Mycobacterium Tuberculosis Aerosol This Information Is Current As Challenge in Mice of September 24, 2021

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Multifunctional, High-Level Cytokine-Producing Th1 Cells in the Lung, but Not Spleen, Correlate with Protection against Mycobacterium tuberculosis Aerosol This information is current as Challenge in Mice of September 24, 2021. Emily K. Forbes, Clare Sander, Edward O. Ronan, Helen McShane, Adrian V. S. Hill, Peter C. L. Beverley and Elma Z. Tchilian

J Immunol 2008; 181:4955-4964; ; Downloaded from doi: 10.4049/jimmunol.181.7.4955 http://www.jimmunol.org/content/181/7/4955 http://www.jimmunol.org/ References This article cites 32 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/181/7/4955.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Boosting bacillus Calmette-Gue´rin (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 Downloaded from 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-␥, 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 http://www.jimmunol.org/ appear to induce Ag-speciﬁc cells with abundant intracellular cytokine staining. The Journal of Immunology, 2008, 181: 4955–4964.

nfection by Mycobacterium tuberculosis is a global epidemic increased number of CD4 85A-specific IFN-␥-secreting T cells in with 1.6 million deaths per year (1). The emergence of HIV- lung lymph nodes (4). I associated mycobacterial infections and increasing frequency IFN-␥ has been shown to be necessary but not sufficient for of multidrug-resistant isolates reinforces the need to develop new protection against M. tuberculosis. Th1 CD4 cells are the main control strategies. Although bacillus Calmette-Gue´rin (BCG)2 con- producers of cytokines following BCG immunization and there- fers a degree of protection against disseminated disease in the very fore the frequency of IFN-␥-producing cells has been widely by guest on September 24, 2021 young, protection against pulmonary disease in older age groups used as a correlate of protection against M. tuberculosis. How- is poor. ever, recent data from mice and cattle show that measurement Because BCG provides partial protection, an attractive strategy of spleen or blood IFN-␥-producing CD4 cells does not corre- is to develop vaccines that can be used as boosters following BCG late with protection (3, 5, 6). Furthermore, although BCG im- primary immunization. Attempts to boost CD4 immunity above munization stimulates long-lasting, predominantly Th1 protec- the level induced by BCG alone have had variable success. For tive immunity in both mice and humans, the use of CD4- or example, mice immunized with recombinant Ag 85B protein are CD8-deficient mice indicates that CD8 cells can also be pro- very poorly protected, although they develop a splenic CD4 T cell tective (7, 8). Increased protection over BCG can be achieved population producing large amounts of IFN-␥ (2, 3). In contrast, also by immunization with a proapoptotic mutant of M. tuber- intranasal (i.n.) BCG priming followed by an i.n. boost with re- culosis or prime boost regimes using recombinant adenovirus combinant modified vaccinia Ankara expressing the abundant and containing Ag 85A (Ad85A) (9–12). These regimes generate immunogenic M. tuberculosis Ag 85A (MVA85A), generates in- strong lung CD8 IFN-␥ responses. In addition, rapid accumu- creased protection against aerosol challenge, associated with an lation of CD8 IFN-␥-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-␥ are important components of protection against The Jenner Institute, University of Oxford, Headington, Oxford, United Kingdom M. tuberculosis, other immune mechanisms can contribute to Received for publication February 12, 2008. Accepted for publication July increased protection. Recently multifunctional CD4 T cells se- 29, 2008. creting IFN-␥, TNF, and IL-2 have been proposed as one such The costs of publication of this article were defrayed in part by the payment of page component and have been shown to correlate with protection in charges. This article must therefore be hereby marked advertisement in accordance Leishmania major infection in mice (13). Furthermore, a high with 18 U.S.C. Section 1734 solely to indicate this fact. frequency of purified protein derivative (PPD)-specific multi- 1 Address correspondence and reprint requests to Dr. Elma Tchilian, The Jenner In- stitute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, functional cells has been demonstrated in BCG vaccinated mice Headington, Oxford OX3 7DQ, U.K. E-mail address: [email protected] and humans, although there is no direct evidence that these cells 2 Abbreviations used in this paper: BCG, bacillus Calmette-Gue´rin; i.n., intrana- provide protection against M. tuberculosis (13). sal; i.d., intradermal; MFI, median fluorescence intensity; PPD, purified protein In our experiments, we took advantage of the observation that derivative. boosting BCG-primed mice with Ad85A by different routes has Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 very different effects on protection against aerosol challenge with www.jimmunol.org 4956 LUNG RESPONSE AND TUBERCULOSIS PROTECTION

