Resident CD49a?CD103?NKG2C? NK Cells Restrict HIV Infection in Human Lymphoid Tissue Explants

Natural killer (NK) cells are pivotal effectors in antiviral immunity, yet their tissue-specific roles during acute HIV infection remain poorly defined. Using an ex vivo human tonsillar tissue model, we profile NK cell responses to early HIV infection and uncover distinct subsets with specialized functions. We identify a previously uncharacterized memory-like NK population (CD16?/?CD69?CD49a?CD103?NKG2C?) associated with reduced viral burden and enriched in cytotoxic mediators (GNLY, PRF1, GZMB), apoptotic ligands (FASLG, TRAIL), cytokine receptors (IL2RA, ILR2B, IL2RG, IL12RB2, IL18R1) and trafficking molecules (CCL3–5, CCR7, SELL). These cells coexpress NKG2A, suggesting a regulatory balance between activation and inhibition. Although functionally capable of clearing HIV-infected CD4? T cells in a tissue-mimetic environment, they show impaired cytotoxicity and transcriptional signs of exhaustion after infection. Conversely, HIV drives the reprogramming of immature CD16?CD69? NK cells toward a more cytotoxic and migratory effector phenotype. These findings reveal dynamic NK cell adaptations in lymphoid tissue during early HIV infection and highlight tissue-resident NK cells as promising targets for immunotherapeutic intervention. Overall design: Tonsils from a 9-year-old and a 10-year-old child were left overnight in dissection medium to inactivate contaminating microorganisms. Dissection medium included RPMI-1640 medium (Gibco) supplemented with 500 U/ml penicillin (Fisher Scientific), 500 µg/ml streptomycin (Fisher Scientific), 1 µg/ml gentamicin (Gibco), and 5 µg/ml amphotericin B (Gibco). Tonsillar tissue explants (8 mm³ blocks) were cultured on the air-liquid interface on absorbable gelatin sponges (Goodwill). The hemostatic sponges were soaked in RPMI-1640 medium supplemented with 20% Fetal Bovine Serum (FBS) (Gibco), 10 mM HEPES (Biowest), 100 U/ml penicillin, 100 µg/ml streptomycin, and 66 µg/ml amikacin (Vall d'Hebron Hospital pharmacy) (R20 medium). The first day of culture, R20 medium also included 310 µg/ml of timentin (Caisson Labs). Half of the tissue blocks were infected with HIV Bal viral stock (TCID50: 625 units) via dropwise application directly onto the tissue. Medium and sponges were refreshed every 2 days. After 5 days of culture, the tissue blocks were mechanically dissociated and filtered sequentially through 70 µm and 30 µm cell strainers to obtain a single-cell suspension. The CD56+ cell population was enriched from both HIV-infected and uninfected tissue suspensions using the MagniSort™ Human CD56 Positive Selection Kit. Subsequently, CD3- CD56+ cells were further purified by Fluorescence-Activated Cell Sorting (FACS) and analyzed using scRNA-seq.