Ethics statement

Peripheral blood mononulear cells was obtained at the University of California, Los Angeles in accordance with UCLA Institutional Review Board (IRB) approved protocols under written informed consent using an IRB-approved written consent form by the UCLA/CFAR Virology Laboratory and was distributed for this study without personal identifying information. Human fetal tissue was purchased from the UCLA/CFAR Gene Therapy Core, was obtained without identifying information and did not require IRB approval for use. Animal research carried out in this manuscript was performed under the written approval of the UCLA Animal Research Committee (ARC) in accordance to all federal, state and local guidelines. Specifically, the experiments were performed strictly according to the guidelines in The Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the accreditation and guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC) International under UCLA ARC Protocol Number 1997-176-53.

Cells

U1 cells (NIH AIDS Reagent Program) are a subclone of the promonocytic cell line U937 that is chronically infected with HIV and makes replication competent virus upon activation53. J.RT3-T3.5 (ATCC, Manassas, VA) are a subclone of leukemia T cell line Jurkat mutated in the T cell receptor beta chain locus such that these cells fail to express surface CD3 and TCR. U1 and J.RT3-T3.5 cells were maintained in culture media containing RPMI 1640 (Invitrogen, Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Omega Scientific, Tarzana, CA) and 100 U/ml penicillin and 100 μg streptomycin (GIBCO, Life Technologies).

Virus stocks

For all the studies, we used HIV-1 89.6 , a dual tropic HIV strain that infects cells expressing CXCR4 and/or CCR5 co-receptors. The strain was selected to ensure infection of all susceptible cell populations. Stocks of HIV-1 molecular clone 89.6 were obtained from 24-h harvests of supernatants from infected CEMx174. Supernatants were filtered and treated with DNase (2 μg/ml) (Worthington, Lakewood, N.J.) for 30 min at room temperature in the presence of 0.01 M MgCl 2 . Viral infectivity was determined by limiting dilution titration on GHOST (3) X4/R5 (NIH AIDS Reagent Program). Lentiviral vectors expressing a TCR specific for the HIV Gag epitope SL9 were produced by calcium phosphate transfection of 293FT cells with the pCCL.PPT.SFFV.1.9.IRES. dLNGFR54 in conjunction with the lentiviral packaging vectors pMDLg/pRRE and pCMV-VSV-G. Supernatants were harvest on day 2 and passed through a 0.45 micron filter and concentrated by ultracentrifugation. Infectivity was assessed by titration of lentiviral vectors on J.RT3-T3.5 cells and flow cytometric analysis for the co-expression of SL9TCR (SL9 iTAg MHC Class-I tetramer-PE, Beckman Coulter, Brea, CA) and dLNGFR (CD271-FITC, Stem Cell Technologies, Vancouver, British Columbia, Canada).

Flow cytometry

Fluorochrome-conjugated mAb, specific for human CD4, CD8, CD25, CD38, CD45, CD69, CCR5, CXCR4, HLA-DR were obtained from BD Biosciences (San Diego, CA). For each analysis, cells were labeled with mAb, fixed with 2% paraformaldehyde and analyzed using BD LSRII Fortessa flow cytometer (BD Biosciences) using FACSDiva software. Gating on human anti-CD45-stained cells was used to exclude contaminating murine cells. Subsequent analyses were performed using FlowJo software (Tree Star, Ashland, OR).

Serum cytokine assay

Blood samples from our cohorts were recovered via puncture of the retro-orbital venous plexus using EDTA coated capillaries. Samples were spun at 3000 rpm for 5 min and serum was collected. Cytokine levels were determined using a cytometric bead array assay (BD CBA Human Th1/Th2/Th17 Cytokine Kit, BD Biosciences) specific for human IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ and IL-17A. Samples were acquired on a BD LSR Fortessa flow cytometer (BD Biosciences) with FACSDiva software and subsequent analyses were performed using FCAP Array software.

Generation of BLT mice and treatment

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were initially purchased from Jackson Laboratories and bred and maintained under laminar flow conditions in the Mouse/Human Chimera Core Facility at the University of California, Los Angeles (UCLA). Humanized mice were prepared as previously described44. Mice were then monitored for human cell engraftment 6 - 10 weeks post-injection. Upon reconstitution the animals were treated with cocaine for 5 days. Cocaine hydrochloride (5 mg/ml in saline) was obtained from the National Institute on Drug Abuse [NIDA; National Institutes of Health (NIH), Bethesda, MD] and diluted in saline prior to use. This dose of cocaine was selected on the basis of prior dose/response experiments (0.1, 5 or 10 mg/kg)7,55 in which the 5 mg/kg dose was shown to have no effects on engraftment yet significantly enhanced HIV infection22. Cocaine was delivered by intraperitoneal (i.p.) injection beginning 5 days pre-infection (4 - 8 weeks post human tissue implantation). After the 5-day pretreatment, a subset of animals was infected with HIV-1 89.6 , followed by continuous cocaine administration until day 14 post-infection when the animals were sacrificed. Each experiment utilized humanized mice that were made from human tissue from the same donor and the donor tissue was unique experiment to experiment. Animals exhibiting any symptoms of graft versus host disease (GVHD) are removed from our analyses.

Viral load assay

Peripheral blood was collected by cardiac puncture and transferred into microcentrifuge tubes containing 330 mM EDTA. Viral RNA was extracted from plasma using the QIAamp Viral RNA Mini Kit (Qiagen). Quantitative RT-PCR was performed using the following primers/probe specific for gag sequences: NG1F (position 453–480) 28 bp (5′-GAGCTAGAACGATTCGCAGTTAATCCTG-3′), NG1R (position 570–534) 37 bp (5′-ATAATGATCTAAGTTCTTCTGATCCTGTCTGAAGGGA-3), NG1Z probe (position 482–520) 39 bp (FAM-5′ -CCTTTTAGAGACATCAGAAGGCTGTAGACAAATACTGGG-3-BHQ). Reverse transcription was performed using the Superscript II kit (Invitrogen). Real-time, quantitative PCR was performed on a BioRad CFX96 thermocycler. Results from samples were interpolated within the quantitation derived from the RNA standards.

