WO2015074083A1 - Méthodes de diagnostic d'une inflammation médiée par les lymphocytes t - Google Patents

Méthodes de diagnostic d'une inflammation médiée par les lymphocytes t Download PDF

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WO2015074083A1
WO2015074083A1 PCT/US2014/066238 US2014066238W WO2015074083A1 WO 2015074083 A1 WO2015074083 A1 WO 2015074083A1 US 2014066238 W US2014066238 W US 2014066238W WO 2015074083 A1 WO2015074083 A1 WO 2015074083A1
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inflammatory
cell
exosomes
allograft
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Nicole Suciu-Foca
George Vlad
Zheng Xu
Chih-Chao Chang
Eric Koonming HO
Elena VASILESCU
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The Trustees Of Columbia University In The City Of New York
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • T-cell- mediated inflammation also referred to acute cellular rejection
  • Such early detection is particularly important to prevent allograft rejection, graft rejection, and graft versus host disease.
  • Heart allografts are a very common treatment for heart disease, yet at present, repetitive invasive heart biopsies are the only way to determine if a recipient is rejecting the transplant.
  • FIG. 1 ILT3.Fc decreases inflammatory miRNA expression in exosomes from primary MLC with or without ILT3.Fc. Mean + standard deviation of one out of 3 representative experiments is shown.
  • FIG. 2 Comparison of miRNAs contained by exosomes from CD8 or CD4 T cells primed alone in 7-day MLC. Mean + standard deviation of one out of 3 experiments are shown.
  • FIG. 3 is a graph that illustrates transfection of inflammatory miRNA that mimics reverse T suppressor's inhibitor effect on MLC, according to an embodiment.
  • FIG.4 shows the proliferative responses of CD4 Th cells to an allogeneic stimulator in the presence of CD8+ Ts pretreated with various exosomes.
  • Fig. 4A The inhibitory capacity of allospecific CD8+ Ts was tested after a 24h preincubation with control medium (blue bars), or exosomes isolated from 7 day primary MLC cultured in the absence (red bars) or presence of 50mg/ml ILT3Fc (green bars).
  • Fig.4 B Exosomes were isolated from 7 day cultures of CD4 Th cells, CD8 cells or total CD3 cells with an allogeneic CD2 depleted stimulator. These exosomes were then tested for their ability to reverse the inhibitory capacity of CD8+ Ts, after 24 hours of preincubation. The bars represent proliferative responses of CD4 Th in the presence of the pre- treated Ts.
  • Embodiments of the invention include a method, comprising a) obtaining a biological sample from a subject, b) isolating exosomes from the subject sample and determining a level of one or more inflammatory miRNAs selected from the group consisting of miR21, 30b, 146a, 155, 365, and Let-7a in the isolated exosomes, c) comparing the level of the one or more inflammatory miRNAs in the subject sample to a corresponding predetermined control level of the one or more inflammatory miRNAs in exosomes isolated from biological samples taken from a normal population, and d) determining that the subject has T cell-mediated inflammation if the inflammatory mRNA level in the subject sample is significantly higher by at least 40% than the corresponding control level.
  • This method can include e) if the subject has T cell-mediated inflammation then treating the inflammation.
  • the subject has had an allograft or has an autoimmune disease; if such a subject has inflammation then treatment for allograft rejection, inflammation or the autoimmune disease is begun.
  • a biopsy is performed to determine whether or not the allograft is being rejected.
  • the method is done using pre- and post-allograft subject samples.
  • the subject having an allograft presents with a symptom of rejection.
  • Allografts in various embodiments include heart, kidney, skin, muscles, lung, liver, eye, pancreas, bone marrow, bone, intestine, heart valves, nerves, veins and thymus.
  • a multiple biological samples from the subject are obtained at different times and the method is conducted to determine if the subject has T cell-mediated inflammation.
  • the biological sample is blood or serum and the subject is human.
  • the subject has had an autologous bone marrow graft and in other embodiments the subject is at risk of developing a cardiac disorder, or has a neurodegenerative disorder.
  • Another embodiment includes a method for determining whether a therapeutic amount of a pharmaceutical agent causes T cell-mediated inflammation, by a) obtaining a biological sample from a subject being treated with the pharmaceutical agent b) isolating exosomes from the subject sample and determining a level of one or more inflammatory miRNAs selected from the group consisting of miR21, 30b, 146a, 155, 365, and Let-7a in the isolated exosomes, c) comparing the level of the one or more inflammatory miRNAs in the subject sample to a corresponding predetermined control level of the one or more inflammatory miRNAs in exosomes isolated from biological samples taken from a normal population not treated with the pharmaceutical agent, d) determining that the subject has T cell-mediated inflammation if the inflammatory mRNA level in the subject sample is significantly higher by at least 40% than the corresponding control level, and e) if the subject has inflammation then reducing the amount of the pharmaceutical or discontinuing treatment with the pharmaceutical agent.
  • inflammatory miRNA control level and "control level of inflammatory miRNA” are used interchangeable herein to mean a predetermined level based on a range of levels for the particular miRNA found in a population of normal subjects who are disease- and inflammation-free. Methods for calculating a normal range are known in the art and include for example determining the 95% confidence interval for a population of normal, healthy subjects (i.e. not suffering from any diagnosed acute or chronic inflammatory conditions). A normal range would have to be calculated for the level of expression of each inflammatory miRNA.
  • a baseline expression level can be calculated for any given subject and in the case of allografts, includes a single determination of the expression level of a particular miRNA, obtained before the onset of T cell-mediated inflammation (before the allograft, or before any T cell-mediated inflammation i.e. in the absence of rejection).
