Key role of macrophages in tolerance induction via T regulatory type 1 (Tr1) cells

Abstract

T regulatory type 1 (Tr1) cells are a class of regulatory T cells (T_regs ) participating in peripheral tolerance, hence the rationale behind their testing in clinical trials in different disease settings. One of their applications is tolerance induction to allogeneic islets for long-term diabetes-free survival. Currently the cellular and molecular mechanisms that promote Tr1-cell induction in vivo remain poorly understood. We employed a mouse model of transplant tolerance where treatment with granulocyte colony-stimulating factor (G-CSF)/rapamycin induces permanent engraftment of allogeneic pancreatic islets in C57BL/6 mice via Tr1 cells. The innate composition of graft and spleen cells in tolerant mice was analyzed by flow cytometry. Graft phagocytic cells were co-cultured with CD4⁺ T cells in vitro to test their ability to induce Tr1-cell induction. Graft phagocytic cells were depleted in vivo at different time-points during G-CSF/rapamycin treatment, to identify their role in Tr1-cell induction and consequently in graft survival. In the spleen, the site of Tr1-cell induction, no differences in the frequencies of macrophages or dendritic cells (DC) were observed. In the graft, the site of antigen uptake, a high proportion of macrophages and not DC was detected in tolerant but not in rejecting mice. Graft-infiltrating macrophages of G-CSF/rapamycin-treated mice had an M2 phenotype, characterized by higher CD206 expression and interleukin (IL)-10 production, whereas splenic macrophages only had an increased CD206 expression. Graft-infiltrating cells from G-CSF/rapamycin-treated mice-induced Tr1-cell expansion in vitro. Furthermore, Tr1-cell induction was perturbed upon in-vivo depletion of phagocytic cells, early and not late during treatment, leading to graft loss suggesting that macrophages play a key role in tolerance induction mediated by Tr1 cells. Taken together, in this mouse model of Tr1-cell induced tolerance to allogeneic islets, M2 macrophages infiltrating the graft upon G-CSF/rapamycin treatment are key for Tr1-cell induction. This work provides mechanistic insight into pharmacologically induced Tr1-cell expansion in vivo in this stringent model of allogeneic transplantation.

Keywords: macrophage; regulatory T cells; tolerance; transplantation.

Fig. 1

Early surge in phagocytic cells in the grafts of granulocyte colony‐stimulating factor (G‐CSF)/rapamycin‐treated and transplanted mice. Diabetic C57BL/6 mice were transplanted with Balb/c‐derived pancreatic islets and treated for 30 days with G‐CSF/rapamycin. Grafts and spleens were harvested and cells were collected and detected by flow cytometry. One representative dot‐plot (out of four per group) of flow cytometry staining of graft‐infiltrating F4/80⁺CD11b⁺ macrophages gated on CD11c⁻ cells [untreated mice: upper left panel (a), treated mice: upper right panel (a)] and dendritic cells (DC) gated on live cells [untreated mice: lower left panel (a), treated mice: lower right panel (a)] infiltrating the spleens (b) and the grafts (c) of G‐CSF/rapamycin‐treated (closed circles) and untreated (open circles) diabetic C57BL/6 mice over time. Data are presented as mean ± standard error of the mean (s.e.m.), n = 4–6/group, **P < 0·01, n.s. = non‐significant, Mann–Whitney test.

Fig. 2

Depletion of early and not late phagocytic cells leads to graft loss. Phagocytic cell‐depleting clodronate liposomes were administered to transplanted mice at several time‐points during the course of granulocyte colony‐stimulating factor (G‐CSF)/rapamycin treatment. Graph shows graft survival curves without () or with clodronate liposome treatment at day 0 (), day 14 (), day 21 () and day 30 (); n = 4–6/group.

Fig. 3

Alternatively activated and interleukin (IL)‐10‐producing macrophages predominate in the grafts of granulocyte colony‐stimulating factor (G‐CSF)/rapamycin‐treated mice. Diabetic C57BL/6 mice were transplanted with Balb/c‐derived pancreatic islets and treated for 3 or 7 days with G‐CSF/rapamycin before euthanizing on days 3 or 7. Grafts and spleens were harvested and cells were collected and detected by flow cytometry. One representative dot‐plot (out of three to five per group) of flow cytometry staining of CD206⁺ graft‐infiltrating macrophages F4/80⁺CD11b⁺CD11c⁻ cells (untreated mice: upper left panel, treated mice: upper right panel) and IL‐10⁺ graft‐infiltrating macrophages F4/80⁺CD11b⁺CD11c⁻ cells [untreated mice: lower left panel (b), treated mice: lower right panel (a)]. Data on percentages of CD206⁺ macrophages and IL‐10⁺ macrophages infiltrating the islet allografts (b) and the spleens (c) of G‐CSF/rapamycin‐treated (closed circles) and untreated (open circles) diabetic C57BL/6 mice is presented as mean ± standard error of the mean (s.e.m.), n = 3–5/group, *P < 0·05,**P < 0·01, n.s. = non‐significant, Mann‐Whitney test.

Fig. 4

Macrophages infiltrating the grafts early during granulocyte colony‐stimulating factor (G‐CSF)/rapamycin treatment induce T regulatory type 1 (Tr1)‐cell expansion in vitro. Graft‐infiltrating cells harvested earlier were pooled by group [G‐CSF/rapamycin‐treated (closed circles) and untreated (open circles) mice] and used for co‐culture experiments. A total of 100 000 cells were cultured with CD4⁺ cells, isolated by negative immunomagnetic selection from naive splenocytes of CD45.1 C57BL/6 mice, at a 1 : 1 ratio in 96‐well round‐bottomed plates in complete medium for 5 or 7 days. Upon collection, Tr1 cells were detected by flow cytometry: LAG3⁺CD4⁺CD25⁻CD45.1⁺ and represented over time.

Fig. 5

Early phagocytic cell depletion in vivo leads to a halt in T regulatory type 1 (Tr1)‐cell induction. Clodronate liposomes were administered at 14 or 21 days during the course of granulocyte colony‐stimulating factor (G‐CSF)/rapamycin treatment of transplanted mice. Mice were euthanized 1 week after clodronate treatment, their spleens were harvested and the frequency of Tr1 cells was detected by flow cytometry. One representative dot‐plot (out of six) of flow cytometry staining of Tr1 cells gated on CD4⁺ cells in mice treated [left panel (a)] or untreated [right panel (a)] with clodronate liposomes (CLO) at days 14 or 28 post‐transplant (open forms) or no treatment (closed forms) (b). Data are presented as mean ± standard error of the mean (s.e.m.), n = 3–6/group, *P < 0·05, Mann–Whitney test.