Kaposi’s sarcoma (KS) lesions are characterized by a prominent leukocyte infiltrate composed of mononuclear phagocytes and T cells. KS-associated CD4+ and CD8+ cells showed predominantly a type II cytokine profile. The CC chemokine viral macrophage inflammatory protein-II (vMIP-II) encoded by the KS-associated herpes virus 8 was a selective chemoattractant for T helper 2 (Th2 cells) and for monocytes, whereas it was inactive on other leukocytes, including Th1 cells, dendritic cells, and natural killer (NK) cells. vMIP-II was an agonist for CCR8, a chemokine receptor selectively expressed on CD4+ and CD8+ cells with a type II cytokine profile. Hence, vMIP-II has agonist activity for a chemokine receptor (CCR8), which is preferentially expressed on polarized Th2 cells. The capacity of vMIP-II to attract type II T cells selectively is likely to be a component of the virus strategy to subvert the host immune response.

HUMAN HERPES VIRUS 8 (HHV8), also known as Kaposi’s sarcoma virus, is associated with Kaposi’s sarcoma (KS), body cavity–based lymphoma, and Castelman’s disease.1-4The HHV8 genome includes three open reading frames coding for proteins with considerable (∼40%) identity to human CC chemokines and one coding for a chemokine receptor, ORF74.5 KS is an opportunistic tumor characterized by prominent angiogenesis and leukocyte infiltration,6 7 including T cells and monocytes.

The current concept of the multistep process of leukocyte recruitment into tissues envisions chemotactic agonists as one of the key effector molecules.8-10 Chemokines are a superfamily of chemotactic proteins that can be divided in four groups on the basis of a cysteine structural motif. Most of the chemokines fall in two subfamilies: the α (or CXC) chemokines, mainly active on neutrophils and lymphocytes and the β (or CC) chemokines active on multiple subsets of mononuclear cells. Lymphotactin (γ or C chemokines) and fractalkine (δ or CX3C chemokines) may define two additional groups of this superfamily.10,11 Recent results indicate that polarized T helper 1 (Th1) and Th2 populations differentially express chemokine receptors and respond to chemotactic agonists.12 13 

Here we show that CD4 and CD8 cells infiltrating KS have predominantly a type II cytokine profile and that the KS chemokine viral macrophage inflammatory protein-II (vMIP-II) is a selective attractant for type II T cells and interacts as an agonist with a receptor (CCR8) selectively expressed on this polarized subset.

Preparation of effector cells.

Human monocytes and neutrophils were obtained from buffy coats of healthy blood donors by density gradients on Ficoll (Biochrom) Percoll (Pharmacia, Uppsala, Sweden), as previously described.14Monocyte-derived dendritic cells (mono-DC) and CD34-derived dendritic cells (CD34-DC) were obtained as previously described.15Stable transfectants of CCR8 (TER1) were prepared by electroporation of Jurkat cells with pcDNA3-HA/TER1 and subsequent selection in G418.16 Th1 and Th2 cultures were obtained as previously described.13 

Migration assay.

Cell migration was evaluated using a chemotaxis microchamber technique as previously described.14 Monocytes, neutrophils, DCs, and Jurkat cells were tested using a 5-μm pore-size polycarbonate filter. For Jurkat cells filters were previously coated with murine collagen type IV. At the end of the incubation, filters were removed, stained with Diff-Quik (Baxter s.p.a., Rome, Italy), and five high-power oil-immersion fields were counted. T-cell cultures were tested with the leading front methods using nitrocellulose filters, and migration was evaluated as distance (μm) migrated by the two fastest cells.14 Human recombinant MCP-3 (MCP-3) was a kind gift of Dr A. Minty (Sanofi Elf Bio Recherches, Labège, France). vMIP-II was chemically synthesized as previously described17 and was a kind gift of Dr Ian Clark-Lewis (University of British Columbia, Vancouver, Canada).

Generation of Th1, Th2, Tc1, and Tc2 lines from cord blood leukocytes.

