ORIGINAL RESEARCH

Immunological profiles of immune restoration diseasepresenting as mycobacterial lymphadenitis andcryptococcal meningitisDBA Tan,1 YK Yong,2 HY Tan,2 A Kamarulzaman,2 LH Tan,2 A Lim,1 I James,3 M French1,4 and P Price1,4

1School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia, 2University of MalayaMedical Center, Kuala Lumpur, Malaysia, 3Center for Clinical Immunology and Biomedical Statistics, Murdoch Universityand Royal Perth Hospital, Australia and 4Department of Clinical Immunology and Immunogenetics, Royal Perth Hospital,Australia

ObjectivesA proportion of HIV patients beginning antiretroviral therapy (ART) develop immune restorationdisease (IRD). Immunological characteristics of IRD were investigated in a cohort of HIV patientsbeginning therapy in Kuala Lumpur, Malaysia.

MethodsPeripheral blood mononuclear cells were collected at weeks 0, 6, 12, 24 and 48 of ART from fivepatients experiencing IRD [two with cryptococcal and three with Mycobacterium tuberculosis (Mtb)disease], eight non-IRD controls who had begun ART with CD4 T-cell counts of o100 cells/mL and17 healthy controls. Leukocytes producing interferon-gamma (IFNg) were quantified by enzyme-linked immunospot assay after stimulation with purified protein derivative (PPD), early secretoryantigenic target-6 (ESAT-6), Cryptococcus neoformans or Cytomegalovirus antigens. Plasmaimmunoglobulin (IgG) antibodies reactive with these antigens were assessed by enzyme-linkedimmunosorbent assay. Proportions of activated (HLA-DRhi) and regulatory (CD25 CD127lo andCTLA-41 ) CD4 T-cells were quantified by flow cytometry.

ResultsPlasma HIV RNA declined and CD4 T-cell counts rose within 8–27 weeks on ART. Mtb IRD patientsdisplayed elevated IFNg responses and/or plasma IgG to PPD, but none responded to ESAT-6.Cryptococcal IRD occurred in patients with low baseline CD4 T-cell counts and involved clear IFNgand antibody responses to cryptococcal antigen. Proportions of activated and regulatory CD4 T-cellsdeclined on ART, but remained higher in patients than in healthy controls. At the time of IRD,proportions of activated CD4 T-cells and regulatory CD4 T-cells were generally elevated relative toother patients.

ConclusionsCryptococcal and Mtb IRD generally coincide with peaks in the proportion of activated T-cells,pathogen-specific IFNg responses and reactive plasma IgG. IRD does not reflect a paucity ofregulatory CD4 T-cells.

Keywords: antiretroviral therapy, CMV, cryptococcus, regulatory T-cells, tuberculosis

Received: 9 December 2007, accepted 1 February 2008

Introduction

Successful antiretroviral therapy (ART) suppresses thereplication of HIV and allows the recovery of CD4 T-cellnumbers within a few months [1,2]. However, recovery ofantigen-specific CD4 T-cell responses is variable amongHIV patients on ART [3–6]. Approximately 10–40% of

Correspondence: Patricia Price, Level 2, Medical Research FoundationBuilding, Rear 50 Murray Street, Perth, WA 6000, Australia. Tel: 1 61 89224 0378; fax: 1 61 8 9224 0204; e-mail: patricia.price@uwa.edu.au

DOI: 10.1111/j.1468-1293.2008.00565.xr 2008 British HIV Association HIV Medicine (2008), 9, 307–316

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patients beginning ART with advanced immunodeficiencyexperience immune restoration disease (IRD).

Clinically, IRD patients have atypical presentation ofdisease associated with common opportunistic pathogenssuch as Mycobacterium avium complex (MAC), Mycobac-terium tuberculosis (Mtb), Cytomegalovirus (CMV), Varicellazoster virus (VZV), hepatitis C virus (HCV) or Cryptococcusneoformans [7]. The timing of these events coincides withincreases in CD4 T-cell counts on ART, suggesting thatrestored immune responses against antigens of viable ornon-viable pathogens can be immunopathological ratherthan protective [7]. However, few longitudinal sample setshave been available to characterize these responses.

