A method for inducing tolerant dendritic cells in vitro by combining immunosuppressants

Abstract

The present invention provides a method for inducing tolerant dendritic cells in vitro by combining immunosuppressants, wherein the combined immunosuppressants used are sCD83, TGF-β1 and IL-10. By using the method provided by the present invention, tolerant DCs with high activity and strong immunosuppressive function can be obtained from peripheral blood. Such DCs can be stored in liquid nitrogen and have good activity after resuscitation, which can meet the clinical demand for tolerant DC cell products and are used for preventing and treating autoimmune diseases and preventing transplant rejection.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a method for inducing tolerant dendritic cells in vitro in combination with immunosuppressants. Background Art

[0002] Autoimmune disease (AID) is characterized by multiple tissue and organ damage and immune disorders, and is listed by the World Health Organization (WHO) as one of the killers that endanger human health. The disease has a high morbidity and disability rate, and a low cure rate. The main feature of autoimmune disease is that the body lacks tolerance to self-antigens, resulting in overactivation of the immune system, inducing damaging immune responses and causing tissue damage. Currently, the main treatment for autoimmune diseases is the use of immunosuppressants. However, long-term use of immunosuppressants has significant side effects and increases the risk of infection and tumors.

[0003] Cell therapy is a treatment option that uses autologous or allogeneic somatic cells to induce in vitro culture to produce cells with a certain immune tolerance function, which are then infused back into the body of patients with autoimmune diseases to inhibit overactivation of the immune system and tissue damage. Cell therapy is highly safe and targeted, so it is increasingly gaining attention, making it a promising treatment option that is expected to become a new option for treating autoimmune diseases from the perspective of regulating the homeostasis of the immune system.

[0004] Dendritic cells (DC cells) are the key to activating adaptive immunity. They not only have immune coordination functions, such as clearing foreign invading viruses, bacteria and other microbial infections, but also can regulate the immune system and induce immune tolerance in a stable state, thereby maintaining the balance of the immune system. Mononuclear-derived tolerant DC cells derived from peripheral blood can eliminate pathological autoimmune or inflammatory responses without interfering with protective immunity, so that the immune system tends to balance. Therefore, this therapy may provide lasting beneficial effects, or even be therapeutic.

[0005] It has been reported that tissue matrix cells, cytokines (IL-10, TGF-β1) and soluble molecules (sCD40L, sCTLA-4) can regulate the functional state of DC cells. Studies have found that DC cells stimulated by IL-10 and TGF-β1 in vitro express lower levels of co-stimulatory molecules than unstimulated DC cells and are in an inhibitory state; such DC cells can induce allogeneic T cells to produce lower antigen-specific immune responses by secreting IL-10, IDO, etc. in vitro; such DC cells that express low co-stimulatory molecules and inhibitory cytokines and can induce low T cell responsiveness in vivo and in vitro are collectively referred to as tolerant DC cells. Tolerant DCs have also been used in mouse experiments to study the treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and autoimmune diabetes. However, whether IL-10, TGF-β1 or both induce tolerant DCs, they are susceptible to microenvironmental influences, have poor stability, and poor immunosuppressive effects. The optimization of the preparation of tolerant DCs with strong inhibitory function is the main problem that needs to be solved at present.

[0006] Soluble CD83 (sCD83) molecule is a glycoprotein belonging to the immunoglobulin class. sCD83 is the extracellular region of the CD83 molecule (CD83 ext), which is produced by the hydrolysis of CD83 on the cell membrane under the action of enzymes and has the function of inhibiting immune response; it has been reported that sCD83 can inhibit the maturation of DC cells and the expression of co-stimulatory molecules on the membrane in vitro, induce changes in the cytoskeleton of DC, promote the secretion of IDO, thereby promoting the production of regulatory T cells, and regulating the body's immune balance. Many studies have shown that sCD83 cannot directly affect the activity of T cells, but indirectly inhibits T cell activation by inhibiting DC cells.

