The active E rosette test: correlation with delayed cutaneous hypersensitivity.

An active subpopulation of peripheral blood T lymphocytes, characterized by rapid (5-min) rosette formation with sheep erythrocytes (A-RFC), was measured in normal individuals after they were skin tested with microbial antigens. A significant rise in A-RFC occurred in all individuals who developed positive delayed cutaneous hypersensitivity (DCH) reactions, whereas skin test nonresponders showed no significant rise in A-RFC. No similar consistent changes occurred in populations of total T cells, characterized by longer (60-min) rosette formation with sheep erythrocytes, or in B cells, measured by immunofluorescence of surface immunoglobulin. The A-RFC response paralleled the DCH response in timing, but not in intensity. These results provide in vivo evidence for a biologically distinct T cell subpopulation, and focus attention on the A-RFC as immunologically active cells.     

Migration inhibitory factor (MIF) and proliferative responses by human lymphocyte subpopulations separated by sheep erythrocyte rosette formation.

Human peripheral blood lymphocytes were separated by a combination of rosette formation with sheep erythrocytes and differential density centrifugation into subpopulations of rosette positive (T-enriched) cells and rosette negative (T depleted) cells. These were then tested in vitro for the production of macrophage migration inhibitory factor (MIF) and for incorporation of ³H-thymidine in response to specific antigens. Both T enriched and T depleted cell populations produced MIF but only T enriched cells exhibited significant antigen-induced ³H-thymidine incorporation. These findings using a T cell surface marker as the basis for cell separation, a technique which should not alter the B cell surface, confirm an earlier report in which human cells were separated on the basis of surface immunoglobulin, a B cell marker.     

Enumeration and isolation of rabbit T and B lymphocytes by using antibody-coated erythrocytes.

Rosette formation with antibody-coated erythrocytes (Ab-E) was employed for the enumeration and isolation of rabbit B cells (Ig+T-) and T cells (Ig-T+). The cells bearing surface Ig (Ig+ cells) were enumerated by a direct immunocytoadhesion technique utilizing anti-rabbit IgG antibody-coated erythrocytes (Ab-E). To enumerate cells bearing thymus cell antigen (T+ cells), an indirect rosette technique was used in which lymphocytes were first sensitized with guinea pig anti-rabbit thymus cell antiserum and then rosetted with anti-guinea pig IgG Ab-E. To demonstrate the specificity of the anti-thymus cell antiserum, a 51Cr radioimmunoassay for counting rosettes was employed along with visual counting to enumerate Ig+ and T+ cells in lymph node cell populations. When Ig+ and T+ lymph node cells were rosetted simultaneously with sheep and human erythrocytes, no mixed rosettes (less than 1%) were observed. Ficoll-Hypaque gradient centrifugation was used to obtain purified Ig+T- and Ig-T+ cells by removing rosetted T+ and Ig+ cells, respectively. The purity of isolated Ig-T+ cells was indicated by 94 to 95% indirect rosetting with anti-thymus cell antiserum and by 0 to 3% direct rosetting with anti-rabbit IgG Ab-E. The purity of isolated Ig+T- cells was indicated by 90 t0 94% direct rosetting with anti-rabbit IgG Ab-E and by 2 to 3% indirect rosetting with anti-thymus cell antiserum. The percentage of Ig+T- and Ig-T+ cells were determined in peripheral blood and in various lymphoid organs. The isolated Ig+T- and Ig-T+ cells were also characterized by their responses to mitogens. Thus, nearly pure Ig+T- and Ig-T+ cells were isolated by "negative selection," which should minimize functional changes of the cells, and thereby facilitate the study of their biologic properties, e.g., their response to mitogens.     

Identification of T and B cell subpopulations in human peripheral blood: electrophoretic mobility distributions associated with surface marker characteristics.

