Neonatal innate TLR-mediated responses are distinct from those of adults.

The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from TLRs and other environmental sensors. We set out to provide a comprehensive analysis defining differences in response to TLR ligation between human neonates and adults. In response to most TLR ligands, neonatal innate immune cells, including monocytes and conventional and plasmacytoid dendritic cells produced less IL-12p70 and IFN-alpha (and consequently induced less IFN-gamma), moderately less TNF-alpha, but as much or even more IL-1beta, IL-6, IL-23, and IL-10 than adult cells. At the single-cell level, neonatal innate cells generally were less capable of producing multiple cytokines simultaneously, i.e., were less polyfunctional. Overall, our data suggest a robust if not enhanced capacity of the neonate vs the adult white-blood cell TLR-mediated response to support Th17- and Th2-type immunity, which promotes defense against extracellular pathogens, but a reduced capacity to support Th1-type responses, which promote defense against intracellular pathogens.

Identification of B cells through negative gating-An example of the MIFlowCyt standard applied.

Polychromatic flow cytometric analysis takes advantage of the increasing number of available fluorophores to positively identify and simultaneously assess multiple parameters in the same cell (1). Additional parameters may be analyzed through negative identification (i.e., through exclusion of particular stains or antibodies employed). In this report, we tested whether such negative-gating strategy would identify human B lymphocytes in innate immune phenotyping studies. To this end, B cells were identified as the negatively-stained subpopulation from the CD123 vs. CD11c plot of the CD14(neg-low), MHC II(high) human peripheral blood mononuclear cells. To test the specificity of this negative gating approach, we confirmed that negatively gated B cells indeed expressed CD19, the bona fide marker for human B cells. However, a small number of unidentified cells were contained in the negatively-gated B cells. Furthermore, a small percentage cells expressing markers used to identify monocytes and myeloid dendritic cells (mDC) coexpressed CD19. This identifies such negative B-cell gating approach as potentially problematic. When applied to the analysis of Toll-like receptors (TLR) stimulation experiments, we were however able to interpret the results, as B-cells respond to TLR stimulation in a qualitative different pattern as compared to monocytes and DC. This report is presented in a manner that is fully compliant with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, which was recently adopted by the International Society for Advancement of Cytometry (ISAC) (2) and incorporated in the publishing policies of Cytometry and other journals. We demonstrate how a MIFlowCyt-compliant report can be prepared with minimal effort, and yet provide the reader with a much clearer picture of the portrayed FCM experiment and data.