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The Journal of Immunology, 2008, 181, 4955 -4964
Copyright © 2008 by The American Association of Immunologists, Inc.
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Multifunctional, High-Level Cytokine-Producing Th1 Cells in the Lung, but Not Spleen, Correlate with Protection against Mycobacterium tuberculosis Aerosol Challenge in Mice

Emily K. Forbes, Clare Sander, Edward O. Ronan, Helen McShane, Adrian V. S. Hill, Peter C. L. Beverley and Elma Z. Tchilian1

The Jenner Institute, University of Oxford, Headington, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Boosting bacillus Calmette-Guérin (BCG)-primed mice with a recombinant adenovirus expressing Mycobacterium tuberculosis Ag 85A by different administration routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to Ag 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast, intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to Ag 85A and an increased response to purified protein derivative. This effect is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensity and integrated median fluorescence intensity for IFN-{gamma}, IL-2, and TNF. In contrast, mice immunized with BCG alone have few Ag-specific cells in the lung and a low proportion of multifunctional cells, although individual cells have high median fluorescence intensity. Successful immunization regimes appear to induce Ag-specific cells with abundant intracellular cytokine staining.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection by Mycobacterium tuberculosis is a global epidemic with 1.6 million deaths per year (1). The emergence of HIV-associated mycobacterial infections and increasing frequency of multidrug-resistant isolates reinforces the need to develop new control strategies. Although bacillus Calmette-Guérin (BCG)2 confers a degree of protection against disseminated disease in the very young, protection against pulmonary disease in older age groups is poor.

Because BCG provides partial protection, an attractive strategy is to develop vaccines that can be used as boosters following BCG primary immunization. Attempts to boost CD4 immunity above the level induced by BCG alone have had variable success. For example, mice immunized with recombinant Ag 85B protein are very poorly protected, although they develop a splenic CD4 T cell population producing large amounts of IFN-{gamma} (2, 3). In contrast, intranasal (i.n.) BCG priming followed by an i.n. boost with recombinant modified vaccinia Ankara expressing the abundant and immunogenic M. tuberculosis Ag 85A (MVA85A), generates increased protection against aerosol challenge, associated with an increased number of CD4 85A-specific IFN-{gamma}-secreting T cells in lung lymph nodes (4).

IFN-{gamma} has been shown to be necessary but not sufficient for protection against M. tuberculosis. Th1 CD4 cells are the main producers of cytokines following BCG immunization and therefore the frequency of IFN-{gamma}-producing cells has been widely used as a correlate of protection against M. tuberculosis. However, recent data from mice and cattle show that measurement of spleen or blood IFN-{gamma}-producing CD4 cells does not correlate with protection (3, 5, 6). Furthermore, although BCG immunization stimulates long-lasting, predominantly Th1 protective immunity in both mice and humans, the use of CD4- or CD8-deficient mice indicates that CD8 cells can also be protective (7, 8). Increased protection over BCG can be achieved also by immunization with a proapoptotic mutant of M. tuberculosis or prime boost regimes using recombinant adenovirus containing Ag 85A (Ad85A) (9, 10, 11, 12). These regimes generate strong lung CD8 IFN-{gamma} responses. In addition, rapid accumulation of CD8 IFN-{gamma}-secreting cells in the lungs after challenge correlates with BCG-induced protection (2).

Taken together these data indicate that although CD4 Th1 cells and IFN-{gamma} are important components of protection against M. tuberculosis, other immune mechanisms can contribute to increased protection. Recently multifunctional CD4 T cells secreting IFN-{gamma}, TNF, and IL-2 have been proposed as one such component and have been shown to correlate with protection in Leishmania major infection in mice (13). Furthermore, a high frequency of purified protein derivative (PPD)-specific multifunctional cells has been demonstrated in BCG vaccinated mice and humans, although there is no direct evidence that these cells provide protection against M. tuberculosis (13).

In our experiments, we took advantage of the observation that boosting BCG-primed mice with Ad85A by different routes has very different effects on protection against aerosol challenge with M. tuberculosis. We used these contrasting immunization regimes to determine what features of the immune response induced by boosting correlate with protection over and above that afforded by BCG immunization alone. We show that the presence of large numbers of multifunctional T cells in the lung, but not spleen, correlates with a reduction in mycobacterial burden after M. tuberculosis aerosol challenge.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and immunizations

All experiments were performed with 6- to 8-wk-old female BALB/c mice (Harlan Orlac) were approved by the Animal Use Ethical Committee of Oxford University and fully complied with the relevant Home Office guidelines. BCG (Pasteur 1173P2 D2) provided by Dr. Lagranderie (Pasteur Institute, Paris, France) was administered s.c. in the left hind footpad at a dose of 2 x 105 CFU in 30-µl volume.

