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Clinical and Diagnostic Laboratory Immunology, January 2005, p. 157-164, Vol. 12, No. 1
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.1.157-164.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Induction of Thymus-Derived T Cells by Escherichia coli Enterotoxin B Subunit in Peritoneal Cavities of Mice

Toshiyasu Shimizu,¹ Keiko Sasaki,¹ Michio Kato,¹ Hideyuki Arimitsu,¹ Sadayuki Ochi,¹ Naoko Shigemori,¹ Eddy Bagus Wasito,² Takashi Yokochi,³ and Takao Tsuji^1*

Department of Microbiology, Fujita Health University School of Medicine, Tokyoake,¹ Department of Microbiology, Aichi Medical University, Nagakute, Aichi, Japan,³ Department of Microbiology, Tropical Disease Center, Airlangga University, Unair, JL, Mulyorejo, Surabaya, Indonesia²

Received 20 July 2004/ Returned for modification 10 September 2004/ Accepted 23 September 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G. The numbers of both and ß T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM₁ ganglioside-binding ability. Early after administration a small number of T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing T cells were detected. T cells induced by the B subunit did not express a characteristic V gene over the time course of the study. The induction of T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like ß T cells. T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen. The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice. These results suggest that the B subunit activates intraperitoneal and ß T cells in a manner dependent upon its ability to bind to GM₁ ganglioside. T cells induced by the B subunit are Th2-type cells derived from the thymus. These T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of ß T cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In mice, T-cell-receptor (TCR) T cells develop through multiple pathways (23). T cells expressing V5/V1 or V6/V1 first appear in the fetal thymus (7, 11). V/5⁺ T cells later migrate to the epidermis and exclusively colonize this peripheral site (2, 7). V/6⁺ T cells also colonize the mucosal epithelia of the tongue, uterus, vagina, and lung (12); but later, they can also distribute to other, peripheral organs (22, 25, 27). The adult thymus can also generate T cells expressing primarily V1 or V4 (11, 24). A third extrathymic developmental pathway for T cells generates T cells preferentially expressing V1, V4, or V7 (16, 24, 35). These pathways create a diverse range of T cells expressing many types of V within the peritoneal cavities of adult mice (20).

Particularly intriguing aspects of T cells are their distinctive localization at sites of infection, in addition to normal epithelial tissues. This association at sites of first contact with infectious agents or their products implicates T cells in frontline defense reactions. The preferential expression of T cells can be due to the selective homing and proliferation of cells controlled by a number of factors unique to T cells and a particular site or inflammatory conditions, such as microbial infection (2).

The contribution of T cells to host defenses against bacteria has been examined by infection of T-cell-depleted mice with intracellular bacteria (4, 17, 28). Mice rendered deficient for T cells by homologous recombination of the TCR- chain gene exhibited diminished host defenses against Mycobacterium tuberculosis (17). T cells, however, may have heterogeneous functions during infectious disease. Mice deficient for TCR- suffered intestinal damage upon oral infection with Eimeria verformis, suggesting that intraepithelial T cells function in the resolution of intestinal inflammation (26). Moreover, although protection against extracellular bacteria depends primarily on neutrophils and antibodies (Abs) (32), T cells are also important for host defense against pathogens such as Escherichia coli (20, 21, 30).

Multiple different bacterial antigens, such as mycobacterial purified protein derivative, tetanus toxoid, staphylococcal enterotoxin A, heat shock protein 65, isopentenyl pyrophosphate, and hemolysin from Listeria monocytogenes or E. coli, serve to initiate T-cell-mediated host defenses against bacteria (2). Lipopolysaccharide (LPS), which induces a potent host defense response against E. coli infection through the induction of cytokines such as gamma interferon (IFN-) and interleukin-15 (IL-15), also stimulates T cells (20, 21, 30).

