Immunology 1992 75 420-426
B-cell maturation in chronic lymphocytic leukaemia IV. T-CELL-DEPENDENT ACTIVATION OF LEUKAEMIC B CELLS BY STAPHYLOCOCCAL ENTEROTOXIN 'SUPERANTIGENS'
X. DUAN, C. NERL,*
0.
JANSSEN & D. KABELITZ Institutfiir Immunologie, Universitit Heidelberg, Heidelberg and *Stddtisches Krankenhaus Mfinchen-Schwiabing, Mfinchen, Germany
Accepted for publication 14 October 1991
SUMMARY
Staphylococcal enterotoxins (SE) are potent T-lymphocyte activators that stimulate T cells by directly cross-linking HLA-DR molecules on antigen-presenting cells with the V/# gene products of the T-cell receptor. The different SE activate all T cells expressing a given Vfl, and, therefore, have been termed 'superantigens'. Here we show that SE are potent activators of leukaemic B cells from patients with chronic lymphocytic leukaemia (CLL). Purified B cells from seven of eight CLL patients with high WBC counts ( > 80,000/l) responded to one or several of the tested SE (SEA, SEB, SEC,, SED, SEE) by proliferation ([3H]TdR incorporation) and/or Ig secretion. In several instances, the response of leukaemic B cells to SE was much stronger than was the response to other known B-cell activators including EBV, pokeweed mitogen (PWM), phorbolester (TPA), and Staphylococcus aureus Cowan I (SAC). The activation of leukaemic B cells by SE was strictly dependent on the addition of irradiated T cells isolated from healthy donors. FACS analysis of cultured cells ensured that the proliferating cells were indeed B cells. Taken together, these results demonstrate that SE are strong T-cell-dependent B-cell activators that, in some cases, can stimulate maturation of leukaemic B cells which are refractory to other activation signals. INTRODUCTION
TSST-1 of monocyte-derived IL-i and tumour necrosis factor-
Some bacterial toxins such as toxic shock syndrome toxin-I (TSST- 1) or StaphYlococcus aureuls cnterotoxins (SE) have been known for some time to be strongly mitogenic to T lymphocytes."2 Recently, these toxins have attracted much attention, due to the identification of their unusual mode of action. Thus, it was found that SE bind directly to major histocompatibility complex (MHC) class II molecules on the surface of antigenpresenting cells (APC), without need of being processed.3 6 Individual SE such as SEA, SEB, SEC,, SED or SEE, activate all T cells expressing a given V/I gene segment of the T-cell receptor (TcR), and a selective specificity for certain V,3 can be assigned to some SE.7 9 Therefore, these toxins have been termed 'superantigens'."' It appears that superantigens act by directly cross-linking HLA-DR and TcR molecules. While it is clear that SE-mediated cross-linking of HLA-DR+ APC and T cells strongly activate the latter, it is possible that APC are similarly activated during this process. In fact, stimulation by
alpha (TN F-oc) production has been demonstrated by Parsonnet and co-workers."1"12 More recently, Mourad et al.13 reported that TSST- 1 is a T-ccll-dependent activator of human B-cell proliferation and differentiation. In the present study we have investigated possible effects of staphylococcal enterotoxins on the in vitro differentiation of leukaemic B cells from patients with B-cell chronic lymphocytic leukaemia (CLL). Ig secretion has been successfully induced in these cells by a variety of polyclonal B-cell activators. 14 1' Under certain circumstances, leukaemic B cells could also be triggered to proliferate in ,itr-o.' 23 Results of many studies published over the last few years indicate that there is a considerable clonal heterogeneity of leukaemic CLL B-cell responses to polyclonal B-cell activators and defined (recombinant) cytokines.24 1' Here we show that SE are potent T-cell-dependent activators of leukaemic CLL B-cell proliferation and differentiation. In some instances, SE were by far more effective than other well-known (T-cell-dependent or -independent) B-cell activators such as EBV, pokeweed mitogen (PWM), phorbolester (TPA), or Staphylococcus aureus Cowan I (SAC).
