MOESM1 of Increased susceptibility of CD4+ T cells from elderly individuals to HIV-1 infection and apoptosis is associated with reduced CD4 and enhanced CXCR4 and FAS surface expression levels

Additional file 1: Figure S1. Increased levels of apoptosis in lymphocytes from elderly donors. Gating strategy of flow cytometry analysis of death and apoptosis rates from mock or HIV-1 NL4-3 reporter virus infected GFP+ or GFP− cells. Figure S2. Expression of activation markers and viral LTR activity in HIV-1 infected PBMC cultures from young and elderly individuals. (A, B) Representative primary data and statistical evaluations of the expression levels of (A) CD69 and (B) CD25 on X4 or R5 HIV-1 NL4-3 reporter virus infected GFP+ or GFP− cells from young (Y) and elderly (O) blood donors. (C) GFP expression levels in PBMC cultures from young and elderly donors infected with X4 or R5 HIV-1 NL4-3 reporter constructs. Each symbol represents the result obtained for one individual PBMC donor from the young (blue) or elderly (red) groups

Backcrossing of B6N (hGFAP-ECFP)GCFD mice to FVB for a single generation re-activated transgenic ECFP expression.

<p>(A and B) Cerebellar slices of 8-week-old mice were immunostained with anti-GFP and anti-S100β antibodies and analyzed. Only single ECFP expressing Bergmann glia (S100β positive cells) were detected in B6N(hGFAP-ECFP)_GCFD mice (A, upper panel), while ∼91.5% of Bergmann glia were ECFP positive in FVB(hGFAP-ECFP)_GCFD mice (A, lower panel). Backcrossing of B6N(hGFAP-ECFP)_GCFD for one generation with FVB WT mouse led to increased ECFP expression in B6NxFVB1 littermates (A, middle panel). (C) GFAP and ECFP mRNA levels in B6N, FVB and B6NxFVB1 mice (8 w). Relative expression is normalized to GFAP mRNA level in B6N mice. *: p<0.05 and ***: p<0.001. Data are obtained from three independent experiments with samples from three mice (n = 3) in every experiment. Scale bars indicate 100 µm.</p

hGFAP promoter controlled transgene expression in five different mouse lines.

<p>(A) Transgenic constructs used for oocyte injection. (B) Widespread expression of ECFP in FVB(hGFAP-ECFP)_GCFD mice with high levels in the cerebellum. Scale bar indicates 1 mm. (C) Abundant fluorescent signals from Bergmann glia of FVB(hGFAP-ECFP)_GCFD, FVB(hGFAP-EGFP)_GFEA/C, B6N(hGFAP-AmCyan)_GCYM and FVB(hGFAP-CT2_GCFT × R26tdTom). Transgene copy numbers are indicated below the respective mouse lines. Scale bars indicate 100 µm.</p

Immunohistochemical analysis of reporter protein expression in different transgenic mouse lines showed lower expression in the B6N background when compared to FVB.

<p>Cerebellar vibratome slices (cb) of 8-week-old mice were immunolabeled with anti-GFP (A and C) and anti-S100β antibodies (A, C and E), endogenous fluorescence of tdTomato in E. Upper panels depict transgene expression in B6N, lower panels in FVB. The S100β staining indicates all Bergmann glia. Results of comparative analysis in B6N and FVB mice are presented as percentage of transgene expressing Bergmann glia (S100β positive cells) (B, D and F). ***: p<0.001, **: p<0.01. Scale bars indicate 50 µm.</p

Quantitative RT-PCR analysis of transgene and endogenous GFAP mRNA levels in FVB and B6N mice.

<p>(A) Cerebellar GFAP mRNA levels in wild type B6N and FVB mice (1 w and 8 w). (B-D) Transgenic mRNA levels compared to endogenous GFAP mRNA levels in the cerebellum of B6N and FVB mice (1 w and 8 w). (B) hGFAP-ECFP_GCFD. (C) hGFAP-EGFP_GFEC. (D) hGFAP-CT2_GCTF. Relative expression is normalized to GFAP mRNA level in 1 w B6N mice. *: p<0.05, **: p<0.01, ***: p<0.001. Data are obtained from three independent experiments with samples from three mice (n = 3) in every experiment.</p

Comparative Western blot analysis of endogenous GFAP and transgenic proteins in B6N and FVB mice.

<p>Cerebellar homogenates of transgenic and wild type mice (1 w and 8 w) were probed with anti-GFP (to detect ECFP or EGFP), anti-human estrogen receptor α (ER α, recognizing CT2), and anti-GFAP and anti-α-tubulin antibodies. (A) Western blot analysis of transgene expression. (B) Western blot analysis of endogenous GFAP expression in WT and transgenic mice (hGFAP-ECFP)_GCFD. (C) Western blot analysis of endogenous GFAP expression in WT and five transgenic mouse lines (hGFAP-ECFP_GCFD; hGFAP-EGFP_GFEA; hGFAP-EGFP_GFEC; hGFAP-CT2_GCTF; hGFAP-AmCyan_GCYM) in both FVB and B6N background.</p

Inhibition of IFNβ promoter activity by SIVcol and SIVolc Vpr.

