises a stretch of about 10 to about 30 nucleotides in length that are complementary to a V-, D- or J- gene segment, and capable of hybridizing in close proximity of, preferably within 5 to 15 nucleotides, a junction of a D segment with either the V or J gene segment.
  • the ligated (and covalently bound) labeled annealer oligonucleotides can be detected on the array using standard detection techniques for DNA micro-array analysis such as fluorescent microscopy and imaging.
  • the DNA molecules on the arrays used for the invention can be arranged both in 5' to 3 'direction or 3' to 5' in order to detect (-) and/or (+) strands of the CDR amplification reaction.
  • a ligase any DNA ligase, but preferably phage T4 or a bacterial ligase may be used, optionally a thermostable DNA ligase.
  • the free 5' end of the annealer probes and/or oligonucleotides on the array are to be phosphorylated to enable the ligation reaction.
  • the annealer probes used for detection purposes are preferably labeled with a fluorochrome, but radioactive, enzymatic or immuno -haptens may also be used.
  • More than one labeled annealer sequence may be used simultaneously on one array, provided that different labels are used per annealer, such as fluorochromes like Cy3 or Cy5 that vary in color and can be distinguished.
  • multiple annealers may be used in a process on a single array in a first step, followed in a second step by further typing of annealer or sequence only of spots selected in this first step.
  • the first embodiment of the invention is modified such that in the process according to the first embodiment, the predetermined random oligonucleotide sequences on the solid carrier array in step d) are preceded by a 3' or 5' sequence of 8-100 nucleotides, preferably 10-30, that are complementary or identical to part or all of a V, D or J gene segment and wherein the labeled annealer sequence comprises a population of random oligonucleotide sequences of 3 to 10 nucleotides in length, preferably random hexamers in all possible permutations.
  • detection of amplified CDR3 fragments from TCR or BCR rearranged gene transcripts takes place by hybridization to an oligonucleotide array, which contains sequences capable of hybridizing to specific V and/or D and/or J gene segments, followed by a stretch of random nucleotides.
  • an oligonucleotide array which contains sequences capable of hybridizing to specific V and/or D and/or J gene segments, followed by a stretch of random nucleotides.
  • the location of the random sequences on the array is known.
  • the length of the additional, non-random sequences may vary in length from about 100 to 8 nucleotides, preferably are between 30 and 10 nucleotides, and having 90 to 100% identity with a V, D, or J segment.
  • a labeled annealer sequence is added during or after hybridization of the amplified CDR fragments on the array.
  • the annealer oligonucleotide comprises a random sequence that is detected only if it is capable of hybridizing directly on a NDN hypervariable region of the CDR3 domain of a TCR or BCR.
  • the labeled and hybridized annealers will only be ligated to the hybridized complexes on the array if they are annealed directly adjacent to the random oligonucleotide sequence, preferably a random hexamer.
  • the annealer sequence can be a random sequence between 3 and 10 bases in length, most preferably a hexamer, with a fluorescent label covalently attached. Attached to the random annealer sequence an additional constant nucleotide sequence may be added, e.g. to serve as primer sequence in subsequent reactions.
  • the third embodiment of the invention is a further modification of the previous embodiments one and two wherein either the V-segment specific primer or the J- or C- segment specific primer for the amplification of CDR sequences in step b) is provided with a fluorescent label to facilitate detection of hybridized sequences to the oligonucleotide solid carrier array, wherein the nucleotides complementary or identical to part or all of a V, D or J gene segment on the solid carrier oligonucleotide array are between 6 and 30 nucleotides in length and a ligation and denaturation step e) is omitted.
  • either the V or the J/C specific primers in the amplification step of CDR fragments from TCR or BCR rearranged genes or transcripts is provided with a label, preferably a fluorescent label, to facilitate detection of hybridized sequences on the array.
  • a label preferably a fluorescent label
  • both primers may be labeled but with a different, distinguishable label, for instance a fluorochrome emitting at a different wavelength.
  • labeled nucleotides are incorporated during amplification. A ligation reaction is not required in this embodiment.
  • the 3' or 5' V-, D- or J- gene segment homologous sequences preceding predetermined random oligomer e.g.
  • hexamer sequences on the array are preferably between 10 and 30 nucleotides in length, this achieves optimal results in terms of resolution of the TCR and/or BCR repertoires.
  • the detection of specifically hybridized complexes on the array takes place to visualize diversity in the repertoire, as visualized in figure 6.
  • the stringency of the washing conditions of the hybridized oligonucleotide array can be readily adapted by the skilled artisan to achieve an optimal signal on the array. In this description the terms method and process are used interchangeably.
  • the method or process visualizes on an oligonucleotide array format, the end-result of the V(D)J recombination process and in particular the deletion process that takes place in the NDN region, where 1 up to 13 nucleotides are deleted from the V-, D- and/or J-gene segments. Furthermore, the variation in the NDN region is enhanced by N-insertions, which is also displayed by the method of the invention.
  • the method or process according to all embodiments can be applied, when generic V- and J- or C- gene segment specific primers are used to amplify the complete spectrum of all TCR or BCR gene rearrangements, visualize the complete spectrum on an array.
  • the method of the invention may preferably display a smaller subset of the TCR or BCR spectrum when the solid carrier oligonucleotide array comprises an array of predetermined sequences specific for at least 1 to up to 47 V- gene segments and/or one up to three D and/or 1 up to 13 J gene segments of the TCR ⁇ , extended with permutations of 3 up to 6 random nucleotides, most preferably 5 or 6 random nucleotides.
  • the method of the invention may be used to detect the presence of one or more specific TCR or BCR gene rearrangements in a qualitative way; i.e. the presence or the absence of a specific rearrangement.
  • the method may also be used to quantify the presence of specific rearrangements, or may be adapted to only detect the presence of predominant rearrangements in a sample.
  • Qualitative determination of TCR or BCR gene rearrangements may be the relative quantity to the other rearrangements present in a sample or may be quantified relative to a reference TCR or BCR rearrangement that is present in a predetermined quantity.
  • One or more reference-CDR rearrangements may also be added to the sample in a known quantity / concentration in order to make other rearrangements quantifiable.
  • a quantitative reference nucleic acid is applied to the methods of the invention in the T- or B-arrays.
