NK cells influence both innate and adaptive immune responses after mucosal immunization with antigen and mucosal adjuvant - PubMed (original) (raw)

NK cells influence both innate and adaptive immune responses after mucosal immunization with antigen and mucosal adjuvant

Lindsay J Hall et al. J Immunol. 2010.

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Abstract

NK cells were found to be recruited in a temporally controlled manner to the nasal-associated lymphoid tissue and the cervical lymph nodes of mice after intranasal immunization with Ag85B-early secreted antigenic target 6 kDa from Mycobacterium tuberculosis mixed with Escherichia coli heat-labile toxin as adjuvant. These NK cells were activated and secreted a diverse range of cytokines and other immunomodulators. Using Ab depletion targeting anti-asialo GM1, we found evidence for altered trafficking, impaired activation, and cytokine secretion of dendritic cells, macrophages, and neutrophils in immunized NK cell-depleted mice compared with control animals. Analysis of Ag-specific immune responses revealed an attenuated Ab and cytokine response in immunized NK cell-depleted animals. Systemic administration of rIL-6 but not rIFN-gamma significantly restored immune responses in mice depleted of NK cells. In conclusion, cytokine production, particularly IL-6, via NK cells and NK cell-activated immune populations plays an important role in the establishment of local innate immune responses and the consequent development of adaptive immunity after mucosal immunization.

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Figures

Figure 1

Figure 1. Characterisation of NK cells isolated from the NALT and CLN 5, 24 and 72 h post i.n. immunisation

Cells were isolated from NALT and CLN of Balb/c mice 5, 24 and 72 h after i.n. immunisation with PBS (naïve mice) or 1μg LT + 25μg Ag85B-ESAT6, and stained with flurochrome-labelled mAb and analysed by flow cytometry in which 20,000-100,000 events were recorded. (A) Plots shown are representative of at least ten mice, and the mean NK cell percentages are indicated. (B) Total NK cell number is shown for each organ. Columns represent the mean number ± SD. (C) For analysis, gates were set on DX5+/CD3− cells and the MFI of CD25 and CD69 expression was determined. Columns represent the mean MFI ± SD. The * indicates significant values of p < 0.05; **, p < 0.01; ***, p < 0.001, as determined by one-way ANOVA followed by Bonferroni’s multiple comparison test compared to negative control animals (i.e. PBS immunised, naïve mice).

Figure 2

Figure 2. In situ visualisation of NK cells within the NALT and CLN 5, 24, and 72 h after i.n. immunisation

Balb/c mice were i.n. immunised with PBS (naïve) or LT plus Ag85B-ESAT6. Both the NALT and CLN were collected 5, 24 and 72 h after immunisation. Tissue sections from six individual mice were analysed at each time-point by confocal microscopy. For NALT and CLN, 6μm serial frozen sections were fixed in acetone/ethanol and stained for CD49b (green), PNAd (red) and nuclei (blue). A representative picture for each group is shown. Green stained cells indicate NK cells; TZ, T cell zone; BZ, B cell zone. (Original magnification, x28.)

Figure 3

Figure 3. Characterisation of surface marker expression on IL-6+ and IL-6− mouse NK subsets

Balb/c mice were i.n. immunised with 1μg LT + 25μg Ag85B-ESAT6 or PBS (data not shown) and NALT and CLN cells isolated at 72 h. Cells were stained with DX5, CD3 and IL-6 together with the indicated surface marker. Histograms represent expression level of the indicated surface markers on the IL-6− and IL-6+ subsets, which are gated on DX5+/CD3− NK cells. The percentages are averaged from at least five mice and represent the mean percent positive cells for surface marker indicated above each histogram, compared with isotype control. The histograms shown are representative of at least five mice.

Figure 4

Figure 4. NK cell depletion impacts on other innate populations in the NALT and CLN 24 h post immunisation

Balb/c mice were treated with anti-AGM1 or normal rabbit IgG on days -7, -4, -2. Mice were then i.n. immunised with PBS (naïve) or 1μg LT and 25μg Ag85B-ESAT6 on day 0. Cells were isolated from the NALT and CLN of naive and immunised animals 24 h after i.n. immunisation, and stained with flurochrome-labelled mAb and analysed by flow cytometry in which 20,000-100,000 events were recorded. All data represent ten animals per group from two independent experiments. (A) Columns represents the mean percentage of CD11c+, F4/80+ and Ly6G+ cells ± SD. (B) For analysis, gates were set on the innate subset marker positive (CD11c+ or F4/80+) cells and the MFI of VCAM-1, and CD86 expression was determined. A gate was also set around Ly6G+ cells and the level of CD69 expression was determined. Data represents mean MFI ± SD. (C) Cells were stimulated for 6 h with BD Leukocyte Activation Cocktail plus GolgiPlug in vitro, stained with surface mAb to determine CD11c+, F4/80+ or Ly6G+ populations and then permeabilised and stained with anti-cytokine flurochrome-labelled mAb. Data represent percent of cytokine positive cells out of total specific cell population ± SD. The * indicates p < 0.05; **, p < 0.01 and ***, p < 0.001 using one-way ANOVA followed by Bonferroni’s Multiple Comparison Test. Representative dots plots are shown in supplementary Fig. S2B.

