Flow Cytometry Data: High-dimensional Immunophenotyping Reveals Skewed NK Cell Differentiation in Adult Hemophagocytic Lymphohistiocytosis

Abstract: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome driven by impaired cytotoxic lymphocyte function and uncontrolled immune activation. While defective NK cell cytotoxicity is central to primary HLH, the role of NK cell differentiation in adult secondary HLH remains unclear. We performed high-dimensional spectral flow cytometry of peripheral blood NK cells from six adults with HLH and 39 healthy donors to resolve subset composition and chemokine receptor architecture. HLH was characterized by a pronounced skewing toward immature CD56dim NKG2A+ CD57− KIR− NK cells, accompanied by contraction of downstream mature cytotoxic subsets, indicating disruption of the differentiation trajectory. Unsupervised clustering further subdivided this expanded compartment into two populations with distinct chemokine receptor repertoires: an innate-like subset enriched for CX3CR1, CXCR1, and CXCR2, and an adaptive-like subset expressing high levels of CCR5, CXCR3, and CXCR4, with the latter significantly increased in HLH patients. Although CCR5 upregulation likely reflects inflammatory cytokine exposure, the selective expansion of immature NKG2A+ CD57− KIR− subsets to our knowledge has not been reported in severe viral infections, suggesting a perturbation of NK maturation distinct from viral inflammatory responses. These are consistent with a differentiation bottleneck that may link impaired cytotoxic surveillance with sustained inflammatory amplification characteristic in adult HLH. Methods: Cells were thawed and rested in R10 medium (RPMI 1640, 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 20 mM HEPES, pH 7.0) for 2 hours, counted, and then stained for flow cytometric analysis using an appropriate combination of antibodies for 15 min in FACS buffer (phosphate-buffered saline (PBS) pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA) in the dark, at room temperature. Finally, cells were washed three times. Samples were acquired using Cytek Northern Lights equipped with a plate loader. The list of used fluorochrome-labelled antibodies can be found in the Supplementary Table S1. All mAbs were titrated and used at dilutions, ensuring saturated staining of 1 × 106 cells. Dead cells were labeled using LIVE/DEAD Fixable Aqua (Thermo Fisher; cat#L34957) with 1:800 dilution. Biotin-conjugated antibody was secondary stained with Streptavidin-Brilliant Violet 570 (Biolegend; cat#405227). The gates for chemokine receptors were set using appropriate fluorescence minus all chemokine receptors control. Fluorochrome Antigen Clone Dilution Vendor VioBlue KIR3DL1 REA1005 1:50 Miltenyi V500 CD14 M5E2 1:50 BD Bioscience V500 CD19 HIB19 1:50 BD Bioscience V500 CD3 UCHT1 1:50 BD Bioscience Biotin CXCR1 42705 1:50 R&D BV570-SA Biotin Streptavidin 1:100 Biolegend BV605 CD56 HCD56 1:50 Biolegend BV650 CX3CR1 2A9-1 1:50 Biolegend BV711 CCR5 J418F1 1:50 Biolegend BV785 CXCR4 12G5 1:50 Biolegend VioGreen CD57 TB03 1:100 Miltenyi PE NKG2C REA205 1:100 Miltenyi PE-Dazzle594 CXCR2 5E8 Biolegend PerCp CCR7 G043H7 1:50 Biolegend PE-Cy5.5 KIR2DL2/L3/S2 GL183 1:50 Beckman Coulter PE-Vio770 KIR2DL1/S1 REA1010 1:50 Miltenyi APC NKG2A Z199 1:25 Beckman Coulter Alexa Fluor 700 CXCR3 G025H7 1:50 Biolegend APC-Vio770 KIR2DL1 1:50 Miltenyi LIVE/DEAD Fixable Aqua Dead cell stain L34957 1:800 Thermo Fisher