WO2003016355A1 - Pre-t cell receptor (pre-tcr) and the characterisation and regulation of the expression and function thereof during the development of t cells in humans - Google Patents

Abstract

The invention relates to technical tools that are used to identify and select subspecies of pre-T lymphocytes using the pre-T cell receptor (pT alpha ). Moreover, said invention relates to methods of regulating the expression and function of said receptor during the development of human T lymphocytes. In one particular embodiment, the invention relates to monoclonal antibody K5G3 which recognises the pT alpha <a> isoform in a specific manner, thereby enabling the expression of the pT alpha chain on the cellular surface to be analysed for the first time.

Description

TITLE

PRE-RECEIVER OF T CELLS (PRE-TCR). CHARACTERIZATION AND REGULATION OF ITS EXPRESSION AND FUNCTION DURING THE DEVELOPMENT OF T-CELLS IN HUMANS

TECHNICAL SECTOR

Biomedicine. Immunology. Identification and selection of T lymphocytes during their intrathymic development.

STATE OF THE ART

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). This TCR receptor is a heterodimer specific to each T cell clone, which is expressed on the surface associated with the CD3 complex. Approximately 90% of peripheral blood T cells (Tαβ) express a TCR consisting of a TCRα polypeptide chain and a TCRβ. The remaining 10% (Tγδ) express a TCR consisting of a TCRγ chain and a TCRδ. 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). In each T cell clone, the combination of the V, D and J domains in both β and γ chains and in both γ and δ chains participate in antigen recognition specifically by defining a single point of attachment (idiotype) . In contrast, domain C does not participate in antigen binding. Although both types of TCR are highly polymorphic, the degree of polymorphism is much higher in the case of TCRαβ.

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 Tαβ 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 TCRαβ, 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. Cell 1994; 76: 219-28; 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). In the thymus, genes encoding the TCRβ chain are rearranged and expressed earlier than those corresponding to the TCRα locus. Thymocytes that productively rearrange the TCRβ locus first express a pre-TCR complex, independent of TCRα, consisting of a TCRβ chain covalently linked to a invariant pre-TCRα (pTα) chain, which associates with the CD3 complex and promotes control point generally referred to as "β selection". (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; Saint-Ruf C, Ungewiss K, Groettrup M, Bruno L, Fehling HJ, von Boehmer H. Analysis and expression of a cloned pre-T cell receptor gene. Science 1994; 266: 1208-12; van Oers NS, von Boehmer H, Weiss A The pre-T cell receptor (TCR) complex is functionally coupled to the TCR-zeta subunit. J Exp Med 1995; 182: 1585-90; Berger MA, Dave V, Rhodes MR, et al. Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required. J Exp Med 1997; 186: 1461-7). Β selection induces the survival and proliferative expansion of those thymocytes in which a correct rearrangement of TCRβ 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. Productive 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. Immunity 1999; 11: 91-101; Kruisbeek AM, Haks MC, Carleton M, Wiest DL, Michie AM, Zuniga-Pflucker JC. Branching out to gain control: how the pre-TCR is linked to multiple functions. Immunol Today 2000; 21: 637-44; von Boehmer H, Aifantis I, Feinberg J, et al. Pleiotropic changes controlled by the pre-T-cell receptor. Curr Opin Immimol 1999; 11: 135-42). Pre-TCR-mediated cell expansion abruptly ends, and in the resulting population of resting small thymocytes rearrangements begin at the TCRα 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). Following rearrangement at the TCRα locus and substitution of pTα for TCRα, the TCRαβ 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 Tαβ cells takes place. By means of an additional "negative selection" process, the clonal elimination of those thymocytes with autoreactive TCRαβ receptors will be induced, thus guaranteeing tolerance to the proper components. Most of the knowledge about the functional expression of pre-TCR during intrathymic development in mice comes from experimental approaches aimed at the breakdown of genes that encode for critical components involved in the generation and assembly of pre-TCR (Malissen B, Ardouin L, Lin SY, Gillet A, Malissen M. Function of the CD3 subunits of the pre-TCR and TCR complexes during T cell development. Adv Immunol 1999; 72: 103-48; Fehling HJ, von Boehmer H. Early alpha beta T cell development in the thymus of normal and genetically altered mice.Curr Opin Immunol 1997; 9: 263-75; Haks MC, Oosterwegel MA, Blom B, Spits HM, Kruisbeek AM. Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR. Semin Immunol 1999; 11: 23-37). These studies suggest that pre-TCR activity begins at the CD44 ^{+ / "} CD25 ⁺ thymocyte stage, where peak transcription of pTα is achieved (Saint-Ruf C, Ungewiss K, Groettrup M, Bruno L, Fehling HJ , von Boehmer H. Analysis and expression of a cloned pre-T cell receptor gene. Science 1994; 266: 1208-12) and induces functional rearrangement of the TCRβ chain (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; Godfrey DI, Kennedy J , Mombaerts P, Tonegawa S, Zlotnik A. Onset of TCR-beta gene rearrangement and role of TCR-beta expression during CD3-CD4-CD8-thymocyte differentiation. J Immunol 1994; 152: 4783-92). However, there is no direct evidence of pre-TCR expression in thymocytes from normal mice, probably due to its low levels of expression on the cell surface and the lack of antibodies that specifically recognize it (von Boehmer H, Aifantis I, Feinberg J, et al. 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 TCRα 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. Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required. J Exp Med 1997; 186 : 1461-7). The presence of barely detectable levels of pre-TCR on the cell surface has led to the question of whether the expression of pre-TCR in the thymocyte membrane would be essential for the β selection process. In this sense, it has been observed that the TCRβ chain must leave the endoplasmic reticulum (RE) compartment to induce cell differentiation and proliferation, and allelic exclusion of TCRβ (O'Shea CC, Thornell AP, Rosewell IR, Hayes B, Owen MJ. Exit of the pre-TCR from the ER / cis-Golgi is necessary for signaling differentiation, proliferation, and allelic exclusion in immature thymocytes. Immunity 1997; 7: 591-9). However, the binding of the pre-TCR to a possible ligand seems to be dispensable for its function, since the development of T cells is independent of the extracellular immunoglobulin (Ig) domains of the pre-TCR (Jacobs H, Iacomini J, van d, N, Tonegawa S, Berns A. Domains of the TCR beta-chain required for early thymocyte development. J Exp Med 1996; 184: 1833-43; Irving BA, Alt FW, Killeen Ν. Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains. Science 1998; 280: 905-8). In this sense, it has recently been suggested that the pre-TCR could be endowed with properties of constitutive activation, as it has been found to spontaneously recruit into lipid domains or membrane rafts enriched in activated signal-translating molecules (Saint-Ruf C, Panigada M, Azogui O, Debey P, von Boehmer H, Grassi F Different initiation of pre-TCR and gammadeltaTCR signaling. Nature 2000; 406: 524-7).

