Vol. 58, No. 11
INFECTION AND IMMUNITY, Nov. 1990, p. 3711-3716
0019-9567/90/113711-06$02.00/0 Copyright C) 1990, American Society for Microbiology
Activation of Cholera Toxin-Specific T Cells In Vitro CHARLES
0.
ELSONl*
AND
SCOTT SOLOMON2
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294,' and Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 232982 Received 23 April 1990/Accepted 21 August 1990
Cholera toxin (CT) and its B subunit (CT-B) are potent oral immunogens in vivo, although both strongly inhibit polyclonal lymphocyte activation in vitro. In order to help understand this paradox, we have studied the activation and proliferation of CT-specific T cells in vitro, by using CT-B-primed lymph node T cells as responders, concanavalin A-stimulated peritoneal macrophages as antigen-presenting cells (APCs), and various forms of CT-B as antigen. The results indicate that in many ways CT-specific T cells respond in a manner similar to that of T cells specific for other protein antigens: the degree of proliferation was proportional to the dose of antigen and APCs in the cultures, was antigen specific, and was H-2 restricted. APCs from genetic high-responder strains to CT stimulated significantly more proliferation in F1 (high x low) responder T cells than did APCs from low responder strains. However, there was a marked difference in the activation of CT-specffic T cells when different forms of CT-B were used. Native CT-B stimulated little or no T-cell proliferation, whereas denatured CT-B or CT-B blocked by its ligand, GM1 ganglioside, stimulated T cells well. Addition of native CT-B to cocultures of primed T cells, APCs, and these latter stimulatory forms of CT-B inhibited the specific proliferative response to CT-B to varying degrees, depending on the ratio of the two forms in culture. We conclude that the ability of CT-B to inhibit T cells extends even to T cells specific for CT itself. Because of these inhibitory properties, processing of CT to nonbinding molecular forms or fragments must be an important prerequisite for the immune response to CT to occur in vivo, and such processing is likely to be important in the immune response to a variety of other enterotoxins as well. Cholera toxin (CT) is an 85-kDa protein composed of an A and B subunit (13, 37). The A subunit is a 27-kDa protein that catalyzes the ADP ribosylation of a guanine nucleotidebinding protein, Gs, which activates adenylyl cyclase. The resulting intracellular increase of the second messenger cyclic AMP results in a variety of biological effects, depending on the cell involved (30). The B subunit protein (CT-B), also called choleragenoid, is a 58-kDa homopentamer that binds to GM1 ganglioside on the cell surface, enabling the A subunit to come into contact with the cell (16). CT-B consists of five identical peptides of 11.6 kDa, noncovalently arranged in a pentameric ring (35). The primary amino acid structure of the B subunit has been determined (20, 21). CT is the most potent nonreplicating oral immunogen known. Microgram amounts delivered into the intestine of rodents result in both mucosal and systemic immunization (7, 9). In contrast, oral administration of other protein antigens such as keyhole limpet hemocyanin, a good parenteral immunogen, fails to produce intestinal and systemic antibodies and instead induces a state of partial tolerance (6). The mucosal immune response to CT is T cell dependent (25) and genetically restricted in the mouse by the I-A subregion of the H-2 major histocompatibility complex (MHC) (8) and shows extended memory (27). CT can alter the immune response to unrelated protein antigens when both are delivered to the gut simultaneously (6, 26). The mechanism of these remarkable properties of CT remains unclear. The ability to activate cyclic AMP is one possibility but cannot be the entire explanation, in that the B subunit shares most of the same properties with the holotoxin. These in vivo effects of CT and its B subunit contrast strikingly with their effects on lymphocyte activation in vitro where both CT and CT-B have proven to be potent inhibitors *
of T-cell proliferation (15, 38), inhibiting both polyclonal responses to mitogens and antigen-specific T-cell responses to keyhole limpet hemocyanin (38). T cells seem to play a central role in the immunogenicity (25) and adjuvanticity of CT in vivo, but little is known about the properties of CT-specific T cells. This prompted the present study, which focused on the T-cell response to the nontoxic B subunit. The working hypothesis for this set of experiments, one which could reconcile the conflicting in vivo and in vitro effects of these molecules, was that CT-specific T cells would be refractory to inhibition by CT-B. We found that CT-specific T-cell activation was similar in many ways to that seen with other, more conventional protein antigens. However, the molecular form of CT-B used in the cultures was crucial because, contrary to the hypothesis, native CT-B did inhibit T cells specific for itself in vitro. MATERIALS AND METHODS Animals. Mice of the C57BL/6, C3H/He, B6C3Fj, B10, B10.T, B10.BR, and B10.D2 strains were obtained from the Jackson Laboratories, Bar Harbor, Maine. B10.S mice were a gift of Donald Shreffler at Washington University, St. Louis, Mo. Materials. CT-B used in the experiments was a gift of Dr. J. Armand of the Institut Merieux, Marcy l'Etoile, France, or was purchased from List Biological Laboratories, Inc., Campbell, Calif. Urea, Tris, dithiothreitiol, 2-mercaptoethanol, EDTA, thioglycolate, carbonyl iron, paraformaldehyde, sodium pyruvate, GM1 ganglioside, and iodoacetic acid were obtained from Sigma Chemical Co., St. Louis, Mo. L-Glutamine and penicillin-streptomycin were obtained from GIBCO Laboratories, Grand Island, N.Y. Concanavalin A was obtained from Boehringer Mannheim Corp., Indianapolis, Ind. Incomplete and complete Freund adjuvants were obtained from Difco Laboratories, Detroit, Mich.
