PDCD1 and IFNL4 genetic variants and risk of developing hepatitis C virus‐related diseases

Abstract Background Genetic variants of IFNL4 and PDCD1 genes have been shown to influence the spontaneous clearance of hepatitis C virus (HCV) infection. We investigated the IFNL4 rs12979860 and the PDCD1 polymorphisms in 734 HCV‐positive patients, including 461 cases with liver disease of varying severity and 273 patients with lymphoproliferative disorders to determine the association of these genes with patient's outcome. Methods Expression levels of PDCD1 mRNA encoded by haplotypes were investigated by quantitative PCR in hepatocellular carcinoma (HCC) tissue and peripheral blood mononuclear cells. Flow cytometry was used to detect PD‐1 and its ligand PD‐L1. Results The frequency of IFNL4 rs12979860 C/T or T/T genotypes was significantly higher in patients with HCV‐related diseases than blood donors (P < .0001). Patients expressing the IFNλ4 variant with one amino acid change that reduces IFNλ4 secretion was found increased in frequency in HCV‐related diseases compared to HCC PDCD1 mRNA levels in HCC tissue were significantly higher in cases carrying the PD‐1.3 A or the PD‐1.7 G allele (P = .0025 and P = .0167). Linkage disequilibrium (LD) between PD‐1.3 and IFNL4 was found in patients with mixed cryoglobulinaemia (MC) only (LD = 0 in HCC; LD = 72 in MC). PBMCs of MC patients expressed low levels of PD‐L1 in CD19+IgM+B cells and of PD‐1 in CD4+T cells suggesting the involvement of regulatory B cell‐T cell interaction to the pathogenesis of MC. Conclusion Collectively, our data indicate an important contribution of IFNλ4 expression to the development of HCV‐related HCC and an epistatic contribution of IFNL4 and PDCD1 in MC. Lay summary Studies of IFNL4 and PDCD1 genes are helpful to better understand the role of host genetic factors and immune antigens influencing the outcome of HCV‐related diseases. Our data support an association between the expression of IFNλ4, which prevents the expression of IFNλ3, with all the different HCV‐related diseases studied, and besides, evidence that a higher IFNλ4 expression is associated with hepatocellular at a younger age. The expression pattern of low PD‐L1 on B cells and high PD‐1 on CD4+T‐cells in patients with HCV‐positive cryoglobulinaemia suggests a critical role of the PD‐1/PD‐L1 signaling in modulating B cell‐T cell interaction in this lymphoproliferative disease.


| INTRODUC TI ON
and as cause of a variety of autoimmune diseases. 8 Immunotherapies that inhibit the interaction between PD-1 and its ligands PD-L1 and PD-L2 have been shown to confer a substantial survival benefit in patients with HCC 9,10 as well as in those with haematological 11 or infectious diseases. 12 The IFNL4 gene encodes the IFNλ4 protein that increases the expression of IFN-stimulated genes (ISGs) in the liver. The receptors for IFNλ4 are expressed on selective cell types like hepatocytes and some immune cells. 13 IFNλ4 is expressed only in individuals carrying the IFNL4 rs368234815 ∆G allele, which creates an open reading frame. In contrast, individuals with the rs368234815 T/T allele do not express the protein because of a premature stop codon. Other IFNL4 polymorphisms include a common variant (rs117648444) resulting in a P70S amino acid change and two others (L79F and K154E), which lead to lower IFNλ4 secretion and antiviral activity. 14,15 High IFNλ4 expression is strongly associated with spontaneous HCV clearance and a lower response to IFN-based treatment. 16,17 The IFNL4 rs12979860[T] allele variant may be used as an alternative to IFNL4 rs368234815 ΔG variant to test for IFNL4 expression since their almost complete linkage is well established. 18 IFNλ4 was recently found to have selective pressure on the HCV genome. 19 Genome-wide association studies of patients with hepatitis infection identified polymorphisms in IFNL4 and PDCD1 genes conferring a higher risk of chronic infection, HCV-related diseases and treatment response. 8,20,21 Data from patients with HCV-related lymphoproliferative diseases are limited, and there is a clear need to verify the role of IFNλ4 in the Caucasian population. The rs11568821 (PD-1.3) was associated with the development of autoimmune diseases in Europeans and Mexicans but not in African Americans. 22 In addition, the incidence of HCC is higher among Asian Americans than White Americans 23 , while a high incidence of HCV-related MC was present in the Mediterranean basin, 24 suggesting that genetic polymorphisms and environmental risk factors may influence the risk of these diseases. Investigation of IFNL4 and PDCD1 immunogenetic profiles in patients with different HCV-related diseases could help to elucidate these pathologies. We therefore studied IFNL4 and PDCD1 genetic variants distribution and their relative expression in Italian patients diagnosed with different HCV-related diseases. Results could underlie the complex immune interactions leading to different clinical outcomes in chronic HCVpositive patients.

