INFECTION
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Vol. 59, No. 3
IMMUNITY, Mar. 1991, p. 1210-1214
0019-9567/91/031210-05$02.00/0 Copyright © 1991, American Society for Microbiology
Cell and Receptor Requirements for Streptococcal Pyrogenic Exotoxin T-Cell Mitogenicity BETTINA A. B. LEONARD, PETER K. LEE, MARC K. JENKINS, AND PATRICK M. SCHLIEVERT* Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received 30 July 1990/Accepted 30 November 1990
Streptococcal pyrogenic exotoxins (SPEs) A, B, and C, like other members of the pyrogenic toxin family, are able to cause toxic shock-like syndromes. One of the major properties of these toxins is the ability to induce Tcell proliferation. Characterization of T cell mitogenicity associated with SPEs A, B, and C was undertaken. SPEs A, B, and C were mitogenic for C57BL10/SnJ and BALB/cWAT T cells, with activities differing in intensity depending on the mouse strain and toxin employed. SPE-induced, T-cell-proliferative activity was dependent on class II major histocompatibility complex molecules expressed on antigen-presenting cells. The abilities of SPEs A, B, and C to preferentially stimulate murine cells with certain T-cell receptor Vis were investigated by fluorescence-activated cell sorter analysis. SPE A preferentially activated T cells expressing VD 8 but not VD 3, 6, or 11, while SPEs B and C preferentially stimulated T cells which did not express any of the tested V,s.
Streptococcal pyrogenic exotoxin (SPE) serotypes A, B, and C, produced by group A streptococci, are members of a larger pyrogenic toxin family which also includes the staphylococcal enterotoxins (SEs A through E) and toxic shock syndrome toxin 1 (TSST-1) (5, 41, 43). These toxins show various degrees of sequence similarity but share many biological activities (5, 31, 37, 41, 43). For example, SPE A shares highly significant amino acid sequence similarity with SEs B and C, particularly in the carboxyl half of the molecule, while SPE C is less closely but significantly related to SPE A (37). In contrast, TSST-1 and SPE B are relatively distinct from the rest of the toxins (4, 16). Indeed, SPE B is the only one of the toxins made as a large precursor. Although structurally diverse, all of the pyrogenic toxins are able to cause toxic shock syndrome or related illnesses, such as scarlet fever (2, 5, 8-10, 21, 23, 30, 31, 33, 39, 41-43). All of the toxins share the ability to induce T-cell mitogenicity, tumor necrosis factor release, pyrogenicity, B-cell immunosuppression, and erythrophagocytosis. They also can enhance delayed-type hypersensitivity and lethal endotoxin shock (5, 11, 12, 31, 37, 41, 43). Recently, the T-cell mitogenicity of the SEs and TSST-1 has been characterized. The toxins are mitogenic for both CD4- and CD8-positive cells (14, 35). Like classical antigens, proliferation depends on interaction of the toxins with the T-cell receptor (TCR) through antigen-presenting cells (APCs) and class II major histocompatibility complex (MHC) molecules (6, 7, 13, 15, 18, 19, 22, 26-29, 40, 42-45). However, processing does not appear to be required, and mitogenicity occurs regardless of antigen and class II MHC haplotype specificities (27, 29, 45). There is some specificity, however, since proliferation is dependent on TCR VP usage, with preferential stimulation of murine VIs 3, 7, 8.1, 8.2, 8.3, and 17 by SE B and of VIs 3, 15, and 17 by TSST-1, for example (27, 45). The mitogenic activities of the SPEs have also been examined but not in as much detail as for the other members of the toxin family (1, 35, 38). All three SPE types are potently mitogenic for rabbit and human cells but have been *
reported to be only weakly mitogenic for BALB/cWAT mouse lymphocytes (1, 38). In addition, in two previous studies a macrophage requirement for T-cell proliferation induced by SPEs was not seen (35, 38). In this report, mitogenicity induced by SPE has been characterized. (This work was presented in part at the 90th Annual Meeting of the American Society for Microbiology [23a]). The mouse strains utilized in this study were C57BL10/ SnJ (H-2b) purchased from either Jackson Laboratories (Bar Harbor, Maine) or the National Institutes of Health and BALB/cWAT (H-2d) mice from the Department of Microbiology mouse colony maintained at the University of Minnesota. Monoclonal antibodies utilized for fluorescence-activated cell sorter (FACS) staining were 500-A-2 (anti-CD3), KJ25a (anti-VP 3), KJ16-133 (anti-VP 8.1 and VP 8.2), RR4-7 (anti-VP 6), and RR3-15 (anti-V,B 11). Monoclonal antibody M5/114 (anti-Epk:Eotk, Apb:AcXb) was utilized for class II MHC blocking studies. In order to prepare the toxins, large cultures (20 liters) of Streptococcus pyogenes 594 (SPE A), 86-858 (SPE B), or 86-104 (SPE C) were grown at 37°C with slow stirring in a dialyzable beef heart medium until stationary phase; each of these strains makes one pyrogenic toxin type (36, 39). Toxins were purified by precipitation by addition of 4 volumes of absolute ethanol, resolubilization in pH 4.5 acetate-buffered saline, and preparative thin-layer isoelectric focusing (36, 39). The toxins obtained were tested for reactivity by Ouchterlony immunodiffusion with hyperimmune rabbit antisera against each SPE and for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 10 ,ug of each toxin migrated as a homogeneous protein band that corresponded to the molecular weight of the appropriate toxin. The lyophilized toxins were stored. SPEs are potently mitogenic for rabbit and human T cells but were reported to be only weakly or not mitogenic for murine lymphocytes (1, 38). On the basis of evidence that other members of the larger pyrogenic toxin family stimulate only certain VP-expressing T-cell subsets, it was proposed that the low mitogenicity of the SPEs for murine lymphocytes could result from murine cells' not having the necessary V,B repertoire. The mitogenic activities of SPEs A, B,
Corresponding author. 1210
VOL. 59, 1991
NOTES
Stimulation of C57BL1O/SnJ Splenocytes
TABLE 1. Murine TCR VP usage in T-cell populations stimulated with SPE A, B, or Ca
100 80
1211
Region antibody directed against
-
% of cells labeled' when stimulated with: PMA-I SPE A SPE B SPE C
.o =
7*
60-
-0--
SPE A SPEB
40-
* -O-
SPEC
.01
c
80
-
as
60
-
95.3
91.4
93.7
4.6 9.3
1.7 1.2
1.5 2.8
0.2 0.6
18.1 7.5
42.0 2.7
1.9 1.3
0.7 0.02
V,6
.1
1
10
ug/mi toxin
a C57BL10/SnJ mice were employed in this study, and results reported are representative of seven independent experiments. b Determined by FACS analysis.
