differential susceptibility of leukocyte subsets to cytotoxic t cell killing: implications for hiv...

Original Article

Differential Susceptibility of Leukocyte Subsetsto Cytotoxic T Cell Killing: Implications

for HIV Immunopathogenesis

Jie Liu and Mario Roederer*ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland

Received 21 September 2006; Revision Received 8 November 2006; Accepted 20 November 2006

Background: Cytotoxic T lymphocytes (CTL) are crucialfor the host defense against viral infection. In many cases,this anti-viral immune response contributes to host patho-genesis, through inflammation and tissue destruction. Fewstudies have explored the relative susceptibility of in-fected cells to CTL killing, and the range of cell types thatmay be effectively killed by CTLs in vivo, both of whichare key to understanding both immune control of infec-tion and immune-related pathogenesis.Methods: We developed and optimized a highly sensitivemethod to quantify the relative susceptibility of leukocytesubsets to CTL-mediated killing. Maximal sensitivity wasachieved by uniquely measuring cell death occurring dur-ing the assay culture.Results: We found that leukocyte subsets have a widerange of susceptibility to antigen-specific CTL-mediatedlysis. Generally, T cells were more susceptible than B or

NK cells, with CD4 T cells being more susceptible thanCD8 T cells. In all lymphocyte lineages, susceptibility wasgreater for more differentiated subsets compared withtheir na€ıve counterparts; however, for dendritic cells,immature cells are more susceptible than mature cells. Wefocused on the susceptibility of T cell subsets, and foundthat na€ıve cells are far more resistant than memory cells,and in particular, CCR51 or HLA-DR1 memory cells arehighly susceptible to CTL-mediated killing.Conclusions: These results provide an explanation forthe observation that certain subsets of CD4 T cells areablated during chronic HIV infection, and indicate whichsubsets are most likely to contain the persistent viral reser-voir. Published 2007 Wiley-Liss, Inc.�

Key terms: cytotoxic T cells; CCR5; HIV; leukocytes

Cytotoxic T lymphocytes (CTL) are one of the key com-ponents of host defense against viral infection and for tumorcell elimination. Strong virus-specific cytotoxic CD8 T cellresponses are observed in most HIV, EBV, CMV and HCVinfections and correlate with the control of viral replicationand disease progression. Thus, the assessment of CD8 T cellcytotoxic function becomes a crucial measurement to moni-tor antigen specific immune responses for evaluating the ef-ficacy of vaccines aimed at eliciting cellular immunity.

CTL control viral replication by killing infected cellsand/or releasing soluble anti-viral factors. In many cases,this anti-viral response also contributes to host pathologythrough inflammation and tissue destruction. Indeed, thelatter may significantly impact immune function itself: forexample, the CD8 T cell-mediated killing of HIV-infectedCD4 memory T cells might alter the landscape of CD4 Tcell immunity, as, for example, HIV-specific and CMV-spe-cific CD4 T cells are preferentially infected by HIV (1).Neither HIV nor the herpesviruses are completely eradi-

cated from the host; these viruses hide in privileged reser-voirs, escaping immune control and leading to persistentinfections (2–13). It remains to be determined what char-acteristics of the reservoir make it resistant to immunecontrol, particularly, the mechanism of its apparent insen-sitivity to CTL.Most transmitted forms of HIV and SIV (the nonhuman

primate equivalent of HIV) infect CD4 T cells by bindingto both the CD4 molecule as well as the chemokine recep-tor CCR5. Thus, the primary target for these viruses ismemory CD4 T cells, since na€ıve cells do not express

�This article is a US government work and, as such, is in the public do-

main in the United States of America.

*Correspondence to: Mario Roederer, 40 Convent Dr., Room 5509,

Bethesda, MD 20895, USA.

E-mail: [email protected] online 2 January 2007 in Wiley InterScience (www.

interscience.wiley.com).