  • the term "Significantly elevated” or “significantly increased” as used herein means an increase of at least about 40% in the level of a particular miRNA in exosomes isolated from a subject compared to a control level of the particular inflammatory miRNA or the subject's individual baseline level of the particular miRNA in a non-inflamed state.
  • raft means a graft of tissue obtained from a donor of the same species as, but with a different genetic make-up from, the recipient. Also called a “homograft,” an example is a tissue or organ transplant between two humans. Allograft and organ transplant are used interchangeably.
  • ILT3 means "immunoglobulin-Like Transcript-3", and is synonymous with “ILT-3", “LIR-5”, “CD85K” and “LILRB4.”
  • the mRNA coding sequence for human ILT3 is provided under GenBank No. U82979.
  • ILT3Fc means a potent immunosuppressive agent that includes the extracellular domain of ILT3 (which includes the ILT3 ligand binding site) bound to Fc that specifically targets activated T cells which it converts into T suppressor cells.
  • the "Extracellular domain of ILT3" as used herein, means the N-terminal 258 amino acid residues of ILT3 (e.g. , human ILT3 having the sequence of GenBank Accession No. U82979).
  • Micromalian cell as used herein, means any mammalian cell including, without limitation, cells which are normal, abnormal and transformed, and are exemplified by T cells and immune cells. Human cells are preferred.
  • Inflammation means either acute or chronic inflammation. It is a local response to cellular injury that is marked by capillary dilatation, leukocyte infiltration, redness, heat, and pain and that serves as a mechanism initiating the elimination of noxious agents and of damaged tissue.
  • T cell-mediated inflammation 'T cell-mediated acute cellular rejection
  • T cell-mediated inflammatory response and 'T cell-mediated inflammatory disorders
  • T cell-mediated inflammatory disorders include all such diseases mediated by T cells, such as autoimmune diseases, allograft rejection, graft vs. host disease, allergies, hypersensitivity and others described herein.
  • the embodiments of the invention are directed to diagnosing and treating T cell- mediated inflammatory disorders.
  • exosomes as used herein, is also referred to in the literature as microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes,
  • RNA molecules in exosomes include mRNA and miRNA.
  • exosomes RNA that is shuttled from one cell to another, known as "exosomal shuttle RNA," potentially affects protein production in the recipient cell.
  • Exosomes are remarkably stable in bodily fluids strengthening their case as reservoirs for disease biomarkers.
  • exosomes can be isolated from a subject's biological sample, for example using techniques described in the materials and methods including isolating total exosomes using Invitrogen Total Exosome Isolation Kit.
  • Flow cytometry is an applied optical method to detect exosomes in suspension. Methods to detect single exosomes include atomic force microscopy, nanoparticle tracking analysis, Raman microspectroscopy, resistive pulse sensing, and transmission electron microscopy and microarrays.
  • inflammatory exosome means an exosome containing one or more inflammatory miRNAs, including but not limited to those selected from the group consisting of miR21, 30b, 146a, 155, 365, and Let-7a.
  • miR or "micro RNA” or “miRNA” are used interchangeably used herein to mean a class of small non-coding RNAs (mature microRNAs are approximately 21-23 nucleotides in length) that are key negative regulators of gene expression.
  • miRs are typically transcribed by RNA polymerase II and their expression is controlled by transcriptional factors.
  • the mature miRs Mature microRNAs are partially complementary to the 3'UTR of one or more target messenger RNAs (mRNAs) and they inhibit target mRNA translation, for example by directly binding to specific miR binding sites in the 3'- untranslated region (3' UTR) of target genes.
  • mRNAs target messenger RNAs
  • Inflammatory miRNAs means miRNAs that are known to be increased during inflammation (hereafter "inflammatory miRNAs) including but not limited to miR21, 30b, 146a, 155, 365, and Let-7a.
  • biological sample means a blood or serum sample from a mammal, preferably a human subject.
  • subject means any mammal such as a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate. In the preferred embodiment, the subject is a human being.
  • Organs as used herein means a collection of cells and tissues joined in a structural unit to serve a common function.
  • Organs include any organ that can be transplanted including the heart, kidney, lung, liver, eye, pancreas, bone, intestine and thymus.
  • Tissues include tendons (both referred to as musculoskeletal grafts), composite tissue allografts, cornea, muscle, skin, heart valves, bone marrow, nerves and veins.
  • alloactivated means the activation or initiation of an immune response against a foreign "alio" antigen from the same species.
  • the body attacks especially transplanted tissue.
  • An alloimmune response results in graft rejection, which is manifested as deterioration or complete loss of graft function.
  • Allografts in this context include, for example, heart, kidney, skin, lung, liver, eye, lung, pancreas, bone marrow and bone.
  • Transplanted tissue in this context includes, for example, tendons (both referred to as musculoskeletal grafts), composite tissue allografts, cornea, muscle, skin, heart valves, bone marrow, nerves and veins.
  • Inflammation is an indication of allograft rejection.
  • Grade 2R/3A rejection shall mean herein that in Grade 2 R/3A, two or more foci of mononuclear cells (lymphocytes/macrophages) with associated myocyte damage are present. Eosinophils may be present. The foci may be distributed in one or more than one biopsy fragment. Intervening areas of uninvolved myocardium are present between the foci of rejection.
  • treating a subject afflicted with a disorder as used herein means shall mean causing the subject to experience a reduction, remission, mediation or regression of the disorder and/or its symptoms.
