CN117731782A

CN117731782A - Application of CD11c expression promoter in preparation of tumor treatment drugs

Info

Publication number: CN117731782A
Application number: CN202311604246.5A
Authority: CN
Inventors: 李林凤; 陆虹旻; 耿小川; 尤倩
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2023-11-28
Filing date: 2023-11-28
Publication date: 2024-03-22

Abstract

The invention provides application of an expression promoter of CD11c in preparing a medicine for treating tumors. The invention proves that CD11c is an important medium for producing IFN-gamma by NKDCs, and endows the NKDCs with strong IFN-gamma production capacity, and the expression level of CD11c is linearly related to the IFN-gamma production of the NKDCs, thereby providing a new thought and progress for developing new anticancer immunotherapy medicaments clinically.

Description

Application of CD11c expression promoter in preparation of tumor treatment drugs

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of an expression promoter of CD11c in preparation of a medicine for treating tumors.

Background

Natural killer cells (NK) are considered a critical population involved in the innate immune response. Until recently, NK cells were not widely considered as the major cell population of the innate immune system producing Interferon (IFN) -gamma during viral infection and tumorigenesis. Since the discovery of natural killer dendritic cells (NKDCs; NK1.1 ⁺ CD11c ⁺ ) And a subset thereof, i.e., killer dendritic cells that produce IFN- γ (IKDCs; NK1.1 ⁺ CD11c ⁺ B220 ⁺ ) Researchers have demonstrated that NKDCs are the predominant IFN- γ producing cell during the innate immune response against infectious diseases and tumorigenes. Studies have shown that NKDCs have greater IFN- γ production capacity than NK cells after activation, although the relationship of IKDCs as a subset of NKDCs to NK cells in terms of development and function remains controversial. Demonstration of the expression of CD11c by activated NK cells by genetically engineered mice ^low B220 ⁺ Markers, and IKDCs are functionally and developmentally similar to NK cells. Thus, IKDCs are considered an erroneous concept in terms of function and development. However, IKDCs have recently been redefined as intermediates for human NK cells or immature NK cells. In addition, the human NK cell marker CD56 has been found to be expressed on myeloid and plasmacytoid (p) DCs. These new findings support our opinion that NKDCs are a deterministic population with common features of NK cells and DCs.

NKDCs, as a well-established subgroup, have not been able to define their exact functional role and role in immunization. There is currently no unification in many reports. NKDCs have been considered by researchers to be an activated state of NK cells, since only a small fraction of activated NK cells secrete IFN- γ, whose surface expresses the B220 molecule; while most of the NKDCs also express B220. The ability to secrete IFN-gamma is often associated with natural killing. IFN-gamma plays a key role in anti-tumor immunity, but the source of IFN-gamma is still unclear, particularly during the induction phase of anti-tumor immune responses.

