Trimeric HIV-1 envelope (Env) immunogens are attractive due to their ability to display quaternary epitopes targeted by broadly neutralizing antibodies (bNAbs) while obscuring unfavorable epitopes. Results from the RV144 trial highlighted the importance of vaccine-induced HIV-1 Env V1V2-directed antibodies, with key regions of the V2 loop as targets for vaccine-mediated protection. We recently reported that a trimeric JRFL-gp120 immunogen, generated by inserting an N-terminal trimerization domain in the V1 loop region of a cyclically permuted gp120 (cycP-gp120), induces neutralizing activity against multiple tier-2 HIV-1 isolates in guinea pigs in a DNA prime/protein boost approach. Here, we tested the immunogenicity of cycP-gp120 in a protein prime/boost approach in rabbits and as a booster immunization to DNA/modified vaccinia Ankara (MVA)-vaccinated rabbits and rhesus macaques. In rabbits, two cycP-gp120 protein immunizations induced 100-fold higher titers of high-avidity gp120-specific IgG than two gp120 immunizations, with four total gp120 immunizations being required to induce comparable titers. cycP-gp120 also induced markedly enhanced neutralizing activity against tier-1A and -1B HIV-1 isolates, substantially higher binding and breadth to gp70-V1V2 scaffolds derived from a multiclade panel of global HIV-1 isolates, and antibodies targeting key regions of the V2-loop region associated with reduced risk of infection in RV144. Similarly, boosting MVA- or DNA/MVA-primed rabbits or rhesus macaques with cycP-gp120 showed a robust expansion of gp70-V1V2-specific IgG, neutralization breadth to tier-1B HIV-1 isolates, and antibody-dependent cellular cytotoxicity activity. These results demonstrate that cycP-gp120 serves as a robust HIV Env immunogen that induces broad anti-V1V2 antibodies and promotes neutralization breadth against HIV-1.IMPORTANCE Recent focus in HIV-1 vaccine development has been the design of trimeric HIV-1 Env immunogens that closely resemble native HIV-1 Env, with a major goal being the induction of bNAbs. While the generation of bNAbs is considered a gold standard in vaccine-induced antibody responses, results from the RV144 trial showed that nonneutralizing antibodies directed toward the V1V2 loop of HIV-1 gp120, specifically the V2 loop region, were associated with decreased risk of infection, demonstrating the need for the development of Env immunogens that induce a broad anti-V1V2 antibody response. In this study, we show that a novel trimeric gp120 protein, cycP-gp120, generates high titers of high-avidity and broadly cross-reactive anti-V1V2 antibodies, a result not found in animals immunized with monomeric gp120. These results reveal the potential of cycP-gp120 as a vaccine candidate to induce antibodies associated with reduced risk of HIV-1 infection in humans. While great progress has been made in developing antiretroviral therapies, and preventative measures, such as preexposure prophylaxis, have aided in reducing new infections, an effective vaccine remains crucial to the eradication of human immunodeficiency virus type 1 (HIV-1) . While uThe main target of antibodies in an HIV-1 vaccine is the HIV-1 Envelope (Env) glycoprotein, which mediates the initial attachment and fusion to host target cells. HIV-1 Env is first expressed as a precursor peptide, gp160, which forms homotrimers before furin-mediated cleavage separates gp160 into its gp120 and gp41 subunits, resulting in a noncovalently linked heterotrimeric spike . Monomer12–de novo N terminus is generated within the V1 loop region with the native N and C termini joined via an amino acid linker chain  . No neutralizing activity against BaL.26 was observed in gp120-immunized rabbits, and no neutralizing antibodies against tier 2 pseudoviruses, including JRFL, were observed in any of the immunized animals , moderate (BaL.26), or low  neutralization sensitivity . Two cycP-gp120 immunizations generated a strong cross-clade NAb response against tier-1A clade B (SF162.LS) and clade C (MW965.26) viruses  . In cont animals . However animals –23.Antibody avidity plays an important role in protection from HIV/SIV infection –28. To m–Recent vaccine studies in both nonhuman primates and humans have emphasized the importance of antibodies targeting the V1V2 loop region of gp120 in protecting from HIV-1 infection , 12–14. 12–Rabbits immunized twice with cycP-gp120 or four times with gp120 showed comparable binding to gp120/gp140 proteins from multiple isolates, including cross-clade reactivity against non-clade B isolates . The cyc4β7 binding site motif  and potential SHIV strains  . As withThe results from the VAX003 and RV144 trials suggest that the implementation of a poxvirus vector prime in the RV144 trial is important for promoting protective immune responses, and recent studies suggest these responses can be attributed to the promotion of distinct IgG subclasses between the two trials . There iTo test the use of MVA-HIV and cycP-gp120 in a poxvirus prime/protein boost scheme, we immunized rabbits intramuscularly with MVA-HIV at weeks 0 and 8 , followeA major effector function of nonneutralizing antibody responses is antibody-dependent cellular cytotoxicity ADCC), and recent studies examining antibody responses from the RV144 trial suggest that ADCC is involved with vaccine efficacy , with alCC, and rOur clade B DNA/MVA vaccine has completed phase 2a immunogenicity studies in humans, and plans are under way for phase 2b efficacy studies . To testRecent efforts in HIV-1 vaccine design have focused on the design and implementation of novel trimeric HIV-1 Env immunogens with the goal of generating protective antibody responses, such as bNAbs  and V1V2A striking feature of cycP-gp120 is the ability to induce broadly cross-reactive V1V2-directed antibody responses, a major correlate of decreased HIV-1 risk in the RV144 trial , 11, 31,4β7 expressed on activated CD4+ T cells, mediating viral uptake and infection , protocol YER-2002080. Experiments were carried out in accordance with USDA regulations and recommendations derived from the  Animals . Rhesus 8 PFU MVA-62BSm (weeks 0 and 8), followed by boosting with cycP-gp120 (weeks 16 and 32). JRFL-gp120 and cycP-gp120 immunizations were given with the adjuvant Adjuplex (Sigma). The production of cycP-gp120 was as described previously  New Zealand White rabbits  were immunized intramuscularly with 20 μg of recombinant JRFL-gp120  or JRFL-hCMP-V1cyc-gp120 (cycP-gp120) (weeks 0 and 8). Both JRFL-gp120 and cycP-gp120 contain the E168K mutation. An additional group was immunized intramuscularly with 1 × 10eviously . JRFL-gpFour Indian rhesus macaques were immunized intramuscularly with two DNA primes (weeks 0 and 12) followed by two MVA-62Bsm boosts (weeks 16 and 24), with both vaccines encoding HIV-1 clade B HxB2 gag/pol and ADA env. In addition, animals were boosted subcutaneously with 100 μg of JRCSF-cycP-gp120 with Adjuplex at weeks 56 and 58.450 reading. Rhesus macaque serum IgG binding to recombinant HIV-1 SF162 (clade B) gp140 (Immune-Tech) was measured by ELISA against serial dilutions of sera and detected with anti-rhesus IgG HRP (Southern Biotech) before quantification against a standard curve against rhesus IgG.Rabbit serum IgG binding to recombinant JRFL-gp120 was measured using JRFL-gp120 (Immune-Tech)-coated ELISA plates and reacted against serial dilutions of sera before detection with anti-rabbit IgG horseradish peroxidase (HRP) (Southern Biotech). Endpoint titers were measured by calculating the serum dilution required to reach an optical density (OD) of 0.16, four times the background level of detection. Antibody avidity was calculated by incubating JRFL-gp120- or cycP-gp120-coated ELISA plates with serial dilutions of rabbit sera before incubation for 10 min with PBS or 1.5 M sodium thiocyanate (NaSCN). Plates then were washed and bound antibodies detected using anti-rabbit IgG HRP. The avidity index was determined by measuring the ratio of the area under the concentration-time curve (AUC) for PBS- or NaSCN-treated sera. For rabbit sera binding to V2 peptides, 15-mer peptides corresponding to the HIV-1 C.1086 V2 region were synthesized (Genemed Synthesis Inc.) and diluted in PBS before coating onto ELISA plates (1 μg/ml). Serial dilutions of rabbit sera were reacted against V2 peptides, and AUC analysis was used to measure binding. Additional peptides corresponding to the V2 hot-spot region of HIV-1 strains JRFL-E168K (N-RDKVQKEYALFYKLD-C), ADA (N-RDKVKKDYALFYRLD-C), SF162P3 (N-GNKMQKEYALFYRLD-C), 1157 (N-RDKKQKVYALFYRLD-C), and 1157-Y173H (N-RDKKQKVHALFYRLD-C)  were synNeutralizing antibody activity was measured in 96-well culture plates by using Tat-regulated luciferase (Luc) reporter gene expression to quantify reductions in virus infection in TZM-bl cells. TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program, as contributed by John Kappes and Xiaoyun Wu. Assays were performed with HIV-1 Env-pseudotyped viruses as described previously . Test saBinding antibody multiplex assays (BAMA) were performed as described previously , 44. BriADCC activity was measured using a Luc-based assay against HIV-1 subtype B JRFL IMC-infected target cells performed as previously described . Target t test (Mann-Whitney test) was used for all comparisons unless otherwise noted . Spearman correlations were used to analyze relationships between multiple variables.Statistical analysis was performed using GraphPad Prism, v7.0. A two-tailed nonparametric


Kip1 plays a pivotal role in the control of cell growth and metastasis formation.The tumor suppressor protein p27Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27kip1 regulates H-Ras activity via its interaction with stathmin.Several studies pointed to different roles for p27in vitro and in vivo models, we show that p27kip1 is an important regulator of Ras induced transformation. In H-RasV12 transformed cells, p27kip1 suppressed cell proliferation and tumor growth via two distinct mechanisms: 1) inhibition of CDK activity and 2) impairment of MT-destabilizing activity of stathmin. Conversely, in K-Ras4BV12 transformed cells, p27kip1 acted mainly in a CDK-dependent but stathmin-independent manner.Here, using Using human cancer-derived cell lines and primary breast and sarcoma samples, we confirmed in human models what we observed in mice.Overall, we highlight a pathway, conserved from mouse to human, important in the regulation of H-Ras oncogenic activity that could have therapeutic and diagnostic implication in patients that may benefit from anti-H-Ras therapies. Interversus K-Ras mutations. In tumors driven by K-Ras mutations, such as the urethane-induced [G12D-induced [V12-induced breast and salivary gland cancers [V12/p27 null tumors displayed features characteristic of highly aggressive tumors [V12-driven transformation via not only the inhibition of the cyclin/CDK/RB pathway but also via RB-independent pathways [A large body of literature exists focusing on the cooperation between Ras and p27 during tumor onset and progression. It is widely accepted that p27 expression impacts on Ras-driven tumor progression. Interesting differences have been noted when p27 knock-out mice have been challenged with tumorigenic models relying on H-Ras -induced , 14 or K-induced  lung can cancers  or the D cancers , p27 acte tumors . Accordipathways .versus K-Ras-driven tumor progression. Here, we address this hypothesis using different in vitro and in vivo model systems.We have recently observed that p27 participates to the regulation of H-Ras activity by modulating its recycling and ubiquitination . Since HV12-transformed WT and p27KO 3T3 fibroblasts [V12 overexpressing-p27WT 3T3 cell clones - -  and in tal (3D)- , but notal (3D)- .6 WT H-RasV12 cells in nude mice (n = 5) were not sufficient to give rise to tumors, while the same number of p27KO H-RasV12 cells determined the