T cell contamination in flow cytometry gating approaches for analysis of innate lymphoid cells

Innate lymphoid cells (ILCs) differ from T and B cells as they do not express genetically rearranged antigen receptors. The most prominent member of this group, NK cells, can be identified by numerous surface receptors such as natural cytotoxicity receptors (NCRs). However, novel groups of ILCs have recently been described and classified based on fate-determining transcription factors and cytokines being produced, similarly to T helper cells. Due to the lack of exclusive markers, ILCs are primarily defined by the paucity of lineage markers. Using RORc-fate-mapping mice, we found that the common lineage exclusion using CD3 yields an ILC population containing a large proportion of T cells with recombined TCR loci and low expression of CD3. Thus, we suggest adding CD5 as a marker for thorough elimination of T cells to avoid erroneous interpretations of ILC function in immunity

CD5⁺ positive cells within the ILC3 gate express high levels of <i>TRAC</i>.

<p>Normalized fold expression for <i>TRAC</i> and <i>TRDC</i> comparing CD5⁺ and CD5⁻ cells within the ILC3 gate (“CD5⁺ ILC3s”, pregated on CD45⁺ B220⁻ CD11b⁻ CD3⁻ TCRβ⁻ YFP⁺ cells), αβ and γδ T cells, monocytes (Mono) purified from <i>RORc</i>-eYFP mice and ILC3s derived from <i>RORc</i>-eYFP <i>RAG1</i>^−/− mice (ILC3s <i>RAG1</i>^−/−). Results are presented relative to those obtained from ILC3s of <i>RORc</i>-eYFP <i>RAG1</i>^−/− mice (mean +/− s.e.m.).</p

Flow cytometric analysis of ILC3s using <i>RORc</i>-eYFP fatemap mice.

<p>(a) Gating strategy for ILC3s in <i>RORc</i>-eYFP mice: after exclusion of doublets, live CD45⁺ B220⁻ CD3⁻ TCRβ⁻ YFP⁺ cells were selected. (b) The population gated in panel a contains a significant fraction of CD5⁺ cells both in spleen, lung and colon. (c) In <i>RORc</i>-eYFP-RAG^−/− mice all YFP⁺ CD45⁺ live cells stain negative for CD3, TCRβ and also CD5. Representative plots for n = 3 (mean +/− s.e.m.).</p

Interleukin-12 bypasses common gamma-chain signalling in emergency natural killer cell lymphopoiesis

Differentiation and homeostasis of natural killer (NK) cells relies on common gamma-chain (γc)-dependent cytokines, in particular IL-15. Consequently, NK cells do not develop in mice with targeted γc deletion. Herein we identify an alternative pathway of NK-cell development driven by the proinflammatory cytokine IL-12, which can occur independently of γc-signalling. In response to viral infection or upon exogenous administration, IL-12 is sufficient to elicit the emergence of a population of CD122+CD49b+ cells by targeting NK-cell precursors (NKPs) in the bone marrow (BM). We confirm the NK-cell identity of these cells by transcriptome-wide analyses and their ability to eliminate tumour cells. Rather than using the conventional pathway of NK-cell development, IL-12-driven CD122+CD49b+ cells remain confined to a NK1.1lowNKp46low stage, but differentiate into NK1.1+NKp46+ cells in the presence of γc-cytokines. Our data reveal an IL-12-driven hard-wired pathway of emergency NK-cell lymphopoiesis bypassing steady-state γc-signalling

Arm swing asymmetry in overground walking

Abstract Treadmill experiments suggest that left-dominant arm swing is common in healthy walking adults and is modulated by cognitive dual-tasking. Little is known about arm swing asymmetry in overground walking. We report directional (dASI) and non-directional arm swing symmetry indices (ndASI) from 334 adults (mean age 68.6 ± 5.9 y) walking overground at comfortable (NW) and fast (FW) speeds and while completing a serial subtraction task (DT). dASI and ndASI were calculated from sagittal shoulder range of motion data generated by inertial measurement units affixed to the wrist. Most (91%) participants were right-handed. Group mean arm swing amplitude was significantly larger on the left in all walking conditions. During NW, ndASI was 39.5 ± 21.8, with a dASI of 21.9 ± 39.5. Distribution of dASI was bimodal with an approximately 2:1 ratio of left:right-dominant arm swing. There were no differences in ndASI between conditions but dASI was smaller during DT compared to FW (15.2 vs 24.6; p = 0.009). Handedness was unrelated to ndASI, dASI or the change in ASI metrics under DT. Left-dominant arm swing is the norm in healthy human walking irrespective of walking condition or handedness. As disease markers, ndASI and dASI may have different and complementary roles

Arm swing asymmetry in overground walking

Treadmill experiments suggest that left-dominant arm swing is common in healthy walking adults and is modulated by cognitive dual-tasking. Little is known about arm swing asymmetry in overground walking. We report directional (dASI) and non-directional arm swing symmetry indices (ndASI) from 334 adults (mean age 68.6 ± 5.9 y) walking overground at comfortable (NW) and fast (FW) speeds and while completing a serial subtraction task (DT). dASI and ndASI were calculated from sagittal shoulder range of motion data generated by inertial measurement units affixed to the wrist. Most (91%) participants were right-handed. Group mean arm swing amplitude was significantly larger on the left in all walking conditions. During NW, ndASI was 39.5 ± 21.8, with a dASI of 21.9 ± 39.5. Distribution of dASI was bimodal with an approximately 2:1 ratio of left:right-dominant arm swing. There were no differences in ndASI between conditions but dASI was smaller during DT compared to FW (15.2 vs 24.6; p = 0.009). Handedness was unrelated to ndASI, dASI or the change in ASI metrics under DT. Left-dominant arm swing is the norm in healthy human walking irrespective of walking condition or handedness. As disease markers, ndASI and dASI may have different and complementary roles