Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+ T-Lymphocyte Reactivity during Combination Antiretroviral Therapy in HIV-1-Infected Patients with Advanced Immunodeficiency

JOURNAL OF VIROLOGY,0022-538X/00/$04.0010

May 2000, p. 4127–4138 Vol. 74, No. 9

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD81

T-Lymphocyte Reactivity during CombinationAntiretroviral Therapy in HIV-1-InfectedPatients with Advanced Immunodeficiency

CHARLES R. RINALDO, JR.,1,2* XIAO-LI HUANG,1 ZHENG FAN,1 JOSEPH B. MARGOLICK,3

LUANN BOROWSKI,1 AKI HOJI,1 CHRISTINE KALINYAK,1 DEBORAH K. MCMAHON,1,2

SHARON A. RIDDLER,1,2 WILLIAM H. HILDEBRAND,4 RICHARD B. DAY,1

AND JOHN W. MELLORS1,2,5

Graduate School of Public Health1 and School of Medicine,2 University of Pittsburgh, and the Veterans Affairs MedicalCenter,5 Pittsburgh, Pennsylvania 15261; Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland

212053; and University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731904

Received 17 December 1999/Accepted 29 January 2000

The long-term efficacy of combination antiretroviral therapy may relate to augmentation of anti-humanimmunodeficiency virus type 1 (HIV-1) CD81 T-cell responses. We found that prolonged treatment of late-stage HIV-1-infected patients with a protease inhibitor and two nucleoside reverse transcriptase inhibitorsfailed to restore sustained, high levels of HIV-1-specific, HLA class I-restricted, cytotoxic-T-lymphocyte pre-cursors and gamma interferon (IFN-g) production by CD81 T cells. In some patients, particularly thoseinitiating three-drug combination therapy simultaneously rather than sequentially, there were early, transientincreases in the frequency of anti-HIV-1 CD81 T cells that correlated with decreases in HIV-1 RNA andincreases in T-cell counts. In the other patients, HIV-1-specific T-cell functions either failed to increase ordeclined from baseline during triple-drug therapy, even though some of these patients showed suppression ofplasma HIV-1 RNA. These effects of combination therapy were not unique to HIV-1 specific T-cell responses,since similar effects were noted for CD81 T cells specific for the cytomegalovirus pp65 matrix protein. The leveland breadth of CD81 cell reactivity to HLA A*02 HIV-1 epitopes, as determined by IFN-g production and HLAtetramer staining after combination therapy, were related to the corresponding responses prior to treatment.There was, however, a stable, residual population of potentially immunocompetent HIV-1-specific T cellsremaining after therapy, as shown by tetramer staining of CD81 CD45RO1 cells. These results indicate thatnew strategies will be needed to target residual, immunocompetent HIV-1-specific CD81 T cells to enhance theeffectiveness of antiretroviral therapy in patients with advanced immunodeficiency.

Combination antiretroviral therapy with two nucleoside re-verse transcriptase (RT) inhibitors and a potent protease in-hibitor can produce sustained suppression of human immuno-deficiency virus type 1 (HIV-1) RNA in blood to fewer than 50copies per ml (16, 17). The success of triple combination ther-apy has led to the hypothesis that HIV-1 could eventually beeliminated from infected individuals. The finding of persistent,latent HIV-1 in patients despite virus suppression with therapy(6, 10, 11, 13, 53, 54), however, indicates that such treatmentstrategies alone may not be sufficient for control or eliminationof viral infection.

The ability to generate high levels and a broad specificity ofanti-HIV-1 cytotoxic T lymphocytes (CTL) is considered to bea critical component of the host immune response to HIV-1 (2,5, 23, 24, 29, 43, 44, 52). As for other chronic infectious dis-eases, it is possible that the long-term efficacy of combinationantiretroviral therapy will require augmentation of host im-mune responses such as anti-HIV-1 CTL reactivity. Recentstudies, however, have yielded contradictory evidence on theimpact of combination antiretroviral therapy on anti-HIV-1CTL reactivity. Several reports have shown that the numbers of

anti-HIV-1 CTL precursors (CTLp), as measured in vitro in a2-week, limiting dilution assay, increase with suppressive anti-retroviral therapy in acute (8) and chronically infected patients(21, 37). In contrast, others have reported that levels of circu-lating, CD81 CD381 T cells that bind HLA A2 tetramericHIV-1 Gag p17 and RT peptide complexes decrease afterinitiation of antiretroviral therapy in patients with advancedimmunodeficiency (15, 21, 30, 31). In addition, none of thesereports have addressed the breadth of the HIV-1-specific,CD81 T-cell reactivity that recovers with virus suppression.

Significant differences in the CTLp and tetramer bindingassays may account for the discrepant findings (33). The CTLpassay is dependent on cell growth in vitro in response to anti-gen stimulation, which is subject to many experimental vari-ables. In contrast, tetramer staining is a direct measure of thenumber of CD81 T cells specific for CTL epitopes that doesnot require in vitro cell growth, although it is not a directmeasure of CD81 T-cell function. Single cell production ofgamma interferon (IFN-g) has recently been reported to be asensitive assay for both the quantity and function of antiviralCTL (25, 33). The antiviral and immunomodulatory effects ofIFN-g are important in host control of viral infections (9). Thismeasure of immune reactivity does not depend on the capacityof the CD81 T cells to replicate and become active cytotoxiccells during prolonged in vitro culture. IFN-g production isalso a result of both antigen-specific T-cell binding and conse-

* Corresponding author. Mailing address: A427 Crabtree Hall, Uni-versity of Pittsburgh Graduate School of Public Health, 130 DeSotoSt., Pittsburgh, PA 15261. Phone: (412) 624-3928. Fax: (412) 624-4953.E-mail: [email protected].

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quent immune reactivity rather than just a measure of HLAclass I tetramer binding to T cells.

We have therefore performed a detailed, longitudinal as-sessment of the effects of prolonged treatment with a simulta-neous or sequential combination of a protease inhibitor (indi-navir [IDV]) and two nucleoside RT inhibitors (zidovudine[ZDV] and lamivudine [3TC]), on multiple parameters ofHIV-1-specific, CD81 T-cell function in patients enrolled inthe Merck 035 trial (16, 17). Our results show that combinationantiretroviral therapy did not lead to sustained recovery ofhigh levels of CTLp and IFN-g-producing CD81 T cells spe-cific for HIV-1 Gag, Pol, and Env proteins. Thus, it may benecessary to use additional therapeutic approaches to augmentHIV-1-specific CD81 cells in patients with advanced immuno-deficiency on suppressive antiretroviral therapy.

