T and B lymphocytes in myasthenia gravis.

Peripheral blood lymphocytes from seventeen non-thymectomized and nine thymectomized patients with myasthenia gravis (MG) and thirteen healthy controls were examined for the presence of surface markers characteristic of T and B lymphocytes by rosette formation with sheep red blood cells (SRBC). T cells were identified by their capacity to spontaneously form rosettes with SRBCs. The percentage of B lymphocytes was determined by the erythrocyte antibody complement (EAC) rosette-forming test. The EAC complex was prepared with either whole rabbit anti-SRBC serum or with the IgM fraction of rabbit anti-SRBC serum. The two kind of erythrocyte complement rosette-forming cells (EAC-RFC) are designated erythrocyte-haemolysin-complement RFC (EA(H)C-RFC), and erythrocyte-IgM-complement RFC (EA(M)C-RFC). The percentage of total lymphocytes and T cells was not altered in MG patients. The percentage of 'active' T cells, which have been considered to be more actively involved in cellular immunity, was also similar in MG patients and controls. A significant increase in EA(H)C-RFC occurred in both thymectomized and non-thymectomized MG patients, while in B cells detected by EA(M)C-RFC no alterations were found. The increase in EA(H)C-RFC in lymphocytes from MG patients may be due to an increase in the 19S antibody-forming B lymphocytes or to an increase in T cells which have Fc receptors on their surface.     

The effect of complement in adherent immune complexes on Fc and C3 receptor expression in human monocytes.

The effect of complement in surface-bound immune complexes on the expression of Fc and C3 receptors in membranes of adherent human monocytes was examined. Monocytes were isolated from mononuclear leucocyte preparations by adherence to substrates containing fibrin, fibrin with immune complexes (containing rabbit IgG antibodies), or fibrin with immune complexes and mouse complement. Fc or C3 receptors on the top or exposed surface of the monocytes were detected by rosette formation with sheep erythrocytes coated with IgG (EA) or IgM and complement (EAC). Monocytes adherent to surface-bound immune complexes exhibited an absence of EA rosette-forming ability without any change in EAC rosettes. This specific loss of Fc receptor function was induced more easily in freshly-isolated monocytes than in cells maintained in suspension culture for up to 7 days. The presence of complement in the immune complex substrates did not reverse the decrease in Fc receptors seen with freshly-isolated or cultured monocytes. Monocytes adherent to immune complexes and complement exhibited a decrease in C3 receptor function. This decrease was more readily induced in cells cultured for three days in the presence of serum than in freshly-isolated monocytes. Experiments performed with EAC or immune complex substrates relatively enriched in C3b or C3bi indicated that C3b in the substrate induced a decrease in monocyte C3b receptors and C3bi led to a decrease in C3bi receptors. No evidence was found for C3d receptors on the human monocytes although these receptors on a subpopulation of human lymphocytes appeared to be altered in a similar fashion.     

Interaction of anti-staphylococcal protein A antisera with Fc receptor-bearing human normal lymphocytes.

Staphylococcal protein A (SpA) is known to bind the Fc region of IgG of most mammalians and to possess biologic activity both in vivo and in vitro, where it acts as a lymphocyte polyclonal mitogen. Its binding to the Fc gamma portion bears many features of the antibody-antigen interaction, such as the dissociation constant, lattice formation, and complement activation. Moreover, SpA seems to compete with membrane Fc receptors for IgG so that the possibility of an interaction with the same CH domain(s) of IgG can be considered. In the present study, evidence is given that anti-SpA antisera obtained from chickens and rabbits are able to inhibit EA rosette formation by normal human lymphocytes and that they are able to recognize, with immunofluorescent staining, a subpopulation of normal human peripheral blood lymphocytes (PBL) that closely resembles that of EA rosette-forming cells (RFC). Moreover, the depletion of EA RFC by means of a single gradient centrifugation is accomplished by the parallel depletion of PBL stainable by anti-SpA antisera. The relevance of these results in the hypothesis of a similarity between the combining sites of SpA and membrane Fc receptor(s) for IgG is discussed.     

Vaccination with Mycobacterium bovis BCG affects the distribution of Fc receptor-bearing T lymphocytes in experimental pulmonary tuberculosis.

