Generation of donor-specific Tr1 cells to be used after kidney transplantation and definition of the timing of their in vivo infusion in the presence of immunosuppression
- Bechara Mfarrej†1View ORCID ID profile,
- Eleonora Tresoldi†1, 3,
- Angela Stabilini1,
- Alessia Paganelli2,
- Rossana Caldara2,
- Antonio Secchi2 and
- Manuela Battaglia1Email author
© The Author(s) 2017
Received: 8 December 2016
Accepted: 3 February 2017
Published: 21 February 2017
Operational tolerance is an alternative to lifelong immunosuppression after transplantation. One strategy to achieve tolerance is by T regulatory cells. Safety and feasibility of a T regulatory type 1 (Tr1)-cell—based therapy to prevent graft versus host disease in patients with hematological malignancies has been already proven. We are now planning to perform a Tr1-cell—based therapy after kidney transplantation.
Upon tailoring the lab-grade protocol to patients on dialysis, aims of the current work were to develop a clinical-grade compatible protocol to generate a donor-specific Tr1-cell—enriched medicinal product (named T10 cells) and to test the Tr1-cell sensitivity to standard immunosuppression in vivo to define the best timing of cell infusion.
We developed a medicinal product that was enriched in Tr1 cells, anergic to donor-cell stimulation, able to suppress proliferation upon donor- but not third-party stimulation in vitro, and stable upon cryopreservation. The protocol was reproducible upon up scaling to leukapheresis from patients on dialysis and was effective in yielding the expected number of T10 cells necessary for the planned infusions. The tolerogenic gene signature of circulating Tr1 cells was minimally compromised in kidney transplant recipients under standard immunosuppression and it eventually started to recover 36 weeks post-transplantation, providing rationale for selecting the timings of the cell infusions.
These data provide solid ground for proceeding with the trial and establish robust rationale for defining the correct timing of cell infusion during concomitant immunosuppressive treatment.
KeywordsTolerance induction Cell therapy Kidney transplantation Clinical-grade compatible protocol
Circulating T regulatory type 1 (Tr1) cells with an alloantigen-specific regulatory function have been consistently associated with operational tolerance after transplantation . Alloantigen-specific Tr1 cells can be induced in vitro in the presence of exogenous IL-10 or by tolerogenic IL-10-producing dendritic cells (DC-10) and they are hyporesponsive (anergic) to the alloantigen used for their generation [2, 3]. These IL-10-anergized T cells have been tested as medicinal product in a proof-of-concept trial in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT) to provide immune reconstitution in the absence of severe graft versus host disease (GvHD) (the ALT-TEN trial) .
The ONE Study—a European Commission FP7-funded consortium-aims to test several distinct haematopoietic immunoregulatory cells as therapies after kidney transplantation from living donors by initiating a series of independent clinical trials based on the same general design . Our group participates in this consortium to test donor-specific Tr1 cells. The ALT-TEN trial already performed was certainly instrumental although the know-how that was developed in this first clinical experience did not necessarily allow performing The ONE Study faster and more efficiently. We overcame the first hurdle of tailoring the Tr1-cell generation protocol to patients on dialysis, yet only at a lab-scale . In this study, we aimed at: (1) defining a reproducible and clinical-grade compatible protocol for the generation of a Tr1-cell—enriched medicinal product for kidney transplant recipients, (2) characterizing the final cell product, and (3) testing the sensitivity of circulating Tr1 cells to immunosuppressive therapy to determine the ideal timing of the medicinal product infusion.
Healthy donors and patients
Peripheral blood, buffy coat or leukapheresis were obtained from healthy donors or renal transplant recipients enrolled in The ONE Study Reference Group Trial (i.e., control group in which patients were treated with standard immunosuppressive therapy) (NCT01656135) after written informed consent in accordance with the Declaration of Helsinki under the protocol approved by the San Raffaele Hospital’s Ethics Committee (IRB #OSR-TheOne).