Bourgarit et al. [8] described dramatic interferon-g(IFNg) responses by cells from seven patients withpreviously treated tuberculosis (TB) who developed MtbIRD (‘paradoxical’ IRD). Purified protein derivative (PPD)induced Th1-related cytokines and chemokines, includingIL-2, IL-12, IFNg and IP-10 (CXCL10). Production of TNFa,IL-6, IL-1b, IL-10, RANTES and MCP-1 by culturedperipheral blood mononuclear cells (PBMCs) in responseto PPD and CMV were also elevated at the time of IRD,suggesting broad-based immune activation. Their resultsare consistent with our finding that MAC IRD reflectsrestoration of delayed-type hypersensitivity (DTH) re-sponses to mycobacterial antigens [7,9]. Bourgarit et al.demonstrated that cells from Mtb IRD patients did notrespond to secreted mycobacterial proteins such as theearly secretory antigenic target (ESAT)-6 and 85B protein[8]. These antigens are only released by viable bacteria, so‘paradoxical’ IRD may be induced by residual antigensfrom treated infections. This requires confirmation.

Elevated humoral immune responses have been asso-ciated with IRD. Patients who developed CMV retinitis asan IRD had a steady increase in plasma IgG reactive toCMV antigens in the first year of ART [10]. Similarly, HIV–HCV co-infected patients who developed hepatotoxicityand clinical hepatitis had increased HCV core-specific IgG[11]. Serum antibodies against several mycobacterialantigens (notably PGL-Tb1) mark symptomatic Mtb[12,13], but have not been addressed in an IRD. Levels ofpathogen-specific antibodies before and during ART mayhelp to predict and/or diagnose IRD, or shed light onpathogenic mechanisms.

In patients with no previous diagnosis of cryptococcalinfection, presentations of cryptococcal disease in thecontext of increased CD4 T-cell counts and decreasedplasma HIV RNA on ART are defined as ‘unmasking’ IRD[14,15]. They usually present as central nervous systemdisease (meningitis or focal enhancing mass lesions) earlyafter ART or as lymphadenitis within 15 months on ART[14–16]. Histological examination of lymph nodes may

show granulomatous inflammation, necrosis or suppura-tion [7]. Cryptococcal IRD patients may have higherbaseline HIV-1 RNA levels and initial cerebrospinal fluid(CSF) cryptococcal antigen titres than patients with no IRD.They may also have higher lumbar puncture openingpressures and leukocyte counts than untreated patientswith AIDS-related cryptococcal disease [14]. Cellular andhumoral immune responses have not been monitored incryptococcal IRD.

Patients who experienced IRD provoked by CMV, MACand/or HCV infections retain elevated levels of plasma andcellular markers of immune activation for several years.This includes CCR3, CCR5, plasma interleukin-(IL)-6 andsoluble IL-6 receptor (sIL-6R) [17–19]. Natural regulatoryCD25CD4 T-cells (T-regs) can suppress the activation,proliferation and production of cytokines by CD4 and CD8T-cells [20]. Depletion of T-regs from PBMCs of HIV-infected patients increased HIV- and CMV-specific re-sponses by CD4 and CD8 T-cells [21]. Hence a deficiency inT-regs may promote the excessive T-cell responsescharacteristic of an IRD. However, proportions of T-regsand activated CD4 T-cells are correlated during the firstyear of ART [22], so T-reg populations may increase afteran IRD as a consequence of immune activation.

In this study, regulatory T-cells were identified asCD4CD25CD127lo and CD4CTLA-4 1 T-cells. Low expres-sion of IL-7 receptor (CD127) with co-expression of CD25identifies CD4 T-regs expressing the transcription factorFoxP3 [23,24]. These markers differentiate T-regs fromactivated T-cells, which may also express CD25. CD4T-cells expressing the inhibitory receptor cytotoxicT-lymphocyte-associated protein-4 (CTLA-4) were assayedbecause they influence down-regulation of effector T-cellactivation [25].

We conducted a longitudinal study in a cohort of HIVpatients beginning ART with a range of opportunisticinfections to investigate the immunological characteristicsof IRD; specifically pathogen-specific IFNg responses, IgGantibody responses and the proportions of activated (HLA-DRhi) and regulatory (CD25CD127lo or CTLA-4 1 ) CD4T-cells. This is the first report of the immunology ofcryptococcal IRD.

Patients and methods

Study subjects

Five Chinese male HIV patients with cryptococcal (n 5 2) orMtb (n 5 3) IRD were identified from a cohort of 47 patientsenrolled into an ongoing study of immune reconstitution inpatients at the Infectious Disease Out-patient Clinics atUniversity Malaya Medical Centre (UMMC), Kuala Lumpur,

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Malaysia. Other IRDs observed in the cohort includedhistoplasmosis, VZV disease and Kaposi’s sarcoma. Eightpatients with no evidence of IRD were selected forcomparison. These were matched with the IRD patients bysex (male) and ethnicity (Chinese). Healthy Chinese donors(mostly hospital staff, n 5 17) residing in Kuala Lumpurwere included as controls. They are presumed to beHIV-negative.

