B7+CTLA4+ T cells engage in T–T cell interactions that mediate apoptosis: a model for lentivirus-induced T cell depletion

B7þCTLA4þ T cells engage in T–T cell interactions that mediateapoptosis: a model for lentivirus-induced T cell depletion

Thomas W. Vahlenkampa,*, Marta E. Bulla, Janet L. Dowa, Ellen W. Collissonb,Barbara J. Winslowc, Anagha P. Phadkeb, Wayne A.F. Tompkinsa,

Mary B. Tompkinsa

aImmunology Program, North Carolina State University, Raleigh, NC 27606, USAbDepartment of Veterinary Pathobiology, College of Veterinary Medicine,

Texas A&M University, College Station, TX 77843-4467, USAcSchering-Plough Animal Health Corporation, San Diego, CA 92121, USA

Received 21 September 2003; received in revised form 9 December 2003; accepted 9 December 2003

Abstract

Apoptosis in lymph node (LN) T cells of feline immunodeficiency virus (FIV)-infected cats is associated with cells co-

expressing B7.1 and B7.2 costimulatory molecules, and their ligand CTLA4. To study the possibility of B7.1/B7.2–CTLA4

mediated T–T interactions and the predicted induction of T cell apoptosis in vitro, costimulatory molecules were up-regulated on

CD4þ and CD8þ T cells by mitogen stimulation. B7.1 expression on in vitro stimulated CD4þ and CD8þ cells increased within

24 h; B7.2 and CTLA4 expression increased after 48–72 h. Apoptosis, as analyzed by terminal deoxynucleotidyl transferase

(transferase nick end labeling, TUNEL)-based staining followed by three color flow cytometric analysis, correlated to the cells

expressing B7 and/or CTLA4. Blocking experiments revealed that CD4þ and CD8þ T cell apoptosis could be significantly

inhibited with anti-B7 antibodies. As FIV infection results in immune activation with a T cell phenotype similar to that of the in

vitro activated T cells, the data support the hypothesis that the chronic expansion of B7þCTLA4þ LN T cells in infected cats

allows for T–T cell interactions resulting in T cell depletion and eventually the development of AIDS.

# 2004 Elsevier B.V. All rights reserved.

Keywords: FIV; Apoptosis; B7 costimulatory molecules; CTLA4

1. Introduction

The outcome of T cell antigen receptor occupancy

is strongly influenced by costimulatory signals pro-

vided by B7.1 (CD80) and B7.2 (CD86) on the antigen

presenting cell (APC) (Lenschow et al., 1996; Gimmi

et al., 1993). The B7 molecules interact with CD28

and CTLA4 (CD152) on the T cell surface. Engage-

ment of CD28 that is constitutively expressed on T

cells leads to T cell activation and proliferation by the

induction of IL-2 transcription (Fraser et al., 1991)

stabilization of cytokine mRNA (Lindsten et al., 1993)

and the expression of bcl-xL (Boise et al., 1995).

Following activation, the T cells up-regulate CTLA4,

which upon engagement of B7 inhibits T cell

responses by antigen specific clonal deletion of acti-

vated T cells via the suppression of IL-2 (Krummel

Veterinary Immunology and Immunopathology 98 (2004) 203–214

* Corresponding author. Tel.: þ1-919-513-6339;

fax: þ1-919-513-6464.

E-mail address: [email protected]

(T.W. Vahlenkamp).

0165-2427/$ – see front matter # 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2003.12.006

and Allison, 1996; Walunas et al., 1994; Gribben et al.,

1995). As binding of B7.1 and B7.2 for CTLA4 is 20–

100� greater than for CD28 (Greenfield et al., 1998)

negative signaling dominates on activated CTLA4

expressing T cells, thereby terminating the immune

response.

Although B7 molecules are generally expressed on

professional APCs, both B7.1 and B7.2 molecules are

also expressed on activated T cells (Azuma et al.,

1993; Sansom and Hall, 1993; Wyss-Coray et al.,

1993). Freshly isolated human and murine T cells

express low levels of B7.2, and both the B7.1 and B7.2

molecules are up-regulated following activation with

anti-CD3 (Azuma et al., 1993; Hathcock et al., 1994).

Activated human and mouse T cells also up-regulate

MHC class II molecules, suggesting to some that they

are capable of antigen presentation to MHC class II-

restricted T cells (Barnaba et al., 1994). In contrast to

antigen presentation by professional APCs, several

studies have indicated that T cells expressing B7

and MHC Class II molecules prime responding T

cells for anergy and apoptosis upon subsequent anti-

gen presentation by professional APC (Lombardi et al.,

1994; Greenfield et al., 1997).

Apoptosis is mediated by interactions of cell sur-

face receptors with their ligands (Fas/FasL, TNFaR/

TNFa, CTLA4/B7) that transduce an intracellular

signal resulting in cell death. In contrast to the Fas

and TNF receptors, which induce caspase-mediated

apoptosis in many different tissues, the CTLA4/B7

apoptotic pathway appears to be T cell specific. We

recently reported that a significant fraction of lymph

node (LN) T cells from FIV-infected cats are char-

acterized by a marked up-regulation of B7.1, B7.2 and

CTLA4 on both CD4þ and CD8þ cells (Tompkins

et al., 2002). Three-color flow cytometry revealed that

a high number of T cells from FIV positive cats co-

express B7.1, B7.2 and CTLA4. This highly unusual T

cell phenotype should enable frequent T–T interac-

tions mediated by B7.1/B7.2–CTLA4 ligation that

could potentially transduce a signal for anergy and

apoptosis. We therefore proposed that the progressive

loss of immune function in FIV infection might be the

result of chronic B7–CTLA4 interactions between

activated T cells. Anergy and apoptosis resulting from

negative signaling via the B7/CTLA4 pathway would

be consistent with three main immune correlates of

immunosuppression observed in FIV, simian immu-

nodeficiency virus (SIV), and human immunodefi-

ciency virus (HIV) infections in animals and

humans: (i) disease progression correlates with the

immune activation and not, e.g. with viral load or

number of infected target cells (Anderson et al., 1998;

Leng et al., 2001; Muro-Cacho et al., 1995), (ii)

inability of T cells to produce IL-2 and proliferate

in vitro in response to MHC class II-restricted recall

antigens during the asymptomatic stage of the infec-

tion (Lawrence et al., 1992; Midema, 1992), and (iii) a

high degree of T cell apoptosis in LN of infected

individuals (Muro-Cacho et al., 1995; Finkel et al.,

1995; Gougeon et al., 1993; Guiot et al., 1997;

Meyaard et al., 1992; Sarli et al., 1998).

To investigate B7/CTLA4-mediated T–T cell inter-

actions, we developed an in vitro model using mitogen

stimulated T cells from uninfected spf cats. Costimu-

latory molecules B7.1, B7.2 and CTLA4 were up-

regulated by stimulation of CD4þ and CD8þ cells with

Concanavalin A (ConA) or the combination of phor-

bol myristate acetate (PMA) and ionomycin. T cell

interactions in this autologous system were analyzed

by measuring T cell apoptosis in the presence or

absence of anti-B7 antibodies. The results further

support the hypothesis that the unique T cell pheno-

type capable of T–T interactions resulting in anergy

and apoptosis may be an important component in

negatively regulating immune responses. This T–T

immunoregulatory process is co-opted and amplified

in FIV-infected cats leading to chronic T cell deple-

tion.

2. Materials and methods

2.1. Cats

Spf cats were obtained from Liberty Labs (Liberty

Corners, NJ, USA) or Cedar River Laboratory (IA)

and housed at the Laboratory Animal Resource Facil-

ity at the College of Veterinary Medicine, North

Carolina State University. At the time samples were

taken cats ranged in age between 3 and 5 years.

2.2. Blood collection

Whole blood (20–30 ml) was collected into EDTA

vacutainer tubes. PBMC were isolated by Percoll

204 T.W. Vahlenkamp et al. / Veterinary Immunology and Immunopathology 98 (2004) 203–214

(Sigma, St. Louis, MO) density centrifugation fol-

lowing the manufacturer’s instructions. Briefly,

whole blood was spun for 5 min at 400 � g, the

plasma removed and the cells resuspended in Hanks

buffer containing 3% EDTA. Cells were layed

on a 43–60% Percoll grandient and spun for 5 min

at 400 � g followed by 20 min centrifugation at

800 � g. Lymphocytes were harvested from the gra-

dient’s interface, washed twice with phosphate buf-

fered saline containing 3% EDTA and resuspended in

culture medium.