Contrary to mouse studies, most of the information obtained on the expression of pre-TCR in human thymocytes comes from direct analysis of ex vivo isolated cell populations. Such experimental approaches have recently made it possible to successfully detect the expression of the pTα chain on the surface of primary human thymocytes, using a specific antiserum (Trigueros C, Ramiro AR, Carrasco YR, from Yebenes VG, Albar JP, Toribio ML. Identification of a late stage of small noneyeling pTa. J Exp Med 1998; 188: 1401-12), observing that the expression of pTα is confined in vivo to a limited fraction of large double-positive CD4 + CD8 + thymocytes (DP) size, which are in division, and which co-express low levels of CD3 on the surface. These large " ^low CD3" cells constitute the immediate progeny of pre-T CD4 + CD8αα + cells (Carrasco YR, Trigueros C, Ramiro AR, Yebenes VG, Toribio ML. Beta-selection is associated with the onset of CD8beta chain expression on CD4 (+) CD8alphaalpha (+) pre-T cells during human intrathymic development. Blood 1999; 94: 3491-8), in which the first human TCRβ rearrangements are initiated (Blom B, Nerschuren MC, Heemskerk MH, et al. TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation. Blood 1999; 93: 3033-43) and thus represent the most immature population of post-T cell pre-T cells. β selection. At a later stage, pre-TCR expression is lost from the cell surface in a population of large thymocytes that are still dividing, and which subsequently give rise to the population of small resting thymocytes in which rearrangements are initiated. at the TCRα 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). Despite the fact that the expression of the pre-TCR on the cell surface is restricted to a specific intrathymic maturation stage, it has been observed that the transcription of the pTα gene covers, in both mice and humans, all stages of pre-T cells and decreases only immediately before CD4 + or CD8 + stage of "simple positive" mature thymocytes (SP) (Saint-Ruf C, Ungewiss K, Groettrup M, Bruno L, Fehling HJ, von Boehmer H. Analysis and expression of a cloned pre-T cell receptor gene Science 1994; 266: 1208-12; 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. J Exp Med 1996; 184: 519-30). Therefore, the regulation of the surface expression of the pre-TCR is for now an open question. Recently a second isomorph of pTα has been described, originating from the alternative processing or "splicing" of the pTα mRNA, called pTα, which lacks the second exon of pTα that codes for most of the Ig extracellular domain, but which retains the residue cistern presumably involved in covalent binding with TCRβ (Barber DF, Passoni L, Wen L, Geng L, Hayday AC. The expression in vivo of a second isoform of pT alpha: implications for the mechanism of pT alpha action. J lmrnunol 1998; 161: 11-6). The pTα isoform is co-expressed in the mouse thymus along with the conventional isoform, called pTα ^a , albeit at significantly lower levels (about 10-fold) (Barber DF, Passoni L, Wen L, Geng L, Hayday AC. The expression in vivo of a second isoform of pT alpha: implications for the mechanism of pT alpha action. J lmmunol 1998; 161: 11-6). Conservation of this alternative pTα transcript in humans (Saint-Ruf C, Lechner O, Feinberg J, von Boehmer H. Genomic structure of the human pre-T cell receptor alpha chain and expression of two mRNA isoforms. Eur J Immunol 1998; 28 : 3824-31) suggests that this could play an important role in the development of thymocytes. However, the regulation of pTα ^b expression during T cell development has not been directly studied at this time. As of today, the only data available on the possible function of pTα come from transfection experiments in mouse cells in which each of the pTα isoforms has been observed to have functionally different effects on the expression of the TCRβ chain in the cell surface (Barber DF, Passoni L, Wen L, Geng L, Hayday AC. The expression in vivo of a second isoform of pT alpha: implications for the mechanism of pT alpha action. J Immunol 1998; 161: 11-6) . From these studies, Barber and colleagues have proposed that pTα ^b may be more efficient than pTα ^a in transporting TCRβ to the cell surface, allowing for increased expression of TCRβ as part of an alternative CD3-associated pre-TCR. However, the physiological relevance of such an alternative form of pre-TCR remains to be determined. DESCRIPTION Brief description

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 Tα ^a and pTα isoforms of the pre-TCR receptor show a different gene expression pattern throughout the intrathymic development of these T lymphocytes, and that the isoform pTα ^b , in contrast to pTα ^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 TCRβ intracellularly, providing evidence for its independent regulation and support the individual role of both isoforms during the T cell differentiation process.

Thus, the present invention provides a series of techniques that allow identifying and selecting the different pre-T lymphocyte cell subtypes. In the first place, real-time PCR from these T lymphocyte cell populations, with specific oligonucleotides and Taqman probes that allow the simultaneous expression of both pTα ^a and pTα ^b isoforms ^{to be} distinguished; and second, the in vivo identification of pre-T lymphocytes by a monoclonal antibody that specifically recognizes the pTα ^a isoform and forms part of the present invention. These methods provided by this invention allow, among other applications, to detect and identify 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.

As a particular embodiment of the present invention, the monoclonal antibody K5G3 is presented that specifically recognizes the Ig extracellular domain of the human pTα chain and, therefore, reacts exclusively with the pTα ^a isoform, allowing for the first time to analyze the expression of the pTα 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. On the other hand, monoclonal antibodies that specifically recognize the pTα isoform and that have been produced from fragments of this extracellular Ig domain of the human pTα chain form part of the present invention. Finally, the 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 pTα are part of the present invention.

