Characterization of Human Null Cells Isolated from Peripheral Lymphocytes by a Simultaneous Double-Rosetting Procedure

Human null cells were isolated from peripheral blood lymphocytes (PBL) by differential centrifugation on a Ficoll-Hypaque gradient of the PBL following simultaneous rosetting with erythrocyte indicators specific for B and T lymphocytes. Specifically, the T lymphocytes were rosetted with 2-aminoethylisothiuronium bromide-treated sheep erythrocytes (ShE), whereas the B lymphocytes were either rosetted with ShE coated with xenoantibodies against human gamma globulin or first sensitized with monoclonal antibodies to human Ig-like antigens and then rosetted with ShE coated with xenoantibodies against mouse gamma globulin. Approximately 90% of the lymphocytes isolated were null cells that did not bear detectable B-cell markers-that is, surface immunoglobulin and/or Ia-like antigens-or T-cell markers-that is, ShE receptors. The large majority of the null cells expressed receptors for the IgG Fc fragment (53-93%), C3 component (65-92%) and monkey erythrocytes (60-91%) but lacked receptors for the IgM Fc fragment and murine erythrocytes. The null cells exhibited high natural killer cell activity and antibody-dependent cellular cytotoxicity and were two- to four-fold active than the total PBL and the T-enriched cell fractions. The null cells, however, did not respond to stimulation with phytohaemagglutinin and failed to function as either stimulators or responders in an unidirectional mixed lymphocyte reaction.     

Interaction of human lymphocytes with fluid phase human C3b detected by immunofluorescence

Human lymphocytes obtained from tonsils and peripheral blood were found to bind human fluid phase C3b, obtained by trypsin treatment. This binding was detected by indirect immunofluorescence (IIF) using specific anti-C3 antisera. Lymphocytes isolated from thymus tissue scored low percentages in IIF, indicating that the main population of thymus-derived lymphocytes are T cells. The distribution pattern of C3b-binding cells was compared with that of cells forming rosettes with sheep erythrocytes coated with antibody and complement (EAC) and with sheep erythrocytes (E) only, as well as with that of Ig- bearing lymphocytes, as detected by direct immunofluorescence. It appeared that the distribution pattern of lymphocytes which can bind fluid phase C3b is similar to that of EAC rosette-forming and of Ig-bearing lymphocytes. Pre- incubation of the lymphocytes with C3b and pretreatment of the cells with trypsin decreased the capacity to form rosettes and to bind C3b to their sur- face. Human monocytes, granulocytes and erythrocytes did not bind fluid phase C3b, as judged by IIF. Therefore, the selective binding of fluid phase C3b to lymphocytes provides a specific method for the detection of complement-reactive lymphocytes in lymphoid cell preparations.     

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.     

Lymphocyte membrane receptors in human lymphoid leukemias.

Membrane-bound immunoglobulins, receptors for the Fc fragment of IgG and receptors for the third component of human or murine complement were used as B cell membrane markers to study peripheral blood lymphocytes from twenty-two patients with chronic lymphatic leukemia (CLL), five patients with acute lymphoblastic leukemia (ALL) and one patient with Sézary syndrome. The capacity of human T cells of forming "spontaneous rosettes" with sheep erythrocytes was employed as T cell membrane marker. In nineteen out of twenty-seven CLL or ALL cases tested a larger percentage of cells than that found in normal individuals expressed at least one of the three B cell membrane markers studied. In the patient with Sézary syndrome the percentage of cells forming "spontaneous rosettes" with sheep erythrocytes was larger than the normal, while cells bearing B cells markers were below the normal values.     

Separation and properties of EA-rosette-forming lymphocytes in humans.

Human peripheral blood lymphocytes were separated into subpopulations enriched or depleted with respect to B lymphocytes (Ig-bearing cells), T lymphocytes, (cell forming rosettes with sheep erythrocytes: E-RFC) and Fc receptor-bearing lymphocytes (EA-RFC). From the distributions and recoveries of the various cell types it could be concluded that there was very little overlap between Ig-bearing lymphocytes and EA-RFC. The latter cells partly belonged to "null" (non-T, non-B) cells; it was however demonstrated that 30 % of the EA-RFC were T cells (E-RFC). The lytic capacity in antibody-dependent lymphocytotoxicity (ADL) was shown to correspond with the proportions of EA-RFC in the various fractions. Non-T cells showed enhanced ADL activity when compared to the unseparated cells. Purified T cells populations also displayed ADL activity. Since the latter could not be due to contaminating non-T cells, this activity was ascribed to Fc receptor-bearing T lymphocytes.     

