Figure legends
Figure 1. IL-35 is abundantly expressed in HCC patients. (A-B)
The level of mRNA expression of IL-35 and IL-35R was calculated by the
log2 array data downloaded from GSE14520. We used the mean expression of
IL-12p35 and Ebi3 to reflect IL-35 and the mean expression of IL-12Rβ2
and gp130 to reflect IL-35R. (C) Serums from 30 HCC cases and 30 heath
volunteers were isolated by centrifuging and IL-35 level was detected
using an AimPlex bead-based immunoassay.
Figure 2. Detection of IL-35 in HCC tissues. (A) Representative
images of immunohistochemical (IHC) staining for of IL-12p35 and Ebi3 in
HCC tumor and non-tumor tissues (50× and 200× respectively). The left
panel was tumor samples and the right panels were non-tumor samples. (B)
Western blot analysis of IL-12p35 and Ebi3 in 30 paired HCC tumor and
non-tumor tissues. Three representative results were shown. (C)
Quantitative data represent the mean ± standard deviation. * represent p
< 0.05, ** represent p < 0.01.
Figure 3. Differentially expressed genes in IL-35-high and -low
expressed groups. (A) The hierarchical clustering of the 4238 genes
differentially expressed in non-tumor samples. (B) The hierarchical
clustering of the 1625 genes differentially expressed in tumor samples.
Each column represents individual tissue samples. Genes and samples were
ordered by centered correlation and complete linkage. The scale
represents the level of gene expression from −3.0 to 3.0 on a log2
scale.
Figure 4. Majority of the differentially expressed genes
correlated with T cell immune function. (A) Venny plot of the
differentially expressed genes showed that there were 1270 genes
overlapped between the tumor and non-tumor samples. (B) Input of all
1270 genes into the online IPA software and the analysis results showed
that most of the canonical pathway was related to T cell immunity. The
top 20 signal pathway names and the log(p-value) are listed.
Figure 5. Characteristics of IL-35 high and low subgroups
determined by the comparison of the gene array transcriptome data.(A–B) The comparison of Foxp3 and IL-10 expression levels between the
IL-35 high and low subgroups. (C) Immune cytolytic activity which
reflected T cell cytotoxic capability was calculated based on transcript
levels of two key cytolytic effectors [granzyme A (GZMA) and perforin
(PRF1)]. (D) Comparison of IFN-γ, the main T cell-derived effective
cytokine, expression between the IL-35 high and low subgroups. (E–F)
The expression comparisons of T cell inhibitory receptors, which reflect
the T cell exhaustion status.