CN114832014B

CN114832014B - Application of NK (Natural killer) cells, NK cell reinfusion preparation and combined preparation

Info

Publication number: CN114832014B
Application number: CN202210776222.7A
Authority: CN
Inventors: 黄晓军; 赵翔宇; 商倩楠; 余星星
Original assignee: Peking University Peoples Hospital
Current assignee: Peking University Peoples Hospital
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2022-10-21
Anticipated expiration: 2042-07-04
Also published as: CN115887493A; CN114832014A

Abstract

The invention provides an application of NK cells, an NK cell reinfusion preparation and a combined preparation, wherein NK cell reinfusion can prevent cytomegalovirus infection, eliminate cytomegalovirus infection and promote immune reconstitution of NK cells, is safe and well-tolerated, does not have serious side effects related to infusion, does not cause virus-resistant strains by adopting NK cell reinfusion, does not cause a series of drug side effects such as obvious bone marrow inhibition and renal function damage, and has mature and stronger functions of NK cell phenotypes reconstructed in vivo of patients after NK cell reinfusion.

Description

Application of NK (Natural killer) cells, NK cell reinfusion preparation and combined preparation

Technical Field

The invention relates to the field of biomedicine, in particular to application of NK cells, an NK cell reinfusion preparation and a combined preparation.

Background

Following hematopoietic stem cell transplantation or solid tumor transplantation, the most common complication is Cytomegalovirus (CMV) infection, accounting for nearly 50% of graft-related deaths. Since the number and function of early immune cells are not reconstructed to normal level after transplantation, cytomegalovirus infection is easy to occur, the death risk related to transplantation is increased, and the transplantation result is poor. The antiviral drugs have limited prevention and treatment effects, and long-term application of the antiviral drugs can cause virus-resistant strains, and can cause a series of drug side effects such as obvious bone marrow suppression, renal function damage and the like. Prevention and treatment of CMV infection remains a major challenge to be addressed at present.

Disclosure of Invention

In order to solve the problems in the background art, the invention provides an application of NK cells, an NK cell reinfusion preparation and a combined preparation, so as to prevent cytomegalovirus infection or eliminate cytomegalovirus infection or promote NK cell immune reconstitution.

In a first aspect, the invention provides a use of NK cells for preparing a preparation for preventing cytomegalovirus infection, wherein the preparation for preventing cytomegalovirus infection comprises a first preparation; the first formulation comprises expanded NK cells in vitro, the first formulationA preparation contains NK cells not less than 1 × 10 ⁷ /kg, said first formulation is for use in the prevention of cytomegalovirus infection.

Alternatively, the first formulation is administered twice each on 20 days and 27 days after hematopoietic stem cell transplantation.

Optionally, the agent for preventing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M ² The second formulation is administered after administration of the first formulation, the second formulation for aiding the first formulation in preventing cytomegalovirus infection.

In a second aspect, the present invention provides a use of NK cells in the preparation of a cytomegalovirus-clearing formulation comprising a first formulation; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 ⁷ Kg, said first formulation is for use in clearing cytomegalovirus infection.

Optionally, the agent for clearing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M ² The second formulation is administered after administration of the first formulation, the second formulation being used to aid the first formulation in clearing cytomegalovirus infections.

In a third aspect, the present invention provides the use of NK cells in the preparation of an agent for promoting immune reconstitution, said agent for promoting immune reconstitution comprising a first agent; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 ⁷ /kg, said first formulation is for promoting immune reconstitution of NK cells.

Optionally, the agent that promotes immune reconstitution further comprises a second agent; the second formulation comprises IL-2, the second formulationThe IL-2 content in the preparation is 40 ten thousand IU/M ² A second formulation for administration after administration of the first formulation, the second formulation for aiding the first formulation in promoting NK cell immune reconstitution.

In a fourth aspect, the present invention provides an NK cell transfusion preparation comprising: a first formulation comprising ex vivo expanded NK cells, the content of ex vivo expanded NK cells in the first formulation being not less than 1 x 10 ⁷ /kg, the first formulation is for preventing cytomegalovirus infection, or for clearing cytomegalovirus infection in vivo, or for promoting immune reconstitution of NK cells.

Optionally, the NK cell reinfusion formulation further comprises a second formulation; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M ² Said second formulation being administered after administration of said first formulation.

In a fifth aspect, the invention provides a combined preparation for preventing cytomegalovirus infection after hematopoietic stem cell transplantation, which comprises an NK cell return preparation and IL-2, wherein the content of in-vitro amplified NK cells in the NK cell return preparation is not less than 1 x 10 ⁷ /kg, the content of the IL-2 is 40 ten thousand IU/M ² (ii) a The NK cell reinfusion formulation was administered twice on days 20 and 27 after hematopoietic stem cell transplantation, and IL-2 was administered three times per week after the first administration of the NK cell reinfusion formulation.

In a sixth aspect, the invention provides a combined preparation for eliminating cytomegalovirus infection after hematopoietic stem cell transplantation, which comprises an NK cell return preparation and IL-2, wherein the content of in-vitro amplified NK cells in the NK cell return preparation is not less than 1 x 10 ⁷ Per kg, the content of the IL-2 is 40 ten thousand IU/M ² (ii) a The NK cell reinfusion formulation was administered twice on days 20 and 27 after hematopoietic stem cell transplantation, and IL-2 was administered three times per week after the first administration of the NK cell reinfusion formulation.

In a seventh aspect, the present invention provides a combined preparation for promoting immune reconstitution of NK cells including an NK cell reinfusion preparation and IL-2 after hematopoietic stem cell transplantationThe content of in vitro amplified NK cells in the back infusion preparation is not less than 1 × 10 ⁷ /kg, the content of the IL-2 is 40 ten thousand IU/M ² (ii) a The NK cell reinfusion formulation was administered twice on days 20 and 27 after hematopoietic stem cell transplantation, and IL-2 was administered three times per week after the first administration of the NK cell reinfusion formulation.

Compared with the prior art, the invention has the following advantages:

the NK cell feedback is safe and good in tolerance, no serious side effect related to infusion occurs, virus-resistant strains cannot be caused by adopting the NK cell feedback, a series of drug side effects such as obvious bone marrow suppression and renal function damage cannot be caused, the HCMV infection can be prevented, HCMV elimination can be promoted, the immune reconstruction of NK cells can be promoted, and the NK cell phenotype in a patient body after the NK cell feedback is mature and has stronger function.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments of the present invention will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without inventive labor.

FIG. 1 shows the proportion of NK cells in liver, spleen, lung, periphery after NK cell reinfusion for 14 d;

figure 2 shows HCMV clearance in liver, spleen, lung, periphery following NK cell reinfusion for 14 d;

figure 3 shows a comparison of NK reinfusion groups with control groups for HCMV infection and refractory HCMV infection;

figure 4 shows a comparison of the duration of HCMV infection in NK reinfusion groups versus control groups;

FIG. 5 shows a comparison of NK reconstitution of NK cells in NK reinfused group and control group;

FIG. 6 shows NK cell monitoring in vivo after 20 patient NK cell reinfusion;

FIG. 7 shows a comparison of HCMV and refractory HCMV infection in IL-2-injected and non-injected IL-2 groups;

FIG. 8 shows the percentage and absolute number of NK cells for the IL-2-injected group and the IL-2-non-injected group;

FIG. 9 shows the absolute numbers of NKG2C + NK subpopulations versus the expression of the activation receptor NKP30 for the IL-2-injected and non-injected IL-2 groups.

Detailed Description

The following examples are provided to better understand the present invention, not to limit the best mode, and not to limit the content and protection scope of the present invention, and any product that is the same or similar to the present invention and is obtained by combining the present invention with other features of the prior art and the present invention falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents and other instruments used are not indicated by manufacturers, and are conventional reagent products which can be purchased in the market.

NK cells are the earliest reconstituted immune cells after hematopoietic stem cell transplantation. The reconstitution of HCMV-specific NK cells 180 days after transplantation was found to be inversely correlated with the incidence of CMV infection. Our previous studies also showed that patients with rapid IFN-. Gamma. + NK cell reconstitution had a low HCMV infection rate 15 days after transplantation. Although there is much clinical evidence that there is a correlation between NK cell function and CMV infection, whether NK cells have anti-CMV activity in vivo to directly kill viruses or inhibit the spread of viruses has not been experimentally confirmed.

