The authors have declared that no competing interests exist.
Conceived and designed the experiments: SKM HD SGT ANH ALC NN. Performed the experiments: SKM RAB UK LM DS NN HJ. Analyzed the data: SKM HD UK LM DS KS ALC RJS RLM. Contributed reagents/materials/analysis tools: SKM UK LM DS SGT NN. Wrote the paper: SKM HD RJS RLM ALC. Developed the virus propagation and microvesicle removal methods: SGT NN.
HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level
Dendritic cells (DCs) are vital for immune recognition of pathogens as they capture, internalise, degrade and present foreign peptides to T lymphocytes. It is thought that HIV-1 hijacks the DCs functions, such as migration and maturation, to increase contact with the major target cell CD4+ T lymphocytes leading to dissemination throughout the body. Currently there is still some controversy over the ability of HIV-1 to infect and mature DCs, which may be due to differences in the inoculum used. Here we examined the effect of contaminating microvesicles (MVs) identified in HIV-1 preparations on HIV-1 modulation of DC function. We show that when MVs are present with HIV-1, the inoculum induces greater DC maturation and adhesion probably via cellular HSP90α and β and viral nef within the MVs. The functional consequences are reduced
Dendritic cells (DC), located throughout the body, but in particular in the male foreskin and the anogenital and cervical mucosa, are susceptible to HIV-1 infection
Both immature and mature DCs are able to transfer HIV-1 to T lymphocytes by different mechanisms during 2 sequential phases of uptake, the first following vesicular uptake and the second after de novo infection of DCs transfer
In this study we have investigated the relative roles of HIV-1 and associated MVs on the maturation status of DCs and viral transfer to T lymphocytes using HIV-1 inocula containing or stripped of MVs. We have shown that MVs present within an HIV-1 inoculum enhance MDDC maturation which in turn induces more DC∶T lymphocyte clusters and results in higher levels of HIV-1 transfer to T lymphocytes. This induced maturation limits the productive infection of monocyte-derived DCs (MDDC). We propose that these MVs play a role in cell to cell spread of HIV-1
Previous work from our laboratory showed partial maturation of dendritic cells in response to HIV-1BaL, however we were unable to rule out the role of MVs in non-viral replication dependent DC maturation
MDDCs were infected with either HIV-1BaL(CD45−) or HIV-1BaL(pellet) at a MOI of 3 measured as TCID50. Levels of productively infected cells were assessed by flow cytometry for intracellular HIV-1 p24 antigen at 48 and 120 hours post infection (
MDDCs were infected for 48-1BaL(pellet) or HIV-1BaL(CD45−) at an MOI of 3 determined by TCID50, and assessed for intracellular HIV-1 p24 antibody by flow cytometry and HIV-1 proviral DNA by Q-PCR. A) Representative dot plots of untreated and HIV-1BaL(CD45−) exposed DCs identifying infected (p24+ cells in box on the right) and exposed but non-infected (p24−) DCs at 120 hours. B) The level of infection at 48 and 120 hours for both HIV-1BaL(pellet) and HIV-1BaL(CD45−) viruses by flow cytometry (mean +/− SEM, n = 12, *p<0.05 **p<0.01) and C) by Q-PCR (HIV-1 proviral DNA normalised by albumin values) (mean +/− SEM, n = 4, *p<0.05).
The susceptibility of DCs to become productively infected with HIV-1 is related to the maturation status of the DCs
MDDCs were exposed to high titre HIV-1BaL(pellet), HIV-1BaL(CD45−) at a MOI of 3 or a potent maturation cocktail for 48 hours and expression of maturation determined by flow cytometry. A) Representative histograms for CD83 and CD86 shown (isotype grey tint, immature solid grey line, HIV-1BaL(CD45−) dashed line, HIV-1BaL(pellet) dotted line and mature solid black line). B) The levels of surface maturation marker expression with flow cytometry MFI results expressed as a ratio of normalised to untreated (mock/immature) controls as log (2) fold change (mean +/− SEM, n = 12, *p<0.05 **p<0.01). C) Kinetics over time of ICAM-1 expression determined by flow cytometry MFI for HIV-1BaL(pellet) treated MDDCs (mean +/− SEM, n = 3). D) Productively infected MDDCs are more mature than uninfected MDDCs for both virus stocks. Infected cells were identified by p24 antigen staining and the expression of CD83, CD86 and ICAM-1 was compared to that for the HIV-1 exposed uninfected cells. Representative histograms for the markers of p24+ (dashed line) and p24− (dotted line) compared to isotype control (grey tint) for HIV-1BaL(pellet) and HIV-1BaL (CD45−). Bar graphs delineating the expression of normalised maturation and co-stimulatory marker expressed as log (2) fold change to mock treated cells (mean +/− SEM, n = 5, *p<0.05).