Abbreviations: APC, antigen-presenting cell: CLL, chronic lymphocytic leukaemia; PBMC, peripheral blood mononuclear cells, SE, staphylococcus enterotoxin; TcR, T-cell receptor. Correspondence: Dr D. Kabelitz, Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305. D-6900 Heidelberg, Germany.
MATERIALS AND METHODS
lymphocyte populations Heparinized blood was obtained from healthy donors or CLL patients after informed consent. None of the CLL patients (six Isolation ot
420
B-cell maturation in chronic lymphocyte leukaemia
Wiebaden, Germany). All cultures were incubated at 37 in a humidified atmosphere of 5'S, CO2 in air.
Table 1. Characterization of PBMC from CLL patients
Response of purified leukaemic B cells to enterotoxins Patient
Sex
1. 2. 3. 4. 5. 6. 7. 8.
m f f f f m f f
WE LA
NI KU KO RE HU ER
WBC/pl 80,000
128,000 129,000 150,000 160,000 200,000 200,000 300,000
{M, CD3+ 2-6 46
30 28 1.0 39 68 2.1
sig
1gM IgM IgM IgM IgM IgG IgM IgG
Proliferation*
Ig secretiont
-
+ + + + +
+ +
+
421
+ +
* [3H]TdR incorporation > 5000 c.p.m. per 2 x 105 seeded cells. t IgM or IgG in cell culture supernatant > 400 ng/ml.
females, two males) was on treatment within the last 3 months before blood was taken for the present study. White blood cell (WBC) counts ranged from 80,000 to 300,000/11d. All patients included in this study were diagnosed as suffering from B-celltype CLL based on low percentages of circulating CD3 t T cells and the expression of either K- or .-Ig light chain on the vast majority of peripheral blood lymphocytes (see Table 1). Peripheral blood mononuclear cells (PBMC) were isolated by FicollHypaque density gradient centrifugation, and were stored in liquid nitrogen until use. T cells were separated from adherent cell-depleted PBMC by rosette formation with sheep erythrocytes (E) as described.34 Leukaemic B cells were purified from CLL PBMC by two consecutive depletions of E-rosette-forming T cells as previously described.35 The resulting CLL E cell fraction was contaminated with 5000 c.p.m. per 2 x 1I05 seeded cells; Ig production: > 400 ng `ml), proliferation was induced in B cells from three of eight patients, and Ig secretion in B cells from seven of eight patients. Only in one patient (RE) were the purified leukaemic B cells refractory to activation by SE. DISCUSSION SE have been known for a long time to be strong T-cell mitogens.2 However, SE differ from other polyclonal T-cell mitogens such as plant lectins in that they specifically activate some but not other T cells.7 Recently published evidence indicates that a given SE activates all T cells expressing the
/lymplhocyte leukaeinia
425
'correct' Vfl gene segment of the TcR.5'" Due to their stimulatory activity for a whole subset of T cells expressing a particular Vfl, SE (and related bacterial toxins) have been termed 'superantigens'.") As far as APC are concerned, superantigens can bind directly to MHC class 11 antigens without need of being processed.4 6 Inasmuch as CLL B cells express H LA-DR and, to a varying degree, other class II molecules on their surface,36 we considered the possibility that CLL B cells are activated in a Tcell-dependent fashion by SE, similarly to what has been described for the activation of normal B cells by TSST-1.'3 Our present results demonstrate that SE are indeed potent activators of CLL B cells in vitro. As with other B-cell activators,'4"16'20127'33 there was considerable heterogeneity in the capacity of the various CLL B cells to respond to SE stimulation. However, several of the investigated clonal B-cell populations responded much better to SE than to conventional B-cell activators including BV, PWM, SAC or TPA (see Figs 1 and 2). Thus, a significant B-cell proliferation was induced in three of eight CLL samples, while Ig production (> 400 ng/ml) was triggered in seven of eight cases. The phenotypic analysis of cultured cells revealed that the proliferating cells were indeed B cells. Of interest, the five tested SE were not in every case equally efficient in stimulating CLL B-cell maturation. Although SEA, SEB, SEC, SED and SEE all stimulated comparable levels of Ig production and proliferation in some patients' B cells, Ig secretion in other leukaemic B-cell populations was selectively induced by one, two or three of the enterotoxins. The reason why certain leukaemic B cells respond to some but not other SE is not readily apparent. As shown by Tcell depletion and reconstitution with normal irradiated T cells, the activation of leukaemic B cells by SE was totally dependent on T cells. Therefore, the clonally variable response of CLL B cells to different SE might be dictated by the responsiveness of the irradiated T cells to the various SE. It is remarkable, however, that the very same irradiated normal T-cell population provided 'help' to one leukaemic B-cell clone in response to only one SE, and to another B-cell clone in response to three different SE (compare Figs 2b and 4b). In this context, it is of interest that Herman et al/.37 recently reported that HLA-DR alleles differ in their ability to present staphylococcal enterotoxins to T cells. Therefore, it is possible that the HLA-DR allele expressed by the leukaemic B-cell clone might be important in determining the responsiveness to different SE. Clearly, more work with HLAtype leukemic B- and normal T-cell populations needs to be done to clarify this point. In conclusion, we have shown that enterotoxins are potent T-cell-dependent activators of leukaemic CLL B cells. These toxins will prove useful in further cellular and molecular analyses of leukaemic B-cell maturation.
