Separation of antibody helper and antibody suppressor human T

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1 Proc. Natl. Acad. Sci. USA Vol. 77, No. 11, pp , November 198 Immunology Separation of antibody helper and antibody suppressor human T cells by using soybean agglutinin (human lymphocyte subpopulation/differential agglutination) YAIR REISNER*, SAVITA PAHWA*, J. W. CHIAO*, NATHAN SHARONW, ROBERT L. EVANS*, AND ROBERT A. GOOD* *From the Developmental Immunobiology, Cellular Engineering and Cancer Immunotherapy Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 121; and tthe Department of Biophysics, Weizmann Institute for Science, Rehovoth, Israel Contributed by Robert A. Good, July 15, 198 ABSTRACT Soybean agglutinin (SBA) binds specifically to mouse B cells and has been used in the past to separate mouse B and T spleen cells by differential agglutination of the B cells. In the present study it was found that a major T-cell subpopulation of human peripheral blood mononuclear cells is agglutinated by SBA along with the B cells and monocytes. Tests of such cell surface markers as Fc receptors for IgG and IgM, as well as functional assays of antibody production by B cells, revealed that the SBA-agglutinated cell fraction contains the antibody helper T cells whereas the unagglutinated fraction is enriched with antibody suppressor T cells. Similar observations were made in tests of the proliferative response to mumps antigen. A recently prepared monoclonal antibody, anti-leu 2a, which recognized the same thymus-dependent antigen previously defined by a heterologous anti-human T cell serum (atth2), was found to define by indirect immunofluorescence a subpopulation of SBA- cells of intermediate staining intensity which was not detectable in the SBA+ population. The selective agglutination of lymphocytes by various lectins has been used in the past few years for the isolation of lymphocyte subpopulations (1). This approach is feasible because clumps of the agglutinated cells can be separated from the unagglutinated cells by layering the cell mixture in a viscous medium such as bovine serum albumin solution or fetal calf serum and allowing the cells to sediment. After separation the agglutinated cells can be dissociated to viable single cells by washing with the sugar inhibitor of the lectin. By using this method, mature murine T splenocytes were separated from B cells by soybean agglutinin (SBA) (2). Moreover, from binding studies with radioactively labeled SBA it was found that spleen T cells do not bind the lectin. In this regard they differ from B cells or thymocytes which bear substantial numbers (16 sites per cell) of receptors for this lectin on the cell surface (unpublished observations). We have now found that, in contrast to the mouse T splenocytes, a major human T-cell subpopulation in the peripheral blood is agglutinated together with the B cells by SBA. Thus, human B cells cannot be isolated by this method. However, analysis of differences in biological function and cell surface markers between the cells agglutinated by SBA and those not agglutinated revealed that two distinct T-cell subpopulations, the helper and the suppressor T cells for antibody production, are separated by SBA. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact MATERIAL AND METHODS Cell Suspensions. Heparinized human blood was diluted with an equal volume of balanced salt solution (3), layered over 15 ml of Ficoll/Hypaque (Lymphoprep, Nyegaard, Oslo, Norway) in 5-ml plastic centrifuge tubes (Falcon, no. 227), and centrifuged for 3 min at 4 X g at room temperature. Peripheral blood mononuclear cells (PBM) were collected from the interface, washed twice with RPMI-164 medium, and resuspended in the required medium. Purification of T cells by erythrocyte rosetting with sheep erythrocytes (SRBC) was performed as described (4). Lectin. SBA was purified by affinity chromatography (5). Polyclonal Antibody Production Assay. This assay was performed as described (6) with pokeweed mitogen (PWM) for the mitogenic stimulation of B cells, and detecting Ig-secreting cells by a reverse plaque assay after 6 days. of each type were at 5 X 15 cells per ml. Cocultures consisted of 2.5 X 15 cells of each type per ml. The final volume was kept constant at 1 ml. Antigen-Specific Assay of Antibody Production. This assay was performed as described by Hoffman (7). of each type (1-3 X 15) were in microtiter plates at a final volume of.1 ml, conditions which were found by Hoffman (7) to give optimal plaque-forming cell (PFC) response in this assay. In the cocultures the different cell types were mixed at a 1:1 ratio and the final volume was kept at.1 ml. Mumps Virus-Induced Proliferative Response. (15 cells in.1 ml of RPMI-164 medium) were placed in roundbottom microtiter plates as described (8), and the antigen was added in various concentrations to a final volume of.2 ml. After 5 days of cell culture in a 37 C (5% C2/95% air, humidified), cells were pulsed with.2,ci (1 Ci = 3.7 X 11 becquerels) of [3H]thymidine for 16 hr. The cells were then harvested with a multiple cell harvester and the uptake of 3H was measured by liquid scintillation counting. Cell Surface Markers. Surface membrane Igs were detected by immunofluorescence, with a rabbit IgG-F(ab')2 reagent with antibody specificity for humans Igs (9). T lymphocytes were identified by spontaneous rosetting capacity with uncoated SRBC (1). Myeloperoxidase stain was used to determine the presence of monocytes (11). T cells with IgG or IgM receptors were detected with a mixed rosette method that detected simultaneously the SRBC rosetting capacity and the Ig receptors Abbreviations: SBA, soybean agglutinin; SRBC, sheep erythrocytes; PBM, peripheral blood mononuclear cells; PWM, pokeweed mitogen;- PFC, plaque-forming cells.

