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HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

Appay V, Nixon DF, Donahoe SM, Gillespie GM, Dong T, King A, Ogg GS, Spiegel HM, Conlon C, Spina CA, Havlir DV, Richman DD, Waters A, Easterbrook P, McMichael AJ, Rowland-Jones SL - J. Exp. Med. (2000)

Bottom Line: However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells.This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor.Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.

ABSTRACT
The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

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Tetramer staining and cell activation. PBMCs from a CMV-positive B7 donor were incubated for 12 h with brefeldin A in the presence of PBS (no activation control) (A and D), OKT3 (100 ng/ml) (B and C), or specific CMV B7 peptide (10 μM) (E and F). Tetramer staining was carried out either after overnight incubation (A, B, D, and E) or before addition of the activators (C and F). Percentages for tetramer-staining cells are shown. Cells were fixed and analyzed by flow cytometry after staining for CD8 surface molecules.
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Figure 1: Tetramer staining and cell activation. PBMCs from a CMV-positive B7 donor were incubated for 12 h with brefeldin A in the presence of PBS (no activation control) (A and D), OKT3 (100 ng/ml) (B and C), or specific CMV B7 peptide (10 μM) (E and F). Tetramer staining was carried out either after overnight incubation (A, B, D, and E) or before addition of the activators (C and F). Percentages for tetramer-staining cells are shown. Cells were fixed and analyzed by flow cytometry after staining for CD8 surface molecules.

Mentions: A potential hindrance to functional studies of tetramer-staining cells is that activation procedures needed to stimulate cytokine production will also lead to rapid downregulation of the TCR 29, with which the tetrameric complexes interact to stain antigen-specific T cells. This problem can be overcome by tetramer staining of the cells before activation. Fig. 1 shows the staining of PBMCs from a healthy CMV-positive HLA-B7 donor, in whom ∼1% of the CD8+ T cell population stains with an HLA-B7 CMV tetramer. PBMCs were incubated overnight in the presence of either OKT3 or the CMV B7-restricted epitope peptide. Significant downregulation of the TCR assessed by the level of tetramer staining was seen both in cells treated with OKT3 and, more dramatically, in the cells stimulated with peptide. However, cells stained with tetramers before activation maintained reasonable (although slightly reduced) levels of tetramer staining despite activation, probably due to tetramer internalization 30. A clear feature of the activated CD8+ T cells is partial downregulation of CD8, which is also most marked with peptide stimulation; this can be used as an indicator of activation. Similar data were obtained with donors of different HLA type and for CD8+ T cells of different specificity (HIV or CMV; data not shown).


HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

Appay V, Nixon DF, Donahoe SM, Gillespie GM, Dong T, King A, Ogg GS, Spiegel HM, Conlon C, Spina CA, Havlir DV, Richman DD, Waters A, Easterbrook P, McMichael AJ, Rowland-Jones SL - J. Exp. Med. (2000)

Tetramer staining and cell activation. PBMCs from a CMV-positive B7 donor were incubated for 12 h with brefeldin A in the presence of PBS (no activation control) (A and D), OKT3 (100 ng/ml) (B and C), or specific CMV B7 peptide (10 μM) (E and F). Tetramer staining was carried out either after overnight incubation (A, B, D, and E) or before addition of the activators (C and F). Percentages for tetramer-staining cells are shown. Cells were fixed and analyzed by flow cytometry after staining for CD8 surface molecules.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887711&req=5

Figure 1: Tetramer staining and cell activation. PBMCs from a CMV-positive B7 donor were incubated for 12 h with brefeldin A in the presence of PBS (no activation control) (A and D), OKT3 (100 ng/ml) (B and C), or specific CMV B7 peptide (10 μM) (E and F). Tetramer staining was carried out either after overnight incubation (A, B, D, and E) or before addition of the activators (C and F). Percentages for tetramer-staining cells are shown. Cells were fixed and analyzed by flow cytometry after staining for CD8 surface molecules.
Mentions: A potential hindrance to functional studies of tetramer-staining cells is that activation procedures needed to stimulate cytokine production will also lead to rapid downregulation of the TCR 29, with which the tetrameric complexes interact to stain antigen-specific T cells. This problem can be overcome by tetramer staining of the cells before activation. Fig. 1 shows the staining of PBMCs from a healthy CMV-positive HLA-B7 donor, in whom ∼1% of the CD8+ T cell population stains with an HLA-B7 CMV tetramer. PBMCs were incubated overnight in the presence of either OKT3 or the CMV B7-restricted epitope peptide. Significant downregulation of the TCR assessed by the level of tetramer staining was seen both in cells treated with OKT3 and, more dramatically, in the cells stimulated with peptide. However, cells stained with tetramers before activation maintained reasonable (although slightly reduced) levels of tetramer staining despite activation, probably due to tetramer internalization 30. A clear feature of the activated CD8+ T cells is partial downregulation of CD8, which is also most marked with peptide stimulation; this can be used as an indicator of activation. Similar data were obtained with donors of different HLA type and for CD8+ T cells of different specificity (HIV or CMV; data not shown).

Bottom Line: However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells.This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor.Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.

ABSTRACT
The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Show MeSH
Related in: MedlinePlus