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.     

Receptors for Helix pomatia A haemagglutinin on leukaemic lymphocytes from patients with chronic lymphocytic leukaemia (CLL).

Blood lymphocytes from thirteen patients with CLL were studied for surface-bound Ig (SIg), Fc receptors (EA rosettes), receptors for sheep erythrocytes (E rosettes) and receptors for Helix pomatia A haemagglutinin (HP), a carbohydrate-binding protein with specificity for N-acetyl-D-galactosamine and related sugars. Fluorescein-labelled HP binds to subpopulations of human peripheral blood lymphocytes (PBL) treated with neuraminidase. In normal peripheral blood, HP binds to the T-lymphocytes while the majority of the B cells bearing surface-bound immunoglobulin do not have receptors for HP. In untreated CLL, HP binds to 90-100 percent of the neuraminidase-treated PBL. Almost all of the SI-G-POSITIVE CELLS IN CLL patients also have receptors for HP. Two groups of patients were found: in one the total fraction of SIg+ cells was less than or equal to 50 percent and about 30 percent of these lost their Ig during incubation at 37 degrees C. No such loss of SIg was revealed in the remaining patients where total SIg+ fraction was approximately 70 percent. These patients usually had higher blood lymphocyte counts, probably reflecting a more advanced disease. CLL patients in remission with low numbers of leukaemic cells also had low numbers of blood lymphocytes carrying both SIg and HP-receptors. It is concluded that leukaemic cells carry both HP receptors and SIg. Testing of this combination therefore provides a valuable new tool for monitoring patients with CLL.     

Spontaneous cell-mediated cytotoxicity in humans. Distribution and characterization of the effector cell.

When lymphocytes from healthy donors were tested as effector cells, the cytotoxic activities observed in spontaneous and in antibody-dependent cell-mediated cytotoxicity were positively correlated. However, with lymphocyte preparations obtained from renal patients, a dissociation between the two activities was occasionally observed. Human natural killer cells are lymphocytes, with receptors for the Fc fragment of IgG molecules, but with no surface immunoglobulin. Their cytotoxicity is reduced by the presence of granulocytes or monocytes. After separation of rosetting and non-rosetting cells with AET- (2-aminoethylisothiouronium bromide hydrobromide) or neuraminidase-treated sheep erythrocytes, the majority of the activity was recovered in the non-rosetting fraction, but a portion of it was present consistently in the rosetting cell fraction. Cells in the latter fraction also displayed receptors for the Fc fragment of immunoglobulin G.     

Concomitant presence of tumor-specific cytotoxic and inhibitor lymphocytes in patients with osteogenic sarcoma.

The lack of detectable tumor-specific cytotoxicity by the peripheral blood lymphocytes of patients with cancer may be due to a lack of cytotoxic lymphocytes or the presence of suppressor lymphocytes that inhibit cytotoxic cells. Unfractionated peripheral blood lymphocytes from 12 of 28 patients with osteogenic sarcoma were cytotoxic to osteogenic sarcoma cells in vitro (P less than 0,001). When the peripheral blood lymphocytes from patients whose lymphocytes were not cytotoxic underwent fractionation, a tumor-specific cytotoxic subpopulation was isolated from 11 of 13 patients (P less than 0.0001). Lymphocytes that inhibited cytotoxic activity of autologous tumor-specific cytotoxic lymphocytes were found in four of 10 patients with osteogenic sarcoma but not in six normal controls. Inhibitor lymphocytes form rosettes with sheep erythrocytes and adhere to nylon, whereas cytotoxic lymphocytes have a receptor for C3 but no surface immunoglobulin. The lack of tumor-specific lymphocytotoxicity in some patients can be due to inhibitor lymphocytes.     

Receptors for immunoglobulin and complement on sheep RBC-binding lymphocytes in newborn infants.

Sheep erythrocyte-binding human peripheral blood lymphocytes bearing receptors for the Fc-part of IgG and/or complement were studied in twenty newborn pre-term and term infants and fifteen adult controls. A technique with mixed rosettes and fluorescein-labelled sheep erythrocytes was used. The newborn infants had a significantly lower proportion of lymphocytes with receptors for sheep erythrocytes and the Fc-part of IgG (1.6%) than the adults (6.7%). Also the percentage of lymphocytes binding both sheep erythrocytes and complement-coated indicator cells was significantly lower in the newborn infants (2.6%) than in the adults (8.9%). A significantly higher proportion of lymphocytes had receptors for sheep erythrocytes and complement than for sheep erythrocytes and the Fc-part of IgG in both newborn infants and adults. There was no correlation between the values of lymphocytes binding sheep erythrocytes, or the Fc of IgG or complement and the proportion of mixed rosettes.     

