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International Immunology Advance Access originally published online on September 11, 2006
International Immunology 2006 18(11):1531-1539; doi:10.1093/intimm/dxl086
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CD27 contributes to the early systemic immune response to Mycobacterium tuberculosis infection but does not affect outcome

Catharina W. Wieland1,2, Marjolein E. Kerver1,2, Sandrine Florquin3, Martijn A. Nolte1,4, Jannie Borst5, Rene van Lier1,4, Marinus H. J. van Oers1,6 and Tom van der Poll1,2

1 Center of Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
2 Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
3 Department of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
4 Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
5 Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
6 Department of Hematology, Academic Medical Center, University of Amsterdam, The Netherlands

Correspondence to: C. W. Wieland; E-mail: c.wieland{at}amc.uva.nl


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The development of a strong Th1-mediated adaptive immune response is considered of main importance for host defense against the intracellular pathogen Mycobacterium tuberculosis. The induction of a cellular immune response is not only dependent on the engagement of the TCR but also requires co-stimulation. In order to study the role of the co-stimulatory molecule of the tumor necrosis factor receptor family member CD27 during murine M. tuberculosis infection, we intranasally infected wild-type (WT) and CD27 knockout (KO) mice with 105 colony-forming units M. tuberculosis. Whereas there were no differences in bacterial growth, inflammation and IFN{gamma} production by CD4+ and CD8+ lymphocytes in the lungs early after infection, the number of splenic CD8+ T cells producing the key Th1 cytokine IFN{gamma} was lower in CD27 KO mice than in WT mice. After 6 weeks, CD27 KO mice had 3.6-fold higher mycobacterial counts in their lungs and displayed more pulmonary inflammation and increased numbers of infiltrated leukocytes. Despite these differences early in infection, an equal number of WT and CD27 KO mice died during a 43-week observation period and lung bacterial loads and inflammation were comparable in the surviving animals. Our data suggest that CD27 does not contribute to the local IFN{gamma}-mediated response and long-term protection against M. tuberculosis.

Keywords: bacterial, co-stimulation, lung, rodent, T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Mycobacterium tuberculosis and its associated disease tuberculosis is one of the three leading causes of death by infectious disease worldwide (1, 2). In the majority of healthy individuals, a strong Th1 response develops, but although this Th1 response is able to contain the infection in granulomas and prevent disease, M. tuberculosis bacilli are not eradicated from the lungs and remain a potential threat to the infected individual. When host immune defenses become compromised, infected individuals are at risk of reactivation of the disease and developing tuberculosis.

It is widely accepted that proper CD4+ and CD8+ T cell function is pivotal for control of M. tuberculosis infection (3). Interactions between T cells and antigen-presenting cells like M. tuberculosis-infected macrophages are essential and tightly regulated. T cell activation not only depends on engagement of the TCR but also requires co-stimulation. One of the important co-stimulatory receptors during the adaptive immune response is the tumor necrosis factor receptor (TNFR) family member CD27 (4, 5). In mice, CD27 is expressed on most CD4+ and CD8+ T cells in peripheral lymphoid organs and on a large proportion of NK cells (6–8). The contribution of CD27 to the immune response is tightly regulated by the expression of its ligand CD70, which is controlled by antigen presentation and Toll-like receptor stimulation on T cells, B cells and dendritic cells (9, 10). From in vitro studies, CD27 has long been known as a co-stimulator of T cell and B cell responses and engagement of CD27 with antibody or its ligand CD70 has been demonstrated to affect T cell proliferation, cytokine production, IgG production by B cells and plasma cell differentiation in the presence of other co-stimulatory signals (7, 8,11–15). Several earlier studies focused on the role of CD27 during viral infection and herein the most dramatic defect in CD27 knockout (KO) mice concerned reduced T cell memory, in particular of CD8+ T cells (16–19). The role of CD27 during bacterial infection is less well defined.

