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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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Comparison among peptide, OKT3, and tetramer activation. PBMCs from a CMV-positive B7 donor were incubated for 12 h with brefeldin A in the presence of PBS (no activation control), specific CMV B7 peptide (10 μM), OKT3 (100 ng/ml), or CMV B7 tetramers. Tetramer staining was carried out after overnight incubation for the no activation control or before addition of the activators for the “activated” cells. Cells were stained for CD69 (A), IFN-γ (B), and MIP-1β (C) and analyzed by flow cytometry. Data show cells gated on the CMV B7 tetramer-positive population. Mean fluorescence intensity is shown for each condition.
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Figure 2: Comparison among peptide, OKT3, and tetramer activation. PBMCs from a CMV-positive B7 donor were incubated for 12 h with brefeldin A in the presence of PBS (no activation control), specific CMV B7 peptide (10 μM), OKT3 (100 ng/ml), or CMV B7 tetramers. Tetramer staining was carried out after overnight incubation for the no activation control or before addition of the activators for the “activated” cells. Cells were stained for CD69 (A), IFN-γ (B), and MIP-1β (C) and analyzed by flow cytometry. Data show cells gated on the CMV B7 tetramer-positive population. Mean fluorescence intensity is shown for each condition.

Mentions: Different methods of T cell activation were studied to determine the optimal protocol for intracellular cytokine staining. OKT3 is an antibody that activates T cells by binding to the CD3 molecule and cross-linking the TCR complex 31. Specific peptides are presented by the HLA molecules on APCs among the PBMC population to the TCRs of specific CD8+ T cells and induce activation. Since this interaction is reproduced when tetramers bind to the TCR-appropriate T cells, we examined whether they could directly influence T cell activation or interfere with activation of the cells induced by either peptide or OKT3. Fig. 2 A shows the extent of activation of the CMV B7 tetramer-staining population treated with tetramers alone or in combination with either OKT3 or peptide, using the early activation marker CD69. Similar levels of CD69 upregulation were observed using all three methods, confirming that tetramers can directly activate T cells. Whereas activation levels appeared to be identical, different profiles of IFN-γ and MIP-1β secretion were observed using intracellular staining. This was particularly striking for IFN-γ release, which was very low using OKT3 or tetramers, but maximal when triggered by specific peptide on APCs (Fig. 2 B). Compared with IFN-γ, higher levels of MIP-1β secretion were observed in response to OKT3 or tetramers, but were maximal if stimulated by the peptide-pulsed cells (Fig. 2 C). Thus, the optimal method of inducing cytokine release from antigen-specific CD8+ T cells appears to be the use of specific peptides. Similar results were obtained with PBMCs from different donors (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)

Comparison among peptide, OKT3, and tetramer activation. PBMCs from a CMV-positive B7 donor were incubated for 12 h with brefeldin A in the presence of PBS (no activation control), specific CMV B7 peptide (10 μM), OKT3 (100 ng/ml), or CMV B7 tetramers. Tetramer staining was carried out after overnight incubation for the no activation control or before addition of the activators for the “activated” cells. Cells were stained for CD69 (A), IFN-γ (B), and MIP-1β (C) and analyzed by flow cytometry. Data show cells gated on the CMV B7 tetramer-positive population. Mean fluorescence intensity is shown for each condition.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Comparison among peptide, OKT3, and tetramer activation. PBMCs from a CMV-positive B7 donor were incubated for 12 h with brefeldin A in the presence of PBS (no activation control), specific CMV B7 peptide (10 μM), OKT3 (100 ng/ml), or CMV B7 tetramers. Tetramer staining was carried out after overnight incubation for the no activation control or before addition of the activators for the “activated” cells. Cells were stained for CD69 (A), IFN-γ (B), and MIP-1β (C) and analyzed by flow cytometry. Data show cells gated on the CMV B7 tetramer-positive population. Mean fluorescence intensity is shown for each condition.
Mentions: Different methods of T cell activation were studied to determine the optimal protocol for intracellular cytokine staining. OKT3 is an antibody that activates T cells by binding to the CD3 molecule and cross-linking the TCR complex 31. Specific peptides are presented by the HLA molecules on APCs among the PBMC population to the TCRs of specific CD8+ T cells and induce activation. Since this interaction is reproduced when tetramers bind to the TCR-appropriate T cells, we examined whether they could directly influence T cell activation or interfere with activation of the cells induced by either peptide or OKT3. Fig. 2 A shows the extent of activation of the CMV B7 tetramer-staining population treated with tetramers alone or in combination with either OKT3 or peptide, using the early activation marker CD69. Similar levels of CD69 upregulation were observed using all three methods, confirming that tetramers can directly activate T cells. Whereas activation levels appeared to be identical, different profiles of IFN-γ and MIP-1β secretion were observed using intracellular staining. This was particularly striking for IFN-γ release, which was very low using OKT3 or tetramers, but maximal when triggered by specific peptide on APCs (Fig. 2 B). Compared with IFN-γ, higher levels of MIP-1β secretion were observed in response to OKT3 or tetramers, but were maximal if stimulated by the peptide-pulsed cells (Fig. 2 C). Thus, the optimal method of inducing cytokine release from antigen-specific CD8+ T cells appears to be the use of specific peptides. Similar results were obtained with PBMCs from different donors (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