E-rosette Formation Research Articles

Rapid enumeration of T lymphocytes by a flow-cytometric immunofluorescence method.

Combined with current advances in microprocessor-controlled flow cytometers, monoclonal antibodies provide a rapid means of phenotyping individual cell surface markers for a large number of clinical samples accurately and reproducibly, which may provide useful information in diagnosing disease and monitoring patients. We have developed a one-step flow-cytometric immunofluorescence procedure for enumerating E-rosette lymphocytes from whole blood by using the monoclonal antibody OKT11. This antibody recognizes the sheep erythrocyte receptor on the lymphocyte surface and can block sheep E-rosette formation. The flow cytometer we use, an Ortho Spectrum III, distinguishes lymphocytes from other leukocytes by measuring the narrow forward and right-angle light-scattering properties of the cells. The instrument further differentiates T lymphocytes frm non-T lymphocytes by measuring the green fluorescence signal of the OKT11-positive lymphocytes. In a typical sample, 1500--2500 lymphocytes are counted in 25 s. In a study of 158 patient samples, ranging from 1% to greater than 90% E-rosette-positive lymphocytes, the correlation coefficient between the manual E-rosette count and the flow immunofluorescence measurement is 0.943.

Evaluation of E-rosetting human lymphocytes with OKT11 and other monoclonal antibodies

Monoclonal antibody OKT11 was found to compete with sheep red blood cells for binding sites on human lymphocytes. Preincubation of lymphocytes with OKT11 eliminated E-rosette formation. In a study of 142 peripheral blood samples ranging from 1% to over 90% E-rosette-positive cells, comparison to the percent OKT11-positive cells yielded a correlation coefficient of 0.93. In normal donors, subsets of OKT11+ cells were identified using two-color immunofluorescent staining methods with OKT3, OKT4, and OKT8. On the average, approximately 13% of OKT11+ lymphocytes were OKT3- and 13% of OKT11+ lymphocytes were OKT4- and OKT8-. Based on our double antibody fluorescence intensity data, low antigen density OKT11+ lymphocytes were OKT3-. OKT4+ and OKT8+ lymphocytes in normal peripheral lymphocytes have similar OKT11 antigen density.

Clinical experience with antilymphocyte serum.

Many different antilymphocytic antisera have been used clinically, and the properties of any particular type of ALS are not necessarily identical to those of any other type. Nevertheless, it is possible to draw certain general conclusions about the effects of ALS in human subjects. ALS administration has often been shown to reduce the number of circulating E-rosette-positive lymphocytes, although the precise mechanisms by which this reduction occurs are not known. Using a combined technique of E-rosette formation and immunofluorescence, heterologous immunoglobulin has been demonstrated on T and non-T lymphocytes from patients receiving non-selective ALS. Fifteen years' experience has failed to provide convincing support for the view that ALS (including immunoglobulin prepared from the whole antiserum) prolongs human renal allograft survival. It is not yet known whether ALS is a useful immunosuppressive agent in cardiac transplantation. One observation of possible clinical interest is that bone marrow regeneration has occurred in a number of patients with aplastic anemia who have been treated with ALS. No satisfactory method has been developed for monitoring the dose of ALS in human subjects. Appropriate studies may determine whether monoclonal antilymphocytic antibodies are clinically useful, for example in prolonging the survival of transplanted organs, in preventing or treating graft-versus-host disease, or in treating lymphoma, leukemia, or aplastic anemia.

Induction of differentiation in human T-lymphoblastic leukemia cell lines by 12- O-tetradecanoylphorbol 13-acetate (TPA): Studies with monoclonal antibodies to T cells

