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Keywords:

  • BAL;
  • CD57+ T cells;
  • sarcoidosis;
  • Th1 cells

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

T cells expressing CD57 (a natural killer cell marker) with interferon-γ (IFN-γ) producing capacity increase under various conditions. CD57+ T cells are also present in the bronchoalveolar lavage fluid (BALF) of sarcoidosis, and several phenotypical and functional analyses of these cells have been reported. In the present study, BALF T cells obtained from 52 patients with sarcoidosis were classified further into CD4+CD57+ T cells, CD4+CD57 T cells, CD8+CD57+ T cells and CD8+CD57 T cells and their phenotypes and functional characteristics were assessed. Substantial proportions of these T cell subsets expressed natural killer cell markers CD161 and CD122. The biased expansion of Vβ2 T cells was observed in both CD4+CD57+ T cells and CD4+CD57 T cells in BALF from most patients, while the expansion of other Vβ T cells was also observed in some patients. Unexpectedly, the biased expansion of certain Vβ T cells was also seen in either CD8+CD57+ T cells or CD8+CD57 T cells, while the expanded Vβ T cells in CD8+ T cells differed substantially among individuals. BALF T cells showed a remarkably lower T cell receptor (TCR) intensity than that of peripheral blood T cells. Both CD8+ T cell subsets in BALF of sarcoidosis expressed the intracellular perforin/granzyme B, while all four subsets expressed intracellular IFN-γ after in vitro activation, and CD4+ T cells, especially CD4+CD57+ T cells, expressed tumour necrosis factor-α. These findings indicate that CD57+ T cells as well as CD57 T cells in the BALF are phenotypically and functionally different from peripheral blood T cells and may play an important role in the Th1 dominant state and inflammation in pulmonary sarcoidosis.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Natural killer (NK) cells and T cells with NK cell markers, such as NK1·1 or interleukin (IL)-2 receptor β (CD122) in mice and CD56 or CD57 in humans, are abundant in the liver and these NK-type T cells and NK cells play an important role in the defence against bacterial infections and tumours by producing interferon (IFN)-γ[1–4] (reviewed in [1]). Although CD56+ T cells and CD57+ T cells are relatively minor populations in the peripheral blood (PB) lymphocytes of humans, they produce a large amount of ΙFΝ-γ and acquire a potent antitumour cytotoxicity by stimulation with anti-CD3 antibody, T helper 1 (Th1) cytokines [5] or bacterial superantigens [6]. In addition, CD57+ T cells in PB increase steadily in proportion with ageing and showed a biased Vβ T cell receptor (TCR) usage [5,7]. They are considered to be thymus-independent T cells [8–11] and may play a crucial role in Th1 immune responses against infections and tumours in aged hosts based on by their IFN-γ-producing potential [1,5,6].

On the other hand, in the human lung, which has extensive mucosal surfaces exposed to the ambient environment and is one of the first-defence line organs, T cells have been known to exist with alveolar macrophages based on the findings of a bronchoalveolar lavage fluid (BALF) analysis [12,13]. Sarcoidosis is known as one of the Th1 cytokine diseases and lymphocytic alveolitis represents a common finding in this disease [14–17]; Th1 cytokines as well as chemokines may play an important role during the process of sarcoid granuloma formation [18,19]. CD57+ T cells reportedly expand in BALF of sarcoidosis [20,21] and are suggested to play a role in Th1 cytokine production. However, the TCR repertoire and functional analyses of BALF T cells have been carried out so far in T cells that have not yet been characterized based on a subset analysis of the CD57, CD4 or CD8 expression [22–25].

In the present study, we focused therefore on four subsets of BALF T cells, CD4+CD57+ T cells, CD4+CD57 T cells, CD8+CD57+ T cells and CD8+CD57 T cells, and demonstrate the detailed TCR Vβ repertoires of BALF T cells using a flowcytometric analysis. In addition, we show other phenotypical characteristics and the expression of the intracellular perforin/granzyme B, as well as Th1 cytokines in these four subsets. Our findings show the unique features of BALF T cells, which will hopefully help to further elucidate the T cell subsets in BALF of sarcoidosis.

METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Study population

Fifty-two patients with sarcoidosis participated in the study (Table 1). Forty-seven had histological evidence consistent with sarcoidosis (showing non-caseating epitheloid cell granuloma) with no evidence of mycobacterial, fungal, parasitic infection or inorganic material known to cause granulomatous diseases. The remaining five had typical clinical and chest radiographic features with bilateral hilar lymphoadenopathy. No patient had ever received either therapy with systemic corticosteroid or cytotoxic agents. The assessment of disease activity included clinical features, chest radiography, chest high-resolution computed tomography, pulmonary function tests, 67Ga scintigram and routine blood studies. The period from the detection of the disease (symptomatic onset and/or abnormality of chest X-ray screening) to the time when the study was performed was 1 month to 7 years. The duration after detection of the disease in 10 patients was less than 2 years (acute disease) and the other 42 had been followed-up for more than 2 years (chronic disease) (Table 1). At the time of the study, 27 patients showed normal chest X-ray findings (stage 0) (Table 1); however, almost all of them had been classified as stage I or stage II at the time of diagnosis. In addition, among these stage 0 patients, lymphocyte percentages less than 10 (among BALF cells) were seen in only nine patients and 15 patients showed lymphocyte percentages of more than 20, thus suggesting that the most patients in this group still had the some disease activity in the lungs. Among the 42 patients with chronic disease, 31 patients whose chest X-ray showed stage 0 or I (spontaneous remission or unchanged) were clinically stable without any symptoms, six patients had repeated spontaneous remission and progression and five patients had progressive disease without any remission (Table 1). To compare the findings of our patients with normal individuals, five healthy subjects (three non-smokers and two smokers) were entered into the study. Informed written consent was obtained from all patients. The protocol was reviewed and approved by the ethics committee for human studies of National Defence Medical College.

Table 1.  Clinical characteristics of sarcoidosis patients
Subjects (n)52
 Male/female24/28
Age (year)  48 ± 15 (21–84)
Smoker/non-smoker (n)14/38
Acute (< 2 years)/chronic (> 2 years) disease (n)10/42
Chest X-ray stages (n)
 027
 I13
 II 3
 III, IV 9
Activities in chronic disease patients (n)
 Stable31
 Unstable 6
 Progressive 5
Lesions other than lung (n)
 Uveitis22
 Skin 9
 Stomach 2
 Liver and spleen 1
BALF (mean ± s.e.m.) (range)
 Cellular concentration ( × 105 cells/ml) 1·2 ± 0·1 (0·4–3·7)
 % of lymphocytes (%)30·8 ± 2·8 (1·5–77·4)

Bronchoalveolar lavage (BAL) analysis

BAL was performed under local anaesthesia on 52 sarcoid cases and five controls according to standard procedure [12]. Briefly, four 50-ml aliquots of sterile saline were instilled into a bronchus in the right middle lobe through a flexible fibre-optic bronchoscope. BALF was recovered by gentle suction and then kept on ice. BALF was filtered through a two-layer sterile gauze. The total cell number was counted using a Neubauer haemocytometer (Brand, Wertheim, Germany). After centrifugation (4°C, 500 g, 10 min), the cell pellets were washed twice and resuspended in RPMI-1640 medium (Sigma-Aldrich, Irvine, UK) supplemented with 10% heat-inactivated (56°C, 30 min) FBS, 2 mm l-glutamine and 1% antibiotic–antimycotic (GibcoBRL, Paisley, UK) (complete medium). Total cell viability (median = 95%) was determined by trypan blue exclusion (stained cells were almost all macrophages). Smears for differential counts were prepared by cytocentrifugation (Cytospin 2; Shandon, Runcorn, UK) at 22 g for 3 min; thereafter the cells were stained with May–Giemsa staining, and then 500 cells per slide were counted.

