Development and validation of the first consensus gene-expression signature of operational tolerance in kidney transplantation, incorporating adjustment for immunosuppressive drug therapy.

BACKGROUND: Kidney transplant recipients (KTRs) with "operational tolerance" (OT) maintain a functioning graft without immunosuppressive (IS) drugs, thus avoiding treatment complications. Nevertheless, IS drugs can influence gene-expression signatures aiming to identify OT among treated KTRs. METHODS: We compared five published signatures of OT in peripheral blood samples from 18 tolerant, 183 stable, and 34 chronic rejector KTRs, using gene-expression levels with and without adjustment for IS drugs and regularised logistic regression. FINDINGS: IS drugs explained up to 50% of the variability in gene-expression and 20-30% of the variability in the probability of OT predicted by signatures without drug adjustment. We present a parsimonious consensus gene-set to identify OT, derived from joint analysis of IS-drug-adjusted expression of five published signature gene-sets. This signature, including CD40, CTLA4, HSD11B1, IGKV4-1, MZB1, NR3C2, and RAB40C genes, showed an area under the curve 0⋅92 (95% confidence interval 0⋅88-0⋅94) in cross-validation and 0⋅97 (0⋅93-1⋅00) in six months follow-up samples. INTERPRETATION: We advocate including adjustment for IS drug therapy in the development stage of gene-expression signatures of OT to reduce the risk of capturing features of treatment, which could be lost following IS drug minimisation or withdrawal. Our signature, however, would require further validation in an independent dataset and a biomarker-led trial. FUNDING: FP7-HEALTH-2012-INNOVATION-1 [305147:BIO-DrIM] (SC,IR-M,PM,DSt); MRC [G0801537/ID:88245] (MPH-F); MRC [MR/J006742/1] (IR-M); Guy's&StThomas' Charity [R080530]&[R090782]; CONICYT-Bicentennial-Becas-Chile (EN-L); EU:FP7/2007-2013 [HEALTH-F5-2010-260687: The ONE Study] (MPH-F); Czech Ministry of Health [NV19-06-00031] (OV); NIHR-BRC Guy's&StThomas' NHS Foundation Trust and KCL (SC); UK Clinical Research Networks [portfolio:7521].

Development of a multivariable gene-expression signature targeting T-cell-mediated rejection in peripheral blood of kidney transplant recipients validated in cross-sectional and longitudinal samples.

BACKGROUND: Acute T-cell mediated rejection (TCMR) is usually indicated by alteration in serum-creatinine measurements when considerable transplant damage has already occurred. There is, therefore, a need for non-invasive early detection of immune signals that would precede the onset of rejection, prior to transplant damage. METHODS: We examined the RT-qPCR expression of 22 literature-based genes in peripheral blood samples from 248 patients in the Kidney Allograft Immune Biomarkers of Rejection Episodes (KALIBRE) study. To account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene-expression residuals from linear mixed-effects models in stable patients. To select genes, we used penalised logistic regression based on 27 stable patients and 27 rejectors with biopsy-proven T-cell-mediated rejection, fulfilling strict inclusion/exclusion criteria. We validated this signature in i) an independent group of stable patients and patients with concomitant T-cell and antibody-mediated-rejection, ii) patients from an independent study, iii) cross-sectional pre-biopsy samples from non-rejectors and iv) longitudinal follow-up samples covering the first post-transplant year from rejectors, non-rejectors and stable patients. FINDINGS: A parsimonious TCMR-signature (IFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A) showed cross-validated area-under-ROC curve 0.84 (0.77-0.88) (median, 2.5th-97.5th centile of fifty cross-validation cycles), sensitivity 0.67 (0.59-0.74) and specificity 0.85 (0.75-0.89). The estimated probability of TCMR increased seven weeks prior to the diagnostic biopsy and decreased after treatment. Gene expression in all patients showed pronounced variability, with up to 24% of the longitudinal samples in stable patients being TCMR-signature positive. In patients with borderline changes, up to 40% of pre-biopsy samples were TCMR-signature positive. INTERPRETATION: Molecular marker alterations in blood emerge well ahead of the time of clinically overt TCMR. Monitoring a TCMR-signature in peripheral blood could unravel T-cell-related pro-inflammatory activity and hidden immunological processes. This additional information could support clinical management decisions in cases of patients with stable but poor kidney function or with inconclusive biopsy results.

