Advanced Genomics-Based Approaches for Defining Allograft Rejection With Single Cell Resolution

Abstract

Solid organ transplant recipients require long-term immunosuppression for prevention of rejection. Calcineurin inhibitor (CNI)-based immunosuppressive regimens have remained the primary means for immunosuppression for four decades now, yet little is known about their effects on graft resident and infiltrating immune cell populations. Similarly, the understanding of rejection biology under specific types of immunosuppression remains to be defined. Furthermore, development of innovative, rationally designed targeted therapeutics for mitigating or preventing rejection requires a fundamental understanding of the immunobiology that underlies the rejection process. The established use of microarray technologies in transplantation has provided great insight into gene transcripts associated with allograft rejection but does not characterize rejection on a single cell level. Therefore, the development of novel genomics tools, such as single cell sequencing techniques, combined with powerful bioinformatics approaches, has enabled characterization of immune processes at the single cell level. This can provide profound insights into the rejection process, including identification of resident and infiltrating cell transcriptomes, cell-cell interactions, and T cell receptor α/β repertoires. In this review, we discuss genomic analysis techniques, including microarray, bulk RNAseq (bulkSeq), single-cell RNAseq (scRNAseq), and spatial transcriptomic (ST) techniques, including considerations of their benefits and limitations. Further, other techniques, such as chromatin analysis via assay for transposase-accessible chromatin sequencing (ATACseq), bioinformatic regulatory network analyses, and protein-based approaches are also examined. Application of these tools will play a crucial role in redefining transplant rejection with single cell resolution and likely aid in the development of future immunomodulatory therapies in solid organ transplantation.

Mesenchymal Stromal Cell Therapy in Lung Transplantation

“…Co-stimulatory signals and cytokine-mediated lymphocyte differentiation and expansion are also needed to mount an efficient alloimmune response, and the effector mechanisms involve CD4 + helper T cells, CD8 + cytotoxic T cells, B cells, antibody production, and complement-mediated damage. Importantly, Tregs function to limit the alloimmune response [ 84 ].…”

The Value of Single-cell Technologies in Solid Organ Transplantation Studies

“…20,22 Analysis of such formalin-fixed paraffin embedded biopsies opens up new avenues to study the allograft, for example, by studying biobank samples retrospectively. 11 Yet,…”

“…[6][7][8][9][10] This technique profiles thousands of predefined transcripts simultaneously through hybridization. 11 More recently, bulk RNA sequencing, which can quantify all transcripts in a cell population rather than a targeted set of genes, 12 has been developed. Still, both techniques measure pooled RNA and thus reflect the average gene expression of cells.…”

“…20,22 Analysis of such formalin-fixed paraffin embedded biopsies opens up new avenues to study the allograft, for example, by studying biobank samples retrospectively. 11 Yet, it remains difficult to analyze gene expression patterns of individual cells using spatial transcriptomics. Combining spatial transcriptomics with single-cell transcriptomics may be a way to overcome this challenge, and may further increase our knowledge on the roles of specific cell subsets and their interactions during alloimmune responses.…”

Transcriptome profiling of immune rejection mechanisms in a porcine vascularized composite allotransplantation model

“…To address these knowledge gaps, we turned to the potent tool of RNA sequencing (RNA-Seq) coupled with bioinformatics analysis in a porcine VCA model. This technique is widely recognized for its capacity to comprehensively profile the transcriptome with exceptional sensitivity ( 22 – 25 ). While RNA-Seq has been widely used in solid organ transplantation studies, its application in VCA research, especially in large animal models, remains underutilized ( 26 – 29 ).…”