Autorosette formation in humans: study of the specificity of the T cell receptors for autologous erythrocytes

Spontaneous autorosette formation has been described as being restricted to a subpopulation of the circulating helper/inducer T cell subset. In order to study the specificity of the binding between human lymphocytes and autologous red blood cells (auto-RBC), we have investigated the relationship between autorosette forming cells (auto-RFC) and rosettes formed with allogeneic (allo-) or xenogeneic (xeno-) RBC. Using a mixed rosette assay in which the origin of the erythrocytes was assessed by the FITC labeling of one type of erythrocyte, we have shown that auto-RFC and allo-RFC belong to the same T cell subset, and that the T cells which rosette with auto-RBC can also bind xenogeneic (pig, sheep, rabbit) RBC, although a disparate incidence of rosettes is found depending upon the origin of the erythrocytes. Whether T lymphocytes co-expressed distinct receptors for RBC of different species was then investigated. Preincubation of lymphocytes with monoclonal antibody OKT11A (directed against the T cell receptors for sheep RBC) completely abrogated rosette formation with auto- or allo-RBC, indicating that auto- and allo-RBC interact with the lymphocytes by their receptors for sheep RBC. Therefore, the auto-RFC may represent T lymphocytes having high affinity receptors for sheep RBC.     

Rosette formation with goat erythrocytes. A marker for human T lymphocytes.

We demonstrate the use of goat erythrocytes in a rosette procedure for the classification of human lymphocytes. The population is almost perfectly overlapping with the lymphocytes which form rosettes with sheep red blood cells. 70·2 ± 7·5% of peripheral lymphocytes form rosettes with goat erythrocytes and less than 1% of these cells have surface immunoglobulins. Enrichment of goat rosette-forming cells results in a population with an increased percentage of both goat and sheep rosettes. This population retains activity to the T-cell mitogens Con A and PHA, while the cells depleted of goat rosettes have greatly diminished responses to these same mitogens. Tonsil and spleen lymphocytes form 50·2 ± 6·8% and 24% of goat rosettes respectively, while peripheral blood lymphocytes from patients with CLL rarely form goat rosettes. Cell lines maintained in vitro rosetted with goat cells in a parallel fashion to sheep cells. Thus T-cell lines, such as Molt-3, which form rosettes with SRBC also rosette with GRBC, while sheep rosette-negative lines, i.e. Molt-4, are negative for both erythrocytes. B-lymphoid cell lines were negative, as were several lymphoma cell lines. There was a slight variation in the binding of goat cells, depending on the source of the goat. Thus, as in sheep rosettes, some animals were better sources than others, although all the animals tested formed rosettes.Human lymphocytes are capable of binding goat red cells. The cells which bind to the erythrocytes seem identical to those binding sheep red blood cells, and should be considered as a T-cell population. Preliminary inhibition data suggests that the receptor on T cells is the very same structure for both erythrocytes.     

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.     

Antibody Dependent Cellular Cytotoxicity and Mitogen Responsiveness of Human Peripheral Blood Lymphocytes Differing in Avidity for Sheep Erythrocytes

To characterize the effector cells (K-cells) in antibody-dependent cellular cytotoxicity (ADCC) against a nucleated target cell (El 4), human peripheral blood lymphocytes (PBL) were fractionated by roseue sedimentation into subpopulations differing in avidity for sheep erythrocytes (E). The fractions obtained were assayed for surface markers, and as a functional T cell marker, for responsiveness to the mitogen leucoagglutinin (La). By depleting PBL of E-receptor-bearing cells (E_l+), approximately half of the cytotoxic potential was found in the E_L+-depleted fraction. While the E_L+ fraction had low activity on a per cell basis, it nevertheles contained a significant proportion of the original cytotoxicity. By sequential E-rosetting, fractions binding E with high avidity (E_L+), low avidity (E_L+) or not at all (E ^-) were obtained. Both E-binding fractions consisted primarily of T-cells, as judged from their surface marker profiles. Both fractions responded well to La, but with different dose optima. The E^- fraction contained primarily B-and null cells and did not respond to La. Significant K-cell activity was foundl in all these fractions. The results show that a significant fraction of the K-cells have receptors for E and these can be of either high or low avidity. Since both E_H+ and E_L+ fractions respond well to La and contain K-cells, a T-cell origin of the later is suggested. However, whether or not cytotoxicity and La-responsiveness are functions of the same cells remains to be established.     

