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The Journal of Immunology, 2006, 177: 1780-1785.
Copyright © 2006 by The American Association of Immunologists

Phenotypical and Functional Analysis of Memory and Effector Human CD8 T Cells Specific for Mycobacterial Antigens1

Nadia Caccamo, Serena Meraviglia, Carmela La Mendola, Giuliana Guggino, Francesco Dieli2 and Alfredo Salerno

Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Palermo, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mycobacterium tuberculosis infects one-third of the global population and claims two million lives every year. Because memory CD8 T cells exhibit a high heterogeneity in terms of phenotype and functional characteristic, we investigated the frequency, phenotype, and functional properties of Ag85A epitope-specific HLA-A*0201 CD8 T cells in children affected by tuberculosis (TB) before and 4 mo after chemotherapy and healthy contact children. Using Ag85A peptide/HLA-A*0201 pentamer, we found a low frequency of blood peptide-specific CD8 T cells in tuberculous children before therapy, which consistently increased after therapy to levels detected in healthy contacts. Ex vivo analysis of the expression of CD45RA and CCR7 surface markers indicated a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of T central memory cells and a decrease of terminally differentiated T cells, which was reversed after therapy. Accordingly, pentamer-specific CD8 T cells from tuberculous patients produced low levels of IFN-{gamma} and had low expression of perforin, which recovered after therapy. The finding of an elevated frequency of pentamer-specific CD8 T cells with T effector memory and terminally differentiated phenotypes in the cerebrospinal fluid of a child with tuberculous meningitis strongly indicates compartmentalization of such CD8 effectors at the site of disease. Our study represents the first characterization of Ag-specific memory and effector CD8 T cells during TB and may help to understand the type of immune response that vaccine candidates should stimulate to achieve protection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tuberculosis (TB)3 is a leading infectious disease affecting millions of people worldwide. Mycobacterium tuberculosis, the primary etiologic agent of TB, causes ~8 million new cases each year and the disease is responsible for at least 2 million deaths annually (1). Many parameters, notably socioeconomic factors, coinfection with HIV, the increasing incidence of antibiotics resistance, and genetic predisposition of the host influence the susceptibility to TB.

Although a role for CD4 T cells in protection against M. tuberculosis is well reported, there is also a large body of evidence derived from both human and nonhuman models that suggests an essential role for CD8 T cells (2, 3, 4, 5). These cells are able to secrete IFN-{gamma} and TNF-{alpha} upon recognition of mycobacterial Ags. Moreover, CD8 T cells can kill infected cells via a granule-dependent mechanism involving perforin and granulysin, which also possess a direct antimicrobial activity (6). The anatomy of the adaptively immune response to infection with M. tuberculosis suggests that CD8 T cells are intimately involved in the host response. Also, CD8 T cells are present within the granuloma where they have access to infected cells and are poised to prevent bacillary dissemination. Finally, CD8 T cells specific for numerous mycobacterial Ags can be isolated at high frequency from human and mouse models, consistent with the hypothesis that CD8 T lymphocytes are constantly being stimulated with Ag (7).

In this study, we have analyzed the frequency, the phenotype, and the functional properties of CD8 T cells specific for the mycobacterial Ag 85A (Ag85A) in children affected by TB and in healthy tuberculin-positive and -negative control children.

Ag85A is one of three distinct but highly conserved proteins (85-A, -B, and -C) that together make up the Ag85 complex, a highly cross-reactive Ag found in all mycobacterial species tested so far. Interest in Ag85A is based on the grounds that it is a secreted Ag that constitutes a major portion of the secreted proteins present in the culture filtrate of both M. tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) (8, 9). Furthermore, CD8 CTL responses have been observed against this Ag (10, 11). The Ag85 complex has also been shown to induce good proliferative, IFN-{gamma} production and cytolytic responses in BCG-vaccinated, M. tuberculosis- and Mycobacterium leprae-infected individuals (12, 13, 14, 15). Moreover, murine experiments have shown that plasmid DNA vaccination encoding Ag85A generates strong Th1 CD4 T cell and CD8 T cell-mediated CTL responses (16, 17, 18) and a recent phase 1 study using recombinant modified vaccinia virus Ankara expressing Ag85A has shown that it induces high levels of Ag-specific IFN-{gamma}-producing T cells when used alone or in combination with BCG in naive healthy volunteers (19).

