Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
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Originally published online as doi:10.1189/jlb.0505244 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1086-1096.)
© 2005 by Society for Leukocyte Biology

Distribution and leukocyte contacts of {gamma}{delta} T cells in the lung

J. M. Wands*, Christina L. Roark*, M. Kemal Aydintug*, Niyun Jin*, Youn-Soo Hahn{dagger}, Laura Cook*, Xiang Yin*, Joseph Dal Porto*, Michael Lahn*, Dallas M. Hyde{ddagger}, Erwin W. Gelfand§, Robert J. Mason, Rebecca L. O’Brien* and Willi K. Born*,1

* Departments of Immunology,
§ Pediatrics, Division of Cell Biology, and
Medicine, National Jewish Medical and Research Center, Denver, Colorado;
{dagger} Department of Pediatrics, Chungbuk National University and College of Medicine, Cheongju, Korea; and
{ddagger} California National Primate Research Center, University of California, Davis

1Correspondence: Department of Immunology at National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: bornw{at}njc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pulmonary {gamma}{delta} T cells protect the lung and its functions, but little is known about their distribution in this organ and their relationship to other pulmonary cells. We now show that {gamma}{delta} and {alpha}ß T cells are distributed differently in the normal mouse lung. The {gamma}{delta} T cells have a bias for nonalveolar locations, with the exception of the airway mucosa. Subsets of {gamma}{delta} T cells exhibit further variation in their tissue localization. {gamma}{delta} and {alpha}ß T cells frequently contact other leukocytes, but they favor different cell-types. The {gamma}{delta} T cells show an intrinsic preference for F4/80+ and major histocompatibility complex class II+ leukocytes. Leukocytes expressing these markers include macrophages and dendritic cells, known to function as sentinels of airways and lung tissues. The continuous interaction of {gamma}{delta} T cells with these sentinels likely is related to their protective role.

Key Words: T cell receptor • {gamma}{delta} T cell subsets • mouse


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Substantial populations of {gamma}{delta} T cells have been found in the epidermis of rodents [1 ], in the intestinal epithelia [2 , 3 ], in the mammary gland and uterine epithelium, and in the placenta [4 5 6 ]. Because of their prevalence in epithelia, it was suggested that {gamma}{delta} T cells survey epithelial cells and form a first line of defense against infections [7 ]. Newer studies have indicated that {gamma}{delta} T cells also play roles in inflammation [8 ], epithelial growth, and repair [9 , 10 ], and in the prevention of epithelial malignancy [11 ] and that their responses may be triggered by autologous stress-induced determinants [12 ], including the human MIC proteins [13 ]. Although mechanisms in vivo remain to be resolved, experiments in vitro suggest that {gamma}{delta} T cells not only receive signals from but also alter development and functional capabilities of myeloid cells that include dendritic cells (DC) and macrophages [14 15 16 17 18 ].

The lung contains {gamma}{delta} T cells as well [19 20 21 ]. This population is comparatively small, but it seems to protect lung function [22 23 24 ]. This may be of importance in the remarkable resilience of the lung against environmental stimuli. Expanded populations associated with infections of the lung [25 , 26 ] might also contribute to host resistance and the resolution of pulmonary inflammation at later stages [25 , 27 , 28 ]. Likewise, noninfectious pulmonary inflammation associated with injury or allergic responses is influenced by {gamma}{delta} T cells [22 , 29 , 30 31 32 33 34 ] and chronic obstructive pulmonary disease has been associated with a diminished {gamma}{delta} T cell response [35 ]. However, information about pulmonary {gamma}{delta} T cells in situ is extremely limited. Some studies were performed in association with human diseases (emphysema, cancer) and in cigarette smokers [36 37 38 39 ] but not in the normal lung [40 ]. Taking advantage of a modified histological technique, we now report a systematic investigation of resident {gamma}{delta} T cells in the normal lung of healthy mice, characterizing their tissue distribution, contacts with other leukocytes, and comparisons with {alpha}ß T cells. Most resident pulmonary {gamma}{delta} T cells were found in nonalveolar locations with exception of the mucosa where they appear to be extremely rare. Frequent contacts suggest that the {gamma}{delta} T cells monitor the far more numerous macrophages and DC of the lung.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
C57BL/6, B6.T cell receptor (TCR)-ß–/–, and B6.TCR-{delta}–/– mice, ages 8–12 weeks, were obtained from Jackson Laboratories (Bar Harbor, ME). The mice used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center (Denver, CO).

Preparation of lung tissue
Mice were killed by lethal injection of Nembutal [Pentobarbital, 300 µl of a 1:4 mix (80–100 mg/kg) in saline, intraperitoneally]. To inflate the lungs, mice were tracheostomized, the trachea was cannulated (22-gauge blunt-end needle), and the diaphragm was punctured. Undiluted optical cutting temperature (OCT) compound freezing medium was then injected into the lung (1–2 ml) until the lung filled the pleural cavity. The trachea was then tied and clamped, and the lungs were dissected out and placed in ice-cold phosphate-buffered saline (PBS). Subsequently, the lung was separated into eight pieces as follows; the right lung was cut into two pieces, and the left lung into its four respective lobes. These lung pieces were then placed into plastic freezing molds and covered with OCT. Filled molds were snap-frozen (on a slurry of crushed dry ice and 100% ethanol), and frozen blocks were stored at –70°C. Sections were cut from blocks (equilibrated to –20°C) at a thickness of 5–10 µm and mounted on SuperFrost Plus microscope slides (Fisher Scientific, Loughborough, UK, Cat. #12-550-15). The slides were air-dried for at least 1 h before staining or storing at –20°C (for up to 2 weeks).