MATERIALS AND METHODS

Patients. Of 27 HIV-1-seropositive adults who participated in the Pittsburghportion of the Merck 035 trial (17), 14 were enrolled in this immunology sub-study. Each patient gave written, informed consent approved by the University ofPittsburgh Institutional Review Board. Patients in the Merck 035 trial received atleast 6 months of prior ZDV treatment; prior therapy with 3TC or a proteaseinhibitor was not allowed. Samples were also available from several years beforethe trial from 3 of the 14 patients who were previously enrolled in the Pittsburghportion of the Multicenter AIDS Cohort Study (MACS), a longitudinal investi-gation of the natural history of HIV-1 infection. The 14 study participants wererandomized to one of three treatment regimens as previously described (45).Seven patients (group A) received 800 mg of IDV (Crixivan; Merck, West Point,Pa.) every 8 h plus 200 mg of ZDV (Retrovir; Glaxo-Wellcome, ResearchTriangle Park, N.C.) every 8 h and 150 mg of 3TC (Epivir; Glaxo-Wellcome)every 12 h concurrently; three patients (group B) received 200 mg of ZDV every8 h and 150 mg of 3TC every 12 h, and four patients (group C) received 800 mgof IDV every 8 h. Matching placebos for these drugs were administered tosubjects in the latter two groups.

The duration of the original clinical trial was to be 52 weeks, but because of thepreliminary finding of greater antiviral activity of the triple-drug regimen, thestudy was amended during the trial to reduce the randomized, blinded treatmentto at least 24 weeks, followed by open label triple-drug therapy for all studyparticipants.

Six of the seven group A patients remained in this immunology substudy for its148-week duration, while one (A1155) discontinued the substudy after 48 weeksbut remained in the Merck 035 trial. One group B patient (B1171) and one groupC patient (C1180) discontinued from the substudy due to virologic failure (a 2-to 3-log10 rebound in viral RNA load) after 100 weeks.

Viral serology and load. Determination of HIV-1 antibody was done by en-zyme immunoassay and immunoblotting. All of the patients were confirmedpositive for prior cytomegalovirus (CMV) infection by an immunofluorescenceimmunoglobulin G antibody assay of serum (Immunopathology Laboratory, Uni-versity of Pittsburgh Medical Center). Serum samples were assayed for HIV-1RNA by the ultrasensitive quantitative RT-PCR assay (Amplicor; Roche) (16,41). Data are presented as copies of HIV-1 RNA per ml of serum, with the lowerlimit of detection being 50 copies of RNA per ml of serum.

T-cell phenotyping. T-cell subsets were quantified by flow cytometric analysis(Profile II; Coulter, Miami, Fla.) after staining with monoclonal antibodies(MAb) specific for CD3, CD4, and CD8 T cells (Becton-Dickinson, MountainView, Calif.). Peripheral blood mononuclear cells (PBMC) were assessed for theproportions of CD81 and CD41 memory (CD45RO1) and naive (CD45RA1)subsets by three-color fluorescence using MAb conjugated to fluorescein isothio-cyanate (FITC), phycoerythrin (PE), and phycoerythrin-cyanin 5.1 (PECy5);antibody combinations were CD45RO/CD45RA/CD8 or /CD4, HLA-DR/CD38/CD8 or /CD4, and CD4/CD28/CD8 or /CD4. Three-color analyses were per-formed on an Elite ESP flow cytometer (Coulter).

CTLp assay. PBMC were prepared from cryopreserved cells for use as effec-tors in anti-HIV-1 CTLp frequency and bulk lysis assays (19). Assay results fromcryopreserved PBMC are similar to those using fresh PBMC (19). There is alsominimal variation in cytotoxic activity between replicates and split samples and infresh PBMC obtained within several months from the same, untreated individ-uals (19). A median of 10 samples (range, 8 to 13) obtained at baseline throughup to 148 weeks of the trial were tested from each patient. PBMC were stimu-lated with psoralen-treated, UV light-irradiated, autologous, Epstein-Barr virus-transformed B-lymphocyte cell lines (B-LCL) that had been infected overnightwith vaccinia virus (VV) containing the combined Gag-Pol and Env codingsequences from the BH10 and HXB2 strains of HIV-1 LAI (VVgpe) (TherionBiologics, Cambridge, Mass.). This results in a consistent expression of theHIV-1 vector in .70% of the B-LCL (19). The precursor frequencies weredetermined by limiting-dilution assay of PBMC seeded in complete mediumcontaining 15% fetal calf serum (FCS). PBMC were seeded at 0 (medium

control) and at 250, 500, 1,000, 3,000, 6,000, 12,000, and 16,000 cells per well in24 replicate wells of 96-well round-bottom microtiter plates. To each well wereadded 2.5 3 104 gamma-irradiated allogeneic PBMC from one or two HIV-1-seronegative normal donors as feeder cells, 100 U of recombinant interleukin-2(rIL2; Chiron, Emeryville, Calif.), and stimulator cells (1,600 stimulator cells perwell) from VV-GPE-infected, inactivated B-LCL. The cells were cultured for 14days at 37°C in a 5% CO2 atmosphere, with fresh complete medium containing15% FCS and rIL2 added every 5 days. On day 14, the cells in culture weredivided, transferred to two new wells, and adjusted to 100 ml with completemedium containing 15% FCS. The numbers and viability of the cells weremonitored by trypan blue dye exclusion. Cytotoxicity was measured against51Cr-labeled, autologous B-LCL (104) infected with recombinant VVgag, VVpol,VVenv, or the NYCBH strain of VV as a control (VVvac). The fraction ofnonresponding wells was the number of wells in which the 51Cr release did notexceed the mean spontaneous release plus 10% of the incorporated 51Cr (total51Cr release 2 spontaneous 51Cr release) divided by the number of wells assayed.

The precursor frequency was estimated by the maximum-likelihood methodwith a statistical program provide by S. Kalams (Boston, Mass.). CTLp activitywas expressed as the net precursor frequency per 106 PBMC, i.e., the number ofCTLp/106 PBMC specific for HIV-1 antigen minus the number of CTLp/106

PBMC specific for non-HIV-1-expressing target cells. The mean (6 standarderror [SE]) number of CTLp in the VVvac control was 65 (69) (n 5 158).

For bulk lysis assays, PBMC were stimulated as in the precursor frequencyassay and assessed against the same targets at three effector/target cell ratios(40:1, 20:1, and 10:1). For each determination, the value for the lysis of targetsinfected with the VV control was subtracted from the value for the HIV-1protein-expressing targets. Data were calculated as lytic units per 107 cells de-rived from an exponential regression analysis of the multiple effector/targetratios (4), since these were highly correlated with the percent lysis for theindividual effector/target cell ratios (43).

