Pathogenesis Of Acute Graft-versus-host Disease Research Articles

Sigle Agent of Posttransplant Cyclophosphamide Without Calcineurin Inhibitor Controls Severity of Experimental Chronic GVHD.

Chronic graft-versus-host disease (GVHD) is a major cause of late death and morbidity following allogeneic hematopoietic cell transplantation (HCT), but its pathogenesis remains unclear. Recently, haplo-identical HCT with post-transplant cyclophosphamide (Haplo-HCT with PTCY) was found to achieve a low incidence rate of acute GVHD and chronic GVHD. However, while the pathogenesis of acute GVHD following Haplo-HCT with PTCY has been well investigated, that of chronic GVHD remains to be elucidated, especially in HLA-matched HCT with PTCY. Based on its safety profile, PTCY is currently applied for the human leucocyte antigen (HLA)-matched HCT setting. Here, we investigated the mechanisms of chronic GVHD following HLA-matched HCT with PTCY using a well-defined mouse chronic GVHD model. PTCY attenuated clinical and pathological chronic GVHD by suppressing effector T-cells and preserving regulatory T-cells compared with a control group. Additionally, we demonstrated that cyclosporine A (CsA) did not show any additional positive effects on attenuation of GVHD in PTCY-treated recipients. These results suggest that monotherapy with PTCY without CsA could be a promising strategy for the prevention of chronic GVHD following HLA-matched HCT.

Role of Defibrotide in the Prevention of Murine Model Graft-versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Vascular endothelial cells are entirely exposed and damaged during the pathogenesis of acute GVHD. Defibrotide is a mixture of single-stranded oligonucleotides that has several pharmacological effects that contribute to its endothelial protective properties. B10.BR mice were conditioned followed by the infusion of donor C57BL/6J T-cell depleted (TCD) bone marrow cells with or without splenocytes. Animals were either treated with defibrotide or appropriate controls daily for the first week and then three times per week thereafter. Allogeneic defibrotide-treated recipients demonstrated significantly better survival with reduced clinical GVHD. Significantly reduced organ pathology in the gut was associated with significantly decreased T-cell infiltration in the ileum and colon on day +28. Serum cytokine analysis revealed significantly reduced level of TNF and IL-6 at day +7 and TNF at day +28 in allogeneic defibrotide treated recipients. Significantly reduced levels of ICAM-1, Angiopoietin-2 in serum and reduced VCAM-1 and HCAM in the ileum and colon of allogeneic defibrotide-treated recipients were observed. Improved survival was seen in the GVL model (C3H.SW into C57BL/6J mice with C1498-luc). Defibrotide treatment through its anti-inflammatory and endothelial-protective effects reduces the severity of acute GVHD, while not impairing GVL activity.

Acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation

Acute graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic stem cell transplantation determining the prognosis of hematopoietic stem cell recipients because of the impairment of multiple organs and systems and subsequent secondary immunodeficiency caused by combination immunosuppressive therapy. Current strategies are focused on the prevention of this life-threatening complication and on the timely start of treatment with appropriate selection of medications based on the knowledge of acute GVHD pathogenesis and grading as well as of risk factors for its development / progression in different patients. Here we provide a brief overview of acute GVHD and the existing approaches to its prevention and treatment.

Role of endothelial cells in graft-versus-host disease.

To date, the only curative treatment for high-risk or refractory hematologic malignancies non-responsive to standard chemotherapy is allogeneic hematopoietic transplantation (allo-HCT). Acute graft-versus-host disease (GVHD) is a donor T cell-mediated immunological disorder that is frequently fatal and the leading cause of non-relapse mortality (NRM) in patients post allo-HCT. The pathogenesis of acute GVHD involves recognition of minor and/or major HLA mismatched host antigens by donor T cells followed by expansion, migration and finally end-organ damage due to combination of inflammatory cytokine secretion and direct cytotoxic effects. The endothelium is a thin layer of endothelial cells (EC) that line the innermost portion of the blood vessels and a key regulator in vascular homeostasis and inflammatory responses. Endothelial cells are activated by a wide range of inflammatory mediators including bacterial products, contents released from dying/apoptotic cells and cytokines and respond by secreting cytokines/chemokines that facilitate the recruitment of innate and adaptive immune cells to the site of inflammation. Endothelial cells can also be damaged prior to transplant as well as by alloreactive donor T cells. Prolonged EC activation results in dysfunction that plays a role in multiple post-transplant complications including but not limited to veno-occlusive disease (VOD), transplant associated thrombotic microangiopathy (TA-TMA), and idiopathic pneumonia syndrome. In this mini review, we summarize the biology of endothelial cells, factors regulating EC activation and the role of ECs in inflammation and GVHD pathogenesis.

