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Peripheral invariant NKT cells (iNKT) and CD8 + tissue-resident memory T cells (T RM ) express high levels of the extracellular ATP receptor P2RX7 in mice. High extracellular ATP concentrations or NAD-mediated P2RX7 ribosylation by the enzyme ARTC2.2 can induce P2RX7 pore formation and cell death. Because both ATP and NAD are released during tissue preparation for analysis, cell death through these pathways may compromise the analysis of iNKT and CD8 + T RM Indeed, ARTC2.2 blockade enhanced recovery of viable liver iNKT and T RM The expression of ARTC2.2 and P2RX7 on distinct iNKT subsets and T RM is unclear, however, as is the impact of recovery from other nonlymphoid sites. In this study, we performed a comprehensive analysis of ARTC2.2 and P2RX7 expression in iNKT and CD8 + T cells in diverse tissues, at steady-state and after viral infection. NKT1 cells and CD8 + T RM express high levels of both ARTC2.2 and P2RX7 compared with NKT2, NKT17, and CD8 + circulating memory subsets. Using nanobody-mediated ARTC2.2 antagonism, we showed that ARTC2.2 blockade enhanced NKT1 and T RM recovery from nonlymphoid tissues during cell preparation. Moreover, blockade of this pathway was essential to preserve functionality, viability, and proliferation of both populations. We also showed that short-term direct P2RX7 blockade enhanced recovery of T RM , although to a lesser degree. In summary, our data show that short-term in vivo blockade of the ARTC2.2/P2RX7 axis permits much improved flow cytometry-based phenotyping and enumeration of murine iNKT and T RM from nonlymphoid tissues, and it represents a crucial step for functional studies of these populations.

ARTC2.2/P2RX7 Signaling during Cell Isolation Distorts Function and Quantification of Tissue-Resident CD8+ T Cell and Invariant NKT Subsets