Fig 1.
Sort 2 HSPCs are depleted for HSCs.
A. Diagrammatic representation of HSPC purification. FT, column flow-through; CB, cord blood; BMMC, bone marrow mononuclear cells. B. Representative flow cytometric analysis of HSPC samples from an HIV+ individual. C. Gates for each HSPC population phenotype and lineage output according to Doulatov et al [9]. (HSC, hematopoietic stem cell; MPP, multipotent progenitor; MLP, multilymphoid progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte/erythrocyte progenitor; GMP, granulocyte/monocyte progenitor; B-NK, B and NK cell progenitor; MDC, macrophage and dendritic cell; EMK, erythroid and megakaryocyte; G, granulocyte.) D. Representative flow cytometric analysis of differentiation markers expressed on bone marrow HSPCs purified as described in A. For the two right-most panels, numbers indicate percentage of total CD34+ events from each sort falling into that gate. E. Flow cytometric plot comparing relative numbers of HSCs (CD34+CD38- cells that are also CD90+) in Sort 1 versus Sort 2. F-I. Summary graph showing the relative frequency of the indicated progenitor in each sort, n = 3 uninfected donors. To facilitate comparison, results were normalized to Sort 2 (F and G) or Sort 1 (H—I). Mean ± standard deviation is indicated. J. Summary plots of methylcellulose colony formation assays from three uninfected donors. Mean ± standard deviation is indicated. Colony forming unit (CFU)-E, erythroid; CFU-GM, granulocyte/macrophage and CFU-GEMM, multilineage. (*p<0.05 and **p<0.01, 2-tailed Student’s t-test).
Table 1.
Donor characteristics.
Fig 2.
Near-full-length HIV provirus in HSPCs preparations is unlikely to be from T cell contamination.
A. Flow cytometric analysis of HSPCs from three donors that harbored near full-length genomes. Numbers indicate the frequency of events falling into each quadrant. CD133 or CD34 were assessed for Sort 1 or Sort 2 respectively. B. Summary table showing data for Fisher’s exact test, which compares calculated frequencies of provirus assuming all provirus originates from T cells. PCRs were performed at limiting dilution. Significantly discrepant frequencies in sorted and flow-through samples indicate T cells are an unlikely source of provirus in sorted samples. Conservative estimate compares the top of the 95% confidence interval for the calculated infection rate for flow-through CD3+ T cells with the bottom of the 95% confidence interval for the calculated infection rate for CD3+ T cells in the sorted HSPCs. C. Agarose gel analysis of rearranged T cell receptor gene PCR. 50, 100 or 200 cell equivalents of DNA from the first round PCR reaction that yielded each provirus were added to each reaction. For “T cells”, 200 cell equivalents of DNA from purified CD4+ T cells were added to the reaction. The expected size of the amplicons is 250–300 bp. Numbers to the left of each gel indicate the location of molecular weight markers.
Table 2.
Summary of donor env amplicons in Sort 1 and 2 HSPCs.
Table 3.
Analysis of env amplicons isolated from HIV+ donors.
Table 4.
Summary of Env phenotypes with V3 regions matching HSPC-associated env sequences.
Fig 3.
Expression of HIV receptors in progenitor cells.
A. Affymetrix array data of gene expression in human bone marrow stem and differentiated cell types extracted from a published data set [12] accessed via the NCBI Gene Expression Omnibus database (GSE42519). Left panel, heat map representing the z-score (scaled for each column) of the log transformed gene expression data. Right panel, relative expression levels of HIV receptors in each subset. The original microarray data went through background correction, normalization, and log transformation via the RMA method. For the right panel, data was converted from log transformed to linear. B. RNA seq analysis of mouse bone marrow and differentiated cell types. Left panel, heat map representing the z-score (scaled for each column) of the log transformed gene expression data. Right panel, relative expression levels of HIV receptors in each subset. Mean ± SEM is shown. (Abbreviations: FC, fold change; HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythrocyte progenitor; PM, promyelocyte; MY, myelocyte; MM, metamyelocytes; BC, band cell; PMN, polymorphonuclear cells; GR, granulocyte; ProB, pro-B cell; PreB, pre-B cell; BM, unfractionated bone marrow. FPKM, Fragments Per Kilobase Of Exon Per Million Fragments Mapped) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, 2-tailed unpaired t test).