Moreover, we have determined the ex vivo frequency of epitope-specific CD8 T cells in the peripheral blood and in the cerebrospinal fluid (CSF) obtained from one tuberculous child to detect compartmentalization of these cells at the site of disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Human subjects

Peripheral blood was obtained from five HLA-A*0201 children with TB (three boys, two girls; age range, 6–12 years) from the Children’s Hospital G. Di Cristina (Palermo, Italy); six tuberculin (purified protein derivative (PPD))-positive, naturally infected healthy children (three boys, three girls; age range, 5–11 years); and seven tuberculin (PPD)-negative healthy children (four boys, three girls; age range, 5–11 years). All of the groups consisted of children from the same geographical area (Sicily, southern Italy) and of similar socioeconomic background. Four of the five tubercolous patients were affected by pulmonary TB, as established by the presence of clinical symptoms of TB, by the positivity of the tuberculin (PPD) skin test, and by chest radiography, whereas one patient was affected by TB meningitis. Tuberculous children were treated with 2 wk of daily isoniazid, rifampin, and pyrazinamide; and then 6 wk of the same combination twice weekly, followed by 16 wk of twice-weekly isoniazid and rifampin without pyrazinamide. Response to therapy was defined as improvement in symptoms, weight gain, improvement by physical examination, chest radiography (reduction of the size of both pulmonary infiltrates and adenopathy), laboratory parameters (erythrocyte sedimentation rates, C-reactive protein levels, granulocyte numbers, and hemoglobin levels), and adherence to treatment. Peripheral blood and CSF were collected before and 4 mo after chemotherapy. None of the TB children had been vaccinated in infancy with BCG or had evidence of HIV infection or was being treated with steroid or antitubercular drugs at the time of sampling. Tuberculin (PPD) skin tests were considered positive when the induration diameter was >5 mm at 72 h since injection of 1 U of PPD (Statens Seruminstitut). Informed consent was given by the TB children’s parents. The following nomenclature was used throughout the text for the different groups of children: TB0, children affected by TB before starting chemotherapy; TB4, children affected by TB, 4 mo after chemotherapy; H-PPD+, healthy tuberculin-positive children; H-PPD–, healthy tuberculin-negative children.

Subjects were HLA typed serologically. The HLA subtype, A*0201, was confirmed by PCR amplification technique using sequence-specific oligonucleotide primers.

Pentamer staining

PBMC or CSF mononuclear cells were isolated from heparinized blood by centrifugation on Ficoll-Hypaque (Pharmacia). The medium used throughout was RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% heat-inactivated pooled human AB+ serum, 2 mM L-glutamine, 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 10–5 M 2-ME. PBMC or CSF cells were washed in complete medium and incubated with FITC-labeled anti-CD8 mAb and PE-labeled pentamers (10 µl), washed, and analyzed by flow cytometry on a FACSCalibur analyzer. Viable lymphocytes were gated by forward and side scatter, and analysis was performed on 100,000 acquired events for each sample. HLA-A2 pentamer complexes loaded with the M. tuberculosis Ag85A epitope GLPVEYLQV, the 16-kDa epitope GILTVSVAV, and the ESAT-6 epitope AMASTEGNV were purchased from ProImmune. To assess the phenotype of pentamer+ T cells, cells were stained with allophycocyanin-labeled anti-CCR7 mAb and PE-Cy5-labeled anti-CD45RA mAb (all from BD Biosciences) in incubation buffer (PBS containing 1% FCS and 0.1% sodium azide) for 30 min at 4°C. Cells were then washed twice in PBS with 1% FCS and analyzed by flow cytometry. To study expression of other cell surface markers, four-color FACS analysis was performed on PBMC upon staining with PE-labeled pentamers, allophycocyanin-labeled anti-CCR7 mAb, PE-Cy5-labeled anti-CD45RA mAb, and FITC-labeled anti-CD62L or FITC-labeled anti-CD27 or FITC-labeled anti-CD28 or FITC-labeled anti-CD57 mAbs (all from BD Biosciences).

