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Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with  M. bovis . Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ + IL-2 + TNF-α +  and IFN-γ +  TNF-α +  response in naturally infected cattle. These multifunctional CD4 T cells express a CD44 hi CD45RO + CD62L lo  T-effector memory (T EM ) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced ‘quality of response’ as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T EM  cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

Development of an Antibody to Bovine IL-2 Reveals Multifunctional CD4 TEM Cells in Cattle Naturally Infected with Bovine Tuberculosis