CN114377133A - Targeting renal CD8+Application of TRM formation and activation in glomerular injury - Google Patents

Targeting renal CD8+Application of TRM formation and activation in glomerular injury Download PDF

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CN114377133A
CN114377133A CN202210066616.3A CN202210066616A CN114377133A CN 114377133 A CN114377133 A CN 114377133A CN 202210066616 A CN202210066616 A CN 202210066616A CN 114377133 A CN114377133 A CN 114377133A
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renal
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易凡
李亮
唐伟
贾蒙
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Shandong University
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Abstract

The present invention provides targeting renal CD8+TRMThe application of formation and activation in glomerular injury belongs to the technical field of biological medicine and molecular biology. The invention discovers that T exists in kidney through researchRMCD8 in mouse glomerular injury disease model and in humans+Is not CD4+TRMThe proportional amount of (c) increases. In pathological conditions, the kidney CD8+TRMFrom peripheral blood infiltration. Wherein the increase in IL-15 levels in the renal cortex promotes renal CD8+TRMThe formation and activation of which in turn aggravate glomerulosclerosis and podocyte injury. Use of blocking antibodies to IL-15 and its receptor CD122 for the IL-15 signaling pathway significantly improved glomerulosclerosis symptoms in a mouse model and thus had good resultsThe value of practical application.

Description

Targeting renal CD8+TRMUse of formation and activation in glomerular injury
Technical Field
The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to targeted kidney CD8+TRMThe use of formation and activation in glomerular injury.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Podocytes, i.e., epithelial cells of the glomerular visceral layer, together with the glomerular endothelial cells and the glomerular basement membrane, form the glomerular filtration barrier. Podocytes are highly differentiated epithelial cells that attach to the outside of the basement membrane. Since the podocyte membrane is the last barrier for plasma protein filtration and the pore size is the smallest in the filtration barrier, it is the most vulnerable site in the glomerular structure.
Although there is strong evidence that podocyte injury is a key mediator of the pathogenesis of glomerular diseases such as Focal Segmental Glomerulosclerosis (FSGS), Minimal Change Disease (MCD), Diabetic Nephropathy (DN), and Lupus Nephritis (LN), effective treatment of podocytes remains a significant challenge. However, the inventors found that no effective drug for treating or preventing glomerulosclerosis and podocyte injury exists at present, and only symptomatic treatment is available, and improvement of renal function is limited.
Disclosure of Invention
In response to the deficiencies of the prior art, it is an object of the present invention to provide a targeting of renal CD8+TRMThe use of formation and activation in glomerular injury. The invention discovers that T exists in kidney through researchRMCD8 in mouse glomerular injury disease model and in humans+Is not CD4+TRMThe proportional amount of (c) increases. In pathological conditions, the kidney CD8+TRMFrom peripheral blood infiltration. Further research shows that the increase of IL-15 level of renal cortex promotes renal CD8+TRMThe formation and activation of which in turn aggravate glomerulosclerosis and podocyte injury. While the use of blocking antibodies to IL-15 and its receptor CD122 for the IL-15 signaling pathway significantly improved the symptoms of glomerulosclerosis in a mouse model. The present invention has been completed based on the above results.
In a first aspect of the invention, a target is providedTo inhibit renal CD8+TRMThe use of the formed and activated substance of (a) in at least one of a1) -a5) as follows:
a1) alleviating symptoms of proteinuria and/or preparing a product that alleviates symptoms of proteinuria;
a2) inhibiting mesangial proliferation and/or preparing a product for inhibiting mesangial proliferation;
a3) reducing podocyte damage and/or preparing a product for reducing podocyte damage;
a4) reducing glomerulosclerosis and/or preparing a product for reducing glomerulosclerosis;
a5) treating diseases related to glomerular injury and/or preparing products for treating diseases related to glomerular injury.
In a second aspect of the invention, a composition is provided, the active ingredients of which at least comprise a targeted kidney CD8 inhibition+TRMThe formation and activation of (a).
The targeting inhibits renal CD8+TRMThe substance for formation and activation of (a) may be an IL-15 signal blocker; the IL-15 signal blocker can be a small molecule compound or an antibody, preferably an antibody, and in one embodiment of the invention, the IL-15 signal blocker can be an IL-15 signal blocking antibody, including Anti-IL-15/CD 122.
In a third aspect of the invention, there is provided the use of a composition as described above in any one or more of:
a1) alleviating symptoms of proteinuria and/or preparing a product that alleviates symptoms of proteinuria;
a2) inhibiting mesangial proliferation and/or preparing a product for inhibiting mesangial proliferation;
a3) reducing podocyte damage and/or preparing a product for reducing podocyte damage;
a4) reducing glomerulosclerosis and/or preparing a product for reducing glomerulosclerosis;
a5) treating diseases related to glomerular injury and/or preparing products for treating diseases related to glomerular injury.
In a fourth aspect of the invention, there is provided a method of treating a disease associated with glomerular injury, the method comprising administering to a subject a therapeutically effective amount of an IL-15 signal blocker or a composition as described above.
