CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. CARs in vitro , but hade clinic –9. Theree clinic –13, incre clinic , promotee clinic  and impro models . CARs cosistence –19. 2G Csistence . Howeversistence –26. The esponses . CARs coing CD28 . The pering CD28 , 30. DesIn the present study, we compare 2G CAR T cells containing CD28 to a 3G CAR containing both CD28 and 4-1BB to generate a rationale for the use of the latter in clinical trials. We investigated the functional capacity of 3G compared to 2G CARs and have initiated a mapping of the intracellular signaling capacity post antigen stimulation in both 2G and 3G CARs.PBMCs were isolated from blood of patients with CLL (n = 4) or healthy donors (n = 2) using Ficoll paque gradient centrifugation . Written consent was obtained from all patients in concordance with the Helsinki Declaration and the study was approved by the Uppsala Regional Ethical Review Board, Uppsala, Sweden (DNr: 2006:145). Peripheral blood from healthy donors was obtained from the blood bank at Uppsala University Hospital. Deidentified cord blood (CB) units were obtained through the MD Anderson Cord Blood Bank  on a Baylor College of Medicine (BCM) IRB-approved protocol.CD19+ Daudi   and the following day (day 3) transduced on RetroNectin-coated  plates pre-incubated with retroviral supernatant and kept in 100IU IL-2/ml.CAR T cells were generated from peripheral mononuclear blood cells (PBMCs) and cultured in RPMI medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptavidin, all purchased from Life Technologies. B cells were isolated using αCD20 magnetic beads  and T cells were subsequently purified from the CD20 negative fraction using pan T magnetic beads , both from Miltenyi Biotec GmbH . T cells were activated with 1μg OKT-3/mL  at day 0. On day 2, T cells were stimulated with 100IU IL-2/mL  once a week  and 3 days after the last B cell stimulation (endpoint of co-culture) and used for Western blot and cell signaling multiplex assay. Cell culture supernatants were collected 24h after B cell stimulation (once/week), and analyzed for IFNγ  and Granzyme B  using ELISA.B cells were isolated from PBMCs through αCD20 magnetic bead isolation. CD20 negative cells were subsequently purified using pan T magnetic beads to generate CAR T cells (Miltenyi Biotec GmbH). Due to the slow proliferative capacity of T cells generated from leukemic patients, the expansion was ongoing for 14 days. On day 14, 1x10e a week  similarl2 Fragment Goat Anti-Human IgG (H+L) purchased from Jackson ImmunoResearch Europe LTD (cat no 109-496-088), or negative isotype control antibodies (Biolegend). Cells were washed with PBS. Analyzed on BD Canto II  and evaluated with FlowJo . The transduction efficiency of 3G CAR was lower than that of 2G CAR. Therefore, a correction factor was calculated based on the CAR expression of the 2G and 3G T cells from the same donor at the same time point. This correction factor was then used to normalize the values of the functional assays.Cells were incubated with 1% BSA in PBS for 10 min, centrifuged and stained for 15 min with the following antibodies: CD3-FITC (cat no 300406), CD4-APC/Cy7 (cat no 300518), CD8-PE/Cy7 (cat no 301012), CD19-PerCP (cat no 302228), CCR7-BV421 (cat no 353208), CD45RA-APC/Cy7 (cat no 304128), CD27-PE (cat no 302808), CD28-PE/Cy7 (cat no 302926), Tim-3-BV421 (cat no 345007), PD-1-PE (cat no 329906) all purchased from Biolegend, α-CAR-DyLight649 AffiniPure F(ab')B cells were isolated from PBMCs through αCD20 magnetic bead (Miltenyi Biotec GmbH) isolation and the CD20 negative fraction was subsequently purified using pan T magnetic beads all according to manufacturer’s protocol. Pan T purified T cells were used to generate Mock, 2G and 3G CAR T cells. The T cells were stimulated with αCD20-labelled B cells (4:1 ratio) for 1 and 3h. At these time-points the co-culture was run through a LS column (Miltenyi Biotec GmbH) and the T cell fractions (99.9% CD3+) were washed with cold PBS and resuspended in lysis buffer containing M-PER mammalian protein extraction reagent (cat no 78503) supplemented with 1% Halt phosphatase inhibitor cocktail (cat no 78415) and 1% protease inhibitor cocktail (cat no 1861277) all from Thermo Scientific. Protein lysates were assayed for kinase tyrosine phosphorylation analyzed on PamStation12 .The MILLIPLEX® MAP T-Cell Receptor Signaling Magnetic Bead Kit 7-Plex from Millipore Merck (cat no 48-690MAG) was used to detect changes in phosphorylated CREB (Ser133), Erk/MAP kinase 1/2 (Thr185/Tyr187), and phosphorylated tyrosine residues on CD3 epsilon chain, Lck, ZAP-70, LAT, and Syk in T cell lysates from day 14, hence before and 3 days after the last B cell stimulation (endpoint of the co-culture). The assay was set up according to manufacturer’s instructions loading 15μg protein/well and analyzed on the Luminex® system.51Cr, PerkinElmer, cat no NEZ030S005MC) for 2 hours and subsequently washed prior to co-culture with CAR T cells at different ratios for 4 hours. Co-culture supernatants were mixed with Optiphase supermix  and analyzed on the beta counter Wallac Trilux, 1450 microbeta . Cytotoxicity (% lysis) was calculated as follows: (sample-spontaneous release)/(maximum release–spontaneous release) x 100.Target cells (Daudi and K562) were labeled with radioactive chromium  and treated upon measurable tumor (2 weeks post tumor cell injection) with 5x106 2G, 3G or mock transduced human T cells via i.p. injection (6 mice per group). Survival and tumor size was monitored throughout the experiment using a caliper. Mice were euthanized if tumor ≥1cm2. Animals were housed at the Rudbeck Animal Facility and cared for by the staff according to local regulations. The in vivo experiments were approved by Uppsala Animal Ethics Committee (ref no: C319/9).C57BL/6J nu/nu mice  were injected subcutaneously with 5x105 CD34+ cells selected from umbilical cord blood (UCB) units . Engraftment was confirmed within 6 weeks by measuring human CD45+ and CD19+ cells in the peripheral blood by flow cytometry. Engrafted mice received via tail injection T cells derived from the corresponding UCB negative fraction, transduced with 2G or 3G vector construct and further labeled with FFluc (3x106/mouse). In vivo expansion of T cells was monitored over time by IVIS imaging , while the elimination of CD19+ cell was measured by flow cytometry of peripheral blood samples. By day 60 or 100 mice were euthanized and spleen, bone marrow and blood collected for flow cytometry analysis (human CD45+ and CD3+). CAR expression by T cells was detected using the Cy-5-conjugated goat anti human IgG (H+L) antibody (Jackson ImmunoResearch) that recognizes the human IgG1-CH2-CH3 component incorporated as a spacer region within the CARs. Samples were analyzed in a FACSCalibur (BD Pharmingen) and were analyzed by CellQuest pro software (BD Biosciences). At least 10000 positive events were acquired in each sample. The NSG mice were maintained at Baylor College of Medicine Animal Facility on a BCM IACUC approved protocol.NSG mice 6 weeks of age (Jackson laboratories) were sublethally irradiated (280Gy) and engrafted with 2x10Statistical evaluations were performed using GraphPad Prism . Differences between groups generating p-values <0.05 were considered significantly different.Activated T cells from healthy donors and CLL patients were efficiently transduced with Mock, 2G or 3G virus (MLV vector), resulting in a median 2G CAR expression of 33% ranging from 5 to 90% and a 3G median CAR expression of 27.1% ranging from 3 to 87% at day 14,prior to stimulation. There was a difference in CAR expression when comparing 2G and 3G CAR and also between donors. Therefore, to enable comparison of 3G CAR T cells to 2G, CAR expression was normalized for all functional assays. T cells were expanded with autologous B cells, IL-2 or a combination of the two. After one month’s expansion, CAR T cells stimulated with both B cells and IL-2 had a significantly larger fraction of CAR positive cells  as well 3G CAR T cells were harvested and phenotyped using multicolor flow cytometry before and after one month’s stimulation with IL-2 +/- B cells. As controls, 2G CAR T cells and Mock transduced T cells were used. The majority of cells were of CD8 lineage, yet there were CD4+ T cells present in all cultures  . CAR posAn extended T cell phenotype was determined in the different groups before and after expansion. The phenotypes were defined as follows; naïve: CD45RA+ CCR7+, effector: CD45RA+ CCR7-, central memory: CD45RA- CCR7+ and effector memory: CD45RA- CCR7-. CAR or Mock transduced T cells had a similar phenotype prior to co-culture independently of CAR expression. The majority of T cells had a memory-like phenotype of either central or effector type . After eTo evaluate the activation status of intracellular signaling pathways downstream CAR and signaling pathways activated secondary to CAR stimulation, CAR T cells were stimulated with autologous B cells and analyzed by the PamGene tyrosine kinase array. The Tyrosine Kinase PamChip® Array by PamGene consists of 144 peptides with known phosphorylation sites derived from 100 different proteins. Intact cells or cell lysates can be stimulated and analyzed for the presence of active kinases that will phosphorylate the Array peptides. Hence, an increased phosphorylation of a certain peptide do not mean that the cells contained that certain peptide, but has activated the kinases that can act on those peptides. CAR T cells showed an overall increased activation status compared to Mock transduced cells and 3G T cells were generally more activated than 2G T cells. The activation status in 3G CARs was also maintained over time compared to 2G CARs. Nevertheless, the pattern of what kinases that were activated was similar between 2G and 3G. As expected, signaling molecules downstream of the TCR were phosphorylated also by stimulating CAR receptors such as CD3 zeta, LAT, LCK and ZAP-70. Interestingly, also other proteins involved in regulation of the cell cycle (CDK2), cell adhesion  and exocytosis (Annexins) were phosphorylated. Stimulating cell adhesion may be of importance especially for treatment of solid tumors, and increased exocytosis may result in enhanced serial killing capacity. The most interesting phosphorylation targets are listed in Since the PamGene array only demonstrate the capacity of cells to phosphorylate a library of peptides, we confirmed some of the data using the MILLIPLEX® MAP T-Cell Receptor Signaling Magnetic Bead Kit 7-Plex and Western blots. LAT, ZAP-70, SYK and Erk all showed a similar pattern of higher levels of phosphorylated proteins in 3G CAR T cells compared to 2G T cells . Hence, Cytotoxic capacity was evaluated with a chromium release assay before and after one month’s expansion. 