  • the reference nucleic acid is a set of nucleic acid molecules comprising a constant sequence linked to a random hexamer sequence. Preferably in this set all of the 4096 random hexamer sequences are present in equimolar ratios.
  • the random hexamer sequence is preferably linked at the 3 '-end of the sequence to a constant sequence. Preferably the random hexamer sequence is linked directly to the 3'-end of the constant sequence.
  • the reference nucleic acid preferably is a synthetic nucleic acid molecule, e.g.
  • the reference nucleic acid preferably is a single stranded nucleic acid molecule.
  • the reference nucleic acid sequence may be a sequence complementary to or contain an annealer sequence as described for the analysis of a BCR or TCR sample.
  • the reference nucleic acid may then be hybridized to a fiuorescently labeled annealer sequence, preferably in equimolar ratios. This combination (i.e. the ds-hybrid of the reference nucleic acid and the labeled sequence complementary to the constant part of the reference nucleic acid) is referred to herein as a "reference probe".
  • the reference probe can be added to a T- or B-array sample (i.e. TCR or BCR sample hybridized with the annealer of interest) and hybridized to the array.
  • a T- or B-array sample i.e. TCR or BCR sample hybridized with the annealer of interest
  • the reference probe and the annealer used in the TCR or BCR sample are labeled with different fluorochromes.
  • Figure 11 illustrates this for the situation where the constant region of the reference probe is identical to the annealer used for the analysis of a TCR sample. Addition of a reference probe allows assessment and corrections of inhomogeneities in signal intensities in the array. Furthermore it allows to study sequence specific effects and to correct for specific hybridization/ligation effects and potential interactions between hexamer sequences. The skilled person will appreciate that each individual annealer will require its own specific reference probe.
  • the reference nucleic acid is modified in such a way that the reference nucleic acid can be added to the RNA or cDNA of the original TCR and/or BCR sample before amplification and that it can subsequently be co-amplified with this sample. In this way potential skewing of the repertoire due to sequence differences during amplification can be investigated and corrected.
  • this modification consists of addition of primers on either side of the reference nucleic acid sequence, allowing co-amplification with the BCR or TCR sample in a polymerase chain reaction.
  • the invention relates to use of a reference probe in the analysis of universal microarrays to correct for skewing in the analysis of complex nucleic acid samples due to skewed amplification or inhomogenieties in microarrays
  • the reference probe preferably consists of a set of nucleic acid molecules comprising a constant sequence linked to a random hexamer sequence. Additionally nucleotide sequence might be added on one or both ends to allow coamplification with the sample to be analyzed. Preferably in this set all of the 4096 random hexamer sequences are present in equimolar ratios.
  • annealers used analyze subsequent Jdels from a single J gene segment, these annealers will show strong sequence homology. This may induce interference between these different annealers, e.g. since annealers tailored to high Jdels, i.e. number of nucleotides deleted >6, will also hybridize to low Jdels, e.g. J gene segments with only 2 or 3 deleted (resp Jdel(2) or Jdel(3)) and interfere with signal production by Jdel(2) or Jdel(3) specific annealers. This might be prevented by only combining annealers on one array that show little sequence homology, preferably specific for different J segments.
  • a Jdel(n) specific annealer detects a Jdel(n- ⁇ T) TCR the first d nucleotides of J-oriented part of the hexamer signal (counting from the position where the hexamer is ligated to the Jdel(n) sequence) will be identical to the first d nucleotides starting from position n in the J-germline segment, with a maximum of 6 nucleotides (see Figure 12). This will present itself as a germline signal, recognizable in the analysis based on the hexamer sequence if the J-specificity and deletion status of the annealer is known.
  • each T-array will show germline signals that on average increase in intensity with increasing d. This phenomenon likewise occurs in the analysis of V or D gene segments, both in T- and in B-cell receptor analysis. Depending on the array structure it might be advantageous to blocks these germline signals. Germline signals may hinder analysis if annealers are pooled on the same array, since germline signals from one annealer might block signals from another annealer. In a preferred method of the invention, therefore, this is prevented by extension of the annealer using a ddNTP specific for the J-segment analyzed with the annealer.
  • the annealer is hybridized to the DNA or cDNA of the TCR pool analyzed. Subsequently an extension reaction is performed using a nucleotide variant that prevents subsequent ligation or elongation, e.g. dideoxy nucleotide, and using a suitable DNA polymerase. Probes in which within the TCR sequence the germline sequence extends beyond the sequence complementary to the annealer a ddNTP specific for the sequence will be incorporated. This will prevent subsequent ligation in the hybridization/ligation reaction on the universal microarray (see Figure 13). The same technology may also be used for the analysis of B-cell receptor repertoires.
  • kits of parts for carrying out the method or process of the invention, or for carrying out individual steps of the invention.
  • a kit of parts may comprise sets of specific primers suitable for amplifying rearranged TCR or BCR nucleotide sequences to produce polynucleotides that contain the NDN-region. These primers may be labeled.
  • the kit may also comprise a solid carrier comprising random oligonucleotides (e.g. hexamers), optionally provided with 3' or 5' sequences complementary to a part of or the complete sequence of one or more V, D or J segments of a TCR or BCR chain, for use according to the method of the invention, and a document with instructions in written or in electronic form on a data-carrier.
  • the solid carrier may be divided in subsections that each may be individually loaded with one or more annealer oligonucleotides in a design that is intended to analyze the complete receptor repertoire of one or more individual BCR or TCR chains using the method described in the first embodiment of the invention.
  • the kit preferably also comprises labeled annealer oligonucleotides that are complementary to V, D or J specific sequences.
  • the kit may also comprise a mixture of random oligonucleotides between 3 and 10 nucleotides in length, preferably random hexamers labeled with a fluorescent label.
  • kit of parts according to the invention may be designed and adapted for the detection of specific rearrangements known to be linked to or associated with certain infectious or autoimmunity diseases, by providing primers and arrays that allow amplification and detection of specific BCR or TCR gene rearrangements that are associated with the presence of a disease or infection.
  • the first embodiment is modified such that the target of amplification is not CDR3, but is any part of the J or V gene segments of the B-cell receptor, including CDRl or CDR2, using a similar approach as described for CDR3. It is well within the capabilities of the skilled person to select suitable primers for amplification CDR fragments.