M. tuberculosis. We used these contrasting immunization regimes 85A70–78aa peptide (MPVGGQSSF) tetramers (supplied by the National to determine what features of the immune response induced by Institutes of Health Tetramer Facility, NIAID, Bethesda, MD), the domi- boosting correlate with protection over and above that afforded by nant 85A epitope in H-2D mice. Three individual mice were analyzed per group. Cytokine frequency and BCG immunization alone. We show that the presence of large number presented are after background subtraction of an identically gated numbers of multifunctional T cells in the lung, but not spleen, population of cells from the same sample, incubated with medium only. correlates with a reduction in mycobacterial burden after M. tuberculosis aerosol challenge. ELISPOT and cytometric bead array assays ELISPOT IFN-␥ assay was conducted as previously described (4) using Materials and Methods coating and detecting Abs from Mabtech. Spleen and lung cells were as- Animals and immunizations sayed following 18–20 h of stimulation with PPD, individual peptides, or pool of peptides. Three individual mice were tested in each group, and each All experiments were performed with 6- to 8-wk-old female BALB/c mice condition was tested in duplicate. (Harlan Orlac) were approved by the Animal Use Ethical Committee of For cytometric bead array assay (BD Biosciences), 1 ϫ 107 spleno- Oxford University and fully complied with the relevant Home Ofﬁce guide- cytes were stimulated for 18 h with 20 ␮g/ml PPD or 8 ␮g/ml pooled lines. BCG (Pasteur 1173P2 D2) provided by Dr. Lagranderie (Pasteur 85A peptides. The level of cytokines in the supernatant was measured Institute, Paris, France) was administered s.c. in the left hind footpad at a using mouse inﬂammation and Th1/Th2 cytometric bead array kits, fol- dose of 2 ϫ 105 CFU in 30-␮l volume. lowing the manufacturer’s instructions. Three individual mice were The construction, design, and preparation of human adenovirus type 5 tested for each group. Controls with cells cultured in medium alone AdHu5 E1/E3 deleted, expressing M. tuberculosis Ag 85A (Ad85A), us- were included and the background cytokine production from these cul- ing the human CMV promoter containing intron A, were previously tures subtracted.

described (14). The construct was generated by the group of Dr. J. Downloaded from Wilson (University of Pennsylvania School of Medicine, Philadelphia, Mycobacterial growth inhibition assay PA). In some experiments, mice were immunized with an empty control The assay for the ability of lung and splenic cells to inhibit the intracellular adenovirus, consisting of a modified Gateway entry vector growth of BCG was adapted from Hoft et al. (16). Briefly, lung and splenic pENTR4.CMV.BGH cloned into a human adenovirus type 5 destination cells from BA i.d and BA i.n. immunized mice were isolated as described vector pAd-PL DEST (Invitrogen). above and plated in 6 well plates at a concentration of 5 ϫ 105/ml in Ten weeks after the BCG prime the Ad85A booster immunization was ␣-MEM, 10% FBS. They were cultured in medium alone or with PPD or administered either intradermally (i.d.) or i.n. Mice were anesthetized and recombinant Ag 85A at 20 ␮g/ml for 6 days. ␮ ␮ ϫ 9 http://www.jimmunol.org/ immunized i.d. with 25 l in each ear (total 50 l containing 2 10 viral Peritoneal macrophages were obtained from BALB/c mice plated at particles of Ad85A per mouse) or for the i.n. administration allowed slowly 15,000 cells/well in U-bottom 96-well microtiter plates in ␣-MEM and ␮ ϫ 9 to inhale 50 lof2 10 viral particles of Ad85A. incubated at 37°C overnight. The next day, nonadherent cells were re- M. tuberculosis aerosol challenge moved 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 anti- Four weeks after the Ad85A booster immunization, mice were challenged biotic-free medium to remove extracellular BCG and the stimulated splenic by aerosol with M. tuberculosis (Erdman strain; FDA Center for Biologics or lung cells were added at an E:T ratio of 10:1. After 72 h coculture, 0.2% Evaluation and Research, Bethesda, MD), using a modified Henderson saponin was added for1htorelease BCG from the infected macrophages, apparatus (15). Deposition in the lungs was measured 24 h after challenge and viable organisms were quantitated by plating on 7H11 agar. Results are and was ϳ200 CFU/lung. Mice were sacrificed 4 wk (one experiment) or expressed as a percentage of inhibition of mycobacterial growth calculated

