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Keywords:

  • Bacterial infection;
  • Cytokine receptor;
  • Cytokines;
  • Rodent;
  • T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

CD8+ T cells are involved in protection against Mycobacterium tuberculosis infection and represent a promising target for new vaccine strategies. Because IL-15 is important for the homeostasis of CD8+ T cells, we studied the immune response in IL-15-deficient mice during tuberculosis. In the absence of IL-15, CD8+ T cells failed to efficiently accumulate in draining lymph nodes and at the site of infection. The expression of antigen-specific effector functions, such as the production of interferon-γ and cytotoxicity, were impaired in CD8+ T cells, but not CD4+ T cells, from IL-15-deficient mice. This defect was associated with an increased mortality of IL-15-deficient mice during the chronic phase of infection. The lectin-like stimulatory receptor natural killer group 2D (NKG2D) was up-regulated on CD8+ T cells only from wild-type mice, but not from IL-15-deficient mice. Mechanistically, blocking NKG2D function with an mAb inhibited M. tuberculosis-directed CD8+ T cell responses in vitro. We conclude that in addition to regulating the expansion of CD8+ T cells, IL-15 is also necessary for inducing effector mechanisms in CD8+ T cells that depend on NKG2D expression. Hence, our results implicate IL-15 and NKG2D as promising targets for modulating CD8+ T cell-mediated protection against tuberculosis.

Abbreviations:
Mtb:

Mycobacterium tuberculosis

NKG2D:

natural killer group 2D

TB:

tuberculosis

VSV:

vesicular stomatitis virus

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Human tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is responsible for eight million new cases and two million deaths annually 1. Improved vaccination strategies will need to target all mechanisms that contribute to restricting the growth of Mtb 2. Although the cell-mediated immune response is known to be critical in host defense against infection with mycobacteria, the relative contribution of T cell subsets and the mechanisms by which T cells participate in the control of infection are still not completely defined. It is generally accepted that both, CD4+ and CD8+ T cells, are an essential component of protective immunity against TB 3. CD4+ T cells are particularly critical during the early phase of infection, while CD8+ T cells appear to contribute mostly at later stages 4, 5. Both, CD4+ and CD8+ T cells, produce interferon-γ (IFN-γ) which in turn stimulates the anti-microbial activity of macrophages. Intracellular pathogens are then killed through reactive nitrogen intermediates produced by the inducible nitric oxide synthase 6 or through effector mechanisms mediated by the newly described member of the 47-kD guanosine triphosphatase family, LRG-47 7.

CD8+ T cells can also cause death of both target cells and their intracellular bacterial cargo, either through perforin-dependent cytolysis by the release of granzymes and granulysin, or by ligation of Fas ligand (FasL) on their surface with Fas on infected macrophages 5, 810. Because CD8+ T cells are involved in protection during the chronic stage of TB 4, 5, 11, and participate in memory immune responses to Mtb infection 12, generating a more robust CD8+ T cell response may provide an effective vaccination strategy to the control and clearance of infection. Thus, exploring the mechanisms which exclusively promote the expansion of activated effector and memory CD8+ T cells may improve vaccine-induced immune protection against TB.

One cytokine which is of particular interest for the expansion of effector and memory CD8+ T cells is IL-15. IL-15 is a T cell-stimulating cytokine that, while sharing some properties with IL-2, also mediates unique functions 13, 14. IL-15 is a potent chemoattractant 15 and controls both proliferation and survival of naive and memory-phenotype CD8+ T cells 16, 17. Furthermore, mice lacking IL-15 or IL-15Rα are deficient in the generation of memory-phenotype CD8+ T cells 18, 19 and are highly susceptible to infection with viruses 20, 21. Treatment with exogenous IL-15 or overexpression of endogenous IL-15 results in enhanced protection against mycobacterial infections 2225. However, the mechanisms by which IL-15 mediates protection during experimental TB are not completely understood.

We sought to define whether IL-15 would be essential for the development of anti-mycobacterial immunity and therefore analysed the immune response of IL-15-deficient (IL-15–/–) mice in response to aerosol infection with Mtb. Our results demonstrate that relevant protective CD8+ T cell responses during chronic infection are dependent on IL-15. Mechanistically, we provide evidence that IL-15 induces expression of natural killer group 2D (NKG2D) on CD8+ T cells whose costimulatory properties are involved in both cytokine secretion and cytolysis of CD8+ T cells.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Protection against TB is dependent on IL-15

To determine the functional significance of IL-15, the course of infection with 300 or 100 colony-forming units (CFU) Mtb via the aerosol route was monitored in wild-type C57BL/6 and IL-15–/– mice. During the course of infection with 300 CFU, the bacterial loads in lungs from IL-15–/– mice were found to be significantly increased already on days 42 and 98 (Fig. 1A). After a low-dose infection with 100 CFU, bacterial loads in the lungs of Mtb-infected IL-15–/– mice were comparable to those observed in infected C57BL/6 mice until day 98 after infection (Fig. 1B). During the chronic phase of infection, however, CFU in lungs from IL-15–/– mice infected with the lower dose were also found to be significantly increased. In line with this elevated bacterial load, IL-15–/– mice succumbed to Mtb infection significantly earlier than wild-type mice (Fig. 1C). Whereas C57BL/6 mice started to die after day 320 of aerosol infection with 100 CFU Mtb, death of IL-15–/– mice was observed as early as 250 days of infection (Fig. 1C). All mutant mice died before day 350 of infection and had increased bacterial loads immediately prior to death (Fig. 1D).

