Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using a-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1’s BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from nonhomologous end-joining to homologous recombination

Application to the Analysis of Germinal Center Reactions In Vivo

Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method

Epistatic Interactions Between Mutations of Deoxyribonuclease 1-Like 3 and the Inhibitory Fc Gamma Receptor IIB Result in Very Early and Massive Autoantibodies Against Double-Stranded DNA

Autoantibodies against double-stranded DNA (anti-dsDNA) are a hallmark of systemic lupus erythematosus (SLE). It is well documented that anti-dsDNA reactive B lymphocytes are normally controlled by immune self-tolerance mechanisms operating at several levels. The evolution of high levels of IgG anti-dsDNA in SLE is dependent on somatic hypermutation and clonal selection, presumably in germinal centers from non-autoreactive B cells. Twin studies as well as genetic studies in mice indicate a very strong genetic contribution for the development of anti-dsDNA as well as SLE. Only few single gene defects with a monogenic Mendelian inheritance have been described so far that are directly responsible for the development of anti-dsDNA and SLE. Recently, among other mutations, rare null-alleles for the deoxyribonuclease 1 like 3 (DNASE1L3) and the Fc gamma receptor IIB (FCGR2B) have been described in SLE patients and genetic mouse models. Here, we demonstrate that double Dnase1l3- and FcgR2b-deficient mice in the C57BL/6 background exhibit a very early and massive IgG anti-dsDNA production. Already at 10 weeks of age, autoantibody production in double-deficient mice exceeds autoantibody levels of diseased 9-month-old NZB/W mice, a long established multigenic SLE mouse model. In single gene-deficient mice, autoantibody levels were moderately elevated at early age of the mice. Premature autoantibody production was accompanied by a spontaneous hyperactivation of germinal centers, early expansions of T follicular helper cells, and elevated plasmablasts in the spleen. Anti-dsDNA hybridomas generated from double-deficient mice show significantly elevated numbers of arginines in the CDR3 regions of the heavy-chain as well as clonal expansions and diversification of B cell clones with moderate numbers of somatic mutations. Our findings show a strong epistatic interaction of two SLE-alleles which prevent early and high-level anti-dsDNA autoantibody production. Both genes apparently synergize to keep in check excessive germinal center reactions evolving into IgG anti-dsDNA antibody producing B cells

Plasma cell output from germinal centers is regulated by signals from Tfh and stromal cells

Germinal centers (GCs) are the sites where B cells undergo affinity maturation. The regulation of cellular output from the GC is not well understood. Here, we show that from the earliest stages of the GC response, plasmablasts emerge at the GC-T zone interface (GTI). We define two main factors that regulate this process: Tfh-derived IL-21, which supports production of plasmablasts from the GC, and TNFSF13 (APRIL), which is produced by a population of podoplanin+CD157highfibroblastic reticular cells located in the GTI that are also rich in message for IL-6 and chemokines CXCL12, CCL19, and CCL21. Plasmablasts in the GTI express the APRIL receptor TNFRSF13B (TACI), and blocking TACI interactions specifically reduces the numbers of plasmablasts appearing in the GTI. Plasma cells generated in the GTI may provide an early source of affinity-matured antibodies that may neutralize pathogens or provide feedback regulating GC B cell selection

A model for interclonal competition in the germinal center: Dynamic selection processes by-pass the affinity dead-end of low affinity anti-NP specific B cells

The GC reaction is a highly competitive process in which high affinity B cells are favored to survive and proliferate in order to generate Abs of high affinity. To further analyze this competition, focusing on the fate of low affinity B cells, we made use of a transgenic transfer system. The mice used for this model express a transgenic heavy chain which, when paired with a λ1 LC, generate NP-specific Abs of low affinity. When splenocytes of such mice were transferred into wt recipients, B1-8lo GC B cells are reproducibly outcompeted between day 6 and 9 of the anti-NP response. This competitive phase was accompanied by an accumulation of B1-8lo GC B cells in the LZ together with a decreased frequency of proliferative B1-8lo cells in the DZ. We were further able to shed light on the molecular mechanisms leading to the disadvantage of B1-8lo GC B cells. A W33L mutation which is canonically found to be selected in C57BL/6 mice in response to NP is known to enhance affinity 10-fold. As expected, affinity maturation of endogenous GC cells led to a positive selection of cells bearing this mutation. In contrast, B1-8lo GC B cells did not show any selection of W33L, resulting in a selective disadvantage due to the absence of an increased affinity. Through generating recombinant Abs and measuring their affinity to NP, we were able to show that one of the four pre-existing amino acid exchanges within the B1-8lo heavy chain sequence was sufficient to reduce the affinity of an unmutated B1-8 Ab 10-fold. Hence, this amino acid change solely explains the low affinity of the B1-8lo Ab. Furthermore, the same amino acid exchange was found to interfere with an affinity enhancing effect of W33L, preventing B1-8lo B cells to stay competitive, being outcompeted by endogenous cells. To shed light on the fate of B1-8lo GC B cells during competition, we analyzed apoptosis and memory B cell (MBC) differentiation. Although the frequency of apoptotic B1-8lo GC B cells was increased compared to endogenous cells, the majority of these cells did not bind NP. It remains unclear whether this is due to increased apoptosis of B1-8lo GC B cells that exhibit another specificity than NP or due to the fact that surface molecules are downregulated on apoptotic cells, which might lead to impaired Ag binding. Surprisingly, we found B1-8lo cells to largely contribute to CD38hiFas-NP+ B cells, which are supposed to mainly contain MBCs. Interestingly, the frequency of this population remained fairly constant at all time points analyzed, although NP-specific B1-8lo B cells were concurrently outcompeted in the GC. Although it needs to be proven that this population indeed contains MBCs, a contribution of low affinity B cells to the MBC pool has been described before. Nevertheless, the extent of this contribution might give new insights into the fate of low affinity B cells which are unable to compete in the GC reaction

Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix

Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach