Takahisa Nakanishi

IL-33 Promotes the Induction and Maintenance of Th2 Immune Responses by Enhancing the Function of OX40 Ligand

BACKGROUND In Th2 immune responses, TSLP is a key player by induction of OX40-ligand (OX40L) on dendritic cells (DCs), which is the trigger to induce Th2 cell-mediated allergic cascade. Thus, TSLP-DC-OX40L axis might be the principal pathway in the inflammatory cascades in atopic dermatitis and asthma. IL-33, which is produced by epithelial cells, has been implicated in the Th2 immune responses and pathogenesis of the allergic disorders. However, the role of IL-33 in the Th2-polarizing TSLP-DC-OX40L axis still remains largely elusive. We focused on the ability of IL-33 to promote OX40L-mediated Th2 responses. METHODS Purified human naïve or memory CD4+ T cells were stimulated with recombinant OX40L or TSLP-treated DCs (TSLP-DCs) in the presence of IL-33, and the cytokine production by the primed T cells was examined. We also performed immunohistochemical analyses for the expression of IL-33 in specimens of lymph node and skin from the patients with atopic dermatitis. RESULTS IL-33 remarkably enhanced TSLP-DCs-driven or OX40L-driven Th2 responses from naïve T cells and the Th2 functional attributes of CRTH2+ CD4+ Th2 memory cells by the increased production of IL-5, IL-9, and IL-13. In addition, IL-33 was expressed in the nuclei of epithelial cells in not only skin lesion but also lymph nodes of the patient with atopic dermatitis, suggesting a specialized role in adaptive T cell-priming phase. CONCLUSIONS IL-33 works as a positive regulator of TSLP-DC-OX40L axis that initiates and maintains the Th2 cell-mediated inflammatory responses, and therefore, it would be a new therapeutic target for the treatment of allergic disorders.BACKGROUND In Th2 immune responses, TSLP is a key player by induction of OX40-ligand (OX40L) on dendritic cells (DCs), which is the trigger to induce Th2 cell-mediated allergic cascade. Thus, TSLP-DC-OX40L axis might be the principal pathway in the inflammatory cascades in atopic dermatitis and asthma. IL-33, which is produced by epithelial cells, has been implicated in the Th2 immune responses and pathogenesis of the allergic disorders. However, the role of IL-33 in the Th2-polarizing TSLP-DC-OX40L axis still remains largely elusive. We focused on the ability of IL-33 to promote OX40L-mediated Th2 responses. METHODS Purified human naïve or memory CD4+ T cells were stimulated with recombinant OX40L or TSLP- treated DCs (TSLP-DCs) in the presence of IL-33, and the cytokine production by the primed T cells was examined. We also performed immunohistochemical analyses for the expression of IL-33 in specimens of lymph node and skin from the patients with atopic dermatitis. RESULTS IL-33 remarkably enhanced TSLP-DCs-driven or OX40L-driven Th2 responses from naïve T cells and the Th2 functional attributes of CRTH2+ CD4+ Th2 memory cells by the increased production of IL-5, IL-9, and IL-13. In addition, IL-33 was expressed in the nuclei of epithelial cells in not only skin lesion but also lymph nodes of the patient with atopic dermatitis, suggesting a specialized role in adaptive T cell-priming phase. CONCLUSIONS IL-33 works as a positive regulator of TSLP-DC-OX40L axis that initiates and maintains the Th2 cell-mediated inflammatory responses, and therefore, it would be a new therapeutic target for the treatment of allergic disorders.

The correlation between platelet activation markers and HMGB1 in patients with disseminated intravascular coagulation and hematologic malignancy