  • Subjects receiving an allograft/transplant require immunosuppression, therefore treating a subject that has had an allograft/transplant who is showing inflammation which is an abnormally high immune response, can reduce the inflammation and thereby prevent or delay the onset of transplant rejection.
  • terapéuticaally effective amount means an amount sufficient to inhibit the progression of an enumerated disease in a subject.
  • (MLC)-primed alloactivated CD4+ T cells make and release inflammatory exosomes that contain one or more inflammatory miRNAs in the group miR21, 30b, 146a, 155, 365, and Let-7a.
  • this release was inhibited if alloactivation was carried out in the presence of ILT3.Fc.
  • ILT3.Fc decreased the level of expression of the inflammatory miRNAs in exosomes and exposure to inflammatory exosomes diminished the suppressive activity of ILT3-Fc-induced CD8+ T suppressor cells ("Ts”) at high effector- to- suppressor T cell ratios.
  • T cell alloactivation is a necessary step for T cell- mediated inflammation. It has been discovered that T cell-mediated inflammation in a subject can be diagnosed if there is a significant increase of at least 40% in the level of one or more inflammatory miRNAs in exosomes isolated from a biological sample from the subject. Results presented here further show that the inflammatory miRNA 21 was significantly increased in vivo in humans undergoing heart stage grade 2R/3A allograft rejection (described in Example 4).
  • T cell-mediated inflammation is an indication of allograft rejection.
  • allografts transplants
  • Transplanted organs include, for example, pancreas heart, kidney, skin, lung, liver, eye, bone, and bone marrow.
  • Transplanted tissues include, for example, vascular tissue and islets.
  • Transplanted cells include stem cells, e.g., umbilical cord stem cells or adult stem cells, pancreatic islet cells, epithelial cells, endothelial cells, and liver cells.
  • stem cells e.g., umbilical cord stem cells or adult stem cells
  • pancreatic islet cells e.g., pancreatic islet cells
  • epithelial cells e.g., endothelial cells
  • liver cells e.g., liver cells.
  • a prosthetic device e.g., stent can initiate T cell-mediated inflammation.
  • biopsies are used to determine if a subject is undergoing an allograft rejection.
  • T cell-mediated inflammation that underlies allograft rejection can be detected with a blood test, and invasive biopsies can be reserved for subjects shown that have T cell-mediated inflammation.
  • T cell-mediated inflammation is also associated with most if not all autoimmune diseases. Autoimmune diseases are chronic, but undergo periods of active disease and remission. Using the methods of the present invention to detect T cell-mediated inflammation enables one to determine if a subject that appears to be in remission is actually developing active disease requiring treatment.
  • T suppressor cells also called regulatory T cells (T reg )
  • T suppressor cells are a specialized subpopulation of T cells that suppress activation of the immune system thereby maintaining tolerance to self-antigens.
  • DC dendritic cells
  • the DC can instruct T cells to become suppressor/regulatory cells (Vlad G, Cortesini R, Suciu-Foca N. License to heal: bidirectional interaction of antigen- specific regulatory T cells and tolerogenic APC. J Immunol 2005;174:5907-14.).
  • ILT3 is a prototype of such inhibitory molecules which is characteristically increased on the membrane of human tolerogenic DC and induces the differentiation of human T cells into T suppressor cells.
  • Adaptive T suppressor cells induce the upregulation of ILT3 on dendritic cells that in turn trigger the differentiation of new waves of antigen- specific T suppressor cells.
  • the human immunoglobulin like transcript (ILT) 3 and 4 also known as
  • LIRB4/LIR5/CD85k and LIRB2/LIR2/CD85D belong to a family of innate immune receptors which are expressed by DC and monocytes (Cella M, et al., J, et al. J Exp Med 1997; 185: 1743- 51). These ILT receptors display a long cytoplasmic tail containing immunoreceptor tyrosine- based inhibitory motifs (ITIMs) that mediate inhibition of cell activation by recruiting tyrosine phosphatase SHP-1, and an extracellular domain that contains the ILT3 ligand binding site.
  • ITIMs immunoreceptor tyrosine- based inhibitory motifs
  • Tolerogenic human DC are characterized by high expression of ILT3/ILT4 on their membrane and by their capacity to induce T cell anergy and the differentiation of regulatory/suppressor T cells (Chang CC, et al. Nat Immunol 2002;3:237-43; Manavalan JS, Rossi PC, Vlad G, et al. High expression of ILT3 and ILT4 is a general feature of tolerogenic dendritic cells. Transpl Immunol 2003; 11:245-58.3,4).
  • knockdown (KD) of ILT3 from DC increases their TLR responsiveness (Chang CC, Liu Z, Vlad G, et al.
  • Ig-like transcript 3 regulates expression of proinflammatory cytokines and migration of activated T cells. J Immunol 2009;182:5208-16.), as reflected in synthesis and secretion of proinflammatory cytokines (IL-1 alpha and beta, IL-6 and type I IFN) and migration factors CXCL10 and CXCL11. ILT3KD-DC enhance T cell proliferation and secretion of IFN-g and IL-17 when pulsed with CMV or used as alio stimulators in MLC.
  • the extracellular domain of ILT3 retains the T cell inhibitory function even upon deletion of the cytoplasmic, ITIM-containing tail, since DC transfected with a construct comprising only the extracellular portion were still capable to elicit the differentiation of CD8+ T suppressor cells (Kim-Schulze S, Scotto L, Vlad G, et al. J Immunol 2006;176:2790-8.; Suciu- Foca, US Serial No. 11/661,877.).
