Additional file 3: of Increased ex vivo cell death of central memory CD4 T cells in treated HIV infected individuals with unsatisfactory immune recovery

Figure S2. A model for CD4 homeostasis in Immunodiscordant individuals. Immunoconcordant individuals show a full recovery of CD4 T-cell counts with a high representation of naïve cells, and balanced frequencies of different memory subsets (upper panels). The profile of CD4 T-cell maturation in immunoconcordant individuals (green line) overlaps with that of HIV uninfected individuals (blue line in upper right plot) in which CCR7− CD4 T cells show increased turnover and short live [22]. Conversely,immunodiscordant individuals (lower panels) show a reduced naïve subset with no signs of altered turnover [14] that limits the generation of new memory cells. Among memory subsets, central memory cells are also reduced and subjected to homeostatic pressure to generate new cells [14] increasing TCM→TTM transition (and lowering TCM/TTM ratios) and increasing TCM cell death. TTM and TEM cells emerging from TCM cells also show higher sensitivity to cell death (Figure 2D), explaining the lack of accumulation of terminally differentiated cells in these subjects. This scenario results in a shifted profile of CD4 T-cell maturation (red line) compared to healthy individuals (blue line in lower right plot)

HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4-7

Ls appear as an unstained population with high forward scatter values (in orange); while CD4 T cells were identified by fluorescent staining and low forward scatter values (in purple). Events displaying bright fluorescence and forward scatter values consistent with MOLT cells were defined as cellular conjugates (in red, gate MOLT-CD4 in the figure). The percentage of CD4 T cells forming conjugates in the absence or the presence of Leu3a, or under continuous shacking condition is shown in each plot. Panel B shows the quantification of cellular conjugates in the presence of the following inhibitors: mAbs against the adhesion molecules ICAM-1 (RM3A5), ICAM-3 (140.11) and LFA-1 (R7.1), coreceptor antagonists AMD3100 and TAK779 (all at 10 μg/ml), gp41 inhibitor C34 (1 μg/ml) or Leu3a (5 μg/ml). Data are Mean ± SD of 3 independent experiments including cells from 3 different donors. Asterisks indicate a significant inhibition in the formation of conjugates in the presence of Leu3a and under shaking conditions.<p><b>Copyright information:</b></p><p>Taken from "HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4"</p><p>http://www.retrovirology.com/content/5/1/32</p><p>Retrovirology 2008;5():32-32.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2359761.</p><p></p

Additional file 2: of Increased ex vivo cell death of central memory CD4 T cells in treated HIV infected individuals with unsatisfactory immune recovery

Table S1. A. The multivariate relationship between TN, TCM, TTM, TEM, TTD and Total Death were determined by multiple linear regression. B. Linear regression model shows no statistical differences between group of patients (Discordant/Concordant) in the association of Total Death with TTM

Additional file 1: of Increased ex vivo cell death of central memory CD4 T cells in treated HIV infected individuals with unsatisfactory immune recovery

Figure S1. Gating strategy followed to identify T-cell maturation stages. Panel A. CD4 and CD8 T cells were gated and analyzed for the expression of CD27 and CD28. For CD4 T cells, terminally differentiated (TTD) cells were defined as CD28–CD27−, while effector memory (TEM) cells were CD28+CD27−. Double positive cells were further analyzed for CCR7 and CD45RA expression to identify naïve (TN) cells (CCR7+CD45RA+), central memory (TCM) cells (CCR7+CD45RA−) and transitional Memory (TTM) cells (CCR7–CD45RA−). Panel B. For CD8 T cells, the general strategy was similar, unless for the definition of TEM cells that was CD27+CD28– cells. The expression of CD57 was analyzed in the whole CD4 or CD8 T-cell population (lower left dot plot in each panel) to define replicative senescence (CD28–CD57+ cells). In addition the expression of CD57 in each subset was also evaluated (lower dot plots in each panels)

Additional file 3: of Increased ex vivo cell death of central memory CD4 T cells in treated HIV infected individuals with unsatisfactory immune recovery

Figure S2. A model for CD4 homeostasis in Immunodiscordant individuals. Immunoconcordant individuals show a full recovery of CD4 T-cell counts with a high representation of naïve cells, and balanced frequencies of different memory subsets (upper panels). The profile of CD4 T-cell maturation in immunoconcordant individuals (green line) overlaps with that of HIV uninfected individuals (blue line in upper right plot) in which CCR7− CD4 T cells show increased turnover and short live [22]. Conversely,immunodiscordant individuals (lower panels) show a reduced naïve subset with no signs of altered turnover [14] that limits the generation of new memory cells. Among memory subsets, central memory cells are also reduced and subjected to homeostatic pressure to generate new cells [14] increasing TCM→TTM transition (and lowering TCM/TTM ratios) and increasing TCM cell death. TTM and TEM cells emerging from TCM cells also show higher sensitivity to cell death (Figure 2D), explaining the lack of accumulation of terminally differentiated cells in these subjects. This scenario results in a shifted profile of CD4 T-cell maturation (red line) compared to healthy individuals (blue line in lower right plot)

Mapping humoral responses.

<p>(A) Plasma from RP (n = 5) and VNP (n = 8) individuals were tested in triplicate for the recognition of consensus peptide V in ELISA. Data are arbitrary absorbance units per ml of plasma, boxes represent mean+/−SD. (B) Plasma from VNP patients 8 (upper left), 9 (upper right), 11 (lower left) and 16 (lower right) were assayed for the recognition of five different peptides displaying a consensus sequence covering 609–624 residues of gp41 or the equivalent patient-based envelope sequences. Two samples were analyzed for each patient, the earliest sample available (empty bars) and the sample (or closest sample for patient 9) of Env cloning (dark bars). Peptide sequences used in ELISA assays are shown in the left axis of each plot. The autologous population sequence from each patient obtained at the indicated timepoints (year-month) is also shown in top of each panel. Intrapatient sequence changes are highlighted. Data are mean+/−SD of triplicate samples.</p

Expression of NKp44L in CD4 T cells from RPs and VNPs.

<p>The <i>in vivo</i> expression of NKp44L was assessed in gated CD3+ CD4+ cells after staining thawed PBMC from RP (panel A) and VNP patients (panel B, upper plots). A representative healthy donor was also analyzed (left plot in panel B). PBMC from VNP individuals or from the healthy donor were also incubated for 5 h in the presence of the 3S consensus peptide V (lower plots in panel B). In each plot, values indicate the % of positive cells.</p

HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4-3

Ted in each histogram. Control histogram corresponds to background of staining. Panel B shows HIV transfer from 293T cells, transfected to produce HIV particles, to target CD4 T cells in the absence (light bars) or presence (grey bars) of NL4-3 Env expression. The effect on the addition of blocking antibodies against CD4 (Leu3a) and the gp41 inhibitory peptide C34 was also tested. Data (Mean ± SD) were obtained using cells from 3 different donors. Asterisk indicates a significant inhibition in HIV transmission in the presence of Leu3a within NL4-3 Env transfected 293T cells.<p><b>Copyright information:</b></p><p>Taken from "HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4"</p><p>http://www.retrovirology.com/content/5/1/32</p><p>Retrovirology 2008;5():32-32.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2359761.</p><p></p

HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4-6

ICAM-3, total LFA-1, the activated form of LFA-1 and CD4. Staining of the cell surface molecules was performed using monoclonal antibodies RM3A5, 140.11, R7.1, mAb24 and Leu3a respectively. The expression of HIV Env was evaluated in MOLT cells using pooled serums from HIV infected individuals. Figure shows the expression of each individual antigen (empty peaks) with the negative control of staining (solid peaks). Histograms show a single representative experiment.<p><b>Copyright information:</b></p><p>Taken from "HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4"</p><p>http://www.retrovirology.com/content/5/1/32</p><p>Retrovirology 2008;5():32-32.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2359761.</p><p></p

HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4-5

Ure. Cells were gated as CD3+/CD8-/CD4+ PBMCs (non-productively infected cells, upper histograms) or CD3+/CD8-/CD4- PBMCs (productively infected cells, lower histograms). Histograms show a representative experiment displaying the expression of each individual antigen (solid peaks) with the negative control of staining (empty peaks). In panel B, purified productively HIV infected CD3+/CD8-/CD4- PBMCs were cocultured with CMFDA-labeled primary unstimulated CD4 T cells. After 24 hours of coculture cells were stained with anti-CD45RO and anti-HIV CA p24 antibodies. HIV transmission was measured in both memory CD45RO+ (RO+ solid bars) and naive (RO-, empty bars) subsets in the presence of Leu3a, C34 and a panel of blocking agents against adhesion molecules used at the same concentrations as in Figure 3 (whole IgGs against ICAM-1, LFA-1 and ICAM-3, soluble ICAM-1 or Fab fragments of the anti-ICAM-3 mAb 140.11). Values are Mean ± SD of data corresponding to up to 6 different donors. Asterisks indicate significant differences (p < 0.05) from control or between divalent and monomeric blocking agents.<p><b>Copyright information:</b></p><p>Taken from "HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4"</p><p>http://www.retrovirology.com/content/5/1/32</p><p>Retrovirology 2008;5():32-32.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2359761.</p><p></p