[0007] Currently, it has been reported that cytokines (IL-10 and / or TGF-β1) or combined hormone drugs can induce DC tolerance, which is used in the prevention and treatment of transplantation, autoimmune diseases, etc. There are also reports on the use of drugs to induce DCs tolerance by inhibiting the expression of co-stimulatory molecules on the surface of mouse DCs, or genetic modification of IL-10 and TGF-β1 to promote DC inhibitory function. However, the DC tolerance obtained by these methods has the disadvantages of poor stability, genetic or drug intervention affecting the state of the cells themselves, and low viability, which affects the use of tolerant DCs. Summary of the invention

[0008] In view of the above-mentioned deficiencies of the prior art, the present invention provides a method for inducing tolerant dendritic cells in vitro with a combined immunosuppressant, wherein the combined immunosuppressant used is sCD83, TGF-β1 and IL-10. Using the method provided by the present invention, tolerant DCs with high activity and strong immunosuppressive function can be obtained from peripheral blood. Such DCs can be stored in liquid nitrogen and have good activity after resuscitation, which can meet the clinical demand for tolerant DC cell products and are used for preventing and treating autoimmune diseases and preventing transplant rejection.

[0009] The technical solution of the present invention is as follows:

[0010] A method for inducing tolerant dendritic cells in vitro by combining immunosuppressants, wherein the combined immunosuppressants are sCD83, TGF-β1 and IL-10.

[0011] Preferably, the steps of the above method are as follows:

[0012] (1) Collect peripheral blood and place it in an anticoagulant tube containing 50u / mL sodium heparin, and dilute it with PBS + 2% FBS diluent, the volume ratio of peripheral blood to diluent is 1:1, separate it by density gradient centrifugation with lymphocyte separation fluid to obtain peripheral blood mononuclear cells, wash the cells with PBS + 2% FBS buffer, add culture medium and culture for 1-2 hours, discard the supernatant, and take the adherent DC precursor cells in the culture dish;

[0013] (2) Inducing DC precursor cells to differentiate into DCs under the action of cytokines IL-4 and GM-CSF, and in this process, adding combined immunosuppressants (sCD83, TGF-β1 and IL-10) to the culture dish to induce their differentiation into tolerant DCs; the resulting tolerant DCs have a purity of >95% and a survival rate of >95%, express the DC marker CD11c and low expression of costimulatory molecules CD80, CD83, CD86 and HLA-DR, and are able to secrete IL-10 and IDO;

[0014] (3) The induced tolerant DCs were functionally identified and stored in liquid nitrogen.

[0015] Preferably, in step (1), the culture medium is 1640 culture medium containing 10-15% by volume of autologous inactivated plasma; wherein the autologous inactivated plasma is plasma homologous to the cells induced by the culture.

[0016] Preferably, in step (2), the process of cytokine-induced DC precursor cell differentiation into DC is as follows: adherent DC precursor cells are cultured in 1640 culture medium containing 10-15% autologous inactivated plasma by volume for 7 days, and cytokines IL-4 and GM-CSF are added on days 0, 3, and 5, respectively.

[0017] Preferably, on day 0, the amount of cytokine IL-4 added is 500u / mL, and the amount of GM-CSF added is 1000u / mL.

[0018] Preferably, in step (2), combined immunosuppressants are added on the 5th day and the 7th day, respectively, wherein the added amount of sCD83 is 10 ng / mL, the added amount of TGF-β1 is 0.2 ng / mL, and the added amount of IL-10 is 10 ng / mL.

[0019] Preferably, in step (3), after pretreatment with cryopreservation solution, the tolerant DCs are cultured at 2×10 6 / mL density was divided into cryopreservation tubes and stored at -196°C liquid nitrogen; wherein the cryopreservation solution consisted of 10% DMSO and 90% FBS.

[0020] Tolerogenic dendritic cell products are dendritic cell products used to prevent and treat autoimmune diseases and prevent transplant rejection.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. By using the method provided by the present invention, tolerant DC with high activity and strong immunosuppressive function can be obtained from peripheral blood. Such DC can be stored in liquid nitrogen and has good activity after resuscitation, which can meet the clinical demand for tolerant DC cell products and be used for preventing and treating autoimmune diseases and preventing transplant rejection reactions.

[0023] 2. In the present invention, sCD83 combined with IL-10 and TGF-β1 can reduce the expression of co-stimulatory molecules in DC cells, promote DC to secrete IL-10 and IDO, and has the effect of inhibiting T cell activation; and the tolerant DC obtained by this method has the advantages of good stability and good activity after cryopreservation and resuscitation.

