Molecular Signature Associated With Acute Rejection in Vascularized Composite Allotransplantation

Abstract

Background: A deeper understanding of acute rejection in vascularized composite allotransplantation is paramount for expanding its utility and longevity. There remains a need to develop more precise and accurate tools for diagnosis and prognosis of these allografts, as well as alternatives to traditional immunosuppressive regimens.

Methods: Twenty-seven skin biopsies collected from 3 vascularized composite allotransplantation recipients, consisting of face and hand transplants, were evaluated by histology, immunohistochemistry staining, and gene expression profiling.

Results: Biopsies with clinical signs and symptoms of rejection, irrespective of histopathological grading, were significantly enriched for genes contributing to the adaptive immune response, innate immune response, and lymphocyte activation. Inflammation episodes exhibited significant fold change correlations between the face and hands, as well as across patients. Immune checkpoint genes were upregulated during periods of inflammation that necessitated treatment. A gene signature consisting of CCL5, CD8A, KLRK1, and IFNγ significantly predicted inflammation specific to vascularized composite allografts that required therapeutic intervention.

Conclusions: The mechanism of vascularized composite allograft-specific inflammation and rejection appears to be conserved across different patients and skin on different anatomical sites. A concise gene signature can be utilized to ascertain graft status along with a continuous scale, providing valuable diagnostic and prognostic information to supplement current gold standards of graft evaluation.

FIGURE 1.

Acute rejection and inflammatory reaction biopsies share DEGs. Clinical images demonstrating (A) NR and (B) AR. Clustering by (C) PCA and (D) unsupervised hierarchical heatmap clustering demonstrating colocalization of inflamed biopsies separate from NR. Volcano plots annotated with top DEGs comparing (E) AR to NR and (F) IR to NR. G, Pathway enrichment of DEGs from aggregated AR/IR samples. Printed with permission and copyrights retained by Eduardo D. Rodriguez, MD, DDS. AR, acute rejection; DEG, differentially expressed gene; IL, interleukin; IR, inflammatory reaction; NR, nonrejection; PCA, principal component analysis.

FIGURE 2.

Inflammatory mechanisms are conserved across anatomical sites. A, Unsupervised hierarchical heatmap clustering depicting fold changes of inflamed biopsies compared with NR in patient 1. B, Pearson correlation coefficients comparing NanoString panel fold changes between inflamed samples in patient 1. Volcano plots comparing (C) face inflamed vs NR, (D) hand inflamed vs NR, and (E) face vs hand inflamed exclusively in patient 1. F, Venn diagram of mutually enriched DEGs. DEG, differentially expressed gene; NR, nonrejection.

FIGURE 3.

Inflammatory mechanisms are conserved across patients with VCA. A, Unsupervised hierarchical heatmap clustering depicting fold changes of face inflamed biopsies compared with NR across patients. B, Pearson correlation coefficients comparing NanoString panel fold changes between face inflamed samples across patients. Volcano plots comparing (C) patient 1 face inflamed vs all NR, (D) patients 2/3 face inflamed vs all NR, and (E) patient 1 vs patients 2/3 face inflamed. F, Venn diagram of mutually enriched DEGs. DEG, differentially expressed gene; NR, nonrejection; VCA, vascularized composite allotransplantation.

FIGURE 4.

Concise Inflammation Score distinguishes graft status. Normalized and log-transformed counts for (A) immune checkpoint genes. Elastic net regression genes with Inflammation Score trained on (B) internal dataset and tested on (C) external dataset. Dotted line represents threshold discriminating significantly inflamed from noninflamed samples. D, ROC curve of Inflammation Score validated on external dataset of VCA and non-VCA biopsies. *P_adj < 0.05, **P_adj < 0.01, ***P_adj < 0.001. AR, acute rejection; DEG, differentially expressed gene; IR, inflammatory reaction; NR, nonrejection; ROC, receiver operating characteristic; VCA, vascularized composite allotransplantation.