Sensitive and Viable Identification of Antigen-specific CD8+ T Cells

www.elsevier.com/locate/jim

Journal of Immunological Methods 281 (2003) 65–78

Sensitive and viable identification of antigen-specific CD8+ T cells

by a flow cytometric assay for degranulation

Michael R. Betts*, Jason M. Brenchley, David A. Price, Stephen C. De Rosa,Daniel C. Douek, Mario Roederer, Richard A. Koup

Laboratory of Immunology, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,

40 Convent Drive, Bethesda, MD 20892, USA

Received 22 January 2003; received in revised form 25 June 2003; accepted 7 July 2003

Abstract

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or

intracellular cytokine production, neither of whichmeasure the cytolytic potential of these cells. Here we present a novel technique

to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced

degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of

cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with

loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is

dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate

peptide, concordant with production of intracellular IFNg. Finally, CD107-expressing CD8+ T cells are shown to mediate

cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined withMHC-class

I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8 +Tcells

expressing cognate T cell receptors (TCR).

D 2003 Elsevier B.V. All rights reserved.

Keywords: Degranulation; T lymphocyte; Intracellular cytokine; CD107a; CD107b

0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0022-1759(03)00265-5

Abbreviations: APC, allophycocyanin; CFSE, carboxyfluorescein diacetate succinimidyl ester; CMTMR, chloromethyl-benzoyl-amino-

tetramethyl-rhodamine; CMV, cytomegalovirus; CTL, cytotoxic T lymphocyte; ELISpot, enzyme-linked immunospot; FCS, fetal calf serum;

FITC, fluorescein isothiocynate; ICS, intracellular cytokine staining; HIV, human immunodeficiency virus; HLA, human leukocyte antigen;

IFNg, interferon-gamma; LAMP, lysosomal associated membrane protein; MHC, major histocompatibility complex; MIP1h, macrophage

inflammatory protein-1-beta; NK, natural killer cell; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; PerCP, peridinin chlorophyll

protein; PHA, phytohemagglutinin; qPCR, quantitative polymerase chain reaction; SEB, staphylococcus enterotoxin-B; TNFa, tumor necrosis

factor alpha.

* Corresponding author. Tel.: +1-301-594-8612; fax: +1-301-480-2779.

E-mail address: [email protected] (M.R. Betts).

M.R. Betts et al. / Journal of Immunological Methods 281 (2003) 65–7866

1. Introduction

After MHC-mediated recognition of cognate pep-

tide, CD8+ cytotoxic T lymphocytes exhibit two gen-

eral effector functions: production of soluble factors,

including cytokines and chemokines, and target cell

killing. Recent technological advances in multiparam-

eter flow cytometry have enabled the enumeration of

antigen-specific CD8+ T cells (MHC-class I tetramer

staining) and a direct assessment of their ability to

produce cytokine (intracellular cytokine staining

(ICS)) (Altman et al., 1996; Kern et al., 1998; Appay

et al., 2000; Betts et al., 2000). These techniques have

advantages over other methods, such as ELISpot anal-

ysis, in that they allow for precise phenotypic charac-

terization of the T cell populations of interest. CD8+ T

cell-mediated target cell killing, however, has histori-

cally been assessed by the standard chromium (51Cr)

release assay (Brunner et al., 1968), or, more recently,

by methods that monitor the release of fluorescent dyes

from target cells (Sheehy et al., 2001; Liu et al., 2002).

These techniques are cumbersome, semi-quantitative,

and potentially insensitive. Importantly, none of these

methods directly examine the CD8+ T cells that medi-

ate killing; rather, they examine the death of target

cells, essentially the aftermath of CD8+ T cell effector

function.

Cytotoxic CD8+ T lymphocytes (CTL) mediate the

killing of target cells via two major pathways, a

granule-dependent (perforin/granzyme) and indepen-

dent (ligand–ligand induced cell death, e.g. fas-fasL)

mechanism (reviewed in Trapani and Smyth, 2002).