  • the annealers are chosen to be specific for regions in the V or J gene segments and to hybridise in close proximity to (hyper)variable regions to detect presence and frequency of somatic mutations.
  • the second embodiment is modified such that the target of amplification is not CDR3, but can be any part of the J or V gene segments of the B- cell receptor, including CDRl or CDR2, using a similar approach as described for CDR3.
  • the oligonucleotides on the array are adapted for on specific regions in the V or J gene segments to detect presence and frequency of somatic mutations in the adjacent nucleotide sequences.
  • the fluorescent random oligonucleotide that encodes the germline sequence may carry a different tag than the other random oligonucleotides.
  • the third embodiment is modified such that the target of amplification is not CDR3, but can be any part of the J or V gene segments of the B- cell receptor, including CDRl or CDR2, using a similar approach as described for CDR3.
  • the oligonucleotides on the array are focused on specific regions in the V or J gene segments to detect presence and frequency of somatic mutations in the adjacent nucleotide sequences.
  • this array can discriminate between 1,000,000 different T cell clones, in our terms: has a resolution of 1,000,000 different clones. If all background T cell clones would have the same size we would expect on average expect 25 different T cells clones in each spot. Following a poisson distribution the expectation is that due to this random background no spot would contain more than 52 clones. We therefore expect that the increase in signal due to the 10,000-fold expanding T cell clone is much higher than this background noise. This signal may be even more clearly observed if ratios of spot signals before and after expansion are compared.
  • T cell clones may have expanded considerably. In models of chronic disease it has been reported that up to 25% of the repertoire may be antigen specific (10).
  • Muraro et al (13) reported that in HTLV-I associated myelopathy an individual T cell clone accounted for 2% of the CD8 T cells, and 14.2% of the Taxl l- 19-specific, HLA- A2 restricted CD8+ T cells in the peripheral blood.
  • the frequency of a MBP83-99-specific T cell clone P2-10 in the peripheral blood was 1:7000 PBMCs during remission and 1: 4000 PBMCs resp. 1:1800 PBMCs during two exacerbations of the disease (two years apart).
  • T beta chains seem to pair with a single alpha chain only. If a T cell clone makes up 2% of the T cells, among a sample of 25,000,000 T cells this clone would have expanded to 500,000 cells, all localizing on the same spot in our array. This is substantially higher than the average 25 cells per spot, and is well detectable using current techniques.
  • the current invention in all embodiments and variants thereof, allows detection on a solid carrier of the repertoire of rearranged T-cell or B-cell receptors, partially or fully. It allows detection of the full repertoire or a selected part of the repertoire, by choosing and combining primers that are specific for certain gene segments, V, J and/or C gene segments flanking the CDR on either side.
  • the method of the invention may thus be used or applied for the diagnosis of an immune response against a pathogen, allergen or auto-allergen.
  • the repertoire of T- and B-cells will change in response to stimulation of the immune system upon exposure to various external and internal stimuli, ranging from allergens, toxins, autoantigen to pathogens.
  • the results of the VDJ rearrangement, nucleotide deletion and insertion, and hypermutation pathway in response to these stimuli can now be visualized in a convenient way.
  • the current invention provides new diagnostic tools for detection of immune responses to infections, allergies and immune disorders, in particular autoimmune disorders.
  • the method of the invention allows detection of both predominant rearrangements that are induced in response to a certain agent, but by choosing specific primer pairs, may also be used to detect TCR or BCR rearrangements that are relatively rare.
  • Each of the embodiments reveals the V, D or J gene segment used, the number of nucleotides deleted and sequence information regarding the first few nucleotides of the NDN region. This information can be used to selectively amplify and fully sequence the rearrangement under study.
  • the method of the invention allows identification of patterns of TCR and/or BCR rearrangements which are associated with immune responses upon exposure to a given toxin, pathogen or (auto)allergen. Once a pattern of rearrangements has been established by analysis of micro-array data, T- and/or B-cell repertoires of subjects may be diagnosed using the method of the invention to detect an immune response, which immune response may be associated with clinical symptoms or a disease, allergy or infection.
  • TCR or BCR pattern Once a TCR or BCR pattern has been established, more targeted and specific tests may be designed by choosing a more discriminatory and limited primer set that allows detection of those specific TCR or BCR rearrangements indicative of a certain disease, condition or infection. Screening of the full repertoire would then no longer be required.
  • the method of the invention allows both identification and monitoring of T cell clones without a priori knowledge of variable sequence, antigen specificity, or T cell phenotype.
  • the method has sufficient resolution to detect single clones and sufficient sensitivity to pick up expansion of T cell clones early after antigenic exposure or stimulation or infection.
  • the method is scalable and automatable for T- and B cell receptor repertoire analysis of blood and tissue samples from humans and other species.
  • the method of the invention can be used for rapid, complete, unbiased screening of the B- and T cell repertoire for the presence of dominant clones or changes in the BCR or TCR repertoire or composition.
  • full nucleotide sequences of dominant BCR or TCR chains can be obtained using primers consisting of part the annealer sequence followed by the nucleotide sequence specific for the dominant spot. The resulting information regarding repertoire constellation, repertoire changes and dominant clones will find applications in diagnostics and medicine.
  • the method of the invention can for instance be applied for modeling of T cell responses where the antigenic determinant is unknown.
  • the CDR3 region of the TCR-beta and possibly also the TCR-alpha each extend over half of the peptide in the antigen binding groove.
  • the TCR residues that contact residues of the peptide seem to make up the center part of the CDR3 region.
  • the TCR sequence information acquired in the T-array can be combined with existing 3-D structural data accessible in public databases to model MHC / T cell receptor complexes in silico.
  • the method and means of the invention are also advantageous for use in the diagnosis of infectious diseases, autoimmune disease, and cancer.
  • Dedicated TCR arrays can be developed that can be used as diagnostic test and as monitoring tool for the immune response.
  • These dedicated arrays according to the invention find application in chronic rheumatic diseases, diabetes, M. Crohn and other chronic diseases, in particular autoimmune diseases.
  • Major infectious diseases like AIDS and hepatitis, meningitis, cholera, typhus, lepra and tuberculosis may be diagnosed according to this invention.
