Method for providing information necessary for prediction of likelihood of developing rheumatoid arthritis or for prognosis of same

Abstract

The present invention relates to a novel marker for predicting the likelihood of developing rheumatoid arthritis or for prognosis of same, and a novel therapeutic target for preventing or treating rheumatoid arthritis. As such, the likelihood of developing rheumatoid arthritis or the likely course of same can be predicted, and a method for screening for a preventative or therapeutic agent can be provided.

Description

Methods for providing information necessary to predict the likelihood of developing or prognosticating rheumatoid arthritis

[0001] The present invention relates to a method for providing information necessary for predicting the possibility of developing or prognosing rheumatoid arthritis.

[0002]

[0003] Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of unknown cause. It causes inflammation, swelling, and pain in the joints throughout the body, starting with the peripheral joints of the fingers, toes, and wrists. It is a disease that begins with inflammation and infiltration of immune cells into the synovium surrounding the joints, followed by the creation of new blood vessels, destruction of articular cartilage, and bone damage, resulting in functional impairment and deformation of the joints.

[0004] Rheumatoid arthritis is thought to be caused by genetics, viral and bacterial infections, physical and mental stress, and smoking. Complications of rheumatoid arthritis include organ involvement (lungs, kidneys, heart), osteoporosis, and vasculitis. The resulting joint deformation and chronic pain are closely related to the patient's physical disability and reduced quality of life. It is a disease with a significant economic burden, ranking 5th in disease burden expenditures and increasing medical expenses by more than 10% every year, so appropriate treatment is essential.

[0005] The rheumatoid arthritis treatment market is a huge disease market worth approximately KRW 32 trillion, with four drugs occupying the top five in total global pharmaceutical sales, and the largest autoimmune disease market. The global rheumatoid arthritis drug market is expected to grow at a compound annual growth rate (CAGR) of 4.34% from 2021 to 2030, reaching a value of over USD 39.26 billion by 2030.

[0006] Currently, rheumatoid arthritis is primarily treated with steroids and analgesics after diagnosis, focusing on disease-modifying antirheumatic drugs. However, these drugs have limitations such as insufficient therapeutic efficacy and various side effects due to immunosuppressive effects. Recently, the widespread use of various biological agents, such as TNF-α inhibitors, IL-6 receptor inhibitors, T-cell inhibitors, and B-cell inhibitors, has improved the treatment outcome of rheumatoid arthritis. However, insufficient therapeutic efficacy is still observed in 30% of patients, and side effects such as a 5-15-fold increase in the incidence of tuberculosis and a 2-4-fold increase in the incidence of serious infections occur. In particular, the fact that rheumatoid arthritis relapses in more than 50% of patients after discontinuation of the drug is a fundamental limitation of treatment. There is a persistent need to develop new treatments for rheumatoid arthritis that can overcome the problems of existing rheumatoid arthritis treatments: 1) increased incidence of infectious side effects, 2) presence of patients who are refractory to treatment, 3) relapse after discontinuation, 4) decreased compliance due to decreased preference for injections, and 5) reduced injection side effects and efficacy.

[0007]

[0008] The present invention aims to provide a new information providing method capable of accurately predicting the possibility of developing or prognosing rheumatoid arthritis.

[0009] The present invention aims to provide a method for screening a candidate substance for the prevention or treatment of rheumatoid arthritis.

[0010]

[0011] The present invention relates to a method for detecting CD25 in the small intestine low Foxp3 + The present invention relates to a method for providing information necessary for predicting the possibility of developing rheumatoid arthritis, which provides information that if the number of T cells is greater than that of the control group, the possibility of developing rheumatoid arthritis in the subject is greater than that of the control group.

[0012] In the method of the present invention, the small intestine may be a small intestine mucosa.

[0013] In addition, the present invention relates to a method for detecting CD25 in the small intestine in a subject low Foxp3 + The present invention relates to a method for providing information necessary for predicting the prognosis of rheumatoid arthritis, which provides information that if the number of T cells is greater than that of the control group, the subject will have a worse prognosis of rheumatoid arthritis than the control group.

[0014] In the method of the present invention, the small intestine may be a small intestine mucosa.

[0015] In addition, the present invention provides Foxp3 in the small intestine + Increase the expression of CD25 in T cells, or CD25 low Foxp3 + The present invention relates to a screening method for selecting a substance that reduces the number of T cells or suppresses their differentiation into pathological Th17 cells (T helper 17 cells) as a candidate substance for preventing or treating rheumatoid arthritis.