The construction, design, and preparation of human adenovirus type 5 AdHu5 E1/E3 deleted, expressing M. tuberculosis Ag 85A (Ad85A), using the human CMV promoter containing intron A, were previously described (14). The construct was generated by the group of Dr. J. Wilson (University of Pennsylvania School of Medicine, Philadelphia, PA). In some experiments, mice were immunized with an empty control adenovirus, consisting of a modified Gateway entry vector pENTR4.CMV.BGH cloned into a human adenovirus type 5 destination vector pAd-PL DEST (Invitrogen).

Ten weeks after the BCG prime the Ad85A booster immunization was administered either intradermally (i.d.) or i.n. Mice were anesthetized and immunized i.d. with 25 µl in each ear (total 50 µl containing 2 x 109 viral particles of Ad85A per mouse) or for the i.n. administration allowed slowly to inhale 50 µl of 2 x 109 viral particles of Ad85A.

M. tuberculosis aerosol challenge

Four weeks after the Ad85A booster immunization, mice were challenged by aerosol with M. tuberculosis (Erdman strain; FDA Center for Biologics Evaluation and Research, Bethesda, MD), using a modified Henderson apparatus (15). Deposition in the lungs was measured 24 h after challenge and was ~200 CFU/lung. Mice were sacrificed 4 wk (one experiment) or 6 wk (two experiments) after M. tuberculosis challenge. Spleens and lungs were homogenized and the bacterial load was determined by plating 10-fold serial dilutions of tissue homogenates on Middlebrook 7H11 agar plates (E&O Laboratories). Colonies were counted after 3–4 wk of incubation at 37°C.

Cell isolations and peptides

Spleen and lung cells were cultured in {alpha}-MEM supplemented with 10% heat inactivated FBS, L-glutamine, 2-ME, and penicillin and streptomycin. Lungs were perfused with PBS via the right ventricle, cut into small pieces, and digested with 0.7 mg/ml collagenase type I (Sigma-Aldrich) and 30 µg/ml DNase I (Sigma-Aldrich) for 45 min at 37°C. Lung fragments were then crushed through a 70-µm Falcon strainer using a 5-ml syringe plunger, washed, and enumerated.

Cells were stimulated with PPD at 20 µg/ml (Statens Serum Institut) or a pool of 66 15mer peptides overlapping by 10 aa and covering the entire sequence of Ag 85A. Each peptide was at a final concentration of 8 µg/ml during the stimulation. In some experiments, cells were stimulated with the CD4 (Ag85A99–118aa TFLTSELPGWLQANRHVKPT) and CD8 (Ag85A70–78aa MPVGGQSSF) dominant peptide epitopes of Ag 85A at 2 µg/ml. Peptides were synthesized by Peptide Protein Research.

Flow cytometry

Cells harvested from spleen, lungs, or blood were stimulated with PPD or pooled 85A peptides for 1 h at 37°C. GolgiPlug (BD Biosciences) was added according to the manufacturer’s instruction and cells were incubated for an additional 5 h before intracellular cytokine staining. Cells were washed and incubated with CD16/CD32 mAb to block Fc binding. Subsequently the cells were stained for CD4, CD8, {alpha}4 integrin chain and IFN-{gamma}, IL-2, and TNF (eBioscience) using the BD Cytofix/Cytoperm kit according to the manufacturer’s instructions. Cells were fixed with PBS 1% paraformaldehyde, run on a CyAN ADP Analyzer (DakoCytomation), and analyzed using FlowJo software. Median fluorescence intensity (MFI) and integrated MFI were calculated using Spice 4.1.6, provided by Dr. M. Roederer (Vaccine Research Centre, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD). In some experiments, Ag-specific CD8 cells were enumerated using H-2Ld 85A70–78aa peptide (MPVGGQSSF) tetramers (supplied by the National Institutes of Health Tetramer Facility, NIAID, Bethesda, MD), the dominant 85A epitope in H-2D mice.

Three individual mice were analyzed per group. Cytokine frequency and number presented are after background subtraction of an identically gated population of cells from the same sample, incubated with medium only.

ELISPOT and cytometric bead array assays

ELISPOT IFN-{gamma} assay was conducted as previously described (4) using coating and detecting Abs from Mabtech. Spleen and lung cells were assayed following 18–20 h of stimulation with PPD, individual peptides, or pool of peptides. Three individual mice were tested in each group, and each condition was tested in duplicate.

For cytometric bead array assay (BD Biosciences), 1 x 107 splenocytes were stimulated for 18 h with 20 µg/ml PPD or 8 µg/ml pooled 85A peptides. The level of cytokines in the supernatant was measured using mouse inflammation and Th1/Th2 cytometric bead array kits, following the manufacturer’s instructions. Three individual mice were tested for each group. Controls with cells cultured in medium alone were included and the background cytokine production from these cultures subtracted.

