Tuberculosis-Associated Immune Restoration Syndrome in HIV-1-Infected Patients Involves Tuberculin-Specific CD4 Th1 Cells and KIR-Negative δγ T Cells This information is current as of October 5, 2021. Anne Bourgarit, Guislaine Carcelain, Assia Samri, Christophe Parizot, Matthieu Lafaurie, Sophie Abgrall, Veronique Delcey, Eric Vicaut, Daniel Sereni and Brigitte Autran

J Immunol 2009; 183:3915-3923; ; Downloaded from doi: 10.4049/jimmunol.0804020 http://www.jimmunol.org/content/183/6/3915

References This article cites 42 articles, 14 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/183/6/3915.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 5, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Tuberculosis-Associated Immune Restoration Syndrome in HIV-1-Infected Patients Involves Tuberculin-Specific CD4 Th1 Cells and KIR-Negative ␥␦ T Cells1

Anne Bourgarit,*† Guislaine Carcelain,* Assia Samri,* Christophe Parizot,* Matthieu Lafaurie,‡ Sophie Abgrall,§ Veronique Delcey,¶ Eric Vicaut,ሻ Daniel Sereni,† Brigitte Autran,2* and the PARADOX Study Group

Tuberculosis (TB)-associated immune restoration syndrome (IRS) is a frequent event (10 to 30%) in HIV-1-infected patients receiving antiretroviral treatment and is associated with an increased number of IFN-␥-producing tuberculin-specific cells. To further understand the immune mechanisms of TB-IRS and to identify predictive factors, we prospectively analyzed the Th1 and TCR␥␦ T cells known to be involved in mycobacterial defenses and dendritic cells at baseline and after antiretroviral and TB Downloaded from treatment in 24 HIV-1؉ patients, 11 with and 13 without IRS. At baseline, these two groups differed by significantly lower proportions of TCR␥␦ and V␦2؉ T cells displaying the inhibitory receptors CD94/NKG2 and CD158ah,b in IRS patients. The two groups did not differ in the baseline characteristics of CD8 or CD4 T cells or TLR-2 expression on monocytes or myeloid/ plasmacytoid dendritic cells. During IRS, the increase in tuberculin-specific IFN-␥-producing cells involved only highly activated effector memory multifunctional (IFN-␥؉TNF-␣؉IL-2؊) CD4 T cells, whereas activated HLA-DR؉ CD4؉ T cells also increased

during IRS. In contrast, dendritic cells decreased significantly during IRS and there were no changes in TLR-2 expression. Finally, http://www.jimmunol.org/ the V␦2؉ T cells, mostly killer Ig-related receptor (KIR) (CD94/NKG2؊ and CD158؊), significantly peaked during IRS but not in non-IRS patients. In conclusion, IRS is associated with an increase in the number of activated tuberculin-specific effector memory CD4 T cells and of KIR؊V␦2؉ TCR␥␦؉ T cells. Higher proportions of V␦2؉TCR␥␦؉ T cells lacking KIR expression are present as baseline and distinguish patients who will develop IRS from those who will not. The Journal of Immunology, 2009, 183: 3915–3923.

mmune restoration syndrome (IRS)3 is observed in 10 to 30% skin test reaction in previously anergic patients suggests the res- of patients coinfected with HIV-1 and Mycobacterium tuber- toration of effective antimycobacterial immunity (3). We previous I culosis (Mtb) after they begin highly active antiretroviral reported a strong increase in the number of tuberculin-specific T by guest on October 5, 2021 therapy (HAART) (1). It is characterized by clinical symptoms cells in patients developing IRS, compared with patients without revealing exacerbation of granulomatous lesions (2). The patho- this syndrome (4). This phenomenon was associated with a mas- physiology of IRS is not well understood but is thought to reflect sive inflammatory and Th1 storm. The nature of these the sudden restoration of immune competence against a coexisting IFN-␥-producing cells is thus far unknown, and the involvement of , such as Mtb, in HIV-1-infected patients with profound other cells in this IFN-␥- and Th1-mediated granuloma formation immune deficiency. During IRS, recovery of a strong tuberculin remains hypothetical. Nonetheless, anti-tuberculosis (TB) immune reaction and granuloma formation classically involve macro- phages, dendritic cells (DC), NK cells, TCR␥␦, and CD4ϩ and *Laboratory of Cellular Immunology, INSERM, Pitie-Salpetriere Hospital, Assis- CD8ϩ ␣␤ T cells (5). Both HIV-1 and TB impair this tance Publique des Hoˆpitaux de Paris (APHP), Universite´Pierre et Marie Curie, Paris, France; †Department of Internal Medicine, and ‡Department of Infectious Diseases, cellular immunity, which recovers after the are con- Saint-Louis Hospital, APHP, Universite´Denis-Diderot Paris, France; §Department of trolled by both antiviral and antimycobacterial therapies. Infectious Diseases, Avicenne Hospital, APHP, Bobigny, France; and ¶Internal Med- icine, ࿣Lariboisiere Hospital, Clinical Research Unit, Fernand Widal Hospital, APHP, Tuberculin-specific T cells are known to recover rapidly, both Paris, France qualitatively and quantitatively, within 3 to 6 mo of HAART ini- Received for publication December 2, 2008. Accepted for publication June 14, 2009. tiation and successful HIV-1 control (6, 7). In TB-coinfected pa- The costs of publication of this article were defrayed in part by the payment of page tients, these cells have been shown to reach proportions up to 33% charges. This article must therefore be hereby marked advertisement in accordance of the peripheral CD4ϩ T cells after 9 mo of HAART and to have with 18 U.S.C. Section 1734 solely to indicate this fact. an effector memory CD45RAϪCD62Lϩ phenotype (8). Moreover, 1 This work was supported by the Agence Nationale pour la Recherche sur le Sida et les he´patites virales. A.B. was a SIDACTION fellow. recovery is associated with recovery of delayed-type hyper- 2 sensitivity to tuberculin (9). Taken together, these findings suggest Address correspondence and reprint requests to Dr. Brigitte Autran, Cellular and ϩ Tissular Immunology Laboratory, Hoˆpital Pitie-Salpetriere, Batiment CERVI, 47-83, that acute restoration of functional CD4 T cells specific for Mtb Boulevard de l’Hopital, 75013 Paris, France. E-mail address: brigitte.autran@ may be massively involved in the pathophysiology of IRS. psl.aphp.fr The stark increase in tuberculin-specific T cells may also reveal 3 Abbreviations used in this paper: IRS, immune restoration syndrome; DC, dendritic ϩ Ϫ cell; HAART, highly active antiretroviral therapy; IQR, interquartile range; KIR, the existence of a restoration process for APCs. CD11c CD123 killer Ig-related receptor; mDC, myeloid DC; MFI, mean fluorescence intensity; Mtb, myeloid DC (mDC) are involved in early activation of specific Mycobacterium tuberculosis; pDC, plasmacytoid DC; PPD, purified deriva- anti-TB Th1 immune response (10). The mycobacterial TLR-2 re- tive; SFC, spot-forming cell; TB, tuberculosis. ceptor involvement is responsible for DC maturation (CD83) and Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 activation (CD86) and IL-12 production. Interaction of Mtb with www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804020 3916 IMMUNITY OF TB-HIV-1 IMMUNE RESTORATION SYNDROME