As previously indicated, the pTα ^a isoform specifically induces the expression on the cell surface of all the components of the pre-TCR, TCRβ, pTα 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. As a particular embodiment 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. Likewise, as a particular embodiment, the constitutive cellular activity of pre-T lymphocytes can be induced by transfecting the pTα ^a isoform into said pre-T lymphocytes as described in the present invention, further forming part of the present invention. the pTα ^to -GFP gene construct. As a particular embodiment of the present invention, the regulation of pre-human T lymphocytes can be carried out by transfection of the pTα ^b isoform that induces intracellular retention in the ER of TRCβ chains, the pTα gene construct also forming part of the present invention. -GFP. Detailed Description As mentioned above, in both mice and humans, a second mRNA isoform of pTα, pTα ^b , has been described, the function, regulation and biochemical properties of which are largely unknown. The analysis of the pTα ^a -GFP and pTα ^b -GFP transfectants carried out in the present invention has provided important keys to the expression and function of both isoforms. First, the results of the present invention provide the first formal evidence that expression on the cell surface of all components of the pre-TCR, TCRβ, pTα ^a and CD3 complex is specifically induced after transfection with pTα ^a (see Example 1). It is important to mention that these data constitute the first formal evidence of the expression of a pTα chain on the cell surface by detection with a specific monoclonal antibody (K5G3), result of the present invention, and cytofluorimetric techniques. Second, the trace of GFP in the cells transfected with pTα ^a has led ^{to the} conclusion that TCRα-deficient human mature T cells, specifically the mimic T JR3.11 cell line, are capable of expressing the pre-TCR on the surface. cell (Figure 1C). This is an unexpected result because previous data showed that transfection of pTα ^a into TCRα-deficient mature murine T cells was not sufficient to promote pre-TCR expression in the cell membrane (Saint-Ruf C, Ungewiss K, Groettrup M, Bruno L, Fehling HJ, von Boehmer H. Analysis and expression of a cloned pre-T cell receptor gene. Science 1994; 266: 1208-12). In addition to the parallelism with pre-BCR (Borst J, Jacobs H, Brouns G. Composition and function of T-cell receptor and B-cell receptor complexes on precursor lymphocytes. Curr Opin lmmunol 1996; 8: 181-90), these results are the main experimental argument against the existence of an additional string "surrogate" NpreT, the identification of which has not occurred until now. Thus, our data suggests that, at least for the mature JR3.11 cell line, this hypothetical NpreT chain does not exist in humans, or at least is not absolutely necessary during assembly and surface expression of the preTCR. Most importantly, the present invention shows evidence of the functionality and competence of the pre-TCR receptor expressed on the cell surface of the transfectants in terms of CD69 induction (Figure 2B) and intracytosolic Ca ²⁺ mobilization (Figure 2A). Also 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-pTα K5G3 antibody that binds pTα ^a and triggers both signals. Therefore, the reagent generated in the present invention (anti pTα 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.

Neither of these functional or phenotypic effects were observed when the pTα ^b chain was expressed in JR3.11 cells instead of the pTα ^a isoform, indicating that the pTα ^b chain is unable to promote surface expression of a pre-TCR complex ( Figure IB and 1C). Also, it could be demonstrated the specificity of the monoclonal antibody K5G3 generated in this invention in the selective recognition of isofomia pTα ^a. These results were further confirmed by examination by confocal microscopy, which revealed the existence of a surface complex in cells transfected with pTα ^a but not with pTα (Figure 3B).

Interestingly, despite their differences in surface expression, these studies demonstrated that both isoforms are preferentially located in the endoplasmic reticulum (ER) of the cell (Figure 3A). This result may reflect the existence of a specific retention mechanism which could be located in the cytoplasmic domain of pTα. Confocal microscope studies further revealed that, according to previous studies, (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; 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. Rmmunity 1994; 1: 83-93; Kruisbeek AM, Haks MC, Carleton M, Wiest DL, Michie AM, Zuniga-Pflucker JC. Branching out to gain control: how the pre-TCR is linked to multiple functions. Immunol Today 2000; 21: 637-44; 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; Wiest DL, Berger MA, Carleton M. Control of early thymocyte development by the pre-T cell receptor complex: A receptor without a ligand? Semin hnmunol 1999; 11: 251-62), only extremely low levels of pr e- TCR are able to reach the plasma membrane of cells transfected with pTα, despite all the amount of protein found in the cytoplasm, according to the trace of GFP expression (data not shown). Most importantly, these low levels of pre-TCR surface expression reveal a distribution associated with glycolipid-rich membrane microdomains (rafts), as revealed by a typical pattern of dicontinuous (or patchy) expression of CD3, which was observed to collocalize with the lipid vesicle marker CD59. This patchy colocalization of CD3 and CD59 on the surface of human cells transfected with pTα ^a closely resembles that seen in mouse pre-T cells (Saint-Ruf C, Panigada M, Azogui O, Debey P, von Boehmer H, Grassi F. Different initiation of pre-TCR and gammadeltaTCR signaling. Nature 2000; 406: 524-7), and further reinforces the idea that pre-TCR is spontaneously recruited into lipid vesicles and reaches the plasma membrane as a constitutively active complex in human cells. This is again related to the apparently dispensable (or non-existent) ligand of preTCR (Irving BA, Alt FW, Killeen N. Thymocyte development in the absence of pre- T cell receptor extracellular immunoglobulin domains. Science 1998; 280: 905-8 ; Saint-Ruf C, Panigada M, Azogui O, Debey P, von Boehmer H, Grassi F. Different initiation of pre-TCR and gammadeltaTCR signaling. Nature 2000; 406: 524-7). In contrast to the findings described in the present invention, Barber and colleagues have previously proposed that the murine pTα isoform is more efficient than pTα ^a in driving a pre-TCR complex to the cell surface (Barber DF, Passoni L, Wen L, Geng L, Hayday AC. The expression in vivo of a second isoform of pT alpha: implications for the mechanism of pT alpha action. J Immunol 1998; 161: 11-6). These results were based on the finding that pTα ^a , but not pTα ^b , was capable of reducing surface levels of the transfected TCRβ chain, which was independently expressed by pTα in a TCR-deficient T cell line and, therefore, The discrepancies between mice and humans are likely to depend on the particular experimental system used in this study. In this regard, it should be noted that the results of the present invention have been obtained from two different human cell lines, JR3.11 and SupT 1.

The absence of preTCR complexes on the surface of pTα transfected cells in the present invention raises the possibility that, despite maintaining the cistern residue involved in binding to TCRβ, pTα ^b might be unable to form dimers with the TCRβ chain. We note that this is not the case, since TCRβ-pTα ^b dimers can be precipitated from the cytoplasm of transfected cells (Figure 4A). Therefore, other reasons for altered expression of pTα-containing complexes in the plasma membrane must be considered. An attractive possibility is that one or more CD3 chains may be displaced or absent in the pTα complexes, thereby preventing their release from the endoplasmic reticulum and their transport to the cell surface. A series of experiments are currently being carried out by the inventors of the present invention to determine the exact composition of the cytoplasmic heterodimer pTα ^b -TCRβ in terms of association with CD3, and preliminary results suggest that CD3γ may be associated with pTα ^a -TCRβ, but not with the pTα -TCRβ heterodimer (data not shown). The result described in the present invention regarding that overexpression of pTα inhibits or reduces the expression of endogenous pre-TCR on the surface of pre-T SupTl cells (Figure 4B) could suggest a possible competitive or regulatory role of pTα, which may be unable to reach the plasma membrane, but is capable of intracellularly binding and retaining TCRβ and / or CD3 chains.

