In vivo PD-1 blockade increases resistance to <i>L. sigmodontis</i>.

<p>(A) Timeline of <i>L. sigmodontis</i> infection showing approximate timings of the molts from larval (L3/L4) to adult stages and the development of patency in relation to in vivo antibody treatments and autopsies. (B–D) <i>L. sigmodontis</i>-infected BALB/c IL-4gfp reporter mice were treated with a blocking anti-PD-1 mAb (triangles) or rat IgG (squares) from d28–d43 and their adult parasite burdens assessed at d 60 pi, and their blood Mf levels at d 68 pi. (B) Mf counts per ml of peripheral blood. (C) Number of adult parasites within the PC. (D) Number of live eggs within the uteri of individual female parasites recovered from IgG and anti-PD-1 treated hosts. Panels show one representative experiment of two (B) or pooled data from four independent experiments (C & D). Symbols represent individual mice (B & C) or female parasites (D), and lines represent means (B & C) or medians (D). *** p<0.001 (ANOVA performed on combined data from two (B) or four (C & D) independent experiments).</p

CD4⁺ Th2 cells are conditioned towards a functionally hypo-responsive phenotype during <i>L. sigmodontis</i> infection.

<p>PC, tLN, and splenic CD4⁺ T cells from naïve (open symbols) and <i>L. sigmodontis</i> infected (closed symbols) BALB/c IL-4gfp mice were analyzed at d20, d40 and d60 pi for expression of GFP, IL-4, IL-5 and IL-2. (A) Representative flow plots showing expression of IL-4 protein by PC IL-4gfp⁺CD4⁺ T cells. (B–J) Percentage of PC (B–D), tLN (E–G), and splenic (H–J) IL-4gfp⁺CD4⁺ Th2 cells producing IL-4 (B, E, H), IL-5 (C, F, I), and IL-2 (D, G, J) protein upon stimulation with PMA and ionomycin. Symbols represent individual animals and lines represent means. Panels show one representative experiment of two with 4–6 mice per group. ***p<0.001, ** p<0.05, significant change over time (ANOVA performed using combined data from infected mice from two experiments), ¶ significant pair-wise comparison (p<0.05, Tukey's HSD).</p

<i>L. sigmodontis</i>-elicited alternatively activated macrophages do not suppress T cells via the PD-1 pathway.

<p>PC macrophages from <i>L. sigmodontis</i> infected or naïve BALB/c mice were assessed for expression of PD-L1 and PD-L2 and suppressive function at d 60 pi. (A) Representative histograms showing expression of PD-L1 and PD-L2 on PC F4/80^high macrophages. Grey shaded histograms represent isotype controls, dotted lines show naïve macrophages and solid lines show infected macrophages. (B) Geometric mean fluorescent intensity minus isotype controls of PD-L1 and PD-L2 on F4/80^high macrophages from naïve (open circles) and infected (closed squares) mice. Symbols represent individual animals and lines show mean. Panels show one representative experiment of two with at least 5 mice per group. *** Significant effect of infection, (p<0.001, ANOVA based on combined data from two independent experiments). (C & D) Adherent PC macrophages were purified from individual naïve and <i>L. sigmodontis</i> infected mice and tested in vitro for their ability to inhibit the Ag-specific proliferation of pooled PC IL-4gfp⁺ Th2 cells purified 60 d pi (C) or naïve DO11.10 T cells (D) in the presence of control IgG or blocking antibodies against PD-1, PD-L2, and PD-L2. Panels show mean and SD with 6 mice per group. Open bars show medium control and closed bars represent the presence of LsAg (C) or OVA (D). ** p<0.001 (ANOVA using combined data from two independent experiments).</p

Th2 cell hypo-responsiveness is associated with PD-1.