Fig 4.
A. Schematic of full-length HIV used in C-E. B. Flow cytometric plots of CD133-sorted cord blood HSPCs cultured for 7 days and stained as in Fig 1D. Cells gated in left plot were overlaid on the total live population on the right. C. Representative flow cytometric plots of cord blood-derived CD133-sorted cells expanded for four days, infected with the indicated virus and harvested 2 days post-infection. Gag + cells are overlaid onto CD34 versus CD133 plots for the total live cell population and the percentage of Gag+ cells in the CD133high and CD133low regions is indicated in the overlay. D. Summary graph of relative CD133 expression by mean fluorescence intensity (MFI) on HSPCs infected with the indicated HIV. Results were normalized to those for YU-2 infected cells for each experiment. E. Summary graph showing the frequency of Gag+ cells falling into CD133bright gate. Data was compiled from four cord blood experiments; mean ± standard deviation is indicated (****p < 0.0001, 2-tailed paired t-test).
Fig 5.
Targeting of intermediate progenitors by CCR5-tropic Envs is a conserved property extending to a transmitted/founder virus.
A. Schematic of HIV-7/SF-GFP construct and HIV envelope plasmid used to construct pseudotyped viruses used in C-E. B. Summary table of envelope proteins used to pseudotype HIV-7/SF-GFP virus. C. Representative flow plots of cord blood-derived CD133-sorted cells expanded for four days, transduced with the indicated virus and harvested 3 days post-infection for flow cytometric analysis. In each right panel, GFP+ cells were overlaid onto plots of the total cell population and the percentage of GFP+ cells in the CD133high and CD133low regions is indicated. Gates were determined based on isotype control antibody staining (top panel). D. Summary graph of CD133 MFI for experiments performed as in C. Results are compiled from 11 cord blood experiments. Mean ± standard deviation is indicated; n≥3 for each envelope. 2-tailed Student’s t-test indicates significance for each HIV envelope with respect to VSV-G (**p < 0.01,***p < 0.001, ****p < 0.0001). E. Data from (D) compiled by tropism. Mean ± standard deviation is indicated; one-way ANOVA, p = 0.0002, with Tukey’s Multiple Comparisons Test indicated (**p < 0.01 and ***p < 0.001).
Fig 6.
HSPCs with greater CD4 expression are preferentially infected by HIV Envs.
A. Representative flow cytometry plots and gating strategy for cord blood-derived CD133-sorted cells infected with virus containing the indicated envelope protein and harvested 3 days post-infection. Gating for CD4 was determined by the inclusion of 1% of cells stained with an isotype control antibody (gray). For GFP plots, numbers indicate the percentage of GFP+ events. B. Summary graphs depicting the ratio of infected cells in CD4high versus CD4low/- subsets of cord blood-derived HSPCs infected and analyzed as in part A. For SV16, two replicates had 0.0% infection in the CD4low/- gate leading to an undefined ratio, so 30.0 was used as a conservative estimate of the ratio. Data from 11 uninfected cord blood experiments. Mean ± standard deviation is indicated; n≥3 for each envelope. Results were compared to infection by VSV-G pseudotyped viruses and p values were determined using 2-tailed Student’s t-test (***p<0.001, ****p<0.0001). C. Data from B but compiled by tropism. Mean ± standard deviation is indicated; one-way ANOVA, p<0.0001, with Tukey’s Multiple Comparisons Test indicated (***p<0.001 and ****p<0.0001).
Fig 7.
CD4high HSPCs include progenitors with multi-lineage potential.
A. Representative flow plots of Sort 1 and Sort 2 bone marrow-derived HSPCs sorted using fluorescence activated cell sorting into CD4high and CD4low/- subsets. B and C. Sorted HSPCs were analyzed by methylcellulose colony formation assays. Mean ± standard deviation is indicated for counts from three blinded investigators; 2-tailed Student’s t-test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). CFU-E, erythroid; CFU-GM, granulocyte/macrophage and CFU-GEMM, multilineage.
Fig 8.
CD4high HSPCs include progenitors with multi-lineage potential.