Generation and characterization of dendritic cells
Peripheral blood mononuclear cells (PBMC) were isolated by density-gradient centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway). IL-10-producing dendritic cells (DC-10) and mature DC (mDC) were generated from healthy donors . Monocytes were isolated by harvesting the adherent fraction of PBMC or by selection with CD14+ microbeads using the AutoMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany) and following manufacturer’s instructions. Monocytes were cultured with 10 ng/ml rhIL-4 (GMP-grade, Miltenyi Biotec) and 100 ng/ml rhGM-CSF (GMP-grade, Miltenyi Biotec) for 7 days in the presence (DC-10) or absence (mDC) of 10 ng/ml rhIL-10 (GMP-grade, CellGenix GmbH, Freiburg, Germany). The culture medium was supplemented with GMP-grade fetal bovine serum (FBS) (Lonza, Basel, Switzerland) or GMP-grade human AB sera (Lonza). mDC were matured during the last 2 days of culture with 5 µg/ml of rMPL-A (GMP-grade, Invivogen, Toulouse, France). At the end of the 7-day cultures, DC-10 and mDC were harvested and irradiated at 60 Gy with a Cs137 source Biobeam 2000 irradiator (Gamma-Service Medical GmbH, Leipzig, Germany). DC-10 yield was measured as: 100× [no. of generated DC-10 cells/no. of plated cells].
Supernatants were collected 48 h after culturing DC-10 in the presence or absence of stimulation by lipopolysaccharide (LPS) from Escherichia coli (5μg/ml, Sigma Chemicals, St. Louis, MO). IL-10 released into the supernatant was quantified by ELISA (BD Pharmingen, San Diego, CA). The detection limit of IL-10 was 15 pg/ml.
Generation and characterization of Tr1- cell enriched product: T10 cells
Ability of T10 cells to suppress the proliferation of autologous CD4+ T cells upon donor or third party mDC stimulation was assessed by 3H-thymidine incorporation in the last 16–18 h of a 5-day culture.
The immune phenotype of in vitro generated DC, T10 and Tm cells was tested by flow cytometry as previously described . The TCR Vβ repertoire was determined with the IOTest® Beta Mark TCR V beta Repertoire Kit (Beckman Coulter, Inc, Brea, CA, USA) following manufacturer’s instructions.
Cells were analyzed with the BD FACS Canto II (Beckton Dickinson, San Jose, CA, USA) within few hours after staining. Data was analyzed using FCS 3.0 (DeNovo Instruments, Los Angeles, CA, USA).
Dual IFNγ/IL-10 ELISPOT
Dual IFNγ/IL-10 ELISPOT (Diaclone, Besancon, France) was performed according to manufacturer’s instructions with a slight modification: visualization of IL-10 was performed using Vector Blue Alkaline Phosphatase substrate kit (Vector Labs, Burlingame, CA, USA) and the A.EL.VIS 4-Plate ELISPOT Reader (A.EL.VIS GmbH, Hannover, Germany) was used. Analysis was performed using ImageJ (version 1.48, NIH, USA) to quantify IFNγ-producing cells (red spots), IL-10-producing cells (blue spots) or dual IFNγ/IL-10-producing cells (purple spots).
Transcript analysis of purified Tr1 cells
Peripheral blood was collected and PBMC were frozen from patients enrolled in The ONE Study Reference Group Trial at our center at the following time points: 4-weeks pre-transplant, 8- 36- and 60-weeks post-transplant. PBMC were thawed and Tr1-cell sorting was performed using MoFlo Legacy Cell Sorter (Beckman Coulter, Indianapolis, IN, USA). To verify the expression of anti-inflammatory genes characteristic of Tr1 cells (as previously described ) and pro-inflammatory genes characteristic of T effector cells (i.e., il-17a, il-1b, tnf, il-6 and ifnγ), QuantiGene 15-plex assay (Affymetrix, Santa Clara, CA, USA) was performed following manufacturer’s instructions. Mean fluorescence intensity from the measured beads per gene was reported using Bio-Plex 200 system (BioRad Laboratories, Hercules, CA, USA). Probe set information is provided in Additional file 1.
Comparisons between groups were performed using Student’s t test, Mann–Whitney test or Wilcoxon matched pairs test depending on the experiments. For all analyses, a two-tailed p value ≤0.05 was considered significant. Comparison of variances was performed using the F-test. Statistical analyses were performed using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA).
DC-10 generation in compliance with clinical-grade manufacturing
T10 cell generation in compliance with clinical-grade manufacturing
A protocol for the generation of cell products to be infused into patients has to be solid and highly reproducible to have the highest chance to be used in clinical trials. Accordingly, buffy coats from eight healthy donors were used for DC-10 generation and buffy coats from eight more donors were used for the isolation of CD4+ T cells. Flasks were used as clinical-grade compatible culture containers to generate T10 cells.