Non-IRD patients and healthy controls had no knownhistory of cryptococcal disease, but several patients had ahistory of Mtb and vaccination with the mycobacterialantigen Bacille Calmette-Guerin (BCG) is standard practicein Malaysia. IRD patients, non-IRD patients and controlswere 37–56 (median 43) years old, 33–64 (median 39) yearsold and 21–71 (median 31) years old, respectively. HIVpatients were older than the healthy controls (P 5 0.02), butthe IRD and non-IRD groups were similar in age (P 5 0.22).Institutional ethics approval was obtained for the study andinformed consent was given by all participants.

Sample collection, plasma HIV RNA level and CD4 T-cellcounts

Whole blood was collected into lithium heparin tubes frompatients at weeks 0, 6, 12, 24 and 48 after commencement

of ART, and once from control donors. Plasma was storedat � 80 1C. PBMCs were obtained by Ficoll gradientcentrifugation and then cryopreserved in liquid nitrogen.Plasma HIV RNA levels and CD4 T-cell counts wereperformed by the routine clinical laboratories at UMMC.Plasma HIV-1 RNA was measured using the COBASAmplicor HIV-1 Monitor Test, v. 1.5 (Roche Diagnostics,Indianapolis, IN, USA). The cut-off defining an undetect-able viral load was o50 HIV-1 RNAcopies/mL. CD4 T-cellcounts were performed by flow cytometry.

Enzyme-linked immunospot assay of IFNg producingcells

Enzyme-linked immunospot (ELISpot) assay was per-formed as described in [26]. Cells were stimulatedfor 20 h with PPD (10 mg/mL), ESAT-6 (10 mg/mL) (StatensSerum Institute, Copenhagen, Denmark), CMV or crypto-coccal antigen. A mannoprotein preparation of C. neofor-mans (acapsular strain Cap67) was provided by DrStuart M Levitz (University of Massachusetts MedicalSchool, Worcester, MA, USA) [27] and used at 50 mg/mL.Spots were counted using AID ELISpot Reader v. 2.9software (Autoimmun Diagnostika GmbH, Strassberg,Germany).

Table 1 Laboratory diagnosis of cryptococcal and Mycobacterium tuberculosis (Mtb) immune restoration disease (IRD)

Patient C1 C2 C2

Clinical presentation Cryptococcal meningitis on ART (IRD)

Time of assay Week 1 2 (IRD) Week 1 3 (IRD)Week 1 5(recovery phase)

C. neoformans culture (blood) Negative Positive NegativeCryptococcal Ag titre (serum) 1:128 1:512 Not doneC. neoformans culture (CSF) Positive Positive NegativeCryptococcal Ag titre (CSF) 1:32 1:16 1:4

Patient T1 T2 T3

Initial presentation Disseminated TB before IRD

Mycobacterial culture andAFB (at presentation)

Positive (week�7) Positive (week�6) Positive (week�4)

Mycobacterial culture andAFB (on anti-TB therapy)

Negative (week�4) Not done Negative (week 1 3)

Clinical presentation at IRD Increased lymphadenitis on ART

Time of IRD Week 1 2 Week 1 12 Week 1 12Mycobacterial cultureand AFB (after IRD)

Negative (week 1 4 and week 1 8) AFB-positive only (week 1 12) Not done

AFB, acid-fast bacilli; ART, antiretroviral therapy; CSF, cerebrospinal fluid; TB, tuberculosis.Times are shown as the week before (� ) or after ( 1 ) the initiation of ART. Mycobacterial culture and AFB were assessed in pus from affected lymph nodes.

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Enzyme-linked immunosorbent assay for pathogen-specific IgG

Enzyme-linked immunosorbent assay (ELISA) was per-formed as described in [10]. Half-volume 96-well plateswere coated with a predetermined optimal concentration ofCMV antigen, C. neoformans antigen or PPD (antigensdescribed earlier). Pooled plasma samples from CMV,Cryptococcal and PPD-seropositive individuals were usedas standards and assigned an arbitrary value of 100 units/mL of antigen-specific IgG. Coefficients of variance werebelow 15%.

Three-colour flow cytometry to measure proportions ofT-cell sub-sets

Flow cytometry was performed as described in Lim et al.[22]. Thawed PBMCs were surface-stained with CD4-PECy5, CD25-FITC, CD127-PE and/or HLA-DR-PE (CoulterImmunotech, Marseille, France). CTLA-4-PE (Coulter) werestained intracellularly and processed using Intraprepreagents (Coulter); 50 000–100 000 events were acquiredusing a Becton Dickinson FACSCalibur flow cytometer andanalysed using the FlowJo program v. 5.7.2 (Tree Star,Ashland, OR, USA).

