Proper evaluation of the immune status of liver transplant recipients is crucial for appropriate post-transplant management. Current monitoring of graft function relies mainly on non-specific liver enzyme tests and immunosuppressive drug levels. Alterations in hepatic enzyme levels occur for multiple reasons including acute rejection (AR). Currently the gold standard for diagnosis of AR is liver biopsy. Identifying an immune signature that differentiates AR patients through a minimally invasive blood test would be preferable.

To define an immune signature of AR, we utilized cytometry by time-of-flight (mass cytometry) to comprehensively characterize the peripheral blood mononuclear cells (PBMC) from pediatric recipients of liver allografts (n=11) on conventional immunosuppression (tacrolimus monotherapy). PBMC from patients with biopsy-proven AR prior to treatment (3.8±5.9 years post-transplant, mean age 9.6±8.0 y) were compared to stable liver recipients (13.0±1.5 years post-transplant, mean age 15.2±4.9 y).

Correlation analyses from mass cytometry data was performed using Citrus, a method for unsupervised identification of significant cellular populations, with cell subsets identified using hierarchical clustering of live cells using R. Cells were clustered on the basis of expression of 22 markers and significant changes in cell frequency inferred using the “glmnet” package in R. When the two patient populations were compared, two clusters of significance, a CD4⁺CD5⁺CD25⁺CD27⁺CD44⁺ T cell population and a CD56⁺CD16⁺ NK cell population, were significantly increased in the AR patients as compared to the stable group. Manual gating of the data revealed that CD3^–CD19^–CD20^–CD8a⁺CD16⁺CD56⁺ NK cells were significantly increased in the AR group (p≤0.05) and that the NK:CD3⁺ T cell ratio as well as the NK:CD4⁺CD5⁺CD25⁺CD27⁺ T cell ratio were significantly increased in AR compared to stable patients (p≤0.01 for both).

Additional studies to further define the phenotype and function of these NK and T cell populations are necessary. In summary, using mass cytometry, we have demonstrated that NK cells and a specific T cell subset are significantly increased during AR in pediatric transplant recipients. These results demonstrate the power of mass cytometry to discover significant immune cell populations that may have diagnostic potential.

CITATION INFORMATION: Lau A, Haas K, Shawler T, Esquivel C, Martinez O, Castillo R, Krams S. Mass Cytometry Reveals NK Cell and T Cell Subsets in Pediatric Liver Transplant Patients with Acute Rejection. Am J Transplant. 2016;16 (suppl 3).

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