  • a soluble form of ILT3 comprising the extracellular domain expressed as an ILT3Fc fusion protein retains immunomodulatory activity. This recombinant protein inhibited primary and secondary T cell responses in MLC and blocked the differentiation of CD8 + cytotoxic T cells (CTL).
  • Both membrane and soluble ILT3 have potent immunosuppressive activity, inhibiting the differentiation of CD4+ and CD8+ effector cells from primed CD3 T cells [11-15].
  • CD8+ Ts cells primed in MLC in the presence of recombinant soluble ILT3 (ILT3.Fc) strongly upregulate the expression of BCL6, a transcription factor belonging to a class of zinc finger transcriptional repressors including BCL6, a known inhibitor of IFN-gamma, IL-2, IL-17, IL-5 and granzyme B expression (Chang CC, et al. J Immunol 2010;185:5714-22; Vlad G, et al., Hum Immunol 2011;72: 107-14).
  • ILT3.Fc such as dual specific phosphatase (DUSP10), TGF-Beta receptor 2 (TGFBR2), CXCR4 and the suppressor of cytokine signaling SOCS 1.
  • ILT3.Fc inhibits the expression of inflammatory miRNAs which target the 3'UTR of these ILT3.Fc upregulated genes.
  • MicroRNA are small non-coding, single stranded RNA molecules that regulate gene expression at the post-transcriptional level by degrading or blocking translation of messenger RNA (mRNA). MiRNAs can also interact directly with proteins [18]. Multiple miRNA, such as miR-146a and miR-155, have been reported to be important regulators of the immune system [1-3]. Recently, miRNA have been found in exosomes, which are vesicles of endocytic origin released by many cells. Exosomes can mediate communication between Antigen Presenting Cells (APC) and suppressor or effector T cells signaling the inhibition or activation of immune responses [4,5].
  • APC Antigen Presenting Cells
  • Exosomes may contribute to cell-cell communication at the level of synapses or by delivering mRNA or miRNA that can modify recipient cell protein production and gene expression not only within the microenvironment but also at a distance, by traffic through the systemic circulation.
  • the ability of exosomes to deliver nucleic acids to cells at a distance makes them ideal candidates for immune-monitoring purposes as well as for use as vectors for gene therapy.
  • Isolation of total exosomes from a biological sample is routine in the art and can be accomplished for example with Invitrogen's Total Exosome Isolation Kit.
  • the extraction of the total RNA from exosomes was performed by using Trizol reagent and miRNeasy Mini Kit (Qiagen), a procedure that is also routine. Quantification of miRNA expression level in exosomes was performed using TaqMan Small RNA assays (Applied Biosystems). The sequence of miRs are avail on line at http://www.mirbase.org/.
  • the miR detection kits such as those used herein are commercially available and are directed to particular miRs such as the inflammatory miRs herein.
  • Real-Time PCR were performed using Applied Biosystems 7300 Real Time PCR system and analyzed with Sequence Detection Software (Version 1.4).
  • ILT3.Fc completely inhibits (97 + 3%) T cell proliferation in MLC at 50 lg/ml and induces the differentiation of CD8+ Ts within 6 days.
  • Certain experiments were designed to test whether exosomes released in the supernatant of primary MLC grown in the presence or absence of ILT3 (50 lg/mL) can alter the inhibitory effect of ILT3-Fc-induced CD8+ Ts.
  • Purified miRNA-containing exosomes were isolated from supernatants collected on day 5 from MLC in which CD3+ T cells from responder A had been stimulated with allogeneic APC from stimulator B in the presence or absence of ILT3.Fc.
  • Example 1 shows that ILT3.Fc decreased inflammatory miRNA expression in exosomes.
  • the results described in Example 2 showed that ILT3Fc inhibited the release into the medium of inflammatory miRNA-containing exosomes (also herein "inflammatory exosomes") by T cells primed in MLC. It was also discovered that Ts pre-treated with exosomes from MLC alloactivated without ILT3.Fc were less inhibitory than Ts pre-treated with fresh medium, allowing T cell proliferation to return to levels seen in MLC without Ts. (Table 1).
  • Alloactivated CD4+ T cells were the major contributors of exosomes containing inflammatory microRNA and ILT3.Fc inhibits the production of inflammatory miRNA by CD4 T cells (FIG. 2).
  • Quantitative Real-Time PCR showed that there was a dramatic increase in six inflammatory miRNAs (miR-146a, miR-155, miR-21, miR-30b, miR-365 and Let-7a) contained in exosomes released from the CD4 T cells [1-5,16, 19-23]. See Example 3.
  • ILT3.Fc inhibited by 90% or more the release into exosomes of miR-146a, miR- 155, and miR-21; however, ILT3.Fc was less suppressive for miR-30b, miR-365 and miR-7a, which were inhibited by 50%, 30% and 30% respectively (FIG. 3). It was also discovered that that CD4+ T cells are the major contributors of inflammatory miRNAs in exosomes from in vitro-activated T cells, and that ILT3.Fc inhibited their production (Table 2).
  • certain embodiments are directed to methods for detecting and treating T cell-mediated inflammation in a subject by determining if there is a significant increase of at least about 40% in the level of one or more inflammatory miRNAs (in the group miR21, 30b, 146a, 155, 365 and Let-7a) in exosomes isolated from a biological sample, preferably blood, taken from the subject, compared to the corresponding control level of inflammatory miRNA. If T cell-mediated inflammation is detected, then it can be treated using methods known in the art.
