Effects of HIV-1 Infection on Lymphocyte Phenotypes in ... · Key Words: CD8+ T lymphocytes,...

BASIC SCIENCE

Effects of HIV-1 Infection on Lymphocyte Phenotypesin Blood Versus Lymph Nodes

Otto O. Yang, MD,*† John J. Ferbas, PhD,* Mary Ann Hausner, MS,* Lance E. Hultin, BS,*

Patricia M. Hultin,* David McFadden, MD,‡ Mark Sawicki, MD,‡ Roger Detels, MD,§

Martin Majchrowicz, MPH,* Jose L. Matud, MS,* Janis V. Giorgi, PhD,*†k

and Beth D. Jamieson, PhD*

Summary: Most immunopathogenesis studies of HIV-1 use periph-

eral blood. Most lymphocytes reside in lymphoid tissues, however,

and the extent to which blood mirrors tissues is unclear. Here, we

analyze lymphocytes in blood and lymph nodes of HIV-1–uninfected

and –infected persons. Baseline comparison of node and blood

lymphocytes in seronegative persons demonstrates a lower ratio of

CD8+ versus CD4+ T lymphocytes, a lower number of effector cells

(CD282) within the CD8+ compartment, and greater activation

(D-receptor [DR+]) within the CD4+ compartment. In infected versus

uninfected persons, nodes exhibit elevated CD8+ T lymphocytes with

an increased memory-effector phenotype (CD62L2/CD45RA2) and

activation (CD38+ and DR+) but minimal differences in the CD4+

compartment. Changes attributable to HIV-1 infection are markedly

greater in node lymphocytes than in blood. Comparisons of CD8+

T-lymphocyte parameters and viremia in infected persons reveal

positive correlations of CD38+ expression on cells in blood and nodes

and a negative correlation of terminal effector cells (CD62L2/CD45RA+)

in the nodes to viremia. Multiple linear regression analysis indicates

that CD38 expression on node (not blood) CD8+ T lymphocytes is the

sole independent predictor for viremia. Thus, blood indirectly reflects

processes in lymphoid tissues, and caution should be applied when

interpreting immunopathogenesis studies of blood.

Key Words: CD8+ T lymphocytes, peripheral blood, lymph node,

HIV-1

(J Acquir Immune Defic Syndr 2005;39:507–518)

The accessibility of peripheral blood has limited most HIV-1pathogenesis studies in humans to assays of blood, under

the assumption that this compartment adequately reflects theoverall interactions of virus, target cells, and immune re-sponses. Indeed, valuable correlations have been determined,such as the usefulness of the blood CD4+ T-lymphocyte con-centration in predicting the risk of infection and viremia levelin predicting the rate of disease progression. Such findingshave suggested that peripheral blood can serve as a window forviewing systemic pathologic processes in HIV-1 infection.

The CD4+ T lymphocytes that are the predominant sourceof HIV-1 replication in vivo mostly reside in lymphoid tissues,however. It has been estimated that only 2% of the total lym-phocyte population is found in peripheral blood1; therefore,the blood is probably a minor site of viral replication within thehost. Data suggest that more vigorous viral replication occursin lymphoid tissues than is reflected by the degree of viremia,2,3

indicating that tissues are the major reservoir of viral replication.Furthermore, infected CD4+ T lymphocytes are rare in the pe-ripheral circulation even when viremia is high,4 suggesting thatvirion production occurs outside this compartment.

Although CD8+ T lymphocytes are believed to have animportant role in reducing HIV-1 replication in vivo, quantita-tive correlates of immunity have remained elusive. Qualitativeexperiments have shown that CD8+ T-lymphocyte depletion ofsimian immunodeficiency virus (SIV)–infected macaquesresults in uncontrolled viral replication in vivo,5–7 but compre-hensive attempts to correlate the magnitude or breadth of theHIV-1-specific CD8+ T-lymphocyte response in blood to viremiahave been disappointing.8,9 A more global measure of CD8+ Tlymphocytes, the activation status of these cells (in blood) asreflected by CD38 expression, has been noted to correlate withviremia and to predict disease progression.10,11 Viremia isa better predictor of disease than CD38 expression on CD8+

T lymphocytes,12 however, and the relative relationships ofCD38 expression, CD8+ T-lymphocyte activity, and viralreplication are unknown.

An important factor that is poorly understood is theextent to which measurements of CD8+ lymphocytes in bloodreflect antiviral activity at the major sites of viral replication.Although some data indicate that HIV-1–specific CD8+

T-lymphocyte targeting is similar between blood and lymph nodecompartments,13 the potential differences remain poorly under-stood. Here, we perform detailed assessments of T-lymphocyte

Received for publication February 11, 2005; accepted April 27, 2005.From the *Department of Medicine, Geffen School of Medicine, UCLA

Medical Center, University of California, Los Angeles, CA; †Departmentof Microbiology, Immunology, and Molecular Genetics, University ofCalifornia, Los Angeles, CA; ‡Department of Surgery, Geffen School ofMedicine, UCLA Medical Center, University of California, Los Angeles,CA; and §UCLA School of Public Health, University of California, LosAngeles, CA.

kDeceased.Supported by National Institute of Allergy and Infectious Diseases grants

AI037613 (J. V. Giorgi) and AI043203 (O. O. Yang).Reprints: Otto O. Yang, Division of Infectious Diseases, 37-121 CHS, UCLA

Medical Center, 10833 LeConte Avenue, Los Angeles, CA 90095(e-mail: [email protected]).

Copyright � 2005 by Lippincott Williams & Wilkins

J Acquir Immune Defic Syndr � Volume 39, Number 5, August 15 2005 507

subpopulations and compare blood and lymph nodes in HIV-1–infected and –uninfected persons.

MATERIALS AND METHODS

Study SubjectsThe HIV-1–seropositive subjects were men in the Los

Angeles arm of the Multicenter AIDS Cohort Study (MACS),with the exception of 2 women (Table 1). The seronegativecontrol subjects were men in the MACS cohort with recent andpast high-risk exposure to HIV-1 selected on the basis of highinfection–risk exposure, defined as exceeding the number ofanal insertive partners corresponding to the 90th percentile ofall seronegative men in the MACS during the 2.5-year intervalfrom 1982 through 1985, and continued high-risk exposure, asindicated by reported sexual behavior at MACS visits from1993 through 1994, where exposures are defined as unpro-tected anal receptive intercourse with an HIV-1–infected sex-ual partner. High-risk seronegative subjects were chosen as theappropriate control group, because the sexual exposure statusof individuals (independent of HIV-1 infection) has been cor-

related with changes in T-lymphocyte subsets compared withlow-risk persons.14 All subjects provided informed consentunder University of California Institutional Review Board(IRB)–approved protocols.

Collection of Peripheral BloodMononuclear Cells

Peripheral blood mononuclear cells (PBMCs) were ob-tained from heparinized blood specimens within 1 hour ofphlebotomy by density gradient centrifugation on Lympho-prep (Nycomed Pharma, Oslo, Norway), and all processing forimmunologic assays was completed within 6 hours.

Isolation of Lymph Node Mononuclear CellsSuperficial inguinal lymph nodes were removed surgi-

cally through sterile operative procedures for subsequent iso-lation of mononuclear cells. Each node was bisected, and thecut surface of each half was mechanically disrupted through50-gauge stainless steel mesh. The resulting lymph node mono-nuclear cell (LNMC) suspension was passed through a 70-mmcell strainer (Becton Dickinson, Franklin Lakes, NJ).

HIV-1 Quantitation in BloodHIV-1 RNA in plasma was quantitated by using the

Amplicor HIV-1 Quantitation Kit (Roche, Branchburg, NJ) perthe manufacturer’s recommended protocol.

Phenotypic Analysis of T Lymphocytes byFlow Cytometry

T-cell subset phenotyping by cell surface staining offreshly isolated cells was performed as previously described.15

All monoclonal antibodies were purchased as conjugates of fluo-rescein isothiocyanate (FITC), phycoerythrin (PE), or peri-dinin chlorophyll protein (PerCP) from Becton DickinsonImmunocytometry Systems (San Jose, CA). Two-color (FITC/PE)or 3-color (FITC/PE/PerCP) antibody combinations wereused to stain for expression of CD3/CD4, CD3/CD8,CD4/CD28/CD3, CD8/CD28/CD3, CD45RA/CD62L/CD4,CD45RA/CD62L/CD8, human leukocyte antigen (HLA)–D-related (DR)/CD38/CD4, and HLA-DR/CD38/CD8 and wereimmediately analyzed on a FACScan flow cytometer (BDIS,San Jose, CA). Cell surface CD38 molecule numbers wereestimated as previously reported,16 using a bead standard.To ensure accurate quantitation of staining, the cytometer wasstandardized for fluorescence intensity each day usingglutaraldehyde-fixed chicken red blood cells (Biosure, GrassValley, CA).

For intracellular detection of perforin, 150 mL of wholeblood or 5 3 105 node cells were first surfaced stained withanti–CD3-PE and anti–CD8-PerCP. After surface staining,OrthoPermaFix solution (Ortho Diagnostics, Raritan, NJ) wasadded for 40 minutes at room temperature, followed by 2washes. The cells were then stained for 40 minutes at roomtemperature with perforin-FITC or IgG2-FITC isotype control(Ancell, Bayport, MN) in the presence of human AB serum.The cells were washed twice and run immediately ona FACSCalibur flow cytometer (BDIS). Because of high var-iability in background staining, only CD8+ T cells that ex-pressed high levels of perforin were graded as positive for this

TABLE 1. Clinical Characteristics of Subjects

Subject HIV AgeDuration(years) Treatment CD4

PlasmaViral Load

40014 + 35 11 None 910 ,100

40808 + 46 .14 d4T/3TC/saquinavir 343 ,100

41038 + 53 .12 d4T/3TC/indinavir 457 983

41266 + 44 .11 None 512 ,100

41459 + 47 3 None 300 977,816

41617 + 38 .12 AZT 618 4066

41696 + 38 .11 d4T/3TC/indinavir 596 ,100

41977 + 43 .10 AZT/3TC/ddI/ritonavir/saquinavir

564 ,100

42805 + 39 ? d4T/3TC/indinavir 187 4333

42808 + 34 11 AZT/3TC 453 65,443

42825 + 35 13 d4T/3TC/saquinavir 275 61,209

42827 + ? 11 d4T/3TC/ddI/delavirdine 805 1219

42828 + 47 8 None 739 6796

42832 + ? 9 AZT/3TC 513 2673

42836* + 38 10 None 656 2245

42839* + 40 .12 ddC/nevirapine 225 520

50272 + 43 .7 AZT 768 5881

10478 2 47 — — 1011 ,100

40290 2 54 — — 524 ,100

40294 2 45 — — 738 ,100

40381 2 47 — — 1137 ,100

40514 2 57 — — 636 ,100

40729 2 50 — — 1057 ,100

41001 2 51 — — 678 ,100

41455 2 41 — — 734 ,100

42801 2 ? — — 636 ,100

42819 2 28 — — 947 ,100

42830 2 51 — — 842 ,100

*Female subject; all other subjects are male.AZT indicates zidovudine; ddC, dideoxycytidine; ddI, didanosine; d4T, stavudine;

3TC, lamivudine.

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Yang et al J Acquir Immune Defic Syndr � Volume 39, Number 5, August 15 2005

study. The cutoff was determined in previous control studiesby gating on CD282/CD8+ natural killer (NK) lymphocytes,which express high levels of perforin.

Statistical AnalysisMost comparisons between HIV-1–seropositive and

–seronegative groups were performed using bootstrap resam-pling,17 adjusting traditional t test P values (designated Padj)for multiple comparisons (14 variables for each CD4+ andCD8+ T-lymphocyte subset measured in peripheral blood andlymph node sample types), interdependence of certain variables,and nonnormal distribution of several variables (Table 2). Allcompared arms included at least 7 independent measurementsfor each group (see Table 2). Comparisons were performedusing the Multitest procedure in SAS (SAS Institute Cary,NC). This program also was used to evaluate for correlationsamong immune markers in the peripheral blood, lymph nodes,and viremia (Table 3). For these comparisons, viremia wasquantified as the logarithm10 of HIV-1 genomes per milliliterof plasma; measurements in infected persons below the limitof detection were assigned a value of 2 log10 units. Identifiedcorrelations were confirmed by additional testing using aMarkov Chain/Monte Carlo simulation (WinBUGS; ImperialCollege of Science, Technology, and Medicine, London,England), in which values for viremia below the limit ofdetection (400 copies/mL of plasma) were considered cen-sored data in the range of 0 to 400.18

RESULTS

HIV-1 Infection Preferentially Affects CD8+ butNot CD4+ T-Lymphocyte Phenotypes inPeripheral Blood

Several markers on the peripheral blood CD8+ andCD4+ T lymphocytes of HIV-1–seronegative and –seropositivepersons were assessed and compared. The seronegative per-sons were highly exposed yet uninfected men from the MACSserving as controls for the infected persons, most of whomwere also men from the MACS (see Table 1). The peripheralCD8+ T-lymphocyte pool was larger in the seropositive versusthe seronegative persons (median 6 SD absolute CD8+

T-lymphocyte counts of 1098 6 473 cells/mm3 vs. 719 6281 cells/mm3; P = 0.024, Padj = 0.173), and the CD4+

T-lymphocyte pool was significantly smaller (524 6213 cells/mm3 vs. 812 6 200 cells/mm3; P = 0.001, Padj =0.016). This was also reflected in the percentages of CD8+

(P , 0.001, Padj , 0.001) and CD4+ (P = 0.001, Padj =0.010) cells within the total lymphocyte pool (Fig. 1).These populations then were analyzed individually for subsetsof CD62L/CD45RA, CD28, and CD38/DR expression.

Within the blood CD8+ T-lymphocyte compartment (seeFig. 1A), the major difference was the activation status. Thememory and naive populations defined by CD62L/CD45RAwere similar between seropositive and seronegative persons.Differentiation of effector cells, as reflected by CD28 status,was also similar. The activation status, as defined by CD38/DRmarkers of activation, showed that the CD38+ subsets werehigher in seropositive persons, however, as well as the total

percentage of cells that were CD38+ (P = 0.003, Padj = 0.029).In contrast to CD38, total DR+ cell percentages showed a weaktrend toward higher DR+ cells in seropositive persons, possiblysuggesting differential activation attributable to infection.

Interestingly, the blood CD4+ T-lymphocyte compart-ment (see Fig. 1B) was more similar between seropositive andseronegative subjects. Again, memory and naive subsets andCD28 status were similar. There was a trend toward higherCD38+ but not DR+ subsets in the seropositive subjects(P = 0.039, Padj = 0.275 for total CD38+ percentage). Thesedata indicated that HIV-1 infection results in more dramaticchanges within the CD8+ T-lymphocyte compartment thanwithin the CD4+ T-lymphocyte compartment, suggesting pre-ferential responsiveness of CD8+ T lymphocytes to infection.

CD8+ and CD4+ T-Lymphocyte Concentrationsand Phenotypes Vary Between PeripheralBlood and Lymph Nodes inUninfected Subjects

To define the baseline status of T lymphocytes in lymphnodes in the absence of perturbation by HIV-1 infection, thesubpopulations of CD8+ and CD4+ T lymphocytes werecompared in the blood and lymph nodes of the HIV-1–seronegative control subjects (Fig. 2). Overall, the percentagesof CD8+ T lymphocytes were lower and those of CD4+ Tlymphocytes were higher in lymph nodes compared with blood(Padj , 0.001 for each). In the CD8+ compartment (see Fig.2A), the CD62L/CD45RA and CD38/DR subsets were similar,but a markedly lower percentage of CD282 effector cells werefound in lymph nodes versus blood (Padj , 0.001). Bycontrast, the CD4+ compartment showed more differences (seeFig. 2B), revealing higher CD38+/DR+ (P = 0.001, Padj =0.012), lower CD382/DR2 (P = 0.006, Padj = 0.042), higherCD382/DR+ (P = 0.002, Padj = 0.018), and higher total DR+

(P = 0.001, Padj = 0.012) cell percentages in lymph nodes,suggesting more activation marked by DR but not CD38expression. Although there was a suggestion for a trend ofmore CD282/CD4+ effector cells in blood (P = 0.0269, Padj =0.132), this difference was less marked than that noted forCD8+ T lymphocytes. Overall, these observations suggestedthat lymph nodes contain a lower percentage of CD8+ Tlymphocytes than blood and these tend to be CD28+ and thatlymph nodes contain a higher percentage of CD4+ T lym-phocytes than blood and these tend to be activated as dem-onstrated by DR but not CD38 expression.

HIV-1 Infection Preferentially IncreasesActivated Effector Subsets of CD8+ but NotCD4+ T Lymphocytes in Lymph Nodes

These cell subsets were also evaluated in lymph nodesfrom HIV-1–seropositive persons to examine the effects ofinfection. The percentage of CD8+ T lymphocytes overall wasincreased (Padj , 0.001), and there were marked differencesamong differentiation and activation parameters of lymphnode CD8+ T lymphocytes as a result of infection (Fig. 3A).Percentages of CD8+ memory-effector lymphocytes asdefined by CD62L2/CD45RA2 (P = 0.003, Padj = 0.023)and CD282 (P = 0.009, Padj = 0.060)19–21 phenotypes wereincreased. Activation, as represented by percentages of CD38+

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TABLE 2. Statistical Calculations

Peripheral Blood

Seronegative SeropositiveSeronegative vs.Seropositive

n Mean SD n Mean SD P Padj

CD8+ T lymphocytes

Cells/mm3 11 719 281 17 1098 473 0.024 0.173

Lymphocytes (%) 11 33.8 10.8 17 51.4 8.9 ,0.001 ,0.001

CD62L+/CD45RA2 9 24.5 9.2 14 26.0 9.2 0.703 0.953

CD62L+/CD45RA+ 9 29.1 11.8 14 23.0 9.8 0.194 0.694

CD62L2/CD45RA2 9 24.2 13.8 14 29.9 12.7 0.283 0.796

CD62L2/CD45RA+ 9 22.2 12.3 14 21.2 12.9 0.828 0.953

CD282 10 47.7 20.0 17 52.9 13.8 0.330 0.796

CD28+ 10 52.4 20.0 17 47.1 13.8 0.330 0.796

CD38+/DR2 10 6.4 3.4 17 11.4 6.0 0.005 0.052

CD38+/DR+ 10 5.7 3.4 17 18.7 12.1 0.015 0.123

CD382/DR2 10 61.9 21.9 17 46.0 14.7 0.016 0.126

CD382/DR+ 10 26.1 20.1 17 23.9 10.9 0.672 0.953

Total CD38+ 10 12.1 4.9 17 30.1 14.2 0.003 0.029

Total DR+ 10 31.8 22.5 17 42.6 15.6 0.106 0.534

CD38 molecules 10 531 226 17 2193 1421 0.174 0.694

CD4+ T lymphocytes

Cells/mm3 11 812 200 17 524 213 0.001 0.016

Lymphocytes (%) 11 38.7 7.3 17 25.7 8.6 0.001 0.010

CD62L+/CD45RA2 9 45.0 7.2 14 39.3 13.1 0.254 0.728

CD62L+/CD45RA+ 9 33.8 12.0 14 42.8 15.4 0.101 0.519

CD62L2/CD45RA2 9 18.8 7.2 14 14.5 6.0 0.148 0.572

CD62L2/CD45RA+ 9 2.4 1.4 14 3.5 2.7 0.753 0.977

CD282 9 10.0 10.3 14 10.3 10.0 0.937 0.993

CD28+ 9 90.0 10.3 14 89.7 10.0 0.937 0.993

CD38+/DR2 9 21.4 9.7 14 28.9 9.3 0.068 0.410

CD38+/DR+ 9 1.7 0.8 14 5.2 4.3 0.102 0.519

CD382/DR2 9 66.4 8.9 14 57.0 15.0 0.107 0.519

CD382/DR+ 9 10.5 7.1 14 9.1 4.9 0.623 0.977

Total CD38+ 9 23.1 9.2 14 34.0 11.5 0.039 0.275

Total DR+ 9 12.2 7.6 14 14.2 8.7 0.614 0.977

CD38 molecules 9 983 788 14 2242 1490 0.042 0.293

Lymph NodePeripheral Blood vs. Lymph Node

Seronegative SeropositiveSeronegative vs.Seropositive Seronegative Seropositive

n Mean SD n Mean SD P Padj P Padj P Padj

CD8+ T lymphocytes

Cells/mm3

Lymphocytes (%) 10 13.1 4.6 17 29.9 6.4 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001

CD62L+/CD45RA2 7 28.8 13.6 12 20.1 6.4 0.061 0.247 0.464 0.884 0.076 0.256

CD62L+/CD45RA+ 7 27.1 14.0 12 19.9 8.9 0.163 0.488 0.769 0.888 0.407 0.645

CD62L2/CD45RA2 7 33.5 9.3 12 52.0 11.5 0.003 0.023 0.152 0.549 ,0.001 0.001

CD62L2/CD45RA+ 7 10.6 12.6 12 8.1 6.2 0.631 0.860 0.085 0.410 0.004 0.023

CD282 8 2.4 1.0 16 18.3 11.3 0.009 0.060 ,0.001 ,0.001 ,0.001 ,0.001

CD28+ 8 97.6 1.0 16 81.7 11.3 0.009 0.060 ,0.001 ,0.001 ,0.001 ,0.001

CD38+/DR2 9 4.2 2.0 17 7.7 3.5 0.051 0.247 0.098 0.431 0.033 0.147

CD38+/DR+ 9 4.0 1.4 17 32.9 19.0 ,0.001 ,0.001 0.183 0.584 0.014 0.075

CD382/DR2 9 58.5 7.4 17 33.7 16.3 ,0.001 0.004 0.671 0.888 0.028 0.139

CD382/DR+ 9 33.3 8.2 17 25.7 11.8 0.163 0.488 0.331 0.797 0.646 0.653

Total CD38+ 9 8.1 2.7 17 40.6 20.1 ,0.001 ,0.001 0.049 0.272 0.089 0.256



(Padj , 0.001) and DR+ (Padj = 0.023) cells, was increased, inlarge part because of a larger CD38+/DR+ subset (Padj ,0.001). By contrast, there was a lower percentage of totalCD4+ T lymphocytes in the lymph nodes of infected versusuninfected persons (Padj , 0.001), but the phenotypes wereremarkably similar, with the exception of an elevatedpercentage of the CD38+/DR+ (P = 0.001, Padj = 0.016)subset (Figure 3B). These findings further confirmed greaterphenotypic changes in the CD8+ than CD4+ T lymphocytecompartment attributable to HIV-1 infection.

HIV-1–Induced Changes in CD8+

T-Lymphocyte Phenotypes are Much Greaterin Lymph Nodes Than in Blood

To compare quantitatively the degree of change invarious T-lymphocyte subsets in infected versus uninfectedpersons, the ratio of each subset in infected versus uninfectedpersons was calculated for CD8+ (Fig. 4A) and CD4+ (Fig. 4B)

T lymphocytes in the blood and lymph nodes. This analysisrevealed that the changes in the CD8+ T-lymphocytecompartment attributable to infection occurred in the samesubsets in the blood and lymph nodes but that they were ofgreater magnitude in the lymph nodes. In particular, thepercentages of CD282, CD38+/DR+, and total CD38+ subsetswere increased many fold in the lymph nodes versus the blood.There were fewer changes in the CD4+ T lymphocytes, which,overall, were similar between the blood and lymph nodes.

