Visualization of Early APC/T Cell Interactions in the Mouse Lung Following Intranasal Challenge

Mice

C57BL/6 and BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). DO11.10 TCR Tg mice (28) had previously been backcrossed to BALB/c for more than 10 generations and then bred to RAG2-deficient mice (RAG2^−/−), also on a BALB/c background. All mice used were female and 6–10 wk of age at the time of Ag challenge. Mice were housed in microisolator cages in a specific pathogen-free facility and provided with food and water ad libitum, according to protocols approved by the Washington University Institutional Animal Care and Use Committee. All mice appeared healthy, and regular monitoring of sentinels showed no serological or histological evidence of respiratory tract infection.

Fluorescent beads

The 0.431-μm carboxylate-modified fluorescent beads were obtained from Sigma-Aldrich (St. Louis, MO) at a concentration of ∼2.3 × 10¹² particles/ml (catalog nos. L3280 and L4030). Before i.n. administration, beads were washed three times in sterile PBS by centrifugation at 16,000 × g for 12 min and were resuspended in sterile PBS (Life Technologies, Grand Island, NY) at a final concentration of ∼7.5 × 10¹⁰ particles/ml. Conjugation of beads to OVA was conducted in 25 mM MES (pH 6.1), 1 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, with beads at 7.5 × 10¹⁰ beads/ml, and 2 mg/ml OVA (all from Sigma-Aldrich). Conjugation reactions were rotated overnight in the dark at room temperature followed by two washes in 25 mM MES. Immediately before use, beads were washed twice in sterile PBS and resuspended at ∼7.5 × 10¹⁰ particles/ml. After conjugation, beads were stained with rabbit polyclonal anti-chicken egg albumin (Accurate Chemical and Scientific, Westbury, NY), and then with a FITC-conjugated anti-rabbit Ig (Sigma-Aldrich) to assess the fraction of beads conjugated to OVA. More than 40% of beads were positive for OVA as assessed by flow cytometry.

Recovery of lung and airway leukocytes

Mice were anesthetized with 75 mg/kg ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and 1 mg/kg medetomidine (Orion, Espoo, Finland), and were humanely sacrificed. The trachea was cannulated and cells were recovered by flushing the lungs with four aliquots of 0.8 ml of sterile-filtered 2% FCS (HyClone Laboratories, Logan, UT) in PBS. RBCs were lysed using lysis buffer (0.15 M NH₄Cl, 10 mM Tris (pH 7.5)), and nucleated cells were counted using a hemacytometer. Parenchymal lung cells from the left lobe were obtained after lavage by mincing the tissue in 23 μg/ml Liberase (Boehringer Mannheim, Indianapolis, IN) with 30 μg/ml DNase I (Sigma-Aldrich), incubating at 37° for 30 min and pressing through a 70-μm filter (BD Biosciences, Franklin Lakes, NY).

Histology and immunohistochemistry

In all cases, BAL was performed before harvesting lung tissue. After tying off the left mainstem bronchus, the left lung was removed for enzymatic digestion. The right lung was inflated with 50% OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA) in sterile PBS and frozen on dry ice. Seven-micrometer sections were cut, fixed in acetone for 10 min, and stored at −20°C until staining. Where indicated, sections were stained with a biotin-conjugated monoclonal anti-CD11c mAb (BD PharMingen, San Diego, CA) and then with streptavidin-FITC (BD PharMingen). Frozen sections of peribronchial LNs were stained with FITC-conjugated monoclonal anti-CD4 (BD PharMingen) and biotin-conjugated monoclonal anti-B220 (BD PharMingen) and then with Alexa-350-conjugated neutravidin (Molecular Probes, Eugene, OR). Sections were examined using a fluorescence microscope (BX-60; Olympus, Melville, NY). T cells that were 5-(and 6)-carboxytetramethylrhodamine (TAMRA)-positive and bead⁺ APCs were counted as colocalizing if, at ×400 magnification in a 7-μm section, they showed cell-cell contact. Analysis of slides for determination of colocalization was performed in a blinded fashion with a random selection of sections. Statistical significance between groups was assessed using an unpaired Student’s t test with equal variance.

i.n. administration of beads

To deliver fluorescent beads i.n., mice were lightly anesthetized with either Metofane (methoxyflurane; Schering-Plough Animal Health, Union, NJ) or a ketamine/xylene mixture, given 40 μl of beads (∼3 × 10⁹ beads) in sterile PBS on the nostrils, and allowed to recover in a supine position.

Analysis of cell phenotype by flow cytometry

Flow cytometry was performed using a FACSCalibur and data were analyzed with CellQuest software (BD Biosciences, Mountain View, CA). All Abs, with the exception of the biotin-conjugated monoclonal anti-F4/80 (Caltag Laboratories, Burlingame, CA) and rat anti-NLDC-145 (Bachem, Torrence, CA), were purchased from BD PharMingen and include FITC-conjugated anti-CD11c, anti-CD4, and anti-CD11b; PE-conjugated anti-CD11b; and biotin-conjugated anti-CD11c and anti-B220. Biotin conjugates were detected using streptavidin-conjugated APCs (BD PharMingen). After lysis of red cells, nonspecific Ab staining via FcR was blocked with supernatant from hybridoma 2.4G2 (American Type Culture Collection, Manassas, VA). Stained cells were stored at 4°C in 1% paraformaldehyde/PBS until just before analysis.

Assessment of lymphocyte proliferation in vitro

CD4⁺ lymphocytes were purified from the spleens of RAG2^−/− DO11.10 TCR Tg mice (kindly provided by O. Kanagawa, Washington University, St. Louis, MO) using anti-mouse CD4 Dynabeads and the mouse CD4 DETACHaBEAD system (Dynal BioTech, Lake Success, NY), according to the manufacturer’s instructions. CD11c⁺ cells were purified from the Liberase-digested lungs of naive mice using a FITC-conjugated anti-CD11c mAb (BD PharMingen), anti-FITC magnetic beads, and a magnetic column (Miltenyi Biotec, Auburn, CA). Purity of CD11c⁺ cells after magnetic sorting, as assessed by flow cytometry, was >94%. Purified T cells (3 × 10⁵) were cultured in 200 μl of Iscove’s medium supplemented with 10% FCS (HyClone Laboratories), 0.1 mM nonessential amino acids, 2 mM sodium glutamate, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5.5 μM 2-ME (all from Life Technologies) together with 2 × 10⁴ CD11c⁺ cells and 20 μg of soluble OVA, 10 μl of fluorescent beads only, or 10 μl of fluorescent beads conjugated to OVA. Cells were cultured for 3 days at 37°C under 5% CO₂. One microcurie of [³H]thymidine (Amersham, Arlington Heights, IL) was then added to each well, and the culture was continued for an additional 24 h. Cells were harvested on a PHD cell harvester (Cambridge Technology, Watertown, MA), and incorporation was measured on a scintillation counter (LS3801; Beckman Coulter, Fullerton, CA).

Adoptive transfer of T cells

OVA-specific T cells were differentiated in vitro into Th1 or Th2 cells as previously described (29, 30, 31). On day 7 of culture, cells were centrifuged over Histopaque 1119 (Sigma-Aldrich) to remove dead cells, and they were washed twice in PBS. Before labeling, a sample of Th1 and Th2 cells were stained for expression of CD25 and CD69. Levels of these activation markers were the same between the two groups, suggesting that there was no gross difference in the activation status of the two cell populations. Cells were then fluorescently labeled by incubation for 15 min at 37°C with 6 μg/ml TAMRA-succinimidyl ester (SE) (Molecular Probes) in Ca²⁺- and Mg²⁺-free PBS. Fluorescently labeled cells were washed twice in sterile PBS and transferred to anesthetized, naive animals i.v. via retro-orbital injection. Naive CD4⁺ T cells were recovered from spleens and LNs of RAG2^−/− DO11.10 Tg mice, and sorted using anti-mouse CD4 Dynabeads and mouse CD4 DETACHaBEAD (Dynal BioTech). Then they were incubated with TAMRA-SE and injected as described above. Purity of naive CD4⁺ cells was >95% as determined by flow cytometry.