To the editor Disseminated intravascular coagulation (DIC) frequently complicates hematologic malignancy and infections [1]. Coagulation abnormalities and thrombocytopenia are common in DIC, and the extent of hemostatic disorders appears to correlate with disease severity [2]. In particular, low platelet count is predictive of poor outcome [3, 4]. In DIC patients, the thrombin generated may react with thrombin receptors (PAR-1) located on the platelets and result in platelet activation. On the other hand, high mobility group-B1 DNA binding protein (HMGB1) is a nuclear architectural chromatin binding protein released by necrotic cells that has been identified as a mediator of endotoxin-induced lethality, and acts at least in part as a proinflammatory cytokine [5–7]. Moreover, HMGB1 also plays a role in the pathogenesis of DIC because plasma HMGB1 levels correlate with DIC scores [8]. However, the significance of activated platelets related to HMGB1 in DIC patients is poorly understood. Here, we describe the correlation between platelet activation markers and HMGB1 in DIC patients with hematologic malignancy. In addition, we investigated the effect of recombinant thrombomodulin (rTM) on these markers. Patients with DIC associated with hematologic malignancy were recruited. DIC was diagnosed according to the diagnostic criteria established by the Japanese Ministry of Health, Labor and Welfare [9]. In total, 45 patients comprised of 27 males and 18 females were enrolled. The median age was 54 years (range 21–78 years). Serum samples collected at baseline and 14 days after rTM therapy were tested by ELISA for antibodies against the markers. All ELISA kits were used according to the manufacture’s instructions. For DIC patients with hematologic malignancy, univariate analysis showed that prothrombin time (PT), fibrinogen, C-reactive protein (CRP), plateletderived microparticle (PDMP), soluble CD40 ligand (sCD40L), interleukin (IL)-6, and tissue necrotizing factor (TNF)! regulated on activation of normal T-cell expressed and secreted (RANTES) and monocyte chemotactic peptide-1 (MCP-1) were significantly associated withHMGB1 (Table I). In addition, fibrinogen, CRP, PDMP, sCD40L, TNF! and RANTES were significant factors in the multivariate model with HMGB1 (Table I). DIC patients exhibited a significant decrease inHMGB1 levels after rTM therapy (before vs. after, 26.3! 8.4 vs. 12.1! 5.9 ng/ ml, p< 0.001). In addition, the levels of platelet activation markers and cytokines were significantly decreased after rTM therapy in DIC patients (before vs. after, PDMP: 19.9! 3.3 vs. 11.7! 2.9 U/ml, p< 0.001; sCD40L: 3.45! 1.18 vs. 2.00! 0.79 ng/ml, p< 0.001; RANTES: 92.6! 14.5 vs.

Plasmacytoid dendritic cells have a cytokine-producing capacity to enhance ICOS ligand-mediated IL-10 production during T-cell priming

Plasmacytoid dendritic cells (pDCs) have the potential to prime CD4(+) T-cells to differentiate into IL-10-producing T regulatory cells through preferential expression of inducible co-stimulatory ligand (ICOS-L). Although pDCs produce cytokines such as type-I IFNs, TNF-α, or IL-6 accompanying up-regulation of ICOS-L expression during activation in response to toll-like receptor (TLR)-ligands or IL-3, the roles of the pDC-derived cytokines in T-cell priming remain largely elusive. Therefore, we investigated the functional involvement of these cytokines in generating IL-10-producing T regulatory cells. We found that either IFN-α or IL-6 enhanced the pDC- or ICOS-L-driven generation of IL-10-producing T-cells from naive CD4(+) T-cells and their regulatory functions. However, IFN-α stimulation in the absence of ICOS-L showed only a marginal tendency to increase the T-cell production of IL-10 and thus pDC-derived type-I IFNs in response to CpG could function together with ICOS-L. In addition, IL-6 functioned to generate IL-10-producing T-cells only on T-cell priming by pDCs activated by IL-3 or under IL-4-mediated T(h)2 conditions. Thus, type-I IFNs and IL-6 act as supplementary factors for the ICOS-L-dependent IL-10-producing T-cell differentiation in pDCs activated along the TLR-dependent and IL-3-dependent pathways, respectively. We also showed that pDC-derived TNF-α induced ICOS-L expression on pDCs in an autocrine manner and that IL-6 promoted ICOS expression on T-cells, contributing to the ICOS/ICOS-L-mediated T-cell response. Our results suggest that the ICOS-L-mediated tolerogenic pDC function in adaptive immunity is backed up by the elaborate cytokine-producing ability of pDCs.

Can recombinant thrombomodulin play a preventive role for veno-occlusive disease after haematopoietic stem cell transplantation?

Can recombinant thrombomodulin play a preventive role for veno-occlusive disease after haematopoietic stem cell transplantation? -

Platelet-derived microparticles cause CD154-dependent activation of dendritic cells

