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B lymphocyte precursor cells are the target cells for the major subtype of paediatric cancer, acute lymphoblastic leukaemia. Using a murine IL-7-dependent clonogenic assay for normal B cell precursors as a model, we have investigated the sensitivity of these cells versus other normal and leukaemic haemopoietic cells to alpha-particle radiation. We find that B cell precursors are remarkably susceptible to the lethal effects of alpha-particles and have a very low probability of surviving a single alpha-track. B cell precursors are also very sensitive to the lethal effects of low LET X-rays. The mutation frequency in a marker gene (HPRT) does not, however, appear to be greater in B cell precursors that survive X-radiation than in other haemopoietic cells.

Glucocorticoid receptor levels and cellular responses to glucocorticoid treatment in vitro were evaluated for the malignant cells from patients with leukaemia. Cells from all cases of lymphoid and myeloid leukaemia contained glucocorticoid receptors; receptor levels and the extent of in vitro responses appeared to depend primarily on cell type and differentiation status. For acute lymphoblastic leukaemia (ALL), high receptor levels were associated with the common ALL phenotype, intermediate levels with null (unclassified) ALL and low levels with T-ALL. Considerable variation in receptor levels and in responses in vitro was observed for acute myeloid leukaemia; receptor levels measured by whole-cell assay did not relate to patient survival.

The neutral glycolipids and gangliosides of sheep thymus and of sheep peripheral lymphocytes were compared. The patterns of both of these major classes of glycolipids were more complex in thymus than in the lymphocytes. The incorporation of radioactivity from d -[1- 14 C]galactose into the individual glycolipids of control and concanavalin A-stimulated sheep peripheral lymphocytes was also studied. A marked enhancement of incorporation into trihexosylceramide and an alteration of the pattern of incorporation into gangliosides were noted in the mitogen-treated cells. The results suggest that significant alterations of glycolipid composition and metabolism may occur during at least certain stages of lymphocyte differentiation.

This paper provides a summary and analysis of information presented by speakers at a special symposium in the Sixth International Workshop on Chromosomes in Leukemia (London, 1987). Some updated references are provided but inevitably in such a fast moving field, much new data has been generated since the Symposium. The main conclusions reached, however, have not significantly changed.

The origin of rosette-forming cells of mice immunized with sheep red blood cells has been investigated. By the use of antigenic markers on thymus cells (H-2, θ) it was shown that a considerable proportion of RFC formed in the spleens in irradiated animals reconstituted with thymus and bonemarrow cells, as well as in intact normal and immunized animals were of thymus origin. The possible limitations of the test systems and the significance of the results have been discussed.

The immunological significance of antigen-binding cells (“rosette”-forming cells) in mice injected with sheep erythrocytes has been investigated in different experimental situations by a comparative quantitative study of antibody-producing cells and antigen-binding cells as well as of antigen-sensitive cells utilizing a cell-transfer system. “Passive” antibody administration to animals immunized with sheep red cells caused a considerable depression of antibody-producing cells, whereas “rosette”-forming cells as well as antigen-sensitive cells were much less suppressed. The dose response of mice to sheep erythrocytes was studied and it was found that very low doses of antigen induced immunological priming or memory and a marked response with regard to rosette-forming cells in the absence of any significant development of antibody-producing cells. These results suggest that the antigen-binding lymphoid cells in the spleens of mice immunized against sheep erythrocytes include antigen-sensitive cells as well as cells actively engaged in antibody secretion. A considerable proportion of the former are thymus derived, possessing the thymus-specific antigen θ, and with immunoglobulin-like binding sites for antigen, as judged by inhibition of antigen binding by treatment of the cells with an anti-Fab and anti-light chain antibody.

Summary The evidence for the existence of distinct populations of thymic-dependent T - lymphocytes and thymic - independent (bursa - equivalent) B-lymphocytes is reviewed. Both populations contain antigen-sensitive cells, probably with specific antibody on their surface. B-lymphocytes can differentiate, proliferate, and mature into plasma-cells which synthesise humoral antibody. On contact with antigen (which may have to be macrophage-processed), the T-lymphocytes transform into large blast-cells and divide both in vivo and in tissue-culture. These T-lymphoblasts, which do not have demonstrable intracellular immunoglobulin, subserve many functions: ( a ) They divide further to form an expanded population of primed antigen-sensitive cells which provide immunological memory because of their long life-span. ( b ) They are " killer " cells which are cytotoxic for graft target-cells. ( c ) They release a number of soluble factors which increase vascular permeability, are mitogenic for other lymphocytes, and activate macrophages, &c. These characteristics taken together with ( b ) form the basis for the phenomena of cell-mediated (delayed) hypersensitivity and cellular immunity. ( d ) They may cooperate during the immune response to certain (" thymus-dependent ") antigens by stimulating the antigen-sensitive B-lymphocyte. The cooperative function of T-lymphoblasts would account for the relationship often observed between delayed hypersensitivity and humoral-antibody formation, and for the occurrence of allergen-induced blastcell transformation of lymphocytes from allergic patients with anaphylactic - type hypersensitivity. Lymphoproliferative disorders, primary immunological deficiency, and autoimmune diseases can also be analysed in terms of T-lymphocyte and B-lymphocyte populations.

Lymphocytes from atopic patients allergic to grass pollen transform in culture in response to the specific allergen. A small proportion of the transformed cells were shown to contain IgE. A comparable number of cells were stained by fluorescein-conjugated pollen extract. The relationship of the lymphocyte transformation to delayed-type hypersensitivity in atopic individuals is discussed.

