Background/Aims: Hypoxia occurs in many pathological conditions, including inflammation and cancer. Within this context, hypoxia was shown to inhibit but also to promote T cell responses. Due to this controversial function, we aimed to explore whether an insufficient anti-tumour response during colitis-associated colon cancer could be ascribed to a hypoxic microenvironment. Methods: Colitis-associated colon cancer was induced in wildtype mice, and hypoxia as well as T cell immunity were analysed in the colonic tumour tissues. In addition, CD4⁺ effector T cells and regulatory T cells were cultured under normoxic and hypoxic conditions and examined regarding their phenotype and function. Results: We observed severe hypoxia in the colon of mice suffering from colitis-associated colon cancer that was accompanied by a reduced differentiation of CD4⁺ effector T cells and an enhanced number and suppressive activity of regulatory T cells. Complementary ex vivo and in vitro studies revealed that T cell stimulation under hypoxic conditions inhibited the differentiation, proliferation and IFN-γ production of T_H1 cells and enhanced the suppressive capacity of regulatory T cells. Moreover, we identified an active role for HIF-1α in the modulation of CD4⁺ T cell functions under hypoxic conditions. Conclusion: Our data indicate that oxygen availability can function as a local modulator of CD4⁺ T cell responses and thus influences tumour immune surveillance in inflammation-associated colon cancer.

Background & AimsLiver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.MethodsHepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.ResultsCell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×107 hepatocytes, 1.8±0.5×106 Kupffer cells, 4.3±1.9×105 liver sinusoidal endothelial cells, and 3.2±0.5×105 stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146+ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence.ConclusionsOur isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Antiviral CD8 ؉ T cells are a key component of the adaptive immune system against hepatitis C virus (HCV).For the development of immune therapies, it is essential to understand how CD8 ؉ T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8 ؉ epitopes in NS5B (LLRHHNMVY 2450-2458 and SQRQKKVTF 2466-2474 ), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs.A ntiviral CD8 ϩ T cells are one of the key components of the adaptive immune system against hepatitis C virus (HCV). During acute infection, HCV-specific CD8 ϩ T cells are activated in the majority of patients (24, 38). Nevertheless, viral persistence is observed in 50 to 80% of patients. These patients with chronic HCV infection are at risk of progressive liver disease and liver cancer. For the development of immune therapies against HCV, it is essential to understand how CD8 ϩ T cells contribute to clearance of infection and why they fail so often. Different mechanisms for CD8 ϩ T cell failure have been described. The proposed mechanisms include primary failure by lack or impairment of priming of specific CD8 ϩ T cells upon HCV infection, as well as secondary failure after the initial priming (reviewed in reference 40). Studies of patients with acute HCV infection suggest that CD8 ϩ T cells are activated in most patients, irrespective of the outcome of infection (6), which supports an important role for secondary failure of HCV-specific CD8 ϩ T cells.Upon transition from acute to chronic HCV infection, the frequency of specific CD8 ϩ T cells declines (6). Moreover, CD8 ϩ T cells from patients with chronic HCV infection produce fewer cytokines and p...

CD8؉ T cells are an essential component of successful adaptive immune responses against hepatitis C virus (HCV). A major obstacle to vaccine design against HCV is its inherent viral sequence diversity. Here, we test the hypothesis that different sequence variants of an immunodominant CD8 ؉ T cell epitope, all binding with high affinity to HLA class I, target different T cell receptor repertoires and thereby influence the quality of the CD8 ؉ T cell response. The impacts of sequence differences in the HLA-A*02-restricted HCV NS3 1406 -1415 epitope on in vitro priming of naive CD8 ؉ T cells from seronegative donors and cross-reactivity of primed T cells with other epitope variants were characterized. Although the six epitope variants tested were all high-affinity binders to HLA-A*02:01, substantial differences in priming and cross-reactivity of CD8 ؉ T cells were observed. The variant associated with the most reproducible priming and induction of T cells with broad cross-reactivity was a genotype 1b variant (KLSAL GLNAV) that is more common in HCV isolates collected in Asia but is rare in sequences from Europe and North America. The superior immunogenicity and cross-reactivity of this relatively rare epitope variant were confirmed by using HCV-specific memory CD8 ؉ T cells from people who inject drugs, who are frequently exposed to HCV. Collectively, the data suggest that sequence differences at the epitope level between HCV isolates substantially impact CD8 ؉ T cell priming and the degree of cross-reactivity with other epitope variants. IMPORTANCEThe results have important implications for vaccine design against highly variable pathogens and suggest that evidence-based selection of the vaccine antigen sequence may improve immunogenicity and T cell cross-reactivity. Cross-reactive CD8 ؉ T cells are likely beneficial for immune control of transmitted viruses carrying epitope variants and for prevention of immune escape during acute infection. To this end, rare epitope variants and potentially even altered epitope sequences associated with priming of broadly cross-reactive T cell receptors should be considered for vaccine design and need further testing.

