AU2021105782A4

AU2021105782A4 - Application of TNFSF15 protein in preparing medicine for treating tumor

Info

Publication number: AU2021105782A4
Application number: AU2021105782A
Authority: AU
Inventors: Luyuan Li; Qiangzhe Zhang
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2021-10-21
Anticipated expiration: 2029-08-18

Abstract

The invention discloses an application of TNFSF15 protein in preparing drugs for treating tumors. In the application, TNFSF15 protein is used as an immunopotentiator of immune cells, which could change bone marrow-derived macrophages induced by mouse M-CSF or GM CSF, mouse peritoneal macrophages and macrophage Raw264.7 cell line into M1 subtype in vitro. Meanwhile, TNFSF15 could also enhance its function of killing tumor cells, as well as promote lymphocyte immune cells such as B cells and CD8' T cells to infiltrate tumors, to promote anti-tumor immune response. The invention has obvious effects on expansion of immunotherapy and killing activation of T cells, and provides more possibilities for immunotherapy of tumors.

Description

Application of TNFSF15 protein in preparing medicine for treating tumor

TECHNICAL FIELD

The invention relates to the technical field of medicines, in particular to an

application of TNFSF15 protein in preparing medicines for treating tumors.

BACKGROUND

Cancer is a major disease faced by human beings. With the increase of

environmental pollution and life pressure, the incidence of cancer is increasing year by

year. The methods of treating tumors mainly include surgical resection and radiation

killing for local tumors, and the methods of killing tumor cells by chemical drugs, all of

which have the disadvantages of being unable to eradicate diseases, easy to relapse, and

great side effects. With the development of modem immunology, people realize that the

stability of immune function plays an important role in the occurrence, development,

metastasis, reversal and regression of tumors. Tumor is a complex system. Besides its

own tumor cells, its microenvironment also contains many resident cell types, such as

adipocytes, fibroblasts, macrophages, neutrophils and mast cells. Macrophages often play

a vital role in the occurrence, development and metastasis of tumors.

Tumor necrosis factor superfamily-15,TNFSF15, also known as TL1A, is a vascular

growth inhibitor secreted by mature vascular endothelial cells. Studies have found that it

is not only a negative vascular regulator, but also can inhibit angiogenesis and tumor

growth in tumors; It can also be used as an immune activator to promote the activation of

T cells and the maturation of dendritic cells. At present, there is no report about the

relationship between TNFSF15 and immune cells, especially macrophages, B cells and T

cells.

SUMMARY

The purpose of the invention is to provide an application of TNFSF15 protein in

preparing drugs for treating tumors, so as to solve the problems existing in the prior art,

the protein can enhance the polarity of macrophage M1, make it have killing effect,

regulate and control the function of B cells, activate T cells to promote tumor immunity

and exert anti-tumor effect.

To achieve the above purpose, the present invention provides the following scheme:

The invention provides an application of TNFSF15 protein in preparing drugs for

treating tumors.

Furthermore, that TNFSF15 protein is used as an immunopotentiator of immune

cells, and the immune cell include macrophages and lymphocytes.

Further, the lymphocytes include B cells and T cells.

Furthermore, that TNFSF15 protein can increase the proportion of macrophage in

tumors and inhibit tumor growth.

Furthermore, the TNFSF15 protein can promote the proliferation of B cells and the

killing of T cells.

The invention also provides a method for amplifying immune cells in vitro, which

comprises the step of culturing the immune cells in a polarized medium containing

TNFSF15 protein.

Furthermore, the concentration of the TNFSF15 protein in the polarized medium is

not higher than 10 [g/mL.

Furthermore, the method specifically comprises the following steps: culturing

immune cells in an incubator at 37°C and 5% C02, discarding the culture medium, adding

fresh culture medium containing TNFSF15, and continuing to culture.

The invention also provides a culture medium for expanding immune cells in vitro,

and the culture medium comprises TNFSF15 protein.

