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 (n= 10), immune responders (IRs, n= 14), and healthy controls (HCs, n= 9). We developed scGeneANOVA, a novel mixed model differential gene analysis tool, to detect differentially expressed genes and pathways. We performed flow cytometry and bulk RNA-seq to PLWH cohort (23 INRs and 37 IRs) to validate the corelation. In addition, we developed the Viral Identification and Load Detection Analysis (VILDA) tool to quantify HIV-1 transcripts and investigate their relationship with host gene expression.
Results
We had indentified 8 major clusters and 51 subclusters within 232,369 PBMC cells across 33 samples. The scGeneANOVA tool identified critical genes and pathways that were missed by traditional analysis methods, and revealed that INRs exhibit a dysregulated interferon (IFN) expression profile. RNA velocity and TCR tracing showed an increased tendency toward an exhaustion-prone differentiation trajectory in the CD4+ T cells of INRs, with fewer cells differentiating into effector cells. Transcriptional analysis on bulk RNA-seq cohort and public data validated the role of IFN in driving CD4+ T cell exhaustion. The VILDA tool showed a higher levels of HIV-1 transcripts in INRs, suggesting that the size and transcriptional activity of the HIV-1 reservoir are closely linked to IFN signaling activation in INRs.
Conclusions
This study revealed that INRs exhibit a dysregulated IFN response, closely associated with CD4+ T cell exhaustion. This immune exhaustion was identified as a key factor contributing to immune recovery failure. These findings point to the central role of IFN signaling and HIV-1 reserviors in INR-related immune dysfunction. The identification of key genes and pathways offers potential biomarkers and therapeutic targets for improving immune recovery in this vulnerable population.
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