179. Single cell transcriptomic analysis of T helper cells in PR3 ANCA associated vasculitis
Creators: name: Seerapani Gopaluni1
name: Ferdinand1, John
name: Clatworthy1, Menna
name: Jayne1, David
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background: </strong>CD4 T cells in ANCA associated vasculitis (AAV) patients, irrespective of their disease status, were shown to be in a state of persistent activation. We have shown that a subset of CD4 T cells, circulatory peripheral T helper cells (cPTH) expressing CD4+CXCR5+PD1-, were increased in the peripheral blood of PR3-AAV patients and that phenotypically similar cells were present in the kidney tissue and urine of nephritic patients. Bulk transcriptomic analysis indicated that cPTH cells were proliferating and were cytotoxic cells expressing chemokine receptors supporting migration to sites of inflammation. The primary aim of this study was to understand the composition of the cPTH cell subset, its heterogeneity, and the clonal relationship between circulatory TFH (cTFH) and cPTH cells. <strong>Methods: </strong>Three patients with PR3-ANCA associated vasculitis (PR3 AAV), in stable remission on minimal immunosuppression with rising PR3-ANCA levels and three healthy volunteers (HV) were recruited. 100 mL of blood was obtained from each subject and PBMCs extracted. The samples were enriched for CD4 T cells by positive selection using magnetic cell sorting. For each subject, 160,000 cPTH cells, 80,000 cTFH and 300,000 each of CXCR5+PD1- and double negative (cDN) T cells were flow sorted and processed using 10x Genomics’ single-cell RNA-seq (scRNA-seq) technology combined with feature barcoding technology. Cells were pooled in a ratio of 1:1:1:1 <strong>Results: </strong>After performing pre-processing and quality control steps, 19997 cells were obtained from the 6 subjects (3 patients and 3 HVs). The total number of cells from HVs was 13353 and from patients was 6644. Leiden clustering analysis resulted in 10 clusters and based on differential gene expression analysis each cluster was annotated. From bulk transcriptomic analysis (our previous work), a gene set (n=171) of significantly upregulated genes by the cPTH subset was used to derive enrichment scores. Clusters 7, 8 and 9 had higher enrichment scores, with cluster 9 scoring the most. Further analysis of cluster 9 revealed these cells to be expressing low levels of CD28 (CD28low), higher levels of CX3CR1, genes of recent activation such as HLA-DR and CD69 and higher levels of cytotoxic genes (also suggested on reactome pathway analysis). Cluster 9 from patients when compared to HVs had higher expression of cytotoxic (GZMB, PRF1) and chemokine receptors (CX3CR1). TCR clonal diversity index was lower in patients compared to HVs across all clusters implying restricted repertoire. The diversity indices fell steeply for clusters 8 and 9 for both patients and HVs but the drop was more pronounced in patients, suggesting greater clonal expansion in these clusters. Clonal overlap analysis showed that TCR clones in cluster 9 frequently shared clones with T cells in cluster 8 and cluster 7, suggesting that cytotoxic CD4 T cells in cluster 9 possibly originated from cluster 7 (predominantly Th1 subset without cytotoxic gene enrichment), through cluster 8 (predominantly Th1 subset with cytotoxic gene enrichment). CD4 T cells of patients had low levels of expression of IL-7a possibly due to chronic antigenic stimulation of T cells. <strong>Conclusions: </strong>We have shown that cPTH cells express cytotoxic genes and that cells within clusters 7, 8 and 9 expressing GZMB, PRF1, CCL5, NKG7, GZMA are the predominant subset that contributed to the genes identified on bulk transcriptomics. Further, cells in cluster 9 were differentially expressed with patients having a higher expression of activation markers, CD69, HLA-DR and chemokine receptors such as CX3CR1, CXCR4, CXCR3. cPTH cells were not a homogenous group of cells and consisted of at least five different clusters, each expressing a predominant gene sets characteristic for either Th1, Th17, cytotoxic, TFH-like or CD28null subsets. TCR clonality analysis showed that these cells were clonally expanded and that they share clones mostly with Th1 phenotype subsets suggesting that they have common origins with other cPTH cells. <strong>Disclosures:</strong> None