CN111321116A

CN111321116A - In vitro immune synapse system and method for in vitro evaluation of immune response

Info

Publication number: CN111321116A
Application number: CN201811525844.2A
Authority: CN
Inventors: 庄秀琪; 锺文彬; 陈映安; 吴美莉
Original assignee: National Pingtung University of Science and Technology
Current assignee: National Pingtung University of Science and Technology
Priority date: 2018-12-13
Filing date: 2018-12-13
Publication date: 2020-06-23

Abstract

The invention relates to an in vitro immune synapse system and a method for evaluating immune response in vitro. The in vitro immune synapse system comprises antigen presenting cells derived from the same individual pig, and at least one of a specific number of T cell subsets isolated from peripheral blood mononuclear cells. When the sample to be tested and the in-vitro immune synapse system are subjected to a co-culture step for a preset time, whether the sample to be tested has immunogenicity or immune stimulation can be judged according to the immune related change degree of the cells, and the sample to be tested has potential to replace some animal experiments.

Description

In vitro immune synapse system and method for in vitro evaluation of immune response

[ technical field ] A method for producing a semiconductor device

The invention relates to an in vitro simulated immune system and a method for evaluating immune response by using the same, in particular to an immune synapse system for evaluating immune response in vitro by using various immune cells and a method for evaluating immune response in vitro by using the same.

[ background of the invention ]

The vaccine is the best strategy for controlling all diseases, so that the vaccine which is safe, effective and timely in supply is developed to prevent pathogens, and the vaccine is the first key issue of global public health.

At present, animal vaccines are still developed, and through animal experimental tests and screening, antigens and adjuvants with good immune effects can be found out. However, the cost of developing animal experiments for vaccines and immunoadjuvants is relatively high. Secondly, the development of vaccines by animal experiments is limited by the species specificity of the animal immune system, and the experimental results between one species of animal and other pathogens are not necessarily applicable to other species of animals or other pathogens.

Although some in vitro cell evaluation platforms are available in the market, the cells of the platforms are mostly derived from mice or commercially available cell lines, and thus the evaluation results cannot replace animal experiments at present.

In other words, the size of the mice is small, and the number of immune cells available to each mouse is quite limited, and it is necessary to pool immune cells from different individuals to adequately test. However, since co-culture of different types of immune cells derived from different individuals is likely to cause various immune rejection reactions, evaluation of immune cells derived from different individuals can be performed only with the same type of immune cells, and evaluation of T cell epitopes cannot be performed.

As for commercially available immune cell lines, although the number of cells is sufficient, these cell lines are not primary cells, and their physiological metabolism and immune response are different from those of normal cells, so that the immune response results obtained therefrom are also less reliable.

In view of the above, it is desirable to develop a system for evaluating immune response in vitro to overcome the disadvantages of the conventional in vitro cell assay and further replace the animal assay.

[ summary of the invention ]

It is therefore an object of the present invention to provide an in vitro immune synapse system comprising antigen presenting cells derived from the same individual pig and at least one of a specific number of T cell subsets isolated from peripheral blood mononuclear cells.

Another objective of the present invention is to provide a method for evaluating an immune response in vitro, which comprises performing a co-culture step on a sample to be tested and the in vitro immune synapse system for a period of time, and then detecting immune-related changes of the immune synapse system to determine whether the sample to be tested has immunogenicity or immune-stimulating properties, so as to replace animal experiments.

In accordance with the above objects of the present invention, an in vitro immune synapse system comprising an antigen presenting cell and at least one of a regulatory T cell, a killer cell and a helper T cell is provided. The antigen presenting cell, the regulatory T cell, the killer cell and the helper T cell are primary cells derived from the same individual, and the number of the antigen presenting cell may be, for example, greater than the sum of the numbers of the regulatory T cell, the killer cell and the helper T cell.

According to one embodiment of the present invention, the same individual is a Specific Pathogen Free (SPF) pig.

According to an embodiment of the present invention, the antigen presenting cell may be, for example, an alveolar macrophage.