Only data from the CTLp assays are shown because of the quantitative natureof CTLp determinations and the similar patterns of CTL lytic activity delineatedby both the CTLp and bulk lysis methods (19, 43). We have found that lyticactivity was mediated by purified CD81 T cells and not by CD41 T cells and wasnot seen against HLA class I mismatched targets, indicating that the anti-HIV-1CTLp response was mediated by HLA class I-restricted, CD81 T cells (19, 43).

Single cell IFN-g assay. A single cell-based enzyme immunoassay (ELISPOT)was done to enumerate the number of IFN-g producing cells (19a) by a modi-fication of the methods of Tanguay and Killion (47) and Lalvani et al. (25). Amedian of 11 (range, 7 to 13) cryopreserved samples were available from 11 ofthe 14 patients in this assay. Nitrocellulose membranes in 96-microwell polyvi-nylidene difluoride-backed plates (Millipore, Bedford, Mass.) were coated over-night at 4°C with 50 ml of anti-IFN-g MAb (10 mg/ml, 1-DIK; Mabtech, Stock-holm, Sweden) per well. The antibody-coated plates were then washed four timeswith phosphate-buffered saline (PBS; Biowhittaker, Walkersville, Md.) andtreated with 180 ml of RPMI medium (Life Technologies, Grand Island, N.Y.)per well containing 10% human serum (Sigma, St. Louis, Mo.) for 1 h at 37°C.The responder cells for this assay were either PBMC or, when sufficient cellswere available, CD81 cells enriched by negative selection of PBMC with anti-body-coated magnetic beads (anti-CD4, anti-CD19, and anti-CD16 MAb; Dynal,Lake Success, N.Y.) to remove CD41 T cells, B cells, and natural killer cells,respectively. We have found that CD81 T cells are the predominant cell typeproducing IFN-g in PBMC used in our assay (19A). A total of 105 to 106 of thesePBMC or CD81 cells (98% pure) were stimulated with 104 to 105 VVgpe-infected, inactivated B-LCL as in the CTLp assay, in 200 ml of AIM V Medium(Life Technologies) and incubated overnight at 37°C in 5% CO2. B-LCL infectedwith a VV expressing CMV pp65 matrix phosphoprotein (VVcmv; a gift from S.Riddell, University of Washington), known to be a major target antigen forCD81 CTL (27), were used for comparative stimulation of IFN-g-producing cellsby a non-HIV-1 antigen. In certain experiments, PBMC or CD81 cells wereincubated overnight at 37°C in 5% CO2 with HLA A*02-associated, HIV-1peptides (10 mg/ml) in nitrocellulose membrane 96-well plates. These peptideswere Gag p1777–85 SLYNTVATL (49), Gag p24151–159 TLNAWVKVV (36), PolRT476–484 ILKEPVHGV (50), and Env gp120192–199 KLTSCNTSV (3). Theplates were washed four times with PBS containing 0.05% Tween 20 (Sigma),and 2 ml of the secondary antibody (biotin-conjugated anti-IFN-g MAb 7-B6-1;Mabtech) per ml was added in 100 ml to each well; the plates were then incubatedfor 2 h at 37°C in CO2. The plates were washed four times with PBS containing0.05% Tween 20 and treated with avidin-bound, biotinylated horseradish perox-idase H (Vectastain Elite Kit; Vector Laboratories, Burlingame, Calif.) for 1 h atroom temperature. The plates were then washed three times with PBS containing0.05% Tween 20 and three times with PBS, followed by a 5-min incubation with100 ml of 3-amino-9-ethylcarbazole (Sigma) per well. The reaction was stoppedwith running tap water. The red-brown spots, representing single CD81 T cellsproducing IFN-g, were counted with a dissecting microscope. PBMC or CD81 Tcells stimulated with the phorbol ester, phorbol 12-myristate 13-acetate (1 ng/ml), and the calcium ionophore, ionomycin (1 mM/ml) (PMA-ionomycin; Sig-ma), were the positive control. PBMC or CD81 T cells were stimulated withB-LCL infected with the VVvac as the negative control for cells stimulated withVV–HIV-1 protein-expressing B-LCL and with medium alone as the negativecontrol for the peptide-stimulated cells. The number of antigen-specific, CD81

T-cell-producing IFN-g was calculated by subtracting the values for the cells

4128 RINALDO ET AL. J. VIROL.

stimulated with control VVvac-infected B-LCL from the cells stimulated with theB-LCL infected with either VVgag, VVpol, VVenv, or VVcmv or by subtractingthe number of spot-forming cells in the medium control from the peptide-stimulated cells. The mean (6 the SE) numbers of spot-forming cells in themedium controls and in the VVvac control were 15 (64) (n 5 97) and 100 (614)(n 5 130), respectively.

Staining with peptide-HLA tetramers. To assess the association between fre-quency of cells producing IFN-g and binding the tetramers (1), we performed aparallel experiment using HLA A*0201 tetramer refolded around Gag p1777–85SLYNTVATL and HLA A*0201 tetramer refolded around Pol RT476–484ILKEPVHGV to stain cells from the same samples that were tested by theELISPOT assay. PBMC from 6 of the 14 patients who were HLA-A*0201(A1160, A1166, A1169, B1178, and C1164) and HLA-A*0212 (C1180), as typedby high-resolution, reference-strand-mediated conformation analysis (51), werestained and analyzed for tetramer-positive cells according to a protocol obtainedfrom the NIH Tetramer Synthesis Facility. Briefly, approximately 106 freeze-thawed cells were surface stained with FITC-conjugated MAb against CD45 RO(Coulter), PE-conjugated HLA-A2 tetramers for Gag p1777–85 or Pol RT476–484(NIH Tetramer Synthesis Facility), and PECy5-conjugated MAb against CD8(Coulter). As a negative control, a similar number of cells were stained withFITC- or PECy5-conjugated isotype-matching MAb and PE-conjugated avidin(Coulter). After incubation and washing, cells were fixed with 1% paraformal-dehyde and analyzed in an EPICS XL-MCL flow cytometer (Coulter) within24 h. Approximately 20,000 events were collected within a CD81 lymphocytegate. The data were calculated as the percentage of tetramer-staining cells perCD81 cell and reported in this study as the number of tetramer-positive cells per106 CD81 cells to allow direct comparisons with other immunologic parameters.We determined a threshold level of tetramer-positive cells of .200/106 (0.02%)CD81 cells based on the mean (63 SD) staining of CD81 T cells of HLA A*02HIV-1-seronegative donors.