Profiles of interferon-gamma and interleukin-2 in patients after allogeneic hematopoietic stem cell transplantation.

BACKGROUNDAllogeneic hematopoietic stem cell transplantation (allo-HSCT) may be related to the occurrence of complications, including graft-versus-host disease (GvHD) and infections. The pathogenesis of acute GvHD is connected with T lymphocytes, which identify alloantigens on host's antigen-presenting cells, activate production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), and act on the immune effector cells and damage tissues and organs.AIMThe aim of the study was to investigate and distinguish serum concentration profiles of IFN-gamma and IL-2 within a 30-d period after allo-HSCT.METHODSWe enrolled 62 patients, i.e., 30 (48%) male and 32 (52%) female subjects [median age 49.5 (19-68) years], after allo-HSCT from siblings (n = 12) or unrelated donors (n = 50) due to acute myeloid leukemia with myeloablative conditioning (n = 26; 42%) and with non-myeloablative conditioning (n = 36; 58%). All patients were given standard immunosuppressive therapy with cyclosporin-A and methotrexate and pre-transplant antithymocyte globulin in the unrelated setting. Blood samples were collected pre-transplant before and after (on day -1) the conditioning therapy and on days +2,+4, +6, +10, +20, and +30 after allo-HSCT. Serum levels of IL-2 and IFN-gamma were determined using ELISA. RESULTSPatients were divided into four groups depending on the presence of acute GvHD and clinical manifestations of infection. Group I included patients with neither acute GvHD nor infections [n = 15 (24%)], group II consisted of patients with infections without acute GvHD [n = 17 (27%)], group III was comprised of patients with acute GvHD without infections [n = 9 (15%)], and group IV included patients with both acute GvHD and infections [n = 21 (34%)]. IFN-gamma concentrations were higher in Group II than in other groups on days +20 (P = 0.014) and +30 (P = 0.008). Post-hoc tests showed lower concentrations of IFN-gamma on day +30 in groups I (P = 0.039) and IV (P = 0.017) compared to group II. The levels of IL-2 were mostly undetectable.CONCLUSIONSerum levels of IFN-gamma following allo-HSCT progressively escalate. High serum levels of IFN-gamma are related to infectious complications rather than acute GvHD. Serum concentrations of IL-2 in most patients are undetectable.

Calprotectin Expressing Donor-Derived Macrophages Increase in Acute Gastrointestinal Graft-Versus-Host Disease

Acute graft versus host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). GVHD is therefore the main obstacle for a more widespread use of this highly effective and potentially curative therapy. Although donor T cells are believed to be key mediators in the pathogenesis of acute GVHD, recent reports have suggested that monocyte-derived macrophages also contribute. However, data to support a role for macrophages in acute GVHD in the gastrointestinal tract are sparse. Here we performed a spatiotemporal in situ study to determine the presence of donor and recipient macrophage subsets in colon biopsies from allo-HSCT patients with and without GVHD. Our study was a retrospective study examining colon biopsies from 31 allo-HSCT patients (10 females), of which 21 (5 females) had clinical and histologically-verified GVHD. To distinguish host from donor macrophages we examined gender mismatched donors applying a combination of immunostaining and fluorescence in situ hybridization with probes to X and Y chromosomes. The density of colonic mucosal macrophages was significantly increased (P = .0031) in patients with acute GVHD (n = 21) compared with patients without GVHD (n = 10). Most macrophages were of donor origin in both groups; however, in acute GVHD there was a fivefold increase in donor-derived macrophages expressing the antimicrobial protein calprotectin; reminiscent of recently emigrated proinflammatory monocytes. Moreover, colonic macrophages were found in close proximity to both host and donor T cells. Together, our results suggest that donor-derived proinflammatory macrophages are involved in the immunopathology of colonic acute GHVD in humans.

