Human autologous rosette-forming cells. V. Study of MHC control in erythrocyte-lymphocyte interaction

The role of the major histocompatibility complex (MHC) in human autorosette formation was studied. On a large series of healthy subjects who were typed for HLA antigens, we tested in blind rosette formation with 90 autologous and 295 allogeneic red blood cells (RBC). We found that the mean levels of auto- and allorosettes were similar, being significantly higher in females than in males. However, we failed to find any role for blood group antigens and any involvement of HLA antigens in the interactions between lymphocytes and RBC in rosette formation. Moreover, high or low autorosette levels were not associated with a particular HLA allele. The comparison of individual percentages of auto- and allorosettes indicated that 51% of the subjects displayed identical levels of auto- and allorosettes whereas 29% formed preferentially rosettes with auto- rather than allo-RBC and 20% exhibited lower autorosette than allorosette levels. Among the group of subjects who were better responders for autorosettes than allorosettes, we found an increased frequency of the haplotype A29, B44. Taken together these findings suggest that in contrast to the murine situation, the autorosetting phenomenon in humans is not HLA restricted.     

Human T cell receptors for monkey erythrocytes.

To test the specificity of T cell receptors, erythrocytes and lymphocytes of man and rhesus monkey (Macaca mulatta) and erythrocytes of sheep were mixed in four different combinations to observe the rosette formation. During the study, a major proportion of human T cells formed spontaneous rosettes with the erythrocytes of rhesus monkey. A small number of monkey lymphocytes formed rosettes with human group O Rh-negative cells while T cells both of man and rhesus monkey formed rosettes with sheep red blood cells.     

Mouse red blood cell rosettes: human B and some T lymphocytes express receptors for mouse erythrocytes in the presence of Ficoll.

Inclusion of 14% Ficoll in the assay of rosette formation of human lymphocytes with mouse red blood cells (RBC) markedly enhances the cellular interaction. Both the strength of the rosettes (number of attached RBC) and the number of reacting lymphocytes are increased significantly, irrespective of the lymphocyte source--blood, lymph node and tonsil. In the presence of Ficoll, most of the sIg-bearing B lymphocytes [expressing receptors for Fc(IgG) and/or C3] and a significant number of the sheep RBC-rosetting T lymphocytes (particularly the nylon wool non-adherent subset from the tonsil), express receptors for the mouse erythrocyte.     

Differential effect of vinblastine and colchicine on human active and total T rosettes.

The effect of vinblastine and colchicine on human blood T cell rosette formation with sheep erythrocytes was assessed. For some concentrations, both drugs increased the percentage of active T rosettes. In contrast, the total T rosette percentage was decreased by the same drugs. These experiments further confirm the heterogeneity of T cell rosettes and suggest the existence of three types of sheep red blood cell receptors on human blood T lymphocytes.     

Characterization of pig lymphocyte receptors for allogeneic and non-allogeneic erythrocytes. I. Apparent common identity of both receptors.

In the pig thymus, the proportion of allogeneic (or autologous) erythrocyte rosette forming cells (P-RFC) is always lower than that of sheep erythrocyte (non-allogeneic) rosette forming cells (S-RFC) even under saturated RBC/lymphocyte ratios and optimal dextran concentration. This difference accounted for lymphocytes rosetting with sheep erythrocytes and not with pig erythrocytes (P-S+ cells), as opposed to those lymphocytes which are able to bind both types of erythrocytes (P+S+ cells). Since formation of both sheep and pig erythrocyte rosettes is inhibited similarly by anti-T receptor serum, is inhibited reciprocally by sheep and pig erythrocyte membrane fragment and is similarly trypsin sensitive, it was concluded that the same receptor was responsible for both sheep and pig rosette formation. Furthermore it was found that P+S+ cells had a higher avidity for sheep erythrocytes (and lower for pig erythrocytes) than the other subset which did not bind pig erythrocytes.     

The lack of specificity of the sheep erythrocyte-T lymphocyte rosetting phenomenon.

One subpopulation of lymphocytes, thymus-derived (T) cells, is identified by its characteristic ability to form spontaneous rosettes with sheep erythrocytes (SRBC). However, the mechanism by which the rosettes form remains unknown. To gain more insight into the specificity of this phenomenon, the ability of SRBC to rosette with cells other thah lymphocytes was studied. All of the cell types utilized in this study (L cells, monkey liver cells, HeLa cells, and human liver, bladder, lung, parathyroid, and dermal fibroblasts), except neoplastic B lymphocytes, rosetted with SRBC. Viability was not a factor in the rosette formation. These findings suggest that the process of T lymphocyte-SRBC rosette formation is not due to a T cell-specific membrane receptor or antigen but may be due to a widely distributed basic substructure of the cell membrane.     