Corresponding author. 3711
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HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) was obtained from United States Biochemical, Cleveland, Ohio. Goat anti-mouse immunoglobulin G (IgG) antibody was obtained from TAGO, Inc., Burlingame, Calif. Tritiated thymidine was obtained from ICN Radiochemicals, Irvine, Calif. Magnetic goat anti-mouse IgG was purchased from Advanced Magnetics, Inc., Bedford, Mass. Culture media. RPMI 1640 and Hanks balanced salt solution without Ca2' and Mg2+ were obtained from GIBCO Laboratories. Fetal calf serum was obtained from M. A. Bioproducts, Walkersville, Md. Complete media for cell culture consisted of RPMI 1640 containing 10% fetal calf serum, 2 mM L-glutamine, 25 mM HEPES, 0.05 mM 2-mercaptoethanol, 2 mM sodium pyruvate, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml. Hanks balanced salt solution media for harvesting peritoneal macrophages consisted of Hanks balanced salt solution containing 10 mM HEPES, 2 mM sodium pyruvate, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml. Modifications of CT-B. (i) Denatured CT-B. Denatured CT-B was prepared by reduction and S-carboxymethylation in a modification of previously described methods (5, 12). Briefly, CT-B was dialyzed into 8 M urea-0.005 M EDTA0.1 M Tris (pH 8.2). To the dialyzed CT-B was added a 100 molar to mole cysteine (in CT-B) excess of dithiothreitol under a nitrogen barrier. The reaction was allowed to proceed 24 h at room temperature. Subsequently, a 2.5 M excess of iodoacetic acid (to moles of dithiothreitiol) was added under a nitrogen barrier and sealed in the dark. After this reaction was allowed to proceed 24 h at room temperature, the solution was dialyzed sequentially against 4, 2, and 1 M urea and phosphate-buffered saline. Then it was incubated with paraformaldehyde-fixed murine T-cell lymphoma cells at 4°C for 24 h on a tilter in order to remove any remaining native CT-B from the preparation. Protein concentration was measured via BCA protein assay (Pierce Chemical Co., Rockford, Ill.). (ii) GM1 ganglioside-blocked CT-B. GM1 ganglioside was diluted in phosphate-buffered saline and added to CT-B at a 5:1 molar ratio prior to addition to the lymphocyte cultures to block all the binding sites. In some experiments, other molar ratios were used to block only a fraction of the binding sites. Immunization. Mice were injected subcutaneously at the base of the tail and in the foot pads with CT-B in incomplete Freund adjuvant as a mixed emulsion containing 50 to 100 pug of antigen in a total volume of 100 to 200 ,ul (11). Cell isolations. Antigen-primed peripheral lymph node (PLN) T cells were prepared by a modification by Richman et al. (34) of the method of Corradin et al. (4). Eight to fourteen days after priming, the inguinal, periaortic, and popliteal lymph nodes were removed and placed into cell suspensions by straining through a small mesh strainer or by teasing apart between the ends of sterile frosted microscope slides (28) into complete media. After two washes, no more than 2 x 108 cells in 2 ml of warm (37°C) media containing 5% fetal calf serum was applied to a nylon wool column (600 mg of nylon wool in a 12-ml syringe) (18). After incubation at 37°C for at least 45 min, the cells were eluted from the column. Cells were reconstituted at 0.5 x 107 to 1 x 107 cells per ml in complete media containing 5% fetal calf serum and added to a flask containing magnetic goat anti-mouse IgG particles (2 ml of washed particles per 5 x 107 cells). After incubation of the flask at 37°C for 20 min with gentle mixing, the adherent cells were removed by magnetic separation. The nonadherent cells were placed in another flask, and
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magnetic separation was carried out again in order to ensure complete removal of the particles. The nonadherent cells were reconstituted at 4 x 106 to 10 x 106 cells per ml in complete media for use in cell culture. In some experiments, the nylon wool-passed PLN T cells were instead reconstituted at 107 cells per ml in complete media along with 200 mg of carbonyl iron particles and incubated at 37°C for 30 min. Phagocytic cells were removed with a magnet. The nonphagocytic cells were reconstituted at 1.5 x 106 cells per ml in complete media and incubated overnight at 37°C in 5% CO2 and humidified air in wells precoated with S ,ug of goat anti-mouse IgG antibody per ml to remove any B cells or any remaining adherent macrophages. The nonadherent cells were reconstituted at 4 x 106 to 10 x 106 cells per ml in complete media for use in cell culture. Peritoneal macrophages for use as antigen-presenting cells (APCs) were prepared as previously described (33). Animals were injected intraperitoneally with 1.5 ml of 40 ,ug/ml concanavalin A. Four to five days later, the mice were sacrificed and their peritoneal cavities were lavaged with Hanks balanced salt solution media. The harvested cells were further purified by glass adherence, then washed twice in complete media, and adjusted to 5 x 105 cells per ml for addition to culture. Assay of mitogen-induced proliferation of T cells. Triplicate cultures were performed with or without CT-B, denatured CT-B, and concanavalin A in wells of a 96-well Costar tissue culture plate. Spleen cells or PLN T cells isolated as described above were incubated at 37°C for 2 days in 5% CO2 humidified air. After 2 days of incubation, 0.5 mCi of [3H]thymidine was added, and after 5 additional h at 37°C, the cells were harvested in an automated collecting device (PHD cell harvester; Cambridge Technology, Inc., Watertown, Mass.). Proliferation was assessed as the amount of incorporation of [3H]thymidine into cell DNA, as measured by beta scintillation counting (Beckman Instruments, Palo Alto, Calif.) of the harvested samples. Assay of antigen-specific proliferation of T cells. PLN T cells (2 x 105 to 5 x 105) and macrophages (2.5 x 104), prepared as described above, were cultured in a final volume of 200 ml in flat-bottomed wells of a 96-well tissue culture plate. Triplicate cultures were performed with and without the addition of different forms of CT-B. The plate was incubated at 37°C for 4 days, at which time 0.5 mCi of [3H]thymidine was added, and after an additional 18 h in culture, the cells were harvested as described above. Proliferation was estimated by measurement of [3H]thymidine incorporation into T-cell DNA. RESULTS Response to different molecular forms of CT-B. When CT-B-primed purified lymph node T cells were cultured in vitro with APCs and native CT-B, little or no proliferative response was observed in repeated experiments over a wide dose range (Table 1). Because T cells recognize the primary structure of protein antigens and because of previous data indicating that CT-B was able to inhibit polyclonal T-cell activation (38), CT-B was denatured by reduction and alkylation and then absorbed on paraformaldehyde-fixed tumor cells to remove any remaining native CT-B. The same T cells that failed to respond to the native form of CT-B now had a brisk response to the denatured form of CT-B (Table 1). In independent experiments, these two molecular forms of CT-B were tested for their ability to inhibit polyclonal T-cell
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TABLE 1. Proliferative response of primed PLN T cells to denatured versus native CT-B
[3H]TdR incorporation' (mean cpm + SD)
CT-B dose (,ug/ml)
Denatured CT-B
0 1 3 10 30
4,485 + 1,617 10,850 ± 1,489 11,206 ± 2,874 35,906 ± 12,949 50,326 ± 8,363
a
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Native CT-B
± ± ± ± 11,096 ±
4,485 2,654 4,016 7,013
1,617 325 754 967 1,394
10,000
TdR, Thymidine.
0 0
responses; native CT-B was able to inhibit concanavalin A-stimulated polyclonal T-cell responses, whereas the denatured form was not (data not shown), which is consistent with observations of others that S-carboxymethylated CT is unable to bind to GM1 ganglioside (23). CT-B-specific T cells could also be stimulated if the CT-B was preincubated with its specific ligand, GM1 ganglioside, to block its binding sites or if APCs were first pulsed with native CT-B and washed prior to being added to primed T-cell cultures (Fig. 1). Interestingly, there was little difference in the dose-response between the CT-B-pulsed APCs and the GM1-blocked CT-B added to culture, despite the ability of CT-B to actively bind to the macrophage surface in the latter situation but not the former. Effect of antigen and APC dose on T-cell responses. In these experiments, primed T cells were exposed to various doses of antigen and APCs. The T-cell response to CT-B increased as increasing numbers of APCs were added at any given dose of antigen, and the T-cell response also increased as the antigen dose increased at any given number of APCs (Fig. 2). CT-B primed T cells did not proliferate when irrelevant antigens such as ovalbumin or hen egg lysozyme were added to the cultures (data not shown). Genetic restriction of T-cell response to CT-B. The question of genetic restriction of T-cell response to CT-B was examined from two directions. In the first approach, B10 mice were immunized with CT-B, and the draining PLN T cells were isolated. These were cocultured with APCs from a variety of H-2 congenic strains. The H-2-compatible APCs
1 40 20 5 10 Macrophages added (x 1000)
FIG. 2. The effect of antigen and APC dose on the in vitro T-cell response to CT-B. CT-B-primed T cells at 4 x 105 cells per well were cultured with the numbers of APCs shown plus GM1 ganglioside-blocked CT-B at the following concentrations: 100 (G), 30 (0), 10 (O), and 0 (A) ,ug/ml.
from the B10 strain were able to present CT-B to T cells from the B10 strain, whereas APCs from H-2 disparate B10 congenic strains were not (Fig. 3). The second approach was based on previous observations that the in vivo immune response to CT varies among the strains, with variation determined by the I-A subregion of the H-2 complex. In these experiments, C3B6F1 mice were immunized with CT-B in vivo and the draining PLN T cells were cultured in vitro in the presence of antigen plus either high-responder (C3B6F1, C57BL/6) or low-responder (C3H/HeJ) APCs. These results paralleled the results seen with in vivo immunizations in that the F1 T cells responded poorly to antigen presented by APCs from the C3H/HeJ strain but responded well to antigen presented by APCs from the high-responder C3B6F1 and C57BL/6 strains (Fig. 4). Native CT-B inhibits CT-B-specific T-cell activation. One explanation for the poor response of the primed T cells to native CT-B was that the native CT-B was able to bind directly to the T cells and thus inhibit them in a similar 12 10
x
0
6
2
.1
.01
.1
1
10 Dose
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FIG. 1. Proliferative response of primed T cells to different forms of CT-B. CT-B-primed PLN T cells at 4 x 105 cells per well were cultured with 2.5 x 104 APCs plus different forms of CT-B at the doses shown. Symbols: *, native CT-B was added directly to culture; A, native CT-B was preincubated with five times an equimolar concentration of GM1 ganglioside prior to addition to culture; 0, APCs were adhered for 1 h, pulsed for 2 h with native CT-B at the doses shown, and washed before T cells were added to the wells. The background proliferation in control cultures receiving no added antigen was 5,728 cpm.
