Foxp3⁺ Treg do not mediate suppression of humoral response in <i>L. sigmodontis</i>-infected mice.

<p><b>(A)</b> Diagram of the experimental setup. Six- to eight-week-old BALB/c DEREG mice and littermates were naturally infected with <i>L. sigmodontis</i> or left non-infected. To deplete T_reg, all mice received diphtheria toxin (DT, 0.5 µg i.p.) on three consecutive days starting two days before infection. At day 60 p.i. mice were vaccinated with 100 µg DNP-KLH/Alum i.p. <b>(BE)</b> Peripheral blood lymphocytes were stained for CD4 and Foxp3 to control depletion of T_reg at the day of the third DT injection. <b>(C)</b> Quantification of DNP-specific IgG1, IgG2a and IgG2b in sera of DNP-KLH-vaccinated non-infected BALB/c (white squares, n = 7) and DEREG (light blue squares, n = 10) and <i>L. sigmodontis</i>-infected BALB/c (black squares, n = 9) and DEREG (dark blue squares, n = 9) mice. <b>(D)</b> Diagram of the experimental setup. Six- to eight-week-old BALB/c DEREG mice and littermates were naturally infected with <i>L. sigmodontis</i> or left non-infected. To deplete T_reg, all mice received DT (0.5 µg i.p.) on three consecutive days starting one day before vaccination. At day 60 p.i. mice were vaccinated with 100 µg DNP-KLH/Alum i.p. <b>(F)</b> Quantification of DNP-specific IgG1, IgG2a and IgG2b in sera of DNP-KLH-vaccinated non-infected BALB/c (white squares, n = 7) and DEREG (light blue squares, n = 9), and <i>L. sigmodontis</i>-infected BALB/c (black squares, n = 9) and DEREG (dark blue squares, n = 6) mice. Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Black asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected BALB/c mice. Blue asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected DEREG mice (Two-way ANOVA).</p

Reduced numbers of vaccine-specific B cells in <i>L. sigmodontis</i>-infected mice.

<p>Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> or left non-infected and vaccinated with 30 µg DNP-KLH/Alum s.c. at day 60 p.i. PopLN cells from DNP-KLH-vaccinated non-infected (white bars) and <i>L. sigmodontis</i>-infected (black bars) mice were stained for CD19, PNA-binding, DNP-binding, IgM and IgG at day 14 post vaccination. Mice had been infected for 60 days at the time point of vaccination. <b>(A)</b> Gating strategy of CD19⁺ B lymphocytes. <b>(B)</b>Total cell numbers of DNP⁺PNA⁺ lymph node cells within the CD19⁺ (total), within the IgG⁺IgM⁺CD19⁺ and within the IgG⁺CD19⁺ gate (n = 10–14). Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Asterisks indicate significant differences between non-infected and infected mice (student's t-test).</p

Infection with <i>L. sigmodontis</i> does not suppress humoral response to TI vaccination.

<p>Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> (blue squares, n = 10) or left non-infected (white squares, n = 10) and vaccinated with 100 µg NIP-Ficoll i.p. at day 60 p.i. An additional control group was infected but not vaccinated (closed circles, n = 2). NIP-specific IgM, IgG1 and IgG3 was quantified in sera. Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Asterisks indicate significant differences of the mean of NIP-specific Ig in non-infected and infected mice after vaccination with NIP-Ficoll (Two-way ANOVA).</p

Spatial separation of nematode- and vaccine-specific responses.

<p><b>(A)</b> Diagram of the experimental setup. Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> or left non-infected and vaccinated with 30 µg DNP-KLH/Alum s.c. at day 60 p.i. <b>(B)</b> Diagram of spatial separation of immune responses. <b>(C)</b> Splenocytes and draining popLN cells were cultured for 21 days at 37°C. Supernatants were analysed for DNP-specific and <i>L. sigmodontis</i>-specific IgG1, IgG2a and IgG2b. <b>(D)</b> Quantification of DNP-specific IgG1 and IgG2b in sera of <i>L. sigmodontis</i>-infected (blue squares, n = 14) and non-infected (white squares, n = 10) vaccinated mice. Control mice were infected but not vaccinated (black circles, n = 2). Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected mice (Two-way ANOVA).</p

Reduced numbers and frequency of vaccine-induced T_FH in <i>L. sigmodontis</i>-infected mice.

<p>Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> or left non-infected and vaccinated with 30 µg DNP-KLH/Alum s.c. at day 60 p.i. PopLN cells derived from DNP-KLH-vaccinated non-infected (white bars) and <i>L. sigmodontis</i>-infected (black bars) mice were stained for CD4, CD44, PD1, CXCR5, ICOS and Foxp3 at day 14 post vaccination. <b>(A)</b> Gating strategy of T_FH. <b>(B)</b> Frequency of CD4⁺ T cells (n = 20; pooled data derived from four independent experiments). <b>(C)</b> Frequencies and total cell numbers of PD1⁺CXCR5⁺ T_FH cells within the CD4⁺CD44⁺ activated T helper cells (n = 14; pooled data derived from three independent experiments). <b>(D)</b> ICOS expression of PD1⁺CXCR5⁺ T_FH cells within the CD4⁺CD44⁺ activated T helper cells (n = 10; pooled data derived from two independent experiments). <b>(E)</b> Foxp3 expression of PD1⁺CXCR5⁺ T_FH cells within the CD4⁺CD44⁺ activated T helper cells (n = 5; data from one experiment representative for two independent repeats). Asterisks indicate significant differences between non-infected and infected mice (student's t-test).</p

Opposing impact of <i>L. sigmodontis</i> adults and MF on humoral response to TD vaccination.