According to another object of the invention, a method for assessing an immune response in vitro is provided. In one embodiment, the in vitro immune synapse system is provided, wherein the in vitro immune synapse system comprises, for example, antigen presenting cells, at least one of regulatory T cells, killer cells and helper T cells, and control cells, and the antigen presenting cells, regulatory T cells, killer cells, helper T cells and control cells (control cells) are primary cells derived from the same individual. Then, the sample to be tested and the immune synapse system are co-cultured for 24-48 hours. Immune-related changes in the immune synaptic system are then detected, wherein the immune-related changes may be, for example, TLR gene expression and/or cytokine concentration. When the immune-related change of any one of the regulatory T cells, killer cells and helper T cells is significantly different from the immune-related change of the control cells, the sample to be tested is judged to have immunogenicity or immunostimulation.

According to an embodiment of the present invention, the sample to be tested may be, for example, an antigen or an adjuvant.

According to an embodiment of the present invention, the control cell may be a monocyte, for example.

The in vitro immune synapse system comprises antigen presenting cells from the same individual pig and a plurality of T cell subgroups separated by peripheral blood mononuclear cells, and the T cell antigen determinant of a sample to be tested is evaluated. The animal immune experiment proves that the in vitro immune synapse system has the same result as the animal experiment. Therefore, the in vitro immune synapse system is expected to replace animal experiments when new antigens or new adjuvants are developed in the future.

[ description of the drawings ]

The foregoing and other objects, features, and advantages of the invention will be apparent from the following more particular description of the embodiments of the invention, as illustrated in the accompanying drawings in which:

FIGS. 1A-1D show images of transmission electron microscope (FIGS. 1A-1C) and scanning electron microscope (FIG. 1D) after pairing porcine alveolar macrophages with regulatory T cells (FIGS. 1A and 1B) or helper T cells (FIGS. 1C and 1D) according to an embodiment of the present invention.

FIGS. 2A to 2C are histograms of the relative expression levels of mRNA of antigen presenting cells, regulatory T cells, killer cells, helper T cells, and toll-like receptors (TLR) 3 (FIG. 2A), TLR7 (FIG. 2B), and TLR8 (FIG. 2C) of control cells co-cultured in vitro, respectively, after immunization of individual pigs with different Porcine Reproductive and Respiratory Syndrome (PRRS) antigens according to an embodiment of the present invention.

FIGS. 3A-3B are histograms of the content of IL-2 (FIG. 3A) and IL-10 (FIG. 3B) in antigen presenting cells co-cultured with regulatory T cells, killer cells, helper T cells and control cells, respectively, after immunization of a swine individual with different PRRS antigens according to one embodiment of the present invention.

FIG. 4 is a histogram of the relative expression level of TLR mRNA in screening immune adjuvants of in vitro immune synapse systems (including porcine helper T cells and alveolar macrophages) in accordance with one embodiment of the present invention.

FIG. 5 is a histogram of IL-2 content of the in vitro immune synapse system screening immune adjuvants according to one embodiment of the invention.

[ detailed description ] embodiments

As mentioned above, the present invention provides an in vitro Immune Synapse (IS) system and a method for in vitro immune response assessment, which comprises antigen-presenting cells (APC) from the same pig and several T cell subsets isolated from Peripheral Blood Mononuclear Cells (PBMCs), and IS used for the T cell epitope (T-cell epitope) assessment of a sample to be tested.

In other words, the present invention IS referred to herein as an Immunological Synapse (IS) in which a T cell subset of APC and PBMCs form an immunological synapse in vitro. The above-mentioned Immunological Synapse (IS) IS a hypothesis proposed by recent researchers, and IS defined as an interaction between two or more kinds of cells in the immune system, including stable and cooperative interaction, and causes at least one kind of subsequent reaction of the cells. The IS system fully encompasses essential elements of the immune system response in animals, including the integration of messages between APCs and various T cell subsets (e.g., antigen recognition systems), soluble factors that direct action (e.g., cytokines), and intermolecular interactions that form specific immune responses (e.g., intercellular receptor and ligand binding systems).