ACKNOWLEDGMENTS We would like to thank Dr Gerhard Moldenhauer for kindly providing monoclonal antibodies. D Kabelitz was supported by the Alfried Krupp Award for young professors.
REFERENCES 1. CALVANO S.E.. QUIMBY F.W., ANTONACCI A.C.. REISER R.F.. BERGDOLL M.S. & DINEEN P. (1984) Analysis of the mitogenic effects of toxic shock toxin on human peripheral blood mononuclear cells in ritro. (Clint Immnunol. Inmunopat/hlO. 33, 99.
426
X. Duan et al.
2. LANGFORD M.P., STANTON G.J. & JOHNSON H.M. (1978) Biological effects of staphylococcal enterotoxin A on human peripheral lymphocytes. Inle(t Immun. 22, 62. 3. FLEISCHER B., SCHREZENMEIER H. & CONRADT P. (1989) T lymphocyte activation by staphylococcal enterotoxins: role of class 1I molecules and T cell surface structures. Cell. Inmununl. 120, 92. 4. MOLLICK J.A., COOK R.G. & RICH R.R. (1989) Class II MHC molecules are specific receptors for staphylococcus enterotoxin A. Science, 244, 817. 5. FRASER J.D. (1989) High affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature, 339,221. 6. FISCHER H., DOHLSTEN M., LINDVALL M., SJIGREN H-O. & CARLSSON R. (1989) Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines. J. Inmmunol. 142, 3151. 7. FLEISCHER B. & SCHREZENMEIER H. (1988) T cell stimulation by staphylococcal enterotoxins. Clonally variable response and requirement for major histocompatibility complex class 1I molecules on accessory or target cells. J. exp. Med. 167, 1697. 8. KAPPLER J., KOTZIN B., HERRON L., GELFAND E.W., BIGLER R.D., BOYLSTON A., CARREL S., POSNETT D.N., CHOI Y. & MARRACK P. (1989) Vfl-specific stimulation of human T cells by staphylococcal toxins. Science, 244, 81 1. 9. CHOI Y., KOTZIN B., HERRON L., CALLAHAN J., MARRACK P. & KAPPLER J. (1989) Interaction of Staphyllococcus aureus toxin superantigens' with human T cells. Proc. nail. Acad. Sci. U.S.A. 86, 8941. 10. MARRACK P. & KAPPLER J. (1990) The staphylococcal enterotoxins and their relatives. Science, 248, 705. 11. PARSONNET J., HICKMAN R.K., EARDLEY D.P. & PIER G.B. (1985) induction of human interleukin- 1 by toxic shock syndrome toxin- 1. J. Infect. Dis. 151, 514. 12. PARSONNET J. & GILLIS Z.A. (1988) Production of tumor necrosis factor by human monocytes in response to toxic shock syndrome toxin-l. J. Infect. Dis. 158, 1026. 13. MOURAD W., SCHOLL P., DIAz A., GEHA R. & CHATILA T. (1989) The staphylococcal toxic shock syndrome toxin I triggers B cell proliferation and differentiation via major histocompatibility complex-unrestricted cognate T/B cell interaction. J. exyp. Med. 170, 2011. 14. FU S.M., CHIORAZZI N. & KUNKEL H.G. (1979) Differentiation capacity and other properties of the leukemic cells of chronic lymphocytic leukemia. lInmunol. Revl. 48, 23. 15. ROBERT K.H. (I 979) Induction of monoclonal antibody synthesis in malignant human B cells by polyclonal B cell activators. Relationship between B cell subsets and prognosis. Inmniunol. Rev 48, 123. 16. TOTTERMAN T.H., NILSSON K. & SUNDSTROM C. (1980) Phorbol ester-induced differentiation of chronic lymphocytic leukemia cells. Nature, 288, 176. 17. BLOEM A.C., VAN HOOFF C.O.M., BAST E.J.E.G. & BALLIEUX R.E. (I 984) Mitogenic stimulation of malignant B cells. Chronic lymphocytic leukaemia: secretion of monoclonal IgM by in vitro-induced plasmablasts. Clin. exp. Immunol. 