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Section 3. Physico-Chemical Biology
Gudkov Georgy Vladimirovich, State Federal-Funded Educational Institution of Higher Professional Training Kuban State Medical University of Ministry of Health of the Russian Federation,
holder of post-doctoral degree in medicine, professor of clinical immunology, allergology and laboratory diagnosis of advanced training and professional retraining faculty E-mail: pol09@mail.ru Filippov Evgeny Fedorovich, State Federal-Funded Educational Institution of Higher Professional Training Kuban State Medical University of Ministry of Health of the Russian Federation, holder of post-doctoral degree in medicine, head of clinical immunology, allergology and laboratory diagnosis of advanced training
and professional retraining faculty E-mail: filippovef@mail.ru
Тen Flora Paksunovna, State Federal-Funded Educational Institution of Higher Professional Training Kuban State Medical University of Ministry of Health
of the Russian Federation Piven Alexander Vladimirovich, State Federal-Funded Educational Institution of Higher Professional Training Kuban State University Biologist, Postgraduate student E-mail: alexander_piven@mail.ru Krutenko Dmitry Viktorovich, Ph.D., Biology
MODELING OF THE CYTOKINE ENVIRONMENT PROVIDING THE OPTIMAL IMMUNE RESPONSE OF ANTITUMOR VACCINES BASED ON DENDRITIC CELLS
Abstract: The inclusion of interferons and TLR ligands in the maturation cocktail provides a mature phenotype of DC with the predominance of costimulatory molecules over co-inhibitory, active directed migration in response to chemokines (CCR7 ligands), and also a high level of secretion of IL-12p70 as a response to CD40L stimulation. In a mixed medium of lymphocytes, these signals
are functionally interpreted by polarizing the immune response toward IFNy-secreting Th1 cells and suppressing the concomitant activation of regulatory T-cells. As a contrast to standard DC vaccines, the proposed maturation protocol creates a aDCl culture with the cells that simultaneously combine the necessary, often contradictory, properties of antitumor vaccines.
Keywords: DENDRITIC CELLS, ANTITUMOR VACCINES, TH1- CELL, PGE2, IL - 12, CCL19, CCL21, CCR7, TLR LIGANDS.
Dendritic cells (DC) are used in cancer immunotherapy for more than 20 years because of their ability to induce antitumor immunity [1]. The clinical effectiveness of DC remains limited, although the antigen-specific immune response has been detected in most cases [2; 3]. Autologous monocytes of peripheral blood are used to create DC vaccines in the majority of studies, which undergo a two-stage process of differentiation and maturation [4]. The composition and concentration of cocktail components for maturation have crucial significance for the future DC. The "gold standard" is the combination of TNFa, IL-1^, IL-6 and PGE2, however, different sources suggest a wide variety of cytokine composition variants for maturation of DC [5].
The ability of the DC to secrete IL- 12p70 has crucial importance for the polarization of the immune response in the direction of Th1, which is most needed for an effective reaction against tumor cells. However, bioactive IL-12p70 cannot be produced by DC, which has matured under the conditions of a standard combination of cytokines [6]. This negative effect is mostly created by PGE2, which prevents the production of IL-12p70 and in parallel induces the production of IL-12R antagonist (IL-12p40 homodimer) [7]. Moreover, PGE2 selectively disrupts production IL-2 and IFNy by T-cells, inhibits the activation of T-cells in response to Th1-cytokines such as IL-2 and IL-12p70, affects the appearance of the ability to attract regulatory T-cells (Treg), and also stimulates naive CD4+ T-cells to differentiate towards Treg. At the same time, in the presence of obvious suppressor activity, PGE2 shows a synergy with TNFa and promotes maturation of DC expressing CCR7. The latter provides the reaction of DC to chemokines CCL19
and CCL21 (both are CCR7 ligands) and active migration of DC to the lymph nodes [10].
