T lymphocytes are the central cell type of the specific immune system. The ma-jority of these CD+ cells (95 %) express a T-cell receptor (TCR) composed of an α and a β chain (αβTCR+) associated (or not) with the expression of either CD4 or CD8. The biggest difference between CD+CD4+ T cells and CD+CD8+ T cells is probably the type of MHC used for the recognition of the processed antigen, MHC class II (CD4) and MHC class I (CD8), respectively. In addition to these “classi-cal” cells, some unconventional cells link innate and adaptive immunity, such as γδ T cells and CD1 restricted T cells.
CD4+ T cells
During M. tuberculosis infection, CD4+ T cells are actively recruited to the site of infection and its corresponding drain-ing lymph nodes (15). The importance of CD4+ T cells in the protective immune response against intracellular bacteria is illustrated by both experimental ani-mal studies and clinical conditions as-sociated with CD4+ T cell deficiency (e.g.
AIDS patients). For example, depletion of CD4+ T cells after the establishment of M. tuberculosis infection in mice, is as-sociated with rapid exacerbation of TB.
In addition, infection of CD4+ T cell-de-ficient mice with M. tuberculosis leads to rapid death, despite the effective main-tenance of inducible NO synthase and RNI expression and the increase of IFN-γ secretion by CD8+ T cells (16). There-fore, CD4+ T cells must elaborate ad-ditional protective mechanisms, which are independent of IFN-γ and RNI. These mechanisms are poorly characterized, but could include the release of
addi-tional interleukins, the conditioning of the APC or even the induction of apop-tosis of infected cells.
Activation of CD4+ T cells relies on an-tigenic presentation by MHC class II molecules expressed by the APC and even by non-professional APC upon activation by cytokines such as IFN-γ.
In general, antigens that are presented by MHC class II molecules are found in the phagosomal compartment and have followed a classical way from the endoplasmic reticulum to the surface of the membrane. However, M. tubercu-losis evolved several mechanisms that interfere with this process of antigenic presentation. The inhibition of the pha-go-lysosomal fusion is one of the best-known examples (105).
The cytokine repertoire expressed by primed CD4+ T cells is an important de-terminant in the fight between host and pathogen. Th1 CD4+ T cells notably produce IFN-γ and IL- and dominate in cell-mediated immunity against intrac-ellular pathogens. In contrast, Th CD4+ T cells secrete predominantly IL-4, IL-5 and IL-1 and are involved in immediate or allergic type-1 hypersensitivity.
Th1 responses and especially IFN-γ are central effectors in acquired resistance against M. tuberculosis by stimulating the microbicidal functions of macro-phages and cytotoxic CD8+ T cells. IFN-γ is secreted by a somewhat restricted category of cells: CD4+ and CD8+ αβ T cells, γδ T cells, CD1+ cells and NK(T) cells. By activation of effector cells, IFN-γ enhances Th1 CD4+ protective immune responses and cytotoxic functions of CD8+ T cells. It also induces type NO synthase and other effector molecules in infected alveolar macrophages (17).
IL-1 (sustained by IL-18) is a key regula-tor of IFN-γ production via the induction and the maintenance of Th1 effector functions of CD4+ T cells in M. tubercu-losis-infected mice (18-140). IL-1 and IL-18 are mainly produced by macro-phages in response to several microbial components from mycobacteria and are potent IFN-γ-inducing factors. Alto-gether, these cytokines link innate and adaptive immune responses by promot-ing Th1 differentiation and enhancpromot-ing cytolytic activities of NK cells, macro-phages and cytotoxic CD8+ T cells. In ad-dition to their role in the establishment of the primary resistance in humans and animal models, IL-1 and IFN-γ have also been associated with recall responses in mice upon vaccination (94, 141).