A. Flow cytometric analysis of differentiation markers expressed on bone marrow HSPCs purified as described in Fig 1A. For the two right-most panels, numbers indicate percentage of total CD34+ events in each sort falling into that gate. B. Summary table of frequencies for each phenotypic gate as shown in A. Lineage outputs based upon Doulatov et al [9]. (Abbreviations: HSC, hematopoietic stem cell; MPP, multipotent progenitor; MLP, multilymphoid progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte/erythrocyte progenitor; GMP, granulocyte/monocyte progenitor; B-NK, B and NK cell progenitor; MDC, macrophage and dendritic cell; EMK, erythroid and megakaryocyte) C. Summary graphs depicting the percentage of each subset of the total CD34+ cells in each sort. Cells were isolated from cord blood (n = 5, circles) or bone marrow (n = 2, squares). For three experiments (2 cord blood and 1 bone marrow), lineage-positive cells were physically or analytically excluded from analysis (open symbols). Mean ± standard deviation is indicated; 2-tailed Student’s t-test (*p<0.05, ***p<0.001, ****p<0.0001).
Table 5.
HIV isolation from CD4-negative lineages from a subset of ten donors.
Fig 9.
Evidence for transmission of proviral genomes from multipotent CD4+ HSPCs to differentiated peripheral blood cells.
A. Flow cytometric plots showing purity of CD4-negative lineages containing provirus identical to HSPC-derived provirus. “Pre” indicates the cell population post CD4-bead depletion and prior to fluorescence activated cell sorting (FACS). “Post” indicates the cell populations following FACS. Numbers in the upper right corner indicate the frequency of cells in that quadrant. The frequency of CD4+ cells that were also CD3+ by gating was 0% (see also Table 5). B and C. Phylogenetic trees showing genetic relationships amongst amplicons. HIV RNA shown is cell-associated (Fig 10B). Arrows indicate location of identical amplicons shown in Fig 10. Red lines indicate identical sequences. Scale indicates nucleotide substitutions per site. ACH2, 89.6, BaL, YU-2, HXB2 and NL4-3 are subtype B HIVs. 84ZR085 (84ZR) and 94UG114 (94UG) are subtype D HIV molecular clone outgroups [32]. Phylogenetic analysis was performed by maximum likelihood method using MEGA7[33] and history was inferred based on the Hasegawa-Kishino-Yano model [34]. The tree with the highest log likelihood is shown. Abbreviations: PBMC, unfractionated peripheral blood mononuclear cells; BMMC, bone marrow mononuclear cell (column flow-through).
Fig 10.
Identical HIV proviral genomes in CD4-negative progeny.
A and C. Diagrammatic representation of clonal, defective proviral genomes from the indicated donor 431000 cell types. Colored bars indicate regions of identity. Dotted lines indicate location of deletion. White space indicates region where no sequence information is available. mRNA is cell associated from part B. B, (upper panel) Flow cytometric analysis of purified CD4-negative and CD4+ populations isolated from donor 431000 PBMCs. (Lower panels) Summary graphs of HIV-1 RNA isolated from supernatant (left) and cells (right) following treatment with PMA and ionomycin. Dashed line indicates limit of detection. *p<0.05 D and E. Provirus found in CD4-negative progeny can be recovered in two independent donations separated by months. D. Flow cytometric plots showing purity of CD4-negative lineages. “Pre” indicates the cell population post CD4-bead depletion and prior to fluorescence activated cell sorting (FACS). “Post” indicates the cell populations following FACS. Numbers in the upper right corner indicate the frequency of cells in that quadrant. The frequency of CD4+ cells that were also CD3+ by gating was 0% (see also Table 5). E. Phylogenetic tree showing genetic relationships amongst amplicons from two separate donations (408 and 428). Arrow indicates location of identical 693 bp gag amplicon from CD8 cells. (The region of identity extended through the non-translated LTR region of the amplicon, which was not included in the phylogenetic analysis.) Red lines indicate identical sequences. Scale indicates nucleotide substitutions per site. 89.6, BaL, YU-2, HXB2 and NL4-3 are subtype B HIVs. 84ZR085 (84ZR) and 94UG114 (94UG) are subtype D HIV molecular clone outgroups [32]. Phylogenetic analysis was performed by maximum likelihood method using MEGA7 [33] and history was inferred based on the Hasegawa-Kishino-Yano model [34]. The tree with the highest log likelihood is shown. Abbreviations: PBMC, unfractionated peripheral blood mononuclear cells; BMMC, bone marrow mononuclear cell (column flow-through).