Cernunnos/Xlf Deficiency Results in Suboptimal V(D)J Recombination and Impaired Lymphoid Development in Mice

Xlf/Cernunnos is unique among the core factors of the non-homologous end joining (NHEJ) DNA double strand breaks (DSBs) repair pathway, in the sense that it is not essential for V(D)J recombination in vivo and in vitro. Unlike other NHEJ deficient mice showing a SCID phenotype, Xlf−/− mice present a unique immune phenotype with a moderate B- and T-cell lymphopenia, a decreased cellularity in the thymus, and a characteristic TCRα repertoire bias associated with the P53-dependent apoptosis of CD4+CD8+ DP thymocytes. Here, we thoroughly analyzed Xlf−/− mice immune phenotype and showed that it is specifically related to the DP stage but independent of the MHC-driven antigen presentation and T-cell activation during positive selection. Instead, we show that V(D)J recombination is subefficient in Xlf−/− mice in vivo, exemplified by the presence of unrepaired DSBs in the thymus. This results in a moderate developmental delay of both B- and T-lymphocytes at key V(D)J recombination dependent stages. Furthermore, subefficient V(D)J recombination waves are accumulating during TCRα rearrangement, causing the typical TCRα repertoire bias with loss of distal Vα and Jα rearrangements

Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination

V(D)J recombination and immunoglobulin class switch recombination (CSR) are two somatic rearrangement mechanisms that proceed through the introduction of double-strand breaks (DSBs) in DNA. Although the DNA repair factor XRCC4 is essential for the resolution of DNA DSB during V(D)J recombination, its role in CSR has not been established. To bypass the embryonic lethality of XRCC4 deletion in mice, we developed a conditional XRCC4 knockout (KO) using LoxP-flanked XRCC4 cDNA lentiviral transgenesis. B lymphocyte restricted deletion of XRCC4 in these mice lead to an average two-fold reduction in CSR in vivo and in vitro. Our results connect XRCC4 and the nonhomologous end joining DNA repair pathway to CSR while reflecting the possible use of an alternative pathway in the repair of CSR DSB in the absence of XRCC4. In addition, this new conditional KO approach should be useful in studying other lethal mutations in mice

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

A truncation variant of the cation channel P2RX5 is upregulated during T cell activation

Members of the P2X family of ligand-gated cation channels (P2RX) are expressed by various cell types including neurons, smooth- and cardiac muscle cells, and leukocytes. The channels mediate signalling in response to extracellular ATP. Seven subunit isoforms (P2RX1-P2RX7) have been identified and these can assemble as homo- and heterotrimeric molecules. In humans, P2RX5 exists as a natural deletion mutant lacking amino acids 328-349 of exon 10, which are part of transmembrane (TM) 2 and pre-TM2 regions in other organisms like rat, chicken and zebrafish. We show that P2RX5 gene expression of human T lymphocytes is upregulated during activation. P2RX5 is recruited to the cell surface. P2RX5-siRNA-transfected CD4+ T cells produced twofold more IL-10 than controls. Surface and intracellular P2RX5 expression was upregulated in activated antigen-specific CD4+ T cell clones. These data indicate a functional role of the human P2RX5 splice variant in T cell activation and immunoregulation

Immunomodulatory effects of the ether phospholipid edelfosine in experimental autoimmune encephalomyelitis

The 2-lysophosphatidylcholine analog edelfosine induces apoptosis in highly proliferating cells, e.g. activated immune cells. We examined mechanisms of action of edelfosine on immune functions in experimental autoimmune encephalomyelitis, a well-accepted animal model for multiple sclerosis. We observed activated caspase-3 expression in lymphoid organs and the central nervous system; however, edelfosine did not induce global apoptosis. Edelfosine improved the disease course and led to reduced frequencies of CD4(+) T cells infiltrating into the central nervous system. Our data suggest edelfosine as an interesting treatment candidate for multiple sclerosis

Edelfosine impact on HLA-DR/DP/DQ expression on human T cells.

