Nematode-Derived Proteins Suppress Proliferation and Cytokine Production of Antigen-Specific T Cells via Induction of Cell Death

<div><p>In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of <i>Onchocerca</i><i>volvulus</i>-derived excreted/secreted proteins. Addition of two recombinant <i>O</i><i>. volvulus</i> proteins, abundant larval transcript-2 (<i>Ov</i>ALT-2) and novel larval transcript-1 (<i>Ov</i>NLT-1) to cell cultures of T cell receptor transgenic CD4⁺ and CD8⁺ T cells suppressed antigen-specific stimulation <i>in vitro</i>. Ovalbumin-specific CD4⁺ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of <i>Ov</i>ALT-2 or <i>Ov</i>NLT-1 displayed reduced DNA synthesis quantified by ³H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8⁺ OT-I T cells was suppressed by <i>Ov</i>ALT-2 and <i>Ov</i>NLT-1. In contrast, another recombinant <i>O</i><i>. volvulus</i> protein, microfilariae surface-associated antigen (<i>Ov</i>103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from <i>O</i><i>. volvulus</i>, which interfere with the function of antigen-specific T cells <i>in vitro</i>.</p> </div

Nematode-derived ESP suppress OVA-specific T cell function.

<p>A) Coomassie blue-staining of recombinant proteins after purification and separation by SDS-PAGE. B) Spleen cells from TCR transgenic DO11.10 (B, C), OT-II (D) or OT-I (E, F) mice were stimulated with 10 ng/mL OVA_323-339 (B-D) or OVA_257-264 (E, F) peptide in the presence of ESP or LPS in increasing concentrations as indicated on the x-axis. OVA-specific proliferation of DO11.10 (B, C) and OT-II (D) T cells was measured after 72 h as ³H-thymidine incorporation. IL-2 (E) and IFN-γ (F) secretion by OT-I T cells was analyzed by ELISA. Error bars show SD of triplicates. Data shown are representative for five (B, C) or two (D, E, F) independent experiments. Asterisks indicate significant differences in the mean of <i>Ov</i>103 or LPS to <i>Ov</i>7 or <i>Ov</i>ALT-2 or <i>Ov</i>NLT-1 (*** p < 0.001).</p

No impact of <i>Ov</i>7 on proliferation of OT-II T cells <i>in vivo</i>.

<p>CFSE-labeled OT-II spleen cells were adoptively transferred i.v. into C57BL/6 mice. 20 µg <i>Ov</i>7 was injected i.p. at the day of the transfer and one day later. Mice were sacrificed 2 d after <i>in vivo</i> activation with 50 µg OVA_323-339 peptide and proliferation was analyzed. A) Representative dot blot showing the gating strategy. We gated on CD4⁺CFSE⁺ cells to calculate the frequency of OT-II T cells in the spleen (B) and analyzed the number of cell divisions OT-II T cells have undergone (C). Shown is the frequency of OT-II T cells that did not divide (M₀) or divided once (M₁), twice (M₂), or three times and more (M₃) after stimulation in the presence of PBS (white bars) or <i>Ov</i>7 (red bars). Shown are combined results from two independent experiments with six mice per group, error bars show SEM. Analysis with students t test revealed no significant statistical difference of the mean (p = 0,73 (B) and p = 0,13 (C, three and more division cycles). This result was reproduced in two other independent experiment using five control and seven <i>Ov</i>7-treated mice.</p

Nematode-derived proteins <i>Ov</i>NLT-1 and <i>Ov</i>ALT-2 induce apoptosis in spleen cells.

