Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion

Abstract

Recent evidence indicates that the secreted Helicobacter pylori vacuolating toxin (VacA) inhibits the activation of T cells. VacA blocks IL-2 secretion in transformed T cell lines by suppressing the activation of nuclear factor of activated T cells (NFAT). In this study, we investigated the effects of VacA on primary human CD4(+) T cells. VacA inhibited the proliferation of primary human T cells activated through the T cell receptor (TCR) and CD28. VacA-treated Jurkat T cells secreted markedly diminished levels of IL-2 compared with untreated cells, whereas VacA-treated primary human T cells continued to secrete high levels of IL-2. Further experiments indicated that the VacA-induced inhibition of primary human T cell proliferation was not attributable to VacA effects on NFAT activation or IL-2 secretion. We show here that VacA suppresses IL-2-induced cell-cycle progression and proliferation of primary human T cells without affecting IL-2-dependent survival. Through the analysis of a panel of mutant VacA proteins, we demonstrate that VacA-mediated inhibition of T cell proliferation requires an intact N-terminal hydrophobic region necessary for the formation of anion-selective membrane channels. Remarkably, we demonstrate that one of these mutant VacA proteins [VacA-Delta(6-27)] abrogates the immunosuppressive actions of wild-type VacA in a dominant-negative fashion. We suggest that VacA may inhibit the clonal expansion of T cells that have already been activated by H. pylori antigens, thereby allowing H. pylori to evade the adaptive immune response and establish chronic infection.

Fig. 1.

VacA inhibits activation-induced proliferation of primary human CD4⁺ T_h cells. (A) Purified primary human T_h cells were labeled with CFSE and treated with acid-activated VacA (10 μg/ml), acidified-PBS (PBS), or medium alone for 1 h, followed by TCR/CD28 stimulation for 48 h, as described in Materials and Methods. Control cells were treated with medium alone, without TCR/CD28 stimulation. Activated T cells were expanded in IL-2-containing media, and T cell proliferation was analyzed at day 5 postactivation by flow cytometry. (B) Graphic representation of the histograms shown in A. (C) Dose-response analysis of VacA effects on primary human CD4⁺ T cell proliferation. T_h cells were CFSE-labeled and treated with different concentrations of acid-activated (pH 3) VacA for 1 h. Cells were then stimulated and analyzed as in A. (D) Effects of acid-activated VacA (pH 3) and nonacid-activated VacA (pH 7.5) on T cell proliferation. T_h cells were CFSE-labeled and treated with acid-activated or nonactivated VacA (10 μg/ml), as described above. All of the results are representative of three experiments using cells from different donors and different toxin preparations.

Fig. 2.

VacA inhibits activation-induced proliferation of primary human T_h cells independent of effects on IL-2 secretion and NFAT activation. (A) Purified primary human T_h cells or Jurkat T cells were pretreated with medium alone, wild-type (WT) VacA, VacA-Δ(6–27), or cyclosporine A (CspA) for 1 h, followed by TCR/CD28 stimulation (CD3/CD28; Upper) or stimulation with PMA (50 ng/ml) and ionomycin (500 ng/ml) (Lower), as indicated. IL-2 secretion was measured at 24 h after stimulation by using a CBA, as described in Materials and Methods. Results represent the mean ± SD from triplicate samples. (B) Purified primary human T_h cells were CFSE-labeled and pretreated with wild-type VacA or PBS in the presence or absence of supplemental IL-2 (200 units/ml) for 1 h as indicated. Cells were then TCR/CD28 stimulated in the presence or absence of supplemental IL-2 for 48 h, expanded in IL-2-supplemented media, and subjected to flow cytometric analysis at day 5 posttreatment. (C) Primary human CD4⁺ T cells stably transduced with a GFP reporter under the control of NFAT (NFAT-GFP T_h cells; see Materials and Methods) were pretreated with different additives as in A for 1 h before TCR/CD28 stimulation (CD3/CD28). GFP expression was assessed by flow cytometric analysis 24 h after stimulation. Results represent the mean ± SD from triplicate samples and are expressed as the percentage of cells demonstrating inducible expression of GFP, relative to the PBS-treated cells. CspA, cyclosporine A (50 nM); WT, wild-type VacA toxin (10 μg/ml); and Δ6–27, VacA-Δ(6–27) mutant toxin (10 μg/ml).

Fig. 3.

VacA inhibits IL-2-driven proliferation of primary human T_h cells. (A) Wild-type (WT) VacA (10 μg/ml) or VacA-Δ(6–27) (10 μg/ml) were added to CFSE-labeled purified primary human CD4⁺ T cells at the indicated time points either preceding or after (pre- or post-) TCR/CD28 stimulation. Activated cells were expanded in IL-2-containing media, and cell proliferation was analyzed by flow cytometry at day 5 after stimulation. (B) Primary human T_h cells were TCR/CD28 stimulated for 48 h and expanded in the presence of IL-2 for 2 additional days. At day 4 after stimulation, T cells were removed from IL-2 and treated with PBS, wild-type VacA (10 μg/ml), or rapamycin (Rap; 200 ng/ml) for 24 h. After 24 h, IL-2 was added back to the media as indicated, and cells were treated again with the different additives and expanded in fresh media containing supplemental IL-2 for 3 days. Cell proliferation was assessed by cell counting with a hemacytometer. Results represent the mean ± SD from triplicate samples. ^*, P < 0.001 when compared with the PBS-treated cells. (C) Viability of T_h cells was determined by flow cytometric gating for viable cells based on forward and side scatter properties at days 1, 2, and 3 after TCR/CD28 stimulation. (D) For cell-cycle analysis, primary human T_h cells were treated as described in B. Cell-cycle distribution was analyzed at 36 h after IL-2 stimulation by using PI staining and flow cytometry analysis, as described in Materials and Methods. The percentages of total cells in S phase and G₂M phase are shown. Results represent the mean ± SD from triplicate samples. ^*, P < 0.01 when compared with the PBS-treated cells. Results are representative of at least two experiments using cells from different donors and different toxin preparations.

Fig. 4.

Analysis of VacA mutant proteins demonstrates that an intact N-terminal hydrophobic domain is required for VacA-mediated effects on T cell proliferation. Purified primary human T_h cells were CFSE-labeled and treated with wild-type VacA (WT VacA, 10 μg/ml), one of three different mutant toxins (each 10 μg/ml), or PBS for 1 h. Cells were then TCR/CD28 stimulated for 48 h, expanded in IL-2-containing media, and analyzed by flow cytometry at day 5 after stimulation, as described in Materials and Methods. Results are representative of three experiments using cells from different donors and different toxin preparations.

Fig. 5.

Effects of a dominant-negative mutant VacA toxin. (A) Primary human T_h cells were CFSE-labeled and then treated for 1 h with wild-type VacA (10 μg/ml) and VacA-Δ(6–27) in different ratios (WT:Δ6–27) as indicated. Cells were then TCR/CD28 stimulated and expanded in IL-2-supplemented media, as described in Materials and Methods. Cell proliferation was analyzed by flow cytometric analysis on day 5 after stimulation. (B) Jurkat T cells were pretreated with wild-type VacA and VacA-Δ(6–27) as in A for 1 h as indicated, activated with PMA (50 ng/ml) and ionomycin (500 ng/ml) and then incubated for 24 h. Culture supernatants were assayed for IL-2 secretion (pg/ml) by using a CBA assay as described in Materials and Methods. Results are representative of three experiments using cells from different donors, different preparations of Jurkat T cell lines, and different toxin preparations.