Establishment of a BRAF V595E-mutant canine prostate cancer cell line and the antitumor effects of MEK inhibitors against canine prostate cancer.

Canine prostate cancer (cPCa) is a malignant neoplasm with no effective therapy. The BRAF V595E mutation, corresponding to the human BRAF V600E mutation, is found frequently in cPCa. Activating BRAF mutations are recognized as oncogenic drivers, and blockade of MAPK/ERK phosphorylation may be an effective therapeutic target against BRAF-mutated tumours. The aim of this study was to establish a novel cPCa cell line and to clarify the antitumor effects of MEK inhibitors on cPCa in vitro and in vivo. We established the novel CHP-2 cPCa cell line that was derived from the prostatic tissue of a cPCa patient. Sequencing of the canine BRAF gene in two cPCa cell lines revealed the presence of the BRAF V595E mutation. MEK inhibitors (trametinib, cobimetinib and mirdametinib) strongly suppressed cell proliferation in vitro, and trametinib showed the highest efficacy against cPCa cells with minimal cytotoxicity to non-cancer COPK cells. Furthermore, we orally administered 0.3 or 1.0 mg/kg trametinib to CHP-2 xenografted mice and examined its antitumor effects in vivo. Trametinib reduced tumour volume, decreased phosphorylated ERK levels, and lowered Ki-67 expression in xenografts in a dose-dependent manner. Although no clear adverse events were observed with administration, trametinib-treated xenografts showed osteogenesis that was independent of dosage. Our results indicate that trametinib induces cell cycle arrest by inhibiting ERK activation, resulting in cPCa tumour regression in a dose-dependent manner. MEK inhibitors, in addition to BRAF inhibitors, may be a targeted agent option for cPCa with the BRAF V595E mutation.

Pulmonary solid adenocarcinoma in a dog.

A 12-year-old castrated male Beagle dog presented with a 1-month history of progressive loss of appetite and cough. One month after the initial visit, a detailed clinical examination was performed due to weight loss and persistent cough. Computed tomography demonstrated diffuse opacification of the entire right lung and cranial lobe of the left lung. Samples of the pulmonary lesions obtained by fine-needle aspiration (FNA) were highly cellular with scattered and clustered foci of large round cells, suggestive of a round cell tumour. Ten days after the FNA, the dog was euthanized due to decreased activity and severe respiratory symptoms. At necropsy, enlargement of the entire right lung and cranial lobe of the left lung was seen. The external and cut surfaces of the lungs were homogeneously grey-white. Histopathological examination of sections of the right lung and the cranial lobe of the left lung revealed proliferation of large round or polygonal neoplastic cells arranged in nests of variable size separated by a thin fibrous stroma. Neoplastic cells were immunopositive for cytokeratin and thyroid transcription factor-1 but negative for vimentin, CD204, chromogranin A and synaptophysin. On the basis of these findings, the tumour was diagnosed as pulmonary solid adenocarcinoma.

A feline case of multiple myeloma treated with bortezomib.

BACKGROUND: Multiple myeloma (MM) is an uncommon neoplasm in cats. There is no established standard of treatment due to the rare occurrence of this disease in cats. Bortezomib is a proteasome inhibitor that serves as the first-line drug for MM in humans, but its effectiveness currently is unknown in feline MM. We present here the case report of a feline MM that exhibited a favorable response to bortezomib. CASE PRESENTATION: The case was an 11-year-old non-castrated male domestic cat with light-chain MM presenting with clinical symptoms (anorexia, fatigue, and vomiting), mild azotemia, and pancytopenia. The cat failed on melphalan with prednisolone (MP), so bortezomib (Velcade) was initiated on Day 88. A total of 6 cycles of the treatment was performed, with each treatment cycle consisting of twice-weekly subcutaneous administration for 2 weeks followed by a 1-week rest. The dose of bortezomib was 0.7 mg/m2 for first week and 1.0 mg/m2 for second week in the first cycle. A dose of 0.7 mg/m2 was used for subsequent cycles. Prednisolone was used concomitantly in the first 2 cycles. Following treatment with bortezomib, clinical symptoms disappeared and a decrease in serum globulin and recovery of pancytopenia were noted. A monoclonal gammopathy, overproduction of serum immunoglobulin light chain, and Bence-Jones proteinuria that existed at diagnosis were undetectable on Day 123. A monoclonal gammopathy also was not detectable at the end of the bortezomib treatment (Day 213). Anorexia, fatigue, and marked bone marrow toxicity were experienced when bortezomib was administrated at a dose of 1.0 mg/m2, while no recognizable toxicity was observed at a dose of 0.7 mg/m2 throughout the treatment period. The case was placed on follow-up and there was no evidence of relapse as of Day 243. CONCLUSIONS: Bortezomib was effective and durable for the treatment of this case of feline MM after failure with MP. Bortezomib was well-tolerated in this cat at a dose of 0.7 mg/m2, but not at 1.0 mg/m2. Bortezomib appears to be a drug worthy of further study for the treatment of feline MM.

