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WO2013093170A1 - Procédés et compositions permettant de déterminer la diversité du répertoire de lymphocytes t d'un individu - Google Patents

Procédés et compositions permettant de déterminer la diversité du répertoire de lymphocytes t d'un individu Download PDF

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Publication number
WO2013093170A1
WO2013093170A1 PCT/ES2012/070910 ES2012070910W WO2013093170A1 WO 2013093170 A1 WO2013093170 A1 WO 2013093170A1 ES 2012070910 W ES2012070910 W ES 2012070910W WO 2013093170 A1 WO2013093170 A1 WO 2013093170A1
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seq
primers
tcr
chain
primer
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Alejandro VALLEJO TILLER
Manuel Alejandro GONZÁLEZ-SERNA MARTÍN
Manuel Leal Noval
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Universidad de Sevilla
Fundacion para la Investigacion Biomedica del Hospital Universitario Ramon Y Cajal
Servicio Andaluz de Salud
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Universidad de Sevilla
Fundacion para la Investigacion Biomedica del Hospital Universitario Ramon Y Cajal
Servicio Andaluz de Salud
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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
    • C12Q1/6876—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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to a method for determining the diversity of the repertoire of T lymphocytes of an individual, as well as to compositions for carrying out said method.
  • the T-cell receptor is the molecule that is involved in antigen recognition and that gives these cells their ability to discriminate.
  • the variability in the TCR chains is centralized in the binding region of the antigenic peptide, which is encoded by the gene segments V and J in the ⁇ chain, or V, D and J in the ⁇ chain.
  • V and J in the ⁇ chain
  • V, D and J in the ⁇ chain
  • the resulting gene regions code for the CDR3 loops, which will be of varying length and that form the center of the antigen binding site.
  • TCRs unlike immunoglobulins, do not suffer somatic hypermutation and, therefore, all diversity is concentrated in CDR3.
  • Disturbances in the repertoire of TCR have been observed in subjects infected with HIV-1, possibly reflecting responses of cytotoxic T lymphocytes (in English cytotoxic T lymphocyte, CTL) energetic but not completely effective, mainly composed of CD8 T cells resulting from a oligoclonal expansion that share common antigenic determinants. These disturbances frequently occur either by clonal expansion of T cells or by deletion in one or more families of ⁇ . Multiple or persistent clonal expansions of CD8 T cells have been observed in individuals with chronic or progressive HIV-1 infections. HIV-1 infection induces disturbances in the repertoire of peripheral T-cell receptors during primary infection.
  • Epitope-specific responses are the sum of individual responding clones of T cells and a repertoire of structurally diverse TCRs is likely necessary for optimal elimination of viremia in infections capable of rapidly developing escape mutations.
  • a varied matrix of TCRs could reduce the likelihood of CTL escape due to cross recognition of mutated epitopes and is more likely to contain high affinity T cell clones capable of recognizing specific epitopes efficiently and, therefore, mediate Better control of virus replication.
  • Oligoclonal expansions and deletions within subpopulations of T cells can be measured by analysis of the CDR3 hypervariable region of the TCR (Raaphorst et al., 2002, Hum. Immunol. 63: 51-60). Variation in CDR3 length reflects changes in the TCRVP repertoire during HIV-1 infection
  • the variation in the length of the CDR3 is the basis of the spectratypin ⁇ technique where said region is amplified and the distribution of the different lengths is an indicator of the variety of the TCR repertoire.
  • This technique is useful to detect clonal expansions in response to certain pathological situations, such as with tumor-infiltrated T cells (Puisieux et al, 1994, J. Immunol. 153: 2807-2818), or to analyze variations that may occur in the repertoire during the course of certain diseases, such as immunodeficiencies or autoimmune diseases.
  • a difficulty associated with this technique is the high number of necessary reactions and a complicated interpretation of the results. In Monteiro et al. (Monteiro et al, 1995, Mol. Med.
  • a method of analyzing the diversity of the repertoire of T and / or B lymphocytes by multiplex PCR is described in US 2011/0003291.
  • the total number of primers defined is 119, which are specific for regions of the V or D genes, and J, so that they allow the detection of V (D) J or D-J rearrangements.
  • Fernandes et al. they developed a system for spectratyping by multiplex PCR for the evaluation of the repertoire of the ⁇ chain of the TCR (Fernandes et al, 2005, Clin. Diagn. Lab. Immunol.
  • the invention relates to a composition or kit for analyzing the repertoire of T cells in a sample comprising:
  • each of said primers specifically hybridizes with segment V of a gene encoding a ⁇ chain of the TCR
  • the invention relates to a method for analyzing the repertoire of T cells in a sample comprising the steps of
  • the invention relates to the use of the composition or kit, or of the method of the above aspects in the diagnosis of a pathology.
  • the invention in a final aspect, relates to a method for monitoring the evolution of a pathology in a patient in response to a treatment comprising determining the complexity of the repertoire of T cells in a sample of said patient after being subjected to said treatment, where an increase in the complexity of said repertoire with respect to complexity before starting treatment is indicative that the patient responds to said treatment.
  • Figure 1 describes the expression ratios of each of the TCRVP families at different points in time. Families with a significantly disturbed expression rate at the time of treatment discontinuation are highlighted.
  • FIG. 2 describes the proportion of the expression of disturbed families of TCRVP.
  • A The expression of TCRVplO, TCRVpl4 and TCRVpl5 was increased at the time of treatment discontinuation, and
  • B the expression of TCRVP20, TCRVP28 and TCRVP29 was decreased. Black lines represent average values.
  • Figure 3 describes the proportion of the expression of families of TCRVP disturbed according to the time of antiretroviral treatment. Empty tables, patients with ⁇ 100 months of antiretroviral treatment; full pictures, patients with> 100 months of antiretroviral treatment. Black lines represent average values.
  • Figure 4 describes the proportion, cell activation and apoptosis levels of Vpl4, Vp20 and Vp2 cells.
  • Figure 5 describes recent recent thymus migration cells and memory effector cells of ⁇ 14, ⁇ 20 and ⁇ 2 cells.
  • the inventors have developed a new CDR3 spectratyping technique to simplify the study of the ⁇ repertoire of T cells.
  • This technique is a new approach to the study of the cell repertoire in the first place due to the original design of a group of primers that amplify the sequences representative of the wide cellular repertoire, unlike previous works that focused their analysis in other regions of the TCRp.
  • the design of the test that facilitates and simplifies both the obtaining of the data is also novel, thanks to the different size ranges of the amplification products and the two markers used, which allows the products to be summarized in only three PCR reactions in format multiplex, as well as the interpretation of the same without the need to amplify internal controls to which to refer the results as in previous works.
  • compositions or kits to analyze the repertoire of T cells are provided.
  • composition or kit of the invention relates to a composition or kit for analyzing the repertoire of T cells in a sample, hereinafter composition or kit of the invention, comprising
  • T cell or "T lymphocyte” refers to those leukocytes that have ovoid-shaped nuclei that occupy most of the intracellular space. T lymphocytes are responsible for coordinating the cellular immune response, making up 70% of the total lymphocytes that secrete proteins or cytokines. They also deal with the cooperation to develop all forms of immune responses, such as the production of antibodies by B lymphocytes. They differ from B lymphocytes and killer cells (natural killer or K) by having a special receptor in the membrane surface, the T cell receptor (called TCR by T cell receptor).
  • TCR T cell receptor
  • CD4 + lymphocytes for expressing the surface CD4 marker. They are responsible for initiating the cascade of the coordinated immune response by interacting with the "peptide-CMH- ⁇ " complex, which is expressed on the surface of antigen presenting cells. When activated, CD4 + lymphocytes specialize, differentiating themselves into effector lymphocytes, which are distinguished by the type of cytokines they produce:
  • Thl which migrate to infected tissues and assist in the activation of macrophages, since Thl fundamentally secrete ⁇ interferon; Thl are important in defense against intracellular microbes and inflammation;
  • Th2 which remain primarily in lymphoid tissues and assist in the activation of B lymphocytes; mainly secrete IL-4 (which stimulates the secretion of IgE, which in turn activates mast cells) and IL-5 (which activates eosinophils); Th2 are important in allergic reactions and in defense against parasites; - Thl 7, so named because they secrete IL-17, in addition to IL-22; They are the main mediators in some allergic reactions, and appear to be involved in the development of diseases such as multiple sclerosis, rheumatoid arthritis and inflammatory bowel disease.
  • Thl, Th2 or Thl7 The differentiation in Thl, Th2 or Thl7 is not random, but depends on the stimuli received by the virgin T4 lymphocyte when it contacts a foreign antigen.
  • CTL Cytotoxic T lymphocytes
  • CD8 + lymphocytes for expressing the surface CD8 marker. They are responsible for the effector functions of cellular immunity, through interaction with a “peptide-CMH-I” complex; CTLs recognize the cells infected by the pathogen for which they are specific or tumor cells, and destroy them by secreting a series of molecules (perforin, granzymes, FasL) that activate apoptosis of the target cell.
  • Memory T lymphocytes are cells that are generated after activation of T lymphocytes, by exposure to a foreign antigen (a pathogen).
  • Memory cells can be both CD4 + and CD8 +, and typically express the CD45RO cell surface protein. They have a long life, are functionally inactive, and can circulate for months or years, prepared to respond to new exposures to the same antigen. The re-exposure to the antigen they recognize causes a rapid expansion of effector T cells, thus providing the immune system with "memory" against past infections.
  • Treg cells Regulatory T lymphocytes (Treg cells)
  • Regulatory T lymphocytes formerly known as suppressor T cells: express the surface CD4 marker and are distinguished from CD4 + lymphocytes by the presence of the FoxP3 intracellular marker. Its main function is to eliminate cell-mediated immunity at the end of the immune reaction and eliminate self-reactive T cells that escaped the thymus negative selection process.
  • Two main classes of CD4 + Treg cells have been described: - “natural Treg cells” or “CD4 + CD25 + FoxP3 + Treg cells” arise in the thymus and have been related to the interactions between developing T cells and dendritic cells, both myeloid (CDl lc +) and plasmocytoid (CD123 +);
  • Treg adaptation cells or “Th3 cells” or “Trl cells”, which can be generated during a normal immune response.
  • “Gamma / delta T cells” are a small group of T cells that have a specific TCR on their surface. Most lymphocytes have a TCR composed of two glycoprotein chains called ⁇ and ⁇ . However, in gamma / delta cells, the TCR is formed by a ⁇ chain and a ⁇ chain. This group of lymphocytes is very rare (2% of the total), but they are abundant in the intestinal mucosa, forming part of a lymphocyte population called intraepithelial lymphocytes.
  • T lymphocyte receptor or "TCR” (for T cell receptor in English) is a cellular receptor associated with an intracellular signaling pathway characterized by belonging to the family of receptors with intrinsic enzymatic activity and having peptide ligands small associated with CMH molecules in the plasma membrane of macrophages and other antigen presenting cells.
  • the molecular characteristics of said receptor comprise the possession of an individual transmembrane ⁇ helix, although there are several kinase proteins associated with cytosolic domains (present only in T lymphocytes), and their signal transduction pathway involves the activation of cytosolic tyrosine kinase proteins. , via PI-3 kinase, via IP3 / DAG and via Ras / MAPK. In this way, its activation by means of an external stimulus causes a cascade of internal enzymatic reactions that facilitates the adaptation of the cell to its environment, by means of second messengers.
  • the TCR is made up of two chains similar to immunoglobulins, but associated with the cell membrane.
  • the two chains are called TCRa and TCRP, they are arranged side by side joined by disulfide bonds.
  • Certain molecules on the surface of T cells stabilize both interactions mediated by TCR as well as intracellular communication, including CD3, CD4 and CD8.
  • the TCR chains comprise a constant domain (C) and a variable domain (V), which is located in the apical region of the extracellular portion.
  • the variable domain has 3 small hypervariable regions (CDRs) that are directly responsible for the interaction with the peptide complex: CMH, in a highly complementary way. The most specific is CDR3.
  • CDRs hypervariable regions
  • the ⁇ and ⁇ chains (and in rare cases the alternative combination of ⁇ : ⁇ chains) are associated on the cell membrane with a group of molecules without morphological variability called together CD3. That TCR: CD3 membrane complex is required for the stable interaction of T cells with antigen presenting cells and is responsible for much of the intracellular communication mediated by TCR.
  • the CD3 complex is composed of three monomers called ⁇ , ⁇ and ⁇ spatially bound but not covalently.
  • the CD3 complex further comprises an intracellular portion composed of two ⁇ chains, which is responsible for transmitting the signals resulting from the TCR: CMH-antigen binding.
  • Bone marrow hematopoietic stem cells, as well as early lymphoid progenitor cells that will give rise to lymphocytes (T and B) contain genes for immunoglobulins (Ig) and for T-cell receptors in germinal configuration ( or inherited), which is different from the configuration found in mature lymphocytes.
  • Ig immunoglobulins
  • T-cell receptors in germinal configuration ( or inherited), which is different from the configuration found in mature lymphocytes.
  • both the loci for the Ig (heavy and light chains) and the loci for the TCRs ( ⁇ and ⁇ chains) each contain multiple genes for the variable region (V), up to several hundred, and one or few genes for the constant region (C). Between the V genes and the C genes there are small nucleotide sequences, which are called gene segments of union (J, by joining) and diversity (D). All loci contain V, J and C genes; Ds are only found in the locus of the Ig heavy chain and in the TCR ⁇ chain.
  • the decision of a progenitor lymphoid cell to become a T lymphocyte is associated with recombination in the locus of the TCR ⁇ chain (located on chromosome 14) of a specific V segment gene with a specific J segment gene, to form a single exon VJ.
  • the selection of a specific segment V and J is random.
  • the VJ exon is linked by the splicing mechanism with the C region.
  • a segment V, one D and one J are also randomly selected. then a VDJ exon that then binds to the C region.
  • This DNA recombination and RNA splicing sequence generates the ⁇ and ⁇ chains of the TCR of mature lymphocytes.
  • the diversity of the antigen receptors is due to the use of different combinations of segments V, D and J in different lymphocyte clones, called combinatorial diversity, which is also increased by introducing changes in the nucleotide sequence at the junctions of segments V , D and J, called diversity of unions.
  • combinatorial diversity is also increased by introducing changes in the nucleotide sequence at the junctions of segments V , D and J, called diversity of unions.
  • combinatorial diversity is limited to the number of sequences available for each segment, but the diversity of the unions is almost unlimited. This diversity of unions can be produced by three types of mechanisms:
  • TdT deoxyribonucleotidyl transferases
  • the nucleotide sequence in the V (D) J zone of a TCR and the length of it in a lymphocyte clone is very different from the same zone of any other clone.
  • the junction zones constitute the most variable zone of the CDR3, and it is the most important for antigen recognition.
  • the term "repertoire of T cells”, as used in the present invention, refers to the set of T lymphocytes that express TCRs with different specificities and that are present in a subject at a given time.
