COMBINED FORMULATION KIT FOR ANALYZING PHENOTYPE AND FUNCTION OF CD1C+DENRTIC CELL SUBSET AND USE THEREOF

Abstract

Disclosed are a combined formulation kit for analyzing the phenotype and function of a CD1c.sup.+ dendritic cell subset and the use thereof, wherein the detection objects of the kit include CD1c, CD40, IL-6 and IL-10. The kit can be used to efficiently and quickly identify the phenotype of a CD1c.sup.+ dendritic cell subset in peripheral blood and analyze the function thereof, thereby ensuring accuracy and reducing the economic cost produced by detecting a large number of surface antigen molecules, and the detection method is also simple to implement.

Claims

1. A combined formulation design for identifying the phenotype and function of a CD1c+ dendritic cell subset, comprising CD1c, CD40, IL-6 and IL-10.

2. A method for identifying and/or preparing a product for identifying the phenotype and function of a CD1c.sup.+ dendritic cell subset comprising using the combined formulation design of claim 1.

3. The method according to claim 2, wherein the product comprises a kit and/or a detection reagent.

4. A kit for identifying the phenotype and function of a CD1c.sup.+ dendritic cell subset, comprising an anti-CD1c antibody, an anti-CD40 antibody, an anti-IL-6 antibody and an anti-IL-10 antibody, wherein the anti-CD1c antibody, the anti-CD40 antibody, the anti-IL-6 antibody and the anti-IL-10 antibodies are labeled with four different fluorochromes, respectively.

5. The kit according to claim 4, wherein the fluorochrome label is selected from FITC, PE-Cy7, PerCP-Cy5.5, Amcyan, APC-Cy7 or Q-Dot.

6. A method for identifying the phenotype and function of a CD1c.sup.+ dendritic cell subset, which adopts a kit of claim 4 for detection, wherein the method comprising the following steps: (1) pretreatment of peripheral blood: separating dendritic cells, adding a leukocyte-stimulating factor and incubating; (2) staining the blood cells obtained in step (1), then adding an anti-CD40 antibody and an anti-CD1c antibody that are labeled with different fluorochromes, carrying out a first incubation, staining again, and then fixing the obtained dendritic cells with a formalin solution, and carrying out a second incubation for later use; (3) resuspending the cells obtained in step (2) in a cell-penetrating solution, centrifuging and discarding the supernatant, resuspending the precipitated cells in a cell-penetrating solution, adding an anti-IL-6 antibody and an anti-IL-10 antibody that are labeled with different fluorochromes, and incubating; and (4) resuspending the incubated cells in step (3) in a cell-penetrating solution, centrifuging and discarding the supernatant, resuspending the precipitated cells in a cell-staining solution, and analyzing and detecting with a flow cytometry.

7. The method according to claim 6, wherein the volume of the peripheral blood in step (1) is 10-100 μL.

8. The method according to claim 7, wherein the volume concentration of the leukocyte-stimulating factor is 0.1%-0.3%.

9. The method according to claim 7, wherein the incubation in step (1) is carried out for 4-6 h.

10. The method according to claim 7, wherein the incubation in step (1) is carried out at a temperature of 37-40° C.

11. The method according to claim 6, wherein the first incubation in step (2) is carried out at room temperature for 30-60 min.

12. The method according to claim 11, wherein the mass fraction of the formalin solution in step (2) is 2-4%.

13. The method according to claim 6, wherein the incubation in step (3) is carried out for 12-24 h at 4° C. in the dark.

14. The method according to claim 6, wherein, the analysis and detection comprise the following steps: analyzing the proportion of a dendritic cell subset having phenotype CD1c.sup.+ though the expression of CD1c, analyzing the differentiation and maturation status of the CD1c.sup.+ dendritic cell subset though the expression of CD40 molecule, and analyzing the function of the CD1c.sup.+ dendritic cell subset though the secretion and expression of IL-6 and IL-10.

