Involvement of local regula- regula-tory T cells

Rationale: Local PPD-specific IFN-γ⁺ CD4⁺ T cells were described in both pleural TB and pulmonary TB (04-07).

Similarly, significant proportions of IFN-γ⁺ T cells were detected locally upon stimulation with HBHA. This is in sharp contrast to the concomitant low HBHA-specific IFN-γ secretion in the periphery (S. Place, Manuscript in preparation).

However, high concentrations of pro-inflammatory T cells within a confined space, such as the pleural compartment, may be associated with subsequent risk of significant host tissue damage. Be-tween 0 and 50 % untreated patients with pleural TB will develop active pa-renchymal TB in their life. Despite this delayed risk of TB disease, pleural TB is often associated with an early spon-taneous ad integrum resolution (08), so that we hypothesized that the local inflammatory anti-TB response may be regulated.

Objectives: To investigate the possi-ble presence of Treg cells within BAL and pleural fluids during active TB and to characterize the suppressive effect mediated by these cells on the IFN-γ responses upon antigenic stimulation with PPD, HBHA or ESAT-6.

Material and methods: About 50 ml of heparinized pleural fluids were col-lected from 9 patients suffering from newly diagnosed, untreated TB

pleu-risy (mean age = 7.4 years; range, 1.8 – 68. years; 1 Caucasian, 6 patients from Africa and from Asia) and from 1 naive subject suffering from a bacterial pneumonia and taken as a control. This uninfected subject (64. years) was not vaccinated with BCG and had no known exposure to M. tuberculosis. Diagnosis of pleural TB was confirmed either by the presence of granuloma (n=4), by a posi-tive smear and/or culture (n=4) or by a favourable clinical evolution upon anti-TB treatment. About 10-15 mL of BAL fluid were also collected on RPMI 1640 from 6 other patients suffering from cul-ture-confirmed untreated pulmonary TB (mean age = 6.4 years; range, 1.

– 5.8 years; Caucasian, from Africa, 1 from Asia and 1 from Central America).

All the patients were recruited from Bel-gian hospitals linked to the Université Libre de Bruxelles hospital network. All the individuals were living in Belgium at the time of enrolment and were HIV-sero-negative. None presented muno-depressive illness or received im-muno-suppressive treatment. The local Ethical Committee of the hospital, where the patients were enrolled, approved the study, and all individuals gave in-formed consent. For 5 of them, 0 ml of heparinized peripheral blood was also collected. Pleural and BAL mononuclear cells were filtrated (100 µM) and isolated after simple centrifugation or after cen-trifugation on a density gradient if the sample was bloody. PBMC were isolated and both pleural mononuclear cells and PBMC were stimulated as described elsewhere (00). CD5^high T cells were de-pleted from pleural mononuclear cells and PBMC by positive immunomagnetic selection using anti-CD5 antibodies

according to the manufacturer’s instruc-tions (CD5 microbeads II, Miltenyi Bio-tec, Germany). CD5 and FOXP expres-sion by CD⁺CD4⁺ lymphocytes from pleural and BAL fluids were analysed as previously described (00).

Preliminaryresults:

HBHA-specific IFN-γ secretion during TB pleurisy

The IFN-γ secretion induced by the dif-ferent mycobacterial antigens was com-pared for six patients with TB pleurisy between circulating and pleural lym-phocytes. Pleural lymphocytes secreted significantly more IFN-γ in response to the different mycobacterial antigens than circulating lymphocytes, confirm-ing previous results from our laboratory (Table S2 and S. Place, manuscript in preparation).

Proportions of pleural and BAL CD4⁺CD25^highFOXP3⁺ T cells

The local proportion of CD5^highFOXP⁺ lymphocytes among CD4⁺ T cells was analysed for patients with pleural TB and for 6 patients with pulmonary TB (Table S3). Whereas similar proportions of CD5^highFOXP⁺ lymphocytes were found among the pleural lymphocytes and among the PBMC, their proportions were significantly higher among BAL (P=0.00), raising to 5.69 % in the TB pa-tients.

Depletion of pleural CD4⁺CD25^high T cells

As the number of lymphocytes collect-ed from BAL was lower than the number collected from pleural fluids for obvious ethical reasons (lower volume of the col-lected liquid), most depletion studies were performed on pleural lymphocytes

Table S2. IFN-γ responses from pleural mononucleated cells and from PBMC

Antigen N

Pleural: Median (P25; P75)

(pg/ml)

PBMC: Median (P25; P75)

(pg/ml)

P value*

Culture medium 6 72 (15 – 415) 0 (0 – 16) 0.03

PHA 5 20474 (6360 – 46608) 16603 (5297 – 25127) 0.44 PPD 6 38507 (16566 – 53922) 4895 (3095 – 18468) 0.03

HBHA 6 2451 (835 – 17362) 178 (45 – 310) 0.03

ESAT-6 5 699 (216 – 3813) 31 (9 – 1346) 0.06

*Wilcoxon signed rank test

Table S3. Proportions of local CD4⁺CD25^highFOXP3⁺ T cells

Pleural fluid BAL PBMC

N = 3 6 18

Median 1.67 5.69 2.07

P25-75 - 3.71 – 8.65 1.90 – 2.93

Min-Max 1.53 – 2.67 3.65 – 8.75 1.18 – 5.60

to analyse the suppressive function of the local CD4⁺ Treg lymphocytes. We depleted the CD5^high cells, as most Treg cells are CD4⁺CD5^high T cells. This pro-cedure allowed us to efficiently remove the CD4⁺CD5^high T cells, as the median proportion of Treg cells decreased from 4.69 % of CD4⁺ T cells before depletion (P5-75: 4.4-6.0 %) to 0.40 % after de-pletion (0.8-1.8 %) (P=0.0). The pro-portions of pleural CD14⁺ monocytes were low, both among total and CD5 -depleted cells (< .5 %; n= ).

