Double Rosette Research Articles

Peripheral Blood Lymphocyte Receptors for B-Lymphoblastoid Cell Lines (B-LCL)

AbstractBecause interactions between B cells and T lymphocytes are of fundamental importance in the generation of the immune response to most antigens, we attempted to identify the cells capable of binding B-lymphoblastoid cell lines (B-LCL), their tissue distribution, and their presence in other species. Cells bearing a surface receptor for B-LCL were found in human peripheral blood, tonsil, and bone marrow, as well as mouse and rat spleen. Binding cells are phenotypically heterogeneous. The majority are T cells as defined by their ability to bind sheep red blood cells (E-rosettes). However, a subpopulation of non-T-lymphocytes were capable of binding B-LCL. This was demonstrated by depleting T cells with an E-rosette centrifugation technique and then performing a double rosette assay. The wide distribution of T lymphocytes with receptors for B-lymphoblastoid cells within peripheral lymphoid organs and their presence in several species suggest that these surface molecules may represent one of the means by which T cells and B cells interact in the induction of the immune response to T-dependent antigens.

Human lymphocyte/human erythrocyte rosettes. I. Blood “H” rosettes are high‐affinity E‐rosette‐forming T lymphocytes occurring in high frequency

Human lymphocytes will rosette with human red blood cells under certain conditions (H rosettes). The work presented in this paper documents the following: (1) a reproducible short-term assay for the detection of H rosettes in proportions similar to those reported recently in the literature (25-30%); (2) technical improvements on the basic assay system demonstrating that, in fact, 40-65% of human lymphocytes form H rosettes; (3) the fact that, in normal donors, auto and allo H rosettes are formed by identical lymphocyte subpopulations; (4) that H rosettes are formed independently of the ABH, Rh and Thomsen-Freidenreich (TF) red-cell antigens on the indicator red cells; and (5) that H rosette-forming cells consist almost entirely of T lymphocytes having high-affinity receptors for sheep erythrocytes. The large difference in size between sheep and human RBC made it possible to do double rosette assays in which high- and low-affinity E-rosette-forming T cells could be visualized on the same slide. As with T lymphocytes in general, a large proportion of HRFC was found to have Fc-IgM receptors while a smaller proportion has Fc-IgG receptors. Studies done on a small group of leukemic patients supported the T-cell nature of the H rosette-forming cell.

Studies on Fc Receptor Bearing T Cells in Infectious and Immunological Diseases of Children: Part 1. Fc Receptor Bearing T Cells in Rubella and Other lnfectious Diseases

Receptors for the Fc portion of IgG (FcR) has been demonstrated on a small population of T cells using various reagents such as aggregated IgG and erythrocyte-antibody rosette. The immunological role of T cells bearing the FcR (Ty cells) is still unestablished, but the possibility to be suppressor and/or killer has been suggested. In the present study, Ty cells were detected by using double rosette formation consisting of sheep red blood cell (SRBC) and chicken erythrocyte sensitized with IgG (chick-EA). In order to clarify the effects of virus on T cells, the ratio of Ty cells to total T cells was estimated in cases of infectious diseases and discussed. The ratio of Ty cells to total T cells was increased in cases of rubella and measles, however, did not show significant difference in cases of bacterial and mycoplasma infections. This may suggest that the Ty cells reflect the appearance of killer to the virus infected target cells, which is known as one of the important host defence mechanisms against viral infection. The haemagglutination inhibition (HI) antibody titer of rubella and the ratio of Ty cells examined on the 14th day of illness showed statistical inverse correlation (n=22, p<0.05). This may suggest that the Ty cells function as suppressor in vivo, at least on the 14th day of illness.