Protocol reproducibility with the optimized clinical-grade compatible conditions using buffy coats from eight donor pairs
CD14+ purity (%)
DC-10 yield (%)
CD4+ purity (%)
T10-cell yield (%)
Donor-specific anergy (%)
CD4+ T cells isolated from buffy coats with lab-grade magnetic columns had a mean purity of 98 ± 1% (mean ± SD). Average T10-cell yield after 10 days of co-culture with allogeneic DC-10 was 53 ± 31% (mean ± SD) and T10-cell donor-specific anergy was 80 ± 10% (mean ± SD) (Table 1). The T10-cell product was constituted of 96 ± 4% (mean ± SD) CD4+ T cells; the remaining non-CD4+ T cells were donor-derived DC-10 cells that were irradiated and therefore dead or prone to die (Additional file 2). Coefficients of variation were high for both DC-10 and T10-cell yield, probably due to intrinsic differences among donors. The cut off anergy value for classifying T10 cells as anergic towards donor antigens was determined utilizing the “mean minus 2× SD” as statistical method . This method was chosen based on our previous experience in the ALT-TEN trial . Based on the eight T10-cell preparations generated from eight different donor pairs, the anergy cut off of T10 cells was 60%. Thus, T10-cell products for clinical-grade-compatible use will be considered anergic when the value is ≥60%.
In vitro characterization of the medicinal product
Gene signature of circulating Tr1 cells is transiently affected by immunosuppressive drugs
These data suggest that Tr1 cells expand along with the CD4+ T-cell memory population and that the Tr1-cell tolerogenic gene expression profile remains stable even under active immunosuppressive treatment. Data are limited to two patients (being the only patients we enrolled in the reference group trial) but they attempted to dissect the Tr1-cell sensitivity to immunosuppression in vivo, a relevant concern that was—to our knowledge—never addressed before. Importantly, these data suggest that the best timing of ex vivo-generated Tr1-cell infusion could be right at the moment of transplant (to reduce inflammation and control alloreactivity) and around 36 weeks post-transplant, when the Tr1-cell signature starts to recover.
Optimization of a clinical-grade compatible protocol for generating donor-specific Tr1-enriched cell medicinal products is a pre-requisite for the planned clinical trial in kidney transplant recipients . In this study we described a clinical-grade compatible protocol that enabled the production of donor-specific Tr1-cell enriched medicinal product (named T10 cells) by coculturing recipient CD4+ T cells with tolerogenic donor DC-10 in the presence of exogenous IL-10 for 10 days. The generated T10 cells are anergic and suppressive towards donor stimulation in vitro, maintain a stable function upon cryopreservation and are successfully produced in clinically sufficient amounts starting from leukapheresis from patients on dialysis. We also demonstrated that circulating Tr1 cells have a limited sensitivity (in terms of viability and gene expression profile) to standard immunosuppressive treatment in vivo.
Several hurdles are encountered when attempting to perform cell therapy clinical trials . First, the protocol for generating the medicinal product needs to be clinical-grade. To that end, we surpassed all the obstacles. Second, a sufficient number of cells to be infused is a prerequisite. Up scaling to leukapheresis could, in some instances, represent a hurdle in terms of sample collection and protocol adaptability . Here we proved that leukapheresis can be collected with no medical contra-indications from patients on dialysis and that both tolerogenic DC-10 and T10 cells can be generated from leukapheresis products.
Another key aspect is the definition of lab tests that ensure safety of the cell product. Within The ONE Study consortium, some groups are using polyclonal Tregs. We decided to invest in the donor-specific Tr1-cell-based therapy in an attempt to promote antigen-specific tolerance. However, the generation of T10 cells with donor-derived DC, although they are tolerogenic and well characterized [3, 7], contains an intrinsic risk of generating alloreactive T cells that, once infused, could be potentially risky for the patient leading to graft rejection. The ONE Study Cell Therapy Trials are feasibility and safety trials . Thus, in vitro assays that prove medicinal-product safety are mandatory. Donor-specific anergy is an optimal assay to test T10-cell safety, while the suppression assay provides indications on the possible efficacy of Tr1 cells in vivo. Accordingly, T10 cells that show no suppressive capacity in vitro but retain donor-specific anergy are considered safe and therefore will be infused in patients participating in The ONE Study cell therapy trial at our institute.
Some groups working with FOXP3+ Tregs as cell therapy products, reported problems with cryopreservation, thus requiring—for instance—further cell manipulation upon thawing [16, 17]. The demonstration that T10 cells are stable and conserve their Tr1-cell content and donor-specific anergy properties upon cryopreservation, allows for flexibility in their preparation and feasibility for more than one infusion.