The granule-dependent pathway does not require de

novo synthesis of proteins by the effector CD8+ T cell,

which instead utilize pre-formed lytic granules located

within the cytoplasm (Trapani and Smyth, 2002). The

lytic granules are membrane-bound secretory lyso-

somes that contain a dense core composed of various

proteins, including perforin and granzymes (Peters et

al., 1991). The core is surrounded by a lipid bilayer

containing lysosomal associated membrane glycopro-

teins (LAMPs), including CD107a (LAMP-1),

CD107b (LAMP-2), and CD63 (LAMP-3) (Peters et

al., 1991). These proteins are not normally found on the

surface of Tcells, although they have been observed on

the cell surface of PHA-activated lymphocytes

(CD107a and b), and ionomycin treated CD4+ and

CD8+ CTL clones (CD63) (Kannan et al., 1996; Bossi

and Griffiths, 1999). Although CD107a and b expres-

sion on the cell surface of peripheral blood cells has

been shown to enhance lymphocyte vascular adhesion

(Kannan et al., 1996), the function of these proteins, if

any, on the surface of activated T cells remains to be

determined. The presence of CD107a and b in the

cytotoxic granular membrane has been proposed to

protect against the leakage of contents from the granule

itself by coating the interior of the membrane (Peters et

al., 1991). CD107a, CD107b, and CD63 are constitu-

tively expressed on the surface of activated platelets,

and are found ubiquitously in lysosomal and endosomal

membranes of numerous other cell types (reviewed in

Fukuda, 1991).

Degranulation of activated CD8+ T cells occurs

rapidly after TCR stimulation, as a result of the polar-

ized mobilization of microtubules that transport the

lytic granules towards the immunological synapse

formed between the CTL and the target (reviewed in

Barry and Bleackley, 2002). Once the granules reach

the plasma membrane of the CTL, the membranes fuse

(Peters et al., 1991), releasing the granule contents into

the immunological synapse, ultimately resulting in the

death of the target cell. Degranulation is a requisite

process of perforin-granzymemediated killing, and is a

critical step required for immediate lytic function

mediated by responding antigen-specific CD8+ T cells.

To date, there are no flow cytometric assays to monitor

this process directly. Monitoring the presence or ab-

sence of perforin and granzymes A and B gives no

indication as to the ability of T cells to degranulate.

Furthermore, identifying a cell as lacking perforin does

not mean it had perforin prior to stimulation. Finally,

CD8+ T cells specific for certain viral antigens have

low baseline levels of perforin (Appay et al., 2000,

2002; Sandberg et al., 2001), causing difficulty in

measuring a further loss after antigen stimulation.

We developed a novel assay that directly measures

degranulation in primary responding antigen-specific

CD8+ T cells by multi-parameter flow cytometry. This

assay measures the cumulative exposure of granular

membrane proteins (CD107a and b) on the cell surface

of responding antigen-specific T cells, providing a

positive marker of degranulation. Significant expres-

sion of cell surface CD107a and b can be observed as

early as 30 min following stimulation of primary CD8+

T cells, and reaches maximum by 4 h. As expected,

inhibition of degranulation dramatically reduces the

M.R. Betts et al. / Journal of Immunological Methods 281 (2003) 65–78 67

acquisition of cell surface CD107a and b. This assay

can be combined with existing methods that assess

cytokine production in responding antigen-specific

CD8+ T cells directly ex vivo, thus providing simulta-

neous assessment of two critical CD8+ T cell effector

functions.

2. Materials and methods

2.1. Patient samples

Peripheral blood mononuclear cells (PBMC) were

obtained from 11 anonymous healthy donors at the

National Institutes of Health Department of Transfu-

sion Medicine. PBMC from two HIV-1 infected

patients were obtained from both the Amelia Court

HIV Clinic at the University of Texas Southwestern

Medical Center and the National Institutes of Health

OP-8 and OP-11 clinics. These donors signed informed

consent required by the institutions’ Review Boards.

2.2. Antibodies and reagents

The following anti-human monoclonal antibody

reagents were obtained from BD Pharmingen, San

Diego, CA (purified antibodies to some cell-surface

markers were conjugated to various fluorochomes in

our laboratory (Roederer), as indicated by an asterisk

following the fluorochrome used): anti-CD28, anti-

CD49d, anti-IFN-g [fluorescein isothicyanate (FITC),

allophycocyanin (APC)], anti-CD3 [phycoerythrin

(PE), peridinin chlorophyll protein (PerCP)], anti-

CD4 (FITC), anti-CD8 (PerCP), anti-CD16 (FITC),

anti-CD20 (FITC), anti-CD107a (FITC, PE*, APC*),

anti-CD107b (FITC, PE*, APC*), anti-CD63 (PE),

and anti-perforin (PE). Anti-granzyme B conjugated

to PE was obtained from Caltag, Burlingame, CA.

CMV-A2 tetramers were obtained from Beckman

Coulter Immunomics, San Diego, CA.

2.3. Cell stimulation

Fresh PBMC were isolated using Hypaque-Ficoll

(Pharmacia, Uppsala, Sweden) density centrifugation.

