RU2486238C1 - METHOD OF LOADING DENDRITIC CELLS WITH ANTIGEN OF INFECTIOUS ORIGIN Opistorchis felineus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: monocytes are isolated from venous blood MNCs using the Percoll cushion bi-gradient of density: 47.5% SIP and 15% SIP, respectively, at cooling to +4°C. The monocytes are placed in a completely nutritional culture medium with adding 20 ng/ml IL-4 and 20 ng/ml GM-CSF. The completely nutritional culture medium is replaced on the day 3 of cultivation. On the day 4 of cultivation the antigen of infectious origin Opisthorchis felineus is added at a dose of 40 mcg/ml and maturation of dendritic cells is simultaneously induced with lipopolysaccharide E.coli of serotype 055: B5 in a dose of 1 mcg/ml. On the day 6 of cultivation the dendritic cells are washed and analysed.

EFFECT: use of the method provides obtaining the mature, antigen-loaded dendritic cells.

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Description

The invention relates to medicine, specifically to immunology, and can be used to obtain activated dendritic cells capable of initiating an antigen-specific immune response.

The proposed dose of the helminth antigen Opisthorchis felineus is optimal for modeling the immune response based on the culture of dendritic cells in response to an infectious stimulus in order to develop approaches for the treatment of allergic diseases based on cell technologies.

Numerous facts indicate the ability of helminth infections to modify the host's immune response, which has a significant impact on the risk of development and the clinical course of allergies. The nature and extent of this modifying effect depends on specific conditions, including the genetic properties of individuals. The current information on the immune mechanisms of modeling the risk of allergic diseases in individuals with parasitic diseases is insufficient and requires an in-depth analysis of their molecular basis. The study of the modifying effect of helminth antigens on the formation of the immune response in patients with allergic diseases is especially important for regions with a high prevalence of parasitic infestations [1].

According to the immuno-epidemiological study carried out by L.M. Ogorodova et al. in natural foci of opisthorchiasis of the Tomsk region, in patients with bronchial asthma against the background of chronic opisthorchiasis, a relative decrease in the level of pathogenetically significant pro-inflammatory cytokines (IL-4, IL-5) and a decrease in specific IgE levels compared with patients with bronchial asthma without opisthorchiasis are recorded [2]. The revealed signs of a decrease in the tension of immediate hypersensitivity reactions against the background of opisthorchiasis invasion are of considerable interest in terms of studying the molecular immune mechanisms that control various parts of the pathogenesis of allergies.

Currently, there are no effective methods for the prevention of allergic diseases. The only pathogenetic method of treatment and limiting the expansion of the spectrum of causally significant allergens in patients with allergic diseases is allergen-specific immunotherapy (ASIT). One of the main disadvantages of this method is its specificity only for specific allergens, which complicates the treatment of patients with polyvalent sensitization. Adverse reactions during ASIT are the main problem when using allergen extracts. The most severe adverse effects include anaphylaxis caused by IgE-mediated mast cell degranulation leading to the release of vasoactive mediators and IgE-mediated capture of antigens by dendritic cells. A more advanced area of ASIT is allergen-specific vaccines based on dendritic cells, the creation of which is at the stage of clinical trials.

In this regard, the study of the antigenic, immunogenic, and allergenic properties of Opisthorchis felineus and the molecular and cellular mechanisms for modifying immediate hypersensitivity reactions under the influence of the Opisthorchis felineus antigen are relevant. The first activation (loading) of dendritic cells using the helminth antigen Opisthorchis felineus will allow us to study the immunogenic, antigenic and allergenic properties of the pathogen to develop approaches to the creation of anti-allergic vaccines based on dendritic cells.

A number of research centers are conducting research on the creation of vaccines based on dendritic cells for the immunotherapy of oncological, infectious and autoimmune diseases. The effect of dendritic cells activated by various infectious antigens on the functional properties of cells of the immune system, and the further formation of the immune response are studied.