Heat-labile enterotoxin (LT) is produced by enterotoxigenic E. coli and causes severe diarrhea in animals and humans in a manner similar to that of cholera toxin (CT), produced by Vibrio cholerae. Both toxins are composed of A and B subunits. The A subunit is toxic, while the B subunit allows the toxin bind to the GM₁ ganglioside. The B subunit exhibits a high level of antigenicity (9); controversy remains, however, as to whether the molecule exhibits an adjuvant action (3, 6, 19, 33) against antigens coadministered nasally, peritoneally, orally, or intramuscularly or whether it has a tolerative effect (1, 29, 34).

In this study, we examined the effect of the B subunit of LT (LT-B) on the proliferation of T cells. As the induction of T cells occurs at the site of first contact with infectious agents or their products and the peritoneal cavity of mice is a tissue site for the preferential expression and induction of T cells, we examined whether LT-B might induce T cells by injecting LT-B into the peritoneal cavity. We also evaluated the relationship between T cells activated by LT-B administration and the antigenicity of LT-B and its function as an adjuvant stimulus.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice, plasmids, and bacteria. All experiments were performed with BALB/c and ICR female mice (Shizuoka Animal Co., Ltd.) aged 4 to 5 months. Age-matched female mice served as controls. The athymic nu/nu mice and fetal AKR mice were purchased from Charles River Co., Ltd.

The Bluescript II SK⁺-1 plasmid carrying the LT-B gene (Toyobo Co., Ltd.) was constructed from strain EWD 299 by PCR, as described previously (14). The G33D B subunit, a mutant form with an aspartate in place of the endogenous glycine at position 33 from the N terminus, was prepared as described previously (31).

Preparation of normal and mutant LT-B subunits. Recombinant LT-B (rLT-B) was purified by immobilized D-galactose affinity column chromatography in TEAN buffer (50 mM Tris-HCl, 1 mM EDTA, 3 mM NaN₃, 0.2 M NaCl [pH 6.8]), as described previously (14). Bacterial culture in CAYE medium (2.0% Casamino Acids, 0.6% yeast extract, 0.25% NaCl, 0.871% K₂HPO₄, 0.25% glucose, 0.1% trace salt solution [5.0% MgSO₄, 0.5% MnCl₂, 0.5% FeCl₃, 0.0001% H₂SO₄]), isolation of crude cell extracts, and rLT-B purification by successive chromatography steps have been reported previously (14).

The G33D LT-B mutant bound to the GM₁ ganglioside receptor at a 10,000-fold lower affinity than normal LT-B, as determined by the GM₁ immunosorbent assay. G33D was purified by affinity chromatography with anti-LT-B Abs, as reported previously (31).

Determination of antibody titers in serum. The antibody titers in serum were determined by enzyme-linked immunosorbent assay. Briefly, polystyrene microtiter plates were coated with 50 or 20 µg of bovine immunoglobulin G (IgG) (BGG) or LT-B per ml in coating buffer. After incubation, phosphate-buffered saline (PBS)-1% bovine serum albumin was added to each well, and the plate was incubated for 18 h at 4°C. The wells were washed, and serially diluted sera were added to the wells. The plates were incubated at 37°C for 60 min. Bound Abs were detected by using alkaline phosphatase-conjugated rabbit anti-mouse IgG, IgG1, IgG2a, IgG2b, and IgG3 Abs at a dilution of 1/1,000 and visualization with 4-nitrophenylphosphate (1 mg/ml). The titer was defined as the absorbance that yielded for each IgG a value of 1.0 above the background value.

Preparation of peritoneal cells. After intraperitoneal (i.p.) injection of rLT-B (20 µg in PBS [pH 7.2]) into mice, we injected 2 ml of RPMI (Gibco BRL, Co., Ltd.) containing 10% fetal calf serum (FCS) and 10 U of heparin per ml into the peritoneal cavity. Peritoneal cells were collected by centrifugation with a swing rotor (Tomy RL-131) at 1,000 rpm for 5 min. After extensive washing in PBS containing 2% FCS, the cells were passed through a nylon filter prior to experimentation.