2 Immunology: Reisner et al. PBM (O5ml,4x 18/ml) Autologous RBC ( 5,ul, 4 x 19/ml ) 2 mm W \ i \ 5% in PB' SBA+ Agglutinated D -Goloctose, 2M + SB1Am SBA- SBA (.5ml, 2mg/mi) 3~~~~SA Unagglutinated Cel Is 5min FIG. 1. Procedure for fractionation of PBM cells by SBA. BSA, bovine serum albumin; PBS, phosphate-buffered saline. (12). Rabbit IgG-coated guinea pig erythrocytes or rabbit IgM-coated ox erythrocytes were used with uncoated SRBC. The method for enumerating the cells in these subclasses and the isolation of rabbit IgG and IgM have been described (12). The binding of anti-leu-2 was detected by indirect immunofluorescence with fluorescein-conjugated goat anti-mouse antiserum. Staining of the cells and analysis by fluorescenceactivated cell sorter were carried out as described (13). RESULTS AND DISCUSSION Separation of PBM by SBA. The PBM suspension prepared by Ficoll/Hypaque fractionation (4 X 18 cells/ml,.5 ml) was incubated in a 17 X 1 mm polystyrene tube for 5 min at room temperature with autologous erythrocytes (4 X 19 cells/ml, 5,l) and SBA (2 mg/ml,.5 ml) (Fig. 1). The cells were then layered, with a pasteur pipette, on top of 8 ml of 5% bovine serum albumin in phosphate-buffered saline in a 15-ml conical plastic tube. After 2 min at room temperature, most of the agglutinated cells had sedimented, whereas the unagglutinated single cells remained on the surface. The bottom and top fractions were removed separately with pasteur pipettes and transferred to 15-ml conical plastic tubes. The cells were then suspended in 1 ml of.2 M galactose in phosphate-buffered saline. After 5 min at room temperature the cells were collected by centrifugation (2 X g, 5 min) and washed twice more with galactose. This washing procedure dissociated all aggregates into a fully dispersed single-cell suspension. Finally, the erythrocytes were lysed by treatment with.83% ammonium chloride solution, and the lymphocytes were washed twice with RPMI-164 medium. The agglutinated fraction contained approximately 79-87% of the cells (mean of five experiments, 83%) and the total recovery ranged between 6% and 8%. A trypan blue dye exclusion test showed that both fractions contained more than 95% viable cells. PBM from most normal human subjects can be separated by SBA without the addition of autologous erythrocytes. However, for reasons not yet fully understood, PBM from some subjects are poorly agglutinated and the use of autologous erythrocytes as carriers maximizes efficiency of separation of PBM of those subjects. We therefore introduced the addition of autologous erythrocytes as a standard feature of our method. We noted that SBA preparations from different sources varied remarkably in specific activity. We therefore defined M Proc. Natl. Acad. Sci. USA 77 (198) 6779 the specific activity of our SBA preparation by an agglutination assay using normal human erythrocytes as follows: 1 IA1 of SBA (2 mg/ml in phosphate-buffered saline) was serially diluted (1:1) in wells of round-bottom microtiter plates (Linbro/Titertek, Flow Laboratories, Hamden, CT) to which 1,Ml of thrice-washed human erythrocytes (5 X 17 cells per ml in phosphate-buffered saline) were added. The end point of agglutination was defined as the lectin concentration at which all erythrocytes in a well sedimented and formed a discrete button surrounded by a clear area. The end point of agglutination of the SBA preparations used in this study was ug/ml. Separation of T by SBA. T cells, previously purified by SRBC-rosetting, were fractionated with SBA as described above for PBM. In these experiments the unagglutinated fraction contained about 69-85% of the cells (mean of six experiments, 78%). Surface Markers of PBM Fractionated with SBA. Most of the B cells (surface Ig-positive cells; 14%) and monocytes (peroxidase-positive cells; 22%) as well as a major subpopulation of T cells (55%) were agglutinated by SBA, whereas the unagglutinated cell fraction