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Expression of a 26,000-dalton glycoprotein on activated human T cells
CELLULAR
IMMUNOLOGY
64, 192- 199 ( 1981)
Expression of a 26,000~Dalton Glycoprotein on Activated Human T Cells’ THIERRY HERCEND,~ LEE M. NADLER, JOHN M. PESANDO, ELLIS L. REINHERZ, STUART F. SCHLOSSMAN, AND JEROME RITZ Division
of Tumor Immunology, Sidney Farber Cancer Institute 44 Binney Street, Boston, Massachusetts
and Harvard 02115
Medical
School.
Received May 22, 1981; accepted July 2, 1981
In the present study, we describe the expression of a 26,000-dalton glycoprotein on human T cells following stimulation by either mitogen or alloantigen. This glycoprotein, which is the target antigen of a monoclonal antibody designated 52, is distinct from Ia-like molecules and is not present on resting T cells. We demonstrate GP 26 expression in both major immunoregulatory subsets, i.e., T4+ (inducer) and T8+ (cytotoxic/suppressor) following activation and show that the GP 26-bearing cells are not directly responsible for the cytotoxicity generated in MLR. The relationship of the 52 target antigen to other glycoproteins with similar molecular weight which have been described on activated T cells is discussed.
INTRODUCTION Cell activation has been shown to be an important aspect of the response of T cells to specific antigens or mitogens and results in the acquisition of specific functions. The characterization of antigens which appear on T cells during activation provides one method of studying the mechanisms involved in T-cell responses. In this regard, it has been previously shown that human T cells express Ia antigens following activation by a variety of stimuli. However, not all activated T cells express Ia antigens and it has been demonstrated that within the major immunoregulatory subsets, i.e., T4+ (inducer) and T8+ (cytotoxic/suppressor), there are subsets of cells which become Ia positive whereas others remain Ia negative (l-4). Subsequently, these phenotypic differences have been used to further separate cells within the T4+ and T8+ subsets, and functionally distinct subpopulations have been defined ( 12, 13). In this report we examine the expression of a glycoprotein on activated T lymphocytes and describe some basic characteristics of the T cells which express this antigen. The molecular weight of this glycoprotein varies from 25,000 to 28,000 daltons depending on the conditions used to prepare it, and we designated it GP 26. In normal unstimulated peripheral blood, GP 26 does not appear to be present on lymphocytes, monocytes, granulocytes, or red cells, but is present on platelets. ’ This work was supported by NIH Grants CA 28740, CA 19589, CA 25369, and Al 12069. * To whom requests for reprints should be sent at: Sidney Farber Cancer Institute, 44 Binney St., Boston, Mass. 02115. 192 OOOS-8749/81/150192-08$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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This glycoprotein is recognized by several monoclonal antibodies recently developed as a result of immunization with leukemic cells from a patient with lymphoblastic leukemia (J. Ritz, submitted for publication). For the purpose of this study, we utilized one of these monoclonal antibodies designated 52. MATERIALS
AND METHODS
Monoclonal antibodies. The method for generation and characterization of a series of monoclonal antibodies specific for a 26,000-dalton cell surface glycoprotein is described elsewhere ( 19). Briefly, Balb/c mice were immunized with acute lymphoblastic leukemia cells and immune spleen cells were fused with NS-1 myeloma cells using polyethylene glycol as described by Kennett et al. (6). A series of monoclonal antibodies of different isotypes were found to have identical reactivity with ALL3 cells and were found to be specific for a 26,000-dalton cell surface glycoprotein. One of these reagents, designated 52, was used in the present studies. 52 is a murine IgM antibody by standard typing reagents (Meloy Laboratories). The specificities of other monoclonal antibodies Anti-T3, Anti-T4, Anti-T8 and IZ (anti-Ia) have been described previously (7, 13, 18). Isolation of lymphocytes populations. Human peripheral blood mononuclear cells were isolated from healthy volunteer donors by Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, N.J.). Subsequently, lymphocytes were separated into E-rosette-positive (E+) and E-rosette-negative (E-) subpopulations by use of 5% sheep erythrocytes (Microbiological Associates, Walkerville, Md.) (4). Normal human macrophages were obtained from the Epopulation by adherence to plastic plates (4). T-Cell stimulation by alloantigens, and mitogens. E+ cells were stimulated with either EB transformed B lymphoblastoid cells (mitomycin-treated Laz 388) phytohemagglutinin (4 pg/ml), or concanavalin A (12.5 pug/ml) as described (7). Culture medium was RPM1 1640 containing 20% AB human serum (American Red Cross Blood Services, Boston, Mass.), 1% sodium pyruvate (Grand Island Biological Company, Grand Island, N.Y.) and 1% nonessential amino acid mixture (Microbiological Associates). Cells were cultured in 75-cm2 growth area tissue culture flasks (Falcon) at an initial concentration of lo6 cells/ml. Five percent macrophages were added to the ES cell suspensionsat the initiation of the cultures. Proliferative responses were measured by a ‘[ Hlthymidine incorporation test (4). Cytofluorographic analysis. Cytofluorographic analysis of all cell populations were performed by indirect immunofluorescence with fluorescein-conjugated goat anti-mouse FAB IgG as described (4) on a cytofluorograph FC 200/4800A (Ortho Instruments). Background staining was obtained by substituting a 0.1-ml aliquot of 1:1000 ascites from a BALBjc mouse injected intraperitioneally with a nonspecific clone (designated J-O). Percentage of positive cells was calculated by subtracting the number of cells which were reactive with J-O from the number reactive with specific antibody. Plate technique for separation of T-cell subsets. A modification of the plate 3 Abbreviations used: ALL, acute lymphoblastic leukemia; EB, Epstein-Barr; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; Con A, concanavalin A; MLR, mixed lymphocyte reactions.