Tat/rev real time RT-PCR

Expression of viral genomic RNA was measured by quantitative real-time RT-PCR and compared to in vitro-transcribed RNA standards specific for multiply spliced Tat-Rev RNA as previously described56. The 18S primer/probe set was obtained from Applied Biosystems specific for eukaryotic 18S rRNA as an endogenous control to allow relative gene expression quantification. The reaction conditions were carried out using the iScript One-Step RT-PCR Kit for Probes (Bio-Rad)56.

Quantitative real time PCR

We used quantitative real-time PCR to detect the presence of viral DNA as previously described56. Briefly, cells were harvested and DNA was subsequently isolated to be used in a quantitative real time PCR using primers specific for HIV-1 sequences56. A primer-probe pair specific for the human β-globin gene was utilized to determine the input of cellular DNA as an endogenous control to allow relative gene quantification56. The reactions were carried out using the TaqMan Core Reagents Kit (ABI Biosystems)56.

Intracellular cytokine assay

1 × 106 cells/well were seeded in 48-well plates in RPMI-10 media and stimulated for 6 hours with PMA/Inonomycin at 37 °C, 5% CO2. Following incubation, the cells were washed and stained with antibodies against CD3, CD4, CD8, CD45, CD56 and IFN-γ (BDBiosciences) to measure levels of cytokine release. Samples were run on an LSRII Fortessa (BD Biosciences) and analyzed using FlowJo.

Generating HIV-specific CTL

CD8+ T cells were isolated by magnetic bead isolation (EasySep Human CD8+ Selection Kit, Stem Cell Technologies) from the PBMC of healthy donors obtained by the UCLA CFAR Virology Core and activated overnight in 50 ng/ml anti-CD3 (clone OKT3, Imgenex, San Diego, CA) and 300 U/ml IL-2 (NIH AIDS Reagent Program). Activated CD8+ were then transduced with a lentiviral vector containing HIV SL9-specific TCR and cultured in AIM-V media (Invitrogen, Life Technologies) supplemented with 5% human AB serum (Omega Scientific), 20 ng/ml IL-7 and 20 ng/ml IL-15 (both Invitrogen, Life Technologies). CD8+ T cells expressing the SL9-specific TCR were purified by magnetic bead separation based on dLNGFR expression (EasySep Human CD271 Selection Kit, Stem Cell Technologies) and confirmed >85% by flow cytometric analysis.

Cytotoxicity Assay

Purified SL9TCR-expressing CD8+ T cells were incubated in culture media (described above) for 3 days with 10 μM cocaine (NIDA Drug Supply Program) or not. On the second day, they were labeled with 2 μM Vybrant CFDA-SE (Invitrogen, Life Technologies) according to manufacturer’s suggested protocol and maintained in fresh culture media with 10 μM cocaine overnight. On the same day, target U1 cells were labeled with 5 μM CellTrace| Violet (Invitrogen, Life Technologies) according to manufacturer’s suggested protocol and activated to produce HIV with 10 μM prostratin (LC Labs, Woburn MA). The following day, cocaine or mock treated CTL were co-incubated with prostratin-activated U1 cells for 4 hr at the described effector to target ratios. Target cell sensitivity to CTL was assessed by the intracellular expression of cleaved caspase 3 via flow cytometric analysis. Percent specific killing was assessed by subtracting the %cleaved caspase 3 in target cells cultured with CTL from the %cleaved caspase 3 in target cells cultured alone. Conjugates of effector CTL and target U1 cells were assessed by flow cytometric analysis of Vybrant CFDA-SE and CellTrace Violet double positive for cells.

IFN-γ Production and CD107a Degranulation Assays

Purified SL9TCR-expressing CD8+T cells were incubated in culture media (described above) for 3 days with 10 μM cocaine or not. On the second day, they were labeled with 2 μM Vybrant CFDA-SE (Invitrogen, Life Technologies) according to manufacturer’s suggested protocol and maintained in fresh culture media with 10 μM cocaine overnight. On the same day, target U1 cells were labeled with 5 μM CellTrace Violet (Invitrogen, Life Technologies) according to manufacturer’s suggested protocol and activated to produce HIV with 10 μM prostratin (LC Labs, Woburn MA). The following day, cocaine or mock treated CTL were co-incubated with prostratin-activated U1 cells for 6 hr at an 1:1 effector to target ratio in the presence of GolgiStop and GolgiPlug (BD Biosciences, San Jose, CA) and PerCP/Cy5.5-anti-CD107a (1:50, Biolegend, San Diego, CA). Some CTL were also treated with phytohemagglutinin, PHA, (10 μg/ml) and IL-2 (100 U/ml) to induce maximal expression of CD107a and IFN-γ. After 6 hr, IFN-γ expression was assessed by intracellular cytokine staining.

Statistical analysis

All statistical analyses were carried out using GraphPad Prism 6. For two groups, we used Mann-Whitney test or one-tailed Student’s t-test, while for groups of 3 or more we used a Kruskal-Wallis (with Dunn’s multiple comparisons test) or one-way ANOVA (with a Tukey post-test). For most of our analysis, unless otherwise indicated, we ran non-parametric tests. For our proportion analysis, we used a Fisher’s exact test.