To assess the kinetics of ICAM-1 up-regulation, the protein expression was determined at 0, 16, 24, 48 and 96 hours post infection by flow cytometry. We found that while ICAM-1 expression peaked at 16 hours post infection, the expression was still above baseline levels at 48 hours post infection (
When the MDDC population was divided into the infected (p24+) and exposed but uninfected (p24−) populations it became apparent that within the HIV-1BaL(CD45−) treated cells, maturation was restricted to the p24+ productively infected cells and very little bystander maturation of p24− cells was seen. This contrasted with the HIV-1BaL(pellet) treated DCs which showed increased CD83, CD86 and ICAM-1 expression in both infected and bystander cells (
To confirm that the differences induced by HIV-1BaL(pellet) compared to HIV-1BaL(CD45−) virus were a result of the MVs that were removed, we next devised a MV replacement experiment. The bead bound CD45+ MVs could not be used for these replacement assays as the CD45 microbeads caused DC maturation (data not shown). Additionally, it was not possible to remove the CD45 beads from the depleted fraction without damaging the MVs. We therefore generated MVs from SUPT1.CCR5-CL.30 cells used for propagation of HIV-1 stocks by stimulation with CD3 and CD28 antibodies for 3 days. The supernatant was subsequently concentrated as for HIV-1BaL virus stock generation. This method of activating SUPT1.CCR5-CL.30 cells resulted in similar cytopathic phenotypic changes to those cells infected with HIV-1BaL. MVs from untreated SUPT1.CCR5-CL.30 cells had no effect on HIV-1 induced DC maturation or ICAM-1 expression (
MDDCs were either treated with HIV-1BaL(pellet), HIV-1BaL(CD45−) alone, CD3/CD28 activated MVs (20 µl/3.5×105 cells) alone or HIV-1BaL(CD45−) with the addition of CD3/CD28 activated MVs for 48 hours. To calculate the MV ‘inoculum’ the CD45 concentration of the MVs preparation was matched to the CD45 concentration of the HIV-1BaL(pellet) stock (see
Once MV preparations were generated they were added back to the HIV-1BaL(CD45−) virus stock and then used to infect MDDCs. HIV-1BaL(pellet) was used as a positive control and the amount of MVs added was matched to the CD45 concentration of the HIV-1BaL(pellet) virus (determined by western blot densitometry,
HIV-1BaL (pellet) | HIV-1BaL (CD45−) | HIV-1BaL (CD45+) bead | |
100+/−0.01 | 6.343+/−0.04 | 93.363+/−0.41 |
Net intensity normalised to HIV-1BaL (pellet) +/− SE n = 3. Image J analysis.
In addition to the enhanced maturation, adding back MVs resulted in significantly less HIV-1 infection of MDDCs at 120 hours post infection (3.72+/−1.1% SEM, p = 0.05,
In contrast to MVs from activated SUPT1.CCR5-CL30 cells, MVs from primary CD4+ lymphocytes activated by anti-CD3 and anti-CD28 antibodies induced significant maturation of MDDCs alone, without accompanying HIV-1BaL(CD45−) (
In addition, exposure of primary blood myeloid BDCA1+ DCs to HIV-1BaL(pellet) showed enhanced maturation (up-regulated CD83) but this was diminished when they were exposed to HIV-1BaL(CD45−) and restored when MVs from activated SUPT1.CCR5-CL.30 cells were added to HIV-1BaL(CD45−) (
ICAM-1 was markedly up-regulated in the MDDCs infected with HIV-1BaL(pellet) compared to maturation mix or HIV-1BaL(CD45−) virus (
MDDCs were either untreated or exposed to HIV-1BaL(pellet), HIV-1BaL(CD45−) (MOI 3) or a potent maturation cocktail for 48 hours and co-cultured with autologous CD4+ T lymphocytes at a ratio of 5 T lymphocytes∶1 DC for 45 minutes. A) Representative dot plot of the flow cytometry gating strategy for MDDC selection by FSC and SSC. B) Clusters were identified as double positive for CD3 (T lymphocyte marker) and CD1a (DC marker). Representative dot plots are shown. C) The percentage of DC clusters following treatment (mean +/− SEM, n = 10, *p<0.05 **p<0.01). D) DC:CD3+ cell clusters were identified in the p24− and p24+ fractions of the co-culture and compared by box and whisker plots for the HIV-1BaL(pellet) virus (mean +/− SEM, n = 10, **p<0.01). E) Cluster formation following addition of neutralising antibody to ICAM-1 (mean +/− SEM, n = 3, *p<0.05).
DCs were then infected with either HIV-1BaL(pellet) or HIV-1BaL(CD45−) virus for 48 hours and co-cultured with autologous CD4+ T lymphocytes. The percentage of DCs that clustered with CD4+ T lymphocytes was significantly different between the two virus stocks (p<0.02). The HIV-1BaL(CD45−) treated DCs, which showed very little up-regulation of co-stimulatory markers or of ICAM-1, had levels of clustering with CD4+ lymphocytes (17.2+/−2.9% SEM) similar to the untreated negative control DCs. In contrast, the HIV-1BaL(pellet) treated DCs with higher ICAM-1 and co-stimulatory marker expression showed an increased cluster formation with CD4+ T lymphocytes (30.44+/−4.8% SEM), similar to mature DCs (
After defining infected and bystander MDDCs separately, significantly higher clustering was seen in the p24+ DCs than in p24− DCs for HIV-1BaL(pellet) (
Next, the effects of differential DC maturation by the two HIV-1BaL stocks upon T lymphocyte proliferation were examined. MDDCs were exposed to HIV-1BaL(pellet) or HIV-1BaL(CD45−) virus for 48 hours and then added to CFSE labelled allogeneic PBMCs from HIV-1 seronegative subjects at a ratio of 1 DC∶10 PBMC and co-cultured for a further 5 days. Following culture, cells were assessed for proliferation by flow cytometry. CD4+ T lymphocyte and MDDC populations were identified by size gating and the level of proliferation was assessed by CFSE dilution (
MDDCs exposed to HIV-1BaL(pellet) or HIV-1BaL(CD45−) for 48 hours were co-cultured with allogeneic PBMC at a ratio of 1 DC∶10 PBMC for 5 days. Proliferation was determined by CFSE dilution. The cells were stained for CD4+ T lymphocytes and analysed by flow cytometry. A) Representative dot plots of CD4+ T lymphocytes for each treatment are shown. B) The bar graph compares the proliferation between each treatment determined by adding the percentage proliferation normalised to live cell number of the CD4+ lymphocyte population and the MDDC population (mean +/− SEM, n = 3, *p = 0.05 **p = 0.01).
As DCs are very efficient at transferring HIV-1 to T lymphocytes, we investigated the capacity of MDDC infected with the two different virus stocks to induce T lymphocyte activation and to transmit virus to T lymphocytes. MDDCs were infected with either HIV-1BaL(pellet) or HIV-1BaL(CD45−) virus for 48 hours and added to resting autologous CD4+ T lymphocytes for up to 96 hours. The levels of CD69 expression on CD4+ T lymphocytes, as a marker for T lymphocyte activation (
A) MDDCs treated with HIV-1BaL(pellet) or HIV-1BaL(CD45−) at (MOI 3) for 48 hours were co-cultured with unstimulated autologous CD4+ T lymphocytes. The expression of CD69 on T lymphocytes was determined by flow cytometry at 24, 48, 72 and 96 hours post T lymphocyte addition (mean +/− SEM, n = 3, *p<0.05). B) The level of infection of T lymphocytes was determined by gating on CD3+ cells (to exclude DCs) followed by p24 staining determined at 24, 48, 72 and 96 hours post T lymphocyte addition (mean +/− SEM, n = 3, *p<0.05).
