Long-Term Maintenance of Virus-Specific Effector Memory CD8⁺ T Cells in the Lung Airways Depends on Proliferation

Viruses, animals, and infections

The Enders strain of Sendai virus was grown, stored, and titrated as previously described (4). Female C57BL/6, B6.PL-Thy1a/Cy (Thy1.1⁺), and B6.SJL-Ptprca Pep3b/BoyJ (Ly5.1⁺) mice were purchased from the Animal Breeding Facility at the Trudeau Institute (Saranac Lake, NY). Mice (6–12 wk) were anesthetized by i.p. injection with 2,2,2-tribromoethanol and infected intranasally with either 125 or 500 50% egg infectious doses (EID₅₀)⁴ of Sendai virus.

Peptides

Sendai virus peptides (nucleoprotein (NP) 324–332) and influenza virus (NP_366–374) peptides were purchased from New England Peptide (Fitchburg, MA). Peptide purity was evaluated using reversed-phase HPLC analysis.

In vivo 5-bromo-2-deoxyuridine (BrdU) labeling

BrdU (Sigma-Aldrich, St. Louis, MO) was administered either in sterile drinking water (0.8 mg/ml) or by i.p. injection of 0.2 ml of PBS/BrdU solution (4 mg/ml) at the indicated times during infection. In experiments where mice were fed water containing BrdU, the solution was replaced daily. For pulse-chase experiments, animals were given normal drinking water at the end of the pulse period.

Tissue preparation

Single-cell suspensions were prepared from spleens and mediastinal lymph nodes (MLN) by passage through cell strainers. Spleen cells were depleted of erythrocytes by treatment with buffered ammonium chloride solution. Bronchoalveolar lavage (BAL) cells were collected by lavage of the lungs three to four times with 1 ml of HBSS. T cells were isolated from the remaining lung tissue by mechanical disruption through cell strainers. The cells were subsequently resuspended in 80% isotonic percoll and layered with 40% isotonic percoll. After centrifugation at 400 × g for 25 min, the cells at the 80%/40% interface were collected, washed, and counted.

Intratracheal cell transfers and in situ labeling of cells in the lung airways

BAL cells were collected from C57BL/6 mice at >30 days after Sendai virus infection. Where indicated, donor cells were labeled with CFSE (Molecular Probes, Eugene, OR) by incubation in HBSS containing 0.5–0.7 μM CSFE for 10 min in the dark. The cells were then washed and resuspended in PBS at a concentration of 0.5–2 × 10⁷ cells/ml before transfer. Thy1.1⁺ C57BL/6 recipient mice were anesthetized and 100 μl (0.5–2 × 10⁶ cells) of the cell suspension or PBS were instilled into the lungs (via the trachea) using a 1-ml syringe fitted with a blunted 20-gauge needle. In some experiments, CFSE-labeled cells were subsequently isolated from the lung airways and restimulated for 4–5 days in vitro, using either the Sendai (Sen) NP_324–332 or influenza (Flu)-NP_366–374 peptides (2 μg/ml final concentration), human rIL-2 (10 U/ml final concentration) (R&D Systems, Minneapolis, MN), and a cell density of 1 × 10⁶/well in 24-well plates. All wells received 1 × 10⁶ gamma-irradiated (4000 rad) Ly5.1⁺ naive spleen cells as APCs. For in situ labeling of cells in the lung airways, 80 μl of PBS containing CFSE (0.2 mM) was instilled into the trachea using a blunted 20-gauge needle.