Intracellular FACS analysis

To study intracellular production of IFN-{gamma}, PBMC were stimulated with the Ag85A peptide GLPVEYLQV, in the presence of monensin for 6 h at 37°C in 5% CO2. The cells were harvested, washed, and stained with allophycocyanin-labeled anti-CCR7 mAb, PE-Cy5-labeled anti-CD8 mAb, and PE-labeled pentamers in incubation buffer (PBS containing 1% FCS and 0.1% Na azide) for 30 min at 4°C. The cells were washed twice in PBS with 1% FCS and fixed with PBS containing 4% paraformaldehyde overnight at 4°C. Fixation was followed by permeabilization with PBS containing 1% FCS, 0.3% saponin, and 0.1% Na azide for 15 min at 4°C. Staining of intracellular cytokine was performed by incubation of fixed permeabilized cells with FITC-labeled anti-IFN-{gamma} mAb. Viable lymphocytes were gated by forward and side scatter, and analysis was performed on 100,000 acquired events for each sample.

For detection of perforin, PBMC were stained directly ex vivo (i.e., without any Ag stimulation in vitro) in calcium-free medium with allophycocyanin-labeled anti-CCR7 mAb, PE-Cy5-labeled anti-CD8 mAb, and PE-labeled pentamers as above described, washed, and fixed with PBS containing 4% paraformaldehyde for 30 min at 4°C. After two washes in permeabilization buffer, cells were stained with FITC-conjugated anti-perforin Ab ({delta}G2; Alexis; 2 mg/ml final concentration). After two more washes in PBS containing 1% FCS, the cells were analyzed by FACSCalibur.

Statistics

Values of p were derived from two-tailed ANOVA tests. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ex vivo analysis of circulating Ag85A epitope-specific CD8 T cells

To determine the ex vivo frequency of peptide-specific CD8 T cells, PBMC from HLA-A*0201 TB patients and PPD+ and PPD– healthy children were stained with HLA-A*0201/Ag85A peptide (GLPVEYLQV) pentamer and analyzed by FACS.

The mean frequency of pentamer-specific CD8 T cells was 0.14% in TB patients before chemotherapy and raised to 0.57% at 4 mo after therapy; the frequency of pentamer CD8 T cells was 0.76% in PPD+ healthy children and 0.001% in PPD– healthy children, thus confirming the specificity of the pentamer used (Fig. 1A). Fig. 1B shows FACS analysis of HLA-A*0201/Ag85A peptide-specific CD8 T cells of one representative subject for each tested group.


Figure 1
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  		FIGURE 1. Ex vivo analysis of frequency of peptide-specific CD8 T cells. A, Cumulative data on the frequencies of the Ag85A pentamer-specific CD8 T cells in peripheral blood of TB children before (TB0) and 4 mo after therapy (TB4), and PPD+ (H-PPD+) and PPD– (H-PPD–) healthy children. Values indicate the percentage ± SD of CD8+ Ag85A pentamer+ cells. B, FACS analysis of Ag85A epitope-specific CD8 T cells of one representative subject for each tested group. Values indicate the percentage of CD8+ pentamer+ cells.

 
These results indicate that the frequencies of Ag85A epitope-specific CD8 T cells are low in TB children, when compared with PPD+ healthy children, but increase after antimycobacterial therapy.

Distribution of Ag85A epitope-specific CD8 T cell subsets

Four populations of memory CD8 T cells have been identified based on the expression of CD45RA and CCR7 (20): T naive (Tnaive, CD45RA+CCR7+), T central memory (TCM, CD45RA–CCR7+), T effector memory (TEM, CD45RA–CCR7–), and terminally differentiated T effector memory (TEMRA, CD45RA+CCR7–). In preliminary analyses, we have compared the distribution of CD8 T cell subsets in blood of HLA-A*0201 TB patients, PPD+ and PPD– healthy children, with the data summarized in Table I. In TB patients before therapy, 25% of blood CD8 T cells were CCR7+CD45RA– (TCM), whereas in other groups this subset accounted for 15% or less of CD8 T cells. In contrast, the CCR7–CD45RA+ TEMRA population was less than the other groups. The distribution of CCR7+CD45RA+ (Tnaive) and CCR7–CD45RA– (TEM) were not different in tested groups. These results provide experimental evidence for a large accumulation of CD8 TCM and a consistent reduction of CD8 T cells with potential effector functions (TEMRA) in peripheral blood of TB patients, which reverses after therapy.


