Material & methods

For the reasons described in the “Ra-tionale and Objectives”, we focused in this work on some parameters of the cellular immune responses of M. tuber-culosis-infected subjects. These param-eters were 1) the induction of an IFN-γ synthesis/secretion and ) the induction of a lymphocyte proliferation by differ-ent mycobacterial antigens.

Several antigens were used, but we fo-cused mainly on HBHA provided by C.

Locht (INSERM U69; Institut Pasteur de Lille, France). Other antigens were used for comparison, PPD (batch RT49; 4 µg/

ml; Statens Serum Institute, Denmark), ESAT-6 (10 µg/ml, provided by T.M. Do-herty; Statens Serum Institute, Den-mark), Ag85 (5 µg/ml, provided K. Huy-gen; Institut Pasteur Brabant, Belgium) and Candidin (50 IC/ml; Stallergènes;

Belgium). Phytohemagglutinin A ( µg/

ml; Remel, Lenexa-KS 6615 USA) was used as a positive control.

The IFN-γ secretions induced by the PBMC upon stimulation with these an-tigens were measured by ELISA in 96 hours cell culture supernatants. The ELISA technique was chosen to meas-ure the IFN-γ concentrations for several reasons, namely its good sensitivity, its cost-effectiveness and the know-how developed in the host laboratory. For some experiments, the cell proliferation was used as a read-out of specific cel-lular immune responses, and we chose the carboxyfluorescein diacetate, suc-cinimidyl ester (CFSE) labelling method to analyse this parameter. This method offers a practical way to specifically as-sess the T cell proliferation, in contrast to the classical H-thymidine incorporation test that reflects the global proliferation of all the cells present in the assay (e.g.

T cells, B cells, monocytes, etc…). The CFSE labelling has also the advantage to be a non-radioactive method.

A major technical development made in this work was the characterization of the CD4⁺CD5⁺ Treg cells and their in-volvement in active TB in humans. This characterization included:

- A phenotypical characterization by means of the intracellular ex-pression of FOXP and the cell surface expression of several markers

- A depletion of CD5⁺ cells or of CD4⁺CD5⁺ T cells

- The functional testing of Treg cells in vitro

Phenotypicalanalysis(surfacemark-ers): The expression of several surface molecules expressed by the PBMC was analysed by flow-cytometry. Briefly, cells are stained with fluorochrome-labelled monoclonal antibodies, which are spe-cific for a given surface molecules (e.g.

CD, CD4). As several fluorochromes are available for the same antibody, it is pos-sible to obtain an accurate phenotypical characterization of the target cells.

FOXP3 staining: FOXP intracellular staining is a relatively new tool that al-lows the simultaneous characteriza-tion of different phenotypic markers of the cells. This is a major technical ad-vantage compared to the assessment of the FOXP expression done by Re-verse-transcriptase Polymerase Chain Reaction (9). After the staining of the cells of interest (1.106/ml) with selected surface markers (e.g. CD, CD4, CD5, CD17), they were fixed, permeabilized and APC-labelled anti-FOXP antibody

was added (10 µl/ml, Rat IgGa, Clone PCH101; eBiosciences, San Diego, CA, USA). Other antibodies directed to intra-cellular antigens were sometimes added (e.g. CD15, GITR).

DepletionofCD25⁺cells:The CD5^high cells were depleted by using the EasySep system according to the recommanda-tions from the manufacturer (StemCell;

Grenobles, France). This procedure al-lowed us to positively select the CD5^high cells from isolated PBMC (figure M1). The CD5^high–depleted PBMC were further suspended in culture medium at a final concentration of x10⁶ cells/ml, whereas the CD5^high cells (positive fraction) were kept for further phenotypical analysis.