Statistical analyses

Mann–Whitney U-tests were used to compare groups ofindividuals. No longitudinal analyses were applied to theIRD patients because the groups were too small. Long-itudinal data from the eight non-IRD patients wereanalysed on a log10 scale using continuous piecewise-linear mixed models with a change point at week 15 of ART.The slope estimates and P-values of the plots relate to thesetwo segments, testing for divergence from a slope of zero.Statistically significant differences are shown in Figs 1–3.Analyses were carried out in S-Plus 7.0 for Windows(Insightful Corporation, Seattle, WA, USA). P-values below0.05 were considered to be statistically significant.

Results

Clinical presentations and microbiological assessment ofIRD patients

The five cases of IRD presented as atypical opportunisticinfections or inflammatory disease after ART [7]. Twopatients experienced ‘unmasking’ cryptococcal IRD andthree patients experienced ‘paradoxical’ Mtb IRD. Theclinical presentations of IRD and the diagnostic tests

Fig. 1. Interferon-gamma (IFNg) responses in immune restoration disease (IRD) patients (left panels) and non-IRD patients (right panels). IFNgresponses to Cryptococcus neoformans (a) and purified protein derivative (PPD) antigens (b) increased during Cryptococcal IRD andMycobacterium tuberculosis (Mtb) IRD, respectively, in IRD patients. IFNg responses to early secretory antigenic target-6 (ESAT-6) (c) increasein Cryptococcal IRD patients but not in Mtb IRD patients. (d) IFNg responses to Cytomegalovirus (CMV) increased in all IRD patients aftercommencing ART. Significant increases in PPD (b) and CMV (d) IFNg responses was observed in non-IRD patients within 15 weeks ofcommencing ART.

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performed are summarized in Table 1. One cryptococcalIRD patient (denoted C1) presented with meningitis 2 weeksafter beginning ART, while the other (denoted C2)presented with cryptococcal meningitis and cryptococcae-mia after 3 weeks on ART. In both patients, symptoms

began about 1 week before diagnosis, which was based onexamination of CSF. Cryptococcal antigen was titred bylatex agglutination and C. neoformans was cultured fromthe CSF. C. neoformans was isolated from blood in C2 only.Patients were treated with amphotericin B and fluconazole.

Fig. 2. Plasma immunoglobulin (IgG) antibody titres in immune restoration disease (IRD) (left panels) and non-IRD (right panels) patients.Cryptococcal IRD patients have highest plasma IgG reactive to Cryptococcus neoformans antigens (a) during IRD. Mycobacteriumtuberculosis (Mtb) IRD patients (T2 and T3) have higher anti-purified protein derivative (PPD) (b) and anti-Cryptococcus neoformans antigens(a) plasma IgG than non-IRD patients. All IRD patients have increased anti-Cytomegalovirus (CMV) plasma IgG (c) after commencingantiretroviral therapy (ART). Significant decrease in IgG reactive to PPD (b) was seen in non-IRD patients within 15 weeks on ART.

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Symptoms resolved in C1 with no further investigations. InC2, a second lumbar puncture had elevated openingpressure and lower titre of cryptococcal antigen in theCSF. There was no fungal growth in CSF or blood culturesat that time.

All three Mtb IRD patients had presented with active TBinfection 4–7 weeks before starting ART and receivedantibiotic therapy. IRD was diagnosed at 3 weeks (T1), 12weeks (T2) and 14 weeks (T3) after commencing ART. Allthree patients presented with fever and one or more

Fig. 3. Proportions of activated (HLA-DRhi) (a) and regulatory CD25CD127lo (b) or CTLA-4 1 CD4 T-cells in immune restoration disease (IRD)(left panels) and non-IRD (right panels) HIV-infected patients remained higher than healthy controls in the first year of antiretroviral therapy(ART). Proportions of activated CD4 T-cells (a) and CTLA-4 1 CD4 T-cells (b) decreased significantly in non-IRD patients after 15 weeks on ART.Proportions of CD25CD127lo CD4 T-cells did not change significantly within the first year of ART.

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enlarged lymph nodes 1–2 weeks before diagnosis, whichwas based on exacerbations of cervical lymphadenitis,lymphadenopathy and lymph nodes evolving into ab-scesses or cold abscess enlargement. T1 had a worseningleft parotid abscess.

