Kyoichi Tamura

Mutations in the fifth immunoglobulin‐like domain of kit are common and potentially sensitive to imatinib mesylate in feline mast cell tumours

The purpose of the current study was to investigate the mutation status of KIT in feline mast cell tumours (MCTs) and to examine the effects of tyrosine kinase inhibition on the phosphorylation of mutant kit in vitro and in clinical cases of cats. Sequence analysis of KIT identified mutations in 42/62 MCTs (67·7%). The vast majority of the mutations were distributed in exons 8 and 9, both of which encode the fifth immunoglobulin‐like domain (IgD) of kit. All five types of kit with a mutation in the fifth IgD were then expressed in 293 cells and examined for phosphorylation status. The mutant kit proteins showed ligand‐independent phosphorylation. The tyrosine kinase inhibitor imatinib mesylate suppressed the phosphorylation of these mutant kit proteins in transfectant cells. In a clinical study of 10 cats with MCTs, beneficial response to imatinib mesylate was observed in 7/8 cats that had a mutation in the fifth IgD of kit in tumour cells. Mutations in the fifth IgD of kit thus appear to be common and potentially sensitive to imatinib mesylate in feline MCTs. These data provide an in vivo model for paediatric mastocytosis where mutations in the fifth IgD of kit also occur.

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.

The DC-HIL/Syndecan-4 Pathway Regulates Autoimmune Responses through Myeloid-Derived Suppressor Cells

Having discovered that the dendritic cell (DC)–associated heparan sulfate proteoglycan–dependent integrin ligand (DC-HIL) receptor on APCs inhibits T cell activation by binding to syndecan-4 (SD-4) on T cells, we hypothesized that the DC-HIL/SD-4 pathway may regulate autoimmune responses. Using experimental autoimmune encephalomyelitis (EAE) as a disease model, we noted an increase in SD-4+ T cells in lymphoid organs of wild-type (WT) mice immunized for EAE. The autoimmune disease was also more severely induced (clinically, histologically, and immunophenotypically) in mice knocked out for SD-4 compared with WT cohorts. Moreover, infusion of SD-4−/− naive T cells during EAE induction into Rag2−/− mice also led to increased severity of EAE in these animals. Similar to SD-4 on T cells, DC-HIL expression was upregulated on myeloid cells during EAE induction, with CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) as the most expanded population and most potent T cell suppressor among the myeloid cells examined. The critical role of DC-HIL was supported by DC-HIL gene deletion or anti–DC-HIL treatment, which abrogated T cell suppressor activity of MDSCs, and also by DC-HIL activation inducing MDSC expression of IFN-γ, NO, and reactive oxygen species. Akin to SD-4−/− mice, DC-HIL−/− mice manifested exacerbated EAE. Adoptive transfer of MDSCs from EAE-affected WT mice into DC-HIL−/− mice reduced EAE severity to the level of EAE-immunized WT mice, an outcome that was precluded by depleting DC-HIL+ cells from the infused MDSC preparation. Our findings indicate that the DC-HIL/SD-4 pathway regulates autoimmune responses by mediating the T cell suppressor function of MDSCs.

DC-HIL-expressing myelomonocytic cells are critical promoters of melanoma growth

A major barrier to successful cancer immunotherapy is the tumor’s ability to induce T-cell tolerance by exploiting host regulatory mechanisms. Having discovered the DC-HIL receptor, which inhibits T-cell responses by binding to syndecan-4 on effector T-cells, we posited the DC-HIL/syndecan-4 pathway to play an important role in cancer promotion. Among DC-HIL+ myelomonocytic cells, during growth of implanted mouse melanoma, CD11b+Gr1+ cells were the most expanded population and the most potent at suppressing T-cell activation. Deletion of the DC-HIL gene or infusion of anti-DC-HIL mAb abrogated these cells’ suppressor function and expansion, and markedly diminished melanoma growth and metastasis. IL-1β and IFN-γ were elevated in mice bearing melanoma, and concurrent exposure to both cytokines optimally induced DC-HIL expression by tumor-infiltrating CD11b+Gr1+ cells. Ligation of DCHIL transduced phosphorylation of its intracellular immunoreceptor tyrosine-based activation motif (ITAM), that in turn induced intracellular expression of IFN-γ and inducible nitric oxide synthase (iNOS), known to mediate T-cell suppression by CD11b+Gr1+ cells. Thus DC-HIL is the critical mediator of these cells’ suppressor function in melanoma-bearing mice and a potential target for improving melanoma immunotherapy.