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Figure 1. IL-15 mediates protection against chronic Mtb infection. C57BL/6 (black circles) and IL-15–/– (white circles) mice were infected with (A) 300 or (B–D) 100 CFU Mtb via the aerosol route. (A–C) Mycobacterial colony enumeration assays in lungs (A, B) during the course of infection and (D) in moribund IL-15–/– mice. (C) During the course of infection survival of ten infected mice per group was monitored. Animals that lost more than 25% of their original body weight were sacrificed. Differences between C57BL/6 and IL-15–/– mice in (A, B, D) were defined as significant (*p⩽0.05, **p⩽0.01). Differences in survival kinetics between C57BL/6 and IL-15–/– mice were highly significant (p⩽0.001; log rank test).

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IL-15 supports the expansion of CD8+ T cells after Mtb infection

A prerequisite for antigen-specific T cell activation and the development of a protective T cell response is the presence of a sufficient T cell repertoire in the lymph node draining the challenge site. In order to examine how pulmonary infection of IL-15–/– mice affects the recruitment and activation of T cells in lymph nodes, wild-type and IL-15–/– mice were challenged with Mtb by aerosol, and the number and activation status of CD4+ and CD8+ T cells in the draining lymph nodes were assessed by flow cytometry during the course of infection.

In both, C57BL/6 and IL-15–/– mice, the total number of lymphocytes present in the mediastinal lymph node increased steadily and to a similar extent during the course of infection (Fig. 2A). Whereas the relative amount of CD4+ T cells in IL-15–/– mice was apparently not impaired compared to wild-type mice, the CD8+ T cell subset was significantly reduced during the course of infection (Fig. 2B). Considering the total amounts of cells in the draining lymph nodes (Fig. 2A), the difference between C57BL/6 and IL-15–/– mice in expanding CD8+ T cells steadily increased during the course of infection. When the percentages of activated cells within the CD4+ and CD8+ T populations were determined by the analysis of activation markers such as high levels of CD44 and low levels of CD62L, it became apparent that C57BL/6 and IL-15–/– mice had similar increasing percentages of activated CD4+ T cells in mediastinal lymph nodes after infection with Mtb (Fig. 2C). Although the relative amount of activated CD8+ T cells was not significantly reduced in draining lymph nodes from IL-15–/– mice, the degree of increase in CD44high CD62Llow CD8+ T cells was lower.

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Figure 2. Expansion and activation of CD8+ T cells in mediastinal lymph nodes from Mtb-infected IL-15–/– mice. C57BL/6 (black circles) and IL-15–/– (white circles) mice were infected with 100 CFU Mtb via the aerosol route. For total cell counts and flow cytometric analysis of CD4+ and CD8+ T cells, single-cell suspensions from mediastinal lymph nodes were prepared at different time points. (A) The amount of total cells was counted and (B, C) surface markers were stained for flow cytometric analysis of (B) CD3+ CD4+ and CD8+ T cells and (C) activated (CD44+ CD62Llow CD3+) CD4+ and CD8+ T cells. Data represent means and standard deviations of four mice. One experiment representative of two performed is shown. Statistical analysis was performed using the unpaired Student's t-test defining differences between C57BL/6 and IL-15–/– as significant (*p⩽0.05, **p⩽0.01, ***p⩽0.001).

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Effector mechanisms against mycobacteria carried out by macrophages, CD4+ and CD8+ T lymphocytes take effect in granulomas at the site of infection. To assess the formation and cellular composition of granulomas in lungs after aerosol infection with Mtb, tissue sections from C57BL/6 and IL-15–/– mice were histologically examined 98 days after infection (Fig. 3).

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Figure 3. CD4+ and CD8+ T cells differentially accumulate in the lungs of Mtb-infected IL-15–/– mice. C57BL/6 (left panel) and IL-15–/– (right panel) mice were infected with 100 CFU Mtb via the aerosol route for 42 days. (A, B) The granulomatous response was assessed in formalin-fixed sections stained with hematoxylin and eosin (bar = 0.5 mm). (C–F) The accumulation of (C, D) CD4+ and (E, F) CD8+ T cells was analysed by immunohistochemistry (brown staining) in sections from frozen lung tissue (bar = 0.5 mm; arrows indicate CD4+ or CD8+ T cells, respectively).