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated and challenged 6 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. By using a double rosette assay with isotype-specific antibody-coated ox and uncoated rabbit erythrocytes, the proportions of T lymphocytes bearing Fc receptors for immunoglobulin G (IgG) (T gamma cells) or IgM (T mu cells) were quantified in tissues taken from animals that were killed within 4 weeks postchallenge. Tuberculin reactivity in vivo and in vitro and antimycobacterial resistance were also measured. BCG vaccination protected the guinea pigs and resulted in significantly enhanced proportions of T mu cells in the blood during the first 3 weeks and in the spleen during weeks 2 and 3 postchallenge. Levels of T gamma cells declined in all tissues during the first 3 weeks of infection and were unaffected by prior vaccination with BCG. Increased proportions of T mu cells in the blood were accompanied by dramatic tuberculin skin reactions and purified protein derivative-induced lymphoproliferation in BCG-vaccinated guinea pigs during the first 2 weeks following virulent pulmonary challenge. Peak levels of T mu cells in the spleens of vaccinated animals at 2 weeks coincided with the first appearance of virulent mycobacteria in that organ. BCG vaccination appears to influence immunoregulatory events in pulmonary tuberculosis through effects on the distribution of IgM Fc receptor-bearing (T mu cell) T lymphocytes.     

Effect of radiotherapy on lymphocyte cytotoxicity in vitro.

The cytotoxic functions of highly purified blood lymphocytes from patients with breast cancer were studied before and after radiotherapy. Addition of PHA or of rabbit antibodies to target cells (chicken erythrocytes) were chosen as two means of inducing lymphocyte cytotoxicity in vitro. The proportion of T and non-T-lymphocytes was determined by means of E and EAC rosette tests. The antibody-induced cytotoxocity of lymphocytes decreased following radiotherapy while that mediated by PHA remained unchanged. There was some reduction in the percentage of EAC rosette-forming cells. These results, as well as our earlier observations, suggest that the decrease in the peripheral blood of the proportion of lymphocytes with receptors for activated complement is responsible for changes in the antibody-mediated lymphocyte cytotoxicity.     

Different rosette assays for detecting Fc receptor-bearing lymphocytes measure different subpopulations

The capacity of purified lymphocytes from human peripheral blood to bind the Fc portion of IgG was investigated by the rosette technique using ox erythrocytes sensitized with rabbit anti-ox IgG (EA_ox) and human erythrocytes sensitized with anti-CD IgG (EA_CD). With unfractionated lymphocytes EA_ox always detected more rosette-forming cells (RFC) than did EA_CD; however, in lymphocyte populations specifically depleted of B lymphocytes by passage through copolymer styrene bead columns, the proportion of rosettes formed with EA_ox and with EA_CD was the same. Double labelling for Fc receptors and surface immunoglobulin (SIg) demonstrated that most of the lymphocytes which formed rosettes with either EA_ox or EA_CD also carried SIg. Pre-incubation of lymphocytes at 37° to remove heatlabile SIg did not affect their ability to form EA rosettes but reduced the proportion of SIg-bearing cells. Following pre-incubation a significant proportion of EA_ox RFC still remained SIg positive but the lymphocytes which formed rosettes with EA_CD no longer carried SIg.

These studies suggest that rosette formation with EA_CD detects only `K' lymphocytes (non-T, non-B cells bearing heat-labile SIg) while EA<sub>ox</sub> detects some B lymphocytes as well. By reducing the amount of IgG bound to ox erythrocytes sensitivity was reduced to the point where EA<sub>ox</sub> no longer formed rosettes with B lymphocytes but still detected `K' lymphocytes indicating either a qualitative or quantitative difference between the Fc receptors on B and `K' lymphocytes. Treatment of lymphocytes with trypsin decreased the percentage of rosettes formed with EA<sub>ox</sub> but not with EA<sub>CD</sub> supporting the conclusion that there is a structural difference between the Fc receptors on B and `K' lymphocytes although a difference in receptor density is not excluded.

When EA_CD and fluorescein-labelled ox erythrocytes sensitized with a low concentration of rabbit anti-ox IgG were mixed, most RFC bound both the indicator erythrocytes simultaneously suggesting that the Fc receptors on `K' lymphocytes do not exhibit species specificity.