In summary, and taken together, the results described in the present invention are compatible with two different roles of pTα ^b in the differentiation of thymocytes. First, it can be considered a competitive function, responsible for the inhibition or regulation of the expression on the surface of preTCR with pTα ^a . Although there are no data on the regulation or stability of the pTα ^b protein in vivo, the mRNA of pTα ^a has been found in ixna on average ten times more abundant than pTα ^b in the thymus both by other authors (Barber DF, Passoni L, Wen L, Geng L, Hayday AC. The expression in vivo of a second isoform of pT alpha: implications for the mechanism of pT alpha action. J In munol 1998; 161: 11-6) as in the present invention at least in population studied. This result may argue against the competitive role of pTα in thymic differentiation, however this possibility can only be excluded when there are adequate reagents to carry out protein analysis in individual cells. Secondarily, there would still be the possibility that pTα plays a signaling function independent of surface expression, from an intracellular compartment of thymocytes. This second possibility is especially attractive in light of recent data that pTα (as well as pTα ^a ) can constitutively promote NF-κB activation in transfected mouse cells (Voll RE, Jimi E, Phillips RJ, et al. NF-kappaB activation by the pre-T cell receptor serves as a selective sxrrvival signal in T lymphocyte development. Immunity 2000; 13: 677-89). The model of intracellular signaling from an internal compartment fits well with the absence of pTα on the cell surface, and also with the idea that preTCR complexes can be constitutively activated on the cell surface and a ligand is not required. Therefore, it seems reasonable to think that the expression of the complex in the cell membrane could be dispensable, as long as a functional complex could be formed inside the cell, as is the case with the pTα molecule.

Finally, in the present invention, a possible function of pTα ^a and pTα was evaluated by studying their expression during the development of T cells in the thymus. In this regard, the quantitative RT-PCR studies allowed to accurately compare the changes in the expression of pTα ^a and pTα ^b in detailed and representative intrathymic subtypes of independent stages of maturation pre- and post-selection β (see Example 4, Figure 6). In fact, this technique revealed that while both pTα isoforms reach maximum expression levels at the β selection checkpoint, the peak of expression of pTα is delayed with respect to pTα ^a and decreases less dramatically (about 5 times above the mean). ) than pTα ^a after β selection, which implies an increase in the pTα ^b / pTα ^a ratio during thymic development. On the one hand, this differential transcription pattern indicates that the expression of both isoforms (both at the level of mRNA processing itself and at the level of its stability) is strongly controlled during the development of T cells, reflecting the functional differences between the two. A similar finding has been published for the products of Pax-5 alternative splicing during B cell development (Zwollo P, Arrieta H, Ede K, Molinder K, Desiderio S, Pollock R. The Pax-5 gene is alternatively spliced during B-cell development. J Biol Chem 1997; 272: 10160-8). On the other hand, the data described in the present invention suggest that the function of pTα may be restricted in a narrow range of development, either coincidentally or immediately after the expression of the TCRβ chain and the β selection. Finally, although previous data indicates that stimulation induced by anti-CD3 antibodies through preTCR could silence the pTα locus (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. rmmunity 1994; 1: 83-93), this is the first demonstration that normal cells ex vivo have lost pTα expression (pTα ^a and pTα) after β selection. As β selection results simultaneously with activation of TCRα 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. J Exp Med 1996; 184: 519-30), a transcriptional regulatory mechanism could, in turn, limit pre-TCR expression to stages prior to the expression of the TCRα chain. Therefore, the competitive displacement of the pTα chain by TCRα during the assembly of TCRαβ, although it seems to be relevant in vitro in cell lines (Trop S, Rhodes M, Wiest DL, Hugo P, Zuniga-Pflucker JC. Competitive displacement of pTalpha by TCR- alpha during TCR assembly prevenís surface coexpression of pre-TCR and alphabeta TCR [In Process Citation]. J Immunol 2000; 165: 5566-72), does not appear to be required for the control of in vivo expression of pre-TCR on the cell surface. This proposal is in agreement with our finding that pTα transcription drops abruptly in pre-T cells without TCRα immediately after PMA-induced activation. Furthermore, the fact that pTα is only slightly reduced under identical experimental conditions may reflect the relative increase in pTα ^b transcripts observed in post-selection β cells in vivo. Due to the role of T lymphocytes in the immune response, the characterization and analysis of the different groups or subgroups of T lymphocytes involved in local or systemic immune responses is taking on an important role in view of better monitoring and modulation of the immune system. Some of these efforts have been directed to the analysis of T cells by PCR directed to the amplification of V regions of the α and β 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). Likewise, the possibility of characterizing and selecting pre-T cell populations at different stages of intrathymic development, pre or post-β 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. Likewise, the functional implication of 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.

DESCRIPTION OF THE FIGURES Figure 1.- The surface expression of the pre-TCR is detected in pTα ^a transfectants but not in pTα. A) Schematic representation of the pTα ^a - GFP and pTα -GFP constructs employed in the transfection experiments, EC, TM, CYT and EGFP indicate the extracellular, transmembrane, cytoplasmic and Enhanced Green Fluorescent Protein region, respectively. B) Expression on the cell surface of CD3 in transient transfectants pTα ^a and pTα. 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). C) Expression on the cell surface of components of the pre-TCR complex in representative lines of stable transfectants of pTα (al 3.2) and pTα (b71.2) Surface expression of CD3, TCRβ and pTα is shown by staining with monoclonal antibodies anti-CD3 (Ieu4-PE), anti-TCRβ (anti-Vβ8) and anti-pTα (K5G3) in relation to the expression of GFP. The results are representative of more than 30 different stable transfectants of pTα ^a and pTα ^b and of ten independent experiments. Figure 2.- pTα ^a , but not pTα ^b , promotes the expression on the cell surface of a pre-TCR competent in the transmission of intracellular signals. A) Analysis of intracellular Ca ²⁺ increases in pTα and pTα ^b transfectants after stimulation. The mature Jurkat T lines, and the transfectants pTα ^a (al 0.4) and pTα ^b (bl .4) were treated with fura-2AM, and after stimulation with anti-CD3 (OKT3) and anti-pTα (K5G3) monoclonal antibodies. ), the mobilization of Ca ²⁺ was assessed by fluorimetry. B) Analysis of CD69 expression after stimulation of the pTα ^a and pTα transfectants. The pTα ^a and pTα cell lines were stimulated with the anti-CD3, anti-pTα or PMA monoclonal antibodies. After 12 hours, CD69 expression was analyzed by flow cytometry. CD69-PE versus GFP figures are presented. The results are representative of five experiments and four distinct stable transfectants of pTα ^a and pTα ^b .

Figure 3.- Analysis by confocal microscopy of stable transfectants pTα ^a -GFP and pTα ^b -GFP .. Transfectants pTα (al 3.2) and pTα ^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 pTα ^a and pTα ^b transfectants are shown. B) 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.