  • T cell-mediated inflammation is an indication of allograft rejection. If T cell-mediated inflammation is detected in an allograft subject who is otherwise symptom free, it can be treated before rejection advances thereby facilitating early intervention. If T cell-mediated inflammation is detected in an allograft recipient, then the recipient can also be given a biopsy to definitively determine if the subject is undergoing rejection and the stage of allograft rejection. By screening subjects for T cell-mediated
  • the subject is a mammal, preferably a human.
  • the donor in an allograft is from the same species as the subject being treated. Transplantation can be performed across species (i.e., xenogeneic transplantation or xenograft).
  • the subject has an autoimmune disease or other T cell- mediated inflammatory disease.
  • Other embodiments are set forth in more detail in the summary of the invention.
  • Autoreactive T cells are also pivotal in the pathogenesis of organ- specific autoimmune disease where proinflammatory T cells can induce tissue inflammation.
  • a subject with an autoimmune disease undergoes periods of remission where treatment is discontinued or reduced.
  • Such subjects are ideal candidates for the present methods to monitor T cell-mediated inflammation. If such a subject in remission is determined to have T cell-mediated inflammation using the methods of the present invention during a period of apparent remission, then treatment of the inflammation and the underlying autoimmune disease should be initiated.
  • T cell-mediated inflammation may be acute or chronic (an excessive or unregulated inflammatory response for weeks, months, years, or indefinitely).
  • Autoimmune diseases include chronic inflammatory bowel diseases, ulcerative colitis, Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, lupus vulgaris, antiphospholipid syndrome, alopecia areata, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune peripheral neuropathy, autoimmune urticaria, dermatomyositis, diabetis type 1, , narcolepsy, pemphigus vulgaris, temporal arteritis, transverse myelitis, Wegener's granulomatosis, Goodpasture's syndrome, dermatitis, allergic encephalomyelitis, neuriti
  • Systemic lupus erythematosus (lupus). People with lupus develop autoimmune antibodies that can attach to tissues throughout the body. The joints, lungs, blood cells, nerves, and kidneys are commonly affected in lupus. Treatment often requires daily oral prednisone, a steroid that reduces immune system function.
  • IBD Inflammatory bowel disease
  • the immune system attacks the lining of the intestines, causing episodes of diarrhea, rectal bleeding, urgent bowel movements, abdominal pain, fever, and weight loss. Ulcerative colitis and Crohn's disease are the two major forms of IBD. Oral and injected immune-suppressing medicines can treat IBD.
  • MS Multiple sclerosis
  • the immune system attacks nerve cells, causing symptoms that can include pain, blindness, weakness, poor coordination, and muscle spasms.
  • Various medicines that suppress the immune system can be used to treat multiple sclerosis.
  • Type 1 diabetes mellitus Immune system antibodies attack and destroy insulin- producing cells in the pancreas. By young adulthood, people with type 1 diabetes require insulin injections to survive.
  • Rheumatoid arthritis The immune system produces antibodies that attach to the linings of joints. Immune system cells then attack the joints, causing inflammation, swelling, and pain. If untreated, rheumatoid arthritis causes gradually causes permanent joint damage. Treatments for rheumatoid arthritis can include various oral or injectable medications that reduce immune system overactivity.
  • Guillain-Barre syndrome The immune system attacks the nerves controlling muscles in the legs and sometimes the arms and upper body. Weakness results, which can sometimes be severe. Filtering the blood with a procedure called plasmapheresis is the main treatment for Guillain-Barre syndrome.
  • T-cells collect in the skin.
  • the immune system activity stimulates skin cells to reproduce rapidly, producing silvery, scaly plaques on the skin.
  • Graves' disease The immune system produces antibodies that stimulate the thyroid gland to release excess amounts of thyroid hormone into the blood (hyperthyroidism). Symptoms of Graves' disease can include bulging eyes as well as weight loss, nervousness, irritability, rapid heart rate, weakness, and brittle hair. Destruction or removal of the thyroid gland, using medicines or surgery, is usually required to treat Graves' disease.
  • Hashimoto's thyroiditis Antibodies produced by the immune system attack the thyroid gland, slowly destroying the cells that produce thyroid hormone. Low levels of thyroid hormone develop (hypothyroidism), usually over months to years. Symptoms include fatigue, constipation, weight gain, depression, dry skin, and sensitivity to cold. Taking a daily oral synthetic thyroid hormone pill restores normal body functions.
  • Myasthenia gravis Antibodies bind to nerves and make them unable to stimulate http://autoimmunediseaselist.com/ muscles properly. Weakness that gets worse with activity is the main symptom of myasthenia gravis. Mestinon (pyridostigmine) is the main medicine used to treat myasthenia gravis.
  • Vasculitis The immune system attacks and damages blood vessels in this group of autoimmune diseases. Vasculitis can affect any organ, so symptoms vary widely and can occur almost anywhere in the body. Treatment includes reducing immune system activity, usually with prednisone or another corticosteroid.
  • Goodpasture syndrome also known as Goodpasture's disease, anti-glomerular basement antibody disease, or anti-GBM disease
  • Anti-GBM disease is a rare autoimmune disease in which antibodies attack the lungs and kidneys, leading to bleeding from the lungs and to kidney failure. It may quickly result in permanent lung and kidney damage, often leading to death. It is treated with immunosuppressant drugs such as corticosteroids and cyclophosphamide, and with plasmapheresis, in which the antibodies are removed from the blood.