The inconsistency of these effects lead to an active search for alternative ways of generating mature DC, which lead to a discovery that Toll-like receptor agonists (TLR) induce maturation of DC capable of inducing Th1 polarization [11]. These agents have been used in cocktails for maturation of DC, and TLR4-ligand-lipopolysaccharide (LPS), TLR3-ligand-polyI: C [13] and TLR7/8-ligand-R848 (resiquimod) are the most known among them [12; 14]. It has been shown that the combination of pro-inflammatory cytokines, interferons and TLR ligands can lead to generation of DC, which actively secret IL-12p70 [15].
Mature DCs express costimulatory (CD80 = = B7-1 and CD86 = B7-2) and co-inhibitory (CD274 = PD-L1) molecules, and the interaction of corresponding T-cell ligands with them influences the direction of differentiation of the latter [16]. Various influences of these molecules on the phenotype and function of T-cells have been found, but their manifestation pattern depends on the conditions of maturation of the DC, needs further research and clarification. Not sufficiently studied are the questions about the effect of different maturation protocols on the migration properties of DC, their ability to attract T-cells and induce Treg. In this relation, test of DC in a mixed leukocytes culture (MLC) with autologous T-cells without the influence of exogenous cytokines and allogeneic stimulation will reveal more subtle differences in the nature of the immune response induced by DC, matured under different conditions.
Therefore, the purpose of this study was to research the various protocols ofDC generation which,
through the expression of a pattern of costimulatory molecules and secreted cytokines, could provide the optimal functional effect of these signals on the activation of T-cells. In particular, the effect of various TLR ligands and pro-inflammatory cytokines on the properties of DC was compared to determine the type of polarization of the immune response of T-cells.
Materials and techniques
Cultivation of DC and acquisition of naive CD4+ ^cells.
Reagents: interleukin-4 (IL-4, Thermo Fisher Scientific) granulocyte-macrophage colony-stimulating factor (GM-CSF, Neostim, Russia), interleukin-1^ (lL-1^, Betaleikin, Russia), interleukin-6 (IL-6, Thermo Fisher Scientific), interferon-a2 (iFNa, Ro-feron-A, Roche, Switzerland), interferon-y (iFNy, Inharon, Russia), lipopolysaccharide (LPS, E.coli 0111: B4, Sigma-Aldrich), prostaglandin-E2 (PGE2, Sigma-Aldrich), tumor necrosis factor-a (TNFa, Life Technologies), polyinosinic-polycytidylic acid (poly-I: C, Sigma-Aldrich), resiquimod (R848, Sig-ma-Aldrich).
To cultivate DC of 14 conditionally healthy donors (age group from 25 to 37 years), samples of peripheral heparinized venous blood were obtained in a volume of up to 20 ml, which was processed no later than 6 hours after sampling. Peripheral mononuclear cells (PMC) were isolated in accordance with standard protocol in Histopaque-1077 density gradient (density 1.077 g/ml, Sigma-Aldrich). Monocytes were obtained from the BMD fraction by adhesion to plastic, followed by culturing for 6 days in a serum-free medium Panserin 413 (PAN Biotech GmbH, Aidenbach, Germany) containing 15% Panexin basic serum replacement (PAN Biotech GmbH, Aidenbach, Germany) and recombinant human cytokines (GM-CSF-50 ng/ml and IL-4-25 ng/ml), in a humidified atmosphere with 5% CO2 at 37 °C. On the 6th day for 48 hours, a different combination of pro-inflammatory inter-leukins, interferons and TLR ligands was used to induce maturation of two DC populations: standard
DCs-sDC: TNFa (50 ng/ml), IL-1ß (12 ng/ml), IL-6 (25 ng/ml) and PGE2 (l yg/ml); Polarized DCs inducing Type I immune responses of T-help-ers (aDCl): TNFa (50 ng/ml), IL-lß (12 ng/ml), IL-6 (25 ng/ml), IFNa (3000 U/ml), IFNy (1000 U/ml), LPS (2.5 yg/ml), poly-I: C (20 yg/ml) and R848 (3 yg/ml). Mature cultures of the DC were washed twice and used for further research.