Generation of T helper cell lines was performed by stimulating cord blood mononuclear cells with 2 μg/mL PHA (Wellcome, Beckenham, UK) in polarizing conditions as described.18 Differentiation of Th1 cells was obtained by addition of 2 ng/mL of IL-12 and 1,000 U/mL of interferon-α (IFN-γ; Hoffmann-La Roche Inc, Nutley, NJ) together with 200 ng/mL of neutralizing anti–interleukin-4 (IL-4) antibodies (Pharmingen, San Diego, CA), whereas differentiation of Th2 cells was obtained by addition of 200 U/mL of IL-4 (Pharmingen) together with 2 μg/mL of neutralizing anti–IL-12 antibodies 17F7 and 20C2 (a gift of M. Gately, Hoffmann-La Roche Inc). Cells were evaluated for their cytokine production profiles by intracellular staining as previously described.13 Polarized CD4+ Th1 and Th2 cells or CD8+ Tc1 and Tc2 cells were separated by immunomagnetic negative selection by incubating the cells with anti-CD4 or anti-CD8 monoclonal antibodies (Pharmingen).

In vitro generation of T-lymphocyte clones from skin biopsies.

Skin biopsy specimens were incubated in IL-2 (20 U/mL)-containing medium for 7 to 10 days to expand in vivo–activated IL-2 receptor–expressing T cells as previously described.19T-cell suspensions obtained from each skin specimen were then cloned under limiting dilution conditions (0.5 cell/well) in the presence of phytohemagglutinin (PHA; 1% vol/vol), IL-2 (20 U/mL), and irradiated peripheral blood mononuclear cells (PBMC) as feeder cells. The cell surface phenotype of clonal T-cell blasts was assessed with flow cytometry by using fluorescein isothiocyanate (FITC)-conjugated anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies (MoAbs). T blasts (1 × 106/mL) from each clone were then stimulated with PMA plus ionomycin for 24 hours, and the production of IL-4 and IFN-γ was measured in cell-free culture supernatants by using appropriate enzyme-linked immunosorbent assay (ELISA) assays.

T-cell clones were generated under the same experimental conditions from skin biopsy specimens of one normal volunteer, two patients with alopecia areata, three patients with atopic dermatitis, and three patients with KS. Higher proportions of CD8+ than CD4+ T-cell clones were generated from the skin of KS patients (369 and 53 clones, respectively), whereas CD4+T-cell clones were prevalent in controls (53 and 14 clones for CD4+ and CD8+, respectively). As reported in Fig 1, the majority of CD4+T-cell clones generated from healthy skin or the skin of alopecia areata patients showed a Th1-skewed profile, whereas those generated from the skin of atopic dermatitis patients showed a more heterogeneous cytokine profile. However, virtually no CD8+ T-cell clones with Tc2 profile, except in one patient with atopic dermatitis, were found. By contrast, high proportions of both CD4+ and CD8+ T-cell clones generated from the skin of KS patients showed a Th2-skewed phenotype (Fig 1).

Fig. 1.

Th2- and Tc2-skewed cytokine profile of CD4+ and CD8+ T-cell clones generated from the neoplastic skin of patients with KS. Clones were obtained from skin biopsy specimens, taken for diagnostic purpose from three HIV-infected patients with KS. As controls, cytokine production by T-cell clones generated under the same experimental conditions from skin biopsy specimens of one healthy volunteer, two patients with alopecia areata (AA), and three patients with atopic dermatitis (AD) was evaluated. Each symbol represents the amounts of IL-4 and IFN-γ produced by a single CD4+ (open squares) or CD8+ (closed circles) T-cell clone. Lines represent cutoff values (IFN-γ, 0.8 ng/mL; IL-4, 0.2 ng/mL), calculated as 5 SD over values found in cultures containing feeder cells alone.

Fig. 1.

