-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

4056 Single-Cell Multi-Omics Analysis Reveals Dysfunctional Features of the Immune Responses in Vexas Syndrome

Program: Oral and Poster Abstracts
Session: 503. Clonal Hematopoiesis, Aging, and Inflammation: Poster III
Hematology Disease Topics & Pathways:
Research, Acquired Marrow Failure Syndromes, Autoimmune disorders, Translational Research, Bone Marrow Failure Syndromes, Genetic Disorders, Diseases, Immune Disorders, Computational biology, Biological Processes, Technology and Procedures, Molecular biology, Omics technologies
Monday, December 9, 2024, 6:00 PM-8:00 PM

Hiroki Mizumaki, MD1*, Shouguo Gao, PhD1*, Zhijie Wu, MD, PhD1*, Xingmin Feng, PhD1*, Fernanda Gutierrez-Rodrigues, PhD1, Lemlem Alemu, BS1*, Diego Quinones Raffo, BS2*, Ivana Darden, RN1*, Sachiko Kajigaya, PhD1*, Emma M. Groarke, MD1, Neal S. Young, MD1 and Bhavisha A. Patel, MD1

1Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
2National Heart, Lung, and Blood Institute (NHLBI)/NIH, Bethesda, MD

Background

VEXAS syndrome is a newly-described adult-onset autoinflammatory disease caused by somatic UBA1 mutations. In VEXAS, UBA1 mutations are restricted to hematopoietic stem cells and mature myeloid cells, which initiate severe systemic autoinflammation. In contrast, UBA1 mutations are not present in patients’ mature lymphocytes, and circulating lymphocytes are decreased in peripheral blood (PB) of VEXAS patients, implying that wild-type UBA1 (wtUBA1) lymphoid cells are not actively involved in the pathophysiology of VEXAS. However, monoclonal B-cell lymphocytosis and plasma cell dyscrasias are observed in VEXAS despite the absence of UBA1 mutations, suggesting that autoinflammation initiated by UBA1-mutated (mtUBA1) cells impacts residual lymphoid cells. The complexity of immune cell responses in VEXAS has not been unraveled. We employed multi-modal single-cell analysis--single-cell RNA sequencing (scRNA-seq) combined with single-cell V(D)J sequencing (scV(D)J-seq) and genotyping of transcriptomes (GoT)--to obtain an unbiased and comprehensive visualization of immunological responses in PB immune cells in VEXAS.

Method

Nine VEXAS patients (all males, median age, 70 [range, 60-74]) and 5 age-matched healthy male donors (HDs) were enrolled. Their peripheral blood mononuclear cells (PBMCs) isolated were subjected to scRNA-seq and scV(D)J-seq using Chromium Single Cell 5' Reagent Kits (10x genomics, Pleasanton, CA) according to the manufacturer’s instructions. GoT libraries were constructed from cDNA samples from patients with UBA1 M41 mutations using a previously described method with some modifications. The constructed libraries were sequenced with the NovaSeq 6000 system (Illumina, San Diego, CA).

Results

After a quality control, we obtained 129,759 high-quality cells from VEXAS patients and 54,373 cells from HDs for further analysis. GoT identified UBA1 transcripts in 16.8% (19,542 out of 116,547) cells. mtUBA1 transcripts were enriched in monocytes, dendritic cells, and NK cells, but depleted in B and T cells. Differential abundance analysis by Milo identified a prominent decrease in NK cells, B cells, mucosal-associated invariant T cells and dendritic cells in VEXAS. Differential gene expression (DGE) analysis identified many differentially upregulated genes in most cell types in VEXAS, particularly genes related to inflammatory response pathways such as IFN-α, IFN-γ, and TNF-α, indicating broad immune cell activation in VEXAS. Unexpectedly, we observed monocytes in VEXAS to exhibit striking dysfunctional transcriptional features characterized by low expression of HLA class II genes with high expressions of S100A alarmins. Both mtUBA1 and wtUBA1 monocytes were abnormal, suggesting extrinsic rather than cell intrinsic mechanisms.

GoT was also applied to NK cells. DGE analysis between mtUBA1 and wtUBA1 NK cells identified upregulation of genes associated with inflammatory response pathways and cytotoxic activity in mtUBA1 NK cells: inflammation in VEXAS appeared driven by mtUBA1 NK cells in addition to mtUBA1 myeloid cells.

To better characterize adaptive immune response in VEXAS, we examined V(D)J sequences of TCR and BCR repertoires in each single adaptive cell. Large clonal expansions were observed in effector memory CD8+ T cells, more extreme in VEXAS than in HDs. When GLIPH2, an algorithm for clustering TCRs based on amino-acid level similarities, was used to identify common TCR groups, TCR groups from several VEXAS patients and HDs were identified, but no TCR groups specific to VEXAS, and thus absence of shared driver target antigens. In B lineage cells, differential abundance analysis identified prominent decreases in transitional B cells and naïve B cells, and an increase in plasmablasts among B cell subtypes in VEXAS. Increased numbers of expanded BCR clonotypes were present in plasmablasts from VEXAS patients. Plasmablasts from VEXAS patients displayed significant upregulation of genes involved in protein processing in endoplasmic reticulum (ER), the ER stress response pathways, and cell-cycling pathways.

Conclusions

Comprehensive single-cell multi-omics analyses provides a high-resolution view of the immunological landscape of VEXAS syndrome, and molecular dysregulation in T cell subsets, monocytes, NK cells, and B lineage cells.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH