Interleukin-4 promotes human CD8+ T cell expression of CCR7

Interleukin-4 promotes human CD8+ T cell expression of CCR7

Introduction

Differentiation of human T cells reflects changes in the

expression of proteins that control cell cycle, survival,

migration and effector functions. Numerous models have

been proposed with considerable controversy regarding the

phenotypic associations.1 Although it is broadly agreed that

naive T cells express CD45RA, CCR7, CD62L, CD28 and

CD27, the patterns with which these are lost (or subse-

quently re-acquired) following antigen encounter are under

debate. For example, expression of the lymph node homing

receptor CCR7 has been suggested to define progression

from central memory (CCR7+) to effector memory

(CCR7)) populations, and although these terms are widely

employed, it is becoming increasingly clear that CCR7 can

be re-acquired.2,3 The analysis of markers associated with

T cell activation has also contributed to the controversy. It

is relatively well accepted that several cell surface molecules

are expressed transiently after interaction with cognate

antigen including CD25, HLA-DR, CD38 and CD71.

More recently, a combination of CD28, CD27, CCR7

and CD45RA has been used to define a linear model of

progression of antigen-experienced CD8+ T cells from

early (CD27+, CD28+, CCR7), CD45RA)) to intermediate

(CD27+, CD28), CCR7), CD45RA+/)) to late (CD27),

CD28), CCR7), CD45RA+/)) differentiation, but it is

recognized that the markers can be variable.1 Little is

known of the mechanisms of regulation of expression of

markers of human T cell differentiation, but transforming

growth factor (TGF)-b is thought to promote the expres-

sion of CCR74 and interleukin (IL)-12 promotes murine

CD62L.5

We have previously shown that cytomegalovirus

(CMV)-specific CD8+ T cells express markers associated

with activation and effector function, and putatively

follow the linear differentiation pathway described above,

Suranjith L. Seneviratne,1 Antony P.

Black,1 Louise Jones,1 Kati di

Gleria,1 Abigail S. Bailey1 and

Graham S. Ogg1,2

1MRC Human Immunology Unit and Univer-

sity of Oxford, Weatherall Institute of Molecu-

lar Medicine, Oxford, United Kingdom, and2Department of Dermatology, Churchill

Hospital, Oxford, United Kingdom

doi:10.1111/j.1365-2567.2006.02478.x

Received 19 January 2006; revised 23 August

2006; accepted 23 August 2006.

Correspondence: Dr Graham Ogg, Cutaneous

Immunology Group, MRC Human

Immunology Unit and University of Oxford,

Weatherall Institute of Molecular Medicine,

Oxford OX3 9DS, United Kingdom.

Email: graham.ogg@clinical-medicine.

ox.ac.uk

Senior author: Dr Graham Ogg

Summary

Despite strong evidence supporting a pathway of human T cell differenti-

ation characterized by changes in the expression of CCR7, CD28, CD27

and CD62L, few studies have addressed the mechanisms of pathway regu-

lation. Cutaneous lymphocyte-associated antigen (CLA)-positive skin-

homing CD8+ T cells expressed significantly elevated levels of activation

markers compared with CLA) CD8+ T cells in individuals (n = 27) with

cutaneous atopic disease. Despite such an activated phenotype, CLA+ T

cells expressed significantly higher levels of CCR7 than a CLA) T cell sub-

set. Interleukin (IL)-4 was found to dramatically promote CCR7 expres-

sion by antigen-specific CD8+ cells. Furthermore, skin-homing CD8+ T

cells from individuals with severe disease produced significantly less IL-10

than those derived from mildly affected atopic subjects. Thus in a

T-helper 2 dominated disease, tissue-specific CD8+ T cells show altered

CCR7 expression and cytokine production, which may contribute to con-

tinued lymph node homing, antigen presentation and disease. IL-4 pro-

motes expression of CCR7, a marker linked to existing models of CD8+ T

cell differentiation.

Keywords: cytokines; cell differentiation; T cells; cutaneous lymphocyte

associated antigen

Abbreviations: AD, atopic dermatitis; CLA, cutaneous lymphocyte-associated antigen; CMV, cytomegalovirus; HIV, humanimmunodeficiency virus; PBMCs, peripheral blood mononuclear cells.

