Viral load, CD4+ and CD8⁺ T-cell counts (n = 2).

<p>Viral load (triangles; 46731 and 41023 viral RNA copy/ml of plasma), absolute CD4⁺ T-cell count (squares) and absolute CD8⁺ T-cell count (circles) are shown for two time points. The grey arrows show the time points in which the virus was sequences and the black arrows indicate the time points in which the leukapheresis blood was taken and PBMCs were used in this study.</p

Greater frequency of polyfunctional IFN-γ producing CD8⁺ T-cells with autologous HIV-1 Nef peptides.

<p>(a) The frequency of CD8⁺ IFN-γ⁺ Gr-B⁺ CD107a⁺ detected by autologous HIV-1 Nef peptide and clade-B consensus Nef peptide stimulation. Functional subpopulations were defined by sequential gating of Gr-B and CD107a on CD3⁺ CD8⁺ IFN-γ⁺ cells. Each point in the graphs represents the response to one overlapping peptide. The responses of both subjects in both time points are shown together in each graph. (b) CD8⁺ IFN-γ⁺ Gr-B⁺ CD107a⁺ and CD8⁺ IFN-γ⁺ Gr-B⁻ CD107a⁺ T-cells detected by using autologous HIV-1 Nef and clade-B consensus Nef peptides in the chronic phase of infection. Each point in the graphs represents the response to one overlapping peptide. The responses of both subjects are represented in each graph. (c) Percentage of mono-, oligo- and polyfunctional CD8⁺ T-cells targeting pairs of consensus and autologous HIV-Nef peptides in an ICS assay. The graph represents an example of the difference in the function of stimulated CD8⁺ T-cells between the two subjects.</p

Autologous HIV-1 Clade-B Nef Peptides Elicit Increased Frequency, Breadth and Function of CD8⁺ T-Cells Compared to Consensus Peptides

<div><h3>Objective</h3><p>To determine the function and phenotype of CD8⁺ T-cells targeting consensus and autologous sequences of entire HIV-1 Nef protein.</p> <h3>Methods</h3><p>Multiparameter flow cytometry-based analysis was used to evaluate the responses of two treatment naïve HIV-infected individuals, during primary and the chronic phases of infection.</p> <h3>Results</h3><p>A greater breadth and magnitude of CD8 IFN-γ responses to autologous compared to clade-B consensus peptides was observed in both subjects. Cross recognition between autologous and consensus peptides decreased in both subjects during progression from primary to chronic infection. The frequencies of TEMRA and TEM CD8⁺ T-cells targeting autologous peptides were higher than those targeting consensus peptides and were more polyfunctional (IFN-γ⁺ Gr-B⁺ CD107a⁺).</p> <h3>Conclusions</h3><p>Our data indicate superior sensitivity and specificity of autologous peptides. The functional and maturational aspects of “real” versus “cross-recognized” responses were also found to differ, highlighting the importance of a sequence-specific approach towards understanding HIV immune response.</p> </div

Greater frequency of terminally differentiated IFN-γ producing CD8⁺ T-cells responding to autologous HIV-1 Nef peptides.

<p>(a) The frequency of terminally differentiated effector (CD8⁺ IFN-γ⁺ CD45RA⁺ CCR7⁻ CD27⁻) and effector memory (CD8⁺ IFN-γ⁺ CD45RA⁻ CCR7⁻ CD27⁻) T-cells targeting autologous HIV-1 Nef and clade-B consensus HIV-1 Nef peptides in both subjects. The phenotypes of IFN-γ producing cells were detected by sequential gating of CD45RA, CCR7 and CD27 on CD3⁺ CD8⁺ IFN-γ⁺ T-cells. Each point in the graphs represents the response to one overlapping peptide. The responses of both subjects in both time points are represented in each graph. (b) The percentage of central memory (TCM), transitional memory (TTEM), effector memory (EM) and terminally differentiated effector (TEMRA) CD8⁺ T-cells targeting pairs of consensus and autologous HIV-Nef peptides in an ICS assay. The graph represents an example of the difference in the phenotype of stimulated CD8⁺ T-cells between the two subjects.</p

Comparison of the trend of increase or decrease of CD8⁺ IFN-γ⁺ response to autologous and clade-B consensus HIV-1 Nef peptides in the two subjects.

<p>All responses ≥0.01% are taken into account.</p>*<p>IFN-γ responses of CD8⁺ T-cells from primary to chronic phases.</p>$<p>Chi-square test.</p

Comparison of peptide clade-B consensus sequence and autologous sequence (WT and mutants) corresponding to the Nef-specific CD8 T-cell responses detected in study subjects.

*<p>shows that the consensus and the autologous peptides had similar a. a. sequences, and a.a differences between consensus and autologous sequences are shown in bold.</p

<i>In vitro</i> exposure of circulating progenitor cells to CSL-111.

<p>Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (n = 7) and plated on fibronectin-coated plates in the absence or presence of CSL-111 (1 mg/mL) from day 0 to day 4 (D0-4), 4 to 7 (D4-7) or 0 to 7 (D0-7). After 7 days of culture, adherent cells were harvested and analyzed by flow cytometry. (A) All adherent cells were quantified by flow cytometry using cell counting beads for enumeration. (B) CSL-111 treatment increases the total number of CD34⁺ cells when added to cell culture media at D0-4 and D0-7; CD34⁺ cells were quantified by flow cytometry. (C) CSL-111 treatment reduces basal apoptosis in eEPCs when added to cell culture media at D0-4 and D0-7. Apoptosis was measured by flow cytometry using Annexin V labeling. Each box plot shows the median, the interquartile range, the maximum and the minimum of the relative change. * indicates p < 0.05 between groups from Wilcoxon signed-rank tests.</p

Representative example of sequential gating strategy for flow cytometric analysis of endothelial progenitor cells.

<p>A modified ISHAGE strategy was applied for EPC quantification. 1) Representative sample stained with CD45-FITC. Region R6 represents lymphocytes. 2) Anti-CD34-PE staining of cells from R1. Region R2 represents CD34⁺ cells. 3) Region R3 is placed to include the low Side Scatter and low to intermediate CD45 staining. 4) R4 represents all events from regions R1, R2 and R3 displayed on a FSC vs SSC dot plot to confirm that the selected events fall into a lymph-blast region. 5) Displays the events included in regions R1, R2, R3 and R4. A quadrant was positioned to separate the positive and the negative cells for VEGFR2 staining. An appropriate isotype control was used to adequately place the quadrant. Region R5 represents the total EPCs (CD34+/VEGFR2+ cells). 6) Events from region R6. This region is used to set the region R4. 7) All events. This histogram is useful to establish the lower limit of CD45 expression for the CD34⁺ events. The region R8 is placed in the right top of the histogram to count all Stem-count fluorospheres accumulated for each sample for absolute quantification. 8) Events from region R8. This region includes the Stem-count fluorospheres singlet population.</p

Decreased levels of serum stromal cell-derived factor-1 (SDF-1) in patients with acute coronary syndrome following treatment with reconstituted high-density lipoprotein (rHDL) compared to controls.

<p>Relative changes from baseline in serum SDF-1 in the control group and in the rHDL-treated group. Each box plot shows the median, the interquartile range, the maximum and the minimum. p < 0.05 between groups from Mann-Whitney test.</p