E rosette formation at 37°:A property of mitogen-stimulated human peripheral blood lymphocytes

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C.Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture.This observation also demonstrates technical problems associated with the use of SRBC to quantitate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.     

Receptors for IgM on certain human B lymphocytes.

A receptor for IgM was demonstrated on the surface of human B lymphocytes by using a rosette technique with ox erythrocytes coated with rabbit IgM antibody (EAM). Lymphocytes forming rosettes with EAM did not bind sheep red cells, had membrane Ia-like antigens and, in some instances, surface immunoglobulin. The specificity of EAM rosettes was confirmed by inhibition experiments with purified human Ig. IgM but not IgG molecules inhibited the rosette reaction. In addition, inhibition of EAM rosettes with IgM fragments showed that the receptor has affinity for a part of the molecule located in the Fc portion. By analogy with the receptors previously found on certain human T cells, receptors for IgM were not detected on freshly isolated B cells, but were expressed after overnight culture in IgM-free media. Studies on different human lymphoid tissues showed that IgM receptors are expressed on a limited percentage of both circulating and noncirculating B cells. In addition to normal B cells, the malignant B cells of a majority of cases of chronic lymphocytic leukemia expressed the receptors for IgM.     

A human lymphoid cell line with receptors for both sheep red blood cells and complement

The human lymphoid cell line MOLT 4, from a patient with acute lymphocytic leukemia, was initially considered to be derived from T lymphocytes, on the basis of rosette formation with sheep erythrocytes (E). This cell line has now also been found to form rosettes with sheep erythrocytes sensitized with rabbit antibody and mouse complement (EAC). Evidence is presented that the formation of both E and EAC rosettes is due to two separate receptors on the MOLT cells: (a) EAC rosettes were formed more rapidly and were more stable than E rosettes; (b) preincubation of MOLT with an EAC membrane preparation inhibited resetting with EAC and not with E; (c) MOLT formed rosettes with EAC prepared from trypsinized E, but did not bind to trypsin-treated E alone. The implications of this finding, in regard to the derivation of this cell line, are discussed.     

The significance of varying SRBC/lymphocyte ratio in T cell rosette formation.

The incubation ratio of sheep red blood cells (SRBC) to lymphocytes is a critical factor in rosette formation, whereas the length of time SRBC and lymphocytes are incubated together does not significantly affect the percentage of lymphocytes forming rosettes. The graph obtained by plotting percentage of rosette formation against the ratio of SRBC to lymphocytes is similar to that resulting from the formation of bimolecular complexes. If rosette formation is analogous to formation of bimolecular complexes, maximal rosette formation occurs when the system is saturated, i.e., with excess SRBC, and is a measure of the total capacity of a lymphocyte population to form rosettes. In addition, the percentage of rosette formation observed at a limiting SRBC/lymphocyte ratio gives an indication of the avidity of the lymphocytes for SRBC. This interpretation may provide an explanation for the difference between the "active" and "total" rosettes. When the log of the SRBC/lymphocyte ratio is plotted against percentage of rosette formation, a straight line is obtained, suggesting that within a given normal lymphocyte sample, T cell subsets with different avidities are not detected by rosette formation at different SRBC/lymphocyte ratios.     

Inhibition of T-lymphocyte rosetting by HL-A alloantisera and complement.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.     

Human lymphocyte subpopulations: giant SRBC rosettes.