Steroid regulation: An overlooked aspect of tolerance and chronic rejection in kidney transplantation.

Steroid conversion (HSD11B1, HSD11B2, H6PD) and receptor genes (NR3C1, NR3C2) were examined in kidney-transplant recipients with "operational tolerance" and chronic rejection (CR), independently and within the context of 88 tolerance-associated genes. Associations with cellular types were explored. Peripheral whole-blood gene-expression levels (RT-qPCR-based) and cell counts were adjusted for immunosuppressant drug intake. Tolerant (n = 17), stable (n = 190) and CR patients (n = 37) were compared. Healthy controls (n = 14) were used as reference. The anti-inflammatory glucocorticoid receptor (NR3C1) and the cortisol-activating HSD11B1 and H6PD genes were up-regulated in CR and were lowest in tolerant patients. The pro-inflammatory mineralocorticoid gene (NR3C2) was downregulated in stable and CR patients. NR3C1 was associated with neutrophils and NR3C2 with T-cells. Steroid conversion and receptor genes, alone, enabled classification of tolerant patients and were major contributors to gene-expression signatures of both, tolerance and CR, alongside known tolerance-associated genes, revealing a key role of steroid regulation and response in kidney transplantation.

Reduced TCR Signaling Contributes to Impaired Th17 Responses in Tolerant Kidney Transplant Recipients.

BACKGROUND: The development of spontaneous kidney transplant tolerance has been associated with numerous B cell-related immune alterations. We have previously shown that tolerant recipients exhibit reduced B-cell receptor signalling and higher IL-10 production than healthy volunteers. However, it is unclear whether cluster of differentiation (CD)4 T cells from tolerant recipients also display an anti-inflammatory profile that could contribute to graft maintenance. METHODS: CD4 T cells were isolated from kidney transplant recipients who were identified as being tolerant recipients, patients with chronic rejection or healthy volunteers. CD4 T cells from the 3 groups were compared in terms of their gene expression profile, phenotype, and functionally upon activation. RESULTS: Gene expression analysis of transcription factors and signalling proteins, in addition to surface proteins expression and cytokine production, revealed that tolerant recipients possessed fewer Th17 cells and exhibited reduced Th17 responses, relative to patients with chronic rejection or healthy volunteers. Furthermore, impaired T-cell receptor signalling and altered cytokine cooperation by monocytes contributed to the development of Th17 cells in tolerant recipients. CONCLUSIONS: These data suggest that defective proinflammatory Th17 responses may contribute to the prolonged graft survival and stable graft function, which is observed in tolerant recipients in the absence of immunosuppressive agents.

Increased CD40 Ligation and Reduced BCR Signalling Leads to Higher IL-10 Production in B Cells From Tolerant Kidney Transplant Patients.

BACKGROUND: An increased percentage of peripheral transitional B cells producing IL-10 has been observed in patients tolerant to kidney allografts. In healthy volunteers, the balance between the CD40 and B-cell receptor (BCR) signalling modulated IL-10 production by B cells, with stimulation via the BCR decreasing CD40-mediated IL-10 production. In this study, we evaluate whether in tolerant kidney transplant patients, the increased IL-10 production by B cells was due to an altered CD40 and/or BCR signalling. METHODS: B cells obtained from a new cohort of tolerant renal transplant recipients and those from age- and sex-matched healthy volunteers were activated via CD40 and BCR, either alone or in combination. RESULTS: In tolerant patients, we observed higher percentages of B cells producing IL-10 after CD40 ligation and higher expression of CD40L on activated T cells compared with healthy controls. Furthermore, B cells from tolerant recipients had reduced extracellular signal-regulated kinase signalling after BCR-mediated activation compared with healthy controls. In keeping with this, combining BCR signalling with CD40 ligation did not reduce IL-10 secretion as was observed in healthy control transitional B cells. CONCLUSIONS: Altogether, our data suggest that the altered response of B cells in tolerant recipients may contribute to long-term stable graft acceptance.