Immunobiological classification of acute lymphoblastic leukaemia (ALL).
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Biomedical subjects
Publications and source records attributed to M Ferrarini.
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Human T blasts, obtained by stimulation of peripheral blood mononuclear cells (PBM) with tetanus toxoid, diphtheria toxoid or Candida albicans, were expanded in long-term culture using alternate periods of antigen restimulation and growth in media containing interleukin 2. The cells gave a proliferative response only to the antigen originally used for stimulation. Such as response was strictly dependent upon the presence of autologous but not of allogeneic mitomycin C-treated mononuclear cells. When added to autologous PBM depleted of E-rosetting cells together with the specific antigen, the T blasts induced a polyclonal proliferation and differentiation of B cells. Allogeneic B cells were activated by antigen-stimulated T blasts only in the presence of irradiated mononuclear cells autologous to the responding T blasts. The above responses seemed not to be regulated solely by the release of soluble factors; apparently cell to cell interactions had to take place to obtain an efficient B cell activation.
Large granular lymphocytes (LGL) are nonadherent cells with cytoplasmic azurophilic granules, avid receptors for the Fc portion of IgG, and a paranuclear localization of alpha-naphthyl acid esterase or acid phosphatase. LGL constitute the bulk of TG cells (cells with receptors for sheep erythrocytes and for IgG molecules) and null cells (non-T, non-B cells). In the present study we demonstrate that 20-33% of the circulating human LGL express receptors for the third complement component (C3R). When TG cell or null cell fractions from normal individuals or non-T cells from a patient with infantile agammaglobulinaemia (which contained almost exclusively LGL) were rosetted with erythrocytes coated with antibody and complement, a variable number of C3R-bearing cells were detected. Such cells were isolated and analysed further; the great majority of them displayed the cytochemical and ultrastructural features of LGL.
Malignant lymphocytes from 30 B-cell chronic lymphocytic leukemia (B-CLL) patients were studied for the cytochemical localization of two acid hydrolases, alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AT). The large majority of the cells stained for both ANAE and AP in 7 cases, for AP only in 18 cases, and were negative for both the enzymes in 5 cases. Ultrastructural analysis revealed that the cells that displayed more mature morphological features, such as well developed smooth and rough membrane compartments, were those positive for acid hydrolases. That ANAE and AP are expressed by B cells at late stage of maturation was confirmed by the finding that some lymphocytes and all of the plasmacytoid lymphocytes and plasma cells from Walderström's macroglobulinemia, from mixed cryoglobulinemia, and from multiple myeloma patients stained strongly for both ANAE and AP. Using the expression of acid hydrolases and certain ultrastructural features as markers of cell differentiation, it was possible to demonstrate a process of maturation within the single B-CLL clones with accumulation of the cells at stages that differed in the various cases.
The colony-forming capacity of peripheral blood T-cells isolated from B-cell chronic lymphocytic leukemia patients was studied and compared with that of peripheral blood T-cells isolated from 2 groups of normal controls (young and aged healthy donors). Upon stimulation with phytohemagglutinin or pokeweed mitogen. T-cells from both the patients and the controls developed into colonies but only in the presence of the appropriate conditioned media. When the responses of the 3 groups were compared, no statistically significant differences were detected. However, the number of patients who failed to respond to phytohemagglutinin was larger than that of normal controls. Studies on a selected group of patients demonstrated that such unresponsiveness was unrelated to the presence of suppressor cells or to inadequate conditions used for stimulation in culture. Rather, this unresponsiveness may be connected with an intrinsic T-cell defect present in certain B-cell chronic lymphocytic leukemia patients.
Large granular lymphocytes (LGL) are defined as nonadherent mononuclear cells with cytoplasmic azurophilic granules, avid receptors for the Fc portion of IgG, and cytotoxic functions (NK or ADCC activities). In the present study, the granules of LGL isolated from human peripheral blood have been analyzed by enzyme cytochemistry and electron microscopy. It had been found that: (1) in the single cells, granules at different stages of maturation could be detected: in addition, packaging of the granules took place in the proximity of the Golgi apparatus, which is similar to that seen in secretory cell types. (2) Acid phosphatase (AP) was observed within the granules and the vesicles located in the Golgi area: the Golgi apparatus identified through its thiamine pyrophosphatase-positivity was consistently negative for AP. (3) Alpha naphthyl-acetate esterase (ANAE) activity was localized in the granules as well as on the membrane of LGL and monocytes. (4) The ANAE activity of LGL was of the monocytic and not of the lymphocytic type, as shown by NaF inhibition. (5) The LGL granules, although identifiable as primary lysosomes, were not involved in the process of phagocytosis, since LGL failed consistently to ingest latex particles or opsonized red cells.
Rosettes with ox erythrocytes coated with purified IgG antibody were used to detect Fc receptors on neutrophils from 60 patients with solid neoplasias and from 55 normal controls. The mean average of the rosettes in the patients was 48.10%, and that in normal controls was 79.42%, with a highly significant difference according to the Wilcoxon test [negative probability, P(w)-4.47 . 10(-5)]. The low proportion of patients' rosettes ws related to the presence of a serum factor, which also inhibited normal neutrophil rosette formation. Patient neutrophils (or normal neutrophils treated with patient sera) recovered their rosetting capacity when cultured in vitro. No correlation was found between low percentages of rosette-forming cells and the level of circulating immune complexes of the individual patients. Additional evidence also supported the finding that IC and the serum factor are probably unrelated.