appearance of tumor masses within 10-12 days (data not shown). Using 2×106 cells, all mice injected with WT H-RasV12 cells developed slow growing tumors within 15-17 days, but p27KO H-RasV12 cells formed fast growing tumors within 5-10 days , with no appreciable difference among the clones utilized  [n vitro experiments using growth curves  or not (in vivo) affected in their growth by either p27WT or p271-170. However, p27WT, but not p271-170, reduced the ability of p27KO H-RasV12 transformed fibroblasts to move in 3D-Matrigel   , 12, 21 s Figure  and softs Figure  and in vs Figure  using sul Figure  and to i) Figure  and 2F.in vitro and in vivo growth observed in WT versus p27KO H-RasV12 transformed fibroblasts, we used 3T3 fibroblasts derived from WT, p27KO and also double knock-out (DKO) for both, p27 and stathmin C57BL/6 embryos [V12 cells displayed an intermediate growth rate  are highly homolog proteins (83% aminoacid identity in the first 165 aminoacids) with an hyper variable C-terminus (24 aa) which comprises the membrane targeting sequence  activities were significantly higher in p27KO when compared to WT and DKO transformed cells  in rescue-experiments, we re-performed some of the same assays in p27 null H-RasV12 transformed cells, reintroducing the mouse p27, either wild type (m-p27WT) or mutated in the binding to Cyclin/CDKs complexes [CK-)  . In lineoft agar  but signoft agar .V12  and with H-Ras2 Figure  or K-Ras2 Figure  primary V12 grew at higher extent than the correspondent WT and DKO cells both in culture and in anchorage independent manner  as human counterpart of transformed fibroblasts. Following proliferative stimuli, MES-SA cells (established from primary STS) displayed high levels of p27 coupled with low levels of ERK1/2 activation and EGR1 expression, while HS-913T cells (established from metastatic STS) displayed low/null p27 levels, high ERK1/2 activation and EGR1 expression Figure  and 8B. ormation ) we next- Figure . Since N- Figure  and, when Figure .Finally, using BRAF-mutated colorectal carcinoma cells , overall confirming the observation made using the Sv129 3T3 cells  that we have characterized here and in previous publications [The role(s) played by p27 when located in the cytoplasm are at the center of an interesting scientific debate. It has been considered either a cellular lear p27 , 32 or alear p27 , 23, 29,lear p27 , 34 or clear p27 , breast lear p27 , 37, intlear p27  and proslear p27 , the CDKlear p27 -39. One 7 [®Stop , 37 thatications , 21.Our data show that p27 and stathmin regulate in concert H-Ras but not K-Ras activity. This evidence could explain the contradictory tumor suppressor roles ascribed to p27 in different mouse models of cancer. In the presence of K-Ras mutations, p27 acts invariably as a haploinsufficient tumor suppressor gene -15. In tHigh levels of stathmin expression have often been linked to the acquisition of a metastatic phenotype . The useSuccessful targeting of the Ras-MAPK pathway represents a promising tool to treat aggressive human cancers, but optimal selection is needed to identify patients who would benefit from such therapies. Our findings suggest that evaluation of p27 and stathmin expression may contribute to this selection. This is particularly relevant for breast cancer, where Ras oncogenes are infrequently mutated but often hyperactive  and p27 Detailed description of the material and methods used is provided in supplementary material.Sarcoma and breast cancer tissues were collected at CRO Aviano, Italy and stored in the Institutional Biobank, provided that the specific informed consent was obtained from the patient. Scientific use of biological materials was approved by the Internal Review Board (IRB) of CRO Aviano (# IRB-07/2015).All animal experimentation were reviewed and approved by the CRO institutional Animal Care and Use Committee (OPBA), authorized by Italian Ministry of Health (# 616/2015-PR) and conducted according to that OPBA's guidelines.6 or 2×106 transformed cells into the flanks of female athymic nude mice . Tumor growth was monitored every other day for up to 26 days from injection.Primary tumors were established by subcutaneous injection of 1×10Primary wild type (WT), p27 knock-out (p27KO) and p27/stathmin double KO (DKO) mouse embryo fibroblasts (MEF) were prepared from embryos at day 13.5, according to standard procedures , 7. 3T3 V12 (gently provided by Dr. R. Baserga) and K-Ras4BV12  cDNAs both cloned in pMSCV-hygro retroviral vector (Clontech). Primary MEFs were transformed using concomitantly SV40 Large TAg (provided by Dr. R. Maestro) and pMSCV-Hygro-H-RasV12 or pMSCV-Hygro-K-Ras4BV12. Human p27WT or p271-170 were described [WT and p27CK- cDNAs were provided by Dr. Bruno Amati.3T3 fibroblasts were transformed using H-Rasescribed , 22; mouProliferation assays include: growth curve experiments, using the Trypan Blue exclusion test and the MTS assay (Promega); cell cycle distribution using flow cytometry; BrdU incorporation assay (Roche); soft agar assays; and tissues staining with Ki67.Motility assays include were performed essentially as described , 21, 22 n = 8) or metastatic samples (n = 9). Breast cancer specimens derived from locally advanced primary tumors, as better specified in A total of 17 sarcoma (leiomyosarcomas and fibromyosarcomas) and 37 breast tumor specimens were collected and diagnosed at Centro di Riferimento Oncologico (CRO) of Aviano , according to the World Health Organization (WHO) criteria. Sarcoma samples were described elsewhere [


HDAC9 copy number gains in 50% of diffuse large B-cell lymphoma (DLBCL). To study the role of HDAC9 in lymphomagenesis, we generated a genetically engineered mouse (GEM) model that constitutively expressed an HDAC9 transgene throughout B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eμ). Here, we report that the Eμ-HDAC9 GEM model develops splenic marginal zone lymphoma and lymphoproliferative disease (LPD) with progression towards aggressive DLBCL, with gene expression profiling supporting a germinal center cell origin, as is also seen in human B-NHL tumors. Analysis of Eμ-HDAC9 tumors suggested that HDAC9 might contribute to lymphomagenesis by altering pathways involved in growth and survival, as well as modulating BCL6 activity and p53 tumor suppressor function. Epigenetic modifications play an important role in the germinal center response, and deregulation of the B-cell epigenome as a consequence of mutations and other genomic aberrations are being increasingly recognized as important steps in the pathogenesis of a variety of B-cell lymphomas. A thorough mechanistic understanding of these alterations will inform the use of targeted therapies for these malignancies. These findings strongly suggest a role for HDAC9 in B-NHL and establish a novel GEM model for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches based on histone deacetylase inhibitors.Histone deacetylase 9 (HDAC9) is expressed in B cells, and its overexpression has been observed in B-lymphoproliferative disorders, including B-cell non-Hodgkin lymphoma (B-NHL). We examined HDAC9 protein expression and copy number alterations in primary B-NHL samples, identifying high HDAC9 expression among various lymphoma entities and  Summary: This study demonstrates that aberrant expression of HDAC9 in B cells promotes development of lymphoproliferative disease and lymphoma through altering expression of genes involved in the cell cycle and survival, and modulating the activity of key B-lineage factors such as BCL6 and p53. Non-Hodgkin lymphoma (NHL) are a heterogeneous group of cancers of B, T or natural-killer cells, and constitute 4-5% of all cancers , with diHDAC9 has been implicated in diverse conditions, including ischemic stroke, schizophrenia and obesity  in B-NHL  cells  cells .TP53, thus modulating DNA-damage-induced apoptotic responses  and non-HATs (HDAC9 transgene was constitutively expressed in B cells under the control of the immunoglobulin heavy chain (Eμ) enhancer (Eµ-HDAC9). Eµ-HDAC9 mice developed B-lymphoproliferative disorders with progression towards B-NHL. This is consistent with the hypothesis that deregulated protein acetylation plays a pathological role in B-NHL, and provides a model for preclinical evaluation of HDAC inhibitors (HDACIs).Underlining a potential role in B-NHL, HDAC9 interacts with BCL6 , a transesponses . Evidencnon-HATs . These mnon-HATs , and we reg cell function has previously been described , marginal zone lymphoma (MZL) (n=5), follicular lymphoma (FL) (n=9), classical Hodgkin lymphoma (CHL) (n=3) and mantle cell lymphoma (MCL) (n=6). Highest levels of HDAC9 expression were observed in the most aggressive lymphomas, such as DLBCL (both GC and non-GC subtypes) and MCL . In contrast, low-grade B-cell lymphomas, as well as CHL, showed low HDAC9 expression in tumor cells when nuclear intensity was compared with that of adenocarcinoma cells as a positive control  (HDAC9 locus (chr. 7p21.1) has been observed in B-NHL  of DLBCL patients . A total of 46% (13/28) of samples with HDAC9 copy number gains presented trisomy 7 , whereas 43% (12/28) of cases reported with smaller regions of amplification within the chromosome that contained the HDAC9 gene . Here, one case displayed a specific amplification of HDAC9  .Within the immune system, a role for HDAC9 in the control of Tescribed , and we n the GC   in the B-cell compartment from an early stage of B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eμ) . We generated three independent transgenic lines   and monitored a total of 124 mice (78 Eµ-HDAC9 and 46 wild type) for tumor formation and overall survival. We found expression of the TGHDAC9 in the bone marrow and spleen but not in the liver  and spleen , with greatest expression of TGHDAC9 found in the splenic marginal zone , compared to 0/10 wild-type littermates. Histopathology and fluorescence-activated cell sorting (FACS) analysis revealed evidence of abnormal B-cell expansion in the spleen, compatible with the development of lymphoproliferative disorder (LPD) (n=1), and splenic MZL (SMZL) (n=2); no abnormalities were observed in control mice (0/10) (P<0.0001) . Analysis of immunoglobulin (Ig) gene rearrangements in these mice confirmed monoclonal expansions of B-cell populations in 2/3 young-adult Eµ-HDAC9 mice at 8 months of age  of wild-type littermates that exhibited evidence of B cell malignancies (P<0.0001)  . Eµ-HDAC9 B-cell tumors were primarily of splenic origin (Eµ-HDAC9 mice either developed splenic B-cell lymphoma/leukemia (SBCL/L) or another hematological malignancy, such as histiocytic sarcoma (With age (i.e. past 14 months), <0.0001) B. Flow cc origin C,D, withc origin . An addi sarcoma .Fig. 3.Eµ-HDAC9 B-NHL samples confirmed their monoclonal origin . Thus, we concluded that Eµ-HDAC9 lymphomas and LPDs were derived from B-cell precursors, with evidence of transit through the GC or having experienced the GC reaction. In mice, the lymphomas and LPDs exhibited both GC and post-/non-GC immunophenotypes , a marker for GC B cells and plasma cells, and typically found in non-GC-type DLBCL . Among the upregulated genes, pathway analysis using KEGG and gene ontology (GO) annotated databases revealed enrichment for genes involved in the cell cycle, cell division and response to DNA damage (Tables S3 and S4). Pathway-based hierarchical clustering  confirmed regulation of cell-cycle-related genes and pointed to modulation of MAPK/ERK pathways. Furthermore, analysis of predicted protein–protein interactions between KEGG-classified genes from TGHDAC9 B-cell tumors (Table S2) showed high network connectivity  signaling (Table S2). Additionally, signal transduction pathways revealed substantial enrichment for genes involved in G1/S and G2/M transition followed by the Polo-like kinase 1 (PLK1) pathway  led to reductions in HDAC9, consistent with a decrease in gene dosage -binding