DOI: 10.1002/cyto.a.20363

Published 2007 Wiley-Liss, Inc.� Cytometry Part A 71A:94–104 (2007)

CCR5. Indeed, CCR51CD4 memory T cells are dramati-cally reduced, compared with CCR5–CD4 memory T cells,during chronic infection (14–17). Thus, it was a surprisethat both CCR51 as well as CCR52 memory T cells wereequivalently infected by SIV during acute infection (16).This paradox is explained by the fact that essentially allmemory T cells express CCR5; the ‘‘CCR52’’ memory Tcells simply do not express sufficient quantities to be de-tectable by flow cytometry. This raises the question: if allmemory CD4 T cells are CCR51 and equivalently infectedby SIV (or HIV), then why are the cells expressing highlylevels of CCR5 selectively depleted? We hypothesized thatthis depletion may be a consequence of immune mechan-isms, i.e., that ‘‘CCR51’’ CD4 T cells, once infected, aremore susceptible to immune control than their ‘‘CCR52’’counterparts.

There are few studies on the relative susceptibility ofinfected cells to CTL killing and the range of cell typesthat may be effectively killed by CTLs in vivo. Quantifyingtarget susceptibility is key to understanding both immunecontrol of infections during (persistence), as well asimmune-mediated pathogenesis. However, studying CD8T cell function by conventional CTL assays provides lim-ited information on target cell susceptibility. Traditionalassays such as the 51Cr- or 3H-release assays (18,19) onlymeasure the total cell death and mask any heterogeneitywithin the target cells. Some studies have shown that dif-ferent cell lines exhibit different susceptibilities to specificCTL cytolysis (20), suggesting that there could indeed beheterogeneity in target susceptibility to CTL.

Recently, a number of flow cytometry-based assays forCTL killing have been developed; flow-based assays havethe advantage of identifying targets on a cell-by-cell basisand thus revealing underlying heterogeneity in suscepti-bility. Such assays include the use of 7-AAD to stain CFSElabeled target cells (21); propidium iodide (PI) incorpora-tion into GFP transfected P815 cells (22); DAPI uptakeby CMTMR-labeled target cells (23); detection of acti-vated fluorogenic caspases (24); or combining Annexin Vwith PI to determine tumor cell apoptosis vs. necrosis(25). These assays, using fluorochromes instead of radio-active isotopes, provide a simple, non-radioactive alterna-tive to classical cytolysis assays, with reasonable sensitiv-ity and relatively low cell number requirement. To date,most of these assays studied immortalized cell lines orclones and provided limited information regarding thesusceptibility of primary cells to cytolytic killing. Weundertook to adapt the advantages of the flow cyto-metric-based fluorescence assays to quantify CTL suscep-tibility of target cells ex vivo.

Based on our polychromatic flow cytometry technol-ogy, we developed a novel method to study the suscept-ibilities of peripheral blood leukocytes to CD8 T cell-mediated killing. The essential point of this assay is theability to discriminate effector from target cells and fur-ther discriminate subsets of targets to reveal heterogeneityin target susceptibility. We found there is a diverse rangeof susceptibilities among peripheral blood leukocytesassociated with cell lineage, differentiation stage and acti-

vation status. Generally, differentiated cells, such as differ-entiated T, B and NK cells, are more susceptible than less-differentiated cells. Similarly, activated T cells are moresusceptible than resting T cells. Importantly, CCR51memory T cells are considerably more sensitive to CTLkilling than CCR52 memory T cells. Thus, quantificationof CTL susceptibility reveals a rich biology of immune-mediated control of viral infections, and provides an ex-planation for the observation that CCR51 memory T cellsare profoundly depleted in SIV and HIV infection.