Once the adaptive immune response has been induced, Th1-polarized CD4+ and CD8+ T cells constitute the predomi-nant source of IFN-γ. The importance of IFN-γ in the control of TB infection was illustrated both in mice and humans:
IFN-γ-/- mice are unable to control the infection with impaired granuloma for-mation and profound susceptibility to infection with M. tuberculosis (5). Upon vaccination with M. bovis BCG, humans with a IFN-γ receptor mutation will suc-cumb from disseminated BCG infection (14, 14).
During the 80’s, the Th1-Th imbalance was thought to be important in the origin of the TB disease. This hypoth-esis was based on the correlation be-tween the clinical course of M. leprae infection and the dichotomy between Th1 and Th responses. Th1 responses are strongly protective and are associ-ated with a limited form of the disease (the tuberculoid form). In contrast, Th
responses are correlated with diffused and uncontrolled disease (the leproma-tous form). Whether or not the balance of Th1-Th responses is also important with regard to the evolution of M. tuber-culosis infection is controversial. Rook and co-workers have published that the upregulation of genes related to type cytokines during active TB correlates to some extent with the disease (144).
This is in agreement with the hypothesis that exacerbated Th-like or regulatory processes could undermine the efficacy of the protective response. From their point of view, the presence of IL-4, in as-sociation with a simultaneous decrease of its potential antagonist IL4δ, could have a dominant role in the progression toward active disease (145). According to these authors, most of the unfavour-able effects of IL-4 are mediated by in-appropriate macrophage activation and decreased TLR and iNOS expression.
However, other authors failed to associ-ate Th responses and TB disease (146).
In consequence, the debate about the Th1-Th balance is still open. It becomes even more complex after the recent de-scription of an additional role for other cellular subsets, such as regulatory T cells and Th17 T cells (see below).
CD8+ T cells
The contribution of CD8+ T cells to the host resistance against M. tuberculosis is multifaceted, including the release of cytokines, the lysis of target cells, the in-duction of target cell apoptosis and di-rect antimicrobial activity (147). CD8+ T cells release cytokines, such as IFN-γ and TNF-α, albeit at a lower level compared to CD4+ T cells (148). More importantly, CD8+ T cells mediate essential cytotoxic
functions based on the release of cyto-toxic granules upon antigenic stimula-tion through TCR/MHC-I interacstimula-tions (figure 15). In contrast to MHC-II mol-ecules, the broad majority of host cells expresses MHC-I molecules. The ubiq-uitous expression of MHC-I molecules greatly increases the area under the control of CD8+ T cells and significantly reduces the chance that M. tuberculosis passes unnoticed. From this point of view, CD8+ T cells should be important in the control of TB infection.
In mice, the role of CD8+ T cells during TB was reported several years ago. Indeed, the absence of functional CD8+ T cells in mice is associated with a poor prognosis after M. tuberculosis infection (149). This study relied on targeted mutations of the ß-microglobulin gene, which is a major chain of the MHC class I molecule.
However, other cellular subtypes (i.e. γδ T cells) also express the ß-microglobu-lin. Accordingly, unconventional T cells were also probably absent in this mod-el. In addition, the ß-microglobulin is also involved in the metabolism of iron, which is required for optimal mycobac-terial growth. Therefore, this model of ß-microglobulin-/- mouse is probably not ideal. However, the importance of CD8+ T cells was later confirmed by showing that the survival of mice with selective mutation of MHC-I molecules was reduced upon TB infection (150).
Cytotoxic granules of CD8+ T cells con-tain perforin, which is a pore forming protein able to mediate a lytic activity.