As illustrated in table S4, depletion of CD5^high T cells from pleural mononu-clear cells increased their IFN-γ respons-es to ESAT-6. Interrespons-estingly, the IFN-γ concentrations secreted in the absence of in vitro stimulation with antigens, also rose upon Treg cell depletion. Con-trasting with these results and also with previous results published on PBMC (00), depletion of pleural Treg cells did not increase the IFN-γ secretion neither in response to HBHA, nor to PPD. A poly-clonal stimulation with phytohemagglu-tinin-A was also not associated with an increased IFN-γ secretion upon deple-tion of Treg cells. The same experiment was performed on the pleural fluid from

a control patient suffering from a non-TB pneumonia. In this case, we did not observe any change in the antigen-in-duced IFN-γ secretion when comparing total to CD5-depleted pleural cells.

Most patients with local regulation against ESAT-6 (Fig S5C; #1, #, #4) did not show a local or a peripheral regula-tion against HBHA (Fig S5B; #, #4). This could be explained by a recirculation of both effector T cells and Treg cells from the site of infection. In contrast, patients with a peripheral regulation against HBHA (Fig S5B; # and #5) may be con-sidered as suffering from a more ad-vanced disease. Of interest, one of these patients did not show local regulation against ESAT-6 (Fig S5C; #).

Discussion:

PBMC are may move from the site of an-tigenic priming to the infectious sites.

Therefore, local and peripheral immune responses may differ during active TB.

Thanks to their accessibility, TB-associ-ated pleural effusions provide an oppor-tunity to study local immune responses during disease. We have observed that pleural IFN-γ responses were

consist-Table S4. Effect of CD25⁺ cells depletion on IFN-γ secretion by pleural mononuclear cells

Antigen N

Median before Treg depletion (P25; P75) (pg/ml)

Median after Treg depletion (P25; P75) (pg/ml)

P value*

Culture medium 8 33 (15 – 134) 121 (16 – 360) 0.02

PHA 8 18883 (9399 – 37024) 18033 (9224 – 40390) 0.53 PPD 8 40733 (20152 – 53050) 23970 (18984 – 36347) 0.08

HBHA 8 2824 (1147 – 6302) 3419 (545 – 6188)) 0.37

ESAT-6 7 977 (151 – 4683) 1641 (603 – 11068) 0.05

*Wilcoxon signed rank test, one tailed

ently stronger than those observed in the periphery. This raised the possibility of a preferential accumulation/expan-sion of M. tuberculosis-specific effector T cells within the pleural compartment where antigenic stimulations are pre-dominant. In front of this considerable T cell activation, the host has to regulate T cell responses in order to limit collateral damages from excessive inflammation.

Treg cells may offer one of these regula-tory mechanisms.

We have shown that

CD4⁺CD5^highFOXP⁺ T cells were present locally, both in pulmonary and pleural TB. Upon depletion of pleural CD5^high T cells, a significant rise of ESAT-6-specific IFN-γ secretion was observed. This con-firms that ESAT-6-specific T cell respons-es are down-regulated by Treg cells. In contrast, HBHA-specific and

PPD-spe-cific IFN-γ secretions were not modi-fied after depletion of Treg cells. These observations were the most surprising findings of this study, as they contrast with previously published data about peripheral immune responses during TB (00). They clearly indicate that the antigenic specificity of the regulation is variable according to the anatomical site that is analysed.

Preliminary data suggested that TB pleurisy was associated with lower per-centages of local Treg cells than pulmo-nary TB. Interestingly, TB pleurisy is of-ten self-resolving, whereas pulmonary TB is not (08). Moreover, it was shown that excessive amounts of Treg cells pre-vent the resolution of disease (58, 09).

Therefore, the proportions of Treg cells during TB may be a surrogate marker of disease severity.

Figure S5.Fold increases in IFN-γ secretions upon depletion of CD5^high cells from pleural cells (blue columns) and from PBMC (red columns) of 5 patients (numbered #1 to

#5). The pleural cells and the PBMC were stimulated in parallel with PPD (S5.A.), HBHA (S5.B.) or ESAT-6 (S5.C.). The ratios between the amounts of secreted IFN-γ before and after depletion were established for 5 patients. A fold-increase higher than 1 indicates that the IFN-γ secretion is increased upon depletion of CD5^high cells.

#1

#2

#3

#4

#5

Fold increase in PPD-specific IFN-γ

Pleural cells PBMC

Patients

#1

#2

#3

#4

#5

Patients

A.

0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8

#1

#2

#3

#4

#5

Patients

0 1 2 3 4

B.

Fold increase in HBHA-specific IFN-γ

C.

Fold increase in ESAT-6-specific IFN-γ

4.3. In vitro expansion of CD4

CD25

FOXP3