<p>(A–D) PC and tLN CD4⁺ T cells from naïve (open symbols) and <i>L. sigmodontis</i> infected (closed symbols) BALB/c IL-4gfp mice were analyzed at d20, d40 and d60 pi for expression of GFP, PD-1, CXCR5, and IL-4. (A–B) Percentage (A) and fluorescence intensity (B) of PD-1 expression by PC IL-4gfp⁺CD4⁺ Th2 cells. (C) Representative plots showing expression of PD-1 versus intra-cellular IL-4 by PC IL-4gfp⁺ Th2 cells. (D) Representative staining for GFP and CXCR5 on tLN CD4⁺ T cells. (E) Percentage of tLN CD4⁺GFP⁺CXCR5⁻ Th2 cells expressing PD-1. Symbols represent individual mice, and lines represent means. Panels show one representative experiment out of two. *** Significant increase over time (p<0.001, ANOVA performed using combined data from two experiments), ¶ significant pair-wise comparison, (p<0.05, Tukey's HSD). (F & G) In vitro proliferation minus medium controls (F) and IL-5 production (G) by PC IL-4gfp⁺ Th2 cells purified from <i>L. sigmodontis</i> infected mice 60 d pi and restimulated with LsAg in presence of anti-PD-1 mAb or control rat IgG. IL-4gfp⁺ Th2 cells were pooled from 10–15 mice, and panels show one representative experiment of two. Bars represent means. Due to the necessity for pooling samples, error bars show SD of triplicate cultures for proliferation (F), and it was not possible to calculate error bars or statistics for IL-5 (G). ΔΔΔ p<0.025, (ANOVA performed using combined data from two experiments).</p

The proportion and total number of IL-4gfp⁺ Th2 cells increase as infection progresses.

<p>PC, tLN, and splenic CD4⁺ T cells from naïve (open symbols) and <i>L. sigmodontis</i> infected (closed symbols) BALB/c IL-4gfp mice were analyzed at d20, d40 and d60 pi for expression of GFP. (A) Representative flow plots showing expression of CD4 versus GFP by PC CD4⁺ T cells. (B–G) Percentage of CD4⁺ T cells expressing GFP (B, D, F) and total numbers of IL-4gfp⁺ Th2 cells (C, E, G) within the PC (B & C), tLN (D & E) and spleen (F & G). Symbols represent individual mice and lines represent means. Panels show pooled data from two independent experiments, with 4–6 mice per group. *** Significant increase over time (p<0.001, ANOVA using combined data from two experiments), ¶ significant pair-wise comparison (p<0.05, Tukey's HSD).</p

In vivo PD-1 blockade increases the expansion of IL-4gfp⁺ T cells within the tLN immediately post-treatment.

<p><i>L. sigmodontis</i>-infected BALB/c IL-4gfp reporter mice were treated with a blocking anti-PD-1 mAb (closed triangles) or rat IgG (closed squares) from d 28–37 and IL-4gfp⁺ T cells from the PC (A–D) and tLN (E–H) analysed at d 40. The proportion (A & E) and total number (B & F) of CD4⁺ T cells expressing GFP, as well as the percentage of IL-4gfp⁺ Th2 cells producing IL-4 (C & G) and IL-5 (D & H) protein following simulation with PMA and ionomycin was assessed. Panels show one representative experiment of three with 4–6 mice per group. Symbols denote individual mice and lines represent means. *** Significant effect of infection independent of treatment (p<0.001, ANOVA performed on combined data from three independent experiments). ¶ Significant pair-wise comparison (p<0.05, Tukey's HSD).</p

In vivo PD-1 blockade results in a long-term restoration of Th2 cell functional quality.

<p><i>L. sigmodontis</i>-infected IL-4gfp reporter mice were treated with a blocking anti-PD-1 mAb (closed triangles) or rat IgG (closed squares) from d 28–d43 and Th2 cell quantity and functional quality assessed at d 60. Open symbols represent naïve untreated controls. (A–D) The proportion (A & C) and total number of IL-4gfp⁺ Th2 cells (B & D) in the PC (A & B) and tLN (C & D). Panels show one out of two representative experiments with 4–6 mice per group. Symbols denote individual mice and lines represent means. *** Significant effect of infection independent of treatment (p<0.001, ANOVA performed on combined data from two independent experiments). (E–G) IL-4gfp⁺ Th2 cells were purified from the PC (E & F) and tLN (G) and their ability to proliferate (E) and produce IL-5 (F & G) following in vitro restimulation with LsAg was assessed. Panels show one representative experiment of two. Within each experiment IL-4gfp⁺ Th2 cells were pooled from 6–10 mice per group. Bars show means (E–G). Due to the pooled samples error bars show SD of triplicate cultures for proliferation (E), and there are no error bars or statistics for IL-5 (F & G). ** Significant effect of treatment upon restimulation with LsAg (p<0.01, ANOVA performed using combined data from two independent experiments).</p

In vivo PD-1 blockade results in a reduced incidence of hosts with blood MF and of parasites with uterine Mf.

*<p>Significant difference in incidence compared to IgG controls, p<0.001 (GLM using combined data from two or three experiments).</p