Figure 4.- The pTα ^b isoform retains the TCRβ chain intracellularly. A) JR3.11 cells transiently transfected with the constructs pTα ^a -GFP or pTα ^b -GFP, were metabolically labeled. Cell lysates were immunoprecipitated with a rabbit anti-pTα antiserum (CT) and resolved on SDS-PAGE in gels of two dimensions (non-reducing / reducing conditions). B) CD3 surface expression in pre-T SupTl cells transiently transfected with constructs pTα ^to -GFP or pTα ^b -GFP. 24 hours after transfection the cells were labeled with the anti-CD3 monoclonal antibody Ieu4-PE and analyzed by flow cytometry. CD3 expression is shown in "untransfected GFP cells ^" (unshaded histograms) and in GFP ⁺ transfected cells (shaded histograms).

Figure 5.- Specific detection of pTα ^a and pTα ^b by quantitative real-time RT-PCR. A) Design of the amplicons pTα ^a and pTα. Oligonucleotides are represented as arrows and Taqman probes as black bars. The distribution of exons is also described. B) The oligonucleotides for pTα ^b and the Taqman probe were used to amplify the plasmid templates pTα and pTα ^a . PCR reactions were performed in triplicate. As negative control, the amplification curves obtained in the absence of template are shown. Figure 6.- Differential regulation of the pTα ^a and pTα ^b isoforms during human intrathymic development. A) Representative intrathymic cell subtypes of different consecutive stages of maturation were isolated as described in Methods, and their phenotypic characterization is shown at the bottom of the figure: proT CD34 +, pre-T CD4 +, CD4 + CD8αα, Large (large in division) CD8αβ and Small (small, at rest) CD8αβ. The mRNAs were isolated and subjected to reverse transcription and quantitative RT-PCR. Specific amplifications of pTα, pTα ^b (see Figure 5A) and GAPDH were performed in parallel in triplicate reactions for each of the cDNA samples. The pTα ^a and pTα values represented in the upper figure were obtained after normalization with the GAPDH values. The pTα ^b / pTα ^a ratio is shown in the graph below. B) Different susceptibility of pTα and pTα mRNAs to cellular activation. Activation of pre-T SupTl cells was assessed by expression of CD69, using an anti-CD69-PE antibody and flow cytometry. The histogram on the left shows the expression of CD69 in SupTl cells before (unshaded) and after treatment (shaded). Expression of pTα ^a and pTα ^b mRNAs was determined by quantitative RT-PCR before (white bars) and after (black bars) of SupTl activation, as shown in a representative experiment in the graph on the right.

EXAMPLES OF THE INVENTION Example 1.- Deficient expression on the cell surface of the human protein pTα ^b as part of a functional pre-TCR complex.

RT-PCR was performed from RNA isolated from unfractionated human thymocytes, and the complete cDNAs of pTα ^a and pTα ^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 pTα gene, which allowed the amplification of two different fragments, one with the expected size corresponding to pTα ^a , and the other with 320 base pairs less (data not shown). The cloning and sequencing of these smaller products confirmed that they correspond to the previously described human isoform of pTα ^b , in which exon 2 does not exist which codes for most of the extracellular domain "Ig-like" (Saint- Ruf C, Lechner O, Feinberg J, von Boehmer H. Genomic structure of the human pre-T cell receptor alpha chain and expression of two mRNA isoforms. Eur J Iminunol 1998; 28: 3824-31). In order to obtain some progress in the function of an alternative pre-TCR complex containing a putative pTα, a series of transfection experiments were carried out using a human T cell line deficient in the TCRα chain, JR3.11, derived from from Jurkat, and where transfection of the TCRα chain had previously been shown to reconstitute the cell surface expression of mature functional CD3-associated TCRαβ xm (Arnaud J, Huchenq A, Nernhes MC, et al. The interchain disulfide bond between TCR alpha beta heterodimers on human T cells is not required for TCR-CD3 membrane expression and signal transduction. ht Immunol 1997; 9: 615-26.). The cDΝAs from ρTα ^a and pTα ^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).

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 µg of plasmid DNA was transfected at 264V and 975 µF in a Gene Pulser II (Bio-Rad laboratories, Riclimond, CA). Cell surface expression of the CD3 complex was analyzed 24h later by staining with the anti-CD3 monoclonal antibody Ieu4-PE (BD Biosciences (Franklm Lakes, NJ) and flow cytometry in the transfected JR3.11 cells, which were controlled by the expression of GFP, following the methodology previously described by Trigueros et al. (Trigueros C, Ramiro AR, Carrasco YR, Yebenes VG, Albar JP, Toribio ML. Identification of a late stage of small noneyeling pTa. J Exp Med 1998; 188:. 1401-1412) in an Epics XL (Coulter Corp., Hialeah, FL) As shown in Figure IB, CD3 expressed on the surface of cells transfected GFP ⁺ pTα ^to, albeit at very low levels, similar to those observed in pre-T primary cells (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), while CD3 was not detected in GFP cells. ^" The results The results were markedly different in GFP ⁺ cells transfected with pTα ^b , where CD3 surface expression could not be detected, suggesting that, unlike pTα ^a , transfection with pTα ^b is not sufficient to induce expression in the cell surface of the pre-TCR complex. These results were further confirmed in stable transfection experiments in JR3.11 cells with pTα ^to -GFP and pTα -GFP. Stable transfectants were generated by G418 selection, 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). As seen in Figure 1C, 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. Again, pTα ^to -GFP ⁺ cells express CD3, but CD3 expression was not found in GFP ⁺ cells in any of analyzes performed on more than 30 pTα transfected cell lines. It should be noted that all the pTα ^a transfectants originated in this study (around 35 cell lines) present low levels of CD3, similar to that found in transient transfections. In addition, cell surface marking was performed with an anti-TCRβ monoclonal antibody (1734-14 anti-Vβ8 monoclonal antibody plus goat anti-mouse Igs-PE, obtained from Dr A. Alcover (Institute Pasteur, Paris, France) and Caltag Laboratories (Burlingame, CA), respectively More importantly, this anti-Vβ8 labeling demonstrated that GFP ⁺ CD3 cells co-express the endogenous chain TCRβ and CD3 in stoichiometric amounts (Figure 1C), suggesting that both CD3 and TCRβ proteins are expressed on the surface of the pTα ^a transfectants as part of a conventional pre-TCR complex pTα -TCRβ.

