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CHEMOKINES
IL-8 responsiveness defines a subset of CD8 T cells poised to kill
Christoph Hess, Terry K. Means, Patrick Autissier, Tonia Woodberry, Marcus Altfeld, Marylyn M. Addo, Nicole Frahm,
Christian Brander, Bruce D. Walker, and Andrew D. Luster
CD8 T cells play a key role in host defense
against intracellular pathogens. Efficient
migration of these cells into sites of infection is therefore intimately linked to their
effector function. The molecular mechanisms that control CD8 T-cell trafficking
into sites of infection and inflammation
are not well understood, but the chemokine/chemokine receptor system is
thought to orchestrate this process. Here
we systematically examined the chemokine receptor profile expressed on human
CD8 T cells. Surprisingly, we found that
CXC chemokine receptor 1 (CXCR1), the
predominant neutrophil chemokine receptor, defined a novel interleukin-8/CXC ligand 8 (IL-8/CXCL8)–responsive CD8 Tcell subset that was enriched in perforin,
granzyme B, and interferon-␥ (IFN␥), and
had high cytotoxic potential. CXCR1 expression was down-regulated by antigen
stimulation both in vitro and in vivo, suggesting antigen-dependent shaping of the
migratory characteristics of CD8 T cells.
On virus-specific CD8 T cells from persons with a history of Epstein-Barr virus
(EBV) and influenza infection, CXCR1 expression was restricted to terminally dif-
ferentiated effector memory cells. In HIV-1
infection, CXCR1-expressing HIV-1–specific CD8 T cells were present only in
persons who were able to control HIV-1
replication during structured treatment
interruptions. Thus, CXCR1 identifies a
subset of CD8 T cells poised for immediate cytotoxicity and early recruitment into
sites of innate immune system activation.
(Blood. 2004;104:3463-3471)
© 2004 by The American Society of Hematology
Introduction
A central feature of the adaptive immune response is the generation
of antigen-specific effector and memory T cells. Naive T cells are
induced to proliferate (clonal expansion) and differentiate upon
encounter with cognate antigen presented by dendritic cells in
secondary lymphoid structures.1,2 Activation and subsequent differentiation lead to the formation of several CD8 T-cell memory
subsets with different functional and migratory properties, classified as central memory, effector memory, and terminally differentiated effector memory cell subsets.3,4 Terminally differentiated
effector memory CD8 T cells express high levels of perforin,
granzyme A and B, and Fas ligand; are highly cytotoxic; and home
to sites of infection and inflammation. Effector memory CD8 T
cells are thought to allow for a recall response in the tissue, as these
cells survey nonlymphoid organs in search of cognate antigen and,
while able to produce interferon-␥ (IFN␥) upon antigen recognition, are only moderately cytotoxic. In contrast, central memory
cells recirculate between the blood and the lymphoid compartment
and are thought to provide a pool of antigen-experienced cells with
a high proliferative capacity but a lower activation threshold than
naive cells, thus allowing for a more rapid generation of effector
cells during a recall response.3,5,6
Distinct migratory properties are a determining factor in T-cell
subset function. Homing characteristics are thought to be controlled by the expression pattern of adhesion molecules and
chemokine receptors acquired by specialized T-cell subsets during
the process of differentiation and polarization. For example, central
memory T cells retain expression of the lymph-node homing
receptors CC chemokine receptor 7 (CCR7) and L-selectin. In
contrast, effector memory and terminally differentiated effector
memory cells lose expression of CCR7 and L-selectin, and acquire
receptors allowing homing to peripheral tissue and to sites of
infection/inflammation, respectively.3 Although not systematically
examined in any detail, inflammatory chemokine receptors, such as
CXC chemokine receptor 3 (CXCR3), CCR5, and CX3C chemokine receptor 1 (CX3CR1), have been described on CD8 T-cell
subsets with effector function and are thought to enable these
subsets to migrate into the periphery.7-10
In this study, we systematically analyzed the chemokine receptor
profile of human CD8 T-cell subsets as defined by expression of the
lymph-node homing chemokine receptor CCR7 and the cell-surface
tyrosine phosphatase CD45RA. Naive cells were defined as
CCR7⫹CD45RAhigh, central memory cells (CMs) as CCR7⫹CD45RA⫺,
effector memory cells (EMs) as CCR7⫺CD45RA⫺, and terminally
differentiated effector memory cells as CCR7⫺CD45RAhigh (CD45RA
re-expressing EMs [EMRAs]).3,4,11 Subset-specific analysis allowed for
the unambiguous identification of a population of CD8 T cells expressing functional CXCR1. CXCR1 was differentially expressed on EpsteinBarr virus (EBV)– and influenza-specific CD8 T cells that had higher
levels of perforin and IFN␥ and greater cytotoxic activity. On HIV-1–
specific CD8 T cells, expression of CXCR1 was unique to treated
From the Center for Immunology and Inflammatory Diseases, Division of
Rheumatology, Allergy and Immunology, Massachusetts General Hospital,
Harvard Medical School, Charlestown, MA; the Howard Hughes Medical
Institute, Partners AIDS Research Center, Massachusetts General Hospital
and Division of AIDS, Harvard Medical School, Charlestown, MA; and the
Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, MA.
M.M.A., T.W., P.A., and T.K.M.); the Doris Duke Charitable Foundation (B.D.W.
and M.A.); the FAIR-foundation (A.D.L. and M.A.); and the Swiss National
Science Foundation (C.H.).
Reprints: Andrew D. Luster, Massachusetts General Hospital, Bldg 149, 13th
St, Charlestown, MA 02129; e-mail: [email protected].