<p>(A) Schematic representation of the canonical NF-κB signaling pathway and the IFNβ promoter reporter constructs used in the present study. The wild type IFNβ core promoter contains an NF-κB binding site that was mutated by introducing three nucleotide changes (highlighted in red). (B) HEK293T cells were cotransfected with different <i>vpr</i> alleles, a <i>Gaussia</i> luciferase construct for normalization, and one of the firefly luciferase reporter constructs described in (A) (with wild type or mutated NF-κB binding site). To activate the IFNβ promoter, cells were stimulated with Sendai virus. Luciferase activities were determined 40 hr post-transfection. Vprs were categorized into three groups: Vprs from lentiviruses encoding <i>vpu</i> (HIV/SIV_<i>vpu</i>, green), downmodulating CD3 via Nef (HIV/SIV_CD3, blue), or lacking a <i>vpu</i> gene and the CD3-downmodulation activity (SIVolc/col, orange) (**p < 0.01; ***p < 0.001; n.s. p>0.05).</p

NF-κB inhibition by SIVolc and SIVcol Vpr reduces LTR-driven gene expression.

<p>(A, B) HEK293T cells were cotransfected with the indicated <i>vpr</i> alleles, a firefly luciferase reporter construct under the control of the HIV-1 M, SIVcol or SIVolc LTR promoter, and a <i>Gaussia</i> luciferase construct for normalization. Cells were (A) stimulated with TNFα or (B) cotransfected with a constitutively active mutant of IKKβ (c.a. IKKβ). Luciferase activities were determined 40 hr post-transfection. Mean values of four independent experiments in triplicates ± SEM are shown (*p<0.05; **p < 0.01; ***p < 0.001).</p

SIVcol and SIVolc Nef fail to efficiently downmodulate the T cell receptor CD3.

<p>(A) Human PBMCs were transduced with VSV-G pseudotyped NL4-3 constructs coexpressing the indicated Nef proteins and eGFP via an IRES. 72 hr post-transduction, CD3 surface expression was quantified by flow cytometry. Mean values of six infections ± SEM are shown on the left. The panel on the right shows examples for primary flow cytometry data. (B) Due to the lack of an antibody detecting SIVcol and SIVolc Nef, counteraction of human SERINC5 was analyzed to verify functional Nef expression. HEK293T cells were cotransfected with increasing amounts of a SERINC5 expression plasmid and the NL4-3-based proviral constructs also used in (A). 40 hr post-transfection, infectious virus yield was quantified by infection of TZM-bl reporter cells. Mean values of three independent experiments in triplicates ± SEM are shown. (C) HEK293T cells were cotransfected with the proviral constructs described in (A) and expression vectors for CD3-CD8 fusion proteins comprising the cytoplasmic domain of human or colobus CD3ζ and the extracellular and transmembrane domain of human CD8. 40 hr post transfection, CD3ζ downmodulation was determined by monitoring CD8 surface levels via flow cytometry. Mean values of three to four independent experiments ± SEM are shown. In (A) and (C), asterisks indicate statistically significant differences compared to the <i>nef</i>-defective control (**p < 0.01; ***p < 0.001).</p

Vpr-mediated modulation of NF-κB activity and immune activation in infected T cells.

<p>(A) Genomic organization of a chimeric infectious molecular clone (IMC) of HIV-1 M CH293.1 expressing heterologous <i>vpr</i> alleles. The CH293 <i>vpr</i> open reading frame was replaced by XbaI and MluI restriction sites (green), allowing the insertion of heterologous AU1-tagged <i>vpr</i> alleles (yellow). The original <i>vpr</i> start codon in <i>vif</i> was silenced (blue) to prevent expression of a truncated CH293 Vpr. Expression of Vpu was abrogated by inserting a premature stop codon (pink). (B) Expression of heterologous Vpr proteins from the CH293.1 chimeras described in (A). HEK293T cells were cotransfected with CH293.1 IMCs encoding the indicated <i>vpr</i> alleles. 40 hr post-transfection, cells and supernatants were harvested and virions in the supernatant were purified by centrifugation through a sucrose cushion. Subsequently, Western blotting was performed to detect AU1-tagged Vpr, Env and Gag. Detection of GAPDH served as loading control. (C) HEK293T cells were cotransfected with the indicated CH293.1 chimeras, a firefly luciferase reporter construct under the control of three NF-κB binding sites, and a <i>Gaussia</i> luciferase construct for normalization. Luciferase activities were determined 40 hr post-transfection. Mean values of three independent experiments in triplicates ± SEM are shown. (D) The SupD1 NF-κB reporter cell line was transduced with the indicated VSV-G pseudotyped CH293.1 chimeras. Cells were harvested at various time points post-transduction to determine the activation levels of NF-κB. The mean values of triplicate infections ± SD of a representative experiment are shown. (E) PBMCs of three different donors were transduced with VSV-G pseudotyped CH293.1 chimeras encoding the indicated <i>vpr</i> alleles. Cells were harvested 72 hr post-transduction and total cellular RNA was isolated and reversely transcribed. IFNβ mRNA levels were determined by quantitative RT-PCR and normalized to GAPDH mRNA. The mean values ± SEM are shown. In (C) to (E), asterisks indicate statistically significant differences compared to CH293.1 wild type transfected or infected cells (*p < 0.05; **p < 0.01; ***p < 0.001).</p