MHC tetrameric reagents, staining, and analysis

MHC class I peptide tetramers were generated by the Molecular Biology Core Facility at the Trudeau Institute as described previously (16). The Sen-NP_324–332/K^b tetramer is highly specific for CD8⁺ T cells that recognize the Sendai virus NP_324–332/K^b epitope (2, 17). Staining with tetrameric reagents was performed for 1 h at room temperature. After washing, the cells were stained with anti-CD8 Tricolor (Caltag Laboratories, Burlingame, CA) or anti-CD8-CyChrome and biotinylated, FITC-conjugated, or APC-conjugated Abs specific for Ly5.1 (CD45.1) or Thy1.2 (BD PharMingen, San Diego, CA) on ice for 20 min. Stained samples were run on either a BD Biosciences FACScan or FACSCalibur flow cytometer and data were analyzed using CellQuest software (BD Immunocytometry Systems, San Jose, CA). In some experiments, B cells were depleted before staining by panning on anti-Ig-coated flasks.

BrdU staining was performed as previously described (18). Briefly, 1–2 × 10⁶ cells were surface stained as indicated above and treated with 0.9 ml of FACS Lysing Solution (BD Biosciences) for 15 min at room temperature. The cells were then fixed overnight at 4°C in 1 ml of 1% paraformaldehyde/PBS containing 0.05% Nonidet P-40. After washing, cellular DNA was denatured with 50 Kunitz units of DNase I (bovine pancreas; Sigma-Aldrich) for 30 min at 37°C. The cells were then washed with PBS containing 5% FCS and 0.5% Nonidet P-40 and incubated with 5 μl of anti-BrdU/FITC (BD Biosciences) for 45 min on ice. The cells were washed twice before analysis by flow cytometry.

The percentage of tetramer-positive cells among total live cells was calculated by dividing the number of tetramer⁺/CD8⁺ events by the total number of events in a live cell gate.

Cell cycle analysis

T cells were isolated from the lung airways and surface stained as indicated above. After washing, the cells were resuspended in 1 ml of PBS containing 2% FCS, 0.1% NaN₃, 0.5% saponin, and 10 μg of Hoechst 33342 for 30–60 min on ice. Data were acquired using a FACSVantage SE cell sorter fitted with an argon laser emitting at 488 nm and a krypton laser with UV optics. The data were analyzed using FlowJo software (Treestar, San Carlos, CA), and doublets were excluded by width and area analysis.

Two distinct populations of memory CD8⁺ T cells remain in the lungs following recovery from a Sendai virus infection

Substantial numbers of NP_324–332/K^b-specific CD8⁺ T cells persist in the lung airways of C57BL/6 mice following recovery from an intranasal Sendai virus infection (5, 6). These cells are located primarily in the airway epithelium and the lamina propria (data not shown) and express markers that are generally associated with acute activation (CD25⁺ and CD69⁺) (3). The absolute numbers of Ag-specific memory CD8⁺ T cells in the airways decline slowly over the first 6 mo postinfection (with a t_1/2 of ∼40 days) and then stabilize thereafter (5). To identify the mechanisms that control this biphasic decline in numbers of memory T cells in the lung airways, we first asked whether the cells were proliferating. Mice were given BrdU in the drinking water for a period of 8 days during either the acute phase of infection (days 0–8) or after a stable memory population had been established (days 25–32, Sendai virus is cleared by day 10). T cells were then isolated from the lung airways and tissues on day 33 and NP_324–332/K^b-specific T cells were analyzed for the incorporation of BrdU. As shown in Fig. 1⇓, when BrdU was administered between 0 and 8 days of the infection, ∼90% of the NP_324–332/K^b-specific memory T cells in the lung airways were BrdU positive on day 33. This indicates that virus-specific CD8⁺ T cells had divided during the acute infection and incorporated BrdU, but the majority had not divided substantially after that time. However, at least 10% of the cells were BrdU negative, indicating that some cells had continued to divide after the virus had been cleared. To confirm this, we analyzed BrdU incorporation later in the response after infectious virus was cleared (days 25–32 postinfection). As shown in Fig. 1⇓, 14% of the NP_324–332/K^b-specific T cells in the lung airways on day 33 were BrdU positive, indicating that some cell division at this late time point. Similar data were obtained for cells isolated from the lung tissues (Fig. 1⇓). The reciprocal nature of the BrdU profiles in these experiments suggested that there were two populations of NP_324–332/K^b-specific memory T cells in the lung airways. One was a large static population of nonproliferating cells (90%) that persisted in the lung airways following the acute infection. The other was a smaller population of cells that continued to divide after resolution of the primary infection (10%).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