It is noted that the treatment of the diseases associated with glomerular injury includes at least improvement of glomerular sclerosis and podocyte injury.
The beneficial technical effects of one or more technical schemes are as follows:
1. the technical proposal utilizes an animal model to scientifically evaluate and prove the exact improvement and treatment effect of the IL-15 signal blocking antibody (Anti-IL-15/CD122) on the chronic glomerular disease on the biochemical function and the histiocyte level.
2. The Anti-IL-15/CD122 related to the technical scheme has no obvious influence on relevant pathological indexes of the normal control group mice, and has good safety.
3. The technical proposal utilizes the evaluation of Transwell experimental science and proves that the IL-15 signal blocking antibody (Anti-IL-15/CD122) improves the activated CD8+TRMCausing damage to podocytes, thus having good practical application value.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a diagram of the results of mass spectrometry flow detection of the immune microenvironment of the kidney region at physiological levels of human and mouse in the example of the present invention.
Wherein, FIG. 1A shows mass spectrometric detection of CD45 of kidney of normal human+Dimension reduction map of constituent members of cells.
FIG. 1B is a mass spectrometric detection of kidney CD45 in normal mice+Dimension reduction map of constituent members of cells.
FIG. 1C shows normal human and mouse kidney CD45+Thermographic analysis of cells.
FIG. 1D is a dimension reduction chart of normal human kidney CD3, CD69, CD45RA, CCR 7.
FIG. 1E is a graph of the dimension reduction analysis of normal mouse kidney CD3, CD69, CD 44.
FIG. 2 shows an example of flow cytometry for detecting CD69 in kidney+Memory T cells and CD69-Chemotaxis and activity of memory T cells are compared to a statistical map.
Wherein, FIG. 2A shows the detection of renal CD69 by flow cytometry+Memory T cells and CD69-Statistical profiles of expression of CXCR3 and CD62L for memory T cells.
FIG. 2B is a statistical graph of the flow cytometry analysis of kidney IFN-. gamma.and perforin expression.
FIG. 3 is a graph of the kidney CD8 in the doxorubicin nephropathy and db/db mouse model in accordance with an embodiment of the present invention+TRMAnd (4) a proportional activity result chart.
Wherein, FIG. 3A shows the mouse kidney CD8 of doxorubicin nephropathy detected by flow cytometry+TRMSchematic and statistical diagrams of (a).
FIG. 3B shows mouse kidney CD4 for detecting adriamycin nephropathy by flow cytometry+TRMA statistical map of (2).
FIG. 3C is a graph showing flow cytometry detection of db/db mouse kidney CD8+TRMSchematic and statistical diagrams of (a).
FIG. 3D is a schematic representation of flow cytometry for CD4 detection+TRMA statistical map of (2).
FIG. 3E shows mouse kidney CD8 for detecting doxorubicin nephropathy by flow cytometry+TRMStatistical profiles of IFN-. gamma.expression and Granzyme B expression.
FIG. 3F is a graph showing flow cytometry detection of db/db mouse kidney CD8+TRMStatistical plots of IFN-. gamma.expression and Perforin.
FIG. 4 shows the statistics of periglomerular CD8 by staining pathological sections of kidney of FSGS and DKD patients in the present example+,CD4+TRMFigure of quantitative results.
Wherein FIG. 4A is kidney CD8 of normal human, FSGS, DN patient+TRMAnd (5) cellular immunofluorescence staining result graph.
FIG. 4B shows kidney CD4 of normal human, FSGS, DN patient+TRMAnd (4) an immunofluorescence staining result graph.
FIG. 5 shows doxorubicin nephropathy in an embodiment of the inventionDemonstration of Kidney CD8 in pathological conditions after mice use FTY720+TRMResults of peripheral infiltration rather than self-proliferation are shown.
Wherein FIG. 5A is a schematic representation of the administration of FTY720 to doxorubicin nephropathy model mice.
FIG. 5B is a flow cytometry assay for renal CD8+TRMPattern and statistical plots of Ki-67 expression.
FIG. 6 shows the nude mice back transfusion CD8 of adriamycin nephropathy in the embodiment of the invention+CD69-CD44+Detection of renal CD8 post-T cell+Phenotype results of T cells.
Wherein FIG. 6A shows the reinfusion of CD8 into nude mice with doxorubicin nephropathy+CD69-CD44+Pattern of T cells.
FIG. 6B is a flow chart of detecting kidney CD8+Statistical plots of the proportion of CD69 expressed by T cells.
FIG. 6C shows the flow detection of kidney CD8+Statistical plots of the proportion of CXCR3 expressed by T cells.
FIG. 7 is a graph showing the results of significantly elevated levels of renal cortical IL-15 in the mouse doxorubicin nephropathy model in accordance with an embodiment of the present invention.
Among them, FIG. 7A is a statistical graph of the levels of renal cortical IL-15 in Elisa test con group and ADR group mice.
