Mechanisms Regulating Natural Suppressor Cell Activity under Normal Conditions and During Tumor Growth

The dissertation investigates the mechanisms regulating the immunosuppressive and antitumor activities of natural suppressor cells under normal conditions and in various models of tumor growth. Particular attention is paid to the role of nitric oxide, the effects of tumor cells and T lymphocytes, and the dependence of natural suppressor cell activity on cellular origin, the content of immature hematopoietic cells, and the properties of target cells.

The study considers bone marrow cells, embryonic liver cells, and other myelocaryocyte populations. It examines transplanted, spontaneous, chemically induced, and virus-induced models of tumor growth, as well as responses under different forms of antigenic stimulation in vitro. The work compares immunosuppressive and antitumor effects, identifies the soluble factors and contact interactions involved, and examines the relationship between natural suppressor cell activity and the functional state of T lymphocytes.

As is well known, a defective immune response, manifested in inadequate initiation and, consequently, in the development of immunosuppression, is one of the important pathogenetic factors of tumor growth. It has been shown that tumor cells express so-called tumor-associated antigens [Whiteside T.L., 2000], and when these are recognized adequately, the immune system can successfully prevent tumor growth or induce its regression [Cheever M.A. et al., 1995; Bocchia M. et al., 1996; Boon T. et al., 1996]. The mechanisms by which tumors escape immunological surveillance are diverse. By producing immunosuppressive cytokines (TGF-β, prostaglandins, nitric oxide, IL-10, alpha-2-macroglobulin, vascular endothelial growth factor [VEGF]), tumor cells affect both T lymphocytes and cells of the microenvironment [Куртенков О.А. et al., 1983; Ключарева Т.Е. et al., 1988; Black R.N. et al., 1980; Ladisch S. et al., 1983; Miescher S. et al., 1986; Cukrova V. et al., 1987; Miescher S. et al., 1988; Ebert E.C. et al., 1990; Roszman T. et al., 1991; Tada T. et al., 1991; Grégoire M. et al., 1992; Yoshino I. et al., 1992; Alexander J.P. et al., 1993; Lalim H. et al., 1993; Alleva D.G. et al., 1994; Chen Q. et al., 1994; Lieubeau B. et al., 1994; Hersh E.M. et al., 1995; Weller M. et al., 1995; Lagadec P. et al., 1996; Tsushima H. et al., 1996; Wojtowicz-Praga S. et al., 1997; Santin A.D. et al., 1999; Zou J.P. et al., 1999; Sharma S. et al., 1999; Kudo D., 2003; Abrahams V.M. et al., 2003; Andreola G. et al., 2002; Shurin G.V. et al., 2001; Peguet-Navarro J. et al., 2003; Mullins D.W. et al., 2003].

The initiation and development of an immune response depend substantially on the microenvironment of immunocompetent cells, and cytokines play a key role in regulating this process. Certain hematopoietic cells—one of the important sources of immunoregulatory substances (NO, TGF-β, IL-10, and others)—are natural suppressor cells (NSCs) [Moore S.C. et al., 1992a; Belsky Y.P. et al., 1993; Angulo I. et al., 1995; Kusmartsev S.A. et al., 1995; Sennikov S.V. et al., 1996; DeKoter R.P. et al., 1997; Seledtsova G.V. et al., 1997; Yanagie H. et al., 1997; Mori T. et al., 1998; Sennikov S.V. et al., 2001]. They represent immature cells of various hematopoietic lineages. NSCs are an extremely heterogeneous population of cells possessing natural suppressor activity, that is, the ability to suppress the proliferation of immunocompetent cells nonspecifically (without prior contact with targets and without restriction by histocompatibility-complex antigens) [Michelson J.D. et al., 1988; Noga S.J. et al., 1988; Saffian D.C. et al., 1991; Hoskin D.W. et al., 1992; Sugiura K. et al., 1992].

