Ekaterina Sidorova

Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)

Monoclonal expansion of B cells and plasma cells, producing antibodies against ‘self’ molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström’s macroglobulinaemia and B‐type of chronic lymphocytic leukaemia (B‐CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN‐MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin‐specific B cell clones from peripheral blood of patients with PN‐MGUS, by selection of cells bearing specific membrane Ig‐receptors for myelin protein P0, using beads coated with P0. P0‐coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein–Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti‐P0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5+ positive, although the expression declined in vitro over time. The anti‐P0 antibodies were of IgM‐λ type. The antibodies belonged to the VH3 gene family with presence of somatic mutations. The IgM reacted with P0 and myelin‐associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5+ clones included in the study as controls. The expanded clones expressed CD80 and HLA‐DR, which is compatible with properties of antigen‐presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P0‐specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen‐presentation by these cells followed by T cell activation.

Role of different B-cell subsets in the specific and polyclonal immune response to T-independent antigens type 2

Role of different B-cell subsets in the immune response to T-independent antigen type 2 (TI-2) was studied. BALB/c and C57BL/6 mice were immunized by polyvinylpyrrolidon (PVP), and the numbers of antibody- and Ig-forming cells (AFC and IFC, respectively) were determined by ELISPOT method. The number of cells producing non-specific Ig (nIFC) was calculated as the difference between the number of IFC and AFC; the number of nIFC induced by PVP was calculated as the difference between the number of nIFC in immune and control splenocytes. Immunization by PVP induced not only the AFC appearance, but also the increase in the number of the antigen-induced nIFC. The treatment of splenocytes by anti-CD5 antibodies and guinea pig complement reduced the increase in the numbers of newly formed AFC and nIFC to approximately 40% of control level. It means that CD5+ cells play an important role not only in the specific, but also in polyclonal immune response to non-self TI-2. To be sure that the decrease of AFC and nIFC numbers is due to depletion for CD5+ B-cells, but not CD5+ T-cells, splenocytes were separated to B-1 and B-2 subsets, and the numbers of AFC, IFC and nIFC were determined in each B-cell subpopulation separately. The overwhelming majority of newly formed AFC and nIFC was detected in B-1 subset. The numbers of AFC and nIFC in B-1 compartment was approximately 10-fold greater than in B-2 cells. A close parallelism between AFC and nIFC formation was observed. It is concluded that specific and polyclonal immune response to non-self TI-2-PVP-depends mainly on CD5+ B-1 subset.

Human monoclonal antibodies to viral peptides

A new approach to the developing of human monoclonal antibodies to viral peptides is described. The method is based on the positive selection of B cells specific to viral peptides from normal human tissues. To isolate B cells bearing immunoglobulin receptors specific to viral epitopes magnetic beads coated by viral peptides were used. Human B lymphocytes specific to the neutralizing epitope of gp120 of human immunodeficiency virus and to the major epitope of Epstein-Barr nuclear antigen-I were isolated from freshly removed human tonsils. Preselected cells were transformed by Epstein-Barr virus and cultivated on irradiated human embryonic lung fibroblasts in RPMI 1640-OptiMEM medium with 10% fetal calf serum and all necessary supplements. The cultures of B cells producing antibodies to viral epitopes used for selection were obtained. Two cultures secreting IgM antibodies to the peptide MN-24 (neutralizing epitope of immunodeficiency virus) and to the peptide p107 of Epstein-Barr nuclear antigen-1 were expanded, and the specificity of the antibodies and antibody-producing cells was analyzed. The method of positive selection of B cells specific to viral peptides may be used for the preparation of human monoclonal antibodies to viral antigens.

Model system for study of cell interactions and mechanisms of immune response to T-independent antigens of type 2 in vitro

