Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection - PubMed (original) (raw)

Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection

Shabaana A Khader et al. J Exp Med. 2006.

Abstract

Migration of dendritic cells (DCs) to the draining lymph node (DLN) is required for the activation of naive T cells. We show here that migration of DCs from the lung to the DLN after Mycobacterium tuberculosis (Mtb) exposure is defective in mice lacking interleukin (IL)-12p40. This defect compromises the ability of IL-12p40-deficient DCs to activate naive T cells in vivo; however, DCs that express IL-12p40 alone can activate naive T cells. Treatment of IL-12p40-deficient DCs with IL-12p40 homodimer (IL-12(p40)(2)) restores Mtb-induced DC migration and the ability of IL-12p40-deficient DCs to activate naive T cells. These data define a novel and fundamental role for IL-12p40 in the pathogen-induced activation of pulmonary DCs.

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Figures

Figure 1.

Figure 1.

T cell activation after Mtb infection requires IL-12p40. (a) B6 (filled circle), IL-23p19–deficient (open triangle), IL-12p35–deficient (open square), and IL-12p40–deficient (open circle) mice were infected with ∼75 CFU Mtb via the aerosol route, and the number of antigen-specific IFN-γ–producing CD4 T cells in the lung on day 21 was determined by ELISPOT. Data points represent the mean number of Th1 cells/time point (n = 4). One experiment representative of three total experiments is shown. The dashed line represents the limit of detection for this assay. **, P ≤ 0.001 relative to B6 value determined by Student's t test. (b) The number of activated CD4 T cells in the lungs of mice infected for 21 d was determined by flow cytometry. Data points represent the mean number of T cells/time point (n = 8–16). p-values were determined by the Student's t test.

Figure 2.

Figure 2.

IL-12p40–deficient BMDCs can stimulate naive T cells if not required to migrate. BMDCs were generated from B6 and IL-12p40–deficient (p40KO) mice and either treated with Mtb (filled bars) or left uninfected (striped bars) and pulsed with OVA323–339. Naive OVA-specific TCRTg CD4 T cells were cultured with the BMDCs for 3 d. The number (a) and phenotype (b) of cells present on day 3 were determined. Data points represent the mean of each group (n = 3). One experiment representative of two total experiments is shown. p-values were determined by the Student's t test. (c) BMDCs were generated as described for (a) and delivered intravenously into mice that had previously received naive TCRTg OVA-specific T cells. The number of T cells was determined at day 4. Data points represent the mean number of T cells (n = 3–4). One experiment is shown. (d) BMDCs from B6 (filled circles), IL-12p40–deficient (open circles), and IL-12p35–deficient (open squares) mice were cultured with Mtb, and the amount of IL-10 released over time was determined by ELISA. Data points represent the mean for each group (n = 3). One experiment representative of two total experiments is shown. *, P ≤ 0.01 for the difference between B6 and IL-12p40–deficient BMDCs as determined by the Student's t test.

Figure 3.

Figure 3.

IL-12p40–deficient steady-state lung DCs are not compromised. The number of total live cells (a), the total number of CD11c+ cells (b), and the total number of low autofluorescence CD11c+ cells was determined (c). Data points represent the mean for each group (n = 5). One experiment representative of two total experiments is shown. The expression MHC class II was analyzed on low-autofluorescence CD11c+ cells (d). Black histogram represents the isotype control, gray histogram represents B6 cells, and the open line shows expression of class II on IL-12p40–deficient (p40KO) cells. One mouse representative for each group is shown.

Figure 4.

Figure 4.

Pathogen-activated DCs fail to migrate from the lungs of IL-12p40–deficient mice. CFSE and either LPS (a) or irradiated Mtb (b) were delivered intratracheally to B6 and gene-deficient mice, and the frequency of CFSE-labeled CD11c+ cells in the DLN and lung was determined by flow cytometry after 18 h. Data points represent the mean for each group (n = 3–4). One experiment representative of two total experiments is shown. p-values were determined by the Student's t test. (c) Representative histogram illustrating the population of CFSE+CD11c+ cells within the DLNs of B6 and gene-deficient mice at 18 h after delivery of Mtb to the lungs of CFSE-treated mice.

Figure 5.

Figure 5.

BMDCs from IL-12p40–deficient mice fail to migrate from the lung when activated by Mtb. BMDCs from B6 and gene-deficient mice were exposed to irradiated Mtb for 3 h, washed, labeled with TAMRA orange, and delivered intratracheally to B6 mice. The frequency (a) and number (b) of CD11c+/TAMRA-labeled cells within the DLN was then determined over time using flow cytometry. The frequency and number of CD11c+/TAMRA-labeled cells within the lung were also determined on day 2 (filled bars) and day 4 (striped bars) (c). Data points represent the mean for each group (n = 3–4). One experiment representative of two total experiments is shown. p-values were determined by the Student's t test.

