Regulatory T cells for minimising immune suppression in kidney transplantation: phase I/IIa clinical trial

Abstract

Objective: To assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model.

Design: Investigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13).

Setting: Charité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union).

Participants: Recipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9).

Interventions: CD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×10⁶ cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48.

Main outcome measures: The primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio.

Results: For all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire.

Conclusions: The application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies.

Trial registration: NCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).

Fig 1

ONE study ONEnTreg13 clinical trial design. Upper panel: time schedule of ONEnTreg13 clinical trial: patient enrolment, cell collection, living donor kidney transplantation, nTreg adoptive cellular therapy (dose escalation of 0.5, 1.0, or 2.5-3.0×10⁶ fresh cells/kg of body weight), and primary 60 week study follow-up. Lower panel: overview of protocol immunosuppressive regimen of ONEnTreg13 clinical trial with doses adjusted to specified levels at indicated time points: first steroid reduction until week 14, followed by MMF reduction at week 36-48, with continuation of tacrolimus monotherapy in nTreg group until study end at week 60 and three year follow-up. GMP=good manufacturing practice; IV=intravenous; KTx=kidney transplantation; MMF=mycophenolate mofetil; nTreg=natural regulatory T cell

Fig 2

Autologous nTreg infusion might enable minimisation of immunosuppression in patients with kidney transplant. Tapering of immunosuppression in the nTreg trial group (switch to monotherapy) compared with reference group (continuous dual or triple drug regimen) with monitoring of patients’ tacrolimus levels (median±interquartile range, shown on lower panel). KTx=kidney transplantation; MMF=mycophenolate mofetil; nTreg=natural regulatory T cell

Fig 3

Long term follow-up of renal allograft function (median±interquartile range): glomerular filtration rate (GFR, mL/min), serum creatinine (mg/dL), proteinuria (mg/day), and C reactive protein (CRP, mg/dL)

Fig 4

No immune complications after infusion: monitoring of cytokine release after infusion by measuring inflammatory cytokine serum or urinary levels (box plot, minimum-maximum range) with respective typical normal values (serum: TNFα<15.0 pg/mL, IFNγ=10-30 pg/mL, IL-6<5.0 pg/mL, IL-1<5.0 pg/mL, IL-8<10.0 pg/mL, IL-10<5.0 pg/mL, IP-10<200 pg/mL) and monocyte HLA-DR (molecules per cell) either 24 hours before or 24 hours after systemic nTreg infusion, including two week follow-up for HLA-DR. No significant differences between groups for any assessed parameters. HLA=human leukocyte antigen; IFNγ=interferon γ; IL=interleukin; IP-10= urinary interferon induced protein 10; nTreg=natural regulatory T cell; TNFα=tumour necrosis factor α

Fig 5

Exploratory biomarker analysis of therapy response to nTreg treatment. More than 100 biomarkers were analysed in patients receiving nTregs, with a more comprehensive summary presented in figures S5-S6. Patients receiving nTregs were evaluated according to cell dose applied and compared with reference group. Transiently enhanced Treg levels and favourable Treg:Teff ratio. Upper panels: multiparameter flow cytometry profiling of nTregs in patient blood either comparing nTreg with reference group or showing the infused cell dose (0.5, 1.0, and 2.5-3.0×10⁶ cells/kg of body weight); table shows significance levels. Lower panels: ratio between CD4+ CD25^high CD127^low Treg levels relative to either CCR7– CD4+ or CCR7– CD8+Teffs shown as representative result for almost 60 immune cell subsets shown in figure S6; table shows significance levels. nTreg=natural regulatory T cell; Teff= effector T cell; Treg=regulatory T cell

Fig 6

Decreasing clonal diversity by oligoclonal nTreg expansion. Next generation sequencing based TCR repertoire analysis of individual nTreg GMP products before infusion (GMP columns) and ex vivo Tregs isolated at indicated patient visits (week 4, 12, 36, and 60, according to sample availability) for all 11 patients receiving nTregs, with a more detailed summary shown in figure S5. GMP=good manufacturing practice; nTreg=natural regulatory T cell; TCR=T cell receptor; Treg=regulatory T cell