CD4+ lymphocytes co-cultured with HIV-1BaL(pellet) exposed MDDCs resulted in higher and more rapid kinetics of infected CD4+ lymphocytes than when co-cultured with HIV-1BaL(CD45−) exposed MDDCs. Thus suggesting that DC maturation induced by MVs as well as virus in HIV-1BaL preparations acts to enhance HIV-1 transmission.
We next characterised the MVs in the HIV-1 and activated SUPT1.CCR5-CL.30 stocks. Initially, key candidate proteins present in MVs from the HIV-1BaL(pellet) and the MVs obtained from CD3/CD28 activated and non-activated SUPT1.CCR5-CL.30 cells were investigated by Western blot and compared to HIV-1BaL(CD45−), parent SUPT1.CCR5-CL.30 cells as well as the anti-CD45 bead bound HIV-1BaL preparation (
30 µg of protein from MDDCs, SUPT1.CCR5-CL.30, bead bound HIV-1BaL(CD45+), HIV-1BaL(CD45−), HIV-1BaL(pellet), non-activated MV and activated MV preparations were compared for the expression of A) CD45, B) Alix, C) CD81, D) H2A, E) HSP90α F) HSP90β by western blot using SDS-page gel, nitrocellulose membrane and developed by chemiluminescence.
MDDC | SUPT-1 | Bead HIV-1BaL (CD45+) | HIV-1BaL (CD45−) | HIV-1BaL (pellet) | Vesicles (activated) | Vesicles (non-act) | |
+++ | ++ | ++ | − | + | + | + | |
+ | − | − | − | − | − | − | |
+ | − | + | + | + | ++ | + | |
− | − | − | ++ | ++ | + | − | |
++ | ++ | + | + | + | + | − | |
+ | ++ | − | − | − | − | − | |
+ | − | − | − | − | − | − | |
+++ | + | − | − | − | − | − | |
− | − | + | + | − | − | − | |
+ | + | + | + | + | − | − | |
− | − | − | − | − | − | − | |
+ | + | − | − | − | − | − | |
− | − | − | − | − | − | − |
+++ high protein expression, − below detection limit.
positive in PBMCs,
positive in transfected lysate.
To fully characterize the protein complement of the MVs from the two HIV-1 inocula and also the anti-CD45 bead bound material compared to those from activated and non-activated SUPT1.CCR5-CL.30 cells we separated the protein bands by gel electrophoresis and subjected them to Tandem Mass Spectrometry. After filtering for non-human contaminating proteins present in the fetal calf serum, 266 proteins with >0.01% share of the total were detected in HIV-1BaL(pellet), 255 in HIV-1BaL(CD45−), 274 in the anti-CD45 bead bound preparation, 462 in MVs from activated SUPT1.CCR5-CL.30 and 143 from non-activated SUPT1.CCR5-CL.30 MVs. The key proteins characteristic of MVs which were identified are shown in
Bead HIV-1BaL (CD45+) | Vesicles (activated) | Vesicles (non-activated) | |
0.95 | 0.569 | 0.149 | |
0.716 | 0.28 | 0.029 | |
0.072 | 0.071 | 0.032 | |
0.052 | 0.052 | 0.032 | |
0.39 | 0.063 | 0.028 |
HIV-1BaL (pellet) | HIV-1BaL (CD45−) | MVs (activated) | MVs (non-activated) | |
0.32 | 0.33 | 0.40 | 0.13 | |
0.20 | 0.63 | 0.25 | 0.04 | |
0.03 | 0.00 | 0.00 | 0.00 | |
0.023 | 0.00 | |||
2.17 | 3.82 | |||
0.021 | 0.073 |
The HIV-1 accessory protein nef was present in HIV-1BaL(pellet) (at lower levels than gag or env), but completed depleted from HIV-1BaL(CD45−) virus. However, Gag/pol and env peptides were concentrated 1.5 to 3 fold in HIV-1BaL(CD45−) compared to HIV-1BaL(pellet) (
To directly examine the effect of HSPs 90α and β and nef on MDDC maturation, graded concentrations of recombinant HSP 90α (
MDDCs (0.5×106 cells/mL) were treated with recombinant proteins, A) HSP90β and B) nef at variable concentrations for 48 hours. The effects on the maturation markers, CD80, CD83 and CD86, were measured by flow cytometry and expressed as log (2) fold change in MFI compared to mock treated MDDCs (mean +/− SEM, n = 3, *p<0.05, **p<0.01). C) MDDCs (0.5×106 cells/mL) were treated with recombinant nef at 10 nM (lowest significant effective concentration) and recombinant HSP90β at 0.5 nM (completely ineffective concentration on MDDC maturation alone) or maturation mix for 48 hours and expression of CD80, CD83 and CD86 measured by flow cytometry and expressed as log (2) fold change in MFI compared to mock treatment (mean +/− SEM, n = 3, *p<0.05, **p<0.001).
