Lymphocyte binding of aggregated IgG and surface Ig staining in chronic lymphocytic leukaemia

Eleven selected patients with chronic lymphocytic leukaemia were evaluated for lymphocyte binding of aggregated IgG and surface Ig staining in order to classify them into B and T cell types. Ten of the eleven patients bound aggregates and stained for surface Ig. In the individual ten patients the number of cells binding aggregates was high (88–100%, mean 96%) whereas the number staining for surface Ig was more variable (8–100%, mean 62%). Parallel and double labelling experiments with aggregates and sheep red blood cell rosettes, a human T cell marker, provided evidence that aggregates were binding to B cells only, even when surface Ig was not detectable. Aggregates did not bind to human thymocytes. Evidence was presented that lymphocytes from some cases of CLL have low but not absent amounts of surface Ig that may be only partially detected by fluorescence techniques. Aggregate binding appears to be a more sensitive method for the detection of B lymphocytes than surface Ig staining.In one of the eleven patients the leukaemic cells were negative in the aggregate binding test. Separate studies on this case also indicated an absence of surface Ig staining and a high percentage of cells forming sheep red blood cell rosettes. It would appear that this case represented a T cell leukaemia.     

Electrophoresis of lymphocytes from normal human subjects and patients with chronic lymphocytic leukaemia

The electrophoretic mobility (EM) of peripheral blood lymphocytes from twelve normal subjects and four patients with chronic lymphocytic leukaemia (CLL) was studied in relation to the thymus-derived (T) lymphocytes and bone marrow-derived (B) lymphocytic system. An attempt was made to correlate electrophoretic results with other methods of identifying T and B lymphocytes—T cells by the formation of sheep-cell rosettes and B cells by the formation of erythrocyte–antibody–complement (EAC') rosettes and by staining for surface immunoglobulin.

In the normal subjects the majority of cells migrated quickly with a small `tail' of slower cells. It is suggested that the faster populations are T cells and the slower, B. In CLL the majority of the cells were slow migrators. There was agreement between the percentage of fast cells as assessed by electrophoresis with that of T cells by sheep-cell rosetting; there was also some agreement between the percentage of electrophoretically slow cells to B cells by EAC' rosettes or surface immunoglobulin.

It was possible to remove some of the B cells by density centrifugation after forming EAC' rosettes. This further defined the T cell peak on electrophoresis.

It is concluded that T and B cells carry different surface charge densities which permit them to be separated by electrophoresis and that the malignant B lymphocytes of CLL migrate electrophoretically in a similar fashion to normal B cells.

     

Evidence of Aberration of T-cell Subsets in Aged Individuals

In the present study, T-cell subsets from aged individuals were examined by using anti-BAT (brain-associated thymocyte antigen) serum. Anti-BAT serum was raised against the human fetal brain at 28 weeks of gestation. After absorption wit AB erythrocytes, B-cell lines, and leukaemic cells, anti-BAT serum was T cell-specific but unreactive to normal B cells. The ability of anit-BAT serum-treated lymphocytes from aged individuals to respond to concanavalin A, phytohaemagglutinin, and pokeweed mitogen (PWM) was unaltered even at a high concentration. In PWM-stimulated Ig synthesis, T lymphocytes lacking the anti-BAT serum-reactive T-cell subset enhanced the PWM-stimulated Ig synthesis of autologous B lymphocytes from young individuals. The Con A-induced suppressor function of lymphocytes from aged individuals was not significantly abolished by treatment with anti-BAT serum and complement. In the autologous mixed lymphocyte reaction, the decrease in response was minimal when responder cells from aged individuals was treated with anti-BAT serum even at a high concentration. It is concluded that the T-cell subset with suppressor function is defective in aged individuals.     