<p>ESP were separated into protein and "flow through" using Amicon filter. Spleen cells were isolated from BALB/c mice and incubated with increasing concentration of <i>Ov</i>103, <i>Ov</i>ALT-2 and <i>Ov</i>NLT-1 or the corresponding low molecular weight filtrate ("flow through"). After 6 h of incubation cells were stained with 7-AAD and annexin V. Apoptotic cells (annexin V⁺) were further identified by 7-AAD expression as early apoptotic cells (7-AAD^-) or late apoptotic (7-AAD⁺). Representative dot blots are shown for spleen cells after incubation with 5 µg/mL of nematode-derived <i>Ov</i>103 (A) as control peptide, <i>Ov</i>ALT-2 (B) or <i>Ov</i>NLT-1 (C). Statistical analysis of the frequencies of early (D) and late (E) apoptotic cells. Results show one individual experiment and are representative for two independent experiments.</p

<i>Ov</i>ALT-2 suppresses OVA-specific OT-II T cell proliferation <i>in</i><i>vitro.</i>

<p>Spleen cells were isolated from OT-II mice and labeled with CFSE. Cells were stimulated with 50 ng/mL OVA_323-339 peptide in the presence of nematode-derived proteins <i>Ov</i>103, <i>Ov</i>ALT-2 or <i>Ov</i>7 in increasing concentrations as indicated on the x-axis. After 72 h cells were stained with anti-CD4 mAb and proliferation of CD4⁺CFSE⁺ T cells was analyzed by flow cytometry as percentage of dividing T cells (marker M1). Shown is a representative dot blot of OT-II spleen cells after incubation with 7.5 µg/mL <i>Ov</i>103 (A) or <i>Ov</i>ALT-2 (B) in the presence of OVA-peptide. C) Graph shows percentage of dividing T cells in the presence of indicated concentration of ESP. Data are presented as the mean of combined results derived from two independent experiments, error bars show SEM. Asterisks indicate significant differences in the mean of <i>Ov</i>103 to <i>Ov</i>7 or <i>Ov</i>ALT-2, respectively (** p < 0.01; *** p < 0.001).</p

Suppression of OVA-specific T cell proliferation by proteinase K digested <i>O</i>. <i>vovlvulus</i> ESPs.

<p>A) ESPs were separated using a 3 kDa Amicon filter into the protein and the "flow through" containing molecules smaller than 3 kDa. Spleen cells derived from DO11.10 mice were labeled with CFSE and stimulated with 10 ng/mL OVA_323-339 peptide in the presence of ESP or "flow through". Proliferation was analyzed after 72 h by flow cytometry and is depicted as frequency of dividing CD4⁺ T cells. B) SDS-PAGE and Coomassie blue staining of recombinant nematode-derived proteins that were either native or digested with proteinase K. C) Thymidine incorporation of DO11.10 cells after incubation with native or proteinase K digested nematode-derived proteins. Shown are results from one experiment representative for two independent repeats. Error bars show SEM of triplicates and asterisks indicate significant differences of the mean (*** p < 0.001).</p

Immune response in the presence and absence of Treg during <i>S. ratti</i> infection.

<p>BALB/c (white bars), BALB/c DEREG (black bars), C57BL/6 (light grey bars), and C57BL/6 DEREG (dark grey bars) mice were treated with DT on three consecutive days starting one day before <i>S. ratti</i> infection. <b>A</b>: Polyclonal IgE in the serum was quantified at the indicated time points. Shown is one experiment (n = 6 per time point) that is representative for one independent repeat. Asterisks indicate significant difference of the mean analyzed by one-way ANOVA with Bonferroni post test (*** p≤0.001). <b>B: </b><i>S. ratti</i>-specific IgM was quantified in the serum on day 7 p.i. Shown are the combined results of three independent experiments (n = 9–11). Asterisks indicate significant difference of the mean analyzed by students <i>t</i> test (* p≤0.05). <b>C–G:</b> Mice were sacrificed on day 6 p.i. and splenocytes (2×10⁵) were stimulated for 72 h with <i>S. ratti</i> lysate (upper panel) or with α-CD3 (lower panel). IL-13 (<b>C</b>), IL-4 (<b>D</b>), IL-10 (<b>E</b>), IL-3 (<b>F</b>) and IL-9 (<b>G</b>) in the supernatant were quantified by ELISA. Splenocytes that were cultivated in the presence of medium only did not secrete cytokines (data not shown). Shown are the combined results of two independent experiments (n = 8) for <b>C–F</b> and combined results of four independent experiments (n = 14–16) for <b>G</b>. Asterisks indicate significant difference of the mean analyzed by one-way ANOVA with Bonferroni post test (* p≤0.05, ** p≤0.01, *** p≤0.001).</p

