This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Umemura, M.
Right arrow Articles by Yoshikai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Umemura, M.
Right arrow Articles by Yoshikai, Y.

 Previous Article  |  Next Article 

Infection and Immunity, October 2003, p. 6045-6048, Vol. 71, No. 10
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.10.6045-6048.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Interleukin-15 as an Immune Adjuvant To Increase the Efficacy of Mycobacterium bovis Bacillus Calmette-Guérin Vaccination

Masayuki Umemura,1 Hitoshi Nishimura,2* Kimika Saito,2 Toshiki Yajima,2 Goro Matsuzaki,1 Satoru Mizuno,3 Isamu Sugawara,3 and Yasunobu Yoshikai2

Molecular Microbiology Group, Center of Molecular Biosciences, University of the Ryukyus, Okinawa 903-0213,1 Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582 ,2 Department of Molecular Pathology, the Research Institute of Tuberculosis, Kiyose 204-0022, Japan3

Received 17 March 2003/ Returned for modification 7 May 2003/ Accepted 30 June 2003


arrow
ABSTRACT
 
Interleukin-15 (IL-15) transgenic mice which had been inoculated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) 24 weeks previously showed resistance against airborne infection with Mycobacterium tuberculosis H37Rv accompanied by an increased CD8+-Tc1-cell response. IL-15 may be used as an immune adjuvant given with BCG vaccination to enhance its biologic efficacy.


arrow
TEXT
 
Mycobacteriumbovis bacillus Calmette-Guérin (BCG) is the live vaccine approved for prevention of Mycobacterium tuberculosis infection, especially in infants (17). However, it confers incomplete protection against tuberculosis, at least in adults (11). Protection against chronic pulmonary tuberculosis requires sustained cellular immunity mediated by CD8+ Tc1 cells (1, 2, 5, 13), and it is not clear that BCG is effective for inducing such long-term cellular immunity sufficient for protection against the pulmonary disease.

Antigen (Ag)-specific memory T-cell responses are of vital importance in establishment of protective immunity to microbial infection. Interleukin-15 (IL-15) has been reported to have an important function in the proliferation and survival of memory CD8+ T cells of the CD44high CD62L+ Ly6C+ phenotype (6, 7, 14). It has previously been found that IL-15 transgenic (Tg) mice have an increased number of Ag-driven memory CD8+ T cells following microbial exposure (9, 12, 15). To provide implications for future strategies on development of immune adjuvants to increase efficacy of BCG vaccination, we examined the vaccine's effectiveness in IL-15 Tg mice for inducing protection against airborne infection with M. tuberculosis H37Rv.

IL-15 Tg mice with the C57BL/6 background, which were constructed by using previously described IL-15 gene cDNA under the control of a major histocompatibility complex (MHC) class I promoter, have been described previously (9). In each experiment, age- and sex-matched C57BL/6 mice, purchased from Charles River Japan Inc. (Hino, Japan), were used as controls. All mice were used at 6 to 8 weeks of age. M. bovis BCG (Tokyo strain) and M. tuberculosis H37Rv were dissolved in 7H9 medium (Difco, Detroit, Mich.) supplemented with ADC enrichment (Difco). Mice were intraperitoneally (i.p.) immunized with 5 x 106 CFU of M. bovis BCG and 24 weeks later were challenged with M. tuberculosis by being placed into the exposure chamber of an aerosol generator (Glas-Col, Inc., Terre Haute, Ind.) in which the nebulizer compartment was filled with 5 ml of a suspension containing 106 CFU of tubercle bacilli under conditions that would introduce about 100 bacteria into the lungs of each animal (16). Bacterial counts in the lungs were determined 10 weeks after aerosol infection. For histological examination, the left lobes of the lungs were excised and fixed with 20% formalin-buffered methanol solution, Mildform 20NM (containing 8% formaldehyde and 20% methanol; Wako Pure Chemical Co., Osaka, Japan), dehydrated with a graded ethanol series, treated with xylene, and embedded in paraffin. Sections 5 µm thick were cut from each paraffin block and stained with hematoxylin and eosin. For intracellular cytokine staining, splenocytes (2 x 106 cells/ml) were incubated without any stimulation or with 5 µg of purified protein derivative (PPD; Japan BCG association, Tokyo, Japan)/ml and 100 pg of recombinant IL-2 (Takeda Chemical, Osaka, Japan)/ml for 6 h at 37°C and 5% CO2, with 10 µg of brefeldin A (Sigma)/ml added for the last 2 h, in 24-well flat-bottomed plates (Falcon; Becton Dickinson, Oxford, United Kingdom) in a volume of 1 ml of RPMI medium containing 10% fetal calf serum. After 6 h of culture, the cells were harvested, washed once in Hanks balanced salt solution containing 2.5% newborn horse serum and 0.1% NaN3 (staining buffer), and surface stained with staining buffer with Cy-Chrome-conjugated anti-CD4 monoclonal antibody (MAb) and phycoerythrin-conjugated anti-CD8 MAb. After surface staining, cells were subjected to intracellular cytokine staining by using the Fast Immune Cytokine System (Becton Dickinson) according to the manufacturer's instructions. For intracellular cytokine staining, we used fluorescein isothiocyanate (FITC)-anti-gamma interferon (IFN-{gamma}) MAbs or FITC-conjugated rat immunoglobulin G1 or immunoglobulin G2b as an isotype control. Samples were processed in a FACSCalibur flow cytometer and analyzed by using CELLQuest software.

