HIV 1 Infects Multipotent Progenitor Cells Causing Cell Death and Establishing Latent Cellular Reservoirs Christoph C. Carter, Adewunmi Onafuwa Nuga, Lucy A. M c Namara, James Riddell IV, Dale Bixby, Michael R. Savona and Kathleen L. Collins Supplementary Figure 1. (a) and (b) Infection of HPCs by HIV molecular clones. (a) UCB CD34 + HPCs were infected with NL4 3 1, 89.6 2, 94UG114.1 3, MJ4 4 or YU 2 5. 48 hours post infection, cells were analyzed for CD34 and intracellular Gag expression. Numbers represent the percentage of CD34 + Gag + cells. The shaded overlays represent mock treated CD34 + cells stained with an isotype matched control antibody. (b) Cells were infected as in (a) and cultured in CC110 medium. Gag expression was analyzed periodically by flow cytometry. Data are shown as percent Gag + cells. (c) Infection of HPCs with HIV generates infected cells with light scattering properties of dead cells. Bone marrow derived CD34 + HPCs were infected with HIV 89.6. 48h post infection, the cells were analyzed for forward scatter (FSC) side scatter (SSC) and intracellular HIV Gag expression. Cells were first analyzed for FSC and HIV Gag expression (left panels). Gag + events (red) were then overlaid on FSC vs. SSC plots in the right panels to show the light scattering properties of the Gag + cells. Of the Gag + cells, 36% fell outside the expected light scattering range for HPCs (red dots outside the oval gate). (d) Gag + cells were rapidly depleted from cell culture. CD34 + HPCs were infected with 89.6 ΔE SF GFPenv 89.6 (Fig. 1c). Within the culture, some cells expressed GFP only from the internal promoter and did not express HIV gene products from the
viral LTR. As shown, expression of HIV gene products correlated with a striking loss of infected cells from the culture. In contrast, expression of GFP from the internal promoter did not.
Supplementary Figure 2. (a d) HPC purification. (a) Bone marrow mononuclear cells were adherence depleted and sorted for CD133 using commercially available immunomagnetic cell separation kits, as described in Methods. Antibodies to CD133 were included during cell labeling to assess sort purity. These cells were used for the experiment depicted in Fig. 3a. (b) Purified bone marrow CD34 + HPCs were purchased commercially, and the purity of the CD34 + cells was assessed by staining with an antibody to CD34 and a control antibody and analyzing by flow cytometry. These cells were used for the experiment depicted in Fig. 4a. (c e) Bone marrow CD34 + HPCs were purified from whole bone marrow as described in Methods. Sort purity was assessed by staining input cells and sorted cells with antibody to CD34 and analyzing by FACS. (c) Cells used for the experiment depicted in Fig. 4c. (d) Cells used for the experiment depicted in Fig. 4d. (e) Cells used for the experiment depicted in Fig. 4g. (f) and (g) Infection of immature phenotype bone marrow derived HPCs. (f) Bone marrow derived HPCs (>90% CD133 + ) were infected with HIV 7SF GFPenv 89.6 and analyzed for CD34 and GFP expression 3 d post infection. CD34 + cells were gated in the left panels and GFP + cells were quantified in the right panels. (g) HIV 7SF
GFPenv 89.6 infects immature phenotype HPCs. Cells shown in (f) were analyzed for CD133, CD34, CD38 and GFP expression. CD133 + cells that were also CD34 + CD38 were analyzed for GFP expression. Numbers represent the percent of CD133 + CD34 + CD38 cells expressing GFP. Gray histograms and events represent isotype control staining.
Supplementary Figure 3. (a) and (b) Expression of HIV receptors on bone marrow HPC subsets. (a) Fresh whole bone marrow from a healthy donor was obtained as described in Methods. Adherence depleted bone marrow mononuclear cells (BMMCs) were stained for CD34, CD38, CD4, CXCR4 and CCR5 and analyzed by flow cytometry. The CD34 + CD38 + and CD34 + CD38 subpopulations were analyzed for expression of CD4 vs. CXCR4 or CD4 vs. CCR5. Isotype control antibody staining is shown in the top panels. Numbers represent the frequency of cells within each gate or quadrant. (b) Five healthy donors were analyzed as described above. The graph depicts the frequency of CD4 + CXCR4 + and CD4 + CCR5 + cells within the CD34 + CD38 + and CD34 + CD38 subpopulations. Data are shown as mean ± standard deviation.
