KR20130002100A

KR20130002100A - Composition and method for regulating t cell proliferation using cxcl3 chemokine and cxcr2

Info

Publication number: KR20130002100A
Application number: KR1020110063141A
Authority: KR
Inventors: 김동구; 김진회; 이용수
Original assignee: 건국대학교 산학협력단
Priority date: 2011-06-28
Filing date: 2011-06-28
Publication date: 2013-01-07

Abstract

The present invention relates to a composition for regulating T cell proliferation using CXCL3 chemokine and CXCR2 and a method thereof.

Description

Composition and method for regulating T cell proliferation using CBCL3 chemokine and CBCL2 {Composition and method for regulating T cell proliferation using CXCL3 chemokine and CXCR2}

Mesenchymal stem cells (hereinafter referred to as 'MSCs') have differentiation capacity into chondrocytes, tendon cells (tenocytes), skeletal myocytes, and neurons, and differentiate human hematopoietic stem cells. , Survival and engraftment can be improved [G. Almeida-Porada, A.W. Flake, H.A. Glimp, and E.D. Zanjani, Exp Hematol 27 (1999) 1569-75; M. Angelopoulou, E. Novelli, J.E. Grove, H.M. Rinder, C. Civin, L. Cheng, and D.S. Krause, Exp Hematol 31 (2003) 413-20; P.S. in 't Anker, W.A. Noort, A.B. Kruisselbrink, S.A. Scherjon, W. Beekhuizen, R. Willemze, H.H. Kanhai, and W.E. Fibbe, Exp Hematol 31 (2003) 881-9; Z. J. Liu, Y. Zhuge, and O. C. Velazquez, J Cell Biochem 106 (2009) 984-91].

MSCs have immunomodulatory activity; they induce epoptosis in activated T cells, inhibit T cell responses, and contribute to the successful regulation of severe graft-versus-host disease [B. Maitra, E. Szekely, K. Gjini, M.J. Laughlin, J. Dennis, S.E. Haynesworth, and O.N. Koc, Bone Marrow Transplant 33 (2004) 597-604; A. Uccelli, V. Pistoia, and L. Moretta, Mesenchymal stem cells: Trends Immunol 28 (2007) 219-26].

Chemokines are cytokine-like proteins that selectively regulate the gathering and trafficking of leukocyte subsets to the site of inflammation through chemoattraction [K. Ebnet, and D. Vestweber, Histochem Cell Biol 112 (1999) 1-23]. Growth-related oncogene (GRO), one of the CXC chemokine subfamily members, plays an important role in inflammation and wound healing.

CXC chemokines are known to be involved in tumorigenesis, angiogenesis and metastasis [J. Luan, R. Shattuck-Brandt, H. Haghnegahdar, JD Owen, R. Strieter, M. Burdick, C. Nirodi, D. Beauchamp, KN Johnson, and A. Richmond, J Leukoc Biol 62 (1997) 588-97; DR Smith, PJ Polverini, SL Kunkel, MB Orringer, RI Whyte, MD Burdick, CA Wilke, and RM Strieter, J Exp Med 179 (1994) 1409-15; B. Wang, DT Hendricks, F. Wamunyokoli, and MI Parker, Cancer Res 66 (2006) 3071-7]. GRO chemokines consist of CXCL1, 2, and 3 and bind to its common receptor, CXC chemokine receptor (CXCR) 2.

The present invention has been made by the above necessity, an object of the present invention is to provide a composition for inhibiting T cell proliferation.

Another object of the present invention is to provide a method of inducing expression of CXCR2 in T cells.

In order to achieve the above object, the present invention provides a composition for inhibiting T cell proliferation comprising (C-X-C motif) ligand 3 (hereinafter referred to as 'CXCL3') chemokine as an active ingredient.

In one embodiment of the present invention, the CXCL3 chemokine preferably has an amino acid sequence of SEQ ID NO: 1, but all substitutions, deletions, inversions, etc. mutants intended by the present invention by one or more substitutions to these sequences are also present invention Included in the range of chemokines.

In another aspect, the present invention provides a composition for inhibiting T cell proliferation comprising a gene encoding CXCL3 chemokine as an active ingredient.

In one embodiment of the present invention, the gene preferably has a nucleotide sequence of SEQ ID NO: 2, but considering the degeneracy of the gene codons, all substitutions intended to the present invention by one or more substitutions in these sequences, Mutant genes such as deletions, inversions, and the like are also included in the scope of the chemokines of the present invention.

The present invention also provides a method of inducing the expression of CXC chemokine receptors (CXCR) 2 in T cells by co-culturing mesenchymal stem cells and T cells.

Hereinafter, the present invention will be described.