CD4 T-cell counts and HIV RNA levels confirm that allpatients responded to ART

All HIV patients (IRD and non-IRD) had plasma HIV RNAlevels 4100 000 copies/mL prior to ART and o100 copies/mL after 8–27 weeks on ART. All patients had lowerproportions of CD4 T-cells (expressed as % of lymphocytes)prior to ART compared to healthy controls [median (range)3.8% (0.5–11%) vs. 33% (20–41%); Po0.001]. IRD patientsC1, C2 and T3 and one non-IRD patient began ART witho2.5% CD4 T-cells. Proportions of CD4 T-cells in all HIVpatients increased during the year of study [12% (4.1–20%)at their last sample collection], but remained lower thanhealthy controls [33% (20–41%), Po0.001]. The increase inproportions of CD4 T-cells was significant in non-IRDpatients over the first 15 weeks on ART (slope 5 0.02,P 5 0.002). A similar increase was observed in the IRDpatients.

IFNg responses to antigens of provoking pathogenspeaked at the time of IRD

Samples were collected on two to six (median five)occasions from each patient over a period of 14–53(median 47) weeks after commencement of ART, with theexception of T1 (who was recruited at week 3 of ART).Antigen-specific IFNg responses were measured to assessthe hypothesis that IRD is caused by excessive cell-mediated responses to antigens of opportunistic pathogens.The upper limit of the ELISpot assay was 2000 spots per200 000 cells. The median and interquartile ranges ofELISpot counts recorded in healthy control donors (n 5 17)are presented for comparison.

IFNg responses to C. neoformans antigens peaked at thetime of IRD in C1 and C2 (Fig. 1a). These responses wereapproximately 10-fold higher than those seen in any of thenon-IRD patients or healthy controls. After the IRDepisodes, responses declined over the year of study.

Two Mtb IRD patients (T1 and T2) exhibited strong IFNgresponses to PPD; these remained high throughout thestudy (Fig. 1b). T3 exhibited a small increase in his PPDresponse on ART (13–443 spots per 200 000 cells), whichpeaked prior to the presentation of IRD. The low responsemay reflect his low baseline proportions of CD4 T-cells(data not shown). Tuberculin skin tests performed on T2

and T3 remained Mantoux-negative during their IRDepisode.

IFNg responses to PPD in non-IRD patients increasedsignificantly within 15 weeks of ART (Fig. 1b) with nosignificant change thereafter. One non-IRD patient hadpeak IFNg responses above 400 spots per 200 000 PBMCswith no signs of TB disease pre- and post-ART. This isabove the median for healthy controls (120 spots). HisMantoux tests were negative at baseline and after 24 weekson ART. However, he had higher proportions of CD4 T-cellsbefore ART than all other patients, so we suspect a robustbut protective response to mycobacterial antigen. In othernon-IRD patients, PPD responses increased to levels similarto healthy controls during the first year of ART.

IFNg responses to ESAT-6 antigen in Mtb IRD patients(Fig. 1c) did not follow the pattern seen with PPD or reachthe median response of healthy controls. The cryptococcalIRD patients (C1 and C2) displayed increased responses onART, while there was no significant increase in responses ofnon-IRD patients.

CMV was included as a control antigen because nopatients experienced CMV IRD. IFNg responses to CMVincreased in IRD patients, reaching levels similar tocontrols within the first year on ART. CMV IFNg responsesincreased in non-IRD patients after 15 weeks on ART withno significant change thereafter. C1, C2 and two non-IRDpatients had low IFNg responses to CMV before and afterART (Fig. 1d).

The results demonstrate IFNg responses to the initiatingantigens pre-ART and during IRD. This was compared withplasma IgG antibody levels reactive to PPD and C.neoformans as an alternative marker of these IRDs. CMVwas again included as a control antigen.

Levels of plasma IgG reactive with Cryptococcus peakedduring an IRD

Levels of plasma IgG antibodies reactive with C. neofor-mans, PPD and CMV were measured in longitudinalsamples from IRD and non-IRD patients. Results arepresented in arbitrary units (Fig. 2). This allows compar-isons between patients but not between antigens.

Levels of antibodies reactive with cryptococcal antigenspeaked at the time of IRD. C2 had higher levels of antibody(Fig. 2a) and higher antigen titres (Table 1) than C1, despitehaving a lower IFNg response. Two Mtb IRD patients (T2and T3) had intermediate and stable levels of anti-cryptococcal IgG. T1 and all non-IRD patients retainedlow levels throughout.

Mtb IRD patients (T2 and T3) had higher anti-PPDantibody levels than non-IRD patients (Fig. 2b), but levelswere low in T1. Anti-CMV antibody levels began low in C1,

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C2 and T3, but increased thereafter. No significant changewas observed in non-IRD patients (Fig. 2c).

The results show increased IFNg or IgG responses to theinitiating antigen in all five IRD patients, with concurrentincreases in responses to CMV, PPD or ESAT-6 in severalpatients who did not experience IRD associated with thesepathogens. This could reflect non-specific immune activa-tion and/or expansion of memory T-cells present atthe start of ART. Hence proportions of activated andregulatory CD4 T-cells were investigated in our long-itudinal sample sets.