FCM. The following monoclonal Abs (MAbs) coupled to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or biotin were used for flow cytometry: biotin-anti-CD3 (clone 145-2C11), FITC-anti-mouse ß TCR (clone H57-597), FITC-anti-mouse TCR (clone GL3), PE-anti-IL-4 (clone 11B11), PE-anti-mouse IFN- (clone XMG1.2), PE-anti-mouse IL-10 (clone JES5-2A5), PE-anti-mouse Thy-1.1 (clone HIS51), and PE-anti-mouse Thy-1.2 (clone 53-2.1). Anti-Thy-1.1 and anti-Thy-1.2 clones were purchased from Bioscience Co., Ltd. Additional MAbs were purchased from Pharmingen Co., Ltd. Species and isotype-matched Igs coupled to FITC, PE, or biotin (Bioscience or Pharmingen Co., Ltd.) were used as staining controls.

To stain the surface antigens on peritoneal cells, 10⁵ cells were treated with 5% skim milk in PBS at 4°C for 30 min. The samples were then incubated with a mixture of the designated anti-CD IgG MAb at 4°C for 30 min, as described previously (13). For intracellular cytokine staining, we added 2 ml of fluorescence-activated cell sorter (FACS) lysing solution (Becton Dickinson) to the cells, followed by blocking in 5% skim milk. After incubation at room temperature for 10 min, the cells were resuspended in 500 µl of FACS permeabilizing solution (Becton Dickinson) and incubated for 10 min at room temperature. Following a wash in PBS containing 2% FCS, the samples were stained with the designated MAb at 4°C for 30 min. Flow cytometric analysis was performed with a FACScan instrument (FACSCaliber; Becton Dickinson); the data were analyzed with Lysis II software.

Analysis of V-gene segment use by reverse transcription-PCR (RT-PCR). Total RNA was extracted from mouse peritoneal cells by the acid-guanidinium-phenol-chloroform method. DNA synthesis was performed by PCR. RNA was primed by using a cDNA cycle kit (Invitrogen Corp., San Diego, Calif.) with 10 µg of total RNA in the presence of either 20 pmol of the -chain C-region (c) primer (5'-CTT ATG GAG GAT TTG TTT CAG C-3') or 6.7 pmol of the -chain J-region (J) primer (5'-TTG GTT CCA CAG TCA CTT GG-3') in 21-µl reaction mixtures. PCR was performed with a PCR thermal cycler (Takara Corp., Tokyo, Japan), with PCR cycles composed of 1 min at 94°C, 1 min at 54°C, and 30 s at 72°C. Prior to the first cycle, the samples were incubated for 7 min at 94°C. After 35 cycles, extension was performed for an additional 4 min at 72°C. The 5' V-gene-specific primers have been described previously (30) and are as follows: V1/2, 5'-ACA CAG CTA TAC ATT GGT AC-3'; V2, 5'-CGG CAA AAA ACA AAT CAA CAG-3'; V4, 5'-TGT CCT TGC AAC CCC TAC CC-3'; V5, 5'-TGT GCA CTG GTA CCA ACT GA-3'; V6, 5'-GGA ATT CAA AAG AAA ACA TTG TCT-3'; V7, 5'-AAG CTA GAG GGG TCC TCT GC-3'; V1, 5'-ATT CAG AAG GCA ACA ATG AAA G-3'; V2, 5'-AGT TCC CTG CAG ATC CAA GC-3'; V3, 5'-TTC CTG GCT ATT GCC TCT GAT-3'; V4, 5'-CCG CTT CTC TGT GAA CTT CC-3'; V5, 5'-CAG ATC CTT CCA GTT CAT CC-3'; V6, 5'-TCA AGT CCA TCA GCC TTG TC-3'; V7, 5'-CGC AGA GCT GCA GTG TAA CT-3'; and V8, 5'-AAG GAA GAT GGA CGA TTC AC-3'.