The majority of Sendai virus NP_324–332/K^b-specific memory CD8⁺ T cells in the lung undergo minimal proliferation following resolution of the primary infection. Three groups of C57BL/6 mice were intranasally infected with 500 EID₅₀ Sendai virus. One group was given normal drinking water (No BrdU, n = 3), a second group was given BrdU water from days 0 to 8 postinfection (BrdU days 0–8, n = 3), and a third group was given BrdU water from days 25 to 32 postinfection (BrdU days 25–32, n = 3). At day 33 after infection, BAL cells and cells isolated from the lung tissue (after lavage) were pooled within each group and stained with Sen-NP_324–332/K^b tetramer-PE, anti-CD8-Tricolor, and anti-BrdU-FITC Abs. The data are shown as anti-BrdU incorporation among CD8⁺/tetramer⁺ gated T cells. The data are representative of two independent experiments.

To further investigate this hypothesis, we analyzed the stability of these two BrdU-defined populations over time. As shown in Table I⇓, memory T cells that had been labeled during days 1–8 declined from 90% of the recovered cells at day 33 to 0% by day 368 (Table I⇓). This decline in the frequency of BrdU-labeled NP_324–332/K^b-specific memory cells paralleled the early decline (over the first 6 mo) in absolute numbers of cells in the lung airways that has been reported previously (5). Similarly, when mice were given BrdU in the drinking water at late time points postinfection, such as 1 year, there was a steady accumulation of BrdU-labeled NP_324–332/K^b-specific memory T cells in the lung airways over 21 days (Fig. 2⇓B). This rate of accumulation was identical to that observed at 1 mo after infection (Fig. 2⇓A) and was also very similar to that observed in the spleen. These observations are consistent with the idea that a subpopulation of memory CD8⁺ T cells in the lung airways are maintained over the long term by homeostatic proliferation of the memory T cell pool. Together, the data support the hypothesis that memory CD8⁺ T cells in the lung are comprised of a pool of nondividing cells that remain static in the lung after the resolution of the infection and a pool of slowly dividing cells that may be recruited from the lymphoid organs.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Sendai virus NP_324–332/K^b-specific memory CD8⁺ T cells in the lung airways and spleen are maintained by homeostatic proliferation. C57BL/6 mice were intranasally infected with 500 EID₅₀ Sendai virus and then given BrdU by continuous feeding in the drinking water starting at either 1 mo (A) or 12 mo (B) after infection. On various days after BrdU initiation, cells were isolated from the lung airways (○) or spleen (▪) and stained with Sen-NP_324–332/K^b tetramer-PE, anti-CD8-Tricolor, and anti-BrdU-FITC Abs. The data are shown as BrdU incorporation among CD8⁺/tetramer⁺ gated T cells and the error bars indicate the SD of three individual mice. The full time course was done once, but the day 6 and day 21 time points were repeated several times with similar results.