The electrophoretic mobility distributions of human peripheral blood lymphocytes isolated on Ficoll-Hypaque gradients were characterized by laser Doppler spectroscopy. Three major subpopulations were spectrally resolved, due to differences in their mobility, when an electric field was applied to the scattering cuvette. The fastest component (centered at 2.35 mum/sec/V/cm for 25 degrees C, 0.28 M sucrose medium of 0.005 ionic strength) passed through nylon fiber columns and was identified as a T cell subpopulation. The slowest component (1.85 mum/sec/V/cm) which was further enriched by a one-step rosette procedure with sheep erythrocytes, reacted with activated-complement (C3) and antiserum to human immunoglobulins and was therefore identified as a B cell subpopulation. The intermediate component (centered at 2.15 mum/sec/V/cm) appears to be another T cellsubpopulation. Although cells of this component did not pass through nylon fiber columns, they did rosette with sheep erythrocytes. Furthermore, these cells did not appear to have surface immunoglobulins or complement receptors.     

Human autologous rosette-forming cells. III. Binding of erythrocytes from different species to the T-cell receptors for autologous red blood cells

Spontaneous rosette formation in humans is restricted to a subpopulation of the circulating T cells. We have previously shown that the interaction between lymphocytes and autologous red blood cells (auto-RBC) is not mediated by a self-recognition mechanism, since allogeneic (allo-) RBC bind to T cells through the same receptors. In this work, we have extended these observations to thymocytes. Using a mixed-rosette assay in which one type of erythrocyte was identified by FITC labeling, we have shown that almost all the thymocytes which attached auto-RBC could also fix allo-RBC. However, as for the peripheral blood lymphocytes (PBL), binding of human RBC to thymocytes occurred with varying affinities according to the erythrocyte's origin. In order to further study the specificity of the erythrocyte to lymphocyte binding in rosette formation, PBL were mixed with auto-RBC and erythrocytes of xenogeneic (xeno-) origin. Although very disparate incidences of rosettes were found according to the species from which the RBC were derived, most of the autorosetting lymphocytes also had receptors for xeno-RBC. In addition, preincubation of PBL with monoclonal antibody OKT11A (directed against the sheep RBC receptors on T cells) completely abrogated rosette formation with all the erythrocytes tested (human auto- and allo-, sheep, pig, and rabbit) except mouse RBC. Taken together these data strongly suggest that human auto- or allo-, as well as sheep or some other xeno-RBC, bind to T lymphocytes by a single receptor and that the combining sites are expressed with different densities or varying affinities depending upon the RBC's origin. Therefore, spontaneous autorosettes may represent T lymphocytes having high-affinity receptors for sheep RBC.     

Alteration of T and null lymphocyte frequencies in the peripheral blood of human opiate addicts: in vivo evidence for opiate receptor sites on T lymphocytes

Street opiate addiction produces a significant depression in the absolute number of total T lymphocytes in peripheral blood as measured by the ability of the lymphocytes to rosette sheep red blood cells (SRBC). Associated with the decrease in T cells, there is an increase in the absolute number of null lymphocytes but no significant changes in B lymphocytes or total white blood cell count. The T cell values for 2 different populations of addicts (n = 12 and 32) are 31.8% and 23.1%, whereas the null cell values are 51.1% and 57.6%, respectively. The values for comparable control populations (n = 18 and 10) are: T% = 70.7% and 67.4%, and null % = 9.2% and 14.5%. Self-reported use of marihuana does not significantly alter the distribution of cell populations. A 1- to 3-hr incubation of addicted-derived lymphocytes with 10(-6) to 10(-7) M Naloxone reverses both T cell depression and null cell increase by allowing the null cells to express SRBC receptors. Cyclic AMP and dibutyryl cyclic AMP can also convert the null cells to T cells. The conversion of null to T lymphocytes has additionally been measured by monitoring the increase in PHA-stimulated growth in 72-hr cultures as determined by tritiated thymidine incorporation into DNA. These results support the hypothesis that opiates can alter T lymphocyte number and function in vivo, and that this alteration may produce a significant degeneration in the immune competence of street opiate addicts.     

Functional activities of rosette separated human peripheral blood leukocytes.