I
1
I100
CT-B ()/ml)
FIG. 3. Response of CT-B-primed T cells from B10 mice to CT-B presented by APCs from the B10 strain and from various H-2 congenic B10 strains. CT-B-primed B10 T cells at 4 x 105 cells per well were cultured with 2 x 105 APCs from B10 and H-2 congenic B10 strains at multiple doses of GM1 ganglioside-blocked CT-B. Symbols: O, B10 (H-2b); *, B10.S (H-2s); 0, B10.T (6R) (H-2q); A, B10.BR (H-2k; *, B10.BR (H-2). The data shown represent the stimulation index derived by dividing the counts per minute at any given dose of antigen by the counts per minute of cultures receiving no antigen.
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Gm 1 ganglioside Native blocked-CT-B CT-B E 0)
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100
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60
_ 0
50
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LL
0 10 20 30 40 50 0 0
90 80
F
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.1
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_
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FIG. 4. Effect of H-2 restriction on CT-B-specific T-cell proliferation. A total of 2 x 105 CT-B-primed B6C3F, PLN T cells were cultured for 5 days in the presence of denatured CT-B in the doses shown and with 2 x 104 APCs from either high- or low-responder strains. Symbols: A, B6C3F1 (H-2blk); 0, C57BL/6 (H_2b); *, C3H/He (H-2k).
fashion to its inhibition of polyclonal T-cell responses. This possibility was directly tested by adding native CT-B back to cultures of primed T cells, APCs, and immunogenic forms of CT-B. Similar results were obtained whether the immunogenic form of CT-B was denatured CT-B (data not shown) or CT-B blocked by GM1 ganglioside (Fig. 5). In both instances, the addition of native CT-B back to these cocultures inhibited the activation of CT-B-specific T cells in vitro. This inhibition was explored further by two types of experiments. In the first, various ratios of GM1-blocked CT-B to native CT-B were added to primed T cells plus APCs, holding the total amount of antigen in the system constant (Fig. 6). Cultures receiving more than 20% of total antigen as native CT-B were significantly inhibited. In a second experiment, the same point was addressed by varying the amount of blocking of the native CT-B by preincubating the CT-B with various molar ratios of GM1 ganglioside. Because CT-B is a pentamer, a 5:1 molar ratio of GM1 ganglioside to CT-B blocks all of the binding sites and results in stimulation of the T cells (Fig. 7). A decrease of the molar ratio from 5:1 to 4:1, i.e., a reduction of 20% in the receptor blocking, resulted in a significant inhibition of CT-B-specific
10,000 20,000 30,000 40,000 Mean CPM ± SD
FIG. 6. Effect of different ratios of GM1-blocked to native CT-B on the proliferation of CT-B-primed T cells. A total of 4 x 105 CT-B-primed T cells plus 2.5 x 105 APCs were cocultured with different amounts of GM1 ganglioside-blocked CT-B and native CT-B, with the total amount of CT-B added to the cultures constant at 100 p.g/ml. For comparison, the degrees of T-cell proliferation with 50 ,ug of GM1 ganglioside-blocked CT-B added per ml and with no antigen added are shown.
T-cell activation, an inhibition which slowly increased further as the molar ratio was further reduced. DISCUSSION CT is a potent stimulator of secretory IgA responses when delivered into the intestine and thus has been an important probe for our understanding of the mucosal immune system and of the interaction of pathogenic toxins with host defenses. The intestinal immunogenicity of CT has been taken advantage of in the form of an effective oral vaccine against cholera (14, 17). As with most protein antigens, the immune response to cholera toxin is T cell dependent (25); however, little is known about the activation, properties, or cellular immunology of CT-specific T cells. This study represents a first attempt to define the T-cell response to CT. Much of our understanding of how T cells are triggered by antigens comes from studies on secondary activation of T
20,000 70,000 60,000 50,000
oa- 10,000-
T
-
40,000a-E 30,00020,000-
10,000 .1
1
10
100
Dose (mg/ml) FIG. 5. Addition of native CT-B to cultures of CT-B-primed T cells, APCs, and a stimulatory form of CT-B. A total of 4 x 105 CT-B primed PLN T cells were cultured with 2.5 x 105 APCs plus various doses of CT-B blocked by GM1 ganglioside; duplicate cultures received in addition native CT-B at 3 j±g/ml. Symbols: Cl, cultures contained only CT-B blocked by GM1 ganglioside at doses shown; *, cultures contained CT-B blocked by GM1 ganglioside plus native CT-B.
0
5 4 3 2 1 Gm 1 ganglioside: CT-B molar ratio
FIG. 7. CT-B-specific T-cell proliferation in the presence of CT-B blocked by various amounts of GM1 ganglioside. A total of 4 X 105 CT-B-primed T cells were cultured with 2.5 x 104 APCs, plus either 100 (_) or 30 (=) ,g of CT-B per ml, as shown. Prior to culture, the CT-B was preincubated with 5 x, 4 x, 3 x, 2 x, 1 x, or 0 equimolar concentrations of GM1 ganglioside in order to block all, part, or none of its GM1 binding sites.
