David Game

Generation and expansion of human CD4+CD25+ regulatory T cells with indirect allospecificity : Potential reagents to promote donor-specific transplantation tolerance

Background. Harnessing naturally arising CD4+CD25+ regulatory T cells (Tregs) for potential adoptive cell therapy is hampered by their innate autoreactivity and their limited number. Methods. CD4+CD25+ Tregs were purified from peripheral blood of human leukocyte antigen (HLA) DR1*0101+A2− individuals, and stimulated with autologous monocyte-derived dendritic cells (DCs). Results. Here we show that CD4+CD25+ Tregs specific for an HLA A2 (103–120) peptide can be selected from the peripheral blood CD4+CD25+ T cell population of a healthy individual and detected using a tetramer comprised of HLA DRB1*0101 and the A2 peptide. The selected cells can be expanded substantially (i.e., a 1600-fold increase over a two-week period) by T-cell receptor (TCR) stimulation and high-doses of interleukin-2 (IL-2). The CD4+CD25+ Tregs with indirect allospecificity for the A2 peptide showed more potent antigen-specific suppression than polyclonal CD4+CD25+ Tregs. Conclusions. These data may pave the way for clinical studies using CD4+CD25+ Tregs with indirect allospecificity as therapeutic reagents for the induction of donor-specific transplantation tolerance.

CD4+CD25+ Regulatory T Cells Do Not Significantly Contribute to Direct Pathway Hyporesponsiveness in Stable Renal Transplant Patients

CD4(+)CD25(+) regulatory T cells have been shown to regulate a variety of autoimmune and allogeneic responses in mice and humans. The role of CD4(+)CD25(+) cells in regulating alloresponses in human transplant recipients remains uncertain. Previous research has demonstrated a reduced frequency of direct pathway donor-specific T cells in renal transplant recipients when compared with the frequency of T cells reactive to an HLA-matched third party. A number of mechanisms have been proposed to account for this finding; the purpose of this study was to determine whether CD4(+)CD25(+) cells play a significant role. Twelve stable renal transplant patients were investigated using limiting dilution assay (LDA) and ELISPOT for interferon-gamma to determine the effect of depleting CD4(+)CD25(+) cells on the direct pathway alloresponse. The percentage of CD4(+)CD25(+) cells in the peripheral blood of the study patients was equivalent to that of healthy controls. Furthermore, in no case did depletion of CD4(+)CD25(+) cells significantly increase the frequency of donor-specific T cells detected by LDA. This was also found with ELISPOT in all except one patient, in whom depletion revealed an increased frequency of alloreactive T cell to both donor and third party. Finally, kinetic analysis of the LDA data did not indicate regulation against donor when compared with third party. It is concluded that the action of CD4(+)CD25(+) regulatory cells is not the main mechanism of donor-specific hyporesponsiveness in the direct pathway of allorecognition.

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.

Improving distress in dialysis (iDiD): a feasibility two-arm parallel randomised controlled trial of an online cognitive behavioural therapy intervention with and without therapist-led telephone support for psychological distress in patients undergoing haemodialysis.

Introduction Psychological distress is common in end-stage kidney disease (ESKD) and is associated with poorer health outcomes. Cognitive behavioural therapy (CBT) is recommended in UK clinical guidelines for the management of depression in people with long-term conditions. Access to skilled therapists competent in managing the competing mental and physical health demands of ESKD is limited. Online CBT treatments tailored to the needs of the ESKD population offers a pragmatic solution for under-resourced services. This study examines the feasibility and acceptability of implementing a two-arm parallel randomised controlled trial of online CBT with (intervention arm) and without (control arm) therapist support to improve psychological distress in patients undergoing haemodialysis. Methods Patients will be screened for depression and anxiety while attending for their haemodialysis treatments. We aim to recruit 60 adult patients undergoing haemodialysis who meet criteria for mild to moderately severe symptoms of depression and/or anxiety. Patients will be randomised individually (using a 1:1 computerised sequence ratio) to either online CBT with therapist telephone support (intervention arm), or online CBT with no therapist (control arm). Outcomes include feasibility and acceptability descriptive data on rates of recruitment, randomisation, retention and treatment adherence. Self-report outcomes include measures of depression (Patient Health Questionnaire-9), anxiety (Generalised Anxiety Disorder-7), quality of life (Euro-QoL), service use (client service receipt inventory) and illness cognitions (brief illness perception questionnaire). A qualitative process evaluation will also be conducted. The statistician will be blinded to treatment allocation. Ethics and dissemination A National Health Service (NHS) research ethics committee approved the study. Data from this study will provide essential information for the design and testing of further interventions to ameliorate distress in patients undergoing dialysis. Any amendments to the protocol will be submitted to the NHS committee and study sponsor. Trial registration number NCT023528702; Pre-results.