MATERIALS AND METHODSCulture Medium, Antibodies, and Dyes

Cells were cultured in RPMI 1640 (Invitrogen, CA) sup-plemented with 10% FBS, L-glutamine, streptomycin, andpenicillin. rIL-2 (NCI, MD) at 100 U/ml and rIL-7 (PeproTech, NJ) at 10 ng/ml were added for CD8 T clones. AllmAb reagents, either purified or pre-conjugated wereobtained from BD PharMingen (San Diego, CA); anti-CD4and anti-CD8 were conjugated in our laboratory and CCR7was obtained from R&D Systems (Minneapolis, MN). Fluo-rescein Isothiocyanate (FITC), 5-(and -6)-carboxyfluores-cein diacetate succinimidyl ester (CFSE), Alexa 680, 7-Ami-noactinomycin D (7-AAD), Ethidium Monoazide (EMA),and Cascade Blue were obtained from Molecular Probes(Eugene, OR). Phycoerythrin (PE) and Allophycocyanin(APC) were obtained from ProZyme (San Leandro, CA).Cy5 and Cy7 were obtained from Amersham Life Sciences(Pittsburgh, PA). Qdot 605, Qdot 655, and Qdot 705were obtained from Quantum Dot (Hayward, CA). PIwas from Aldrich-Sigma (St. Louis, MO). Fluorescent con-jugations were carried out following standard protocols(http://www.drmr.com/abcon); conjugates were validatedby comparison with commercial conjugates. Annexin V-FITC was from BD PharMingen and Annexin V-CascadeBlue was conjugated in our laboratory. Monomeric pep-tide-major histocompatibility class I (pMHCI) proteinswere produced as described previously with minor modi-fications (26,27). For the purposes of this study, the CMVpeptide was synthesized to >95% purity (Research Genet-ics, NY) and refolded with the HLA-A2 molecules. Conju-gation to streptavidin (SA)-coated PE was carried out at afinal pMHCI:SA molar ratio of either 4:1 or 3:1 assumingsteric hinderance of one free biotin-binding site on eachSA molecule; equal aliquots of SA-PE were added sequen-tially to soluble pMHCI monomer to ensure completesaturation.

Generation of CD8 T Cell Clones

CD31CD81 cells that bind the CMV-peptide-MHC mul-timer were sorted from PBMC of CMV seropositivehealthy HLA-A2 donor with a FACSVantage (BD Bio-sciences, CA). Sorted cells were seeded into 96-well platesat one cell per well and cultured with PBMCs from healthydonor as feeder cells, which were pre-stimulated withPHA at 2.5 lg/ml (Sigma-Aldich, MO) and then irradiatedwith 40 Gy. Anti-CD3 antibody was added at a final con-centration of 1 lg/ml to stimulate expansion. Cells in

95DIFFERENTIAL SUSCEPTIBILITY OF PBMC TO CTL KILLING

Cytometry Part A DOI 10.1002/cyto.a

wells that showed clonal expansion were collected andtransferred to 24-well plates for further proliferation.Three weeks later, the expanded CD8 T cell clones werestimulated with CMV peptide by loading an HLA-A21 Blymphoma cell line with the peptide at 100 lg/ml (thecell line was irradiated with 160 Gy). The CMV-specificproliferating CD8 T cell clones were further expanded.The clones were re-stimulated with CMV peptide every3–4 weeks depending on proliferation.

RT-PCR

Examination of the TcR Vb usage by RT-PCR was doneas previously described (53). BV primers were the kindgift of Dr. Daniel Douek (VRC, NIH).

Microscopic Assay

CMV-loaded PBMCs were coated onto microculturewells to form a single layer on the glass bottom of micro-well dishes (Mat Tek, MA). CTL clones that were stainedwith anti-CD8 conjugated with Alexa 488 were seededinto the culture. PI was added into the culture medium asa cell death indicator. The culture was placed into a cli-mate control chamber with a constant temperature of37�C and humidified. Images were taken sequentially with30 s intervals, using a computer controlled Axiovert 200Mmicroscope (Zeiss, NY).

Cytotoxicity Assay

Target cell preparation: Whole blood from healthyHLA-A21, CMV sero-negative donors were obtainedfrom the Blood Bank, National Institutes of Health (Be-thesda, MD). Peripheral blood mononuclear cells(PBMCs) were isolated by Ficoll gradient-centrifugationand resuspended in RPMI 1640 culture medium. ThesePBMCs were pulsed with CMV peptide (NLVPMVATV) at10 lg/ml, in 37�C for 1 hr. PBMCs without CMV peptidewere used as negative control. Cells were then stainedwith fluorescently-conjugated antibodies and FITC-con-jugated Annexin V at room temperature for 15 min.Effector cell preparation: CD8 T clone cells were stainedwith CFSE (Molecular Probes, OR) at 0.25 lM. Assay:Effector and target cells at indicated E:T ratios were cul-tured in U-bottom 96-well plates. Cells were centrifugedat 300g for 2 min. At selected time intervals, cells wereharvested and stained with Annexin V-Cascade Blue.Flow cytometric analysis was carried out on an LSR-II(BD Biosciences, CA).