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cells in vitro after priming in vivo by using a system developed by Corradin et al. (4). Such studies have shown that protein antigens are taken up and processed by APCs in such a way that denatured peptides and peptide fragments are expressed on the cell surface in association with class II MHC molecules (36). The peptide fragments appear to lie in a cleft of the class II MHC molecules (3), and the T-cell receptor recognizes both the foreign peptide and adjacent facets of the self-class II MHC molecule (1). In many ways, the T-cell response to CT-B in the present study was similar to that which has been established previously for other protein antigens in this system. The T-cell proliferative responses to CT-B were antigen specific and proportional to the numbers of APCs and the doses of antigen in the cultures. The importance of class II MHC molecules for the CT-B-specific T-cell response was demonstrated by a lack of antigen presentation by APCs from H-2 incompatible congenic inbred strains and by the relative inability of APCs from a mouse strain harboring a low-responder Ia gene (H-2k) to stimulate CT-B-specific F1 (high x low responder) T cells. In other aspects, however, the response to CT-B was quite distinctive. The molecular form of CT-B used in the cultures was critical. In particular, native CT-B appeared not to stimulate T-cell activation, whereas denatured CT-B or CT-B blocked with its specific ligand, GM1 ganglioside, stimulated it very well. Subsequent experiments showed that the apparent lack of stimulation with native CT-B was due to an active inhibition of CT-B-specific T-cell proliferation. In previous work, we have shown that native CT-B inhibits T-cell activation induced by polyclonal activators or by specific, but unrelated, antigens (38). The molecular mechanism of this inhibition is not yet known, but it does require binding of CT-B to the T-cell surface (38). Consistent with these previous observations, forms of CT-B that were unable to bind to T cells were stimulatory in vitro, whereas the native form was inhibitory. Moreover, when both forms were present in culture, the ratio of the native to the stimulatory form was important in determining whether and to what degree T-cell activation occurred. Concentrations of native CT-B equal to or greater than 1 ,ug/ml were inhibitory in vitro, an amount likely to be physiologically relevant in vivo. CT itself is a much more potent inhibitor than is CT-B, with inhibition being seen with nanogram concentrations in vitro and effects on T cells in vivo being expected to be that much more profound. It is interesting to consider the results of this study in the context of the events that must take place in vivo in the intestine for a response for CT to occur. Certainly the intestinal lumen is a hostile environment for any antigen, considering digestion by enzymes, denaturation by bile, dilution by secretions, and a limited duration of exposure due to intestinal motility, trapping by the mucus coat, etc. The present study illustrates that many of these nonspecific defenses would not necessarily limit sensitization of T cells in that denatured CT-B, in essence a long peptide, was an effective immunogen for CT-B-specific T cells. Degradation of CT in the lumen may reduce its ability to bind, however, and the ability of CT and CT-B 'to bind to GM1 ganglioside on epithelial cells has been thought to be very important in its intestinal immunogenicity, perhaps by sequestering it and prolonging its time in the intestine. Indeed, there is some data indicating that intestinal epithelial cells not only express class II MHC antigens but also can present antigens in vitro (2), although it is unproven whether they function as APCs in vivo. The ability of the toxin to bind to GM1 gangliosides
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present on the M cells of the follicle-associated epithelium is likely a very important feature of its immunogenicity, because M cells represent a critical portal of entry for enteric antigens, including CT (32), into gut lymphoid follicles. CT does not appear to interfere with the ability of APCs to take up antigen, process it, or present it in an immunogenic form recognizable by T cells. For example, in the present study, APCs pulsed with CT-B were effective in activating CT-B-primed T cells. In previous studies, neither CT nor CT-B interfered with APC uptake, processing, or presentation of keyhole limpet hemocyanin antigen to primed T cells (38). CT has been reported to increase interleukin-1 secretion by APCs (24) and so may enhance this aspect of immune induction. In the present study, APCs were also effective in taking up GM1 ganglioside-blocked CT-B and presenting that form to primed T cells. Interestingly, in experiments in which APCs were pulsed with either native CT-B or GM1 ganglioside-blocked CT-B, equivalent T-cell responses were found. This is curious because of evidence that the ability of an antigen to specifically bind to APCs, especially antigenspecific B cells (22), greatly enhances the efficiency of antigen presentation. From these results, the form of antigen reaching APCs in gut lymphoid follicles is not likely to be a limiting factor in the induction of the immune response to CT. The types of T-cell subsets being stimulated in this system in vitro or in gut-associated lymphoid tissue (GALT) in vivo are not yet known. Murine CD4 helper T cells have been subdivided into Thl and Th2 subtypes on the basis of the pattern of lymphokines they secrete (29). Th2 cells produce interleukin 4 and interleukin 5, which are important in generating IgA responses (10, 31). Whether one or the other of these subtypes is preferentially stimulated by CT-B is, at present, unknown; there are no surface markers by which these two subsets can be distinguished. However, previous work in vivo has shown that CT stimulates not only the secretory IgA response, but also a fairly striking plasma IgG response (7), a Thl function. These and other results (6, 7, 8) would suggest that there may be no preferential stimulation of either the Thl or the Th2 helper cell subtypes by CT. However, the present results do indicate that the ratio of the native to the stimulatory form of CT and CT-B is likely to be a very important determinant of the extent of T-cell activation in GALT. The approach used here is relevant to the study of the mucosal immune response to other bacterial toxins. For example, shiga toxin has recently been reported to stimulate a strong secretory IgA response when administered into the intestines of mice (19). Shiga toxin is a true cellular poison, and one can predict it will be extremely toxic in vitro, which will make its study difficult. However, the approaches used here should allow a detailed exploration of antigen uptake, processing, and presentation and of the T-cell response to this as well as to other pathogenic toxins whose cellular immunology is largely unexplored. ACKNOWLEDGMENTS This work was supported by Public Health Service grant DK28623 from the National Institute of Diabetes and Digestive and Kidney Diseases. We wish to acknowledge the expert technical assistance of Hazel Bowden. LITERATURE CITED 1. Allen, P. M., G. R. Matsueda, R. J. Evans, J. B. Dunbar, G. R. Marshall, and E. R. Unanue. 1987. Identification of the T-cell
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18. 19.
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and Ia contact residues of a T-cell antigenic epitope. Nature (London) 327:713-715. Bland, P. W., and L. G. Warren. 1986. Antigen presentation by epithelial cells of the rat small intestine. I. Kinetics, antigen specificity and blocking by anti-Ia antisera. Immunology 58:14. Brown, J. H., T. Jardetzky, M. A. Saper, B. Samraoui, P. J. Bjorkman, and D. C. Wiley. 1988. A hypothetical model of the foreign antigen binding site of class II histocompatibility antigens. Nature (London) 332:845-850. Corradin, G., H. M. Etlinger, and J. M. Chiller. 1977. Lymphocyte specificity to protein antigens. I. Characterization of the antigen-induced in vitro T cell-dependent proliferative response with lymph node cells from primed mice. J. Immunol. 119:10481053. Crestfield, A. M., S. Moore, and W. H. Stein. 1963. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. Biol. Chem. 238:622-627. Elson, C. O., and W. Ealding. 1984. Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen. J. Immunol. 133:2892-2897. Elson, C. O., and W. Ealding. 1984. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J. Immunol. 132:2736-2742. Elson, C. O., and W. Ealding. 1987. Ir gene control of the murine secretory IgA response to cholera toxin. Eur. J. Immunol. 17:425-428. Fujita, K., and R. A. Finkelstein. 1972. Antitoxic immunity in experimental cholera: comparison of immunity induced perorally and parenterally in mice. J. Infect. Dis. 125:647-655. Harriman, G. R., D. Y. Kunimoto, J. F. Elliott, V. Paetkau, and W. Strober. 1988. The role of IL-5 in IgA B cell differentiation. J. Immunol. 140:3033-3039. Herbert, W. J. 1965. Multiple emulsions. A new form of mineral-oil antigen adjuvant. Lancet ii:771. Hirs, C. H. W. 1967. Reduction and S-carboxymethylation of proteins. Methods Enzymol. 11:199-203. Holmgren, J. 1981. Actions of cholera toxin and treatment of cholera. Nature (London) 292:413-417. Holmgren, J., J. Clemens, D. A. Sack, and A. M. Svennerholm. 1989. New cholera vaccines. Vaccine 7:94-96. Holmgren, J., L. Lindholm, and I. Lonnroth. 1974. Interaction of cholera toxin and toxin derivatives with lymphocytes. I. Binding properties and interference with lectin-induced cellular stimulation. J. Exp. Med. 139:801-819. Holmgren, J., I. Lonnroth, and L. Svennerholm. 1973. Tissue receptor for cholera enterotoxin: postulated structure from studies with GM1 ganglioside and related glycolipids. Infect. Immun. 8:208-214. Holmgren, J., A. M. Svennerholm, J. Clemens, D. Sack, R. Black, and M. Levine. 1987. An oral B subunit-whole cell vaccine against cholera: from concept to successful field trial. Adv. Exp. Med. Biol. 216B:1649-1660. Julius, M. H., E. Simpson, and L. A. Herzenberg. 1973. A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3:645-649. Keren, D. F., J. E. Brown, R. A. McDonald, and J. S. Wassef. 1989. Secretory immunoglobulin A response to shiga toxin in rabbits: kinetics of the initial mucosal immune response and inhibition of toxicity in vitro and vivo. Infect. Immun. 57:18851889.