NSCs may belong to the granulocyte-macrophage, erythroid, or lymphoid lineage [Чеглякова В.В., 1989; Sugiura K., 1992; Brooks-Kaiser J.C., 1993; Кусмарцев С.А., 1994; Kusmartsev S.A. et al., 2003b; Bronte V. et al., 2003]. What they share is, first, a null phenotype and, second, pronounced immunosuppressive activity both in vitro and in vivo (they suppress T- and B-lymphocyte responses to antigens and mitogens) [McGarry R.C., 1982]. In addition, NSCs have been shown to possess antitumor activity by inhibiting the proliferation of certain tumor cells in vitro [Seledtsov V.I., 1995]. It should be noted that although the immunosuppressive role of NSCs during tumor growth has been demonstrated, the literature contains no data on their antitumor properties. The significance of the antitumor activity of NSCs in the development of malignant neoplasms therefore remains an open question.

The important role of NSCs in immune regulation has been demonstrated in in vivo studies of tumor growth [Young M., 1994], the graft-versus-host reaction (GVHR) [Hertel-Wulff B., 1987], and immunization with Salmonella typhimurium [Schleifer K.W., 1993]. During certain physiological states (pregnancy) [Brooks-Kaiser J.C., 1993], in disease (tumor growth) [Kusmartsev S.A., 1989; Young M.R., 1996], and in certain experimental models (bone marrow cell transplantation [Imamura M.E., 1986], GVHR [Holda J.H., 1988], and cyclophosphamide administration [Maier T., 1989]), NSC activity increases substantially. In situations of stimulated hematopoiesis, NSC activity also increases substantially; the cells migrate from the bone marrow to foci of ectopic hematopoiesis, particularly the spleen, where they exert a pronounced immunosuppressive effect.

The mechanism of the immunosuppressive action of NSCs is based on their secretion of soluble mediators—suppressor factors (SFs)—which may include TGF-β, nitric oxide, prostaglandins, certain nucleosides, and other substances, including those not yet identified [Moore S.C., 1992; Angulo I., 1995; Kusmartsev S.A., 1995; DeKoter R.P., 1997; Yanagie H., 1997; Mori T.E., 1998]. Data in the literature on the immunosuppressive factors produced by NSCs during tumor growth are limited and contradictory. TGF-β [Young M.R., 1991] and NO have been identified, in particular, as SFs. It is unclear whether these discrepancies are attributable to specific features of the tumor-growth model used, whether NSCs produce only one SF, or whether the factors change as the tumor grows. The immunosuppressive activity of bone marrow NSCs in intact mice is known to be attributable to nitric oxide production [Angulo I., 1995]. Whether the level of NSC nitric oxide production changes during tumor growth, and whether this depends on the type of carcinogenesis or the dynamics of tumor progression, remains unanswered.

It is known that the tumor itself produces hematopoiesis-stimulating factors (GM-CSF, IL-3), thereby activating NSCs [Young M.R., Newby M. et al., 1987; Young M.R., Wright M.A. et al., 1992; Young M.R. et al., 1996]. It is therefore relevant to study the direct effect of tumor factors on NSC activity and nitric oxide synthesis and to compare the properties of NSCs from tumor-bearing animals with those of NSCs obtained after exposure to factors produced by that tumor.

T lymphocytes have been shown to be important regulators of hematopoiesis [Гольдберг Е.Д. et al., 1982; Гольдберг Е.Д. et al., 1983; Гольдберг Е.Д., Дыгай А.М., 1985; Гольдберг Е.Д. et al., 1988; Дыгай А.М. et al., 1989; Бабаева А.Г., 1990]. Moreover, a T-lymphocyte cytokine, interferon-γ, is an inducer of one of the main suppressor factors (NO) of NSCs. Despite this, researchers have paid insufficient attention to the regulation of NSC activity by T cells. It is relevant to study the effect of T-cell factors on NSCs and to compare the qualitative and quantitative characteristics of NSCs with the functional state of T lymphocytes under different immunization procedures.