To investigate whether overexpression of IL-15 can increase the efficacy of BCG vaccination for protection against M. tuberculosis infection, we examined bacterial growth in lungs of BCG-immunized IL-15 Tg mice or non-Tg mice 7 or 10 weeks after aerosol infection with M. tuberculosis H37Rv. Both groups of mice had been i.p. immunized with BCG 24 weeks before aerosol infection with M. tuberculosis. There was no difference in bacterial numbers in lungs of non-Tg mice and IL-15 Tg mice without BCG immunization at 7 weeks (data not shown) or 10 weeks (Fig. 1) postinfection. However, the bacterial numbers in lungs were significantly lower in BCG-immunized IL-15 Tg mice than in BCG-immunized non-Tg mice 10 weeks postinfection (Fig. 1) (P < 0.05).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 1. Mycobacterial growth in BCG-immunized IL-15 Tg mice after airborne infection with M. tuberculosis H37Rv. IL-15 Tg and non-Tg mice were challenged i.p. with 5 x 106 CFU of M. bovis BCG. Twenty-four weeks after immunization with BCG, mice were infected with M. tuberculosis H37Rv by the airborne route in the exposure chamber of an airborne infection apparatus. Ten weeks later, the numbers of bacteria recovered from the lungs were determined. Data are expressed as the means ± SD of results for five mice and are representative of results from three separate experiments. Statistical analysis was performed with Student's t test (*, P < 0.05; **, P < 0.001). +, immunized; -, not immunized.

We next examined histological changes in lungs 10 weeks after airborne infection with M. tuberculosis. No significant difference was found in granuloma numbers and sizes between non-Tg mice and IL-15 Tg mice, both of which had not been immunized with BCG. Although BCG immunization reduced the histopathological changes in lungs of both groups, the granuloma numbers and sizes were significantly smaller in BCG-immunized IL-15 Tg mice than in BCG-immunized non-Tg mice (Fig. 2). Thus, the efficacy of BCG vaccination for protection against M. tuberculosis infection was augmented in IL-15 Tg mice.



View larger version (111K):
[in this window]
[in a new window]
  		FIG. 2. Results of histologic examination of lung tissues from BCG-immunized IL-15 Tg mice after airborne infection with M. tuberculosis H37Rv. Twenty-four weeks after immunization with 5 x 106 CFU of M. bovis BCG, IL-15 Tg and non-Tg mice were infected with M. tuberculosis H37Rv by the airborne route in the exposure chamber of a Middlebrook airborne infection apparatus. Mice were sacrificed 10 weeks after airborne infection with M. tuberculosis H37Rv, and formalin-fixed sections were stained with hematoxylin and eosin. The granuloma regions in lung tissues from nonimmunized non-Tg mice (A), nonimmunized IL-15 Tg mice (B), BCG-immunized non-Tg mice (C), and BCG-immunized IL-15 Tg mice (D) are shown. Magnification, x100. An example from one of three separate experiments is shown. +, immunized; -, not immunized.