Figure 5

Figure 5. Anti-AGM1 treatment influences the development of antigen-specific antibody and cytokine responses

Balb/c mice were treated with anti-AGM1 or normal rabbit IgG on days -7, -4, -2 then i.n. immunised with 1μg LT and 25μg Ag85B-ESAT6 or PBS on day 0. (A) Mice were further treated ever 3-4 days, and then bled on days 14 and 21 for evaluation of primary antigen-specific antibody synthesis by ELISA. Titres from day 21 are shown here and reflect those seen on day 14 (data not shown). Data reflect ten individual mice per group from two independent experiments (minus background naïve animal titres) expressed as total antibody titre using a cut off of OD 0.2., with the black bar showing the geometric mean. (B) Mice were also bled on days 1, 7, 14 and 21 after immunisation and serum cytokine responses were evaluated using ELISA. Graph represents cytokine levels (pg/mL) ± SD. (C) Mice were sacrificed on day 21 when spleens were removed for T-cell assays. Cytokine responses were measured upon in vitro stimulation with Ag85B-ESAT6 (5μg/mL) for 36-48 h. Cells were also stimulated with 5μg/mL Con A (positive control) indicated by solid black line and RPMI+ media (negative control) indicated by dashed line. Columns represent the mean (± SD) stimulation indices of splenocytes from ten immunised animals per group from two individual experiments (minus background naïve cytokine levels). For all cytokines we found significant difference between naïve and immunised mice (data not shown). The differences in cytokine levels between immunised IgG control or anti-AGM1 treated animals is shown by **, p < 0.01 and ***, p < 0.001 using the Mann-Whitney U-Test.

Figure 6

Figure 6. Anti-AGM1 antibody treatment alters number and cytokine profile of systemic immune populations

Balb/c mice were treated with either anti-AGM1 or control IgG antibody and then immunised with either PBS (naïve) or Ag85B-ESAT6 and LT. Mice were sacrificed on day 21 when spleens were removed for T-cell assays. (A) Splenocytes were stained with flurochrome-labelled mAb and analysed by flow cytometry in which 200,000 events were recorded. Data represents the mean percentage of DX5+/CD3−, CD11c+, F4/80+ Ly6G+, CD19+ and CD3+ cells ± SD. Cytokine responses were measured upon in vitro stimulation with Ag85B-ESAT6 (5μg/mL) for 36-48 h. Cells were also stimulated with 5μg/mL Con A or RPMI+ media (data not shown). Stimulated cells were permeabilised and stained with anti-cytokine flurochrome-labelled mAb to determine cytokine profiles. (B) Data represent percent of cytokine positive NK cells out of total NK cell population ± SD in those mice treated with IgG control and i.n. immunised. Immunised mice treated with anti-AGM1 had less than 1% splenic NK cells and therefore cytokine analysis was not performed. (C) Data indicates the percent of cytokine positive adaptive cellular populations (CD19+ and CD3+) in both IgG control and anti-AGM1 naïve and immunised mice. The * indicates significant values of p < 0.05; **, p < 0.01; ***, p < 0.001, as determined by one-way ANOVA followed by Bonferroni’s multiple comparison test for immunised mice when compared to appropriate naive animals and IgG control immunised vs. anti-AGM1 treated immunised mice. Representative dots plots are shown in supplementary Fig. S3.

Figure 7

Figure 7. Effect of rIL-6 or rIFN-γ on antibody titres and cytokine levels in immunised mice depleted of NK cells

Animals were treated with either anti-AGM1 or rabbit IgG control antibody and were then i.n. immunised with LT and Ag85B-ESAT6. At days 1 and 2 post immunisation mice were further treated with rIL-6, rIFN-γ or PBS (control). (A) Mice were then bled on day 21 for evaluation of primary antigen-specific antibody synthesis by ELISA. Data reflect five individual mice per group (minus background naïve animal titres) expressed as total antibody titre using a cut off of OD 0.2., with the black bar showing the geometric mean. (B) On day 21 spleens were removed for T-cell assays and cytokine responses were measured upon in vitro stimulation with Ag85B-ESAT6 (5μg/mL) for 36-42 h. Cells were also stimulated with 5μg/mL Con A (positive control) and RPMI+ media (negative control) (see Fig. 5 for average values). Columns represent the mean (± SD) from five individual mice. Significant difference is indicated by; *, p < 0.05; and **, p < 0.01 using a Kruskal-Wallis test followed by Dunn’s Multiple Comparison test.

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