Statistical analysis. Comparisons of the different cumulative parameters weredone by the paired and unpaired Student’s t test. Data among different groupswere assessed by analysis of variance (ANOVA) with the Scheffe multiple com-parison test and then analyzed for associations between different parameters bythe Pearson correlation coefficient test.

RESULTS

Suppression of HIV-1 load and changes in T-cell numberswith triple-combination antiretroviral therapy. The baselinecharacteristics prior to study entry were similar among the 14patients in the three treatment groups in this immunologystudy (P . 0.05 for T-cell numbers and HIV-1 RNA levels)(Table 1). By 14 weeks, all of the 7 patients who received thetriple-drug combination from the study onset (group A) had,500 copies of HIV-1 RNA per ml of serum (Fig. 1). HIV-1RNA further decreased to ,50 copies/ml by 24 weeks in sev-eral patients (A1155, A1160, A1162, and A1177). Virus wasstill intermittently detectable, however, at low copy numbers insamples from each of these patients during the trial. Only onepatient in group A (A1169; Fig. 1) had a breakthrough of virusabove 500 copies/ml late in the trial. The numbers of circulat-ing CD41 T cells increased in all of these patients during the2 years of combination therapy (Fig. 1) (45). In contrast, aspreviously reported (16, 17), there was less effect on viral loadand CD41 T cell numbers in patients who first received ZDV-3TC or IDV (groups B and C; Fig. 2) for 24 to 42 weeks beforebeing switched to open-label, triple combination therapy. Pa-tients B1159, B1178, C1158, and C1176 had ,50 copies ofHIV-1 RNA, whereas there was much less suppression of virus

in the other three patients, after the switch to the triple-drugregimen. The four patients with ,50 copies of HIV-1 RNAhad increases in CD41 T-cell numbers after the switch toopen-label, triple therapy. There were no significant changes inthe numbers of CD81 T cells in the three groups throughoutthe course of treatment (Fig. 1 and 2).

Effect of triple-combination antiretroviral therapy on thenumbers of CTLp specific for HIV-1 proteins. We examinedCD81 CTLp function specific for the three major HIV-1 struc-tural proteins with a conventional limiting-dilution assay (19).The results show that several of the group A patients, whoreceived the triple combination from the study onset, had in-creases in CTLp, particularly to Pol and Env, at consecutivetimes, peaking at weeks 8 to 12 (A1162, A1166, A1169, A1177,and A1179; Fig. 1). Changes in HIV-1-specific, bulk CTL lysiswere comparable to that with CTLp (data not shown). PatientsA1155 and A1160 had very low numbers of anti-HIV-1 CTLp(Fig. 1) and bulk CTL lysis (data not shown). Only two of theseseven patients, however, maintained elevated CTLp activitythroughout most of the study (A1166, anti-Gag, -Pol, and -Env;A1169, anti-Pol), with eventual decline to baseline levels after2 years. Although there was a gap in CTLp data available frompatient A1177 from weeks 8 to 52, results from the bulk lysisassay at all times before week 12 and at weeks 12, 24, and 36showed that high levels of CTL activity against Gag, Pol, andEnv were maintained through 36 weeks, with a concurrentdecrease in both bulk lysis and CTLp responses at 52 weeks(data not shown).

The seven patients in groups B and C received ZDV-3TC(group B) or IDV alone (group C) for a median of 45 weeks(range, 24 to 50 weeks) before being switched to the triple-drug combination. After initiation of the three-drug regimen,group B and C patients had heterogeneous and variablechanges in the numbers of CTLp. Three patients (B1178,C1176, and C1180) had transient increases in anti-Pol andanti-Gag CTLp during triple-drug therapy (Fig. 2), as with thegroup A patients. These patients also had transient increases inCTLp responses during the double-combination or single-com-bination drug treatment phase of the trial (Fig. 2). Althoughthere was a gap in data available for CTLp from weeks 8through 36 for patient B1171, results from the bulk CTL lysisexperiments during this time indicated that there was no CTLresponse to any HIV-1 protein (data not shown).

Effect of combination antiretroviral therapy on the numberof IFN-g-producing CD81 T cells in response to HIV-1 andCMV proteins. We quantified the number of IFN-g-producing,CD81 T cells specific for the three major HIV-1 structuralproteins and to the CMV pp65 matrix protein by a methodrecently developed in our laboratory (19a). The study was doneon a subset of 11 of the 14 patients who had sufficient numbersof cryopreserved PBMC for testing. Group A patients A1166,A1162, and A1179 had early, persistent elevations in IFN-g-producing, CD81 cells specific for at least one HIV-1 proteinthat decreased to, or below, baseline levels by approximately 2years of triple-drug therapy (Fig. 1). Patients A1160 and A1169had lower and fewer persistent elevations in IFN-g-producingcells in response to the HIV-1 proteins. The number of IFN-g-producing CD81 T cells in the group A patients also corre-lated with the levels of CTLp specific for Gag, Pol, and Env(Table 2).

Sequential initiation of triple-drug therapy was not associ-ated with consistent recovery of HIV-1-specific, CD81-cellIFN-g production in four of the six group B and C patientseither before or after receiving the triple-drug regimen (i.e.,B1178, C1158, C1164, and C1180) (Fig. 2). Patients B1159 andC1176, however, had a high, sustained number of IFN-g-pro-

TABLE 1. Baseline characteristics of patients in the immunologysubstudy of the Merck 035 triala

Group No. of patients(sex)

Serum HIV-1 RNA(copies/ml)

T cells/mm3

CD41 CD81

A 7 (M) 54,535 6 10,089 147 6 42 711 6 177B 3 (M) 83,996 6 43,766 241 6 60 907 6 81C 3 (M), 1 (F) 89,001 6 19,814 205 6 46 1,197 6 124

a Values are means 6 the SE. P was not significant for all comparisons, asdetermined by one-way ANOVA.

VOL. 74, 2000 ANTI-HIV-1 T-CELL REACTIVITY IN ANTIRETROVIRAL THERAPY 4129

ducing cells specific for at least one 1 HIV-1 protein during thepre-open-label portion of the drug trial and after the three-drug combination therapy (Fig. 2). In further contrast to thegroup A patients, there were no significant correlations be-tween the numbers of IFN-g-producing CD81 cells and theCTLp levels in the group B and C patients (data not shown).

