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Telomere erosion in memory T cells induced by telomerase inhibition at the site of antigenic challenge in vivo.

Reed JR, Vukmanovic-Stejic M, Fletcher JM, Soares MV, Cook JE, Orteu CH, Jackson SE, Birch KE, Foster GR, Salmon M, Beverley PC, Rustin MH, Akbar AN - J. Exp. Med. (2004)

Bottom Line: Furthermore, significant telomere erosion occurred in specific T cells that respond in the skin, but not in those that are found in the blood from the same individuals.Antibody inhibition studies indicated that type I interferon (IFN), which was identified at high levels in the tissue fluid and by immunohistology, was responsible in part for the telomerase inhibition.Furthermore, the addition of IFN-alpha to PPD-stimulated CD4+ T cells directly inhibited telomerase activity in vitro.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Immunology and Molecular Pathology, Div. of Infection and Immunity, University College London, 46 Cleveland St., London W1T 4JF, England, UK.

ABSTRACT
The extent of human memory T cell proliferation, differentiation, and telomere erosion that occurs after a single episode of immune challenge in vivo is unclear. To investigate this, we injected tuberculin purified protein derivative (PPD) into the skin of immune individuals and isolated responsive T cells from the site of antigenic challenge at different times. PPD-specific CD4+ T cells proliferated and differentiated extensively in the skin during this secondary response. Furthermore, significant telomere erosion occurred in specific T cells that respond in the skin, but not in those that are found in the blood from the same individuals. Tissue fluid obtained from the site of PPD challenge in the skin inhibited the induction of the enzyme telomerase in T cells in vitro. Antibody inhibition studies indicated that type I interferon (IFN), which was identified at high levels in the tissue fluid and by immunohistology, was responsible in part for the telomerase inhibition. Furthermore, the addition of IFN-alpha to PPD-stimulated CD4+ T cells directly inhibited telomerase activity in vitro. Therefore, these results suggest that the rate of telomere erosion in proliferating, antigen-specific CD4+ T cells may be accelerated by type I IFN during a secondary response in vivo.

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Antigen-specific CD4+ T cell infiltration and proliferation in the skin during the MT. PBMCs and blister cells were stimulated with PPD, control antigen (tetanus toxoid), or left unstimulated for 15 h in the presence of brefeldin A. Representative dot plots of intracellular IFN-γ expression in days 3 and 7 blister cells are shown (a). The number in the top right quadrant of each dot plot indicates the percentage of CD4+ T cells producing IFN-γ. (b) The proportion of IFN-γ–secreting (PPD-specific) CD4+ T cells after PPD injection. The graph illustrates intracellular IFN-γ expression in PBMCs stimulated with PPD (gray diamonds) and blister cells stimulated with PPD (black squares) or tetanus toxoid (white squares). The mean ± SEM of 3–11 experiments performed at each time point is shown. Proliferating CD4+ T cells in skin sections after PPD injection (c). Double immunofluorescence staining shows CD4+ cells (green) and proliferating Ki67+ cells (red; magnification, 20), whereas double positive Ki67+CD4+ cells are shown in yellow. (d) The percentage of proliferating Ki67+ CD4+ T cells in the skin after PPD challenge was determined by flow cytometry using blister cells (gray bars) and by double immunofluorescence in skin sections (white bars). The proportion of apoptotic (TUNEL+) T cells in the perivascular infiltrates of skin sections was determined by double immunofluorescence (diamonds and dashed line). The mean ± SEM of three to five experiments performed at each time point is shown.
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fig2: Antigen-specific CD4+ T cell infiltration and proliferation in the skin during the MT. PBMCs and blister cells were stimulated with PPD, control antigen (tetanus toxoid), or left unstimulated for 15 h in the presence of brefeldin A. Representative dot plots of intracellular IFN-γ expression in days 3 and 7 blister cells are shown (a). The number in the top right quadrant of each dot plot indicates the percentage of CD4+ T cells producing IFN-γ. (b) The proportion of IFN-γ–secreting (PPD-specific) CD4+ T cells after PPD injection. The graph illustrates intracellular IFN-γ expression in PBMCs stimulated with PPD (gray diamonds) and blister cells stimulated with PPD (black squares) or tetanus toxoid (white squares). The mean ± SEM of 3–11 experiments performed at each time point is shown. Proliferating CD4+ T cells in skin sections after PPD injection (c). Double immunofluorescence staining shows CD4+ cells (green) and proliferating Ki67+ cells (red; magnification, 20), whereas double positive Ki67+CD4+ cells are shown in yellow. (d) The percentage of proliferating Ki67+ CD4+ T cells in the skin after PPD challenge was determined by flow cytometry using blister cells (gray bars) and by double immunofluorescence in skin sections (white bars). The proportion of apoptotic (TUNEL+) T cells in the perivascular infiltrates of skin sections was determined by double immunofluorescence (diamonds and dashed line). The mean ± SEM of three to five experiments performed at each time point is shown.