[0016] In the method of the present invention, the small intestine may be a small intestine mucosa.

[0017]

[0018] The present invention relates to CD25 low Foxp3 + Based on the differentiation of T cells into pathological Th17 cells, this can be used as a marker to predict the likelihood of developing or prognosis of rheumatoid arthritis.

[0019] The present invention provides a new therapeutic target for preventing or treating rheumatoid arthritis, and can screen candidate substances for preventing or treating the disease.

[0020]

[0021] Figure 1. CD25 in peripheral blood of patients with rheumatoid arthritis (RA). low Foxp3 + T cell profile. CD25 in peripheral blood of patients with severe RA with DAS28 / CRP 5.1 or higher. low Foxp3 +The pattern of T cells was analyzed. There was a higher number of CD25 in the peripheral blood of RA patients compared to normal people. low Foxp3 + The presence of T cells was observed.

[0022] Fig. 2. CD25 of the Minister's LP low Foxp3 + T cell profile and changes in arthritis development. Foxp3 in various lymphoid organs (spleen, lymph nodes, lamina propria (LP) of the small and large intestine) present in the body. + We evaluated the CD25 expression pattern of T cells. As a result, Foxp3 present in the LP of the small intestine + T cells in other lymphoid organs express Foxp3 + Unlike T cells, CD25 expression was low. CD25 was expressed in the LP of the small intestine. low Foxp3 + T cells were present in high proportions (Fig. 2A-C). In addition, CD25 cells were present in the LP of the small intestine during RA induction. low Foxp3 + T cells were found to increase (Figure 2D).

[0023] Figure 3. Fibrous synovial cells (FLS) and intestinal LP CD25 in RA patients. low Foxp3 + Analysis of the T cell-specific chemokine-chemokine receptor axis. Using data uploaded to the NCBI genome database, we produced data by comparing and analyzing the expression of genes specifically expressed when exposed to an inflammatory environment in RA patient FLS (Figure 3A, top). In addition, Foxp3 in the spleen and lymph nodes was analyzed. + Unlike T cells, Foxp3 in small intestinal LP +Gene expression data specifically expressed by T cells were generated (Figure 3A, bottom). Analysis of these generated data, focusing on the chemokine-chemokine receptor axis, revealed an interaction relationship between FLS-specific chemokines in RA patients and chemokine receptors of small intestinal LP Tregs (Figure 3B). In the future, CD25 of small intestinal LP low Foxp3 + T cells may be used to elucidate the mechanisms of arthritis development and screen therapeutic targets during the onset of RA.

[0024] Figure 4. Spleen and intestine LP CD25 - Foxp3 + Evaluation of T cell division inhibition capacity. CD25 in the spleen (Sp) of Foxp3-GFP mice. - Foxp3 - T cells (Tresp) were isolated and stimulated with anti-CD3 / CD28 for 3 days, while simultaneously expressing CD25 of Sp. + Foxp3 + , CD25 - Foxp3 + , CD25 of the small intestine (Si) - Foxp3 + Evaluation of anti-mitotic activity by co-culture with regulatory T cells (Tregs). CD25 + Foxp3 + CD25 in small intestinal LP compared to regulatory T cells - Foxp3 + T cells lost the ability to suppress the division of responding cells (Fig. 4A), and the levels of inflammatory cytokines secreted in the culture supernatant were higher (Fig. 4B).

[0025] Figure 5. Spleen and intestine LP CD25 - Foxp3 + Verification of T cell differentiation into Th17 cells. Sp CD25 in Foxp3-GFP mice. + Foxp3 + , CD25 - Foxp3+ , small intestine (Si) CD25 - Foxp3 + After isolating Tregs and stimulating them with anti-CD3 / CD28 for 3 days under Th17 differentiation conditions, the degree of differentiation into Th17 cells was compared. As a result, CD25 of small intestine LP was higher than that of spleen Treg cells. - Foxp3 + Increased differentiation of Treg cells into Th17.

[0026]

[0027] The present invention is described in detail below.

[0028]

[0029] The present invention relates to a method for providing information necessary for predicting the possibility of developing rheumatoid arthritis.

[0030] The method of the present invention is to detect CD25 in the small intestine of a subject low Foxp3 + If the number of T cells is higher than that of the control group, it provides information that the subject is more likely to develop rheumatoid arthritis than the control group.