Modifying a Xenogeneic Gvhd Model to Allow for Use of CRISPR-Treated Primary Human T Cells

Background:Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia, immunodeficiencies, and bone marrow failure syndromes. Therapeutic use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues. We have previously shown that GVHD-causing T cells increase activation of AMP-activated protein kinase (AMPK), a cellular energy sensor, and that T cell-specific ablation of AMPK in murine models decreases GVHD severity.To study human T cell biology, we modified a previous xenogeneic model. Current models transplant whole peripheral blood mononuclear cells (PBMCs) from healthy human donors into lightly irradiated immunodeficient NOD-scid IL2Rgamma null (NSG) mice. However, in our hands, CRISPR-treatment of primary human T cells requires up to 10 days of culturing to obtain sufficient cells for transplantation. Therefore, prior to assessing GVHD severity using AMPK-deficient human T cells, we optimized the xenogeneic GVHD model to allow time for the manipulation and subsequent injection of CRISPR-engineered cells.Aim:To develop a xenogeneic model compatible with CRISPR/Cas9 manipulated T cells to determine whether changes in human T cells decrease GVHD severity similar to what is seen using murine T cells.Results:We first demonstrated that expanded T cells alone cause minimal xenogeneic GVHD, but that disease could be significantly facilitated with addition of non-T cell antigen presenting cells (APCs). Transplanting T cells plus non-T cell APCs increased numbers of human CD45+CD3+ T cells recovered on day 25 post-transplant (Figure 1A-B) and elevated levels of human interferon (IFN)-γ (Figure 1C). Liver sections from recipients of T cells + APCs subjectively had more perivascular infiltrates than mice receiving T cells alone (data not shown). Additionally, as seen in murine T cells, xenogeneic human T cells increased fatty acid oxidation, additional evidence that our model recapitulates the murine findings (Figure 1D).We next wished to fix the number of co-administered APCs and optimize the number of activated T cells to reliably reproduce xenogeneic GVHD without inducing overt toxicity. To accomplish this goal, we performed xenogeneic transplants with serially decreasing numbers of expanded human T cells (starting with 6×10 6/recipient) and administered 1×10 6 APCs in all cohorts. We also trialed inclusion of recently thawed non-T cell APCs in place of freshly derived cells. Reassuringly, the number of human CD45+CD3+ cells recovered on day 25 post-transplant remained proportional to the number of cells injected (Figure 2A-B), as did levels of human IFN-γ (detected by serum ELISA (Figure 2C). These data indicate that robust xenogeneic GVHD can be induced with as few as 2×10 6 expanded T cells and 1×10 6 autologous APCs, with a concomitant increase in GVHD-associated proinflammatory cytokines. Importantly, these data also demonstrate that 1×10 6 APCs are sufficient to cause reproducibly severe disease and may be recovered from a cryopreserved source.Finally, we wished to extend the assessment of GVHD severity following administration of varying doses of donor T cells. Serum from recipient mice on day 25 post-transplant was analyzed for the production of murine-derived cytokines via a LEGENDplex assay. Of 13 cytokines tested, both murine MCP-1 and TNF-alpha were proportional to the number of T cells injected (Figure 3A-B), with the MCP-1 result confirmed by ELISA (data not shown). Thus, both MCP-1 and TNF-α, cytokines commonly implicated in acute GVHD pathogenesis, provide additional host-derived soluble factors that can be utilized to quantitate the severity of GVHD in our modified xenogeneic model. Expression of these proteins will serve as valuable biomarkers in the assessment of xenogeneic GVHD using CRISPR-treated cells.Conclusions:We have successfully adapted a xenogeneic model of GVHD using in vitro expanded T cells and cryopreserved APCs, thereby allowing for expanded testing of genetically manipulated human T cells in an in vivo model. Future studies will compare the necessity of genes in human donor T cells using CRISPR-mediated gene editing and compare CRISPR techniques with novel pharmacological inhibition using this modified xenogeneic approach. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interleukin-23 receptor signaling by interleukin-39 potentiates T cell pathogenicity in acute graft-versus-host disease