Human thymus-derived lymphocytes have the ability to form rosettes with sheep red blood cells (SRBC) in vitro. In the investigation of rosettes of peripheral blood lymphocytes of 10 normal subjects, the number of SRBC adhering to the lymphocyte in each of 100 rosettes was assessed. The percentage of rosettes with SRBC greater than or equal to 36 per rosette was only 1.2 +/- 0.5. These were defined as giant SRBC rosettes. Peripheral blood lymphocytes were stimulated in vitro by four mitogens: sodium periodate, neuraminidase plus galactose oxidase, pokeweed mitogen, and concanavalin A. The lymphocytes were then cultured at 37 degrees C. The giant rosette-forming lymphocytes became significantly increased 4 to 24 hr after stimulation, prior to the appearance of lymphoblasts or increased incorporation of tritiated thymidine. The giant rosettes were not caused by the hemagglutinating properties of pokeweed mitogen and concanavalin A that were adsorbed on the lymphocyte surfaces. This was shown by the fact that, on removal of the receptors by trypsinization, they were regenerated on culture in vitro in the absence of the mitogens. It was concluded that giant SRBC rosettes constituted a marker for some of the activated lymphocytes. Their appearance was independent of the increase in size of the cells or of DNA synthesis. These receptors were intrinsic to lymphocytes and not caused by mitogens adsorbed on their surfaces.     

Numbers of rosette formine cells in human peripheral blood.

Thymus-derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood were quantitated by assaying percentages of cells forming erythrocyte rosettes. T-cell rosettes were formed with neuraminidase treated sheep erythrocytes. B-cell rosettes were formed with complement coated sheep erythrocytes. Large differences in the percentages of T-rosette forming cells were noted depending on the method used to assay these cells. When rosette forming cells (RFC) and non-RFC were counted concurrently the percentage of T-cell rosettes were 53–75% whereas methods involving the separate counting of RFC and total cells gave T-cell RFC percentages of 23–40%. These differences were due to the “co-rosetting” of non-RFC into the T-cell rosette clusters. This occurred because of the gentleness required to resuspend the fragile T-cell rosettes. “Co-rosetting” was demonstrated by forming stable complement receptor rosettes with complement-coated human erythrocytes and resuspending them either gently or vigorously. Significantly higher percentages of rosettes were noted with gentle cell suspension than with vigorous resuspension. The percentages of rosette forming T-cells in human peripheral blood are therefore lower than previously estimated.     

Studies of the sheep red blood cell receptor using anti-lymphocyte antibodies

Human thymus derived lymphocytes (T cells) interact with sheep red blood cells (SRBC) to form rosettes. We wanted to determine whether the lymphocyte's receptor for SRBC is associated with serologically detectable cell surface antigens. Antisera were prepared by immunizing horses with either fresh human thymus (ATG) or with B lymphocytes from an established lymphoid cell line in culture (ALG). ATG, ALG or Concanavalin A (Con A) were added to lymphocyte preparations to determine their effect on rosetting. The results showed that ATG inhibited rosettes in a dose dependent manner. In contrast, both the Con A and ALG had no effect. By immunofluorescence, Con A and ALG staining cells were able to form rosettes. ATG staining cells were unable to form rosettes. Removal of the ATG receptor by capping could not restore the rosette forming capacity suggesting that inhibition was not due to steric hindrance. We conclude that antibody directed against T cells but not B cells binds to surface antigens which appear to be identical with or in close proximity to the specific SRBC receptor.     

Involvement of human membrane-associated complement components in the rosette formation between marmoset red blood cells and human leukocytes

The majority of human monocytes, a subpopulation of B lymphocytes, and all B lymphoblastoid cell lines (B-LCL) tested but one form rosettes with Marmoset red blood cells (MaRBC). None of the human peripheral T cells, T-LCL, and B chronic lymphoid leukemia cells (B-CLL) used bind to MaRBC. The binding could not be correlated with any membrane markers or antigens present on cultured cells or peripheral blood leukocytes (PBL). Blocking of the rosette formation by preincubation of MaRBC with purified human complement (C) components and cobra venom or by pretreatment of leukocytes and cultured cells with antisera to human C components suggested that membrane-associated C components present on PBL or B-LCL are involved in the binding to MaRBC.     

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.     

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     

Cultivation with K562 cells leads to blastogenesis and increased cytotoxicity with changed properties of the active cells when compared to fresh lymphocytes.

