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WO2003016355A1 - Pre-recepteur des lymphocytes t (pre-tcr) et caracterisation et regulation de son expression et de sa fonction au cours du developpement des lymphocytes t chez les humains - Google Patents

Pre-recepteur des lymphocytes t (pre-tcr) et caracterisation et regulation de son expression et de sa fonction au cours du developpement des lymphocytes t chez les humains Download PDF

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WO2003016355A1
WO2003016355A1 PCT/ES2002/000387 ES0200387W WO03016355A1 WO 2003016355 A1 WO2003016355 A1 WO 2003016355A1 ES 0200387 W ES0200387 W ES 0200387W WO 03016355 A1 WO03016355 A1 WO 03016355A1
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ptα
expression
cells
tcr
lymphocytes
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Ma. Luisa Toribio Garcia
Graciela Edith Carrillo Rosales
Almudena Rodriguez Ramiro
Virginia Pilar Garcia De Yebenes Mena
Yolanda Rodriguez Carrasco
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Consejo Superior de Investigaciones Cientificas CSIC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • T lymphocytes interact with antigens through the T-cell antigen receptor (TCR), which recognizes their processed and peptide-presented target antigen by molecules of the major histocompatibility complex (MHC).
  • TCR receptor is a heterodimer specific to each T cell clone, which is expressed on the surface associated with the CD3 complex.
  • T ⁇ peripheral blood T cells
  • T ⁇ express a TCR consisting of a TCR ⁇ polypeptide chain and a TCR ⁇ .
  • T ⁇ express a TCR consisting of a TCR ⁇ chain and a TCR ⁇ .
  • Each of these TCR chains in each T cell clone, is composed of a unique combination of the domains called variable (V), [diversity (D)], binding (J) and constant (C).
  • V variable
  • D diversity
  • J binding
  • C constant
  • the combination of the V, D and J domains in both ⁇ and ⁇ chains and in both ⁇ and ⁇ chains participate in antigen recognition specifically by defining a single point of attachment (idiotype) .
  • domain C does not participate in antigen binding.
  • both types of TCR are highly polymorphic, the degree of polymorphism is much higher in the case of TCR ⁇ .
  • TCR receptor genes like immunoglobulin genes, consist of a series of regions (Y, [D], J, and C) that are rearranged during the development of T cells in the thymus.
  • the intrathymic progenitors of T ⁇ lymphocytes have to face at least two selection processes that are mediated by signals through two different molecules, the T-cell pre-receptor or pre-TCR, and the mature receptor or TCR ⁇ , that are expressed sequentially during intrathymic development (Borst J, Jacobs H, Brouns G. Composition and function of T-cell receptor and B-cell receptor complexes on precursor lymphocytes. Curr Opin Immunol 1996; 8: 181-90; von Boehmer H. Positive selection of lymphocytes.
  • T cell receptor (TCR) -beta locus allelic exclusion and initiation of TCR-alpha locus rearrangement in immature thymocytes by signaling through the CD3 complex.
  • TCR T cell receptor
  • Thymocytes that productively rearrange the TCR ⁇ locus first express a pre-TCR complex, independent of TCR ⁇ , consisting of a TCR ⁇ chain covalently linked to a invariant pre-TCR ⁇ (pT ⁇ ) chain, which associates with the CD3 complex and promotes control point generally referred to as " ⁇ selection".
  • pT ⁇ pre-TCR ⁇
  • ⁇ selection control point generally referred to as " ⁇ selection”.
  • ⁇ selection induces the survival and proliferative expansion of those thymocytes in which a correct rearrangement of TCR ⁇ takes place and, simultaneously, determines the inhibition of other rearrangements in this locus, or allelic exclusion (Dudley EC, Petrie HT, Shah LM, Owen MJ, Hayday AC. T cell receptor beta chain gene rearrangement and selection during thymocyte development in adult mice. Immunity 1994; 1: 83-93; Hoffinan ES, Passoni L, Crompton T, Leu TM, Schatz DG, Koff A , Owen MJ, Hayday AC.