site, which, when bound by MEF2 family members, activates HDAC9 gene expression (MEF2B (which occur in 11% of DLBCL and 12% of FL) have been reported to directly upregulate expression of BCL6  in GC B cells and drives DLBCL proliferation  have emerged as clinical candidate therapeutic agents , includi ongoing . The upr ongoing . PLK1 pl ongoing  and inhi ongoing . TherefoHDAC9 cDNA was cloned into the pEμSR vector, placing the HDAC9-sequence-containing oligonucleotide cassette downstream of the immunoglobulin heavy chain (IgH) enhancer (Eμ) and the SRα potent promoter . Kaplan–Meier cumulative survival and the log-rank (Mantel–Cox) test were used to determine tumor-free survival and the χ2 test was used to compare B-NHL incidence in Eµ-HDAC9 mice versus wild-type controls. P<0.05 was considered statistically significant. All experimental protocols were monitored and approved by The Institute of Cancer Research Animal Welfare and Ethical Review Body, in compliance with guidelines specified by the UK Home Office Animals (Scientific Procedures) Act 1986 and the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research (in vivo experiments (FLAG-epitope-tagged full-length human promoter . The Eμ-research . ARRIVE eriments .H rearrangements of the heavy chain (JH) locus were amplified and detected in a multiplex PCR reaction using two upstream primers, DFL/DSP and DQ52, together with one reverse primer positioned downstream of JH4 , VH7183b (5′-GTGAAGCCTGGAGGGTCCC-3′), VHQ52 (5′-CAGGTGCAGCTGAAACAGTCA-3′) and reverse JH4 primer (5′-TGAGGAGACGGTGACTGAGGTTCC-3′). The primers amplified all rearrangement products between JH4 and VHJ558, VH7183 or VHQ52 where four different bands would be expected by the combination of each primer set. PCR cycling conditions were: 95°C for 5 min, 95°C for 30 s, 63°C for 30 s and 72°C for 2 min, for 35 cycles. PCR products were gel-purified using the QIAQuick method (Qiagen) and directly sequenced using the ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). Sequences were aligned to those in the international ImMunoGeneTics information system (IMGT) database.Genomic DNA was isolated from tumor specimens and prepared using All Prep Kit (Qiagen). Primers for detection of Ig rearrangements were described previously . D-JH rem of JH4 . For mutm of JH4 . V-DJH rSingle-cell suspensions were obtained from dissected tissues, washed in phosphate buffered saline (PBS) supplemented with 0.2% FBS, filtered through a 45 µm cell strainer and red blood cells were lysed using ammonium chloride solution (STEMCELL Technologies). The antibody combination used for tumor analysis included: CD23, CD19, IgM, IgD, CD21 and B220. Anti-mouse conjugated antibodies were obtained from eBioscience and BioLegend. Cell sorting of splenic CD19-positive mouse B cells was carried out using immunomagnetic isolation with CD19-labeled beads  and purity assessed using anti-mouse PE-conjugated CD19 antibody (eBioscience). Data were acquired on a FACS LSRII analyzer (BD Biosciences) and analyzed using FlowJo software (Tree Star).HDAC9 transcripts in B-cell subsets: F: 5′-TCTGGATGTTCACCATGGAA-3′ and R: 5′-CACTGCCAGGGAAAAAGTTC-3′. Primer sets to detect mouse Hdac9: F: 5′-GGTGATGATTCTCGGAAATTCT-3′ and R: 5′-GAAGCCAGCTCAATGACACA-3′. Abl1 mRNA expression was used as normalization control, F: 5′-CAGCGGCCAGTAGCATCTGACTT-3′ and R: 5′-GCTTCACACCATTCCCCATT-3′.Total RNA from mouse bone marrow and spleen was extracted using TRIzol reagent  following the manufacturer's instructions. Reverse transcription was performed following standard protocol by using AccessQuick Master Mix . Real-time quantitative PCR was carried out using Fast SYBR Green Master Mix . Primer sets used to detect mouse and human n=3) and bone marrow (n=5) from 8- to 12-week-old mice (transgenic and wild type) were prepared as previously described (Single-cell suspensions from spleen (escribed . Bone maescribed . Anti-moTGHDAC9 tumors was performed using hematoxylin and eosin (H&E)-stained sections. Immunophenotypic characterization of the TGHDAC9 lymphomas was performed using antibodies against B220, PAX5, CD3, BCL6 and IRF4 as previously described . After pre-treatment, tissue sections were blocked in Tris-NaCl buffer, washed in PBS and permeabilized in 0.5% Triton X-100. Slides were washed in Tris-NaCl-Tween buffer and reacted with primary antibody overnight at 4°C. Preliminary experiments were performed to determine optimal dilutions for each primary antibody used. Detection methods included: standard IHC, Vectastain Elite ABC system, Vector Laboratories or EnVision system (Dako) DAB staining and ABC-tyramide signal amplification  (see below). Histopathological evaluation of escribed . Human Hescribed . The B-NCell lysates were prepared using RIPA buffer supplemented with protease inhibitors (Roche). Rat monoclonal (IgM) antibody specific to the C-terminus of human HDAC9 (clone 45a7b5b) was developed using a synthetic peptide (DVEQPFAQEDSRTAG) conjugated to Diphtheria toxoid (Mimotopes), corresponding to unique amino acids 1046-1060. Validation was performed using Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad). Other antibodies used for immunoblots included mouse monoclonal anti-FLAG M2 antibody , β-actin loading-control antibody (BA3R)  and acetyl-p53 (Lys379) .Formalin-fixed paraffin-embedded tissue sections were dried for 45 min at 58°C, followed by deparaffinization and hydration in Histoclear  and a graded series of ethanol, respectively. Samples were pretreated by microwave incubation in a pH 6.0 citrate-based antigen-unmasking solution  followed by 2×5 min washes in PBS and permeabilization in 0.5% Triton X-100 (in PBS) for 20 min at room temperature (RT). Samples were washed in PBS prior to blocking for 30 min at RT in Tris-NaCl (TNB) blocking buffer with subsequent incubation in the monoclonal anti-HDAC9 antibody  overnight at 4°C. Primary antibody was followed by biotinylated rabbit anti-rat secondary antibody for 30 min at RT. Endogenous peroxidase activity was inactivated by incubation with 3% hydrogen peroxide in methanol for 15 min at RT. After washing in Tris–NaCl–Tween-20 (TNT) buffer, samples were incubated in streptavidin (SA)-horseradish peroxidase (HRP)  for 45 min at RT. Following this, FITC fluorophore tyramide  was added for 10 min at RT, which results in the deposition of numerous fluorophore labels adjacent to the HRP. This fluorescent signal was then converted to a chromogenic signal by the addition of HRP-labeled anti-FITC , 1/200 dilution in TNB for 45 min at RT, 3×5 min washes in TNT and incubation in chromogen 3-amino-9-ethylcarbazole (AEC) . Hematoxylin QS  was utilized for nuclear counterstaining and the sections mounted in Vectamount aqueous mounting medium . Images were collected on a Zeiss Axioskop 2 light microscope utilizing Axiovision 4.4 software.The following immunolabeling reactions were applied using ABC-TSA method as described elsewhere . PrelimiFormalin-fixed paraffin-embedded tissue sections were dried for 45 min at 58°C followed by deparaffinization and hydration in Histoclear  and a graded series of ethanol, respectively. Samples were pre-treated by microwave incubation in a pH 6.0 citrate-based antigen-unmasking solution  followed by 2×5 min washes in PBS and permeabilization in 0.5% Triton X-100 in PBS for 20 min at RT. Samples were then washed in PBS prior to blocking for 30 min at RT in TNB blocking buffer with subsequent incubation in the monoclonal anti-HDAC9 antibody  overnight at 4°C. Primary antibody was followed by biotinylated rabbit anti-rat secondary antibody  for 30 min at RT. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide (in methanol) for 15 min at RT. After washing in TNT buffer, samples were incubated in SA-HRP  for 45 min at RT. Next, a Cy3 fluorophore tyramide  was added (1/50 dilution prepared in the supplied amplification buffer) for 10 min at RT, which results in the deposition of numerous fluorophore labels adjacent to the HRP. Slides were washed 3×5 min in TNT buffer, any remaining HRP was deactivated by incubating in 3% hydrogen peroxide (in methanol) for 15 min at RT. A second primary antibody was added sequentially  and the protocol was repeated with the FITC fluorophore tyramide  utilized to detect acetylated p53. Slides were counterstained with To-pro 3 iodide  and mounted in Vectashield  mounting medium. Negative control slides were prepared by excluding the primary antibody, by excluding the conjugated secondary antibody-fluorophore and by excluding TSA reagents; negative controls showed no immunoreactivity. Single-labeling experiments carried out to observe the patterns of staining of each primary antibody validated the double-immunostaining results.First, the primary antibodies monoclonal rat anti-HDAC9  and rabbit polyclonal anti-acetylated lysine  were added together and left overnight at 4°C. Following this, secondary anti-rat biotinylated antibody (1/200 dilution) was added and the TSA protocol was carried out using Cy3 fluorophore tyramide . Next, HRP was inactivated before the secondary antibody against acetylated lysine was added (anti-rabbit biotinylated) and the TSA protocol followed using FITC fluorophore tyramide . The HRP was once again inactivated with 3% hydrogen peroxide (in methanol) for 15 min at RT and rabbit polyclonal anti-BCL6 antibody (ab19011) (1/1000 dilution) was added (overnight at 4°C). Following this, the TSA protocol was applied using Cy5 fluorophore tyramide . Secondary-antibody controls were performed to detect any non-specific background staining. Single-staining controls were carried out as in the same-species double-labeling experiments.The EnVision detection system HRP/DAB+ (Dako) was used as previously described . BrieflyPrimary antibodies included polyclonal rabbit anti-Ac-p53 (Lys379) (Thermo Scientific), monoclonal rat anti-HDAC9 antibody (clone 45a7b5b), monoclonal mouse anti-FLAG M2 (Sigma), polyclonal rabbit anti-acetylated lysine (Millipore 06-933), polyclonal rabbit anti-BCL6 (ab19011), anti-CD45R(B220) (ab64100) and anti-CD3 (ab5690). Secondary antibodies included: biotinylated rabbit anti-rat secondary antibody , biotinylated horse anti-mouse secondary  and biotinylated goat anti-rabbit secondary . Fluorochromes and chromogens included SA-HRP , FITC fluorophore tyramide , FITC-HRP , Cy3 fluorophore tyramide , Cy5 fluorophore tyramide  and AEC .http://projects.tcag.ca/cgi-bin/variation/gbrowse/hg18/).Genome-wide DNA profiles were obtained from high-molecular-weight genomic DNA of DLBCL patients using the Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix) following the manufacturer's instructions. Image data analysis and quality control for the hybridized samples were performed using the Affymetrix Genotyping Console 3.0.1 software, and only samples passing the Affymetrix recommended contrast QC and SNP call rates threshold  were considered for analysis. Affymetrix CEL files and corresponding SNP genotype call files generated by the Affymetrix Genotyping Console tool were then analyzed using the dCHIP software. Model-based expression was performed using the perfect-match/mismatch (PM/MM) model to summarize signal intensities for each probe set. Probe intensity data for each array were normalized using a diploid reference set of three normal (non-tumor) DNA samples that had been processed and hybridized in the same experiment as the tumor samples. The standard invariant-set normalization approach in dCHIP was implemented by a karyotype-guided normalization method as previously described . To idenTGHDAC9-derived lymphomas versus normal murine mature B-cell subpopulations, including GC and non-GC  B cells (GeneChip Mouse Gene 1.0 ST Arrays). Data from normal B-cell subsets were obtained from the Immunological Genome Project (GSE15907) (www.immgen.org). Only probes with minimal expression level equal to 10 and minimal standard deviation of 1.5 (log2 transformed) were considered. The hierarchical clustering algorithm is based on the average-linkage, Pearson correlation.Affymetrix GeneChip Mouse Gene 1.0 ST hybridizations were performed using biotin end-labeled cDNA prepared from CD19-positive B cells isolated from tumors. Unsupervised hierarchical clustering was performed on gene expression data from representative https://mygga.genomatix.de), the Search Tool for the Retrieval of Interacting Genes/Proteins STRING v9.1 (http://string-db.org) to develop interactomes or networks . The ZFN binding-cutting site (in lowercase) was 5′-CTCTGGTCCCAGTTCACCaaacaATGGGCCAACTGGAAGTG-3′. Delivery of ZFN was performed following the manufacturer's protocol (Sigma-Aldrich). The human Burkitt's lymphoma cell line Raji (ATCC CCL-86) was used for the study. Cells were maintained in RPMI medium 1640 (1×) (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and grown in a 5% CO2 incubator at 37°C. Cells were transfected by nucleofection (electroporation) using Amaxa Cell Line Nucleofector Kit V (Lonza), and grown for 48 h followed by single-cell sorting in a 96-well format using BD FACS Aria (BD Biosciences). After 3-4 weeks, single-cell-derived clones were screened and analyzed using CEL-I assay (SURVEYOR mutation detection assay) following the manufacturer's instructions (Transgenomic). Genomic DNA was obtained by high-throughput HotSHOT DNA preparation method in 96-well plates. ZFN mutant clones were confirmed by sequencing.ZFNs targeting human