To study the stimulating and polarizing effects of different DC populations (sDC and aDC1) on autologous naive CD4+ T-cells under MLC, the latter were isolated from the suspension of the non-adhesive PMC depleted monocyte fraction by negative immuno-magnetic separation using the EasySep CD4+ kit (StemCell Technolog ies) in according to the manufacturer's protocol.
Flow cytometry using marked monoclonal antibodies (BD Biosciences) and the corresponding isotypic controls was carried out for the phenotyp-ing of DC-CD14 (FITC, M5E2), CD83 (APC, HB15e), HLA-DR (PerCP, L243), CD80 (FITC, L307), CD86 (PE, 233), CD274 (APC, MIH1), CCR7 (FITC, 3D12), CCL19 (PE, T50-867) and regulatory T-cells — CD4 (FITC, RPA-T4), CD25 (PE-Cy7, M-A251), CD 127 (AlexaFluor 647, HIL-7R-M21), FoxP3 (PE, 259D/C7). Expression of intracellular cytokines in different T-cell populations was determined after 4 days of incubation of MLC according to protocols of BD FastImmune™ CD69/CD8/CD3 (#340365) reagents, BD FastImmune™ Anti-Human IL-4 (#340451) and FastImmune™ IFNyCD69/CD8/CD3 (#346048). Cell population analysis was performed on a BD FAC-SCanto flow cytometer (Becton Dickinson, USA), with 3 to 6 color marks and data analysis in the BD FACSDiva v.6 program (Becton Dickinson, USA). Expression was assessed for the mean fluorescence intensity (MFI), the ratio of the MFI value for coloring with a specific marked antibody to MFI for the corresponding isotype.
Cytokine secretion and T-cell proliferative response were evaluated in autologous MLC, where
naive CD4+ T-cells (1 x 106/cavity) were used as the responding cells and cultured in 24-well plates in serum-free Panserin 413 medium supplemented with 10% serum replacer Panexin basic in humid atmosphere at 37 °C in 5% CO2. DC served as stimulants, which were added to autologous T-cells in the ratio 1:10. Secretory and proliferative response of T-cells was evaluated at the end of 4 days of incubation. The concentration of soluble cytokines IFNy and IL-4 was evaluated using the Multi-Analyte Profiling (xMAP) technology on a Luminex 200 2-beam laser analyzer (Luminex Corporation, USA), according to the manufacturer's methodology for a commercial test system (Procarta® Mix & Match Assays, eBiosci-ence) and using ProcartaPlex Analyst 1.0 software (concentration range from 1 to 32000 pg/ml).
The proliferative response of T-cells in MCL was evaluated by the incorporation of bromodeoxy-uridine (BrdU) in DNA synthesis according to the protocol for the BD Pharmingen™ BrdU Flow Kits reagent kit.
The secretion of IL-12p70 and IL-10 by dendritic cells was stimulated during 24-hour incubation in a neutral medium (without cytokines) with CD40L. MEGACD40L® Protein (Enzo Life Sciences Inc.) was used as the latter, which is an oligomeric construct that efficiently mimics the membrane-associated aggregation of CD40L observed in vivo. Stimulation with CD40L recreates the interaction of DC with naive CD4+ T-cells expressing CD40 receptor. The concentration of IL-12p70 and IL-10 in the supernatant was measured using xMAP technology (see above).
Chemotaxis of DC and naive T-cells in vitro
was performed on the basis of the Migratest (Gly-cotope Biotechnology) kit using 24-cavity plates equipped with membranes with a pore diameter of 3 ^m. The lower chamber was filled with a medium of chemokine CCL21 (Thermo Fisher Scientific) in a volume of 350 ^l, and 100 ^l of a DC suspension (5 x 104) was added to the top, after which the plate was incubated for 2 hours at 37 °C. Chemotaxis of
naive CD4+ T-cells was examined in similar 24-cavity membrane plates. A suspension of naive CD4+ T-cells (2 x 105) in a volume of100 ^l was introduced into the upper chamber and the lower chamber was filled with supernatant from DC culture (5 x 105 DC in 1 ml of neutral medium stimulated with CD40L for 24 hours), after which the plate were incubated for 3 hours at 37 °C. To study the CCR7-dependent migration component of naive CD4+ T-cells for 30 minutes prior to chemotaxis, they were incubated with blocking unlabeled anti-CCR7 antibodies (Purified, 3D12, BD Biosciences).