APCs play a critical role in the initiation and maintenance of immune responses. APCs recognize pathogen structure molecular patterns (PAMPs) through the Pathogen Recognition Receptor (PRR) of the immune system, and initiate specific immune pathways through signal transmission. After APCs are activated by antigen or adjuvant, different cytokines or pro-inflammatory cytokines can be generated by recognition of PRR, which affects differentiation and proliferation of different T cell subsets and determines which immune cell or pro-inflammatory cell response pattern the immune response is inclined to.

The Toll-like receptor (TLR) is the main family of PRR, and the research on APCs and TLRs in recent years provides a lot of new information on immune mechanisms and vaccine/adjuvant development of TLR effectors (agonst). TLR agonists and cytokines can alter the microenvironment in which APCs come into contact with T cells, affecting stability during IS formation between the two. The deeper the formation of TLR agonists and cytokines on specific antigens or infected APCs and T cell IS IS, the more effective the TLR agonists and cytokines can be used as immunological adjuvants for animal vaccines.

Although many TLR agonists have been tried as immunomodulators or as active molecules in immunoadjuvants, the microenvironment in which APCs are in contact with T cells requires the involvement of specific cytokines to form a stable IS. However, the study of the effects of TLR-effectors interacting with cytokines on the development of IS very demanding.

In one embodiment, the in vitro immune synapse system of the invention may comprise antigen presenting cells and specific T cell subsets isolated from PBMCs, examples of which may include, but are not limited to, at least one of regulatory T (Treg) cells, killer (NK) cells, and helper T (T helper; Th) cells. In other embodiments, the in vitro immune synapse system may also comprise antigen presenting cells, at least one of regulatory T cells, killer cells and helper T cells, and control cells (control cells).

One of the technical features of the present invention is that antigen presenting cells, regulatory T cells, killer cells, helper T cells and control cells are primary cells derived from the same individual, thereby overcoming immune rejection. In one embodiment, the same individual can be, for example, a Specific Pathogen Free (SPF) swine, regardless of breed. The primary generation of cells from SPF pigs can exclude interference from other antigens and antibodies. Without SPF pigs, the results of testing specific T cell subsets isolated from peripheral blood mononuclear cells, antigen presenting cells, may be disturbed or even not consistent with animal experiments.

The antigen presenting cell referred to herein may be derived from any site, depending on the sample to be tested, such as alveolar macrophages (alveolar cells), orchis cells (langerhans' cells), interdigital cells (IDC) of the thymus, Dendritic Cells (DC) in the spleen and lymph nodes, activated B cells, macrophages, and the like.

The regulatory T cells of the invention, as referred to herein, are preferably labeled as having at least CD4+/CD25+ cells. The killer cells are preferably labeled as having at least CD 4-/CD 25 +. Helper T cells are preferably labeled as having at least the CD4+/CD 25-cell. As for the cell type of the control cell, it is preferable to use a cell having at least CD 4-/CD 25-cells as a marker, and a monocyte is exemplified.

In the above-mentioned in vitro immune synapse system, the number of antigen presenting cells is preferably greater than the sum of regulatory T cells, killer cells and helper T cells, and more preferably, the number of antigen presenting cells is 2-fold to 10-fold the sum of regulatory T cells, killer cells and helper T cells. In other embodiments, the number of cells of the antigen presenting cell may be greater than the sum of the number of cells of the regulatory T cell, the killer cell, the helper T cell and the control cell, and preferably, the number of cells of the antigen presenting cell may be 2-fold to 10-fold the sum of the number of cells of the regulatory T cell, the killer cell, the helper T cell and the control cell.

The in vitro immune synapse system can be used in a method for evaluating immune response in vitro. In one embodiment, the above-described in vitro immune synapse system is provided.

Secondly, the sample to be tested and the immune synapse system are subjected to a co-culture step for a preset time. The sample to be tested referred to herein can be, for example, an antigen or an adjuvant, wherein the types of the antigen and the adjuvant are well known to those skilled in the art of the present invention and are not described herein.

The co-culturing step, as referred to herein, of the invention may comprise pairing of the antigen presenting cells with any one of a specific number of subpopulations of T cells isolated from the PBMCs, e.g., pairing of antigen presenting cells with regulatory T cells, pairing of antigen presenting cells with helper T cells, pairing of antigen presenting cells with killer cells.

