Jonathan S. Serody

Bone marrow myeloid-derived suppressor cells (MDSCs) inhibit graft-versus-host disease (GVHD) via an arginase-1–dependent mechanism that is up-regulated by interleukin-13

Myeloid-derived suppressor cells (MDSCs) are a well-defined population of cells that accumulate in the tissue of tumor-bearing animals and are known to inhibit immune responses. Within 4 days, bone marrow cells cultured in granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor resulted in the generation of CD11b(+)Ly6G(lo)Ly6C(+) MDSCs, the majority of which are interleukin-4Rα (IL-4Rα(+)) and F4/80(+). Such MDSCs potently inhibited in vitro allogeneic T-cell responses. Suppression was dependent on L-arginine depletion by arginase-1 activity. Exogenous IL-13 produced an MDSC subset (MDSC-IL-13) that was more potently suppressive and resulted in arginase-1 up-regulation. Suppression was reversed with an arginase inhibitor or on the addition of excess L-arginine to the culture. Although both MDSCs and MDSC-IL-13 inhibited graft-versus-host disease (GVHD) lethality, MDSC-IL-13 were more effective. MDSC-IL-13 migrated to sites of allopriming. GVHD inhibition was associated with limited donor T-cell proliferation, activation, and proinflammatory cytokine production. GVHD inhibition was reduced when arginase-1-deficient MDSC-IL-13 were used. MDSC-IL-13 did not reduce the graft-versus-leukemia effect of donor T cells. In vivo administration of a pegylated form of human arginase-1 (PEG-arg1) resulted in L-arginine depletion and significant GVHD reduction. MDSC-IL-13 and pegylated form of human arginase-1 represent novel strategies to prevent GVHD that can be clinically translated.

In vitro-differentiated TH17 cells mediate lethal acute graft-versus-host disease with severe cutaneous and pulmonary pathologic manifestations

The morbidity and mortality associated with graft-host-disease (GVHD) is a significant obstacle to the greater use of allogeneic stem cell transplantation. Donor T cells that predominantly differentiate into TH1/Tc1 T cells and generate pro-inflammatory cytokines such as interferon-gamma (IFN-gamma) mediate GVHD. Although numerous studies have described a pathogenic role for IFN-gamma, multiple reports have demonstrated that the lack of IFN-gamma paradoxically exacerbated GVHD lethality. This has led to speculation that another subset of T cells may significantly contribute to GVHD mortality. Several groups have demonstrated a new lineage of CD4+ T helper cell development distinct from TH1 or TH2 differentiation. This lineage is characterized by production of interleukin (IL)-17A, IL-17F, IL-22, and IL-21 and has been termed TH17 cells. Here, we demonstrate that a highly purified population of TH17 cells is capable of inducing lethal GVHD, hallmarked by extensive pathologic cutaneous and pulmonary lesions. Upon transfer, these cells migrate to and expand in GVHD target organs and secondary lymphoid tissues. Finally, we demonstrate differential roles for tumor necrosis factor-alpha (TNF-alpha) and IL-17A in the clinical manifestations of GVHD induced by TH17 cells. Our studies demonstrate that cells other than TH1/Tc1 can mediate acute GVHD.

Differential Roles for CCR5 Expression on Donor T Cells during Graft-versus-Host Disease Based on Pretransplant Conditioning

The coordinated expression of chemokines and receptors may be important in the directed migration of alloreactive T cells during graft-vs-host disease (GVHD). Recent work demonstrated in a murine model that transfer of CCR5-deficient (CCR5−/−) donor cells to nonconditioned haploidentical recipients resulted in reduced donor cell infiltration in liver and lymphoid tissues compared with transfer of CCR5+/+ cells. To investigate the function of CCR5 during GVHD in conditioned transplant recipients, we transferred CCR5−/− or wild-type C57BL/6 (B6) T cells to lethally irradiated B6D2 recipients. Unexpectedly, we found an earlier time to onset and a worsening of GVHD using CCR5−/− T cells, which was associated with significant increases in the accumulation of alloreactive CD4+ and CD8+ T cells in liver and lung. Conversely, the transfer of CCR5−/− donor cells to nonirradiated recipients led to reduced infiltration of target organs, confirming previous studies and demonstrating that the role of CCR5 on donor T cells is dependent on conditioning of recipients. Expression of proinflammatory chemokines in target tissues was dependent on conditioning of recipients, such that CXCL10 and CXCL11 were most highly expressed in tissues of irradiated recipients during the first week post-transplant. CCR5−/− T cells were shown to have enhanced migration to CXCL10, and blocking this ligand in vivo improved survival in irradiated recipients receiving CCR5−/− T cells. Our data indicate that the effects of inhibiting CCR5/ligand interaction on donor T cells during GVHD differ depending on conditioning of recipients, a finding with potentially important clinical significance.

