However, DNA-Ad5 dramatically improved CD8+ T cell sensitivity by increasing T cell receptor (TCR) functional avidity for GUCY2C (~20-fold). Preventing enhanced CD8+ T cell TCR avidity, while preserving the enhanced CD8+ T cell quantity, eliminated antitumor efficacy. Thus, a GUCY2C DNA-Ad5 prime-boost strategy synergizes to produce highly potent CD8+ T cell responses and therapeutic CRC activity, which may translate to patients at risk for metastatic CRC.Heterologous prime-boost immunizations with plasmid DNA and viral vaccines synergize to produce effective immune responses against tumor and infectious disease antigens. Yet, mechanisms underlying their synergy remain incompletely defined. Here, we used the colorectal cancer (CRC) antigen guanylyl cyclase C (GUCY2C) to explore the synergy of DNA-Ad5 (adenovirus type 5) prime-boost immunizations. GUCY2C is selectively expressed by intestinal epithelial cells and its expression is maintained by metastatic CRC cells. The compartmentalized expression of GUCY2C, normally in the mucosa but extending systemically in metastases, makes GUCY2C a target for CRC immunotherapy. Adenoviral vector expressing GUCY2C (Ad5-GUCY2C) is safe and effective in mice, producing immune responses and antitumor immunity, but heterologous prime-boost strategies may be required to maximize therapeutic efficacy. DNA vaccines are advantageous in heterologous prime-boost strategies reflecting the absence of vector-specific immunity which limits repetitive viral delivery. However, low transfection rates and immunogenicity characterize DNA vaccine strategies. Here, combining CCL20 and CCL21 as chemokine adjuvants to recruit dendritic and T cells, respectively, with electroporation (EP) of GUCY2C-expressing plasmids to increase transfection rates, optimizes GUCY2C immune responses. Primary immunization with DNA plasmids encoding chemokines and GUCY2C followed by boosting with Ad5-GUCY2C (DNA-Ad5) produced curative antitumor responses, in contrast to immunization with either component alone. Interestingly, DNA-Ad5 produced only a modest increase (<2-fold) in the magnitude of GUCY2C-specific CD8


We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.HIV infection induces phenotypic and functional changes to CD8 HIV-1 infection contributes substantially to global morbidity and mortality, with no immediate promise of an effective vaccine. One major obstacle to vaccine development and therapy is to understand why HIV-1 replication persists in a person despite the presence of viral specific immune responses. The emerging consensus has been that these immune cells are functionally ‘exhausted’ or anergic, and thus, although they can recognize HIV-1 specific target cells, they are unable to effectively keep up with rapid and dynamic viral replication in an individual. We have identified a novel combination pathway that can be targeted, TIGIT and PD-L1which may be responsible, at least in part, for making these immune cells dysfunctional and exhausted and thus unable to control the virus. We show that by blocking the TIGIT and PD-L1 pathway, we can reverse the defects of these viral specific immune cells. Our findings will give new directions to vaccines and therapies that will potentially reverse these dysfunctional cells and allow them to control HIV-1 replication, but also serve in “Shock and Kill” HIV curative strategies. During chronic viral infections, high antigenic loads continually stimulate T cells leading to progressive loss of function termed “T cell exhaustion” . Through+ T cell exhaustion [in vivo demonstrated efficacy in restoring simian immunodeficiency virus (SIV)-specific T cell and humoral immunity, and led to a reduction of SIV viremia and in immune activation. However, this did not completely control virus, suggesting that additional therapies are needed. Importantly, not all features of the exhausted T-cells are restored by interfering with single pathways [Indeed, blocking the interaction of T cell negative checkpoint receptor pathways using targeted reagents against PD-1/Programmed Death-Ligand 1 (PD-L1), Tim-3, Lymphocyte-activation gene 3 (Lag-3) and CD160 has shown promise in reversing CD8haustion ,8,10–12.haustion  as well haustion –19. A sihaustion . Blockadpathways ,8,20. Sypathways ,22.+ T cells and CD4+ T cell subsets [+ T cell effector activity were generated following synergistic dual blockade of PD-L1 and TIGIT in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection [ex-vivo in patients with advanced melanoma [T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) is a recently described immune checkpoint receptor that belongs to the CD28 family and contains an extracellular IgV domain, a transmembrane domain, and a cytoplasmic tail containing two-immunoreceptor tyrosine-based inhibitory motif (ITIM) . It has  subsets  and is i subsets –27. TIGI subsets ,24,28. S subsets ,29–31. A subsets . This in subsets ,31. Furt subsets . Recentlnfection  and ex-vmelanoma .+ T cell responses by synergistic modulation of negative checkpoint receptors, we investigated the expression and function of TIGIT in HIV disease pathogenesis, and in the SIV non-human primate model of HIV/AIDS.To date, these results have not been replicated in any human viral disease, but over-expression of both TIGIT and PD-1 on virally exhausted T cells suggests that this is a promising avenue of exploration as a viable strategy to increase control or eliminate viral infections through T cell modulation. Given the potential to restore anti-HIV-specific CD8+ CD8+ T cells in HIV-infected participants , even among those with viral suppression, relative to HD  relative to HD (+ CD4+ T cells were significantly elevated among NC (24.5%) compared to HD (16.05%)  from HIV-infected individuals that were either acutely infected (AI), non-controllers (NC), cART suppressed (AS), or elite controllers (EC), and compared these results to age-matched HIV-uninfected healthy donors (HD) . We obseve to HD . TIGIT+ (16.05%) .+ CD8+ T cells inversely correlated with CD4 cell counts, but not with CD8+ T cell activation or plasma viral load , Polymerase (54.7%), Envelope (54.3%), Nef (52%), and also for CMV pp65 (57.8%) (+ T cells were observed on a per cell basis as measured by Geometric Mean Fluorescence Intensity (GMFI)  . Comparay (GMFI) .+ T cells to determine whether they retain features of immune exhaustion. We found that most of the TIGIT expressing CD8+ T cells co-expressed PD-1 with the frequency of TIGIT+ PD-1+ CD8+ T cells significantly expanded in chronic HIV infection  compared to HD (13.65%)  phenotype  . The fre4 counts  and posi4 counts  among alom PBMCs . Furtherhenotype . These r+ CD8+ T cells, we evaluated the functional status of the TIGIT expressing cells. We stained T cells with the nuclear antigen Ki-67, which is associated with cellular proliferation, and observed that TIGIT+ cells expressed significantly more Ki-67 than TIGIT- CD8+ T cells  robustly upregulated TIGIT upon stimulation (+PD-1- and TIGIT+PD-1+) cells only marginally increased TIGIT expression . These data suggest that HIV-specific CD8+ T cell proliferation can be markedly improved with simultaneous combination blockade of TIGIT and PD-L1.Single blockade of PD-L1 significantly enhanced HIV-specific CD8feration . When bode alone . Though + T cells derived from the lymph node (LN) (38.6%) and splenic (60.9%) compartments when compared to SIV-uninfected macaques (LN 10.82% and spleen 25.55%), but not in PBMCs  (GenBank: KR534505) and observed that it shares 88.11% sequence homology with human TIGIT . We reasMCs Figs  and S6B.ral load .-CD95+), and central memory  CD8+ T cells when compared to naïve  CD8+ T cells from PBMCs, LN and from the spleen   .+ and rhTIGIT- CD8+ T cells from SIV-infected rhesus macaques (+ T cells lacking rhTIGIT from PBMC produced significantly more IFN-γ compared to rhTIGIT+ CD8+ T cells when stimulated with either phorbol 12-mysistate 13-acetate (PMA) + ionomycin or SIV Gag181-189 CM9 peptide  observe that TIGIT appears to associate with the cellular viral reservoir in CD4+ T cells, (3) we found that co-blockade of TIGIT and PD-L1 lead to a greater restoration of T cell function compared with a single blockade, and (4) by successfully cloning rhTIGIT (GenBank: KR534505) we reveal the similarities in expression and function of rhTIGIT on T cells in the non-human primate model of HIV/AIDS. Our findings reveal a novel inhibitory pathway involved in the suppression of T cell responses during chronic viral infection, the blockade of which may contribute to the reversal of T cell dysfunction in the control or elimination of HIV infection.In this report we profiled TIGIT expression on T cells in HIV-infected participants with various degrees of viral control and in SIV-infected rhesus macaques. We (1) unveil a role for TIGIT+ T cells tracked disease progression (depletion of CD4 T cells and T cell activation), this was not evident across the various HIV-infected groups. There was a significant difference in TIGIT+ memory CD8+ T cells in acute infection compared to the uninfected group, but not in the global CD8+ T cell population. This suggests there may be a gradient, with an increase in global TIGIT+ CD8+ T cells in acute infection that becomes greater over time, which is distributed among the differentiated CD8+ T cell populations and may differ when compared to other negative checkpoint receptors which are found elevated during the early stages of HIV infection [While TIGIT levels on CD8nfection ,39.+ T cells did not increase TIGIT expression after HIV-1 Gag peptide stimulation as a group, however a subset of individuals with moderate levels of TIGIT increased expression after stimulation, and individuals that expressed high levels of TIGIT retained expression after stimulation. TIGIT remained elevated despite antigen in cART-suppressed individuals; previous studies have also shown that common γ-chain cytokines maintain the ability to regulate immune checkpoint receptor expression in the absence of antigenic stimulation [TIGIT induction appears to be driven by polyclonal TCR stimulation and this is a common feature among immune checkpoint receptors ,40. We omulation ,41. Our + T cell loss [TIGIT expression was found to be associated with T cell activation principally among EC who represent a small population of HIV-infected individuals able to spontaneously suppress their viral load (<50 copies/ml) in the absence of cART for prolonged periods of time . Howeverell loss ,44. In aell loss ,46. Highell loss ,48. Giveell loss ,50.+ tumor infiltrating lymphocytes in patients with advanced melanoma synergistically improves potent anti-tumor responses [+ T cells in HIV infection despite pharmacological or immunological viral suppression. Our data shows the presence of TIGIT and PD-1 dual expressing HIV and SIV-specific CD8+ T cells and co-blockade of TIGIT and PD-L1 better enhanced proliferation of HIV and SIV-specific CD8+ T cell responses compared to single blockade. Although we see a significant increase among all HIV-infected individuals, it was evident that subsets of weak-responders exist and appear heterogeneous irrespective of stage of infection, viral load levels or viral suppression. Indeed, combinational blockade of CTLA-4 and PD-1 revealed a subset of weak-responders to anti-tumor activity. Different or expanded combinations of immune checkpoint blockers with anti-TIGIT may need to be considered in the arsenal to improve anti-viral T cell immunity in all individuals.Viral clearance of the chronic strain of LCMV (clone 13) in mice by combined blockade of TIGIT and PD-L1 provided the first evidence of the advantages of targeting these two pathways . In addiesponses . Here wein vivo studies yielding activity in reactivating of latent virus. However, the ‘Kill’ component is less well developed. Shan and colleagues demonstrate that after reactivation of latent virus from CD4+ T cells, CD8+ T cells’ activity had the capacity to kill latently infected CD4+ T cells if appropriate pre-stimulation of HIV peptide and IL-2 was provided an in vitro latency assay using Bcl-2 as a survival signal to prolong the longevity of the latent CD4+ T cells [+ T cells responses given that these pathways persist in the setting of viral suppression. Furthermore, given our findings showing the relationship with TIGIT expression on CD4+ T cells and the total cell associated HIV DNA, it remains unclear what role TIGIT may play in the establishment of the reservoir in CD4+ T cells, however it is likely related to the capacity of TIGIT’s ability control T cell activation or proliferation.Persistence of the cellular latent HIV reservoir has been a major barrier to the eradication of HIV . One pro T cells . Further T cells . Blockin+ T cell functions may further aid in the ‘Shock and Kill’ approaches being considered