Surface markers on human lymphocytes: studies of normal subjects and of patients with primary immunodeficiencies

Peripheral blood lymphocytes of twenty normal controls and twelve patients with primary immunodeficiencies were examined for surface membrane Ig and receptors for C3 complement (B cell markers) and for spontaneous rosette formation with sheep erythrocytes (T cell markers). In patients with defects in T cell function no lymphocytes forming spontaneous rosettes were seen. In patients with B cell deficiency they were normal or increased. Lymphocytes with membrane immunoglobulins were normal in patients with T cell defect and absent in patients with severe agammaglobulinaemia. Lymphocytes with receptors for C3 complement were increased in patients with T defect and normal in patients with most other forms of immunodeficiency studied.     

T and B lymphocytes in the marmoset: a natural haemopoietic chimera.

The thymus-derived (T) lymphocyte and bone marrow-derived (B) lymphocyte populations of the marmoset were characterized using specific cell surface markers Approximately 85% of the thymocyates formed rosettes with neuraminidase-treated sheep erythrocytes (En). The percentage (approximately 69%) of peripheral blood lymphocytes (PBL) forming rosettes with En was the same as that which stained with fluorescently labelled goat anti-marmoset thymocyte serum (ATS). These two assays identified the same cell population since treatment of cells with ATS and complement resulted in a concomitant decrease in En rosette formation. Marmoset PBL also formed rosettes with human erythrocytes sensitized with antibody and complement (HEAC); since the percentage (approximately 20%) HEAC rosette was the same as that of cells stained with fluorescently labelled goat anti-marmoset IgG, these cells were considered to be B cells. A small percentage of cells (approximately 1-5%) possessed both types of receptors. The mean percentages of T and B cells present in PBL of single-born, presumably non-chimeric animals, were the same as that of iso-sexual and heterosexual chimeras.     

Glutaraldehyde and human T cell rosettes

Blood mononuclear cells from ten healthy donors and cells from fifty cultured human lymphoblastoid lines were tested for the capacity to form rosettes with sheep erythrocytes. The effect of adding glutaraldehyde before scoring rosettes and the effect of substituting glutaraldehyde-fixed for fresh sheep cells were both examined.The results suggest that glutaraldehyde increases the apparent proportion of rosette-forming cells by altering the sheep erythrocytes in such a way that they will adhere to some B lymphocytes.Although a small number of cells carry both surface immunoglobulins and receptors for fresh sheep erythrocytes, the rosette test with fresh sheep cells identifies T lymphocytes more accurately in the absence of glutaraldehyde than in its presence.     

Human blood lymphocyte fractionation with special attention to their cytotoxic potential.

In studies concerning the natural cytotoxic activity of human blood lymphocytes we have found that: 1. T cell, which rosette with sheep erythrocytes can be separated by centrifuging on Ficoll the lymphocyte and erythrocyte mixture without previously pelleting and incubating in the cold. 2. Lysis of marker erythrocytes with ammonium chloride impairs the cytotoxic activity of lymphocytes. Incubation at 37 degrees C for 12 hr prior to the test restores the activity to some extent. 3. A high proportion of the so call "null" fraction i.e. cells remaining in the interface after removal of B cells (passage on nylon column) and sedimentation of E rosettes, sediments as rosettes with sheep EAC'indicating that these cells carry low density E and C3 receptors. Rosetting with SRBC or ox EAC' gave significantly lowr values. On a per cell basis the "null" fraction was the most efficient one in natural cytotoxicity. Depletion of cells with low affinity E or E and C3 receptors left highly active subfractions.     

Sheep erythrocyte rosettes in pigs, sheep, cattle and goats demonstrated in the presence of dextran

Formation of rosettes with sheep RBC by blood lymphocytes from young pigs was increased from 24.4% +/- 2.2 (mean +/- S.E.) in PBS to 54.1% +/- 1.9 in the presence of dextran. This increase was achieved without inducing appreciable rosette formation with other RBC which do not form rosettes in PBS. Lymphocytes which rosette only in dextran are predominant in pigs between 20 and 160 days old, when the peripheral lymphocyte pool is increasing very rapidly. The use of dextran revealed major populations of blood lymphocytes rosetting with SRBC in adult sheep (30.7% +/- 2.0), adult cattle (37.3% +/- 4.2) and adult goats (13.3% +/- 1.2). Proportions of rosette-forming lymphocytes tended to increase with age. In calf lymphoid tissues the distribution of rosette-forming lymphocytes suggested that these were T cells. In the improvement of rosette formation with SRBC, dextran was more effective than foetal calf serum, papain treatment of the SRBC or combinations of these treatments.     

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.     

Receptors for IgG molecules on human lymphocytes forming spontaneous rosettes with sheep red cells

A mixed rosette techinque with sheep red blood cells (SRBC) and ox erythrocytes heavily coated with rabbit antibody (EA) was employed to simultaneously idnetify human peripheral blood T lymphocytes and IgG receptor-bearing cells. The findings of a noticeable proportion of ?mixed resettes”? in peripheral blood lymphocytes freshly drawn from normal individuals and of an even higher number of these mixed rosettes in cell suspension kept in culture media supplemented with fetal calf serum provide evidence for the capacity iof human T cells to express membrane receptors for antigen-antibody complexes.     

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.     

A receptor for ''self'' on lymphocytes.