<p>(A) Gating strategy to identify viable CD4⁺ and CD8⁺ T cells and their naïve (CD27⁺CD45RA⁺) and memory (CD27⁺CD45RA⁻) subsets after the incubation of PBMCs for 24 h in the absence of edelfosine or in the presence of 3.3 µg/ml and 10 µg/ml edelfosine, respectively. (B) Representative histograms of one donor display the considerably low expression of HLA-DR/DP/DQ on the previously described T cell subsets. (C) Summary of MedFI values determined for each treatment within each T cell subset (n = 3 donors (represented by symbols •, ▪, ▴), + edelfosine added as indicated, − no edelfosine added). For CD27⁺CD45RA⁺ and CD27⁺CD45RA⁻ populations of CD4⁺ and CD8⁺ T cells no significant reduction of HLA-DR/DP/DQ expression was observed after Bonferroni post-hoc analysis (depicted P-value: as determined by repeated measures ANOVA). Histogram legends for B: no edelfosine (black line), 3.3 µg/ml edelfosine (green line), 10 µg/ml edelfosine (red line), isotype control (blue line).</p

Human T cell proliferation was affected by edelfosine.

<p>(A) Reduced PBMC proliferation upon addition of edelfosine on cell seeding was independent of the addition of PHA. Notably, PHA-activated cells appeared to be susceptible to edelfosine at 10-fold lower concentrations. (B) The inhibitory effect of edelfosine was observed if the drug was added to already activated, proliferating T cells, i.e. two days after cell seeding and PHA addition. Here, a significant reduction of proliferation in unstimulated cells was only detectable with 33.3 µg/ml edelfosine. (C) Preincubation of PBMCs with at least 3.3 µg/ml edelfosine interfered with the cells' capacity to proliferate upon PHA stimulation. No effect was detected in preconditioned, but unstimulated cells (experiments A, B, C: sample size n = 3 donors, each approach was seeded in triplicates and means for each donor are represented by symbols •, ▪, ▴). (D) 1 µg/ml edelfosine or higher concentrations profoundly diminished proliferation in MBP_(83–99)-specific TCLs. One representative TCL of two is shown. Cells were incubated in quadruplicates. • stimulated, ▪ unstimulated (E) PBMCs of one donor were cultured without addition of a stimulus. Proliferation was detectable after seven days. The presence of anti-HLA-DR- and anti-MHC class I-blocking antibodies or 3.3 µg/ml edelfosine inhibited cellular proliferation (• untreated, ▪ blocking antibodies added, ▴ 3.3 µg/ml edelfosine-treated). Bars represent mean values ± SEM, *P<0.05, **P<0.01 and ***P<0.001 after repeated measures ANOVA succeeded by Bonferroni post-hoc analysis.</p

Clustering of up- or downregulated genes to determine biological pathways affected by edelfosine in human CD4⁺ T cells after gene expression analysis.

<p>(A) The incubation of unstimulated cells with 10 µg/ml edelfosine resulted in the upregulation of apoptosis- and cell death-associated genes. Genes involved in immune response and antigen processing and presentation were downregulated. (B) In the case of stimulated cells which were cultured in presence of 3.3 µg/ml edelfosine the downmodulation of cell cycle progression-related genes was found. Additionally, edelfosine resulted in the upregulation of genes assigned to immune response- and virus response-pathways characterized by type I IFN-regulated genes.</p

Edelfosine impact on HLA-DR/DP/DQ expression on human B cells.

<p>(A) Gating strategy to identify viable CD19⁺ B cells and their IgD⁺CD27⁻, IgD⁺CD27⁺ and IgD⁻ CD27⁺ subsets after the incubation of PBMCs for 24 h in the absence of edelfosine or in the presence of 3.3 µg/ml and 10 µg/ml edelfosine, respectively. (B) Histograms, exemplary of one donor, display the edelfosine-induced downmodulation of HLA-DR/DP/DQ on the previously described B cell subsets in comparison to the respective untreated control approach. (C) Summary of MedFI values determined for each treatment within each B cell subset (n = 3 donors (represented by symbols •, ▪, ▴), + edelfosine added as indicated, − no edelfosine added). For CD19⁺, IgD⁺CD27⁻ and IgD⁺CD27⁺ populations significant reductions of HLA-DR/DP/DQ expression were observed (*P<0.05, **P<0.01, ***P<0.001 after repeated measures ANOVA and Bonferroni post-hoc analysis). Histogram legends for B: no edelfosine (black line), 3.3 µg/ml edelfosine (green line), 10 µg/ml edelfosine (red line), isotype control (blue line).</p