Mycobacterial growth inhibition assay

The assay for the ability of lung and splenic cells to inhibit the intracellular growth of BCG was adapted from Hoft et al. (16). Briefly, lung and splenic cells from BA i.d and BA i.n. immunized mice were isolated as described above and plated in 6 well plates at a concentration of 5 x 105/ml in {alpha}-MEM, 10% FBS. They were cultured in medium alone or with PPD or recombinant Ag 85A at 20 µg/ml for 6 days.

Peritoneal macrophages were obtained from BALB/c mice plated at 15,000 cells/well in U-bottom 96-well microtiter plates in {alpha}-MEM and incubated at 37°C overnight. The next day, nonadherent cells were removed and the adherent cells were infected with BCG for 24 h at a MOI of 5:1. The infected cells were then washed three times with warm antibiotic-free medium to remove extracellular BCG and the stimulated splenic or lung cells were added at an E:T ratio of 10:1. After 72 h coculture, 0.2% saponin was added for 1 h to release BCG from the infected macrophages, and viable organisms were quantitated by plating on 7H11 agar. Results are expressed as a percentage of inhibition of mycobacterial growth calculated by the following formula: (C – E)/C x 100, where C is the CFU in cocultures of infected macrophages with T cells rested for 5 days in medium alone and E is CFU in cocultures containing T cells stimulated with Ag.

Statistical analysis

All results are representative of at least two independent experiments with similar results. Data were analyzed using the nonparametric Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Intranasal but not intradermal boosting with Ad85A improves protection afforded by BCG

We used a human adenovirus type 5 containing Ag 85A (Ad85A), to boost immunity in mice following BCG priming. The boost was either i.n. (BA i.n. immunization) or i.d. (BA i.d. immunization) administered 10 wk after BCG. Mycobacterial CFU were enumerated at 6 wk after M. tuberculosis aerosol challenge. Boosting by i.n. but not i.d. administration reduced mycobacterial CFU in the lungs compared with BCG alone (Fig. 1A). This result is consistent with previous studies comparing parenteral (i.m.) and i.n. administration of another adenovirus vector expressing Ag 85A (9, 17). In the spleen, BA i.d. and BA i.n. immunization give equivalent CFU to BCG (Fig. 1B).


Figure 1
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  		FIGURE 1. Mycobacterial CFU after Ad85A boosting of BCG primed mice. BALB/c mice were immunized with BCG and 10 wk later boosted with Ad85A given i.d. (BA i.d.) or i.n. (BA i.n.). Mice were challenged with M. tuberculosis by aerosol 4 wk after the boost. Mice were sacrificed 6 wk later and lung (A) and spleen (B) CFU enumerated. Data represent mean ± SD of n = 6–8 mice per group. *, p < 0.05 vs naive and **, p < 0.05 vs BCG and BA i.d. primed. Similar results were obtained in two other experiments one harvested at 4 wk and another at 6 wk.

 
Spleen responses

Having identified two boosting regimes with very different outcomes, we next asked what are the hallmarks of protective and nonprotective responses to booster immunizations. Because it has been suggested that cells simultaneously producing IFN-{gamma}, IL-2, and TNF may provide optimal effector function and protection against another intracellular pathogen (13) we defined the proportions of multifunctional cells in our immunized animals.

We performed intracellular staining for IFN-{gamma}, IL-2, and TNF on splenocytes stimulated with a pool of peptides covering the whole of Ag 85A. The BA i.d. regime induced the strongest IFN-{gamma}, TNF, and IL-2 responses by CD4 cells (~50,000 or 0.2% Ag-specific cells) almost five times greater than in BCG or BA i.n. immunized mice (Fig. 2A). The pattern of multifunctional cytokine-producing cells is similar for all regimes, with the highest proportion being 3+ cells producing IFN-{gamma}, IL-2, and TNF, followed by 2+ (IFN-{gamma}+TNF+ and IL-2+TNF+) and 1+ (TNF+) (Fig. 2B). There is no difference between the BCG and BA i.n. immunized CD4 response.


Figure 2
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  		FIGURE 2. Cytokine responses of splenic T cells to Ag 85A. Mice were primed with BCG and boosted with Ad85A either i.d. (BA i.d.) or i.n. (BA i.n.) administration. Splenocytes were isolated 4 wk after the boost and stimulated with pooled 85A peptides for 6 h. The frequency of IFN-{gamma}-, IL-2-, and TNF-producing cells were determined by flow cytometry on CD4 (A) or CD8 (C) gated cells and total number of cells per organ calculated. The number of cells expressing each of the seven possible combinations of the cytokines is also shown for CD4 (B) and CD8 (D) cells. Results are expressed as mean ± SEM of n = 3 mice per group, representative of two independent experiments. *, p < 0.05 compared with BA i.n. primed mice.