[0024] 3. In the present invention, the purity of tolerant DC induced by combined immunosuppressants (sCD83, TGF-β1 and IL-10) is above 95%, the survival rate is above 95%, and the tolerant DC can secrete more IL-10 and IDO, and has a strong inhibitory effect on T cells. Compared with the induction of IL-10, TGF-β1 or IL-10 and TGF-β1 alone, a method for inducing tolerant DC with stronger inhibitory stability is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 After 8 days of induction, the activity and purity of tolerogenic DCs were detected by flow cytometry.

[0027] Figure 2 To detect the expression of CD80, CD86, CD83 and HLA on tolerant DCs and uninduced DCs after 8 days of induction with IL-10, TGF-β1, IL-10+TGF-β1 and sCD83+IL-10+TGF-β1 (A), as well as the secretion of IL-10 and IDO by DCs (B).

[0028] Figure 3 After 8 days of induction with IL-10, TGF-β1, IL-10+TGF-β1 and sCD83+IL-10+TGF-β1, mixed lymphocyte assay was used to detect the effects of tolerant DCs and uninduced DCs on the expression of CD69 and Ki67 on T cells.

[0029] Figure 4 Flow cytometry was used to detect the activity of DCs after recovery. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0031] Example 1

[0032] A method for inducing tolerant dendritic cells in vitro in combination with immunosuppressants, comprising the following steps:

[0033] (1) Collect peripheral blood, pass the transfusion-transmitted infectious disease test, place the peripheral blood in a tube containing 50u / mL heparin sodium anticoagulant, add PBS + 2% FBS (1:1) to dilute and resuspend, and separate it by density gradient centrifugation with lymphocyte separation fluid to obtain peripheral blood mononuclear cells. The conditions of density gradient centrifugation are 20°C, 500g, horizontal centrifugation for 30min, and the brake speed is set to 0;

[0034] The cells were diluted and washed with PBS + 2% FBS buffer to ensure cell viability, and then cultured in 1640 culture medium containing 10% autologous inactivated plasma at 1×10 7 / mL per dish were inoculated in 60 mm culture dishes, and after adhering to the wall for 1 hour in an incubator at 37°C, >5% CO2 and saturated humidity, the suspended cells were washed away to obtain DC precursor cells;

[0035] Among them, autologous inactivated plasma is plasma homologous to the cultured induced cells;

[0036] The supernatant was discarded, and 3 mL / dish of clinical-grade 1640 culture medium was added to the culture dish again, and cultured overnight at 37°C, 5% CO2, and saturated humidity;

[0037] Clinical-grade 1640 culture medium + 10% autologous inactivated filtered plasma (the plasma and cells are from the same patient) is used to facilitate the induction of cultured cells for clinical treatment and ensure better status and activity of DCs;

[0038] (2) Inducing DC precursor cells under the action of cytokines:

[0039] The process of cytokine-induced DC precursor cell differentiation into tolerant DC is as follows: adherent DC precursor cells are cultured in 1640 culture medium containing 10% autologous inactivated plasma for 7 days, and cytokines IL-4 and GM-CSF are added on days 0, 3, and 5, respectively;

[0040] Among them, the amount of cytokine IL-4 added on day 0 (next day) was 500u / mL, and the amount of GM-CSF added was 1000u / mL; on days 3 and 5, full amounts of IL-4 and GM-CSF were supplemented;

[0041] In addition, combined immunosuppressants of 10 ng / mL sCD83, 0.2 ng / mL TGF-β1, and 10 ng / mL IL-10 were added on days 5 and 7 to induce their differentiation into tolerant DCs, and tolerant DC cells were collected on day 8;

[0042] The purity of the obtained tolerant DCs was >95%, and the survival rate was >95%. Figure 1 ; express DC marker CD11c and low expression of costimulatory molecules CD80, CD83, CD86 and HLA-DR, see Figure 2 , can secrete IL-10 and IDO; compared with the induction with IL-10, TGF-β1, or IL-10 and TGF-β1 alone, combined immunosuppressant stimulation can downregulate more co-stimulatory molecules on the DC surface and induce DC to secrete more IL-10 and IDO, see Figure 2 ;

