WO2009081254A1 - Cells belonging to the adaptive immune system that express paq isoform of tmem126b - Google Patents
Cells belonging to the adaptive immune system that express paq isoform of tmem126b Download PDFInfo
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- WO2009081254A1 WO2009081254A1 PCT/IB2008/003508 IB2008003508W WO2009081254A1 WO 2009081254 A1 WO2009081254 A1 WO 2009081254A1 IB 2008003508 W IB2008003508 W IB 2008003508W WO 2009081254 A1 WO2009081254 A1 WO 2009081254A1
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- Prior art keywords
- cells
- seq
- ligand
- immune system
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0635—B lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
Definitions
- the present invention relates to ex vivo cells belonging to the hematopoietic system, that present on their surface the proteins TMEM126B, paq isoform and uses of the same and ligands against the protein TMEM126B, paq isoform.
- the gene TMEM126B is known in the art thanks to its gene sequence.
- the gene for Homo sapiens is represented by the number GeneID 55863 according to the Entrez gene designation (http://www.ncbi.nlm.nih. gov/entrez) .
- the gene TMEM126B is present on the chromosome 11.
- the acronym of the protein tmem I26b/TMEM126B means transmembrane protein 126b.
- the expression of the gene TMEM126B has been described for specific kinds of cells, among these are B cells. By microarray type experiments, the expression of TMEM126B has been shown to be involved in different pathologies, among which the lung and breast cancers and the Alzheimer disease.
- TMEM126B exists in 4 iso- forms resulting from an alternative "splicing" of the transcript. Said isoforms are herein fully reported in the attached description according to the internation- al standard WIPO ST.25 and produced using the program _o _
- Patent-In 3. 3
- the Applicant has surprisingly found that the expression of one of the "splice" isoforms of the gene TMEM126B on cells belonging to the hematopoietic sys- tern responds to the requirements mentioned above.
- Figure 1 shows different kinds of cells belonging to the immune system and the percentage of expression of the protein SEQ ID. NO. 2 on their surface, as de- tected by flow cytometry (Fluorescent-activated cell sorting-FACS) (see Example 1 for the description of such method) .
- Figure 1 (a) shows the result, in a graphic form, of a FACS experiment on peripheral blood mononuclear cells (PBMC) where the quantity of cells expressing the protein CD3, identification marker of T cells, and that also express the protein SEQ ID NO. 2, is measured.
- Figure 1 (b) shows the result, in a graphic form, of a FACS experiment on peripheral blood mononuclear cells (PBMC) where the quantity of cells expressing the protein CD19, identification marker of B cells, and that also express the protein * SEQ ID NO. 2, is measured.
- PBMC peripheral blood mononuclear cells
- Figure l(c) shows the result, in a graphic form, of a FACS experiment on peripheral blood mononuclear cells (PBMC) where the quantity of cells expressing the protein CD56, identification marker of NK cells, and that also express the protein SEQ ID NO. 2, is measured.
- PBMC peripheral blood mononuclear cells
- the absence of a second peak on the axis of the ordi- nates permits to conclude that none of the NK cells expresses SEQ ID NO. 2.
- the slight fluorescence displacement with respect to the negative control can be explained by background noise being present due to the method of producing the antibodies ' for SEQ ID NO. 2 used in the FACS experiment.
- Figure 1 (d) shows the result, in a graphic form, of a FACS experiment on peripheral blood mononuclear cells (PBMC) where the quantity of cells expressing the protein CD14, a marker of monocytes, and that also ex- press the protein SEQ ID NO. 2, is measured.
- PBMC peripheral blood mononuclear cells
- the absence of a second peak on the axis of the ordinates permits to conclude that none of the NK cells expresses SEQ ID NO. 2.
- the slight fluorescence displacement with respect to the negative control can be explained by background noise being present due to the method of producing the antibodies for SEQ ID NO. 2 used in the FACS experiment.
- Figure 2 shows schematically the mRNA sequences corresponding to SEQ ID NO. 1-8, and the positions where primers bind (pairs of primers a, b, c, 314 and the single primer a bis) as used in the PCR experiment given in the Example 2.
- Figure 3 shows the results of a series of experiments, as given in the Example 2, with the different primers schematically represented in Figure 2.
- Figure 3 shows the absence of expression of the paq isoform (SEQ ID NO 2) in cells that express CD56 and CD14.
- Figure 4 shows the typical distribution of the paq isoform of TMEM126B (SEQ ID NO. 2) in lymphocyte cells, as investigated in the experiment given in Ex- ' ample 3.
- Figure. 4 (a) shows the selection of a population of lymphocytes existing in the peripheral blood obtained from an average of ten donors, wherein the presence of the isoform SEQ ID NO. 2 of TMEM126B on the total of lymphocyte cells, being represented on the basis of their side scatter physical parameter (SCC-A axis) , is shown.
- -figure 4 (d) shows that the population of T cells that expresses SEQ -JD NO. 2 is equally divided between helper T cells (CD4 + ) and cytotoxic T cells (CD8 + ) .
- Figure 5 shows the results of an experiment, as re- • ported in the Example 4, in which the stimulation effect of the lymphocyte cells (the results are the av- erage obtained from lymphocytes obtained from blood of two separated donors) on the number of cells that express SEQ ID NO. 2 is measured.
- ⁇ hematopoie- tic cells means nucleated cells existing in various tissues or districts, including the blood and lymphoid systems, or that result from said host and come from the dendogramatic lineage starting from the hematopoietic stem cell existing in the bone marrow up to mature cells, such as for example a mature leukocyte.
- state of activity of a cell is intended as the state in which a cell finds itself from those phases known in the art .(-S, Go, Gi, G2 and M) and/or the predisposition to start an apoptotic cell cascade and/or the ability of a cell to respond to stimuli and emit stimulating factors or compounds which modify or form part of a physiological response, such as for example cytokines that form part of an immune response.
- stimulating factors or compounds which modify or form part of a physiological response, such as for example cytokines that form part of an immune response.
- metabolic state of a cell is intended as the reactions that occur within a single cell and which are involved in the production of stimulating proteins/factors/compounds or in the expansion of the number of cells.
- stimulation of said metabolic state or said state of activity is intended as the process at a molecular or cellular level which increases the metabolic or activation state. Said stimulation can be started by a molecular stimulus. Therefore, the "stimulated” cells are cells wherein the activation or metabolic state is increased.
- immune response is intended as any kind of physiological re- sponse, namely a series of biochemical reactions, developed by the host following contact with and/or the presentation of an antigen to cells belonging to the immune system in the context of the present invention.
- immune system a combination of cells and chemical components, among which cytokines, which originate from the hematopoietic system" of a mammal is intended.
- Said cells and chemical components belonging to the immune system can belong to the innate or adaptive immune system.
- innate immune system is intended as a part of the immune system that forms the first defensive line against infections. It consists of elements of the organism pre- existing to the infection, it acts in a rapid manner but it cannot be “educated” and therefore when a same infection occurs again, it will respond in an identical manner.
- Cells which form the innate immune system are "the cells having a phagocytic activity, such as granulocytes, monocytes and macrophages and the cells having a natural cytotoxic activity (NK) .
- adaptive immune system is intended as a part of the immune system characterized by the ability of discriminating and “recognizing” in a specific way a very high number of different macromolecules (antigens) and the ability of "remembering” an antigen against which the system has previously responded. These features allow to educate the adaptive immune system and to obtain faster and more effective responses to a following reinfection with a pathogen.
- Cells that form the adaptive immunity are T cells and B cells.
- the expression of a protein "on the cell surface” is intended as the expression of a protein which crosses the membrane or is fixed in the membrane and which shows at least a part of its three-dimensional structure on the external surface of the cell membrane.
- the term “cells” comprises any maturation phases in which the cell finds itself, such as, for example, the term “B cells” comprises all the possible phases of a B cell from pro-B cells (CD34 + CD19 + CD20 ⁇ Ig ⁇ ) up to, for example, a plasma cell (CD38 + CD27 + CDl9 +/ ⁇ CD20 ⁇ HLA ⁇ DR ⁇ ) .
- Object of the present invention are ex vivo cells belonging to the hematopoietic system which present on their surface proteins SEQ ID NO. 2.
- Said cells according to the invention are preferably cells belonging to the immune system, still more pre- ferably belonging to the adaptive immune system.
- Said cells according to the invention are even more preferably selected from the group consisting of B cells and T cells.
- Said cells are preferably mature B and T cells which present markers CD19 and CD3, re- spectively, on the surface of said lymphocytes.
- the cells according to the invention preferably originate from a human being.
- Cells according to the invention can originate from any sources of hematopoietic cells known in the art, pre- ferably.
- T cells comprised ' in the cells according to the invention are preferably - from 5 to 20%, preferably from 10 to 17.5% and even more preferably from 14 to 15% of the total population of T cells.
- B cells comprised in the cells according to the invention are preferably - from 1 to 80%, preferably from 15 to 70% and even more preferably from 20 to 60% of the total population of B cells.
- Cells according to the invention are maintained alive ex vivo by using any system known in the art for main- taining and preserving in vitro hematopoietic cells.