To investigate whether Ag-specific T cells were sustained more efficiently in IL-15 Tg mice after BCG vaccination, cytokine fluorescence-activated cell sorter analysis of the expression of CD4, CD8, and intracellular IFN-{gamma} in response to PPD and IL-2 was carried out with T cells. Typical results of fluorescence-activated cell sorter analysis are shown in Fig. 3, and relative amounts of lymphocytes having intracellular IFN-{gamma} in each group are shown in Table 1, with values expressed as means ± standard deviations (SD). A large number of CD8+ T cells from the BCG-infected IL-15 Tg mice produced IFN-{gamma} in response to PPD and recombinant IL-2. These results indicated that the generation of CD8+ Tc1 cells was significantly increased in BCG-immunized IL-15 Tg mice following aerosol infection with M. tuberculosis.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 3. Intracellular expression of IFN-{gamma} in splenocytes from BCG-immunized IL-15 Tg mice after airborne infection with M. tuberculosis H37Rv. IL-15 Tg and non-Tg mice were i.p. inoculated with 5 x 106 CFU of M. bovis BCG. Twenty-four weeks after immunization with BCG, mice were infected by the airborne route in the exposure chamber of a Middlebrook airborne infection apparatus. Splenocytes (2 x 106 cells) were collected from mice of each group 10 weeks after airborne infection and cultured with 5 µg of PPD/ml and 100 pg of recombinant human IL-2 (rIL-2)/ml for 6 h at 37°C, with 10 µg of brefeldin A/ml added for the last 2 h. After culture, the cells were surface stained with Cy-Chrome-conjugated anti-CD4 and phycoerythrin-conjugated anti-CD8 MAbs. Surface-stained cells were subjected to intercellular cytokine staining. For intracellular cytokine staining, we used FITC-conjugated anti-IFN-{gamma} MAb. Samples were analyzed by flow cytometry. An example of results from one of three separate experiments is shown.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Intracellular IFN-{gamma}-producing cells among spleen T cells from IL-15 Tg mice after M. tuberculosis H37Rv infection

By promoting cell survival and proliferation, IL-15 plays an important role in long-term maintenance of Ag-specific memory CD8+ T cells following microbial exposure (15). CD8+ Tc1 cells are known to play a requisite role in resistance to M. tuberculosis infection during the latent phase (6). The findings presented here indicate that BCG-immunized IL-15 Tg mice had augmented Tc1 responses at 10 weeks after aerosol infection with M. tuberculosis. It is suggested that Ag-specific memory CD8+ T cells are efficiently generated and/or sustained for a long period following BCG vaccination under circumstances of IL-15 overexpression and that the memory CD8+ T cells rapidly respond to the aerosol infection with M. tuberculosis, conferring strong protection against M. tuberculosis infection in the lungs of IL-15 Tg mice. Maeurer et al. reported that IL-15 enhanced survival of M. tuberculosis-infected mice when it was delivered 3 weeks postinfection but not when it was administrated at the time of infection (8). IL-15 may play a more important role in maintenance than that of generating memory CD8+ T cells.

CD8+ T cells from BCG-infected IL-15 Tg mice produced IFN-{gamma} in response to exogenous PPD in the presence of Ag-presenting cells (APCs). Although exogenous particulate Ags such as PPD are usually present in association with MHC class II, Ags in the extracellular milieu can also be processed and presented in association with MHC class I (10), which is usually required for activation of CD8+ T cells. Therefore, it is possible that PPD may be presented by MHC class I on APCs in an alternative processing pathway. However, brefeldin A inhibits the TAP(transporters associated with antigen processing)-dependent MHC class I pathway and TAP-deficient mice are reported to be susceptible to M. tuberculosis infection. These data suggest that TAP-dependent Ag presentation, a major pathway for activation of CD8+ T cells, is critical for protection against M. tuberculosis infection (1). PPD may contain Ags which can bind directly to MHC class I on APCs, which activate Tc1 cells in BCG-immunized IL-15 Tg mice infected with M. tuberculosis.

It has previously been reported that IL-15 Tg mice showed resistance against an avirulent strain of BCG as assessed by measuring bacterial loads 3 weeks after i.p. infection with BCG (12). On the other hand, the present study revealed that there was no difference in bacterial loads at 10 weeks after aerosol infection with M. tuberculosis between non-Tg mice and IL-15 Tg mice that had not been given BCG vaccination. These results suggest that overexpression of IL-15 alone cannot confer protection against M. tuberculosis. It has been recently reported that virulent but not avirulent M. tuberculosis can evade the host defense mechanisms of Th1 and Tc1 responses (4). Components other than T-cell receptor {alpha}ß T cells are known to contribute to immunity of mice to infection with avirulent BCG (3). It is plausible, therefore, that the difference in susceptibilities of nonimmunized IL-15 Tg mice to BCG and M. tuberculosis is due at least partly to differences in virulence levels of BCG and M. tuberculosis H37Rv. We have found that Ag-specific CD8+-Tc1-cell responses capable of producing IFN-{gamma} persist for more than 24 weeks after BCG vaccination in IL-15 Tg mice, protecting them against airborne infection with M. tuberculosis. IL-15 can be used together with BCG as an immune adjuvant to increase efficacy of BCG vaccination.


arrow
ACKNOWLEDGMENTS
 
We thank C. Yamada, K. Itano, and A. Nishikawa for providing excellent technical support.