The changes in IFN-g production by CD81 T cells duringcombination antiretroviral therapy were not unique for HIV-1proteins. That is, the numbers of IFN-g-producing, CD81 cellsspecific for the CMV pp65 matrix antigen were comparable tothe IFN-g responses to at least one HIV-1 protein duringtriple-drug treatment in most of the group A patients (Fig. 1)and the group B and C patients (Fig. 2).

Relation of HIV-1- and CMV-specific, CD81 T-cell re-sponses to viral load and T-cell counts during triple combina-tion antiretroviral therapy. We investigated whether anti-HIV-1 CTLp and IFN-g reactivity after triple-drug therapy wasrelated to changes in HIV-1 RNA levels and T-cell counts. Wefound that increases in CTLp correlated with decreases inHIV-1 plasma load during the first 12 weeks of simultaneoustriple drug treatment in the group A patients (Table 2). Asignificant although progressively weaker inverse correlation

with HIV-1 RNA levels was maintained for 48 weeks for allthree CTLp HIV-1-specific activities and for 132 weeks offollowup for anti-Gag and anti-Pol CTLp in these patients(data not shown). There was no significant correlation betweenthe CTLp levels and the number of T-cell subsets in group Apatients or with viral load and T-cell subsets in group B and Cpatients during combination therapy (data not shown).

The only significant correlation for IFN-g production wasthe response to Pol and the number of CD41 and CD81 T cellsand the response to CMV with the CD81 T-cell counts duringthe first 12 weeks of combination therapy in the group Apatients (Table 2). These correlations were progressivelyweaker at later time points (data not shown).

Effect of combination antiretroviral therapy on reactivity ofCD81 T cells to HIV-1 peptides assessed by single-cell IFN-gproduction. We next examined the breadth of the IFN-g re-sponse of CD81 T cells using HIV-1 peptides representingknown HLA A*02 CTL epitopes in the subset of six HLAA*02 patients in this cohort. Some but not all of these peptideswere recognized by CD81 T cells during the combined anti-retroviral therapy. Thus, high, persistent numbers of IFN-g-producing, CD81 cells were induced during triple-drug ther-

FIG. 1. Effect of treatment with IDV (I), ZDV (Z), and 3TC (L) initiated simultaneously for seven individually numbered group A patients on serum HIV-1 RNAand T-cell numbers (top rows), anti-HIV-1 CTLp (middle rows), and IFN-g-producing CD81 cells (bottom rows). Data on IFN-g production were not available frompatients A1155 and A1177. Longitudinal data are given for MACS participant A1166 from seroconversion (SC) through the Merck 035 drug trial.


apy by Gag p1777–85 in three of five patients tested (A1166,A1169, and B1178), by Pol RT476–484 in two of five patients(A1166 and C1180), by Env gp120192–199 in two of three pa-tients (A1166 and B1178), and by Gag p24151–159 in one of thethree patients (C1180) (Fig. 3).

The ability of the CD81 T cells to produce IFN-g to theseCTL epitopes was not directly associated with the suppressionof virus. This was demonstrated by the lack of any detectableIFN-g response to the A*02 peptides in patient A1160 (Fig. 3),who had prolonged suppression of HIV-1 (Fig. 1), and therelatively robust IFN-g activity in C1180 (Fig. 3), whose viruswas not completely suppressed (Fig. 1).

The number of CD81 T cells producing IFN-g in responseto Gag p1777–85 peptide was higher than that for the PolRT476–484 peptide in these patients (Gag p1777–85 5 1,085 6419; Pol RT476–484 5 165 6 48; n 5 52; P 5 0.03). IFN-greactivity to the Gag p1777–85 peptide, but not to the PolRT476–484 peptide, correlated with the IFN-g and CTLp re-sponses to the corresponding HIV-1 proteins (Table 3). Therewere insufficient data for such comparisons of different types ofT-cell reactivity to the other HIV-1 peptides.

Specificity of CD81 T cells for HIV-1 peptides during com-bination antiretroviral therapy as assessed by HLA tetramerstaining. We compared CD81 T-cell CTLp and IFN-g re-sponses to the number of CD81 T cells expressing T-cell re-ceptors for HIV-1 Gag p1777–85 and Pol RT476–484 by stainingwith HLA A*02 tetramers in the subset of six HLA A*02patients. The levels of Gag p1777–85 tetramer staining in the sixpatients were higher than those for Pol RT476–484 (mean 6 theSE 5 8,236 6 947 and 3,402 6 280 per 106 CD81 T cells,respectively; n 5 69; P , 1025). These levels were greater thanthe number of IFN-g-producing, CD81 cells induced by thecorresponding peptides (IFN-g for Gag p1777–85 5 1,085 6419 and for Pol RT476–484 5 165 6 48; n 5 52; P , 1028 forboth compared to the tetramer levels). The number of CD81

cells producing IFN-g in response to the Gag p1777–85 and PolRT476–484 peptides, however, correlated with those stainingwith the matching tetramers (Table 3). Similar associationswere noted for the Gag p1777–85 and RT476–484 tetramers andthe number of CTLp and IFN-g producing, CD81 cells stim-ulated by p55 Gag and Pol.

The median of the baseline levels before triple-drug treat-

FIG. 2. Effect of treatment with IDV (I), ZDV (Z), and 3TC (L) given sequentially after treatment with ZDV-3TC (group B) or IDV (group C) for threeindividually numbered group B patients and four group C patients on serum HIV-1 RNA and T-cell numbers (top rows), anti-HIV-1 CTLp (middle rows), and IFN-gproducing CD81 cells (bottom rows). Data on IFN-g production were not available from patient B1171. Longitudinal data are given for MACS participants B1178 andC1158 from seroconversion (SC) through the Merck 035 drug trial.


ment for the six patients was 8,900 (range, 2,400 to 16,300) per106 CD81 cells for the Gag tetramer and 3,300 (range, 1,000 to7,500) per 106 CD81 cells for the Pol tetramer. The number ofCD81 cells staining with the p17 tetramer initially declined andthen stabilized in the first 8 to 16 weeks of triple combinationtherapy in patients A1160, A1169, and B1178 and were rela-tively stable from the onset of the trial in patients A1166,C1164, and C1180 (Fig. 3). This was also reflected in therelatively stable number of CD81 CD45RO1 cells stainingwith the tetramers (Fig. 4). Notably, patient A1160 had thelowest numbers of tetramer-positive cells; these levels werejust above the level of detection (200 per 106 cells, or 0.02%)after the initial decline.