In the above examples, the co-culturing step may be performed in a cell culture solution suitable for the antigen-presenting cells and the peripheral blood mononuclear cells. In general, the cell culture solution is not limited in kind, and commercially available products such as RPMI 1640 culture solution, to which 10% pig serum may or may not be added, can be used.

In the above embodiment, the predetermined time of the co-cultivation step can be, for example, 6 hours to 24 hours, or 12 hours to 24 hours, preferably 18 hours to 24 hours, and more preferably 24 hours.

Thereafter, the degree of immune-related changes of the paired cells after the co-culture step was evaluated.

Thereafter, an immune-related change of the immune synapse system is detected. In general, items for detecting an immune-related change are not limited, and in one embodiment, the items include, for example, the amount of toll-like receptor (TLR) gene expression, cytokine concentration, and the like. The TLR gene expression level referred to herein in the present invention may include, but is not limited to, a TLR3 gene expression level, a TLR7 gene expression level, and/or a TLR8 gene expression level. The cytokine concentration referred to herein in the present invention may be, for example, a concentration of interleukin including, but not limited to, a concentration of IL-2 and/or a concentration of IL-10.

When the immune-related change of any one of the regulatory T cell, killer cell and helper T cell is significantly different from the immune-related change of the control cell, it is determined that the sample to be tested has immunogenicity or immunostimulation. A significant difference as referred to herein is a statistically significant difference, typically P < 0.05.

Taking Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) as an example, PRRSV is the main cause of severe respiratory symptoms in Taiwan pigs. After a pig is infected by PRRSV only, the death rate is higher, and the production performance of a pig farm is extremely lower. PRRSV mainly infects APCs of pigs, especially the immunity between virus-infected APCs and regulatory T cells (Treg), changes the activity of immunocytes, induces the differentiation and proliferation of different T cell subsets, leads to the reduction of the antiviral immune response of the host, and leads to the PRRSV escaping from the immunity of the host. The recent High Pathogenic (HP) PRRSV has caused severe respiratory symptoms in pigs and significant economic losses in the pig industry. Therefore, in the respiratory tract mucosal immune system, the activation of the immune activity of the antigen presenting cells of the respiratory tract mucosal immune system is the main topic of enhancing the development of mucosal immune vaccines. The primary first defense line cell of the respiratory tract mucosa immune system IS alveolar macrophage, which IS related to the interaction of TLR (toll-like receptor) agonist/cytokine and different T cell epitope, and the formation and the regulation mechanism of the animal mucosa immune IS need to be cleared. However, the in vitro immune synapse system of the present invention was confirmed by animal immune experiments to have results consistent with those of animal experiments. Therefore, the in vitro immune synapse system is expected to replace animal experiments when new antigens or new adjuvants are developed in the future.

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Example one in vitro immune synapse System

1.1 isolation of Peripheral Blood Mononuclear Cells (PBMC) from pigs

This example utilizes conventional means to isolate porcine PBMCs from 4-week-old SPF pigs (breed Lance crossed with Yorkshire breed). It should be noted that the age and breed of the SPF pig are only for illustrative purposes, and are not limited to the above, and other breeds or breeds of other ages may be used.

First, blood was collected from the neck of an SPF pig, and was collected from the jugular vein using a 20mL blood collection syringe containing 1mL of 0.5M EDTA. Then, the blood was dispensed and centrifuged for 25 minutes, and a leukocyte cell layer (buffy coat) was taken out, mixed with Hank's Balanced Salt Solution (HBSS; containing no calcium and magnesium ions) at a ratio of 1: 2, mixing uniformly. Then, the cell mixture was slowly injected into Ficoll-Hypaque^TMThe lymphocyte separation solution was centrifuged at 900Xg for 30 minutes at 4 ℃. Thereafter, the PBMC layer was removed and washed with HBSSThe cell concentration was adjusted by using 10% pig serum-containing RPMI-1640 cell culture medium (referred to as PS-RPMI above) 3 times, and the cells were cultured in a 37 ℃ incubator for further use.

