Search

Background. Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disease (GVHD) is often accompanied by severe side effects such as opportunistic infections and cancers. Several approaches have been developed to suppress transplantation reactions using tolerogenic cells, including induction of FoxP3+ Tregs with antigen-loaded dendritic cells (DCs) and induction of CD4+IL-10+ cells with interleukin IL-10-producing DCs. Here, we assessed the effectiveness of both approaches in the suppression of graft rejection and GVHD. Methods. IL-10-producing DCs were generated by the transfection of DCs with DNA constructs encoding mouse IL-10. Antigen-loaded DCs from C57BL/6 mice were generated by transfection with DNA constructs encoding antigenic determinants from the H2 locus of CBA mice which differ from the homologous antigenic determinants of C57BL/6 mice. Results. We found that both IL-10-producing DCs and antigen-loaded immature DCs could suppress graft rejection and GVHD but through distinct nonspecific and antigen-specific mechanisms, respectively. Discussion. We provide data that the novel approach for DCs antigen loading using DNA constructs encoding distinct homologous determinants derived from major histocompatibility complex genes is effective in antigen-specific suppression of transplantation reactions. Such an approach eliminates the necessity of donor material use and may be useful in immunosuppressive therapy side effects prevention.

Melanoma is a tumor that forms as a result of malignant transformation of melanin-producing pigment cells (melanocytes). It is the most aggressive form of skin cancer and is characterized by high resistance to chemotherapeutic drugs, which results in a need to explore alternative methods for this disease therapy. Currently, new approaches to cancer treatment are being intensely developed, oncolytic immunotherapy being one of them. This approach consists in using viruses as targeted tumor-specific cytolytic agents capable of stimulating both tumor-specific and nonspecific immune responses. A considerable body of research is currently aimed on improving the immunostimulatory properties of viruses by inserting the genes encoding immunomodulatory proteins or tumor-specific antigenic determinants into viral genomes. For melanoma, the highest number of tumor-associated antigens (TAAs) has been identified, which serve as the basis for the development of anti-tumor DNA vaccines. The immunogenicity and efficacy of these drugs, however, remain low. The bottlenecks in using DNA vaccines to treat cancer are considered to be imperfect design of polyepitope constructs, as well as inefficient delivery of therapeutic molecules directly to the target cells. A partial solution to these problems may be represented by the use of oncolytic viruses as vectors for the delivery of artificial immunogens. The recombinant vaccinia virus was obtained by transient dominant selection. The cytolytic activity of the obtained virus was tested using the MTT assay. The oncolytic activity of the virus was assessed in the mouse xenograft model obtained using malignant SK-Mel-28 cells. This paper reports the production of a recombinant L-IVP_oncoM virus, the oncolytic virus for the delivery of anticancer therapeutic genes into the cells, on the basis of the vaccinia virus strain L-IVP. Toward this end, the gene encoding the Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the artificial gene encoding a polyepitope immunogen containing the epitopes of the antigens over-expressed in melanoma cells were inserted into the virus genome. These insertions were located in the proximity of the genes encoding thymidine kinase (J2R) and viral growth factor (C11L), respectively. The properties of L-IVP_oncoM were studied in in vitro experiments using cell cultures of various origin and in the in vivo experiments using the mouse xenograft model. The basic experiments to assess the biological properties of the obtained L-IVP_oncoM, which are necessary to characterize the oncolytic virus, have been carried out.

Tolerogenic dendritic cells (tolDCs) and T-regulatory cells (Tregs) are involved in maintaining tolerance to self-antigens and foreign antigens. The cells are used as therapeutic tools for inducing tolerance to transplanted organs or tissues. We investigated the possibility of inducing Tregs in splenocyte cultures using DCs transfected with a DNA construct encoding mouse interleukin-10 (DCpIL-10). DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4 and electroporated with a plasmid encoding mouse IL-10. Furthermore, DCpIL-10 was cocultured with syngeneic splenocytes. The CD4+CD25hiFoxP3+ Treg frequency, IL-10 expression, and inhibition of the mixed lymphocyte reaction were evaluated. C57Bl/6 and CBA mice differ in their initial frequency of CD4+CD25hiFoxP3+ Tregs and baseline IL-10 production. Also, the effectiveness of CD4+CD25hiFoxP3+ Treg upregulation by tolDCpIL-10 was different. In this study, DCpIL-10 from C57Bl/6 mice induced CD4+CD25hiFoxP3+ Tregs in syngenic splenocytes, which was accompanied by an increase in the IL-10 production and a decrease in the proliferation of splenocytes in response to the alloantigen. DCpIL-10 may be used to induce CD4+CD25hiFoxP3+ Tregs and the regulatory potential of splenocytes.

Background B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection and central tolerance induction by the capturing of self-antigens in peripheral tissues and further migration to the thymus using the CCL25-CCR9 chemotaxis axis. Aim Here we investigate the possibility of DCs migration stimulation to the thymus by the transfection with plasmid DNA-constructs encoding CCR9(pmaxCCR9) to develop a system for desired antigen delivery to the thymus for central tolerance induction. Methods Dendritic cells(DCs) cultures were generated from UBC-GFP mice bone marrow cells expressing green fluorescent protein using the rmFlt3-L. DCs cultures were transfected with pmaxCCR9 by electroporation. The efficiency of electroporation was confirmed by RT-qPCR and flow cytometry. The migration of electroporated DCs was assessed in vitro and in vivo. Results Dendritic cells(DCs) cultures obtained from UBC-GFP mice contained both B220+pDCs and SIRPa+cDC2. According to the RT-qPCR assay, the electroporation of obtained DCs cultures with pmaxCCR9 resulted in a 94.4-fold increase of RNA encoding CCR9 compared with non-electroporated cultures. Flow cytometry data showed that DCs cultures electroporated with pmaxCCR9 contained a significantly higher frequency of DCs carrying significantly higher levels of surface CCR9. Migration dynamics of obtained DCs analyzed in vitro showed that pmaxCCR9 electroporated DCs migrated significantly more active to CCL25 and thymic cells than non-electroporated and mock-electroporated DCs. In vivo, 30 days after injection, the relative amount of the DCs electroporated with pmaxCCR9 and pmaxMHC encoding antigenic determinants in the mice thymuses was 2.02-fold higher than the relative amount of the DCs electroporated with control plasmid. Conclusion Thus, the electroporation of murine DCs with pmaxCCR9 stimulated its migration to CCL25 and thymic cells in vitro as well as to the thymus in vivo. The obtained DCs loaded with a desired antigen may be suggested for further evaluation of central tolerance induction ability in in vivo models of autoimmune diseases and transplantation.

