Safety analysis of ex vivo-expanded canine natural killer cells in a xenogeneic mouse model of graft-versus-host disease.

Canine natural killer (NK) cells are large, granular lymphocytes that are neither B lymphocytes nor T lymphocytes. However, it has been reported that canine NK cells share some of the phenotypic characteristics of T lymphocytes, such as CD3 and CD5. Studies are needed to assess the safety of canine NK cells for immunotherapy, especially because the safety of using allogeneic NK cells as an immunotherapy for dogs has yet to be shown. In this study, the safety of cultured canine NK cells was assessed using a xenogeneic mouse model of graft-versus-host disease (GVHD). Mice were injected with either canine peripheral blood mononuclear cells (PBMCs) or cultured NK cells for 2 or 3 weeks. Data were then collected on changes in mice body weights, disease severity scores, and survival rates. Histopathological and immunohistochemical evaluations were also performed. All mice injected with canine PBMCs died within 45 days after injection. Severe clinical signs were caused by GVHD. The histopathological and immunohistochemical evaluations showed that mice injected with canine PBMCs had multiple lesions, including necrosis in their lungs, livers, kidneys, and stomachs, and the injected cells were present around the lesions. By contrast, no mice injected with cultured NK cells without removing the CD3+ TCR- cells exhibited any clinical abnormalities. Moreover, they all survived the 90-day experimental period without exhibiting any histopathological changes. Accordingly, the results of this study suggest that canine NK cells do not cause significant side effects such as GVHD and allogeneic NK cells can safely be used for cancer immunotherapy in dogs.

Safety and immunological effects of recombinant canine IL-15 in dogs.

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays pivotal roles in innate and adaptive immunity. It is also a promising cytokine for treating cancer. Despite growing interest in its use as an immunotherapeutic, its safety and immunological effects in dogs have not been reported. In this study, healthy dogs were given recombinant canine IL-15 (rcIL-15) intravenously at a daily dose of 20 µg/kg for 8 days and monitored for 32 days to determine the safety and immunological effects of rcIL-15. The repeated administration of rcIL-15 was well tolerated, did not cause any serious side effects, and promoted the selective proliferation and activation of canine anti-cancer effector cells, including CD3+CD8+ cytotoxic T lymphocytes, CD3+CD5dimCD21-, and non-B/non-T NK cell populations, without stimulating Treg lymphocytes. The rcIL-15 injections also stimulated the expression of molecules and transcription factors associated with the activation and effector functions of NK cells, including CD16, NKG2D, NKp30, NKp44, NKp46, perforin, granzyme B, Ly49, T-bet, and Eomes. These results suggest that rcIL-15 might be a valuable therapeutic adjuvant to improve immunity against cancer in dogs.

Canine non-B, non-T NK lymphocytes have a potential antibody-dependent cellular cytotoxicity function against antibody-coated tumor cells.

BACKGROUND: The antibody-dependent cellular cytotoxicity (ADCC) is a cell-mediated immune defense mechanism in which effector immune cells actively lyse antibody-coated target cells. The ADCC of tumor cells is employed in the treatment of various cancers overexpressing unique antigens, and only natural killer (NK) cells are known to be major effectors of antibody mediated ADCC activity. Canine NK cells are still defined as non-B, non-T large granular lymphocytes because of the lack of information regarding the NK cell-restricted specific marker in dogs, and it has never been demonstrated that canine NK cells have ADCC ability against tumor cells. In the present study, we investigated whether canine non-B, non-T NK cells have ADCC ability against target antibody-coated tumor cells, using cetuximab and trastuzumab, the only human antibodies reported binding to canine cancer cells. RESULTS: Activated canine non-B, non-T NK cells (CD3-CD21-CD5-TCRαß-TCRγδ-) for 13~17 days ex vivo showed ADCC ability against trastuzumab- or cetuximab-coated target tumor cells expressing various levels of human epidermal growth factor receptor 2 (HER-2) and epidermal growth factor receptor (EGFR). Trastuzumab and cetuximab induced significant ADCC responses of canine NK cells even in CMT-U334 and CF41.Mg cells expressing low levels of HER-2 and/or EGFR, as well as in SKBR3 and DU145 cells overexpressing HER-2 and/or EGFR. The trastuzumab-mediated ADCC activity of NK cells was significantly enhanced by treatment with rcIL-21. CONCLUSIONS: The results of this study suggest that canine non-B, non-T NK lymphocytes have a potential ADCC function and that combinational strategies of monoclonal antibodies with either cytokines, which activate NK cells in vivo, or adoptive transfer of NK cells may be a feasible method for amplifying the efficacy of immunotherapy against malignant cancers even with very low expression of target molecules in dogs.

Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3+CD5dimCD21- Cytotoxic Large Granular Lymphocytes.

Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation, and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3-CD21-) large granular lymphocytes. However, a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes, and that CD3+CD5dimCD21- lymphocytes are putative canine NK cells. Based on our previous reports, we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study, we investigated the phenotypic and functional differences between CD3+CD5dimCD21- (cytotoxic large granular lymphocytes) and CD3-CD5-CD21- NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3+CD5dimCD21- lymphocytes can be differentiated into non-B, non-T NK (CD3-CD5-CD21-TCRαß-TCRγδ-GranzymeB+) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3+CD5dimCD21- cells showed that CD3-CD5-CD21- cells are derived from CD3+CD5dimCD21- cells through phenotypic modulation. CD3+CD5dimCD21- cells share more NK cell functional characteristics compared with CD3-CD5-CD21- cells, including the expression of T-box transcription factors (Eomes, T-bet), the production of granzyme B and interferon-γ, and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion, the results of this study suggest that CD3+CD5dimCD21- and CD3-CD5-CD21- cells both contain a subset of putative NK cells, and the difference between the two populations may be due to the degree of maturation.