[0043] In this example, human sCD83 protein was prepared by overexpressing the extracellular segment (22-133aa) of human recombinant protein CD83 with a His tag in eukaryotic cells HEK293, and then the protein was purified by passing through a nickel column for in vitro induction experiments;

[0044] (3) Tolerant DCs (2×10 6 / mL) was added with freezing solution, and then divided into programmed cooling boxes, kept in a -80℃ refrigerator for 20 hours, and stored in liquid nitrogen at -196℃. After recovery, the activity was detected to be more than 95%; wherein, the freezing solution was composed of 10% DMSO and 90% FBS.

[0045] All the above processes are carried out in a sterile environment.

[0046] Combination Figure 1-Figure 2 It can be seen that the purity of the tolerant DC induced and cultured by the combined immunosuppressants sCD83, TGF-β1 and IL-10 in the present invention is above 95%, the survival rate is 95%, and the DC marker CD11c is expressed, and the co-stimulatory molecules CD80, CD83, CD86 and HLA-DR are lowly expressed. And the tolerant DC induced and cultured by the combined immunosuppressants sCD83, TGF-β1 and IL-10 in the present invention has a stronger immunosuppressive function than the tolerant DC induced by cytokine IL-10 or / and TGF-β1.

[0047] Figure 1 After 8 days of induction culture, flow cytometry was used to detect the activity and purity of tolerant DCs. Figure 1 In the middle, the left picture shows 7AAD staining after induction, detecting activity of 95%, and the right picture shows DC purity of 95% detected by CD11c and HLA staining after induction;

[0048] Figure 2 After 8 days of induction with IL-10, TGF-β1, IL-10+TGF-β1 and sCD83+IL-10+TGF-β1, the expression of CD80, CD86, CD83 and HLA on tolerant DCs and uninduced DCs (A), as well as IL-10 and IDO secreted by DCs (B). *p<0.05, **p<0.01, ***p<0.001.

[0049] Example 2

[0050] Flow cytometry was used to analyze the immune phenotype of tolerogenic DCs. FITC-labeled CD11c antibodies, CD80 antibodies, CD83 antibodies, CD86 antibodies, and HLA-DR antibodies were used for cell surface labeling, and 7AAD staining was used to detect cell survival (see Figure 1); Tolerant DCs induced by combined immunosuppressants such as sCD83, TGF-β1, and IL-10 expressed the DC marker CD11c, expressed lower co-stimulatory molecules CD80, CD83, CD86, and HLA-DR than mature DCs, and expressed lower co-stimulatory molecules than tolerant DCs induced by IL-10 / TGF-β1 alone, but secreted more IL-10 and IDO (see Figure 2 The survival rate of tolerant DCs induced by combined immunosuppressive agents was above 95%.

[0051] The application of tolerant DCs after induced culture or cryopreservation as third-party unrelated donor cells in the prevention and treatment of autoimmune diseases and prevention of transplant rejection.

[0052] Example 3

[0053] Peripheral blood mononuclear cells were separated from peripheral blood by density gradient centrifugation and sorted using a CD4+T cell immunomagnetic bead sorting kit (Miltenyi) according to the manufacturer's instructions. CD4+T cells were used as effector T cells. Effector T cells were stimulated and activated with tolerant DCs or allogeneic mature DCs treated with mitomycin to form a mixed lymphocyte reaction system at a ratio of 1:10; cultured at 37°C, >5% CO2 and saturated humidity, and cells cultured for 4, 5, and 6 days were collected and analyzed by flow cytometry to detect the expression of CD69 and Ki67 in T cells, see Figure 3 .like Figure 3 As shown, tolerogenic DCs cultured with a combination of immunosuppressive agents (sCD83, TGF-β1, and IL-10) inhibited the expansion of allogeneic T effector cells and had an immunosuppressive effect.

[0054] Figure 3 After induction with IL-10, TGF-β1, IL-10+TGF-β1 and sCD83+IL-10+TGF-β1 for 8 days, the cells were co-cultured with sorted T cells, and the effects of tolerant DC and uninduced DC on the expression of CD69 and Ki67 of T cells were detected by flow cytometry. *p<0.05, **p<0.01, ***p<0.001.

