Background
The lung is a mucosal organ that is continuously exposed to the outside environment, rendering it one of the major sites of primary bacterial and viral infections. It is generally believed that upon pathogenic microbial invasion, antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages present in the lung intercept these microbes and participate in the initiation of ensuing innate and adaptive T cell responses. However, the lung is divided into two major compartments: the alveolar space and the parenchyma. In the steady state, the vast majority of cells in the alveolar space, which can be harvested by bronchoalveolar lavage (BAL), are alveolar macrophages (AMs), which are the first line of leukocytes in the lung taking up incoming microbes or microbial antigens [
1‐
4]. On the other hand, the lung parenchymal tissue including the airway epithelium comprises a variety of APC types including DCs and B cells [
5,
6]. Traditionally, it is the lung DCs that are deemed the most critical to transporting antigens from tissue to the local draining lymph nodes to activate naïve T cells [
7,
8].
The issue regarding whether alveolar macrophages are capable of naïve T cell activation has remained controversial [
2,
4,
9‐
15]. While AMs are generally thought to be poor T cell stimulators or even immunosuppressive as shown in some studies [
11,
16,
17], they have also been shown to be able to activate certain T cell subsets [
9]. The possibility for AMs to act as functional APCs is further supported by the evidence that macrophages could migrate to the local lymphoid tissues or acquire DC characteristics [
13,
18‐
20]. Furthermore, local non-migratory residential APCs including macrophages are thought to play a role in presenting antigen to and activating antigen-experienced or effector/memory T cells within the lung parenchyma [
21]. In comparison, there has been well-established evidence to support an indisputable role by lung parenchymal DCs in transporting antigens to the secondary lymphoid tissue and activating naïve T cells [
22‐
24]. However, several important questions remain to be elucidated: 1] to which extent the alveolar space CD11c+ APCs and lung parenchymal CD11c+ APCs differ in their phenotype and ability to activate naïve T cells; 2] whether they differ in their ability to activate antigen-experienced or -primed T cells; and 3] whether these lung CD11c+ APC populations differ from the splenic CD11c+ counterparts which have been often used for understanding APC biology. The lack of such knowledge is due to the fact that the majority of studies have thus far examined the phenotypes and functions of APC populations from various tissue compartments in isolation [
25‐
28] or the direct comparative studies were carried out by using bone marrow- or spleen-derived macrophages and DCs [
29‐
31]. Emerging evidence suggests that the knowledge generated at one site may not apply to another site as different anatomical locations or tissue microenvironments profoundly influence the phenotype and function of APC populations [
5,
32‐
34]. The knowledge regarding the relative T cell activation capacities of APCs within different tissue compartments will help us understand the immune regulatory mechanisms and develop APC-based strategies for immune manipulation.
In the present study, by using various approaches we have directly compared the phenotype of alveolar space CD11c+ APCs and the lung parenchymal CD11c+ APCs and their capability to activate both naïve and antigen-primed T cells. We have also compared these lung CD11c+ APC populations with the splenic CD11c+ APCs. We report that CD11c+ APCs from the alveolar space, lung parenchyma, and the spleen display differential co-stimulatory molecule expression and cytokine responsiveness. In addition, alveolar space APCs are poor activators of naïve T cells compared to lung parenchymal and splenic CD11c+ APC populations. However, alveolar APCs are able to potently activate the in vivo microbial antigen-primed T cells to a similar extent as lung parenchymal and splenic CD11c+ APCs.
Discussion
Antigen presenting cells play a pivotal role in initiating T cell responses against foreign pathogens. Their role becomes even more important at mucosal sites of the body including the skin, gut and lung where maximal microbial encounter occurs. Despite the recognized importance of APCs in eliciting T cell responses in the lung, our understanding regarding the relative T cell activation capacity of alveolar space (BAL), lung parenchymal, and splenic CD11c+ APCs still remains largely to be understood due to the lack of side-by-side comparative studies. In the present study we have evaluated and compared the phenotype of BAL, lung parenchymal, and splenic CD11c+ APC populations and their capabilities to activate both naïve and antigen-primed T cells. We report here that CD11c+ APCs from the BAL, lung parenchyma, and the spleen display differential co-stimulatory molecule expression and cytokine responsiveness. Furthermore, BAL CD11c+ APCs are incapable of markedly activating naïve T cells in contrast to lung parenchymal and splenic CD11c+ APC populations. However, these alveolar space CD11c+ APCs are able to potently activate the in vivo microbial antigen-primed T cells to a similar extent as lung parenchymal and splenic CD11c+ APCs.