The PCR products (5 µl) were subjected to electrophoresis on a 1.5% agarose gel (GIBCO Co., Ltd.). The PCR products were extracted from the agarose and sequenced with an automated DNA sequencer (Applied Biosystems Co., Ltd.) and a DNA sequencing kit (Applied Biosystems Co., Ltd.).

Transplantation of fetal thymus into BALB/c nu/nu mice. A thymic graft was obtained from either fetal AKR mice (16 or 18 days after pregnancy) or 24-h-old AKR mice. The thymus was transplanted between the kidney and peritoneal membrane of BALB/c nu/nu mice (Child Liver Co., Ltd.) at 6 weeks of age. One month after transplantation, the recipient nu/nu mice were injected with LT-B i.p., prior to determination of the expansion of T cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Specific serum IgG subclass responses to BGG coadministered with LT-B i.p. IgG responses were determined to detect which type of T cell specific for BGG was induced by coadministration of LT-B-, BGG-, or LT-B-specific serum. Significant IgG1 and IgG2b anti-BGG Ab responses were evident by i.p. coadministration of BGG and LT-B but not IgG2a and IgG3 (Fig. 1).

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FIG. 1. Serum IgG subclass response to LT-B and BGG in ICR mice immunized once i.p. with LT-B and BGG. rLT-B (10 µg) and BGG (100 µg) were coadministered via the i.p. route to six ICR female mice. The mice were bled 4 weeks after the vaccination. The anti-LT-B and BGG titers were defined with an LT-B- or BGG-coated enzyme-linked immunosorbent assay plate. Values represent means ± standard deviations. Asterisks indicate that the values are significantly greater (P < 0.05) for the LT-B- and BGG-treated groups than for the control group or the group treated with BGG alone. The data shown are representative of two independent analyses with six mice, with similar results obtained in each analysis.

Kinetics of and ß T-cell migration into the peritoneal cavity after injection of LT-B or G33D. To examine the and ß T-cell compartment in the peritoneal cavity after LT-B injection, we analyzed the changes in the kinetics of the peritoneal cell composition in BALB/c mice injected with LT-B i.p. The percentage of cells in the lymphocyte fraction that were positive for both CD3 and the TCR or ß antigen increased significantly after i.p. injection of LT-B into the mice (Fig. 2). The absolute numbers of peritoneal cells and and ß T cells also increased significantly in a time-dependent manner. A mutant LT-B molecule (G33D) defective in GM₁ ganglioside binding did not significantly increase the numbers of T cells (Fig. 3). Moreover, although we examined whether 40 µg of G33D increased the numbers of peritoneal cells and both types of T cells, there was no difference in the levels of induction of these cells by 20 and 40 µg. As LT-B appears to induce the influx of peritoneal T cells, we characterized the T cells recruited by LT-B.

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FIG. 2. Kinetics of peritoneal and ß T cells after i.p. injection of rLT-B into BALB/c mice. rLT-B (20 µg) in PBS was injected i.p. into two BALB/c mice on day 0. Each day after injection, peritoneal cells from two mice were pooled, stained with labeled anti-CD3 and anti-TCR- or anti-TCR-ß MAbs, and analyzed by flow cytometry. The number in each panel indicates the percentage of and ß T cells among the total peritoneal lymphocytes. The data shown are representative of two independent analyses with two mice, with similar results obtained in each analysis.

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FIG. 3. Kinetics of the absolute numbers of total peritoneal cells and and ß T cells after i.p. injection of rLT-B or G33D. The numbers of total peritoneal cells (a), T cells (b), and ß T cells (c) were analyzed each day after i.p. injection of 20 µg of either rLT-B or G33D into six BALB/c mice. Peritoneal cells obtained from mice for each set of experimental conditions were pooled, stained with the designated MAb, and then analyzed by flow cytometry. The means and standard errors for six mice are shown. Asterisks indicate the significant differences in the values for each condition for the mice at day 0 (P < 0.05) (*) and mice injected with G33D (P < 0.05) (**).