Effector CD4+ T cells, the cytokines they generate, and GVHD: something old and something new

GVHD is a syndrome that results from minor and major histocompatibility complex incompatibilities between the donor and recipient. More than 50 years after its initial description, the pathophysiology of GVHD remains poorly understood. Nonetheless, donor T cells have been shown to be critical to the pathophysiology of acute and chronic GVHD, yet precisely how they function remains unclear. The effector mechanisms by which donor T cells mediate tissue inflammation is even less well understood. Identification of several new lineages of CD4(+) T cells made in the past decade and their roles in the pathophysiology of T cell-mediated diseases has shed new light on these effector mechanisms. In this review, we summarize the recent descriptions of these T-cell lineages and the current data supporting their role in acute and to a lesser extent chronic GVHD. Investigations into the activity of these new T-cell lineages may provide more rationale approaches to the treatment or prevention of GVHD.

T Cell Activity After Dendritic Cell Vaccination Is Dependent on Both the Type of Antigen and the Mode of Delivery

Previous work in both human and animal models has shown that CTL responses can be generated against proteins derived from tumors using either peptide-pulsed dendritic cells (DCs) or nucleic acids from the tumor transfected into autologous DCs. Despite the efficacy of this approach for vaccine therapy, many questions remain regarding whether the route of administration, the frequency of administration, or the type of Ag is critical to generating T cell responses to these Ags. We have investigated methods to enhance CTL responses to a peptide derived from the human proto-oncogene HER-2/neu using mice containing a chimeric HLA A2 and H2Kb allele. Changes in amino acids in the anchor positions of the peptide enhanced the binding of the peptide to HLA-A2 in vitro, but did not enhance the immunogenicity of the peptide in vivo. In contrast, when autologous DCs presented peptides, significant CTL activity was induced with the altered, but not the wild-type, peptide. We found that the route of administration affected the anatomic site and the time to onset of CTL activity, but did not impact on the magnitude of the response. To our surprise, we observed that weekly administration of peptide-pulsed DCs led to diminishing CTL activity after 6 wk of treatment. This was not found in animals injected with DCs every 3 wk for six treatments or in animals initially given DCs weekly and then injected weekly with peptide-pulsed C1R-A2 transfectants.

Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans

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.

Ibrutinib treatment ameliorates murine chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Brutons tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell-driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell-mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials.

CD200 is induced by ERK and is a potential therapeutic target in melanoma

Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM.

The Role of Lactoferrin as an Anti-Inflammatory Molecule

The formation of hydroxyl radical via the iron catalyzed Haber-Weiss reaction has been implicated in phagocyte-mediated microbicidal activity and inflammatory tissue injury. The fact that neutrophils contain lactoferrin and mononuclear phagocytes have the capacity to acquire exogenous iron has suggested that iron bound to lactoferrin may influence the nature of free radical products generated by these cells. Over the years the iron-lactoferrin complex has been heralded as both a promoter and inhibitor of hydroxyl radical formation. This manuscript is intended to provide an overview of work performed to date related to this controversy and to present results of a number of preliminary studies which shed further light on the role of lactoferrin in inflammation.

CD30/CD30 Ligand (CD153) Interaction Regulates CD4+ T Cell-Mediated Graft-versus-Host Disease

CD30, a TNFR family member, is expressed on activated CD4+ and CD8+ T cells and B cells and is a marker of Hodgkin’s lymphoma; its ligand, CD30L (CD153) is expressed by activated CD4+ and CD8+ T cells, B cells, and macrophages. Signaling via CD30 can lead to proliferation or cell death. CD30-deficient (−/−) mice have impaired thymic negative selection and increased autoreactivity. Although human alloreactive T cells preferentially reside within the CD30+ T cell subset, implicating CD30 as a regulator of T cell immune responses, the role of CD30/CD153 in regulating graft-vs-host disease (GVHD) has not been reported. We used a neutralizing anti-CD153 mAb, CD30−/− donor mice, and generated CD153−/− recipient mice to analyze the effect of CD30/CD153 interaction on GVHD induction. Our data indicate that the CD30/CD153 pathway is a potent regulator of CD4+, but not CD8+, T cell-mediated GVHD. Although blocking CD30/CD153 interactions in vivo did not affect alloreactive CD4+ T cell proliferation or apoptosis, a substantial reduction in donor CD4+ T cell migration into the gastrointestinal tract was readily observed with lesser effects in other GVHD target organs. Blockade of the CD30/CD153 pathway represents a new approach for preventing CD4+ T cell-mediated GVHD.