  • sample refers to any biological sample capable of containing T lymphocytes that can be obtained from a subject;
  • Illustrative, non-limiting examples of said biological sample include biopsy samples, tissues, cells or fluids, for example blood, milk, plasma, saliva, serum, etc.
  • said biological sample is peripheral blood, preferably peripheral blood enriched in CD4 + and CD8 + T lymphocytes.
  • various methods are available for obtaining enrichment in CD4 + and CD8 + T lymphocytes.
  • said methods can be based on a positive selection, where T cells are isolated through antibodies specific for CD2 or CD3, or on a negative selection, where unwanted cells are removed from the sample by the use of antibodies specific for markers of other cell types, including, but not limited to, CD14, CD16, CD19, CD20, CD36, CD56, CD57, CD66b, CD94, CD123, CD244 and / or glycophorin A. They are available to those skilled in the art.
  • kits include RosetteSep Human T Cell Enrichment Cocktail (StemCell Technologies), EasySep human T cell Enrichment Kit (StemCell Technologies), BioMag SelectaPure Human CD3 + T cell Enrichment System (Polysciences, Inc.), BioMag® SelectaPure Human T cell Enrichment System (Polysciences, Inc.), Pan T Cell Isolation Kit human (Miltenyi Biotec), CD2 MicroBeads human (Miltenyi Biotec), Whole Blood CD3 MicroBeads human (Miltenyi Biotec), Dynabeads CD2 Pan T (Invitrogen), Dynabeads CD3 (Invitrogen) , Dynabeads Untouched Human T Cells (Invitrogen), and Human CD3 + T Cell Enrichment Column (R&D Systems).
  • primer refers to a nucleotide sequence that is complementary to a nucleotide sequence of the genes encoding for the ⁇ chain of the TCR.
  • Each hybrid primer specifically with its target nucleotide sequence and acts as a starting point from which DNA polymerase begins the polymerization of DNA.
  • the primers are short nucleotide sequences, approximately 15-24 nucleotides in length that can be aligned with a strand of target DNA thanks to the complementarity of bases to form a hybrid between the primer and the target strand of DNA. Then, the DNA polymerase enzyme can extend the primer along the DNA strand.
  • the primers can be prepared by any suitable method, including, for example, but not limited to direct chemical synthesis. Methods for preparing and using primers are described, for example, in Sambrook et al., 2001. "Molecular cloning: a Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3.
  • the primer is a deoxyribose oligonucleotide, but may also comprise at least one modified sugar moiety selected from the group that includes but is not limited to arabinose, 2-fluoroarabinous, xylulose and hexose.
  • the oligonucleotide may also comprise at least one modified phosphate main structure.
  • Non-limiting examples of modified oligonucleotides include oligodeoxyribonucleotide selenoate, oligodeoxyribonucleotide phosphorothioate (TPO), oligodeoxyribonucleotide phosphoramidate, oligodeoxyribonucleotide methylphosphonate, peptide nucleic acid (PNA).
  • TPO oligodeoxyribonucleotide phosphorothioate
  • PNA peptide nucleic acid
  • antisense primer refers to a primer that hybridizes with a specific sequence of the constant segment C segment of the gene encoding the ⁇ chain of the TCR, in the 3'-5 sense '.
  • hybridization refers to the process by which two chains of antiparallel nucleic acids and complementary base sequences are associated, in a single double-chain molecule where the nitrogenous bases remain hidden inside. Hybridization can be carried out under conditions of high, medium or low stringency allowing the association of chains with high, medium or low homology, respectively. In a preferred embodiment, the hybridization is carried out under high or medium stringency conditions.
  • hybridization under conditions of high stringency can be carried out in 6xSSC at approximately 45 ° C followed by one or more washes in 0.1 x SSC / 0.2% SDS at approximately 68 ° C.
  • Other conditions of high stringency include, for example, washings in 6xSSC / 0.05% sodium pyrophosphate at 37 ° C, 48 ° C, 55 ° C and 60 ° C, depending on the particular primer.
  • “moderate conditions” is used for hybridization carried out in 6x SSC at approximately 45 ° C followed by one, preferably 3-5 washes in 0.2xSSC / 0.1% SDS at approximately 42-65 ° C.
  • Tm 81.5 ° C + 16.6Log [Na +] + 0.41 (% G + C) - 0.63 (% formamide) -600 / # of bp in the duplex.
  • [Na +] [0.368] and 50% formamide, with a GC content of 42% and an average probe size of 200 bases, the Tm is 57 ° C.
  • the Tm of a DNA duplex decreases by 1-1.5 ° C with each 1% decrease in homology. Therefore, targets with a sequence identity greater than 75% would be observed using a hybridization temperature of 42 ° C.
  • “Tm” or “melting temperature” means that temperature at which 50% of the DNA molecules are dissociated.
  • detecttable label refers to a molecular label whose purpose is to allow the visualization or detection of the molecules to which it is anchored by proper procedures and equipment for the detection of tide.
  • the marking of amplification products can be carried out by conventional methods. Said marking can be direct, for which fluorophores can be used, for example, Cy3, Cy5, fluorescein, alexa, etc., enzymes, for example, alkaline phosphatase, peroxidase, etc., radioactive isotopes, for example, 33P, 1251, etc., or any other marker known to the person skilled in the art.
  • said marking can be indirect through the use of chemical, enzymatic methods, etc .
  • the amplification product may incorporate a member of a specific binding pair, for example, avidin or streptavidin conjugated with a fluorochrome (locus), and the probe binds to the other member of the specific binding pair, for example, biotin (indicator), the reading being carried out by fluorimetry, etc.
  • the amplification product may incorporate a member of a specific binding pair, for example, an anti-digoxigenin antibody conjugated to an enzyme (locus), and the The probe binds to the other member of the specific binding pair, for example, digoxigenin (indicator), etc., the enzyme substrate is transformed into a luminescent or fluorescent product and the reading is carried out by chemo-luminescence, fluorimetry, etc.
  • the detectable labels are fluorophores.
  • fluorescent materials that can be used in oligonucleotide mapping include, but are not limited to, 5-carboxyfluorescein (5-FAM), 6-FAM, t-FAM tetrachlorinated analog (TET), 6-FAM hexachlorinated analog (HEX) ), 6-carboxytetramethylrodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein (JOE), ED, Cy-3, Cy -5, Cy-5.5, fluorescein-6-isothiocinate (FITC) and tetramethylrodamine-5-isothiocinate (TRITC).
  • 5-FAM 5-carboxyfluorescein
  • 6-FAM t-FAM tetrachlorinated analog
  • HEX 6-FAM hexachlorinated analog
  • the detectable labels can be distinguished based on the emission wavelength.
  • the detectable labels are 5-FAM and TAMRA.
  • the composition or kit according to the first aspect of the invention comprises 24 sense primers, each of said sense primers hybridizing with a specific sequence of one of the 24 families of V segments that are part of the gene coding for the ⁇ chain of the TCR.
  • the TCR is an ⁇ and ⁇ chain homodimer, each of which is encoded by a gene formed by gene segments V, D, J and C. There are multiple variants of said gene segments distributed in clusters, the corresponding segments being located at the ⁇ chain on chromosome 7.
  • the number of V segments of the TCR ⁇ chain in the human beings are between 64 and 67 segments in 30 families, called TCRpVl to TCRpV30.
