Purpose: The graft-versus-tumor (GVT) effect is a potent form of immunotherapy against many hematologic malignancies and some solid tumors. The beneficial GVT effect after allogeneic bone marrow transplantation (BMT) is tightly linked to its most significant complication, graft-versus-host disease (GVHD). The role of interleukin-6 (IL-6) after allogeneic BMT is not well understood. This study used a series of complementary knockout and antibody blockade strategies to analyze the impact of IL-6 in multiple clinically relevant murine models of GVHD and GVT.Experimental Design: We examined the effect of the source of IL-6 by analyzing the role IL-6 deficiency in donor T cells, donor bone marrow or in host tissues. We confirmed and extended the relevance of IL-6 deficiency on GVHD and GVT by treating BMT recipients with anti-mouse IL-6 receptor (IL-6R), MR16-1.Results: Deficiency of IL-6 in donor T cells led to prolongation of survival. Total inhibition of IL-6 with MR16-1 caused an even greater reduction in GVHD-induced mortality. The reduction in GVHD was independent of the direct effects on T effector cell expansion or donor regulatory T cells. GVT responses were preserved after treatment with MR16-1.Conclusion: MR16-1 treatment reduced GVHD and preserved sufficient GVT. Tocilizumab, a humanized anti-IL-6R monoclonal antibody (mAb), is approved in several countries including the United States and European Union for the treatment of rheumatoid arthritis and other inflammatory diseases. Blockade of IL-6 with anti-IL-6R mAb therapy may be testable in clinical trials as an adjunct to prevent GVHD in BMT patients without a significant loss of GVT.

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Acute graft-versus-host disease (GvHD) is a major complication that prevents successful outcomes after allogeneic bone marrow transplantation (BMT), an effective therapy for hematological malignancies. Several studies demonstrate that donor T cells and host antigen-presenting cells along with several proinflammatory cytokines are required for the induction of GvHD and contribute to its severity. Increasing evidence demonstrates that human serumderived αalpha-1-anti-trypsin (AAT) reduces production of proinflammatory cytokines, induces anti-inflammatory cytokines, and interferes with maturation of dendritic cells. Using well-characterized mouse models of BMT, we have studied the effects of AAT on GvHD severity. Administration of AAT early after BMT decreased mortality in three models of GvHD and reduced serum levels of proinflammatory cytokines in the allogeneic recipients compared with vehicle (albumin) treated animals. AAT treatment reduced the expansion of alloreactive T effector cells but enhanced the recovery of T regulatory T cells, (Tregs) thus altering the ratio of donor T effector to T regulatory cells in favor of reducing the pathological process. However, despite altering the ratio in vivo, AAT had no direct effects on either the donor T effector cells or T regulatory cells Tregs in vitro. In contrast, AAT suppressed LPSinduced in vitro secretion of proinflammatory cytokines such as TNF-α and IL-1β, enhanced the production of the anti-inflammatory cytokine IL-10, and impaired NF-κB translocation in the host dendritic cells. In light of its long history of safety in humans, these findings suggest that administration of AAT represents a novel unique and viable strategy to mitigate clinical GvHD.inflammation | interleukin

