PMC

iNKT cells that chemotaxed toward CCL17 were enriched in the lungs. Whole-cell suspensions from the lungs (a), spleen (b), or liver (c) of naive mice were allowed to migrate in Transwell inserts (5-μm pores) for 1.5 h to assess chemotaxis in response to medium, CCL17, or CXCL12 (as a positive chemotactic control). iNKT were identified by staining with α-GalCer-loaded CD1d:Ig dimer and anti-TCRβ mz Ab. Data are expressed as the fraction of the input iNKT cell population which migrate, with the mean ± SD reported for three pooled experiments. Statistical analysis is shown as the pairwise comparison of percent migrant values of CCL17 or CXCL12 vs medium control using the Mann-Whitney U test with p ≤ 0.05 (*) and p ≤ 0.01 (**).

Although CCR4^−/− mice showed reduced cytokine production in response to α-GalCer challenge in vivo, purified iNKT cells from CCR4^−/− mice produced more IL-4 than wild-type iNKT cells when activated in vitro, but did not reconstitute AHR when transferred into glycolipid-challenged Jα18^−/− mice. Statistical analysis is shown as the results of the pairwise Student t test between groups with p ≤ 0.05 (*) and p ≤ 0.01 (**). a, Either WT (solid line) or CCR4^−/− (dashed lines) were given α-GalCer i.v. at time 0. Serum was collected over 48 h and analyzed for concentrations of IL-4 (upper panel) and IFN-γ (lower panel) by ELISA. b, iNKT cells from CCR4^−/− mice produced both IL-4 and IFN-γ. Splenocytes were stained with CD1d tetramers (or CD1d:Ig dimers), magnetically enriched, and then purified by FACS, resulting in >95% purity. Sorted iNKT cells from CCR4^−/− or WT mice were cultured for 24 h with α-GalCer (50 ng/ml) with or without blocking anti-CD1d mAb. Supernatants were analyzed for cytokine content by ELISA. Data are representative of three separate experiments shown as mean ± SEM of four replicates. c, iNKT cells from CCR4^−/− mice failed to reconstitute the development of α-GalCer-induced AHR in Jα18^−/− mice. Sorted iNKT cells from CCR4^−/− or WT mice (1 × 10⁶ cells/recipient) were obtained by selection over magnetic columns to > 70% purity and transferred into Jα18^−/− recipients 30 min before i.n. challenge with α-GalCer. AHR was measured (as in ) 24 h later. Data are the mean ± SEM of enhanced pause (Penh) values from three to four mice per group representative of three experiments.

CCL17 is required for α-GalCer-induced AHR. Statistical analysis is shown as the results of the pairwise Student t test between α-GalCer experimental and vehicle control groups with p ≤ 0.05 (*) and p ≤ 0.01 (**). a, CCL17 and CCL22 expression in the lungs increased significantly 6 and 24 h after i.n. challenge with α-GalCer (2 μg). Lung lysates were assayed for CCL17 and CCL22 by ELISA. Data are representative of three experiments with at least four mice per group. b, Neutralization of CCL22 reduced and neutralization of CCL17 blocked the development of α-GalCer-induced AHR. Anti-CCL17, anti-CCL22, or isotype control mAbs (100 μg) were administered 1 day before i.n. challenge with α-GalCer (1.5 μg). AHR was assessed as in at 24 h after i.n. challenge. Data are the enhanced pause (Penh) values (mean ± SEM) from three to five mice per group representative of three experiments. c, Neutralization of CCL17 and CCL22 significantly reduced α-GalCer-induced airway eosinophilia. BAL fluid from the mice in b was analyzed at 3 h after airway measurements. Results of one representative experiment are shown as the mean ± SEM number of cells per milliliter of BAL fluid, TCC, Total cell count; MO, monocytes; EOS, eosinophils; LYM, lymphocytes; NEU, neutrophils. d, Neutralization of CCL17 and CCL22 significantly reduced the number of iNKT cells in the BAL fluid in mice challenged with α-GalCer. Individual BAL fluid collected from mice in (as in c) was stained for iNKT cells using anti-TCRβ mAb and α-GalCer-loaded CD1d tetramer, gated lymphocytes FSC and SSC. Absolute number of BAL iNKT was calculated (see Materials and Methods) and presented as mean ± SD.