Lung tissue preparation and staining procedure
Sections were allowed to dry at room temperature prior to staining. Directly conjugated anti-TCR antibodies did not stain well enough to distinguish positive staining from background. Signal amplification was achieved by using un-conjugated primary antibodies and species-specific biotinylated or fluorochrome-conjugated secondary anti-immunoglobulin (Ig) antibodies. The staining protocol included the following steps: dehydration (in acetone at –20°C for 20’); hydration (in phosphate-buffered saline, PBS, for 5’); block (using 5% normal mouse serum, NMS/Avidin* for 20’); wash (in 0.05% Tween 20 in PBS, 2x10 min each); biotin (using 40 µg/ml biotin for 10’); 1° antibody [anti-TCR monoclonal antibodies (mAb) for 40’]; wash (in 0.05% Tween 20 in PBS, 2x10 min each); 2° antibodies (anti-hamster-biotin/anti-rat-CY5 for 45’); wash (in 0.05% Tween 20 in PBS, 2x10 min each); streptavidin-CY3 for 45’; wash (in 0.05% Tween 20 in PBS, 2x10 min each). Stained sections were mounted with GelMount (Biomeda Laboratories, Malaysia, Code #BM-M01), and coverslips sealed with clear nail polish. Slides were viewed using a Leica (Knowlhill, UK) DMRXMA upright fluorescent microscope, and digital images were generated on an Apple MacIntosh computer connected with the microscope, using the SlideBook imaging program (3I Inc., Atlanta, GA).

Antibodies; specificity of staining
The following antibodies were used as primary reagents for the detection of TCRs: mAb KT3 (rat anti-mouse CD3{epsilon}) [41 ]; mAb GL3 (hamster anti-mouse TCR-{delta}) [42 ]; mAb H57.597.2 (hamster anti-mouse TCR-ß) [43 ]; mAb UC3 (hamster anti-mouse V{gamma}4, GV3S1) [44 ]; mAb 2.11 (hamster anti-mouse V{gamma}1, GV5S1) [45 ]; mAb 536 (anti-V{gamma}5) [46 ] and mAb 17D1 (anti-V{gamma}5/V{delta}1 and conditionally anti-V{gamma}6/V{delta}1) [47 , 48 ]. Note: Throughout the text, we have used the simple numbering system for murine V{gamma} genes, V{gamma}1-7, introduced by S. Tonegawa and collaborators [49 ]. For detection of the primary antibodies, we used the following secondary reagents: anti-hamster-biotin at 1:400 dilution (Jackson ImmunoResearch Laboratories, West Grove, PA, biotin-SP-conjugated AffiniPure goat anti-armenian hamster IgG (H+L), cat. # 127-065-160) and anti-rat-Cy5 at 1:600 dilution (Jackson ImmunoResearch Laboratories, Cy5-conjugated AffiniPure F(ab')2 fragment donkey anti-rat IgG (H+L), cat. # 712-176-153). To use mAb 17D1 for the detection of V{gamma}6/V{delta}1+ cells, we first treated the tissue sections with anti-TCR-{delta} mAb GL3, as described previously for flow cytometry of freshly isolated cells [48 ]. For comparison, we also stained lung tissue with the V{gamma}5-specific mAb 536, which cannot detect V{gamma}6/V{delta}1+ cells [48 ]. Additional antibodies included: rat anti-mouse CD45R/B220 (clone RA3-6B2, BD PharMingen, San Diego, CA) [50 ]; rat anti-mouse DC antigen DEC-205 (ATCC #HB-290) [51 ], rat anti-mouse macrophage antigen F4/80 (clone BM8, eBioscience, San Diego, CA, cat. #4-4801) [52 , 53 ]; rat anti-mouse I-A/I-E (clone M5/114.15.2, BD PharMingen) [54 ]. We examined frozen, acetone-dehydrated sections of lung tissue, stained with TCR-specific mAb followed by secondary fluorescent dye- or biotin-labeled reagents. For the detection of biotin, we used streptavidin directly conjugated to Cy3 (Jackson ImmunoResearch Laboratories, Cy3-conjugated streptavidin, code # 016-160-084). We used lung tissue genetically deficient in {gamma}{delta} or {alpha}ß T cells (derived from B6.TCR-{delta}–/– or B6.TCR-ß–/– mice, respectively) to establish staining conditions using TCR-{delta}- and TCR-ß-specific antibodies. We stained simultaneously with mAb specific for CD3{epsilon}, a protein present in the CD3 complexes associated with {alpha}ß and {gamma}{delta} TCR heterodimers. This dual staining protocol was adopted to show in each case the frequency of all CD3{epsilon}-positive cells ({alpha}ß and {gamma}{delta} T cells, taken together) and as a control to demonstrate coincidence of CD3{epsilon}- and TCR-ß or -{delta} expression. We quantitatively confirmed the specificity for these antibodies, e.g., the TCR-{delta}-specific mAb GL3 stained 0% of CD3{epsilon}+ cells in TCR-{delta}–/– mice (0/93) but 100% in TCR-ß–/– mice (87/87). In C57BL/6 mice, 100% of TCR-{delta}+ cells were also CD3{epsilon}+(88/88), and 100% of V{gamma}4+ cells were also CD3{epsilon}+ in C57BL/6– (43/43) and in TCR-ß–/– mice (19/19). Based on coincidence of TCR-ß- and CD3{epsilon} staining, the TCR-ß-specific mAb H57-597 was also specific (100% of TCR-ß+ cells in C57BL/6 mice were CD3{epsilon}+ (171/171). Traces of nonspecific staining were found with anti-TCR-ß mAb H57.597.2 and with the V{gamma}4-specific mAb UC3.