Preparation of Dendritic Cells

Isolation of myeloid dendritic cells (DC) was performedas described previously (28). Isolated DCs were culturedin the presence of IL-3 (1 ng/ml; R&D Systems, Minneapo-lis, MN) and GM-CSF (2 ng/ml; PeproTech, Rocky Hill, NJ).Lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO)was used at 100 ng/ml to mature DCs for 24 h at 37�C.

Data Collection and Analysis

Cells were analyzed on a modified Becton DickensonLSR II (BD Biosciences, San Jose, CA). Flow cytometricdata were analyzed using FlowJo version 8.1.1 (Tree Star,Inc., Ashland, OR). Statistical comparisons of distributionswere performed using the Student’s t-Test in JMP version5.1 (SAS Institute, Cary, NC).

RESULTSCytotoxic Potential of CMV-Specific

CD8 Cell T Clones

CMV-specific CD8 T cell clones were generated fromsingle CD31CD81HLA-A2/CMV multimer-binding lym-phocytes that had been sorted into wells and expanded bypeptide stimulation. After expansion, we studied the phe-notype, cytotoxic enzyme content, and TCR Vb usage ofthese clones. All the generated T cell clones were specificfor the CMV peptide (both by proliferative responses andby HLA-A2/CMV multimer binding), CD31CD81CD42,and expressed a single TCR Vb allele. Most expressed per-forin and granzyme B. Data shown in Figure 1 are forclone 0920CMVD1 (D1 clone). D1 cells express TCR BV7(Vb6 in older nomenclature) by RT-PCR, and have aneffector-memory phenotype. In addition, after resting 1week post-stimulation, the cells are CD562, CD572, HLA-DR high, and CD951 (not shown).We characterized the cytotoxic potential of the D1

clone against CMV peptide-loaded PBMCs from HLA-A21,CMV seronegative donors by flow cytometry and micros-copy. A substantial portion of CMV peptide-loaded PBMCsstained with 7-AAD after co-culture with D1 clone for15min, indicating that many of the peptide-loaded cells werekilled in this short time frame. PBMCs not loaded withpeptide (and distinguished by being pre-stained with a flu-orescent anti-CD45 antibody) showed essentially no deathduring the same time frame (Fig. 2A). PBMC from an HLA-A2 negative donor showed no death whether or notthey were loaded with CMV peptide (Fig. 2D). Thus,clone D1 exhibits peptide-specific, HLA-A2-restricted cyto-toxicity.Microscopic evaluation revealed that target cells stained

with PI after a brief (5 min) contact with D1 clone (Fig.2B), showing that the killing began as soon as cell-cell con-tact was initiated, and could not mediated by Fas/FasLpathways. Further characterization of the assay conditions(Fig. 2C) showed that the proportion of the 7-AAD-stainedPBMCs correlated with the culture period and the ratio ofD1 cells to PBMCs (E:T ratio). There was little effect ofcell density on the assay, although killing was mildly betterat lower cell densities. Based on these results, we selectedthe following conditions as a standard for our cytotoxicityassay experiments: CMV peptide concentration 5 10 lg/ml; E:T ratio 5 1:1; cell density 5 1 3 106/well (5 3106/ml). Finally, we determined that staining withAnnexin V under these conditions revealed the samepopulation as 7-AAD (Fig. 2E), but with better discrimina-tion; for this and other reasons mentioned below, wechose to use Annexin V staining to identify the killed tar-

96 LIU AND ROEDERER


get cells. Various controls demonstrate that the killing isspecific to the properly matched peptide and MHC on thetargets (Fig. 2D).

Assay Optimization: Exclusion of Pre-AssayDead Cells

As with most existing CTL assays, flow cytometry-basedCTL assays also have a background of cell death in testand control samples; the variability in this backgrounddeath significantly impacts on the sensitivity of the assay(ability to detect low levels of killing). Most of the back-ground dead cells in the assay were dead prior to culture(data not shown); hence we reasoned that by pre-stainingthese cells we could eliminate this source of background.To maintain consistency in how we assessed cell death,we chose to use Annexin V conjugated with either FITCor Cascade Blue to identify cells that had died at differenttimes.

Before addition of effectors, target cells were stainedwith FTIC-conjugated Annexin V after loading with CMVpeptide, and then cultured with CTL clones. CascadeBlue-conjugated Annexin V was added at the end of theco-culture. Any cells that were positive for FITC-AnnexinV were dead prior to the initiation of the assay and couldbe excluded by gating. Cells stained only with CascadeBlue-Annexin V must then have died during the brief cul-ture period and were counted as specific killing (Fig. 3A).

