Trypanosoma cruzi subverts host cell sialylation and may compromise antigen-specific CD8+ T cell responses

Abstract

Upon activation, cytotoxic CD8(+) T lymphocytes are desialylated exposing beta-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8(+) T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8(+) T cell surface, thereby dampening antigen-specific CD8(+) T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8(+) T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8(+) T cell surface. The cytotoxic activity of antigen-experienced CD8(+) T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase-mediated resialylation in vitro and in vivo. Inhibition of the parasite's native trans-sialidase activity during infection strongly decreased CD8(+) T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8(+) T cell interactions with peptide-major histocompatibility complex class I complexes. CD8(+) T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism.

FIGURE 1.

aTS resialylates CD8⁺ T cells from T. cruzi-infected mice. A, PNA binding profile of splenic CD8⁺ T cells isolated from C57BL/6 uninfected mice (a), P. berghei-infected C57BL/6 mice (b), uninfected BALB/c mice (c), T. cruzi-infected BALB/c mice treated with PBS (d), or T. cruzi-infected BALB/c mice treated with aTS (e) and PNA binding profile of splenic CD4⁺ T cells isolated from uninfected BALB/c mice (f), T. cruzi-infected BALB/c mice treated with PBS (g), or T. cruzi-infected BALB/c mice treated with aTS (h). Spleen cells were isolated on dpi 8, stained for flow cytometry, gated for CD8⁺ (a–e) or CD4⁺ (f–h) cells, and analyzed for PNA binding. B, mean fluorescence intensity (MFI) of PNA binding to CD8⁺ T and CD4⁺ T cells isolated from mice treated as in A. Bars, the average of three mice. Results are shown as mean of each group of mice ± S.D. *, p < 0.05; **, p < 0.001 compared with other groups. Note the decrease in PNA binding to CD8⁺ T cells retrieved from infected mice treated with aTS. C, TS activity was detected in the sera of uninfected BALB/c mice treated with aTS (■), T. cruzi-infected BALB/c mice (▴), and T. cruzi-infected BALB/c mice treated with aTS (●). Each symbol represents data obtained from three mice. Mice were inoculated intraperitoneally on day 0 with 1 × 10⁴ trypomastigotes from T. cruzi Y-strain and/or injected intravenously with 30 μg of aTS 1 h before the infection, as well as on dpi 2 and 3. Untreated controls received only PBS. Blood was collected from the tail vein on the days indicated. TS activity was determined as described under “Experimental Procedures.”

FIGURE 2.

aTS resialylates activated CD8⁺ T cells. Splenic CD8⁺ T cells were isolated from uninfected BALB/c mice (A), T. cruzi-infected BALB/c mice treated with PBS (B), or T. cruzi-infected BALB/c mice treated with aTS (C), as described in the legend to Fig. 1C. CD8⁺ T cells were isolated on dpi 15 and stained with MAA (which recognizes α2,3-bound sialic acid) and with anti-CD44 (an activation marker). The numbers indicate the percentage of cells in each quadrant. Mean fluorescence intensity (MFI) of MAA and anti-CD44 are shown at the right and top of each panel, respectively. D, the bar graph shows the mean of mean fluorescence intensities from each group of three mice treated as in A ± S.D. a, CD8⁺ T cells from uninfected BALB/c mice; b, CD8⁺ T cells from T. cruzi-infected BALB/c mice treated with PBS; c, CD8⁺ T cells from T. cruzi-infected BALB/c mice treated with aTS. *, p < 0.05; **, p < 0.001 compared with other groups. Observe the increase in the MAA binding to CD8⁺ T cells from infected mice treated with aTS, indicating that aTS resialylates activated CD8⁺ T cells.

FIGURE 3.

CD43 is a sialic acid acceptor on CD8⁺ T cell surface. A, purified CD8⁺ T cells from uninfected C57BL/6 wild type mice (a), uninfected ST3Gal-I sialyltransferase-deficient C57BL/6 mice (b), and T. cruzi-infected ST3Gal-I sialyltransferase deficient C57BL/6 mice (c) were stained with the mAb S7, which recognizes sialylated forms of CD43. d, isotype control. B, results are shown as the mean of each group of mice ± S.D. *, p < 0.05; **, p < 0.06. Note that infection of ST3Gal-I sialyltransferase-deficient mice with T. cruzi partially restores the anti-CD43 S7 mAb epitope.