3G CARs were equally efficient as 2G CAR T cells in specific killing of CD19+ target cells . The celProliferation was analyzed weekly during expansion, prior to the addition of autologous B cells and IL-2. After one week’s stimulation, 3G CAR T cells proliferated better than Mock transduced cells (p = 0.0302) . The proCell supernatants were collected 24 hours post stimulation and analyzed for IFNγ and Granzyme B using ELISA. IFNγ was mainly seen in supernatants from the T cell expansion cultures where the CAR T cells had been stimulated with antigen, while Mock and untransduced cells showed low or undetectable levels . Granzym+ cells  were injected subcutaneously with Daudi cells and mice with established tumors were treated with one 3G or 2G CAR T cell infusion. Both 3G and 2G CAR T cells efficiently reduced tumor growth . Additio+ cells  were use T cells . In addiefficacy . Mice euefficacy  or day 1efficacy  also shoEx vivo engineering of the patients’ own T cells with CAR receptors to generate an army of trained killers is now under evaluation for both leukemia, lymphoma as well as for other malignancies [The recent success stories of immunotherapy have demonstrated the potency of the immune system to specifically target and kill tumor cells. The induction of Th1 immunity with T cells, M1 macrophages and NK cells as effectors has proven important for potent and sustained responses. gnancies . There agnancies . 4-1BB ignancies . 2G and gnancies . Howevergnancies , 39, 40 gnancies .In this study we aimed to evaluate the functional capacity of CD19-targeting 3G CAR T cells with CD28, 4-1BB and CD3 zeta signaling domains and compare their capacity to 2G CARs lacking 4-1BB as well as to Mock transduced T cells. CAR T cells could be generated from both healthy donors and CLL patients but the CAR expression of 2G CARs was always slightly higher than 3G CAR expression both related to number of positive cells and number of CAR molecules per cell as judged by a higher mean fluorescent intensity upon analysis of CAR expression. Hence, for some of the functional assays we correlated the data to the number of CAR+ cells to be able to more appropriately compare the ability of the 2G and 3G cells. Further, the T cells were analyzed before and after repeated stimulation with autologous B cells to determine the persistence of CAR T cells.in vivo response. For example, in a primate model genetically modified T cells derived of central memory phenotype was shown to persist longer than effector cells derived from effector memory cells [in vivo expansion of CD19-targeting CAR T cells in lymphoma patients [Co-culture of CAR T cells with autologous B cells stimulated an accumulation of both 3G and 2G CAR positive cells in the cultures, which was not seen in cultures with IL-2 alone. Further, antigen-driven proliferation generated a memory-like phenotype of the cultured CAR T cells with low expression of the exhaustion markers PD-1 and Tim-3. There is emerging evidence that such a phenotype will generate a better ry cells . Furtherry cells . Furtherry cells . Xu et apatients . In our patients  and can ex vivo expansion phase of manufacturing, suggesting in the light of recent studies that proliferation capacity might be more important than cytotoxic capacity [in vivo since the complex network of stimulatory and inhibitory pathways are difficult to mimic in preclinical settings. Tammana et al showed that CARs with costimulatory molecule 4-1BB alone or combined with CD28 provided a more robust antitumor response in vivo compared to CARs with CD28 alone or without costimulatory molecules [The proliferative capacity of 3G CAR T cells upon IL-2 stimulation was higher than that of 2G CAR T cells. 2G CARs were not significantly different from Mock or untransduced cells. Upon repeated stimulation with autologous B cells, both 2G and 3G CAR T cells showed increased proliferation capacity compared to Mock and untransduced cells. With the exception of one donor, 3G CARs had still a higher proliferative capacity than 2G CARs. Barrett et al emphasizes the importance of maintaining proliferation capacity during the capacity . Yet, diolecules . Zhong eolecules . 2G and olecules , yet CARThe intracellular signaling events downstream different CARs affect the efficiency and longevity of the T cells. Considering the use of the CD3 zeta domain in the CAR, TCR-associated proteins will likely participate in CAR signaling but it is not known to what extent other signaling pathways are affected in different types of CAR T cells. In this study, the PamGene station was utilized to investigate phosphorylation status of multiple targets. Upon antigen stimulation both 3G and 2G T cells could rapidly activate kinases that have the capacity to phosphorylate a number of proteins. 3G T cells showed a higher activation status than 2G but both were increased compared to Mock T cells. After 3 hours the activation status declined but more slowly in 3G T cells. As expected, all the common proteins downstream the TCR signaling pathway were activated such as CD3 zeta, LAT, LCK and ZAP-70. These data were confirmed also by analyzing the T cells using the MILLIPLEX® MAP T-Cell Receptor Signaling kit. 3G CAR T cells had a significantly higher phosphorylation status on antigen-mediated signaling molecules compared to 2G CAR T cells. The PamGene array also revealed other possibly phosphorylated molecules that will need further confirmation. For example, Calmodulin phosphorylation was increased post antigen stimulation. Calmodulin is a calcium-binding messenger protein important for many Ca2+ regulated pathways in lymphocytes and other cells. For example, upon Ca2+ release from the endoplasmatic reticulum (ER), calcium-bound Calmodulin activates the phosphatase calcineurin, which in turn promotes NFAT-mediated IL-2 transcription . FurtherCDK2 is a protein involved in the progression of the cell cycle as CDK binding to cyclin E during G1 phase promotes the transition from G1 to S phase. Antigen stimulation together with costimulation activates a CDK cascade including CDK2. However, in the absence of costimulation the T cells are entering a state of anergy, which is enforced by the CDK inhibitory protein p27kip1. The major target of p27kip1 is CDK2. In a study of allograft rejection it was shown that CDK2 was highly active in T cells that infiltrated the allograft . InteresThere were also enhanced phosphorylation of molecules that can potentially be involved in self-regulation of the antigen-induced activation such as Crk, Protein 4.1R, FAK and DYRK1A. Crk adaptor molecules interact with a number of signaling molecules in the cell. Their function is not fully understood but there is one report stating that T cells lacking both Crk and CrkL have a defective chemotactic response to multiple chemokines and may show defective diapedesis. In a cutaneous DTH model, Crk and CrkL were required for efficient T cell migration to sites of inflammation. Crk proteins also controlled T cell activation . NeverthDespite of CAR T cell expansion, there was a remaining CAR+ naïve T cell population in the cultures. Ras is activated by TCR stimulation and has a role in activating the MAP kinases leading towards cell growth. Ras is inactivated by Ras GTPase-activating proteins (RasGAPs) such as RASA1. In a paper by Lapinski PE et al it was shown that RASA1 is an important regulator of thymocyte survival during positive selection as well as promoting the survival of naïve T cells in the periphery . In RAFAOther proteins that can be phosphorylated in CAR T cells post activation are Paxillin, PECAM-1 and EGFR. Paxillin is a cytoskeletal adaptor protein that localizes to the microtubule-organizing center in T cells and is part of the immunological synapse of cytotoxic T cells . Paxilliin vivo. 3G CAR T cells had an increased activation of intracellular signaling pathways concerning both TCR signaling and adhesion molecules compared to 2G CARs. However, the tyrosine kinase array indicates potentially interesting pathways that can be activated or blocked to generate even more efficient CAR designs.Taken together, these data demonstrates that antigen-driven expansion of CAR T cells generates memory-like T cells with maintained cytotoxic capacity. Further, while the phenotype and cytotoxic capacity post repeated antigen stimulation were similar between 3G and its predecessor (2G). The 2G and 3G CAR T cells also showed efficient killing S1 FigA. Experimental outline of the co-culture experiment. 3G, 2G CAR T, mock or untransduced T cells were cultured with autologous B cells, IL-2 or the combination of the two. Proliferation was assessed every week. CAR T cell phenotype and cytotoxic capacity was analyzed before and after co-culture using flow cytometry and chromium release assay, respectively. B. Overview of cell preparation. Autologous T and B cells were isolated from healthy donors and CLL patients using MACS beads. The purity of the B cell population (αCD20-isolation) was confirmed with αCD19 FACS staining (mean expression was 96.4% ranging from 90.5 to 99.5). T cells were isolated from the CD20- fraction using pan T beads, rendering an unlabeled CD3+ population with a mean CD3 expression of 99.3% (range 99–99.7).(TIF)Click here for additional data file.S2 FigCAR- T cells were gated as viable singlet CD3+ CAR- cells. Memory phenotype defined as; naïve: CD45RA+, CCR7+, effector: CD45RA+, CCR7-, central memory (CM): CD45RA-, CCR7+, effector memory (EM): CD45RA-, CCR7. CAR negative T cells show similar expression pattern of memory markers as CAR positive cells before co-culture. However, after co-culture with antigen, the increase in memory phenotype and decrease in effector cells was only seen in the CAR positive population.(TIF)Click here for additional data file.S3 FigComparable expression levels of FF-luciferase was seen in 2G and 3G CAR+ T cells as measured by GFP expression (A). NSG Mice were euthanized on day 60 (B) or day 100 (C). CD19+ B cells were completely eliminated and longterm persistence of CAR+ T cells was seen. T and B cells are gated from the CD45+ population. Panels show phenotype from a representative mouse for each construct.(TIF)Click here for additional data file.S1 TableThe table demonstrates mean values from the tyrosine kinase array by PamGene. CAR T cells (2G and 3G) as well as Mock T cells were stimulated with autologous B cells for 1 and 3 hours. The cells were purified using MACS beads and lysed prior to analysis. Data was normalized for CAR expression.(DOCX)Click here for additional data file.