Cellular mechanisms of immune response to type 2 T-independent antigens (TI-2 antigens) are not fully elucidated up till now. In vitro system is the most convenient model for such studies. However, in vitro model requires relatively high cell density in the cultures. It hampers the study of minor lymphocyte subsets like CD5+ B-1 splenocytes, which play the main role in the immune response to TI-2 antigens. The use of cell mixtures of normal and immunodeficient congenic animals may help to resolve this problem. In this work, immune responses to TI-antigens of type 1 (TI-1 antigens) and to TI-2 antigens in vitro were studied in the mixtures of cells of normal (CBA) and congenic xid-mice (CBA/N). CBA/N mice lack CD5+ B-1 cells and do not respond to TI-2 antigens. Therefore, their splenocytes can be used as “filler” cells to create the optimal cell density in the cell cultures. Spleen and peritoneal cells of CBA mice and B-1 and B-2 lymphocytes isolated from peritoneum and spleen, respectively, were cultured in different proportions with CBA/N splenocytes with or without antigens. LPS and polyvinylpyrrolidone (PVP) were used as TI-1 and TI-2-antigens, respectively. Antibody- and immunoglobulin-forming cells (AFC and IFC, respectively) were determined by the ELISPOT method on the 4th day of cultivation. It was shown that CBA and CBA/N cells in mixed cell cultures retained their functional activity. Splenocytes of CBA mice responded to both TI-antigens. Splenocytes of CBA/N mice responded to TI-1 antigen (LPS) only. It means that in vitro B-1 cells play the main role in the immune response to TI-2 antigens, as they do in vivo. Thus, the developed model system can be used to study cellular mechanisms of immune response to TI-1 and TI-2 antigens in vitro.

Antigen-specific receptors. Generation of the diversity from lamprey to human

In the last century it was established that the diversity of the antigen-recognizing receptors of Band T-lymphocytes and Ig/antibodies in mice and humans is due to the random recombination of DNA segments organized in clusters and located in fetal genome far apart. During somatic rearrangement of genome these segments combine and form functional V-genes, coding antigen-specific receptors. In birds and some other animals the diversity is provided or increased by gene conversion, which leads to the diversification of nucleotide sequences in pre-rearranged functional V-genes. Recently it was shown that the generation of the diversity might be realized by an entirely different way. In most primitive and living now agnathan vertebrates, lamprey and hagfishes, Ig-genes are absent, and somatic diversification of the antigen-specific receptors is due to a stepwise assembly of functional V-genes from separate modules. These modules coding leucine-rich repeats (LRR) adjust to a single (or two) “incomplete” germ-line V-gene and insert into it by gene conversion. LRR modules lodge in so called DNA “cassettes”. The number of LRR in the agnathan genome reaches 2–3 thousands; primary structure of LRR is very variable. The properties of lamprey and hagfish antibodies differ from that of other vertebrates. It is extremely interesting that similar LRR are found in Toll-like receptors of insects, mollusks and even plants, where they provide the resistance to different diseases. The data obtained are very important for the evolutionary immunology. The review deals with the mechanisms of generation of diversity of the antigen-specific receptors in vertebrates, insects, and plants.

Effect of Microenvironment on Functional Activity of Murine B-Lymphocytes

In contrast to B-splenocytes, murine peritoneal B-cells do not produce or secrete immunoglobulins (Ig). Twenty-four hours after intraperitoneal transfer of splenocytes containing Ig-forming cells (IFC), IFC content in the peritoneal cavity of recipient mice decreases dramatically. This decrease does not depend on the migration of transferred IFC to the spleen (homing); splenectomy has no effect on this process. In order to check whether the lack of IFC in the peritoneal cavity is due to inhibition of synthesis or secretion of Ig, peritoneal cells (contained 4360 IFC/106 cells) of CBA mice were intraperitoneally transferred to CBA/N mice after a 4-day preincubation in vitro. On the next day, ∼30% of in vitro transferred IFC were detected in the peritoneal cavity of recipient mice, but on day 4 the IFC content in peritoneum returned to the background level. Repeated in vitro incubation of peritoneal cells of recipient mice restored the IFC number in cultured cells. It means that peritoneal microenvironment inhibits functional activity of murine B-lymphocytes.

Role of different lymphocyte subpopulations in the formation of non-specific immunoglobulins induced by antigen injection.

The formation of antibody and non-specific immunoglobulin under the influence of T-dependent (TD) and type 2 T-independent (TI-2) antigens in mice of two congenic strains CBA (Lyb5-, Lyb5+) and CBA/N (Lyb5-) was studied. TD antigens induced in mice of both strains not only the appearance of antibody-forming cells (AFC), but also a great increase in the number of cells producing non-specific immunoglobulins (nIFC). TI-2 antigens induced the AFC and antigen-dependent nIFC formation in CBA mice only. It is concluded that during immune response to TI-2 antigens not only the AFC appearance but the increase in nIFC formation (polyclonal activation) is due mainly to the mature Lyb5+ B cells.