Perforin alone has no anti-mycobacte-rial activity. However, it allows cyto-toxic compounds, such as granzymes and granulysin, to be delivered into the infected cell, which in turn induces
cell death. In contrast to the absence of functional MHC-I molecules, the ab-sence of granzymes or perforin did not compromise the survival of M. tubercu-losis-infected mice (151). Cytotoxic CD8+ T cells that use a granule exocytosis pathway also release granulysin, a sa-posin-like protein (15). In contrast to perforin, granulysin interacts with lipid membranes and activates specific lipid-degrading enzymes (e.g. glucosylceram-idase). Once activated, these enzymes increase the concentration of intracel-lular ceramide, a compound involved in the activation of the caspases apoptotic pathway. Granulysin was shown to be microbicidal against several pathogens, including Gram-positive and negative bacteria, fungi and parasites. Moreover, granulysin was very efficient in killing extracellular M. tuberculosis and even in-tracellular M. tuberculosis if perforin gains
access to the intracellular compartment . Interestingly, plasma granulysin levels decrease during active pulmonary TB, but anti-TB therapy results in a signifi-cant increase (15). Accordingly, some authors suggest that plasma granulysin levels correlate with curative host re-sponses to pulmonary TB (15).
CD8+ T cells also mediate the induc-tion of apoptosis of the infected cells by Fas-FasL interaction. Apoptosis was associated with the inhibition of myco-bacterial growth. This was attributed to improved antigenic presentation upon uptake of the apoptotic particles by pro-fessional APCs. Conversely, apoptosis has been reported to have little impact on M. tuberculosis survival (154).
In humans, the role of CD8+ T cells in the control of M. tuberculosis infection is suggested by indirect arguments. For
CD8
NO CTL
IFN-γ
Perforin Granulysin TNF-α
TNF-α TNF-α
Macrophage activation
Kill host cell and bacteria Kill bacteria
Apoptosis
Lysis/Necrosis
Phagocytosis by other cells
Dissemination Fas-L
Fas
Kill bacteria ?
Figure 15. Major functions of CD8+ T cells duringM. tuberculosis infection.Through the release of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-a), CD8+ T cells activate microbicidal functions of macrophages (i.e. NO production). CD8+ T cells also act as cytotoxic T lymphocytes (CTL) by mediating apoptosis (Fas-L/Fas interactions) and necrosis (Perforin/Granulysin release). Adapted from Kaufmann S.H.E. CD8+ T cells during tuberculosis. In: Cole ST, editor. Tuberculosis and the tubercle bacillus.
Washington DC: ASM Press;005. p. 465-474
instance, the severity of TB was inverse-ly correlated with cytotoxic activity of PBMC from TB patients (155). Recently, HBHA-specific CD8+ T cells from LTBI subjects were shown to be cytotoxic against HBHA-loaded macrophages, via a granule-dependent mechanism (156). HBHA-induced perforin+CD8+ T cells were distinct from the IFN-γ+CD8+ T cells. In addition, microbicidal activity of these HBHA-specific CD8+ T cells was demonstrated (156).
Th17 CD4+ T cells
Th17 cells are IL-17-producing CD4+ T cells. IL-17 is a potent inflammatory cy-tokine that plays a significant role in the recruitment of cells to tissues (e.g. Th1 CD4+ T cells). Th17 cells require TGF-ß and IL-6 for differentiation. In addition, they require the presence of IL- to sus-tain their terminal differentiation that is absolutely required for IL-17 production during M. tuberculosis infection (157).
Moreover, IL- promotes the secretion of TNF-α and IL-6 but not of IFN-γ. Inter-estingly, IFN-γ negatively regulates Th17 cells. IL-17 is involved in autoimmune diseases and contributes to inflamma-tory responses. This is mediated by their capacity to induce chemokines, growth factors and adhesion molecules, and by powerful neutrophil recruitment (158).
The description of Th17-cells is of impor-tance for a better understanding of the heterogeneity of helper T cell fates (fig-ure 16) (159). For instance, differentiation of Th17-cells depends on the presence of IL-6 plus TGF-ß. In the absence of IL-6 but in the presence of TGF-ß, the naive precursors differentiate into regulatory T cells (Treg), which suppress immune responses (see below). Hence, there is
a balance between the pro-inflamma-tory profile mediated by Th-17 cells and the anti-inflammatory profile mediated by Treg cells. Moreover, this balance depends on the cytokine environment, as the presence of IL-6 switches off the pro-regulatory function of TGF-ß.