Functional immunosuppressive as-saysin vitro: The in vitro-induced or expanded Treg cells were co-cultured in the presence of autologous freshly iso-lated PBMC from a LTBI subject. Freshly isolated PBMC were labelled with CFSE (0.5 µM; Vibrant CFDA SE-Cell Tracer kit, Molecular Probes, Eugene, OR). The PBMC containing the in vitro-induced Treg cells were added at different ratios (ranging from 1:10 target cells to 1:1 tar-get cells) to the autologous CFSE⁺ PBMC.

The mixed lymphocyte population was then stimulated with anti-CD (1 ng/ml, Orthoclone OKT, Murunomab, Janssen-Cilag, Anvers, Belgium) and anti-CD8 (5 µg/ml, Immunotech, Marseille, France) monoclonal antibodies for 7h at 7°C in a 5 % CO-containing atmosphere.

The proliferation of the CD⁺CD4⁺ T cells was finally assessed by flow cytometry.

Similar experiments were performed in parallel adding CD4⁺CD5⁺-depleted PBMC.

Conventional T cells CD25 low/med FOXP3 negative

Regulatory T cells CD25 bright FOXP3 positive

Total PBMC

Treg depleted PBMC

α-CD25 µ-beads

strong magnetic field

Cell

EasySep™ magnetic nanoparticle CD25

T.A.C.

T.A.C. = tetrameric antibody complex α-CD25α-dextran

FigureM1.SelectivedepletionofCD25⁺cellsthanks to the use of immuno-magnetic beads (see text).

IV. Results

4.1. HBHA-Induced IFN-γ Release as a Diagnostic Tool for Latent Tuberculosis

A

^{s most} M. tuberculosis-infected in-dividuals will not develop active disease throughout their live, one may consider that they have developed ef-fective protective immune responses against the disease in contrast to the infected subjects that develop active TB. Therefore, extensive comparison of the immune responses of LTBI subjects and of TB patients should help to iden-tify possible correlates of protection. In 00, the HBHA-induced IFN-γ secretion was identified as one possible correlate of protection, as the PBMC from LTBI se-creted significantly higher IFN-γ concen-trations in response to HBHA compared to those from patients with TB disease (5).

In view of the high IFN-γ secretions in-duced by HBHA from the PBMC of LTBI subjects and of the major importance of a rapid detection of LTBI in control strategies of M. tuberculosis infection (94), we evaluated the clinical value of the HBHA-IGRA for the detection of LTBI. Both recently infected subjects and remotely infected subjects (“true”

LTBI) were evaluated, as data published for IGRA using other mycobacterial an-tigens have suggested waning of im-mune responses with time (95). Thanks to ROC curve analysis, we have deter-mined objective cut-off points for posi-tivity affording the best compromise between sensitivity and specificity. The HBHA-IGRA allowed us to detect more than 90 % of the LTBI. Similar sensitivi-ties were found for both recent and re-mote LTBI. In contrast, detection of LTBI with an ESAT-6-IGRA has a low

sensitiv-ity, both with a commercial test (QFT-IT: 50 % of LTBI positive) and with an in-house ESAT-6-IGRA (40.74 %). These low sensitivities were especially noted for LTBI with a remote infection. These observations have important potential clinical implications, as the use of QFT-IT could miss the diagnosis of remote LTBI that should be done before starting therapeutic immune suppression (i.e.

infliximab).

Contrasting with LTBI, active TB disease was consistently associated with low HBHA-specific IFN-γ secretions. Roughly 45 % of TB patients did not secrete sig-nificant amount of IFN-γ in response to HBHA stimulation. This discrepancy be-tween TB disease and LTBI was not obvi-ous upon stimulation with other myco-bacterial antigens such as ESAT-6 or PPD.

Therefore, like the TST, the HBHA-IGRA is not recommended for the diagnosis of active TB. However, as for the TST, in some clinical settings, the HBHA-IGRA may still be useful to help the clinician in his diagnostic decision when active TB is suspected.

Heparin-Binding-Hemagglutinin-Induced IFN-c Release

Dans le document Je dédie ma thèse à mon épouse, (Page 78-83)