NKDCs are similar to NK cells, play an important role in natural immunity, can secrete IFN-gamma like NK cells and T cells, and can trigger the immune response of organisms; and can pass through granzyme B, fas-, TRAIL-, NKG2D, TNF-alpha and Ca ²⁺ Relying on mechanisms and the like to kill the target cells. Since NKDCs have both NK cell and DCs phenotypes, previous studies suggested that activated NK cells and DCs cells can secrete large amounts of IFN-gamma for tumor action. We have previously found that traditional NK cells produce IFN-. Gamma.which is actually associated with NKDCs. At NKl.1 ⁺ NKl.1 in cells ⁺ CDllc ⁺ Is the major subgroup for IFN-gamma production. Regardless of whether the NKDCs are dendritic cells (mDCs) differentiated from NK cells or that can differentiate into monocyte-derived, we speculate that the NKDCs more closely resemble an intermediate transition phase, at least NK cellsThe cells activate a differentiated cell population. Under appropriate conditions, NK cells can differentiate into NKDCs, even acting as DCs to perform antigen presenting functions. It is highly likely that the anti-tumor effect is exerted, at least in part, by NKDCs, rather than by NK cells or DCs. Because of the greater ability of NKDCs to secrete IFN- γ, it was demonstrated to have higher antitumor activity in animal tumor models. The mode of cell killing by NKDCs is very heterogeneous. In vitro experiments confirm that CD11c ^int CD45R ⁺ CD49 ⁺ The mouse-derived NKDCs can kill NK cell-specific target cells YAC-1 as well as tumor cells without any exogenous stimulus, and this effect can be potentiated by the ligand of Flt3 (Flt 3L). Whereas cDCs have no such cytotoxic effects. CD11c is an important marker to distinguish NKDCs from NK cells. CD11c is an integrin-alpha X chain protein, belongs to type I transmembrane protein, and has higher content in mouse and human DCs. This protein binds to the beta 2 chain (ITGB 2) to form a leukocyte specific integrin, termed inactivated C3b (iC 3 b) receptor 4 (CR 4). Thus, CD11c may mediate IFN- γ production by NKDCs by binding to iC3 b. However, the biological significance of CD11c expression in NKDCs has not been studied, i.e., the role played by CD11c in the production of IFN-gamma by NKDCs is not known.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides application of an expression promoter of CD11c in preparing a medicine for treating tumors.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides the use of an expression enhancer for CD11c in the manufacture of a medicament for the treatment of a tumour.

Further, the above-mentioned expression promoter of CD11c plays a role in treating tumors by promoting IFN-gamma production ability of NKDCs.

Further, the expression promoter of CD11c includes an over-expression vector having CD11c gene and/or a small molecule drug targeting CD11c protein and promoting its expression, stability and/or activity increase.

Further, the medicament also comprises a pharmaceutically acceptable carrier or excipient.

Further, the dosage form of the medicament is injection, tablet, pill, capsule, solution, suspension or emulsion.

In a second aspect, the present invention provides a medicament for the treatment of a tumor comprising an expression enhancer for CD11 c.

Compared with the prior art, the invention has the following technical effects:

the invention proves that CD11c is an important medium for producing IFN-gamma by NKDCs, and endows the NKDCs with strong IFN-gamma production capacity, and the expression level of CD11c is linearly related to the IFN-gamma production of the NKDCs, thereby providing a new thought and progress for developing new anticancer immunotherapy medicaments clinically.

Drawings

FIG. 1 shows that CD11c and B220 are at NK1.1 ⁺ Proportional up-regulation in cell populations; flow cytometry analysis was performed on spleen cells isolated from 4-6 (n=3) and 12 week old (n=4) C57BL/6 mice, data were analyzed using flowjo7.6.1 software, bar graphs represent mean ± SEM; (A) NK1.1 ⁺ Cell populations were divided into 4 sub-populations, designated as R1, R2, R3 and R4, respectively, based on the expression level of CD11c, and analyzed for the expression of B220 in NK cells (R1 and R2) and NKDCs (R3 and R4), with B220 expression increasing gradually from R1 to R4; (B) Percentage of B220 expressing cells in subpopulations R1, R2, R3 and R4 in 4-6 and 12 week old C57BL/6 mice; (C) CD11C expression is linearly related to B220 expression, MFI: average fluorescence intensity of CD11 c; (D) DC (N)K1.1 ^- CD11 ^chi ) Expressing the percentage of B220 cells in the population;

FIG. 2 shows B220 ⁺ And B220 ^- Distribution of subpopulations in NK cells and NKDCs; splenocytes isolated from 5-8 week old C57BL/6 mice (n=3) were stimulated with CpG-ODN for 6h and collected for surface staining or intracellular IFN- γ staining; data were analyzed using FlowJo7.6.1 software, bar graphs representing mean ± SEM; (a) a representative scatter plot from a mouse; (B) Percentage of NK cells (black) and NKDCs (grey) producing IFN- γ in spleen cells stimulated with or without CpG-ODN; (C) IFN-gamma producing B220 with or without CpG-ODN stimulation ⁺ (Black) and B220 ^- Percentage of (grey) NK cells ("x" means p.ltoreq.0.0001); (D) IFN-gamma producing B220 with or without CpG-ODN stimulation ⁺ (Black) and B220 ^- Percentage of (grey) NKDCs; (E) B220 ⁺ And B220 ^- Different responses of NKDCs to CpG-ODN stimulation to IFN-gamma; dotted line: the reaction is weak; solid line: the reverberation is strong; "indicates that p is 0.05 or less," indicates that p is 0.01 or less, "" indicates that p is 0.001 or less;