The combination of recombinant nef at its lowest effective concentration (10 nM) with HSP90β at a concentration (0.5 nM) tenfold below its minimum effective concentration (5 nM) enhanced maturation above nef alone, especially for CD80 (
Purification of HIV-1 preparations released from infected T lymphocytes by removal of the contaminating MVs can be achieved by CD45 depletion
Several HIV-1 restriction factors have been described for DCs, including the constitutively expressed APOBEC3G
In this study we show that productively infected DCs are more mature than exposed bystander cells, confirming our previous findings that the maturation was a result of HIV-1 replication, as well as exposure
The adhesion molecule ICAM-1 was investigated due to its role in immunological and viral synapse formation
As well as different levels of DC maturation seen when cells were treated with HIV-1BaL(pellet) or HIV-1BaL(CD45−), corresponding rates of T lymphocyte proliferation and HIV-1 transfer were observed. DCs treated with HIV-1BaL(pellet) led to greater CD4+ and CD8+ T lymphocyte proliferation than those treated with HIV-1BaL(CD45−). In addition, HIV-1BaL(pellet) led to transfer of HIV-1 to both activated and resting CD4+ T lymphocytes, while HIV-1BaL(CD45−) was unable to activate contacting CD4+ T lymphocytes and was only able to be transferred to activated CD4+ T lymphocytes, probably due to decreased ICAM-1 expression and clustering. This indicated a significant role for infected and especially bystander DC maturation due to presence of MVs, on HIV-1 transfer. These results together are important as, until now, it has not been possible to determine the individual roles of maturation and infection on the spread of HIV-1 from DCs to T lymphocytes.
The viral inoculum
Our results show that
Diagram of MDDCs infected with either A) HIV-1BaL(pellet) or B) HIV-1BaL(CD45−) virus. MDDCs exposed to HIV-1BaL(pellet) had increased maturation, limited infection and when co-cultured with T lymphocytes, T lymphocyte activation and HIV-1 transfer was observed. When the MDDCs were assessed based on their p24 status to delineate between infected (p24+) and uninfected bystanders (p24−), there were notable differences with infected MDDCs showing greater maturation, ICAM-1 expression and clustering. However, as only 1–2% of the population were considered infected, T lymphocytes activated by bystander MDDC interactions could also then interact with the small infected population resulting in HIV-1 transfer to these cells. In contrast, MDDCs exposed to the HIV-1BaL(CD45−) had limited MDDC maturation and increased HIV-1 infection. The infected MDDCs did show some maturation, ICAM-1 expression and clustering to T lymphocytes, whereas the bystander cells did not. When co-cultured with T lymphocytes, little T lymphocyte activation and HIV-1 transfer was observed, possibly due to a small number of exposed MDDCs able to activate the T lymphocytes (5–10%) compared to the majority of MDDCs when exposed to HIV-1BaL(pellet). The differences seen between the two virus stocks were a due to the presence of MV in the HIV-1BaL(pellet) virus. up arrow = up-regulated compared to mock, down arrow = down-regulated compared to mock and left-right arrow = no change compared to mock.