Subpopulations of human peripheral blood leukocytes were isolated by rosette formation and tested for functional activity. E -rosette-forming cells (E-RFC) and EAC-RFC were separated from non-resetting cells by sedimentation on Ficoll-Hypaque gradients. The efficiency of the method and the purity of the resulting subpopulations were high. E-RFC responded to PHA Con A, allogeneic leukocytes, and PPD, with higher levels of proliferative reactivity than the unseparated lymphocytes while E-RFC depleted, EAC-RFC, and null cells showed only low levels of reactivity. Reactivity to PWM and tetanus toxoid was also restricted to the E-RFC subpopulation, but was lower than that of unseparated cells. A staphylococcal antigen preparation triggered lymphoproliferative reactivity in the E-RFC, E-RFC depleted, EAC-RFC, and the null cell subpopulations. 51Cr release lymphocyte cytotoxicity against a human lymphoblast target cell line was found in the E-RFC and null cell fractions but was not observed with the EAC-RFC subpopulation.     

Histidine-rich glycoprotein blocks T cell rosette formation and modulates both T cell activation and immunoregulation

Histidine-rich glycoprotein (HRGP) is a plasma and platelet protein with undefined function in vivo. It has been reported to inhibit rosette formation between murine T cells and erythrocytes. We have shown that HRGP binds specifically to human T lymphocytes but not sheep erythrocytes and have demonstrated a 56-kDa HRGP-binding protein on the T cell surface which is distinct from the CD2 sheep erythrocyte receptor. We have now investigated whether HRGP can inhibit human T cell-sheep erythrocyte rosette formation and whether HRGP can modulate T cell activation. HRGP at physiologic concentrations specifically inhibited rosette formation between human T lymphocytes and sheep erythrocytes. HRGP suppressed proliferation of antigen receptor (CD3)-triggered T cells induced by interleukin 2; this suppression was specifically reversed by prior incubation of HRGP with affinity-purified anti-HRGP IgG. Addition of HRGP 12-24 h after CD3 triggering no longer suppressed T cell proliferation, suggesting HRGP suppressed T cell division by interfering with one or more early events in the process of T cell activation. Human serum (containing 100-150 micrograms/ml HRGP) was also capable of suppressing T cell proliferation; serum which had been immunodepleted of HRGP no longer inhibited T cell proliferation. Furthermore, HRGP inhibited interleukin 2 receptor expression on activated T cells, causing decreased T cell interferon-gamma release and altered T cell-dependent inhibition of erythropoiesis. HRGP is thus capable of modulating T cell activation and T cell immunoregulation; HRGP may function as a natural suppressive regulator of human T lymphocyte activation.     

Studies of complement receptors on cytotoxic effector cells in human peripheral blood

The question of whether cells bearing complement receptors (CR) mediate cytotoxicity in vitro against allogeneic Chang liver cell targets was investigated by assessing peripheral blood mononuclear cells (PBMC) from normal humans for cell surface characteristics and cytotoxic capacity before and after depletion of CR+ cells capable of forming rosettes with sheep erythrocytes coated with 19S antibody and mouse complement (EAC) and depletion of Fc receptor-bearing cells capable of forming rosettes with human O+ erythrocytes coated with Ripley antibody (EA-Ripley). PBMC depleted of CR+ cells by density centrifugation contained markedly reduced proportions of phagocytes and sIg + cells and increased proportions of both sIg ?, FcR+ cells as well as cells forming rosettes with sheep erythrocytes (E). PBMC depleted of CR+ cells mediated cytotoxicity to an extent equal to or greater than that mediated by unfractionated PBMC in assays of spontaneous cell-mediated cytotoxicity (SCMC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). Cells harvested from the EAC-rosette enriched pellet mediated cytotoxicity 5- to 10-fold less than unfractionated PBMC; however, the cytotoxic activity of the pellet could not be attributed to CR + effector cells since similar cytotoxic activity was present in cell pellets obtained by density centrifugation of PBMC which had been incubated with E coated with 19S antibody or E alone. PBMC depleted of EA-Ripley rosette-forming cells contained decreased proportions of sIg?, FcR+ cells and increased proportions of CR+ cells; PBMC so depleted contained virtually no SCMC and ADCC effector cell activity. These findings indicate that at least the majority of effector cells which mediate SCMC, ADCC, and MICC do not bear CR.     

Fc receptors on mouse effector cells mediating natural cytotoxicity against tumor cells.