100
Stimulation of BALB/c Splenocytes -
95.3
VP3
V,B8.1-8.2 V 11
as 20
100
CD3
SPEA SPEB
40
SPEC
x
20
.01
9
10
.1
100
ug/mi toxin
FIG. 1. Lymphocyte-mitogenic activities of SPEs A, B, and C in BALB/cWAT and C57BL10 mice. Splenocytes from BALB/cWAT and C57BL10/SnJ mice were incubated with either SPE A, B, or C or PMA-I. Proliferation was determined by [3H]thymidine uptake by the dividing cells. Proliferation is reported as percent PMA-I stimulation + standard deviation, with PMA-I counts per minute set at 100%. These values represent the average of three independent experiments.
and C were therefore examined, with both BALB/cWAT and C57BL10/SnJ mice used as sources of lymphocytes. BALB/ cWAT and C57BL10/SnJ mice have different T-cell repertoires; BALB/cWAT mice, but not C57BL10/SnJ mice, express I-Ed and Mls-2a molecules and thus delete T cells expressing VP 3 and VP 11 TCR , chains. Proliferation of unseparated splenic lymphocytes was used as a measure of the mitogenic activities of SPEs A, B, and C. Splenocytes were incubated in the presence of various toxins in 96-well microtiter plates for 3 days at 37°C with 7% CO2 and then labeled with 1 ,uCi of [3H]thymidine and incubated for an additional 16 h. Cells were harvested onto glass fiber filters, and [3H]thymidine uptake was measured by liquid scintillation counting. Positive controls consisted of cells stimulated with 10 ng of phorbol myristic acid per ml (Sigma Chemical Co., St. Louis, Mo.) and 0.67 ,uM ionomycin (Calbiochem Corp., San Diego, Calif.) (PMA-I). Results of proliferation assays (counts per minute in four replicates + standard deviation) were reported as the percentage of PMA-I control counts per minute. All three types were mitogenic for murine lymphocytes, with activities differing in intensity depending on the mouse strain and toxin employed (Fig. 1). For example, SPE A was able to stimulate BALB/c lymphocytes to 78% of the level seen with PMA-I but stimulated C57BL10 lymphocytes to only 37% of the level seen with PMA-I. Conversely, SPE C stimulated both BALB/c and C57BL10 lymphocytes approximately equally, with slight variations
depending on the toxin batch employed. SPE B's mitogenic activity was intermediate for BALB/c lymphocytes and approximately equal to that of SPEs A and C for C57BL10/ SnJ lymphocytes. On the basis of evidence that the SEs stimulate only certain VP subsets, it was possible that the differences in mitogenic activity were due to differential V,B usage among SPEs A, B, and C. The VP usage patterns can be deduced by comparing the percentages of the T-cell population made up by each V,B subset for PMA-I-stimulated cells and toxin-stimulated cells, assuming that only specific Vps are stimulated. Therefore, lymph node cells from C57BL10/SnJ mice were stimulated with either SPE or PMA-I for 3 days to allow the mitogens to activate the T cells. After 3 days, the cells were washed and incubated with 50 U of recombinant interleukin 2 (Boehringer Mannheim, Indianapolis, Ind.) per ml for 4 days to expand T cells activated in the initial culture (29). On the last day, viable cells were harvested on a Ficoll-Paque 1077 density gradient (Pharmacia, Piscataway, N.J.). The viable T cells were incubated with anti-TCR monoclonal antibodies and then stained with the appropriate fluorescein isothiocyanate-conjugated antibodies (Caltag, San Francisco, Calif., or Cappel, Malvern, Pa.). Negative controls consisted of cells stained with the fluorescein isothiocyanate-conjugated antibodies alone. The intensity of staining was quantified by using a FACS IV (Becton Dickinson and Co., Cockeysville, Md.) (Table 1). The distribution of V,s expressed in PMAI-stimulated cells is nearly identical to that observed in unstimulated T cells from the same strain, suggesting that all T cells were stimulated equally by the pharmacological agents (3, 20, 34). Upon SPE A stimulation, 42% of the activated cells expressed the VP 8.1-8.2 chain of the TCR, while only 18% of PMA-I-stimulated cells expressed VP 8.1-8.2. The numbers of T cells expressing VP 3, 6, or 11 were reduced in SPE A-stimulated populations in comparison with PMA-I-stimulated populations. Therefore, it is concluded that, like SEs B and C, which share highly significant sequence similarity with SPE A, SPE A preferentially stimulated VP 8-expressing T cells. Since VP 8 does not account for all the CD3-positive cells stimulated, other T cells expressing V,s not tested were probably also stimulated. However, there may be relatively few subpopulations stimulated, since V,B 8.1-8.2 accounted for 42% of all the activated T cells; this is in agreement with a recent report by Imanishi et al., who found that V,B 8.2 account for 80% of SPE A-stimulated T cells from C57BL/6 mice (17). Since V,B 8.1-8.2 accounts for 18.0% of the T cells in BALB/c mice as opposed to 16.8% in C57BL10 mice, the use of VP 8 by SPE A probably does not account for the entire difference in the SPE A reactivities of these two mouse strains (20, 25).
1212
NOTES
INFECT. IMMUN.
