Haso W.,U.S. National Cancer Institute |
Lee D.W.,U.S. National Cancer Institute |
Shah N.N.,U.S. National Cancer Institute |
Stetler-Stevenson M.,U.S. National Institutes of Health |
And 10 more authors.
Blood | Year: 2013
Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3l constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL. © 2013 by The American Society of Hematology. Source
Reed P.E.,Wildlife Conservation Society |
Mulangu S.,U.S. National Institutes of Health |
Cameron K.N.,Wildlife Conservation Society |
Ondzie A.U.,Wildlife Conservation Society |
And 12 more authors.
PLoS Neglected Tropical Diseases | Year: 2014
Central Africa is a “hotspot” for emerging infectious diseases (EIDs) of global and local importance, and a current outbreak of ebolavirus is affecting multiple countries simultaneously. Ebolavirus is suspected to have caused recent declines in resident great apes. While ebolavirus vaccines have been proposed as an intervention to protect apes, their effectiveness would be improved if we could diagnostically confirm Ebola virus disease (EVD) as the cause of die-offs, establish ebolavirus geographical distribution, identify immunologically naïve populations, and determine whether apes survive virus exposure.Here we report the first successful noninvasive detection of antibodies against Ebola virus (EBOV) from wild ape feces. Using this method, we have been able to identify gorillas with antibodies to EBOV with an overall prevalence rate reaching 10% on average, demonstrating that EBOV exposure or infection is not uniformly lethal in this species. Furthermore, evidence of antibodies was identified in gorillas thought previously to be unexposed to EBOV (protected from exposure by rivers as topological barriers of transmission).Our new approach will contribute to a strategy to protect apes from future EBOV infections by early detection of increased incidence of exposure, by identifying immunologically naïve at-risk populations as potential targets for vaccination, and by providing a means to track vaccine efficacy if such intervention is deemed appropriate. Finally, since human EVD is linked to contact with infected wildlife carcasses, efforts aimed at identifying great ape outbreaks could have a profound impact on public health in local communities, where EBOV causes case-fatality rates of up to 88%. © 2014. Source
David V.A.,Frederick National Laboratory for Cancer Research |
Menotti-Raymond M.,Frederick National Laboratory for Cancer Research |
Wallace A.C.,Frederick National Laboratory for Cancer Research |
Roelke M.,Frederick National Laboratory for Cancer Research |
And 9 more authors.
G3 (Bethesda, Md.) | Year: 2014
The Dominant White locus (W) in the domestic cat demonstrates pleiotropic effects exhibiting complete penetrance for absence of coat pigmentation and incomplete penetrance for deafness and iris hypopigmentation. We performed linkage analysis using a pedigree segregating White to identify KIT (Chr. B1) as the feline W locus. Segregation and sequence analysis of the KIT gene in two pedigrees (P1 and P2) revealed the remarkable retrotransposition and evolution of a feline endogenous retrovirus (FERV1) as responsible for two distinct phenotypes of the W locus, Dominant White, and white spotting. A full-length (7125 bp) FERV1 element is associated with white spotting, whereas a FERV1 long terminal repeat (LTR) is associated with all Dominant White individuals. For purposes of statistical analysis, the alternatives of wild-type sequence, FERV1 element, and LTR-only define a triallelic marker. Taking into account pedigree relationships, deafness is genetically linked and associated with this marker; estimated P values for association are in the range of 0.007 to 0.10. The retrotransposition interrupts a DNAase I hypersensitive site in KIT intron 1 that is highly conserved across mammals and was previously demonstrated to regulate temporal and tissue-specific expression of KIT in murine hematopoietic and melanocytic cells. A large-population genetic survey of cats (n = 270), representing 30 cat breeds, supports our findings and demonstrates statistical significance of the FERV1 LTR and full-length element with Dominant White/blue iris (P < 0.0001) and white spotting (P < 0.0001), respectively. Copyright © 2014 David et al. Source