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Compared to C57BL/6 mice, IL-15–/– mice showed similar kinetics and magnitude of cellular infiltration into lung tissue (Fig. 3A, B). Both groups of mice showed epitheloid cell differentiation, lymphoid follicle formation and highly structured, compact granuloma development. However, the cellular composition of granulomas was different between wild-type and IL-15–/– mice. Whereas granulomas in lungs from C57BL/6 and IL-15–/– mice contained similar numbers of infiltrating CD4+ T cells (Fig. 3C, D), the amount of CD8+ T cells was reduced in the lungs of IL-15–/– mice when compared to C57BL/6 mice (Fig. 3E, F). Quantitative analysis of NK and T cell populations in perfused lungs from Mtb-infected C57BL/6 and IL-15–/– mice revealed that during the course of infection the overall cellular infiltration (Fig. 4A) as well as the accumulation of CD4+ T cells (Fig. 4C) was not significantly different in lungs from either mouse strain. In contrast, the amount of DX5+ (Fig. 4B) and NK1.1+ (data not shown) NK cells as well as of CD8+ T cells (Fig. 4C) was significantly lower in the absence of IL-15.

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Figure 4. Expansion, proliferation and apoptosis of CD8+ T cells in lungs from Mtb-infected IL-15–/– mice. C57BL/6 (black circles) and IL-15–/– (white circles) mice were infected with 100 CFU Mtb via the aerosol route. For total cell counts and flow cytometric analysis of NK, CD4+ and CD8+ T cells, single-cell suspensions from lungs were prepared at different time points. (A) The amount of total cells was counted and (B, C) surface markers were stained for flow cytometric analysis of (B) DX5+ NK cells and (C) CD3+ CD4+ and CD8+ T cells. (D) BrdU incorporation in proliferating CD44+ CD3+ CD4+ and CD8+ T cells. (E) Quantification of apoptotic annexin V+ PI CD44+ CD4+ and CD8+ T cells. Data represent means and standard deviations of four mice. One experiment representative of two performed is shown. Statistical analysis was performed using the unpaired Student's t-test defining differences between C57BL/6 and IL-15–/– as significant (*p⩽0.05, **p⩽0.01, ***p⩽0.001).

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Analysis of BrdU incorporation and annexin V binding revealed that while proliferation was not affected in activated CD4+ T cells from lungs of IL-15–/– mice, activated CD8+ T cells from mutant mice showed a significantly reduced proliferating capacity during the course of infection when compared to C57BL/6 mice (Fig. 4D). The lack of IL-15 had no effect on apoptosis of activated CD4+ and CD8+ T cells, although apoptosis of activated CD8+ T cells from IL-15–/– mice was consistently higher than of cells from wild-type mice, but this difference did not reach statistical significance (Fig. 4E). Together, the increased susceptibility of IL-15–/– mice during the chronic phase of Mtb infection was accompanied by the reduced presence and proliferation of CD8+ T cells at the site of infection.

IL-15 is a key cytokine for the expression of Mtb-specific effector mechanisms in CD8+ T cells

In the lung, activated effector/memory CD4+ and CD8+ T cells are responsible for an efficient protective effector response to Mtb infection 3. Whereas homeostasis of effector/memory CD4+ T cells is regulated by IL-7 26, IL-15 promotes effector/memory CD8+ T cells and is thought to play an important role in regulating the turn-over of these cells in vivo16, 17, 20. Kinetic analysis of effector/memory phenotypes (CD44high CD62Llow, Fig. 5A; CD44high CD45RBlow, Fig. 5B) of CD4+ and CD8+ T cells in lungs from Mtb-infected C57BL/6 and IL-15–/– mice revealed that the absence of IL-15 had no effect on the relative amount of an effector/memory phenotype within the CD4+ T cell compartment. Most importantly, the relative expression of an effector/memory phenotype within the CD8+ T cell population was also not affected in the absence of IL-15 (Fig. 5).

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Figure 5. CD8+ T cells display normal activation and memory phenotypes in the lungs of Mtb-infected IL-15–/– mice. C57BL/6 (black circles) and IL15–/– (white circles) mice were infected with 100 CFU Mtb. At different time points, effector/memory phenotypes (A; CD44+ CD62Llow; B; CD44+ CD45RBlow) of lung CD3+ CD4+ (left panel) and CD8+ (right panel) T cells were determined by flow cytometric analysis. Data represent means and standard deviations of four mice. One experiment representative of three performed is shown.

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Production of IFN-γ and cytotoxicity are key effector mechanisms against Mtb mediated by antigen-specific CD4+ and CD8+ T cells at the site of infection 3. Quantitiative RT-PCR of enriched CD4+ and CD8+ T cells revealed that whereas IFN-γ mRNA levels in CD4+ T cells from C57BL/6 and IL-15–/– mice were comparably high (Fig. 6A), IFN-γ gene expression was significantly reduced in CD8+ T cells from IL-15–/– mice during the course of Mtb infection (Fig. 6B).