Furthermore, the composition of the culture medium is RPMI 1640 + 10% FBS + 3

pg/mL TNFSF15 protein.

The invention discloses the following technical effects:

1. The invention inhibits tumor growth by activating B cells and T cells with

TNFSF15. Specifically as follows:

1)TNFSF15 can promote the invasion of tumor by immune cells such as B cells and

CD8 mT cells, promote anti-tumor immune response and inhibit tumor growth. The

invention provides more possibilities and guiding significance for immunotherapy and

adoptive therapy of tumors.

2)TNFSF15 can promote the proliferation of B cells, activate and express ICOSL by

activating NF-B signal, and then promote the secretion of GZB by CD8 m T cells to play

a tumor killing role. Meanwhile, P13K signaling pathway is involved in TNFSF15

promoting the secretion of GZB by CD8m T cells. The invention has obvious effects on in

vitro expansion of immunotherapy and killing activation of T cells, and provides more

possibilities for immunotherapy of tumors.

2. The present invention analyzes the function and mechanism of TNFSF15

regulating bone marrow cells to participate in tumor development by constructing a bone

marrow transplantation tumor-bearing mouse model. Specifically as follows:

1)TNFSF15 upregulates lymphocytes in bone marrow of tumor-bearing mice, which

has a regulatory effect on immune effect of bone marrow cells infiltrating tumor.

2) Promote red fluorescence (td-Tomato') bone marrow-derived B cells and T cells

to infiltrate tumor tissues.

3)TNFSF15 induced differentiation of bone marrow cells into B cells through NF

KB and its expression of ICOSL.

4)TNFSF15 promoted the differentiation of bone marrow cells into CD8' T cells

and CD4' T cells.

3. The invention enhances the polarity of macrophage M1 in tumor

microenvironment through TNFSF15 protein, so as to achieve the purpose of tumor

treatment, and can be widely applied to tumor treatment. Specifically as follows:

1) Compared with chemotherapy, TNFSF15, as a biological protein, has the

characteristics of less toxicity and higher safety.

2) The TNFSF15 can effectively enhance the M1 polarity of macrophages from

different sources, and can be applied to related diseases with excessively strong M2

polarity, such as asthma, fibrosis, parasitic infection and bacterial infection.

3) The purification method of TNFSF15 of the present invention is simple and

numerous, and the economic cost is low, which can be combined with other treatment

methods and has a wide application prospect.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the change of polarity of bone marrow-derived macrophages treated

with TNFSF15 protein;

Figure 2 shows the polarity changes of peritoneal macrophages treated with

TNFSF15 protein;

Figure 3 shows the change of polarity of macrophage line Raw264.7 cells treated

with TNFSF15 protein;

Figure 4 shows the changes of phagocytosis of Escherichia coli by macrophages

Raw264.7 cells treated with TNFSF15 protein, in which A is the fluorescence photos of

Escherichia coli in macrophage Raw264.7 treated group and untreated group, and B is the

statistical analysis of the average fluorescence intensity of macrophages phagocytosis of

Escherichia coli in A;

Figure 5 shows the killing effect of TNFSF15 protein on tumor cells, in which A is

the change of phagocytosis of LLC tumor cells after TNFSF15 protein treatment, and B is

the change of apoptosis caused by supernatant of TNFSF15 protein treatment on LLC

tumor cells;

Figure 6 shows that TNFSF15 protein upregulates the proportion of M1

macrophages to inhibit tumor growth, where a is the effect of TNFSF15 protein on LLC

transplanted tumor; B is the effect of TNFSF15 protein on M1 macrophages in tumor; C

is the effect of TNFSF15 protein on M2 macrophages in tumor; D is the effect of

TNFSF15 protein on the ratio of M1/M2 macrophages in tumor;