     

Expression of a receptor for IgM by human T cells in vitro

The capacity of human peripheral blood lymphocytes (PBL) to bind antigen-IgG antibody (EA(IgG)) and antigen-IgM antibody (EA(IgM)) complexes was investigated using a rosette technique with ox erythrocytes coated with rabbit IgG or IgM antibody. It was found that while EA(IgG)-rosette-forming cells (RFC) were detected on PBL freshly drawn from normal individuals, EA(IgM)-RFC were present only in suspensions kept in culture for 24 h in mediá supplemented with sera containing very low or no amounts of IgM. Experiments of simultaneous detection of EA(IgG)-RFC or EA(IgM)-RFC and other membrane markers for human T or B cells together with experiments on purified T or B cell populations indicated that EA(IgG)-RFC were formed by both T and B cells, while T cells only were capable of EA(IgM) rosette formation. The specificity of the receptors for IgG and IgM was determined by studying the inhibitory capacity of purified human IgM and IgG in the rosette assay. The receptor for IgG was inhibited by IgG and not by IgM, while the reverse was true for the receptor for IgM.     

Anti-DNA antibodies: perspectives and perplexities.

B lymphocytes from the rabbit spleen were freed of T cells by removal of cells which formed rosettes with papain-treated rabbit erythrocytes. Additional purification could be achieved if fractionation by rosette removal was preceded by removal with a magnet of cells which adhered to or ingested poly L-lysine coated iron core particles. Cell yield and purification were assessed by complement mediated cytotoxic kill of B and T cells with antibody directed against RABELA and RTLA, respectively. Other criteria depended on determination of the number of Fc receptor bearing cells and of thymidine uptake by cells which were stimulated with concanavalin A, PHA or with antibody directed against the allotypic specificity of receptor Ig light chains. Purified preparations of B cells were obtained in a yield of about 20% of the B cells in the original spleen and contained less than 10% of cells which were not B cells. This method allows purification which does not interfere with the membrane of the isolated cells.     

Evidence for antibody-dependent cell-mediated cytotoxicity by T cells bearing receptors for IgG.

Human lymphocyte populations comprising T cells, T depleted lymphocytes, and T cells enriched for, or depleted of, IgG Fc receptor-bearing (TG) cells, were separated using rosette techniques. All lymphocytes were assessed for the ability to lyse antibody-coated chicken erythrocytes and SL2 mouse lymphoma cells. Their activity was compared with that of monocytes and neutrophil-enriched preparations. IgG Fc receptor positive cells within the T population were highly active in both cytotoxicity assays; the activity could not be ascribed to contamination by monocytes or neutrophils. The TG cells forming junctions with the target cells possessed a characteristic ultrastructure.     

Receptors for IgM and IgM-antigen complexes on human T-lymphocytes reacting with specific anti-human-T-cell antiserum.

A receptor on T lymphocytes that can bind IgM-type antibodies, without, as well as after prior complex formation between the antibody and Esh (sheep red blood cells) has been detected. No prior in vitro incubation of freshly drawn and isolated peripheral blood lymphocytes is required for the expression of the IgM antibody binding receptor on T cells. Results obtained in experiments designed for the simultaneous detection of lymphocytes reacting with specific rhodamine labelled antihuman-T cell antiserum and forming rosettes with EA-IgM complexes of Esh and the IgM antiserum fraction showed the receptor for IgM antibody to be present exclusively on T cells. Pre-incubation of T cells with free IgM or its Fc fragments leads to inhibition of rosette formation with EA-IgM. Treatment of the IgM type pentameric antibody with dithioerythritol to cleave pentameric IgM reduces the percentage of EA-IgM rosette forming cells (RFC) significantly. The possible biological significance of this receptor is discussed.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Surface markers and size of lymphocytes in human umbilical cord blood stimulated into deoxyribonucleic acid synthesis by Epstein-Barr Virus.

We characterized subpopulations of lymphocytes in human umbilical cord blood which are stimulated into deoxyribonucleic acid synthesis by Epstein-Barr virus. Lymphocytes were examined simultaneously for deoxyribonucleic acid synthesis by autoradiography and for surface markers by rosette formation with sheep erythrocytes or erythrocytes coated with antibody and mouse complement (EAC). The subpopulation which incorporated [3H]thymidine after exposure to virus consisted mainly of cells which formed rosettes with EAC. Lymphocytes were enriched or depleted of thymus-derived lymphocytes (T cells), null cells, or cells forming rosettes with EAC. The extent of sensitivity of the cells to stimulation by Epstein-Barr virus correlated with the proportion of the population which formed rosettes with EAC. When mononuclear cell populations were depleted of T lymphocytes and then fractionated by size, small lymphocytes showed higher rates of deoxyribonucleic acid synthesis after virus exposure and higher transformation frequency than did larger cells or unfractionated cells. Thus, the cells which are stimulated into deoxyribonucleic acid synthesis by Epstein-Barr virus appear to be the same as cells which are ultimately transformed.     