Mouse effector cells mediating natural cytotoxicity against tumor cells have been previously thought to be lymphocytes that lack any detectable cell surface markers. The present study presents evidence for receptors for the Fc portion of IgG on these cells. By adsorption of cytotoxic spleen cells on monolayers of sheep erythrocytes (E) plus IgG antibodies to sheep erythrocytes (EA), 50 to 96% of the total cytotoxic reactivity could be removed. Parallel adsorption of cells on E monolayers or on EA monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of cytotoxic activity. The presence of Fc receptors on the NK cells was confirmed by combining EA rosette formation with velocity sedimentation at unit gravity. Peak cytotoxicity occurred at the same sedimentation velocity as the peak of Fc-positive cells. After EA rosette formation, there was a shift to a higher sedimentation velocity in the Fc-positive cells and in the natural cytotoxic activity. The increase in sedimentation velocity of NK activity that was observed in these experiments indicated that most of the cells had only bound a small number (three or four) of antibody-coated erythrocytes. Together, these data indicate that cells with Fc receptors account for most of the total lytic activity of normal mouse spleen cells.     

Lymphocyte populations of Callithrix jacchus marmosets.

A lymphocyte population of common marmosets (Callithrix jacchus) was identified by rosette formation with African green monkey erythrocytes; the rosette-forming cells appeared to be T lymphocytes, as approximately 62% of circulating lymphocytes and 85% of thymus cells formed rosettes with African green monkey erythrocytes. In addition, common marmoset lymphoid cells carrying T-lymphotropic Herpesvirus saimiri or Herpesvirus ateles formed rosettes with African green monkey erythrocytes and treatment of common marmoset circulating lymphocytes with an anti-T cell serum and complement (C′) eliminated rosette-forming cells. Common marmoset T lymphocytes apparently carry a surface receptor for African green monkey erythrocytes, but unlike humans and other closely related nonhuman primates, T lymphocytes of common marmosets fail to form rosettes with sheep erythrocytes.     

Antigenic characterization of a T-CLL with heteroantisera and monoclonal antibodies: evidence for the T cell lineage of an Ia-positive, Fc-IgG--positive, suppressor-cell subpopulation

In a previous report, peripheral blood mononuclear T cells from a patient with T-chronic lymphocytic leukemia (T-CLL) were shown to bear receptors for the Fc portion of IgG (T gamma). Moreover, the ability of these cells to rosette with sheep erythrocytes was strongly inhibited by a preincubation of the cells with theophylline. These data indicated that they represent a highly purified subpopulation of Fc-IgG receptor-positive, low-affinity rosetting cells with in vitro suppressor activity on lectin-induced proliferation of normal lymphocytes. They also were reactive in antibody-dependent cell-mediated cytotoxicity but had no reactivity in natural killer cell assays. These cells were studied in this report with several heteroantisera and monoclonal antibodies. Results indicate that these T-CLL cells express a T cell antigenic pattern (OKT-3+) and the majority are Ia positive. They also react with the OKT-8 reagent (a reagent detecting the subset of T cells that contains the cytotoxic/suppressor cells), whereas they are negative with OKT-4 (which reacts with the subset of T cells that contains helper cells) and OKT-6 (thymocyte) antibodies. Heteroantisera also support the results obtained with monoclonal reagents. Despite some recent evidence showing that a high percentage of T gamma cells may belong to the monocyte-myeloid lineage, these T-CLL cells were negative with OKM-1, a monoclonal antibody reported to detect a monomyeloid antigen. These results suggest that a distinct subpopulation of suppressor T cells can be identified by membrane-marker phenotyping.     

Different maturation capabilities of chronic lymphocytic leukaemia lymphocytes in vitro

Peripheral blood lymphocytes from five patients with B-derived chronic lymphocytic leukaemia were stimulated by Staphylococcus aureus strain Cowan together with T cell mitogen phytohaemagglutinin in 5-9 days suspension cultures. The responses of B lymphocytes were studied on a T cell depleted subpopulation, obtained from harvested lymphocyte cultures using the sheep red blood cell rosette technique. Proliferation tests were performed using a 3H-TdR blast cell index. The maturation process of B-lymphocytes was examined with cytoplasmic Ig studied by FITC-conjugated antisera. Results analysed indicate various degrees of maturation of B cells in different patients.     