66 � 2006 The Authors Journal compilation � 2006 Blackwell Publishing Ltd, Immunology, 120, 66–72

I M M U N O L O G Y O R I G I N A L A R T I C L E

being largely within the CCR7), CD27), CD28) late dif-

ferentiated subset.6 However, human immunodeficiency

virus (HIV)-specific CD8+ T cells frequently show partial

defects in effector function and dominate within the

CCR7+/), CD27+, CD28+/) intermediate subset.3,6 To our

knowledge, there are no data examining the influence of

T helper (Th2) cytokines on markers linked to the pro-

gression of human CD8+ T cell differentiation. Under-

standing the events linked to T cell differentiation is

important in order to define the role of T cells in disease

pathogenesis and also to direct T cell-mediated therapies

(e.g. vaccination) to the optimal induction or modifica-

tion of particular relevant T cell subsets.

Having derived peripheral blood mononuclear cells

(PBMCs) from individuals (n ¼ 27) with cutaneous

atopic disease, we observed that cutaneous lymphocyte-

associated antigen (CLA)-positive skin-homing CD8+

T cells expressed significantly elevated levels of activation

markers compared with CLA) CD8+ T cells. For classical

viral-specific CD8+ T cells, this phenotype was associated

with loss of CCR7 expression. However, by contrast, the

activated CLA+ T cells expressed significantly higher levels

of CCR7 than the CLA) T cell subset. We tested the hypo-

thesis that Th2 cytokines might influence the expression

of markers associated with differentiation. Human leuco-

cyte antigen (HLA)-peptide tetrameric complexes were

used to facilitate phenotypic analysis of human antigen-

specific T cells following stimulation with cognate antigen

in the presence or absence of IL-4, IL-5 and IL-13. IL-4

was found to dramatically promote CCR7 expression and

to polarize CD8+ T cells towards a Tc2 phenotype. Fur-

thermore, skin-homing CD8+ T cells were enriched for an

allergen-specific subset, and cells from individuals with

severe disease produced significantly less IL-10 than those

derived from mildly affected atopic subjects. Thus in con-

clusion, IL-4 promotes the expression of CCR7, a marker

currently linked to CD8+ T cell differentiation.

Materials and methods

Subjects

Fifteen severely symptomatic, 12 mildly symptomatic

atopic individuals and 10 non-atopic controls were

recruited through the Department of Dermatology, Chur-

chill Hospital, Oxford under the ethical approval of the

Oxfordshire Clinical Research Committee. Atopic derma-

titis was defined according to the UK refinements of the

Hanifin and Rajka diagnostic criteria for atopic dermatitis

(AD), which are specific and sensitive for AD.7,8 The cri-

teria require the presence of an itchy skin condition plus

three or more of the following: onset below 2 years

of age; history of skin crease involvement; history of

generally dry skin; visible flexural dermatitis, and personal

history of another atopic disease (or history of atopic

disease in a first-degree relative). All severely symptomatic

atopic patients had, by definition, a disease severity

SASSAD (six-area, six-sign atopic dermatitis) score > 54

(of a possible total of 108).9 All individuals in the mildly

affected group had a SASSAD score < 10. The SASSAD

score is graded on a scale of 0–3 (absent to severe) for

each of the six clinical features (erythema, exudate, excor-

iation, lichenification, dryness, cracking) at six sites (head/

neck, thorax/proximal limbs, mid upper limbs, mid lower

limbs, hands, feet). All the atopic subjects had positive

skinprick tests and IgE radioallergosorbent tests (RAST)

to housedust mite. None of the non-atopic subjects had a

personal or family history of atopic disease and all had

negative housedust mite skinprick tests and IgE RASTs.

There were no statistically significant differences between

the groups in the distribution of age, sex and racial origin.

None of the patients had received systemic immuno-

suppressive treatment for the preceding 6 months.

HLA-peptide tetrameric complexes

Complexes were synthesized as previously described.10,11

Purified HLA heavy chain and b2 microglobulin were

synthesized by means of a prokaryotic expression sys-

tem (pET Novagen; EMD Biosciences, San Diego, CA).