In some instances PBMC were frozen (90% fetal calf

serum (FCS)/10% DMSO) at � 140 jC until use. 106

PBMC were incubated with 1 Ag/ml each of anti-

CD28 and anti-CD49d and 2 Ag/ml of appropriate

peptide (when used) in a 1-ml volume. In some experi-

ments, staphylococcus enterotoxin B (SEB, 1 Ag/ml,

Sigma, St. Louis, MO) or anti-CD3 (clone HIT3a, BD

Pharmingen, 5 Ag/ml) was used to activate the cells.

Conjugated antibodies to the granular membrane pro-

teins CD107a and CD107b were added to the cells

prior to stimulation, unless otherwise noted. In every

experiment a negative control (anti-CD28/CD49d)

was included to control for spontaneous production

of cytokine and/or expression of CD107a/b. The

cultures were incubated for 1 h at 37 jC in a 5%

CO2 incubator, followed by an additional 4–5 h in the

presence of the secretion inhibitor monensin (BD

Pharmingen) or Brefeldin A (Sigma). In some experi-

ments, colchicine (Sigma) was added to inhibit granule

release.

2.4. Immunofluorescent staining/analysis

Immediately following stimulation, PBMC were

washed once, and surface stained with directly con-

jugated antibodies. The cells were washed and then

fix/permeabilized using 750 Al of fixation/permeab-

lization solution, consisting of FacsLyse (Becton

Dickinson Immunocytometry Systems, San Jose,

CA) diluted to a 2� concentration in dH2O and

0.05% Tween-20 (Sigma). After permeabilization,

the cells were washed twice, and stained with

directly conjugated antibodies specific for intracellu-

lar markers. The cells were washed a final time and

resuspended in 1% paraformaldehyde (Electron Mi-

croscopy Systems, Fort Washington, PA) in PBS.

Six-parameter flow cytometric analysis was per-

formed using a FACSCalibur flow cytometer (Becton

Dickinson Immunocytometry Systems). List mode

data files were analyzed using FlowJo software (Tree

Star, San Carlos, CA). In all cases at least 100,000

live events were collected for analysis.

2.5. Direct ex vivo cytotoxicity assay

Autologous B cells were isolated from PBMC by

positive selection using magnetic Microbeads coated

with anti-CD19 monoclonal antibody according to the

manufacturer’s instructions (MACS, Miltenyi Biotec,

Germany). Purified B cells were then labeled with

either carboxyfluorescein diacetate succinimidyl ester


(CFSE) or chloromethyl-benzoyl-amino-tetramethyl-

rhodamine (CMTMR) (Molecular Probes, Eugene,

OR). For CFSE staining, 5� 106 cells/ml were incu-

bated with 0.25 AM CFSE in PBS at 37 jC for 7 min,

then washed three times in RPMI-1640 (BioWhittaker,

Walkersville, MD)/20% FCS. For CMTMR staining,

2� 106 cells/ml were incubated in pre-warmed RPMI/

10% FCS (R10) supplemented with 5 AM CMTMR at

37 jC for 30 min, washed twice in pre-warmed R10,

and then incubated in R10 alone for 1 h prior to a final

wash. Labeled cells were protected from light during

all subsequent procedures. Cells labeled with CFSE

were either pulsed for 90 min with 200 nM CMV pp65

peptide 495–503 (NLVPMVATV) and then washed

three times in R10, or mock-pulsed in parallel; all cells

labeled with CMTMR were mock-pulsed. Cytotoxic-

ity assays used 200,000 each of CFSE and CMTMR-

labeled B cells in FACS tubes; PBMC were added in

R10 to give a range of effector to target (E/T) ratios.

Assays were incubated at 37 jC with 5% CO2 with the

tubes placed at an angle. The elimination of peptide-

pulsed CFSE-labeled cells relative to the unpulsed

CMTMR-labeled internal negative control served as

a measure of specific cytotoxicity. Parallel assays with

mock-pulsed CFSE-labeled cells were set up for each

E/T ratio to control for non-specific toxic effects of

CFSE itself.

3. Results

3.1. CD107a and b are expressed on the surface of

CD8+ T cells that degranulate in response to SEB

In order to identify degranulation by activated

CD8+ T cells, we examined the expression of

Fig. 1. Characterization of CD107a and b staining. (A) PBMC from a nor

antibodies to CD107a and b FITC as shown in the presence of Brefeldin A

(where appropriate). Events shown are gated for CD3 and CD8. (B) A co

monensin (solid line) on the fluorescence of FITC CD107a and b in SEB

FITC anti-CD107a and b for 6 h in the presence of either inhibitor, then sta

and CD8. Values shown represent the mean fluorescence of the indicated p

Cells from one donor were SEB-stimulated, pre-stained with anti-CD107a

for 6 h, followed by staining of CD3 and CD8 molecules. Events shown ar

on SEB stimulated cells. Cells from the same donor used in (C) were surfac

described in (C) and stained with CD3 FITC and CD8 PerCP. Events

degranulation in SEB stimulated PBMC. Cells were stimulated, stained, an

concentrations of colchicine, as depicted on the figure.

