1. Volume 166, Issue 4, Pages 1004-1015 (August 2016)
Multiple Origins of Virus Persistence during Natural Control of HIV Infection 
Eli A. Boritz, Samuel Darko, Luke Swaszek, Gideon Wolf, David Wells, Xiaolin Wu, Amy R. Henry, Farida Laboune, Jianfei Hu, David Ambrozak, Marybeth S. Hughes, Rebecca Hoh, Joseph P. Casazza, Alexander Vostal, Daniel Bunis, Krystelle Nganou-Makamdop, James S. Lee, Stephen A. Migueles, Richard A. Koup, Mark Connors, Susan Moir, Timothy Schacker, Frank Maldarelli, Stephen H. Hughes, Steven G. Deeks, Daniel C. Douek 
Cell 
Volume 166, Issue 4, Pages (August 2016)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
HIV DNA and RNA Levels in Circulating CD4 T Cell Subsets from HIV Controllers
(A) HIV DNA copies detected by FCA per 106 TN, TCM, TTM, or TEM cell equivalents. The participant color code at right applies to all figures. Horizontal bars indicate median values in all figures.
(B) Numbers of HIV-infected TN, TCM, TTM, and TEM cells per milliliter of blood, calculated by adjusting values in (A) for CD4 counts and proportions of CD4 T cells in each subset.
(C) Copies of unspliced (circles) and spliced (diamonds) HIV RNAs in TCM, TTM, and TEM cells from HIV controllers, measured by qRT-PCR and normalized to values in (A). Undetectable values are plotted at the assay’s limit of detection (LOD) with open symbols. Wilcoxon signed-rank test p values are shown.
In (A) and (B), all Wilcoxon signed-rank test p values for comparisons between TN and memory subsets in HIV controllers are <0.0001, and Mann-Whitney p values for comparisons between HIV controllers and non-controllers are <0.001 for all cell subsets. In (C), all Wilcoxon signed-rank test p values for comparisons of unspliced or spliced RNA between subsets are >0.05.
See also Figures S1 and S2.
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4. Figure 2
HIV DNA Sequence Analysis in Circulating CD4 T Cell Subsets from HIV Controllers
(A) Number of distinct HIV DNA sequences detected in blood CD4 T cells from each HIV controller, with number of sequences matching one or more plasma virus in red.
(B) Average genetic distances between plasma HIV RNA sequences and HIV DNA sequences in TCM, TTM, or TEM cells in HIV controllers and non-controllers. Mann-Whitney p values are shown.
(C) Phylogenetic analysis of all sequences in the study, with labels colored by participant. The arrow shows one clade in which G-to-A hypermutated sequences from multiple participants are intermingled.
(D) Genetic compartmentalization between HIV DNA sequences in TCM, TTM, or TEM cells and plasma viruses in HIV controllers and non-controllers, as determined by Slatkin-Maddison testing with Bonferroni correction. Only 13 participants are shown for TCM cells because participant S1270 had no detectable HIV DNA in TCM cells. Fisher’s exact test p values for comparisons between controllers and non-controllers are shown.
(E) Normalized Shannon diversities of plasma viruses and HIV DNA sequences in TCM, TTM, and TEM cells from HIV controllers and non-controllers. Mann-Whitney p values for comparisons between controllers and non-controllers are in black. Wilcoxon signed-rank test p values for comparisons between subsets in HIV controllers are in green. All Wilcoxon signed-rank test p values for comparisons between subsets in non-controllers are >0.05.
See also Figures S3 and S4.
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5. Figure 3
Clonality of Cells and HIV DNA Sequences in Circulating CD4 T Cell Subsets from HIV Controllers
(A) Normalized Shannon diversities of T cell receptor beta (TCRB) sequences from TCM, TTM, and TEM cells in HIV controllers. Wilcoxon signed-rank test p values are shown.
(B) Average genetic distances of plasma virus and TCM, TTM, and TEM cell-associated HIV DNA sequences from most recent common ancestral (MRCA) sequences in HIV controllers and non-controllers. Wilcoxon signed-rank test p values are shown.
(C) Number of recurrent HIV DNA sequences detected in circulating CD4 T cells of each HIV controller, with number of distinct sequences matching one or more plasma virus sequence in red.