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technique reported by Wysocki and Sato (7) was utilized to separate T-cell subsets. Briefly, plastic plates (Fisher Catalog No. 8-757-120) were coated with 10 Fg of affinity-purified goat anti-mouse Ig in 10 ml of PBS and kept at 4°C overnight. The next morning plates were washed. T cells were incubated for 30 min with either anti-T4 or anti-T8 monoclonal antibodies, washed, and then 5 X lo6 cells in a 3-ml volume were applied to a single goat anti-mouse Ig-coated plate. After 60 min of incubation at 4°C supernatants were removed. In plates incubated with anti-T4-coated T cells, the nonadherent population contained less than 5% of T4+ cells, similar purification was obtained with anti-T8-coated cells. Cells from these supernatants were respectively designated as T8+ and T4+ subsets. Complement-mediated lysis of cultured Ef ceils. Ef cells (20 X 106) were incubated for 1 hr at 4°C with 1 ml of antibody at the appropriate dilution. Subsequently cells were washed two times, and 0.4 ml fresh rabbit complement (1:2 dilution) added, and cells were incubated for an additional hour in a shaking water bath at 37°C. Cells were then washed once, layered on Ficoll-Hypaque, spun for 15 min at lOOOg,washed twice more, and analyzed on the cytofluorograph. Cell-mediated lymphocytoxicity. Cell-mediated lysis was performed in microplate assay. Laz 388 or K562 cells (5 X lo6 of either) were treated with 0.2 ml sodium [ “Crlchromate (292 Ci/ml) (New England Nuclear) and incubated in a 37°C shaking water bath for 90 min. Cells were washed twice and diluted to 105/ ml. Labeled target cells ( 100 ~1) were added to microplate wells with 100 ~1 of responding cells at various dilutions and incubated at 37°C for 6 hr. After incubation plates were spun for 5 min at 300g and 100 ~1 of supernatant was aspirated and counted on a gamma scintillation counter (Packard Instrument Co., Downers Grove, Ill.). Specific cytotoxicity and natural killer activity were calculated using the formula: Exp. - SR Percentage specific lysis = FT _ SR X 100, where Exp. = mean of the observed triplicate, SR = spontaneous release, and FT = freeze-thaw. RESULTS Expression of GP 26 on Activated T Cells following Mtogen and Alloantigen Stimulation The first goal of this study was to determine if GP 26 is consistantly expressed on activated T lymphocytes following various stimuli. For this purpose, E+ cells were isolated from peripheral mononuclear cells from numerous healthy donors. T-Cell activation was induced by either mitogens (PHA and Con A) or alloantigens (Laz 388-EB virus-transformed B-cell line). Activated T cells were tested for expression of GP 26 7 days after the initiation of each culture. GP 26-bearing cells were found in 21 out of 22 experiments (Table 1). A similar percentage of GP 26bearing cells was found following either PHA or Con A stimulation. In contrast, there were one third as many JZreactive cells in MLR cultures. Although the mechanisms underlying this differential expression remain unclear, it is noteworthy since this was not observed with expression of Ia antigens. To the contrary, after 6 days of stimulation, MLR cultures contain greater percentages of Ia+ cells than mitogen-activated cultures (4).