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  		Table I. Comparative analysis of the expression of CCR7 and CD45RA in CD8 T cells isolated from blood of TB patients, healthy PPD+ contacts, and healthy PPD– individualsa

 
We then used simultaneous staining with CD45RA and CCR7 mAbs to separate functionally distinct subpopulations of HLA-A0201*/Ag85A epitope-specific CD8 T cells in peripheral blood of TB patients, before and after 4 mo of therapy and PPD+ healthy children (Fig. 2). Interestingly, in healthy PPD+ children, the majority (46%) of blood pentamer-specific CD8 T cells was composed of terminally differentiated (TEMRA, CD45RA+CCR7–) cells. In these individuals, TEM cells accounted for 24% of the Ag85A-epitope specific T cell population, whereas Tnaive and TCM together were ~25%. In TB patients before therapy a large proportion (64%) of blood pentamer-specific memory CD8 T cells were composed of TCM cells. In these patients, Tnaive cells accounted for ~20% and TEM cells for 15% of pentamer-specific CD8 T cells, whereas the pool of terminally differentiated (TEMRA, CD45RA+CCR7–) cells was consistently reduced (2%). However, this Ag-specific CD8 T cell distribution pattern consistently changed 4 mo after therapy, with a significant proportion of TEMRA cells (from 2 to 28%) and a decrease of TCM (from 64 to 29%). Fig. 3A shows a representative FACS analysis of subsets of blood Ag85 epitope-specific CD8 T cells of one subject for each tested group.


Figure 2
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  		FIGURE 2. Cumulative data on the distribution of Ag85A epitope-specific CD8 T cells in peripheral blood of TB children before (TB0) and 4 mo after therapy (TB4), and PPD+ (H-PPD+) healthy children. PBMC were stained with Ag85A pentamer, CD8, CD45RA, and CCR7 mAbs to separate functionally distinct subpopulations. After gating on pentamer+CD8+ cells, the percentage of cells expressing CD45RA and CCR7 was determined. The value reported are the average percentage of the different subset analyzed for each group tested ± SD.

 

Figure 3
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  		FIGURE 3. Analysis of the distribution of Ag85A epitope-specific CD8 T cell subsets. A, PBMC were stained with Ag85A pentamer, CD8, CD45RA, and CCR7 mAbs to separate functionally distinct subpopulations in peripheral blood of TB children before (TB0) and 4 mo after therapy (TB4), and PPD+ (H-PPD+) healthy children and analyzed by FACS. In the flow analyses, at least 106 events were acquired. CD45RA and CCR7 expression are shown upon gating on pentamer+CD8+ cells. Numbers indicate the percentage of cells in each quadrant. B, Phenotype of Ag85A pentamer+CD8+ T cell subsets. Values indicate the percentage of Ag85A pentamer+CD8+ T cells in TB children before (TB0) and 4 mo after (TB4) therapy, expressing the indicated cell surface markers. Numbers indicate the percentage of positive cells.

 
To further define the cell surface phenotype of these Ag85A pentamer+ CD8 T cell subsets, PBMC from TB children before therapy (TB0) and 4 mo after therapy (TB4) were analyzed for CD62L, CD27, CD28, and CD57 expression by four-color immunofluorescence (Fig. 3B).

In TB patients before therapy, Ag85A pentamer+CD8+ cells showed characteristics of unprimed cells, because most of them were CD62L+, CD27+, CD28+, and had low CD57 expression. Four months after therapy, Ag85A pentamer+CD8+ cells had lower expression of CD62L and CD27, and increased CD57 expression, whereas CD28 expression did not change after therapy.