To the EditorPlatelets represent an important linkage betweeninflammation, thrombosis and atherosclerosis [1].Platelet-induced chronic inflammatory processes atthe vascular wall result in development of atheroscle-rotic lesions and atherothrombosis [2]. In addition,accumulating evidence from recent studies indicatesan additional role in modulating adaptive immuneresponses [3, 4]. Platelets express many immunemediators such as cytokines and chemokines.Furthermore, only a few researchers have suggestedthatplatelet-derivedCD154hastheabilitytopromoteprotective adaptive immune responses [5].Dendritic cells (DCs) sense immune adjuvantsthrough pathogen sensors and are critical for linkinginnate and adaptive immunity [6, 7]. In humans,myeloid DCs and plasmacytoid DCs represent twomajor subsets of DCs. They play distinct roles ininnate and adaptive immune responses throughexpression of their specialized cytokines and mole-cules [8].Platelet activation by various agonists or shearstress results in the shedding of platelet-derivedmicroparticles (PDMPs) [9]. Early studies showedthat PDMPs are an important source of integrins andselectins for leukocyte attachment to endothelialcells, which is a process that is important forrecruitment to and transmigration at sites of injury[10, 11]. In addition, platelets and PDMPs areknown to modulate the activity of cells with whichthey interact, including monocytes, neutrophils, andendothelial cells [9–11]. However, the role of PDMPon DC activation is poorly understood. We investi-gated the effect of PDMPs in DC activation focusingon the adaptive immune responses properties ofPDMPs.Human peripheral blood DC subsets (plasmacy-toid DCs and myeloid DCs) were isolated fromperipheral blood mononuclear cells from healthyadult donors, as described previously [12]. Weexamined whether DC subsets are activated in thepresence of thrombin receptor activating peptide(TRAP)-stimulated platelets or PDMPs. DC subsetswere cultured with TRAP-stimulated platelets orPDMPs for 24h, and the expression of somemolecules such as CD40, CD80, CD83, CD86 andHLA-DR was analysed by flow cytometry.We found that, although TRAP-stimulated plate-lets could induce up-regulation of some moleculessuch as CD40, CD80, CD83, CD86 and HLA-DRto some extent, the addition of PDMPs furtherincreased this expression compared with expressioninduced by TRAP-stimulated platelets (Table I). Inaddition, the enhancement of expression of thesemolecules in response to TRAP-stimulated plateletsor PDMPs was completely inhibited by anti-CD154antibody (data not shown).Several reports have suggested that T cell-derivedCD154 contributes to the modulation of adaptiveimmunity. For example, CD154 is crucial for thedevelopment of T cell-dependent humoral immuneresponses during CD4

TGFβ1 and sCTLA-4 levels are increased in eltrombopag-exposed patients with ITP

Some thrombopoietin receptor-agonists (TPR-As) have been developed and shown to be highly effective in the treatment of immune thrombocytopenic purpura (ITP). Soluble cytotoxic T-lymphocyte-associated antigen 4 (sCTLA-4) can modulate and terminate the immune response. Several reports have shown that sCTLA-4 levels are elevated in patients with some autoimmune disorders. However, sCTLA-4 levels have not previously been investigated in TPR-A exposed patients with ITP. We investigated the levels of transforming growth factor (TGF) β(1) and sCTLA-4 in ITP patients to determine the clinical association with TGFβ(1) and sCTLA-4 in TPR-A-exposed patients with ITP. Thirty-seven ITP patients were divided into 2 groups (TPR-A-exposed: 13 patients; unexposed: 24 patients). Doses of eltrombopag ranging from 12.5mg to 50mg were administered daily, and biochemical data obtained before and after eltrombopag administration were compared. Eltrombopag therapy significantly increased sCTLA-4 and TGFβ(1) levels relative to baseline in patients with ITP. In addition, plasma TGFβ(1) was positively correlated with platelet counts and sCTLA-4 in patients with ITP in the eltrombopag-exposed group. However, no significant change in the detection rate for anti-glycoprotein antibody was observed before and 24 weeks after eltrombopag treatment. These results suggest that eltrombopag can partially modulate some immune responses by TGFβ(1) and sCTLA-4, but it does not induce immune tolerance by 24 weeks after treatment.

Delayed HBV reactivation in rituximab-containing chemotherapy: How long should we continue anti-virus prophylaxis or monitoring HBV-DNA?

Reactivation of hepatitis B virus (HBV) infection is a well-recognized and potentially fatal complication in patients treated with chemotherapy for lymphoid malignancies. Although several guidelines recommend antiviral prophylaxis and/or monitoring for HBV-DNA, there is no consensus over what time period these should occur. Clinically, we have encountered delayed reactivation of HBV infections and have reported 12 cases of reactivation in patients. Among them, five patients developed HBV reactivation more than a year after they completed their chemotherapy. This means there can be a delayed HBV reactivation and prolonged monitoring of more than a year after cessation of chemotherapy may be needed. Hence, the current recommendation of stopping antiviral prophylaxis 6-12 months after the cessation of chemotherapy may not fully protect all patients from HBV reactivation. The optimal duration of follow-up needs to be determined, and until better guidelines are set, there is no choice but to keep monitoring patients for reactivation for as long as practicable.