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Expression of GP 26 on Activated
1
T Lymphocytes”
7 Days after Stimulation Number GP 26-positive cells’
Stimulation*
Number of cultures tested
PHA Con A LAZ 388“
12 5 5
Mean% +
Range
33 f 37 f 12+
O-78 8-52 IO-16
38 21 2
’ Anti-T3 monoclonal antibody reacted with more than 95% of cells in each experiment. b Agglutination, increased cell counts, and high [3H]thymidine incorporation were used as activation criteria. ’ Positive cells were identified by indirect immunofluorescence reactivity with 52 monoclonal antibody. 10.000 cells were analyzed on the cytofluorograph in each sample. ’ Allogeneic EB virus-transformed cell line.
Kinetics of GP 26 Expression on PHA- and Alloantigen-Activated
T Cells
Since it has been shown that the expression of membrane proteins on activated T cells varies dramatically with time (9), the observed difference between mitogen activated and alloreactive cells could have been due to differences in the time of expression of GP 26 following stimulation. Therefore, we investigated the kinetics of expression of GP 26 in both systems. E+ cells from nine additional donors were stimulated either by PHA (6) or alloantigens (3) over a lo-day period. Each day cultures were tested for three parameters: (i) percentage of JZpositive cells; (ii) Number of viable cells/mm3; and (iii) [3H]thymidine incorporation. As shown in Fig. 1, the percentage of GP 26-positive cells found daily with each type of activation remained quite different, but the time of expression of GP 26 antigen was similar. With both mitogen and alloantigen activation, the percentage of J2-positive cells reached a peak on Days 5-6 and then decreased to Day 10. This peak correlated
FIG. 1. Kinetics of GP26 expression on PHA-activated and alloreactive T cells. 0 -0, Expression of GP26 on PHA-activated T cells. Each point represents the mean and SEM for six different cultures. n -n , Expression of GP26 on alloreactive T cells. Each point represents the mean and SEM for three different cultures. 0 - - - 0 = Expression of Ia-like molecules on PHA-activated T cells. Each point represents the mean and SEM for six different cultures. The small percentage of J2-reactive cells on Day 0 is due to platelets which are adherent to macrophages added at the initiation of the cultures.
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201 1
Y
b
I
Yll
1
3
5
7
9
11
Days FIG. 2. Expression of GP26 in T4+ (inducer) and T8+ (cytotoxic/suppressor) subsets after PHA stimulation.
with the highest [3H]thymidine incorporation which occurred on Days 5-6 but preceded the peak in cell counts which occured on Days 7-9. The range of expression (Table 1) and consequently the standard deviations (Table 1, Fig. 1) were very wide in PHA cultures but much tighter in MLR, which suggests strong individual variations in the response to this mitogen. In the six cultures stimulated by PHA, the expression of Ia-like molecules was also tested using the IZ monoclonal antibody (18). As shown in Fig. 1, the pattern of expression of Ia antigen is quite different from that of GP 26. Expression of GP 26 on both T4+ (Inducer) and T8+ (Cytotoxic/Suppressor) Cells following PHA Stimulation To further characterize the GP 26-bearing cells, we separated E+ cells in peripheral blood into two major functional subgroups: T4+ (inducer) and T8+ (cytotoxic/suppressor). Cells were separated using the plate technique described under Materials and Methods. Subsequently, T4+ and T8+ cells were each cultured for 10 days after PHA stimulation and expression of GP 26 in each subset was determined during this culture period. As shown in Fig. 2, similar percentages of GP 26-bearing cells were found in both cultures with the only difference being that there were 37% JZreactive cells still present in the T4+ subset on Day 9. Furthermore, these percentages were comparable to those obtained with whole E+ cells (Fig. 1, Table 1). These results suggest that GP 26 expression in not strongly modified by T4-T8 cell interactions during mitogen induced T-cell activation. This was confirmed by further experiments in which PHA-activated E+ cells were treated on Days 4 and 7 by either anti-T4 or anti-T8 plus rabbit complement, and subsequently analyzed on the cytofluorograph for GP 26 expression. The percentages of J2-reactive cells in the surviving populations, i.e., T4- (as T8 subset); T8- (as T4 subset), were similar to those shown in Fig. 2.
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t i:
c
J
Fluorescence
Intensity
Fluorescence
Intensity
EflectorlTarget
Ratio
FIG. 3. Cytotoxic activity of 7-day MLR-activated T cells. Dotted line represents background fluorescence of control ascites and GM/FITC; solid line represents reactivity with 52 antibody. (A) Cytofluorograph analysis of reactivity of 32 with alloreactive T cells used in (C). (B) Cytofluorograph analysis of reactivity of 32 with alloreactive T cells following treatment by 32 plus rabbit complement, and subsequently used in (C). (C) Specific cytotoxicity of whole (0) and GP26-depleted (m) alloreactive cells against chromium labeled Laz 388 cells. Identical results were found in three different experiments.