  • the invention provides for the use and development of small dedicated arrays specific for any clinical problem (e.g.
  • TCR and BCR arrays may become relevant to examine an immune response against malignant cells (or a lack thereof).
  • the technique may have important veterinary applications, e.g. in the detection of infections, and use of hormones.
  • a further application of the method of the invention is in the search for T- or B- cell clone specific targets for novel therapeutic treatments.
  • the method of the invention allows identification of new targets (such as specific TCR-rearrangements) for new methods and means for treatment and/or diagnosis. In particular it provides for detection of allergic reactions and (early onset) auto-immune responses.
  • the method can be used to detect disease associated T- or B-cell clones in animal disease models. These animal models could then be used to test the efficacy of specific therapeutic interventions on the detected T- or B-cell clone activity.
  • this may be applied to find novel targets in human disease.
  • the method of the invention also finds its application in vaccine development and testing.
  • the (lack of) efficacy of vaccines is often poorly understood.
  • the method of the invention can be used to rapidly select important epitopes on microbial products or analogues thereof.
  • the method of the invention can also be applied in fundamental research on T- and B-cell development. Currently, large efforts are invested in order to understand how T- and B-cell develop into various phenotypes. The ability to trace and quantify particular clones is critical in this effort.
  • the method described allows monitoring of the relevant T- and B-cell population in a rapid, sensitive, and in high- resolution.
  • FIG. 1 The T-array protocol, an example of the first embodiment of the method of the invention, (a) During development, VDJ recombination causes enormous variability in TCR ⁇ chain by randomly selecting different combinations of 23 V, 2 D, and 13 J gene segments, by nucleotide insertion (arrow up), and by nucleotide deletion from V( arrow pointing left), D, and J (arrow pointing right) genes. This results in a diversity of an estimated 10 6 different ⁇ chains per individual, (b) N-deletion causes shortening of the V ⁇ and J ⁇ segments. The number of nucleotides deleted from V ⁇ and J ⁇ germline DNA is limited. N-deletion of 192 published TCR ⁇ mRNAs was analyzed.
  • the figure shows the cumulative percentage of CDR3 ⁇ s for the number of nucleotides deleted.
  • the T-array protocol (cl) cDNA from T-cells is generated.
  • CDR3 ⁇ regions are PCR amplified using biotinylated V ⁇ -specific or V ⁇ -generic primers (not shown here) and a C ⁇ or J ⁇ specific primer.
  • Biotinylated strands may be removed after alkaline denaturation using streptavidin coated beads.
  • FIG. 3 Spectratyping and T-array for Jurkat T-cell clone mixed with peripheral blood CD4 + T-cells.
  • Jurkat cells were added in different dilutions to a background of peripheral blood CD4 + T-cells.
  • panel a CDR3 spectratyping with V ⁇ l2-specific primer. The arrow indicates a length identical to 14 amino acids, which is the length of the Jurkat CDR3 ⁇ .
  • panel b A detail of the T-array scans. Arrows indicate hexamer sequence GTTCGG, which is complementary to the first six nucleotides of the Jurkat NDN ⁇ region,
  • panel c Signal intensities of all 4096 spots of the T-array. Black arrows indicate hexamer sequence GTTCGG.
  • FIG. 1 Diversity of the human TCR ⁇ repertoire and methods for repertoire analysis.
  • the human T-cell repertoire contains an estimated 10 6 rearrangements per individual. The resolution of various methods is given in number of separation units per assay.
  • RNA of the T-cell receptor beta chain is amplified in a RT-PCR reaction using a biotinylated V-beta primer and a J-beta primer.
  • the top strand is removed using Streptavidin coated magnetic beads. Together with short fluorescently labeled annealers the bottom strand is added to an array encoding part of the TCR V-beta chain hexamer array linked to a spot-specific random nucleotide sequence. Subsequently a ligation reaction is performed. After washing signals from the individual spots in the array are quantified.
  • RNA of the T-cell receptor beta chain is amplified in a RT-PCR reaction using a V-beta primer and a fluorescently labeled J-beta primer. After denaturation the PCR product is added to the array encoding part of the TCR V-beta chain linked to a spot-specific random nucleotide sequence. After washing signals from the individual spots in the array are quantified.
  • CCTTTT representing the first six nucleotides on the J-oriented side of the NDN ⁇ region of the dominant T-cell clone that was identified in this screen.
  • FIG. 8 T-array analysis of CMV-specific cells in vivo,
  • Peripheral blood was drawn 9 weeks after primary CMV infection of a CMV-seronegative renal transplant recipient, and sorted for CMV+ IFN ⁇ -producing T-cells.
  • cDNA was amplified using anchored PCR (ref. 16). Universal, anchored sequences are crosshatched.
  • V ⁇ 's were analyzed by spectratyping and 11 V ⁇ -families were cloned and sequenced (Van Leeuwen, E.M.M. et al. Blood, 2006).
  • V ⁇ 6.1/J ⁇ 2.7+ pool was amplified by V ⁇ 6.1-and J ⁇ 2.7- primers and loaded on a hexamer array, (e) CDR3 regions, clonal frequencies, and joining sequences of the identified V ⁇ .l /J ⁇ 2.7+ T-cell clones, (f) V ⁇ 6.1/J ⁇ 2.7-specific T-array.
  • FIG. 10 B-array for Ramos B-cell clone mixed with peripheral mononuclear blood cells.
  • Ramos cells were added in different dilutions to a background of peripheral mononuclear blood cells (PBMCs) in a ratio of 1 Ramos cell in 100.000 PBMCs ( Figure 10a) and 1 Ramos cell in 1.000.000 PBMCs ( Figure 10b), respectively.
  • PBMCs peripheral mononuclear blood cells
  • Figures show signal intensities of all 4096 spots of the B-array. Arrows indicate hexamer sequence TAATAA which is complementary to the 5' end of the Ramos
  • Figure 11 Illustration of reference probes that may be used for assessment and corrections of inhomogeneities in signal intensities in the array and to correct for specific hybridization/ligation effects and potential interactions between hexamer sequences.
  • FIG. 1 Schematic depiction of interference by germline signals.
  • Figure 13 Schematic depiction of blocking of germline signals by extension of annealers using dideoxy nucleotides.
