Mechanisms of T cell contraction after immune response resolution.

<p>T cell contraction after resolution of an immune response is usually accomplished through a combination of mitochondria- and death receptor–dependent mechanisms. As a result of T cell expansion, survival factors as IL-2 become scarce, and signalling through survival pathways, like the phosphoinositide 3-kinase (PI3-K)/Akt pathway, ceases, allowing FoxO3-dependent Bim induction. Bim promotes mitochondrial outer membrane permeabilization (MOMP) by relieving the inhibitory effect that antiapoptotic Bcl-2 and Bcl-xL exert on proapoptotic Bax and Bak. MOMP results in cytochrome-c release from the mitochondria, enabling activation of a supramolecular complex, the apoptosome that activates caspase-3. By processing numerous cellular substrates, activated caspase-3 ensures completion of the execution phase of apoptosis. T cell activation also induces Fas ligand expression in T cells, which, by engaging the death receptor Fas, enables caspase-8 activation at the death-inducing signalling complex (DISC). Caspase-8 then activates caspase-3. If the levels of caspase-8–activated caspase-3 are not sufficient to undertake apoptotic cell death, a mitochondrial amplification loop may occur through caspase-8–mediated Bid cleavage. This generates tBid, a proapoptotic Bcl-2 family member that promotes MOMP by activating Bax and Bak.</p

T cell anergy versus T cell exhaustion.

<p>T cell anergy versus T cell exhaustion.</p

Impact of targeted inhibition of suppressive or apoptotic T cell pathways in the outcome of parasitic infection.

<p>Impact of targeted inhibition of suppressive or apoptotic T cell pathways in the outcome of parasitic infection.</p

The uptake of apoptotic T lymphocytes by parasite-hosting phagocytes contributes to the remodelling of the parasite-hosting tissue as a bona fide protective niche.

<p>Increased rates of T cell apoptosis occur during parasite infection, mediated either by death receptor– or mitochondria-dependent mechanisms. Upon clearance, these apoptotic cells induce an alternative state of activation in phagocytes associated with production of suppressive mediators as TGF-β and IL-10, as well as promoting parasite growth. Suppressive cytokines act on effector T cells and, together with antigen persistence and inhibitory T cell receptors, induce exhaustion of these cells. Additionally, inhibition of antigen presentation and costimulation, acting along with suppressive cytokines or enzymes (as IDO, which catabolizes tryptophan), may render naïve T cells anergic and unresponsive throughout infection. Eventually effector, anergic, or exhausted T cells undergo programmed cell death, fuelling the pool of apoptotic corpses and aiding perpetuation of the suppressive state.</p

HIV-1 sensitizes monocyte-derived macrophages and monocyte-derived DCs for Death receptor ligands.

<p>(<b>A</b>) Monocyte-derived macrophages (MØ) or (<b>B</b>) monocyte-derived DCs were incubated with R5 HIV-1_Bal (100 pg/ml of p24) or Mock at day 1 after the initiation of APC differentiation. At day 5, the cells were then cultured overnight in the absence or presence of recombinant TNF-α, TRAIL and FasL (100 ng/ml). A positive control of cell death was performed in the presence of Actinomycin D (10 µg/ml). Apoptosis was determined by flow cytometry using FITC-labeled Annexin V. Percentages of apoptotic cells shown are means ± SEM (n = 3). Statistical significant differences are indicated by an asterisk (p<0.05). (<b>C</b>) Dose response of FasL and Trail. Percentages of apoptotic cells shown are means ± SEM (n = 3). Statistical significant differences as compared to untreated cells are indicated by an asterisk (p<0.05). (<b>D</b>) Cells, before infection, were incubated in the absence or presence of ddI (5 µM). At day 5, the cells were then cultured overnight in the absence or presence of either TRAIL or FasL (100 ng/ml). (<b>E</b>) Cells incubated with R5 HIV-1_Bal were stimulated at day 5 with LPS (10 ng/ml) and IFN-γ (10³ U/ml) overnight in the absence or presence of death receptor antagonists: TNF-R1, TRAIL-R1/TRAIL-R2, Fas-Fc (10 µg/ml). Preventive effect was calculated as follows: ((% of MØ/DC apoptosis - % of MØ/DC apoptosis in the presence of decoy receptors)/(% of MØ/DC apoptosis)) X 100. Values are means ± SEM (n = 3). Statistical significant differences as compared to untreated cells are indicated by an asterisk (p<0.05).</p

Impact of HIV infection on cytokine secretion and maturation of monocyte-derived macrophages and monocyte-derived DCs.

<p>(<b>A</b> and <b>B</b>) HIV-1 infection of monocyte-derived macrophages (MØ) or monocyte-derived DCs. Cells were infected at day 1 after the initiation of APC differentiation without (Mock) or with the R5 HIV-1_Bal (100 pg/ml of p24). At day 5, (<b>A</b>) the percentage of p24⁺ cells was determined by flow cytometry. Values shown are means ± SEM (n = 6). Significant differences are indicated by an asterisk (p<0.05). (<b>B</b>) HIV viral proteins were detected by western blotting using HIV-1⁺ sera. (<b>C</b>) HIV-1 decreases pro-inflammatory cytokines production. Cells at day 5 were stimulated with LPS (10 ng/ml) and IFN-γ (10³ U/ml) overnight. Cells incubated in the absence of R5 HIV-1_Bal and in the absence of stimulation represent the negative control (Med). Supernatants were collected and assessed for the presence of IL-1β, IL-6, IL-8, and TNF-α by flow cytometry using bead array. Values shown are means ± SEM (n = 3). Significant differences are indicated by an asterisk (p<0.05). (<b>D</b> and <b>E</b>) HIV-1 decreases CD86 expression at the surface of stimulated (<b>D</b>) MØ or (<b>E</b>) DCs. Cells were stained with specific CD86 mAbs, and cell surface density was assessed by flow cytometry. One representative experiment out of three is shown; the mean of fluorescence intensity is indicated. CD86 expression values shown are means ± SEM (n = 3). Significant differences are indicated by an asterisk (p<0.05).</p

Increased apoptosis of monocytes and DCs during primary SIV infection.

<p>PBMC from healthy (<b>SIV⁻</b>) and SIV-infected RM at day 14 (<b>SIV⁺</b>) were incubated overnight in the absence or presence of death ligands. (<b>A</b>) The percentages of apoptotic CD4⁺ T cells were analyzed by flow cytometry using FITC-labeled Annexin V. (<b>B</b>) Gating strategy to analyze apoptotic HLA-DR⁺ and CD14⁺ cells. Cells were first analyzed on HLA-DR <i>versus</i> SSC (gate R1) and HLA-DR <i>versus</i> Lin+(CD3⁺CD14⁺CD20⁺) cells (gate R2), or CD14 <i>versus</i> Lin+(CD3⁺CD20⁺) cells (gate R3) (<b>C</b>) The percentages of apoptotic HLA-DR⁺Lin⁻ (CD3⁻CD14⁻CD20⁻) cells was determined by flow cytometry using FITC-labeled Annexin V gated on R1 and R2; (<b>D</b>) Percentage of apoptotic CD4⁺ and HLA-DR⁺Lin⁻ cells. Values shown are means ± SEM (n = 4 for SIV⁻ and SIV⁺); Significantly different compared to medium controls (*, p<0.05). (<b>E</b>) Percentage of apoptotic monocytes (MØ) and DCs at days 0, 11, 14 and 60; values are means ± SEM (n = 6); Significantly different from day 0 (*, p<0.05). (<b>F</b>) PBMC from healthy and SIV-infected AGM at day 14. The percentages of apoptotic CD4⁺ T cells and HLA-DR⁺CD3⁻CD20⁻ cells were analyzed by flow cytometry using FITC-labeled Annexin V. (<b>G</b>) Quantitation of FasL in the sera of SIV-infected RMs and AGM at different time points post-infection. Statistical significant differences as compared to day 0 are indicated by an asterisk. (<b>H</b>) Preventive effect of death receptor antagonists. PBMC from SIV-infected RMs (day 14) were incubated overnight with antagonists of death receptors: TRAIL-R1, TRAIL-R2, Fas-Fc and TNF-R1 (10 µg/ml). Apoptosis of monocytes and DCs was quantified using FITC-Annexin-V. Preventive effect was calculated as follows: ((% of MØ/DC apoptosis - % of MØ/DC apoptosis in the presence of decoy receptors)/(% of MØ/DC apoptosis)) X 100. Values are means ± SEM (n = 5); Significantly different from samples incubated with medium alone (*, p<0.05).</p