FIG. 7B is a statistical chart of the correlation analysis between IL-15 and UACR in renal cortex of mice with Adriamycin nephropathy.
FIG. 8 is a graph showing that IL-15 and not IL-2 in the present example can induce CD8 in vitro+TRMThe formation and activation results are shown.
Wherein, FIG. 8A shows that spleen CD8 is detected by flow cytometry+CD44+CD69 of cells-To CD69+Transformation ratio statistical chart.
FIG. 8B is a flow cytometry assay for spleen CD8+CD69+CD44+Cell expression IFN-gamma ratio statistical chart.
FIG. 9 shows that Anti-CD122 blocking antibody inhibits IL-15 induced CD8 in vitro in the present example+TRMThe formation and activation results are shown.
WhereinFIG. 9A shows CD122 on kidney CD8+TRMSchematic representation of the expression of (a).
FIG. 9B shows that Anti-CD122 blocking antibody inhibits CD8+TRMA statistical map of formation and activation.
FIG. 10 shows that Anti-IL-15/CD122 antibody can be used to inhibit the kidney CD8 of Adriamycin nephropathy mice in the present invention+TRMThe formation and activation of the compounds and the improvement of glomerulosclerosis and podocyte injury.
Wherein, FIG. 10A shows the reinfusion of CD8 into nude mice with doxorubicin nephropathy+CD69-CD44+Pattern of T cells, concurrently with Anti-IL-15/CD122 antibody treatment.
FIG. 10B is a flow cytometry assay of mouse kidney CD8+TRMStatistical map of cells.
FIG. 10C is a flow cytometry analysis of mouse kidney CD8+TRMStatistical plots of cell expression of IFN- γ.
Figure 10D is a statistical plot of the ratio of urine creatinine to urea nitrogen (UACR) for mouse urine.
FIG. 10E is a schematic representation of PAS staining and transmission electron microscopy of pathological sections of mouse kidney.
Fig. 10F is a schematic illustration of mouse kidney pathological section nephrin, podocin immunostaining.
FIG. 11 is a graph showing the results of significant increases in renal cortical IL-15 levels in db/db mice in accordance with the present invention.
Wherein, FIG. 11A is a statistical chart of the renal cortex IL-15 levels of db/db mice detected by flow cytometry.
FIG. 11B is a statistical chart of the correlation analysis between IL-15 and UACR in the renal cortex of db/db mice.
FIG. 12 is a graph showing the effect of Anti-IL-15/CD122 antibody on the inhibition of kidney CD8 in db/db mice according to an embodiment of the present invention+TRMThe formation and activation of the compounds and the improvement of glomerulosclerosis and podocyte injury.
Among them, FIG. 12A is a graph of the pattern of db/db mice given Anti-IL-15/CD122 treatment.
FIG. 12B is a flow cytometry method for detecting mouse kidney CD8+TRMStatistical map of cells.
FIG. 12C is a flow cytometry assay of mouse kidney CD8+TRMStatistical plots of cell expression of IFN- γ.
Figure 12D is a mouse UACR statistical plot.
FIG. 12E is a representation of PAS staining and transmission electron microscopy of pathological sections of mouse kidney.
Fig. 12F is a schematic illustration of mouse kidney pathological section nephrin, podocin immunostaining.
FIG. 13 is a graph showing the significant reduction of activated renal CD8 using Anti-IL-15/CD122 antibody in vitro in accordance with an embodiment of the present invention+TRMThe result of the induced podocyte injury is shown.
Fig. 13A is a schematic diagram and a statistical diagram of the detection of mouse podocyte apoptosis ratio by flow cytometry.
FIG. 13B is a qPCR analysis of mouse podocyte podocin expression statistics.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Interpretation of terms:
focal Segmental Glomerulosclerosis (FSGS) is a nephrotic syndrome that frequently occurs in children and adolescents and is also a major cause of renal failure in adults.
Diabetic Nephropathy (DN) renal disease caused by diabetes belongs to the most common microvascular complications of diabetes. At present, the disease is considered to be related to various factors and is attacked under the combined action of certain genetic background and risk factors. It can cause proteinuria, edema, hypertension, etc., and severe cases can cause renal failure, endangering life.
As described above, there is no drug effective for the treatment or prevention of glomerulosclerosis and podocyte injury at present, and only symptomatic treatment is available, and improvement of renal function is limited.
Tissue-colonizing memory T cells (T)RM) Is a new type of memory T cell discovered in recent years. T in contrast to circulating T cellsRMIs absent from peripheral blood and is significantly different from circulating T cell populations in phenotype, function and metabolism. After antigen-stimulated T cell differentiation into effector T cells, they migrate into many non-lymphoid organs and become established by differentiating in situ into memory T cells. The response occurs rapidly when the same antigenic stimulus is encountered in the tissue. T isRMPlays an important role in pathogen infection, tumor immunity and autoimmune diseases. Recent studies have shown that podocyte damage is associated with the disruption of immune homeostasis. Various immune cells, especially T lymphocytes, are involved in the development of chronic kidney disease. Based on the above background, the inventors believe that the kidney TRMPossibly participating in podocyte injury, and simultaneously providing theoretical basis for searching potential therapeutic targets of the glomerular diseases.