- cells are resuspended in an isotonic nutritive medium containing salts, vitamins, co-factors and proteins (for example media as RPMI1640 or D-MEM) additioned with growth factors (for example, 10% of the volume of cultures of fetal bovine serum or normal human serum is used) .
- growth factors for example, 10% of the volume of cultures of fetal bovine serum or normal human serum is used
- A. further object of the present invention is a method for targeting and/or selecting hematopoietic cells ac- cording to the invention, preferably cells belonging to the immune system, still more preferably cells belonging to the adaptive immune system, from a ' group of ex vivo hematopoietic cells, which comprises at least a step in which the presence of the protein SEQ ID NO. 2 on the surface of the cells according to the invention is used.
- a ligand for the protein SEQ ID NO. 2 more preferably a proteinic ligand, such as for example an antibody or a lectin protein.
- a further object of the present invention is an ex vivo ligand specific to the protein SEQ ID NO. 2.
- said ligand is specific for the protein isoform SEQ ID NO. 2 and is not specific for TMEM126B, isoforms SEQ ID NO.- 4, 6 and 8; even more preferably only specific for the protein SEQ ID NO. 2.
- Said ligand is preferably a polyclonal or monoclonal antibody against the protein SEQ ID NO. 2.
- a monoclonal antibody against the . protein SEQ ID NO. 2 is preferred.
- the monoclonal antibody can be produced with methods known in the art, such as for example recombination methods or methods which use the Kohler and Midstein technology. .
- Said me- thod preferably comprises the following steps: i) immunizing an animal having a spleen, with. the protein SEQ ID NO. 2 so as to obtain an immune response, preferably in combination with an adjuvant; ii) removing the spleen from the animal and processing the same so as to obtain a suspension of intact cells and isolating the leukocytes therefrom, such as for example B cells; iii) forming a hybridoma, for example through fusion, from a leukocyte cell isolated from the suspension resulting in (ii) with an immortalized cell, such as for example cells from a myeloma lineage HGRP "/ ⁇ , iv) enriching the number of cells formed in (iii) with a suitable medium, such as for example a cell feeder layer; v) selecting, by a negative selection method, the cells which have formed a working hybridoma, such as for example by growing the cells formed in (iii) on a HAT medium
- SEQ ID NO. 2 through methods known in the art, such as for example using SEQ ID NO. 2 linked to a marker, for example a probe;
- Said method for selecting the cells according to the invention preferably comprises the following steps:
- Said ligands can be used in separation protocols known in the art, for example magnetic separation or other methods.
- the method for selecting the cells according to the invention or the specific cell subpopulations can include positive selection protocols and/or negative selection protocols known in the art.
- a preferred protocol to be used in the targeting and/or isolation of said subpopulation is a flow cytometry protocol which is able to isolate the cells ac- cording to the invention by discriminating between cells that express and cells that do not express SEQ ID NO. 2.
- a targeting and/or isolation protocol in which flow cytometry with fluo- rochromes is used (FACS ® from Beckton-Dickinson) , pre- ferably as a final step and/or following to an enrich- ment protocol, such as for example a protocol which includes the use of magnetic balls with specific antibodies linked thereon.
- said method can target and/or select cells according to the invention from a sample of cells, preferably from a sample of hematopoietic cells.
- Example 1 a detailed method for identifying cells belonging to the adaptive immune system that express the protein SEQ ID NO. 2 on their surface, starting from peripheral blood collected from an adult human being, is described only for exemplifying purposes and is in no way limiting.
- Another object of the present invention is the use of ex vivo cells belonging to the hematopoietic system that express SEQ ID NO. 2 on their surface as a medicament ⁇
- the cells according to the invention are used in the preparation of a medicament for restoring or improving the effectiveness of the hematopoietic system.
- the expression of SEQ ID NO. 2 in the cells of the immune system is specific to the adaptive immune system. Therefore, anoth- er object of the present invention is the use of the ex vivo cells belonging to the adaptive immune system that express SEQ ID NO. 2 on their surface as a medicament .
- the ex vivo cells belonging to the adaptive immune system are used in the preparation of a medicament for restoring or improving the effectiveness of the adaptive immune system in a patient.
- Said medicament can be preferably used in patients with a reduced immune system, such as HIV-positive patients, or patients which have received treatments wherein lymphocytes have been ablated, such as for example radiotherapy.
- said medicament further contains exci- pients and/or physiological buffer solutions and/or adjuvants.
- Said medicament can be administered enter- ally, preferably by an intra-arterial or intravenous injection or infusion.
- J the cells according to the invention is that the metabolic and/or physiologic state of the hematopoietic cells can be correlated with the percentage of hematopoietic cells that express SEQ ID NO. 2 on the surface of said cells.
- said metabolic and/or physiologic state of the hematopoietic cells is the metabolic state of the adaptive immune system, more preferably when activated through the known growth factor interleukin-2 (IL-2) .
- IL-2 growth factor interleukin-2
- the separation and/or identification of the cells according to the invention from a sample of ex vivo hematopoie- tic cells, preferably with the ligand as above described can be used in order to detect the metabolic and/or physiologic state of the hematopoietic system, preferably of the metabolic and/or physiologic state of the adaptive immune system.
- the cells according to the invention can be used ex vivo in diagnostic tests which comprise at least a step in which a specific antigen is presented to said cells and a step in which the immune response developed by said presentation of the antigen is measured.
- Said immune response from said diagnostic test provides information to the person skilled in the art concerning the immune state of the host from which the cells according to the invention derive.
- Said information concerning the immune state comprises information regarding to the antigenic memory existing in the lymphocytic cells and the probability of the adaptive immune system in developing an immune response to the specific antigen which has been presented in the diagnostic test.
- Said use for diag- nostic tests is especially useful when the specific antigen is a possible vaccine that one desires to examine .
- said use includes a method comprising the following steps: a) preparing an ex vivo sample of hematopoietic cells comprising B cells and/or T cells, b) determining the percentage of B cells and/or T cells existing in said sample that express SEQ ID NO. 2, c) presenting an antigen to said sample and then d) determining the difference in percentage of B cells and/or T cells that express SEQ ID NO. 2 with respect to the percentage determined in the step (b) . .
- the percentage difference of B cells and/or T cells that express SEQ ID NO. 2 between before and after the presentation of an antigen can be correlated to the development of the immune response consequent to the presentation of said antigen.
- the determination of said percentages can be carried out with methods known in the art, such as for example through the use of Ii- gands for SEQ ID NO. 2 and with FACS protocols as detailed above.
- Protocols to be used are elaborated according to the teachings of the person skilled in the art as a func- tion of the antigen presented, such as for example by measuring the immune response through the detection of specific cytokines produced by the cells.
- the antigen according to methods known in the art as a simple introduction of the antigen in a medium/solution containing the cells or introducing a autologous cell to the cells according to the invention (for example a macrophage) which has processed the antigen and presents the same on its surface in a MHC complex.
- Said use includes a method comprising the following steps : a) preparing an ex vivo sample of hematopoietic cells comprising B cells and/or T cells, b) determining the percentage of B cells and/or T cells existing in said sample that express SEQ ID NO. 2, c) introducing said active ingredient in said sample, then d) introducing IL-2 to said sample and then e) determining the percentage difference of B cells and/or T cells that express SEQ ID NO. 2 with respect to the percentage determined in the step (b) and then f) correlating said difference with the action of IL- 2.
- step (a- f) the protocol (steps (a- f) ) as above set forth is repeated, except for the step (c) and the control result is obtained; namely, the physiological one, without the introduction of said active ingredient.
- the effect of the active ingredient on the action of IL-2 is then determined by a comparison of the two results obtained.
- the advantage of said method is that the difference in the percentage of B cells and/or T cells that express SEQ ID NO. 2 is correlated to the action of IL-2. Resulting signals from the action of IL-2 are a central nexus for later cascades of a response of the adaptive immune system and therefore said diagnostic data can be also related to an immune response as already reported above.
- Another object of the invention is the ligand which binds to the protein SEQ ID NO. 2, as mentioned above.
- Said ligand can be prepared as described above.
- said ligand is in a pharmaceutical composition with excipients and/or adjuvants.
- the ligand according to the invention can be used as a medicament .
- said ligand can be used in the preparation of a medicament for stopping, preferably inhibiting, or slowing the metabolic and/or physiologic state of the hematopoietic cells, preferably of the immune system, still more preferably of the adaptive immune system.
- the medicament containing the ligand according to the invention is preferably administered parenterally, prefera- bly by injection and still more preferably by intravenous or intra-arterial administration. It is still more preferable if said medicament contains excipients or immunosuppressants.
- autoimmune etiology 2 can be used for treating or preventing pathologies considered of an autoimmune etiology, such as inflammations, diabetes, multiple sclerosis or pathologies in which one wants to eliminate the immunologic distinction between self and non-self, such as for example in pathologies as Graft vs Host Disease (GVHD) , or it can be used for preventing an attack from the adaptive immune system, such as for example in pregnant women.
- pathologies consi- dered of an autoimmune etiology are characterized by the presence of memory B cells which produce autoantibodies .
