Polyfunctionality of CD4⁺ T Lymphocytes in Buffaloes and Cattle: Comparative Antigen-Specific Cytokine Responses in Bovine Tuberculosis Infection

Susana Flores-Villalva¹Giovanna De Matteis^2*FRANCESCO GRANDONI²Maria Carmela Scata'²Anna Donniacuo³Lorena Schiavo³Giulia Franzoni⁴Piera Mazzone⁵Mahmoud Elnaggar⁶Giorgio Galiero³GIORGIO GALIERO³William Davis⁷Alessandra Martucciello³

• ¹Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal, Instituto Nacional de Investigación Forestal, Agropecuaria (INIFAP), Mexico City, México, Mexico
• ²Council for Agricultural Research and Agricultural Economy Analysis | CREA, Rome, Italy
• ³Experimental Zooprophylactic Institute of Southern Italy (IZSM), Portici, Italy
• ⁴Experimental Zooprophylactic Institute of Sardinia (IZS), Sassari, Italy
• ⁵Experimental Institute of Zooprophylaxis of Umbria and Marche (IZSUM), Perugia, Umbria, Italy
• ⁶A’Sharqiyah University, Ibra, Ash Sharqiyah North, Oman
• ⁷Washington State University, Pullman, Washington, United States

Bovine tuberculosis (BTB) is a significant disease that affects a wide range of animals, including water buffalo. Due to its zoonotic potential and economic impact, BTB is considered a disease of global importance. Although both buffaloes and cattle belong to the Bovidae family, they exhibit notable differences in their immune responses to pathogens. However, limited information is available on the immune response of buffaloes to BTB infection. This study aims to address this gap by comparing antigen-specific cytokine-expressing cells in CD4+ T lymphocyte from buffaloes and cattle. For this purpose, a multicolor flow cytometry assay has been developed. Blood samples were collected from a total of 35 buffaloes and 10 cattle, classified based on IGRA test results and herd status. Buffaloes were divided into two groups: IGRA-positive animals (n = 17) from BTB outbreak farms, and IGRA-negative animals (n = 18), including 13 from BTB outbreak farms and 5 from an Officially Tuberculosis-Free. Similarly, cattle were divided into IGRA-positive animals (n = 6) from a BTB outbreak farm and IGRA-negative animals (n = 4) from an OTF herd. Flow cytometry was employed in this study as a key technique to assess the functional immune responses in whole blood samples from both species. After six hours of in vitro stimulation with PPD-B or PBS, intracellular cytokine staining was performed to identify and quantify cytokine-producing CD4⁺ T cell subsets. This method enabled high-resolution, single-cell analysis of multiple cytokines simultaneously, including IFN-γ, TNF-α, and IL-17A. The results revealed distinct cytokine expression patterns across species and infection status. While factor analysis of mixed data showed overlapping immune response profiles between buffalo and cattle, specific CD4⁺ T cell subsets—such as those producing IL-17A⁺, IFN-γ⁺IL-17A⁺, and TNF-α⁺IL-17A⁺—contributed to species differentiation. Additionally, certain cytokine combinations, particularly IFN-γ⁺ and TNF-α⁺ producing cells, were associated with IGRA test status, distinguishing exposed or infected animals from non-infected controls. T In conclusion, our results highlight similarities in the immune response of buffaloes and cattle suggesting comparable antigen-specific cytokine expression patterns. T