  • T-cell receptor beta-chain gene rearrangement coincident regulation of cell cycle and clonality during development in vivo. Genes Dev 1996; 10: 948-62; Aifantis I, Buer J, von Boehmer H, Azogui O. Essential role of the pre-T cell receptor in allelic exclusion of the T cell receptor beta locus. I munity 1997; 7: 601-7; Haks MC, rimpenfort P, van den Brakel JH, Kruisbeek AM. Pre-TCR signaling and inactivation of p53 induces crucial cell survival pathways in pre-T cells.
  • Pre-TCR-mediated cell expansion abruptly ends, and in the resulting population of resting small thymocytes rearrangements begin at the TCR ⁇ locus (Petrie HT, Livak F, Schatz DG, Strasser A, Crispe IN, Shortman K. Multiple rearrangements in T cell receptor alpha chain genes maximize the production of useful thymocytes. J Exp Med 1993; 178: 615-22; Trigueros C, Ramiro AR, Carrasco YR, de Yebenes VG, Albar JP, Toribio ML. Identification of a late stage of small noncycling pTa. J Exp Med 1998; 188: 1401-12).
  • the TCR ⁇ receptor is expressed in association with CD3 on the cell membrane, and thymocytes undergo a second selection step, known as "positive selection", are rescued from death programmed, and the final differentiation of these pre-T cells into mature T ⁇ cells takes place.
  • a second selection step known as "positive selection”
  • the clonal elimination of those thymocytes with autoreactive TCR ⁇ receptors will be induced, thus guaranteeing tolerance to the proper components.
  • Pleiotropic changes controlled by the pre-T-cell receptor Curr Opin Immunol 1999; 11: 135-42) and, to date, only unambiguous biochemical evidence of the presence of pre-TCR has been obtained in the cell surface of thymocytes from TCR ⁇ deficient mice (Groettrup M, Ungewiss K, Azogui O, et al.
  • a novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein. Cell 1993; 75: 283-94; Berger MA, Dave V, Rhodes MR, et al.
  • TCR ⁇ locus Trigueros C, Ramiro AR, Carrasco YR, de Yebenes NG, Albar JP, Toribio ML. Identification of a late stage of small noneyeling pTa. J Exp Med 1998; 188: 1401-12).
  • pre-T cell receptor pT alpha-TCR beta
  • the present invention is directed to the identification and selection of subspecies of human pre-T lymphocytes.
  • the solution provided by the present invention is based on the fact that the inventors have determined that the T ⁇ a and pT ⁇ isoforms of the pre-TCR receptor show a different gene expression pattern throughout the intrathymic development of these T lymphocytes, and that the isoform pT ⁇ b , in contrast to pT ⁇ a , is unable to promote the expression of a human pre-TCR complex on the surface of transfected cells, although it retains the ability to bind TCR ⁇ intracellularly, providing evidence for its independent regulation and support the individual role of both isoforms during the T cell differentiation process.
  • the present invention provides a series of techniques that allow identifying and selecting the different pre-T lymphocyte cell subtypes.
  • real-time PCR from these T lymphocyte cell populations with specific oligonucleotides and Taqman probes that allow the simultaneous expression of both pT ⁇ a and pT ⁇ b isoforms to be distinguished; and second, the in vivo identification of pre-T lymphocytes by a monoclonal antibody that specifically recognizes the pT ⁇ a isoform and forms part of the present invention.
  • pre-T lymphocyte subspecies in particular, pre-T lymphocytes of leukemias representative of the pre-T maturation stage, to analyze the responsiveness of such leukemias to chemotherapeutic compounds and design personalized therapies.
  • the monoclonal antibody K5G3 is presented that specifically recognizes the Ig extracellular domain of the human pT ⁇ chain and, therefore, reacts exclusively with the pT ⁇ a isoform, allowing for the first time to analyze the expression of the pT ⁇ chain on the cell surface.
  • Another particular embodiment of the present invention are hybridoma cells that produce said monoclonal antibody K5G3 and that has been deposited at the European Collection of Cell Cultures, Center for Applied Microbiology and Research, Portón Down, Salisbury, Wiltshire SP4 OJG, United Kingdom, on August 8, 2001, corresponding to the number of deposit 01080805.
