by JoAnn Castelli, Elaine K

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Mature dendritic cells can enhance CD8+ cell noncytotoxic anti-HIV responses: the role of IL-15 by JoAnn Castelli, Elaine K. Thomas, Michel Gilliet, Yong-Jun Liu, and Jay A. Levy Blood Volume 103(7):2699-2704 April 1, 2004 ©2004 by American Society of Hematology

CD8+ cell noncytotoxic antiviral response after stimulation of CD8+ cells by different approaches. CD8+ cell noncytotoxic antiviral response after stimulation of CD8+ cells by different approaches. CD8+ cells from HIV-infected subjects were treated with 10 U/mL recombinant human IL-2 (control) (▪), polyclonally stimulated by anti-CD3 and anti-CD28 antibodies (▴), or cocultured with allogeneic CD40L-matured monocyte-derived DCs (•) (1:5 DC/CD8+ cell ratio) for 6 days. The CD8+ cells were then removed and cocultured with HIV-1-infected CD4+ cells, at a ratio of 1:1 CD8+/CD4+ cells.5 Culture supernatants were assayed for reverse-transcriptase (RT) activity on days 5 and 7.24 The extent (percent) of CNAR was determined by comparing the average percentage of RT activity in supernatants from duplicate cocultures with the average RT activity in fluids from HIV-infected CD4+ cells cultured alone. Bars indicate average suppression resulting from each treatment. JoAnn Castelli et al. Blood 2004;103:2699-2704 ©2004 by American Society of Hematology

Optimization of the number of DCs for enhancement of CNAR Optimization of the number of DCs for enhancement of CNAR. Monocytes were cultured with GM-CSF and IL-4 for 3 days and matured with CD40L trimer. Optimization of the number of DCs for enhancement of CNAR. Monocytes were cultured with GM-CSF and IL-4 for 3 days and matured with CD40L trimer. In a 96-well plate, DCs at 3-fold dilutions were then added to 50 000 CD8+ cells/well. After 6 days of coculture, the CD8+ cells were removed and transferred into a coculture with HIV-1-infected CD4+ cells. Supernatants from duplicate cultures were assayed for RT activity.26 The extent of CNAR (▦) was determined by comparing these RT results with the average RT activity in supernatants of HIV-infected CD4+ cells cultured alone. Cell proliferation (▧) was measured as described in “Patients, materials, and methods.” Error bars indicate SD from the average suppression observed. Results are representative of 5 separate experiments. JoAnn Castelli et al. Blood 2004;103:2699-2704 ©2004 by American Society of Hematology

Comparison of CNAR after stimulation with allogeneic compared with autologous DCs. CD8+ cells from HIV-infected subjects were either control (10 U/mL recombinant human IL-2; ▦), or cocultured with allogeneic (▧) or autologous (▪) CD40L-matu... Comparison of CNAR after stimulation with allogeneic compared with autologous DCs. CD8+ cells from HIV-infected subjects were either control (10 U/mL recombinant human IL-2; ▦), or cocultured with allogeneic (▧) or autologous (▪) CD40L-matured monocyte-derived DCs for 6 days. The CD8+ cells were removed and cocultured with HIV-1-infected CD4+ cells, at ratios of 0.5:1 and 1:1 (CD8+/CD4+ cells). Error bars indicate SD from the average suppression observed. Results are representative of 7 separate studies. JoAnn Castelli et al. Blood 2004;103:2699-2704 ©2004 by American Society of Hematology

Role of IL-15 in mDC enhancement of CNAR Role of IL-15 in mDC enhancement of CNAR. CD8+ cells from HIV-infected subjects were either untreated or treated (10 U/mL recombinant human IL-2), as indicated for 6 days. Role of IL-15 in mDC enhancement of CNAR. CD8+ cells from HIV-infected subjects were either untreated or treated (10 U/mL recombinant human IL-2), as indicated for 6 days. The mature allogeneic DCs (mDC) were cocultured with CD8+ cells at a ratio of 1:5. The CD8+ cells were then removed and cocultured with HIV-1-infected heterologous CD4+ cells, at a CD8+/CD4+ cell ratio of 0.5:1 (▦) or 2:1 (▪) in the presence or absence of antibodies to IL-15 or IL-12. Supernatants from duplicate cultures were assayed for RT activity. The extent of CNAR was determined by comparing these average RT results with the average RT activity in supernatants of duplicate cultures of HIV-infected CD4+ cells cultured alone. Error bars indicate SD from the average suppression resulting from each treatment. Results are representative of 7 separate experiments. JoAnn Castelli et al. Blood 2004;103:2699-2704 ©2004 by American Society of Hematology

Phenotype and activation changes in CD8+ cells after stimulation by allogeneic DCs with and without anti-IL-15 antibodies. Phenotype and activation changes in CD8+ cells after stimulation by allogeneic DCs with and without anti-IL-15 antibodies. CD8+ cells from HIV-infected subjects were either (A) control (10 U/m recombinant human IL-2), (B) polyclonally stimulated by anti-CD3 and anti-CD28 antibodies, (C) cocultured with allogeneic DCs (1:5 DC/CD8+ cell ratio), or (D) cocultured with allogeneic DCs in the presence of 10 μg/mL anti-IL-15 antibody for 6 days. CD8+ cells were removed and assessed by FACS for activation markers after staining with fluorophore-conjugated antibodies to CD28, CD25, HLA-DR, and CD45RO. Percentages reflect the number of CD8+ cells expressing that phenotype marker. Results are representative of 5 separate experiments. JoAnn Castelli et al. Blood 2004;103:2699-2704 ©2004 by American Society of Hematology

Effect of IL-15 treatment on CNAR by CD8+ cells from HIV-infected subjects. Effect of IL-15 treatment on CNAR by CD8+ cells from HIV-infected subjects. (A) CD8+ cells from HIV-infected subjects were either treated (control) with 10 U/mL recombinant human IL-2, or treated with IL-12 (1 ng/mL) or with IL-15 (150 ng/mL) for 6 days, and then cocultured with infected CD4+ cells at a 0.5:1 ratio. (B) CD8+ cells were treated with the indicated concentrations of IL-15. Cells were then removed and cocultured with HIV-1 acutely infected CD4+ cells, at a ratio of 0.5:1 in panel A and in panel B (▦) and 1:1 in panel B (▧) (CD8+/CD4+ cells). Supernatants from duplicate cultures were assayed for RT activity. The extent (percent) of CNAR was determined by comparing these average RT results with the average RT activity from supernatants of duplicate cultures of HIV-infected CD4+ cells cultured alone. A bar indicates standard deviation from the average suppression of duplicate cultures. (C) Proliferation of cultured CD8+ cells either untreated (10 U/mL IL-2) or treated with the indicated doses of IL-15 was measured on day 5 after a 16-hour incubation with 3H-thymidine. Results are representative of 5 separate experiments. Error bars indicate SD from the average suppression observed. JoAnn Castelli et al. Blood 2004;103:2699-2704 ©2004 by American Society of Hematology