First described in HIV-infected patients with Mycobacterium avium complex infection receiving zidovudine monotherapy , immune restoration syndrome (IRS) in patients suffering from tuberculosis (TB) became extremely frequent (29–36%)  with the HAART era. Classically, IRS occurs in patients co-infected with TB and HIV, naive of HAART, with advanced HIV disease, when both therapies are initiated simultaneously or at a short interval. Usually, clinical symptoms include fever, lymphadenopathy, worsening of respiratory and other initial TB symptoms . Major criteria defining IRS have been proposed  whatever the underlying opportunistic event, including an atypical presentation of opportunistic infection (OI) or tumours in patients responding to HAART (decrease in plasma HIV RNA level more than 1 log10 copies/ml), without any alternative explanation (drug resistance, other OI) and with evidence for immune restoration, as minor criteria. However IRS raises three clinically relevant issues: first, diagnosis is difficult as IRS should be distinguished from treatment failure or resistance or other opportunistic diseases; second, IRS treatment in some life-threatening cases is still empirically based on steroids, which are uneasy to introduce in such immunocompromised patients; third, determination of IRS risk factors could help clinicians to determine their therapeutic strategy, i.e., delaying or not HAART introduction .
The pathophysiology of IRS is not yet totally elucidated. Acute proinflammatory cytokine [interleukin (IL)-6] production has been demonstrated during IRS [5,6] but its source remains unknown. In addition, a strong and dominant hypothesis is that IRS would be associated with restoration of mycobacteria-specific T-lymphocyte response with either recirculation or proliferation of memory T cells after HAART has reduced the plasma viral load (VL) [7,8]. This hypothesis is only suggested but not yet demonstrated, except for the restoration of delayed-type hypersensitivity (DTH) skin test to purified protein derivative (PPD) .
To determine whether IRS is due to an acute restoration of a Th1 TB-specific response that causes its characteristic major inflammatory syndrome we conducted a prospective multicenter study of patients co-infected with HIV and TB. We describe our preliminary results here.
Patients and methods
Twenty-nine consecutive TB-HIV co-infected untreated patients were prospectively included when beginning anti-TB treatment. Inclusion criteria were: HIV-1 infection, no previous HAART, CD4 cell count below 200 cells/μl, anti-TB therapy initiated within a maximum of 1 week before inclusion and indication to further HAART initiation. Inclusion was secondly confirmed when M. tuberculosis infection was proved (positive culture or histological findings). Patients were evaluated when initiating antimycobacterial therapy (TBK) and HAART (M0) and at 1, 3, 6 and 12 months after HAART initiation (M1,3,6,12). In addition, patients with IRS (IRS+) were evaluated at IRS time (TIRS) and 20 days later. IRS was defined as follows : recurrence of inflammatory reaction [fever, elevated c-reactive protein (CRP)], enlargement of pre-existing lesions, or development of new lesions (lymph nodes, pleuritis) with no mycobacterium resistance, no positive culture specimen, no other diagnosis and with a response to HAART (HIV RNA decrease > 1 log copies/ml). Patients who did not experience IRS within 3 months after HAART initiation were defined as IRS−.
The study was accepted by the Saint-Louis Hospital Institutional Ethical Committee and all patients signed an informed consent.
ELISPOT assay for quantification of mycobacterum-specific Th1 cells
Antigen-specific Th1 cells producing interferon (IFN)-γ were prospectively quantified on fresh peripheral blood mononuclear cells (PBMC) by ELISpot as described , after a 40-h stimulation with mycobacterial extracts (PPD, 1 μg/ml; Statens Serum Institute, Copenhagen, Denmark), ESAT-6 recombinant protein (1 μg/ml, kindly provided by Statens Serum Institute) and 85B (2 μg/ml; Lionex-Diagnostics and Therapeutics GmbH, Braunschweig, Germany). Controls included cytomegalovirus (CMV) extracts (Behring, Liederbach, Germany), HIV-1 p24 (Protein-Sciences, Meriden, USA), phytohaemagglutinin (Murex, Paris, France) and medium alone. Spots were counted using an ELISpot reader (Zeiss, Le Pecq, France) and data were expressed as spot forming cells (SFC)/106 PBMC. Results were considered as positive if above a 50 SFC/106 PBMC after subtraction of the mean background obtained with cells alone.
Cytokine/chemokine production quantification
Fresh PBMC were stimulated for cytokine/chemokine production with the same antigens: PPD and CMV as above. Culture supernatants were collected at day 2 and cryopreserved. Quantification of 25 inflammatory and immunomodulatory cytokines/chemokines [IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-13, IL-15, IL-17, IFN-γ, tumour necrosis factor (TNF)-α, granulocyte-macrophage colony stimulating factor (GM–CSF), MIP-1α, MIP-1β, MCP-1, monokine-induced by IFN-γ (MIG), RANTES, IP-10, Eotaxin] was performed for four patients (three IRS+, one IRS−) using the multiplexed sandwich immunoassay (Human-25-plex, Biosource, Montrouge, France) based on flowmetric Luminex technology (Luminex-100, Clinisciences, Montrouge, France) as published . The IFN-γ production was quantified on the same supernatants by a Biosource ELISA kit.