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Figure 6. Impaired IFN-γ production by CD8+ T cells from Mtb-infected IL-15–/– mice. C57BL/6 (black symbols) and IL-15–/– (white symbols) mice were infected with 100 CFU Mtb via the aerosol route and CD4+ and CD8+ T cells were purified after 3 wk of infection. (A, B) IFN-γ gene expression in (A) CD4+ and (B) CD8+ T cells based on expression of β2 m. (C, D) Analysis of IFN-γ production by (C) CD4+ and (D) CD8+ T cells 72 h after restimulation with antigen-presenting macrophages. Data represent means and standard deviations of three mice. One experiment representative of three performed is shown. (E) Analysis of IFN-γ production by CD8+ T cells from Mtb-infected C57BL/6 mice 72 h after restimulation with antigen-presenting macrophages from wild-type (filled bars) and IL-15–/– (hatched bars) mice. Data represent means and standard deviations of three mice. Statistical analysis was performed using the unpaired Student's t-test defining differences between C57BL/6 and IL-15–/– mice as significant (*p⩽0.05, **p⩽0.01).

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To further examine the capacity of effector/memory T cells to accomplish antigen-specific effector mechanisms in the absence of IL-15, we conducted in vitro recall stimulation assays using antigen-pulsed peritoneal macrophages and enriched CD4+ and CD8+ T cells from Mtb-infected mice (Fig. 6C–E). CD4+ T cells from C57BL/6 and IL-15–/– mice produced comparably high levels of IFN-γ in response to incubation with antigen-presenting macrophages (Fig. 6C). By contrast, antigen-restimulated CD8+ T cells from infected IL-15–/– mice secreted significantly lower amounts of IFN-γ when compared to CD8+ T cells from infected C57BL/6 mice (Fig. 6D).

To test whether IL-15–/– macrophages were deficient in inducing antigen-specific IFN-γ production by CD8+ T cells, we restimulated CD8+ T cells from infected C57BL/6 mice with antigen-presenting cells isolated from wild-type or IL-15–/– mice (Fig. 6E). Whereas restimulation of CD8+ T cells from Mtb-infected C57BL/6 mice with antigen-pulsed wild-type macrophages resulted in a profound release of IFN-γ, restimulation with IL-15–/– antigen-presenting cells induced significantly less IFN-γ in wild-type CD8+ T cells. However, neither in CD8+ T cells from Mtb-infected C57BL/6 nor from IL-15–/– mice the addition of exogenous IL-15 had any effect on the antigen-specific production of IFN-γ (data not shown). These results indicate that IL-15 promotes antigen-specific effector responses in CD8+ T cells at the level of accessory cells.

To evaluate the influence of IL-15 on the antigen-specific cytotoxic activity of CD8+ T cells after Mtb infection, we performed a 5-h 51Cr-release assay using enriched CD8+ T cells and antigen-pulsed macrophages at different effector-to-target ratios (Fig. 7A). Whereas antigen-specific CD8+ T cells from infected C57BL/6 mice efficiently lysed target cells, IL-15 deficiency ablated the cytotoxic activity of CD8+ T cells. Quantitiative RT-PCR of enriched CD8+ T cells revealed that gene expression of perforin (Fig. 7B), granzyme B (Fig. 7C) and FasL (Fig. 7D) was significantly reduced in CD8+ T cells from IL-15–/– mice, whereas gene expression of TNF (Fig. 7E) was comparable to that observed in C57BL/6 mice during the course of Mtb infection. In summary, although the development of an effector/memory phenotype within the CD8+ T cell population was apparently not affected by the absence of IL-15, the expression of antigen-specific effector mechanisms was severely impaired in CD8+ T cells.

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Figure 7. Reduced cytotoxic effector functions of CD8+ T cells from Mtb-infected IL-15–/– mice. C57BL/6 (black symbols) and IL-15–/– (white symbols) mice were infected with 100 CFU Mtb via the aerosol route. CD8+ T cells were purified after 3 wk of infection. (A) Specific lysis of antigen-pulsed macrophages by enriched CD8+ effector T cells at different effector-to-target ratios. Data represent means and standard deviations of quadruplicates from pooled CD8+ T cells purified from five mice. One experiment representative of two performed is shown. Gene expression of (B) perforin, (C) granzyme B, (D) FasL and (E) TNF in CD8+ T cells based on expression of β2 m. Data represent means and standard deviations of three mice. One experiment representative of three performed is shown. Statistical analysis was performed using the unpaired Student's t-test defining differences between C57BL/6 and IL-15–/– mice as significant (*p⩽0.05, **p⩽0.01).

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CD8+ T cell NKG2D expression depends on the presence of IL-15 and is required for Mtb-specific effector functions

Recently, the lectin-like receptor NKG2D has been implicated in a novel costimulatory pathway of NK and CD8+ T cells leading to an enhancement of innate and adaptive immunity 27. NKG2D signalling is mediated by the adaptor molecule DAP10 28 through a YXXM motif similar to that of CD28, a costimulator of naive T cells. Importantly, the expression of NKG2D and DAP10 was described to be dependent on IL-15 29, 30.

