U. Hellström

The Interaction of Nonmitogenic and Mitogenic Lectins with T Lymphocytes: Association of Cellular Receptor Sites

The relationship between the surface receptors on neuraminidase‐treated human blood lymphocytes for the mitogenic lectins Phaseolus vulgaris leukoagglutnin (La), concanavalin A (Con A), and soy bean agglutinin (SBA) and the nonmitogenic lectin Helix Pomatia A hemagglutinin (HP) was investigated. Two different techniques, co‐capping with different fluorochrome‐labeled lectins and cell binding‐inhibition experiments with 125 I‐labled lectins, were used. The results demonstrated that the nonmitogenic lectin HP and the mitogenic lectins SBA, La, and Con A bind either to the same macromolecule(s) or to different but physically linked macromolecules on the surface of human T lymphocytes. In contrast, only part of β2‐microglobulin (β2‐m), or β2‐m‐bearing complexes, appear to be physically linked to the lectin receptor complex(es). On the lectin‐binding substance(s) at least two saccharide structures were recognized, one of which binds both HP and SBA and another which hinds SBA and La (and probably also Con A) but not HP.

Receptors for Helix pomatia A Haemagglutinin (HP) on a Subpopulation of Human B Cells

Receptors for Helix pomatia A haemagglutinin (HP) have earlier been found on neuraminidase treated T‐lymphocytes in human peripheral blood. In contrast, the majority of the B‐lymphocytes, characterized by surface bound IgM and/or IgD (SIg) lack these receptors. Double marker experiments with fluorochrome labelled reagents have now shown that a minor fraction (3–24%) of the IgM/D bearing lymphocytes in normal human blood also have HP‐receptors. These HP+ B‐cells constitute approximately 1% of the HP+ lymphocytes in adult blood. Fractionation on HP‐Sepharose columns showed that the HP+ B‐cells are readily eluted with buffer containing 0.1 mg N‐acetyl‐D‐galactosamine. In contrast, the majority of the HP+‐Sig− cells require higher concentrations of the competing hapten for elution (1 mg d‐Ga1NAc/ml buffer). This indicates that the HP‐receptors on these B‐cells differ qualitatively or quantitatively from those on the majority of the T‐cells. Previous findings of HP‐receptors on the Sig+ leukaemic cells in the blood of patients with chronic lymphocytic leukaemia suggested that these structures are expressed on an immature variety of B‐cells. This assumption is favoured by the present finding that approximately 80% of the lymphocytes with surface bound IgM/D in cord blood also have HP‐receptors. Therefore, the HP‐receptor seems to fall in the category of differentiation markers and constitutes a useful tool for characterization and separation of human lymphocytes within both the T‐ and the B‐compartments.

Surface markers of human natural killer cells as analyzed in a modified single cell cytotoxicity assay on poly-L-lysine coated cover slips.

A modified single cell cytotoxicity assay using poly-L-lysine coated cover slips (PLL-SCCA) was employed to study the frequency and surface marker profile of human peripheral blood lymphocytes (PBL) with NK reactivity against K 562 target cells. When compared with the previously described agarose single cell cytotoxicity assay (A-SCCA) identical results were obtained. For 13 donors tested 18.1 +/- 4.4% of the PBL formed conjugates with K 562 and 2.7 +/- 1.6% displayed NK reactivity. In contrast to the A-SCCA, the PLL-modified assay permits direct identification of both conjugate forming (TBC) and cytolytic PBL (NK) by means of surface markers. Indirect immunofluorescence studies with monoclonal anti-PBL antibodies revealed that neither the plating procedures nor the incubation conditions employed affected the expression of the antigens recognized by these reagents. This method of directly identifying NK cells showed that OKM1+ cells were enriched among the NK cells as compared to PBL and TBC (55% vs. 23% and 43%, respectively). In contrast, the OKT3+ or Leu1+ fraction of the NK cells was reduced as compared to PBL and TBC. However, using this method of identification at the effector cell level, a substantial proportion of the NK cells were OKT3+ or Leu1+ (57% or 58% respectively, 7 donors). Approximately 25% of the NK cells were Leu2a+ and 30% were Leu3a+, respectively. However, the size of the Leu3a+ fraction varied considerably with individual donors and the size of this fraction appeared to be inversely related to that of the donors NK pool.

