[The treatment by expanded ex vivo autologous regulatory T-cells CD4+CD25+FoxP3+CD127low restores the balance of immune system in patients with remitting-relapsing multiple sclerosis]. / Lechenie vyrashchennymi ex vivo autologichnymi regulyatornymi T-kletkami CD4+CD25+FOXP3+CD127low vosstanavlivaet balans immunnoi sistemy patsientov s remittiruyushchim rasseyannym sklerozom.

AIM: To assess clinical efficacy and safety of the autologous (own) regulatory T-cells (Tregs)CD4+CD25+Foxp3+CD127low isolated from the blood of patients with remitting-relapsing multiple sclerosis. Patients with autoimmune diseases have the decreased number of peripheral Tregs (pTreg) and impaired suppressive ability. In order to restore levels of pTreg, it is possible to isolate precursor cells, enter expanded ex vivo autologous Treg cells and introduce an expanded amount of autologous cells as Treg vaccine. MATERIAL AND METHODS: A method of ex vivo Tregs expansion by 30-40 times within 5-7 days has been developed. Expanded ex vivo Tregs are more than 90% CD4+CD25+Foxp3+CD127low and have high suppressor activity. Fourteen patients with remitting-relapsing multiple sclerosis were included in pilot studies.Ex vivoTregs were introduced subcutaneously in dosefrom 2.8 to 4.5 108 cell per injection. The duration of follow-up was 1 year. RESULTS AND CONCLUSION: The numbers of pTregs in the blood of these patients elevated by 1.5-2 times. No adverse-effects, a decrease of relapses and stabilization of disability index were observed. It has been suggested that ex vivo expanded Tregs can compensate the impaired function of pTregs and can be used for adoptive immunotherapyof multiple sclerosis.

[Clinical significance of T-regulatory cells in multiple sclerosis].

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system that develops due to the activation of selfreactive T-cells specific for myelin components. Regulatory T-cells CD4+CD25+Foxp3+ (T-reg) play an important role in the autoimmunity control and inhibition of T-cells-mediated myelin destruction. The aim of the study was to determine a number of T-reg in the blood of patients in different stages of disease, to evaluate their functional activity and to obtain T-reg induced ex vivo. The phenotypic analysis revealed the 2-3 fold reduction of T-reg in the exacerbation phase of MS and the increase of their content in the remission while the level of T-reg in the donor's blood remained significantly higher. The inverse correlation between the degree of severity and duration of MS and the number of T-reg was found. The suppression function of T-reg of MS patients was substantially decreased in the exacerbation and remission stages. The induction of ex vivo T-reg allows to increase the number of these cells by 30-90 times during 6-8 days. The induced T-reg have phenotypic and functional characteristics of native T-reg. The adaptive cell treatment using induced T-reg may become an instrument for correction of immune dysfunction in MS.

[The role of regulatory T cells in the development of autoimmune process in multiple sclerosis].

In the maintenance of immunological tolerance important role belongs to the recently discovered population of regulatory T-cells CD4+CD25+FoxP3 +. These cells have potential in suppressing pathologic immune responses observed at various autoimmune diseases including multiple sclerosis. We have shown a reduction in the number and functional activity of T-reg in peripheral blood of patients with multiple sclerosis in the acute stage, the increase in their number during remission, duration of the relationship of the autoimmune process and the degree of disability of patients with the contents of T-reg. The possibility of using the grown ex vivo T-reg for the correction of immunopathological process in multiple sclerosis.

[The role of regulatory cells CD4+CD25+ in the development of chronic infective diseases].