In order to obtain direct evidence that pTα ^a is in fact expressed together with TCRβ and CD3, the reactivity of different pTα ^a -GFP transfected cell lines against a specific monoclonal antibody (K5G3) produced in the present invention against the extracellular region of the pTα chain (Figure 1C). For the production of the anti-pTα monoclonal antibody K5G3, a cDNA encoding the extracellular region of the human pTα chain was first generated by amplification with the sense oligonucleotide 5'-

CCGGATCCATATGCTACCCACAGGTGTGGGC-3 '(SEQ ID NO 3) and the antisense 5' -CCCCGGATCCTCACAGCGCCCCACCCGGTGT-3 '(SEQ ID NO 4). The result of the amplification was digested with BamHI and ligated into the BamHI site of vector pQE32, which contains IP TG inducible His-tag (Qiagen, Hilden, Germany). A 15 KDa recombinant protein was obtained from transformed E. coli MI 5 cells after induction with IPTG, which was purified under denaturing conditions with 8M urea following the manufacturer's instructions. Subsequently, Balb / c mice were immunized intraperitoneally with 50 µg of KLH-coupled antigen in combination with Freund's complete adjuvant (DIFCO, Detroit, MI). After 30 days a second immunization was performed with 50 µg of antigen and incomplete Freund's adjuvant (DIFCO), and 50 days later a third immunization was performed with 70 µg of antigen only. Lymph node cells from immunized mice fused with the murine cell line of Ag8653 myeloma following the conventional procedure. The anti-pTα 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 pTα ^a -GFP, but not against cells transfected with pTα ^b -GFP (data not shown).

As seen in Figure 1C, additional flow cytometric analyzes revealed that the monoclonal antibody K5G3 also showed reactivity against the plasma membrane in pTα ^to -GFP ⁺ cells, and not in pTα ^b -GFP ⁺ cells. These results provide the first formal evidence of expression of the pTα chain on the cell surface and that transfection with pTα specifically induces membrane expression of all components of the pre-TCR complex: TCRβ, pTα ^a and CD3. Example 2.- pTα ^a promotes the expression on the cell surface of a TCR receptor competent in the transmission of intracellular signals. On the other hand, and continuing the previous argument, it is possible that a pre-TCR complex containing an alternative isoform pTα ^b could be expressed on the cell surface, although at levels below the detection limit of flow cytometry. To rule out this possibility, higher sensitivity assays were performed in the present invention to detect surface expression of a functional pre-TCR. First, intracytosolic Ca ²⁺ increases were analyzed to distinguish between pTα and pTα ^b transfectants. The analysis of intracellular Ca ²⁺ mobilization was carried out as previously described (Sahuquillo AG, Roumier A, Teixeiro E, Bragado R, Alarcon B. T cell receptor (TCR) engagement in apoptosis-defective, but interleukin 2 (IL-2) -producing, T cells results in impaired ZAP70 / CD3-zeta association. J Exp Med 1998; 187: 1179-92). Briefly, the Jurkat lines, ρTα ^a (alθ.4) and ρTα (bl.4) (10 ⁷ cells / ml) were treated with fura-2AM (Sigma Chemical Co., Saint Louis, MO), and stimulated with the antibodies monoclonal anti-CD3 (OKT3) and anti-pTα (K5G3) and Ca ²⁺ mobilization was assessed by fluorimetry. The fluorometric responses were measured in a Perkin-Elmer fluorimeter after stimulation with lOμg / ml of the mentioned antibodies. As seen in Figure 2A, no Ca ²⁺ flux was observed in pTα ^b transfectants in response to anti-CD3 treatment. In contrast, pTα ^a transfectants were observed mobilization of Ca ²⁺ after stimulation with anti-CD3, although the response was clearly less than that induced in the Jurkat parental cells after stimulation of the mature CD3-TCRαβ complex with anti-CD3. Furthermore, mobilization of Ca ²⁺ was induced after specific recognition of the pTα ^a chain with K5G3, which corroborates the expression on the cell surface of a functional pre-TCR complex and demonstrates the functionality of the monoclonal antibody K5G3 generated in this invention in the induction of functional responses mediated by pre-TCR.

Finally, the induction of CD69 expression was analyzed as an indicator of cellular activation after crosslinking the pre-TCR complex with antibodies (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). CD69 expression was analyzed by flow cytometry after 12 hours of incubation of the cells in plates previously coated with 20 µg ml of anti-CD3 antibody (OKT3) or anti-pTα (K5G3), or with the presence of 20 ng / ml PMA (Sigma) and lμM ionophore (Sigma). As seen in Figure 2B, CD69 expression was invariably induced by both anti-CD3 and anti-pTα ^a antibodies in GFP ⁺ pTα transfected cells, while CD69 levels remained unchanged in GFP ⁺ pTα ^b transfected cells. As activation with PMA induced high levels of CD69 in the cell membrane of both transfectants, regardless of GFP expression, we can conclude that JR3.11 cells transfected with pTα ^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. Taken together, these functional and phenotypic approaches provide the first evidence that the human pTα protein is unable to be expressed on the cell membrane as part of a CD3-associated functional pre-TCR complex. Example 3.- The pTα ^b protein retains the TCRβ chain intracellularly.

To determine the intracellular location of the pTα -GFP chimeric protein, several of the stable pTα -GFP transfectants were analyzed by confocal microscopy and compared with the pTα ^to -GFP transfectants. 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. Cells were stained by incubation for 30 minutes with 4 µg / ml of monoclonal antibodies anti-Protein Disulfide Isomerase (PDI) (Stressgen, Victoria, Canada) or leu-4 anti-CD3ε (BD Biosciences) in combination with the antibody. E43 anti-CD59 (generously provided by Dr. V. Horejsi, lnstitute of Molecular Genetics, Prague, Czech Republic). Secondary reagents included anti-rabbit Igs-Rhodamine (TRITC) (Southern Biotechnology, Birmingham, AL), anti-mouse IgGl-TRITC and anti-mouse Igs-Cy5 (Jackson ImmunoResearch Laboratories, Lie, West Grove, PA). 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.

Examination of the green fluorescence of non-permeabilized cells (Figure 3A) revealed that GFP was predominantly cytosolic in both pTα transfectants (left panel, GFP), indicating that both pTα ^a and pTα ^b isoforms are located intracellulantly, probably in the reticulum endoplasmic (RE). This possibility was confirmed by staining with an antibody that recognizes the ER-resident PDI protein. Most of both chimeric proteins pTα ^a -GFP and pTα ^b -GFP show a localization pattern similar to that observed with PDI staining (intermediate panel, PDI). The overlap of both fluorescences (right panel, GFP / PDI) revealed that both collocalize, and therefore that the vast majority of both pTα ^a and pTα ^b chains remain retained in the RE in the transfected cell lines. As seen in Figure 3B, staining of the cell surface with an anti-CD3 monoclonal antibody in non-permeabilized cells confirms the results previously obtained by flow cytometry, the selective expression of CD3 in pTα transfectants also being observed by immunofluorescence, but not in the pTα (left panel, CD3). It is interesting to note that the expression of CD3 in pTα ^a transfectants show uneven distribution in one or more spots around the cell which collocalize with GFP expression (left middle panel, GFP / CD3). To discern whether this pattern could suggest a preferential recruitment of pre-TCR complexes in lipid rafts, the pTα ^a and pTα ^b cell lines were stained with an antibody against the CD59 marker, specifically located in these membrane structures. As seen in the CD3 / CD59 and GFP / CD3 / CD59 overlays on the right panel, CD59 remains dispersed with a typical pattern of vesicles or lipid rafts in pTα ^b transfectants, and in the form of large protein pools in transfectants. pTα ^a . It should be noted that CD59 collocalized with CD3 on the membrane of pTα ^a cells, suggesting that pre-TCR complexes containing pTα ^a may be spontaneously recruited into lipid rafts or microdomains in transfected cell lines.