Fractionation of Human Blood Lymphocytes on Helix pomatia A Haemagglutinin Coupled to Sepharose Beads

Abstract. Treatment of human blood lymphocytes with neuraminidase has previausly been shown to uncover receptors for the A haemagglutinin of the snail Helix pomatia (HP). Neuraminidase‐treated lymphocytes were now fractionated on columns charged with large Sepharose particles to which HP had been coupled covalently. HP‐receptor negative (HP‐) lymphocytes passed the columns while HP‐receptor positive (HP+) lymphocytes were retained. The latter cells were eluted by addition of the competitive hapten N‐acetyl‐D‐galactosamine (D‐GalNAc). The total yield of cells recovered after fractionation was 60‐80% Surface marker studies indicated that there was no selective loss of any of the major lymphocyte subpopulations. The fraction that passed the columns (fraction I) consisted of approximately 10% of all lymphocytes. It contained ˜ 1% HP+ cells and ˜3% of all lymphocytes forming rosettes with sheep erythrocytes (E+ cells) present before fractionation. 50‐55% of the lymphocytes in this fraction had surface‐bound immunoglobulin (SIg+ cells) and complement receptors (EAC+ cells). Of the SIg+ cells, ˜60% were true B cells while the remaining 40% had IgG adsorbed to their surface. The majority of the B cells were recovered in this fraction. The lymphocytes of this fraction responded poorly to T‐cell mitogen but had an enhanced K‐cell activity to chicken erythrocytes. Elution of the cells retained on the column with 0.1 mg/ml D‐GalNAc gave a fraction II, consisting of, ˜15% of all lymphocytes. This fraction had a mixed composition. The majority of the cells (˜45%) were recovered by subsequent elution with 1.0 mg/ml D‐GalNAc. This fraction III was strongly enriched with HP+ and E+ cells (T cells). About 10% of the HP+ cells in this fraction were SIg+. However, on the majority of these cells this surface‐bound immunoglobulin was probably externally adsorbed IgG. These HP+‐SIg+ cells were also EAC+ and had Fc receptors, as shown by rosette formation with IgG‐coated bovine erythrocytes. The lymphocytes of fraction III responded most strongly to T‐cell mitogen while their K‐cell activity was weak.

A New Rat Lymphocyte Surface Marker: Characterization and Separation of Cells with Receptors for Helix pomatia Hemagglutinin

Thirty‐two percent of neuraminidase‐treated DA rat spleen lymphocytes and 18% of lymph node lymphocytes possess receptors for Helix pomatia hemagglutinin (HP). Moreover, these HP‐receptor‐bearing cells can be separated from B cells by affinity chromatography on HP‐Sepharose columns. The virtual absence of immunoglobulin (Ig) receptors and the close correlation with reported T‐cell content of these lymphoid tissues surest that HP‐receptor lymphocytes are probably T cells and that HP may provide a convenient marker, for both the identification and the purification of rat T lymphocytes.

Proliferative and Cytolytic Reactivities of Human Lymphocytes Fractionated on Wheat Germ Agglutinin‐Sepharose Columns before or after Alloactivation in Mixed Lymphocyte Culture

Human T‐lymphocyte preparations from peripheral blood with either high‐ or low‐avidity receptors for wheat germ agglutinin (WGA) were obtained by fractionation on WGA‐Sepharose columns. Both fractions contained progenitors of alloreactive T cells, proliferating in mixed lymphocyte culture (MLC) and acting as effector cells in cell‐mediated lympholysis (CML). Proliferation and CML activity of the two fractions were equal and similar to those of the unfractionated cells. However, when the lymphocytes were fractionated after 5 days’ MLC, most of the proliferating and cytolytic cells were found in the lymphocyte fraction enriched in cells with high‐avidity receptors for WGA. The reactivity of the fractions was correlated to their content of blast‐transformed cells. The binding of MLC‐activated lymphocytes to WGA was specific, since it was inhibited by the competitive hapten d‐GlcNAc and no cells were retained on control columns charged with human serum albumin–Sepharose. B cells and monocytic cells were enriched in the fraction with low‐avidity receptors for WGA. As indicated by experiments in which cells were mixed in different proportions, the low MLC–CML activity of the lymphocytes in the fraction with low‐avidity WGA receptors was not caused by suppressor cells present in that fraction.