The possibility of creating such vaccines is due to the fact that dendritic cells are the most effective antigen-presenting cells with a specific ability to present antigens to naive T cells and cause subsequent differentiation of the immune response. Dendritic cells isolated from peripheral blood, after in vitro treatment with the selected antigen, become mature and, in vivo, after returning to the body via intravenous administration, they can initiate an immune response.

A significant amount of research by foreign colleagues is devoted to the study of the structural and functional features of the helminth antigen Shistosoma mansoni, as well as its effect on the initiation of the immune response by dendritic cells obtained from human peripheral blood. A number of authors have shown that Shistosoma mansoni induces a pro-inflammatory phenotype of dendritic cells with subsequent differentiation of the Th2 immune response pathway [8]. Antigen isolated from Fasciola hepatica trematode is able to suppress the maturation and further functioning of dendritic cells [7].

According to the scientific literature, the M. tuberculosis antigen has been studied in sufficient detail, the phenotypic characteristics of dendritic cells loaded with this antigen have been analyzed, and work has been done to determine the effectiveness of the immune response to M. tuberculosis [5].

A known method of producing stimulated dendritic cells for the induction of an immune response against human tuberculosis. The method is based on the collection of immature dendritic cells from the patient, their cultivation ex vivo for maturation and the formation of allostimulatory activity. Cells are additionally harvested by antigen and administered to the same patient to form adaptive immunity. When ex vivo is cultured, immature dendritic cells are introduced into the culture medium with fragmented allogeneic double-stranded genomic DNA with fragments 200-6000 bp in size. in the amount of 5 μg / ml of medium [3].

As for helminth infestation caused by opisthorchisis, in all, at least 20 million people are affected by it in the world. In Russia, more than 60% of all recorded biohelminthoses are accounted for by opisthorchiasis caused by the trematode of the family Opisthorhidae (Opisthorchis felineus). In Southeast Asia, there is a related species, Opisthorchis viverrini, and in the Far East, Clonorchis sinensis [13]. In experimental in vitro cell models, the immunological aspects and molecular biology of infections caused by Opisthorchis viverrini and Clonorchis sinensis are well understood [6, 9].

However, in the literature available for analysis, not a single study is found that describes a method for loading dendritic cells with a helminth antigen prepared from Opisthorchis felineus.

Closest to the proposed is a method for producing mature dendritic cells to induce an immune response. The known method allows to obtain mature dendritic cells expressing on their surface CD1a, CD14 ^low , CD83, CD86, IL-10 ^low , CCR7 and IL-12p70 and having a stable phenotype of low IL-10 production. For this, precursor cells (monocytes) and immature dendritic cells are cultured in the presence of maturation and differentiation factors, such as GM-CSF, IL-4 and TNF-a, at a temperature of 31 to 37 ° C. The known method has a limited scope, since the obtained mature dendritic cells are used to induce an immune response only against malignant tumors and infectious diseases [4].

A new technical task is the development of a new method for producing dendritic cells activated by the infectious antigen Opisthorchis felineus.

To solve the problem in a method of loading dendritic cells with an antigen of infectious origin Opisthorchis felineus, including the isolation of a fraction of peripheral mononuclear cells (MNCs) from venous blood, the procedure for isolating monocytes from MNCs, placing the isolated monocytes in a complete culture medium with cytokines and growth factors, induction of maturation dendritic cells using E.coli lipopolysaccharides; monocyte isolation from venous blood MNCs is performed using a double density gradient ne call 47.5% SIP and 15% SIP, respectively, when cooled to + 4 ° C, after which the isolated monocytes are placed in a complete culture medium with the addition of 20 ng / ml IL4 and 20 ng / ml GM-CSF, while replacing the full nutrient culture media with cytokines and growth factors are carried out on 3 knockings of cultivation, in addition, an antigen of infectious origin Opisthorchis felineus at a dose of 40 μg / ml is added on the 4th day of cultivation simultaneously with E. coli lipopolysaccharide serotype 055: B5 at a dose of 1 μg / ml, then 6 day cultivation dendritic cells The lathes are washed and analyzed by flow cytometry.