The pre-staining had no impact on the quantitation ofkilling when the specific killing was large (e.g., >10%;

Fig. 3B). However, elimination of the pre-existing deadcells increased sensitivity by almost 10-fold, so that lysiscould be measured at E:T ratios as low as 1:64 (Fig. 3C).

Quantifying Susceptibility of PBMC Subsetsto CTL Lysis

We compared the susceptibilities of peripheral leuko-cytes to D1 clone-induced antigen specific cytolysis. Afterloading with CMV peptide and staining with a panel offluorescently-conjugated antibodies and FITC-Annexin V,the target PBMCs from a healthy CMV seronegative donorwere co-cultured with clone D1 CD81 CTL cells used asan effector population. The cultures were harvested andincubated with Cascade Blue-conjugated Annexin V atselected intervals and subjected to flow cytometric analy-sis. Pre-staining targets with antibody conjugates did notalter the kinetics or extent of killing (data not shown). Asshown in Figure 4, specific killing of PBMC subsets wasquantified by first gating on targets by forward- and side-scatter, then eliminating the effectors (stained with CFSE)and pre-existing dead cells (stained with FITC-Annexin V).Then other markers were used to identify the subset of in-terest; the specific killing is revealed by the proportion ofCascade Blue-Annexin V positive cells.Figure 5 illustrates this process for non-T mononuclear

cells. B lymphocytes were identified by the expression ofCD19; these cells were divided into CD27– na€ıve B cellsand CD271 memory B cells (29). During the standardCTL killing assay culture, a greater fraction of CD271 B

FIG. 1. Phenotype, cytotoxic enzyme and TCR Vb expression of clonally-expanded CMV-specific CD8 T cells. After clonal expansion of sorted singleCMV-tetramer1CD81 T cells, the phenotype and cytotoxic enzyme expression were assessed by flow cytometry. Illustrated is a typical phenotype, shownfor clone 0920CMVD1 (D1). For study of TCR BV expression, total RNA was isolated from cloned cells and analyzed by RT-PCR with specific primers fromall 24 BV genes. M represents DNA ladder marker. The PBMC control shows identification of all genes; the clone expresses only TCR BV7.



cells were killed than their CD27– na€ıve counterparts. Thisdifference could not be ascribed to differences in MHCclass I expression (data not shown); rather, it represents anintrinsic difference of the cells to lytic susceptibility.

Within the NK subsets (identified as CD3–CD19–lympho-cytes expressing either CD56 or CD16), there are four pheno-

typically distinct subsets comprising different differentiationstages (30). As seen for B lymphocytes, there is progressivelygreater susceptibility to CTL lysis with increasing differentia-tion stage (Fig. 5B). Thus, earliest NK cells, CD561CD162exhibit the greatest resistance to CTL; the most differentiated,CD562CD161, are the most susceptible.

FIG. 2. Cytolytic function of a CMV-specific CD8 T clone. (A) Target cells were PBMC from an healthy HLA-A21 donor loaded with CMV-peptide; negativecontrol was the same cells without CMV-peptide. The negative control cells were also stained with anti-CD45 and then mixed 1:1 with the peptide-loaded tar-get cells. In this assay, cell death was assessed by 7-AAD staining. (B) CMV-peptide loaded PBMC were cultured with CFSE-stained CD8 T cell clone in the pre-sence of 7-AAD. Using fluorescence microscopy, the 7-AAD-stained dead target cells (red-stained cell) could be identified within minutes of contacting theCFSE-stained effector. (C) The CD8 T cell clone was cultured with PBMC loaded with or without CMV-peptide for different periods of time; at different E:Tratios; with various CMV peptide concentrations; or at different densities of cell culture. (D) Specificity of the killing (top left) is shown by the requirementfor effectors (top right); the requirement for cognate peptide (bottom left); and the requirement for the appropriate MHC allele matching the peptide andeffector (bottom right). (E) Target cells were cultured with CTL effectors and co-stained with 7-AAD and Annexin V. Data shown is gated on target cells.Except as indicated in each panel, the conditions used were the ‘‘standard’’ conditions: E:T5 1:1, culture time5 60 min; peptide concentration5 10 lg/ml,and cell density5 13 106/200 ll.

