Cytokine expression profiles following stimulation.

<p>Isolated pDCs from HIV negative donors were rested overnight, and treated with a panel of stimuli as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037052#s4" target="_blank">materials and methods</a>. Cytokines were quantified in culture supernatants by cytometric bead array. Mean ± SEM concentrations of (<b>A</b>) MIP-1β, (<b>B</b>) IP-10, and (<b>C</b>) TNF-α from 5 donors are shown at various time points following stimulation by HIV_BAL, (red), HIV_NL43 (orange), Influenza (yellow), HSV (green), Imiquimod (light blue), Sendai virus (dark blue), and medium control (white). Neither IL-10 nor IL-12p70 was detected in any of the supernatant samples.</p

Kinetics of IFN-αproduction.

<p>Isolated pDCs from five HIV negative donors were rested overnight, and treated with a panel of stimuli as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037052#s4" target="_blank">materials and methods</a>. Levels of IFN-α in supernatants were quantified by CBA as part of a panel of other cytokines. (<b>A</b>) Quantified IFN-α (pg/ml) present in culture supernatant of 3–5 donors (N1-N5) at various time points (represented by blue shaded bars) following stimulation by HIV_BAL, HIV_NL43 or Influenza (<b>B</b>) Mean ± SEM concentrations of IFN-α present in supernatants at various time points following stimulation by HIV_BAL, (red), HIV_NL43 (orange), Influenza (yellow), HSV-1 (green), Imiquimod (light blue), Sendai virus (dark blue), and medium control (white) from data from 5 donors.</p

IFN-αproduction from pDCs following stimulation with HIV or Influenza.

<p>(A) Gating strategy used to identify CD14 negative BDCA-2 positive CD123 positive pDCs. (B) PBMCs from HIV negative individuals were treated with either Influenza (M.O.I = 0.1) or HIV_BAL (M.O.I = 0.1) in the presence of GolgiPlug™ for 8 hours and stained for intracellular IFN-α expression. Shown is a representative flow cytometry plot. (C) Summary data of pDCs from 3 individuals measuring intracellular IFN-α expression following stimulation with Influenza (M.O.I = 0.1) or HIV_BAL (M.O.I = 0.1) (D) Isolated pDCs from 3 HIV negative patients were stimulated with Influenza (M.O.I = 0.5) and HIV_BAL (M.O.I = 0.5) for 8 hours, and levels of IFN-α in supernatant were quantified by ELISA. Mean ± SEM, * = p<0.05.</p

HIV induces upregulation of pSYK.

<p>(<b>A</b>) Purified pDCs were treated with Influenza and HIV_BAL for various time points. Positive control with anti-BDCA-2 antibody (1∶100) treated for 30 minutes is shown. Cells were harvested, permeabalized and stained for pSYK (Y352) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037052#s4" target="_blank">materials and methods</a>. Shown are representative histograms from 3 independent experiments. (<b>B</b>) PDCs were stimulated with HIV_BAL, HIV_NL43, Influenza, or medium alone for 30 minutes and percentages of pDCs staining positive for pSYK (Y352) and pSYK (Y525/526) were evaluated by flow cytometry (n = 3). In (<b>C</b>) gp120 induces pSyk signaling in pDCs. Purified pDCs were stimulated for 90 minutes in the presence of 1 mg/ml monomeric gp120, AN1, (blue); 20 µg/ml imiquimod, (red); or the absence of stimulation (black). Cells were harvested and stained for pSyk (pY348) and analyzed by flow cytometry. Shown is a representative flow cytometric plot of two experiments. (<b>D</b>) pDCs were stimulated with 0.5 M.O.I. of HIV_BAL, HIV_NL43, Influenza, and imiquimod, or medium alone for 24 hours in the presence or absence of anti-BDCA2 antibody or an IgG1 isotype control and IFN-α was measured by ELISA (n = 2, performed with two independent replicates, *<i>p</i><0.001, **<i>p</i><0.00005). BDCA2 treated pDC completely abrogated IFN-α production in the presence of HIV (bars not seen).</p

Kinetics of CD83 and CD86 upregulation.

<p>Isolated pDCs from HIV negative donors were treated with Influenza (M.O.I = 0.5), imiquimod (20 µg/ml), or HIV (M.O.I = 0.5) for various time points and harvested for analysis by flow cytometry for surface expression of CD83 and CD86. (A) Representative contour plots of CD83 and CD86 upregulation at various time points post stimulation by Influenza, showing distinct kinetics of CD83 and CD86 upregulation. (B) Percentage of pDCs that are CD83 single positive (SP, Blue bars), CD83/CD86 double positive (DP, Purple bars), CD86SP (Red bars), and double negative for both markers (DN, gray bars) at time points post stimulation with Influenza, imiquimod or HIV_BAL. (C) Percentages of pDCs that are CD83⁺ or CD86⁺ at time points post stimulation. Data shown are representative of at least 3 different donors stimulated and stained in duplicate.</p

Tim-3 Negatively Regulates Cytotoxicity in Exhausted CD8⁺ T Cells in HIV Infection

<div><p>Cytotoxic CD8⁺ T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell ‘exhaustion’ is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8⁺ T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3⁺ CD8⁺ T cells to make perforin and 2) the direct ability of Tim-3⁺ CD8⁺ T cells to kill autologous HIV infected CD4⁺ target cells. Surprisingly, Tim-3⁺ CD8⁺ T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8⁺ T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4⁺ T cells and d) their ability to suppress HIV infection of CD4⁺ T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8⁺ T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8⁺ T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.</p> </div

Tim-3 pathway blocking increases the antigen specific CD8⁺ T cells cytotoxicity and HIV suppression in chronic HIV infection.