FIG. 3 shows that CD11c is linearly related to IFN-. Gamma.production in NKDCs, both in steady state and activated by CpG-ODN; splenocytes were collected and stained for surface CD11C, NK1.1 and B220 and intracellular IFN-. Gamma.NK 1.1 when splenic mononuclear cells isolated from 5 to 8 week old C57BL/6 mice were stimulated in vitro with CpG-ODN for 6h ⁺ Each subpopulation of cells; (a) representative scatter plot from mice: NK1.1 ⁺ The cell population was divided into 4 sub-populations; (B) Percentage of IFN- γ producing cells in each subpopulation (R1, R2, R3 and R4) stimulated with or without CpG-ODN; (C) CD11c expression levels are linearly related to the increase in the percentage of IFN- γ expressing cells, MFI: average fluorescence intensity (MFI) of CD11 c; (D-G) B220 ⁺ And B220 ^- Distribution of IFN- γ expressing cells in R1 (D), R2 (E), R3 (F) and R4 (G) subpopulations, data shown from a collection of 3 independent experiments (n=6); error bars represent mean SEM, "×" represents p.ltoreq.0.01;

FIG. 4 shows that CD69 expression in NK cells and NKDCs was up-regulated after activation by CpG-ODN; (A) Small from CpG-ODN stimulationRepresentative scatter plots of murine splenic mononuclear cells, the lower plot shows CD69 expression levels for each subpopulation (R1-R4); (B) Comparison of NK1.1 stimulated by CpG-ODN or not stimulated by CpG-ODN ⁺ CD69 in subpopulations ⁺ Percentage of cells; (C) NK1.1 stimulated or unstimulated in CpG-ODN ⁺ Correlation of CD11c expression with CD69 expression in a cell population; MFI: average fluorescence intensity of CD11c, data shown from a set of 3 independent experiments (n=4); error bars represent mean SEM, "+" represents p.ltoreq.0.001;

FIG. 5 shows that NKDCs, but not NK cells, proliferate actively after stimulation with CpG-ODN, whether in vivo or in vitro; (A) Representative scatter plots showing NK cells, NKDCs and DCs in spleen cells of CpG-ODN treated mice or untreated control mice; (B) Percentages of NK cells, NKDCs and DCs from spleen cells of mice treated or untreated with CpG-ODN (n=3); (C) In vitro, with or without CpG-ODN stimulation, IL2 stimulation as positive control, proliferation of NK cells and NKDCs; error bars represent mean ± SEM, data shown from 3 independent experiments (n=6), "x" represents p <0.05;

FIG. 6 shows that CD11c mediates IFN-gamma production by NKDCs; (A) For CD3 ^- NK1.1 ⁺ Cells were gated and analyzed for expression of CD11c and IFN-gamma; (B-E) stimulation of NK1.1 with anti-CD 11c mab, cpG-ODN and isotype IgG, respectively ^- CD11 ^- Production of IFN-gamma following four cell subsets of cells (B), DCs (C), NK cells (D) and NKDCs (E); the data shown are from 3 independent experiments; ". Times." means that p.ltoreq.0.01; ", indicates that p is less than or equal to 0.001.

Detailed Description

The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.

The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.

Example 1

1. Experimental procedure

(one) mice, culture Medium and reagents

C57BL/6 mice, female and male, 6-8 weeks old (unless otherwise indicated), purchased from Shanghai Jiesjie laboratory animal Co. Mice were kept in SPF class animal houses from Shanghai Renzhi hospitals in china.