In the present invention, we found that MSCs significantly induce the expression of CXCR2 receptor on T cells and significantly induce the expression of GRO, CXCL1, CXCL2, and CXCL3 when co-cultured with T cells. We found that MSCs modulate immunosuppressive activity through CXCL3 signaling by reducing the activation of AKT, JAK2, and STAT3 signaling through CXCR2 receptors in T cells.

The inventors confirmed that MSCs significantly inhibited the proliferation of T cells in NOD-SCID mice injected with cord blood mononuclear cells. To identify factors that inhibit T cell proliferation, we performed cytokine array analysis of culture media derived from co-culture of MSCs and T cells and established that chemokines CXCR1, 2 and 3 were induced. MSCs induced expression of CXCR2 receptor on the cell surface of T cells. In particular, CXCL3 plays an important role in the inhibition of T cell proliferation through CXCR2 signaling and inhibitors of CXCR2 rescued the anti-proliferative effect of CXCL3 stimulation. The inventors have determined that the inhibitory activity of CXCL3 on T cells results in arrest of T cell proliferation due to inhibition of survival and proliferation by JAK 2, STAT-3, and AKT activation blocking. Using NOD / SCID mice with human tumors, we found that MSCs inhibited the proliferation of T cells from tumor tissue and inhibited antitumor activity by T cells. Overall these data show that MSCs directly regulate T cell proliferation by inducing CXCL3 chemokine and its receptor CXCR2 on the surface of T cells.

Hereinafter, the present invention will be described in detail.

Cord blood Hematopoietic For engraftment MSCs In vivo inhibitory effect of

We administered cord blood mononuclear cells (CB-MNCs) by intraperitoneal injection to neonatal NOD / SCID mice with or without MSCs. We determined the engraftment efficiency of CB hematopoietic cells in peripheral blood 4,6 and 8 weeks after injection by flow cytometry. NOD / SCID mice not treated with MSCs had a high percentage of CD45 + human hematopoietic cells at 4 to 8 weeks after injection. In contrast, NOD / SCID mice treated with MSCs showed a significant reduction in CD45 + human hematopoietic cells engrafted in peripheral blood during all test periods. Through flow cytometry, we found that the majority of CD45 + human hematopoietic cells were CD3 + T cells but some of the CD19 + B cells were detected but not in CD56 + NK cells and CD33 + bone marrow cells in NOD / SCID mice injected without MSC ( 1A).

However, while the percentage of human CD45 + cells increased in mice not treated with MSCs, no difference was observed in mice injected with MSCs (FIG. 1B). Bone marrow, spleen, and lymph nodes of NOD / SCID mice without or with MSCs were analyzed 10 weeks after the next CB-MNCs injection. NOD / SCID mice not treated with MSC had higher levels in bone marrow, spleen, lymph nodes than treated mice. Human CD45 + cells are shown (FIG. 2A). The percentages of human CD45 + cells in bone marrow, spleen, lymph node and liver in mice not treated with MSC were 3.2 ± 2.1%, 21.7 ± 21.4%, 24.2 ± 16.9%, and 24.1 ± 21%, respectively, and 0.1 in treated mice. ± 0.1%, 0.2 ± 0.1%, 0.5 ± 0.2%, and 0.4 ± 0.2% (FIG. 2B). Flow cytometry analysis showed that most engrafted human CD45 + cells were CD3 + T cells, including CD4 + T cells and CD8 + T cells, similar in morphology to cells in PBs derived from NOD / SCID mice (Table 1). Histological analysis of the spleen of mice not treated with MSCs showed high engraftment of human CD4 + T cells and CD8 + T cells, but no engraftment was detected for MSC-treated mice (FIG. 2C).

We also identified CD45RA and CD45RO expression profiles in T cells and tested whether CD3 + T cells grafted in NOD / SCID mice can differentiate into immature phenotypes from immature T cells in cord blood. From flow cytometry we found that most of the CD3 + T cells were mature phenotypes (CD45RO + T cells) and the number of naive CD45RA + T cells was reduced (FIG. 10). These results suggest that they regulate the inhibition of engraftment of cord blood hematopoietic cells in NOD / SCID mice.

Table 1 shows the engraftment of human hematopoietic lineage cells in the trachea of NOD / SCID mice injected with umbilical cord blood MNC with or without MSCs.

For T cell proliferation MSCs In vitro inhibitory effect of

Next we tested the immunosuppressive effects of MSCs on T cell proliferation in vitro. CD3 + T cells were purified from splenocytes, human PB and CB-MNCs of NOD / SCID mice injected with umbilical cord blood-MNC and co-cultured with or without MSC in the presence of IL-2. Proliferation of T cells co-cultured with MSCs was significantly reduced compared to that of MSC untreated cells. This was the case for both TD-derived T cells and those derived from NOD / SCID mice injected with CB-MNCs (FIGS. 3A, B). Inhibition rates were similar between T cells and PB-T cells derived from NOD / SCID mice. In order to investigate the inhibitory mechanism in terms of cytokine production of T cells, the present inventors investigated intracellular cytokine by flow cytometry in splenic cells of NOD / SCID mice pre-established with CB-MNCs or T cells co-cultured with MSCs. Expression levels of the Cain IFN-gamma and IL-2 were determined. When cells were stimulated with anti-CD3 / CD28 antibody, IFN-gamma + cells or IL-2 + in CD4 + T cells (FIG. 4A) or CD8 + T cells (FIG. 4B) in the presence of MSCs compared to in the absence of MSCs The proportion of cells was significantly reduced.