The cell lines used in this study were provided as follows: human breast cancer MDA-MB-231 organotropic lines 4175, 1833 and 831 by J. Massagué; human breast cancer 4173 and 4180 cells by A. Minn; human breast cancer 231BR cells by P. Steeg; liver metastasis enriched uveal melanoma cells by V. Rajasekhar; human osteosarcoma 143B cells by A. Narendran; human melanoma 131/4-5B2 and 131/8-2L cells by R. Kerbel; human melanoma SB1B cells by C. F. Verschraegen; human rhabdomyosarcoma CT10 and RD cells by from R. Gladdy; and human Wilms tumour CCG9911 and CLS1 cells by A. Ketsis. Human breast cancer cell lines MDA-MB-231 and MDA-MB-468, human breast epithelial cells MCF10A, human pancreatic cancer cell lines, gastric cancer cell lines and colorectal cancer cell lines were purchased from American Type Culture Collection (ATCC). Although HT29 is commonly misidentified, we purchased this cell line directly from ATCC and the cell line was certified by this repository, therefore we are confident that it is indeed a colon cancer cell line. The C57BL/6 mouse pancreatic adenocarcinoma Pan02 was purchased from the National Cancer Institute Tumour Repository (DTP/DCTD, Frederick National Laboratory for Cancer Research). For in vitro education of human lung fibroblasts WI-38 (ATCC), human bronchial epithelial cells HBEpC (PromoCell), and human Kupffer cells (Life Technologies), cells were maintained in culture for 14 days, with media containing 0, 5 or 10 μg ml−1 of exosomes, replenished every other day. Kupffer cells were cultured in RPMI and WI-38 cells were cultured in alpha-MEM, both supplemented with 10% exosome-depleted FBS (Gibco, Thermo Fisher Scientific) and penicillin-streptomycin. HBEpC cells were cultured in airway epithelial cell growth medium (PromoCell). All cells were maintained in a humidified incubator with 5% CO at 37 °C. FBS was depleted of bovine exosomes by ultracentrifugation at 100,000g for 70 min. All cells lines were routinely tested for mycoplasma and were found to be negative. Exosomes were purified by sequential centrifugation as previously described46. In brief, cells were removed from 3–4-day cell culture supernatant by centrifugation at 500g for 10 min to remove any cell contamination. To remove any possible apoptotic bodies and large cell debris, the supernatants were then spun at 12,000g for 20 min. Finally, exosomes were collected by spinning at 100,000g for 70 min. Exosomes were washed in 20 ml PBS and pelleted again by ultracentrifugation (Beckman 70Ti rotor). Exosome preparations were verified by electron microscopy. Exosome size and particle number were analysed using the LM10 or DS500 nanoparticle characterization system (NanoSight, Malvern Instruments) equipped with a blue laser (405 nm). Normal mammary fat pad tissue-derived exosomes were obtained by culturing five mammary fat pads isolated from healthy 4–6-week-old C57BL/6 mice in 3 ml of FBS-free RPMI for 12 h. The final exosome pellet was resuspended in PBS and protein concentration was measured by BCA (Pierce, Thermo Fisher Scientific). Mass spectrometry analyses of exosomes were performed at the Rockefeller University Proteomics Resource Center using 20 μg of exosomal protein. Samples were denatured using 8 M urea, reduced using 10 mM dithiothreitol (DTT), and alkylated using 100 mM iodoacetamide. This was followed by proteolytic digestion with endoproteinase LysC (Wako Chemicals) overnight at room temperature, and subsequent digestion with trypsin (Promega) for 5 h at 37 °C. The digestion was quenched with formic acid and resulting peptide mixtures were desalted using in-house made C18 Empore (3M) StAGE tips47. Samples were dried and solubilized in the sample loading buffer containing 2% acetonitrile and 2% formic acid. Approximately 3–5 μg of each sample was analysed by reversed phase nano-liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Ultimate 3000 coupled to QExactive, Thermo Scientific). After loading onto the C18 trap column (5 μm beads, Thermo Scientific) at a flow rate of 3 μl min−1, peptides were separated using a 75-μm inner diameter C18 column (3 μm beads, Nikkyo Technos Co.) at a flow rate of 200 nl min−1, with a gradient increasing from 5% buffer B (0.1% formic acid in acetonitrile)/95% buffer A (0.1% formic acid) to 40% buffer B/60% buffer A, over 140 min. All LC–MS/MS experiments were performed in data-dependent mode. Precursor mass spectra were recorded in a 300–1,400 m/z mass range at 70,000 resolution, and 17,500 resolution for fragment ions (lowest mass: m/z 100). Data were recorded in profile mode. Up to 20 precursors per cycle were selected for fragmentation and dynamic exclusion was set to 45 s. Normalized collision energy was set to 27. MS/MS spectra were extracted and searched against Uniprot complete human or mouse proteome databases (January 2013) concatenated with common contaminants48 using Proteome Discoverer 1.4 (Thermo Scientific) and Mascot 2.4 (Matrix Science). All cysteines were considered alkylated with acetamide. N-terminal glutamate to pyroglutamate conversion, oxidation of methionine, and protein N-terminal acetylation were allowed as variable modifications. Data were first searched using fully tryptic constraints. Matched peptides were filtered using a Percolator49 based 1% false discovery rate (FDR). Spectra not being matched at a FDR of 1% or better were re-searched allowing for semi-tryptic peptides. The average area of the three most abundant peptides for a matched protein50 was used to gauge protein amounts within and between samples. LC–MS/MS data from three technical replicates of six organ-tropic samples were analysed using MaxQuant (version 126.96.36.199) and Perseus software (version 188.8.131.52)51, searching against a Uniprot human database (July 2014). Oxidation of methionine and protein N-terminal acetylation were allowed as variable modifications, and cysteine carbamidomethyl was set as a fixed modification. Two missed cleavages were allowed for specificity: trypsin/P. The ‘match between runs’ option was enabled. FDR values at the protein and peptide level were set to 1%. Protein abundance is expressed as LFQ values. Only proteins quantified in at least two out of three replicates in at least one group were retained, and missing values were imputed. A multiple sample ANOVA test was performed and corrected for multiple hypotheses testing using a permutation-based FDR threshold of 0.05. To assess lung, liver and bone exosome distribution, exosomes were injected via the retro-orbital venous sinus, the tail vein or intracardially. Exosome distribution patterns were consistent regardless of the route of injection. For brain distribution, exosomes were only observed in the brain after intracardiac injection. For 24-h exosome treatments, 10 μg of total exosomal protein were injected via the retro-orbital venous sinus, the tail vein, or intracardially in a total volume of 100 μl PBS. For exosome-tracking purposes, purified exosomes were fluorescently labelled using PKH67 (green) or PKH26 (red) membrane dye (Sigma-Aldrich) or FM1-43FX dye (Life Technologies) for the photo-conversion experiment. Labelled exosomes were washed in 20 ml of PBS, collected by ultracentrifugation and resuspended in PBS. When performing peptide blocking experiments, exosomes were incubated with 0.06 μM RGD or HYD-1 (peptide sequence: KIKMVISWKG) peptides for 30 min at 37 °C before exosome injection. An average of five random fields was counted per sample at 20× magnification, and representative pictures were taken at 40× magnification. For education experiments, mice received 10 μg of exosomes retro-orbitally every other day for 3 weeks. To measure exosome uptake by specific cell types, labelled exosomes were injected 24 h before tissue collection and tissues were analysed for exosome-positive cells by immunofluorescence. Pictures were taken at 60 × magnification. For in vitro uptake assays, the membrane of WI-38 cells was labelled with PKH67 dye while 4175-LuT exosomes were labelled with PKH26 dye. Exosomes (10 μg ml−1) were first incubated with PBS or HYD-1 peptide for 30 min at 37 °C, followed by an incubation for 1 h with WI-38 cells at 37 °C. Excess exosomes were washed off and pictures were taken by Nikon confocal microscope (Eclipse TE2000U). The amount of exosomes localizing to the lung was analysed by immunofluorescence or using the Odyssey imaging system (LI-COR Biosciences). In brief, NIR dye-labelled exosomes were injected 24 h before tissue collection and tissues were analysed for