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. 2025 Nov;44(6):960-973.
doi: 10.23876/j.krcp.23.317. Epub 2024 Jun 13.

Single-cell RNA sequencing revealed the role of the Th17 pathway in the development of anti- human leukocyte antigen antibodies in a highly sensitized mouse model

Hanbi Lee  1   2 , Yoo-Jin Shin  2 , Xianying Fang  2 , Sheng Cui  2 , Sun Woo Lim  2 , Seon-Yeong Lee  3   4 , Sang Hun Eum, Ji-Won Min  2   5 , Chang-Won Hong  6 , Hae-Ock Lee  7 , Mi-La Cho  3   4 , Eun-Jee Oh  8 , Chul Woo Yang  1   2 , Byung Ha Chung  1   2
Affiliations

Single-cell RNA sequencing revealed the role of the Th17 pathway in the development of anti- human leukocyte antigen antibodies in a highly sensitized mouse model

Hanbi Lee et al. Kidney Res Clin Pract. 2025 Nov.

Abstract

Background: The aim of this study is to investigate the specific pathway involved in human leukocyte antigen (HLA) sensitization using single-cell RNA-sequencing analysis and an allo-sensitized mouse model developed with an HLA.A2 transgenic mouse.

Methods: For sensitization, wild-type C57BL/6 mouse received two skin grafts from C57BL/6-Tg(HLA-A2.1)1Enge/J mouse (allogeneic mouse, ALLO). For syngeneic control (SYN), skin grafts were transferred from C57BL/6 to C57BL/6. We performed single-cell RNA-sequencing analysis on splenocytes isolated from ALLO and SYN and compared the gene expression between them.

Results: We generated 9,190 and 8,890 single-cell transcriptomes from ALLO and SYN, respectively. Five major cell types (B cells, T cells, natural killer cells, macrophages, and neutrophils) and their transcriptome data were annotated according to the representative differentially expressed genes of each cell cluster. The percentage of B cells was higher in ALLO than it was in SYN. Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the highly expressed genes in the B cells from ALLO were mainly associated with antigen processing and presentation pathways, allograft rejection, and the Th17 cell differentiation pathway. Upregulated genes in the T cells of ALLO were involved in the interleukin (IL)-17 signaling pathway. The ratio of Th17 cluster and Treg cluster was increased in the ALLO. On flow cytometry, the percentage of Th17 (IL-17+/CD4+ T) cells was higher and regulatory T cells (FOXP3+/CD4+ T) was lower in the ALLO compared to those in the SYN.

Conclusion: Our results indicate that not only the B cell lineage but also the Th17 cells and their cytokine (IL-17) are involved in the sensitization to HLA.

Keywords: HLA-A2 antigen; Single-cell gene expression analysis; Th17 Cells; Transplantation.

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Conflict of interest statement

Conflicts of interest

All authors have no conflicts of interest to declare.

Figures

Figure 1.
Figure 1.. Experimental groups and HLA-specific IgG MFI titers in allogeneic and syngeneic transplantation.
(A) The study protocol and definition of experimental groups. (B) MFI titers of HLA.A2-specific IgG measured at week 9. ALLO, allogeneic; DSA, donor-specific antibody; HLA, human leukocyte antigen; IgG, immunoglobulin G; MFI, mean fluorescence intensity; SYN, syngeneic; Tx, transplantation.
Figure 2.
Figure 2.. Single-cell transcriptional profile of splenocytes from allogeneic and syngeneic mice.
(A) UMAP plot for visualization of transcriptional atlas of 18,081 splenocytes. (B) Clusters identified by CellMarker 2.0 database. (C) Percentages of cell fractions. ALLO, allogeneic; NK, natural killer; SYN, syngeneic; UMAP, uniform manifold approximation and projection.
Figure 3.
Figure 3.. Single-cell analysis of B cells.
(A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of highly expressed genes in the allogeneic mouse. (B) KEGG pathway map of Th17 differentiation.
Figure 4.
Figure 4.. Single-cell analysis of T cells.
(A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of highly expressed genes in the allogeneic mouse. (B) KEGG pathway map of interleukin (IL)-17 signaling pathway. (C) The ratio of Th17 cells cluster and Treg cells cluster. ALLO, allogeneic; cAMP, cyclic adenosine monophosphate; COVID-19, coronavirus disease 2019; PI3K, phosphoinositide 3-kinase; SYN, syngeneic.
Figure 5.
Figure 5.. T cell population at week 9 (4 weeks after the second skin graft) in the recipient spleen analyzed using flow cytometry.
(A) Gating strategy and fractions of (B) CD4+/INFγ+ Th1 cells, (C) CD4+/IL4+ Th2 cells, (D) CD4+/IL-17+ Th17 cells, and (E) CD4+/CD25+/Foxp3+ Treg cells. Error bars represent two standard errors. ALLO, allogeneic; FSC, forward scatter; IL, interleukin; INF, interferon; SSC, side scatter; SYN, syngeneic.
Figure 6.
Figure 6.. RT-qPCR analysis of cytokines in the spleen.
The mRNA expression of (A) interleukin (IL)-10, (B) Foxp3, (C) IL-23, and (D) interferon (IFN)-γ. Relative messenger RNA expression levels were calculated after normalization to β-actin expression. Expression level in the SYN was considered as the control. ALLO, allogeneic; RT-qPCR, quantitative real-time polymerase chain reaction; SYN, syngeneic. ap < 0.05 vs. SYN.
Figure 7.
Figure 7.. IgG production assessed using ELISA in co-cultures of T cells with non-T cells.
Concentration of (B) total IgG and (B) IgG2A. ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G. ap < 0.05 vs. Th0, bp < 0.05 vs. Th17, cp < 0.05 vs. nonT, dp < 0.05 vs. Th0 + nonT, ep < 0.05 vs. Th17 + nonT.
Figure 8.
Figure 8.. Schematic figure to outline the pathway of reciprocal activation of B cells and Th17 cells.
None
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