Immunophenotyping and Gene Rearrangement Analysis in Dogs with Lymphoproliferative Disorders Characterized by Small-Cell Lymphocytosis

Lymphocytosis caused by neoplastic proliferation of small lymphocytes is occasionally difficult to distinguish by morphological examination from nonneoplastic lymphocytosis. To examine the clinical utility of gene rearrangement analysis for demonstrating neoplastic proliferation of small lymphocytes, gene rearrangement analysis was performed in comparison with immunophenotyping using peripheral lymphocytes in dogs with small lymphocytosis. Thirty-one dogs with small-cell lymphocytosis (8,100–884,300/μl) were enrolled. By immunophenotyping, lymphocytosis of all dogs was suggested to be neoplastic in nature based on the detection of marked expansion of phenotypically homogeneous lymphocytes or the presence of an aberrant antigen-expressing population of lymphocytes. In contrast, gene rearrangement analysis represented clonality in 27 dogs (detection rate of 87%). From the present study, gene rearrangement analysis was considered to be worthwhile to strengthen the evidence of neoplastic proliferation of small lymphocytes when coupled with immunophenotyping and to be a suitable diagnostic substitute if immunophenotyping is not available in clinical practice.

Neuronal expression of keratinocyte-associated transmembrane protein-4, KCT-4, in mouse brain and its up-regulation by neurite outgrowth of Neuro-2a cells.

One group of proteins that regulates neurite outgrowth and maintains neuronal networks is the immunoglobulin superfamily (IgSF). We previously identified a new member of the IgSF, keratinocyte-associated transmembrane protein-4 (KCT-4), by the signal sequence-trap method from primary cultured human keratinocytes. The KCT-4 mRNA has been found to be highly expressed in the adult human brain, although it is also distributed in various tissues. In the present study, to gain insight into the role of KCT-4 in the nervous system, we examined the expression profile and localization of KCT-4 mRNA in mouse brain. We also evaluated changes in KCT-4 mRNA expression in the differentiation of the neuroblastoma cell line Neuro-2a as the in vitro model of neurite outgrowth. KCT-4 mRNA was detected broadly in various regions of the adult mouse brain by RT-PCR. In situ hybridization revealed that it was expressed highly selectively by neurons but not by glial cells. Moreover, expression of KCT-4 mRNA was induced by neurite outgrowth of Neuro-2a. These data suggest that KCT-4 participates in the regulation of neurite outgrowth and maintenance of the neural network in the adult brain.

Light-Chain Multiple Myeloma in a Cat

A diagnosis of light-chain multiple myeloma was made in an 11-year-old male American Shorthair cat. The cat showed atypical plasma cell infiltration in the bone marrow, biclonal gammopathy caused by polymerization of myeloma protein (M-protein), and Bence-Jones proteinuria. The M-protein in the serum of the cat was analyzed by using 12% sodium dodeyl sulfate (SDS) polyacrylamide gel electrophoresis with Coomassie brilliant blue staining. An intense band with a size of 27 kDa, the size of the immunoglobulin light chain, was clearly observed, whereas the band corresponding to the immunoglobulin heavy chain (59 kDa) was undetectable. The 27-kDa band was confirmed to be an immunoglobulin light chain by Western blotting by using antibodies for feline immunoglobulin. These data suggested that the neoplastic plasma cells produce light chain only, leading to the diagnosis of light-chain multiple myeloma in the cat.