Memory T cells are able to persist in the lung airways for at least 2 wk

The BrdU data suggested that the biphasic kinetics of NP_324–332/K^b-specific memory T cell persistence in the lungs may be attributable to the deposition of a large bolus of nondividing memory cells during the acute response, which slowly declined over time, and a parallel slow continual recruitment of recently divided cells from secondary lymphoid organs. To distinguish between persistence vs continual recruitment, memory T cells were labeled in situ in the lung airways by intratracheal administration of CFSE (day 16 after Sendai virus infection). Flow cytometry was then used to follow the survival of the labeled cells over the succeeding 2 wk. Analysis 1 day after labeling indicated that ∼30% of the total cells in the airways became labeled with CFSE (Fig. 3⇓A). Although the range of staining intensity was quite broad, it was possible to specifically gate on CFSE-positive cells and demonstrate that they included a substantial fraction of NP_324–332/K^b-specific T cells (Fig. 3⇓, A and B). Further analysis on days 4 and 15 postlabeling (Fig. 3⇓, C and D, representing days 20 and 31 postinfection), demonstrated that CFSE-positive, NP_324–332/K^b-specific T cells could be readily recovered 2 wk after labeling. The absolute percentage of CFSE-positive cells in the airways declined over time from 30% at day 1 postlabeling to <5% at day 15 postlabeling (data not shown). However, the percentage of NP_324–332/K^b-specific T cells remained stable within this CFSE-labeled population. These data confirmed that a substantial number of Ag-specific memory CD8⁺ T cells are able to persist in the lung airways for at least 2 wk. These recovered cells expressed CD69 and CD25 markers, consistent with the activated phenotype found in previous studies on memory CD8⁺ T cells recovered from the lung airways (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Sendai virus-specific memory CD8⁺ T cells persist in the lung airways for >2 wk. C57BL/6 mice were infected intranasally with Sendai virus and on day 16 after infection CFSE (80 μl) was introduced directly into the lung airways via the trachea. On days 1 (A and B), 4 (C), and 15 (D) after CFSE labeling, cells were isolated from the lung airways (BAL) and stained with Sen-NP_324–332/K^b tetramer-PE and anti-CD8-Tricolor. The data in A are a histogram of CFSE fluorescence with the frequency of CFSE⁺ cells among the lymphocyte population. The data in B–D are gated on CFSE⁺ lymphocytes and show Sen-NP_324–332/K^b tetramer vs CD8 staining. The percentages in these panels indicate the frequencies of tetramer⁺ cells among CD8⁺ T cells. The frequencies of CFSE⁺ lymphocytes in the spleen and MLN at 1 day after CFSE labeling were <0.7%. The data are representative of four individual experiments.

Together, the BrdU and CFSE data suggested that some memory T cells persist in the lung airways for substantial lengths of time without proliferating. Consequently, we speculated that the majority of the cells were in a resting phase of the cell cycle. To test this idea, we analyzed the DNA content of NP_324–332/K^b-specific T cells in the lung airways by flow cytometry throughout the acute infection and after memory had been established. As shown in Fig. 4⇓A, ∼17% of NP_324–332/K^b-specific CD8⁺ T cells in the lung airways were in the G₂-M or S phase of the cell cycle at the peak of the infection (day 8 postinfection). However, this percentage decreased to <1% by day 15 postinfection and then persisted at this level for >24 days (Fig. 4⇓A). A similar pattern of cell cycle kinetics was observed in tetramer-negative CD8⁺ T cells, except that a lower percentage of the cells were in cycle at day 8 (Fig. 4⇓B). Together these data confirm that the effector memory CD8⁺ T cells that persist in the lung following recovery from a Sendai virus infection are not in the cell cycle.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Sendai virus NP_324–332/K^b-specific memory CD8⁺ T cells in the lung airways persist predominantly in the G₀-G₁ stage of the cell cycle. BAL and spleen cells were isolated from C57BL/6 mice at various times after Sendai virus infection and stained with Sen-NP_324–332/K^b and anti-CD8-Tricolor. The cells were then stained with Hoechst 33342 and analyzed as described in Materials and Methods. The data show the percentages of Sen-NP_324–332/K^b tetramer⁺/CD8⁺ (A) cells or Sen-NP_324–332/K^b tetramer-negative/CD8⁺ (B) cells that were in G₂-S phase of the cell cycle. The line represents the average of two experiments.