2.3. Purification of T cells

To investigate single lymphocyte subsets, CD4þ

and CD8þ cells were enriched by biomagnetic separa-

tion using goat anti-mouse IgG coated beads (Dyna-

beads1 M-450, Dynal, Oslo, Norway). Beads were

coated with antibodies over night at 4 8C on a rotary

shaker. The number of beads was calculated as four

times the estimated number of target cells. B cells

were depleted with beads coated with a cross-reacting

antibody to canine CD21 (P. Moore, University of

California, Davis). Monocytes were depleted with

beads coated with a cross-reacting antibody to human

CD14 (TUK4) obtained from Dako (Carpinteria, CA).

CD4þ or CD8þ cells were depleted with beads coated

with monoclonal antibodies 30A and 3.357 (Tompkins

et al., 1990), respectively. PBMC were incubated with

the antibody coated beads for 3 h at 4 8C on a rotary

shaker. The magnetic separation was performed for

3 min at room temperature. For the enrichment of T

cells, PBMC were depleted for B cells and monocytes.

For the enrichment of CD4þ or CD8þ cells, PBMC

were first depleted for B cells and monocytes followed

by the depletion of the CD4þ or CD8þ cell population.

The purity of the enriched CD4þ or CD8þ cell popu-

lation was verified by two-color flow cytometric ana-

lysis and determined to be >95%.

2.4. Cell culture and stimulation

PBMC or purified T cells (2 � 106 cells/ml) were

cultured in 24-well plates in growth medium (RPMI

containing 10% fetal bovine serum, 1% penicillin-

streptomycin, 1% sodium bicarbonate, 1% sodium

pyrovate, 1% L-glutamine, and 1 mM HEPES buffer)

in the presence or absence of 2 mg/ml ConA or 50 ng/

ml Ionomycin and 10 ng/ml PMA. The expression of

costimulatory molecules was analyzed on unstimu-

lated T cells and after 24, 48 and 72 h of stimulation.

Apoptosis was analyzed on unstimulated T cells and

after 24 and 48 h of culture. Blocking experiments

using anti-B7.1, anti-B7.2 and anti-CTLA4 antibodies

were performed by adding the antibodies after 24 h of

T cell stimulation and the cells were analyzed after

additional 24 h of culture.

2.5. Flow cytometry staining

PBMC were stained using a polyclonal rabbit anti-

feline B7.1, B7.2 or CTLA4 antibodies (Tompkins

et al., 2002) followed by incubation with a phycoer-

ythrin (PE)-labeled donkey anti-rabbit IgG F(ab0)2

(Jackson ImmunoResearch, West Grove, PA). Cells

were stained with the fluorescein isothiocyanate

(FITC)-conjugated anti-feline CD4 (30A) or allophy-

cocyanin (APC)-conjugated anti-feline CD8a (3.357)

antibody (Tompkins et al., 1990). For three-color

analysis, biotinylated CD4þ and CD8þ cells were

stained using streptavidin-conjugated APC (Becton

Dickinson, Los Angeles, CA). The expression of cell

surface molecules and apoptosis were measured on a

FACSCalibur1 flow cytometer (Becton Dickinson,

Los Angeles, CA). At least 20,000 cells were acquired

using Becton Dickinson CellQuest1 software.

2.6. Measurement of apoptosis

PBMC were stained using polyclonal rabbit anti-

feline B7.1, B7.2 or CTLA4 antibodies and subse-

quent incubation with PE-labeled donkey anti-rabbit

IgG. CD4þ and CD8þ cells were stained using bio-

tinylated antibodies and streptavidin-APC. Stained

cells were fixed in 2% paraformaldehyde for 15 min

and permeabilzed using 0.1% Na-citrate, 0.1% Tween

20 for 2 min on ice. The terminal deoxynucleotidyl

transferase nick end labeling (TUNEL)-based assay

(Roche Laboratories) was performed and T cells

analyzed by three-color cytometry as described pre-

viously (Tompkins et al., 2002).

2.7. Statistical analysis

The student’s t-test was used to compare the number

of apoptotic T cells in the antibody treated cultures.