  • monoclonal antibodies that specifically recognize the pT ⁇ isoform and that have been produced from fragments of this extracellular Ig domain of the human pT ⁇ chain form part of the present invention.
  • monoclonal antibodies described above and which are obtained by techniques other than that described in the present invention, and which are part of the existing state of knowledge, are part of the present invention, as well as fragments of these antibodies that maintain their ability to Specific binding to pT ⁇ are part of the present invention.
  • the pT ⁇ a isoform specifically induces the expression on the cell surface of all the components of the pre-TCR, TCR ⁇ , pT ⁇ and CD3 complex, making it a key point in the regulation of pre-T lymphocyte development. and therefore the object of potential methods of control and regulation of said development and are part of the present invention.
  • the use of the K5G3 antibody or its fragments for the induction of the constitutive cellular activity of pre-T lymphocytes is described.
  • the constitutive cellular activity of pre-T lymphocytes can be induced by transfecting the pT ⁇ a isoform into said pre-T lymphocytes as described in the present invention, further forming part of the present invention. the pT ⁇ to -GFP gene construct.
  • the regulation of pre-human T lymphocytes can be carried out by transfection of the pT ⁇ b isoform that induces intracellular retention in the ER of TRC ⁇ chains, the pT ⁇ gene construct also forming part of the present invention. -GFP.
  • the specific role of pre-TCR in the induction of both cellular activation signals has been demonstrated thanks to the generation in this invention of the anti-pT ⁇ K5G3 antibody that binds pT ⁇ a and triggers both signals. Therefore, the reagent generated in the present invention (anti pT ⁇ K5G3 antibody) is efficient in the phenotypic characterization of human pre-T cells that they express the pre-TCR on the cell surface, as well as in the activation of the same by crosslinking of the pre-TCR.
  • the results described in the present invention are compatible with two different roles of pT ⁇ b in the differentiation of thymocytes.
  • it can be considered a competitive function, responsible for the inhibition or regulation of the expression on the surface of preTCR with pT ⁇ a .
  • the mRNA of pT ⁇ a has been found in ixna on average ten times more abundant than pT ⁇ b in the thymus both by other authors (Barber DF, Passoni L, Wen L, Geng L, Hayday AC.
  • TCR ⁇ locus transcription As ⁇ selection results simultaneously with activation of TCR ⁇ locus transcription, assessed by TEA transcription (Levelt CN, Wang B, Ehrfeld A, Terhorst C, Eichmann K. Regulation of T cell receptor (TCR) -beta locus allelic exclusion and initiation of TCR-alpha locus rearrangement in immature thymocytes by signaling through the CD3 complex. Eur J Immunol 1995; 25: 1257-61; Ramiro AR, Trigueros C, Márquez C, San Millan JL, Toribio ML. Regulation of pre- T cell receptor (pT alpha- TCR beta) gene expression during human thymic development.
  • T cells have been directed to the analysis of T cells by PCR directed to the amplification of V regions of the ⁇ and ⁇ genes of the TCR receptor that distinguish tissue lesions rejected or not in cardiac allogeneic transplants (Oaks et al., Am Jmed Sci , 309: 26-34 (1995)) or to determine T-cell immunoproliferative conditions (US6.087.096, Method of intrafamily gragment analysis of the cell receptor alpha and beta chain CDR3 regions) or by TCR-specific monoclonal antibodies for the diagnosis and treatment of immunological diseases such as rheumatoid arthritis (US Patent 6,048,526, Monoclonal antibodies reactive with defined regions of the T cell antigen receptor).
  • pre-T cell populations at different stages of intrathymic development, pre or post- ⁇ selection, described in the present invention can be an important contribution for future uses of these cells or the proper fact of their characterization in the framework of an immunological process, whether physiological or pathological, and are part of the present invention.
  • pre-TCR in the induction of proliferation of intra-thymic pre-T cells, a process that determines the enormous cell expansion (of the order of 100 times) that occurs in the thymus during the differentiation of T lymphocytes, suggests that the surface expression of the pre-TCR constitutes an important checkpoint during the development of T lymphocytes, which must be subject to strict regulatory mechanisms, whose alteration could be the origin of uncontrolled lymphoproliferative processes that would determine the generation of leukemias , therefore, the potential methods of regulation of its gene expression and its activity described in the present invention are part of the same.