Peripheral blood samples

Heparinized PB samples were obtained from patients with sarcoidosis prior to the bronchoscopy, if available. Lymphocyte separation medium (ICN Biomedicals Inc., Aurora, OH, USA) gradient centrifugation was used to separate PB mononuclear cells, which were washed twice and diluted in RPMI-1640 complete medium.

Phenotyping with flow cytometric analysis

Surface phenotypes of BALF lymphocytes and PB lymphocytes (if obtained) were identified by monoclonal antibodies (MoAb) in conjunction with three-colour immunofluorescence tests. MoAbs used in this study were as follows: fluoroscein isothiocyanate (FITC) or phycoerythrin (PE)-anti-CD3 antibody (UCHT1), FITC or phycoerythrin-cyanin 5·1 (PC5)-anti-CD4 antibody, FITC or PC5-anti-CD8 antibody, PE-anti-CD19 antibody, PE or PC5-anti-CD56 antibody, FITC or PE-anti-CD57 antibody or biotin-anti-CD57 antibody with PC5-streptavidin, FITC or PC5-anti-αβ TCR antibody, PE-anti-CD161 antibody and PE-anti-CD122 antibody. Lymphocytes were gated by both a forward scatter and a side scatter and were analysed by FACSCalibur (Becton Dickinson, Mountain View, CA, USA).

Analysis of Vβ TCR repertoires of CD57+ T cells and CD57 T cells

BALF lymphocytes and PB lymphocytes were analysed by four-colour flow cytometry using allophycocyanin (APC) conjugated anti-CD4 antibody, biotin conjugated anti-CD57antibody (with PC5 conjugated streptavidin for the second stain) and 24 various anti-Vβ TCR antibodies conjugated with FITC and PE (Beta Mark TCR Vβ Repertoire Kit, Beckman Coulter). The percentage of each Vβ T cell population was determined in CD4+CD57+ T cells, CD4+CD57 T cells, CD8+CD57+ T cells and CD8+CD57 T cells as follows:

  • % of Vβ T cells in CD4+CD57+αβ T cells = (% CD4+CD57+Vβ T cells/% CD4+CD57+αβ T cells) × 100

  • % of Vβ T cells in CD4+CD57αβ T cells = (% CD4+CD57Vβ T cells/% CD4+CD57αβ T cells) × 100

  • % of Vβ T cells in CD8+CD57+αβ T cells = (% CD4CD57+Vβ T cells/% CD4CD57+αβ T cells) × 100

  • % of Vβ T cells in CD8+CD57αβ T cells = (% CD4CD57Vβ T cells/% CD4CD57αβ T cells) × 100

Intracellular staining of perforin and granzyme B

For intracellular staining, BALF cells were stained first with cell surface FITC, PE or PC5 MoAb, washed, and then fixed and permeabilized in 250 µl of Cytofix/CytopermTM (PharMingen, San Diego, CA, USA) for 20 min at 4°C, washed twice and incubated with intracellular MoAb, i.e. FITC-antihuman perforin antibody (Ancell, Bayport, MN, USA) or PE-antihuman granzyme B antibody (Caltag, Burlingame, CA, USA) for 30 min at 4°C. The cells were then washed and applied immediately to a flow cytometric analyser (FACSCalibur).

Intracellular staining of IFN-γ and TNF-α

BALF cells resuspended in RPMI-1640 medium (1 × 106 cells/ml) were stimulated with 50 ng/ml PMA (Sigma, Deisenhofen, Germany) and 500 ng/ml ionomycin (Sigma) in the presence of 0·7 µl/ml GolgiStopTM (monensin, PharMingen), which blocks the transport of newly synthesized cytokines from the Golgi apparatus. They were then incubated in flat-bottomed 24-well plates for 5 h at 37°C with 5% CO2[26]. The cells were then harvested and washed with cold PBS. The cells were next processed for surface marker staining (anti-CD4, CD8 and CD57), permeabilization and intracellular staining as described above for intracellular staining of perforin and granzyme B. FITC-conjugated anti-IFN-γ antibody and R-PE-conjugated anti-TNF-α antibody were used for intracellular staining. The cytokine expression was then determined by a flow cytometric analyser (FACSCalibur).

Statistical analysis

The data are presented as mean ± s.e.m. For comparisons of the paired and unpaired group data, Student's t-test was used to evaluate statistical significance. Calculations were performed using the StatView program (Abacus Concepts, Inc. Berkeley, CA, USA). The level of critical significance was assumed to be P < 0·05.

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Lymphocyte subpopulations in BALF and the phenotypical characterization of CD57+ T cells and CD57 T cells

The representative sarcoidosis case of BALF and PB lymphocytes is shown in Fig. 1. The proportions of the various subsets in BALF lymphocytes are shown in Table 2. Overall, the proportions of T cells and B cells, and CD4/8 ratio were similar to those in previous reports [17]. αβ TCR+ T cells are mainly dominant and among them, the percentages of CD57+ T cells and CD57 T cells were 16·15 ± 0·89, 83·85 ± 0·89, respectively, while the proportion of CD57+ T cells in the control subjects was lower than that in the sarcoidosis patients (9·71 ± 1·62%, P < 0·05). The proportion of CD3CD56+ NK cells is 1·89 ± 0·17, which is much smaller than that of PB lymphocytes [5]. Next, we investigated various surface markers in CD57+ T cells and CD57 T cells (Table 3), which showed similar CD4/8 ratios. Interestingly, approximately one-third of CD57+αβ TCR+ T cells and CD57αβ TCR+ T cells expressed CD161 (NKR-P1) (Table 3), which is usually expressed on NK cells but not on T cells in PB T cells [5]. A substantial proportion of CD57+αβ TCR+ cells and CD57αβ TCR+ cells also expressed another NK cell marker CD122 [interleukin (IL)-2 receptor β] (Table 3). The percentages of CD161+ cells in CD4+CD57+ T cells, CD4+CD57 T cells, CD8+CD57+ T cells and CD8+CD57 T cells were 34·8 ± 4·1% (mean ± s.e.), 35·4 ± 2·5%, 22·0 ± 3·5% and 35·6 ± 8·4%, respectively.

image

Figure 1. The surface phenotypes of T cells in BALF and PB. BALF lymphocytes and PB lymphocytes were stained with FITC-anti-CD3 antibody and with either biotin-anti-CD57 antibody developed with PC5-streptavidin, or with PE-anti-CD56 antibody (upper panels). They were also stained with FITC-anti-CD4 antibody and PC5-anti-CD8 antibody (lower panels). The numbers indicate percentage of respective populations in total lymphocytes. A representative case is shown.

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Table 2.  Proportion and ratio of various subsets in BALF lymphocyte in sarcoidosis
TCR+αβ T cells* (%)CD19+ B cells* (%)CD3 CD56+ NK cells* (%)CD4/8* ratioCD57+ T cells (%)CD57 T cells (%)
  1. TCR = T cell receptor; NK = natural killer. Data were obtained from 52 patients with sarcoidosis. Values are expressed as the mean ± s.e.m. *Proportion or ratio of gated lymphocytes. †Percentage of TCRαβ-positive T cells.

92·52 ± 0·511·54 ± 0·261·89 ± 0·175·30 ± 0·7816·15 ± 0·8983·85 ± 0·89
Table 3.  Phenotypical characterization of CD57+αβ T cells or CD57αβ T cells in BALF
 CD4 (%)CD8 (%)CD4/8 ratioCD56 (%)CD161* (%)CD122 (%)
  1. Data were obtained from 52, *30 or †20 patients with sarcoidosis. Values are expressed as the mean ± s.e.m.