Fluorescent beads are taken up by cells of the lung

To investigate factors that control Ag uptake, trafficking, and presentation in the mouse lung, we have used fluorescent latex beads as a model inhaled Ag. The beads are well suited for this purpose because they are readily taken up by phagocytic cells, visualized by fluorescence microscopy, and quantitated by flow cytometry. Furthermore, they can also be modified by covalent coupling to immunogens and followed in parallel to immunogen-specific T cell responses. To test their behavior as particulate Ags, we administered 3 × 10⁹ 0.4-μm fluorescent latex beads in 40 μl of sterile PBS to lightly anesthetized C57BL/6 mice. At various times later, the mice were sacrificed, their lungs were harvested, and frozen sections of lung were analyzed by fluorescence microscopy to determine the locations of the beads.

Beads were detected in lung cells as early as 2 h after i.n. instillation and remained present until at least 46 h later. This observation is congruent with other studies demonstrating that lung DCs harvested after Ag challenge retain the ability to present Ag in vitro up to 7 days after initial administration of the Ag (11). The majority of beads detected in this fashion resided within cells of the lung, and not free in the airways, based upon their localization adjacent to 4′,6′-diamidino-2-phenylindole-positive nuclei (Fig. 1⇓, A and B). Beads were also found in large quantities in cells recovered by BAL (Fig. 1⇓, C and D). Fluorescence microscopy in conjunction with Wright-Geimsa staining demonstrates that beads could be found in >50% of BAL cells and that the majority of these cells were of the monocyte/myeloid phenotype.

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FIGURE 1.

Fluorescent beads are taken up by cells in the lung. Mice were administered fluorescent beads i.n., and at various times later their lungs were frozen and sectioned. A, Typical lung section showing beads in yellow-red and lung nuclei counterstained in blue using 4′,6′-diamidino-2-phenylindole. Magnification is ×200 (A) and ×400 (B). C and D, Cells recovered from the BAL 12 h after administration of beads were spun onto a slide, stained with Wright-Giemsa, and observed at a magnification of ×400 (C) or ×1000 (D). E, Lung sections from 12 h stained with biotin-conjugated anti-CD11c and then streptavidin-FITC. Beads are yellow-red (F) and colocalize with CD11c >30% of the time.

Bead⁺ lung cells are CD11c⁺ and bead⁺ BAL cells are CD11c⁺F4/80⁺CD11b⁻

To identify phenotypic characteristics of bead⁺ lung cells, we performed Ab staining of frozen lung sections followed by dual fluorescence microscopy. Using an anti-CD11c Ab, we determined that >30% of the cells in the lung that had taken up fluorescent beads were also CD11c⁺ (Fig. 1⇑, E and F). Analysis by confocal microscopy confirmed that beads were present within the CD11c⁺ cells (data not shown). To permit broader characterization of bead⁺ cells, we performed flow cytometry on cells positive for bead uptake. Bead⁺ cells are easily differentiated from those that have not taken up beads (Fig. 2⇓). By gating on bead⁺ cells, we were able to then test for expression of CD11c and CD11b, F4/80, or NLDC-145 on these same cells (Fig. 2⇓ and Tables I⇓ and II⇓). The markers CD11b, F4/80, and NLDC-145 were chosen because of their suggested differential expression among the myeloid and lymphoid DC subsets (32, 33, 34).

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FIGURE 2.

Bead-positive cells are characterized using flow cytometry. A, Bead⁺ cells from the BAL (gated), were assayed for expression of CD11c and CD11b (B, upper panel). A, lower panel, BAL sample from a mouse receiving i.n. PBS only. B, lower panel, Bead⁺ cells stained with a control Ab.