CD4 and CD8 T-lymphocyte phenotype was analysed in fresh whole blood by four-colour flow cytometry using standard methods  with the following antibodies: anti-HLA-DR-FITC (Immunotech), anti-CD25-PE (Becton-Dickinson), anti-CD4-PerCPCy5 (Becton-Dickinson) or anti-CD8-PerCP (Becton-Dickinson) and anti-CD3-APC (Becton-Dickinson). Stained cells were analysed on a FACScalibur (Becton-Dickinson) as described.
Comparisons between groups were made using non-parametric tests (Fischer exact and Mann–Whitney test) as appropriate.
Twenty-nine consecutive TB–HIV co-infected untreated patients were prospectively included when they began anti-TB treatment. Ten were then excluded for the following reasons: four had unproven tuberculosis, five moved and one was rapidly transferred to the intensice care unit. The other 19 were followed for at least 3 months after initiating HAART. Seven of them (39%) experienced IRS within a median of 23 (range, 12–85) days after M0. IRS clinical manifestations are listed in Table 1. Three patients received oral steroid therapy with rapid recovery; at time of IRS, HAART was transiently stopped for one of them. Patients with (IRS+) and without (IRS−) IRS did not differ in terms of ethnicity, age, sex, dissemination of TB or time between TBK and M0 (27 versus 45 days respectively; non-significant). Median CD4 cell counts at M0 were lower, though not significantly, in IRS+ patients (median 32/μl for IRS+ versus 60/μl for IRS−; P = 0.052) and rose at M3 by 107/μl in IRS+ versus 51/μl in IRS− patients (P = 0.02). The decrease in plasma VL at M1 did not differ between the groups (−5.7 and 5.2 log10 HIV RNA/ml for IRS+ and IRS− respectively).
The number of PPD-specific cells producing IFN-γ did not differ at baseline (M0) between the groups (44 versus 36 SFC/106 PBMC IRS+/IRS−, non-significant) but their proportion increased sharply during IRS with a median 35-fold amplification and reached a maxima median of 2970 (range, 616–3744) SFC/106 PBMC in the seven IRS+ patients (Fig. 1a and b, left panel). This explosive reactivity did not affect the CMV-specific responses from IRS+ patients. The IRS− patients did not display a similar acute increase in PPD-specific responses (maxima median of 430 SFC/106PBMC, P = 0.002) even in the subgroup (n = 6) with a CD4 gain above 50 cells/mm3 at M3 (P = 0.015). One IRS− patient, however, had an acute increase of 1676 SFC/106PBMC at M3 without clinical IRS nor TB relapse; HAART at this time was no longer effective. Two other IRS− patients had high PPD-specific responses at baseline that were maintained under HAART (Fig. 1a, right). In addition, in IRS− patients CMV and PPD responses were similar, though with a trend toward higher CMV-specific responses than in IRS+ patients ((maxima median) 415 SFC/106PBMC versus 150 SFC/106PBMC, P = 0.20) (Fig. 1a and b).
When comparing the reactivity against the three mycobacteria antigens tested, we found that only three IRS+ patients had ESAT-6-specific positive responses during IRS with very low frequencies of 58, 142 and 318 SFC/106 PBMC (Fig. 1b). Furthermore, only two IRS− patients, both with CD4 cell counts > 100/μl, recognized both ESAT-6 and PPD during follow-up (data not shown). In contrast, neither IRS+ nor IRS− patients displayed 85B-specific response at baseline and during follow-up.
An extensive proteomic analysis was performed to evaluate the in vitro production of 25 inflammatory and immunomodulatory cytokines/chemokines in antigen-stimulated PBMC supernatants from four patients (three IRS+, one IRS−). Two distinct patterns were observed during IRS. First, PPD-specific IFN-γ production peaked together with other PPD-specific Th-1 related cytokines/chemokines (IL-2, IL-12, IP10 and MIG) (Fig. 1c), but no Th-2 cytokine peak occurred (IL-4, IL-5, IL-13, IL-15). Second, inflammatory cytokines/chemokines (TNF-α, IL-6, IL-1β, IL-10, RANTES and MCP-1) (Fig. 1d) peaked, produced by both unstimulated and antigen-stimulated cells. In addition, PPD-specific cytokine/chemokine production far exceeded the CMV-specific response in IRS+ patients, while the CMV-reactivity predominated in the IRS− patient (Fig. 1d, right).
