Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality
Tiffany Shi, … , E. Steve Woodle, David A. Hildeman
Tiffany Shi, … , E. Steve Woodle, David A. Hildeman
Published May 25, 2023
Citation Information: J Clin Invest. 2023;133(14):e170191. https://doi.org/10.1172/JCI170191.
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Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality

Abstract

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/β sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/β cDNAs from CD8EXP into Jurkat 76 cells (TCR–/–) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

Authors

Tiffany Shi, Ashley R. Burg, J. Timothy Caldwell, Krishna M. Roskin, Cyd M. Castro-Rojas, P. Chukwunalu Chukwuma, George I. Gray, Sara G. Foote, Jesus A. Alonso, Carla M. Cuda, David A. Allman, James S. Rush, Catherine H. Regnier, Grazyna Wieczorek, Rita R. Alloway, Adele R. Shields, Brian M. Baker, E. Steve Woodle, David A. Hildeman

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Figure 5

Temporal scRNA-Seq analysis of the response to antirejection therapy under tacrolimus maintenance IS.

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Temporal scRNA-Seq analysis of the response to antirejection therapy und...
A participant on tacrolimus IS (TAC_3) was diagnosed with ACR 1B on PTD 217, and a biopsy was obtained prior to antirejection treatment with rATG and steroids. A second biopsy was obtained on PTD 232, and the participant was diagnosed with a borderline lesion. A third biopsy was taken at PTD 295, and the participant was diagnosed with no rejection. (A) Pie charts display number and frequency of expanded clonotypes found in the index biopsy (PTD 217) and subsequent follow-up biopsies (PTD 232, PTD 295). Bar graph shows overlapping clonotypes across the 3 time points. (B) UMAP shows CD8+ clusters in an integrated analysis of all time points. (C) Violin plots show relative expression levels of indicated genes selected to characterize cell cluster phenotypes as activated, exhausted, and memory. (D) Temporal analysis of CD8EXP following antirejection therapy. UMAP plots show clustering of CD8EXP (colored dots) versus CD8UNEXP (gray dots) cells from the participant at PTD 217 (left plot), PTD 232 (middle plot), or PTD 295 (right plot). CD8EXP first expanded on PTD 217 are shown in pink, those first expanding on PTD 232 are shown in green, and those first expanding on PTD 295 are shown in blue. (E) Heatmap shows average expression of unsupervised DEGs (P < 0.05) found between CD8EXP at each time point.