(D) Correlation between the abundance in blood of each recurrent HIV DNA sequence in circulating CD4 T cells from HIV controllers (x axis) and the average genetic distance of that sequence to plasma viruses (y axis). Each symbol represents one distinct sequence and is colored by participant. Spearman r and p values are shown.
(E and F) HIV DNA sequences associated with expanded cellular clones in HIV controllers V907 (E) and S1349 (F), illustrated using dashed borders within phylogenetic trees. Gene locus names corresponding to the HIV integration sites in these expanded clones are shown. Each number in parentheses represents the percentage of all copies of HIV DNA in blood CD4 T cells from the individual deriving from the indicated HIV integrant. Sequences from participant V907 showing G-to-A hypermutation and associated with expanded cellular clones are shown as detached branches; other hypermutated sequences are omitted for clarity. The large sequence cluster in participant S1349 is shown separated from the tree for ease of viewing; an arrowhead shows the position of this sequence on the tree.
See also Table S2.
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6. Figure 4
Subset Distribution and Genetic Attributes of HIV DNA Sequences in Circulating CD4 T Cells
(A) Subset distribution of HIV sequences from presumptive expanded CD4 T cell clones in HIV controllers.
(B) Distribution of HIV DNA sequences from presumptive expanded clones across subsets within TCM, TTM, and TEM populations defined by CXCR5, CCR6, and CD57. Each row represents one sequence, labeled by participant and a unique number. Yellow indicates that the sequence was detected in the given subset. Italics indicate G-to-A hypermutated sequences.
(C) Levels of HIV DNA sequences in circulating CD4 T cells from HIV controllers and non-controllers categorized according to the number of occurrences (Repeat, >1 occurrence; Singlet, one occurrence) and the presence or absence of G-to-A hypermutation (HM, hypermutation detected; no mut., no lethal genetic defect detected). To allow display of these wide-ranging values—including several values of zero—on a logarithmic scale, each plotted value represents the measured value + 1. Mann-Whitney p values are shown. Sequences with lethal genetic defects other than hypermutation were rarely detected.
See also Table S2.
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7. Figure 5
RNA Sequences of Virions Induced from Circulating TCM, TTM, and TEM Cells in HIV Controllers
(A) x-y plots: proximity of each HIV DNA sequence from circulating CD4 T cells or virions induced from TCM, TTM, and TEM cells to the participant’s MRCA (x axis) and nearest genetic neighbor (NN) from plasma virus (y axis). HIV DNA sequences detected once are shown as gray dots; recurrent HIV DNA sequences are shown as black circles scaled by the abundance of the sequence; and sequences detected in induced virion RNA but not in DNA from a second aliquot of cells (i.e., “unique induced” viruses) are shown as stars. Where an induced virion RNA sequence matched a recurrent HIV DNA sequence from blood cells, the circle corresponding to that DNA sequence is filled. The arrow shows one sequence from participant S1270 containing a lethal deletion within gp120. For alignment production, this deletion was filled with the participant’s consensus sequence; the measured genetic distance of this sequence to the plasma virus NN is therefore an underestimate. Hemispheres: quantities of unique induced proviruses and all other HIV DNA sequences in circulating CD4 T cells from HIV controllers. Plots are scaled to show relative levels of HIV DNA in circulating CD4 T cells from the five participants. For participants with undetectable unique induced viruses from blood cells, the LOD of this measurement is shown.
(B) Sequences in virions induced from blood cells in participant S1270. Cyan indicates sequences from an initial experiment; magenta indicates sequences from a repeat experiment. Sequences in which relative insertions were excised or deletions filled in alignment production are shown with gray arrows.
See also Figure S5.
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8. Figure 6 HIV DNA and RNA Levels in LN CD4 T Cell Subsets
(A) Levels of HIV DNA measured by FCA in LN non-GC TFH and GC TFH; non-follicular LN subsets (CD57+ subset collected only for participant LIR02); and blood TCM, TTM, and TEM subsets.
(B and C) Copies of unspliced (B) and spliced (C) HIV RNAs in LN memory CD4 T cell subsets, measured by qRT-PCR and normalized to values in (A). Each cell subset is shown with a unique shape and colored by participant. Undetectable values are plotted at the assay LOD with open symbols.
