Mizuki Tomihari

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses

ABSTRACT Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2+ cells after intravenous injection and in CD11c+ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8+ T-cell response in HLA-A2 transgenic mice and stimulated a CD4+ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-Ab. As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.

Gpnmb is a melanosome-associated glycoprotein that contributes to melanocyte/keratinocyte adhesion in a RGD-dependent fashion.

Abstract:  Gpnmb is a glycosylated transmembrane protein implicated in the development of glaucoma in mice and melanoma in humans. It shares significant amino acid sequence homology with the melanosome protein Pmel‐17. Its extracellular domain contains a RGD motif for binding to integrin and its intracellular domain has a putative endosomal and/or melanosomal‐sorting motif. These features led us to posit that Gpnmb is associated with melanosomes and involved in cell adhesion. We showed that human Gpnmb is expressed constitutively by melanoma cell lines, primary‐cultured melanocytes and epidermal melanocytes in situ, with most of it found intracellularly within melanosomes and to a lesser degree in lysosomes. Our newly developed monoclonal antibody revealed surface expression of Gpnmb on these pigment cells, albeit to a lesser degree than the intracellular fraction. Gpnmb expression was upregulated by UVA (but not UVB) irradiation and by α‐melanocyte‐stimulating hormone (MSH) (but not β‐MSH); its cell surface expression on melanocytes (but not on melanoma cells) was increased markedly by IFN‐γ and TNF‐α. PAM212 keratinocytes adhered to immobilized Gpnmb in a RGD‐dependent manner. These results indicate that Gpnmb is a melanosome‐associated glycoprotein that contributes to the adhesion of melanocytes with keratinocytes.

The DC-HIL/syndecan-4 pathway inhibits human allogeneic T cell responses

T‐cell activation is regulated by binding of ligands on APC to corresponding receptors on T cells. In mice, we discovered that binding of DC‐HIL on APC to syndecan‐4 (SD‐4) on activated T cells potently inhibits T‐cell activation. In humans, we now show that DC‐HIL also binds to SD‐4 on activated T cells through recognition of its heparinase‐sensitive saccharide moiety. DC‐HIL blocks anti‐CD3‐induced T‐cell responses, reducing secretion of pro‐inflammatory cytokines and blocking entry into the S phase of the cell cycle. Binding of DC‐HIL phosphorylates SD‐4s intracellular tyrosine and serine residues. Anti‐SD‐4 Ab mimics the ability of DC‐HIL to attenuate anti‐CD3 response more potently than Ab directed against other inhibitory receptors (CTLA‐4 or programmed cell death‐1). Among leukocytes, DC‐HIL is expressed highest by CD14+ monocytes and this expression can be upregulated markedly by TGF‐β. Among APC, DC‐HIL is expressed highest by epidermal Langerhans cells, an immature type of dendritic cells. Finally, the level of DC‐HIL expression on CD14+ monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF‐β reduced this capacity, whereas knocking down the DC‐HIL gene augmented it. Our findings indicate that the DC‐HIL/SD‐4 pathway can be manipulated to treat T‐cell‐driven disorders in humans.

DC-HIL/glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells.

DC-HIL/glycoprotein nmb (Gpnmb) expressed on antigen-presenting cells attenuates T-cell activation by binding to syndecan-4 (SD-4) on activated T cells. Because DC-HIL/Gpnmb is expressed abundantly by mouse and human melanoma lines, we posited that melanoma-associated DC-HIL/Gpnmb exerts similar inhibitory function on melanoma-reactive T cells. We generated small interfering RNA-transfected B16F10 melanoma cells to completely knock down DC-HIL/Gpnmb expression, with no alteration in cell morphology, melanin synthesis, or MHC class I expression. This knockdown had no effect on B16F10 proliferation in vitro or entry into the cell cycle following growth stimulation, but it markedly reduced the growth of these cells in vivo following their s.c. injection into syngeneic immunocompetent (but not immunodeficient) mice. This reduction in tumor growth was due most likely to an augmented capacity of DC-HIL-knocked down B16F10 cells (compared with controls) to activate melanoma-reactive T cells as documented in vitro and in mice. Whereas DC-HIL knockdown had no effect on susceptibility of melanoma to killing by cytotoxic T cells, blocking SD-4 function enhanced the reactivity of CD8(+) T cells to melanoma-associated antigens on parental B16F10 cells. Using an assay examining the spread to the lung following i.v. injection, DC-HIL-knocked down cells produced lung foci at similar numbers compared with that produced by control cells, but the size of the former foci was significantly smaller than the latter. We conclude that DC-HIL/Gpnmb confers upon melanoma the ability to downregulate the activation of melanoma-reactive T cells, thereby allowing melanoma to evade immunologic recognition and destruction. As such, the DC-HIL/SD-4 pathway is a potentially useful target for antimelanoma immunotherapy.