FIGURE 4.

aTS-mediated resialylation decreases CD8⁺ T cell-mediated cytotoxicity in vitro. A, CD8⁺ T cells were isolated from uninfected mice (a, ■), or T. cruzi-infected mice and were either treated with PBS (b, ●), or with 50 milliunits of aTS plus 100 μg/ml of fetuin as sialic acid donor for 60 min at 37 °C (c, ♦). A2OJ cells (H-2^d), used as targets, were previously treated with the fluorogenic dye CFSE (1 and 10 μm). The population treated with 10 μm was additionally incubated with the immunodominant H-2K^d-restricted synthetic peptide IYNVGQVSI. The population treated with 1 μm (control cells) remained untreated. After incubation for 20 h over a range of effector/target ratios, lysis of target cells was quantified by flow cytometry. B, cPNA binding profile of CD8⁺ T cells isolated from uninfected mice (a) or from T. cruzi-infected mice either treated with PBS (b) or with 0.05 units of aTS plus 100 μg/ml of fetuin as in A (c).

FIGURE 5.

aTS-mediated resialylation decreases Ag sensitivity in vivo. BALB/c mice were either uninfected (a) or infected with T. cruzi and treated with PBS (b) or aTS (c), as described under “Experimental Procedures.” A, three groups of mice were subjected to an in vivo Ag-specific CD8⁺ T cell cytotoxicity assay. For this purpose, target cells loaded with the synthetic peptide IYNVGQVSI were inoculated, and Ag-specific lysis was assessed on the basis of their disappearance (CFSE, 10 μm); a control population not loaded with peptide and inoculated in parallel was spared (CFSE, 1 μm), confirming Ag specificity. B, quantitation of the cytotoxic activity in A. Cytotoxicity of CD8⁺ T cells from infected mice treated with PBS (b) was significantly higher than that of cells from mice treated with aTS (c). *, p < 0.05. C, glycophenotypic analysis of CD8⁺ T cells from the three experimental groups, evaluated by FCM using the lectin PNA. Note that treatment with aTS decreases PNA binding to CD8⁺ T cells.

FIGURE 6.

An inactive TS inhibits resialylation mediated by endogenous aTS. A, incorporation of Neu⁷(³H)Ac by T. cruzi epimastigotes incubated with [Neu⁷(³H)Ac]fetuin (3.26 × 10⁶ cpm/ml) in the presence of TS_Y342H (0, 10, 50, 100, and 200 μg/ml). B, PNA binding to T. cruzi epimastigotes incubated with fetuin (2 mg/ml) in the presence of 0 μg (gray histogram), 10 μg (long dashed line), 50 μg (dashed line), 100 μg (heavy dashed line), and 200 μg (solid line) of TS_Y342H. C, PNA binding to splenic CD8⁺ T cells isolated from uninfected mice (a), from T. cruzi-infected mice treated with PBS (b), from T. cruzi-infected mice treated with TS_Y342H (c), from T. cruzi-infected mice treated with aTS (d), and from uninfected mice treated with TS_Y342H (e). Mice were inoculated intraperitoneally on day 0 with 1 × 10⁴ trypomastigotes from T. cruzi Y-strain. Treated mice were injected intravenously with 30 μg of aTS or 30 μg of TS_Y342H 1 h before the infection as well as on dpi 2 and 3. Untreated controls received only PBS. D, bar graph showing the mean of mean fluorescence intensities (MFI) from each group of three mice treated as in C ± S.D. *, p < 0.05; **, p < 0.001 compared with control group. Note the increase in PNA binding to CD8⁺ T cells from infected mice treated with TS_Y342H.

FIGURE 7.

TS_Y342H decreases T. cruzi virulence. A, parasitemia of T. cruzi-infected mice treated with PBS (◊), TS_Y342H (□), or aTS (●), as described under “Experimental Procedures.” Data are the mean ± S.D. of the parasites counting on 10 mice/group. Shown is one of three experiments performed. B, survival index of T. cruzi-infected mice treated as above. Survival data are pooled from two different experiments with 10 mice each. BALB/c mice received 30 μg of aTS or TS_Y342H 1 h before and at 2 and 3 dpi with T. cruzi. Data were compared with a control-infected group treated with PBS. *, p < 0.001.