IMMUNE PROFILING OF RENAL LYMPH NODES IN A MURINE MODEL OF IgA NEPHROPATHY USING SINGLE-CELL RNA SEQUENCING AND FLOW CYTOMETRY

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Abstract Title *

IMMUNE PROFILING OF RENAL LYMPH NODES IN A MURINE MODEL OF IgA NEPHROPATHY USING SINGLE-CELL RNA SEQUENCING AND FLOW CYTOMETRY

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Co-author 1

Eriko Kosuge e.kosuge.ak@juntendo.ac.jp Juntendo University Department of nephrology Bunkyo-ku Japan *

Co-author 2

Yoshihito Nihei y-nihei@juntendo.ac.jp Juntendo University Department of nephrology Bunkyo-ku Japan -

Co-author 3

Hitoshi Suzuki shitoshi@juntendo.ac.jp Juntendo University Department of nephrology Bunkyo-ku Japan -

Co-author 4

Yusuke Suzuki yusuke@juntendo.ac.jp Juntendo University Department of nephrology Bunkyo-ku Japan -

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Introduction

Recently, we showed the presence of IgA autoantibodies (auto-Abs) against mesangial cells in the serum of patients with IgA nephropathy (IgAN), suggesting that IgAN is an autoimmune disease with tissue-specific auto-Abs (Sci. Adv. 2023, Life Sci. Alliance. 2024, Kidney Int. Reports.2025). In some autoimmune diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), draining lymph nodes of target organs have been reported to be involved in pathogenesis.

This study aims to investigate the role of renal lymph nodes (RLNs) in the pathogenesis of IgAN.

Methods

RLNs from male and female spontaneous IgAN model mice (gddY) were analyzed. As controls, BALB/c mice were used for comparison. RLNs were evaluated using flow cytometry (FCM) at 4, 8, and 12 weeks of age. The number of CD4+ and CD8+ T cells and B cells were analyzed. The frequency of B cell subsets in the RLNs- naïve B cells and germinal center B cells (GCB cells) – were confirmed using FCM. To identify which B cell subsets were increasing, we performed single-cell RNA sequencing (scRNA-seq) on CD45+ cells isolated from male BALB/c mice and gddY mice at 8 weeks of age.

Results

The number of B cells in RLNs from gddY mice increased at 4 weeks of age compared to BALB/c mice (p=0.0005), whereas that of CD4+ and CD8+ T cells did not. scRNA-seq was performed on 9,428 CD45+ cells from two BALB/c mice and 27,678 CD45+ cells from three gddY mice, resulting in the classification of these cells into 17 clusters. Among them, cluster 7 was observed only in RLNs from gddY mice. The differentially expressed genes analysis revealed that cluster 7 were GCB cells (Figure 1). FCM analysis also confirmed the increase of GCB cells in RLNs from gddY mice (Figure 2). The dominant immunoglobulin subtype of GCB cells was IgG.

Figure 1. Cluster 7 was observed only in RLNs from gddY mice. Figure 2. Representative FACS analysis of mice at 4 and 8 weeks of age. The increase of GCB cells in RLNs from gddY mice. The dominant immunoglobulin subtype of GCB cells was IgG.

Conclusion

We found that IgG+ GCB cells increased in RLNs of gddY mice. Given that earlier paper reported that IgG antibodies against mesangial cell antigens are present in the serum of patients with active nephritis in IgAN (J. Clin. Invest. 1991, Nephrology Dialysis Transplantation.1992). The presence of IgG+ GCB cells in RLNs may contribute to production of IgG auto-Abs against glomerular antigens.

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