Enhanced TLR7-induced interferon responses in women living with HIV

Introduction: Biological sex is a key modifier of HIV pathogenesis, with women more frequently achieving spontaneous viral control than men. Toll-like receptor 7 (TLR7), an endosomal RNA sensor encoded on the X chromosome that escapes X-inactivation, plays a pivotal role in antiviral immunity and is increasingly targeted in HIV cure strategies aimed at reversing viral latency. However, it remains unclear whether TLR7-driven immune responses differ by sex in the context of HIV infection. Methods: We characterized sex-specific immune responses to TLR7 stimulation in a cohort of 1,326 antiretroviral therapy (ART)-suppressed individuals living with HIV (192 women, 1,134 men), including 50 spontaneous HIV controllers, and in 43 people living without HIV (28 women, 15 men). Peripheral blood mononuclear cells (PBMCs) were stimulated ex vivo with the TLR7 agonist imiquimod (IMQ), followed by cytokine profiling and transcriptome analysis by RNA sequencing. To investigate transcriptional priming at baseline, we additionally analyzed single-cell RNA-sequencing (scRNA-seq) data from unstimulated PBMCs of 76 women and 214 men living with HIV. Results: PBMCs of women living with HIV (WLWH) released significantly lower amounts of IL-1β and MIP-1α (p < 0.01) following IMQ stimulation than PBMCs of men living with HIV (MLWH), with trends toward reduced IL-8 and IL-1Ra (p < 0.06), while IL-6 and MCP-1 production was similar across sexes. Transcriptomic analysis revealed sex-dependent gene programs following TLR7 activation. Women living without HIV (WLWoH) showed selectively higher IFNγ (type II interferon) signaling and downregulated B cell-associated transcripts compared to men living without HIV (MLWoH). In contrast, WLWH exhibited a pronounced induction of both IFNα (type I) and IFNγ (type II) pathways, marked by elevated expression of interferon-stimulated genes including IRF7, ISG15, MX1, and APOBEC3A, alongside reduced antibacterial and inflammatory signatures compared to MLWH. Single-cell RNA sequencing further identified IRF7 as a key ISG selectively upregulated in plasmacytoid dendritic cells (pDCs) of WLWH. These transcriptional responses were independent of pDC frequency and did not differ between HIV controllers and non-controllers. Discussion: Despite the male-biased cohort, these findings demonstrate that women mount stronger interferon-driven responses upon TLR7 activation compared to men, accompanied by attenuated inflammatory cytokine production. These sex-based immunological differences may contribute to improved viral control in women and highlight the importance of incorporating sex as a biological variable in TLR7-targeted HIV immunotherapies.