Proportions of activated (HLA-DRhi) and regulatory CD4T-cells were generally high during an IRD

High expression of HLA-DR was used to mark CD4 T-cellactivation. Proportions of activated CD4 T-cells werehigher in patients at baseline, compared to healthy controls[median (range) 40% (19–72%) vs. 4.9% (1.4–12%);Po0.001]. Levels declined significantly after 15 weeks onART in non-IRD patients, but peaked at the time of IRD infour of the five IRD patients and remained high (Fig. 3a).Hence immune activation is a general feature of IRD.

Patients had higher proportions of CD25CD127loCD4T-cells at baseline than healthy controls [13% (1.3–34%)vs. 6.3% (3.8–8.6%); P 5 0.002]. Baseline proportions ofCTLA-4 1 CD4 T-cells were also higher in patients than incontrols [17% (2.7–30%) vs. 2.9% (1.4–4.8%); Po0.001].There was no significant change in proportions ofCD25CD127lo CD4 T-cells in non-IRD patients withinthe first year of ART (Fig. 3b). However, proportions ofCTLA-4 1 CD4 T-cells in non-IRD patients declined after 15weeks on ART (Fig. 3c). Proportions of T-cells defined byboth sets of markers remained higher in patients than incontrols throughout.

In cryptococcal IRD patients (C1 and C2), proportions ofregulatory T-cells peaked at the time of IRD and werehigher than levels in non-IRD patients. However, Mtb IRDpatients had levels similar to the non-IRD patients. The lownumbers precluded statistical analyses, but it is clear thatIRD does not reflect low proportions of regulatory T-cells.

Discussion

In this article, we present the first data associatingcryptococcal IRD with elevated IFNg and humoralresponses to cryptococcal antigen. Cryptococcal and MtbIRD were associated with T-cell activation but not adeficiency of regulatory T-cells. While non-IRD patientsand healthy controls were not selected by prior crypto-coccal or mycobacterial diseases, all subjects were recruitedlocally to ensure a similar spectrum of exposure.

All cases of Mtb IRD presented as a worsening of TBlymphadenitis and enlarged abscesses, fitting the definitionof a ‘paradoxical’ IRD. Mtb IRDs have been associated withTh1 and pro-inflammatory responses to PPD [8]. Here,patients T1 and T2 had large increases in CD4 T-cell countsand much higher IFNg responses to PPD than were seen innon-IRD patients. IFNg responses remained high after theIRD episode. Patient T3 had persistently low CD4 T-cellcounts with a moderate IFNg response to PPD that declinedrapidly after his IRD. This was seen in one non-IRD patientwho had no evidence of active Mtb infection before orduring ART. Because only T2 and T3 had clear IgGresponses to PPD before and during therapy, the threepatients present distinct immunological profiles.

No Mtb IRD patients responded to the Mtb-specificprotein ESAT-6. This has been reported [8] and suggeststhat ‘paradoxical’ IRD can reflect responses to non-viablebacteria. Mycobacteria could not be cultured from T1before ART or at the time of his IRD (Table 1). T2 was nottested but T3 was culture-positive before his IRD. Hence thefailure of T3 to respond to ESAT-6 may reflect persistentimmunodeficiency because his CD4 T-cells counts re-mained low and his responses to PPD were poor. Thisdemonstrates two scenarios where TB-diagnosis testsbased on ESAT-6 would miss acute responses associatedwith IRD.

Diagnoses of cryptococcal IRD were based on the overtpresentation of cryptococcosis (antigen and culturableorganisms in CSF) in the context of a virological andimmunological response to ART, meeting the definition of‘incident’ or ‘unmasking’ IRD. We demonstrate increasedIFNg responses and IgG responses to cryptococcal antigensat the time of the IRD. Antibody levels were higher in thepatient with a disseminated infection and elevated serumantigen levels (C2), but both patients showed a clearresponse.

No individuals who had prior cryptococcal disease wereavailable for comparative studies. Five HIV patients treatedfor cryptococcal infection prior to successful ART dis-played no response to cryptococcal antigen (assessed byexpression of CD69 and TNFa) and only one patientdisplayed a small response to the antigen [28]. Theseauthors did not discuss IRD, but no patients relapsed afterwithdrawal of anti-fungal therapy.