Adoptive reinfusion of NK cells holds great promise in the treatment of tumors. However, because the number of NK cells in human peripheral blood is limited, at present, in clinical studies, the in vitro amplified NK cells are mainly used for adoptive feedback of NK, and the inventor discovers that the NK cells amplified by the mbIL21/4-1BBL have stronger expression of activating receptors through in vitro experiments, such as NKP30, NKG2D, DNAM-1 and the like. However, CMV has strict species specificity and in humans NK cell deficient patients are more susceptible to relapse after recovery from herpes virus infection and it is unknown whether NK cells have anti-HCMV effects in clinical applications.

The inventor further explores and discovers that: the NK cell feedback can prevent cytomegalovirus infection, eliminate cytomegalovirus infection and promote the immune reconstitution of NK cells.

Based on the above, in a first aspect, the embodiments of the present invention provide a use of NK cells in preparing a preparation for preventing cytomegalovirus infection, wherein the preparation for preventing cytomegalovirus infection comprises a first preparation; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 ⁷ /kg, said first formulation is for use in the prevention of cytomegalovirus infection.

Alternatively, the first formulation is administered two times each 20 days and 27 days after hematopoietic stem cell transplantation.

The inventors have also found that interleukin-2 (IL-2) can enhance NK cell proliferation, stimulate NK cell activation and cytotoxicity.

In an alternative embodiment of the invention, it is proposed that the agent for preventing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M ² The second formulation is administered after administration of the first formulation, the second formulation being used to assist the first formulation in preventing cytomegalovirus infection.

In the embodiment of the invention, the agent for preventing cytomegalovirus infection can be applied after all operations (including hematopoietic stem cell transplantation and tumor transplantation) which can cause cytomegalovirus infection, so as to prevent the cytomegalovirus infection caused by the operations.

In the embodiment of the invention, the preparation for preventing cytomegalovirus infection can be applied to all people with low immunity (including HIV infectors, immunodeficiency patients and patients with rheumatic immunity) so as to prevent the people with low immunity from infecting cytomegalovirus.

Based on the same inventive concept, in a second aspect, the invention provides an application of NK cells in preparing a preparation for eliminating cytomegalovirus,the cytomegalovirus-clearing agent comprises a first agent; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 ⁷ /kg, said first formulation is used to clear cytomegalovirus infections.

Optionally, the agent for clearing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of the IL-2 in the second preparation is 40 ten thousand IU/M ² The second agent is administered after administration of the first agent, the second agent for aiding the first agent in clearing cytomegalovirus infection.

In the embodiment of the invention, the preparation for eliminating cytomegalovirus infection can be applied after all operations (including hematopoietic stem cell transplantation and tumor transplantation) which can cause cytomegalovirus infection, so as to eliminate the cytomegalovirus infection caused by the operations.

In the embodiment of the invention, the preparation for eliminating cytomegalovirus infection can be applied to all people with low immunity (including HIV infectors, immunodeficiency patients and rheumatoid immunity patients) so as to eliminate cytomegalovirus infected by people with low immunity.

Based on the same inventive concept, in a third aspect, the present invention provides a use of NK cells for preparing an agent for promoting immune reconstitution, the agent for promoting immune reconstitution comprising a first agent; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 ⁷ /kg, said first formulation is for promoting immune reconstitution of NK cells.

Alternatively, the first preparation is administered two times on 20 days and 27 days after the hematopoietic stem cell transplantation, respectively.

Optionally, the agent that promotes immune reconstitution further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M ² Said second formulation being administered after administration of said first formulation, said second formulation being used to assist in promoting the action of the first formulationAnd (4) immune reconstitution of the NK cells.

In embodiments of the invention, the agent for eliminating cytomegalovirus infection can be administered after all surgeries (including hematopoietic stem cell transplantation and tumor transplantation) which may cause cytomegalovirus infection, so as to promote the immune reconstitution of NK cells in patients after the surgeries.

In the embodiment of the invention, the preparation for eliminating cytomegalovirus infection can be applied to all people with low immunity (including HIV infected people, immunodeficiency patients and patients with rheumatic immunity) so as to promote the immune reconstruction of NK cells in people with low immunity.

Based on the same inventive concept, in a fourth aspect, the present invention provides an NK cell transfusion preparation, comprising: a first preparation comprising ex vivo expanded NK cells, the content of the ex vivo expanded NK cells in the first preparation being not less than 1 x 10 ⁷ /kg, the first formulation is for preventing cytomegalovirus infection, or for clearing cytomegalovirus infection in vivo, or for promoting immune reconstitution of NK cells.