The heavy chain was modified by deletion of the trans-

membrane/cytosolic domain and C-terminal addition of a

sequence containing the BirA enzymatic biotinylation site.

Heavy chain, b2 microglobulin were refolded by dilution

with Epstein Barr virus (EBV) BMLF1 peptide 280–8

GLCTLVAML. The 45 kDa refolded product was isolated

by fast protein liquid chromatography (FPLC), and then

biotinylated by BirA (Avidity, Denver, CO) in the pres-

ence of biotin (Sigma, Gillingham, UK), ATP (Sigma)

and Mg2+ (Sigma). Streptavidin-phycoerythrin conjugate

(Sigma) was added in a 1 : 4 molar ratio and the tetra-

meric product was concentrated to 1 mg/ml.

Flow cytometry

Four-colour flow cytometric analysis was performed using

a fluorescence-activated cell sorter (FACS) Calibur (Becton

Dickinson, Oxford, UK) with CELLQUEST software (Becton

Dickinson). 106 PBMC were centrifuged at 300 g for 5 min

and resuspended in a volume of 50 ll R10 (RPMI-1640

medium (Gibco BRL, Paisley, UK) supplemented with 10%

fetal calf serum (FCS) (Globepharm Ltd, Guildford, UK),

2 mM L-glutamine, 50 U/ml penicillin and 50 lg/ml strepto-

mycin). Tetrameric complex was added and incubated at

37� for 20 min. Directly conjugated antibodies (including

anti-CD8, anti-CLA, anti-CD62L, anti-CCR7, anti-CD28,

anti-CD27, anti-CD45RA, anti-CD25, anti-HLA-DR) were

added and the samples incubated for 60 min at 4�.

After two washes in cold phosphate-buffered saline, the

samples were fixed in 2% formaldehyde. Cytokine-specific

� 2006 The Authors Journal compilation � 2006 Blackwell Publishing Ltd, Immunology, 120, 66–72 67

IL-4 promotes CCR7 expression

antibodies were used as follows: phycoerythrin (PE) con-

jugated mouse anti-human IL-4, PE rat anti-human

IL-13, PE rat anti-human IL-5, PE and fluorescein isothio-

cyanate (FITC) mouse anti-human interferon (IFN)-c, PE

mouse anti-human tumour necrosis factor (TNF)-a,

PE-CD25, PE and FITC mouse IgG1, mouse IgG2b, rat

IgG1, rat IgM, PerCP (peridinin chlorophyll protein) con-

jugated CD3 and allophycocyanin (APC) conjugated CD8

(all from Becton Dickinson), FITC rat anti-human IL-4

and PE rat anti-human IL-10 (all from Caltag, San Fran-

cisco, CA). For the phorbol myristate acetate (PMA)/ion-

omycin experiments, cells were stimulated for 4 hr in R10

medium containing 25 ng/ml PMA, 1 lg/ml ionomycin

and 10 mg/ml Brefeldin A.

Statistics

Statistical analyses were performed using the chi-square,

Fisher’s exact and related t-tests and Pearson’s correlation

coefficient.

Antigen-specific T cell culture

PBMC were centrifuged for 5 min at 300 g and the super-

natant removed. 100 ll of 200-lM filtered peptide was

added and the cells incubated for 1 hr at 37� in 5% CO2.

The cells were resuspended in R10 medium with IL-7

(25 ng/ml) in 24 well plates at 2 · 106 cells/ml. On day 4,

IL-2 was added to a final concentration of 50 IU/ml. Other

cytokines were used at the concentrations specified in the

text. Peptides used for T cell culture and for synthesizing

major histocompatibility complex (MHC)–peptide tetra-

meric complexes were synthesized at the laboratory

in-house facility on an automated peptide synthesizer (396

MPS; Advanced Chemtech, Louisville, KY) by conven-

tional solid phase Fmoc chemistry. These peptides were all

analysed for purity by reverse phase high-performance

liquid chromatography (HPLC). Cells were grown at 37�in 5% CO2, in R10 medium, unless otherwise stated. Cell

lines were regularly screened for mycoplasma infection.