Submitted March 23, 2004; accepted July 13, 2004. Prepublished online as
Blood First Edition Paper, August 3, 2004; DOI 10.1182/blood-2004-03-1067.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by the National Institutes of Health (A.D.L., B.D.W., C.B., M.A.,
© 2004 by The American Society of Hematology
BLOOD, 1 DECEMBER 2004 䡠 VOLUME 104, NUMBER 12
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BLOOD, 1 DECEMBER 2004 䡠 VOLUME 104, NUMBER 12
HESS et al
persons able to control HIV-1 replication during periods of structured
treatment interruptions. CXCR1 expression was uniquely downmodulated upon antigen-specific stimulation in vitro, and during periods
of uncontrolled viral replication in vivo. CXCR1 is the predominant
chemokine receptor expressed on human neutrophils, and its ligand,
interleukin-8 (IL-8), is one of the earliest and most abundant chemokines produced during acute inflammatory responses.12,13 Responsiveness to IL-8 is essential during the earliest phase of the host immune
response to infection.14,15 Thus, CXCR1 defines a novel subset of highly
cytotoxic CD8 T cells that, following innate immune system activation, may provide the first wave of antigen-specific protection
during a recall response.
Patients, materials, and methods
Flow cytometry
Antibodies recognizing CCR1 (53504.111), CXCR1 (42705.111), CCR2
(48607.121), CXCR2 (48311.211), CCR3 (61828.111), CXCR5 (51505.111),
CCR6 (53103.111), CXCR6 (56811), CCR7 (150503), CCR9 (112509),
CD27 (57703), and CD28 (37407) were purchased from R&D Systems
(Minneapolis, MN). CXCR3 (1C6), CCR4 (1G1), CXCR4 (12G5), CCR5
(3A9), CCR6 (11A9), CCR7 (2H4), CD3 (UCHT1), CD4 (SK3), CD8
(RPA-T8), and CD19 (SJ25C1) were purchased from BD PharMingen (San
Diego, CA). Antibody recognizing CX3CR1 (2A9-1) was purchased from
MBL (Nakaku Nagoya, Japan). Fluorogenic caspase substrate was purchased from OncoImmunin (Gaithersburg, MD). The following human
leukocyte antigen (HLA) tetramers were used: for influenza, HLA-A2
GILGFVFTL (Immunomics, San Diego, CA); for EBV, HLA-B8–restricted
FLRGRAYGL (EBNA3A) and QAKWRLQTL (EBNA3A), HLA-A2–
restricted GLCTLVAML (BMLF1), and HLA-B8–restricted RAKFKQLL;
for HIV-1, HLA-B8–restricted FLKEKGGL (nef), HLA-B8–restricted
EIYKRWII (p24), HLA-B7–restricted IPRRIRQGL (gp41), and HLA-A2–
restricted SLYNTVATL (p17) (all synthesized and refolded as described
previously).16 Control experiments were performed to exclude binding of
tetramer to natural killer (NK) cells. No such binding was observed (data
not shown). For fluorescence-activated cell scanner (FACS) analysis,
peripheral blood mononuclear cells (PBMCs) were prepared by centrifugation through a density gradient on Histopaque-1077 (Sigma-Aldrich, St
Louis, MO). When appropriate, CD8⫹ cells were selected by means of
anti-CD8 magnetic beads (MACS system; Miltenyi Biotec, Auburn, CA).
Purity of CD8⫹ cells, as confirmed by flow cytometry, was at least 97%
(data not shown). Cells were blocked with 10% human serum before
staining with antibodies to individual chemokine receptors. Staining and
analysis were performed in phosphate-buffered saline (PBS) with 1% fetal
calf serum (FCS). For intracellular perforin analyses, after surface-marker
labeling, cells were fixed and permeabilized with PermeaFix (Ortho
Diagnostic Systems, Raritan, NJ) before intracellular staining with antiperforin monoclonal antibody (27-35; BD PharMingen). Isotype-matched
controls were used for both surface and intracellular staining. Data were
acquired on a FACS Calibur or FACSvantage SE system (system expanded
for 5-color analysis) and analyzed by means of CellQuest software (Becton
Dickinson). To accumulate several hundred antigen-specific CD8 T cells
within the various subsets, up to several million events were acquired.
Cell sorting
Samples were NK-cell depleted with anti-CD56 magnetic beads by means
of the MACS system (Miltenyi Biotec). Depletion of at least 99% of CD56⫹
lymphocytes was found not to change the percentage of CXCR1⫹ CD8 T
cells (data not shown). Alternatively, 5-color flow cytometry including
anti-CD3 monoclonal antibodies was performed. Cells were sorted on
Cytomation MoFlo (Dako Cytomation, Fort Collins, CO) or FACSvantage
SE high-speed cell sorters. The following populations of CD8 T cells
were sorted: for quantitative polymerase chain reaction (PCR) analysis,
CCR7⫹ CD45RAhigh, CCR7⫹ CD45RA⫺, CCR7⫺CD45RA⫺, and
CCR7⫺CD45RAhigh; for cytotoxicity assays; CXCR1⫹, CX3CR1⫹,
CCR7⫺CD45RAhigh, and bulk; for intracellular cytokine staining,
CCR7⫺CD45RA⫺, CCR7⫺CD45RAhigh, CXCR1⫹. For each cell population, 20 000 to 500 000 cells were sorted. Purity was evaluated by postsort
flow cytometry. Less than 1% contamination was observed in all T-cell
subsets (data not shown).
Quantitative PCR (QPCR)
CD3/CD8 double-positive naive (N), CM, EM, and EMRA subsets were
sorted as described. Purity of subsets was assessed by postsort flow analysis
and typically was 99%. Total RNA was extracted by means of the RNeasy
kit (Qiagen, Valencia, CA), and after DNase I (Invitrogen, Carlsbad, CA)
treatment, total RNA from each sample was used as template for the
reverse-transcription reaction. QPCR was performed as described, by
means of the Mx4000 Multiplex Quantitative PCR System (Stratagene, La
Jolla, CA).13 Briefly, all samples were reverse transcribed under the same
conditions (25°C for 10 minutes, 48°C for 30 minutes) and from the same
reverse-transcription master mix to minimize differences in reversetranscription efficiency. The QPCR reaction contained 2 ␮L cDNA, 12.5
␮L 2 ⫻ SYBR Green master mix (Stratagene), and 500 nmol sense and
antisense primer. Emitted fluorescence for each reaction was measured
during the annealing/extension phase, and amplification plots were analyzed with the MX4000 software version 3.0 (Stratagene). Quantity values
(ie, copies) for gene expression were generated by comparing the fluorescence generated by each sample with standard curves of known quantities,
and the calculated number of copies was divided by the number of copies of
the constitutively active gene GAPDH or ␤2-microglobulin. In control
experiments, no significant difference in QPCR results were observed when
comparing data normalized with the use of the housekeeping gene
␤2-microglobulin versus GAPDH.