IL-12 (p35/p40) and IL-23 (p19/p40) signal through IL-12R (IL-12Rβ2/β1) and IL-23R (IL-23Rα/IL-12Rβ1), respectively, which can promote pathogenic T lymphocyte activation, differentiation, and function in graft-versus-host disease (GVHD). With the use of murine models of allogeneic hematopoietic cell transplantation (HCT), we found that IL-12Rβ1 on donor T cells was dispensable to induce acute GVHD development in certain circumstances, while IL-23Rα was commonly required. This observation challenges the current paradigm regarding IL-12Rβ1 as a prerequisite to transmit IL-23 signaling. We hypothesized that p19/EBI3 (IL-39) may have an important role during acute GVHD. With the use of gene transfection and immunoprecipitation approaches, we verified that p19 and EBI3 can form biological heterodimers. We found that IL-39 levels in recipient serum positively correlated with development of acute GVHD in experimental models and in clinical settings, thereby implicating IL-39 in the pathogenesis of acute GVHD. Furthermore, we observed that human T cells can signal in response to IL-39. In chronic GVHD, IL-23Rα and IL-12Rβ1 were similarly required for donor T cell pathogenicity, and IL-39 levels were not significantly different from controls without GVHD. Collectively, we identify a novel cytokine, IL-39, as a pathogenic factor in acute GVHD, which represents a novel potential therapeutic target to control GVHD and other inflammatory disorders.

Inhibition of Bromodomain and Extra Terminal (BET) Domain Activity Modulates the IL-23R/IL-17 Axis and Suppresses Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (GVHD) is the leading cause of non-relapse mortality following allogeneic hematopoietic cell transplantation. The majority of patients non-responsive to front line treatment with steroids have an estimated overall 2-year survival rate of only 10%. Bromodomain and extra-terminal domain (BET) proteins influence inflammatory gene transcription, and therefore represent a potential target to mitigate inflammation central to acute GVHD pathogenesis. Using potent and selective BET inhibitors Plexxikon-51107 and -2853 (PLX51107 and PLX2853), we show that BET inhibition significantly improves survival and reduces disease progression in murine models of acute GVHD without sacrificing the beneficial graft-versus-leukemia response. BET inhibition reduces T cell alloreactive proliferation, decreases inflammatory cytokine production, and impairs dendritic cell maturation both in vitro and in vivo. RNA sequencing studies in human T cells revealed that BET inhibition impacts inflammatory IL-17 and IL-12 gene expression signatures, and Chromatin Immunoprecipitation (ChIP)-sequencing revealed that BRD4 binds directly to the IL-23R gene locus. BET inhibition results in decreased IL-23R expression and function as demonstrated by decreased phosphorylation of STAT3 in response to IL-23 stimulation in human T cells in vitro as well as in mouse donor T cells in vivo. Furthermore, PLX2853 significantly reduced IL-23R+ and pathogenic CD4+ IFNγ+ IL-17+ double positive T cell infiltration in gastrointestinal tissues in an acute GVHD murine model. Our findings identify a role for BET proteins in regulating the IL-23R/STAT3/IL-17 pathway. Based on our preclinical data presented here, PLX51107 will enter clinical trial for refractory acute GVHD in a Phase 1 safety, biological efficacy trial.

Inhibition of inositol kinase B controls acute and chronic graft-versus-host disease

AbstractT-cell activation releases inositol 1,4,5-trisphosphate (IP3), inducing cytoplasmic calcium (Ca2+) influx. In turn, inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) phosphorylates IP3 to negatively regulate and thereby tightly control Ca2+ fluxes that are essential for mature T-cell activation and differentiation and protection from cell death. Itpkb pathway inhibition increases intracellular Ca2+, induces apoptosis of activated T cells, and can control T-cell–mediated autoimmunity. In this study, we employed genetic and pharmacological approaches to inhibit Itpkb signaling as a means of controlling graft-versus-host disease (GVHD). Murine-induced, Itpkb-deleted (Itpkb−/−) T cells attenuated acute GVHD in 2 models without eliminating A20-luciferase B-cell lymphoma graft-versus-leukemia (GVL). A highly potent, selective inhibitor, GNF362, ameliorated acute GVHD without impairing GVL against 2 acute myeloid leukemia lines (MLL-AF9-eGFP and C1498-luciferase). Compared with FK506, GNF362 more selectively deleted donor alloreactive vs nominal antigen-responsive T cells. Consistent with these data and as compared with FK506, GNF362 had favorable acute GVHD and GVL properties against MLL-AF9-eGFP cells. In chronic GVHD preclinical models that have a pathophysiology distinct from acute GVHD, Itpkb−/− donor T cells reduced active chronic GVHD in a multiorgan system model of bronchiolitis obliterans (BO), driven by germinal center reactions and resulting in target organ fibrosis. GNF362 treatment reduced active chronic GVHD in both BO and scleroderma models. Thus, intact Itpkb signaling is essential to drive acute GVHD pathogenesis and sustain active chronic GVHD, pointing toward a novel clinical application to prevent acute or treat chronic GVHD.