Lymphocytes from healthy donors were cultivated with K562, a cell line sensitive to natural cytotoxicity and lacking HLA antigens. Blastogenesis and cytotoxicity were generated in the presence of the serum of the lymphocyte donor. The anti K562 killing effect of the cocultivated populations exceeded considerably that of the fresh lymphocytes. In the majority of cases lymphocytes cultured alone had no cytotoxic activity.We have attempted to characterize the active lymphocyte subset, using the fractionation procedure designed for fresh lymphocytes. Separation of T cells on the basis of SRBC rosetting was not efficient because a high proportion of E rosettes remained in the interface when centrifuged on the Ficoll. Furthermore a high number of E rosetting cells adhered to nylon wool.The cytotoxic potential of various fractions correlated to the presence of blasts and EA positive cells. The least active population was the non-adherent, E rosette sedimented one. The cytotoxicity was not restricted to the sensitizing K562 cell line.     

Inhibition of human T lymphocyte E rosette formation by neutrophils and hydrogen peroxide. Differential sensitivity between helper and suppressor T lymphocytes

Co-incubation of human mononuclear cells with small numbers of autologous polymorphonuclear leukocytes resulted in a ratio-dependent inhibition of T lymphocyte rosette formation with sheep erythrocytes (SRBC). Inhibition by polymorphonuclear leukocytes was reversed with catalase, implicating H2O2 or associated products as the suppressive agent(s). Exposing T lymphocytes directly to micromolar concentrations of H2O2 also inhibited subsequent rosette formation. Inhibition was dose-dependent between 40 and 320 microM H2O2 and was not due to cytotoxicity at those H2O2 concentrations. Kinetic studies demonstrated that after exposing T lymphocytes to H2O2 a proportion of cells appeared to be relatively resistant in that they retained their ability to form E rosettes. T cell phenotype analysis of these cells showed that, in contrast to untreated T cells, H2O2-treated T rosette-forming cells were almost exclusively of the helper/inducer phenotype. Analysis with the monoclonal antibody 4B4 revealed that H2O2-resistant T rosette-forming cells contained significantly fewer 4B4-positive cells than predicted for the T helper population, indicating that H2O2-resistant T cells might be a subset of helper/inducer T lymphocytes. The interaction between H2O2 and T cells was rapid but did not lead to loss of Leu-5b binding sites. Preliminary functional analysis indicates that interleukin 2 production and phytohemagglutinin-induced proliferation by the relatively H2O2-resistant T cells is similar to untreated T cells. In contrast, H2O2-treated T cells which lost their ability to form E rosettes produced undetectable levels of interleukin 2 and proliferated poorly in response to phytohemagglutinin. Finally, mononuclear cells pretreated with H2O2 and subsequently stimulated with mitogens demonstrated a preferential expansion of the T helper population with concomitant loss of T suppressors. Our results show that, although neutrophil-released H2O2 inhibits effective interactions between SRBC and T lymphocyte sheep erythrocyte receptors, there appears to be a population of T helper cells which is relatively resistant to H2O2 both in terms of SRBC rosette formation and response to mitogens. These data suggest that at sites of inflammation phagocyte-released H2O2 could exert immunoregulatory effects on T cells by altering T cell subset survival and allowing the function of a particular lymphocyte population to predominate.     

The assay, properties, and kinetics of human T cells forming clusters with sheep erythrocytes (CFC) in stimulated cultures.