Regulatory T-cells CD4+CD25+Foxp3 (Treg) present a small subpopulation of CD4+ T lymphocytes, which develop in the thymus and are disseminated into peripheral lymphoid organs on the 3rd or the 4th day of the neonatal period. Treg play a decisive role in the pathogenesis of autoimmune diseases and the development of tolerance to transplantation antigens, regulate the immune response to allergens, and suppress antimicrobial immunity. Treg suppress proliferation as well as the cytotoxic effect and the secretion of Th1 and Th2 cytokines by effectory T lymphocytes, thus limiting the strength of the immune response of effectory T-cells, which makes them impossible to adequately control viral and bacterial infections. Recognition via antigen-presenting cells and the subsequent induction of the proliferation of antigen-reactive T and B lymphocytes, directed towards infectious agent elimination, is accompanied by the activation of regulatory T cells as well, which leads to immune response suppression; repeated microbial infections are not only able to strengthen T-cell immunity by generating memory T-cells, but can also strengthen the suppressive activity of endogenous T regulators CD4+CD25+. Moreover, T reg are capable of the direct recognition of a microbial product; these cells selectively express Toll-like receptors (TLR)-2, -4, -5, -7, and -8. Under normal conditions T reg are anergic, but are capable of direct proliferation in response to stimulation by TLR ligands, expressed on microbes and parasites. Treg removal enforces protective immune response to contagious microorganisms, including bacteria, viruses, and fungi, which leads to the elimination of pathogens from the host organism. The removal of Treg population will help to accomplish infectious pathogen elimination and diminish inflammation within a short period of time.

[The role of regulatory T-cells in autoimmune rheumatic diseases].

Regulatory T-cells CD4+CD25+Foxp3, possessing suppressory activity, play the key role in the development of autoimmune diseases, maintenance of peripheral tolerance in transplantation immunity, and the prevention of a pathological immune response to intestinal microflora or microbial infection. A decrease in the total number of circulating CD4+CD25+ regulatory T-cells and their suppressive activity have been found in patients suffering from various autoimmune diseases, such as type 1 diabetes, multiple sclerosis, autoimmune hepatitis, psoriatic arthritis, juvenile idiopathic arthritis, and systemic lupus erythematosus (SLE). The authors of this study investigated the phenotypic characteristics of peripheral blood lymphocytes in 31 SLE patients and the effect of treatment on the content of CD4+CD25+ T-cells before and after pulse therapy with methylprednisolone and cyclophosphan. The total number of regulatory T-cells in the group of untreated patients was almost twice lower vs. the group of healthy donors. As a result of the therapy, the proportion of regulatory T-cells increased significantly, although it did not reach the values in the control group. The data from this research confirm the development of a defect of CD4+CD25+ T-cells at the active phase of SLE, and a possibility to partially correct this defect with an effective therapy.

Human small cell lung carcinoma and carcinoid tumor regulate dendritic cell maturation and function.

The induction of apoptosis in dendritic cells (DC) is a key mechanism by which tumors escape immune recognition and elimination. In fact, a number of studies have showed the correlation between the number of DC within the tumor and the clinical prognosis, suggesting that increased infiltration of tumor tissue by DC was associated with better patient survival and low incidence of metastatic disease. We compared the number of DC and their distribution pattern in human small-cell lung carcinoma and bronchial carcinoid tumor (CT) tissues. Immunohistochemical analysis revealed the presence of cells expressing DC markers CD1a and CD83 in small-cell lung carcinoma tissues and the complete absence of these cells in CT samples. Next, we examined whether human lung tumor cells produce soluble factors that inhibit differentiation of hematopoietic precursors into mature DC. The addition of small-cell lung carcinoma-conditioned medium to CD34+ precursor cell cultures significantly inhibited colony-forming units of DC formation when compared with nontreated control DC cultures. Furthermore, DC generation and differentiation was completely abrogated in CD34+ cell cultures treated with CT-conditioned medium, suggesting that CT-derived factors blocked CD34+ cell differentiation into DC or induced their apoptosis. Finally, flow cytometry analysis of cultured DC confirmed these results. Thus, analysis of our data suggests that human lung tumors produce factors that inhibit DC generation or maturation and may also induce apoptotic death of DC precursors in vitro.

Cytokine-mediated protection of human dendritic cells from prostate cancer-induced apoptosis is regulated by the Bcl-2 family of proteins.