1.2 sorting and culturing of subsets of porcine T cells

The separated porcine PBMC is taken, after the cell concentration is adjusted, an anti-porcine (anti-porcine) CD4 antibody and an anti-porcine CD25 antibody are added into cell sap, and then a secondary antibody is added for action, and then the cell sap is centrifuged at the rotating speed of 1000Xg for 5 minutes. After removal of the supernatant, the cell pellet was broken up by adding Phosphate Buffered Saline (PBS) and cell surface marker analysis and cell sorting (sorting) were performed by flow Cytometry (Coulter Epics AltraFlow Cytometry, Beckman Coulter, Calif., USA). Centrifuging to remove supernatant, washing the cells with PBS for 3 times, adding PS-RPMI culture solution to suspend the cells, and culturing in a 37 deg.C cell culture box; in addition, the cell sap is added with antibodies anti-porcine CD4 and anti-porcine CD25, and the cell markers CD4+/CD 25- (Th cells), CD 4-/CD 25+ (NK cells), CD4+/CD25+ (Treg cells) and CD 4-/CD 25- (control cells) are obtained by setting the selection limits in a computer. Centrifuging the cells to remove supernatant, washing the cells with PBS for 3 times, adding PS-RPMI culture solution to suspend the cells, and culturing in a 37 ℃ cell culture box.

1.3 isolation of porcine alveolar macrophages

In this example, SPF pigs were anesthetized and the lungs were removed immediately. The lung was gently massaged with 200mL of PBS through a tracheal catheter to wash the lung and the wash solution was collected. The washing solution was collected in a 50mL plastic centrifuge tube, centrifuged at 200Xg for 10 minutes at 4 ℃ and the bottom pellet cells were collected, the cells were suspended in PS-RPMI cell culture medium, and the cell concentration was adjusted for antigen presenting cells.

1.4 evaluation of in vitro immune synapse systems

In this example, alveolar macrophages were cultured in RPMI-1640 medium containing 10% FBS in 24-well cell culture plates, 0.5mL of antigen presenting cell suspension (1 × 107 cells/mL) per well was placed in an incubator containing 5% CO2 at 37 ℃ for 24 hours, and then separately added to selected different T cell subsets (Treg cells, Th cells, NK cells and control cells) to adjust to 1 × 106cells/mL, 0.5 mL/well (well) was CO-cultured, antigen protein (2. mu.g/10. mu.L/well) was added, RPMI-1640 medium was added to a total volume of 1mL, and after CO-culturing was performed for 24 hours, 2 repeats were used to identify formation of different culture times and antigen concentrations on IS, cells were first exposed to 4 Pa (pa) in 2% paraformaldehyde (para) in 4 volumes, added with an equal volume of ammonium chloride (amonium chloride), exposed to IS for 20 minutes at room temperature, and then the cells were centrifuged at a rotation speed of 500 XIS for 10 minutes, and then analyzed by electron microscope.

Immune synapses are the stable association of antigen presenting cells with target T cells and are also important keys to the activation of the immune system. The interaction between macrophages and T cells and the formed synapse structure can be observed by transmission electron microscopy and scanning electron microscopy.

Please refer to FIGS. 1A-1D, which are transmission electron microscope (FIGS. 1A-1C) and scanning electron microscope (FIG. 1D) images of porcine alveolar macrophages (FIG. 101) paired with regulatory T cells (FIG. 103) (FIGS. 1A and 1B) or helper T cells (FIG. 1C and 1D) according to an embodiment of the present invention.

The results in FIGS. 1A-1D show that, in the first stage after co-culture, regulatory T cells 103 extend pseudopodia (pseudopodia) towards alveolar macrophage 101 (see FIG. 1A). In the second phase, alveolar macrophages 101 and regulatory T cells 103 approach the center (centroole proximity) (FIG. 1B). In the third phase, the high basal body (Golgi complex) of alveolar macrophage 101 becomes large, the two cell interfaces become flat (FIG. 1C), and an immune synapse is formed (FIG. 1D), which indeed forms an immune synapse system in vitro.