[0031] The subject is any subject without rheumatoid arthritis whose risk of developing rheumatoid arthritis is to be predicted. This subject can be a mammal, including humans, or a human.

[0032] CD25 of the small intestine low Foxp3 + The number of T cells is a marker for predicting the likelihood of developing rheumatoid arthritis.

[0033] The small intestine as a sample may be, for example, the small intestinal mucosa, which may be the small intestinal lamina propria. The small intestine, the small intestinal mucosa, or the small intestinal lamina propria may be targeted, in whole or in part.

[0034] Foxp3 + T cells are T cells that express Foxp3.

[0035] CD25 low Foxp3 +T cells Foxp3 + Among T cells, these cells have low expression of CD25. Foxp3 + When measuring the expression of CD25 on T cells, cells are divided into those with high CD25 expression and those without, depending on the level of expression. Among them, cells with low CD25 expression are CD25 low Foxp3 + It is a T cell.

[0036] Foxp3 + When CD25 expression is confirmed in T cells, Foxp3 + CD25 among T cells low Foxp3 + T cell ratio can be confirmed. And confirmed CD25 low Foxp3 + When counting T cells, CD25 low Foxp3 + The number of T cells can be checked.

[0037] The comparison group is any subject without rheumatoid arthritis whose risk of developing rheumatoid arthritis is being compared. This can be any mammal, including humans, or even a human. It can also be of the same species as the subject.

[0038] The sample of the control group may be identical to the sample of the subject.

[0039] CD25 low Foxp3 + T cells are significantly increased in the peripheral blood and small intestine of patients with rheumatoid arthritis compared to healthy controls, and these cells can differentiate into pathological Th17 cells, which can induce and worsen rheumatoid arthritis. Therefore, CD25 in the small intestine of the subject low Foxp3 + If the number of T cells is higher than that of the control group, the subject can be judged to have a higher risk of developing rheumatoid arthritis than the control group.

[0040]

[0041] In addition, the present invention relates to a method for providing information necessary for predicting the prognosis of rheumatoid arthritis.

[0042] The method of the present invention is to detect CD25 in the small intestine of a subject low Foxp3 + If the number of T cells is higher than that of the control group, it provides information that the subject will have a worse prognosis for rheumatoid arthritis than the control group.

[0043] The subject is a patient with rheumatoid arthritis whose prognosis is to be predicted. This subject may be a mammal, including a human, or a human.

[0044] Small intestine, Foxp3 + T cells, CD25 low Foxp3 + T cells, the ratio of which may be the same as described above.

[0045] The control group is any subject with rheumatoid arthritis whose prognosis is to be compared. This group can be any mammal, including humans, or even a human. It can also be of the same species as the subject.

[0046] CD25 low Foxp3 + T cells are significantly increased in the peripheral blood and small intestine of patients with rheumatoid arthritis compared to healthy controls, and these cells can differentiate into pathological Th17 cells, which can induce and worsen rheumatoid arthritis. Therefore, CD25 in the small intestine of the subject low Foxp3 + A higher number of T cells than the control group may provide information that the subject will have a worse prognosis for rheumatoid arthritis than the control group.

[0047] Pathological Th17 cells can be Th17 cells that secrete both IFN-γ and IL-17A, or Th17 cells that secrete both GM-CSF and IL-17A.

[0048]

[0049] In addition, the present invention relates to a method for screening a candidate substance for preventing or treating rheumatoid arthritis.

[0050] The method of the present invention is Foxp3 in the small intestine + Increase the expression of CD25 in T cells, or CD25 low Foxp3 + Substances that reduce the number of T cells or inhibit their differentiation into pathological Th17 cells are selected as candidates for preventing or treating rheumatoid arthritis.

[0051] The small intestine can be, for example, the small intestinal mucosa, which can be the small intestinal mucosa lamina propria.

[0052] Foxp3 + T cells, CD25 low Foxp3 + T cells, the ratio of which may be the same as described above.

[0053] CD25 low Foxp3 + T cells can differentiate into pathological Th17 cells and induce and worsen rheumatoid arthritis. Therefore, Foxp3 + Increase the expression of CD25 in T cells, or CD25 low Foxp3 + Reducing the number of T cells may prevent or treat rheumatoid arthritis. Therefore, such substances could be selected as candidates for the prevention or treatment of rheumatoid arthritis.

