Critical contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to apoptosis of human CD4+ T cells in HIV-1-infected hu-PBL-NOD-SCID mice

Abstract

Apoptosis is a key for CD4+ T cell destruction in HIV-1-infected patients. In this study, human peripheral blood lymphocyte (PBL)-transplanted nonobese diabetic (NOD)-severe combined immunodeficient (SCID) (hu-PBL-NOD-SCID) mice were used to examine in vivo apoptosis after HIV-1 infection. As the hu-PBL-NOD-SCID mouse model allowed us to see extensive infection with HIV-1 and to analyze apoptosis in human cells in combination with immunohistological methods, we were able to quantify the number of apoptotic cells with HIV-1 infection. As demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), massive apoptosis was predominantly observed in virus-uninfected CD4+ T cells in the spleens of HIV-1-infected mice. A combination of TUNEL and immunostaining for death-inducing tumor necrosis factor (TNF) family molecules indicated that the apoptotic cells were frequently found in conjugation with TNF-related apoptosis-inducing ligand (TRAIL)-expressing CD3+CD4+ human T cells. Administration of a neutralizing anti-TRAIL mAb in HIV-1-infected mice markedly inhibited the development of CD4+ T cell apoptosis. These results suggest that a large number of HIV-1-uninfected CD4+ T cells undergo TRAIL-mediated apoptosis in HIV-infected lymphoid organs.

Figure 1

Apoptosis occurs predominantly in HIV-1–uninfected CD4⁺ T cells. (a) Triple immunofluorescent staining for HIV p24^gag (FITC, green), human CD4 (Cy5, blue), and TUNEL (TRITC, red) of spleens from HIV-1–infected hu-PBL-NOD-SCID mice 2 wk after inoculation of human PBMCs (9 d after infection). Original magnification ×25 (left panel), ×100 (middle panel), and ×1,000 (right two panels). TUNEL (red) and CD4 (blue) double-positive cells are shown as pink in lower magnification (left panel). CD4 (blue) and HIV p24^gag (green) double-positive cells are shown as light green. Arrow shows a small artery. Right upper panel indicates TUNEL⁺, CD4⁺, and p24^gag+ cells, which represented <2% of both TUNEL⁺ and CD4⁺ cells. Right lower panel indicates TUNEL⁺, CD4⁺, and p24^gag− cells, which accounted for >90% of both TUNEL⁺ and CD4⁺ cells. (b) Dual color detection for GFP-expressing HIV-1 (GFP, green) and TUNEL staining (TRITC, red) of spleens from GFP/HIV-1–infected hu-PBL-NOD-SCID mice 2 wk after human PBL transplantation (9 d after infection). The majority of TUNEL⁺ cells were GFP/HIV-1 negative. Original magnification ×100.

Figure 1

Apoptosis occurs predominantly in HIV-1–uninfected CD4⁺ T cells. (a) Triple immunofluorescent staining for HIV p24^gag (FITC, green), human CD4 (Cy5, blue), and TUNEL (TRITC, red) of spleens from HIV-1–infected hu-PBL-NOD-SCID mice 2 wk after inoculation of human PBMCs (9 d after infection). Original magnification ×25 (left panel), ×100 (middle panel), and ×1,000 (right two panels). TUNEL (red) and CD4 (blue) double-positive cells are shown as pink in lower magnification (left panel). CD4 (blue) and HIV p24^gag (green) double-positive cells are shown as light green. Arrow shows a small artery. Right upper panel indicates TUNEL⁺, CD4⁺, and p24^gag+ cells, which represented <2% of both TUNEL⁺ and CD4⁺ cells. Right lower panel indicates TUNEL⁺, CD4⁺, and p24^gag− cells, which accounted for >90% of both TUNEL⁺ and CD4⁺ cells. (b) Dual color detection for GFP-expressing HIV-1 (GFP, green) and TUNEL staining (TRITC, red) of spleens from GFP/HIV-1–infected hu-PBL-NOD-SCID mice 2 wk after human PBL transplantation (9 d after infection). The majority of TUNEL⁺ cells were GFP/HIV-1 negative. Original magnification ×100.