to eliminate latent virus and improve T cell mediated vaccine responses to prevent or limit infection [Our study provides a novel role for TIGIT during HIV disease pathogenesis and our demonstration of a role of rhTIGIT in the non-human primate model of HIV/AIDS provides a platform to investigate our understanding of the complex networks of co-inhibition that can be tailored to each individual or viral infection. Improving CD8nfection .n = 80) were selected from the following groups: untreated non-controllers (n = 20) , treated virologic controllers (participants who had an undetectable plasma HIV-1 RNA level for the previous six months while on cART) (n = 20), spontaneous “elite” virologic controllers  (n = 20), Some of these participants had persistent plasma HIV-1 RNA levels <75 copies/ml) and HIV-infected “cART initiators” (n = 20) who meet strict selection criteria and well documented persistent viral suppression for over 1.5 years. Participants with acute HIV infection (n = 24) were obtained for the OPTIONS cohort of primary HIV infection [n = 20)  SCOPE and OPTIONS cohorts  the Hawa[n = 20)  and chroMacaca mulatta) housed at the Oregon National Primate Research Center (ONPRC), which were SIV infected for other ongoing, unrelated studies. Oregon Health and Science University (OHSU) Institutional Animal Care and Use Committee (IACUC) Protocol #: 0989. The OHSU Institutional Animal Care and Use Committee reviewed and approved all study protocols. All macaques in this study were managed according to the ONPRC animal husbandry program, which aims at providing consistent and excellent care to nonhuman primates. This program is based on the laws, regulations, and guidelines set forth by the United States Department of Agriculture , Institute for Laboratory Animal Research , Public Health Service, National Research Council, Centers for Disease Control, and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. The nutritional plan utilized by the ONPRC is based on National Research Council recommendations and supplemented with a variety of fruits, vegetables, and other edible objects as part of the environmental enrichment program established by the Behavioral Management Unit. Paired/grouped animals exhibiting incompatible behaviors were reported to the Behavioral Management staff and managed accordingly. All efforts were made to minimize suffering through the use of minimally invasive procedures, anesthetics, and analgesics when appropriate. Animals were painlessly euthanized with sodium pentobarbital and euthanasia was assured by exsanguination and bilateral pneumothorax, consistent with the recommendations of the American Veterinary Medical Guidelines on Euthanasia (2013)All persons gave written informed consent to participate in the study and approval for the study was obtained from the University of Hawaii Committee of Human Subjects. Samples were obtained from Indian rhesus macaques . Cellular RNA and DNA from PBMC T-cell subsets cells were purified using the AllPrep DNA/RNA kit  as specified by the manufacturer, quantified using a Nanodrop (ND-1000) spectrophotometer and normalized to cell equivalents by qPCR using human genomic TERT for DNA and GAPDH or RPLP0 expression for RNA . Total cellular HIV-1 DNA (integrated and unintegrated) and RNA (unspliced and multiply spliced) was quantified with a qPCR TaqMan assay using LTR-specific primers F522-43  and R626-43  coupled with a FAM-BQ probe (5’ CCA GAG TCA CAC AAC AGA CGG GCA CA 3) [Cryopreserved PBMCs were rapidly thawed and enriched for CD4CA CA 3)  on a SteCryopreserved PBMC were rapidly thawed in warm 10% cRPMI  supplemented with 10% fetal bovine serum (FBS) (Hyclone), 1% penicillin-streptomycin (Hyclone), 10 mM HEPES (Hyclone), 2 mM L-glutamine (Hyclone), and 10 μg/ml DNase I , washed with PBS + 2% FBS (Hyclone) (complete RPMI). Cells were stained for viability with an aqua amine reactive dye , then incubated with panels of conjugated anti-human monoclonal antibodies (mAbs) The following directly conjugated mAbs used in this study were obtained from BD biosciences : PE-Cy5-conjugated anti-CD38 (Clone: HIT2), V450-conjugated anti-CD45RA (HI100), FITC-conjugated anti-CD45RA (HI100), PerCP-Cy5.5-conjugated anti-CD27 (M-T271), Alexa Flour 700-conjugated anti-CD4 (RPA-T4), FITC-conjugated anti-HLA-DR (G46-6), APCH-7-conjugated anti-CD8 (SK1), FITC-conjugated anti-CD57 (NK-1), APC-conjugated CD107α (H4A3). mAb obtained from Beckman Coulter  ECD-conjugated anti-CD3 (UCHT1). mAbs obtained from eBioscience  PE-Cy7-conjugated anti-CD28 (CD28.2), PerCP-eFluor 710-conjugated anti-TIGIT (MBSA43), PE-conjugated anti-TIGIT (MBSA43), Mouse IgG1 Kappa isotype control PerCP-eFluor 710 (P3.6.2.8.1), mouse IgG1 K isotype control PE (P3.6.2.8.1). mAbs obtained from Biolegend  Brilliant Violet 605-conjugated anti-CCR7 (G043H7), APC-conjugated anti-PD-1 (EH12.2H7), mouse IgG1 Kappa isotype control PE (MOPC-21). Qdot 605-conjugated anti-CD8 (3B5) was obtained from Invitrogen . In some experiments cells were fixed with 1X Lyse buffer (BD Biosciences) followed by 1X BD FACS Permeabilizing solution 2 (BD Biosciences) and stained with FITC-conjugated Ki-67 (35/Ki-67), FITC-conjugated interferon gamma (IFN-γ) (25723.11), Alexa 700-conjugated Granzyme B (GB11), PE-conjugated perforin (B-D48) . All cells were washed with PBS + 2% FBS and then fixed in 1% paraformaldehyde  before acquiring (within 18 hours) on a custom four laser LSRFortessa flow cytometer (BD Biosciences). Between 100,000 to 500,000 lymphocyte events were collected for each sample. Isotype controls or fluorescence minus one (FMO) samples were prepared to facilitate gating. Anti-mouse or anti-rat IgG-coated beads (BD Biosciences) were individually stained with each fluorochrome-conjugated antibody and used for software-based compensation. Data were analyzed using Flowjo Software version 9.5 .+ T cells to high purities with an EasySep Human CD8+ T cell negative selection enrichment kit (Stemcell). Cells were surface stained with the following combination of mAbs: BV711-conjugated anti-CD3, Alexa 700-conjugated anti-CD4 (BD Biosciences), PerCP-eFluor 710-conjugated anti-TIGIT (eBioscience), Qdot605-conjugated anti-CD8, APC-conjugated anti-PD-1 (Invitrogen). Cells were sorted on a BD FACS ARIA and checked for purity. Gating was facilitated by isotype controls.Cryopreserved PBMCs were rapidly thawed and enriched for CD8+PD-1+, TIGIT+PD-1-, TIGIT-PD-1+, TIGIT-PD-1-) were seeded at 100,000 cells per well in a 96 well culture plate with 200 μl of 10% cRPMI. Sorted cells were stimulated with anti-CD3 + anti-CD28 Dynabeads (Life Technologies) for 48 hours in an incubator at 37°C with 5% CO2, supernatants were harvested from the cultures and processed according to recommended manufacture procedure with a Milliplex MAP Human High Sensitivity T cell Panel  for GM-CSF, TNF-α, IL-13, IL-12 (p70), IL-10, IL-8, IL-7, IL-6, IL-5, IL-4, IL-2, IL-1β, IFN-γ. Samples were acquired on a Luminex 200 (EMD Milipore). Samples were run in duplicate. The intra-assay CV% for the conditions of each cytokines were <10%.The four-sorted populations  (Life Technologies) followed recommended manufacture procedure.2 with 5 μg/ml brefeldin A and 5 μg/ml monensin (Sigma-Aldrich) culture media, DMSO alone, pooled HIV-1 Gag peptides, or anti-CD3/CD28 dynabeads (Life Technologies) in the presence or absence of purified isotype IgG control, anti-TIGIT and/or anti-PD-L1 mAbs. After stimulation, the cells were washed and stained for viability with AARD and cultured with surface phenotype panel against CD8, TIGIT or an isotype control antibody, followed by intracellular staining of CD3 and IFN-γ and acquisition on the flow cytometer as above. For the proliferation assay, thawed PMBCs were washed two times and resuspended in PBS supplemented with 0.01% BSA at a concentration of one million cells per milliliter. Cells were labeled with 1mM Carboxyfluorescein succinimidyl ester (CFSE) violet trace (Invitrogen) using protocols previously described [2. At the end of the stimulation, cells were washed and stained for viability with AARD and cultured with surface phenotype panel against CD3, CD8, TIGIT or an isotype control antibody and acquired on the flow cytometer as above.In the intracellular cytokine stimulation assay studies, thawed cryopreserved PBMCs were stimulated for 12 hours in an incubator at 37°C with 5% CO2. After stimulation, the cells were washed and stained for viability with AARD cultured with surface phenotype panel against CD3, CD4, CD8, TIGIT or isotype control antibody and acquired on the flow cytometer as above.PBMCs were thawed and one million cells were plated per stimulation condition. Stimulation conditions included media alone, 25 ng/ml IL-2 (Roche), 50 IU/ml of IL-12 , 50 IU/ml of IL-18  and 25 ng/ml IL-15 . Cells were stimulated for six days in an incubator at 37°C with 5% CO2. Stimulation conditions contained culture media, DMSO alone or pooled HIV-1 gag peptides, in the presence of brefeldin A (Sigma-Aldrich), monensin (Sigma-Aldrich), anti-TIGIT mAb anti-PD-L1 mAb or mouse IgG1 isotype control. After stimulation, cells were washed and stained for cellular viability with AARD and conjugated antibodies against CD8 and CD4, followed by intracellular staining of CD3 and IFN-γ and acquired on a flow cytometer as described above.HIV-infected cryopreserved PBMC from individuals identified in p-value of less than 0.05.The repeated-measures, one-way ANOVA followed by Tukey’s multiple comparison, Wilcoxon matched-pairs signed ranked test was used for paired tests and the Spearman’s rho test was used for correlation analyses. Measures of central tendency are expressed as medians and interquartile ranges . Statistical analyses were conducted using Prism Graphpad release 5.0d  or SPSS 22.0  and the statistical significance of the findings was set at a Indian rhesus macaque (Macaca mulatta) TIGIT (rhTIGIT): GenBank KR534505.S1 Text(DOCX)Click here for additional data file.S1 Fig+ and CD4+ T cells. Gated on singlets, excluded dead cells, gated on lymphocytes, gated on CD3+ T cells, and gated on expression of CD8 or CD4. (B) Representative histograms of TIGIT Isotype, TIGIT FMO and TIGIT expression on CD8+ or CD4+ T cells . Graphs show the association of the frequency (%) of (C) TIGIT+ CD8+ or (D) TIGIT+ CD4+ T cells against clinical CD4 count for cART suppressed  and elite controllers . Graphs show the association of the frequency (%) of (E) TIGIT+ CD8+ or (F) TIGIT+ CD4+ T cells against T cell activation (% CD38+HLA-DR+) for cART suppressed  and non-controllers . Graphs show the association of the frequency (%) of (G) TIGIT+ CD8+ or (I) TIGIT+ CD4+ T cells against viral load log10 (copies/ml) for non-controllers . Graphs show the association of the frequency (%) of (H) TIGIT+ CD8+ or (J) TIGIT+ CD4+ T cells against copies of cell associated HIV RNA per million CD4+ T cells for L-AS . Spearman’s rho tests were performed for correlations.(A) Representative flow cytometry plots showing gating scheme to isolate CD8(TIF)Click here for additional data file.S2 Fig+ T cells subsets grouped by disease category. HIV-Uninfected , acute infected , cART suppressed , elite controller , and non-controllers . Repeated-measures one-way ANOVA, followed by Tukey’s multiple comparisons test were used for comparison . Cryopreserved PBMCs from chronically HIV-infected individuals were phenotyped for TIGIT expression on CD8+ T cell subsets. (B) Representative flow cytometry plots showing gating scheme to isolate CD8+ T cell subsets. Live lymphocytes gated for CD8+ T cells, subset into CD45RA+ and CD45RA-, further stratified by expression of CCR7 and CD27. (C) Representative flow cytometry plots showing CD28 expression on CD8+ T cell subsets. (D) Representative flow cytometry plots showing TIGIT expression on CD8+ T cell subsets. (E) Graph shows compiled frequency (%) of TIGIT expression on CD8+ T cell subsets (n = 20).(A) Graph shows compiled frequency (%) of TIGIT expression on CD8(TIF)Click here for additional data file.S3 FigEx vivo CD8+ T cells from chronically HIV-infected individuals were FACS sorted into populations according to their expression of TIGIT and PD-1. (A) Representative flow cytometry plot of TIGIT and PD-1 expression PRE-SORT. Gating was facilitated by isotype controls for TIGIT and PD-1. (B) Representative flow cytometry plots of CD8+ T cells sorted into TIGIT+PD-1+, TIGIT+PD-1-, TIGIT-PD-1+, and TIGIT-PD-1-. No stimulation (left panel) and stimulated with anti-CD3 + anti-CD28 Dyanbeads for 48 hours (right panel). (C) Graphs show compiled data of phenotypes of sorted populations with no stimulation (open box) and anti-CD3 + anti-CD28 Dyanbeads (filled box) (n = 2). Supernatants were harvested and cytokine production was assessed 48 hours post anti-CD3 + anti-CD28 stimulation by high sensitivity multiplex bead array. (D) Graphs show concentrations of cytokines produced from sorted populations.