These results indicate a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of TCM and a decrease of TEMRA cells, which is reversed after therapy.

Functional analysis of Ag85A-specific CD8 T cells

To address the functional properties of different pentamer specific CD8 T cell subsets, PBMC from TB patients before and after therapy and healthy PPD+ children were stimulated with Ag85A peptide and assessed for the ability to produce IFN-{gamma} or tested immediately ex vivo (i.e., without any Ag or peptide stimulation in vitro) for intracellular perforin. Cumulative data are shown in Fig. 4. Results clearly indicate that a substantial proportion of pentamer-specific CCR7– CD8 T cells has the ability to produce IFN-{gamma} and to express perforin, both in healthy PPD+ children and in TB patients, although in the latter the percentages of IFN-{gamma}+ and perforin+ Ag85A epitope-specific CCR7– CD8 T cells were significantly lower at the time of diagnosis (TB0), but increased 4 mo after therapy (TB4).


Figure 4
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  		FIGURE 4. Cumulative data of functional analysis of Ag85A epitope-specific CD8 T cells. The data are expressed as the percentage of pentamer+CD8+ T cells producing IFN-{gamma} and expressing perforin within CCR7– ({blacksquare}) and CCR7+ ({square}) cell populations.

 
Conversely, a lower proportion of Ag85A epitope-specific CCR7+ CD8 T cells have the ability to produce IFN-{gamma} and to express perforin in healthy PPD+ children and TB patients after therapy, whereas very low IFN-{gamma} and perforin content was detected in pentamer-specific CCR7+ CD8 T cells from TB patients before therapy.

Analysis of Ag85A-specific CD8 T cell subsets at the site of disease

As one of the above-studied TB patients presented with TB meningitis, we determined the frequency and subset distribution of Ag85A peptide-specific CD8 T cells in both PBMC and CSF. As shown in Fig. 5, the frequency of Ag85A-specific CD8 T cells was greater in CSF (1.30%) than in PBMC (0.21%), indicating compartmentalization of mycobacteria-specific T cells at the site of disease. No Ag-specific bias in the repertoire of the polyclonal T responses in CSF was evident because the frequency of HLA-*A0201 pentamer complexes loaded with M. tuberculosis 16-kDa epitope GILTVSVAV demonstrated a similar enrichment in CSF compared with PBMC (0.14 and 1.56% in PBMC and CSF, respectively), and the frequency of HLA-A*0201 pentamer complexes loaded with ESAT-6 epitope AMASTEGNV was 0.18 and 0.97% in PBMC and CSF, respectively. In contrast, undetectable levels of CD8 T cells specific for the CMV pp65 495–503 (NLVPMVATV) epitope were found in both PBMC and CSF (data not shown). The distribution of Ag85A-specific CD8 T cells was different in PBMC and in CSF of this patient (Fig. 5): although a large proportion (93%) of blood pentamer-specific memory CD8 T cells were mostly composed of TCM (23%) and Tnaive (70%) cells, these subsets were almost absent from the CSF, where TEM (30%) and TEMRA (65%) accounted for the vast majority of Ag85A-specific CD8 T cells. Of note, a similar subset distribution was observed in freshly isolated CSF cells and in CSF-derived T cell line, thus confirming that the method used to generate the cell line did not cause any skewing in the distribution of CD8 T cell subset.


Figure 5
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  		FIGURE 5. The frequency and the distribution analysis of Ag85A pentamer+ CD8 T cells in CSF and in PBMC. Frequency and subset distribution of Ag85A pentamer+ CD8 T cells obtained from PBMC and CSF of one child affected by TB meningitis. In the flow analyses, at least 106 events were acquired and gated on pentamer+ cells.

 
These results demonstrate compartmentalization of Ag-specific effector memory CD8 T cell subsets at the site of disease during active TB, which might explain, at least in part, their consistent reduction in peripheral blood.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A protective role of MHC class I-mediated CD8 CTLs has been suggested earlier in experimental TB in mice, because mice lacking functional CD8 T cells are more susceptible to mycobacterial infection (2, 21).