Results

Phenotypic analysis of skin-homing T cells

The expression of CD25, HLA-DR, CD38 and CD71 by

CLA+ CD3+ T cells was significantly (P < 0�05) greater in

individuals with severe atopic disease than those with

mild disease. Specifically, the expression by CLA+ CD3+ T

cells in those with severe disease compared with those

with mild disease was: 43�1% vs. 25% CD25; 29% vs.

19�7% HLA-DR; 24�8% vs. 18�4% CD38, and 39�1% vs.

29�2% CD71. There was some variation in the intensity

of CLA expression within the CLA+ population, and

greater intensity of anti-CLA staining associated with

higher levels of HLA-DR, CD38 and CD71 expression.

CD69 expression on CLA+ CD3+ T cells was also investi-

gated but the cells were virtually all negative, consistent

with the finding that CD69 is an early marker of activa-

tion that is only briefly expressed. Furthermore, the

CLA+ CD8+ T cells derived from atopic subjects (mild

and severely affected) expressed significantly (P < 0�05)

greater levels of activation markers than the CLA) CD8+

T cells. Specifically, the levels expressed by CLA+ CD8+ T

cells compared with CLA) CD8+ T cells were: 32�2%

(4�3) vs. 11�9% (5�3) CD25; 21�1% (6�2) vs. 9�2% (3�2)

HLA-DR; 25�2% (1�9) vs. 8% (2�0) CD38, and 30% (4�3)

vs. 15�9% (3�7) CD71 (standard deviations shown in

brackets). Furthermore, the levels of activation markers

expressed by CLA+ CD8+ T cells from atopic individuals

(mild and severely affected) were significantly (P < 0�05)

higher than those from non-atopic subjects (Table 1).

Overall, these data suggest that CLA+ CD8+ T cells isola-

ted from individuals with AD express markers of activa-

tion more frequently than CLA) CD8+ T cells, and are

consistent with T cell recognition of persistent cutaneous

atopic antigens.

Although both CD28 and CD27 expression by

CLA+ CD3+ T cells in individuals with severe atopic dis-

ease were significantly (P < 0�05) lower than in those

with mild disease, the differences were small, with the

majority expressing both CD28 and CD27. Specifically,

the levels in those with severe disease compared with

those with mild disease were: 87% vs. 97% CD28, and

75% vs. 87% CD27. The levels of expression of CD28 and

CD27 by CLA+ CD8+ T cells were not significantly dif-

ferent from levels expressed by CLA) CD8+ T cells

(Table 2). However, the levels of CD62L and CCR7 were

significantly higher (P < 0�01) in CLA+ CD8+ T cells

than in CLA) CD8+ T cells (Table 2). By contrast, the

EBV)tetramer binding cells expressed significantly lower

levels of CD62L and CCR7 (Table 2). These levels of

CCR7 and CD62L expressed by EBV-specific T cells were

significantly (P < 0�01) lower than the levels for

Table 1. Percentage (standard deviation in

brackets) of specified T cells expressing mark-

ers of activation

CD25 HLA-DR CD38 CD71

CLA+ CD8+ atopics 32�2% (4�3)1 21�1% (6�2)1 25�2% (1�9)1 30�0% (4�3)1

CLA+ CD8+ non-atopics 12�2% (4�0) 6�0% (2�1) 7�5% (3�5) 14�3% (6�0)

1Significant (P < 0�05) difference in expression of marker between CLA+ CD8+ T cells from

atopic and non-atopic subjects.


S. L. Seneviratne et al.

CLA+ CD8+ T cells. Therefore, in individuals with AD,

tissue-specific CD8+ T cells do not show the expected loss

of lymph node homing markers with increasing expres-

sion of markers of activation. We proceeded to test the

hypothesis that Th2 cytokines could influence the expres-

sion of markers associated with differentiation.

Influence of Th2 cytokines on expression ofdifferentiation markers by antigen-specific T cells

It is known that IL-4 induces downregulation of CLA by

antigen-specific T cells12 and therefore we were unable to

examine the influence of IL-4 on the expression of differ-

entiation markers by CLA+ CD8+ T cells. Therefore,

fluorescent HLA-peptide tetrameric complexes (tetramers)

were generated based on HLA-A*0201 and refolded

with the EBV BMLF1 A*0201-binding peptide 280–8

GLCTLVAML. Following incubation with cognate anti-

gen, the cells were cultured with or without IL-4, IL-5,

IL-9 and IL-13 (10 ng/ml) and analysed longitudinally.