(A)–(C) include results from blood CD4 TCM, TTM, and TEM subsets that are also shown in Figure 1. Mann-Whitney p values are shown.
(D) The percentage of all HIV DNA copies in LN memory CD4 T cells from each participant detected in each subset.
(E) HIV RNA+ LN cells detected by in situ hybridization using 35S-labeled riboprobes in two study participants. White arrows indicate examples of HIV RNA+ cells. Some such cells were associated with areas of diffusely increased signal corresponding to the follicular dendritic cell network (follicular); others were outside such areas (extrafollicular).
See also Figures S6 and S7 and Table S3.
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9. Figure 7
Analysis of HIV DNA Sequences and Host Gene Expression in LN CD4 T Cell Subsets from HIV Controllers
(A) Left: proximity of each HIV DNA sequence from LN GC and non-GC TFH cells to each participant’s MRCA (x axis) and plasma virus NN sequence (y axis). HIV DNA sequences from blood cells are shown as in Figure 5; HIV DNA sequences from GC and non-GC TFH cells are shown as green-filled circles scaled by their relative abundance in LN. Sequences from GC and non-GC TFH cells were compared to those from blood cells for genetic distance to plasma virus NN and MRCA sequences. Mann-Whitney p values for these comparisons are shown. Right: proximity of each HIV DNA sequence from non-TFH TCM and TEM LN cells and TCM, TTM, and TEM blood cells to the NN sequence from GC and non-GC TFH cells. All Mann-Whitney p values for comparisons between LN cell subsets and blood cell subsets are <0.05.
(B) PCA of transcriptomes from blood (PB) and LN CD4 T cell subsets. Clusters of symbols representing samples of the same cell subset from multiple study participants are demarcated with dashed boundaries.
See also Figures S6 and S7 and Table S3.
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10. Figure S1
Gating for Fluorescence-Activated Cell Sorting of CD4 T Cell Subsets from Blood, Related to Figure 1
(A–F) Leukocytes (A) not part of multi-cell conjugates (B) that were viable and stained with the T cell marker CD3 (C) but not myeloid cell markers CD14 and CD11c (D), the cytotoxic T cell marker CD8 (E), or lineage dump markers including CD20, CD56, and TCR-γδ (F) were divided by CD27 and CD45RO staining and collected as TN (G, top-left gate) or TEM (G, bottom gate) subsets.
(H) Cells that were double-positive (DP; G, top-right gate) for CD27 and CD45RO were further divided by CCR7 staining (H) into TCM (top) and TTM (bottom) subsets. Both CD4high and CD4low cells (E) were included in sorted subsets to ensure collection of any cells with CD4 downregulation due to active HIV infection and Nef activity. Numbers on plots represent percentages of plotted cells falling within the gates shown.
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11. Figure S2
Fluorescence-Assisted Clonal Amplification, Related to Figure 1
Cell-associated HIV DNA or plasma virion cDNA was plated in 384-well plates at an estimated 0.3 copies/well and amplified by PCR using primers flanking a fragment of the env gene including HXB2 base positions , in the presence of SYBR Green I as a marker of dsDNA.
(A and B) Wells showing fluorescence amplification (A) and amplicon melting temperatures between °C (B) were considered to be positive.
(C) Specificity of FCA analyzed by agarose gel electrophoresis. Lanes corresponding to FCA wells from (A) and (B) that contained amplification products with melting temperatures in the target range are indicated with “+” symbols. M, molecular weight marker. Gel images were cropped to include only the relevant size range. Results are shown for a sample consisting of linearized HIV plasmid diluted into HIV-negative PBMC DNA at 1 copy per 1,000 cell equivalents.
(D) The sensitivity of FCA was similar to the sensitivity of HIV gag taqman PCR for single copies of HIV in equal aliquots of this sample run in 96 PCR wells for each test (D).
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12. Figure S3
Phylogenetic Trees Representing Single-Copy HIV Sequence Results Obtained by FCA from Plasma Virions and from Cell-Associated DNA of CD4 TCM, TTM, and TEM in HIV Controllers, Related to Figure 2
Each tree represents results from a single study participant, rooted on the HXB2 sequence. Sequences from each participant showing G-to-A hypermutation are indicated as detached branches in the box associated with that participant’s tree. Sequences in which relative insertions were excised or deletions filled in alignment production are shown with gray arrows.