Figure 6.

Figure 6.

IL-12p40–deficient DCs fail to become responsive to homeostatic chemokines when stimulated by Mtb. BMDCs from B6 and gene-deficient mice were either left uninfected or infected with Mtb for 3 h. BMDCs were then placed in the upper chamber of a transwell plate and CCL19 was added to the lower chamber. The cells that migrated from the upper to the lower chamber within 90 min were then counted. The data are shown as chemotaxis index, which is the fold increase in induced migration over spontaneous migration. (a) The chemotaxis index was determined for B6 and gene-deficient mice. (b) The mRNA from untreated B6 and IL-12p40–deficient (p40KO) BMDCs treated with Mtb for 3 h was analyzed for the relative induction of mRNA for CCR7 by real-time PCR. The level of ccr7 gene expression in unstimulated B6 and IL-12p40–deficient (p40KO) BMDCs was not significantly different (not depicted). (c) The chemotaxis index for Mtb-activated IL-12p40–deficient (p40KO) BMDCs treated with IL-12(p40)2 or Mtb-activated BMDCs producing IL-12p40 alone was determined. Data points represent the mean for each group (n = 3–4). (d) The chemotaxis index for Mtb-activated p40-deficient BMDCs was determined when IL-12(p40)2, denatured IL-12(p40)2, or IL-12p70 was added to p40-deficient BMDCs. (e) The chemotaxis index for Mtb-activated BMDCs was determined in the presence and absence of exogenous IL-10. (f) The production of IL-12p40 from B6 BMDCs activated by Mtb for 3 h in the presence or absence of exogenous IL-10 was determined by ELISA. One experiment representative of two total experiments for each of panels a–f is shown. p-values were determined by the Student's t test.

Figure 7.

Figure 7.

DCs from IL-12p40–deficient mice are unable to activate naive T cells when delivered into the lung. BMDCs generated from B6 and gene-deficient mice were exposed to irradiated Mtb for 3 h, pulsed with OVA323–339, and delivered intratracheally into mice that had received intravenous delivery of naive TCRTg CD4 T cells 48 h earlier. The frequency of CD4+CD90.1+ OVA-specific cells in the DLN cells was determined by flow cytometry for mice receiving BMDCs from each of the groups (a, top). The frequency of activated CD4+CD90.1+ cells in the DLN was also determined by flow cytometry (a, bottom). The total number of CD4+CD90.1+ T cells (b) and the total number of activated CD4+CD90.1+ T cells (c) were calculated. ELISPOT was used to determine the number of IFN-γ–producing OVA-specific T cells within the DLN (d). Data points represent the mean for each group (n = 3–4). One experiment representative of two total experiments is shown. p-values were determined by the Student's t test. CFSE-labeled naive TCRTg CD4 T cells were delivered to mice, and the loss of CFSE 4 d after intratracheal delivery of B6 or IL-12p40-deficient (p40KO) BMDCs was determined by flow cytometry (e). Graphs are representative of three to seven mice and of two experiments Numbers indicate the mean frequency of cells within the undivided and divided gates for each group. **, P ≤ 0.005 compared with the B6 values by Student's t test.

Figure 8.

Figure 8.

DCs expressing IL-12p40 alone or IL-12p40–deficient DCs treated with IL-12(p40)2 can activate naive T cells when delivered to the lung. BMDCs from B6 or IL-12p40–deficient (p40KO) mice were treated with irradiated Mtb alone or Mtb in the presence of IL-12(p40)2 for 3 h. The cells were pulsed with OVA323–339 and transferred intratracheally into B6 mice that had received a bolus of naive CD4 TCRTg T cells. The number (a) and activation phenotype (b) of CD4+CD90.1+ T cells within the DLN at 4 d were determined by flow cytometry. BMDCs from B6, IL-12p40–deficient (p40KO), or IL-12p35/IL-23p19–deficient (p35/p19KO) mice were treated as described above and delivered to mice, and the number (c) and activation phenotype (d) of transferred naive T cells were determined at day 4 by flow cytometry. ELISPOT was used to determine the total number of antigen-specific IFN-γ–producing cells in the DLN for IL-12(p40)2–treated IL-12p40–deficient (p40KO) DCs (e). Data points represent the mean for each group (n = 3–4). One experiment representative of three total experiments for each of panels a–e is shown. p-values were determined by the Student's t test.

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