Paula Mobillo

Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell–related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group—namely, the tolerant recipients—were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS‐independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross‐validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

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.

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.

Absence of Anti-Donor Specific Antibodies in Drug-Free Tolerant Kidney Transplant Recipients: 1920

Mobillo P.1, Norris S.1, Rebollo-Mesa I.1, Nova-Lamperti E.1, Kamra Y.2,3, Lord G.2,3,4, Vaughan R.2,5, Lechler R.2,4, Hernandez-Fuentes M.1,3, GAMBIT Consortium; Immune Tolerance Network; Indices of Tolerance Consortium 1King‘s College London, MRC Centre for Transplantation, London, United Kingdom, 2King‘s College London, London, United Kingdom, 3NIHR Comprehensive Biomedical Research Centre at Guy‘s and St Thomas‘s Hospital. NHS Foundation Trust in partnership with King‘s College London and King‘s College Hospital, London, United Kingdom, 4King‘s Health Partners, London, United Kingdom, 5Guy‘s and St Thomas‘ NHS foundation Trust, Clinical Transplantation laboratory, London, United Kingdom

Differential effect of memory, naive and transitional B cells in autologous CD4(+) T cell proliferation, activation and cytokine production

CD200 is a cell-surface glycoprotein that is normally expressed in tissues of the immune system, where its role is to protect immune privileged sites. We previously established CD200 to be frequently over-expressed and associated with poor AML patient outcome. In this study, we investigated the possibility that CD200 expression may mediate suppression of T-cell function in this disease. Using multiparameter flow cytometry, we compared PMA/ionomycin stimulated CD8+ T-cell cytotoxic potential (CD107a expression) and the frequency of intracellular TNFa, IL-2 and IFNc producing CD4+/CD8+ memory T-cells between CD200hi and CD200lo patients. We demonstrated that both the magnitude of the CD8+ memory cytotoxic T-cell response and the Th1 cytokine producing CD4+ memory helper T-cells was significantly inhibited in CD200hi AML patients (P < 0.05). Further, using ELISPOT assays to measure IFNg release we showed that the Th1 memory response to common viral antigens was significantly reduced by 75% in CD200hi versus CD200lo AML patients(P < 0.05). Recovery of IFNc release in response to recall antigens was observed in CD4+ memory T-cells incubated with a blocking antibody to CD200R. In conclusion, this study shows a correlation between T-cell dysfunction and expression of CD200 which suggests targeting this axis could be therapeutically beneficial for AML CD200hi patients.