Despite major improvements in allogeneic hematopoietic cell transplantation over the last decades, corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) cause high mortality. Pre-clinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib. In this retrospective survey, 19 stem cell transplant centers in Europe and the United States reported outcome data from 95 patients who had received ruxolitinib as salvage-therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grade III or IV) or SR-cGVHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1–7) and SR-cGVHD (1–10). The ORR was 81.5% (44/54) in SR-aGVHD including 25 CRs (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3%–90.7%,95% CI) and 97.4% (92.3%–100%,95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and CMV-reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Ruxolitinib may constitute a promising new treatment option for SR-aGVHD and SR-cGVHD that should be validated in a prospective trial.

Key Points• T follicular helper cells and germinal center B cells are increased and strongly correlate with the development of cGVHD in a murine model. • Blocking mAbs for IL-21, ICOS, and CD40L are potential novel therapeutics for cGVHD.Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD. (Blood. 2014;123(25):3988-3998)

Chronic GVHD (cGVHD) poses a significant risk for HSCT patients. Preclinical development of new therapeutic modalities has been hindered by models with pathologic findings that may not simulate the development of human cGVHD. Previously, we have demonstrated that cGVHD induced by allogeneic HSCT after a conditioning regimen of cyclophosphamide and total-body radiation results in pulmonary dysfunction and airway obliteration, which leads to bronchiolitis obliterans (BO), which is pathognomonic for cGVHD of the lung. We now report cGVHD manifestations in a wide spectrum of target organs, including those with mucosal surfaces. Fibrosis was demonstrated in the lung and liver and was associated with CD4 ؉ T cells and B220 ؉ B-cell infiltration and alloantibody deposition. Donor bone marrow obtained from mice incapable of secreting IgG alloantibody resulted in less BO and cGVHD. Robust germinal center reactions were present at the time of cGVHD disease initiation. Blockade of germinal center formation with a lymphotoxin-receptor-immunoglobulin fusion protein suppressed cGVHD and BO. We conclude that cGVHD is caused in part by alloantibody secretion, which is associated with fibrosis and cGVHD manifestations including BO, and that treatment with a lymphotoxin-␤ receptorimmunoglobulin fusion protein could be beneficial for cGVHD prevention and therapy. (Blood. 2012;119(6):1570-1580) IntroductionChronic GVHD (cGVHD) is a significant complication of allogeneic HSCT. 1 Progress in developing interventional strategies to counter cGVHD has been hampered by variable onset and pathologic manifestations of cGVHD, now better defined by the National Institutes of Health consensus conference, 2 and a dearth of robust preclinical venues that closely mimic conditions in which cGVHD is generated and manifested. 3 Although the exact causes of cGVHD are unknown, higher antibody levels have been associated with autoimmunity and implicated in cGVHD. 4,5 Studies of newly diagnosed patients with extensive cGVHD showed that they had elevated soluble B-cell activating factor (BAFF) levels and anti-ds-DNA antibodies. 6,7 Increased soluble BAFF in cGVHD was associated with higher circulating levels of pre-germinal center (GC) B cells and post-GC plasmablasts. 8 B cells from cGVHD patients are hyperresponsive to TLR-9 signaling and have up-regulated CD86 levels, 9 which suggests an important participatory role for B cells in establishing cGVHD and emphasizes the need for further investigation into the immunologic role of B cells in cGVHD pathogenesis.Existing murine cGVHD models simulate one or more of the pathologic manifestations, such as increased serum antibodies (typically anti-DNA antibodies), scleroderma, and fibrosis of skin and liver, and the less common immune complex deposition in kidneys and glomerulonephritis. [10][11][12] The type of multiorgan involvement and alloantibodies seen in cGVHD patients often has not been well represented in these preclinical models. Moreover, some models do not involve conditioning regimens, whereas ot...

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• The ROCK2 inhibitor, KD025, decreases chronic GVHD pathology in multiple murine models.• KD025 inhibits STAT3 phosphorylation to decrease RORgt and Bcl6 expression in both murine and human cells.Chronic graft-versus-host disease (cGVHD) remains a major complication following allogeneic bone marrow transplantation (BMT). The discovery of novel therapeutics is dependent on assessment in preclinical murine models of cGVHD. Rho-associated kinase 2 (ROCK2) recently was shown to be implicated in regulation of interleukin-21 (IL-21) and IL-17 secretion in mice and humans. Here, we report that the selective ROCK2 inhibitor KD025 effectively ameliorates cGVHD in multiple models: a full major histocompatibility complex (MHC) mismatch model of multiorgan system cGVHD with bronchiolitis obliterans syndrome and a minor MHC mismatch model of sclerodermatous GVHD. Treatment with KD025 resulted in normalization of pathogenic pulmonary function, which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. Spleens of mice treated with KD025 had decreased frequency of T follicular helper cells and increased frequency of T follicular regulatory cells, accompanied by a reduction in signal transducer and activator of transcription 3 (STAT3) and concurrent increase in STAT5 phosphorylation. The critical role of STAT3 in this cGVHD model was confirmed by data showing that mice transplanted with inducible STAT3-deficient T cells had pulmonary function comparable to the healthy negative controls. The therapeutic potential of targeted ROCK2 inhibition in the clinic was solidified further by human data demonstrating the KD025 inhibits the secretion of IL-21, IL-17, and interferon g along with decreasing phosphorylated STAT3 and reduced protein expression of interferon regulatory factor 4 and B-cell lymphoma 6 (BCL6) in human peripheral blood mononuclear cells purified from active cGVHD patients. Together these data highlight the potential of targeted ROCK2 inhibition for clinical cGVHD therapy. (Blood. 2016; 127(17):2144-2154