Cytokine array

From in vivo and in vitro data, we hypothesized that new soluble factors produced by MSCs may be involved in immunosuppressive effects on T cells and inhibition of engraftment. To identify new inhibitory effectors produced by MSCs, we performed cytokine array analysis using culture media from co-culture of T cells and MSCs from splenic cells of NOD / SCID mice engrafted with CB-T cells. Was performed. Compared with cells cultured in other media, co-culture of MSCs and T cells significantly induced expression of growth-related oncogene (GRO) family cytokines (FIG. 11). GRO chemokines consisted of GRO alpha, beta, and gamma chemokines (referred to as CXCL1, CXCL 2, and CXCL 3, respectively) and bound to their specific receptors, CXCR2. We therefore measured the expression levels of the independent chemokines CXCL1, 2, and 3 using an ELISA assay for co-culture of MSCs and T cells. Compared to culture medium containing only MSCs or CD3 + T cells, we found significant up-regulation of the expression of CXCL1, 2, and 3 in T cells derived from NOD / SCID mice and human PB engrafted with CB-MNCs Was observed. In fact, CXCL1 expression was at least about 30-fold higher than PB-T cells alone, and about 4 to 8-fold higher in co-culture medium of PB-T cells and MSCs for CXCL2 or CXCL3 (FIG. 5A). The inventors also observed that the expression of CXCL1, CXCL2, and CXCL3 was 4- to 7-fold higher in co-culture medium than in other culture media (FIG. 5B).

The present inventors then tested whether co-culture with MSCs induced the expression of CXCL1, CXCL2, and CXCL3 co-receptors, CXCR2, on the surface of T cells. CXCR2 was expressed in myeloid cells, and less was expressed in T cells of PB, but was not significantly expressed in NOD / SCID mice and CB derived T cells engrafted with CB-MNCs (FIG. 12). When NOD / SCID mouse-derived T cells engrafted with CB-MNCs were co-cultured with MSCs and anti-CD3 / CD28 antibodies, we observed upregulation of expression of CXCR2 in CD4 + T and CD8 + T cells. The proportion of CXCR2 + cells was 4.5-fold higher in CD4 + T cells (FIG. 6A) and 2.3-fold higher in CD8 + T cells in MSC co-culture compared to cultures treated only with anti-CD3 / CD28 (FIG. 6B).

We confirmed the expression of CXCR2 by immunofluorescence analysis and T cells derived from co-culture with MSCs showed a significantly higher proportion of CXCR2-positive cells compared to control cells (FIG. 6C).

These results strongly suggest that MSCs express CXCL1, CXCL2, and CXCL3 chemokines during co-culture with T cells and induce expression of its specific receptor, CXCR2 at the T cell surface.

CXCL3 Inhibits T cell proliferation

To define the biological activity of CXCL1, 2, and 3 chemokines, NOD / SCID mouse derived T cells pre-grafted with CB-MNCs in the presence of anti-CD3 / CD28 antibody to induce T cell proliferation and CXCR2 expression Co-culture with MSC. After isolation of T cells, the cells were treated with CXCL1, 2, and 3 and their proliferative effect was measured 2 days after stimulation. T cells treated with CXCL1 or CXCL2 showed no difference in cell proliferation compared to control cells, but CXCL3-treated T cells showed a significant decrease in cell proliferation (FIG. 7A). To obtain further evidence that this inhibitory effect of CXCL3 was derived from direct signaling through the CXCR2 receptor, we treated the cells with a specific CXCR2 receptor-specific inhibitor, SB225002, under the same culture conditions. Pretreatment of T cells with SB225002 disrupted CXCL3-induced inhibition of T cell proliferative activity induced by anti-CD3 / CD28 antibody stimulation (FIG. 7B).

These results suggest that CXCL3 signaling directly affects the antiproliferative activity of T cells through the CXCR2 receptor.

In order to identify the mechanism by which CXCL3 inhibits the proliferation of T cells, we identified whether CXCL3 stimulated T cells undergo cell death using flow cytometry of Annexin V and PI. After co-culture of MSCs and anti-CD3 / CD28 antibodies, T cells were isolated and treated with CXCL3 in the presence or absence of a CXCR2 inhibitor, SB225002. Flow cytometry showed a significant increase in Annnexin V + epopotic cells in CXCL3-treated cells compared to untreated control cells. However, cell death was recovered in cells treated with SB225002 in response to CXCL3 signaling at the levels observed in control cells (FIG. 7C). Proliferation data indicate that CXCL3, but not CXCL1 or CXCL2, induces cell death and directly affects the regulation of T cell proliferation.