55, 636. 18. YOSHIZAKI K., NAKAGAWA T., KAIEDA T., MURAGUCHI A., YAMAMURA Y. & KISHIMOTO T. (1982) Induction of proliferation and Ig production in human B leukemic cells by anti-immunoglobulins and T cell factors. J. Intmunol. 128, 1296. 19. LARSON R.A. & YACHNIN S. (1983) Cytochalasin is a potent mitogen for chronic lymphocytic leukemia cells in vitro. J. clin Invest. 72, 1268. 2t). KABELITZ D., PFEFFER K., VON STELDERN D., BARTMANN P., BRUDLER 0., NERL C. & WAGNER H. (1985) In 1itro maturation of B cells in chronic lymphocytic leukemia. 1. Synergistic action of phorbol ester and interleukin 2 in the induction of Tac antigen expression and interleukin 2 responsiveness in leukemic B cells. J. Iummunol. 135, 2876.
21. LANTZ O., GRILLOT-COURVAIN C., SCHMITT C., FERMAND J.-P. & BROUET J.-C. (1 985) Interleukin 2-induced proliferation of leukemic human B cells. J. exp. Med. 161, 1225. 22. BAEKER T.R. & ROTHSTEIN T.L. (1986) Effects of anti-lgM on mitogen-induced proliferation of human B-lymphocyte malignancies. C/in. Ihnniunol. InununoI0athol. 39, 285. 23. CALVERT J.E., PROCTOR S.J. & JEFFERIS R. (1987) Activation of B chronic lymphocytic leukaemic cells by Branlhamella catarrhalis. Imtntunology', 60, 45. 24. MAYER L., CROW M.K. & THOMPSON C. (1985) Synergy between B cell differentiation factors and interleukin 2, using a monoclonal system. J. Ininninol. 135, 3272. 25. OSTLUND L., EINHORN S., ROBERT K.-H., JULIUSSON G. & BIBERFELD P. (1986) Chronic B-lymphocytic leukemia cells proliferate and differentiate following exposure to interferon in litro. Blood, 67, 152. 26. EMILIE D., KARRAY S., MERLE-BERAL H., DEBRE P. & GALANAUD P. (1988) Induction of differentiation in human leukemic B cells by interleukin 2 alone: differential effect on the expression of p and J chain genes. Eur. J. Immunol. 18, 1479. 27. PFEFFER K., NERL C. & KABELITz D. (1987) B cell maturation in chronic lymphocytic leukemia. II. Clonal heterogeneity in the response to various B cell activator and recombinant cytokines (rIFN-c, rIFN-a, rI12). Innnunohiologv,, 175, 172. 28. Touw I., DORSSERs L. & LOWENBERG B. (1987) The proliferative response of B cell chronic lymphocytic leukemia to interleukin 2: functional characterization of the interleukin 2 membrane receptors. Blood, 69, 1667. 29. TOTTERMAN T.H., CARLSSON M. & NILSSON K. (I1988) Induction of IgM secretion by chronic lymphocytic leukaemia cells in serum-free medium: effects of interferon-a. -c and phorbol ester. Clin. exrp. IflimlUn1ol. 71, 187. 30. KARRAY S., DELFRAISSY J.-F., MERLE-BERAL H., WALLON C., DEBRE P. & GALANALD P. (1988) Positive effects of interferon-a on B celltype chronic lymphocytic leukemia proliferative response. J. Imnmutin!. 140, 774. 31. NERL C., JANSSEN 0. & KABELITZ D. (1988) B cell maturation in chronic lymphocytic leukemia. Ill. Effect of recombinant cytokines on leukemic B cell proliferation. Leukemia, 2 (suppl.), 50S. 32. KAWANO M., IWATO K., ASAOKU H., TANABE O., TANAKA H., ISHIKAWA H., IMAMURA N. & KURAMOTO A. (1989) Heterogenous response of B cell chronic lymphocytic leukaemia (B-CLL) cells to anti-human IgM antibody (anti-L) and B cell stimulatory factor (BSF- 1). Br. J. Haematol. 71, 47. 33. CARLSSON M., SLUNDSTR6M C., BENGTSSON M., T6TTERMAN T.H., ROSEN A. & NILSSON K. (1989) Interleukin 4 strongly augments or inhibits DNA synthesis and differentiation of B-type chronic lymphocytic leukemia cells depending on the costimulatory activation and progression signals. Eur. J. Immuno/. 19, 913. 34. KABELITZ D., HERZOG W.R., ZANKER B. & WAGNER H. (1985) Human cytotoxic T-lymphocytes. 1. Limiting dilution analysis of alloreactive cytotoxic T-lymphocyte precursor frequencies. Scand. J. Inmmunol. 22, 329. 35. JANSSEN O., NERL C. & KABELITZ D. (1989) T cells in B-cell chronic lymphocytic leukemia: quantitative assessment of cytotoxic and interleukin-2-producing lymphocyte precursors by limiting dilution analysis. Blood, 73, 1622. 36. DREXLER H.G., GIGNAC S.M., BRENNER M.K., COUSTAIN-SMITH E., JANOSSY G. & HOFFBRAND A.V. (1988) Differential expression of MHC class 11 antigens in chronic B-cell disorders. C/in. exp. Immunol. 71, 217. 37. HERMAN A., CROTEAU G., SEKALY R.-P., KAPPLER J. & MARRACK P. (1990) H LA-DR alleles differ in their ability to present staphylococcal enterotoxins to T cells. J. evp. Med. 172, 709.
B-cell maturation in chronic lymphocytic leukaemia IV. T-CELL-DEPENDENT ACTIVATION OF LEUKAEMIC B CELLS BY STAPHYLOCOCCAL ENTEROTOXIN 'SUPERANTIGENS'
X. DUAN, C. NERL,*
0.
JANSSEN & D. KABELITZ Institutfiir Immunologie, Universitit Heidelberg, Heidelberg and *Stddtisches Krankenhaus Mfinchen-Schwiabing, Mfinchen, Germany
Accepted for publication 14 October 1991
SUMMARY
Staphylococcal enterotoxins (SE) are potent T-lymphocyte activators that stimulate T cells by directly cross-linking HLA-DR molecules on antigen-presenting cells with the V/# gene products of the T-cell receptor. The different SE activate all T cells expressing a given Vfl, and, therefore, have been termed 'superantigens'. Here we show that SE are potent activators of leukaemic B cells from patients with chronic lymphocytic leukaemia (CLL). Purified B cells from seven of eight CLL patients with high WBC counts ( > 80,000/l) responded to one or several of the tested SE (SEA, SEB, SEC,, SED, SEE) by proliferation ([3H]TdR incorporation) and/or Ig secretion. In several instances, the response of leukaemic B cells to SE was much stronger than was the response to other known B-cell activators including EBV, pokeweed mitogen (PWM), phorbolester (TPA), and Staphylococcus aureus Cowan I (SAC). The activation of leukaemic B cells by SE was strictly dependent on the addition of irradiated T cells isolated from healthy donors. FACS analysis of cultured cells ensured that the proliferating cells were indeed B cells. Taken together, these results demonstrate that SE are strong T-cell-dependent B-cell activators that, in some cases, can stimulate maturation of leukaemic B cells which are refractory to other activation signals. INTRODUCTION
TSST-1 of monocyte-derived IL-i and tumour necrosis factor-