The statistical processing of data was carried out using the "Statistica 6.0 for Windows" application software package. To detect significant differences in the compared indicators, the non-parametric Wilcoxon-Mann-Whitney U-test was used. Differences were considered significant at a significance level of p < 0.05.
Results
Migration activity of the DC
DC of monocytic origin maturing with introduction (sDC) or in the absence of PGE2 (aDC1), represent two types of mature DC that are usually cultured in serum-free media for clinical use.
Although the DC maturation protocols using PGE2 are associated with the suppression of IL-12p70 production [17], the ability of PGE2 to enhance CCR7 expression and, accordingly, the migration activity of DC as a response to chemokines CCL19 and CCL21 (CCR7 receptor ligands that attract DC to lymph nodes) justifies the use of PGE2 in standard DC maturation protocols (sDC) when creating cellular vaccines [18].
In contrast to sDC, the use of another maturation protocol involving interferons and TLR ligands leads to the formation of mature polarized DCs (aDC1) capable of secreting high levels of IL-12 and inducing tumor-specific cytotoxic T-lymphocytes (CTL) in vitro. However, aDC1 is characterized by low migration activity in vitro in response to chemokines that bind to CCR7 (CCL19 and CCL21).
In accordance with the data obtained (Fig. 1), DC, which matured in the presence of PGE2 (sDC) showed not only a statistically significant higher level of CCR7 expression than aDC1, but also high migration activity in response to CCL21. However, even significant differences in the expression of CCR7 in sDC and aDC1 cells rapidly disap-
peared after being transferred from the maturation medium to the neutral medium containing only GM-CSF. This equalization of the differences in the expression of CCR7 after transfer to a neutral medium was naturally reflected in the disappearance of the differences in migration activity in sDC and aDC1.
Figure 1. CCR7 expression (A) and in vitro migration capacity as a response to chemokine CCL21 (B) in two types of DC populations (sDC and aDC1)
To analyze the causes of differences in the expression of CCR7 in two types of DC (sDC and aDC1), the levels of its surface and intracellular expression were compared. Surprisingly, despite the marked predominance of surface expression of CCR7 in sDC, the determined total content of this receptor (surface and intracellular) did not show significant differences between the two types of DC. Taking into account the existence of the mechanism of ligand-induced internalization of CCR7, the possibility of leveling differences in the levels of surface expression of this receptor in sDC and aDC1 was studied by adding exogenous CCL19 (it is known that CCL21 does not affect internalization).
According to the data obtained, the addition of exogenous CCL19 (100 ng / ml) significantly reduced the level of surface expression of CCR7
only in the aDC1 culture, which initially actively expressed CCR7 due to its maturation conditions without the addition of PGE2. At the same time, in both cultures of DC (sDC and aDC1), the overall expression level of CCR7 (surface and intracellular) did not change under the influence of exogenous CCL19.
PGE2 is a potent inhibitor of CCL19 DC products (sDC)
Given the selective increase in the surface (but not general) expression of CCR7 in DC culture maturing in the presence of PGE2 (sDC), and the ability of exogenous CCL19 to eliminate it, the effect of PGE2 on the endogenous production of CCL19 in DC cultures was compared (Fig. 2).