CXCL3 The AKT , JAK2 , And STAT3 Inhibits T cell proliferation by inhibiting pathways

To confirm how CXCL3 inhibits T cell proliferation, T cells were cultured in the presence of MSCs, exposed to anti-CD3 / CD28 antibody stimulation, and then treated with CXCL3. Phosphorylation of AKT, JAK2, and STAT3 was assessed by flow cytometry with specific antibody staining. CXCL3 treatment reduced JAK2, STAT-3, and AKT phosphorylation compared to untreated control cells. SB225002 treatment disrupted CXCL3-induced reduction of phosphorylation of AKT, STAT3, and JAK2 compared to control cells (FIG. 8A, B).

These data suggest that CXCL3 regulates the inhibitory effect of T cell proliferation via AKT, STAT3, and JAK2 signaling through the CXCR2 receptor.

T cell Mediated Antitumor Active MSCs Suppressed by.

We tested the ability of MSCs to directly modulate anti-tumor activity by inhibiting T cells against tumors in a tumor animal model consisting of a mixture of T cells, tumors, and MSCs. To establish this animal model, we transplanted CB-MNCs into NOD / SCID mice and confirmed the engraftment of human T cells in the PB of NOD / SCID mice. Human cervical tumor (HeLa) cells were then co-injected with MSCs in the left flank of pre-established NOD / SCID mice and injected without MSCs in the right flank. Five weeks after tumor injection, the mice were sacrificed and the tumors measured and measured to determine the antitumor effect of MSCs. Tumors co-injected with MSCs were larger than those of mice not injected with MSCs (FIGS. 9A-C). In immunohistochemical analysis, we determined that tumor tissue had a high level of infiltration by CD8 + T cells that were not injected with MSC but only few CD8 + T cells were present in tumor tissue of mice co-injected with MSCs. (FIG. 9D). Using immunohistochemistry, we confirmed the presence of MSCs (CD90 + and CD105 + double positive) in tumor tissues (FIG. 9E). These data suggest that MSCs can inhibit anti-tumor immune responses by controlling T cell proliferation or invasion in tumor tissues.

The present invention shows that human MSCs inhibit CXC chemokine signaling, T cell proliferation and survival via CXCL3 through specific binding to CXCR2 on T cells, and this MSC-mediated T cell inhibitory effect is anti-tumor activity in vivo. It affects.