  • TCR ⁇ -CDR3 mRNA sequences of 192 human T-cell clones were collected from the public database of NCBI at NIH. V ⁇ -, J ⁇ -, D ⁇ -segments were identified using the V-QUEST algorithm from the international ImMunoGeneTics information system 6 . A number of 50 sequences were validated manually, and assignment errors were identified only for N-deletions larger than 8 nucleotides. To exclude other assignment errors all CDR3 ⁇ sequences with N-deletions larger than 7 nucleotides were therefore assigned manually.
  • Jurkat cell line clone E6-1 ATCC, Manassas, VA
  • the Ramos cell line EBV negative Burkitt's lymphoma cell line (31)
  • DMEM culture medium Sigma- Aldrich, St. Louis, MO
  • FCS 5% FCS
  • IMDM IMDM supplemented with 10% FCS
  • Human peripheral blood mononuclear cells PBMC ( Figure 3) were isolated from buffy coats of healthy blood donors by density centrifugation with Ficoll-Isopaque (Pharmacia Biotech, Uppsala, S). Informed consent was obtained from blood donors.
  • CD4 + T cells were isolated by using anti-CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, D), followed by positive selection with the VarioMACS (Miltenyi Biotec) according to the manufacturer's protocol. The purity of the CD4+ cells isolated was measured using anti-CD4 PerCP-conjugated antibodies (BD Biosciences, San Jose, CA).
  • PBMCs For flow cytometry, thawed PBMCs ( Figure 7) were resuspended in IMDM (BioWhittaker, Verviers, Belgium), containing 10% FCS and antibiotics (100 U/ml sodium penicillin G and 100 ⁇ g/ml streptomycin sulfate). Cells were washed in PBS containing 0.01% (w/v) NaN 3 and 0.5% (w/v) BSA (PBA). A total of 250,000 PBMCs were incubated with an appropriate concentration of tetrameric HLA-peptide complexes in a small volume for 10 min at 4°C.
  • CMV-specific IFN- ⁇ -producing CD4+ cells from a renal transplant recipient were isolated using an IFN ⁇ Secretion Assay Detection Kit (PE) (Miltenyi Biotec, Amsterdam, The Netherlands) according to the manufacturer's conditions.
  • PE IFN ⁇ Secretion Assay Detection Kit
  • PBMCs peripheral blood mononuclear cells
  • IFN ⁇ Catch Reagent for 5 minutes at 4°C
  • IFN ⁇ Detection Antibody PE
  • CD4 APC BD Pharmingen, San Diego, USA
  • FACsARIA FACsARIA
  • PBMCs from a CMV seropositive, HLA- A2 + healthy volunteer donor were used for expansion of CMV specific CD8 + cells. Informed consent was obtained from the blood donor.
  • PBMCs were stimulated in IMDM supplemented with 10% human pool serum, antibiotics, and 2-ME with CMVpp65-A2 peptide NLVPMVATV (1.25 ⁇ g/ml) and IL-2 (50 U/ml Biotest, Dreieich, D) in 24-well plates. After one week, cells were restimulated on a weekly basis with irradiated (30 Gy) CMV-pp65-A2 peptide loaded EBV transformed cell-lines expressing HLA- A2 + (5x10 4 cells/ml) in the presence of IL-2.
  • V ⁇ PCR products were purified and ligated into pGEM-T Easy Vector (Promega, Madison, WI) and cloned by transformation of competent DH5 ⁇ E. coli. Selected colonies were amplified by PCR using M13 primers (Invitrogen - Life Technologies, Breda, NL) and then sequenced on the ABI Prism 3730 DNA automatic sequencer (Applied Biosystem, Foster City, CA) using the dye terminator cycle sequencing chemistry (vl.l) (Perkin Elmer, Foster City, CA).
  • PCR was performed with TCR V ⁇ primers (Ref. Doumaid, K. et al. Transpl. Imm. 8, 83-94 (2000)) in combination with a TCR C ⁇ primer, labelled with fluorescent dye fluorophore fluorescamine (FAM).
  • TCR V ⁇ primers Ref. Doumaid, K. et al. Transpl. Imm. 8, 83-94 (2000)
  • FAM fluorescent dye fluorophore fluorescamine
  • Each amplification reaction was performed with 4 ⁇ l cDNA in the presence of 25 pmol 5' sense TCR V ⁇ primer, 25 pmol 3' antisense TCR C ⁇ primer, 0.5 mM MgCl 2 , 0.5 mM dNTP, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 4 mM KCl, 2.5 units AmpliTaq DNA polymerase (Perkin Elmer/Roche Molecular Systems Inc., Branchburg, NJ) in a total volume of 40 ⁇ l. PCR was performed in a Tl Thermocycler (Biometra, Goettingen, D).
  • the FAM-labelled PCR products were analyzed on the ABI Prism 3100 Genetic Analyzer capillary system (Applied Biosystem, Foster City, CA) using POP6 as separation matrix, filter set D for the detection of fluorescent signals, and ROX500 as internal size standard. Genescan Software (Applied Biosystem, Foster City, CA) was used for size determination and quantification. T-array protocol
  • cDNA synthesis (Fig IcI) is followed by PCR amplification (Ic2), isolation of polyclonal, single strands (Ic3), hybridization of annealer oligonucleotides (Ic4), and ligation, washing, scanning, and quantification of hexamer arrays (lc5-7).
  • Biotinylated PCR products were obtained using sense biotinylated V ⁇ primers against reverse, antisense C ⁇ or J ⁇ primers (PCR conditions as described above).
  • cDNA was synthesized using the smart PCR cDNA synthesis kit (Clontech, Mountain View, CA) and PCR amplified using a biotinylated primer with the 5 'Primer II A sequence given by the supplier and a reverse J ⁇ primer. Isolation of single strands (Fig. IcS).