By making use of CD27 KO mice, we studied the role of CD27 during pulmonary infection with M. tuberculosis. We found that although CD27 KO mice were as susceptible to tuberculosis as WT animals in the long term, the host response of CD27 KO mice was altered during the first 6 weeks of infection: CD27 KO mice displayed higher pulmonary bacterial counts, enhanced pulmonary inflammation and a reduced IFN{gamma} response after M. tuberculosis-specific stimulation of splenocytes but not of local T cells. Our data suggest that CD27 plays a minor role during the initiation of the systemic adaptive immune response against M. tuberculosis.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Eight- to ten-week-old age and sex-matched CD27 KO mice backcrossed eight times to a C57BL/6 background were generated as described previously (16). Wild-type (WT) C57BL/6 control mice were purchased from Harlan Spague Dawley (The Hague, The Netherlands). All animal experiments were performed according to institutional and national guidelines and approved by the Animal Care and Use Committee of the University of Amsterdam.

Experimental infection
Mycobacterium tuberculosis H37Rv was grown for 4 days in liquid Dubos medium containing 0.01% Tween 80. A replicate culture was incubated at 37°C, harvested at mid-log phase and stored in aliquots at –70°C. For each experiment, a vial was thawed and washed with sterile 0.9% NaCl. Lung infection was induced as described previously (20–22). Briefly, mice were anesthetized by inhalation with isoflurane (Abott Laboratories Ltd, Kent, UK) and infected intranasally with 105 colony-forming units (CFU) M. tuberculosis in 50 µl saline. After 1 day, 3 and 6 weeks of infection, mice were killed, lungs and part of liver were removed aseptically and homogenized in five volumes of sterile 0.9% NaCl. Ten-fold dilutions were plated on Middlebrook 7H11 agar plates to determine bacterial loads. Colonies were counted after 3 weeks at 37°C. Detection limit of bacterial growth is 100 CFU g–1 organ.

Histology
Lungs were harvested for histology 3 and 6 weeks after infection, fixed in 10% buffered formalin and embedded in paraffin. Four-micrometer sections were stained with hematoxylin and eosin (H&E), and analyzed by a pathologist who was blinded for groups. To score lung inflammation and damage, the entire lung surface was analyzed with respect to the following parameters: interstitial inflammation, endothelialitis, bronchitis, granuloma formation, edema and pleuritis. Each parameter was graded on a scale of 0–4, with 0: absent; 1: mild; 2: moderate; 3: severe and 4: very severe. The total ‘lung inflammation score’ was expressed as the sum of the scores for each parameter, the maximum being 24. Confluent (diffuse) inflammatory infiltrate was quantified separately and expressed as percentage of the lung surface.

Characterization of inflammatory infiltrates in the lungs
Pulmonary cell suspensions were obtained by crushing lungs through a 40-µm cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA) as described previously (20, 22). Erythrocytes were lysed with ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4); the remaining cells were washed twice with RPMI 1640 (Bio Whittaker, Verviers, Belgium), and counted by using a hemocytometer. The percentages of macrophages, polymorphonuclear cells and lymphocytes were determined using cytospin preparations stained with H&E.

Splenocyte stimulation
Single-cell suspensions were obtained by crushing spleens through a 40-µm cell strainer (Becton Dickinson) as described (20, 22). Erythrocytes were lysed with ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4); the remaining cells were washed twice with RPMI 1640 (Bio Whittaker) supplemented with 10% FCS and 1% antibiotic–antimycotic (GiboBRL, Life Technologies, Rockville, MD, USA). Cells were seeded in 96-well round-bottom culture plates at a cell density of 1 x 106 cells per well in quadruplicate, and stimulated with 20 µg ml–1 purified protein derivative (PPD). Supernatants were harvested after 48-h incubation at 37°C in 5% CO2, filter sterilized and stored until assays were performed. For intracellular IFN{gamma} staining, a similar approach was used. Splenocytes and lung cells from infected mice were stimulated with PPD (20 µg ml–1) in medium supplemented with anti-CD28 (2.5 µg ml–1; PharMingen, San Diego, CA, USA) for 6 h at 37°C in 5% CO2. After 1 h of stimulation, GolgiPlug (PharMingen) was added to prevent secretion of the produced cytokines. Cells were then fixed in 2% PFA, aspecific binding was blocked using FcR blocker (PharMingen; 20 min at 4°C) and cells were incubated with FITC-conjugated antibody against CD4 and allophycocyanin (APC)-conjugated antibody against CD8 for 30 min at 4°C (antibodies; PharMingen). After washes with PBS with BSA (0.5%), cells were permeabilized using Cytofix/Cytoperm solution (PharMingen), washed again with Perm/wash solution (PharMingen) and incubated with a PE-conjugated antibody against IFN{gamma} (PharMingen). Cells were washed, re-suspended in PBS/0.5% BSA and analyzed by flow cytometry. The percentage of IFN{gamma}-positive cells was determined in the lymphocyte and CD4+ or CD8+ gates. Absolute number of double-positive cells was calculated per stimulation of 106 splenocytes. As a control, isotype IgG was used for the IFN{gamma} staining.