- said medicament containing the ligand specific to SEQ ID NO. 2 can be used for treating or preventing lymphoid leukemias, preferably those associated to the lineage of B cells, such as for example hairy cell leukemia.
- the ligand according to the invention is linked to a compound which damages the cell expressing SEQ ID NO. 2.
- Said damaging compound can be toxic or anyhow adapted for the purpose of eliminating the target of the ligand; namely, the immune cell expressing SEQ ID NO. 2 on its surface.
- said immune cell expressing SEQ ID NO. 2 on its surface is a memory B cell.
- said toxic substance are a toxin or a radioactive atom, such as for example iodine 131.
- the damaging compound can be an enzyme which can be successively involved in a monoclonal therapy system known in the art as ADEPT.
- said ligand is linked to a marker, such as for example ⁇ a secondary antibody associated with a probe, such as for example a fluorescent, phosphorescent or radioactive probe, con- 5 nected on the secondary antibody.
- Said ligand linked to a marker can be used for the preparation of a medicament in order to qualitatively or quantitatively evaluate the metabolic and/or physiologic status of cells belonging to the hematopoietic system, prefera-
- SEQ ID NO. 2 shows how the meta- ' bolic and/or physiologic active the cells according to the invention are.
- the distribution of SEQ ID NO. 2 on each cell shows how the metabolic and/or physiologic state of the cells according to the invention is ac-
- the in vivo position of the ligands which is related to the position of the cells according to the invention, shows sites in a body of greater affluence for -the cells of ' the hematopoietic system, preferably of the immune system, still more preferably of the
- Example 1 Isolation of subpopulations of cells according to the invention that express SEQ ID NO. 2 in peripheral blood
- the antiserum SEQ ID NO. 2 was made according to methods known in the art, by immunizing mice with the whole primary structure of TMEM126B. Samples for the negative control were incubated for 10 min in ice with a non immunized mouse antiserum, in order to set the negativity of the final staining of the imagine resulting from FACS. 4. Cells of the centrifuged samples were washed twice with PBS with 5% NHS, removing the supernatant after a 3 min centrifugation at 1500 rpm and resuspending with PBS 5% NHS. 5.
- the protein SEQ ID NO. 2 is clearly present on the surface of a percentage of T cells and B cells, but is not clearly present on the surface of other cells belonging to the immune system, such as for example NK cells (cells marked with CD56 and reported in fig. c of Figure 1) .
- the slight fluorescence displacement of fluorescence (the peak) relative to the negative can be ascribed to a background noise due to the production method of the antibodies for SEQ ID NO. 2 used in the FACS experiment.
- the slight fluorescence displacement with re- spect to the negative control can be explained by background noise being present due to to the production method of the antibodies for SEQ ID NO. 2 used in the FACS experiment.
- Figure (d) of Figure 1 shows the result, in a graphic form, of the quantity of cells that express the protein CD14, marker _ of monocytes, which also express the protein SEQ ID NO. 2.
- the absence of a second peak on the axis of ordinates permits the conclusion that none of the monocytes expresses SEQ ID NO. 2.
- the slight fluorescence displacement with respect to the negative control can be explained by background noise being present due to the production method of the antibodies for SEQ ID NO. 2 used in the FACS experiment.
- the slight fluorescence displacement with respect to the negative control can be explained by background noise being present due to the production method of the antibodies for SEQ ID NO. 2 used in the FACS experiment.
- Example 2 - The expression of proteins SEQ ID NO. 2 in cells belonging to the peripheral blood detected through PCR reactions
- each primer " has a precise position in correspondence with the gene TMEM126B and some primers can only cover certain isoforms.
- Cells purified using Ficoll, as described in the Exam- pie 1, were used for the enrichment of 4 groups of hematopoietic cells having the markers • CD3, CD14, CD19 or CD56 present on their surface. The enrichment was performed through specific antibodies conjugated to magnetic balls ' (Miltenyi Biotech, cat# 130-046-7.02) according to the supplier protocol.
- RNA was extracted through the Qiagen kit (cat# 74104), according to the supplier protocol and the cDNA was produced starting from 100 ng RNA, through the enzyme Retro- Script (Ambion, cat# 1710) according to the supplier protocol .
- the molecular weight of the different PCR products is evaluated using the DNA molecular weight marker XVI (250 bp ladder) by Roche Applied Science. Results are shown in Figure 3, where expression of the three isoforms pan, pap and pao, SEQ ID NO. 4, 6 and 8, is clearly seen in all the tested cells, whereas the isoform SEQ ID NO. 2, paq, is not present in the cells expressing CD56 (NK cells) or cells expressing CD14 (monocytes) .
- Example 3 Isolation and distribution of different populations of lymphocyte cells that express SEQ ID NO. 2 Some peripheral blood mononuclear cells were isolated, as described in the Example 1, from 10 independent do- nors .
- the cells obtained were stained with the antiserum against SEQ ID NO. 2 diluted 1:150 and with monoclonal antibodies directed against CD3, CD19 and CD56, as de- scribed in Example 1.
- m-anti-h CD27FITC a known monoclonal antibody with the fluo- rochrome fluorescein (FITC) linked thereon
- FITC fluo- rochrome fluorescein
- m-anti- hCD4APC-Cy7 a known monoclonal antibody with the flu- orochrome Allphycocyanin-Cyanin7 linked thereon
- m-anti-h CD8PE-Cy5 a known monoclonal antibody with the fluorochrome phycoerythrin-Cyanin ⁇ linked thereon.
- FIG. 4 shows an exemplifying and representative representation of the expression of the protein TMEM126B isoform paq (SEQ ID NO. 2) in the to- tal lymphocyte population of the 10 donors analyzed.
- the "gate" performed for analyzing how SEQ ID NO. 2 is distributed within the T, B and NK lymphocyte subpopulations .
- helper T cells CD4 +
- cytotoxic T cells CD8 +
- Example 4 Correlation between expression of SEQ ID NO. 2 on B cells and T cells and the metabolic state of the same lymphocytes .
- PBMCs Peripheral blood mononuclear cells
- FIG 5 (a) the effect of the stimuli above de- scribed on the expression of the protein SEQ ID NO. 2 in the subpopulation of B cells is graphically shown.
- Each bar represents the average of two independent donors and from the figure it results clearly that the stimulus with IL-2 alone is sufficient for inducing an increase of the expression of the protein TMEM126B isoform paq in the B cells.
- Such increase is slightly higher if the cells are stimulated by IL-2 in the presence of the mitogen PHA, whereas the stimulus with PHA alone is not able to induce an increase of the ex- pression of the protein TMEM126B isoform paq.
- FIG 5 (b) the effect of the stimuli above described on the expression of the protein SEQ ID NO. 2 in the subpopulation of the T cells is graphically- shown.
- Each bar represents the average of two indepen- dent donors and from the figure it clearly results that the stimulus with IL-2 alone is sufficient for inducing an increase of the expression of the protein TMEM126B isoform paq in the T cells.
- Such increase is higher if the cells are stimulated by IL-2 in the presence of the mitogen PHA, whereas the stimulus with PHA alone is not able to induce an increase of the expression of the protein TMEM126B isoform paq.
- Gl y Thr Thr Al a Gl y Phe Ser Gl y lie Phe ser Asn Phe Leu Phe Arg 50 55 60
- Phe Gly lie Leu Asn Gly Leu Tyr His Tyr Ala VaI phe GIU GIU Thr 180 185 190
- Gin Thr Gin Met Lys Leu Met Ala lie Pro Leu VaI phe Gin lie Met 165 170 175
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Abstract
The present invention relates to ex vivo cells belonging to the hematopoietic system, that present on their surface, proteins having sequences TMEM126B, paq isoform, and uses of the same and ligands against the protein TMEM126B, paq isoform.
Description
"CELLS BELONGING TO THE ADAPTIVE IMMUNE SYSTEM THAT EXPRESS PAQ ISOFORM OF TMEMl26B"
The present invention relates to ex vivo cells belonging to the hematopoietic system, that present on their surface the proteins TMEM126B, paq isoform and uses of the same and ligands against the protein TMEM126B, paq isoform.
The gene TMEM126B is known in the art thanks to its gene sequence. The gene for Homo sapiens is represented by the number GeneID 55863 according to the Entrez gene designation (http://www.ncbi.nlm.nih. gov/entrez) . The gene TMEM126B is present on the chromosome 11. The acronym of the protein tmem I26b/TMEM126B means transmembrane protein 126b. The expression of the gene TMEM126B has been described for specific kinds of cells, among these are B cells. By microarray type experiments, the expression of TMEM126B has been shown to be involved in different pathologies, among which the lung and breast cancers and the Alzheimer disease.
It is also known the protein TMEM126B exists in 4 iso- forms resulting from an alternative "splicing" of the transcript. Said isoforms are herein fully reported in the attached description according to the internation- al standard WIPO ST.25 and produced using the program
_o _
Patent-In 3. 3 .
There is a need in the art to improve the procedures of isolation and recognition of specific cells belonging to the hematopoietic system, preferably the immune system.