Some bacterial toxins such as toxic shock syndrome toxin-I (TSST- 1) or StaphYlococcus aureuls cnterotoxins (SE) have been known for some time to be strongly mitogenic to T lymphocytes."2 Recently, these toxins have attracted much attention, due to the identification of their unusual mode of action. Thus, it was found that SE bind directly to major histocompatibility complex (MHC) class II molecules on the surface of antigenpresenting cells (APC), without need of being processed.3 6 Individual SE such as SEA, SEB, SEC,, SED or SEE, activate all T cells expressing a given V/I gene segment of the T-cell receptor (TcR), and a selective specificity for certain V,3 can be assigned to some SE.7 9 Therefore, these toxins have been termed 'superantigens'."' It appears that superantigens act by directly cross-linking HLA-DR and TcR molecules. While it is clear that SE-mediated cross-linking of HLA-DR+ APC and T cells strongly activate the latter, it is possible that APC are similarly activated during this process. In fact, stimulation by
alpha (TN F-oc) production has been demonstrated by Parsonnet and co-workers."1"12 More recently, Mourad et al.13 reported that TSST- 1 is a T-ccll-dependent activator of human B-cell proliferation and differentiation. In the present study we have investigated possible effects of staphylococcal enterotoxins on the in vitro differentiation of leukaemic B cells from patients with B-cell chronic lymphocytic leukaemia (CLL). Ig secretion has been successfully induced in these cells by a variety of polyclonal B-cell activators. 14 1' Under certain circumstances, leukaemic B cells could also be triggered to proliferate in ,itr-o.' 23 Results of many studies published over the last few years indicate that there is a considerable clonal heterogeneity of leukaemic CLL B-cell responses to polyclonal B-cell activators and defined (recombinant) cytokines.24 1' Here we show that SE are potent T-cell-dependent activators of leukaemic CLL B-cell proliferation and differentiation. In some instances, SE were by far more effective than other well-known (T-cell-dependent or -independent) B-cell activators such as EBV, pokeweed mitogen (PWM), phorbolester (TPA), or Staphylococcus aureus Cowan I (SAC).
Abbreviations: APC, antigen-presenting cell: CLL, chronic lymphocytic leukaemia; PBMC, peripheral blood mononuclear cells, SE, staphylococcus enterotoxin; TcR, T-cell receptor. Correspondence: Dr D. Kabelitz, Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305. D-6900 Heidelberg, Germany.
MATERIALS AND METHODS
lymphocyte populations Heparinized blood was obtained from healthy donors or CLL patients after informed consent. None of the CLL patients (six Isolation ot
420
B-cell maturation in chronic lymphocyte leukaemia
Wiebaden, Germany). All cultures were incubated at 37 in a humidified atmosphere of 5'S, CO2 in air.
Table 1. Characterization of PBMC from CLL patients
Response of purified leukaemic B cells to enterotoxins Patient
Sex
1. 2. 3. 4. 5. 6. 7. 8.
m f f f f m f f
WE LA
NI KU KO RE HU ER
WBC/pl 80,000
128,000 129,000 150,000 160,000 200,000 200,000 300,000
{M, CD3+ 2-6 46
30 28 1.0 39 68 2.1
sig
1gM IgM IgM IgM IgM IgG IgM IgG
Proliferation*
Ig secretiont
-
+ + + + +
+ +
+
421
+ +
* [3H]TdR incorporation > 5000 c.p.m. per 2 x 105 seeded cells. t IgM or IgG in cell culture supernatant > 400 ng/ml.
