Polyclonal stimulation of resting B lymphocytes by antigen-specific T lymphocytes (original) (raw)
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Histopathology, 1993
A number of different factors can profoundly influence the quantification of immunostained cells. Given the characteristics of immunohistological detection systems with non-linearity of signal and antigen concentration, we investigated the relationship of signal (number of stained cells) to the dilution of antibody employed. Three antibodies were studied which have been advocated as being effective in fixed material as markers of cell proliferation: PClO (anti-proliferating cell nuclear antigen (PCNA)), Ki-S1 and MIBl (a novel anti-Ki-67). Serial sections of tonsil were immunostained with a range of antibody dilutions using a fixed detection system and the number of stained cells quantified. Similar experiments were performed on tumour xenografts with known growth fraction and, in vitro, on human diploid fibroblasts in logarithmic growth phase. With both PClO and Ki-S1 the number of stained cells increased with decreasing antibody dilution with no plateau being identified. In contrast, MIBl showed a clear plateau. Immunocytological data indicate that PCNA and Ki-S1 antigen are present at low (but detectable) levels in at least some non-cycling cells and thus an artificial 'cut-off' has to be employed in assessing the number of proliferating cells with these antibodies. The superiority of MIBl probably reflects the rapidity of catabolism of the Ki-6 7 antigen at the end of M phase. Taken together, these data point to the importance of carefully considering fundamental immunochemical properties such as antibody concentration (as well as antibody affinity and sensitivity of detection system) when employing immunological markers of cell proliferation in quantitative procedures. 18. Galand P. Degraef C. Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues. Cell Tissue Kinet. 1989: 22; 383-392. 19. Rogers S. Wells R. Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 1986: E M B O J . 1985: 4: 655-661. 92; 5 3 1-540. 234; 364-367.
Journal of Immunological Methods, 1979
Based on the observation that binding of IgM cytophilic antibodies to lymphocytes is temperature dependent, a direct plaque forming cell (PFC) assay was developed to detect IgM-receptor bearing human peripheral blood T and B lymphocytes. Lymphocytes were passively sensitized with IgM anti-SRBC molecules at 4°C, added to SRBC monolayers, then incubated at 37°C with guinea pig complement to develop the plaques. The PFC assay has methodological advantages over rosetting methods which demonstrate IgM receptors, and under certain conditions is more sensitive than these rosette techniques. A mean of 17% of freshly isolated uncultured lymphocytes, enriched for B cells, formed direct plaques while a mean of 3% of T-enriched preparations formed direct plaques. However, if the lymphocytes were preincubated with vibrio cholerae neuraminidase (VCN) these figures increased to 46% and 35% respectively. The specificity of plaque formation by VCN-treated lymphocytes was established. SRBC sensitized with a F(abr)2 preparation of an IgG anti-SRBC reagent failed to bind to VCN-treated lymphocytes, inclusion of IgM, but not other Ig molecules in the test medium, inhibited plaque formation, and, most important, plaque formation by T and B cells was inhibited by F(c)sP but not by Fabp fragments. These results indicate that T and B lymphocytes express IgM-class specific membrane receptors, that these receptors may be hidden on normal lymphocytes but are revealed by treatment with VCN and that the IgM receptor on VCN-treated lymphocytes is F(c)~ specific. These findings are discussed briefly with regard to other and partly contradictory data obtained after overnight in vitro lymphocyte culture. As demonstrated by direct PFC assay, the B cell IgM receptor is trypsin sensitive.
European Journal of Immunology, 1977
A previous study of primary and secondary immune responses of rats and mice immunized against various protein antigens allowed us t o describe a population of immunocytes containing, synthesizing and secreting immunoglobulins without detectable antibody function against the antigen injected (IFC). We here report their T cell-dependence. Mice, thymectomized, irradiated and reconstituted with bone marrow or fetal liver cells, generally show neither ant ibody-forming cells (AFC) nor cells synthesizing immunoglobulin without detectable antibody function. In some mice, probably only partly T cell-deprived, antibody-containing cells were seen, and at that time they were associated with cells synthesizing immunoglobulin without detectable antibody function. For most of the animals studied in primary response, however, the IFC population remained higher than the AFC population throughout the immune response. In normal animals immunoglobulin-synthesizing cells were predominant a t the beginning of the immune response, then decreased and progressively replaced by antibody-synthesizing cells. After the first injection or after two stimuli, the number of responders among T celldeprived mice increased progressively. Finally, these experiments indicate that both cells synthesizing immunoglobulins without detectable antibody function and specific AFC are thymus-dependent and rule o u t the hypothesis according t o which horseradish peroxidase is a polyclonal B cell activator.
A Study of the Proliferative Response of Rabbit T Cells Using the Brdu-Hoechst Method
Cell Proliferation, 1984
Con-A-and PHA-induced proliferation of cells from rabbit thymus, spleen and mesenteric lymph node was studied with the DNA-fluorescent probe 33258 Hoechst. The fluorescence of this probe is quenched when 5-bromo-2'-deoxy-uridine is incorporated into nascent DNA during the S phase. Fluorescence decreased with increasing content of newly formed DNA per cell. Proliferation kinetics and the number of Con-A-and PHA-reactive cells (Ct and Pt cells) were determined cytofluorometrically. Lymphocytes from control and dexamethasone (DX)-treated animals start their proliferation early: after 42 hr about 25% of the control and the majority of the DX-resistant cells finished their second cell division. Small numbers of C+ (12.0%) and Pt (3.5%) cells were found in control thymus, while these percentages were enhanced in DX thymus: 32.5 and 27.0% respectively; 50% of the spleen T cells in control and DX animals are C+ or P+ and 75% of the lymph-node T cells are C+ (after DX 45%) and 50% are P+ (after DX also 50%). It is concluded that in thymus and lymph nodes, a steroid sensitive (Ss) C+P-, and in lymph nodes a Ss C+P+ cell pool is present. A mitogen non-proliferative cell pool (C-P-) is present in control and DX thymus. Stimulation of lymphocytes by mitogens in vitro is widely used in experimental and clinical immunology. Con-A and PHA are mitogenic lectins able to transform T lymphocytes into proliferative blasts (Oppenheim & Rosenstreich, 1974). The DNA synthetic capacity of these cells is usually quantified with radioactive DNA precursors (usually 3H-thymidine) and the amount of label incorporated has been used as a measure of the proliferative capacity. For both Con-A and PHA large differences in the extent of stimulation have been reported, and heterogeneity among T cells in this respect has therefore been considered (Stobo, 1975). Using elimination of mitogen-reactive subsets by photolysis Touraine ef al. (1976) have demonstrated that a large population of T cells in human peripheral blood is proliferative both to Con-A and well as to PHA (C+ and P+ respectively). Other authors have reported that C+ and P+ cells differ in a number of physical (