Furthermore, there is a need of being able to differentiate between cells belonging to the adaptive immune system and the innate immune system. There is also the need of improving the use of cells belonging to the adaptive immune system and the innate immune systems in the applications within the therapeutic/diagnostic/prognostic field.
Finally, there is the need in the art of being able to define the metabolic and/or physiologic state of a cell belonging to the hematopoietic system, preferably the immune system.
The Applicant has surprisingly found that the expression of one of the "splice" isoforms of the gene TMEM126B on cells belonging to the hematopoietic sys- tern responds to the requirements mentioned above.
The present invention is further illustrated hereinafter by the use of the attached figures .
Figure 1 shows different kinds of cells belonging to the immune system and the percentage of expression of the protein SEQ ID. NO. 2 on their surface, as de-
tected by flow cytometry (Fluorescent-activated cell sorting-FACS) (see Example 1 for the description of such method) .
Figure 1 (a) shows the result, in a graphic form, of a FACS experiment on peripheral blood mononuclear cells (PBMC) where the quantity of cells expressing the protein CD3, identification marker of T cells, and that also express the protein SEQ ID NO. 2, is measured. Figure 1 (b) shows the result, in a graphic form, of a FACS experiment on peripheral blood mononuclear cells (PBMC) where the quantity of cells expressing the protein CD19, identification marker of B cells, and that also express the protein *SEQ ID NO. 2, is measured. Figure l(c) shows the result, in a graphic form, of a FACS experiment on peripheral blood mononuclear cells (PBMC) where the quantity of cells expressing the protein CD56, identification marker of NK cells, and that also express the protein SEQ ID NO. 2, is measured. The absence of a second peak on the axis of the ordi- nates permits to conclude that none of the NK cells expresses SEQ ID NO. 2. The slight fluorescence displacement with respect to the negative control can be explained by background noise being present due to the method of producing the antibodies 'for SEQ ID NO. 2 used in the FACS experiment.
Figure 1 (d) shows the result, in a graphic form, of a FACS experiment on peripheral blood mononuclear cells (PBMC) where the quantity of cells expressing the protein CD14, a marker of monocytes, and that also ex- press the protein SEQ ID NO. 2, is measured. The absence of a second peak on the axis of the ordinates permits to conclude that none of the NK cells expresses SEQ ID NO. 2. The slight fluorescence displacement with respect to the negative control can be explained by background noise being present due to the method of producing the antibodies for SEQ ID NO. 2 used in the FACS experiment.
Figure 2 shows schematically the mRNA sequences corresponding to SEQ ID NO. 1-8, and the positions where primers bind (pairs of primers a, b, c, 314 and the single primer a bis) as used in the PCR experiment given in the Example 2.
Figure 3 shows the results of a series of experiments, as given in the Example 2, with the different primers schematically represented in Figure 2. Figure 3 shows the absence of expression of the paq isoform (SEQ ID NO 2) in cells that express CD56 and CD14. Figure 4 shows the typical distribution of the paq isoform of TMEM126B (SEQ ID NO. 2) in lymphocyte cells, as investigated in the experiment given in Ex- '
ample 3. Figure. 4 (a) shows the selection of a population of lymphocytes existing in the peripheral blood obtained from an average of ten donors, wherein the presence of the isoform SEQ ID NO. 2 of TMEM126B on the total of lymphocyte cells, being represented on the basis of their side scatter physical parameter (SCC-A axis) , is shown. When isolated and investigated for lineage "marker" (CD56, CD19 and CD3) , the distribution of the lymphocyte population that expresses SEQ ID NO. 2, as schematized in figure 4 (b) , in cells having a natural cytotoxic activity (NK cells) is so little to be considered as negligible, being a value due to practical inaccuracies of the experiment. Figure 4 (b) also shows that 17% of the lymphocyte cells ex- pressing SEQ ID NO.' 2 are T cells (CD3+) and 81% are B cells (CD19+) . Figure 4 (c) shows that the majority of B cells that express SEQ ID NO. 2 (between 70% and'
80%) are memory B cells, whereas -figure 4 (d) shows that the population of T cells that expresses SEQ -JD NO. 2 is equally divided between helper T cells (CD4+) and cytotoxic T cells (CD8+) .
Figure 5 shows the results of an experiment, as re- •ported in the Example 4, in which the stimulation effect of the lymphocyte cells (the results are the av- erage obtained from lymphocytes obtained from blood of
two separated donors) on the number of cells that express SEQ ID NO. 2 is measured.
In Figure 5 (a), one sees that the percentage of B cells without stimulation that express SEQ ID NO. 2 is 35%. After they have been stimulated with phytohemaggluti- nin (PHA) together with interleukin-2 (IL-2) , the percentage of B cells rises to 85%. The stimulation with IL-2 alone, a known growth factor capable of metaboli- cally stimulating both B and T cells, increases the percentage to 75%, whereas with PHA alone, known not to be a B cell activator, the percentage is indeed shown to decrease and not rise.
In figure 5 (b) one sees that the percentage of T cells without ^stimulation that express SEQ ID NO. 2 is 2%. After having been stimulated with phytohemagglutinin (PHA) together with interleukin-2 (IL-2), the percentage of T cells rises to 23%. The stimulation with IL- 2 alone, a known growth factor capable of metabolical- Iy stimulating both T and B cells, increases the per- centage to 14%, whereas with PHA alone, a known activator of T cells only (and not B cells) , it is seen that the percentage increases but it is of an experimentally negligible value (the difference is 1%) . In the context of the present invention, Λλhematopoie- tic cells" means nucleated cells existing in various
tissues or districts, including the blood and lymphoid systems, or that result from said host and come from the dendogramatic lineage starting from the hematopoietic stem cell existing in the bone marrow up to mature cells, such as for example a mature leukocyte. In the context of the present invention, "state of activity" of a cell is intended as the state in which a cell finds itself from those phases known in the art .(-S, Go, Gi, G2 and M) and/or the predisposition to start an apoptotic cell cascade and/or the ability of a cell to respond to stimuli and emit stimulating factors or compounds which modify or form part of a physiological response, such as for example cytokines that form part of an immune response. In the context of the present invention, "metabolic state" of a cell is intended as the reactions that occur within a single cell and which are involved in the production of stimulating proteins/factors/compounds or in the expansion of the number of cells. In the context of the present invention, "stimulation" of said metabolic state or said state of activity is intended as the process at a molecular or cellular level which increases the metabolic or activation state. Said stimulation can be started by a molecular stimulus.
Therefore, the "stimulated" cells are cells wherein the activation or metabolic state is increased. In the context of the present invention, "immune response" is intended as any kind of physiological re- sponse, namely a series of biochemical reactions, developed by the host following contact with and/or the presentation of an antigen to cells belonging to the immune system in the context of the present invention. In the context of the present invention, by "immune system", a combination of cells and chemical components, among which cytokines, which originate from the hematopoietic system" of a mammal is intended. Said cells and chemical components belonging to the immune system can belong to the innate or adaptive immune system.
In the context of the present invention, "innate immune system" is intended as a part of the immune system that forms the first defensive line against infections. It consists of elements of the organism pre- existing to the infection, it acts in a rapid manner but it cannot be "educated" and therefore when a same infection occurs again, it will respond in an identical manner. Cells which form the innate immune system are "the cells having a phagocytic activity, such as granulocytes, monocytes and macrophages and the cells
having a natural cytotoxic activity (NK) . In the context of the present invention, "adaptive immune system" is intended as a part of the immune system characterized by the ability of discriminating and "recognizing" in a specific way a very high number of different macromolecules (antigens) and the ability of "remembering" an antigen against which the system has previously responded. These features allow to educate the adaptive immune system and to obtain faster and more effective responses to a following reinfection with a pathogen. Cells that form the adaptive immunity are T cells and B cells.
Said components of the immune system, and their responses, are well known in the art. It is also well known that the different components of the immune system interact one with the other to result in • a complete immune system.
In the context of the present invention, the expression of a protein "on the cell surface" is intended as the expression of a protein which crosses the membrane or is fixed in the membrane and which shows at least a part of its three-dimensional structure on the external surface of the cell membrane. In the context -of the present invention, the term "cells" comprises any maturation phases in which the
cell finds itself, such as, for example, the term "B cells" comprises all the possible phases of a B cell from pro-B cells (CD34+CD19+CD20~Ig~) up to, for example, a plasma cell (CD38+CD27+CDl9+/~CD20~HLA~DR~) . Object of the present invention are ex vivo cells belonging to the hematopoietic system which present on their surface proteins SEQ ID NO. 2.
Said cells according to the invention are preferably cells belonging to the immune system, still more pre- ferably belonging to the adaptive immune system.
Said cells according to the invention are even more preferably selected from the group consisting of B cells and T cells. Said cells are preferably mature B and T cells which present markers CD19 and CD3, re- spectively, on the surface of said lymphocytes.
Still more preferably, the cells according to the invention preferably originate from a human being. Cells according to the invention can originate from any sources of hematopoietic cells known in the art, pre- ferably. a source including cells belonging to the adaptive immune system and preferably in vivo. Said source is preferably the peripheral blood. T cells comprised 'in the cells according to the invention are preferably - from 5 to 20%, preferably from 10 to 17.5% and even
more preferably from 14 to 15% of the total population of T cells.