  • each of the 24 sense primers specifically hybridizes with a segment V that are part of the gene that codes for the ⁇ chain of TCRs 2, 3.1, 4.1, 5.1, 6.1, 7.1, 9, 10.1, 11, 12.3, 13, 14, 15, 16, 18, 19, 20, 23 24, 25, 27, 28, 29 and 30.
  • the 24 sense primers comprise variants of the sequences identified in SEQ ID NO: 24 (Table 1) that retain the ability to specifically hybridize each of said sense primers with a specific sequence from one of the 24 families of V segments that are part of the gene that codes for the ⁇ chain of the TCR, where the variants have an identity with the sequences SEQ ID NO: 24 of at least 99%, at least 98%, at least 97% ), at least 96%>, at least 95%>, at least 90%>, at least 85%>, at least 80%), at least 75%>, at least 70%> , at least 65%>, at least 60%>, at least 55%>, at least 50%>, at least 45%>, at least 40%>, at least 35%>, at least 30%>.
  • the 24 sense primers comprise the sequences identified in SEQ ID NO: 1 to 24 (Table 1).
  • composition or kit of the invention further comprises a first antisense primer labeled with a first detectable label, wherein said hybrid primer specifically with segment C of the gene encoding the ⁇ chain of the TCR, and a second antisense primer that hybrid specifically with the same region as said first antisense primer labeled with a first detectable label, which is marked with a second detectable label different from the first detectable label.
  • said first antisense primer labeled with a first detectable label comprises the sequence identified in SEQ ID NO: 25 or a variant thereof that retain the ability to hybridize with the same region of a specific sequence of the constant C segment segment of the gene encoding the ⁇ chain of the TCR as the sequence identified in SEQ ID NO: 25.
  • Such variants may have an identity with the sequence SEQ ID NO: 25 of at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90% at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at minus 45%, at least 40%, at least 35%, at least 30%.
  • sequence of said first antisense primer labeled with a first detectable label comprises the sequence identified with SEQ ID NO: 25 (Table 1).
  • said first antisense primer is labeled with 5-FAM or TAMRA, preferably 5-FAM.
  • said second antisense primer that hybridizes specifically with the same region as said first antisense primer, which is labeled with a second detectable tag different from the first detectable tag comprises variants of the sequence identified in SEQ ID NO: 25 ( Table 1), which retain the ability to hybridize with a specific sequence of the constant segment C segment of the gene encoding the ⁇ chain of the TCR, where the variants have an identity with the sequence SEQ ID NO: 25 of at least 99 %, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%), at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at minus 30%
  • the sequence of said second antisense primer labeled with a second detectable label comprises the sequence identified with SEQ ID NO: 25 (Table 1).
  • said second antisense primer is labeled with 5-FAM or TAMRA, preferably TAMRA.
  • the composition or kit of the invention further comprises at least one additional primer that hybridizes with the segment C constant region of the gene encoding the ⁇ chain of the TCR.
  • the two antisense primers comprise variants of the sequences identified in SEQ ID NO: 26 and SEQ ID NO: 27 (Table 1) that retain the ability to hybridize each of said antisense primers with a region specific sequence.
  • the variants have an identity with the sequences SEQ ID NO: 26 and SEQ ID NO: 27 of at least 99%, at least 98%, at least 97%, at least 96%), at least 95%, at least 90%, at least 85%, at least 80%, at least 75%), at least 70%, at least 65%>, at least 60%>, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%.
  • the first antisense primer comprises the sequence SEQ ID NO: 26 and / or the second antisense primer comprises the sequence SEQ ID NO: 27 (Table 1).
  • ⁇ 2 CCACAAGCTGGAGGACTC (SEQ ID NO: 1)
  • ⁇ 16 GCT TGAGGAT TCAGCAGTG (SEQ ID NO: 5)
  • ⁇ 12 ATGCCCGAGGATCGAT TCTCA (SEQ ID NO: 10)
  • ⁇ 14 AAACAGGATGAGTCCGGTATGC (SEQIDNO: 15)
  • the composition or kit of the invention further comprises the reagents necessary to carry out the cDNA amplification reaction and / or the reagents necessary to perform RNA reverse transcription reactions, including but not limited to a, deoxynucleotides triphosphate (dNTPs), divalent and / or monovalent ions, a buffer solution (buffer) that maintains the proper pH for the functioning of DNA polymerase, DNA polymerase or mixture of different polymerases, reverse transcriptase or mixture of different reverse transcriptases , etc.
  • dNTPs deoxynucleotides triphosphate
  • buffer solution buffer solution
  • the kit of the invention does not comprise the reagents necessary to practice the method of the invention, these are commercially available and can be found as part of a kit. Any commercially available kit containing the reagents necessary to carry out an amplification reaction can be used successfully in the practice of the method of the invention. Methods to analyze the repertoire of T cells
  • the invention relates to a method for analyzing the repertoire of T cells in a sample, hereinafter the first method of the invention, comprising the steps of
  • step (ii) analyze the number of amplification products obtained in step (i).
  • said sample will be any biological sample capable of containing T lymphocytes that can be obtained from a subject.
  • said biological sample include biopsy samples, tissues, cells or fluids, for example blood, milk, plasma, saliva, serum, etc.
  • the sample is a peripheral blood sample.
  • peripheral blood relates to the volume of circulating blood distant from the heart, that is, the blood circulating through the organism of a subject.
  • said peripheral blood sample is enriched in CD4 + or CD8 + cells.
  • CD4 + and CD8 + T lymphocytes As one skilled in the art can understand, various methods are available for obtaining enrichment in CD4 + and CD8 + T lymphocytes.
  • Various commercial kits are available to the person skilled in the art for positive or negative enrichment of CD4 + or CD8 + T cells by gradient, columns, magnetic particles, etc.
  • kits include StemSep® Human CD4 Positive Selection Kit (StemCell Technologies), StemSep® Human CD4 + T Cell Enrichment Kit (StemCell Technologies), RosetteSep TM Human CD4 + T Cell Enrichment Cocktail (StemCell Technologies), StemSep® Human CD8 Positive Selection Kit (StemCell Technologies), StemSep® Human CD8 + T Cell Enrichment Kit (StemCell Technologies), RosetteSep TM Human CD8 + T Cell Enrichment Cocktail (StemCell Technologies) , CD4 + T Cell Isolation Kit II, human (Miltenyi Biotec), CD8 + T Cell Isolation Kit II, human (Miltenyi Biotec), Dynal® CD4 Positive Isolation Kit (Invitrogen), Dynal® CD8 Positive Isolation Kit (Invitrogen), Human CD4 + T Cell Enrichment Column (R&D Systems), Human CD8 + T Cell Enrichment Column (R&D Systems), BD IMag TM CD4
  • RNA refers to the mRNA or messenger RNA.
  • the mRNA is the ribonucleic acid that contains the genetic information from the DNA to be used in protein synthesis, that is, it determines the order in which the amino acids will bind.
  • RNA from the biological sample Prior to the extraction of the RNA from the biological sample, it can be treated physically or mechanically to break the tissue or cellular structures and release the intracellular components to an aqueous or organic solution to prepare the nucleic acids for extraction.
  • RNA extraction can be performed by any of the procedures known to those skilled in the art, including, but not limited to, Trizol, guanidinium salts, phenol chloroform, etc. Such procedures can be found, for example, in Sambrook et al., 2001. "Molecular cloning: a Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3. There are also commercial kits that allow the extraction of RNA from a sample, such as the Qiagen RNA extraction kit. As the person skilled in the art knows, when working with RNA, maximum precautions must be taken to avoid contamination with RNAs and the degradation of RNA.