Figure 2. Secretion of CCL19 by DC populations (sDC and aDC1) under different incubation conditions: in a maturation medium; in a neutral medium; in a neutral medium after reactivation with CD40L; (*, p < 0.05; **, p < 0.01)
By taking in consideration, that CCL19 can affect the intensity of internalization of CCR7, we detected a significantly higher level of endogenous secretion of CCL19 in mature aDCl culture compared to sDC. Considering that in vivo the source of the chemokine CCL21 is the cells of the lymphatic endothelium, we did not find its significant secretion in both types of DC cultures. Analysis of factors inducing maturation of DC and their effect on the regulation of CCL19 production in cultures of sDC and aDC1 showed that the main inducers of CCL19 are TNFa, IFNa, poly-I: C (TLR3 ligand), LPS (TLR4 ligand) and R848 (TLR7/8-ligand), while PGE2 exerts a potent inhibitory effect on the secretion of this chemokine. In addition, PGE2 also suppresses the production of CCL19 originally induced by LPS (ligand TLR4).
The stability of CCL19 secretion by both populations of mature DC was evaluated after transferring them to a neutral medium (without stimulating factors), as well as in the presence of a CD40L stimulator. Despite the fact that after the transfer to a neutral medium, the secretion of CCL19 at the end of the 24 hour incubation sharply decreased in both populations that had differently matured DC, it was restored to a much greater extent after their reactiva-
tion with the help of CD40L. The population sDC, in contrast to aDC1, despite the absence of PGE2 in the medium, weakly secreted CCL19 both in a neutral medium and after CD40L stimulation. The data obtained indicate that the maturation conditions of DC play a decisive role in their ability to secrete CCL19 in a neutral medium. By means of this chemokine, the DC are able to attract recirculating T-cells (naive and central memory) that drain into the drainage lymph nodes, which express the corresponding receptor (CCR7).
To determine the functionality of the chemokine CCL19 secreted by aDC1, the ability of sDC and aDC1 to target naive CD4+ T-cells expressing CCR7 was evaluated. For a more revealing assessment of the contribution of the CCR7 pathway to the migration of naive T-cells, blocking monoclonal antibodies against CCR7 were used. While a large number of naive CD4+ T-cells migrated towards the supernatant aDC1, the ability of the supernatant sDC to cause migration of naive CD4+ T-cells did not exceed the control level corresponding to a pure medium. Expectedly, the migration activity of naive CD4+ T-cells completely disappeared in the presence of blocking antibodies against CCR7.
Profile of costimulatory molecules
At the end of the maturation stage, both DC populations (sDC and aDCl) exhibited a mature phe-notype, which manifested itself in significant expression of such markers as CD83 and HLA-DR against
the background of the disappearance of CD14. However, analysis of the expression of costimulatory and inhibitory markers (CD40, CD80 = B7-1, CD86 = = B7-2, CD274 = PD-L1) revealed significant differences between them (Fig. 3).
Figure 3. Profile of the expression of costimulatory molecules by populations of dendritic cells sDC and aDCl (*, p < 0.05; **, p < 0.01)
As we can see, in the aDC1 population, the expression level of CD40 was more than 2 times the corresponding value (MFI) for sDC. The expression of the CD80 and CD86 costimulation molecules showed the highest MFI values in both DC populations, but a significant prevalence was observed in aDC1 87.2 and 97.6, respectively (versus 41.9 and 60.8 in the sDC population). The expression of the inhibitory marker CD274 was higher in the sDC population (17.1 vs. 23.4), with the MFI CD86/ CD274 ratio, which characterizes the degree of positive costimulation potential, significantly prevailed in the aDC1 population (5.4 vs. 3.1, p = 0.005).
Secretion of IL-12p70
Analysis of the supernatant obtained after a 24 hour stimulation of DC with CD40L under neutral conditions showed a high IL-12p70 content (2.5 x 103 pg/ml) in the aDC1 population, while in the sDC supernatant its concentration was extreme-
ly low (38 pg/ml, p = 0.005) and slightly exceeded the detectable threshold (Fig. 4).
The result of measuring the concentration of IL-10 was at the boundary of the detected level and did not exceed 8 pg/ml in the supernatants ofboth DC populations. The difference in the secretion of these inter-leukins in two DC populations demonstrates the ratio of concentrations of IL-12p70 / IL-10 (p = 0.007).
aDCl induce the activation of Thl cells producing IFN-y
The tolerogenic activity of various DC populations was assessed by their ability to induce proliferation of regulatory and non-regulatory activated T-cells in MLC for 4 days. The surface marker CD127 and intracellular FoxP3 were used to isolate subpopulations: CD4+CD25highFoxP3+CD127- is a phenotype of regulatory T-cells (T-reg); CD4+CD25highFoxP3" CD127+ is a phenotype of non-regulatory activated T-cells (Fig. 5).