Advances in understanding the pathogenesis of graft-versus-host disease.

Acute graft-versus-host disease (GVHD) remains a major complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT). The emergence of different immuno-prophylaxis strategies, such as post-transplant cyclophosphamide or anti-thymocyteglobulin has reduced the incidence of acute GVHD in recent years. The biology of the acute GVHD we observe in the clinic may change due to the use of novel immuno-stimulatory agents, including immune checkpoint inhibitors or anti-neoplastic immune-modifiers, like lenalidomide, given before or after allo-HSCT. Here we discuss the recent advances in our understanding of acute GVHD with a focus on early events of the disease, including tissue damaging factors, innate immune cells, costimulatory pathways, immune cell signalling, immuno-regulatory cell types, biomarkers of GVHD and regenerative approaches. New insight in the pathogenesis of acute GVHD has revealed the role of pro-inflammatory intracellular signalling, defects in intestinal tissue regeneration and anti-bacterial defence, as well as a reduced diversity of the microbiome, which will be the basis for the development of novel therapies.

CD38brightCD8+ T Cells Associated with the Development of Acute GVHD Are Activated, Proliferating, and Cytotoxic Trafficking Cells.

We have previously reported that a peripheral blood absolute CD38brightCD8+ effector memory T cell (TEM) population expansion of >35 cells/µL predicts the development of acute graft-versus-host disease (GVHD). We hypothesized that these T cells are activated, proliferating, and cytotoxic trafficking cells that are not a response to viral reactivation and may be involved in acute GVHD. We characterized peripheral blood T cell populations at the time of maximum CD38brightCD8+ TEM expansion in patients from our originally reported pediatric allogeneic hematopoietic cell transplantation recipient cohort. Samples were incubated with fluorochrome-conjugated antibodies directed against CD3, CD8, CD38, HLA-DR (T cell activation), Ki-67 (T cell proliferation), granzyme B (marker of cytotoxic T cells), CLA (skin trafficking), CCR5 (visceral trafficking), and CXCR6 (liver trafficking). We also incubated samples with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) peptide pools and measured IFN-γ production by flow cytometry and performed EBV and CMV tetramer staining. Higher median proportions of cell expression of HLA-DR, Ki-67, granzyme B, CLA, CCR5, and CXCR6 were observed for CD38brightCD8+ T cells compared with CD38nonbrightCD8+ T cells in patients with acute GVHD (P < .05) but not in patients without acute GVHD (P not significant). No IFN-γ production was observed after incubation with CMV and EBV peptide pools. EBV-specific tetramer populations of 6.85% and 3.17% were detected in 2 patients with acute GVHD, whereas a CMV-specific tetramer population of 3.77% was detected in 1 patient with acute GVHD. No EBV- or CMV-specific tetramer populations were detected in any patient without acute GVHD. We conclude that CD38brightCD8+ T cells associated with the development of acute GVHD are activated, proliferating, and cytotoxic trafficking cells that do not appear to respond to CMV or EBV reactivation. Further studies are needed to determine whether these cells are directly involved in acute GVHD pathogenesis.

Neutrophils provide cellular communication between ileum and mesenteric lymph nodes at graft-versus-host disease onset

AbstractConditioning-induced damage of the intestinal tract plays a critical role during the onset of acute graft-versus-host disease (GVHD). Therapeutic interference with these early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. By using a novel photoconverter reporter system, we show that neutrophils that had been photoconverted in the ileum postconditioning later migrated to mesenteric lymph nodes (mLN). This neutrophil migration was dependent on the intestinal microflora. In the mLN, neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. Pharmacological JAK1/JAK2 inhibition reduced neutrophil influx into the mLN and MHC-II expression, thereby interfering with an early event in acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation. JAK1/JAK2-inhibition can interfere with this process, which provides a potential therapeutic strategy to prevent early events of tissue damage-related innate immune cell activation and, ultimately, GVHD.