CD107a and b on the cell surface of CD8+ T cells

following activation with SEB. PBMC from a normal

donor were stimulated with SEB and incubated for 6

h in the presence of the secretion inhibitor Brefeldin

A. After stimulation, the cells were stained with

antibodies to T cell markers (CD3 and CD8) and a

mixture of FITC-labeled antibodies to CD107a and b.

A total of 8.4% of CD8+ T cells expressed surface

CD107 after stimulation (Fig. 1A, left panel), indicat-

ing that degranulation had likely occurred within the

responding CD8+ T cells. While this initial result

appeared promising, the frequency of the responding

population was not comparable to that observed by

intracellular cytokine staining for IFNg (approximate-

ly 12%, data not shown). We reasoned that it was

likely that cell surface expression of CD107 on T cells

may be transient. Previous studies have shown that

CD107a and b are targeted primarily to lysosomal/

endosomal membranes, and that any CD107a and b

externalized to the cell surface is rapidly retrieved via

the endocytic pathway (Fukuda, 1991). This sug-

gested that as the cells degranulate, they would

become positive for cell surface CD107 for a brief

period of time before those proteins were internalized.

Therefore, we included the antibodies to CD107a and

b for the duration of the stimulation, rather than just

staining post-stimulation. Any transient surface ex-

pression of CD107 would lead to antibody binding

and either surface retention or uptake of the CD107/

antibody complex. In either case, even transient sur-

face expression of CD107 would lead to fluorescent

labeling of that cell. This modification enhanced the

detection of responding cells, such that 12% of the

CD8+ T cells were observed to express surface

CD107a and b in response to SEB (Fig. 1A, center

panel). This protocol did not result in an increase of

mal donor were stimulated with SEB and incubated with or without

for 6 h, then stained with CD3, CD8, and CD107a and b antibodies

mparison of the differential effects of Brefeldin A (dashed line) and

stimulated CD8+ T cells. Cells were stimulated in the presence of

ined with CD3 and CD8 antibodies. Events shown are gated for CD3

opulation (C) Comparison of different CD107 antibody conjugates.

and b FITC, PE, or APC, and incubated in the presence of monensin

e gated for CD3 and CD8. (D) Coordinate staining of CD107a and b

e stained with CD107a APC and CD107b PE, and then stimulated as

shown are gated for CD3 and CD8. (E) Effect of colchicine on

d analyzed as described in (C) in the presence or absence of varying


background staining for CD107a and b (Fig. 1A, right

panel).

Our initial experiments utilized antibodies to

CD107a and b conjugated to FITC, the fluorescence

of which is sensitive to acidic pH (Roederer et al.,

1987). Because CD107 is internalized from the cell

surface following degranulation, likely into an acidic

endosomal or lysosomal compartment, the fluores-

cence of FITC would be quenched. Therefore we

examined the differential effects of Brefeldin A and

monensin on the mean fluorescence of the responding

cells (Fig. 1B). While both Brefeldin A and monensin

serve as inhibitors of secretion, monensin also neu-

tralizes the pH within endosomes and lysosomes

(Mollenhauer et al., 1990). The mean fluorescence

of CD107a and b-FITC in activated cells incubated


with monensin (mean fluorescence = 78.3) was sub-

stantially higher than cells incubated with Brefeldin A

(mean fluorescence = 41.8)(Fig. 1B). This result sug-

gests that a substantial proportion of the FITC-labeled

CD107a and b antibodies were internalized into an

endosomal or lysosomal compartment, further sup-

porting the need to label these proteins during stim-

ulation and use monensin to achieve optimal detection

of responding CD8+ T cells.

In order to further optimize the staining of CD107,

we conjugated the CD107a and b antibodies to PE and

APC. The PE and APC conjugates were substantially

brighter than the FITC conjugate, without greatly

affecting the fluorescence of the non-responding pop-

ulation (Fig. 1C). One observation of note is that the

PE-CD107 antibody conjugate appears to have a

higher background in non-stimulated cells than the

FITC and APC conjugates (Fig. 1C, upper row),

perhaps related to the hydrophobicity of PE. We

therefore utilized either the FITC or APC CD107

conjugates in our remaining experiments. In general,

background expression of CD107a and b in unstimu-

lated CD8+ T cells varied between 0.05% and 0.5%.

