117. Peripheral T helper like cells in secondary lymphoid organs in PR3 ANCA associated vasculitis
Creators: name: Seerapani Gopaluni1
name: Moyle2, Penelope
name: Carr1, Edward
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> Only 2% of the lymphocytes circulate in the blood stream whilst 40% reside in lymph nodes. We have shown that a specific CD4 T cell subset expressing PD1+CXCR5- (referred to as circulatory PTH cells), was hyper-expanded in the peripheral blood of patients. This subset was cytotoxic in nature and was identified in the inflamed kidney tissue and urine of patients with active nephritis. Using single cell transcriptomic analysis, the cytotoxic gene signature predominantly came from a CD28low cluster that had higher expression of cytotoxic, activation and chemokine gene sets. cPTH cells were clonally expanded and had features consistent with persistent antigenic stimulation, a core feature of chronic auto-immune and inflammatory conditions. In this study, the presence of phenotypically similar cells within secondary lymphoid organs (SLOs), their transcriptomic profile and clonal relationship in comparison to cPTH cells was investigated. <strong>Methods:</strong> The most easily accessible lymph node (LN) in the inguinal region was selected by ultrasound and samples obtained by core needle biopsy and/or fine needle aspiration under local anaesthesia. Paired blood samples were obtained for PBMC extraction. Lymph node biopsy samples were mashed and run through a 70um strainer and made into single cell suspension. Samples from lymph nodes (prefixed with “ln”) and PBMC (prefixed with “c”) were stained with fluorochromes for phenotyping. PTH, T follicular helper cells (TFH), CXCR5+PD1- and double negative (DN) subsets from LN and PBMC were flow sorted from samples that had at least 400 cells of each subset and were subjected to RNA sequencing (SMART-Seq v4 Ultra low input RNA kit for sequencing). <strong>Results:</strong> In 17 subjects, paired pre and post-induction therapy lymph node and peripheral blood samples were available. Another 11 subjects provided samples either at the time of active disease (n=7) or when in remission (n=4). In total, 45 procedures were performed, and 42 samples were obtained. 20 samples were obtained from patients in remission and 22 samples were obtained from patients with active disease. Procedure was well tolerated with no complications other than minor bruising. Cell yield was highly variable and usually low (median of 2102 (range: 21 to 65000)). LNPTH cells (PD1+CXCR5-), were identified in LN lymphocytes in similar proportions to that observed in the peripheral blood. The proportion of lnPTH cells was higher than conventional lymph node T follicular helper (lnTFH) cells (0.93 vs 0.19, p &lt; 0.001). Transcriptomic data from the 4 subsets from 5 AAV patients (in remission) was available for analysis. Differential gene expression analysis of lnPTH cells against cDN cells revealed enrichment for cytotoxic genes (GZMK, GZMA, NKG7, CCL5, PRF1) and chemokine receptor genes (CCR5, CXCR3 and CXCR6) but not CCR2 or CX3CR1 unlike cPTH cells. CRTAM, a precursor gene of cytotoxic effector cells, was also upregulated in lnPTH cells, possibly indicating that the circulating cytotoxic CD4 T cells develop within the SLOs and lnPTH cells could be their precursors. In TCR clonality analysis, the overlap coefficient between cPTH and lnPTH cells was highest in comparison to other subsets. Similarly, cTFH cells have higher overlap coefficients with lnTFH cells. This data suggests that the cPTH cells and lnPTH cells may have a common precursor or that lnPTH cells could be the precursors of cPTH cells. <strong>Conclusions</strong>: The safety and tolerability of percutaneous lymph node biopsy as research tool was demonstrated. Cell yield, though poor, was adequate to perform transcriptomic analysis. Cells that were phenotypically similar to cPTH cells were present within the lymph nodes in similar proportions to peripheral blood. Transcriptomic analysis showed that lnPTH cells share cytotoxic gene signatures with cPTH cells. TCR clonality analysis demonstrated evidence for clonal relationship amongst cPTH, lnPTH and lnTFH cells. <strong>Disclosures:</strong> The authors have no financial disclosures relevant to the submitted work