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HIV-Infected Individuals T Cells in+Expansion of Effector CD8

Loss of CD127 Expression Defines an

SilvestriAltman, Donald L. Sodora, Mark B. Feinberg and GuidoMaria Montroni, Susan M. Kaech, Amy Weintrob, John D. Henry Radziewicz, Giuseppe Piedimonte, Mauro Magnani,Alagarraju Muthukumar, Richard Dunham, Shari Gordon, Mirko Paiardini, Barbara Cervasi, Helmut Albrecht,

http://www.jimmunol.org/content/174/5/2900doi: 10.4049/jimmunol.174.5.2900

2005; 174:2900-2909; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Loss of CD127 Expression Defines an Expansion of EffectorCD8� T Cells in HIV-Infected Individuals1

Mirko Paiardini,2*‡§ Barbara Cervasi,2*‡� Helmut Albrecht,* Alagarraju Muthukumar,¶

Richard Dunham,*‡ Shari Gordon,*‡ Henry Radziewicz,*‡ Giuseppe Piedimonte,�

Mauro Magnani,§ Maria Montroni,# Susan M. Kaech,†‡ Amy Weintrob,* John D. Altman,†‡

Donald L. Sodora,¶ Mark B. Feinberg,*†‡ and Guido Silvestri3*‡

The immunodeficiency that follows HIV infection is related to the virus-mediated killing of infected CD4� T cells, the chronicactivation of the immune system, and the impairment of T cell production. In this study we show that in HIV-infected individualsthe loss of IL-7R (CD127) expression defines the expansion of a subset of CD8� T cells, specific for HIV as well as other Ags, thatshow phenotypic (i.e., loss of CCR7 and CD62 ligand expression with enrichment in activated and/or proliferating cells) as wellas functional (i.e., production of IFN-�, but not IL-2, decreased ex vivo proliferative potential and increased susceptibility toapoptosis) features of effector T cells. Importantly, in HIV-infected individuals the levels of CD8�CD127� T cells are directlycorrelated with the main markers of disease progression (i.e., plasma viremia and CD4� T cell depletion) as well as with the indicesof overall T cell activation. In all, these results identify the expansion of CD8�CD127� effector-like T cells as a novel feature ofthe HIV-associated immune perturbation. Further studies are thus warranted to determine whether measurements of CD127expression on CD8� T cells may be useful in the clinical management of HIV-infected individuals. The Journal of Immunology,2005, 174: 2900–2909.

N aive CD8� T (TN)4 cells that encounter their cognateAg undergo a complex process of maturation and dif-ferentiation that ultimately leads to the generation of a

sizeable pool of long-lived memory CD8� T (TM) cells that me-diate immune protection from subsequent challenge with the sameAg (reviewed in Ref. 1). A series of elegant studies in the murinemodel of lymphocytic choriomeningitis virus infection show thatAg-experienced TN cells differentiate first into short-lived, rapidlyproliferating effector CD8� T (TE) cells, which express a numberof activation markers (i.e., CD25, CD44), have down-regulated thelymph node homing receptors (i.e., CD62L, CCR7), and displaystrong immediate effector function (i.e., IFN-� production andCTL activity) (2–4). Within a few weeks a subset of these TE cellsfurther differentiate into long-lived, slowly proliferating memoryCD8� T (TM) cells, which re-express CD62 ligand (CD62L) andCCR7, show limited immediate effector function, but exhibit a

high proliferative potential upon antigenic restimulation (2–4).Adoptive transfer experiments indicate that Ag-specific TM cellsare more effective than TE cells in mediating protection from chal-lenge (2–4).

In humans, TM cell differentiation is likely regulated in a dif-ferent and more complex manner, with specific features of TM celldifferentiation that may vary following infection with differentpathogens, such as HIV, EBV, CMV, and hepatitis C virus (5–10).Essentially, two main models of TM cell differentiation in humanshave been proposed. In the first model, Ag-specific CD8� T cellsdifferentiate from CCR7�CD62L�CD45RA� TN cells to CCR7�

CD62L�CD45RA� central memory T (TCM) cells, then to CCR7�

CD62L�CD45RA� effector memory (TEM) cells and finally toCCR7�CD62L�CD45RA� TEM cells (TEMRA or terminallydifferentiated effectors) (5–8). In the second model, CD27high

CD28� TN cells differentiate into early (i.e., CD27�CD28�), in-termediate (CD27�CD28�), and late (CD27�CD28�) memory Tcells (9–10). Above and beyond the use of distinct sets of markersto define specific subsets of TM cells, these two models differ inthat the first describes T cell differentiation as a linear process,while the second allows for branches in cell lineage (5–10).

HIV infection is characterized by progressive CD4� T cell de-pletion and immunodeficiency that paradoxically occur in the con-text of a chronic state of immune system activation. Increasingevidence suggests that the generalized immune activation that fol-lows HIV infection plays a prominent, if not predominant, role inthe pathogenesis of AIDS (11–15) and that the extent of this im-mune activation is as good, or perhaps even better, a predictor ofdisease progression than the level of viral replication (16–21).These observations are consistent with the report that, in the non-pathogenic SIV infection of sooty mangabeys (a natural host spe-cies for SIV infection), minimal levels of immune activation andno evidence of immunodeficiency are observed despite chronichigh levels of virus replication (22). However, the mechanisms bywhich chronic immune activation precipitates the development of

Departments of *Medicine and †Microbiology and Immunology, and ‡Emory VaccineCenter, Emory University School of Medicine, Atlanta, GA 30322; §Department ofBiochemistry, University of Urbino, Urbino, Italy; ¶Division of Infectious Diseases,Department of Internal Medicine, University of Texas Southwestern Medical Center,Dallas, TX 75390; �Department of Public Health, University of Messina, Messina,Italy; and #Department of Internal Medicine, University Politecnica delle Marche,Ancona, Italy

Received for publication May 26, 2004. Accepted for publication November21, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants R01-AI52755 (toG.S.), R01-AI049155 (to M.B.F.), and AI35522 (to D.L.S.).2 M.P. and B.C. have contributed equally to this work.3 Address correspondence and reprint requests to Dr. Guido Silvestri, Emory VaccineCenter, Emory University School of Medicine, 954 Gatewood Road NE, Atlanta, GA30329. E-mail address: gsilves@rmy.emory.edu4 Abbreviations used in this paper: TN, naive CD8� T cell; TE, effector CD8� T cell;TM, memory CD8� T cell; TCM, central memory CD8� T cell; TEM; effectormemory CD8� T cell; HAART, highly active antiretroviral therapy.

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immune system compromise and susceptibility to opportunistic in-fections following HIV infection are still poorly understood. Thislack of understanding is partly related to the fact that the functionalrole of the markers of T cell activation that are associated withHIV disease progression, such as the expression of CD38 andHLA-DR on CD8� T cells, is still largely unknown.