B cells comprised in the cells according to the invention are preferably - from 1 to 80%, preferably from 15 to 70% and even more preferably from 20 to 60% of the total population of B cells.
Cells according to the invention are maintained alive ex vivo by using any system known in the art for main- taining and preserving in vitro hematopoietic cells. In a preferred embodiment, after a separation step using FICOLL, cells are resuspended in an isotonic nutritive medium containing salts, vitamins, co-factors and proteins (for example media as RPMI1640 or D-MEM) additioned with growth factors (for example, 10% of the volume of cultures of fetal bovine serum or normal human serum is used) . When resuspended .in such culturemedium, cells result viable and in good conditions for a period of many hours (up to 24 hours) . The advantage of said culture medium is that the cells can also be subjected to stimuli of a different kind (for example the treatment with the mitogen PHA-L) and the behavior thereof can be monitored for many days. A. further object of the present invention is a method for targeting and/or selecting hematopoietic cells ac-
cording to the invention, preferably cells belonging to the immune system, still more preferably cells belonging to the adaptive immune system, from a ' group of ex vivo hematopoietic cells, which comprises at least a step in which the presence of the protein SEQ ID NO. 2 on the surface of the cells according to the invention is used. In said method for targeting and/or selecting the cells according to the invention, it is preferable to use a ligand for the protein SEQ ID NO. 2, more preferably a proteinic ligand, such as for example an antibody or a lectin protein.
Therefore, a further object of the present invention is an ex vivo ligand specific to the protein SEQ ID NO. 2. Preferably, said ligand is specific for the protein isoform SEQ ID NO. 2 and is not specific for TMEM126B, isoforms SEQ ID NO.- 4, 6 and 8; even more preferably only specific for the protein SEQ ID NO. 2. Said ligand is preferably a polyclonal or monoclonal antibody against the protein SEQ ID NO. 2. Among said ligands, a monoclonal antibody against the . protein SEQ ID NO. 2 is preferred. The monoclonal antibody can be produced with methods known in the art, such as for example recombination methods or methods which use the Kohler and Midstein technology.. Said me-
thod preferably comprises the following steps: i) immunizing an animal having a spleen, with. the protein SEQ ID NO. 2 so as to obtain an immune response, preferably in combination with an adjuvant; ii) removing the spleen from the animal and processing the same so as to obtain a suspension of intact cells and isolating the leukocytes therefrom, such as for example B cells; iii) forming a hybridoma, for example through fusion, from a leukocyte cell isolated from the suspension resulting in (ii) with an immortalized cell, such as for example cells from a myeloma lineage HGRP"/~, iv) enriching the number of cells formed in (iii) with a suitable medium, such as for example a cell feeder layer; v) selecting, by a negative selection method, the cells which have formed a working hybridoma, such as for example by growing the cells formed in (iii) on a HAT medium if a myeloma HGRP"7" cell is used; vi) isolating cells which produce antibodies against
SEQ ID NO. 2 through methods known in the art, such as for example using SEQ ID NO. 2 linked to a marker, for example a probe;
- vii) isolating and multiplying the selected cells for ■ producing the monoclonal antibodies against' SEQ ID. NO.
2 .
Said method for selecting the cells according to the invention preferably comprises the following steps:
- preparing an ex vivo sample of cells comprising he- matopoietic cells,
- determining the presence of SEQ ID NO. 2 on the cell surface of said sample of cells with a ligand for SEQ ID NO. 2 and
- isolating cells on which SEQ ID NO. 2 is present from the sample.
Said ligands can be used in separation protocols known in the art, for example magnetic separation or other methods. The method for selecting the cells according to the invention or the specific cell subpopulations can include positive selection protocols and/or negative selection protocols known in the art. A preferred protocol to be used in the targeting and/or isolation of said subpopulation is a flow cytometry protocol which is able to isolate the cells ac- cording to the invention by discriminating between cells that express and cells that do not express SEQ ID NO. 2. Even more preferred is a targeting and/or isolation protocol in which flow cytometry with fluo- rochromes is used (FACS® from Beckton-Dickinson) , pre- ferably as a final step and/or following to an enrich-
ment protocol, such as for example a protocol which includes the use of magnetic balls with specific antibodies linked thereon.
In a preferred embodiment, said method can target and/or select cells according to the invention from a sample of cells, preferably from a sample of hematopoietic cells.
In Example 1, a detailed method for identifying cells belonging to the adaptive immune system that express the protein SEQ ID NO. 2 on their surface, starting from peripheral blood collected from an adult human being, is described only for exemplifying purposes and is in no way limiting. Another object of the present invention is the use of ex vivo cells belonging to the hematopoietic system that express SEQ ID NO. 2 on their surface as a medicament ■
In a preferred embodiment of the use as a medicament, the cells according to the invention are used in the preparation of a medicament for restoring or improving the effectiveness of the hematopoietic system. In another aspect of the invention, the expression of SEQ ID NO. 2 in the cells of the immune system is specific to the adaptive immune system. Therefore, anoth- er object of the present invention is the use of the
ex vivo cells belonging to the adaptive immune system that express SEQ ID NO. 2 on their surface as a medicament .
In a preferred embodiment of the use as a medicament, the ex vivo cells belonging to the adaptive immune system are used in the preparation of a medicament for restoring or improving the effectiveness of the adaptive immune system in a patient. Said medicament can be preferably used in patients with a reduced immune system, such as HIV-positive patients, or patients which have received treatments wherein lymphocytes have been ablated, such as for example radiotherapy. Preferably, said medicament further contains exci- pients and/or physiological buffer solutions and/or adjuvants. Said medicament can be administered enter- ally, preferably by an intra-arterial or intravenous injection or infusion.
An advantage of Jthe cells according to the invention is that the metabolic and/or physiologic state of the hematopoietic cells can be correlated with the percentage of hematopoietic cells that express SEQ ID NO. 2 on the surface of said cells. Preferably, said metabolic and/or physiologic state of the hematopoietic cells is the metabolic state of the adaptive immune system, more preferably when activated through the
known growth factor interleukin-2 (IL-2) . Therefore, in an embodiment of said invention, the separation and/or identification of the cells according to the invention from a sample of ex vivo hematopoie- tic cells, preferably with the ligand as above described, can be used in order to detect the metabolic and/or physiologic state of the hematopoietic system, preferably of the metabolic and/or physiologic state of the adaptive immune system. In another embodiment, the cells according to the invention can be used ex vivo in diagnostic tests which comprise at least a step in which a specific antigen is presented to said cells and a step in which the immune response developed by said presentation of the antigen is measured. Said immune response from said diagnostic test provides information to the person skilled in the art concerning the immune state of the host from which the cells according to the invention derive. Said information concerning the immune state comprises information regarding to the antigenic memory existing in the lymphocytic cells and the probability of the adaptive immune system in developing an immune response to the specific antigen which has been presented in the diagnostic test. Said use for diag- nostic tests is especially useful when the specific
antigen is a possible vaccine that one desires to examine .
Preferably, said use includes a method comprising the following steps: a) preparing an ex vivo sample of hematopoietic cells comprising B cells and/or T cells, b) determining the percentage of B cells and/or T cells existing in said sample that express SEQ ID NO. 2, c) presenting an antigen to said sample and then d) determining the difference in percentage of B cells and/or T cells that express SEQ ID NO. 2 with respect to the percentage determined in the step (b) . . The percentage difference of B cells and/or T cells that express SEQ ID NO. 2 between before and after the presentation of an antigen can be correlated to the development of the immune response consequent to the presentation of said antigen. The determination of said percentages can be carried out with methods known in the art, such as for example through the use of Ii- gands for SEQ ID NO. 2 and with FACS protocols as detailed above.
Protocols to be used are elaborated according to the teachings of the person skilled in the art as a func- tion of the antigen presented, such as for example by
measuring the immune response through the detection of specific cytokines produced by the cells. Alternatively, it is possible to present the antigen according to methods known in the art as a simple introduction of the antigen in a medium/solution containing the cells or introducing a autologous cell to the cells according to the invention (for example a macrophage) which has processed the antigen and presents the same on its surface in a MHC complex. In another preferred embodiment, the presence of SEQ ID NO. 2 can be used on a sample of cells which comprises B cells and/or T cells for determining the inhibitory or synergic effect of an active ingredient on the action of IL-2. Preferably, the effect of said ac- tive ingredient as an antagonist of the action of IL-2 can be determined. Said use includes a method comprising the following steps : a) preparing an ex vivo sample of hematopoietic cells comprising B cells and/or T cells, b) determining the percentage of B cells and/or T cells existing in said sample that express SEQ ID NO. 2, c) introducing said active ingredient in said sample, then d) introducing IL-2 to said sample and then
e) determining the percentage difference of B cells and/or T cells that express SEQ ID NO. 2 with respect to the percentage determined in the step (b) and then f) correlating said difference with the action of IL- 2.