Because infection induces the expression of MHC class I-like ligands for NKG2D on macrophages such as Rae-1 3133, we first analysed whether infection with Mtb induces the expression of the NKG2D ligand Rae-1 on pulmonary macrophages (Fig. 8A). Flowcytometric analysis of PI F4/80+ macrophages in cell suspensions of perfused lungs revealed that infection with Mtb up-regulates Rae-1 on macrophages from both C57BL/6 and IL-15–/– mice to a similar extent (Fig. 8A). In contrast, whereas we found NKG2D to be expressed on most NK cells and activated CD8+ T cells in the lungs of Mtb-infected C57BL/6 mice, its expression was significantly reduced on DX5+ NK cells (Fig. 8B) and activated CD8+ T cells (Fig. 8C) in IL-15–/– mice during the course of infection. Together, IL-15 appeared to be indispensable for the induction of a functional NKG2D receptor complex during Mtb infection.

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Figure 8. After infection with Mtb, NKG2D expression on CD8+ T cells is dependent on IL-15. C57BL/6 (black symbols) and IL-15–/– (white symbols) mice were infected with 100 CFU Mtb via the aerosol route. (A) Expression of NKG2D ligands on pulmonary macrophages was assessed by flow cytometric analysis of Rae-1 gated on PI F4/80+ cells in single-cell suspensions of perfused lungs from C57BL/6 (black line) and IL-15–/– (red line) mice that have been infected with Mtb for 96 days. Representative histogram of one out of three mice per group (gray histogram, isotype control). NKG2D expression on (B) DX5+ NK cells and (C) activated CD44+ CD8+ T cells from lungs of infected mice was determined at different time points by flow cytometric analysis. Data represent means and standard deviations of three mice. One experiment representative of two performed is shown. Statistical analysis was performed using the unpaired Student's t-test defining differences between C57BL/6 and IL-15–/– mice as significant (***p⩽0.001).

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Blocking the interaction of NKG2D with its ligands H60 and Rae-1 affects the cytotoxic activity of CD8+ T cells 34. Therefore, we analysed the role of this interaction during in vitro recall responses of antigen-specific CD8+ T cells from Mtb-infected wild-type mice. The incubation with an isotype control had no effect on either the production of IFN-γ (Fig. 9A) or the cytotoxic activity (Fig. 9B) of CD8+ T cells in response to antigen-pulsed macrophages. In contrast, blocking the interaction of NKG2D and its ligand with a monoclonal antibody (mAb) resulted in a significantly reduced production of IFN-γ (Fig. 9A) and an impairment in cytotoxicity (Fig. 9B) by antigen-specific CD8+ T cells. Together, our results revealed NKG2D-mediated signalling as a key event for expression of antigen-specific CD8+ T cell-mediated effector mechanism and protection during TB.

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Figure 9. NKG2D mediates Mtb-specific responses of CD8+ T cells. C57BL/6 mice were infected with 100 CFU Mtb via the aerosol route and CD8+ T cells were purified after 3 wk of infection. (A) IFN-γ production by CD8+ T cells 72 h after restimulation with antigen-presenting macrophages in the presence of a neutralizing anti-NKG2D antibody (black bars) or an isotype control (hatched bars). Data represent means and standard deviations of three mice. One experiment representative of two performed is shown. Statistical analysis was performed using the unpaired Student's t-test defining differences between C57BL/6 and IL-15–/– as significant (**p⩽0.01). (B) Specific lysis of antigen-pulsed macrophages by enriched CD8+ effector T cells at different effector-to-target ratios in the presence of a neutralizing anti-NKG2D antibody (solid circles) or an isotype control (stars). Data represent means and standard deviations of quadruplicates from pooled CD8+ T cells purified from five mice.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Our data show that IL-15 is critical for the development of full-blown protective immunity against experimental TB in mice. The lack of IL-15 was associated with a dramatic reduction in the numbers and function of NK and CD8+ T cells, most notably in a deficiency of NKG2D expression and of NKG2D-dependent effector mechanisms. Because it regulates the quantity of CD8+ T cells and the quality of CD8+ T cell-mediated anti-mycobacterial functions, IL-15 is a key player in the protective immune response to Mtb.

An essential role for CD8+ T cells in long-term protection against Mtb has been established 35. However, the factors responsible for the induction and maintenance of the memory/effector CD8+ T cell immune response have remained elusive 35. Recently, attention was drawn to the role of IL-15 in induction and maintenance of memory/effector CD8+ T cells 36. In accordance with the importance of CD8+ T cells for protection against a number of pathogens, IL-15 was shown to improve the proliferation of memory/effector CD8+ T cells and protective CD8+ T cell-mediated immune responses such as IFN-γ production and cytotoxicity 3739. For example, a deficiency in IL-15 or IL-15Rα led to an impaired CD8+ T cell-mediated immune response and decreased protection from infection with herpes simplex virus type 2 21, 40, vesicular stomatitis virus (VSV) 41, vaccinia virus 18 or Toxoplasma gondii42. However, in some cases the role of IL-15 appeared to be redundant since IL-15–/– mice proved capable of developing protective immune responses after infection with gamma herpes virus 68 43, lymphocytic choriomeningitis virus 20 or T. gondii44.

