Donor-Specific Antibody Is Associated with Increased Expression of Rejection Transcripts in Renal Transplant Biopsies Classified as No Rejection

Abstract

Background: Donor -specific HLA antibody (DSA) is present in many kidney transplant patients whose biopsies are classified as no rejection (NR). We explored whether in some NR kidneys DSA has subtle effects not currently being recognized.

Methods: We used microarrays to examine the relationship between standard-of-care DSA and rejection-related transcript increases in 1679 kidney transplant indication biopsies in the INTERCOMEX study (ClinicalTrials.gov NCT01299168), focusing on biopsies classified as NR by automatically assigned archetypal clustering. DSA testing results were available for 835 NR biopsies and were positive in 271 (32%).

Results: DSA positivity in NR biopsies was associated with mildly increased expression of antibody-mediated rejection (ABMR)-related transcripts, particularly IFNG-inducible and NK cell transcripts. We developed a machine learning DSA probability (DSAProb) classifier based on transcript expression in biopsies from DSA-positive versus DSA-negative patients, assigning scores using 10-fold cross-validation. This DSAProb classifier was very similar to a previously described "ABMR probability" classifier trained on histologic ABMR in transcript associations and prediction of molecular or histologic ABMR. Plotting the biopsies using Uniform Manifold Approximation and Projection revealed a gradient of increasing molecular ABMR-like transcript expression in NR biopsies, associated with increased DSA (P<2 × 10-16). In biopsies with no molecular or histologic rejection, increased DSAProb or ABMR probability scores were associated with increased risk of kidney failure over 3 years.

Conclusions: Many biopsies currently considered to have no molecular or histologic rejection have mild increases in expression of ABMR-related transcripts, associated with increasing frequency of DSA. Thus, mild molecular ABMR-related pathology is more common than previously realized.

Keywords: gene expression; kidney biopsy; rejection; renal transplantation; transplantation.

Copyright © 2021 by the American Society of Nephrology.

Figures

Figure 1.

Distribution of n=1679 biopsies using principal component analysis (PCA) and colored by archetype groups and DSA status. The main population of 1679 kidney transplant indication biopsy samples colored by (A) previously defined six-class rejection-based archetypal group assignment (NR, TCMR, mixed, early-stage ABMR, fully developed ABMR, and late-stage ABMR), and (B) the known DSA status of each biopsy sample (biopsy samples where the status is unknown are marked in gray). Biopsy specimens are distributed from NR to rejection across PC1 (x axis of [A]), and from ABMR to TCMR across PC2 (y axis of [A] and [B]). Therefore, biopsy samples with low probability of molecular rejection will be located to the left in PC1.

Figure 2.

AUCs showing the predictive performance of selected classifiers for ABMR, TCMR, and DSA status. ROC curves demonstrating the predictive performance of the DSAProb classifier score, the ABMRpm classifier score, and the TCMR classifier score for prediction of MMDx ABMR sign-out diagnoses, any of the three ABMR stages described by rejection archetype groups, histologic ABMR as assigned by the local center, DSA status, and MMDx TCMR sign-out diagnoses.

Figure 3.

A scatterplot showing the ABMRpm classifier scores versus the DSAProb classifier scores for each biopsy sample in the full (n=1679) population. Biopsy specimens are colored by their assigned rejection-based archetype groups. The blue dotted regression line shows the best fit through the data. Red dotted horizontal and vertical lines show the cutoffs for the ABMRpm classifier score and DSAProb classifier score.

Figure 4.

Gradients in 1679 biopsies over archetype groups, and DSAProb/ABMRpm classifier scores. All 1679 indication kidney transplant biopsy specimens distributed using UMAP, colored by (A) assigned rejection-based archetypal class, (B) increasing DSAProb classifier score, and (C) increasing ABMRpm score. Biopsy samples with low probability of molecular rejection are located toward the bottom of Component 2 in (A–C). Biopsy samples with rejection are located toward the upper region of Component 2, with ABMR on the left and TCMR on the right of Component 1. This same population was also distributed using PCA. The distribution of PC1 versus PC2 was colored by increasing DSAProb classifier score in (D) and by increasing ABMRpm classifier score in (E). PC1 distributed biopsy samples from NR to rejection (x axis of [D] and [E]), and PC2 separates ABMR from TCMR (y axis of [D] and [E]). Rolling average plots for frequency of percentage positive DSA status (y axis) versus increasing average DSAProb classifier score are shown for all 1679 biopsy samples (F) and in 1012 NR samples (G). (H) The percentage positive DSA versus the rolling average for the ABMRpm classifier in the 1012 NR biopsy samples. Rolling average plots are segmented, and colored on the basis of the same cutoffs for the DSAProb and ABMRpm used in (A–C).

Figure 5.

Mean DSAProb and ABMRpm classifier scores differ between DSA positive and DSA negative biopsies. Beeswarms and boxplots showing the (A–C) DSAProb and (D–F) ABMRpm classifier scores divided by DSA status (DSA-positive versus DSA-negative). The center line of the box shows the median score in each group, whereas the box shows the data between the first and third quartiles of the scores (interquartile range or IQR). Whiskers mark 1.5×IQR. DSAProb classifier scores are shown divided into DSA-positive and DSA-negative groups in (A) all 1679 biopsy samples, (B) 1012 biopsy samples with no molecular rejection (NR), and (C) 764 biopsy samples with no molecular or histologic rejection. ABMRpm scores divided into DSA-positive and DSA-negative groups are shown in (D) all 1679 biopsy samples, (E) 1012 NR biopsy samples, and (F) 764 biopsy samples with no molecular or histologic rejection. All P values are calculated from Welch’s two-sample t tests comparing the DSA-positive and DSA-negative groups.

Figure 6.

Three year graft survival compared between DSA positive and DSA negative biopsies, DSAProb high and DSAProb low biopsies, and ABMRpm high versus ABMRpm low biopsies. Horizontal ticks mark censoring events by 3 years postbiopsy. The survival curves for DSA-positive and DSA-negative groups are shown in (A) all 1679 biopsy specimens and in (B) 1012 NR biopsy specimens. Survival curves are also shown for high versus low DSAProb classifier score in (C) all 1679 biopsy samples and in (D) 1012 NR biopsy samples. Finally, survival curves are shown for high versus low ABMRpm classifier score in (E) all 1679 biopsy samples and in (F) 1012 NR biopsy samples.