Toshio Kitawaki

Natural Alpha Interferon-Producing Cells Respond to Human Immunodeficiency Virus Type 1 with Alpha Interferon Production and Maturation into Dendritic Cells

ABSTRACT Natural alpha interferon (IFN-α)-producing cells (IPCs) are now recognized as identical to plasmacytoid dendritic cell (DC) precursors in human blood and are thought to play an important role in antiviral immunity. In the present study, we examined the susceptibility as well as the cellular responses of IPCs to human immunodeficiency virus type 1 (HIV-1) infection. HLA-DR+ CD11c− lineage-negative cells (IPCs) were purified from peripheral blood mononuclear cells by magnetic-bead separation and cell sorting. We substantiated that IPCs expressing the major HIV-1 coreceptors, CXCR4 and CCR5, are susceptible to infection of both T-cell-line-tropic NL4-3 and macrophage-tropic JR-CSF HIV-1 by quantification of HIV-1 p24 in the culture supernatants and by provirus integration assay using human conserved Alu-HIV-1 long terminal repeat PCR. To evaluate the cellular response of IPCs to HIV-1, we examined IFN-α production and their differentiation into DCs. After incubation with either NL4-3 or JR-CSF, IPCs produced a large amount of IFN-α and at the same time underwent morphological differentiation into DCs with upregulation of CD80 and CD86. Heat inactivation of the supernatants containing HIV-1 did not affect the IFN-α production and maturation, whereas removal of virions by ultracentrifugation completely nullified both biological effects, indicating that these cellular responses do not require actual HIV-1 infection but are elicited by interaction with HIV-1 virions or certain viral components. In conclusion, these data strongly suggest that IPC can directly recognize and respond to HIV-1 with IFN-α production, which is crucial for preventing progress of HIV-1 infection and occurrence of opportunistic infection.

Depletion and impaired interferon-alpha-producing capacity of blood plasmacytoid dendritic cells in human T-cell leukaemia virus type I-infected individuals.

Dendritic cells (DCs) play an important role in innate and adaptive immunity. There are two major populations of blood DCs, myeloid DCs (myDCs) and plasmacytoid DCs (pcDCs). pcDCs are particularly important in antiviral as well as in general host defence, as they are the principal producers of type I interferons (IFNs). In this study, we analysed myDCs and pcDCs in healthy controls, human T‐cell leukaemia virus type I (HTLV‐I)‐infected asymptomatic carriers (ACs), and patients with adult T‐cell leukaemia (ATL). ATL patients had significantly decreased number of pcDCs and myDCs compared with controls. IFN‐α production by peripheral blood mononuclear cells (PBMCs) was markedly reduced in ATL patients. Purified pcDCs from ACs were found to have impaired IFN‐α‐producing capacity, suggesting a functional defect in pcDCs in HTLV‐I‐infected individuals. Interestingly, pcDCs were shown to be susceptible to HTLV‐I infection. Thus, impaired IFN‐α production by pcDCs may contribute to the immunodeficiency observed in ATL. Furthermore, IFN‐α‐producing capacity was inversely correlated with HTLV‐I proviral load in PBMCs from ACs, suggesting a role for pcDCs in maintaining the carrier state. Taken together, we hypothesize that the depletion and impaired IFN‐α‐producing capacity of blood DCs may contribute to the immunodeficiency in ATL and/or the development of ATL.

Distribution and kinetics of SR‐PSOX/CXCL16 and CXCR6 expression on human dendritic cell subsets and CD4+ T cells

Dendritic cells (DCs) coordinate T cell responses by producing T cell‐attracting chemokines and by inducing the expression of chemokine receptors on T cells. Scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR‐PSOX)/CXC chemokine ligand 16 (CXCL16) is a unique chemokine that also functions as an endocytic receptor and an adhesion molecule in its membrane‐bound form. SR‐PSOX/CXCL16 is the only known ligand of CXC chemokine receptor 6 (CXCR6) that is expressed on activated T cells and thus, may play an important role in enhancing effector functions of T cells. Here, we investigated the expression of SR‐PSOX/CXCL16 on human DC subsets and that of CXCR6 on T cell subpopulations to elucidate the dynamics of CXCL16/CXCR6 interaction in DC/T cell responses. Membrane‐bound SR‐PSOX/CXCL16 was expressed on macrophages, monocyte‐derived DCs, and blood myeloid DCs, and the expression increased after DC maturation. Myeloid antigen‐presenting cells constitutively secreted SR‐PSOX/CXCL16 for an extended period, suggesting the involvement of CXCL16 in peripheral and lymphoid tissues. Plasmacytoid DCs hardly expressed SR‐PSOX/CXCL16 on their surfaces but secreted significant amounts of SR‐PSOX/CXCL16. A subset of CD4+ effector memory T (TEM) cells constitutively expressed CXCR6, whereas central memory T cells (TCM) and naïve T cells did not. Upon stimulation with mature DCs, however, the expression of CXCR6 on TCM cells was markedly up‐regulated, whereas the expression on naïve T cells was induced only weakly. These results suggest that the interaction between SR‐PSOX/CXCL16 and CXCR6 plays an important role in enhancing TCM cell responses by mature DCs in lymphoid tissues and in augmenting TEM cell responses by macrophages in peripheral inflamed tissues.