Flow cytometric analysis
Lung and mediastinal lymph node cell suspensions obtained from infected mice were analyzed by flow cytometry using FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) as described previously (20, 22). Cells were brought to a concentration of 4 x 106 cells ml–1 of FACS buffer (PBS supplemented with 0.5% BSA, 0.01% NaN3 and 0.35 mM EDTA). Immunostaining for cell-surface molecules was performed for 30 min at 4°C using directly labeled antibodies against CD3 (CD3–PE), CD4 (CD4–APC), CD69 (CD69–FITC) and CD8 (CD8–PerCP). All antibodies were used in concentrations recommended by the manufacturer (PharMingen). After staining, cells were fixed in 2% PFA, and T cell-surface molecules were analyzed on CD3+ cells within the lymphocyte gate.

Cytokine and chemokine measurements
For cytokine measurements, lung homogenates were diluted 1:2 in lyses buffer containing 300 mM NaCl, 30 mM Tris, 2 mM MgCl2, 2 mM CaCl2, 1% Triton X-100, and Pepstatin A, Leupeptin and Aprotinin (all 20 ng ml–1, pH 7.4) and incubated on ice for 30 min. Homogenates were centrifuged at 1500 x g at 4°C for 15 min; supernatants were sterilized using a 0.22-µm filter (Corning Incorporated, Corning, NY, USA) and stored at –20°C until assays were performed. IFN{gamma}, IL-4, IL-12p70, TNF, IL-1ß, IL-6, cytokine-induced neutrophil chemoattractant (KC) and Macrophage Inflammatory Protein 2 were measured using ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The detection limits were 62 pg ml–1 for IFN{gamma}, IL-4, IL-12p70, TNF, IL-1ß and MIP-2, 31 pg ml–1 for IL-4, 37 pg ml–1 for KC and 7.8 pg ml–1 for IL-6.

Statistical analysis
All values are expressed as mean ± SEM unless indicated otherwise. Comparisons were done with Mann–Whitney U-tests using GraphPad Prism version 4.00, GraphPad Software (San Diego, CA, USA). Survival curves were compared by log-rank test. When comparing two groups at multiple time points, two-way analysis of variance (ANOVA) was used. If appropriate, ANOVAs were followed by Bonferroni post-test. Statistical analyses of bacterial counts were performed after log transformation. Values of P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Mycobacterial growth
In order to obtain insight in the growth of M. tuberculosis in the presence or absence of CD27, we determined mycobacterial counts in the lungs 1 day, 3 and 6 weeks after infection (Fig. 1). At 1 day and 3 weeks post-infection, the numbers of bacteria in the lungs were comparable in WT and CD27 KO mice. At 6 weeks of infection, however, the lungs of CD27 KO mice contained 3.6-fold more M. tuberculosis than the lungs of WT mice (P < 0.01). In contrast to loads in the lungs, mycobacterial counts in the liver were comparable in WT and CD27 KO mice at all time points (Fig. 1).


Figure 1
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  		Fig. 1 Enhanced pulmonary bacterial growth in lungs of CD27 KO mice after 6 weeks of infection. Mycobacterial loads in lungs: WT (closed symbols) and CD27 KO (open symbols) were infected intranasally with 105 CFU of Mycobacterium tuberculosis. After 1 day, 3 and 6 weeks of infection, mice were sacrificed and bacterial loads were determined in lung (A) and liver (B) homogenates. After 1 day, M. tuberculosis could not be recovered from liver (<102 CFU g–1). Data are means ± SEM of three (1 day) or eight mice per group and time point. **P< 0.01.