Human studies have provided evidence that MHC class I-restricted CD8 T recognize M. tuberculosis-infected cells and can release IFN-{gamma}, lyse infected target cells, and kill intracellular bacteria (22, 23, 24, 25, 26, 27). For these reasons, there has been an intense effort to define M. tuberculosis epitopes that can be presented by MHC class I molecules to CD8 T cells. In the present study, we have analyzed the functional properties and the phenotype of Ag85A-epitope HLA-A*0201 CD8 T cells in BCG vaccinated subjects, TB patients before and after antimycobacterial therapy, and PPD+ healthy donors using soluble HLA-A*0201/Ag85A peptide (GLPVEYLQV) pentamer. HLA-pentameric complexes have been developed for use in determining Ag-specific CD8 T cell frequencies to be detected by FACS analysis. The great advantage of this technique is that it is able to detect Ag-specific T cells independent of their effector functions. In addition, through multicolor analysis, it is possible to obtain cell surface and intracellular phenotype data and to directly sort the cells to generate purified populations that can be functionally analyzed.

Taking advantage of pentamer technology, Ag-specific T cells are now being studied in different situations such as viral infection, aging, cancer, and intracellular pathogen (28).

We have studied the specificity of CD8 T cells toward Ag85A of M. tuberculosis, because it induces strong Th1 CD4 and CD8 T cell responses (16, 17, 18). It was therefore of particular interest to assess the proportion of circulating CD8 T cells that would recognize peptides from Ag85A.

Data here reported show quantitative and qualitative differences between TB children and healthy PPD+ children in the pool of Ag85-specific CD8 T cells. The frequency of blood Ag85A-epitope specific CD8 T cells is reduced in tuberculous children before chemotherapy, when compared with the frequency detected in PPD+ healthy children, but a partial recovery of Ag85 epitope-specific T cells could be demonstrated 4 mo after therapy.

Additionally, although in healthy PPD+ children the majority of blood pentamer-specific CD8 T cells was composed of effector memory TEMRA and TEM cells, in TB children before therapy a large proportion (~85%) of blood pentamer-specific memory CD8 T cells was mostly composed of TCM and TEM cells, whereas the pool of terminally differentiated (TEMRA) cells was consistently reduced or absent. However, this Ag-specific CD8 T cell distribution pattern consistently changed 4 mo after therapy, with a significant recovery of TEMRA cells and decreased frequencies of TCM cells. This assumption is supported by the analysis of expression of many different markers on pentamer-specific CD8 T cells (including CD45RA, CCR7, CD27, CD28, CD57, and CD62L) and by functional data showing reduced production of intracellular IFN-{gamma} and expression of perforin by pentamer-specific CD8 T cells, both in the CCR7– population and, even if at a lower extent, in the CCR7+ population.

The reason for the reduction of blood effector (TEM and TEMRA) pentamer-specific CD8 T cells during active TB and their recovery after therapy is unknown. One possibility is that such mycobacteria-specific CD8 T cells are sequestered at sites of disease but then appear in blood after successful therapy. This phenomenon of sequestration of Ag-specific cells has been widely observed in TB for both CD4 and CD8 (29, 30, 31) population, and although a detailed phenotypic analysis is generally not possible in such cases, the reduced IFN-{gamma} production in the peripheral blood upon Ag stimulation in vitro is strongly suggestive of the loss/reduction of effector memory CD4 and CD8 T cells. Evidence for Ag85 epitope-specific CD8 T cell sequestration is provided here. A significantly high percentage of Ag85A epitope-specific CD8 T cells was found in the CSF of a child with TB meningitis, when compared with the peripheral blood. Furthermore, the vast majority of these T cells was composed of TEM and TEMRA cells. Alternatively, it is possible that the reduced effector memory Ag85A-specific CD8 T cells in TB children could be the consequence of generalized illness, and they recover upon disease improvement. Another possibility comes from a study of mouse lymphocytic choriomeningitis virus-specific CD8+ T cells, showing a reversion of TEM to TCM cells, which were then stably maintained (32). Thus, reversion from TEM to TCM might explain the reduction of the former and the increase of the latter in children with active disease. Also, M. tuberculosis is known to have evolved potent immunomodulatory capability; thus, it is possible that M. tuberculosis retards the proper maturation of TB-specific T cells through specific interactions during Ag presentation. Perhaps during active TB infection, the presence of high levels of Ag actively prevents proper maturation, i.e., inducing apoptosis in TB-specific T cells.