Localizing pulmonary T cells
The following terms were established to describe the distribution of {gamma}{delta} T cells within the lung: lamina propria smooth muscle (LP/SM): a cell location defined by the basal lamina, the loose connective tissue of the lamina propria, and the smooth muscle layer of the muscularis. These layers can be as few as three cells thick to greater than 10 but are always bounded by the smooth muscle and the basal lamina surrounding the bronchioles. These T cells may appear to be in contact with or within a few cell diameters of the epithelial cell layer of the bronchiole. Connective tissue bronchiole (CT/BR): a cell location defined by the loose connective tissue of the submucosa and extending to the adventitia surrounding the bronchioles. These T cells are seen around the bronchioles outside the smooth muscle layer. Connective tissue blood vessels (CT/BV): a cell location extending from the endothelium of the veins and arteries to the adventitia surrounding the vessels. It includes the supporting connective tissue and smooth muscle. These T cells are not only seen in the connective tissues but sometimes appear to be in contact with the endothelium. Alveolar (ALV): a cell location in the respiratory area of the lung. It is bordered by the connective tissue and adventitia of the airways and vessels and extends to, but does not include, the visceral pleura. This locale includes the alveoli, capillaries, and associated tissues not included in the other categories. These T cells may appear to be in contact with the alveolar septum and the capillaries of the respiratory area of the lung. Visceral pleura (VPL): a cell location that is in contact with the mesothelium or the connective tissue of the visceral pleura.

We have also examined the epithelium adjacent to the lumen of the airways (mucosa). Here, {gamma}{delta} T cells and {alpha}ß T cells were essentially absent. Therefore, the mucosa was not included in the quantitative comparison of the lung tissues.

Confocal microscopy and image analysis
Stained sections were mounted and slides were viewed using a Leica DMRXMA upright fluorescent confocal microscope. Digital images were captured using a Cooke (Romulus, MI) SensiCam, and processed on an Apple MacIntosh Computer using the SlideBook imaging program (3I Inc.).

Statistical analysis
The continuity-adjusted {chi}2 test was used for comparison of cell distribution percentages and cell-to-cell contact frequencies. Distribution percentages and cell-to-cell contact frequencies were obtained by summing the cell numbers from a minimum of three mice/sample. The two-sample t-test was used to compare distributions of partner cells in the vicinity of {gamma}{delta} and {alpha}ß T cells. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The population of resident pulmonary {gamma}{delta} T cells is small but widely distributed
By comparison with epidermis, intestines, or spleen, the population of resident pulmonary {gamma}{delta} T cells is very small. Based on cytofluorometric analyses of TCR-{delta}+ cells retrievable from enzymatically digested and mechanically dispersed lung tissue [23 , 24 ], we estimate a population size of {gamma}{delta} T cells in the lung of normal adult C57BL/6 mice of 2–5 x 104 cells, further divisible into subsets expressing different TCR-V{gamma} genes (V{gamma}4+, ~45%; V{gamma}1+, ~15%; others are predominantly V{gamma}6+, V{gamma}7+ cells are rare, and V{gamma}5+ cells are almost absent). The entire {gamma}{delta} T cell population represents 5–10% of all T lymphocytes in the normal lung.

To investigate the distribution of pulmonary {gamma}{delta} T cells, we examined antibody-stained lung tissue of normal untreated adult C57BL/6 mice. Specificity of the staining with anti-TCR mAb was confirmed by comparing lung tissue of B6.TCR-ß–/– and B6.TCR-{delta}–/– mice (Materials and Methods). {gamma}{delta} T cells were detected at relative frequencies similar to those predicted by the cytofluorometry. Figure 1 shows TCR-{delta}+ and CD3{epsilon}+ lymphocytes in the C57BL/6 lung. Cells co-expressing TCR-{delta} and CD3{epsilon} ({gamma}{delta} T cells) were found in and around the airway walls but not in the mucosa (Fig. 1A and 1E) , in the perimeter of the bronchioles (Fig. 1A and 1E) , in the perimeter of the blood vessels (Fig. 1A and 1D) , associated with the alveolar epithelium (Fig. 1A 1B 1C) , and at or in the vicinity of the visceral pleura (Fig. 1F) . CD3{epsilon}+ cells that do not express TCR-{delta} ({alpha}ß T cells) were far more frequent than {gamma}{delta} T cells in the pulmonary parenchyma while {gamma}{delta} T cells were present in locales where {alpha}ß T cells appeared to be relatively infrequent (e.g., in and around airway walls). To confirm this apparent difference in cellular distributions, we next examined separately each of the locales listed above and determined relative frequencies of {gamma}{delta} T cells among total T cells (Fig. 2 ). As expected, we found large variations between locales, with the highest relative frequency of {gamma}{delta} T cells at the lamina propria/smooth muscle of the airways (LP/SM, 49%) and the lowest associated with the alveolar epithelium (ALV, 8%). Because the locales examined differ in quantity of tissue or surface area in the tissue sections, with the parenchyma (alveolar epithelium) by far exceeding all others, we also determined allocations of the T cells to these sites relative to their total number in the entire sections (Fig. 3 , larger pie charts). We found that the vast majority of {alpha}ß T cells localizes in the parenchyma, with much smaller fractions present in the other four regions. In marked contrast, {gamma}{delta} T cells are broadly distributed and total cells in nonparenchymal locations well exceed those within the parenchyma. These data show that {gamma}{delta} T cells are present in all regions of the lung, with exception of the airway mucosa. Relative frequencies are highest at select locations near the airways, blood vessels and visceral pleura.



