The response to phytohemagglutinin or to concanavalin A as a probe for subpopulations of human peripheral blood lymphocytes

CELLULARIMMUNOLOGY17, 310--314 (1975)

The Response to Phytohemagglutinin or to Concanavalin A as a Probe for Subpopulations of Human Peripheral Blood Lymphocytes 1 ARNOLD J. RAWSON

AND

T. C. I-IUANG

Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19174 Received December 23, 1974 Subtotal killing of human peripheral blood Iymphocytes responsive to P H A leaves a residual population which will respond ten times as vigorously to Con A as to PI-IA. By contrast, after subtotal killing of Con A-responsive cells, there is no relative enhancement of the response to P H A . This is interpreted as being suggestive of two populations of human peripheral blood lymphocytes: one responsive to either P H A or Con A, and the other principally responsive to Con A alone.

INTRODUCTION Evidence has been accumulating which suggests the presence of at least two T lymphocyte subpopulations in mice (1-5). In this species it appears that the ability to respond to stimulation by either phytohemagglutinin ( P H A ) or concanavalin A (Con A) is limited to T lymphocytes (2). The experiments of Stobo and co-workers (1) appear to define two subpopulations of T lymphocytes on the basis of the differential responsiveness to P H A and Con A. Their results are compatible with the hypothesis that one subpopulation responds well to stimulation either by P H A or Con A, whereas the other responds almost exclusively to stimulation by Con A. These subpopulations appear to differ with respect to their distribution in lymph nodes, spleen, and bone marrow, and also appear to have different biological functions. To our knowledge, no comparable information of human lymphocytes is available. It was the purpose of the present study to use the response to stimulation by P H A or Con A as a probe for subpopulations of human peripheral blood Iymphocytes. Advantage was taken of the ability to selectively eliminate cells, responding to a given mitogen, by treatment with 5-bromodeoxyuridine (BUdR) and light subsequent to stimulation (6), Human peripheral blood lymphocytes were stimulated with one of the two mitogens, and then exposed to BUdR and light to destroy the responding population. Subsequently the residual cells were tested for their ability to respond to the other mitogen. M A T E R I A L S AND M E T H O D S

Lymphocytes Lymphocytes were obtained by venopuncture of normal volunteers from 20 to 60 yr of age, the blood being drawn directly into heparinized tubes. Blood was 1 Supported by a Grant from the Barsumian Memorial Fund. 310 Copyright © 1975 by Academic Press Inc. All rights of reproduct on in any form reserved.

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diluted 2:1 with 0.85% sodium chloride solution, and centrifuged over FicollHypaque. The lymphocytes were recovered from the interface, washed with Hanks' solution, and suspended in MEM (GIBCO) with 10% normal human A, B serum at a concentration of 0.25 X 106/ml. Cultures were kept at 37°C in a moist chamber containing 5 ~ carbon dioxide and 9 5 ~ air. They were maintained for 1 wk. All experiments were done in triplicate.

Mitogens PHA-M (Difco) was reconstituted, and kept frozen. Con A (soluble, Calbiochem) was used as received and kept refrigerated. Both mitogens were titrated with suspensions of different normal lymphocytes to determine the optimum stimulating concentration.

BUdR Technique With minor modifications, the technique of Zoschke and Bach (6) was followed. BUdR (Sigma) was used at a concentration of 10-6 M, and was introduced to the culture on the second day (considering the culture to have been set up on day zero). On day 3 the culture was exposed to light (fluorescent daylight bulb) for 60 min. On day 4 the culture was exposed to light for 45 min. The medium was then aspirated and replaced with fresh medium, and the culture was again exposed to light for an additional 30 min. If the culture was to be restimulated, the secondary mitogen was introduced at this point.

Pulsing and Counting Sixteen hours before termination of the cultures on day 7, 0.25 /~Ci of tritiated thymidine (6.7 Ci/mM, New England Nuclear) was added. The degree of incorporation of tritium into the 5 ~ trichloroacetic acid precipitable fraction of the sediment was measured in a scintillation counter. The efficiency of counting was approximately 33~. RESULTS Table 1 summarizes the results of three different experiments performed at different times. Each experiment studied the lymphocytes of two normal subjects in parallel, and each test was performed upon an aliquot of the same cell suspension at the same time. Cultures were initially stimulated with an optimum concentration of PHA or Con A, or were not stimulated initially. The mitogens were added immediately after the establishment of the culture (day zero). On day 2, BUdR 10 6 M was added as shown in the table, and on days 3 and 4 the cultures to which BUdR was added were exposed to light. After changing the medium on day 4, a second mitogen was added as indicated in the table. Test no. 5 shows relatively little nonspecific killing of the reactive cells by BUdR and light. Estimated specific killing of PHA-responding cells, averaged 74~. That of Con A-responding cells was much lower, averaging 58~, although 9 0 ~ killing was achieved in one experiment. These estimates are arrived at by calculat-