Adenovirus (Ad) vectors are being investigated as vaccine candidates, but baseline antivector immunity exists in human populations to both human Ad (HuAd) and chimpanzee Ad (ChAd) vectors. In this study, we investigated the immunogenicity and cross-reactivity of a panel of recently described rhesus adenoviral (RhAd) vectors. RhAd vectors elicited T cells with low exhaustion markers and robust anamnestic potential. Moreover, RhAd vector immunogenicity was unaffected by high levels of preexisting anti-HuAd immunity. Both HuAd/RhAd and RhAd/RhAd prime-boost vaccine regimens were highly immunogenic, despite a degree of cross-reactive neutralizing antibodies (NAbs) between phylogenetically related RhAd vectors. We observed extensive vector-specific cross-reactive CD4 T cell responses and more limited CD8 T cell responses between RhAd and HuAd vectors, but the impact of vector-specific cellular responses was far less than that of vector-specific NAbs. These data suggest the potential utility of RhAd vectors and define novel heterologous prime-boost strategies for vaccine development.IMPORTANCE To date, most adenoviral vectors developed for vaccination have been HuAds from species B, C, D, and E, and human populations display moderate to high levels of preexisting immunity. There is a clinical need for new adenoviral vectors that are not hindered by preexisting immunity. Moreover, the development of RhAd vector vaccines expands our ability to vaccinate against multiple pathogens in a population that may have received other HuAd or ChAd vectors. We evaluated the immunogenicity and cross-reactivity of RhAd vectors, which belong to the poorly described adenovirus species G. These vectors induced robust cellular and humoral immune responses and were not hampered by preexisting anti-HuAd vector immunity. Such properties make RhAd vectors attractive as potential vaccine vectors. Adenoviruses (Ads) have been explored as vaccine vectors due to their safety profile and immunogenicity , 2. Howe4–1–7–Many different adenovirus serotypes exist, and most human adenoviruses are from species A to F . ChAds t20–We previously described three rhesus adenoviruses (RhAds) that cluster into the poorly characterized adenovirus species G . In addin = 8 to 12/group) were injected intramuscularly (i.m.) with 109 viral particles (vp) of ChAd24-Gag, RhAd52-Gag, RhAd53-Gag, RhAd56-Gag, Ad5-Gag, or Ad26-Gag, and cellular immune responses were assessed by Db/AL11 tetramer binding and intracellular staining (ICS) assays (We initiated studies to investigate the immunogenicity of a panel of RhAd vectors in comparison with that of the chimpanzee Ad serotype 24 (ChAd24) and human Ad5 and Ad26 vectors  18). Gr. Gr18). b/AL11+ CD8+ T cells at the set point after day 28, with a mean of 9.5% tetramer-positive CD8+ T cells for Ad5-Gag at day 49 compared to a mean of 5.3% to 6.7% for all other vectors . However, Ad26-Gag, ChAd24-Gag, and all RhAd-Gag vectors expressed lower levels of PD-1 (3.6 to 17.1%) than did Ad5-Gag (25.9%) at the terminal time point  (+) vaccine-elicited T cells than Ad26-Gag, ChAd24-Gag, and RhAd-Gag vector vaccination , which suggests a more effector-like, rather than memory-like, phenotype (+) CD8+ T cells (As shown in  < 0.03) , 25. Morhenotype . The reshenotype . ICS on  T cells . These d T cells , 25.n = 40 to 50) were primed with 109 vp Ad26-Gag or RhAd52-Gag at week 0. At week 8, mice were boosted (n = 8 to 10/group) with 109 vp of ChAd24-Gag, RhAd52-Gag, RhAd53-Gag, RhAd56-Gag, or Ad26-Gag, and CD8+ T cell responses were assessed by Db/AL11 tetramer binding assays. As shown in P = 0.0142; ChAd24-Gag versus Ad26-Gag, P = 0.0625), followed by RhAd56-Gag, RhAd52-Gag, and Ad26-Gag. These data show the potency of RhAd/ChAd and RhAd/RhAd vaccine regimens.We next sought to evaluate the immunogenicity of HuAd/RhAd and RhAd/RhAd heterologous prime-boost vaccine regimens. Groups of C57BL/6 mice  were immunized with ChAd24-Env, RhAd52-Env, RhAd53-Env, RhAd56-Env, Ad5-Env, or Ad26-Env encoding human immunodeficiency virus type 1 (HIV-1) clade C Env 459C gp140 (n = 5/group) were primed with Ad26-Env at week 0 and boosted with ChAd24-Env, RhAd52-Env, RhAd53-Env, RhAd56-Env, or Ad26-Env at week 8. As shown in 10 titer at week 10   . These dThe extent of immunologic cross-reactivity between the Ad5 and RhAd vectors is not yet known, but it has been previously reported that preexisting Ad5 immunity can impede the immune responses generated by certain nonhuman adenovirus vectors . Moreoven = 5/group) were injected twice with 109 vp of Ad5-empty at weeks −8 and −4  Gag or Env 459C gp140, and the responses were evaluated by Db/AL11 tetramer binding assays and Env-specific enzyme-linked immunosorbent assays (ELISAs). As shown in To model the effects of Ad5 preexisting immunity on RhAd vector immunogenicity, C57BL/6 mice (8 and −4 . As shown = 40) were preimmunized with two injections of 109 vp Ad5-empty at week −8 and week −4 prior to vaccination . At 4 weeks after the second Ad5-empty injection, mice were primed with Ad26-Gag or RhAd52-Gag at week 0 and were boosted with ChAd24-Gag, RhAd52-Gag, RhAd53-Gag, Ad26-Gag, or Ad5-Gag at week 8 (n = 4/group). Priming responses were pooled for Ad26-Gag and RhAd52-Gag and depicted on the graph in b/AL11 tetramer binding responses, reaching 31.9% of CD8+ T cells at week 10 . In RhAd52-Gag-primed mice, all vectors except for Ad5-Gag resulted in effective boosting responses, reaching 26.0% CD8+ T cells at week 10; this was particularly the case for the heterologous vectors RhAd53-Gag and ChAd24-Gag . As shown in We next conducted prime-boost immunization experiments in mice with high levels of baseline Ad5 immunity. C57BL/6 mice (cination . All micn = 5/group) were preimmunized with either one or two injections of 109 vp Ad26-empty, RhAd52-empty, RhAd53-empty, RhAd56-empty, or phosphate-buffered saline (PBS) . However, we observed substantial suppression of the homologous RhAd52-Gag vector with one RhAd52-empty preimmunization and complete suppression with two RhAd52-empty preimmunizations  . At 4 weizations , which rizations . We alsoizations  and C. T9 vp of RhAd52-empty . Serum collected 1 day after IgG transfer but prior to Ad-Gag vaccination verified RhAd52 NAbs in mice that received RhAd52 IgG but not in mice that received sham IgG  and partially suppressed RhAd56-Gag  but did not significantly impair RhAd53-Gag or Ad26-Gag, thus confirming the suppressive potential of these cross-reactive NAbs.To explore the suppressive potential of cross-reactive RhAd-specific NAbs in greater detail, we conducted adoptive transfer studies with purified IgG. Donor mice were immunized twice, 4 weeks apart, with 1052-empty . IgG wassham IgG . As shown = 5/group) were injected twice with 109 vp of Ad5-empty, Ad26-empty, RhAd52-empty, or RhAd53-empty 4 weeks apart. At 4 weeks after the final injection, spleens were harvested and stimulated with overlapping 15-mer hexon peptides spanning the entire hexon region of each serotype. As shown in + T cells exhibited extensive cross-reactivity to homologous and heterologous peptide pools. In contrast, CD8+ T cells were more restricted in their cross-reactivity. These data suggest broad cross-reactivity for the CD4 responses and less extensive cross-reactivity for the CD8 responses induced by RhAd vectors.Cross-reactive cellular responses have previously been reported to be extensive among HuAd serotypes , 8. To i9 vp RhAd52-empty or saline 4 weeks apart, raising median NAb log10 titers of 2.3 among the RhAd52-empty groups (n = 5/group) received 5 × 107 pooled splenocytes from RhAd52-immune or naive donor mice and then were vaccinated with either RhAd52-Gag or RhAd56-Gag. As shown in Finally, we performed a cellular adoptive transfer study to evaluate the biological significance of these cross-reactive T cell responses in this model. C57BL/6 mice were injected twice with 10y groups . Groups Adenoviridae . Whole-genome and hexon DNA sequences were aligned using the ClustalW program. Maximum likelihood phylogenetic trees were based on the general time-reversible model and were bootstrapped 50 times. The trees with the highest log likelihoods are shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.Phylogenetic trees were constructed using MEGA (version 7) software  were used for all immunization experiments. Mice were vaccinated with the E1/E3-deleted Ad5, Ad26, ChAd24, RhAd52, RhAd53, or RhAd56 vector . The vec9C gp140  transgenEnzyme-linked immunosorbent assays (ELISAs) were performed as described previously . Brieflyb tetramer loaded with the immunodominant AL11 peptide (AAVKNWMTQTL) as described previously  was added and samples were incubated for 5 h at 37°C. After the incubation, cells were washed and stained with the surface stain antibodies (mentioned above), permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with anti-IFN-γ (clone XMG1.2) antibodies for half an hour. Vital exclusion dye was purchased from Invitrogen. All antibodies were purchased from either BioLegend or BD Biosciences. All samples were acquired using an LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo (version 9.6.4) software (TreeStar).Splenocytes were stimulated with 1 μg/ml of an overlapping SIV2. After incubation, the medium was removed and 100 μl of phosphate-buffered saline (PBS) and 100 μl of Steady-Glo substrate (Promega) were added to the wells. The luciferase (Luc) activity in the cells was measured with a Victor 3 multilabel counter . Neutralization titers were defined as the maximum serial dilution where 90% of the virus was neutralized by the serum.Adenovirus-specific neutralization antibody (NAb) titers using mouse serum samples were conducted as previously described . BrieflyIgG was purified from mouse serum using IgG purification NAb spin kits (Thermo Scientific) according to the manufacturer's instructions. Serum was bound to the spin column and washed with binding buffer (Thermo Scientific). Bound IgG was then eluted using elution buffer  and neutralized with neutralization buffer . IgG was then buffer exchanged into 1× PBS via spin columns (Amicon Ultra 10k device).7 splenocytes or purified IgG from either the RhAd52 donor mice or naive donor mice via the intravenous route. One day following adoptive transfer, mice were vaccinated with the RhAd52, RhAd53, RhAd56, or Ad26 vector expressing SIV Gag. Following vaccination, mice were followed weekly for tetramer binding responses as mentioned above.Adoptive transfer studies were performed essentially as previously described . Donor mt test and GraphPad Prism (version 7.0) software (GraphPad Software).Statistical analyses were performed using a two-tailed nonparametric Mann-Whitney U