Stimulating capacity of fresh and cultured human leukaemic lymphoid and myeloid cells in `one-way'' mixed lymphocyte reaction

Fresh normal peripheral blood B lymphocytes possess a strong stimulating capacity while fresh thymus cells or fresh peripheral T lymphocytes possess a weak, but significant stimulating capacity on allogeneic lymphocytes in `one-way' mixed lymphocyte reaction. Fresh leukaemic T lymphoid cells from patients with T-cell ALL or T-cell CLL exert little or no stimulation on allogeneic lymphocytes. Fresh leukaemic B lymphoid cells from patients with B-cell CLL or B-cell HCL, on the other hand, exert a lesser stimulation on allogeneic lymphocytes, as compared to that of normal B lymphocytes. Leukaemic myeloblasts from patients with AML or Ph<sup>1</sup>(+) CML-BP exert significantly higher stimulation than leukaemic lymphoid cells in `one-way' mixed lymphocyte reaction (P<0.05). Cultured leukaemic T lymphoid cells (MOLT-4) possess no stimulating capacity, cultured leukaemic B lymphoid cells (BALM-2) possess a moderate degree of stimulating capacity and cultured leukaemic, possibly myeloid, cells (NALM-1 and K562) possess vigorous stimulation on allogeneic lymphocytes. The stimulating capacity of NALM-1 or K562 cells is significantly higher than that of BALM-2 cells (P<0.01 or P<0.05, respectively) and that of MOLT-4 cells (P<0.001). These observations suggest that the stimulating capacity of leukaemic T or B lymphoid cells may have been completely or partially lost during the process of leukaemogenesis. Since we do not have an opportunity to study the stimulating capacity of normal myeloblasts, it is not known whether the stimulating capacity of leukaemic myeloblasts, which is found to be very strong on allogeneic lymphocytes, may have been modified during the process of leukaemogenesis.     

Mitogenic stimulation of malignant B cells. Chronic lymphocytic leukaemia: relationship between stimulation and surface phenotype.

Peripheral blood lymphocytes (PBL) from 14 patients with chronic lymphocytic leukaemia (CLL) were stimulated with pokeweed mitogen (PWM), formalinized Staphylococcus aureus (Sta) and a combination of both mitogens. The leukaemic B cells were characterized by rosetting techniques (using mouse erythrocytes and complement coated erythrocytes) and immunofluorescence for membrane bound immunoglobulin (mIg). No clear correlation between phenotype and the reactivity with PWM could be found. Results of stimulation with Sta however, indicate that lymphocytes carrying membrane bound IgM (mIgM) and IgD (mIgD) and the receptor of the third complement component (C3R) can be induced to differentiate into immunoglobulin (Ig) containing cells. Addition of PWM to these cultures often enhanced this response. Some leukaemic B cells are able to differentiate after challenge with the appropriate stimulus.     

Studies on non-H-2 linked lymphocyte activating determinants. I. Description of the cell type bearing the Mls product.

The non-H-2 linked, lymphocyte activating determinants (LADs), which are coded by the minor (histocompatibility) lymphocyte stimulating locus (Mls), were detected on B lymphocytes, but not on immunoglobuin (Ig) negative or on theta positive T lymphocytes. These determinants, like the complement receptor and surface IgD, develop late in neonatal life. Thus, the Mls coded LADs mark a population of B lymphocytes which are not present in the spleens of newborn to 2-week-old mice. Studies of B lymphocytes derived from the spleens of adult mice indicated that the cells bear the Mls coded LADs have a high density of surface Ig and possess the complement receptor.     

STUDIES ON NON-H-2 LINKED LYMPHOCYTE ACTIVATING DETERMINANTS I. DESCRIPTION OF THE CELL TYPE BEARING THE MLS PRODUCT

The non-H-2 linked, lymphocyte activating determinants (LADs), which are coded by the minor (histocompatibility) lymphocyte stimulating locus (Mls), were detected on B lymphocytes, but not on immunoglobulin (Ig) negative or on theta positive T lymphocytes. These determinants, like the complement receptor and surface IgD, develop late in neonatal life. Thus, the Mls coded LADs mark a population of B lymphocytes which are not present in the spleens of newborn to 2-week-old mice. Studies of B lymphocytes derived from the spleens of adult mice indicated that the cells that bear the Mls coded LADs have a high density of surface Ig and possess the complement receptor.     