The surface marker CD11c has been widely used for identifying and/or purifying antigen presenting DCs and macrophages [
22,
35‐
40,
44] although it may also be expressed by other leukocyte subsets [
37]. We believe that the majority of alveolar space CD11c+ APCs isolated by lavaging naïve mouse lung we used in our study are alveolar macrophages, different from the BAL of infected lungs which may contain heterogenous CD11c+ APC populations. This conviction is also supported by other published studies demonstrating that the vast majority (> 96%) of BAL CD11c+ cells are alveolar macrophages [
39,
40]. However, it is likely that a small fraction of these naïve BAL CD11c+ APCs may be DCs as it has previously been shown that a very minute fraction (< 2%) of BAL CD11c+ APCs are DCs [
39]. On the other hand, the lung interstitial CD11c+ APCs used in this study are most likely a mixture of pulmonary DCs and macrophages. It is noteworthy that although the use of additional surface markers such as F4/80 and DEC-205 in conjunction with CD11c may aid in differentiating macrophages from DCs [
22,
35‐
40,
44], it has been reported that F4/80 can also be expressed by DCs [
37] and that DEC205 can also be expressed by pulmonary macrophages [
45]. Our findings highlight that the tissue microenvironment or the anatomical location influences the function of APCs. Von Garnier et al. have shown that dendritic cells within the main conducting airway tissue (the trachea and main bronchi) and the lung parenchyma differ in their capacity to activate OVA-specific CD4+ T cell activation [
5]. Similarly, we have shown that the CD11c+ APCs from the alveolar space are weak allogenic stimulators and are incapable of activating naive OVA-specific CD4+ and CD8+ T cells contrast to lung parenchymal and splenic CD11c+ APCs. However, we demonstrate that alveolar space CD11c+ APCs were able to activate antigen-primed type 1 CD4+ and CD8+ T cells to a similar extent as the lung parenchymal and splenic CD11c+ APCs. To our knowledge, our current study is the first to have directly compared alveolar space, lung parenchymal, and splenic CD11c+ APCs for their phenotypes and capacities to activate both naïve and primed T cells and concluded that alveolar space or BAL CD11c+ APCs are much poorer activators of naïve T cells than those isolated from the lung parenchyma and the spleen. This observation is in general agreement with the finding that alveolar macrophages may be immunosuppressive [
25]. The fact that contrast to the relatively strong naïve T cell-activating capability of lung parenchymal and splenic CD11c+ APCs, BAL CD11c+ APCs is a poor activator of naïve T cells, suggests that a small fraction of DCs, if any, present in the BAL CD11c+ APC population plays a negligible role. However, we cannot completely rule out the possibility that under steady state conditions (naïve), the T cell activating capacity of contaminating DCs (if any) may be suppressed by the alveolar macrophages and with antigen-primed T cells, these BAL DCs may overcome the inhibitory effect of the alveolar macrophages to more readily activate antigen-primed T cells which may have decreased activation threshold. However, we feel that this is unlikely since the lung interstitial or splenic CD11c+ APCs activated antigen-primed T cells to a similar extent as they did to naïve T cells. It has also been demonstrated that alveolar macrophages can indeed act as accessory cells for mycobacterial antigen experienced γδ T cells [
9]. Our results suggest that naïve T cells and antigen-primed effector T cells may have differential activation requirements. Furthermore, our results imply that alveolar macrophages and lung interstitial APCs play a distinct role in the early phase of T cell priming and activation with the latter playing the most critical role. However, during the effector phase of T cell responses, both alveolar macrophages and lung APCs could go on to present antigens and activate antigen-primed effector T cells. Given the large number of alveolar macrophages at the site of infection, these APCs may play an even greater role than DCs in effector T cell activation within the respiratory tract. We have recently demonstrated that airway luminal T cells are critical to immune protection against intracellular bacterial infection such as pulmonary tuberculosis [
46,
47].