High affinity IL-4 receptors are expressed on resting T

cells, and increase following activation. In addition, they

are present on numerous other cell types, including hae-

matopoietic stem cells, mast cells, macrophages, endothel-

ial cells, fibroblasts, muscle cells and neuroblasts. We had

not anticipated that IL-5 would induce direct changes,

as CD8+ T cells are not known to express IL-5R, but

IL-13Ra1 is widely expressed, including by heart, liver

and skeletal muscle B and T cells. The levels of CD62L

and CCR7 did not change significantly at days 5, 7,

10 and 14 when incubated in the absence of IL-4, IL-5 or

IL-13. However, there were large significant (P < 0�01)

increases in the expression of CCR7 in the presence of

IL-4 (Fig. 1), which were maximal by day 5 and subse-

quently sustained. We were unable to examine earlier

time-points due to the known T-cell receptor downregu-

lation following ligation. Only minor changes were

observed in the tetramer-negative population, suggesting

that the influence of IL-4 is greatest following activation

with cognate antigen. These findings were also confirmed

using tetramer binding cells specific for a different virus,

namely influenza (matrix peptide 58–66 GILGFVFTL).

There was no influence on the expression of CD62L,

CD28, CD27 or CD45RA by the cytokines. Figure 2

shows the dose–response of IL-4, IL-5 and IL-13 on the

expression of CCR7 by antigen-specific CD8+ T cells.

Levels of � 10 ng/ml of IL-4 induced a significantly

(P < 0�01) increased proportion of CCR7-expressing anti-

gen-specific CD8+ T cells. EBV-specific CD8+ T cells were

Table 2. Percentage (standard deviation in

brackets) of specified T cells expressing mark-

ers of differentiation/homing

CD62L CCR7 CD28 CD27

EBV-specific CD8+ 7�6% (1�8) 15�8% (2�8) 67�4% (19�9) 93�0% (5�4)

CLA+ CD8+ 65�0% (7�6)1,2 72�8% (8�7)1,2 80�0% (5�4) 82�6% (6�2)

CLA) CD8+ 25�0% (6�2) 35�0% (4�2) 83�0% (5�0) 90�8% (4�1)

1Significant (P < 0�01) difference in expression of marker between CLA+ CD8+ T cells and

CLA)CD8+ T cells. 2Significant (P < 0�01) difference in expression of marker between

CLA+ CD8+ T and Epstein Barr virus (EBV)-specific CD8+ T cells.

EBV Tetramer

Tetramer-negative Tetramer-positive

CCR7

Ant

i-CD

8

CD

62L

control

IL-13

IL-5

IL-4

7% 19%

8% 20%

6% 14%

6% 43%

nil cytokine A.013 nil cytokine A.013

il13 100 ng/ml A.012 il13 100 ng/ml A.012

il5 100 ng/ml A.008 il5 100 ng/ml A.008

il4100 ng/ml A.004 il4 100 ng/ml A.004

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

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104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

Figure 1. Examples of expression of CD62L and CCR7 by CD8+ T

cells after antigen-specific T cell culture in the presence of 100 ng/ml

interleukin (IL)-4, IL-5 or IL-13. The top panel shows the sources of

tetramer-negative and tetramer-positive CD8+ T cells for the remain-

ing panels. The figures in the lower right corner of each histogram

represent the proportion of cells in the CD62L) CCR7+ quadrant.



derived from both atopic and non-atopic subjects, and all

showed significant increases in the proportion of CCR7-

expressing CD8+ T cells in the presence of IL-4. Further-

more, consistent with previous reports, such levels of IL-4

were observed to polarize the EBV-specific CD8+ T cells

towards a Tc2 phenotype with significantly increased pro-

duction of IL-4 and less IFN-c (data not shown). Overall,

these data show that, in addition to known effects of IL-4

polarization towards a Tc2 phenotype, IL-4 promotes

expression of CCR7 by viral-specific CD8+ T cells.