| PATIENTS AND ME THODS
This study investigated data and biological samples obtained from Lay summary: Studies of IFNL4 and PDCD1 genes are helpful to better understand the role of host genetic factors and immune antigens influencing the outcome of HCV-related diseases. Our data support an association between the expression of IFNλ4, which prevents the expression of IFNλ3, with all the different HCV-related diseases studied, and besides, evidence that a higher IFNλ4 expression is associated with hepatocellular at a younger age. The expression pattern of low PD-L1 on B cells and high PD-1 on CD4+T-cells in patients with HCV-positive cryoglobulinaemia suggests a critical role of the PD-1/PD-L1 signaling in modulating B cell-T cell interaction in this lymphoproliferative disease.

K E Y W O R D S
cryoglobulinaemia, hepatitis virus C, hepatocellular carcinoma, IFNλ4, PD-1 we obtained from clinical records the following data: age at diagnosis, sex, HCV positivity, HCV viral load and genotype, and specific diagnosis. Data on blood donors' age and sex were collected.
In agreement with the Centre for Disease Controls and Prevention (CDC) guidelines, we defined as CHC a patient with long-lasting HCV infection who had persistent viraemia for more than 6 months after initial exposure diagnosed by blood test. Such test is based on the detection of serum anti-HCV antibodies performed by an enzyme immunoassay (III-generation EIA) against antigens from the HCV-core and from the HCV-nonstructural regions and is confirmed by the qualitative analysis that detects the presence of HCV RNA (Cobas Amplicor HCV assay). All patients we have included in the CHC group were positive to both these tests. The viral load was determined in 192 patients (Table 1) to monitor their response to treatment. Data reported in Table 1 were referred to tests performed before treatment.
The amino acid changes G58R and P70S in the IFNλ4 protein flank residue N61, whose glycosylation is required for secretion of active IFNλ4 protein. 50 Consequently, these variants lower the antiviral activity of the reference IFNλ4. 51

| Genotyping and sequencing of IFNL4 and PDCD1
Genotyping of IFNL4 rs12979860 and rs117648444 (C>T, P70S) was performed using custom TaqMan SNP genotyping assays (Applied Biosystems) on a 7900HT Fast Real-Time PCR system (Applied Biosystems).
To sequence IFNL4 (from intron 1 to exon 5), we first amplified the gene from genomic DNA by PCR in a reaction volume of 25 μl (200 ng dNTPs and 0.5 U GoTaq DNA Polymerase, Promega) using primers as reported in Table S2 and cycling conditions as described in reference 4. Sequencing was performed as previously reported. 52 To genotype PDCD1 SNPs, genomic DNA (30-300 ng) was amplified in a 50 μL reaction mixture containing 10 pmol of each primer (

| LD analysis
Linkage disequilibrium (LD) was estimated among PD-1, that is, PD-1.3, PD-1.5, PD-1.7 and IFNL4 rs12979860 SNP. Distribution of the Lewontin's coefficient D′ and correlation coefficient r 2 was calculated as the measures of LD using the SHEsis online tool (http://analy sis.bio-x.cn). LD within each genomic region was explored and the extent of statistical significance of each pairwise association was represented by a scale of colour intensity.