females, two males) was on treatment within the last 3 months before blood was taken for the present study. White blood cell (WBC) counts ranged from 80,000 to 300,000/11d. All patients included in this study were diagnosed as suffering from B-celltype CLL based on low percentages of circulating CD3 t T cells and the expression of either K- or .-Ig light chain on the vast majority of peripheral blood lymphocytes (see Table 1). Peripheral blood mononuclear cells (PBMC) were isolated by FicollHypaque density gradient centrifugation, and were stored in liquid nitrogen until use. T cells were separated from adherent cell-depleted PBMC by rosette formation with sheep erythrocytes (E) as described.34 Leukaemic B cells were purified from CLL PBMC by two consecutive depletions of E-rosette-forming T cells as previously described.35 The resulting CLL E cell fraction was contaminated with 5000 c.p.m. per 2 x 1I05 seeded cells; Ig production: > 400 ng `ml), proliferation was induced in B cells from three of eight patients, and Ig secretion in B cells from seven of eight patients. Only in one patient (RE) were the purified leukaemic B cells refractory to activation by SE. DISCUSSION SE have been known for a long time to be strong T-cell mitogens.2 However, SE differ from other polyclonal T-cell mitogens such as plant lectins in that they specifically activate some but not other T cells.7 Recently published evidence indicates that a given SE activates all T cells expressing the
/lymplhocyte leukaeinia
425
'correct' Vfl gene segment of the TcR.5'" Due to their stimulatory activity for a whole subset of T cells expressing a particular Vfl, SE (and related bacterial toxins) have been termed 'superantigens'.") As far as APC are concerned, superantigens can bind directly to MHC class 11 antigens without need of being processed.4 6 Inasmuch as CLL B cells express H LA-DR and, to a varying degree, other class II molecules on their surface,36 we considered the possibility that CLL B cells are activated in a Tcell-dependent fashion by SE, similarly to what has been described for the activation of normal B cells by TSST-1.'3 Our present results demonstrate that SE are indeed potent activators of CLL B cells in vitro. As with other B-cell activators,'4"16'20127'33 there was considerable heterogeneity in the capacity of the various CLL B cells to respond to SE stimulation. However, several of the investigated clonal B-cell populations responded much better to SE than to conventional B-cell activators including BV, PWM, SAC or TPA (see Figs 1 and 2). Thus, a significant B-cell proliferation was induced in three of eight CLL samples, while Ig production (> 400 ng/ml) was triggered in seven of eight cases. The phenotypic analysis of cultured cells revealed that the proliferating cells were indeed B cells. Of interest, the five tested SE were not in every case equally efficient in stimulating CLL B-cell maturation. Although SEA, SEB, SEC, SED and SEE all stimulated comparable levels of Ig production and proliferation in some patients' B cells, Ig secretion in other leukaemic B-cell populations was selectively induced by one, two or three of the enterotoxins. The reason why certain leukaemic B cells respond to some but not other SE is not readily apparent. As shown by Tcell depletion and reconstitution with normal irradiated T cells, the activation of leukaemic B cells by SE was totally dependent on T cells. Therefore, the clonally variable response of CLL B cells to different SE might be dictated by the responsiveness of the irradiated T cells to the various SE. It is remarkable, however, that the very same irradiated normal T-cell population provided 'help' to one leukaemic B-cell clone in response to only one SE, and to another B-cell clone in response to three different SE (compare Figs 2b and 4b). In this context, it is of interest that Herman et al/.37 recently reported that HLA-DR alleles differ in their ability to present staphylococcal enterotoxins to T cells. Therefore, it is possible that the HLA-DR allele expressed by the leukaemic B-cell clone might be important in determining the responsiveness to different SE. Clearly, more work with HLAtype leukemic B- and normal T-cell populations needs to be done to clarify this point. In conclusion, we have shown that enterotoxins are potent T-cell-dependent activators of leukaemic CLL B cells. These toxins will prove useful in further cellular and molecular analyses of leukaemic B-cell maturation.
ACKNOWLEDGMENTS We would like to thank Dr Gerhard Moldenhauer for kindly providing monoclonal antibodies. D Kabelitz was supported by the Alfried Krupp Award for young professors.
REFERENCES 1. CALVANO S.E.. QUIMBY F.W., ANTONACCI A.C.. REISER R.F.. BERGDOLL M.S. & DINEEN P. (1984) Analysis of the mitogenic effects of toxic shock toxin on human peripheral blood mononuclear cells in ritro. (Clint Immnunol. Inmunopat/hlO. 33, 99.
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X. Duan et al.
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