List of primary antibodies and antigen retrieval methodologies.

<p>A list of ID numbers for genes and proteins used in the paper: Desmin: 1674, CD3: 916, Interleukin-7: 3574, CD45RA: 151460, Caspase3: 600636, Collagen Type I: 120150, CD4: 186940, CD8: 925, Ki-67: 176741.</p

Incomplete LT restoration is associated with high level of apoptosis in naïve T cell populations and incomplete restoration of naïve T cells.

<p>A. Confocal images of LN sections from uninfected subjects and patients receiving 6 months of HAART at either acute or AIDS phase of infection, immunofluorescently stained for CD45RA (red) and CD3 (blue), showing the different extent of restoration of naïve T cells. Scale bar, 10 µm. B. Quantification of number of naïve T cells before and after HAART at each stage of infection, showing the different kinetics of restoration of naïve T cells. Error bars represent the s.d. C. Confocal images of LN sections from patients receiving 6 months of HAART at either acute or AIDS phase of infection immunofluorescently stained for CD45RA (red), CD3 (blue) and TUNEL staining of apoptotic cells (green), showing the higher level of apoptosis in naïve T cell populations after 6 months of HAART when HAART was started during chronic phase of infection. Scale bar, 10 µm. D. Quantification of average number of apoptotic naïve T cells before and after HAART at each stage of infection, showing the different kinetics of decrease of apoptotic naïve T cells.</p

SIV vRNA+ Cells in the Lungs of Macaques Infected with SIV¹.

<p>¹Totally 38 infected and 6 uninfected rhesus macaques were used in this study, and vRNA+ cells were quantified by ISH.</p><p>²DPI = Days post infection.</p><p>³Virus load in plasma was detected by real-time RT-PCR.</p><p>⁴SIV vRNA⁺ cells in lungs were detected using ISH with SIV-specific riboprobes:</p><p>−ith SIV-s⁺ cells/mm²</p><p>+, 5–50 vRNA⁺ cells/mm²</p><p>++, 51–100 vRNA⁺ cells/mm²</p><p>++++, >200 vRNA⁺ cells/mm².</p><p>SIV vRNA+ Cells in the Lungs of Macaques Infected with SIV<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125500#t001fn001" target="_blank">¹</a>.</p

Naïve T cells need to contact FRCs to get access to IL-7 for survival.

<p>(A–C) IL-7 is produced and presented on the surface of stromal cells. A-B. Confocal images of monolayer of fixed and permeablized stromal cells isolated from human tonsil immunofluorescently stained for (A) IL-7 (green) or (B) desmin (green) and DAPI (blue) at one-day post passage. Scale bar, 30 µm. C. Confocal image of live stromal cells (DAPI: blue) staining showing the IL-7 (green) on the surface of stromal cells. Scale bar, 10 µm. D-E. FRC-like stromal cells enhance the survival of naïve T cells via IL-7. D. Triple fluorescently stained activated caspase 3⁺ (green), CD45RA⁺ (red) and CD3⁺ (blue) cells in an ex vivo culture system showing that stromal cells enhance the survival of naïve T cell by mechanisms dependent on IL-7 and cell contact. 2x10⁵ lymphocytes from human tonsil were cultured with or without stromal cells for 2–3 days. Naïve T cell apoptosis is reduced in co-cultures with stromal cells (+ stromal cells) compared to cultures without stromal cells. Apoptosis in the naïve T cell population increases with IL-7 blocking antibody (anti-IL-7) or when lymphocytes are separated from stromal cells by a transwell filter (Filter) compared to co-cultures with stromal cells. Scale bar, 60 µm. E. Quantification of the percentages of activated caspase 3⁺CD45RA⁺CD3⁺ naïve T cells in total T cell population at day 2 and day 3 cultures. Values are the mean of the percent apoptotic naïve T cells ± s.d. ANOVA comparison was done on the average percentages of day 2 and day 3.</p

The depletion of CD4+ T cells, but not macrophages, in lung tissues during SIV infection.

<p>CD4+ T cells (A-F) and CD68+ macrophages (G-I) in the lung tissues of uninfected and infected macaques detected using immunohistochemical staining and quantified using Aperio Spectrum Plus analysis program. The A-E are representative images of CD4+ T cells in uninfected lung tissues (A-C) and, infected at 10 days (D) and 12 weeks (E) PI respectively. (A) Digitized whole tissue section. (B) Magnified image from the box in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125500#pone.0125500.g003" target="_blank">Fig 3A</a>, where the brown corresponds to the stained CD4+ T cells. (C) The red/yellow marked-up regions correspond to the stained CD4+ T cells in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125500#pone.0125500.g003" target="_blank">Fig 3B</a> used for quantification with Aperio Spectrum Plus analysis program. The G-H are the representative images of pulmonary CD68+ macrophages in uninfected lung tissues (G) and infected at 12 weeks PI(H). The <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125500#pone.0125500.g003" target="_blank">Fig 3F and 3I</a> are the histograms of CD4+ T cell and macrophage quantification in lung tissues, respectively. X-axis shows the time of infection at 0 (n = 6), 3 (n = 6), 6 (n = 6), 10 (n = 4) days and 12 weeks (n = 4) PI, and y-axis shows the cell number expressed as per square millimeter of lung tissue. *Indicates significant differences from controls (*P<0.05, **P<0.01). Statistical analysis of cell amount per mm² tissue was performed with non-parametric Mann-Whitney U test.</p

Macrophages are the main SIV RNA+ cells in the lungs from very early (A) at 10 days PI to chronic infection at 5 months PI (B).

<p>SIV vRNA+ macrophages were distinguished from T cells by immunohistochemical staining for CD163. The red arrows indicate CD163+ vRNA+ cells identified by the overlying collection of silver grains (black dots); blue arrows indicate SIV RNA+ cells that are not macrophages in lung associated lymphatic tissue (C).</p

Collagen deposition and loss of the FRC network impede access to and source of IL-7 in HIV-1 infection.

<p>A. FRCs are the major producers of IL-7. LN sections (representative image for one HIV negative subject of 5) stained for desmin (red) and IL-7 (green). Merged confocal image shows co-localization of IL-7 and FRCs in T cell area. Scale bar, 10 µm. B–C. Collagen deposition and loss of the FRC network disrupts interaction between T cells and FRCs. Confocal images of LN sections from an uninfected subject (representative image for one subject of 5) immunofluorescently stained for desmin (green), collagen (red) and CD3 (blue). The merged image shows that FRCs colocalize with collagen and T cells are in contact with the FRC network (B). Confocal images of LN sections from a subject at AIDS stage (representative image for one subject of 6). The merged image shows that the loss of FRCs and associated collagen deposition leads to loss of the contact between FRCs and T cells, which instead contact mainly extra-FRC collagen. Scale bar, 20 µm (C). D. Confocal images of LN sections from HIV negative subjects and from subjects at different stage of HIV infection immunofluorescently stained for desmin (green) and collagen (red), showing the gradual loss of FRCs in the T cell zone within LTs, which is associated with extensive collagen deposition during HIV infection. Scale bar, 20 µm. E. Quantification of average amount of FRCs and collagen deposition at each stage of infection, showing the gradual loss of FRCs and collagen deposition. Error bars represent the s.d. F. Confocal images of LN sections from HIV negative subjects and from subjects at different stage of HIV infection immunofluorescently stained for IL-7 (green), showing that the gradual loss of IL-7 in the T cell zone is associated with gradual depletion of FRCs (E). Scale bar, 20 µm.</p

SIVmac251-specific CD8 T cell responses in selected animals of the study.

<p>^aND: Not Done</p><p>^bEC: Elite Controller</p><p>^cOI: Occult Infection.</p><p>SIVmac251-specific CD8 T cell responses in selected animals of the study.</p

Administration of GML (or placebo) for all study animals was once per day, except on each challenge day when treatment was given 1 hour prior to each of two separate inoculations of SIVmac251, resulting in two doses.

<p>There were 3 cohorts consisting of both treatment and control animals indicated by degree of shading (cohort 1 = lightest; cohort 2 = medium; cohort 3 = darker). Cohorts only differed in the scheduled time of pre-treatment and first and second challenge days. All cohorts received the same dose continuously for the time indicated. In addition, viral loads for uninfected animals were followed for at least one year after challenge 2.</p><p>Administration of GML (or placebo) for all study animals was once per day, except on each challenge day when treatment was given 1 hour prior to each of two separate inoculations of SIVmac251, resulting in two doses.</p