Re-examination of the EA rosette assay (Ripley) for Fc receptor leucocytes.

Numerous investigations has utilized rosette formation with Ripley antibody-coated human erythrocytes (EA) to identify or deplete Fc receptor-bearing K lymphocytes in whole mononuclear cell preparations. This study examines the interaction between Ripley EA and purified preparations of human lymphocytes, monocytes and neutrophils and demonstrates that this technique is not specific for K lymphocytes. Indeed, 100% of blood monocytes rosette these EA target cells. Moreover, data from both rosetting studies and antibody-dependent cytotoxicity (ADCC) reactions suggest that the avidity of Ripley EA is actually greater for monocytes than for lymphocytes. In contrast to previous reports, 100% human neutrophils were found to possess Fc receptors, as determined by their ability to rosette Ripley EA. Thus, the extent of rosetting and ADCC by all three Fc receptor-bearing leucocytes depends significantly on the degree of antibody sensitization with neutrophils requiring the greatest, and monocytes the least, amount of target-bound antibody for Fc receptor-mediated interaction.     

Fc receptors on rabbit lymphocytes. Identification and organ distribution of rosette-forming cells; cocapping with surface immunoglobulin

Fc receptor (FcR)-bearing cells were demonstrated using ox erythrocytes coated with homologous IgG-type antibodies (EAY) in rabbit peripheral blood leukocytes (PBL) and in various lymphoid organs. Discrimination of the rosette-forming cells (RFC) is carried out after prior ingestion of tetramethylrhodamine isothiocyanate-labeled latex particles and in transmission electron microscopic studies. Most of the nonlymphoid cells (5-10%) in PBL and spleen cell suspensions expose FcR. These nonlymphoid cells are almost absent in other lymphoid organs, except in bone marrow. The average percentage of cells rosetting with IgG-sensitized erythrocytes (EAγRFC) in lymphoid cell preparations of the various tissues was as follows: PBL 25%, bone marrow 65%, appendix 37%, spleen 40%, Peyer's patches 44%, thymus 2% and peripheral lymph node 27%. The nature of FcR-bearing PBL was further studied using F(ab′)₂ anti-IgM, anti-IgA or anti-T cell conjugates. About half of the population of B cells, bearing IgM or IgA, express FcR. Moreover, about 80% of the RFC are found within the B cell population. Only a few T cells were found rosetting with EAγ suggesting that most of the non-B lymphoid RFC are “null” cells. In different lymphoid organs, the percentages of EAγRFC and B cells are comparable but not identical. A greater part of the EAγRFC also expresses the receptor for the third component of complement. After capping of membrane IgM determinants, FcR is located in the same cap on the majority (60%) of the FcR-positive IgM-capped cells.     

Some parameters of the erythrocyte-antibody-complement (EAC) rosette system for recognition of bovine B-cells

A population of bovine lymphocytes formed EAC rosettes with sheep RBCs sensitized with rabbit antiserum and several species of complement. Mouse complement gave best results. Horse anti-sheep RBC serum and bovine anti-rabbit RBC serum did not support rosette formation. Optimum working concentrations of antiserum (1 : 400) and complement (1 : 20) were determined by ‘checkerboard’ titration. The occurrence of E (direct) rosettes, EA rosettes (using RBCs sensitized with rabbit antiserum but no complement), EAC rosettes and cells carrying surface immunoglobulin (sIg) (observed by a direct fluorescent antibody test) in blood and organs was observed. In a series of 33 blood samples from animals 1 week to 26 months of age the mean occurrence of these markers was: E (tested in foetal calf serum) = 2.9%; E (in CF buffer with 0.1% gelatin) = 4.3%; EA = 19.4%; EAC = 29.2%; sIg = 27.7%. The distribution of these markers in lymphoid organs of four calves and two adult heifers was examined. E rosettes occurred mostly among thymus and lymph node lymphocytes, while blood, lymph nodes and spleen contained many cells forming EA and EAC rosettes and carrying sIg. Only a few bone marrow cells carried surface markers. Cells from peripheral blood of two heifers and spleen and lymph nodes from a 3 months old calf were fractionated on nylon wool columns and contaminating macrophages and platelets were removed by treatment with carbonyl iron and adenosine diphosphate. These treatments showed that EAC and sIg cells usually occurred in the same population (presumably B-cells) and E rosette-forming cells occurred in a second, distinct population (T-cells). EA rosettes were not confirmed to B- or T-cells and their occurrence varied between organs. It was concluded that in general the EAC rosette and sIg are markers for B-cells but that probably many cell types can form EA rosettes.     