CD57+αβ T cells74·13 ± 3·0920·84 ± 2·175·54 ± 1·132·68 ± 0·3033·00 ± 1·9820·03 ± 4·18
CD57αβ T cells75·77 ± 2·0223·07 ± 2·265·47 ± 0·832·23 ± 0·2635·59 ± 1·9814·00 ± 2·72

TCR Vβ repertoire of CD4+CD57+ T cells, CD4CD57+ (CD8+CD57+) T cells and CD4+CD57 T cells, CD4CD57 (CD8+CD57) T cells in BALF of sarcoidosis patients and controls

Because eight of 24 Vβ T cell subsets, including those expressing Vβ1, Vβ7·2, Vβ11, Vβ12, Vβ13·1, Vβ20, Vβ21·3 and Vβ23, did not show any skewed expansion and comprised less than 10% of T cells of either subsets from all donors, only the percentages for the other 16 Vβ T cells are shown (Fig. 2). In most cases (15 of 20), the biased usage of TCR Vβ2 was dominant in both CD4+CD57+ T cells and/or CD4+CD57 T cells (Fig. 2), while the expansion of other Vβ T cells was also observed in some cases. In addition, TCR repertoires of CD4+CD57+ T cells were relatively parallel to those of CD4+CD57 T cells (Fig. 2). On the other hand, whereas the biased expansions of several Vβ T cells, such as Vβ2, Vβ3, Vβ5·1, Vβ7·1, Vβ9 and Vβ17, were observed commonly in either CD8+CD57+ T cells or CD8+CD57 T cells from some patients, the proportions of expanded Vβ T cells differed between both subsets. In addition, other expanded Vβ T cells differed substantially among patients and also differed between CD8+CD57+ T cells and CD8+CD57 T cells (Fig. 2).

image

Figure 2. TCR Vβ expressions in four different T cell subsets of the BALF from patients. The proportions (%) of various Vβ T cells in CD4+CD57+ T cells, CD4+CD57 T cells, CD8+CD57+ T cells and CD8+CD57 T cells were evaluated as described in the Methods section. The analyses were performed in the 20 latest consecutive patients.

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A noteworthy patient was case 5, whose Vβ13·2 T cells expanded dramatically both in CD8+CD57+ T cells and CD8+CD57 T cells (Fig. 2). This case was a 50-year-old female and the chest X-ray findings have progressed over the previous 2 years; the proportion of CD57+ T cells had also increased gradually (3·9–19·8%), while the CD4/CD8 ratio had decreased rapidly (10·9–1·6) during the same period. In addition, approximately 35% of BALF CD8+ T cells were CD8+CD57+ T cells (not shown). Therefore, the patient was considered to have active progressive sarcoidosis.

We also examined the TCRβ repertoires in the BALF T cells from controls. The data from one subject were not available due to the small number of lymphocytes in the BALF. In contrast to the sarcoidosis cases, no biased usage of TCR Vβ2 was observed in either subsets of CD4+ T cells in the BALF from the control subjects (Fig. 3). However, the biased expansion of some Vβ T cells was observed in these four subsets and differed substantially among individual controls (Fig. 3).

image

Figure 3. TCR Vβ expressions in four different T cell subsets of the BALF from the controls. The proportions (%) of various Vβ T cells in CD4+CD57+ T cells, CD4+CD57 T cells, CD8+CD57+ T cells and CD8+CD57 T cells were evaluated as described in the Methods section.

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Both CD57+ T and CD57 T cells in BALF express low levels of αβTCR

Because we noted that CD57+ T cells in PB lymphocytes express a lower intensity of TCR than that of CD57 T cells (Fig. 4a), we also examined the intensity of TCR of either CD57+ T cell or CD57 T cell-subsets in BALF by gating them (Fig. 4a). Interestingly, not only CD57+Τ cells but also CD57Τ cells in BALF express a much lower level of TCR than those of either CD57+Τ cells or CD57Τ cells in PB (Fig. 4a), thus indicating that the number of surface TCRs of BALF T cells was much smaller than that of PB T cells. The mean fluorescence intensities of αβ TCR were similar between the sarcoidosis patients and normal subjects, irrespective of CD57 expression (Fig. 4b). Regardless of CD4 or CD8 expression (data not shown), the low TCR intensity is also essentially similar.

image

Figure 4. (a) TCR intensities of BALF T cells and PB T cells. BALF lymphocytes and PB lymphocytes were stained with PC5-anti-TCRαβ antibody and with PE-anti-CD57 antibody and TCR intensities of CD57+ subsets and CD57 subsets are shown in the histograms. A representative case is shown. (b) The mean fluorescence intensities of TCRαβ were similar between the sarcoidosis patients and normal subjects irrespective of the CD57 expression.