  • FIGURES Figure 1 The surface expression of the pre-TCR is detected in pT ⁇ a transfectants but not in pT ⁇ .
  • JR3.11 cells were transfected with the constructs described in A) and analyzed 24 hours later by flow cytometry after staining with the anti-CD3 monoclonal antibody Ieu4-PE. Both histograms show CD3 expression in GFP + cells (thick line histogram) compared to CD3 expression in GFP " cells (fine line histograms).
  • FIG. 3 Analysis by confocal microscopy of stable transfectants pT ⁇ a -GFP and pT ⁇ b -GFP .
  • Transfectants pT ⁇ (al 3.2) and pT ⁇ b (bl.4) were fixed to the slides as described in the Methods.
  • A) The cells were permeabilized and incubated with an specific antibody against the ER-resident PDI protein, plus a second TRITC-labeled antibody. Representative images of GFP and PDI and overexposed GFP / PDI of both pT ⁇ a and pT ⁇ b transfectants are shown.
  • Non-permeabilized cells were incubated with the anti-CD3 monoclonal antibody (leu-4) plus a second TRITC-labeled antibody, and anti-CD59 (E43) plus a second Cy5-labeled antibody. Representative images of the membrane expression patterns of CD3, and overlapping GFP / CD3, CD3 / CD59 and GFP / CD3 / CD59, are shown.
  • FIG. 4 The pT ⁇ b isoform retains the TCR ⁇ chain intracellularly.
  • CT rabbit anti-pT ⁇ antiserum
  • Figure 5. Specific detection of pT ⁇ a and pT ⁇ b by quantitative real-time RT-PCR.
  • Figure 6. Differential regulation of the pT ⁇ a and pT ⁇ b isoforms during human intrathymic development.
  • the mRNAs were isolated and subjected to reverse transcription and quantitative RT-PCR. Specific amplifications of pT ⁇ , pT ⁇ b (see Figure 5A) and GAPDH were performed in parallel in triplicate reactions for each of the cDNA samples.
  • the pT ⁇ a and pT ⁇ values represented in the upper figure were obtained after normalization with the GAPDH values.
  • the pT ⁇ b / pT ⁇ a ratio is shown in the graph below.
  • RT-PCR was performed from RNA isolated from unfractionated human thymocytes, and the complete cDNAs of pT ⁇ a and pT ⁇ b were subsequently generated by PCR amplification with the oligonucleotides 5'- GGGCCCGGATCCATATGGCCGGTACATGGCTG-3 '(SEQ ID NO 1) and antisense 5'- GGGGGATCCCCGGCAGCTCCAGCCTGCAG-3 '(SEQ ID NO 2). These oligonucleotides are specific for exon 1 and 4 of the pT ⁇ gene, which allowed the amplification of two different fragments, one with the expected size corresponding to pT ⁇ a , and the other with 320 base pairs less (data not shown).
  • the cD ⁇ As from ⁇ T ⁇ a and pT ⁇ b were fused to the Green Fluorescent Protein (GFP) coding sequence by digestion and ligation at the BamHI site of the pEGFP-vectorl vector (Clontech, Palo Alto, CA) ( Figure 1A) and were independently transfected into JR3.11 cells for comparison. JR3.11 cells they were grown in RPMI 1640 medium (Biowhittaker, Walkersville, MD) supplemented with 10% FCS (Gibco BRL, Paislay, UK).
  • GFP Green Fluorescent Protein
  • Transfections were carried out by electroporation as previously described (Trigueros C, Ramiro AR, Carrasco YR, de Yebenes VG, Albar JP, Toribio ML. Identification of a late stage of small noneyeling pTa. J Exp Med 1998; 188 : 1401-12). Briefly, 50 ⁇ g of plasmid DNA was transfected at 264V and 975 ⁇ F in a Gene Pulser II (Bio-Rad laboratories, Riclimond, CA).
  • the heterogeneous expression of GFP in each of the transfected cell lines served to trace the specificity of CD3 expression on the cell surface by two-color flow cytometry.