The role of Th-17 cells during TB remains controversial. Khader et al. showed some evidence for a Th17 cell-mediated establishment of an early recall response within the lung (160). These authors vac-cinated mice with a peptide comprising amino-acid 1-0 of ESAT-6. After vac-cination, these mice were challenged with M. tuberculosis H7Rv. As expected, vaccination was associated with a reduc-tion of CFU within the lungs in compari-son with unvaccinated mice. Strikingly, these vaccinated mice also showed an early infiltration of their lung by IFN-γ-producing cells, which was preceded by an infiltration of IL-17-producing cells.
ILp19-deficient mice lack IL-17-pro-ducing cells. Upon vaccination of these mice with ESAT-6, M. tuberculosis infec-tion was associated with both deficient granulomatous structures and reduced accumulation of activated CD4+ T cells in the lung. Moreover the addition of exogenous IL-17 significantly increased the accumulation of INF-γ-producing CD4+ T cells in the lung of these deficient mice. Upon vaccination, the absence of memory Th17 cells was associated with impaired recall responses and poor pro-tection against M. tuberculosis infection (160).
However, another group demonstrated that the absence of a functional IL- pathway or the absence of IL-17 did not compromise the control of a massive BCG infection in mice (161). Based on
their data, Khader and co-workers pro-posed that Th17 cells act as “immune surveillance cells”, which are preferen-tially found in the periphery and which mediate a rapid recall response rely-ing on the arrival of IFN-γ-producrely-ing cells. Once the production of IFN-γ in response to mycobacterial antigens is triggered, IFN-γ itself will turn down the production of IL-17.
Unconventional T cells
Unconventional T cells are T cells that are activated by other ways than the conventional MHC presentation sys-tem. Gamma-delta (γδ) T cells and CD1-restricted T cells (including some γδ T cells) are the most well described.
γδ T cells
In humans, γδ T cells recognize their target antigens without presentation by the MHC complex, in contrast to con-ventional αβ T cells. Vγ9/Vδ T cells, a subtype of γδ T cells, directly recognize non-protein antigens or metabolites (e.g. phosphoesters derived from my-cobacteria) However, the antigen-pre-senting molecule, if it is necessary, still remains unknown. This particularity allows rapid responses and therefore, γδ T cells are able to participate in early immune responses. Hence, γδ T cells are classified as innate-like lymphocytes (16). Other subtypes of γδ T cells inter-act with non-classical molecules such as CD1c (which allows presentation of lip-ids and glycoliplip-ids (see below)) or MHC
Naive T cell Dendritic cell
TGF-β
COUNTER-REGULATION
PROTECTION EARLY RECALL
RESPONSE
COUNTER-REGULATION
Treg IL-2
IL-2R FOXP3
- IL-6 IFN-γ
Th1 IL-12
IL-12R T-bet
+ IL-6
Th17 IL-23
IL-23R RORγt
IL-4
Th2
Gata-3
Figure16.HelperTcellfatesduringinfectionwithM. tuberculosis.Differentiation of Th17 cells depends on the combination of Transforming Growth Factor-Beta (TGF-ß) and interleukin-6 (6). TGF-ß without IL-6 promotes the differentiation of naive T cells into regulatory T cells (Treg) and inhibits the differentiation and function of Th1/Th T cells. Adapted from: Reiner, SL. Development in motion : Helper T cells at work. Cell 007; 19:-6.
class I-related molecules (e.g. MICA and MICB, that are induced on the surface of stressed cells).
Vγ9/Vδ T cells from the PBMC of LTBI subjects selectively expand in vitro dur-ing M. tuberculosis infection and are able to kill both extracellular and intracellu-lar M. tuberculosis (16). After activation by phosphoantigens, γδ T cells also pro-duce a large amount of cytokines (e.g.