Competitive localization of WT vs CCR4^−/− iNKT and conventional T cells in allergen-sensitized BM chimeric mice. Irradiated host mice were reconstituted with CD45.1⁺ WT and CD45.2⁺ CCR4^−/− BM cells (WT: CCR4^−/− chimera) or with CD45.1⁺ WT and CD45.2⁺ WT BM cells (WT:WT chimera). The mice were sensitized to and challenged with OVA as described in Materials and Methods. a, Upper panel, Analysis of CCR4^−/− BM-derived iNKT cell competitive homing disadvantage D in mice at 24 h after final i.n. challenge with OVA. See Materials and Methods for calculation of disadvantage D. The relative disadvantage of CCR4^−/− BM iNKT cells was 2-fold in allergen-sensitized and -challenged mice in the BAL fluid compartment but not other tissue as calculated by flow cytometric assessment of CD45.1 and CD45.2⁺ cells in CCR4^−/−:WT chimeras, compared with CD45.1⁺ and CD45.2⁺ WT cells in control WT:WT chimeras and represent the mean ± SD of pairwise comparisons normalized to engraftment ratios for four chimeric pairs. Statistical analysis shown as pairwise comparison of the D values derived from the BAL fluid or lungs to that of blood using the Mann-Whitney U test (p < 0.05 (*) and p ≤ 0.01 (**)). Lower panel, Conventional T cells derived from CCR4^−/− BM are disadvantaged only in the BAL fluid of CCR4^−/−:WT resting chimeric mice as determined by flow cytometry and identification of anti-TCRβ mAb-positive cells excluding CD1d tetramer⁺, represents the mean ± SD of pairwise comparisons (four pairs). b, CCR4 influences the BAL/airway compartment size of iNKT but not conventional T cells. The relative compartment size is the ratio of the absolute number of iNKT cells in the indicated compartment of CCR4^−/−:WT chimeras divided by that of WT:WT chimeras. Upper panel, Allergen-challenged CCR4^−/−:WT chimeras have three-fifths the total iNKT cells in the BAL fluid/airways but not blood or lung tissue when compared with WT:WT chimeras. Lower panel, In contrast, CCR4^−/−:WT chimeras had slightly reduced numbers of total conventional T cells in the BAL fluid/airways, lung tissue, and blood when compared with WT:WT chimeras. Data are presented as mean ± SEM ratios for pairwise comparisons (four pairs). Statistical analysis shown as pairwise comparison of the relative compartment size values for the BAL fluid or lungs to that of blood using the Mann-Whitney U test (p < 0.05 (*) and p ≤ 0.01 (**)).

AHR induced with α-GalCer failed to occur in CCR4^−/− mice. Statistical analysis is shown as the results of the pairwise Student t test between α-GalCer experimental and vehicle control groups with p ≤ 0.05 (*) and p ≤ 0.01 (**). a, Significant AHR assessed at 24 h following i.n. challenge with α-GalCer (2.0 μg/mouse) in WT mice did not occur in CCR4^−/− mice. Data are the enhanced pause (Penh) values (mean ± SEM) of at least four mice per group, representative of four experiments. b, Invasive measurement of airway resistance confirmed CCR4^−/− mice failed to develop AHR following α-GalCer at 24 h after 1.5 μg of i.n. challenge of α-GalCer, shown as dynamic compliance C_dyne (left, in ml per cm H₂O) and airway resistance R_L (right, in cm H₂O per ml per s). Data represent the mean ± SEM from two experiments (n = 3–4). c, Airway eosinophilia in response to α-GalCer failed to occur in CCR4^−/− mice. BAL fluid from the mice in a was analyzed 3 h after airway measurements. One representative experiment of four is shown as mean ± SEM of the number of cells per milliliter of BAL fluid. TCC, Total cell count; MO, monocytes; EOS, eosinophils; LYM, lymphocytes; NEU, neutrophils. d, iNKT cells in the BAL fluid of CCR4^−/− mice sensitized were significantly reduced in percentage when compared with those of WT mice. Pooled BAL fluid collected from four mice in a were evaluated for iNKT cells using anti-TCRβ mAb and unloaded CD1d tetramer (negative control; left panel) or α-GalCer-loaded CD1d tetramer from WT (middle panel) vs CCR4^−/− mice (right panel) or WT iNKT cells (rightmost panel), with percentage of lymphocytes indicated above the iNKT cell gate. e, iNKT cell counts in the BAL fluid was significantly reduced in CCR4^−/− vs WT mice. BAL fluid was collected from individual mice (as in b) and stained for iNKT cells using anti-TCRβ mAb and α-GalCer-loaded CD1d tetramer, gated on lymphocytes by FSC and SSC. Absolute number of BAL iNKT was calculated and pooled across experiments, presented as mean ± SEM.