In general, levels of activated (HLA-DRhi) CD4 T-cells innon-IRD patients declined on ART but remained abovelevels found in healthy controls. Elevated cellular andplasma immune activation markers associated with thedevelopment of mycobacterial IRD [29] and persistentelevation is described after viral IRD [17,19,30]. Here,proportions of HLA-DRhi CD4 T-cells peaked at the time ofIRD. However, four non-IRD patients also showed transient

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increases at week 6 and proportions of these cells atbaseline did not predict IRD. Activation markers may riseas a consequence of IRD, rather than being a cause.

We show for the first time that increased IFNg and IgGresponses and induction of HLA-DR expression at the timeof IRD do not reflect reduced proportions of T-cells withphenotypes associated with regulation. However, theregulatory functions were not investigated. HIV infectionchanges the tissue distribution of T-regs [31], so theproportions of regulatory T-cells in PBMCs may not reflectproportions at the site of IRD (lymph nodes in Mtb IRDpatients or meninges in cryptococcal IRD patients).

In conclusion, we show that IRD involves increasedpathogen-specific IFNg responses and plasma IgG, withhigh proportions of activated (HLA-DRhi) and regulatory(CD25CD127lo and CTLA-4 1 ) CD4 T-cells. Further studiesof the immunological profiles of IRD are warranted.

Acknowledgements

The authors thank Professors Ng Kee Peng and ShamalaDevi for laboratory facilities, and the patients and controlswho contributed to this study. The project was supported byInfectious Diseases Research Fund, UMMC and theUniversity of Western Australia. This is publication 2007-33 (Clinical Immunology and Immunogenetics, Royal PerthHospital).

References

1 Battegay M, Nuesch R, Hirschel B, Kaufmann GR.

Immunological recovery and antiretroviral therapy in HIV-1

infection. Lancet Infect Dis 2006; 6: 280–287.

2 Le Guillou-Guillemette H, Renier G, Vielle B et al. Immune

restoration under HAART in patients chronically infected with

HIV-1: diversity of T, B, and NK immune responses. Viral

Immunol 2006; 19: 267–276.

3 Burgess K, Price P, James IR et al. Interferon-gamma responses

to Candida recover slowly or remain low in immunodeficient

HIV patients responding to ART. J Clin Immunol 2006; 26:

160–167.

4 Jansen CA, De Cuyper IM, Steingrover R et al. Analysis of the

effect of highly active antiretroviral therapy during acute

HIV-1 infection on HIV-specific CD4 T cell functions. AIDS

2005; 19: 1145–1154.

5 Lederman HM, Williams PL, Wu JW et al. Incomplete immune

reconstitution after initiation of highly active antiretroviral

therapy in human immunodeficiency virus-infected patients

with severe CD4 cell depletion. J Infect Dis 2003; 188:

1794–1803.

6 Schluger NW, Perez D, Liu YM. Reconstitution of immune

responses to tuberculosis in patients with HIV infection who

receive antiretroviral therapy. Chest 2002; 122: 597–602.

7 French MA, Price P, Stone SF. Immune restoration disease after

antiretroviral therapy. AIDS 2004; 18: 1615–1627.

8 Bourgarit A, Carcelain G, Martinez V et al. Explosion of

tuberculin-specific Th1-responses induces immune restoration

syndrome in tuberculosis and HIV co-infected patients. AIDS

2006; 20: F1–F7.

9 French MA, Lenzo N, John M et al. Immune restoration disease

after the treatment of immunodeficient HIV-infected patients

with highly active antiretroviral therapy. HIV Med 2000; 1:

107–115.

10 Stone SF, Price P, Tay-Kearney ML, French MA.

Cytomegalovirus (CMV) retinitis immune restoration disease

occurs during highly active antiretroviral therapy-induced

restoration of CMV-specific immune responses within a

predominant Th2 cytokine environment. J Infect Dis 2002;

185: 1813–1817.

11 Stone SF, Lee S, Keane NM, Price P, French MA. Association of

increased hepatitis C virus (HCV)-specific IgG and soluble CD26

dipeptidyl peptidase IV enzyme activity with hepatotoxicity

after highly active antiretroviral therapy in human

immunodeficiency virus-HCV-coinfected patients. J Infect Dis

2002; 186: 1498–1502.

12 Lyashchenko K, Colangeli R, Houde M, Al Jahdali H, Menzies

D, Gennaro ML. Heterogeneous antibody responses in

tuberculosis. Infect Immun 1998; 66: 3936–3940.

13 Simonney N, Chavanet P, Perronne C et al. B-cell immune

responses in HIV positive and HIV negative patients with

tuberculosis evaluated with an ELISA using a glycolipid

antigen. Tuberculosis (Edinb) 2007; 87: 109–122.