T cells
Antigen Presenting Cells
T cells - PMA/I
APC - PMA/I
T cells - SPE C
APC - SPE C
T cells - SPE A
APC - SPE A
T ccils
APC
I T
0
60 40 80 % PMAJI Stimulation
20
T cells
+
0
Antigen Presenting Cells
T/APC - SPE C
T/APC SPE C
T/APC - SPE C - M5/114
T/APC SPE A
T/APC - SPE A
T/APC
T/APC - SPE A - M5/114
-
-
-
0
20
40
60
80
100
120
% PMA/I Stimulation
.
61)
X0 * PMA/I Stimulation
40
0(X)
120)
Inhibition by Class I1 MoAb M5/114
E[1
T/APC PMA/I
.
21)
0
20
40 80 60 % PMA/I Stimulation
100
120
FIG. 2. Roles of APCs and class II MHC molecules in the mitogenic activities of SPEs A and C. Purified T cells and adherent APCs from C57BL10/SnJ mice were incubated either separately or together in the presence or absence of toxin (50 ,ug/ml) or PMA-I. Proliferation was measured by uptake of [3H]thymidine by the dividing cells. The values are reported as percent stimulation standard deviation, with PMA-I set at 100%. The results reported are representative of four independent experiments. MoAb, Monoclonal antibody.
SPEs B and C may also preferentially stimulate V,B-containing T-cell subsets, since they are mitogens as potent as SPE A for C57BL10/SnJ lymphocytes. However, the identities of the VP subpopulations stimulated could not be determined in these experiments, since after activation by SPE B or C, V,Bs 3, 6, 8, and 11 were all reduced in stimulated populations. It is interesting that SPE A, which is structurally similar to the SEs (35), shares the SEs' ability to stimulate VP 8-containing T cells (27, 45), whereas SPEs B and C, which may be structurally distinct, do not. To investigate the role of APCs in the stimulation of T cells, populations greatly enriched for T cells or APCs were obtained and incubated either together or separately in the presence of toxin. T-cell populations were depleted of macrophages and B cells by a series of three treatments. A majority of the adherent APCs were removed from splenocytes from C57BL10/SnJ mice by plating the cells on plastic for 1 h at 37°C with 7% CO2 and then passing the nonadherent cells over a Sephadex G-10 (Sigma) column (24, 32). B cells and any remaining adherent cells were removed by passage over a T-cell column containing antibodies to B cells according to the manufacturer's specifications (Beckman Instruments, Inc., Fullerton, Calif.). The T-cell populations were then labeled with anti-CD3 monoclonal antibodies followed by fluorescein isothiocyanate-conjugated goat antirat immunoglobulin G and found to be greater than 98% by FACS analysis with a Becton Dickinson FACS IV. T-celldepleted adherent APCs were prepared by plating splenocytes from C57BL10/SnJ mice on plastic for 1 h at 37°C with 7% CO2. The nonadherent cells were removed by washing, and the adherent cells were recovered by scraping. SPEs A and C were chosen as representative toxins for these studies. The cells and toxins were incubated in various combinations, and proliferation was determined as described above. Neither purified T cells nor purified APCs were stimulated
strongly with either SPE A or SPE C (Fig. 2A and B, respectively). However, when T cells were combined with APCs, the proliferative response was restored (Fig. 2C), indicating that the mitogenic response depended on the presence of APCs. Previously, it was reported that SPEinduced mitogenicity was independent of macrophages (35, 38). To determine whether the small amount of proliferation seen in the purified T cells was due to a small number of contaminating APCs or whether it was due to mitogenic activity independent of APCs, the anti-class II MHC monoclonal antibody M5/114 was added to the SPE A- and SPE C-induced cells. If the residual activity was independent of APCs, the addition of M5/114 should not interfere with the residual mitogenic response since murine T cells do not express class II MHC molecules. Monoclonal antibody M5/114, specific for only the class II MHC molecule expressed on C57BL10/SnJ cells, blocked all proliferation (Fig. 2D), suggesting that not only APCs but also class II MHC interactions were needed for SPEs to induce T-cell mitogenesis. The antibody itself was not toxic to the cells, since it did not interfere with PMA-I stimulation. Additionally, the blocking appeared to be specific for class II since MKS4 monoclonal antibody, specific for I-AS molecules, did not interfere with presentation of SPE A or C to C57BL10/SnJ mice (H-2k) (data not shown). SPEs A and C stimulated both CD4- and CD8-positive T cells (data not shown), so it is interesting that the class II MHC monoclonal antibodies blocked all proliferation. This could indicate one of two mechanisms for blocking proliferation of CD8-positive T cells. Either (i) CD8-positive-cell proliferation is dependent on help from the CD4-positive cells or (ii) the CD8 cells are of a subset that recognizes class II MHC molecules. Overall, this report shows that SPEs A, B, and C stimulate T-cell proliferation by a mechanism similar to those of the SEs and TSST-1. Mitogenicity is V,B specific and requires
VOL. 59, 1991