INFECT. IMMUN. 20. Kurosky, A., D. E. Markel, and J. W. Peterson. 1977. Covalent structure of the B chain of cholera enterotoxin. J. Biol. Chem. 252:7257-7264. 21. Lai, C.-Y. 1977. Determination of the primary structure of cholera toxin B subunit. J. Biol. Chem. 252:7249-7256. 22. Lanzavecchia, A. 1985. Antigen-specific interaction between T and B cells. Nature (London) 314:537-539. 23. Lonnroth, I., and J. Holmgren. 1975. Protein reagent modification of cholera toxin: characterization of effects on antigenic, receptor-binding, and toxic properties. J. Gen. Microbiol. 91: 263-277. 24. Lycke, N., A. K. Bromander, L. Ekman, U. Karlsson, and J. Holmgren. 1989. Cellular basis of immunomodulation by cholera toxin in vitro with possible association to the adjuvant function in vivo. J. Immunol. 142:20-27. 25. Lycke, N., L. Erikson, and J. Holmgren. 1987. Protection against cholera toxin after oral immunization is thymus-dependent and associated with intestinal production of neutralizing IgA antitoxin. Scand. J. Immunol. 25:413-419. 26. Lycke, N., and J. Holmgren. 1986. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology 59:301-308. 27. Lycke, N., and J. Holmgren. 1987. Long-term cholera antitoxin memory in the gut can be triggered to antibody formation associated with protection within hours of an oral challenge immunization. Scand. J. Immunol. 25:407-412. 28. Mishell, B. B., and S. M. Shigii. 1980. Selected methods in cellular immunology. W. H. Freeman, San Francisco. 29. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman. 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2348-2357. 30. Moss, J., D. L. Burns, J. A. Hsia, E. L. Hewett, R. L. Guerrant, and M. Vaughn. 1984. Cyclic nucleotides: mediators of bacterial toxin action in disease. Ann. Intern. Med. 101:653-666. 31. Murray, P. D., D. T. McKenzie, S. L. Swain, and M. F. Kagnoff. 1987. Interleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression. J. Immunol. 139:2669-2674. 32. Neutra, M. R., T. L. Phillips, E. L. Mayer, and D. J. Fishkind. 1987. Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rabbit Peyer's patch. Cell Tissue Res. 247:537-546. 33. Phipps, R. P., and D. W. Scott. 1983. A novel role for macrophages: the ability of macrophages to tolerize B cells. J. Immunol. 131:2122-2127. 34. Richman, L. K., R. J. Klingenstein, J. A. Richman, W. Strober, and J. Berzofsky. 1979. The mouse Kupffer cell. I. Characterization of the cell-serving accessory function in antigen specific T cell proliferation. J. Immunol. 123:2602-2608. 35. Spangler, B. D., and E. M. Westbrook. 1989. Crystallization of isoelectrically homogeneous cholera toxin. Biochemistry 28: 1333-1340. 36. Unanue, E. R., and J. C. Cerottini. 1989. Antigen presentation. Fed. Am. Soc. Exp. Biol. J. 3:2496-2502. 37. Van Heynigan, S. 1982. Cholera toxin. Bioscience Reports 2:135-146. 38. Woogen, S. D., W. Ealding, and C. 0. Elson. 1987. Inhibition of murine lymphocyte proliferation by the B subunit of cholera toxin. J. Immunol. 139:3764-3770.
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0019-9567/90/113711-06$02.00/0 Copyright C) 1990, American Society for Microbiology
Activation of Cholera Toxin-Specific T Cells In Vitro CHARLES
0.
ELSONl*
AND
SCOTT SOLOMON2
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294,' and Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 232982 Received 23 April 1990/Accepted 21 August 1990
Cholera toxin (CT) and its B subunit (CT-B) are potent oral immunogens in vivo, although both strongly inhibit polyclonal lymphocyte activation in vitro. In order to help understand this paradox, we have studied the activation and proliferation of CT-specific T cells in vitro, by using CT-B-primed lymph node T cells as responders, concanavalin A-stimulated peritoneal macrophages as antigen-presenting cells (APCs), and various forms of CT-B as antigen. The results indicate that in many ways CT-specific T cells respond in a manner similar to that of T cells specific for other protein antigens: the degree of proliferation was proportional to the dose of antigen and APCs in the cultures, was antigen specific, and was H-2 restricted. APCs from genetic high-responder strains to CT stimulated significantly more proliferation in F1 (high x low) responder T cells than did APCs from low responder strains. However, there was a marked difference in the activation of CT-specffic T cells when different forms of CT-B were used. Native CT-B stimulated little or no T-cell proliferation, whereas denatured CT-B or CT-B blocked by its ligand, GM1 ganglioside, stimulated T cells well. Addition of native CT-B to cocultures of primed T cells, APCs, and these latter stimulatory forms of CT-B inhibited the specific proliferative response to CT-B to varying degrees, depending on the ratio of the two forms in culture. We conclude that the ability of CT-B to inhibit T cells extends even to T cells specific for CT itself. Because of these inhibitory properties, processing of CT to nonbinding molecular forms or fragments must be an important prerequisite for the immune response to CT to occur in vivo, and such processing is likely to be important in the immune response to a variety of other enterotoxins as well. Cholera toxin (CT) is an 85-kDa protein composed of an A and B subunit (13, 37). The A subunit is a 27-kDa protein that catalyzes the ADP ribosylation of a guanine nucleotidebinding protein, Gs, which activates adenylyl cyclase. The resulting intracellular increase of the second messenger cyclic AMP results in a variety of biological effects, depending on the cell involved (30). The B subunit protein (CT-B), also called choleragenoid, is a 58-kDa homopentamer that binds to GM1 ganglioside on the cell surface, enabling the A subunit to come into contact with the cell (16). CT-B consists of five identical peptides of 11.6 kDa, noncovalently arranged in a pentameric ring (35). The primary amino acid structure of the B subunit has been determined (20, 21). CT is the most potent nonreplicating oral immunogen known. Microgram amounts delivered into the intestine of rodents result in both mucosal and systemic immunization (7, 9). In contrast, oral administration of other protein antigens such as keyhole limpet hemocyanin, a good parenteral immunogen, fails to produce intestinal and systemic antibodies and instead induces a state of partial tolerance (6). The mucosal immune response to CT is T cell dependent (25) and genetically restricted in the mouse by the I-A subregion of the H-2 major histocompatibility complex (MHC) (8) and shows extended memory (27). CT can alter the immune response to unrelated protein antigens when both are delivered to the gut simultaneously (6, 26). The mechanism of these remarkable properties of CT remains unclear. The ability to activate cyclic AMP is one possibility but cannot be the entire explanation, in that the B subunit shares most of the same properties with the holotoxin. These in vivo effects of CT and its B subunit contrast strikingly with their effects on lymphocyte activation in vitro where both CT and CT-B have proven to be potent inhibitors *
of T-cell proliferation (15, 38), inhibiting both polyclonal responses to mitogens and antigen-specific T-cell responses to keyhole limpet hemocyanin (38). T cells seem to play a central role in the immunogenicity (25) and adjuvanticity of CT in vivo, but little is known about the properties of CT-specific T cells. This prompted the present study, which focused on the T-cell response to the nontoxic B subunit. The working hypothesis for this set of experiments, one which could reconcile the conflicting in vivo and in vitro effects of these molecules, was that CT-specific T cells would be refractory to inhibition by CT-B. We found that CT-specific T-cell activation was similar in many ways to that seen with other, more conventional protein antigens. However, the molecular form of CT-B used in the cultures was crucial because, contrary to the hypothesis, native CT-B did inhibit T cells specific for itself in vitro. MATERIALS AND METHODS Animals. Mice of the C57BL/6, C3H/He, B6C3Fj, B10, B10.T, B10.BR, and B10.D2 strains were obtained from the Jackson Laboratories, Bar Harbor, Maine. B10.S mice were a gift of Donald Shreffler at Washington University, St. Louis, Mo. Materials. CT-B used in the experiments was a gift of Dr. J. Armand of the Institut Merieux, Marcy l'Etoile, France, or was purchased from List Biological Laboratories, Inc., Campbell, Calif. Urea, Tris, dithiothreitiol, 2-mercaptoethanol, EDTA, thioglycolate, carbonyl iron, paraformaldehyde, sodium pyruvate, GM1 ganglioside, and iodoacetic acid were obtained from Sigma Chemical Co., St. Louis, Mo. L-Glutamine and penicillin-streptomycin were obtained from GIBCO Laboratories, Grand Island, N.Y. Concanavalin A was obtained from Boehringer Mannheim Corp., Indianapolis, Ind. Incomplete and complete Freund adjuvants were obtained from Difco Laboratories, Detroit, Mich.