  • Agents for treating autoimmune diseases with a T-cell inflammatory component include: orencia , ridaura, rheumatrex, dexamethasone, intensol, aspirin, adalimumab, certolizumab pegol, solu-medrol, methylprednisolone, cuprimine, ansaid, ecotrin, anaprox, tolmetin, methylprednisolone sodium succ intravenous, motrin, abatacept, ntravenous, solu-medrol, cimzia, solu-cortef, sandimmune, ecotrin, prednisolone sodium phosphate, pediapred, ibuprofen, ib, millipred ora, tocilizumab intravenous, mediproxen, aristospan, hydrocortisone, auranofin, penicillamine, fenoprofen , azathioprine, ana
  • minocycline- wipes minocin kit with wipes, flanax (naproxen), aristospan intralesional, solu- cortef, abatacept, golimumab, tocilizumab, meloxicam, mobic, naproxen, remicade, plaquenil, diclofenac, indomethacin, kenalog, etodolac, nabumetone, humira
  • the present methods are especially valuable to detect T cell-mediated inflammation in subjects at risk of developing allograft/transplant rejection, and graft-versus-host disease. If inflammation is detected in an otherwise symptom-free subject, it is a sign of rejection that should be treated, and followed in most cases with a biopsy.
  • Immunosuppressive agents used to treat allograft recipients include cyclosporine, OKT3 Antibody, rapamycin, Campath I, anti- CD69 antibody, thymoglobulin, and anti-thymocytic antibody.
  • Symptoms associated with rejection of a transplant include increased blood urea nitrogen (BUN) levels for kidney, increased glycemia for pancreas, lymphocyte infiltrates for heart, and increased levels of enzymes such as aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) for liver.
  • BUN blood urea nitrogen
  • SGOT aspartate aminotransferase
  • SGPT alanine aminotransferase
  • Exosomes can have, but not be limited to, a diameter of greater than about 10, 20, or 30 nm. They can have a diameter of about 30-1000 nm, about 30-800 nm, about 30-200 nm, or about 30-100 nm. In some embodiments, the exosomes can have, but not be limited to, a diameter of less than about 10,000 nm, 1000 nm, 800 nm, 500 nm, 200 nm, 100 nm or 50 nm. As used throughout, the term "about,” when referring to a value or to an amount is meant to encompass variations in some embodiments .+-.10% from the specified amount, as such variations are appropriate.
  • exosomes can also include any shed membrane bound particle that is derived from either the plasma membrane or an internal membrane.
  • CONFIRM Exosomes can also include cell-derived structures bounded by a lipid bilayer membrane arising from both herniated evagination (blebbing) separation and sealing of portions of the plasma membrane or from the export of any intracellular membrane-bounded vesicular structure containing various membrane-associated proteins of tumor origin, including surface-bound molecules derived from the host circulation that bind selectively to the tumor-derived proteins together with molecules contained in the exosome lumen, including but not limited to tumor-derived microRNAs or intracellular proteins.
  • CTEs Circulating tumor-derived exosomes
  • CTEs Circulating tumor-derived exosomes
  • CTEs typically have unique biomarkers that permit their isolation from bodily fluids in a highly specific manner.
  • Exosomes can be directly assayed from the biological samples, such that the level of exosomes is determined or the one or more biomarkers of the exosomes are determined without prior isolation, purification, or concentration of the exosomes. Alternatively, exosomes may be isolated, purified, or concentrated from a sample prior to analysis. Isolation of Exosomes
  • An exosome may be purified or concentrated prior to analysis.
  • total exosomes are isolated from a biological sample such as serum and blood, for example using Invitrogen Total Exosome Isolation Kit.
  • Analysis of an exosome can include quantitating the amount one or more exosome populations of a biological sample. For example, a heterogeneous population of exosomes can be quantitated, or a homogeneous population of exosomes, such as a population of exosomes with a particular biomarker profile, a particular bio-signature, or derived from a particular cell type (cell-of-origin specific exosomes) can be isolated from a heterogeneous population of exosomes and quantitated. Analysis of an exosome can also include detecting, quantitatively or
  • An exosome can be stored and archived, such as in a bio-fluid bank and retrieved for analysis as necessary.
  • An exosome may also be isolated from a biological sample that has been previously harvested and stored from a living or deceased subject.
  • an exosome may be isolated from a biological sample which has been collected as described in King et al., Breast Cancer Res 7(5): 198-204 (2005) or isolated from an archived or stored sample.
  • an exosome may be isolated from a biological sample and analyzed without storing or archiving of the sample.
  • a third party may obtain or store the biological sample, or obtain or store the exosomes for analysis.
  • exosomes can be obtained from a biological sample.
  • exosomes may be concentrated or isolated from a biological sample using size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof. These methods can be used to separate exosomes from contaminants.
  • exosomes can be isolated by differential centrifugation, anion exchange and/or gel permeation chromatography (for example, as described in U.S. Pat. Nos. 6,899,863 and 6,812,023), sucrose density gradients, organelle electrophoresis (for example, as described in U.S. Pat. No. 7,198,923), magnetic activated cell sorting (MACS), or with a nanomembrane ultrafiltration concentrator.
  • Various combinations of isolation or concentration methods can be used.
  • exosomes may hinder isolation of exosomes from a biological sample.
  • exosomes may be isolated from a biological sample using a system that utilizes multiple antibodies that are specific to the most abundant proteins found in blood. Such a system can remove up to several proteins at once, thus unveiling the lower abundance species such as cell- of- origin specific exosomes.
  • This type of system can be used for isolation of exosomes from biological samples such as blood.