The induction of differentiation in human malignant T-lymphoblastic cell lines MOLT-3 and Jurkat by the tumor promoter, 12- O-tetradecanoylphorbol 13-acetate (TPA) was examined using the monoclonal antibodies OKT3, OKT4, OKT6, and OKT8 which are known to react with human T-cell differentiation antigens. It was found that in the presence of nanomolar concentrations of TPA the proportion of OKT3 + (mature T-cell marker) cells increased while the proportion of OKT4 +, OKT6 +, and OKT8 + (relatively immature T-cell markers) cells decreased. These changes in the distribution of the OKT antigens in MOLT-3 cells were found to be more prominent with MOLT-3 cells than when the Jurkat cells were used. In studies using a double labeling approach it was found that although the OKT3 + and E-rosette-positive (E +) cells appeared to belong to the same subpopulations of MOLT-3 cells, the OKT3 antigen was probably not related to the receptor for sheep erythrocytes because adsorption of the OKT3 antibody did not block E-rosette formation. Studies using the DNA synthesis inhibitor, arabinosylcytidine (ara-C) also indicate that DNA synthesis was not required for the induction of more mature T-cell antigens in the malignant T-cell lines by TPA. These studies, taken together with our earlier reports, support the conclusion that namomolar concentrations of TPA can induce differentiation in these malignant T-cell lines. Furthermore we have shown that the T-cell hybridoma antibodies are useful markers to detect differentiation changes in human T cells.

Heterogeneity in a lymphoid tumor: coexpression of T and B surface markers

Heterogeneity of leukemic cells was defined in a case of lymphoma. Four phenotypically distinct subpopulations of leukemic cells were identified. One subpopulation was observed to simultaneously express B- and T-cell characteristics. B-cell characteristics included monoclonal IgM (lambda) surface immunoglobulin, HLA-DR antigens, and expression of the B-cell antigen identified by the BA-1 monoclonal antibody. T-cell characteristics included E-rosette formation, expression of the pan-T- associated antigens recognized by the Leu-1 and OKT-11 monoclonal antibodies, and expression of the suppressor cytotoxic T-cell- associated antigen recognized by the Leu-2 and OKT-8 monoclonal antibodies. In addition to this subpopulation, three other phenotypically distinct subpopulations were identified, two of which expressed monoclonal IgM (lambda) surface immunoglobulin. The results of this investigation indicates that three phenotypically distinct lymphoid subpopulations bearing B-cell characteristics, and probably a fourth T-cell subgroup, were derived from a common lineage. These findings suggest that the malignancy involved a lymphoid progenitor cell that may possess diverse maturational capacity.

Heterogeneity in a Lymphoid Tumor: Coexpression of T and B Surface Markers

Heterogeneity of leukemic cells was defined in a case of lymphoma. Four phenotypically distinct subpopulations of leukemic cells were identified. One subpopulation was observed to simultaneously express B- and T-cell characteristics. B-cell characteristics included monoclonal IgM (lambda) surface immunoglobulin, HLA-DR antigens, and expression of the B-cell antigen identified by the BA-1 monoclonal antibody. T-cell characteristics included E-rosette formation, expression of the pan-T-associated antigens recognized by the Leu-1 and OKT-11 monoclonal antibodies, and expression of the suppressor cytotoxic T-cell-associated antigen recognized by the Leu-2 and OKT-8 monoclonal antibodies. In addition to this subpopulation, three other phenotypically distinct subpopulations were identified, two of which expressed monoclonal IgM (lambda) surface immunoglobulin. The results of this investigation indicates that three phenotypically distinct lymphoid subpopulations bearing B-cell characteristics, and probably a fourth T-cell subgroup, were derived from a common lineage. These findings suggest that the malignancy involved a lymphoid progenitor cell that may possess diverse maturational capacity.

Lymphokine production in T-lymphocyte subsets defined by active E-rosette formation.

Human T-cell subsets, defined by active E rosette formation, were examined for their ability to produce leucocyte migration inhibitory factor (LIF) in response to mitogen or specific antigen. It was determined that T cells enriched for active rosette-forming (A+) cells produced LIF in response to concanavalin A, whereas T cells depleted of active rosette-forming (A-) cells did not. Similarly, A+ cells from a tuberculin-sensitive donor produced LIF in response to tuberculin purified protein derivative, whereas A- cells from the same donor failed to produce the mediator. Thus, T-cell production of LIF in humans appears to be restricted to those T cells capable of active rosette formation.

Phenomenon of human T cells rosetting with sheep erythrocytes analyzed with monoclonal antibodies. "Modulation" of a partially hidden epitope determining the conditions of interaction between T cells and erythrocytes.