In order to determine the inhibitory or synergic effect of the active ingredient, the protocol (steps (a- f) ) as above set forth is repeated, except for the step (c) and the control result is obtained; namely, the physiological one, without the introduction of said active ingredient. The effect of the active ingredient on the action of IL-2 is then determined by a comparison of the two results obtained. The advantage of said method is that the difference in the percentage of B cells and/or T cells that express SEQ ID NO. 2 is correlated to the action of IL-2. Resulting signals from the action of IL-2 are a central nexus for later cascades of a response of the adaptive immune system and therefore said diagnostic data can be also related to an immune response as already reported above. The determination of said percentages • can be carried out with methods known in the art, such as for example through the use of ligands for SEQ ID NO. 2 and with FACS protocols, as set forth above." Another object of the invention is the ligand which
binds to the protein SEQ ID NO. 2, as mentioned above. Said ligand can be prepared as described above. In a preferred embodiment, said ligand is in a pharmaceutical composition with excipients and/or adjuvants. The ligand according to the invention can be used as a medicament .
In another preferred embodiment, said ligand can be used in the preparation of a medicament for stopping, preferably inhibiting, or slowing the metabolic and/or physiologic state of the hematopoietic cells, preferably of the immune system, still more preferably of the adaptive immune system. In said embodiment, the medicament containing the ligand according to the invention is preferably administered parenterally, prefera- bly by injection and still more preferably by intravenous or intra-arterial administration. It is still more preferable if said medicament contains excipients or immunosuppressants. Said medicament containing the specific -ligand for SEQ ID NO. 2 can be used for treating or preventing pathologies considered of an autoimmune etiology, such as inflammations, diabetes, multiple sclerosis or pathologies in which one wants to eliminate the immunologic distinction between self and non-self, such as for example in pathologies as Graft vs Host Disease
(GVHD) , or it can be used for preventing an attack from the adaptive immune system, such as for example in pregnant women. Preferably, in said use of the Ii- gand as just above described, the pathologies consi- dered of an autoimmune etiology are characterized by the presence of memory B cells which produce autoantibodies .
In another preferred embodiment, said medicament containing the ligand specific to SEQ ID NO. 2 can be used for treating or preventing lymphoid leukemias, preferably those associated to the lineage of B cells, such as for example hairy cell leukemia. In a preferred embodiment of said use, the ligand according to the invention is linked to a compound which damages the cell expressing SEQ ID NO. 2. Said damaging compound can be toxic or anyhow adapted for the purpose of eliminating the target of the ligand; namely, the immune cell expressing SEQ ID NO. 2 on its surface. Preferably, said immune cell expressing SEQ ID NO. 2 on its surface is a memory B cell. Examples of said toxic substance are a toxin or a radioactive atom, such as for example iodine 131. Alternatively, the damaging compound can be an enzyme which can be successively involved in a monoclonal therapy system known in the art as ADEPT.
In another preferred embodiment, said ligand is linked to a marker, such as for example ■ a secondary antibody associated with a probe, such as for example a fluorescent, phosphorescent or radioactive probe, con- 5 nected on the secondary antibody. Said ligand linked to a marker can be used for the preparation of a medicament in order to qualitatively or quantitatively evaluate the metabolic and/or physiologic status of cells belonging to the hematopoietic system, prefera-
10 bly to the immune system, still more preferably to the adaptive immune system, as already described above for ex vivo cells. Said evaluation of the metabolic and/or physiologic state of the cells according to the invention can be performed ex vivo or in vivo. The number
15 of cells that express SEQ ID NO. 2 shows how the meta- ' bolic and/or physiologic active the cells according to the invention are. The distribution of SEQ ID NO. 2 on each cell shows how the metabolic and/or physiologic state of the cells according to the invention is ac-
20 tive. The in vivo position of the ligands, which is related to the position of the cells according to the invention, shows sites in a body of greater affluence for -the cells of ' the hematopoietic system, preferably of the immune system, still more preferably of the
25. adaptive immune system.
Example 1 - Isolation of subpopulations of cells according to the invention that express SEQ ID NO. 2 in peripheral blood
Isolation of mononuclear cells from the peripheral blood
1. A 10 ml sample of peripheral blood of a healthy donor was diluted 1:50 in a phosphate-buffered saline
(PBS) .
2. 15 ml of Ficoll-Hypaque (density 1.077 g/1) was in- troduced in a 50 ml Falcon and then 30 ml of peripheral blood of a healthy donor was layered thereon. The ■ blood was dropped very slowly in order to not disturb the interface. The operation was repeated until all the sample was used up. 3. The Falcon was then centrifuged at 1600 rpm for 30 min at room temperature, without brake. Mononuclear cells (PBMC) positioned themselves at the interface between Ficoll-Hypaque and plasma. Said PBMC ring was collected and transferred in a 50 ml Falcon. 4. PBMCs were washed twice with 50 ml PBS containing 5% normal human serum. (NHS) by centrifuging for 10 min at 1200 rpm. •
5. The pellet was then washed with 50 ml PBS 5% NHS by centrifuging for 10 min at 800 rpm. 6. PBMCs resulting in a pellet at the end of the step
5 were then resuspended in 10-30 ml PBS 5% NHS at room temperature .
Isolation of cells according to the invention from PBMC 1. Cells were counted with a Burker chamber and 5xlO5- IxIO6 PBMC per sample were stained.
2. Samples were incubated for 20 min at room temperature with PBS 50% NHS.
3. Samples were centrifuged for 3 min at 1500 rpm and, without washing, were incubated for 10 min in an ice
■ bath with the antiserum against SEQ ID NO. 2 diluted 1:50 and 1:150 in 100 microlitres of PBS with 5% NHS. The antiserum SEQ ID NO. 2 was made according to methods known in the art, by immunizing mice with the whole primary structure of TMEM126B. Samples for the negative control were incubated for 10 min in ice with a non immunized mouse antiserum, in order to set the negativity of the final staining of the imagine resulting from FACS. 4. Cells of the centrifuged samples were washed twice with PBS with 5% NHS, removing the supernatant after a 3 min centrifugation at 1500 rpm and resuspending with PBS 5% NHS. 5. Said resuspended cells were then again incubated for 10 min in an ice bath with Goat-anti-mouse IgG-PE
( Southern Biotech®) , a known "secondary" antibody with the fluorochrome phycoerythrin (PE) linked thereon, diluted 1:100 in 100 microlitres PBS with 5% NHS.
6. Cells were then washed twice with PBS with 5% NHS, by centrifuging for 3 min at 1500 rprα, and resuspend- ing with PBS with 5% NHS.
7. To the resuspended pellet 12 micrograms per sample of mlgG (mouse immunoglobulin) were added and incubated for at least 60 min in ice. 8. Cells were incubated for 10 min in an ice bath with m-anti-hCD19Cychrome (BD Biosciences®) , a known monoclonal antibody with the fluorochrome PE-Cy5 linked thereon and with mouse-anti-hCD3FITC (BD Biosciences®) , a known monoclonal antibody with the fluo- rochrome fluoresceine (FITC) linked thereon and with mouse-anti-hCD56APC (BD Biosciences®) , a known monoclonal antibody with the fluorochrome Allophycocyanin linked thereon.
9. Finally, stained cells were washed (by centrifuging at 1500 rpm for 3 min) with PBS 10% NHS and resuspended in 500 microlitres for FACSCalibur® acquisition.
10. The BecktonDickinson-FACS® machine was operated according to the protocols known in the art and de- scribed in Current Protocols in Immunology (2001) ,
John Wiley and Sons Inc., Units 5.4.1-5.4.22, to give the obtained results which are shown in Figure 1, figs. a-d.
From the results, one can see that the protein SEQ ID NO. 2 is clearly present on the surface of a percentage of T cells and B cells, but is not clearly present on the surface of other cells belonging to the immune system, such as for example NK cells (cells marked with CD56 and reported in fig. c of Figure 1) . The slight fluorescence displacement of fluorescence (the peak) relative to the negative can be ascribed to a background noise due to the production method of the antibodies for SEQ ID NO. 2 used in the FACS experiment. The slight fluorescence displacement with re- spect to the negative control can be explained by background noise being present due to to the production method of the antibodies for SEQ ID NO. 2 used in the FACS experiment. Figure (d) of Figure 1 shows the result, in a graphic form, of the quantity of cells that express the protein CD14, marker _ of monocytes, which also express the protein SEQ ID NO. 2. The absence of a second peak on the axis of ordinates permits the conclusion that none of the monocytes expresses SEQ ID NO. 2. The slight fluorescence displacement with respect to the negative
control can be explained by background noise being present due to the production method of the antibodies for SEQ ID NO. 2 used in the FACS experiment. The slight fluorescence displacement with respect to the negative control can be explained by background noise being present due to the production method of the antibodies for SEQ ID NO. 2 used in the FACS experiment. Example 2 - The expression of proteins SEQ ID NO. 2 in cells belonging to the peripheral blood detected through PCR reactions
For the purpose of demonstrating the presence of SEQ ID NO. 2 on the surface of cells belonging to the adaptive immune system, the expression of the different isoforms was shown through PCR amplification expe- riments with specific primers.