was composed mainly of T cells (88%) and was depleted of B cells (2%) and monocytes (3%) (Fig. 2). Antibody Production in the Separated Cell Fractions of PBM. In accordance with the test for surface markers, in which we found enrichment with B cells in the agglutinated fraction, stimulation of B cells to produce antibodies with either a specific antigen (SRBC) or with PWM revealed an enrichment for this activity in the agglutinated fraction and a marked depletion in the unagglutinated one relative to unseparated PBM (Fig. 3). Recombining of the separated cell fractions showed that addition of unagglutinated cells to the agglutinated cells resulted in a suppressive effect (Table 1). Suppressor and Helper Activity in T-Cell Subpopulations Separated with SBA. The experiments described above suggested that two major regulatory T-cell subpopulations may be separated with SBA: antibody helper cells which are agglutinated by the lectin (SBA+), and antibody suppressor cells which are not agglutinated by the lectin (SBA-). This hypothesis was tested further by experiments in which purified T cells (erythrocyte-rosetted cells) were separated with SBA and the SBA+ and SBA- cells were tested for their regulatory effect on antibody production in two different assay systems: (i) antigen-specific assay against SRBC and (ii) polyclonal assay using PWM to induce B cells to synthesize and secrete Ig. The regulatory effect of the agglutinated and unagglutinated T cell fractions was tested in three groups of mixing experiments in which different B-cell preparations were used as precursors of antibody-producing plasma cells: (a) B cells purified by removal of erythrocyte-rosetting T cells, (b) whole PBM cells, and (c) SBA-agglutinated fraction of PBM which is enriched for B cells but also contains monocytes and SBA+ T cells (Fig. 2). Table 1. Antibody production by PBM cell fractions separated by SBA: Effect of mixing cell fractions ISC/16 cells PBM 16,4 SBA- PBM 1,8 SBA+ PBM 32,6 PBIM + SBA- PBM 9,8 PBM + SBA+ PBM 45,6 SBA+ PBM + SBA- PBM 4,5

3 678 Immunology: Reisner et al. a b c FIG. 2. Incidence of T cells (yin), B cells (-), and monocytes (E) in PBM fractionated by SBA. (a) Unseparated; (b) unagglutinated by SBA; (c) agglutinated by SBA. These are the results of a typical experiment. The first group of experiments was designed to test helper activity rather than suppressor activity because the background of antibody-producing cells of this B-cell population is low in the absence of added T cells. In the antigen-specific assay in which this background was negligible, we found that all the helper activity was contained in the agglutinated fraction and none was found in the unagglutinated fraction (Table 2). Similar results were obtained in the polyclonal assay with PWM in which a small but significant background of antibody production was present even without added T cells. Thus, the SBA+ T cells enhanced antibody production and the SBA- T cells suppressed the small preexisting background of antibody production. The low values of antibody production in the two isolated T-cell fractions (Table 2) rule out the possibility that the helper effect was attributable to addition of antibody-producing cells. The second group of experiments, in which whole PBM cells were used as a source of B cells, was designed to assay suppressor T cells because whole PBM cell suspensions contain both types of regulatory T cells and therefore the effect of adding helper cells would be expected to be less pronounced than the effect of adding suppressor cells. Indeed, in the polyclonal assay with PWM, most of the suppressive activity was found in the SBA- T-cell fraction whereas the SBA+ cells exerted a minor helper effect (Table 3). I 9 c) * C-) -5 C/) soo 3 a b c a b c FIG. 3. Antibody production in PBM cells fractionated by SBA. Columns: a, unseparated; b, unagglutinated; c, agglutinated. (Left) Polyclonal stimulation of antibody