Intratracheal instillation of L-selectin-deficient (L-Sel−/−) mice with an adenovirus 2 (Ad2) vector resulted in the lack of respiratory Ad2- or β-galactosidase-specific CTLs with concomitant long-lived β-galactosidase transgene expression in the lungs. The absence of Ag-specific CTLs was attributed to a deficiency in lymphoid CD11c+CD8+ dendritic cells (DCs) in the lower respiratory lymph nodes (LRLNs). To enable L-Sel−/− CTL activity, cell-sorted L-Sel−/−CD8+ T cells were cocultured with cell-sorted L-Sel+/+CD8+ or CD8 DCs or L-Sel−/−CD8 DCs. Only the CD8+ DCs restored CTL activity; L-Sel−/−CD8 DCs failed to support L-Sel+/+ CTLs because these remained immature, lacking the ability to express costimulatory molecules CD40, CD80, or CD86. Although no lung CD8+ DCs were detected, the DC environment remained suppressive in L-Sel−/− mice evident by the lack of CTL responses following adenoviral challenge with OVA in recipient L-Sel−/− adoptively transferred with OT-1 CD8+ T cells. To assess whether the L-Sel−/−CD8 DCs could be induced into maturity, microbial stimulation studies were performed showing the failure of L-Sel−/− LRLN to make matured DCs. When L-Sel−/− mice were subjected in vivo to microbial activation before Ad2 vector dosing, CTL activity was restored stimulating the renewed presence of LRLN CD8+ DCs in L-Sel−/− mice. These studies show that impairment of L-Sel−/− DC maturation results in insufficient mature DCs that require microbial activation to restore increases in respiratory CD8+ DCs to support CTL responses.

Few studies have assessed the role of L-selectin in mucosal responses despite the importance of L-selectin in Peyer’s patches (1, 2, 3, 4), nasal-associated lymphoid tissue (5, 6), head and neck lymph nodes (HNLNs3; Refs. 5 , 7 , 8), and human tonsils (9). The homing receptor, L-selectin, mediates naive lymphocyte binding through its interaction with peripheral node addressin (PNAd), which is the predominant addressin expressed in peripheral LNs (10, 11). It is also responsible for some binding in mucosal tissues, including the mesenteric LNs (12) and the various HNLNs (5). Because PNAd is a composite of carbohydrate moieties expressed on different glycoprotein backbones (13), PNAd-L-selectin interactions have also been shown to mediate some binding of naive lymphocytes to the Peyer’s patches and nasal-associated lymphoid tissue through the expression of PNAd carbohydrate on the mucosal addressin cellular adhesion molecule-1 glycoprotein backbone (6, 14, 15).

The development of L-selectin-deficient (L-Sel−/−) mice has enabled testing of L-selectin’s importance to Ag-specific memory responses. These mice show diminished delayed-type hypersensitivity responses (16) and primary Ag-specific T cell proliferative responses (16), but not mitogen-stimulated T cell proliferative responses (17). This failure in Ag-specific T cell proliferative responses is not attributed to defective Ag presentation because this aspect remains functionally intact and capable of presenting Ag to wild-type T cells (18). Interestingly, defects in secretory IgA responses were observed in L-Sel−/− mice that were mucosally immunized. When L-Sel−/− mice were immunized intranasally with the potent mucosal adjuvant, cholera toxin, systemic IgG and gut IgA responses were not affected, but upper respiratory and vaginal IgA responses were delayed (7, 8). L-Sel−/− mice orally immunized with an attenuated Salmonella vaccine vector expressing the fimbriae from enterotoxigenic Escherichia coli showed no fimbriae-specific mucosal IgA Abs despite elevations in systemic IgG Ab titers (19).

Although most studies have focused on L-selectin expression on B and T lymphocytes, few studies have evaluated the role of L-selectin expression on dendritic cells (DCs; Refs. 20, 21, 22). In particular, L-Sel−/− mice were found to show a reduced expression of CD11c+CD11b DCs in their LN when compared with L-Sel+/+ BALB/c mice (22), but the functional consequence of this observation was not pursued. In this current study, in addition to the L-selectin deficiency, these mice showed an overall deficiency in DC numbers, with the CD8+ DC subset being particularly affected in the lower respiratory LNs (LRLNs), as evidenced by an overall reduction in their numbers, resulting in a concomitant loss of CTL function. The absence of these CD11c+CD8+ DCs resulted in the loss of Ad2- and β-galactosidase (βgal)-specific CTL responses despite the presence of other lymphoid DCs.

The nonreplicating Ad2/βGal-2 vector (23) was provided by the Virus Production Unit of Genzyme and was subsequently expanded in 293 cells (CRL-1573; American Type Culture Collection (ATCC)) and purified by cesium chloride-gradient centrifugation (23, 24). Wild-type adenovirus 2 (Ad2) stock was purchased from Quantum Biotechnologies and grown in HeLa cells (CCL-2.2; ATCC) for 48 h following infection. The virus was harvested by repeated freeze thawing and titered by serial dilutions. A stock of Ad5 CMV Trf-OVA (25) was provided by the University of Iowa Gene Transfer Vector Core, referred to here as Ad5-OVA.

C57BL/6N mice (Frederick Cancer Research Facility, National Cancer Institute, Frederick, MD) and C57BL/6 mice (Charles River Laboratories) were used throughout this study. L-Sel−/− and OT-I breeder pairs were obtained from The Jackson Laboratory to establish our colony. All mice were maintained at the Montana State University Animal Resources Center under pathogen-free conditions in individually ventilated cages under HEPA-filtered barrier conditions and were fed sterile food and water ad libitum. Mice (5–10/group) at 6–8 wk of age were anesthetized with isofluorane (Abbott Laboratories) inhalation to effect and received a single intratracheal (i.t.) dose of 1 × 109 PFU of Ad2/β Gal-2 vector in 50 μl (24, 26). In some instances, mice were dosed i.t. with 0.1, 10, or 100 μg of E. coli O55:B5 LPS (Sigma-Aldrich) 3 days before or 2 days after i.t. instillation with Ad2 vector. Most evaluations were conducted after 12 days, except for the kinetic studies.

The lungs, LRLNs, HNLNs, and spleens were used throughout these studies for cytolytic assays or FACS analysis. Lymphocytes were isolated from the LN and spleens by mechanical disruption followed by Ficoll-Hypaque (Lymphocyte M; Accurate Chemical) density gradient centrifugation (24, 26). Lung mononuclear cells were isolated and subjected to collagenase (Worthington) digestion (24, 26) with >95% cell viability recovered.

DCs were isolated from LRLN by collagenase (50 U/ml type IV; Sigma-Aldrich) digestion plus DNase (0.8 U/ml; Promega) in Teflon flasks under gentle stir for 30 min at 37°C. The digested LRLN were passed through Nitex (Fairview Fabrics) and incubated at 37°C for 30 min. Cell suspensions were washed in complete medium (CM) consisting of RPMI 1640 medium supplemented with 0.2 mM l-glutamine, 10 mM HEPES, 0.1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS (Invitrogen Life Technologies). Total lymphocytes were resuspended in 2.0 ml of HBSS (Invitrogen Life Technologies) and then added to 1.0 ml of Optiprep (Axis-Shield PoC) and mixed gently. This was then layered with a 1:3.2 (14.3%) solution of diluent, which consisted of 0.88% NaCl, 1.0 mM EDTA, and 0.5% (w/v) BSA, and 10 mM HEPES-NaOH (pH 7.4) and Optiprep. The gradient was then topped with 3.0 ml of HBSS. Lymphocytes were subjected to density gradient centrifugation for 15 min at 20°C. DCs were removed from the top of the 12% Optiprep gradient and washed in CM. Typically, DCs were enriched to >80% purity, as evaluated by immunofluorescent staining with anti-CD11c (BD Pharmingen) and DEC205 (Serotec) mAbs.

To assess conversion of CD8 to CD8+ DCs, DCs were isolated using a magnetic labeling kit to isolate CD8+ DC (Miltenyi Biotec). Total LRLN and lung lymphocytes were subfractionated to remove non-DCs. Following enrichment, CD8+ DCs were isolated by positive selection, and the CD8 DC fraction was passed through the column. Both the CD8+ and CD8 DCs were evaluated by FACS and showed >80% purity, and the CD8 DCs contained ≤2.2% CD8+ DCs. The purified CD8+ DCs were stained with 1.25 μM carboxy-fluorescein diacetate succinimidyl ester (CFDA; Molecular Probes-Invitrogen) in RPMI 1640 for 5 min at room temperature in the dark and then washed three times. The purified CD8 DCs were stained with 2 μl of CM-DiI (Molecular Probes-Invitrogen) in 1 ml of RPMI 1640 for 20 min at 37°C and then washed three times. The CFDA-labeled CD8+ and CM-DiI-labeled CD8 DCs were cocultured without or with 10 μg/ml LPS for 24 h. Some cultures were then infected with Ad2/βgal-2 at a 500:1 ratio for additional 24 h. DCs were harvested, washed, and stained for CD11c, CD8α, MHC class II, and TCRβ.