Chemotaxis
CD8 T cells were negatively selected by means of anti-CD4, anti-CD19,
anti-CD14, and anti-CD56 magnetic beads with the use of the MACS
system. Purity as confirmed by flow cytometry was at least 95% (data not
shown). CD8 T cells were suspended in RPMI with 1% bovine serum
albumin and loaded in duplicate into modified Boyden chamber inserts
(pore size, 5 ␮m) for 24-well cell-culture dishes. IL-8 and macrophagederived chemokine (MDC) were added to RPMI with 1.5% bovine serum
albumin in the lower wells at various concentrations. After incubation for 2
hour at 37°C, cells that migrated into the lower chamber were washed and
stained for CD3, CD8, CD45RA, and CCR7, and analyzed by flow
cytometry. By gating on CD3/CD8 double-positive lymphocyte subsets
migrating in response to a given chemotactic gradient, potentially contaminating NK cells were excluded from the analysis. The CD8 T-cell
phenotype with respect to expression of CCR7 and CD45RA ex vivo was
compared with the respective phenotype after 2 hours at 37°C (in presence
and absence of IL-8 and MDC), and no significant changes were observed
(data not shown). In control experiments, a known amount of polystyrene
counting beads (diameter, 15.8 ␮m) (Polysciences, Warrington, PA) was
added to the cells that migrated into the lower well, allowing their
quantitation by flow cytometry.17 Background migration of naive
(CCR7⫹CD45RAhigh) CD8 T cells (not expressing CCR4 or CXCR1)
(Figure 1A-B) was not affected by either IL-8 or MDC (data not shown).
Migration of CM CD8 T cells, EM CD8 T cells, and EMRA CD8 T cells
is presented as fold increase/decrease using naive CD8 T cells as
internal standard.
Ligand-induced internalization
CD8 T cells were negatively selected as described. CD8 T cells were
suspended in RPMI with 1% normal human serum and incubated for 45
minutes at 37°C with IL-8 and MDC at various concentrations. After
incubation, cells were washed, and expression of CXCR1 and CCR4 on
subsets of CD8 T cells was determined by flow cytometry.
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BLOOD, 1 DECEMBER 2004 䡠 VOLUME 104, NUMBER 12
CXCR1 DEFINES A HIGHLY CYTOTOXIC CD8 T-CELL SUBSET
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Cytotoxicity assay
Statistical analysis
CXCR1⫹ CD8 T cells, CX3CR1⫹ CD8 T cells, CCR7⫺CD45RAhigh CD8
T cells, and bulk CD8 T cells were sorted (as described) and used as
effector cells. EBV-immortalized B cells were used as target cells. The
caspase-3–activity assay was performed as previously described.18 Briefly,
effector cells were incubated in duplicates with target cells at a 1:1 ratio. To
activate effector cells, 4 ␮g/mL anti-CD3 monoclonal antibody (mAb)
(OKT3, immunoglobuln G2a [IgG2a]) (eBioscience, San Diego, CA) was
used. Plates were centrifuged at 25g for 30 seconds and incubated for 90
minutes at 37°C prior to adding caspase-3 substrate for an additional 30
minutes. The percentage of caspase-3–activity–positive target cells was
analyzed by flow cytometry. Caspase-3 activity induction was almost
entirely dependent on anti-CD3 mAb activation (less than 5% killing in
absence of anti-CD3 mAb) (Figure 3C), excluding potentially contaminating
NK cells from contributing to killing.
Student t test analyses were performed with JMP Version 3 software (SAS
Institute, Cary, NC). Mean values of caspase activity were used from
individuals tested twice. Data are presented as mean ⫾ standard
deviation (SD).
Intracellular cytokine staining
CCR7⫺CD45RA⫺ CD8 T cells, CCR7⫺CD45RAhigh CD8 T cells, and
CXCR1⫹ CD8 T cells were sorted (as described). Sorted CD8 T-cell subsets
were incubated in presence/absence of 2 ␮g/mL anti-CD3 mAb (OKT3,
IgG2a) at 37°C, 5% CO2. Brefeldin A (GolgiPlug; BD PharMingen) was
added after 1 hour, and after a total incubation period of 6 hours, cells were
washed, fixed, and permeabilized with PermeaFix (Ortho Diagnostic
Systems), and then incubated for 30 minutes at 4°C with anti-IFN␥ mAb
(25723.11; BD PharMingen). The percentage of IFN␥⫹ CD8 T-cell subsets
was analyzed by flow cytometry. IFN␥ production by CD8 T-cell subsets
was entirely dependent on anti-CD3 mAb activation, excluding potentially
contaminating NK cells from contributing to the signal observed for IFN␥.
Enzyme-linked immunospot
Peptides corresponding to described optimal HIV-1, EBV, and influenza
CD8 T-cell epitopes were synthesized on an automated peptide synthesizer.
Fresh or frozen PBMCs were plated in 96-well polyvinylidene difluoride–
backed plates (MAIP S45; Millipore, Billerica, MA) previously coated with
0.5 ␮g/mL anti-IFN␥ monoclonal antibody 1-D1k (Mabtech, Stockholm,
Sweden) overnight at 4°C. PBMCs were added to the wells at 50 000 to
200 000 cells per well, and plates were processed as described.19 IFN␥producing cells were counted by direct visualization and expressed as
spot-forming cells (SFCs). Negative controls were subtracted and in all
cases were fewer than 60 SFCs per 106 input cells.
In vitro peptide stimulation
PBMCs were incubated in RPMI with 10% FCS in the presence of antigen,
control peptide (2 ␮g/mL each), or no peptide for 4 hours at 37°C. After
incubation, cells were washed, stained with tetramers and antibodies, and
analyzed by flow cytometry.
Figure 1. Chemokine receptor expression on CD8
T-cell subsets. (A) Protein and mRNA levels of chemokine receptors were assessed on naive, central memory
(CM), effector memory (EM), and CD45RA re-expressing
effector memory (EMRA) CD8 T cells. CXCR1 and CX3CR1
were selectively expressed on EMRA CD8 T cells, while
CCR4 was selectively expressed on CM and EM CD8
T-cell subsets. Note the generally tight association of protein
expression and mRNA levels. (B) Representative examples
of cell surface expression of CXCR1 and CCR4 on naive,
CM, EM, and EMRA CD8 T cells. (C) Distribution of
CXCR1⫹ CD8 T cells among subsets of CD8 T cells
defined by expression/absence of CD27 and CD28.
CXCR1⫹ CD8 T cells were highly enriched within the
CD27⫺ and CD28⫺ subpopulations of CD8 T cells. Data
represent the mean (⫾ SD) of 3 to 12 healthy blood
donors. Several donors were tested more than once. n.d.
indicates not done.
Study subjects
Influenza- and EBV-specific CD8 T cell responses were analyzed in healthy
volunteers. For influenza, 5 donors with a vaccine-induced CD8 T
cell-response and 1 donor with an infection-induced response were studied.
For EBV, all donors (n ⫽ 6) were studied in the chronic/latent phase of
infection. HIV-specific CD8 T-cell responses were characterized in individuals (n ⫽ 15) followed at the Massachusetts General Hospital ([MGH],
Boston, MA). The study was approved by the Institutional Review Board,
and we abided by the tenets of the Helsinki protocol. Informed consent was
obtained from all study participants.
Results
Expression of chemokine receptors on CD8 T-cell subsets
To begin to understand the homing patterns of human CD8
T-cell subsets, we defined the chemokine receptor profile
on naive (CCR7 ⫹ CD45RA high ), central memory (CM)
(CCR7⫹CD45RA⫺), effector memory (EM) (CCR7⫺CD45RA⫺),
and CD45RA re-expressing effector memory CD8 T cells (EMRA)
(CCR7⫺CD45RAhigh) from healthy donors. The relative proportions of these CD8 T-cell subsets were as follows: naive, 53% ⫾ 9%;
CM, 15% ⫾ 4%; EM, 20% ⫾ 7%; EMRA, 12% ⫾ 4% (n ⫽ 8).
These subsets were examined for cell-surface expression of
CCR1–CCR6, CCR9, CXCR1–CXCR6, CX3CR1, and the cytotoxic effector molecule perforin with the use of flow cytometry
(Figure 1A). Several different patterns of expression were found.
CXCR4 was expressed on more than 90% of naive CD8 T cells, but
fewer than 50% of antigen-experienced CD8 T cells. In contrast,
CCR5 was absent from naive CD8 T cells, but expressed on
approximately 70% of EM CD8 T cells and only 20% to 40% of
CM and 20% to 40% of EMRA CD8 T cells. CXCR3 expression
was not selective for any subset, while CCR4 was selectively
expressed on CM and EM CD8 T cells, but not expressed on naive
or EMRA CD8 T cells. CCR6 and CXCR6 were both expressed at
high levels (ie, bright stain; data not shown), but on only a small
percentage of CM and EM CD8 T cells, and not on EMRA CD8 T
cells. In contrast, CX3CR1 and CXCR1 were preferentially
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3466
HESS et al
expressed on EMRA CD8 T cells. The percentages of CXCR1⫹
CD8 T cells within CD8 T-cell subsets were as follows: naive,
0.8% ⫾ 0.5%; CM, 0.3% ⫾ 0.4%; EM, 4.5% ⫾ 2.8%; and EMRA,
28.1% ⫾ 11.1% (n ⫽ 12). These same CD8 T-cell subsets were
sorted by means of flow cytometry and, with the use of quantitative
PCR (QPCR), examined for levels of chemokine receptor mRNA
(Figure 1A). A generally tight association of mRNA levels and
chemokine receptor expression was observed. Representative flow
cytometry data demonstrating the selective expression of CXCR1
on EMRA, and CCR4 on CM and EM CD8 T cells are shown in
Figure 1B. Down-regulation of CD27 and CD28 has also been used
to distinguish memory- and effector-type CD8 T cells.20,21 Consistent with our findings demonstrating the enrichment of CXCR1⫹
CD8 T cells in a subset with effector function defined by CCR7
and CD45RA, CXCR1 was highly enriched in the effector-type
CD8 T-cell subsets defined by loss of CD27 or CD28 expression
(Figure 1C).
BLOOD, 1 DECEMBER 2004 䡠 VOLUME 104, NUMBER 12
Figure 3. MDC- and IL-8–induced cell-surface down-regulation of CCR4 and
CXCR1 on CD8 T-cell subsets. CD8 T cells were exposed to increasing concentrations of MDC and IL-8, and cell-surface expression was assessed by flow cytometry.
MDC selectively induced a dose-dependent internalization of CCR4 on CM and EM
CD8 T cells, while IL-8 selectively induced a dose-dependent internalization of
CXCR1 on EM and EMRA CD8 T cells. Representative data from 4 independent
experiments are shown.
CXCR1 is functional on CD8 T cells
Antigen-experienced CD8 T cells with effector function respond
quickly to chemotactic signals in order to efficiently find and
eliminate invading pathogens. We therefore assessed the chemotactic response of CD8 T cells to gradients of IL-8 (CXCL8, CXCR1
ligand) and (as an internal control) MDC (CCL22, CCR4 ligand),
using a transwell chemotaxis assay. CXCR1⫹ CD8 T cells and
CCR4⫹ CD8 T cells could not be directly tracked since exposure to
their ligands induced rapid and complete internalization of their
respective receptors. Since expression of CCR7 and CD45RA was
not affected by exposure to IL-8 and MDC (data not shown), we
determined chemotaxis of CD8 T-cell subsets defined by these
markers along a gradient of IL-8 and MDC. We found a selective
and dose-dependent enrichment of EMRA greater than EM CD8 T
cells (expressing CXCR1) (Figure 1A-B) in response to a chemotactic gradient of IL-8 (Figure 2A), whereas CM and EM CD8 T
cells (expressing CCR4) (Figure 1A-B) selectively migrated in
response to a chemotactic gradient of MDC (Figure 2B).
