Monitoring pharmacologically induced immunosuppression by immune repertoire sequencing to detect acute allograft rejection in heart transplant patients: a proof-of-concept diagnostic accuracy study

Abstract

Background: It remains difficult to predict and to measure the efficacy of pharmacological immunosuppression. We hypothesized that measuring the B-cell repertoire would enable assessment of the overall level of immunosuppression after heart transplantation.

Methods and findings: In this proof-of-concept study, we implemented a molecular-barcode-based immune repertoire sequencing assay that sensitively and accurately measures the isotype and clonal composition of the circulating B cell repertoire. We used this assay to measure the temporal response of the B cell repertoire to immunosuppression after heart transplantation. We selected a subset of 12 participants from a larger prospective cohort study (ClinicalTrials.gov NCT01985412) that is ongoing at Stanford Medical Center and for which enrollment started in March 2010. This subset of 12 participants was selected to represent post-heart-transplant events, with and without acute rejection (six participants with moderate-to-severe rejection and six without). We analyzed 130 samples from these patients, with an average follow-up period of 15 mo. Immune repertoire sequencing enables the measurement of a patient's net state of immunosuppression (correlation with tacrolimus level, r = -0.867, 95% CI -0.968 to -0.523, p = 0.0014), as well as the diagnosis of acute allograft rejection, which is preceded by increased immune activity with a sensitivity of 71.4% (95% CI 30.3% to 94.9%) and a specificity of 82.0% (95% CI 72.1% to 89.1%) (cell-free donor-derived DNA as noninvasive gold standard). To illustrate the potential of immune repertoire sequencing to monitor atypical post-transplant trajectories, we analyzed two more patients, one with chronic infections and one with amyloidosis. A larger, prospective study will be needed to validate the power of immune repertoire sequencing to predict rejection events, as this proof-of-concept study is limited to a small number of patients who were selected based on several criteria including the availability of a large number of samples and the absence or presence of rejection events.

Conclusions: If confirmed in larger, prospective studies, the method described here has potential applications in the tailored management of post-transplant immunosuppression and, more broadly, as a method for assessing the overall activity of the immune system.

Conflict of interest statement

CV, IDV, and SRQ have a patent S14-048 (STAN-1110PRV) 61/951,908: Provisional Application pending. HAV, HL, CS, GC, and KKK have nothing to declare.

Figures

Fig 1. Monitoring overall immunosuppression.

Organ transplant recipients receive immunosuppressive therapy, which usually includes calcineurin inhibitors. These drugs inhibit T cell activation, leading to fewer B cells expressing class-switched and mutated IGH transcripts. We measured overall immunosuppression using immune repertoire sequencing (IGH-Seq). A schematic of the IGH-Seq protocol and data analysis is shown on the right. C_Region, primer pool specific to constant region; CDR3, complementary determining region 3; PBMC, peripheral blood mononuclear cell; UID, unique identifier; V_FR3, primer pool specific to V segments.

Fig 2. Using immune repertoire sequencing to measure immunosuppression.

(A) Relative IGH sequence composition at increasing expression levels (molecules/sequence) is shown across all individuals (n = 12) in a histogram for days 1 and 180 post-transplant. Bar colors indicate the contribution of different immunoglobulin isotypes. Inserts show rescaled histograms. Highly expressed (HE) sequences (sequences represented by more than one molecule) are labeled in the insert. (B) ABS level (defined as the ratio of all highly expressed IgA, IgD, IgE, IgG, and mutated IgM sequences to the total number of molecules) as a function of tacrolimus trough level (n = 130) (average ± standard error of the mean). (C) Median ABS level (colors indicate isotype contribution), tacrolimus trough level, and total anellovirus load for all study patients (n = 12) during the first year after transplant.

Fig 3. Reduced level of overall immunosuppression correlates with acute rejection events.

ABS levels are shown for two individuals with (C and D) and two individuals without (A and B) moderate-to-severe acute rejection events (top). Colors indicate isotype contribution. cfdDNA levels are shown for the same individuals (bottom). Biopsy grades are shown as vertical blue lines (bottom). Thin lines indicate mild ACR events (grade 1R), and thick lines indicate moderate-to-severe ACR events (grade ≥ 2R).

Fig 4. ABS levels correlate well with cfdDNA and endomyocardial biopsy results.

(A) ABS levels plotted for all study participants. Acute rejection events as diagnosed by endomyocardial biopsy (ACR, violet; acute antibody-mediated rejection [AMR], green) or cfdDNA level (pink) are shown in color. (B) Samples are classified by rejection status diagnosed by biopsy as no evidence of rejection, mild ACR (biopsy grade 1R), or moderate-to-severe ACR (biopsy grade ≥ 2R), and by rejection status diagnosed by the cfdDNA assay. Samples are colored as in (A) if at any point the organ recipient experienced a serious rejection event. As biopsy grades are independent of cfdDNA measurements, there is overlap between samples in the elevated cfdDNA and biopsy-proven rejection bins. The groups were compared using a one-sided Mann-Whitney U test, which has high efficiency for both normally and non-normally distributed datasets. (C) Receiver operating characteristic (ROC) curves for ABS levels compared to cfdDNA (pink) and endomyocardial biopsy (violet) results. FPR, false rejection rate; TPR, true positive rate. (D) ABS levels measured before and after rejection diagnosis. The black line shows the single-exponent fit of ABS levels measured prior to rejection, y = a × (exp[b × t]), with best fit values (least squares) of a = 0.0317 and b = 0.00468. The gray line shows the single-exponent fit of ABS levels after diagnosis and during treatment of rejection: y = a × (exp[b× t]), with best fit values (least squares) of a = 0.0344 and b = −0.0324. Green lines show the median ABS levels in the covered time periods.

Fig 5. Reduced net immunosuppression correlates with acute rejection.

ABS level is shown for three individuals: (A) a heart transplant recipient without moderate-to-severe rejection events, (B) a heart transplant recipient with infectious complications, and (C) a heart transplant recipient with AL amyloidosis. For the patient with AL amyloidosis (C), the approximate schedule of drug treatment is indicated at the top of the panel. Colors indicate isotype contribution. cfdDNA level is shown for the same individuals (bottom). Biopsy grades are shown as vertical blue lines (bottom): thin lines indicate mild rejection events (grade 1R), and thick lines indicate moderate-to-severe rejection events (grade ≥ 2R). SCT, stem cell transplant.