 
BA i.d. immunization induced the strongest CD8 response (~1.1 x 106 or 10% Ag-specific cells) followed by the BA i.n. regime (~1 x 105 cells) (Fig. 2, C and D). The vast majority of the CD8 cells in the BA i.d. and BA i.n. immunized mice are 2+ (IFN-{gamma}+TNF+) and 1+ (IFN-{gamma}+) because only 0.2% of the CD8 cells produce IL-2. Nevertheless in the BA i.d. immunization group there are nearly as many 3+ CD8 as 3+ CD4 cells (~23,000 CD4 vs ~20,000 CD8 cells). The most effective BA i.n. immunization regime induces nearly 3000 CD4 3+ vs 10,000 CD8 3+ cells. BCG alone induces very few 85A Ag-specific CD8 cells.

Following PPD stimulation in vitro no significant differences are detected in the CD4 responses between the regimes (Fig. 3). The largest proportion of the CD4-responding cells produce TNF only, followed by 1+ cells (IFN-{gamma}+) and 3+ cells. This proportion is not dissimilar from the data of Darrah et al. (13), although they showed somewhat higher proportion of 3+ cells in a different mouse strain, C57BL/6. The pattern of cytokine production in response to PPD differs from that induced by Ag 85A probably because only the response to 85A has been boosted. Boosting changes the pattern and increases the proportion of 3+ cells. A very low number of CD8 cells responding to PPD are detected (less than 1000 cytokine-producing cells per spleen) (data not shown).


Figure 3
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  		FIGURE 3. Cytokine responses of splenic T cells to PPD. Mice were primed with BCG and boosted with Ad85A either i.d. (BA i.d) or i.n. (BA i.n.) administration. Splenocytes were isolated 4 wk after the boost and stimulated with PPD for 6 h. The frequency of IFN-{gamma}-, IL-2-, and TNF-producing cells was determined by flow cytometry on CD4-gated (A) cells and the total number of cells per organ was calculated. The number of cells expressing each of the seven possible combinations of the cytokines is shown for CD4 cells (B). CD8 responses are very low and not shown. Results are expressed as mean ± SEM of n = 3 mice per group, representative of two independent experiments.

 
The magnitude and nature of responses in the spleen was also confirmed by the actual secretion of cytokines measured by cytometric bead array assay after 18 h of stimulation with 85A or PPD (Table I). The BA i.d. regime induces more IFN-{gamma}, TNF, IL-2, and IL-6 following 85A stimulation than the other regimes. However following PPD stimulation although more IFN-{gamma}, TNF, IL-2, and IL-6 were produced, there are no significant differences between the regimes. No significant differences in MCP-1, IL-10, IL-4, IL-5, or IL-12 production are detected after 85A or PPD stimulation (data not shown).


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  		Table I. Spleen cell PPD and 85A Ag-specific cytokine responsesa

 
Finally it is interesting to note that the 85A Ag-specific response differs very little in mice immunized with Ad85A alone or BCG primed Ad85A boosted mice (data not shown), suggesting that BCG does not prime a response to 85A efficiently in BALB/c mice. Furthermore, the PPD response is not boosted by administration of Ad85A, yet 85A is a major component of PPD. A possible explanation is that the epitopes recognized by CD4 and CD8 cells of BALB/c mice are not present in commercially available PPD. Alternatively, there may be competition for binding to APCs from the numerous other peptides present in PPD during the in vitro restimulation.

Taken together these data show that the magnitude of the response to Ag 85A in spleen, measured either as number of IFN-{gamma} or multifunctional cells or secreted cytokines, does not correlate with protection assessed by enumeration of CFU. Conversely the response to PPD is unaltered by i.d. or i.n. boosting with Ad85A and yet these groups have different levels of protection.

Lung responses

Lung responses differ from those in the spleen. The most effective BA i.n. regime induces very large CD4 and CD8 85A Ag-specific responses (Fig. 4A). Nearly ~22,000 (0.5%) CD4 cells produce IFN-{gamma}. However, less cells produce IL-2 compared with the spleen, so that most of the response is 2+ cells (IFN-{gamma}+TNF+), or 1+ cells (IFN-{gamma}+) and there are only ~3000 3+ cells (Fig. 4B).


Figure 4
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  		FIGURE 4. Cytokine responses of lung T cells to Ag 85A. Mice were primed with BCG and boosted with Ad85A either i.d. (BA i.d) or i.n. (BA i.n.) administration. Lung cells were isolated 4 wk after the boost and stimulated with pooled 85A peptides for 6 h. The frequency of IFN-{gamma}-, IL-2-, and TNF-producing cells was determined by flow cytometry on CD4-gated (A) or CD8-gated (C) cells and the total number of cells per organ was calculated. The number of cells expressing each of the seven possible combinations of the cytokines is shown for CD4 (B) and CD8 (D) cells. Results are expressed as mean ± SEM of n = 3 mice per group, representative of two independent experiments. *, p < 0.05 compared with BA i.d. primed mice.