IFN-γ, TNF-α, IL-4), and chemokines (e.g.
MIP1-β, lymphotactin). In addition, the protection mediated by γδ T cells could be achieved by the killing of infected cells. This process depends on several mechanisms, such as Fas-FasL interac-tion, TNF-α production and the release of cytotoxic granules (containing per-forin, granzyme and granulysin) (164).
The importance of γδ T cells during TB in humans is not clearly defined. For instance, neither the relative percent-age, nor the absolute numbers of pe-ripheral blood γδ T cells from adult TB patients are altered in comparison with uninfected controls (165, 166). However, a substantial amount of γδ T cells was found within the TB lesions where they exhibited strong reactivity toward my-cobacterial antigens (165, 166). Results obtained in murine models are also
con-troversial. It was demonstrated that γδ T cell-deficient mice are more susceptible to infection, supporting the idea that γδ T cells are protective during TB infection (167). Other authors showed that myco-bacterial growth was identical in γδ T cell-deficient and wild-type mice (168).
However, in this study, polymorphonu-clear infiltrates were more pronounced with a small lymphocytic response in γδ T cell-deficient mice, suggesting that γδ T cells can regulate the infiltration of polymorphonuclear cells, which medi-ate tissue damage without inducing good levels of microbicidal activity.
CD1-restricted T cells
The CD1-restricted T cells are interest-ing because they recognise glycolipids, which are abundant in the mycobacte-rial cell wall. The CD1 denomination in-cludes several related proteins that are associated with the ß-microglobulin and that are encoded by genes located outside the MHC locus. In humans, five CD1 genes are described (from CD1a to CD1e). CD1 molecules are structur-ally related to the MHC class I molecules but show a particular hydrophobic anti-gen-binding groove that allows them to bindi highly hydrophobic ligands such
Table n°4 : CD1 molecules
CD1a, b and c (group 1) CD1d (group 2)
Species Human Human, mouse
Genomic location Non-MHC Non-MHC
Major cellular expression B cells (CD1c) and DC (immature)
B cells, monocytes, macrophages, DC Antigens presented Endogenous/exogenous glycolipids T cells subsets DN, CD8+, CD4+,
TCRaβ, TCR γδ NKT, DN, CD4+, TCRaβ
as lipids. Cell types that express CD1 molecules are divided in two groups in function of the CD1 involved (table n°4, adapted from reference (169)). CD1 mol-ecules of group 1 present certain myco-bacterial lipid antigens to T cells, such as mycolic acids and LAM, which are criti-cal components of the mycobacterial cell wall (170, 171).
Most of the studies that suggest a pro-tective role of the CD1-dependent anti-genic presentation focus on the group 1 CD1 molecules. Once activated, group 1 CD1-restricted T cells are able to secrete Th1 cytokines like IFN-γ (171), but they also exert some cytotoxic activities that can lyse M. tuberculosis-infected cells (17). As a consequence, it is thought that CD1-restricted T cells play a protec-tive role during the infection with M.
tuberculosis. Unfortunately, M. tubercu-losis is able to down-regulate the CD1 expression (11) and therefore, to con-tribute to the disruption of the antigenic presentation.
Until now, the protective role of group CD1-restricted T cells during TB has not been demonstrated. NKT cells are the most famous subset of group CD1-re-stricted T cells. NKT cells are CD+ cells that also express markers of NK cells, such as CD16 or CD56. Even though their proportion increases during active TB (17), the role of these cells during M. tu-berculosis infection remains practically unexplored.
In mice, NKT cells respond to myco-bacterial cell wall components and are involved in early granuloma formation (174). NKT also produce IFN-γ during early phases of M. bovis BCG infection in mice (175). However, CD1d-deficient mice that lack NKT cells show no
in-creased susceptibility to M. tuberculosis infection (176).