With respect to mycobacterial infections, there are discrepant reports on the role of IL-15. Following infection with M. bovis BCG or Mtb H37Rv, treatment of infected mice with recombinant IL-15 or overexpression of IL-15 resulted in enhanced anti-mycobacterial protection which was attributed to the ability of this cytokine to promote NK and CD8+ T cell-mediated immune responses 2325. In contrast, a more recent report showed that administration of exogenous IL-15 did not influence the course of Mtb infection in wild-type mice, and that Mtb-infected IL-15–/– mice had only mild defects in protective CD8+ T cell-mediated immune responses 45. Our own study in aerosol-infected IL-15–/– mice documents an essential role for IL-15 in generating and maintaining long-term protection during Mtb infection. The increased susceptibility of IL-15–/– mice was accompanied by a profound defect of NK and CD8+ T cell expansion in infected tissues whereas CD4+ T cells were not affected.

NK cells are considered to be innate immune cells whereas CD8+ T cells belong to the adaptive immune systems. However, NK cells have previously been shown to have no impact on early resistance to Mtb infection 46, and the lack of an early effect on mycobacterial proliferation or survival in IL-15-knockout mice is compatible with this notion. In contrast, CD8+ T cells have been implicated in protective immune responses during the chronic phase of TB 4, 5, 11. Because survival kinetics of Mtb-infected IL-15–/– mice were comparable to the outcome of infection in mice lacking MHC class I or perforin 3, 4, 47, our results support the interpretation that IL-15 may selectively promote CD8+ T cell-mediated protection during TB. However, we have not formally excluded the possibility that the profound defciency of NK cell expansion may have contributed to the enhanced susceptibility of Mtb-infected IL-15–/– mice.

The fact that mice devoid of CD8+ T cells still succumb to Mtb infection despite the development of fully functional CD4+ T cell responses, corroborates results from other investigators showing that CD8+ T cells play an important role in controlling latent and chronic infection 11, 35. Maintenance of CD8+ T cells is critically dependent upon the two common γ chain cytokines IL-7 and IL-15. Whereas IL-7 promotes the survival of both naive and memory CD8+ T cells, IL-15 uniquely supports basal memory CD8+ T cell proliferation 20, 41, 48. Thus, in the absence of proliferative IL-15 signals, effector/memory CD8+ T cells undergo slow atrophy in number until they become essentially undetectable under certain conditions 20, 49. After infection with Mtb, we now show that the accumulation of CD8+ T cells in the lungs of IL-15–/– mice was indeed substantially reduced. This impaired expansion was accompanied by a significantly reduced proliferation of activated CD8+ T cells, indicating that during Mtb infection in mice IL-15 is indispensable for maintaining the CD8+ T cell population.

The generation of immunological memory is the end result of a productive immune response and is the ultimate goal of vaccination. Memory CD8+ T cells appear to be more sensitive to IL-15-induced proliferation than either naive-phenotype CD8+ T cells or naive or memory CD4+ T cells 50. Moreover, an important clue that IL-15 is required for memory CD8+ T cell generation or maintenance is the lack of memory-phenotype CD8+ T cells in IL-15Rα–/– and IL-15–/– mice 18, 19. Previous work has also shown that under certain conditions only long-term maintenance of effector/memory-phenotype CD44high CD8+ T cells is controlled by IL-15 20. After infection with VSV, the clonal expansion of tetramer-positive VSV-specific CD8+ T cells was strikingly decreased in IL-15–/– mice 41.

However, during Mtb infection of IL-15–/– mice, the significantly reduced accumulation of CD8+ T cells was not limited to CD44high CD8+ T cells expressing an effector/memory phenotype. The relative proportion of effector/memory cells among the CD8+ T cell population was rather unaffected by the absence of IL-15. Because IL-15Rα–/– and IL-15–/– mice contain approximately half of the normal number of naive CD8+ T cells 18, 19, IL-15 is also essential for naive CD8+ T cell production or survival. Therefore, it appears that during TB, IL-15 promotes homeostasis of both, naive and effector/memory CD8+ T cells.

There are a few experimental details that may have impacted on the different results obtained by different researchers using Mtb-infected IL-15–/– mice. Lazarevic et al.45 used a very low dose (30 CFU) for aerosol infection with a strain of Mtb (Erdman) that, in most investigators’ hands, is less virulent than the standard laboratory strain H37Rv, and followed the course of infection only until 15 wk post-inoculation. In our own experiments using a slightly higher infectious inoculum (100 CFU) of the virulent H37Rv strain, enhanced mycobacterial replication and accelerated death of IL-15–/– mice was not evident until 4 and 8 months post-infection, respectively. Using an increased inoculum of 300 CFU, however, infection with Mtb H37Rv resulted in enhanced bacterial loads in the lungs from IL-15–/– mice already on day 42 of infection. It is possible, therefore, that the more pronounced phenotype of IL-15–/– mice in our own experiments was a function of both initial bacterial load and time.