Alvocidib decreased MCL-1, and/or increased pro-apoptotic proteins such as BIM or NOXA, often synergistically with venetoclax. Over-expression of BCL-XL diminished synergy, while knock-down of BIM almost entirely abrogated synergy, demonstrating that the synergistic interaction between alvocidib and venetoclax is primarily dependent on intrinsic apoptosis. CDK9 inhibition predominantly mediated venetoclax sensitization, while CDK4/6 inhibition with palbociclib did not potentiate venetoclax activity. Combined, venetoclax and alvocidib modulate the balance of BCL-2 family proteins through complementary, yet variable mechanisms favoring apoptosis, highlighting this combination as a promising therapy for AML or high-risk MDS with the capacity to overcome intrinsic apoptosis mechanisms of resistance. These results support clinical testing of combined venetoclax and alvocidib for the treatment of AML and advanced MDS.More effective treatment options for elderly acute myeloid leukemia (AML) patients are needed as only 25–50% of patients respond to standard-of-care therapies, response duration is typically short, and disease progression is inevitable even with some novel therapies and ongoing clinical trials. Anti-apoptotic BCL-2 family inhibitors, such as venetoclax, are promising therapies for AML. Nonetheless, resistance is emerging. We demonstrate that venetoclax combined with cyclin-dependent kinase (CDK) inhibitor alvocidib is potently synergistic in venetoclax-sensitive and -resistant AML models  Targeting of intrinsic apoptosis via inhibition of B-Cell Lymphoma 2 (BCL-2) family proteins is a promising therapeutic strategy. BCL-2-selective inhibitor venetoclax ABT-199) has demonstrated clinical activity in chronic lymphocytic leukemia (CLL) and has recently received FDA-approval for some relapsed CLL patients 99 has de and highin vitro, we selected MOLM-13 and MV4-11 cell lines, which have low single-agent venetoclax half-maximal effective concentration (EC50) values (in assays assessing relative cell number) of 9.0 ± 1.6 and 7.8 ± 2.1 nM, respectively. These low nM venetoclax EC50 values are consistent with values for primary AML samples sensitive to venetoclax  (BD Biosciences) and propidium iodide (Sigma Aldrich) added to [5 µg/mL]. After incubating 15 minutes, cells were analyzed on LSR Fortessa (BD Biosciences).www.imagej.nih.gov).After treatment with alvocidib and/or venetoclax at indicated doses/times, protein was harvested with lysis buffer  containing 1 mM PMSF. Lysates were quantified by BCA (Pierce/Thermo Fisher), 26–30 µg total protein resolved by 4–15% SDS-PAGE, transferred to PVDF membranes (80V/90 minute wet-transfer), and blocked with 5% non-fat dry milk before primary antibody incubation, 4°C/overnight. MCL-1 (#4527), pMCL-1-Thr163 (#1476), BCL-2 (#2872), BCL-XL (#2762), BIM (#2819), PUMA (#4976), β-tubulin (#2128) , NBK , NOXA , and β-actin . Densitometry was performed with Image J software  was amplified by PCR and cloned into plasmid pSC11CMVFlag, with a CMV promoter and 5′3Xflag tag. BCL-XL was then cloned into lentiviral vector pWPI (Addgene plasmid #12254) resulting in plasmid pWPIS6FBCL-XL. Plasmid pWPIS6FBCL-XL, together with packaging plasmid psPAX2 (plasmid #12660) and pMG2.g (plasmid #12259) were transfected into 293T cells and rescued into lentivirus.MicroRNAs were isolated using Directzol RNA mini-prep kit (Zymo Research). Reverse transcription was performed using TaqMan MicroRNA RT Kit, resulting cDNA underwent pre-amplification with TaqMan PreAmp Master Mix, and RTqPCR was performed with TaqMan MicroRNA assays and TaqMan Universal Master Mix II containing UNG (Life Technologies).siRNA were purchased from Qiagen. For target protein knock-down validation, 0.04 nmol siRNA was mixed with 750 µL RNAiMax transfection reagent (ThermoFisher) diluted in OPTI-MEM (Invitrogen) (dilutions optimized per cell line and reagent batch), after 30 minutes 2.6 to 4.3E5 cells (optimized per cell line) were added in 1 mL unsupplemented RPMI-1640, spiked with FBS to [2.2%], and harvested at 48 hours. For siRNA drug dose response assays, siRNA were pre-printed on 384-well plates, OPTI-MEM-diluted transfection reagent was added at 20 µL/well, after 30 minutes 1000–2000 cells/well were added in 20 µL of unsupplemented RPMI-1640, and spiked with FBS to [2.2%]. For BIM siDDRs, plates were incubated for 24 hours prior to simultaneous dosing of venetoclax and alvocidib and read 48 hours later. For BCL-XL siDDRs, plates were incubated for 48 hours prior to simultaneous addition of venetoclax or ABT-737 and alvocidib and read 48 hours later.scidHrhr) . Study animals were housed under standard conditions, and given food and water ad libitum. 1E7 OCI-AML3 cells were suspended in serum-free media, mixed 1:1 in matrigel (Corning), and injected subcutaneously into the hind flank. Randomization and treatment was initiated once tumor volumes had reached approximately 200 mm3. Alvocidib was formulated in saline, while venetoclax was formulated in Phosal 50PG: PEG400: Ethanol. Mice were administered vehicle or drug treatment by intraperitoneal injection, (qdx5)x3 with alvocidib (2.5 mg/kg), by oral gavage (qdx5)x3 with venetoclax (100 mg/kg), or both alvocidib and venetoclax. Tumor length and width measurements were made with digital calipers, and tumor volumes were calculated as follows: length x width x width/2, where length was the longest diameter. Tumor volume and body weights were measured and recorded twice weekly.Animal studies were reviewed and approved for ethical consideration by an internal review committee at Tolero Pharmaceuticals, Inc. OCI-AML3 cells were transplanted into 6–8 week old, female, hairless outbred SCID mice (Crl:SHO-PrkdcP values were calculated using two-tailed Student’s t-test. For correlative comparisons of drug EC50 values, or drug activity with protein level, regression analysis was used to calculate R2 and corresponding p values. R values shown in Figure


Estrogen receptor α positive (ERα+) breast cancer accounts for most breast cancer deaths. Both prolactin (PRL) and extracellular matrix (ECM) stiffness/density have been implicated in metastatic progression of this disease. We previously demonstrated that these factors cooperate to fuel processes involved in cancer progression. Culture of ERα+ breast cancer cells in dense/stiff 3D collagen-I matrices shifts the repertoire of PRL signals, and increases crosstalk between PRL and estrogen to promote proliferation and invasion. However, previous work did not distinguish ECM stiffness and collagen density. In order to dissect the ECM features that control PRL signals, we cultured T47D and MCF-7 cells on polyacrylamide hydrogels of varying elastic moduli (stiffness) with varying collagen-I concentrations (ligand density). Increasing stiffness from physiological to pathological significantly augmented PRL-induced phosphorylation of ERK1/2 and the SFK target, FAK-Y925, with only modest effects on pSTAT5. In contrast, higher collagen-I ligand density lowered PRL-induced pSTAT5 with no effect on pERK1/2 or pFAK-Y925. Disrupting focal adhesion signaling decreased PRL signals and PRL/estrogen-induced proliferation more efficiently in stiff, compared to compliant, extracellular environments. These data indicate that matrix stiffness shifts the balance of PRL signals from physiological (JAK2/STAT5) to pathological (FAK/SFK/ERK1/2) by increasing PRL signals through focal adhesions. Together, our studies suggest that PRL signaling to FAK and SFKs may be useful targets in clinical aggressive ERα+ breast carcinomas. Estrogen receptor alpha positive (ERα+) breast cancers constitute the most plentiful breast cancer subtype , and metThe discrepancies in these studies present an apparent conflict in PRL actions in breast cancer: PRL activity has been correlated with aggressive ERα+ tumors, yet activation of the canonical PRL signaling mediator correlates with favorable outcomes. A possible explanation may be that PRL can also signal through other effectors, including Focal Adhesion Kinase (FAK), Src-Family Kinases (SFKs), and ERK1/2 –17, whicin situ [The ECM is increasingly recognized as an active participant in breast cancer. Increased mammographic density, which is comprised of both increased cellular density and fibrillar collagen, correlates with increased breast cancer risk –20. Incrin situ , and thein situ . One of in situ , 26, whiin situ , 28. Orgin situ .in vitro system [Utilizing a 3-dimensional floating collagen-I gel o system , we receo system . Additioo system . PRL sigo system ). Howeveo system . These do system , indicatTo distinguish the impacts of matrix stiffness and ligand density on PRL signals in breast cancer cells, we examined PRL-induced signaling in ERα+, PRLR+ breast cancer cell lines cultured on well-characterized polyacrylamide hydrogels , 37 coatin situ 16-26 kPa, and invasive carcinoma 35-100 kPa [MMP2 and MMP9, and the progenitor marker ITGA6 (CD49f) were increased in response to PRL only in the stiff matrix environment (p<0.05)  Figure . Despite) Figure , indicat) Figure .In order to determine the effect of collagen-I ligand density on PRL-induced signals, we cultured T47D cells on polyacrylamide hydrogels at 25 kPa stiffness and varied the collagen-I ligand concentration from 50 to 800 μg/ml. Altered collagen-I ligand density also did not affect PRL signals to pERK1/2 or pFAK Y925 Figure . HoweverStiff extracellular environments increase formation of focal adhesion complexes that contain both FAK and SFKs , 45, andDirectly downstream of collagen ligand/β1 integrin complexes is FAK, which autophosphorylates at Y397 when integrins engage the ECM . To testSFKs are a family of oncogenes that contribute to progression of breast cancer , 54, andOn 2-D tissue culture plastic, PRL is mitogenic for breast cancer cells , 55, andThe desmoplastic response during breast cancer progression is well characterized . Dein vivo [Here we demonstrated that the physical stiffness (elastic modulus) of the ECM, but not collagen I ligand density, controls PRL-induced signals to the pro-tumorigenic FAK/SFK/ERK1/2 signaling pathway, with only modest effects on PRL-induced STAT5 signals. In contrast, collagen density moderates PRL signals to STAT5. Although hormone-induced proliferation was only slightly higher in stiff compared to compliant matrices, the lack of effect of matrix stiffness on PRLR expression suggests a greater portion of these hormonal signals become routed through FAK in stiff matrices, and are more susceptible to inhibition of this pathway. These findings indicate that a stiff extracellular environment promotes PRL signals through focal adhesions, fueling tumor progression Figure . Notablyin vivo . We woulin vivo , 47, 63.Focal adhesion complexes are large sites of cell-matrix interactions, containing numerous receptors, scaffolding proteins, and kinases that transduce extracellular cues to cells . A key cRecent evidence indicates that the intracellular domain of PRLR is intrinsically disordered, and can associate with the plasma membrane through distinct lipid modifications . This suOur findings begin to resolve the apparent dichotomy of PRL actions in breast cancer: PRL can reduce aggressive tumor behavior through STAT5, but increase tumor progression through FAK/SFK/ERK1/2. Activation of the canonical PRL signal mediator, STAT5, is a positive prognostic factor in breast cancer that predicts sensitivity to anti-estrogen therapies and favorable outcomes –14. Thesin vitro [We have shown that physical rigidity of the ECM is a major determinant of the spectrum