APCs and class II MHC molecules. These results are not surprising, since the streptococcal toxins are part of the larger family of pyrogenic toxins which are biochemically and biologically related and which are able to produce similar diseases. This research was supported by Public Health Service grant HL36611 from the National Heart, Lung, and Blood Institute to P.M.S. and by Public Health Service grants AI27998 and A128635 from the Institute of Allergy and Infectious Diseases to M.K.J. M.K.J. is the recipient of a Pew Scholar Award. B.A.B.L. was supported by training grant 5T32CA09138 from the National Cancer Institute of the National Institutes of Health. REFERENCES 1. Barsunmian, E. L., P. M. Schlievert, and D. W. Watson. 1978. Nonspecific and specific immunological mitogenicity by group A streptococcal pyrogenic exotoxins. Infect. Immun. 22:681688. 2. Bergdoll, M. S., B. A. Crass, R. F. Reiser, R. N. Robbins, and J. P. Davis. 1981. A new staphylococcal enterotoxin, enterotoxin F, associated with toxic shock syndrome. Lancet i:10171021. 3. Bill, J., 0. Kanagawa, B. Woodland, and E. Palmer. 1989. The MHC molecule I-E is necessary but not sufficient for the clonal deletion of Vpll-bearing T cells. J. Exp. Med. 169:1405-1419. 4. Blomster-Hautamaa, D. A., B. N. Kreiswirth, J. S. Kornblum, R. P. Novick, and P. M. Schlievert. 1986. The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1. J. Biol. Chem. 261:15783-15786. 5. Blomster-Hautamaa, D. A., and P. M. Schlievert. 1988. Nonenterotoxic staphylococcal toxins, p. 297-330. In M. C. Hardegree and A. T. Tu (ed.), Handbook of natural toxins, vol. 4. Bacterial toxins. Marcel Dekker, Inc., New York. 6. Carlsson, R., H. Fischer, and H. 0. Sjogren. 1988. Binding of staphylococcal enterotoxin A to accessory cells is a requirement for its ability to activate human T cells. J. Immunol. 140:24842488. 7. Chatila, T., N. Wood, J. Parsonnet, and R. S. Geha. 1988. Toxic shock syndrome toxin 1 induces inositol phospholipid turnover, protein kinase C translocation, and calcium mobilization in human T cells. J. Immunol. 140:1250-1255. 8. Cone, L. A., D. R. Woodard, P. M. Schlievert, and G. S. Tomory. 1987. Clinical and bacteriologic observations of a toxic shock-like syndrome due to Streptococcus pyogenes. N. Engl. J. Med. 317:146-149. 9. de Azavedo, J. C. S., and J. P. Arbuthnott. 1984. Toxicity of staphylococcal toxic shock syndrome toxin 1 in rabbits. Infect. Immun. 46:314-317. 10. de Azavedo, J. C. S., T. J. Foster, P. J. Hartigan, J. P. Arbuthnott, M. O'Reilly, B. N. Kreiswirth, and R. P. Novick. 1985. Expression of the cloned toxic shock syndrome toxin 1 gene (tst) in vivo with a rabbit uterine model. Infect. Immun. 50:304-309. 11. Fast, D. J., P. M. Schlievert, and R. D. Nelson. 1988. Nonpurulent response to toxic shock syndrome toxin 1 producing Staphylococcus aureus: relationship to toxin-stimulated production of tumor necrosis factor. J. Immunol. 140:949-953. 12. Fast, D. J., P. M. Schlievert, and R. D. Nelson. 1989. Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production. Infect. Immun. 57:291-294. 13. Fleisher, B. 1989. Bacterial toxins as probes for the T cell antigen receptor. Immunol. Today 10:262-264. 14. Fleisher, B., and H. Schrezenmeier. 1988. T cell stimulation by staphylococcal enterotoxins. Clonally variable response and requirement for major histocompatibility complex class II molecules on accessory or target cells. J. Exp. Med. 167:1697-1707. 15. Fraser, J. D. 1989. High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature (London) 339:221223. 16. Hauser, A. R., and P. M. Schlievert. 1990. Nucleotide sequence
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of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor. J. Bacteriol. 172:4536-4542. 17. Imanishi, K., H. Igarashi, and T. Uchiyama. 1990. Activation of murine T cells by streptococcal pyrogenic exotoxin type A: requirement for MHC class II molecules on accessory cells and identification of VP elements in the T cell receptor of toxin reactive T cells. J. Immunol. 145:3170-3176. 18. Janeway, C. A., Jr., J. Yagi, P. J. Conrad, M. E. Katz, B. Jones, S. Vroegop, and S. Buxser. 1989. T cell responses to Mls and to bacterial proteins that mimic its behavior. Immunol. Rev. 107:61-88. 19. Kappler, J., B. Kotzin, L. Herron, E. W. Gelfand, R. D. Bigler, A. Boylston, S. Carrel, D. N. Posnett, Y. Choi, and P. Marrack. 1989. VP specific stimulation of human T cells by staphylococcal toxins. Science 244:811-813. 20. Kappler, J. W., U. Staerz, J. White, and P. Marrack. 1988. Self tolerance eliminates T cells specific for Mls-modified products of the major histocompatibility complex. Nature (London) 332: 35-40. 21. Kohler, W., D. Gerlach, and H. Gnoll. 1987. Streptococcal outbreaks and erythrogenic toxin type A. Zentralbl. Bakteriol. Mikrobiol. Hyg. A 266:104-115. 22. Langford, M. P., G. J. Stanton, and H. M. Johnson. 1978. Biological effects of staphylococcal enterotoxin A on human peripheral lymphocytes. Infect. Immun. 22:62-68. 23. Lee, P. K., and P. M. Schlievert. 