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  		Figure 1. Resident {gamma}{delta} and {alpha}ß T cells in the normal lung. Sections of frozen lung tissue (C57BL/6, adult, untreated) were stained with antibodies. Blue, Anti-CD3{epsilon}; red, anti-TCR-{delta}; pink, CD3{epsilon}/TCR-{delta} coincidence; green/yellow, tissue autofluorescence. (A) Overview, terminal bronchiole (I), blood vessels (II), and parenchyma (III). Most of the {alpha}ß T cells (blue) are found in alveolar locations, whereas the less frequent {gamma}{delta} T cells (pink) are found adjacent to the airway wall to blood vessels and in alveolar locations. (B–F) Examples of tissue localizations; (B, C) alveolar (ALV); (D) blood vessel wall (CT/BV); (E) peri-bronchial connective tissue (CT/BR); (F) visceral pleura (VPL).

 


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  		Figure 2. Distribution density of {gamma}{delta} T cells relative to total T cells in five major areas of the lung. Frozen, antibody-stained sections from untreated adult C57BL/6 lung (as shown in Fig. 1 ) were examined for T cell distributions in five separate locations including alveolar (ALV); peri-bronchial, except locations directly at the lamina propria (CT/BR) and locations directly at the lamina propria/smooth muscle (LP/SM); around blood vessels, including locations within the blood vessel wall (CT/BV); and at the visceral pleura (VPL). In each case, multiple sections of the lungs of at least three mice were examined for CD3{epsilon}+/TCR-{delta}+ (solid columns, first number) and CD3{epsilon}+/TCR-{delta}– cells (open columns, second number). Numbers of cells counted: CT/BR (29/67), CT/BV (43/62), LP/SM (24/24), ALV (12/138), VPL (67/103). Cell distributions in all four nonalveolar areas are significantly different from that in ALV (P<0.0005). The data are expressed in percent of total CD3{epsilon}+ cells, as mean of individual mice ± SEM.

 


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  		Figure 3. Partitioning of {gamma}{delta} and {alpha}ß T cell populations within the lung tissues. Frozen sections from untreated, adult C57BL/6 lung (as shown in Fig. 2 ) were stained with antibodies specific for CD3{epsilon}, TCR-{delta}, TCR-ß, V{gamma}1, V{gamma}4, and V{gamma}6. The lung was divided into five locations, including alveolar (ALV); peri-bronchial, except locations directly at the lamina propria (CT/BR) and locations directly at the lamina propria/smooth muscle (LP/SM), around blood vessels, including locations within the blood vessel wall (CT/BV); and at the visceral pleura (VPL). The airway mucosa (*) was also analyzed, but T cells were not found. In each case, multiple sections of the lungs of at least three mice were examined. Total numbers of identified and allocated cells were 699 (total {gamma}{delta} T cells); 4656 (total {alpha}ß T cells); 58 (V{gamma}1+ {gamma}{delta} T cells); 89 (V{gamma}4+ {gamma}{delta} T cells). Significant differences in cell distributions determined include those between {gamma}{delta} and {alpha}ß T cells in the parenchymal tissue (ALV, P<0.0005), between each of the included subsets and total {gamma}{delta} or {alpha}ß T cells (P<0.0005 for all four comparisons), and between V{gamma}4+ and Vg1+ or total {gamma}{delta} T cells around blood vessels (CT/BV, P<0.01 and P<0.0001, respectively), among others. Percent values represent the mean of individual mice.

 
Subsets of resident {gamma}{delta} T cells are allocated to different pulmonary locales
In mice and other vertebrates, subsets of {gamma}{delta} T cells develop sequentially in ontogeny and colonize different tissues [4 , 55 ]. We determined histologically the allocations of two major TCR-V{gamma}-defined pulmonary subsets, as was done for total {gamma}{delta} T cells (Fig. 3 , smaller pie charts). It is interesting that the bulk of V{gamma}1+ and V{gamma}4+ populations was found in the parenchyma. This difference to the distribution of total {gamma}{delta} T cells implies the presence of other subpopulations, which are primarily allocated to nonparenchymal locations. A substantial fraction of pulmonary {gamma}{delta} T cells expresses V{gamma}6+ [21 ]. These cells can now be detected with an antibody (mAb 17D1) that binds the V{gamma}6V{delta}1 invariant TCR only in the presence of an anti-TCR-{delta} antibody, presumably due to a required change in TCR conformation [48 ]. The antibody distinctly labeled some cells in lung sections (Fig. 4 ), although at a reduced efficiency by comparison with cells in solution. These putative V{gamma}6+ cells were found mostly in nonparenchymal locations, indicating that V{gamma}6+ cells contribute to the broader tissue distribution of total {gamma}{delta} T cells in the lung. V{gamma}5+ cells (detected by mAb 536) were not found (not shown). As a further indication of tissue segregation among the subsets, we found that V{gamma}1+ and V{gamma}4+ cells differed in their distributions around the blood vessels, with 17% of V{gamma}1+ but only 3% of V{gamma}4+ cells allocated to this area.