(A–N) Participant identifiers are as follows: (A) S1492, (B) S1270, (C) S1788, (D) S1548, (E) LIR04, (F) V907, (G) S1475, (H) S1349, (I) S1495, (J) S1541, (K) LIR02, (L) LIR01, (M) V912, and (N) LIR03.
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13. Figure S4
Phylogenetic Trees Representing Single-Copy HIV Sequence Results Obtained by FCA from Plasma Virions and from Cell-Associated DNA of CD4 TCM, TTM, and TEM in Non-controllers, Related to Figure 2
Each tree represents results from a single study participant, rooted on the HXB2 sequence. Sequences from each participant showing G-to-A hypermutation are indicated as detached branches in the box associated with that participant’s tree. Sequences in which relative insertions were excised or deletions filled in alignment production are shown with gray arrows.
(A–F) Participant identifiers are as follows: (A) V016, (B) V257, (C) V621, (D) V667, (E) V965, and (F) V723.
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14. Figure S5
Calculated Numbers of Cells from Presumptive Expanded CD4 T Cell Clones Included in Virion Induction Cultures of CD4 TCM, TTM, and TEM Cells from HIV Controllers, Related to Figure 5
Each presumptive expanded clone is defined by a distinct HIV DNA sequence (y axis labels). Blue bars indicate TCM cultures, green bars indicate TTM cultures, and red bars indicate TEM cultures. Filled bars indicate sequences also detected in virions induced from the given CD4 T cell subset.
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15. Figure S6 Study Participant LIR01, Related to Figures 6 and 7
(A–H) Gating for fluorescence-activated cell sorting (FACS) of CD4 T cell subsets from lymph node. Leukocytes (A) not part of multi-cell conjugates (B) that were viable and stained with the T cell marker CD3 (C) but not B cell marker CD20 (D), myeloid cell markers CD14 and CD11c (E), or the cytotoxic T cell marker CD8 (F) were divided by CD27 and CD45RO staining and collected as TN (G, top-left quadrant) or TEM (G, bottom quadrants) subsets. Cells that were double-positive for CD27 and CD45RO (G, top-right quadrant) were further divided by CD57 and PD1 staining (H) into CD57+ TCM (top-left quadrant), GC TFH (top-right quadrant) and non-GC TFH (bottom-right quadrant) subsets. Both CD4high and CD4low cells (F) were included in sorted subsets to ensure collection of any cells with CD4 downregulation due to active HIV infection and Nef activity. Numbers on plots represent percentages of plotted cells falling within the gates or quadrants shown.
(I) Ex vivo quantification of HIV DNA in memory CD4 T cell subsets from lymph node and blood. Undetectable values are plotted at the assay LOD with open bars.
(J) Average pairwise genetic distances among plasma virus sequences from all study donors, with the value for participant LIR01 shown in dark blue.
(K) Average genetic distances to MRCA of plasma virus sequences from all study donors, with the value for participant LIR01 shown in dark blue. C, HIV controllers; NC, non-controllers.
(I) includes results from blood CD4 TCM, TTM, and TEM subsets that are also shown in Figure 1. (K) shows results that are also shown in Figure 3.
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16. Figure S7
Correlation between the Relative Genetic Proximity of Blood TCM Cell-Associated HIV DNA Sequences to the Plasma Virus and Plasma HIV RNA Levels in HIV Controllers, Related to Figures 6 and 7
Genetic proximity to the plasma virus was defined as the reciprocal of the average genetic distance between HIV DNA sequences in a given subset and plasma virus RNA sequences. The genetic proximity to the plasma virus of HIV DNA sequences from the TCM subset was normalized to the corresponding value for HIV DNA sequences from the TEM subset in the same individual to determine the relative genetic proximity of TCM cell-associated HIV DNA sequences to the plasma virus for that individual. For participant S1270, who had no detectable HIV DNA in blood TCM cells, the relative genetic proximity of blood TCM cell-associated HIV DNA sequences to the plasma virus was set equal to the lowest value among the remaining participants. This was done to reflect the absence of HIV DNA sequences closely related to the plasma virus within the blood TCM cell subset in this individual. Spearman r and p values are shown.
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