Nonproliferating memory T cells in the lung retain the capacity to mediate a potent proliferative response to Ag

The BrdU and CFSE studies suggested that large numbers of nonproliferating memory CD8⁺ T cells persisted in the lung airways after recovery from a Sendai virus infection. We further investigated the longevity of memory T cells in the airways using an intratracheal transfer system. BAL cells were isolated from Thy1.2⁺ mice that had recovered from a Sendai virus infection and CFSE labeled in vitro. The labeled cells were then transferred to the airways of naive Thy1.1⁺ mice. After 8 days, the BAL, lung tissue, MLN, and spleens were recovered from recipient mice and donor cells were identified by Thy1.2 staining. As shown in Fig. 5⇓ (left panels), substantial numbers of Thy1.2⁺ donor cells could be recovered from the airways, but not the lung tissue, spleens, or MLN, indicating that the donor cells had not colonized other sites. Analysis of the CFSE levels on NP_324–332/K^b tetramer-positive cells at various days (days 1–8) posttransfer further demonstrated that the cells did not proliferate over this time frame (Fig. 5⇓, right panels). Similar results were obtained when memory T cells were transferred into the lungs of mice that had previously recovered from a Sendai virus infection (data not shown). In this case, the host and donor NP_324–332/K^b-specific CD8⁺ T cells were distinguished using the Thy1 marker. Although the memory cells from the lung airways did not proliferate in situ, they had not lost their capacity to respond to Ag. Thus, cells that had been reisolated from the lung airways 8 days after transfer proliferated in vitro in response to the NP_324–332 peptide and IL-2 (Fig. 6⇓). These cells were also able to produce IFN-γ and mediate cytolytic activity in response to target cells coated with the NP_324–332 peptide (data not shown). Taken together, the data demonstrate that memory CD8⁺ T cells can survive in the lung airways for substantial periods of time without proliferating and still retain the capacity to respond to Ag.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

Sendai virus-specific CD8⁺ T cells isolated from the lung airways do not proliferate after intratracheal transfer into naive mice. BAL cells were collected from C57BL/6 (Thy1.2⁺) mice at day 31 after Sendai virus infection, labeled with CFSE, and transferred into naive C57BL/6 (Thy1.1⁺) mice via the trachea. On day 8 posttransfer, cells isolated from the spleen, MLN, BAL, or lung tissue were analyzed for donor cell Thy1.2 expression (left panels). In addition, at days 1, 2, 4, and 8 after transfer, BAL cells were isolated and stained with Sen-NP_324–332/K^b tetramer-PE, anti-CD8-CyChrome, and anti-Thy1.2-allophycocyanin (right panels). The data are gated on Thy1.2⁺/CD8⁺ lymphocytes and are representative of five independent experiments. The absolute numbers of donor cells recovered from the BAL was quite variable between experiments. However, in every experiment, donor cells were only recovered from the BAL, and not other tissues.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Sendai virus-specific memory CD8⁺ T cells remain functional following intratracheal transfer. BAL cells were collected from C57BL/6 (Thy1.2⁺) mice at day 42 after Sendai virus infection and transferred into naive C57BL/6 (Thy1.1⁺) mice via the trachea. At 8 days posttransfer, BAL cells were reisolated from the lung airways and stimulated in vitro with IL-2 and either Flu-NP_366–374 (control) or Sen-NP_324–332 peptides. After 5 days, the cells were stained with Sen-NP_324–332/K^b tetramer-PE, anti-CD8-Tricolor, and anti-Thy1.2-allophycocyanin. The upper panel represents Sen-NP_324–332/K^b tetramer-PE staining and CFSE intensity among control peptide-stimulated Thy1.2⁺/CD8⁺ lymphocytes and the lower panel represents Sen-NP_324–332/K^b tetramer-PE staining and CFSE intensity among Sen-NP_324–332-stimulated Thy1.2⁺/CD8⁺ lymphocytes. The data are representative of three independent experiments. The level of expansion of tetramer-positive cells induced by the Sen-NP_324–332 peptide was variable between experiments, but was always much greater than that induced by the control Flu-NP_366–374 peptide.