Lymphocytes with parallel tubular structures: morphologically a distinctive subpopulation

Peripheral blood lymphocytes were fractionated in T, B and Null cell enriched subsets by means of sheep red blood cell rosette (ESRBC) sedimentation and nylon wool adherence. The ultrastructural features of these subpopulation were investigated. The T cell fraction in which the sheep erythrocytes were removed from the ESRBC rosette-forming cells (ESRBC-RFC) by lysis with ammonium chloride, consisted mainly of two morphologically distinctive subsets. The majority of the cells (80%) displayed a smooth surface membrane and had a high nuclear to cytoplasmic ratio with few cytoplasmic organelles. The other cell type (18%) had a relatively rough surface membrane, a low nuclear to cytoplasmic ratio, often an indented nucleus and numerous cytoplasmic organelles such as characteristic amorphous granules and sometimes parallel tubular structures (p.t.s.). If the T cells were obtained after mechanical vibration of the ESRBC-RFC, the majority of these cells appeared morphologically identical to this latter cell type. Cells with p.t.s. and amorphous granules were also demonstrated within the Null and B cell enriched fractions (50% and 25% respectively), though in the B cell enriched fraction this cell type is probably due to a contamination of Null cells. Previous observations had already demonstrated that these cells in the three fractions represent the Fc gamma receptor-bearing lymphocytes. The similarities suggest that the Fc gamma receptor-bearing and p.t.s. containing lymphocytes form a morphologically distinct subpopulation.     

Activated T lymphocytes within human solid tumors

Summary The majority of lymphocytes separated from tumor cell suspensions were T cells. Conjugates of T lymphocytes and tumor cells were often seen. Variable numbers of T cells exhibited signs of activation such as the ability to form stable E rosettes and attachment to normal and malignant cells (a phenomenon designated natural attachment: NA). A proportion of T cells activated in vitro by allogeneic stimulation regularly exhibit these properties. The T cell-tumor conjugates in the suspensions may represent the NA phenomenon, but they could also be the product of T cells that adhere on the basis of specific recognition of cell surface antigens.Abbreviations BBS balanced salt solution - E rosettes rosettes formed with sheep erythrocytes - EA rosettes rosettes formed with ox erythrocytes coated with anti-ox IgG - FCS fetal calf serum - MLC mixed lymphocyte cultures - NA natural attachment - PBL peripheral blood lymphocytes - SRBC sheep erythrocytes - T lymphocytes thymusderived lymphocytes     

Human T and B lymphocyte rosette tests: Effect of enzymatic modification of sheep erythrocytes (E) and the specificity of neuraminidase-treated E

Sheep erythrocytes (E) were treated with papain or neuraminidase to evaluate what effect these enzymes would have on the E rosette test for human T lymphocytes. Few or no rosettes were detected with sheep erythrocytes treated with papain (E_P). Sensitization of E_P with rabbit antibody and mouse complement resulted in a rosette indicator (E_PAC) which could be used to detect peripheral blood complement receptor lymphocytes (CRL) and thymocyte CRL without having to perform the rosette assay at 37 °C. Neuraminidase treatment of E (E_N) enabled the detection of approximately 20% more rosette-forming cells (RFC) in human peripheral blood lymphocyte (PBL) suspensions compared to untreated E. Rosette studies on a patient with severe combined immunodeficiency disease and on human lymphoblastoid cell lines showed that the additional rosettes detected with E_N were T lymphocytes.     

Cytotoxicity from cultured cells: analysis of precursors involved in generation of human cells mediating natural and antibody-dependent cell-mediated cytotoxicity.

Natural cell-mediated cytotoxicity of human peripheral blood lymphocytes natural killer (NK) against K-562 and antibody-dependent cellular cytotoxicity (ADCC) against Chang cells, as measured in a 4-hr ⁵¹Cr release assay, were both completely removed by depletion of Fc receptor-positive (FcR+) cells. After in vitro culture for 7 days, however, NK- and ADCC-like activities spontaneously regenerated. The nature of precursor cells was studied by examination of lymphocyte subpopulations required for generation of this cytotoxicity. After depletion of FcR+ cells from PBL, the following subpopulations were prepared: sheep erythrocyte rosette-forming cells (E+), surface membrane immunoglobulin-positive cells (SmIg+), and null cells (lacking E+, SmIg+, or FcR+ markers). Separate cell types or mixtures were cultured in vitro in medium containing 10% fetal calf serum for 7 days and then tested for NK and ADCC. Whereas unseparated FcR-depleted cells developed substantial cytotoxic activity, each of the subpopulations cultured alone was negative or had low activity. Addition of SmIg+ cells to other cell types had no effect; however, mixture of 80% E+ and 20% null cells resulted in optimal NK and ADCC. It is not presently clear which population the precursors were in. However, the requirement for proliferation by the null cell population but not by the E+ cells (as indicated by sensitivity to radiation and mitomycin C) suggested that the precursors for NK cells may be null cells.     

