Bianca E. Teal

Identification and expansion of highly suppressive CD8 +FoxP3 + regulatory T cells after experimental allogeneic bone marrow transplantation

FoxP3(+) confers suppressive properties and is confined to regulatory T cells (T(reg)) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4(+) T(reg) are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8(+) population of FoxP3(+) T(reg) that convert from CD8(+) conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8(+) T(reg) undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4(+)FoxP3(+) population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8(+)FoxP3(+) T(reg) are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8(+)FoxP3(+) T(reg) thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation.

CSF-1-dependant donor-derived macrophages mediate chronic graft-versus-host disease.

Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17-dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS-). Cutaneous cGVHD developed in a CSF-1/CSF-1R-dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r-/- mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti-CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD.

Induced regulatory T cells promote tolerance when stabilized by rapamycin and IL-2 in vivo

Natural regulatory T cells (nTregs) play an important role in tolerance; however, the small numbers of cells obtainable potentially limit the feasibility of clinical adoptive transfer. Therefore, we studied the feasibility and efficacy of using murine-induced regulatory T cells (iTregs) for the induction of tolerance after bone marrow transplantation. iTregs could be induced in large numbers from conventional donor CD4 and CD8 T cells within 1 wk and were highly suppressive. During graft-versus-host disease (GVHD), CD4 and CD8 iTregs suppressed the proliferation of effector T cells and the production of proinflammatory cytokines. However, unlike nTregs, both iTreg populations lost Foxp3 expression within 3 wk in vivo, reverted to effector T cells, and exacerbated GVHD. The loss of Foxp3 in iTregs followed homeostatic and/or alloantigen-driven proliferation and was unrelated to GVHD. However, the concurrent administration of rapamycin, with or without IL-2/anti–IL-2 Ab complexes, to the transplant recipients significantly improved Foxp3 stability in CD4 iTregs (and, to a lesser extent, CD8 iTregs), such that they remained detectable 12 wk after transfer. Strikingly, CD4, but not CD8, iTregs could then suppress Teff proliferation and proinflammatory cytokine production and prevent GVHD in an equivalent fashion to nTregs. However, at high numbers and when used as GVHD prophylaxis, Tregs potently suppress graft-versus-leukemia effects and so may be most appropriate as a therapeutic modality to treat GVHD. These data demonstrate that CD4 iTregs can be produced rapidly in large, clinically relevant numbers and, when transferred in the presence of systemic rapamycin and IL-2, induce tolerance in transplant recipients.

Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions

Graphical abstract

Lung parenchyma-derived IL-6 promotes IL-17A-dependent acute lung injury after allogeneic stem cell transplantation.

Idiopathic pneumonia syndrome (IPS) is a relatively common, frequently fatal clinical entity, characterized by noninfectious acute lung inflammation following allogeneic stem cell transplantation (SCT), the mechanisms of which are unclear. In this study, we demonstrate that immune suppression with cyclosporin after SCT limits T-helper cell (Th) 1 differentiation and interferon-γ secretion by donor T cells, which is critical for inhibiting interleukin (IL)-6 generation from lung parenchyma during an alloimmune response. Thereafter, local IL-6 secretion induces donor alloantigen-specific Th17 cells to preferentially expand within the lung, and blockade of IL-17A or transplantation of grafts lacking the IL-17 receptor prevents disease. Studies using IL-6(-/-) recipients or IL-6 blockade demonstrate that IL-6 is the critical driver of donor Th17 differentiation within the lung. Importantly, IL-6 is also dysregulated in patients undergoing clinical SCT and is present at very high levels in the plasma of patients with IPS compared with SCT recipients without complications. Furthermore, at the time of diagnosis, plasma IL-6 levels were higher in a subset of IPS patients who were nonresponsive to steroids and anti-tumor necrosis factor therapy. In sum, pulmonary-derived IL-6 promotes IPS via the induction of Th17 differentiation, and strategies that target these cytokines represent logical therapeutic approaches for IPS.

Donor colonic CD103+ dendritic cells determine the severity of acute graft-versus-host disease

Koyama et al. show that GVHD markedly enhances alloantigen presentation within the mesenteric lymph nodes, mediated by donor CD103+CD11b− DCs that migrate from the colon under the influence of CCR7. This antigen presentation imprints gut-homing integrin signatures on donor T cells, leading to their migration to the GI tract where they mediate fulminant disease.

Promoting regulation via the inhibition of DNAM-1 after transplantation

Donor T cells play pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects following bone marrow transplantation (BMT). DNAX accessory molecule 1 (DNAM-1) is a costimulatory and adhesion molecule, expressed mainly by natural killer cells and CD8(+) T cells at steady state to promote adhesion to ligand-expressing targets and enhance cytolysis. We have analyzed the role of this pathway in GVHD and GVL. The absence of DNAM-1 on the donor graft attenuated GVHD in major histocompatibility complex (MHC)-mismatched and MHC-matched BMT following conditioning with lethal and sublethal irradiation. In contrast, DNAM-1 was not critical for GVL effects against ligand (CD155) expressing and nonexpressing leukemia. The effects on GVHD following myeloablative conditioning were independent of CD8(+) T cells and dependent on CD4(+) T cells, and specifically donor FoxP3(+) regulatory T cells (Treg). The absence of DNAM-1 promoted the expansion and suppressive function of Treg after BMT. These findings provide support for therapeutic DNAM-1 inhibition to promote tolerance in relevant inflammatory-based diseases characterized by T-cell activation.

Cross-Dressing by Donor Dendritic Cells after Allogeneic Bone Marrow Transplantation Contributes to Formation of the Immunological Synapse and Maximizes Responses to Indirectly Presented Antigen

The stimulation of naive donor T cells by recipient alloantigen is central to the pathogenesis of graft-versus-host disease after bone marrow transplantation (BMT). Using mouse models of transplantation, we have observed that donor cells become “cross-dressed” in very high levels of recipient hematopoietic cell–derived MHC class I and II molecules following BMT. Recipient-type MHC is transiently present on donor dendritic cells (DCs) after BMT in the setting of myeloablative conditioning but is persistent after nonmyeloablative conditioning, in which recipient hematopoietic cells remain in high numbers. Despite the high level of recipient-derived alloantigen present on the surface of donor DCs, donor T cell proliferative responses are generated only in response to processed recipient alloantigen presented via the indirect pathway and not in response to cross-dressed MHC. Assays in which exogenous peptide is added to cross-dressed MHC in the presence of naive TCR transgenic T cells specific to the MHC class II–peptide combination confirm that cross-dressed APC cannot induce T cell proliferation in isolation. Despite failure to induce T cell proliferation, cross-dressing by donor DCs contributes to generation of the immunological synapse between DCs and CD4 T cells, and this is required for maximal responses induced by classical indirectly presented alloantigen. We conclude that the process of cross-dressing by donor DCs serves as an efficient alternative pathway for the acquisition of recipient alloantigen and that once acquired, this cross-dressed MHC can assist in immune synapse formation prior to the induction of full T cell proliferative responses by concurrent indirect Ag presentation.

Autophagy is required for stem cell mobilization by G-CSF

Granulocyte colony-stimulating factor (G-CSF) is widely used clinically to prevent neutropenia after cytotoxic chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Autophagy, a process of cytoplasmic component recycling, maintains cellular homeostasis and protects the cell during periods of metabolic stress or nutrient deprivation. We have observed that G-CSF activates autophagy in neutrophils and HSCs from both mouse and human donors. Furthermore, G-CSF-induced neutrophil and HSC mobilization is impaired in the absence of autophagy. In contrast, autophagy is dispensable for direct HSC mobilization in response to the CXCR4 antagonist AMD3100. Altogether, these data demonstrate an important role for G-CSF in invoking autophagy within hematopoietic and myeloid cells and suggest that this pathway is critical for ensuring cell survival in response to clinically relevant cytokine-induced stress. These findings have direct relevance to HSC transplantation and the increasing clinical use of agents that modulate autophagy.

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

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.