Prostate cancer is the most common cancer in men in the United States, and second in cancer-induced mortality. It is likely that tumour-induced immunosuppression is one of the reasons for low treatment efficacy in patients with advanced prostate cancer. It has been recently demonstrated that prostate cancer tissue is almost devoid of dendritic cells (DC), the major antigen-presenting cells responsible for the induction of specific antitumour immune responses. In this study, we have tested the hypothesis that prostate cancer induces progressive suppression of the DC system. We found that co-incubation of human DC with three prostate cancer cell lines led to the high levels of premature apoptosis of DC, which were significantly higher than in DC cultures co-incubated with normal prostate cells or blood leucocytes. Stimulation of DC for 24 hours with CD40 ligand (CD154), IL-12 or IL-15 prior to their co-incubation with prostate cancer cells resulted in a significant increase in DC survival in the tumour microenvironment. Furthermore, activation of DC with these cytokines was also accompanied by increased expression of the anti-apoptotic protein Bcl-x(L) in DC, suggesting a possible mechanism involved in DC protection from apoptotic death. In summary, our data demonstrate that prostate cancer induces active elimination of DC in the tumour microenvironment. Stimulation of DC by CD154, IL-12 or IL-15 leads to an increased expression of the anti-apoptotic protein Bcl-x(L) and increased resistance of DC to prostate cancer-induced apoptosis. These results suggest a new mechanism of tumour escape from immune recognition and demonstrate the cytokine-based approaches which might significantly increase the efficacy of DC-based therapies for cancer.

Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6.

Defensins contribute to host defense by disrupting the cytoplasmic membrane of microorganisms. This report shows that human beta-defensins are also chemotactic for immature dendritic cells and memory T cells. Human beta-defensin was selectively chemotactic for cells stably transfected to express human CCR6, a chemokine receptor preferentially expressed by immature dendritic cells and memory T cells. The beta-defensin-induced chemotaxis was sensitive to pertussis toxin and inhibited by antibodies to CCR6. The binding of iodinated LARC, the chemokine ligand for CCR6, to CCR6-transfected cells was competitively displaced by beta-defensin. Thus, beta-defensins may promote adaptive immune responses by recruiting dendritic and T cells to the site of microbial invasion through interaction with CCR6.

The generation of human dendritic and NK cells from hemopoietic progenitors induced by interleukin-15.

Interleukin-15 (IL-15) is a pleiotropic cytokine that induces the generation and differentiation of lymphoid cells and shares many biological activities with IL-2. We have shown here the development of dendritic cells (DC) from human CD34+ hemopoietic precursor cells cultured for 2-4 weeks with IL-15 alone. DC generated with IL-15 have typical morphological, immunocytochemical, phenotypic, and functional characteristics of mature DC. Dual flow cytometry analysis performed weekly demonstrated increasing co-expression of CD1a or CD83 with HLA-DR, CD80, CD86, IL-2R alpha, beta, and gamma. Two populations of cells were distinguished among CD34+ progeny. Small and medium-size cells were mainly natural killer (NK) cells (72.6-85.2% CD56+) and low numbers of DC (9.1-21.3% CD1a+). Large cells were mostly DC (75.4-95.4% CD1a+). Isolated CD34+ cells did not express IL-2R subunits but after 2-3 days in culture with IL-15, they were found to express IL-2Rgamma. Induced expression of IL-2Rgamma on CD34+ cells may explain the primary mechanism of IL-15-regulated differentiation of hemopoietic precursor cells. Thus, our data suggest that IL-15 stimulates CD34+ cells to differentiate into NK and DC and may represent a new growth and survival factor for lymphoid DC.

[The effect of a clover extract on the proliferation of human and murine lymphocytes in vitro]. / Vliianie ékstrakta klevera na proliferatsiiu limfotsitov cheloveka i myshi in vitro.

The extracts were prepared from meadow clover harvested at the stages of blossoming and budding. The major biological activity of such extracts is represented by flavonoid compounds. The influence of extracts on the proliferation of peripheral blood mononuclear cells obtained from healthy donors and of inbred mouse splenocytes in vitro was analyzed. Both preparations stimulated cellular proliferation. The lever of stimulating activity correlated with the stage-dependent concentration of flavonoids.

[The prophylactic administration of interleukin-2 increases the survival of mice with an acute intra-abdominal infection caused by Staphylococcus aureus]. / Profilakticheskoe vvedenie interleikina-2 uvelichivaet vyzhivaemost' myshei s ostroi vnutribriushnoi infektsiei, vyzvannoi Staphylococcus aureus.

