116 Peripheral T helper cell gene signature in patients with PR3 ANCA associated vasculitis
Creators: name: Seerapani Gopaluni1
name: Carr1, Edward
name: Duvuru2, Geetha
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> A subset of T cells with the phenotype CD4+PD1+CXCR5- (called peripheral T helper cells (PTH)) were shown to infiltrate renal tubules and in the urine of PR3 ANCA associated vasculitis (PR3 AAV) patients with active nephritis. Circulating PTH cells (cPTH) were shown to correlate with disease activity and PR3 AAV patients had higher proportion of these cells compared to age matched healthy volunteers and disease controls. This study evaluated the nature of these cells by studying the transcriptome of flow sorted cPTH, circulating TFH (cTFH) (CXCR5+PD1+) and double negative (cDN) CD4 T cells in greater detail. The gene signature of cPTH cells was validated by studying candidate genes expressed by urinary lymphocytes obtained from AAV patients with nephritis. <strong>Methods:</strong> Peripheral blood mononuclear cells (PBMC) from 5 independent PR3 AAV patients in remission were flow sorted to obtain cPTH, cTFH and cDN samples. At least 1000 – 10,000 cells were sorted from PBMCs and subjected to RNA sequencing (SMART-Seq v4 Ultra low input RNA kit for sequencing). In a parallel and independent experiment, urine samples from patients with active nephritis were tested for the expression of a set of genes (TGFB1, PRF1, VIM, FOXP3, CD25, CCL5 (RANTES), GZMB, CD3, TBX21, CD20, CCL2, CXCR3, CDH1 (E-cadherin), NKCC2, VEGFA, CD46). <strong>Results:</strong> Differential gene expression analysis between cPTH and cDN cells resulted in 832 upregulated and 702 down-regulated genes. The cPTH cell transcriptome, when compared to cDN T cells, was enriched for cytotoxic genes such as Granzyme A (GZMA), Granzyme B (GZMB), Granzyme K (GZMA), Perforin-1 (PRF1), C-C Motif Chemokine Ligand 5 (CCL5), Natural Killer Cell Granule Protein 7 (NKG7) and genes implicated in cell proliferation such as RRM2, DLGAP5, CDC20, TOP2A, BIRC5. Further, these cells were also enriched for chemokine receptor genes, such as CX3CR1, CCR2, CCR5, CXCR3 that aid the trafficking of these cells to sites of inflammation. Similarly, cPTH cells differentially expressed cytotoxic and chemokine receptors when compared to cTFH cells. mRNA expression of urinary lymphocytes from an independent cohort of patients with and without active nephritis revealed differential expression of similar cytotoxic genes (GZMB, PRF1, CCL5, VIM). Genes that aid B cell differentiation such as CXCL13, IL-21 were not enriched in cPTH cells. <strong>Conclusions:</strong> Transcriptomic analysis of flow-sorted cPTH cells from patients suggested, these cells to be proliferating activated CD4 T cells when compared to cTFH or cDN cells. This supports the hypothesis that cPTH cells could be pathogenic effector cells. Surprisingly, they were enriched for cytotoxic genes (GZMH, GZMK, PRF1, NKG7, CCL5, GNLY, SLAMF7, CD160) and chemokine receptors (CCR2, CCR9, CCR5, CX3CR1, CXCR3, CXCR6) that may aid their migration to sites of inflammation and partake directly in tissue inflammation and damage. Genes that help B cell differentiation were not enriched in cPTH cells. All the samples for the transcriptomic study were from patients in remission and this signal could be specific to patients with active disease or to the cells exclusively found at the sites of inflammation. <strong>Disclosures</strong>: The authors have no financial disclosures relevant to the submitted work