In a transplanted non-small cell lung cancer model, E-M remarkably decreased the density of intratumoral macrophages and blood vessels, leading to tumor regression. This study unravels a novel mechanism of endostatin on regulating TAM recruitment and polarization, and suggests that E-M is a remarkably promising and multifunctional anti-tumor agent.Endostatin recently was reported by our laboratory to possess ATPase activity that is indispensable for its anti-angiogenesis and anti-tumor effects. An engineered endostatin, E-M, which owns higher ATPase activity exhibits stronger inhibitory effects on angiogenesis. Tumor-associated macrophages (TAMs), especially M2-polarized TAMs, contribute to tumor progression by promoting tumor cell proliferation, metastasis, angiogenesis, and immunosuppression, thus emerging as crucial targets for therapeutic intervention. Endostatin reportedly modulated functions of TAMs, but the detailed mechanisms remain unclear. Here, in our study, we demonstrated that E-M exhibited stronger inhibitory effects on macrophages than endostatin and other low ATPase mutants, which indicates that the ATPase activity is required for the inhibitory effects of endostatin on TAMs. Moreover, we elucidated that endostatin co-receptor, nucleolin and integrin α5β1, overexpressed on the surface of M2 macrophages, facilitated the internalization of E-M via the caveolae/lipid raft- and clathrin-dependent pathways. E-M inhibited the migration of TAMs through blockade of p38 MAP kinase and Erk1/2 signaling pathways, and prevented the alternative activation of TAMs. As a result, TAM-induced tumor cell proliferation and angiogenic activities  Macrophages are important tissue-resident immune cells which participate in maintaining homeostasis and defensing against pathogens . Plastic+CD163+ macrophage infiltration in patients with diffuse-type giant cell tumor and increases CD8+ T cells and NK cells in tumor tissues . The recombinant murine M-CSF and IL-4 were purchased from Sino Biological Inc . The recombinant murine IL-13 was purchased from PeproTech . Antibodies such as p38, p-p38, Erk1/2, p-Erk1/2 and Hsp90 were from Santa Cruz Biotechnology . Akt, p-Akt, 4E-BP1, p-4E-BP1, and Arginase-1 antibodies were from Cell Signaling Technology . Integrin α5 and nucleolin antibodies were from Abcam . Rabbit polyclone antibody for endostatin was from Oncogene Research Products . CD31 and F4/80 antibodies were from BD Bioscience . CD11b-FITC and CD11b-PE-Cy7 antibodies for flow cytometry were from eBioscience . F4/80-FITC and CD206-APC antibodies were from Biolegend . HIF-1α antibody was from GeneTex . FITC-linked anti-rat, FITC-linked anti-rabbit, TRITC-linked anti-mouse and Dylight 649-linked anti-rabbit IgG antibodies were from Beijing Cowin Biotech .A549, Raw 264.7, B16-F10, L929, SVEC4-10, and MS1 murine endothelial cells were purchased from the American Type Culture Collection (ATCC). A549 cells were cultured in RPMI-1640  supplemented with 10% FBS. RAW 264.7, B16-F10, L929, SVEC4-10, and MS1 cells were maintained in DMEM supplemented with 10% FBS. All cells were cultured under the condition of 37°C and 5% COCells were fixed with 4% paraformaldehyde and washed with ice-cold PBS twice. For tumor tissues, slides of cryo-section were fixed in ice-cold acetone and washed in PBS. Then these samples were blocked with 10% goat serum and stained with primary antibodies. At last, fluorescein-conjugated secondary antibodies were added and fluorescent images were captured by Nikon A1 laser scanning confocal microscope. These images were then analyzed with Nikon image software NIS-Elements AR 3.0.Bone marrow-derived macrophages (BMDMs) were treated with 20 and 40 μg/mL E-M for 24 h. Then these cells were harvested and washed in cold PBS. After centrifugation, 100 μL binding buffer was added to these cells. Then 5 μL Annexin V-FITC and 5 μL PI staining solution  were added to the cell suspension. These cells were incubated for 15 min at room temperature. At last, 400 μL binding buffer was added to the cell suspension and cell apoptosis was analyzed by BD Calibur .Total RNA from cells was isolated with TRIzol  and subjected to cDNA synthesis with the First Strand cDNA Synthesis Kit . Quantitative RT-PCR (qRT-PCR) was used to analyze mRNA expression levels. It was performed with 2×TransStart Green qPCR SuperMix . The reaction was run on the Mx3000P system . GAPDH or 18S was used as the control. Relative quantification was analyzed with the ΔΔCt method. The primers for qRT-PCR were listed in Supplementary Table Western blot was performed following the previous report . In brie3 cells were seeded in each well of 96-well plate. Cells were treated with E-M for 72 h. After the treatment, 10 μL MTT solution  in 100 μL DMEM was added to each well and the whole plate was incubated at 37°C for 4 h. Then 100 μL formazan solution was added to each well and incubated for another 4 h. At last, the absorbance of each well at 570 nm (OD570) was measured under Varioskan Flash (Thermo Fisher Scientific).4 × 106 cells in 100 μL PBS containing 3% FBS were stained with primary antibodies on ice for 30 min and washed with ice-cold PBS once. Then these cells were stained with fluorescein-conjugated secondary antibodies for 30 min and washed with ice-cold PBS twice. For fluorescein-labeled primary antibodies, cells were just stained with these antibodies and washed with PBS once. At last, 500 μL PBS was added to these cells and cells were analyzed with FACSAria III (BD Biosciences).1 × 105 cells were seeded in the upper chamber of 5 μm Millicell (Millipore). Recombinant proteins including WT endostatin, E-M, E176A, K96A, and K96R were added to the medium containing concentrated A549 conditioned medium (CM) or 2% FBS in the lower chamber. Cells were allowed to migrate for 6 h at 37°C. Then cells were fixed with 4% paraformaldehyde and stained with crystal violet . Migrated cells were counted randomly in five independent fields under the Olympus IX71 optical microscope.Raw 264.7 cells or BMDMs were pre-treated with endostatin or different ATPase mutants for 1 h and 1 × 10SVEC4-10 cells were plated in the 6-well plate and allowed to grow to confluence. Then 10 μL pipette tips were used to scratch lines. BMDM CM from different treatment groups was added to these wells and cells were incubated at 37°C for 6 h. Images were taken with optical microscope and five independent were analyzed with Image-Pro Plus 6.0 software .4 MS1 cells in the medium containing 2% FBS were added to each well and cultured at 37°C. After 3.5 h, tubule formation was observed and five independent fields were captured under the optical microscope. The tubule length was analyzed with Image-Pro Plus 6.0 software.The tubule formation assay was performed as previously described . In brieCells in the tibia and femur of C57BL/6 mice were flushed out with 10 mL DMEM. After red blood cells lysis, myeloid cells were incubated in medium containing 10% FBS and 100 ng/mL M-CSF or L929 CM. At the 4th day, fresh medium was added to these cells and incubated for another 3 days.6 A549 cells were inoculated subcutaneously into the right axilla of female BALB/c nude mice . When these tumors grew to 100 mm3, mice were separated randomly into two groups (n = 5 mice/group). For the E-M treatment group, 12 mg/kg E-M was administered to mice intravenously every other day and the treatment was lasted for 12 days. For liposome treatment group, 200 μL PBS or clodronate liposomes were injected to mice (n = 5 mice/group) 6 days before the tumor implantation. The injection was given every 3 days and tumors were allowed to grow for 12 days. Tumor growth was monitored and tumor volumes were calculated by the formula: volume = 0.52ab2 (a represents the long diameter and b indicates the short diameter).All animal experiments were approved by the Institutional Animal Care and Use Committees of Tsinghua University . 1 × 10PBS and clodronate liposomes were prepared following the previous report . Under tt-test was used to assess the statistical differences for two-group comparisons. For comparing three or more groups, an one-way analysis of variance (ANOVA) was applied to assess the statistical differences. P < 0.05 was regarded to be significant.All experimental data were presented as mean ± SD or SEM. A two-tailed Student’s Figure 1A and Supplementary Figure Figure 1B and Supplementary Figure in vitro migration assay. Compared to endostatin, E-M exhibited a much stronger inhibitory effect on macrophage migration, whereas these low-ATPase mutants had no or only minor effects on macrophage migration . We also linked E-M to CNBr-activated Sepharose 4B column and incubated it with BMDM lysates. After elution, nucleolin and integrin α5 were detected in the eluted solution which further confirmed the interaction between E-M and nucleolin or integrin α5 . Taken together, the ATPase activity is necessary for the biofunctions of endostatin on macrophage functions, and nucleolin and integrin α5β1 expressed on macrophage surface were also E-M receptors.As the integrin α5β1-nucleolin-uPAR co-receptor complex was identified to mediate endostatin internalization and nuclear translocation of endostatin in endothelial cells , we examFigure 2A). Flow cytometric results showed that rhodamine-conjugated E-M (Rh-E-M) was internalized into BMDMs and Raw264.7 cells in a time-dependent manner . Our group previously reported that blockade of caveolae/lipid raft pathway accelerated endostatin internalization through the more efficient clathrin pathway . In the Matrigel plug assay, A549 CM and B16-F10 CM recruited a large number of F4/80+ macrophages into plugs but E-M blocked the macrophage recruitment . During the polarization switch process, STAT3 and STAT6 were selectively activated in macrophages . Compared to WT endostatin, E-M was also found to have stronger inhibitory effects on M2 polarization  cell co-culture system, Arg-1 was also up-regulated in BMDMs but E-M reversed this effect . Moreover, BMDMs enhanced the proliferation of A549 cells after 12 h in the co-culture system but the proliferation rate was reduced in the presence of E-M . To exclude the direct influence on A549 cells, MTT assay was used to examine the effects of E-M on A549 cells and we found that E-M had no effects on A549 cell proliferation . Moreover, B16-F10 CM induced alternative activation of BMDMs was suppressed by E-M . In conclusion, E-M can indeed inhibit the alternative activation of macrophages and M2 macrophage-induced tumor cell proliferation.To induce M2-polarized macrophages, we treated BMDMs with A549 CM and observed that the expression of M2 markers, including rophages . However Figures ,B. Durins Figure . CompareFigure 5A). After A549 CM treatment, we collected the BMDM CM in different treatment groups and treated endothelial cells with these CM. The tube formation ability of MS1 cells was enhanced in A549 CM pre-treatment group but this effect was abolished in the E-M pre-treatment group . Moreover, consistent results were obtained in endothelial cell migration and wound healing assays. BMDM CM from A549 CM pre-treatment group enhanced the motility of endothelial cells but CM from E-M pre-treatment group had no such effect . It was reported before that the transcription factor HIF-1α participated in regulation of pro-angiogenic factors production . PI3K/Akt and mTOR signaling pathways were known to regulate the HIF-1α expression in endothelial cells . After the blockade of activation of PI3K/Akt and mTOR with their respective inhibitors LY294002 and KU0063794, the expression of HIF-1α induced by A549 CM in BMDMs was significantly down-regulated . Taken together, E-M can abolish the pro-angiogenic effects of tumor cell-induced TAMs.Tumor-associated macrophages are well-known to promote tumor angiogenesis through production of different pro-angiogenic factors . To deteoduction . Our final cells . Then wein vivo, we firstly implanted A549 cells into mice subcutaneously and treated mice with PBS and E-M. Six days after implantation, tumor volumes in both groups displayed no significant differences so impacts of tumor volume differences were excluded . However, macrophage recruitment was notably inhibited in E-M-treated group, accompanied by decreased tumor angiogenesis . Moreover, when the tumor volumes reached 100 mm3, we treated A549 tumor-bearing mice with E-M for 12 days. After the treatment, the tumor growth was significantly inhibited by E-M . And we observed that the recruitment of macrophages was remarkably blocked after E-M treatment . However, the density of macrophages in other organs including liver, spleen, kidney and colon from E-M treated tumor-bearing mice was not affected . We also confirmed these results in a syngeneic tumor model. We treated B16-F10-bearing mice with E-M and got the similar results. E-M inhibited B16-F10 tumor growth and the density of intratumoral macrophages and blood vessels was significantly decreased by E-M (Supplementary Figures To examine whether E-M could inhibit the recruitment of macrophages ATPase activity is indispensable for the biological functions of endostatin. Nowadays, endostatin is also discovered to exhibit functions on other cell types, not only on endothelial cells. Our study, for the first time, elucidate that the engineered endostatin derivative E-M, with much higher ATPase activity, influence the recruitment and alternative activation of TAMs. Compared to the WT endostatin and other mutants with low ATPase activity, E-M exhibits much stronger inhibitory effects on macrophage recruitment. Moreover, it also inhibits the alternative activation of TAMs and M2 TAM-induced pro-angiogenic effects. These effects result in decreased intensity of intratumoral macrophages and blood vessels, which accumulatively lead to tumor regression.In different tumor ecosystems, TAMs are the most prominent among the innate immune cells . These m+ T cells via the production of immunosuppressive factors such as IL-10, TGF-β, and HLA-G (+ T cells and weaken the tumoricidal activity of cytotoxic T cells. Inhibition of TAMs recruitment with CSF-1R inhibitors improves the immune cell response in tumor tissues and enhances the therapeutic efficacy of chemotherapy, radiation therapy and immune checkpoint blockade therapy (+ T cells and NK cells in tumors from IL-2 treated mice and regulate macrophage polarization (Immunosuppression is a huge challenge for anti-tumor therapy. M2-polarized TAMs are well-known to directly inhibit the immune response of CD8nd HLA-G . Moreovend HLA-G . Inhibit therapy . Rolny a therapy . Based orization . In our Tumor angiogenesis is an important hallmark of cancer , and it Nucleolin and integrin α5β1 are well-known co-receptor mediating the internalization of endostatin . These pIn summary, our study demonstrates for the first time that E-M, an engineered endostatin with higher ATPase activity, can impede the recruitment and alternative activation of TAMs. Suppression of TAM activities and tumor angiogenesis simultaneously endows E-M a stronger ability to inhibit tumor growth. Our findings illustrate that E-M is most likely to be a novel potential agent for cancer therapeutics and the ATPase activity of endostatin may provide a new approach for drug design in the future.MX, SZ, YF, and YL conceived and designed experiments. MX, SZ, and LJ carried out the experiments. All authors contributed to the acquisition of data and analyzing the data. All authors were involved in writing the manuscript and had final approval of the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vaccine-induced responses were identified in both central and effector memory (EM) compartments. EM CD4+ T cells expressing IL-4 and IL-21 were detected recognizing both vaccine antigens. Consistently higher response rates to both antigens in RTS,S/AS01E-vaccinated than comparator-vaccinated children were observed. RTS,S/AS01E induced polyfunctional CSP- and HBsAg-specific CD4+ T cells, with a greater degree of polyfunctionality in HBsAg responses. In conclusion, RTS,S/AS01E vaccine induces T cells of higher functional heterogeneity and polyfunctionality than previously characterized. Responses detected in memory CD4+ T cell compartments may provide correlates of RTS,S/AS01-induced immunity and duration of protection in future correlates of immunity studies.Comprehensive assessment of cellular responses to the RTS,S/AS01E vaccine is needed to understand potential correlates and ultimately mechanisms of protection against malaria disease. Cellular responses recognizing the RTS,S/AS01E-containing circumsporozoite protein (CSP) and Hepatitis B surface antigen (HBsAg) were assessed before and 1 month after primary vaccination by intracellular cytokine staining and 16-color flow cytometry in 105 RTS,S/AS01-vaccinated and 74 rabies-vaccinated participants (controls) in a pediatric phase III trial in Africa. RTS,S/AS01E-vaccinated children had significantly higher frequencies of CSP- and HBsAg-specific CD4 A highly efficacious vaccine can substantially contribute to control and eventual elimination of malaria. This life-threatening disease caused an estimated 429,000 deaths in 2015  and it is coexpressed with HBsAg alone. In the Phase III trial, RTS,S was formulated with AS01E liposomal adjuvant containing monophosphoryl lipid A and QS21 and was designed to induce strong anti-CSP antibody and T helper (TH) 1 cell responses. Accordingly, in past clinical trials in endemic areas, RTS,S consistently induced high anti-CSP IgG titers  of G titers –11 and mesponses –10. IgG esponses , 13. How+ T cells expressing IL-2, TNF-α, and IFN-γ (and CD40L in naïve adults) have been detected by intracellular cytokine staining (ICS) or ELISpot , or memory phenotypes that may be induced by RTS,S/AS01E and could be correlated with antibody responses and involved in vaccine-induced protection. Interestingly, in the Phase III trial, we detected TH1 responses in supernatants of CSP-stimulated cells associated with protection in RTS,S/AS01E vaccinees, whereas a TH2 cytokine, IL-5, was associated with increased risk for malaria  cells and effector/effector memory T cells from RTS,S-vaccinated adults produced IL-2 after ex vivo CSP stimulation and frequencies were higher in protected vs. non-protected subjects  isolated from a subset of children aged 5–17 months at enrollment from Tanzania and Mozambique and following receipt of either the RTS,S/AS01E vaccine or a comparator rabies vaccine.The aim of this study was to analyze RTS,S/AS01E cellular immunogenicity after primary vaccination using two qualified 16-color multiparametric ICS assays that allow the assessment of memory cell subsets and regulatory, cytotoxic, TClinicalTrials.gov NCT00866619), described elsewhere , and Ifakara Health Institute and Bagamoyo Research and Training Centre . The two sites had low-medium malaria transmission intensity at the time of the study  tubes. PBMCs were isolated by density gradient centrifugation, cryopreserved and shipped to the Fred Hutchinson Cancer Research Center where the PBMC were thawed and stained .Blood was collected in 5 ml sodium citrate (BD Vacutainer2 incubator overnight. The resting step increases the sensitivity of the assay (data not shown), probably by decreasing the stress and activation of PBMC due to the thawing process and exposure to the toxic cryopreservation agent. PBMC were stimulated with peptide pools covering the HBsAg or the CSP antigen present in the RTS,S vaccine . Negative controls contained 0.5% DMSO, the diluent for peptide pools, and Staphylococcal enterotoxin B was used as positive control stimulation at 1 µg/ml. Cultures were incubated 6 h at 37°C, 5% CO2. This short incubation time increases the sensitivity and specificity of the assay to detect antigen-specific cells, avoiding non-specific and secondary immune responses. Further details are found in Supplementary Material.Thawed PBMC were rested in a 37°C, 5% COFH-like cells were excluded from the analysis. Flow cytometry analysis was performed using FlowJo software (Version 9.9 Tree Star). Gating strategies for both panels are shown in Figures S1 and S2 in Supplementary Material.Peripheral blood mononuclear cells were stained with one of two 16-color ICS panels that were designed for the study and that had previously undergone assay qualification with a formerly validated panel , 18. CelThe raw FCS files and manual gates were imported into the R environment  using thFor the analysis of the effect of vaccination on the frequencies of cells expressing the functional markers, a multivariate linear mixed effect model was fitted using logarithm-10 transformed cell frequencies  as an outcome. These models are commonly used –24 and aTo define positivity of responses in each cell subset and subject, we identified the functional markers that were differentially expressed between the antigen (CSP or HBsAg) stimulations and their corresponding background (DMSO control stimulation) for each cell subset, subject, and at each timepoint. Thus, the proportion of cells expressing that marker was compared between the two stimulation conditions for each subject and timepoint. The statistical method used was mixture models for single-cell assays (MIMOSA) , using tPolyfunctional responses  were analyzed using combinatorial polyfunctionality analysis of antigen-specific T cell subsets (COMPASS) . COMPASSStatistical tests were considered significant at 0.05 α-level. Adjustments for multiple testing were done using Holm  and Benj+ and CD8+ T cells , NK and NK-T cells (CD56), the TH1 cytokines IFN-γ, IL-2, and TNF-α, the TH2 cytokine IL-4, the costimulatory molecule CD154 (CD40L), and the cytotoxic marker granzyme B . P1 additionally had CD45RA and CCR7 markers that allowed the identification of memory cell subsets, and IL-21, a cytokine related to TFH. Panel 2 (P2) included γδ TCR for the identification of γδ T cells and several functional markers: the regulatory cytokine IL-10, the TH2 cytokine IL-13, the TH17 cytokine IL-17, and cytotoxicity marker CD107a. The gating strategy is detailed in Figures S1 and S2 in Supplementary Material. The common set of core markers  allowed the pooled analysis for these markers. Males and females were similarly represented in both study sites and vaccine groups, with a proportion of 59 and 56.8% females in the RTS,S/AS01E and comparator groups, respectively.We used two different multiparameter ICS panels to assess cellular immune responses specific to the RTS,S/AS01E vaccine antigens. PBMC from 55 RTS,S and 41 comparator vaccinees, and 50 RTS,S and 33 comparator vaccinees were stained with antibody panel 1 (P1) and panel 2 (P2), respectively. The two panels had the same set of core markers, which allowed the exclusion of dead cells, monocytes (CD14), identification of CD4+ T cells expressing IL-2 (217% increase), TNF-α (72.3%), and CD154 (101.4%) from baseline to post-vaccination in contrast to the rabies vaccinees , TNF-α (346%), and CD154 (268.3%)  and CD4+ TEM (CD45RA− CCR7−) cells recognized both vaccine antigens. After accounting for baseline frequencies, RTS,S/AS01E vaccinees had more CSP-specific CD4+ TEM cells producing IL-2 and TNF-α, respectively, than comparator vaccinees , naïve CD4+ T cells (CD45RA+ CCR7+), other analyzed cell subsets  or on other functional markers .Therefore, RTS,S/AS01E vaccination induced CSP and HBsAg-specific CD4+ T cells producing IL-2 or TNF-α or CD154, IL-4, and CD107a  revealed non-specific responses  upon RTS,S/AS01E vaccination Figure . Accounta Figure A, i. In a Figure B, i.ex vivo antigen stimulations when not accounting for background. RTS,S/AS01E-vaccinated children had CD4+ T cells and CD8+ T cells expressing IL-4 and CD107a, as was observed for the background alone and, thus, likely reflecting non-specific responses . Additionally, RTS,S/AS01E vaccination increased the frequencies of CD4+ T cells expressing IL-13 and CD8+ T cells expressing CD154 and IL-2 or TNF-α or CD154 following CSP stimulation when not taking background into account . These additional responses in RTS,S/AS01E-vaccinated children were no longer significant when taking into account the background and likely reflect a non-specific effect of RTS,S/AS01E vaccination.Since we detected an effect of RTS,S/AS01E vaccination in the background, we looked at the +, TNF-α+, and CD154+ CD4+ T cell positive CSP-specific responses in RTS,S/AS01E than in comparator vaccinees post-vaccination , we determined the positivity of the responses for each subject comparing the frequencies of T cells expressing a functional marker in the antigen-stimulated and unstimulated control conditions for each participant using the MIMOSA statistical method. If a response is truly vaccine specific, one would expect to detect it after vaccination but not at baseline or in the comparator vaccinees. We found a significantly higher proportion of IL-2+ and IL-17+ CD4+ T cell positive responses in RTS,S/AS01E than comparator vaccinees at post-vaccination. Rates of HBsAg responders ranged from 26.7 to 76.1% in RTS,S/AS01E vaccinees , and no significant differences were detected between vaccination groups for any of the two vaccine antigens .When looking at CD4+ T cells than comparator vaccinees , and two markers  in RTS,S/AS01E vaccinees, whereas almost no responses were detected in comparator vaccinees or at baseline  or IL-21 (for HBsAg) in addition to IL-2, TNF-α and CD154 . This further highlights the induction of highly polyfunctional CD4+ T cell subsets with a TH2 and TFH functions by RTS,S/AS01E. No polyfunctional responses were detected in CD8+ T cells for either vaccine antigen.When the COMPASS analysis was performed using all the functional makers included in each antibody panel separately, we identified additional highly polyfunctional CD4ex vivo antigen-specific T cell response induced by RTS,S/AS01E vaccination in a pediatric Phase III trial  and effector memory (EM) compartments, and observed polyfunctional CD4+ T cell responses, which may be a key feature of a protective response. Although this analysis was limited to vaccine recipients who did not become infected with P. falciparum, this study has identified key immune responses that can be examined in a larger case-control study within this trial.We provide a detailed characterization of the II trial –4. In adesponses –10, we h+ T cell populations in a subset of children: CSP-specific IL-2, TNF-α, and CD154 CD4+ T cell responses and HBsAg-specific IFN-γ, IL-2, TNF-α, CD154, and IL-17 CD4+ T cell responses. Frequencies of cytokine-positive CD4+ T cells were consistent with the frequencies of IL-2 and TNF-α CSP-specific responses reported previously in RTS,S Phase II trials in endemic areas  were similar to the estimated VE in the Phase III trial (50% after a year of follow-up or 28% till study end)  , 33, 34,udy end) . Contrarmulation , 14. Thiugh IL-2 . Remarka studies , 7.+ T cell responses were detected in CM and EM compartments. Memory phenotypes are relevant to define high-quality and long-lasting responses, for instance, the higher proliferative potential of CM cells has been associated with protection from infection  and TFH (IL-21) effector functions together with CD40L could provide help to B cells for humoral responses and may correlate with antibody responses .+ T cell and CD8+ T cell responses were detected after vaccination compared with baseline. Since these responses included IL-4 and CD107a, it could be indicating a bias to a TH2 status and higher cytolytic potential of T cells in RTS,S/AS01E-vaccinated children. There is increasing evidence of non-specific effect of vaccines  in order to assess antigen-specific effects.RTS,S/AS01E may have a non-specific effect since higher background CD4vaccines , 48, butIn this study, we only included children without experiencing a documented malaria episode during the 18 months of follow-up; therefore, the clinical relevance of our findings will be assessed in future correlates studies. The exclusion of malaria cases is unlikely to bias results and affect study conclusions since malaria transmission intensity was low at that time in Manhiça and Bagamoyo and lack of malaria is not likely to be due solely to vaccine-induced protection, but also to lack of exposure to the mosquito-bearing parasite. Another potential limitation of our study relates to the toxicity of HBsAg peptide concentration used. Although this could impact cellular responsiveness, we did exclude dead and dying cells in the analysis, and it is likely that any impact would affect both vaccine groups.The breadth of functions, patterns, and variability of responses to CSP, together with memory phenotypes of responding cell subsets described in our study, reflects a complex response to the RTS,S/AS01 vaccine. These responses, together with anti-CSP IgG data, may provide insights into the lack of protection in a substantial proportion of vaccinees, and may be key in providing correlates for VE and duration of protection.Approval for the study protocol was obtained from the Ethical Committee of the Hospital Clínic in Barcelona , the National Health and Bioethics Committee , the Ethikkommission Beider Basel , the National Institutional Review Board , the Ifakara Health Institute IRB , and the PATH’s Research Ethics Committee . Written informed consent was obtained from parents or guardians of participating children in accordance with the Declaration of Helsinki.Substantial contributions to the conception or design of the work: GM, SR, CDa, CDo, and MJM. Acquisition of samples/data: GM, AN, MM, CJ, TR, JC, CDa, and CDo. Supervision analysis of samples: SR, KC. Analysis of data: GM, AA, JH, JA, DM, and CV. Data management: HS. Project management and coordination: ND-P and NW. Interpretation of data for the work and drafting the manuscript: GM, SR, CDo. Revising it critically for important intellectual content; final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: GM, SR, AA, AN, MM, KC, CJ, TR, JC, JH, HS, ND-P, NW, DM, JA, CV, CDa, CDo, MJM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Matrix metalloproteinase-9 (Mmp-9) is involved in different general and cell-type–specific processes, both in neuronal and non-neuronal cells. Moreover, it is implicated in an induction or progression of various human disorders, including diseases of the central nervous system. Mechanisms regulating activity-driven Mmp-9 expression in neurons are still not fully understood. Here, we show that stabilization of Mmp-9 mRNA is one of the factors responsible for the neuronal activity-evoked upregulation of Mmp-9 mRNA expression in hippocampal neurons. Furthermore, we demonstrate that the molecular mechanism related to this stabilization is dependent on the neuronal seizure-triggered transiently increased binding of the mRNA stability-inducing protein, HuR, to ARE1 and ARE4 motifs of the 3′UTR for Mmp-9 mRNA as well as the stably augmented association of another mRNA-stabilizing protein, HuB, to the ARE1 element of the 3′UTR. Intriguingly, we demonstrate further that both HuR and HuB are crucial for an incidence of Mmp-9 mRNA stabilization after neuronal activation. This study identifies Mmp-9 mRNA as the first HuB target regulated by mRNA stabilization in neurons. Moreover, these results are the first to describe an existence of HuR-dependent mRNA stabilization in neurons of the brain. Electrical activity is an important component of neuronal behavior. It influences multiple cellular phenomena, including gene expression, and participates in neuronal response to external stimuli. The neuronal activity-regulated protease Mmp-9 plays an important role in synaptic plasticity, learning, and memory as well as in many central nervous system (CNS) diseases, including stroke, epilepsy, schizophrenia, neurodegenerative disorders, and so on  were obtained from Sigma-Aldrich, USA. Papain latex was from Worthington, USA. The electroporation Buffer R was procured from the Neon Transfection System 100 μL Kit, Invitrogen, USA.The Gibco™ Minimum Essential Medium (MEM), Gibco™ MEM Non-Essential Amino Acids, GlutaMAX, penicillin, streptomycin, Neurobasal medium, glutamine, TranscriptAid T7 High Yield transcription Kit, Pierce™ RNA 3′ End Biotynylation Kit, LightShift® Chemiluminescent RNA EMSA Kit, and HuR siRNA  were purchased from Thermo Fisher Scientific, USA. Poly-ad libitum. Prior to the experiments, all rats were habituated to handling for a week. The animal studies were approved by the IV Warsaw Ethical Committee on Animal Research at the Centre of Postgraduate Medical Education. All procedures on rats were conducted in accordance with the rules established by the IV Local Ethical Committee on Animal Research of the Centre of Medical Postgraduate Education on the basis of national laws. All efforts were made to minimize animal stress and sufferings.Adult male Wistar rats weighing approximately 200–230 g were obtained from Mossakowski Medical Research Centre Polish Academy of Sciences . They were individually caged in a temperature- and humidity-controlled room , on a 12/12 light/dark cycle. Food and water were available Rats were handled and received intraperitoneal injections of saline once a day for three consecutive days before commencement of the experimental treatment. Animals were randomly divided into two groups: experimental and control. Then, rats from the experimental group received an intraperitoneal injection of PTZ . Behavioral seizures were scored according to a modified Racine's scale . Only animals displaying seizures and scored as 4 or 5 were used for further experiments; these animals were sacrificed 2 or 4 h after the onset of convulsions, and both hippocampi were dissected for further analyses. Animals with seizures scored as 1–3 and those which died before the appropriate time points were not included in further studies.5 cells/mL in Gibco™ MEM supplemented with 10% bovine calf serum, 1% Gibco™ MEM Non-Essential Amino Acids, 1% GlutaMAX, 1% sodium pyruvate, 0.3% glucose, 100 units/mL penicillin, and 0.1 mg/mL streptomycin and were maintained in a humidified incubator with 95% O2/5% CO2 at 37°C on 3.5 cm2/well plates that were previously coated with 50 μg poly-d-lysine/well. Then, 2–3 h after plating, MEM was replaced with Neurobasal Medium that supplemented with 2% B27 supplement, 100 units/mL penicillin, 0.1 mg/mL streptomycin, 1% glutamine, 0.5% glutamic acid, and 25 μM β-mercaptoethanol. On the third day after seeding, 5 mM cytosine-β-d-arabinofuranoside was added to the cultures to inhibit the proliferation of non-neuronal cells. To maintain the cultures, it was necessary to replace the medium containing supplements every 3–4 days.Rat primary hippocampal neurons were obtained from neonatal (P0–P1) Wistar rats. Animals were decapitated, and both of the hippocampi were dissected out and dissociated using papain latex. Then, neuronal cells were plated at a density of 2 × 102+ and Mg2+. Then, these cells were resuspended at high cell density in Electroporation Buffer R to a final concentration of 1 × 107 cells/mL. Electroporations were undertaken in a 100-μL tip containing 1 × 106 cells. Optimal results of electroporation were achieved using a single 20-ms pulse at 1,400 V. Immediately after electroporation, cells were plated with high density (5 × 105 cells/well) on 12-well poly-d-lysine-coated cell plates containing the Neurobasal Medium without antibiotics. After 24 h of plating, the Neurobasal Medium without antibiotics was replaced with Neurobasal Medium supplemented with 100 units/mL penicillin and 0.1 mg/mL streptomycin.DNA electroporation was done using the Neon Transfection System. Neuronal cells were isolated and dissociated (as described earlier) and washed with phosphate-buffered saline without Cain vitro culture (DIV 7), neuronal cultures were treated with bicuculline (50 μM) dissolved in dimethyl sulfoxide. Bicuculline was added for 30 min and then washed away by replacing the medium three times. Cultures were placed back in the incubator for the next 3.5 h before addition of the TRI Reagent.On Day 7 of 1/2 for Mmp-9 mRNA was estimated in the primary rat hippocampal neuronal cultures induced by bicuculline (50 μM) as described earlier. Four hours after bicuculline stimulation, a new medium containing actinomycin D (5 μg/mL) was added. Starting from the time of actinomycin D addition, RNA was isolated from the cultures (unstimulated as well as stimulated with bicuculline) at the following time points 0, 1, 2, 4, 6, 8, and 12 h. RNA was isolated and analyzed by reverse transcription quantitative PCR (RT-qPCR).The TTotal RNA was extracted from the rat hippocampi and primary rat hippocampal neuronal cultures using TRI Reagent according to the manufacturer's protocol.RT-qPCR was carried out as described previously . HuR siRNA was purchased from Ambion (Elavl1 Silencer® Select siRNA). Constructs expressing siRNA for HuB were obtained by inserting the following oligonucleotides specific to the 340–358 bp region of the rat HuB at the BglII/HindIII site of pSUPER: 5′-GATCCCCGTGAACTACATTGATCCCATTCAAGAGATGGGATCAATGTAGTTCACTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGTGAACTACATTGATCCCATCTCTTGAATGGGATCAATGTAGTTCACGGG-3′.Constructs expressing siRNA for HuC were obtained by inserting the following oligonucleotides specific to the 116–134 bp region of the rat HuC at BglII/HindIII site of pSUPER:5′-GATCCCCCCAACCTCATCGTCAACTATTCAAGAGATAGTTGACGATGAGGTTGGTTTTTGGAAA-3′.5′-AGCTTTTCCAAAAACCAACCTCATCGTCAACTATCTCTTGAATAGTTGACGATGAGGTTGGGGG-3′.The impact of HuR, HuB, or HuC on the degradation of Mmp-9 mRNA was tested by the addition of neutralizing anti-HuR, anti-HuB and anti-HuC antibodies to samples comprising cytoplasmic extracts obtained from the unstimulated (control) and PTZ-treated  rat hippocampi. We added 0.4 μg antibody to 130 μg cytoplasmic extracts and the mixture was pre-incubated at room temperature for 1 h before the addition of the rat hippocampal RNA (20 μg). RNA used for neutralization experiments was extracted from the hippocampi of rats sampled 4 h after the PTZ-induced seizures. To exclude any nonspecific inhibitory effects induced by the serum, normal isotype sera were tested in parallel. Mixtures of antibody, RNA, and the protein extracts were incubated for 2 h. Then, RNA was extracted using TRI Reagent and analyzed quantitatively by RT-qPCR. Primers and conditions used for RT-qPCR are listed in Table U-test (to compare two groups), or the Kruskal–Wallis test with Dunn's post hoc test (for three or more groups) for data with non-normal distribution. Parametric Student's t-test (to compare two groups) or one-way ANOVA with Tukey's post hoc pairwise test (for three or more groups) was used for data with normal distribution. Data from RNA degradation assay and Mmp-9 mRNA half-life time were tested using two-way ANOVA with Tukey or Bonferroni post hoc tests. Results are shown as mean ± SEM. Differences were considered significant at p < 0.05. Descriptive and analytical statistical analysis with parametric and non-parametric tests was conducted using GraphPad Prism 7 .The Kolmogorov–Smirnov and Shapiro–Wilk tests were used to validate the assumption of normality. Statistical significance was determined using a nonparametric, Mann–Whitney To evaluate changes of Mmp-9 mRNA after neuronal activation, we isolated RNA from the unstimulated as well as PTZ-stimulated (2 and 4 h after PTZ-induced seizures) rat hippocampi and analyzed Mmp-9 mRNA content by RT-qPCR. Our results demonstrated the occurrence of substantial neuronal seizure-dependent upregulation of Mmp-9 mRNA expression Figure .To check whether the Mmp-9 mRNA stabilization can be induced by neuronal activation, we measured the rate of Mmp-9 mRNA degradation in the hippocampus by an RNA degradation assay (RDA). We mixed cytoplasmic protein lysates isolated from the unstimulated as well as PTZ-stimulated (2 and 4 h after PTZ-induced seizures) rat hippocampi with equal amounts of rat hippocampal RNA and incubated the samples for 0, 30, 60, and 120 min. We found that Mmp-9 mRNA underwent gradual degradation in the unstimulated hippocampus Figure . In contAdenylate-uridylate-rich elements (AREs) of mRNA 3′-UTRs are one of the main mRNA-stabilizing motifs found in mammalian mRNAs  are the main mRNA stabilizers binding to AREs  rat hippocampi with anti-HuR, anti-HuB, or appropriate normal isotype antibodies as well as with total RNA extracted from the rat hippocampus at 4 h after PTZ-induced seizures. After 60 min of incubation, we extracted RNA and evaluated Mmp-9 mRNA expression by RT-qPCR. We found that the neutralization of HuR or HuB completely blocks PTZ-induced Mmp-9 mRNA stabilization in the rat hippocampus, leading to strongly elevated decay of Mmp-9 mRNA Figure . In contAbnormal Mmp-9 enzymatic activity participates in the induction or progression of many disorders, including CNS diseases . Consequently, it is well established that Hu proteins play important roles in multiple steps of neurodevelopment. It has been revealed that HuB regulates neurogenesis through engagement in the promotion of neuronal progenitor cell differentiation (Akamatsu et al., Hu proteins can regulate posttranscriptional gene expression in the brain by acting via different mechanisms, depending on the specific target mRNA and the regulatory context. The binding of HuR or ectotopically expressed HuB to ARE-containing mRNAs can result in the stabilization and increased translatability of mRNA transcripts (Jain et al., In summary, our studies define a new mechanism regulating the neuronal activity-evoked upregulation of Mmp-9 expression by acting through Hu-induced increased stabilization of Mmp-9 mRNA and, thereby, delineate a critical dependence of the stabilization on HuR and HuB actions.MR, KZ-B, and MW-G designed the study and wrote the paper. KZ-B and MW-G designed, conducted, and analyzed the experiments. MR analyzed the data. AC, MA-L, AB, and JW-D conducted and analyzed the experiments. All authors analyzed the results and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This cytotoxicity is blocked by the caspase inhibitor Z-VAD. Analysis of the application of these reagents for the treatment of inflammatory arthritis in vivo suggests that FasL-expressing LVP could be useful for therapy in autoimmune diseases such as rheumatoid arthritis, where there is an excess of Fas-expressing activated T cells in the joint. LVP could be a vehicle not only for mFasL but also for other membrane-bound proteins that maintain their native conformation and might mediate biological activities.During budding, lentiviral particles (LVP) incorporate cell membrane proteins in the viral envelope. We explored the possibility of harnessing this process to generate LVP-expressing membrane proteins of therapeutic interest and studied the potential of these tools to treat different pathologies. Fas-mediated apoptosis is central to the maintenance of T cell homeostasis and prevention of autoimmune processes. We prepared LVP that express murine FasL on their surface. Our data indicate that mFasL-bearing LVP induce caspase 3 and 9 processing, cytochrome C release, and significantly more cell death than control LVP  Several examples of their use to correct genetic defects in have been described in both human disease /FasL CD178) receptor/ligand interaction is a key factor in the induction of programmed cell death . Fas/Fas8 recepto+ cytotoxic T cells as well as a proportion of activated CD4+ T cells and NK cells. mFasL is processed by metalloproteases to generate soluble FasL (sFasL) that although still able to bind Fas, is much less efficient in killing target cells  and tested their efficiency in specific killing of Fas-expressing cells. Exposure of Fas-expressing cells to the FasL-LVP triggered activation of caspase 3 and subsequent apoptosis, as shown by annexin V (AV)-propidium iodide (PI) incorporation and cell cycle analysis, with no crosslinking requirement. FasL-LVP injection in the footpad of mice with established collagen-induced arthritis caused a reduction in paw inflammation, of cells infiltrating the tissue, as well as clear improvement in the clinical score compared to controls. Our l-glutamine; for BaF/3, 10% conditioned medium of an IL-3-producing WEHI-3B cell line (DSMZ ACC26) was added to medium.HEK293T cells (human embryonic kidney cells) were obtained from American Type Culture Collection . The IL-3-dependent murine pro-B cell line, BaF/3, was obtained from German Collection of Microorganisms and Cell Cultures , and the murine pre-B lymphoma cell line L1.2  was cult2.Thymus tissue from C57BL/6 mice  was disaggregated with a potter homogenizer and the isolated thymocytes cultured in RPMI, 50 µM β-mercaptoethanol and 10 mM HEPES (pH 7.4). All cells were maintained at 37°C with 5% COvia a glycosylphosphatidylinositol (GPI) anchor the following oligonucleotides: mFasLFor—AGAGTCGACGCCACCATGCAGCAGCCCATGAATTAC; mFasLRev—GCAAGCTTAGAGCTTATACAAGCCGAAAAAGG; FasLFor—AGAGTCGACGCCACCATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTACACCCTC; mFasLGPIFor—ACGTCTGTTGCTACACCCTCTGAAAAAAAAGAGC; and mFasLGPIRev—GCAAGCTTGCCACCAGAGCTTGAACTGAGCTTATACAAGCCGAAAAAGG. DAFGPIFor—GCAAGCTTCCAAATAAAGGAAGTGGAACC; DAFGPIRev—GCATGCGGCCGCTAAGTCAGCAAGCCCATGGTTAC were used to amplify fragments of mFasL, murine DAF, and a linker sequence from mouse spleen cDNA, and then different combinations of these oligonucleotides were used to amplify overlapping fragments from the first round of PCR to generate a full length mFasL-GPI fragment that was cloned into the pBJ-Neo vector  followed by ultracentrifugation in a Beckman SW55 rotor  through a 20% sucrose cushion. The LVP-containing pellet was concentrated, resuspended in PBS, and stored in aliquots at −80°C. Control LVP were produced as above using HEK293T cells transfected with empty LVTHM/GFP and PAX2 plasmids (control HEK293T cells).Lentiviral expressing murine FasL (FasL-LVP) were produced by transient co-transfection of HEK293T cells with mFasL-GPI-LVTHM/GFP plasmid and, 24 h later, with PAX2 plasmid  at a 1:1 ratio using JetPEI . At 72 h posttransfection, cell supernatant was collected and centrifuged  with appropriate antibodies diluted in 50 µl staining buffer . After incubation , cells were washed twice with staining buffer and analyzed in a Gallios cytometer . Experiments were analyzed using FlowJo software .For cell staining, cells were incubated , -mCD95L-PE (BD Biosciences), and -CXCR4-biotin . Streptavidin PE was from Beckman Coulter.To evaluate FasL-expressing LVP, we used flow cytometry of LVP coupled to latex beads. Aldehyde/sulfate latex beads  were sonicated  and mixed in a 1:3 v/v ratio with LVP . PBS (1 ml) was added, and the final suspension was incubated  with continuous rocking. Free reactive groups were blocked using glycine . Beads coupled to LVP were washed with PBS/BSA 0.5%. For FACS analysis, the pellet was resuspended in PBS staining buffer and stained with specific antibodies as described above.5) were incubated  with 10 µg/ml control LVP or FasL-LVP . After washing with PBS, cells were resuspended in 50 µl detergent  containing 10 ng/ml PI . Cell cycle phases were analyzed by flow cytometry. sFasL  crosslinked with anti-6-His antibody  was used as control.Thymocytes or BaF/3 cells (5 × 105) were incubated  with 10 µg/ml control LVP or FasL-LVP . Cells were stained with AV-FITC and with PI following the manufacturer protocols . All samples were analyzed in a Gallios cytometer. The number of early apoptotic (AV+/PI−) and necrotic/late apoptotic (AV+/PI+) cells was expressed as a percentage of total cells.Thymocytes, BaF/3, or L1.2 cells , protein concentration in lysates was determined using BCA . Thymocyte lysates (40 µg) and LVP  were resolved by 12% SDS-PAGE, transferred to nitrocellulose membrane , blocked with 5% dry milk in TBS , and incubated  with primary antibodies: anti-mFasL (BD Biosciences), -caspase 8 , -caspase 3 , or -GFP . After incubation with appropriate peroxidase (PO)-conjugated secondary antibody , proteins of interest were detected by chemiluminescence . ImageJ software was used for quantitation.Cells were lysed in 100 µl lysis buffer containing triethanolamine  and 2% digitonin, supplemented with protease inhibitors, aprotinin and leupeptin (20 µg/ml each), PMSF (1 mM), and sodium orthovanadate (10 µM). Lysis was carried out with agitation . After centrifugation  on microscope slides coated with poly-l-lysine . Cells were fixed , permeabilized , and blocked with blocking buffer . Anti-cytochrome C antibody was added  followed by anti-mouse Alexa 647 . After extensive washing with PBS, samples were incubated with DAPI  and analyzed in an Olympus FV1000 confocal microscope with a 63× NA 1.4 oil objective.Cells were incubated with Mitotracker  and seeded (1.5 × 100) were inoculated on day 0, 2, 4, 6, and 9 in the footpad with FasL-LVP or LVP  and the clinical score re-evaluated (Cst). As control, unaffected limbs were treated in parallel with FasL-LVP or LVP .Two-month-old DBA/1J mice were immunized intradermally at the tail base with an emulsion of chicken type II collagen (CII) in Freund’s complete adjuvant . ArthritAt the end of the experiment, the score was monitored, and the paws were removed, fixed in 4% formalin, decalcified with 10% EDTA, and paraffin-embedded. Sections (4 µm thick) were stained with hematoxylin and eosin or alkaline phosphatase.Microtiter plates were coated with chicken type II collagen . After blockade of remaining protein-binding sites with 0.5% BSA in PBS, plates were incubated with serial dilutions of serum samples of immunized DBA/1J mice, followed by PO-labeled subclass-specific rabbit anti-mouse antisera. When needed, LVP- and FasL-LVP-coated microtiter plates (1 µg/ml) were incubated with serial dilutions of serum samples of mice injected with LVP in the footpad (time 0 and 14 days after treatment), followed by PO-rabbit anti-mouse IgG (Dako) antisera.p < 0.0001, **p < 0.001, *p < 0.05). We used one-way ANOVA analysis and Tukey’s multiple comparison test. Data are shown as mean ± SEM.Results were analyzed with Prism 5.0 . Transfection efficiency was controlled by assessing GFP expression  Figures A,B. Flow Figures C. To con6 cells/ml) were plated in RPMI medium supplemented with 5% FCS and IL-3, and exposed to serial dilutions of FasL-LVP, control LVP, or sFasL plus crosslinker as a positive control. Cell cycle analysis by PI incorporation and flow cytometry showed that treatment with FasL-LVP significantly increased cell death compared to control , autoprocessing of the initiator procaspase 8 and activation of the effector procaspase-3, which triggers the extrinsic pathway of apoptosis  and/or cp < 0.01)  Figure A was obs) Figure B, sugges) Figure C. We non) Figure D. The usin vitro and in vivo.As they bud from the membrane of the cell, LVP incorporate components of the plasma membrane, including embedded proteins in their lipid environment, into the virus particle . Here, wThe Fas molecule belongs to a subset of the TNFR family called death receptors, which can trigger apoptosis by inducing caspase activation. Ligation of Fas triggers apoptosis in activated T cells; this mechanism is involved, for example, in regulation of the clonally expanded T cell population after antigen clearance . Fas-medWe engineered and transiently expressed a chimeric FasL-GPI in HEK293T cells and used them to produce LVP that express FasL, but lack viral envelope proteins. Replacement of C-terminal transmembrane domains by GPI-addition signal peptides allows expression of the proteins on the plasma membrane and provides stable association of the construct with the lipid bilayer , 34. PlaIn vitro, the virus-like particles induced cell death in Fas-positive cells, including the B cell line, BAF/3 and primary thymocytes. The absence of apoptosis in L1.2 cells, which do not express cell surface Fas, and in cells treated with control LVP showed that this effect is specific; it indicated that FasL-LVP-induced cell death is mediated by the FasL expressed on the particles and not by other components expressed at the membrane or by other LVP components. In this context, when VSV-G pseudotyped LVP were used in similar experiments, increased cell death was observed independently of FasL, probably due to the ability of these LVP to infect cells .JMR-F and MM designed research; JMR-F, AG, PL, LM-M, RV, GC, and HR performed research; GC, DB, and HR contributed new reagents/analytic tools; JMR-F, RV, and MM analyzed data; and JMR-F, HR, and MM wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, an influx of T cells to the lungs results in similar absolute numbers of specific CD4+ T cells, compared with uninfected individuals.Human immunodeficiency virus infection leads to lower frequencies of CD4 Mycobacterium tuberculosis–specific CD4+ T cells in blood during early HIV infection, but little is known about responses in the lungs at this stage. Given that mucosal organs are a principal target for HIV-mediated CD4+ T-cell destruction, we investigated M. tuberculosis–specific responses in bronchoalveolar lavage (BAL) from persons with latent M. tuberculosis infection and untreated HIV coinfection with preserved CD4+ T-cell counts. M. tuberculosis–specific CD4+ T-cell cytokine  responses were discordant in frequency and function between BAL and blood. Responses in BAL were 15-fold lower in HIV-infected persons as compared to uninfected persons (P = .048), whereas blood responses were 2-fold lower (P = .006). However, an increase in T cells in the airways in HIV-infected persons resulted in the overall number of M. tuberculosis–specific CD4+ T cells in BAL being similar. Our study highlights the important insights gained from studying M. tuberculosis immunity at the site of disease during HIV infection.Human immunodeficiency virus type 1 (HIV) infection substantially increases the risk of developing tuberculosis. There is extensive depletion of  Tuberculosis is one of the leading causes of infectious disease globally, with >10 million cases and 1.8 million deaths in 2015 [+ T-cell counts [Whilst the majority of opportunistic infections develop during advanced HIV-associated immunodeficiency, tuberculosis occurs over a wide range of CD4l counts . The risl counts  and incrl counts , 3, 6.Mycobacterium tuberculosis immunity in HIV–M. tuberculosis coinfection focus on characterizing T-cell responses to M. tuberculosis in blood. Both quantitative and qualitative defects in the CD4+ T-cell response to M. tuberculosis have been reported in HIV-infected persons with latent M. tuberculosis infection [M. tuberculosis and depletion of CD4+ T cells in the airways are considerably less well characterized during HIV infection. HIV has been described as a disease of the mucosal immune system, owing to profound and early CD4+ T-cell depletion at mucosal sites such as the gastrointestinal tract [+ T cells in the lungs, sampled by bronchoalveolar lavage (BAL), are CCR5+ memory cells [+ CD4+ T cells has been reported to be relatively maintained in BAL during HIV infection [The majority of studies of nfection . Lung real tract , 13. Thery cells , 15, thery cells , the frenfection , 20.M. tuberculosis–specific CD4+ T-cell responses in the lungs in HIV infection have been examined in only a few studies. A lower frequency of BAL CD4+ T-cell responses in immunosuppressed HIV-infected individuals as compared to HIV-uninfected individuals has been reported, and the frequency and function of these cells is distinct from those in blood [M. tuberculosis–specific CD4+ T cells are preferentially and substantially depleted in blood early in HIV infection [M. tuberculosis responses and CD4+ T-cell depletion in the airways prior to substantial immunodeficiency and their relationship with M. tuberculosis–specific responses in blood. We characterized the frequency and absolute number of T cells, the HIV load, and M. tuberculosis–specific T-cell responses in BAL and blood specimens from moderately immunosuppressed and untreated HIV-infected and HIV-uninfected individuals with latent M. tuberculosis infection.in blood , 17, 21.nfection , 22, we + T-cell counts of >400 cells/mm3  and 25 HIV-seronegative persons . HIV RNA levels were determined using an Abbott m2000 RealTime HIV-1 assay, and blood CD4+ T-cell counts were determined by the Flow-CARE PLG CD4 test. All volunteers were sensitized to M. tuberculosis, based on positive results of an interferon γ (IFN-γ) release assay , and tuberculosis was excluded on the basis of symptoms, radiological evidence, and BAL fluid culture results. Previous history of tuberculosis was excluded in all participants except for 2, in whom tuberculosis had occurred >10 years prior to enrollment. This study was approved by the Research Ethics Committees of the University of Cape Town (REF158/2010) and Stellenbosch University (N10/08/275). All participants provided written, informed consent.Participants were recruited from Cape Town, South Africa, into 2 groups: 25 ART-naive HIV-seropositive persons with CD4Blood specimens were collected and processed within 4 hours. Flexible bronchoscopy was performed while participants were conscious and sedated. Saline (160 mL) was instilled into the right middle lobe in 20-mL aliquots, aspirated, and stored on ice until processing. BAL samples were centrifuged, and the cell pellet was washed with cold phosphate-buffered saline (Sigma) and filtered through a 100-μm cell strainer . Acellular BAL fluid was stored at −80°C, and cells underwent immunological analysis while fresh. To correct for epithelial lining fluid (ELF) dilution due to variable fluid volumes recovered , the urea method was used  as described elsewhere . BAL vir6 cells per BAL specimen , or 8.7 × 106 cells/mL ELF, were obtained (Supplementary Table 1). The absolute number of T lymphocytes in BAL fluid was calculated using the frequencies of live CD3+, CD4+, or CD8+ T cells from an ex vivo flow cytometry–based phenotyping panel (for specimens from 38 participants) and by microscopy.BAL cells were counted using Trypan Blue exclusion and differentially stained . A median of 10.6 × 10M. tuberculosis (20 μg/mL) or phorbol 12-myristate 13-acetate (0.01 μg/mL) and ionomycin (1 μg/mL), in the presence of anti-CD28 and anti-CD49d . Unstimulated cells were incubated with costimulatory antibodies only. Brefeldin A (5 μg/mL) was added after 7 hours. After incubation, red blood cells were lysed, and the cell pellet was stained with a violet viability dye, ViViD (Molecular Probes), treated with FACS Lyse (BD), and cryopreserved in 10% dimethyl sulfoxide in fetal calf serum.Whole-blood stimulation was performed as previously described . Briefly6 total live BAL cells and/or <2 × 105 total lymphocytes, based on Trypan and differential counts, respectively).Fresh BAL cells underwent similar stimulation in R10 medium  with the addition of 0.02 mg/mL DNase I, 50 U/mL of penicillin-streptomycin, and 0.8 mg/mL of Fungin. BAL cells were stained with ViViD, treated with FACS Lyse, and stained. BAL cytokine data are reported for 30 of 50 participants (16 with and 14 without HIV infection). The remaining 20 participants had insufficient BAL lymphocyte yields to perform T-cell stimulation assays (<10 × 10Unstimulated BAL cells were stained ex vivo with anti-CD3-PE-Cy7 and CCR5-PE (both from BD) and with CD4-PE-Cy5.5 and CD8-Qdot705 (both from Invitrogen). Freshly stimulated BAL cells and stimulated cryopreserved blood cells were washed and stained with anti-CD4-PE-Cy5.5 and CD8-Qdot705 (both from BD), permeablized, and stained intracellularly with CD3-APC-H7, IFN-γ-Alexa700, and interleukin 2 (IL-2)-APC  and with tumor necrosis factor α (TNF-α)-PE-Cy7 (eBiosciences). Cells were acquired on a BD Fortessa, using FACSDiva software, and data were analyzed using FlowJo (TreeStar) and Pestle and Spice . A positU test, the Wilcoxon matched pairs test, and the Spearman rank test) were used for all comparisons. A P value of < .05 was considered statistically significant.Statistical analyses were performed using Prism 5 (GraphPad). Nonparametric tests , and exhibited a wide range of plasma viral loads . HIV RNA was detectable in BAL fluid from 20 of 25 participants, with a median load of 10700 RNA copies/mL ELF . There was a significant positive correlation between viral load in plasma and BAL , consistent with published studies [Blood and bronchoalveolar lavage (BAL) samples were collected from 25 HIV-infected and 25 HIV-uninfected persons sensitized to  studies , 19.P = .031) and a higher proportion of lymphocytes . As expected, HIV-infected participants had significantly lower frequencies of CD4+ T cells in BAL and blood specimens, compared with HIV-uninfected participants . In HIV-uninfected persons, the ratio of CD4+ to CD8+ T cells in BAL fluid was lower than in blood specimens , consisting of 26-fold more CD8+ T cells (P = .001) and 7-fold more CD4+ T cells (P = .03). When we examined CCR5 expression on BAL CD4+ T cells, a median of 76% expressed CCR5, regardless of HIV status (data not shown), implying that these cells are potential targets for HIV, despite their higher absolute number in BAL fluid as compared to the value for HIV-uninfected individuals. Absolute numbers of BAL CD4+ T cells  and CD8+ T cells  correlated positively with BAL viral load .The cellular content of BAL consisted primarily of alveolar macrophages (>90%) and smaller populations of lymphocytes and neutrophils (Supplementary Table 1). Compared with HIV-uninfected individuals, HIV-infected individuals had a significantly lower proportion of macrophages  and 50% of HIV-infected individuals  in HIV-infected participants as compared to uninfected individuals  or IL-2 (P = .019), compared with HIV-uninfected participants  likely reflected relatively early infection. A representative example of CD4+ T-cell cytokine production in blood in response to PPD is depicted in P = .0067; r = 0.38; data not shown). Similar to our observations in BAL fluid, the total frequency of M. tuberculosis–specific CD4+ T cells in blood specimens was significantly lower (by 2-fold) in HIV-infected individuals as compared to HIV-uninfected subjects . After adjustment for absolute CD4+ T-cell count in blood, this was more pronounced, with 3-fold lower numbers of M. tuberculosis–specific cells , suggesting that the decrease in M. tuberculosis CD4+ T-cell responses was related to overall CD4+ T-cell depletion. We found no relationship between blood M. tuberculosis response frequencies and plasma viral load , or between BAL responses to M. tuberculosis and either BAL CD4+ T-cell count or BAL viral load .We next analyzed nfection , 22. Alt+ T-cell counts in the absence of ART, the frequency of peripheral M. tuberculosis–specific CD4+ T cells was significantly lower than in HIV-uninfected subjects.Overall, our data show that even in HIV-infected individuals with well-maintained peripheral CD4M. tuberculosis responses in blood are frequently measured in vaccine trials, but whether they are a surrogate for responses in the airways is not clear. Thus, we directly compared the frequencies of M. tuberculosis–specific CD4+ T-cell responses between blood and BAL specimens in the subgroup of 30 participants from whom we had data for both compartments. There was no difference in the median frequency of M. tuberculosis responses between BAL and blood specimens, regardless of HIV infection status . Furthermore, HIV-infected individuals had a significantly higher proportion of cells expressing IFN-γ, IL-2, and TNF-α; IL-2 and TNF-α only; and TNF-α only , compared with HIV-uninfected individuals. We also noted a significant difference in the median fluorescent intensity of TNF-α in M. tuberculosis–specific CD4+ T cells, with a higher value in HIV-infected individuals as compared to uninfected individuals .Finally, we investigated the functional quality of CD4pecimens  and 5B. nctional . MoreoveM. tuberculosis–specific CD4+ T cells during HIV infection, and they highlight distinct HIV-induced changes in blood compared to BAL. Collectively, these data emphasize that M. tuberculosis responses in blood do not necessarily reflect the quantity or quality of the M. tuberculosis immune responses in the airways.Taken together, these results suggest specific perturbations in cytokine production in + T cells are progressively depleted [+ T-helper type 1 (Th1) cells play a critical role in immunity to M. tuberculosis [+ T-cell responses to M. tuberculosis during HIV infection has been described in many studies [M. tuberculosis in blood, the effect of HIV on immunity at the site of infection is considerably less well studied. We examined M. tuberculosis–specific lung immunity in HIV-infected persons with moderate immunosuppression. We found a decreased frequency of M. tuberculosis–specific CD4+ Th1 responses in blood specimens from HIV-infected participants as compared to uninfected persons, consistent with published studies of advanced HIV disease [+ T-cell frequencies in BAL fluid, they had a 7-fold greater absolute CD4+ T-cell count in these specimens, compared with HIV-uninfected individuals; (2) increased CD4+ T-cell numbers in BAL fluid were associated with BAL viral load; (3) in 30 participants in whom we measured lung immunity, one third had no detectable M. tuberculosis–specific responses in BAL fluid, despite detectable T-cell responses in blood specimens; (4) although HIV-infected participants had significantly lower frequencies of M. tuberculosis–specific CD4+ T cells, when those with detectable BAL responses were adjusted for CD4+ T-cell numbers in BAL fluid, no significant differences were observed from HIV-uninfected participants. These data highlight disparities in the effect of HIV on CD4+ T-cell dynamics in the blood and the lungs and underscore the compartmentalization of M. tuberculosis–specific responses between the 2 sites.HIV infection is the greatest recognized risk factor for the development of tuberculosis in high-burden settings . The risdepleted , 27. CD4rculosis , and dep studies , 29–31.  disease , 17, 21.M. tuberculosis response frequency in blood specimens as compared to BAL fluid, regardless of HIV infection. Although there was a weak positive association between blood and BAL response frequencies, undetectable responses in BAL fluid were unrelated to the magnitude of M. tuberculosis–specific CD4+ T-cell frequencies in blood specimens. The absence of responses to M. tuberculosis in BAL fluid was more pronounced in HIV-infected persons, with 50% having no detectable responses. In addition to HIV-associated depletion, several possibilities could account for the absence of BAL responses. We sampled a single site, and M. tuberculosis immunity may not be uniform throughout the lung [M. tuberculosis–exposed individuals, among whom the spectrum of M. tuberculosis history could range from contained latent infection to cleared infection without persisting M. tuberculosis–specific memory responses in the lungs [M. tuberculosis–specific CD4+ T cells were also functionally distinct between the compartments, with a negligible percentage of polyfunctional PPD-specific CD4+ T cells within the BAL CD4+ T-cell population, compared with approximately 25% in blood. This is in contrast to studies using mitogens and other pathogens, in which highly polyfunctional CD4+ T cells were detected in BAL fluid [Adaptive immunity in blood is used as a surrogate for lung responses, emphasizing the importance of understanding differences between blood and BAL T-cell responses. Approximately two thirds of individuals had a higher the lung . Furtherhe lungs . M. tubeAL fluid , 20, andM. tuberculosis–specific responses may have important implications for tuberculosis vaccine development. Recent trials of the first novel tuberculosis vaccine, MVA85A, failed to demonstrate protection against M. tuberculosis infection or tuberculosis [+ T-cell responses in blood. An effective tuberculosis vaccine may need to induce immunity to M. tuberculosis in the airways. Although BAL is expensive to perform and requires specialized personnel, early vaccine trials could investigate local pulmonary responses in a subset of individuals. Additionally, vaccine delivery into the airways may be a promising strategy to induce long-lived pulmonary immunity and increase vaccine efficacy [This compartmentalization in frequency and function between blood and lung rculosis , 35 despefficacy .+ T-cell numbers in BAL fluid during HIV infection despite moderate peripheral CD4+ T-cell depletion. The marked increase of both CD4+ and CD8+ T-cell counts in the airways that we observed was reflective of a degree of lymphocytic alveolitis characteristic of HIV infection [+ memory T cells supports the hypothesis that the lung is a mucosal site distinct from the gastrointestinal tract when it comes to the effects of HIV [M. tuberculosis CD4+ T cells may be offset by an influx of CD4+ T cells that normalizes the absolute number of M. tuberculosis–specific CD4+ T cells and thus keeps the tuberculosis risk relatively low, thus resolving the apparent discrepancy between a substantially decreased M. tuberculosis–specific CD4+ T-cell frequency but only a doubling of tuberculosis risk in early infection [We found increased CD4nfection . The rels of HIV  and may s of HIV , 16, 20.nfection .+ Th1 responses, considered the cornerstone of adaptive M. tuberculosis immunity. There is, however, a growing body of evidence that IFN-γ–independent mechanisms may also provide protection against tuberculosis [M. tuberculosis. In addition, early defects in innate immunity in both blood and BAL may influence tuberculosis risk. Last, although BAL is a relatively noninvasive technique for studying immunity in the lungs, a major limitation is the low yield of lymphocytes, enabling us to perform functional immune assays in only 60% of our cohort. Thus, further studies will be required to confirm our findings.It is worth noting that we restricted our analyses to blood and BAL CD4rculosis . Furtherrculosis , in protM. tuberculosis–exposed HIV-infected persons with moderately well-preserved CD4+ T-cell numbers had a significant decrease in the frequency of CD4+ T-cell responses to M. tuberculosis in blood and BAL specimens. However, an increased T-cell count in the airways related to the HIV load may have resulted in a similar absolute number of M. tuberculosis–specific CD4+ T cells in BAL fluid. Our novel findings emphasize the differences between blood and lung responses to M. tuberculosis and highlight the need to better understand M. tuberculosis responses in the lungs.In summary, this study showed that antiretroviral-untreated, The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Table S1Click here for additional data file.Fig S1Click here for additional data file.


Lagenaria siceraria plants infected with Cucumber green mottle mosaic virus (CGMMV) were comprehensively characterized and compared. Many more vsiRNAs were present in infected leaves than in fruits. vsiRNAs from both leaves and fruits were mostly 21- and 22-nt in size as previously described in other virus-infected plants. Interestingly, vsiRNAs were predominantly produced from the viral positive strand RNAs in infected leaves, whereas in infected fruits they were derived equally from the positive and negative strands. Many leaf-specific positive vsiRNAs with lengths of 21-nt (2058) or 22-nt (3996) were identified but only six (21-nt) and one (22-nt) positive vsiRNAs were found to be specific to fruits. vsiRNAs hotspots were only present in the 5′-terminal and 3′-terminal of viral positive strand in fruits, while multiple hotspots were identified in leaves. Differences in GC content and 5′-terminal nucleotide of vsiRNAs were also observed in the two organs. To our knowledge, this provides the first high-resolution comparison of vsiRNA profiles between different tissues of the same host plant.RNA silencing is an evolutionarily conserved antiviral mechanism, through which virus-derived small interfering RNAs (vsiRNAs) playing roles in host antiviral defense are produced in virus-infected plant. Deep sequencing technology has revolutionized the study on the interaction between virus and plant host through the analysis of vsiRNAs profile. However, comparison of vsiRNA profiles in different tissues from a same host plant has been rarely reported. In this study, the profiles of vsiRNAs from leaves and fruits of  In general, 21-nt vsiRNAs usually predominate in the population, there is a strong A/U bias at the first nucleotide of vsiRNAs, and vsiRNA-producing hotspots can be identified within the viral genome  is a member of the genus Lagenaria siceraria, accession “Hangzhou gourd”) were sown in soil rich in organic matters in a greenhouse with the ambient temperatures between 20 and 25°C, and watered every 3 days to maintain ample soil moisture. At the two and a half leaf stage plants were mechanically inoculated with CGMMV virions on the two expanding leaves using sap from a previously infected plant. Approximately 100 mg of tissue was homogenized in 20 volumes of inoculation buffer , while the mock plants were only inoculated with inoculation buffer.Seeds of bottle gourd  according to the manufacturer's instructions. The presence of CGMMV infection in the tissues was confirmed with a One Step RT-PCR Kit  following the product's protocol and using CGMMV specific primers . The RNA quantity and quality from each sample was evaluated by denaturing agrose gel electrophoresis and a 2100 Bioanalyzer .Approximately 5 μg of total RNA was extracted for the preparation of a small RNA library according to the protocol of TruSeq Small RNA Sample Prep Kits . Briefly, total RNA was resolved using denatured 8% polyacrylamide gel electrophoresis (PAGE) and small RNA fragments were isolated. After ligation of the 5′ and 3′ adaptors, the short RNA fragments were reverse transcribed using SuperScript II Reverse Transcriptase  and amplified by PCR. Finally, single-end sequencing (36 bp) was performed on an Illumina Hiseq2500 at LC-BIO  following the protocol of the manufacturer.http://hannonlab.cshl.edu/fastx_toolkit/). The remaining sRNA reads were collapsed to uniread sets and the reads of > 30-nt or <18-nt were discarded. Clean sRNA reads were used for further bioinformatics analysis.After parsing small RNA sequences from the 3′ adaptor sequence, low quality and junk sequences, including transfer RNAs (tRNAs), ribosomal RNA (rRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), and repetitive sequences, were removed using the FASTX-Toolkit (L. siceraria libraries were mapped to the CGMMV reference genome (NCBI Accession No: KP868654) using Bowtie software (http://bowtie-bio.sourceforge.net) with one mismatch. To facilitate comparisons across different libraries, vsiRNA read numbers were normalized to “Reads Per Million” (RPM) based on the total small RNA read numbers of the corresponding library. All of the downstream analyses were performed using custom perl scripts and linux (Cent OS 6.5) bash script. For statistical analysis of the three biological replicates, one-way ANOVA analysis using Originpro 8.5 software was performed and values of p < 0.01 were considered significant. To avoid the inaccuracy of low copy sequences, sequences with <10 raw reads in each of the three replicates were removed . Specific (Unique) vsiRNAs were extracted from the three replicates of each sample during this analysis. RNA secondary structures were predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) with default parameters.To identify CGMMV-derived siRNAs, processed reads from each of the 12 Total RNA was isolated from plants with Trizol  according to the manufacturer's instructions. For northern blot of CGMMV RNAs, a DNA probe targeting CGMMV CP was synthesized with primers (5′-GCTTACAATCCGATCACAC-3′ and 5′-ATTATCTATCTCAGCCCTAG-3′) and labeled with DIG according to the manufacturer's protocol . For northern blot of positive-stranded CGMMV RNAs in leaves, a sequence (5′-CAACACAGGACCGTTGAGGAAAGCGTAAAAACCCGCACCTGGGAATCTAGAATTAATATCTACGACAGACGAGGGTAACGCA-3′) was synthesized and labeled as DNA probe, and its complementary sequence was used for detecting negative-stranded CGMMV RNAs. Another sequence (5′-CATAGCTCTGAGCTTTAACTACACTAAAGTCAGTTATAGATAAATACTTAAGAATGGAAAAATAGTTAGGGAGCAACTTATC-3′) was used for detecting positive-stranded CGMMV RNAs in fruits, and its complementary sequence was used for detecting negative -stranded CGMMV RNAs in fruits. Pre-hybridization, hybridization and signal detection were done according to the protocol of the DIG High Prime DNA Labeling and Detection Starter Kit II .Tissue immunoblot was carried out as described previously -infected leaves of bottle gourd showed the typical green mottle mosaic symptom 14 days after inoculation Figure , while tAfter the removal of the junk, adapter and repeat reads, total numbers of small RNAs 18–30 nt long obtained from the three virus-infected fruits were 8,558,357 , 9,240,205  and 5,654,646 . Corresponding numbers from the mock fruits were 9,442,756 , 9,432,345  and 9,268,640 . From three virus-infected leaves, totals were 10,250,660 , 7,513,459  and 12,265,181  and from three mock leaves, there were 8,766,823 , 4,064,336  and 6,644,494 . An overview of the deep sequencing results is presented in Table The size distribution of these 12 small RNA libraries was similar. Reads with 24-nt length accounted for most (60–70%) of the total sRNAs, followed by 23-nt Figure . HoweverN. benthamiana plants infected with Beet necrotic yellow vein virus (BNYVV) showed that the antiviral response was more effective in leaves than in roots; vsiRNAs accumulated more in leaves than in roots, whereas BNYVV mRNA levels were lower in leaves than in roots  of the total sRNAs of virus-infected fruits, while the corresponding figures for unique vsiRNAs were 0.95–1.15% (1.06 ± 0.10%). These values are much lower than those from infected leaves where total vsiRNAs accounted for 20.59–30.99% (27.49 ± 5.98%) of the total sRNAs and the corresponding figures for unique vsiRNAs were 2.24–3.47% (2.89 ± 0.62%)  Figures . The incThe numbers of vsiRNAs derived from positive or negative strand viral RNA were also compared. In infected leaves, many more vsiRNAs were produced from the positive strand viral RNA irrespective of vsiRNA length Figures , which iL. siceraria according to the size and density of the bands  cover the entire CGMMV genome Figure , consistPrevious studies have shown that vsiRNAs are preferentially produced from GC-rich regions and vsiRNAs tend to have a higher GC content than that of the entire viral genome , the International Science & Technology Cooperation Program of China (2015DFA30700) and Fund for Research in the Public Interest of Zhejiang Province (2015C32042).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Along with crop growth, there were active interconversions among Olsen-P, Org-P, Fe-P and O-Al-P in the soil, and some available P converted into Ca10-P, with O-Fe-P possibly being a transitional form for this conversion. The oilseed rape/corn system had less runoff TP at the early stage, and wheat/sweet potato system had a lower runoff P at the late stage. Intercropping corn with sweet potato in the field with oilseed rape as a previous crop may be helpful for alleviating runoff P load during the summer in this region.Soil phosphorus (P) fractions and runoff P concentration were measured to understand the fate of soil P entering surface runoff water during summer cropping season of different double cropping systems under two fertilizer regimes. The dominant form of runoff P was particulate P (PP). Runoff total P (TP) was higher at the vegetative growth stage and lower at the crop reproductive stage. TP and PP were derived mainly from soil Olsen-P, Al-P and Fe-P and amounts increased with sediment content in runoff water. Runoff P discharge was closely related to the changes in soil P forms. Soil Olsen-P, mainly consisting of some Ca Phosphorus (P) enrichment has been recognized as the most critical stimulator to water eutrophication –3, and a4C1-extractable labile P), Al-P (NH4F-extractable Al phosphates), Fe-P (NaOH-extractable Fe phosphates), O-P (P occluded within oxides extracted with sodium citrate-Na2S2O4 solution) and Ca-P (H2SO4-extractable Ca phosphates) in acid and neutral soils [2-P [CaHPO4·nH2O], Ca8-P [Ca8H2(PO4)6·nH2O] and Ca10-P [Ca10(PO4)6·(OH)2] based on their availability and solubility [2-P, Al-P, Org-P, Fe-P, O-Al-P, O-Fe-P and Ca10-P [Phosphorus in soils exists in many complex chemical forms that differ markedly in their behavior, mobility, and resistance to bioavailability in the soils . Therefoal soils . Howeverlubility , 20. A clubility , Lei et d Ca10-P . Comparid Ca10-P  and Gu aThe response and availability of different soil P fractions to fertilizer application, crop types and rotation patterns has been studied in Northern China , 20 and The field experiment of our study did not involve any endangered or protected species and no specific permissions were required for our cropping experiments.Zea mays L.), sweet potato (Ipomoea batatas Lam.), winter wheat (Triticum aestivum L.) and oilseed rape (Brassica napus L.) with the major conventional double cropping systems as wheat and corn (wheat/corn), wheat and sweet potato (wheat/sweet potato) and oilseed rape and corn (rape/corn).A continuous double cropping experiment was conducted at Xijiadian , Danjiangkou City, Hubei Province. The experimental site was about 5 km from Danjiangkou Reservoir that is the second largest artificial freshwater lake in Asia and also the headwater of the middle route of the South-to-North Water Transfer Project of China. From there, the water is transferred to major populous areas in northern China such as Beijing and Tianjin for drinking water. However, the water tends to be eutrophic with increasing total N and P concentrations in the reservoir  and the The climate is typical in the semi-humid northern subtropical zone, with average annual mean temperature of 15.9°C, average monthly mean temperature of 3.1°C (January) - 31.9°C (August) and frost-free period of 180–250 days. The long-term average annual precipitation is 834 mm with most of the rain falling in July to September.Sesamum indicum L.) was grown without fertilizer application in the season prior to the experiment to control weeds and ensure the area was uniform for the experiment. The basic fertility and P fractions of the yellow brown clay were determined as described by Bao [2-P, 12.86 mg/kg of Al-P, 83.96 mg/kg of Fe-P, 19.71 mg/kg of O-Al-P, 197.21 mg/kg of Ca10-P and 12.96 mg/kg of Org-P in the soil. The pH and CaCO3 content of the soil was 6.97 and 3.5%, respectively.The plot experiment was set up on a typically sloped farmland with a slope of 9.9° from October 2008 to October 2011. Sesame  with 3 replicates. There were 3 continuous double cropping systems including winter oilseed rape/corn, wheat/corn and wheat/sweet potato under 2 fertilizer rates, i.e. a lower rate that was commonly used by farmers in the region, and a higher rate that mimicked potential fertilizer use in the future. The 3 cropping systems were combined with 2 fertilizer rates to form 6 factorial treatments: (1) rape/corn + lower fertilizer rate (RCL); (2) rape/corn + higher fertilizer rate (RCH); (3) wheat/corn + lower fertilizer rate (WCL); (4) wheat/corn + higher fertilizer rate (WCH); (5) wheat/sweet potato + lower fertilizer rate (WPL); (6) wheat/ sweet potato + higher fertilizer rate (WPH).2). A concrete barrier about 15 cm wide was inserted 35 cm into bedrock and 25 cm above ground between individual plots to prevent lateral flow. A waterproof cement catchment grooves, which was at the same level as the topsoil on the top and 2 cm deep, was installed across the lower edge of each plot. One end of the catchment grooves was channeled to a water tank that had a total volume of 1 m3 and was covered to prevent rainfall entry and evaporation. A 55 L plastic bucket was placed in the tank to collect runoff water and sediment. The infrastructure was set up in the summer of 2008 and artificially adjusted soil slope of the plots after completion of construction.There were 18 plots in total constructed in 3 rows in the field, each with 7.2 m in length, and 3.2 m in width  was sown with row spacing of 30 cm in the first ten days of October, and thinned out into 20 cm between plants at 5 leaf stage. Winter wheat (cv. Yumai 9203) was sown with row spacing of 25 cm in the second ten days of October. Corn (cv. Yanyu 18) was sown with row spacing of 60 cm in the last ten days of May to the first ten days of June, and thinned out into 30 cm between plants in a row at 4–5 leaf stage. Sweet potato  was transplanted in furrow-ridge tillage with a width of 40 cm (ridge height = 30 cm and furrow width = 30 cm). The distance between transplants was 30 cm on each ridge and the transplanting was undertaken in the last ten days of May up to mid June depending on rainfall. Oilseed rape and winter wheat was harvested in the middle and last ten days of May, respectively. Corn was harvested in the period from the end of September to the tenth of October, and sweet potato in the second ten days of October.2O5) and potassium chloride (59% K2O). The rest of the management was based on the local conventional standard of field production.The fertilizer applied for summer crops included 120.8 kg N/ha, 19.6 kg P/ha and 52.3 kg K/ha for the lower rate, and 169.1 kg N/ha, 27.5 kg P/ha and 73.2 kg K/ha for the higher rate; while, those for winter crops were 138.0 kg N/ha, 19.6 kg P/ha and 56.0 kg K/ha for the lower rate, and 193.5 kg N/ha, 27.5 kg P/ha and 78.4 kg K/ha for the higher rate. All phosphorus and potassium fertilizers were applied as base fertilizer prior to sowing both winter and summer crops. N fertilizer was split into 43% base fertilizer and 57% topdressing fertilizer for summer crops, and 64% base fertilizer and 36% topdressing fertilizer for winter crops. Topdressing N fertilizer was applied after thinning in late June for corn and 7 days after transplanting for sweet potato in summer crop season. In winter crop season, topdressing N fertilizer was applied at 5-leaf stage (12%) and bolting stage (24%) for oilseed rape and jointing stage for winter wheat. The fertilizers used in this study were urea (46% N), calcium superphosphate  was measured at 3 random locations within a plot, using AccuPAR LP-80  at the vegetative growth (23 July) and reproductive stage (24 August), respectively. Crop economic organs (grain for corn and root tuber for sweet potato) and straw including corncob were sampled and oven-dried at 80°C to determine the dry yields and P contents after harvest.In the present study, we focused on the summer cropping season in the fourth year (2011) of the experiment with high surface runoff in the entire cropping season and stabilized and uniformed soils under different treatments in plots. The amount of runoff water/sediment was recorded after rainfall events on 27 July and 23 August 2011, at the vegetative growth and reproductive stage of crops, respectively. All water samples were taken to the laboratory in iceboxes for determining the concentrations of sediment, total phosphorus (TP), particulate phosphorus (PP), total dissolved phosphorus (TDP), and dissolved inorganic P (DIP) in runoff. Three 4.8 cm diameter cores were taken to a depth of 20 cm from each plot and bulked to determine the concentration of Olsen-P, Org-P, CaSamples of runoff water were analyzed using the method of SEPA . After t2SO4  and HClO4  for determining the P content. Soil samples were air-dried and ground to pass a 150-μm mesh prior to analysis. Olsen-P was determined by the method of Olsen et al. [The dried plant samples were ground into fine powder and then digested with concentrated Hn et al. . Soil P n et al. . Phosphon et al. .P = 0.05. Pearson correlation and linear regression analysis was performed between Olsen-P and different P fractions in soil, and runoff TP and PP and soil Olsen-P and P fractions. All data analyses were performed using SPSS 17.0 .Data on the runoff amount, sediment and P concentrations, and the contents of soil Olsen-P and P fractions and crop LAI and P uptake of all treatments were analyzed using 2-way analysis of variance (ANOVA) with cropping systems in combination with different fertilizer rates as random factor and blocks (replicates) as fixed factor in a generalized linear model. Duncan’s multiple comparison test was subsequently used to determine significance of differences between treatments at P < 0.01) differences among different cropping systems in crop leaf area index (LAI) at vegetative growth and reproductive stages, economic yield and P uptake, but not in straw yield of summer crops  differences were detected in the concentrations of runoff TP and DIP at the vegetative growth, PP at the reproductive stage, and TDP and DOP at both stages  associated with soil Olsen-P (R2 = 0.46 for TP and 0.50 for PP), Al-P (R2 = 0.49 for TP and 0.54 for PP) and Fe-P (R2 = 0.44 for both TP and PP), but negatively (P < 0.01) with Ca10-P (R2 = 0.71 for TP and 0.73 for PP)  .The relative importance of surface and subsurface flows for pollutant transport has been studied at plot scale , 32 and 10-P. Higher fertilizer rate tended to increase Olsen-P as well as Ca2-P in all cases and Al-P, Fe-P, O-Al-P and Org-P in most cases, compared to the lower fertilizer application rate. From the vegetative growth to reproductive stage, there was a consistent decrease in soil Olsen-P, Ca2-P and Al-P but an increase in Ca10-P. Nevertheless, Fe-P, O-Al-P and O-Fe-P decreased only by relatively smaller amounts or even increased in some cases. From this and together with the correlation analysis, it was concluded that Olsen-P was mainly from Ca2-P and partially from Al-P and there were active interconversions among Olsen-P, Org-P, Fe-P and O-Al-P, and some Olsen-P was converted into Ca10-P along with crop growth. Apart from the effects of crop growth and P uptake, Ca10-P formation contributes to reduced P load in runoff flow because of its immobility; however, this may result in low soil P available to plant [10-P.Numerous studies have shown that runoff P concentration is highly related to soil P status and P application , 8, 48. to plant . Meanwhi2-P showed the largest availability to plant followed by Al-P and Fe-P in different soil Pi fractions [2-P, Org-P and O-Al-P were insignificantly related to runoff P loss, although they could convert to Olsen-P. Soil Ca2-P is the Pi fraction preferred by crops [Our study showed that runoff TP and PP were derived mainly from Olsen-P, Al-P and Fe-P in the soil. Yu et al. indicated that Caractions . This inby crops , which mby crops .R2 values in this study, although all regression analyses are significant. This may be attributed to the methods used to measure P in soils and runoff water in this study. Do Nascimento et al. [In this study, higher fertilizer rate did not significantly increase the runoff TP in cropping systems. Phosphorus was applied as basal fertilizer, and the nutrients in the fertilizer applied not only included P but N and K. These might have stimulated plant growth with relatively more P uptake, leading to less loss of the nutrient as crops grew. Runoff increased and TP concentration tended to decrease at the reproductive stage under the higher fertilizer rate for wheat/sweet potato, compared to the other cropping systems. In addition to the dilution of P by more runoff water, this was also related to the characteristics of the creeping growth behavior of sweet potato. When sweet potato branches, the ground coverage increases rapidly, the progress of which is enhanced by higher fertilizer rate. This may have reduced infiltration velocity of rain water , leadingo et al.  recentlySharpley et al. reported that runoff water is considered as degraded if its total P exceeds a guideline concentration of 0.10 mg P/L . Total P2-P and Al-P was increased by elevating fertilizer rate. Along with crop growth, there were active interconversions among Olsen-P, Org-P, Fe-P and O-Al-P in the soil, and some available P changed to Ca10-P, with O-Fe-P as a transitional form. Runoff TP was lower at the early stage in oilseed rape/corn and at the late stage in wheat/sweet potato. Intercropping corn with sweet potato following oilseed rape may help reduce runoff P load during the summer in this region.There were differences in runoff P loss in the summer crop season under different cropping systems and fertilizer rates. Runoff TP concentration was relatively higher at the vegetative stage but decreased at the reproductive stage for all cropping systems. Particulate P was the major form in runoff. Higher runoff sediment yields resulted in higher runoff TP and PP. Runoff TP and PP were mainly derived from soil Olsen-P, Al-P and Fe-P. Soil Olsen-P that was mainly consisted of some CaS1 FileRCL: rape/corn + lower fertilizer rate; RCH: rape/corn + higher fertilizer rate; WCL: wheat/corn + lower fertilizer rate; WCH: wheat/corn + higher fertilizer rate; WPL: wheat/sweet potato + lower fertilizer rate; WPH: wheat/ sweet potato + higher fertilizer rate.(XLS)Click here for additional data file.S2 FileRCL: rape/corn + lower fertilizer rate; RCH: rape/corn + higher fertilizer rate; WCL: wheat/corn + lower fertilizer rate; WCH: wheat/corn + higher fertilizer rate; WPL: wheat/sweet potato + lower fertilizer rate; WPH: wheat/ sweet potato + higher fertilizer rate.(XLS)Click here for additional data file.S3 FileRCL: rape/corn + lower fertilizer rate; RCH: rape/corn + higher fertilizer rate; WCL: wheat/corn + lower fertilizer rate; WCH: wheat/corn + higher fertilizer rate; WPL: wheat/sweet potato + lower fertilizer rate; WPH: wheat/ sweet potato + higher fertilizer rate.(XLS)Click here for additional data file.S4 FileRCL: rape/corn + lower fertilizer rate; RCH: rape/corn + higher fertilizer rate; WCL: wheat/corn + lower fertilizer rate; WCH: wheat/corn + higher fertilizer rate; WPL: wheat/sweet potato + lower fertilizer rate; WPH: wheat/ sweet potato + higher fertilizer rate.(XLS)Click here for additional data file.