CD83 Modulates B Cell Function In Vitro: Increased IL-10 and Reduced Ig Secretion by CD83Tg B Cells

The murine transmembrane glycoprotein CD83 is an important regulator for both thymic T cell maturation and peripheral T cell responses. Recently, we reported that CD83 also has a function on B cells: Ubiquitous transgenic (Tg) expression of CD83 interfered with the immunoglobulin (Ig) response to infectious agents and to T cell dependent as well as T cell independent model antigen immunization. Here we compare the function of CD83Tg B cells that overexpress CD83 and CD83 mutant (CD83mu) B cells that display a drastically reduced CD83 expression. Correlating with CD83 expression, the basic as well as the lipopolysaccharide (LPS) induced expression of the activation markers CD86 and MHC-II are significantly increased in CD83Tg B cells and reciprocally decreased in CD83mu B cells. Wild-type B cells rapidly upregulate CD83 within three hours post BCR or TLR engagement by de novo protein synthesis. The forced premature overexpression of CD83 on the CD83Tg B cells results in reduced calcium signaling, reduced Ig secretion and a reciprocally increased IL-10 production upon in vitro activation. This altered phenotype is mediated by CD83 expressed on the B cells themselves, since it is observed in the absence of accessory cells. In line with this finding, purified CD83mu B cells displayed a reduced IL-10 production and slightly increased Ig secretion upon LPS stimulation in vitro. Taken together, our data strongly suggest that CD83 is expressed by B cells upon activation and contributes to the regulation of B cell function

PEDIA: prioritization of exome data by image analysis.

PURPOSE: Phenotype information is crucial for the interpretation of genomic variants. So far it has only been accessible for bioinformatics workflows after encoding into clinical terms by expert dysmorphologists. METHODS: Here, we introduce an approach driven by artificial intelligence that uses portrait photographs for the interpretation of clinical exome data. We measured the value added by computer-assisted image analysis to the diagnostic yield on a cohort consisting of 679 individuals with 105 different monogenic disorders. For each case in the cohort we compiled frontal photos, clinical features, and the disease-causing variants, and simulated multiple exomes of different ethnic backgrounds. RESULTS: The additional use of similarity scores from computer-assisted analysis of frontal photos improved the top 1 accuracy rate by more than 20-89% and the top 10 accuracy rate by more than 5-99% for the disease-causing gene. CONCLUSION: Image analysis by deep-learning algorithms can be used to quantify the phenotypic similarity (PP4 criterion of the American College of Medical Genetics and Genomics guidelines) and to advance the performance of bioinformatics pipelines for exome analysis

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

CD4⁺ T Cells Are as Protective as CD8⁺ T Cells against <i>Rickettsia typhi</i> Infection by Activating Macrophage Bactericidal Activity

<div><p><i>Rickettsia typhi</i> is an intracellular bacterium that causes endemic typhus, a febrile disease that can be fatal due to complications including pneumonia, hepatitis and meningoencephalitis, the latter being a regular outcome in T and B cell-deficient C57BL/6 RAG1^-/- mice upon <i>Rickettsia typhi</i> infection. Here, we show that CD4⁺ T_H1 cells that are generated in C57BL/6 mice upon <i>R</i>. <i>typhi</i> infection are as protective as cytotoxic CD8⁺ T cells. CD4⁺- as well as CD8⁺-deficient C57BL/6 survived the infection without showing symptoms of disease at any point in time. Moreover, adoptively transferred CD8⁺ and CD4⁺ immune T cells entered the CNS of C57BL/6 RAG1^-/- mice with advanced infection and both eradicated the bacteria. However, immune CD4⁺ T cells protected only approximately 60% of the animals from death. They induced the expression of iNOS in infiltrating macrophages as well as in resident microglia in the CNS which can contribute to bacterial killing but also accelerate pathology. <i>In vitro</i> immune CD4⁺ T cells inhibited bacterial growth in infected macrophages which was in part mediated by the release of IFNγ. Collectively, our data demonstrate that CD4⁺ T cells are as protective as CD8⁺ T cells against <i>R</i>. <i>typhi</i>, provided that CD4⁺ T_H1 effector cells are present in time to support bactericidal activity of phagocytes via the release of IFNγ and other factors. With regard to vaccination against TG <i>Rickettsiae</i>, our findings suggest that the induction of CD4⁺ T_H1 effector cells is sufficient for protection.</p></div

Immune CD4⁺ T cells induce NO release by <i>R</i>. <i>typhi</i>-infected macrophages <i>in vitro</i> and inhibit bacterial growth via IFNγ and TNFα.