Whether infused T10 cells retain their viability and function in vivo under treatment with immunosuppressive drugs remains an important open question. One approach to address this issue was by testing the effect of immunosuppressive drugs in vitro on the ex vivo-expanded human FOXP3+ Tregs or by using humanized mouse models . This showed detrimental dose-dependent effects of immunosuppressive treatments on viability and proliferative capacity while sparing the immunosuppressive function of FOXP3+ Tregs. We approached this issue by analyzing the frequency and the gene signature of circulating Tr1 cells collected from renal transplant recipients under active immunosuppressive treatment. Our data suggest that immunosuppressive drugs do not affect Tr1 cells since the cells remain in circulation and a transient change in the intensity of the gene signature is observed. Based on these data, we chose two different timings of T10-cell infusion to increase the chance of obtaining in vivo immune regulation. The first dose will be infused at the time of transplant (for Tr1-cell enrichment just around the transplant period). The second dose will be infused at 36 weeks post-transplant, timepoint in which the Tr1-cell signature is recovering.
Taken together, our results demonstrate the reproducibility of an optimized clinical-grade-compatible protocol for generating Tr1-enriched T10 cells. The necessary following steps for performing the trial in patients are underway.
We describe the steps undertaken to achieve and validate a reproducible optimized clinical-grade compatible protocol capable of generating donor-specific Tr1 cells in sufficient numbers. Additionally, selecting the timing of infusion of Tr1 cells to patients under immunosuppression remains an open question. We provide data assessing the viability and gene signature of circulating Tr1 cells in the presence of active immunosuppression thus supporting our rationale for selecting the timing of the planned infusions. We believe that this study highlights the importance of optimizing and validating Tr1 cell manufacturing protocols to bring them closer to the bedside.
antigen presenting cells
advanced-therapy medicinal products
coefficient of variation
IL-10-producing dendritic cells
fetal bovine serum
good manufacturing practice
graft versus host disease
hematopoietic stem cell transplantation
mature dendritic cells
medicinal product constituted of donor PBMC anergized with irradiated host DC-10
peripheral blood mononuclear cells
effector T mature cells
T regulatory type 1
- T10 :
medicinal product enriched in Tr1 cells
BM participated in research design, performance of the research, contributed analytic tools, participated in data analysis and writing of the paper. ET participated in research design, performance of the research, contributed analytic tools, participated in data analysis. ASt participated in research design, contributed new reagents and performance of the research. AP participated in performance of the research. RC and ASe participated in performance of the research and writing of the paper. MB corresponding author, participated in research design, data analysis and writing of the paper. All authors read and approved the final manuscript.
We thank Maria Grazia Roncarolo, Rosa Bacchetta (Stanford University, CA) and Silvia Gregori (San Raffaele Telethon Institute for Gene Therapy, Milano) for helpful scientific discussion. We also thank members of the lab of Lorenzo Piemonti (Diabetes Research Institute, San Raffaele Hospital) and FRACTAL facility (San Raffaele Hospital) for technical assistance. We also thank all the donors who actively participated to this study.
The authors declare that they have no competing interests.
Availability of data and materials
Ethics approval and consent to participate
Written informed consent in accordance with the Declaration of Helsinki under the protocol approved by the San Raffaele Hospital’s Ethics Committee (IRB #OSR-TheOne) was obtained.
This project was funded through a grant to MB from the 7th framework programme of the EU (FP7-HEALTH-2010 The ONE Study; Grant Agreement 260687). BM was supported by the 7th framework programme of the EU (Marie Curie Initial Training Network-FP7-PEOPLE-2011-ITN), under the Marie Skłodowska-Curie Grant Agreement No. 289903: EUTRAIN.
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- Roncarolo MG, Gregori S, Bacchetta R, Battaglia M. Tr1 cells and the counter-regulation of immunity: natural mechanisms and therapeutic applications. Curr Top Microbiol Immunol. 2014;380:39–68.PubMedGoogle Scholar
- Groux H, Bigler M, de Vries JE, Roncarolo MG. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J Exp Med. 1996;184(1):19–29.View ArticlePubMedGoogle Scholar
- Gregori S, Tomasoni D, Pacciani V, Scirpoli M, Battaglia M, Magnani CF, et al. Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway. Blood. 2010;116(6):935–44.View ArticlePubMedGoogle Scholar
- Bacchetta R, Lucarelli B, Sartirana C, Gregori S, Lupo Stanghellini MT, Miqueu P, et al. Immunological outcome in haploidentical-HSC transplanted patients treated with IL-10-anergized donor T cells. Front Immunol. 2014;5:16.View ArticlePubMedPubMed CentralGoogle Scholar
- Geissler EK, The ONE. Study compares cell therapy products in organ transplantation: introduction to a review series on suppressive monocyte-derived cells. Transplant Res. 2012;1(1):11.View ArticlePubMedPubMed CentralGoogle Scholar
- Petrelli A, Tresoldi E, Mfarrej BG, Paganelli A, Spotti D, Caldara R, et al. Generation of donor-specific T regulatory type 1 cells from patients on dialysis for cell therapy after kidney transplantation. Transplantation. 2015;99(8):1582–9.View ArticlePubMedGoogle Scholar
- Bacchetta R, Gregori S, Serafini G, Sartirana C, Schulz U, Zino E, et al. Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy. Haematologica. 2010;95(12):2134–43.View ArticlePubMedPubMed CentralGoogle Scholar
- Petrelli A, Tresoldi E, Mfarrej BG, Paganelli A, Spotti D, Caldara R, et al. Generation of donor-specific T regulatory type 1 cells from patients on dialysis for cell therapy after kidney transplantation. Transplantation. 2015;99:1582–9.View ArticlePubMedGoogle Scholar
- Meyer-Wentrup F, Burdach S. Efficacy of dendritic cell generation for clinical use: recovery and purity of monocytes and mature dendritic cells after immunomagnetic sorting or adherence selection of CD14+ starting populations. J Hematother Stem Cell Res. 2003;12(3):289–99.View ArticlePubMedGoogle Scholar
- Babatz J, Röllig C, Oelschlägel U, Zhao S, Ehninger G, Schmitz M, et al. Large-scale immunomagnetic selection of CD14+ monocytes to generate dendritic cells for cancer immunotherapy: a phase I study. J Hematother Stem Cell Res. 2003;12(5):515–23.View ArticlePubMedGoogle Scholar
- Singh G. Determination of cutoff score for a diagnostic test. Internet J Lab Med. 2006. http://ispub.com/IJLM/2/1/9884. Accessed 9 Jan 2016.
- Gagliani N, Jofra T, Stabilini A, Valle A, Atkinson M, Roncarolo MG, et al. Antigen-specific dependence of Tr1-cell therapy in preclinical models of islet transplant. Diabetes. 2010;59(2):433–9.View ArticlePubMedGoogle Scholar
- Thurner B, Röder C, Dieckmann D, Heuer M, Kruse M, Glaser A, et al. Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. J Immunol Methods. 1999;223(1):1–15.View ArticlePubMedGoogle Scholar
- Gagliani N, Magnani CF, Huber S, Gianolini ME, Pala M, Licona-Limon P, et al. Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med. 2013;19(6):739–46.View ArticlePubMedGoogle Scholar
- Brusko TM, Hulme MA, Myhr CB, Haller MJ, Atkinson MA. Assessing the in vitro suppressive capacity of regulatory T cells. Immunol Invest. 2007;36(5–6):607–28.View ArticlePubMedGoogle Scholar
- Peters JH, Preijers FW, Woestenenk R, Hilbrands LB, Koenen HJ, Joosten I. Clinical grade Treg: GMP isolation, improvement of purity by CD127 depletion, Treg expansion, and Treg cryopreservation. PLoS ONE. 2008;3(9):e3161.View ArticlePubMedPubMed CentralGoogle Scholar
- Golab K, Leveson-Gower D, Wang XJ, Grzanka J, Marek-Trzonkowska N, Krzystyniak A, et al. Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications. Int Immunopharmacol. 2013;16(3):371–5.View ArticlePubMedGoogle Scholar
- Trzonkowski P, Bacchetta R, Battaglia M, Berglund D, Bohnenkamp HR, tenBrinke A, et al. Hurdles in therapy with regulatory T cells. Sci Transl Med. 2015;7(304):304–18.View ArticleGoogle Scholar
- Tchao NK, Turka LA. Lymphodepletion and homeostatic proliferation: implications for transplantation. Am J Transpl. 2012;12(5):1079–90.View ArticleGoogle Scholar
- Strasser EF, Eckstein R. Optimization of leukocyte collection and monocyte isolation for dendritic cell culture. Transfus Med Rev. 2010;24(2):130–9.View ArticlePubMedGoogle Scholar
- Scottà C, Fanelli G, Hoong SJ, Romano M, Lamperti EN, Sukthankar M, et al. Impact of immunosuppressive drugs on the therapeutic efficacy of ex vivo expanded human regulatory T cells. Haematologica. 2016;101(1):91–100.View ArticlePubMedPubMed CentralGoogle Scholar