Successful treatment with mogamulizumab followed by allogeneic hematopoietic stem-cell transplantation in adult T-cell leukemia/lymphoma: a report of two cases.

A humanized anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, mogamulizumab (MOG), has been shown to be safe and effective in the treatment of relapsed/refractory adult T-cell leukemia/lymphoma (ATLL). MOG depletes ATLL cells as well as regulatory T cells (Tregs), as CCR4 is expressed on these cells as well. In this context, pretransplant treatment with MOG may induce severe graft-versus-host disease (GVHD) in allogeneic hematopoietic stem-cell transplantation (HSCT). However, the influence of MOG on allogeneic HSCT, including its induction of GVHD, is unclear. In this report, we describe two patients treated with MOG who subsequently underwent allogeneic HSCT. They did not develop severe GVHD or treatment-related complications. In addition, we examined the kinetics of Tregs in the second case. Finally, we suggest that the detrimental effects of MOG can be avoided, which should be prospectively evaluated in future studies.

Evaluation of thrombosis-related biomarkers before and after therapy in patients with multiple myeloma

Background Thrombosis is one of the complications in the clinical course of multiple myeloma (MM). Vascular endothelial cells and/or the hemostatic-coagulatory system are thought to play an important role in thrombosis of MM. In addition to melphalan-prednisone (Mel-P) therapy, several new therapeutic drugs such as lenalidomide or bortezomib have been developed and show effectiveness against MM. However, these new drugs also have risk of therapy-related thrombosis. Methods We assessed 103 MM patients and 30 healthy controls, using enzyme-linked immunosorbent assays to evaluate five biomarkers: platelet-derived microparticles (PDMP), plasminogen activator inhibitor-1 (PAI-1), high mobility group box protein-1 (HMGB1), endothelial protein C receptor (EPCR), and soluble vascular cell adhesion molecule-1 (sVCAM-1). The effects of Mel-P, bortezomib, and lenalidomide on the plasma concentrations of these biomarkers were investigated. Results The plasma concentrations of PDMP, PAI-1, HMGB1, EPCR, and sVCAM-1 were higher in MM patients than in healthy controls. Mel-P, bortezomib, and lenalidomide therapies all reduced biomarker levels after treatment. However, when only patients with higher levels of EPCR were compared, differences were seen between the three therapies in the elevation of PDMP, HMGB1, and PAI-1. Conclusion These results suggest that both MM and therapies for MM can induce a hypercoagulable state. The elevated risk of thrombosis conferred by hypercoagulability increases patient morbidity and mortality. Attention should be paid to therapy-related thrombosis when new therapeutic regimens are selected for MM patients.

Evaluation of a biosimilar granulocyte colony-stimulating factor (filgrastim XM02) for peripheral blood stem cell mobilization and transplantation: a single center experience in Japan

Background Biosimilar granulocyte colony-stimulating factor (G-CSF) has recently been introduced into clinical practice. G-CSFs are used to mobilize CD34+ cells and accelerate engraftment after transplantation. However, in Asia, particularly in Japan, data for peripheral blood stem cell (PBSC) mobilization by this biosimilar G-CSF are currently lacking. Therefore, the clinical efficacy and safety of biosimilar G-CSF for hematopoietic stem cell transplantation needs to be evaluated in a Japanese context. Materials and methods The subjects included two groups of patients with malignant lymphoma and multiple myeloma. All patients received chemotherapy priming for the mobilization of PBSCs. All patients were treated with chemotherapy followed by the administration of either the biosimilar G-CSF, filgrastim XM02 (FBNK), or the originators, filgrastim, or lenograstim. Results There were no significant differences among FBNK, filgrastim, and lenograstim treatments in the numbers of CD34+ cells in harvested PBSCs, the scores for granulocyte/macrophage colony forming units, or for malignant lymphoma and multiple myeloma patients evaluated as separate or combined cohorts. In addition, there were no significant differences in safety, side effects, complications, or the time to engraftment after autologous hematopoietic stem cell transplantation. Conclusion Biosimilar FBNK shows the same efficacy and safety as originator G-CSFs for facilitating bone marrow recovery in Japanese malignant lymphoma and multiple myeloma patients undergoing stem cell transplantation. In addition, it is less expensive than the originators, reducing hospitalization costs.