A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

The concept of regulatory T cell (Treg)-based immunotherapy has enormous potential for facilitating tolerance in autoimmunity and transplantation. Clinical translation of Treg cell therapy requires production processes that satisfy the rigors of Good Manufacturing Practice (GMP) standards. In this regard, we report our findings on the implementation of a robust GMP compliant process for the ex vivo expansion of clinical grade Tregs, demonstrating the feasibility of this developed process for the manufacture of a final product for clinical application. This Treg isolation procedure ensured the selection of a pure Treg population that underwent a 300-fold expansion after 36 days of culture, while maintaining a purity of more than 75% CD4+CD25+FOXP3+ cells and a suppressive function of above 80%. Furthermore, we report the successful cryopreservation of the final product, demonstrating the maintenance of phenotype and function. The process outlined in this manuscript has been implemented in the ONE study, a multicenter phase I/IIa clinical trial in which cellular therapy is investigated in renal transplantation.

Screening for psychological distress using the Patient Health Questionnaire Anxiety and Depression Scale (PHQ-ADS): Initial validation of structural validity in dialysis patients

OBJECTIVE To validate the factor structure of the Patient Health Questionnaire Anxiety and Depression Scale (PHQ-ADS)- which is a composite measure of depression and anxiety using the Patient Health Questionnaire-9 and Generalised Anxiety Disorder Scale (GAD-7), in a sample of haemodialysis patients. METHOD Screening data (n=182) used to select entry into a feasibility study of an online cognitive-behavioural therapy intervention for distress in dialysis patients were analysed here. Structural validity of the PHQ-ADS was evaluated using confirmatory factor analysis (CFA), assessing alternative models including a bi-factor model. In the bi-factor model all items from the PHQ-9 and GAD-7 (16-items in total) were loaded onto a general distress factor. Respective items of the PHQ-9 and GAD-7 were specified as subgroup factors. Omega-hierarchical was calculated to indicate the level of saturation of a multidimensional scale by a general factor. Construct validity was determined against the Brief Illness Perception Questionnaire. RESULTS A bi-factor PHQ-ADS model had good fit to the data (chi-square=96.1, p=0.26, CFI=0.99; TLI=0.99; RMSEA=0.02). The general distress factor accounted for approximately 84% of the explained variance (omega-h=0.90). Distress scores were significantly higher in females compare with males. There was a significant association between distress and negative illness perceptions (r=0.58, p<0.01). CONCLUSIONS The PHQ-ADS appears to have good structural validity in haemodialysis patients and is sufficiently unidimensional to warrant the use of a total distress score. A full psychometric analysis of the PHQ-ADS in a larger sample of dialysis patients is warranted.

Tailored online cognitive behavioural therapy with or without therapist support calls to target psychological distress in adults receiving haemodialysis: a feasibility randomised controlled trial

BACKGROUND Psychological distress is prevalent in haemodialysis (HD) patients yet access to psychotherapy remains limited. This study assessed the feasibility and acceptability of online cognitive-behavioural therapy (CBT) tailored for HD patients, with or without therapist support, for managing psychological distress. METHODS This feasibility randomised controlled trial recruited patients from a UK HD centre. Following psychological distress screens, patients with mild-moderate psychological distress (Patient Health Questionnaire PHQ-9; score: 5-19 and/or Generalised Anxiety Disorder; GAD-7 score: 5-14) who met remaining inclusion criteria were approached for consent. Consenters were individually randomised (1:1) to online-CBT or online-CBT plus three therapist support calls. Outcomes included recruitment, retention, and adherence rates. Exploratory change analyses were performed for: psychological distress, quality of life (QoL), illness perceptions, and costs. The statistician was blinded to allocation. RESULTS 182 (44%) out of 410 patients approached completed psychological distress screens. 26% found screening unacceptable; a further 30% found it unfeasible. Psychological distress was detected in 101 (55%) patients, 60 of these met remaining inclusion criteria. The primary reason for ineligibility was poor computer literacy (N=17, 53%). Twenty-five patients were randomised to the supported (N=18) or unsupported arm (N=7); 92% were retained at follow-up. No differences in psychological distress or cost-effectiveness were observed. No trial adverse events occurred. CONCLUSION Online CBT appears feasible but only for computer literate patients who identify with the label psychological distress. A definitive trial using the current methods for psychological distress screening and online care delivery is unfeasible. ClinicalTrials.gov Identifier: NCT02352870.