Through a series of experimental researches, the inventor obtains the following research results:
specifically, the mass flow analysis technology proves that the proportion of the T lymphocytes is larger no matter the kidney of a normal human or a mouse. Co-existing with CD69+Is marked TRMA cell.
The chemotaxis and activity characteristics of mouse kidney non-resident memory T cells and resident memory T cells are compared by flow cytometry.
The invention constructs a mouse model of adriamycin nephropathy and db/db (30 weeks) type II diabetes, and discovers the kidney CD8 of the model group through flow cytometry detection+Is not CD4+TRMThe ratio of (a) to (b) is increased,the activity is enhanced.
The present invention finds CD8 by FSGS and DN patient kidney tissue section staining+Is not CD4+TRMIncreased around the glomeruli.
The invention proves that the CD8 of the kidney under pathological conditions+TRMDerived from peripheral blood and not proliferated by itself.
The invention finds that the IL-15 level secreted by the renal cortex of the model group is increased by detecting the IL-15 level of the renal cortex tissue of the doxorubicin nephropathy and the db/db mouse model. IL-15 promotes CD8 at the same time+TRMDifferentiation and activation of (3).
According to the invention, the Anti-IL-15/CD122 antibody of the IL-15 signal path impedance body is used, and two antibodies are detected to obviously improve the proteinuria symptoms, glomerulosclerosis and podocyte injury of an adriamycin nephropathy model and a db/db model.
The invention constructs a Transwell simulated renal microenvironment and finds that only activated CD8 is present+TRMObviously promotes the damage of podocyte. Meanwhile, an IL-15 signal blocking antibody Anti-IL-15/CD122 is used, and the use of the two antibodies is detected to obviously improve the damage of the podocytes.
Based on the above findings, in one exemplary embodiment of the present invention, targeted inhibition of renal CD8 is provided+TRMThe use of the formed and activated substance of (a) in at least one of a1) -a5) as follows:
a1) alleviating symptoms of proteinuria and/or preparing a product that alleviates symptoms of proteinuria;
a2) inhibiting mesangial proliferation and/or preparing a product for inhibiting mesangial proliferation;
a3) reducing podocyte damage and/or preparing a product for reducing podocyte damage;
a4) reducing glomerulosclerosis and/or preparing a product for reducing glomerulosclerosis;
a5) treating diseases related to glomerular injury and/or preparing products for treating diseases related to glomerular injury.
In the present invention, the diseases associated with glomerular injury include glomerulosclerosis and podocyte injury.
In yet another embodiment of the invention, the targeted inhibition of renal CD8+TRMThe substance for forming and activating comprises an IL-15 signal blocker, the IL-15 signal blocker can be a small molecule compound or an antibody, preferably an antibody, and in one embodiment of the invention, the IL-15 signal blocker can be an IL-15 signal blocking antibody, including Anti-IL-15/CD 122.
In still another embodiment of the present invention, in a1) -a5), the product can be a drug or a reagent for basic research, for example, in the present application, basic research can be performed on relevant disease mechanisms and the like by constructing doxorubicin nephropathy model mouse and db/db mouse model and applying IL-15/CD122 blocking antibody thereto, observing physiological and pathological changes thereof.
In yet another embodiment of the present invention, a composition is provided, the active ingredients of which comprise at least a targeted renal CD8 inhibition+TRMThe formation and activation of (a).
In yet another embodiment of the invention, the targeted inhibition of renal CD8+TRMThe substance for formation and activation of (a) may be an IL-15 signal blocker; the IL-15 signal blocker can be a small molecule compound or an antibody, preferably an antibody, and in one embodiment of the invention, the IL-15 signal blocker can be an IL-15 signal blocking antibody, including Anti-IL-15/CD 122.
In a further embodiment of the invention, there is provided the use of the above composition in any one or more of:
a1) alleviating symptoms of proteinuria and/or preparing a product that alleviates symptoms of proteinuria;
a2) inhibiting mesangial proliferation and/or preparing a product for inhibiting mesangial proliferation;
a3) reducing podocyte damage and/or preparing a product for reducing podocyte damage;
a4) reducing glomerulosclerosis and/or preparing a product for reducing glomerulosclerosis;
a5) treating diseases related to glomerular injury and/or preparing products for treating diseases related to glomerular injury.
In the present invention, the diseases associated with glomerular injury include glomerulosclerosis and podocyte injury.
The product can be a drug or an experimental reagent, and the experiment can be actually used for basic research.
According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.
Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.
In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.
In yet another embodiment of the present invention, the medicament of the present invention may be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
In still another embodiment of the present invention, the subject to which the medicament is administered may be a human or non-human mammal, such as a mouse, rat, guinea pig, rabbit, dog, monkey, orangutan, or the like.