Longitudinal anti-HIV-1 CD81 T-cell responses from sero-conversion through combination antiretroviral therapy. Threeof the patients receiving either the triple-drug therapy simul-taneously at baseline (A1166) or the three-drug combinationsequentially (B1178 and C1158) were also part of the MACS.This provided an unusual opportunity to assess anti-HIV-1CD81 T-cell responses in relation to the complete course ofHIV-1 infection from seroconversion through different coursesof antiretroviral therapy. None of the HIV-1- or CMV-specificT-cell functional parameters correlated with changes in T-cellnumbers or HIV-1 RNA load during the MACS study (datanot shown). However, there were strong correlations betweenthe number of anti-HIV-1 and anti-CMV IFN-g-producingCD81 cells in these three MACS participants prior to theMerck 035 trial (Fig. 5).

Patient A1166 had a progressive increase in numbers ofCTLp and IFN-g-producing CD81 cells specific for Gag, Pol,and Env and for IFN-g production to CMV, from seroconver-sion to approximately 2 years after infection, associated with aconsistently high level of HIV-1 RNA (Fig. 1). There was asimilar increase in IFN-g-producing cells in response to thep17, RT, and gp120 peptides, whereas the number of tetramer-positive cells was relatively high and stable during this time(Fig. 3). By 4.4 years after infection, when the patient beganthis drug treatment trial, the numbers of CD81 T cells specificfor HIV-1 proteins and CMV had declined severalfold. After

placement on triple-drug therapy, the viral load decreased to,500 copies by 4 weeks. The number of anti-HIV-1 and anti-CMV CD81 cells increased concurrently in 24 weeks of com-bination antiretroviral therapy to numbers similar to the peakpretreatment level. These T-cell functions eventually de-creased, however, to undetectable levels by 114 weeks of triple-drug therapy, while the virus remained suppressed.

Patient B1178 had a more prolonged delay in developingCTLp specific for HIV-1 proteins after seroconversion (Fig. 2).In contrast, there was a robust IFN-g response by 0.8 yearsafter seroconversion to all three HIV-1 proteins and CMV(Fig. 2), as well as to the p17 peptide (400 spots/106 CD81

cells), with concurrently higher levels of p17 tetramer-positivecells (10,700/106 CD81 cells, or 1.07%) (Fig. 3). These T-cellparameters declined by 8 years after infection. There were noCD81 IFN-g responses to the p24, RT, or Env peptides, al-though there were low numbers of Pol RT476–484 tetramer-positive cells maintained for years after seroconversion. Therewas a sharp, transient rise in anti-HIV-1 and CMV CD81

T-cell reactivity in the 40 weeks of the dual-drug therapy,during which time the viral load rebounded to high, baselinelevels. T-cell reactivity to HIV-1 proteins and CMV recoveredafter the switch to triple-drug therapy, with suppression of theviral load, but most of these immune parameters decreased by132 weeks.

Patient C1158 developed CTLp against Gag and Env soonafter seroconversion but subsequently had very low or unde-tectable numbers of CTLp precursors in the 5 years prior toentry into this drug therapy trial (Fig. 2). Interestingly, therewere high levels of IFN-g-producing CD81 cells early afterseroconversion to all three HIV-1 proteins and CMV thatdeclined only by the start of this drug trial. This patient alsomaintained a high viral load during the 5 years of infectionbefore the treatment trial, except for a transient decline duringZDV monotherapy. This participant showed an excellent viro-logic response to treatment with IDV alone, with suppressionby 12 weeks in the trial. There were variable, fluctuating levelsof CD81 T-cell responses to HIV-1 proteins and CMV antigenduring the pre-open-label period and during the triple-drugtreatment, even though the virus load remained suppressed.

Alteration in T-cell lineage and activation phenotypes bycombination drug therapy. We next determined whetheranti-HIV-1 CD81 cell reactivity was related to changes inCD81 memory and activated T cells. The percentage of CD81

CD45RO1 memory T cells decreased and that of CD81

CD45RA1 naive T cells increased during the first severalmonths of treatment in the group A patients and then stabi-lized (Fig. 6A). A similar pattern was noted for CD41

CD45RO1 memory T cells and CD41 CD45RA1 naive T cellsin these patients (data not shown). Group A patients alsoshowed decreases in activated CD81 CD381 HLA-DR1 cellsthat correlated with decreases in HIV-1 RNA (r 5 0.63, P 50.006), increases in CD41 cells (r 5 20.65, P , 0.001), anddecreases in CD81 CD282 cells (r 5 0.55, P 5 0.001).

Group B and C patients showed changes in T-cell subpopu-lations when switched to triple-drug therapy that were differentfrom those in group A. Thus, there was a rise in memory andactivated CD81 T cells and a concurrent decrease in naiveCD81 T cells in group B patients after the start of combinationantiretroviral therapy (Fig. 6B). There was little change fromthe baseline level in the memory and naive CD81 T cells ingroup C patients, whereas there were decreases in activatedCD81 T cells during triple-drug therapy (Fig. 6C). This may berelated to the heterogeneous effects of the triple combinationantiretroviral therapy on their viral load, i.e., one of three

TABLE 2. Correlations between CD81 T-cell reactivities to HIV-1proteins and various T-cell and viral parameters in group A patients

on combination antiretroviral therapy

Test combination Duration(wk) Antigen

Parameter

Test 1 Test 2 na rb P

CTLp IFN-g 0–128 Gag 45 0.48 ,0.01Pol 45 0.45 ,0.01Env 45 0.61 ,0.01

HIV-1 RNA 0–12 Gag 25 20.41 ,0.05Pol 25 20.47 ,0.05Env 25 20.52 ,0.05

IFN-g CD41 T cells 0–12 Gag 17 20.18 NSc

Pol 17 0.68 ,0.05Env 17 20.27 NSCMV 12 0.87 ,0.01

CD81 T cells 0–12 Gag 17 20.13 NSPol 17 0.61 ,0.05Env 17 0.36 NSCMV 12 0.38 NS

a n, number of determinations.b Pearson correlation coefficient.c NS, not significant (P . 0.05).


group B patients and two of four group C patients tested didnot show complete suppression of HIV-1 RNA (Fig. 2).

We found no correlation of anti-HIV-1 CTLp or IFN-g-producing cell numbers with the percentages of these CD81

T-cell subpopulations (data not shown). It should be noted,however, that the HIV-1-stimulated, cultured cells used in theCTLp assessments were .80% CD81 CD381 HLA-DR1 andCD81 CD282 regardless of the patients’ treatment group orlevels of anti-HIV-1 CTLp and IFN-g-producing cells. More-over, there was no difference in the growth of the cells in theCTLp cultures among the three groups of patients throughoutthe 2 years of followup (e.g., mean increases [6 the SE] in cellnumbers from an initial 25,000 cells per culture were 10.9[61.3]-, 16.8 [60.7]-, and 14.8 [64.1]-fold in groups A, B, andC, respectively, during the first 36 weeks of treatment; P .0.05).