The antibody-independent cytotoxic activity of normal circulating human leucocytes. II. Failure to demonstrate effector cell-target cell interaction and target cell specificity of the circulating cytotoxic-enhancing factor.

The naturally-occurring antibody-independent cellular cytotoxic activity (NOCC) of normal circulating human monocytes and neutrophils was investigated employing a number of erythrocytes and the K-562 cell line as target cells simultaneously. The identity of the effector cell(s) was shown to be dependent upon or be a function of the type of target cell selected for the assay system. A number of erythrocyte targets (rabbit, horse, sheep and ox erythrocytes) were lysed to varying degrees by neutrophils and monocytes and not by lymphocytes. Irrespective of the red blood cell (RBC) target, the effector monocyte invariably possessed receptors for both C'3 and the Fc of IgG. In contrast, the cytotoxic cells using the K-562 target cell were lymphocytes. Monocytes and neutrophils were inactive. The cytotoxic-enhancing activity in normal human serum exhibits specific and non-specific properties which suggests that more than one factor is involved. With respect to the monocyte cytotoxic cells, only the rabbit erythrocytes could totally absorb the serum factor in a specific fashion. Absorption of the serum with horse, sheep or ox erythrocytes resulted in a significant loss of potentiating activity with respect to all of the erythrocyte targets but a more marked loss of activity using the absorbing erythrocytes as targets. With respect to the polymorphonuclear leucocyte effector cells, only the rabbit RBC were capable of specifically absorbing out the cytotoxic-enhancing factor present in the normal human serum. Absorption of the serum with sheep, horse or ox RBC resulted in total cross-absorption of the enhancing factor. Chicken and human RBC, which do not serve as targets for the NOCC assay, could not absorb out the cytotoxic-enhancing factor with respect to any of the target erythrocytes. The composition of the soluble serum factor(s) is under current investigation but it is not an immunoglobulin since pure serum albumin can substitute for normal serum in the NOCC assay. The mechanism of erythrocyte lysis by the cytotoxic monocyte was investigated. Mononuclear cells were incubated with target cell monolayers and with target cells under optimal rosetting conditions. No interaction between the effector and target cells could be detected. The monocytes did not adhere to the target cell monolayer nor did they form rosettes with the target cells. Thus, the results fail to corroborate or support the assumption that the cytotoxic activity of the monocyte is dependent upon conventionally-detectable receptors. Erythrophagocytosis was not observed to any significant degree under the assay conditions used. Therefore, the nature of the interaction between the cytotoxic monocyte and the erythroid target cell which results in lysis of the target cell remains to be elucidated.     

In vitro effect of murine-derived transfer factor on Salmonella-specific rosette formation.

The splenic lymphocytes from Salmonella-immune ICR Swiss or C3H/HeJ mice formed greater than 0.2% antigen-specific rosettes with sheep erythrocytes coated with a spent-medium protein antigen of Salmonella typhimurium. These rosette-forming lymphocytes were found to be sensitive to the effects of antithymocyte serum plus complement. Transfer factor prepared from the Salmonella-immune splenic lymphocytes of ICR Swiss mice was active in sensitizing nonimmune ICR Swiss or C3H/HeJ lymphocytes to form greater than or equal to 0.2% rosettes with salmonella antigen-coated sheep erythrocytes. These rosettes were also sensitive to antithymocyte serum and complement. Few rosettes were formed between the transfer factor-treated lymphocytes and sheep erythrocytes coated with a Listeria protein antigen. A nonimmune dialysate preparation was inactive in sensitizing nonimmune lymphocytes, as indicated by a lack of rosette formation. Neither the immune transfer factor nor the nonimmune dialysate had any enhancing or abrogating effect upon rosette formation by splenic lymphocytes from Salmonella-immune mice. The enumeration of antigen-specific rosettes may be a useful means of assaying for transfer factor activity.     