Mitogenic Responsiveness of Human T‐Lymphocyte Subpopulations: Regulation by Suppressive Fc‐Receptor‐bearing T Cells and Influence of Fractionation Procedures

The proliferative response induced by leucoagglutinin (La) in different subpopulations of human I lymphocytes was studied Subpopulations enriched in cells with either high‐ or low‐avidity receptors for sheep erythrocytes (SRBC) (EH.+, Et+) were prepared by sequential E‐rosetting. In addition, T lymphocytes prepared by E‐rosetting under optimal conditions (E+ror) were fractionated on wheat germ agglutin (WGA)‐Sepharose columns, rendering fraction enriched in lymphocytes with either low‐ or high‐avidity receptors for WGA. The T lymphocytes were found to comprise at least three functionally distinct subpopulations, differing with respect to mitogen responsiveness. Cell characterized by high‐avidity receptors for WGA and SRBC were highly responsive to La simulation, regardless of the method used for purification. In contrasts cell with low‐avidity receptors for WGA and probably also for SRBC hut lacking Fe receptors for IgG responded only marginally but were conditioned to respond when subjected to E‐rosetting under optimal conditions. This response was suppressed by lymphocytes with Fc receptors for IgG, which probably also had low‐avidity receptors for WGA and SRBC. The lymphocytes with high‐avidity receptors for WGA and SRBC did not appear to be susceptible to suppression by Fcγ+ cells.

Regulation of the immune response to hepatitis B virus and human serum albumin. I. Hepatitis B surface antigen-induced secretion of antibodies with specificity for human serum albumin in hepatitis B-immune donors in vitro.

The circulatory pool of B cells, from donors immune lo hepatitis B (HB) through natural infection, contained sensitized B cells with the capacity to secrete antibodies with specificity for human serum albumin (USA) when stimulated with purified hepatitis B surface antigen (HBsAg) in vitro The immunoglobulin secretion was dependent upon and regulated by T cells and specifically induced, since it was not obtained in cell cultures from HB‐susceptible donors. Culture supernatants with anti‐USA reactivity also contained specific antibodies to HBsAg (anti‐HBs). indicating that the outer coat of HBV normally provokes an immune response to both the viral antigen and a self component. Perturbation in the regulation of the immune response triggered by USA in association with HBV/HBsAg particles may involve a putative risk for development of chronic HBsAg carriership.

A new surface marker on equine peripheral blood lymphocytes. I. Subpopulations of lymphocytes with receptors for Helix pomatia A hemagglutinin (HP)

Untreated and neuraminidase-treated equine peripheral blood lymphocytes were analysed for binding of the A hemagglutinin of the snail Helix pomatia (HP). For optimal staining by direct immunofluorescence, the concentration of neuraminidase had to be increased as compared to that needed for other species. Moreover, higher concentrations of HP were required for optimal staining of equine lymphocytes as compared to lymphocytes from other species. Even so, the maximal number of equine lymphocytes exhibiting positive staining was only about 20%. No, or very few, HP-positive lymphocytes were seen when neuraminidase treatment was omitted. However, when the more sensitive method of indirect immunofluorescence was used, approximately 60% of the lymphocytes were HP positive without prior treatment with this enzyme. Neuraminidase treatment significantly increased this figure to about 75%. In all instances, HP binding was specific since it was inhibited by the competitive sugar hapten N-acetyl-D-galactosamine (D-GalNAc) while addition of D-glucose (D-Glc) gave no inhibition. HP binding to neuraminidase-treated lymphocytes was also investigated quantitatively by means of 125I-labeled HP. The number of HP molecules bound per HP-positive cell was approximately 3 X 10(5) and the apparent association constant for the binding of HP to its cellular receptors was approximately 8 X 10(7) 1/mol. No binding of HP to untreated lymphocytes could be recorded in these experiments.

The use of supernatants from neuraminidase and galactose oxidase (NAGO)-treated lymphocytes as a source of T cell growth factor

Human peripheral blood or tonsil lymphocytes produce T cell growth factor (TCGF), when activated with neuraminidase (NA) and galactose oxidase (GO). Partial purification of NAGO-TCGF on Sepharose G-100 columns gave a TCGF-active fraction within the same molecular weight range as the conventional lectin-induced TCGF (approximately 15,000 Da). Human T cells, activated in mixed lymphocyte culture (MLC) with irradiated allogeneic EB-virus transformed B-cells (LCL) could be maintained in continuous culture for several months with retained functional activities. The cells showed similar growth patterns when cultured in the presence of either NAGO-TCGF or PHA-TCGF. The growing cells were characterized by means of monoclonal antibodies. After 4 weeks of culture 98% of these were OKT3+ and 87% were also OKT8+. The cytolytic activities of the cultures were tested in cell-mediated lympholysis (CML) against allogeneic LCL as target cells, in natural cytotoxicity (NK) against K562 cells and in antibody dependent cytotoxicity (ADCC) against bovine erythrocytes. Cultures displaying one or several of these functions were obtained. The results indicate, that TCGF obtained from supernatants of NAGO-activated lymphocytes is as potent as the T cell growth promoting factor obtained by lectin stimulation. One major advantage of using NAGO-generated TCGF is that contamination with lectin is avoided.