1 shows the engraftment of human hematopoietic cells in peripheral blood of NOD / SCID mice injected with human CB monocytes with or without MSC. Cells were collected on the days described from neonate NOD-SCID mice. (A) Human cells of injected NOD / SCID mice with or without MSC were detected by staining with human leukocyte marker, CD45, and CD3, CD19, CD33, and CD56 antibodies. Assays were performed with a flow cytometer in 4 week mice. (B) Percentages of human CD45 + cells were determined by flow cytometry in PB of MSC treated (n) or untreated (O) mice at 4, 6 and 8 weeks post injection. Data is representative of five mice in each group.
2 shows flow cytometry analysis of bone marrow, spleen, and lymph nodes of NOD / SCID mice injected with human CB monocytes with or without MSC.
CB-MNC cells (10 × 10 ⁶ / mouse) were transplanted into neonatal NOD / SCID mice with or without 1 × 10 ⁶ MSCs and cells were collected from bone marrow, spleen and lymph nodes of mice 10 weeks after injection. Cells were stained with anti-human CD45 and CD3 antibodies and analyzed by flow cytometry. (A) Representative flow cytometry data from bone marrow (BM), spleen, and lymph nodes (LN) of NOD / SCID mice treated with or without MSC (B) Percentage of human CD45 + hematopoietic cells in BM, spleen and lymph nodes Determined by the analyzer. (C) Immunofluorescence assays were performed on spleens of NOD / SCID mice treated with or without MSC using anti-human CD4-FITC and anti-human CD8-PE antibodies. Hoechst dye staining was used as counter staining. Data is representative of five mice in each group.
Figure 3 shows the in vitro immunosuppressive effect of MSC on T cells derived from cord blood, human peripheral blood or NOD / SCID mice injected with human CB monocytes. T cells were purified by NOD / SCID mice injected with cord blood mononuclear cells or MACS derived from cord blood, PB. CD3 + T cells (1 × 10 ⁵ ) were stimulated with IL-2 (10 ng / ml) cytokine in the presence or absence of 2 × 10 ⁴ MSCs in 96 well plates for 2 days and analyzed for CCK. Cultivation was performed three times, showing the average of four independent experiments. The histogram shows the mean ± SEM of cpm. * Mean statistically significant compared to control cells (P <0.05).
Figure 4 shows the regulation of IL-2 and IFN-gamma expression by MSC co-culture. CD3 + T cells were purified from splenocytes of NOD-SCID mice injected with CB-MNCs using the EasySep Selection Kit. Cells were cultured with or without MSC in the presence of anti-CD3 / CD28 antibody stimulation. Expression of IL-2 or IFN-gamma was determined by intracellular staining of CD4 + T cells (A) or CD8 + T cells (B) using a flow cytometer. The number in the quadrant box represents the positive percentage of the target cell. The results are representative of three experiments.
5 shows an ELISA assay of chemokines, CXCL1, CXCL 2, or CXCL 3;
CD3 + T cells were prepared from splenocytes or human PBs of NOD-SCID mouse-T cells co-cultured with MSC. (A) Human PB derived CD3 + T cells were co-cultured with MSC or not at the indicated volume. Chemokines were calculated in an ELISA assay using an ELISA kit. (B) CD3 + T cells were purified from splenocytes of NOD / SCID mice injected with CB-MNCs and not co-cultured or cultured with MSCs. Expression of the described chemokine CXCL1, 2, or 3 was determined in an ELISA assay using an ELISA kit. The data show an fold increase in the expression level (volume) of chemokines compared to that of the medium containing only MSCs or T cells. The results are representative of three experiments.
Figure 6 shows the induction of CXCR2 expression by co-culture of T cells and MSC. Purified CD3 + T cells from splenocytes of NOD / SCID mice injected with CB-MNCs were not co-cultured or cultured with MSC for 2 days in the presence of anti-CD3 / CD28 antibody stimulation. Expression levels of CXCR2 receptors were determined by flow cytometry and staining with CD4 (A) or CD8 (B) antibodies. The number of histograms represents the percentage of positive cells in CD4- or CD8-positive gated cells. (C) Immunohistochemical analysis was performed to investigate the expression of CXCR2 receptor on the cell surface of T cells with or without MSCs.
Figure 7 shows the anti-proliferative effect of CXCL3 on T cells. (A) Purified CD3 + T cells obtained from splenocytes of NOD / SCID mice were treated with CB-MNCs. T cells were co-cultured with MSC in the presence of anti-CD3 / CD28 antibody stimulation to induce expression of CXCR2 receptor on T cells. After stimulation, T cells were collected and treated with CXCR1, 2, or 3 for 2 days and the effect on proliferation was determined. (B) After culturing T cells with anti-CD3 / CD28 antibody and MSCs, the cells were collected and treated with CXCL3 and SB225002 (N- (2-Bromophenyl) -N '-(2-hydroxy-4-nitrophenyl) urea) And cell growth was quantified. (C) The effect on cell death was determined by measuring Annexin V and PI staining following treatment with CXCL3 and / or SB225002 inhibitors. The culture was repeated three times. Significant differences compared to the group of CXCL3-treated cells are indicated. All data are representative of three experiments. (* P <0.05).
8 shows that CXCL3 is involved in the survival or proliferation signaling pathway of T cells. CD3 + T cells obtained from splenocytes of NOD / SCID mice were injected with CB-MNCs and then co-cultured with MSC in the presence of anti-CD3 / CD28 antibody. After 2 days T cells were collected and stimulated with CXCL3 alone (A) or with CXCL3 and SB 225002 (B). Flow cytometry showed the percentage of phosphorylated -JAK2, -STAT3, and -AKT positive cells compared to that of the isotype control (unfilled histogram). In all cases, the data are representative of three independent experiments.
Figure 9 shows the inhibitory effect of MSC on the inhibition of tumor growth by T cells. Prior to establishing a tumor model, all NOD / SCID mice were injected into cord blood mononuclear cells and engraftment of the cord blood T cells was confirmed by flow cytometry. Mice were injected subcutaneously with 2 × 10 ⁶ human cervical cancer (HeLa) cells in the right flank posterior or in the posterior posterior flank with a combination of HeLa and 2 × 10 ⁶ MSCs. (A) Tumors are shown in untreated (control) or MSC-treated mice 8 weeks after tumor injection. Tumor size (B) and weight (C) were measured after 8 weeks of injection. Histogram data represent mean ± SEM of cpm. * Means statistically significant compared to control cells (P <0.05). (D) Immunofluorescence analysis indicates the presence or absence of CD4 + T or CD8 + T cells in cervical cancer tissue. (E) The presence of MSCs was confirmed by staining with anti-human CD105 antibody with tumor tissue and MSC-specific marker, anti-human CD90. Arrows indicate CD90 and CD105 double positive cells. All data are representative of three replicates in three mice per group.
10 is a phenotypic analysis of human T cells engrafted in the spleen of NOD / SCID mice injected with CB-MSC. Cells were characterized by staining with antibodies against CD45RO (activated T cell marker) and CD45AR (naive T cell marker) using splenocytes derived from NOD / SCID mice treated with or without MSC. Umbilical cord blood leukocytes were used as a control. Immunohistochemical analysis was performed on spleens of mice treated with or without MSC by staining with CD4-FITC and CD8-PE antibodies.
FIG. 11 shows cytokine antibody arrays of NOD / SCID mouse derived human T cells and MSC coculture, culture medium derived from MSC or T cells alone. FIG. Purified CD3 + T cells were obtained from NOD / SCID mouse derived T cells and cultured with or without MSCs. After 48 hours of incubation, supernatants were collected from each cell culture to confirm cytokine expression profiles. Cytokine arrays showed significant induction of expression (box) of growth-related oncogene (GRO) family in coculture medium.
Figure 12 shows CXCR2 expression in NOD / SCID mice engrafted with CB-MNCs and human hematopoietic cells of CB and PB. Cells were stained with anti-human CXCR2 antibody, anti-human CD45 antibody, and anti-human CD3 antibody for flow cytometry. The number of positive cells in total white blood cells of the described origin is indicated. CB is umbilical cord blood; PBL is peripheral blood leukocytes; NOD / SCID mouse-T cells represent NOD / SCID mouse T cells injected with CB-MNCs.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example 1: primary cells and cell lines