  • annealer oligonucleotides Hybridization of annealer oligonucleotides (Fig. Ic4) . Single strands were then incubated with 1 pmol Cy5-labeled, 5' phosphorylated annealer oligonucleotide (Biolegio, Maiden, NL) at a starting temperature of 90 0 C. The heated water bath was (passively) cooled to ambient temperature. Sequences of used annealer oligonucleotides are CTAACTATGGCTACACCTTCGGTTT (Fig. 2,3), AAACTGCTGGCACAGAAGTACACTT (Fig 2d,e), ACTATGGCTACACCTTCGGTT (Fig. 7,9) and CTACGAGC AGTACTTCGGG (Fig. 8). B-array protocol
  • the B-array protocol is identical to the T-array protocol ( Figure 1), with the exception that the PCR-primers and annealers that are used are specifically designed for the (BCR) IgHV, IgHJ, and IgHC-segment.
  • the forward primer was designed for the IgHV4-34-segment (TTGAACGCCGCGGACAC, FAM labelled) and the reverse primer for the IgHJ6-segment (TTGTCCCTTGGCCCCAGAC, biotin- labelled) sense (according to the IMGT classification (6)).
  • the sense strand was used in this experiment as template for the annealer, and as input for the array.
  • the sequence of the annealer used in Fig 10 was complementary to the IgHV4-34 sense strand (CTCTCGCACAGT AATAC ACATT, 3'-Cy-5-labelled, 5 '-phosphorylated). Ligation, washing, scanning, and quantification (Fig. lc5-7).
  • Ligation on arrays was performed at 30 0 C in a volume of 90 ⁇ l (B-arrays) or 125 ⁇ l (T-arrays) in Ix BSA (NEB, Ipswich, MA), 25 ⁇ l 5x DNA Ligase buffer, 12 units T4 DNA ligase. After ligation slides were washed in 0.1 % SDS at 90 0 C, ddH2O at RT, and dried by 500 x g centrifugation for 3 minutes. Ligated arrays were scanned with a GSI Lumonics ScanArray 5000 (Perkin-Elmer Life Sciences, Boston, MA). Spot intensities were quantified using ArrayVision 6.0 software (Image Research, St. Catharines, Ontario, CDN).
  • Ligation in solution For experiments shown in Fig. 2b-e, ligation was performed in solution with single hexamer oligonucleotides. 1 pmol of hexamers, 4 units of T4 DNA ligase and 2 ⁇ l 5x DNA Ligase buffer (Invitrogen - Life Technologies, Breda, NL) and template/annealer complex were added in a total volume of 10 ⁇ l and incubated for 45 minutes at 16°C, followed by a 10 minutes denaturation step at 65°C. Ligation products were analyzed on the ABI Prism 3100 Genetic Analyzer capillary system and Genescan software as described above. Reference probe
  • control-polynucleotide was annealed using 5 pmol of control annealer (5'-P- CTAACTATGGCTACACCTTCGGTTT-3', Cy5 end-labeled). Both reaction products were mixed after annealing.
  • control annealer 5'-P- CTAACTATGGCTACACCTTCGGTTT-3', Cy5 end-labeled.
  • CDR3 complementarity determining region 3
  • VDJ-recombination by random deletion and addition of nucleotides at the V-, D-, and J-junctions and produces the hypervariable NDN region, which can be used as a signature for each TCR (Fig. Ia).
  • Fig. Ic T-array protocol to interrogate the first six nucleotides of the NDN region and the length of the J ⁇ -gene segment. Resolution is created in three subsequent steps by: i) V ⁇ -specific PCR amplification of the CDR3 ⁇ (Fig. Ic2), U) hybridization of a labelled oligonucleotide ("annealer") specific for the J ⁇ -family and for the number of J ⁇ -nucleotides deleted (Fig.
  • the resolution of this T-array protocol depends on the number of V ⁇ and J ⁇ segments, the size of the microarrays, and the number of J ⁇ -nucleotides deleted.
  • To predict the potential resolution of the assay we analyzed the distribution of N-deletion in a random selection of 192 published CDR3 ⁇ mRNA sequences. For more than 99% of the sequences a maximum of 10 nucleotides is deleted from the V ⁇ genes, and a maximum of 11 from J ⁇ genes (Fig. Ib). Within these limits, an almost uniform distribution of the TCRs was observed over the number of nucleotides deleted. This enabled us to predict the potential resolution of the assay.
  • the beta-chain repertoire contains approximately 10 6 unique sequences 10 , each of which pairs with a limited number of CC chains. Based on these numbers we calculated that -dependent on the format used - on average 0.1 to 1.6 CDR3 ⁇ sequences from the complete repertoire of a human individual will ligate to a single sequence on the universal hexamer microarray. In theory, the assay should therefore have sufficient resolution to detect single CDR3 ⁇ sequences.
  • the specificity of the protocol was tested using the T-cell clone Jurkat E6-1, for which the CDR3 is known. After PCR amplification of the Jurkat CDR3 ⁇ region, we isolated the antisense strand and hybridized it to a fluorescent Iy labelled oligonucleotide encoding the NDN-oriented end of the J ⁇ l-2 sequence. Specificity of the ligation reaction for Jurkat NDN sequence was then tested with hexamers either complementary or not complementary to this NDN sequence (Fig. 2b-c).
  • the annealer oligonucleotide was elongated, indicating that the ligation is sequence specific for the Jurkat CDR3 ⁇ .
  • the sense strand was used as a template, the annealer was elongated only with the hexamer sequence complementary to the 5 '-end of NDN ⁇ (Fig. 2d-e).
  • the Jurkat NDN sequence GTTCGG gave the strongest signal (Fig. 2f-h). This shows that the protocol is sequence specific for the T-cell clone analyzed.
  • Example 3 Determination of sensitivity T-array
  • T-array is specific for the NDN sequence of the analyzed T-cell clone
  • sensitivity of the assay was determined.
  • Jurkat cells were diluted in different concentrations in a background of peripheral blood CD4 + T-cells. Quantitative PCR showed that TCR transcripts in Jurkat cells were not more abundant than in CD4 + cells obtained from a healthy blood donor.
  • Jurkat/CD4 + mixtures were then PCR amplified with a V ⁇ l2-sense (according to the IMGT classification (6), corresponding to V ⁇ 8 in the Arden classification (5)) primer and a fluorescamine-labelled C ⁇ reverse primer and size separated by capillary electrophoresis (Fig. 3a).
  • the antisense strands were isolated and hybridized to a J ⁇ -l-2-specific, Cy5- labeled oligonucleotide and ligated on a universal hexamer microarray (Fig. 3b-c).
