Activation of human CD141+ and CD1c+ dendritic cells in vivo with combined TLR 3 and TLR 7/8 ligation

Mice reconstituted with human hematopoietic stem cells are valuable models to study aspects of the human immune system in vivo. We describe a humanized mouse model (hu mice) in which fully functional human CD141+ and CD1c+ myeloid and CD123+ plasmacytoid dendritic cells (DC) develop from human cord blood CD34+ cells in immunodeficient mice. CD141+ DC are the human equivalents of murine CD8+ /CD103+ DC which are essential for the induction of tumor-inhibitory cytotoxic T lymphocyte (CTL) responses, making them attractive targets to exploit for the development of new cancer immunotherapies. We used CD34+ -engrafted NSG-A2 mice to investigate activation of DC subsets by synthetic dsRNA or ssRNA analogues polyinosinic-polycytidylic acid/poly I:C and Resiquimod/R848, agonists for TLR3 and TLR8 respectively, both of which are expressed by CD141+ DC. Injection of hu mice with these agonists resulted in upregulation of costimulatory molecules CD80, CD83 and CD86 by CD141+ and CD1c+ DC alike, and their combination further enhanced expression of these molecules by both subsets. When combined, poly I:C and R848 enhanced serum levels of key cytokines associated with cross-presentation and the induction of CTL responses including IFN-α, IFN-β, IL-12 and CXCL10. These data advocate a combination of poly I:C and R848 TLR agonists as means of activating human DC for immunotherapy. This article is protected by copyright. All rights reserved

Donor T-cell–derived GM-CSF drives alloantigen presentation by dendritic cells in the gastrointestinal tract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has recently emerged as an important pathogenic cytokine in acute graft-versus-host disease (GVHD), but the nature of the T-cell lineages secreting the cytokine and the mechanisms of action are less clear. Here we used interleukin 17A-fate reporter systems with transcriptional analysis and assays of alloantigen presentation to interrogate the origins of GM-CSF-secreting T cells and the effects of the cytokine on antigen-presenting cell (APC) function after experimental allogeneic stem cell transplantation (SCT). We demonstrated that although GM-CSF-secreting Th17 and non-Th17 cells expanded in the colon over time after SCT, the Th17 lineage expanded to represent 10% to 20% of the GM-CSF secreting T cells at this site by 4 weeks. Donor T-cell-derived GM-CSF expanded alloantigen-presenting donor dendritic cells (DCs) in the colon and lymph nodes. In the mesenteric lymph nodes, GM-CSF-dependent DCs primed donor T cells and amplified acute GVHD in the colon. We thus describe a feed-forward cascade whereby GM-CSF-secreting donor T cells accumulate and drive alloantigen presentation in the colon to amplify GVHD severity. GM-CSF inhibition may be a tractable clinical intervention to limit donor alloantigen presentation and GVHD in the lower gastrointestinal tract

IL-6 dysregulation originates in dendritic cells and initiates graft-versus-host disease via classical signaling

Graft-versus-host-disease (GVHD) after allogeneic stem cell transplantation (alloSCT) is characterized by interleukin 6 (IL-6) dysregulation. IL-6 can mediate effects via various pathways, including classical, trans and cluster signaling. Given the recent availability of agents that differentially inhibit these discrete signaling cascades, understanding the source, signaling and cellular targets of this cytokine is paramount to inform the design of clinical studies. Here we demonstrate that IL-6 secretion from recipient dendritic cells (DC) initiates the systemic dysregulation of this cytokine. Inhibition of DC-driven classical signaling following targeted IL-6 receptor (IL-6R) deletion in T-cells eliminated pathogenic donor Th17/Th22 cell differentiation and resulted in long-term survival. Following engraftment, donor DC assume the same role, maintaining classical IL-6 signaling-dependent GVHD responses. Surprisingly, cluster-signaling was not active after transplant, while inhibition of trans-signaling with sgp130Fc promoted severe, chronic cutaneous GVHD. The latter was a result of exaggerated polyfunctional Th22 cell expansion that was reversed by IL-22 deletion or IL-6R inhibition. Importantly, inhibition of IL-6 classical-signaling did not impair the graft-versus-leukemia effect. Together, these data highlight IL-6 classical-signaling and downstream Th17/Th22 differentiation as key therapeutic targets after alloSCT

Inhibition of the cytolytic protein perforin prevents rejection of transplanted bone marrow stem cells in vivo

Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically-mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and non-malignant disorders, but when the body recognises the graft as foreign it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of 16; the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function

Th17 plasticity and transition toward a pathogenic cytokine signature are regulated by cyclosporine after allogeneic SCT

T-helper 17 (Th17) cells have been widely implicated as drivers of autoimmune disease. In particular, Th17 cytokine plasticity and acquisition of an interleukin-17A(+)(IL-17A(+)) interferon gamma(IFN gamma)(+) cytokine profile is associated with increased pathogenic capacity. Donor Th17 polarization is known to exacerbate graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT); however, donor Th17 cytokine coexpression and plasticity have not been fully characterized. Using IL-17 "fate-mapping" mice, we identified IL-6-dependent Th17 cells early after allo-SCT, characterized by elevated expression of proinflammatory cytokines, IL-17A, IL-22, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor. This population did not maintain lineage fidelity, with a marked loss of IL-17A and IL-22 expression late posttransplant. Th17 cells were further segregated based on IFN gamma coexpression, and IL-17A(+)IFN gamma(+) Th17 displayed an enhanced proinflammatory phenotype. Th17 cytokine plasticity and IFN gamma production were critically dependent upon donor-derived IL-12p40, and cyclosporine (CsA) treatment regulated this differentiation pathway. This observation was highly concordant with clinical samples from allo-SCT recipients receiving CsA-based immune suppression where although the IFNgnegative- Th17 subset predominated, IFN gamma(+)-Th17 cells were also present. In sum, Th17 polarization and ensuing differentiation are mediated by sequential inflammatory signals, which are modulated by immunosuppressive therapy, leading to distinct phenotypes within this lineage