Supplementary Material for: Potential Role of Gr-1⁺ CD8⁺ T Lymphocytes as a Source of Interferon-γ and M1/M2 Polarization during the Acute Phase of Murine <b><i>Legionella pneumophila</i></b> Pneumonia

In this study, we analyzed interferon (IFN)-γ-producing cells and M1/M2 macrophage polarization in <i>Legionella pneumophila</i> pneumonia following anti-Gr-1 antibody treatment. Anti-Gr-1 treatment induced an M1-to-M2 shift of macrophage subtypes in the lungs and weakly in the peripheral blood, which was associated with increased mortality in legionella-infected mice. CD8⁺ T lymphocytes and natural killer cells were the dominant sources of IFN-γ in the acute phase, and anti-Gr-1 treatment reduced the number of IFN-γ-producing CD8⁺ T lymphocytes. In the CD3-gated population, most Gr-1-positive cells were CD8⁺ T lymphocytes in the lungs and lymph nodes (LNs) of infected mice. Additionally, the number of IFN-γ-producing Gr-1⁺ CD8⁺ T lymphocytes in the lungs and LNs increased 2 and 4 days after <i>L. pneumophila</i> infection, with anti-Gr-1 treatment attenuating these populations. Antibody staining revealed that Gr-1⁺ CD8⁺ T lymphocytes were Ly6C-positive cells rather than Ly6G, a phenotype regarded as memory type cells. Furthermore, the adoptive transfer of Gr-1⁺ CD8⁺ T lymphocytes induced increases in IFN-γ, M1 shifting and reduced bacterial number in the <i>Legionella</i> pneumonia model. These data identified Ly6C⁺ CD8⁺ T lymphocytes as a source of IFN-γ in innate immunity and partially associated with reduced IFN-γ production, M2 polarization, and high mortality in anti-Gr-1 antibody-treated mice with <i>L. pneumophila</i> pneumonia