Antiviral CD8؉ T cells are a key component of the adaptive immune response against HCV, but their impact on viral control is influenced by preexisting viral variants in important target epitopes and the development of viral escape mutations. Immunodominant epitopes highly conserved across genotypes therefore are attractive for T cell based prophylactic vaccines. Here, we characterized the CD8 ؉ T cell response against the highly conserved HLA-B*51-restricted epitope IPFYGKAI 1373-1380 located in the helicase domain of NS3 in people who inject drugs (PWID) exposed predominantly to HCV genotypes 1a and 3a. Despite this epitope being conserved in both genotypes, the corresponding CD8 ؉ T cell response was detected only in PWID infected with genotype 3a and HCV-RNA negative PWID, but not in PWID infected with genotype 1a. In genotype 3a, the detection of strong CD8؉ T cell responses was associated with epitope variants in the autologous virus consistent with immune escape. Analysis of viral sequences from multiple cohorts confirmed HLA-B*51-associated escape mutations inside the epitope in genotype 3a, but not in genotype 1a. Here, a distinct substitution in the N-terminal flanking region located 5 residues upstream of the epitope (S1368P; P ‫؍‬ 0.00002) was selected in HLA-B*51-positive individuals. Functional assays revealed that the S1368P substitution impaired recognition of target cells presenting the endogenously processed epitope. The results highlight that, despite an epitope being highly conserved between two genotypes, there are major differences in the selected viral escape pathways and the corresponding T cell responses. IMPORTANCE HCV is able to evolutionary adapt to CD8؉ T cell immune pressure in multiple ways. Beyond selection of mutations inside targeted epitopes, this study demonstrates that HCV inhibits epitope processing by modification of the epitope flanking region under T cell immune pressure. Selection of a substitution five amino acids upstream of the epitope underlines that efficient antigen presentation strongly depends on its larger sequence context and that blocking of the multistep process of antigen processing by mutation is exploited also by HCV. The pathways to mutational escape of HCV are to some extent predictable but are distinct in different genotypes. Importantly, the selected escape pathway of HCV may have consequences for the destiny of antigen-specific CD8 ؉ T cells. Based on phylogenetic analysis, hepatitis C virus (HCV) can be classified into at least seven genotypes and multiple subtypes that differ up to 20% at the amino acid level (1). The HCV genotypes have distinct epidemiological characteristics, they are associated with different transmission risk factors, and their frequencies in a population are regionally different. In Europe and North America the HCV genotypes 1 and 3 are most common (2, 3). Since routine screening via nucleic acid amplification for HCV eliminated the risk for infection through blood products, the most important risk group for incident HC...

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Summary Adaptation of hepatitis C virus (HCV) to CD8+ T cell selection pressure is well described; however, it is unclear if HCV differentially adapts in different populations. Here, we studied HLA class I‐associated viral sequence polymorphisms in HCV 1b isolates in a Chinese population and compared viral substitution patterns between Chinese and German populations. We identified three HLA class I‐restricted epitopes in HCV NS3 with statistical support for selection pressure and found evidence for differential escape pathways between isolates from China and Germany depending on the HLA class I molecule. The substitution patterns particularly differed in the epitope VTLTHPITK1635‐1643, which was presented by HLA‐A*03 as well as HLA‐A*11, two alleles with highly different frequencies in the two populations. In Germany, a substitution in position seven of the epitope was the most frequent substitution in the presence of HLA‐A*03, functionally associated with immune escape and nearly absent in Chinese isolates. In contrast, the most frequent substitution in China was located at position two of the epitope and became the predominant consensus residue. Moreover, substitutions in position one of the epitope were significantly enriched in HLA‐A*11‐positive individuals in China and associated with different patterns of CD8+ T cell reactivity. Our study confirms the differential escape pathways selected by HCV that depended on different HLA class I alleles in Chinese and German populations, indicating that HCV differentially adapts to distinct HLA class I alleles in these populations. This result has important implications for vaccine design against highly variable and globally distributed pathogens, which may require matching antigen sequences to geographic regions for T cell‐based vaccine strategies.

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