This study describes a new method of detecting the in vitro stimulation of lymphocytes based on the appearance of cells having the property to cluster several layers of SRBC around themselves (CFC). The formation of multilayer rosettes is temperature dependent and requires trypan-blue-excluding, metabolically active blastoid cells. Non-separated cells as well as purified T cells stimulated with allogeneic leucocytes (MLR), specific antigens, or polyclonal mitogens (PWM, Con A) gave rise to CFC. Multilayer rosettes were not formed by separate B cells. In the MLR, CFC were detected 48 hr after the beginning of cultures and increased in number thereafter just like thymidine incorporation. The response to Con A and PWM was detected earlier and gave rise to two or three peaks, the first rise in the number of CFC coinciding with the peak of thymidine incorporation but the maximum increase occuring two or three days later. Treatment of blastoid cells with a serum specific for T cells, but not with an anti-immunoglobulin serum, abolished their ability to form ordinary or multilayer rosettes. When separated, however, on a nylon wool column, CFC were more adherent than the bulk of T cells. It is suggested that CFC form a subpopulation of T cells with distinct characteristics, allowing a direct assessment of membrane changes which take place when T lymphocytes are activated in vitro.     

树鼩(Tupaia belangeri)淋巴细胞的自体花结

参与人自体花结(A花结)形成的分子(如CD2/LFA-3),与免疫细胞的粘附和激活有关。我们曾发现,人和猴淋巴细胞表面的树鼩红细胞(TRBC)受体不同于绵羊红细胞(SRBC)受体(CD2),可能是一种新的白细胞分化抗原。花结试验表明,树鼩的外周血淋巴细胞(TPBL)和胸腺细胞都能形成A花结,结花率分别为20.9％和11.1％;而绵羊红细胞花结(E花结)形成率分别是20.9％和1.1％。以四种单克隆抗体(McAb)(Leu 5,0-275,AICD2.1和E2 McAb)进行树鼩A花结和E花结的抑制与抗原调变试验,结果表明,这些抗体对树鼩的A花结都没有明显的抑制或调变作用,但对E花结的抑制及调变作用明显。说明TPBL表面的TRBC受体不同于SRBC受体,与CD2/LFA-3及E2分子无关。因此,TPBL的A花结与E花结形成机制不同。     

Carbohydrate specificity of T-cell cytophilic chicken anti-SRBC IgM antibodies.

Fixation of passively sensitized lymphocytes by formaldehyde resulted in amplified erythrocyte rosette formation. The cytophilic anti-SRBC antibodies were of the IgM class and they attached selectively to thymocytes and T lymphocytes. Anti-SRBC sera from approximately 30% of immunized chickens gave rosette counts between 500–1500 per 10⁴ cells whereas the remainder gave values considerably lower. The onset and peak of the cytophilic IgM response were reached later than that of IgM haemagglutinating antibodies and there was little correlation between the count of cytophilic rosettes and haemagglutinjn titres in individual chickens. On the basis of competitive inhibition of rosette formation by a hog blood group substance it is suggested that the cytophilic antibodies have binding site specificity for a saccharide on the surface of SRBC.     

Human lymphocyte receptor movement induced by sheep erythrocyte binding: effect of temperature and neuraminidase treatment

The formation of sheep red blood cells (SRBC) rosettes by human lymphocytes was promoted by incubation at 4 °C and by treatment of the lymphocytes or SRBC by neuraminidase. On incubating the untreated SRBC rosettes at 37 °C, the rosettes dissociated by capping in which rings were converted into horseshoes and then caps. This capping was inhibited by incubation of the rosettes at 4 °C and partially by treatment of the cells with neuraminidase. During rosette formation, the proportion of caps decreased progressively during 4 °C incubation. This decrease of capping was also promoted by neuraminidase treatment. We concluded that the main reason why 4 °C and neuraminidase treatment facilitated rosette formation was by inhibition of capping.     

