Regulation of Foxo1 expression is critical for central B cell tolerance and allelic exclusion.

Resolving the molecular mechanisms of central B cell tolerance might unveil strategies that prevent autoimmunity. Here, using a mouse model of central B cell tolerance in which Forkhead box protein O1 (Foxo1) is either deleted or over-expressed in B cells, we show that deleting Foxo1 blocks receptor editing, curtails clonal deletion, and decreases CXCR4 expression, allowing high-avidity autoreactive B cells to emigrate to the periphery whereby they mature but remain anergic and short lived. Conversely, expression of degradation-resistant Foxo1 promotes receptor editing in the absence of self-antigen but leads to allelic inclusion. Foxo1 over-expression also restores tolerance in autoreactive B cells harboring active PI3K, revealing opposing roles of Foxo1 and PI3K in B cell selection. Overall, we show that the transcription factor Foxo1 is a major gatekeeper of central B cell tolerance and that PI3K drives positive selection of immature B cells and establishes allelic exclusion by suppressing Foxo1.

CD19 Is Internalized Together with IgM in Proportion to B Cell Receptor Stimulation and Is Modulated by Phosphatidylinositol 3-Kinase in Bone Marrow Immature B Cells.

Newly generated immature B cells that bind self-antigen with high avidity arrest in differentiation and undergo central tolerance via receptor editing and clonal deletion. These autoreactive immature B cells also express low surface levels of the coreceptor CD19, a key activator of the PI3K pathway. Signals emanating from both CD19 and PI3K are known to be critical for attenuating receptor editing and selecting immature B cells into the periphery. However, the mechanisms that modulate CD19 expression at this stage of B cell development have not yet been resolved. Using in vivo and in vitro models, we demonstrate that Cd19 de novo gene transcription and translation do not significantly contribute to the differences in CD19 surface expression in mouse autoreactive and nonautoreactive immature B cells. Instead, CD19 downregulation is induced by BCR stimulation in proportion to BCR engagement, and the remaining surface IgM and CD19 molecules promote intracellular PI3K-AKT activity in proportion to their level of expression. The internalized CD19 is degraded with IgM by the lysosome, but inhibiting lysosome-mediated protein degradation only slightly improves surface CD19. In fact, CD19 is restored only upon Ag removal. Our data also reveal that the PI3K-AKT pathway positively modulates CD19 surface expression in immature B cells via a mechanism that is independent of inhibition of FOXO1 and its role on Cd19 gene transcription while is dependent on mTORC1.

Elevated Detection of Dual Antibody B Cells Identifies Lupus Patients With B Cell-Reactive VH4-34 Autoantibodies.

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B2R) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/lpr mice; and 2) to determine whether B2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ+λ+ B2R cells in one fourth of the SLE patients. Abnormal numbers of κ+λ+ B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of "B cells that are naïve IgD+" (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ+λ+ SLE patients harbored normal frequencies of κ+λ+ and other B2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity.

Factors affecting detection of bimodal sour-savory mixture and inter-individual umami taste perception.

While basic taste interactions have been the subject of many research studies, there is one combination where data is limited in the literature: sour and umami. This combination is universal in culinary preparations and of key interest to the food industry. Therefore, the primary goal of the present study is to assess how increasing concentrations of acidity (citric acid) affect, if at all, the intensity of a constant concentration of umami (monosodium glutamate, MSG). The secondary goal is to investigate other possible factors in umami taste perception. Here, a crowdsourced cohort of 734 individuals (age range 8-81) tasted and rated the intensity of 50 mM MSG alone, and in combination with citric acid at varying concentrations (1.25 mM, 6.25 mM, 31.25 mM). Participants were also genotyped for the single nucleotide polymorphism rs34160967 in the T1R1 gene. The results show a significant decrease in the intensity perception of umami as sour concentration increases (low: p = 0.005, medium: p < 0.001, high: p < 0.001). Situational factors such as participant hunger level and time since last eating also have a significant effect on umami intensity perception. Neither the biological factors of sex, age, and ancestry appear to play a role in umami perception, nor does variation in gene TAS1R1 at rs34160967. These new data contribute to the growing field of taste and sensory interaction by giving evidence that sour suppresses umami taste perception in bi-model samples.

Self-reported Smoking Status, TAS2R38 Variants, and Propylthiouracil Phenotype: An Exploratory Crowdsourced Cohort Study.

TAS2R38 gene variants, which confer sensitivity to specific bitter tastants (e.g., 6-n-propylthiouracil), have been repeatedly associated with lower alcohol use via greater bitterness perception, but research exploring TAS2R38 variation in relation to smoking shows mixed results. In both, the working hypothesis is that 1 or more copies of the functional allele increases bitterness and may provide a barrier to early use. Such a barrier to initiation may, conceivably, manifest as differential rates of current use across diplotypes. Here, an age-diverse convenience sample (n = 886) of Denver Museum of Nature and Science guests was used to explore cross-sectional relationships between TAS2R38 diplotype, self-reported tobacco use (current, former, never smokers), and a rapid measure of 6-n-propylthiouracil phenotype (bitterness of filter paper discs). TAS2R38 diplotypes were determined by Sanger sequencing. After excluding rare diplotypes, data from 814 participants were analyzed. A mix of current (~10%), former (25%), and never smokers (65%) were included. As expected, there was a relationship between TAS2R38 diplotype and 6-n-propylthiouracil bitterness. However, contrary to our hypothesis, there was no evidence of a relationship between diplotype and smoker status among participants with common TAS2R38 diplotypes. Notably, we observed a relationship between of 6-n-propylthiouracil bitterness and smoking status, but the effect was opposite of what was expected: current smokers perceived higher (not lower) bitterness than never smokers. When all the various factors (diplotype, age, sex, and smoking status) were included in ANOVA, all remained predictive of 6-n-propylthiouracil bitterness. Reasons for greater phenotypic bitterness among current smokers are unknown and merit further study.