DC-HIL+ CD14+ HLA-DR no/low cells are a potential blood marker and therapeutic target for melanoma.

Melanoma growth and metastasis depend on a battle between the cancer’s invasive properties and the host’s capacity to counter such attributes. Immunosuppression is a potent promoter of cancer progression that not only counters host control of tumor spread but also prevents anti-cancer treatments from achieving their full benefit (Ilkovitch and Lopez, 2008). Because CD11b+Gr1+ cells are most potent at suppressing T-cell function (Frey, 2006), their exponential proliferation in cancer patients severely limits efficacy of immunotherapy (Diaz-Montero et al., 2009).

Molecular Characterization of the Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) Gene Associated with the Feline AB Blood Group System

Cat’s AB blood group system (blood types A, B, and AB) is of major importance in feline transfusion medicine. Type A and type B antigens are Neu5Gc and Neu5Ac, respectively, and the enzyme CMAH participating in the synthesis of Neu5Gc from Neu5Ac is associated with this cat blood group system. Rare type AB erythrocytes express both Neu5Gc and Neu5Ac. Cat serum contains naturally occurring antibodies against antigens occurring in the other blood types. To understand the molecular genetic basis of this blood group system, we investigated the distribution of AB blood group antigens, CMAH gene structure, mutation, diplotypes, and haplotypes of the cat CMAH genes. Blood-typing revealed that 734 of the cats analyzed type A (95.1%), 38 cats were type B (4.9%), and none were type AB. A family of three Ragdoll cats including two type AB cats and one type A was also used in this study. CMAH sequence analyses showed that the CMAH protein was generated from two mRNA isoforms differing in exon 1. Analyses of the nucleotide sequences of the 16 exons including the coding region of CMAH examined in the 34 type B cats and in the family of type AB cats carried the CMAH variants, and revealed multiple novel diplotypes comprising several polymorphisms. Haplotype inference, which was focused on non-synonymous SNPs revealed that eight haplotypes carried one to four mutations in CMAH, and all cats with type B (n = 34) and AB (n = 2) blood carried two alleles derived from the mutated CMAH gene. These results suggested that double haploids selected from multiple recessive alleles in the cat CMAH loci were highly associated with the expression of the Neu5Ac on erythrocyte membrane in types B and AB of the feline AB blood group system.

Molecular changes associated with the development of resistance to imatinib in an imatinib-sensitive canine neoplastic mast cell line carrying a KIT c.1523A>T mutation.

Although imatinib has therapeutic activity for certain subsets of patients with mastocytosis, it is not always curative. Here, molecular mechanisms that confer imatinib resistance to neoplastic mast cells were investigated using an imatinib‐sensitive canine neoplastic mast cell line VI‐MC carrying a KIT c.1523A>T activating mutation. Two imatinib‐resistant sublines were established by culturing VI‐MC cells in increasing concentrations of imatinib (1 μM resistant, rVI‐MC1; 10 μM resistant, rVI‐MC10). Both sublines had a second KIT mutation c.2443G>C. Recombinant KIT with the second mutation was insensitive to 1 μM but sensitive to 10 μM imatinib. The effect of imatinib on the phosphorylation of KIT and its downstream signalling proteins was then examined using these sublines. KIT and ERK were constitutively phosphorylated in both sublines, and their phosphorylation was suppressed by 10 μM imatinib in rVI‐MC1 cells. However, KIT but not ERK phosphorylation was suppressed in rVI‐MC10 cells. The phosphorylation of ERK in rVI‐MC10 cells was also not diminished by the Src family kinase (SFK) inhibitor dasatinib. This second mutation in KIT may play an important role in imatinib resistance in neoplastic mast cells. Furthermore, KIT/SFK‐independent activation of ERK would be involved in imatinib resistance when the neoplastic cells are exposed to higher concentrations of imatinib.