  • the hexamer sequence GTTCGG which is complementary to the 3' end of Jurkat NDN region, was quantitatively picked up (Fig. 3c).
  • the GTTCGG signal was similarly intense as the hexamer spots TGTCGG and CTTCGG.
  • Example 4 Detection of expanding T-cell clones after viral antigen stimulation
  • Human peripheral blood cells from a healthy HLA- A2 + donor latently infected with the ⁇ herpes virus CMV were isolated and stimulated with the CMV-peptide NLVPMVATV.
  • This 9-amino acid motif from the viral structural protein pp65 dominates the cytotoxic T-lymphocyte response against CMV (Ref: Wills, M.R. et al.
  • the human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T- cell receptor usage of pp65- specific CTL. J. Virol. 70, 7569-7579 (1996)).
  • the CD8 + response to NLVPMVATV is V ⁇ -restricted, in particular for but not limited to V ⁇ 6-1 + T-cells (IMGT classification (6); V ⁇ l3 + in Arden classification (5)), which was in agreement with spectratyping analysis of our donor (data not shown).
  • T-cell clones Tolerance of T-cell clones is associated with membrane antigen changes. Nature 303, 625-627 (1983)). During the next 10 days, the fraction of tetramer-positive cells slowly increased to -60%. From day 6, the V ⁇ l3 + compartment became restricted to a CDR3 length correlating to 14 amino acids (Fig. 7b), suggesting that either a single T-cell clone or only a limited number of clones in the V ⁇ l3 compartment had expanded.
  • sequences that have a 3' CTA-end identical to the germ-line end sequence of J ⁇ l-2. These sequences derive from TCRs that had no nucleotides deleted from the germline J ⁇ l-2 gene and therefore give 5' -(NNN)CT A-3' signals. Similarly, sequences that have a (NNNN)T A-end derive from TCRs encoding the germline J ⁇ l-2 gene with only one terminal nucleotide deleted. Indeed, these signals derive from such TCR sequences as shown by complete sequencing of these TCR ⁇ 's (see also below).
  • Example 5 Validating T-array data by sequencing of multiple T-cell clones To test whether the T-array signal matches the frequency of these T-cell clones as estimated by repetitive cloning and sequencing, we sequenced V ⁇ 6-l + /J ⁇ l-2 + TCRs
  • T-array signal well above background. Eleven clones were detected with a frequency of only 1 out 52. Three of these gave T array signals above background. Eight clones gave signals similar to background, suggesting that the concentration of these clones in the blood sample is below the detection limit of the T-array. The clonal frequencies measured at day 3 were in agreement with the expansion measured by the T-array, showing that the T-array protocol quantitatively detects clonal expansion.
  • Example 6 Application of T-array protocol for in vivo detection
  • T-array protocol for detection of clones in vivo
  • This sample of 11,600 sorted CMV-specific T-cells was pre- amplified by anchored PCR, which was used here as pre- amplification step to generate sufficient cDNA from a relatively small amount of RNA (Fig. 8a-b).
  • Spectratyping indicated a relatively broad repertoire.
  • V ⁇ .l Within the repertoire 11 V ⁇ families, among which V ⁇ .l, were extensively analyzed by cloning and sequencing. In the V ⁇ .l pool, 60 clones were sequenced, revealing 12 unique sequences of which 4 were J ⁇ 2.7 + .
  • a T- array was performed to screen the V ⁇ .l- J ⁇ 2.7 subpopulation with an annealer oligonucleotide that detects J ⁇ 2.7 sequences with 3 or less nucleotides deleted from the J ⁇ 2.7 gene (Fig. 8f). All 3 clones that meet these criteria were picked up with the T- array (Fig. 8e). In addition, the T-array signal matched the clonal frequency of the T- cell clones identified.
  • Example 7 V ⁇ -generic protocol for screening the T-cell repertoire
  • Figure 7 shows hexamer arrays for V ⁇ /J ⁇ -specific TCR sequences which cover only 0.03% of the repertoire. Full-repertoire screening would not be feasible with this approach due to the large amount of hexamer arrays required. To analyze a significantly larger fraction of the repertoire on one single
  • Figure 9 shows a V ⁇ -generic T-array obtained after anchored-PCR amplification (Fig. 9f) compared to a V ⁇ -6-1 specific T-array (Fig. 9e) of the same sample.
  • the generic T-array shows expansion of the same V ⁇ 6-1 clone (IMGT classification (6); V ⁇ l3 + in Arden classification (5)) identified earlier in the V ⁇ l3- specific array (hexamer CCTTTT).
  • the signal of this clone is approximately 12-fold lower than the signal obtained in the V ⁇ 6-1 -specific array.
  • FIG. 9f shows that in the generic T-array protocol other expanded T-cell clones, apparently V ⁇ 6-1 -negative, are identified. A number of these sequences have a 3' (C)TA-end, identical to the germ- line end sequence of J ⁇ l-2, which indicates that these sequences probably derive from
  • Example 8 Determination of sensitivity ofB-array The same principle of the T-array was also evaluated for B-cell receptor (BCR) analysis. An experiment was performed which was similar to example 3 but now adapted for the BCR (B-array). For this experiment cells of the B-cell clone Ramos (31) were diluted in a background of peripheral mononuclear blood cells (PBMC). Ramos/PBMC mixtures were then PCR amplified with an IgHV4-34 (according to IMGT classification) fiuorescamine-labelled- sense primer and a biotin-labelled IgHJ6 (according to IMGT classification) reverse primer.
  • PBMC peripheral mononuclear blood cells
  • the sense strands were isolated as described in the material and methods section and hybridized to an IgHV4-34 specific, Cy5-labelled oligonucleotide and ligated on a universal hexamer microarray (Fig 10).
  • the hexamer sequence TAATAA which is complementary to the 5 '-end of Ramos NDN region, was quantitatively picked up as the strongest signal (Fig 10a).
  • the sequence TAATAA was still picked up as fourth strongest signal (Fig 10b).
  • the reference probe spiked with Jurkat synthetic CDR3 DNA (control DNA), was annealed to a Cy3-labeled annealer.
  • the control polynucleotide was annealed to a Cy5-labeled annealer. After the annealing reaction both probes were mixed and incubated on the microarray.
