FcγRIIb gene knockout MDSC and its application as anti-tumor drug

Abstract

The present invention belongs to the field of immunology, and specifically relates to an FcγRIIb gene knockout MDSC and its application as an anti-tumor drug. ‑ / ‑ ) of MDSCs, comprising the following steps: knocking out the FcγRIIb gene (FcγIIRb ‑ / ‑ ) mice were injected subcutaneously with 1×10 6 The lung cancer cell line 3LL cells or melanoma cell line B16F10 cells were isolated and obtained from the mouse spleen after 3 weeks. The spleen was ground with a sterile needle core and filtered through a 400-mesh steel mesh to collect single cells. Tris-NH 4 Red blood cells were lysed with Cl, washed twice with PBS, labeled with CD11b magnetic beads at 4°C for 15 min, and then positively selected for CD11b by column selection. + Cells can be used to prepare MDSCs with FcγRIIb gene knockout.

Description

Technical Field

[0001] The present invention belongs to the field of immunology, and specifically relates to an FcγRIIb gene knocked-out MDSC and an application thereof as an anti-tumor drug. Background Art

[0002] When tumors occur, the differentiation process of common myeloid progenitor cells is blocked, causing myeloid progenitor cells and immature myeloid cells at different differentiation stages to proliferate and activate into myeloid-derived suppressor cells (MDSCs) with immunosuppressive functions, which accumulate in large numbers in the bone marrow, peripheral blood, spleen, lymph nodes, and tumor tissues. MDSCs mainly exert their powerful immunosuppressive functions by inhibiting T cells, such as inhibiting T cell proliferation by expressing arginase and inducible nitric oxide synthase (iNOS); and inducing the production of regulatory T cells by secreting IL-10 or TGF-β. Animal experiments and clinical studies have shown that MDSC expansion is negatively correlated with the prognosis of various tumors and reduces the effect of tumor immunotherapy. Therefore, regulating the differentiation and phenotypic function of MDSCs is a new strategy for tumor immunotherapy. Recent studies have shown that under certain circumstances, such as anti-phosphatidylserine antibodies, MDSCs in tumor tissues can be polarized to an immunostimulatory phenotype; using SHP-1 inhibitors, MDSCs in tumor tissues and spleens can be polarized to an immunostimulatory phenotype and carry out anti-tumor responses. Therefore, changing the polarization state of MDSCs to polarize them to an immunostimulatory phenotype is a new idea for tumor immunotherapy. Summary of the invention

[0003] The present invention provides a FcγRIIb gene knockout (FcγIIRb - / - ) of MDSCs.

[0004] Another embodiment of the present invention provides a method for preparing the above-mentioned FcγRIIb gene knockout MDSC, characterized by comprising the following steps:

[0005] FcγRIIb knockout (FcγIIRb - / - ) mice were injected subcutaneously with 1×10 6 The lung cancer cell line 3LL cells or melanoma cell line B16F10 cells were isolated from mice after 3 weeks. The spleen was ground with a sterile needle, filtered through a 400-mesh steel mesh and single cells were collected. The red blood cells were lysed with Tris-NH4Cl, washed twice with PBS, labeled with CD11b magnetic beads at 4°C for 15 minutes, and then positively selected for CD11b by separation column. + Cells can be used to prepare MDSCs with FcγRIIb gene knockout.

[0006] In the above preparation method, FcγIIRb - / -The mice are preferably mice with a C57BL / 6 background.

[0007] The FcγRIIb gene knockout MDSCs prepared above were detected by flow cytometry for CD11b + Gr-1 + The purity of MDSC is above 90%.

[0008] Another embodiment of the present invention provides the use of the above-mentioned FcγRIIb gene knockout MDSC in the preparation of an anti-tumor drug. The anti-tumor drug is preferably used for preventing and / or treating lung cancer and melanoma.