6 wk (two experiments) after M. tuberculosis challenge. Spleens and lungs by the following formula: (C Ϫ E)/C ϫ 100, where C is the CFU in by guest on September 24, 2021 were homogenized and the bacterial load was determined by plating 10- cocultures of infected macrophages with T cells rested for 5 days in me- fold serial dilutions of tissue homogenates on Middlebrook 7H11 agar dium alone and E is CFU in cocultures containing T cells stimulated plates (E&O Laboratories). Colonies were counted after 3–4 wk of incu- with Ag. bation at 37°C. Statistical analysis Cell isolations and peptides All results are representative of at least two independent experiments with Spleen and lung cells were cultured in ␣-MEM supplemented with 10% similar results. Data were analyzed using the nonparametric Mann-Whit- heat inactivated FBS, L-glutamine, 2-ME, and penicillin and streptomycin. ney U test. 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 Results ␮ g/ml DNase I (Sigma-Aldrich) for 45 min at 37°C. Lung fragments were Intranasal but not intradermal boosting with Ad85A improves then crushed through a 70-␮m Falcon strainer using a 5-ml syringe plunger, washed, and enumerated. protection afforded by BCG ␮ Cells were stimulated with PPD at 20 g/ml (Statens Serum Institut) or We used a human adenovirus type 5 containing Ag 85A (Ad85A), 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 ␮g/ml to boost immunity in mice following BCG priming. The boost was during the stimulation. In some experiments, cells were stimulated with the either i.n. (BA i.n. immunization) or i.d. (BA i.d. immunization)

CD4 (Ag85A99–118aa TFLTSELPGWLQANRHVKPT) and CD8 administered 10 wk after BCG. Mycobacterial CFU were enumer- (Ag85A70–78aa MPVGGQSSF) dominant peptide epitopes of Ag 85A at 2 ated at 6 wk after M. tuberculosis aerosol challenge. Boosting by ␮ g/ml. Peptides were synthesized by Peptide Protein Research. i.n. but not i.d. administration reduced mycobacterial CFU in the Flow cytometry lungs compared with BCG alone (Fig. 1A). This result is consistent with previous studies comparing parenteral (i.m.) and i.n. admin- Cells harvested from spleen, lungs, or blood were stimulated with PPD or pooled 85A peptides for1hat37°C. GolgiPlug (BD Biosciences) was istration of another adenovirus vector expressing Ag 85A (9, 17). added according to the manufacturer’s instruction and cells were incubated In the spleen, BA i.d. and BA i.n. immunization give equivalent for an additional 5 h before intracellular cytokine staining. Cells were CFU to BCG (Fig. 1B). washed and incubated with CD16/CD32 mAb to block Fc binding. Sub- ␣ sequently the cells were stained for CD4, CD8, 4 integrin chain and Spleen responses IFN-␥, IL-2, and TNF (eBioscience) using the BD Cytofix/Cytoperm kit according to the manufacturer’s instructions. Cells were fixed with PBS 1% Having identified two boosting regimes with very different out- paraformaldehyde, run on a CyAN ADP Analyzer (DakoCytomation), and comes, we next asked what are the hallmarks of protective and analyzed using FlowJo software. Median fluorescence intensity (MFI) and nonprotective responses to booster immunizations. Because it integrated MFI were calculated using Spice 4.1.6, provided by Dr. M. ␥ Roederer (Vaccine Research Centre, National Institute of Allergy and In- has been suggested that cells simultaneously producing IFN- , fectious Diseases (NIAID), National Institutes of Health, Bethesda, MD). IL-2, and TNF may provide optimal effector function and pro- In some experiments, Ag-specific CD8 cells were enumerated using H-2Ld tection against another intracellular pathogen (13) we defined The Journal of Immunology 4957 Downloaded from