As it is seen from Figure 2, each primer" has a precise position in correspondence with the gene TMEM126B and some primers can only cover certain isoforms. Cells purified using Ficoll, as described in the Exam- pie 1, were used for the enrichment of 4 groups of hematopoietic cells having the markers • CD3, CD14, CD19 or CD56 present on their surface. The enrichment was performed through specific antibodies conjugated to magnetic balls' (Miltenyi Biotech, cat# 130-046-7.02) according to the supplier protocol.
From the cells obtained after the enrichment, RNA was extracted through the Qiagen kit (cat# 74104), according to the supplier protocol and the cDNA was produced starting from 100 ng RNA, through the enzyme Retro- Script (Ambion, cat# 1710) according to the supplier protocol .
2 μl of cDNA were used for a RT-PCR analysis, with the primers represented in Figure 2. Primers used were the following: a fw: " SEQ ID NO. 9 a rev: SEQ ID NO. 10 a bis fw: SEQ ID NO. 11 b fw: SEQ ID NO. 12 b rev: SEQ ID NO. 13 c fw: SEQ ID NO. 14 c rev: SEQ ID NO. 15
314 fw: SEQ ID NO. 16
314 rev: SEQ ID NO. 17
Sequences were entirely reported in the attached de- scription according to the international standard WIPO ST.25 and developed with the program Patent-In 3.3. Conditions used for RT-PCR with the specific primers for the different isoforms of the protein TMEM126B were the following: cDNA: 2 microlitres
fw primer (10 micromolar) : 1 microlitre rev primer (10 micromolar) : 1 microlitre 2X Taq PCR Master Mix (Qiagen, cat# 201443) : 25 micro- litres Sterile water: enough to reach a final volume of 50 microlitres .
Conditions of the thermal cycles of the PCR: 94°C, 3 min 30 cycles of 94°C/30 sec, 55°C/30 sec and 72°C/30 sec. 72°C, 10 min. oo, 4°C
The molecular weight of the different PCR products is evaluated using the DNA molecular weight marker XVI (250 bp ladder) by Roche Applied Science. Results are shown in Figure 3, where expression of the three isoforms pan, pap and pao, SEQ ID NO. 4, 6 and 8, is clearly seen in all the tested cells, whereas the isoform SEQ ID NO. 2, paq, is not present in the cells expressing CD56 (NK cells) or cells expressing CD14 (monocytes) .
Example 3 - Isolation and distribution of different populations of lymphocyte cells that express SEQ ID NO. 2 Some peripheral blood mononuclear cells were isolated, as described in the Example 1, from 10 independent do-
nors .
The cells obtained were stained with the antiserum against SEQ ID NO. 2 diluted 1:150 and with monoclonal antibodies directed against CD3, CD19 and CD56, as de- scribed in Example 1.
To such antibodies already described in the Example 1, the following antibodies were added: m-anti-h CD27FITC, a known monoclonal antibody with the fluo- rochrome fluorescein (FITC) linked thereon, m-anti- hCD4APC-Cy7, a known monoclonal antibody with the flu- orochrome Allphycocyanin-Cyanin7 linked thereon, and m-anti-h CD8PE-Cy5, a known monoclonal antibody with the fluorochrome phycoerythrin-Cyaninδ linked thereon. Finally, stained cells were washed (by centrifuging at 1500 rpm for 3 min) with PBS 10% NHS and resuspended in 500 microlitre"s for the FACSCanto® acquisition. The BecktonDickinson-FACS® machine was operated according to the protocols known in the art and mentioned in Current Protocols in Immunology (2001) , John Wiley and Sons Inc., Units 5.4.1-5.4.22 to give the obtained results which are shown in Figure 4, figs, a- d.
Figure (a) of figure 4 shows an exemplifying and representative representation of the expression of the protein TMEM126B isoform paq (SEQ ID NO. 2) in the to-
tal lymphocyte population of the 10 donors analyzed. In the figure, there is shown the "gate" performed for analyzing how SEQ ID NO. 2 is distributed within the T, B and NK lymphocyte subpopulations . In figure 4 (b) , the average of the distribution of the protein SEQ ID NO. 2 on T, B and NK cells, as measured in the 10 analyzed donors, is represented in a bar graph form. From figure 4 (b) it is deduced that the protein SEQ ID NO. 2 is mainly expressed on B cells, much less on T cells and the expression on NK cells is so little to be considered as negligible, as a value due to practical inaccuracies of the experiment. In figure 4 (c) , the average of the distribution of the protein SEQ ID NO. 2 on the subpopulations of "virgin" B cells (CD27") and memory B cells (CD27+) , as measured in the 10 analyzed donors, is represented in form of a bar graph. From the' figure it is deduced that the protein TMEM126B isoform paq is mainly expressed on memory B cells. In figure 4 (d) , the average of the distribution of the protein SEQ ID NO. 2 on the subpopulations of helper T cells (CD4+) and cytotoxic T cells (CD8+) , as measured in the 10 analyzed donors, is represented in form of a bar graph. From the figure one sees that the protein TMEM126B isoform paq is present in an equivalent way
on helper T cells and cytotoxic T cells.
Example 4 — Correlation between expression of SEQ ID NO. 2 on B cells and T cells and the metabolic state of the same lymphocytes . Peripheral blood mononuclear cells (PBMCs) , isolated using Ficoll as described in the example 1, are plated in U-bottomed 9β-well plates (2xlO5 cells/ well) and stimulated under the following conditions :
- 1 μg/ml PHA (PHA-L, Roche) in the presence ■ of 100 U/ml IL-2 (recombinant human IL-2, Chiron)
- 100 U/ml IL-2 (recombinant human IL-2, Chiron)
- 1 μg/ml PHA (PHA-L, Roche)
- no stimuli (negative control)
2. Cells are incubated in the presence of the stimuli for 48 hours.
3. Cells are collected, stained and analyzed as described at points 2-17 of example 1.
The results obtained are shown in Figure 5.
In figure 5 (a) , the effect of the stimuli above de- scribed on the expression of the protein SEQ ID NO. 2 in the subpopulation of B cells is graphically shown. Each bar represents the average of two independent donors and from the figure it results clearly that the stimulus with IL-2 alone is sufficient for inducing an increase of the expression of the protein TMEM126B
isoform paq in the B cells. Such increase is slightly higher if the cells are stimulated by IL-2 in the presence of the mitogen PHA, whereas the stimulus with PHA alone is not able to induce an increase of the ex- pression of the protein TMEM126B isoform paq.
In figure 5 (b) , the effect of the stimuli above described on the expression of the protein SEQ ID NO. 2 in the subpopulation of the T cells is graphically- shown. Each bar represents the average of two indepen- dent donors and from the figure it clearly results that the stimulus with IL-2 alone is sufficient for inducing an increase of the expression of the protein TMEM126B isoform paq in the T cells. Such increase is higher if the cells are stimulated by IL-2 in the presence of the mitogen PHA, whereas the stimulus with PHA alone is not able to induce an increase of the expression of the protein TMEM126B isoform paq.