Moreover, the ability of APCs to activate T cells is related to their co-stimulatory molecules expression as demonstrated by studies in which T cell proliferation is strongly inhibited by abrogation of either CD80 (B7.1) or CD86 [
48,
49], signifying that CD11c+ cell populations from the alveolar space, lung parenchyma, and the spleen all have the capacity to activate T cells since they all express MHC II and the co-stimulatory molecules B7.1 and CD40, albeit at different levels. This may explain the differential activation of naïve T cells by BAL, lung parenchymal and splenic CD11c+ APCs. In general, we observed relatively robust responses of both transgenic CD8 and CD4 T cell responses to the APCs loaded with OVA peptides. This is likely due to the efficiency of direct MHC loading and presentation of OVA peptides, particularly the CD8 T cell peptide. In comparison, the transgenic CD4 T cell responses to the APCs infected with an adenovirus expressing OVA protein were considerably lower than CD8 T cell responses. This is in accord with the understanding that virus infection preferentially target the MHC I pathway (CD8 T cell activation). This observation was noted across all three CD11c+ APC populations, suggesting that despite coming from different anatomical sites, these CD11c+ APC populations process viral antigens in a similar manner for naïve T cell activation. We have also reported here that freshly isolated BAL CD11c+ cells produce significantly greater levels of the pro-inflammatory cytokine TNF-α than their parenchymal and splenic counterparts, which is in agreement with the current understanding that alveolar macrophages are a great source of TNF-α [
50]. However, parenchymal CD11c+ APCs produce a greater amount of the type 1 cytokine IL-12 compared to BAL CD11c+ APCs, which is in agreement with the stronger ability of the parenchymal CD11c+ APCs to activate naïve T cells. This disparate levels of TNF-α and IL-12 released by BAL and lung parenchymal CD11c+ APCs to LPS or mycobacterial antigens may be due to their use of distinct TLRs and differential TLR expression [
29,
51,
52], which is likely influenced by the different tissue microenvironments [
53,
54]. IL-10, a potent Th1 opposing cytokine, was undetectable in our CD11c+ APC cultures from all the three compartments, suggesting that this cytokine does not account for the differential type 1 cytokine responses seen in our study. Our study reinforces that the knowledge generated by using APCs from one tissue site cannot be generalized and applied for all due to the heterogeneity of APCs tailored to different anatomic locations and specialized functions.
In conclusion, we have provided the evidence that the CD11c+ APC populations existing within the alveolar space, lung parenchyma, and the spleen differ much in their co-stimulatory molecule expression, cytokine responsiveness, and antigen presentation and T cell activation capacities. Furthermore, we have shown that alveolar space macrophages are poor activators of naïve T cells but can activate antigen experienced T cells to a similar extent as lung parenchymal and splenic CD11c+ APC populations. The findings presented in this study thus enhance our understanding about the relative capacity of alveolar space, lung parenchymal and splenic CD11c+ APCs to activate T cells, and further highlights the importance of tissue microenvironment influencing antigen presenting function of different APC populations.
Methods
Mice
Six- to 10-wk-old female C57Bl/6 and Balb/c mice were purchased from Harlan Laboratories. Female/male OT-I and OT-II transgenic mice were bred at McMaster University Central Animal Facility. All mice were housed in a specific pathogen-free level B facility. All experiments were conducted in accordance with the McMaster Animal Research Ethics board.
Mycobacterial preparation
Mycobacterium bovis BCG (Connaught strain) was prepared as previously described in our lab [
55,
56]. Briefly, BCG was grown in Middlebrook 7H9 broth (Difco) supplemented with Middlebrook OADC enrichment (Invitrogen), 20% glycerol, and 0.05% Tween 80 for 10 to 15 days, and samples were then divided into aliquots and stored at -70°C. BCG was washed twice with phosphate-buffered saline (PBS) containing 0.05% Tween 80 and resuspended in PBS. It was then passed through a 27-gauge needle 10 times to disperse clumps and then diluted with PBS to the desired concentration before use.
Isolation of CD11c+ APCs from the alveolar space, lung parenchyma, and spleen
Naïve mice were sacrificed by bleeding the abdominal vessels. Mouse lungs were then removed aseptically and lavaged with PBS to isolate bronchoalveolar lavage (BAL) cells from the alveolar space. The mouse lung was exhaustively lavaged 5 times to a total volume of 1.8 ml PBS through a polyethylene cannulated into the trachea to ensure maximal cell recovery. Extra effort was made to ensure that only the lung parenchyma was collected and any trachea and main bronchi including all associated hilar lymph nodes were removed. The lavaged lungs and the spleens were infused with collagenase type 1 (Sigma) and then cut up into small pieces and subsequently incubated in collagenase at 37°C; 1 hour for lungs and 45 minutes for spleens. The collagenase digested lungs and spleens were then passed through 100 μm cell strainers, and a 3 ml needle plunger was used to mash the cells through the strainer. After red blood cell lysis with a mouse erythrocyte lysing kit (R&D Systems), lungs and spleens were filtered and resuspended in complete RPMI (cRPMI) media (RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamine). Cells were counted, and viability (always > 90%) was measured by trypan blue exclusion. Depending on the experiment, BAL, lung, and spleen cells from a number of mice were pooled and incubated with CD11c microbeads (Miltenyi biotec) according to the manufacturer's instructions. CD11c labelled cells were then passed through an MS column on the OctoMACS separator (Miltenyi biotec). Samples were run through MACS separation columns twice to achieve higher purity. Cells were counted, and viability was measured by trypan blue exclusion. Purity of cell preparations were determined using flow cytometry, and the purity of CD11c+ populations was consistently > 90%.