[0054] CD25 low Foxp3 + T cells differentiate into pathological Th17 cells, secreting cytokines such as IL-17A, IL-17F, and IL-21. This can induce and worsen rheumatoid arthritis. Therefore, CD25 low Foxp3 + Since inhibition of differentiation of T cells into pathological Th17 cells blocks the factors that cause and aggravate rheumatoid arthritis, such substances can be selected as candidates for the prevention or treatment of rheumatoid arthritis.

[0055] The substances are not limited in type, including natural products, compounds, polymers, proteins, peptides, and nucleic acids.

[0056]

[0057] The present invention will be described in more detail with reference to the following examples.

[0058]

[0059] Example

[0060] Materials and Methods

[0061] CD25 in peripheral blood of patients with rheumatoid arthritis (RA) low Foxp3 + T cell analysis (Figure 1)

[0062] CD25 in peripheral blood low Foxp3 + Blood samples for T cell count analysis were provided by patients visiting Gyeongsang National University Hospital. Ten samples were from patients with rheumatoid arthritis (RA) with severe disease activity, defined as a Disease Activity Score 28 (DAS28)-C-reactive protein (CRP) level of 5.1 or higher, and four samples were from healthy controls (HC).

[0063] Blood cell separation was performed using MColl (RadianQbio) to separate white blood cells, then centrifuged (400 g, 5 min, room temperature) to remove the supernatant, and 9 mL of RPMI medium (Gibco) was added to suspend the cells. The total cell number was then measured using a cell counting device (Bio-rad).

[0064] Leukocytes isolated from blood were stained with fluorescently conjugated antibodies (Table 1) at 4°C for 30 minutes and then analyzed using LSRFortessa TM LiveCD4 using X-20 flow cytometer (BD Biosciences) + CD25 low Foxp3 +The percentage of CD25 T cells per mL of blood was analyzed in RA patients and HC. low Foxp3 + T cell count ({total white blood cell count / mL Х LiveCD4 + CD25 low Foxp3 + %} / 100) were compared and analyzed.

[0065]

[0066] AntibodiesSourceFixable Viability Dye eFluor TM 506eBioscienceAnti-human CD4, FITC, clone OKT4BD BioscienceAnti-human Foxp3, APC, clone PCH101eBioscienceAnti-human CD25, BV605, clone M-A251BioLegend

[0067]

[0068] CD25 in lymphoid organs of normal and rheumatoid arthritis mice low Foxp3 + T cell analysis (Figure 2)

[0069] 1) Construction of a rheumatoid arthritis mouse model (collagen-induced arthritis; CIA): A rheumatoid arthritis mouse model (collagen-induced arthritis mouse model; CIA mouse model) was constructed using collagen. Briefly, 2 mg / mL of bovine type 2 collagen (CII) and 1 mg / mL of complete Freund's adjuvant (CFA) were mixed and intradermally injected into the tail of 7-8 week-old male DBA / 1J mice at 100 μL per mouse (Day 0). Twenty-one days later, arthritis was induced by boosting the mixture by intradermally injecting 100 μL per mouse into the tail of DBA / 1J mice at 1 mg / mL of CII and 1 mg / mL of incomplete Freund's adjuvant (IFA) (Day 21). After boosting, the arthritis index was assessed, and mice were sacrificed at the peak of arthritis (Day 42), after which lymphoid organs (spleen, lymph nodes, small intestine, and large intestine) were collected. DBA / 1J mice of the same age without arthritis were used as controls.

[0070] 2) Isolation of immune cells in mouse lymphoid organs and CD25 low Foxp3 + T cell analysis: Spleen (Sp), inguinal lymph nodes (Inguinal LN), and mesenteric lymph nodes (Mesenteric LN) from normal and CIA mice were removed, pulverized, and centrifuged at 1,500 rpm for 5 minutes to harvest cells. For spleen cells, red blood cells were hemolyzed by reacting with ACK lysis buffer for 5 minutes, and then centrifuged at 1,500 rpm for 5 minutes to harvest cells free of red blood cells.