The result of the developed method is the production of activated dendritic cells capable of initiating an antigen-specific immune response.

The method is as follows.

1) Isolation of the fraction of peripheral mononuclear cells (MNC)

Peripheral venous blood diluted 1: 4 with RPMI-1640 serum-free medium was added to a 50 ml sterile plastic tube (Falcon, USA). At the bottom of the tube with blood layered 10 ml of ficoll solution with a density of 1.077 g / cm ³ . Centrifuged at 2000 rpm in a centrifuge "NF400R" (Turkey) for 25 minutes at room temperature. Interphase ring cells are harvested using a Pasteur plastic transfer pipette (Sigma-Aldrich, USA). The collected cells are transferred to a sterile 50 ml plastic tube and washed twice using serum-free medium RPMI-1640 or Hanks solution (PanEco, Russia) at 2000 rpm for 10 minutes. After a second wash, the cells were resuspended in complete RPMI-1640 medium containing fetal calf serum (ETS) (Sigma-Aldrich, USA).

2) The procedure for isolating monocytes from MNCs of venous blood.

The isolation of monocytes from blood MNCs is carried out using a double gradient of percoll density (1.131 g / cm ³ , pH = 8.5-9.5, Sigma, USA). Prior to the monocyte isolation procedure, a solution of standard Isotone Percoll (SIP) is prepared. For this, to 1 ml of a 10-fold buffered phosphate solution (25 g of NaCl, 0.25 g of Na ₂ HPO4 and 4.2 g of KH ₂ PO _{4 is} adjusted with distilled water to 400 ml, pH = 7.2-7.4) add 9 ml of percoll density 1,131 g / cm ³ . Two percoll density gradients are prepared using SIP and an incomplete RPMI medium: 47.5% SIP (5226 μl SIP mixed with 5776 μl RPMI per 2 samples) and 15% SIP (4250 μl RPMI per 2 samples added to 750 μl SIP) .

The volume of the MNC precipitate obtained after washing twice was measured and RPMI was added in such an amount that the total volume of the cell suspension in the sample was 1 ml. 1.5 ml of SIP is added to the resulting cell volume. The suspension is then transferred to sterile 15 ml tubes. 5 ml of 47.5% SIP are slowly layered on the surface of the cell suspension along the wall of the tube, and then 2 ml of 15% SIP are also deposited on top of the wall. Samples are centrifuged for 45 min at 3500 rpm and upon cooling to 4 ° C. After centrifugation, 2 rings are obtained: the upper ring is monocytic, the MNCs are in the lower ring, red blood cells are deposited on the bottom of the tube. The monocytic ring is collected in a separate sterile tube with a Pasteur transfer pipette and washed twice with serum-free RPMI-1640 at 2000 rpm for 10 minutes.

The collected monocytes are used to prepare a suspension with a concentration of 5 × 10 ⁵ cells / ml. The suspension is prepared on the basis of RPMI-1640 medium containing 10% ETS, 50 μM β-mercaptoethanol (Sigma-Aldrich, USA), 2 mM L-glutamine (Paneko, Russia), 50,000 units / liter of penicillin (" Paneko ”, Russia), 50 g / l of streptomycin (Paneko, Russia), 11 mg / l of sodium pyruvate (Paneko, Russia).

3) Cultivation of dendritic cells from monocytes

Isolated monocytes with a concentration of 5 × 10 ⁵ cells / ml are placed in 24-well plastic culture plates (Nunclon, Denmark) in a volume of 1 ml per well. Cells are incubated for 1 hour in a CO ₂ incubator in a humid atmosphere of 5% CO ₂ at + 37 ° C. Then, 1 ml of medium is taken from each well, and 1 ml of complete RPMI medium containing IL-4 (ProSpec, USA; 20 ng / ml) and GM-CSF (ProSpec, USA; 20 ng / ml) is added in exchange ) A nutrient medium with cytokines is replaced with a fresh one on the 3rd day of cultivation. On day 4, dendritic cell maturation was induced using a lipopolysaccharide of bacterial origin (L2880-055: B5, Sigma-Aldrich, USA). Stimulation of dendritic cells with the infectious antigen Opisthorchis felineus is also carried out on day 4 according to the scheme in figure 1. The working dose of the Opisthorchis felineus antigen in the culture well is 40 μg / ml. Figure 2. shows a diagram of the cultivation of dendritic cells from peripheral blood monocytes in the conditions of activation of Opisthorchis felineus

