Defining successful intrahepatic CD4+ T cell responses against the hepatitis C virus

Approximately 180 million people globally, including 2-3 million in the United States, live with chronic hepatitis C virus infection. Persistent HCV replication causes progressive liver injury including cirrhosis and cancer. Direct acting antivirals (DAA) that cure chronic HCV infection have been available for a decade but new infections outpace treatment. New cases of HCV infection doubled between 2013 and 2022 and it is unlikely that treatment alone will interrupt virus transmission. A vaccine to prevent chronic HCV infection could provide a cost-effective solution to this problem and is supported by substantial evidence. Spontaneous resolution of infection in 25-30% of humans and chimpanzees provides life-long immunity that sharply reduces the risk of persistent infection with diverse HCV genotypes. Vaccine-mediated protection will likely require broadly neutralizing antibodies and a T cell response that is sustained until after apparent clearance of the virus. The objective of this proposal is to define the protective CD4+ T cell signature that supports effective CD8+ T cell immunity. Human CD4+ T cells are challenging to study. CD4+ T cells target multiple class II epitopes and circulate at low frequency. They are therefore difficult to visualize and enrich with class II tetramers. Blood sampling is typically infrequent in prospective studies of those at risk of infection and liver is rarely sampled during acute hepatitis C. Here, protective T cell immunity will be defined with historical blood and liver samples collected from chimpanzees between 2005 and 2013 with NIH approval and funding. Preliminary studies support this approach. Co-expression of the PD-1 and ICOS activation antigens was established as surrogate marker for direct ex vivo visualization and enrichment of HCV-specific CD4+ T cells, a significant advance for feasibility of the proposed studies. The blood response coincided with peak ALT and infiltration of multifunctional HCV-specific CD4+ T cells into liver. HCV-specific CD4+ T cell responses will be defined by single cell RNA sequencing (scRNAseq) and spatial genomic analysis of liver, technologies that did not exist when the samples were collected. An innovative Central Hypothesis was developed from preliminary scRNAseq studies: CD4+ T cells infiltrate liver to form lymphoid aggregates, where they attract and regulate intrahepatic CD8+ T cell responses to HCV. Lymphoid aggregates were first defined in liver during the non-A,non-B hepatitis era. Their function and significance in acute hepatitis C remains unexplained. Here, we propose that they are a site for close range CD4+:CD8+ T cell communication that determines infection outcome. This hypothesis is supported by identification of a unique HCV-specific CD4+ T cell population that differentially expressed CXCL13 and LGALS9, which encodes the galectin-9 inhibitory ligand implicated in CD8+ T cell exhaustion. These CD4+ T cells had a transcriptional profile consistent with formation of tertiary lymphoid structures. Transcriptional and spatial characterization of HCV- specific CD4+ T cells in blood and liver, facilitated by identification of a key surrogate marker of the response, is expected to fill a substantial gap in knowledge critical for understanding HCV control and vaccination of humans. Project Number: 1R01AI190069-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Christopher Walker | Institution: RESEARCH INST NATIONWIDE CHILDREN'S HOSP, COLUMBUS, OH | Award Amount: $489,389 | Activity Code: R01 | Study Section: Immunity and Host Defense Study Section[IHD] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19006901