TLR-2 on monocytes can both provoke and inhibit proinflamma- ELISPOT reader (Zeiss), and data were expressed as spot-forming cells tory macrophage activation (11–13). Both HIV-1 and Mtb can reg- (SFC)/106 PBMCs. Results were considered positive when they exceeded 6 ulate immune response orientation by DC; their interaction affects 50 SFC/10 PBMCs after subtraction of the mean background obtained with cells alone. the balance of the TLR-2/DC-SIGN pathway (14). HIV-1 has been associated with defects in circulating DC, both myeloid Membrane phenotyping and plasmacytoid (15), defects that are partially restored by anti- PBMCs were phenotyped on fresh whole blood by four-color flow cytom- retroviral and anti-tuberculosis therapy (16–18). TLR-2 membrane etry with standard methods (5). The different circulating populations were expression seems to be modulated by proinflammatory cytokine defined and characterized as follows. ␣␤ production (19). This raises the question, not yet studied, of the T cells were stained with the following Ab combinations: anti- HLA-DR-FITC (Beckman Coulter), anti-CD25-PE (BD Biosciences), involvement of these cells and their receptors in the IRS anti-CD4-PerCPCy5 (BD Biosciences); or anti-CD8-PerCP (BD Bio- phenomenon. science) and anti-CD3-allophycocyanin (BD Bioscience), anti-45RA- Finally, TCR␥␦ T cells, another T cell subset, appear to play a FITC (Beckman Coulter), anti-62L-PE (Pharmingen), and anti-CD7-PE (Beckmann Coulter). predominant role against Mtb infection. These T cells produce ϩ ␥ ␥␦ T lymphocytes were defined as CD3 lymphocytes with pan-␥␦ TCR IFN- strongly and early, in response to mycobacterial phospho- FITC or PE-CY5 (Beckman Coulter). The subpopulations were character- antigens (20–23). They are quantitatively and functionally im- ized by anti-TCR␦2-FITC (Beckman Coulter) and their function determined paired by both HIV-1 (24) and Mtb infection (25), which create an by HLA-DR expression (allophycocyanin and PeCy5; BD Biosciences) imbalance between the V␦1 and the normally predominant V␦2ϩ and by membrane expression of NKR CD94/NKG2-PE (HP-3B1; Beck- subsets (26) and V␦2ϩ anergy (27, 28). After HAART and anti-TB man Coulter), CD158a,h-PE (A09778, anti-KIR2DL1, 2DS1), and CD158b-PE (IM1846, anti-KIR 2DL2, 2DL3, 2DS2) (Beckmann Coulter). treatment, these defects, already associated with inflammatory syn-

For one patient, we were able to have the kinetics of the functional sub- Downloaded from dromes in SIV-infected macaques (29), recover, except for the populations characterized on fresh blood by CD45RA-CyC5 (Beckmann amplification of the V␦1ϩ population (30). Moreover, the TCR␥␦ Coulter) and CD27-PE (BD Biosciences). ϩ Ϫ T cell effector function is known to be modulated by inhibitory and DC were defined as HLA-DR Lin1-(CD3-14-16-19-20-CD56 ) PBMC (anti-HLA-DR-PeCy5, -allophycocyanin (BD Biosciences); anti- activating MHC class-I ligand receptors that belong to the NK Lin1-FITC, (BD Biosciences)). Subpopulations were characterized as receptor family. These modulations permit pathogens to escape the CD11cϩCD123Ϫ for mDC and CD11cϪCD123ϩ for plasmacytoid DC immune reaction. TB increases NKG2A inhibitor expression on (pDC) (anti-CD123 PE and anti-CD11c; BD Biosciences). TLR-2 mem- V␦2ϩ cells (31), whereas HIV-1 increases NKG2C activation on brane expression was measured as mean fluorescence intensity (MFI) (anti- http://www.jimmunol.org/ ϩ TLR-2 PE, eBioscience). V␦1 T cells (32). These characteristics raised the question of the ϩ Ϫ Monocyte-macrophages were characterized as CD14 CD3 PBMCs killer Ig-related receptor (KIR) involvement in the pathophysiol- (anti-CD14 FITC; Beckman Coulter; anti-CD3 allophycocyanin; BD Bio- ogy of IRS. sciences). The TLR-2 membrane expression was measured as MFI (anti- To improve our understanding of the pathophysiology of TB- TLR2 PE; eBioscience). associated IRS and identify predictive factors for it, we prospec- Flow cytometry analyses were performed on a FACSCalibur with CellQuest Pro software (BD Biosciences). A mean of 10,000 events was tively analyzed the characteristics of the major actors of Mtb-re- acquired. When populations of interest were identified, subsequent analysis lated granulomatous responses in patients with and without IRS, of differentiation or activation status of the rarest populations was consid- both before and after initiation of HAART. ered only if at least 100 positive cells were detectable in the by guest on October 5, 2021 appropriate gate. Materials and Methods Intracytoplasmic cytokine staining Patients ␣␤ T cells. Frozen PBMCs with viability above 80% after thawing were cultured for4hinRPMI 1640 medium (5% FCS). Cells were then washed Thirty-five consecutive untreated patients coinfected with TB and HIV-1 ϫ 6 ␮ were prospectively included when they began anti-TB treatment. Inclusion and 1 10 cells were stimulated for 2 h with tuberculin (10 g/ml) in a 24-well flat-bottom plate. Cells stimulated with medium alone and PHA (1 criteria were: HIV-1 infection, no previous HAART, CD4 count below 200 3 ␮g/ml) were used as negative and positive controls, respectively. Brefeldin cells/mm , anti-TB therapy initiated, and indications for HAART. Inclu- ␮ sion was confirmed when the Mtb infection was proven (positive culture or A (20 g/ml) was added after 2 h and the incubation continued for another histological findings). Patients were evaluated when they began antimyco- 18 h. After washings, cells were incubated for 15 min with the following fluorescent-labeled conjugated mAbs: anti-CD27-FITC (BD Biosciences), bacterial therapy (T ) and HAART (M ) and at 1, 3, 6, and 12 mo after BK 0 anti-CD8-FITC, anti-CD45RA-PE (Beckman Coulter), anti-HLA-DR-PE beginning HAART (M ). In addition, patients with IRS were evalu- 1,3,6,12 (BD Biosciences), anti-TNF-␣-PE, anti-CD4-PECy5 (BD Biosciences), ated when it was first diagnosed (TIRS) and 20 days later. IRS was defined (33) as recurrence of inflammatory reaction (fever and elevated C-reactive and anti-CD8-PercP (BD Bioscience). After washings, cells were fixed protein), enlargement of pre-existing lesions, or development of new le- with PFA 4%, washed in PBS-0.5% BSA-0.1% saponin buffer and then stained for 15 min at room temperature with allophycocyanin-conjugated sions (lymph nodes or pleuritis) with no mycobacterium resistance and no ␥ other apparent cause, in a patient responding to HAART (HIV-1-RNA IFN- Ab (BD Biosciences). Flow cytometric analyses were performed on Ͼ a FACSCalibur with CellQuest Pro software (BD Biosciences) on decrease 1 log copies/ml). Patients who did not experience IRS within 3 ϳ months of HAART initiation were considered not to have it. Eleven pa- 500,000 events. tients initially included were excluded: TB infection was not confirmed for TCR␥␦-T cells. Frozen PBMCs with a cell viability above 80% after thaw- four, four changed medical center, two were lost to follow-up within three ing were cultured overnight in RPMI 1640 medium (5% FCS). Cells were 6 months of starting HAART, and one was transferred early to ICU for then washed and 1 ϫ 10 cells were stimulated for 4 h with coated anti- seizures. The immunological investigations were therefore performed on CD3 (5 ␮g/ml) in a 24-well flat-bottom plate. Cells stimulated with me- the 24 patients who could be analyzed. dium alone were used as negative controls. Brefeldin A (20 ␮l/ml) was The study was approved by the institutional review board at Saint Louis immediately added. Staining was performed as above with the following Hospital, and all patients provided written informed consent. Preliminary fluorescent-labeled conjugated mAbs: anti-TCRV␦2-FITC (BD Biosciences), data of some of these patients have already been published (4). anti-CD94/NKG2-PE, anti-CD158ah-PE, anti-CD158b-PE (Beckman Coulter), anti-TCR␥␦-biotin (eBioscience), anti-streptavidin-PE-CY7 Methods (eBioscience), and then with allophycocyanin-conjugated IFN-␥ Ab (BD ELISPOT assay for quantification of mycobacterial-specific Th1 Biosciences). cells Statistical analysis Ag-specific Th1 cells producing IFN-␥ were prospectively quantified on Groups were compared with nonparametric tests (Fisher’s exact test and fresh PBMCs by ELISPOT as described (4), after a 40-h stimulation with Mann-Whitney as appropriate) at baseline, M1/TIRS, and at the data peak or mycobacterial extracts (tuberculin, 1 ␮g/ml; Statens Serum Institute). Con- nadir after treatment began. Generalized linear models were used to com- trols were PHA (Murex) and medium alone. Spots were counted with an pare the changes of different parameters with time. Two-sided significant The Journal of Immunology 3917