Finally, loss of effector memory Ag85A epitope-specific CD8 T cells might be the consequence of sustained in vivo mycobacterial stimulation, which causes their apoptosis. For example, high levels of bacteria (such as occurs in TB patients) due to the inability to contain and prevent their spread, would presumably result in chronic stimulation of effector CD8 T cells and their apoptosis, thus providing an explanation for why this population is lost in children with active disease but recovers after therapy.

In agreement with this possibility, it has been shown that TEMRA CD8 T cells have an enhanced susceptibility to cell death and die upon Ag activation, and this is associated with a lower expression of the antiapoptotic Bcl-2 protein, supporting the notion that the high susceptibility to cell death is an intrinsic feature of effector memory CD8 T cell subsets (20).

TEMRA cells are an enigmatic population of the CD8 memory pool. Various lines of evidence, such as the loss of CD27 and CCR7, the low proliferative capacity, the high susceptibility to apoptosis, and the presence of high levels of perforin and Fas ligand indicate that they represent the most differentiated type of memory cells. This notion is consistent with a recent report showing that EBV-specific effector cells that proliferate in response to persistent Ag have an even lower expansion potential and are CD45RA+. TEMRA cells have been reported to appear after the acute phase of viral infection, contain cells specific for lytic but not latent EBV Ags (33), and are absent in persistent HIV infection (34, 35).

Intriguingly, although CD8 TEMRA cells cannot be generated by antigenic stimulation, they are efficiently generated rather exclusively by a CD8 TCM subset upon cytokine stimulation. Importantly, CD8 TEMRA generation can be completely prevented by TCR stimulation, suggesting that it requires homeostatic proliferation in the absence of Ag and IL-15 has been demonstrated to be a key cytokine (36, 37). This is an important feature that might explain the reduction of the size of circulating Ag85A epitope-specific CD8 TEMRA cells in TB, as IL-15 produced by IFN-{gamma}-stimulated monocytes (38, 39, 40), maintain the frequency of M. tuberculosis-responsive CD8 IFN-{gamma}+ (very likely effector memory CD8 T cells) and mice lacking IL-15 have a defect in the CD8 recall response to M. tuberculosis (41). Finally, IL-15 mRNA and protein are more strongly expressed in immunologically resistant tuberculoid patients than in unresponsive and susceptible lepromatous patients.

Thus, a reduction of IL-15 production during active TB may cause a block in the differentiation of TCM to TEMRA and thus explain an increase of the former and loss of the latter.

The observations reported in the present study provide new insights in the distribution and the functional characterization of M. tuberculosis-specific memory CD8 T cells in different anatomical compartments in humans. Although our study has only assessed the CD8 T cell response to one well-defined Ag of M. tuberculosis, the availability of pentamers of known mycobacterial epitopes with HLA-class I binding specificities might allow us to extend these findings to other important Ags such as ESAT-6, CFP-10, and the 16-kDa acr, to provide a better evaluation of the global CD8 T cell response during active TB and its modification after therapy.


   Acknowledgments
 
We thank Dr. H. M. Vordermeier for critically reviewing the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work has been supported by grants from the Italian Ministry for Instruction, University and Research (to F.D.). N.C., F.D., and A.S. were supported by grants from the University of Palermo. Back

2 Address correspondence and reprint requests to Dr. Francesco Dieli, Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy. E-mail address: dieli{at}unipa.it Back

3 Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Guérin; PPD, purified protein derivative; CSF, cerebrospinal fluid; TCM, T central memory; TEM, T effector memory; TEMRA, terminally differentiated T effector memory. Back

Received for publication December 21, 2005. Accepted for publication May 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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