This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas, Japan Society for the Promotion of Science.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Division of Host Defense, Medical Institute of Bioregulation, Kyushu University 3-1-1, Maidashi, Higashiku, Fukuoka 812-8582, Japan. Phone: 81-92-642-6962. Fax: 81-92-642-6973. E-mail: nishihit{at}bioreg.kyushu-u.ac.jp. Back

Editor: J. D. Clements


arrow
REFERENCES
 
    1
  1. Behar, S. M., C. C. Dascher, M. J. Grusby, C. R. Wang, and M. B. Brenner. 1999. Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J. Exp. Med. 189:1973-1980.[Abstract/Free Full Text]
    2. 2
  2. Flynn, J. L., M. M. Goldstein, K. J. Triebold, B. Koller, and B. R. Bloom. 1992. Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. USA 89:12013-12017.[Abstract/Free Full Text]
    3. 3
  3. Izzo, A. A., and R. J. North. 1992. Evidence for an alpha/beta T cell-independent mechanism of resistance to mycobacteria. Bacillus-Calmette-Guérin causes progressive infection in severe combined immunodeficient mice, but not in nude mice or in mice depleted of CD4+ and CD8+ T cells. J. Exp. Med. 176:581-586.[Abstract/Free Full Text]
    4. 4
  4. Jung, Y.-J., R. LaCourse, L. Ryan, and R. J. North. 2002. Virulent but not avirulent Mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1-dependent, nitric oxide synthase 2-independent defense in mice. J. Exp. Med. 196:991-998.[Abstract/Free Full Text]
    5. 5
  5. Kaufmann, S. H. E. 1993. Tuberculosis: the role of the immune response. Immunologist 1:109-114.
    6. 6
  6. Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J. L. Viney, M. Embers, N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, K. Brasel, P. J. Morrissey, K. Stocking, J. C. Schuh, S. Joyce, and J. J. Peschon. 2000. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J. Exp. Med. 191:771-780.[Abstract/Free Full Text]
    7. 7
  7. Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, and A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669-676.[CrossRef][Medline]
    8. 8
  8. Maeurer, M. J., P. Trinder, G. Hommel, W. Walter, K. Freitag, D. Atkins, and S. Storkel. 2000. Interleukin-7 or interleukin-15 enhances survival of Mycobacterium tuberculosis-infected mice. Infect. Immun. 68:2962-2970.[Abstract/Free Full Text]
    9. 9
  9. Nishimura, H., T. Yajima, Y. Naiki, H. Tsunobuchi, M. Umemura, K. Itano, T. Matsuguchi, M. Suzuki, P. S. Ohashi, and Y. Yoshikai. 2000. Differential roles of interleukin 15 mRNA isoforms generated by alternative splicing in immune responses in vivo. J. Exp. Med. 191:157-170.[Abstract/Free Full Text]
    10. 10
  10. Pfeifer, J. D., M. J. Wick, R. L. Roberts, K. Findlay, S. J. Normark, and C. V. Harding. 1993. Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature 361:359-362.[CrossRef][Medline]
    11. 11
  11. Roche, P. W., J. A. Triccas, and N. Winter. 1995. BCG vaccination against tuberculosis: past disappointments and future hopes. Trends Microbiol. 3:397-401.[CrossRef][Medline]
    12. 12
  12. Umemura, M., H. Nishimura, K. Hirose, T. Matsuguchi, and Y. Yoshikai. 2001. Overexpression of IL-15 in vivo enhances protection against Mycobacterium bovis bacillus Calmette-Guerin infection via augmentation of NK and T cytotoxic 1 responses. J. Immunol. 167:946-956.[Abstract/Free Full Text]
    13. 13
  13. Van Pinxteren, L. A. H., J. P. Cassidy, B. H. C. Smedegaard, E. M. Agger, and P. Anderson. 2000. Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells. Eur. J. Immunol. 30:3689-3698.[CrossRef][Medline]
    14. 14
  14. Waldmann, T. A., and Y. Tagaya. 1999. The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens. Annu. Rev. Immunol. 17:19-49.[CrossRef][Medline]
    15. 15
  15. Yajima, T., H. Nishimura, R. Ishimitsu, T. Watase, D. H. Busch, E. G. Pamer, H. Kuwano, and Y. Yoshikai. 2002. Overexpression of IL-15 in vivo increases antigen-driven memory CD8+ T cells following a microbe exposure. J. Immunol. 168:1198-1203.[Abstract/Free Full Text]
    16. 16
  16. Yamada, H., S. Mizuno, M. Reza-Gholizadeh, and I. Sugawara. 2001. Relative importance of NF-{kappa}B p50 in mycobacterial infection. Infect. Immun. 69:7100-7105.[Abstract/Free Full Text]
    17. 17
  17. Young, D. B., and K. Duncan. 1995. Prospects for new interventions in the treatment and prevention of mycobacterial disease. Annu. Rev. Microbiol. 49:641-673.[CrossRef][Medline]