The background expression of CD107a and b can be a

result of several factors, including the presence of

dead/apoptotic cells, platelets, granulocytes, mono-

cytes, or B cells within the cultures. In particular,

removal of monocytes and B cells through the use of

a dump channel can dramatically reduce the back-

ground CD107 labeling observed (data not shown).

We next examined the coordinate expression of

CD107a and b on the cell surface of SEB-activated

CD8+ T cells. Both CD107a and b are coordinately

expressed on the majority of the responding cells,

although the CD107a signal appears to be stronger in

most cells than CD107b with these reagents (Fig. 1D).

CD107a and b are likely differentially regulated within

all cell types, as they are encoded on different chro-

mosomes, and expressed at different copy numbers

within the cell (Fukuda, 1991). It remains to be deter-

mined if these two proteins are differentially expressed

in cytotoxic granules within individual CD8+ Tcells or

subpopulations of CD8+ T cells. Therefore, to ensure

greatest sensitivity, we chose to use amixture of the two

antibodies in future experiments.

In order to ensure that CD107a and b expression on

the cell surface occurred as a result of degranulation,

we examined the effect of the microtubule inhibitor

colchicine on the expression of CD107a and b.

Colchicine has a potent effect on the expression of

cell surface CD107a and b following activation of

CD8+ T cells with SEB (Fig. 1E). This result, along

with the observation that CD107a and b are expressed

on the cell surface in the presence of the secretion

inhibitors Brefeldin A and monensin, indicates that

CD107a and b are indeed expressed on the cell

surface as a result of degranulation.

3.2. Cell surface expression of CD107a and b is

associated with the loss of intracellular perforin

Perforin is released from cytotoxic granules during

the degranulation process; therefore, acquisition of

cell surface CD107a and b on CD8+ T cells after

stimulation should be associated with a loss of intra-

cellular perforin. To address this, we stimulated

PBMC with anti-CD3, and performed a time course

to compare the levels of intracellular perforin with cell

surface CD107a and b. As shown in Fig. 2, CD107a

and b was rapidly expressed after stimulation, con-

comitant with a loss of perforin expression. After a 5-

h stimulation, nearly every perforin expressing cell

had degranulated, indicating that acquisition of cell

surface CD107a and b occurs as a result of degranu-

lation, rather than de novo production.

3.3. Comparison of CD107a and b expression with

CD63 expression following degranulation

CD63 (LAMP-3) can also be found within the

membrane of cytotoxic granules (Peters et al., 1991),

and has been previously used as a positive marker

associated with the deposition of fas ligand on the cell

surface during degranulation in ionomycin-stimulated

CD4+ and CD8+ T cell clones (Bossi and Griffiths,

1999). We examined the expression of CD63 in

comparison with CD107a and b in CD8+ T cells that

degranulate in response to SEB (Fig. 3). While a

portion of the responding CD8+ T cells expressed

CD63, CD107a, and CD107b, the majority of respond-

ing cells were CD63 low or negative. Furthermore, the

CD63 background was substantially higher than that

observed for CD107a and b. These results show that

CD107 expression on ex vivo activated CD8+ T cells

provides a more accurate assessment of degranulation

than does CD63 expression.

Fig. 2. Acquisition of cell surface CD107 is correlated with a loss of intracellular perforin. PBMC were stimulated with anti-CD3 (5 Ag/ml), and

incubated for up to 5 h in the presence of anti-CD107a and b FITC and monensin. Baseline perforin expression was approximately 20% (data

not shown). At the time-points designated on the figure, aliquots were removed, washed, and permeabilized, followed by staining for perforin,

CD3, and CD8. Events shown are gated for live CD3+ CD8+ T cells.


3.4. Cytotoxic-granule containing lymphocytes

express higher levels of CD107a and b than

non-granule containing lymphocytes

While all lymphocytes express CD107a and b on

endosomal and lysosomal membranes, not all lympho-

cytes have cytotoxic granules that contain perforin.

Such granules are expressed by CD8+ Tcells, NK cells,

and a subset of CD4+ T cells. We therefore examined

the relationship between intracellular expression of

CD107 and perforin expression. Nearly all lympho-

cytes express detectable CD107a and b when staining

for these molecules following permeabilization (data

not shown). Cells that contain perforin, however,

express even higher levels of CD107a and b. For

example, NK cells, which typically express high levels

Fig. 3. Comparison of CD63 expression with CD107a and b expression. PBMC were stimulated with SEB in the presence of antibodies to CD63

PE and CD107a and b FITC and monensin, incubated for 6 h, and then stained with CD3 and CD8 antibodies. Events shown are gated CD3+

CD8+ T cells.


of perforin, have higher CD107a and b expression than

B cells or CD4+ T cells. Interestingly, although only a

subset of CD8+ T cells is perforin+, the CD8+ T cell

population as a whole expresses more CD107a and b

than B cells or CD4+ T cells. This suggests that CD8+

T cells have a higher granular content, similar to NK

cells. To support this conclusion, we also compared

granzyme B, another granular component expressed by

a higher percentage of CD8+ Tcells than perforin, with

CD107a and b content. CD8+ T cells and NK cells that

express granzyme B also have high levels of CD107a

and b (data not shown).

3.5. CD107a and b are expressed on the cell surface

of activated peptide-specific CD8+ T cells

The standard for examining antigen-specific CD8+

T cell effector responses by flow cytometry is mea-

surement of intracellular cytokine production, typical-

ly IFNg, after stimulation with cognate peptide. We

therefore compared production of IFNg in response to

peptide stimulation with acquisition of cell surface

CD107a and b (Fig. 4A–D). PBMC isolated from

four different patients were stimulated with peptides

derived from either CMVor HIV, stained for CD107a

and b, then incubated for 5 h in the presence of

monensin. After stimulation, the cells were permeabi-

lized and stained for intracellular IFNg. As can be seen

in Fig. 4, nearly all of the CD8+ T cells that produce

IFNg in response to specific peptide also express

CD107a and b. This indicates that acquisition of cell

surface CD107a and b occurs in an antigen specific

manner, and that nearly all CD8+ T cells which

produce cytokine in response to cognate antigen

degranulate. As shown in Fig. 4C, a mixture of

peptides can also be used to stimulate CD8+ T cells

to degranulate, suggesting that measurement of cell

surface CD107a and b could be used in peptide

response mapping procedures. Interestingly, in some

patients a population of CD107a+ and b+ cells that did

not produce IFNg could be observed (Fig. 4B and C).

This suggests that measuring CD8+ T cell responses

by IFNc production alone may underestimate the total

response.

3.6. Kinetics of CD107a and b expression as

compared to IFNg production

Having established that cell surface expression of

CD107a and b occurs in an antigen specific manner,

we examined in more detail the rate at which cells

became surface positive for CD107a and b after

stimulation. Intracellular expression of IFNg has

previously been shown to plateau between 5 and 6

h, thus we previously had stimulated the cells for this

length of time to compare CD107 and IFNg expres-

sion. It is known, however, that degranulation occurs

very rapidly following triggering of the T cell recep-

tor complex. We therefore expected to detect acqui-

sition of cell surface CD107a and b rapidly follo-

Fig. 4. CD107a and b is expressed by ex vivo activated antigen-specific CD8+ T cells. PBMC from four different donors were stimulated with

specific peptides in the presence of anti-CD28/49d, anti-CD107a and b FITC or APC, and monensin for 6 h. Peptides used include HLA-A2

restricted CMV pp65 NLVPMVATV (A, B), overlapping HIV-Gag derived 15-mers (15-mer peptides overlapping by 11 amino acids, C), and

HLA-B57 restricted HIV Gag KAFSPEVIPMF (D). All peptides were used at a final concentration of 2 Ag/ml. Events shown are gated CD3+

CD8+ T cells.


wing stimulation. Expression of cell surface CD107

can be detected as early as 30 min to 1 h follow-

ing stimulation with anti-CD3 (Fig. 2) or specific

peptide (Fig. 5), and peaks between 4 and 5 h. IFNg,

which requires de novo synthesis following activa-

tion, also peaked at 4–5 h post stimulation, although

no IFNg production was detected at 1 h post-stimu-

lation. Thus, optimal expression of both cell surface

CD107a and b and IFNg occurs between 4 and 6 h

post-stimulation.

3.7. CD107a and b can be used to measure

degranulation in activated MHC-class I

tetramer+cells

The functionality of tetramer-binding CD8+ T cells

can be examined by staining for IFNg production after

the stimulation of tetramer stained cells with cognate

peptide. However, since cytokine production alone

does not provide a full functional assessment of the

tetramer binding cells, we examined the ability of

tetramer stained cells to degranulate, an example of

which is shown in Fig. 6. Approximately 0.8% of

CD8+ T cells in this individual are capable of binding

to the CMV-A2 MHC-class I tetramer (Fig. 6, left

panel). After stimulation with peptide, only 25% of the

tetramer + cells produced IFNg (Fig. 6, center panel),

while a substantially higher proportion (>50%) of the

same tetramer + cells degranulated following stimula-

tion (Fig. 6, right panel). Similar results were observed

in four additional individuals (data not shown). These

data demonstrate that examination of cytokine produc-

tion alone may not provide sufficient information

regarding the full functionality of tetramer + cells.