In an alternative embodiment of the invention, the NK cell back-infusion formulation further comprises: a second formulation comprising IL-2, the IL-2 content of the second formulation being 40 ten thousand IU/M ² And a second formulation for aiding the first formulation in preventing cytomegalovirus infection, or in clearing cytomegalovirus infection in vivo, or in promoting NK cell immune reconstitution, administered after the first formulation is administered.

The NK cell transfusion preparation provided by the embodiment of the invention can be applied after all operations (including hematopoietic stem cell transplantation and tumor transplantation) which can cause cytomegalovirus infection, so as to prevent the cytomegalovirus infection caused by the operations, eliminate the cytomegalovirus infection caused by the operations, or promote the immune reconstruction of NK cells in a patient after the operations.

The NK cell return preparation provided by the embodiment of the invention can also comprise: HIV-infected persons, immunodeficiency patients, and patients with rheumatic immunity) to prevent cytomegalovirus infection of immunocompromised persons, or to eliminate cytomegalovirus infection of immunocompromised persons, or to promote NK cell reconstitution in immunocompromised persons.

Specifically, when the NK cell reinfusion formulation of the present embodiment is used for a patient after hematopoietic stem cell transplantation, the administration method may specifically include: the first preparation is administered two times on 20 days and 27 days after the hematopoietic stem cell transplantation, and the second preparation is administered three times per week after the first preparation to prevent cytomegalovirus infection after the hematopoietic stem cell transplantation, or to eliminate cytomegalovirus infection after the hematopoietic stem cell transplantation, or to promote immune reconstitution of NK cells after the hematopoietic stem cell transplantation.

Based on the same inventive concept, in a fifth aspect, the invention provides a combined preparation for preventing cytomegalovirus infection after hematopoietic stem cell transplantation, which comprises an NK cell transfusion preparation and IL-2, wherein the content of in-vitro amplified NK cells in the NK cell transfusion preparation is not less than 1 x 10 ⁷ Per kg, the content of the IL-2 is 40 ten thousand IU/M ² (ii) a The NK cell reinfusion formulation was administered twice on days 20 and 27 after hematopoietic stem cell transplantation, and IL-2 was administered three times per week after the first administration of the NK cell reinfusion formulation.

Based on the same inventive concept, in a sixth aspect, the invention provides a combined preparation for eliminating cytomegalovirus infection after hematopoietic stem cell transplantation, which comprises an NK cell reinfusion preparation and IL-2, wherein the content of in-vitro amplified NK cells in the NK cell reinfusion preparation is not less than 1 x 10 ⁷ Per kg, the content of the IL-2 is 40 ten thousand IU/M ² (ii) a The NK cell transfusion preparation was administered twice on 20 days and 27 days after hematopoietic stem cell transplantation, respectively, and IL-2 was administered three times per week after the first administration of the NK cell transfusion preparation.

Based on the same inventive concept, in a seventh aspect, the present invention provides a combined preparation for promoting immune reconstitution of NK cells after hematopoietic stem cell transplantation, the combined preparation comprising an NK cell reinfusion preparation in which the content of in vitro expanded NK cells is not less than 1X 10 and IL-2 ⁷ /kg, the content of the IL-2 is 40 ten thousand IU/M ² (ii) a The NK cell transfusion preparation was administered twice on 20 days and 27 days after hematopoietic stem cell transplantation, respectively, and IL-2 was administered three times per week after the first administration of the NK cell transfusion preparation.

In order to make the present invention better understood by those skilled in the art, the application of the NK cells, the NK cell transfusion preparation and the combination preparation provided by the present invention are illustrated by specific examples below.

Example 1 mouse model experiment:

(ii) infusing HCMV-IGG seropositive G-CSF mobilized donor peripheral blood stem cells 1X 106/mouse through the tail vein after irradiation of 6-8 week female NSG mice with X-ray sublethal dose; 2 weeks later, MRC-5 cells infected with HCMV strain AD169 were intraperitoneally injected; 4 weeks after transplantation, adoptive reinfusion of expanded NK cells or primary NK cells 1X 10 ⁷ One, and 5 ten thousand units of IL-2 were intraperitoneally injected every other day starting with the reinfusion of NK cells. Taking the liver, spleen and lung of the mouse 14 days after the NK cells are back-transfused, and detecting the proportion of the NK cells by using a flow cytometry; based on the in situ hybridization method, the number of HCMV positive cells was detected using an HCMV-RNA probe.