FIGURE 1. Mycobacterial CFU after Ad85A boosting of BCG primed mice. BALB/c mice were immunized with BCG and 10 wk later http://www.jimmunol.org/ boosted with Ad85A given i.d. (BA i.d.) or i.n. (BA i.n.). Mice were FIGURE 3. Cytokine responses of splenic T cells to PPD. Mice were challenged with M. tuberculosis by aerosol 4 wk after the boost. Mice primed with BCG and boosted with Ad85A either i.d. (BA i.d) or i.n. (BA were sacrificed 6 wk later and lung (A) and spleen (B) CFU enumerated. i.n.) administration. Splenocytes were isolated 4 wk after the boost and -p Ͻ 0.05 vs stimulated with PPD for 6 h. The frequency of IFN-␥-, IL-2-, and TNF ,ء .Data represent mean Ϯ SD of n ϭ 6–8 mice per group p Ͻ 0.05 vs BCG and BA i.d. primed. Similar results were producing cells was determined by flow cytometry on CD4-gated (A) cells ,ءء naive and obtained in two other experiments one harvested at 4 wk and another at and the total number of cells per organ was calculated. The number of cells 6 wk. 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 Ϯ SEM of n ϭ 3 mice per group, repre- by guest on September 24, 2021 the proportions of multifunctional cells in our immunized sentative of two independent experiments. animals. We performed intracellular staining for IFN-␥, IL-2, and TNF on splenocytes stimulated with a pool of peptides covering the kine-producing cells is similar for all regimes, with the highest whole of Ag 85A. The BA i.d. regime induced the strongest IFN-␥, proportion being 3ϩ cells producing IFN-␥, IL-2, and TNF, fol- TNF, and IL-2 responses by CD4 cells (ϳ50,000 or 0.2% Ag- lowed by 2ϩ (IFN-␥ϩTNFϩ and IL-2ϩTNFϩ) and 1ϩ (TNFϩ) specific cells) almost five times greater than in BCG or BA i.n. (Fig. 2B). There is no difference between the BCG and BA i.n. immunized mice (Fig. 2A). The pattern of multifunctional cyto- immunized CD4 response.

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-␥-, IL-2-, and TNF-producing cells were determined by ﬂow cytometry on CD4 (A)orCD8(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 Ϯ SEM of n ϭ 3 mice per group, representative of two inde- p Ͻ 0.05 ,ء .pendent experiments compared with BA i.n. primed mice. 4958 LUNG RESPONSE AND TUBERCULOSIS PROTECTION

Table I. Spleen cell PPD and 85A Ag-specific cytokine responsesa The magnitude and nature of responses in the spleen was also confirmed by the actual secretion of cytokines measured by cyto- N (pg/ml) B (pg/ml) BA i.d. (pg/ml) BA i.n. (pg/ml) metric bead array assay after 18 h of stimulation with 85A or PPD (Table I). The BA i.d. regime induces more IFN-␥, TNF, IL-2, and 85A IFN-␥ 13 70 1653b 435 IL-6 following 85A stimulation than the other regimes. However IL-2 5 12 157b 52 following PPD stimulation although more IFN-␥, TNF, IL-2, and TNF 24 53 189b 51 IL-6 were produced, there are no significant differences between b IL-6 7 11 123 39 the regimes. No significant differences in MCP-1, IL-10, IL-4, PPD IL-5, or IL-12 production are detected after 85A or PPD stimula- IFN-␥ 73 1895 2896 3290 IL-2 6 316 294 245 tion (data not shown). TNF 56 647 854 759 Finally it is interesting to note that the 85A Ag-specific response IL-6 14 150 130 126 differs very little in mice immunized with Ad85A alone or BCG a Mice were primed with BCG and boosted with Ad85A either i.d. (BA i.d.) or i.n. primed Ad85A boosted mice (data not shown), suggesting that (BA i.n.) administered. Splenocytes were isolated 4 wk after the boost and stimulated BCG does not prime a response to 85A efficiently in BALB/c with PPD or pooled 85A peptides for 18 h. IFN-␥, IL-2, TNF, and IL-6 production was determined using CBA assay. Results shown are mean of three mice per group, mice. Furthermore, the PPD response is not boosted by adminis- representative of two independent experiments. Overall interassay and intraassay vari- tration of Ad85A, yet 85A is a major component of PPD. A pos- ation is less than 15%. sible explanation is that the epitopes recognized by CD4 and CD8 b p Ͻ 0.05 compared with B and BA i.n. immunization groups. cells of BALB/c mice are not present in commercially available