A rapid method for the isolation of human peripheral null lymphocytes

We describe here a new technique for the isolation of human peripheral null lymphocytes: cells lacking detectable surface immunoglobulin as well as receptors for sheep erythrocytes. The double rosette technique described employs a combined negative selection for Ig + cells by specific anti-Ig-coated sheep erythrocytes and E rosette selection for T cells. This singlestep procedure facilitates the isolation of null cells from large cell populations and can be completed in less than 2 hr. The isolated null subpopulation represents less than 5% of the total peripheral mononuclear cells and is at least 85–90% pure by sensitive surface marker analysis. The null subpopulation is shown to be highly enriched for effectors of both antibody-dependent cytotoxicity against autologous lymphocytes and spontaneous cytotoxicity against the K562 leukemia blast cell line. Application of this technique will allow further characterization of the effector populations for ADCC and NK as well as differentiation of T and B cell precursors.

Age-Related Changes in the Subsets and Functions of Human T Lymphocytes

Abstract The purpose of this study was to investigate changes in immunocompetence associated with aging in humans by the use of various immunologic parameters. Volunteers examined in this study were healthy or apparently healthy without diseases affecting the immune system and ranged from 14 to 89 years of age. The results of this study are summarized in Table I. The absolute number of peripheral blood lymphocytes (PBL) decreased with aging. The mean count was 2043 in the adult group (less than 60 years of age) and 1659 in the aged group (over 60 years of age). When T lymphocytes were assayed by rosette formation with neuraminidase-treated sheep erythrocytes in the presence of fetal calf serum after 1 hr incubation at 4°C, there was no difference in the proportions of T lymphocytes between the groups, whereas the proportions of T cells as assayed by rosette formation with sheep erythrocytes in the absence of fetal calf serum were significantly depressed in the aged group (p &amp;lt; 0.01). On the other hand, the proportion of T lymphocytes assayed by rosette formation with sheep erythrocytes after centrifugation at 1500 RPM for 5 min at room temperature was higher in the aged group than in the adult group (p &amp;lt; 0.02). The proportion of IgG-Fc receptor-bearing T lymphocytes detected by double rosette formation with sheep erythrocytes and chicken erythrocytes sensitized with rabbit IgG antibody was also higher in the aged than in the adult group (p &amp;lt; 0.05). The proportion of complement receptor-bearing lymphocytes did not change with aging. In addition, the proportion of surface Ig-bearing lymphocytes examined after being treated in pH 4.0 acetate buffer for 1 min to remove nonspecifically bound Ig from the cell surface showed no differences between the two groups. Response to PHA and concanavalin A was impaired in the aged (aged 70 to 83 years) compared to the young adult group (aged 20 to 40 years) (p &amp;lt; 0.01), whereas the response to pokeweed mitogen was of the same magnitude. PHA response was negatively correlated with the proportion of IgG-Fc receptor-bearing T cells. The mixed lymphocyte culture reaction with mitomycin-treated AL1 cells derived from Burkitt's lymphoma was also impaired in the aged group (p &amp;lt; 0.01) as was cell-mediated cytotoxicity to AL1 cells as target cells (p &amp;lt; 0.05). Levels of IgG and IgA in plasma were significantly higher in the aged than in the adult group (p &amp;lt; 0.001), whereas those of IgM and IgE were not different. Radioimmunoassay determination of Ig classes revealed that the geometric means of IgG, but not IgM or IgA, synthesized in vitro was significantly increased in the aged compared to the young adult group (p &amp;lt; 0.05) when PBL were cultured in the presence of PWM for 7 days. This increase in IgG synthesis in the aged group was attributed to enhanced T helper activity.

683 E-ROSETTE-FORMING CELLS (E RFC) AND Tγ CELLS (SUPPRESSOR T) PRECURSORS IN CORD BLOOD LYMPHOCYTES (CBL). RESPONSIVENESS TO THYMOSIN FRACTION