Anti-D66 is a monoclonal antibody able to inhibit E-rosette formation of T cells both at 4 degrees C and at 37 degree C but that does not inhibit T cell rosette formation with neuraminidase or 2-amino-ethylisothiouronium bromide (AET)-pretreated E. As demonstrated by capping experiments, it defines an epitope, D66, that is directly involved in E-rosette formation. D66 is distinct from the epitope defined by 9.6 because 9.6, a previously defined "pan-T" monoclonal antibody, inhibits E(AET) rosette formation and because no cross-blocking occurred between both antibodies fixation. However, 9.6 and D66 are carried by the same molecule, as demonstrated by sequential immunoprecipitation assays performed on two different T cell lines. On the thymocyte surface, also, 9.6 and D66 are most probably carried by the same molecule, as indicated by cocapping and colysostripping experiments. D66 is present at higher densities on thymocytes and activated T cells than on peripheral blood T cells. Investigation of numerous T cell populations, both normal and malignant, showed a straightforward correlation between elevated D66 density and ability to form 37 degrees C stable E-rosettes. Neuraminidase treatment of thymocytes and peripheral blood lymphocytes forming E-rosettes unmasked a large fraction of D66 not readily accessible on their surface. These hidden D66 epitopes appear to be responsible for a surprising observation: the ability of anti-D66 to inhibit E-rosette formation could be totally reversed by fixation on anti-D66 of an antibody to mouse immunoglobulin or an Fab fragment anti-mouse immunoglobulin. This would induce microdisplacement with emergence of hidden D66, as documented by fluorometric studies. Finally, malignant T cells with a differentiative status of mature T cells, but forming no (or low numbers of) E-rosettes, could be induced both to display D66 and to form E-rosettes by neuraminidase treatment.

E-rosette formation at 37 °C: Analysis with monoclonal OKT antibodies

When cultured in the presence of PHA, a proportion of human peripheral blood mononuclear cells acquires the capacity to form E rosettes with sheep erythrocytes that are resistant to incubation at 37 °C. The nature of this 37 °C stable E-rosette formation was investigated using a panel of monoclonal OKT antibodies directed to human T-lymphocyte surface antigens. OKT11A antibody, at a concentration of 0.2–0.4 μg/ml, markedly blocked 37 °C E rosetting. OKT1, OKT3, OKT4, OKT6, and OKT8 antibodies, when tested at 10 μg/ml, show no such inhibiting activity. Quantitative studies with 125I-labeled OKT11A indicated that the antibody interacted strongly with both 37 °C E-rosetting and nonrosetting cells, the association constant being 1.6–2.0 × 10 9 M −1 . However, on the average, a threefold higher concentration of OKT11A receptor sites was found on 37 °C E-rosette-forming cells (14.8 × 10 4 sites/cell) than on nonrosetting cells (4.8 × 10 4 sites/cell). Our data suggest that 37 °C E-rosette formation is governed by a lymphocyte surface determinant recognized by OKT11A antibody. “Overexpression” of OKT11A antigenic sites on a proportion of PHA-stimulated lymphocytes may explain their capacity to form 37 °C stable E-rosettes.

Effect of prostaglandins and aspirin on active E-rosette formation in patients with multiple sclerosis

Active E-rosette-forming lymphocytes were examined in patients with multiple sclerosis, patients with other neurological diseases, and patients with rheumatoid arthritis. Significantly fewer ( P < 0.01) active E rosettes were found in multiple sclerosis and RA patients than in controls. The lower levels of active E rosettes in MS could be enhanced by physiological levels of prostaglandins E 1 and E 2 (10 −4 and 10 −11 M). MS patients appar to have a circulating population of immature E-rosetting lymphocytes sensitive to modulation with prostaglandin E.

E-rosette formation with SRBC and changes in cAMP level of hog lymphocytes--a study of certain affecting factors.

A further study of cAMP change in porcine lymphocytes after E-rosette formation is presented in the present report using peripheral blood lymphocytes of pig mixed with different concentrations of SRBC membrane as well as SRBC treated with different enzymes for modification of the rosette formation. It has been found that cAMP enhancement in lymphocyte follows the increase in the concentration of SRBC membrane used, and that a similar relation appeared between modification of cAMP and enzyme-treated SRBC in the rosette formation. It has also been found that after rosetting with SRBC, cAMP level of lymphocytes began to rise within 1 min, reached its peak at 10 min and then returned to control level in about 8 hr.