Extracellular Vesicles as Potential Biomarker for Acute Graft-Versus-Host-Disease

INTRODUCTION: Graft-versus-Host-Disease (GVHD) is the main cause of non-relapse mortality after Hematopoietic Stem Cells Transplantation (HSCT). Identyfing potential biomarkers of GVHD could be crucial to define patients at high risk of GVHD development and to better assess GVHD grading. One potential attractive biomarker may be represented by extracellular vesicles (EVs). EVs are membrane-enclosed structures secreted by many cell types and may be represented by exosomes, shedding vesicles (microvesicles; MVs) and apoptotic bodies. EVs are detectable in body fluids, in particular peripheral blood (PB) and urine; EVs play a key role in the regulation of physiological and pathological processes. The aim of this study was to investigate EVs in PB of allotransplanted patients (EVs count, size and phenotype) and analyze a potential correlation with acute and chronic GVHD (aGVHD, cGVHD).METHODS: At our center, between 2000 and 2008, 41 multiple myeloma patients underwent an allograft. Median age of the patients was 53 years (range 34-65). Donors were HLA-identical siblings in 83% of the transplants. Conditioning regimen was mostly non-myeloablative (32/41,78%) and PB stem cells were used as source in all patients. GVHD Prophylaxis consisted of cyclosporine and mycophenolic acid in 34/41 patients (83%). Disease status was partial or less than partial remission in 95% of the patients. For standard policy, serum samples were collected before and monthly after transplant up to 6 months or disease relapse. EVs extraction was feasible due to high EVs stability in frozen serum samples. EVs were extracted using a protamine-based precipitation method and analyzed by flow-cytometry (Guava EasyCyte Flow Cytometer) without using latex-activated beads. We investigated a panel of fluorescence antibodies against specific membrane proteins (CD44, CD138, CD146, KRT18, CD120a, CD8, CD30, CD106, CD25, CD31, CD144, CD86, and CD140a). At each time-point, we determined for each sample: total EVs concentration, fluorescence distribution and percentage of positive EVs for a given marker. Data were computed by logistic regression analysis; Odds Ratio (OR) was calculated as proportional change as compared with pre-transplant baseline level of each marker.RESULTS: In our cohort, the cumulative incidence of aGVHD and cGVHD was 56% (95% 40.7-71.8%) and 71% (95% CI 56.3-85.2%) at day 100 and 24 months, respectively.Four biomarkers (CD146, CD25, CD106, CD31) showed a potential correlation with acute GVHD development. Two biomarkers (CD146 and CD25) were associated with an increased risk of developing aGVHD (CD146 fluorescence, OR 2.94, p=0.040 and CD25 fluorescence, OR 1.61, p<0.001). Furthermore, CD106 positive concentration (OR 0.23, p=0.077) and CD31 fluorescence and positive concentration (OR 0.24, p=0.052; OR 0.50, p=0.067, respectively) were associated with a decreased risk of aGVHD. All the biomarkers associated with aGVHD showed a proportional change from baseline in signal level before GVHD onset (an increase in case of CD146 and CD25, a reduction with CD106 and CD31, respectively).No statistically significant association was observed between our panel of biomarkers and cGVHD.CONCLUSIONS: In this study, we observed a potential association between 4 biomarkers expressed on EVs' surface and aGVHD. CD146, CD31 and CD106 belong to Cell Adhesion Molecule family (MCAM-1, PECAM-1 and VCAM-1, respectively), which are crucial for endothelium and immune cells interaction. CD25 is IL2 Receptor and is a marker of immune activation and inflammation. All these proteins mentioned above, could have a role in acute GVHD pathogenesis. A perspective study is currently ongoing at our Center to validate these data. DisclosuresBoccadoro:CELGENE: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Abbivie: Honoraria; Mundipharma: Research Funding; SANOFI: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Antibiotic-mediated modification of the intestinal microbiome in allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is curative for many patients with severe benign and malignant hematologic disorders. The success of allogeneic HSCT is limited by the development of transplant-related complications such as acute graft-versus-host disease (GvHD). Early pre-clinical studies suggested that intestinal microflora contribute to the pathogenesis of acute GvHD, and that growth suppression or eradication of intestinal bacteria prevented the development of acute GvHD even in MHC-mismatched transplants. These observations led to the practice of gut decontamination (GD) with oral non-absorbable antibiotics in patients undergoing allogeneic HSCT as a method of acute GvHD prophylaxis. Microbiome studies in the modern sequencing era are beginning to challenge the benefit of this practice. In this review, we provide a historical perspective on the practice of GD and highlight findings from the limited number of clinical trials evaluating the use of GD for acute GvHD prevention in allogeneic HSCT patients. In addition, we examine the role of the gut microbiota in allogeneic HSCT in the context of recent studies linking the microflora to regulation of intestinal immune homeostasis. We discuss the implications of these findings for future strategies to reduce acute GvHD risk by selective manipulation of the microbiota.