CBL “null” cells prepared by nylon column filtration and E rosette depletion constitute roughly 10% of the total Ficoll-Hypaque separated mononuclear cells. This subset, although heterogeneous, is constituted mainly of cells (80%) which possess human T lymphocyte differentiation antigens and/or receptors for the Fc portion of IgG (45% to 58%). Following incubation of CBL*with thymosin (Fraction V), the following changes were seen: E RFC increased from 9% ± 10 to 28% ± 15 (p < .05); Tγ increased from 58.5% ± 14 to 66. 8% ± 12 (p < .05). Employing a double rosette assay using a chicken erythrocyte-IgG complex (CEA) developed in our laboratory, instead of sheep erythrocyte-IgG complex (sheep EA), there was an increase only in the Tγ precursors from 45% ± 7 to 88% ± 5.6 (p < .02), but not in E RFC nor in double rosettes. Since 5 adult peripheral blood specimens tested showed an average of 5.5% double rosettes, we doubt that steric hindrance is the cause of this observation. Rather, the Tγ precursor appears to be more avid for CEA than the mature Tγ cell, or Fc receptors induced by thymosin appear before E receptors. This subset could be a regulator of maternal-fetal immune responses and play an important role in plasma cell differentiation in the fetus.

Lymphoblasts With Both T and B Markers in Childhood Leukemia and Lymphoma

A modification of the double rosette method of Mendes et al. was used to examine lymphoblasts from 22 children with acute lymphoblastic leukemia and 1 child with lymphoblastic lymphoma. This method was used since it directly and simultaneously detected T, B, and null lymphocytes as well as monocytes. In addition it detected the D lymphocyte which has both T- and B- cell surface markers. Based on surface characteristics, lymphoblasts from the patients studied could be divided into three groups: (1) null cells with no detectable markers; (2) cells with T-cell markers; or (3) D or double cells with both the T-cell receptor for sheep erythrocytes and the B-cell receptor for activated complement. No lymphoblasts were found which had easily detectable surface immunoglobulins or complement receptors alone. Eighteen patients had null-cell lymphoblasts; four patients, including the child with lymphoma, had two lymphoblast populations, T and D cells. No circulating D lymphoblasts were detected when these patients went into remission. This simple, reproducible method utilizes easily prepared, stable reagents for surface-marker detection and has the advantage of detecting directly another lymphoid subpopulation, the D cell.

A Thrice Repeated Ossuary Inscription from French Hill, Jerusalem

During the 1970-1971 AIAR salvage excavation of Late Hellenistic and Herodian tombs at French Hill (Karm el-Wiz) north of Jerusalem, one of the thirteen ossuaries discovered was inscribed.' Being the second one found in kokh I of tomb 1, this ossuary was designated TI/K1-OsB. Its 70 cm. length and 27 cm. width, and 40 cm. height, placed it in group II, the largest group of ossuaries found when grouped according to size. Its face was decorated with the conventional incised double geometric rosette, but these finely executed rosettes, each with sixteen heart-shaped petals, were placed between unusual stylized columns whose capitals and bases were depicted with circles (fig. 1:1). The same Aramaic inscription was incised in clear, easily identified characters by at least two different hands on three different surfaces of this ossuary, and read: 4;n M: :CoW'

Rosette-forming cell and cytophilic antibody.

AbstractImmunization of mice with two non‐cross‐reacting erythrocytes results in the appearance of double rosettes. Injection of sera from doubly immunized animals into normal mice also results in double rosette appearance when spleen cells are examined after rosette formation and incubation at 0 °C for 1 h. However, passive rosettes entirely disappear after overnight incubation at 4 °C or after incubation for 1 h at 37 °C, whereas active ones persist in significant numbers. These treatments therefore provide an accurate discrimination between cells passively sensitized by cytophilic antibody and cells actively forming antibody.Removal of cells adhering to plastic surfaces from spleen cells of doubly immunized animals provokes a decrease of simple rosettes and a disappearance of doubles among non‐adherent cells.When adherent cells are tested for existence of hemolytic plaque‐forming cells (PFC), it was found that PFC also adhere. Conversely, the non‐adherent cell population is severely depleted of such cells. Moreover, double PFC, again found in initial cell populations, were recovered with adherent cells but were absent in the final non‐adherent population.