Finally, prospective flow cytometry analysis showed increased expression of T-cell activation markers at time of IRS. Indeed, CD4/DR T cells increased from 55% at M0 up to 79% at IRS and 81% at M3, following the same kinetics than the PPD-specific IFN-γ-producing responses (data not shown). As expected, CD4/DR T cells decreased during HAART in IRS− patients, from 55% down to 40% at M3 (P = 0.06, at M3 between IRS+ and IRS− patients).
This prospective sequential study of HIV and TB co-infected patients initiating anti-TB and HAART therapies provides the first demonstration that, in this setting, IRS do correspond to an exacerbation of TB-specific Th1 responses with proinflammatory events during the immune reconstitution allowed by HAART.
It is well known that recovery of a PPD-specific response is a characteristic of immune reconstitution with HAART [12–15]. Our prospective study differs from others by clearly showing an exceptionally acute, intense and reproducible kinetic of the PPD-specific response concurrent with the acute inflammatory clinical symptoms described as IRS. This abrupt response was absent in only one IRS + patient who stopped HAART during IRS and received corticosteroids. One IRS− patient showed a similar peak though with no clinical diagnosis of IRS but with poor HAART observance and immunovirological control. This kinetics of acute PPD-specific response was not correlated to the amount of CD4 cell recovery.
Though the hypothesis had been previously formulated, similar findings had not been demonstrated yet, possibly due to the retrospective characteristics of previous studies. In addition, we show the exacerbated mycobacteria-specific response was limited to some persistent antigens contained in PPD but did not affect the mycobacterial proteins ESAT-6 and 85B, suggesting differences in kinetics of persistence or expression levels between antigens involved in IRS. This finding is critical for diagnosis because new tuberculosis-diagnostic tests based on ESAT-6  would have missed this acute response.
The sharp amplification of IFN-γ producing mycobacteria-specific T cells was associated with an acute burst of other Th-1 and proinflammatory cytokines/chemokines, in accordance with Stone et al.  showing high serum IL-6 levels during IRS. Both phenomena correlate with the abrupt onset of the clinical findings : lymph node enlargement and functional granuloma formation . We therefore propose that an excessive restoration of PPD-specific Th1 response with no Th2 balance is responsible for enlargement of TB granuloma lesions and is associated with an acute release of non-specific pro-inflammatory cytokines and chemokines inducing the systemic inflammatory syndrome. This latter findings support the use of systemic anti-inflammatory and/or immunosuppressive agents in severe IRS cases. However these results are still preliminary and need to be confirmed on a larger scale.
This mechanism can probably be extended to non-HIV patients experiencing equivalent paradoxical reactions during anti-TB therapy. In some non-HIV patients, IFN-γ secretion, missing during active TB infection, reappears during anti-mycobacterial treatment . This IFN-γ production may be particularly strong concurrently to the restoration of immune response in profoundly immune-suppressed patients. No identified factors explain why such physiologic response to mycobacterial antigens becomes so intense, sometimes life-threatening. However diverse genetic factors affecting the Th1–Th2 balance might determine predisposition to IRS and its severity in various infections as already proposed by P. Price .
In conclusion, an acute exacerbation of Th1 responses against some mycobacterial antigens appears to cause IRS in patients co-infected with HIV and TB. This key event provides new evidence valuable for the diagnosis and treatment of IRS.
This work was supported by Agence Nationale pour la Recherche sur le Sida et les hépatites virales (ANRS). A. Bourgarit is a SIDACTION fellow. We are especially grateful to the PARADOX TB Study group: S. Abgrall, A. Baakili, A-M Béglé, F. Besse, D. Bollens, O. Bouchaud, P. Bursachi, J. Cadranel, J. Camuset, C. Chakvetadze, J. Delgado, M. Diemer, B. Dupont, S. Elmarsafy, O. Fain, L. Fonquernie, A. Furco, P.-M. Girard, C. Grillot-Courvalin, J.F. Grivois, A. Guignet, M.C. Guilleminot, J.-L. Herrmann, V. Jeantils, V. Joly, V. Jouis, P. Klutse, K. Lacombe, M. Lafaurie, R. Lahoulou, A. Lavolé, B. Lefebvre, A. Lefort, E. Letellier, O. Lortholary, A. Metro, M. Trumeau, J.-L. Meynard, M.-C. Meyohas, J.-M. Molina, G. Obenga, M. Parrinello, O. Pelet, C. Pintado, D. Ponscarme, A. Rami, W. Rozenbaum, H. Sahli, P. Sellier, L. Slama, S. Courtial, R. Tubiana, J. Stirnemann, S. Tassi, O. Taulera, H. Touitou, I. Vacher, F. Vincent, P. Yeni. We are also grateful to A. Samri for her monitoring. The authors wish to thank Cliniscience for their lending the Luminex automat.
Special acknowledgement to J.A. Cahn for her editing of the manuscript.
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