Competitive localization of WT vs CCR4^−/− iNKT in BM chimeric mice. Irradiated host mice were reconstituted with CD45.1⁺ WT and CD45.2⁺ CCR4^−/− BM cells (WT: CCR4^−/− chimera) or with CD45.1⁺ WT and CD45.2⁺ WT BM cells (WT:WT chimera). a, Resting CCR4^−/−:WT chimeras had fewer iNKT cells only in the BAL fluid compartment. Six to 8 wk after BM reconstitution, iNKT cells were identified by staining with α-GalCer -loaded CD1d tetramer and anti- TCRβ mAb. The numbers in plots indicate the percentage of iNKT cells of total lymphocytes in the blood, lung digest (after BAL fluid harvest), or BAL fluid. Results are representative of five chimeric pairs. b, CCR4^−/−:WT chimeras 24 h after i.n. challenge with α-GalCer (2 μg) have fewer iNKT cells only in the BAL fluid compartment. CCR4^−/−:WT and WT:WT chimeras were assessed as in a and results are representative of six chimeric pairs. c, Upper left panel, analysis CCR4^−/− BM-derived iNKT cell competitive homing disadvantage D. See Materials and Methods for calculation of disadvantage D. The relative disadvantage of CCR4^−/− BM iNKT cells was 2- to 3-fold in resting mice in the BAL fluid compartment but not other tissue as calculated by flow cytometric assessment of CD45.1 and CD45.2⁺ cells in CCR4^−/−:WT chimeras, compared with CD45.1⁺ and CD45.2⁺ WT cells in control WT:WT chimeras and represent the mean ± SD of pairwise comparisons normalized to engraftment ratios for five chimeric pairs. Statistical analysis shown as pairwise comparison of the D values derived from the BAL fluid or lungs to that of blood using the Mann-Whitney U test (p < 0.05 (*) and p ≤ 0.01 (**)). Upper right panel, The disadvantage of CCR4^−/− BM-derived cells was greatest in the BAL fluid compartment of α-GalCer -challenged chimeric and also in the lung tissue but not blood from flow cytometric assessment as in b for six chimeric pairs. Lower left and lower right panels, Conventional T cells derived from CCR4^−/− BM were disadvantaged only in the BAL fluid of CCR4^−/−:WT resting chimeric mice as determined by flow cytometry and identification of anti-TCRβ mAb-positive cells excluding CD1d tetramer⁺, representative of the mean ± SD of pairwise comparisons above in resting (five pairs) and α-GalCer -challenged (six pairs) mice. d, CCR4 influenced the BAL/airway compartment size of iNKT but not conventional T cells. The relative compartment size is the ratio of the absolute number of iNKT cells in the indicated compartment of CCR4^−/−:WT chimeras divided by that of WT:WT chimeras. Upper panels, Resting and α-GalCer -challenged CCR4^−/−:WT chimeras had half the total iNKT cells in the BAL fluid/airways but not blood or lung tissue when compared with WT:WT chimeras. Lower panels, In contrast, CCR4^−/−:WT chimeras had equal numbers of total conventional T cells in the BAL fluid/airways, lung tissue, and blood when compared with WT:WT chimeras. Data are presented as mean ± SEM ratios for pairwise comparisons (five resting and six challenged). Statistical analysis shown as pairwise comparison of the relative compartment size values for the BAL fluid or lungs to that of blood using the Mann-Whitney U test (p < 0.05 (*) and p ≤ 0.01 (**)).