Binding of DC-HIL to dermatophytic fungi induces tyrosine phosphorylation and potentiates antigen presenting cell function.

APCs express receptors recognizing microbes and regulating immune responses by binding to corresponding ligands on immune cells. Having discovered a novel inhibitory pathway triggered by ligation of DC-HIL on APC to a heparin/heparan sulfate-like saccharide of syndecan-4 on activated T cells, we posited DC-HIL can recognize microbial pathogens in a similar manner. We showed soluble recombinant DC-HIL to bind the dermatophytes Trichophyton rubrum and Microsporum audouinii, but not several bacteria nor Candida albicans. Dermatophyte binding was inhibited completely by the addition of heparin. Because DC-HIL contains an ITAM-like intracellular sequence, we questioned whether its binding to dermatophytes can induce tyrosine phosphorylation in dendritic cells (DC). Culturing DC with T. rubrum (but not with C. albicans pseudohyphae) induced phosphorylation of DC-HIL, but not when the tyrosine residue of the ITAM-like sequence was mutated to phenylalanine. To examine the functional significance of such signaling on DC, we cross-linked DC-HIL with mAb (surrogate ligand), which not only induced tyrosine phosphorylation but also up-regulated expression of 23 genes among 662 genes analyzed by gene-array, including genes for profilin-1, myristoylated alanine rich protein kinase C substrate like-1, C/EBP, LOX-1, IL-1β, and TNF-α. This cross-linking also up-regulated expression of the activation markers CD80/CD86 and heightened APC capacity of DC to activate syngeneic T cells. Our findings support a dual role for DC-HIL: inhibition of adaptive immunity following ligation of syndecan-4 on activated T cells and induction of innate immunity against dermatophytic fungi.

The DC‐HIL ligand syndecan‐4 is a negative regulator of T‐cell allo‐reactivity responsible for graft‐versus‐host disease

Acute graft‐versus‐host disease (GVHD) is the most important cause of mortality after allogeneic haematopoietic stem cell transplantation. Allo‐reactive T cells are the major mediators of GVHD and the process is regulated by positive and negative regulators on antigen‐presenting cells (APC). Because the significance of negative regulators in GVHD pathogenesis is not fully understood, and having discovered that syndecan‐4 (SD‐4) on effector T cells mediates the inhibitory function of DC‐HIL on APC, we proposed that SD‐4 negatively regulates the T‐cell response to allo‐stimulation in acute GVHD, using SD‐4 knockout mice. Although not different from their wild‐type counterparts in responsiveness to anti‐CD3 stimulation, SD‐4−/− T cells lost the capacity to mediate the inhibitory function of DC‐HIL and were hyper‐reactive to allogeneic APC. Moreover, infusion of SD‐4−/− T cells into sub‐lethally γ‐irradiated allogeneic mice worsened mortality, with hyper‐proliferation of infused T cells in recipients. Although there my be little or no involvement of regulatory T cells in this model because SD‐4 deletion had no deleterious effect on T‐cell‐suppressive activity compared with SD‐4+/+ regulatory T cells. We conclude that SD‐4, as the T‐cell ligand of DC‐HIL, is a potent inhibitor of allo‐reactive T cells responsible for GVHD and a potentially useful target for treating this disease.

Depleting Syndecan-4 + T Lymphocytes Using Toxin-Bearing Dendritic Cell-Associated Heparan Sulfate Proteoglycan-Dependent Integrin Ligand: A New Opportunity for Treating Activated T Cell-Driven Disease

Because syndecan-4 (SD-4) is expressed by some (but not all) T cells following activation and serves as the exclusive ligand of dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL), we envisioned the DC-HIL/SD-4 pathway to be a therapeutic target for conditions mediated by selectively activated T cells. We conjugated soluble DC-HIL receptor with the toxin saporin (SAP; DC-HIL-SAP) and showed it to bind activated (but not resting) T cells and become internalized by and deplete SD-4+ T cells. In hapten-sensitized mice, DC-HIL-SAP injected i.v. prior to hapten challenge led to markedly suppressed contact hypersensitivity responses that lasted 3 wk and were restricted to the hapten to which the mice were originally sensitized. Such suppression was not observed when DC-HIL-SAP was applied during sensitization. Moreover, the same infusion of DC-HIL-SAP produced almost complete disappearance of SD-4+ cells in haptenated skin and a 40% reduction of such cells within draining lymph nodes. Our results provide a strong rationale for exploring use of toxin-conjugated DC-HIL to treat activated T cell-driven disease in humans.