RPMI1640 medium, L-glutamine, penicillin and streptomycin were purchased from Gibco, life Technologies. Fetal Bovine Serum (FBS) was purchased from Biological Industries. Beta-mercaptoethanol was purchased from Sigma. RPMI1640 medium was supplemented with 10% FBS, 50. Mu.M beta. -mercaptoethanol, 2mM L-glutamine and 1% streptomycin/penicillin as complete medium (R10F). Antibodies used in the experiments included: FITC-conjugated rat anti-mouse CD11C (clone N418; biolegend, cat.117306), FITC-conjugated rat anti-mouse NK1.1 (PK 136 clone; sungene Biotech Co., ltd., cat.M100N 2-02E), PE-conjugated rat anti-mouse B220 (clone RA3-6B2 clone;Tonbo Biosciences,cat.55-0452-U100 or Biolegend, cat.103207), PE-conjugated rat anti-mouse CD69 (H1.2F3 clone; BD Pharmingen, cat.55337), PE-conjugated rat anti-mouse IFN-gamma (XMG 1.2 clone; biolegend, cat.50808), perCP-Cy5.5-conjugated rat anti-mouse CD3 (145-2C11 clone;Sungene Biotech Co, ltd., cat.10032-32C), perCP-Cy5.5-conjugated rat anti-mouse CD11C (N418; ebiosingen, BD.45-conjugated rat anti-7482, APC) and PE-conjugated rat anti-mouse IFN-gamma (XMG 1.2 clone; biolegend, cat.5059), perCP-Cy5.5-conjugated rat anti-mouse CD3 (145-2C11 clone;Sungene Biotech Co, ltd., cat.10032C); CFSE (Invitrogen, cat.4341058).

(II) spleen cell and Bone Marrow (BM) cell separation

C57BL/6 mice were sacrificed by cervical dislocation, and spleens were crushed. Individual splenocytes were filtered through a 40 micron nylon reticulocyte filter (BD Falcon) to obtain a single cell suspension and transferred to a 15ml EP tube containing RPMI1640 medium (containing 10% fbs,50 μm β -mercaptoethanol and 1% streptomycin/penicillin). The samples were centrifuged and washed with PBS. After the washing step, 3ml of 1×ack lysis buffer (Beyotime Biotechnology) was added to the lysed blood cells. After incubation for 3min at room temperature, 3ml of RPMI1640 medium was added to block ACK lysis buffer activity. The sample was centrifuged again and washed with PBS. Counting for standby.

(III) in vitro CpG-ODN stimulation

Will prepare 2X 10 ⁶ Spleen mononuclear cells from individual mice were inoculated into 24-well plates and cultured in RPMI1640 medium (10% FBS, 50. Mu.M. Beta. -mercaptoethanol and 1% streptomycin/penicillin). The CpG-OND was stimulated at a concentration of 5. Mu.g/ml, while BFA was added simultaneously at the beginning of the culture, at a final concentration of 4. Mu.g/ml. At 37℃and 5% CO ₂ After 6h of incubation in the incubator of (2), the cells were harvested and washed with PBS.

(IV) in vivo CpG-ODN stimulation

C57BL/6 mice at 11 weeks of age were intraperitoneally injected with 35. Mu.g CpG-ODN. Control mice were injected with PBS. Mice were sacrificed 12h after CpG-ODN injection. Isolated spleen cells were seeded in 24 well plates, BFA was added to the culture broth at a final concentration of 4. Mu.g/ml, and after 5h incubation the cells were harvested and washed with PBS for flow analysis.

(V) staining of cell surface and intracellular cytokines

For cell surface staining, spleen monocytes were first transferred to 96-well plates and cells were incubated at 4℃with 1. Mu.l 2.4G2 Fc receptor blocker added to 50. Mu.l staining buffer (PBS containing 1% FBS). After incubation for 30min, the cells were washed 2 times with staining buffer and stained with the corresponding antibody in 50 μl of staining buffer. The samples were incubated at 4℃for 30-40min in the absence of light. A single positive control for each fluorescent dye was used for appropriate color compensation. After incubation, the cells were washed 2 times with staining buffer.