(TIF)Click here for additional data file.S4 Fign = 8) TIGIT expression frequency (%) on CD4+ T cells with or without cytokine stimulation for six days. P values were calculated with repeated-measures one-way ANOVA, followed by Tukey’s multiple comparisons test (*p < 0.05). (B) Compiled data of HIV-Infected individuals  TIGIT expression frequency (%) on CD8+ T cells (right panel) and CD4+ T cells (left panel) after six days of IL-21 stimulation (n = 6). P values were calculated by Wilcoxon matched-pairs signed ranked test.(A) Compiled data of HIV-Infected individuals Click here for additional data file.S5 FigEx vivo PBMCs from chronically HIV-infected individuals were stimulated with HIV Gag peptide pool in the presence of mAb blocking antibodies. Representative flow cytometry plots gated on (A) CD8+ or (C) CD4+ T cells, showing IL-2 responses from an HIV-infected individual. No HIV-1 Gag stimulation with an isotype control, HIV-1 Gag stimulation with an isotype control, HIV-1 Gag stimulation with anti-TIGIT, HIV-1 Gag stimulation with anti-PD-L1, HIV-1 Gag stimulation with dual blockade (anti-TIGIT + anti-PD-L1) and a positive control (anti-CD3 + anti-CD28 Dynabeads). Graphs show compiled data showing variation in the frequency (%) of (B) CD8+ or (D) CD4+ T cell IL-2 in responses to HIV-1 Gag peptide pool with isotype control or mAb blockade; TIGIT blockade (left panel), PD-L1 blockade (middle panel), and dual blockade (right panel) (n = 16).(TIF)Click here for additional data file.S6 Fig+ T cells from PBMCs, LNs and spleen in representative non-infected and SIV-infected animals. (C) Representative flow cytometry plots depict rhTIGIT expression frequency (%) on naïve (N) (CD28+CD95-), effector memory (EM) (CD28-CD95+), and central memory (CM) (CD28+CD95+) cells from PBMCs, LNs and spleens in representative SIV-infected animals. Graphs show frequency (%) of rhTIGIT+ N, EM and CM CD8+ cells from SIV-infected PBMCs , LNs , and spleens . P values were calculated with repeated-measures one-way ANOVA, followed by Tukey’s multiple comparisons test. (D) Graph shows compiled frequency (%) of rhTIGIT+ CD8+ T cells after stimulation with IL-2, IL-12 or IL-15 for six days. NS, no stimulation. P values were calculated with repeated-measures one-way ANOVA, followed by Tukey’s multiple comparisons test. (E) Representative flow cytometry plot showing secondary antibody only against CM9 tetramer staining to facilitate rhTIGIT gating. (F) Representative flow cytometry plot showing PD-1 FMO and secondary antibody only to facilitate rhTIGIT and PD-1 gating. (G) Representative flow cytometry plots of rhTIGIT and PD-1 expression on Mamu-A*01 SIV-Gag CM9 tetramer specific CD8+ T cells. Graphs show compiled data of rhTIGIT and PD-1 expression frequency (%) on Mamu-A*01 SIV-Gag CM9 tetramer specific CD8+ T cells (n = 4) from PBMC (open circle), LNs (open square), and spleen (open triangle). (H) Representative flow cytometry plots of rhTIGIT and PD-1 expression on Mamu-A*01 SIV-Tat SL8 tetramer specific CD8+ T cells. Graphs show rhTIGIT and PD-1 expression frequency (%) on Mamu-A*01 SIV-Tat SL8 tetramer specific CD8+ T cells (n = 4) from PBMC (open circle), LNs (open square), and spleen (open triangle).(A) Alignment shows amino acid sequences of human TIGIT (Hu TIGIT) and Rhesus TIGIT (Rh TIGIT). Highlighted sequences indicate homology between human and rhesus TIGIT. Dashes indicate gaps in alignment. (B) Representative flow cytometry plots depict rhTIGIT expression frequency (%) on CD8(TIF)Click here for additional data file.S7 Fig+ Ki-67+ and rhTIGIT- Ki-67+ CD8+ T cells in PBMCs  and LNs  from SIV-infected animals. Wilcoxon matched-pairs signed- rank test was performed for statistical analysis (C) Representative flow cytometry plots depict Ki-67 and PD-1 expression in PBMCs and LNs from SIV-infected animals. (D) Graphs show frequency (%) of PD-1+ Ki-67+ and PD-1- Ki-67+ CD8+ T cells in PBMCs  and LNs  from SIV-infected animals. Wilcoxon matched-pairs signed- rank test was performed for statistical analysis.(A) Representative flow cytometry plots depict Ki-67 and rhTIGIT expression in PBMCs and LNs from SIV-infected animals. (B) Graphs show frequency (%) of rhTIGIT(TIF)Click here for additional data file.


Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 (Th17) and interleukin (IL)-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmunediseases by Th17 and IL-17 suppression. In the present study we have created geneticengineered mesenchymal stem cells (MSCs) that mediate with lentiviral vectors to releaseIL-27 as an adequate vehicle for ex vivo gene therapy in the reduction of inflammation andautoimmune diseases.In this experimental study, we isolated adipose-derived MSCs(AD-MSCs) from lipoaspirate and subsequently characterized them by differentiation. Twosubunits of IL-27 (p28 and EBI3) were cloned in a pCDH-513B-1 lentiviral vector. Expressions of p28 and EBI3 (Epstein-Barr virus induced gene 3) were determined by real timepolymerase chain reaction (PCR). MSCs were transduced by a pCDH-CMV-p28-IRES-EBI3-EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10expression.Cell differentiation confirmed true isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified successful cloning of both p28and EBI3 in the pCDH-513B-1 lentiviral vector. Real time PCR showed high expressions level of IL-27 and IL-10 as well as accurate activity of IL-27.The results showed transduction of functional IL-27 to AD-MSCs by means of alentiviral vector. The lentiviral vector did not impact MSC characteristics. In recent years stem cell therapy has become a primaryaspect of numerous research and clinical projects. Stem ceAutoimmune diseases are multi-factorial disorderswith complicated immune system dysregulationmediated by immune cytokines andimmune cells . In manyThere are many autoimmune diseases which allhave the same mechanism of pathogenicity, thusone approach can be used as a general treatmentfor these diseases . In the In this experimental study, we purchased twosubunits of mouse IL-27 (p28 and EBI3) cDNAfrom Open Biosystems . Both genes were cloned in pCDH-513B-1  combined with an internalribosome entry site (IRES) sequence arrangedas p28-IRES-EBI3 under a cytomegalovirus(CMV) promoter. pCDH-513B-1 have copGFP(copepod green fluorescent protein) for fluorescenttagging and puromycin for selectingstably transduced cells. All cloning procedureswere performed according to the commondigestion-ligation protocol. Polymerasechain reaction (PCR) was carried out for threefragments with Xba1-p28-Nhe1, Nhe1-IRESBamH1and BamH1-EBI3-Not1 overhanging.Then, all fragments were separately cloned inpCDH-513B-1. We verified the pCDH-CMVp28-IRES-EBI3-EF1-copGFP-Pur construct bydigestion and subsequent sequencing.6 HEK-293Tcells were cultured in a 10 cm plate in Dulbecco’sModified Eagle’s Medium (DMEM)  with 10% Fetal bovineserum (FBS) one day prior to transfection. We replaced themedium 2 hours before transfection with freshmedium. Ca3(PO4)2 buffer that contained 21 μgof pCDH-CMV-p28-IRES-EBI3-EF1-copGFPPur,21 μg of pCMV-dR8.2, 10.5 μg of pMD2,33 μl of TE 1X, 105 μl of 2.5 M CaCl2, and 1050μl of 2x Hank’s buffered salt solution (HBSS)was used for one 10 cm plate. We added the 2XHBSS during the time the solution was vortexed.Transfection medium was replaced withfresh medium within 14 hours after transfection.Medium with viruses was collected after24, 48 and 72 hours and centrifuged at 15000rpm, then filtered through a 0.45 μm filter beforetransduction.Recombinant lentivirus was produced accordingto the TRONOLAB protocol with somemodifications . BrieflyAdipose tissue was obtained from lipoaspirate plastic surgery performed at clinics accordingto a Bioethics Agreement of the ShahidBeheshti University of Medical Science andStem Cell Research Center Committee. Adiposetissue was washed three times with phosphatebuffered saline (PBS) that contained 3Xpenicillin/streptomycin and amphotericin untila clear tissue was attained. DMEM mediumthat contained dispase (50 U/ml)-CollagenaseI were added to the adipose tissue, after whichthe solution was shaken for 30 minutes at 37˚C.The solution was centrifuged at 1500 rpm andthe supernatant was discarded. The plated cellswere kept. RBC was lysed by erythrocyte lysisbuffer for 5 minutes at 37˚C, and then centrifugedat 1200 rpm for 5 minutes. The platedcells were suspended in DMEM and distributedin flasks with DMEM that contained 10% FBSfor 3 days. For adipogenic differentiation, cellswere cultured in DMEM that contained 10%FBS, 0.5 mM isobutylmethylxanthine (IBMX),dexamethasone (10-7 M), insulin (66 nM), andindomethacin (0.2 mM). For osteocyte differentiationthe cells were cultured in DMEM thatcontained 10% FBS, dexamethasone (10-7M),β-glycerol-phosphate (10 mM), and ascorbicacid 2-phosphate (50 μg/ml).Second passage AD-MSCs were cultured in sixwellcell culture plates, and then washed with PBSbefore adding fresh recombinant virus. In orderto remove all FBS proteins to enable better transductionwe used the spinfection method at 2000rpm for 60 minutes at a temperature of 25˚C. Aftercentrifuging, the plate was placed in a 37˚C incubator;the medium was changed 14-20 hours afterspinfection.6 AD-MSCs and lentiviral engineered ADMSCswere carried out by Qiagen , RNA extraction and cDNA kits, respectively, accordingto the manufacturers’ protocols. cDNAwas used for quantitative real time PCR. Expressionsof octamer-binding transcription factor 4(Oct-4), IL-27 and EBI3 were evaluated in ADMSCsand lentiviral engineered AD-MSCs. TATA-binding protein (TBP) expression was consideredto be the endogenous reference gene. Primersequences used this studied are provided in table 1.Total RNA extraction and cDNA synthesis from2×10Secretion and function of IL-27 were examined bybioassay using naive T cells from a C57BL/6 mousethat was co-cultured with COS-7 cells transducedwith recombinant virus derived from pCDHCMV-p28-IRES-EBI3-EF1-copGFP-Pur and selectedfor puromycin at a concentration of 2 μg/cc.The C57BL/6 mouse was killed according to thelaboratory animal protocol. The spleen was removedand cut in small pieces then digested with dispasecollagenase(100U/ml) for 10 minutes and passedthrough 0.45 μM filter. Cells were collected by centrifugationat 1200 rpm and RBCs were lysed by aRBC lysis buffer. After three days, naive T cells werecultured in fresh RPMI 1640 medium and co-culturedwith COS-7 that was engineered by a recombinantvirus (COS7/IL-27). Total RNA was extractedfrom T cells co-cultured with COS-7 and COS-7/IL-27. Expression of IL-10 was appraised as a downstreamgene in the IL-27 signal transduction pathwayby real time PCR. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) expression was used as anendogenous reference gene. The following primerswere used for IL-10 and GAPDH: IL-10 forward:5´AATAAGAGCAAGCCAGTG3´ and reverse:5´CCAGCAGACTCAATACAC3'; and GAPDHforward: 5'CCACAACTCTTCCATTCTC3' and reverse:5'CCAAGATTCACGGTAGATAC 3'.Human adipose tissue was obtained following informedconsent in accordance with the Declarationsof the Shahid Beheshti University of Medical Scienceand Stem Cell Research Center Committee.3(PO4)2protocol. The transfect efficiency was morethan 90% (6.We cloned mouse p28 and EBI3 cDNA in apCDH-513B-1 lentiviral vector. Digestion withXbaI-NotI showed that cloning was successful. The conthan 90% . The virAdipocyte cells were isolated from liposuctiontissue with mechanical and enzyme digestion.Multipotency of the cells was confirmed by theirdifferentiation into adipocyte and osteocyte cells.Alizarin Red staining confirmed the presence ofosteocytes  and oil Real time PCR showed expression of p28 increased2000-fold and EBI3 increased 650-fold intransduced AD-MSCs compared with the controlAD-MSCs . IRES seWe examined the biological activity of IL-27that was secreted from COS-7 cells. Naive T cellsproduced larger amounts of IL-10 when co-culturedwith COS-7/IL-27 cells compared to naiveT cells co-cultured with COS-7 . Real tiin vitro visualization (Autoimmune diseases are complex disorderswith an immunological basis that are dependenton cytokines that are good targets for gene therapy. A greatlization . The finReal time PCR verified overexpressed IL-27because of the leniviral CMV promoter. P28 andEBI3 combined with IRES, thus the expression ofthe EBI3 subunit decreased by one third comparedto p28. This result was similar to other publishedstudies related to IRES effectiveness on gene expression. p28 is Our results and those of other similar studies, 20 did Murugaiyan et al. have shown that human IL-27induces generation of T cells which secrete largeamounts of IL-10 . Real tiIL-27 can be successfully transduced to ADMSCsby means of a lentiviral vector. IL-27 wasfunctional. The lentiviral vector did not affectMSCs characteristics.


In intubated acute respiratory failure patients, inhomogeneity of ventilation-perfusion matching  might determine extreme pH shifts and increase the risk of developing ventilator-induced injury. Positive end-expiratory pressure (PEEP) redistributes tidal ventilation towards more perfused dependent lung regions, potentially improving homogeneity of ventilation-perfusion matching.In this study, we used Electrical Impedance Tomography (EIT) to assess global and regional lung ventilation and perfusion at different PEEP levels and to verify the effects of PEEP on homogeneity of ventilation-perfusion matching.2/FiO2≤300 mmHg and PEEP≥5 cmH2O. We randomly applied two PEEP levels  for 20 minutes each and collected ventilation and EIT data at the end of each step. From EIT, we measured: 1. regional ventilation heterogeneity ; 2. regional homogeneity of ventilation-perfusion matching (HV/P); 3. regional compliance; 4. cumulated regional lung hyperdistension.We enrolled 20 intubated critically ill patients undergoing controlled mechanical ventilation, sedated, paralyzed, with PaO2/FiO2 was 197 ± 52 mmHg, lower PEEP was 7 (7-9) cmH2O while higher PEEP was 12 (12-14) cmH2O (p < 0.001). At higher PEEP, VtHet was reduced (1.8 (1.5-2.4) vs. 2.2 (1.8-2.6), p < 0.001). Regional HV/P improved at higher PEEP in non-dependent areas  as well as in the dependent ones, albeit non-significantly   and cumulative hyperdistension of the same areas significantly increased .Finally, by applying higher PEEP, regional compliance of non-dependent lung decreased (31 ± 12 vs. 37 ± 13 mL/cmHImproved homogeneity of ventilation-perfusion matching might represent one of the protective mechanisms associated with the use of higher PEEP. On the other hand, such benefits must be balanced with increased risk of hyperdistension of non-dependent lung.Institutional.


Mps1 is a dual specificity protein kinase that regulates the spindle assembly checkpoint and mediates proper microtubule attachment to chromosomes during mitosis. However, the molecular mechanism that controls Mps1 protein level and its activity during the cell cycle remains unclear. Given that sumoylation plays an important role in mitotic progression, we investigated whether Mps1 was SUMO-modified and whether sumoylation affects its activity in mitosis. Our results showed that Mps1 was sumoylated in both asynchronized and mitotic cell populations. Mps1 was modified by both SUMO-1 and SUMO-2. Our further studies revealed that lysine residues including K71, K287, K367 and K471 were essential for Mps1 sumoylation. Sumoylation appeared to play a role in mediating kinetochore localization of Mps1, thus affecting normal mitotic progression. Furthermore, SUMO-resistant mutants of Mps1 interacted with BubR1 more efficiently than it did with the wild-type control. Combined, our results indicate that Mps1 is SUMO-modified that plays an essential role in regulating Mps1 functions during mitosis. The spindle assembly checkpoint (SAC) is a conserved surveillance mechanism that regulates partitioning of duplicated genome into two daughter cells during mitosis . Extensimono-polar spindle 1) is an evolutionarily conserved protein that functions as a key component of SAC [Cdc20 [Mps1 . To further confirm the slow mobility shifts were sumoylated Mps1, HEK293T cells were transfected with plasmids expressing Myc-tagged Mps1 alone or along with constructs either expressing His6-SUMO-1 and FLAG-tagged wild-type SENP1 or His6-SUMO-1 and FLAG-tagged enzymatically defective SENP1 (SENP1-mut). Forty-eight hours post-transfection, cells were treated with nocodazole for 16 h. Equal amounts of cell lysates prepared under the denature condition were subjected to Ni-IDA affinity pull-down analysis. Precipitated proteins were then blotted with antibodies against the Myc-tag. The slower mobility shifts of Mps1 were completely abolished in cells expressing wild-type SENP1, but not SENP1-mut  with the highest scores were subjected to mutagenic analysis. Mutant constructs were made that expressed Myc-tagged Mps1 with lysine residues replaced with arginines Figure . HEK293TSumoylation plays a role in regulating the subcellular localization of target proteins –27. To tKinetochore localization is required for the mitotic function of Mps1 , 29. To S10 accelerated in cells expressing Myc-Mps1-mut .HeLa and HEK293T cell lines were obtained from the American Type Culture Collection. HeLa cells stably expressing His6-SUMO-1 or His6-SUMO-2 were kindly provided by R.T. Hay . Cells wTransfection of plasmids or siRNAs was achieved with either Fugene HD (Roche Diagnostic) or Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Mitotic shake-off cells were obtained from gentle tapping of cells treated with nocodazole or taxol (50 ng/ml) (Sigma-Aldrich) for 14 h. Both types of shake-off cells were used for mitotic release in the presence or absence of nocodazole as specified.Rabbit polyclonal antibodies for BubR1 were developed in the laboratory. Antibodies for Myc-Tag, p-H3S10, Cyclin B1, and β-actin were purchased from Cell Signaling Technology Inc. Mouse monoclonal antibody for Mps1 was purchased from Invitrogen. GFP antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-FLAG and anti-α-Tubulin were purchased from Sigma Aldrich. Mouse anti-SUMO-1 and SUMO-2/3 antibodies were kindly provided by Dr. Michael J. Matunis (John Hopkins University). Human IgGs (CREST) against centromere proteins were purchased from Antibodies Incorporated .Various Mps1 mutants with lysine residues  replaced with arginines (R) were generated using the QuickChange Lightning Multi Site-directed Mutagenesis kit (Stratagene). Individual mutations were confirmed by DNA sequencing (Seqwright). His6-SUMO-1 plasmid was purchased from Addgene. FLAG-SENP-1 and its mutant expression plasmids were kindly provided by J. Cheng . UBC9 cDSDS-PAGE was carried out using the mini-gel system from Bio-Rad. Proteins were transferred to PVDF (Polyvinylidene fluoride) membranes. After blocking with TBST buffer  containing 5% nonfat dry milk for 1 h, the membranes were incubated overnight with primary antibodies, followed by incubation with horse-radish peroxidase-conjugated secondary antibodies  for 1 h at room temperature. After thorough washing with TBST buffer, signals were developed with an enhanced chemiluminescent system (Pierce).For immunoprecipitation, cells were lysed in a lysis buffer  and cleared by centrifugation. 1 μg of antibody and 25 μl of protein G-agarose resin  were then added to 2 mg cell lysates and incubated at 4°C overnight followed by five washing with the lysis buffer. Proteins bound to resin were eluted with SDS sample buffer and then subject to analysis by SDS-PAGE followed by Western blot with appropriate antibodies.2HPO4/NaH2PO4 (pH 7.4), 300 mM NaCl, 0.1% Triton X-100) containing 20 mM imidazole. Ni-IDA Superflow Resin (Clontech) was added to the cell lysates and incubated with gentle agitation at room temperature for 2 h. The resin was then washed 3 times at room temperature with the lysis buffer containing 40 mM imidazole. After the last wash, His6-tagged proteins were eluted in the lysis buffer containing 300 mM imidazole and blotted for Mps1, the MYC-tag, SUMO-1, or SUMO-2.For pull-down assays under denaturing conditions, cells were lysed in a lysis buffer  and left on ice for 10 min. After centrifugation (2000 × g × 10 min) the supernatant (the cytoplasmic extract) were collected and the pellet fraction was re-suspendend in buffer 2  glycerol, 0.2 mM EDTA) followed by gentle agitation for 1 h at 4°C. The supernatants (the nucleoplasmic/loose chromatin binding fraction) were harvested after centrifugation . The pellets (chromatin binding) were re-suspended in a lysis buffer , 300 mM NaCl, 0.1% Triton X-100).Cells washed twice with cold PBS were lysed in buffer 1  prior to spreading onto microscope slides (Fisher Scientific). Chromosome spreads were incubated overnight with antibodies to Myc-tag and CREST. After incubation with Alex Fluor 488-conjugated goat anti-mouse IgG and Alex Fluor 555-conjugated goat anti-human IgG, cells were counterstained with 4, 6-diamidino-2-phenylindole . Chromosomal images were captured with a Leica TCS SP5 confocal microscope or a Leica AF6000 fluorescence microscope.2-independent medium (GIBCO-BRL). GFP-positive cells were subject to time-lapse video imaging on a Leica TCS SP5 confocal microscope.For time-lapse video imaging, HeLa cells seeded onto chamber slides (Lab-TeK) were transfected with various expression constructs for 24 h. The transfected cells were then cultured in a humidified chamber at 37°C in CO


Hbs) of seed coats is caused by high temperatures which discolors the seed coats of many legumes, affecting the visual appearance and quality of seeds. The genetic determinants underlying Hbs in cowpea are unknown.Heat-induced browning  x CB46 (hbs negative) and IT84S-2246 (Hbs positive) x TVu14676 (hbs negative). Hbs-1 was identified in both populations, accounting for 28.3% -77.3% of the phenotypic variation. SNP markers 1_0032 and 1_1128 co-segregated with the trait. Within the syntenic regions of Hbs-1 in soybean, Medicago and common bean, several ethylene forming enzymes, ethylene responsive element binding factors and an ACC oxidase 2 were observed. Hbs-1 was identified in a BAC clone in contig 217 of the cowpea physical map, where ethylene forming enzymes were present. Hbs-2 was identified in the IT93K-503-1 x CB46 population and accounted for of 9.5 to 12.3% of the phenotypic variance. Hbs-3 was identified in the IT84S-2246 x TVu14676 population and accounted for 6.2 to 6.8% of the phenotypic variance. SNP marker 1_0640 co-segregated with the heat-induced browning phenotype. Hbs-3 was positioned on BAC clones in contig512 of the cowpea physical map, where several ACC synthase 1 genes were present.We identified three QTL associated with the heat-induced browning of seed coats trait, hbs alleles into cowpea varieties, contributing to higher quality seeds.The identification of loci determining heat-induced browning of seed coats and co-segregating molecular markers will enable transfer of The online version of this article (doi:10.1186/1471-2164-15-328) contains supplementary material, which is available to authorized users. Heat-induced browning is a phenomenon caused by high temperatures which discolor the seed coats of many legumes. The brown discoloration affects the visual appearance and quality of seeds which reduces its commercial value. The heat-induced brown discoloration affects the seed coats of soybean , common In cowpea, the brown discoloration only appears on the seed coat and does not affect the underlying cotyledons . In geneHbs trait is controlled by a single nuclear gene dominant to normal non-browning seed coat phenotype (hbs) [Hall and Patel 1985) studied the genetic inheritance of heat-induced browning in three cowpea populations derived from crosses with the heat-induced browning genotype, Tvu 4552; Tvu 4552 x CB5, Tvu 4552 x Bambey 21 and Tvu 4552 x PI 204647. It was confirmed with backcross populations that the  studied Medicago truncatula, Glycine max, Phaseolus vulgaris and Arabidopsis thaliana, and a cowpea EST sequence collection housed in HarvEST:Cowpea database [Molecular genetic tools and genomic resources have been developed for cowpea with an objective of enhancing breeding programs for the improvement of cowpea varieties for the United States, India, Brazil and numerous countries in Africa and Asia. These genomic resources have been integrated by using a 1536-SNP genotyping platform and include an EST-derived SNP consensus genetic map –11, knowdatabase . The cowdatabase . In addidatabase . These rHbs-1, Hbs-2 and Hbs-3, associated with heat-induced browning of seed coats using the cowpea RIL populations IT93K-503-1 x CB46 and IT84S-2246 x TVu14676. SNP markers were identified which co-segregated with the heat-induced browning of seed coats phenotype in the Hbs-1 and Hbs-3 loci, and could be used for indirect selection in breeding a higher quality cowpea grain. Additionally, ethylene forming enzymes were identified as a cowpea candidate gene for the Hbs-1 locus and an ACC synthase 1 gene was identified as a cowpea candidate gene for the Hbs-3 locus.In this study, we identified three QTL, Hbs) x CB46 (hbs) population identified two loci for the heat-induced browning phenotype. We designated the major locus as Hbs-1 and the minor locus as Hbs-2. Hbs-1 spanned 35.21 cM to 76.13 cM on linkage group 8 of the IT93K-503-1 x CB46 individual genetic map  spanned from 45.27 cM to 45.76 cM  and 1_0437 (57.41 cM) which were previously identified within the stage II heat-tolerance QTL Cht-3[Cht-3 in soybean [Hbs-2 locus with the Cht-3 locus does not concur with the findings of Hall and Patel (1988) in which the heat-induced browning of seed coats trait was not linked with heat tolerance in early floral bud development. However, this QTL mapping study and Lucas et al. (2013) used different cowpea populations than the study by Hall and Patel (1988).QTL analysis of two phenotypic datasets for the IT93K-503-1 (QTL Cht-3. Heat sh soybean . The oveHbs) x TVu14676 (hbs) population using two phenotypic datasets, wherein one major and one minor QTL were identified. The major locus was found to overlap directly with the IT93K-503-1 x CB46 Hbs-1 locus on the cowpea consensus genetic map, spanning from 34.66 cM to 56.58 cM on linkage group 5  and hbs (negative) genotypes , Medtr5g092480 was annotated as ERF11 and Medtr5g092760 was annotated as an EFE  shared the same marker bin as SNP 1_0396 (37.96 cM) on the cowpea consensus genetic map, so it was considered the closest marker to the Hbs-3 locus (SAM) to ACC by the enzyme (ACC) synthase (ACS) [Synteny between cowpea, soybean, Medicago and common bean as well as the integrated genomic resources for cowpea were used to identify ethylene forming enzymes (EFE) as the cowpea candidate gene for the se (ACS) . Thus, Ase (ACS) , 19.Arabidopsis showed that ethylene, abscisic acid (ABA) and salicylic acid (SA) were key to protecting against heat-induced stress; an ethylene-insensitive mutant ethylene resistant 1 (etr-1) showed an increase of susceptibility to heat [Arabidopsis insensitive mutants to ethylene signaling, etr-1 and ethylene insensitive 2 (ein2), showed a significant reduction in tolerance to basal-thermotolerance compared to wild-type [The importance of ethylene closely associated with heat stress has been indicated by several studies. Researchers examining heat-induced oxidative damage in  to heat . In anotild-type . Munne-Bild-type . Ethylenild-type . Additioild-type . Investiild-type . Similarild-type . The facHbs-1, Hbs-2 and Hbs-3, associated with heat-induced browning of seed coats in cowpea. The major heat-induced browning locus, Hbs-1, was observed in both RIL populations, IT93K-503-1 x CB46 and IT84S-2246 x TVu14676. Hbs-2 was a minor locus identified in the IT93K-503-1 x CB46 population, while Hbs-3 was a minor locus observed in the IT84S-2246 x TVu14676 population. Parental lines IT93K-503-1 and IT84S-2246 both exhibited the heat-induced browning of seed coats trait. The genetic and physical mapping and identity of candidate genes for the Hbs-1 and Hbs-3 loci were conducted utilizing integrated cowpea consensus genetic and physical maps as well as syntenic relationships with soybean, Medicago and common bean. The major locus, Hbs-1, was narrowed to cowpea BAC clone CM018C23 of BAC contig 217 on the cowpea physical map, where ethylene-forming enzymes (EFE) were present and considered as putative cowpea candidate genes. The minor locus, Hbs-3 was positioned on BAC clones CM042F21, CH047M01 and CM014K16 of contig512 of the cowpea physical map where ACC synthase 1 genes were present and considered as potential candidate genes.In this study, we report the identity of three loci, Hbs-1 phenotype and SNP marker 1_0640 which co-segregated with the Hbs-3 phenotype. The heat-induced browning phenotype is present in about 20% of the elite IITA breeding lines. Since the Hbs phenotype is not manifested unless the breeding material is exposed to the appropriate heat conditions, having markers to screen against the trait at the seedling stage would limit the number of plants needed to be grown to maturity. This would enable an efficient selection process to ensure that cowpea cultivars being bred do not carry the heat-induced browning trait. These approaches should expedite variety development by at least halving the current traditional breeding selection process which relies on time-consuming and costly phenotyping under heat stress conditions. Future goals include functional analysis of cowpea Hbs-1 and Hbs-3 candidate genes, which enhance our understanding of the heat-induced browning phenomenon as well as provide a ‘perfect marker’ which would further improve marker-assisted breeding efficiency.The practical outcome of this study was the identification of molecular markers 1_0032 and 1_1128 co-segregating with the Hbs) x California Blackeye ‘46’ (hbs) population consisted of 113 lines which were advanced to the F10 generation using single seed decent. IT93K-503-1 is an advanced breeding line developed by International Institute for Tropical Agriculture (IITA) which features several important traits, including drought tolerance [Fusarium oxysporum f.sp. tracheiphilum (Fot) race 3 [Macrophomina phaseolina[9 and F10 generation were phenotyped for the heat-induced browning trait using a set of 99 RILs.The IT93K-503-1  race 3  and 4 [2) race 3 , and reshaseolina. Califorhaseolina. The F9 Hbs) x TVu14676 (hbs) consisted of 136 RILs which were advanced to the F8 generation using single seed descent. IT84S-2246 is an IITA breeding line which has strong resistance to several root-knot nematodes including Rk-virulent M.incognita and Rk-aggressive M. javanica[Striga gesnerioides races SGl, SG2, SG3 and SG5 [9 and F10 generation were phenotyped for the heat-induced browning of seed coats trait using 131 and 134 out of 146 RILs. All cowpea materials were available from the University of California Riverside cowpea germplasm collection.The IT84S-2246 . The cowpea cultivars used for the marker-trait association study were SNP genotyped at the F8 or higher generation.The IT93K 503–1 x CB46 and IT84S-2246 x TVu14676 populations were genotyped at the FThe genetic map for the IT93K 503–1 x CB46 RIL population was previously created and is included in the cowpea consensus genetic map vs.2 , and vs.Kruskal-Wallis and Interval Mapping analysis packages of MapQTL 5.0 software were used to conduct the QTL analysis . A QTL wG. max, cowpea and M. truncatula and cowpea and P. vulgaris using EST-derived SNP markers previously BLASTed and aligned to the sequenced genomes. Syntenic relationships between the different genomes can be examined in HarvEST:Cowpea database [Synteny was examined between cowpea and database . The soydatabase . Syntenidatabase . Due to Genotypic data comprised of cowpea varieties and their SNP call for each locus of the cowpea consensus genetic map were visualized using Flapjack software .HindIII and MboI . Contigs were assembled using the snapshot method of DNA fingerprinting [The physical map was developed using an advanced African breeding line IT97K-499-35 . It consprinting  and asseprinting . The cowSequences were generated for cowpea BACs using an Illumina HiSeq 2000 sequencer by John Weger at the Institute of Integrative Genomics Biology, University of California, Riverside (UCR) from DNA samples prepared by Yaqin Ma (UCR). Sequences of each BAC were generated from paired-end 100 base reads using the combinatorial pooling method described previously . A NODE QTL analysis ofHbs-1in IT93K-503-1 x CB46 population. (DOCX 16 KB)Additional file 1: Hbs-1, Hbs-2 andHbs-3on the cowpea consensus genetic map. Heat-induced browning of seed coats QTLs were positioned on the cowpea consensus genetic map using SNP markers identified in the QTL analyses. Hbs-1 and Hbs-2 (labeled light blue) were identified in the IT93K-503-1 x CB46 population. Hbs-1 and Hbs-3 (labeled magenta) were identified in the IT84S-2246 x TVu14676 population. The most significant SNP marker for each QTL is highlighted in the corresponding color on the linkage group. SNP marker 1_0032 is labeled red since it was the most significant marker for both the Hbs-1 locus identified in IT93K-503-1 x CB46 and the IT84S-2246 x TVu14676 population. (PDF 47 KB)Additional file 2: Hbs-1in IT93K-503-1 x CB46 and IT84S-2246 x TVu14676 individual maps, cowpea consensus genetic map, and the cowpea physical map. (DOCX 14 KB)Additional file 3: QTL analysis ofHbs-2in IT93K-503-1 x CB46 population. (DOCX 12 KB)Additional file 4: Hbs-2in IT93K-503-1 x CB46 genetic map, cowpea consensus genetic map, and the cowpea physical map. (DOCX 16 KB)Additional file 5: QTL analysis ofHbs-1in the IT84S-2246 x TVu14676 population. (DOCX 16 KB)Additional file 6: QTL analysis ofHbs-3in the IT84S-2246 x TVu14676 population. (DOCX 12 KB)Additional file 7: Hbs-3in the IT84S-2246 x TVu14676 population, cowpea consensus genetic map and cowpea physical map. (DOCX 14 KB)Additional file 8: Origins of genotypes used for the marker-trait association. (DOCX 12 KB)Additional file 9: a. Synteny figure ofHbs-1locus withG. max. Synteny was examined for the Hbs-1 locus between cowpea and G. max using EST-derived SNP markers previously BLASTed and aligned to the sequenced genome. The Hbs-1 locus which spanned 45.27 cM to 47.18 cM on the cowpea consensus genetic map linkage group 5 was determined to be syntenic with soybean chromosomes 2 and 14. The syntenic locus in soybean chromosome 2 extended from soybean locus Glyma02g42560 to Glyma02g43640 which corresponded to 44.42 cM to 46.51 cM of the Hbs-1 locus. The syntenic locus on soybean chromosome 14 spanned from Glyma14g05250 to Glyma14g06330 which corresponded to 44.42 cM to 47.18 cM of the Hbs-1 locus on the cowpea consensus genetic map. Ethylene responsive element binding factor 3 and 11 and ethylene forming enzymes were observed in the syntenic regions of soybean and were considered candidate genes for the Hbs-1 locus. b. Synteny figure of Hbs-1 locus with M. truncatula. Synteny was examined for the Hbs-1 locus between cowpea and M. truncatula using EST-derived SNP markers previously BLASTed and aligned to the sequenced genome. The Hbs-1 locus which spanned 45.27 cM to 47.18 cM on the cowpea consensus genetic map linkage group 5 was determined to be syntenic with M. truncatula chromosome 5 where it spanned from Medicago locus Medtr5g018870 to Medtr5g093060. Ethylene response factor 3 (ERF3) and an ethylene forming enzyme were present in the locus and were considered candidate genes. (TIFF 2 MB)Additional file 10: Synteny table ofHbs-1inGlycine maxchromosomes 2 and 14. (DOCX 12 KB)Additional file 11: Synteny table ofHbs-1inMedicago truncatulachromosome 5. (DOCX 12 KB)Additional file 12: Synteny ofHbs-1withP. vulgaris. Synteny was examined for the Hbs-1 locus between cowpea and P.vulgaris using EST-derived SNP markers previously BLASTed and aligned to the sequenced genome. The Hbs-1 locus was translated from the cowpea consensus genetic map vs.4 (45.27 cM to 47.18 cM) to vs.6 (49.9 cM to 51.5 cM) on linkage group 5, which corresponded to Phvul.008G213300.1 locus to Phvul.008G214300.1 locus. An ethylene-forming enzyme and an ACC oxidase gene were observed in the region and were considered candidate genes for the Hbs-1 locus. (TIFF 845 KB)Additional file 13: Synteny table ofHbs-1inP. vulgarischromosome 8. (DOCX 11 KB)Additional file 14: Heat-induced browning of seed coats phenotype. Cowpea genotypes which are positive for the Hbs trait manifest a brown discoloration either partially or over the entire surface of the seed coat when exposed to high temperature heat during flowering. A. RIL number 9 from the IT93K-503-1 x CB46 population which is positive for the heat-induced browning of seed coats (Hbs) trait is shown. B. RIL number 8 from the IT93K-503-1 x CB46 population which is negative for the heat-induced browning of seed coats (hbs) trait is shown. (TIFF 3 MB)Additional file 15:


There is an error in the first sentence and fourth paragraph of the Introduction. The following sentence is incorrect:Optineurin (tineurin (TE.DATA the MAVS-TRAOptn), also called NRP (NEMO-related protein) or FIP-2 (adenovirus E3-14.7K-interacting protein) exhibits 53% of sequence similarities with NEMO .The correct sentence is:Optineurin (“optic neuropathy inducing” or Optn), also called NRP (NEMO-related protein) or FIP-2 (adenovirus E3-14.7K-interacting protein) is closely related to NEMO with 53% of sequence similarity .


EMRA compared to CD45RO-CCR7+ TNAIVE-cells. Moreover, miR-21 but not miR-223 levels were significantly increased in CD45RO-CD31- post-thymic TNAIVE cells as compared to thymic CD45RO-CD31+ TNAIVE cells. Upon activation of CD45RO- TNAIVE cells we observed a significant induction of miR-21 especially in CD4+ T cells, while miR-223 levels significantly decreased only in CD4+ T cells. Besides composition and activation-induced changes, we showed a borderline significant increase in miR-21 levels upon an increasing number of population doublings in CD4+ T-cell clones. Together, our results show that ageing related changes in miRNA expression are dominant in the CD45RO- T-cell compartment. The differential expression patterns can be explained by age related changes in T-cell composition, i.e. accumulation of CD8+ TEMRA and CD4+ post-thymic expanded CD31- T cells and by cellular ageing, as demonstrated in a longitudinal clonal culture model.MicroRNAs (miRNAs) have emerged as important players in the regulation of T-cell functionality. However, comprehensive insight into the extent of age-related miRNA changes in T cells is lacking. We established miRNA expression patterns of CD45RO- naïve and CD45RO+ memory T-cell subsets isolated from peripheral blood cells from young and elderly individuals. Unsupervised clustering of the miRNA expression data revealed an age-related clustering in the CD45RO- T cells, while CD45RO+ T cells clustered based on expression of CD4 and CD8. Seventeen miRNAs showed an at least 2-fold up- or downregulation in CD45RO- T cells obtained from young as compared to old donors. Validation on the same and independent samples revealed a statistically significant age-related upregulation of miR-21, miR-223 and miR-15a. In a T-cell subset analysis focusing on known age-related phenotypic changes, we showed significantly higher miR-21 and miR-223 levels in CD8+CD45RO-CCR7- T Advanced age has been associated with defects of the immune system to mount appropriate antigen specific responses to pathogens. The most profound age-associated changes are observed in T cells. Due to thymus involution with age, the output of naïve T cells is reduced, while the proportion of memory T cells increases, thereby compromising the diversity of the T-cell pool.NAIVE), CCR7+CD45RA-(CD45RO+) central memory (TCM), CCR7-CD45RA-(CD45RO+) effector memory (TEM) and CCR7-CD45RA+(CD45RO)- late-stage effector memory (TEMRA) T-cell subsets [EMRA cells is higher in elderly than in young individuals [NAIVE cells increases with age, while the fraction of CD31+ T cells progressively decreases [in vivo situation of chronic antigenic stress. Long-term cultured T-cell clones may thus represent a model for cellular ageing [Naïve T cells express CD45RA, while being negative for CD45RO . Within  subsets . Whetherividuals . Within ecreases . In eldeecreases . Human Tr ageing .MiRNAs are a class of small non-coding RNAs that bind to mRNA transcripts of protein-coding genes in a sequence-specific manner. Based on the degree of sequence complementarity they induce degradation of the mRNA or repress translation . A singlin vitro and in in vivo models of CD8 T-cell ageing [NAIVE cells [The contribution of changes in miRNA expression patterns to the age-associated decreased functionality of the immune system is largely unexplored. Differential miRNA expression patterns were shown in replicative l ageing . Some ofl ageing . A role VE cells . In addiVE cells .EMRA and CD31- T cells, T-cell activation and by cellular ageing. Finally, we assessed changes longitudinally in a clonal T cell culture model and found alterations in miRNA expression as a function of population doublings in vitro.There are no comprehensive studies of differences in miRNA expression patterns in primary human T cells from younger and older donors. To address this issue, we analyzed miRNA expression in CD4+ and CD8+ T cells sorted based on expression of CD45RO from peripheral mononuclear blood cells (PBMCs) of young and elderly individuals. We observed age-related differences in miRNA expression predominantly within the CD45RO- T-cell compartment. These differences could be explained by a combination of age-associated accumulation of TIn total 27 healthy young (≤ 30 yrs) and 24 healthy elderly (≥ 55 yrs) participants were included in this study. Demographic characteristics of all donors and miRNA analysis strategies are summarized in Table A in T-cell clones were generated from phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) by limiting dilution in the presence of IL-2 using irradiated PBMCs as feeder cells ,18. In tAll participants provided written informed consent according to the Declaration of Helsinki to participate in this study that was approved by The Medical Ethical Committee (METC) (project number: 2009.118) of the University Medical Center Groningen UMCG.NAIVE (CD45RO-CCR7+) and terminally differentiated (TEMRA) cells (CD45RO-CCR7-); as well as CD3+ and CD31- populations were isolated using combinations of the following anti-human monoclonal antibodies: CD3-e450, CD4-A647 , CD45RO-FITC, CCR7-PE, CCR7-PECY7 and CD31-PE . See Figure A in in Peripheral blood was collected in heparin-containing vacutainer tubes  and peripheral blood mononuclear cells (PBMC) were freshly isolated by density gradient centrifugation using Lymphoprep  according to the manufacturer’s protocol. CD4 T cells were sorted by fluorescence-activated cell sorting (FACS) as CD3+CD4+ and CD8 T cells as CD3+CD4-. Within the CD4 and CD8 T-cell subsets, naïve (CD45RO-) and memory (CD45RO+), truly TTM chromatography columns, supplied with Bio-Gel P-6 polyacrylamide gel matrices, were applied for efficient purification of RNA samples . RNA concentration was measured on a NanoDrop ND-1000 Spectrophotometer . RNA samples with 260 / 280 and 260 / 230 ratio of ≥ 1.90 were used for further analysis.Total RNA was extracted using the miRNeasy Mini Kit  following the manufacturer’s instructions. Micro Bio-Spin2 transformed and normalized using a 90th percentile shift using GeneSpring software (v.11.5.1). We excluded control probes and probes detecting viral miRNAs. MiRNAs detected in at least 7 out of 8 samples were included for further analysis. Visualization of normalized data and hierarchical clustering was performed with Pearson correlation distance and complete linkage clustering parameters using Genesis software (v.1.7.6)  [Equal amounts of RNA samples from FACS sorted CD3+CD4+CD45RO+/- and CD3+CD4-CD45RO+/- T cells from healthy young (n = 5) and elderly (n = 5) donors were pooled for miRNA expression profiling. Microarray profiling was performed using the SurePrint Human miRNA Array Kit with 8x15K format  from Agilent (G447013) according to manufacturer’s protocol . Arrays were scanned using an Agilent scanner according to the manufacturer's instructions (Agilent Technologies). Array images were analyzed using Agilent feature extraction software. For data analysis, raw data were logAustria) . The micMiRNA expression levels were determined by quantitative (q)RT-PCR. Multiplexed cDNA synthesis for up to 6 specific miRNAs was performed using Taqman MicroRNA Reverse transcription kit with a multiplexed reverse transcription primers of TaqMan microRNA Assays : for miR-21 (000397), miR-223 (002295), miR-451 (001141), miR-22 (000398), miR-15a (000389), miR-197 (000497), miR-766 (001986), miR-574-3p (002349), miR-328 (000543), miR-483-3p (CSS07FR), miR-885-5p (241451_mat), miR-28-5p (000411) and RNU49 (001005) as described earlier . RNU49 s–ΔCt (Δ Ct = Ct gene—Ct reference gene).All PCR reactions were run in triplicate. Mean cycle threshold (Ct) values were quantified with the Sequence Detection Software . Relative expression levels were quantified using the 26 cells/mL in RPMI medium  supplemented with 5% V/V mouse anti-human CD28 hybridoma , 2% HSA . Cells were split and placed in fresh medium supplemented with αCD3/αCD28 on day 3, 5 and 7. On day 10 cells were harvested and stained for FACS analysis or lysed for RNA isolation.Culture plates were coated with goat-anti-mouse IgG2a Ab  and mouse anti-human CD3 hybridoma supernatant . CD3+CD4+CD45RO- and CD3+CD4-CD45RO- T cells were seeded at a density of 0,5x10in vitro activated T cells were assessed using mAbs against human CD25-PE (BC96)  and CD45RO-FITC (UCHL1) . Cells were analyzed on BD LSR-II Flow Cytometer by Diva software (BD Biosciences). Data analysis was performed on Kaluza Flow Analysis Software (1.2) .Cell surface markers on Results obtained from qRT-PCR are expressed as median respectively. Unpaired samples were compared using the Mann-Whitney test and paired samples using the Wilcoxon signed-rank test. For comparisons of paired unstimulated and stimulated CD4+ and CD8+ T cells, we applied the Wilcoxon matched pairs test. T-cell clones with low and high PD were compared using a paired Wilcoxon signed-rank test. Statistical analyses were performed with GraphPad Prism version 5.0  and SPSS Statistics version 22.0 . P < 0.05 was considered statistically significant.To identify