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Resting memory B cells can be divided into classical or atypical groups, but the heterogenous marker expression on activated memory B cells makes similar classification difficult. Here, by longitudinal analysis of mass cytometry and CITE-seq data from cohorts with COVID-19, bacterial sepsis, or BNT162b2 mRNA vaccine, we observe that resting B cell memory consist of classical CD45RB + memory and CD45RB lo memory, of which the latter contains of two distinct groups of CD11c + atypical and CD23 + non-classical memory cells. CD45RB levels remain stable in these cells after activation, thereby enabling the tracking of activated B cells and plasmablasts derived from either CD45RB + or CD45RB lo memory B cells. Moreover, in both COVID-19 patients and mRNA vaccination, CD45RB lo B cells formed the majority of SARS-CoV2 specific memory B cells and correlated with serum antibodies, while CD45RB + memory are activated by bacterial sepsis. Our results thus identify that stably expressed CD45RB levels can be exploited to trace resting memory B cells and their activated progeny, and suggest that atypical and non-classical CD45RB lo memory B cells contribute to SARS-CoV-2 infection and vaccination. Activated memory B cells express a variety of markers. Here, by mass cytometry and CITE-seq, the authors identify differential expression of CD45RB as a marker distinguishing classical and atypical/non-classical memory B cells, with the former being more prominent during sepsis, while the latter being more abundant in COVID-19 infection or vaccination.

Consecutive mRNA vaccinations against SARS-CoV-2 reinforced both innate and adaptive immune responses. However, it remains unclear whether the enhanced innate immune responses are mediated by epigenetic regulation and, if so, whether these effects persist. Using mass cytometry, RNA-Seq, and ATAC-Seq, we show that BNT162b2 mRNA vaccination upregulated antiviral and IFN-stimulated gene expression in monocytes with greater effects after the second vaccination than those after the first vaccination. Transcription factor–binding motif analysis also revealed enriched IFN regulatory factors and PU.1 motifs in accessible chromatin regions. Importantly, although consecutive BNT162b2 mRNA vaccinations boosted innate immune responses and caused epigenetic changes in isolated monocytes, we show that these effects occurred only transiently and disappeared 4 weeks after the second vaccination. Furthermore, single-cell RNA-Seq analysis revealed that a similar gene signature was impaired in the monocytes of unvaccinated patients with COVID-19 with acute respiratory distress syndrome. These results reinforce the importance of the innate immune response in the determination of COVID-19 severity but indicate that, unlike adaptive immunity, innate immunity is not unexpectedly sustained even after consecutive vaccination. This study, which focuses on innate immune memory, may provide novel insights into the vaccine development against infectious diseases.

Sialic acid sugars cover the surface of dendritic cells (DCs) and have been suggested to impact several aspects of DC biology. Research into the role of sialic acids in DCs, however, is complicated by the limited number of tools available to modulate sialic acid expression. Here we report on a synthetic, fluorinated sialic acid mimetic, Ac53FaxNeu5Ac, which potently blocks sialic acid expression in human monocyte‐derived DCs (moDCs). Sialic acid blockade enhanced the responsiveness of moDCs to Toll‐like receptor (TLR) stimulation as measured by increased maturation marker expression and cytokine production. Consequently, the T‐cell activation capacity of Ac53FaxNeu5Ac‐treated moDCs was strongly increased. In addition to sialic acids, moDCs also expressed the sialic acid‐binding immunoglobulin‐like lectins (Siglecs) ‐3, ‐5, ‐7, ‐9 and ‐10, immune inhibitory receptors recognizing these sialic acids. Treatment with Ac53FaxNeu5Ac abrogated putative cis and trans interactions between sialic acids and Siglec‐7/‐9. Together, these data indicate that sialic acids limit the activation of moDCs via the TLR pathway, potentially by interacting with Siglec‐7 or Siglec‐9. Metabolic sialic acid blockade with Ac53FaxNeu5Ac could therefore potentially be used to generate more potent DC‐based vaccines for induction of robust anti‐viral or anti‐tumor immune responses.

An integrated β-camera and microfluidic chip was developed that is capable of quantitative imaging of glycolysis radioassays using (18)F-FDG in small cell populations down to a single cell. This paper demonstrates that the integrated system enables digital control and quantitative measurements of glycolysis in B-Raf(V600E)-mutated melanoma cell lines in response to specific B-Raf inhibition. The β-camera uses a position-sensitive avalanche photodiode to detect charged particle-emitting probes within a microfluidic chip. The integrated β-camera and microfluidic chip system was calibrated, and the linearity was measured using 4 different melanoma cell lines (M257, M202, M233, and M229). Microfluidic radioassays were performed with cell populations ranging from hundreds of cells down to a single cell. The M229 cell line has a homozygous B-Raf(V600E) mutation and is highly sensitive to a B-Raf inhibitor, PLX4032. A microfluidic radioassay was performed over the course of 3 days to assess the cytotoxicity of PLX4032 on cellular (18)F-FDG uptake. The β-camera is capable of imaging radioactive uptake of (18)F-FDG in microfluidic chips. (18)F-FDG uptake for a single cell was measured using a radioactivity concentration of 37 MBq/mL during the radiotracer incubation period. For in vitro cytotoxicity monitoring, the β-camera showed that exposure to 1 μM PLX4032 for 3 days decreased the (18)F-FDG uptake per cell in highly sensitive M229 cells, compared with vehicle controls. The integrated β-camera and microfluidic chip can provide digital control of live cell cultures and allow in vitro quantitative radioassays for multiple samples simultaneously.