In yet another embodiment of the present invention, there is provided a method for treating a disease associated with glomerular injury, the method comprising administering to a subject a therapeutically effective amount of an IL-15 signaling blocker or the above composition.
It is noted that the treatment of the diseases associated with glomerular injury includes at least improvement of glomerular sclerosis and podocyte injury.
As mentioned above, the IL-15 signal blocker may be a small molecule compound or an antibody, preferably an antibody, and in one embodiment of the present invention, the IL-15 signal blocker may be an IL-15 signal blocking antibody, including Anti-IL-15/CD 122.
The subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It will be appreciated that the optimum dosage and interval for administration of the active ingredients of the invention will be determined by the nature and external conditions, such as the form, route and site of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a nominal period of time, may be determined by methods known in the art.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out under conventional conditions.
Examples
Firstly, experimental steps
1. Analysis of immune microenvironment composition in renal tissue region
1.1 Kidney region immune microenvironment composition (FIG. 1A, C left)
1.1.1 immune microenvironment composition of human Kidney region
Selecting a tissue beside a cancer of a patient with clinical renal cancer, grinding the tissue, digesting, and extracting a renal mononuclear cell. And preparing a staining antibody by using a machine buffer solution, and performing mass spectrum flow type machine. The high dimensional data is visualized in two dimensions using the dimensionality reduction algorithm t-SNE, showing the distribution of each cluster and the expression of the markers, as well as the differences between the groups.
1.1.2 mouse Kidney region immune microenvironment (right of C, FIG. 1B)
Selecting normal WT Balb/c mouse kidney tissue, grinding the tissue, digesting, and extracting kidney mononuclear cells. And preparing a staining antibody by using a machine buffer solution, and performing mass spectrum flow type machine. The high dimensional data is visualized in two dimensions using the dimensionality reduction algorithm t-SNE, showing the expression of the distribution of each cluster, as well as the differences between the groups.
1.2 distribution of T lymphocyte markers in the kidney in the immune microenvironment of the renal region
1.2.1 distribution of CD3, CD69, CD45RA and CCR7 in the immune microenvironment of the human renal region (FIG. 1D)
Operating according to the procedure of 1.1.1, the high dimensional data is visualized in two dimensions using the dimension reduction algorithm t-SNE, showing the distribution and expression of each marker, and the differences between the groups.
1.2.2 distribution of CD3, CD69, CD44 in the immune microenvironment of the mouse Kidney region (FIG. 1E)
Operating according to the procedure of 1.1.2, the high dimensional data is visualized in two dimensions using the dimension reduction algorithm t-SNE, showing the distribution and expression of each marker, and the differences between the groups.
2. Kidney TRMAnalysis of phenotypic characteristics
2.1 Normal mouse Kidney TRMCharacterization of chemotactic and homing Properties of (1) (FIG. 2A)
The kidney of a normal WT Balb/c mouse is selected, fully ground, digested with collagenase, and centrifuged by percoll density gradient to obtain the mononuclear cell of the kidney. The cell membranes of the mononuclear cells are stained by a flow staining technology. Flow-type on-machine detection of kidney TRM(CD3+CD69+CD44+) Non-resident T cells with renal memory (CD 3)+CD69-CD44+) Expression abundance of CXCR3 and CD 62L.
2.2 Normal mouse Kidney TRMCharacterization of cell Activity (FIG. 2B)
The kidney of a normal WT Balb/c mouse is selected, fully ground, digested with collagenase, and centrifuged by percoll density gradient to obtain the mononuclear cell of the kidney. The cell membranes of the mononuclear cells are stained by a flow staining technology. Then, the membrane is broken to carry out intracellular staining, and the kidney T is detected on a flow type computerRM(CD3+CD69+CD44+) Non-resident T cells with renal memory (CD 3)+CD69-CD44+) The expression abundance of IFN-. gamma.and perforin.
3. T in glomerular injury in mice and humansRMProportional Activity characterization (FIG. 3)
3.1T in glomerular injury in miceRMCharacterization of the proportional Activity
Constructing an adriamycin nephropathy model and a db/db type II diabetes mouse model (30 weeks old), taking out the kidney of the mouse, fully grinding and digesting with collagenase, obtaining mononuclear cells of the kidney by percoll density gradient centrifugation, and carrying out cytomembrane staining on the mononuclear cells by utilizing a flow staining technology. Flow-type on-machine detection of kidney CD4+And CD8+TRMIn the same time, intracellular staining was carried out to detect kidney CD8+TRMActivity of (2).
3.2 FSGS and DN patient Kidney TRMDistribution of (2)
Immunofluorescence detection of peri-glomerular T in FSGS and DN patient kidneysRMDistribution of (D) and simultaneous comparison of T with paracancerous kidneyRMThe number of (2) varies.