EL4 thymoma (TIB39) and E.G7-OVA (CRL2113) (H-2b) cells were obtained from ATCC. E22 cells are EL4 cells transfected with the lacZ gene and were provided by Dr. Y. Paterson (University of Pennsylvania School of Medicine, Philadelphia, PA) (27).

To assess CTL activity in mononuclear cell suspensions from lungs, LRLN, and spleens following a single i.t. dose of Ad2/βGal-2, a standard 51Cr-release assay was adapted (24). For Ad2-infected targets, E22, and EL-4 cells were prepared and loaded with 100 mCi of Na251CrO4 (NEN Life Science Products/DuPont), and cytotoxic assays were performed identically to those previously described (26).

To determine the supportive DCs for Ad2- and βgal-specific CTLs, L-Sel+/+ and L-Sel−/− LRLN and HNLN CD8+ T cells, L-Sel+/+ LRLN CD11c+CD8+, and L-Sel+/+ and L-Sel−/− LRLN CD11c+CD8 were sorted by flow cytometry to >98% purity. DCs were cocultured with CD8+ T cells at a 1:5 ratio and restimulated with mitomycin C-treated Ad2-infected splenocytes for 5 days.

To assess the role of lung DC support, 5 × 106 purified splenic and respiratory LN-transgenic OT-1 anti-OVA CD8+ T cells (obtained to >95% purity) were adoptively transferred into L-Sel+/+ or L-Sel−/− mice, and 1 day later, mice were dosed i.t. with 1 × 109 PFU of Ad5-OVA. L-Sel+/+ and L-Sel−/− lungs were evaluated 2 and 3 days after Ad5-OVA challenge for OVA257–264 H-2Kb pentamer+ (PE-labeled Pro5 MHC pentamer; Proimmune) CD8+ T cells by flow cytometry and CTL responses to OVA using the E.G7-OVA as target cells in a chromium release assay, as described above.

Mice dosed i.t. with Ad2/βGal-2 were evaluated for the expression of βgal according to the modified method by Parsons et al. (28) at varying intervals between 3 and 28 days. Following exsanguination, lungs were perfused with 2 ml of cold 2% paraformaldehyde in Dulbecco’s PBS (DPBS) containing 2 mM MgCl2 and 1.25 mM EGTA. The fixed, inflated lungs were dissected from the chest cavity and immersed in paraformaldehyde at 4°C overnight. Lungs were rinsed in rinse buffer (DPBS containing 2 mM MgCl2 and 0.1% Triton X-100) for three changes, 30 min each on a rotator. XGAL (40 mg/ml; Fisher-Scientific) dissolved in dimethylformamide was diluted to 1.0 mg/ml in XGAL staining buffer (5 mM potassium ferrocyanide and 5 mM potassium ferricyanide in rinse buffer). Rinsed lungs were immersed into XGAL staining solution up to 3 days at 30–32°C. After the blue color developed, stained lungs were immersed into 30% sucrose in DPBS overnight at 4°C, and then embedded in OCT (Sakura Finetek), snap-frozen, and cryosectioned at −26°C. Five-micrometer frozen sections were mounted on Plus Charge (Erie Scientific) microscope slides, air dried at room temp, counterstained with 10 dips in nuclear fast red, rinsed with water, air dried, and coverslipped using permanent mounting medium.

To determine the type of CD11c+ DCs recruited to the LRLN following one i.t. instillation with Ad2 vector, lymphocytes from lungs and LRLN were assessed. Fluorochrome-conjugated mAbs (BD Pharmingen) for mouse CD11c (N418), TCRβ (H57-597), Mac-3, CD4 (RM4-5), CD8α (53-6.7), B220 (RA3-6B2), CD11b (M1/70), L-Sel (Mel-14), Gr1 (RB6-8C5), CD40 (3/23), CD80 (16-10A1), and CD86 (GL-1). The mAbs for plasmacytoid DC (pDC) included cJF05-1C2.4.1 (mPDCA-1 clone; Miltenyi Biotec) and anti-Siglec H mAb (29; clone 440c; Cell Sciences). Immunofluorescent staining was then measured by flow cytometry using a forward scatter gate set for lymphocytes in the LRLN and a forward scatter gate set for lymphocytes and myeloid cells in the lungs.

For cell sorting of CD8+ T (CD8+TCRβ+) cells, LRLN lymphocytes were labeled with fluorochrome-conjugated anti-CD4, -CD8, and -TCRβ mAbs for 30 min on ice. For cell sorting of DCs (CD11clowCD8+ vs CD8TCRβ), LRLN lymphocytes were labeled with anti-CD11c, -CD8α, and -TCRβ mAbs. The excess label was washed, and cells were subjected to cell sorting using a FACSVantage (BD Biosciences). Greater than 98% purity of single-positive populations was isolated for in vitro cell culture.

The Student t test was used to evaluate the differences between experimental parameters in each experiment.

Intratracheal instillation with the Ad2/βGal-2 vector elevates perforin- and FasL-dependent cytolytic Ad2- and βgal-specific CD8+, not CD4+, T cells (24). To learn the DCs that support these CD8+ T cell responses, L-Sel+/+ and L-Sel−/− mice were given a single i.t. dose of 1 × 109 infectious units of Ad2/βGal-2. L-Sel+/+ mice gave the expected dose-dependent (varying E:T ratios) CTL response against Ad2-infected targets using freshly isolated lung, mediastinal, and hilar LNs, referred to as LRLN, HNLN, and splenic lymphocytes as effector cells (Fig. 1,A). Ag-specific reactivity was determined because no lysis of mocked-infected cells (Fig. 1,A) nor lysis of YAC-1 cells (data not shown) occurred. Analysis of lymphocytes freshly isolated from Ad2/βGal-2-dosed L-Sel−/− mice showed no cytolytic activity against Ad2- or mock-infected targets (Fig. 1,B) nor after in vitro Ag restimulation for 5 days in any of the tissues tested (Fig. 1,D), which contrasted with Ag-restimulated L-Sel+/+ lymphocytes (Fig. 1,C). This observed inactivity was not time-dependent because weekly sampling to 6 wk postinstillation of L-Sel−/− mice failed to reveal CTL responsive in any of the tested tissues (Fig. 1, E–J) nor by Ag restimulation assays (data not shown). In contrast, L-Sel+/+ lung and splenic CTLs showed increasing cytolytic activity against Ad2-infected targets and βgal-expressing (E22 cells) targets for at least to 6 wk postinstillation (Fig. 1, E–J).

FIGURE 1.

L-Sel−/− mice fail to develop CTL responses to Ad2 after i.t. instillation with Ad2 vector even after 6 wk post-i.t. instillation, resulting in sustained lung βgal transgene expression. L-Sel+/+ and L-Sel−/− mice were i.t. instilled with the Ad2/βGal-2 vector, and (A and B) freshly isolated mononuclear cells from lungs, LRLNs, HNLNs, and spleens were assessed 12 days later for CTL activity against wild-type Ad2- or mock-infected targets. C and D, Following in vitro restimulation for 5 days with Ad2-infected targets, LRLN, HNLN, and splenic CTL activity was assessed. Varying E:T cell ratios were tested and showed that despite Ag restimulation, lymphocytes from L-Sel−/− mice failed to lyse virus-infected cells. The percent cytotoxicity was expressed as the level of cytotoxicity obtained at each E:T cell ratio corrected for spontaneous release divided by total release of 51Cr corrected for spontaneous release. Data are representative of eight experiments and depicted as mean ± SD. To determine time dependence, L-Sel+/+ and L-Sel−/− mice were given a single i.t. dose Ad2/βGal-2 vector and freshly isolated lymphocytes (ex vivo) from the lungs, LRLNs, and spleens were evaluated 3, 4, or 6 wk later against (E, G, and I) Ad2-infected targets or (F, H, and J) βgal-expressing EL-4 (E22) cells at varying E:T cell ratios. L-Sel−/− mice remained unresponsive to Ad2 or βgal at any of the time points examined. Lungs from (K and L) 3 and (N and O) 28 day post-i.t.-instilled L-Sel−/− and L-Sel+/+ mice were evaluated for βgal expression. The βgal activity diminished after 3 days in L-Sel+/+ mice resembling (M) normal lungs, but endured for (N) at least 28 days in L-Sel−/− mice. The kinetic CTL data (mean ± SD) are representative of two experiments and the depicted histology are representative of five mice per group.

