Characterizing the cellular microenvironment of fibrotic regions in lymph nodes (LN) may further our understanding of LN fibrosis during chronic HIV disease.
Inguinal LN excisions were done by a board-certified surgeon on 6 virally-suppressed persons living with HIV (PLWH) on ART and 4 HIV-uninfected participants (controls) recruited at the University Clinics at Kaka’ako at the John A. Burns School of Medicine. A portion of each LN was formalin-fixed, paraffin-embedded (FFPE) and slide-mounted tissue sections were prepared. H&E staining was performed on LN slides and fibrotic regions were quantified by a board-certified pathologist. Digital spatial profiling (DSP) was performed on LN slides (NanoString Inc.) using the GeoMx DSP platform. LN slides were stained with fluorescent antibodies targeting CD3, CD8, and CD68 to identify cells of interest (COI): CD8 and CD4 T cells and macrophages (M?). Two fibrotic and two non-fibrotic regions were identified for each LN slide remotely by a board-certified pathologist and protein expression profiling for each COI in each region was performed using nCounter barcoding technology. Statistical analyses performed were T-tests and Linear Mix Model tests.
LNs excised from PLWH had slightly higher amounts of fibrosis as compared to controls (HIV+ vs. HIV- median LN fibrosis: 25% vs. 18%), however not statistically different (p=0.511). Although we did not observe significant fold change (log2FC) differences in protein expression profiles in each LN COI between PLWH and controls, we did find significant log2FC in protein expression profiles in each LN COI between fibrotic and non-fibrotic regions in both groups. In PLWH: M? in fibrotic LN regions had a higher protein profile of CD14 (log2FC=1.035; p=0.001, FDR=0.037), B7-H3 (log2FC=0.728; p=0.012, FDR=0.119), ?-SMA (log2FC=0.439; p=0.035, FDR=0.216), and CD163 (log2FC=1.372; p=0.045, FDR=0.233); CD8 T cells had higher OX40L (log2FC=0.905; p=0.002, FDR=0.009), fibronectin (log2FC=1.247; p=0.006, FDR=0.020), and CD34 (log2FC=1.026; p=0.009, FDR=0.027); CD4 T cells had higher CD34 (log2FC=1.047; p=0.005, FDR=0.033), fibronectin (log2FC=1.275; p=0.005, FDR=0.033), OX40L (log2FC=0.880; p=0.009, FDR=0.034) and B7-H3 (log2FC=0.444; p=0.044, FDR=0.104). Controls had similar increases in protein profiles for each COI in fibrotic regions.
DSP analyses reveal distinct immune cell protein expression profiles in fibrotic LN microenvironments, which may inform targeted fibrosis treatments.