Concentration of Perforin-Expressing CD8+

T Lymphocytes in Blood and Lymph Nodesof Infected Subjects Is Not Less Than Thatof Uninfected Subjects

Because reduced perforin expression in lymph nodeCD8+ T lymphocytes has been proposed as a mechanism ofimmune failure in HIV-1 infection,22–26 this parameter wasassessed in the blood and lymph nodes of the subjects (Fig. 5).Intracellular staining of CD8+ T lymphocytes in the blood andlymphs node of uninfected persons revealed markedly lowerpercentages of perforin-expressing cells in the lymph nodescompared with the blood (mean of 0.2% vs. 27.2% in the4 tested subjects; P = 0.007). In HIV-1–infected persons, therewas a similar relation between the lymph nodes and blood(mean of 1.5% vs. 35.9% in the 11 tested subjects; P , 0.001).Comparison of the uninfected and infected persons indicateda trend for increased perforin-expressing CD8+ T lymphocytesin both compartments, particularly the lymph node (blood, P =0.177; lymph node, P = 0.032). Further evaluation of theinfected subjects revealed that freshly isolated unstimulated

TABLE 2. (continued ) Statistical Calculations

Lymph NodePeripheral Blood vs. Lymph Node

Seronegative SeropositiveSeronegative vs.Seropositive Seronegative Seropositive

n Mean SD n Mean SD P Padj P Padj P Padj

Total DR+ 9 37.3 8.4 17 58.6 16.5 0.003 0.023 0.498 0.884 0.007 0.038

CD38 molecules 9 439 265 17 4596 5092 0.002 0.016 0.428 0.884 0.070 0.256

CD4+ T lymphocytes

Cells/mm3

Lymphocytes (%) 10 63.1 7.5 16 37.6 11.7 ,0.001 ,0.001 ,0.001 ,0.001 0.002 0.015

CD62L+/CD45RA2 7 41.5 12.5 12 36.2 11.5 0.337 0.915 0.496 0.852 0.531 0.774

CD62L+/CD45RA+ 7 27.7 9.9 12 36.0 10.3 0.174 0.737 0.297 0.685 0.205 0.493

CD62L2/CD45RA2 7 21.3 6.8 12 19.4 7.7 0.581 0.990 0.503 0.852 0.076 0.281

CD62L2/CD45RA+ 7 9.5 13.5 12 8.4 10.1 0.771 0.999 0.138 0.414 0.093 0.294

CD282 7 0.2 0.1 12 0.5 0.4 0.930 0.999 0.026 0.132 0.003 0.016

CD28+ 7 99.8 0.1 12 99.5 0.4 0.930 0.999 0.026 0.132 0.003 0.016

CD38+/DR2 7 18.0 6.4 12 24.8 10.3 0.133 0.660 0.444 0.811 0.303 0.573

CD38+/DR+ 7 4.8 2.1 12 12.8 7.5 0.001 0.016 0.001 0.012 0.004 0.017

CD382/DR2 7 51.3 9.8 12 43.6 15.6 0.233 0.827 0.006 0.042 0.036 0.153

CD382/DR+ 7 25.8 8.8 12 18.8 7.6 0.039 0.280 0.002 0.018 0.001 0.004

Total CD38+ 7 22.9 6.9 12 37.6 15.8 0.013 0.118 0.955 0.951 0.513 0.774

Total DR+ 7 30.6 10.3 12 31.6 10.1 0.832 0.999 0.001 0.012 ,0.001 0.001

CD38 molecules 7 1549 382 12 3182 1901 0.019 0.158 0.104 0.351 0.170 0.453

n indicates number of individuals tested; P = raw t test value, Padj = bootstrap-adjusted value. Significant P values are highlighted (P , 0.05 and Padj , 0.05), and borderline P valuesare in bold font (P , 0.05 and Padj . 0.05).

TABLE 3. Multiple Linear Regression Analysis of Viremia andCD8+ T-Lymphocyte Parameters

Parameter Estimate SE t P

Intercept 26.86 4.90 21.40 0.199

Blood CD38 molecules 0.32 1.11 0.29 0.781

Node CD62L2/CD45RA+ 20.34 1.34 20.25 0.808

Node CD38 molecules 2.65 1.12 2.36 0.046

Multiple r2 = 0.76.All parameters are examined in log10 units.



bulk CD8+ T lymphocytes in both compartments possessedHIV-1–specific lytic activity directed against Gag, Pol, Env,and Nef (by chromium release assays using recombinantvaccinia virus–infected autologous transformed B cells, datanot shown). Neither perforin levels nor bulk HIV-1–specificcytolytic activity related to viremia or CD8+ T-lymphocytephenotypes in these subjects (not shown). Overall, these datadid not demonstrate any quantitative reduction of perforinexpression in the blood or lymph node CD8+ T lymphocytes of

HIV-1–infected subjects, although they did not exclude thepossibility that this unaltered perforin expression was in-appropriately low in the setting of infection.

Blood CD8+ T-Lymphocyte CompartmentImprecisely Mirrors That in Lymph Nodesof Infected Subjects

Because most prior phenotypic studies of CD8+ Tlymphocytes in HIV-1 infection have been performed usingblood, the relationships of the CD8+ T-lymphocyte subsets inthe lymph node to those in the peripheral blood were thereforeassessed for the HIV-1–infected persons (Fig. 6). The overall

FIGURE 1. Comparison of T-lymphocyte subsets in blood ofHIV-1–seropositive and –seronegative subjects. Peripheralblood lymphocytes from HIV-1–seronegative (white boxes)and –seropositive (shaded boxes) subjects were analyzed byflow cytometry to assess subsets of CD8+ and CD4+ lympho-cytes. These T lymphocytes were characterized for expressionof CD62L/CD45RA, CD38/HLA-DR, and CD28. Box plots(spanning the 25th–75th percentiles) indicate the average(dotted line), median (solid line), and range (brackets).*Bootstrap-adjusted P , 0.05 (see Table 2). A, Percentage ofCD8+ T lymphocytes within total lymphocytes and percen-tages of various subsets within CD8+ T lymphocytes. B, Per-centage of CD4+ T lymphocytes within total lymphocytes andpercentages of various subsets within CD4+ T lymphocytes.

FIGURE 2. Comparison of T-lymphocyte subsets in blood andlymph nodes of seronegative subjects. Peripheral blood (whiteboxes) and lymph node (shaded boxes) lymphocytes from theHIV-1–seronegative subjects were analyzed by flow cytometryto assess subsets of CD8+ and CD4+ lymphocytes as in Figure 1.*Bootstrap-adjusted P , 0.05 (see Table 2). A, Percentage ofCD8+ T lymphocytes within total lymphocytes and percen-tages of various subsets within CD8+ T lymphocytes. B, Per-centage of CD4+ T lymphocytes within total lymphocytes andpercentages of various subsets within CD4+ T lymphocytes.



percentages of total CD8+ T lymphocytes (see Fig. 6) wereroughly correlated between the 2 compartments (r2 = 0.323,P = 0.018). The subsets defined by CD62L/CD45RA, CD28,and CD38/DR were also roughly correlated (see Fig. 6;r2 range: 0.271–0.684). Despite these correlations, however,subset interrelations within each compartment showed varia-tion. Comparing CD38 expression with the percentage ofCD62L2/CD45RA+ (terminal effector) cells (Fig. 7), a signif-icant correlation was seen in the lymph nodes but not in blood(r2 , 0.001, P = 0.970 for blood; r2 = 0.644, P = 0.002 forlymph node). These results indicated that although thepercentages of T-lymphocyte subsets in the blood roughly

reflect the populations in the lymph nodes, interrelations in thelymph node may be not be evident in the blood.

CD8+ T-Lymphocyte Phenotypes in LymphNodes Correlate More Closely to ViremiaThan Those in Blood

Because most viral replication occurs in lymphoidtissues, the relationship of viremia to CD8+ T-lymphocytesubsets in the blood and lymph nodes of seropositive subjectswas evaluated (Fig. 8). Examination of CD38 expressionconfirmed a weakly positive correlation of expression on bloodCD8+ cells to viremia (see Fig. 8A; r2 = 0.220, P = 0.058) but a

FIGURE 3. Comparison of T-lymphocyte subsets in lymphnodes of HIV-1–seronegative and –seropositive subjects.Lymph node lymphocytes from the HIV-1–seronegative (whiteboxes) and seropositive (shaded boxes) subjects were analyzedby flow cytometry to assess subsets of CD8+ and CD4+

lymphocytes as in Figure 1. *Bootstrap-adjusted P , 0.05(see Table 2). A, Percentage of CD8+ T lymphocytes withintotal lymphocytes and percentages of various subsets of CD8+

T lymphocytes. B, Percentage of CD4+ T lymphocytes withintotal lymphocytes and percentages of various subsets of CD4+

T lymphocytes.

FIGURE 4. Ratios of T-lymphocyte subsets between seropos-itive and seronegative subjects in blood and lymph nodes.Blood and lymph node lymphocytes from the HIV-1–seropos-itive and –seronegative subjects were analyzed by flowcytometry to assess subsets of CD8+ and CD4+ lymphocytesas in Figures 1 and 2. For each subset in the blood or lymphnode, the ratio of mean values for seropositive versusseronegative subjects was calculated (1 SD in brackets). A,Ratios for CD8+ T lymphocytes are plotted. B, Ratios for CD4+ Tlymphocytes are plotted.



much stronger association of lymph node CD8+ cells (seeFig. 8B; r2 = 0.505, P = 0.001). Further examining thepercentage of terminal effector CD8+ T lymphocytes(CD62L2/CD45RA+) in blood, there was no clear correlationto viremia (see Fig. 8C; r2 = 0.080, P = 0.374), whereas thepercentages in the lymph node showed a robust negativecorrelation (see Fig. 8D; r2 = 0.520, P = 0.008), indicating thatthe lymph node but not blood concentration of these effectorcells is an important determinant of antiviral activity. Thememory-effector subsets (CD62L2/CD45RA2) in the bloodand lymph nodes were somewhat positively associated withviremia (r2 = 0.250 and 0.288, P = 0.098 and 0.072 for bloodand lymph node, respectively; not shown), suggesting anti-genic dependence of the frequency of these cells but notantiviral activity.

A multiple linear regression model was applied to CD38expression on lymphocytes in the blood and lymph nodes, thepercentage of CD622/CD45RA+ lymphocytes in the lymphnode, and viremia (see Table 3). This model suggested thatCD38 expression on CD8+ T lymphocytes in the lymphnode (but not blood) was the sole significant predictor ofviremia (P = 0.046). This agreed with a subsequent MarkovChain/Monte Carlo simulation. This model therefore sug-gested that the activation status of CD8+ T lymphocytes asreflected by CD38 expression is an independent predictor ofimmune containment of HIV-1 replication in lymph nodes.

DISCUSSIONThe interaction of CD8+ T lymphocytes with HIV-1

in vivo, although believed to be crucial in pathogenesis,remains obscure. One necessary assumption for most studiesof this interaction is that examinations of cells and virus in theblood provide an accurate representation of processes in the

whole in vivo environment. Because lymphocytes, which aremajor targets of infection and effectors of cellular immunityagainst HIV-1, traffic between blood and tissues, it is rea-sonable to assume that T lymphocytes in tissue and bloodcompartments somewhat mirror each other. Supporting thisview, a detailed study of CD8+ T lymphocytes in HIV-1–infected individuals demonstrates that the targeting of HIV-1–specific CD8+ T lymphocytes is highly similar in blood andlymph nodes.13 It is less clear, however, whether the functionalstatus of the T lymphocytes in the blood and lymph nodes isalso similar. Multiple studies have sought to investigate CD8+

T-lymphocyte function or phenotype as a determinant for theefficacy of the immune response against HIV-1, but most havebeen performed using peripheral blood, which is not the majorsite of viral replication.

