Dendritic cells are the most potent antigen-presenting cells, and play a key role in the regulation of the immune system since they are the only cells capable of priming naive T cells. Their potency in antigen presentation has led several investigators to use them as vaccine adjuvants in therapy against tumors.

Since dogs are considered as a very interesting experimental model for immune-mediated diseases, graft rejections and cancers, a better characterization of canine dendritic cells (caDC) is required. caDC can be derived from monocytes (Mo) in the presence of canine GM-CSF (caGM-CSF) and canine IL-4 (caIL-4) in a 7 day culture. To date, no specific marker of caMo-DC has been described in contrast to human Mo-DC, for which several markers are available for characterizing the different subsets of DC and the different steps of differentiation and maturation.

The first part of our study consisted in the development of an elutriation technique to obtain large quantities of pure canine monocytes. Canine peripheral blood mononuclear cells were isolated from whole blood by Ficoll gradient, then separated by an elutriation process. We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64%±7.9 when labeled with anti-CD14 antibody. This purity increased to 83%±2.2 by the use of magnetic anti-CD14 microbeads after elutriation. Cell viability was more than 95% and apoptosis was less than 10%. The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR).

In the second part, we demonstrated that caMo-DC were labeled with three anti-human costimulatory molecule CD86 (FUN-1, BU63 and IT2.2 clones), while resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by caIL-4 and was upregulated during the differentiation of the caMo-DC, with a maximum at day 7. Furthermore, caMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 monoclonal antibody (mAb) was able to completely block the caMo-DC-induced proliferation in MLR. We also observed that caMo-DC highly expressed MHC class II and CD32, but we failed to determine their maturation state due to the lack of commercially available canine markers.

In the third part, we investigated the expression of toll-like receptor 3 (TLR3), which was shown to be specifically expressed in human DC. TLR is a family of functionally important receptors for recognition of pathogen-associated molecular pattern, since they trigger the pro-inflammatory response and upregulation of costimulatory molecules. We demonstrated the cross-reactivity of three TLR3 mAb (619F7, 722E2 and 713E4 clones, Dendritics, Lyon, France) towards canine PBMC and caMo-DC. Using flow cytometry, TLR3 expression was low to moderate in caMo and lymphocytes depending on the anti-TLR3 clone used, with the 722E2 clone remaining always more intense. After culture of caMo in the presence of caGM-CSF and caIL-4 for 7 days, the non-adherent caMo-DC obtained strongly expressed TLR3 with the 3 anti-TLR3 clones. These results slightly differ from those in human, where TLR3 was shown to be exclusively expressed in DC but absent in precursor monocytes, by means of total RNA extraction and northern blot analysis.

Indeed, the caMo-DC we generated in the presence of caGM-CSF and caIL-4 could have already initiated their maturation since they were found to express CD86 and to be competent to stimulate lymphocyte proliferation in MLR, but we do not have enough criteria, such as CD83 and the DC-Lamp in humans, to define if these caMo-DC are mature or immature.

At least this first approach of canine TLR3 protein expression could be useful to investigate canine innate immune defence and its role in adaptative immunity. However, since there is a lack of canine specific markers, these results will contribute to a better characterization of canine dendritic cells, and could perhaps advance the use of caMo-DC in immunotherapy.

To date, canine DC have been studied as models for graft rejection and for their role in the presentation of minor histocompatibility antigens. Canine DC were also produced from CD34+ bone marrow progenitor cells and generated from PBMC from dogs with oral malignant melanoma. Recently, bone marrow-derived DC were used to vaccinate dogs with stage I and III oral melanoma after surgical excision and treatment with radiation therapy (Gyorffy et al., 2005). Among the 3 dogs receiving 3 subcutaneous vaccinations over a 4-month period, one dog has displayed no clinical signs of recurrent melanoma 48 months after initial DC injection, and another relapsed 22 months after vaccination. Although this therapeutic approach warrant some additional investigation, ex vivo DC expansion seems feasible for immunotherapy of spontaneous cancers in outbred dogs.

References

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