Human cord blood (CB) samples were obtained from umbilical cord and placental tissues according to the guidelines of the Ethics Committee of the primary hospital. Monocytes derived from CB were isolated by density gradient centrifugation in Ficoll-Paque ^™ Plus (Amersham Biosciences). Purified cells were washed and suspended in PBS containing 2% fetal bovine serum (FBS). After two washes, cord blood-derived mononuclear cells (CB-MNCs) were stored in ice until transplantation into neonatal NOD / SCID mice. HeLa cell lines were purchased from the Korean Cell Line Bank (KCLB) and were treated with EMEM and 10% blood inactivated FBS. Cultured in supplemented DMEM medium. Human MSCs were purchased from Chambrex Bioscience. MSCs were cultured in minimal alpha medium (α-MEM), 10% fetal calf serum and 2 mM L-glutamine (Gibco). Medium was changed every 3 days and cells were subcultured at 70-80% confluent stages. MSCs were used at 4-8 passages for all experiments.

Example 2: animal model mouse

Non-obese diabetic-severe combined immunodeficient (NOD / SCID) mice were purchased from an animal laboratory in KKIBB, Korea, and maintained in an animal room at the Stem Cell Research Institute of Primary Medical University. Six to ten week old mice were used for transplantation and tumorigenesis experiments of CB-MNCs. To reconstruct NOD-SCID mice-T cells, we injected intraperitoneally with 1 × 10 ⁷ CB-MNCs in 1-3 day old NOD / SCID mice. Treatment with 1 × 10 ⁶ MSCs commenced with injection of CB-MNCs in NOD / SCID mice. To investigate the effect of MSC on human cervical cancer tumorigenesis, 2 × 10 ⁶ HeLa cells were subcutaneously injected into the right back plate without MSCs, MSCs (4 × 10 ⁵ ) of NOD / SCID mouse-T cells previously injected with CB-NMC were injected subcutaneously in the left dorsal plate. Tumor volume and weight were determined at the expense of animals 5 weeks after injection into tumor cells with or without MSCs to determine MSC mediated inhibition and tumor growth of T cells in vivo.

Example 3: Flow Cytometry Analysis

To determine the chimerism of human hematopoietic cells in peripheral blood, engraftmented human hematopoietic cells were examined 4,6, and 8 weeks after injection of CB-MNCs. Mice were sacrificed and bone marrow, spleen, lymph nodes and liver were collected to confirm the presence of human hematopoietic cells in the trachea. The tissue was dismantled and passed through a nylon filter to remove debris. Samples were prepared as single cell suspension in staining medium with PBS and 2% FBS. The cells were stained with the listed labeled antibodies (Abs): FITC-attached anti-human CD45 (HI30), CD4 (RPA-T4), CD45RO (UCHL1), CD8 (HIT8a), CD45RA (HI100), and CD182 (CXCR2); PE-attached anti-human CD34 (581), CD33 (WIM53), CD19 (HIB19), CD3 (UCHT1), CD4 (RPA-T4), CD8 (HIT8a), and CD182 (CXCR2); And APC-attached anti-human CD56 (B159) and CD3 (UCHT1). Activated and isotype control Abs were purchased from BD Pharmingen. Stained cells were analyzed with a VantageSE flow cytometer (BD-Biosciences) fluorescence-activated cell sorter (FACS). Data were live gated by deficiency and propagation of propidium iodide uptake and side scatter. The frequency at the quadrant corner is given as the percentage of gated cells. The collected data were analyzed by CELLQUEST software (Becton Dickinson).