Example two verification of immune synapse systems in animal experiments

The 12 SPF pigs at 4 weeks of age were divided into 3 groups (4 per group), and a pig immunization program was performed with a negative control (control) group, and PRRSV antigen 1 (without T cell epitope; PRRSV-1) and antigen 2 (with T cell epitope; PRRSV-2) without T cell epitope (T cell epitope), respectively, for the first immunization at 4 weeks of age and the second immunization after 6 weeks of age. Antigen 1 contained no additional specific T cell epitope and antigen 2 contained specific additional designed T cell epitope, antigen 2 used the T cell epitope of PRRSV that was the sequence designed by the inventors. However, PRRSV-1 and PRRSV-2 are only examples for verifying the immune synapse system, and other conventional sequences may be used, and are not described herein.

The SPF pigs are sacrificed at 8 weeks of age, alveolar macrophages and PBMCs are respectively separated, the alveolar macrophages are used as Antigen Presenting Cells (APC), the APC and specific T cell subsets (namely Th cells, NK cells, Treg cells and control cells) obtained after blood PBMCs are sorted by a flow cytometer are cultured together, the functional influence of animal immunity on in-vitro immune synapses is evaluated, and TLR gene expression and cytokines are respectively analyzed to be used as verification of a subsequent screening platform.

2.1 assessment of changes in TLR Gene expression

The expression level of the TLR gene in the specific subset of immunostimulatory T cells is determined in the following manner or in a conventional manner. First, utilize

Fitting together

Total RNA of the specific T cell subset was extracted with a column, and cDNA was synthesized using the procedure attached to a commercially available kit [ e.g., PrimeScript RT reagent kit (Takara) ] after quantitative determination by spectrophotometry briefly, 0.5. mu.g of RNA was taken, 2. mu.L of 5-fold (×) PCR buffer solution, 0.5. mu.L of PrimeScript reverse transcriptase mix (RT enzyme mix) I, 0.5. mu.L of oligo dT primer (oligo dT primer; 50. mu.M), 0.5. mu.L of random primer (random primer; 100. mu.M) were added, each reaction volume was made up to 10. mu.L with secondary water (dd H2O), and the mixture was treated at 42 ℃ for 30 minutes.

The TLR3 gene upstream primer is shown as SEQ ID NO 1 sequence, and the TLR3 gene downstream primer is shown as SEQ ID NO 2 sequence. The TLR7 gene upstream primer is shown as SEQ ID NO 3, and the TLR7 gene downstream primer is shown as SEQ ID NO 4. The TLR8 gene upstream primer is shown as SEQ ID NO 5 sequence, and TLR8 gene downstream primer is shown as SEQ ID NO 6 sequence.

Please refer to fig. 2A to 2C, which are histograms of relative expression levels of mRNA of toll-like receptor (TLR) 3 (fig. 2A), TLR7 (fig. 2B) and TLR8 (fig. 2C) of antigen presenting cells, regulatory T cells, killer cells, helper T cells and control cells after in vitro co-culture of individual pigs immunized with different PRRS antigens according to an embodiment of the present invention. The relative expression amounts of TLR3, TLR7 and TLR8 mrnas in fig. 2A to fig. 2C are represented as- Δ Δ Ct in the geometric mean (2- Δ Δ Ct). The groups with different letters marked above the bars in fig. 2A-2C represent significant differences between the two groups (P < 0.05).

The results in fig. 2A to fig. 2C show that after the PRRSV-1 and PRRSV-2 are used to immunize pigs, the functional expression of the immunological synapses of the antigen presenting cells and different T cell subsets is indeed effectively promoted, and the gene expression of TLR3, TLR7 and TLR8 related to the virus presenting ability is increased, and the difference is statistically significant.

2.2 evaluation of changes in the content of cytokines

This example quantifies the IL-2 and IL-10 content of porcine cell supernatants using commercially available porcine IL-2 and IL-10 kits. The attached standard solution was diluted for use according to the procedure of the kit, and the procedure thereof is briefly described below. First, 100. mu.L of the sample or diluted standard solution was added to a 96-well plate and allowed to act for 1 hour. Subsequently, 200. mu.L of biotinylated antibody reagent (biotinylated antibody reagent) was added to the 96-well plate and allowed to act for an additional 1 hour. Then, 100. mu.L of streptavidin (streptavidin-HRP) labeled with horseradish peroxidase (HRP) was added thereto and reacted for 30 minutes, and 100. mu.L of a tetramethylbenzidine (3,3 ', 5, 5' -tetramethylbenzidine; TMB) substrate solution was added thereto and reacted for 30 minutes in the absence of light. Then, 100. mu.L of stop solution (stop solution) was added to terminate the reaction to measure the absorbance. After each reaction, the reaction solution was washed with wash buffer (wash buffer) 3 to 5 times. All steps were performed at room temperature.