Mentions: We evaluated the proportion of antigen-specific CD4+ T cells that were isolated from the skin during the MT by their ability to synthesize IFN-γ after restimulation with PPD in vitro (Fig. 2 a). Minimal IFN-γ was synthesized when PPD was not added to the blister cells or when the cells were stimulated with tetanus toxoid (Fig. 2, a and b). Therefore, the cells that expressed IFN-γ were Ag-specific (PPD). We found a significant increase in these cells after intradermal challenge with PPD (Fig. 2 b; Kruskal-Wallis test; P < 0.0001). There was no increase in Ag-specific CD4+ T cells in the blood, and these remained relatively constant at 0.5% of the CD4+ T cell pool throughout the course of the response (Fig. 2 b). The maximum proportion of Ag-specific CD4+ T cells detected after antigen challenge (32% of total CD4+ T cells) was considerably higher than that predicted from previous studies (0.5–2%; reference 24). The proportion of CD8+ Ag-specific T cells in the blisters did not increase during the course of the response and was <1% at all time points.


Telomere erosion in memory T cells induced by telomerase inhibition at the site of antigenic challenge in vivo.

Reed JR, Vukmanovic-Stejic M, Fletcher JM, Soares MV, Cook JE, Orteu CH, Jackson SE, Birch KE, Foster GR, Salmon M, Beverley PC, Rustin MH, Akbar AN - J. Exp. Med. (2004)

Antigen-specific CD4+ T cell infiltration and proliferation in the skin during the MT. PBMCs and blister cells were stimulated with PPD, control antigen (tetanus toxoid), or left unstimulated for 15 h in the presence of brefeldin A. Representative dot plots of intracellular IFN-γ expression in days 3 and 7 blister cells are shown (a). The number in the top right quadrant of each dot plot indicates the percentage of CD4+ T cells producing IFN-γ. (b) The proportion of IFN-γ–secreting (PPD-specific) CD4+ T cells after PPD injection. The graph illustrates intracellular IFN-γ expression in PBMCs stimulated with PPD (gray diamonds) and blister cells stimulated with PPD (black squares) or tetanus toxoid (white squares). The mean ± SEM of 3–11 experiments performed at each time point is shown. Proliferating CD4+ T cells in skin sections after PPD injection (c). Double immunofluorescence staining shows CD4+ cells (green) and proliferating Ki67+ cells (red; magnification, 20), whereas double positive Ki67+CD4+ cells are shown in yellow. (d) The percentage of proliferating Ki67+ CD4+ T cells in the skin after PPD challenge was determined by flow cytometry using blister cells (gray bars) and by double immunofluorescence in skin sections (white bars). The proportion of apoptotic (TUNEL+) T cells in the perivascular infiltrates of skin sections was determined by double immunofluorescence (diamonds and dashed line). The mean ± SEM of three to five experiments performed at each time point is shown.
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Related In: Results  -  Collection