Sle1.Sle2.Sle3 (TC) mice results in the secretion of autoantibodies. TC dendritic cells (DCs) enhance B cell proliferation and antibody secretion in a cytokine-dependent manner. However, the specific cytokine milieu by which TC DCs activate B cells was not known. In this study, we compared TC and C57BL/6 (B6) control for the distribution of DC subsets and for their production of cytokines affecting B cell responses. We show that TC DCs enhanced B cell proliferation through the production of IL-6 and IFN-γ, while antibody secretion was only dependent on IL-6. Pre-disease TC mice showed an expanded PDCA1+ cells prior to disease onset that was localized to the marginal zone and further expanded with age. The presence of PDCA1+ cells in the marginal zone correlated with a Type I Interferon (IFN) signature in marginal zone B cells, and this response was higher in TC than B6 mice. In vivo administration of anti-chromatin immune complexes upregulated IL-6 and IFN-γ production by splenic DCs from TC but not B6 mice. The production of BAFF and APRIL was decreased upon TC DC stimulation both in vitro and in vivo, indicating that these B cell survival factors do not play a role in B cell modulation by TC DCs. Finally, TC B cells were defective at downregulating IL-6 expression in response to anti-inflammatory apoptotic cell exposure. Overall, these results show that the TC autoimmune genetic background induces the production of B cell-modulating inflammatory cytokines by DCs, which are regulated by the microenvironment as well as the interplay between DC.The breakdown in tolerance of autoreactive B cells in the lupus-prone NZM2410-derived B6. Sle1.Sle2.Sle3 (TC) lupus-prone mouse to investigate how DCs contribute to B cell dysfunction. TC mice are C57BL/6 (B6) congenic mice that express the three lupus susceptibility loci  that are necessary and sufficient to induce a full clinical disease similar to that of NZM2410. The TC autoimmune phenotypes include early pre-disease lymphocyte activation, the production of high titers of anti-dsDNA IgG by 5 month of age, and lupus nephritis by 7 month of age Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the loss of tolerance to self-antigens by B cells, resulting in the production of pathogenic autoantibodies (autoAbs). Dendritic cells (DC) can be classified into two main categories, classical DCs (cDC) and plasmacytoid DCs (pDC). Both pDCs and cDCs promote autoAb secretion by B cells. pDCs produce type I IFN in response to apoptotic cell autoantigens, and cDCs activated either by type I IFN or T cells secrete IL-6. Furthermore, both type I IFN and IL-6 can contribute directly or indirectly to autoAb production We have previously shown that anti-CD40 stimulated bone-marrow derived DCs (BMDC) from TC mice secreted high levels of IL-6 and induced B cell proliferation and Ab production pDCs selectively express TLR7 and TLR9 which recognize RNA or DNA lpr model, including a decreased plasmablast numbers and autoAb production, suggesting a direct effect on B cells Shp1, a phosphatase highly expressed by DCs, led to a lupus-like phenotype with significantly elevated secretion of inflammatory cytokines by DCs Depletion of both cDCs and pDCs ameliorated lupus pathogenesis in the MRL/In this study, we explore the effects of DCs from lupus-prone TC mice on B cell function. Our results show that anti-CD40 activated BMDCs from TC lupus-prone mice induce a greater B cell proliferation in an IL-6 and IFN-γ dependent manner. In addition, IL-6, but not IFN-γ produced by DCs, enhances the secretion of IgM by B cells. Confirming these results, splenic DCs from TC mice produce elevated levels of IL-6 and IFN-γ in the presence of anti-chromatin ICs. Total splenic DCs from TC mice have an expanded pDC population that is largely concentrated in the marginal zone (MZ). This expansion correlated with a type I IFN signature in TC MZ B cells, suggesting an interaction between pDCs and MZ B cells in the lupus mice. Finally, MZ B cells from TC mice maintained a higher IL-6 secretion in response to anti-inflammatory apoptotic cells than MZ B cells from B6 mice. Overall, these results demonstrate that the dysregulation of cytokine networks in DCs from lupus mice, in which IL-6 and both type I and II IFNs play a major role, contribute to the activation of pathogenic B cells.NZM2410/Aeg Sle2NZM2410/Aeg Sle3NZM2410/AegSle1/LmoJ (TC in this paper) congenic strain has been previously described The B6.NZM-Experiments using mice reported here were approved by the Institutional Animal Care and Use Committee of the University of Florida (UF #201303860).4Cl for 5 min to lyse red blood cells and were passed through a pre-separation filter (Miltenyi Biotec) to remove debris. Cells were blocked on ice for 30 min with 10% rabbit serum and anti-CD16/32 (2.4G2) in staining buffer . Cells were then stained for 30 min on ice with pre-determined amounts of the following fluorophore-conjugated or biotinylated Abs: CD21 (4E3), CD23 (B3B4), IgM (II/41), CD11c (HL3), CD8 (53-6.7), DCIR2 (33D1), DEC205 (205yekta), PDCA-1 (927), IFNγ (XMG1.2), all purchased from either eBiosciences or BD Biosciences. Dead cells were gated out with the Fixable Viability Dye eFluor 780. Intracellular staining for IFN-γ was performed on cells fixed and permeabilized with eBiosciences reagents. Stained cells were processed on a CyAn flow cytometer (Beckman Coulter) and at least 100,000 cells were acquired per sample. Flow cytometry data sets were analyzed with the FCS Express software (De Novo).Single cell suspensions of splenocytes were treated with 155 mM of NH− B cells were isolated from B6 splenocytes by negative selection (Miltenyi Biotec). For BMDC-B cell co-cultures, 2×104 BMDCs from either TC or B6 mice were cultured for 5 d with 105 B cells and 10 ug/ml anti-CD40 Ab (1C10) (eBioscience) in duplicate as previously described 4 BMDCs were cultured with 10 ug/ml anti-CD40 Ab for 24 h, after which RNA was extracted for qRT-PCR and gene array analyses, and supernatants were collected from the cultures for ELISA. IFN-γ was also quantified by intracellular flow cytometry, and BMDCs stimulated with either IL-12 (5 ng/ml) or IL-18 (20 ng/ml) were used as positive controls. Mice used in these experiments were 2–3 months of age.Bone marrow (BM)-derived DCs (BMDCs) were differentiated with 10 ng/ml GM-CSF (Peprotech) and 5 ng/ml IL-4 (Peprotech), then purified with anti-CD11c beads (Miltenyi Biotec), as previously described 7 cells from the PL2-8 hybridoma (anti-chromatin IgG2b) b) Two month old mice were first injected i.p. with 250 ul of pristane (Sigma) on d0 and d7. On d10, they were injected with 10Gapdh was used as internal control. The results were normalized to the average unstimulated or 2 month old B6 values. The primers used are listed in Irf7 (Mm00516788_m1) expression relative to Ppia (Mm02342429_g1) endogenous control. ELISA kits were used to quantify IL-6, IL-10, IFN-γ (BD Biosciences), and BAFF (R&D Systems) from the culture supernatants. Additional cytokines from culture supernatants were assessed using the Mouse Autoimmune Response Multi-Analyte ELISArray Kit (Qiagen), all according to the manufacturers' instructions. Microarray gene expression profiling was performed from B6 B cells cultured for 5 d with the supernatant of anti-CD40-activated BMDCs from either B6 or TC mice (N = 4 in each group), as previously described t tests.Gene expression was quantified by qPCR from RNA extracted from BMDCs, splenic DCs or from sorted MZ/FO B cells using Sybr Green (Applied Biosystems) as previously described + cells was computed for the whole splenic section as well as for the marginal zone.Spleens from 2 and 7 month old B6 and TC mice were snap-frozen in Tissue TeK freezing medium (Fisher). Seven micrometer thick frozen sections were fixed to slides in ice-cold acetone for 15 min, air dried for 30 sec and blocked with 1.5% BSA in PBS for 30 min at room temperature. The sections were then stained for 30 min at room temperature in a humidified chamber with purified rat anti mouse PDCA-1 antibody  and followed by Alexa 555–conjugated goat anti-rat IgG (Life Technologies) for another 30 min. Sections stained only with fluorescence labeled secondary antibody were used as control. All tissue sections were mounted in ProLong Gold Antifade Reagent (Life Technologies) and viewed with a Leica DM IRBE inverted Nomarski/epifluorescence microscope outfitted with Leica TCS NT laser confocal optics. Imaging quantitation was performed with MetaMorph 7.5, image analysis . The number of PDCA-1+ apoptotic thymocytes and in 1% of 7AAD+ Annexin V- necrotic thymocytes. Marginal zone and follicular B cells were sorted from purified splenic CD43− B cells as IgM+CD21+CD23− for MZ B cells and IgM+CD21−CD23+ for FO B cells, using a FACS Aria-II cell sorter (BD Biosciences). Post-sorting purity of either subset was greater than 90%. The sorted MZ or FO B cells were co-cultured at a 1∶7 ratio with apoptotic thymocytes and 1 ug/ml CpG-B (Invivogen) for 3 d at 37°C. Cytokines were quantified from extracted RNA and culture supernatant by qPCR and ELISA, respectively.To generate apoptotic cells, thymocytes were cultured with 1 uM Dexamethasone (Sigma) for 4 h at 37°C. Staining with 7AAD and Annexin V (BD Biosciences) determined that this treatment typically resulted in 45% of Annexin Vt tests (paired when appropriate) if the data was normally distributed. Multiple comparison test corrections were applied when needed. When indicated, results were normalized to average values for control B6 samples. Significance levels in figures were labeled as * for p<0.05, ** for p<0.01, and *** for p<0.001.Data analysis was performed with GraphPad Prism 6.0 software. Unless indicated, graphs show means and standard errors of the mean (SEM). Statistical significance between strains was determined by two-tailed Mann-Whitney tests or in vitro  expression. We also compared the expression of type 1 IFN inducible genes that have been reported to be increased in DCs from young TC mice Mx1 and Isg-15 than B6 BMDCs . To determine the mechanisms by which TC DC-produced cytokines regulated B cell functions, we compared the gene expression in B6 B cells that had been stimulated with the supernatant of either B6 or TC anti-CD40-stimulated BMDCs. As previously shown Il17a message expression in B cells exposed to TC-produced supernatant , as well as in the expression of genes induced by Il17a  and lymphoid (CD11c+ CD8+) splenic DCs (+ DEC205- DCs that have been shown to play a role in the activation of extrafollicular B cells + (+ splenic DCs (data not shown). This indicated that this recently described DC subset is not involved in the TC model.We characterized splenic cDC and pDC subsets in young TC and B6 mice to assess whether the differences observed between B6 and TC BMDCs precede disease onset enic DCs . Since p cells + , or DEC2mid PDCA-1+ pDCs was significantly higher in young TC mice than in B6  in TC than in B6 MZ B cells , which confirms a recent study showing low expression of Mx1 by splenic B cells pDCs are known for their capacity to secrete high levels of Type I IFNs  strains . In addi B cells , but Isg B cells . Meanwhiin vivo, given that TLR7 and TLR9 are expressed at high levels in pDCs In addition to Type I IFNs, splenic DCs produce various cytokines in response to TLR7/TLR9 stimulation by anti-chromatin immune complexes (IC) in vitro with stimulated BMDCs. Meanwhile, Isg-15 has been shown to be expressed at higher levels in B6 pDCs than in TC pDCs Isg-15 expression in B6 than in TC splenic DCs Click here for additional data file.


We therefore investigated the in vivo effects of chronic and binge ethanol ingestion and compared to chronic ethanol followed by three repeat binge ethanol on the liver of male C57/BL6 mice fed ethanol in liquid diet (4%) for four weeks followed by binge ethanol . Chronic followed by binge ethanol exacerbated fat accumulation, necrosis, decrease in hepatic SAM and SAM:SAH ratio, increase in adenosine levels, and elevated CYP2E1 levels. Histone H3 lysine acetylation (H3AcK9), dually modified phosphoacetylated histone H3 (H3AcK9/PS10), and phosphorylated H2AX increased after binge whereas phosphorylation of histone H3 ser 10 (H3S10) and H3 ser 28 (H3S28) increased after chronic ethanol-binge. Histone H3 lysine 4 and 9 dimethylation increased with a marked dimethylation in H3K9 in chronic ethanol binge group. Trimethylated histone H3 levels did not change. Nuclear levels of histone acetyl transferase GCN5 and histone deacetylase HDAC3 were elevated whereas phospho-CREB decreased in a distinctive manner. Taken together, acute on chronic ethanol ingestion caused amplification of liver injury and elicited characteristic profiles of histone modifications, metabolic alterations, and changes in nuclear protein levels. These findings demonstrate that chronic ethanol exposure renders liver more susceptible to repeat acute/binge ethanol induced acceleration of alcoholic liver disease.Chronic alcoholics who also binge drink ( Binge drinking associated with chronic alcohol consumption is a major public health concern in the United States and around the globe ,2,3,4. BOxidative stress, increase in adenosine levels, and dysregulated methionine metabolism are considered to be contributing factors for the progression of liver injury ,11,12,13We have shown in a clinically relevant rat model that chronic ethanol followed by repeat ethanol binge is accompanied by augmentation of liver injury . In the Chronic ethanol administration or acute repeat binge caused only a moderate increase in liver steatosis, which was mainly microvascular, whereas there was a marked macrosteatosis in the chronic-binge ethanol group A. Liver s-adenosyl methionine (SAM) and s-adenosyl homocysteine (SAH). Under the experimental conditions used in the present study, chronic ethanol increased SAM levels but a subsequent binge in chronic ethanol treated group resulted in a decrease that was statistically significant  has been shown to stimulate CBP/p300 mediated acetylation of H3K9 and is one of the important transcriptional regulator of hepatic metabolic and inflammatory responses ,37,38.Phosphorylation of CREB that usually occurs in the cytosol facilitates its translocation into the nucleus ,38. Therin vivo and in primary hepatocytes thus contributing to ethanol induced oxidative stress [Heavy binge drinking in patients who are chronically consuming alcohol is the most common cause for the worsening of liver damage in chronic alcoholic liver disease ,8,9. To e stress ,11,33. Ae stress ,31,32. He stress ,40. In tEnhanced adenosine release from hepatic slices from alcohol treated mice and increased expression of adenosine receptors have been reported in animal models of alcoholic liver injury and in humans ,13. Mores-adenosyl methionine (SAM) levels increased after chronic ethanol but decreased by chronic ethanol binge. Although increased SAM levels indicate adaptive response to chronic ethanol administration, the significant decrease in SAM levels and decreased ratio of SAM/SAH suggest increased cytotoxic effects of chronic ethanol-binge combination under the experimental protocols utilized in the present study. Decreases in SAM levels are associated with impairment in essential methylation reactions through inhibition of various methyltransferases including phosphatidylethanolamine methyltransferase, isoprenylcysteine carboxyl methyltransferase and protein L-isoaspartate methyltransferase. Decreased activity of these enzymes is associated with increased steatosis, increased apoptosis and increased accumulation of damaged proteins seen in alcoholic liver disease [Dysregulation of methionine metabolism is one of the hepatotoxic effects of ethanol . Hepatic disease ,40.Chronic ethanol intake or binge alone caused insignificant increase in histone H3S10 and H3S28 phosphorylation whereas binge after chronic ethanol increased histone H3S10 and H3S28 phosphorylation. In contrast to histone phosphorylation, histone H3K4 and H3K9 dimethylation increased in chronic, binge, and chronic binge groups. H3K4 dimethylation is usually associated with induction of gene expression and H3K9 dimethylation is associated with silencing of gene expression . This suIn contrast to H3S10 and H3S28 phosphorylation, H3K9 acetylation was increased in the binge ethanol but not in the chronic ethanol-binge group. Histone H3K9 acetylation is dependent on a balance between the activation of histone acetyl transferases and histone deacetylases. We have reported a role of GCN5 in ethanol induced H3K9 acetylation in ethanol metabolizing human hepatoma cell line (VA13 cells) . Levels CREB is a key transcription factor playing a pivotal role in liver regeneration, hepatic carbohydrate metabolism, suppression of hepatic lipogenesis, and hepatic inflammatory responses. The levels of phospho-CREB in the whole cell extract were significantly increased in chronic ethanol, binge and chronic ethanol binge but nuclear levels were markedly decreased with most pronounced decrease noted in the ethanol binge group. Thus, dysregulation of nuclear P-CREB levels may be an underlying mechanism in alcohol induced liver injury by both binge and chronic ethanol intake. One mechanism that may contribute to transcriptional regulation is CREB dependent CBP/p300 mediated histone acetylation ,37.Antibodies to histone H3AcK9, phospho-H3 ser 10, phospho-H3 ser 10, phospho-H3 ser 28, histone H3 protein, dimethyl H3-lys4, dimethyl H3-lys9, phopsho-H2AX were obtained from Chemicon . Antibodies to GCN5 and CYP2E1 were procured form Santa Cruz . Antibody to HDAC3, phospho-CREB, and CREB protein were procured from Cell signaling . Triglyceride assay kit (Cat #F6428), protease inhibitors cocktail (p8340), and anti β-actin antibody were purchased from Sigma-Aldrich .v/v) in sterile water and orally gavaged to the stomach using a 20-gauge stainless steel blunt tipped needle. The average amount of intragastric alcohol solution was about 0.45 mL. The three ethanol binge were given consecutively each 12 h apart. Four hours after the last binge ethanol administration blood and liver samples were collected. Liver was perfused with cold phosphate buffered saline containing phosphatase inhibitors before collection. A small portion of the liver was placed in formalin for immunohistochemistry and the reminder was frozen in liquid nitrogen and stored at −70 °C. The animal care and protocol used in this study was approved by the University of Missouri Animal Care Committee (Protocol #8175) and complied with NIH guide for the care and use of laboratory animals.Seven week old C57BL/6J male mice, were purchased from Harlan  and housed under a 12-h/12-h light/dark cycle. Animals were permitted ad libitum consumption of standard laboratory chow for one week to acclimatize. This was followed by feeding animals with Lieber-DeCarli liquid diet . Ethanog for 5 min. The supernatant was used for triglyceride estimation using the assay kit and protocol provided by the supplier .Serum ALT levels were measured by kinetic ALT assay in an automated analyzer . The levels of Liver SAM, SAH, adenosine and GSH were measured by high performance liquid chromatography as described earlier ,30. Form2, 5 mM glycerophosphate, 1 mM EDTA, 1 mM Na-orthovanadate, 1 mM EGTA, 1 mM DTT, and Sigma protease inhibitor cocktail (p8340)), a small portion of the whole cell extract was saved. The remainder of the homogenate was layered over 1.0 M sucrose cushion. After centrifugation at 1600× g for 10 min at 4 °C, the cytosolic fraction (supernatant) was transferred to a precooled microcentrifuge tube, frozen in liquid nitrogen and stored at −70 °C. The nuclear pellet was further suspended in 1.0 M sucrose containing 0.25% NP-40 and passed through a 26-gauge needle 6 times and centrifuged at 1600× g for 10 min. This step removed contaminated endoplasmic reticulum and plasma membrane. The nuclear pellet was again re-suspended in 1.0 M sucrose and centrifuged at 1600× g for 10 min followed by washing the pellet once with homogenization buffer. The purity of the nuclear fraction was verified by the absence of b-tubulin and calreticulin and presence of histone H3 [The whole cell extract and nuclear extracts were prepared at 4 °C as described earlier . After hstone H3 . The nucThe whole liver lysate protein or nuclear fraction (40 µg) was separated by 10% or 15% SDS-PAGE followed transfer onto nitrocellulose membrane using Bio-Rad Trans-Blot apparatus. Membrane was washed with 20 mM Tris, pH 7.5, containing 0.1% Tween 20 and 150 mM NaCl (TBST) and blocked with TBST containing 5% nonfat dry milk for 2 h at room temperature. Membrane was incubated with primary antibody overnight at 4 °C in blocking buffer and washed with TBST, followed by incubation with corresponding secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. Immunoblots were visualized using chemiluminescent reagent . LSA-3000 imaging system  was used to capture chemiluminescence and Multi Gauge™ software (Fujifilm Life Sciences) was used for quantitation of immune blots. The chemiluminescence intensity was within the linear range of detection. Beta actin and histone H3 protein immune blots were used for equal protein loading of whole cell extracts and nuclear extracts respectively. Repeat immunoblotting was done by stripping the membrane using Restore Western blot stripping buffer (Pierce).p-value of <0.05 were considered statistically significant.Data obtained from 4 animals in each group were used for analysis. The data are expressed as mean ± S.E.M. ANOVA was used for statistical analysis and differences with a In conclusion, augmentation of liver injury by repeat acute binge in chronic ethanol treated mice is accompanied by increased levels of CYP2E1 protein, ALT, triglyceride, decrease in the SAM:SAH ratio, marked increase in hepatic adenosine levels, and increased H3 phosphorylation , a histone modification known to enhance TNFα gene expression. The association between gene polymorphism for CYP2E1, methionine metabolism, and TNFα has been considered in the alcoholic liver injury in humans . Analogo


During the early stage of the avian influenza A(H7N9) epidemic in China in March 2013, a strain of the virus was identified in a 4-year-old boy with mild influenza symptoms. Phylogenetic analysis indicated that this strain, which has similarity to avian subtype H9N2 viruses, may represent a precursor of more-evolved H7N9 subtypes co-circulating among humans. Influenza A(H7N9) virus infected >400 persons in China during March 2013–April 2014  (SH/JS01), which was detected in a patient with mild influenza symptoms in Shanghai during March 2013, during the very early stage of the influenza A(H7N9) epidemic. Phylogenetic analysis indicates that this strain may represent an earlier precursor of the more evolved H7N9 subtypes co-circulating at low levels at the time of isolation in March 2013 thus providing insight into the evolution of H7N9 subtypes.A mild case of influenza A(H7N9) virus infection was identified in a 4-year-old boy in a rural area of Jinshan District, Shanghai, reported on March 31. The patient had been exposed to poultry. His signs and symptoms included acute fever (maximum 39° axillary), cough, nasal drainage, and tonsillitis. A diagnosis of upper respiratory tract infection was made, and the child recovered after 5 days of antiviral drug therapy (http://platform.gisaid.org/epi3/frontend#57f951) and GenBank . Strict controls were used during PCR amplification; results were confirmed by another laboratory to exclude contamination with laboratory strains. We constructed maximum likelihood trees of each gene segment sequence using the general time-reversible model implemented in MEGA 5.1 , A/Shanghai/2/2013 (SH/2), and A/Anhui/1/2013 (AH/1). In the HA gene of SH/JS01, the only mammalian adapting substitution observed was 138A (H3 numbering); amino acid residues involved in receptor-binding specificity showed avian-like signatures, including 186G and 226Q, which were similar to SH/1 but distinctive from SH/2 and AH/1. In the internal genes of SH/JS01, we observed some human-like and mammalian-adapting signatures, including 89V in polymerase basic subunit (PB)2, 368V in PB1, 356R in the RNA polymerase acidic subunit, 42S in nonstructural gene 1, and 30D and 215A in matrix gene 1; however, some hallmark changes involved in mammalian adaptation still showed avian signatures, including 627E and 701D in PB2 and 100V and 409S in the RNA polymerase acidic subunit . Most stPhylogenetic analysis and divergence time estimation showed that the SH/JS01 HA gene diverged in October 2005 and was closely related to SH/1; nucleotide similarity was 99.7% online Technical Appendix Figure 1). However, the NA gene, which is most closely related to A/northern shoveler/Hong Kong/MPL133/2010(H2N9) and A/duck/Jiangxi/21714/2011(H11N9) with nucleotide similarity of 99% and 99.3%, respectively, are estimated to have diverged in September 2010, earlier than that of known strains of the 2013 H7N9 subtype (estimated to have occurred in January 2011)  and major (M) clades in the phylogenetic trees, and first 9, then 27 genotypes  Figure 3SH/JS01, a distinct H7N9 virus strain identified in 2013 during the early stage of the influenza A(H7N9) epidemic in China, provided information to define the evolution of the H7N9 subtype. Although identified in an infected human, SH/JS01 has more avian-prone properties and fewer mammalian-adapting mutations than other known human 2013 H7N9 subtypes. SH/JS01 has a waterfowl-like NA gene characterized by the absence of a deletion in the NA stalk and most of its internal genes are more closely related to avian H9N2 subtype strains isolated during the 2011–2012 influenza season than to other recently emerged strains of the H7N9 subtype. Molecular clock analysis further predicted an earlier divergence time in most genes of SH/JS01. These findings indicate that SH/JS01 might be a precursor strain of the H7N9 virus that co-circulated with more evolved viruses, although we cannot exclude that SH/JS01 may have been generated independently from the other H7N9 strains by reassortment of waterfowl strains with avian H9N2 strains and then transmitted directly to a human.The sequences of SH/JS01 contained more avian-like signatures than those of other H7N9 isolates from humans; this underscores the potential of these viruses to infect humans. The phenotypic characteristics of SH/JS01, which might describe its zoonotic potential, remain to be investigated. It is unclear whether other SH/JS01–like viruses are still circulating in poultry in China and if so, what the potential is for their evolution and ability to infect humans. Intensive influenza surveillance and additional influenza A virus genome sequences isolated from poultry and from humans with severe and mild manifestations of infection are needed to clarify the population dynamics of H7N9 viruses.Phylogenetic analysis of avian influenza viruses, 1988–2013


A novel avian H7N9 virus with a high case fatality rate in humans emerged in China in 2013. We evaluated the immunogenicity and protective efficacy of a candidate Vero cell culture-derived whole-virus H7N9 vaccine in small animal models.Antibody responses induced in immunized DBA/2J mice and guinea pigs were evaluated by hemagglutination inhibition (HI), microneutralization (MN), and neuraminidase inhibition (NAi) assays. T-helper cell responses and IgG subclass responses in mice were analyzed by ELISPOT and ELISA, respectively. Vaccine efficacy against lethal challenge with wild-type H7N9 virus was evaluated in immunized mice. H7N9-specific antibody responses induced in mice and guinea pigs were compared to those induced by a licensed whole-virus pandemic H1N1 (H1N1pdm09) vaccine.The whole-virus H7N9 vaccine induced dose-dependent H7N9-specific HI, MN and NAi antibodies in mice and guinea pigs. Evaluation of T-helper cell responses and IgG subclasses indicated the induction of a balanced Th1/Th2 response. Immunized mice were protected against lethal H7N9 challenge in a dose-dependent manner. H7N9 and H1N1pdm09 vaccines were similarly immunogenic.The induction of H7N9-specific antibody and T cell responses and protection against lethal challenge suggest that the Vero cell culture-derived whole-virus vaccine would provide an effective intervention against the H7N9 virus. A novel influenza A/H7N9 virus emerged in February in China in 2013 . Despiteex vivo lung and trachea explant cultures [The novel H7N9 virus binds both to avian  and human  receptors [cultures . H7N9 vicultures , associacultures . Moreovecultures . H7N9 iscultures , and limcultures ,14. Sevecultures , and to Before the emergence of the novel H7N9 virus, transmission of H7 viruses from birds to mammals had been reported only rarely, and human infections with N9 subtype viruses had not been reported. Accordingly, in a seroepidemiological study, no pre-existing immunity to H7N9 was detected in any age groups , we compAll animal experiments were reviewed and approved by the Baxter Bioscience Institutional Animal Care and Use Committee (IACUC Vienna/ Orth) and animal welfare officers. Animal experiments were conducted in accordance with Austrian laws on animal experimentation and approved by Austrian regulatory authorities (permit number LF1 TVG-38/009–2011). Experiments were conducted according to guidelines set out by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animals were housed according to EU guidelines, in housing facilities accredited by the AAALAC, and monitored daily. For blood sampling and virus challenge, all animals were anaesthetized with isoflurane using a UNO—Univentor Anaesthesia Unit according to the manufacturer`s protocol. Humane endpoints were used during the study, based on weight loss—animals with >25% weight loss were sacrificed using carbon dioxide treatment. All efforts were made to minimize suffering.The non-adjuvanted Vero cell culture-derived inactivated whole-virus H7N9 vaccine was manufactured using a H7N9 A/Anhui/1/2013 virus  obtained from the National Institute for Biological Standards and Control, UK. Vaccine virus was grown in Vero cell culture and double inactivated with formalin and UV irradiation, then purified by continuous sucrose gradient centrifugation and ultra/diafiltration steps prior to formulation, as previously described for licensed H1N1pdm09 and H5N1 vaccines ,20.5 TCID50 (5000 LD50) of H7N9 virus in 50 μl buffer (25 μl in each nostril). Challenged mice were monitored each work day for 14 days after challenge. Clinical symptoms were categorised as ‘ruffled fur’, ‘hunched back’, or ‘lethargic’; each category being assigned a numerical value of 1, and scored cumulatively for individual mice. Animals dying within the 14 day observation period were assigned a value of 6. For T cell analyses and IgG subclass determination in H7N9-immunized mice, blood was drawn and spleens were harvested from 5 of 10 mice 6 days after the first immunization and from the remaining 5 mice three weeks after the second immunization. Animal experiments were conducted in accordance with Austrian laws and guidelines set out by the Association for Assessment and Accreditation of Laboratory Animal Care International.Groups of 10 female DBA/2J mice (Janvier), aged 6–9 weeks, or groups of 5 female Duncan-Hartley guinea pigs , aged 6–9 weeks, were injected subcutaneously twice, three weeks apart, with six different 5-fold dilutions of vaccine ranging from 3.75 μg to 0.0012 μg HA antigen (in a volume of 0.5 mL) or buffer. For the H7N9 vaccine, due to the non-availability of standardized reagents, HA antigen content was estimated based on total protein content, whereby 10 μg total protein is estimated to contain 3.75 μg HA antigen, based on the HA content of the H1N1pdm09 comparator vaccine, as measured by SRD analysis. Additional characterization of the content of the H7N9 and H1N1pdm09 viral proteins HA, NA, NP and M1 was carried out by SDS gel analysis followed by densitometric analysis of coomassie-stained SDS-PAGE gels. Individual bands were identified by excision and in-gel digested with trypsin. Tryptic peptides were then analyzed by liquid chromatography-mass spectroscopy and the masses of the peptides searched against virus specific protein databases  using the search engine “BioworksBrowser”. Densitometry measurements were calibrated against a purified GAPDH protein standard. Viral RNA content was determined by extracting RNA from vaccine preparations using hot guanidinium thiocyanate-phenol-chloroform-based extraction method , followed by analysis using the Agilent Small RNA Kit and Agilent Bioanalyzer 2100 (Agilent Biotechnologies), according to the manufacturer’s instructions. Blood was drawn three weeks after the second immunization for serological analyses. In addition, three weeks after the second immunization with H7N9 vaccine, mice were challenged intranasally with 1 x 10Blood was drawn 3 weeks after the second immunization for serological analyses. HI antibody titers were determined using chicken or horse erythrocytes as previously described . Sera wiThe frequency of H7N9-specific IFN-γ- or Interleukin-4 (IL-4)-secreting cells was analyzed using mouse IFN-γ and IL-4 enzyme-linked immunospot (ELISPOT) kits , as previously described , using vELISA plates were coated overnight at 4°C with either recombinant H7N9 HA (rHA) or polyclonal anti-mouse Fab2 IgG (Sigma). After blocking non-specific binding and subsequent washing, diluted sera or serial dilutions of purified murine IgG1, IgG2a or IgG2b (Sigma) were added to wells containing H7N9 rHA  or Fab2 IgG. Plates were incubated for 1 h at room temperature, and washed again, prior to further incubation for 1 h with IgG subclass-specific peroxidase-conjugated goat anti-mouse IgG antibodies. Bound IgG subclass antibodies were detected colorimetrically using TMB substrate.50  and PD50  values were calculated using in-house software based on the one-hit model [EDit model . StatistResults of the characterization of the content of the H7N9 and H1N1pdm09 viral proteins by SDS gel and densitometric analyses of coomassie-stained SDS-PAGE gels are shown in The ability of the whole-virus H7N9 vaccine to elicit H7N9-specific HI, MN and NAi antibodies was investigated by immunization of guinea pigs and DBA/2J mice. To investigate the hypothesis that H7N9 vaccines would be less immunogenic than other influenza vaccines , we also50 values for the individual serological assays were calculated as 16 ng for HI (horse erythrocytes), 16 ng for HI (chicken erythrocytes), 19 ng for MN, and 35 ng for NAi.Immunization with the H7N9 vaccine induced dose-dependent antibody responses in guinea pigs, as measured by HI, MN and NAi assays , 169 ng for MN, and 401 ng for NAi. Assessment of H7N9 vaccine-induced HI antibody titers using horse erythrocytes in the HI assay resulted in consistently higher titers than using chicken erythrocytes; differences were highly significant (p<0.001) for the three highest doses.Antibody responses induced by the H7N9 whole-virus vaccines in mice are shown in There was a highly significant correlation of MN titers with HI titers in guinea pigs and mice for HI assays utilizing horse erythrocytes  and chicken erythrocytes . A highly significant correlation  between vaccine-induced NAi titers and HI or MN titers was also calculated in both species, indicating that immune responses directed against both the HA and NA proteins of the whole-virus H7N9 vaccine increase proportionally in a dose-dependent manner.50 values calculated for the H7N9 and H1N1pdm09 vaccines were very similar, with overlapping 95% CI values for all comparisons. Taken together, these data indicate that the whole-virus H7N9 and H1N1pdm09 vaccines are similarly immunogenic in mice and guinea pigs.Antibody responses induced by the H1N1pdm09 vaccine in guinea pigs and mice are shown in 50 = 20 TCID50). No consistent symptoms or death were seen in any of these models after challenge with H1N1pdm09, so no challenge studies were done with that virus. Consequently, the ability of the whole-virus H7N9 vaccine to protect against high titer lethal H7N9 virus challenge was assessed in immunized DBA/2J mice. Mice which had received two immunizations, three weeks apart, were challenged with high titer  of wild-type H7N9 virus. 50 value for the H7N9 vaccine was calculated to be 0.008 to 0.01 μg HA.Pilot challenge experiments (data not shown) using CD1, BALB/c, and DBA/2J mice, as well as guinea pigs indicated that only DBA/2 mice exhibited visible disease symptoms and death after challenge with H7N9 virus  cell responses induced by the whole-virus H7N9 vaccine, DBA/2J mice were immunized twice, three weeks apart, with the 3.75 μg HA vaccine dose. To distinguish between Th1- and Th2-type T cell responses, spleen cells from spleens harvested one week after the first immunization and three weeks after the second immunization were evaluated by IFN-γ  and IL-4To further substantiate the differences in the Th cell responses, immune sera collected at the same time points were evaluated by ELISA to distinguish between HA-specific IgG1, characteristic of a Th2 type response, and IgG2a and IgG2b responses, which are indicative of a Th1 type response . Three wA non-adjuvanted, Vero cell culture-derived whole-virus H7N9 vaccine was produced using identical methodologies previously used for the manufacture of licensed H1N1pdm09 and H5N1 vaccines ,20. CharPilot experiments using CD1, BALB/c, and DBA/2J mice demonstrated that visible disease symptoms and death were seen only after challenge of DBA/2J mice with H7N9. This result is in contrast to previous studies which have demonstrated that 100% lethality could be achieved in BALB/c mice after challenge with a mouse-adapted A/Anhui/01/2013 H7N9 strain . HoweverThe whole-virus H7N9 vaccine was highly immunogenic in guinea pigs and mice and provided dose-dependent protection of mice against lethal challenge with wild-type H7N9 virus. Antibody responses induced by the H7N9 vaccine were very similar to those induced by a licensed whole-virus H1N1pdm09 vaccine.High HI titers were induced by the H7N9 vaccine in both animal models as measured in HI assays using either chicken or horse erythrocytes. Previous studies had indicated that chicken erythrocytes are insensitive for the detection of H7 HA antibodies, as a result of the relatively low density of α2,3-linked sialic acid receptors on chicken compared to horse erythrocytes ,24. The 50 values of 1:5, that is, below the level of detection in the HI and MN assays [Seasonal influenza vaccines are licensed on the basis of HI antibody titers, which have been shown to correlate with clinical protection ,32. HoweN assays .50 challenge dose. The reason for this is not known. However, in previous studies, the use of a challenge dose of 105 H5N1 also did not result in consistent death of all the buffer control mice [Surprisingly, 100% lethality was not seen in the control challenge groups, despite the use of a 5000 LDrol mice . It coul+ T cell responses are important for the activation of CD8+ T cells, macrophages and natural killer cells, which have a direct role in prevention of severe disease and mortality [+ T cell responses provide cognate help to B cells, a prerequisite for immunoglobulin switch, affinity maturation and the establishment of B-cell memory [In addition, the survival curves following challenge show an unusual pattern in that animals die continuously from Day 4 to Day 14, in contrast to data previously published for H7N9 in the BALB/c model . This maortality , whereasl memory . A balanTaken together, comparison of HI, MN and NAi antibody responses induced by the whole-virus H7N9 vaccine and the licensed whole-virus H1N1pdm09 vaccine indicate that both vaccines are similarly highly immunogenic. Moreover, the induction of a potent, balanced T cell response by the H7N9 vaccine in DBA/2 mice, as was demonstrated earlier for an H1N1pdm vaccine in BALB/c mice , provideLimited data for other H7N9 vaccines have been reported to date. An H7N9 virus like particle (VLP) vaccine, either non-adjuvanted or adjuvanted with a novel saponin-based adjuvant formulation, was reported to induce HI and NAi antibodies in mice , but theThe same Vero cell technology used to manufacture the whole-virus H7N9 vaccine has previously been used to manufacture licensed H1N1pdm09 –18,50 an


In vitro, the purified RAD51 G151D protein directly and significantly enhances DNA strand exchange activity in the presence of RPA. In concordance with this result, co-incubation of G151D with BRCA2 resulted in a much higher level of strand-exchange activity compared to WT RAD51. Strikingly, the RAD51 G151D variant confers resistance to multiple DNA damaging agents, including ionizing radiation, mitomycin C, and doxorubicin. Our findings demonstrate that the RAD51 G151D somatic variant has a novel hyper-recombination phenotype and suggest that this property of the protein is important for the repair of DNA damage, leading to drug resistance.The RAD51 protein plays a key role in the homology-directed repair of DNA double-strand breaks and is important for maintaining genome stability. Here we report on a novel human RAD51 variant found in an aggressive and therapy-refractive breast carcinoma. Expression of the RAD51 G151D variant in human breast epithelial cells increases the levels of homology-directed repair. Expression of RAD51 G151D in cells also promotes high levels of chromosomal aberrations and sister chromatid exchanges.  Therapeutic resistance is a major hurdle for the treatment and eradication of cancer. Furthermore, the development of therapeutic resistance significantly decreases patient survival and negatively impacts the quality of life of patients battling cancer. Cancer cells utilize a number of previously described mechanisms in order to overcome sensitivity to cancer therapeutics, including overexpression of RAD51. However, in this study we report a novel gain-of-function heterozygous somatic variant, RAD51 G151D, identified in a highly refractory and aggressive breast adenocarcinoma. RAD51 G151D induces a hyper-recombination phenotype in human cells resulting in increased resistance to therapeutics via enhanced HDR of DSBs. We further demonstrate enhanced DNA strand exchange activity in the presence of RPA, providing a possible mechanism for the hyper-recombination phenotype observed in cells. Our study presents a novel hyper-recombinant RAD51 tumor-associated variant (RAD51 G151D), providing the first evidence that links altered RAD51 function with therapeutic resistance as well as a novel genetic marker to identify patients at high risk for aggressive and refractory disease. Human RAD51 is a RecA-like recombinase required for HDR (homology-directed repair) of DSBs (double-strand breaks), forming helical nucleoprotein filaments on DNA in an ATP-dependent manner and catalyzing strand exchange between homologous sequences. RAD51 is an essential protein for genome maintenance with roles in both HDR and replication fork maintenance . Both geCurrently, the association between cancer incidence and/or progression and RAD51 is strictly correlative. Previous studies have shown elevated RAD51 expression levels in prostate cancer, invasive breast cancer and small cell lung cancers –22; howeIn this study, we investigated the cellular effects of a heterozygous somatic tumor variant, RAD51 G151D, we identified in 1 out 32 breast tumors analyzed by DNA sequencing. Previously, we demonstrated that the RAD51 G151D protein possesses altered biochemical and biophysical properties . LocatedRAD51 G151D was identified in an African-American woman with early-onset infiltrating ductal adenocarcinoma. After failed radiation and chemotherapy treatment at the primary site, the patient developed metastatic disease only a year after the initial diagnosis. Attempts to eradicate the sites of metastasis using radiation and chemotherapy were unsuccessful. Given the potential linkage of RAD51 with chemo-resistance and genome instability –35, we pSceI results in a chromosomal DSB at an integrated I-SceI recognition site in a gene encoding for GFP (SceGFP). Repair of the I-SceI-induced double strand break via HDR, using a truncated GFP repeat (iGFP) downstream as a template, results in restoration of a functional GFP gene subsequently measured by flow cytometry. As shown in SceI-induced DNA DSB.To determine the HDR efficiency of RAD51 G151D activity at DSBs, we generated MCF-7 DR-GFP cells with stable and equivalent expression of RAD51 WT or G151D  and perf-SceI-induced DNA DSB restores GFP expression, repair by other pathways such as non-homologous end-joining (NHEJ) or single-strand annealing (SSA) can occur. Repair by HDR, NHEJ or SSA each result in loss of the I-SceI recognition site, therefore I-SceI site loss can be used to query overall DSB repair . We. We-SceIng cells  using a ng cells  37]. Si. Si-SceIAs an outcome of HDR, elevated sister chromatid exchanges (SCEs) can be indicative of increased levels of HDR as well as increased illegitimate or inappropriately regulated HDR . TherefoRAD51 forms discrete nuclear foci in response to endogenous and exogenous DNA damage and these foci are considered sites of HDR repair of damage . PreviouTo visualize DSB repair after exposure to IR, we monitored gamma-H2AX (γ-H2AX) and p53 binding protein 1 (53BP1) foci using immunofluorescence. Phosphorylation of H2AX occurs rapidly when DSBs are present; the resultant γ-H2AX product forms foci at sites of DSBs, participating in the recruitment of DNA repair proteins to the break site. Similarly, 53BP1 rapidly forms discreet foci upon IR exposure at sites of DSBs, as compared to the diffuse localization demonstrated in undamaged cells. Therefore, both γ-H2AX and 53BP1 are surrogate markers for DSBs. MCF10A cells expressing RAD51 G151D had significantly fewer γ-H2AX foci compared to WT cells at 4 and 8 hours post IR exposure  as well Enhanced repair via HDR is likely to confer resistance to DNA damaging agents that result in DSBs. From a translational perspective, the tumor from which RAD51 G151D was identified turned out to be largely refractory to a range of therapeutic interventions including ionizing radiation (IR) and mitomycin C (MMC). Therefore, we characterized the response of MCF10A cells expressing either RAD51 WT or RAD51 G151D to IR, MMC, and doxorubicin. IR can lead to both DNA SSBs and DSBs through the formation of hydroxyl radicals or by direct ionization. MMC is an alkylating agent that forms interstrand DNA cross-links leading to inhibition of DNA replication and collapse of replication forks thereby resulting in the formation of DSBs . Cells ein vivo. However, it remained unclear whether the fidelity of HDR is affected by RAD51 G151D expression. To address this question, we prepared metaphase spreads from early passage MCF10A cells expressing RAD51 WT or G151D and scored chromosomal aberrations. As shown in The data thus far indicate that expression of RAD51 G151D resulted in increased HDR The mechanism by which RAD51 G151D induces a hyper-recombination phenotype was pursued with particular interest since the mutation is not located in regions vital to RAD51 recombinase function, including the single- and double-stranded DNA binding surfaces (loops L1 and L2) or Walker A and B motifs. The location of the mutation in a loop on the outer surface of the RAD51 monomer and RAD51 filament indicated the potential for changes in protein-protein contact. We initially hypothesized that RAD51 G151D may possess altered affinity for pro-recombination mediators such as BRCA2 or PALB2 resulting in increased loading efficiency of RAD51 G151D onto ssDNA. However, as shown in Enhanced DNA strand exchange in the presence of RPA provides a plausible mechanistic framework for the hyper-recombination phenotype observed in cells expressing the G151D variant. However, in our cell models, the G151D variant is expressed in the presence of endogenous RAD51 potentially mimicking the heterozygous state present within the patient’s tumor. We previously demonstrated that RAD51 WT and G151D directly interact in a yeast two-hybrid system and presumably assemble into mixed filaments in a biochemically reconstituted EMSA analysis . Taken tFRET) approaching 1.0 (FRET approaching 0 (FRET). The addition of 400 nM of RAD51 WT or G151D in the presence of 2 nM ATP decreases the EFRET, indicating efficient filament formation on the ssDNA tail   for 20 min followed by treatment with 0.5 