98 LIU AND ROEDERER


We also examined myeloid cells to determine their sus-ceptibility to CTL lysis (data not shown). We identifiedmonocytes by expression of CD14 and high side scatter;these cells show a similar susceptibility to killing as lymph-oid subsets (approximately 15–20% killed within an hour).

DC are the primary antigen-presenting cells for generat-ing new T cell responses. Conceivably, an effective CTLresponse to an epitope expressed by a DC would kill thatcell and lead to the inability to generate novel T cellresponses to other epitopes presented by the same cell(leading to original antigenic sin). Thus, we examined thesusceptibility of purified immature myeloid DC and LPS-matured DC (Fig. 5C). Unlike all other cell types we exam-ined, mature DC showed decreased susceptibility to CTLcompared with immature. This may reflect a mechanismof DC to enable them to present antigen as long as possi-ble to drive immune responses even in the face of aneffective CTL response.

Susceptibility of T Cell Subsets to CTL Killing

Of all of the PBMC compartments, the T cells show thegreatest phenotypic heterogeneity; much work has beendone to define the functional correlates of the differentphenotypes, and models have been proposed that dividethese cells into na€ıve, central memory, effector memory,

and terminally-differentiated memory cells based on theexpression of markers such as CD45RA and CCR7 (31,32).As shown in Figure 6, subsets of CD4 T cells are generallyslightly more susceptible to CTL lysis than the correspond-ing CD8 T cell subsets. As was the case for NK cells and Bcells, we observed that the more differentiated subsetsshowed a greater susceptibility to CTL lysis, with na€ıvecells being virtually completely resistant to killing. Wecould not attribute these differences to expression levelsof MHC class I (not shown), suggesting that recognition ofthe cells by the CTL was likely equivalent.Among CD45RA- memory T cells, we could identify

CCR51 and CCR52 T cells, as well as HLA-DRbright andHLA-DRlow cells. HLA-DR identifies cells which are in anactivated state (although not mitotically active); highCCR5 expression may likely also be a correlate of activa-tion status. We noted a substantial difference in the sus-ceptibility of ‘‘resting’’ vs. ‘‘activated’’ memory T cells (Fig.6). Thus, for example, CCR51 CD4 memory T cells wouldbe killed five times more efficiently than CCR52 CD41memory T cells, if both were expressing foreign antigen.

DISCUSSION

We sought to quantify the differences in susceptibilityto CTL lysis among PBMC subsets. Using polychromatic

FIG. 3. Eliminating pre-assay dead cells significantly improves sensitivity. CMV-peptide loaded (open symbols) or unloaded (closed symbols) were cul-tured together with the CTL effectors at different E:T ratios. Panel A shows the staining using the two different conjugates of Annexin V; FITC Annexin Videntifies cells that were dead prior to assay; CasBlue Annexin V identifies cells that were dead at the conclusion of the assay. Hence, CasBlue1FITC– cellsare those that died during the assay and indicate assay-related cell death. Quantification of cell death during the culture for total cells (B) is limited by thefraction of cells that were dead prior to assay. By pre-staining cells with FITC-Annexin V and eliminating these from quantification, sensitivity is dramaticallyimproved (C). Data are shown as mean 6 1 standard deviation for replicates from one subject; data is representative of three experiments.



flow cytometry, we developed a sensitive, rapid assay tocharacterize the degree to which CTL can kill phenotypi-cally-identifiable subsets. As a model for CTL killing, weused CMV-specific CTL clones, as these are functionallyand phenotypically homogeneous; however, the assay caneasily be performed with fresh ex vivo isolated CTL. As wedemonstrate, the assay is highly specific to antigenic pep-tide presented by the cognate MHC allele, and proceedsin a kinetically predictable fashion that depends onthe effector:target (E:T) ratio, culture time, and peptideconcentration.

We use Annexin V as the primary probe to assess celldeath in this assay. Annexin V binds to the membrane phos-pholipid phosphatidylserine (PS), which is translocatedfrom the inner to the outer leaflet of the plasma membraneduring cell damage. Because this translocation happens atthe early stage of cell death, Annexin V can be used todetect apoptosis at a very early time point (33,34). Anotheradvantage of using Annexin V is that it can be conjugatedto variety of dyes to fit into different staining panels-and, infact, our assay uses two different conjugates of Annexin Vin the same panel. In addition, Annexin V can be combinedwith PI or 7-AAD to distinguish early apoptotic cell deathfrom late stages of apoptosis or necrosis (34,35).