ϩ Table I. Clinical characteristics of patients with and without IRS activation marker. As expected, the balance between the V␦2 and V␦2Ϫ TCR␥␦ϩ T cells was inverted, with more V␦2Ϫ T cells than ϩ ϩ IRS (n ϭ 11) Non-IRS (n ϭ 13) p V␦2 in both groups. However, the proportions of V␦2 cells observed in patients who later developed IRS were significantly Gender (M/F) 6/5 9/4 NS higher than in the patients who did not (median, 23% (IQR, 11– Age, yr 41 (30–56) 37 (26–63) NS ϩ TB infection 42) of TCR␥␦ T cells vs 12% (IQR, 9–24), respectively ( p ϭ Pulmonary TB 2 4 NS 0.02); Table II, Fig. 3E). Moreover, surface expression of the lec- Disseminated TB 9 9 NS tin-like heterodimer CD94/NKG2 and the KIRs CD158a,h and Smear positive 5 4 NS 158b was measured on all subsets of CD3ϩ/Ϫ ␥␦ϩ/Ϫ and V␦2ϩ/Ϫ HIV infection (M0) CD4 (/mm3) 37 (3–123) 56 (13–330) NS cells (Fig. 2). CD158a,h and b expression did not differ on Ϫ ϩ ϩ Ϫ ϩ Ϫ VL (log) 5.7 (4.6–6.5) 5.2 (4.3–5.9) NS V␦2 ␥␦ CD3 , ␥␦ CD3 ,orCD3 T cells between the two M0HAART groups. In contrast, patients who later developed IRS had signif- ϩ Days from TBK 36 (7–77) 50 (14–111) NS icantly lower frequencies of V␦2 ␥␦ T cells positive for TIRS/M 1 CD158a,h and 158b ( p ϭ 0.03) than those who did not (Fig. 2A, Days from M0HAART 26 (7–85) ϩ ϩ CD4 (/mm3) 108 (59–430) 163 (9–580) NS Table II). Likewise, CD94 ␥␦ T cells were less frequent among ⌬ Ϫ Ϫ CD4 from M0HAART 54 ( 1; 393) 77 ( 50; 250) NS IRS-to-be patients (median, 41% (IQR, 24–49) vs 61% (39–71), M3 p ϭ 0.02; Table II, Fig. 2B), as were CD94bright cells (data not CD4 (/mm3) 117 (58–399) 132 (49–410) NS ⌬ Ϫ Ϫ shown). These characteristics were already present before anti-TB CD4 from M0HAART 86 ( 74; 367) 73 ( 88; 354) NS VL Ͻ 200 cp/ml (n) 7/10 8/11 NS treatment (median, 38% (IQR, 16–48) vs 63 (IQR, 46–77) for IRS and non-IRS respectively, p ϭ 0.001) for CD94 but not CD158a,h Downloaded from Mann-Whitney nonparametric tests comparing patients with and without IRS. VL, ␥ Viral load. and b. The stronger IFN- production induced by CD3 triggering in CD94/NKG2Ϫ or CD158Ϫ V␦1ϩ and V␦2ϩ TCR␥␦ϩ T cells compared with CD94/NKG2ϩ or CD158ϩ cells demonstrates the level was fixed at 5%. All calculations were made using SAS software negative CD94/NKG2 and CD158 signaling (Fig. 2D). version 9.1.3 from SAS Institute. In conclusion, at baseline the only immunological difference between patients who will and will not develop IRS was that the http://www.jimmunol.org/ Results ϩ former had a lower proportion of TCR␥␦ T cells displaying the Patients inhibitory receptors CD158 and CD94/NKG2, particularly among Our prospectively analysis showed that 11 (46%) of the 24 patients V␦2ϩ TCR␥␦ϩ T cells involved against Mtb. These groups did developed IRS symptoms in a median of 26 days (range, 7–85) not, however, differ for the conventional CD4ϩ Th1 cells specific after HAART began. As shown in Table I, patients with and with- for tuberculin. out IRS did not differ for age, sex ratio, country of birth, HIV-1 Characterization of IFN-␥-producing tuberculin-specific T cells infection status, baseline (TBK and M0) CD4 count and HIV-1 viral load, TB clinical presentation, percentage of smear-positive pa- during IRS by guest on October 5, 2021 tients, or resistance to anti-TB treatment. Patients with and without We first confirmed in a larger group of patients (n ϭ 24) that IRS did not differ for quantitative CD4 T cell restoration or viral TB-associated IRS is associated with a peak in tuberculin-specific control at IRS onset or M1, respectively. cells producing IFN-␥; these cells, measured by ELISPOT, reached a median value of 3462 (IQR, 1422–3562) vs 453 (120– Baseline characteristics of immune cells involved in Th1 1676) SFC/106 PBMC in patients without IRS ( p Ͻ 0.005, Fig. pathways 1A). The peak values occurred within a median of 197 days (range All immunologic characteristics were studied at initiation of 23–390) after HAART initiation in IRS vs 119 (range, 28–377) in HAART in a median of 40 (range, 7–111) days after initiation of non-IRS compared with a median of 26 days for clinical symp- TB treatment. In conventional T cells, we detected similar levels toms. This acute restoration of purified protein derivative (PPD)- of tuberculin-specific IFN-␥ producing T cells in both groups, with specific T cells was significantly more rapid in IRS patients as an overall median of 52 (interquartile range (IQR), 19–182) SFC/ assessed by a significantly higher increase of PPD-specific T cells 6 10 PBMCs (Fig. 1A). Intracellular staining showed those cells at time of IRS (in IRS patients) compared with M1 (in control were CD4ϩ (Fig. 1B). In addition, we observed no difference in non-IRS patients) with a median increase between baseline and 6 Ϫ ϩ activation (HLA-DR) or differentiation markers (CD45RA and T-IRS or M1 of 593 SFC/10 PBMC (range, 42– 3286) in IRS CD62L) on either CD4ϩ or CD8ϩ T cells (Table II). vs 58 SFC/106 PBMC in non-IRS patients (range, Ϫ102–ϩ2136; When analyzing innate immune cells such as APCs, circulating p ϭ 0.034; Fig. 1A). We then used intracellular staining to inves- DCs accounted for a median of 0.46% PBMCs overall with a tigate the nature of these cells that produce IFN-␥ in response to CD11cϩCD123ϪmDC/CD11cϪCD123ϩpDC subpopulation ratio tuberculin. At the time of clinical IRS, tuberculin-specific IFN-␥ϩ of 4:1. Neither parameter differed between the two groups (Table T cells were mostly CD4ϩ T cells representing a median of 73% II). Similarly, the maturation/activation status of DC, as measured (59, 70, 76, 89, 97) of all IFN-␥ϩ CD3 T cells. They represented by the level of membrane CD83/CD86 expression, did not differ from 0.8 to 22% (median, 3.2% (0.8, 2, 3.2, 13.2, 21.8); Fig. 1C) (Table II), nor did the number of peripheral CD14ϩ monocytes. of all CD4ϩ T cells. In addition, all these cells coproduced TNF-␣ Membrane expression of TLR-2, as shown by the MFI, also did (Fig. 1D) but not IL-2. These tuberculin-specific CD4ϩ T cells had not differ between IRS and non-IRS patients on both monocytes an extremely homogeneous differentiation profile of activated (me- (MFI, 30 (IQR, 17–47) vs 31 (23–40)) and mDC (MFI, 11 (IQR, dian, 99% (38, 97, 99, 99, 99) HLA-DRϩ) effector memory (84% 5–20) vs 9 (6–18); Table II). (79, 84, 89) CD45RAϪCD27Ϫ) IFN-␥ϩTNF-␣ϩIL-2Ϫ T cells as Finally, TCR␥␦ϩ peripheral T cells accounted for a median of observed in three to five IRS patients. 5.6% (IQR, 1.5–14.4) of the CD3ϩ cells at baseline in the entire This involvement of activated tuberculin-specific IFN-␥ produc- study group. This proportion did not differ between patients who ing CD4 T cells was associated with changes in the proportions of did and did not develop IRS, nor did expression of the HLA-DR total CD4ϩ and CD8ϩHLA-DRϩ-activated T cells (Fig. 3A). The 3918 IMMUNITY OF TB-HIV-1 IMMUNE RESTORATION SYNDROME Downloaded from http://www.jimmunol.org/ by guest on October 5, 2021