[0009] The preparation method of the PBS of the present invention is as follows: NaCl (16 g), NaHPO4 (5.78 g), KH2PO4 (0.27 g) and KCl (0.4 g) are dissolved in 2000 mL of triple distilled water, the pH is adjusted to 7.4 with hydrochloric acid, the solution is sterilized by high pressure, and stored at 4° C. for later use.

[0010] The specific method used in the present invention is to respectively give wild type (WT) mice and FcγRIIb gene knockout (FcγIIRb - / - ) mice were injected subcutaneously with 1×10 6 The lung cancer cell line 3LL cells or melanoma cell line B16F10 cells were used. The spleens of the mice were taken out 3 weeks later and CD11b + Gr-1 + MDSC. Arginase activity was detected after lysis of some MDSCs, and supernatants were collected 24 hours after plating of some MDSCs to detect the secretion of nitric oxide (NO), IL-10, and TNF-α in the supernatants. Results showed that in both tumor-bearing models, compared with WT MDSCs, FcγIIRb - / - MDSC arginase activity and IL-10 secretion were significantly decreased, and iNOS activity and TNF-α secretion were significantly increased, suggesting that FcγRIIb deficiency induces MDSC polarization toward immune stimulatory type. - / - MDSCs have anti-tumor effects and were given to WT mice and FcγIIRb - / - Mice were injected subcutaneously with 1×10 6 3LL cells were obtained from the spleen of the mice 3 weeks later, and WT MDSC and FcγIIRb were separated by magnetic beads. - / - MDSCs were used as spare. 1×10 5 WT MDSCs and FcγIIRb were intravenously infused into tumor-bearing mice on the 7th, 14th, and 21st days after the injection of 3LL cells. - / -The results showed that adoptive transfer of WT MDSCs promoted tumor growth, while adoptive transfer of FcγIIRb - / - MDSCs slow down tumor growth ( Figure 1 ), suggesting that FcγIIRb - / - MDSCs have anti-tumor effects.

[0011] The present invention proposes for the first time that FcγIIRb plays an important regulatory role in the polarization of MDSC to an immunostimulatory phenotype, thereby affecting the progression of tumors, and provides a basis for the development of new tumor prevention and treatment methods targeting FcγIIRb. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 FcγRIIb - / - Cytokine secretion of MDSC in B16F10 tumor-bearing mice

[0013] Figure 2 FcγRIIb - / - Cytokine secretion of MDSC in 3LL tumor-bearing mice

[0014] Figure 3 Splenic MDSCs in FcγRIIb-deficient tumor-bearing mice have anti-tumor effects

[0015] Figure 4 FcγRIIb deficiency reduces mortality in 3LL tumor-bearing mice DETAILED DESCRIPTION

[0016] In order to facilitate further understanding of the present invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the invention and are not intended to limit the scope or implementation principles of the present invention. The implementation methods of the present invention are not limited to the following contents.

[0017] Description of raw materials:

[0018] (1) 3LL and B16F10 cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in our laboratory.

[0019] (2) Mouse CD11b sorting magnetic beads were purchased from Metronidex, Germany.

[0020] (3) ELISA detection kits for IL-10 and TNF-α were purchased from Dakoway Company.

[0021] (4) C57BL / 6 mice were purchased from the Center for Comparative Medicine of Yangzhou University.

[0022] (5) The FcγIIRb gene-deficient mice were obtained from the Jackson Laboratory in the United States and maintained by our laboratory.

[0023] (6) Griess reagent for detecting nitric oxide was purchased from Sigma, USA.

[0024] (7) The kit for detecting arginase activity was purchased from BioAssay Company, USA.

[0025] (I) Polarization of spleen MDSCs toward an immunostimulatory phenotype in FcγRIIb-deficient tumor-bearing mice

[0026] (1) Acquisition of MDSCs

[0027] WT mice and FcγIIRb mice on a C57BL / 6 background were given - / - 1×10 6 3LL cells or B16F10 cells were obtained from mice after 3 weeks of sterile separation. The spleen was ground with a sterile needle, filtered through a 400-mesh steel mesh and single cells were collected. Red blood cells were lysed with Tris-NH4Cl, washed twice with PBS, labeled with CD11b magnetic beads at 4°C for 15 min, and then positively selected for CD11b by sorting column. + Cells were detected by flow cytometry for CD11b + Gr-1 + The purity of MDSC is above 90%.