exosome-positive areas. Whole-lung images were analysed using image J software, quantifying red fluorescence area in arbitrary units. Cryostat sections prepared at a 15-μm thickness were placed on glass slides and re-fixed in 0.075 M sodium cacodylate, pH 7.4, containing 2.5% glutaraldehyde. For photoconversion, slides were washed twice in 0.1 M sodium cacodylate buffer, pH 7.4. Autofluorescence was quenched using 100 mM NH Cl in cacodylate buffer for 45 min. On the basis of optimization experiments, sections were photoconverted for 2 h by incubation in 5.4 mg ml−1 3,3′-diaminobenzidine in 0.1 M sodium cacodylate buffer, pH 7.4, and exposure to the light of an Intensilight C-HGFI 130-W mercury lamp and a 4×/0.1 NA objective (Nikon Inverted Microscope Eclipse Ti). For electron microscopy processing, sections were post-fixed in 1% osmium tetroxide buffer for 15 min on ice. After washing with water, slides were placed in 1% aqueous uranyl acetate for 30 min. Sections were washed with water, dehydrated in a graded series of ethanol concentrations and subsequently in acetone for 10 min at room temperature. Samples were embedded in Eponate. Serial sections were cut at 70 nm in thickness and transferred to formvar-coated slot grids and imaged on a JEOL 100CX at 80 kV with an AMT XR41 digital imaging system. Cell lines were analysed for specific genes using pre-designed TaqMan assays (Applied Biosystems). In brief, RNA was extracted from tissues or cells using the RNeasy kit (Qiagen), and reverse transcribed using Superscript Vilo (Life Technologies). qRT–PCR was performed on a 7500 Fast Real Time PCR System (Applied Biosystems), using TaqMan Universal PCR Master Mix (Applied Biosystems). Relative expression was normalized to β-actin levels. For shRNA-mediated knockdown of ITGβ and ITGβ , specific interfering lentiviral vectors containing GFP reporter and puromycin resistance gene cassettes were used. In brief, oligonucleotide 5′-CCGGGAGGGTGTCATCACCATTGAACTCGAGTTCAATGGTGATGACACCCTCTTTTTG-3′ targeting the 5′-GAGGGTGTCATCACCATTGAA-3′ sequence in the human ITGB4 gene (EntrezGene ID: 3691) or oligonucleotide 5′-CCGGAGCTTGTTGTCCCAATGAAATCTCGAGATTTCATTGGGACAACAAGCTTTTTTG-3′ targeting the 5′-AGCTTGTTGTCCCAATGAAAT-3′ sequence in the human ITGB5 gene (EntrezGene ID: 3693) were cloned into the pLKO.1 vector. As a control, we used the empty pLKO.1 vector. For retrovirus production for integrin overexpression, the pWZL and pBabe vectors systems were used. pWZL-hygro-ITGB and pBabe-puro-ITGB were provided by F. Giancotti. Lentiviral and retroviral particles were packaged using 293T cells. Infected target cells were selected using 500 μg ml−1 hygromycin B or 2 μg ml−1 puromycin (Invitrogen). Bone marrow was prepared for flow cytometry as previously described1. For analysis of lung, tissues were minced and then digested at 37 °C for 20 min with an enzyme cocktail (collagenase A, dispase and DNaseI, Roche Applied Science). Single-cell suspensions were prepared by filtering through a 70-μm strainer and passing through an 18G syringe. Lung fibroblasts were identified by flow cytometry using an anti-mouse rabbit polyclonal S100A4 (1:50, Abcam; ab27957), or SPC (1:100, Santa Cruz; FL-197), revealed by Alexa Fluor 568-conjugated goat anti-rabbit secondary (A-11011, Life Technologies, 1:400). For liver, tissues were mechanically dissociated, and single-cell suspensions were filtered through a 40-μm strainer. Allophycocyanin-conjugated F4/80 (1:100, eBioscience; clone BM8) was used to identify liver macrophages by flow cytometry. Cell fluorescence indicating fluorescently labelled exosome uptake was analysed using a FACSCalibur or a FACSCanto (Beckton Dickinson). FACS data was analysed with FlowJo software (TreeStar Inc.). Twenty-thousand cells were plated in 24-well transwell plates with inserts (8-μm pore size, Corning) and were incubated at 37 °C for 6 h. Cell inserts were fixed with 4% paraformaldehyde (PFA) for 10 min, followed by PBS wash and haematoxylin staining to allow visualization and counting. Nine random fields were counted per well at 20× magnification and the average number of migrated cells per field was calculated. Human peripheral blood samples were obtained from control healthy subjects and cancer patients with lung or liver metastasis, or