Figure 7 shows the effect of eliminating TNFSF15 protein on tumor macrophages

and tumor growth, in which A is the effect of TNFSF15 protein and macrophage

scavenger clodronate on MHC I M1 macrophages in LLC transplanted tumor, B is the

effect of TNFSF15 protein and macrophage scavenger clodronate on CD86* M1

macrophages in LLC transplanted tumor, C is the picture of LLC transplanted tumor with different treatment groups of TNFSF15 protein and macrophage scavenger clodronate, D is the change of tumor volume of LLC transplanted tumor with different treatment groups of TNFSF15 protein and macrophage scavenger clodronate;

Figure 8 shows TNFSF15 stimulating lymphocyte gene expression; (A-B)qPCR was

used to detect mRNA levels of lymphocyte proliferation genes ccnbl and bub1; The

mRNA levels of fasl, icos and icosl were detected by (C-E)qPCR;

Figure 9 shows the regulation of TNFSF15 on the proliferation (A, B220 & CD19)

and activation (B, B220 & ICOSL) of B cells;

Figure 10 shows the ratio of TNFSF15 activating T cells (CD8&GZB) in different

antibodies or inhibitors;

Figure 11 shows TNFSF15 promoting B cells and CD8* T cells to infiltrate LLC

tumor and inhibiting tumor growth; (A) Infiltration of B cells in tumors and statistics of

the proportion of B cells in tumors; (B) Detecting CD8* T cell infiltrating tumor and the

proportion statistics of CD8' T cell infiltrating tumor; (C) Tumor volume monitoring

curve; (D) Tumor weight on day 19;

Figure 12 shows that TNFSF15 promotes the activation of B cells and T cells in

tumor tissues; (A) B cells in control group and TNFSF15 treatment group expressed

ICOSL; (B) Counting the proportion of ICOSL* B cells in tumor tissues; (C) CD8' T

cells in control group and TNFSF15 treatment group expressed GZB; (D) Counting the

proportion of CD8' GZB* T cells in tumor tissues; (E) CD4' T cells in tumor of control

group and TNFSF15 treatment group expressed T-bet; (F) Counting the proportion of

CD4' T-bet* T cells in tumor tissues; (G) CD8' T cells in tumor of control group and

TNFSF15 treatment group expressed IFN-y; (H) Counting the proportion of CD8m IFN-yf

T cells in tumor tissues;

Figure 13 shows TNFSF15 promoting bone marrow cells to infiltrate tumors; (A)

The proportion of red fluorescence td-Tomatobone marrow-derived cells invading

tumor; (B) The ratio of red fluorescence td-Tomato bone marrow-derived cells in tumor

was statistically shown;

Figure 14 shows the tumor tissue of bone marrow transplanted tumor-bearing mice,

co-locating bone marrow-derived cells and B cells by immunofluorescence; Green: B220

(TNFSF15 treatment promoted the number increase); Red: td-BMC(TNFSF15 treatment

promoted the number increase); Blue: DAPI(TNFSF15 treatment promotes the increase

of merge number); Scale: 20 m; Td-BMC: Td-Tomato bone marrow cells;

Figure 15 shows the co-localization of CD8 T cells and td-Tomato bone marrow

derived cells in tumor tissues, tumor marginal tissues and tumor peripheral tissues;

Green: CD8 (TNFSF15 treatment promoted the increase of the number of tumor interior

and edge); Red: td-BMC (TNFSF15 treatment promoted the increase of the number of

tumor interior and edge); Blue: DAPI (TNFSF15 treatment promotes the increase of

merge number inside and at the edge of tumor); Scale: 20 m; Td-BMC: Td-Tomato

bone marrow cells;

Figure 16 shows bone marrow cells differentiating into B cells and ICOSL B cells;

(A) Bone marrow cells differentiate into B cells under the action of Vehicle or TNFSF15;

(B) Bone marrow cells differentiate into B cells under the condition of Vehicle containing

NF-KB inhibitor or TNFSF15; (C) Statistics of differentiation of bone marrow cells into