Absence of GP 26 on Human Cytotoxic T Cells As demonstrated above, T8+ cells can express GP 26 following activation. Therefore, we studied the possibility that the cytotoxic cells generated in MLR, which have a controversial phenotype ( 11, 12) would be recognized by 52 antibody. Ef cells were stimulated by the Laz 388 cell line in a one-way MLR and responding cells were assayed for cytotoxicity against chromium-labeled Laz 388 cells either directly or after treatment with 52 plus complement. As shown in Fig. 3, complement lysis removed any detectable GP 26-positive cells (B) and the surviving cells were slightly more cytotoxic (C) than the control (concentration effect). Identical results were found using K562 cells as targets (data not shown). These results indicate that activated T cells defined by a GP 26+ phenotype are not directly responsible for specific cytotoxicity or natural killer-like activity ( 16). Failure of J2 Monoclonal Antibody to Block T-Cell Responses Finally, we investigated a possible functional role of the GP 26 molecule in a series of blocking experiments. A 1/ 100 dilution of 52 antibody added at the initiation of the cultures failed to block proliferation induced by either mitogens, alloantigens, or soluble antigens. This did not modify either generation of cytotoxicity in MLR or immunoglobulin secretion in a pokeweed mitogen driven system (data not shown). DISCUSSION The present study suggests that GP 26 is a monomorphic antigen which is present on activated T cells. Peripheral blood from more than 20 donors was examined and
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in all casesexcept one, JZ-positive cells were found following stimulation; cells from the single negative donor were retested and in this subsequent examination, 15% of J2-positive cells were present on Day 4 but diminished to 2% of 52 positive on Day 7 following PHA stimulation. Thus, T cells from all donors tested thus far have been shown to express GP 26. In addition, our data demonstrate that following PHA stimulation, GP 26 is expressed earlier on activated T cells and is consequently distinct from Ia-like molecules. Although some characteristics of GP 26-bearing cells are demonstrated, the biological function of this molecule remains unknown. Kersey et al. (10) have recently identified a cell surface structure in leukemic cells from 77% of patients with null-ALL which has a similar molecular weight of 24,000 daltons. This protein, recognized by a monoclonal antibody designated BA2, was thought to represent a surface marker of lymphohematopoietic progenitor cells in normal bone marrow. The reactivity of 52 with leukemic cells is very similar to that of BA-2 and it is possible that both antibodies are specific for the same surface molecule. The reactivity of BA-2 with activated T cells and platelets has not been described. In studies utilizing [%]methionine labeling techniques, Udey and Parker (14) have identified the synthesis of a family of 28,000 MW cell surface glycoprotein by Con A-activated T cells. This glycoprotein could also be similar to the target antigen identified by J2 antibody, although the kinetics of expression of GP 26 seems different since it was found on activated T cells within several hours of mitogen stimulation. In other studies (15), a 28,00 MW glycoprotein was identified 3-4 days after mitogen stimulation but the expression of this protein by activated T cells was relatively weak. Further studies will be necessary to determine the functional significance of the expression of this glycoprotein (or family of glycoproteins) in T-cell activation. ACKNOWLEDGMENTS We would like to thank Herb Levine and John Daley for assistance in performing Daniela Colangelli for preparing this manuscript.
these studies and
REFERENCES I. Evans, R. L., Faldetta, T. J., Humphreys, R. E., Pratt, D. M., Yunis, E. J., and Schlossman, S. F., J. Exp. Med. 148, 1440, 1978. 2. Fu, S. M., Chiotazzi, Wang, C. Y., Montazeri, G., Kunkel, H. G., Ko, H. S., and Gottlieb, A. B., J. Exp. Med. 148, 1423, 1978. 3. Socu-Foca, N., Sunsinmo, E., Kiernam, P., Rohowsky, C., Weiner, J., and Rubinstein, P., Transplant. Proc. 10, 845, 1978. 4. Reinherz, E. L., Kung, P. C., Pesando, J. M., Ritz, J., Goldstein, G., and Schlossman, S. F., J. Exp. Med. 150, 1472, 1979. 5. Reinherz, E. L., and Schlossman, S. F., J. Immunol. 122, 1335-1341, 1979. 6. Kennet, R. H., Denis, R. A., Tung, A. S., and Klinman, N. R., “NIH Workshop: Lymphocyte Hybridomas.” Bethesda, Md. (Springer, Berlin) pp. 77, 91, 1978. 7. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F., Proc. Nat. Acad. Sci. USA 78, 4061, 1979. 8. Reinherz, E. L., Morimoto, C., Penta, A., and Schlossman, S. F., J. Imtnunol. 126, 67, 1981. 9. Boto, W. O., and Humphreys, R. E., J. Immunol. 126, 759, 1981. 10. Kersey, J. H., LeBien, T., Abramson, C. S., Newman, R., and Greaves, M., J. Exp. Med. 153, 726, 1981. 11. Moretta, A., Mingari, M. C., Haynes, B. F., Sekaly, R. P., Moretta, L., and Fauci, A. S., J. Exp. Med. 153, 213, 1981.
SHORT 12. 13. 14. 15. 16. 17. 18. 19.
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Reinherz, E. L., Hussey, R., and Schlossman, S. F., Immunogenetics 11, 421, 1980. Reinherz, E. L., Hussey, R., and Schlossman, S. F., Eur. J. Immunol. 10, 758, 1980. Udey, M. C., and Parker, C. W. J. Immunol. 126, I 106, 1981. Andersson, C., Gahmberg, C. G., Kimura, A. K., and Wigzell, H., Proc. Nut. Acad. Sci. USA 7, 3455, 1978. Seeley, J. K., Masucci, G., Poros, A., Klein, E., and Golub, S. B. J. Immunol. 123, 1303, J979. Wysocki, L. J., and Sato, V. L., Proc. Nat. Acad. Sci. USA 75, 2844, 1978. Nadler, L. M., Stashenko, P., Hardy, R., Pesando, J. M., Yunis, E. J., and Schlossman, S. F., Human Immunol. 1, 77, 198 1. Ritz, J., Pesando, J. M., Notis-McConarty, J., Lazarus, H., and Schlossman, S. F., Nature (London) 283, 583, 1980.
IMMUNOLOGY
64, 192- 199 ( 1981)
Expression of a 26,000~Dalton Glycoprotein on Activated Human T Cells’ THIERRY HERCEND,~ LEE M. NADLER, JOHN M. PESANDO, ELLIS L. REINHERZ, STUART F. SCHLOSSMAN, AND JEROME RITZ Division
of Tumor Immunology, Sidney Farber Cancer Institute 44 Binney Street, Boston, Massachusetts
and Harvard 02115
Medical
School.
Received May 22, 1981; accepted July 2, 1981
In the present study, we describe the expression of a 26,000-dalton glycoprotein on human T cells following stimulation by either mitogen or alloantigen. This glycoprotein, which is the target antigen of a monoclonal antibody designated 52, is distinct from Ia-like molecules and is not present on resting T cells. We demonstrate GP 26 expression in both major immunoregulatory subsets, i.e., T4+ (inducer) and T8+ (cytotoxic/suppressor) following activation and show that the GP 26-bearing cells are not directly responsible for the cytotoxicity generated in MLR. The relationship of the 52 target antigen to other glycoproteins with similar molecular weight which have been described on activated T cells is discussed.
INTRODUCTION Cell activation has been shown to be an important aspect of the response of T cells to specific antigens or mitogens and results in the acquisition of specific functions. The characterization of antigens which appear on T cells during activation provides one method of studying the mechanisms involved in T-cell responses. In this regard, it has been previously shown that human T cells express Ia antigens following activation by a variety of stimuli. However, not all activated T cells express Ia antigens and it has been demonstrated that within the major immunoregulatory subsets, i.e., T4+ (inducer) and T8+ (cytotoxic/suppressor), there are subsets of cells which become Ia positive whereas others remain Ia negative (l-4). Subsequently, these phenotypic differences have been used to further separate cells within the T4+ and T8+ subsets, and functionally distinct subpopulations have been defined ( 12, 13). In this report we examine the expression of a glycoprotein on activated T lymphocytes and describe some basic characteristics of the T cells which express this antigen. The molecular weight of this glycoprotein varies from 25,000 to 28,000 daltons depending on the conditions used to prepare it, and we designated it GP 26. In normal unstimulated peripheral blood, GP 26 does not appear to be present on lymphocytes, monocytes, granulocytes, or red cells, but is present on platelets. ’ This work was supported by NIH Grants CA 28740, CA 19589, CA 25369, and Al 12069. * To whom requests for reprints should be sent at: Sidney Farber Cancer Institute, 44 Binney St., Boston, Mass. 02115. 192 OOOS-8749/81/150192-08$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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This glycoprotein is recognized by several monoclonal antibodies recently developed as a result of immunization with leukemic cells from a patient with lymphoblastic leukemia (J. Ritz, submitted for publication). For the purpose of this study, we utilized one of these monoclonal antibodies designated 52. MATERIALS
AND METHODS
Monoclonal antibodies. The method for generation and characterization of a series of monoclonal antibodies specific for a 26,000-dalton cell surface glycoprotein is described elsewhere ( 19). Briefly, Balb/c mice were immunized with acute lymphoblastic leukemia cells and immune spleen cells were fused with NS-1 myeloma cells using polyethylene glycol as described by Kennett et al. (6). A series of monoclonal antibodies of different isotypes were found to have identical reactivity with ALL3 cells and were found to be specific for a 26,000-dalton cell surface glycoprotein. One of these reagents, designated 52, was used in the present studies. 