[0028] (2) Detection of MDSC arginase and secreted cytokines

[0029] Take 1×10 6 MDSCs were lysed with cell lysis buffer and the arginase activity was detected using the arginase detection kit of BioAssay. MDSCs obtained by magnetic bead sorting were resuspended to 1×10 in 1640 culture medium containing 10% FBS. 6 / mL density, plated on cell culture plates, 24 hours later, the supernatant was collected, Griess reagent was used to detect the NO level in the supernatant, and ELISA was used to detect the expression levels of IL-10 and TNF-α. It was found that FcγIIRb was higher in MDSCs from 3LL and B16F10 tumor-bearing mice than in WT MDSCs. - / - MDSC arginase activity and IL-10 secretion were significantly decreased, and iNOS activity and TNF-α secretion were significantly increased ( Figure 1 , Figure 2 ), suggesting that FcγRIIb deficiency induces MDSC polarization toward the immunostimulatory type.

[0030] (II) Splenic MDSCs from FcγRIIb-deficient tumor-bearing mice have anti-tumor effects

[0031] (1) Acquisition of MDSCs

[0032] WT mice and FcγIIRb mice on a C57BL / 6 background were given - / - 1×10 6 3LL cells were obtained from mice after 3 weeks of sterile separation. The spleen was ground with a sterile needle, filtered through a 400-mesh steel mesh and single cells were collected. Red blood cells were lysed with Tris-NH4Cl, washed twice with PBS, labeled with CD11b magnetic beads at 4°C for 15 min, and then positively selected for CD11b by sorting column. + Cells were detected by flow cytometry for CD11b + Gr-1 + The purity of MDSC is above 90%.

[0033] (2) Adoptive transfer of MDSCs into 3LL tumor-bearing mice

[0034] C57BL / 6 mice were injected subcutaneously with 1×10 5 3LL cells were intravenously infused into tumor-bearing mice on the 7th, 14th, and 21st days after the injection of 3LL cells. 5×10 6 WT MDSC or FcγIIRb - / - The results showed that adoptive transfer of WT MDSCs promoted tumor growth, while adoptive transfer of FcγIIRb - / - MDSCs delayed tumor growth, suggesting that FcγIIRb-deficient MDSCs have anti-tumor effects ( Figure 3 ).

[0035] (3) FcγRIIb deficiency reduces the mortality of 3LL tumor-bearing mice

[0036] While detecting the growth of 3LL tumors, we also observed the mortality of WT and FcγIIRb- / - tumor-bearing mice. The results showed that compared with WT mice, the mortality of FcγIIRb- / - mice was significantly reduced, suggesting that FcγIIRb deficiency has a protective effect against tumor-induced death in mice ( Figure 4 ).

Claims

1. Use of FcγRIIb gene knockout MDSC in the preparation of anti-tumor drugs, characterized in that The method for preparing FcγRIIb gene knockout MDSCs comprises the following steps: Knockout of FcγRIIb gene - / - Mice were injected subcutaneously with 1×10 6 The spleen of the mouse was obtained by sterile separation after 3 weeks. The spleen was ground with a sterile needle core, filtered through a 400-mesh steel mesh and single cells were collected. The red blood cells were lysed with Tris-NH4Cl, washed twice with PBS, labeled with CD11b magnetic beads at 4℃ for 15 minutes, and then positively selected for CD11b by selection column. + cells, and thus MDSCs with FcγRIIb gene knockout can be prepared; the FcγIIRb - / - The mice were selected from C57BL / 6 background mice; the prepared FcγRIIb gene knockout MDSCs were detected by flow cytometry for CD11b + Gr-1 + The purity of MDSC is above 90%; the anti-tumor drug is selected from the group used for preventing and / or treating lung cancer.

Images