Dose-response of absolute numbers of peritoneal T cells after i.p. injection of each dose of LT-B. We examined the responses of T cells to increasing doses of LT-B (Fig. 4). Following injection, the absolute numbers of T cells increased proportionately in a dose-dependent manner. Treatment of LT-B-treated samples with 10 µg of polymyxin B per ml, a quantity capable of neutralizing more than 10 µg of LPS per ml, did not affect the induction of T cells. Treatment of LT-B at 100°C for 10 min reversed the induction of T cells in the peritoneal cavity, suggesting that the LT-B itself and not the contaminating LPS induces the recruitment of peritoneal T cells.

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FIG. 4. Dose-response of the absolute numbers of peritoneal T cells after i.p. injection of each dose of rLT-B with or without treatment with polymyxin B or heating. rLT-B (10 mg/ml) suspended in PBS was treated with 10 µg of polymyxin B per ml at 37°C for 30 min or by heating at 100°C for 10 min. After dilution of rLT-B to 1 mg/ml, rLT-B-treated samples were injected i.p. into six BALB/c mice. Three days later, peritoneal cells were obtained from each mouse. Pooled cells were stained with labeled anti-CD3 and anti-TCR- MAbs and then analyzed by flow cytometry. Asterisks indicate significant differences in the values from the values for the control mice (P < 0.05). , rLT-B treated with polymyxin B; •, rLT-B not treated with polymyxin B; , rLT-B heated at 100°C for 10 min.

Cytokine production by T and ß T cells in the peritoneal cavity induced by LT-B. To elucidate the function of T cells induced by LT-B, we examined the production of cytokines, such as IL-4, IL-10, and IFN-, by T cells. Following intracellular cytokine staining, a small number of T cells stained with anti-IL-4 and anti-IFN- Abs at 3 days. At 5 and 7 days, however, T cells stained with anti-mouse IL-4 and IL-10 Abs, respectively (Fig. 5), demonstrating a change in the quality of the T-cell response.

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FIG. 5. Cytokine production by peritoneal T cells induced by injection of rLT-B into mice. rLT-B (20 µg) in PBS was injected i.p. into two BALB/c mice. On each day after administration, peritoneal cells were obtained from the two mice and pooled. These cells were stained extracellularly with anti-CD3 and anti-TCR- MAbs and intracellularly with an anti-IL-4, -IL-10, or -IFN- MAb, as described in Materials and Methods. We analyzed the positive cells for cells double positive for CD3+ and . The data shown are representative of three independent analyses with two mice, with similar results obtained in each analysis. PBS, PBS injection; LT-B, rLT-B (20 µg) injection.

Moreover, we also examined the pattern of cytokine production by ß T cells induced by LT-B (Fig. 6). ß T cells produced cytokines identical to those synthesized by T cells.

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FIG. 6. Cytokine production by peritoneal ß T cells induced by injection of rLT-B into mice. rLT-B (20 µg) in PBS was injected i.p. into two BALB/c mice. On each day after administration, peritoneal cells were obtained from the two mice and pooled. These cells were stained extracellularly with anti-CD3 and anti-TCR-ß MAbs and intracellularly with an anti-IL-4, -IL-10, or -IFN- MAb, as described in Materials and Methods. We analyzed the positive cells for cells double positive for CD3+ and ß. The data shown are representative of three independent analyses with two mice, with similar results obtained in each analysis. PBS, PBS injection; LT-B, rLT-B (20 µg) injection.

V- and V-gene expression by T cells in the peritoneal cavities of mice injected i.p. with LT-B. To examine V-gene expression by T cells in the peritoneal cavity following LT-B injection, we extracted total RNA from peritoneal cells. V-gene expression was analyzed by RT-PCR. T cells from the peritoneal cavities of mice injected with PBS exhibited bands corresponding to V1/2, V2, V4, and V6. T cells recruited following LT-B administration demonstrated the same bands at 1, 3, 5, and 7 days after LT-B injection. DNA sequencing of each band confirmed that these sequences corresponded to portions of the V1/2, V2, V4, and V6 genes, as described previously (8).