[0071] In addition, immune cells were isolated from the lamina propria (LP) of the small intestine (Si) and large intestine (Li) of normal and CIA mice. The isolation method was as follows. The intestinal tissues, from which fat and Peyer's patches had been removed, were cut lengthwise to expose the intestinal mucosa, and then washed with PBS to remove the intestinal contents. After washing, the intestinal tissues were cut into 1-2 cm lengths and placed in PBS containing 1 mM DTT, 30 mM EDTA, and 10 mM HEPES. The intestinal tissues were shaken at 37℃ for 10 minutes to collect them. The collected intestinal tissues were further shaken in PBS containing 30 mM EDTA and 10 mM HEPES at 37℃ for 10 minutes, and then placed in RPMI medium containing collagenase (100 U / mL) and DNase (150 μg / mL) to perform enzyme digestion at 37℃ for 60 minutes. Lamina propria mononuclear cells (LPMC) were isolated by centrifugation (1000 g, 20 min, room temperature) using 80% and 40% percoll gradient solutions from the cells recovered from the intestinal tissue.

[0072] Total cell count was measured using a cell counter (Bio-rad), and then the cells were stained with fluorescently conjugated antibodies (Table 2) at 4°C for 30 minutes and analyzed for LiveCD4 using an LSRFortessaTM X-20 flow cytometer (BD Biosciences). + CD25 - Foxp3 + Wow LiveCD4 + CD25 + Foxp3 + The percentage of T cells in each tissue was analyzed. CD25 - Foxp3 + Wow CD25 - Foxp3 +After converting the T cell count ({total number of cells X corresponding cell %} / 100), the counts were compared and analyzed between each tissue or between normal and CIA mice.

[0073]

[0074] AntibodiesSourceFixable Viability Dye eFluor TM 506eBioscienceAnti-mouse CD4, BV421, clone GK1.5BioLegendAnti-mouse CD25, PE, clone PC61.5eBioscienceAnti-mouse Foxp3, APC, clone FJK-16seBioscience

[0075]

[0076] Fibroblasts (FLS) and intestinal LP CD25 in RA patients low Foxp3 + Analysis of the T cell-specific chemokine-chemokine receptor axis (Figure 3)

[0077] Analysis was performed using a data analysis tool called ImaGEO, and the data used were public data released by NCBI, including GSE71316, GSE58145, GSE20366, GSE41229, and GSE120785, and fibrous synovial cells (FLS) and intestinal LP CD25 of RA patients. low Foxp3 + We performed a T cell-specific chemokine-chemokine receptor axis analysis.

[0078]

[0079] Spleen and Small Intestine LP CD25 - Foxp3 + Evaluation of T cell division inhibition ability (Figure 4)

[0080] 1) Isolation of mouse spleen and small intestine cells: Foxp3-GFP mice (C.Cg-Foxp3 tm2Tch / J, Strain #: 006769, RRID: IMSR_JAX: 006769) were isolated from the spleen and small intestine, and the cell isolation method was the same as described in the above mouse lymphoid organ immune cell isolation and CD25lowFoxp3+ T cell analysis.

[0081] 2) T cell sorting according to CD25 and Foxp3 expression: CD4 was isolated from cells prepared from the mouse spleen and small intestine cells using a CD4 isolation kit (Miltenyi Biotec). + T cells were isolated. Isolated CD4 + After staining with fluorescently conjugated antibodies (Table 3) for 30 min at 4°C, splenic CD4 cells were sorted using a sorter (CytoFlex SRT). + CD25 among T cells - Foxp3 - T cells, CD25 + Foxp3 + , CD25 - Foxp3 + Regulatory T (Treg) cells, small intestinal CD4 + CD25 among T cells - Foxp3 + Treg cells were isolated individually.

[0082] AntibodiesSourceFixable Viability Dye eFluor™780eBioscienceAnti-mouse CD4, PerCP-Cyanine5.5, clone GK1.5BioLegendAnti-mouse CD25, APC, clone PC61BD Bioscience

[0083] 3) Evaluation of the division inhibition ability of sorted T regulatory cells: CD25 cells from the spleen isolated from T cell sorting according to the above CD25 and Foxp3 expression - Foxp3 - After centrifugation and removal of supernatant, T cells were centrifuged at 4X10 6Cells were suspended by adding PBS containing 0.1% bovine serum albumin (BSA) to the cell pellet to obtain cells / mL (preparation of responding T cells). Responding T cells were stained by mixing PBS (+0.1% BSA) containing 10 μM CellTrace Violet (CTV, Invitrogen) 1:1 with the responding T cell suspension and incubating for 20 minutes in a darkroom (CTV labeling of responding cells). Complete RPMI media (Gibco, +10% fetal bovine serum [FBS] and 1X penicillin / streptomycin [P / S]) equivalent to 5 times the volume of the cell staining mixture was added and incubated for 5 minutes (stopping the staining reaction), and the supernatant was removed by centrifugation at 300 g for 10 min. Afterwards, 2 X 10 6 Complete RPMI media was added to the pellet to suspend the cells at 100 cells / mL (preparation of CTV-stained responding T cells).