B cells under the action of Vehicle or TNFSF15 (with or without stimulation of NF-KB inhibitor); (D) Bone marrow cells differentiated into ICOSL' B cells under the action of

Vehicle or TNFSF15; (E) Bone marrow cells differentiated into ICOSL' B cells under the

condition of Vehicle containing NF-KB inhibitor or TNFSF15; (F) Statistics of

differentiation of bone marrow cells into ICOSL'B cells under the action of Vehicle or

TNFSF15 (NF-KB inhibitor stimulates or does not stimulate cells);

Figure 17 shows bone marrow cells differentiating into CD8' T cells and CD4' T

cells; (A) Under the action of TNFSF15 or Vehicle, bone marrow cells differentiate into

CD8' T cells; (B) Statistics on the proportion of bone marrow cells differentiating into

CD8' T cells; (C) bone marrow cells differentiated into CD4' T cells under the action of

Vehicle or TNFSF15; (D) Statistics on the proportion of bone marrow cells

differentiating into CD4' T cells.

DESCRIPTION OF THE INVENTION

Various exemplary examples of the present invention will now be described in

detail, which should not be regarded as a limitation of the present invention, but rather as

a more detailed description of certain aspects, characteristics and examples of the present

invention.

Example 1 Effect of TNFSF15 protein on macrophage activation

1) Preparation of Raw264.7 cells

The mouse macrophage line Raw264.7 cells were resuspended in (RPMI 1640 +

% FBS) culture medium, and the cell concentration was adjusted to 2 x 10' cells/mL, 1

mL per well, and then added to a well plate (such as a 12-well plate) and cultured in an

incubator (37°C, 5% C0 2 ).

2) Induction of M1 macrophages

Macrophage culture medium was replaced by induction medium containing

TNFSF15 protein (RPMI 1640 + 10% FBS + 3 pg/mL TNFSF 15 protein) and cultured

for 24 h.

3) Result analysis:

It can be seen from Figure 1 that compared with the control group, TNFSF15 protein

could change the bone marrow-derived macrophages into M1 subtype; It can be seen

from Figure 2 that TNFSF15 protein could change the peritoneal macrophages into M1

subtype; It can be seen from Figure 3 that TNFSF15 protein could change the

macrophage cell line Raw264.7 into M1 subtype. Therefore, TNFSF15 protein can

change macrophages into M1 subtype.

Example 2 Effect of TNFSF15 protein on phagocytosis of bacteria by macrophages

1) Treatment of Raw264.7 cells

Raw264.7 was spread in a 24-well plate with a climbing wafer on the bottom, and

after 12 h, it was treated with Buffer and TNFSF15 for 24 h

2) Phagocytosis operation

Phagocytosis index (PI)= total number of phagocytized fluorescent particles / total

number of macrophages, because phagocytized fluorescent particles cannot be counted

clearly, fluorescence intensity is often used instead.

3) Result analysis:

It can be seen from Figure 4 that TNFSF15 protein significantly increases the

phagocytosis of Raw264.7 cells to Escherichia coli, so TNFSF15 protein can enhance the

phagocytosis of macrophages.

Example 3 Effect of TNFSF15 protein on killing tumor cells by macrophages

1) The effect of TNFSF15 protein on macrophage phagocytosis of tumor cells

Phagocytosis percentage = macrophage phagocytizing tumor cells / total

macrophagex 100%.

2) The effect of TNFSF15 protein on macrophage promoting apoptosis of tumor

cells

Get Raw264.7 supernatant.

Adjust the density of Raw264.7 to 5 x 10' cells per well and spread it in a six-well

plate, after 12 h, add the corresponding Buffer and TNFSF15 to stimulate the well, after

24 h, suck the cell supernatant in a centrifuge tube, centrifuge at 500 g for 10 min,

remove the precipitate, transfer the supernatant to a new centrifuge tube and store it at

°C for later use.