IntroductionAg presentation on radiosensitive host hematopoiesis-derived APCs to the alloreactive donor T cells is considered to be obligatory for the induction of acute GVHD. 1-9 However, under certain conditions, whether clinically relevant minor Ags can induce GVHD in the absence of functional radiosensitive host hematopoietic APCs is not known. [6][7][8]10 Clinical data from MHC-matched BMT show that male recipients from female donors (F3M) are at a greater risk of developing GVHD 9 and show H-Y-specific alloresponses. [11][12][13][14] These clinical data suggest a strong correlation between H-Y Ag disparity and GVHD. However, in the context of HLA-matched clinical F3M BMT, the donors are also likely to be mismatched with the recipients at multiple minor Ags. Therefore, whether H-Y disparity alone is sufficient for causing clinical acute GVHD is not known. The experimental evidence for the causative role of H-Y Ags in GVHD and mortality has not been reported. Furthermore, the relevance of donor T-cell alloreactivity against a single minor Ag and mechanisms of its presentation in causing GVHD are not known. [1][2][3][4][5] Although some studies have suggested that high doses of TCR transgenic (Tg) T cells can cause GVHD, its severity was limited and was in the context of MHC mismatch or against minor Ags with unknown clinical relevance. 15,16 With the use of both H-Y-specific Tg and non-Tg T cells in multiple well-established BM chimeras we demonstrate, in contrast to the existing notion, that presentation of clinically relevant minor H-Y Ag by host radiosensitive hematopoieticderived APCs is not obligatory for induction of acute GVHD. 3,[6][7][8]10,17 Our data further suggest that in the absence of radiosensitive host hematopoietic-derived APCs, when sufficient numbers of alloreactive donor T cells are infused, nonhematopoietic-derived cells such as endothelial and certain epithelial cells activate alloreactive T cells, might induce GVHD. Methods MiceMale and female C57BL/6 (B6, H-2 b , CD45.2 ϩ ), B6 Ly5.2 (H-2 b , CD45.1 ϩ ), and BALB/c (H-2 d ) mice were purchased from The Jackson 17 and MataHari (RAG-1 Ϫ background, CD8 ϩ Tg, H-2 b , CD45.2 ϩ , H-2D b -restricted) mice 18 were obtained from Taconic. All animals were cared for under regulations reviewed and approved by the University Committee on Use and Care of Animals of the University of Michigan, based on University Laboratory Animal Medicine guidelines. Generation of BM chimerasWe administered 1100 cGy total body irradiation ( 137 Cs source) to mice and then injected them intravenously with 5 ϫ 10 6 BM cells with 5 ϫ 10 6 whole spleen cells from donor mice on day Ϫ1. For generating MHC class I-deficient (␤2m-KO) BM chimeras, recipient mice were treated with 200 g of anti-NK1.1 mAb (PK136) on days Ϫ2 and Ϫ1. 6 The peripheral blood from sentinel mice were analyzed for donor chimerism at 3 months and found to show Ͼ 98% donor chimerism in all cell lineages. The CD11c ϩ cells in the splenocytes from these animals also showed Ͼ 95% donor chimerism. BMTBMT...

Histone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep IntroductionPathogenic T-cell responses can be detrimental to the host. For example, GVHD is a life-threatening complication after allogeneic BM transplantation (BMT). 1-3 GVHD is caused by donor T cells that attack normal tissues of the recipient. 1-3 Standard immunosuppressive therapy for GVHD lacks efficacy and impairs the antitumor activity. 1,2,4 New approaches are needed to control GVHD.Epigenetic modifications are thought to be important for T-cell immune responses. 5,6 These modifications include histone methylation, DNA methylation, and histone acetylation. 7-10 Histone methylation is the modification of certain amino acids of a histone by adding methyl groups. [8][9][10] Depending on the site and degree of methylation, histone methylation can be activating or repressive. For example, trimethylation of histone H3 at lysine 4 (H3K4me3), H3K36me3, and H3K79me3 are associated with transcriptional activation, whereas H3K9me3, H3K27me3, and H4K20me3 are related to gene repression. 8,9 Recent studies have reported that histone methylation may play important roles in regulating the expression of genes associated with survival, proliferation, and differentiation of Ag-activated T cells. 11,12 Unlike histone methylation, DNA methylation results in global silencing of gene expression, 10 whereas histone acetylation is associated with a relaxing chromatin structure that facilitates transcription. 10 It has been shown that the DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-AzaC) and the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) may prevent GVHD in mice through a mechanism of modulating regulatory T cells (Tregs) 13,14 and APCs, 15-17 respectively. However, global modifications of DNA and chromatin structures are found to be associated with toxicities and adverse effects. 10 Thus, novel epigenetic approaches capable of targeting a specific set of genes in alloreactive T cells are desirable for controlling GVHD while minimizing adverse effects.3-Deazaneplanocin A (DZNep) possesses the potent ability to selectively inhibit some histone methylation, such as H3K27me3, H3K4me3, and H4K20me3. 18,19 DZNep is an inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. AdoHcy hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine. 20,21 When this enzyme is inhibited, AdoHcy accumulates in cells, leading to inhibition of the histone methyltransferease (HMT) activity and the subsequent histone methylation inhibition. 20 DZNep acts through a different pathway than DNA methylation inhibitors and HDAC inhibitors. 10,18,19 An Inside Blood analysis of this article appears at the fron...