Bands related to V1, V4, V5, V6, and V7 were detected from RNA prepared from peritoneal cells from normal mice. At 1 day after LT-B administration, however, the amount of a band representing V1 paired with V6 (V6/1) could be seen to be relatively increased. However, there was not a characteristic usage of the V gene over the time course of the study. The small number of peritoneal T cells recruited by G33D administration showed that V and V usage was identical to that seen in normal mice.

Changes in numbers of peritoneal T cells following LT-B stimulation in vitro. To elucidate whether LT-B acts directly on the proliferation of peritoneal T cells or indirectly through the stimulation of other peritoneal cells, we examined the changes in the numbers of T cells in vitro following stimulation with LT-B. LT-B did not affect the numbers of T cells over a 7-day period of stimulation.

Lack of induction of and ß T cells by injection of LT-B into athymic nu/nu mice and restoration of induction by AKR fetal thymus graft. T cells develop through multiple pathways, and these pathways are both dependent and independent of thymic selection. We therefore determined the effect of the thymus on the induction of and ß T cells following i.p. injection of LT-B into athymic nu/nu mice. LT-B was unable to induce the accumulation of either type of T cell in the peritoneal cavities of athymic nu/nu mice. Upon transplantation of fetal ARK thymus tissue into BALB/c nu/nu mice, however, the induction of and ß T cells following LT-B stimulation could be partially restored in the mice. The -type T cells used V and V, identical to those seen in the normal mice.

Moreover, both T-cell subsets induced by LT-B expressed the Thy-1.1 antigen derived from the donor AKR mice but did not express Thy-1.2, which is expressed by the recipient BALB/c mice (Fig. 7).

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FIG. 7. Detection of Thy-1.1 and Thy-1.2 antigens on and ß T cells induced by rLT-B in nu/nu mice grafted with fetal AKR thymus tissue. Three days after three nu/nu mice grafted with fetal thymus tissue were injected i.p. with PBS (closed areas) or rLT-B (20 µg; open areas); and peritoneal cells were isolated, stained with anti-Thy-1.1 or anti-Thy-1.2 Abs, and then analyzed by flow cytometry. We analyzed the positive cells for cells double positive for CD3+ and or ß. Representative data from two independent analyses are shown, with similar results obtained in each analysis.

No induction of or ß T cells in athymic nu/nu mice by coinjection of LT-B and peritoneal cells. We examined the induction of T cells from extrathymic tissues by LT-B by coinjection of normal peritoneal cells. When normal peritoneal cells prepared from BALB/c mice (Fig. 8a) or AKR mice (Fig. 8b) were coinjected i.p. into BALB/c nu/nu mice, T cells were not induced by LT-B in either mouse strain. Upon i.p. injection of peritoneal cells from AKR mice treated with LT-B into BALB/c nu/nu mice, peritoneal ß and T cells expressed the Thy-1.1 antigen derived from the donor AKR mice (Fig. 8c).

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FIG. 8. Lack of induction of or ß T cells by rLT-B in athymic nu/nu mice by i.p. injection of peritoneal cells from normal mice. Normal peritoneal cells were obtained from either three BALB/c mice (a) or three AKR mice (b and c). Normal peritoneal cells were coadministered with PBS (left) or rLT-B (right) (20 µg) to three BALB/c nu/nu mice. Three days after injection, pooled cells were stained with labeled anti-CD3, anti-TCR-, or anti-TCR-ß (a and b) and anti-Thy-1.1 or Thy-1.2 (c) MAbs and analyzed by flow cytometry. The results are representative of those from two independent experiments, with similar results obtained in each experiment.

