Single-cell multi-omics profiling uncovers the immune heterogeneity in HIV-infected immunological non-responders

Abstract

Background: Immunological non-responders (INRs) are people living with HIV-1 who fail to achieve full immune reconstitution despite long-term effective antiretroviral therapy (ART). This incomplete recovery of CD4⁺ T cells increase the risk of opportunistic infections and non-AIDS-related morbidity and mortality. Understanding the mechanisms driving this immune dysfunction is critical for developing targeted therapies.

Methods: We performed single-cell RNA sequencing (scRNA-seq) and single-cell VDJ sequencing (scVDJ-seq) on peripheral blood mononuclear cells (PBMCs) from INRs, immune responders (IRs), and healthy controls (HCs). We developed scGeneANOVA, a novel mixed model differential gene analysis tool, to detect differentially expressed genes and pathways. In addition, we developed the Viral Identification and Load Detection Analysis (VILDA) tool to quantify HIV-1 transcripts and investigate their relationship with interferon (IFN) pathway activation.

Findings: Our analysis revealed that INRs exhibit a dysregulated IFN response, closely associated with CD4⁺ T cell exhaustion and immune recovery failure. The scGeneANOVA tool identified critical genes and pathways that were missed by traditional analysis methods, while VILDA showed higher levels of HIV-1 transcripts in INRs, which may drive the heightened IFN response. These findings support a potential contribution of IFN signalling in INR-related immune dysfunction.

Interpretation: Our study provides new insights into the pathogenic mechanisms behind immune recovery failure in INRs, suggesting that IFN signalling might be involved in the development of CD4⁺ T cell exhaustion. The identification of key genes and pathways offers potential biomarkers and therapeutic targets for improving immune recovery in this vulnerable population.

Funding: This study was supported by the grants from Special Research Fund for the Central High-level Hospitals of Peking Union Medical College Hospital (Grant No. 2022-PUMCH-D-008), Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (Grant No. 2021-I2M-1-037), National Key Technologies R&D Program for the 13th Five-year Plan (Grant No. 2017ZX10202101-001). The funders played no role in the design, experiment conduction, data analysis and preparation of the manuscript of this work.

Keywords: Acquired immunodeficiency syndrome; CD4(+) T cells; Human immunodeficiency virus-1; Immunological non-responders; Interferon; Single cell RNA sequencing.