The absorbance measurement is generally performed by reading the absorbance at a wavelength of 450/540nm using a commercially available continuous spectrophotometer, establishing a standard curve with the absorbance of a standard solution serially diluted at different concentrations, calculating the concentration of the cytokine contained in each sample according to the regression equation of the standard curve, and calculating the significant difference between the average values of each group using commercial statistical software (e.g., SAS statistical program), and the results are shown in fig. 3A to 3B.

Please refer to fig. 3A-3B, which are histograms of the content (pg/mL) of IL-2 (fig. 3A) and IL-10 (fig. 3B) of antigen presenting cells co-cultured with regulatory T cells, killer cells, helper T cells and control cells, respectively, after a pig individual is immunized with PRRS antigen according to an embodiment of the present invention. The groups with different letters marked above the bars in fig. 3A-3B represent significant differences between the two groups (P < 0.05).

The results from FIG. 3A to FIG. 3B show that after immunization of pigs with PRRSV-1 and PRRSV-2, the IL-2 (with immunopotentiation) content and the IL-10 (with immunosuppression) content were increased, especially after immunization of pigs with PRRSV-2 containing T cell epitope, the IL-10 content was inhibited more significantly, and the difference was statistically significantly different.

Example III application of in vitro immune synapse System

3.1 assessment of immunogenicity of antigens

First, alveolar macrophages and sorted T cell subsets were isolated from 6 4-week-old Specific Pathogen Free (SPF) pigs and co-cultured in 24-well plates to establish an in vitro immune synapse system in the same manner as in example one.

Secondly, the above-mentioned in vitro immune synapse system is co-cultured with antigen protein (treatment group, 2. mu.g PRRSV-1 or PRRSV-2) or without antigen protein (control group) in a carbon dioxide incubator at 37 ℃ for 24 hours, after the culture is completed, the 24-well plate is centrifuged at 300 × g for 15 minutes, and the cells are separately harvestedThe supernatant was taken for cytokine detection. At the same time, utilize

reagent (thermofisher scientific) lysed the cell pellet and TLR gene expression was detected in the same manner as in the example, and the results are shown in fig. 4.

Please refer to fig. 4, which is a histogram illustrating the relative expression of TLR mRNA in vitro immune synapse system (including T-helper cells and alveolar macrophages) screening immune adjuvants according to an embodiment of the present invention. The groups with different letters marked above the bars in FIG. 4 represent significant differences between the two groups (P < 0.05).

The results in fig. 4 show that PRRS antigens (i.e. PRRSV-1 or PRRSV-2) do significantly promote gene expression of TLR3, TLR7, and TLR8 of immune synapses, and that this difference is statistically significantly different.

3.2 evaluation of the immunostimulatory Properties of the immunological adjuvants

In addition, the in vitro immune synapse system (NK/APC or Treg/APC) of example one was co-cultured with or without immune adjuvant (control group), and IL-2 content was determined in the same manner as in example two. The aforementioned immunoadjuvants were poly IC (Sigma-Aldrich) and IL-18(Sigma-Aldrich Co.). Add 2. mu.L of LPoly IC (1mg/mL) (Sigma-Aldrich Co.) (2. mu.g/well) or 100. mu.L of IL-18(1ng/mL) (Sigma-Aldrich) (0.1ng/well) per well cytosol. The detection methods of poly IC and IL-18 are described in J.Immunol.162(10):6114-6121 (15.5.1999) and J.Immunol.176: 1348-1354 (2006), which are incorporated herein by reference.

Please refer to fig. 5, which is a histogram of IL-2 content (pg/mL) of the in vitro immune synapse system screening immune adjuvant according to one embodiment of the invention. The groups with different letters marked above the bars in fig. 5 represent significant differences between the two groups (P < 0.05).