The proposed dose of the helminth antigen Opisthorchis felineus is optimal for modeling the immune response based on the culture of dendritic cells in response to an infectious stimulus in order to develop approaches for the treatment of allergic diseases based on cell technologies.

Percoll is a balanced medium for centrifugation in a gradient of cell density and subcellular structures. Centrifugation leads to a heterogeneous distribution of cells and particles of different sizes in the medium. The concentrations of percoll density gradients (47.5% and 15%) for isolating monocytes from peripheral blood MNCs were selected experimentally by the authors. To select the optimal density of SIP solutions, we measured the refractive indices of the prepared solutions on a laboratory refractometer with respect to the sucrose gradient. Experimental work using SIP concentrations of 47.5% and 34%, respectively, resulted in a lower monocyte yield after centrifugation compared to the number of cells at the stated density of SIP solutions.

LPS - lipopolysaccharides of the bacterial cell wall of E. coli (strain L2880-055: B5) are added to antigen-activated dendritic cells to induce maturation of the latter. The optimal LPS dose of 1 μg / ml for the induction of dendritic cell maturation was selected by the authors in the experiment. This dose is sufficient in comparison with the use of lower concentrations of LPS to stimulate DC in the conditions of activation of the Opisthorchis felineus antigen and to obtain the optimal number of mature DCs.

The cultivation time, the concentration of cytokines used, as well as the dose of the helminth antigen Opisthorchis felineus were selected by the authors experimentally in order to obtain the maximum number of mature activated dendritic cells that can cause an antigen-specific immune response. The cultivation of monocytes for 6 days in a complete nutrient medium with cytokines and growth factors resulted in mature DCs. With a reduction in cultivation time (4 days), the yield of mature DCs decreased, most of the cells possessed the properties of immature DCs. Cultivation of DC for more than 6 days led to the appearance of aging and non-viable cells.

Adding to the culture medium IL4 and GM-CSF in a concentration of less than 20 ng / ml led to a decrease in the differentiation of monocytes in DC, and in general, to obtain a smaller amount of DC on the 6th day of cultivation. Selected doses of IL4 and GM-CSF are sufficient for growing DC. The use of large doses of IL4 and GM-CSF is impractical due to the high cost of these reagents.

The phenotype of dendritic cells was experimentally confirmed by flow cytometry using a FacsCalibur cytofluorimeter (Becton Dickinson, USA). The study of the expression of surface clusters of dendritic cell determination was carried out using the appropriate monoclonal antibodies. The expression level of CD209 (PerCP, BD PharmingenTM, USA), costimulatory molecule CD86 / B7-2 (FITC, BD PharmingenTM, USA) was determined; molecules of the antigenic presentation of MHC classes I and II (HLA-DR, FITC, BD Biosciences, USA) and a marker for terminal differentiation of dendritic cells - CD83 (PE, BD PharmingenTM, USA). The analysis of dead cells was performed using a dye to assess the viability of 7-AAD cells (BD Bioscinces, USA). The principle of action of 7-AAD is to stain dead cells by penetrating only through the damaged cell membrane and binding to double-stranded DNA.

The cultivation time, the concentration of cytokines used, as well as the dose of the helminth antigen Opisthorchis felineus 40 μg / ml were selected empirically by the authors as a result of a series of experiments in order to obtain the maximum number of mature activated dendritic cells that can cause an antigen-specific immune response.