T.W. Vahlenkamp et al. / Veterinary Immunology and Immunopathology 98 (2004) 203–214 205

3. Results

3.1. Up-regulation of B7 costimulatory molecules

and CTLA4 on Ionomycin/PMA or ConA-stimulated

T cells

As reported previously (Tompkins et al., 2002),

analysis of freshly isolated PBMC revealed that

B7.1 was constitutively expressed on unstimulated

CD4þ (Fig. 1A), but not on CD8þ cells (Fig. 1B).

Upon stimulation with I/PMA, the number of CD4þ

cells expressing costimulatory molecules increased

over 48 h of culture (Fig. 1A). In contrast to the

B7.1 expressing cells, the number of cells expressing

B7.2 and CTLA4 continued to increase over the 72 h

culture period but did not reach the number of B7.1

expressing cells. On CD8þ cells all the costimulatory

molecules increased over 72 h of culture (Fig. 1B).

The number of CD8þ cells expressing B7.1 was high-

est, followed by cells expressing B7.2 and CTLA4.

After ConA stimulation, the number of CD4þ cells

expressing the costimulatory molecules increased

over 48 h of culture and remained unchanged for up

to 72 h of culture (Fig. 1C). Similar numbers of cells

stained positive for B7.1 and B7.2. On CD8þ cells, the

number of B7.2 expressing cells reached twice that of

B7.1 expressing cells at 48 h of culture (Fig. 1D).

After 72 h, the number of B7.2 expressing CD8þ cells

had decreased, but still exceeded the number of B7.1

expressing cells, which remained unchanged between

the 48 and 72 h time point. Data on unstimulated cells

are for 0 h in culture and did not significantly change

at 24, 48 and 72 h in culture (data not shown).

To determine if other cells cooperated in the induc-

tion of costimulatory molecules on mitogen stimulated

CD4þ or CD8þ cells, the two T cell subsets were

enriched by cell depletion of B cells, monocytes

and CD4þ or CD8þ cells with antibody-coated mag-

netic beads (purity >95% CD4þ or CD8þ cells) and

stimulated with I/PMA. The expression of all three

0

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Unstimulated 24h 48h 72h

Per

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otal

CD

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ells

(A) (C)0

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30

40

50

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70

24h 48h 72h

0

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70

24h 48h 72h

B7.1B7.2CTLA4

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70


Per

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otal

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ells

(B) (D)

Unstimulated

Unstimulated

Fig. 1. Increased expression of B7.1, B7.2 and CTLA4 costimulatory molecules on CD4þ and CD8þ T cells upon in vitro stimulation of feline

PBMC. PBMC (2 � 106) were stimulated with ionomycin (50 ng/ml) and PMA (10 ng/ml). Two-color flow cytometric analysis was performed

after 24, 48 and 72 h of culture to determine the expression of B7.1, B7.2 and CTLA4 molecules on both CD4þ (A) and CD8þ (B) cells. The

up-regulation of costimulatory molecule expression was most pronounced for B7.1 followed by B7.2 and CTLA4. Stimulation of PBMC with

2 mg/ml ConA also caused an increase in the expression of costimulatory molecules on CD4þ (C) and CD8þ (D) cells. After 48 h of

stimulation B7.2 up-regulation exceeded the expression of B7.1 most prominently found on CD8þ T cells. Note that B7.1 is constitutively

expressed on feline CD4þ cells. The mean of the data � standard deviation obtained from six independent experiments is shown.


costimulatory molecules on purified CD4þ or purified

CD8þ cells progressively increased over the 72 h cul-

ture suggesting that other cell types did not contribute to

up-regulation of these molecules (Fig. 2).

3.2. Co-localization of B7.1 costimulatory molecules

on B7.2 and CTLA4 expressing cells

Three-color flow cytometry was performed for co-

localization analysis (Fig. 3A–D). After 24 h of sti-

mulation at least 35% of the CD4þB7.2þ cells and

CD8þB7.2þ cells simultaneously express B7.1 mole-

cules. Approximately 40% of the CD4þCTLA4þ cells

and 35% of the CD8þCTLA4þ cells co-express B7.1.