  • RNA After obtaining the RNA, a reverse transcription (RT) reaction of the mRNA is carried out followed by amplification by polymerase chain reaction (PCR) [RT-PCR] to obtain the double helix cDNA corresponding to the RNA present in the sample.
  • PCR polymerase chain reaction
  • the cDNA preparation has been obtained using a primer that hybridizes with the constant C segment segment of the gene encoding the ⁇ chain of the TCR.
  • the sequence of said at least one additional primer comprises SEQ ID NO: 26 and / or SEQ ID NO: 27.
  • cDNA or "complementary DNA”, as used in the present invention, refers to single stranded DNA that is synthesized from a single strand of RNA.
  • RNA Ribonucleic acid
  • the method of the invention comprises a first stage where at least one amplification reaction of said cDNA is carried out by using specific sense primers of the DNA, where each one of said hybrid primers specifically with segment V of the gene encoding the ⁇ chain of the TCR and two antisense primers that specifically hybridize with the CP constant region of the TCR where each of said antisense primers is labeled with a distinct detectable label.
  • amplify refers to the use of techniques or methods to increase the number of copies of a specific nucleic acid segment.
  • amplification product refers to the nucleic acid generated as a result of an amplification.
  • Many amplification methods are based on chain enzymatic reactions, such as a polymerase chain reaction or PCR, a ligase chain reaction, or self-sustained sequence replication, rolling circle amplification assays, invasive excision assays , primer extension assay, enzymatic breakdown methods, NASBA, hybridization methods in sandwich, methods in which molecular markers are employed, and the like.
  • Real-time PCR also called RT-PCR, quantitative PCR, quantitative real-time PCR or RTQ-PCR
  • RT-PCR quantitative PCR
  • RTQ-PCR quantitative real-time PCR
  • the DNA is specifically amplified by a polymerase chain reaction.
  • DNA is quantified after each round of amplification.
  • Common methods of quantification include the use of fluorescent markers that are interspersed with double stranded DNA and modified DNA oligonucleotides (called probes) capable of emitting fluorescence when hybridizing with a complementary DNA.
  • amplification can be carried out by appropriately labeled primers, and products amplified by primer extension can be detected by suitable procedures and equipment for the detection of the marking.
  • the probes of the present invention are labeled with at least one detectable residue, wherein the residue or detectable residues are selected from the group consisting of: conjugates, branched detection system, chromophores, fluorophores, labels (spin label), radioisotopes, enzymes, haptens, an acridinium ester and luminescent compounds.
  • the primers are labeled with a fluorophore.
  • the amplification of step (i) is carried out in several reactions using in each of the pairs reactions of sense-antisense primers that give rise to amplification products that are distinguishable from the other amplification products obtained in the same reaction based on size and / or based on the detectable label.
  • the multiplicity of sense primers comprises 24 primers.
  • each of the 24 sense primers hybridizes specifically with a segment V that is part of the gene that codes for the ⁇ chain of TCRs 2, 3.1, 4.1, 5.1, 6.1, 7.1, 9, 10.1, 1 1, 12.3, 13, 14, 15, 16, 18, 19, 20, 23 24, 25, 27, 28, 29 and 30.
  • the 24 sense primers comprise variants of the sequences identified in SEQ ID NO: 24 (Table 1) that retain the ability to specifically hybridize each of said sense primers with a specific sequence from one of the 24 families of V segments that are part of the gene that codes for the ⁇ chain of the TCR, where the variants have an identity with the sequences SEQ ID NO: 24 of at least 99%, at least 98%, at least 97% ), at least 96%>, at least 95%>, at least 90%>, at least 85%>, at least 80%), at least 75%>, at least 70%> , at least 65%>, at least 60%>, at least 55%>, at least 50%>, at least 45%>, at least 40%>, at least 35%>, at least 30%>.
  • the 24 sense primers comprise the sequences identified in SEQ ID NO: 1 to 24 (Table 1).
  • the two antisense primers that specifically hybridize with the constant CP region of the TCR where each of said antisense primers is labeled with a distinct detectable label comprises variants of the sequence identified in SEQ ID NO: 25 (Table 1), which retain the ability to hybridize with a specific sequence of the constant segment C segment of the gene encoding the ⁇ chain of the TCR, where the variants have an identity with the sequences SEQ ID NO: 25 of at least 99%>, at least 98%>, at least 97%>, at least 96%>, at least 95%>, at least 90%>, at least 85% or, at at least 80%>, at least 75%>, at least 70%>, at least 65%>, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%.
  • said two antisense primers comprise the sequence identified as SEQ ID NO: 25 (Table 1).
  • said two antisense primers are labeled with 5-FAM or TAMRA.
  • said first antisense primer is labeled with 5-FAM and said second antisense primer is labeled with TAMRA.
  • cDNA amplification is performed for 24 ⁇ families using eight RT-PCR reactions in multiplex format (R1 to R8), taking at least one primer corresponding to a 70-90 bp molecular weight amplification product ( SEQ ID NO: 1-8), or variants thereof that retain the ability to specifically hybridize each of corresponding V segments that are part of the gene encoding the ⁇ chain of the TCR; at least one primer corresponding to a 120-160 bp molecular weight amplification product (SEQ ID NO: 9-16), or variants thereof that retain the ability to specifically hybridize each of corresponding V segments that are part of the gene coding for the ⁇ chain of the TCR; at least one primer corresponding to a 180-210 bp molecular weight amplification product (SEQ ID NO: 17-24), or variants thereof that retain the ability to specifically hybridize each of corresponding V segments that are part of the gene coding for the ⁇ chain of the TCR; and at least one labeled antisense primer (SEQ ID NO: 1
  • the eight RT-PCR reactions in multiplex format are performed following the primer mixtures of Table 2, that is, the following combinations of primers:
  • - Rl SEQ ID NO: l + SEQ ID NO: 11 + SEQ ID NO: 17 + SEQ ID NO: 25 marked with 5-FAM;
  • - R2 SEQ ID N05 + SEQ ID NO: 13 + SEQ ID NO: 22 + SEQ ID NO: 25 marked with TAMRA;
  • Table 2 Example of mixtures of sense-antisense primers.
  • Rl Vp2 (SEQ ID NO: 1)
  • R5 Vp4 (SEQIDNO: 3)
  • Vpl5 (SEQ ID NO: 11) Vpl2 (SEQIDNO: 10) Vpll (SEQ ID NO: 17) Vp25 (SEQ ID NO: 19) Cl (SEQIDNO: 25) Cl (SEQIDNO: 25)
  • Vpl0 (SEQIDNO: 13)
  • Vpl4 (SEQIDNO: 15)
  • Vp9 (SEQIDNO: 22)
  • Vp23 (SEQIDNO: 23) C2 (SEQ ID NO: 25) C2 (SEQ ID NO: 25)
  • Vp6 (SEQ ID NO: 9)
  • Vp28 (SEQ ID NO: 12)
  • Vp27 (SEQIDNO: 20)
  • Vp24 (SEQ ID NO: 18) Cl
  • SEQIDNO: 25 Cl
  • R4 Vpl8 (SEQ ID NO: 6)
  • R8 Vp29 (SEQ ID NO: 8)
  • Vpl3 (SEQ ID NO: 14)
  • Vp20 (SEQ ID NO: 16)
  • Vp30 (SEQ ID NO: 24)
  • Vp7 (SEQ ID NO: 21)
  • the PCR is carried out in a thermocycler that performs the cycles in the exact times and temperatures programmed, such as the hybridization temperature, which depends on the melting temperature of each of the primers used in the reaction or the temperature of extension.