A large population of lymphocytes is able to form rosettes with syngeneic, allogeneic or closely related xenogeneic erythrocytes. Similar results were found with spleen cells from mice, rats and rabbits. The highest numbers were found in mice where up to 30% of lymphocytes bound autologous erythrocytes. Rosette formation is probably due to stereospecific cell surface receptors since erythrocytes of distant xenogeneic origin were not recognized. Rosette forming cells do not seem to be restricted to the B-cell or T-cell compartment since mouse thymus cells as well as spleen cells from congenitally athymic (nude) mice bound erythrocytes to a similar degree.     

Inhibitory effect of sheep erythrocyte fragments on rosette formation of human T lymphocytes with sheep red blood cells.

The effect of sheep red blood cells (SRBC) fragments on rosette formation of human peripheral T lymphocytes with SRBC was evaluated on the active and total T-rosette tests. The rosetting capacity of active rosette-forming cells was selectively and nearly completely inhibited by the pretreatment of lymphocytes with SRBC fragments. The decrease in total rosettes by blocking with SRBC fragments was almost parallel to that of active rosettes. SRBC fragments had no inhibitory effect on the rosetting capacity of a lymphocyte population in which active rosette-forming cells were removed by gradient centrifugation. These results suggested that active rosette-forming cells in human T lymphocytes have the receptors of high affinity for SRBC and these receptors readily bind SRBC fragments, resulting in block of rosette formation.     

T- and B-lymphocyte subpopulations following radiotherapy for invasive squamous cell carcinoma of the uterine cervix.

Lymphocyte subpopulations in patients with cervical carcinoma were studied before and after radiotherapy. T lymphocytes were recognized by their ability to form spontaneous rosettes with sheep erythrocytes (E rosettes). Two surface marker characteristics were used to detect B lymphocytes: receptors for activated complement responsible for erythrocyte--antibody--complement (EAC) rosette formation, and surface membrane immunoglobulin (SmIg), readily stainable by immunofluorescence. We have demonstrated a significant depression of total lymphocytes after radiotherapy which persists for more than 5 years. This reduction in lymphocytes is due to a loss of E rosette-forming T cells, SmIg-bearing B cells and null cells. Absolute numbers of EAC rosette-forming B cells are not altered by treatment, and there is a rise in this cell type when the results are expressed as percentages of the total lymphocyte count. The possible functional immunological significance of these changes is discussed.     

All CD2-positive human lymphocytes rosette with sheep erythrocytes in the presence of polyethylene glycol

The efficiency of rosette formation between human peripheral resting lymphocytes and sheep erythrocytes (E) is remarkably increased in the presence of 1-4% polyethylene glycol of MW 10,000 (PEG). In fact, all CD2 ('sheep E receptor')-positive lymphocytes formed rosettes. CD2 positivity was assayed by immunofluorescence staining with the monoclonal antibody (mAb) Leu 5b. PEG also induced autologous E rosetting which otherwise did not take place under the usual conditions. The specificity of the rosettes was assessed by blocking CD2 with mAb Leu 5b and by blocking the CD2 target structure (T11TS) on the sheep E with the mAb L180/1. The mAb Leu 5b abolished all rosette formation, but mAb L180/1 only abolished the sheep E rosettes as this mAb is specific for sheep E and does not cross-react with human E. The results indicate that sheep E in the presence of PEG specifically rosette with all CD2-positive lymphocytes.     

Spontaneous autorosette-forming cells in man. A marker for a subset population of T lymphocytes?

A subpopulation of human peripheral blood lymphocytes are capable of binding with human (autologous or allogeneic) erythrocytes, forming rosettes. The conditions which lead to autorosette formation are similar to those required for sheep red-cell rosetting. Ageing human erythrocytes are shown to bear less of the determinants involved in the phenomenon than younger ones. Evidence is presented that autorosetting is a T-cell marker. As autorosette-forming cells are very sensitive to the inhibiting effects of ATG they could therefore belong to a T-cell subpopulation.     

In vitro differentiation of human marrow cells into T lymphocytes by thymic extracts using the rosette technique.

The manner by which human and calf thymic extracts induce precursor cells of human marrow to differentiate in vitro into T lymphocytes has been studied using as a T-cell marker the spontaneous rosette formation technique of human T lymphocytes with sheep erythrocytes (E rosette). These findings confirm previous observations made in the study of the same process using a different T-cell marker, specific antigenicity recognizable by a heterologous anti-human T-cell serum in a microcytotoxicity test. The number of cells revealing evidence of differentiation demonstrated by the E rosette formation technique is smaller than that obtained with the anti-human T-cell serum, indicating perhaps that a different stage of maturation of T lymphocytes is recognized by the antiserum from the one detected by spontaneous rosette formation. Based on the effects of specific inhibitors of nucleic acids and protein synthesis, it can be concluded that these thymic extracts ultimately act by influences exerted in the cell nucleus and that RNA and protein synthesis are required for the differentiation of precursor cells into T lymphocytes induced by thymic extracts. In addition, continued protein synthesis appears to be required for maintenance of receptors for sheep erythrocytes on the cell surface.