For staining intracellular cytokines, cells were first incubated in 100 μl of fixation/permeation working fluid for 0.5h. Then, the cells were washed twice with 1 Xpermeabilized buffer. Mu.l of anti-mouse IFN-. Gamma.antibody was added to 100. Mu.l of 1 Xpermeabilization buffer and the cells were incubated at 4℃for 30min, after which the cells were washed twice with 1 Xpermeabilization buffer. All samples were fixed in 100 μl 1% paraformaldehyde solution and then detected by flow cytometry.

CFSE proliferation assay for NK cells and NKDCs

NK cells and NKDCs obtained by flow cytometry sorting are inoculated into a 24-well plate, the NK cells and the NKDCs are stimulated and cultured for 48 hours by IL2 (with the final concentration of 40 ng/mL) and CpG-ODN (with the final concentration of 5 mu g/mL) respectively, the cells are collected and incubated in CFSE staining solution with the final concentration of 3 mu M, the cells are subjected to light-resistant ice bath for 10 minutes, then the staining is stopped by using a whole culture medium, and the proliferation curve is detected by an up-flow cell analyzer after the ice bath for 5 minutes.

(seventh) statistical analysis

Statistical analysis was performed using Rstudio version 0.97.551 and GraphPad Prism 6. First, the normalization of all samples was checked using a Shapiro-Wilk test. If normalization (p > 0.05) is demonstrated, student's t-test is used to test for significant differences between conditions. If the samples are not normally distributed (p.ltoreq.0.05), the significance of the difference between the conditions is checked using a non-parametric Mann-Whitney U test. The correlation between the parameters was checked by the Pearson Correlation test. P is less than or equal to 0.05, and the difference is considered to be obvious; p.ltoreq.0.01 is considered to be very different.

2. Experimental results

1. Expression of CD11c in IKDCs correlates with B220 expression

In mice, CD11c is NK1.1 ⁺ Specific markers for NKDCs in the population, B220 was a specific marker for IKDCs, a subset of NKDCs, reflecting the possible correlation of CD11c expression with B220 expression in IKDCs differentiation under steady state conditions. To verify this hypothesis, monocytes (MNCs) were isolated from spleens of 4-6 and 12 week old C57BL/6 mice, stained with fluorochrome-conjugated monoclonal antibodies NK1.1, CD11C, B220 and CD3, and analyzed by flow cytometry. Exclusion of CD3 ⁺ Cell to eliminate NK1.1 in sample ⁺ CD3 ⁺ (NKT) cells. For NK1.1 ⁺ CD3 ^- The population was gated and divided into 4 subgroups (R1, R2, R3 and R4) based on the expression level of CD11c (fig. 1A). In these subgroups, the level of CD11c expression in NK cells (R1 and R2) was almost negative (CD 11c ^neg ) But from low levels in NKDCs (R3: CD11c ^low ) Gradually increasing to a medium level (R4: CD11c ^int ). B220 in these subgroups ⁺ Percentage of cells with NK1.1 ⁺ Up-regulation of CD11C expression on cells increased (fig. 1C). About 10%, 20%, 38% and 58% of B220 was found in the R1, R2, R3 and R4 sub-populations, respectively ⁺ Cells (Picture)1B&C) A. The invention relates to a method for producing a fibre-reinforced plastic composite Thus, under steady state conditions, about 50% of NKDCs (r3+r4) in the spleen are IKDCs. Although B220 in NK cells (R1 and R2) in aged mice (12 weeks old) compared to young mice (4-6 weeks old) ⁺ The percentage of cells was significantly reduced, but in NKDCs (R3 and R4) B220 ⁺ The percentage of cells was not significantly different between young and old mice (fig. 1B and 1C). In addition, CD11c expression levels and NK1.1 of young and old mice ⁺ B220 in subpopulation ⁺ The percentage of cells was linearly dependent (young mice: p=0.0176, r ² = 0.9652,Pearson's R = 0.9824 old mice: p=0.0299, r ² = 0.9411,Pearson's R = 0.9701) (fig. 1C), indicating that IKDCs differentiation is not affected by changes in steady state conditions (from young to old), whereas IKDCs differentiation follows NK1.1 ⁺ Upregulation of CD11c in the population was enhanced (NKDCs in R3: 62% B220) ^- NKDCs v.s.38% IKDCs; NKDCs in R4: 42% B220 ^- NKDCs v.s.58% IKDCs). In contrast, DC population (NK 1.1 ^- CD11c ^hi ) In both young and old mice<5% of B220 ⁺ Cells (pDCs: NK1.1-CD11 c) ^hi B220 ⁺ ). The percentage of pDCs in the aged mice appeared to be lower than in the young mice (fig. 1D). These results indicate that the differentiation of IKDCs is dependent on CD11c and B220 at NK1.1 ⁺ Related expression on the cell; CD11c is critical for NKDCs differentiation; b220 is critical for the differentiation of a subset of NKDCs, IKDCs.