Rapid internalization of many chemokine receptors upon ligand
binding is thought to allow a cell to continuously sense small
changes in ligand concentration gradients,22,23 and ligand-induced
chemokine receptor internalization and chemotaxis can occur
independently.24 As another test of CXCR1 responsiveness in CD8
T-cell subsets, CD8 T cells isolated from healthy donors were
incubated with increasing concentrations of IL-8 and (as an internal
control) MDC. Subsets of CD8 T cells were then examined for
cell-surface expression of CXCR1 and CCR4. In a specific and
Figure 2. Chemotaxis of CD8 T-cell subsets. Chemotaxis of CD8 T-cell subsets in
response to IL-8 (A) and MDC (B). Migration of CM, EM, and EMRA CD8 T cells is
shown relative to background migration of naive CD8 T cells. Migration of naive CD8
T cells (CCR7⫹CD45RAhigh) was not affected by IL-8 or MDC. Subset migration
relative to migration of naive CD8 T cells in the absence of chemokine is defined as a
chemotactic index of 1. Note that the selective migration of EMRA is greater than that
of EM CD8 T cells in response to IL-8, versus the selective migration of CM that is
greater than that of EM CD8 T cells in response to MDC. Representative data from 3
independent experiments are shown.
dose-dependent manner, IL-8 induced internalization of CXCR1 on
EMRA and EM CD8 T cells, whereas MDC induced internalization
of CCR4 on CM and EM CD8 T cells (Figure 3). No effect of IL-8
and MDC on cell surface staining of their respective receptors was
observed when experiments were repeated at 4°C, thus excluding
competition of IL-8 and MDC for antibody binding (data not
shown). Thus, EMRA CD8 T cells selectively migrate toward IL-8,
and IL-8 selectively induces CXCR1 internalization on EMRA
CD8 T cells.
Cytotoxicity and IFN␥ production of CXCR1ⴙ CD8 T cells
To determine if CXCR1 defined a unique CD8 T-cell subset, we
compared expression of perforin in CXCR1⫹ CD8 T cells with the
entire population of EMRA CD8 T cells. Perforin was expressed in
approximately 60% more CXCR1⫹ CD8 T cells than EMRA CD8
T cells, suggesting that CXCR1⫹ CD8 T cells are more immediately cytotoxic (Figure 4A). Likewise, when mRNA levels of
perforin and granzyme B were compared in sorted bulk CD8 T
cells, EMRA CD8 T cells, and CXCR1⫹ CD8 T cells, the highest
amount of perforin and granzyme B message was detected in the
CXCR1⫹ CD8 T-cell subset (Figure 4B). Since CX3CR1 is also
preferentially expressed on CD8 T cells with an effector phenotype,10 we also compared expression of perforin in CX3CR1⫹ CD8
T-cell with expression in CXCR1⫹ CD8 T cells. As was true for
EMRA CD8 T cells, approximately 60% more CXCR1⫹ CD8 T
cells expressed perforin compared with the CX3CR1⫹ subset
(Figure 4A). Thus, in contrast to the CXCR1⫹ subset, the CX3CR1⫹
subset was not enriched for perforin compared with the bulk
EMRA cell population. To directly assess cytotoxic activity, we
compared ex vivo cytotoxicity of sorted EMRA CD8 T cells,
CX3CR1⫹ CD8 T cells, CXCR1⫹ CD8 T cells, and bulk CD8 T
cells in an apoptosis assay based on detection of caspase-3 activity
at the single-cell level. As shown in Figure 4C, cytotoxic activity
varied significantly among individuals, but was always highest in
the CXCR1⫹ subset of CD8 T cells. Cytotoxicity of the CX3CR1⫹
subset was not higher than cytotoxicity of EMRA CD8 T cells,
although both were more cytotoxic than bulk CD8 T cells. In
addition to perforin/granzyme–mediated cytotoxicity, IFN␥ is also
an important component of the CD8 T cell–mediated immune
response.25 Consistent with increased cytotoxicity of the CXCR1⫹
CD8 T-cell subset, there was a significantly higher percentage of
IFN␥-producing CD8 T cells in the CXCR1⫹ subset compared with
the EMRA subset (Figure 4D). As was true for cytotoxicity, the
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BLOOD, 1 DECEMBER 2004 䡠 VOLUME 104, NUMBER 12
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Figure 4. Perforin, granzyme B, and IFN␥ expression and ex vivo cytotoxic activity of CD8 T-cell subsets. (A) The percentage of EMRA CD8 T cells and CX3CR1⫹ CD8
T cells expressing perforin was compared with the percentage of CXCR1⫹ CD8 T cells expressing perforin. Significantly more CXCR1⫹ CD8 T cells expressed perforin than
either EMRA CD8 T cells (77% ⫾ 9% versus 48% ⫾ 12%) or CX3CR1⫹ CD8 T cells (77% ⫾ 9% versus 44% ⫾ 8%). Data represent the mean (⫾ SD) from at least 3
independent experiments. (B) Comparison of perforin and granzyme B mRNA expression levels of bulk CD8 T cells, EMRA CD8 T cells, and CXCR1⫹ CD8 T cells. The mRNA
levels of perforin and granzyme B were 2-fold and 0.25-fold higher, respectively, in the CXCR1⫹ CD8 T-cell subset than in the EMRA CD8 T-cell subset. Data represent the
mean (⫾ SD) from 3 independent experiments. (C) Direct ex vivo cytotoxic activity of bulk CD8 T cells, CX3CR1⫹ CD8 T cells, EMRA CD8 T cells, and CXCR1⫹ CD8 T cells was
compared in 6 individuals, by means of a sensitive caspase-activity assay. Representative data from 4 independent experiments are shown, and data are expressed as the
percentage of caspase-positive target cells induced by each indicated group. The relative difference between CXCR1⫹ CD8 T cells and EMRA CD8 T cells and between
CXCR1⫹ CD8 T cells and CX3CR1⫹ CD8 T cells is indicated in the figure (⌬). Statistical analysis of the pooled data (n ⫽ 6 individuals; 2 of them tested twice) confirmed that
cytotoxicity was significantly higher in the CXCR1⫹ CD8 T-cell subset (42% ⫾ 15% caspase-positive target cells) than in bulk CD8 T cells (21% ⫾ 14% caspase-positive target
cells), EMRA CD8 T cells (30% ⫾ 12% caspase-positive target cells), or CX3CR1⫹ CD8 T cells (28% ⫾ 6% caspase-positive target cells) (P ⬍ .005). In the absence of
anti-CD3 antibody stimulation, fewer than 5% of target cells were caspase-positive. (D) IFN␥ production of anti-CD3–activated EM CD8 T cells, EMRA CD8 T cells, and
CXCR1⫹ CD8 T cells was analyzed by intracellular cytokine staining. Four independent experiments are shown, and data are expressed as the percentage of IFN␥⫹ cells within
the indicated CD8 T-cell subset. The relative difference between cytotoxic activity of CXCR1⫹ CD8 T cells and EMRA CD8 T cells and between CXCR1⫹ CD8 T cells and EM
CD8 T cells is indicated in the figure (⌬). Statistical analysis of the pooled data (n ⫽ 4) confirmed that IFN␥ production was significantly higher in the CXCR1⫹ CD8 T-cell subset
(37% ⫾ 7% IFN␥⫹ CD8 T cells) than in EMRA CD8 T cells (25% ⫾ 5% IFN␥⫹ CD8 T cells) and EM CD8 T cells (19% ⫾ 4% IFN␥⫹ CD8 T cells) (P ⬍ .005). No IFN␥ production
was observed in absence of anti-CD3 antibody stimulation (data not shown).
number of IFN␥-producing cells varied among individuals, but was
always highest in the CXCR1⫹ subset of CD8 T cells (Figure 4D).
These data demonstrate that CXCR1 uniquely defines a subset of
IL-8–responsive CD8 T cells with increased cytotoxicity and
IFN␥ production.
CXCR1 expression on influenza-, EBV-, and HIV-1–specific
CD8 T cells
We next examined CXCR1 expression on antigen-specific CD8 T
cells directed against influenza, EBV, and HIV-1 epitopes in
individuals with known responses as determined by enzyme-linked
immunospot (ELISpot) analyses. Patient characteristics are summarized in Figure 5. Major histocompatibility complex (MHC) class I
tetramers were used to identify antigen-specific CD8 T cells.
Distribution of influenza-specific CD8 T-cell maturation subsets
(CM, EM, EMRA) was as follows: CM, 34 ⫾ 22; EM, 39 ⫾ 15;
EMRA, 27 ⫾ 11. Distribution of EBV-specific CD8 T-cell subsets
was as follows: CM, 8 ⫾ 6; EM, 49 ⫾ 5; EMRA, 44 ⫾ 11.
Similarly to the distribution on bulk CD8 T cells, on EBV- and
influenza-specific CD8 T cells, CXCR1 was expressed on 25% to
30% of antigen-specific EMRA CD8 T cells, while CXCR1 was
detected on only approximately 10% of EM and approximately 2%
of CM CD8 T cells. This expression pattern was observed for
antigen-specific EMRA CD8 T cells targeting an HLA-A2–
restricted matrix-derived influenza epitope (GILGFVFTL), as well
as 2 latent and 2 lytic EBV epitopes (latent, HLA-B8–restricted
FLRGRAYGL [EBNA3A] and QAKWRLQTL [EBNA3A]; lytic,
HLA-A2–restricted GLCTLVAML [BMLF1] and HLA-B8–
restricted RAKFKQLL [BZLF1]) (Figure 6A-B). As expected, no
EBV DNA was detectable by PCR in the plasma of any of the
individuals studied (data not shown). Representative flowcytometry profiles of CXCR1 expression on influenza- and EBVspecific CD8 T-cell subsets are shown in Figure 6C.
For our HIV studies, we analyzed individuals belonging to 2
clinical categories: (i) persons able to control HIV-1 replication at
relatively low levels (fewer than 7500 copies per milliliter plasma)
without highly active antiretroviral therapy (HAART) (controllers), and (ii) persons unable to control viral replication off
HAART (noncontrollers). The group of controllers was recruited from the Boston, MA, acute–structured treatment interruption (STI) cohort, which is following persons induced to
control HIV-1 replication through treatment of acute HIV-1
infection, followed by supervised treatment interruptions (induced controllers, H1-H4).26 These induced controllers offered
the unique opportunity to study persons able to control HIV-1
replication both on and off HAART. The group of noncontrollers
was subdivided into persons on HAART (H5-H11) and persons
off HAART (H12-H15). The following tetramers were used to
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3468
HESS et al
BLOOD, 1 DECEMBER 2004 䡠 VOLUME 104, NUMBER 12
the percentage of CXCR1⫹ HIV-1–specific CD8 T cells decreased
to a mean of 3% (Figure 7A, right nonshaded panel). Expression of
CXCR1 on HIV-1–specific CD8 T cells in medically controlled
HIV-1 infection thus differentiated persons able to control HIV-1
replication from persons unable to do so when HAART was
discontinued. Representative flow-cytometry profiles of CXCR1
expression on HIV-1–specific CD8 T cells in noncontrollers and
induced controllers, both on and off HAART, are shown in
Figure 7B.
Other chemokine receptors expressed on HIV-1–specific CD8 T
cells isolated from induced controllers (eg, CX3CR1, CXCR3,
CCR5, CXCR4) did not vary significantly with viral load. In
contrast, HLA-DR, which is commonly used as a CD8 T-cell
activation marker,27,28 displayed an expression pattern inverse to
that of CXCR1, being expressed on approximately 3-fold more
HIV-1–specific CD8 T cells at times when persons were off
HAART, compared with when they were on HAART (Figure 7C).