Effect of histamine, H1 and H2 agonists on E-rosette formation in normal and atopic subjects.

The effect of histamine on the capacity of human peripheral blood lymphocytes to form E rosettes was studied in normal and atopic subjects. Histamine had little effect on E-rosette formation (E-RF) in atopic subjects, but significantly inhibited E-RF in normal subjects in a dose-dependent fashion. To investigate this phenomenon further, specific histamine type 1 and 2 receptor agonists were used in the rosette assay. The H1 receptor agonist caused significant inhibition of E-RF in both normal subjects and atopics, whereas the H2 agonist produced significant inhibition of E-RF in normal but not atopic subjects. The results indicate a histamine type 2 receptor dysfunction in a subpopulation of atopic peripheral blood T lymphocytes.

Appearance of three lymphokines in culture supernatants harvested at different times

Peritoneal cells from mice with methylcholanthrene-induced tumours were reacted with the corresponding tumour extract (antigen) and supernatants were prepared after incubation times of 1, 3.5 and 24 h. All the supernatants were then tested simultaneously on the same pools of normal indicator cells for their effects on leucocyte adherence, E-rosette formation and macrophage migration. The leucocyte adherence inhibition factor (LAIF) was consistently detected only in the 1-h supernatant, E-rosette augmentation factor (E-RAF) only in the 3.5-h supernatant and migration inhibition factor (MIF) only in the 24-h supernatant. It is concluded that LAIF, E-RAF and MIF are probably distinct factors, having different stabilities to degradation or inhibition under the conditions of cell culture. A less likely interpretation is that a single factor with multiple activities is affected by a series of distinct inhibitors. In practical terms, the 3 assays correlate only if the appropriate incubation times are used.

E-rosette formation of lymphocytes from patients with atopic dermatitis

The total number of circulating T lymphocytes in 47 patients with atopic dermatitis was a significantly lower than in 98 healthy persons. However, when the T lymphocytes were investigated for their affinity to sheep erythrocytes (E-rosette formation), no difference was found between patients and healthy persons, when using several different techniques varying in length of incubation of sheep erythrocytes and lymphocytes, presence of fetal calf serum, glassware, media, increased mechanical force at resuspension, and different observers. Our findings are discussed with reference to earlier publications of a reduced percentage of E-rosette forming lymphocytes in patients with atopic dermatitis.

Effects of two synthetic serum thymic factor analogues and four thymic factor fragments on the low E-rosette forming cells in patients with chronic renal failure.

Two new analogues of serum thymic factor (STF), [Dab3]- and [Asn5, Gln9]-STF and four thymic factor fragments, H-Lys-Ser-Gln-OH, H-Lys-Ser-Gln-Gly-OH, H-Lys-Ser-Gln-Gly-OH and H-Lys-Ser-Gln-Gly-Gly-Ser-OH, were synthesized by the solution method, and were tested to determine their effects on the low E-rosette forming cells in patients with chronic renal failure. The analogue [Dab3]-STF and three fragments, H-Lys-Ser-Gln-Gly-OH, H-Lys-Ser-Gln-Gly-Gly-OH and H-Lys-Ser-Gln-Gly-Gly-Ser-OH, increased E-rosette forming capacity when incubated in vitro with patient's blood, but [Asn5, Gln9]-STF and the tripeptide, H-Lys-Ser-Gln-OH, had no effect.

Reactivity of Antiserum to Human Brain with Peripheral Blood Lymphocytes

Antisera to human brain (AHBS) and human thymocytes (AHTS) were produced in rabbits and selectively absorbed to render them specific for T cells. After absorption AHBS, but not AHTS, lost most of its cytotoxic activity against T cells. Absorbed AHBS bound up to 95% of peripheral blood T lymphocytes as detected by indirect immunofluorescence and inhibited up to 46% of the lytic activity of AHTS; however, it was incapable of inhibiting the E-rosette formation of T lymphocytes. All 10 samples of human peripheral blood lymphocytes, pretreated with AHBS, were significantly suppressed in their response to antigens, but fewer samples were affected in their response to mitogens and to allogeneic stimulation, indicating diversity in the nature of the receptors involved in the cellular responses.