Improved resistance in Treg-depleted BALB/c DEREG mice in the absence of granulocytes.

<p>BALB/c (white bars), BALB/c DEREG (black bars), C57BL/6 (light grey bars), and C57BL/6 DEREG (dark grey bars) mice were treated with DT on 3 consecutive days starting one day before <i>S. ratti</i> infection. <b>A:</b> Percentage of Gr1⁺CD11b⁺ cells in PBL was analyzed at the indicated time points. <b>BC:</b> BALB/c (white bars), BALB/c DEREG (black bars) were treated with DT and received an additional injection of anti-Gr1 mAb or isotype control one day before <i>S. ratti</i> infection. Percentage of Gr1⁺CD11b⁺ cells in PBL of infected non-depleted mice was measured on day 1 p.i. (<b>B</b>) and number of adult parasitic females in the small intestine was counted on day 6 p.i. (<b>C</b>). Shown are the combined results of two independent experiments (n = 6). Asterisks indicate significant difference of the mean analyzed by students <i>t</i> test (* p≤0.05).</p

Depletion of IL-9 during <i>S. ratti</i> infection in BALB/c DEREG mice at different time points.

<p><b>A:</b> Experimental setup for α-IL-9 treatment either early (blue arrows) or late (grey arrows) in <i>S. ratti</i> infection is shown. <b>B:</b> Graph shows number of parasitic adults in the small intestine of BALB/c (white), Treg-depleted, isotype treated BALB/c DEREG (black), Treg-depleted early α-IL-9 treated BALB/c DEREG (dark blue) and Treg-depleted late α-IL-9 treated BALB/c DEREG (dark grey) mice on day 6 p.i. Shown are the combined results of three independent experiments (n = 14). <b>C:</b> Concentrations of MMCP-1 in the serum of infected mice on day 4 and day 6 p.i. are shown as combined results of two independent experiments (n = 9). Asterisks indicate significant difference of the mean analyzed by one-way ANOVA with Bonferroni post test (* p≤0.05, *** p≤0.001).</p

IL-9 administration or depletion during <i>S. ratti</i> infection.

<p><b>A:</b> Experimental setup for IL-9 treatment is shown. <b>B–C:</b> Number of parasitic adults in the small intestine of infected non-treated BALB/c (<b>B</b>, white) and C57BL/6 (<b>C</b>, light grey) mice or infected and IL-9 treated BALB/c (<b>B</b>, black) and C57BL/6 (<b>C</b>, dark grey) mice on day 6 p.i. Shown are the combined results of three independent experiments (n = 12). <b>D:</b> Experimental setup for α-IL-9 treatment is shown. <b>E–F:</b> Number of parasitic adults in the small intestine of infected isotype treated BALB/c (<b>D</b>, white) and C57BL/6 (<b>E</b>, light grey) mice or infected and α-IL-9 treated BALB/c (<b>D</b>, black) and C57BL/6 (<b>E</b>, dark grey) mice on day 6 p.i. <b>G–H</b>: Concentration of MMCP-1 in the serum of infected isotype treated or α-IL-9 treated BALB/c (<b>G</b>) and C57BL/6 (<b>H</b>) mice on day 4 and day 6 p.i. Shown are the combined results of two independent experiments (n = 8). Asterisks indicate significant differences of the mean analyzed by students <i>t</i> test (** p≤0.01, *** p≤0.001).</p