Influence of transfusion of lymphokine-activated T killer cells on inflammatory responses in dogs after laparotomy

The influence of transfusion of lymphokine-activated T killer cells (T-LAK) on inflammatory responses was examined in dogs after laparotomy. Plasma C-reactive protein (CRP) level, cell numbers of peripheral blood lymphocytes (PBLs) and T lymphocyte subsets (CD3+, CD4+ and CD8+) and mRNA expression levels of cytokines including interleukin (IL)-2, IL-12, IL-4, IL-10 and transforming growth factor (TGF)-β in peripheral blood mononuclear cells (PBMCs) were measured in dogs with (T-LAK group) or without (control group) a single T-LAK administration immediately after laparotomy. The plasma CRP level initially increased and then decreased to the normal range at 7 days after laparotomy in the T-LAK group, which was earlier than in the control group. The expression level of IL-10 mRNA showed a marked postoperative increase and was significantly higher than the preoperative level on day 7 (P<0.05), whereas the level in the control group showed no clear change after laparotomy. A significant increase in IL-2 mRNA expression level in the T-LAK group was observed on day 14, which was two weeks earlier than in the control group (P<0.05). These results suggest that T-LAK therapy in dogs after laparotomy leads to earlier resolution of postoperative inflammation by production of an anti-inflammatory cytokine (IL-10) in the early phase of the postoperative period and earlier restoration of cell-mediated immunity related to cytokine production by PBMCs.

A comparison of the immunological effects of propofol and isoflurane for maintenance of anesthesia in healthy dogs.

Most anesthetics have an immuno-suppressive effect on cellular and neurohumoral immunity, and research shows that total intravenous anesthesia (TIVA) with propofol has a greater immuno-protective effect than inhalational anesthesia in human medicine. However, in veterinary clinics, these effects remain ambiguous. To clarify the details, we focused on propofol and isoflurane, investigating clinical blood hematology and immunological profiles drawn from healthy dogs under and after two anesthesia techniques. Twelve healthy adult beagles were included in this study, randomly assigned to the propofol anesthesia group (group P: n=6) or the isoflurane anesthesia group (group I: n=6). In both groups, the number of lymphocytes in peripheral blood decreased after 2 hr of anesthesia (2 hr), but group P showed significantly less decrease than group I. For T-lymphocyte subsets examined by flowcytometry, the ratio of CD3+, CD4+ and CD8+ lymphocytes in the peripheral blood mononuclear cell (PBMC) of group P at 2 hr also exhibited a high level compared to group I. Moreover, for mRNA expression of cytokines measured by real-time PCR, the IL2 (pro-inflammatory cytokine) of group P showed no decrease like group I. The IL10 (anti-inflammatory cytokine) of group P also showed no increase like group I, while both cytokines maintained nearly the same level until 2 hr. These results suggest that, compared to propofol, isoflurane had more strongly immuno-suppression caused by anesthesia, and propofol itself might have some immuno-protective effects. Thus, TIVA with propofol might benefit immunological support in the perioperative period of dogs.

Gene and protein expression of a soluble form of CTLA-4 in a healthy dog

Cytotoxic T lymphocyte associated gene-4 (CTLA-4) is a costimulatory molecule, expressed on the surface of activated T cells that negatively regulates T cell activation. In humans, alternative splicing of the CTLA-4 gene generates two major isoforms of mRNA, and a soluble form of CTLA-4 (sCTLA-4) was detected in normal human serum. We describe alternatively spliced mRNA expressed in peripheral blood mononuclear cells obtained from a healthy dog lacking the transmembrane domain coded by exon 3 of the CTLA-4 gene. Immunoprecipitation and western blotting of dog serum revealed a band of approximately 23-kDa, which is consistent with the predicted size, based on the amino acid sequence of the canine sCTLA-4 obtained in this study.