Thus, CXCR1 appears to be uniquely regulated on HIV-1–specific
CD8 T cells.
Antigen-induced down-regulation of CXCR1
Figure 5. Summary of clinical, virologic, and immunologic parameters of study
participants. Shaded indicates patient on highly active antiretroviral therapy (HAART);
not shaded indicates off HAART. — indicates undetectable; ND, not done; NA, not
applicable. *Symbols indicate study subjects within clinical category and correspond
to symbols used in Figures 6A-B and 7A.
analyze HIV-1–specific CD8 T cells: HLA-B8–restricted,
FLKEKGGL (nef); HLA-B8–restricted, EIYKRWII (p24); HLAB7–restricted, IPRRIRQGL (gp41); and HLA-A2–restricted,
SLYNTVATL (p17).
In noncontrollers (both on and off HAART), a mean of only
approximately 5% of HIV-1–specific CD8 T cells expressed
CXCR1 (Figure 7A, left panels). In contrast, a significant percentage of HIV-1–specific CD8 T cells isolated from induced controllers on HAART (viral loads fewer than 50 copies per milliliter
plasma) expressed CXCR1 (mean 16%) (Figure 7A, right shaded
panel). However, when these same persons were off HAART and
viral loads increased (range, 357-7290 copies per milliliter plasma),
We hypothesized that antigen exposure and hence activation in
vivo, as suggested by increased expression of HLA-DR off
HAART, down-modulated CXCR1 on antigen-specific effector
CD8 T cells of induced controllers. To confirm this in vitro, we
pulsed autologous antigen-presenting cells of study participants
H1, H3, H4, and E3 with optimal epitope peptides, and
examined the effect of antigen-specific activation on levels of
CXCR1, CX3CR1, and CXCR3 expression on antigen-specific
EMRA CD8 T cells (Figure 8). The percentage of EMRA CD8 T
cells that expressed CXCR3 on HIV-1–specific CD8 T cells
increased after a 4-hour incubation period, irrespective of
addition of antigen, control peptide, or no peptide (Figure 8A).
The percentage of EMRA CD8 T cells that expressed CX3CR1
did not significantly change following 4 hours of incubation
with antigen, control peptide, or no peptide (Figure 8B). In
contrast, the percentage of both HIV-1–specific and EBVspecific EMRA CD8 T cells (but not bulk CD8 T cells; data not
shown) that expressed CXCR1 on their cell-surface was markedly decreased following a 4-hour incubation with their cognate
antigen, but not following incubation with control peptide or no
Figure 6. CXCR1 expression on subsets of EBV- and influenza-specific CD8 T cells. The percentage of antigen-specific CD8 T-cell subsets expressing CXCR1 was
assessed by means of HLA class I tetramers combined with antibody staining. CD8 T-cell responses were identified by ELISpot analyses using optimal epitope peptides and
are given in Figure 5. (A) Percentage of CXCR1 expression on subsets of influenza-specific CD8 T cells. (B) Percentage of CXCR1 expression on subsets of EBV-specific CD8
T cells. Both lytic and latent antigen-specific CD8 T-cell responses were analyzed (Figure 5). (C) Representative flow-cytometry profiles of CXCR1 expression on influenza- and
EBV-specific CD8 T-cell subsets.
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BLOOD, 1 DECEMBER 2004 䡠 VOLUME 104, NUMBER 12
CXCR1 DEFINES A HIGHLY CYTOTOXIC CD8 T-CELL SUBSET
3469
Figure 7. CXCR1 expression on HIV-1–specific CD8 T
cells in noncontrollers and controllers. (A) The percentage of antigen-specific CD8 T cells expressing
CXCR1 was assessed by means of HLA class I tetramers
combined with antibody staining. The 2 left columns
compare CXCR1 expression on HIV-1–specific CD8 T
cells in noncontrolling individuals on HAART (shaded
column, H5-H11; viral loads below detection limit) with
noncontrolling individuals off HAART (nonshaded column, H12-H15; viral load range, 18 000-209 193 copies
per mL plasma) (cross-sectional comparison). The 2 right
columns longitudinally compare CXCR1 expression on
HIV-1–specific CD8 T cells in induced controllers (H1-H4)
on HAART (shaded column, viral loads below detection
limit) and off HAART (nonshaded column; viral load
range, 357-7290 copies per mL plasma). Significant
expression of CXCR1 on HIV-1–specific CD8 T cells was
observed only in persons on HAART with treated acute
HIV-1 infection (induced controllers). (B) Representative
flow-cytometry profiles of CXCR1 expression on HIV-1–
specific CD8 T cells in noncontrollers and induced controllers, both on and off HAART. (C) In contrast to CXCR1,
no significant differences were observed in the percentage of HIV-1–specific CD8 T cells that expressed CCR5,
CXCR4, CXCR3, or CX3CR1 when induced controllers
were studied on or off HAART. The percentage of
HIV-1–specific CD8 T cells that expressed HLA-DR,
however, was significantly higher when these were analyzed while people were off, as opposed to on, HAART
(60% ⫾ 6% versus 21% ⫾ 6%). Data represent the mean
(⫾ SD) of at least 4 independent measurements.
peptide (Figure 8C-D). These data demonstrate that cell-surface
expression of CXCR1 is uniquely down-modulated upon antigenspecific activation of EMRA CD8 T cells.
Figure 8. Antigen-specific stimulation of HIV-1– and EBV-specific CD8 T cells in
vitro. PBMCs from study subjects H1, H3, H4 (all HIV-1), and E3 (EBV) were pulsed
with antigen (HIV-1 or EBV peptide) or control peptide (c-peptide) or were left
unpulsed (no peptide). After a 4-hour incubation, expression of CXCR3 (A), CX3CR1
(B), and CXCR1 (C-D) was assessed on antigen-specific EMRA CD8 T cells. Note
that CXCR3 cell-surface expression increased, while CX3CR1 levels did not change
following the 4-hour incubation, irrespective of incubation conditions. In contrast,
CXCR1 expression decreased on HIV-1– and EBV-specific EMRA CD8 T cells
stimulated with cognate antigen. Data represent the mean (⫾ SD) of 3 and 2
independent experiments for HIV-1 and EBV, respectively.