Recent evidence indicates that expression of IL-7R �-chain(CD127) on lymphocytic choriomeningitis virus-specific CD8� Tcells during the generation of primary antiviral immune responsesidentifies a subset of TE cells that differentiate successfully intofully protective TM cells (4). In analogy with this model, we hy-pothesized that a determinant of the chronic immune activationobserved in HIV-infected individuals is the expansion of activat-ed/TE cells that do not express of CD127 and thus fail to properlydifferentiate in TM cells. In addition, given the known role of IL-7in regulating T cell homeostasis, a dysregulation of the IL-7/IL-7Rsystem may also support a pathogenic model in which the HIV-associated CD4� T cell depletion is associated with impaired Tcell production (23). We report that HIV infection is consistentlyassociated with the expansion of CD8�CD127� T cells (specificfor HIV as well as for other Ags) that show immunophenotypicand functional features of TE cells, and that the levels ofCD8�CD127� T cells in HIV-infected patients correlate withmarkers of disease progression (i.e., plasma viremia and CD4� Tcell depletion) as well as with the overall levels of immune systemactivation. We propose that in HIV-infected individuals thechronic expansion of CD8�CD127� TE-like cells may be both adeterminant and a consequence of the heightened levels of immuneactivation and bystander apoptosis that are associated with CD4�

T cell depletion and progression to AIDS.

Materials and MethodsPatient population

All subjects included in this study were enrolled at the HIV Infection Clinicof the Crawford and Long Hospital, the Hope Clinic of the Emory VaccineCenter, and the Grady Infectious Disease Program (all affiliated withEmory University, Atlanta, GA), or at the Service of Clinical Immunology,University of Ancona (Ancona, Italy). Individuals included in this studywere: 1) HIV-1-infected therapy-naive adults with CD4� T cell counts�50 per �l and levels of plasma viremia �5000 HIV-1 RNA copies/ml; 2)HIV-1-infected adults treated with highly active antiretroviral therapy(HAART; at least 2 reverse transcriptase inhibitors and 1 protease inhib-itor) for at least 6 mo and with �400 HIV-1 RNA copies/ml; and 3)healthy, age-matched HIV-uninfected controls. The clinical, immunologic,and virologic characteristics of the studied patients are displayed in TableI. These studies were approved by the Institutional Review Board of EmoryUniversity and all participants gave written informed consent.

FACS analysis and sorting

PBMCs were isolated from whole blood by density gradient centrifugationusing standard procedures. In some cases purified PBMCs that had beencryopreserved in liquid nitrogen were thawed before analysis; in thesecases the staining involved using Abs directed against CD3, CD8, CD127,and HLA class I tetramers only. PBMCs were analyzed by four-color flu-orescent Ab staining to determine the percentage and absolute number ofspecific cell subpopulations (calculated from the total white blood cellcount and the percentage of lymphocytes in the white blood cell differen-tial). Intracellular and surface staining were performed using the followingAbs: anti-CD4 PerCP, anti-CD8 PerCP, anti-CD62L FITC, anti-CCR5 Cy-Chrome, anti-CXCR4 CyChrome, anti-CD45RO FITC, anti-HLA-DR al-lophycocyanin, anti-CD95 allophycocyanin, anti-Ki-67 FITC (all from BDPharmingen), anti-CD45RA TriColor (Caltag Laboratories), anti-CD127-PE (Corixa). Isotype-matched controls were used in all experi-ments. To stain for CCR7 we used the allophycocyanin-labeled tetramericMIP3� complex (24). Flow cytometric acquisition and analysis of sampleswere performed on at least 10,000 acquired events, gated on lymphocytes,on a FACSCalibur flow cytometer driven by the CellQuest software pack-age (BD Pharmingen). Data analyses were performed using the FlowJosoftware (TreeStar). Cell sorting experiments were performed on a Mo-Flow (Cytomation).

Tetrameric HLA molecules

Tetrameric HLA class I molecules were produced as previously described(25). Prokaryotic expression vectors encoding the extracellular portion ofthe HLA-A2*0201 and �2-microglobulin molecules (tagged to a birArecognition sequence) were separately expressed in Escherichia coliafter isopropyl �-D-thiogalactoside-induction and isolated from inclu-sion bodies. HLA-A*0201 and �2-microglobulin chains were folded inthe presence of the appropriate peptides (HIVgag-p17SLYNTVATL,CMVpp65NLVPMVATV, EBVbmfl-1GLCTLVAML). Folded trimolecu-lar complexes were biotinylated with BirA enzyme and FPLC purified ona MonoQ column. PE-streptavidin was finally mixed with biotinylatedmonomers to obtain the tetrameric HLA complex.

In vitro cytokine production

Intracellular cytokine production by PBMCs was assessed by flow cytom-etry. FITC-conjugated Abs against human IL-2, IFN-�, and TNF-� (allfrom BD Pharmingen) were used, and staining with a pool of appropriate

Table I. Clinical, immunologic, and virologic characteristics of thestudied patients

CodeAge(y) Sex

CD4�

T Cell Viral Load HAART

1 38 M 389 100,000 No2 57 M 111 43,400 Yes3 41 M 286 289 Yes4 43 M 454 110 Yes5 46 M 450 291,000 No6 52 M 431 370 Yes7 38 M 427 85,300 No8 47 M 1250 �50 Yes9 63 M 92 100,000 No

10 31 M 385 200 Yes11 46 F 237 �50 Yes12 56 M 260 460 Yes13 60 M 400 333 Yes14 36 M 464 46,700 No15 40 M 282 �50 Yes16 36 M 332 30,100 No17 57 M 393 �50 Yes18 29 M 325 234,000 No19 39 M 392 15,500 No20 26 M 597 1300 No21 36 M 695 8600 Yes22 41 M 603 400 Yes23 29 M 312 85,500 No24 40 M 363 82,200 No25 49 M 372 502,000 No26 46 M 182 675,000 No27 45 F 586 2,100 No28 41 M 200 148,000 No29 45 M 190 88,800 No30 35 M 144 154,000 No31 34 F 600 750 No32 38 F 304 15,000 Yes33 43 F 325 74,900 No34 22 M 314 110,000 No35 35 M 179 �50 Yes36 32 M 765 16,663 No37 31 M 350 891 Yes38 34 F 195 143,000 No39 45 M 631 91,960 No40 31 F 338 10,715 Yes41 33 M 213 102,329 No42 41 M 88 400 No43 36 M 580 400 Yes44 38 M 470 60 No45 39 M 434 31,486 No46 37 M 306 150 No47 64 F 578 1,235 Yes48 29 F 637 7,033 Yes49 60 M 479 3,656 Yes50 44 F 495 8,106 No51 40 M 140 �50 Yes

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FIGURE 1. Decreased CD127 expression on TM (i.e., non-naive) cells from HIV-infected patients correlates with disease progression and immuneactivation, but not with prevailing IL-7 plasma levels. A, Mean expression of CD127 (IL-7R �-chain) on total, memory, and naive CD4� and CD8� T cells.Naive cells are defined as CD45RA�CD62L�, and all non-naive cells are considered memory. The analysis was performed on 18 untreated HIV-infectedpatients (f), 13 HAART-treated (u), and 10 healthy controls (�). Statistical analyses were performed between controls and untreated HIV-infectedpatients, and between HIV-infected patients (untreated vs HAART-treated). B, Representative dot plots showing the expression of (Figure legend continues)

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isotype-matched Abs was included as a negative control. PBMCs wereincubated for 4 h in medium containing phorbol 12-myristate 13-acetate(10 ng/ml), the calcium ionophore A23187 (200 ng/ml), and either mo-nensin (10 nM) or brefeldin A (5 nM) as inhibitors of Golgi transport (allfrom Sigma-Aldrich). Cells were first surface stained with anti-CD4 allo-phycocyanin, anti-CD8 PerCP, and anti-CD127 PE mAbs, and then fixedand permeabilized using the CytoFix/CytoPerm kit (BD Pharmingen).

In vitro lymphocyte studies

Studies of the expression of CD127 after ex vivo stimulation were per-formed on PBMCs treated either with rIL-7 (10 ng/ml; BD Pharmingen) orPHA (10 �g/ml; Sigma-Aldrich). Staining for CD127 was performed at 7,22, and 72 h posttreatment. In vitro proliferation after treatment with PHAwas determined in sorted CD8�CD127� and CD8�CD127� cells usingCFSE labeling. The fraction of proliferating cells was measured by flowcytometry at 72 and 120 h postactivation. Studies of spontaneous and ac-tivation-induced apoptosis were performed in CD127� and CD127� Tcells after a 48-h incubation either with no stimulus (spontaneous apopto-sis) or with PHA (activation-induced apoptosis). Rates of apoptotic cellswere determined by flow cytometry as a percentage of cells reactive toAnnexin VFITC (BD Pharmingen).

IL-7 plasma levels

IL-7 levels in the plasma of HIV-infected patients and controls were esti-mated using the commercial sandwich enzyme immunoassay kit (R&DSystems). Anticoagulants other than heparin, which may interfere withplasma IL-7 measurements, were used at the time of blood collection. Inpreliminary experiments the specificity of the IL-7 ELISA was ascertainedby blocking with anti-IL-7 antisera (R&D Systems). The reproducibility ofthe IL-7 ELISA was assessed through the incorporation of a control plasmasample in each assay, and variations were between 0.1 and 5%.

Statistical analysis

The performed analyses include the two-tailed Student t test for the com-parison between groups, whereas correlations involving different sets ofdata within the same group were determined using the Spearman’s rankcorrelation coefficient. Significance was assessed at the p � 0.05 level. Allanalyses were performed using SAS software.

ResultsDecreased CD127 expression on TM cells from HIV-infectedpatients

To determine whether HIV infection is associated with aberrantregulation of CD127 expression on T cells, we used multipara-metric flow cytometry to perform an immunophenotypic analysisof peripheral blood lymphocytes from both HIV-infected patients(either untreated or treated with HAART) and healthy uninfectedcontrols. The clinical, immunologic, and virologic characteristicsof the studied patients are displayed in Table I. As shown in Fig.1A, untreated HIV-infected patients exhibited a significant de-crease in CD127 expression (measured as a percentage of CD127�

cells) on both CD4� and CD8� T cells, with a more pronounceddecrease observed in the CD8� T cell population, a result consis-tent with previous studies (26–28). These described results are alsoconsistent with the observation that the levels of CD127 mRNAare �4- to 6-fold decreased in sorted CD8� T cells isolated fromHIV-infected patients as compared with CD8� T cells of unin-fected controls (data not shown). When the expression of CD127was examined in TN (CD45RA�CD62L�) and TM (i.e., non-

naive) cells, we found that the decline of CD127 expression pri-marily involved TM lymphocytes (Fig. 1, A and B), although asmall but significant reduction in CD127 expression was observedin TN cells as well. To then determine whether the decreased per-centage of CD8�CD127� memory T cells reflected an absolutereduction of this cell subset or, alternatively, an expansion ofCD8�CD127� memory T cells, we calculated the absolute countsper cubic millimeter of CD127� and CD127� cells in the totalCD8� T cell population as well as in the TN and TM cell subsets.As shown in Fig. 1C, the absolute numbers of CD8� T cells ex-pressing CD127 were similar in HIV-infected and uninfected in-dividuals, whereas the absolute counts of CD8�CD127� T cellswere increased in untreated HIV-infected patients compared withboth uninfected controls and HAART-treated HIV-infected pa-tients. Importantly, a marked absolute increase of CD8�CD127�

memory T cells was consistently observed in HIV-infectedpatients, whereas only a small increase in the number ofCD8�CD127� naive T cells was present in the same group ofpatients (Fig. 1C). In all, these results indicate that HIV infectionis associated with a significant expansion of TM (i.e., non-naive)cells that have lost the expression of CD127.

Expansion of CD8�CD127� T cells correlates with markers ofdisease progression and immune activation

We next investigated a possible correlation between the level ofexpansion of CD8�CD127� memory T cells and the main biolog-ical markers of disease progression during HIV infection, i.e., HIVplasma viremia and severity of CD4� T cell depletion. As shownin Fig. 1D, the level of expansion of CD127� TM cells was di-rectly correlated with plasma viremia and inversely correlated withCD4� T cell count. Consistent with the finding that the expansionof CD8�CD127� memory T cells is directly correlated with levelsof HIV replication is the observation that HIV-infected patientsthat are treated with HAART show lower levels of CD8�CD127�

memory T cells than untreated HIV-infected patients (Fig. 1, A andC). Interestingly, a significant correlation was also found betweenthe percentage of CD8�CD127� memory T cells and the level ofimmune activation as measured by the expression of the prolifer-ation marker Ki-67 in CD8� (Fig. 1D) and CD4� T cells (data notshown). In addition, a direct correlation was found between thenumber of CD8�CD127� memory T cells and the level of T cellsexpressing the activation markers HLA-DR and CD95 (data notshown). In contrast, no correlation was found between plasma lev-els of IL-7 and either the percentage (Fig. 1D) or the absolutecount (data not shown) of CD8�CD127� memory T cells. Theseresults suggest that the prevailing plasma levels of IL-7, which areinversely correlated with CD4� T cell counts in HIV-infected pa-tients (29–30), do not determine the levels of IL-7R expression onCD8� T cells. Because IL-7 has the potential to down-modulateCD127 expression both in vitro and in vivo (4, 31–34), wesequentially measured CD127 expression on CD8� T cells afterex vivo treatment with either rIL-7 or the mitogen PHA. Asshown in Fig. 1E, we found that a transient down-modulation ofCD127 follows treatment with rIL-7, whereas a more persistent

CD127 on CD8� T cells in one untreated HIV-infected patient, one HAART-treated, and one control (gate on lymphocyte region). Numbers in the upperright quadrant indicate the percentage of CD8� T cells that are CD127� and CD127�, respectively. C, Absolute counts per cubic millimeter of total,memory, and naive CD8� T cells expressing or not expressing CD127 are shown. The analysis was performed on the same individuals as in A. D, Expansionof CD8�CD127� T cells is correlated directly with plasma viremia (top left), inversely with CD4� T cell counts (top right), and directly with expressionof Ki-67 on CD8� T cells (bottom left). No correlation was found between CD8�CD127� T cells and plasma levels of IL-7 (bottom right). All analyseswere performed on the 31 HIV-infected patients described in A. Best fitting curves are shown. E, Sequential analysis of CD127 expression on CD8� T cells(measured as percentage of initial expression) treated with PHA (dashed line), rIL-7 (solid line), or medium alone (dotted line) for 96 h. Results representthe mean of five independent experiments performed using PBMCs from healthy donors.

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loss of CD127 was observed when CD8� T cells were treatedwith PHA. Taken together with the observation that the expan-sion of CD8�CD127� T cells correlates with the levels of im-mune activation but not with plasma levels of IL-7 (Fig. 1D),this result suggests that chronic antigenic stimulation may bemore important than the plasma level of IL-7 in determining theunusual abundance of CD8�CD127� T cells seen in HIV-in-fected individuals. An additional alternative possibility, giventhe role of IL-7 in regulating T cell homeostasis, is that theperipheral (i.e., postthymic) expansion of memoryCD8�CD127� T cells is related, as a compensatory homeo-static mechanism, to a state of impaired intrathymic T cell pro-duction that may follow HIV infection (23).

Expansion of CD8�CD127� T cells involves CD8� T cellsspecific for HIV as well as other Ags

We next sought to determine whether the loss of CD127 expres-sion on TM (i.e., non-naive) cells involved predominantly HIV-specific lymphocytes. To this end, we examined, in a subset ofHLA-A*0201-positive HIV-infected patients, the level of CD127expression on HIV-specific CD8� T cells using the HIVgag-p17SLYNTVATL tetramer. As shown in Fig. 2, we foundsimilarly reduced levels of CD127 expression in the HIV-spe-cific CD8� T cells as in the bulk population of TM cells of thesame patients. Interestingly, CD127 expression on CD8� T cells

specific for other Ags, such as the HLA-A*0201-restrictedCMVpp65NLVPMVATV and EBVbmfl-1GLCTLVAML epito-pes, was significantly reduced in HIV-infected patients as com-pared with EBV- and CMV-specific CD8� T cells isolated fromhealthy HIV-uninfected controls (Fig. 2B). These results indicatethat the HIV-associated expansion of CD8�CD127� memory Tcells does not involve exclusively HIV-specific TM cells.

Immunophenotypic features of CD8�CD127� andCD8�CD127� T cells

To better characterize the immunophenotypic features of the ex-panded CD8�CD127� T cells we used multiparametric flow cy-tometric analysis to assess the expression of memory T cell dif-ferentiation markers on both the CD8�CD127� and theCD8�CD127� T cell subsets. Specifically, we used staining withCD45RA, CD62L, and CCR7 to determine the distribution ofCD8�CD127� and CD8�CD127� T cells within the subsets ofTN (CD62L�, CD45RA�, and CCR7�), TCM cells (CD62L�,CD45RA�, CCR7�), TEM cells (CD62L�, CD45RA�, CCR7�),and CD45RA� TEMRA CELLS (CD62L�, CD45RA�, CCR7�)(5–8). As shown in Fig. 3A, we found that in both HIV-infectedpatients and controls CD8�CD127� T cells comprised essentiallycells expressing markers of TN and TCM differentiation (i.e.,CCR7� and either CD62L�CD45RA� or CD62L�CD45RA�),whereas CD8�CD127� T cells included predominantly TEM cells

FIGURE 2. Decreased CD127 expression on Ag-specific CD8� T cells. A, CD127 expression in HIVgag-p17SLYNTVATL- and EBVbmfl-1GLCTLVAML-spe-cific CD8� T cells from a representative HIV-infectedpatient (top four dot plots), and from an uninfectedhealthy control (bottom two dot plots) is shown. TheCD127 vs tetramer dot plots are pregated on total CD8�

T cells. Numbers in the top quadrants indicate the per-centage of CD127� cells among the tetramer-positiveand the tetramer-negative populations, respectively. B,Mean expression of CD127 on CD8� T cells specific forHIVgag-p17SLYNTVATL, CMVpp65NLVPMVATV,and EBVbmfl-1GLCTLVAML in 19 HLA-A*0201-positive HIV-infected patients and 5 HLA-A*0201-poshealthy controls.

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(i.e., CCR7� and either CD62L�CD45RA� or CD62L�CD45RA�).The most striking and consistent association in this set of immu-nophenotypical analyses was found between CD127 and CCR7expression (Fig. 3A). Consistent with the fact that CD8�CD127�

T cells show immunophenotypic features of TEM differentiation,we observed that this cell subset is enriched for the expression ofactivation markers such as HLA-DR and CD95 (Fig. 3A). Inter-estingly, CD8�CD127� T cells, but not CD8�CD127� T cells,included significant numbers of cells expressing the CXCR4 re-ceptor (Fig. 3A), whereas in HIV-infected patients, no difference inthe expression of CD45RO or CCR5 was observed betweenCD8�CD127� and CD8�CD127� T cells (Fig. 3A). In all, thesefindings indicate that CD8�CD127� T cells include predomi-nantly TN and TCM cells, whereas CD8�CD127� T cells includepredominantly TEM (i.e., CCR7� and either CD62L�CD45RA�

or CD62L�CD45RA�) cells, a subset of which express markers ofT cell activation.

CD8�CD127� T cells are primed to produce high levels ofIFN-� and low levels of IL-2

The immunophenotypic features of CD8�CD127� andCD8�CD127� T cells appear to recapitulate those of TN/TCM

and TEM/TEMRA cells, respectively. In addition, CD8�CD127�

T cells are enriched in activated T cells. We next tried to determinewhether and to what extent the functional features ofCD8�CD127� and CD8�CD127� T cells are consistent with theTCM/TEM paradigm of memory T cell differentiation described inhumans (5–8) and/or the TM/TE paradigm of TM cell differenti-ation described in mice (2–4). As shown in Fig. 3, B and C, themeasurement of cytokine production by CD8� T cells from bothHIV-infected patients and controls following a brief ex vivo stim-ulation with PMA and A23187 showed that CD8�CD127� T cellsare enriched in cells capable of producing IFN-� and almost totallydepleted in cells capable of IL-2 synthesis. Conversely,CD8�CD127� T cells showed more limited IFN-� productionwhile including a larger fraction of IL-2-secreting cells (Fig. 3, Band C). No differences were observed between CD8�CD127� andCD8�CD127� T cells with respect to TNF production (Fig. 3C).Importantly, no significant changes were observed in the percent-age of CD8�CD127� and CD8�CD127� T cells under theseexperimental conditions (data not shown), indicating that brieftreatment with PMA and A23187 did not induce major down-regulation of CD127.

FIGURE 3. CD8�CD127� T cells showphenotypic and functional features of TEcells whereas CD127�CD8� T cells showfeatures of either TN or TM cells. A, Expres-sion of the phenotypic markers CD45RA,CD62L, CD45RO, CCR7, CCR5, CXCR4,CD95, and HLA-DR on CD8�CD127� (f)and CD8�CD127� (�) T cells from 31HIV-infected patients (left) and 10 healthycontrols (right). B, Production of IL-2 andIFN-� by CD8�CD127� (solid line) andCD8�CD127� (dotted line) in one represen-tative HIV-infected patient after a 4-h stim-ulation with PMA and A23187. C, Mean ofthe percentages of CD8�CD127� (f) andCD8�CD127� (�) T cells producing IL-2,IFN-�, and TNF-� in 10 HIV-infected pa-tients and 10 healthy uninfected controls.

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In all, these results are consistent with the hypothesis that theexpanded CD8�CD127� T cells of HIV-infected patients showcharacteristic identifiers of TE/TEM cells, including the pattern ofcytokine production as well as immunophenotypic markers of Tcell differentiation. Interestingly, the finding of an expansion ofCD8�CD127� T cells that produce IFN-� but not IL-2 is consis-tent with previous observations indicating that advanced HIV in-fection is associated with a defect in IL-2 production (35–38) andnormal or increased IFN-� production (35, 39, 40) by T cells.

CD8�CD127� T cells show increased levels of in vivoproliferation, but decreased ex vivo proliferative potential

To further examine the relationship between loss of CD127 ex-pression and the functional features of TM cell differentiation, weassessed the proliferative potential of the CD8�CD127� T cellsboth in vivo and in vitro. We first examined the prevailing levelsof in vivo proliferation, as assessed by expression of the Ki-67proliferation marker, and found, in both HIV-infected patients andcontrols, significantly higher levels of proliferation inCD8�CD127� as compared with CD8�CD127� T cells (Fig. 4A).However, when the ex vivo proliferative capacity of sortedCD8�CD127� and CD8�CD127� T cells from uninfected donorswas measured in response to mitogenic stimulation (Fig. 4B), aninverse phenomenon was observed with levels of proliferationmarkedly higher in CD8�CD127� as compared withCD8�CD127� T cells. The fact that CD8�CD127� T cells areenriched in cells that are proliferating in vivo (in both HIV-in-fected patients and controls) but exhibit a reduced proliferativepotential in response to ex vivo restimulation is consistent with thehypothesis that CD8�CD127� T cells are comprised predomi-nantly of cells that display functional features typical of TE and/orTEM cells.

CD8�CD127� T cells show increased susceptibility tospontaneous and activation-induced apoptosis

We next sought to determine the level of susceptibility to sponta-neous and activation-induced apoptosis in CD8�CD127� andCD8�CD127� T cells by measuring the level of annexin V-pos-itivity after a 48-h incubation with either medium alone or PHA,respectively. As shown in Fig. 4C, sorted CD8�CD127� T cellsderived from both HIV-infected patients and healthy uninfectedcontrols showed a 2- to 5-fold increase in the level of both spon-taneous and activation-induced apoptosis as compared with sortedCD8�CD127� T cells from the same individuals. Because TEcells generated during an acute viral infection manifest a shortlifespan in vivo unless they successfully differentiate into TM cells(2–4), the increased susceptibility to apoptosis shown byCD8�CD127� T cells is also consistent with the hypothesis thatthis population is enriched in cells showing functional featurestypical of TE cells.

DiscussionThe processes underlying the differentiation of Ag-specific mem-ory CD8� T cells that follow viral infections have been the subject

FIGURE 4. CD8�CD127� T cells show increased levels of in vivo pro-liferation, but decreased ex vivo proliferative potential and increased sus-ceptibility to apoptosis. A, Expression of the proliferation marker Ki-67 onCD8�CD127� (f) and CD8�CD127� (�) in 31 HIV-infected patients(left) and 10 healthy controls (right). B, Efficiency of ex vivo proliferationafter PHA treatment (4 days) in sorted CD8�CD127� (top dot plots) and

CD8�CD127� (bottom dot plots) T cells from an uninfected donor. Dataare representative of seven experiments. C, Levels of baseline (i.e., beforeculture), spontaneous (i.e., medium alone), and PHA-induced apoptosismeasured as a percentage of annexin V-positive cells in CD8�CD127� (f)and CD8�CD127� (�) lymphocytes from seven uninfected donors andfive HIV-infected patients. D, Summary of phenotypical and functionalfeatures of CD8�CD127� (TE-like) and CD8�CD127� (TN- and TM-like) T cells.

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of intense investigation in both the murine (1–4) and the human(5–10) systems. Although significant differences have been de-scribed between the two species, the results of these complex im-munophenotypic and functional studies have allowed for the iden-tification of certain common features of memory CD8� T celldifferentiation in mice and humans. More specifically, it is nowpossible to draw a tentative analogy between the memory (i.e.,Ag-experienced or non-naive) CD8� cells that are defined as TEand TM (in the murine system) and those that are defined as TEMand TCM (in the human system), respectively. Following this anal-ogy and in an attempt to simplify and summarize the somewhatcomplex nomenclature used to define the various stages of mem-ory CD8� T cell differentiation by different authors, we will refer,in discussing the above-presented findings, to the CD8�CD127�

cells that show phenotypic and functional features of TE and/orTEM cells as “TE-like cells”, and to CD8�CD127� cells thatshow features of TM and/or TCM cells as “TCM-like cells”.

During chronic HIV infection, persistent viral replication coex-ists with high levels of CD8� T cell activation and turnover (41–45). We hypothesized that the loss of CD127 expression on CD8�

T cells serves as a novel marker of the HIV-associated generalizedimmune activation and accelerated T cell turnover, and that inHIV-infected patients the chronic expansion of CD8�CD127�

TE-like lymphocytes may have pathogenic relevance as these cellsmight be impaired, due to their lack of responsiveness to IL-7signaling, in their ability to differentiate and/or revert into restingCD8� TM/TCM cells. We found that HIV-infected patients con-sistently show an expansion of CD8�CD127� T cells that corre-lates with markers of disease progression such as the level of viralreplication and the severity of CD4� T cell depletion. In addition,we found that in both HIV-infected patients and healthy controls,the analysis of a series of phenotypic and functional features ofCD8�CD127� and CD8�CD127� T cells is compatible with thehypothesis that CD8�CD127� T cells include TN and TCM-likecells, while CD8�CD127� T cells are comprised of TE-like cells.Fig. 4D summarizes the immunophenotypical and functional fea-tures of CD8�CD127� and CD8�CD127� T cells that we havedescribed in this report. Importantly, the marked expansion ofCD8�CD127� T cells seen in HIV infection was associated withabsolute counts of CD8�CD127� TCM-like cells that were similarto those observed in uninfected healthy individuals, thus failing tosupport the hypothesis that a generalized deficit of CD8� TCMcells occurs during HIV infection.