Ultrastructural and serological studies on the resistance of activated B cells to the cytotoxic effects of anti-immunoglobulin serum. Patch and cap formation of surface immunoglobulin on mitotic B lymphocytes.

Previous studies have shown that rabbit anti-mouse immunoglobulin sera (anti-Ig) which kill non-activated B lymphocytes in the presence of complement, are incapable of doing so when the cells are activated by antigen or mitogen into mitosis. Results reported here indicate that the resistance is not dependent on either the source of antiserum or complement, or on the presence of a mitotic inhibitor, colcemid. Immunoperoxidase staining-electron microscopy techniques were applied to assess whether there was any conspicuous difference between unstimulated versus mitogen-stimulated, mitotic cells with respect to density or distribution of cell surface Ig. No such differences were found; furthermore, mitotic cells showed rapid classical ''patch and cap'' formation of cell surface Ig when incubated with anti-Ig at room temperature, indicating the retention of fluid membrane dynamics by lymphocytes in this stage of the cell cycle. In contrast to this cytotoxic resistance, T or B lymphocytes in mitosis were found to be as sensitive, or more so, to lysis by various other antisera when compared to non-mitotic cells. Thus the resistance of mitotic B cells to the cytotoxic effects of anti-Ig serum seems unique and appears independent of any conspicuous quantitative or qualitative change in cell surface Ig.     

Activation of human B lymphocytes induced by Robinia pseudoacacia lectin in the presence of T cells.

Robinia pseudoacacia seed lectin is a potent human lymphocyte activator which is capable of activating pure T cells but not pure B lymphocytes. However, when B and T cells were cultured together, the thymidine incorporation was found to be higher than that expected from B- or T-cell cultures alone. Killing of T cells by anti-human-T-lymphocyte antigen (HTLA) serum and complement at the time of thymidine incorporation was found to be unable to suppress completely the thymidine uptake whereas treatment by anti-human-B-lymphocyte and monocyte antigen (HBLMA) serum reduced the response to some extent. Moreover, stimulated lymphoblasts were shown to bear B-cell markers (surface Ig and complement receptors) in about the same proportion as B lymphocytes present in the cultures. These results show that B cells proliferate in the presence of T cells and Robinia lectin. Finally, activation of B cells by Robinia lectin in the presence of T cells led to their maturation to plasma cells in the same way as PWM.     

Separation of T lymphocytes from normal individuals and patients with B lymphocyte chronic lymphocytic leukaemia.

Previous studies have shown that lymphocytes from patients with chronic lymphocytic leukaemia have a diminished response to mitogens which stimulate T cells. Chronic lymphocytic leukaemia is most often a disease of accumulating B cells so that T lymphocytes are diluted by large numbers of leukaemic cells. Direct comparison with the responses of normal lymphocytes to mitogenic stimulation is therefore suspect. To circumvent this difficulty, a method of isolating T cells from normal individuals and patients with chronic lymphocytic leukaemia was developed. Lymphocytes containing an average of 16.1 per cent B cells from normal individuals were applied to IgG-anti-IgG-coated Degalan bead columns and held at 4 degrees for 2 hours. The eluted cells contained less than 2 per cent B cells. When chronic lymphocytic leukaemic lymphocytes, containing an average of 68.6 per cent B cells, were applied to IgG-anti-IgG columns, the eluted cells contained 36.4 per cent B cells. To improve the purification of T lymphocytes, columns of uncoated Degalan beads were used to remove non-specifically adherent cells. Eluted lymphocytes were then applied to IgG-anti-IgG columns. This resulted in the recovery of purified populations of T cells with less than 2 per cent contamination with B cells. Patients with chronic lymphocytic leukaemia were found to have lymphocytes with either a normal density or a low density of surface immunoglobulins. B cells were successfully removed from lymphocyte suspensions in all cases of chronic lymphocytic leukaemia with a normal density of lymphocyte surface immunoglobulins. In the three cases of chronic lymphocytic leukaemia with low density surface immunoglobulins, separation by this method was unsuccessful. However, an enriched T-cell population was obtained when leukaemic lymphocytes which had lost all detectable surface immunoglobulins were passed through a column coated with heat-aggregated IgG.     