To investigate whether the deficient expression on the cell surface of pTα was due to a defective association with TCRβ, transient pTα ^a and pTα ^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 pTα, region, therefore, shared by both isoforms of pTα. Immunoprecipitated proteins resolved on two-dimensional gels. Specifically, 24 hours after transfection, cells were washed twice in PBS and incubated for 30 minutes at 37 ° C in Met / Cys-free DMEM medium (10 ⁷ cells / ml). Labeling was carried out for 30 minutes by adding 300 µCi / ml of a combination of ³⁵ S Met / Cys (Amersham Pharmacia Biotech, Uppsala, Sweden). After an additional hour of incubation with RPMI 1640 medium (Biowhittaker), supplemented with 10%> FCS, the cells were washed twice with PBS and screened in a lysis solution with 1% Brij 96. After a prewashed with preimmune rabbit serum bound to G-Shepharose protein beads (Amersham), the lysates were immunoprecipitated with a rabbit antiserum against the cytoplasmic region of pTα (CT-1) coupled to G-Sepharose protein beads. Immunoprecipitates were extensively washed with a lysis solution and resolved on a two-dimensional SDS-PAGE gel. The CT-1 antiserum immunoprecipitated one pTα dimer -TCRβ of pTα transfectants migrated off-diagonal (Figure 4A, left). More importantly, pTα ^b -TCRβ dimers were also observed in the precipitates of pTα ^b transfectants (Figure 4A, right), indicating that poor expression on the cell surface of pre-TCR complexes containing the pTα isoform is not due to poor association with TCRβ.

To analyze whether the pTα protein is directly responsible for the inhibition of the expression of the pre-TCR complex on the cell surface, a series of transfection experiments were performed on the human pre-T cell line, SupTl. SupTl cells lack TCRα, but not TCRβ, express pTα 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). To analyze the effect of pTα on membrane expression of pre-TCR, SupTl cells grown under the same conditions described for JR3.11 in Example 1, were transfected with pTα ^to -GFP or with pTα ^b -GFP. A moderate increase of expression of CD3 on the surface of the GFP ⁺ cells was observed when overexpressed pTα ^a. In contrast, when the pTα ^b isoform was transfected, CD3 expression was reduced in GFP cells (shaded histogram) compared to GFP cells ^" (unshaded histogram) (Figure 4B). Therefore our data indicates that the absence of pTα expression on the cell surface is not due to an altered association with TCRβ, and they suggest that pTα may be controlling the levels of pre-TCR expression on the cell membrane by binding and retaining the TCRβ chain in the cytoplasm Example 4.- Differential regulation of the expression of pTα ^a and pTα ^b during human intrathymic development.

In order to obtain information on a possible function of pTα ^b in vivo, in the present invention it was decided to establish the expression pattern of both isoforms of pTα ^a and pTα during intrathymic development. This issue was addressed using the real-time quantitative PCR technique, which provides the ideal system to connect quantify the transcription process, since the detection of the product Amplification takes place throughout the entire process, in real time, through the use of a combined detector and thermocycler system. This method allows quantification to be performed from the early stages of amplification, when the amount of the product is proportional to the amount of the starting target DNA. To carry out this analysis, the total 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 pTα and pTα isoforms using Primer Express software (Applied Biosystems, Foster City, CA). Taqman probes were labeled with 6-FAM (Applied Biosystems). For the amplification of pTα ^a 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 pTα ^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 pTα ^b were designed in the first exon and at the junction between exon 1 and exon 3. The TaqMan probe for pTα binds to the first exon. Since the specificity of this amplicon corresponds exclusively to the junction between exon 1 and exon 3, which is partially conserved at different splicing sites, it was necessary to test different 3 'oligonucleotides to satisfy the required specificity. Oligonucleotides and probes were used at xma final concentration of 300nM and 200nM, respectively. GAPDH amplifications were performed with the specific Pre-Developed Taqman Assay Reagent for the quantification of the expression of the human GAPDH gene. (Applied Biosystems), according to the manufacturer's instructions. All PCR reactions were performed in triplicate using the TaqMan Universal PCR Master Mix (Applied Biosystems). 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 pTα ^b cDNA is detectable from the first cycles of the reaction. In contrast, when a plasmid containing the pTα cDNA was used as a template, the amplification curves overlapped with those obtained for controls where the template was not added. This result ensures that pTα ^{a is} not detected by cross recognition with pTα oligonucleotides in thymic samples. The specificity of the oligonucleotides for pTα ^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. Therefore, the cDNA samples from the different thymic populations were amplified in parallel for GAPDH, pTα ^a and pTα ^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 pTα ^a / GAPDH and pTα / GAPDH values for each sample.

The cDNAs synthesized, as described above, from the RNA obtained from different populations of representative thymocytes of different maturation stages. Said populations were isolated from human thymus waste samples, resected during cardiac connective surgery from patients aged between 1 month and 3 years, as previously described (Carrasco YR, Trigueros C, Ramiro AR, de Yebenes VG , Toribio ML. Beta-selection is associated with the onset of CD8beta chain expression on CD4 (+) CD8alphaalpha (+) pre-T cells during human intrathymic development. Blood 1999; 94: 3491-8). For this, centrifugations were carried out in discontinuous Percoll density gradients (LKB, Uppsala, Sweden), from which the density fractions of 1,068 and 1.08 were recovered, with responses to large thymocytes, or small thymocytes, respectively. Subsequently, mature T, B, NK and myeloid cells were removed from each fraction, as previously described (Trigueros C, Ramiro AR, Canasco YR, de Yebenes VG, Albar JP, Toribio ML. Identification of a late stage of small noneyeling pTa. J Exp Med 1998; 188: 1401-12). CD34 + and CD4 + CD8 + cells (DP) 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 CD8αα ⁺ and CD8αβ ⁺ cells by selection by flow cytometry in an EPICS Elite Cell Sorter (Coulter Electronics, Inc) after labeling with anti-CD8β (2ST8-5H7, kindly provided by Dr EL Reinherz, Dana-Farber Cancer Institute, Boston, MA) plus PE-labeled goat anti-mouse IgG2a antibody (Caltag).