 
CD27 KO mice display more pulmonary inflammation
Histopathological analysis of the lungs 3 weeks after infection showed a prominent interstitial infiltrate consisting of macrophages/monocytes, lymphocytes and occasional neutrophils surrounding mainly small airways and vessels in both WT and CD27 KO mice (Fig. 2A and B). At this time point, both groups of mice displayed equal granuloma formation, inflammation of the pleura and a comparable percentage of confluently inflamed lung (Fig. 2 and Table 1). In line, lung inflammation scores and right lung weights were similar at 3 weeks after infection (Table 1). After 6 weeks of infection, the inflammatory infiltrate became more diffuse and dense in both groups, but more pronounced in CD27 KO mice than in WT mice (Fig. 2 and Table 1); in line, lungs of CD27 KO mice were heavier than lungs from WT mice.


Figure 2
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  		Fig. 2 Enhanced inflammation after 6 weeks of infection in CD27 KO mice. Histopathology: representative slides of lungs of WT (A and C) and CD27 KO mice (B and D) infected with 105 CFU of Mycobacterium tuberculosis 3 weeks (A and B) and 6 weeks (C and D) earlier. H&E staining; original magnification x10.

 

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  		Table 1 Inflammation scoresa

 
Cellular composition of lung infiltrates
To obtain more detailed insight into the cellular composition and function of the pulmonary infiltrates, we determined whole lung leukocyte and differential counts and further analyzed lymphocyte subsets and activation status by flow cytometry (Tables 2 and 3). In accordance with the histopathology, lungs of infected CD27 KO mice contained more leukocytes than the lungs of WT mice at 6 weeks, but not at 3 weeks post-infection. This difference in leukocyte counts was mainly due to the enhanced recruitment of macrophages and neutrophils, whereas the number of lymphocytes only tended to be higher in the CD27 KO mice. There were no differences in the distribution of CD3+/CD4+ and CD3+/CD8+ lymphocyte populations in lungs of CD27 KO mice compared with WT mice. The activation status of CD4+ and CD8+ T lymphocytes was comparable as shown by similar percentages of CD69+ cells (Table 3). Moreover, when we investigated functional differences of lung lymphocytes during infection by stimulating lung cells ex vivo and measuring IFN{gamma} production by CD4+ or CD8+ cells, we were not able to detect any differences in the percentage of IFN{gamma}+ cells (Fig. 3) the amount of IFN{gamma} produced per cell [mean fluorescence intensities (MFIs); data not shown] or the absolute number of IFN{gamma}+ cells per lung (data not shown). In addition to lung leukocyte recruitment, we studied the cellularity of a local and systemic lymphoid organ and did not detect any differences in the total number of mediastinal draining lymph node cells and spleen (data not shown). When flow cytometric analysis of draining lymph node was performed, no differences between CD27 KO and WT mice were detected in the T cell populations and their activation status were detected in the draining lymph node of infected lungs (Table 3).


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  		Table 2 Total and differential cell counts lungsa

 

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  		Table 3 T cell subsets in lungs, spleen and draining lymph nodea

 

Figure 3
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  		Fig. 3 Similar IFN{gamma}+ lung lymphocytes upon PPD stimulation. Intracellular IFN{gamma} production in response to the Mycobacterium tuberculosis-specific antigen PPD by CD8+ (upper panel) and CD4+ (lower panel). After 2 weeks of infection with 105 CFU M. tuberculosis, lung cells were isolated from WT (left panels) and CD27 KO (right panels) mice and stimulated with PPD (20 µg ml–1) for 6 h. IFN{gamma}-positive cells were quantified within gated CD4+ or CD8+ cell populations. Representative FACS figures for groups of five mice.