Corresponding author. 3711
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HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) was obtained from United States Biochemical, Cleveland, Ohio. Goat anti-mouse immunoglobulin G (IgG) antibody was obtained from TAGO, Inc., Burlingame, Calif. Tritiated thymidine was obtained from ICN Radiochemicals, Irvine, Calif. Magnetic goat anti-mouse IgG was purchased from Advanced Magnetics, Inc., Bedford, Mass. Culture media. RPMI 1640 and Hanks balanced salt solution without Ca2' and Mg2+ were obtained from GIBCO Laboratories. Fetal calf serum was obtained from M. A. Bioproducts, Walkersville, Md. Complete media for cell culture consisted of RPMI 1640 containing 10% fetal calf serum, 2 mM L-glutamine, 25 mM HEPES, 0.05 mM 2-mercaptoethanol, 2 mM sodium pyruvate, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml. Hanks balanced salt solution media for harvesting peritoneal macrophages consisted of Hanks balanced salt solution containing 10 mM HEPES, 2 mM sodium pyruvate, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml. Modifications of CT-B. (i) Denatured CT-B. Denatured CT-B was prepared by reduction and S-carboxymethylation in a modification of previously described methods (5, 12). Briefly, CT-B was dialyzed into 8 M urea-0.005 M EDTA0.1 M Tris (pH 8.2). To the dialyzed CT-B was added a 100 molar to mole cysteine (in CT-B) excess of dithiothreitol under a nitrogen barrier. The reaction was allowed to proceed 24 h at room temperature. Subsequently, a 2.5 M excess of iodoacetic acid (to moles of dithiothreitiol) was added under a nitrogen barrier and sealed in the dark. After this reaction was allowed to proceed 24 h at room temperature, the solution was dialyzed sequentially against 4, 2, and 1 M urea and phosphate-buffered saline. Then it was incubated with paraformaldehyde-fixed murine T-cell lymphoma cells at 4°C for 24 h on a tilter in order to remove any remaining native CT-B from the preparation. Protein concentration was measured via BCA protein assay (Pierce Chemical Co., Rockford, Ill.). (ii) GM1 ganglioside-blocked CT-B. GM1 ganglioside was diluted in phosphate-buffered saline and added to CT-B at a 5:1 molar ratio prior to addition to the lymphocyte cultures to block all the binding sites. In some experiments, other molar ratios were used to block only a fraction of the binding sites. Immunization. Mice were injected subcutaneously at the base of the tail and in the foot pads with CT-B in incomplete Freund adjuvant as a mixed emulsion containing 50 to 100 pug of antigen in a total volume of 100 to 200 ,ul (11). Cell isolations. Antigen-primed peripheral lymph node (PLN) T cells were prepared by a modification by Richman et al. (34) of the method of Corradin et al. (4). Eight to fourteen days after priming, the inguinal, periaortic, and popliteal lymph nodes were removed and placed into cell suspensions by straining through a small mesh strainer or by teasing apart between the ends of sterile frosted microscope slides (28) into complete media. After two washes, no more than 2 x 108 cells in 2 ml of warm (37°C) media containing 5% fetal calf serum was applied to a nylon wool column (600 mg of nylon wool in a 12-ml syringe) (18). After incubation at 37°C for at least 45 min, the cells were eluted from the column. Cells were reconstituted at 0.5 x 107 to 1 x 107 cells per ml in complete media containing 5% fetal calf serum and added to a flask containing magnetic goat anti-mouse IgG particles (2 ml of washed particles per 5 x 107 cells). After incubation of the flask at 37°C for 20 min with gentle mixing, the adherent cells were removed by magnetic separation. The nonadherent cells were placed in another flask, and
INFECT. IMMUN.
magnetic separation was carried out again in order to ensure complete removal of the particles. The nonadherent cells were reconstituted at 4 x 106 to 10 x 106 cells per ml in complete media for use in cell culture. In some experiments, the nylon wool-passed PLN T cells were instead reconstituted at 107 cells per ml in complete media along with 200 mg of carbonyl iron particles and incubated at 37°C for 30 min. Phagocytic cells were removed with a magnet. The nonphagocytic cells were reconstituted at 1.5 x 106 cells per ml in complete media and incubated overnight at 37°C in 5% CO2 and humidified air in wells precoated with S ,ug of goat anti-mouse IgG antibody per ml to remove any B cells or any remaining adherent macrophages. The nonadherent cells were reconstituted at 4 x 106 to 10 x 106 cells per ml in complete media for use in cell culture. Peritoneal macrophages for use as antigen-presenting cells (APCs) were prepared as previously described (33). Animals were injected intraperitoneally with 1.5 ml of 40 ,ug/ml concanavalin A. Four to five days later, the mice were sacrificed and their peritoneal cavities were lavaged with Hanks balanced salt solution media. The harvested cells were further purified by glass adherence, then washed twice in complete media, and adjusted to 5 x 105 cells per ml for addition to culture. Assay of mitogen-induced proliferation of T cells. Triplicate cultures were performed with or without CT-B, denatured CT-B, and concanavalin A in wells of a 96-well Costar tissue culture plate. Spleen cells or PLN T cells isolated as described above were incubated at 37°C for 2 days in 5% CO2 humidified air. After 2 days of incubation, 0.5 mCi of [3H]thymidine was added, and after 5 additional h at 37°C, the cells were harvested in an automated collecting device (PHD cell harvester; Cambridge Technology, Inc., Watertown, Mass.). Proliferation was assessed as the amount of incorporation of [3H]thymidine into cell DNA, as measured by beta scintillation counting (Beckman Instruments, Palo Alto, Calif.) of the harvested samples. Assay of antigen-specific proliferation of T cells. PLN T cells (2 x 105 to 5 x 105) and macrophages (2.5 x 104), prepared as described above, were cultured in a final volume of 200 ml in flat-bottomed wells of a 96-well tissue culture plate. Triplicate cultures were performed with and without the addition of different forms of CT-B. The plate was incubated at 37°C for 4 days, at which time 0.5 mCi of [3H]thymidine was added, and after an additional 18 h in culture, the cells were harvested as described above. Proliferation was estimated by measurement of [3H]thymidine incorporation into T-cell DNA. RESULTS Response to different molecular forms of CT-B. When CT-B-primed purified lymph node T cells were cultured in vitro with APCs and native CT-B, little or no proliferative response was observed in repeated experiments over a wide dose range (Table 1). Because T cells recognize the primary structure of protein antigens and because of previous data indicating that CT-B was able to inhibit polyclonal T-cell activation (38), CT-B was denatured by reduction and alkylation and then absorbed on paraformaldehyde-fixed tumor cells to remove any remaining native CT-B. The same T cells that failed to respond to the native form of CT-B now had a brisk response to the denatured form of CT-B (Table 1). In independent experiments, these two molecular forms of CT-B were tested for their ability to inhibit polyclonal T-cell
CHOLERA TOXIN-SPECIFIC T CELLS
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TABLE 1. Proliferative response of primed PLN T cells to denatured versus native CT-B
[3H]TdR incorporation' (mean cpm + SD)
CT-B dose (,ug/ml)
Denatured CT-B
0 1 3 10 30
4,485 + 1,617 10,850 ± 1,489 11,206 ± 2,874 35,906 ± 12,949 50,326 ± 8,363
a
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Native CT-B
± ± ± ± 11,096 ±
4,485 2,654 4,016 7,013
1,617 325 754 967 1,394
10,000
TdR, Thymidine.