Infection and Immunity, October 2003, p. 6045-6048, Vol. 71, No. 10
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.10.6045-6048.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Tang, C., Yamada, H., Shibata, K., Yoshida, S.-i., Wajjwalku, W., Yoshikai, Y. (2009). IL-15 protects antigen-specific CD8+ T cell contraction after Mycobacterium bovis bacillus Calmette-Guerin infection. J. Leukoc. Biol. 86: 187-194 [Abstract] [Full Text]  
  • Saikh, K. U., Kissner, T. L., Nystrom, S., Ruthel, G., Ulrich, R. G. (2008). Interleukin-15 Increases Vaccine Efficacy through a Mechanism Linked to Dendritic Cell Maturation and Enhanced Antibody Titers. CVI 15: 131-137 [Abstract] [Full Text]  
  • Bolesta, E., Kowalczyk, A., Wierzbicki, A., Eppolito, C., Kaneko, Y., Takiguchi, M., Stamatatos, L., Shrikant, P. A., Kozbor, D. (2006). Increased Level and Longevity of Protective Immune Responses Induced by DNA Vaccine Expressing the HIV-1 Env Glycoprotein when Combined with IL-21 and IL-15 Gene Delivery. J. Immunol. 177: 177-191 [Abstract] [Full Text]  
  • Toka, F. N., Rouse, B. T. (2005). Mucosal application of plasmid-encoded IL-15 sustains a highly protective anti-Herpes simplex virus immunity. J. Leukoc. Biol. 78: 178-186 [Abstract] [Full Text]  
  • Kutzler, M. A., Robinson, T. M., Chattergoon, M. A., Choo, D. K., Choo, A. Y., Choe, P. Y., Ramanathan, M. P., Parkinson, R., Kudchodkar, S., Tamura, Y., Sidhu, M., Roopchand, V., Kim, J. J., Pavlakis, G. N., Felber, B. K., Waldmann, T. A., Boyer, J. D., Weiner, D. B. (2005). Coimmunization with an Optimized IL-15 Plasmid Results in Enhanced Function and Longevity of CD8 T Cells That Are Partially Independent of CD4 T Cell Help. J. Immunol. 175: 112-123 [Abstract] [Full Text]  
  • Lazarevic, V., Yankura, D. J., Divito, S. J., Flynn, J. L. (2005). Induction of Mycobacterium tuberculosis-Specific Primary and Secondary T-Cell Responses in Interleukin-15-Deficient Mice. Infect. Immun. 73: 2910-2922 [Abstract] [Full Text]  
  • Blaser, B. W., Roychowdhury, S., Kim, D. J., Schwind, N. R., Bhatt, D., Yuan, W., Kusewitt, D. F., Ferketich, A. K., Caligiuri, M. A., Guimond, M. (2005). Donor-derived IL-15 is critical for acute allogeneic graft-versus-host disease. Blood 105: 894-901 [Abstract] [Full Text]  
  • Hovav, A.-H., Fishman, Y., Bercovier, H. (2005). Gamma Interferon and Monophosphoryl Lipid A-Trehalose Dicorynomycolate Are Efficient Adjuvants for Mycobacterium tuberculosis Multivalent Acellular Vaccine. Infect. Immun. 73: 250-257 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Umemura, M.
Right arrow Articles by Yoshikai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Umemura, M.
Right arrow Articles by Yoshikai, Y.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»