3) Result analysis:

It can be seen from Figure 5 that TNFSF15 protein increases the phagocytosis of

LLC tumor cells by Raw264.7 cells, and Caspase3 of LLC cells is sheared and activated

in the supernatant of Raw264.7 treated with TNFSF15, so it can be seen that TNFSF15

protein can enhance the killing ability of macrophages.

Example 4 Effect of TNFSF15 protein on macrophages in tumor

1) Obtaining a cell line with high expression of TNFSF15 or prepare TNFSF15

recombinant protein

(1) Constructing plvx-puro-TNFSF15 plasmid; Or the patent CN107541536A for

preparing TNFSF15 recombinant protein (Here is its new medicinal function.).

(2) After plvx-puro-TNFSF15, pspAX2 and pMD.2G were transfected into 293T

cells, virus supernatant was collected and transfected into LLC cells.

Furthermore,TNFSF15 over-expressing cell line was screened by limited dilution

method.

2) Establishment of tumor model with TNFSF15 overexpression

(1) The LLC overexpressing hTNFSF15 constructed above and the control LLC cell

line were resuscitated and cultured, grew to a certain amount, digested, washed twice

with PBS, and then resuspended in serum-free medium at a density of 5 x 10' cells /mL.

(2) According to the amount of 5xIO cells /100 L per mouse, the cells were

planted under the skin of C57BL/6J mice. The tumor size was measured every two days,

and the weight of mice was recorded until the tumor was collected. Tumor volume

(mm)= length x width x width / 2.

3) Analyze that polarity of macrophage in tumor

Tumor tissues were removed from mice, and then processed and analyzed by

computer.

It can be seen from Figure 6 that the overexpression of TNFSF15 in tumor tissue

inhibits tumor growth, increases the proportion of M1 macrophages and inhibits the

proportion of M2 macrophages, therefore, it can be seen that TNFSF15 protein increases

the proportion of M1/M2 macrophages in tumor and improves tumor immune

microenvironment.

Example 5 The relationship between TNFSF15 protein and macrophage in tumor

and tumor inhibition

1) Establishing a tumor model of TNFSF15 overexpression LLC cleared by

macrophages

(1) The LLC overexpressing hTNFSF15 constructed above and the control LLC cell

(2) According to the amount of 5 x 10' cells /100 L per mouse, the cells were

planted under the skin of C57BL/6J mice. after the tumor grew to 7d, LLC-TNFSF15 and

LLC-Mock mice were randomly divided into two groups;

(3) Take out the Clodronate Liposomes (CL) and sterile PBS from the refrigerator

and naturally return to room temperature. Mix up and down, and inject 200 L into the

abdominal cavity of each mouse according to the group;

(4) Once every three days, the tumor size was measured every two days, and the

weight of mice was recorded until the tumor was collected. Tumor volume (mm)= length

x width x width / 2).

2) Analyze that polarity of macrophage in tumor

It can be seen from Figure 7 that the overexpression of TNFSF15 in tumor tissues

increases the proportion of M1 macrophages and inhibits tumor growth, but with the use

of macrophage scavenger, the tumor inhibitory effect of TNFSF15 is weakened, so it can

be seen that TNFSF15 inhibits tumor growth by enhancing the immune activity of

macrophages in tumors.

Example 6 TNFSF15 can promote the proliferation and activation of lymphocytes

The isolated spleen lymphocytes were added to a 6-well plate, with 5 x 106 cells per

well and 3 mL of culture medium (RPMI 1640 containing 15% FBS and 1%

streptomycin mixture (1OX)). 3 g/mL TNFSF15 stimulated the cells, and the control

group was added with corresponding volume of buffer as Vehicle. Lymphocyte proliferation was detected by qPCR (cyclin B1 gene ccnb; Serine/threonine protein kinase BUB Igene bubI) and activation (Icos, Icosl and Fasl genes) in mitotic detection points. The result of flow cytometry analysis is shown in Figure 15, which indicates that

TNFSF15 stimulation significantly promotes the increase of mRNA expression of

lymphocyte proliferation genes ccnbl and bub1, and lymphocyte immune promotion

genes fasl, icos and icosl.