1989. Quantification and toxicity of group A streptococcal pyrogenic exotoxins in an animal model of toxic shock syndrome-like illness. J. Clin. Microbiol. 27:1890-1892. 23a.Leonard, B., M. K. Jenkins, and P. M. Schlievert. 1990. Characterization of streptococcal pyrogenic exotoxin-induced T-cell mitogenicity, abstr. B251, p. 129. Abstr. 90th Annu. Meet. Am. Soc. Microbiol. 1990. American Society for Microbiology, Washington, D.C. 24. Ly, I. A., and R. I. Mishell. 1974. Separation of mouse spleen cells by passage through columns of Sephadex G10. J. Immunol. Methods 5:239-247. 25. MacDonald, H. R., R. Schneider, R. K. Lees, R. C. Howe, H. Acha-Orbea, H. Festenstein, R. M. Zinkernagel, and H. Hengartner. 1988. T cell receptor VP use predicts reactivity and tolerance to Mlsa-encoded antigens. Nature (London) 332:4045. 26. Marrack, P., M. Blackman, E. Kushnir, and J. Kappler. 1990. The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells. J. Exp. Med. 171:455-464. 27. Marrack, P., and J. Kappler. 1990. The staphylococcal enterotoxins and their relatives. Science 248:705-711. 28. Mollick, J. A., R. G. Cook, and R. R. Rich. 1989. Class II MHC molecules are specific receptors for staphylococcus enterotoxin A. Science 244:817-820. 29. Norton, S. D., P. M. Schlievert, R. P. Novick, and M. K. Jenkins. 1990. Molecular requirements for T cell activation by the staphylococcal toxic shock syndrome toxin-1. J. Immunol. 144:2089-2095. 30. Parsonnet, J., Z. A. Gillis, A. G. Richter, and G. B. Pier. 1987. A rabbit model of toxic shock syndrome that uses a constant, subcutaneous infusion of toxic shock syndrome toxin 1. Infect. Immun. 55:1070-1076. 31. Poindexter, N. J., and P. M. Schlievert. 1985. The biochemical and immunological properties of toxic shock syndrome toxin-1 (TSST-1) and association with TSS. J. Toxicol. Toxin Rev. 4:1-39. 32. Poindexter, N. J., and P. M. Schlievert. 1985. Toxic shock syndrome toxin-i-induced proliferation of lymphocytes: a comparison of mitogenic response of human, murine, and rabbit lymphocytes. J. Infect. Dis. 151:65-72. 33. Pullen, A. M., P. Marrack, and J. W. Kappler. 1988. The T cell repertoire is heavily influenced by tolerance to polymorphic self antigens. Nature (London) 335:796-801. 34. Rasheed, J. K., R. J. Arko, J. C. Feeley, F. W. Chandler, C. Thornsberry, R. J. Gibson, M. L. Cohen, C. D. Jeffries, and C. V. Broome. 1985. Acquired ability of Staphylococcus aureus
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in a rabbit model. Infect. Immun. 47:598-604. Regelmann, W. E., E. D. Gray, and L. W. Wannamaker. 1982. Characterization of the human cellular immune response to purified group A streptococcal blastogen A. J. Immunol. 128: 1631-1636. Schlievert, P. M., K. M. Bettin, and D. W. Watson. 1977. Purification and characterization of group A streptococcal pyrogenic exotoxin type C. Infect. Immun. 16:673-679. Schlievert, P. M., G. A. Bohach, C. J. Hovde, B. N. Kreiswirth, and R. P. Novick. 1990. Molecular studies of toxic shock syndrome associated staphylococcal and streptococcal toxins, p. 313-326. In R. P. Novick (ed.), Molecular biology of the staphylococci. VCH Publishers, Inc., New York. Schlievert, P. M., D. J. Schoettle, and D. W. Watson. 1979. Nonspecific T-lymphocyte mitogenesis by pyrogenic exotoxins from group A streptococci and Staphylococcus aureus. Infect. Immun. 25:1075-1077. Schlievert, P. M., K. N. Shands, B. B. Dan, G. P. Schmid, and R. D. Nishimura. 1981. Identification and characterization of an exotoxin from Staphylococcus aureus associated with toxic shock syndrome. J. Infect. Dis. 143:509-516. Scholl, P., A. Diez, W. Mourad, J. Parsonnet, R. S. Geha, and T.
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AND
Vol. 59, No. 3
IMMUNITY, Mar. 1991, p. 1210-1214
0019-9567/91/031210-05$02.00/0 Copyright © 1991, American Society for Microbiology
Cell and Receptor Requirements for Streptococcal Pyrogenic Exotoxin T-Cell Mitogenicity BETTINA A. B. LEONARD, PETER K. LEE, MARC K. JENKINS, AND PATRICK M. SCHLIEVERT* Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received 30 July 1990/Accepted 30 November 1990
Streptococcal pyrogenic exotoxins (SPEs) A, B, and C, like other members of the pyrogenic toxin family, are able to cause toxic shock-like syndromes. One of the major properties of these toxins is the ability to induce Tcell proliferation. Characterization of T cell mitogenicity associated with SPEs A, B, and C was undertaken. SPEs A, B, and C were mitogenic for C57BL10/SnJ and BALB/cWAT T cells, with activities differing in intensity depending on the mouse strain and toxin employed. SPE-induced, T-cell-proliferative activity was dependent on class II major histocompatibility complex molecules expressed on antigen-presenting cells. The abilities of SPEs A, B, and C to preferentially stimulate murine cells with certain T-cell receptor Vis were investigated by fluorescence-activated cell sorter analysis. SPE A preferentially activated T cells expressing VD 8 but not VD 3, 6, or 11, while SPEs B and C preferentially stimulated T cells which did not express any of the tested V,s.