Soluble heat shock protein 70 members in patients undergoing allogeneic hematopoietic cell transplantation

BackgroundHeat shock proteins (HSP) are highly conserved immunogenic proteins serving as potent danger signals. They are upregulated under stress conditions like fever and hypoxia. Extracellular HSP are involved in antigen presentation, cytokine release and maturation of antigen presenting cells. MethodsThe release of the inducible members of the HSP70 family, Hsp72 and Hsp70B′, into the serum of 20 patients undergoing allogeneic hematopoietic cell transplantation and 20 healthy donors was evaluated using enzyme linked immunosorbent assay (ELISA) kits. ResultsEight patients (40%) did not receive anti-thymocyte globulin (ATG) for prophylaxis of graft versus host disease (GvHD). These patients had no detectable or low serum levels of Hsp72 (n=3, 0.03 to 1.92ng/ml) which were in line with levels detected in 20 healthy individuals (p=0.07). Measurable HSP was not associated with any medication or transplantation-related procedures. In twelve patients (60%) receiving ATG, detected high levels of HSP reflected cross-reactivity of the rabbit-derived ATG with the anti-rabbit antibody used in the ELISA. ConclusionsAssumed HSP70 expression detected such ELISA has to be regarded carefully after ATG application. Neither radiochemotherapy, nor inflammation or sepsis during aplasia induced HSP70 release into the serum. Thus, soluble HSP70 may not be involved in the pathogenesis of acute GvHD.

Absence of Coronin 1B in Donor T Cells Diminishes Acute Gvhd By Impairing T Cell Accumulation in Secondary Lymphoid Tissue

Allogeneic stem cell transplant is a standard treatment for patients with high-risk and relapsed myeloid and lymphoid malignancies. However, donor T cells from the stem cell graft mediate graft-versus-host disease (GVHD), which is a common cause of morbidity and mortality for transplant recipients. Our group and others have shown that migration of donor T cells into secondary lymphoid tissue (SLT) and subsequent migration to target organs is critical to the pathogenesis of acute GVHD.The Coronin family of proteins consists of actin-binding proteins, which regulate filament formation by interacting with the Arp2/3 complex. Coronin 1B, a ubiquitously expressed member of the Coronin family, is required for lamellipodial protrusion and effective cell migration. Previous work has not evaluated a role for this protein in the function of T lymphocytes or during acute GVHD.To evaluate the effect of Coronin 1B in acute GVHD pathogenesis, we transplanted B6 T cell depleted bone marrow cells with wild type or Coronin 1B-/- T cells to lethally irradiated B6D2 and BALB/c recipient mice and evaluated clinical score of GVHD and overall survival. B6D2 recipients of Coronin 1B-/- T cells demonstrated 100% survival (Figure 1A. p< .001 as determined by Log-rank (Mantel-Cox) test) and significantly decreased clinical scores after transplant. This was confirmed with improvement in survival in BALB/c recipients of Coronin 1B-/- T cells. Additionally, Coronin 1B-/- T cells were capable of eliminating P815 tumor cells, indicating that loss of Coronin 1B does not inhibit graft-versus-tumor activity. By day 12 post- transplant, all mice receiving bone marrow alone developed tumor compared to none of the mice receiving Coronin 1B-/- T Cells. However, protection was not complete as 40% of Coronin 1B-/- T cell recipients developed tumor by day 23.To determine the effect of Coronin 1B on T cell migration during GVHD, B6D2 recipients were given GFP-expressing wild type or Coronin 1B-/- T cells along with T cell depleted bone marrow. Lymphoid tissue and target organs were harvested and analyzed by flow cytometry or GFP ELISA. We observed decreased accumulation of Coronin 1B-/- CD4+ (Figure 1B. p< .01 as determined by Student's t -test) and CD8+ T cells in the inguinal lymph node, mesenteric lymph node, and the spleen 4 days after transplant with no difference in accumulation in lymphoid tissue on days 7 and 14 after transplant. Additionally, we found decreased accumulation of Coronin 1B-/- donor T cells in the lung, colon and spleen 14 days after transplant (Figure 1C. p< .05 by Student's t -test). We also quantified the amount of cytokine in target organs by ELISA, and observed a decrease in IFN-γ and TNF-α in the colon 14 days after transplant.Our data demonstrate that Coronin 1B-/- T cells elicit reduced GVHD compared to wild type T cells. This was correlated with decreased accumulation of Coronin 1B-/- T cells in SLT early after transplant. These data indicate that targeting the migration of T cells to SLT is a viable approach to prevent acute GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cytotoxic T Lymphocyte Antigen-4 Down-Regulates T Helper 1 Cells by Increasing Expression of Signal Transducer and Activator of Transcription 3 in Acute Graft-versus-Host Disease.