3.8. CD107a and b expression directly correlates with

cytotoxic activity

While measurement of the ability of CD8+ T cells

to degranulate provides an indication of cytotoxic

potential, it still does not prove that degranulating

cells are capable of killing targets. To address this

question more directly, we examined the cytotoxic

activity of a CMV-specific CD8+ T cell population

known to degranulate in response to an HLA-A2

restricted CMV-derived peptide (NLVPMVATV,

Fig. 5. Time course of CD107a and b expression as compared to IFNg production. PBMC were stimulated with CMV-A2 NLVPMVATV

peptide (2 Ag/ml) for up to 6 h in the presence of anti-CD28/49d, anti-CD107a and b FITC and monensin. At 1, 2, 3, 4 and 6 h post stimulation,

aliquots were removed, washed, permeabilized and stained for intracellular IFNg, CD8, and CD3. The frequency of responding CD8+ cells was

determined at each time point and the % maximal response was calculated for each time point. (x, CD3+ CD8+ IFNg+ cells; n, CD3+ CD8+

CD107a and b+ cells; D, CD3+ CD8+ CD107a and b+ IFNg+ cells).


CMV-A2). PBMC were isolated from an individual

with a high CMV-specific CD8+ T cell frequency (Fig.

7A, left panel). Approximately 10–15% of the circu-

lating CD8+ T cells in this individual are specific for

the CMV-A2 peptide as measured by direct tetramer

binding, and 80% of the tetramer + cells degranulate in

response to the CMV-A2 peptide (Fig. 7A, right

panel). We then examined the cytotoxic ability of these

cells directly by using a flow cytometry-based killing

assay, as described in the Materials and methods.

CFSE labeled cells, depicted in green, are selectively

Fig. 6. CD107a and b are expressed by functional MHC-class I tetramer

complexes, then activated with CMV-A2 peptide (NLVPMVATV) in the pr

5 h. Following stimulation, the cells were washed, permeabilized, and sta

killed by the CD8+ T cells only when CMV-A2

peptide loaded, as demonstrated by a shift into the

dead cell population determined by the forward/side

scatter profiles (Fig. 7B). The selective loss of peptide

loaded CFSE labeled B cells in the presence of CD8+

T cells can also be directly observed by comparing the

frequency of CMTMR and CFSE labeled cells within

the live cell gate (Fig. 7C). Taken together these results

indicate that antigen-specific CD8+ T cells that degra-

nulate, as measured by CD107 expression, mediate

cytotoxic activity.

+ cells. PBMC were stained with CMV-A2 MHC-class I tetrameric

esence of anti-CD28/49d, anti-CD107a and b APC and monensin for

ined for CD8 and IFNg. All events shown are gated CD8+ cells.

Fig. 7. Correlation between CD107a and b expression and cytotoxic activity. (A) PBMC were stained with a CMV-A2 MHC-class I tetramer,

and anti-CD107a and b APC and treated as follows: Left panel: stained with anti-CD3 and anti-CD8, and analyzed without further incubation.

Right panel: stimulated with cognate peptide (NLVPMVATV), incubated for 5 h in the presence of monensin, washed, then stained with anti-

CD3 and anti-CD8. All events shown are live-gated on CD3 and CD8. (B and C) Multiple parameter overlay plots showing the distribution of

CFSE and CMTMR-labeled autologous B cells within the forward/side scatter profile (B, green events = CFSE, red events = CMTMR) or live

gate (as depicted in B) only (C). In the example shown, 2� 106 PBMC were incubated with 200,000 target cells for 60 h. Left panel: neither

population of fluorescent dye-labeled cells was pulsed with cognate peptide. Right panel: the target cells labeled with CFSE only were pre-

pulsed with cognate peptide (NLVPMVATV) at 200 nM.



4. Discussion

We present here a novel assay that measures de-

granulation in response to antigen-specific stimulation,

an essential CD8+ T cell effector function. We dem-

onstrate that the granular membrane proteins CD107a

and b are expressed on the cell surface of activated

CD8+ T cells due to degranulation. Expression of cell

surface CD107a and b on CD8+ T cells often occurs in

concert with production of IFNg in response to stim-

ulation, but both functions do not necessarily occur in

all antigen-specific CD8+ T cells. Additionally, we

show that the same CD8+ T cells which degranulate

are capable of cytotoxic activity.