production by PWM. (Right) Specific assay after incubation with SRBC (PFC, plaque-forming cells). Results are shown as mean + SD. Jy Proc. Natl. Acad. Sci. USA 77 (198) Table 2. Antibody production by isolated B cells: Effect of addition of T-cell fractions ISC/16 Anti-RBC PFC/16 cells cells B 22 SBA- T 24 SBA+T 8 5 B+SBA-T 88 B+SBA+T B 132 SBA-T 16 SBA+T 28 B+SBA-T B+SBA+T Experiment 3 B ND SBA-T ND SBA+T ND 3 B+SBA-T ND B+SBA+T ND 332 ND, not done. Finally, the third group of experiments, in which the agglutinated fraction of PBM was used as the source of antibody producing cells, also supports the conclusion that helper cells are agglutinated by SBA and suppressor cells are not (Table 4). Table 3. Antibody production by PBM cells: Effect of addition of T cells separated by SBA Anti-SRBC ISC/16 ISC/16 PFC/16 cells cells PBM 24,8 399 SBA- T 24 SBA+T 8 PBM + SBA- T 8, 21 PBM + SBA+ T 16, 2466 PBM 32,6 99 SBA- T 49 SBA+T 9 PBM + SBA- T 1, 46 PBM + SBA+ T 1, Experiment 3 PBM 6, 4 SBA- T 22 SBA+T 6 PBM + SBA- T 16,8 182 PBM + SBA+T 56,8 127 Experiment 4 PBM ND 434 SBA-T ND SBA+T ND PBM + SBA- T ND 11 PBM + SBA+ T ND 968 ND, not done.

4 Table 4. Antibody production of SBA+ PBM cells: Effect of mixing with T cells fractionated by SBA ISC/16 cells SBA+ PBM 32,6 T, total 3, SBA- T 1,6 SBA+ T 2,8 SBA+ PBM + total T 44, SBA+ PBM + SBA- T 1,6 SBA+ PBM + SBA+ T 56,6 Also, these experiments demonstrated a possible clinical application of the SBA separation technique for evaluation of regulatory T cell anomalies in immunodeficiencies. For example, simple fractionation of patients' whole PBM with SBA (which should take no longer than 1 hr) could yield an agglutinated fraction containing B cells, monocytes, and helper T cells and devoid of suppressor T cells, which would be ideal for antibody production. Therefore, it is to be expected that, in patients with an excessive number of suppressor cells, antibody production should be increased in the agglutinated fraction that has been depleted of suppressor cells. Fc Receptors for IgG and IgM on the Surface of T Separated by SBA. Both the agglutinated and the unagglutinated T-cell fractions contain cells that bear Fc receptors for Igs on their surfaces. The agglutinated T cell fraction contains about 3fold more of these cells than does the unagglutinated fraction. As shown in Table 5, which summarzes results of two experiments, the unagglutinated fraction is enriched with Ty cells, which specifically bind IgG (7.1% in Exp. 1 and 53.9% in Exp. 2) relative to the unseparated T cells (6.2% and 18.2%, respectively). In contrast, the Fc receptor-bearing cells in the agglutinated fraction are almost exclusively Tu cells (97.7% and 98.9%, respectively). These results are in agreement with previous suggestions (14, 15) that Ty cells act as suppressor cells for antibody production whereas TMu cells act as helper cells in these assays. It remains to be determined whether these activities can also be regulated by cells in the separated fractions which do not bear Fc receptors. Fluorescent Staining with Monoclonal Antibody to TH2 Antigen. Analysis of the T cells separated by SBA agglutination ) E Immunology: Reisner et al. Fluorescence Intensity FIG. 4. Fluorescence of intensity scan of T cells separated by SBA and stained with the anti-leu 2a monoclonal antibody from the serum of a nude mouse bearing the relevant hybrid tumor: *, unseparated;, agglutinated; -, unagglutinated. The peak of intermediate intensity associated with the SBA- population was not seen when these cells were treated with the appropriate background controls, including the serum from a tumor-free nude mouse. E Q. Proc. Natl. Acad. Sci. USA 77 (198) 6781 FIG. 5. Mitogenic response to mumps virus antigen of T cells separated by SBA. *, Unseparated; *, agglutinated; *, unagglutinated. revealed that the SBA- fraction is enriched with a unique subpopulation of T cells that stains to intermediate fluorescence