PPD. Alternatively, there may be competition for binding to APCs Downloaded from BA i.d. immunization induced the strongest CD8 response from the numerous other peptides present in PPD during the in (ϳ1.1 ϫ 106 or 10% Ag-specific cells) followed by the BA i.n. vitro restimulation. regime (ϳ1 ϫ 105 cells) (Fig. 2, C and D). The vast majority of Taken together these data show that the magnitude of the the CD8 cells in the BA i.d. and BA i.n. immunized mice are response to Ag 85A in spleen, measured either as number of ϩ ϩ ϩ ␥ 2ϩ (IFN-␥ TNF ) and 1ϩ (IFN-␥ ) because only 0.2% of the IFN- or multifunctional cells or secreted cytokines, does not CD8 cells produce IL-2. Nevertheless in the BA i.d. immuni- correlate with protection assessed by enumeration of CFU. Con- http://www.jimmunol.org/ zation group there are nearly as many 3ϩ CD8 as 3ϩ CD4 cells versely the response to PPD is unaltered by i.d. or i.n. boosting (ϳ23,000 CD4 vs ϳ20,000 CD8 cells). The most effective BA with Ad85A and yet these groups have different levels of i.n. immunization regime induces nearly 3000 CD4 3ϩ vs protection. 10,000 CD8 3ϩ cells. BCG alone induces very few 85A Ag- specific CD8 cells. Lung responses Following PPD stimulation in vitro no significant differences are Lung responses differ from those in the spleen. The most ef- detected in the CD4 responses between the regimes (Fig. 3). The fective BA i.n. regime induces very large CD4 and CD8 85A largest proportion of the CD4-responding cells produce TNF only, Ag-specific responses (Fig. 4A). Nearly ϳ22,000 (0.5%) CD4 by guest on September 24, 2021 followed by 1ϩ cells (IFN-␥ϩ) and 3ϩ cells. This proportion is cells produce IFN-␥. However, less cells produce IL-2 com- not dissimilar from the data of Darrah et al. (13), although they pared with the spleen, so that most of the response is 2ϩ cells showed somewhat higher proportion of 3ϩ cells in a different (IFN-␥ϩTNFϩ), or 1ϩ cells (IFN-␥ϩ) and there are only mouse strain, C57BL/6. The pattern of cytokine production in re- ϳ3000 3ϩ cells (Fig. 4B). sponse to PPD differs from that induced by Ag 85A probably be- There is a huge CD8 response in the BA i.n. immunized cause only the response to 85A has been boosted. Boosting group, with ϳ1.1 ϫ 106 (ϳ30%) of the CD8 cells producing changes the pattern and increases the proportion of 3ϩ cells. A cytokines. As in the spleen, a very low proportion of the Ag- very low number of CD8 cells responding to PPD are detected specific CD8 cells produce IL-2 and therefore most of the cy- (less than 1000 cytokine-producing cells per spleen) (data not tokine-producing cells are 2ϩ (IFN-␥ϩTNFϩ) and 1ϩ (IFN- shown). ␥ϩ) producing cells. But nevertheless there are more 3ϩ CD8

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-␥-, 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 Ϯ SEM of n ϭ 3 mice per group, representative of two p Ͻ 0.05 ,ء .independent experiments compared with BA i.d. primed mice. The Journal of Immunology 4959