Example 4: Human T Cell Isolation

To isolate purified T cells, human CD3 ⁺ T cells were selectively isolated from splenocytes of NOD / SCID mice injected with CB-MNCs for 4 to 6 weeks prior to sacrifice or selectively from human peripheral blood. Splenocytes were resuspended in PBS supplemented with 2% FBS and stained with anti-human FITC CD3 (UCHT1) antibody (BD Bioscience), and the EasySep FITC Selection Kit (StemCell Technologies) was used to isolate CD3-positive T cells. It was. Enrichment of purified cells was confirmed by flow cytometry and it showed a> 90% positive cell population, which was used for further experiments.

Example 5: proliferation Assay

To determine the anti-proliferative effect of MSCs, 2 × 10 ⁴ MSCs / cells were seeded in 96 well dishes containing alpha MEM with 10% fetal calf serum (FBS). After 24 hours of incubation, the medium was removed and the MSCs were washed with PBS (-) and further incubated in alpha MEM medium supplemented with 10% FBS, and human CD3 + T cells (1 × 10 ⁵ / well) were intraperitoneally treated with CB-MNCs. Purification from splenocytes, human PB leukocytes, CB-MNCs derived from NOD / SCID mice previously established by intra-injection. To induce T cell proliferation, cells were stimulated with 10 ng / ml IL-2 (BD Bioscience) for 2 days and cell proliferation capacity was measured using CCK Solution Kit (Dojindo). To investigate the effect of chemokines on T cells, MSCs were incubated for 24 hours and then washed twice with PBS (-). Purified CD3 + T cells obtained from NOD / SCID mice, human PB leukocytes, previously established with CB-MNCs, were co-cultured with MSCs and anti-CD3 / CD28 antibodies. After 2 days of culture, T cells were isolated and transferred to 96-well plates in serum-free medium containing 100 ng / ml of CXCL1, CXCL2, and CXCL3 (R & D) chemokines, respectively. Inhibition testing of CXCR2 signaling was performed using 200 nM CXCR2-specific antagonists, SB225002 (Calbiochem), 30 minutes prior to treatment with CXCL3 in serum-free medium. Two days after the treatment, cell proliferation was measured with a CCK Cell Counting Kit (Dojindo) using a microimmunore reader at 450 nm wavelength.

Example 6: cytokine antibody array

MSCs (4 × 10 ⁵ / well) were seeded in 60 mm dishes containing alpha-MEM with 10% FBS. After 24 hours of culture, MSCs were further cultured in serum-free alpha-MEM with purified CD3 + T cells (2 × 10 ⁶ / well) derived from NOD / SCID mice injected with PBS (−) and injected with CB-MNCs. After 1 day, MSCs alone, T cells alone, and cell media derived from MSC and T cell coculture were collected for cytokine testing using ChemiArray ^™ Human Antibody Array I (Chemicon) according to the manufacturer's instructions. In summary, cytokine array membranes were blocked with blocking buffer at room temperature and overnight incubated at 4 ° C. After incubation, the membrane was washed three times with 2 ml Wash Buffer I at room temperature with stirring and twice with 2 ml Wash Buffer II. The membrane was then incubated with 2 ml biotin-attached antibody (1: 500 dilution) for 2 hours at room temperature and washed as above; This was incubated with 1 ml streptavidin-attached peroxidase (1: 1000 dilution) for 1 hour at room temperature. After sufficient washing, the membrane was exposed to peroxidase substrate (R & D) in the dark for 5 minutes before imaging. The film was exposed to X-ray film within 10 minutes exposure to the substrate.

Example 7: ELISA Assay

MSCs (2 × 10 ⁵ / ml) were seeded in 24-well plates containing alpha-MEM with 10% FBS medium. After 25 hours, cells were washed with PBS (-) and further cultured in serum-free alpha-MEM with CD3 + T cells purified from splenocytes or human PB of NOD / SCID mice previously injected 5 to 8 weeks with CB-MNCs. . After 48 hours of incubation, the culture medium was obtained from T cells only, MSCs only, or T cells and MSC coculture to analyze the concentration of chemokines, CXCL1, CXCL 2, or CXCL 3. Chemokines, CXCL1 and CXCL2 were measured using commercial human CXCL1 ELISA Kit (R & D) and human CXCL2 ELISA Kit (IBL), respectively, according to the manufacturer's instructions. To measure CXCL3 levels, we coded 96 well plates with 100 μl / well of 5 μg / ml goat anti-human CXCL3 capture antibody in coating buffer (eBioscience). After overnight incubation at 4 ° C., each well was washed sufficiently and blocked with 200 μl / well blocking buffer (R & D) at room temperature for 1 hour. After three washes, the culture medium was added to CXCL3 specific capture antibody coated plates and incubated overnight at 4 ° C. After sufficient washing, 100 μl / well rabbit anti-human CXCL3 capture antibody (0.5 g / ml) was added to the 96-well plate. After incubation with HRP-attached Dunky anti-rabbit IgG antibodies, 100 μl / well of substrate solution was added to each well. The absolute absorbance was measured with a microplate reader at 450 nm.