mM hydroxyurea (HU), IR (8 GY) or untreated then pulsed with chlorodeoxyuridine (CIdU) for 20 min , mitomycin C, and 5-fluorouracil, along with ionizing radiation (IR), eventually succumbing to metastatic disease. In light of the hyper-recombination phenotype induced by RAD51 G151D presented in this study, we suggest that RAD51 G151D contributed to the refractory and aggressive nature of the breast cancer from which it was identified.rad55 and rad57 mutant yeast strains [The DNA strand exchange activity of RAD51 G151D was higher than WT in both the oligonucleotide DNA strand exchange assay and the smFRET analysis, providing a mechanistic explanation for the hyper-recombination phenotype observed in cellular assays, including increased RAD51 foci, increased HDR, and enhanced DSB repair. Gain-of-function mutations in yeast Rad51, predominantly in the L2 loop in Rad51, were previously shown to decrease sensitivity to IR in  strains . The Rad strains ,52. Wher strains . Increas strains . FurtherFRET distributions produced by the respective proteins. In conjunction with the measured decrease in ATP hydrolysis, it is conceivable that G151D may form a more stable filament. A more stable RAD51 filament could promote the formation of double Holliday junctions (dHJs) resulting in increased crossover events (increased SCEs) with mutagenic outcomes .Increased filament stability is another possible contributing factor to the hyper-recombinant activity of RAD51 G151D. We previously demonstrated a 6-fold decrease in catalytic efficiency of ATP hydrolysis by RAD51 G151D compared to WT . The dec1, utilization of imprecise templates (e.g. homologous chromosome or repetitive sequences), and improper dissolution of Holliday junction intermediates can lead to mutations, loss-of-heterozygosity, and other genomic abnormalities [HDR is a cell cycle regulated process utilizing multiple mechanisms at each step to prevent inappropriate engagement or erroneous recruitment of HDR proteins to the site of DNA damage ,54. Inapmalities ,55–57. Fmalities ,40,58–60malities . ChangesRAD51 G151D contributes significantly to increased genomic instability in both non-transformed and transformed cells. In this report we demonstrate increased SCEs in cells expressing RAD51 G151D. Human mitotic cells preferentially process HDR intermediates in favor of non-crossover (NCO) events to prevent removal of genetic information in the form of loss-of-heterozygosity (LOH) –63. TherG151D mutation was identified failed to respond to a number of therapeutics, including IR, MMC and doxorubicin. In total, our data lead us to conclude that RAD51 G151D expression directly contributed to the therapeutic resistance of the primary and metastatic tumors in the patient. Previous work has demonstrated multiple mechanisms by which tumor cells can acquire multidrug resistance [We propose that RAD51 G151D contributes significantly to the refractory phenotype of cancer cells, by conferring resistance to therapeutics as a result of its hyper-recombinant phenotype. We have shown that expression of RAD51 G151D in two independent human cell models confers increased resistance to DNA damaging agents. The patient from whom the sistance –69. Oversistance ,34,70,71RAD51 G151D mutation as clinical guide for predicting therapeutic response and disease progression could have a significant impact on the survival outcome of cancer patients harboring this somatic tumor variant. Furthermore, our results provide support for future studies to identify RAD51 mutations and other HDR-associated variants associated with disease progression and therapy resistance. We anticipate that further detailed mechanistic insights into the role of HDR factors in cancer will lead to novel pharmaceutical targets and improved clinical outcomes for those patients with refractory tumors and metastatic disease.Genetic diversity and adaptation in cancer cells is driven, in part, by loss-of-heterozygosity and copy number variation, which generate populations of cells with increased proliferative capacity, resistance to DNA damaging agents, and invasive and metastatic properties. We propose that RAD51 G151D increases the levels of cellular genomic instability and resistance to mainline DNA damaging agent therapy, driving the clinically aggressive disease exhibited by this patient. Utilization of the NotI, BamHI sites in the pRVY-Tet retroviral vector. The pCBAIsceI expression vector was a kind gift from the Jasin laboratory.The RAD51 G151D variant was generated as previously described . The RAD2 incubator. Pools of MCF10A, MCF-7, MCF-7 DRGFP, and U2OS-DRGFP cells with stable expression of RAD51 WT or RAD51 G151D were generated as previously described [MCF10A cells, an immortalized, non-transformed mammary epithelial cell line, were obtained from ATCC. MCF10A cells were maintained in DMEM/F12 medium  supplemented with 5% horse serum (HyClone), 1% penicillin-streptomycin (Gibco), epidermal growth factor (EGF) , hydrocortisone , insulin , Cholera Toxin . GP2-293 cells (Clonetech), a retroviral packaging cell line, MCF-7 DRGFP cells (a kind gift from the Jasin lab), and U2OS-DRGFP cells (a kind gift from the Bindra lab) were maintained in DMEM  10% fetal bovine serum (FBS) (Invitrogen) supplemented with 1% penicillin-streptomycin. All cells were maintained at 37°C in a humidified 5% COescribed , using aSynchronized cells were collected in cell lysis buffer  then centrifuged at 10,000xg for 10 minutes. Samples were mixed with 6x loading buffer , separated on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF-FL) membranes . The membrane was blocked in Odyssey blocking buffer (PBS) (Millipore) for 1hr at room temperature (RT) with gentle shaking. The membrane was then probed rabbit polyclonal RAD51 primary antibody (1/300)  and mouse monoclonal tubulin primary antibody (1/10000)  overnight at 4°C with gentle shaking. After 3 washes in PBS/0.1% Tween, the membrane was probed with IRDye 800CW goat anti-mouse IgG (H+L)  and IRDye 680RD goat anti-rabbit IgG (H+L)  for 1hr at RT. The membrane was washed with PBS/0.1% Tween and visualized using the Odyssey CLx Infrared Imaging System. Expression levels were quantified using Image Studio Software version 2.1.10.6 cells per well in a 6-well plate. The next day, the cells were nucleofected using the Amaxa Cell Line Nucleofector Kit V (Lonza) as per the manufacturers instructions with either no DNA, 2 μg of pmaxGFP, or 1–4 μg of the I-SceI expression vector pCBASce [SceI site loss for cells nucleofected with no DNA or the I-SceI expression vector. To amplify the DR-GFP sequence surround the I-SceI site, PCR reactions with a final volume of 50 μL included: 1 μg of genomic DNA as a template, dNTPs (1 mM), MgSO4 (1 mM), Pfx amplification buffer (1x), PCRx Enhancer solution (1x), Platinum Pfx DNA Polymerase (Invitrogen) and primers at 0.2 μM. The primer sequences were DR-GFP-RS-F 5’ CGTGCTGGTTATTGTGCTGTCTCA and DR-GFP-RS-R 5’ TGCTGCTTCATGTGGTCGGGGTAG. Amplification was performed as previously described [SceI (NEB) for 20 hours, separated on a 2.5% agarose gel containing SYBR Safe DNA gel stain (Invitrogen) and visualized using the BioRad VersaDoc Imaging System. Signals of the +I-SceI and–I-SceI bands were quantified using Quantity One software version 4.6.5. The percent HDR was calculated by dividing the percent GFP+ cells by percent I-SceI site loss.MCF-7 DR-GFP cells expressing RAD51 WT or RAD51 G151D were plated at 2x10 pCBASce . The perescribed  using a SceI site was created in the luciferase ORF that completely disrupts luciferase activity. The second luciferase ORF inserted downstream lacks a promoter but can be utilized as a donor in HDR of the first luciferase ORF upon generation of a DSB by expression of the I-SceI nuclease. The pSce-MJ mammalian I-SceI expression vector was a kind gift from Dr. Fen Xia. To perform the assay, cells were seeded into 6-well plates at 5x105 cells/well. 24 hours later, cells were nucleofected with 500 ng of gWiz.Lux-5’-3’Luc vector and 500 ng of the I-SceI expression vector using Amaxa Cell Line Nucleofector Kit L (program T020). As negative and positive controls respectively, gWiz.Lux-5’-3’Luc or gWiz. Lux vector were transfected alone. Cells were harvested 24 and 48 hours post-nucleofection. Luminescence was measured using an integration time of 5 seconds with 40 μL of the lysate plus 100 μL of luciferin substrate . Luciferase values were measured as independent triplicates in each experiment. The data presented is the average of two independent experiments.The HDR luciferase assay was performed as previously described . Briefly2HPO4 and 0.1 M KH2PO4 (pH 6.8), sealed with rubber cement then placed under one 100-W lamp  that was placed at a distance of 20 cm from the surface of the slides for 25 minutes. Slides were incubated in pre-warmed 1X SSC  for 1hr at 50°C, washed in water then stained in 5% KaryoMax Giemsa  (Invitrogen) for 20 minutes. Slides were washed in water, dried and mounted in Permount mounting media. Spreads were imaged under a 100x objective using an Olympus BX50 Light Microscope with QImaging Retiga 2000R digital camera and software.The sister chromatid exchange assay was performed as previously described ,75 with Metaphase spreads were prepared and chromosomal aberrations were analyzed as described .For ionizing radiation (IR) sensitivity and mitomycin C (MMC), cells seeded at various low densities were exposed to 0, 2, 4, 6, 8 GY of IR (X-irradiation) or 0, 0.6, 0.8, 1.0 or 1.2 uM of MMC for 4 hours then rinsed twice with PBS. The media was replaced with DMEM/F12 complete growth media and the cells were incubated for 10–12 days before being washed with 1xPBS and stained with crystal violet . Colonies with more than 50 cells were scored by eye. For both IR and MMC experiments, plating efficiency was calculated by dividing the number of colonies counted by the number of cells plated; clonogenic survival was determined by dividing the plating efficiency of treated cells by the plating efficiency of untreated cells. The data is representative of 4 independent experiments.3 cells were plated per well in triplicate in a 96 well plate. The following day, the cells were treated with 0, 100, 200, 400 or 600 nM for 1 hour after which the media was replaced with DMEM/F12 complete media. Cell viability was measured 72 hours post-treatment using the Vibrant MTT Cell proliferation assay kit (Invitrogen) as per the manufacturers instructions. Absorbance was measured using a BIO-TEK Synergy HT micro-titer plate reader. Percent cell death was calculated by dividing the absorbance of treated cells by the absorbance of untreated cells, subtracting that value from 1 then multiplying by 100. The data is representative of 4 independent experiments.To measure doxorubicin sensitivity, 10MCF10A RAD51 WT or G151D expressing cells plated in 8 well chamber slides (Millipore) were exposed to 0 or 8GY of IR (X-rays) then allowed to recover for 0, 2, 4, 8 or 24 hours post-exposure. Cells were washed 2 times with PBS then fixed  for 15 minutes at room temperature. Cells were rinsed with PBS then incubated with blocking/permeabilization solution  for 1 hour at with gentle shaking. The blocking/permeabilization solution was then replaced with blocking/permeabilization solution containing diluted primary antibody as follows; rabbit polyclonal RAD51: 1/100  and mouse monoclonal anti-phospho-Histone H2A.X (Ser139): 1/200  and incubated overnight at 4°C with gentle shaking. The next day, the cells were washed with PBS/0.5% TritonX-100 followed by 2 washes with PBS. Cells were then incubated with AlexaFlour 488 goat anti-mouse IgG (H+L) antibody (1/1000) (Invitrogen) and AlexaFlour 647 goat anti-rabbit IgG (H+L) antibody (1/1000) (Invitrogen) diluted in blocking/permeabilization solution for 1 hour with gentle shaking. The cells were washed with PBS/0.5% TritonX-100 followed by 2 washes with PBS then slides were mounted in Prolong Gold Antifade reagent with DAPI (Invitrogen). Cells were imaged using a Zeiss LSM 510 META confocal scanning laser microscope. For spontaneous foci formation, 1243 nuclei of MCF10A RAD51 WT expressing cells and 1168 nuclei of MCF10A RAD51 G151D expressing cells were counted. For RAD51 and gamma H2A.X foci formation post-IR exposure, at least 1500 nuclei were counted for both cell lines.5 