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The NOTCH1/CD39 axis: a Treg trip-switch for GvHD

Leukemia volume 30, pages 1931–1934 (2016)Cite this article

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Regulatory T cells (Tregs) suppress alloimmune reaction such as graft versus host disease (GvHD)1 and promote tolerance induction to allogeneic organ transplants.2 In high-risk acute leukaemia patients undergoing full-haplotype mismatched transplantation we demonstrated that adoptive immunotherapy with Tregs conventional T cells (Tcons) almost completely prevented acute and chronic GvHD, favoured post-transplant immunological reconstitution and was associated with a powerful graft-versus-leukaemia (GvL) effect.3, 4, 5 Interestingly, GvHD severity and mortality was markedly reduced by inactivation of NOTCH signalling in donor T cells by means of humanised antibodies and conditional genetic models.6, 7, 8 The present study attempted to unravel the connection between Tregs and NOTCH signalling in Tcons for GvHD prevention. We discovered that NOTCH1 downregulation on Tcons is a new Treg mechanism of action and showed that Tregs use the CD39 pathway to modulate NOTCH1 expression on Tcons.

T cells were collected from healthy donors after obtaining informed consent. Tregs and Tcons were selected as described using CliniMACS procedure and reagents (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).9, 10 Phenotypes and Tregs suppressive capacity were determined as already described10 (Supplementary Methods). Tcons were stained with 0.5 μm carboxyfluorescein diacetate succinimidyl ester (CFSE) and activated with antiCD3 and antiCD28 microbeads (Milteny) at a bead:cell ratio of 1:1 or with irradiated peripheral blood mononuclear cells, a mixed lymphocyte reaction (MLR)-type co-cultures. The cells were cultured alone or with Tregs in a 1.1 ratio (10 experiments). In another set of Treg/Tcon co-cultures anti-CD39 mAB (10 μg/ml, clone A1, Biolegend, San Diego, LA, USA) or IgG control was added (10 experiments). After 5 days, Tcons from all co-cultures were analysed for NOTCH1 expression. CFSE+activated Tcons were sorted by Facs ARIA III (BD Becton, Dickinson and Company, San Jose, CA, USA), and used for suppression assays and further molecular studies. mRNA expression levels of NOTCH1, HES1 and NOTCH1 ligands on Tcon-CFSE+ cells was performed using the 7900Ht Fast Real Time PCR System (Applied Biosystems, Warrington, UK) with Power SYBR Green kits (Applied Biosystems). Primers are listed in Supplementary Table S1. To quantify cAMP, Tcons were collected from different setting of Tcon and Treg/Tcon co-cultures and intracellular cAMP levels were analysed using a commercially available assay (Direct cAMP Elisa Kit, EnzoLife Science, Farmingdale, NY, USA) according to the manufacturer’s instructions. BALB/c recipient mice were irradiated at day −2 with total body irradiation 2 doses of 4 Gy, 4 h apart with 200-kV X-ray source. Overall 5 × 105 donor-type (C57BL/6) GFP+ Treg were administered intravenously on day −2, 5 × 106 T-cell-depleted bone marrow cells (TCD BM) and 1 × 106 Tcons from C57BL/6 mice were injected intravenously on day 0. Mice were killed on day 5 after transplantation. Spleen and lymph nodes were harvested and disassociated into cell suspensions, filtered, stained and FACS analysed (Supplementary Methods). Mice were weighed weekly and GvHD score was calculated.11 In each experiment a group of mice that received TCD BM, Tcon and Treg were treated with the selective CD39 inhibitor polyoxometalate-1 (POM1, Tocris Bioscience, Bristol, UK)12 at the dose of 10 mg/kg/day from day −1 to day +5 after transplantation.

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Acknowledgements

We thank Dr Geraldine Anne Boyd for her help. This work was supported by ‘Associazione Umbra Leucemie e Linfomi’, Perugia and by ‘Associazione Italiana Leucemie, Linfomi e Mieloma’, L’Aquila Section, Italy.

Author contributions

BDP, AP, FF and MDI designed the study and oversaw the results; BDP, MFM and MDI drafted the paper; PS, ER, LR, AC, AV and RN contributed to the design and interpretation of the study; BDP, AP, SB, DC, ED, PA, TZ and RIO performed the in vitro and in vivo studies and interpreted the results.

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Author notes

  1. F Falzetti and M Di Ianni: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Hematology and Clinical Immunology, Department of Medicine, University of Perugia, Perugia, Italy

    B Del Papa, A Pierini, P Sportoletti, D Cecchini, E Dorillo, P Aureli, T Zei, R Iacucci Ostini, L Ruggeri, A Carotti, A Velardi, M F Martelli & F Falzetti

  2. Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University, Stanford, CA, USA

    A Pierini & R Negrin

  3. Department of Life, Hematology Section, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy

    S Baldoni & M Di Ianni

  4. Department of Experimental Medicine, Biosciences and Medical Embryology Section, University of Perugia, Perugia, Italy

    E Rosati

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  1. B Del Papa
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Correspondence to M Di Ianni.

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Del Papa, B., Pierini, A., Sportoletti, P. et al. The NOTCH1/CD39 axis: a Treg trip-switch for GvHD. Leukemia 30, 1931–1934 (2016). https://doi.org/10.1038/leu.2016.87

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