Example 7 TNFSF15 promotes proliferation and activation of B cells through NF

KB

well mixed with 2 x 10' cancer cells, and 3 mL of culture medium (2 mL RPMI 1640

containing 15% FBS, 1% double antibody + 15% FBS, 1% double antibody high glucose

DMEM). 3 g/mL TNFSF15 stimulated the cells, and the control group was added with

corresponding volume of buffer as Vehicle.

Co-cultured cancer cells (LLC) were incubated with CFSE at 37°C CFSE 10 min

before mixing lymphocytes, and then put into ice bath with 40% FBS for 10 min to stop

the reaction. Add PBS to wash twice, discard supernatant, and culture to resuspend cells.

The inhibitors NF-KB(1 [M) and PI3K(1 [M) stimulated immune cells in advance

for 1 h, and then cancer cells containing 3 g/mL TNFSF15 or Vehicle were added.

ICOSL neutralized antibody (10 [g/mL), pre-incubated lymphocytes at 37°C for 2 h,

and added cancer cells containing 3 g/mL TNFSF15 or buffer.

Cells were cultured in a constant temperature incubator at 37°C and 5% C02 for 72

h.

Cells were collected, washed once with PBS, and stained with PI. The cells were

washed with PBS, resuspended with PBS, and passed through a 300 mesh cell screen.

The death of cancer cells and the proliferation and activation of lymphocytes were

detected by flow analysis.

Results as shown in Figure 16, TNFSF15 significantly promoted the percentage of

B220+CD19' double positive B cells and B220+ICOSL* double positive B cells,

indicating that TNFSF15 promoted the proliferation and activation of B cells. PDTC, an

inhibitor of NF-KB, significantly inhibited the percentage of B220+CD19' double positive

B cells and B220+ICOSL* double positive B cells, indicating that TNFSF15 promoted the

proliferation and activation of B cells through NF-KB signal.

Example 8 TNFSF15 plays a tumor killing role by activating T cells through ICOSL

and P13K

The method is as shown in example 7, and the results are shown in Figure 17, which

shows that TNFSF15 can significantly promote the percentage of CD8*GZB* double

positive T cells, and TNFSF15 can promote the proliferation and activation of CD8' T

cells. Treatment with ICOSL neutralizing antibody or Wortmannin, an inhibitor of P3K,

significantly inhibited the percentage of CD8*GZB* double positive T cells, indicating

that TNFSF15 activated T cells through ICOSL and P3K.

Example 9 TNFSF15 regulates B cells and T cells against tumor in vivo

LLC(Lewis Lung Cancer) tumor-bearing mouse model: When the confluence in the

culture dish reaches 80%, it is passaged. After digestion into single suspension cells, the

cells were collected by centrifugation and washed twice with 10 times volume PBS.

Finally, PBS was discarded, and the basal medium without serum and antibody was added, and plasma cells were resuspended to 3 x 106 mL- cell suspension. Cells were inoculated subcutaneously on the right hind leg of C57BL/6 mice aged 6-8 weeks, and each mouse received 100 L of the above cell suspension.

On the fourth day of tumor inoculation, when the tumor can be observed by naked

eyes, the mice were randomly divided into two groups, which were given medicine

beside the tumor. Every two days, the drug was given and the weight and tumor volume

of mice were monitored (the longest and shortest diameters were measured by vernier

caliper). Tumor tissues were collected on the 19th and 30th days after tumor inoculation.

Experimental group: each mouse was injected with 5 mg/kg TNFSF15 beside the

tumor; Control group: Each mouse was injected with the same volume of Vehicle beside

the tumor.