Surface markers on lymphocytes of multiple sclerosis patients.

Peripheral blood lymphocytes of twenty-two multiple sclerosis (MS) patients and thirty-five healthy controls were examined for the presence of surface markers characteristic for B lymphocytes (surface immunoglobulin, receptor for C3 (EAC), reporter for Fc (EA) and the spontaneous rosette-forming capacity characteristic of T cells. The results obtained indicate that the number of B and T cells in MS is similar to controls, as evaluated by the presence of surface immunoglobulin and E rosette-forming capacity. However, a statistically significant reduction in the percentage of lymphocytes bearing C3 receptors has been found in MS patients. It might have resulted from a reduction in the lymphocyte population bearing C3 receptor but no surface immunoglobulin. The EA rosette test revealed the greatest difference between the groups. The difference indicated a reduction in the density of the receptor for 7S Fc on lymphocytes from MS patients. The results obtained are consistent with the hypothesis of an immune deficit in multiple sclerosis.     

Human T cell receptor for IgM: specificity for the pentameric Fc fragment.

The specificity of the IgM receptor expressed by human T cells cultured in IgM-free media has been investigated. IgM receptors have been detected using a rosette system with ox erythrocytes coated with rabbit antibody (EA(IgM)), and the inhibitory capacity of different IgM fragments in the rosette system has been tested. It was found that F(c)5mu but not F(ab')2mu, nor monomeric IgM (8 S IgM) inhibited EA (IgM) rosette-forming cells. This indicates that the receptor present on the surface of T cells has affinity for a structure located in the Fc portion of the pentameric IgM.     

Methods for producing T- and B-lymphocyte receptor-specific antisera.

Spent tissue culture medium from two continuous lymphoblastoid cell lines, FL-74 and CT45-S, expressing the T-lymphocyte receptor for guinea pig E and the B-lymphocyte receptor for EAC respectively were used to produce receptor-specific antisera. Anti-E receptor sera blocked E rosette formation on FL-74 cells, canine and feline lymphocytes and canine and feline thymocytes but not EAC rosette formation by CT45-S cells or canine and feline lymphocytes. Anti-EAC receptor sera blocked EAC rosette formation on CT45-S cells and canine or feline lymphocytes. Absorption of antisera will the appropriate lymphoblastoid cell line removed E or EAC-blocking activity. The results of this study suggest that similar methods may be used to produce lymphocyte subpopulation-specific antisera in other species including man.     

Heterogeneity of human lymphocyte Fc receptors. I. Differential susceptibility to proteolysis

To study the possible heterogeneity of human lymphocyte Fc receptors, isolated human peripheral blood lymphocytes (PBL) were enzymatically altered (`stripped') by exposure to pronase or papain. Pronase treatment markedly increased the percentages of PBL binding IgG-sensitized erythrocytes (EA), while simultaneously removing or inactivating their receptors for heat-aggregated IgG (aggG). Papain treatment markedly diminished the ability of PBL to bind both EA and aggG. Essentially identical results were obtained utilizing EA composed of either human Rh-positive type O erythrocytes sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley) or with chicken erythrocytes sensitized with rabbit anti-CRBC IgG (CRBC-A). CRBC sensitized with Fab'₂ fragments of rabbit anti-CRBC IgG were incapable of forming rosettes with normal or with pronase- or papain-stripped PBL. Pre-treatment of normal lymphocytes with aggG totally ablated their ability to rosette with EA.

Incubation of pronase-stripped PBL for 18–20 hr in 5% CO₂-air at 37°C resulted in diminution (to levels originally present) in the percentages of lymphocytes binding EA, but no regeneration of aggG receptors. Similar incubation of papain-stripped PBL resulted in significant reappearance of receptors binding EA, but no regeneration of aggG receptors. These results strongly suggest that: (1) lymphocyte receptors that bind EA complexes differ from those that bind aggG; (2) some lymphocytes possess cryptic receptors for EA that are expressed after proteolysis with pronase; (3) PBL having receptors for EA also have aggG receptors; and (4) there is no evidence that proteolytic stripping of PBL results in the generation of functionally different receptors for complexed IgG, since the Fc specificity of this receptor remains unchanged.