of PRL-induced signals, increasing PRL activation of the tumor progressive FAK/SFK/ERK1/2 signaling cascade in stiff environments through localization of PRLR to focal adhesions. Our data provide a mechanism for how tumor environments can shift PRLR signals away from physiological STAT5, and subsequent positive prognostic outcomes, to the poorer outcomes of increased signals through focal adhesions. Moreover, PRL increases expression of mammary ECM components , and incin vitro , a hallmin vitro . A modelRecombinant hPRL (Lot AFP795) was obtained from Dr. A.F. Parlow . Type-I rat tail collagen (#CB354249) was obtained from Fisher Scientific . Sulfo-SANPAH (#C1111-100) was obtained from ProteoChem . Inhibitors used for these studies were purchased as follows: PP-2 (#ab120308) from Abcam , Dasatinib from Selleckchem , pFAK Y397 inhibitors PF-573228 (#PZ0117) from Sigma Aldrich  and pFAK Y397 inhibitor PF-562271 (#S2890) from Selleck Chemicals . Protein A/G agarose beads (#SC-2003) were obtained from Santa Cruz Biotechnology . Antibodies used in these studies were purchased as follows: PRLR-ECD (#35-9200), pSRC Y418 (#44660G), pFAK Y397 (#44624G), and pSTAT5 (#71-6900) from Invitrogen ; ERK1/2 (#9102), pERK1/2 (#9101), FAK (#3285), and pFAK Y925 (#3284) from Cell Signaling Technology ; cSRC (sc-18), PRLR (sc-20992), and STAT5 (sc-835x) from Santa Cruz Biotechnology ; FAK clone 4.47 (#05-537) from EMD Millipore . Ki-67 (Ab15580) from AbCam ; β1-integrin blocking antibody  and rat IgG2a,k isotype antibody (cat. # 555841) was purchased from BD Biosciences . Donkey anti-rabbit conjugated to Rhodamine (TRITC) secondary antibody (711-025-152) was purchased from Jackson Immuno-Research . All other reagents were obtained from Fisher Scientific or Sigma-Aldrich.2O under high intensity UV light for 5 minutes. Excess sulfo-SANPAH was rinsed off in DI H2O and collagen-I was then added to the functionalized hydrogels at the appropriate concentrations  [Polyacrylamide gel inserts were prepared as described , 37. Bri0 μg/ml) , 39 for 0 μg/ml) . The gel+, PRLR+ T47D and MCF-7 breast cancer cells were maintained as previously described [MMP2 F, 5′-CTG CAA CCT GTT TGT GCT GAA; MMP2 R, 5′-GGC TTG CGA GGG AAG AAG T; MMP9 F, 5′-CGG AGT GAG TTG AAC CAG; MMP9 R, 5′-GTC CCA GTG GGG ATT TAC; ITGA6 F, 5′-CAT ATA GAG AAC TGA GGG CTT TCC; and ITGA6 R, 5′-TCC GAG CTC ACA GTC AGC TT. For proliferation studies, 24 h after plating in phenol-red free charcoal stripped serum media, cultures were serum starved overnight, and then treated ± 17β-estradiol (E2) 1nM and ± PRL 4nM for 24 h. For some experiments, inhibitors were added 1 h prior to hormone treatment at the following concentrations: 5μM PF-573228, 5 μM PF-562271, 5 μM PP-2, 250 nM dasatinib, or 500 ng/ml mAb13.ERαescribed , 80. T47escribed . Brieflyescribed . The folImmunofluorescence was performed as previously described . BrieflyStatistical analyses were performed using GraphPad Prism v.4.0. Independent experiments examining signaling cascades by immunoblotting were analyzed via two-way ANOVA followed by post-hoc paired t-tests. Analysis of Ki67 staining for proliferation studies was assessed by two-way ANOVA, followed by Holm-Sidak multiple comparison tests.


Mycobacterium tuberculosis (Mtb) and helminth infections elicit antagonistic immune effector functions and are co-endemic in several regions of the world. We therefore hypothesized that helminth infection may influence Mtb-specific T-cell immune responses. We evaluated the cytokine profile of Mtb-specific T cells in 72 individuals with pulmonary TB disease recruited from two Sub-Saharan regions with high and moderate helminth burden i.e. 55 from Tanzania (TZ) and 17 from South Africa (SA), respectively. We showed that Mtb-specific CD4 T-cell functional profile of TB patients from Tanzania are primarily composed of polyfunctional Th1 and Th2 cells, associated with increased expression of Gata-3 and reduced expression of T-bet in memory CD4 T cells. In contrast, the cytokine profile of Mtb-specific CD4 T cells of TB patients from SA was dominated by single IFN-γ and dual IFN-γ/TNF-α and associated with TB-induced systemic inflammation and elevated serum levels of type I IFNs. Of note, the proportion of patients with Mtb-specific CD8 T cells was significantly reduced in Mtb/helminth co-infected patients from TZ. It is likely that the underlying helminth infection and possibly genetic and other unknown environmental factors may have caused the induction of mixed Th1/Th2 Mtb-specific CD4 T cell responses in patients from TZ. Taken together, these results indicate that the generation of Mtb-specific CD4 and CD8 T cell responses may be substantially influenced by environmental factors in vivo. These observations may have major impact in the identification of immune biomarkers of disease status and correlates of protection. Mycobacterium tuberculosis (Mtb) and helminth infections are co-endemic in several regions of the world and their immune responses may be mutually antagonistic. We therefore hypothesized that helminth infection would impact and potentially shape Mtb-specific T-cell responses and systemic inflammation in patients suffering from active pulmonary tuberculosis (TB) enrolled from two helminth endemic regions i.e. Tanzania (TZ) and South Africa (SA). In this study, we demonstrate for the first time that TB patients from SA and TZ harbor distinct immune responses to Mtb antigens. Indeed, we showed that Mtb-specific CD4 T-cell responses of TB patients from TZ were composed by a mixed T helper type 1 (Th1) and Th2 responses. In contrast, the cytokine profile of Mtb-specific CD4 T cells of TB patients from SA was dominated by Th1 cells and associated with TB-induced systemic inflammation and elevated serum levels of type I IFN. Taken together, these data indicate that Mtb-specific T-cell responses are diverse in human populations and can be strongly influenced by host and pathogen genetic background, co-infections and yet unknown environmental factors. Identification of correlates of risk and protection from TB disease will help in the rational development of protective T-cell based vaccines against TB, early monitoring TB treatment outcomes and focused follow up of high risk populations. Therefore, co-infections of helminths with Mtb, HIV-1 and/or Plasmodium falciparum occur in a large proportion of the subjects [Helminth infections are endemic in many African countries with different prevalence depending on the geographic region helminth species and age of population . Soil trn faeces . Of notesubjects .Mycobacterium tuberculosis (Mtb) is a facultative intracellular organism, obligate aerobe, infecting primarily lungs via the aerogenic route [Mtb among which 5–15% will develop tuberculosis disease (TB) [M. bovis, the Bacillus of Calmette et Guérin (BCG). While BCG immunization protects from life-threatening disseminated forms of TB disease in children, its efficacy in adults is inconsistent [Mtb-specific immunity are partially defined. Several studies have underscored the essential role of IL-12/IFN-γ axis in the protection against Mtb infection [Mtb infection, since a severe reduction in the CD4 T cell number during HIV infection or the suppression of their function following anti-TNF-α therapy are associated with increased risk of TB reactivation [ic route . It has nsistent . The pronfection –10. In ativation , 12.Mtb-specific T-cell response is usually ascribed to typical Th1 response with CD4 T cells producing cytokines such as IFN-γ or TNF-α that contribute to the recruitment of monocytes and granulocytes and activate the anti-microbial activity of macrophages [The current paradigm of human cellular immunity indicates that functionally-distinct CD4 T-cell populations are specifically involved against a variety of pathogens depending on their size and their intra- or extra-cellular localization. In this model, type 1 helper CD4 T cells (Th1 cells) intervene against viruses and intracellular pathogens, Th2 cells against parasites such as worms and Th17 cells against extracellular pathogens , 14 inclrophages , 17. By rophages –21 assocrophages , 23, clerophages , 25. In rophages .Mtb/helminth co-infected individuals have focused on the impact of helminth infection on 1) TB diagnosis [Mtb. However limited information is available on the cytokine profile of Mtb-specific T-cell immune responses in subjects with Mtb/helminth co-infection.Multiple studies performed in iagnosis –35, 2) riagnosis , 37 and iagnosis –40. In aiagnosis , cellulaiagnosis –44 and hiagnosis  immune rMtb-specific immune response in patients with clinically active TB from two countries i.e. Tanzania (TZ) and South Africa (SA) in the presence or absence of active helminth infections. We provide evidence that the functional profile of Mtb-specific CD4 T cells of TB patients from TZ was characterized by a mixed Th1/Th2 cytokine profile, while that from SA was associated with a typical single IFN-γ and dual IFN-γ/TNF-α Th1 profile.In the present study, we investigated the cytokine profile of i.e. Mtb, in human populations from different geographic areas. Ad hoc designed studies will be needed to define the factors driving the distinct functional profiles as well as to determine whether the distinct functional profiles are associated with variation of the TB pathology and/or response to drug therapy.These results demonstrate that distinct functional profiles of CD4 T-cell responses can be directed against the same pathogen, Mtb-specific T-cell responses in patients with pulmonary tuberculosis (TB) and helminth co-infection from two Sub-Saharan countries, e.g. Tanzania (TZ) and South Africa (SA) and ii) to determine the influence of ongoing or past helminth infections on Mtb-specific T-cells responses. Therefore, we analyzed the cytokine profile and cell lineage T cell transcription factor expression in Mtb-specific CD4 T cells and serum cytokine levels in 72 individuals (The aims of the present study were i) to delineate ividuals . The patividuals .Mtb-specific CD4 T-cell responses were assessed by intracellular cytokine staining (ICS) according to the gating strategy shown in Mtb-specific CD4 T cells to produce IFN-γ, TNF-α, IL-2, IL-4, IL-5 and/or IL-13 in response to ESAT-6 and CFP-10 peptide pools stimulation was assessed by multi-parametric flow cytometry in 25 TB patients and 30 Mtb/helminth co-infected patients from TZ and compared to 17 TB patients from SA. Of note, Th2 cytokines i.e. IL-4, IL-5 and IL-13 were all assessed in the same flow cytometry fluorescence channel, which allowed the assessment of total Th2 cytokine production but prevented direct identification of individual IL-4, IL-5 or IL-13 Mtb-specific CD4 T-cell responses. Cytokine profiles of Mtb-specific CD4 T cells from three representative TB patients from SA (#08) and TZ (TB (#60062) and Mtb/helminth co-infected patient (#60031) are shown in Mtb-specific memory CD4 T cells from TB patients from SA versus TB patients from TZ (+ and IL-4/IL-5/IL-13+Mtb-specific memory CD4 T-cell frequencies in TB patients from TZ compared with TB patients from SA (P<0.05), while the frequencies of IFN-γ and TNF-α producing Mtb-specific memory CD4 T cells were not significantly different between TB patients from TZ and from SA   .Mtb-specific memory CD4 T cells of TB patients from SA and TZ   and in IFN-γ-IL-2-TNF-α-IL-4/5/13+ CD4 T-cell populations (single IL-4/5/13 Mtb-specific CD4 T cells) as compared to TB patients from SA   and in IFN-γ+IL-2-TNF-α-IL-4/5/13- CD4 T-cell populations (single IFN-γ Mtb-specific CD4 T cells) as compared to TB patients from TZ  (Mtb-specific CD4 T-cell cytokine profile of TB patients and Mtb/helminth co-infected patients from TZ (P>0.05)  by polychromatic flow cytometry (n = 7). The results obtained showed that SEA-specific CD4 T-cell responses were dominated by single TNF-α and single IL-4/IL-5/IL-13-producing