<p>1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of 2×10⁶ purified CD4⁺ T cells from either naïve or immune BALB/c mice (day 7 post infection). IFNγ and TNFα were neutralized by the addition of 10 μg/ml anti-IFNγ and/or anti-TNFα as indicated on the x-axis. Cytokines were quantified in the supernatants 72h after T cell addition by LEGENDplex assay. IFNγ (left, y-axis), TNFα (middle, y-axis), IL-22 (right, y-axis) and IL-2 (below, left) are shown. Other cytokines were not detectable (<b>A</b>). In addition, NO was detected 72h after T cell addition (<b>B</b>). Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after T cell addition (<b>C</b>). 1×10⁶ bone-marrow-derived BALB/c macrophages were treated with recombinant IFNγ (1 U/ml) or TNFα (400 U/ml). NO was quantified in the cell culture supernatants after 72h (<b>D</b>). 1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of recombinant IFNγ (1 U/ml) or TNFα (400 U/ml). The cytokines were neutralized by simultaneous addition of either anti-TNFα or anti-IFNγ (10 μg/ml each) as indicated on the x-axis. Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after cytokine addition (<b>E</b>). Graphs show the mean±SEM of combined results from 2 independent experiments (n = 4 T cells from each group of mice (A-C) and n = 2 for the treatment with recombinant cytokines (D-E)). Statistical analysis was performed by One-way ANOVA (Kruskal-Wallis test followed by Dunn´s post test). Asterisks indicate significant differences (*<i>p</i><0.05, **<i>p</i><0.01).</p

Detection of infiltrating T cells, IBA1⁺ cells and iNOS expression in sagittal sections of the brains from <i>R</i>. <i>typhi</i>-infected C57BL/6 RAG1^-/- control mice, CD4⁺ and CD8⁺ recipients (day 7 post transfer).

<p>Sagittal sections were further stained for CD3 to detect infiltrating T cells, for IBA1 that is expressed on infiltrating MΦ as well as by microglia and for iNOS. Sections from the brains of CD4⁺ T cell recipients that were not infected were used as additional control (<b>A</b>). Representative stainings of the brain from a <i>R</i>. <i>typhi</i>-infected C57BL/6 RAG1^-/- control mouse (<b>B</b>), a <i>R</i>. <i>typhi</i>-infected mouse that received CD4⁺ T cells (<b>C</b>) and a CD8⁺ T cell recipient (<b>D</b>) are shown.</p

Enhanced protection by CD4⁺IFNγ^-/- T cells in the absence of TNFα.

<p>1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of 2×10⁶ purified CD4⁺ T cells from either naïve or immune BALB/c IFNγ^-/- mice (day 7 post infection). TNFα was neutralized by simultaneous addition of 10 μg/ml anti-TNFα. Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after T cell addition. Graphs show the mean and SEM of combined results from two independent experiments (n = 4 T cells from each group of mice) (<b>A</b>). CB17 SCID mice (n = 7 for each group) were infected with 1×10⁶ sfu <i>R</i>. <i>typhi</i>. 1×10⁶ purified CD4⁺ T cells from BALB/c IFNγ^-/- mice were adoptively transferred one day prior to the infection with <i>R</i>. <i>typhi</i>. Control groups of mice received PBS instead. TNFα was neutralized by intraperitoneal application of 500 μg anti-TNFα every three days beginning on day 3 post infection. Control animals received equal amounts of isotype antibody. The state of health of the mice was monitored by weight change (y-axis, upper left) and a clinical score (y-axis, upper right) and the survival rates (y-axis, below) were assessed. Dotted lines show the data for surviving animals of the isotype- and anti-TNFα-treated groups of CD4⁺IFNγ^-/- recipients. Statistical analysis of survival rates was performed with Log-rank (Mantel-Cox) test. Asterisks indicate significant differences compared to control animals (**<i>p</i><0.01) (<b>B</b>). Serum GPT levels (y-axis) were assessed from all groups of animals at the time of death and in surviving animals at the end of the experiment (day 34). Combined results are shown. Each dot represents a single mouse. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test) (*<i>p</i><0.05) (<b>C</b>). The bacterial content (y-axis) in the organs was quantified by <i>prsA</i>-specific qPCR from all animals that succumbed to the infection at the time of death and from surviving animals at the end of the experiment (day 34) as indicated on the x-axis (<b>D</b>). Statistical analysis for C and D was performed by One-way ANOVA (Kruskal-Wallis test followed by Dunn´s post test). Asterisks indicate statistically significant differences (*<i>p</i><0.05, **<i>p</i><0.01).</p

BALB/c mice generate cytotoxic CD8⁺ cells that are sporadically reactivated.

<p>BALB/c mice were infected with 1×10⁶ sfu <i>R</i>. <i>typhi</i>. Control mice received PBS instead and were used as "day 0" control. Spleen cells were isolated and stained for CD8, KLRG1 and CD11a or restimulated with PMA/Ionomycin for 4h and stained for CD8 and intracellular IFNγ and Granzyme B. The dot plots show example stainings from day 7 post infection. Mice were analyzed for cytokine and Granzyme B expression on day 0, 7 and 15 (n = 6) and day 35 (n = 4). 3–4 mice were analyzed for KLRG1 and CD11a expression. Graphs show the percentage of KLRG1⁺, CD11a⁺, Granzyme B⁺ and IFNγ⁺ T cells among CD8⁺ T cells (y-axis) at indicated days post infection (x-axis). Graphs show combined results from 2 independent experiments. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test). Asterisks indicate significant differences compared to day 0 (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p