DISCUSSION

This study provides evidence that triple-drug antiretroviraltherapy (IDV plus 3TC plus ZDV) fails to produce a sustainedincrease in anti-HIV-1 CD81 T-cell functions in HIV-1-in-fected patients with advanced immunodeficiency. There weretwo basic patterns of HIV-1-specific T-cell reactivity after com-bination antiretroviral therapy in the 14 patients in our study.One was an early rise from very low pretreatment levels in

anti-HIV-1 CTLp and IFNg-producing, CD81 cells specific forHIV-1 Gag, Pol, and Env during the triple-drug therapy. Thisresponse, however, declined to baseline levels in most of thesepatients by 2 years. This pattern is similar to the temporaryenhancing effect of combination therapy on anti-HIV-1 CTLpin two late-stage patients recently described by Kalams et al.(21). The second pattern of anti-HIV-1 CD81 T-cell responsesthat we observed was a failure of CTLp and IFN-g reactivity toincrease above the low baseline levels throughout the 2 yearsof triple-drug therapy. It is possible that we missed brief, tem-porary rises of anti-HIV-1 T-cell activity in these patients dur-ing the time interval between test samples. However, Pontesilliet al. (37) have also noted that some late-stage patients onseveral different combination therapies for 1 year do not re-cover anti-HIV-1 CTLp reactivity.

Several factors could be related to the changes in anti-HIV-1CD81 T-cell reactivity after combination antiretroviral ther-apy. Group A patients, who initiated the triple-drug combina-tion from the study onset, had a strong correlation early intreatment between an increase in anti-HIV-1 CTLp to all threeHIV-1 proteins and decreases in viral load. Interestingly, anincrease in the number of IFN-g-producing CD81 cells specificfor Pol in the group A patients correlated with an increase inthe number of CD41 and CD81 T cells but not with a decreasein viral load. These correlations were not evident in group B

FIG. 3. Effect of treatment with IDV, ZDV, and 3TC given simultaneously (patients A1166, A1160, and A1169) or sequentially (patients B1178, C1164, and C1180)on the number of IFN-g-producing CD81 cells reactive to HLA A*02 HIV-1 peptides and the number of HLA A*02 tetramer-HIV-1 peptide staining CD81 cells.


and C patients, who initiated treatment with the three drugssequentially. The data suggest that CTLp and IFN-g CD81 cellfunctions, while related, are not identical. Thus, the ability ofCD81 T cells to produce IFN-g in response to HIV-1 antigensmay be a better correlate of recovery of T-cell immunity duringcombination therapy than the number of anti-HIV-1 CTLp.The data also suggest that sequential initiation of the threedrugs, which is known to promote antiviral drug resistance(16), has less restorative effect on functional, anti-HIV-1CD81 cell reactivity than does initiating the three drugs simul-taneously.

The results indicate that the early rise in anti-HIV-1 CD81

T-cell responses in the subgroup of our patients represents atrue, albeit transient, enhancement of CD81 cell function. Itdoes not appear to be simply a result of redistribution ofpreexisting populations of these CD81 memory cells from ex-travascular spaces during therapy (35). That is, although weobserved a decrease in the numbers of memory, CD81

CD45RO1 cells and activated, CD81 CD381 HLA DR1 andCD81 CD282 cells in many of these patients, which includeanti-HIV-1 CTL effectors (12, 18), this did not correlate withchanges in the number of CD81 CTLp or IFN-g-producingcells. Alternatively, the enhancement in anti-HIV-1 CTLpcould have been due to a selective increase in the growthpotential of circulating CD81 T cells in response to HIV-1antigen-presenting cells during combination therapy. This wasnot the case, however, as there was a similar, temporary aug-mentation in HIV-1-specific, IFN-g-producing CD81 T cells inthese patients that is not dependent on cell replication. More-over, there was no difference in outgrowth of CD81 cells inCTLp cultures from patients with or without temporal in-creases in this anti-HIV-1 CD81 T-cell function.

The longitudinal data in the three MACS participants fromseroconversion suggest that persons who develop the strongestand most persistent CD81 T-cell reactivity to HIV-1 afterinfection have the best recovery of such immunologic functions

FIG. 4. Relationship of the number of CD81 CD45RO1 cells binding HLA A*02 tetramers with the duration of triple-combination therapy (mean 6 the SE). Thedata represent a median of four (range, two to six) determinations at each time point for the six patients from Fig. 3.

TABLE 3. Correlations between CD81 T-cell reactivities to HIV-1 peptides in six HLA A*02 group A, B, and C patients during combinationantiretroviral therapy

Test combination Parameter

Test 1 Test 2 na rb P

IFN-g for Gag p1777–85 IFN-g for VVgag p55 53 0.50 ,0.01CTLp for VVgag p55 44 0.33 ,0.05Tetramer for Gag p1777–85 50 0.76 ,0.01

IFN-g for Pol RT476–484 IFN-g for VVpol 50 0.27 NSc

CTLp for VVpol 41 0.24 NSTetramer for Pol RT476–484 51 0.48 ,0.01

Tetramer for Gag p1777–85 CTLp for VVgag p55 52 0.67 ,0.01IFN-g for VVgag p55 59 0.39 ,0.01

Tetramer for Pol RT476–484 CTLp for VVpol 51 0.31 ,0.05IFN-g for VVpol 61 0.09 NS

a n, number of determinations.b Pearson correlation coefficient.c NS, not significant (P . 0.05).


after combination antiretroviral therapy. Both MACS partici-pants with persistently high numbers of CTLp and IFN-g-producing CD81 cells in response to the three HIV-1 proteinsprior to the drug trial (A1166 and B1178) recovered similarlevels of this T-cell reactivity for a prolonged period afterreceiving the triple-drug combination. The third patient(C1158) failed to develop consistently high numbers of CTLpspecific for any of the HIV-1 proteins before or after treat-ment. He did, however, maintain relatively high numbers ofIFN-g-producing CD81 cells specific for HIV-1 during theMACS study that also reached high levels after triple-drugtreatment.