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  		Figure 4. Pulmonary {gamma}{delta} T cells stained with mAb 17D1 in the presence of anti-TCR-{delta} mAb. In the absence of TCR-V{gamma}5+ cells, TCR-V{gamma}6+/V{delta}1+ {gamma}{delta} T cells can be detected with mAb 17D1, in the presence of anti-TCR-{delta} mAb. Frozen sections from untreated, adult C57BL/6 lung (as shown in Fig. 1 ) were stained with mAb GL3 (red, specific for TCR-{delta}) and subsequently, with mAb 17D1 (blue). Green/yellow, Tissue autofluorescence. Putative V{gamma}6+/V{delta}1+ cells (pink) peripheral to a blood vessel (upper) and an airway (lower, possibly a mucosal location) are shown. The lower set of panels also contains a {gamma}{delta} T cell, which is not stained with mAb 17D1 (orange). Because of limitations of the conditional staining procedure, we expect to underestimate numbers of V{gamma}6+ {gamma}{delta} T cells.

 
Many pulmonary T cells are in direct contact with other leukocytes
The low density of leukocytes in the lung provides a unique opportunity for the detection of individual cell–cell interactions (Fig. 5A ). We have examined interactions with pulmonary T cells in normal lung tissue stained for several additional leukocyte markers, including major histocompatibility complex class II I-A (expressed at high levels by certain myeloid cells, especially immature DC, and by B lymphocytes but not T cells), F4/80 and DEC-205 (expressed primarily by macrophages, other myeloid cells and DC), CD45R (B220, expressed by lymphoid cells including B cells, subsets of T and NK cells, and plasmacytoid DC) and CD3{epsilon} (expressed by all T lymphocytes).



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  		Figure 5. Direct cell contacts between {gamma}{delta} or {alpha}ß T cells and other leukocytes in the lung. Frozen sections from untreated, adult C57BL/6 lung (as shown in Fig. 1 ) were stained with anti-TCR-{delta} mAb (GL3) and one of the following additional antibodies: anti-I-A/E (M5/114), anti-F4/80 (BM8), anti-DEC-205, or anti-CD45R (B220). (A) Examples: {gamma}{delta} T cells (orange), other leukocytes (blue). (B) Using the same antibodies and in addition, anti-CD3{epsilon} and anti-TCR-ß mAb, contacts between {gamma}{delta} or {alpha}ß T cells and all other leukocytes were enumerated across the entire lung and expressed as percent of either type of T cell in contacts with individual leukocyte types. In each case, multiple tissue sections of at least three mice were examined. Contacts counted (first number: contacts with a given leukocyte type; second number: number of T cells examined). Contacts with {gamma}{delta} T cells: CD3+(9/166), DEC-205+(10/139), CD45R+(24/245), F4/80+(45/209), I-A+(187/422). Contacts with {alpha}ß T cells: CD3+(15/110), DEC-205+(14/275), CD45R+(75/452), F4/80+(32/379), I-A+(142/446). Significant differences were found with contacts between {gamma}{delta} T cells and CD3+ cells versus F4/80+ cells (P<0.0005) and versus I-A+ cells (P<0.0001) and with contacts between {alpha}ß T cells and CD3+ cells versus DEC-205+ cells (P<0.008) and versus I-A+ cells (P<0.0002). Comparing {gamma}{delta} and {alpha}ß T cells, contact frequencies of the two types of T cells with I-A+ cells, CD45R+ cells, and F4/80+ cells were significantly different (P<0.0002, P<0.02, and P<0.0001, respectively). Data are shown as averages of individual mice (mean±SEM).

 
Across the entire lung, contacts of T cells with other T cells were relatively infrequent, especially those involving {gamma}{delta} T cells (Fig. 5B) . Much larger fractions of T cells were found in direct contact with non-T cells, especially in the case of {gamma}{delta} T cells. This difference was maintained in the parenchyma (Fig. 6A and 6B ). However, because the distribution density of I-A+ non-T cells is higher that that of T cells (Fig. 6C) , it was not clear to what extent frequencies of cell contacts reflect intrinsic preferences vs opportunity.



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  		Figure 6. Contacts of parenchymal T cells with other leukocytes and distributions of the partner cells. Frozen sections from untreated, adult C57BL/6 lung (as shown in Fig. 1 ) were stained with anti-TCR-{delta} mAb (GL3) or anti-TCR-ß mAb (H57) and one of the following additional antibodies: anti-I-A/E (M5/114), anti-CD3{epsilon}, anti-CD45R (B220), anti-F4/80 (BM8), and anti-DEC-205. (A–C) T cell contacts with other leukocytes expressed as percent of total T cells. Contacts were enumerated as in Figure 5 . Total parenchymal contacts with {gamma}{delta} T cells: CD3+(2/52), DEC-205+(3/61), CD45R+(19/114), F4/80+(11/90), I-A+(67/170); total nonparenchymal contacts with {gamma}{delta} T cells for comparison: CD3+(7/114), DEC-205+(7/78), CD45R+(5/131), F4/80+(24/79), I-A+(79/174); total parenchymal contacts with {alpha}ß T cells: CD3+(15/110), DEC-205+(8/277), CD45R+(70/376), F4/80+(16/321), I-A+(108/376). Comparing {gamma}{delta} and {alpha}ß T cells in the parenchyma, contact frequencies of the two types of T cell with I-A+ cells and F4/80+ cells were significantly different (P<0.02 and P<0.03, respectively). (D) Relative frequencies of individual leukocyte types relative to total leukocyte frequency in the parenchyma (percent). Numbers of cells counted are 37 (CD3+), 17 (DEC-205+), 55 (CD45R+), 13 (F4/80+), and 105 (I-A+). (E) Distribution of partner cells in the vicinity of parenchymal {gamma}{delta} and {alpha}ß T cells (around an individual T cell in the center of the visual field). In each case, at multiple tissue sections, at least three mice were examined. Comparing the distributions of F4/80+ and I-A+ cells around the two types of T cells, no significant differences were found (P>0.74 and P>0.68, respectively). Data shown represent averages of individual mice (mean±SEM).

 
Pulmonary {gamma}{delta} T cells preferentially interact with myeloid cells
Nearly half of the total pulmonary {gamma}{delta} T cells, a fraction larger than that of {alpha}ß T cells, were found in contact with I-A+ non-T cells (Fig. 5B) . As I-A+ leukocytes are a heterogeneous population containing lymphoid and myeloid cells, it seemed possible that the two T cell types have different intrinsic preferences for cellular partners. Alternatively, differences in the tissue distribution of the T cells might influence their contact opportunities. To address these possibilities, we first examined contacts of the T cells with lymphoid and myeloid cells identified by other markers, CD45R (B220) and F4/80, respectively. We also included DEC-205, primarily expressed by a subset of DC. Across the entire lung (Fig. 5B) , {gamma}{delta} T cells were found more frequently in contact with F4/80+ cells (25%) and less frequently in contact with CD45R+ cells (<10%). The inverse was true for {alpha}ß T cells, and contacts with DEC-205+ cells were infrequent for either type of T cell (~5%). This comparison suggested that leukocyte contacts with pulmonary {gamma}{delta} T cells preferentially involve myeloid cells, including F4/80+ macrophages and I-A+ DC, whereas contacts with {alpha}ß T cells frequently involve CD45R+ lymphoid cells, including B cells but also some T cells and plasmacytoid DC [56 , 57 ]. Next, to diminish the possible influence of the T cell distribution, we compared leukocyte contacts in one locale (Fig. 6 A, C-E) . We chose the parenchyma, where most {alpha}ß T cells and the two V{gamma}-defined {gamma}{delta} T cell subsets are allocated primarily. For comparison, leukocyte contacts in nonparenchymal locations are shown also (Fig. 6B) . Even within the same location (the parenchyma), differences between {alpha}ß and {gamma}{delta} T cells in contacts with CD3+, I-A+ and F4/80+ cells were maintained, suggesting that they reflect preferences of the T cells rather than different opportunities due to parenchymal vs nonparenchymal cell distributions. Differences in contacts with CD45R+ cells disappeared. In the parenchyma, we also examined relative frequencies of the various leukocytes as a way to compare opportunities for contacts at this site (Fig. 6D) . Given their low relative frequency, F4/80+ myeloid cells emerge as preferred partners of {gamma}{delta} T cells whereas CD3+ lymphoid cells appear to be avoided. In contrast, leukocyte contacts with {alpha}ß T cells seem to follow more closely the relative frequencies of the partner cells and thus could be primarily based on opportunity. However, a comparison of {alpha}ß and {gamma}{delta} T cells for contacts with CD45R+ and CD3+ cells is consistent with some bias of the {gamma}{delta} T cells against lymphoid partners. Finally, we considered the possibility that partner cells might be distributed differently in the immediate "neighborhood" of the two types of T cells, within the parenchyma. Therefore, we compared relative distribution densities from the "perspective" of the T cells, by assessing how many potential partners are present in the surroundings of an individual T cell (Fig. 6E) . No significant differences were found with regard to I-A+ and F4/80+ cells, supporting the notion that these contacts with {gamma}{delta} T cells reflect intrinsic cellular preference rather than opportunity.

Accumulation of the {gamma}{delta} TCR at sites of contact with I-A+ partner cells
Contacts of {gamma}{delta} T cells with bright I-A+ cells were particularly striking (Fig. 7 ). We often found increased {gamma}{delta} TCR-staining at the areas of cell contact (Fig. 7A) , reminiscent of the immunological synapses between {alpha}ß T cells and antigen-presenting cells (APC). However, there was no corresponding accumulation of I-A, suggesting that I-A is not involved in these contacts. V{gamma}1+ and V{gamma}4+ {gamma}{delta} T cells in the parenchyma were found in contact with bright I-A+ cells (Fig. 7 , B and C, respectively), indicating that such interactions are not limited to any particular subset. Finally, in some contacts of {gamma}{delta} T cells and I-A+ cells (Fig. 7D) , traces of TCR-staining could be detected within the partner cells (Fig. 7E) , perhaps indicating synaptic transfer of {gamma}{delta} T cell membrane (trogocytosis) to the I-A+ partners.