15. The method according to claim 6, wherein the method specifically comprising the following steps: (1) subjecting 10-100 μL of peripheral blood to anticoagulation treatment, mixing the whole peripheral blood with 1× red blood cell lysis buffer, rotating and shaking for 10 s, leaving at room temperature in the dark for 15 min, centrifuging at 350 g for 5 min, discarding the supernatant, resuspending the precipitated cells in a cell-staining solution, adding a leukocyte-stimulating factor at a volume concentration of 0.08-0.1% and incubating at 37° C. for 4-6 h; (2) staining the blood cells obtained in step (1), then adding an anti-CD40 antibody and an anti-CD1c antibody that are labeled with different fluorochromes, incubating for 25-35 min at room temperature, staining again, and then fixing the obtained dendritic cells with 2% formalin solution, and incubating at room temperature in the dark for 15 min for later use; (3) resuspending the cells obtained in step (2) in a cell-penetrating solution, centrifuging and discarding the supernatant, resuspending the precipitated cells in a cell-penetrating solution, adding an anti-IL-6 antibody and an anti-IL-10 antibody that are labeled with different fluorochromes, and incubating at 4° C. in the dark for 12 h; and (4) resuspending the incubated cells in step (3) in a cell-penetrating solution, centrifuging and discarding the supernatant, resuspending the precipitated cells in a cell-staining solution, and analyzing and detecting by flow cytometry, analyzing the proportion of a dendritic cell subset having phenotype CD1c.sup.+ though the expression of CD1c, analyzing the differentiation and maturation status of the CD1c.sup.+ dendritic cell subset though the expression of CD40 molecule, and analyzing the function of the CD1c.sup.+ dendritic cell subset though the secretion and expression of IL-6 and IL-10.

16. The method according to claim 11, wherein the second incubation in step (2) is carried out at room temperature in the dark for 15-20 min.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0044] FIG. 1 shows the expression ratio of CD40 on CD1c.sup.+ dendritic cells in lung small cell carcinoma patients in the example;

[0045] FIG. 2 shows the expression ratio of IL-6 on CD1c.sup.+ dendritic cells in lung small cell carcinoma patients in the example;

[0046] FIG. 3 shows the expression ratio of IL-10 on CD1c.sup.+ dendritic cells in lung small cell carcinoma patients in the example;

[0047] FIG. 4 shows the expression ratio of CD40 on CD1c.sup.+ dendritic cells in healthy individuals in the example;

[0048] FIG. 5 shows the expression ratio of IL-6 on CD1c.sup.+ dendritic cells in healthy individuals in the example;

[0049] FIG. 6 shows the expression ratio of IL-10 on CD1c.sup.+ dendritic cells in healthy individuals in the example.

DETAILED DESCRIPTION

[0050] In order to further illustrate the technical means adopted in the present application and effect thereof, the technical solutions of the present application are further described below by detailed description, but the present application is not limited to the scope of the examples.

[0051] Experimental Materials

[0052] Flow cytometry (BD, C6);

[0053] Anti-human CD1c, CD40, IL-6 antibodies (Biolegend) IL-10 antibody (BD).

Example 1 Pretreatment of Peripheral Blood from Non-Small Cell Lung Cancer Patients/Healthy Individuals

[0054] The pretreatment steps are as follows:

[0055] (1) One drop (10-100 μl) of venous peripheral blood was taken from patients with non-small cell lung cancer and healthy adults, respectively, and anticoagulated.

[0056] (2) The whole peripheral blood was mixed in 2 ml 1× Red Blood Cell Lysis Buffer (Biolegend), rotated and shaken for 10 seconds and then left at room temperature for 15 min in the dark.

[0057] (3) Centrifuged in a centrifuge (350 g for 5 min), the supernatant was poured out, and the precipitated cells were suspended in 2 ml of cell-staining solution (PBS solution containing 2.5% fetal bovine serum).

[0058] (4) A leukocyte stimulating factor (BD) was added at a concentration of 0.1% and the cells were incubated at a constant temperature of 37 degrees for 6 hours.

Example 2 Analysis of Degree of Development and Differentiation of CD1c+ Dendritic Cell Subsets in Peripheral Blood from Non-Small Cell Lung Cancer Patients/Healthy Individuals

[0059] The spare blood cells were centrifuged (350 g) for 5 minutes, the supernatant was poured out, and then the cells were suspended in 100 μl of cell-staining solution. Then 2 μl anti-human CD1c antibody and 2 μl anti-human CD40 antibody (Biolegend) were added, incubated for 30 min at room temperature, and then 2 ml of cell-staining solution was added, and centrifuged (350 g) twice, each for 5 min. After the supernatant was poured out, the cells were fixed with 2 ml of 2% formalin solution and incubated for 20 min at room temperature in the dark.

Example 3 Functional Analysis of CD1c+ Dendritic Cell Subsets in Peripheral Blood from Non-Small Cell Lung Cancer Patients/Healthy Individuals

[0060] (1) The fixed spare cells were suspended in 2 ml of cell-penetrating solution (Biolegend) and centrifuged (350 g) for 10 min for twice.

[0061] (2) The precipitated cells were resuspended in 100 μl of cell-penetrating solution after centrifugation, added with 2 μl IL-6 antibody (Biolegend) and 2 μl IL-10 antibody (BD), and incubated for 30 min at room temperature in the dark.

[0062] (3) The incubated cells were suspended in 2 ml of cell-penetrating solution and then centrifuged (350 g) for 5 minutes for twice.

[0063] (4) Finally, the supernatant was poured out and the precipitated cells were resuspended in 0.5 ml of cell-staining solution and tested by flow cytometry analysis.

[0064] Detection and Results Analysis

[0065] 1. The expression of the co-signaling stimulatory molecule CD40 in human peripheral blood CD1c.sup.+ dendritic cell subset was detected by flow cytometry (this data was used to assess the differentiation and maturation status of the human peripheral blood CD1c.sup.+ dendritic cell subset).

[0066] 2. Functional analysis of human peripheral blood CD1c.sup.+ dendritic cells: the secretion and expression of cytokines IL-6 and IL-10 in CD1c.sup.+ dendritic cells were determined (expressed as a ratio in %). The results are shown in FIGS. 1 to 6, wherein the expression of CD40, IL-6, and IL-10 in CD1c.sup.+ dendritic cells in lung small cell carcinoma patients are shown in FIGS. 1 to 3, and the expression of CD40, IL-6, and IL-10 in CD1c.sup.+ dendritic cells in healthy individuals are shown in FIGS. 4 to 6, respectively.

[0067] As shown in FIGS. 1 to 3, the expression ratio of CD40, IL-6, and IL-10 on CD1c.sup.+ dendritic cells in non-small cell lung cancer patients were 5.45%, 2.22%, and 7.4%, respectively, while the expression ratio of CD40, IL-6, and IL-10 on CD1c.sup.+ dendritic cells in healthy individuals were 96.9%, 27.3%, and 3.19%, respectively, demonstrating that the combined formulation design and identification method of the present application can effectively identify CD1c.sup.+ dendritic cell subsets in peripheral blood and analyze their differentiation and maturation as well as their function. For example, it is known that if the expression of CD40 on the surface of DCs is higher, the differentiation and maturation degree of the DCs is higher. Our results showed that the expression of CD40 on the surface of DCs in healthy individuals is significantly more than that in lung cancer patients (FIGS. 1 and 4), indicating that the differentiation and maturation degree of CD1c.sup.+ DCs in lung cancer patients was significantly lower than that of CD1c.sup.+ DCs in healthy individuals. For another example, IL-6 is a cytokine that promotes immune responses, and if DCs can secrete more IL-6, it indicates that the DCs can promote immune responses by secreting more IL-6. Our results showed that CD1c.sup.+ DCs in normal healthy individuals secreted significantly more IL-6 than those in lung cancer patients (FIG. 2 and FIG. 5), which indicates that the ability of CD1c.sup.+ DCs in lung cancer patients to enhance immune responses by secreting IL-6 is not as strong as that in healthy individuals. This is a sign of low CD1c.sup.+ DC-mediated immune function in lung cancer patients. In contrast, IL-10 is a cytokine that suppresses immune function, and if DCs secrete more IL-10, it indicates that the DCs have immunosuppressive efficacy and can suppress immune responses by secreting more IL-10. Our results showed that CD1c.sup.+ DCs in lung cancer patients precisely secreted more IL-10 than CD1c.sup.+ DCs in normal healthy individuals (FIG. 3 and FIG. 6). This indicates that CD1c.sup.+ DCs in lung cancer patients can inhibit immune function by secreting more IL-10 than CD1c.sup.+DCs in healthy individuals, and CD1c.sup.+DCs in lung cancer patients are a kind of DCs with immunosuppressive function compared with CD1c.sup.+DCs in healthy individuals.

[0068] In summary, the assay protocol of the present application can efficiently and rapidly compare the development and differentiation differences as well as functional differences between peripheral blood CD1c.sup.+DCs from patients with non-small cell lung cancer and healthy individuals. The method of the present application uses human whole blood to determine dendritic cell subsets in human peripheral blood and their function, which is simpler and easier to implement, and saves a lot of labow, material and financial resources than the traditional PBMC isolation method. The method of the present application requires only one drop of blood (10-1000 from the patient to obtain the desired full set of information. It saves a lot of time in separating PBMCs, making it simple and fast to determine in one step. It is suitable for testing a large number of samples in clinical.