| Analysis of PDCD1 mRNA expression
The expression levels of haplotypes within PDCD1 gene were evaluated by real-time quantitative PCR using total RNA from PBMCs of selected cases based on their PDCD1 haplotype (n = 12) and from selected HCC tissues (n = 12). Briefly, total RNA (500 g) was reverse transcribed in a 20 μL volume with Superscript II RNase H and an oligo d(T) primer (Life Technologies).

| IFNL4 and PDCD1 genotype frequencies
Patients and controls were genotyped for five SNPs having possible immune-modulating effects, including rs12979860 in IFNL4 and four

PD-1.5 (%) rs2227981
# PD-1.6 (%) rs10204525 log-additive 2.04 (1.33-3.13) P < .0001 SNPs in PDCD1 genes (Table S1). The PDCD1 SNPs, termed PD-1.3 (rs11568821), PD-1.5 (rs2227981), PD-1.6 (rs10204525) and PD-1.7 (rs7421861), had a MAF ≥0.05 in the reported Toscani population in Italy as well as in BD (Table S1). Genotype frequencies of the five SNPs are given in Table 2, in Table S3 according to fibrosis and in Sidak's corrections. The association was more evident in patients with liver diseases, who had a lower frequency of the protective genotype C/C than patients with a lymphoproliferative disease (25% vs 38%; Table 2). T/T genotype showed a positive trend with the increase in liver disease severity (P = .018. Table S5) and apparently not affected by a sustained response to anti-HCV treatment (Table S6).
The four PDCD1 SNPs showed no association when genotype frequencies were compared between all HCV-positive patients and BD ( and CHC with mild-moderate fibrosis (52%); chi-squared test for trend for PD-1.7 = 5.596, P = .018 (Table S5). SVR did not significantly affect the genotype association (Table S6). Patients with a lymphoproliferative disease had a higher frequency of the PD-1.5 C/C allele (34%) than did patients with CHC (26%); in an overdominant model, OR = 0.60, P = .012, P-value remained statistically significant after both Bonferroni and Sidak's corrections.

| IFNL4 mutational analysis
The Consequently, these variants reduce the protein antiviral activity. 51 Our results showed that these two variants associate with the rs12979860  (Table 3). HCC patients with a fully active IFNL4 expression were diagnosed with tumour at younger age than those with reduced expression (Figure 2). These data suggest that IFNλ4 levels are a risk factor for HCC development.

| PDCD1 polymorphisms and mRNA expression
We next examined whether PDCD1 polymorphisms affected mRNA expression in tumour biopsies and PBMCs from HCC patients ( Figure 3A). We found higher PDCD1 mRNA expression in HCC tissues carrying the PD-1.3 A/G genotype than the G/A genotype (P = .0025, Mann-Whitney test; Figure 3B and C).
Similarly, expression was higher in HCC tissues with the PD-1.7 G polymorphism (A/G and G/G genotypes) than with the A/A genotype (P = .0167, Mann-Whitney test; Figure 3B and C).
In addition, a comparison between cirrhosis and HCC has been performed (Table S7). These results suggested that specific haplotypes may influence the susceptibility to or progression of different HCVrelated diseases (Table 4). In addition, linkage disequilibrium analysis (LD) evidenced a linkage that did not occur by chance (LD >50) between PDCD1 SNPs and rs12979860 of IFNL4 in BD and in patients with CHC, MC and B-NHL but not in patients with cirrhosis or HCC. An association between LD-1.3 and IFNL4 was found in MC (LD = 72) but not in HCC (LD = 0) (Figure 4). In addition, real-time polymerase chain reaction (PCR) indicated higher PDCD1 expression in HCC tissue samples carrying a PD-1.3 G/A allele and a PD-1.7 G/A or G/G allele than in those with A/A genotypes, respectively, but not in PBMCs of the same patients ( Figure 3).
Overall these results underline the importance of IFNL4 ex-