  • the amplification of the different TCRVP segments requires specific reaction conditions and procedures for each of the pairs of primers used, which can be achieved by systematic variation of each parameter.
  • the number of cycles and the alignment temperature used in the PCR reaction must be suitable for obtaining reliable results by using each of the pairs of sense-antisense primers of the invention.
  • Parameters such as the concentration of primers are specific to each primer and have been adjusted in the validation of the primer set.
  • the high temperature multiplex RT-PCR reactions are performed in 20 ⁇ of reaction mixture containing 100 ng mRNA template, 1 ⁇ dNTP (Roche Applied Science), 5x buffer [Roche Applied Science], 20 U of RNase inhibitor (Protector, Roche Applied Science), 10 U reverse transcriptase (Transcriptor, Roche Applied Science), and 1 ⁇ of each primer (three sense primers and one antisense primer).
  • the first reaction cycle consists of an incubation at 42 ° C for 45 min, followed by an inactivation step at 85 ° C for 5 min and 35 cycles consisting of 15 s at 94 ° C, 15 s at 58 ° C and 30 s at 72 ° C, with a final extension stage of 7 min at 72 ° C.
  • the first method of the invention comprises a second stage of analysis of the number of amplification products obtained in the first stage. This It is carried out by separating the amplification products or amplicons. Virtually any conventional method can be used within the scope of the invention to separate the amplification products. Techniques for separating amplification products are widely described in the state of the art, such as in Sambrook et al., 2001 (cited ad supra). Techniques for separating amplification products are, for example, submerged electrophoresis with Methafor gels, polyacrylamide gels electrophoresis, capillary electrophoresis, etc.
  • the size of the separated fragments is identified, for which any of the methods of identification of amplification fragments known in the state of the art can be used, such as hybridization with labeled probes (for example with a fluorophore) which will be detected by a detector and processed by a computer system, staining, for example, with ethidium bromide, silver staining, etc.
  • labeled probes for example with a fluorophore
  • staining for example, with ethidium bromide, silver staining, etc.
  • electropherogram can be generated where the size of the amplified fragments can be identified.
  • GeneScan Genetic Analysis System CEQ 8000, GenomeLab, Beckman Coulter.
  • the area under the curve calculated for each CDR3 length in a ⁇ family in a probability distribution. In this way, the distribution of the TCR lengths provides a percentage of the perturbations of the TCR repertoire.
  • the analysis of the amplification products obtained in step (ii) is carried out by determining the size and / or the marking of each of the amplification products in each of the amplification reactions.
  • the analysis of the molecular spectrum of each ⁇ family is performed by combinations of amplifications of different molecular weight and different tides.
  • said combinations are carried out from the amplification products obtained from the Rl and R2 reactions, the amplification products obtained from the R3 and R4 reactions, the products of amplification obtained from reactions R5 and R6 and the amplification products obtained from reactions R7 and R8 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained from the Rl and R2 reactions, the amplification products obtained from the R3 and R4 reactions, the amplification products obtained from the R5 and R8 reactions, and the amplification products obtained from reactions R7 and R6 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R2 reactions, the amplification products obtained from the R3 and R6 reactions, the amplification products obtained from the R5 and R4 reactions, and amplification products obtained from reactions R7 and R8 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R2 reactions, the amplification products obtained from the R3 and R6 reactions, the amplification products obtained from the R5 and R8 reactions, and amplification products obtained from reactions R7 and R4 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R2 reactions, the amplification products obtained from the R3 and R8 reactions, the amplification products obtained from the R5 and R4 reactions, and amplification products obtained from reactions R7 and R6 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R2 reactions, the amplification products obtained from the R3 and R8 reactions, the amplification products obtained from the R5 and R6 reactions, and amplification products obtained from reactions R7 and R4 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R4 reactions, the amplification products obtained from the R3 and R2 reactions, the amplification products obtained from the R5 and R6 reactions, and amplification products obtained from reactions R7 and R8 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R4 reactions, the amplification products obtained from the R3 and R2 reactions, the amplification products obtained from the R5 and R8 reactions, and amplification products obtained from reactions R7 and R6 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R4 reactions, the amplification products obtained from the R3 and R6 reactions, the amplification products obtained from the R5 and R2 reactions, and amplification products obtained from reactions R7 and R8 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R4 reactions, the amplification products obtained from the R3 and R8 reactions, the amplification products obtained from the R5 and R2 reactions, and amplification products obtained from R7 and R6 reactions (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R4 reactions, the amplification products obtained from the R3 and R8 reactions, the amplification products obtained from the R5 and R6 reactions, and amplification products obtained from reactions R7 and R2 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R4 reactions, the amplification products obtained from the R3 and R6 reactions, the amplification products obtained from the R5 and R8 reactions, and amplification products obtained from reactions R7 and R2 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R6 reactions, the amplification products obtained from the R3 and R2 reactions, the amplification products obtained from the R5 and R4 reactions, and amplification products obtained from reactions R7 and R8 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R6 reactions, the amplification products obtained from the R3 and R2 reactions, the amplification products obtained from the R5 and R8 reactions, and amplification products obtained from reactions R7 and R4 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R6 reactions, the amplification products obtained from the R3 and R4 reactions, the amplification products obtained from the R5 and R2 reactions, and amplification products obtained from reactions R7 and R8 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R6 reactions, the amplification products obtained from the R3 and R8 reactions, the amplification products obtained from the R5 and R2 reactions, and amplification products obtained from reactions R7 and R4 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R6 reactions, the amplification products obtained from the R3 and R8 reactions, the amplification products obtained from the R5 and R4 reactions, and amplification products obtained from reactions R7 and R2 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R6 reactions, the amplification products obtained from the R3 and R4 reactions, the amplification products obtained from the R5 and R8 reactions, and amplification products obtained from reactions R7 and R2 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R8 reactions, the amplification products obtained from the R3 and R2 reactions, the amplification products obtained from the R5 and R6 reactions, and amplification products obtained from R7 and R4 reactions (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R8 reactions, the amplification products obtained from the R3 and R2 reactions, the amplification products obtained from the R5 and R4 reactions, and amplification products obtained from reactions R7 and R6 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R8 reactions, the amplification products obtained from the R3 and R4 reactions, the amplification products obtained from the R5 and R2 reactions, and amplification products obtained from reactions R7 and R6 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R8 reactions, the amplification products obtained from the R3 and R6 reactions, the amplification products obtained from the R5 and R2 reactions, and amplification products obtained from reactions R7 and R4 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R8 reactions, the amplification products obtained from the R3 and R4 reactions, the amplification products obtained from the R5 and R6 reactions, and amplification products obtained from reactions R7 and R2 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said combinations are carried out from the amplification products obtained the Rl and R8 reactions, the amplification products obtained from the R3 and R6 reactions, the amplification products obtained from the R5 and R4 reactions, and amplification products obtained from reactions R7 and R2 (according to Table 2), which are separated into their corresponding four electrophoresis strokes.
  • said method further comprises additional amplification of the cDNA using at least one additional primer that hybridizes with the C segment of the gene encoding the ⁇ chain of the TCR.
  • the sequence of said at least one additional primer comprises SEQ ID NO: 26 and / or SEQ ID NO: 27, or variants of said primers that retain the ability to hybridize specifically with the corresponding region of segment C of the gene which codes for the constant region of the ⁇ chain of the TCR.
  • the sample is a peripheral blood sample.
  • the sample is a peripheral blood sample enriched in CD4 + or CD8 + cells.
  • the invention relates to the use of the composition or kit of the first aspect, or of the method of the second aspect, in the diagnosis of a pathology.