52 is a murine IgM antibody by standard typing reagents (Meloy Laboratories). The specificities of other monoclonal antibodies Anti-T3, Anti-T4, Anti-T8 and IZ (anti-Ia) have been described previously (7, 13, 18). Isolation of lymphocytes populations. Human peripheral blood mononuclear cells were isolated from healthy volunteer donors by Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, N.J.). Subsequently, lymphocytes were separated into E-rosette-positive (E+) and E-rosette-negative (E-) subpopulations by use of 5% sheep erythrocytes (Microbiological Associates, Walkerville, Md.) (4). Normal human macrophages were obtained from the Epopulation by adherence to plastic plates (4). T-Cell stimulation by alloantigens, and mitogens. E+ cells were stimulated with either EB transformed B lymphoblastoid cells (mitomycin-treated Laz 388) phytohemagglutinin (4 pg/ml), or concanavalin A (12.5 pug/ml) as described (7). Culture medium was RPM1 1640 containing 20% AB human serum (American Red Cross Blood Services, Boston, Mass.), 1% sodium pyruvate (Grand Island Biological Company, Grand Island, N.Y.) and 1% nonessential amino acid mixture (Microbiological Associates). Cells were cultured in 75-cm2 growth area tissue culture flasks (Falcon) at an initial concentration of lo6 cells/ml. Five percent macrophages were added to the ES cell suspensionsat the initiation of the cultures. Proliferative responses were measured by a ‘[ Hlthymidine incorporation test (4). Cytofluorographic analysis. Cytofluorographic analysis of all cell populations were performed by indirect immunofluorescence with fluorescein-conjugated goat anti-mouse FAB IgG as described (4) on a cytofluorograph FC 200/4800A (Ortho Instruments). Background staining was obtained by substituting a 0.1-ml aliquot of 1:1000 ascites from a BALBjc mouse injected intraperitioneally with a nonspecific clone (designated J-O). Percentage of positive cells was calculated by subtracting the number of cells which were reactive with J-O from the number reactive with specific antibody. Plate technique for separation of T-cell subsets. A modification of the plate 3 Abbreviations used: ALL, acute lymphoblastic leukemia; EB, Epstein-Barr; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; Con A, concanavalin A; MLR, mixed lymphocyte reactions.
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technique reported by Wysocki and Sato (7) was utilized to separate T-cell subsets. Briefly, plastic plates (Fisher Catalog No. 8-757-120) were coated with 10 Fg of affinity-purified goat anti-mouse Ig in 10 ml of PBS and kept at 4°C overnight. The next morning plates were washed. T cells were incubated for 30 min with either anti-T4 or anti-T8 monoclonal antibodies, washed, and then 5 X lo6 cells in a 3-ml volume were applied to a single goat anti-mouse Ig-coated plate. After 60 min of incubation at 4°C supernatants were removed. In plates incubated with anti-T4-coated T cells, the nonadherent population contained less than 5% of T4+ cells, similar purification was obtained with anti-T8-coated cells. Cells from these supernatants were respectively designated as T8+ and T4+ subsets. Complement-mediated lysis of cultured Ef ceils. Ef cells (20 X 106) were incubated for 1 hr at 4°C with 1 ml of antibody at the appropriate dilution. Subsequently cells were washed two times, and 0.4 ml fresh rabbit complement (1:2 dilution) added, and cells were incubated for an additional hour in a shaking water bath at 37°C. Cells were then washed once, layered on Ficoll-Hypaque, spun for 15 min at lOOOg,washed twice more, and analyzed on the cytofluorograph. Cell-mediated lymphocytoxicity. Cell-mediated lysis was performed in microplate assay. Laz 388 or K562 cells (5 X lo6 of either) were treated with 0.2 ml sodium [ “Crlchromate (292 Ci/ml) (New England Nuclear) and incubated in a 37°C shaking water bath for 90 min. Cells were washed twice and diluted to 105/ ml. Labeled target cells ( 100 ~1) were added to microplate wells with 100 ~1 of responding cells at various dilutions and incubated at 37°C for 6 hr. After incubation plates were spun for 5 min at 300g and 100 ~1 of supernatant was aspirated and counted on a gamma scintillation counter (Packard Instrument Co., Downers Grove, Ill.). Specific cytotoxicity and natural killer activity were calculated using the formula: Exp. - SR Percentage specific lysis = FT _ SR X 100, where Exp. = mean of the observed triplicate, SR = spontaneous release, and FT = freeze-thaw. RESULTS Expression of GP 26 on Activated T Cells following Mtogen and Alloantigen Stimulation The first goal of this study was to determine if GP 26 is consistantly expressed on activated T lymphocytes following various stimuli. For this purpose, E+ cells were isolated from peripheral mononuclear cells from numerous healthy donors. T-Cell activation was induced by either mitogens (PHA and Con A) or alloantigens (Laz 388-EB virus-transformed B-cell line). Activated T cells were tested for expression of GP 26 7 days after the initiation of each culture. GP 26-bearing cells were found in 21 out of 22 experiments (Table 1). A similar percentage of GP 26bearing cells was found following either PHA or Con A stimulation. In contrast, there were one third as many JZreactive cells in MLR cultures. Although the mechanisms underlying this differential expression remain unclear, it is noteworthy since this was not observed with expression of Ia antigens. To the contrary, after 6 days of stimulation, MLR cultures contain greater percentages of Ia+ cells than mitogen-activated cultures (4).