We propose that during Mtb infection, CD8+ T cell-mediated effector functions such as IFN-γ production and the expression of cytotoxic effector molecules are critically dependent on the presence of IL-15 and IL-15-producing accessory cells. Moreover, our results suggest a novel link between the expression of the C-type lectin-like receptor NKG2D, shown to be dependent on IL-15 29, 30, and CD8+ T cell effector functions during experimental TB. NKG2D signaling favors the development of effector CD8+ T cells 51 and augments cytotoxic and proliferative responses of CD8+ T cells on antigen encounter 32, 52, thus qualifying NKG2D as an important T cell costimulatory molecule during infection. Indeed, Listeria monocytogenes infection of mice defective in NKG2D-mediated cytotoxicity revealed a significant role of NKG2D engagement for NK and CD8+ T cell-mediated effector mechanisms and protection during infection 53.

After infection with Mtb, we noted an up-regulation of NKG2D ligands on lung macrophages from wild-type mice and speculated that the expression of NKG2D on CD8+ T cells might be involved in mediating CD8+ T cell-mediated effector mechanisms. Indeed, inhibition of NKG2D receptor engagement abrogated effector mechanisms of antigen-specific CD8+ T cells purified from Mtb-infected wild-type mice. However, we cannot exclude the possibility that in addition to NKG2D expression on CD8+ T cells its presence on NK cells may have had an impact on protective anti-mycobacterial effector mechanisms in vivo.

IL-15 has been described to induce NKG2D expression on CD8+ T cells 29 and to suppress its down-regulation in activated CD8+ T cells 51. In support of these data, the present report reveals that the expression of NKG2D in activated CD8+ T cells is dependent on IL-15, indicating that a failure to up-regulate this costimulatory receptor complex may have contributed to the reduced antigen-specific production of IFN-γ and cytotoxicity by CD8+ T cells in the absence of IL-15. Although IL-15 may have directly affected CD8+ T cell-mediated effector functions, our results raise the hypothesis that during experimental TB the expression of protective effector mechanisms through antigen-specific CD8+ T cells depends to some extent on IL-15-induced NKG2D. However, only analysis of mice defective in NKG2D-dependent effector mechanisms 53, 54 will disclose the significance of NKG2D engagement on CD8+ T cell-mediated protection against Mtb infection in vivo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice and bacterial infection

Eight- to 10-wk-old female IL-15–/– mice on a C57BL/6 genetic background 18 were bred under specific pathogen-free conditions at the Free University of Berlin (Germany). C57BL/6 mice of the same age and sex were purchased as wild-type controls from Charles River (Sulzfeld, Germany). For experimental infection, animals were maintained in specific pathogen-free biosafety level 3 facilities at the Research Center Borstel. All experimental and animal handling procedures were in accordance with the German Animal Protection Law and were approved by the Animal Research Ethics Board of the Ministry of Environment (Kiel, Germany). Mtb (H37Rv) was grown and maintained as described previously 55. Aerosol infection of experimental animals was performed using an inhalation exposure system (Glas-Col, Terre-Haute, IN) 55.

Colony enumeration assay and histopathology

At different time points after infection, lungs from mice were harvested, homogenized in PBS/0.05% Tween-80 and plated in tenfold serial dilutions on Middelbrook 7H10 agar containing 10% OADC. After an incubation at 37°C for 3 wk, CFU were enumerated. One lung lobe, a piece of liver and spleen per mouse were fixed in 4% formalin-PBS, set in paraffin blocks and sectioned (2–3 µm). Histology was performed using standard protocols for hematoxylin/eosin staining.

Immunhistochemistry

Frozen lung tissue was cut into 5-µm sections. After acetone and chloroform fixation, CD4+ or CD8+ T cells were detected with a rat anti-mouse CD4 or CD8 mAb (BD Bioscience, Heidelberg, Germany). The CD4 or CD8 mAb was visualized using peroxidase-conjugated secondary IgG antibodies (Dianova, Hamburg, Germany) and diaminobenzidine as substrate.

Preparation of single-cell suspensions

For flow cytometric analysis, antigen-specific restimulation and cytotoxicity assays, single-cell suspensions of mediastinal lymph nodes and lungs were prepared from Mtb-infected mice at different time points. Lymph node cells were isolated by straining through 100-µm pore size cell strainers (BD Biosciences). For lung cell digestion, infected mice were euthanized by a high dose of an anesthetic containing 150 U heparin (Ratiopharm, Ulm, Germany) at different time points of infection. The pulmonary cavity was opened and the lung was perfused through the pulmonary artery with 5 mL of PBS. To prepare single-cell suspensions, perfused lungs were removed and processed as described 55. To obtain enriched CD4+ and CD8+ T cells, single-cell suspensions were sorted by MACS® (Miltenyi, Bergisch Gladbach, Germany) using magnetic anti-CD4 or anti-CD8 microbeads 55. Purity of enriched CD4+ and CD8+ T cells was >95% as determined by flow cytometry.

