Rosetting of human T lymphocytes with goat red blood cells: Effect of treatment with 2-aminoethylisothiouronium bromide (AET) and comparison with AET treated sheep red blood cells (original) (raw)
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Journal of Immunological Methods, 1976
Purified human B and T lymphocytes were obtained by rosetting HPL with AET-SRBC or MRBC and separating the non-rosetted from the rosetted cells on Ficoll-Hypaque gradient. 92 ± 3% of the purified B cells were fluorescent positive for MBIg and 95 ± 2% of the purified T cells rosetted with AET-SRBC. 69 ± 5% of the B cells and 61 ± 8% of the T cells present in the unfractionated HPL were recovered in the purified fractions. * Abbreviations used in this paper: AET, 2-aminoethylisothiouronium bromide hydrobromide; AET-SRBC, SRBC treated with AET; AHG, aggregated human gamma globulin; B cell, bursal equivalent; EAC, complement coated sensitized sheep erythrocytes; EAC1-5 rab, immune adherence positive EA coated with C1-C5 from rabbit serum deficient in C6; EAC3d m°, immune adherence negative EA coated with C1-C3 from mouse serum deficient in C5 ; HPL, human peripheral lymphocytes; MBIg, membrane bound immunoglobulin; MEM, minimum essential medium; MRBC, monkey red blood cells; SRBC, sheep red blood cells; T cell, thymus derived; VBS, veronal-buffered saline.
Rosette formation with goat erythrocytes. A marker for human T lymphocytes
Clinical and experimental immunology, 1978
We demonstrate the use of goat erythrocytes in a rosette procedure for the classification of human lymphocytes. The population is almost perfectly overlapping with the lymphocytes which form rosettes with sheep red blood cells. 70'2 ± 7-5% of peripheral lymphocytes form rosettes with goat erythrocytes and less than 1% of these cells have surface immunoglobulins. Enrichment of goat rosette-forming cells results in a population with an increased percentage of both goat and sheep rosettes. This population retains activity to the T-cell mitogens Con A and PHA, while the cells depleted of goat rosettes have greatly diminished responses to these same mitogens. Tonsil and spleen lymphocytes form 50-2 -4 68% and 24% ofgoat rosettes respectively, while peripheral blood lymphocytes from patients with CLL rarely form goat rosettes. Cell lines maintained in vitro rosetted with goat cells in a parallel fashion to sheep cells. Thus T-cell lines, such as Molt-3, which form rosettes with SRBC also rosette with GRBC, while sheep rosette-negative lines, i.e. Molt-4, are negative for both erythrocytes. B-lymphoid cell lines were negative, as were several lymphoma cell lines. There was a slight variation in the binding of goat cells, depending on the source of the goat. Thus, as in sheep rosettes, some animals were better sources than others, although all the animals tested formed rosettes.
Journal of Experimental Medicine, 1972
By using the two criteria (a) high density of immunoglobulin determinants on the cell surface and (b) presence of receptors for C'3 on the cell surface for defining bone marrow-derived lymphocytes, it is indirectly shown that all or at least a major population of human thymus-derived lymphocytes under certain conditions will form nonimmune rosettes with sheep red blood cells (SRBC). Almost all thymocytes tested from two different donors formed rosettes. The SRBC rosettes are not formed by virtue of immunoglobulin receptors and form only around living cells. Positive bivalent ions are required for rosette formation since EDTA will block rosette formation. Sodium iodoacetate will also block rosette formation demonstrating the dependence on an intact glycolytic pathway. Rosette formation is temperature dependent and will not appear at 37°C. Trypsin treatment of lymphocytes will abolish their SRBC-binding ability which cannot be restored by treating them with fresh donor serum or fe...
Journal of Experimental Medicine, 1973
Sheep red blood cell rosette formation has been demonstrated in a number of laboratories to be a specific marker for human T cells (1-3). A variety of different methods have been utilized which have shown some differences in the percentages of T cells that form these rosettes. The procedure of Jondal and Wigzell gives values close to 100% for peripheral blood T cells and for thymocytes. Recently double labeling experiments utilizing markers for B cells along with the rosette system have indicated that independent cells are involved although rare double labeled cells may be observed (3). The simplicity and specificity of the procedure have led to wide clinical use of this method and valuable information has been obtained (3, 4).
Bovine T lymphocytes — An improved technique of E rosette formation
Journal of Immunological Methods, 1978
The effect of treating sheep red bk)od cells (SRBC) with neuraminidase or 2-aminoethylisothiouronium bromide (AET) and different media (fetal bovine serum, Ficoll or dextran) during E rosetting of bovine lymphocytes was examined. It was found that the use of AET treated SRBC along with 6% dextran medium gave 60% and 83% E rosettes with bovine peripheral blood leukocytes and fetal thymocytes respectively. Furthermore, under these conditions the as.~ay time was greatly reduced. Since these rosettes were shown to be specific markers of bovine T lymphocytes and such rosettes were stable over a 2 h period at 37°C the possible use of the present technique is discussed in regard to the isolation of purified T lymphocyte cr T lymphocyte depleted populations which can subsequently be used for functional studies.
“Active” T cells in guinea pig peripheral blood lymphocytes
Clinical Immunology and Immunopathology, 1981
The ability to form rosettes with rabbit red blood cells (RRBC) has been shown to be characteristic of T but not B cells from guinea pigs. In an attempt to devise an assay system to measure the guinea pig equivalent of human "active" T cells, we have investigated the effects of various conditions on RRBC binding by guinea pig lymphocytes, including incubation time, RRBC:lymphocyte ratio, presence or absence of guinea pig serum or fetal calf serum in the incubation medium, and pretreatment of RRBC with neuraminidase or 2-aminoethylisothiouronium bromide (AET). Without incubation and at a 100: 1 ratio of RRBC to guinea pig lymphocytes, about 35% of guinea pig peripheral blood lymphocytes bound RRBC, similar to the number of "active" T cells in human peripheral blood as measured by rosette formation with sheep red blood cells. Cold incubation increased the number of rosetting cells to over SO'%, presumably the total number of T cells. A relative decrease in the number of RRBC diminished rosetting only at ratios of 1O:l or less. Pretreatment of RRBC with neuraminidase slightly increased, whereas AET pretreatment drastically reduced the number of rosettes. Increasing concentrations of guinea pig serum in the medium up to 100% slightly inhibited rosetting. These results suggest the presence of two T-cell populations in guinea pig peripheral blood, equivalent to the human "active" and "total" rosette-forming T-cell subpopulations.