Biological characterization of T and B lymphocytes separated from normal mouse spleen by a physical adherence column method

The capacity of spleen cell populations enriched for T and B lymphocytes by a physical adherence column method to respond in vitro to phytomitogens and allogeneic lymphocytes was determined. Column filtrate cells (T lymphocytes) responded well to phytohaemagglutinin- and mitomycin-C-treated allogeneic spleen cells, but poorly to pokeweed mitogen. Adherent cell populations from the column (B and some T lymphocytes) responded well to pokeweed mitogen, but poorly to phytohaemagglutinin- and mitomycin-C-treated allogeneic cells.Purified peripheral T lymphocytes prepared from normal mouse spleen by the column method reconstituted the depleted in vitro antibody response to the thymic-dependent SRBC antigen of all B lymphocyte sources tested, namely, spleen cells from congenitally athymic mice, neonatally thymectomized mice, and adult thymectomized mice which had been reconstituted with bone marrow, and a lymphocyte population prepared by incubating spleen cells with anti-θ serum and complement. When transferred with sheep erythrocytes to congenitally athymic mice, purified peripheral T cells restored the in vivo IgM and IgG responses of these animals. These results confirm that the column filtrate is a thymus derived subpopulation of cells capable of cell-mediated immunity and cooperation with B lymphocytes in humoral immunity both in vitro and in vivo.     

The effect of specific anti-rat thymocyte serum on cell-mediated tumor immunity and other lymphocyte functions

A burro anti-rat thymocyte serum (ATS) was rendered specific for thymus-derived lymphocytes (T cells) by repeated absorptions with Murphy-Sturm lymphoma cells. All of the cells in this lymphoma cell line were shown to have surface immunoglobulin and it was presumed to be bone marrow-derived. Treatment of lymphocytes with ATS eliminated stimulation by phytohemagglutinin and concanavalin A, but did not reduce the proportion of cells with surface immunoglobulin or complement receptor. ATS also did not decrease the number of antibody-producing spleen cells from rats immunized against sheep erythrocytes. The ATS was used to determine the nature of the effector cells in the cell-mediated cytotoxicity of immune W/Fu rat spleen cells against a syngeneic Gross virus-induced lymphoma. This cytotoxic response was consistently inhibited, indicating that T cells were needed for reactivity.     

Immunoglobulin-positive mononuclear cells in human peripheral blood: detection by mixed anti-globulin and direct anti-globulin-rosetting reactions.

Ig-bearing mononuclear cells were identified in Ficoll-Hypaque preparations of human peripheral blood by using mixed anti-globulin (MAG) and direct anti-globulin rosettes; indicator cells consisted of sheep erythrocytes coated with human F(ab')2 or anti-F(ab')2 antibody, respectively. Of the cell population isolated from 10 normal subjects, a mean of 68% was lymphocytes. However, fewer than 50% of the cells with detectable surface Ig were lymphocytes. On viable cell preparations using chromic chloride-treated sheep erythrocytes (CrCl3SRBC) coated with anti-F(ab')2 antibody, a mean of 20.1% of the lymphocytes formed rosettes, i.e., were B. Up to 6% of peripheral blood lymphocytes formed mixed Ig-rosettes and E-rosettes. On viable lymphocytes using F(ab')2-coated CrCl3SRBC, MAG rosettes were insensitive in detection of B lymphocytes. Formaldehyde treatment of lymphocytes increased the number of B cells detectable to 25.5% of the lymphocyte population. Study of T-enriched and B-enriched populations showed that the observed increase in B cell reactivity was real and not due to MAG-rosetting T cells. A one-stage procedure for T and B lymphocyte separation is described.