Supplementary Figure 4. (a) Stimulation of infected HPCs with PMA induces the release of HIV particles. Bone marrow derived CD34 + HPCs were mock treated or infected with HXB eplapenv VSVG and treated with 10 ng ml 1 PMA or DMSO. Cell free culture supernatants were periodically collected and frozen. Culture supernatants were then assayed for HIV particle content by reverse transcriptase assay as described in Methods. (b-f) BMMCs immunodepleted for CD34 + cells die under culture conditions used to maintain CD34 + cells. CD34 + UCB derived cells (97% purity) and UCB mononuclear cells immunodepleted for CD34 (CD34, 99.8% purity) were plated at equal density and maintained in CC100 (100 ng ml 1 SCF, 100 ng ml 1 Flt3 L, 20 ng ml 1 interleukin-3 and 20 ng ml 1 interleukin-6) plus GM CSF (100 ng ml 1 ), TNF α (2.5 ng ml 1 ) and PMA (10 ng ml 1 ) as indicated. Images were obtained with light microscopy after 6 (b) or 14 d (c) in culture. (d) and (e) UCB cells immunodepleted for CD34 and cultured as described above were dead based on flow cytometric parameters. Equal numbers of initially CD34 + and CD34 depleted BMMCs were seeded. After culturing, each culture condition was stained with 7AAD and analyzed by flow cytometry. (d) Cells cultured in CC100 or in GM CSF TNF α were analyzed on d 14. (e) Cells cultured in PMA were analyzed on d 6. Numbers indicate the percentage of cells that were alive based on forward scatter (FSC) and 7AAD uptake. (f) Log relative total number of cells present in each culture condition at d 14. Relative numbers of cells were determined by preparing equal volumes of CD34 + and CD34 cells from each condition, adding an equal number of counting beads to each sample, and determining the number of live cells counted by flow cytometry in the time that 12,000 cytometer counting beads were counted.
Supplementary Figure 5. PMA does not stimulate integration of HIV viral genomes. UCB mononuclear cells were adherence depleted, enriched for CD34 + cells by immunomagnetic cell sorting and expanded in STIF media. 4 d after sorting, the cells were infected with HXB eplapenv VSVG in the presence and absence of 8 µm raltegravir, then cultured in STIF media with or without 8 µm raltegravir. 7 d post infection, the cells were split into the indicated condition. PMA was used at 10 ng ml 1 and an equal volume of the solvent, DMSO, was added to the control cells. 2 d after stimulation, genomic DNA was isolated from the cells and the integrated HIV genomes ng -1 of cellular DNA was determined by real time PCR. Error bars are standard error of the mean (n = six replicates). Student s t test was used to compare the means of each sample. * indicates p < 0.05 and *** indicates p < 0.001 that the indicated condition had more integrated HIV DNA than the mock treated cells and the infected cells treated with raltegravir at the time of initial infection. Differences between the infected DMSO and PMA treated conditions were not significant regardless of whether raltegravir was added at the time of induction. In this experiment, PMA treatment caused a two fold increase in the percentage of cells that were PLAP positive. This re activation was observed with or without raltegravir added at the time of induction. No PLAP expression was observed when raltegravir was added during the initial infection (data not shown).
Supplementary Figure 6. (a and b) Induction of HIV gene expression is partially resistant to antiretrovirals. (a) UCB cells were adherence depleted, then enriched for CD34 + cells by immunomagnetic cell sorting. The flow through was used as a source of cells immunodepleted for CD34. The percentage of cells that were positive for CD34 is shown. (b) The purified cells were expanded for 4 d in STIF media, then the CD34 + and cells immunodepleted for CD34 were infected with HIV NL4 3 (0 d), then cultured in CC110 media. Gag expression in infected cells was evaluated beginning 48 hours post infection. Cells were stained with 7AAD (a dead cell exclusion dye), then permeabilized, stained with an antibody to Gag, and analyzed by flow cytometry. When less than 0.1% of live, initially CD34 + cells were Gag positive (d 7), the cells were stimulated with GM CSF TNF α in the presence (GMR) and absence of raltegravir (GM). Gag expression in all conditions was evaluated 3 and 7 d after stimulation. * Indicates that > 99% of the cells were dead in at least one condition at this time point; percentages of Gag + cells were set to zero when at least 99% of cells were dead. Only one point (NL4 3 + GM CSF TNF α + raltegravir, d 10) had a non zero number of Gag positive cells before this adjustment. (c) and (d) Stimulation of infected HPCs with GM CSF
TNF α induces the production of infectious HIV, which triggers a spreading infection. (c) Highly purified bone marrow derived CD34 + HPCs were mock treated or infected with HIV 89.6 and cultured in CC110 medium. 8 d after infection, HPCs were stimulated with 100 ng ml 1 GM CSF and 2.5 ng ml 1 TNF α or maintained in CC110 medium. Half of the samples were treated with 2 µm raltegravir at the time of stimulation. At 2, 4, 8 and 11 d post infection, a portion of the cells were removed and analyzed for MHC Class I (W6/32) and intracellular HIV Gag by flow cytometry. The data are depicted as the frequency of actively infected cells (Gag + W6/32 low ) at each time point. (d) 3 d after stimulation (11 d post infection), cell free culture supernatant was collected from the experiment depicted in panel (c) and was used to infect CEM SS cells. 3 d post infection, the CEM SS cells were analyzed for HIV Gag expression by flow cytometry. The percentage of Gag + CEM SS cells following secondary infection is indicated.
Patient Year of Diagnosis Viral Load CD4 cells/ L Total BMMCs/ 10 ml Yield of CD34 + cells Purity of sample % of CD34 + cells that were Gag +(2) Raltegravir use in donor 1 2006 202,000 840 8.0x10 7 70,000 62% 0.9% no 2 1998 61,000 239 8.5x10 7 87,500 41% 1.8% yes 3 2008 64,900 504 9.0x10 7 80,000 98% 0.0% no 4 1999 172,000 298 4.0x10 7 200,000 19% 4.8% unknown 5 2009 190,000 364 2.8x10 8 380,000 85% 0.0% no 6 2005 191,000 164 1.3x10 8 600,000 92% 0.0% no 7 2006 <48 (1) 705 5.2x10 7 60,000 83% 0.0% no 8 1997 <48 (1) 292 5.0x10 7 150,000 85% 0.0% NA 9 2004 <48 (1) 263 6.6x10 7 112,500 82% 0.1% 2 NA 10 2001 <48 (1) 533 5.4x10 7 120,000 90% 0.1% 2 NA 11 1987 <48 (1) 447 3.6x10 7 200,000 88% 0.0% NA 12 2002 <48 (1) 537 8.8x10 7 200,000 57% 0.0% NA 13 1984 <48 (1) 612 5.1x10 7 220,000 96% 0.0% NA 14 2006 <48 (1) 647 2.4x10 7 90,000 30% 0.0% NA 15 2001 <48 (1) 912 1.0x10 8 200.000 90% ND NA 1 viral load undetectable >6 months 2 isotype controls ranged from 0.0-0.2% Supplementary Table 1. Summary of human subject data
Supplementary Figure 7. The integrase inhibitor raltegravir potently inhibits HIV spread in culture. CEM T Cells were infected with wild type HIV (89.6) spin infected at room temperature for 2 h, after which the virus was removed and fresh media with or without 2 µm raltegravir was added to the cells. Half the media (and drug) was removed and replaced every 3 d. After 10 d in culture, the cells were stained for intracellular Gag and analyzed by flow cytometry. Numbers indicate the percentage of cells expressing Gag. References for Supplementary Figures 1. Adachi, A., et al. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59, 284-291 (1986). 2. Collman, R., et al. An infectious molecular clone of an unusual macrophagetropic and highly cytopathic strain of human immunodeficiency virus type 1. J Virol 66, 7517-7521 (1992). 3. Gao, F., et al. A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1. J Virol 72, 5680-5698 (1998). 4. Ndung'u, T., Renjifo, B. & Essex, M. Construction and analysis of an infectious human Immunodeficiency virus type 1 subtype C molecular clone. J Virol 75, 4964-4972 (2001). 5. Li, Y., et al. Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replicationcompetent and -defective viral genomes. J Virol 65, 3973-3985 (1991).