An important finding of this study is that changes in CD81

T-cell reactivity were not restricted to anti-HIV-1 responses.The results from the three MACS patients were most illustra-tive of this where there were concurrent increases and eventualdeclines in both HIV-1-specific and CMV-specific, IFN-g-pro-ducing CD81 cells after seroconversion and during the drugtrial. We have observed a similar pattern of slow increase ofanti-HIV-1 CD81 CTL after seroconversion, together with apersistently high HIV-1 load in about 75% of MACS subjectstested to date (43). However, the concurrent rise and fall inanti-HIV-1 and anti-CMV CD81 T-cell reactivity during thenatural progression of HIV-1 infection was unexpected. Itcould be a response to an increased burden of CMV antigenwith progressive immunosuppression, although major eleva-tions in systemic CMV load only appear late in HIV-1 infec-tion (39). Alternatively, it is possible that the increases in thenumber of anti-CMV CD81 T cells are due to a bystandereffect where these cells are signaled to expand by cytokines

produced in response to other, possibly HIV-1-specific stimuli(48). The present study suggests that these antiviral CD81

memory T cells are under similar homeostatic control duringboth the natural progression of HIV-1 infection and afterhighly viral suppressive, triple-drug therapy.

We found a limited breadth of CD81 T-cell responses tofour HLA A*02 peptides representing known CTL epitopes inpatients receiving combination therapy. The IFN-g response tothe Gag p1777–85 peptide, one of the most widely recognizedHLA A*02 HIV-1 epitopes (49), was present throughout treat-ment in three patients but was completely absent in the othertwo patients tested. Likewise, T-cell IFN-g reactivity to HLAA*02 epitope Pol RT476–484 was found in only two of fivepatients after combination therapy. The patients also had het-erogenous IFN-g responses to the Gag p24151–159 and Envgp120192–199 HLA A*02 epitopes. This restriction in T-cellspecificity to certain HIV-1 peptides could be related to thepersistence of oligoclonal CD81 T-cell repertoire perturba-tions after treatment (14). In support of this, we found that aselective T-cell reactivity to these known HLA A*02 epitopeswas present throughout the natural history of HIV-1 infectionprior to initiation of triple-drug therapy in the two MACSHLA A*02 patients. Dalod and colleagues (7) have also re-cently shown that restricted CD81 T cell responses to HIV-1peptides persist during natural HIV-1 infection. Our data sug-gest that dominance of T cell epitopes during natural HIV-1infection is not broadened during combination therapy.

We postulate that the late decline in numbers of functional,HIV-1 specific, CD81 T cells during triple combination drugtherapy may be a normal homeostatic response to the persis-tent reductions in HIV-1 antigen. Similar low levels of anti-HIV-1 CTLp have been found in some long-term nonprogres-sors with a very low viral load (23, 38, 43). This finding isdistinct from the loss of anti-HIV-1 CD81 T cells that occursin progressive HIV-1 infection associated with a high viralburden and low CD41 T-cell numbers, as shown in our threeMACS subjects and in other studies (23, 38, 43). Differentmechanisms may underlie this loss in progressive disease, in-cluding T-cell clonal deletion and anergy (28), that are onlypartially reversed during triple-combination drug therapy.

A second basis for the loss of functional anti-HIV-1 CD81 Tcells during treatment with the three-drug combination may bethe lack of recovery of anti-HIV-1 CD41 T-cell responses, aswe have noted in these same trials (45). This notion is sup-ported by reports that long-term maintenance of high levels ofother types of antiviral CD81 CTL requires CD41 T-cell help(26, 42). High CD41 T-cell reactivity to HIV-1 has also beenrelated to lower HIV-1 load in long-term nonprogressors andin patients with early HIV-1 infection who are treated withcombination therapies (20, 22, 46).

The intensity of the T-cell response was also determined bystaining the CD81 cells with HLA A*02 Gag p1777–85 and PolRT476–484 tetramers. The numbers of CD81 cells binding theGag tetramer were higher than for the Pol tetramer, as previ-ously reported (30). Our data show that the tetramer stainingand single-cell IFN-g responses to these peptides were corre-lated but were not identical parameters of immunity. Of inter-est is that there was persistent reactivity of CD81 T cells tothese two tetramers in the two MACS participants in the yearsprior to the trial. These data contrast with a recent report thatlower levels of tetramer-binding CD81 cells are associatedwith disease progression in HIV-1-infected persons not receiv-ing combination therapy (32). As noted in other studies (15, 30,31), we found an early decrease in tetramer staining in somepatients during combination therapy. However, like the twopatients recently studied by Kalams et al. (21), most of the

FIG. 5. Correlations between the IFN-g responses to CMV and HIV-1 Gag,Pol, and Env in the three MACS participants (A1166, B1178, and C1158) afterseroconversion and prior to entry into the Merck 035 trial.


patients on triple-drug therapy in our study maintained rela-tively stable levels of tetramer-positive CD81 cells, includingCD45RO1 memory T cells. This population of CD81

CD45RO1 cells may be an important, residual source of mem-ory T cells specific for HIV-1 in patients on combination ther-apy.

It is known that HIV-1 can break through combination ther-apy resulting in viral load rebound (16) and that latent viruscan persist in resting CD41 T cells and be reactivated (6, 10,11, 53). Our study and others (34, 40) suggest that strategies toincrease T-cell function will be required as adjunct therapy torestore anti-HIV-1 immunity and better control HIV-1 infec-tion. In this regard, we have found that HIV-1-specific, CD81

T cells can be induced in vitro by multiple, repetitive stimula-tions with IL-12 and dendritic cells loaded with HIV-1 proteins(55; Z. Fan, X. Huang, and C. R. Rinaldo, Jr., unpublishedresults). This induction is possible with samples from patientswho have not responded to stimulation with our conventional,VV–HIV-1 and B-LCL system (Fan et al., unpublished). This,together with our present finding of residual CD81 T cellsspecific for HIV-1 immunodominant peptides, suggests thatcompetent T cells specific for HIV-1 are still present afterprolonged combination therapy. Such cells offer a target forimmunomodulation in vivo that could improve the host’s ca-pacity to control HIV-1 infection and enhance the effectivenessof antiretroviral therapy.

ACKNOWLEDGMENTS

We thank A. Zeevi and R. Kaslow for portions of the HLA typing;J. Liebmann, A. Bazmi, E. Molina, S. McQuiston, B. Colleton, H. Li,M. Tseng, W. Jiang, Q. Al-Shboul, and E. Ramirez for technicalassistance; and T. Silvestre, W. Buchanan, L. Johnson, N. Mantz, J.Stewart, A. Sparks, and R. Kudray for clinical assistance.

This project was supported by National Institutes of Health grants

R01-AI41870, U01-AI37984, U01-AI35041, and T32-AI07487 and by agrant from the Merck Research Laboratories.

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Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+ T-Lymphocyte Reactivity during Combination Antiretroviral Therapy in HIV-1-Infected Patients with Advanced Immunodeficiency

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