Tumor volume (mm3)= L x W x W/ 2; In which L represents the longest diameter

of tumor and W represents the shortest diameter of tumor.

Flow detection and analysis of cells, intracellular antibody flow staining, and nuclear

antibody flow staining.

The results are shown in Figure 11-12, indicating that TNFSF15 can promote the

percentage of B220+CD19' double positive B cells and CD8*CD45' double positive T

cells in LLC tumor, and significantly inhibit the volume and weight of LLC tumor,

indicating that TNFSF15 can promote the invasion of B cells and CD8' T cells and

inhibit the growth of LLC tumor. TNFSF15 can also promote the percentage of

B220+ICOSL' double positive B cells, CD8*GZB* double positive T cells, CD4*T-bet*

double positive T cells and CD8*GFN-B* double positive T cells in LLC tumors, indicating that TNFSF15 can play an anti-tumor role by promoting the activation of B cells and T cells in tumors.

Example 10 TNFSF15 promotes bone marrow-derived B and T cells to infiltrate

tumors

1) Prepare RPMI 1640 resuspended cells containing 10% FBS for later use.

2) Establish td-Tomato' bone marrow cell transplantation model mice.

The ratio of td-Tomato' bone marrow cells in peripheral blood and bone marrow

was analyzed by flow cytometry. The channel of td-Tomato in streaming is PE

(Excitation light: 488 nm ; ; Emitted light: 561 nm).

3) Establishment of subcutaneous transplanted tumor LLC model mice

The complete culture medium of LLC cells is high glucose DMEM containing 10%

fetal bovine serum and 1% double antibody. Culture conditions: Incubate the incubator at

37°C and 5% Co 2 .

The cells were observed under microscope, and the LLC cells were passaged (1: 3)

when the confluent degree in the culture dish reached 80%. After the cell line is

resuscitated, the cells after 3-10 passages can be used for experiment or cryopreservation.

When subculturing cells, firstly, remove the culture medium in the culture dish, wash the

cells once with sterile PBS(10 cm cell culture dish plus 4 mL), and discard PBS. Add

pancreatin digestion solution (2 mL in 10 cm cell culture dish), put the culture dish with

pancreatin in 37°C incubator for 2 min, wash the tumor cells digested into single

suspension cells in 10 times volume PBS for 3 times, 200 g, 5 min, discard the

supernatant, and add serum-free and anti-antibody culture medium to suspend the cells to x 106 mL-. Each mouse (two weeks after receiving bone marrow cell transplantation) was subcutaneously inoculated with 100 L of the above-mentioned resuspended cells.

On the fourth day of tumor inoculation, when the tumor can be observed by naked

eyes, the mice were randomly divided into two groups, which were given medicine

Experimental group: each mouse was injected with 5 mg/kg TNFSF15 beside the

the tumor.

Tumor volume (mm3)= L x W x W/ 2; In which L represents the longest diameter

of tumor and W represents the shortest diameter of tumor.

4) Flow detection and analysis, intracellular antibody flow staining and nuclear

antibody flow staining.

5) For tissue morphology, immunofluorescence and immunohistochemistry analysis

of cell expression

The expression and localization of target antibody in tissues were observed under

confocal microscope.

The results are shown in Figure 13-15, indicating that TNFSF15 promotes the

proportion of td-Tomato' bone marrow-derived cells in tumors; TNFSF15 can also

promote B220' B cells and td-Tomato' bone marrow-derived B cells to infiltrate tumors.

TNFSF15 promotes CD8' T cells and td-Tomato'CD8' T cells from bone marrow to

infiltrate tumor.

Example 11 Role of TNFSF15 in regulating bone marrow cell differentiation B cell

and activation

Medium: RPMI 1640 + 10% FBS + 1% double antibody.

Culture conditions: Incubate the incubator at 37°C and 5% Co 2

. 12-well plate, 106 cells were added to each well, and TNFSF15(3 g/mL) was

stimulated for 72 hours.