age related differences in T cells, we compared miRNA expression profiles from sorted T cells from peripheral blood of 5 healthy young (median age 27) and 5 healthy old (median age 64) donors. CD4 and CD8 T cells were sorted from the CD45RO- and CD45RO+ gates  and 7 healthy old (median age 67) donors  and old donors   with a concomitant significant increase in the percentage of TEMRA cells (median percentage 67% in elderly vs 41% in young)  . The yien young) . QRT-PCRVE cells . Hence, As a next step, we studied expression patterns of miR-21 and miR-223 in relation to CD31 expression as it is known that CD31- central naïve T cells increase with age especially in the CD4+ T cell subset. CD31+ and CD31- T cells were sorted from the CD45RO- compartment for both CD4 and CD8 from PBMCs of 8 healthy young (median age 28) and 7 healthy old (median age 75) donors , induction of miR-21 and reduction of miR-223. In vitro activation of CD8+ T cells is also associated with acquisition of the memory phenotype (CD45RO+) and the induction of miR-21, whereas the miR-223 levels remain constant.Prolonged exposure of T cells to pathogens in elderly increases the number of TCR primed antigen-experienced memory T cells. To explore a relation between the increased miR-21 and miR-223 levels in elderly and T cell activation, we sorted both CD4+ and CD8+ CD45RO- T cells from peripheral blood of 7 healthy young donors (median age 27)  (median population doubling (PD) is 28) and a higher (>40) (median PD is 55) number of PDs. Expression of miR-21 tended to be increased in high vs low PD T-cell clones (p = 0.0525), whereas the miR-223 levels did not differ in T-cell clones with low and high population doublings  and CDC25A cell cycle genes [To study whether CD4+ T cell changes in miR-21 and miR-223 could also be related to cellular ageing we used T-cell clones with low and high population doublings as an f ageing . Extensile genes . These TEMRA cells, whereas increase in miR-21 levels are related to the number of cell divisions, the accumulation of TEMRA and CD31- central naïve T cells, the latter likely reflecting post-thymic TCR stimulation.Taken together, our results provide an evidence for an age-related miRNA expression pattern especially in CD45RO- T cells. These changes are most likely defined by a combination of ageing-related changes in T-cell composition, activation and the number of cell divisions. MiR-223 changes are associated specifically with an accumulation of TS1 FileFigure A-Representative scatter plots from T-cell subsets sorting strategies. Figure B- T-cell subsets purity following cell sorting. Figure C-Heatmap of the differentially expressed miRNAs in naïve (CD45RO-) and memory (CD45RO+) T cells from young and old donors. Figure D-Validation of microRNA array. Figure E-Activated T cell phenotype. Table A-Characteristics of donors. Table B-Characteristics of T cell clones. Table C-Normalized expression values of miRNAs. Table D-Differentially expressed miRNAs in CD45RO- (CD4/CD8) T cells.(DOC)Click here for additional data file.


The non-nucleoside reverse transcriptase inhibitor nevirapine is used in the treatment of HIV in many developing countries. Its use is associated with occurrence of hypersensitivity in 6-10% of patients. This hypersensitivity can manifest as a number of phenotypes which include the severe skin blistering reactions Stevens Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). The aim was to undertake a genome wide association study (GWAS) in order to identify genetic variants associated with predisposition to nevirapine-induced hypersensitivity. A total of 333 nevirapine-exposed (151 hypersensitive and 182 tolerant) , HIV-infected Malawian adults were genotyped for 826,551 genotyped SNPs using the Illumina HumanOmni1-Quad_v1 chip. A replication cohort of 62 hypersensitive and 59 tolerant patients from Malawi and Uganda was genotyped for 40 SNPs statistically significantly associated with a hypersensitive phenotype in the main cohort (p<5x10-5) using the Sequenom iPLEX platform or TaqMan allelic discrimination. Logistic regression analysis identified 40 statistically significant SNP signals associated with a nevirapine hypersensitivity phenotype. Only 1 SNP association signal  was statistically significant in both our main discovery (=1.48x10-6) and enriched replication cohort (38 cases and 59 controls) (p=9.6x10-5). Meta-analysis determined the odds ratio as 5.17 (p=2.61x10-10). Data suggest this SNP to be a strong proxy for HLA-C*04:01 carriage (96% co-occurrence). We have confirmed that, in a sub-Saharan African population, HLA-C*04:01 carriage confers a significant risk for nevirapine-induced SJS_TEN though not to less severe hypersensitivity phenotypes. No other significant high penetrance genetic risk factors were identified.


Furthermore, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of LNCaP- and PC3-derived prostate cancer cell variants. Genome-wide transcriptional analysis of prostate cancer cells isolated from xenotransplanted TLR9-positive and -negative tumors revealed a unique gene expression signature, with prominent upregulation of inflammation- and stem cell-related markers. TLR9 signaling orchestrated expression of critical stem cell-related genes such as NKX3.1, KLF-4, BMI-1 and COL1A1, at both mRNA and protein levels. Our further analysis identified that TLR9-induced NF-κB/RELA and STAT3 transcription factors co-regulated NKX3.1 and KLF4 gene expression by directly binding to both promoters. Finally, we demonstrated the feasibility of using TLR9-targeted siRNA delivery to block RELA- and STAT3-dependent prostate cancer cell self-renewal in vivo. The intratumoral administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates inhibited growth of established prostate tumors and reduced clonogenic potential of cancer cells. Overcoming cancer cell self-renewal and tumor-propagating potential by targeted inhibition of TLR9 signaling can provide therapeutic strategy for late-stage prostate cancer patients.Prostate cancer progression was associated with tumorigenic signaling activated by proinflammatory mediators. However, the etiology of these events remains elusive. Here, we demonstrate that triggering of the innate immune receptor, Toll-like Receptor 9 (TLR9), in androgen-independent prostate cancer cells initiates signaling cascade leading to increased tumor growth and progression. Using limited dilution/serial transplantation experiments, we show that TLR9 is essential for prostate cancer cells' potential to propagate and self-renew  In developed countries, prostate cancer is the second leading cause of male cancer-related deaths with lack of effective therapies for late-stage cancer patients . The ini+ prostate cancers are reportedly less differentiated, more aggressive and prone to reoccur [Cellular reactions to infection, tissue stress and injury involve activation of Toll-like receptors (TLRs) in various hematopoietic cells . More re reoccur , 8. TLR9 reoccur . Recent  reoccur . Downstr reoccur . In immu reoccur , 12. STA reoccur , 14. Lit reoccur –18In the current study, we investigated whether inflammatory TLR9 signaling in prostate cancer cells provides a set of molecular targets driving tumor aggressiveness and tumor-propagating potential. These studies provide insights into tumorigenic role of inflammation in advanced prostate tumors.TLR9 cDNA (LNCaP-TLR9+ and LN-TLR9HI) or mock vector (LNCaP-TLR9− and LN-TLR9LO); meanwhile the PC3 cells were transduced with either TLR9 shRNA (PC-TLR9LO) or non-silencing control vector (PC-TLR9HI)  Figure , inlays.) Figure . The in y change . To evals Figure , right. LO tumors showed limited and delayed engraftment in NSG mice, thus preventing us from the TPC assessment within the timeframe of our analysis : KLF-4, SOX-2, OCT-4 and NANOG. [in vivo grown LN-TLR9HI and LN-TLR9LO tumors showed the significant upregulation of majority of the tested stem cell-related genes after TLR9 expression  significantly downregulated of the majority (6/8) of tested genes, except for canonical ESC factors NANOG and OCT4 . Recent reports demonstrated synergism between IL-6/STAT3 and PI3K/Akt or Src pathways in promoting prostate cancer aggressiveness and progression [NKx3.1 [BMI-1 [SOX-4 [KLF4 and SOX2 [NANOG and OCT4. This is in contrast to recently shown direct TLR4/NANOG signaling in liver cancer-initiating cells [Downstream TLR9 signaling involves NF-κB activation and indirectly induces STAT3 signaling. , 9, 11 Igression , 39. Our [NKx3.1 , BMI-1 [1 [BMI-1 , and SOX1 [SOX-4 ; some arand SOX2 . Howeverng cells , which m+ human prostate cancer cells were comparable to our previous observations in blood cancer models [+ prostate tumors confirmed therapeutic efficacy of such strategy. Targeting either of the two tumorigenic transcription factors inhibited tumor growth and reduced clonogenic potential of tumor cells. Overall, our findings suggest that TLR9-mediated siRNA delivery targets prostate cancer-propagating cells, thereby halting tumor progression. Further in vivo studies will elucidate effects of inhibiting TLR9-mediated signaling, which should trigger differentiation of cancer-propagating cells into tumor cells with potentially greater sensitivity to standard therapies [+ tumor-propagating cells alone or in combination with expanding panel of antiproliferative agents, can address an unmet need for treatment of patients with advanced and poorly differentiated prostate cancers.While targeting stem cell-like cells gained interest as a potential method to overcome therapeutic resistance of late-stage cancers, such task remains challenging , 31. TLRr models . The proherapies . TherefoInvestigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the authors' institutional review board. Prostate cancer specimens from patients' samples were acquired from the Biospecimen Repository (COH) with the prior Institutional Review Board approvals (IRB09213), with written, informed consent of all patients. 4 μm sections prepared from the selected 48 prostate cancer specimens were deparafiinized, stained using human TLR9-specific antibodies  and evaluated by a pathologist (COH).LO and PC-TLR9HI cells were generated using lentiviral transduction of pKLO-puro.1-shTLR9 or control pKLO-puro.1 vectors (Sigma-Aldrich) into PC3-luc cells, respectively, followed by selection of stable cell lines in puromycin (1.5 μg/ml). The sequence of shTLR9 was: 5′-CCGGCCACTTCTATAACCGGAACTTCTCGAGAAG TTCCGGTTATAGAACTGGTTTTTG-3′. To generate the LNCaP-TLR9+ and LN-TLR9HI cells, the full length human TLR9 cDNA (Addgene) was cloned into pLVX-EF1α vector and lentivirally transduced into LNCaP and LN-S17 cells to select stable clones.Human prostate cancer cells: DU145, PC3 expressing luciferase and STAT3 PC3-luc; , LNCaP-S4 of freshly isolated tumor cells suspended in the agarose-medium mixture (RMPI/10%(v/v)FCS/0.3%(w/v) agarose). The colonies were counterstained and counted after 1–2 weeks of culture. The clonogenic assay design was reported by others [For the spheroid formation assays, 24-well culture dishes coated with 0.6%(w/v) agarose were overlayed using 1 × 10y others .RELAsiRNA : CpG-siRNA(antisense strand): 5′ G*G*TGCATCGATGCAGG*G*G*G*G-Linker-UCC UUUUACGUUUCUCCUCAAUCCGGU 3′ siRNA(sense strand): 5′ CGGAUUGGAGAAACGUAAAAGGA 3′.NOD/SCID/IL-2RγKO mice (NSG), aged 6–8 weeks, were purchased from the Jackson Laboratory. Mouse care and experimental procedures were performed under pathogen-free conditions following protocols approved by the Institutional Animal Care and Use Committee (COH). The limited-dilution assays to calculate tumor-propagating cell frequency were performed as described elsewhere using the ELDA software to calculate frequencies and probability estimates . The CpGTotal cellular lysates were prepared as previously reported  and analBMI-1, COL1A1, KLF4, NANOG, NKX3.1, OCT-4, SOX2 and SOX4  and the specific pairs of primers designed for For uptake studies, following the incubation with fluorescently-labeled oligonucleotides cells were analyzed using C6 flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).ChIP assays were carried out with a EipTect ChIP OneDay (Qiagen) following the manufacturer's instructions. For ChIP-re-ChIP assay, the immunoprecipitates were subjected to a second round of immunoprecipitation. The presence of specific DNA fragments was quantified using qPCR and specific primers listed in the t test was used to calculate two-tailed P value to estimate statistical significance of differences between two treatment groups. One- or two-way ANOVA plus Bonfeerroni post-test were applied to assess differences between multiple groups or in tumor growth kinetics experiments, respectively. Statistically significant P values were indicated in figures as follows: ***, P < 0.001; **, P < 0.01 and *, P < 0.05. Data were analyzed using Prism software v. 6.01 (GraphPad).Unpaired