Streptococcal pyrogenic exotoxin (SPE) serotypes A, B, and C, produced by group A streptococci, are members of a larger pyrogenic toxin family which also includes the staphylococcal enterotoxins (SEs A through E) and toxic shock syndrome toxin 1 (TSST-1) (5, 41, 43). These toxins show various degrees of sequence similarity but share many biological activities (5, 31, 37, 41, 43). For example, SPE A shares highly significant amino acid sequence similarity with SEs B and C, particularly in the carboxyl half of the molecule, while SPE C is less closely but significantly related to SPE A (37). In contrast, TSST-1 and SPE B are relatively distinct from the rest of the toxins (4, 16). Indeed, SPE B is the only one of the toxins made as a large precursor. Although structurally diverse, all of the pyrogenic toxins are able to cause toxic shock syndrome or related illnesses, such as scarlet fever (2, 5, 8-10, 21, 23, 30, 31, 33, 39, 41-43). All of the toxins share the ability to induce T-cell mitogenicity, tumor necrosis factor release, pyrogenicity, B-cell immunosuppression, and erythrophagocytosis. They also can enhance delayed-type hypersensitivity and lethal endotoxin shock (5, 11, 12, 31, 37, 41, 43). Recently, the T-cell mitogenicity of the SEs and TSST-1 has been characterized. The toxins are mitogenic for both CD4- and CD8-positive cells (14, 35). Like classical antigens, proliferation depends on interaction of the toxins with the T-cell receptor (TCR) through antigen-presenting cells (APCs) and class II major histocompatibility complex (MHC) molecules (6, 7, 13, 15, 18, 19, 22, 26-29, 40, 42-45). However, processing does not appear to be required, and mitogenicity occurs regardless of antigen and class II MHC haplotype specificities (27, 29, 45). There is some specificity, however, since proliferation is dependent on TCR VP usage, with preferential stimulation of murine VIs 3, 7, 8.1, 8.2, 8.3, and 17 by SE B and of VIs 3, 15, and 17 by TSST-1, for example (27, 45). The mitogenic activities of the SPEs have also been examined but not in as much detail as for the other members of the toxin family (1, 35, 38). All three SPE types are potently mitogenic for rabbit and human cells but have been *
reported to be only weakly mitogenic for BALB/cWAT mouse lymphocytes (1, 38). In addition, in two previous studies a macrophage requirement for T-cell proliferation induced by SPEs was not seen (35, 38). In this report, mitogenicity induced by SPE has been characterized. (This work was presented in part at the 90th Annual Meeting of the American Society for Microbiology [23a]). The mouse strains utilized in this study were C57BL10/ SnJ (H-2b) purchased from either Jackson Laboratories (Bar Harbor, Maine) or the National Institutes of Health and BALB/cWAT (H-2d) mice from the Department of Microbiology mouse colony maintained at the University of Minnesota. Monoclonal antibodies utilized for fluorescence-activated cell sorter (FACS) staining were 500-A-2 (anti-CD3), KJ25a (anti-VP 3), KJ16-133 (anti-VP 8.1 and VP 8.2), RR4-7 (anti-VP 6), and RR3-15 (anti-V,B 11). Monoclonal antibody M5/114 (anti-Epk:Eotk, Apb:AcXb) was utilized for class II MHC blocking studies. In order to prepare the toxins, large cultures (20 liters) of Streptococcus pyogenes 594 (SPE A), 86-858 (SPE B), or 86-104 (SPE C) were grown at 37°C with slow stirring in a dialyzable beef heart medium until stationary phase; each of these strains makes one pyrogenic toxin type (36, 39). Toxins were purified by precipitation by addition of 4 volumes of absolute ethanol, resolubilization in pH 4.5 acetate-buffered saline, and preparative thin-layer isoelectric focusing (36, 39). The toxins obtained were tested for reactivity by Ouchterlony immunodiffusion with hyperimmune rabbit antisera against each SPE and for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 10 ,ug of each toxin migrated as a homogeneous protein band that corresponded to the molecular weight of the appropriate toxin. The lyophilized toxins were stored. SPEs are potently mitogenic for rabbit and human T cells but were reported to be only weakly or not mitogenic for murine lymphocytes (1, 38). On the basis of evidence that other members of the larger pyrogenic toxin family stimulate only certain VP-expressing T-cell subsets, it was proposed that the low mitogenicity of the SPEs for murine lymphocytes could result from murine cells' not having the necessary V,B repertoire. The mitogenic activities of SPEs A, B,
Corresponding author. 1210
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NOTES
Stimulation of C57BL1O/SnJ Splenocytes
TABLE 1. Murine TCR VP usage in T-cell populations stimulated with SPE A, B, or Ca
100 80
1211
Region antibody directed against
-
% of cells labeled' when stimulated with: PMA-I SPE A SPE B SPE C
.o =
7*
60-
-0--
SPE A SPEB
40-
* -O-
SPEC
.01
c
80
-
as
60
-
95.3
91.4
93.7
4.6 9.3
1.7 1.2
1.5 2.8
0.2 0.6
18.1 7.5
42.0 2.7
1.9 1.3
0.7 0.02
V,6
.1
1
10
ug/mi toxin
a C57BL10/SnJ mice were employed in this study, and results reported are representative of seven independent experiments. b Determined by FACS analysis.
100
Stimulation of BALB/c Splenocytes -
95.3
VP3
V,B8.1-8.2 V 11
as 20
100
CD3
SPEA SPEB
40
SPEC
x
20
.01
9
10
.1
100
ug/mi toxin
FIG. 1. Lymphocyte-mitogenic activities of SPEs A, B, and C in BALB/cWAT and C57BL10 mice. Splenocytes from BALB/cWAT and C57BL10/SnJ mice were incubated with either SPE A, B, or C or PMA-I. Proliferation was determined by [3H]thymidine uptake by the dividing cells. Proliferation is reported as percent PMA-I stimulation + standard deviation, with PMA-I counts per minute set at 100%. These values represent the average of three independent experiments.