Inhibitor group: Inhibitor NF-KB(1 M) and cells were pre-incubated for 1 h at

37°C. Then, the corresponding culture medium (including Vehicle or TNFSF15) was

added at 37°C for 72 hours.

Neutralizing antibody group: ICOSL neutralizing antibody (10 g/mL) and cells

were pre-incubated for 2 h at 37°C. Then, the corresponding culture medium (including

Vehicle or TNFSF15) was added at 37°C for 72 hours.

Collecting cells, washing with PBS, and analyzing B220' and ICOSL* B220' cells

by flow staining.

The results are shown in Figure 16, indicating that TNFSF15 can promote the

percentage of bone marrow cells differentiating into B220' B cells and the percentage of

bone marrow cells differentiating into B220+ICOSL' double positive activated B cells,

indicating that TNFSF15 can promote the differentiation and activation of bone marrow

cells to B cells. PDTC, an inhibitor of NF-B, significantly inhibited the percentage of

bone marrow cells differentiating into B220' positive B cells and B220+ICOSL' double

positive B cells, indicating that TNFSF15 promoted the differentiation and activation of

bone marrow cells into B cells through NF-KB signals.

Example 12 Role of TNFSF15 in regulating differentiation of bone marrow cells

into T cells

Medium: RPMI 1640 + 10% FBS + 1% double antibody.

Culture conditions: Incubate the incubator at 37°C and 5% Co 2

. 12-well plate, 106 cells were added to each well, and TNFSF15(3 g/mL) was

stimulated for 72 hours.

Inhibitor group: Inhibitor NF-KB(1 M) and cells were pre-incubated for 1 h at

added at 37°C for 72 hours.

Neutralizing antibody group: ICOSL neutralizing antibody (10 g/mL) and cells

Vehicle or TNFSF15) was added at 37°C for 72 hours.

Cells were collected, washed with PBS, and the ratio of CD4* and CD8* cells was

analyzed by flow staining.

The results are shown in Figure 15, indicating that TNFSF15 increased the

percentage of CD8' T cells and CD4' T cells in bone marrow cells, indicating that

TNFSF15 can promote the differentiation of bone marrow cells into CD8' and CD4' T

cells.

The above examples only describe the preferred mode of the invention, but do not

limit the scope of the invention, on the premise of not departing from the design spirit of

the invention, various modifications and improvements made by ordinary technicians in

the field to the technical scheme of the invention shall fall within the protection scope

determined by the claims of the invention.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An application of TNFSF15 protein in preparing drugs for treating tumors.

2. The application according to claim 1, wherein the TNFSF15 protein is used as an

immunopotentiator for immune cells, and the immune cells include macrophages and

lymphocytes.

3. The application according to claim 2, wherein the lymphocytes comprise B cells

and T cells.

4. The application according to claim 2, characterized in that the TNFSF15 protein

can increase the proportion of macrophages in tumors and inhibit tumor growth.

5. The application according to claim 3, characterized in that the TNFSF15 protein

can promote the proliferation of B cells and the killing of T cells.

6. A method for expanding immune cells in vitro, characterized in that the method

comprises the step of culturing immune cells in a polarized medium including TNFSF15

protein.

7. The method according to claim 1, wherein the concentration of the TNFSF15

protein in the polarized medium is not higher than 10 g/mL.

8. The method according to claim 1, which specifically comprises the following

steps: Culturing immune cells in an incubator at 37°C with 5% C02, discarding the

culture medium, adding fresh culture medium containing TNFSF15, and continuing to

culture.

9. A culture medium for expanding immune cells in vitro, characterized in that the

culture medium comprises TNFSF15 protein.

10. The culture medium according to claim 9, characterized in that the composition

of the culture medium is RPMI 1640 + 10% FBS + 3 pg/mL TNFSF15 protein.