4 kidney CD8 in pathological conditions+TRMCD69 mainly derived from peripheral blood infiltration-T cells
4.1 demonstration of Kidney CD8 in vivo in pathological conditions+TRMT cells from peripheral infiltration (FIG. 5)
Injecting FTY720 into Adriamycin nephropathy model mice, taking out the mice kidney after 6 weeks, fully grinding and digesting with collagenase, obtaining mononuclear cells of the kidney by percoll density gradient centrifugation, carrying out cytomembrane staining on the mononuclear cells by using a flow staining technology, then carrying out nuclear rupture treatment, carrying out Ki-67 staining in the nucleus, and detecting CD8 of the kidney+TRMThe level of proliferation of (a).
4.2 demonstration of Kidney CD8 in vivo+TRMCD69 from infiltration-T cells (FIG. 6)
An athymic mouse nude mice (Balb/c germ line) is purchased to construct an adriamycin nephropathy model, and the adriamycin nephropathy model is intravenously returned to CD8+CD69-CD44+T lymphocytes, after 6 weeks, the mouse kidney was removed, ground thoroughly and digested with collagenase, mononuclear cells of the kidney were obtained by percoll density gradient centrifugation, and the phenotype of mouse kidney T lymphocytes was examined by flow cytometry.
5 IL-15 promotes CD8+TRMFormation and activation of
5.1 Adriamycin Kidney disease model mouse Kidney cortex IL-15 assay (FIG. 7)
The IL-15 content of the mouse renal cortex tissue is detected by Elisa. Meanwhile, a full-automatic biochemical analyzer is used for analyzing the ratio (UACR) of the mouse urine microalbumin to the urine creatinine, and Graphpadprism software is used for carrying out correlation analysis.
5.2 IL-15 promotes CD8+TRMFormation and activation of (FIG. 8)
5.2.1 IL-15 promotes CD8+TRMFormation of
Spleen mononuclear cells were extracted and stimulated continuously for 3 days with recombinant mouse IL-15(50 ng/mL). Flow cytometry for detecting CD8 therein+CD44+T lymphocyte CD69+The ratio of (a) to (b).
5.2.2 IL-15 promotes CD8+TRMActivation of
Spleen mononuclear cells were extracted and stimulated continuously for 3 days with recombinant mouse IL-15(50 ng/mL). Flow cytometry for detecting CD8 therein+CD69+CD44+The proportion of IFN- γ production by T cells.
5.3 Anti-CD122 vs. CD8+TRMInfluence of formation and activation of (1) (FIG. 9)
5.3.1 expression of CD122
Extracting kidney mononuclear cells, performing flow cytomembrane staining on CD122, and detecting CD8 in kidney+TRMAnd renal CD8+non-TRMExpression of (2).
5.3.2 Anti-CD122 vs. CD8+TRMInfluence of formation and activation of
Spleen mononuclear cells were extracted and stimulated continuously for 3 days with recombinant mouse IL-15(50 ng/mL). Adding Anti-CD122 at the same time, and detecting CD8 by flow cytometry+CD69+CD44+The proportion of T cells and the proportion of IFN- γ that they produce.
6 IL-15/CD122 blocking antibody vs CD8+TRMThe formation and activation of (B), and the effects on glomerulosclerosis and podocyte injury
6.1 IL-15/CD122 blocking antibody against Adriamycin Kidney disease model mouse CD8+TRMAnd the effects on glomerulosclerosis and podocyte injury (FIG. 10)
The Anti-IL-15/CD122 was injected intraperitoneally into T-cell reinfused doxorubicin renal disease mice. Detection of renal CD8 using flow cytometry+TRMTo the production of IFN-gamma; detecting the ratio (UACR) of microalbumin in mouse urine and urinary creatinine by using a full-automatic biochemical analyzer; detecting the glomerular glycogen accumulation condition of the mice by using a PAS dyeing technology; the expression of nephrin, podocin of podocytes was detected by immunofluorescence staining.
6.2 db/db mice Kidney cortex IL-15 level assay (FIG. 11)
The IL-15 content of the mouse renal cortex tissue is detected by Elisa. Meanwhile, a full-automatic biochemical analyzer is used for analyzing the ratio (UACR) of the mouse urine microalbumin to the urine creatinine, and Graphpadprism software is used for carrying out correlation analysis.
6.3 IL-15/CD122 blocking antibody against kidney CD8 in db/db mice+TRMAnd the effects on glomerulosclerosis and podocyte injury (FIG. 12)
Db/db mice were injected intraperitoneally with Anti-IL-15/CD122 antibody. Detection of renal CD8 using flow cytometry+TRMTo the production of IFN-gamma; detecting microalbumin and urinary creatinine in the urine of the mouse by using a full-automatic biochemical instrument; detecting the glomerular glycogen accumulation condition of the mice by using a PAS dyeing technology; the expression of nephrin, podocin of podocytes was detected by immunofluorescence staining.
6.4 in vitro experiments: IL-15/CD122 blocking antibody Effect against podocyte injury (FIG. 13)
6.4.1 mouse podocyte apoptosis assay
In vitro experiments Using Transwell, mouse podocyte cell line and CD8 from Adriamycin Kidney disease model group+TRMSeparating cells, adding Anti-IL-15/CD122 blocking antibody into the system, and detecting the apoptosis ratio of mouse podocytes by a flow cytometer.