Numerous previous studies have suggested that cytotoxic T lymphocyte antigen-4 (CTLA-4) plays an important role in acute graft-versus-host disease (GVHD). How CTLA-4 acts in regulating acute GVHD remains unknown, however. In the present study, we found that, compared with healthy controls, CTLA-4 plasma and relative mRNA levels in patients with acute GVHD were initially decreased and then markedly elevated after 28days of treatment. CTLA-4 levels were higher in patients with grade I-II acute GVHD compared with those with grade III-IV acute GVHD both before and after treatment. Up-regulation of CTLA-4 significantly increased the luciferase activity and degree of phosphorylation of signal transducer and activator of transcription 3 (STAT3). Meanwhile, T cell activation was significantly inhibited, and levels of IFN-γ, IL-17, and IL-22 decreased. These findings suggest that CTLA-4 might be involved in the pathogenesis of acute GVHD, and may down-regulate T helper 1 cells by increasing STAT3 expression in acute GVHD.

Significance of Th1/Th17 Imbalance in Mice with Acute Graft-versus-Host Disease

To investigate the effects of Th1/Th17 cell imbalance on the pathogenesis of acute graft-versus-host disease (GVHD) in mice. In a murine GVHD model of C57BL/6 (H-2(b)), a low dose of halofuginone (HF) was applied for treating the recipients in order to result in Th1/Th17 imbalance. Rechipient mice were divided into GVHD group (without HF intervention) and GVHD plus HF group (treated by HF). The recipients were monitored for survival rate, clinical scores of acute GVHD, contents of circulatory Th1 and Th17 cells, Th1/Th17 ratio and serum level of IFN-γ and IL-17A. Expression levels of IFN-γ and IL-17A in target organs were analyzed by using real-time PCR, and the target organs were delivered for histological examinations. Recipients treated with HF showed that all the mortality, circulatory Th1/Th17 ratio and clinical score were higher than those in the mice without HF intervention (P < 0.05). Circulatory Th1/Th17 ratio positively correlates with clinical score (P < 0.001). HF administration reduces the expression level of intestinal IL-17A and increases intrahepatic and intestinal IFN-γ level (P < 0.05), HF treatment aggravates GVHD in liver and small intestine with augmented hepatic and intestinal inflammation. Th1/Th17 imbalance contributes to the pathogenesis of acute GVHD.

MicroRNAs as biomarkers for graft-versus-host disease following allogeneic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HCT) is a well-established treatment for many malignant and non-malignant hematological disorders. As frequent complication in up to 50% of all patients, graft-versus-host disease (GVHD) is still the main cause for morbidity and non-relapse mortality. Diagnosis of GVHD is usually done clinically, even though confirmation by pathology is often used to support the clinical findings. Effective treatment requires intensified immunosuppression as early as possible. Although several promising biomarkers have been proposed for an early diagnosis, no internationally recognized consensus has yet been established. Here, microRNAs (miRs) represent an interesting tool since miRs have been recently reported to be an important regulator of various cells, including immune cells such as T cells. Therefore, we could assume that miRs play a key role in the pathogenesis of acute GVHD, and their detection might be an interesting possibility in the early diagnosis and monitoring of acute GVHD. Recent studies additionally demonstrated the implication of miRs in the pathogenesis of acute GVHD. In this review, we aim to summarize the previous reports of miRs, focusing on the pathogenesis of acute GVHD and possible implications in diagnostic approaches.