 
Th1 response of ex vivo-stimulated spleen cells
To obtain insight into the effect of CD27 on the development of the Th1 or Th2 response, the ability of splenocytes harvested from infected mice to produce Th1 (IFN{gamma}) and Th2 (IL-4) cytokines upon antigen-specific stimulation with PPD was assessed. Although the total number of cells per spleen did not differ between WT and CD27 KO mice (data not shown), splenocytes from CD27 KO mice infected with M. tuberculosis 3 weeks earlier produced less IFN{gamma} in response to PPD than splenocytes from WT animals (Fig. 4.) In a next step, we investigated whether the reduction of IFN{gamma} was due to a reduced number of antigen-specific cells or to reduced IFN{gamma} production per antigen-specific cell in the spleen of the CD27 KO mice. As shown in Fig. 5, the percentage and absolute number of IFN{gamma}+ cells were reduced in CD8+ lymphocytes from CD27 KO mice when stimulated with the specific antigen PPD (not significant with 3002 ± 397 CD4+/IFN{gamma}+ T cells from WT and 1745 ± 426 CD4+/IFN{gamma}+ T cells from CD27 KO mice; P < 0.05 for the number CD8+/IFN{gamma}+ T cells with 4274 ± 869 CD8+/IFN{gamma}+ T cells from WT mice and 1210 ± 264 CD8+/IFN{gamma}+ T cells from CD27 KO mice, respectively). When we studied the MFI of the IFN{gamma}+ population, no difference in IFN{gamma}+ MFI was detected in CD4+ and CD8+ T cells, indicating that the ability to produce IFN{gamma} was not different in the splenocytes from CD27 KO mice (154.6 ± 10.4 and 123.7 ± 11.2 MFI IFN{gamma} in CD4+ cells; 116.4 ± 10.0 and 136.9 ± 27.3 MFI IFN{gamma} in CD8+ cells for WT and CD27 KO splenocytes, respectively). At 6 weeks after infection, no difference in IFN{gamma} production was detectable between WT and CD27 splenocytes, suggesting that the systemic Th1 response at this time point was similar in both groups. The Th2 cytokine IL-4 was hardly detectable in the supernatants of PPD-stimulated splenocytes and did not differ between groups (Fig. 4).


Figure 4
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  		Fig. 4 Reduced early IFN{gamma} response by splenocytes from infected CD27 KO mice. IFN{gamma} response to the Mycobacterium tuberculosis-specific antigen PPD by splenocytes. After 3 weeks (A and C) and 6 weeks (B and D) of infection with 105 CFU M. tuberculosis, splenocytes were isolated from WT (filled bars) and CD27 KO (open bars) mice and stimulated with PPD (20 µg ml–1) for 48 h. IFN{gamma} (A and B) and IL-4 (C and D) were measured in supernatants. Data are mean ± SEM of eight mice per group and time point. *P < 0.05.

 

Figure 5
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  		Fig. 5 Reduced percentage of CD8+/IFN{gamma}+ cells upon PPD stimulation. Intracellular IFN{gamma} production in response to the Mycobacterium tuberculosis-specific antigen PPD by CD8+ (upper panel) and CD4+ (lower panel). After 2 weeks of infection with 105 CFU M. tuberculosis, splenocytes were isolated from WT (left panels) and CD27 KO (right panels) mice and stimulated with PPD (20 µg ml–1) for 6 h. IFN{gamma}-positive cells were quantified within gated CD4+ or CD8+ cell populations. Representative FACS figures for groups of seven to eight mice.

 
Pulmonary cytokine and chemokine levels
Local production of cytokines and chemokines plays an important role in cell recruitment, granuloma formation and host defense against pulmonary tuberculosis (3). Therefore, we measured IFN{gamma}, IL-4, IL-12p70, TNF, IL-1ß, IL-6 as well as the CXC chemokines MIP-2 and KC in lung homogenates of WT and CD27 KO mice (Table 4). No major differences between CD27 KO and WT mice were found except for IL-1ß concentrations which were higher in the former mouse strain. Moreover, pulmonary KC levels were reduced in CD27 KO mice at 3 weeks post-infection, whereas MIP-2 levels in the lungs of CD27 KO mice were increased compared with WT lung levels 6 weeks after infection (Table 4).


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  		Table 4 Cytokine and chemokine profilea

 
CD27 KO mice are not more susceptible to M. tuberculosis during long-term infection
To investigate the role of CD27 in the long-term response and outcome of tuberculosis, WT and CD27 KO mice were inoculated with M. tuberculosis and monitored for 43 weeks. As shown in Fig. 6, in both groups, 9 out of 14 (64%) mice died during this period. At the end of the experiment, we sacrificed the remaining mice of both groups and determined bacterial loads in lungs and liver (Fig. 6). At this late time point of infection, no differences were detected in the number of M. tuberculosis CFU in lungs and liver between WT and CD27 KO mice (P > 0.05). In addition, lung histopathology did not differ between CD27 KO and WT mice (data not shown).