Expression of pro- and anti-apoptotic molecules in HIV-1 infected monocyte-derived macrophages and monocyte-derived DCs.

<p>(<b>A</b>) Monocytes-derived MØ or monocytes-derived DCs were incubated with R5 HIV-1_Bal (100 pg/ml of p24) or Mock at day 1 after the initiation of APC differentiation. At day 5, the cells were lysed and then the proteins were detected by immunoblotting with specific antibodies against FLIP and Mcl-1. Actin was used as a control for equal protein loading. Values represent the ratio of the protein bands normalized with respect to the loading control, analyzed with GeneTools (SynGene). Mock (non-infected cells) was considered arbitrary equivalent to 1, and bands are compared between (Mock) and HIV-infected cells (HIV). (<b>B</b>) Ratio of FLIP, Mcl-1 and Mcl-1_Exon-1 proteins. Bars show the mean ± SEM of three independent experiments. Statistical significant differences as compared to non-infected cells are indicated by an asterisk (p<0.05). (<b>C</b>) Expression of pro-apoptotic Bax and Bak molecules in the enriched mitochondrial fraction. Hsp60 was used as a control for equal protein loading. Values represent the ratio of the protein bands normalized with respect to the loading control (Hsp60), analyzed with GeneTools (SynGene). Mock (non-infected cells) was considered arbitrary equivalent to 1, and bands are compared between (Mock) and HIV-infected cells (HIV). (<b>D</b>) Ratio of Bax and Bak proteins. Bars show the mean ± SEM of three independent experiments. Statistical significant differences as compared to non-infected cells are indicated by an asterisk (p<0.05).</p

Expression of pro- and anti-apoptotic molecules in monocytes and mDCs during primary SIV infection.

<p>(<b>A</b>) Expression of FLIP and Mcl-1 in purified CD14⁺ (MØ) from healthy RM (SIV⁻) and SIV-infected RMs (SIV⁺). After isolation, the cells were lysed and the proteins were immunoblotted with specific antibodies against the anti-apoptotic molecules FLIP and Mcl-1. Actin was used as a control for equal protein loading. Values represent the ratio of the FLIP and Mcl-1 bands and normalized with respect to the loading control. (<b>B</b>) Flow cytometric analysis of the active form of the pro-apoptotic molecules Bax and Bak in CD4⁺ T cells, and monocytes (MØ) at days 0 and 14. (<b>C</b>) Percentage of active form of Bax and Bak among monocyte and DC populations at days 0 and 14. Values are means ± sem (n = 6); Significantly different from day 0 (*, p<0.05). (<b>D</b> and <b>E</b>) PBMC from SIV-infected RMs were incubated without or with Q-VD-OPH (10 µM) and then stimulated with LPS (10 ng/ml) overnight. (<b>D</b>) Fold increase in surviving cells incubated with Q-VD-OPH is shown. (<b>E</b>) Numbers of HLA-DR⁺CD3⁻CD20⁻ expressing TNF-α in the absence or presence of Q-VD-OPH after stimulation is shown. Bars show the mean ± SEM of three independent experiments. Statistical significant differences as compared to untreated cells are indicated by an asterisk (p<0.05).</p

Frequency of SIV-DNA⁺ cells in SIV-infected macaques.

<p>Frequency of SIV-DNA⁺ CD4⁺ T cells, CD14⁺ cells and DCs. Prism version 3.0 (GraphPad Software) was used to calculate means ± SD at days 7, 11, 14 and 60 post-infection.</p