<p>The effect of HIV Gag-specific CD8⁺ T cells after blocking the Tim-3 pathway with a Tim-3 blocking antibody (clone 2E2) on eliminating HIV-infected CD4⁺ T cells was tested in a virus suppression assay. Autologous CD4⁺ T cells (targets) are infected with a primary HIV virus isolate. Autologous CD8⁺ T cells (effectors) are added in 1∶1 ratio at the time of infection with 2E2 antagonistic anti-Tim-3 antibody or isotype at 10 µg/ml. The co-culture is incubated at 37°C for three days. The final readout is the percentage of HIV (p24⁺) positive target cells on day three examined by intracellular flow cytometry. In <i>a</i>) are autologous CD4⁺ T cells in the absence of autologous CD8⁺ T cells taken from an HIV infected individual after exogenous infection by HIV in the presence of Tim-3 antibody or isotype. Tim-3 blockade had no effect on total and infected CD4⁺ numbers. In <i>b</i>) shown are CD4⁺ T cells from an HIV uninfected normal volunteer infected with exogenous HIV and co-cultured with autologous CD8⁺ T cells (1∶1 ratio). Again we could not appreciate any difference in survival of CD4⁺ T cells. Tim-3 blockade had no effect on CD8⁺ mediated suppression of HIV Infection. In <i>c</i>), is a representative experiment showing the percentage of infected p24⁺ CD4⁺ T cells in the two different conditions in a chronic HIV infected individual and in <i>d)</i> is a representative experiment showing the percentage of infected p24⁺ CD4⁺ T cells in the two different conditions in a viral controller, Shown in <i>e</i>), are summary data for four chronically HIV infected individuals and in <i>f</i>) are summary data for three viral controllers. Each solid circle represents the average of three independent experiments from each individual showing the percentage of p24⁺ CD4⁺ T cells (<i>p</i> value based on two tailed paired t test).</p

Tim-3 pathway blocking improves the perforin release in antigen specific CD8⁺ T cells only in chronically HIV infected individuals.

<p>2×10⁵ negatively sorted CD8⁺ T cells from <i>ex vivo</i> PBMC from 5 untreated chronically infected (Fig <i>a</i>) and four untreated viral controllers (Fig <i>b)</i> were stimulated with a pool of HIV Gag peptides (final concentration of 2 µg/ml/peptide) for 6 h. Perforin released in supernatant was measured in an ELISA experiment in pg/ml. (αTim-3  =  2E2 Tim-3 blocking antibody, Cntr  =  IgG1 isotype control antibody) (<i>p</i> value based on two tailed paired t test).</p

Perforin content of Tim-3⁺ CD8⁺ T cells as determined by two antibody clones.

<p>In <i>a</i>), a representative experiment showing Tim-3 expression on <i>ex vivo</i> CD8⁺ T cells from a treatment naïve chronically HIV infected subject showing perforin expression by two antibodies detecting different conformations of perforin on Tim-3⁺ or Tim-3<b>⁻</b>CD8⁺ T cells. The δG9 clone has been proposed to predominantly detect stored (granule associated) perforin and clone B-D48 detects stored perforin plus majority of other perforin conformations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040146#pone.0040146-Hersperger2" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040146#pone.0040146-Hersperger3" target="_blank">[29]</a>. In <i>b</i>) a representative figure showing the relationship between two different conformations of perforin and Tim-3 expressions after gating out the naïve and terminally differentiated CD8⁺ T cells by gating in all CD45RA^- memory subsets. In <i>c),</i> summary of data for all 9 chronically HIV infected individuals stratifying perforin antibodies and Tim-3 expression on total CD8⁺ T cells. In <i>d</i>) summary of data for 5 chronically HIV infected individuals showing the perforin expression with two clones and Tim-3 expressions of memory subsets of CD8⁺ T cells (<i>p</i> value based on two tailed paired t test).</p

Tim-3⁺ CD8⁺ T cells cannot degranulate as effectively as Tim-3⁻ CD8⁺ T cells.

<p><i>Ex vivo</i> PBMC from HIV chronically infected subjects were stimulated for 6 hours with a pool of HIV Gag peptides or SEB or DMSO and stained for CD107a and Tim-3. In <i>a), a</i> representative experiment showing higher CD107a expression in Tim-3 negative component of CD8⁺ T cells after stimulation with either pool of HIV antigens or SEB. In <i>b</i>), summary of all data for 10 chronically HIV infected individuals showing higher ability for degranulation in Tim-3 negative subpopulation of CD8⁺ T cells after stimulation with Gag peptides. In <i>c</i>) summary of all data for 8 chronically HIV infected individuals showing higher ability for degranulation in Tim-3 negative subpopulation of CD8⁺ T cells after stimulation with SEB (<i>p</i> value based on wilcoxon signed rank test).</p