Characterization of lung infection–induced TCRγδ T cell phenotypes by CyTOF mass cytometry

Abstract

T cell receptor γδ cells are known to be the primary effector T cells involved in the response to bacterial infections, yet their phenotypic characteristics are not as well established as other T cell subsets. In this study, we used cytometry by time-of-flight mass cytometry to better characterize the phenotypic response of T cell receptor γδ cells to Streptococcus pneumoniae lung infection. Mice were infected, and cells from lung washouts, spleen, and lymph nodes were stained to detect cell-surface, intracellular, and signaling markers. We observed that infection caused a significant increase in T cell receptor γδ cells, which expressed high interferon-γ and interleukin-17A levels. Profiling T cell receptor γδ cells by cytometry by time-of-flight revealed that activated γδ T cells uniquely coexpressed cell-surface Gr-1, cluster of differentiation 14, and cluster of differentiation 274 (programmed death-ligand 1). Further classification of Gr-1 expression patterns on T cell receptor γδ cells demonstrated that Gr-1(+) T cell receptor γδ cells were the primary source of interferon-γ, whereas Gr-1(-) cells mostly expressed interleukin-17A. Gr-1(+) T cell receptor γδ cells also showed higher ζ-chain-associated protein kinase 70, p38, and 4eBP1 signaling in response to infection as compared with Gr-1(-) T cell receptor γδ cells. Taken together, Gr-1 expression patterns on γδ T cells in the lung provide a robust marker to differentiate interferon-γ- and interleukin-17A-producing subsets involved in the early immune response to bacterial pneumonia.

Lung‐resident γδ T cells and their roles in lung diseases

“…Other studies showed that increased γδ T cells in the lungs after S. pneumoniae infection expressed high levels of IFN‐ γ and IL‐17A. Activated γδ T cells uniquely co‐express Gr‐1, CD14 and CD274 (PD‐L1), and Gr‐1 expression patterns on lung γδ T cells provide a marker to differentiate IFN‐ γ (Gr‐1 + )‐ and IL‐17A (Gr‐1 − ) ‐producing subsets . It was also found that the resolution of S. pneumoniae ‐induced lung inflammation was associated with a > 30‐fold increase in γδ T‐cell number, which decreased the number of pulmonary dendritic cells and alveolar macrophages …”

Characterization of pulmonary immune responses to hyperoxia by high-dimensional mass cytometry analyses

“…cannula was inserted into trachea via neck incision. Lungs were lavaged three times with 1 mL of phosphate buffered saline, fluid collected, and cell suspension obtained, as previously described 45 . Cells were cryopreserved as described above for subsequent CyTOF antibody staining.…”

The contrasting roles of Th17 immunity in human health and disease

“…Advantages of this platform offers include tracking kinetics of cellular trafficking and studying interactions between many phenotypically and diverse immune cell subsets . For example, a recent study using CyTOF found that identification of a unique γδ T cell subset expressing Ly‐6G myeloid differentiation antigen enabled distinguishing between IL‐17A‐ and IFN‐γ‐producing cells in the lungs of S. pneumoniae infected mice .…”