Figure 6
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  		Fig. 6 CD27 deficiency does not impact on the outcome of chronic infection. WT (n = 14; closed symbols) and CD27 KO mice (n = 14; open symbols) were intranasally infected with 105 CFU of Mycobacterium tuberculosis and followed for 43 weeks (A). After 43 weeks, remaining mice (n = 5 per group) were sacrificed and bacterial loads were determined in lung (B) and liver (C). CFU were presented as means ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
During adaptive immune responses to pathogens, MHC–peptide ligation of the TCR in combination with potent additional or co-stimulatory signals activates lymphocytes and thereby stimulates their proliferation, differentiation and survival. Several members of the TNFR family, like CD27, OX-40 and 4-1BB have been implicated as potentially important T and B cell co-stimulatory molecules during immune responses to infectious agents (4, 5, 12). In case of human M. tuberculosis infection, adequate activation of T cells results in development of a protective Th1 response which is able to contain the infection in the lung.

Herein, we sought to determine the role of the TNFR family member CD27 in the immune response during murine infection with M. tuberculosis. We demonstrated that CD27 KO mice have a transiently impaired defense against tuberculosis as indicated by increased mycobacterial loads in their lungs at 6 weeks after infection. At an earlier time point, we found that CD27 KO mice have a decreased number of CD8+ cells capable of producing IFN{gamma} in the spleen. We did not investigate whether this reduced systemic IFN{gamma} response was directly related to the enhanced bacterial growth in the lung 4 weeks later. More importantly, IFN{gamma} levels measured locally were similar in WT and CD27 KO mice at both 3 and 6 weeks after infection. Moreover, functional analysis of pulmonary lymphocytes demonstrated that an equal number of local CD4+ and CD8+ cells from infected WT or CD27 KO responded to PPD. Of note, 3.6x higher bacterial loads are not considered to represent a biologically relevant difference. It was shown before that the percentage of IFN{gamma}-positive CD4+ and CD8+ T cells was increased in mice constitutively expressing (transgenic) CD70, suggesting that CD27 ligation drives the accumulation of Th1-type effector cells in C57BL/6 mice (23). Moreover, young (not yet B cell depleted) CD70 transgenic mice showed enhanced CD8+ T cell responses to tumors due to both increased numbers of effector cells as well as IFN{gamma}-based effector function (24). Of interest, consistent with our findings that IFN{gamma} production on a per cell basis was not altered in CD27-deficient T cells upon PPD stimulation, the IFN{gamma} production capacity of infiltrating T cells in the lung during infection with influenza virus was comparable in WT and CD27 KO mice in response to phorbol myristate acetate (PMA)/ionomycin (16). We included PMA/ionomycin stimulation in the IFN{gamma} production experiments and did not detect any differences either (data not shown). In contrast to the pulmonary compartment, the percentage of IFN{gamma}-positive splenic CD8+ T cells in WT mice was higher than CD4+ T cells. Although host defense against mycobacteria is traditionally believed to be dominated by CD4+ T cell IFN{gamma} production (and illustrated by our lung stimulation data), recent data obtained from mouse and human studies suggest that CD8+ T cells complement this by cytolysis and IFN{gamma} production (25–28). Nevertheless, only a minor proportion of lung CD8+ T cells respond to PPD. Despite reduced early systemic IFN{gamma} responses in the CD27 KO mice, CD27 deficiency did not result in differences in local lymphocyte function (IFN{gamma} production capacity) and did not impact on outgrowth of M. tuberculosis in the lungs and distant organs in the long term, IFN{gamma} production locally or in response to recall antigen stimulation later in infection. Ultimately, this leads to a similar survival of the CD27 KO mice and CD27 co-stimulation was not pivotal for the cellular immune response later during infection and thus for the outcome of M. tuberculosis infection.

CD27 signaling has been shown to maintain T cell survival and to enhance T cell accumulation in peripheral tissues: after influenza virus challenge, CD27-deficient mice displayed an impaired expansion of antigen-specific T cells and a reduced accumulation at the effector site (16–18). We tried to address this in our model by studying the numbers of splenocytes and local lymph node cells during infection with M. tuberculosis. It was shown before that naive CD27 KO mice do not differ from WT mice in spleen, lymph node or thymus T cell numbers and the CD4+ or CD8+ subset composition of lymphoid organs was normal in CD27 KO mice (16). Our results suggest that in the case of M. tuberculosis infection, CD27 co-stimulation is not important for T cell expansion in lymphoid organs. Moreover, we tested T cell function in the lung and found no differences in their IFN{gamma} response to PPD. IFN{gamma} production by CD4+ T cells is believed to be the most important effector function of lymphocytes during M. tuberculosis infection.

