IFN-γ and TNF-α producing CD4+ T-cells in the blood after Mycoplasma hyosynoviae challenge of vaccinated pigs

In a vaccine trial against Mycoplasma hyosynoviae infection, pigs were vaccinated with formalin fixed whole-cell-antigen formulated with adjuvant DDA/TDB (SSI). Placebo pigs received adjuvant with saline. Vaccinations were performed at five and eight weeks of age, followed by an intranasal M. hyosynoviae challenge inoculation three weeks later. Vaccination induced both antibodies and a cell-mediated immune response (CMI) in vaccinated pigs compared to placebo pigs as shown by M. hyosynoviae antigen (Ag) specific IFN-γ response in an IL-18 potentiated whole-blood IFN-γ stimulation assay (mean IFN-γ level 1936 pg/ml vs. 82 pg/ml (p=0.0001)). A central memory T cell phenotype with polyfunctional capacity to produce all three cytokines IFN-γ, TNF-α and IL-2 has recently been linked to development of vaccine induced protection in several infections. In a subset of seven vaccinated pigs and four placebo pigs the cytokine production before (day -1), and after (day 15) challenge inoculation was therefore further characterized by flow cytometry. Briefly, PBMC cultures were incubated with Ag, PBS or staphylococcus enterotoxin B (SEB) in the presence of recombinant porcine IL-18 (50 ng/ml). Cultured cells were stained with mAbs against CD4 and CD8, followed by intracellular staining for IFN-γ, TNF-α and IL-2. Between 50,000-100,000 CD4+ lymphocytes were acquired on a FACSCanto II and subsets of CD4+ cells producing combinations of cytokines were measured. For comparison of cytokine responses, the integrated mean fluorescence intensity (iMFI) was calculated. Anti-IL-2 staining showed high background, which makes it difficult to perform reliable identification of the polyfunctional T-cells. Despite this high level of cells apparently producing IL-2, cells producing IL-2 in combination with IFN-γ or TNF-α were rare. However, CD4+ cells producing IFN-γ or TNF-α after Ag-stimulation were detected in vaccinated pigs, and increased IFN-γ level (iMFI) in cells co-producing IFN-γ and TNF-α was more pronounced in vaccinated pigs compared to placebo pigs in response to M. hyosynoviae challenge (day 15 p.i). Polyfunctional cells simultaneously producing IFN-γ, TNF-α and IL-2 were not identified in this trial. Nor did the vaccine show complete protection against M. hyosynoviae infection. Identification of Ag-specific CMI response with quantification of cells co-producing IFN-γ and TNF-α may contribute in a positive manner in evaluation of the vaccine against M. hyosynoviae.