At 6 and 12 h after instillation of beads, most bead⁺ cells from the BAL were also positive for the DC marker CD11c (>80%). In turn, a great majority of these CD11c⁺ cells were negative for the myeloid-specific marker CD11b. For example, at 6 h compare 85.9% CD11c^highCD11b⁻ vs 2.7% CD11c^highCD11b⁺ (Table I⇑). By 24 and 48 h, a greater portion of bead⁺ BAL cells were low or negative for expression of CD11c compared with the populations at 6 or 12 h, although they remained negative for CD11b expression. This CD11c^low/−CD11b⁻ population of cells also showed lower side light scatter and forward light scatter values than the bead-positive population at 6 h. It remains unclear whether this increase in the CD11c^low/−CD11b⁻ subset at 24 and 48 h represents a newly emigrated population of cells entering into the airway lumen or merely indicates the down-regulation of surface markers on cells, which phagocytosed beads at some earlier time. To continue characterization, staining with F4/80 revealed that >85% of bead⁺ BAL cells were also F4/80⁺ at all times examined (Table II⇑). Furthermore, of all bead⁺ cells at 6 and 12 h, ∼65% of these were F4/80⁺CD11c^high.

In the lung tissue itself, regardless of the time after challenge, ∼25% of bead⁺ cells are CD11c⁺CD11b⁻, with a majority (50–60%) being CD11c^low/−CD11b⁻. As in the BAL, <10% of bead⁺ lung leukocytes are either CD11c⁻CD11b⁺ or CD11c⁺CD11b⁺. Flow cytometry data regarding CD11c positivity correlate closely with those obtained using tissue staining and then dual fluorescence microscopy (∼30%). For bead⁺ cells recovered from the lung, nearly equal numbers of CD11c⁺F4/80⁺, CD11c^low/−F4/80⁺, and CD11c^low/−F4/80⁻ populations were observed (Table II⇑). Lastly, at 6 and 12 h, ∼20% of bead⁺ cells from the BAL or lung were also NLDC-145⁺.

OVA covalently conjugated to fluorescent latex beads can stimulate OVA-specific CD4⁺ T cells in vitro

To enhance the utility of fluorescent beads as a model Ag, we tested whether beads would be recognized by Ag-specific CD4⁺ Tg T cells if covalently conjugated to the Ag recognized by the transgenic TCR. For this purpose, we tested whether CD11c⁺ lung cells given OVA-conjugated fluorescent beads could stimulate proliferation of OVA-specific DO11.10 Tg T cells. CD11c⁺ lung cells were recovered from naive, wild-type animals; incubated with OVA-conjugated fluorescent beads, beads alone, or unconjugated OVA (soluble OVA); and then cultured for 96 h with CD4⁺ DO11.10 Tg T cells. When CD4⁺ DO11.10 T cells were stimulated with OVA-conjugated beads using CD11c⁺ cells from the lung as APCs, robust proliferation was observed at 96 h (Fig. 3⇓). Beads alone stimulated no proliferation over background.

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FIGURE 3.

T cells proliferate in vitro in response to Ag delivered by OVA-conjugated beads. CD11c⁺ lung cells were harvested using FITC-conjugated Abs and then sorted with anti-FITC magnetic beads. A total of 2.5 × 10⁴ CD11c⁺ cells per well were incubated for 2 h with soluble ova (OS), OVA-conjugated beads (OB), or beads alone (B) before addition of 3 × 10⁵ CD4⁺ DO11.10 Tg T cells. Cultures were grown for 96 h with [³H]thymidine administered during the final 24 h of culture. Results are the average of triplicate wells plus SD. A second experiment using FACS-sorted CD11c⁺ lung APCs gave similar results.

i.n. administered fluorescent beads traffic to the T cell zone of peribronchial LNs

Other studies have demonstrated that in vitro differentiated DCs traffic from the skin to the draining LN within 24 h (6). To determine whether fluorescent beads administered i.n. exhibit a similar temporal progression, we analyzed the accumulation of beads in peribronchial LNs harvested at varying times after challenge. Small numbers of beads (2–3 bead⁺ cells/section) were detected in peribronchial LN sections as early as 6 h after i.n. administration. These bead⁺ clusters initially localized exclusively in the subcapsular sinus (Fig. 4⇓A). In contrast, 12 h after challenge, peribronchial LNs showed large numbers of bead⁺ clusters (>10 per section) in nearly each section examined (20 sections from eight mice). Furthermore, by this time the majority of fluorescent bead⁺ cells were localized within the T zone of the LN (Fig. 4⇓B). By 24 and 48 h, there was a further increase in the number of bead⁺ cells per LN section, showing similar exclusive localization to the T zone (Fig. 4⇓C).

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FIGURE 4.

Beads traffic to the T zone of the LN by 12 h, but colocalize with Th1 cells in the lung earlier. A, Six hours after administration, beads can be seen in the subcapsular sinus of the peribronchial LN (∗, lower right), whereas by 12 h they reach the T zone (B), where their accumulation peaks at 24 h (C). A–C, Fluorescent beads are yellow-red, green represents staining with FITC-conjugated anti-CD4, and blue is B220-biotin followed by Alexa-350-conjugated neutravidin. In adoptive transfer experiments, TAMRA⁺ cells (red) can be found in the draining LN before challenge in mice receiving naive cells (D) and to a lesser extent Th1 cells (not shown), but beads (blue) do not arrive at the T zone until 12 h post-challenge (E). E, B220⁺ cells are stained with B220-biotin and then streptavidin-FITC. F, Higher magnification highlights the Th1/APC interaction seen in E. In contrast, TAMRA⁺ Th1 cells interact with bead⁺ APCs in the lung parenchyma as early as 6 h after Ag challenge (G and H; higher magnification).

Th1 cells preferentially accumulate in the lung and BAL of mice after i.n. challenge with OVA-conjugated beads

Other groups have shown that naive T cells first encounter their Ag in the regional LN. To test whether previously activated cells follow the same paradigm, we combined analysis of OVA-conjugated fluorescent beads and migration of OVA-specific Tg T cells. In vitro differentiated DO11.10 Th1, Th2, or naive CD4⁺ cells were labeled with the fluorescent dye TAMRA-SE and transferred i.v. to naive BALB/c mice. Three days later, these mice were challenged i.n. with one administration of OVA-conjugated beads and were examined at various times.

The total number of cells recovered by BAL in all three groups of mice increased less than 2-fold over the entire period of evaluation. However, in contrast, the number of TAMRA⁺ cells recovered from the BAL of mice given labeled Th1 increased more than 10-fold, from a value of 1.9 ± 0.37 × 10³ at baseline to over 26.7 ± 0.98 × 10³ at 48 h (Fig. 5⇓A). Significantly, neither mice given Th2 cells nor mice given naive CD4⁺ T cells exhibited a substantial increase in the number of TAMRA⁺ cells in the BAL cells at any time after a single airway challenge. These results are consistent with prior findings from our laboratory (30). Importantly, the relative proportion of Th1 cells did not increase in either the nondraining inguinal LN or the spleen between baseline and 48 h after airway challenge (data not shown). This indicates that the accumulation of Th1 cells in the airway was specifically localized to the region of Ag challenge.

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FIGURE 5.

Th1 cells preferentially accumulate in response to challenge with OVA-conjugated beads. A total of 5 × 10⁶ in vitro differentiated DO11.10 Th1 or Th2 cells or 6 × 10⁶ DO11.10 naive cells were transferred to naive BALB/c recipients. Total number of TAMRA-SE⁺ cells recruited to the BAL (A) and the percent of TAMRA-SE⁺ cells present in enzymatically digested lung samples (B) at various times after challenge with OVA-conjugated beads. C, Number of TAMRA-SE⁺ BAL cells before challenge, highlighting the difference at baseline between mice given Th1, Th2, or naive cells. All numbers are the average ± SE.

Percentages of TAMRA⁺ cells in the lung parenchyma were also analyzed. Th1 cells at baseline were represented at a higher frequency in lung tissue than either Th2 cells or naive, undifferentiated cells (Fig. 5⇑B). There were modest increases in the percentage of lung Th1 cells over the 48 h after OVA challenge and smaller transient changes in Th2 cells. The failure to detect a substantial increase in naive or Th2 cells in the BAL or lung was not due to a gross loss or death of these populations, because both groups could be found at other anatomical locations, including the peribronchial LN, nondraining inguinal LN, and spleen (Fig. 4⇑D and data not shown).