[0055] Example 4

[0056] The tolerant DCs (Example 1) cultured with combined immunosuppressants (sCD83, TGF-β1 and IL-10) were pretreated with cryopreservation solution, divided into cryovials, and stored in liquid nitrogen at -196°C. After thawing, the activity of DCs was detected to be more than 95%, as shown in Figure 1. Figure 4Human peripheral blood is subjected to strict testing procedures (syphilis antibody testing, HIV immune testing, CMV antibody testing, hepatitis B antigen antibody testing, etc.) and then induced to differentiate into tolerant DC in vitro. The tolerant DC obtained by culture with combined immunosuppressants (sCD83, TGF-β1 and IL-10) is frozen in liquid nitrogen after passing the purity and in vitro and in vivo inhibitory function tests for the prevention and treatment of autoimmune diseases and the prevention of transplant rejection.

[0057] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A method for inducing tolerant dendritic cells in vitro in combination with immunosuppressants, characterized in that: The combined immunosuppressants are sCD83, TGF-β1, and IL-10; Under the action of cytokines IL-4 and GM-CSF, DC precursor cells are induced to differentiate into DCs, and in this process, combined immunosuppressants are added to the culture dish to induce their differentiation into tolerant DCs; When combined immunosuppressants were added, the amount of sCD83 added was 10 ng / mL, the amount of TGF-β1 added was 0.2 ng / mL, and the amount of IL-10 added was 10 ng / mL.

2. The method for inducing tolerant dendritic cells in vitro using a combined immunosuppressant according to claim 1, characterized in that: Here are the steps: (1) Collect peripheral blood and place it in an anticoagulant tube containing 50u / mL sodium heparin, and dilute it with PBS + 2% FBS diluent, the volume ratio of peripheral blood to diluent is 1:1, separate it by density gradient centrifugation with lymphocyte separation fluid to obtain peripheral blood mononuclear cells, wash the cells with PBS + 2% FBS buffer, add culture medium and culture for 1-2 hours, discard the supernatant, and take the adherent DC precursor cells in the culture dish; (2) Inducing DC precursor cells to differentiate into DCs under the action of cytokines IL-4 and GM-CSF, and in this process, adding combined immunosuppressants to the culture dish to induce them to differentiate into tolerant DCs. The purity of the tolerant DCs obtained is >95%, the survival rate is >95%, and they express the DC marker CD11c and low expression of costimulatory molecules CD80, CD83, CD86 and HLA-DR, and can secrete IL-10 and IDO; (3) The induced tolerant DCs were functionally identified and stored in liquid nitrogen.

3. The method for inducing tolerant dendritic cells in vitro using a combined immunosuppressant according to claim 2, characterized in that: In step (1), the culture medium is 1640 culture medium containing 10-15% by volume of autologous inactivated plasma; wherein the autologous inactivated plasma is plasma homologous to the cultured cells.

4. The method for inducing tolerant dendritic cells in vitro by combining immunosuppressants according to claim 2, characterized in that: In step (2), the process of cytokine-induced DC precursor cell differentiation into DC is as follows: adherent DC precursor cells are cultured in 1640 culture medium containing 10-15% autologous inactivated plasma for 7 days, and cytokines IL-4 and GM-CSF are added on days 0, 3, and 5, respectively.

5. The method for inducing tolerant dendritic cells in vitro by combining immunosuppressive agents according to claim 4, characterized in that: On day 0, the added amount of cytokine IL-4 was 500u / mL and the added amount of GM-CSF was 1000u / mL.

6. The method for inducing tolerogenic dendritic cells in vitro by combining immunosuppressive agents according to claim 2, characterized in that: In step (2), a combined immunosuppressant is added on the 5th day and the 7th day, respectively.

7. The method for inducing tolerant dendritic cells in vitro by combining immunosuppressive agents according to claim 2, characterized in that: In step (3), after pretreatment with cryopreservation solution, tolerant DCs were cultured at 2×10 6 / mL density was divided into cryopreservation tubes and stored at -196°C liquid nitrogen; wherein the cryopreservation solution consisted of 10% DMSO and 90% FBS.

Images