Blocking ADP-ribosylation of cell surface proteins during cell preparation with a Nanobody against ART2 enhances chemotaxis of T cells during in vitro migration assays. (P3388)

Abstract

Abstract ADP-ribosylation is the enzymatic transfer of the ADP-ribose moiety from NAD+ to a protein, a posttranslational modification that regulates the function of the targeted proteins. Although in normal conditions there is minimal extracellular NAD+, it is released from cells in inflammation and during preparation of primary lymphocytes. ART2 (CD296) is an ADP-ribosyltransferase expressed by most mature T cells, including NKT, and regulatory T cells. A major substrate for ART2 is P2X7, an ion channel that mediates shedding of CD62L, and NAD+-induced cell death (NICD). In this manner, NAD+ released during cell preparation affects the phenotype and survival of cells expressing ART2. In this study, we used a recombinant variable domain of the llama heavy-chain antibody S+16a to block the ADP-ribosyltransferase activity of ART2. The recombinant antibody was administered i.v. to mice and 1-2 h later, the CD4+ T cells from spleen and lymph nodes were isolated. The cell phenotype and functional state was analyzed by flow cytometry and chemotaxis assay respectively. A single dose of S+16a blocked the shedding of CD62L and reduced the number of apoptotic cells about 80%. The higher cell viability resulted in a 50-100% increase in the cell number responding to the chemokines CCL22 and CXCL12. We conclude that S+16a is a useful tool to protect mature T cells from NICD induced by the released NAD+ from cells during purification, thereby enhancing the efficiency of in vitro migration assays.