Discrepancy between biological activity of thymosin and its cyclic nucleotide-modulating activity.

We have studied cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic 3':5'-guanosine monophosphate (cGMP) concentration in cord blood lymphocytes (CBL) and in adult peripheral blood lymphocytes (PBL) after stimulation with thymosin V-1 and V-2. At concentrations of 100 micrograms/ml, thymosin V-1 produced an 85% increase of cAMP in adult PBL, alterations being demonstrated within 1 min, whereas it did not affect the cAMP level in CBL which have a nine times higher basal level than PBL. On the other hand, thymosin V-1 did not have any effect on cGMP in CBL nor in adult PBL. Thymosin V-2 which inhibits E-rosette formation also increased cAMP in adult PBL like thymosin V-1. The increasing effect of thymosin V-1 on cAMP was observed even when cAMP phosphodiesterase was completely inhibited with isobutyl methylxanthine. From these data it was assumed that thymosin probably acts through the stimulation of adenylate cyclase.

Immunological changes following posttraumatic splenectomy.

In 31 splenectomized individuals blood lymphocytes and serum immunoglobulins were examined 1-11 years after splenectomy. The results demonstrate: 1. Increase of the surface immunoglobulin bearing lymphocytes. 2. Reduction of the E-rosette forming lymphocytes, especially in a combined test assay. 3. Reduction of serum IgM. A surface-restoring effect of the RES of the spleen is discussed as a reason for the diminished E-rosette formation in splenectomized individuals.

Lymphoblastic lymphoma/leukemia of T-cell origin: ultrastructural, cytochemical, and immunologic features of ten cases.

Ten cases of T-lymphoblastic lymphoma/leukemia were studied with light and electron microscopy. Cytochemical strains were performed on touch preparations, and mononuclear cell suspensions were tested for spontaneous rosette formation with sheep erythrocytes, C3 receptors, and surface immunoglobulins. The present investigation was performed to evaluate several ultrastructural parameters, mainly the nuclear shape, as diagnostic clues for this group of lymphomas. Characteristic convoluted nuclei were present in 7 to 47% of the lymphoblasts. This percentage correlated with the focal acid phosphatase reaction and E-rosette formation. Acid phosphatase was the best cytochemical marker (70-100% of the lymphoblasts showed focal reaction product). By ultrastructural cytochemistry, the reaction product was demonstrated in the Golgi cisternae and primary lysosomes. The cell suspensions obtained from different sources contained 14 to 95% E-rosette-forming cells. No specific morphologic, cytochemical, or immunologic differences were found between patients with or without mediastinal involvement.

Autologous mixed lymphocyte reaction in man. II. Histamine-induced suppression of the autologous mixed lymphocyte reaction by T-cell subsets defined with monoclonal antibodies

Human peripheral blood mononuclear cells were separted into T and non-T cells by E-rosette formation. The influence of histamine (10−8−10−3 M) on the proliferative response of T cells in autologous and allogeneic mixed lymphocyte cultures (MLC) was studied. Pretreatment of responder T cells but not of stimulator non-T cells with histamine for 24 hr resulted in a markedly diminished proliferative response in both autologous and allogeneic MLC. A minimum of 4 hr of incubation of T cells with histamine was required to inhibit autologous MLC. Furthermore, T cells pretreated with histamine followed by mitomycin C treatment, when cocultured with fresh autologous T cells, suppressed their proliferative response in both autologous and allogeneic MLC. Analysis with OKT4 and OKT8 monoclonal antibodies revealed that histamine-induced suppressor T cells were contained in OKT 8 + -cell subset. Hitamine-treated OKT 4 + cells had no suppressive effect on the proliferative responses of autologous T cells. Supernatants of T cells cultured with histamine for 24 hr also suppressed both autologous and allogeneic MLC responses of fresh T cells, suggesting that the suppression could be mediated by a soluble suppressor factor(s). Experiments with H1 and H2 agonists and antagonists indicated that histamine-induced activation of suppressor T cells and the production of a soluble suppressor factor(s) were mediated through histamine type 2 receptors.