In the setting of HIV infection, a marked and persistent expan-sion of CD8�CD127� TE-like cells might reflect a pathogenicmechanism, even in the presence of normal numbers ofCD8�CD127� TCM-like cells, by contributing to the HIV-asso-ciated chronic immune activation, which in turn is thought to be acausative factor of the CD4� T cell depletion of AIDS patients(11–15). Consistent with this hypothesis is the strong direct cor-relation observed between the expansion of CD8�CD127� cellsand the main biological markers of the HIV-associated chronicimmune activation (i.e., percentage of proliferating CD4� andCD8� T cells and percentage of CD8� T cells expressing activa-tion markers such as HLA-DR and CD95). The fact that CD127down-regulation follows T cell activation (Fig. 1E) (4, 31–33) alsosupports the hypothesis of a relationship between chronic in vivoimmune activation and expansion of CD8�CD127� TE-like cells.

Interestingly, our observation of large numbers ofCD8�CD127� TE-like cells is consistent with, and expands upon,the findings of several previous studies conducted in HIV-infectedindividuals. First, a chronic expansion of CD8�CD127� TE-likecells (that may produce high levels of proinflammatory cytokinessuch as IFN-�) in the setting of declining CD4� T cell counts is

consistent with the notion that in AIDS patients a state of progres-sive and profound immunodeficiency coexists with signs of gen-eralized immune activation (11–15), and that the extent of thisimmune activation is as good, or perhaps even better, a predictorof disease progression than the level of viral replication (16–21).Second, the fact that HAART corrects, at least in part, the HIV-associated expansion of TE-like CD8�CD127� cells is consistentwith the observation that HAART-mediated suppression of vire-mia is associated with a significant reduction of the level of im-mune activation (46). Third, the observation that CD8�CD127� Tcells isolated from healthy controls show reduced in vitro prolif-erative potential and increased susceptibility to apoptosis is con-sistent with the possibility that, in the setting of chronic HIV in-fection, the population of CD8�CD127� cells is also characterizedby short in vivo lifespan. In this case, these expandedCD8�CD127� TE-like cells may include those fast-replicatingand fast-dying CD8� T cells described recently in HIV-infectedpatients using in vivo T cell labeling with deuterium-based meth-ods (45), as well as the fraction of CD8� T cells that appears to beexquisitely sensitive to apoptosis during HIV infection (47–49).Finally, the expansion of CD8�CD127� TE-like cells that we ob-served in HIV-infected patients defined as normal progressors con-trasts with what is seen in HIV-infected long-term nonprogressors(who maintain effective control of HIV replication in associationwith low levels of immune activation) in whom a substantial frac-tion of their HIV-specific CD8� T cells display functional featuresof TCM cells, such as a high proliferative response upon in vitroantigenic restimulation (50), thus supporting the possibility that anexpansion of CD8�CD127� TE-like cells reflects a pathogenicmechanism of immunodeficiency.

An intriguing and somewhat unexpected result of the presentstudy is the relative increase of the fraction of CD8�CD127� Tcells specific for Ags other than HIV (i.e., CMV, EBV) that wasobserved in HIV-infected patients as compared with healthy un-infected controls. The relative expansion of EBV- and CMV-spe-cific CD8�CD127� TE-like cells in HIV-infected patients may beinterpreted alternatively as a consequence of EBV and/or CMVreactivation in an immunodeficient host (i.e., Ag-dependent acti-vation), or as the result of the HIV-associated immune activationon the pattern of differentiation of EBV- and CMV-specific TMcells in absence of EBV and/or CMV reactivation (i.e., bystanderactivation). At present we do not have data to support either pos-sibility, although studies are in progress to determine the level ofCD127 expression on CD8� T cells that are specific for Ags thatare not likely to be reactivated during HIV infection.

It should be noted that increased numbers of CD8�CD127� Tcells in the presence of low CD4� T cell counts may also reflecta “homeostatic”, IL-7-independent attempt to reconstitute the poolof T cells in the context of impaired de novo T cell production bythe thymus. In this perspective, an additional possibility is that thedescribed expansion of CD8�CD127� T cells from multiple Agspecificities (i.e., EBV, CMV) is in fact the consequence of anincreased extrathymic, IL-7-independent, homeostasis-driven pro-liferation of existing TM cell.

In summary, the data reported in this study suggest that theexpansion of CD8�CD127� TE-like cells is a previously unrec-ognized feature of the generalized immune dysfunction that fol-lows HIV infection. In this perspective, the expansion ofCD8�CD127� cells may represent a new marker to be evaluated,in association with other known parameters of T cell activation(i.e., levels of CD38, HLA-DR), to monitor the level of immuneactivation in HIV-infected patients. In contrast to markers such asHLA-DR and CD38, in which in vivo function on T cells is ba-sically unknown, the loss of CD127 expression on CD8� T cells is

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a marker of the HIV-associated immune activation whose biolog-ical function may provide clues as to a pathogenic mechanism ofimmune dysfunction (i.e., loss of responsiveness to IL-7 and fail-ure to differentiate and/or reconvert into TCM-like CD8� cells).The potential pathogenic role of the expanded CD8�CD127� TE-like cells suggests that immune-based interventions in the settingof HIV infection could be aimed not only at directly reconstitutinga sizeable CD4� T cell pool and at reducing the level of immuneactivation (51, 52), but ideally also at favoring an appropriate bal-ance of the different subsets of TM cells (i.e., TCM, TEM,TEMRA). The results we report indicate that further studies arewarranted to determine whether and to what extent measurementsof CD8�CD127� T cells may be useful in the clinical manage-ment of HIV-infected patients as a predictor of disease progressionas well as a marker of immunological response to therapy.

AcknowledgmentsWe thank Drs. Rafi Ahmed, John Wherry, Ann Chahroudi, andSilvija I. Staprans for helpful discussion, Michael Hulsey for technicalassistance with FACS sorting, and the research staff of the Emory Centerfor AIDS Research Clinical Research and Immunology Cores and the HopeClinic of the Emory Vaccine Center for their facilitation of these studies.

DisclosuresThe authors have no financial conflict of interest.

References1. Seder, R. A., and R. Ahmed. 2003. Similarities and differences in CD4� and

CD8� effector and memory T cell generation. Nat. Immunol. 4:835.2. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia,

U. H. von Andrian, and R. Ahmed. 2003. Lineage relationship and protectiveimmunity of memory CD8 T cell subsets. Nat. Immunol. 4:225.

3. Kaech, S. M., S. Hemby, E. Kersh, and R. Ahmed. 2002. Molecular and func-tional profiling of memory CD8 T cell differentiation. Cell 111:837.

4. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, andR. Ahmed. 2003. Selective IL-7R expression identifies effector CD8 T cells thatgive rise to long-lived memory cells. Nat. Immunol. 4:1191.

5. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Twosubsets of memory T lymphocytes with distinct homing potentials and effectorfunctions. Nature 401:708.

6. Champagne, P., G. S. Ogg, A. S. King, C. Knabenhans, K. Ellefsen, M. Nobile,V. Appay, G. P. Rizzardi, S. Fleury, M. Lipp, et al. 2001. Skewed maturation ofmemory HIV-specific CD8 T lymphocytes. Nature 410:106.