FIGURE 1.

L-Sel−/− mice fail to develop CTL responses to Ad2 after i.t. instillation with Ad2 vector even after 6 wk post-i.t. instillation, resulting in sustained lung βgal transgene expression. L-Sel+/+ and L-Sel−/− mice were i.t. instilled with the Ad2/βGal-2 vector, and (A and B) freshly isolated mononuclear cells from lungs, LRLNs, HNLNs, and spleens were assessed 12 days later for CTL activity against wild-type Ad2- or mock-infected targets. C and D, Following in vitro restimulation for 5 days with Ad2-infected targets, LRLN, HNLN, and splenic CTL activity was assessed. Varying E:T cell ratios were tested and showed that despite Ag restimulation, lymphocytes from L-Sel−/− mice failed to lyse virus-infected cells. The percent cytotoxicity was expressed as the level of cytotoxicity obtained at each E:T cell ratio corrected for spontaneous release divided by total release of 51Cr corrected for spontaneous release. Data are representative of eight experiments and depicted as mean ± SD. To determine time dependence, L-Sel+/+ and L-Sel−/− mice were given a single i.t. dose Ad2/βGal-2 vector and freshly isolated lymphocytes (ex vivo) from the lungs, LRLNs, and spleens were evaluated 3, 4, or 6 wk later against (E, G, and I) Ad2-infected targets or (F, H, and J) βgal-expressing EL-4 (E22) cells at varying E:T cell ratios. L-Sel−/− mice remained unresponsive to Ad2 or βgal at any of the time points examined. Lungs from (K and L) 3 and (N and O) 28 day post-i.t.-instilled L-Sel−/− and L-Sel+/+ mice were evaluated for βgal expression. The βgal activity diminished after 3 days in L-Sel+/+ mice resembling (M) normal lungs, but endured for (N) at least 28 days in L-Sel−/− mice. The kinetic CTL data (mean ± SD) are representative of two experiments and the depicted histology are representative of five mice per group.

Close modal

Because of the observed immune unresponsiveness, we questioned whether in vivo transgene expression by the lacZ gene delivered by this Ad2 vector would be sustained. Intratracheal instillation of L-Sel−/− and L-Sel+/+ mice was conducted using the Ad2/βGal-2 vector, and at varying intervals, mice lungs were sampled for βgal expression. When compared with L-Sel+/+ mice, L-Sel−/− lungs showed sustained βgal expression for at least 28 days (Fig. 1,N) contrasting with transgene expression in L-Sel+/+ mice, which lost its βgal expression (Fig. 1 O). Such evidence suggests that the βgal protein was not stimulating a CTL response in L-Sel−/− mice. Thus, these studies show that the Ad2/βGal-2 vector was unable to stimulate a CD8 T cell-dependent response in L-Sel−/− mice.

FACS analysis was performed to determine whether the observed unresponsiveness by L-Sel−/− mice might be attributed to a defect or an absence of a particular DC subset. Determination of DC subsets was based upon descriptions for respiratory DCs (29, 30, 31, 32, 33, 34) in which forward- and side-scatter profiles were evaluated, and to exclude lung macrophages, MHC classlow, CD11clow, and Mac-3+ cells were removed from the analysis. For the lymphocyte gate, CD11c+TCRβ+ cells were also excluded from the analysis. Four DC subsets determined relevant to respiratory tissues (29, 30, 31, 32, 33) were evaluated: conventional DCs (cDCs), CD11chighMHC class II+; lymphoid DCs, CD11c+MHC class II+, CD8+ or CD8; myeloid DCs (mDCs), CD11c+ MHC class IIhigh, CD11bhigh, SiglecH; and pDCs, CD11c+MHC class II+, B220high, SiglecH+ (Figs. 2 and 3).

FIGURE 2.

In naive (B) or in (D) Ad2 vector-dosed L-Sel−/− LRLNs, the CD11clowCD8+ DCs are reduced when compared with (A and C) L-Sel+/+ mice. FACS analysis was performed on (A and C) L-Sel+/+ and (B and D) L-Sel−/− LRLN CD11clowMHC class II+ (TCRβ) DCs to discern CD8 (dashed lines) and CD8+ (solid lines) DC subsets. The majority of the (B and D) L-Sel−/− LRLN DCs were CD11clowCD8 when compared with (B and D) L-Sel+/+ LRLN DCs regardless of instillation with (C and D) Ad2 vector. Cell surface analyses were performed on L-Sel+/+ and L-Sel−/− LRLN to discern the DC phenotypes: mDCs, CD11c+, MHC class II+, CD11bhigh, Siglec-H, B220+/−; lymphoid DCs, CD11c+, MHC class II+, CD8+/−, Gr-1+, B220+; and pDCs, CD11c+, MHC class II+, B220high, Gr-1+, Siglec-H+, CD11b+/−. The depicted data are representative from at least 10 mice per group.

FIGURE 2.

In naive (B) or in (D) Ad2 vector-dosed L-Sel−/− LRLNs, the CD11clowCD8+ DCs are reduced when compared with (A and C) L-Sel+/+ mice. FACS analysis was performed on (A and C) L-Sel+/+ and (B and D) L-Sel−/− LRLN CD11clowMHC class II+ (TCRβ) DCs to discern CD8 (dashed lines) and CD8+ (solid lines) DC subsets. The majority of the (B and D) L-Sel−/− LRLN DCs were CD11clowCD8 when compared with (B and D) L-Sel+/+ LRLN DCs regardless of instillation with (C and D) Ad2 vector. Cell surface analyses were performed on L-Sel+/+ and L-Sel−/− LRLN to discern the DC phenotypes: mDCs, CD11c+, MHC class II+, CD11bhigh, Siglec-H, B220+/−; lymphoid DCs, CD11c+, MHC class II+, CD8+/−, Gr-1+, B220+; and pDCs, CD11c+, MHC class II+, B220high, Gr-1+, Siglec-H+, CD11b+/−. The depicted data are representative from at least 10 mice per group.

Close modal
FIGURE 3.

Differences are found in L-Sel−/− lung DC subsets in naive mice following i.t. Ad2 vector instillation. FACS analysis of naive lung revealed that both (A) L-Sel+/+and (B) L-Sel−/− mice showed the presence of mDCs (CD11c+ MHC class IIhigh, CD11bhigh, B220+Siglec-H), cDCs (CD11chighMHC class II+), lymphoid DCs (CD11c+MHC class II+CD11b+B220+), and pDCs (CD11c+MHC class II+CD11b+B220highSiglec-H+). (C and D) By 12 days post-i.t. instillation with the Ad2 vector, less cDCs were present in (D) L-Sel−/− lungs (Table III). Positive (CD11c+B220highSiglec-H+) pDC histograms for splenic pDCs obtained from Ad2-dosed (C) L-Sel−/− and (D) L-Sel+/+ mice are depicted. The depicted data are representative of at least 10 mice per group.

FIGURE 3.

Differences are found in L-Sel−/− lung DC subsets in naive mice following i.t. Ad2 vector instillation. FACS analysis of naive lung revealed that both (A) L-Sel+/+and (B) L-Sel−/− mice showed the presence of mDCs (CD11c+ MHC class IIhigh, CD11bhigh, B220+Siglec-H), cDCs (CD11chighMHC class II+), lymphoid DCs (CD11c+MHC class II+CD11b+B220+), and pDCs (CD11c+MHC class II+CD11b+B220highSiglec-H+). (C and D) By 12 days post-i.t. instillation with the Ad2 vector, less cDCs were present in (D) L-Sel−/− lungs (Table III). Positive (CD11c+B220highSiglec-H+) pDC histograms for splenic pDCs obtained from Ad2-dosed (C) L-Sel−/− and (D) L-Sel+/+ mice are depicted. The depicted data are representative of at least 10 mice per group.

Close modal

Evaluation of naive L-Sel−/− and L-Sel+/+ LRLN DC subsets revealed that the CD11chigh cDCs were nearly nonexistent, and in fact, for both mice, nearly all the DCs were CD11clow (Fig. 2, A and B). Subsequent evaluations of these DC subsets revealed that the overall percentages of lymphoid, mDCs, and pDCs did not vary between naive L-Sel−/− and L-Sel+/+ LRLNs (Table I); however, of the lymphoid DCs, the CD8+ DC subset was diminished in L-Sel−/− LRLN (Fig. 2,B; Table II). The percentage of the CD11clowCD8+ DCs of the total CD11clow DCs in naive L-Sel−/− LRLN was 14.4%, which was reduced by nearly half when compared with L-Sel+/+ mice (Fig. 2,B; Table II).