14 Shelburne SA III, Darcourt J, White AC Jr et al. The role of

immune reconstitution inflammatory syndrome in AIDS-

related Cryptococcus neoformans disease in the era of highly

active antiretroviral therapy. Clin Infect Dis 2005; 40:

1049–1052.

15 Skiest DJ, Hester LJ, Hardy RD. Cryptococcal immune

reconstitution inflammatory syndrome: report of four cases in

three patients and review of the literature. J Infect 2005; 51:

e289–297.

16 Jenny-Avital ER, Abadi M. Immune reconstitution

cryptococcosis after initiation of successful highly active

antiretroviral therapy. Clin Infect Dis 2002; 35: e128–e133.

17 Price P, Mathiot N, Krueger R, Stone S, Keane NM, French MA.

Immune dysfunction and immune restoration disease in HIV

patients given highly active antiretroviral therapy. J Clin Virol

2001; 22: 279–287.

18 Stone SF, Price P, Keane NM, Murray RJ, French MA. Levels of

IL-6 and soluble IL-6 receptor are increased in HIV patients

with a history of immune restoration disease after HAART. HIV

Med 2002; 3: 21–27.

Immunological profiles of immune restoration disease 315

r 2008 British HIV Association HIV Medicine (2008) 9, 307–316

19 Almeida CA, Price P, French MA. Immune activation in

patients infected with HIV type 1 and maintaining suppression

of viral replication by highly active antiretroviral therapy.

AIDS Res Hum Retroviruses 2002; 18: 1351–1355.

20 Kinter AL, Hennessey M, Bell A et al. CD25( 1 )CD4( 1 )

regulatory T cells from the peripheral blood of asymptomatic

HIV-infected individuals regulate CD4( 1 ) and CD8( 1 ) HIV-

specific T cell immune responses in vitro and are associated

with favorable clinical markers of disease status. J Exp Med

2004; 200: 331–343.

21 Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF.

Human CD4 CD25 regulatory T cells control T-cell responses to

human immunodeficiency virus and cytomegalovirus antigens.

J Virol 2004; 78: 2454–2459.

22 Lim A, Tan D, Price P et al. Proportions of circulating T cells

with a regulatory cell phenotype increase with HIV-associated

immune activation and remain high on antiretroviral therapy.

AIDS 2007; 21: 1525–1534.

23 Liu W, Putnam AL, Xu-Yu Z et al. CD127 expression inversely

correlates with FoxP3 and suppressive function of human

CD4 T reg cells. J Exp Med 2006; 203: 1701–1711.

24 Seddiki N, Santner-Nanan B, Martinson J et al. Expression of

interleukin (IL)-2 and IL-7 receptors discriminates between

human regulatory and activated T cells. J Exp Med 2006; 203:

1693–1700.

25 Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-

associated antigen 4 plays an essential role in the function of

CD25( 1 ) CD4( 1 ) regulatory cells that control intestinal

inflammation. J Exp Med 2000; 192: 295–302.

26 Keane NM, Price P, Stone SF, John M, Murray RJ, French MA.

Assessment of immune function by lymphoproliferation

underestimates lymphocyte functional capacity in HIV patients

treated with highly active antiretroviral therapy. AIDS Res

Hum Retroviruses 2000; 16: 1991–1996.

27 Mansour MK, Schlesinger LS, Levitz SM. Optimal T cell

responses to Cryptococcus neoformans mannoprotein are

dependent on recognition of conjugated carbohydrates by

mannose receptors. J Immunol 2002; 168: 2872–2879.

28 Aberg JA, Price RW, Heeren DM, Bredt B. A pilot study of the

discontinuation of antifungal therapy for disseminated

cryptococcal disease in patients with acquired

immunodeficiency syndrome, following immunologic response

to antiretroviral therapy. J Infect Dis 2002; 185: 1179–1182.

29 Pires A, Nelson M, Pozniak AL et al. Mycobacterial immune

reconstitution inflammatory syndrome in HIV-1 infection

after antiretroviral therapy is associated with deregulated

specific T-cell responses: beneficial effect of IL-2 and

GM-CSF immunotherapy. J Immune Based Ther Vaccines

2005; 3: 7.

30 Stone SF, Price P, Brochier J, French MA. Plasma bioavailable

interleukin-6 is elevated in human immunodeficiency virus-

infected patients who experience herpesvirus-associated

immune restoration disease after start of highly active

antiretroviral therapy. J Infect Dis 2001; 184: 1073–1077.

31 Andersson J, Boasso A, Nilsson J et al. The prevalence of

regulatory T cells in lymphoid tissue is correlated with viral

load in HIV-infected patients. J Immunol 2005; 174:

3143–3147.

316 DBA Tan et al.

r 2008 British HIV Association HIV Medicine (2008) 9, 307–316