  • diagnosis refers to the procedure by which a disease, nosological entity, syndrome or any health condition is identified.
  • pathology refers to a process in which the lymphocyte repertoire is altered. These pathological processes include infectious processes, tumor processes and immunodeficiencies. In a particular embodiment, said pathology is an immunodeficiency. Examples no Limitations of immunodeficiencies include immunodeficiencies caused by diabetes mellitus, liver failure, hepatitis, intestinal lymphangiectasia, aplastic anemia, cancer, host-versus-graft disease, chemotherapeutic drugs, immunosuppressive drugs, corticosteroids, radiotherapy, cytomegalovirus, Epstein-Barr virus, HIV, measles virus, varicella-zoster virus, alcoholism, malnutrition, nephrotic syndrome, renal failure, uremia, rheumatoid arthritis, systemic lupus erythematosus, burns and chromosomal abnormalities.
  • the diagnosis of a pathology comprises a first stage (i), in which the repertoire of T cells in a subject as described in the first method of the invention is analyzed, and a second stage (ii), in which the result of the analysis performed in stage (i) is compared with that obtained for a control sample. If these results differ substantially from each other, then it is indicative that the subject undergoes a pathology, as understood in the present invention. If these results do not differ substantially from each other, then it is indicative that the subject does not suffer a pathology, as is understood in the present invention.
  • control sample refers to a sample obtained from a healthy subject.
  • the term "subject”, as used in the present invention, includes any mammal; Non-limiting examples are domestic animals and livestock, primates and humans.
  • the subject is a human being, male or female, of any age or race.
  • Virtually any subject can be analyzed according to the present invention to analyze its repertoire of T cells.
  • said subject is a subject on which it is desired to diagnose a pathology.
  • said subject is a patient on whom one wants to monitor the evolution of a pathology in response to a treatment.
  • patient refers to a subject suffering from pathology, although said subject has not yet been diagnosed.
  • the term "healthy subject”, as used in the present invention includes any subject that does not have a pathology where the lymphocyte repertoire is altered.
  • the healthy subject is a human being, male or female, of any age or race.
  • the pathology is diagnosed by checking the alterations in the TCR repertoire in CD8 + cells.
  • said alterations in the repertoire of the TCR in CD8 + cells are checked during HIV-1 infection in infected patients.
  • patients who can be analyzed with the method of the present invention include, without limitation, those patients suffering from acquired immunodeficiency syndrome.
  • AIDS immunodeficiency syndrome
  • patients who can be analyzed with the method of the present invention include, without limitation, those patients suffering from acquired immunodeficiency syndrome.
  • the term "acquired immunodeficiency syndrome (AIDS)" refers to a disease that affects HIV-infected humans. It is said that a person suffers from AIDS when their organism, due to the immunodeficiency caused by HIV, is not able to offer an adequate immune response against infections. Note the difference between being infected with HIV and suffering from AIDS. An HIV-infected person is HIV positive and goes on to develop an AIDS picture when their level of CD4 + T cells, cells that attack the virus, drops below 200 cells per milliliter of blood. HIV specifically attacks CD4 + T cells and enters them.
  • the virus transforms its single-stranded genetic material (RNA) to a double-stranded (DNA) one to incorporate it into the host's own genetic material (infected person) and uses it to replicate or make copies of itself.
  • the new copies of the virus leave the cells, which they lysate, into the blood to infect other CD4 + T cells. This cycle is repeated again and again.
  • HIV infection is classified into different categories, according to the symptoms and conditions that the patient has:
  • Category A patients with primary or asymptomatic infection.
  • Category B patients who present or have presented symptoms that do not belong to category C, but are related to HIV infection. These include bacillary angiomatosis, vulvo-vaginal candidiasis, or treatment-resistant oral candidiasis, uterine cervix dysplasia or non-invasive cervical carcinoma, pelvic inflammatory disease (PID), fever less than 38.5 ° C or diarrhea, of more than one Month-long, shingles (more than one episode, or an episode with more than one dermatome), hairy oral leukoplakia, peripheral neuropathy, idiopathic thrombocytopenic purpura.
  • PID pelvic inflammatory disease
  • Bacterial infections recurrent Salmonella septicemia (other than Salmonella typhy), tuberculosis, Mycobacterium avium complex infection (MAI), atypical mycobacterial infections.
  • Viral infections cytomegalovirus infection (retinitis or disseminated), herpes simplex virus infection (HSV types 1 and 2), can be chronic or in the form of bronchitis, pneumonitis or esophagitis.
  • cytomegalovirus infection retinitis or disseminated
  • HSV types 1 and 2 herpes simplex virus infection
  • Fungal infections aspergillosis, candidiasis, both disseminated and of the esophagus, trachea or lungs, coccidiodomycosis, extrapulmonary or disseminated, extrapulmonary cryptococcosis, histoplasmosis, either disseminated or extrapulmonary.
  • Protozoal infections Pneumocystis jiroveci pneumonia, neurological toxoplasmosis, chronic intestinal cryptosporidiosis, chronic intestinal isosporiasis.
  • HIV encephalopathy progressive multifocal leukoencephalopathy, wasting syndrome or wasting syndrome.
  • Tumor processes Kaposi's sarcoma, Burkitt lymphoma, other non-lymphomas
  • Hodgkin especially immunoblastic lymphoma, primary brain lymphoma or B-cell lymphoma, invasive carcinoma of the cervix. Not all patients infected with the HIV virus have AIDS.
  • the criteria for diagnosing AIDS may vary from region to region, but the diagnosis typically requires:
  • Antiretroviral therapy includes, but is not limited to, HAART (High Activity Antiretroviral Therapy), protease inhibitors, fusion inhibitors, integrase inhibitors, specific co-receptor agents, 3TC, AZT, nevirapine, transcriptase inhibitors Reverse non-nucleoside analogs and reverse transcriptase inhibitors nucleoside analogues.
  • TARGA is a combination of three or more antiretroviral drugs.
  • the term "HAART" refers to a combination of highly active antiretroviral agents and usually comprises three drugs.
  • Non-limiting examples of reverse transcriptase inhibitors include nucleoside analog reverse transcriptase inhibitors such as zidovudine (AZT, Retrovir), didanosine (ddl, Videx), stavudine (d4T, Zerit), lamivudine, (3TC, Epivir), abacavir ( ABC, Ziagen), tenofovir (TDF, Viread), combivir (CBV, combination of AZT and 3TC), and non-nucleoside reverse transcriptase inhibitors such as nevirapine (NVP, Viramune), delavirdine (DLV, rescriptor), efavirenz ( EFV, substitute,).
  • nucleoside analog reverse transcriptase inhibitors such as zidovudine (AZT, Retrovir), didanosine (ddl, Videx), stavudine (d4T, Zerit), lamivudine, (3TC, Epivir), abacavir ( ABC, Ziagen
  • Non-limiting examples of protease inhibitors include saquinavir (SQV, Invirase), ritonavir (RTV, Norvir), indinavir (IDV, Crixivan), nelfinavir (NFV, Viracept), fosamprenivir (FPV, Lexiva), kaletra (lopinavir and ritonavir) and fortovase (saquinavir in soft gelatin formulation).
  • HIV refers to the human immunodeficiency virus type-1. HIV-1 includes but is not limited to extracellular virus particles and forms of HIV-1 associated with HIV-1 infected cells.
  • the '' HIV-2 ' 1 calls the human immunodeficiency virus type-2. HIV-2 includes but is not limited to extracellular virus particles and forms of HIV-2 associated with HIV-2 in infected cells.