[0084] Responding T cells stained with CTV were plated at 2 Х 10 in a 96-well plate. 5 cells / well. Afterwards, spleen CD25 + Foxp3 + , CD25 - Foxp3 + Treg cells or small intestinal CD25 - Foxp3 + Treg cells (1 Х 10 5 cells) were added to the wells containing responding T cells and co-cultured (Responding T: Treg = 2:1). At the same time, Dynabeads Mouse T-Activator CD3 / CD28 (Gibco) was added at 1.5 х 10 per well. 5The proliferation of responding T cells was stimulated by culturing them for 3 days with beads (bead-to-cell ratio = 0.5:1).

[0085] On the third day of culture, the culture supernatant was collected and cytokines secreted into the supernatant were measured using a cytometric bead array (CBA) kit. Cells recovered from 96 wells were stained with fluorescently conjugated antibodies (Table 3) at 4°C for 30 minutes and then analyzed for CD25 using an LSRFortessaTM X-20 flow cytometer (BD Biosciences). - Foxp3 - The fluorescence intensity of CTV of responding T cells was analyzed. This analysis utilized the principle that the fluorescence intensity of CTV weakens as responding T cells divide and proliferate. In other words, the division-inhibitory ability of co-cultured splenic and small intestinal Treg cells against responding T cells was evaluated through analysis of CTV fluorescence intensity within responding T cells.

[0086]

[0087] Spleen and Small Intestine LP CD25 - Foxp3 + Evaluation of T cell differentiation into Th17 cells (Figure 5)

[0088] The spleen and small intestine of Foxp3-GFP mice were removed and cells were isolated. The cell isolation method was the isolation of immune cells in the lymphoid organs of the mice and CD25 low Foxp3 + As described in T cell analysis, CD4 isolation and sorting were performed as in the T cell sorting according to CD25 and Foxp3 expression above, and splenic CD25 + Foxp3 + , CD25 - Foxp3 + Treg, small intestine CD25 - Foxp3 + Treg cells were obtained.

[0089] Each Treg cell was seeded at 1 Х 10 in a 96-well plate. 5 Cells were seeded per well, and at the same time, Dynabeads Mouse T-Activator CD3 / CD28 (Gibco) 5Х10 4 Beads / well (bead-to-cell ratio = 0.5:1) and mouse (m) IL-6 (100 ng / mL), mIL-23 (50 ng / mL), mIL-1β (10 ng / mL), and human TGF-β1 (10 ng / mL) included in the Mouse CytoBox Th17 kit (Miltenyi Biotec) were treated and cultured for 3 days to induce differentiation into Th17 cells.

[0090] On the third day of culture, cells recovered from 96 wells were treated with PMA (Sigma-Aldrich, 50 ng / mL), Ionomycin (Sigma-Aldrich, 500 μg / mL), and BD GolgiPlug (BD bioscience, 1 μg / mL) for 5 h, and then stained with fluorescence-conjugated antibodies (Table 4) at 4°C for 30 min. Afterwards, IL-17A in splenic and small intestinal Treg cells was analyzed using an LSRFortessaTM X-20 flow cytometer (BD Biosciences). + The percentage of cells was analyzed to compare and evaluate the degree of differentiation into Th17.

[0091]

[0092] AntibodiesSourceFixable Viability Dye eFluor TM 506eBioscienceAnti-mouse CD4, PE-Cyanine7, clone GK1.5eBioscienceAnti-mouse CD25, PE, clone PC61.5eBioscienceAnti-mouse IL-17A, APC, clone eBio17B7eBioscience

[0093]

[0094] result

[0095] CD25 in peripheral blood of patients with rheumatoid arthritis (RA) low Foxp3 + Increased T cells

[0096] Leukocytes were isolated from the peripheral blood of RA patients with severe disease activity (DAS28-CRP 5.1 or higher) and healthy controls (HC), stained with CD4, Foxp3, and CD25 fluorescent antibodies, and analyzed by flow cytometry.