  • the isolation of exosomes from a biological sample may also be enhanced by high abundant protein removal methods as described in Chromy et al. J. Proteome Res 2004; 3: 1120- 1127.
  • the isolation of exosomes from a biological sample may also be enhanced by removing serum proteins using glycopeptide capture as described in Zhang et al, Mol Cell Proteomics 2005; 4: 144-155.
  • exosomes from a biological sample such as urine may be isolated by differential centrifugation followed by contact with antibodies directed to cytoplasmic or anti-cytoplasmic epitopes as described in Pisitkun et al., Proc Natl Acad Sci USA, 2004; 101: 13368-13373.
  • Isolation or enrichment of exosomes from biological samples can also be enhanced by use of sonication (for example, by applying ultrasound), or the use of detergents, other membrane- active agents, or any combination thereof.
  • Example 1 Materials and Methods Cell isolation
  • PBMC peripheral blood mononuclear cells
  • CD3+ T cells were obtained by depletion of CD14, CD16, CD19, CD36, CD56, CD123, and Glycophorin A positive cells using Pan T cell Isolation Kit (Miltenyi Biotec, Auburn, CA).
  • CD4+ or CD8+ T cells were magnetically sorted from CD3+ T cells to a purity of 93-98% using CD4 or CD8 isolation kits (Miltenyi Biotec) for negative selection.
  • CD8+ T suppressor cells (1 _ 106 cells/ml) from responder A were stimulated with allogeneic APC, consisting of irradiated CD3-depleted PBMCs (0.5 _ 106 cells/ml) from stimulator B, in cultures containing ILT3Fc (50 ⁇ g/mL). 7 days after priming, CD8+ T cells were negatively selected and tested for suppressor activity. For suppressor assays, putative Ts were added to primary A anti-B MLC at a 1: 1, 1:2, 1:4, and 1:8 suppressor to responder cell ratio.
  • the Fetal Bovine Serum used for all cell culture was exosome depleted.
  • Primary MLCs were set up using responding CD3+ T cells (5 _ 104 cells/well) and allogeneic CD3-depleted stimulating APCs (2.5 _ 104 cells/well) irradiated with 3000 rads.
  • CD8+ Ts treated or not treated with exosomes were added to MLCs containing unprimed, autologous CD4+ T cells (from responder A) and APC from the original stimulator (B) used for priming.
  • Cell cultures were labeled with [3H] thymidine on day 5 after priming and harvested after another 18 h.
  • the percent of inhibition of CD4+ T cell proliferation by CD8+ Ts cells was calculated according to the formula: (1 _ cpm of [Ts + Th + APCs]/cpm of [Th + APCs])_100 .
  • CD8+ Ts generated from MLC containing ILT3Fc were collected after 6 days and incubated for an additional 24 h with exosomes isolated from MLC grown in medium without ILT3-Fc (MLC exosomes) or with ILT3Fc (ILT3 exosomes). CD8+ Ts incubated in fresh medium served as a control.
  • Total exosomes were isolated from cell cultures using Invitrogen Total Exosome Isolation Kit (from cell culture media). Briefly, 5 mL of cell cultures were collected and centrifuged at 1200g for 10 min to remove live cells. Supernatants were centrifuged at 13,200 rpm for 1 h at 4 _C to remove cell debris. The final supernatants were mixed with exosome isolation reagents at a ratio of 2: 1 and rolled overnight at 4 _C. The next day, the mixture was centrifuged at 13,200 rpm for 1 h at 4 _C. The supernatant was discarded and the exosome enriched pellet was suspended in PBS buffer and preserved at 4 _C for later use [16].
  • total RNA including miRNA from plasma or sera can be isolated by using the miRNeasy kit (Qiagen) by spinning 200 ⁇ sera/plasma down in a micro-centrifuge tube for 10 min at the highest speed at 4°C.
  • the pellet can be transferred to a clean tube containing 700 ⁇ of Qiazol reagent, and mixed by vertex 5-6 times. Then, it can be spiked with 10 ⁇ diluted (1: 1000) miR-39 from C. elegans (undiluted micorRNA is 5 n mole/lml H20). 140 ⁇ of chloroform can be added, and vigorously mixed for 15 sec. centrifuged at the highest speed at 4C for 15 min.
  • the aqueous phase (500 ⁇ ) can be transferred to a new tube and 750 ⁇ of 100% ethanol can be added.
  • the contents can be loaded onto a RNA column provided by the Qiagen kit, following the protocol by washing with the buffers provided. After spin drying the column, RNA can be eluted by adding 35 ul RNase free water provided in the kit, sitting on a 42- 65°C dry bath for 1 min. Then, the RNA can be spun down at 10,000 RPM at room temp.
  • miRNA mimics miR-21, miR-30b, and miR-155
  • control scrambled RNA oligonucleotide purchased from Ambion. miRNA mimics were added to activated CD8+ T cells (1 _ 106) using Amaxa's Human T cell Nucleofector Kit (Lonza) according to the manufacturer's instruction [16].
  • ILT3Fc inhibits the release into the medium of inflammatory exosomes by T cells primed in MLC.
  • Ts were then tested in doubling dilutions for their effect on the proliferation of naive, autologous T cells from responder A to the allogeneic APC from stimulator B.
  • Ts When transferred to primary MLC at 50,000 and 25,000 Ts per culture, Ts induced >90 and >80 inhibition of MLC reactivity respectively, regardless of whether fresh medium or exosomes from culture grown with or without ILT3.Fc were used for "pre-treatment" .
  • Ts to T responding cell ratio (1:4 and 1:8, i.e.