Molecular characterization of canine SHP2 mutants and anti-tumour effect of SHP2 inhibitor, SHP099, in a xenograft mouse model of canine histiocytic sarcoma.

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic neoplasm. Mutations in src homology 2 domain-containing phosphatase 2 (SHP2; encoded by PTPN11), which recently have been identified in canine HS tumour cells, could be attractive therapeutic targets for SHP099, an allosteric inhibitor of SHP2. Here, molecular characteristics of wild-type SHP2 and four SHP2 mutants (p.Ala72Gly, p.Glu76Gln, p.Glu76Ala and p.Gly503Val), including one that was newly identified in the present study, were investigated. Furthermore, in vivo effects of SHP099 on a HS cell line carrying SHP2 p.Glu76Ala were examined using a xenograft mouse model. While SHP2 Glu76 mutant cell lines and SHP2 wild-type/Gly503 mutant cell lines are highly susceptible and non-susceptible to SHP099, respectively, a cell line carrying the newly identified SHP2 p.Ala72Gly mutation exhibited moderate susceptibility to SHP099. Among recombinant wild-type protein and four mutant SHP2 proteins, three mutants (SHP2 p.Ala72Gly, p.Glu76Gln, p.Glu76Ala) were constitutively activated, while no activity was detected in wild-type SHP2 and SHP2 p.Gly503Val. Activities of these constitutively activated proteins were suppressed by SHP099; in particular, Glu76 mutants were highly sensitive. In the xenograft mouse model, SHP099 showed anti-tumour activity against a SHP2 p.Glu76Ala mutant cell line. Thus, there was heterogeneity in molecular characteristics among SHP2 mutants. SHP2 p.Glu76Ala and perhaps p.Glu76Gln, but not wild-type SHP2 or SHP2 p.Gly503Val, were considered to be oncogenic drivers targetable with SHP099 in canine HS. Further studies will be needed to elucidate the potential of SHP2 p.Ala72Gly as a therapeutic target of SHP099 in canine HS.

A canine case of malignant melanoma carrying a KIT c.1725_1733del mutation treated with toceranib: a case report and in vitro analysis.

BACKGROUND: Canine malignant melanoma is highly aggressive and generally chemoresistant. Toceranib is a kinase inhibitor drug that inhibits several tyrosine kinases including the proto-oncogene receptor tyrosine kinase KIT. Although canine malignant melanoma cells often express KIT, a therapeutic effect for toceranib has yet to be reported for this tumor, with only a small number of patients studied to date. This is a case report of a dog with malignant melanoma that experienced a transient response to toceranib. Furthermore, the KIT expressed in the tumor of this case was examined using molecular analysis. CASE PRESENTATION: A Shiba Inu dog presented with a gingival malignant melanoma extending into surrounding structures with metastasis to a submandibular lymph node. The dog was treated with toceranib (Palladia®; 2.6-2.9 mg/kg, orally, every other day) alone. Improvement of tumor-associated clinical signs (e.g., halitosis, tumor hemorrhage, trismus, and facial edema) with reduced size of the metastatic lymph node was observed on Day 15. The gingival tumor and associated masses in the masseter and pterygoid muscles decreased in size by Day 29 of treatment. Toceranib treatment was terminated on Day 43 due to disease progression and the dog died on Day 54. The tumor of this dog had a novel deletion mutation c.1725_1733del within KIT and the mutation caused ligand-independent phosphorylation of KIT, which was suppressed by toceranib. This mutation was considered to be an oncogenic driver mutation in the tumor of this dog, thereby explaining the anti-tumor activity of toceranib. CONCLUSIONS: This is the first report that presents a canine case of malignant melanoma that responded to toceranib therapy. KIT encoded by KIT harboring a mutation c.1725_1733del is a potential therapeutic target for toceranib in canine malignant melanoma. Further investigation of the KIT mutation status and toceranib therapy in canine malignant melanoma will need to be undertaken.

Commitment toward cell death by activation of autophagy with survivin inhibitor YM155 in two canine squamous cell carcinoma cell lines with high expression of survivin.