0 0
responses; native CT-B was able to inhibit concanavalin A-stimulated polyclonal T-cell responses, whereas the denatured form was not (data not shown), which is consistent with observations of others that S-carboxymethylated CT is unable to bind to GM1 ganglioside (23). CT-B-specific T cells could also be stimulated if the CT-B was preincubated with its specific ligand, GM1 ganglioside, to block its binding sites or if APCs were first pulsed with native CT-B and washed prior to being added to primed T-cell cultures (Fig. 1). Interestingly, there was little difference in the dose-response between the CT-B-pulsed APCs and the GM1-blocked CT-B added to culture, despite the ability of CT-B to actively bind to the macrophage surface in the latter situation but not the former. Effect of antigen and APC dose on T-cell responses. In these experiments, primed T cells were exposed to various doses of antigen and APCs. The T-cell response to CT-B increased as increasing numbers of APCs were added at any given dose of antigen, and the T-cell response also increased as the antigen dose increased at any given number of APCs (Fig. 2). CT-B primed T cells did not proliferate when irrelevant antigens such as ovalbumin or hen egg lysozyme were added to the cultures (data not shown). Genetic restriction of T-cell response to CT-B. The question of genetic restriction of T-cell response to CT-B was examined from two directions. In the first approach, B10 mice were immunized with CT-B, and the draining PLN T cells were isolated. These were cocultured with APCs from a variety of H-2 congenic strains. The H-2-compatible APCs
1 40 20 5 10 Macrophages added (x 1000)
FIG. 2. The effect of antigen and APC dose on the in vitro T-cell response to CT-B. CT-B-primed T cells at 4 x 105 cells per well were cultured with the numbers of APCs shown plus GM1 ganglioside-blocked CT-B at the following concentrations: 100 (G), 30 (0), 10 (O), and 0 (A) ,ug/ml.
from the B10 strain were able to present CT-B to T cells from the B10 strain, whereas APCs from H-2 disparate B10 congenic strains were not (Fig. 3). The second approach was based on previous observations that the in vivo immune response to CT varies among the strains, with variation determined by the I-A subregion of the H-2 complex. In these experiments, C3B6F1 mice were immunized with CT-B in vivo and the draining PLN T cells were cultured in vitro in the presence of antigen plus either high-responder (C3B6F1, C57BL/6) or low-responder (C3H/HeJ) APCs. These results paralleled the results seen with in vivo immunizations in that the F1 T cells responded poorly to antigen presented by APCs from the C3H/HeJ strain but responded well to antigen presented by APCs from the high-responder C3B6F1 and C57BL/6 strains (Fig. 4). Native CT-B inhibits CT-B-specific T-cell activation. One explanation for the poor response of the primed T cells to native CT-B was that the native CT-B was able to bind directly to the T cells and thus inhibit them in a similar 12 10
x
0
6
2
.1
.01
.1
1
10 Dose
100
1000
FIG. 1. Proliferative response of primed T cells to different forms of CT-B. CT-B-primed PLN T cells at 4 x 105 cells per well were cultured with 2.5 x 104 APCs plus different forms of CT-B at the doses shown. Symbols: *, native CT-B was added directly to culture; A, native CT-B was preincubated with five times an equimolar concentration of GM1 ganglioside prior to addition to culture; 0, APCs were adhered for 1 h, pulsed for 2 h with native CT-B at the doses shown, and washed before T cells were added to the wells. The background proliferation in control cultures receiving no added antigen was 5,728 cpm.
I
1
I100
CT-B ()/ml)
FIG. 3. Response of CT-B-primed T cells from B10 mice to CT-B presented by APCs from the B10 strain and from various H-2 congenic B10 strains. CT-B-primed B10 T cells at 4 x 105 cells per well were cultured with 2 x 105 APCs from B10 and H-2 congenic B10 strains at multiple doses of GM1 ganglioside-blocked CT-B. Symbols: O, B10 (H-2b); *, B10.S (H-2s); 0, B10.T (6R) (H-2q); A, B10.BR (H-2k; *, B10.BR (H-2). The data shown represent the stimulation index derived by dividing the counts per minute at any given dose of antigen by the counts per minute of cultures receiving no antigen.
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Gm 1 ganglioside Native blocked-CT-B CT-B E 0)
10,000
D
a-
'a '0 (a
m
5,000
1 10 Denatured CT-B (mg/ml)
100
70
60
_ 0
50
E
50
LL
0 10 20 30 40 50 0 0
90 80
F
0
.1
100
0
_
_ 0
FIG. 4. Effect of H-2 restriction on CT-B-specific T-cell proliferation. A total of 2 x 105 CT-B-primed B6C3F, PLN T cells were cultured for 5 days in the presence of denatured CT-B in the doses shown and with 2 x 104 APCs from either high- or low-responder strains. Symbols: A, B6C3F1 (H-2blk); 0, C57BL/6 (H_2b); *, C3H/He (H-2k).
fashion to its inhibition of polyclonal T-cell responses. This possibility was directly tested by adding native CT-B back to cultures of primed T cells, APCs, and immunogenic forms of CT-B. Similar results were obtained whether the immunogenic form of CT-B was denatured CT-B (data not shown) or CT-B blocked by GM1 ganglioside (Fig. 5). In both instances, the addition of native CT-B back to these cocultures inhibited the activation of CT-B-specific T cells in vitro. This inhibition was explored further by two types of experiments. In the first, various ratios of GM1-blocked CT-B to native CT-B were added to primed T cells plus APCs, holding the total amount of antigen in the system constant (Fig. 6). Cultures receiving more than 20% of total antigen as native CT-B were significantly inhibited. In a second experiment, the same point was addressed by varying the amount of blocking of the native CT-B by preincubating the CT-B with various molar ratios of GM1 ganglioside. Because CT-B is a pentamer, a 5:1 molar ratio of GM1 ganglioside to CT-B blocks all of the binding sites and results in stimulation of the T cells (Fig. 7). A decrease of the molar ratio from 5:1 to 4:1, i.e., a reduction of 20% in the receptor blocking, resulted in a significant inhibition of CT-B-specific
10,000 20,000 30,000 40,000 Mean CPM ± SD
FIG. 6. Effect of different ratios of GM1-blocked to native CT-B on the proliferation of CT-B-primed T cells. A total of 4 x 105 CT-B-primed T cells plus 2.5 x 105 APCs were cocultured with different amounts of GM1 ganglioside-blocked CT-B and native CT-B, with the total amount of CT-B added to the cultures constant at 100 p.g/ml. For comparison, the degrees of T-cell proliferation with 50 ,ug of GM1 ganglioside-blocked CT-B added per ml and with no antigen added are shown.