The aim of the study is to investigate the mechanisms regulating the immunosuppressive and antitumor activities of natural suppressor cells under normal conditions and during tumor growth. The study is also intended to assess the role of nitric oxide in the manifestation of their antiproliferative activity.

Research tasks:

1. To study the role of nitric oxide in the immunosuppressive and antitumor activities of natural suppressor cells from the principal hematopoietic tissues and in mice of different strains.

2. To compare the contribution of nitric oxide to the immunosuppressive and antitumor activities of natural suppressor cells in certain myelocaryocyte populations with varying contents of immature cells (after removal of mature macrophages, after prolonged cultivation, during recovery of hematopoiesis after cyclophosphamide administration, and in fractions with low buoyant density).

3. To investigate the ability of pathologically altered undifferentiated cells (cells from a number of tumor lines) to exhibit natural suppressor activity.

4. To study the mechanisms of induction and manifestation of antitumor activity depending on the properties of tumor target cells. The role of soluble NSC factors and contact interactions between cells.

5. To study the immunosuppressive and antitumor activities of natural suppressor cells in various models of tumor growth (chemical- and virus-induced carcinogenesis, and transplanted and spontaneous tumor growth).

6. To assess the role of nitric oxide as a factor of natural suppressor cells in the mechanism underlying their immunosuppressive activity in various models of tumor growth (chemical- and virus-induced carcinogenesis, and transplanted and spontaneous tumor growth).

7. To study the relationship between the functional state of T lymphocytes (as reflected by their production of interferon-γ and interleukin-2) and the mechanism underlying the immunosuppressive activity of natural suppressor cells in animals with different types of tumor growth.

8. To study the mechanisms of induction and manifestation of the immunosuppressive and antitumor activities of bone marrow natural suppressor cells under the direct action of factors secreted by tumor-line cells.

9. To study the direct effect of T lymphocytes on the induction of natural suppressor cell activity and the mechanisms underlying its manifestation in vitro. To compare natural suppressor cell activity and the mechanisms underlying its manifestation depending on the functional state of T lymphocytes in in vivo models (the graft-versus-host reaction, allogeneic pregnancy, and the development of an immune response to thymus-dependent antigens).

Statements to Be Defended.

1. Natural suppressor activity is characteristic of blast cells of different origins; it is induced by the target cell itself, and the method of induction determines the mechanism of its manifestation.

2. Immunosuppressive activity induced by lymphocytes is mediated by nitric oxide production. Antitumor activity of natural suppressor cells induced by tumor cells is mediated by factors other than nitric oxide.

3. The following are characteristic features of tumor growth, irrespective of the type of carcinogenesis and histological tumor type:

- quantitative and qualitative changes in the immunosuppressive activity of natural suppressor cells (an increase in its level and the appearance of a factor other than nitric oxide);

- enhanced antitumor activity of natural suppressor cells based on an NO-independent mechanism.

4. Ehrlich carcinoma growth is an atypical model of carcinogenesis: the properties of the cells in this tumor line determine the distinctive properties of NSCs.

5. Depending on their functional state, T lymphocytes induce alternative pathways of natural suppressor cell activation. T lymphocytes with increased interferon-γ production activate natural suppressor cells whose mechanism of immunosuppressive action is determined by nitric oxide; T cells with reduced interferon-γ production induce natural suppressor cells that produce other suppressor factors.

Scientific Novelty. The study identified the mechanisms initiating the immunosuppressive and antitumor activities of natural suppressor cells. It was shown for the first time that the induction of these activities differs and depends on target cells, which determine the mechanism of manifestation of natural suppressor activity through the factors they produce. The study also demonstrated the possibility of selectively regulating either the immunosuppressive or the antitumor activity of natural suppressor cells. It was found that the immunosuppressive activity of natural suppressor cells is substantially attributable to nitric oxide, and its synthesis becomes more pronounced as the content of immature hematopoietic cells in the effector-cell population increases. Antitumor activity also increases with such enrichment, but nitric oxide does not participate in its formation.