  • the diverse repertoire of TCR rearrangements can potentially be analyzed using microarrays, which have a high capacity to differentiate and monitor many unique DNA rearrangements in parallel.
  • the size of the TCR ⁇ repertoire at the DNA level is too large for full TCR repertoire analysis at single-clone resolution on a single microarray.
  • the ⁇ receptor diversity is estimated at 10 15 to 10 18 rearrangements, which is formed for a relatively large part by the ⁇ chain.
  • the size of the ⁇ chain repertoire is much more limited (10 6 ), and microarrays could produce sufficient resolution to distinguish single T cell clones in the repertoire of one individual.
  • T-arrays tag individual clones based on the sequence information in the NDN-J, NDN-V, ND or DN junction.
  • the tag for each clone consists of the J, D or V family used, the number of terminal nucleotides deleted from this J, D or V gene segment and the first six nucleotides of the NDN, ND or DN region.
  • This design creates more than 10 6 unique tags, which in theory allows single- clone analysis of the complete TCR ⁇ repertoire on high-density microarrays. The validity of the T-array protocol was shown in several experiments. Jurkat
  • TCR was selectively ligated to hexamer oligonucleotides complementary to its NDN sequence both in solution and using hexamer arrays (Fig. 2).
  • the protocol allowed early, highly specific identification of an expanding T cell clone after in vitro stimulation with CMV-peptide (Fig. 7).
  • IFN ⁇ -secreting CD4+ T cells from a renal transplant patient 9 weeks after CMV infection T-arrays detected the dominant V ⁇ 6.1 + /J ⁇ 2.7 + clones identified earlier by extensive cloning and sequencing (Fig. 8).
  • the sensitivity of the protocol was determined after mixing a Jurkat T-cell clone in a background of peripheral blood CD4+ T-cells in a range of dilutions.
  • the data show that the Jurkat TCR rearrangement was detected in a ratio of at least 1 in 10 6 (Fig. 3). This is two logs more sensitive than V ⁇ /C spectratyping, which can detect a T-cell clone in 1 in 10 4 .
  • V ⁇ -J ⁇ spectratyping an alternative approach which is not widely used, is theoretically 12-fold higher than that of V ⁇ spectratyping and therefore 10-fold less sensitive than the T-array approach.
  • T-arrays make highly sensitive detection and tracking of T cells possible.
  • the protocol allowed quantitative monitoring of T cell clones.
  • the signal clearly decreased (Fig. 3)
  • the increasing frequency of the CMV-specific clone in the in vitro experiment as evidenced by tetramer staining and spectratyping was also clearly reflected in the arrays, and confirmed by the repetitive cloning and sequencing, even as early as day zero.
  • the observed clonal frequencies of the CMV-specific clones in the in vivo experiment were quantitatively reflected in the T-array data.
  • Algorithms based on known ligation patterns have been developed that identify false positives and reduce the loss of resolution when complex mixtures such as full-genome transcripts are analyzed on hexamer arrays. Such algorithms may help further to minimize the effect of cross ligations on the resolution of T-arrays and help to detect less frequent clones.
  • the technology described here can be applied to quantitatively monitor a small selection of the TCR ⁇ repertoire, and sensitively and quantitatively track a subset of T- cell clones. While the combination of spectratyping, cloning and sequencing may take several weeks, the T-array method takes only a single day including scanning and quantification. Furthermore it is sensitive, and allows monitoring of growth kinetics at the clonal level. This rapid and sensitive method may find application in the study of the relation between clonal expansion of T cells and autoimmune phenomena, e.g. responses to immunotherapy, retrospectively and prospectively. Recurrence of autoimmune disease could be predicted in the case of previously identified clones, or the fate of T-cells in adoptive therapy against cancer could be monitored at single-clone level.
  • FIG. 9 shows the feasibility of a protocol in which T-array analysis is preceded by simultaneous amplification of all V ⁇ families in one PCR reaction using anchored PCR 16 .
  • the resulting 144 arrays can then be housed in a high-density matrix of multiple arrays that can be individually loaded.
  • Such matrices have recently become available. Rapid, quantitative and sensitive full repertoire screening would have significant impact in immunological research and on the development of immunotherapeutics.
  • Identical arrays might be built for the analysis of the TCR ⁇ , - ⁇ and : ⁇ repertoires and of the B- cell receptor repertoire in humans and other species.
  • Hennecke J, Wiley DC Structure of a complex of the human alpha/beta T cell receptor (TCR) HAl.7, influenza hemagglutinin peptide, and major histocompatibility complex class II molecule, HLA-DR4 (DRA*0101 and DRBl*0401): insight into TCR cross-restriction and alloreactivity. J Exp Med 2002; 195(5):571-581.
  • TCR human alpha/beta T cell receptor
  • Butz EA Bevan MJ. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 1998; 8(2): 167- 175. (19) Ruiz M, Giudicelli V, Ginestoux C, Stoehr P, Robinson J, Bodmer J et al.
  • the sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments. Proc Natl Acad Sci U S A 1993; 90(9):4319-4323. (24) Ogle BM, Cascalho M, Joao C, Taylor W, West LJ, Platt JL. Direct measurement of lymphocyte receptor diversity. Nucleic Acids Res 2003; 31(22):el39.

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Abstract

La présente invention concerne un procédé de détermination de réarrangements génétiques des récepteurs de TCR ou BCR et de permettre la visualisation du répertoire des domaines CDR3 réarrangés V(D)J sur une matrice d'oligonucléotides d'un support solide. Le procédé permet de visualiser le résultat final du processus de recombinaison V(D)J et, en particulier, du processus de délétion et d'insertion qui a lieu dans la région hypervariable NDN, 1 à 13 nucléotides étant délétés des segments géniques V, D et/ou J par l'utilisation d'un oligonucléotide hybrideur marqué qui s'hybride sur ou à proximité étroite de la région NDN. Le procédé permet la détection du répertoire complet ou d'une partie sélectionnée du répertoire en choisissant et en combinant des amorces qui sont spécifiques de certains segments géniques V, D et/ou J encadrant le CDR3 de chaque côté. Le procédé de l'invention peut être utilisé ou appliqué dans le diagnostic d'une réponse immune contre un agent pathogène, un allergène ou un auto-allergène.
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