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-␥-, 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 Ϯ SEM of n ϭ 3 mice per group, representative of two indepen- -p Ͻ 0.05 com ,ء .dent experiments pared with BA i.d. mice. Downloaded from than 3ϩ CD4 cells (ϳ60,000 CD8 vs ϳ3000 CD4) (Fig. 4, C that BCG alone induces a higher proportion of single cytokine- and D). producing CD4 cells because not only the 85A Ag-specific cells Whereas in the spleen i.n. or i.d. boosting does not alter the but PPD-specific CD4 cells from BCG immunized mice show a response to PPD, in the lung the BA i.n. regime increases the lower proportion of multiple cytokine-producing cells http://www.jimmunol.org/ response considerably (Fig. 5). This response may occur be- (Fig. 6B). cause circulating PPD specific CD4 memory cells are trapped in The MFI of cytokine-producing 85A Ag-specific CD4 and the lung due to the inflammation induced by Ad85A i.n. immu- CD8 cells induced by the BA i.n. regime are higher than those nization. However, we did not see any nonspecific trapping of of 85A Ag-specific cells induced by other regimes but it is not Ag-specific CD8 tetramer-binding cells at 4 wk after the boost, always the 3ϩ cells, which produce the highest amount of cy- when control empty human adenovirus type 5 vector was ad- tokines (Fig. 7). However, the integrated MFI for the 85A Ag- ministered i.n. at the time of i.d. boosting with Ad85A (data not specific cells in the BA i.n. regime are consistently the highest shown). However, nonspecific trapping has been shown to be for all three cytokines, reflecting the large number of Ag-spe- transient and CD4 and CD8 cells may behave differently (12). cific lung cells found after this immunization (Fig. 7). Interest- by guest on September 24, 2021 There are ϳ17,000 CD4 Ag-specific cytokine-producing cells, ingly, the MFI for the CD4 PPD-specific response is highest for of which ϳ5200 are 3ϩ cells and ϳ9300 2ϩ cells (IFN- ␥ϩTNFϩ). The CD8 response to PPD, as in the spleen is weak, but Ad85A i.n. increases it significantly to ϳ5000 CD8 IFN-␥ϩ cells. Because the total number of PPD-specific CD8 cells is low, we did not enumerate multifunctional cells.

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 2ϩ 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-␥ production, despite the fact that this regime does not increase protection. 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, FIGURE 6. Multifunctional cytokine-producing cells in the spleen and BCG or BA i.d. immunization induces very few 85A Ag-spe- lungs. Results indicate the proportions of single (1ϩ green), dual (2ϩ cific CD4 or CD8 cells (Figs. 4 and 5), so that interpreta- blue), and triple (3ϩ red) producers of IFN-␥, TNF, and IL-2 in the spleen tion of these data requires caution. Nevertheless, it appears (A) and lungs (B) of immunized mice. 4960 LUNG RESPONSE AND TUBERCULOSIS PROTECTION Downloaded from

FIGURE 7. MFI and integrated MFI for intracellular cytokines of lung CD4 and CD8 T cells of immunized mice. MFI indicate the in- http://www.jimmunol.org/ tensity 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-␥ (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. by guest on September 24, 2021

TNF and IL-2 in the BCG immunized mice with no clear pattern amounts of intracellular cytokines (the 3ϩ cells are not always the for IFN-␥. The integrated MFI for IL-2 is also highest for CD4 highest), which may account for their protective efﬁcacy. When PPD IL-2 producers in the BCG immunized mice. both the amount of cytokine and the number of cells are taken into In summary, the most effective BA i.n. regime induces a strong account by calculating the integrated MFI (13), the BA i.n. regime lung CD4 and CD8 response to 85A and also boosts the CD4 appears overwhelmingly the most potent in inducing a lung 85A response to PPD. These responses consist of cells with high Ag-speciﬁc response. The Journal of Immunology 4961

FIGURE 8. Multifunctional cells at 4 and 11 wk after Ad85A boost. The number of multifunctional 3ϩ cells expressing IFN-␥, IL-2, and TNF in spleen (A) and lung (B)inre- sponse to pooled 85A peptides or PPD at 4 and 11 wk after boost are shown. Downloaded from http://www.jimmunol.org/