intensity with the anti-leu 2a monoclonal antibody. This antibody defines the same population of lymphocytes recognized by the anti-th2 heterologous antisera. In contrast, the agglutinated and unagglutinated fractions differ little in the number of cells that stain with high intensity with anti-leu 2a (36.2% and 42.5%, respectively) (Fig. 4). The unexpected enrichment in the SBA- fraction both for the suppressor cells and the cells of intermediate fluorescence intensity with anti-leu 2a antibodies suggests that cells comprising this newly detected peak of intermediate fluorescence intensity may play a role either in generating suppressor cells or in mediating suppression in our assays. Mitogenic Response to Phytohemagglutinin, Concanavalin A, Alloantigen, and Mumps Virus Antigen. The separated T-cell fractions did not differ significantly in their proliferative responses to the phytomitogens phytohemagglutinin and concanavalin A or to allogeneic stimulation in mixed lymphocyte culture. However, in agreement with previous reports that the mitogenic response to antigens is produced by helper T cells, we found that the SBA+ T-cell fraction is highly enriched, relative to the unseparated and SBA- fractions, with cells that respond by proliferation to the mumps virus antigen (Fig. 5). Moreover, because most T cells are agglutinated by SBA, it seems that the enrichment in this fraction for antigen response cannot be explained simply by enrichment for helper cells but rather may reflect depletion of suppressor T cells which otherwise would exert a suppressive influence on the helper T cells, as postulated for suppression of antibody production (16). These results, along with those mentioned above, clearly demonstrate that human suppressor and helper T cells can be separated by using SBA. Preliminary studies also indicate that fluorescein-conjugated SBA could be used to stain helper T cells. Such cell staining, as well as the separation by agglutination of Table 5. Incidence of T1A and Ty cells in T cell fractions separated by SBA %T-y %TM T'yas%of T cells cells cells Fc+ cells Unseparated SBA SBA Unseparated SBA SBA

5 6782 Immunology: Reisner et al. the two T-cell subpopulations, could be of clinical importance in the evaluation of immunodeficient patients. This investigation was aided by U.S. Public Health Service Grants CA-8748, CA-19267, CA-1744, AI-11843, NS-11457, and AG-541 and Contract NCI-CB from the National Institutes of Health and by the Zelda R. Weintraub Cancer Fund, the Judith Harris Selig Memorial Fund, the George Fredrick Jewett Foundation, the Jeanne and Robert Rimsky Fund, the Siegfried and Josephine Bieber Foundation, and the J.M. Foundation. 1. Reisner, Y. & Sharon, N. (198) Trends Biochem. Sci. (Pers. Ed.) 5 (2), Reisner, Y., Ravid, A. & Sharon, N. (1976) Biochem. Biophys. Res. Commun. 72, Boyum, A. (1974) Tissue Antigens 4, Wybran, J., Carr, M. C. & Fudenberg, H. H. (1973) CGin. Immunol. Immunopathol. 1, Gordon, J. A., Blumberg, S., Lis, H. & Sharon, N. (1972) FEBS Lett. 24, Gronowicz, E., Coutinho, A. & Melchers, F. (1976) Eur. J. Immunol. 6, Proc. Nati. Acad. Sci. USA 77 (198) 7. Hoffman, M. K. (198) Proc. Natl. Acad. Sci USA 77, Rotter, V., Yakir, Y. & Trainin, N. (1979) J. Immunol. 123, Chiao, J. W., Pantic, V. S. & Good, R. A. (1974) Clin. Exp. Immunol. 18, Bentwich, A., Douglas, S. D., Siegal, F. P. & Kunkel, H. G. (1973) Cin. Immunol. Immunopathol. 1, Kaplow, L. S. (1965) Blood 26, Chiao, J. W., Fried, J., Arlin, Z. A., Freitag, W. B. & Good, R. A. (198) Cell. Immunol. 51, Evans, R. L., Lazarus, H., Penta, A. C. & Schlossman, S. F. (1978) J. Immunol. 12, Moretta, L., Ferrarini, M. & Cooper, M. D. (1978) Contemp. Topics Immunobiol. 8, Gupta, S. & Good, R. A. (1978) in Human Lymphocyte Differentiation: Its Application to Cancer, eds. Serrou, R. & Rosefeld, C. (North-Holland, Amsterdam), pp Harwell, L., Marrack, P. & Kappler, J. W. (1977) Nature (London) 265,57-59.