The results in FIG. 5 show that an in vitro immune synapse system paired with Treg cells and APC cells can form an immune synapse. Poly IC significantly promoted IL-2 levels in immune synapses compared to IL-18, and this difference was statistically significantly different.

In summary, although the present invention is described with reference to the in vitro immune synapse system established by cells from a specific source, a specific analysis mode or a specific evaluation method as an example, the in vitro immune synapse system and the method for evaluating an immune response in vitro of the present invention are not limited thereto, and the in vitro immune synapse system and the method for evaluating an immune response in vitro of the present invention may be established by cells from other sources, other analysis modes or other evaluation methods without departing from the spirit and scope of the present invention.

In view of the above, it is seen that the advantage of the in vitro immune synapse system and the method for in vitro immune response assessment of the present invention is that the in vitro immune synapse system comprises antigen presenting cells from the same individual pig and specific T cell subsets isolated from peripheral blood mononuclear cells is utilized for the T cell epitope assessment of a test sample. The animal immune experiment proves that the in vitro immune synapse system has the same result as the animal experiment. Therefore, the in vitro immune synapse system is expected to replace animal experiments when new antigens or new adjuvants are developed in the future.

While the invention has been described with respect to various embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

[ notation ] to show

101: alveolar macrophages 103: regulatory T cells

105: helper T cell

[ biological Material deposit ] without

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Claims

1. An in vitro immune synapse system comprising:

an antigen presenting cell; and

at least one of a regulatory T cell, a killer cell and a helper T cell, and

wherein the antigen presenting cell, the regulatory T cell, the killer cell and the helper T cell are primary cells derived from the same individual, and the number of the antigen presenting cell is greater than the sum of the regulatory T cell, the killer cell and the helper T cell.

2. The in vitro immune synapse system of claim 1, wherein the same individual is a specific pathogen-free pig.

3. The in vitro immune synapse system of claim 1, wherein the antigen presenting cell is an alveolar macrophage.

4. The in vitro immune synapse system of claim 1, wherein the antigen presenting cell has a cell number 2-fold to 10-fold that of the sum of the regulatory T cell, the killer cell and the helper T cell.

5. The in vitro immune synapse system of claim 1, further comprising a control cell, wherein the control cell is derived from the same individual, and wherein the control cell has at least the CD 4-/CD 25-cell marker.

6. The in vitro immune synapse system of claim 5, wherein the control cell is a monocyte.

7. A method for assessing an immune response in vitro, comprising:

providing an in vitro immune synapse system, wherein said in vitro immune synapse system comprises:

an antigen presenting cell;

at least one of a regulatory T cell, a killer cell, a helper T cell; and

a control cell, and

wherein the antigen presenting cell, the regulatory T cell, the killer cell, the helper T cell and the control cell are primary cells derived from the same individual, and the number of the antigen presenting cell is 2-fold to 10-fold of the total number of the regulatory T cell, the killer cell and the helper T cell;

co-culturing a sample to be tested and the immune synapse system for 24 hours; and

detecting an immune-related change in the immune synaptic system, wherein the immune-related change comprises the level of toll-like receptor gene expression and/or a cytokine concentration, and

wherein the sample is determined to be immunogenic or immunostimulatory when the immune-related change of any of the regulatory T cells, the killer cells, and the helper T cells is significantly different from the immune-related change of the control cells.

8. The method of claim 7, wherein the test sample comprises an antigen or an adjuvant.

9. The method of claim 7, wherein the subject is a specific pathogen-free pig.

10. The method of claim 7, wherein the antigen presenting cell is an alveolar macrophage, the regulatory T cell has at least the CD4+/CD25+ cell marker, the killer cell has at least the CD 4-/CD 25+ cell marker, the helper T cell has at least the CD4+/CD 25-cell marker, the control cell is a monocyte, and the control cell has at least the CD 4-/CD 25-cell marker.

11. The method for evaluating an immune response according to claim 7, wherein the toll-like receptor gene expression level includes a toll-like receptor 3 gene expression level, a toll-like receptor 7 gene expression level and/or a toll-like receptor 8 gene expression level.

12. The method of claim 7, wherein the cytokine concentration comprises a concentration of interleukin.