<p><b>(A)</b> Six- to eight-week-old BALB/c mice received 10,000 MF i.v. (green squares, n = 10) or were left untreated (white squares, n = 10). Both groups were vaccinated with 100 µg DNP-KLH/Alum i.p. immediately. Control mice (black circles, n = 2) received 10,000 MF i.v. but were not immunized. DNP-specific IgG1, IgG2a and IgG2b was quantified in sera. Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Asterisks indicate significant differences of the mean of DNP-specific Ig in non-treated and MF-treated mice (Two-way ANOVA). <b>(B)</b> Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> (closed squares) or left non-infected (white squares, n = 8). Day 60 <i>L. sigmodontis</i>-infected mice received 10,000 MF i.v. additionally (purple squares, n = 10) and all groups were vaccinated with 100 µg DNP-KLH/Alum i.p. immediately. Control mice were also vaccinated with 100 µg DNP-KLH/Alum i.p. and either infected for 60 days (blue squares, n = 2) or received purified MF only (green squares, n = 4). Non-vaccinated control mice were infected for 60 days and received MF i.v. (black squares, n = 2) or just received MF i.v. (black circles, n = 2). DNP-specific IgG2a was quantified in sera. Results are expressed as mean ± SEM of pooled data derived from two experiments. Black asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected BALB/c mice. Purple asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected MF-treated mice. Green asterisks indicate significant differences of the mean of DNP-specific Ig in MF-treated non-infected and MF-treated day 60 <i>L. sigmodontis</i>-infected mice (student's t-test).</p

Suppressed IgG response to TD vaccination 16 weeks after termination of <i>L. sigmodontis</i> infection.

<p><b>(A)</b> Diagram of the experimental setup. Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> (closed squares and circles) or left non-infected (white squares). Mice were allowed to naturally clear the infection. Microfilaraemia was monitored and the time point no MF was detectable in the blood of any previously infected mouse was defined as termination of infection. 16 weeks later non-infected mice (open squares, n = 14) and mice, which had terminated the infection (orange squares, n = 15) were vaccinated with 100 µg DNP-KLH/Alum i.p. An additional control group was allowed to clear the infection, but was not DNP-KLH/Alum-vaccinated (closed circles, n = 2). <b>(B)</b> Microfilaraemia of mice naturally infected with <i>L. sigmodontis</i> of one experiment representative for three independent repeats. <b>(C)</b> DNP-specific IgG1 and IgG2b was quantified in sera. Results are expressed as mean ± SEM of pooled data derived from three independent experiments. Asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected mice (Two-way ANOVA).</p

Infection with <i>L. sigmodontis</i> suppresses humoral response to TD vaccination.

<p><b>(A)</b> Diagram of the experimental setup. Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> (closed squares) or left non-infected (open squares; n = 10) and were vaccinated with 100 µg DNP-KLH/Alum i.p. at <b>(B)</b> day 0 (grey, n = 8–10), <b>(C)</b>) day 14 (black, n = 10), <b>(D)</b> day 30 (red, n = 10) or <b>(E)</b> day 60 (blue, n = 10) p.i. An additional control group was infected but not vaccinated (closed circles, n = 2–4). DNP-specific IgG1, IgG2a and IgG2b was detected in sera. Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected mice after vaccination with DNP-KLH (Two-way ANOVA).</p

Role of IL-9 during <i>S. ratti</i> infection in Treg-depleted BALB/c mice.

<p><b>A:</b> Experimental setup is shown. <b>B:</b> Number of parasitic adults in the small intestine of BALB/c (white), Treg-depleted BALB/c DEREG (black), α-IL-9 treated BALB/c (light blue) and α-IL-9 treated Treg-depleted BALB/c DEREG (dark blue) mice on day 6 p.i. <b>C:</b> Number of parasitic adults in the small intestine of BALB/c (white), Treg-depleted BALB/c DEREG (black), α-IL-13 treated BALB/c (light green) and α-IL-13 treated Treg-depleted BALB/c DEREG (dark green) mice on day 6 p.i. <b>D:</b> Concentration of MMCP-1 in the serum of infected mice at indicated time points. Shown are the combined results of four (<b>BD</b>) or two (<b>C</b>) independent experiments (BD: n = 14, C: n = 8). Asterisks indicate significant difference of the mean analyzed by (<b>BC</b>) students <i>t</i> test or (<b>D</b>) one-way ANOVA with Bonferroni post test (*** p≤0.001).</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