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fig2: Antigen-specific CD4+ T cell infiltration and proliferation in the skin during the MT. PBMCs and blister cells were stimulated with PPD, control antigen (tetanus toxoid), or left unstimulated for 15 h in the presence of brefeldin A. Representative dot plots of intracellular IFN-γ expression in days 3 and 7 blister cells are shown (a). The number in the top right quadrant of each dot plot indicates the percentage of CD4+ T cells producing IFN-γ. (b) The proportion of IFN-γ–secreting (PPD-specific) CD4+ T cells after PPD injection. The graph illustrates intracellular IFN-γ expression in PBMCs stimulated with PPD (gray diamonds) and blister cells stimulated with PPD (black squares) or tetanus toxoid (white squares). The mean ± SEM of 3–11 experiments performed at each time point is shown. Proliferating CD4+ T cells in skin sections after PPD injection (c). Double immunofluorescence staining shows CD4+ cells (green) and proliferating Ki67+ cells (red; magnification, 20), whereas double positive Ki67+CD4+ cells are shown in yellow. (d) The percentage of proliferating Ki67+ CD4+ T cells in the skin after PPD challenge was determined by flow cytometry using blister cells (gray bars) and by double immunofluorescence in skin sections (white bars). The proportion of apoptotic (TUNEL+) T cells in the perivascular infiltrates of skin sections was determined by double immunofluorescence (diamonds and dashed line). The mean ± SEM of three to five experiments performed at each time point is shown.
Mentions: We evaluated the proportion of antigen-specific CD4+ T cells that were isolated from the skin during the MT by their ability to synthesize IFN-γ after restimulation with PPD in vitro (Fig. 2 a). Minimal IFN-γ was synthesized when PPD was not added to the blister cells or when the cells were stimulated with tetanus toxoid (Fig. 2, a and b). Therefore, the cells that expressed IFN-γ were Ag-specific (PPD). We found a significant increase in these cells after intradermal challenge with PPD (Fig. 2 b; Kruskal-Wallis test; P < 0.0001). There was no increase in Ag-specific CD4+ T cells in the blood, and these remained relatively constant at 0.5% of the CD4+ T cell pool throughout the course of the response (Fig. 2 b). The maximum proportion of Ag-specific CD4+ T cells detected after antigen challenge (32% of total CD4+ T cells) was considerably higher than that predicted from previous studies (0.5–2%; reference 24). The proportion of CD8+ Ag-specific T cells in the blisters did not increase during the course of the response and was <1% at all time points.

Bottom Line: Furthermore, significant telomere erosion occurred in specific T cells that respond in the skin, but not in those that are found in the blood from the same individuals.Antibody inhibition studies indicated that type I interferon (IFN), which was identified at high levels in the tissue fluid and by immunohistology, was responsible in part for the telomerase inhibition.Furthermore, the addition of IFN-alpha to PPD-stimulated CD4+ T cells directly inhibited telomerase activity in vitro.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Immunology and Molecular Pathology, Div. of Infection and Immunity, University College London, 46 Cleveland St., London W1T 4JF, England, UK.

ABSTRACT
The extent of human memory T cell proliferation, differentiation, and telomere erosion that occurs after a single episode of immune challenge in vivo is unclear. To investigate this, we injected tuberculin purified protein derivative (PPD) into the skin of immune individuals and isolated responsive T cells from the site of antigenic challenge at different times. PPD-specific CD4+ T cells proliferated and differentiated extensively in the skin during this secondary response. Furthermore, significant telomere erosion occurred in specific T cells that respond in the skin, but not in those that are found in the blood from the same individuals. Tissue fluid obtained from the site of PPD challenge in the skin inhibited the induction of the enzyme telomerase in T cells in vitro. Antibody inhibition studies indicated that type I interferon (IFN), which was identified at high levels in the tissue fluid and by immunohistology, was responsible in part for the telomerase inhibition. Furthermore, the addition of IFN-alpha to PPD-stimulated CD4+ T cells directly inhibited telomerase activity in vitro. Therefore, these results suggest that the rate of telomere erosion in proliferating, antigen-specific CD4+ T cells may be accelerated by type I IFN during a secondary response in vivo.

Show MeSH
Related in: MedlinePlus