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cells participate in early responses to bacterial infection and play a protective role against pathogens such as L. monocytogenes, Salmonella, Mycobacterium, E. coli, and Toxoplasma gondii (2, 4, 17, 20, 21, 28, 30). In such intracellular or extracellular bacterial infections, several antigens, including bacterial components and toxins, activate T cells (2). LT and CT, produced by enterotoxigenic E. coli and V. cholerae, respectively, induce diarrhea, but they also serve as strong adjuvants that stimulate immune responses to other antigens influencing the ß T- and B-cell repertoire (3, 19, 33). The B subunits of these toxins alone are strongly antigenic and appear to have some adjuvant action, but this effect is highly reduced from that seen for the intact holotoxin. Kim et al. (15) reported that holo-LT selectively inhibits the extrathymic T-cell development of intestinal epithelial lymphocytes, including ß and T cells. As yet, however, no reports have demonstrated that CT, LT, or their corresponding B subunits can recruit and activate peritoneal T cells. In this study, therefore, we examined the effect of LT-B on peritoneal T cells.

Upon i.p. injection of LT-B into mice, both the percentages and the absolute numbers of and ß T cells increased in a time-dependent manner (Fig. 4). These increases were dependent on LT-B binding to GM₁ ganglioside (Fig. 3). Our analysis of T cells revealed that this population expressed the same V and V genes as peritoneal T cells without LT-B injection. Coadministration of E. coli (MV1184 strain) and LT-B, prepared for these studies, to mice did not affect the E. coli infection in the peritoneal cavity or the spleen (data not shown). Thus, the T cells induced by LT-B are heterogeneous and do not appear to be a primary defense that minimizes E. coli infection, although LT-B is an agent that induces T cells in the frontline immune response.

Functionally, LT-B-induced T cells produced cytokines of the Th2 type, such as IL-4 and IL-10, like ß T cells do (Fig. 5 and 6). IL-4 is a key cytokine that shifts the Th1-Th2 balance toward Th2-type cells. IL-10 inhibits the production of IL-12 and IFN-, key cytokines that shift the Th1-Th2 type balance to a Th1-like response, and promotes the activation of Th2-type cells (10, 18). As T cells have also been divided into at least two subsets (Th1 and Th2) (5), LT-B shifts the Th1-Th2 balance of T cells to the Th2-type response (Fig. 5). Moreover, significant IgG1 and IgG2b Ab responses but not IgG2a Ab responses to LT-B and BGG were observed by i.p. injection of LT-B and BGG (Fig. 1). Thus, the Th2 types of and ß T cells induced by LT-B may work together, promote the humoral immune response to LT-B itself, and allow LT-B to exhibit its adjuvant action to induce a specific Th2 type of response to BGG.

Next, we examined whether T cells induced by LT-B might originally be thymic or extrathymic. We first examined the stimulatory effect of LT-B on peritoneal cells in vitro. T cells did not proliferate upon in vitro stimulation of peritoneal cells by LT-B, suggesting that LT-B induces the influx of T cells into the peritoneal cavity. T cells could not be induced by LT-B administration in athymic nu/nu mice; induction was restored upon grafting of a normal fetal thymus. Thus, the induction of T cells by LT-B is dependent on the thymus. Moreover, T cells in athymic nu/nu BALB/c mice with a fetal thymic graft expressed the Thy-1.1 antigen derived from the donor thymic graft, not the Thy-1.2 antigen expressed by the host BALB/c mice (Fig. 7). These data suggest that T cells migrate from the thymus and home into the peritoneal cavity following LT-B stimulation but cannot be derived from extrathymic tissues.

In conclusion, LT-B induces the accumulation of peritoneal T cells in a manner dependent on its binding to the GM₁ ganglioside receptor. LT-B-induced T cells derived from the thymus produce Th2-type cytokines. These cells may be functionally involved in the promotion of specific humoral immune responses to LT-B and the adjuvant action that promotes the response of specific Th2-type T cells to coadministered antigens through their various interactions with ß T cells.

 
This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan and by grants from the Open Research Center of Fujita Health University from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

 
* Corresponding author. Mailing address: Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan. Phone: 81-562-93-2432. Fax: 81-562-93-4003. E-mail: ttsuji{at}fujita-hu.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Clinical and Diagnostic Laboratory Immunology, January 2005, p. 157-164, Vol. 12, No. 1
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.1.157-164.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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