Combination Of Regulatory T Cells and Rapamycin As Treatment For Experimental Chronic Graft-Versus-Host Disease

Background Chronic graft-versus-host disease (cGvHD) is one of the main complications of allogeneic hematopoietic cell transplantation (allo-HCT). Donor CD4+ conventional T cells (Tconv) as well as regulatory T cells (Treg) are the key-players in its pathogenesis. Rapamycin (sirolimus, rapa) is a mTor inhibitor that can suppress activation and proliferation of Tconv without inhibiting Treg. Aims To assess the impact of a combined treatment with Tregand rapa on experimental cGvHD. Methods Lethally irradiated BALB/c mice were injected with 10x106 bone marrow cells and 70x106 splenocytes from B10.D2 donor mice. Mice were divided in four groups on day -1 and treatments were started on day 20 with either PBS, rapa 1 mg/kg/Day, Treg 1.106 cells (D+20), or rapa 1 mg/kg/Day + Treg 1.106 cells (D+20). Treg (CD4+ CD25+ cells) were purified from spleen cells from donor B10.D2 mice using the CD4+ CD25+ regulatory T cell isolation kit (Miltenyi Biotec, GmbH, Germany). Treg purity (defined as CD4+ FoxP3+ cells) was ≥ 70% of total cells and > 92% of CD4+T cells. The severity of sclerodermatous cGvHD was assessed with the following clinical scoring system. Briefly, animals were individually scored every 3 days for five parameters: weight loss (1, 10-20%; 2, > 20%), posture (1, kyphosis only at rest; 2, severe kyphosis when the animal moved), activity (1, moderate activity impairment; 2, no move unless stimulated), skin (1, erythema or scaling tail; 2, open lesion on the body surface) and hair loss (1, > 1 cm2; 2, > 2 cm2). Mice which reached a score of 8 were estimated to have terminal GvHD and were sacrificed. Terminal GvHD-free survivals between the 4 groups were compared using the Log-rank test. Results The effects of the treatments were evaluated 7 days after starting the treatments (D+27 post-transplantation). Numbers of total and central memory and effector memory CD4+ T cells/µL were significantly (p<0.05) decreased in rapa-, Treg-, and Treg + rapa- treated mice compared to PBS mice, while numbers of naïve CD4+ T cells/µL were significantly (p<0.05) decreased in Treg-, and Treg + rapa- treated mice. Further, proliferation of CD4+ T cells (assessed by Ki67 expression) was significantly decreased in rapa- (p<0.05) and Treg + rapa- treated mice (p<0.05). In addition, counts and proliferation of CD8+ T cells/µL were significantly (p<0.05) lower in rapa- and Treg+ rapa- treated mice compared to PBS mice. Importantly, terminal-GvHD-free survival was significantly shorter in PBS mice than in Treg (P=0.03), rapa (P=0.04), and Treg+ rapa mice (P=0.02). Conclusion Our results showed that Treg and rapa administration improved cGvHD in this model of cGvHD. Disclosures: No relevant conflicts of interest to declare.

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GM-CSF Therapy Expands Regulatory T Cells and Protects Against Chronic Graft Versus Host Disease

Blood ◽

10.1182/blood.v128.22.2151.2151 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2151-2151

Author(s):

Hideaki Yoshimura ◽

Masaaki Hotta ◽

Atsushi Satake ◽

Shosaku Nomura

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Graft Versus Host Disease ◽

Cd4 T Cells ◽

Intracellular Cytokine Staining ◽

Dependent Manner ◽

Host Disease ◽

Graft Versus Host ◽

Gm Csf ◽

Allogeneic Host

Abstract Regulatory T cells (Tregs) possess the ability to suppress chronic graft-versus-host disease (cGVHD). Hence, the in vivo expansion of Tregs can be used as therapy against cGvHD. In addition to IL-2, Tregs require TCR and costimulatory signals from antigen presenting cells such as dendritic cells (DCs) for their optimal proliferation. Both fms-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony stimulation factor (GM-CSF) induce the development of DCs and promote the proliferation of Tregs in a DC-dependent manner. GM-CSF preferentially increases CD11c+CD8a- DCs, whereas FLT3L more equally supports the development of many DC subsets. However, it is unknown whether GM-CSF-mediated CD11c+CD8a- DC expansion leads to the proliferation of Tregs and contributes to the inhibition of alloimmune responses against host antigens. To test whether the injection of GM-CSF augments Tregs and ameliorates cGVHD, we used a MHC-matched mouse cGVHD model (B10.D2 → Balb/c). Balb/c mice were lethally irradiated (850 cGy) and transplanted with 8 × 106 T cell-depleted bone marrow cells and 3.5 × 106 CD4+ T cells from either syngeneic or B10.D2 mice. Host mice were treated with vehicle or GM-CSF (in the form of immune complexes; GM-CSF ICs) for 3 days (days 17-19) and monitored for skin GVHD score and mortality. We used GM-CSF ICs because the injection of GM-CSF ICs but not GM-CSF itself increased splenic CD11c+CD8a- DCs and Tregs. The administration of GM-CSF to allogeneic host mice significantly protected against GVHD-induced skin diseases (p<0.001) (Fig 1). Similar results were obtained when GM-CSF ICs were administrated at a later stage (days 27-29). Although CD11c+CD8- DCs were decreased in mice transplanted from the allogeneic donor compared with mice transplanted from the syngeneic donor, the administration of GM-CSF increased the CD11c+CD8a-/CD11c+CD8a+ DC ratio. However, the expansion of macrophages was not observed in mice administered GM-CSF. Expectedly, the administration of GM-CSF increased Tregs in the peripheral blood and the peripheral lymph nodes (PLNs) (P<0.05) (Fig 2). We investigated the production of proinflammatory cytokines (IFN-g, IL-17) of CD4+ T cells in the spleen, PLNs, and the skin by intracellular cytokine staining, as these cytokines are important for cGVHD pathogenesis in this model. The proportion of IFN-g+CD4+ T cells in the spleen and PLNs was slightly but not significantly decreased in GM-CSF-administered mice. The proportion of IL-17+CD4+ T cells in the skin was decreased in GM-CSF-administered mice compared to vehicle-administered mice. We next investigated the production of IL-2 and IL-10, as these cytokines are associated with survival and function of Tregs. Skin infiltrating Tregs were not increased in GM-CSF-administered mice compared to vehicle-administered mice; however, the proportion of IL-10+ Tregs was increased in GM-CSF-administered mice. The proportion of IL-2+CD4+ T cells was comparable in the allogeneic host with or without GM-CSF, indicating that GM-CSF-induced Treg expansion did not result from an increase in IL-2 production by CD4+　T cells. Together, these data suggest that GM-CSF induces the proliferation of Tregs by expanding CD11c+CD8a- DCs, and can regulate alloimmune responses in a cGVHD mouse model. Our findings indicate the potential of GM-CSF as a therapeutic strategy to ameliorate cGVHD. Disclosures No relevant conflicts of interest to declare.

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