[0097] CD4 + Foxp3 + After gating T cells, CD25 was determined based on the level of CD25 expression. low Wow CD25 high It was classified into one cell, and based on this, CD4 + CD25 low Foxp3 + (hereinafter simply referred to as CD25 low Foxp3 + The number of T cells was calculated (named as CD25). As a result, CD25 low Foxp3 + We observed that T cells were significantly increased in the peripheral blood of RA patients compared to the peripheral blood of HC (Fig. 1).

[0098]

[0099] CD25 of Mouse Collection LP low Foxp3 + T cell distribution and its relationship to arthritis development

[0100] CD25 found to be increased in the peripheral blood of RA patients low Foxp3 + To determine the extent to which T cells are distributed in the lymphoid organs of the body, cells were isolated from the spleen, inguinal lymph nodes (LN), mesenteric lymph nodes (MNL), small intestine (SI), and large intestine (LI) LP using mice, and then stained with CD25 low Foxp3 +The distribution pattern of T cells was confirmed.

[0101] As a result, Foxp3 in the spleen, inguinal LN, mesenteric LN, and LI-LP of normal mice + T cells are CD25 - CD25 compared to the proportion of cells + While the cell ratio was higher in SI-LP, Foxp3 + T cells are CD25 - Cells were present at a higher ratio (Fig. 2A). Analysis by counting cells showed that, except for SI-LP, the remaining organs were CD25 - CD25 compared to cell count + Unlike those showing higher or similar cell counts, SI-LP showed CD25 - CD25 cell count + CD25 was predominantly increased compared to the number of cells (Fig. 2B). - Foxp3 + The percentage of T cells was also highest in SI-LP (Fig. 2C).

[0102] CD25 - Foxp3 + To confirm the relationship between T cells and RA development, CD25 expression was measured in tissues in normal and CIA mice. + Foxp3 + Wow CD25 - Foxp3 + Comparative evaluation of T cell counts revealed that CD25 was present in all analyzed organs. + Foxp3 + Wow CD25 - Foxp3 + It was confirmed that the number of T cells was increased in CIA mice compared to normal mice. This is thought to be due to the increase in the total cell number due to inflammation and immune response caused by CIA. Among these, it is noteworthy that, unlike other organs, the CD25 of SI-LP - Foxp3 +T cells were significantly increased in CIA mice compared to normal mice. In addition, CD25 was significantly increased in SI-LP of CIA mice. + Foxp3 + CD25 compared to T cells - Foxp3 + A predominant increase in T cells was observed (Fig. 2D). That is, CD25 of SI-LPs was increased with the development of arthritis. - Foxp3 + A marked increase in T cells was observed, which were CD25 positive in SI-LP - Foxp3 + These results suggest a correlation between T cell proliferation and the development of arthritis. CD25 of SI-LP - Foxp3 + T cells are thought to play an important role in the development of arthritis.

[0103]

[0104] Fibroblasts (FLS) and intestinal LP CD25 in RA patients low Foxp3 + Analysis of the T cell-specific chemokine-chemokine receptor axis

[0105] Using public data (GSE120785) uploaded to the NCBI genome database, we obtained specific RA-FLS genome expression data depending on TNF stimulation (Fig. 3A, top). There were genomes that were specifically expressed differently in TNF-stimulated RA-FLS compared to unstimulated RA-FLS, confirming that RA patient FLS have genomes that are specifically expressed when exposed to an inflammatory environment (Fig. 3A, top).

[0106] In addition, public data (GSE71316, GSE58145, GSE20366, GSE41229) uploaded to the NCBI genome database were used to identify Foxp3 in the popliteal lymph node (pLN), mesenteric lymph node (mLN), spleen (Sp), and SI-LP. + We compared and analyzed the genome expression of T cells (Fig. 3A, bottom). As a result, SI-LP Foxp3 + T cells in the rest of the organs express Foxp3 + A specific genome expression pattern was observed that differs from the genome expression of T cells.

[0107] Therefore, as a result of analyzing the relationship centered on the chemokine-chemokine receptor axis among the genes specifically expressed in RA-FLS and SI-LP, we were able to derive the interaction relationship between the FLS-specific chemokines of RA patients and the chemokine receptors of SI-LP Treg (Fig. 3B). This result suggests a relationship between the development of RA and SI-LP Treg, and suggests that SI-LP CD25 may be involved in the development of RA in the future. low Foxp3 + It is expected that Treg cells can be useful in elucidating the mechanism of arthritis development and screening for therapeutic targets.