7. Geginat, J., F. Sallusto, and A. Lanzavecchia. 2001. Cytokine-driven proliferationand differentiation of human naive, central memory, and effector memory CD4�

T cells. J. Exp. Med. 194:1711.8. Geginat, J., A. Lanzavecchia, and F. Sallusto. 2003. Proliferation and differen-

tiation potential of human CD8� memory T-cell subsets in response to antigen orhomeostatic cytokines. Blood 101:4260.

9. Appay, V., P. R. Dunbar, M. Callan, P. Klenerman, G. M. Gillespie, L. Papagno,G. S. Ogg, A. King, F. Lechner. C. A. Spina, et al. 2002. Memory CD8� T cellsvary in differentiation phenotype in different persistent virus infections. Nat. Med.8:379.

10. van Baarle, D., S. Kostense, M. H. van Oers, D. Hamann, and F. Miedema. 2002.Failing immune control as a result of impaired CD8� T-cell maturation: CD27might provide a clue. Trends Immunol. 23:586.

11. Kinter, A., J. Arthos, C. Cicala, and A. S. Fauci. 2000. Chemokines, cytokinesand HIV: a complex network of interactions that influence HIV pathogenesis.Immunol. Rev. 177:88.

12. Hazenberg, M. D., D. Hamann, H. Schuitemaker, and F. Miedema. 2000. T celldepletion in HIV-1 infection: how CD4� T cells go out of stock. Nat. Immunol.1:285.

13. Grossman, Z., M. Meier-Schellersheim, A. E. Sousa, R. M. Victorino, andW. E. Paul. 2002. CD4� T cell depletion in HIV infection: are we closer tounderstanding the cause? Nat. Med. 8:319.

14. Douek, D. C., L. J. Picker, and R. A. Koup. 2003. T cell dynamics in HIVinfection. Annu. Rev. Immunol. 21:265.

15. Silvestri, G., and M. Feinberg. 2003. Turnover of lymphocytes and conceptualparadigms in HIV infection. J. Clin. Invest. 112:821.

16. Giorgi, J. V., L. E. Hultin, J. A. McKeating, T. D. Johnson, B. Owens,L. P. Jacobson, R. Shih, J. Lewis, D. J. Wiley, J. P. Phair, et al. 1999. Shortersurvival in advanced human immunodeficiency virus type 1 infection is moreclosely associated with T lymphocyte activation than with plasma virus burden orvirus chemokine coreceptor usage. J. Infect. Dis. 179:859.

17. Leng, Q., G. Borkow, Z. Weisman, M. Stein, A. Kalinkovich, and Z. Bentwich.2001. Immune activation correlates better than HIV plasma viral load with CD4T-cell decline during HIV infection. J. Acquired Immune Defic. Syndr. 27:389.

18. Sousa, A. E., J. Carneiro, M. Meier-Schellersheim, Z. Grossman, andR. M. Victorino. 2002. CD4 T cell depletion is linked directly to immune acti-

vation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viralload. J. Immunol. 169:3400.

19. Anthony, K. B., C. Yoder, J. A. Metcalf, R. DerSimonian, J. M. Orenstein,R. A. Stevens, J. Falloon, M. A. Polis, H. C. Lane, and I. Sereti. 2003. IncompleteCD4 T cell recovery in HIV-1 infection after 12 months of highly active anti-retroviral therapy is associated with ongoing increased CD4 T cell activation andturnover. J. Acquired Immune Defic. Syndr. 33:125.

20. Hazenberg, M. D., S. A. Otto, B. H. van Benthem, M. T. Roos, R. A. Coutinho,J. M. Lange, D. Hamann, M. Prins, and F. Miedema. 2003. Persistent immuneactivation in HIV-1 infection is associated with progression to AIDS. AIDS17:1881.

21. Hunt, P. W., J. N. Martin, E. Sinclair, B. Bredt, E. Hagos, H. Lampiris, andS. G. Deeks. 2003. T cell activation is associated with lower CD4� T cell gainsin human immunodeficiency virus-infected patients with sustained viral suppres-sion during antiretroviral therapy. J. Infect. Dis. 187:1534.

22. Silvestri, G., D. L. Sodora, R. A. Koup, M. Paiardini, S. P. O’Neil,H. M. McClure, S. Staprans, and M. B. Feinberg. 2003. Non-pathogenic simianimmunodeficiency virus infection of sooty mangabeys is characterized by limitedbystander immunopathology despite high levels viral replication. Immunity18:341.

23. McCune, J. M. 2001. The dynamics of CD4� T-cell depletion in HIV disease.Nature 410:974.

24. Ravkov, E. V., C. M. Myrick, and J. D. Altman. 2003. Immediate early effectorfunctions of virus-specific CD8�CCR7� memory cells in humans defined byHLA and CC chemokine ligand 19 tetramers. J. Immunol. 170:246.

25. Altman, J. D., P. A. Moss, P. J. Goulder, D. H. Barouch, M. McHeyzer-Williams,J. I. Bell, A. J. McMichael, and M. M. Davis. 1996. Phenotypic analysis ofantigen-specific T lymphocytes. Science 274:94.

26. Carini, C., M. F. McLane, K. H. Mayer, and M. Essex. 1994. Dysregulation ofinterleukin-7 receptor may generate loss of cytotoxic T cell response in humanimmunodeficiency virus type 1 infection. Eur. J. Immunol. 24:2927.

27. Vingerhoets, J., E. Bisalinkumi, G. Penne, R. Colebunders, E. Bosmans,L. Kestens, and G. Vanham. 1998. Altered receptor expression and decreasedsensitivity of T-cells to the stimulatory cytokines IL-2, IL-7 and IL-12 in HIVinfection. Immunol. Lett. 61:53.

28. MacPherson, P. A., C. Fex, J. Sanchez-Dardon, N. Hawley-Foss, and J. B. Angel.2001. Interleukin-7 receptor expression on CD8� T cells is reduced in HIV in-fection and partially restored with effective antiretroviral therapy.J. Acquired Immune Defic. Syndr. 28:454.

29. Napolitano, L. A., R. M. Grant, S. G. Deeks, D. Schmidt, S. C. De Rosa,L. A. Herzenberg, B. G. Herndier, J. Andersson, and J. M. McCune. 2001. In-creased production of IL-7 accompanies HIV-1-mediated T-cell depletion: im-plications for T-cell homeostasis. Nat. Med. 7:73.

30. Fry, T. J., E. Connick, J. Falloon, M. M. Lederman, D. J. Liewehr, J. Spritzler,S. M. Steinberg, L. V. Wood, R. Yarchoan, J. Zuckerman, et al. 2001. A potentialrole for interleukin-7 in T-cell homeostasis. Blood 97:2983.

31. Sudo, T., S. Nishikawa, N. Ohno, N. Akiyama, M. Tamakoshi, H. Yoshida, andS. Nishikawa. 1993. Expression and function of the interleukin 7 receptor inmurine lymphocytes. Proc. Natl. Acad. Sci. USA 90:9125.