  • pT ⁇ to -GFP + cells express CD3, but CD3 expression was not found in GFP + cells in any of analyzes performed on more than 30 pT ⁇ transfected cell lines.
  • all the pT ⁇ a transfectants originated in this study (around 35 cell lines) present low levels of CD3, similar to that found in transient transfections.
  • cell surface marking was performed with an anti-TCR ⁇ monoclonal antibody (1734-14 anti-V ⁇ 8 monoclonal antibody plus goat anti-mouse Igs-PE, obtained from Dr A.
  • mice were immunized intraperitoneally with 50 ⁇ g of KLH-coupled antigen in combination with Freund's complete adjuvant (DIFCO, Detroit, MI). After 30 days a second immunization was performed with 50 ⁇ g of antigen and incomplete Freund's adjuvant (DIFCO), and 50 days later a third immunization was performed with 70 ⁇ g of antigen only. Lymph node cells from immunized mice fused with the murine cell line of Ag8653 myeloma following the conventional procedure. The anti-pT ⁇ specificity of the antibodies obtained was determined by immunofluorescence techniques in COSA7 cell transfectants. Among the antibodies obtained, the K5G3 antibody showed reactivity against all COS GFP + cells transfected with pT ⁇ a -GFP, but not against cells transfected with pT ⁇ b -GFP (data not shown).
  • TCR T cell receptor
  • IL-2 interleukin 2
  • CD69 expression was analyzed by flow cytometry after 12 hours of incubation of the cells in plates previously coated with 20 ⁇ g ml of anti-CD3 antibody (OKT3) or anti-pT ⁇ (K5G3), or with the presence of 20 ng / ml PMA (Sigma) and l ⁇ M ionophore (Sigma).
  • CD69 expression was invariably induced by both anti-CD3 and anti-pT ⁇ a antibodies in GFP + pT ⁇ transfected cells, while CD69 levels remained unchanged in GFP + pT ⁇ b transfected cells.
  • JR3.11 cells transfected with pT ⁇ b maintain their intrinsic capacity to express CD69, but are not capable of express the pre-TCR complex that promotes the proper activation of intracellular signals from the cell surface.
  • these functional and phenotypic approaches provide the first evidence that the human pT ⁇ protein is unable to be expressed on the cell membrane as part of a CD3-associated functional pre-TCR complex.
  • the transfected lines JR3.11 were attached to plates pre-coated with Poly-L-Lys (Sigma) (5x10 5 cells / plate) by incubation at 37 ° C for two hours. The plates were washed in PBS, fixed with 2% paraformaldehyde in PBS for 10 min and blocked with 2% BSA / PBS. For intracellular staining, cells were permeabilized for 5 min with 0.05% Triton X-100 (SIGMA) prior to blocking.
  • SIGMA Triton X-100
  • the preparations were visualized using a Radiance 2000 confocal system (Bio-Rad Laboratories, Hercules, CA) coupled to an Axiovert S100TV inverted microscope (Zeiss, Obercochen, Germany).
  • EGFP, TRITC and Cy5 fluorescences were detected using a bandpass filter HQ515 / 30, a longpass filter HQ600 / 50, and a longpass filter HQ660 / LP, respectively.
  • CD3 in pT ⁇ a transfectants show uneven distribution in one or more spots around the cell which collocalize with GFP expression (left middle panel, GFP / CD3).
  • the pT ⁇ a and pT ⁇ b cell lines were stained with an antibody against the CD59 marker, specifically located in these membrane structures.
  • CD59 remains dispersed with a typical pattern of vesicles or lipid rafts in pT ⁇ b transfectants, and in the form of large protein pools in transfectants.
  • transient pT ⁇ a and pT ⁇ b transfectants were metabolically labeled with 35 S-methionine, and their lysates were immunoprecipitated with a rabbit antiserum (CT -1) prepared against a synthetic peptide contained in the cytoplasmic region of the human protein pT ⁇ , region, therefore, shared by both isoforms of pT ⁇ .
  • CT -1 rabbit antiserum
  • SupTl cells lack TCR ⁇ , but not TCR ⁇ , express pT ⁇ mRNA, and have low levels of CD3 on the cell surface, which correlate with the expression of an endogenous preTCR (Trigueros C, Ramiro AR, Carrasco YR, de Yebenes VG, Albar JP, Toribio ML. Identification of a late stage of small noneyeling pTa. J Exp Med 1998; 188: 1401-12).