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TABLE 2 EFFECT OF EXPOSURE TO P H A OR Con A AFTER SUBTOTAL DESTRUCTION OF CELLS RESPONDING TO THESE MITOGENS

Expt

Subject

Destruction of cells responsive to

cpm in excess of control PHA

Con A

354

1 2

PHA PHA

40 303

657 2094

356

3 4

PHA PHA

1094 125

8546 5837

357

5 6

PHA PHA

457 0

1450 2254

354

1 2

Con A Con A

0 0

0 0

356

3 4 5 6

Con A Con A Con A Con A

0 0 0 0

0 0 0 0

357

ing the ratio of test no. 2 to test no. 1, and test no. 8 to test no. 7, after deducting control values (test no. 6). Comparison of test no. 3 with test no. 4 shows stimulation by Con A of residual cells remaining after presumed destruction of most of the PHA-responding population. This is clearly illustrated in Table 2, where cpm in excess of control values (test no. 2) are indicated. It can be seen that in all cases of specific killing of the PHA-responsive population, restimulation of the residual cells by Con A resulted in an uptake of tritiated thymidine several times that resulting from restimulation with P H A . The mean cpm resulting from Con A restimulation is more than ten times that from P H A restimulation. By contrast, after specific killing of the majority of Con A-responsive cells, restimulation by either mitogen failed to achieve a response in excess of control values (test no. 8). DISCUSSION Perphaps the most serious criticism of this method is the failure to achieve total specific killing of responding cells. This is a greater problem in the case of Con Aresponsive cells than in those responding to P H A , and may in fact indicate a biological difference between populations defined in this manner, or more probably, may reflect the fact that response to Con A is somewhat delayed as compared with response to P H A (8). It appears clear, nonetheless, that after subtotal specific killing of P H A responsive cells, a residual population is still able to respond to Con A far better than to freshly introduced P H A . By contrast, after specific killing of Con Aresponsive cells, no relative enhancement of the response to P H A is observed. Several possible explanations for this phenomenon must be considered. Since it is estimated that approximately 2 5 ~ of the cells responsive to P I t A , and 40~b of the cells responsive to Con A may not have been destroyed by the combination

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of mitogen and BUdR, it is possible that the relatively greater response to Con A of the cells remaining after initial exposure to P H A , might reflect a difference in reaction kinetics associated with these two mitogens (8). Such an explanation seems unlikely, however. Under conditions of our experiments, the response to P t t A usually peaks at day 3, whereas that to Con A reaches a maximum at day 4. Thus, on the basis of kinetics alone the response by the secondarily stimulated cells should be higher with P H A . An alternative explanation for the relatively increased response, to Con A, of cells remaining after initial exposure to P H A , might invoke cellular heterogeneity. The failure of PI-tA to produce a relatively enhanced stimulation of cells remaining after initiat Con A exposure makes it unlikely that P H A and Con A stimulate entirely separate cell populations. The data would fit best with a model postulating two cell populations: one responding to both P H A and Con A, and the other largely to Con A alone. According to this model, reduction of cells responding to Con A should not change the relative response of the residual cells to the two mitogens. On the other hand, reduction of the PHA-responsive cells should result in an increased response to Con A as compared with P H A . This model is compatible with the data of Stobo and Paul (1) in the mouse, where it was shown that selective lysis of T lymphocytes with increasing concentrations of anti-0 serum and complement reduced response to both P H A and Con A, but left a residual response to Con A after that to P H A was abolished. Although in the mouse, only T lymphocytes are believed to respond to P H A or Con A (2), it is not yet clear whether this is the case with human lymphocytes. Although it is generally agreed that the predominant responders to these mitogens are T lymphocytes, recent studies have suggested that there may be some response on the part of B lymphocytes (7). An additional comment should be made on the data in Table 1, where the residual cells, after Con-A-specific killing, appear to have consistently more counts if a second mitogen is not added. The mean differences, however, do not appear to be significantly by Student's t test.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Stobo, J. D., and Paul, W. E., Y. Immunol. 110, 362, 1973. Stobo, J. D., and Paul, W. E., Immunology 4, 367, 1972. Stobo, J. D., Paul, W. E., and Henney, C. S., f. lmmunol. 110, 652, 1973. Shortman, K., Byrd, W. J., Cerrottini, J. C., and Brunner, K. T., Cell. Immunol. 6, 25, 1973. Cantor, It., and Asofsky, R., Y. tLvp. Med. 135, 764, 1972. Zoschke, D. C., and Bach, F. H., J. Immunol. Methods 1, 55, 1971. Chess, L, MacDermott, R. P., and Schlossman, S. F., Feder. Proc. 33, 734, 1974. Lindahl-Kiessling, K., Exp. Cell. Res. 70, 17, 1972.