Recent studies have shown that many virally infected cells secrete MVs, including vaccinia virus
Nef was also detected in MVs but depleted in the MV stripped HIV inoculum. Pure reombinant nef was a potent inducer of MDDC maturation. Nef has previously been shown to be secreted in exosomes and induce bystander cell effects,
The complexity of the HIV-1 and MV effects on DCs is dissected into 4 scenarios in panels A and B of
In conclusion,
MDDCs were generated from CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMC) of anonymous blood donors from the Australian Red Cross Blood Service, Sydney using CD14 magnetic beads (Miltenyi Biotech; Gladbach, Germany) as described previously
When required, MDDC were matured for 48 hours in maturation mix consisting of (v/v) final concentration 50 pg/mL IL-1β (R&D Systems; Minneapolis, MN, USA), 5 U/mL IL-6 (R&D Systems), 50 pg/mL TNF-α (R&D Systems) and 5 ng/mL PGE2 (Sigma; Milwaukee, WI, USA) in 0.1% (w/v) bovine serum albumin (BSA, Sigma) PBS.
CD4+ T lymphocyte isolation (97% purity) was performed using magnetic beads (Miltenyi Biotech) according to the manufacturer's protocol.
Primary blood myeloid DCs were isolated from PBMCs collected from whole blood, using a negative selection magnetic bead myeloid DC isolation kit (Miltenyi), as per manufacturer's instructions. Isolated mDCs were cultured at 1×106 cells/mL in RF10 (RPMI supplemented with 10% FCS and no cytokines) and collected at 24 h post treatment.
Phenotype/purity was checked by Flow cytometry. Stained with Live/Dead Aqua (Invitrogen), Lin marker (BD), HLA-DR (Biolegend), BDCA-1 (Miltenyi) and BDCA-3 (Miltenyi). Maturation was checked by staining with Live/Dead, HLA-DR, BDCA3, CD83 (BD) and CD86 (BD). Cells were gated by size, Live, and HLA-DR+ before gating on BDCA-3+ and BDCA-3− (“BDCA-1”). CD83 and CD86 expression was analysed on the separate BDCA-1/BDCA-3 populations.
Recombinant HSP90α and β free of LPS, were purchased from Abcam, UK (at 2.7 mg/mL and 1.7 mg/mL respectively) and recombinant nef from BioAcademia Inc. Japan (at 0.48 mg/mL)
HEK293T cells (Human Embryonic Kidney cells, NIH AIDS Research and Reference Reagent Program) were transfected with pWT/BaL (NIH AIDS Research and Reference Reagent Program, contributed by Dr. Bryan R. Cullen) and pHEF-VsV-g (NIH AIDS Research and Reference Reagent Program, contributed by Dr. Lung-Ji Chang) plasmids using polyethylenimine (PEI, Polyscience; Warrington, PA, USA) to generate VsV-g pseudotyped pBaL. Purified high titre HIV-1BaL stocks in the order of 2×109 TCID50/mL were generated by infection of SUPT1.CCR5-CL.30 cells (Human Non-Hodgkin's T lymphocyte Lymphoma, contributed by Prof. James Hoxie at the University of PA) with VsV-g pseudotyped pBaL. HIV-1 infected supernatants were concentrated using tangential filter concentration using the Millipore Lab scale system (Millipore; Billerica, MA, USA) and 2× Pellicon filters connected in parallel (300 kDa) (Millipore). When required, CD45+ MVs were depleted from supernatant using CD45 magnetic beads (Miltenyi Biotech). Virus (18 mLs) was incubated at room temperature with 2 mLs microbeads for 2 hours before adding to the top of a LS column. CD45 depleted virus that flowed through the column as well as non-depleted supernatants were concentrated further by ultracentrifugation with 1 mL under-layed 20% sucrose cushion and centrifuged at 100,000×g (Beckman Optima XL-100K Ultracentrifuge with 70Ti rotor) at 4°C for 90 minutes. Virus content was determined by p24 gag ELISA as per manufacturer's instructions (Beckman-Coulter; Hialeah, FL). The 50% tissue culture infectious dose (TCID50) values were generated in TZM-BL cells (NIH AIDS Research and Reference Reagent Program, contributed by John Kappes and Xiaoyun Wu) measured by LTR β-galactosidase reporter gene expression after a single round of infection
MVs from activated SUPT1.CCR5-CL.30 or primary T lymphocytes were generated using antibodies to CD3 (1 µg/mL, BD Pharmingen; Becton Dickinson; San Jose, CA) and CD28 (5 µg/mL, BD Pharmingen) added to 100×106 cells, cultured for 3 or 6 days respectively alongside unstimulated cells and the supernatant concentrated as above. The CD45 concentration for virus and MV was determined by western blot. Endotoxin levels for all virus and MV stocks were negative using the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GeneScript; Piscataway, NJ).
Immature day 5 MDDCs were infected with pelleted (HIV-1BaL(pellet)) or CD45depleted (HIV-1BaL(CD45−)) virus in 200 µL media with a MOI of 3 at 37°C for 2 hours before resuspending at 1×106/mL and incubating further as required. MVs were added at 20 µL/mL to match the CD45 concentration to HIV-1BaL(pellet) virus stocks. Recombinant proteins were added at various final concentrations to MDDCs cultured at 0.5×106cells/mL in RF10+cytokines. Harvested cells at 48 hours post treatment for FACS analysis.
Direct conjugated mAbs directed against ICAM-1-fluorescein isothiocyanate (FITC) (Beckman Coulter), CD80-Phycoerythrin-Cy5 (PE-Cy5), CD83-FITC, CD86-PE-Cy5, HLA-DR-Allophycocyanin (APC), MR-APC, CD1a-FITC, CD40-FITC, CD3-APC, CD8-PE-Cy5 and CD69-APC (BD Pharmingen) and CD4-PE (Sigma) were used for surface staining. Antibody staining was performed at 4°C for 30 minutes using fluorescence-activated cell sorter buffer (1% (v/v) Human Ab Serum, 2 mM EDTA and 0.1% (w/v) sodium azide made up in PBS). For intracellular staining HIV-1BaL or mock treated MDDCs were fixed and permeabilised in Cytofix/Cytoperm (BD), re-suspended in permwash buffer (1% (v/v) human AB serum (Sigma), 0.1% (w/v) saponin, 0.1% (w/v) sodium azide, made up in PBS at 4°C) and incubated for 2 hours with PE conjugated p24 (clone KC57-RD1, Beckman Coulter; Fullerton, CA). IgG isotype control antibodies were incubated with cells to control for nonspecific binding. Cells were then analysed with a FACS-Canto flow cytometer (Becton Dickinson) and FlowJo (Tree Star Inc., Ashland, OR).
Blocking of ICAM-1 (clone HA58, 5 µg/2×105 cells, BD Pharmingen) was performed at 37°C for 30 minutes.
2×105 cells were lysed at 60°C for 90 minutes in Q-PCR Lysis Buffer (10 mM Tris-Hydrochloride, 50 mM potassium chloride, 2.5 mM magnesium chloride, 0.45% (v/v) NP-40, 0.45% (v/v) Tween-20, 50 µg/mL Proteinase K (Sigma)) followed by denaturing at 94°C for 15 minutes. HIV-1 proviral DNA was detected using the HIV-long terminal repeat (LTR) gag primer probe set as previously described
In addition, the expressions of selected genes were assessed using reverse transcribed total unamplified RNA as previously described
For the clustering assay, treated MDDC were co-cultured with autologous CD4+ T lymphocytes at a ratio of 1 DC∶5 T lymphocytes and incubated at 37°C for 45 minutes. Cells were subsequently stained for CD1a, CD3 and p24 for flow cytometric analysis as described above.
The proliferation was determined by carboxyfluroescein succinimidyl ester (CFSE) dilution. Briefly, allogeneic PBMCs were stained with 5 µM final concentration CSFE (Molecular Probes; Eugene, OR) for 10 minutes at 37°C, rescued with equal volume of 100% FBS and washed with RPMI+10% FBS. These PBMCs were added to the HIV-1BaL infected MDDCs at a ratio of 1 MDDC∶10 PBMC and incubated at 37°C for 5 days. Cells were subsequently stained for CD3, CD4, CD8 and p24 for flow cytometric analysis as described above.
