Single-cell multi-omics sequencing reveals the functional regulatory landscape of STAT3-V637M Hyper-IgE syndrome

Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by, among others, the excessive production of IgE and repetitive bacterial/fungal infections. Although STAT3 is a known causative factor of HIES, the biological consequences of STAT3 mutation are not completely understood, especially at the epigenetic level. Here, we used multi-omic approaches to decipher the multidimensional immune disturbance in a male HIES patient harboring STAT3-V637M. In his peripheral blood mononuclear cell (PBMC) we found significant clonal expansion of CD8 T cells (whose expression of CD8 subunits was increased, resulting in enhanced responsiveness to MHC I molecules), but not CD4 T cells and B cells in his. The lack of T cells-B cells interaction could be responsible for the limited number of his B cells, although they exhibited a higher potential in producing immunoglobulin, elevated SPIC binding might bias this process toward IgE isotype production. Immune checkpoint inhibitors, including CTLA4, LAG3, were overexpressed in his PBMC-CD4 T cells, accompanied by reduced CD28 and IL6ST (gp130) expression. For his CD4 T cells, integrative analyses predicted upstream transcription factors (ETV6, KLF13, and RORA) for LAG3, IL6ST, and CD28, respectively. The down-regulated phagocytic, and nitric oxide synthetic genes in his PBMC-monocytes seem to be the culprit of his disseminated bacterial/fungal infection. In his bronchoalveolar lavage fluid (BALF), we identified two macrophages with anti-bacterial properties, which were characterized by CXCL8/S100A8/S100A9, or SOD2, respectively. Together, this study comprehensively described how the immune cell landscape was disturbed in STAT3-V637M HIES and provided a resource for further studies on STAT3 biology. We collected PBMC and BALF from an 18-year-old male harboring STAT3-V637M. Paired PBMC and BALF samples collected from the patient were subjected to GEX/V(D)J-seq and scRNA-seq according to the manufacturer’s protocol, respectively. For PBMC, single cell suspension was loaded into 10x Genomics Chromium system using Chromium Next GEM Single Cell 5' v1.1 to generate TCR/BCR libraries and 5' GEX libraries. For BALF sample, Single Cell 3' v3 Reagent Kits (10X Genomics, Pleasanton, CA, USA) were used during library preparation. Next, libraries were sequenced on a NovaSeq 6000 sequencing system (Illumina, San Diego, CA, USA). Sequencing data were processed using the Cell Ranger 6.0.1 pipeline and aligned to GRCh38-1.1.0 reference genome to generate cell-by-gene count matrix (scRNA/GEX) or filtered contig annotations (scVDJ). For the patient’s PBMC, we additionally performed scATAC-seq using 10x Genomic single cell ATAC reagent v1.1 kit in accordance with the manufacturer’s protocol. Briefly, nuclei were isolated from PBMC and counted by Trypan Blue, scATAC-seq libraries were prepared following the 10X Genomics Chromium Single Cell ATAC Reagent Kits User Guide. Next, scATAC-seq libraries were sequenced using PE50 sequencing on NovaSeq 6000. Sequencing reads were processed through the Cell Ranger ATAC 1.1.0 pipeline and aligned to GRCh38-1.1.0 reference genome for mapping, de-duplication, and identification of Tn5 cut sites. **Submitter declares that the raw data will be submitted to another designated repository due to the patient's privacy and national legislation**