 
There is a huge CD8 response in the BA i.n. immunized group, with ~1.1 x 106 (~30%) of the CD8 cells producing cytokines. As in the spleen, a very low proportion of the Ag-specific CD8 cells produce IL-2 and therefore most of the cytokine-producing cells are 2+ (IFN-{gamma}+TNF+) and 1+ (IFN-{gamma}+) producing cells. But nevertheless there are more 3+ CD8 than 3+ CD4 cells (~60,000 CD8 vs ~3000 CD4) (Fig. 4, C and D).

Whereas in the spleen i.n. or i.d. boosting does not alter the response to PPD, in the lung the BA i.n. regime increases the response considerably (Fig. 5). This response may occur because circulating PPD specific CD4 memory cells are trapped in the lung due to the inflammation induced by Ad85A i.n. immunization. However, we did not see any nonspecific trapping of Ag-specific CD8 tetramer-binding cells at 4 wk after the boost, when control empty human adenovirus type 5 vector was administered i.n. at the time of i.d. boosting with Ad85A (data not shown). However, nonspecific trapping has been shown to be transient and CD4 and CD8 cells may behave differently (12). There are ~17,000 CD4 Ag-specific cytokine-producing cells, of which ~5200 are 3+ cells and ~9300 2+ cells (IFN-{gamma}+TNF+). The CD8 response to PPD, as in the spleen is weak, but Ad85A i.n. increases it significantly to ~5000 CD8 IFN-{gamma}+ cells. Because the total number of PPD-specific CD8 cells is low, we did not enumerate multifunctional cells.


Figure 5
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  		FIGURE 5. Cytokine responses of lung T cells to PPD. Mice were primed with BCG and boosted with Ad85A either i.d. (BA i.d) or i.n. (BA i.n.) administration. Lung cells were isolated 4 wk after the boost and stimulated with PPD for 6 h. The frequency of IFN-{gamma}-, IL-2-, and TNF-producing cells was determined by flow cytometry on CD4-gated (A) or CD8-gated (C) cells and the total number of cells per organ was calculated. The number of cells expressing each of the seven possible combinations of the cytokines is shown only for CD4 (B) cells, but not for CD8 because the numbers were too low for reliable analysis. Results are expressed as mean ± SEM of n = 3 mice per group, representative of two independent experiments. *, p < 0.05 compared with BA i.d. mice.

 
Quality of responses

We further studied the quality of spleen and lung responses by computing the MFI of staining for intracellular cytokines (13). Fig. 6A summarizes the proportions of single, double, and triple cytokine-producing cells in the spleen. Although the absolute number is very different (Figs. 2 and 3), the proportion of different cytokine-producing cells is very similar in all immunized mice. However, the large CD8 85A Ag-specific population in the BA i.d. mice has the highest cytokine MFI in the spleen, although it is the 2+ cells, which produce the highest level of cytokines. In contrast, among CD4 cells, 85A- and PPD-specific cells from BCG immunized mice show the highest MFI (data not shown). However, when integrated MFI is considered, it is the BA i.d. regime that shows the largest IL-2, TNF, and IFN-{gamma} production, despite the fact that this regime does not increase protection.


Figure 6
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  		FIGURE 6. Multifunctional cytokine-producing cells in the spleen and lungs. Results indicate the proportions of single (1+ green), dual (2+ blue), and triple (3+ red) producers of IFN-{gamma}, TNF, and IL-2 in the spleen (A) and lungs (B) of immunized mice.

 
In contrast in the lung, the boosting regimes appear to induce a different proportion of single or multiple cytokine-producing 85A Ag-specific cells compared with BCG (Fig. 6B). However, BCG or BA i.d. immunization induces very few 85A Ag-specific CD4 or CD8 cells (Figs. 4 and 5), so that interpretation of these data requires caution. Nevertheless, it appears that BCG alone induces a higher proportion of single cytokine-producing CD4 cells because not only the 85A Ag-specific cells but PPD-specific CD4 cells from BCG immunized mice show a lower proportion of multiple cytokine-producing cells (Fig. 6B).

The MFI of cytokine-producing 85A Ag-specific CD4 and CD8 cells induced by the BA i.n. regime are higher than those of 85A Ag-specific cells induced by other regimes but it is not always the 3+ cells, which produce the highest amount of cytokines (Fig. 7). However, the integrated MFI for the 85A Ag-specific cells in the BA i.n. regime are consistently the highest for all three cytokines, reflecting the large number of Ag-specific lung cells found after this immunization (Fig. 7). Interestingly, the MFI for the CD4 PPD-specific response is highest for TNF and IL-2 in the BCG immunized mice with no clear pattern for IFN-{gamma}. The integrated MFI for IL-2 is also highest for CD4 PPD IL-2 producers in the BCG immunized mice.


Figure 7
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  		FIGURE 7. MFI and integrated MFI for intracellular cytokines of lung CD4 and CD8 T cells of immunized mice. MFI indicate the intensity of staining for intracellular cytokines in single, dual, or triple cytokine-producing cells. Integrated MFI (iMFI) are the product of the intensity of staining and the number of cytokine-producing cells. MFI and integrated MFI are shown for cells producing IL-2 (A), TNF (B), and IFN-{gamma} (C) from mice immunized with BCG alone (red line), BA i.d. (green line), or BA i.n. (blue line) and restimulated in vitro with either pooled Ag 85A peptides or PPD.

 
In summary, the most effective BA i.n. regime induces a strong lung CD4 and CD8 response to 85A and also boosts the CD4 response to PPD. These responses consist of cells with high amounts of intracellular cytokines (the 3+ cells are not always the highest), which may account for their protective efficacy. When both the amount of cytokine and the number of cells are taken into account by calculating the integrated MFI (13), the BA i.n. regime appears overwhelmingly the most potent in inducing a lung 85A Ag-specific response.

The lung and spleen responses are relatively persistent

All of the immunogenicity data described was performed at 4 wk after boost as this time is when the animals are challenged with M. tuberculosis. However, if prime boost regimes are to be useful, it will be important that they should induce persistent responses. We therefore assessed the immune response at a later time point, 11 wk after boost. The number of multifunctional 3+ CD4 and CD8 cells responsive to 85A does not change in the spleen, whereas it is reduced in the lung (Fig. 8). Despite the decrease of 85A Ag-specific cells in the lung at 11 wk, nevertheless ~1000 CD4 and ~5000 CD8 3+ cells remain. Interestingly the CD4 response to PPD is not changed in the lung and increased in the spleen at 11 wk BA i.d. and BA i.n. mice (Fig. 5). Overall the same trends were seen for the 2+ and 1+ producing cells (data not shown). These data show that an Ag-specific (memory) T cell population persists in the BA i.d. and BA i.n. imunization groups at 11 wk, although declining relatively rapidly in the lungs. Consistent with this finding, in a preliminary experiment we found that there is no difference in mycobacterial burden in the lungs between BCG and BA i.n. mice challenged 10 wk after the boost (data not shown).


Figure 8
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  		FIGURE 8. Multifunctional cells at 4 and 11 wk after Ad85A boost. The number of multifunctional 3+ cells expressing IFN-{gamma}, IL-2, and TNF in spleen (A) and lung (B) in response to pooled 85A peptides or PPD at 4 and 11 wk after boost are shown.

 
Properties of 85A Ag-specific cells

Ag specificity and mycobacterial inhibitory capacity of cells

We confirmed using individual peptides and ELISPOT assays as well as tetramer staining for CD8 cells, that the CD4 and CD8 responses in spleen and lung show identical specificity for the dominant IAd-restricted 85A99–118aa peptide and H-2Ld 85A70–78aa peptides, as reported by others (4, 17, 18) (data not shown). These results indicate that the reduction in CFU induced by i.n. immunization is not due to induction of T cells with differing Ag specificity to those induced by systemic immunization.

We next compared the ability of lung cells isolated from BA i.n. and spleen cells from BA i.d. immunized mice to inhibit mycobacterial growth in macrophages. Both lung cells (from BA i.n. mice) and spleen cells (from BA. i.d. mice), inhibit BCG growth to an equal extent, 45% at 10:1 E:T ratio, indicating that both cell types have similar capacity to inhibit mycobacterial growth irrespective of their geographic location. However, the presence of inhibitory cells in the spleen does not correlate with effective immunity in the lung. This result has important implications for development of vaccines against M. tuberculosis.

Integrin expression

As the most effective BA i.n. boost regime is associated with the presence of a very large number of cells in the lung but few in the spleen, we investigated what factors are important for the localization of the immune cells in the lung. It has been shown that VCAM-1, a vascular adhesion molecule, is up-regulated on endothelium in inflamed lungs (19). The integrins {alpha}4β1 and {alpha}4β7 are the counter-receptors on lymphocytes for VCAM-1 (20). We therefore studied the expression of the shared integrin {alpha}4 chain on the CD8 T cells in the lung and spleen of Ad85A boosted mice. When total CD8 cells from lung and spleen are compared, those in the lung consistently express more integrin {alpha}4, irrespective of the immunization of the mice as shown in BA i.n. mice (Fig. 9). However when expression of integrin {alpha}4 is compared on IFN-{gamma}+ and IFN-{gamma}– cells, a different picture emerges. In the spleen, the IFN-{gamma}+ cells are consistently more strongly stained than IFN-{gamma}– cells, whereas in the lung both populations are equally strongly stained (Fig. 9). These data suggest that immmunization with Ad85A generates a population of splenic Ag-specific cells that have up-regulated the {alpha}4 integrin chain and therefore have the potential to migrate efficiently to tissues that express the counter-receptors, VCAM-1 and MAdCAM (20). In contrast, in the lung both 85A Ag-specific and other CD8 T cells express high levels of integrin {alpha}4, suggesting that either all these cells have entered the lungs because they express appropriate addressins and adhesion molecules or that following entry or immunization in situ, lung resident cells up-regulate the integrin {alpha}4 chain (19, 21).