CD4 T cells, while polyfunctional IFN-γ+IL-2+TNF-α+ CD4 T cells represented less than 5% of total SEA-specific CD4 T-cell responses confirming previous observations [We next analyzed the cytokine profile of  P<0.05) . In cont P<0.05) . No sign(P>0.05) . Of notervations , 47  or not  lung migration capacity (Mtb-specific CD4 T cells was compared between TB patients from SA or TZ with evidence of past exposure to helminths versus TB patients with no sign of ongoing or past exposure to helminths (Mtb-specific CD4 T cells from active TB cases was not influenced by 1) polyparasitism  (P>0.05) (To better estimate the influence of 1) different helminth species, 2) infection caused by more than one helminth (polyparasitism), 3) differences in helminth lung migration capacity and 4) past helminth exposure on the generation of Th2  species , or betwcapacity . Similarelminths . The cum(P>0.05) , 2) by h(P>0.05) .Mtb-specific T-cells, multiplex bead array analyses (luminex) were performed on supernatants of ESAT-6/CFP-10 peptide pool (Mtb)-stimulated cell cultures. The cumulative data showed that Mtb-stimulated cell culture supernatants of TB patients from TZ secreted similar levels of IFN-γ, TNF-α and IL-10 (P>0.05), but significantly higher levels of IL-2, IL-4, IL-5, IL-13, IL-17A and IL-17F than Mtb-stimulated cell culture supernatants of TB patients from SA   , by helm(P>0.05) , or by p(P>0.05) .Mtb-specific CD4 T cells from Tanzanian TB patients have a mixed Th1/Th2 cytokine profile which is observed either in patients with active helminth infection or in a large proportion of patients with positive helminth serology. In contrast, Mtb-specific CD4 T cells from South African TB cases have a classical, previously described Th1 cytokine profile.Taken together, our data indicate that a high proportion of Mtb-specific Th2 CD4 T cells was associated with changes in the expression of Th1 and Th2 cell lineage transcription factors, T-bet and Gata-3 respectively [We then determined whether the development of ectively , 49.Mtb/helminth co-infected patients from TZ  compared with TB patients from SA  (high) was significantly lower in TB and Mtb/helminth patients from TZ  as compared to TB patients from SA  (high did not differ between TB patients and Mtb/helminth co-infected patients from TZ (P>0.05) (Mtb/helminth co-infected patients from TZ was not influenced by the helminth species and by polyparasitism (P>0.05)  (Representative examples and cumulative data showed that the percentage of memory CD4 T cells expressing Gata-3 was significantly increased in TB and  P<0.05) . In cont P<0.05) . However(P>0.05) . In addi(P>0.05) , by helm(P>0.05) , or by p(P>0.05) . Interes<0.0001)  thus supMtb-specific CD4 T-cell cytokine profile of TB patients and Mtb/helminth co-infected patients from SA or TZ. To address this issue, we plotted the percentage of Mtb-specific CD4 T cells producing IFN-γ or IL-4/5/13 against the percentage memory CD4 T cells expressing T-bethigh or Gata-3 from the same patients   (We then determined whether the expression of T-bet or Gata-3 by memory CD4 T cells was associated with patients . The cum 0.0085)  and the <0.0088) .high expression.Taken together, these data indicate that TB patients from TZ have a mixed Th1/Th2 cytokine profile associated with increased Gata-3 and reduced T-betMtb-specific CD8 T cells were more frequently detected in patients with TB disease as compared to those with latent Mtb infection [i.e. QuantiFERON TB PLUS proposes optimized CD8-TB-specific-peptides stimulation [Mtb-specific CD8 T cells. To address this issue, the ability of Mtb-specific CD8 T cells to produce IFN-γ, TNF-α, IL-2 and perforin was assessed in 16 TB patients and 23 Mtb/helminth co-infected patients from TZ by flow cytometry. As shown in Mtb-specific CD8 T cells was significantly reduced in Mtb/helminth co-infected patients from TZ as compared to TB patients from TZ    . However P>0.05; . Interes P>0.05) .Taken together, these data suggest that TB patients from SA and TZ showed differences in serum cytokine profile. In comparison to Tanzanian TB cases, South African patients showed a more pronounced pro-inflammatory serum cytokine profile associated with high levels of type I IFNs.Mtb-specific immune signature in TB patients and the influence of ongoing helminth infection, a principal component analysis (PCA) was performed. The results indicated that the Mtb-specific immune profile of TB and Mtb/helminth co-infected patients from TZ clustered away from that of TB patients from SA with a percentage of discrimination reaching about 70%  higher proportion of Mtb-specific Th1 cells , iii) higher serum levels of pro-inflammatory cytokines (IL-1α and TNF-α), IL-12p70 and IL-23 and negatively correlated with a) Gata-3 expression on memory CD4 T cells, b) higher proportion of polyfunctional Mtb-specific CD4 T cells and c) type 2 cytokine secretion (IL-5 and IL-13) (P<0.05)  .Mtb-specific immune signatures of TB patients from SA and TZ are significantly different and that Th1 cytokine profiles are positively associated with TB-induced systemic inflammation and higher serum levels of type I IFNs.Taken together, the data indicate that the serum cytokine profile and the Mtb in the sputum of treated individuals based on sputum smear microscopy. The cumulative data showed a significant reduction in the proportion of patients with detectable Mtb in the sputum following 60 days of drug treatment   , suggesti.e. IL-1α, IL-6, and IL-1β   and significant decrease of serum levels of CRP and pro-inflammatory cytokines  P<0.05; , irrespe P>0.05) .Mtb-specific T-cell immunity in TB and Mtb/helminth patients. The functional profiles of Mtb-specific CD4 T-cell responses and the transcription factor expression profiles were assessed using aforementioned experimental strategies. The cumulative data indicated that the initiation of efficient TB treatment did not significantly influence 1) the Mtb-specific CD4 T-cell functional profiles (P>0.05) (i.e. IL-4 and/or IL-5 and/or IL-13 (P>0.05) (Mtb/helminth infected patients from TZ (P>0.05)  , 2) the (P>0.05)  and 3) t(P>0.05) . Indeed, T cells .Mtb-specific CD4 T cells. These data provide evidence that TB treatment outcome during this follow up period was not influenced by presence or absence of ongoing helminth infection.In summary, two months of TB treatment did not change the functional profile of Mtb are co-endemic in several regions of the world, including Tanzania [Mtb-specific T-cell responses. Our hypothesis is founded on the observations that helminths stimulate Th2 type immune responses and previous studies that demonstrated that the cytokine micro-environment may influence, at least in vitro, the functional profile of antigen-specific CD4 T cell responses [Helminth infections have been shown to impact the control of virus replication in mouse models , 56 and Tanzania , we hypoesponses , 60.Mtb-specific CD4 T cells in patients with active TB disease from TZ have a Th2 cytokine profile as indicated by the production of IL-4/IL-5/IL-13 and expression of the Th2 cell lineage transcription factor Gata-3. Mtb-specific CD4 T-cells comprised Th1 and Th2 cells with a polyfunctional cytokine profile, and the increased frequency of Gata-3+ memory CD4 T cells was associated with reduced frequency of T-bethigh+ memory CD4 T cells. Interestingly, Mtb-specific CD4 T cells in patients with active TB disease from SA had a typical Th1 profile encompassing single IFN-γ and dual IFN-γ/TNF-α cells. Taken together, these results demonstrate that active TB disease induced the generation of mixed Th1/Th2 Mtb-specific CD4 T cells in patients from TZ whereas a typical Th1 Mtb-specific CD4 T cell response was generated in patients from SA. Further investigations would be needed to determine whether TB-uninfected individuals from TZ and SA harbor different basic response to TB antigens.In the present study, we provide evidence that a significant proportion of Mtb-specific CD4 T-cell cytokine profile is consistently proposed to allow the discrimination between active and latent Mtb infections [Mtb-specific CD4 T cells (in association with Th1 cytokines i.e IFN-γ and/or TNF-α) are associated with individuals with LTBI and therefore Mtb containment, while high proportion of Mtb-specific CD4 T cells producing Th1 cytokines in absence of IL-2 is associated with patients suffering from active TB disease [These observations are of high interest since the assessment of fections –64. Amon disease –65. Inde disease , 67. Int disease , 69 and  disease , 70.Mtb-specific CD4 T cells. In this context, about 67% of patients from TZ either had an ongoing active helminth infection or had evidence of previous helminth exposure/infection. However, about 11% of the patients from SA also showed a positive serology for previous helminth exposure/infection with helminth but no sign of generation of Mtb-specific Th2 CD4 T cells. Despite this discordance between positive serology for helminth exposure and lack of the generation of Mtb-specific Th2 CD4 T cells in patients from SA, we cannot exclude that the frequency and/or severity of helminth re-infection/exposure in patients from TZ is higher as when compared to South African patients and therefore responsible for the mixed Th1/Th2 functional profile of Mtb-specific CD4 T cells [Mtb genetic diversity and other yet unknown environmental factors may contribute to the generation of mixed Th1/Th2 functional cytokine profile and/or influence the durability of the Th2 response after parasite infection is cleared [As mentioned above, the study hypothesis was that helminth infection might influence the functional profile of  T cells , 72. In  cleared  in patieMtb-specific CD8 T cell responses detected by polychromatic flow cytometry. Interestingly, this proportion was significantly reduced (about two fold) in the presence of ongoing helminth infections. These results provide additional evidence that helminth infections may significantly influence the generation of distinct T cell subsets in active TB disease. This important finding echoes with recent data demonstrating that ongoing helminth infection impairs virus-specific T-cell immunity via a STAT-6-dependent alternative activation of macrophages differentiation [Consistent with previous studies , 74, abontiation .Mtb-specific CD4 T cells lacking IL-2 co-production. These IFN-β serum levels were inversely correlated with polyfunctional Mtb-specific CD4 T cells and Th2 cytokines detected by polychromatic flow cytometry and in cell culture supernatants. Based on these findings, the so far unappreciated role of IFN-β  in coordinating TB specific immunity needs to be further explored.In contrast to Tanzanian TB cases, the South African TB patients were characterized by significantly higher serum levels of pro-inflammatory cytokines (IL-1α and TNF-α) in combination with IL-12p70 and type I IFNs. Interestingly, statistical analyses revealed that elevated serum levels of IFN-β were associated with elevated serum levels of pro-inflammatory cytokines and Th1 Mtb infection and the progression of TB disease is under intense debate. It has been clearly demonstrated that functional impairment of the IL-12p70/IFN-γ axis predisposes to the development of mycobacterial disease [Mtb-specific CD8 T cells is however still controversial. Some studies indicate that Mtb-specific CD8 T cells may play an important role in protective immunity against TB via the production of perforin and/or cytolysin [Mtb-specific CD8 T cells may be detrimental [Mtb-specific CD8 T cells were enriched in TB patients as compared to individuals with LTBI [Mtb transmission via inflammation mediated mucosal damage enabling access of Mtb bacilli to airways [The role of Th1, Th2 and CD8 T cells in the control of  disease , probabl disease . The rolytolysin –80, whilrimental , since Mith LTBI . Excessi airways .Mtb-induced type I IFNs [Mtb-induced type I IFN might be detrimental, since TB patients with reduced/absent type I IFN signature had reduced bacterial load and/or improved host survival [The dual role of IFN-γ in the protection or progression of TB disease may be linked to macrophage hypo-responsiveness to IFN-γ , also cae I IFNs , 84. Indsurvival –87. Intesurvival , enhancesurvival  and intesurvival , 90, 91,Mtb strains isolated from TZ and SA, the antigen load and the genetic background of the individuals living in TZ versus SA might be associated with this profile, since the level of Mtb-induced type I IFN production might be strain dependent [The reason why TB patients from TZ had lower levels of type I IFNs remains unclear and needs to be further investigated. However, one could postulate that the genetic background of the ependent . Of noteMtb-specific CD4 T-cell cytokine and transcription factor expression profile but strongly reduced CRP serum levels and pro-inflammatory cytokine circulation in combination with improved BMI.Finally, we did not observe any influence of ongoing helminth infection on the efficacy of TB therapy. Interestingly, after sixty days of treatment, we did not observe changes in Mtb-specific CD4 and CD8 T cell responses, may be substantially influenced by co-infectious agents and possibly genetic and environmental factors resulting in pronounced variations in the qualitative and quantitative profile of pathogen-specific responding T cells in human populations.In conclusion, we provide evidence that the generation of Ascaris lumbricoides, Trichuris trichiura) and S. mansoni infections using the Kato-Katz thick smear and FLOTAC methods performed on one stool sample at date of blood sample collection, for active S. stercoralis infection using the Baermann technique and for active S. haematobium infection using the urine filtration method. Binax NOW ICT test card were used to detect W. Bancrofti antigen on blood sample [Echinococcus spp, Fasciola hepatica, Filaria, Schistosoma spp, S. stercoralis, Toxocara spp and Trichinella spp) was performed by ELISA. The serodiagnostic helminth screening ELISA is routinely performed at the diagnostic centre of the Swiss Tropical and Public Health Institute and detects helminth specific IgG. A total of 30 Mtb/helminth co-infected patients were recruited. TB patient co-infected with only one helminth species were infected with S. mansoni (n = 7), W. bancrofti (n = 6), hookworms (n = 2), S. haematobium (n = 2) or S. stercoralis (n = 2). Eleven TB patients (36%) were co-infected with multiple helminth species. Blood samples were collected prior to and following 60 days of anti-mycobacterial treatment ) from 20 TB and 24 Mtb/helminth co-infected patients recruited in TZ. In addition, seventeen subjects were enrolled at the field site of the South African Tuberculosis Vaccine Initiative in the Boland Overberg region of the Western Cape Province of SA (SA). TB disease was diagnosed by positive sputum Xpert MTB/RIF and HIV infection was ruled out by rapid serological test. PBMCs were collected as part of a cross-sectional study, in HIV-negative participants before commencing treatment for TB. Diagnosis of helminth exposure was performed using ELISA detecting helminth-specific IgG .In total, 72 subjects were recruited to participate in this study. No statistical method was used to predetermine sample size. Fifty-five subjects were recruited at the Mwananyamala Hospital, Dar es Salaam, and the TB clinic of Bagamoyo (TZ). TB patients (n = 55) were selected based on sputum smear microscopy confirmed by GeneXpert assay and HIV infection was ruled out by rapid serological test (Alere Determine HIV-1/2 test). The diagnosis of ongoing and/or past helminth infection was based on assays performed on feces , urine (urine filtration), whole blood (Immuno-chromatography) and serum (ELISA) samples at date of blood sample collection and the assay used depended on the helminth species and on the site of sample collection. All TB patients from TZ were screened for active soil-transmitted helminths , the Ifakara Health Institute Institutional Review Board and the National Institute for Medical Research . For SA, the Human Research Ethics Committee of the University of Cape Town granted the study protocol approval.The following monoclonal antibodies (mAbs) were used in different combinations. CD3-APC-H7 (CloneSK7), CD4-PECF594 or CD4-APC (Clone RPA-T4), CD8-PB (Clone RPA-T8), IFN-γ-AF700 or IFN-γ-APC (Clone B27), TNF-α-PeCy-7 (Clone MAb11), IL-4-PE (Clone 3010.211), IL-2-PE (Clone MQ1-17H12), Gata-3-PeCy-7 (Clone L50-823), all from Becton Dickinson (BD); CD45RA-BV711 (Clone HI100), IL-2-PerCpCy5.5 (Clone MQ1-17H12), IL-5-PE (Clone TRFK5), IL-13-PE (Clone JES10-5A2), T-bet-PerCpCy5.5 (Clone 4B10) were purchased from BioLegend; CD8-Efluor625NC (Clone RPA-T8) from eBioscience; perforin-FITC (Clone B-D48) from Diaclone.Mtb-derived CFP-10 and ESAT-6 peptide pools are composed of 15-mers overlapping by 11 amino-acids encompassing the entire sequences of the proteins and all peptides were HPLC purified (>90% purity).Staphyloccocus enterotoxin B  or unstimulated in the presence of Golgiplug  as previously described [PBMCs were stimulated overnight in complete media (RPMI (Invitrogen), 10% fetal calf serum , 100 µg/ml penicillin, 100 unit/ml streptomycin (BioConcept)) with ESAT-6 and CFP-10 peptide pools (1 µg/ml) or with escribed . At the Staphyloccocus enterotoxin B  or unstimulated in the presence of Golgiplug . At the end of the stimulation period, cells were washed and stained  for dead cells using the Aqua LIVE/DEAD stain kit, then permeabilized   and stained  with mAbs to CD3, CD4, CD8, TNF-α, IFN-γ, IL-2 and perforin.PBMCs were stimulated overnight in complete media (RPMI (Invitrogen), 10% fetal calf serum , 100 µg/ml penicillin, 100 unit/ml streptomycin (BioConcept)) with ESAT-6 and CFP-10 peptide pools (1 µg/ml) or PBMCs were washed, stained  for dead cells using the Aqua LIVE/DEAD stain kit, then washed and stained  for CD3, CD4, CD8, CD45RA. Cells were then washed, permeabilized   and stained  with mAbs to T-bet and Gata-3.5 cells) were stimulated for 24 hours in complete media (RPMI (Invitrogen), 10% fetal calf serum , 100 µg/ml penicillin, 100 unit/ml streptomycin (BioConcept)) with ESAT-6 and CFP-10 peptide pools (1 µg/ml) or with Staphyloccocus enterotoxin B  or left unstimulated (negative control). At the end of the stimulation period, culture supernatants were collected and levels of TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-13, IL-10, IL-17A and IL-17F were assessed cells by luminex assay .PBMCs  and CRP was assessed by nephelemetry  as previously described .Mtb-specific T cells and cytokine profile of Mtb-specific T-cell responses were analyzed using SPICE software (version 5.34) following background subtraction. When required, analysis and presentation of distributions was performed using SPICE, downloaded from <http://exon.niaid.nih.gov/spice> [Cells were fixed with CellFix (BD), acquired on an LSRII SORP  and analyzed using FlowJo (version 9.7.7) . Frequencies of cytokine-producing v/spice> .P values) was obtained either using two-tailed Chi-square analysis for comparison of positive proportions or using one-way ANOVA  followed by Mann–Whitney test or Wilcoxon Matched-pairs two-tailed Signed Rank test for multiple comparisons or Spearman rank test for correlations using GraphPad Prism version 7 . Statistical analyses of global cytokine profiles (pie charts) were performed by partial permutation tests using the SPICE software as described [Statistical significance Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigProportion of SEA-specific CD4 T-cell responses producing IFN-γ, IL-4/5/13, TNF-α and/or IL-2 of TB patients from TZ (n = 7). All the possible combinations of the responses are shown on the x axis and the percentage of the functionally distinct cell populations within the SEA-specific CD4 T-cell populations are shown on the y axis. Responses are grouped and color-coded on the basis of the number of functions. The pie chart summarizes the data, and each slice corresponds to the fraction of SEA-specific CD4 T cell response with a given number of functions within the responding CD4 T-cell population. Bars correspond to the fractions of different functionally distinct CD4 T-cell populations within the total CD4 T cells. Red arcs correspond to IL-4/5/13-producing CD4 T-cell populations.(PDF)Click here for additional data file.S4 FigA) Proportion of Mtb-specific CD4 T cells producing IL-4/5/13 among total Mtb-specific CD4 T-cell responses assessed in TB patients from TZ infected with one helminth (n = 19) as compared to TB patients from TZ infected with more than one helminth (n = 11) or (B) between TB patients from TZ infected with helminth species harbouring  or not  lung migration capacity. (C) Proportion of Mtb-specific CD4 T cells producing IL-4/5/13 among total Mtb-specific CD4 T-cell responses assessed in TB patients from SA with (n = 2) or without past helminth exposure (n = 15). (D) Proportion of Mtb-specific CD4 T cells producing IL-4/5/13 among total Mtb-specific CD4 T-cell responses assessed in TB patients from TZ without ongoing helminth infection but with (n = 7) or without past helminth exposure (n = 18). (E) Levels of IL-4, IL-5 and IL-13 secreted in Mtb-stimulated culture supernatants in TB patients from TZ infected with one helminth (n = 19) and TB patients from TZ infected with more than one helminth (n = 10) or (F) between TB patients from TZ infected with helminth species harbouring or not lung migration capacity. (G) Levels of IL-4, IL-5 and IL-13 secreted in Mtb-stimulated culture supernatants in TB patients from SA with (n = 2) or without past helminth exposure (n = 10). (H) Levels of IL-4, IL-5 and IL-13 secreted in Mtb-stimulated culture supernatants in TB patients from TZ without ongoing helminth infection but with (n = 6) or without past helminth exposure (n = 15). (I) Percentage of memory CD4 T cells (CD45RA-) expressing Gata-3 of TB patients from TZ infected with one helminth (n = 8) and TB patients from TZ infected with more than one helminth (n = 5) or (J) between TB patients from TZ infected with helminth species harbouring or not lung migration capacity. (K) Percentage of memory CD4 T cells (CD45RA-) expressing Gata-3 of TB patients from SA with (n = 2) or without past helminth exposure (n = 10). (L) Percentage of memory CD4 T cells (CD45RA-) expressing Gata-3 of TB patients from TZ without ongoing helminth infection but with (n = 6) or without past helminth exposure (n = 8). Helminth species were color coded ; W. bancrofti, orange; hookworms, green; S. mansoni, red, S. haematobium, yellow, S. stercolaris, violet, and patients infected with more than one helminth species in light blue. Statistical significance (P<0.05) was calculated by Mann-Whitney test.((PDF)Click here for additional data file.S5 Fighigh and the percentage of memory CD4 T cell expressing Gata-3 in TB patients from SA (n = 12), TB patient (n = 14) and Mtb/helminth co-infected patients (n = 13) from TZ. TB patients were color coded; TB patients from SA, blue; TB patients from TZ, red and Mtb/helminth co-infected patients, green. Statistical significance (P<0.05) was calculated using Spearman rank test.(A) Correlation between the percentage of memory CD4 T cell expressing Tbet(PDF)Click here for additional data file.S6 FigA) Levels of IL-4, IL-5 and IL-13 produced in Mtb-stimulated culture supernatants of TB (n = 13) and Mtb/helminth co-infected patients from TZ (n = 18) assessed by luminex assay. (B) Percentage of memory (CD45RA-) CD4 T cells expressing Gata-3 or T-bethigh of TB (n = 11) and TB/helminth co-infected patients (n = 8) from TZ. TB patients were color coded; TB patients, red and Mtb/helminth co-infected patients, green.((PDF)Click here for additional data file.