MDDCs infected with HIV-1BaL (pellet or CD45−) for 48 hours were co-cultured with resting CD4+ T lymphocytes for a further 24, 48, 72, 96 and 120 hours at 37°C before staining with p24 antibody for flow cytometry as described above.
Viral, MV and parent cell preparations were lysed for 1 hour at 4°C in SDS lysis buffer (10 mM HEPES, 150 mM NaCl, 1% (v/v) Triton-X-100, 1 µg/mL protease inhibitor cocktail (Sigma) at pH of 7.5), followed by centrifugation for 10 minutes at 16,000×g at 4°C. The protein concentration was determined using the DC Protein Assay (Bio-Rad) according to the manufacturer's instructions. The protein concentration was determined using the DC Protein Assay (Bio-Rad) according to the manufacturer's instructions.
30 µg of protein prepared with 1× NUPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) containing 400 mM dithiothreitol was boiled for 10 minutes before loaded into NUPAGE 4–12% polyacrylamide bis-tris gradient SDS-PAGE gels (Invitrogen) alongside a 10–250 kilo Dalton (kDa) molecular weight marker (Bio-Rad). Electrophoresis was run at 200 V for 50 minutes in NUPAGE 3-(N-morpholino)propanesulfonic acid (MOPS) SDS Running Buffer (Invitrogen). The proteins were transferred to a nitrocellulose membrane (Amersham Biosciences) in transfer buffer (250 mM Tris (pH 8.3), 1.92M Glycine and 0.05% (w/v) SDS) overnight at 55 mA. Non-specific binding sites were blocked by incubating for 1 hour in 250 mM Tris (pH 8), 1.4M NaCl and 30 mM KCl (pH 8) containing 20% (v/v) polyethylene glycol
For Mass Spectrometry, 30 µg of each sample was prepared in SDS lysis buffer, run on 4–12% bis-tris gradient SDS-PAGE gel and stained with Brilliant Blue G (Sigma).
The 1D SDS-Gel lanes were sliced into 31 1 mm×5 mm bands using a disposable grid cutter (The Gel Company, USA) and in-gel digested with trypsin using an automated liquid handling procedure with a TECAN Freedom Evo liquid handling system (Männedorf, Switzerland). The 31 fractions were pooled (2 gel fractions per pool) and analysed by LC-MS/MS in duplicate.
Peptide separation was performed on an Eksigent Nano 2D plus system (ABSciex, USA) employing splitless pumps enabled for nanoflow rates. RP-HPLC Trap and separation columns were prepared in-house (Supplementary data for extended
Mass Spectrometry analysis involved converting Thermo .RAW files to mzXML format using MSConvert
The identified peptides and their inferred proteins and spectral quantities are reported in the appended Microsoft Excel file (Mercier et al peptide-protein proteomic data.xlsx). The raw data for this project (103 Thermo .raw files, 53.5 Gb total) has been deposited in the Tranche proteomics raw data repository
The raw data for this project (103 Thermo .raw files, 53.5 Gb total) has been deposited in the Tranche proteomics raw data repository
Tranche repository using the following hash:
“69iC+dKFUFu7JMcZxCSdI3cS0q37GPeG3yuWB2h32wnh 27xemcTBoEY75tDGOGt0fGFes8Jy 69iC+xf+ST5lv7Uz7lfUY 5UQAAAAAAAAy3A = = ”
(EPS)
(EPS)
The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HEK293T cells and pWT/BaL plasmid from Dr. Bryan R. Cullen and pHEF-VsV-g plasmid from Dr. Lung-Ji Chang. SUPT1.CCR5-CL.30 cells (Human Non-Hodgkin's T lymphocyte Lymphoma) were contributed by Prof. James Hoxie at the University of PA. The authors thank Karen Byth for assistance with statistical analysis and David Ott for advice on preparation of the HIV-1BAL(CD45−) stocks.