In some experiments, Vγ9Vδ2+ T cells were preincubated with anti-TCR Vg9 (clone 7A5; Pierce Endogen) or anti-NKG2D blocking mAbs (clone 149810; R&D Systems) before being added to 51Cr-labeled target cells. Intracellular expression of cytotoxic granules was investigated by intracellular staining and flow cytometry on the same effector cells, using PE-conjugated anti-Granzyme B (clone FGB12, Invitrogen), anti-Granzyme A (clone CB9, BD Biosciences), and anti-perforin (dG9, Ancell) mAbs. Data
were analyzed by GraphPad Prism Software 5.0 (GraphPad Software Inc.) using Mann–Whitney test. A p value of less than 0.05 was considered significant. GSI-IX manufacturer This work was supported by grants from Associazione Italiana Ricerca sul Cancro (A.I.R.C.) Milano, Neratinib Italy (grant number 4014 to I.A.), from Finanziamento Ricerca Corrente, Ministero della Salute, anno 2011 and Progetto Strategico Oncologico 2006 rif070701. The authors declare no financial or commercial conflict of interest. “
“Patrolling Ly6C− monocytes are blood-circulating cells that play a role in inflammation
and in the defense against pathogens. Here, we show that similar to natural killer (NK) cells, patrolling monocytes express high levels of S1PR5, a G-coupled receptor for sphingosine-1 phosphate. We found that S1pr5−/− mice lack peripheral Ly6C− monocytes but have a normal number of these cells in the bone marrow (BM). Various lines of evidence exclude a direct contribution of S1PR5 in the survival of Ly6C− monocytes at the periphery. Rather, our data support a role for S1PR5 in the egress of Ly6C− monocytes from the BM. In particular, we observed a reduced frequency of patrolling monocytes in BM sinusoids of S1PR5 KO mice. Unexpectedly, S1P was not a chemoattractant for patrolling monocytes and had no significant effect on their viability in vitro. Moreover, the disruption of S1P gradients in vivo did not alter Ly6C− monocyte trafficking and viability. These data suggest that S1PR5
regulates the trafficking of monocytes via a mechanism independent of S1P gradients. Blood monocytes are bone marrow (BM) derived phagocytic cells that play an important role in innate immunity against different classes of pathogens [1]. Human and mouse monocytes have been subdivided into at least two subsets on the basis of expression of CD14 and CD16 (human) and Ly6C (mouse) and several functional, migratory old [2] and transcriptomic [3-5] parameters. Mouse Ly6C+ monocytes are classical inflammatory monocytes, equivalent to human CD14+ CD16− monocytes, as recently confirmed by gene profiling experiments [4, 5]. They are rapidly recruited to inflamed tissues in response to CC chemokine Receptor 2 (CCR2) [6] or CCR6 [7] ligands. During infection by various pathogens (intracellular bacteria, parasites, or viruses), they differentiate into TNF/iNOS producing dendritic cells (Tip-DCs) that produce large amounts of TNF-α, reactive oxygen species, and nitric oxide [8].