Diverse immune environments in human lung tuberculosis granulomas assessed by quantitative multiplexed immunofluorescence

The precise nature of the local immune responses in lung tuberculosis (TB) granulomas requires a comprehensive understanding of their environmental complexities. At its most basic level, a granuloma is a compact, organized immune aggregate of macrophages surrounded by myeloid, B and T cells. We established two complementary multiplex immunolabeling panels to simultaneously evaluate the myeloid and lymphocytic contexture of 14 human lung TB granulomas in formalin-fixed paraffin-embedded tissue samples. We observed diverse CD3+ and CD8+ T-cell and CD20+ B lymphocyte compositions of the granuloma immune environment and a relatively homogeneous distribution of all myeloid cells. We also found significant associations between CD8+ T-cell densities and the myeloid marker CD11b and phagocytic cell marker CD68. In addition, significantly more CD68+ macrophages and CD8+ T cells were found in Mycobacterium tuberculosis-infected granulomas, as detected by Ziehl-Neelsen staining. FOXP3 expression was predominately found in a small subset of CD4+ T cells in different granulomas. As the success or failure of each granuloma is determined by the immune response within that granuloma at a local and not a systemic level, we attempted to identify the presence of reactive T cells based on expression of the T-cell activation marker CD137 (4-1BB) and programmed cell death-1 (PD-1). Only a small fraction of the CD4+ and CD8+ T cells expressed PD-1. CD137 expression was found only in a very small fraction of the CD4+ T cells in two granulomas. Our results also showed that multinucleated giant cells showed strong PD-L1 but not CTLA-4 membrane staining. This study offers new insights into the heterogeneity of immune cell infiltration in lung TB granulomas, suggesting that each TB granuloma represents a unique immune environment that might be independently influenced by the local adaptive immune response, bacterial state, and overall host disease status.