FIGURE 1. Characterization of IFN-␥ϩ tuberculin-specific T cells. A, ELISPOT IFN-␥ quantification of tuberculin- (PPD) and control- (CMV Ag) specific IFN-␥ producing cells in IRS (left) and non-IRS (right) before and after HAART initiation. B, Baseline intracellular cytokine staining in one patient. C, Intracellular cytokine staining in five IRS patients during IRS. D, Intracellular cytokine staining of tuberculin-specific cells in one representative IRS patient during IRS.

proportions of activated CD4ϩ T cells increased both in IRS and CD25ϩHLA-DRϩ-activated cells or CD25high T cells neither at non-IRS patients about a month after HAART began, as described time of IRS nor afterward (data not shown). during immune restoration (7), while activated CD8ϩ T cells in- At IRS, the proportion of total DC decreased from a median of creased only in IRS patients. The magnitude of these increases, 0.64% to 0.2% of PBMCs ( p ϭ 0.02; Fig. 3B). This decrease however, differed significantly at peak for patients with and with- involved both mDC and pDC. However, only the proportions of out IRS, with medians of 34% (IQR, 24–42) vs 18% (IQR, 7–25) pDC differed between IRS and non-IRS patients ( p ϭ 0.04; Fig. ( p ϭ 0.02) and 10% (IQR, 2–20) vs 0 (IQR, 0–7) ( p ϭ 0.01) of 3B). Peripheral proportions of CD14ϩ monocytes did not differ CD4ϩ and CD8ϩ T cells, respectively. The CD4ϩHLA-DRϩ T over time or at IRS, and there was no significant modulation of cells remained significantly higher in IRS patients throughout fol- TLR-2 expression on CD14ϩ monocytes (Fig. 3C)oronmDC low-up ( p ϭ 0.02; Fig. 3A). In contrast, CD25 expression on CD4 (data not shown). T cells remained lower in IRS patients ( p ϭ 0.023; Table II) al- Finally, after HAART initiation, circulating V␦2ϩTCR␥␦ϩ T though there was no difference in the number of CD4ϩ cells’ proportions remained higher ( p ϭ 0.02; Fig. 3D) and peaked The Journal of Immunology 3919

Table II. Baseline characteristics of T cells, DCs, and monocytes (36) that did not detect this increase in specific T cells during IRS. It is important to note that in our study the significant differences IRS (n ϭ 11) Non-IRS (n ϭ 13) p in the peak number of IFN-␥-producing cells at restoration did not

