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

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.

Figure 1.

The Mantoux test (MT): a human in vivo model of a memory immune response. Samples were collected between 0 and 19 d after PPD injection (a, left). Skin biopsies were collected for immunohistochemistry, and the top right panel shows a day-3 MT skin section with a perivascular (PV) collection of CD3⁺ T cells (E and D denote the epidermis and dermis, respectively; magnification, 20). Cutaneous lymphocytes were isolated from skin SBs that were induced over the sites of PPD injection at different times (a, bottom right). In panel b, the clinical score resulting from PPD challenge was measured at the time of sampling (□, left y axis), whereas the number of T cells within dermal perivascular infiltrates was determined by indirect immunoperoxidase staining (▪, right y axis). The mean ± SEM of five experiments per time point is shown. In panel c, the percentage of granulocytes (black bars), lymphocytes (gray bars), and other leukocytes (white bars) isolated from SBs raised at different time points after PPD injection is shown. Leukocyte proportions were determined by flow cytometry. The mean ± SEM for three to six experiments per time point is shown. The percentage of CD3⁺ T cells expressing CD4 in the peripheral blood (black bars) and blisters (white bars) during the course of the MT response was determined by flow cytometry (d). The mean ± SEM for four to seven experiments per time point is shown.

Figure 2.

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.

Figure 3.

Clonal analysis of CD4⁺ T cells in the skin during the MT. HDA of purified blood and blister CD4⁺ T cells was performed for the 26 Vβ families (a). Unstimulated blood CD4⁺ T cells (Unstim), blood CD4⁺ T cells stimulated with PPD in vitro for 7 d (PPD), and blister CD4⁺ T cells from a day 7 MT (Blister CD4) collected from the same individual were compared. One representative Vβ family from one out of five volunteers that were investigated is shown. Solid-line arrows indicate clonal bands shared between PPD-stimulated blood CD4⁺ T cells and blister cells. Dashed-line arrows indicate clonal bands that are only present in the PPD-stimulated blood CD4⁺ T cells. HDA was performed for 26 different Vβ families on purified blister CD4⁺ T cells isolated from paired MTs on days 7 and 19 (b). Arrows on the left of each gel indicate clonal bands that are present in the blisters at both time points. Three representative Vβ families from one out of four volunteers are shown. The asterisks on each gel indicate the position of the carrier homoduplex.

Figure 4.

Differentiation of CD4⁺ T cells in the skin during the MT. Representative dot plots of CD45RA and CD45RB expression on blood (PB) and suction blister (SB) CD4⁺ T cells on different days after PPD challenge (a). The dotted line defines the gate between CD45RB high and low expression. (b) The percentage of highly differentiated CD4⁺ memory T cells (defined as CD45RA⁻, CD45RB^lo) in the blood (▪) and skin blisters (▾) during the MT. The horizontal line denotes the mean. p-values were calculated using the paired Student's t test. The telomere length of blood (▪) and skin (▾) CD4⁺CD45RO⁺ T cells was determined during the MT (c). The telomere length of CD4⁺ T cells from blood and SBs at days 7 and 19 after PPD injection was determined by two-color flow-FISH. The horizontal line denotes the mean. p-values were calculated using the paired Student's t test. The telomere length of PPD-specific blood and skin CD4⁺ T cells 19 d after MT induction was determined by three-color flow-FISH (d). The histogram shows the telomere length (mean fluorescence intensity [MFI]) of PPD-specific (as defined by IFN-γ production) CD4⁺ T cells in the blood (shaded histogram) and blister (unshaded histogram). The data represent one out of two experiments with identical results that were performed.

Figure 5.

Low telomerase activity in CD4⁺ T cells in the skin after PPD stimulation in vivo. Telomerase activity in T cells isolated from SBs after PPD injection in two different individuals on days 3 and 7 after PPD injection (in vivo) and also from blood CD4⁺ T cells from one individual at different times after PPD stimulation in vitro (a). Telomerase activity (total product generated) per 500 Ki67⁺ proliferating T cells isolated from MTs (b, white bars) and PBMCs stimulated in vitro with PPD (black bars). The mean ± SEM of three experiments is shown. Skin CD4⁺ T cells can up-regulate telomerase activity when stimulated with PPD in vitro (c). T cells were isolated from day 3 MTs, and telomerase activity was determined in T cells immediately upon isolation (Blister), whereas the remaining cells were stimulated with PPD in vitro for 3 d (In vitro re-stim). Data are representative of four experiments performed. The negative control contains the PCR mix without cell extract, and the positive control contains an extract of a telomerase-positive tumor cell line. TSR8 denotes the internal quantitative control. Day 3 blister fluid suppresses telomerase activity (d). PBMCs were stimulated with PPD in vitro in the presence of autologous serum as a control or autologous blister fluid that was obtained from day 3 MT. Telomerase activity per 500 Ki67⁺ T cells was measured on day 3. The data are representative of three experiments performed.

Figure 6.

Type I IFN in blister fluid inhibits telomerase activity in PPD-stimulated T cells. IFN-α–producing cells present in day 3 MT skin sections were identified using indirect alkaline phosphatase staining (a, top). The bottom panel shows staining with an isotype control (magnification, 20). Keratinocytes (K) and isolated cells within the perivascular infiltrates (PV; indicated by arrows) expressed IFN-α. (b) The presence of type I IFN in blister fluid collected at different times after MT induction. The data represent the mean ± SEM of type I IFN in blister samples from five different individuals assayed per time point. Type I IFN mediates the suppression of telomerase activity induced by day 3 blister fluid (c). PBMCs were stimulated with PPD in vitro in the presence of autologous day 3 blister fluid (+MT BF) or autologous serum (+serum). Blocking antibody reactive with type I IFN receptor 2 (CD118; +anti-IFN R) or isotype control antibody (+cnt Ab) were added to cultures stimulated in the presence of MT blister fluid. Samples were collected 3 d after stimulation, and samples were adjusted to 500 proliferating Ki67⁺ T cells per reaction for telomerase analysis (one out of three representative experiments is shown). Direct inhibition of telomerase activity by type I IFN (d). A PPD-specific CD4⁺ T cell line was restimulated with PPD in the presence of recombinant IFN-α (black bars) or Roferon (white bars), a clinical preparation of IFN-α for 4 d. Samples were adjusted so that telomerase activity was measured in an equivalent number of cycling (Ki67⁺) T cells (one of three representative experiments is shown). Blister-derived CD4⁺ T cells can expand in vitro (e). Cells recovered from a day-19 blister were stimulated in vitro with PPD-pulsed irradiated autologous PBMCs as APCs. Arrows indicate each point of restimulation in vitro. The asterisks indicate points when samples were collected to measure telomerase activity. Telomerase activity of CD4⁺ T cells isolated from day 19 blisters decreases with repeated stimulation in vitro (f). Samples were collected on day 4 after each point of PPD restimulation. Telomerase activity per 500 Ki67⁺ T cells was determined as before. Stim 4, Stim 8, and Stim 9 indicate the 4th, 8th, and 9th stimulations with PPD and irradiated APCs in vitro.