The localization of alpha-naphthyl acid esterase (ANAE) activity in T lymphocytes with receptors for IgM (T(M) cells) have been studied at the electron microscope. The electron-opaque product of the cytochemical reaction was detected around, but never inside, single (or groups of) vesicles, which suggested a possible membrane localization of the enzyme activity. These same vesicles were found to contain acid phosphatase by both light- and electron-microscopic examination and were bound by unit membranes; this data indicates that they likely represent primary lysosomes. The presence of such lysosomes in a restricted paranuclear area is a distinctive feature of T(M) cells.
The ultrastructural and cytochemical features of human peripheral blood TG cells (T cells with receptors IgG) and of the cells of the so-called third population (non-T, non-B cells with high avidity receptors for IgG) have been investigated and compared. Both TG and third-population cells (TPC) contained acid hydrolases with a paranuclear localization of alpha-naphthyl acid esterase, beta-glucuronidase or acid phosphatase. At the electron microscopy level, TG and TPC were indistinguishable and displayed rough cell surface, indented nuclei, abundant cytoplasm with predominance of the smooth over the rough membranes and peroxidase-negative granules. A large proportion of cells of the TPC could form rosettes with sheep erythrocytes after treatment with neuraminidase. The observed close similarities between TG and TPC may suggest that both cell types belong to a special subset of T cells. However, the alternative hypothesis that both TG and TPC are part of a subset unrelated to T cells, such as a new non-T, non-B cell population, or even of the monocytic-macrophage lineage, is also discussed.
A new technique which makes use of anti-delta-specific allo-or heteroantisera coupled to Sepharose 4 B has been employed to investigate the presence of IgD molecules in the sera of three mouse strains. The sera were reacted with the insoluble immunosorbents, and the eluted material was labeled with 125I prior to antigenic or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This material reacted with anti-delta or anti-L chain reagents, but not with other anti-class-specific antisera and, after reduction, was resolved in three discrete bands in SDS-PAGE. The fastest of these bands comigrated with L chain, whereas the other two moved identically to mouse lymphocyte surface delta chains. Immunofluorescence studies on fixed smears of spleen and Peyer's patches' cells revealed the presence of a small but consistent number IgD-containing plasma cells.
Endogenous and surface labeling techniques were used on human lymphoid cells to characterize intracytoplasmic, membrane and secreted IgD, IgD synthesized by lymphocytes and inserted into the cell membrane displayed a single molecular form with the same mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as the previously described slow migrating serum IgDl. Plasma cells produced and secreted IgDl and another IgD corresponding to the faster-moving serum IgD2. Conversion of one molecular form into the other was never observed, thus indicating that neither molecule is a precursor or a degradation product of the other.
Human IgD present in the serum of normal individuals or of patients with Hodgkin's disease (having high IgD concentrations) was characterized and compared with five IgD myeloma proteins. IgD was isolated using a highly specific anti-delta insoluble immunoabsorbent from which the bound material was eluted with sodium dodecyl sulphate (SDS) or urea. The latter reagent could be removed by extensive dialysis, thus making possible the renaturation of the eluted molecules. The purity of the IgD thus isolated was confirmed by antigenic analysis. Both kappa and lambda light chain determinants were present on serum IgD, although lambda light chain was predominant with a ratio over the kappa chain of 2:1. SDS-polyacrylamide slab gel electrophoresis analysis revealed two different molecular forms of serum IgD, one (IgD) migrating identically to monoclonal IgD, the other (IgD2) having a faster mobility. The difference between the two molecules was entirely, due to the different sizes of their constituent delta chains. Peptide mapping of the two chains (delta1 and delta2 respectively) and of the delta chain of an IgD myeloma protein was carried out using 125I-labelled material. The three molecules displayed a high degree of homology, the delta2 chain differing by the presence of three characteristic extra peptides. The significance of these extra peptides is discussed in the light of the peptide mapping technique employed.
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Six different immune complex test systems for the detection of IgG Fc receptors were applied to the study of various human lymphocyte populations. The extent of binding varied widely according to the system and the cell type employed. Two systems bound preferentially to a high proportion of B lymphocytes from peripheral blood or tonsils, one of which bound with only a very few T cells. In contrast, four other test systems which bound well with the Fc receptors on T lymphocytes gave weaker reactions with Fc receptors on B cells. The reactivity of Fc receptors on null or third population lymphocytes was similar to that of the Fc-positive T cells. Pronase digestion experiments showed a graded selective loss of reactivity with the different Fc reagents. No one system was optimal for all of the lymphocyte populations, although aggregated IgG exhibited the broadest spectrum of reactivity. A pronounced effect of temperature was evident on the binding reactions, and native IgG showed strong binding at 4 degrees C, particularly to the Fc receptors on T cells.
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The susceptibility to proteolysis and the mode of re-expression of receptors for IgM or IgG present on two different subpopulations of human T lymphocytes (T.M and T.G cells, respectively) have been investigated. The IgM receptor was highly susceptible to both trypsin and pronase, whereas the IgG receptor was resistant to trypsin and sensitive only to high concentrations of pronase. The receptors have been removed by treating purified human T cells with pronase and their reappearance on the cell surface has been followed in vitro. The IgM receptors on the cell surface were detectable within 2 hr and the resynthesis was completed in 6 hr. IgG receptors were detectable in 4 to 6 hr and the resynthesis completed within 12 hr. When protein synthesis was inhibited by culturing the cells in the presence of cycloheximide for up to 12 hr, only the IgM receptor (which had a higher turnover rate) failed to be expressed. Whereas interaction of IgG immune complex with the IgG receptors was previously shown to induce a modulation of the receptors, contact with antigen-IgM antibody complexes did not alter the mode of expression of IgM receptors.
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