Numerous methods exist to examine both the phys-

ical presence and functionality of antigen-specific

CD8+ T cells. The physical presence of antigen-

specific CD8+ T cells can be monitored with MHC-

class I tetramers or qPCR clonotype analysis (Altman

et al., 1996; Douek et al., 2002). Neither of these

techniques, however, provides any indication as to the

functionality of the cells detected. Functionality can be

assessed using both direct (intracellular cytokine stain-

ing and CFSE-based proliferation assays) and indirect

(e.g. 51Cr release assays, flow-based killing assays and3H proliferation assays) methods (Kern et al., 1998;

Sheehy et al., 2001; Brenchley et al., 2002; Liu et al.,

2002). The direct methods as a whole, however,

provide no information regarding cytotoxic ability,

and conversely the indirect methods provide no indi-

cation as to the identity of the effector cells. The assay

we describe here provides a link between the direct and

indirect methods of CD8+ T cell effector analysis by

enabling precise phenotypic and functional character-

ization of responding CD8+ T cells through flow

cytometry using a marker that is only expressed during

degranulation, the initial event that takes place during

target cell lysis.

Although the CD107 assay does not directly mea-

sure target cell lysis, it does provide an indication of

the cytotoxic potential of the responding CD8+ T

cells. Current methods to assess CD8+ T cell-mediat-

ed target cell killing, including the standard chromium

release and flow-based killing assays do not identify

or quantify effector cells, instead measuring their

downstream effect on target cells. Thus, it has not

been possible to characterize directly the full func-

tional capacity and phenotype of the responding

CD8+ T cells. Our CD107 assay can provide an

assessment of the capacity, frequency and phenotype

of CD8+ T cells that kill in conditions similar to those

used in a standard 51Cr release assay.

More recently, intracellular cytokine staining has

provided a wealth of information regarding the

phenotype and functional status of antigen-specific

CD8+ T cells. The question, however, has remained

whether CD8+ T cells that produce cytokine after

stimulation are cytotoxic. By measuring degranula-

tion in the same cells, it is apparent that the majority

of the CD8+ T cells that respond to antigen by

producing cytokine also degranulate. This suggests,

therefore, that cytokine producing CD8+ T cells that

degranulate should be capable of killing targets,

provided they express the necessary granular comp-

onents to do so.

Interestingly, we observe in some patients that a

substantial population of responding CD8+ T cells

degranulate but do not produce IFNg. It is well

documented that many tetramer + cells do not produce

detectable levels of IFNg after direct ex vivo stimula-

tion with cognate peptide (Goepfert et al., 2000;

Shankar et al., 2000). Therefore, functional heteroge-

neity exists within the population of responding CD8+

Tcells. Thus, in assessing the frequency of the CD8+ T

cell response to any particular antigen, one should also

include measurement of degranulation, lest the re-

sponse frequency be underestimated.

Current data suggests that CD8+ T cell populations

specific for a single antigen can produce multiple

cytokines, but that heterogeneity exists as to the

cytokines produced by individual cells within that

population (data not shown). Importantly, there does

not appear to be a significant population of CD8+ T

cells that produce IFNg without degranulating. Al-

though it remains to be determined if the non-IFNg

producing CD107+ cells produce other cytokines in

response to stimulation, preliminary results indicate

that CD107 is expressed on the surface of MIP1h+ and

TNFa +CD8+ Tcells (data not shown). Thus, CD107a

and b expression after stimulation provides a more

complete assessment of the total frequency of respond-

ing CD8+ T cells than does monitoring production of

any one cytokine alone.

An important methodological aspect of the CD107

assay is that fixation or permeabilization of the

responding cells is not necessary, thus making this a


suitable procedure for sorting of live cells. Previously,

sorting of antigen-specific CD8+ T cells ex vivo has

been limited to methods utilizing either tetramer stain-

ing or IFNg capture systems. Tetramer-based sorts,

while very rapid and specific, are limited by the need

for very specific reagents and restricted peptide-MHC

combinations. While IFNg capture is not limited by

reagent availability, it is inherently more difficult to

perform, and is limited to IFNg producing cells. Unlike

tetramer-based methods, sorting based on CD107a and

b expression is not limited by reagent availability, or to

prior knowledge of HLA type or peptide recognition,

since mixtures of overlapping peptides can be used.

Additionally, sorting based on CD107a and b is not

limited only to those cells capable of producing a

certain cytokine, as is the IFNg capture assay.

In conclusion, we have described a novel method to

assess CD8+ T cell effector function based on the

ability of these cells to degranulate. This assay is sim-

ple, rapid, sensitive, and can be adapted for use in

combination with both tetramer and intracellular cyto-

kine assays. Assessment of degranulation alongside

cytokine production and phenotypic characterization

will greatly enhance our knowledge of the functionality

of antigen-specific CD8+ Tcells both in disease as well

as in vaccine models.

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Sensitive and Viable Identification of Antigen-specific CD8+ T Cells

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