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Expression of GP 26 on Activated
1
T Lymphocytes”
7 Days after Stimulation Number GP 26-positive cells’
Stimulation*
Number of cultures tested
PHA Con A LAZ 388“
12 5 5
Mean% +
Range
33 f 37 f 12+
O-78 8-52 IO-16
38 21 2
’ Anti-T3 monoclonal antibody reacted with more than 95% of cells in each experiment. b Agglutination, increased cell counts, and high [3H]thymidine incorporation were used as activation criteria. ’ Positive cells were identified by indirect immunofluorescence reactivity with 52 monoclonal antibody. 10.000 cells were analyzed on the cytofluorograph in each sample. ’ Allogeneic EB virus-transformed cell line.
Kinetics of GP 26 Expression on PHA- and Alloantigen-Activated
T Cells
Since it has been shown that the expression of membrane proteins on activated T cells varies dramatically with time (9), the observed difference between mitogen activated and alloreactive cells could have been due to differences in the time of expression of GP 26 following stimulation. Therefore, we investigated the kinetics of expression of GP 26 in both systems. E+ cells from nine additional donors were stimulated either by PHA (6) or alloantigens (3) over a lo-day period. Each day cultures were tested for three parameters: (i) percentage of JZpositive cells; (ii) Number of viable cells/mm3; and (iii) [3H]thymidine incorporation. As shown in Fig. 1, the percentage of GP 26-positive cells found daily with each type of activation remained quite different, but the time of expression of GP 26 antigen was similar. With both mitogen and alloantigen activation, the percentage of J2-positive cells reached a peak on Days 5-6 and then decreased to Day 10. This peak correlated
FIG. 1. Kinetics of GP26 expression on PHA-activated and alloreactive T cells. 0 -0, Expression of GP26 on PHA-activated T cells. Each point represents the mean and SEM for six different cultures. n -n , Expression of GP26 on alloreactive T cells. Each point represents the mean and SEM for three different cultures. 0 - - - 0 = Expression of Ia-like molecules on PHA-activated T cells. Each point represents the mean and SEM for six different cultures. The small percentage of J2-reactive cells on Day 0 is due to platelets which are adherent to macrophages added at the initiation of the cultures.
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201 1
Y
b
I
Yll
1
3
5
7
9
11
Days FIG. 2. Expression of GP26 in T4+ (inducer) and T8+ (cytotoxic/suppressor) subsets after PHA stimulation.
with the highest [3H]thymidine incorporation which occurred on Days 5-6 but preceded the peak in cell counts which occured on Days 7-9. The range of expression (Table 1) and consequently the standard deviations (Table 1, Fig. 1) were very wide in PHA cultures but much tighter in MLR, which suggests strong individual variations in the response to this mitogen. In the six cultures stimulated by PHA, the expression of Ia-like molecules was also tested using the IZ monoclonal antibody (18). As shown in Fig. 1, the pattern of expression of Ia antigen is quite different from that of GP 26. Expression of GP 26 on both T4+ (Inducer) and T8+ (Cytotoxic/Suppressor) Cells following PHA Stimulation To further characterize the GP 26-bearing cells, we separated E+ cells in peripheral blood into two major functional subgroups: T4+ (inducer) and T8+ (cytotoxic/suppressor). Cells were separated using the plate technique described under Materials and Methods. Subsequently, T4+ and T8+ cells were each cultured for 10 days after PHA stimulation and expression of GP 26 in each subset was determined during this culture period. As shown in Fig. 2, similar percentages of GP 26-bearing cells were found in both cultures with the only difference being that there were 37% JZreactive cells still present in the T4+ subset on Day 9. Furthermore, these percentages were comparable to those obtained with whole E+ cells (Fig. 1, Table 1). These results suggest that GP 26 expression in not strongly modified by T4-T8 cell interactions during mitogen induced T-cell activation. This was confirmed by further experiments in which PHA-activated E+ cells were treated on Days 4 and 7 by either anti-T4 or anti-T8 plus rabbit complement, and subsequently analyzed on the cytofluorograph for GP 26 expression. The percentages of J2-reactive cells in the surviving populations, i.e., T4- (as T8 subset); T8- (as T4 subset), were similar to those shown in Fig. 2.
