Serum soluble B7-H4 is a prognostic marker for patients with non-metastatic clear cell renal cell carcinoma - Fig 3

<p>Progression-free survival (A) and overall survival (B) of non-metastatic clear cell renal cell carcinoma patients with and without serum soluble B7-H4.</p

Patient characteristics.

<p>Patient characteristics.</p

Detection of serum soluble B7-H4 in patients with non-metastatic clear cell renal cell carcinoma.

<p>The concentration of the soluble B7-H4 in the HDs and the patients diagnosed with renal cell carcinoma. Sera from 108 renal cancer patients and 108 HDs were diluted 1:10 in PBS and tested by ELISA as described in <a href="http://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1000166#s2" target="_blank">Materials and Methods</a>. The data were analyzed using the Mann-Whitney U test followed by multiple regression analysis (<i>p</i><0.005).</p

Univariate and multivariate analyses of risk factors predicting overall survival in patients with renal cancer.

<p>Univariate and multivariate analyses of risk factors predicting overall survival in patients with renal cancer.</p

Univariate and multivariate analyses of risk factors predicting progression free survival in patients with renal cancer.

<p>Univariate and multivariate analyses of risk factors predicting progression free survival in patients with renal cancer.</p

The association between serum soluble B7-H4 and peripheral blood neutrophils.

<p><b>A:</b> The data were analyzed using the Mann-Whitney U test followed by multiple regression analysis (<i>p</i><0.005). <b>B:</b> The data summarize the findings of 68 RA patients and were analyzed using Spearman's rank test. y = 0.07x−19.6, R² = 0.428, <i>p</i><0.001.</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

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

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