Table I.

DC subsets in the LRLNs

DC SubsetaTreatmentbL-SelectincP1dP2e Naive vs Treatment
Ad2/βGal-2LPS−/−+/+−/− vs +/+−/−+/+
Lymphoid − − 72.4 ± 3.1 77.8 ± 2.6 NS − − 
 − 76.8 ± 1.9 81.2 ± 2.1 NS NS NS 
 75.8 ± 2.7 78.0 ± 1.1 NS NS NS 
Plasmacytoid − − 10.8 ± 2.3 11.8 ± 1.8 NS − − 
 − 17.1 ± 1.2 14.6 ± 1.5 NS 0.038 NS 
 15.3 ± 1.7 17.6 ± 2.3 NS NS NS 
Myeloid − − 26.8 ± 3.0 21.5 ± 2.5 NS − − 
 − 21.9 ± 2.0 17.2 ± 2.0 NS NS NS 
 23.6 ± 1.0 21.1 ± 1.1 NS NS NS 
DC SubsetaTreatmentbL-SelectincP1dP2e Naive vs Treatment
Ad2/βGal-2LPS−/−+/+−/− vs +/+−/−+/+
Lymphoid − − 72.4 ± 3.1 77.8 ± 2.6 NS − − 
 − 76.8 ± 1.9 81.2 ± 2.1 NS NS NS 
 75.8 ± 2.7 78.0 ± 1.1 NS NS NS 
Plasmacytoid − − 10.8 ± 2.3 11.8 ± 1.8 NS − − 
 − 17.1 ± 1.2 14.6 ± 1.5 NS 0.038 NS 
 15.3 ± 1.7 17.6 ± 2.3 NS NS NS 
Myeloid − − 26.8 ± 3.0 21.5 ± 2.5 NS − − 
 − 21.9 ± 2.0 17.2 ± 2.0 NS NS NS 
 23.6 ± 1.0 21.1 ± 1.1 NS NS NS 
a

Pulmonary DCs were gated on lymphocytes that were CD11clowTCRαβ; no CD11chigh cDCs were detected in the LNs.

b

DCs were derived from naive, Ad2/βGal-2-dosed, or Ad2/βGal-2 + LPS-dosed mice.

c

−/−, L-Selectin-deficient mice; +/+, B6 mice. Data presented as mean ± SEM.

d

Value of p between L-Sel−/− and L-Sel+/+ DC subsets (n = 10 mice/group).

e

Value of p between treatments within L-Sel−/− or L-Sel+/+ groups.

Table II.

Lymphoid DC subsets in the LRLN

DC SubsetaTreatmentbL-SelectincP1dP2e Naive vs Treatment
Ad2/βGal-2LPS−/−+/+−/− vs +/+−/−+/+
LRLN CD8+ − − 14.4 ± 1.6 28.4 ± 3.6 0.004 − − 
 − 16.7 ± 2.9 29.4 ± 3.1 0.006 NS NS 
 31.7 ± 4.7 36.9 ± 4.3 NS 0.003 NS 
LRLN CD8 − − 84.5 ± 1.6 71.0 ± 3.8 0.007 − − 
 − 81.6 ± 2.8 69.6 ± 3.1 0.009 NS NS 
 67.1 ± 5.0 61.8 ± 4.5 NS 0.004 NS 
DC SubsetaTreatmentbL-SelectincP1dP2e Naive vs Treatment
Ad2/βGal-2LPS−/−+/+−/− vs +/+−/−+/+
LRLN CD8+ − − 14.4 ± 1.6 28.4 ± 3.6 0.004 − − 
 − 16.7 ± 2.9 29.4 ± 3.1 0.006 NS NS 
 31.7 ± 4.7 36.9 ± 4.3 NS 0.003 NS 
LRLN CD8 − − 84.5 ± 1.6 71.0 ± 3.8 0.007 − − 
 − 81.6 ± 2.8 69.6 ± 3.1 0.009 NS NS 
 67.1 ± 5.0 61.8 ± 4.5 NS 0.004 NS 
a

Pulmonary DCs were gated on lymphocytes that were CD11clowTCRαβ.

b

DCs were derived from naive, Ad2/βGal-2-dosed, or Ad2/βGal-2 + LPS-dosed mice.

c

−/−, L-Selectin-deficient mice; +/+, B6 mice. Data presented as mean ± SEM.

d

Value of p between L-Sel−/− and L-Sel+/+ DC subsets (n = 10 mice/group).

e

Value of p between treatments within L-Sel−/− or L-Sel+/+ groups.

Twelve days after dosing with the Ad2/βGal-2 vector revealed that only a slight significant increase in pDCs (CD8Gr-1+Siglec-H+B220+) was observed in L-Sel−/− LRLNs (Table I), and the percentage of CD11c+CD8+ DCs (Gr-1+Siglec-HB220) in the L-Sel−/− or L-Sel+/+ LRLN remained unchanged from their respective naive percentages, leaving the L-Sel−/− CD11c+CD8+ DCs reduced (Fig. 2,D; Table II). Although instillation of L-Sel−/− mice with Ad2/βGal-2 vector did not result in the absence of a specific DC subset, the percentage of CD11c+CD8+ DCs remained less (p = 0.006) than in L-Sel+/+ LRLNs. Thus, the observed failure to induce Ad2- and βgal-specific CTLs may be due to the inability to mobilize L-Sel−/− CD11c+CD8+ allowing the accumulation of CD11c+CD8 DCs (p = 0.009; Table II). No changes in LRLN mDCs (CD11bhighGr-1+Siglec-HB220+) for either L-Sel−/− or L-Sel+/+ mice were observed following instillation with the Ad2 vector (Fig. 2, C and D, and Table I).

Examination of the naive lung DCs revealed minimal differences in the percentages of the various subsets, with the pDCs and cDCs being slightly less in L-Sel−/− mice than L-Sel+/+ mice (Fig. 3, A and B; Table III). By 12 days after i.t. delivery with the Ad2 vector, the lymphoid DCs did decrease in the L-Sel−/− lungs with a concomitant increase in cDCs and no significant change in mDCs (Fig. 3, C and D; Table III). A similar pattern was also observed in the L-Sel+/+ lungs, although more cDCs and less lymphoid DCs were obtained when compared with the L-Sel−/− mice. The pDCs in the L-Sel−/− lungs did increase by ∼70% when compared with naive lungs (p < 0.001), but the percentage of pDCs in these lungs was not significantly different from the percentage of pDCs from the Ad2 vector-dosed L-Sel+/+ lungs (Table III). There were minimal to no CD8+ DCs and these remained unchanged (data not shown).

Table III.

DC subsets in the lungs

DC SubsetaTreatmentbL-SelectincP1dP2e Naive vs Treatment
Ad2/βGal-2LPS−/−+/+−/− vs +/+−/−+/+
Lymphoid − − 57.6 ± 2.0 54.4 ± 4.4 NS − − 
 − 44.5 ± 3.7 33.9 ± 2.7 0.035 0.007 <0.001 
 53.5 ± 2.9 38.6 ± 2.3 <0.001 NS 0.004 
Plasmacytoid − − 10.2 ± 0.7 14.2 ± 0.8 0.001 − − 
 − 17.3 ± 1.5 17.1 ± 1.6 NS <0.001 NS 
 11.9 ± 1.4 14.5 ± 1.5 NS NS NS 
Myeloid − − 11.4 ± 0.9 14.3 ± 2.0 NS − − 
 − 17.5 ± 2.9 17.6 ± 3.8 NS NS NS 
 17.8 ± 1.3 23.4 ± 1.3 0.006 <0.001 <0.001 
Conventional − − 17.8 ± 1.0 24.0 ± 2.4 0.025 − − 
 − 33.8 ± 3.6 46.3 ± 3.9 0.029 <0.001 <0.001 
 19.5 ± 2.5 31.8 ± 3.0 0.004 NS NS 
DC SubsetaTreatmentbL-SelectincP1dP2e Naive vs Treatment
Ad2/βGal-2LPS−/−+/+−/− vs +/+−/−+/+
Lymphoid − − 57.6 ± 2.0 54.4 ± 4.4 NS − − 
 − 44.5 ± 3.7 33.9 ± 2.7 0.035 0.007 <0.001 
 53.5 ± 2.9 38.6 ± 2.3 <0.001 NS 0.004 
Plasmacytoid − − 10.2 ± 0.7 14.2 ± 0.8 0.001 − − 
 − 17.3 ± 1.5 17.1 ± 1.6 NS <0.001 NS 
 11.9 ± 1.4 14.5 ± 1.5 NS NS NS 
Myeloid − − 11.4 ± 0.9 14.3 ± 2.0 NS − − 
 − 17.5 ± 2.9 17.6 ± 3.8 NS NS NS 
 17.8 ± 1.3 23.4 ± 1.3 0.006 <0.001 <0.001 
Conventional − − 17.8 ± 1.0 24.0 ± 2.4 0.025 − − 
 − 33.8 ± 3.6 46.3 ± 3.9 0.029 <0.001 <0.001 
 19.5 ± 2.5 31.8 ± 3.0 0.004 NS NS 
a

Pulmonary DCs were gated on lymphocytes that were CD11clowTCRαβ.

b

DCs were derived from naive, Ad2/βGal-2-dosed, or Ad2/βGal-2 + LPS-dosed mice.

c

−/−, L-Selectin-deficient mice; +/+, B6 mice. Data presented as mean ± SEM.

d

Value of p between L-Sel−/− and L-Sel+/+ DC subsets (n = 10 mice/group).

e

Value of p between treatments within L-Sel−/− or L-Sel+/+ groups.