from patients without distant metastasis at Weill Cornell Medical College, University Medical Center Hamburg-Eppendorf, Oslo University Hospital, Memorial Sloan Kettering Cancer Center and University of Nebraska Medical Center, all pathologically confirmed. All individuals provided informed consent for blood donation on approved institutional protocols (WCMC IRB 0604008488 (DL), MSKCC IRB 12-137A (JB)). Plasma or serum exosomes were isolated as previously described1. ITGβ and ITGα levels in exosomes were measured by ELISA (ABIN417641 and ABIN417609 from Antibodies Online, and LS-F7188 from LifeSpan Biosciences), using 2 μg of exosomes per 100 μl of sample diluent, in duplicate reactions, according to the manufacturer’s instructions. All mouse work was performed in accordance with institutional, IACUC and AAALAS guidelines, by the animal protocol 0709-666A. All animals were monitored for abnormal tissue growth or ill effects according to AAALAS guidelines and euthanized if excessive deterioration of animal health was observed. No statistical method was used to pre-determine sample size. No method of randomization was used to allocate animals to experimental groups. The investigators were not blinded to allocation during experiments and outcome assessment. Mice that died before the predetermined end of the experiment were excluded from the analysis. In none of the experiments did tumours exceed the maximum volume allowed according to our IACUC protocol, specifically 2 cm3. For exosome localization, education and tumour implantation experiments for mouse cell lines, 6-week-old C57BL/6 Mus musculus females purchased from Jackson labs were used. For exosome localization, education and tumour implantation experiments for human cell lines, 6–8-week-old NCr nude (NCRNU-F sp./sp.) females purchased from Taconic were used. For lung metastasis studies using organotropic lines, 6–8-week-old nude female mice were pre-educated with exosomes for 3 weeks followed by tail vein injection of 2 × 105 or intracardiac injection of 1 × 105 luciferase-positive cancer cells resuspended in 100 μl PBS. Four weeks after intracardiac injection and eight weeks after tail vein injection, lung metastasis was measured using the IVIS 200 bioluminescence imaging system (Xenogen, Caliper Life Sciences), and tissues were cut in 6-μm sections and stained with haematoxylin and eosin for histology. To analyse the role of exosome education in tumour metastasis, 6–8-week-old C57BL/6 female mice pre-educated with pancreatic cancer-derived exosomes were injected intrasplenically with 1 × 106 Pan02 mCherry cells resuspended in 30 μl of Matrigel (Corning). One or twenty-one days later, mice were euthanized, and livers were analysed for metastatic lesions by measuring liver weight. To follow the levels of tumour-derived exosomes in plasma of tumour-bearing mice, 1 × 106 4175 lung-tropic cells were injected in the mammary fat pad of nude mice. Mouse blood (250 μl) was drawn from the retro-orbital sinus when tumour size was over 800 mm3, followed by tumour resection. One week after the tumour was resected, mice were analysed by bioluminescence IVIS imaging for luciferase activity and separated into two groups: recurrence/tumour-free and recurrent tumours. Mouse blood was drawn and the plasma of mice within the same group was pooled for exosome isolation. Western blot analysis with anti-human ITGβ antibodies was used to detect tumour-derived exosomes. To assess exosome-induced vascular leakiness, 10 μg of total exosome protein were injected by retro-orbital injection. Then 20 h after exosome treatment, mice were injected with 2 mg of Texas Red-lysine fixable dextran 70,000 MW (Invitrogen) via retro-orbital injection. One hour after dextran injection, mice were euthanized and perfused with PBS. Lungs were dissected and fixed in a mix of 2% PFA and 20% sucrose overnight, then embedded in Tissue-tek O.C.T. embedding compound (Electron Microscopy Sciences) and frozen in a dry-ice/ethanol bath. O.C.T. blocks were sectioned and stained for DAPI, pictures were taken using a Nikon confocal microscope (Eclipse TE2000U). Images were analysed using image J software, quantifying red fluorescence area in arbitrary units. For histological analysis, tissues were dissected and fixed