Our data indicate that there are baseline differencesbetween T lymphocytes in the blood and lymph nodes of HIV-1–uninfected persons. There is a much lower ratio of CD8+ toCD4+ T lymphocytes. There are fewer differentiated CD8+ ef-fector T lymphocytes, as reflected by the higher percentage ofCD28 expression, but the activation status is similar. AmongCD4+ T lymphocytes, there is, again, a lower percentage ofCD282 effector cells but also more marked differences in theactivation status, notable for a greater percentage of HLA-DR–expressing cells. Overall, there are more differences inthe CD4+ T-lymphocyte compartment than in the CD8+

T-lymphocyte compartment when comparing lymph node andblood in the absence of HIV-1 infection.

Comparing the lymph node T-lymphocyte populationsbetween HIV-1–seronegative and –seropositive persons, theCD8+ T lymphocytes are predominantly affected, similar toblood but to a greater degree. The CD282 and CD38+/HLA-DR+ population expansions are proportionally greater in thelymph node versus the blood, indicating that analysis of thesesubsets in the blood of HIV-1–infected persons underestimatesthe changes in the total lymphocyte pool. Changes in the CD4+

lymphocyte phenotypes, by contrast, are far more similarbetween the blood and lymph node. HIV-1 infection thereforeprovokes greater alterations in the CD8+ T lymphocytes oflymph nodes than in those of peripheral blood.

It has been suggested that reduced perforin expression inblood22–25 and lymph node26 CD8+ T lymphocytes may bea mechanism of immune failure in HIV-1 infection. We findthat the mean perforin expression level is much lower in thelymph node than in the blood of HIV-1–infected persons butthat this difference is similar to that in HIV-1–seronegativesubjects. It seems that the percentages of perforin-expressingCD8+ T lymphocytes might, in fact, be somewhat increased inHIV-1 infection; thus, depletion compared with the uninfectedstate does not seem to be a mechanism of immune failure inHIV-1 infection. These data do not exclude inadequate eleva-tion of perforin-expressing cells or reduction of perforinexpression in the HIV-1–specific subset of CD8+ T lympho-cytes, however.

Interestingly, global assessments of CD8+ T-lymphocytephenotypes in the lymph node do correlate with plasmaviremia. There is a positive correlation of CD38 expressionand viremia in agreement with the prior observation ofCD38 expression on peripheral blood CD8+ T lymphocytes

FIGURE 5. Perforin expression in CD8+ T lymphocytes of theblood and lymph nodes of HIV-1–uninfected and –infectedsubjects. Freshly isolated lymphocytes were assessed by flowcytometry for expression of surface CD3 and CD8 as wellas intracellular perforin. The percentage of CD3+/CD8+

T lymphocytes was measured using gates defined byperforin expression of natural killer cells (high perforin).



correlating to viremia.12 Expression levels on cells in the bloodand lymph nodes are correlated, but statistical analysissuggests that the predictive value of CD38 expression oncells in the lymph nodes for viremia is independent of that inblood. Furthermore, the percentage of CD8+ terminal effectorcells of the CD62L2/CD45RA+ phenotype in the lymph nodeis inversely correlated to viremia, but this correlation is notobserved in blood. The percentage of these cells is also closelyinversely correlated with CD38 expression levels within thelymph node but, again, not in blood. Thus, despite general

correlations of cell populations between blood and the lymphnode, the interrelations of populations within the lymph nodeare obscured in blood.

The mechanism behind the relationships of CD38 ex-pression on CD8+ T lymphocytes within the lymph node toviremia is unclear. Prior work has demonstrated a clearphenomenon of a positive correlation between blood CD8+

T-lymphocyte CD38 expression and viremia as well as diseaseprogression.10–12 Our data suggest that this correlation is moredirect in lymph nodes, supporting a relationship based on the

FIGURE 6. Correlations betweenCD8+ T-lymphocyte subsets in theblood and lymph nodes of infectedsubjects. The percentages of totalCD8+ T lymphocytes within lympho-cytes and percentages of subsetswithin CD8+ T lymphocytes in theblood are plotted against the corre-sponding subsets within the lymphnode. Each point represents the valuesfor 1 infected subject, and linearregression statistics (least squares) forthe whole population are indicated.



interaction of these cells with HIV-1. It has been hypothesizedthat the expression of CD38 could be a marker of ineffectiveCD8+ T lymphocytes and therefore directly associated withdefective responding cells or expressed secondary to excessive

immune activation because of general immune failure.27 Priorcorrelative work using blood supports the latter hypothesis,finding that CD38 expression is a predictor of diseaseprogression independent of viremia or CD4+ T-lymphocyte

FIGURE 7. Correlations betweenCD38 expression and the percentageof CD622/CD45RA+ terminal effectorCD8+ T lymphocytes in the bloodand lymph nodes. The median expres-sion of CD38 (number of molecules)on CD8+ T lymphocytes is plottedagainst the percentage of theCD62L2/CD45RA+ subsets withinCD8+ T lymphocytes in the blood(A) or lymph node (B). Each pointrepresents the values for 1 infectedsubject, and linear regression statistics(least squares) for the whole popula-tion are indicated.

FIGURE 8. Correlations betweenHIV-1 viremia and CD8+ T-lympho-cyte subsets in the blood and lymphnodes. The median numbers ofCD38 molecules found on CD8+

T lymphocytes in the blood (A) andlymph node (B) are plotted againstthe level of viremia in each seropos-itive individual. The percentages ofCD62L2/CD45RA+ (terminal effectorcell phenotype) cells within the totalCD8+ T-lymphocyte compartment ofblood (C) and lymph node (D) areplotted against the level of viremia ineach seropositive individual. Linear re-gression statistics (least squares) forthe each population are indicated.



concentration.12 The observation that CD38 expression onCD4+ T lymphocytes also predicts disease progression28,29

lends additional support to this concept of CD38 being ageneral marker for immune failure through immune activa-tion, independent of viral replication.