Fig. 1 shows the proportion of NK cells in liver, spleen, lung, periphery after NK cell reinfusion for 14 d.

As can be seen from fig. 1, after NK cell transfusion 14d, sustained presence of NK cells could be detected in the tissues, and the proportion of NK cells in the in vitro expanded NK transfusion group was significantly higher compared to the primary NK transfusion group in liver, spleen, lung, periphery.

Fig. 2 shows HCMV clearance in the liver, spleen and lung after NK cell reinfusion for 14d, where the deep colored spots in the histopathological map were HCMV infected cells.

As shown in FIG. 2, the HCMV-infected cells were identified by the dark spots indicated by black arrows in the in situ hybridization method of HCMV-RNA. Virus-cleared mice were counted for positive mouse histiocyte status and HCMV clearance was calculated for the mice, showing that the ex vivo expanded NK reinfused had stronger HCMV clearance than the primary NK reinfused, in liver (76.5% vs. 33.3%, p = 0.032), lung (82.4% vs. 44.4%, p = 0.046).

Example 2 clinical study:

collecting peripheral blood 60-80ml (determined by WBC count of peripheral blood of donor) 10 days before transplantation of haplotype hematopoietic stem cells, or collecting peripheral blood stem cells (3-4 ml) after mobilization of donor, separating PBMC, freezing, and recovering PBMC2 x 10 of donor 10 days after transplantation ⁷ The donor-derived NK culture was initiated.

20 patients with haplotype hematopoietic stem cell transplantation were selected as subjects for reinfusion NK under the following conditions: (1) diagnosing as follows: acute leukemia, or MDS, MM or lymphoma. (2) age 16-65 years; (3) seropositive for donor HCMV-IGG; (4) HCMV negative within 20 days after transplantation; (5) 1-4 degrees aGVHD did not occur within 20 days after transplantation, or aGVHD was already relieved and glucocorticoid dose < 0.5mg/kg/d within 72h before NK cell reinfusion; (6) patient consent to group.

20 patients were returned donor-derived ex vivo expanded NK cells (not less than 1X 10 cells per time) 20 days (+ -3 d) and 27 days (+ -3 d) after transplantation ⁷ In terms of/kg). At the same time, from the first reinfusion, IL-2 (40 ten thousand IU/M) is performed according to the patient's wishes ² ) Subcutaneous injections were administered 3 times a week, 3 weeks after the first reinfusion.

20 patients with reinfusion were compared to historical control patients without reinfusion. Comparing the cumulative incidence of HCMV infection, the cumulative incidence of refractory HCMV infection, and the duration of HCMV infection.

The results of the study are as follows:

(1) First, from a safety point of view, no side effects were observed in patients after NK reinfusion, and there was no statistical difference between the reinfusion and control groups in death, disease recurrence, and aGVHD.

(2) Fig. 3 shows the comparison between the NK reinfusion group and the control group for HCMV infection and treatment-refractory HCMV infection, and it can be seen from fig. 3 that the cumulative incidence of HCMV infection and treatment-refractory HCMV infection is reduced in the NK reinfusion group compared to the control group, indicating that NK reinfusion can prevent HCMV infection after transplantation.

In patients following HSCT, HCMV infection develops primarily from primary infection, or in the early stages of immunodeficiency, HCMV is reactivated from pre-existing latency in the transplant or recipient. Chronic use of antiviral drugs results in increased mortality from refractory HCMV infection. The development of refractory HCMV infection means that HCMV infection persists for more than 2 weeks under standard antiviral treatment, reflecting the therapeutic effect of HCMV clearance. The results of the embodiment of the invention show that the mechanism of HCMV infection after NK cell infusion and HSCT resistance is to effectively promote HCMV clearance by remarkably reducing intractable HCMV infection, shortening HCMV duration and reducing HCMV-DNA copy number.

(3) Fig. 4 shows the comparison of the duration of HCMV infection in NK reinfusion group with the control group, and it can be seen from fig. 4 that the duration of HCMV infection is shortened in NK reinfusion group compared to the control group, indicating that NK reinfusion can promote clearance of HCMV infection after transplantation.