Th2- and Tc2-skewed cytokine profile of CD4+ and CD8+ T-cell clones generated from the neoplastic skin of patients with KS. Clones were obtained from skin biopsy specimens, taken for diagnostic purpose from three HIV-infected patients with KS. As controls, cytokine production by T-cell clones generated under the same experimental conditions from skin biopsy specimens of one healthy volunteer, two patients with alopecia areata (AA), and three patients with atopic dermatitis (AD) was evaluated. Each symbol represents the amounts of IL-4 and IFN-γ produced by a single CD4+ (open squares) or CD8+ (closed circles) T-cell clone. Lines represent cutoff values (IFN-γ, 0.8 ng/mL; IL-4, 0.2 ng/mL), calculated as 5 SD over values found in cultures containing feeder cells alone.

Close modal

The capacity of vMIP-II to elicit directional migration of various leukocyte populations was then studied (Fig2). vMIP-II induced migration of human monocytes with an ED50 of 32 ± 4 ng/mL (∼3 nmol/L) and maximal activity at 100 ng/mL. Under the same conditions MCP-3, used as reference chemoattractant, had an ED50 of 15 ± 3, and the maximal response was 1.9 ± 0.3-fold (n = 3) higher than that of vMIP-II. Checkerboard analysis showed that vMIP-II elicited actual chemotactic migration in monocytes. This contrasts with previous data17 in which vMIP-II was a weak monocyte attractant. This difference in vMIP-II efficacy is presumably due to the cell preparation and assay used. Other leukocyte populations, neutrophils, monocyte-derived or CD34-derived dendritic cells (Fig 2), NK cells, unseparated lymphocytes, and naive cord blood T cells (data not shown) showed no substantial response to vMIP-II.

Fig. 2.

Agonist activity of vMIP-II. Monocytes (mono), neutrophils (PMN), monocyte- and CD34-derived dendritic cells (mono-DC and CD34-DC, respectively) and Jurkat (J) cells (A) or Th1 and Th2 cultures (B) were tested for their ability to migrate in response to different concentrations of vMIP-II. (C) Northern blot analysis of CCR8 in Th1, Th2, Tc1 (Th1 CD8+), and Tc2 (Th2 CD8+) cultures.

Fig. 2.

Agonist activity of vMIP-II. Monocytes (mono), neutrophils (PMN), monocyte- and CD34-derived dendritic cells (mono-DC and CD34-DC, respectively) and Jurkat (J) cells (A) or Th1 and Th2 cultures (B) were tested for their ability to migrate in response to different concentrations of vMIP-II. (C) Northern blot analysis of CCR8 in Th1, Th2, Tc1 (Th1 CD8+), and Tc2 (Th2 CD8+) cultures.

Close modal

vMIP-II showed weak chemotactic activity for IL-2–activated, but not resting, T cells (data not shown). These indications prompted a more careful analysis of the capacity of vMIP-II to attract T-lymphocyte subpopulations. As shown in Fig 2A, vMIP-II showed preferential attraction of Th2 versus Th1 cells, both in bulk cultures and in clonal populations. It has recently been shown that chemokine receptors are differentially expressed in Th1 versus Th2 cells.12,13,16,20-22 CCR4 and CCR3 are expressed predominantly in Th2 cells, whereas Th1 cells express CCR5 and CXCR3.13 A similar differential expression of chemokine receptors has been observed recently in CD8+ T cells with different cytokine profiles23 (and unpublished results).

vMIP-II has been shown to interact with multiple chemokine receptors as an antagonist or as an agonist.17,24 It binds CCR3,24 a receptor expressed preferentially on polarized Th2 cells,12,13,21,22 and shows activity on eosinophils.24 As shown in Fig 2, vMIP-II interacts also with CCR8, the I309 receptor,16,25 26 and elicits migration of CCR8-transfected cells (J/CCR8; Fig 2A). Chemotactic index of J/CCR8 for vMIP-II was 5.3 ± 1.5 (n = 5) compared with 2.8 (n = 2) for I-309 (at the peak concentrations of 100 ng/mL and 30 ng/mL, respectively). Chemotactic index to SDF-1 (1 μg/mL), used as reference chemoattractant, through its endogenous receptor (CXCR4), was 4.1 ± 0.3 (n = 4). As shown in Fig 2C, CCR8 was predominantly expressed in CD4+ Th2 cells and in CD8 T cells with a Th2 phenotype (Tc2). Hence, the ability of vMIP-II to act on chemokine receptors, such as CCR8 that is expressed on Th2 cells, underlies the ability of vMIP-II to selectively attract these cells.