2. NKDCs are functionally different from NK cells in terms of IFN-gamma production

It has been reported that NKDCs secrete IFN-gamma at higher levels than NK cells, although the number of NKDCs is not as abundant as NK cells. In steady state, the amount of IFN-gamma produced by NKDCs appears to be equivalent to that of B220 ⁺ NKDCs or IKDCs. To determine B220 ⁺ Whether NKDCs/IKDCs also produced more IFN-gamma than B220-NKDCs in the activated state, spleen MNCs were isolated from 5-8 week old C57BL/6 mice and were capable of aggregating intracellular cytokines IFN-gamma by in vitro stimulation with murine TLR9 ligand CpG oligodeoxynucleotides (CpG-ODNs) for 6h in the presence of BrefeldinA (BFA), a protein transport inhibitor. Then, NK1.1, B was performed on the samples220. IFN-. Gamma.and CD11c staining and analysis by flow cytometry. As shown in FIG. 2, both NKDCs and NK cells were activated by CpG-ODN, producing more IFN-gamma than unstimulated cells. It is possible that in steady state, NKDCs produce more IFN- γ than NK cells. In addition, steady-state NK cells produced more IFN-gamma than CpG-ODN activated NK cells (FIGS. 2A and B). In NK cells producing IFN-. Gamma.they are not affected by B220 expression. Regardless of the state (steady state or activated), about 80% of them are B220 negative (fig. 2C). In contrast, in IFN-gamma producing NKDCs, B220 ⁺ Comparable to the percentage of B220-NKDCs in steady state, but after CpG-ODN stimulation B220-NKDCs instead of B220 ⁺ The percentage of NKDCs increased (fig. 2D). These results indicate that: (a) In the steady and activated state, NKDCs produce IFN- γ much more than NK cells; (b) Whether activated or not, NK cells producing IFN-gamma are mostly B220-cells; (c) In steady state, B220 ⁺ And B220 ^- NKDCs constitutively produce considerable levels of IFN-gamma, but, upon activation by CpG-ODN, B220 ^- NKDCs predominate in the production of IFN- γ (fig. 2E).