A Simple Method for Specific Depletion of Lymphocytes from Bovine Peripheral Blood Mononuclear Cells

The treatment of bovine peripheral blood mononuclear cells with rabbit anti-bovine immunoglobulin, goat anti-rabbit immunoglobulin (GAR) and complement resulted in the specific lysis of all surface immunoglobulin (SIg) bearing B lymphocytes. No SIg lymphocytes were detected after 12 hours of culture following lytic treatment, whereas cells treated with antibody without complement readily stained for SIg. The percentage of cells forming E-rosettes or binding peanut agglutinin (PNA) increased following lysis. The percentage of latex-ingesting monocytes also increased after lysis even though many of these cells had cytophilic Ig. B lymphocyte-depleted (T cell-enriched) populations cultured with phytohemagglutinin (PHA) and concanavalin A (Con A) were never less reactive than unseparated cells. No differences in background mitosis was observed for these 2 cell preparations. These results suggest that bovine SIg^? cells do not require B lymphocytes to respond to PHA and Con A and that lysis of B lymphocytes does not alter the responsiveness of the recovered SIg^? cells.     

Human peripheral lymphocytes bearing both B-cell complement receptors and T-cell characteristics for sheep erythrocytes detected by a mixed rosette method

Human peripheral lymphocyte preparations were tested with a mixed rosette method for the presence of lymphocytes bearing both the complement receptors characteristic of B lymphocytes, and the capacity of T lymphocytes to spontaneously bind with sheep red blood cells (SRBC). The large and oval shaped pigeon red blood cells (PRBC) which do not form T-cell rosettes were utilized as indicators for rosette formation with the complement receptor-bearing B cell. In every individual (an average of 2·06%) lymphocytes were observed which formed rosettes with both SRBC and PRBC indicators. These findings show that independent markers for both T and B cells may be present on the surface of the same lymphocytes.     

Lymphocyte subpopulations in peripheral blood of healthy persons. Characterization by surface markers and lack of selection during purification

Lymphocytes were isolated at 99% purity from peripheral blood of healthy persons by defibrination, gelatine sedimentation, treatment with carbonyl iron powder and centrifugation on Ficoll–Isopaque. Subpopulations were identified by three surface markers: cells forming rosettes with sheep red blood cells (SRBC) (E-binding lymphocytes) as a measure of T lymphocytes; lymphocytes with surface immunoglobulin identified by indirect immunofluorescence (B lymphocytes); lymphocytes with receptors for C3 observed by the rosette method using SRBC treated with rabbit antiserum and human complement (EAC-binding lymphocytes).

The yield of lymphocytes after purification varied from 15 to 65%. No selection of lymphocytes was observed either by counting immunoglobulin-bearing and EAC-binding lymphocytes in whole blood and in purified cells from the same sample, or by statistical analysis of lymphocytes in subpopulations as a function of the yields from twenty-six experiments. In the absence of selection during purification the total numbers of T and B lymphocytes could be calculated from the percentages and the total numbers of lymphocytes. Our normal values are close to those reported using other non-selective methods of purification.

When lymphocytes were simultaneously stained for immunoglobulin and rosetted with EAC, cells bearing either or both markers were found. In total, 27–35% cells were identified by these markers. Since about 70% of the cells were E-binding, practically all lymphocytes could be identified. A small overlap between E-binding and immunoglobulin-bearing/EAC-binding lymphocytes may occur.

Either the IgM or the IgG-containing fractions obtained after fractionation of rabbit anti-SRBC serum on Sephadex G-200 could be used for sensitization of SRBC with complement. Formation of rosettes was not prevented by pretreating the lymphocytes with aggregated IgG, while rosettes formed with EA prepared by high concentrations of IgG antibody (Fc-binding lymphocytes) were abolished. It is concluded that rosettes formed with IgG-EAC (or whole serum EAC) using diluted antiserum identify complement-reactive lymphocytes and are not caused by synergism with Fc receptors. When SRBC were sensitized with varying dilutions of whole antiserum or its IgG fraction identical plateaus for the percentages of EAC-binding lymphocytes were found. Subagglutinating concentrations of the IgM fraction was insufficient to reach the plateau and also consistently resulted in lower values for EAC-binding lymphocytes.