Because CD11c+CD8+ DCs were greatly reduced in L-Sel−/− LRLN, their addition should restore the cytolytic activity by L-Sel−/− CD8+ T cells. To test for this possibility, naive CD11c+CD8+ or CD11c+CD8 DCs were cell-sorted from L-Sel+/+ LRLN and cocultured for 5 days in the presence of Ag and cell-sorted CD8+ T cells obtained from L-Sel−/− mice given a single i.t. dose of Ad2/βGal-2 vector, after which CTL activity was assessed. The cultures containing the CD8+ DCs restored cytolytic activity, whereas those cocultured with CD8 DCs did not (Fig. 4,A). Such evidence suggested that Ad2-specific CTLs may be induced in L-Sel−/− mice, but the frequency of supportive CD11c+ CD8+ DCs was insufficient in these mice, leaving L-Sel−/− mice in an unresponsive state. To test this possibility, L-Sel+/+CD8+ T cells were cocultured without or with L-Sel−/− LRLN CD8 DCs, and after 5 days, CTL responses were measured. The L-Sel+/+ CTL responses were normal; however, the cultures containing the L-Sel−/− LRLN DCs were compromised (Fig. 4 A). Thus, this collective evidence shows that in the absence of L-selectin, LRLN CD11c+CD8 DCs are not supportive, whereas CD11c+CD8+ DCs support CTL responses.

FIGURE 4.

L-Sel−/− CTL activity is restored upon coculture with L-Sel+/+CD11c+CD8+ DCs. Cell-sorted (B) L-Sel−/− or L-Sel+/+ LRLN and HNLN CD8+ T cells from 12 days postinstillation with Ad2 vector were cocultured with (C) cell-sorted L-Sel+/+CD11c+CD8+ or L-Sel+/+ or L-Sel−/−CD11c+CD8 DCs for 5 days and were then (A) evaluated for CTL activity against Ad2-infected targets. L-Sel+/+CD11c+CD8+ DCs restored CTL activity to L-Sel−/− CD8+ T cells contrasting the inability of L-Sel+/+CD11c+CD8 DCs or L-Sel−/−CD11c+CD8 DCs to support L-Sel−/− CTLs. L-Sel+/+CD8+ T cells cocultured with total L-Sel+/+CD11c+ DCs showed CTL activity. Data are representative of four experiments.

FIGURE 4.

L-Sel−/− CTL activity is restored upon coculture with L-Sel+/+CD11c+CD8+ DCs. Cell-sorted (B) L-Sel−/− or L-Sel+/+ LRLN and HNLN CD8+ T cells from 12 days postinstillation with Ad2 vector were cocultured with (C) cell-sorted L-Sel+/+CD11c+CD8+ or L-Sel+/+ or L-Sel−/−CD11c+CD8 DCs for 5 days and were then (A) evaluated for CTL activity against Ad2-infected targets. L-Sel+/+CD11c+CD8+ DCs restored CTL activity to L-Sel−/− CD8+ T cells contrasting the inability of L-Sel+/+CD11c+CD8 DCs or L-Sel−/−CD11c+CD8 DCs to support L-Sel−/− CTLs. L-Sel+/+CD8+ T cells cocultured with total L-Sel+/+CD11c+ DCs showed CTL activity. Data are representative of four experiments.

Close modal

The observed inactivity by L-Sel−/− CD8+ T cells could be attributed to a lack of DC maturation. A kinetics analysis was performed to assess LRLN CD11clow DCs for costimulatory molecule expression of B7.1, B7.2, and CD40 (Fig. 5). Both L-Sel+/+ and L-Sel−/− CD8+ DCs showed enhanced expressions of B7.1 (p < 0.001) on day 3 post-i.t. instillation and no changes in CD40 levels at 3 or 12 days post-i.t. instillation (Fig. 5,C). There was a significant increase in the amount of B7.2 expression on L-Sel+/+ (p < 0.001), but not on L-Sel−/− CD8+ DCs 3 days post-i.t. instillation with the Ad2 vector (Fig. 5,B). Both L-Sel+/+ and L-Sel−/− CD8 DCs showed no changes in B7.1 or B7.2 levels, although the L-Sel+/+ LRLN showed more constitutive B7.2+ DCs (Fig. 5,E). A reduction in CD40 was also observed in L-Sel−/− LRLN CD8 DCs at 3 days post-i.t. instillation (Fig. 5 F). In the lung CD11clow DCs, even fewer changes were observed (data not shown). No differences in B7.1 or B7.2 were observed for either L-Sel+/+ and L-Sel−/− CD8+ or CD8 DCs. These findings collectively show the CD8 DCs are immature, even following i.t. dosing with the Ad2 vector, and the increased presence of CD8 DCs in L-Sel−/− LRLN may pre-empt a responsive phenotype.

FIGURE 5.

Reduced expression of costimulatory molecules by L-Sel−/− LRLN CD11c+ DCs. A kinetic analysis was performed to discern expression levels of costimulatory molecules (A and D) B7.1, (B and E) B7.2, or (C and F) CD40 following i.t. dosing with Ad2/βGal-2 vector. Evaluations were done by cell surface staining of LRLN CD11c+ (A–C) CD8+ and (D–F) CD8 DCs and analyzed by flow cytometry. Both L-Sel−/− and L-Sel+/+CD11c+CD8+ DCs showed increased B7.1 at 3 days post-i.t. instillation with the Ad2 vector. Only L-Sel+/+CD11c+CD8+ DCs showed increased expression of B7.2, and neither mice showed changes in CD40. For both L-Sel−/− and L-Sel+/+CD11c+CD8 DCs, no changes in B7.1 or B7.2 were observed as a consequence of Ad2 vector instillation; however, L-Sel−/−CD11c+CD8 DCs were significantly lower in B7.2 expression and on 3 days post-i.t. instillation were significantly reduced in B7.1 expression when compared with L-Sel+/+ mice. Data depict results from eight individual mice. Differences between L-Sel−/− and L-Sel+/+ CD11c+ DC: *, p ≤ 0.005; **, p = 0.018; ***, p = 0.044. Differences in costimulatory molecule expression within the L-Sel−/− or L-Sel+/+ CD11c+ DC subset: ¶, p ≤ 0.001; ¶¶¶, p = 0.03.

FIGURE 5.

Reduced expression of costimulatory molecules by L-Sel−/− LRLN CD11c+ DCs. A kinetic analysis was performed to discern expression levels of costimulatory molecules (A and D) B7.1, (B and E) B7.2, or (C and F) CD40 following i.t. dosing with Ad2/βGal-2 vector. Evaluations were done by cell surface staining of LRLN CD11c+ (A–C) CD8+ and (D–F) CD8 DCs and analyzed by flow cytometry. Both L-Sel−/− and L-Sel+/+CD11c+CD8+ DCs showed increased B7.1 at 3 days post-i.t. instillation with the Ad2 vector. Only L-Sel+/+CD11c+CD8+ DCs showed increased expression of B7.2, and neither mice showed changes in CD40. For both L-Sel−/− and L-Sel+/+CD11c+CD8 DCs, no changes in B7.1 or B7.2 were observed as a consequence of Ad2 vector instillation; however, L-Sel−/−CD11c+CD8 DCs were significantly lower in B7.2 expression and on 3 days post-i.t. instillation were significantly reduced in B7.1 expression when compared with L-Sel+/+ mice. Data depict results from eight individual mice. Differences between L-Sel−/− and L-Sel+/+ CD11c+ DC: *, p ≤ 0.005; **, p = 0.018; ***, p = 0.044. Differences in costimulatory molecule expression within the L-Sel−/− or L-Sel+/+ CD11c+ DC subset: ¶, p ≤ 0.001; ¶¶¶, p = 0.03.

