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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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Intracellular staining for IFN-γ, MIP-1β, and TNF-α in HIV-specific or CMV-specific CD8+ T cells. PBMCs from HIV-infected patients were incubated for 6 h with brefeldin A in the presence of PBS (no activation) or specific peptides (10 μM) (activation). Intracellular staining for IFN-γ, MIP-1β, and TNF-α was carried out, and the cells were analyzed by flow cytometry. Cytokine staining is shown on CMV (A) and HIV (B) specific CD8+ T cell populations gated using the tetramers (top). Percentages of cells present in quadrants are shown. Representative data are shown (see Table ).
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Figure 4: Intracellular staining for IFN-γ, MIP-1β, and TNF-α in HIV-specific or CMV-specific CD8+ T cells. PBMCs from HIV-infected patients were incubated for 6 h with brefeldin A in the presence of PBS (no activation) or specific peptides (10 μM) (activation). Intracellular staining for IFN-γ, MIP-1β, and TNF-α was carried out, and the cells were analyzed by flow cytometry. Cytokine staining is shown on CMV (A) and HIV (B) specific CD8+ T cell populations gated using the tetramers (top). Percentages of cells present in quadrants are shown. Representative data are shown (see Table ).

Mentions: The optimized protocol for combined tetramer and intracellular cytokine staining described above was used to study the function of circulating CD8+ T cells specific for HIV and CMV. Fig. 4 shows representative results of intracellular staining for IFN-γ, MIP-1β, and TNF-α within the CMV (A) or HIV (B) specific CD8+ T cell population of two different donors. A slight decrease in the percentage of tetramer-positive cells was generally observed after stimulation. A small proportion of the activated T cells may not stain brightly enough to be gated among the tetramer-positive cells, or some cells may have presented antigen to one another and been lysed. However, the independent analysis of the tetramer staining data and cytokine staining data suggest that the gated tetramer-positive activated population is representative of the antigen-specific population as a whole. The cytokine expression profile is remarkably similar between the two viral specificities. After activation of the cells using specific antigens, the great majority of tetramer-staining cells displayed reduced levels of CD8 and TCR expression, and upregulation of CD69, indicating that most of the virus-specific population was adequately activated. 21 HIV-infected donors were studied, using the tetramers shown in Table (in total, 26 different tetramer-positive populations were studied). In most donors, between 50 and 95% of the activated virus-specific CD8+ T cells were producing IFN-γ and MIP-1β (Table ). Interestingly, although most of the antigen-specific CD8+ T cells secreted TNF-α in parallel with the other factors, a distinct subset of tetramer-staining cells was identified that failed to produce TNF-α even though they secreted IFN-γ and MIP-1β and had upregulated CD69. The size of the TNF-α–negative subpopulation varied between 0 and 60% in different tetramer-staining populations. Double cytokine stainings confirmed that these cells could still produce the other cytokines (data not shown). Overall, these results clearly demonstrate that, in chronically HIV-infected donors with a significant population of HIV-specific or CMV-specific tetramer-staining CD8+ cells, the majority of these cells produce a range of antiviral soluble factors in response to specific antigens. However, 2 of the 15 HIV-specific populations we analyzed (H9 and H14) showed much lower numbers of cytokine-secreting cells (∼25% of tetramer-positive cells). We are currently in the process of investigating these cells further to characterize their apparent defect in cytokine secretion.


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)

Intracellular staining for IFN-γ, MIP-1β, and TNF-α in HIV-specific or CMV-specific CD8+ T cells. PBMCs from HIV-infected patients were incubated for 6 h with brefeldin A in the presence of PBS (no activation) or specific peptides (10 μM) (activation). Intracellular staining for IFN-γ, MIP-1β, and TNF-α was carried out, and the cells were analyzed by flow cytometry. Cytokine staining is shown on CMV (A) and HIV (B) specific CD8+ T cell populations gated using the tetramers (top). Percentages of cells present in quadrants are shown. Representative data are shown (see Table ).
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Related In: Results  -  Collection

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Figure 4: Intracellular staining for IFN-γ, MIP-1β, and TNF-α in HIV-specific or CMV-specific CD8+ T cells. PBMCs from HIV-infected patients were incubated for 6 h with brefeldin A in the presence of PBS (no activation) or specific peptides (10 μM) (activation). Intracellular staining for IFN-γ, MIP-1β, and TNF-α was carried out, and the cells were analyzed by flow cytometry. Cytokine staining is shown on CMV (A) and HIV (B) specific CD8+ T cell populations gated using the tetramers (top). Percentages of cells present in quadrants are shown. Representative data are shown (see Table ).
Mentions: The optimized protocol for combined tetramer and intracellular cytokine staining described above was used to study the function of circulating CD8+ T cells specific for HIV and CMV. Fig. 4 shows representative results of intracellular staining for IFN-γ, MIP-1β, and TNF-α within the CMV (A) or HIV (B) specific CD8+ T cell population of two different donors. A slight decrease in the percentage of tetramer-positive cells was generally observed after stimulation. A small proportion of the activated T cells may not stain brightly enough to be gated among the tetramer-positive cells, or some cells may have presented antigen to one another and been lysed. However, the independent analysis of the tetramer staining data and cytokine staining data suggest that the gated tetramer-positive activated population is representative of the antigen-specific population as a whole. The cytokine expression profile is remarkably similar between the two viral specificities. After activation of the cells using specific antigens, the great majority of tetramer-staining cells displayed reduced levels of CD8 and TCR expression, and upregulation of CD69, indicating that most of the virus-specific population was adequately activated. 21 HIV-infected donors were studied, using the tetramers shown in Table (in total, 26 different tetramer-positive populations were studied). In most donors, between 50 and 95% of the activated virus-specific CD8+ T cells were producing IFN-γ and MIP-1β (Table ). Interestingly, although most of the antigen-specific CD8+ T cells secreted TNF-α in parallel with the other factors, a distinct subset of tetramer-staining cells was identified that failed to produce TNF-α even though they secreted IFN-γ and MIP-1β and had upregulated CD69. The size of the TNF-α–negative subpopulation varied between 0 and 60% in different tetramer-staining populations. Double cytokine stainings confirmed that these cells could still produce the other cytokines (data not shown). Overall, these results clearly demonstrate that, in chronically HIV-infected donors with a significant population of HIV-specific or CMV-specific tetramer-staining CD8+ cells, the majority of these cells produce a range of antiviral soluble factors in response to specific antigens. However, 2 of the 15 HIV-specific populations we analyzed (H9 and H14) showed much lower numbers of cytokine-secreting cells (∼25% of tetramer-positive cells). We are currently in the process of investigating these cells further to characterize their apparent defect in cytokine secretion.

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