Interaction of antigen-specific T cell factors with unique "receptors" on the surface of mast cells: demonstration in vitro by an indirect rosetting technique

Picryl (trinitrophenyl) chloride (PCL) contact sensitization of mice induces T cells that release an antigen-binding T cell factor (PCLF) that plays an important role in the initiation of contact sensitivity responses, in part via activation of mast cells. The current study employs an in vitro indirect rosette assay to demonstrate that PCLF can interact with the mast cell surface. Sheep red blood cells (SRBC) were hapten conjugated with trinitrophenyl (TNP), dinitrophenyl (DNP), or oxazolone (OX). When TNP-conjugated SRBC were coated with PCLF, monoclonal anti-DNP IgE, or anti-DNP IgG1, they produced 40 to 50% rosettes with purified normal mouse peritoneal mast cells. Analogous antigen-binding factors, from lymphoid cells of OX and dinitrofluorobenzene contact-sensitized mice, gave similar mast cell rosetting levels with OX-SRBC and DNP-SRBC, respectively. PCLF demonstrated a high degree of hapten specificity in that it formed rosettes with TNP-SRBC but not with DNP-SRBC, unlike IgE and IgG1, or DNPF, which formed rosettes with either SRBC type. Similarly, soluble TNP-BSA could inhibit PCLF rosette-forming capacity, but soluble DNP-BSA could not. In addition to mouse mast cells, PCLF formed rosettes with rat basophil leukemia cells, mouse peritoneal exudate macrophages, mouse alveolar macrophages, and J 774 cultured mouse macrophages; it did not form rosettes with rat mast cells, rat alveolar macrophages, or mouse spleen cells. Thus, PCLF-formed rosettes were antigen specific, relatively species specific, and mast cell/macrophage specific. PCLF-mediated rosette-forming activity could be detected in the presence of nanogram quantities of PCLF. More than 10 times greater IgE was needed to produce IgE-mediated rosettes. Reduction and alkylation eliminated the rosetting activity of IgE, but the rosetting activity of PCLF was not affected. PCLF, but not IgE rosette-forming activity, could be removed by and eluted from affinity columns linked with a monoclonal antibody specific for T cell-derived antigen-binding factors, whereas PCLF rosetting activity was not retained by an anti-immunoglobulin affinity column. Preincubation of mast cells with rat myeloma IgE or mouse monoclonal IgE of various specificities blocked IgE rosettes but not PCLF-induced rosettes. Other immunoglobulin isotypes likewise did not block PCLF rosettes. However, PCLF rosettes could be blocked by preincubation of mast cells with OX factor (OXF),and OXF-mediated rosettes could be blocked similarly by PCLF. These results suggest that the antigen-binding T cell factor PCLF interacts with a unique receptor on the surface of mouse mast cells.     

A human B lymphocyte antigen (P-76) shared by B-cell chronic lymphocytic leukemia cells and hairy cell leukemia cells

A membrane antigen with an apparent specificity to B lymphocytes was detected with immunochemical techniques and its properties were analyzed. Anti-B-CLL serum was raised in a rabbit by immunization with B-cell chronic lymphocytic leukemia (B-CLL) cells. This anti-B-CLL serum was absorbed with erythrocytes, liver homogenate and insolubilized immunoglobulins. After further absorption with T-CLL cells, chronic myelocytic leukemia (CML) cells and acute myelocytic leukemia (AML) cells, the anti-B-CLL serum still reacted with peripheral blood B lymphocytes, B-CLL cells and hairy cell leukemia (HCL) cells. In contrast, no reactivity was seen with peripheral blood T lymphocyte or monocytes, or leukemia cells of non-B cell origin. An immunoprecipitation of radiolabeled cell surface proteins was attempted using the anti-B-CLL serum in the presence of Staphylococcus Aureus Cowan 1 (SaCl), and the precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A membrane antigen with an apparent molecular weight of 76,000 daltons (P-76) was immunoprecipitated with the anti-B-CLL serum from the lysates of normal B lymphocyte, B-CLL cells and HCL cells. The antigen (P-76) is not composed of disulfide-linked subunits and has no structural relationship with HLA-DR (Ia-like) antigens or other known antigens. These results suggest that this antigen is B-lymphocyte specific, and favour the B-lymphocyte nature of HCL cells.