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  		Figure 7. Contacts of {gamma}{delta} T cells with bright I-A+ cells in the parenchyma. Frozen sections from untreated adult lung (C57BL/6) were stained with anti-TCR-{delta} or anti-TCR-V{gamma} mAb (red) and mAb M5/114 (anti-I-A/E; blue). (A) Accumulation of the {gamma}{delta} TCR at the contact with an I-A+ cell (TCR-{delta}, z stack series). (B, C) Contacts involving V{gamma}1+ and V{gamma}4+ {gamma}{delta} T cells, respectively. (D) Contact involving a {gamma}{delta} T cell and an I-A+ cell. (E) Same contact as in D but without tissue fluorescence: Trogocytosis? Apparent transfer of the {gamma}{delta} TCR at the contact with the I-A+ cell (TCR-{delta}, z stack series).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Given the extremely thin distribution of the pulmonary {gamma}{delta} T cells, their strong regulatory influence on lung function seems remarkable [22 23 24 ]. To be sure, substantial expansions of these cells can be seen during infections [25 , 26 , 28 ] and after prolonged airway stimulation with allergen [58 ]. These expanded populations might have different and more diverse roles, but even under conditions that do not substantially change the small resident pulmonary population (challenge of nonsensitized mice with aerosolized OVA), the protective effect of the {gamma}{delta} T cells can be demonstrated [22 , 23 ]. Some mechanism is implied, therefore, that amplifies and projects responses of the few {gamma}{delta} T cells within the tissues of the lung. Our earlier functional studies have ruled out {alpha}ß T cells and B cells as required partners for the regulatory functions of the pulmonary {gamma}{delta} T cells [22 ], suggesting instead a partner-role for cells of the innate system. Consistently, the study described here implicates myeloid cells (in particular macrophages and DC) as partners of the pulmonary {gamma}{delta} T cells, because it provides histological evidence for contact-interactions with these cells in situ. We suspect that such interactions reflect a monitoring activity of the {gamma}{delta} T cells, first because they appear to be continuous (ongoing in the normal lung at all times), and second because the population of pulmonary {gamma}{delta} T cells is far smaller than the population of pulmonary myeloid cells. Conceivably, such contacts could also lead to changes in the partners involved [14 15 16 17 18 ], and thus might stimulate the {gamma}{delta} T cells while providing also a mechanism for projecting their regulatory responses via their contact-partners.

We began this study merely for the purpose of detecting {gamma}{delta} T cells within the normal lung. The extremely low density of distribution (often less than one cell/per field of view) required rigorous controls for the specificity of the antibody staining (see Materials and Methods), and it made quantitative morphometric analysis impractical, at least in the first instance. We therefore divided the lung into five major regions, i.e., the submucosa and adventitia surrounding the bronchioles (CT/BR, see methods for abbreviations), the adventitia up to the endothelium of arteries and veins (CT/BV), the region between the basal lamina and the smooth muscle surrounding the bronchioles (LP/SM), the alveoli (ALV), and the mesothelium, or the connective tissue of the visceral pleura (VPL). The epithelium lining the lumen of the airways (mucosa) was also examined, but we did not find appreciable numbers of T cells in this location. We counted the cells within the five regions separately. This permitted comparing relative densities of {alpha}ß and {gamma}{delta} T cells in defined regions of the lung, as well as relative allocations of these cells to the five major regions. We found that by far most {alpha}ß T cells in the normal lung are allocated to the parenchyma/alveoli, whereas the majority of {gamma}{delta} T cells are allocated to nonalveolar regions, with exception of the mucosa. Consequently, despite their far smaller population size, {gamma}{delta} T cells matched or nearly matched the {alpha}ß T cells in relative density in all four nonalveolar regions. In contrast, in the parenchyma, the largest region in terms of tissue mass or surface area in the tissue sections, the relative density of the {gamma}{delta} T cells was much lower than that of {alpha}ß T cells.

The very different distributions of the two types of T cells likely are related to differences in functional roles. The more narrow, parenchyma-biased distribution of {alpha}ß T cells seems noteworthy because {alpha}ß T cells in the normal lung are far from being a homogeneous population and are divisible into naïve, memory, and recently activated cells, or into CD4+, CD8+, and NKT subsets [59 ]. Their narrow distribution in the lung nevertheless may reflect some measure of functional homogeneity. The broad distribution of the {gamma}{delta} T cells on the other hand may reflect functional heterogeneity. The overall distribution appears to be a composite of several partially overlapping distributions of {gamma}{delta} T cell subsets [60 ]. Thus, we found that two major subsets, V{gamma}4+ and V{gamma}1+ cells, have more parenchyma-biased distributions than total pulmonary {gamma}{delta} T cells. This then implies the existence of other parenchyma-avoiding {gamma}{delta} T cells. So far, we have not been able to characterize these other cells. V{gamma}6+ {gamma}{delta} T cells, a known pulmonary subset [20 , 21 , 48 ], might fill this niche. On the other hand, because of their relative size, V{gamma}4+ and V{gamma}1+ subsets must account for nearly all parenchymal {gamma}{delta} T cells. The biological significance of the subset segregation is not yet clear. Subsets of {gamma}{delta} T cells develop sequentially in ontogeny. Suggestively, two late-developing subsets, V{gamma}4+ and V{gamma}1+ cells, segregate to the peripheral lung known to divert late during organogenesis from the earlier developing proximal airways [61 ]. T cells expressing {alpha}ß TCRs arise even later in ontogeny and are also found in the peripheral lung. The parenchyma-biased distributions of V{gamma}1+ and V{gamma}4+ {gamma}{delta} T cells along with {alpha}ß T cells may be important with regard to the impact of all three on airway hyperresponsiveness [62 , 63 ]. Notably, the parenchymal allocation of {gamma}{delta} T cells remained unchanged following airway challenge with ovalbumin (not shown), suggesting that in the unchallenged normal lung, these cells are already correctly placed for their regulatory function. The parenchyma-avoiding {gamma}{delta} T cells on the other hand likely have different roles. These cells might consist primarily of an early developing subset (V{gamma}6+ cells), perhaps segregating with the proximal airways. Finally, the absence of all {gamma}{delta} T cells in the airway mucosa seems noteworthy given the mucosal localization of {gamma}{delta} T cells in the intestines and reproductive tract.