It was shown for the first time that tumor cells possess immunosuppressive activity that is identical, in the mechanisms of induction and manifestation, to that of transformed poorly differentiated hematopoietic cells.

It was established that the antitumor activity of hematopoietic cells is manifested toward tumor target cells with different properties and, only to some extent, may be mediated by nitric oxide. Natural suppressor cells exert an antitumor effect through soluble mediators whose secretion is induced by the tumor target cell, including as a result of cell-cell contact between the effector and the target.

Using a large panel of tumor-growth models (chemical- and virus-induced carcinogenesis, and transplanted and spontaneous tumor growth), it was shown for the first time that increased natural suppressor activity occurs in animals with a tumor irrespective of the type of carcinogenesis and histological tumor type. The immunosuppressive effect of natural suppressor cells in this setting is substantially attributable to nitric oxide. The mechanism of the observed enhancement of natural suppressor cell activity may also involve the direct stimulating effect of factors produced by tumor cells. It was shown for the first time that, under different types of carcinogenesis, antitumor activity increases in parallel with immunosuppressive activity.

It was shown for the first time that solid growth of Ehrlich carcinoma represents a special model of carcinogenesis. This is attributable to several properties of this tumor: its cells produce nitric oxide without additional stimulation, secrete an inducer of nitric oxide synthesis, and stimulate interferon-γ production by T lymphocytes. These properties presumably account for the spontaneous (without additional stimulation) production of nitric oxide by bone marrow cells of tumor-bearing animals. The pathogenesis of growth of this tumor is also atypical: it is accompanied by increased interferon-γ production by T cells of the tumor host. The immunosuppressive activity of natural suppressor cells also increases in this model of carcinogenesis, but nitric oxide is their main and only suppressor factor in this case.

The relationship between the functional state of T lymphocytes and the quantitative and qualitative characteristics of natural suppressor cells was identified for the first time.

Theoretical and Practical Significance. The data obtained make clearer the mechanisms underlying the induction of natural suppressor cell activity and their functional heterogeneity. This, in turn, enables a deeper understanding of the immunoregulatory action of hematopoietic cells and broadens existing knowledge of the pathogenesis of tumor growth and the mechanisms of interaction between the immune system and hematopoiesis. The aspects of tumor-growth pathogenesis identified in this study may suggest new targets for pharmacological correction in this type of pathology.

Presentation of the Work. The materials included in the dissertation were presented at the IX European Conference on Immunology (Rome, Italy, 1988); at the 1st International Symposium of the Russian Academy of Medical Sciences, “Laboratory Animals in Medical-Biological and Biotechnological Research” (Moscow, 1992); at the 2nd International Congress of the Russian Academy of Medical Sciences (Salerno, Italy, 1993); at the conference “Experimental and Clinical Immunology” (Tomsk, 1995); at conferences dedicated to the 35th anniversary of the Research Laboratory, “Medical-Biological Aspects of Neurohumoral Regulation” (Tomsk, 1997), and to the 15th anniversary of the Research Institute of Pharmacology, “Current Problems in Pharmacology and the Search for New Drug Compounds” (Tomsk, 1999); at “Problems of Experimental and Clinical Pharmacology” (Tomsk, 2000); at “Current Problems of Experimental and Clinical Pharmacology” (Tomsk, 2001); at “Current Problems of Experimental and Clinical Pharmacology” (Tomsk, 2002); at “Current Problems in Pharmacology” (Tomsk, 2004); and at the Russian Scientific-Practical Conference “Current State and Prospects for the Development of Experimental and Clinical Oncology” (Tomsk, 2004).

Publications Resulting from the Study. Thirty printed works on the subject of the dissertation have been published, including 19 in central journals.

Volume and Structure of the Dissertation. The dissertation comprises 420 pages of typewritten text and consists of an introduction, four chapters, conclusions, and a list of references used. The work is illustrated by 92