SEQUENCE LISTING
<110> ISTITUTO NAZIONALE DI GENETICA MOLECOLARE - INGM <120> CELLS BELONGING TO THE ADAPTIVE IMMUNE SYSTEM THAT EXPRESS ISOFORM PAQ
TMEM126B
<130> 21.10197.12. IT.5 <160> 17 <170> Patentln version 3.3
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<220>
<223> cDHA of pap
<400> 7 tcgggtatga ggctgggact aagccaaggg attcaggtgt ggtgccggtg ggaactgagg 60 aagcgcccaa ggttttcaag atggcagcat ctatgcatgg tcagcccagt ccttctctag 120 aagatgcaaa actcagaaga ccaatggtca tagaaatcat agaaaaaaat tttgactatc 180 ttagaaaaga aatgacacaa aatatatatc aaatggcgac atttggaaca acagctggtt 240 tctctggaat attctcaaac ttcctgttca gacgctgctt caaggttaaa catgatgctt 300 tgaagacata tgcatcattg gctacacttc catttttgtc tactgttgtt actgacaagc 360 tttttgtaat tgatgctttg tattcagata atataagcaa ggaaaactgt gttttcagaa 420 gctcactgat tggcatagtt tgtggtgttt tctatcccag ttctttggct tttactaaaa 480 atggacgcct ggcaaccaag tatcataccg ttccactgcc accaaaagga agggttttaa 540 tccattggat gacgctttgt caaacacaaa tgaaattaat ggcgattcct ctagtctttc 600 agattatgtt tggaatatta aatggtctat accattatgc agtatttgaa gagacacttg 660 agaaaactat acatgaagag taaccaaaaa aatgaatggt tgctaactta gcaaaatgaa 720 gtttctataa agaggactca ggcattgctg aaagagttaa aagtaactgt gaacaaataa 780 tttgttctgt gccttttgcc tggtatatag caaatactca aaaagtattc aataattcaa 840 tcaataaata taagtttcat cttacacgta agatacaggt cttatctcct gatggtgtgt 900 ccattttgcc tggtatataa cagataataa atatccagtg tcaataa 947
<210> 8
<211> 200
<212> PRT
<213> Homo sapiens
<400> 8
Met Ala Ala ser Met His Gl y Gin Pro ser Pro ser Leu Gl u Asp Ala 1 5 10 15
Lys Leu Arg Arg Pro Met VaI lie Gl u lie lie Gl u Lys AΞΠ phe Asp 20 25 30
Tyr Leu Arg Lys Gl ϋ Met Thr Gin Asn lie Tyr Gin Met Ala Thr Phe 35 40 45
Gly Thr Thr Ala Gly Phe Ser Gly lie phe Ser Asn Phe Leu Phe Arg 50 55 60
Arg cys Phe Lys val Lys His Asp Ala Leu Lys Thr Tyr Ala ser Leu 65 70 75 80
Ala Thr Leu pro Phe Leu ser Thr VaI VaI Thr Asp Lys Leu Phe VaI 85 90 . 95 lie Asp Ala Leu Tyr ser Asp Asn lie ser Lys Gl u Asn cys VaI Phe 100 105 110
Arg ser ser Leu lie Gly lie VaI cys Gly VaI Phe Tyr Pro ser ser 115 ■ 120 125
Leu Ala phe Thr Lys Asn Gly Arg Leu Ala Thr Lys Tyr His Thr VaI 130 135 140
Pro Leu Pro Pro Lys Gly Arg VaI Leu lie His Trp Met Thr Leu Cys 145 150 155 160
Gin Thr Gin Met Lys Leu Met Ala lie Pro Leu VaI phe Gin lie Met 165 170 175
Phe Gly lie Leu Asn Gly Leu Tyr His Tyr Ala VaI phe Gl u Gl u Thr 180 185 190
Leu Glu Lys Thr lie His Glu Glu 195 200
<210> 9
<211> 19
<212> DNA
<213> Artificial
<220>
<223> Forward primer a
<400> 9 ggaagcgccc aaggaaatg 19
<210> 10
<211> 19
<212> DNA
<213> Artifi ci al
<220>
<223> Reverse primer a
<400> 10 gcagcgtctg aacaggaag 19
<210> 11
<211> 19
<212> DNA
<213> Artificial
<220>
<223> Forward pri mer a bi s
<400> 11 aggctgggac taagccaag 1"
<210> 12
<211> 21
<212> DNA
<213> Arti fi ci al
<220>
<223> Forward primer b
<400> 12 • ,., ttcctgttca gacgctgctt c 21
<210> 13
<211> 22
<212> DNA
<213> Arti fi ci al
<220>
<223> Reverse primer b
<400> 13 gcctcagttt gggttgctta tc 22
<210> 14
<211> 18
<212> DNA
<213> Artificial
<220>
<223> Forward pri mer c
<400> 14 tcagacgctg cttcaagg 18
<210> 15
<211> 18
<212> DNA
<213> Artificial
<220>
<223> Reverse primer c
<400> 15 tggcagtgga acggtatg 18
<210> 16
<211> 18
<212> DNA
<213> Artificial
<22O>
<223> Forward primer 314
<400> 16 ggcgacattt ggaacaac 18
<210> 17
<211> 18
<212> DNA
<213> Artificial
<22O>
<223> Reverse primer 314
<400> 17 tttggtggca gtggaacg 18
Claims
1. Ex vivo cells belonging to the hematopoietic system that present proteins SEQ ID NO. 2 on their surface .
2. The cells according to claim 1, wherein the hematopoietic cells are cells belonging to the immune system.
3. The cells according to claim 2, wherein the cells belonging to the immune system are cells belonging to the adaptive immune system.
4. The cells according to claim 3, wherein the cells are B cells and/or T cells.
5. The cells according to claim 4, wherein the B cells present CD19.
6. The cells according to claim 4, wherein the T cells present CD3.
7. A method for selecting the cells according to any one of the claims 1-6, said method comprising the fol¬ lowing steps: - preparing an ex vivo sample of cells comprising he¬ matopoietic cells,
- determining the presence of SEQ ID NO. 2 on the sur¬ face of the cells of said sample with a ligand for SEQ ID NO. 2 and - isolating from the sample the cells on which SEQ ID NO. 2 is present.
8. The method according to claim 7, wherein the sample of ex vivo cells consists of cells belonging to the immune system.
9. The cells according to any one of the claims 1 to 6 for use as a medicament.
10. The cells according to any one of the claims 1 to 6 for use as a medicament for restoring or improving the hematopoietic system, preferably the immune system, of a patient in need thereof.
11. The cells according to claim 10, wherein the immune system is the adaptive immune system.
12. The cells according to claim 11, wherein the adaptive immune system is of HIV-positive patients or patients who have had chemotherapy.
13. Use of the cells according to any one of the claims 1 to 6 for determining the metabolic and/or physiologic state of a sample of ex vivo cells, preferably the state of the adaptive immune system.
14. A method for determining the development of an immune response to an antigen which comprises the fol¬ lowing steps: a) preparing an ex vivo sample of hematopoietic cells comprising B cells and/or T cells, b) determining the percentage of B cells and/or T cells present in said sample that express SEQ ID NO. 2 , c) presenting an antigen to said sample and then d) determining the percentage difference of B cells and/or T cells that express SEQ ID NO. 2 relative to the percentage determined in the step (b) , e) correlating said difference to the development of the immune response.
15. A method for determining the inhibitory or synergic effect of an active ingredient on the action of IL-2 which comprises the following steps: a) preparing an ex vivo sample of hematopoietic cells comprising B cells and/or T cells, b) determining the percentage of B cells and/or T cells existing in said sample that express SEQ ID NO. 2, c) introducing said active ingredient in said sample, then d) introducing IL-2 in said sample, then e) determining the percentage difference of B cells and/or T cells that express SEQ ID NO. 2 with respect to the percentage determined in the step (b) and then f) correlating said difference with the action of IL-2 g) repeating steps (a)-(f) without the step (c) to ob¬ tain a control result h) comparing the result obtained in step (f) with that obtained in step (g) so as to obtain the inhibitory or synergic effect of the active ingredient.
16. The method according to claim 15, wherein the inhibitory effect of the active ingredient to be deter- mined is as an antagonist of the action of IL-2.
17. A ligand against the protein SEQ ID NO. 2.
18. The ligand according to claim 17, wherein the ligand does not bind to the proteins of SEQ ID NO. 4 or 6 or 8.
19. The ligand according to claim 17 or 18, wherein the ligand is an antibody.
20. The ligand according to claim 19, wherein the antibody is monoclonal.
21. The ligand according to any one of the claims 17 to 20, wherein the ligand is bound to a marker.
22. The ligand according to any one of the claims 17 to 21 for use as a medicament.
23. The ligand according to any one of the claims 17 to 21 for use as a medicament for identifying and/or selecting and/or isolating the cells belonging to the adaptive immune system.
24. The ligand according to any one of the claims 17 to 20 for use as a medicament for inhibiting an adap¬ tive immune response.
25. The ligand according to claim 24, wherein the adaptive immune response is involved in an autoimmune pathology.
26. The ligand according to claim 25, wherein the autoimmune pathology is due to the presence of memory B cells.
27. The ligand according to claim 24, wherein the adaptive immune response is a lymphoid leukemia, preferably a lymphoid leukemia associated with the B cells lineage.
28. The ligand according to any one of the claims 24 to 27, wherein the ligand is bound to a damaging compound, preferably a toxic compound.
29. The ligand according to claim 21 for the preparation of a medicament to qualitatively or quantitative- Iy evaluate the metabolic and/or physiologic state of cells belonging to the hematopoietic system.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ITMI2007A002367 | 2007-12-18 | ||
IT002367A ITMI20072367A1 (en) | 2007-12-18 | 2007-12-18 | CELLS BELONGING TO THE HEMATOPOIETIC SYSTEM EXPRESSES ISOFORMA PAQ OF TMEM126B |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009081254A1 true WO2009081254A1 (en) | 2009-07-02 |
Family
ID=40315604
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IB2008/003508 WO2009081254A1 (en) | 2007-12-18 | 2008-12-16 | Cells belonging to the adaptive immune system that express paq isoform of tmem126b |
Country Status (2)
Country | Link |
---|---|
IT (1) | ITMI20072367A1 (en) |
WO (1) | WO2009081254A1 (en) |
-
2007
- 2007-12-18 IT IT002367A patent/ITMI20072367A1/en unknown
-
2008
- 2008-12-16 WO PCT/IB2008/003508 patent/WO2009081254A1/en active Application Filing
Non-Patent Citations (2)
Title |
---|
KEELING J H ET AL: "In vitro analysis of lymphocytes: Markers and functions", JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY, C.V. MOSBY, ST. LOUIS, MO, US, vol. 8, no. 2, 1 February 1983 (1983-02-01), pages 239 - 251, XP023323794, ISSN: 0190-9622, [retrieved on 19830201] * |
TAYLOR T D ET AL: "Human chromosome 11 DNA sequence and analysis including novel gene identification", NATURE 20060323 NATURE PUBLISHING GROUP GB, vol. 440, no. 7083, 23 March 2006 (2006-03-23), pages 497 - 500, XP002523558 * |
Also Published As
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ITMI20072367A1 (en) | 2009-06-19 |
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