Both CD4+ and CD8+ T cells comprise of different subsets based on the cytokines they produce.27,28Th1 cells predominantly mediate phagocyte-dependent protective immunity as well as inflammatory autoimmune disorders, whereas Th2 cells are responsible for phagocyte-independent protection and are prominent in the pathogenesis of allergic diseases.28 The results presented here show that KS lesions are infiltrated by CD8+and CD4+ T cells with a predominant type II cytokine profile. The viral chemokine encoded by KS-associated HHV8 was a selective attractant for type II T cells and showed agonist activity for CCR8, a receptor selectively expressed on polarized type II T cells. There is evidence that activation of a Th2 response can lead to delayed virus clearance.29 Hence, the capacity of the HHV8-encoded chemokine vMIP-II to selectively attract monocytes and Th2 cells is likely a component of the virus strategy to subvert immunity.

Supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) by 40% Fund (A.M.) and by special project AIDS from Istituto Superiore Sanità, Grants No. 40A.0.66 (A.M.) and 30A.0.72 (A.V.).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

1
Chang
Y
Cesarman
E
Pessin
MS
Lee
F
Culpepper
J
Knowles
DM
Moore
PS
Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma.
Science
266
1994
1865
2
Boshoff
C
Whitby
D
Hatziioannou
T
Fisher
C
Walt
J
Hatzakis
A
Weiss
R
Schulz
T
Kaposi’s-sarcoma-associated herpesvirus in HIV-negative Kaposi’s sarcoma.
Lancet
345
1995
1043
3
Dupin
N
Grandadam
M
Calvez
V
Gorin
I
Aubin
JT
Havard
S
Lamy
F
Leibowitch
M
Huraux
JM
Escande
JP
Agut
H
Herpesvirus-like DNA sequences in patients with mediterranean Kaposi’s sarcoma.
Lancet
345
1995
761
4
Boshoff
C
Schulz
TF
Kennedy
MM
Graham
AK
Fisher
C
Thomas
A
McGee
JO
Weiss
RA
O’Leary
JJ
Kaposi’s sarcoma-associated herpesvirus infects endothelial and spindle cells.
Nat Med
1
1995
1274
5
Arvanitakis
L
GerasRaaka
E
Varma
A
Gershengorn
MC
Cesarman
E
Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation.
Nature
385
1997
347
6
Armes
J
A review of Kaposi’s sarcoma.
Adv Cancer Res
53
1989
73
7
Sciacca
FL
Stürzl
M
Bussolino
F
Sironi
M
Brandstetter
H
Zietz
C
Zhou
D
Matteucci
C
Peri
G
Sozzani
S
Benelli
R
Arese
M
Albini
A
Colotta
F
Mantovani
A
Expression of adhesion molecules, platelet-activating factor, and chemokines by Kaposi’s sarcoma cells.
J Immunol
153
1994
4816
8
Springer
TA
Traffic signal for lymphocyte recirculation and leukocyte emigration: The multistep paradigm.
Cell
76
1994
301
9
Butcher
EC
Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity.
Cell
67
1991
1033
10
Rollins
BJ
Chemokines.
Blood
90
1997
909
11
Baggiolini
M
Dewald
B
Moser
B
Human chemokines: An update.
Annu Rev Immunol
15
1997
675
12
Sallusto
F
Mackay
CR
Lanzavecchia
A
Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells.
Science
277
1997
2005
13
Bonecchi
R
Bianchi
G
Bordignon
PP
D’Ambrosio
D
Lang
R
Borsatti
A
Sozzani
S
Allavena
P
Gray
PA
Mantovani
A
Sinigaglia
F
Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s.
J Exp Med
187
1998
129
14
Sozzani
S
Luini
W
Molino
M
Jı́lek
P
Bottazzi
B
Cerletti
C
Matsushima
K
Mantovani
A