ϩ always coincide exactly with the IRS clinical symptoms. In all LT␣␤CD4 %CD4ϩ/CD3ϩ 5 (2, 11) 9 (3, 10) 0.43 patients tested, both at baseline and during IRS, tuberculin-specific %CD45RAϪCD62Lϩ 66 (51, 69) 60 (52, 66) 0.67 Ϫ Ϫ T cells were, either mainly or only, CD4 T cells. They had all the %CD45RA CD62L 25 (15, 38) 25 (14, 42) 0.93 Ϫ Ϫ %CD45RAϩCD62LϪ 2 (0, 8) 0 (0, 1) 0.19 characteristics, both CD45RA CD27 and multifunctional (IFN- ϩ Ϫ ϩ ϩ Ϫ ϩ %CD4 CD7 17 (11, 23) 14 (8, 19) 0.22 ␥ TNF-␣ IL-2 ), of activated HLA-DR effector memory T %CD4ϩHLADRϩ 44 (27, 73) 42 (27, 52) 0.40 %CD4ϩCD25ϩ 37 (21, 48) 43 (27, 59) 0.02* cells. The mechanism of this expansion may be redistribution or %CD4ϩCD25bright 0.16 (0, 1) 0.4 (0.1, 3.7) 0.25 proliferation, as already shown in immune restoration after ϩ LT␣␤CD8 HAART (6). In accordance with the fact that tuberculin is a com- %CD8ϩ/CD3ϩ 83 (77, 86) 81 (79, 88) 0.82 %CD45RAϪCD62Lϩ 25 (13, 38) 24 (16, 30) 0.52 bination of hundreds of Ags, we could show in one patient that this %CD45RAϪCD62LϪ 36 (20, 44) 45 (32, 51) 0.19 expansion was not clonal (data not shown). During IRS, after treat- %CD45RAϩCD62LϪ 13 (4, 21) 21 (14, 28) 0.14 %CD8ϩHLADRϩ 57 (35, 66) 62 (49, 75) 0.42 ment of these disseminated TB infections in HIV-1 coinfected pa- ␥ϩ ϩ DC tients, we did not find the functional IFN- IL-2 TCM conver- %DC/PBMC 0.64 (0.33, 0.85) 0.44 (0.25, 0.52) 0.16 sion of tuberculin-specific cells that Millington et al. found in TB %mDC/PBMC 0.24 (0.15, 0.45) 0.25 (0.18, 0.33) 0.90 %pDC/PBMC 0.05 (0.03, 0.07) 0.06 (0.02, 0.16) 0.63 independently of HIV-1 and IRS, that might represent the persis- CD83/DC (MFI) 4 (3, 5) 5 (4, 9) 0.13 tent Ag spreading (37). These differences might reflect the pro- CD86/DC (MFI) 58 (49, 90) 49 (44, 62) 0.26 TLR2/mDC (MFI) 11 (5, 20) 9 (6, 18) 0.95 found immune deficiency status of our patients. In addition to this Downloaded from CD14ϩ tuberculin-specific phenomenon, the peak of total activated CD4 %CD14/PBMC 24 (13, 33) 18 (15, 22) 0.67 TLR2 (MFI) 30 (17, 47) 31 (23, 40) 0.99 and CD8 PBMCs was suggestive of additional mechanisms in- LT ␥␦ϩ volving non-Ag-specific T cell activation, as observed in granu- %TCR␥␦ϩ/CD3ϩ 7.3 (3, 9) 5.6 (5, 7) 0.30 ϩ loma lesion (38). %V␦2/␥␦ 23 (11, 42) 12 (9, 24) 0.02* %HLA-DRϩ/␥␦ϩ 29 (8, 37) 26 (13, 40) 0.75 Another major hallmark of these TB-associated IRS in this ex- %CD94/␥␦ϩ 41 (24, 49) 61 (39, 71) 0.02 ploratory study of HIV-1 infected patients is characterized by the ␦ ϩ %CD158a/VV 2 2 (1, 3) 5 (3, 11) 0.03 ϩ http://www.jimmunol.org/ ϩ ␦ ␥␦ %CD158b/VV␦2 6 (3, 10) 14 (11, 22) 0.03 involvement of the V 2 TCR T cells that do not express KIR. As established in HIV-1 infection (24) and in HIV-1-TB coinfec- general linear model ␥␦ϩ ␦ ϩ ,ء ;Medians (Q1, Q3); p, Mann-Whitney non-parametric test comparing patients with and without IRS. tion (25), the TCR T cells V 2 subpopulation was markedly reduced in PBMCs of all patients, whereas the usual tissular V␦2Ϫ subpopulation of ␥␦ T cells expanded. In this paper, we show that with a higher amplitude in IRS compared with non-IRS patients at baseline and throughout the follow-up, patients with IRS had (13 vs 3.5% of TCR␥␦ T cells, respectively; p ϭ 0.02; Fig. 3E). At more circulating V␦2ϩ TCR␥␦ T cells than patients without it, a ϩ that peak, 6% (IQR, 4–10) V␦2 T cells displayed the CD158 highly interesting finding because these cells have been described ϩ receptors and 31% (IQR, 26–43) TCR␥␦ T cells were CD94/ as specific for Mtb (20, 21, 23). We thus hypothesize that they may ϩ by guest on October 5, 2021 NKG2 in the IRS patients. The CD94/NKG2 expression on be involved in IRS pathogenesis, together with conventional CD4 ϩ TCR␥␦ T cells remained significantly lower during follow-up in Th1 cells. Gioia et al. (28) showed the quantitative and qualitative IRS than in non-IRS patients ( p Ͻ 0.0001; Fig. 2C). We did not restoration of V␦2ϩ effector memory T cells after TB control. Shen detect any change in cell activation status, as measured by et al. (29) have already shown a recovery after HAART of immune HLA-DR. responses directed against mycobacterial phosphoantigens, at the Ϫ ϩ In conclusion, IRS is associated with expansion of KIR V␦2 same time that the number of tuberculin-specific IFN-␥-producing ϩ TCR␥␦ T cells in addition to the expansion of activated effector CD4 T cells increases in bacillus Calmette-Gue´rin-SIV coinfected memory CD4 T cells specific for mycobacteria. monkeys receiving antiretrovirals. In this paper, we add to this body of knowledge the recovery of effector V␦2ϩ TCR␥␦ T cells Discussion during IRS, a finding that supports the involvement of these cells Our study shows two major characteristics of IFN-␥ producing T in the inflammatory syndrome. cells associated with TB-IRS: tuberculin-specific T cells are mul- Even more importantly, the significantly lower levels of KIRϩ tifunctional CD4ϩ effector memory T cells and the involvement of and NKR lectin-like CD94/NKG2ϩ TCR␥␦ T cells in IRS patients V␦2ϩ TCR␥␦ T cells that do not express KIR. This latter charac- compared with those without IRS also points to differences in the teristic can distinguish at baseline patients who will develop IRS modulation of signaling pathways in these patients. Because the from those who will not. Abs we used characterize NK cell receptors belonging to the fam- The IRS frequency in this study appears to be higher than that ily of KIR or lectin-like molecules but cannot distinguish between reported in the literature (1, 34), although we used the same cri- the inhibitory or activating properties of these receptors, we con- teria. However, a similar high frequency is currently being ob- ducted a functional analysis of IFN-␥ production after CD3 stim- served in an ongoing clinical trial evaluating a new antiretroviral ulation in those cells. Results clearly show these receptors were strategy in HIV-infected patients with TB France (BKVIR trial) mediating negative signals on all ␥␦ϩ CD3ϩ T cells tested that (O. Lortholary, personal communication) and might reflect a better were CD158ϩ or CD94ϩ but not on the CD158Ϫ or CD94Ϫ coun- knowledge of this now well recognized complication, leading to a terparts and are therefore in accordance with the reported inhibi- more frequent clinical diagnosis. tory function of CD158 or NKG2A in V␦2ϩ ␥␦ T cells during TB Our results provide a clear definition of the T cells involved in (31). As almost 90% of these cells are known to be specific for the the tuberculin-specific IFN-␥ production previously associated mycobacterial Ag isopentenyl pyrophosphate, our results provide a with IRS. We confirm and extend prior findings by our group (4) known mechanism for the amplification of the dysregulated im- and others (35) that IFN-␥-producing tuberculin-specific T cells mune response to TB that would cause the inflammatory syn- are highly amplified in patients with IRS compared with to those drome. This lower expression appeared to be specific to the V␦2ϩ without it. These findings differ, however, from those of groups subset of TCR␥␦ T cells and was not observed on conventional 3920 IMMUNITY OF TB-HIV-1 IMMUNE RESTORATION SYNDROME Downloaded from http://www.jimmunol.org/ by guest on October 5, 2021