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Expression of cytoplasmic perforin and granzyme B in BALF T cells

Becuase experiments have indicated so far that BALF T cells are activated and show some NK cell-like properties, and a majority of PB CD57+ T cells have both cytoplasmic perforin and granzyme B, their expression in each subset of BALF was examined in eight patients. Perforin causes membrane pore formation of target cells and granzyme B enters into target cells through the pores made by perforin, thereby inducing target apoptosis [27,28]. In PB lymphocytes, 90% of NK cells express perforin and granzyme B while a substantial proportion of CD57+ T cells (70%) but not regular T cells express them after in vitro activation [5]. In the BALF, CD8+CD57+ T cells and CD8+CD57 T cells were positive for both perforin and granzyme B (Fig. 5a,b). Only a minor population of CD4+CD57 T cells expressed perforin and granzyme B. However, a substantial population of CD4+CD57+ T cells expressed granzyme B (Fig. 5a–d).

image

Figure 5. (a) Intracellular perforin expressions of four T cell subsets in BALF. (b) Intracellular granzyme B expressions of four T cell subsets in the BALF. Intracellular perforin or granzyme B together with CD57 and CD4 or CD8 were stained in freshly isolated BALF lymphocytes as described in the Methods section and either perforin or granzyme B expression was shown in the histograms. The numbers shown in the histograms represent the percentage of positive cells. The proportions of (c) perforin- or (d) granzyme B-positive cells in four T cell subsets in the BALF. The data represent the means ± s.e.m. from eight patients. *P < 0·05, **P < 0·001.

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Intracellular IFN-γ and TNF-α production in each subset of BALF

After the stimulation of PMA and ionomycin, each subset of BALF was examined for their cytokine production. The results showed that all subsets expressed IFN-γ,while the proportion of IFN-γ-positive CD4+CD57+ T cells were significantly larger than those of CD4+CD57 T cells, both in acute disease patients (Figs 6a, n= 4) and chronic disease patients (Figs 6b, n= 6). The difference in the percentages of IFN-γ-producing cells between CD4+CD57+ T cells and CD4+CD57 T cells tends to be larger in acute patients than in chronic patients. As reported recently [29,30], CD4+ T cells seem to be the main TNF-α producers, while the proportion of TNF-α-producing cells in CD4+CD57+ T cells was significantly larger than that of CD4+CD57 T cells (Fig. 6c, n= 6). However, although two of these six cases were acute patients, while the other four cases were chronic patients, essentially the same patterns of subsets and TNF production levels were observed. No data from the controls could be obtained due to the small number of lymphocytes in the BALF.

image

Figure 6. Proportion of IFN-γ (a) in acute cases, (b) in chronic cases and TNF-α (c) positive cells in four T cell subsets in the BALF. BALF lymphocytes were stimulated with PMA and ionomycin, and cytokines together with CD57 and CD4 or CD8 were stained as described in the Methods section. The data represent the means ± s.e.m. from four patients of acute cases and six of chronic cases for IFN-γ and from six patients for TNF-α. *P < 0·05, **P < 0·01, ***P < 0·005, ****P < 0·0005.