Adoptive transfer of WT but not CCR4^−/− -derived iNKT cells induced the development of AHR in OVA-sensitized and challenged mice. Statistical analysis is shown as the results of the pairwise Student t test between experimental and negative control groups with p ≤ 0.05 (*) and p ≤ 0.01 (**). a, CCR4^−/− mice failed to develop significant OVA-induced AHR. AHR was assessed in OVA-sensitized and -challenged mice (sensitized with alum or Aspergillus Ag (alum sensitized shown)). Data are Penh values (mean ± SEM) of at least four mice per group, representative of three experiments for OVA-sensitized groups (shown) and two for Aspergillus-sensitized groups. b, Airway eosinophilia was reduced in OVA-sensitized and -challenged CCR4^−/− vs WT mice. BAL fluid from mice in a was taken 2 days after the final OVA i.n. challenge. Representative results are shown as the mean ± SEM number of cells per milliliter of BAL fluid. TCC, Total cell count; MO, monocytes; EOS, eosinophils; LYM, lymphocytes; NEU, neutrophils. c, IL-4 production by OVA-restimulated bronchial lymph node cells was measured in CCR4^−/− and WT mice after sensitization and challenge with OVA (see Materials and Methods). d, Adoptive transfer of iNKT cells from WT but not CCR4^−/− mice boosted AHR in CCR4^−/− mice. Splenic iNKT cells were sorted from CCR4^−/− or WT mice (1.5 × 10⁶ cells/recipient) and transferred into OVA/alum-sensitized CCR4^−/− mice 1 day before consecutive i.n. challenges with OVA. AHR was assessed by changes in airway resistance (R_L) and dynamic compliance (C_dyne) in response to methacholine in anesthetized, tracheostomized, intubated, and mechanically ventilated mice. Data represent the mean ± SEM of three to four mice per group from three experiments. e, iNKT cell numbers in the BAL fluid of CCR4^−/− mice sensitized and challenged with OVA were significantly reduced compared with those of WT mice. BAL fluid collected from each mouse in c was evaluated for iNKT cells using anti- TCRβ mAb and unloaded CD1d tetramer (negative control; leftmost panel) or α-GalCer -loaded CD1d tetramer from WT (second panel) vs CCR4^−/− mice that were recipients to either CCR4^−/− iNKT (third panel) or WT iNKT cells (rightmost panel), with percentage of lymphocytes indicated above the iNKT cell gate. f, Adoptive transfer of WT but not CCR4^−/− increased the absolute number of iNKT cells in the BAL fluid of CCR4^−/− mice sensitized and challenged with OVA. Flow cytometric analysis of mice similarly treated as in d were used to calculate the absolute number of iNKT cells in the BAL fluid (see Materials and Methods). g, Adoptive transfer of iNKT cells from WT but not CCR4^−/− mice reconstituted AHR in Jα18^−/− mice. iNKT cells were sorted from the spleens of CCR4^−/− or WT mice (1 × 10⁶ cells/recipient) and transferred into OVA/alum-sensitized Jα18^−/− mice 1 day before consecutive i.n. challenges with OVA. AHR was assessed by changes in airway resistance (R_L) and dynamic compliance (C_dyne, as in 6 days). Data are representative of the mean ± SEM of three to five mice per group from two experiments. h, Adoptively transferred iNKT cells from WT but not CCR4^−/− mice localized into the BAL fluid of Jα18^−/− mice after transfer. BAL fluid cells from individual mice treated as in a were collected 1 day after measurement of AHR and were stained with anti-TCRβ mAb and α-GalCer -loaded CD1d tetramer. One representative experiment of three is shown and data are the mean ± SD of calculated individual BAL iNKT cell numbers.