FIGURE 2. Phenotypic and functional characterization of NKR on CD3ϩ and CD3Ϫ lymphocytes and ␥␦ T cells at baseline. Data were established in ϩ ϩ 11 patients who will (black) and 13 patients who will not (white) experience IRS. A, Baseline (M0) frequencies of CD158a,h (left) and b (right) cells. ϩ ␥␦ϩ B, Baseline (M0) frequencies of CD94/NKG2 heterodimer expression measured as CD94 positive cells. C, Kinetic of CD94/NKG2 TCR T cells in patients with (black) and without (white) IRS (median). D, Anti-CD3 stimulation of TCR ␥␦ϩ T cells in one IRS patient at baseline. p, Mann-Whitney nonparametric test; §, general linear model.

CD3ϩ T cells nor on CD3Ϫ lymphocytes, suggesting this phenom- Although the value of peripheral blood analyses of DC and enon is not genetically determined. However, we cannot exclude a monocytes is limited for these tissular cells, we observed a de- trapping of the KIRϩ TCR␥␦ T cells in the granuloma because our crease rather than the expected quantitative restoration of mDC in observations were made in the peripheral blood cells. It is therefore patients with, but not those without, IRS. We suggest that this difficult to assign this phenomenon a specific pathogenic role. Our phenomenon may reflect their recruitment at inflammatory sites. findings may complement those of Price et al. (39), who found an We saw no other differences in DC, monocyte activation, or increased frequency of activating NKR haplotypes but no differ- pattern recognition receptors at baseline or during IRS that ence in inhibitory haplotypes in patients experiencing - could explain the uncontrolled Th1 reaction storm seen in IRS. associated IRS. Indeed, we saw no modulation of TLR-2 membrane expression The Journal of Immunology 3921 Downloaded from http://www.jimmunol.org/ by guest on October 5, 2021

FIGURE 3. Kinetics of changes in circulating immune competent cells after HAART initiation. Results are expressed as median values in 11 patients with (black) and 13 patients without (white) IRS. A, Median HLA-DRϩ CD4ϩ and CD8ϩ T cells after HAART initiation. Arrow, Peak of HLA-DRϩ CD4ϩ .p ϭ 0.02 (CD4ϩ) and p ϭ 0.01 (CD8ϩ); §, general linear model of CD4ϩHLA-DRϩ between IRS and non-IRS patients, p ϭ 0.02 ,ء ;and CD8ϩ T cells B, DC and pDC subpopulations. Arrow, Significantly different nadir in IRS vs non-IRS at time of IRS, p ϭ 0.02. C, CD14ϩ monocyte frequency and TLR2 membrane expression. D, Proportion of V␦2ϩ and V␦2Ϫ TCR␥␦ T cells in IRS and non-IRS patients (median). E, Proportion of V␦2ϩ TCR␥␦ϩ T cells at baseline, at peak of increase after HAART initiation. The changes from baseline are represented as ⌬. p, Mann-Whitney nonparametric test comparing patients with and without IRS; §, general linear model. during IRS, even in the context of the proinflammatory cytokine therefore cannot definitively identify or rule out roles for these storm already described (4, 19). Of note, we did not measure innate immune-competent cells in triggering this exacerbated DC-SIGN expression, which is known to be up-regulated in TB. inflammatory syndrome. The balance between TLR-2-activation and DC-SIGN-inhibi- Finally, the only immune characteristic that appears to predict tion during IRS may require further exploration (40, 41). We IRS at initiation of HAART is the lower proportion of KIRϩ 3922 IMMUNITY OF TB-HIV-1 IMMUNE RESTORATION SYNDROME

ϩ TCR␥␦ T cells, whereas, unlike Boulware et al. (42) and Tan of macrophage class II MHC expression and antigen processing by 19-kDa li- et al. (35), we found no difference in T cell activation at baseline. poprotein of Mycobacterium tuberculosis. J. Immunol. 167: 910–918. 12. Means, T. K., S. Wang, E. Lien, A. Yoshimura, D. T. Golenbock, and Overall, the number of parameters explored in this relatively small M. J. Fenton. 1999. Human toll-like receptors mediate cellular activation by study does not allow us to draw definitive conclusions on the re- Mycobacterium tuberculosis. J. Immunol. 163: 3920–3927. 13. Stenger, S., and R. L. Modlin. 2002. Control of Mycobacterium tuberculosis spective role of these parameters. However, after adjusting our through mammalian Toll-like receptors. Curr. Opin. Immunol. 14: 452–457. statistical analysis according to the Hochberg and Benjamini 14. Geijtenbeek, T. B., S. J. Van Vliet, E. A. Koppel, M. Sanchez-Hernandez, method (43), the magnitude of tuberculin-specific T cells during C. M. Vandenbroucke-Grauls, B. Appelmelk, and Y. Van Kooyk. 2003. Myco- ϩ ␥␦ϩ target DC-SIGN to suppress dendritic cell function. J. Exp. Med. 197: IRS and the low proportion of CD94/NKG2 TCR T cells at 7–17. baseline still remain significant. 15. Grassi, F., A. Hosmalin, D. McIlroy, V. Calvez, P. Debre, and B. Autran. 1999. In conclusion, we provide two clear and independent mecha- Depletion in blood CD11c-positive dendritic cells from HIV-infected patients. ␥ AIDS 13: 759–766. nisms for the storm of IFN- -producing cells that is associated 16. Lichtner, M., R. Rossi, F. Mengoni, S. Vignoli, B. Colacchia, A. P. Massetti, with IRS in HIV-TB coinfected patients with an acute amplifica- I. Kamga, A. Hosmalin, V. Vullo, and C. M. Mastroianni. 2006. Circulating ␣ tion of conventional tuberculin-specific multifunctional CD4 ef- dendritic cells and interferon- production in patients with tuberculosis: corre- Ϫ ϩ lation with clinical outcome and treatment response. Clin. Exp. Immunol. 143: fector-memory T cells and an amplification of KIR V␦2 329–337. TCR␥␦ϩ T cells that is already present before HAART initiation 17. Schmidt, B., S. H. Fujimura, J. N. Martin, and J. A. Levy. 2006. Variations in plasmacytoid dendritic cell (PDC) and myeloid dendritic cell (MDC) levels in and appears to distinguish patients who will develop IRS from HIV-infected subjects on and off antiretroviral therapy. J. Clin. Immunol. 26: those who will not. 55–64. 18. Zhang, Z., J. Fu, Q. Zhao, Y. He, L. Jin, H. Zhang, J. Yao, L. Zhang, and Acknowledgments F. S. Wang. 2006. Differential restoration of myeloid and plasmacytoid dendritic

