Autophagy-dependent regulatory T cells are critical for the control of graft-versus-host disease
Laëtitia Le Texier, Katie E. Lineburg, Benjamin Cao, Cameron McDonald-Hyman, Lucie Leveque-El Mouttie, Jemma Nicholls, Michelle Melino, Blessy C. Nalkurthi, Kylie A. Alexander, Bianca Teal, Stephen J. Blake, Fernando Souza-Fonseca-Guimaraes, Christian R. Engwerda, Rachel D. Kuns, Steven W. Lane, Michele Teng, Charis Teh, Daniel Gray, Andrew D. Clouston, Susan K. Nilsson, Bruce R. Blazar, Geoffrey R. Hill, Kelli P.A. MacDonald
Laëtitia Le Texier, Katie E. Lineburg, Benjamin Cao, Cameron McDonald-Hyman, Lucie Leveque-El Mouttie, Jemma Nicholls, Michelle Melino, Blessy C. Nalkurthi, Kylie A. Alexander, Bianca Teal, Stephen J. Blake, Fernando Souza-Fonseca-Guimaraes, Christian R. Engwerda, Rachel D. Kuns, Steven W. Lane, Michele Teng, Charis Teh, Daniel Gray, Andrew D. Clouston, Susan K. Nilsson, Bruce R. Blazar, Geoffrey R. Hill, Kelli P.A. MacDonald
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Research Article Immunology Transplantation

Autophagy-dependent regulatory T cells are critical for the control of graft-versus-host disease

Abstract

Regulatory T cells (Tregs) play a crucial role in the maintenance of peripheral tolerance. Quantitative and/or qualitative defects in Tregs result in diseases such as autoimmunity, allergy, malignancy, and graft-versus-host disease (GVHD), a serious complication of allogeneic stem cell transplantation (SCT). We recently reported increased expression of autophagy-related genes (Atg) in association with enhanced survival of Tregs after SCT. Autophagy is a self-degradative process for cytosolic components that promotes cell homeostasis and survival. Here, we demonstrate that the disruption of autophagy within FoxP3+ Tregs (B6.Atg7fl/fl-FoxP3cre+) resulted in a profound loss of Tregs, particularly within the bone marrow (BM). This resulted in dysregulated effector T cell activation and expansion, and the development of enterocolitis and scleroderma in aged mice. We show that the BM compartment is highly enriched in TIGIT+ Tregs and that this subset is differentially depleted in the absence of autophagy. Moreover, following allogeneic SCT, recipients of grafts from B6.Atg7fl/fl-FoxP3cre+ donors exhibited reduced Treg reconstitution, exacerbated GVHD, and reduced survival compared with recipients of B6.WT-FoxP3cre+ grafts. Collectively, these data indicate that autophagy-dependent Tregs are critical for the maintenance of tolerance after SCT and that the promotion of autophagy represents an attractive immune-restorative therapeutic strategy after allogeneic SCT.

Authors

Laëtitia Le Texier, Katie E. Lineburg, Benjamin Cao, Cameron McDonald-Hyman, Lucie Leveque-El Mouttie, Jemma Nicholls, Michelle Melino, Blessy C. Nalkurthi, Kylie A. Alexander, Bianca Teal, Stephen J. Blake, Fernando Souza-Fonseca-Guimaraes, Christian R. Engwerda, Rachel D. Kuns, Steven W. Lane, Michele Teng, Charis Teh, Daniel Gray, Andrew D. Clouston, Susan K. Nilsson, Bruce R. Blazar, Geoffrey R. Hill, Kelli P.A. MacDonald

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Figure 5

G-CSF induces Treg mobilization.

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G-CSF induces Treg mobilization. (A) Representative zebra plot of flow c...
(A) Representative zebra plot of flow cytometry analysis and absolute number (#) of TIGIT+ and TIGITneg CD4+FoxP3-GFP+ Tregs from spleen and BM of BALB/c FoxP3-GFP mice after saline or G-CSF injections (10 μg/mice; 1 injection/day for 6 days) (n = 6 from 2 independent experiments). (B) Representative zebra plot and histogram of flow cytometry analysis of CXCR4 expression on FoxP3+CD4+CD8–CD3+ Tregs in spleen and BM of naive C57BL/6 naive mice (n = 3 from 1 experiment). The isotype control represents staining of pooled BM and spleen cells. (C) Experiments performed at CSIRO. Absolute number (#) of HSPCs (LSK; lineagenegSca-1+c-kit+), HSCs (LSKSLAM; LSKCD150+CD48neg), B220negGr-1negMac-1negCD4+FoxP3+ Tregs, TIGIT+ B220negGr-1negMac-1negCD4+FoxP3+ Tregs, and TIGITneg B220negGr-1negMac-1negCD4+FoxP3+ Tregs from peripheral blood of B6.FoxP3-GFP mice after saline, G-CSF (250 μg/kg, 2 injections/day for 4 days), or AMD3100 injection (n = 4–12 from 3 independent experiments). Data are shown as mean ± SEM. Statistical significance was determined using an unpaired 2-tailed Mann-Whitney U test (*P < 0.05; **P < 0.01; ****P < 0.0001). (D) Geometric mean of LC3 expression in splenic TIGIT+ and TIGITneg CD4+FoxP3+ Tregs from C57BL/6 naive mice incubated in vitro for 2 hours without (Nil) or with (G-CSF) plus CQ (n = 4 from 2 independent experiments). Data are shown as mean ± SEM. Statistical significance was determined using a paired t test (*P < 0.05; **P < 0.01). Statistical analyses were performed using GraphPad Prism version 6.01 software. G-CSF, granulocyte-colony stimulating factor; TIGIT, T cell immunoreceptor with Ig and ITIM domains; HSPC, hematopoietic stem and progenitor cells; HSC, hematopoietic stem cells; LC3, microtubule-associated protein light chain 3; CQ, chloroquine.