3. Production of IFN-gamma by NKDCs correlates with expression of CD11c

Since expression of CD11c correlates with expression of B220 in NKDCs (FIG. 2B), we further investigated whether IFN-. Gamma.produced by NKDCs (R3 and R4) correlates with the expression level of CD11c (FIG. 3A). As shown in FIG. 3, NK cells (R1 and R2) hardly expressed CD11c (MFI<50 Producing considerable levels of IFN- γ in the steady and activated states (fig. 3B and 3C). However, the ability of NKDCs (R3 and R4) to produce IFN-. Gamma.was correlated with the expression level of CD11c, either in steady state or CpG-ODN stimulated activation (R3: MFI)>100，R4：MFI>200 (fig. 3C). In the activated state, IFN- γ producing NK cells were increased mainly in the R1 population, but IFN- γ producing NKDCs were increased mainly in the R4 population (FIGS. 3B and C). Linear regression analysis showed that IFN- γ produced by NKDCs was highly correlated with expression of CD11c under CpG-ODN stimulation (Pearson's R =0.9819 r ² =0.8782, p=0.0181) (fig. 3C). Further separation of the components of IFN-gamma producing NK cells (R1 and R2) and IFN-gamma producing NKDCs (R3 and R4)Analysis showed that B220-cells are the dominant population of IFN- γ -producing NK cells, regardless of their status (FIGS. 3D and E), but B220- γ -producing IFN- γ ^- And B220 ⁺ The proportion of NKDCs was distributed differently in the R3 and R4 populations in the steady state and in the activated state (fig. 3F and G). IFN-. Gamma.producing B220 in the NKDCs population when activated by CpG-ODN ^- NKDCs increased (fig. 3D and G). However, in activated B220 ^- In NKDCs, the increase in IFN-gamma production appeared to be independent of CD11c levels (FIG. 3F&G) Indicating B220 ^- NKDCs to B220 ⁺ NKDCs respond more actively to exogenous stimuli. The results suggest that CD11c may be an important factor in mediating IFN- γ production by NKDCs.

4. NKDCs are not just activated NK cells

IKDCs are reported to be a subset of NKDCs, and possibly also activated NK cells. However, IFN- γ producing activated NK cells did not express CD11c, and most of them also did not express B220 (FIG. 3), suggesting that NKDCs are functionally different from NK cells. To further elucidate the relationship between NKDCs and NK cells, we examined the expression of lymphocyte activation marker CD69 on NKDCs and NK cells after CpG-ODN stimulation. Spleen MNCs isolated from 5 to 8 week old C57BL/6 mice were stimulated in vitro with CpG-ODN for 6h. BFA was not added to the cell culture medium because BFA could inhibit up-regulation of CD69 expression during cell activation. Following stimulation, cells were stained for NK1.1, CD11c, CD3 and CD 69. NK1.1 ⁺ The cell population was divided into R1, R2, R3 and R4 populations as previously defined (fig. 4A). In steady state, CD69 expression seems to be similar to NK1.1 ⁺ The level of CD11c on the cells was positively correlated. After CpG-ODN activation, CD69 expression was significantly upregulated in both NK cells (R1+R2) and NKDCs (R3+R4), but CD69 expression was much higher in NKDCs (R3+R4) than in NK cells (R1+R2) (FIG. 4B). The above results indicate that CpG-ODNs are different in activation effect on NKDCs and NK cells, further confirming that NKDCs are unlikely to be activated NK cells.

5. Stimulation of CpG-ODN promotes proliferation of NKDCs in vivo or in vitro

Considering that CD69 is significantly up-regulated in all NK cells under in vitro CpG-ODN stimulation, we speculate that CpG was in vivoNK cells may expand more than NKDCs under ODN stimulation. We stimulated 11 week old C57BL/6 mice in vivo for 12h by intraperitoneal injection of CpG-ODN. Spleen MNCs were then isolated, NK1.1, CD3, CD11c and B220 stained and NK cells were analyzed for expansion by flow cytometry (fig. 5A). CD3 ⁺ Cells were gated to exclude Natural Killer T (NKT) cells. As shown in FIG. 5, there was no significant difference in the percentage of NK cells in the spleens of mice in the absence of stimulation with CpG-ODN, whereas the percentage of NKDCs in the spleens of mice after stimulation with CpG-ODN was increased 2-fold over the unstimulated group (FIG. 5B). In addition, the percentage of DC in the spleen of mice was also almost unaffected by the stimulation with CpG-ODN (FIG. 5B).