cells in HIV-1-infected children after treatment with highly active antiretroviral Downloaded from We are especially grateful to the PARADOX TB Study group: A. Baakili, therapy. J. Immunol. 176: 5644–5651. A.-M. Be´gle´, F. Besse, D. Bollens, O. Bouchaud, P. Bursachi, J. Cadranel, 19. Flo, T. H., O. Halaas, S. Torp, L. Ryan, E. Lien, B. Dybdahl, A. Sundan, and J. Camuset, C. Chakvetadze, J. Delgado, M. Diemer, B. Dupont, T. Espevik. 2001. Differential expression of Toll-like receptor 2 in human cells. J. Leukocyte Biol. 69: 474–481. S. Elmarsafy, O. Fain, L. Fonquernie, A. Furco, P.-M. Girard, 20. Tanaka, Y., C. T. Morita, Y. Tanaka, E. Nieves, M. B. Brenner, and B. R. Bloom. C. Grillot-Courvalin, J. F. Grivois, A. Guignet, M. C. Guilleminot, J.-L. 1995. Natural and synthetic non-peptide antigens recognized by human ␥␦ T Herrmann, V. Jeantils, V. Joly, V. Jouis, P. Klutse, K. Lacombe, cells. Nature 375: 155–158. R. Lahoulou, C. Lascoux, A. Lavole, B. Lefebvre, A. Lefort, E. Letellier, 21. Shen, Y., D. Zhou, L. Qiu, X. Lai, M. Simon, L. Shen, Z. Kou, Q. Wang, L. Jiang, J. Estep, et al. 2002. Adaptive immune response of V␥2V␦2ϩ T cells during http://www.jimmunol.org/ O. Lortholary, V. Martinez, A. Metro, M. Trumeau, J.-L. Meynard, M.-C. mycobacterial infections. Science 295: 2255–2258. Meyohas, J.-M. Molina, G. Obenga, M. Parrinello, O. Pelet, C. Pintado, 22. Hoft, D. F., R. M. Brown, and S. T. Roodman. 1998. Bacille Calmette-Guerin D. Ponscarme, A. Rami, W. Rozenbaum, H. Sahli, P. Sellier, L. Slama, vaccination enhances human ␥␦ T cell responsiveness to mycobacteria suggestive S. Courtial, R. Tubiana, J. Stirnemann, S. Tassi, O. Taulera, H. Touitou, of a memory-like phenotype. J. Immunol. 161: 1045–1054. I. Vacher, F. Vincent, and P. Yeni. Special acknowledgment to 23. Kabelitz, D., A. Bender, S. Schondelmaier, B. Schoel, and S. H. Kaufmann. 1990. A large fraction of human peripheral blood ␥␦ϩ T cells is activated by Myco- J. A. Cahn for editing of the manuscript. bacterium tuberculosis but not by its 65-kD heat shock protein. J. Exp. Med. 171: 667–679. Disclosures 24. Autran, B., F. Triebel, C. Katlama, W. Rozenbaum, T. Hercend, and P. Debre. 1989. T cell receptor ␥␦ϩ lymphocyte subsets during HIV infection. Clin. Exp. The authors have no financial conflict of interest. Immunol. 75: 206–210. 25. Carvalho, A. C., A. Matteelli, P. Airo, S. Tedoldi, C. Casalini, L. Imberti, by guest on October 5, 2021 References G. P. Cadeo, A. Beltrame, and G. Carosi. 2002. ␥␦ T lymphocytes in the pe- 1. Lawn, S. D., L. G. Bekker, and R. F. Miller. 2005. Immune reconstitution disease ripheral blood of patients with tuberculosis with and without HIV co-infection. associated with mycobacterial infections in HIV-infected individuals receiving Thorax 57: 357–360. antiretrovirals. Lancet Infect. Dis. 5: 361–373. 26. Boullier, S., M. Cochet, F. Poccia, and M. L. Gougeon. 1995. CDR3-independent ␥␦ ␦ ϩ 2. Race, E. M., J. Adelson-Mitty, G. R. Kriegel, T. F. Barlam, K. A. Reimann, V 1 T cell expansion in the peripheral blood of HIV-infected persons. N. L. Letvin, and A. J. Japour. 1998. Focal mycobacterial lymphadenitis follow- J. Immunol. 154: 1418–1431. ing initiation of protease-inhibitor therapy in patients with advanced HIV-1 dis- 27. Rojas, R. E., K. A. Chervenak, J. Thomas, J. Morrow, L. Nshuti, S. Zalwango, ␦ ϩ ␥␦ ease. Lancet 351: 252–255. R. D. Mugerwa, B. A. Thiel, C. C. Whalen, and W. H. Boom. 2005. V 2 3. Narita, M., D. Ashkin, E. S. Hollender, and A. E. Pitchenik. 1998. Paradoxical T cell function in Mycobacterium tuberculosis- and HIV-1-positive patients in the worsening of tuberculosis following antiretroviral therapy in patients with AIDS. United States and Uganda: application of a whole-blood assay. J. Infect. Dis. 192: Am. J. Respir. Crit. Care Med. 158: 157–161. 1806–1814. 4. Bourgarit, A., G. Carcelain, V. Martinez, C. Lascoux, V. Delcey, B. Gicquel, 28. Gioia, C., C. Agrati, R. Casetti, C. Cairo, G. Borsellino, L. Battistini, G. Mancino, E. Vicaut, P. H. Lagrange, D. Sereni, and B. Autran. 2006. Explosion of tuber- D. Goletti, V. Colizzi, L. P. Pucillo, and F. Poccia. 2002. Lack of culin-specific Th1-responses induces immune restoration syndrome in tubercu- CD27ϪCD45RAϪV␥9V␦2ϩ T cell effectors in immunocompromised hosts and losis and HIV co-infected patients. AIDS 20: F1–F7. during active pulmonary tuberculosis. J. Immunol. 168: 1484–1489. 5. Flynn, J. L., and J. D. Ernst. 2000. Immune responses in tuberculosis. Curr. Opin. 29. Shen, L., Y. Shen, D. Huang, L. Qiu, P. Sehgal, G. Z. Du, M. D. Miller, Immunol. 12: 432–436. N. L. Letvin, and Z. W. Chen. 2004. Development of V␥2V␦2ϩ T cell responses 6. Li, T. S., R. Tubiana, C. Katlama, V. Calvez, H. Ait Mohand, and B. Autran. during active mycobacterial coinfection of simian immunodeficiency virus-in- 1998. Long-lasting recovery in CD4 T-cell function and viral-load reduction after fected macaques requires control of viral infection and immune competence of highly active antiretroviral therapy in advanced HIV-1 disease. Lancet 351: CD4ϩ T cells. J. Infect. Dis. 190: 1438–1447. 1682–1686. 30. Poles, M. A., S. Barsoum, W. Yu, J. Yu, P. Sun, J. Daly, T. He, S. Mehandru, 7. Autran, B., G. Carcelain, T. S. Li, C. Blanc, D. Mathez, R. Tubiana, C. Katlama, A. Talal, M. Markowitz, A. Hurley, D. Ho, and L. Zhang. 2003. Human immu- P. Debre, and J. Leibowitch. 1997. Positive effects of combined antiretroviral nodeficiency virus type 1 induces persistent changes in mucosal and blood ␥␦ T therapy on CD4ϩ T cell homeostasis and function in advanced HIV disease. cells despite suppressive therapy. J. Virol. 77: 10456–10467. Science 277: 112–116. 31. Boullier, S., Y. Poquet, F. Halary, M. Bonneville, J. J. Fournie, and 8. Hengel, R. L., M. C. Allende, R. L. Dewar, J. A. Metcalf, J. M. Mican, and M. L. Gougeon. 1998. Phosphoantigen activation induces surface translocation of H. C. Lane. 2002. Increasing CD4ϩ T cells specific for tuberculosis correlate intracellular CD94/NKG2A class I receptor on CD94Ϫ peripheral V␥9V␦2T with improved clinical immunity after highly active antiretroviral therapy. AIDS cells but not on CD94Ϫ thymic or mature ␥␦ T cell clones. Eur. J. Immunol. 28: Res. Hum. Retroviruses 18: 969–975. 3399–3410. 9. Wendland, T., H. Furrer, P. L. Vernazza, K. Frutig, A. Christen, L. Matter, 32. Fausther-Bovendo, H., N. Wauquier, J. Cherfils-Vicini, I. Cremer, P. Debre, and R. Malinverni, and W. J. Pichler. 1999. HAART in HIV-infected patients: res- V. Vieillard. 2008. NKG2C is a major triggering receptor involved in the V␦1T toration of antigen-specific CD4 T-cell responses in vitro is correlated with CD4 cell-mediated cytotoxicity against HIV-infected CD4 T cells. AIDS 22: 217–226. memory T-cell reconstitution, whereas improvement in delayed type hypersen- 33. French, M. A., P. Price, and S. F. Stone. 2004. Immune restoration disease after sitivity is related to a decrease in viraemia. AIDS 13: 1857–1862. antiretroviral therapy. AIDS 18: 1615–1627. 10. Jiao, X., R. Lo-Man, P. Guermonprez, L. Fiette, E. Deriaud, S. Burgaud, 34. Dhasmana, D. J., K. Dheda, P. Ravn, R. J. Wilkinson, and G. Meintjes. 2008. B. Gicquel, N. Winter, and C. Leclerc. 2002. Dendritic cells are host cells for Immune reconstitution inflammatory syndrome in HIV-infected patients receiv- mycobacteria in vivo that trigger innate and acquired immunity. J. Immunol. 168: ing antiretroviral therapy: pathogenesis, clinical manifestations and management. 1294–1301. Drugs 68: 191–208. 11. Noss, E. H., R. K. Pai, T. J. Sellati, J. D. Radolf, J. Belisle, D. T. Golenbock, 35. Tan, D. B., Y. K. Yong, H. Y. Tan, A. Kamarulzaman, L. H. Tan, A. Lim, W. H. Boom, and C. V. Harding. 2001. Toll-like receptor 2-dependent inhibition I. James, M. French, and P. Price. 2008. Immunological profiles of immune The Journal of Immunology 3923

