Highlights d B.1.1.7, B.1.351, and P.1 do not show augmented host cell entry d Entry inhibitors under clinical evaluation block all variants d B.1.351 and P.1 can escape from therapeutic antibodies d B.1.351 and P.1 evade antibodies induced by infection and vaccination

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

Multiple myeloma is an incurable plasma cell neoplasia characterized by the production of large amounts of monoclonal immunoglobulins.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that selectively degrades aberrant mRNAs with premature termination codons (PTCs). Although a small number of cases exist in mammals, where NMD controls levels of physiologic PTC transcripts, it is still unclear whether the engagement of NMD in posttranscriptional control of gene expression is a more prevalent phenomenon. To identify physiologic NMD substrates and to study how NMD silencing affects the overall dynamics of a cell, we stably down-regulated hUPF2, the human homolog of the yeast NMD factor UPF2, by RNA interference. As expected, hUPF2-silenced HeLa cells were impaired in their ability to recognize ectopically expressed aberrant PTC transcripts. Surprisingly, hUPF2 silencing did not affect cell growth and viability but clearly diminished phosphorylation of hUPF1, suggesting a role of hUPF2 in modulating NMD activity through phosphorylation of hUPF1. Genome-wide DNA microarray expression profiling identified 37 novel up-regulated and 57 downregulated transcripts in hUPF2-silenced cells. About 60% of the up-regulated mRNAs carry typical NMD motifs. Hence, NMD is important not only for maintaining the transcriptome integrity by removing nonfunctional and aberrant PTC-bearing transcripts but also for posttranscriptional control of selected physiologic transcripts with NMD features.Eukaryotes have acquired numerous ways to control the integrity and quality of transcripts; one of them is the so-called nonsense-mediated mRNA decay (NMD). This posttranscriptional mRNA surveillance pathway recognizes and degrades aberrant (nonsense) transcripts with premature termination codons (PTCs), thereby preventing accumulation of truncated nonfunctional or potentially noxious polypeptides as well as dissipation of energy for translating aberrant mRNA. The physiological relevance of NMD is demonstrated in several pathological situations, where NMD-resistant nonsense mRNAs accumulate. For example, the presence of stable nonsense mRNAs encoding truncated ␤-globin or ␤-amyloid precursor protein correlates with the onset of ␤ 0 -thalassemia (52) or the formation of neuritic plaques in Alzheimer's patients (65), respectively.NMD was first observed in Saccharomyces cerevisiae (40) and Caenorhabditis elegans (23) and seems to operate in all eukaryotes, including mammals (13). Recently, homologs of the three yeast NMD factors Upf1p, Upf2p, and Upf3p (for Up frameshift protein) have been identified in humans. hUPF1/ rent1 (3,41,45,56,58), the human homolog of yeast UPF1/ regulator of nonsense transcripts, is an ATP-dependent RNA helicase (5), can be phosphorylated at serine residues in SQ motifs (55,70), and resides in the cytoplasm (3), although it can enter the nucleus (46). hUPF1 is supposed to trigger efficient degradation of nonsense transcripts once it has been recruited to the mRNA by hUPF2/rent2. hUPF2 can also be phosphorylated (12), has no known enzymatic activity, shows strong perinuclear staining, and interacts not only with hUPF1 (41) ...

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We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.

The emergence of SARS-CoV-2 variants threatens efforts to contain the COVID-19 pandemic. The number of COVID-19 cases and deaths in India has risen steeply and a SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the ACE2 receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyze whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 enters two out of eight cell lines tested with roughly 50% increased efficiency and is equally inhibited by two entry inhibitors. In contrast, B.1.617 is resistant against Bamlanivimab, an antibody used for COVID-19 treatment. B.1.617 evades antibodies induced by infection or vaccination, although less so than the B.1.351 variant. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.