Our data on T lymphocytes in lymph nodes provideevidence for the possibility that CD38 expression may be moredirectly associated with CD8+ T-lymphocyte antiviral function.CD38+/CD8+ T lymphocytes are believed to be more prone toapoptosis.30–32 Also, multiple functions in T lymphocytes havebeen ascribed to the CD38 molecule itself, including enzy-matic activity of protein ribosylation,32 serving as an adhesiveby binding CD31 on endothelial cells,33 acting as a costimu-latory receptor,34,35 and perhaps assisting in nucleotidescavenging in exhausted cells.35 Whether expression of theCD38 molecule has direct functional consequences or servesas an indirect marker for the status of CD8+ T lymphocytesremains to be determined.

The inverse correlation of lymph node CD8+ T lym-phocytes of the CD62L2/CD45RA1 phenotype to viremia isa novel finding. These cells are believed to be terminally dif-ferentiated effector cells that are high-efficiency killers,19 andskewed maturation of HIV-1–specific CD8+ T lymphocytescausing an arrest at the CD45RA2 preterminally differeniatedstage has been proposed as a specific mechanism of immunefailure in HIV-1 infection.20 Although this potential perturba-tion of CD8+ T-lymphocyte development has been demon-strated by comparing the phenotypes of HIV-1–specific versuscytomegalovirus (CMV)-specific CD8+ T lymphocytes inHIV-1–infected persons, direct evidence of functional conse-quences has been lacking. Our data demonstrate a significantnegative correlation of CD62L2/CD45RA+ CD8+ T lympho-cytes in the lymph node to viremia, suggesting that these cellsmay be involved directly in viral suppression in vivo. This cor-relation, although clear with cells from the lymph node, is notobserved with cells from the blood, supporting the hypothe-sis that the key HIV-1 immune interactions occur in lymphoidtissues and not in blood.

It is unclear to what degree CD38 expression andCD62L2/CD45RA+ differentiation of CD8+ T lymphocytesare independent or interrelated. It has been observed by chro-mium release assays on sorted cells that the CD38+/HLA-DR+

compartment contains more HIV-1–specific CD8+ T-lympho-cyte activity than the CD38+/HLA-DR2 or CD382/HLA-DR+

compartment.36 These data do not exclude the possibility thatCD38 is a marker for ineffective CD8+ T lymphocytes; how-ever, because we find no association of the CD382/DR+

population with reduced viremia, this seems unlikely. Never-theless, these results must be interpreted with caution, becausethe distribution of CD38 and DR expression on HIV-1–specific CD8+ T lymphocytes may not be reflected by that ofthe CD8+ T-lymphocyte population as a whole. More detailedstudies are required to define whether these factors are mech-anistically distinct in their associations with viremia.

Finally, a factor that could affect our results is antire-troviral drug therapy. The individuals in this study had highlysuppressed or varying levels of viremia attributable totreatment. Such treatment can reduce the levels of activation37

and HIV-1–specific cellular immunity.38–40 Thus, it is possible

that treatment of the research subjects blunted cellular acti-vation and reduced the differences we detected. Anotherconsideration is whether drug therapy contributed to the dif-ferences noted. Further study of untreated individuals isnecessary to discriminate these possibilities.

In summary, we find that there are potentially importantdifferences between T lymphocytes in the blood and lymphnodes. Baseline differences are notable in the ratio andphenotypes of CD4+ and CD8+ T lymphocytes in HIV-1–uninfected persons. HIV-1 infection particularly affects thephenotype of the CD8+ lymphocytes, and the changes seen inthe peripheral blood underestimate those seen in lymph nodes.Although there are general correlations between T-lymphocytepopulations in the blood and lymph nodes, the interaction ofCD8+ T lymphocytes with HIV-1 is likely best seen with cellsfrom lymph nodes, which are the predominant source of viralreplication. These data introduce caveats in the interpretationof pathogenesis and vaccine studies of blood.

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APPENDIXThe MACS (available at: http://www.statepi.jhsph.

edu/macs/macs.html) includes the following individuals:

Baltimore, The Johns Hopkins University Bloomberg Schoolof Public Health: Joseph B. Margolick (PrincipalInvestigator), Haroutune Armenian, Barbara Crain,Adrian Dobs, Homayoon Farzadegan, Nancy Kass,Shenghan Lai, Justin McArthur, and Steffanie Strathdee

Chicago, Howard Brown Health Center, The Feinberg Schoolof Medicine, Northwestern University, and CookCounty Bureau of Health Services: John P. Phair(Principal Investigator), Joan S. Chmiel (Co-PrincipalInvestigator), Sheila Badri, Bruce Cohen, Craig Con-over, Maurice O’Gorman, Frank Pallela, Daina Varia-kojis, and Steven M. Wolinsky

Los Angeles, University of California, UCLA Schools ofPublic Health and Medicine: Roger Detels and BethJamieson (Principal Investigators), Barbara R. Visscher(Co-Principal Investigator), Anthony Butch, John Fahey,Otoniel Martınez-Maza, Eric N. Miller, John Oishi, PaulSatz, Elyse Singer, Harry Vinters, Otto Yang, andStephen Young

Pittsburgh, University of Pittsburgh, Graduate School ofPublic Health: Charles R. Rinaldo (Principal Investiga-tor), Lawrence Kingsley (Co-Principal Investigator),James T. Becker, Phalguni Gupta, John Mellors, SharonRiddler, and Anthony Silvestre

Data Coordinating Center (Baltimore), The Johns HopkinsUniversity Bloomberg School of Public Health: AlvaroMunoz (Principal Investigator), Lisa P. Jacobson (Co-Principal Investigator), Haitao Chu, Stephen R. Cole,Janet Schollenberger, Eric Seaberg, Michael Silverberg,and Sol Su

National Institutes of Health (Bethesda), National Institute ofAllergy and Infectious Diseases: Carolyn Williams

National Cancer Institute: Jodi Black



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