The ability of Opisthorchis felineus to modulate the immune response was analyzed by co-cultivation with dendritic cells for 48 hours, which are key antigen-presenting cells responsible for generating and directing the immune response. The determination of the interaction characteristics of the Opisthorchis felineus antigen and dendritic cells by the nature of the change in the phenotypic characteristics of dendritic cells and their ability to mature is necessary for further study of the polarization of the immune response. For cultivation in the proposed method, a working (final) dose of the helminth antigen Opisthorchis felineus - 40 μg / ml, which is optimal for modeling an immune response based on dendritic cells in response to an infectious stimulus, was selected and selected.

The difference of the proposed method is a methodological approach, which includes the implementation of successive stages of the selection of MNCs and monocytes using a density gradient of ficoll (1.077 g / cm ³ ) and a double step density gradient of percoll (1.131 g / cm ³ ), respectively, allowing to obtain a sufficient number of monocytes for subsequent cultivation dendritic cells.

As a result of isolation on a density gradient of ficoll (1.077 g / cm), a peripheral blood MNC fraction was obtained, represented by lymphocytes and monocytes. For the initial characterization of cell populations, side scatter (SSC) and forward scatter (FSC) scatterings obtained using flow cytometry were used.

According to the results of the analysis according to the above parameters, monocytes amounted to about one fifth (14% -23%) of the MNC fraction (Fig. 2a, region R1). Lymphocytes characterized by low SSC / FSC signals represented 70% -80% of the MNC cell mass (Fig. 2a, region R2). After separation of lymphocytes and monocytes using a double gradient of percoll density (1.131 g / cm ³ ), the cell fraction collected from the phase separation contained 74.2 ± 5.5% of cells having average SSC / FSC values characteristic of monocytes (Fig. .2b, region R1).

An example of the method

The material for the study was the venous blood of a healthy volunteer. During centrifugation using a density gradient of ficoll (1.077 g / cm ³ ) and a double step gradient of density of percoll (1.131 g / cm ³ ), respectively, peripheral blood monocytes were obtained. Next, the isolated monocytes at a concentration of 5 × 10 ⁵ cells / ml were placed in 24-well plastic culture plates and cultured in complete RPMI medium containing IL-4 (20 ng / ml) and GM-CSF (20 ng / ml) in CO ₂ incubator in a humid atmosphere of 5% CO ₂ at + 37 ° C.

On the 3rd day of cultivation, a nutrient medium with cytokines was replaced. On day 4, DC maturation was induced in the presence of LPS. To obtain antigen-activated DCs, stimulation with the infectious antigen Opisthorchis felineus was performed on day 4. For phenotypic characterization of DCs, the expression level of CD209 (PerCP) was studied; costimulatory molecule - CD86 / B7-2 (FITC); molecules of the antigenic presentation of MHC classes I and II (HLA-DR, FITC) and a marker of terminal differentiation of dendritic cells - CD83 (PE) by flow cytometry.

As part of the selection of the antigen dose, serial dilutions of Opisthorchis felineus were used in three working concentrations - 160, 40 and 10 μg / ml, while the final concentration of antigen in the culture well was 40, 10 and 2.5 μg / ml. All selected antigen concentrations (40, 10, 2.5 μg / ml) in the presence of LPS were sufficient to induce DC maturation, however, the use of a dose of 40 μg / ml antigen led to the most pronounced expression of phenotypic markers on the surface of DCs. At the same time, the cells remained viable and possessed a mature phenotype. The analysis of cell viability was carried out on the basis of measuring the DNA content using intranuclear dye 7AAD, the level of cell apotosis did not exceed 15%. The expression of the surface marker CD209 on the obtained DC was 69%; 35% and 40% of expression of the terminal differentiation marker (CD83) and costimulatory molecule CD86 were detected, respectively. The severity of the HLA-DR marker was the highest and amounted to 95%, respectively.

application

Fig 1. The method of loading dendritic cells with an infectious antigen (scheme)

Figure 2. The distribution of cells depending on the signals of the lateral (SSC) and front light scattering (FSC)

Note:

a - populations of mononuclear cells of healthy subjects (ficoll gradient 1,077 g / cm ³ )

b - populations of mononuclear cells of healthy subjects (double gradient of percoll, 1.131 g / cm ³ )

R1 -% of monocytes from peripheral blood mononuclear cells

R2 -% of lymphocytes from peripheral blood mononuclear cells

List of sources of information

1. Ilyinsky E.N. Actual questions of studying the problem of opisthorchiasis in Siberia. / E.N.Ilinsky // Bulletin of Siberian medicine. - 2002. - No. 1. - p. 63-69.