The signal transduction pathway involved in the migration induced by a monocyte chemotactic cytokine.
J Immunol
147
1991
2215
15
Power
CA
Church
DJ
Meyer
A
Alouani
S
Proudfoot
AEI
Clark-Lewis
I
Sozzani
S
Mantovani
A
Wells
TNC
Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3 alpha from lung dendritic cells.
J Exp Med
186
1997
825
16
Bernardini
G
Hedrick
J
Sozzani
S
Luini
W
Spinetti
G
Weiss
M
Menon
S
Zlotnik
A
Mantovani
A
Santoni
A
Napolitano
M
Identification of the CC chemokines TARC and macrophage inflammatory protein-1 beta as novel functional ligands for the CCR8 receptor.
Eur J Immunol
28
1998
582
17
Kledal
TN
Rosenkilde
MM
Coulin
F
Simmons
G
Johnsen
AH
Alouani
S
Power
CA
Luttichau
HR
Gerstoft
J
Clapham
PR
Clarklewis
I
Wells
TNC
Schwartz
TW
A broad-spectrum chemokine antagonist encoded by Kaposi’s sarcoma-associated herpesvirus.
Science
277
1997
1656
18
Rogge
L
Barberis-Maino
L
Biffi
M
Pazzinini
N
Presky
DH
Gubler
U
Sinigaglia
F
Selective expression of an IL-12 receptor component by human Th1 cells.
J Exp Med
185
1997
825
19
Maggi
E
Manetti
R
Annunziato
F
Cosmi
L
Giudizi
MG
Biagiotti
R
Galli
G
Zuccati
G
Romagnani
S
Functional characterization and modulation of cytokine production by CD8+ T cells from human immunodeficiency virus-infected individuals.
Blood
89
1997
3672
20
Qin
SX
Rottman
JB
Myers
P
Kassam
N
Weinblatt
M
Loetscher
M
Koch
AE
Moser
B
Mackay
CR
The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions.
J Clin Invest
101
1998
746
21
Gerber
BO
Zanni
MP
Uguccioni
M
Loetscher
M
Mackay
CR
Pichler
WJ
Yawalkar
N
Baggiolini
M
Moser
B
Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils.
Curr Biol
7
1997
836
22
Loetscher
P
Uguccioni
M
Bordoli
L
Baggiolini
M
Moser
B
Chizzolini
C
Dayer
J-M
CCR5 is characteristic of Th1 lymphocytes.
Nature
391
1998
344
23
Sad
S
Marcotte
R
Mossman
TR
Cytokine induced differentiation of precursors mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokine.
Immunity
2
1995
271
24
Boshoff
C
Endo
Y
Collins
PD
Takeuchi
Y
Reeves
JD
Schweickart
VL
Siani
MA
Sasaki
T
Williams
TJ
Gray
PW
Moore
PS
Chang
Y
Weiss
RA
Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines.
Science
278
1997
290
25
Stuber Roos
R
Loetscher
M
Legler
DF
Clark-Lewis
I
Baggiolini
M
Identification of CCR8, the receptor for the human CC chemokine I-309.
J Biol Chem
272
1997
17251
26
Tiffany
HL
Lautens
LL
Gao
JL
Pease
J
Locati
M
Combadiere
C
Modi
W
Bonner
TI
Murphy
PM
Identification of CCR8: A human monocyte and thymus receptor for the CC chemokine I-309.
J Exp Med
186
1997
165
27
Mosmann
TR
Coffman
RL
Th1 and Th2 cells: Different patterns of lymphokine secretion leads to different functional properties.
Annu Rev Immunol
7
1989
145
28
Romagnani
S
The Th1/Th2 paradigm.
Immunol Today
18
1997
263
29
Actor
JK
Shirai
M
Kullberg
MC
Buller
RM
Sher
A
Berzofsky
JA
Helminth infection results in decreased virus-specific CD8+ cytotoxic T-cell and Th1 cytokine responses as well as delayed virus clearance.
Proc Natl Acad Sci USA
90
1993
948

Author notes

Address reprint requests to A. Mantovani, MD, Istituto di Ricerche Farmacologiche ‘Mario Negri,’ Via Eritrea 62, 20157 Milan, Italy; e-mail: [email protected].

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