The lung and spleen responses are relatively persistent BA i.n. mice) and spleen cells (from BA. i.d. mice), inhibit All of the immunogenicity data described was performed at 4 BCG growth to an equal extent, 45% at 10:1 E:T ratio, indi- wk after boost as this time is when the animals are challenged cating that both cell types have similar capacity to inhibit my- with M. tuberculosis. However, if prime boost regimes are to be cobacterial growth irrespective of their geographic location. useful, it will be important that they should induce persistent However, the presence of inhibitory cells in the spleen does not responses. We therefore assessed the immune response at a later correlate with effective immunity in the lung. This result has by guest on September 24, 2021 time point, 11 wk after boost. The number of multifunctional important implications for development of vaccines against 3ϩ CD4 and CD8 cells responsive to 85A does not change in M. tuberculosis. 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 ϳ1000 CD4 and ϳ5000 CD8 3ϩ cells remain. Inter- Integrin expression estingly the CD4 response to PPD is not changed in the lung As the most effective BA i.n. boost regime is associated with and increased in the spleen at 11 wk BA i.d. and BA i.n. mice the presence of a very large number of cells in the lung but few (Fig. 5). Overall the same trends were seen for the 2ϩ and 1ϩ in the spleen, we investigated what factors are important for the producing cells (data not shown). These data show that an Ag- localization of the immune cells in the lung. It has been shown specific (memory) T cell population persists in the BA i.d. and that VCAM-1, a vascular adhesion molecule, is up-regulated on BA i.n. imunization groups at 11 wk, although declining rela- endothelium in inflamed lungs (19). The integrins ␣ ␤ and tively rapidly in the lungs. Consistent with this finding, in a 4 1 ␣ ␤ are the counter-receptors on lymphocytes for VCAM-1 preliminary experiment we found that there is no difference in 4 7 (20). We therefore studied the expression of the shared integrin mycobacterial burden in the lungs between BCG and BA i.n. ␣ chain on the CD8 T cells in the lung and spleen of Ad85A mice challenged 10 wk after the boost (data not shown). 4 boosted mice. When total CD8 cells from lung and spleen are compared, those in the lung consistently express more integrin Properties of 85A Ag-specific cells ␣ 4, irrespective of the immunization of the mice as shown in Ag specificity and mycobacterial inhibitory capacity of cells ␣ BA i.n. mice (Fig. 9). However when expression of integrin 4 ϩ Ϫ We confirmed using individual peptides and ELISPOT assays is compared on IFN-␥ and IFN-␥ cells, a different picture ϩ as well as tetramer staining for CD8 cells, that the CD4 and emerges. In the spleen, the IFN-␥ cells are consistently more Ϫ CD8 responses in spleen and lung show identical specificity for strongly stained than IFN-␥ cells, whereas in the lung both d d the dominant IA -restricted 85A99–118aa peptide and H-2L populations are equally strongly stained (Fig. 9). These data 85A70–78aa peptides, as reported by others (4, 17, 18) (data not suggest that immmunization with Ad85A generates a popula- ␣ shown). These results indicate that the reduction in CFU in- tion of splenic Ag-specific cells that have up-regulated the 4 duced by i.n. immunization is not due to induction of T cells integrin chain and therefore have the potential to migrate effi- with differing Ag specificity to those induced by systemic ciently to tissues that express the counter-receptors, VCAM-1 immunization. and MAdCAM (20). In contrast, in the lung both 85A Ag-spe- ␣ We next compared the ability of lung cells isolated from BA cific and other CD8 T cells express high levels of integrin 4, i.n. and spleen cells from BA i.d. immunized mice to inhibit suggesting that either all these cells have entered the lungs mycobacterial growth in macrophages. Both lung cells (from because they express appropriate addressins and adhesion 4962 LUNG RESPONSE AND TUBERCULOSIS PROTECTION