Bortezomib suppresses function and survival of plasmacytoid dendritic cells by targeting intracellular trafficking of Toll-like receptors and endoplasmic reticulum homeostasis

Dendritic cells (DCs) play a pivotal role in the pathogenesis of inflammatory disorders, so suppressing the activity of DCs is instrumental in treating such diseases. In the present study, we show that a proteasome inhibitor, bortezomib, suppresses the survival and immunostimulatory function of human plasmacytoid DCs (pDCs) by targeting 2 critical points, intracellular trafficking of nucleic acid-sensingToll-like receptors (TLRs) and endoplasmic reticulum (ER) homeostasis. Among the immune cells in blood, pDCs were the most susceptible to the killing effect of bortezomib. This correlates with a decrease in the spliced form of a transcription factor XBP1, which rescues cells from apoptosis by maintaining ER homeostasis. Bortezomib suppressed the production of interferon-α and interleukin-6 by pDCs activated with a TLR9-stimulating CpG DNA and a TLR7-stimulating influenza virus, which appears to be partially independent of apoptosis. Bortezomib inhibited translocation of TLR9 from the ER to endolysosomes but not of an ER membrane protein, Unc93B1, that delivers TLR9 to endolysosomes. Thus, bortezomib suppresses the activity of pDCs by inhibiting intracellular trafficking of TLRs through disrupting the coordinated translocation of TLRs and Unc93B1 and by disturbing ER homeostasis. This study suggests that proteasome inhibitors may alleviate inflammatory disorders such as lupus and psoriasis that involve pDCs.

Dendritic cells that endocytosed antigen-containing IgG-liposomes elicit effective antitumor immunity.

Liposomes represent a promising vehicle to deliver exogenous antigens to dendritic cells (DCs) for tumor immunotherapy. Targeting exogenous antigens to Fcγ receptors on DCs has been shown to result in efficient presentation of antigen-derived peptides on major histocompatibility complex (MHC) class I and class II molecules. In this study, it was investigated whether DCs that endocytosed physicochemically optimized antigen-containing liposomes conjugated with IgG efficiently present antigens on MHC class I and class II molecules, and consequently induce strong antitumor immune responses. IgG-conjugated liposomes that were 200 nm in diameter without attaching polyethylene glycol were most efficiently endocytosed by DCs. Human monocyte-derived DCs that endocytosed tetanus toxoid (TT)-containing IgG liposomes via CD32 stimulated CD4+ T cells more strongly than DCs pulsed with TT-containing bare liposomes or with soluble TT. Immunization of mice with DCs that endocytosed ovalbumin (OVA)-containing IgG liposomes but not OVA-containing bare liposomes or soluble OVA completely prevented the growth of OVA-expressing lymphoma cells. Importantly, administration of DCs that endocytosed OVA-containing IgG liposomes to the mice with established OVA-expressing tumors strongly suppressed tumor growth. This study demonstrates an IgG liposome with physicochemical properties suitable for delivering antigens to DCs and paves the way to the application of IgG liposomes for tumor immunotherapy using DCs.

Cross-priming of CD8+ T cells in vivo by dendritic cells pulsed with autologous apoptotic leukemic cells in immunotherapy for elderly patients with acute myeloid leukemia

OBJECTIVE The prognosis for elderly patients with acute myeloid leukemia (AML) remains dismal. To explore the potential of immunotherapy for improving clinical outcomes for these patients, we performed a phase I clinical trial of dendritic cell (DC)-based immunotherapy for elderly patients with AML. MATERIALS AND METHODS Autologus monocytes were obtained after reducing tumor burden by chemotherapy. Immature DCs induced with granulocyte-macrophage colony-stimulating factor and interleukin-4 were pulsed with autologous apoptotic leukemic cells as antigens. DCs were administered intradermally to four patients five times at 2-week intervals. To facilitate DC migration to lymph nodes, injection sites were pretreated with killed Streptococcus pyogenes OK-432 one day before. DCs were coinjected with OK-432 to induce maturation and interleukin-12 production in vivo. RESULTS Antileukemic responses were observed by an interferon-γ enzyme-linked immunospot assay or a tetramer assay in two of four patients. In a human leukocyte antigen-A∗2402-positive patient, induction of CD8(+) T-cell responses to WT1- and human telomerase reverse transcriptase - derived peptides were observed, indicating cross-priming in vivo. The two patients with antileukemic immunity showed longer periods of disease stabilization than the other two patients. CONCLUSIONS This study demonstrates the immunogenicity of autologous DCs that cross-present leukemia-associated antigens from autologous apoptotic leukemic cells in vivo in elderly patients with AML.