Background Recent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patient’s body. Cytotoxic T lymphocytes (CTLs) are considered the main effectors in cell-mediated antitumor immunity. Approaches based on antigen presentation to CTLs by dendritic cells (DCs) are currently being intensively studied, because DCs are more efficient in tumor antigen presentation to T cells through their initiation of strong specific antitumor immune responses than other types of antigen-presenting cells. Today, it has become possible to isolate CTLs specific for certain antigenic determinants from heterogeneous populations of mononuclear cells. This enables direct and specific cell-mediated immune responses against cells carrying certain antigens. The aim of the present study was to develop an optimized protocol for generating CTL populations specific for epitopes of tumor-associated antigen HER2/neu, and to assess their cytotoxic effects against the HER2/neu-expressing MCF-7 tumor cell line. Methods The developed protocol included sequential stages of obtaining mature DCs from PBMCs from HLA A*02-positive healthy donors, magnet-assisted transfection of mature DCs with the pMax plasmid encoding immunogenic peptides HER2 p369–377 (E75 peptide) and HER2 p689–697 (E88 peptide), coculture of antigen-activated DCs with autologous lymphocytes, magnetic-activated sorting of CTLs specific to HER2 epitopes, and stimulation of isolated CTLs with cytokines (IL-2, IL-7, and IL-15). Results The resulting CTL populations were characterized by high contents of CD8+ cells (71.5% in cultures of E88-specific T cells and 90.2% in cultures of E75-specific T cells) and displayed strong cytotoxic effects against the MCF-7 cell line (percentages of damaged tumor cells in samples under investigation were 60.2 and 65.7% for E88- and E75-specific T cells, respectively; level of spontaneous death of target cells was 17.9%). Conclusions The developed protocol improves the efficiency of obtaining HER2/neu-specific CTLs and can be further used to obtain cell-based vaccines for eradicating targeted tumor cells to prevent tumor recurrence after the major tumor burden has been eliminated and preventing metastasis in patients with HER2-overexpressing tumors. Electronic supplementary material The online version of this article (doi:10.1186/s12865-017-0219-7) contains supplementary material, which is available to authorized users.

Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

A method for identification of fragments with high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions whose fragments are frequent in human proteins, and regions exhibiting high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV infection either through induction of cross-reacting effectors of the immune system or through molecular mimicry of physiologically important human proteins, leading to alteration of homeostasis of the organism, is discussed. Most of the regions identified in HIV-1 proteins contain either T-cell (CD8+ CTL or CD4+ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines that would allow exclusion of epitopes with (immuno)pathogenic potential are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions whose fragments are frequent in human proteins, as well as of regions exhibiting pronounced local similarity to proteins that provide for important physiological functions.

Detailed studies of the pathogenesis of certain neuroviral infections allow for a better understanding of the special role of the olfactory neuroepithelial cells in the invasion of viruses into the CNS. Several studies using animal models demonstrated that neurotropic viruses belonging to various families invade the brain via the olfactory tract after parenteral infection. We suppose that intranasal (i.n.) immunization inducing mucosal and systemic immunity will block neurotropic virus propagation into the brain via the olfactory pathway and neutralize virus multiplication in visceral organs. Subject of the present study was the efficacy of i.n. immunization of Balb/c mice with both killed tick-borne encephalitis (TBE) virus and antigenic peptide 89–119 from the envelope protein E of TBE virus in a nanoemulsion formulation. Intranasal immunization with nanoemulsion containing inactivated TBE virus particles induced specific both neutralizing and HAI antibodies. TBE virus specific IgA antibodies were detected in lung and nasal lavages of mice. The fourth i.n. immunization resulted in a drastic titer increase of the specific antibodies to 1:12,800, which was protective in all i.n. immunized mice against intraperitoneal (i.p.) challenge with 100 LD50 of TBE virus. The ratio of specific IgG2a to IgG1 indicated the occurrence of a Th2 type immune response. We observed a similar balance of TBE virus-specific IgG2a/IgG1 after s.c. immunization of mice with the commercial FSME-Immun Inject® vaccine against TBE virus. Thus, the experimental data obtained for the first time demonstrates the feasibility of using nanoparticles containing inactivated TBE virus for effective protection of i.n. immunized mice against the usually lethal infection. We analyzed a number of fragments of E protein of the TBE virus for antigenic similarity with human proteins by computer analysis. Local antigenic similarity of the fragments of E protein of TBE virus and human proteins allows for the identification of the epitopes, which may induce an autoimmune response if applied in a vaccine construct. One of the candidates, peptide 89–119 of E protein of the TBE virus does not contain any epitopes with local similarities to human protein epitopes. This peptide was synthesized and applied intranasally in nanoparticulate formulation. It induced TBE virus-specific antibodies and led to protection in the case of i.p. challenge with TBE virus.