Cell surface immunostaining and FACS analysis
All monoclonal antibodies (MAbs) used were purchased from BD Pharmingen. Immunostaining and FACS were carried out as previously described [
55,
56]. Briefly, cells were blocked for non-specific binding of their Fc receptors with anti-CD16/CD32 antibodies for 15 min and then stained for 30 min on ice with the appropriate combinations of fluorochrome-conjugated MAbs. Fluorochrome-conjugated MAbs to CD11c, B7.1, CD40, MHC Class II, CD3, CD4, and CD8 were used. Appropriate Isotype controls were used for each antibody. The data was collected with the LSRII (BD Biosciences) flow cytometer using FACSDiva software and analyzed with Flowjo software.
Cell culture and ELISA
Purified CD11c+ APCs (0.1 × 106/well) from the alveolar space, lung parenchyma, and spleen were seeded into 96-well flat bottom plate and cultured at 37°C and 5% CO2 with or without antigen stimulation. The antigens used for stimulation were LPS (1 ng/well) and Mycobacterium tuberculosis Culture Filtrate proteins (Mtb-CF) (2 μg/well). Cells were cultured in a total volume of 250 μl of cRPMI. The culture supernatants were collected at 48 h and stored at -20°C until cytokine measurement. TNF-alpha and IL-12p40 concentrations were measured by using ELISA kits (R&D systems).
Mixed leukocyte reaction (MLR)
MLR was carried out as previously described [
53]. Allogeneic splenic T cells were isolated from naïve Balb/c mice using CD4 and CD8 positive isolation kits (both from Miltenyi biotec) according to the manufacturer's protocol and pooled. T cells were then labelled with 5 μM CFSE (Molecular Probes) in PBS supplemented with 5% FBS and then washed three times after staining. 5 × 10
5 CFSE labelled T cells were co-cultured with a varying number of C57Bl/6 CD11c+ APC populations from the alveolar space, lung parenchyma, and spleen in 96-well culture plate at 37°C for 96 hours. T cell proliferation was examined by flow cytometric analysis of CFSE dilution as previously described [
57].
In vitro transgenic T cell proliferation Assay
CD4 T cells were MACS purified from OT-II transgenic mice and CD8 T cells were purified from OT-I transgenic mice using the CD4+ and CD8+ T cell isolation kits, respectively, according to the manufacturer's instructions (Miltenyi biotec). T cells were labelled with 5 μM CFSE (Molecular Probes) and washed 3 times with PBS supplemented with 5% FBS and resuspended in cRPMI. 5 × 105 CFSE labelled OT-II T cells were co-cultured with varying numbers of C57Bl/6 APC populations from BAL, lung, and spleen that were pre-pulsed with CD4 or CD8 OVA peptide (0.1 ng/μl) for 1 hour. Unpulsed CD11c+ cells co-cultured with T cells and T cells cultured with no CD11c+ APCs were set up in parallel as controls. CD8+ and CD4+ T cell proliferations were examined 48 and 72 hours after culture by flowcytometric analysis of CFSE dilution, respectively. In some experiments, APCs were pre-infected with an adenovirus expressing the OVA protein (AdOVA) (100 pfu/cell) overnight and then co-cultured with OT-I or OT-II T cells. An adenoviral vector (Addl70-3) infected CD11c+ cells and uninfected CD11c+ cells were used as controls in these experiments.
Ex vivo T cell activation assay
In vivo mycobacterium-primed CD4+ T cells and CD8+ T cells were MACS-purified as described above from the spleens of mice that had been infected with live
M. bovis BCG (10
6 cfu) for 17d as previously described [
55]. 0.4 × 10
6 T cells were co-cultured with 4000 naïve BAL, lung, and spleen CD11c+ APC populations that were infected with either BCG (2 cfu/cell) or stimulated with Mtb-CF (2 ug/well) or just culture media (no stimulus). IFN-γ secretion ELISPOT was carried out as previously described [
58]. Briefly, isolated CD4+ and CD8+ T cells (0.4 × 10
6) were seeded into a 96-well PVDF microplate (Millipore Corporation) pre-coated overnight with a mouse IFN-γ capture antibody (R&D system). Cells were incubated for 24 h and then washed and incubated with a detection antibody at 4°C overnight. The plate was developed by using standardized streptavidin-conjugated alkaline phosphatase and chromogen method (R&D system). The number of IFN-γ-releasing cells was determined by using an ELISPOT reader (CTL Cellular Technology Ltd).
Statistical analysis
All statistical analyses were performed using unpaired, two-tailed student's t test with Excel spreadsheet software (Microsoft). Values of p < 0.05 were considered statistically significant.
Authors' contributions
KK carried out and designed the experiments and wrote the manuscript. EKR helped with experiments and data analysis. C–LS assisted in infecting the mice and sample collections. SM helped with FACS data analysis and interpretation. PY provided invaluable advice and protocols on APC purification, staining and phenotyping. ZX crafted the idea of this project and contributed to the overall design and execution of experiments and helped with manuscript drafting and fine-tuning. All authors have read and approved the manuscript.