Coexistent Rearrangements of c- MYC , BCL2 , and BCL6 Genes in a Diffuse Large B-Cell Lymphoma

We present a patient with stage III de novo diffuse large B-cell lymphoma. The lymphoma cells showed mature B-cell immunophenotype but lacked surface immunoglobulin (Ig) expression. Long-distance and long-distance inverse polymerase chain reaction assays to detect the oncogene/Ig gene rearrangement revealed that the cells carried 3 independent fusion genes, namely, c-MYC/Ig heavy chain gene (IgH), BCL2/IgH, and Ig λ light chain gene/BCL6.Thus, the lymphoma cells concurrently carried t(8;14)(q24;q32), t(14;18)(q32;q21), and t(3;22)(q27;q11), which developed in association with class switching, V/D/J recombination, and somatic hypermutation, respectively. The lymphoma responded to chemoradiotherapy, and the patient has been well for 2 years, suggesting that multiple oncogene rearrangements may not necessarily be associated with poor clinical outcome.

Recent Advance in Antigen-Specific Immunotherapy for Acute Myeloid Leukemia

Relapse after chemotherapy is inevitable in the majority of patients with acute myeloid leukemia (AML). Thus, it is necessary to develop novel therapies that have different antileukemic mechanisms. Recent advances in immunology and identification of promising leukemia-associated antigens open the possibilities for eradicating minimal residual diseases by antigen-specific immunotherapy after chemotherapy. Several methods have been pursued as immunotherapies for AML: peptide vaccines, granulocyte-macrophage colony-stimulating factor-secreting tumor vaccines, dendritic cell vaccines, and adoptive T cell therapy. Whereas immunogenicity and clinical outcomes are improving in these trials, severe adverse events were observed in highly avid engineered T cell therapies, indicating the importance of the balance between effectiveness and side effects in advanced immunotherapy. Such progress in inducing antitumor immune responses, together with strategies to attenuate immunosuppressive factors, will establish immunotherapy as an important armament to combat AML.

The tyrosine kinase inhibitor dasatinib suppresses cytokine production by plasmacytoid dendritic cells by targeting endosomal transport of CpG DNA

Plasmacytoid dendritic cells (pDCs) produce a vast amount of interferon (IFN)‐α in response to nucleic acids from viruses and damaged self‐cells through Toll‐like receptor (TLR)7 and TLR9. Pharmaceutical agents that suppress IFN‐α production by pDCs are instrumental in elucidating the mechanisms behind IFN‐α production, and in developing novel therapies for inflammatory disorders that involve pDCs. Here, we show that a tyrosine kinase inhibitor for chronic myeloid leukemia with multiple targets, dasatinib, strongly suppresses production of IFN‐α and proinflammatory cytokines by human pDCs stimulated with multimeric CpG oligodeoxynucleotides (CpG‐A) without reducing viability. In contrast, other tyrosine kinase inhibitors, imatinib, and nilotinib, did not suppress the cytokine production at clinically relevant concentrations. Inhibitors of SRC family kinases (SFKs), which are prominent targets of dasatinib, also suppressed the cytokine production. Notably, however, dasatinib, but not SFK inhibitors, abrogated prolonged localization of CpG‐A in early endosomes, which is a critical step for pDCs to produce a large amount of IFN‐α. This study suggests that dasatinib suppresses IFN‐α production by pDCs by inhibiting SFK‐dependent pathways and SFK‐independent endosomal retention of CpG DNA. Kinases controlling the distinctive endosomal trafficking in pDCs may be exploited as targets to develop novel therapies for pDC‐related inflammatory disorders.

Human CD1c+ Myeloid Dendritic Cells Acquire a High Level of Retinoic Acid–Producing Capacity in Response to Vitamin D3

All-trans-retinoic acid (RA) plays a critical role in maintaining immune homeostasis. Mouse intestinal CD103+ dendritic cells (DCs) produce a high level of RA by highly expressing retinal dehydrogenase (RALDH)2, an enzyme that converts retinal to RA, and induce gut-homing T cells. However, it has not been identified which subset of human DCs produce a high level of RA. In this study, we show that CD1c+ blood myeloid DCs (mDCs) but not CD141high mDCs or plasmacytoid DCs exhibited a high level of RALDH2 mRNA and aldehyde dehydrogenase (ALDH) activity in an RA- and p38-dependent manner when stimulated with 1α,25-dihydroxyvitamin D3 (VD3) in the presence of GM-CSF. The ALDH activity was abrogated by TLR ligands or TNF. CD103− rather than CD103+ human mesenteric lymph node mDCs gained ALDH activity in response to VD3. Furthermore, unlike in humans, mouse conventional DCs in the spleen and mesenteric lymph nodes gained ALDH activity in response to GM-CSF alone. RALDH2high CD1c+ mDCs stimulated naive CD4+ T cells to express gut-homing molecules and to produce Th2 cytokines in an RA-dependent manner. This study suggests that CD1c+ mDCs are a major human DC subset that produces RA in response to VD3 in the steady state. The “vitamin D – CD1c+ mDC – RA” axis may constitute an important immune component for maintaining tissue homeostasis in humans.