Figure 2

Immunofluorescent analysis for death-inducing ligands and their receptors in spleens from HIV-1–infected hu-PBL-NOD-SCID mice. (a) Double staining for TRAIL (Cy5, green) and TUNEL (TRITC, red) (magnification ×50). (b) Triple staining for FasL (FITC, green), TUNEL (TRITC, red), and Fas (Cy5, blue) (magnification ×50). (c) Triple staining for TNF-α (FITC, green), TUNEL (TRITC, red), and TNFR1 (Cy5, blue) (magnification ×50). (d) Triple staining for human CD4 (FITC, green), TUNEL (TRITC, red), and TRAIL (Cy5, blue) (magnification ×100). Merging of green and blue is shown as light blue. Merging of green and red is shown as orange. (e) High magnification (×500) of d showing a TUNEL⁺CD4⁺ T cell conjugated with a TRAIL⁺CD4⁺ T cell (blue). Light blue indicates merging of green and blue. (f ) Triple immunostaining for human CD3 (FITC, green), TUNEL (TRITC, red), and TRAIL (Cy5, blue) (magnification ×200). Merging of green and blue is shown as light blue. Merging of green and red is shown as orange. (g) Quantification of TUNEL⁺ cells conjugated with death-inducing ligand–positive cells. TUNEL⁺ cells conjugated with TRAIL⁺, FasL⁺, or TNF-α⁺ cells (indicated by arrows in a, b, and c) were counted in five randomly selected visual fields at magnification of 100. Data are expressed as percentage of conjugates among total TUNEL⁺ cells (mean ± SD of five fields).

Figure 2

Immunofluorescent analysis for death-inducing ligands and their receptors in spleens from HIV-1–infected hu-PBL-NOD-SCID mice. (a) Double staining for TRAIL (Cy5, green) and TUNEL (TRITC, red) (magnification ×50). (b) Triple staining for FasL (FITC, green), TUNEL (TRITC, red), and Fas (Cy5, blue) (magnification ×50). (c) Triple staining for TNF-α (FITC, green), TUNEL (TRITC, red), and TNFR1 (Cy5, blue) (magnification ×50). (d) Triple staining for human CD4 (FITC, green), TUNEL (TRITC, red), and TRAIL (Cy5, blue) (magnification ×100). Merging of green and blue is shown as light blue. Merging of green and red is shown as orange. (e) High magnification (×500) of d showing a TUNEL⁺CD4⁺ T cell conjugated with a TRAIL⁺CD4⁺ T cell (blue). Light blue indicates merging of green and blue. (f ) Triple immunostaining for human CD3 (FITC, green), TUNEL (TRITC, red), and TRAIL (Cy5, blue) (magnification ×200). Merging of green and blue is shown as light blue. Merging of green and red is shown as orange. (g) Quantification of TUNEL⁺ cells conjugated with death-inducing ligand–positive cells. TUNEL⁺ cells conjugated with TRAIL⁺, FasL⁺, or TNF-α⁺ cells (indicated by arrows in a, b, and c) were counted in five randomly selected visual fields at magnification of 100. Data are expressed as percentage of conjugates among total TUNEL⁺ cells (mean ± SD of five fields).

Figure 2

Immunofluorescent analysis for death-inducing ligands and their receptors in spleens from HIV-1–infected hu-PBL-NOD-SCID mice. (a) Double staining for TRAIL (Cy5, green) and TUNEL (TRITC, red) (magnification ×50). (b) Triple staining for FasL (FITC, green), TUNEL (TRITC, red), and Fas (Cy5, blue) (magnification ×50). (c) Triple staining for TNF-α (FITC, green), TUNEL (TRITC, red), and TNFR1 (Cy5, blue) (magnification ×50). (d) Triple staining for human CD4 (FITC, green), TUNEL (TRITC, red), and TRAIL (Cy5, blue) (magnification ×100). Merging of green and blue is shown as light blue. Merging of green and red is shown as orange. (e) High magnification (×500) of d showing a TUNEL⁺CD4⁺ T cell conjugated with a TRAIL⁺CD4⁺ T cell (blue). Light blue indicates merging of green and blue. (f ) Triple immunostaining for human CD3 (FITC, green), TUNEL (TRITC, red), and TRAIL (Cy5, blue) (magnification ×200). Merging of green and blue is shown as light blue. Merging of green and red is shown as orange. (g) Quantification of TUNEL⁺ cells conjugated with death-inducing ligand–positive cells. TUNEL⁺ cells conjugated with TRAIL⁺, FasL⁺, or TNF-α⁺ cells (indicated by arrows in a, b, and c) were counted in five randomly selected visual fields at magnification of 100. Data are expressed as percentage of conjugates among total TUNEL⁺ cells (mean ± SD of five fields).

Figure 3

Inhibition of apoptosis by anti-TRAIL mAb. HIV-1–infected hu-PBL-NOD-SCID mice were injected intraperitoneally with 1 mg of anti-TRAIL mAb (a), anti-FasL mAb (b), or control mouse IgG (c) 9 d after infection. After 3 d (12 d after infection), spleen sections were stained for apoptotic cells by TUNEL (TRITC, red). Original magnification ×100.