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DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

In the present study, we have demonstrated that the skewed expansion of certain Vβ T cells was seen in each αβ T cell subset in the BALF from sarcoidosis. In the majority of patients, a biased Vβ2 T cell expansion was seen in both CD4+CD57+ T cells and CD4+CD57 T cells, while the expansion of other Vβ T cells was observed in some patients. Vβ2 T cell expansion was also seen in CD8+CD57+ T cells and/or CD8+CD57 T cells in some patients; however, the biased expansions of some individually different Vβ T cells other than Vβ2 T cells were seen in either CD8+CD57+ T cells or CD8+CD57 T cells in the majority of patients. Furthermore, the expanded Vβ T cells tended to differ substantially between the two CD8+ subsets. All T cell subsets of the BALF expressed much smaller numbers of TCRs on their surface than those of T cells in PB lymphocytes. Large proportions of both CD57+ T cells and CD57 T cells in the BALF expressed NK cell markers, CD161 (NKRP-1) and CD122, regardless of their CD4 or CD8 expression. CD8+CD57+ T cells and CD8+CD57 T cells in the BALF expressed intracellular perforin/granzyme B. All four subsets expressed intracellular IFN-γ after in vitro activation while CD4+ T cells, especially CD4+CD57+ T cells, produced intracellular TNF-α.

It has been suggested that CD57+ T cells (90% of them are CD8+) in PB lymphocytes are a distinct T cell subset from regular CD57 T cells and they have also been proposed to differentiate extrathymically [8] and thereby increase with ageing [5,10]. CD57+ T cells in PB lymphocytes can express intracellular perforin/granzyme B, have a potent capacity to produce IFN-γ[5] and are enriched in tumour infiltrating lymphocytes [9], thus suggesting that they play a role in the Th1 immune response.

On the other hand, CD57+ T cells, especially CD4+CD57+ T cells, have been shown to expand in the BALF of sarcoidosis patients (which are very rare in PB lymphocytes) [21,31] and a biased expansion of certain T cell populations, such as Vβ2 T cells and Vα2·3 T cells, has been reported [22–25]. These cells in the BALF reportedly produce Th1 cytokines including IFN-γ and are suggested to be involved in the immunopathogenesis of sarcoidosis. CD57 is originally a natural killer cell marker [32] and CD57+ T cells and CD57 T cells in PB lymphocytes are different either functionally or phenotypically from each other [5,7,10]. Therefore, the present study focused on four T cell subsets based on their CD4, CD8 or CD57 expression. CD4+CD57+ T cells expressed significantly more intracellular granzyme B, IFN-γ and TNF-α than did CD4+CD57 T cells. These findings suggest that CD4+CD57+ T cells are in a more activated state than CD4+CD57 T cells and may be involved more profoundly in Th1 dominant immune response in sarcoidosis. Nevertheless, the TCR repertoires of CD4+CD57+ T cells and CD4+CD57 T cells, including the expansion of Vβ2+ T cells, are very similar to each other. A possibility was also raised that CD4+CD57+ T cells represent an activated state of CD4+CD57 T cells and CD4+CD57 T cells may acquire CD57 as a result of their activation.

A notable finding was that the skewed expansions of some Vβ T cells also were observed not only in CD8+CD57+ T cells but also in CD8+CD57 T cells. The biased TCR β repertoire was observed in CD8+CD57+ T cells in PB lymphocytes but was never seen in regular CD8+CD57 T cells in PB lymphocytes [5]. In addition, expanded Vβ T cells in CD8+CD57+ T cells of BALF were different to Vβ T cells expanded in CD8+CD57+ T cells of PB lymphocytes (unpublished observation). Whereas CD8+CD57+ T cells but not CD8+CD57 T cells in PB lymphocytes expressed the intracellular perforin/granzyme B, large proportions of both CD8+CD57+ T cells and CD8+CD57 T cells in BALF of sarcoidosis expressed intracellular perforin/granzyme B, which is an indicator of NK cell activity. Furthermore, even when CD8+CD57 T cells from PB were activated by cytokines (IL-2, IL-12 and IL-15), only a small proportion of them expressed the intracellular perforin/granzyme B [5]. Therefore, BALF CD8+ T cells may play an important role in defence as well as inflammation/pathogenesis in lungs with sarcoidosis. These findings, together with their biased TCR repertoires, also suggest that not only CD8+CD57+ T cells but also CD8+CD57 T cells in BALF are different from either CD8+CD57+ T cells or CD8+CD57 T cells in PB lymphocytes, and BALF CD8+ T cells in sarcoidosis may thus have been underestimated in the past. Consistent with this speculation, it was reported recently that large proportions of CD8+ T cells as well as CD4+ T cells in BALF from sarcoidosis patients expressed intracellular IFN-γ and TNF-α[29,30,33]. However, expanded Vβ T cells differ substantially among patients and also between the CD8+CD57+ T cells and CD8+CD57 T cells. CD8+ T cells are therefore unlikely to respond to the specific antigen(s). Our findings also raise the possibility that, in contrast to BALF CD4+ T cells, two subsets of CD8+ T cells in BALF may be distinct sets of T cells.