| PD-1 expression on T cells and PD-L1 expression on B lymphocytes of MC patients
A new B-cell subpopulation has recently been described, namely F I G U R E 2 Boxplot describing the relationship of IFNL4 genotype related to a fully active IFNL4 secretion with a younger median age of HCC patients. HCC patients carrying a fully active (rs12979860-T/T combined without P70S mutation, rs117648444 C/C) were diagnosed with tumour at younger age compared to those with a reduced expression (rs12979860-T/T combined with P70S mutation, rs117648444 C/T or T/T), median age 61y and 72y, respectively, P = .02 ANOVA test. Boxes range from the 25th to the 75th percentile with a horizontal black line at the median and vertical lines extending to the 10th and 90th percentiles. These data suggest that IFNL4 levels may represent a risk factor for HCC development PD-L1 hi on IgM + CD19 + B cells in HCC patients without HCV infection ( Figure 5A), but lower than 10% levels of PD-1 hi expression on CD4 + T cells and absence of PD-L1 hi in patients with HCV-related MC ( Figure 5B).

| D ISCUSS I ON
This study confirms the significant associations between the A possible explanatory hypothesis is that fully active IFNλ4-P70 causes a low level of viral replication, which in turn leads to an inefficient adaptive immune response and consequently poor HCV clearance 60 and increased risk of developing HCC.

PD-1 inhibitors have been approved for patients with HCC,
and several pivotal trials are ongoing. 61 We found that PD-1.7 (rs7421861) polymorphism after statistical Bonferroni's correction maintains the significant association with the risk of HCC compared with CHC. Our results suggest that PD-1 is unrelated to chronic infection because the polymorphic frequency of the same allele is similar in CHC and BD but might influence the clinical outcome of HCV infection. We examined the relation between PDCD1 polymorphisms and mRNA expression in liver biopsies and PBMCs of selected patients with HCC. Interestingly, HCC biopsies from patients carrying the PD-1.3 (rs11568821-A allele) and PD-1.7 (rs7421861-G allele) polymorphisms expressed higher levels of PDCD1 mRNA; this association was not found in PBMCs from the same patients. These results suggest that the HCC microenvironment promotes PDCD1 expression, whose level could be related to the PD-1.3 and PD-1.7 genotypes.
Since it is widely accepted that mutations have different effects in combination than individually, we analysed the relationship between the PDCD1 haplotype and IFNL4 in HCC and compared it to that in patients with CHC. The GCAT haplotype, exhibiting lower to P70 mutation), which causes an increase in viral replication. 60 We hypothesize that HCV replication intensifies the antigen-driven immune stimulation that in turn sustains B-cell proliferation. 63 The LD analysis showed an epistatic contribution between IFNL4  Note: Multi-loci genotype frequency with a frequency <0.05 in both control and cases has been dropped. P value significant at Bonferroni's correction (P-value threshold of 0.0125) are in bold text and highlighted in red when positive association was found and in grey when a negative association was found.
CIRRHOSIS Recently, a new B-cell subpopulation, the CD19 + PD-L1 + regulatory B cell (Breg), has shown to require PD-L1 expression to regulate CD4 + PD-1 + T follicular helper (Tfh) cell expansion and differentiation and to mediate humoural immunity. 54 These findings disclosed novel mechanisms by which PD-1-PD-L1 signalling regulates antibody production and helps to understand the role of this pathway in physiological states and in the altered humoural immunity found in some autoimmune diseases. Given that PD-

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. F I G U R E 5 PD-1 and PD-L1 flow cytometry analysis. The lymphocyte population was selected on a forwardversus side-scatterplot for each patient. PBMCs from HCV-negative HCC (n = 2), HCV-associated HCC (n = 2) and HCVassociated MC cases (n = 2) were analysed by flow cytometry after staining with anti-CD4 and anti-PD-1 antibodies, or anti-CD19, anti-IgM and anti-PD-L1 antibodies.

O RCI D
Representative dot plot of one case is shown in (A) and (B) respectively