One stress cells confront in physiological and pathophysiological contexts is hyperosmotic stress. The mechanism by which hyperosmotic stress regulates mTORC1 activity is not well understood. We show here that mild hyperosmotic stress induces a rapid and reversible inactivation of mTORC1 via a mechanism involving multiple upstream signaling pathways. We find that hyperosmotic stress causes dynamic changes in TSC2 phosphorylation by upstream kinases, such as Akt, thereby recruiting TSC2 from the cytoplasm to lysosomes where it acts on Rheb, the direct activator of mTORC1. This work puts together a signaling pathway whereby hyperosmotic stress inactivates mTORC1. The mechanistic target of rapamycin  senses the nutritional status of the cell and is a master regulator of growth and metabolism12112One stress to which cells are exposed under both physiological and pathophysiological conditions is hyperosmotic stress. Cells within the kidney , the gastrointestinal tract, the cornea, the liver, intervertebral discs and joints are exposed to substantial osmolarity fluctuations even under physiological conditions (reviewed in78121311Previous reports investigating the effect of hyperosmotic stress on mTORC1 came to differing conclusions. Most of them found that osmostress inactivates mTORC1 (as depicted by phosphorylation of its downstream substrates S6 kinase and 4E-BP1)151617182122161718192223242527The closest upstream direct activator of mTORC1 is the small GTPase Ras homolog enriched in brain (Rheb). mTORC1 binding to active GTP-bound Rheb is essential for mTORC1 activation293132333429The TSC complex appears to be a central integration point for multiple stress signals to inactivate mTORC1404243Here we show that mild hyperosmotic stress is capable of inactivating mTORC1 in a rapid and reversible manner. In response to activation of a calyculin-A-sensitive upstream phosphatase, osmostress leads to inhibition of Akt, thereby relocalizing TSC2 to the lysosome to act on Rheb, and therefore inhibit mTORC1. This work pieces together a pathway that leads to mTORC1 inhibition in response to hyperosmotic stress.To study the mechanism by which hyperosmotic stress (henceforth referred to as osmostress) inhibits mTORC1, we treated mouse embryonic fibroblasts (MEFs) with medium supplemented with increasing amounts of sodium chloride (NaCl), ranging from 25 mM to 150 mM. The osmolality of normal medium is roughly 330 mOsm/kg so that addition of 100 mM NaCl corresponds to a physiological increase to approximately 500 mOsm/kg15161718We previously reported that the mTORC1 inhibitor TSC2 rapidly accumulates on lysosomes when cells are treated with medium lacking amino acids (nutrient stress)40TSC2 knock-out MEFs with hypertonic medium. Whereas control cells rapidly inactivate mTORC1 within minutes of osmostress induction, TSC2-null MEFs retain significant amounts of mTORC1 activity , and embryonic kidney HEK293FT (Invitrogen), were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) , supplemented with 10% FBS (Biochrom). HeLa and MCF-7 cells were cultured in high-glucose DMEM containing 10% FBS (PAA). MCF-7 cells were also supplemented with 1× non-essential amino acids (Gibco). All media were supplemented with 1× Penicillin-Streptomycin (Gibco). +/+p53−/− and TSC2−/−p53−/− MEF cell lines were verified to be Mycoplasma-free and free of contamination with cells of other species, according to the Multiplex cell Contamination Test Report (Multiplexion GmbH), as described at www.multiplexion.de.The identity of HEK293FT, HeLa, and MCF-7 cells was verified using the Multiplex human Cell line Authentication service , which uses an SNP-profiling approach and was performed as described at www.multiplexion.de. The HEK293FT, HeLa, MCF-7, TSC2Hyperosmotic stress conditions were applied by addition of NaCl or sorbitol to the culture media. The concentration of NaCl in serum-free normal culture media  is 110.35 mM and the overall osmolality is 320–360 mOsm/kg, according to the manufacturer’s specifications. An increase of the NaCl concentration by 100 mM or addition of 200 mM sorbitol to full, serum-containing media raises osmolality to ≤500 mOsm/kgAmino acid starvation was carried out as previously describedPharmacological treatments using Akt inhibitor VIII , MEK1/2 inhibitor U0126 , PKC activator phorbol 12-myristate 13-acetate (PMA) , p38MAPK inhibitor SB203580 , PI3K inhibitor LY294002 , mTOR inhibitor Torin1 , and phosphatase inhibitor Calyculin A , were performed as described in the figure legends.Antibodies against phospho-S6K(T389) (#9205), S6K (#9202), phospho-4E-BP1(T37/46) (#9459), 4E-BP1 (#9452), phospho-Akt(S473) (#9271), phospho-Akt(T308) (#9275), Akt (#9272), phospho-TSC2(T1462) (#3611), phospho-TSC2(S1387) (#5584), phospho-TSC2(S1254) (#3616), TSC2 (#4308), mTOR (#2983), phospho-GSK3β(Ser9) (#5558), phospho-p38(T180/Y182) (#9216), p38 (#9212), phospho-MAPKAPK2(T222) (#3316), phospho-ERK1/2(T202/Y204) (#4370), ERK1/2 (#4695), phospho-AMPKβ1(S108) (#4181), phospho-p90RSK(S380) (#9341), phospho-Erk5(T218/Y220) (#3371), phospho-CaMKII(T286) (#3361), phospho-MST1(T183)/MST2(T180) (#3681), and phospho-YAP(S127) (#4911) proteins were purchased from Cell Signaling Technology. An antibody against phospho-TSC2(S664) was purchased from Abcam (ab133465). The mouse LAMP2 (ABL-93) antibody was obtained from Developmental Studies Hybridoma Bank. A monoclonal antibody recognizing human and mouse α-tubulin (#T9026) and an anti-FLAG (M2) (#F1804) antibody were purchased from Sigma. All antibodies were used in 1:1000 dilution for western blotting, except for the phospho-ERK, total ERK, TSC2, and p38 antibodies that were used in 1:2000.For SDS-PAGE and immunoblotting experiments, cells were lysed in-well with ice-cold Triton lysis buffer  for 10 minutes on ice. Samples were clarified by centrifugation , and SDS loading buffer was added to the supernatant before boiling. The samples were analyzed by 1D gel electrophoresis and phospho- and total protein levels were detected using the appropriate antibodies. Quantification of immunoblots was performed using the LICOR Fc detection system and the ImageStudio software (most panels) or by densitometry of films with ImageJ . The Luciferase gene was amplified from a pGL3 vector (Promega) and cloned in the EcoRI/NotI sites, in frame with the FLAG tag sequence in pcDNA3-FLAG. The integrity of all constructs and the presence of point mutations was verified by sequencing.For the pcDNA3-FLAG-Rheb WT expression vector, full-length The pLJM1-FLAG-Raptor WT and pLJM1-FLAG-Raptor-Rheb15 were a gift from David Sabatini (Addgene plasmids #26633 and 26634) and have been described previouslyPlasmid DNA transfections in HEK293FT cells were performed using Effectene (QIAGEN), according to manufacturer’s instructions.Immunofluorescence experiments were performed as previously describedHow to cite this article: Plescher, M. et al. TSC2 mediates hyperosmotic stress-induced inactivation of mTORC1. Sci. Rep.5, 13828; doi: 10.1038/srep13828 (2015).


Up to 80% of children with Juvenile Systemic Lupus Erythematosus (JSLE) develop lupus nephritis (LN) (1), with the 5-year renal survival rate varying between 44-94% (2-4). Conventional markers of JSLE disease activity fail to adequately predict impending LN flares (5), with significant renal involvement  known to occur with low level proteinuria (6). Cross-sectional adult SLE studies have shown urinary vascular cell adhesion molecule-1 (VCAM-1) to be significantly higher in active LN than inactive LN or healthy controls, correlating with traditional markers of LN disease activity .To investigate the role of VCAM-1 as a urinary biomarker in JSLE.Urinary VCAM-1 concentrations were measured by ELISA (R&D Systems Ltd). The assay demonstrated 108-122% linearity of dilution, and 90-106% recovery using spike and retrieval techniques. Samples were diluted 1 in 80, and re-run at different dilutions where necessary. JSLE patients were classified as ‘JSLE active renal’ or ‘JSLE non-active renal’ based on the renal domain of the British Isles Lupus Assessment Group score (BILAG) (rBILAG A/B vs. D/E). Healthy children (HC), attending for non-inflammatory surgery were recruited as controls. Demographic, clinical and biomarker data were not normally distributed, and expressed as median values and interquartile ranges (IQR). Mann-Whitney U test was used when comparing between groups, and correlations utilized the Spearman rank test.Sixty-seven patients participated in the study . JSLE patients had a median age of 16.5 years (range 10.07-21.91), and 36/50 (72%) were female. All JSLE patients had a median of 5 ACR classification criteria (IQR 4-7), with a median length of disease of 4.66 years (IQR 3.2-7.5). 23 (46%) JSLE patients were classed as JSLE active renal disease and 27 (54%) were JSLE non-active renal. Eleven (22%) JSLE patients had previously undergone a renal biopsy: Class IV LN (n=3), Class III (n=6) and Class II (n=2). The healthy controls had a median age of 12 years (range 4.0-16.0), with 5 being female (29%).p=0.002) and HC's . A statistically significant correlation was seen between VCAM-1 levels, C3  and urinary albumin-to-creatinine (UAUC) ratio .Urinary VCAM-1 levels were significantly higher in JSLE active renal patients (16.65 ng/mgCr [IQR 2.58-51.78]), versus non-active renal patients (2.3ng/mgCr [IQR 0.61-10.01], We have shown for the first time in children, that urinary VCAM-1 is able to identify patients with active renal lupus. Further assessment is required in prospective longitudinal studies.None declared.