CLA+ CD8+ T cell cytokine production in individualswith cutaneous atopic disease

Progression along the intermediate and late differentiation

programme is believed to be associated with acquisition of

rapid effector function.1 Having established that in indi-

viduals with AD there was no significant loss of CCR7

despite increasing disease severity, cytokine production by

CLA+ CD8+ T cells was investigated to determine whether

there were functional associations. The CLA+ CD8+ T cells

from both groups were able to produce both Tc1 and Tc2

cytokines but there were significant differences between

those with severe atopic disease and those with mild atopic

disease. The CLA+ CD8+ T cells derived from both groups

were able to produce IFN-c, TNF-a, IL-4, IL-5 and IL-13

but cells derived from severely affected atopics produced

more IL-4, IL-5 and IL-13 than those derived from mildly

affected individuals (Fig. 3). However, CLA+ CD8+ T cells

from severely affected atopics produced significantly

(P < 0�05) less IFN-c and IL-10 than those derived from

mildly affected atopic subjects.

Discussion

The current study provides the first data to document the

influence of Th2 cytokines on markers linked to CD8+ T

cell differentiation and shows that IL-4 strongly promotes

expression of CCR7. Furthermore, it shows that skin-

homing T cells maintain CCR7 expression despite escalat-

ing severity of atopic dermatitis and activation of the

skin-homing T cells. The cell surface phenotype is associ-

ated with low IL-10 production, a cytokine pattern that

has previously been suggested to contribute to atopic dis-

ease pathogenesis.13–22

The Hanifin and Rajka diagnostic criteria for AD have

been refined and validated in many populations and,

0·01

45

40

35

30

25

20

15

10

5

0

40

30

20

10

00·1

Cytokine concentration (ng/ml)

Tetramer-positive

IL-4

IL-5

IL-13

IL-4

IL-5

IL-13

Tetramer-negative

1 10 100 0·01

Per

cent

age

CC

R7

expr

essi

on (

%)

Per

cent

age

CC

R7

expr

essi

on (

%)

0·1

Cytokine concentration (ng/ml)

1 10 100

Figure 2. Dose–response of the influence of interleukin (IL)-4, IL-5 and IL-13 on the expression of CCR7 by CD62L) tetramer-positive and

tetramer-negative CD8+ T cells following incubation of peripheral blood mononuclear cells with specific peptide.

40

50

60

70

30

20

*

*

10

0

Severe Mild Non-atopic

IL-4

IL-1

3IL

-5IL

-10

IFN-γ

TNF-α IL-4

IL-1

3IL

-5IL

-10

IFN-γ

TNF-α IL-4

IL-1

3IL

-5IL

-10

IFN-γ

TNF-α

Mea

n pe

rcen

tage

of C

LA+

CD

8+ T

cel

lsst

aini

ng fo

r in

divi

dual

cyt

okin

es (

%)

Figure 3. Percentage of ex vivo CD8) T cells that produce cytokines

in response to phorbol myristate acetate/ionomycin. Open columns

represent healthy controls (n ¼ 10); hashed columns represent indi-

viduals with mild atopic disease (n ¼ 12); solid columns represent

individuals with severe atopic disease (n ¼ 15). CLA+ CD8+ T cells

from severely affected atopics produced significantly (*P < 0�05)

less interferon (IFN)-c and interleukin (IL)-10 than those derived

from mildly affected atopics. Non-atopics produced significantly

(P < 0�05) less IL-4, IL-5, IL-13 and more IFN-c and tumour necro-

sis factor (TNF)-a than both mildly and severely affected atopic

subjects.



together with AD disease scoring systems, now allow for

the rapid and reproducible identification and characteri-

zation of affected individuals.7–9 T cells are the dominant

infiltrating population associated with lesional AD skin

and include a significant proportion of CD8+ T cells.23,24

Cutaneous lymphocyte-associated antigen (CLA) is a car-

bohydrate-modified P-selectin glycoprotein ligand-1 and

is believed to be a marker of cells that can interact with

E-selectin.25–27 The latter is expressed on venular endo-

thelial cells of inflamed skin, oral mucosa and the female

genital tract, and provides the initial signals that trigger

the rolling of CLA+ T cells along endothelium. CLA is

expressed by the majority of T cells within cutaneous

inflammatory infiltrates, but only by 5–20% of peripheral

blood T cells. Increased frequencies of both CD4+ and

CD8+ T cells producing type-2 cytokines (including IL-4,

IL-5, IL-13) circulate in the peripheral blood of individu-

als with AD. This is particularly so within the putative

skin-homing (CLA)-positive T cell subset.