cells were plated in 60 mm tissue culture dishes. The next day, the cells were exposed to 8GY of ionizing radiation (X-rays) or untreated and harvested by scraping at 0, 4 and 8 hours post-exposure. Cells were washed in cold PBS (without Ca++/Mg++). Cells were combined with molten agarose at 37°C and 50 ul was spread evenly across the circular sample area on the provided slides. Slides were placed at 4°C for 20 minutes then immersed in cold lysis buffer containing 10% DMSO overnight at 4°C. The next day, slides were immersed in neutral electrophoresis buffer for 30 minutes then aligned in an electrophoresis tank at 4°C, equidistance from the electrodes, filled with neutral electrophoresis buffer no more than 0.6 cm above the slides and 14 volts were applied for 45 minutes. Slides were then immersed in DNA precipitation buffer for 30 minutes at room temperature, followed be 70% ethanol for 30 minutes then dried at 37°C for at least 15 minutes. Slides were stained with DAPI and visualized under a 20x objective by epifluorescence microscopy using an Olympus BX50 Microscope with QImaging Retiga 2000R digital camera and software. Data analysis was performed using OpenComet software plugin for ImageJ 1.45s . At least 50 nuclei were counted per experimental group.The Trevigen CometAssay was performed in order to measure double strand DNA breaks after exposure to ionizing radiation as per the manufacturers instructions. Briefly, 5 x 10Treated and untreated cells were fixed in 1% paraformaldehyde in PBS with 5mM EDTA on ice. Cells were then permeabilized in 70% ethanol for at least 30 minutes at -20°C then rehydrated in 1% BSA/0.1% Triton X-100 in PBS for 30 minutes at 4°C. Rehydrated cells were resuspended in propidium iodide (PI)/RNase staining buffer (BD pharmingen) and filtered through a 44μM pore filter. Labeled cells were analyzed on a MACS VYB flow cytometer using 535nm excitation and PI fluorescence detection at 617nm. Percentages of cells in G0/G1, S, and G2/M were calculated using ModFit LT Version 4.1.7 .5 cells per insert in serum-free DMEM. 48 hours after plating, cells that had not invaded were removed using a cotton-tipped swab. Cells that had invaded to the bottom of the transwell were stained in cell stain solution then washed in water. The relative invasiveness of the cells was measured by dissolving the stained cells in 10% acetic acid and performing a colometric reading (OD) at 560nm.The cell invasion assay (Millipore) was performed as per the manufacturer’s instructions. Briefly, the ECM layer of the insert was rehydrated and placed in the wells containing DMEM 10% FBS. MCF-7 cells expressing RAD51 WT or G151D were plated at 5x1032P (T4 Polynucleotide Kinase) on the 5’-end and annealing it to RJ-Oligo2 at a 1:1 molar ratio. The assay buffer contained: 25 mM TrisOAc (pH 7.5), 1 mM MgCl2, 2 mM CaCl2, 0.1 μg/μL BSA, 2 mM ATP, and 1 mM DTT. All pre-incubations and reactions were at 37°C. The protein and DNA substrates were used at the following concentrations: RAD51 (0.3 μM), 3’ tail DNA (4 nM molecules) and dsDNA (4 nM molecules). RPA and BRCA2 proteins were used at the concentrations indicated in the figure (unless otherwise noted). The 3’ tail DNA was incubated first with RPA for 5 minutes, followed by the addition of RAD51 for 5 minutes (or BRCA2 and RAD51 where indicated), and finally, the radiolabeled donor dsDNA was added for 30 minutes. Where proteins were omitted, storage buffer was substituted. In the case where RAD51 protein was titrated, the RPA protein was omitted. The reaction was terminated with Proteinase K/0.5% SDS for 10 minutes. The reactions were loaded on a 6% polyacrylamide gel in TAE buffer and electrophoresis was at 60 V for 80 minutes. The gel was then dried onto DE81 paper and exposed to a PhosphorImager screen overnight. The screen was scanned on a Molecular Dynamics Storm 840 PhosphorImager and bands quantified using ImageQuant software. The percentage of DNA strand exchange product was calculated as labeled product divided by total labeled input DNA in each lane.All DNA substrates were obtained PAGE purified from IDT. The following oligonucleotides were utilized: RJ-167-mer (5’-CTG CTT TAT CAA GAT AAT TTT TCG ACT CAT CAG AAA TAT CCG TTT CCT ATA TTT ATT CCT ATT ATG TTT TAT TCA TTT ACT TAT TCT TTA TGT TCA TTT TTT ATA TCC TTT ACT TTA TTT TCT CTG TTT ATT CAT TTA CTT ATT TTG TAT TA TCC TTA TCT TAT TTA-3’), RJ-PHIX-42-1 (5’-CGG ATA TTT CTG ATG AGT CGA AAA ATT ATC TTG ATA AAG CAG-3’), RJ-Oligo1 (5’-TAA TAC AAA ATA AGT AAA TGA ATA AAC AGA GAA AAT AAA G-3’), and RJ-Oligo2 (5’-CTT TAT TTT CTC TGT TTA TTC ATT TAC TTA TTT TGT ATT A-3’). The 3’ tail DNA substrate was generated by annealing RJ-167-mer to RJ-PHIX-42-1 at a 1:1 molar ratio. The dsDNA donor was generated by first radiolabeling RJ-Oligo1 with FRET) was calculated as the ratio between the acceptor intensity and the sum of the acceptor and donor intensities. Programs written in Matlab were used to view and analyze FRET trajectories. Histograms were generated using a sample size of over 75 individual molecular trajectories.All reactions were performed at room temperature in a buffer composed of 50 mM Tris-HCL, pH 8.0, 1 mM MgCl 2, 2 mM CaCl 2, 0.1 mg/mL BSA and an oxygen scavenging system  D-glucose, 0.02 mg/ml catalase, and 2 mM Trolox) ,78. In b2O) for 2 minutes. Slides were then tilted at a 15° angle to allow DNA fibers to spread on slide then allowed to air-dry. Slides were fixed in 75% methanol/25% acetic acid then placed in 2.5 M HCl for 3 hours. Slides were washed in ddH2O then blocked in 5% BSA. IdU-incorporated replication tracts were labeled using a mouse anti-BrdU primary antibody (1/400) (BD Biosciences) and CIdU-incorporated replication tracts were labeled using rat anti-BrdU primary antibody (1:25) (Abcam) for 2 hrs at RT. Slides were washed then labeled with goat anti-rat Alexa Flour 488 (Invitrogen) secondary antibody (1/200) and goat anti-mouse Texas Red (Santa Cruz) secondary antibody (1/200) for 2.5 hrs at RT. Slides were mounted in Prolong Gold antifade mounting media (Invitrogen) and visualized under a 60x objective by epifluorescence microscopy using an Olympus BX50 Microscope with QImaging Retiga 2000R digital camera and software. Data analysis was performed using ImageJ v. 2.0.0 . At least 100 elongating replication structures were measured per cell line/per experimental group for replication tract length. The data are representative of 3 independent experiments. ***p<0.001; **p<0.01.Cells were grown in appropriate media until 30–40% confluent. Cells were pulsed with 25 μM  of 5-Iodo-2’-deoxyuridine (IdU) (Sigma) for 20 minutes at 37°C. Cells were washed with PBS then treated with 0.5 mM hydroxyurea (HU) (Sigma) for 2hrs, exposed to 8GY IR or untreated. Cells were then pulsed with 250 μM  of 5-Chloro-2’-deoxy-uridine (CIdU) for 20 minutes at 37°C. Cells were harvested, spotted onto microscope slides then lysed in fiber lysis solution . 36 hours post-transfection, the cells were harvested in 500 μL of buffer ‘BB’: , and 1X EDTA-free protease inhibitor cocktail (Roche). Cell lysates were batch bound to 20 μL of amylose resin for 2 hours. The bound proteins were washed two times with buffer ‘B’: 50 mM HEPES (pH 7.5), 0.5% Igepal CA-630, 0.1% Triton X-100, 0.5 mM EDTA, and 1 mM DTT containing 1M NaCl followed by two washes in buffer B containing 250 mM NaCl. Purified RAD51 at the concentrations indicated in the figure were then added and incubated at 37°C for 30 minutes. The protein complexes were then washed again three times with buffer B containing 250 mM NaCl followed by elution in 20 μL of 10 mM maltose. Loading sample buffer was added, samples were heated at 54°C for 4 minutes, and loaded onto a 4–15% gradient SDS-polyacrylamide gel (Bio-Rad TGX Stain-Free gel). The gel was run for 2 hours at 100 Volts. The proteins were visualized by staining with SyproOrange (Invitrogen) and quantified using ImageQuant software on a Storm 860 PhosphorImager (Molecular Dynamics). RAD51 non-specific binding to amylose beads was negligible.Amylose pull-down assays were performed by transiently transfecting 1 μg of the indicated constructs into 5x1032P at the 5’-end using T4 Polynucleotide Kinase (NEB) and then annealed at a 1:1 molar ratio to RJ-PHIX-42-1. ssDNA was the radiolabeled RJ-167-mer alone and the dsDNA substrate was RJ-Oligo1 labeled at the 5’-end by 32P (T4 Polynucleotide Kinase) and annealed to RJ-Oligo2 at 1:1 molar ratio. RAD51 (at the indicated concentrations) was incubated with 0.2 nM (molecules) of the radiolabeled DNA substrate for 30 min at 37°C in DNA strand exchange buffer . The reactions were resolved by electrophoresis on a 6% polyacrylamide gel in TAE  buffer for 90 minutes at 80 V in the cold room (4°C). The gel was then dried onto DE81 paper and exposed to a PhosphorImager screen overnight. The screen was scanned on a Molecular Dynamics Storm 840 PhosphorImager and bands quantified using ImageQuant software. The percentage of protein-DNA complexes was calculated as the free radiolabeled DNA remaining in a given lane relative to the protein-free lane, which defined the value of 0% complex (100% free DNA).Oligonucleotide substrates were obtained PAGE purified from IDT. To generate the 3’ tail DNA substrate, RJ-167-mer was radiolabeled with S1 FigA. Western blot demonstrates equivalent expression of exogenous (I) RAD51 WT and G151D, as well as the fold increase in expression over endogenous RAD51 (I/R), in their respective U2OS-DRGFP pools. B. Percentage of GFP positive cells was measured by flow cytometry as described in (TIF)Click here for additional data file.S2 FigA,B. MCF10A pools expressing RAD51 WT or G151D were exposed to 8GY ionizing radiation then fixed/permeabilized at 0, 2, 4, 8 post-IR exposure (A) or untreated cells (B) were fixed/permeabilized and both populations were labeled with propidium iodide to measure DNA content by flow cytometry. The percentage of cells in S and G2/M phases of the cell cycle were scored using ModFit analysis. The data are graphed as mean ± SD of 2 independent experiments. **p<0.01; ***p<0.001.(TIF)Click here for additional data file.S3 FigA,B. MCF10A pools expressing RAD51 WT or G151D were harvested and single cell electrophoresis was performed to quantitate DNA damage using the comet assay. A. Data are graphed as mean ± SEM. B. Representative images from RAD51 WT or G151D expressing cells. C,D. MCF10A pools expressing WT or G151D were labeled with a γH2AX antibody (green) then mounted in Prolong Gold mounting media containing DAPI . Labeled cells were visualized using a Zeiss LSM 510 META confocal imaging system. C. The number of nuclei with γH2AX was counted. The data are graphed as mean ± SEM (n>500 nuclei) **** p< 0.0001; *** p<0.001. D. Representative images of γH2AX foci in MCF10A RAD51 WT and RAD51 G151D expressing pools.(TIF)Click here for additional data file.S4 FigSerially diluted RAD51 WT and RAD51 G151D expressing MCF-7 pools were X-irradiated at 0, 2, 4, or 8 GY. After 10 days, colonies were stained with crystal violet and scored. Data are representative of 3 independent experiments and graphed as mean ± SD.(TIF)Click here for additional data file.S5 FigA. SyproOrange stained SDS-PAGE gel depicting either BRCA2 (left) or PALB2 (right) incubated with increasing concentrations of purified WT RAD51 or G151D. B. RAD51 WT and G151D binding to BRCA2 (left graph) or PALB2 (right graph) was quantitated using ImageQuant software and normalized to the band intensities of BRCA2 and PALB2 respectively. This experiment was repeated twice. A representative gel image is shown.(TIF)Click here for additional data file.S6 FigA. Schematic of DNA strand exchange assay utilizing RAD51 protein only. B. Autoradiograms of DNA strand exchange in the presence of increasing concentrations of RAD51 WT (upper gel) or G151D (lower gel). Lanes 1 and 11 are no protein controls. C. Quantification of the gels shown in (B). Error bars are S.D., (n = 3).(TIF)Click here for additional data file.S7 FigA. Autoradiograms of increasing concentrations of RAD51 WT and G151D incubated with 3’ tail DNA, ssDNA, and dsDNA radiolabeled substrates. G151D protein-DNA complexes resolve at a faster mobility than WT RAD51. B. Quantification of the gels shown in (A) depicting the percentage of RAD51-DNA complexes.(TIF)Click here for additional data file.