6.4.2 detection of mRNA levels of podocin, a mouse podocyte
In vitro experiments Using Transwell, mouse podocyte cell line and CD8 from Adriamycin Kidney disease model group+TRMThe cells are separated, Anti-IL-15/CD122 blocking antibody is added into the system, and the qPCR experiment detects the expression level of mouse podocyte podocin.
Second, experimental results
1. Analysis of immune microenvironment composition in renal tissue region
1.1 Kidney region immune microenvironment composition (FIG. 1A, C left)
1.1.1 immune microenvironment composition of human Kidney region
Normal human kidneys are rich in T lymphocytes. This suggests that our T lymphocytes may be involved in the development of kidney disease.
1.1.2 mouse Kidney region immune microenvironment (right of C, FIG. 1B)
Normal mice have abundant T lymphocyte content in the kidney. This suggests that our T lymphocytes may be involved in the development of kidney disease.
1.2 distribution of T lymphocyte markers in the kidney in the immune microenvironment of the renal region
The presence of T in the kidney of normal human and mouseRM
2. Kidney TRMAnalysis of phenotypic characteristics
2.1 Normal mouse Kidney TRMCharacterization of chemotactic and homing Properties of (1) (FIG. 2A)
Normal mouse Kidney TRM(CD3+CD69+CD44+) Compared with the non-resident memory T cells which highly express the chemokine receptor CXCR3, the T cells lowly express the homing receptor CD 62L. Highlighting its colonizing properties.
2.2 Normal mouse Kidney TRMCharacterization of cell Activity (FIG. 2B)
Normal mouse Kidney TRM(CD3+CD69+CD44+) High expression of IFN-gamma and perforin compared to non-resident memory T cells.
3. T in glomerular injury in mice and humansRMProportional Activity characterization (FIG. 3)
3.1T in glomerular injury in miceRMCharacterization of the proportional Activity
Adriamycin nephropathy and CD8 in db/db mice compared to control mice+TRMAnd not CD4+TRMThe proportion is increased, the proportion of IFN-gamma and perforin is increased, and the activity is enhanced.
3.2 FSGS and DN patient Kidney TRMDistribution (fig. 4)
CD8 in the kidney of FSGS and DN patients compared to the paracancerous kidney+TRMAnd not CD4+TRMIncreased in number and concentrated around the glomerulus. Description of CD8+TRMMay be involved in the development of glomerular disease.
4 kidney CD8 in pathological conditions+TRMCD69 mainly derived from peripheral blood infiltration-T cells
4.1 demonstration of Kidney CD8 in vivo in pathological conditions+TRMFrom peripheral infiltrationT cell of (2) (FIG. 5)
FTY720 acts as an antagonist of sphingosine-1-phosphate and inhibits infiltration of peripheral T cells into tissues. After FTY720 is injected, the kidney CD8 of the doxorubicin nephropathy mice is found+TRMThere was no significant proliferation, so we concluded that CD8 was present under pathological conditions+TRMOriginating from peripheral infiltration.
4.2 demonstration of Kidney CD8 in vivo+TRMCD69 from infiltration-T cells (FIG. 6)
The nude mouse feedback experiment further proves that the kidney CD8+TRMPossibly from CD69-T cell to CD69+Differentiation of T cells.
5 IL-15 promotes CD8+TRMFormation and activation of
5.1 Adriamycin Kidney disease model mouse Kidney cortex IL-15 assay (FIG. 7)
What factors in the kidney may cause CD8+TRMIs formation and activation of? IL-15 has been reported to participate in this process. Therefore, the IL-15 of the kidney cortex tissue of the doxorubicin kidney disease mice is detected, and the IL-15 level is increased compared with the mice in a control group, and is positively correlated with the proteinuria level of the mice.
5.2 IL-15 promotes CD8+TRMFormation and activation of (FIG. 8)
In vitro experiments, spleen mononuclear cells are stimulated by recombinant IL-15, and the IL-15 is found to promote CD69+T cell formation, while promoting IFN-gamma production. Whereas IL-2, which has a co-receptor with it, does not have these effects.
5.3 Anti-CD122 vs. CD8+TRMInfluence of formation and activation of (1) (FIG. 9)
5.3.1 expression of CD122
CD122 acts as a receptor for IL-15 signaling in T cells, and CD8 in mouse kidney+TRMHigh expression.
5.3.2 Anti-CD122 vs. CD8+TRMInfluence of formation and activation of
Use of blocking antibodies to CD122 significantly inhibited IL-15 versus CD8+TRMFormation and activation ofThe application is as follows.