     

Lymphoid cells in infectious mononucleosis classified according to T and B cell markers.

It has been demonstrated that peripheral blood lymphocytes, particularly the "atypical" ones, are predominantly of the T type in infectious mononucleosis (IM). This is based on membrane marker studies (E rosettes, receptor for complement, receptor for Fc fragment of immunoglobulins (Ig), and membrane Ig) and by anti-T lymphocyte serum. On the other hand, lymphoblastoid cell lines derived from IM patients show the characteristics of B lymphocytes. This permits the suppostion that EBV infects B lymphocytes and stimulates them to proliferate. The cell proliferation is unlimited in vitro and is probably controlled in vivo by T cells. The reduction of cellular immunity in vivo, which contrasts with the high number of T cells in peripheral blood, could be explained by the fact that T cells are engaged in the regulation of B cell proliferation.     

Rabbits possess conventional T lymphocytes.

Rabbit lymphocytes have been analyzed as to surface Ig markers in relation to the function of the cells. A battery of specific anti-Ig reagents as well as supposed B and T cell-specific mitogens were used, and DNA synthesis as well as high-rate Ig synthesis in vitro were recorded. Using cells from spleen lymph node or blood, surface Ig-negative lymphocytes expressed the expected behavior of T lymphocytes. No evidence was found of significant expression of allotypic markers on the surface of such nonactivated rabbit T lymphocytes. Lymphoid cells from bone marrow constituted an exception to the rule in the sense that they contained a high proportion of cells being surface Ig-negative at the time of anti-Ig column fractionation. They did, however, rapidly express surface Ig molecules as well as B cell markers as judged by B cell mitogenic stimulation shortly after in vitro explanation. In conclusion, we failed to find any constant region Ig markers on rabbit lymphocytes, which in every sense behave like conventional T lymphocytes.     

Recruitment of Rab27a to phagosomes controls microbial antigen cross-presentation by dendritic cells

Polyreactive immunoglobulins (Ig) and complement components are present in tissues and blood of healthy individuals. They facilitate pathogen uptake and inactivation in lysosomes of phagocytes and thereby provide rapid protection against infection. Dendritic cells (DCs) are phagocytes that can acquire peptides from phagocytosed antigen to elicit cytotoxic immune responses by CD8⁺ T lymphocytes. The mechanisms that select peptides for cross-presentation are not fully resolved. Here we investigated the role of polyreactive Ig and complement in directing phagosomal antigen processing for cross-presentation. Phagocytosis facilitated by serum opsonization required the presence of Ig for effective antigen cross-presentation of microbe-derived antigen. The presence of complement C3 in serum promoted phagocytosis, yet phagosomes were defective in antigen degradation. The small GTPase Rab27a was recently implicated in antigen cross-presentation and was rapidly recruited to phagosomes only when Ig was present. Our data suggest that prebinding of antigen by polyreactive Ig potentiates the efficiency of antigen cross-presentation to CD8⁺ T cells through recruitment of Rab27a.     

Phorbol ester-induced differentiation of chronic B lymphocytic leukaemia cells--regulatory impact of autologous and allogeneic accessory cells.