Flow cytometric analysis

For flow cytometric analysis of surface markers, single-cell suspensions of lymph nodes and lungs were stained with optimal concentrations of the following specific antibodies: DX5-FITC, CD4-allophycocyanin, CD8-allophycocyanin, CD3-PerCP, CD44-FITC, CD44-PE, CD62L-PE, CD45RB-PE, hamster IgG-PE (all from BD Biosciences), F4/80-Alexa 647 (Caltag, Hamburg, Germany), Rae-1-PE (R&D Systems, Schwalbach, Germany), and NKG2D-PE (clone C7; Biolegend, San Diego, CA) 54. To analyse proliferation of T cells in lungs from infected mice, 1 mg BrdU (BD Biosciences) was i.p. injected 3 days prior to analysis and single-cell suspensions were prepared and processed as described 55. Apoptotic T cells in lungs from infected mice were detected by staining single-cell suspensions with FITC-labelled annexin V (BD Biosciences) and PI (BD Biosciences). Fluorescence intensity was analyzed on a FACSCalibur (BD Biosciences) gating on lymphocytes or macrophages identified by FSC-SSC profile. Viable macrophages were identified by gating on PI cells.

Antigen-specific restimulation of lymphocytes

As antigen-presenting and target cells, peritoneal exudate cells were collected from uninfected wild-type or IL-15–/– mice, and cultured overnight in Iscove's modified Dulbecco's medium (Gibco, Paisley, UK) supplemented with 10 % FCS (Gibco), 0.05 mM β-mercaptoethanol (Sigma, Deisenhofen, Germany), and penicillin and streptomycin (100 U/mL and 100 µg/mL; Gibco). After removing non-adherent cells, adherent macrophages were used for antigen-specific restimulation of enriched CD4+ and CD8+ T cells.

For stimulation of IFN-γ production, enriched CD4+ or CD8+ T cells were incubated with peritoneal macrophages that were pulsed with 25 µg/mL short-term culture filtrate from Mtb (a kind gift of Peter Andersen, Statens Serum Institut, Copenhagen, Denmark) in Iscove's modified Dulbecco's medium. After 72 h of restimulation, supernatants were collected and frozen at –80°C until production of IFN-γ was quantified by ELISA 55. To measure the cytotoxic activity of enriched CD8+ T cells, a 5-h 51Cr (Amersham, Freiburg, Germany) release assay using Mtb short-term culture filtrate-stimulated peritoneal macrophages as target cells at various effector-to-target ratios was employed. Specific lysis was calculated according to the following formula: % specific lysis = [(experimental release – spontaneous release) / (maximum release – spontaneous release)] × 100. To evaluate the impact of NKG2D on antigen-specific restimulation, some experiments were performed in the presence of an anti-NKG2D or a rat IgG2b control antibody (3 µg/mL; R&D Systems) as described 54.

RT-PCR

Total RNA from infected lungs and from enriched CD4+ and CD8+ T cells was extracted using the High Pure RNA Kit® (Roche, Mannheim, Germany) or the SV Total RNA Isolation Systems® (Promega, Mannheim, Germany), respectively. After reverse transcription into cDNA, gene expression was quantified using a Light Cycler (Roche) 55. The following primer pairs were used: IFN-γ: sense 5′-GCTCTGAGACAATGAACGCT-3′, antisense 5′-AAAGAGATAATCTGGCTCTGC-3′; perforin: sense 5′-AATATCAATAACGACTGGCGTGT-3′, antisense 5′-CATGTTTGCCTCTGGCCTCCTA-3′; granzyme B: sense 5′-TGCTGCTCACTGTGAAGGAA-3′, antisense 5′-TTACCATAGGGATGACTTGCTG-3′; FasL: sense 5′-CCGGTGGTATTTATGGT-3′, antisense 5′-CTTTGGTTGGTGAACTC-3′; TNF: sense 5′-TCTCATCAGTTCTATGGCCC-3′, antisense 5′-GGGAGTAGACAAGGTACAAC-3′; β2 m: sense 5′--TCACCGGCTTGTATGCTATC-3′, antisense 5′-CAGTGTGAGCCAGGATATAG-3′.

Statistical analysis

Quantifiable data are expressed as the means of individual determinations and standard deviations. Statistical analysis was performed using the unpaired Student's t-test defining different error probabilities (*p⩽0.05, **p⩽0.01, ***p⩽0.001).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The authors thank Susanne Metken and Manfred Richter for excellent technical assistance, and Ilka Monath, Sven Mohr and Claus Möller for organising the animal facility and taking care of the mice. This work was supported by the University of Lübeck (Research Grant No. 38/03), the German Research Foundation (Research Grant HO 2145/3-1), and the National Genome Research Network (Workpackage Tuberculosis, NIE-S05T22).

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