6 IL-15/CD122 blocking antibody vs CD8+TRMThe formation and activation of (B), and the effects on glomerulosclerosis and podocyte injury
6.1 IL-15/CD122 blocking antibody against Adriamycin Kidney disease model mouse CD8+TRMEffects of glomerulosclerosis and podocyte injury (FIG. 10)
Mouse CD8 administered to IL-15/CD122 antibody-treated group compared to doxorubicin-only kidney disease mouse model group+TRMObviously reduces the formation and activation of the leucocyte, obviously relieves the symptoms of proteinuria, reduces the proliferation of glomerular mesangium and relieves the injury of podocytes.
6.2 db/db mice Kidney cortex IL-15 level assay (FIG. 11)
The kidney cortex IL-15 level of db/db mice is increased, and the mice and the protein urine level are in positive correlation, which suggests that the kidney IL-15 of the mice can be used as an important marker molecule in diseases and participate in the occurrence and development of the diseases.
6.3 IL-15/CD122 blocking antibody against kidney CD8 in db/db mice+TRMAnd the effects on glomerulosclerosis and podocyte injury (FIG. 12)
Mouse CD8 administered to IL-15/CD122 antibody treated group compared to db/db mouse model group alone+TRMObviously reduces the formation and activation of the leucocyte, obviously relieves the symptoms of proteinuria, reduces the proliferation of glomerular mesangium and relieves the injury of podocytes.
6.4 in vitro experiments: effect of IL-15/CD122 blocking antibody on damage to podocytes (FIG. 13)
In vitro co-culture experiments demonstrated that activated CD8+TRMObviously promotes the apoptosis of podocytes and the expression of functional protein podocin, and the IL-15/CD122 blocking antibody obviously inhibits the damage of the podocytes. This suggests us IL-15/CD122 as a key target for the treatment of podocyte injury.
The invention is not the best known technology.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (10)

1. Targeted inhibition of renal CD8+TRMThe use of the formed and activated substance of (a) in at least one of a1) -a5) as follows:
a1) alleviating symptoms of proteinuria and/or preparing a product that alleviates symptoms of proteinuria;
a2) inhibiting mesangial proliferation and/or preparing a product for inhibiting mesangial proliferation;
a3) reducing podocyte damage and/or preparing a product for reducing podocyte damage;
a4) reducing glomerulosclerosis and/or preparing a product for reducing glomerulosclerosis;
a5) treating diseases related to glomerular injury and/or preparing products for treating diseases related to glomerular injury.
2. The use of claim 1, wherein said targeted inhibition of renal CD8+TRMThe substance for forming and activating comprises an IL-15 signal blocker, and the IL-15 signal blocker is a small molecule compound or an antibody, preferably an antibody.
3. The use of claim 2, wherein said IL-15 signal blocker is an IL-15 signal blocking antibody comprising Anti-IL-15/CD 122.
4. The use of claim 1, wherein the product of a1) -a5) is a medicament or a test agent.
5. A composition, characterized in that the active ingredients of the composition at least comprise the targeted inhibition kidney CD8+TRMThe formation and activation of (a).
6. The composition of claim 5, wherein said targeted inhibition of renal CD8+TRMThe substance for forming and activating comprises an IL-15 signal blocker, and the IL-15 signal blocker is a small molecule compound or an antibody, preferably an antibody.
7. The composition of claim 6, wherein said IL-15 signaling blocking agent is an IL-15 signaling blocking antibody comprising Anti-IL-15/CD 122.
8. Use of a composition according to any one of claims 5 to 7 in any one or more of:
a1) alleviating symptoms of proteinuria and/or preparing a product that alleviates symptoms of proteinuria;
a2) inhibiting mesangial proliferation and/or preparing a product for inhibiting mesangial proliferation;
a3) reducing podocyte damage and/or preparing a product for reducing podocyte damage;
a4) reducing glomerulosclerosis and/or preparing a product for reducing glomerulosclerosis;
a5) treating diseases related to glomerular injury and/or preparing products for treating diseases related to glomerular injury.
9. The use of claim 8, wherein the product is a medicament or a test agent;
preferably, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
10. A method for treating a disease associated with glomerular injury, the method comprising administering to a subject a therapeutically effective amount of a compound that targetedly inhibits renal CD8+TRMOr a composition according to any one of claims 5 to 7;
preferably, the treatment of a disease associated with glomerular injury comprises at least amelioration of glomerulosclerosis and podocyte injury.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20040253587A1 (en) * 1995-02-22 2004-12-16 Immunex Corporation Antagonists of interleukin-15
CN1780856A (en) * 2003-02-26 2006-05-31 根马布股份公司 Human antibodies specific for interleukin 15 (IL-15)
CN102939305A (en) * 2010-04-08 2013-02-20 Jn生物科学有限责任公司 Antibodies to cd122

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Publication number Priority date Publication date Assignee Title
US20040253587A1 (en) * 1995-02-22 2004-12-16 Immunex Corporation Antagonists of interleukin-15
CN1780856A (en) * 2003-02-26 2006-05-31 根马布股份公司 Human antibodies specific for interleukin 15 (IL-15)
CN102939305A (en) * 2010-04-08 2013-02-20 Jn生物科学有限责任公司 Antibodies to cd122

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