and C were therefore examined, with both BALB/cWAT and C57BL10/SnJ mice used as sources of lymphocytes. BALB/ cWAT and C57BL10/SnJ mice have different T-cell repertoires; BALB/cWAT mice, but not C57BL10/SnJ mice, express I-Ed and Mls-2a molecules and thus delete T cells expressing VP 3 and VP 11 TCR , chains. Proliferation of unseparated splenic lymphocytes was used as a measure of the mitogenic activities of SPEs A, B, and C. Splenocytes were incubated in the presence of various toxins in 96-well microtiter plates for 3 days at 37°C with 7% CO2 and then labeled with 1 ,uCi of [3H]thymidine and incubated for an additional 16 h. Cells were harvested onto glass fiber filters, and [3H]thymidine uptake was measured by liquid scintillation counting. Positive controls consisted of cells stimulated with 10 ng of phorbol myristic acid per ml (Sigma Chemical Co., St. Louis, Mo.) and 0.67 ,uM ionomycin (Calbiochem Corp., San Diego, Calif.) (PMA-I). Results of proliferation assays (counts per minute in four replicates + standard deviation) were reported as the percentage of PMA-I control counts per minute. All three types were mitogenic for murine lymphocytes, with activities differing in intensity depending on the mouse strain and toxin employed (Fig. 1). For example, SPE A was able to stimulate BALB/c lymphocytes to 78% of the level seen with PMA-I but stimulated C57BL10 lymphocytes to only 37% of the level seen with PMA-I. Conversely, SPE C stimulated both BALB/c and C57BL10 lymphocytes approximately equally, with slight variations
depending on the toxin batch employed. SPE B's mitogenic activity was intermediate for BALB/c lymphocytes and approximately equal to that of SPEs A and C for C57BL10/ SnJ lymphocytes. On the basis of evidence that the SEs stimulate only certain VP subsets, it was possible that the differences in mitogenic activity were due to differential V,B usage among SPEs A, B, and C. The VP usage patterns can be deduced by comparing the percentages of the T-cell population made up by each V,B subset for PMA-I-stimulated cells and toxin-stimulated cells, assuming that only specific Vps are stimulated. Therefore, lymph node cells from C57BL10/SnJ mice were stimulated with either SPE or PMA-I for 3 days to allow the mitogens to activate the T cells. After 3 days, the cells were washed and incubated with 50 U of recombinant interleukin 2 (Boehringer Mannheim, Indianapolis, Ind.) per ml for 4 days to expand T cells activated in the initial culture (29). On the last day, viable cells were harvested on a Ficoll-Paque 1077 density gradient (Pharmacia, Piscataway, N.J.). The viable T cells were incubated with anti-TCR monoclonal antibodies and then stained with the appropriate fluorescein isothiocyanate-conjugated antibodies (Caltag, San Francisco, Calif., or Cappel, Malvern, Pa.). Negative controls consisted of cells stained with the fluorescein isothiocyanate-conjugated antibodies alone. The intensity of staining was quantified by using a FACS IV (Becton Dickinson and Co., Cockeysville, Md.) (Table 1). The distribution of V,s expressed in PMAI-stimulated cells is nearly identical to that observed in unstimulated T cells from the same strain, suggesting that all T cells were stimulated equally by the pharmacological agents (3, 20, 34). Upon SPE A stimulation, 42% of the activated cells expressed the VP 8.1-8.2 chain of the TCR, while only 18% of PMA-I-stimulated cells expressed VP 8.1-8.2. The numbers of T cells expressing VP 3, 6, or 11 were reduced in SPE A-stimulated populations in comparison with PMA-I-stimulated populations. Therefore, it is concluded that, like SEs B and C, which share highly significant sequence similarity with SPE A, SPE A preferentially stimulated VP 8-expressing T cells. Since VP 8 does not account for all the CD3-positive cells stimulated, other T cells expressing V,s not tested were probably also stimulated. However, there may be relatively few subpopulations stimulated, since V,B 8.1-8.2 accounted for 42% of all the activated T cells; this is in agreement with a recent report by Imanishi et al., who found that V,B 8.2 account for 80% of SPE A-stimulated T cells from C57BL/6 mice (17). Since V,B 8.1-8.2 accounts for 18.0% of the T cells in BALB/c mice as opposed to 16.8% in C57BL10 mice, the use of VP 8 by SPE A probably does not account for the entire difference in the SPE A reactivities of these two mouse strains (20, 25).
1212
NOTES
INFECT. IMMUN.
T cells
Antigen Presenting Cells
T cells - PMA/I
APC - PMA/I
T cells - SPE C
APC - SPE C
T cells - SPE A
APC - SPE A
T ccils
APC
I T
0
60 40 80 % PMAJI Stimulation
20
T cells
+
0
Antigen Presenting Cells
T/APC - SPE C
T/APC SPE C
T/APC - SPE C - M5/114
T/APC SPE A
T/APC - SPE A
T/APC
T/APC - SPE A - M5/114
-
-
-
0
20
40
60
80
100
120
% PMA/I Stimulation
.
61)
X0 * PMA/I Stimulation
40
0(X)
120)
Inhibition by Class I1 MoAb M5/114
E[1
T/APC PMA/I
.
21)
0
20
40 80 60 % PMA/I Stimulation
100
120
FIG. 2. Roles of APCs and class II MHC molecules in the mitogenic activities of SPEs A and C. Purified T cells and adherent APCs from C57BL10/SnJ mice were incubated either separately or together in the presence or absence of toxin (50 ,ug/ml) or PMA-I. Proliferation was measured by uptake of [3H]thymidine by the dividing cells. The values are reported as percent stimulation standard deviation, with PMA-I set at 100%. The results reported are representative of four independent experiments. MoAb, Monoclonal antibody.