Perhaps most interesting is that our histological study revealed continuously ongoing interactions between the pulmonary T cells and other non-T leukocytes. In addition to the TCR-defined cells, we examined CD45R (B220)+ cells (mostly B cells, some T and NK cells and plasmacytoid DC) [56 , 57 , 64 , 65 ], F4/80+ cells (mostly macrophages) [66 ], DEC-205+ cells (a subset of DC) [67 ] and I-A+ cells (bright I-A+ cells in the lung are mostly myeloid DC and B cells) [57 , 68 , 69 ]. We found that large fractions of {alpha}ß and {gamma}{delta} T cells are in direct cell-cell contacts with a variety of other leukocytes, although {gamma}{delta} T cells rarely made contacts with other T cells. The exclusion of T cells as {gamma}{delta} T cell partners seems noteworthy because the distribution density of lung T cells is higher than that of other leukocytes (except I-A+ and CD45R+ cells), so that there should be no lack of contact-opportunity. In terms of relative contact-frequency (fraction of cells involved in contacts), {gamma}{delta} T cells easily equaled and sometimes exceeded the {alpha}ß T cells, even in the parenchyma where their relative density is so much lower. Over the entire lung, the {gamma}{delta} T cells were found mostly in contact with myeloid cells whereas {alpha}ß T cells were found more often in contact with lymphoid cells. Furthermore, our data suggest that this difference in cellular contacts is not merely a consequence of the different distributions of the two T cell types. Comparing contacts in only one of the five regions (we chose the alveolar tissue for a detailed analysis), the bias of the {gamma}{delta} T cells for myeloid partners was maintained. Moreover, comparing the immediate neighborhood of the two types of T cells in this region, we did not find differences in leukocyte distributions (I-A+ and F4/80+ cells) that could explain the differences in contacts with the T cells. We therefore conclude that the preference of {gamma}{delta} T cells for myeloid contacts is in fact intrinsic to these T cells. Whether {alpha}ß T cells actually have a bias for lymphoid contacts is not clear because their leukocyte contacts followed more closely the distributions of their leukocyte partners.

Leukocyte contacts of {gamma}{delta} T cells in parenchymal and nonparenchymal locations were different also, with a strong bias of the nonparenchymal cells in favor of F4/80+ and against CD45R+ partners. Whether this difference reflects intrinsic preferences of the segregated subpopulations of {gamma}{delta} T cells, or merely the distributions of partner cells remains to be determined.

Contacts between I-A+ cells and {gamma}{delta} T cells were particularly frequent, involving roughly every second {gamma}{delta} T cell in the lung. In the areas of cell-contact, accumulation of {gamma}{delta} TCRs was evident, reminiscent of immunological synapses between T cells and APC [70 , 71 ], although I-A molecules did not accumulate and do not appear to serve as TCR-ligands in these contacts. Furthermore, in some of these contacts, TCR-{delta}-staining within the I-A+ partner suggested transfer of membrane material from the {gamma}{delta} T cell to the IA+ cell. In vitro, others have shown that {gamma}{delta} T cells can form synapses with several types of cells and exchange membrane material (trogocytosis) [72 , 73 ]. What if any functional consequences might be connected with such interactions remains to be determined. It seems likely, however, that such contacts contribute to the reported {gamma}{delta} T cell-induced changes in DC and macrophages [14 15 16 17 18 ].

Up to this point, our studies have revealed continuous and widespread contact-interactions between {gamma}{delta} T cells and myeloid cells in the normal lung. If myeloid cells, and pulmonary DC in particular, can be regarded as sentinels [74 ], then pulmonary {gamma}{delta} T cells appear to monitor the sentinels, perhaps also modulating their responses. Intriguingly, such a mechanism should enable very small cell populations to exert widespread regulatory control, as is implied for pulmonary {gamma}{delta} T cells in functional studies [22 , 63 ].


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grants RO1 HL-65410 and AI -40611 (to W. K. B.) and AI-44920 (to R. L. O.) and by a grant from the Environmental Protection Agency (X-83084601) to R. J. M., E. W. G., and W. K. B. C. L. R. is supported by an Investigator Award from the Arthritis Foundation. The authors thank Drs. Avi Kupfer, Azzeddine Dakhama, Peter Henson, and Ling Yi Chang (Departments of Pediatrics and Medicine, National Jewish Medical and Research Center) and Andrew Farr (Department of Biological Structure and Immunology, University of Washington, Seattle) for expert advice on the anatomy and histology of the lung.

Received May 6, 2005; revised June 28, 2005; accepted July 15, 2005.


   REFERENCES
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 DISCUSSION
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