Expression of Fbxo7 in haematopoietic progenitor cells cooperates with p53 loss to promote lymphomagenesis.

Fbxo7 is an unusual F box protein that augments D-type cyclin complex formation with Cdk6, but not Cdk4 or Cdk2, and its over-expression has been demonstrated to transform immortalised fibroblasts in a Cdk6-dependent manner. Here we present new evidence in vitro and in vivo on the oncogenic potential of this regulatory protein in primary haematopoietic stem and progenitor cells (HSPCs). Increasing Fbxo7 expression in HSPCs suppressed their colony forming ability in vitro, specifically decreasing CD11b (Mac1) expression, and these effects were dependent on an intact p53 pathway. Furthermore, increased Fbxo7 levels enhanced the proliferative capacity of p53 null HSPCs when they were grown in reduced concentrations of stem cell factor. Finally, irradiated mice reconstituted with p53 null, but not wild-type, HSPCs expressing Fbxo7 showed a statistically significant increase in the incidence of T cell lymphoma in vivo. These data argue that Fbxo7 negatively regulates the proliferation and differentiation of HSPCs in a p53-dependent manner, and that in the absence of p53, Fbxo7 expression can promote T cell lymphomagenesis

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

Fbxo7 cooperates with p53 mutation to promote lymphomagenesis <i>in vivo</i>.

<p>(<b>A</b>) Graph of Kaplan-Meier survival curve of mice reconstituted with p53 null FL cells infected with retroviruses expressing either MSCV control (n = 10, dashed line) or Fbxo7 (n = 10, solid line). (<b>B</b>) H&E staining and immunohistochemistry was conducted as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021165#pone.0021165-Laman1" target="_blank">[11]</a> for (<b>C</b>) CD3 and (<b>D</b>) Fbxo7 in tissue samples from mice reconstituted with Fbxo7-expressing cells. Size bar is 50 µm. (<b>E</b>) PCR amplification reactions for either the p53 null allele (top) or the GFP gene in the MSCV vector (bottom) performed on genomic DNA isolated from paraffin-embedded tissue samples from mice reconstituted with p53 null HSPCs infected with retroviruses bearing either the empty MSCV vector or the MSCV vector expressing Fbxo7. Numbered samples consist of pooled biopsies from multiple organs (liver, spleen, thymus, intestine, kidney, heart) from mice in the two different cohorts, as indicated. ‘T’ denotes samples which consisted of tumour tissue only. For positive controls for the PCR reactions, in the p53 reaction (top), ‘pos’ denotes a reaction where genomic DNA from a p53 null mouse was added, and for the GFP reaction (bottom) ‘pos’ denotes a reaction where MSCV plasmid DNA was added. ‘Neg’ denotes reactions where no template DNA was added.</p

Fbxo7 expression enhances the proliferation of p53 null cells grown at a reduced SCF concentration.

<p>(<b>A</b>) FACS plot of EdU incorporation, showing forward scatter (FSC) on the x-axis and (SSC) side scatter on the y-axis for the left hand panels, with the gated population used for EdU analysis and anti-EdU conjugated to Alexafluor647 on the x-axis and propidium iodide intensity on the y-axis for the right hand panels. (<b>B</b>) Immunoblotting for the expression of various cell cycle regulators, as indicated, in WT and p53 null cells expressing Fbxo7 or the empty MSCV vector. (<b>C</b>) Immunoblotting for Cdk6 in immunoprecipitations of D-type cyclins from equal numbers of sorted WT cells expressing either Fbxo7 or the empty MSCV vector.</p

Fbxo7 acts as a proliferative factor in p53 nulls grown in reduced SCF.

<p>(<b>A</b>) Table of the quantification of colony number of WT and p53 null cells grown at different concentrations of SCF. (<b>B</b>) Graphs of the total cell number at three concentrations of SCF in WT and p53 null cells expressing Fbxo7 compared to MSCV. (<b>C</b>) Graph of ratio of the number of either WT or p53 null cells expressing Fbxo7 compared to MSCV at different concentrations of SCF. In these experiments, the error is represented as the SD, and quantification is of three independent experiments.</p

Fbxo7 expression reduced the colony forming capacity and number of WT and p53 null cells.

<p>(<b>A</b>) Representative FACS plots of retrovirally infected FL cells showing GFP expression. (<b>B</b>) Expression of Fbxo7 in cells assayed by immunoblotting and Ponceau S staining as a loading control. (<b>C</b>) Table of quantification of the number of colonies at each passage. Number in parentheses is the percent decrease relative to the MSCV control. Error is represented as the SD. Quantification is of two independent experiments for each cell type. (<b>D</b>) Graphs of the quantification of total numbers of either WT (left) or p53 null (right) cells at each passage. Error is represented as the SD. Quantification is of two independent experiments for each cell type. * denotes statistical significance, <i>P</i> value<0.05; ** <i>P</i> value<0.01.</p

Bi-Functional Chicken Immunoglobulin-Like Receptors With a Single Extracellular Domain (ChIR-AB1): Potential Framework Genes Among a Relatively Stable Number of Genes Per Haplotype.

The leukocyte receptor complex (LRC) in humans encodes many receptors with immunoglobulin-like (Ig-like) extracellular domains, including the killer Ig-like receptors (KIRs) expressed on natural killer (NK) cells among others, the leukocyte Ig-like receptors (LILRs) expressed on myeloid and B cells, and an Fc receptor (FcR), all of which have important roles in the immune response. These highly-related genes encode activating receptors with positively-charged residues in the transmembrane region, inhibitory receptors with immuno-tyrosine based motifs (ITIMs) in the cytoplasmic tail, and bi-functional receptors with both. The related chicken Ig-like receptors (ChIRs) are almost all found together on a microchromosome, with over 100 activating (A), inhibitory (B), and bi-functional (AB) genes, bearing either one or two extracellular Ig-like domains, interspersed over 500-1,000 kB in the genome of an individual chicken. Sequencing studies have suggested rapid divergence and little overlap between ChIR haplotypes, so we wished to begin to understand their genetics. We chose to use a hybridization technique, reference strand-mediated conformational analysis (RSCA), to examine the ChIR-AB1 family, with a moderate number of genes dispersed across the microchromosome. Using fluorescently-labeled references (FLR), we found that RSCA and sequencing of ChIR-AB1 extracellular exon gave two groups of peaks with mobility correlated with sequence relationship to the FLR. We used this system to examine widely-used and well-characterized experimental chicken lines, finding only one or a few simple ChIR haplotypes for each line, with similar numbers of peaks overall. We found much more complicated patterns from a broiler line from a commercial breeder and a flock of red junglefowl, but trios of parents and offspring from another commercial chicken line show that the complicated patterns are due to heterozygosity, indicating a relatively stable number of peaks within haplotypes of these birds. Some ChIR-AB1 peaks were found in all individuals from the commercial lines, and some of these were shared with red junglefowl and the experimental lines derived originally from egg-laying chickens. Overall, this analysis suggests that there are some simple features underlying the apparent complexity of the ChIR locus

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

International audienceLife-threatening ‘breakthrough’ cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS-CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals (age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto-Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-α2 and IFN-ω, while two neutralized IFN-ω only. No patient neutralized IFN-β. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population