Figure 4. Concentrations of IL-12p70 and IL-10 in the supernatant after 24-hour stimulation of sDC and aDCI with CD40L in neutral medium
Figure 5. Gating strategy for a subpopulation of regulatory T-cells induced in MLC, by a representative example of one of the donors
Compared to the control, both types of DC induced a significant increase in the relative content of FoxP3+ cells, as well as activated cells with CD4+CD25highCD127- phenotype. Cultivation in the presence of aDC1 provided a higher level of activated CD4+CD25high cells than in the presence of sDC (5.3% vs. 4.1%; p = 0.09). At the same time, sDC significantly induced T-reg compared to aDC1 (2.5% vs. 2.0%, p = 0.037). On the contrary, the percentage of activated non-regulatory T-cells predominated when cultured in the presence of aDC1 (0.8%) and significantly exceeded the percentage of these cells in culture with sDC (0.2%; p = 0.007).
The ratio of the percentage of activated T-cells to regulatory (CD4+CD25high / T-reg) further emphasized this difference (the median ratio for aDC1 is 0.35, for sDC = 0.09, p = 0.005).
At the end of 4 days of cultivation in mixed culture supernatants, the concentration of soluble cytokines IFN-y and IL-4 was determined (Fig. 6). After co-cultivation with the presence of aDC1, compared with sDC, a high concentration of IFN-y(3318 pg /ml vs. 638 pg/ml, p = 0.007) was observed, while IL-4 concentration in both cases was slightly higher than the detection threshold.
Figure 6. The content of various subpopulations of CD4+ cells (A, B) induced in MLC upon co-cultivation with sDC and aDCI after subtracting the corresponding values in the control MLC (not stimulated by DC), as well as the concentration of soluble cytokines IFN-y and IL-4 (C) in the supernatant (*, p < 0.05, **, p < 0.01)
To assess the ability of sDC and aDC1 to activate the Th1 / Th2 response in MLC, the relative number of CD3+ cells expressing intracellular cytokines IFNy and IL-4 was examined (Fig. 7). For this purpose, the last 6 hours of MLC incubation was performed in the presence of a protein transport inhibitor (brefeldin). Compared to the control in culture with aDC1, the CD3+IFNy+T-cells (Th1) content increased by an average of 6.2-fold, and CD3+IL-4+T-cells (Th2) increased 4.1-fold, and in culture with sDC - by 1.5fold and 1.1-fold, respectively.
Proliferative response of T-cells in MLC
One of the integral indicators of functional activity of DC is their ability to stimulate the proliferative response of autologous T-cells in MLC, which is associated with the degree of maturity of the DC, as well as the spectrum and level of cytokines produced by them. Two different populations of DC (sDC and aDC1) and T-cells from the same donor (autologous) were used to formulate MLC to exclude possible differences associated with the expression of HLA antigens.
Figure 7. Gating strategy for T-cell subpopulations expressing intracellular cytokines IFNy and IL-4 in response to PMC incubation in mixed culture with DC (sDC and aDC1) by a representative example of one donor
Figure 8. Gating strategy for proliferating T-cells (A) in different phases of mitosis and the corresponding percentage (B); (*, p < 0.05; **, p < 0.01)
The results of the study indicate that there is a +G2/M) was less than 20%. In response to the pres-
pronounced effect of DC on the proliferation of au- ence of DC in MLC, after 72 hours of culture, the
tologous T-cells. In the absence of DC (control), the formation of clones of daughter cells was observed,
proportion of proliferating lymphocytes (S-phase + the proportion of which in the S-phase and G2/M
for aDC1 was 50.30% and 26.4%, respectively, and for sDC38.4% and 18.2% (Fig. 8).