(4) Figure 5 shows the NK cell reconstitution of NK cells compared to control NK cells. As can be seen from fig. 5, NK cell reconstitution in patients was better in the NK reinfusion group than in the control group, as shown by: the number of NK is more, and the expression of activating receptors NKG2C, NKP30 and NKG2D is stronger, which indicates that NK reinfusion can promote the immune reconstitution of NK cells.

(5) Fig. 6 shows the monitoring of NK cells in vivo after NK cell reinfusion in 20 patients, and it can be seen from fig. 6 that both the percentage and absolute number of NK cells in vivo after NK cell reinfusion increased significantly and peaked and then declined around 2-3 weeks after NK cell reinfusion. In addition to NKP30, the expression of activation receptors NKG2C, NKG2D and DNAM-1 is increased. The first week after NK cell reinfusion, the proliferative capacity of NK cells in vivo increased, as indicated by increased expression of KI-67. In addition, increased secretion of NK cell granzyme B and perforin could be detected. The number of NKG2A + NK subgroups is reduced, and the KIR + NK subgroups are increased, which indicates that NK cell infusion can promote proliferation, activation and maturation of NK cells after allogeneic HSCT.

(6) FIG. 7 shows a comparison of HCMV and refractory HCMV infection in IL-2-injected and non-injected IL-2 groups; in the cohort A-no IL-2 (10) and cohort B-IL-2 (10), no difference was observed in the cumulative incidence of HCMV infection in the two groups, a tendency towards less refractory HCMV infection and a tendency towards shorter duration was observed in the IL-2 group, and no statistical difference was observed due to the limited number of patients in the group.

(7) FIG. 8 shows the percentage and absolute numbers of NK cells in the IL-2-injected group and the IL-2-non-injected group, and FIG. 9 shows the absolute numbers of NKG2C + NK subpopulations and the expression of the activation receptor NKP30 in the IL-2-injected group and the IL-2-non-injected group.

As can be seen in fig. 8 and 9, patients in cohort B exhibited a higher percentage and absolute number of NK cells, a higher absolute number of NKG2C + NK subpopulation and higher expression of the activation receptor NKP30 than patients in cohort a, indicating that NK cells in cohort B patients may have greater function under IL-2 stimulation. The percentage and absolute number of NK cells in cohort B patients were higher than in cohort a patients, and the expression of activation receptors NKP30 and NKG2C on NK cells was also higher than in group a patients. Therefore, IL-2 may reverse NK cell depletion, stimulate NK cell expansion, and enhance NK cell function in vivo.

The experimental results of the examples of the invention show that there was no significant difference in patient mortality, relapse and aGVHD between the NK cell infusion cohort and the control cohort. Therefore, ex vivo expanded NK cell infusion is safe and well tolerated without the occurrence of serious side effects associated with infusion. In addition, the NK cells expanded in vitro can prevent HCMV infection, promote HCMV clearance and promote NK cell immune reconstitution. Combination IL-2 treatment can improve the therapeutic effect of NK cell infusion.

The experimental result also shows that the number of NK cells in the returned NK cell group is obviously more than that of the control group, the expression of activating receptors NKP30 and NKG2D is higher than that of the control group, and the absolute number of NKG2C + NK subgroups is also higher than that of the control group. Further, it was shown that, in the patients in the in vitro amplified NK cell infusion group, the reconstructed NK cell phenotype was more mature and functional than the control group.

The application of the NK cells, the NK cell reinfusion formulation and the combined formulation provided by the present invention are described in detail above, and the principle and the embodiment of the present invention are explained in the present document by applying specific examples, and the description of the above examples is only used to help understanding the method and the core concept of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Claims

1. Use of NK cells for the preparation of an immune reconstitution promoting formulation, wherein said immune reconstitution promoting formulation comprises a first formulation; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 ⁷ /kg；

The agent for promoting immune reconstitution further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M ² A second formulation for administration after administration of the first formulation, the second formulation for aiding the first formulation in promoting immune reconstitution of NK cells;

administering the first formulation two times 20 days and 27 days after hematopoietic stem cell transplantation, respectively;

subcutaneously injecting a second formulation 3 times a week from the first administration of the first formulation to 3 weeks after the first administration of the first formulation;

wherein, following administration of the first formulation, expression of the in vivo activation receptors NKG2C, NKG2D and DNAM-1 is increased, expression of KI-67 is increased, secretion of NK cell granzyme B and perforin is increased, the number of NKG2A + NK subpopulations is decreased, and KIR + NK subpopulations are increased.