Meanwhile, spleen MNCs of 5-8-week-old C57BL/6 mice are separated, NK cells and NKDCs are obtained through sorting by a flow cytometer, and then after the cells are respectively stimulated and cultured for 48 hours by CpG-ODN and IL2, the division and proliferation conditions of the NK cells and the NKDCs are detected by a CFSE (circulating fluid flow cytometer) after staining. The results showed that NK cells proliferated only once in 48h in the CpG-ODN stimulated mice, while a small proportion of NK cells proliferated in the IL2 stimulated and untreated control groups, but that NKDCs stimulated with CpG-ODN proliferated twice in 48h, and two distinct proliferation peaks occurred, while NKDCs stimulated with IL2 and untreated control groups proliferated only once (fig. 5C). These results further demonstrate that NK cells respond quite differently to CpG-ODN stimulation with NKDCs, which significantly promotes proliferation of NKDCs.

6. CD11 c-mediated production of IFN-gamma by NKDCs

Since the level of expression of CD11C is positively correlated with the amount of IFN- γ production in NKDCs (FIG. 3C), we further investigated whether blocking CD11C signaling could prevent IFN- γ production in NKDCs. Spleen cells were isolated from the spleen of C57BL/6 mice and stimulated with CpG-ODN (positive control), anti-CD 11C mab, isotype IgG (anti-CD 11C mab control) for 6h in 96-well plates. Cells were then stained with fluorochrome-conjugated mab CD11c, CD3, NK1.1 and IFN- γ and analyzed by flow cytometry. As shown in fig. 6, for CD3 ^- NK1.1 ⁺ And CD3 ^- NK1.1 ^- Cells were gated (FIGS. 6A and 6B) for sortingThe effect on IFN-gamma production by NK cells and NKDCs under anti-CD 11c mab, igG and CpG-ODN stimulation was analyzed. First, we analyzed the variation of the NK and NKDCs ratios in the different treatment groups. The anti-CD 11c treated group did not significantly alter the ratio of NK and NKDCs compared to the isotype control IgG group. However, the percentage of NK cells and NKDCs was significantly increased in the group stimulated with CpG-ODN (FIG. 6B-C), suggesting that CpG-ODN, but not anti-CD 11C mab, may affect the differentiation of NK cells and NKDCs or promote proliferation of NK cells and NKDCs. Then, we analyzed the effect of anti-CD 11c mab on IFN- γ production by NKDCs and NK cells. CpG-ODN stimulation significantly promoted IFN-gamma production by NK cells compared to isotype control IgG, but anti-CD 11C mab did not promote IFN-gamma production by NK cells (FIG. 6B), but specifically promoted IFN-gamma production by NKDCs, with very significant differences compared to IFN-gamma production by NKDCs stimulated with CpG-ODN (FIG. 6C). Overall, CD11c can mediate the production of IFN- γ by NKDCs.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. It will be apparent to those skilled in the art that any equivalent modifications and substitutions of the present invention are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims

Application of expression promoter of CD11c in preparing tumor treating medicine.
2. The use according to claim 1, wherein the expression promoter of CD11c plays a role in the treatment of tumors by promoting IFN- γ production capacity of NKDCs.
3. The use according to claim 1, wherein the expression enhancer of CD11c comprises an over-expression vector with CD11c gene and/or a small molecule drug targeting CD11c protein and enhancing its expression, stability and/or increased activity.
4. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.
5. The use according to claim 1, wherein the medicament is in the form of an injection, a tablet, a pill, a capsule, a solution, a suspension or an emulsion.
6. A medicament for treating tumors, comprising an expression enhancer for CD11 c.
7. The drug according to claim 6, wherein said expression promoter of CD11c plays a role in the treatment of tumors by promoting IFN- γ production capacity of NKDCs.
8. The agent of claim 6, wherein the expression enhancer of CD11c comprises an over-expression vector having a CD11c gene and/or a small molecule agent that targets CD11c protein and promotes its expression, stabilization and/or increased activity.
9. The medicament of claim 6, further comprising a pharmaceutically acceptable carrier or excipient.
10. The medicament according to claim 6, wherein the medicament is in the form of an injection, a tablet, a pill, a capsule, a solution, a suspension or an emulsion.