restoration disease presenting as mycobacterial lymphadenitis and cryptococcal resistant to immune restoration diseases associated with herpes virus infections. meningitis. HIV Med. 9: 307–316. J. Acquired Immune Defic. Syndr. 45: 359–361. 36. Meintjes, G., K. A. Wilkinson, M. X. Rangaka, K. Skolimowska, K. van Veen, 40. Vannberg, F. O., S. J. Chapman, C. C. Khor, K. Tosh, S. Floyd, M. Abrahams, R. Seldon, D. J. Pepper, K. Rebe, P. Mouton, et al. 2008. Type 1 D. Jackson-Sillah, A. Crampin, L. Sichali, B. Bah, P. Gustafson, et al. 2008. helper T cells and FoxP3-positive T cells in HIV-tuberculosis-associated immune CD209 genetic polymorphism and tuberculosis disease. PLoS ONE 3: e1388. reconstitution inflammatory syndrome. Am. J. Respir. Crit. Care Med. 178: 41. Thuong, N. T., T. R. Hawn, G. E. Thwaites, T. T. Chau, N. T. Lan, H. T. Quy, 1083–1089. N. T. Hieu, A. Aderem, T. T. Hien, J. J. Farrar, and S. J. Dunstan. 2007. A 37. Millington, K. A., J. A. Innes, S. Hackforth, T. S. Hinks, J. J. Deeks, polymorphism in human TLR2 is associated with increased susceptibility to tu- D. P. Dosanjh, V. Guyot-Revol, R. Gunatheesan, P. Klenerman, and A. Lalvani. berculous meningitis. Immun. 8: 422–428. 2007. Dynamic relationship between IFN-␥ and IL-2 profile of Mycobacterium 42. Boulware, D., D. Meya, T. Bergemann, I. Vlasova, A. Kambugu, K. P. McAdam, tuberculosis-specific T cells and antigen load. J. Immunol. 178: 5217–5226. E. Janoff, and P. Bohjanen. 2008. Biomarkers of immune activation are predictors 38. Egen, J. G., A. G. Rothfuchs, C. G. Feng, N. Winter, A. Sher, and R. N. Germain. of HIV Immune Reconstitution Inflammatory Syndrome. In 15th Conference on 2008. Macrophage and T cell dynamics during the development and disintegra- Retroviruses and Opportunistic Infections (CROI), February 3–6, Boston. tion of mycobacterial granulomas. Immunity 28: 271–284. Poster 1007. 39. Price, P., C. Witt, D. de Santis, and M. A. French. 2007. Killer immunoglobulin- 43. Hochberg, Y., and Y. Benjamini. 1990. More powerful procedures for multiple like receptor genotype may distinguish immunodeficient HIV-infected patients significance testing. Stat. Med. 9: 811–818. Downloaded from http://www.jimmunol.org/ by guest on October 5, 2021