  • RNA of the different subtypes of selected thymocytes was isolated following the procedures previously described (RamiroAR., Trigueros C, Márquez C, San Millan JL, Toribio ML. Regulation of pre-T cell receptor (pT alpha- TCR beta) gene expression during human thymic development. J Exp Med 1996; 184: 519-30).
  • CDNA was obtained from each RNA sample (10-50 ng) by reverse transcription, using an oligo-dT according to the manufacturer's instructions (GIBCO BRL), which was subsequently used in the PCR reaction.
  • Oligonucleotides and Taqman probes were designed to independently amplify and detect the pT ⁇ and pT ⁇ isoforms using Primer Express software (Applied Biosystems, Foster City, CA). Taqman probes were labeled with 6-FAM (Applied Biosystems).
  • 6-FAM Applied Biosystems
  • the sense oligonucleotide 5'-GTGTCCAGCCCTACCCAC-3 '(SEQ ID NO 5) and the antisense 5'-ATCCAC CAGCAGCATGATTG-3' (SEQ ID NO 6) were used in combination with the Taqman 5'- probe. TGTGGGCGGCACACCCTTTC-3 '(SEQ ID NO 7).
  • the pT ⁇ b isomorph was independently amplified using the 5'-GCCGGTACATGGCTGCTACT-3 'sense oliogonucleotide (SEQ ID NO 8) and the 5'-CTGTAGAAGCCTCTCCTGTG-3' antisense (SEQ ID NO 9) in conjunction with the Taqman 5'-CCTGGTTGTTGGCTTT 3 '(SEQ ID NO 10).
  • the oligonucleotide-probe combinations for both isoforms are schematically represented in Figure 5A.
  • the oligonucleotides for the amplification of pT ⁇ b were designed in the first exon and at the junction between exon 1 and exon 3.
  • the TaqMan probe for pT ⁇ binds to the first exon.
  • Amplifications, detections, and analyzes were performed on an ABI PRISM 7700 system (Applied Biosystems).
  • the specificity of the chosen pairs of oligonucleotides is described in Figure 5B, where it is shown that the amplification of a plasmid containing the pT ⁇ b cDNA is detectable from the first cycles of the reaction.
  • the amplification curves overlapped with those obtained for controls where the template was not added. This result ensures that pT ⁇ a is not detected by cross recognition with pT ⁇ oligonucleotides in thymic samples.
  • the specificity of the oligonucleotides for pT ⁇ a and the probe were tested in the same way (data not shown).
  • the samples were quantified by interpolating their threshold cycle values (Ct) (that is, the cycle in which the reaction fluorescence first exceeds the background) on a standard curve that was constructed with the values of amplification obtained from serial dilutions of a template (10 times) that cover a concentration range of 10 4 .
  • the connection coefficients of the different standard curves obtained using human thymocyte cDNA as template varied between 0.98 and 1.00 (data not shown). Variations between samples were normalized with respect to the expression of GAPDH, used as an endogenous control.
  • the cDNA samples from the different thymic populations were amplified in parallel for GAPDH, pT ⁇ a and pT ⁇ b , and the quantitative values were obtained by interpolation in the human thymocyte cDNA standard curves.
  • the final quantitative data is presented as the ratio of the pT ⁇ a / GAPDH and pT ⁇ / GAPDH values for each sample.
  • CD34 + and CD4 + CD8 + cells were isolated by immunomagnetic selection with magnetic microspheres covered with anti-CD34 and anti-CD8 antibodies (Dynabeads, Dynal Corp, Oslo, Norway), respectively.
  • the CD4 + CD8- population was isolated from the CD8- fraction with spheres covered with anti-CD4 (Dynal).
  • the DP CD3- large thymocytes were subsequently fractionated into CD8 ⁇ + and CD8 ⁇ + cells by selection by flow cytometry in an EPICS Elite Cell Sorter (Coulter Electronics, Inc) after labeling with anti-CD8 ⁇ (2ST8-5H7, kindly provided by Dr EL Reinherz, Dana-Farber Cancer Institute, Boston, MA) plus PE-labeled goat anti-mouse IgG2a antibody (Caltag).