MicroRNAs have emerged as critical modulators of immune responses, but little is known about their transcriptional regulation and tissue specificity. MicroRNA-142 is specifically expressed in hematopoietic tissues and it plays an important role in regulating immunity. Herein we identified the key transcriptional elements for regulation of miR-142 and its impact on TLR-4 mediated expression of IL-6. The PU-1, C/EBPβ and Runx1 transcription factor (TF) binding sites are conserved and constitutively occupied by the respective transcription factors in the miR-142 gene promoter only in the hematopoietic cells. Specific knock-down experiments in hematopoietic cells and rescue experiments in non-hematopoietic cells show that PU-1 is critical for miR-142 gene expression and that it synergizes with Runx1, C/EBPβ and CBFβ. Furthermore TLR4 stimulation enhanced miR-155 while experiments with knock-down and mimic expression of miR-155 demonstrated that miR-155 negatively regulates miR-142-3p expression by targeting PU-1. Thus TLR4 stimulation represses PU-1 resulting in resulting in down-regulation of miR-142 and increased expression of IL-6. These results collectively reveal the direct cis acting sequences of miR-142 specific promoter and that transcription factor PU-1 is necessary for its exclusive expression in hematopoietic cells and regulation of IL-6.

Key Points Targeting APCs to enhance GVT. CD8+ DCs are important for optimizing antitumor responses after allogeneic bone marrow transplantation.

Allogeneic bone marrow transplantation is an effective treatment for a number of malignant and nonmalignant diseases (Applebaum. 2001. Nature. 411: 385-389 and Copelan. 2006. N Engl J Med. 354: 1813-1826. However, the application of this therapeutic modality has been impeded by a number of confounding side effects, the most frequent and severe of which is the development of graft-versushost disease (GVHD) (Copelan. 2006. N Engl J Med. 354: 1813-1826and Blazar and Murphy. 2005. Philos Trans R Soc Lond B Biol Sci. 360: 1747-1767). Alloreactive donor T cells are critical for causing GVHD (Fowler. 2006. Crit Rev Oncol Hematol. 57: 225-244 and Reddy. 2006. Semin Hematol. 43: 3-10), whereas recent data demonstrated a significant role for the naturally occurring thymic-derived donor CD4+ regulatory T cells (Tregs) Abbas. 2003. Nat Rev Immunol. 3: 253-257 and Shevach. 2006. Immunity. 25: 195-201) in suppressing experimental GVHD after bone marrow transplantation Taylor. 2005. Biol Blood Marrow Transpl. 11: 46-49 and Joffe and van Meerwijk. 2006. Semin Immunol. 18: 128-135) . Host APCs are required for induction of GVHD by the conventional donor T cells. However, it is not known whether they are also obligatory for donor Tregmediated suppression of GVHD. Using multiple clinically relevant MHC-matched and -mismatched murine models of GVHD, we investigated the role of host APCs in the suppression of GVHD by donor Tregs. We found that alloantigen expression by the host APCs is necessary and sufficient for induction of GVHD protection by donor Tregs. This requirement was independent of their effect on the maintenance of Treg numbers and the production of IL-10 or IDO by the host APCs. The Journal of Immunology, 2010, 185: 3866-3872. T he application of allogeneic bone marrow transplantation (BMT), which is an effective therapy against may hematological diseases, has been impeded by the development of its major toxicity, graft-versus-host disease (GVHD) (1). GVHD results from immunologically mediated injury to host tissues (2, 3), and alloreactive donor T effector cells (Teffs) are essential for the induction and pathologic changes characteristic of GVHD (1, 4). Regulation of an immune response is now known to be mediated by multiple subtypes of regulatory/suppressor cells (5-7). The bestdescribed bona fide populations of regulatory cells are CD4 FoxP3+ T cells (Tregs) and Teffs (2, 10, 16-18). Although IL-10 can contribute, very little is known about the mechanisms of Treg-mediated GVHD suppression (17). Specifically, the nature and presentation of its target Ags are not well understood.Significant progress has been made in our understanding of the role of APCs and their interaction with alloreactive donor T cells in the induction of GVHD. Key roles for host APCs in the induction and the donor-derived APCs in the exacerbation of acute GVHD have been established (19). However, it is not known whether the host and/or donor APCs play a crucial role in regulating donor Treg-mediated GVHD suppression.Because natural T...