Monoclonal antibodies described as reacting with particular subsets of haemopoietic cells were screened against a variety of neuronal cell lines to further investigate their true specificity. While some reagents, e.g., monoclonal anti-cALL (J5), were found only reactive with haemopoietic cells, other monoclonals, e.g., BA1, BA2, NA134, OKT6, and OKT9, also bound to neuronal cells. Further investigation into the cross-reactivity of these antibodies to a variety of neural crest derived cell lines indicated that, with the exception of OKT9, differential binding patterns to different lines were obtained. This suggests that haemopoietic cell subsets and neural crest derived tissues can share similar differentiation antigens. For monoclonals OKT9 and BA2 this observation was confirmed biochemically, showing that it is not just antigenic determinants but similar molecular weight cell surface antigens that are shared between subsets of the two major cell types. A similar analysis using monoclonal antibodies raised against human neuronal cells or cell lines again indicates that some antigens appear unique to neural tissue while others are shared by haemopoietic cell subsets.

Peripheral blood lymphocytes from healthy individuals, patients with Graves disease and with chronic lymphocytic leukemia, mixed tonsillary and purified B and T lymphocytes, and thymocytes were assayed for susceptibility to cytotoxic activity of cold agglutinins. Sera containing homogeneous anti-i and anti-I cold agglutinins as well as purified 19S IgM(K) anti-I cold agglutinin, and 7S, 11S, and 19S IgM(λ) anti-i cold agglutinins were used. A 2-hr bithermic cytotoxicity assay followed by E and EAC rosette was employed. Cold agglutinins killed and inhibited rosette formation of both B and T lymphocytes. Anti-i cold agglutinins were more lymphocytotoxic against normal peripheral blood, tonsillary and thymic lymphocytes, whereas anti-I cold agglutinins were more cytotoxic against peripheral blood lymphocytes from patients with chronic lymphocytic leukemia or Graves disease. Therefore, lymphocytes from various sources, both B and T, are susceptible to the cytotoxic activity of anti-i and anti-I cold agglutinins; however, the expression of Ii antigens seems to be variable.

Four monoclonal antibodies, 5/138, 5/32, BD6 and 6/266, are reported which recognize a previously described membrane associated dimeric protein of Mr = 2 X 75,000 and whose expression is correlated with malignant phenotype in HeLa-normal fibroblast hybrids.

Publisher Summary This chapter presents results of the studies performed to analyze membrane-associated antigens of lymphoid cells. The chapter focuses on the immunoglobulin receptors on the surface of lymphoid cells. The study established the concept of phenotypic restriction for immunoglobulin secreting cells. The danger of artifacts arising from the presence of soluble complexes remaining after the absorption of antisera was also mentioned in the study. Immunofluorescent staining and a quantitative radioimmunoassay were used to detect and quantitate the immunoglobuhns present on the lymphoid cells present in various tissues of the mouse. The study also reports that no antigen-binding cells could be found in the spleen of bursectomized chickens. The results of studies in which the effect of proteolytic enzyme treatment of lymphoid cells affected their ability to form rosetteswere also discussed. The interpretation of these findings indicated that the receptors on rosette forming cells in immunized animals had a much more rapid turnover than in unimmunized animals.

Publisher Summary This chapter describes a study analyzing immunoglobulin determinants on the surface of antigen binding T- and B-lymphocytes in mice. In this study, CBA mice or (CBA × C57B1)F 1 between 4 and 16 weeks of age were used. Congenitally athymic (“nude”) mice were bred from a mating pair. The assay of rosette-forming cells (RFC) was performed. Macrophages were routinely eliminated from the test suspensions by cotton filtration. It was concluded that all T-RFC have immunoglobulin-like molecules on their surface (inhibition by anti-Fab). T-RFC in normal mice have light chain determinants (principally kappa) on their surface but have no detectable heavy chain determinants. The results also show that T-RFC in actively immunized mice have either kappa light chains or lambda light chains and μ heavy chains. No γ or α heavy chain determinants could be detected, either early or late (day 30) in the response. The inhibitory activity of anti-IgM 1 sera was directed toward hinge-region determinants of μ chains.

A series of 4 monoclonal antibodies reacting with 4 different markers on the surface of B lymphocytes were reacted with a variety of leukaemic lymphocytes. Reactivity was assessed by indirect immunofluorescence using the fluorescence microscope and fluorescence-activated cell sorter. Monoclonal antibody FMC1 reacts with all normal . B cells and all B cell leukaemias tested. FMC4, which is an antibody against the Ia antigen, reacts with all B and Null type leukaemic cells but does not react with T leukaemias, with one exception. FMC7 reacts with only some normal B cells, and shows a similar selectivity in testing with B cell leukaemias. FMC3 is not specific for B cells, since it reacts with T cells. However, it reacts with only 50% of normal B cells. When tested with B-type CLL’s, FMC3 stained the leukaemic cells of 5 of 24 patients. Although larger numbers of samples must be tested before the diagnostic value of these markers can be assessed, these preliminary results suggest that some of these monoclonal antibodies will be useful markers for different types of leukaemic lymphocytes. These studies were carried out in part at the Imperial Cancer Research Fund Laboratories, London, while H.Z. was a recipient of a Travel Grant from the Royal College of Pathologists of Australia.