T cells are positioned to encounter Ags in the regional LNs early after Ag challenge

As shown in Fig. 4⇑D, naive TAMRA⁺ T cells are present in the T zone of the peribronchial LN before an airway challenge. This is also true, to a lesser extent, for mice given Th1 cells (data not shown). Conjugated fluorescent beads administered to naive, wild-type mice, as shown earlier, traffic to the T zone of the LN between 6 and 12 h. Transfer of Ag-specific Th1, Th2, or naive cells did not affect this time course, and bead⁺ cells do not reach the T zone 6 h after i.n. administration of beads. At 12 h, some bead⁺ cells are found in the T zone and can be seen localized adjacent to fluorescently labeled T cells (Fig. 4⇑E). Higher magnification verifies the proximity of this localization (Fig. 4⇑F). This type of APC/T cell interaction in the LN continues to occur at higher frequency 24 and 48 h after Ag challenge.

Adjacent localization of Th1 cells and APCs in the lung 6 h after Ag challenge

Because large amounts of the Ag remained in the lung over the first several days, we reasoned that APC/T cell interactions might be occurring in the peripheral lung tissue at time points before, or directly concurrent with, initial presentation of Ag to T cells in the draining LN. To test this, we investigated localization of TAMRA-labeled T cells and bead⁺ APCs in the lung 6 h after challenge with OVA-conjugated beads.

As demonstrated in Fig. 6⇓, at 6 h after i.n. Ag challenge, OVA-specific Th1 cells were found in much higher numbers per high power field (HPF) than either Th2 or naive cells (Fig. 6⇓A). In contrast, the numbers of bead⁺ APCs/HPF were relatively constant between groups (Fig. 6⇓B). Significantly, Th1 cells were found colocalizing with bead⁺ APCs 4-fold more frequently than either Th2 or naive cells (Fig. 6⇓C). T cell-APC interactions characterized as colocalization are depicted microscopically in Fig. 4⇑, G and H. The frequency of these interactions appeared to be dependent upon the subset of helper cells transferred, because the average number of interactions per T cell was 0.072 interactions/Th1 cell (7.2%), but only 0.018 interactions/Th2 cell (1.8%). T cells and bead⁺ APCs were also frequently found in close proximity, but without apparent cell-cell contact (data not shown). The latter were consistent with cell-cell interactions in the process of development and were found to occur around vessels or in collections of cells at branching points of alveolar walls. In total, these studies substantiate the hypothesis that in vitro differentiated Th1 cells are capable of localizing adjacent to APCs in the peripheral lung tissue on a time course that precedes detection of the same Ag or APC/T cell interaction in the draining LN and that the frequency of this early colocalization is dependent upon the CD4⁺ T cell subtype, being substantially more common with Th1 cells than Th2 cells.

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FIGURE 6.

Th1 cells interact more frequently with bead⁺ APCs 6 h after airway Ag challenge than do either Th2 or naive cells. A total of 7 × 10⁶ in vitro differentiated OVA-specific DO11.10 Th1 cells, Th2 cells, or naive CD4⁺ T cells were transferred i.v. to naive BALB/c recipients. One day later, mice were challenged with OVA-conjugated fluorescent beads. At 6 h, lungs were harvested and analyzed for localization of labeled T cells and bead⁺ APCs. A, TAMRA⁺ DO11.10 Th1 cells are found in higher numbers/HPF than either Th2 cells or naive cells 6 h after Ag challenge (p < 0.001 and p < 0.0001, respectively), whereas at the same time the number of APCs/HPF is equivalent (B). C, Number of interactions (scored as colocalization of T cells and bead⁺ APCs) between Th1 cells and bead⁺ APCs was significantly higher than between either Th2 or naive cells and APCs (p < 0.004 vs either Th2 or naive cells).