Adaptation of Imaging Mass Cytometry to Explore the Single Cell Alloimmune Landscape of Liver Transplant Rejection

Abstract

Rejection continues to be an important cause of graft loss in solid organ transplantation, but deep exploration of intragraft alloimmunity has been limited by the scarcity of clinical biopsy specimens. Emerging single cell immunoprofiling technologies have shown promise in discerning mechanisms of autoimmunity and cancer immunobiology. Within these applications, Imaging Mass Cytometry (IMC) has been shown to enable highly multiplexed, single cell analysis of immune phenotypes within fixed tissue specimens. In this study, an IMC panel of 10 validated markers was developed to explore the feasibility of IMC in characterizing the immune landscape of chronic rejection (CR) in clinical tissue samples obtained from liver transplant recipients. IMC staining was highly specific and comparable to traditional immunohistochemistry. A single cell segmentation analysis pipeline was developed that enabled detailed visualization and quantification of 109,245 discrete cells, including 30,646 immune cells. Dimensionality reduction identified 11 unique immune subpopulations in CR specimens. Most immune subpopulations were increased and spatially related in CR, including two populations of CD45+/CD3+/CD8+ cytotoxic T-cells and a discrete CD68+ macrophage population, which were not observed in liver with no rejection (NR). Modeling via principal component analysis and logistic regression revealed that single cell data can be utilized to construct statistical models with high consistency (Wilcoxon Rank Sum test, p=0.000036). This study highlights the power of IMC to investigate the alloimmune microenvironment at a single cell resolution during clinical rejection episodes. Further validation of IMC has the potential to detect new biomarkers, identify therapeutic targets, and generate patient-specific predictive models of clinical outcomes in solid organ transplantation.

Spatially resolved immune exhaustion within the alloreactive microenvironment predicts liver transplant rejection

“…S1D) in all clinical groups, which highlights their key role in liver homeostasis, disease, and injury processes ( 26 , 27 ). Macrophages can participate in robust infiltration of the AME during severe rejection episodes; however, their role has rarely been investigated in TCMR and CR in clinical LT ( 36 , 37 ). We have previously shown that CR is characterized by a discrete macrophage phenotype absent in NR ( 37 ).…”

“…Macrophages can participate in robust infiltration of the AME during severe rejection episodes; however, their role has rarely been investigated in TCMR and CR in clinical LT ( 36 , 37 ). We have previously shown that CR is characterized by a discrete macrophage phenotype absent in NR ( 37 ). Thus, to obtain a detailed representation of the macrophages complexity and heterogenous activity in LT, we first divided macrophages M1 and M2 based on their expression of CD163 (fig.…”

“…Immune lineage markers included CD3, CD4, CD8, CD20, CD68, and CD11b, and functional or phenotypic markers included CD279 (PD1), FoxP3, Ki67, and Granzyme B among others (table S3). IMC staining was performed using techniques described previously ( 37 ). Ninety-six ROIs (average of 1.2 ROI per patient) were ablated using the Hyperion Imaging System (Standard Biotools) at a power range of 3.5 to 4.5 with a laser frequency at 200 Hz.…”

Cancer Risk and Mutational Patterns Following Organ Transplantation

“…Furthermore, the cellular heterogeneity of tumors is a major obstacle to understanding and treating oncology. Single-cell sequencing technology can make up for limitations of traditional high-throughput sequencing by sequencing single cells, so as to reveal cancer gene structure and expression more accurately from the cellular level, and can be used to reflect the heterogeneity among tumor cells ( Rantalainen, 2018 ; Malone and Humphreys, 2019 ; Natarajan et al, 2019 ; Winterhoff et al, 2019 ; Ung et al, 2022 ). Li et al mapped the heterogeneity of immune cells after liver transplantation for the first time by single-cell sequencing and other techniques.…”

Defining the T cell transcriptional landscape in pediatric liver transplant rejection at single cell resolution

“…Yet, the liver is an immune organ, housing an array of specialized innate immune cell populations (NK, NKT, Mucosal Associated Invariant T (MAIT)) in addition to cognate immune populations (CD4 + or CD8 + T cells) which are transplanted with the allograft into the recipient (27–31). To date, little has been done to investigate the signaling properties of intragraft T cells in liver transplant rejection beyond bulk RNA sequencing (18, 32, 33), tissue microarray (34), imaging mass cytometry (35), and TCR Vβ gene sequencing (36), which yield little insight into rejection pathogenesis beyond known pathways of antigen presentation to and interferon-ɣ production by T cells. A more detailed understanding of the underlying biology of rejection is needed, specifically in pediatric patients, to ensure long-term graft survival and minimize unintended consequences of immunosuppression.…”