32. Schluns, K. S., W. C. Kieper, S. C. Jameson, and L. Lefrancois. 2000. Interleu-kin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat.Immunol. 1:426.

33. Goldrath, A. W., P. V. Sivakumar, M. Glaccum, M. K. Kennedy, M. J. Bevan,C. Benoist, D. Mathis, and E. A. Butz. 2002. Cytokine requirements for acute andbasal homeostatic proliferation of naive and memory CD8� T cells. J. Exp. Med.195:1515.

34. Fry, T. J., M. Moniuszko, S. Creekmore, S. J. Donohue, D. C. Douek,S. Giardina, T. T. Hecht, B. J. Hill, K. Komschlies, J. Tomaszewski, et al. 2003.IL-7 therapy dramatically alters peripheral T-cell homeostasis in normal and SIV-infected nonhuman primates. Blood 101:2294.

35. Lane, H. C., J. M. Depper, W. C. Greene, G. Whalen, T. A. Waldmann, andA. S. Fauci. 1985. Qualitative analysis of immune function in patients with theacquired immunodeficiency syndrome: evidence for a selective defect in solubleantigen recognition. N. Engl. J. Med. 313:79.

36. Clerici, M., N. I. Stocks, R. A. Zajac, R. N. Boswell, D. R. Lucey, C. S. Via, andG. M. Shearer. 1989. Detection of three distinct patterns of T helper cell dys-function in asymptomatic, human immunodeficiency virus-seropositive patients:independence of CD4� cell numbers and clinical staging. J. Clin. Invest.84:1892.

37. Sieg, S. F., D. A. Bazdar, C. V. Harding, and M. M. Lederman. 2001. Differentialexpression of interleukin-2 and � interferon in human immunodeficiency virusdisease. J. Virol. 75:9983.

38. Paiardini, M., B. Cervasi, D. Galati, G. Cannavo, D. Guetard, M. Montroni,M. Magnani, G. Piedimonte, and G. Silvestri. 2001. Exogenous IL-2 adminis-tration corrects the cell cycle perturbation in lymphocytes from HIV-infectedindividuals. J. Virol. 75:10843.

39. Fuchs, D., A. Hausen, G. Reibnegger, E. R. Werner, G. Werner-Felmayer,M. P. Dierich, and H. Wachter. 1989. Interferon-� concentrations are increasedin sera from individuals infected with human immunodeficiency virus type 1.J. Acquired Immune Defic. Syndr. 2:158.

40. Graziosi, C., G. Pantaleo, K. R. Gantt, J. P. Fortin, J. F. Demarest, O. J. Cohen,R. P. Sekaly, and A. S. Fauci. 1994. Lack of evidence for the dichotomy of TH1and TH2 predominance in HIV-infected individuals. Science 265:248.

41. Hazenberg, M. D., J. W. Stuart, S. A. Otto, J. C. Borleffs, C. A. Boucher,R. J. de Boer, F. Miedema, and D. Hamann. 2000. T-cell division in humanimmunodeficiency virus (HIV)-1 infection is mainly due to immune activation: a

2908 CD127 EXPRESSION LOSS IN CD8� CELLS OF HIV-INFECTED PATIENTS

by guest on February 4, 2018http://w

ww

.jimm

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longitudinal analysis in patients before and during highly active antiretroviraltherapy (HAART). Blood 95:249.

42. Mohri, H., A. S. Perelson, K. Tung, R. M. Ribeiro, B. Ramratnam,M. Markowitz, R. Kost, A. Hurley, L. Weinberger, D. Cesar, et al. 2001. In-creased turnover of T lymphocytes in HIV-1 infection and its reduction by an-tiretroviral therapy. J. Exp. Med. 194:1277.

43. Orendi, J. M., A. C. Bloem, J. C. Borleffs, F. J. Wijnholds, N. M. de Vos,H. S. Nottet, M. R. Visser, H. Snippe, J. Verhoef, and C. A. Boucher. 1998.Activation and cell cycle antigens in CD4� and CD8� T cells correlate withplasma human immunodeficiency virus (HIV-1) RNA level in HIV-1 infection.J. Infect. Dis. 178:1279.

44. Sachsenberg, N., A. S. Perelson, S. Yerly, G. A. Schockmel, D. Leduc,B. Hirschel, and L. Perrin. 1998. Turnover of CD4� and CD8� T lymphocytesin HIV-1 infection as measured by Ki-67 antigen. J. Exp. Med. 187:1295.

45. Hellerstein, M. K., R. A. Hoh, M. B. Hanley, D. Cesar, D. Lee, R. A. Neese, andJ. M. McCune. 2003. Subpopulations of long-lived and short-lived T cells inadvanced HIV-1 infection. J. Clin. Invest. 112:956.

46. Pomerantz, R. J., and D. L. Horn. 2003. Twenty years of therapy for HIV-1infection. Nat. Med. 9:867.

47. Meyaard, L., S. A. Otto, R. R. Jonker, M. J. Mijnster, R. P. Keet, and F. Miedema.1992. Programmed death of T cells in HIV-1 infection. Science 257:217.

48. Finkel, T. H., G. Tudor-Williams, N. K. Banda, M. F. Cotton, T. Curiel,C. Monks, T. W. Baba, R. M. Ruprecht, and A. Kupfer. 1995. Apoptosis occurspredominantly in bystander cells and not in productively infected cells of HIV-and SIV-infected lymph nodes. Nat. Med. 1:129.

49. Gougeon, M. L., H. Lecoeur, A. Dulioust, M. G. Enouf, M. Crouvoiser,C. Goujard, T. Debord, and L. Montagnier. 1996. Programmed cell death inperipheral lymphocytes from HIV-infected persons: increased susceptibility toapoptosis of CD4 and CD8 T cells correlates with lymphocyte activation and withdisease progression. J. Immunol. 156:3509.

50. Migueles, S. A., A. C. Laborico, W. L. Shupert, M. S. Sabbaghian, R. Rabin,C. W. Hallahan, D. Van Baarle, S. Kostense, F. Miedema, M. McLaughlin, et al.2002. HIV-specific CD8� T cell proliferation is coupled to perforin expressionand is maintained in nonprogressors. Nat. Immunol. 3:1061.

51. Kovacs, J. A., S. Vogel, J. M. Albert, J. Falloon, R. T. Davey, Jr., R. E. Walker,M. A. Polis, K. Spooner, J. A. Metcalf, M. Baseler, et al. 1996. Controlled trialof interleukin-2 infusions in patients infected with the human immunodeficiencyvirus. N. Engl. J. Med. 335:1350.

52. Chapuis, A. G., G. Paolo Rizzardi, C. D’Agostino, A. Attinger, C. Knabenhans,S. Fleury, H. Acha-Orbea, and G. Pantaleo. 2000. Effects of mycophenolic acidon human immunodeficiency virus infection in vitro and in vivo. Nat. Med.6:762.

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