In individuals with atopic disease, CLA+ CD8+ T cells

express high levels of activation markers such as CD25,

HLA-DR, CD38 and CD71, which is compatible with the

transition from naive to antigen-experienced status and

their role in the recognition of skin-associated antigens

and contribution to cutaneous inflammation. However,

despite increased expression of activation markers in

those with severe disease, there was no concomitant

loss of CCR7. In the absence of IL-4, antigen stimulation

did not induce an upregulation of CD62L and CCR7 by

CLA+ or CLA) T cells. Only minor changes were

observed in the tetramer-negative population, suggesting

that the influence of IL-4 is greatest following activation

with cognate antigen. Although the expression of both

CD62L and CCR7 were elevated on CLA+ CD8+ T cells

compared with EBV-specific CD8+ T cells, only CCR7

expression was promoted by the addition of IL-4, suggest-

ing that other factors might be important in the regula-

tion of CD62L.5 In addition, IL-4 is known to induce

CLA downregulation by antigen-specific T cells12 and thus

overall may promote homing away from the skin and

towards lymphoid tissue, potentially contributing to per-

sistent T cell activation and expansion.

We have previously shown that HIV-specific T cells

show an altered maturation pattern that associates with

functional sequelae.3,6 Interestingly, chronic HIV infection

has also been linked to a progressive Th2 shift, and thus

the current data raise the possibility that it is the cytokine

profile in infected individuals that influences T cell mat-

uration and function.

The functional activity of CLA+ CD8+ T cells was

investigated directly ex vivo and the cells were found to

be a source of both type 1 and 2 cytokines, including

IFN-c, TNF-a, IL-4, IL-5, IL-13 and IL-10. However, in

individuals with severe atopic disease, the CLA+ CD8+ T

cells produced significantly less IL-10 than those derived

from mildly affected atopics. These data may provide a

mechanism for a number of other existing observations

and are consistent with previous data. For example, IL-10

levels in bronchoalveolar lavage fluid from severely affec-

ted atopic subjects were found to be significantly lower

than levels in mildly affected individuals.13 PBMCs from

asthmatic subjects produced less IL-10 mRNA in response

to lipopolysaccharide stimulation than those isolated

from non-atopic subjects13 and low IL-10 producing

promoter polymorphisms have been associated with

severe asthma.14 Alveolar macrophages from asthmatics

produced less IL-10 than those from non-atopic con-

trols15 and, in Gabon, there was an inverse association

between levels of IL-10 produced by PBMCs incubated

with schistosomal antigens and skin test reactivity to

housedust mite.16 Der p 1-specific CD8+ T cells from

severely affected atopic individuals produce both Tc1 and

Tc2 cytokines, but less IL-10 than those from mildly

affected atopics,21 whereas CLA+ CD8+ T cells from non-

atopic individuals are known to have the capacity to pro-

duce both Tc1 and Tc2 cytokines.28 One consequence of

diminished IL-10 production may be a relative reduction

in the regulatory ability to control immune responses to

ubiquitous environmental allergens.

In summary, IL-4 was found to promote CCR7 expres-

sion by antigen-specific T cells, suggesting that the cyto-

kine microenvironment may influence the differentiation

of T cells with associated consequences for effector func-

tion and immune responses to persistent antigens. Thus

IL-4 strongly influences CCR7, a marker that is linked to

existing models of human CD8+ T cell differentiation,

which requires us to re-evaluate our understanding of

mechanisms of pathway regulation.

Acknowledgements

We are very grateful to the Medical Research Council,

British Skin Foundation and Barrie Trust for their sup-

port. We are also most grateful to all the patients

involved in the studies.

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Interleukin-4 promotes human CD8+ T cell expression of CCR7