The SFU count seen with co-culture of infected CKC with infected splenocytes was close to that seen with cells from infected birds stimulated with PMA/ionomycin (1060 ± 53 SPU/106 cells), suggesting that antigen specific antiviral IFNγ producing cells constitute the majority of those able to rapidly produce IFNγ. It was interesting to note that splenocytes from infected birds have greater SFU responses to PMA in our study (discussed below). To analyze the phenotype of the responding splenocytes from infected birds we performed intracellular

staining on cells from co-culture assays. We first validated antibody (EH9) against a previously published anti IFNγ antibody (mAb80, (Ariaans et al., 2008)) using IFNγ transfected CHO cell lines (Supplementary Fig. 4) and in splenocytes stimulated with PMA/ionomycin (Fig. 4A). There was BMS-354825 solubility dmso no statistically significant difference between results obtained with the two antibodies. Non-specific signal was not detected by isotype control staining (Fig. 4B). We then analyzed the phenotype of IFNγ expressing cells from infected birds, following co-culture with either infected or non-infected CKC. Data shown are for a representative sample from infected and non-infected birds (Fig. 4C) gating in the same FSC/SSC lymphocyte region (Fig. 4A) for all conditions. The greatest number of interferon gamma producing

cells was detected during co-culture of infected CKC with splenocytes from infected birds (0.517%), compared with splenocytes from infected birds co-cultured with non-infected CKC (0.069%), and splenocytes ABT-199 purchase from non-infected birds co-cultured with infected CKC (0.071%). It is important to note that the majority of IFNγ positive splenocytes from infected birds co-cultured with infected CKC were CD8 positive (> 60%, Fig. 4C). Having established the utility of the

co-culture ELISpot we used the technique to analyze influenza antigen specific responses in birds vaccinated (prime and boost) with recombinant Fowlpox (F9) or recombinant Fowlpox-NpM1 (F9-NpM1), and then challenged with an influenza virus with heterologous nucleoprotein and matrix protein. Instead of infecting the CKC with influenza virus we used recombinant MVA carrying either a GFP or NpM1 fusion transgene (homologous to second the Fowlpox recombinant) then irradiated the infected CKC as described. Three of the four F9-NpM1 vaccinated birds challenged with influenza showed IFNγ responses that distinguished them from F9 vaccinated and challenged birds (Fig. 5) (40.0 ± 12.5 vs. 3.0 ± 1.9, p < 0.05). The majority of responses in the F9-NpM1 vaccinated birds were greater with CKC infected with MVA-NpM1 fusion transgene. Some responses were also observed with F9-NpM1 vaccinated birds when APCs were infected with MVA-GFP (although this result was not significant).