T-cell activation, an inhibition which slowly increased further as the molar ratio was further reduced. DISCUSSION CT is a potent stimulator of secretory IgA responses when delivered into the intestine and thus has been an important probe for our understanding of the mucosal immune system and of the interaction of pathogenic toxins with host defenses. The intestinal immunogenicity of CT has been taken advantage of in the form of an effective oral vaccine against cholera (14, 17). As with most protein antigens, the immune response to cholera toxin is T cell dependent (25); however, little is known about the activation, properties, or cellular immunology of CT-specific T cells. This study represents a first attempt to define the T-cell response to CT. Much of our understanding of how T cells are triggered by antigens comes from studies on secondary activation of T
20,000 70,000 60,000 50,000
oa- 10,000-
T
-
40,000a-E 30,00020,000-
10,000 .1
1
10
100
Dose (mg/ml) FIG. 5. Addition of native CT-B to cultures of CT-B-primed T cells, APCs, and a stimulatory form of CT-B. A total of 4 x 105 CT-B primed PLN T cells were cultured with 2.5 x 105 APCs plus various doses of CT-B blocked by GM1 ganglioside; duplicate cultures received in addition native CT-B at 3 j±g/ml. Symbols: Cl, cultures contained only CT-B blocked by GM1 ganglioside at doses shown; *, cultures contained CT-B blocked by GM1 ganglioside plus native CT-B.
0
5 4 3 2 1 Gm 1 ganglioside: CT-B molar ratio
FIG. 7. CT-B-specific T-cell proliferation in the presence of CT-B blocked by various amounts of GM1 ganglioside. A total of 4 X 105 CT-B-primed T cells were cultured with 2.5 x 104 APCs, plus either 100 (_) or 30 (=) ,g of CT-B per ml, as shown. Prior to culture, the CT-B was preincubated with 5 x, 4 x, 3 x, 2 x, 1 x, or 0 equimolar concentrations of GM1 ganglioside in order to block all, part, or none of its GM1 binding sites.
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cells in vitro after priming in vivo by using a system developed by Corradin et al. (4). Such studies have shown that protein antigens are taken up and processed by APCs in such a way that denatured peptides and peptide fragments are expressed on the cell surface in association with class II MHC molecules (36). The peptide fragments appear to lie in a cleft of the class II MHC molecules (3), and the T-cell receptor recognizes both the foreign peptide and adjacent facets of the self-class II MHC molecule (1). In many ways, the T-cell response to CT-B in the present study was similar to that which has been established previously for other protein antigens in this system. The T-cell proliferative responses to CT-B were antigen specific and proportional to the numbers of APCs and the doses of antigen in the cultures. The importance of class II MHC molecules for the CT-B-specific T-cell response was demonstrated by a lack of antigen presentation by APCs from H-2 incompatible congenic inbred strains and by the relative inability of APCs from a mouse strain harboring a low-responder Ia gene (H-2k) to stimulate CT-B-specific F1 (high x low responder) T cells. In other aspects, however, the response to CT-B was quite distinctive. The molecular form of CT-B used in the cultures was critical. In particular, native CT-B appeared not to stimulate T-cell activation, whereas denatured CT-B or CT-B blocked with its specific ligand, GM1 ganglioside, stimulated it very well. Subsequent experiments showed that the apparent lack of stimulation with native CT-B was due to an active inhibition of CT-B-specific T-cell proliferation. In previous work, we have shown that native CT-B inhibits T-cell activation induced by polyclonal activators or by specific, but unrelated, antigens (38). The molecular mechanism of this inhibition is not yet known, but it does require binding of CT-B to the T-cell surface (38). Consistent with these previous observations, forms of CT-B that were unable to bind to T cells were stimulatory in vitro, whereas the native form was inhibitory. Moreover, when both forms were present in culture, the ratio of the native to the stimulatory form was important in determining whether and to what degree T-cell activation occurred. Concentrations of native CT-B equal to or greater than 1 ,ug/ml were inhibitory in vitro, an amount likely to be physiologically relevant in vivo. CT itself is a much more potent inhibitor than is CT-B, with inhibition being seen with nanogram concentrations in vitro and effects on T cells in vivo being expected to be that much more profound. It is interesting to consider the results of this study in the context of the events that must take place in vivo in the intestine for a response for CT to occur. Certainly the intestinal lumen is a hostile environment for any antigen, considering digestion by enzymes, denaturation by bile, dilution by secretions, and a limited duration of exposure due to intestinal motility, trapping by the mucus coat, etc. The present study illustrates that many of these nonspecific defenses would not necessarily limit sensitization of T cells in that denatured CT-B, in essence a long peptide, was an effective immunogen for CT-B-specific T cells. Degradation of CT in the lumen may reduce its ability to bind, however, and the ability of CT and CT-B 'to bind to GM1 ganglioside on epithelial cells has been thought to be very important in its intestinal immunogenicity, perhaps by sequestering it and prolonging its time in the intestine. Indeed, there is some data indicating that intestinal epithelial cells not only express class II MHC antigens but also can present antigens in vitro (2), although it is unproven whether they function as APCs in vivo. The ability of the toxin to bind to GM1 gangliosides
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present on the M cells of the follicle-associated epithelium is likely a very important feature of its immunogenicity, because M cells represent a critical portal of entry for enteric antigens, including CT (32), into gut lymphoid follicles. CT does not appear to interfere with the ability of APCs to take up antigen, process it, or present it in an immunogenic form recognizable by T cells. For example, in the present study, APCs pulsed with CT-B were effective in activating CT-B-primed T cells. In previous studies, neither CT nor CT-B interfered with APC uptake, processing, or presentation of keyhole limpet hemocyanin antigen to primed T cells (38). CT has been reported to increase interleukin-1 secretion by APCs (24) and so may enhance this aspect of immune induction. In the present study, APCs were also effective in taking up GM1 ganglioside-blocked CT-B and presenting that form to primed T cells. Interestingly, in experiments in which APCs were pulsed with either native CT-B or GM1 ganglioside-blocked CT-B, equivalent T-cell responses were found. This is curious because of evidence that the ability of an antigen to specifically bind to APCs, especially antigenspecific B cells (22), greatly enhances the efficiency of antigen presentation. From these results, the form of antigen reaching APCs in gut lymphoid follicles is not likely to be a limiting factor in the induction of the immune response to CT. The types of T-cell subsets being stimulated in this system in vitro or in gut-associated lymphoid tissue (GALT) in vivo are not yet known. Murine CD4 helper T cells have been subdivided into Thl and Th2 subtypes on the basis of the pattern of lymphokines they secrete (29). Th2 cells produce interleukin 4 and interleukin 5, which are important in generating IgA responses (10, 31). Whether one or the other of these subtypes is preferentially stimulated by CT-B is, at present, unknown; there are no surface markers by which these two subsets can be distinguished. However, previous work in vivo has shown that CT stimulates not only the secretory IgA response, but also a fairly striking plasma IgG response (7), a Thl function. These and other results (6, 7, 8) would suggest that there may be no preferential stimulation of either the Thl or the Th2 helper cell subtypes by CT. However, the present results do indicate that the ratio of the native to the stimulatory form of CT and CT-B is likely to be a very important determinant of the extent of T-cell activation in GALT. The approach used here is relevant to the study of the mucosal immune response to other bacterial toxins. For example, shiga toxin has recently been reported to stimulate a strong secretory IgA response when administered into the intestines of mice (19). Shiga toxin is a true cellular poison, and one can predict it will be extremely toxic in vitro, which will make its study difficult. However, the approaches used here should allow a detailed exploration of antigen uptake, processing, and presentation and of the T-cell response to this as well as to other pathogenic toxins whose cellular immunology is largely unexplored. ACKNOWLEDGMENTS This work was supported by Public Health Service grant DK28623 from the National Institute of Diabetes and Digestive and Kidney Diseases. We wish to acknowledge the expert technical assistance of Hazel Bowden. LITERATURE CITED 1. Allen, P. M., G. R. Matsueda, R. J. Evans, J. B. Dunbar, G. R. Marshall, and E. R. Unanue. 1987. Identification of the T-cell
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