SPEs B and C may also preferentially stimulate V,B-containing T-cell subsets, since they are mitogens as potent as SPE A for C57BL10/SnJ lymphocytes. However, the identities of the VP subpopulations stimulated could not be determined in these experiments, since after activation by SPE B or C, V,Bs 3, 6, 8, and 11 were all reduced in stimulated populations. It is interesting that SPE A, which is structurally similar to the SEs (35), shares the SEs' ability to stimulate VP 8-containing T cells (27, 45), whereas SPEs B and C, which may be structurally distinct, do not. To investigate the role of APCs in the stimulation of T cells, populations greatly enriched for T cells or APCs were obtained and incubated either together or separately in the presence of toxin. T-cell populations were depleted of macrophages and B cells by a series of three treatments. A majority of the adherent APCs were removed from splenocytes from C57BL10/SnJ mice by plating the cells on plastic for 1 h at 37°C with 7% CO2 and then passing the nonadherent cells over a Sephadex G-10 (Sigma) column (24, 32). B cells and any remaining adherent cells were removed by passage over a T-cell column containing antibodies to B cells according to the manufacturer's specifications (Beckman Instruments, Inc., Fullerton, Calif.). The T-cell populations were then labeled with anti-CD3 monoclonal antibodies followed by fluorescein isothiocyanate-conjugated goat antirat immunoglobulin G and found to be greater than 98% by FACS analysis with a Becton Dickinson FACS IV. T-celldepleted adherent APCs were prepared by plating splenocytes from C57BL10/SnJ mice on plastic for 1 h at 37°C with 7% CO2. The nonadherent cells were removed by washing, and the adherent cells were recovered by scraping. SPEs A and C were chosen as representative toxins for these studies. The cells and toxins were incubated in various combinations, and proliferation was determined as described above. Neither purified T cells nor purified APCs were stimulated
strongly with either SPE A or SPE C (Fig. 2A and B, respectively). However, when T cells were combined with APCs, the proliferative response was restored (Fig. 2C), indicating that the mitogenic response depended on the presence of APCs. Previously, it was reported that SPEinduced mitogenicity was independent of macrophages (35, 38). To determine whether the small amount of proliferation seen in the purified T cells was due to a small number of contaminating APCs or whether it was due to mitogenic activity independent of APCs, the anti-class II MHC monoclonal antibody M5/114 was added to the SPE A- and SPE C-induced cells. If the residual activity was independent of APCs, the addition of M5/114 should not interfere with the residual mitogenic response since murine T cells do not express class II MHC molecules. Monoclonal antibody M5/114, specific for only the class II MHC molecule expressed on C57BL10/SnJ cells, blocked all proliferation (Fig. 2D), suggesting that not only APCs but also class II MHC interactions were needed for SPEs to induce T-cell mitogenesis. The antibody itself was not toxic to the cells, since it did not interfere with PMA-I stimulation. Additionally, the blocking appeared to be specific for class II since MKS4 monoclonal antibody, specific for I-AS molecules, did not interfere with presentation of SPE A or C to C57BL10/SnJ mice (H-2k) (data not shown). SPEs A and C stimulated both CD4- and CD8-positive T cells (data not shown), so it is interesting that the class II MHC monoclonal antibodies blocked all proliferation. This could indicate one of two mechanisms for blocking proliferation of CD8-positive T cells. Either (i) CD8-positive-cell proliferation is dependent on help from the CD4-positive cells or (ii) the CD8 cells are of a subset that recognizes class II MHC molecules. Overall, this report shows that SPEs A, B, and C stimulate T-cell proliferation by a mechanism similar to those of the SEs and TSST-1. Mitogenicity is V,B specific and requires
VOL. 59, 1991
APCs and class II MHC molecules. These results are not surprising, since the streptococcal toxins are part of the larger family of pyrogenic toxins which are biochemically and biologically related and which are able to produce similar diseases. This research was supported by Public Health Service grant HL36611 from the National Heart, Lung, and Blood Institute to P.M.S. and by Public Health Service grants AI27998 and A128635 from the Institute of Allergy and Infectious Diseases to M.K.J. M.K.J. is the recipient of a Pew Scholar Award. B.A.B.L. was supported by training grant 5T32CA09138 from the National Cancer Institute of the National Institutes of Health. REFERENCES 1. Barsunmian, E. L., P. M. Schlievert, and D. W. Watson. 1978. Nonspecific and specific immunological mitogenicity by group A streptococcal pyrogenic exotoxins. Infect. Immun. 22:681688. 2. Bergdoll, M. S., B. A. Crass, R. F. Reiser, R. N. Robbins, and J. P. Davis. 1981. A new staphylococcal enterotoxin, enterotoxin F, associated with toxic shock syndrome. Lancet i:10171021. 3. Bill, J., 0. Kanagawa, B. Woodland, and E. Palmer. 1989. The MHC molecule I-E is necessary but not sufficient for the clonal deletion of Vpll-bearing T cells. J. Exp. Med. 169:1405-1419. 4. Blomster-Hautamaa, D. A., B. N. Kreiswirth, J. S. Kornblum, R. P. Novick, and P. M. Schlievert. 1986. The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1. J. Biol. Chem. 261:15783-15786. 5. Blomster-Hautamaa, D. A., and P. M. Schlievert. 1988. Nonenterotoxic staphylococcal toxins, p. 297-330. In M. C. Hardegree and A. T. Tu (ed.), Handbook of natural toxins, vol. 4. Bacterial toxins. Marcel Dekker, Inc., New York. 6. Carlsson, R., H. Fischer, and H. 0. Sjogren. 1988. Binding of staphylococcal enterotoxin A to accessory cells is a requirement for its ability to activate human T cells. J. Immunol. 140:24842488. 7. Chatila, T., N. Wood, J. Parsonnet, and R. S. Geha. 1988. Toxic shock syndrome toxin 1 induces inositol phospholipid turnover, protein kinase C translocation, and calcium mobilization in human T cells. J. Immunol. 140:1250-1255. 8. Cone, L. A., D. R. Woodard, P. M. Schlievert, and G. S. Tomory. 1987. Clinical and bacteriologic observations of a toxic shock-like syndrome due to Streptococcus pyogenes. N. Engl. J. Med. 317:146-149. 9. de Azavedo, J. C. S., and J. P. Arbuthnott. 1984. Toxicity of staphylococcal toxic shock syndrome toxin 1 in rabbits. Infect. Immun. 46:314-317. 10. de Azavedo, J. C. S., T. J. Foster, P. J. Hartigan, J. P. Arbuthnott, M. O'Reilly, B. N. Kreiswirth, and R. P. Novick. 1985. Expression of the cloned toxic shock syndrome toxin 1 gene (tst) in vivo with a rabbit uterine model. Infect. Immun. 50:304-309. 11. Fast, D. J., P. M. Schlievert, and R. D. Nelson. 1988. Nonpurulent response to toxic shock syndrome toxin 1 producing Staphylococcus aureus: relationship to toxin-stimulated production of tumor necrosis factor. J. Immunol. 140:949-953. 12. Fast, D. J., P. M. Schlievert, and R. D. Nelson. 1989. Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production. Infect. Immun. 57:291-294. 13. Fleisher, B. 1989. Bacterial toxins as probes for the T cell antigen receptor. Immunol. Today 10:262-264. 14. Fleisher, B., and H. Schrezenmeier. 1988. T cell stimulation by staphylococcal enterotoxins. Clonally variable response and requirement for major histocompatibility complex class II molecules on accessory or target cells. J. Exp. Med. 167:1697-1707. 15. Fraser, J. D. 1989. High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature (London) 339:221223. 16. Hauser, A. R., and P. M. Schlievert. 1990. Nucleotide sequence
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