1. Introduction The importance of dendritic cells (DCs) as antigen-presenting cells has been reported by numerous studies since the first characterization by Steinman [1-4]. DCs are characterized by the absence of lineage markers for T cells, B cells, monocyte/macrophage, and natural killer cells [5, 6], the abundant expression of major histocompatibility complex class II molecules (MHC II) [7], and the ability to prime naïve T cells [8]. Recently, the importance of DCs has been evident in the regulation of immunity against various pathogens in bovine, including Salmonella typhimurium [9,10], Mycobacterium avium subspecies paratuberculosis [11], and bovine respiratory syncytial virus [12]. Nevertheless, it is challenging to purify from tissue or blood due to their low frequency and the lack of monoclonal antibodies (mAbs) that detect molecules exclusively expressed on DCs. Therefore, the development of mAbs that can be used to detect or purify all subsets of DCs from blood or tissue will improve our opportunities to study of the functional activity of bovine DCs. Aim of the study Describe the development and characterization of a set of mAbs (LND25A, LND41A, DC5A, and DC77A, all IgG1 isotype) that react with molecules expressed on DC. Conclusion The mAbs detect bovine dendritic cells (loosely adherent, numerous dendrites, and CD14-MHCII+CD11c+) and subsets of CD2+WC1- and CD2-WC1+ γδ T cells.→The mAbs should facilitate further characterization and function of bovine DCs. Materials and Methods 1. Enrichment of DCs Freshly isolated PBMCs were incubated for two hours in full RPMI. Non-adherent cells were discarded, and cells that adhered on the plate were incubated at 37˚C in CO2. After overnight incubation, non adherent cells were collected and transferred to a new plate. After an additional hour of incubation, the remaining non adherent cells were collected and transferred to a new plate and cultured for 2 days. 2. Cell culture conditions Freshly isolated PBMCs and the cultured non adherent cells were resuspended in full RPMI, and cultured at 37˚C in CO2 for 6 days and 2 days, respectively. Non-adherent cells collected after one hour incubation were cultured with full RPMI in a large plate at 37˚C in CO2 for 2 days. 3. Flow cytometery The mAbs used in this study are listed below. Freshly isolated peripheral blood mononuclear cells and Non adherent cells were labeled the mAbs that detect molecules used to identify DCs. The cells were incubated with primary mAbs and secondary mAbs for 15 minutes each on ice. The cells were washed three times after the first incubation and twice after the second incubation. After labeling, the cells were fixed with 2% formaldehyde. The labeled cells were collected using a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA) and analyzed using FCS Express (De Novo Software, Ontario, Canada). 4. Microscopy A BH-2 phase-contrast microscope (Olympus, Center Valley, PA) was used to examine the morphology of loosely adherent twice-released cells. The pictures were taken at the magnification of 1,000X. References [1] Metlay JP, Pure E, Steinman RM. Control of the immune response at the level of antigen-presenting cells: a comparison of the function of dendritic cells and B lymphocytes. Adv Immunol 1989;47:45-116. [2] Steinman RM. Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation. Mt Sinai J Med 2001;68:160-6. [3] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245-52. [4] Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 1973;137:1142-62. [5] Zhuang Y, Mwangi W, Brown WC, Davis WC, Hope JC, Palmer GH. Characterization of a phenotypically unique population of CD13+ dendritic cells resident in the spleen. Clin Vaccine Immunol 2006;13:1064-9. [6] Miyazawa K, Aso H, Honda M, et al. Identification of bovine dendritic cell phenotype from bovine peripheral blood. Res Vet Sci 2006;81:40-5. [7] Brooks CF, Moore M. Differential MHC class II expression on human peripheral blood monocytes and dendritic cells. Immunology 1988;63:303-11. [8] Inaba K, Metlay JP, Crowley MT, Witmer-Pack M, Steinman RM. Dendritic cells as antigen presenting cells in vivo. Int Rev Immunol 1990;6:197-206. [9] Norimatsu M, Harris J, Chance V, Dougan G, Howard CJ, Villarreal-Ramos B. Differential response of bovine monocyte-derived macrophages and dendritic cells to infection with Salmonella typhimurium in a low-dose model in vitro. Immunology 2003;108:55-61. [10] Vremec D, Pooley J, Hochrein H, Wu L, Shortman K. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol 2000;164:2978-86. [11] Lei L, Hostetter JM. Limited phenotypic and functional maturation of bovine monocyte-derived dendritic cells following Mycobacterium avium subspecies paratuberculosis infection in vitro. Vet Immunol Immunopathol 2007;120:177-86. [12] Werling D, Collins RA, Taylor G, Howard CJ. Cytokine responses of bovine dendritic cells and T cells following exposure to live or inactivated bovine respiratory syncytial virus. J Leukoc Biol 2002;72:297-304. LND25A L N D 4 1 A 337 555 FSC S S C 937 DC77A D C 5 A 241 534 Fig 2. Representative dot plots of 6-day cultured cells labeled with LND41A, LND25A, DC5A, and DC77A. An electronic gate was used to isolate large cells in preparations of PBMCs for analysis. The sharp diagonal pattern of labeling in upper right quadrants shows that the mAbs being compared recognize antigenic determinants present on the same molecule. The frequency of each population is indicated by the numbers in the upper-right quadrant. S S C LND41A 1 99 Fig 1. Representative dot plots of freshly isolated PBMCs. Freshly isolated PBMCs were labeled with LND41A. The frequency of each population is indicated by the numbers in the upper-right quadrant. S S C FSC 00 75 25 L N D 4 1 A CD14 25 81 12 L N D 4 1 A CD11c 50 88 7 L N D 4 1 A MHC II 40 87 9 Fig 3. Representative dot plots of non-adherent cells labeled with LND41A and the mAbs specific for molecules characterizing DCs. An electronic gate was used to isolate large cells for analysis. After 2 days of culture, non adherent cells were stained with LND41A and anti-CD14, anti-MHC-II and anti-CD11c. The frequency of each population is indicated by the numbers in the upper-right quadrant. Fig 4. Representative dot plots of 6-day cultured cells. After 6 days of culture, cells were labeled with LND41A, anti-TCR-δ, anti-CD2, and anti-WC1. An electronic gate was used to isolate LND41A+TCR δ+ for analysis. The frequency of each population is indicated by the numbers in the upper- right quadrant. L N D 4 1 A TCRδ 10 7821 C D 2 WC1 1113 51 25 Fig 5. Representative pictures of the loosely adherent cells with numerous dendrites at 1,000x. The morphology of loosely adherent non- adherent cells was examined under a phase- contrast microscope.  The flow cytometric analysis revealed that LND41A detected less than 1% of PBMCs (Fig. 1).  Cross comparison of specificity of the mAbs using Zenon IgG1 step reagents conjugated with different fluorochromes showed the mAbs recognize 2 different molecules, one recognized by LND25A and LND41A; the other recognized by DC5A and DC77A (Fig. 2).  In vitro studies with cultures of adherent cells derived from PBMC revealed LND41A labeled a population of loosely adherent cells with the phenotypic features of DC. These cells did not express CD14+ population but did co-express MHC II and CD11c+ (Fig. 3)  Comparative studies with LND41A and TCRδ showed that subsets of CD2+WC1-, CD2-WC1+, and CD2+WC1+ γδ T cells co-expressed the molecule detected with LND41A (Fig. 4).  Preliminary use of LND41A to identify DC present in the ileum of cows at the clinical stage of Johne’s disease showed that the mAb recognizes a population that is distinct from macrophages.  Further studies are in progress to show the mAbs recognize molecules expressed on all DC. Acknowledgement Special thanks to C. Davitt (The Franceschi Microscopy and Imaging Center) for advice and assistance in microscopy. Characterization of monoclonal antibodies specific for molecules uniquely expressed on bovine dendritic cells Abdellrazeq* 1, S. Tomida 2, and W. C. Davis 2 1 Alexandria University,Edfina, Behara Province, Egypt, 2 Washington State University, Pullman, WA,USA