  • the HIV-1 virus can include the M group with the main known subtypes (A, B, C, D, E, F, G and H), group O, group N, and recombinant forms, including laboratory strains and primary isolates.
  • the invention in a fourth aspect, relates to a method for monitoring the evolution of a pathology in a patient in response to a treatment, hereinafter second method of the invention, which comprises determining the complexity of the repertoire of T cells in a sample of said patient after being subjected to said treatment using a method of the second aspect of the invention, wherein an increase in the complexity of said repertoire with respect to complexity before starting treatment is indicative that the patient responds to said treatment. .
  • subject includes any mammal; Non-limiting examples are domestic animals and livestock, primates and humans. Preferably, the subject is a human being, male or female, of any age or race. Virtually any subject can be analyzed according to the present invention to analyze its repertoire of T cells. However, in a particular embodiment, said subject is a subject on which it is desired to diagnose a pathology. In another particular embodiment, said subject is a patient on whom one wants to monitor the evolution of a pathology in response to a treatment.
  • patient refers to a subject suffering from pathology, although said subject has not yet been diagnosed.
  • pathology refers to a process in which the lymphocyte repertoire is altered.
  • pathological processes include infectious processes, tumor processes and immunodeficiencies.
  • said pathology is an immunodeficiency.
  • immunodeficiencies include immunodeficiencies caused by diabetes mellitus, liver failure, hepatitis, intestinal lymphangiectasia, aplastic anemia, cancer, host-versus-graft disease, chemotherapeutic drugs, immunosuppressive drugs, corticosteroids, radiotherapy, cytomegalovirus, viruses Epstein-Barr, HIV, measles pathway, varicella-zoster virus, alcoholism, malnutrition, nephrotic syndrome, renal failure, uremia, rheumatoid arthritis, systemic lupus erythematosus, burns and chromosomal abnormalities.
  • patients who can be analyzed with the method of the present invention include, without limitation, those patients suffering from acquired immunodeficiency syndrome.
  • the pathology is acquired immunodeficiency syndrome (AIDS) or a pathology associated with HIV infection.
  • the therapy is HAART.
  • AIDS immunodeficiency syndrome
  • HAV infection HIV infection
  • HBV HIV infection
  • BL start of the study (in English, baseline); IT, treatment interruption; TR, resumption of treatment; Homo, homosexual; Hete, heterosexual; IDU, injecting drug user.
  • the CD4 T cell count was determined in fresh samples by flow cytometry.
  • Plasma HIV-1 RNA was determined by quantitative PCR assay (HIV Monitor TM Test Kit, Roche Molecular System, Hoffman-La Roche, Basel, Switzerland), following the manufacturer's instructions. This assay has a detection limit of less than 50 copies of HIV-1 / mL RNA.
  • TCRVP genes Twenty-four families of TCRVP genes were analyzed in each patient at three different time points, after at least 12 months of effective antiretroviral treatment (referral), at least twelve months of treatment interruption (IT), and at least twelve months after the resumption of treatment (RT). Subset analysis of T cells was performed by flow cytometry at the three time points.
  • PBMCs peripheral blood cells were isolated from heparinized blood samples and cryopreserved in liquid nitrogen.
  • the subset of CD8 T cells was isolated by immunomagnetic separation technique (Dynabeads, Dynal, Paisley, United Kingdom), using Anti-CD3 and anti-CD8 monoclonal antibodies for positive cell isolation (Dynabeads, CD8 positive cell isolation kit, Dynal), according to the manufacturer's instructions.
  • the cell fraction contained at least 5.1 x 10 6 cells with a purity greater than 98%.
  • the CDR3 region of 24 TCRVP families was amplified using a new multiplex RT-PCR strategy to minimize the number of reactions.
  • the inventors designed sense primers for 24 ⁇ families that amplify three different ranges of molecular size (Table 1).
  • the inventors also designed an antisense primer labeled with two different fluorochromes (Cl and C2).
  • Cl and C2 two different fluorochromes
  • optimization was performed with purified cord blood cells from a healthy donor. It was found that an amount of 100 ng guarantees the production of a Gaussian distribution of the CDR3 length repertoires of the three ⁇ families amplified in each RT-PCR reaction ( Figure 1).
  • To normalize and ensure good mRNA template quality a region of the GAPDH gene was amplified. In addition, a missing length was repeated twice to confirm the results.
  • High temperature multiplex RT-PCR reactions were carried out in 20 ⁇ of reaction mixture containing 100 ng mRNA template, 1 ⁇ dNTP (Roche Applied Science), 5x buffer (Roche Applied Science), 20 U inhibitor of RNase (Protector, Roche Applied Science), 10 U reverse transcriptase (Transcriptor, Roche Applied Science), and 1 ⁇ of each primer (three primers sense ⁇ and one antisense primer C).
  • the first reaction cycle was carried out at 42 ° C for 45 min, followed by an inactivation step at 85 ° C for 5 min and 35 cycles consisting of 15 s at 94 ° C, 15 s at 58 ° C and 30 s at 72 ° C, with final extension stage of 7 min at 72 ° C.
  • peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • the cells were washed and fixed with 1% paraformaldehyde, and analyzed with a multiparameter flow cytometer (Gallios, Beckman Coulter).
  • Subsets of T cells were defined as follows way: CD8, CD3 + CD8 + CD45RA + CCR7 + naive cells; memory effector cells (MS), CD3 + CD8 + CD45RA-CCR7- cells, and CD3 + CD8 + CD45RA-CCR7 + central memory (CM) cells.
  • activation levels were analyzed by co-expression of CD38 and HLA-DR, recent thymus production by expression of CD31 in naive CD8 cells (CD45RA + CCR7 +), and apoptosis by PD-1. These parameters were analyzed in CD8 cells with three different TCR families, that is, Vpl4, Vp20 and Vp2.
  • Figure 1C shows the overall mean proportion of the expression of each TCRVP family of all patients at baseline (BL), treatment interruption (IT) and treatment resumption (RT) is shown in Figure 1C .
  • ⁇ families were analyzed according to the duration of the patients' antiretroviral treatment. Patients with a longer initial period of antiretroviral treatment (BL, greater than average) showed significant differences between IR and RT, compared to BL, while patients with a longer initial period of antiretroviral treatment (below the mean) showed no significant differences in these time points with respect to the BL ( Figure 3). In addition, the ⁇ 9 and ⁇ ⁇ families increased significantly in IT only in those patients with a longer initial period of antiretroviral treatment, compared to BL (data not shown).

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Abstract

La présente invention a trait au domaine de la biotechnologie. De manière spécifique, elle concerne un procédé permettant de déterminer la diversité du répertoire de lymphocytes T d'un individu. L'invention concerne également un procédé permettant de surveiller l'évolution d'une pathologie chez un patient en réponse à un traitement. De même, elle concerne des compositions permettant de mettre en oeuvre ledit procédé, comprenant plusieurs amorces sens qui s'hybrident de manière spécifique au segment V d'un gène qui code une chaîne β du TCR (récepteur des lymphocytes T), ainsi qu'une amorce antisens marquée avec une première étiquette détectable, qui s'hybride de manière spécifique au segment C du gène qui code la chaîne β du TCR et une seconde amorce antisens qui s'hybride de manière spécifique à la même région que la première amorce antisens et qui est marquée avec une seconde étiquette détectable.
PCT/ES2012/070910 2011-12-23 2012-12-24 Procédés et compositions permettant de déterminer la diversité du répertoire de lymphocytes t d'un individu Ceased WO2013093170A1 (fr)

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