  • the pre- and post-selection- ⁇ thymocytes analyzed correspond to the populations of CD4 + CD8 ⁇ " and CD4 + CD8 ⁇ + cells selected from TCR ⁇ " thymocytes.
  • the stimulus used was the treatment with PMA plus ionophore, and the activation criterion that we used was the induction of the activation antigen CD69 (Figure 6B, left).
  • Quantitative data are shown in Figure 6B (right) and indicate induced cell activation with PMA plus ionophore treatment of calcium causes a significant reduction in mRNA levels pT ⁇ a.
  • mRNA levels of pT ⁇ b are reduced, but weakly, in activated cells, indicating that pT ⁇ a is more sensitive than pT ⁇ to transcriptional regulation induced by cell activation.

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Abstract

La présente invention concerne des outils techniques d'identification et de sélection de sous-espèces de lymphocytes pré-T humains à partir du pré-récepteur des lymphocytes T (pT alpha ). L'invention concerne, de plus, des méthodes de régulation de l'expression et de la fonction dudit récepteur au cours du développement des lymphocytes T humains. Dans un mode de réalisation particulier, l'invention concerne l'anticorps monoclonal K5G3 qui reconnaît de manière spécifique l'isoforme pT alpha <a>, ce qui permet pour la première fois d'analyser l'expression de la chaîne pT alpha dans la surface cellulaire.
PCT/ES2002/000387 2001-08-14 2002-08-01 Pre-recepteur des lymphocytes t (pre-tcr) et caracterisation et regulation de son expression et de sa fonction au cours du developpement des lymphocytes t chez les humains Ceased WO2003016355A1 (fr)

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ESP200101903 2001-08-14
ES200101903A ES2190880B1 (es) 2001-08-14 2001-08-14 Pre-receptor de las celulas t (pre-tcr). caracterizacion y regulacion de su expresion y funcion durante el desarrollo de las celulas t en humanos.

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WO2003016355A1 true WO2003016355A1 (fr) 2003-02-27

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2867373A1 (es) * 2020-04-17 2021-10-20 Consejo Superior Investigacion Tratamiento terapeutico de leucemias linfoblasticas agudas de celulas t con un anticuerpo monoclonal frente al receptor pre-tcr

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BARBER D.F. ET AL.: "Cutting edge: The expression in vivo of a second isoform of pTalpha: Implications for the mechanism of pTalpha action", J. IMMUNOLOGY, vol. 161, 1998, pages 11 - 16 *
KOSUGI A. ET AL.: "Subunit composition of the pre-T cell receptor complex analysed by monoclonal antibody against the pre-T-cell receptor alpha chain", IMMUNOLOGY, vol. 91, 1997, pages 618 - 622 *
RAMIRO A. ET AL.: "Regulation of Pre-T cell receptor (pTalpha-TCRbeta) gene expression during human thymic development", J. EXP. MED., vol. 184, 1996, pages 519 - 530 *
SAINT-RUF C. ET AL.: "Genomic structure of the human pre-T cell receptor alpha chain and expression of two mRNA isoforms", EUR J. IMMUNOL., vol. 28, 1998, pages 3824 - 3831 *
TRIGUEROS C. ET AL.: "Identification of a late stage of small non-cycling pTalpha pre-T-cells as immediater precursors of cell receptor alpha/beta+ thymocytes", J. EXP. MED., vol. 188, 1998, pages 1401 - 1412 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2867373A1 (es) * 2020-04-17 2021-10-20 Consejo Superior Investigacion Tratamiento terapeutico de leucemias linfoblasticas agudas de celulas t con un anticuerpo monoclonal frente al receptor pre-tcr
WO2021209670A1 (fr) * 2020-04-17 2021-10-21 Consejo Superior De Investigaciones Científicas Traitement thérapeutique de leucémies lymphoblastiques aiguës à lymphocytes t avec un anticorps monoclonal reconnaissant le récepteur pre-tcr

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ES2190880B1 (es) 2004-11-16

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