in a mix of 2% PFA and 20% sucrose in PBS overnight, then embedded in Tissue-tek O.C.T. embedding compound. Blocks were frozen in a dry-ice/ethanol bath. For immunofluorescence, 6 μm O.C.T tissue cryosections were stained with antibodies against F4/80 (1:100, eBioscience; BM8), fibronectin (1:50, Santa Cruz; IST-9), S100A4 (1:100, Abcam; ab27957), SPC (1:100, Santa Cruz; FL-197), laminin (1:50, abcam; ab11575), CD31 (1:100, Santa Cruz; MEC 13.3), EpCAM (1:50, Santa Cruz; HEA125). Secondary antibodies conjugated to Alexa Fluor 488 or 549 were used (A-11001 and A-11007, Life Technologies). Fluorescent images were obtained using a Nikon confocal microscope (Eclipse TE2000U) and analysed using Nikon software (EZ-C1 3.6). Exosomes or cells were lysed with RIPA buffer containing a complete protease inhibitor tablet (Roche). Lysates were cleared by centrifugation at 14,000g for 20 min. Supernatant fractions were used for western blot. Samples were separated on a Novex 4–12% Bis-Tris Plus Gel (Life Technologies), and transferred onto a PVDF membrane (Millipore). Membranes were processed for Ponceau red staining followed by 1 h blocking and primary antibody incubation. The antibodies against the following proteins were used for western blot analysis: ITGβ (1:1,000, Cell Signaling; 4706), ITGβ (1:500, Cell Signaling; 4707), ITGα (1:1,000, Cell Signaling; 3750), ITGα (1:10,000, abcam; ab133557), ITGα (1:1,000, abcam; ab190731), ITGα (1:500, abcam; ab117611), ITGβ (1:500, Cell Signaling; 4708), ITGβ (1:500, Millipore; AB2984) Alix (1:1,000, Cell Signaling; 3A9), and GAPDH (1:10,000, Cell Signaling; 14C10). Anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (1:3,000, Cell Signaling; 7074) and anti-mouse IgG, HRP-linked antibody (1:3,000, Cell Signaling; 7076) were used as secondary antibodies. Cells were plated in a 96-well plate and treated with 10 μg ml−1 exosomes for 2 h and then processed according to the protocol provided by the manufacturer. In brief, cells were fixed with 4% PFA and washed with 0.1% TritonX-100/PBS. Cells were then blocked using Odyssey blocking buffer for 1 h and stained overnight at 4 °C with primary antibody in Odyssey blocking buffer containing 0.1% Tween-20. The next day cells were washed again and incubated with LI-COR secondary antibodies for 1 h at room temperature followed by fluorescent imaging using Odyssey. Antibodies against the following proteins were used: Src (1:100, Cell Signaling; 2109), p-Src (1:100, Cell Signaling; 2101), AKT (1:100, Cell Signaling; 9272), p-AKT (1:100, Cell Signaling; 9271), p38 (1:100, Cell Signaling; 9212), p-p38 (1:100, Cell Signaling; 9211), NF-κB (1:100, Cell Signaling; 3034), p-NF-κB (1:100, Cell Signaling; 3033), NFAT (1:100, Thermo Scientific; PA1-023), ILK (1:100, abcam; ab52480), FAK (1:100, abcam; ab40794) and GAPDH (1:100, Cell Signaling; 14C10). IRDye 800CW anti-rabbit IgG (1:800, LI-COR) were used as secondary antibodies. Error bars in graphical data represent mean ± s.e.m. Mouse experiments were performed in duplicate or triplicate, using 3–6 mice per treatment group. Statistical significance was determined using a two-tailed Student’s t-test and one-way ANOVA, in which P values of P < 0.05 were considered statistically significant. Variance was similar between the groups that were statistically compared.
Goode D.,Center for Biomedical Research |
Truong R.,Center for Biomedical Research |
Villegas G.,Center for Biomedical Research |
Calenda G.,Center for Biomedical Research |
And 8 more authors.
PLoS Pathogens | Year: 2014
The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of α4β7 is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of α4β7 and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIVSF162P3 infection, had higher frequency of α4β7 high CD4+ T cells in the vaginal tissue and higher expression of α4β7 and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIVSF162P3. HSV-2 infection increased the frequencies of α4β7 high CD4+ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of α4β7 high CD4+ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4+ T cells and DCs that express high levels of α4β7 is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of α4β7 positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission. © 2014. Source