  • Ts pre-treated with exosomes from MLC without ILT3.Fc were less inhibitory than Ts pre-treated with fresh medium, allowing T cell proliferation to return to levels seen in MLC without Ts.
  • MLC supernatants therefore, contain inflammatory exosomes which abrogate the inhibitory effect of Ts. This indicates that ILT3Fc inhibits the release into the medium of inflammatory exosomes by T cells primed in MLC.
  • exosomes may contain inflammatory miRNA which abolish the Ts effect and/or enhance T cell proliferation in the presence of limiting numbers of Ts (Table 1).
  • T suppressor cells were generated by sorting CD8+ T cells from a primary MLC containing ILT3.Fc on day 7. T suppressor cells were then transfected with different miRNA mimics (miR-21, miR-30b, miR-155 and scrambled mimics as control) by using Amaxa's
  • ILT3.Fc inhibited by 90% or more the release into exosomes of miR-146a, miR-155, miR-21, yet was less suppressive for miR-30b, miR-365 and miR-7a, which were inhibited by 50%, 30% and 30% respectively (FIG. 3). Comparison of the relative expression level of these miRNAs in
  • CD4+ and CD8+ cells from primary MLR showed 90% higher levels of miR-146a, miR-155 and miR-21 in CD4+ compared to CD8+ T cells.
  • the expression level of miR30b, miR365 and Let- 7a was 30%, 50% and 50% lower respectively, in exosomes from supernatants of primed CD8+ compared to CD4+ T cells
  • Exosomes produced by alloactivated CD4+ T cells contain inflammatory molecules which diminish the inhibitor activity of CD8+ Ts.
  • Exosomes produced by alloactivated CD4 + T cells contain inflammatory molecules which diminish the inhibitory activity of CD8 + Ts.
  • the mir21 data summarized in Table 1 show that none of the 7 patients with negative biopsies had elevated serum miR21 levels in post-transplantation samples. By contrast, 4 out of the 5 patients who had positive biopsies for grade 2R/3A rejection had significantly elevated post-transplantation serum miR21 compared to their respective pre-transplant level. Thus, the Positive Predictive Value (PPV) for grade 2R/3A rejection in 4/4 samples showing elevated serum miR21 was 100%.
  • the sensitivity level for a diagnostic test for grade 2R/3A rejection based on elevated miR in 4/5 patients was 80% (i.e. 1 of the 5 serum samples from patients known to have grade 2R/3A rejection did not have elevated miR21). Thus, reliance on serum miR21 levels to diagnose grade 2R/3A rejection, would have a false negative rate of 20%, based on these results.
  • the negative predictive value (58/59) was 98%.
  • Day 54 (0% 1R/1A), Day 85 (0%, 1R/1B), Day 179 (0%, IR/IB), Day 196 (0%, 0)
  • Day 261 (0%, 0), Day 350 (0%, 0), Day 449 (6%, 0) Day 16 (0%, 0), Day 46 (0%, 1R/1A), Day 88 (0%, 1R/1A),
  • An embodiment is directed to a method for diagnosing grade 2R/3A rejection of a heart allograft in a subject by obtaining a pre-allograft serum sample and a post-allograft sample, and if the miR21 level in the pre-allograft sample is at least 40% higher than the miR21 level in the post-allograft sample, then the subject is diagnosed with a grade 2R/3A rejection.
  • the above embodiment can further comprise performing a biopsy to confirm the diagnosis of 2R/3A rejection, and treating the subject for 2R/3A rejection, for example by administering ILT3-Fc or the extracellular domain of ILT3.
  • transcript 3 inhibits tumor allograft rejection in humanized SCID mice and T
  • Immunoglobulin-like transcript 3-Fc suppresses T-cell responses to

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Abstract

L'invention concerne la découverte selon laquelle des lymphocytes T CD4+ alloactivés amorcés par (MLC) fabriquent et libèrent des exosomes inflammatoires qui contiennent des taux élevés d'un ou plusieurs miARN inflammatoires dans le groupe miR21, 30b, 146a, 155, 365 et Let-7a. L'invention concerne une méthode pour la détection précoce d'une inflammation médiée par les lymphocytes T fondée sur la détection de taux significativement élevés de certains microARN inflammatoires dans des exosomes dans un échantillon biologique isolé après d'un sujet.
PCT/US2014/066238 2013-11-18 2014-11-18 Méthodes de diagnostic d'une inflammation médiée par les lymphocytes t WO2015074083A1 (fr)

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CN110885789A (zh) * 2018-09-05 2020-03-17 中国科学院生物物理研究所 高效可控包装內源核酸的工程化外泌体制备及其应用

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WO2010056337A2 (fr) * 2008-11-12 2010-05-20 Caris Mpi, Inc. Procédés et systèmes d'utilisation d'exosomes pour déterminer des phénotypes
WO2013036282A2 (fr) * 2011-09-07 2013-03-14 The Trustees Of Columbia University In The City Of New York Régulation à la baisse de microarn inflammatoires par l'ilt3

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010056337A2 (fr) * 2008-11-12 2010-05-20 Caris Mpi, Inc. Procédés et systèmes d'utilisation d'exosomes pour déterminer des phénotypes
WO2013036282A2 (fr) * 2011-09-07 2013-03-14 The Trustees Of Columbia University In The City Of New York Régulation à la baisse de microarn inflammatoires par l'ilt3

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110885789A (zh) * 2018-09-05 2020-03-17 中国科学院生物物理研究所 高效可控包装內源核酸的工程化外泌体制备及其应用

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