Phorbol ester (TPA) induction of chronic lymphocytic leukaemia (CLL) cells can be used as a model system for the study of human B cell differentiation. We have analysed the role of accessory cells and the correlation to target cell surface Ig phenotype in TPA-induced morphological and functional differentiation of 12 CLL populations. A more than three-fold increase in secreted IgM was seen in 10 of 12 cases, with the strongest responses in patients having monoclonal serum IgM. CLL populations negative for or having only weak surface mu chain expression were less inducible. The impact of autologous and allogeneic accessory cells on TPA induction was studied in cell enrichment/depletion experiments using both physical and cytotoxic antibody techniques. CLL cells physically depleted of autologous E+ and monocytic (light density fraction) cells still responded to TPA. This response could be enhanced by allogeneic E- light fraction cells. Further depletion of autologous accessory cells by treatment of the E- high density fraction CLL cells with a panel of monoclonal antibodies plus complement demonstrated the permissive role of one or two populations of autologous cells expressing low avidity E receptors and the T3, T4 and T8 antigens. Augmenting T cells of similar phenotype were found among allogeneic cells from normal individuals. Thus, TPA-induced IgM secretion in biopsy B-CLL cells is regulated by minute numbers of autologous helper T cells. Furthermore, the Ig secretory response of CLL populations seems to be correlated with the surface Ig the surface immunoglobulin phenotype of the leukaemic cells.     

Identification of a high molecular weight protein on the surface of murine thymus and thymus-dependent cells.

Rabbit anti-mouse Ig reacted with mouse thymocytes resulting in the formation of caps which were shed into the medium and subsequently injected into rabbits. The antiserum from these animals (AMTP) reacted strongly with thymocytes and peripheral T cells and weakly with B cells. The antiserum did not react via the Thy-1 antigen and could be made specific for T lymphocytes by absorption with B lymphocytes. By surface labeling of lymphocytes with 125I, it could be shown that the major T lymphocyte antigen recognized by AMTP was one, or possibly two, large, single chain molecules with a molecular weight of approximately 200000. This molecule was not Ig and, furthermore, the AMTP did not react with cell surface Ig of B lymphocytes. The implications of this finding for previous reports on the existence of immunoglobulin on T lymphocytes are discussed.     

A Simple Method for Specific Depletion of Lymphocytes from Bovine Peripheral Blood Mononuclear Cells

The treatment of bovine peripheral blood mononuclear cells with rabbit anti-bovine immunoglobulin, goat anti-rabbit immunoglobulin (GAR) and complement resulted in the specific lysis of all surface immunoglobulin (SIg) bearing B lymphocytes. No SIg lymphocytes were detected after 12 hours of culture following lytic treatment, whereas cells treated with antibody without complement readily stained for SIg. The percentage of cells forming E-rosettes or binding peanut agglutinin (PNA) increased following lysis. The percentage of latex-ingesting monocytes also increased after lysis even though many of these cells had cytophilic Ig. B lymphocyte-depleted (T cell-enriched) populations cultured with phytohemagglutinin (PHA) and concanavalin A (Con A) were never less reactive than unseparated cells. No differences in background mitosis was observed for these 2 cell preparations. These results suggest that bovine SIg^- cells do not require B lymphocytes to respond to PHA and Con A and that lysis of B lymphocytes does not alter the responsiveness of the recovered SIg^- cells.     

Heterologous specific antiserum for identification of human T lymphocytes*

Peripheral blood lymphocytes (PBL) from two patients with Bruton-type agammaglobulinaemia were used for the preparation of heterologous anti-human T cell-sera which were absorbed with cultured B lymphoblast cells, peripheral blood lymphocytes from a patient with chronic lymphatic leukaemia and `adherent' cells. Using multiple criteria, one antiserum (ATCS) was shown to be specific for T lymphocytes. This antiserum asserts the existence of human-specific T-lymphocyte antigen(s) (HTLA) and provides another method for identifying human T cells. In the presence of rabbit complement, ATCS was cytotoxic for 65·5% (range 49–78) of normal PBL and 97% of thymocytes (the latter cells having also a higher surface density of HTLA than PBL). The study of PBL from a variety of patients showed that the percentage of ATCS-sensitive cells was high in Bruton-type agammaglobulinaemia, variable from patient to patient and from time to time in common variable hypogammaglobulinaemia and generally low in active lepromatous leprosy, in patients under antilymphocyte globulin therapy and in chronic lymphatic leukaemia. Cultured lymphoblasts from various B cell lines or from a Burkitt lymphoma cell line were resistant to ATCS.