A noteworthy case is case 5 (active and progressive), in which more than 60% of either the CD8+CD57+ T or CD8+CD57 T cells in BALF were Vβ13·2 T cells. As it was reported previously that 3·8% of sarcoidosis patients represented CD8+ T cell-dominant alveolitis [34], the Vβ13·2 CD8+ T cells in this case may indeed be involved in the pathogenesis of sarcoidosis in an antigen-specific manner, thus supporting the speculation that CD8+ T cells can also be involved in the inflammation/pathogenesis of sarcoidosis under certain conditions.

As reported previously in whole T cells [35,36], all T cell subsets in the BALF express extremely small numbers of TCRs on their surface (Fig. 4). In PB lymphocytes CD57+ T cells have a lower TCR intensity than CD57 T cells, whereas BALF T cells show a much lower intensity of TCR (Fig. 4). In addition, as reported previously [36] and as also confirmed in this study, a low TCR intensity was also observed in BALF T cells from the controls, thus suggesting that their low TCR intensity cannot be explained solely by TCR modulation following their activation [36]. Furthermore, a substantial proportion of BALF T cells expressed other NK cell markers and the intracellular perforin/granzyme B and the biased TCR β repertoire was observed in each T cell subset. These characteristics are peculiar to BALF T cells. In addition to human CD57+ T cells in PB, mouse CD8+CD122+ T cells with a lower intensity of TCR and with a potent IFN-γ-producing capacity are known to develop extrathymically in the liver [9,37,38]. Intraepithelial T cells with a capacity to produce IFN-γ in the intestine of both mice and humans, which is another first-line defence organ, also develop thymus independently [39–41]. Therefore, although we cannot rule out the possibility that regular T cells may migrate from circulation and accumulate in the alveolar space and modulate their TCR by their activation, we prefer to hypothesize that BALF T cells may develop in the lung in situ thymus independently.

No parameter in our results correlates significantly with the disease stage/activity, except for the Vβ13·2 CD8+ T cell expansion in case 5. Mollers et al. reported that intracellular IFN-γ and TNF-α expression in BALF T cells from acute disease patients were more predominant than those from chronic disease patients [29]; however, cytokine expression might be unable to help distinguish the disease stages. We also showed in this study that the proportion of IFN-γ producing cells in CD4+CD57+ T cells and the proportional difference of IFN-γ-producing cells between CD4+CD57+ T cells and CD4+CD57 T cells of acute disease patients both tended to be larger than those of chronic disease patients. However, although the number of patients examined was small, we did not find any such difference in either the IFN-γ production of CD8+ T cells or the TNF-α production of each T cell subset between acute and chronic disease patients. A recent report suggests that IL-18 (IFN-γ-inducing factor) can be such a parameter; serum IL-18 levels may correlate with the disease activity [42]. However, it should be noted that IL-18 is not a simple cytokine and can be either Th1 or Th2, depending on the condition of the hosts [43,44], and a bacteria superantigen-induced IFN-γ production from PB CD57+ T cells was IL-12-dependent but IL-18-independent [6].

Focusing on four T cells subsets, we believe that the present study further unveils additional unique characteristics regarding BALF T cells in sarcoidosis.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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