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FIG. 3. Cytotoxic activity of 7-day MLR-activated T cells. Dotted line represents background fluorescence of control ascites and GM/FITC; solid line represents reactivity with 52 antibody. (A) Cytofluorograph analysis of reactivity of 32 with alloreactive T cells used in (C). (B) Cytofluorograph analysis of reactivity of 32 with alloreactive T cells following treatment by 32 plus rabbit complement, and subsequently used in (C). (C) Specific cytotoxicity of whole (0) and GP26-depleted (m) alloreactive cells against chromium labeled Laz 388 cells. Identical results were found in three different experiments.
Absence of GP 26 on Human Cytotoxic T Cells As demonstrated above, T8+ cells can express GP 26 following activation. Therefore, we studied the possibility that the cytotoxic cells generated in MLR, which have a controversial phenotype ( 11, 12) would be recognized by 52 antibody. Ef cells were stimulated by the Laz 388 cell line in a one-way MLR and responding cells were assayed for cytotoxicity against chromium-labeled Laz 388 cells either directly or after treatment with 52 plus complement. As shown in Fig. 3, complement lysis removed any detectable GP 26-positive cells (B) and the surviving cells were slightly more cytotoxic (C) than the control (concentration effect). Identical results were found using K562 cells as targets (data not shown). These results indicate that activated T cells defined by a GP 26+ phenotype are not directly responsible for specific cytotoxicity or natural killer-like activity ( 16). Failure of J2 Monoclonal Antibody to Block T-Cell Responses Finally, we investigated a possible functional role of the GP 26 molecule in a series of blocking experiments. A 1/ 100 dilution of 52 antibody added at the initiation of the cultures failed to block proliferation induced by either mitogens, alloantigens, or soluble antigens. This did not modify either generation of cytotoxicity in MLR or immunoglobulin secretion in a pokeweed mitogen driven system (data not shown). DISCUSSION The present study suggests that GP 26 is a monomorphic antigen which is present on activated T cells. Peripheral blood from more than 20 donors was examined and
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in all casesexcept one, JZ-positive cells were found following stimulation; cells from the single negative donor were retested and in this subsequent examination, 15% of J2-positive cells were present on Day 4 but diminished to 2% of 52 positive on Day 7 following PHA stimulation. Thus, T cells from all donors tested thus far have been shown to express GP 26. In addition, our data demonstrate that following PHA stimulation, GP 26 is expressed earlier on activated T cells and is consequently distinct from Ia-like molecules. Although some characteristics of GP 26-bearing cells are demonstrated, the biological function of this molecule remains unknown. Kersey et al. (10) have recently identified a cell surface structure in leukemic cells from 77% of patients with null-ALL which has a similar molecular weight of 24,000 daltons. This protein, recognized by a monoclonal antibody designated BA2, was thought to represent a surface marker of lymphohematopoietic progenitor cells in normal bone marrow. The reactivity of 52 with leukemic cells is very similar to that of BA-2 and it is possible that both antibodies are specific for the same surface molecule. The reactivity of BA-2 with activated T cells and platelets has not been described. In studies utilizing [%]methionine labeling techniques, Udey and Parker (14) have identified the synthesis of a family of 28,000 MW cell surface glycoprotein by Con A-activated T cells. This glycoprotein could also be similar to the target antigen identified by J2 antibody, although the kinetics of expression of GP 26 seems different since it was found on activated T cells within several hours of mitogen stimulation. In other studies (15), a 28,00 MW glycoprotein was identified 3-4 days after mitogen stimulation but the expression of this protein by activated T cells was relatively weak. Further studies will be necessary to determine the functional significance of the expression of this glycoprotein (or family of glycoproteins) in T-cell activation. ACKNOWLEDGMENTS We would like to thank Herb Levine and John Daley for assistance in performing Daniela Colangelli for preparing this manuscript.
these studies and
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