Close modal

Because CD8+ DCs were absent from the lungs at the time points examined (Fig. 3, C and D), the lack of lung CTL activity in L-Sel−/− mice is suggestive that environment or DC content of L-Sel−/− lungs contributes to the observed unresponsiveness. To test this possibility, transgenic OT-I CD8+ T cells specific for OVA257–264 were isolated and adoptively transferred into L-Sel+/+ or L-Sel−/− mice, and 1 day later, challenged i.t. with 1 × 109 PFU of Ad5-OVA. Total lung lymphocytes were harvested from their lungs and evaluated by flow cytometry for CD8+ T cells capable of binding soluble MHC class I loaded with OVA257–264 peptide (pentamer+) T cells. In the first experiment, no differences in the percentage of pentamer+CD8+ T cells were obtained in L-Sel−/− or in L-Sel+/+ recipient lungs 2 or 3 days postchallenge with Ad5-OVA vector (Table IV). In contrast, the L-Sel−/− lungs remained unresponsive despite receiving transgenic OT-1 T cells, as evidence by the lack of lytic activity against E.G7-OVA target cells for either 2 or 3 days postchallenge (Table IV). OT-I T cells adoptively transferred into L-Sel+/+ mice remained responsive and capable of lysing E.G7-OVA target cells (Table IV). When this experiment was repeated, similar results were obtained in that 2 days after adoptive transfer of OT-1 into L-Sel−/− or in L-Sel+/+ recipients. No difference in pentamer+CD8+ T cells were observed, but after 3 days, less pentamer+CD8+ T cells were observed in the L-Sel+/+ lungs, yet CTL activity only associated with L-Sel+/+, not L-Sel−/− lungs (Table IV). Thus, the lack of activity in L-Sel−/− mice appears to be attributed to differences between the lung DCs.

Table IV.

Transgenic OT-1 CD8+ T cells remain unresponsive in Ad5-OVA-dosed L-Sel−/− lungs

OT-1→Recipient TissueaDay Testedb% Pentamer+CD8+ T Cellsbp ValuecE:Td% Lysisdp Valuee
Experiment 1       
 L-Sel+/+ lungs 24.5 ± 10.1 NS 25:1 15.7 ± 4.3 0.005 
    50:1 36.9 ± 2.3 <0.05 
 L-Sel−/− lungs 21.1 ± 2.7  25.1 −1.1 ± 2.6  
    50:1 8.8 ± 2.9  
 L-Sel+/+ lungs 15.2 ± 4.1 NS 25:1 38.0 ± 8.3 <0.05 
    50:1 66.1 ± 10.2 0.005 
 L-Sel−/− lungs 13.0 ± 0.3  25:1 6.5 ± 1.8  
    50:1 8.5 ± 1.6  
Experiment 2       
 L-Sel+/+ lungs 8.5 ± 0.9 NS 25:1 11.8 ± 3.7 NS 
    50:1 20.0 ± 4.3 NS 
 L-Sel−/− lungs 9.0 ± 2.1  25:1 5.5 ± 3.2  
    50:1 11.8 ± 4.0  
 L-Sel+/+ lungs 4.6 ± 0.4 0.040 25:1 17.0 ± 3.8 0.007 
    50:1 24.0 ± 1.4 <0.001 
 L-Sel−/− lungs 8.4 ± 1.2  25:1 2.1 ± 3.4  
    50:1 5.9 ± 3.3  
OT-1→Recipient TissueaDay Testedb% Pentamer+CD8+ T Cellsbp ValuecE:Td% Lysisdp Valuee
Experiment 1       
 L-Sel+/+ lungs 24.5 ± 10.1 NS 25:1 15.7 ± 4.3 0.005 
    50:1 36.9 ± 2.3 <0.05 
 L-Sel−/− lungs 21.1 ± 2.7  25.1 −1.1 ± 2.6  
    50:1 8.8 ± 2.9  
 L-Sel+/+ lungs 15.2 ± 4.1 NS 25:1 38.0 ± 8.3 <0.05 
    50:1 66.1 ± 10.2 0.005 
 L-Sel−/− lungs 13.0 ± 0.3  25:1 6.5 ± 1.8  
    50:1 8.5 ± 1.6  
Experiment 2       
 L-Sel+/+ lungs 8.5 ± 0.9 NS 25:1 11.8 ± 3.7 NS 
    50:1 20.0 ± 4.3 NS 
 L-Sel−/− lungs 9.0 ± 2.1  25:1 5.5 ± 3.2  
    50:1 11.8 ± 4.0  
 L-Sel+/+ lungs 4.6 ± 0.4 0.040 25:1 17.0 ± 3.8 0.007 
    50:1 24.0 ± 1.4 <0.001 
 L-Sel−/− lungs 8.4 ± 1.2  25:1 2.1 ± 3.4  
    50:1 5.9 ± 3.3  
a

Transgenic OT-1 T cells were purified to >95% CD8+ T cells, and 5 × 106 cells were injected i.v. into L-Sel−/− or L-Sel+/+ mice and challenged 24 h later with 1 × 109 Ad5-OVA. Recipient lungs were harvested 2 or 3 days after challenge.

b

Total lymphocytes were harvested from lungs and stained with OVA257–264 peptide in soluble MHC class I pentamer and with anti-CD8 mAb. Percent pentamer+CD8+ T cells ± SEM were determined by flow cytometry.

c

Value of p between the percentage of pentamer+CD8+ T cells in L-Sel+/+ and L-Sel−/− lungs.

d

CTL experiments were performed using freshly isolated L-Sel+/+ or L-Sel−/− lymphocytes as effector cells measuring their lytic activity against E.G7-OVA target cells (percent lysis) at the indicated E:T cell ratios.

e

Value of p for OVA-specific cytolysis between L-Sel+/+ and L-Sel−/− lung lymphocytes.

To test whether microbial activation by LPS would enable stimulation of functional CTLs, L-Sel−/− mice were i.t. dosed with 0.1, 10, or 100 μg of LPS either 3 days prior or 2 days after i.t. instillation with Ad2/βGal-2 vector, and CTL responses were measured 12 days later (Fig. 6, AF). The lower dose of LPS, 0.1 μg, had no effect upon stimulating Ad2-specific CTLs (Fig. 6,A). The 10-μg dose showed an impact (Fig. 6,B), but clearly the 100-μg dose was effective in priming respiratory APC to become responsive (Fig. 6,C). This priming event had to occur before exposure to the Ad2 vector because when the LPS was administered 2 days after i.t. Ad2 vector instillation, the absence of a CTL response remained (Fig. 6, D–F). Subsequent experiments showed that LPS priming enhanced CTL responsiveness to βgal targets as well (Fig. 6, H, J, L, and N). Although not necessary for L-Sel+/+ mice, the cytolytic activity was unchanged by the LPS activation (Fig. 6, G–N). Furthermore, in vitro Ag restimulation with either Ad2 or βgal markedly improved cytolytic activity by L-Sel−/− respiratory effector cells (Fig. 6, G and H). Such studies suggest that activation with the microbial product, LPS, overcomes the unresponsive state by respiratory APC to Ad2/βGal-2.

FIGURE 6.

In vivo microbial stimulation before i.t. instillation with Ad2 vector promotes Ad2-specific CTLs in L-Sel−/− mice because of restored CD8+ DCs in the LRLN. L-Sel−/− mice were given a single i.t. dose of (A and D) 0.1, (B and E) 10, or (C and F) 100 μg of LPS (A–C) 3 days before or (D–F) 2 days after i.t. instillation with Ad2/βGal-2 vector. Lung and splenic CTL ex vivo analysis was conducted on day 12. LPS exposure before Ad2 vector delivery restored Ad2-specific CTL activity in L-Sel−/− mice, as opposed to exposure subsequent to Ad2 vector administration, which had minimal impact even at the 100-μg dose. Reactivity against mock-infected cells was not detected. Data are representative of three experiments. G–N, Microbial activation restores CTL activity in L-Sel−/− mice similarly with those obtained in L-Sel+/+ mice. CTL responses were measured against (G, I, K, and M) Ad2-infected targets or (H, J, L, and N) βgal-expressing E22 cells. Mice were i.t. dosed 3 days with 100 μg of LPS before i.t. instillation with Ad2/βGal-2 vector, and (G–J) ex vivo lung and splenic and (K–N) in vitro Ag-restimulated LRLN and splenic CTL responses were measured. LPS had a minimal impact upon L-Sel+/+ CTLs in contrast to L-Sel−/− CTLs, which were restored to or near to L-Sel+/+ cytolytic activities. Results depict one representative experiment of three.