Discussion
Chemokine receptor expression is tightly controlled during T-cell
development, differentiation, and activation, controlling homing of
lymphocyte subsets.12,29-31 To begin to understand the molecular
basis for the different trafficking patterns of CD8 T-cell subsets, we
screened CD8 T cells phenotypically characterized as naive, central
memory (CM), effector memory (EM), and CD45RA re-expressing
effector memory (EMRA) cells, using a panel of 16 chemokine
receptor monoclonal antibodies. We found that chemokine receptors were differentially expressed on CD8 T-cell subsets. CXCR4
was preferentially expressed on naive cells; CCR4 and CCR6 on
CM cells; CCR5 and CXCR6 on EM cells; and CXCR1 and
CX3CR1 on EMRA cells. In addition to the functional implications
of CD8 T cells with effector function that respond to IL-8
(discussed in the next few paragraphs), our study identified CXCR1
as defining a novel subset of CD8 T cells with uniquely high levels
of IFN␥, perforin, and cytotoxic activity.
CXCR1-ligand (IL-8/CXCL8 and granulocyte chemotactic protein-2 [GCP-2]/CXCL6) responsiveness will allow this subset of
highly cytotoxic CD8 T cells to respond to signals derived from
innate immune responses. Toll-like receptors (TLRs) have recently
been shown to play an important role in innate immune recognition
of pathogens.32 Upon activation, TLRs lead to a signal transduction
cascade that results in cellular activation and chemokine release,
with one of the earliest and most abundantly produced chemokines
being IL-8.13,33 CXCR1 thus defines a “rapid-responder” subset of
CD8 T cells that bridges the gap between the innate and acquired
immune responses. CXCR1⫹ CD8 T cells may provide highly
cytotoxic antigen-specific effector function early at sites of infection, prior to the generation of a new wave of effector cells that first
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3470
BLOOD, 1 DECEMBER 2004 䡠 VOLUME 104, NUMBER 12
HESS et al
needs to be induced to proliferate and differentiate from CM and
EM pools.
Several earlier reports provided evidence for IL-8 responsiveness of CD8 T cells; however, the notion that IL-8 can act as a
direct T-cell chemoattractant has been controversial.34-39 Our
analysis offers a possible explanation for these seemingly contradictory studies, by demonstrating that functional CXCR1 is expressed
on only a relatively small subset of highly cytotoxic CD8 T cells.
High levels of CXCR1 expression on neutrophils and NK cells
make it difficult to identify expression and function of this receptor
on CD8 T cells in samples that contain even small amounts of these
other cell types. Furthermore, as demonstrated in our study, rapid
down-modulation of CXCR1 by both ligand and antigen may have
further complicated the analysis of CXCR1 expression and function on CD8 T cells.
As was true for bulk CD8 T cells, CXCR1 was also preferentially expressed on influenza- and EBV-specific EMRA CD8 T
cells. The small possibility that certain viruses could alter the
expression pattern of CXR1 on maturation subsets therefore seems
unlikely. To begin to assess the physiologic importance of CXCR1⫹
CD8 T cells in the control of chronic viral infections, the
percentage of CXCR1⫹ CD8 T cells was compared in persons who
were able to control HIV-1 replication and persons who were
unable to control HIV-1 replication. Interestingly, significant
expression of CXCR1 on HIV-1–specific CD8 T cells in individuals on HAART was restricted to persons who were able to control
HIV-1 replication when taken off HAART (Figure 7A; for viral
loads, see Figure 5). These data suggest that HIV-1–specific
CXCR1⫹ CD8 T cells are poised to deliver highly cytotoxic
antigen-specific effector function early at sites of infection, and
therefore may play a role in limiting the spread and/or formation of
viral reservoirs, thus influencing viral load set point and outcome of
HIV-1 infection.
Off HAART, little expression of CXCR1 on HIV-1–specific
CD8 T cells was observed (Figure 7A, nonshaded panels). Several
factors might account for the absence of HIV-1–specific CXCR1⫹
CD8 T cells in controllers studied off HAART. CXCR1⫹ CD8 T
cells might be preferentially recruited into sites of viral replication,
thus being absent from the peripheral blood. Alternatively, CXCR1
expression might be down-regulated via antigen-specific activation. Consistent with this second hypothesis, we found that CXCR1
was selectively down-modulated on HIV-1–and EBV-specific CD8
T cells following antigen-specific activation in vitro. In neutrophils, in addition to ligand-induced internalization, proinflammatory stimuli, such as tumor necrosis factor (TNF) and lipopolysaccharide (LPS), have been shown to down-modulate CXCR1
through proteolytic cleavage.40 It is intriguing to note that levels of
CXCR1 on both neutrophils and effector CD8 T cells are tightly
controlled, suggesting that differential responsiveness to CXCR1
agonists is an important feature in fine-tuning the immune response.
In summary, we have identified an IL-8–responsive, CXCR1⫹
subset of highly cytotoxic CD8 T cells, presumably poised to
rapidly deliver an antigen-specific cytotoxic response to sites of
innate immune system activation. In HIV-1 infection, expression of
CXCR1 was characteristic of persons who, when taken off
HAART, were able (at least transiently) to control HIV-1 replication. Antigen-driven shaping of migratory characteristics of CD8
T-cell subsets, as demonstrated here for CXCR1, provides a novel
mechanism whereby the immune system may adapt to changing
requirements during differing stages of an ongoing immune response.
Acknowledgment
We thank L. M. Henry for excellent technical assistance.
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2004 104: 3463-3471
doi:10.1182/blood-2004-03-1067 originally published online
August 3, 2004
IL-8 responsiveness defines a subset of CD8 T cells poised to kill
Christoph Hess, Terry K. Means, Patrick Autissier, Tonia Woodberry, Marcus Altfeld, Marylyn M.
Addo, Nicole Frahm, Christian Brander, Bruce D. Walker and Andrew D. Luster
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