The effect of RIL-2 on the survival of mice with acute Staphylococcus aureus strain 5/2 intra-abdominal [correction of intraperitoneal] infection was studied. RIL-2 was ineffective when administered simultaneously with the LD100 dose of bacteria. Antibiotics (gentamycin or combination of penicillin and streptomycin) administered in the same fashion cured 100% of animals. However, RIL-2 proved to be effective when administered simultaneously with LD70 dose of bacteria. The prophylactic course of RIL-2 consisting of repeated injections on days 3, 2 and 1 before the challenge with LD100 dose of bacteria also resulted in the marked increase of the survival of mice. The hypothetical mechanisms of action and the prospects of RIL-2 application are discussed.

[The effect of recombinant interleukin-2 on the course of experimental staphylococcal peritonitis in mice]. / Vliianie rekombinantnogo interleikina-2 na techenie éksperimental'nogo stafilokokkovogo peritonita u myshei.

The effect of RIL-2 on the survival of mice with S. aureus--induced peritonitis was studied. Animals received bacterial suspension and RIL-2 as following: bacteria--on days 0, +2, RIL-2--day 0 (group 1); bacteria--days 0, +4, RIL-2--days 0, +2 (group 2); bacteria--days 0, +6, RIL-2--days 0, +2, +4 (group 3). RIL-2 exerted no protective effect in group 1. However, in groups 2 and 3, where the control animals survival was, resp., 56% and 38%, the RIL-2 treatment increased survival up to, resp., 84% and 70%. Antibiotics given instead of RIL-2 in analogous regimen decreased the survival in group 3 to the level of 25%. Thus, RIL-2 proved to be a potent therapeutic agent in the 2nd of 3d studied models of S. aureus--induced peritonitis in mice. The perspectives of RIL-2 use in the treatment of bacterial peritonitis, including porous ones, and of the immunodepression--aggravated conditions are discussed.

[A comparison of the suppressor and cytotoxic activities of the blood mononuclear cells during the adoptive immunotherapy of cancer patients using lymphokine-activated killers with a low dose of recombinant interleukin-2]. / Sravnenie supressornoi i tsitotoksicheskoi aktivnosti mononuklearnykh kletok krovi pri adoptivnoi immunoterapii onkologicheskikh bol'nykh limfokinaktivirovannymi killerami s nizkoi dozoi rekombinantnogo interleikina-2.

The suppressor and cytotoxic activities of mononuclear blood cells (MNC) were studied in 70 cancer patients (melanoma, renal carcinoma) undergoing adoptive immunotherapy (AIT). In the course of AIT the patients' MNC were treated in vitro with the recombinant interleukin-2 (RIL-2) in order to generate the lymphokine-activated killer (LAK) cells. Then patients received i/v 2.5-13.6 10(9) autologous LAK cells and RIL-2 (75000 u). Each course included 2-3 repeated infusions; the patients received 1-5 courses according to their clinical conditions. The cytotoxic activity of MNC was assessed by a routine method; but for evaluation of the suppressor activity we used a new technique based on separation of MNS populations in the Percoll gradient. Twenty-four hours after the completion of each AIT course the suppressor activity of MNC decreased drastically up to the zero level in some patients. The decrease in the suppressor activity inversely correlated with the rise in the cytotoxic activity on Mel-I (LAK-sensitive) and K-562 (natural killer-sensitive) target cells. The level of cytotoxicity in some patients reached 51.2%.

[Influence of recombinant interleukin-2 on the course and pathomorphology of intraperitoneal staphylococcal infection in mice]. / Vliianie rekombinantnogo interleikina-2 na techenie i patomorfologiiu vnutribriushnoi stafilokokkovoi infektsii myshei.

Resistance to the lethal doses of the infectious agent (3 DL100) develops in mice 6 days after the intraperitoneal non-lethal dose of staphylococcus in combination with recombinant interleukin-2 (RIL-2). This effect may be due to the activation of T- and B-dependent zones of the regional lymph nodes and spleen, activation and proliferation of the liver stellate reticulo-endotheliocytes, enhancement of the phagocytic activity of the circulating neutrophil granulocytes. These structural and functional mechanisms may be due to both direct RIL-2 effect in combination with an antigenic stimulation and indirect effect through other interleukins produced by lymphocytes activated by RIL-2.