The data obtained indicate that aDC1 in MLC possessed a more pronounced ability to stimulate the proliferative response of T-cells.
Discussion
Our attempt to improve the ability of DC to induce an antitumor response motivated us to develop a protocol for DC serum-free cultivation in an optimized cytokine environment that allows achieving the key properties of DC necessary for high vaccine efficiency: 1) fully mature DC status; 2) high expression of IL-12p70; 3) responsiveness to chemokines of secondary lymphoid organs.
To attract and interact DC with T-cell subpopulations (naive and central memory), the stability of CCL19 products matured at the periphery of the DC after their migration to regional lymph nodes is of great importance. The data obtained demonstrated that PGE2 acts as a potent inhibitor of the production of chemokine CCL19 in the culture of sDC. At the same time, the ability for increased production of CCL19 in the aDC1 population quickly disappeared as soon as it was transferred from the maturation medium to the neutral medium. However, in response to CD40L stimulation of aDC1, in contrast to sDC, repeated chemokine-inducing signals were obtained that led to a second wave of increased production of CCL19.
It is shown that PGE2 inhibits the proliferation of T-cells, directly and indirectly affects the secretion of proinflammatory and antitumor cytokines, and also facilitates the interaction of DC with regulatory T-cells, enhancing their proliferation. However, it was paradoxical that PGE2 promotes the enhancement of CCR7 expression and thereby the chemotaxis of DC in response to chemokines CCL19 and CCL21. The latter provide directional migration of antigen-primed DCs to the draining lymph nodes, where they interact with T-cells (naive and central memory cells). This attractive property of PGE2 justifies its inclusion in the com-
position of standard cytokine cocktails for the maturation of dendritic cell vaccines.
This study attempts to explain this paradoxical property of PGE2 to cause increased expression of CCR7 on the sDC surface and simultaneously to reduce the production of CCL19. It is known that in high concentrations CCL19 induces ligand-depen-dent internalization of CCR7 and its disappearance from the cell surface. For this reason, the addition of exogenous CCL19 to sDC culture resulted in a dramatic decrease in the surface expression of CCR7. However, the effect of exogenous CCL19 did not alter the level of total (surface and intracellular) expression of CCR7 in the sDC population. It appears that PGE2 inhibits the secretion of CCL19 and, as a consequence, ligand-dependent internalization of the CCR7 receptor, which explains its increased surface expression on sDC. It is important to note that the differences in the surface expression of CCR7 on sD C and aD C1 disappeared rapidly after their transfer from the maturing medium to the neutral one.
The weak migration capacity of aDC1 in vitro was due to the low expression of CCR7, since in the conditions of a maturing medium they actively secrete CCL19, which leads to internalization of this receptor. However, the migration activity of aDC1 was rapidly restored, to a level no lower than that of sDC, after their transfer to a neutral medium that did not contain CCL19.
The results obtained do not abolish the possible benefits of using PGE2, which have been shown in a large number of other studies [18]. In either case, the potential benefits of using PGE2 adversely affect the ability of the DC to secrete CCL19 necessary to attract T-cells. In addition, PGE2 is able to locally program the DC for preferential interaction with Treg, which together can explain the generalized immunosuppression associated with chronic inflammation and the tumor process.
Specific binding of the antigen peptide in the composition of the molecule of the major histocompatibility complex presented by APC with the T-cell
receptor is the main signal for activation and differentiation of T-cells (signal 1). However, the type and degree of expression of the resulting response of T-cells depends both on the nature of the interaction of costimulatory molecules on APC with their ligands on T-cells (signal 2) and on the secretion of the corresponding cytokines (signal 3) [19].