Blocking oncogenic signaling induced by the BRAF.sup.V600E .sup.mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAF.sup.V600E .sup.mutation. Seven out of 10 BRAF.sup.V600E .sup.mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 [mu]M. Among the sensitive cell lines, four were highly sensitive with IC.sub.50 values below 1 [mu]M, and three were moderately sensitive with IC.sub.50 values between 1 and 10 [mu]M. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAF.sup.V600E .sup.mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 muM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 muM, and three were moderately sensitive with IC50 values between 1 and 10 muM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.

Resting memory B cells can be divided into classical or atypical groups, but the heterogenous marker expression on activated memory B cells makes similar classification difficult. Here, by longitudinal analysis of mass cytometry and CITE-seq data from cohorts with COVID-19, bacterial sepsis, or BNT162b2 mRNA vaccine, we observe that resting B cell memory consist of classical CD45RB memory and CD45RB memory, of which the latter contains of two distinct groups of CD11c atypical and CD23 non-classical memory cells. CD45RB levels remain stable in these cells after activation, thereby enabling the tracking of activated B cells and plasmablasts derived from either CD45RB or CD45RB memory B cells. Moreover, in both COVID-19 patients and mRNA vaccination, CD45RB B cells formed the majority of SARS-CoV2 specific memory B cells and correlated with serum antibodies, while CD45RB memory are activated by bacterial sepsis. Our results thus identify that stably expressed CD45RB levels can be exploited to trace resting memory B cells and their activated progeny, and suggest that atypical and non-classical CD45RB memory B cells contribute to SARS-CoV-2 infection and vaccination.

Signaling via inhibitory KIR/Ly49 receptors preserves natural killer (NK) cell self-tolerance but also conveys NK cell reactivity towards MHC class-I low target cells in an education process. Here, we demonstrate that mouse NK cell education by H-2Dd regulates transcription of several genes in Ly49A+ NK cells including Ptpn6, encoding the phosphatase SHP-1. SHP-1 was highly expressed in uneducated NK cells, in which knock-out of Ptpn6 increased responsiveness. Following NKp46 triggering of uneducated NK cells, a higher synaptic abundance of phosphorylated SHP-1 was found relative to educated NK cells, concomitant with reduced phosphorylation of several signaling molecules, including PLC-y2, SLP-76, ZAP70/Syk and ERK1/2. SHP-1 overlapped extensively with F-actin and SLP-76 in the uneducated activating synapse of Ly49A+ NK cells, whereas a greater association between Ly49A and SHP-1 was observed in educated NK cells. Thus, our results indicate that in addition to transcriptional regulation, a distinct SHP-1 patterning in NK cell activating synapses can determine their tolerance. Competing Interest Statement The authors have declared no competing interest.

An integrated β-camera and microfluidic chip was developed that is capable of quantitative imaging of glycolysis radioassays using ^sup 18^F-FDG in small cell populations down to a single cell. This paper demonstrates that the integrated system enables digital control and quantitative measurements of glycolysis in B-Raf^sup V600E^-mutated melanoma cell lines in response to specific B-Raf inhibition. Methods: The β-camera uses a position-sensitive avalanche photodiode to detect charged particle-emitting probes within a microfluidic chip. The integrated β-camera and microfluidic chip system was calibrated, and the linearity was measured using 4 different melanoma cell lines (M257, M202, M233, and M229). Microfluidic radioassays were performed with cell populations ranging from hundreds of cells down to a single cell. The M229 cell line has a homozygous B-Raf^sup V600E^ mutation and is highly sensitive to a B-Raf inhibitor, PLX4032. A microfluidic radioassay was performed over the course of 3 days to assess the cytotoxicity of PLX4032 on cellular ^sup 18^F-FDG uptake. Results: The β-camera is capable of imaging radioactive uptake of ^sup 18^F-FDG in microfluidic chips. ^sup 18^F-FDG uptake for a single cell was measured using a radio-activity concentration of 37 MBq/mL during the radiotracer incubation period. For in vitro cytotoxicity monitoring, the β-camera showed that exposure to 1 µM PLX4032 for 3 days decreased the ^sup 18^F-FDG uptake per cell in highly sensitive M229 cells, compared with vehicle controls. Conclusion: The integrated β-camera and microfluidic chip can provide digital control of live cell cultures and allow in vitro quantitative radioassays for multiple samples simultaneously. [PUBLICATION ABSTRACT]

Blocking oncogenic signaling induced by the BRAF V600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAF V600E mutation. Seven out of 10 BRAF V600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC 50 values below 1 μM, and three were moderately sensitive with IC 50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAF V600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.