FIGURE 6.

In vivo microbial stimulation before i.t. instillation with Ad2 vector promotes Ad2-specific CTLs in L-Sel−/− mice because of restored CD8+ DCs in the LRLN. L-Sel−/− mice were given a single i.t. dose of (A and D) 0.1, (B and E) 10, or (C and F) 100 μg of LPS (A–C) 3 days before or (D–F) 2 days after i.t. instillation with Ad2/βGal-2 vector. Lung and splenic CTL ex vivo analysis was conducted on day 12. LPS exposure before Ad2 vector delivery restored Ad2-specific CTL activity in L-Sel−/− mice, as opposed to exposure subsequent to Ad2 vector administration, which had minimal impact even at the 100-μg dose. Reactivity against mock-infected cells was not detected. Data are representative of three experiments. G–N, Microbial activation restores CTL activity in L-Sel−/− mice similarly with those obtained in L-Sel+/+ mice. CTL responses were measured against (G, I, K, and M) Ad2-infected targets or (H, J, L, and N) βgal-expressing E22 cells. Mice were i.t. dosed 3 days with 100 μg of LPS before i.t. instillation with Ad2/βGal-2 vector, and (G–J) ex vivo lung and splenic and (K–N) in vitro Ag-restimulated LRLN and splenic CTL responses were measured. LPS had a minimal impact upon L-Sel+/+ CTLs in contrast to L-Sel−/− CTLs, which were restored to or near to L-Sel+/+ cytolytic activities. Results depict one representative experiment of three.

Close modal

To determine the impact of LPS upon the L-Sel−/− DC subsets, L-Sel−/− and L-Sel+/+ mice were dosed with 100 μg of LPS 3 days before Ad2/βGal-2 instillation. Evaluation of LRLN DCs 12 days post-i.t. instillation revealed an increase in the percentage of lymphoid CD8+ DCs in L-Sel−/− LRLNs to 31.7 ± 4.7, which was equivalent to that found in L-Sel+/+ LRLNs, 36.9 ± 4.3 (Table II). Stimulation with LPS and Ad2/βGal-2 more than doubled the percentage of lymphoid CD8+ DCs in the L-Sel−/− LRLNs (p = 0.003) with no changes in cell surface expression (data not shown) from mice dosed with only Ad2 vector (Fig. 2). In contrast, this same LPS stimulation did not impact the percentage of CD8+ DCs in the L-Sel+/+ LRLN (Table II). These increases in the CD11c+CD8+ DCs suggest that the inability to induce or expand this DC subset in L-Sel−/− mice may account for the observed immune unresponsiveness after a single i.t. dose of Ad2 vector, whereas in L-Sel competent mice, increases in CD11c+CD8+ DCs were achieved. No significant differences in the percentages of pDCs or mDCs were observed as a consequence of in vivo LPS treatment nor were there differences between L-Sel−/− and L-Sel+/+ LRLNs following LPS treatment (Table I).

Examination of L-Sel−/− and L-Sel+/+ lung DCs revealed that the LPS treatment restored lymphoid, pDCs, and cDCs to nearly the same levels as with naive lungs, each of which were less than mice treated with the Ad2 vector only (Table III). The mDCs increased in both L-Sel−/− and L-Sel+/+ lungs (p < 0.001).

To assess whether LPS could induce conversion of L-Sel−/−CD8 DCs to CD8+ DCs, naive L-Sel+/+ and L-Sel−/− lung and LRLN CD8+ and CD8 DCs were isolated. CD8+ DCs were green labeled with CFDA; CD8 DCs were red labeled with CM-DiI. DC subsets were cocultured and treated with LPS for 24 h before infection with Ad2/βGal-2 vector. Treatment of L-Sel−/− lung CD8 DCs with Ad2/βGal-2 or LPS only or both resulted in the conversion into CD8+ DCs with nearly a 4-fold increase in CM-DiI CD8+ DCs (Fig. 7,A), whereas only a 1.7-fold increase was obtained for L-Sel+/+ lung DCs (Fig. 7,B). Concomitant reductions in lung CM-DiI-labeled CD8 DCs were observed for both L-Sel−/− and L-Sel+/+ lung CD8 DCs. This increase in lung CD8+ DCs was not attributed to increased proliferation of endogenous CD8+ DCs because the percentage of CFDA-labeled CD8+ DCs did not significantly change as a result of treatments, although a slight conversion into CD8 DCs was observed more so for the L-Sel−/− lung DCs (Fig. 7,A) than the L-Sel+/+ lung DCs (Fig. 7,B). Neither the L-Sel−/− nor L-Sel+/+ LRLN CD8 DCs showed conversion into CD8+ DCs (Fig. 7, C and D). Thus, these data suggest that treatment with LPS can cause increased numbers of lung CD8+ DCs, which then can migrate to the draining LN.

FIGURE 7.

In vitro microbial activation of lung DCs results in the expansion of CD8+ DCs derived from CD8 DCs. A and B, Naive lung and, C and D, LRLN CD8+ and CD8 DCs were isolated from (A and C) L-Sel−/− and (B and D) L-Sel+/+ mice. CD8+ DCs were labeled with CFDA (green open circles; residual CD8 DCs are depicted as solid green circles), and CD8 DCs were labeled with CM-DiI (red open circles; residual CD8+ DCs are depicted as solid red circles). Media treatments represent baseline for each lung and LRLN population. DCs were stimulated in vitro without or with LPS for 24 h followed by an additional stimulation with Ad2/βGal-2 for 24 h. DCs were harvested and immunostained for CD8 expression by CD11c+MHC class II+TCRβ DCs. Increases in (A) new L-Sel−/− lung CD8+ DCs were observed by the conversion of CD8 (open red) DCs into CD8+ (solid red) DCs upon microbial stimulation, and not by expansion of CD8+ (open green) DCs. C and D, LRLN DCs showed no conversion of CD8 (open red) DCs into CD8+ (solid red) DCs. Depicted are the combined results from two experiments.

FIGURE 7.

In vitro microbial activation of lung DCs results in the expansion of CD8+ DCs derived from CD8 DCs. A and B, Naive lung and, C and D, LRLN CD8+ and CD8 DCs were isolated from (A and C) L-Sel−/− and (B and D) L-Sel+/+ mice. CD8+ DCs were labeled with CFDA (green open circles; residual CD8 DCs are depicted as solid green circles), and CD8 DCs were labeled with CM-DiI (red open circles; residual CD8+ DCs are depicted as solid red circles). Media treatments represent baseline for each lung and LRLN population. DCs were stimulated in vitro without or with LPS for 24 h followed by an additional stimulation with Ad2/βGal-2 for 24 h. DCs were harvested and immunostained for CD8 expression by CD11c+MHC class II+TCRβ DCs. Increases in (A) new L-Sel−/− lung CD8+ DCs were observed by the conversion of CD8 (open red) DCs into CD8+ (solid red) DCs upon microbial stimulation, and not by expansion of CD8+ (open green) DCs. C and D, LRLN DCs showed no conversion of CD8 (open red) DCs into CD8+ (solid red) DCs. Depicted are the combined results from two experiments.