Like other CTL assays, flow cytometry-based assays areconfounded by nonspecific death. However, a majority ofthis death occurred prior to the co-culture with effectors–it is not atypical to find 5% dead cells in normal cultures.The existence of non-specific cell death significantly com-promises the sensitivity of CTL assays. At low E:T ratios(<1/8), it was impossible to quantify the amount of CTL-induced death over the background.One of the principal advantages of our assay (as

afforded by the polychromatic technology) is the abilityto devote two different detectors to cell death. We usedFITC-Annexin V to stain the target cells before the addi-tion of CTLs and Cascade Blue-Annexin V after the cul-ture. Thus, we could eliminate the cells that were deadprior to culture; this reduced nonspecific killing in theassay by an order of magnitude, and leads to an increasein sensitivity of 5- to 10-fold. Indeed, the assay can bedone at E:T ratio as low as 1/64, making it possible toaccurately quantify CD8 cytotoxicity even for low-fre-quency populations (such as those found in ex vivoblood specimens).It is possible to use other dyes such as PI or 7-AAD to

identify cells which died prior to culture. However, wefound that the nuclear-staining dyes have different staining

FIG. 4. Gating strategy for flow cytometric data analysis. To identify subsets of target cells killed by CTL, two strategies were employed. First, FSC and SSCgating on lymphocytes eliminated most of the effector cells (top left). Second, effector CTL were also labeled with CFSE; only CFSE-negative cells were furtheranalyzed (top middle). Pre-assay dead cells were labeled with FITC-Annexin V and also fall into this exclusion gate. The CFSE–, FITC– cells (bottom left) couldthen be phenotyped, for example according to the expression of CD3, and specific cell death assessed by the staining of CasBlue Annexin V (bottom right).

100 LIU AND ROEDERER


properties and sensitivities than Annexin V (36). Someimprovement was seen by using the combination of thesedyes together with FITC Annexin V; however, we foundusing Annexin V alone to distinguish cells that died beforethe assay provided sufficient sensitivity for our goals.

We developed two staining panels to distinguish periph-eral leukocyte populations. In the T cell panel, we usedanti-CD3, CD4, and CD8 to distinct T cell lineages; anti-CD45RA and CCR7 to classify na€ıve and memory differen-tiation stages; and anti-HLA-DR and CCR5 to distinct rest-ing or activated T cells. In the non-T cell panel, we usedanti-CD3, CD14, CD16, CD19, and CD56 to identify dis-tinct cell lineages and NK differentiation stages, togetherwith anti-CD27 to classify na€ıve or memory B cells. Thesesurface markers and antibodies were selected based ontheir functional correlate as well as their stability duringassay period (data not shown). Indeed, we found that

some markers (such as CD62L) were substantially down-modulated in cells that were killed by the CTL, renderingthis marker unusable for identifying subsets following theculture period.For the lymphocyte subsets, we found that T cells were

more susceptible to killing by CTL than B cells or NK cells,with CD4 T cells being more susceptible than CD8 T cells.Within each of these lineages, we uniformly found thatsusceptibility to CTL lysis was greater for differentiatedsubsets compared with their na€ıve counterparts. This sug-gests an intrinsic mechanism that protects (to someextent) na€ıve cells against CTL killing. Certainly, it isknown that BCL-2 expression and intracellular glutathionelevels are higher in na€ıve T cells (37–43)–however, thisassay quantifies perforin-mediated killing within 30–60 min, killing which is unlikely dependent on either BCL-2 or glutathione levels. Thus, there must be additional

FIG. 5. Comparison of susceptibility of B & NK cellsubsets to CTL. (A) CD32 lymphocytes were dividedinto CD191 (B cells) that were CD272 (na€ıve) orCD271 (mature). (B) CD32CD192 lymphocytes weregated based on anti-CD16 or anti-CD56 antibody stain-ing, and further divided into CD161 CD561,CD161CD562, CD162CD56bright and CD162CD56low

subsets. (C): Myeloid DC were isolated from peripheralblood and a portion of them matured with LPS. Data areshown as mean 6 1 standard deviation for six subjects.



mechanisms expressed by na€ıve T cells that render themrelatively resistant to killing by CTL.