Although we detected more influx of cells into the lungs after 6 weeks of infection, this increase was very likely the result of the enhanced bacterial load at this time point of infection. The higher bacterial burden can also be held responsible for the increased inflammation observed in the lungs of CD27 KO mice after 6 weeks of infection with M. tuberculosis. The higher bacterial load at 6 weeks after infection can result in higher concentrations of IL-1ß and MIP-2 at this time point (more bacteria provide a more potent stimulus for cytokine/chemokine production). However, this does not account for the higher IL-1ß levels observed early in infection.

What is known about the role of TNFR family members in the host defense against mycobacterial infections? To our knowledge, one earlier study investigated the role of CD27 during mycobacterial infection, demonstrating that interference with the CD27–CD70 interaction by blocking CD70 antibodies had no effect on Mycobacterium avium growth in liver, spleen and lung (29). Moreover, anti-CD70 treatment did not influence the amount of IFN{gamma} produced by splenocytes stimulated with M. avium antigens during an 80-day observation period (29). The apparent discrepancy with our current findings can potentially be explained by the possibility that the anti-CD70 antibodies did not entirely prevent the interaction between CD27 and CD70, by differences in the mycobacterial species used (M. avium versus M. tuberculosis) and/or by differences in the route of infection (intravenous versus intranasal). Of note, Florido et al. (29) did demonstrate a role in M. avium infection for another member of the TNFR family, CD30. CD30-deficient mice or WT mice treated with a neutralizing antibody against the CD30 ligand CD153 were more susceptible to M. avium infection and presented with higher bacterial loads in liver, spleen and lung, decreased splenic T cell expansion and reduced IFN{gamma} responses. Another co-stimulatory molecule TNFR family member important for T cell activation is CD40. A study by Lazarevic et al. (30) revealed that CD40 KO mice succumbed to low-dose aerosol infection with M. tuberculosis, whereas host defense in CD40L KO mice was unaltered. Moreover, CD40 KO mice displayed more extensive mycobacterial outgrowth after infection with M. avium, which was associated with an attenuated IL-12 and IFN{gamma} response (31). Two studies focused on the role of the CD40 interaction with its ligand CD40L during tuberculosis in mice by using soluble CD40L or blocking CD40L antibody (32, 33). Both studies demonstrated that protective immunity against M. tuberculosis could be achieved independently from CD40L. These data from the literature point to the idea that co-stimulation by different TNFR family members possibly compensate for one another. In contrast to the acute activation of T cells that is pivotal in viral infections, compensation by other members of the TNFR family in this more chronic setting could explain why CD27 KO mice are not more susceptible to M. tuberculosis infection in the long term. To rule out the possibility that lack of CD27 is compensated for only in the genetically altered CD27 KO mice, antibody treatment against CD27 could be an option. But there are several drawbacks that prevented us from pursuing this approach: the long term of the experiments (up to 6 weeks of treatment), the dose and route of treatment with antibodies. Moreover, it is feasible that the animals generate a humoral immune response against the anti-CD27 antibodies during long-term treatment.

In conclusion, our data suggest that CD27 plays a minor role in the early induction of a systemic protective immune response during pulmonary tuberculosis. Nevertheless, this co-stimulatory receptor is not essential for the outcome of M. tuberculosis infection.


   Acknowledgements
 
We thank Joost Daalhuisen and Marieke ten Brink for expert technical assistance. This work was supported by grants from Willem Bakhuys Roozeboom Foundation (to C.W.W.) and the Netherlands Organization of Scientific Research (to S.F. and M.A.N.).


   Abbreviations
 
ANOVA, analysis of variance
APC, allophycocyanin
CFU, colony-forming unit
H&E, hematoxylin and eosin
KC, cytokine-induced neutrophil chemoattractant
KO, knockout
MFI, mean fluorescence intensity
PMA, phorbol myristate acetate
PPD, purified protein derivative
TNFR, tumor necrosis factor receptor
WT, wild type

   Notes
 
Transmitting editor: S. Swain

Received 27 March 2006, accepted 7 August 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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