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In this study, we describe the importance of L-selectin in the development of lymphoid CD8+ DCs to support CTL responses. Expression of CD8 has been shown to be associated with lymphoid DCs (28). L-Sel−/− mice i.t. dosed with a nonreplicating Ad2 vector failed to stimulate CTL responses, either to the Ad2 vector or its βgal transgene. Failure to display such cytolytic activities was not linked to a delay in responding because L-Sel−/− mice remained unresponsive even after 6 wk from initial i.t. dosing. By contrast, the observed CTL responses by the L-Sel+/+ mice were consistent with previous studies (24, 26). To probe the reason for such attenuation of the Ad2- and βgal-specific CTLs, we hypothesized that the supportive DCs in L-Sel−/− mice may be compromised. In an attempt to obtain a correlative phenotype to explain this lack of CTL reactivity, FACS analysis was performed on both L-Sel−/− and L-Sel+/+ respiratory DCs. In the LRLNs, L-Sel−/− mice shared similar phenotypes and percentages of the different DC subsets with L-Sel+/+ mice. The majority of the DCs in both mice were the lymphoid DCs with some pDCs and mDCs, but no cDCs; however, a notable reduction in the percentage of lymphoid CD8+ DCs was apparent. Ad2-immunized L-Sel−/− mice failed to elicit the CD8+ DC subset in the LRLN, unlike that observed with the L-Sel+/+ mice in which LRLN CTL responses to both Ad2 and βgal were induced with significantly more CD8+ DCs, suggesting that these supported the observed CTL responses. In the lungs, the most notable changes were observed in the percentages of DC subsets following i.t. delivery of the Ad2 vector. Although both L-Sel−/− and L-Sel+/+ mice showed a decrease in the percentage of lymphoid DCs with concomitant increases in cDCs, an ∼70% increase in pDCs, and no changes in mDCs in the L-Sel−/− lungs. The cDCs in the L-Sel+/+ lungs nearly doubled. The lymphoid CD8+ DCs were minimally detectable in the L-Sel−/− lungs, and these mice remained unable to support CTL responses using either Ad2 or Ad5 vectors. It may be that this combination of differences in DC subsets, e.g., more pDCs, and possibly differences in magnitude of changes in cDCs and lymphoid DCs could account for L-Sel−/− mouse unresponsiveness in the lungs. This was further evidenced when even the adoptive transfer of immunocompetent transgenic (L-Sel+/+) OT-1 CD8 T cells into L-Sel−/− mice was still unable to support OVA-specific CTL responses following i.t. challenge with the Ad5-OVA. Thus, using three different Ags, it is clear that L-Sel−/− mice are unresponsive in their respiratory CD8 T cell responses.

The absence of CD8+ DCs in the L-Sel−/− LRLN contributed to the observed impairment in CD8+ T cell responses. Consequently, there was an increased presence of CD8 or immature DCs because these failed to show increased costimulatory molecule expression following i.t. instillation with Ad2/βGal-2 vector, unlike that observed with C57BL/6 LRLN CD8+ or CD8 DCs. The cell-sorting experiments showed that the lymphoid CD8+ DCs were responsible for Ag presentation to CD8+ T cells. Neither the L-Sel+/+ nor the L-Sel−/−CD8 DCs could support cell-sorted CD8+ CTL responses; only the L-Sel+/+ CD8+ DCs were able to support such responses. The L-Sel−/− mice could produce CTL responses, but only when virus was presented by L-Sel+/+ CD8+ DCs. Because the L-Sel−/− mice lacked mature DCs in their LRLNs, the absence of CTL responses manifests as unresponsiveness to Ad2 vectors when in fact the L-Sel−/− mice are responsive to Ad2, but require the presence of CD8+ DCs. This defect in lymphoid DCs was not compensated by mDCs because these remained unchanged in either L-Sel+/+ or L-Sel−/− LRLNs. The CD11chigh (cDC) subset was absent from both L-Sel+/+ and L-Sel−/− LRLNs. Thus, these collective findings suggest that the lack of mature lymphoid CD8+ DCs is the major defect in the L-Sel−/− LRLNs.

It is not surprising that fewer DCs were detected in the L-Sel−/− LRLN because naive LN in these mice tend to be smaller (17, 35), but their ability to induce immunity still remains intact. L-Sel−/− mice have been shown to be responsive to immunization, although dependent upon the type of Ag and its route of administration. No significant differences in their Ab responses to keyhole limpet hemocyanin were observed in L-Sel−/− mice when compared with similarly immunized C57BL/6 (L-Sel+/+) mice (16). Serum Ab responses to T cell-independent type 2 Ag were greatly enhanced when administered i.p. rather than s.c. (35). Oral immunization of L-Sel−/− mice showed that systemic Ab responses to passenger fimbriae were retained, albeit, with a more proinflammatory bias than in L-Sel+/+ mice (19). Because of this redirection of Th cell responses, the S-IgA responses were severely attenuated. Similarly, secretory-IgA responses were compromised in mucosal secretions when L-Sel−/− mice were nasally immunized with cholera toxin (7, 8).

Recent studies suggest that DCs are responsible for priming viral (33, 36) or bacterial CTL responses (37) by CD8+ DCs (38, 39). The rapid migration of respiratory DCs to the pulmonary draining LN after an influenza infection suggests that lung DCs may act a source of viral Ags for the regional LN (33). Thus, an inability to migrate from the site of infection to LN could possibly explain the relevance of functional L-selectin expression by DCs (36). The evidence shows that L-Sel−/− LRLNs have a reduced percentage of CD8+ DCs by 43% following Ad2/βGal-2 delivery when compared with L-Sel+/+ LRLNs; thus, the possibility that lung DC migration to the LRLNs is compromised cannot be excluded. Accumulation of DCs in regional LNs has been observed following upper respiratory infection with influenza virus (33) or footpad infection with mouse mammary tumor virus (40). However, the presence of CD8+ DCs was difficult to identify in the lungs from either L-Sel−/− or L-Sel+/+ mice. Adoptive transfer of L-Sel−/− DCs in L-Sel−/− or L-Sel+/+ mice showed migration to the recipients’ lungs and LRLNs can occur (data not shown), suggesting that the adoptive transfer may bypass the in vivo block in migration. Instead, the defect in L-Sel−/− mice appeared to be contributed by the lack of DC maturation. The L-Sel−/− LRLN DCs showed reduced expression of costimulatory molecules, and only upon microbial activation was there an increase in CD8+ DCs. The in vitro-labeling studies revealed that the lung CD8 DCs served as the source of mature DCs, and these DCs matured upon LPS stimulation to become CD8+ DCs. The Ad2 vector only had a modest impact upon this conversion and required microbial activation to enhance increases in L-Sel−/−CD8+ DCs. This conversion did not appear to take place in the LRLN because no changes were detected with the LRLN DC cultures. Consequently, being unable to mature upon infection with the Ad2 vector, the L-Sel−/−CD8 DCs are unable to support CTL responses. Functionally, the CD8+ DCs are important because subsequent cell sorting experiments showed that these were necessary for supporting Ag-specific CTL responses, and only the CD8+ DCs supported both L-Sel+/+ and L-Sel−/−CD8+ anti-Ad2 CTL responses. Even the sorted L-Sel+/+ CD8 DCs failed to support L-Sel−/−CD8+ T cell cytolytic activity. Fully functional L-Sel+/+ CD8+ T cells were also not supported by L-Sel−/−CD8 DCs, suggesting that these CTLs were unresponsive, which is consistent with CD8 DCs being tolerogenic (41, 42).

Immature DCs have been shown to be important for tolerance induction, resulting in the induction of regulatory T cell subset, whereas mature DCs support Ag-reactive responses (43). This maturation appears to be IFN-α-dependent, as previously shown, when chimpanzee and human Ad vectors were used to infect DCs directly, albeit at very high doses (44). However, we were unable to detect IFN-α secretion by any of these DC subsets.

In summary, in the absence of L-selectin, a defect resulted in the failure to stimulate productive CTL responses attributed to a lack of mature DCs in the LRLN. Microbial activation could reverse this effect, enhancing the numbers of CD8+ DCs in the LRLN. The cell isolation studies revealed that CTLs could be induced in L-Sel−/− mice, but these required the presence of lymphoid CD8+ DCs. Addition of the lymphoid CD8+ DC subset restored cytolytic activity, consistent with the notion that L-Sel−/− DCs have Ag-presenting capabilities (18). Microbial activation increased lung CD8+ DC conversion from CD8 DCs, or possibly due to increased recruitment in vivo, but this additional stimulus was required to obtain sufficient mature CD8+ DCs.

We thank Drs. Johanne M. Kaplan and Kathleen Heir of Genzyme for providing us with the Ad2/βGal-2 vector stocks used in this study, the University of Iowa Gene Transfer Vector Core for providing the Ad5 CMV Trf-OVA vector, Larrisa Jackiw for the cell-sorting experiments, and Nancy Kommers for assistance in preparing this manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Public Health Service Grant AI-55563 and in part by Montana Agricultural Station and U.S. Department of Agriculture Formula Funds. The Veterinary Molecular Biology Flow Cytometry Facility was in part supported by National Institutes of Health/National Center for Research Resources, Centers of Biomedical Excellence P20 RR-020185, and in part from a grant from M. J. Murdock Charitable Trust.

3

Abbreviations used in this paper: LN, lymph node; HNLN, head and neck LN; PNAd, peripheral node addressin; DC, dendritic cell; LRLN, lower respiratory LN; Ad2, adenovirus 2; i.t., intratracheal; CM, complete medium; βgal, β-galactosidase; CFDA, carboxy-fluorescein diacetate succinimidyl ester; DPBS, Dulbecco’s PBS; pDC, plasmacytoid DC; mDC, myeloid DC; cDC, conventional DC.

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