Figure 9
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  		FIGURE 9. Expression of the {alpha}4 integrin chain on spleen and lung CD8 cells. Expression of integrin {alpha}4 on CD8-gated T cells from spleen or lung (upper), on intracellular IFN-{gamma}+ or IFN-{gamma}– splenic CD8 cells stimulated in vitro with pooled 85A peptides (middle), and on IFN-{gamma}+ or IFN-{gamma}– lung CD8 cells (bottom). All cells are from BA i.n. immunized mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our data show that the magnitude of the immune response in the spleen does not correlate with reduction in lung mycobacterial CFU following aerosol challenge with M. tuberculosis in BALB/c mice primed with BCG and boosted with Ad85A i.d. or i.n. The BA i.d. regime induces the largest splenic 85A Ag-specific CD4 and CD8 cytokine response and yet does not reduce CFU compared with BCG alone. A similar lack of correlation between reduction in CFU and the magnitude of the splenic response has been shown following parenteral boosting with a different Ad85A construct (9), modified vaccinia Ankara expressing M. tuberculosis Ag 85A (MVA85A) (5) or recombinant Ag 85B (3). Similarly the magnitude of the blood cytokine response to 85A mirrors that for the spleen and does not correlate with CFU reduction either (data not shown).

Recently multifunctional Th1 cells were defined as a correlate of protection against L. major (13). In the same study, multifunctional CD4 PPD-specific cells were detected in BCG vaccinated mice and humans; however, there is no direct evidence that these are important for protection against M. tuberculosis (13). CD8 multifunctional cells have also been proposed as a correlate of an effective response in HIV, as individuals with better control of infection have an increased frequency of such cells (22). In our experiments, although there is a strong CD4 and CD8 splenic response after 85A stimulation in BA i.d. immunized animals, generating a large number of cytokine-producing cells with a high proportion of double and triple cytokine producing cells (Figs. 2 and 6A), no decrease in mycobacterial burden is observed. Conversely after boosting BCG-primed mice with Ad85A i.n., although there is no change in the magnitude or nature of the CD4 PPD-specific or 85A Ag-specific and only a relatively small splenic CD8 T cell response, there is a reduction in mycobacterial burden against aerosol challenge. Thus our data show that neither the frequency of Ag-specific cells nor the presence of multifunctional cells in the spleen (or blood) correlate with reduction of mycobacterial burden following aerosol challenge, in this prime boost mouse model.

In contrast, we show that the magnitude of the response in the lung following boosting, correlates well with a reduction in CFU compared with immunization with BCG alone. In the best-protected mice, boosted with Ad85A i.n., there is a large lung CD4 and CD8 response to Ag 85A as well as an increase in CD4 PPD-specific responses compared with mice immunized with BCG only (Figs. 4 and 5). It is striking that the number of IFN-{gamma} specific CD4 or CD8 cells in the lungs of the BA i.n. animals are of the same order of magnitude as the number induced in the spleen by the same immunogen given i.d. This amount is reflected in the presence of similar number of multifunctional cells in the two organs, although in the spleen there are an equal number of CD4 and CD8 3+ cells, whereas in the lung the majority of cells are CD8. Intriguingly, the two immunization regimes that are effective, BCG and BA i.n. immunization, both induce cells with high MFI for intracellular cytokines, suggesting that the rate of production may be important for protective immunity (Fig. 7).

A further striking feature of the response to this Ad85A construct, as is the case for others, is the excess of CD8 over CD4 cells (17). In the lung there is an increased total number of CD8 T cells and a high proportion of these (up to 30%) are Ag specific. These data suggests that following Ad85A boosting, the CD8 cells, which include the largest number of multifunctional cells, play an important role in protection against aerosol challenge, consistent with other data (2). Most likely either CD4 or CD8 cells can mediate protection equally well after prime boost immunization because both boosting with recombinant adenoviruses as shown in this study and by others (9), or recombinant vaccinia viruses that induce largely CD4 responses (4), can reduce lung mycobacterial burden if given by an appropriate route (i.n.).

