Lymph nodes are enriched for CD4⁺PD-1^hi T cells.

<p>(a) Representative dot plot b) Mean fluorescence intensity (MFI) of PD-1 expression on lymph node CD4⁺ and CD4^– T cells subsets. c) Relative proportions of CD4⁺PD-1^hi T cells and CD4^– PD-1^hi T cells in LN and d) relative proportions of CD3 gated CD4⁺PD-1^hi T cells in LN and peripheral blood mononuclear cells (PBMC).</p

CD4⁺PD-1^hi T cells significantly correlates with CD20⁺Bcl6⁺Ki-67⁺IgG⁺ B cells in LN.

<p>(a) Representative dot plots showing the gating strategy used for analyzing Bcl6 and Ki-67 expression on IgG⁺ B cells in the lymph node (LN) and b) relative proportions of IgG⁺ B cells in LN expressing Bcl6 and Ki-67. Correlation between LN CD4⁺PD-1^hi T cells and c) CD20⁺Bcl6⁺Ki-67⁺IgG⁺ B cells and d) CD20⁺Bcl6^–Ki-67⁺IgG⁺ B cells in LN.</p

CD4⁺PD-1^hi T cells are predominantly CCR7^lo, Bcl6⁺, IOCS⁺ and express a central memory phenotype.

<p>Representative dot plots showing the expression of a) Bcl6 and ICOS on CD3⁺CD4⁺ T cells and b) CD28 and CD95 expression on lymph node (LN) PD-1^hi and PD-1^–CD4 T cell subsets. c) Relative proportions of Bcl6⁺, ICOS+, CD28⁺CD95⁺ CD4⁺PD-1^hi T cells in LN. d) Representative histograms and e) Mean fluorescence intensity (MFI) of CCR7 expression on LN PD-1^–, PD-1^int and PD-1^hiCD4 T cell subsets.</p

CD4⁺PD-1^hi T cells express significantly high levels of CXCR5 and IL-21 mRNA <i>ex vivo</i>.

<p>(a) Representative dot plots showing the gating strategy and sort purity of CD4⁺PD-1^hi and CD4⁺PD-1^– T cells. b) Fold change in expression of CXCR5 and IL-21 mRNA levels in CD4⁺PD-1^hi T cells relative to CD4⁺PD-1^– T cells. mRNA expression was determined <i>ex vivo</i> in sorted cells by relative qRT-PCR.</p

Protection by SC-administered plant-derived PGT121 against intravaginal SHIVSF162P3 challenge in rhesus macaques as measured by log 10 vRNA (copies/ml of plasma).

<p>(A) Protection in 6/6 macaques that received 3.7–7.1 mg/kg doses of PGT121 given 24 hr prior to SHIV SF162P3 challenge. Insert shows a linear correlation between ID50 and dose (3.5–7.1 mg/kg) at 24 hr; ID50 values at doses of 3.86, 4.7 and 5.6 mg/kg were 1:202, 1: 612 and 1: 738 respectively). (B) Protection in 5/6 macaques administered 5 mg/kg SC immediately after SF162P3 challenge. (C) Four control macaques received no passive bnAb. Application of Fisher’s Exact Test showed that protection of animals given PGT121 prior to challenge (panel A) compared to the control group (panel C) was statistically significant (p = 0.03), while protection of animals given PGT121 after challenge was not (p = 0.19).</p

T-cell immune response of immunized macaques.

<p>IFN-γ ELISPOT assays were performed with PBMC samples from each of five Rhesus macaques immunized with 5 mg of hLAMP/<i>gag</i> DNA plasmid at weeks 0, 4, 14, 22 and 38. Each time point represents the average number of cells activated in separate assays by rGag protein or Gag 15- or 20-aa peptides, performed at two laboratories in duplicate or triplicate. Each bar is the average number of cells activated in 8 to 20 ELISPOT wells+/−S.D. and represents the number of IFN-γ⁺ cells per 10⁶ PBMCs.</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

Expression and trafficking of mouse and human LAMP/<i>gag.</i>

<p>(A) Western blot analysis of human 293 cells transfected with mLAMP/<i>gag</i> and hLAMP/<i>gag</i> plasmids. Samples were probed with an anti-Gag monoclonal antibody. The molecular weight markers are indicated on the right. (B) DCEK cells were transfected with the hLAMP/<i>gag</i> plasmid and stained with anti-Gag (red) or anti-MHC II (green) monoclonal antibodies. Digitally merged image shows co-localization of the hLAMP/<i>gag</i> chimera- and MHC II-containing compartments (yellow).</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

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