[0108]

[0109] Mouse spleen and small intestine LP CD25 - Foxp3 + T cell division inhibition ability

[0110] Spleen (Sp) CD25 from Foxp3-GFP mice + Foxp3 + , CD25 - Foxp3 + , small intestine (Si) CD25 - Foxp3 + Treg cells and CD25 - Foxp3 -After co-culture with responding T cells, the ability of each Treg cell in the spleen and small intestine to suppress the division of responding T cells was evaluated.

[0111] As a result, compared to responding T cells cultured without Tregs (No Tregs), splenic CD25 + Foxp3 + The division of responding T cells co-cultured with Treg cells was significantly inhibited, which indicated that splenic CD25 on responding T cells + Foxp3 + We were able to confirm that the division-inhibiting ability of Treg cells was working well (Fig. 4A). However, splenic CD25 + Foxp3 + and CD25 - Foxp3 + Small intestinal LP CD25 compared to Treg cells - Foxp3 + It was observed that the ability of Treg cells to suppress T cell division was lost (Fig. 4A).

[0112] The concentration of inflammatory cytokines in the culture supernatants of co-cultured splenic and small intestinal Treg cells and responding T cells was measured. Splenic CD25 + Foxp3 + and CD25 - Foxp3 + The culture supernatant of Tregs showed lower levels of TNF-α, IFN-γ, and IL-6 compared to the culture supernatant of No Tregs (Fig. 4B). Small intestinal CD25 - Foxp3 + The culture supernatant of Treg also had reduced levels of TNF-α and IFN-γ compared to the culture supernatant of No Treg, but spleen CD25 + Foxp3 + and CD25 - Foxp3 +Compared to the culture supernatant of Tregs, the levels of TNF-α and IFN-γ were found to be increased (Fig. 4B), indicating that small intestinal CD25 - Foxp3 + It was confirmed that Treg cells have a reduced anti-inflammatory function compared to Treg cells in the spleen.

[0113]

[0114] Mouse spleen and small intestine LP CD25 - Foxp3 + Differentiation ability of T cells into Th17 cells

[0115] Splenic CD25 in Foxp3-GFP mice + Foxp3 + , CD25 - Foxp3 + , CD25 - Foxp3 + Treg cells were isolated and stimulated with anti-CD3 / CD28 for 3 days under Th17 differentiation conditions, and the degree of differentiation into Th17 cells was compared.

[0116] As a result, cells expressing IL-17A, a characteristic cytokine of Th17 cells, were detected in the small intestinal LP CD25 - Foxp3 + Splenic CD25 in Tregs + Foxp3 + , CD25 - Foxp3 + It increased compared to Treg cells (Fig. 5). That is, under Th17 differentiation conditions, small intestinal CD25 - Foxp3 + Treg cells are CD25 present in the spleen + Foxp3 + and CD25 - Foxp3 + It was confirmed that differentiation into Th17 cells can occur more readily than Treg cells. Small intestinal LP CD25 - Foxp3 +Treg cells are considered to be highly plastic cells that can change their phenotypes to promote the development and exacerbation of RA, such as Th17 cells, and therefore, small intestinal LP CD25 - Foxp3 + Treg cells may play a key role in the discovery of factors involved in regulating Treg plasticity and in the development of immune cell therapy through plasticity regulation.

Claims

1. CD25 of the small intestine in the target low Foxp3 + A method for providing information necessary for predicting the likelihood of developing rheumatoid arthritis, which provides information that if the number of T cells is greater than that of the control group, the likelihood of developing rheumatoid arthritis in the subject is greater than that of the control group.

2. In claim 1, the method for providing information necessary for predicting the possibility of developing rheumatoid arthritis in the small intestine mucosa.

3. CD25 of the small intestine in the target low Foxp3 + A method for providing information necessary for predicting the prognosis of rheumatoid arthritis, wherein the information is provided that if the number of T cells is greater than that of the control group, the subject will have a worse prognosis of rheumatoid arthritis than the control group.

4. In claim 3, the method for providing information necessary for predicting the prognosis of rheumatoid arthritis of the small intestine mucosa.

5. Foxp3 in the small intestine + Increase the expression of CD25 among T cells, or CD25 low Foxp3 + A screening method for selecting a substance that reduces the number of T cells or inhibits their differentiation into pathological Th17 cells as a candidate substance for preventing or treating rheumatoid arthritis.

6. A screening method according to claim 5, wherein the small intestine is a small intestinal mucosa.

Images