2. Ogorodova L. Comparative characteristics of the content of IgE in children with atonic diseases occurring against the background of persistent infections. / L.M. Ogorodova, O.V. Kozina, V.F. Raenko and others // Allergology. - 2005. - No. 1. - p.13-17.

3. A method of obtaining stimulated dendritic cells for the induction of an immune response against human tuberculosis: US Pat. 2401664 Russian Federation: IPC: A61K A61P C12N / E.A. Alamkina; declared 08/03/2009; publ. 10/20/2010, Electron, dan. - Access mode: http://bankpatentov.ru/node/46175

4. A method of producing mature dendritic cells to induce an immune response and the use of the obtained cells: EP 200801385: IPC: C12N 5/00 A61K 39/00 A61P 35/00 A61P 31/12 / A. Kirkin; Applicant and patent holder. - Dandrite Biotek A / C; declared 12/08/2005; publ. 10/30/2008, Bull. No. 3 http://www.eapo.org/eng/bulletin/ea201103/HTML/015266.html

5. Shevchenko Yu.A. The effect of antigen-activated dendritic cells on the functional properties of peripheral blood mononuclear cells of patients with tuberculosis: dis. ... cand. biol. Sciences / Yu.A. Shevchenko. - Novosibirsk, 2008 .-- 108 s.

6. Cai X.Q. Sequences and gene organization of the mitochondrial genomes of the liver flukes Opisthorchis viverrini and Clonorchis sinensis (Trematoda). / X.Q. Cai, G.H. Liu, H.Q.Song et al. // Parasitol. Res. - 2011. - May 31. [Epub ahead of print].

7. Hamilton C.M. The Fasciola hepatica tegumental antigen suppresses dendritic cell maturation and function. / C. M. Hamilton, D. J. Dowling, C. E. Loscher, R. M. Morphew et al // Infect. Immun. - 2009 .-- Vol.77 (6). - P.2488-2498.

8. Stijn C.M. Schistosoma mansoni worm glycolipids induce an inflammatory phenotype in human dendritic cells by cooperation of TLR4 and DC-SIGN. / C. M. Stijn, S. Meyer, M. van den Broek, S. C. Bruijns, et al // Mol. Immunol. - 2010 .-- Vol. 47 (7-8). - P.1544-1552.

9. Wongratanacheewin, S. Immunology and molecular biology of Opisthorchis viverrni infection. / S. Wongratanacheewin, R.W. Sermswan, S. Sirisinha // Acta tropica. - 2003. - Vol. 88. - P.195-207.

Claims (1)

A method for loading dendritic cells with an antigen of an infectious origin Opisthorchis felineus, comprising isolating a fraction of peripheral mononuclear cells (MNCs) from venous blood, a procedure for isolating monocytes from MNCs, placing isolated monocytes in a complete culture medium with cytokines and growth factors, inducing maturation of dendritic cells using lipopolysaccharides E.coli, characterized in that the isolation of monocytes from venous blood MNCs is carried out using a double gradient of percoll density of 47.5% SIP and 15% SIP, respectively, when cooled to 4 ° C, after which the isolated monocytes are placed in a complete culture medium with the addition of 20 ng / ml IL4 and 20 ng / ml GM-CSF, while replacing the complete culture medium with cytokines and growth factors they are carried out on 3 knockings of cultivation, in addition, an antigen of infectious origin Opisthorchis felineus at a dose of 40 μg / ml is added on the 4th day of cultivation simultaneously with E. coli lipopolysaccharide serotype 055: B5 at a dose of 1 μg / ml, after which the dendritic cells are washed off on the 6th day of cultivation and analyzed by flow cytometry.

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