It is known that the expression of costimulatory molecules from the B7 family (CD80 and CD86) is a decisive event in the maturation of DC [16]. The signal from the interaction of these molecules with ligands (CD28) on the surface of T-cells is important for the initiation of the cell cycle, IL-2 secretion and clonal expansion. At the same time, molecules from the B7 family partially or completely initiate co-inhibitory signals to the T-cells, thereby regulating the degree of expression of the immune response. Comparison of the maturation conditions of DC in a large number of donors made it possible to demonstrate that a positive costimulatory profile predominated in aDC1, while sDC demonstrated a stronger expression of the co-inhibitory molecule (CD274 = =PD-L1 = B7-H1) from the B7 family. This need to be taken into account in future when receiving DC for adoptive immunotherapy, especially in connection with the fact that most clinical trials used sDC-based vaccines. The unfavorable balance of costimulatory molecules expressed on DC may be one of the reasons for the unsuccessful results of clinical trials.
In addition to costimulation, cytokines secreted by DC after contact with T-cells (signal 3) are also of paramount importance in the implementation of an adequate antitumor immune response. The ability of DC for the secretion of cytokines was modeled with the help of CD40L, which allows simulating the natural interaction of DC with naive CD4+ T-cells. Secreted by mature DC, IL-12p70 is an essential component of the cytokine environment for inducing polarization of naive CD4+ T-cells in the direction of the Th1 response, while IL-10 is responsible for the development of tolerance. In most recent studies, preference has been given to cultivation techniques
that provide mature DC with the highest potential for secretion of IL-12p70 [6; 7; 11; 17]. These publications report the possibility of reaching the concentration of IL-12p70 in the culture supernatant for maturation of the DC at a level of several tens of thousands of pg/ml [20]. However, when using DC in in vivo conditions, it is more reasonable to analyze the secretion of IL-12p70 not in an artificial maturation medium, but in a neutral medium in response to the interaction of DC with naive T-cells (signal 3). The concentration of IL-12p70 induced under such conditions is significantly lower. Dohnal et al. [21] consider a concentration of 100 pg/ml as the lower limit for the use of DC as an antitumoral vaccine, but the real value needed to achieve Th1 of the T-cell response in vivo and clinical efficacy remains unknown. According to our results, sDC in response to stimulation of CD40L in vitro produces a very small amount of IL-12p70, while the secretion of this interleukin aD C1 is many times higher than the above threshold of 100 pg/ml. The level of IL-10 in supernatants of both DC populations was extremely low. The ratio of concentrations of IL-12p70/IL-10, characterizing the "strength" of the Th1-polarizing ability of the DC, was maximal in the aDC1 population.
Stimulating ability of DC for autologous CD4+-T-cells was also examined in terms of their ability to induce regulatory and activated T-cells under autologous MLC. Despite the absence of a significant difference in the total number of CD4+ C25+ cells, the subdivision of this heterogeneous population by the degree of expression of CD127 and FoxP3 showed that sDC mainly induces proliferation of regulatory T-cells, while aDC1 - of non-regulable activated T-cells. The study of such opposite effects of DC on the activation of autologous T-cells is of paramount importance for achieving adequate immunotherapy.
When studying the potential of DC to direct the development of T-cells towards Th1, Th2 or Th17 polarization in the supernatant of MLC with aDC1, we detected a high concentration of IFNy. This effect with respect to aDC1 was expected, since
IL-12p70 is a powerful inducer of Th1 polarization. At the same time, the concentration of IFNg in MLC with sDC was also sighificantly increased, although the latter weakly secreted IL-12p70. An explanation for this may be a highly positive, although different in severity, profile of the expression of costimulatory molecules CD80 and CD86 in both DC populations.
The use of the TLR (LPS, poly-I: C, R848) in the cocktail for maturation of interferons and li-gands provided not only the mature phenotype aDC1, but also the predominance of costimula-tory molecules over co-inhibitory, high secretion
of IL-12p70, but not IL-10, in response to CD40L stimulation, active directed migration, and proliferation of a significant number of CTL in vitro. In contrast to standard DC-based vaccines, which are based on the use of either insufficiently mature DC (highly productive for IL-12 but with low migration and stimulatory activity) or mature DC (with high migration and stimulatory activity but with reduced production of IL-12), the protocol developed in this study makes it possible to obtain a culture of aDC1 cells of which are simultaneously combine all the necessary, often contradictory, properties for antitumor vaccines.
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