There does not appear to be a major difference in Ag specificity or function between splenic and lung-resident cells induced by i.d. or i.n. boosting, respectively. Both the CD4 and the CD8 responses are against the same dominant epitopes of Ag 85A, in line with other data in H-2d mice (4, 17, 18). Therefore the exact specificity of the cells does not provide an explanation why the cells in the lung protect, whereas the splenic cells do not. Although we have demonstrated subtle differences in the MFI of cytokine-producing cells from the spleen and lung in i.n. and i.d. immunized mice, there may be other differences in cytokines that we have not measured and that also contribute to successful protective immunity. For example IL-17 is known to be important for mycobacterial immunity (5, 23), whereas it is also possible that i.d. boosting induces regulatory T cells, which can interfere with effective anti-mycobacterial responses (3, 24).

However, because 85A Ag-specific splenic cells transferred intratracheally can protect (17) and lung lymphocytes from BA i.n. immunized mice and splenic lymphocytes from BA i.d. mice reduce mycobacterial CFU in infected macrophages equally well, we consider that the key element in protection against aerosol challenge afforded by i.n. boosting with Ad85A, is the induction of a lung resident population of Ag specific cells, present at the time of challenge. We suggest that following M. tuberculosis challenge, there is a balance between M. tuberculosis and the immune response. If there is a large immune population in the lung (such as that provided by i.n. immunization), these cells immediately respond to the challenge organisms and can rapidly kill many of them. Abundant evidence indicates that tissue resident populations of T cells specific for other Ags have the properties of effector memory or activated effectors (21, 25, 26) and even central memory cells that enter the lung acquire effector memory phenotype and function (27). The lung cells in our BA i.n. immunized mice also show this phenotype (data not shown) and are therefore poised for effector function. Although purified T cells transferred into the airways cause a reduction in CFU after challenge (17), we cannot exclude the possibility that alterations in lung innate immune responses following i.n Ad85A boosting contribute to the observed reduction in lung CFU. Neither we nor others have yet performed challenge experiments after priming with BCG and boosting with an empty adenovirus (10, 12).

Following BCG or BA i.d. immunization a few Ag-specific cells will enter the lungs (Figs. 4 and 5) because some activated T cells can extravasate into nonlymphoid tissues (28, 29, 30). However the majority of spleen Ag-specific cells in BA i.d. boosted mice clearly do not enter the lungs even though they express high levels of the {alpha}4 integrin chain and the few lung-resident Ag-specific cells in BA i.d. immunized mice do not reduce mycobacterial burden beyond the level found in mice immunized with BCG alone. Thus we suggest that it is likely that the challenge organisms have to induce inflammation to up-regulate VCAM-1 on lung vasculature and promote an influx of Ag-specific effector cells (19). Up-regulation of VCAM takes more than a week (18). By this time there will be more mycobacteria and some may have entered macrophages and deployed evasion strategies so that they are less readily killed. Studies of lung infiltrating lymphocytes post BCG challenge show that there are fewer cells in the lungs of BA i.d. than BA i.n. mice at 3 and 10 days postchallenge (data not shown), supporting the idea that the presence of cells in the lung at the time of, or very early after, challenge is critical for protective immunity (2).

Why is it that i.n. immunization induces such a large population of lung resident cells in the absence of a large systemic population? We suggest that the i.n. boost may have three effects. The first is induction of a local immune response in the upper respiratory nasal-associated lymphoid tissue (NALT) (31), although it is not known whether this plays a role in protection against aerosol mycobacterial challenge (32). The second is that some cells primed in the nasal-associated lymphoid tissue may home to the lung. Finally, it is very likely that much of the Ag is inhaled into the lung, reaching even the periphery of lung lobes, as has been shown for other Ags administered i.n. in a volume of 50 µL (33). Once in the lung, Ad85A induces a vigorous local immune response leading to an influx of immune cells into the airways (9, 12, 17).

Although these experiments leave many aspects of immunity to M. tuberculosis unresolved, they confirm the importance of lung-resident memory T cells (14). Protected lungs contain large numbers of multifunctional cytokine-producing cells. Both PPD-specific and 85A Ag-specific lung cells from well-protected animals have high levels of intracellular cytokines. Additional experiments will be required to show whether this property is important for protection. The data have important implications for the design of human vaccines against M. tuberculosis first because they confirm data of many others, indicating that the magnitude and nature of immune responses measured in the spleen (or blood) do not correlate well with protection. Secondly they suggest that local immunity at the portal of entry is an important mechanism by which booster vaccines confer increased protection against mycobacterial infection. Challenges for the future will include the design of immunization regimes that can safely induce long lasting lung resident memory in humans and domestic animals and that can induce high-level cytokine-producing cells. There is also a pressing need to identify accessible, bloodborne correlates of protection.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 Address correspondence and reprint requests to Dr. Elma Tchilian, The Jenner Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ, U.K. E-mail address: elma.tchilian{at}ndm.ox.ac.uk Back

2 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; i.n., intranasal; i.d., intradermal; MFI, median fluorescence intensity; PPD, purified protein derivative. Back

Received for publication February 12, 2008. Accepted for publication July 29, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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