Figures Data for "Development and Internal Validation of a Novel Pre-Transplant Biomarker Panel to Predict Post-Transplant Mortality in Liver Transplant Recipients"

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Figures Data regarding the development and internal validation of the Liver Immune Frailty Index. This study seeks to identify biomarkers of pre-liver transplant (LT) immune dysfunction that predict early mortality following LT. Plasma biomarkers and clinical variables were assessed 279 waitlisted patients immediately prior to LT (T0). HCV IgG, Fractalkine, and MMP3 were multivariate predictors of 1-year mortality. These were utilized to create the Liver Immune Frailty Index (LIFI), which stratifies recipients into -low, -moderate, and –high risk tertiles of early post-LT mortality. One-year mortality was 1.4%, 12.7%, and 58.3% for LIFI-low, -moderate, and -high, respectively. Internal validation demonstrates LIFI predicts early post-LT mortality (C-statistic=0.84, Brier score=0.04). Stratification into LIFI-high or moderate requires cumulative contribution of elevated MMP3 and Fractalkine levels and is not dependent on HCV status. Excluding HCV IgG+ as a covariate similarly stratifies patients at high-, moderate-, and low-risk of early post-LT mortality (LIFIMF, C-statistic 0.83). LIFI may identify patients at risk for persistent severe immune dysfunction and early mortality post-LT.

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Figure 2 - Retrospective chart review of clinical data was performed on a single center cohort patients (n=779) undergoing LT from 1/1/2007-12/31/17 to assess longitudinal trends in disease severity at the time of transplant, patient survival, and causes of mortality within the first year following liver transplant. Figure 3 - Quantitative cytokine and chemokine analysis was performed via Luminex using commercially available assays per manufacturer guidelines. A final panel of 24 cytokines and chemokines was ultimately analyzed in all 279 patients (sIL6Rb CXCL6, CCL1, CXCL12, MMP2, TNF-a, BAFF, Eotaxin, MMP3, IP-10, IL-10, CTACK, and Fractalkine, BioRad, Hercules, CA; GM-CSF, IFN-y, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, and IL-13, MilliporeSigma, Burlington, MA). Plates were read using the LX200 Luminex instrument (ThermoFisher, Waltham, MA) and were analyzed via Bio-Plex Manager Software version 6.2. Figure 4 - Analysis of clinical outcomes and survival data for all patients included in the analysis as stratified by the Liver Immune Frailty Index (LIFI) score Figure 5 - For cell subset evaluation from PMBCs, absolute leukocyte counts were obtained from included patients base on the clinical differential blood cell analysis on the day of liver transplant. For polychromatic flow cytometric analysis, 2x106 cryopreserved PBMCs obtained at the time of liver transplant (T0) were thawed, washed, and rested for 2 hours at 37° C and 5% CO2 at 1-2x106 /mL of RPMI 1640 Medium, GlutaMAX™ Supplement with 10% human AB serum and 1% Penicillin/Streptomycin (Gibco, Thermo Fisher, Waltham, MA). Cells were then washed resuspended in Flow Cytometry Staining Buffer (Invitrogen, Thermo Fisher, Waltham, MA). Viability dye Ghost Dye Violet 510 (Tonbo Biosciences, San Diego, CA) was added, followed cell surface staining with the following mouse anti-human mAbs: CD3, CD4, CD8a, CD197/CCR7 , CD45RA, CD279/PD-1, CD366/Tim-3, TIGIT, and CD25. Intracellular staining for Fox-P3 was then performed using the Foxp3/Transcriptional Factor Staining kit (Invitrogen, Thermo Fisher, Waltham, MA) for cell fixation and permeabilization and anti-Human FoxP3 mAb staining. Unstained normal healthy control cells controlled for autofluorescence, and NHC cells stimulated for 72 hours with anti-CD3 mAb and anti-CD28 mAb served as positive control. Data collection was performed on a five-laser LSR2 Fortessa X-20 Flow Cytometer (BD Biosciences, Franklin Lakes, NJ). Analysis was performed with FlowJo v10.9 (BD Biosciences, Franklin Lakes, NJ).

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