The results obtained in a representative experiment in which the different intrathymic subpopulations with the same individual are represented in Figure 6A. It was observed that immature thymocytes CD34 ⁺ CD4 ^"have moderate levels of expression pTα ^to, which increased to maximum levels at the later stage CD4 ^CD8" CD3 ^". Levels were also observed elevated transcription CD4 CD8αα which have not yet undergone the β selection process (Carrasco YR, Trigueros C, Ramiro AR, de Yebenes VG, Toribio ML. Beta-selection is associated with the onset of CD8beta chain expression on CD4 (+) CD8alphaalpha (+) pre-T cells during human intrathymic development. Blood 1999; 94: 3491-8) However, transcription of pTα ^a drops sharply after this tipping point, as pTα mRNA levels are reduced at least 10-fold in CD4 thymocytes ⁺ CD8αβ ⁺ either large dividing or small thymocytes at rest, in which functional expression of the pre-TCR complex has already taken place, but mature TCRαβ has not yet been expressed (Canasco YR, Trigueros C, Ramiro AR, de Yebenes VG, Torib io ML. Beta-selection is associated with the onset of CDδbeta chain expression on CD4 (+) CD8alphaalpha (+) pre-T cells during human intrathymic development. Blood 1999; 94: 3491-8). The expression pattern of pTα was different. We observe that the transcription of pTα ^b is delayed in development with respect to the expression of pTα, since the mRNA of pTα increases initially in the transition of CD34 ⁺ CD4 cells ^" to CD4 ⁺ CD8 ^" , giving a peak of maximum expression in the next stage CD4 CD8αα, immediately before the β selection process (Figure 6A, upper graph). Transcription levels of pTα decrease immediately after, although somewhat less dramatically than pTα, in CD4 ⁺ CD8αβ thymocytes after β selection. As a consequence of this apparently independent regulation of the expression of pTα ^a and pTα, the ratio pTα ^b / pTα ^a increases throughout the

V »intrathymic development, indicating a relative accumulation of the pTα isoform in late stages of pre-T cells (Figure 6A, lower graph). This relative increase in pTα ^b / pTα ^a in selected thymocytes (CD4 ⁺ CD8 ⁺ ) vs. Unselected thymocytes (CD4 ⁺ CD8 ^" ) are shown in Table 1, based on data from four mutually independent experiments.

Table 1.- Relative proportion of the transcriptional expression pTα ^b / pTα ^a in thymocytes before vs. post-selection β in four independent experiments.

Pre-selection -β * post-selection- β * post / prc! -Selection-β *

1 1.12 7.11 6.3

2 0.40 2.20 4.6

3 1.25 3.62 2.9

4 0.23 1.13 4.9

* The pre- and post-selection-β thymocytes analyzed correspond to the populations of CD4 ⁺ CD8αβ ^" and CD4 ⁺ CD8αβ ⁺ cells selected from TCRαβ ^" thymocytes.

The downregulation of the expression of both pTα isoforms, and more dramatically of the pTα ^a isoform, immediately after β selection, suggests that both events are related, so that the cellular activation inherent in the β selection process could promote quantitatively different responses with respect to decreased pTα ^a and pTα mRNA levels. To analyze For this possibility, we used the pre-T SupTl cell line, in which both isoforms are constitutively expressed, and in the present invention, we proceeded to evaluate whether cell activation involves the induction of quantitative changes in pTα mRNA levels (Figure 6B) . 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 pTα ^a. In contrast, mRNA levels of pTα ^b are reduced, but weakly, in activated cells, indicating that pTα ^a is more sensitive than pTα to transcriptional regulation induced by cell activation.

Claims

1. Identification and regulation of the function of human pre-T lymphocytes characterized in that it is performed by stopping the expression of the isoforms of the pTα gene of the T, pTα ^a and pTα pre-receptor. 2. A method of identification and selection of human pre-T lymphocytes according to claim 1 characterized in that it is carried out in vivo by a monoclonal antibody capable of specifically recognizing the expression on the cell surface of the pTα ^a isoform of the pre-receptor associated with TCRβ already CD3. 3. A method of identification and selection of human pre-T lymphocytes according to claim 2 characterized in that the monoclonal antibody is the antibody K5G3 or a fragment thereof. 4. A method of identification and selection of human pre-T lymphocytes according to claim 1 characterized in that the determination of the expression of the isoforms of the pTα gene of the T lymphocyte preceptor, pTα ^a and pTα, is performed by the technique of real-time quantitative RT-PCR comprising the following steps: a) RNA isolation from said cells, b) cDNA synthesis from said RNA, c) real-time quantitative PCR performed with oligonucleotides and probes Taqman designed to amplify and independently detect the pTα ^a and pTα ^b isoforms, and d) the determination of the expression pattern of said isoforms and their connection with a certain subtype of pre-T cells. 5. An identification and selection method of human pre-T lymphocytes according to claim 4, because the Taqman oligonucleotides and probes for the amplification and detection of the pTα ^a and pTα ^b isoforms are respectively: sense oligonucleotide 5'-GTGTCCAGCCCTACCCAC-3 ' (SEQ ID NO 5) and the antisense 5'-ATCCAC CAGCAGCATGATTG-3 '((SEQ ID NO 6) in combination with the Taqman 5'- TGTGGGCGG CACACCCTTTC-3' probe (SEQ ID NO 7) and the oliogonucleotide sense 5 ' - GCCGGTACATGGCTGCTACT-3 '(SEQ ID NO 8) and antisense 5'- CTGTAGAAGCCTCTCCTGTG-3' (SEQ ID NO 9) together with the Taqman 5'-CCTGGCCCT TGGGTGTCCAGC-3 'probe (SEQ ID NO 10). 6. A method of identification and selection of human pre-T lymphocytes according to claims 2 to 5 characterized in that the pre-T lymphocytes to be identified come from, among other sources, normal or pathological human thymus, and lymphocytes from patients with representative leukemias of the pre-T maturational stage 7. A method of identification and selection of human pre-T lymphocytes according to claim 6 characterized in that it allows to assess the presence of pre-T lymphocytes before and after a chemotherapeutic treatment of said leukemia. 8. Regulation of the function of human pre-T lymphocytes according to claim 1 characterized in that the cellular activation of the pre-T lymphocytes is induced by recognition with the K5G3 antibody or a fragment thereof through the pre-T receptor. 9. Regulation of the function of pre-T human lymphocytes according to claim 1 characterized in that the constitutive cellular activation of pre-T cells is induced by transfecting the pTα isoform in said pre-T lymphocytes. 10.- Regulation of pre-TCR expression in human pre-T lymphocytes according to claim 1 characterized in that the intracellular retention in the ER of TCRβ chains is induced by transfection of the pTα ^b isoform.