Human lymphocyte subpopulations. Human thymus-lymphoid tissue (HTL) antigen-positive lymphocytes forming rosettes with sheep erythrocytes and HTL antigen-negative lymphocytes interacting with antigen-antibody-complement complexes

Human lymphocytes from various lymphoid tissues were studied for the relationship between the existence of HTL (human thymus-lymphoid tissue) antigen, and binding of sheep erythrocytes (E) or sheep erythrocyte–antibody-complement complexes (EA(IgM)C43). E adhered to the majority of thymus lymphocytes and formed rosettes. These lymphocytes were shown to be HTL antigen positive by immunofluorescence performed simultaneously. In the peripheral lymphoid tissues, 10–30% of lymphocytes formed E rosettes and almost all E rosette-forming lymphocytes were HTL antigen positive. Conversely HTL antigen-negative cells did not form E rosettes.

In contrast, the cells binding EA(IgM)C43 were always HTL antigen negative.

There were very few HTL antigen-positive or rosette-forming lymphocytes either with E or EA(IgM)C43 in bone marrow.

From these data we conclude that E-rosette-forming and HTL antigen-positive lymphocytes are of thymus origin and EA(IgM)C43-rosette-forming cells are not thymus-dependent cells.

     

Identification of an anti-monocyte monoclonal antibody that is specific for membrane complement receptor type one (CR1)

A monoclonal antibody (E11) was produced by immunization of mice with intact human cells of monocyte lineage. Despite the finding that E11 did not inhibit rosettes with C3b-coated sheep erythrocytes (EC3b), several lines of evidence indicated that E11 was specific for complement receptor type one (CR1). All monocytes, neutrophils, lymphocytes and erythrocytes that reacted with E11 formed EC3b rosettes. The E11 antigen on these cells was shown to be a molecule of 222 +/- 10 kDa. Treatment of lymphocytes, monocytes, and neutrophils with E11 followed by fluorescein-coupled F(ab')2 anti-mouse-IgG at 37 degrees C in buffer lacking sodium azide, led to capping or apparent endocytosis of the E11 antigen and a diminution in CR1 activity of 88%, 59% and 25%, respectively. This same treatment had no detectable effect on monocyte or neutrophil CR3 activity (EC3bi rosettes). Furthermore, with E11-capped lymphocytes, the residual EC3b rosetting was capped directly over the E11-fluorescence cap, whereas EC3d,g rosetting (CR2 specific) was undiminished and distributed evenly around the circumference of cells containing E11-fluorescence caps. Finally, the binding of E11 to cells was inhibited by the prior treatment of these cells with a well characterized rabbit polyclonal anti-CR1. These data indicated that E11 was specific for a site in CR1 that was distal from the C3b-binding site, so that E11 was unable to block CR1 activity. E11 proved to be useful for identifying CR1 on various cells in tissue sections, and for quantitating CR1 on erythrocytes and neutrophils. Erythrocytes and neutrophils from normal individuals were found to bind an average of 610 and 4.6 X 10(4) 125I-labeled E11 molecules per cell. When E11 was visualized in tissues by immunoperoxidase staining, the cells that apparently contained the greatest amounts of CR1 were dendritic reticulum cells and kidney podocytes. The E11 reactive dentritic reticulum cells were characteristic of both follicular and diffuse follicular center cell tumors. Lymphocytes from patients with chronic lymphocytic leukemia (CLL) characteristically expressed little E11, confirming earlier studies that CLL cells lacked CR1 activity detected by EAC1-3b rosette formation. Because normal B cells have been shown to express CR1 at a very early stage of maturation, the absence of CR1 on CLL cells is discordant with the immature nature of CLL cells defined by immunoglobulin expression.     

Receptors for the Third Complement Component on a Proportion of Large Granular Lymphocytes from Human Peripheral Blood

Large granular lymphocytes (LGL) are nonadherent cells with cytoplasmic azurophilic granules, avid receptors for the Fc portion of IgG, and a paranuclear localization of alpha-naphthyl acid esterase or acid phosphatase. LGL constitute the bulk of TG cells (cells with receptors for sheep erythrocytes and for IgG molecules) and null cells (non-T, non-B cells). In the present study we demonstrate that 20-33% of the circulating human LGL express receptors for the third complement component (C3R). When TG cell or null cell fractions from normal individuals or non-T cells from a patient with infantile agammaglobulinaemia (which contained almost exclusively LGL) were rosetted with erythrocytes coated with antibody and complement, a variable number of C3R-bearing cells were detected. Such cells were isolated and analysed further; the great majority of them displayed the cytochemical and ultrastructural features of LGL.     

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.