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)

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)

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 CD4-1

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

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 CD4-4

Fied primary CD4 T cells. After 2 h of coculture, cells were stained with anti-CD45RO and anti-HIV p24 antibodies. Analysis of HIV transfer in the absence (left graph) or presence (right graph) of the RM3A5 blocking mAb against ICAM-1 was performed after gating separately CD45RO and CD45RA CD4 T cells. Panel B shows the expression of HIV Env (right) and ICAM-1 (left) in 293T transfected cells (empty peaks), solid peaks correspond to negative controls of staining. Panel C shows HIV transfer from 293T cells transfected with an Env defective and an NL4-3 Env plasmids, to CD45RO- (RO-) and CD45RO+ (RO+) target cells in the absence (light bars) or presence (grey bars) of ICAM-1 expression. HIV transmission was again measured in the absence (left graph) or the presence (right graph) of the blocking RM3A5 antibody against ICAM-1. Values are Mean ± SD of 3 experiments performed with CD4 T cells from 3 different donors. Asterisks denote significant differences in HIV transmission to the CD45RA CD4 T subset compared to CD45RO cells (Panel A and C). Significant differences intrasubsets induced by ICAM-1 expression are also indicated by asterisks in panel C, while ns denotes no statistical significance.<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 CD4-2

Ry CD4 T cells with uninfected MOLT (white bars), MOLT NL4-3 (grey bars) or MOLT BaL cells (dark bars). Concentrations were 10 μg/mL for all mAbs against cell surface molecules, except for Leu3a (5 μg/mL) and C34 (1 μg/mL). Data are Mean ± SD of 3 independent experiments employing cells of 3 different donors. Asterisks indicate a significant inhibition in HIV transmission in the presence of Leu3a.<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 CD4-3

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

Evolution of VL and CD4 T cell counts in selected viremic non-progressor patients.

<p>The time course of absolute CD4 T cell counts (grey circles) and plasma VL (black diamonds) are plotted for the different periods off treatment corresponding to patients 8,9,11 and 16. For patient 8 the pre and post treatment periods are also depicted (grey areas). Arrows indicate plasma samples used for Env cloning.</p

Analysis of the NKp44L induction by cloned envelopes.

<p>(A) Graphic representation of sequence variability in gp41 regions surrounding the 3S epitope. The picture has been generated using the weblogo software (<a href="http://weblogo.berkeley.edu/" target="_blank">http://weblogo.berkeley.edu/</a>), representing polar amino acids in red, basic in blue, acidic in green and hydrophobic in black. (B) NKp44L expression in CD4 T cells from representative donors after incubation with Env-defective pseudoviruses (pSG3, negative control), with the 3S consensus peptide V, or with pseudoviruses bearing the following HIV Env: NL4-3, BaL (positive controls). The effect of selected Env clones from VNPs (upper panels) or RPs (lower panels is shown) Empty peaks correspond to control staining of untreated CD4 T cells. (C) The effect of a polyclonal anti 3S antibody on NKp44L expression induced by several Env clones is shown. Values represent the relative expression of NKp44L in the surface of CD4 T cells normalized to the effect of the 3S synthetic peptide (100%). Data show the effect of NL4-3 and BaL envelopes (Ctrl-Env) and a total of 8 patient-derived Env clones.</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 CD4-0

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