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, gen- erating 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. Con- versely 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 multifunc- Downloaded from tional 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-pro- http://www.jimmunol.org/ tected 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-␥ 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 by guest on September 24, 2021 3ϩ cells, whereas in the lung the majority of cells are CD8. In- triguingly, 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). ␣ FIGURE 9. Expression of the 4 integrin chain on spleen and lung CD8 A further striking feature of the response to this Ad85A con- ␣ cells. Expression of integrin 4 on CD8-gated T cells from spleen or lung struct, as is the case for others, is the excess of CD8 over CD4 cells (upper), on intracellular IFN-␥ϩ or IFN-␥Ϫ splenic CD8 cells stimulated in (17). In the lung there is an increased total number of CD8 T cells ϩ Ϫ vitro with pooled 85A peptides (middle), and on IFN-␥ or IFN-␥ lung and a high proportion of these (up to 30%) are Ag specific. These CD8 cells (bottom). All cells are from BA i.n. immunized mice. 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 molecules or that following entry or immunization in situ, lung with other data (2). Most likely either CD4 or CD8 cells can me- ␣ resident cells up-regulate the integrin 4 chain (19, 21). diate protection equally well after prime boost immunization because both boosting with recombinant adenoviruses as shown in Discussion this study and by others (9), or recombinant vaccinia viruses that Our data show that the magnitude of the immune response in the induce largely CD4 responses (4), can reduce lung mycobacterial spleen does not correlate with reduction in lung mycobacterial burden if given by an appropriate route (i.n.). CFU following aerosol challenge with M. tuberculosis in BALB/c There does not appear to be a major difference in Ag specificity mice primed with BCG and boosted with Ad85A i.d. or i.n. The or function between splenic and lung-resident cells induced by i.d. BA i.d. regime induces the largest splenic 85A Ag-specific CD4 or i.n. boosting, respectively. Both the CD4 and the CD8 responses and CD8 cytokine response and yet does not reduce CFU com- are against the same dominant epitopes of Ag 85A, in line with pared with BCG alone. A similar lack of correlation between re- other data in H-2d mice (4, 17, 18). Therefore the exact specificity duction in CFU and the magnitude of the splenic response has been of the cells does not provide an explanation why the cells in the shown following parenteral boosting with a different Ad85A con- lung protect, whereas the splenic cells do not. Although we have struct (9), modified vaccinia Ankara expressing M. tuberculosis demonstrated subtle differences in the MFI of cytokine-producing Ag 85A (MVA85A) (5) or recombinant Ag 85B (3). Similarly the cells from the spleen and lung in i.n. and i.d. immunized mice, magnitude of the blood cytokine response to 85A mirrors that for there may be other differences in cytokines that we have not mea- the spleen and does not correlate with CFU reduction either (data sured and that also contribute to successful protective immunity. not shown). For example IL-17 is known to be important for mycobacterial The Journal of Immunology 4963 immunity (5, 23), whereas it is also possible that i.d. boosting cific and 85A Ag-specific lung cells from well-protected animals induces regulatory T cells, which can interfere with effective anti- have high levels of intracellular cytokines. Additional experiments mycobacterial responses (3, 24). will be required to show whether this property is important for However, because 85A Ag-specific splenic cells transferred in- protection. The data have important implications for the design of tratracheally can protect (17) and lung lymphocytes from BA i.n. human vaccines against M. tuberculosis first because they confirm immunized mice and splenic lymphocytes from BA i.d. mice re- data of many others, indicating that the magnitude and nature of duce mycobacterial CFU in infected macrophages equally well, we immune responses measured in the spleen (or blood) do not cor- consider that the key element in protection against aerosol chal- relate well with protection. Secondly they suggest that local im- lenge afforded by i.n. boosting with Ad85A, is the induction of a munity at the portal of entry is an important mechanism by which lung resident population of Ag specific cells, present at the time of booster vaccines confer increased protection against mycobacterial challenge. We suggest that following M. tuberculosis challenge, infection. Challenges for the future will include the design of im- there is a balance between M. tuberculosis and the immune re- munization regimes that can safely induce long lasting lung resi- sponse. If there is a large immune population in the lung (such as dent memory in humans and domestic animals and that can induce that provided by i.n. immunization), these cells immediately re- high-level cytokine-producing cells. There is also a pressing need spond to the challenge organisms and can rapidly kill many of to identify accessible, bloodborne correlates of protection. them. Abundant evidence indicates that tissue resident populations of T cells specific for other Ags have the properties of effector Disclosures memory or activated effectors (21, 25, 26) and even central mem- The authors have no financial conflict of interest. ory cells that enter the lung acquire effector memory phenotype Downloaded from and function (27). The lung cells in our BA i.n. immunized mice References also show this phenotype (data not shown) and are therefore poised 1. World Health Organization. 2007. Global tuberculosis control: surveillance, for effector function. Although purified T cells transferred into the planning, financing. WHO/HTM/TB/2007.376, Geneva, Switzerland. 2. Mittrucker, H. W., U. Steinhoff, A. Kohler, M. Krause, D. Lazar, P. Mex, airways cause a reduction in CFU after challenge (17), we cannot D. Miekley, and S. H. Kaufmann. 2007. 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clearance of Mycobacterium tuberculosis. J. Immunol. 178: 2661–2665. tract impacts on the immune response. Immunology 122: 316–325. http://www.jimmunol.org/ by guest on September 24, 2021