Example 8: organization Immunofluorescence

To see human cells in tissues, frozen sections were fixed with 100% ethanol, the tissues were blocked by incubating for 30 minutes at 4 ° C. in 1% bovine serum albumin (BSA) buffer and FITC- or PE-attached anti-human CD45 (HI30), CD3 (UCHT1), CD4 (RPA-T4), CD8 (HIT8a), CD33 (WIM53), CD19 (HIB19), CD56 (B159), CD90 (5E10), and CD105 (439-9B) The antibody was directly stained for 1 hour at room temperature. All sections were counterstained with 1 g / ml Hoechst 33342 (Sigma-Aldrich) to observe cell nuclei. After incubation with antibody, sections were washed twice and analyzed by immunofluorescence microscopy using Apotome (Carl Zeiss, Germany).

Example 9: Apoptosis Assay

To confirm the epopotic effect of CXCL3, 1 × 10 ⁶ purified CD3 + T cells were co-cultured with MSCs and anti-CD3 / CD28 antibodies for 2 days. T cells were isolated and transferred to 96-well plates and treated with 10 mg / ml CXCL3 with or without 200 nM CXCR2 antagonist SB225002. After 2 days of culture, cells were stained with Annexin V and propidium iodide (PI) and analyzed using a flow cytometer.

Example 10: Intracellular dyeing

To identify the CXCL3 signaling pathway through the CXCR2 receptor, MSCs (2 × 10 ⁵ / well) were seeded in 24-well plates and 2 × 10 ⁶ CD3 + T cells and 2 mg / ml anti-CD3 / CD28 antibody were added for 2 days. . T cells were collected and stimulated with or without 100 ng / ml CXCL3 for 30 minutes. Cells were pretreated with SB205002 before CXCL3 treatment. After immobilization with BD Cytofix / Cytoperm Kit (BD Pharmingen), the cells were signaled with the listed signaling protein antibodies according to the manufacturer's instructions: anti-phospho-JAK2 (Tyr 1007 / Tyr 1008), anti-phospho-STAT3 (ser727 Intracellular staining with anti-phospho-AKT (ser473) (Cell Signaling Technology). Flow cytometry was performed using FACSvantageSE (BD Pharmingen).

Data of the present invention are shown as mean ± SD. Statistical significance of the differences between the groups was assessed by Student's t test. Statistical significance was determined at the P <0.05 level.

<110> Konkuk University Industrial Cooperation Corp. <120> Composition and method for regulating T cell proliferation using CXCL3 chemokine and CXCR2 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 107 <212> PRT <213> Homo sapiens <400> 1 Met Ala His Ala Thr Leu Ser Ala Ala Pro Ser Asn Pro Arg Leu Leu 1 5 10 15 Arg Val Ala Leu Leu Leu Leu Leu Leu Val Ala Ala Ser Arg Arg Ala 20 25 30 Ala Gly Ala Ser Val Val Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr 35 40 45 Leu Gln Gly Ile His Leu Lys Asn Ile Gln Ser Val Asn Val Arg Ser 50 55 60 Pro Gly Pro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn 65 70 75 80 Gly Lys Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Gln Lys Ile 85 90 95 Ile Glu Lys Ile Leu Asn Lys Gly Ser Thr Asn 100 105 <210> 2 <211> 321 <212> DNA <213> Homo sapiens <400> 2 atggcccacg ccacgctctc cgccgccccc agcaatcccc ggctcctgcg ggtggcgctg 60 ctgctcctgc tcctggtggc cgccagccgg cgcgcagcag gagcgtccgt ggtcactgaa 120 ctgcgctgcc agtgcttgca gacactgcag ggaattcacc tcaagaacat ccaaagtgtg 180 aatgtaaggt cccccggacc ccactgcgcc caaaccgaag tcatagccac actcaagaat 240 gggaagaaag cttgtctcaa ccccgcatcc cccatggttc agaaaatcat cgaaaagata 300 ctgaacaagg ggagcaccaa c 321

Claims

Composition for inhibiting T cell proliferation comprising CXCL3 chemokine as an active ingredient.

The composition for inhibiting T cell proliferation according to claim 1, wherein the CXCL3 chemokine has an amino acid sequence represented by SEQ ID NO: 1.

Composition for inhibiting T cell proliferation comprising a gene encoding CXCL3 chemokine as an active ingredient.

The composition for inhibiting T cell proliferation according to claim 3, wherein the gene has a nucleotide sequence of SEQ ID NO: 2.

A method of co-culturing mesenchymal stem cells and T cells to induce expression of CXC chemokine receptors (CXCR) 2 in T cells.