Canine squamous cell carcinoma (SCC) is difficult to treat if local therapy is not feasible. Recently, survivin inhibitor YM155 was shown to have growth inhibitory activity on high-survivin-expressing canine SCC cell lines HAPPY and SQ4. Here, the mechanisms underlying the effect of YM155 on these cell lines were investigated. YM155 induced cleavage of poly(ADP-ribose) polymerase (PARP) in HAPPY, but not in SQ4 cells. Analyzing two autophagy markers, the level of microtubule-associated protein 1 light chain 3 (LC3)-II and the LC3-II/LC3-I ratio, indicated that YM155 activates autophagy in both cell lines, and this activation occurs prior to PARP cleavage in HAPPY cells. Moreover, inhibition of autophagic flux by chloroquine almost completely prevented the toxic effect of YM155 in both cell lines. Although there are differences in their eventual cell death type, both cell lines may be committed to cell death by activation of autophagy with YM155. Activation of autophagy is likely to be a key mechanism in the growth-inhibitory effects of YM155 in these lines. These data provide new insights into the cytotoxic mechanism of YM155 in canine SCC cells.

Nimustine Treatment of 11 Cases of Canine Histiocytic Sarcoma.

The objective of this retrospective study was to report treatment outcomes in dogs with histiocytic sarcoma (HS) that were treated with nimustine (ACNU). This study evaluated data from 11 dogs including 5 with macroscopic tumors that were treated in the primary setting and 6 that underwent aggressive local therapy while being treated in the adjuvant setting. The median ACNU starting dose was 25 mg/m2 (range, 20-30 mg/m2; 3- to 5-wk intervals, 1-8 administrations). The median overall survival in the primary and adjuvant settings was 120 days (median progression-free survival [PFS], 63 days) and 400 days (median PFS, 212 days), respectively. Neutropenia was observed in eight cases (grade 1, n = 1; grade 2, n = 2; grade 3, n = 2; grade 4, n = 3) with nadir neutrophil count at 1 wk after ACNU administration. Mild gastrointestinal toxicity (grade 1-2) was observed in three cases. ACNU was well tolerated and showed a similar outcome to that seen for lomustine, which is a drug commonly used to treat canine HS, in terms of overall survival and PFS in the current study population. Further investigations will need to be undertaken to definitively determine if ACNU is an appropriate alternative to lomustine for the treatment of HS.

Big Insulin-like Growth Factor 2-Producing Tumor in a Hypoglycemic Dog.

A 10-year-old female Papillon dog that had previously developed a mammary tumor was admitted for treatment of a hypoglycemic attack. Blood examination showed severe hypoglycemia and decreased blood insulin concentration. Computed tomography indicated multiple tumors in the cranial and caudal lobes of the right lung. These tumors were resected surgically and diagnosed as pulmonary adenocarcinomas by histopathologic examination. Hypoglycemia was temporarily improved after the resection, but a hypoglycemic event occurred 2 months after the surgery. Immunohistochemistry of the tumor demonstrated the expression of insulin-like growth factor 2 in tumor cells. Western blot analysis revealed the expression of high-molecular-weight (big)-insulin-like growth factor 2 in the tumor region. Insulin-like growth factor 2 mRNA expression was also confirmed in the tumor using reverse transcription-polymerase chain reaction. These findings indicate the diagnosis of non-islet cell tumor-induced hypoglycemia caused by big-insulin-like growth factor 2 produced by the tumor in the dog. This report provides information on differentiating tumors that cause paraneoplastic hypoglycemia.

Canine histiocytic sarcoma cell lines with SHP2 p.Glu76Gln or p.Glu76Ala mutations are sensitive to allosteric SHP2 inhibitor SHP099.

Some canine cases of histiocytic sarcoma (HS) carry an activating mutation in the src homology two domain-containing phosphatase 2 (SHP2) encoded by PTPN11. SHP099 is an allosteric inhibitor of SHP2 that stabilizes SHP2 in a folded, auto-inhibited conformation. Here, we examined the expression and mutation status of SHP2 in five canine HS cell lines and evaluated the growth inhibitory properties of SHP099 against these cell lines. All five of the canine HS cell lines expressed SHP2, with three of the lines each harbouring a distinct mutation in PTPN11/SHP2 (p.Glu76Gln, p.Glu76Ala and p.Gly503Val). In silico analysis suggested that p.Glu76Gln and p.Glu76Ala, but not p.Gly503Val, promote shifting of the SHP2 conformation from folded to open-active state. SHP099 potently suppressed the growth of two of the mutant cell lines (harbouring SHP2 p.Glu76Gln or p.Glu76Ala) but not that of the other three cell lines. In addition, SHP099 suppressed ERK activation in the cell line harbouring the SHP2 p.Glu76Ala mutation. The SHP2 p.Glu76Gln and p.Glu76Ala mutations are considered to be activating mutations, and the signal from SHP2 p.Glu76Ala is inferred to be transduced primarily via the ERK pathway. Moreover, SHP099-sensitive HS cells, including those with SHP2 p.Glu76Gln or p.Glu76Ala mutations, may depend on these mutations for growth. Therefore, targeting cells harbouring SHP2 p.Glu76Gln and p.Glu76Ala with SHP099 may be an approach for the treatment of canine HS.

Genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib in a canine mast cell tumor cell line.

One of the potential mechanisms underlying acquired resistance to toceranib in canine mast cell tumor (MCT) is the emergence of a secondary mutation in the KIT gene. Here, genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib were investigated in the toceranib-susceptible canine MCT cell line VI-MC, which carries a KIT-activating mutation resulting in a predicted p.(Asn508Ile) amino acid change in the receptor tyrosine kinase protein KIT. Two sublines were cloned from VI-MC and toceranib-resistant sublines then were established by continuous exposure to toceranib. The mutation status of KIT in parental VI-MC and its sublines was investigated using next-generation sequencing (NGS). Additionally, effects of secondary mutations on toceranib sensitivity in p.(Asn508Ile)-mutant KIT were examined. KIT secondary mutations, including those encoding p.(Asn679Lys)-, p.(Asp819Val)-, and p.(Asp819Gly)-mutant KIT, that confer toceranib insensitivity to p.(Asn508Ile)-mutant KIT emerged only in toceranib-resistant VI-MCs. These mutations were not detected by NGS in the parental VI-MC line or in the toceranib-naive cloned VI-MCs, although the parental line and sublines exhibited genetic heterogeneity in KIT that may have been caused by genetic evolution during clonal expansion. VI-MC clones with these secondary mutations in KIT appear to have arisen from subclones during treatment with toceranib rather than being pre-existing. However, further study using a higher resolution technique will be needed to confirm the developmental mechanism of KIT secondary mutation in canine MCT cells with acquired resistance to toceranib.

Establishment and characterization of a cell line from a feline histiocytic sarcoma.

Feline histiocytic sarcoma (HS) is an aggressive and uncommon tumor originating from dendritic cells/macrophages. Here, a feline HS cell line, FHS-1, was established from a case of feline HS and characterized. Immunohistochemically, FHS-1 cells were positive for vimentin and Iba-1, and negative for MHC class II and CD163. FHS-1 cells were positive for α-naphthyl butyrate esterase staining, which was clearly inhibited by sodium fluoride. FHS-1 cells had phagocytic and antigen uptake/processing activities. Moreover, FHS-1 cells were tested for susceptibility to feline infectious peritonitis virus (FIPV) strain 79-1146; however, this cell line was not susceptible to this viral strain. Although FHS-1 cells lost the expression of MHC class II and CD163, our findings indicate that FHS-1 is a feline HS cell line that retains functional properties of dendritic cells/macrophages in terms of phagocytic and antigen uptake/processing activities. While FHS-1 cells are not suitable for in vitro study of FIP using strain 79-1146, they may be applicable for studies aimed at developing new diagnostic and therapeutic strategies for feline HS.

Melanoma-Derived Soluble DC-HIL/GPNMB Promotes Metastasis by Excluding T-Lymphocytes from the Pre-Metastatic Niches.

Soluble factors from the primary tumor induce recruitment of bone marrow-derived progenitors to form tumor-supportive microenvironments or pre-metastatic niches in distal organs before metastasis. How tumor-secreted factors condition the sites for tumor progression remains ambiguous. B16 melanoma produces the secreted form of T cell-inhibitory DC-HIL (sDC-HIL) that travels to distal organs and potentiates the metastatic capacity of tumor cells. We studied the molecular mechanisms and found that sDC-HIL binds to select endothelial cells that co-localize with the sites where bone marrow-derived progenitors and tumor cells migrate. sDC-HIL-bound endothelial cells exist at a similar frequency in mice with or without tumors, and they are strongly associated with survival of intravenously injected tumor cells in the lung. sDC-HIL binding conferred T-cell suppressor function on the ECs and awakened the angiogenic property by inducing vascular endothelial growth factor expression, resulting in enhanced transendothelial migration of bone marrow-derived progenitors and tumor cells, but not for T cells. This selectivity is achieved by the T-cell binding of sDC-HIL, which prevents formation of the leading edges required for chemotaxis. Finally, inducing tumor expression of sDC-HIL significantly reduced tumor-infiltrated T cells. Therefore, the highly metastatic attribute of B16 melanoma can be explained by the endothelial gatekeeper function of sDC-HIL that limits lymphocyte transmigration to pre-metastatic niches.

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re­treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

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.

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 have 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-1ß 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 DC-HIL transduced phosphorylation of its intracellular immunoreceptor tyrosine-based activation motif, which 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.

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.

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.

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.