The only exception to this rule was for DC: LPS-matured DC were more resistant than their immature

counterparts. Because of the importance of DC to initiatena€ıve T cell responses, this resistance may reflect a needby the immune system to preserve as much as possiblethe ability to generate novel T cell responses even in the

FIG. 6. Comparison of susceptibility of T cell subsets.(A) CD4 or CD8 T cells were identified by gating onCD31 lymphocytes. (B) Either CD4 and CD8 T cellwere classified into na€ıve (NA), central memory (CM),effector memory (EM) and terminal effector (TE) sub-sets, depending on their expression of CCR7 andCD45RA. The graphs to the right of these panels showthe relative susceptibility for these differentiation sub-sets within CD4 (upper) or CD8 (lower) T cells. (C)CD45RA2CD41 memory cells were divided intoCCR51 or CCR5- subsets; susceptibility for only theCD45RA- memory T cells is shown to the right. (D)CD45RA-CD41 memory cells were divided into HLA-DR1 or HLA-DR- subsets; susceptibility for the CD45RA-subsets is shown to the right. Error bars represent 61standard deviation for n 5 6 subjects; significant differ-ences are shown with corresponding P-values.



face of a pre-existing effective CTL response to other epi-topes of the same pathogen. It is also known that matureDC express more serpin proteinase inhibitor (PI) 9 andserine protease inhibitor (SPI) 6, both of which are gran-zyme B inhibitors that can protect cells from CTL-inducedcytolysis (44,45). These two types of DCs have distinctroles in immune system (46). Immature DC capture, pro-cess antigen and can induce tolerance; mature DC initiateeffective antigen specific T cell responses (47–49). Thus,the preferential killing of immature antigen-bearing DCmight help to maintain a sustained and positive anti-viralimmunity. The persistence of matured DC is also requiredfor memory T cell development (50,51).

Recently, it was demonstrated that essentially all mem-ory CD4 T cells express CCR5; the ‘‘CCR52’’ memory Tcells do not express sufficient quantities of CCR5 to beidentified by flow cytometric phenotyping. Nonetheless,both CCR51 and CCR52 memory T cells are roughlyequivalently susceptible to infection by SIV (16), whichuses CCR5 as its coreceptor. This observation led to a co-nundrum: if both CCR51 and CCR52 CD4 memory Tcells were infected by SIV, why is there such a predomi-nant loss of CCR51 cells (particularly, during the chronicphase)?

In this study, we found that CCR51 memory T cells arenearly 10-fold more susceptible to CTL killing. Thus, givena heterogeneous population of CD4 memory T cells, forwhich the subsets are equivalently infected by SIV, aneffective CTL response will eliminate the CCR51 cells farmore quickly. In this way, an effective CTL response candramatically impact on the equilibrium representation ofCD4 memory T cell subsets, and thereby shape theimmune system.

Resting CD4 T cells have been repeatedly demon-strated to be a reservoir of HIV (3–5,9,12,13), leading topersistent HIV infection. Here we show that restingmemory T cells (and na€ıve T cells) are highly resistant toCTL lysis. This may explain why CTL have a limited effecton viral replication in the latent viral reservoir (52): virusharbored in those cells can escape the specific immuneattack.

In summary, we describe an optimized, highly-sensitiveflow cytometric-based assay to quantify very low levels ofCTL, as well as to quantify the heterogeneity of suscepti-bility of target populations to CTL killing. The assay ishighly flexible, and can be adapted to studying CTL activ-ity directly ex vivo. Using this assay, we characterized thesusceptibility of PBMC subsets to CTL killing, and therebyprovide an explanation for the loss of CCR51 memory Tcells during chronic HIV infection. This reveals yetanother complexity in HIV disease pathology: the remo-deling of the immune system as effected by the antigen-specific CD8 response.

ACKNOWLEDGMENTS

We thank Dr. Karin Lore for help with the preparationof DC, Steve Perfeto for expert advice and help with cellsorting, Joanne Yu for assistance with sample processing

and antibody conjugation, and Drs. Stephen De Rosa, Jo-seph Mattapallil, Yolanda Mahnke, and Pratip Chattopad-hyay for comments and discussion.

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