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].

Sonic Hedgehog Is a Determinant of γδ T-Cell Differentiation in the Thymus.

Here we investigate the function of Hedgehog (Hh) signaling in thymic γδ T-cell maturation and subset differentiation. Analysis of Hh mutants showed that Hh signaling promotes γδ T-cell development in the thymus and influences γδ T-cell effector subset distribution. Hh-mediated transcription in thymic γδ cells increased γδ T-cell number, and promoted their maturation and increased the γδNKT subset, whereas inhibition of Hh-mediated transcription reduced the thymic γδ T-cell population and increased expression of many genes that are normally down-regulated during γδ T-cell maturation. These changes were also evident in spleen, where increased Hh signaling increased γδNKT cells, but reduced CD27-CD44+ and Vγ2+ populations. Systemic in vivo pharmacological Smoothened-inhibition reduced γδ T-cell and γδNKT cells in the thymus, and also reduced splenic γδ T-cell and γδNKT populations, indicating that Hh signaling also influences homeostasis of peripheral γδ T-cell populations. Taken together our data indicate that Sonic Hedgehog is an important determinant of γδ T-cell effector subset differentiation.

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.

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.

PD-1 expression on natural killer cells and CD8(+) T cells during chronic HIV-1 infection.

Programmed death receptor 1 (PD-1) is an important marker of T-cell exhaustion during HIV-1 infection. Natural killer (NK) cells lose their functional capacity during HIV-1 infection, and PD-1 is expressed on NK cells during other chronic viral and bacterial infections. Here, PD-1 expression was increased on NK cells from both viremic and aviremic HIV-1-seropositive individuals, compared to seronegative controls. However, PD-1 was expressed on a small subset of NK cells and at lower frequency than that observed for CD8(+) T cells. PD-1 was also induced on a minor fraction of NK cells and CD8(+) T cells after long-term culture with IL-15. Raised levels of PD-1 were associated with limited NK cell proliferation, which may have consequences for their maintenance during chronic HIV-1 infection.