After 48 h of stimulation, co-expression of B7.1 on

B7.2, as well as CTLA4 positive cells exceeded 75%

on both CD4þ and CD8þ cells, suggesting that the

major fraction of B7.2þ and CTLA4þ T cells co-

express B7.1 on their surface (Fig. 3E) and that a

large fraction of both CD4þ and CD8þ T cells

co-express all three costimulatory molecules.

3.3. Correlation of apoptosis with T cells expressing

B7.1, B7.2 or CTLA4

PBMC were analyzed fresh and upon culturing for

24 h with or without ConA for T cell apoptosis. After

24 h in culture with ConA, CD4þ and CD8þ cells

showed an increase in apoptosis (Fig. 4A), consistent

with the observation that stimulated cells expressed

higher numbers of costimulatory molecules on their

surface (Fig. 1). Three-color flow cytometry analysis

revealed that TUNEL positive CD4þ and CD8þ cells

were largely restricted to those cells expressing B7.1,

B7.2 and CTLA4 (Fig. 4B).

3.4. B7 receptor blockade inhibits apoptosis in

ConA-activated T cells

To investigate whether the B7 costimulatory path-

way is involved in the induction of apoptosis, anti-

B7.1, anti-B7.2 or anti-CTLA4 antibodies were added

to ConA stimulated PBMC (Fig. 5A). Treatment of

CD4þ cells with anti-B7.1 antibodies resulted in a

significant decrease in the number of apoptotic cells

by approximately 50%. Anti-B7.2 and anti-CTLA4

antibodies reduced the incidence of apoptotic cells by

23 and 18%, respectively. Anti-B7.1 and anti-B7.2

antibodies had no additional effect compared with the

inhibition seen after the treatment with anti-B7.1

antibodies alone. On CD8þ cells, a similar pattern

in the reduction of apoptotic cells was found, and the

combination of anti-B7.1 and anti-B7.2 antibodies

resulted in the most prominent reduction in number

of apoptotic cells by approximately 70%.

To verify that these results are due to T–T cell

interactions, highly enriched CD4þ or CD8þ cell

populations were prepared by antibody-coated mag-

netic bead depletion of CD21 and CD14 positive cells

from the PBMC. Treatment of CD4þ and CD8þ

enriched PBMC with anti-B7.1 antibodies reduced

the number of apoptotic CD4þ and CD8þ cells by

approximately 40 and 60%, respectively (Fig. 5B). The

combination of anti-B7.1 and anti-B7.2 antibodies

showed the most prominent reduction of the number

of apoptotic CD4þ and CD8þ cells (60 and 80%,

respectively). Anti-B7.2 and anti-CTLA4 antibody

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60

70


Per

cent

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CD

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ells

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30

40

50

60

70


Per

cent

of t

otal

CD

8+ c

ells

B7.1B7.2CTLA4

(A)

(B)

Fig. 2. Increased expression of B7.1, B7.2 and CTLA4 molecules

on purified CD4þ and CD8þ cells upon in vitro stimulation.

Magnetic bead purified T cell subsets (2 � 106) were stimulated

with 50 ng/ml ionomycin and 10 ng/ml PMA and the expression of

B7.1, B7.2 and CTLA4 molecules determined by two-color flow

cytometric analysis after 24, 48 and 72 h of culture. The up-

regulation of costimulatory molecule expression and the percentage

of cells expressing these molecules was comparable to the results

obtained from similarly stimulated PBMC cultures. The results of

one representative experiment are shown.


treatment alone did not significantly reduce the number

of apoptotic cells. These results suggest that T cell

apoptosis is the result of B7–CTLA4 and in particular

B7.1 mediated T–T interactions.

4. Discussion

LN from FIV-infected cats display increased num-

bers of B7þCTLA4þ T cells (Tompkins et al., 2002).

Up-regulation of B7 and CTLA4 on T cells correlates

with disease progression, as the highest numbers of

B7þCTLA4þ CD4þ and CD8þ cells are seen in

animals with advanced infection. As LN T cell apop-

tosis is a characteristic of FIV-infected cats, and as

apoptosis is associated with B7þCTLA4þ T cells,

we hypothesized that T–T interactions mediated by

B7.1/B7.2–CTLA4 ligation may be responsible for T

cell apoptosis and the eventual development of AIDS.

The in vitro mitogen stimulation assays presented here

were intended to simulate the mitogenic microenvir-

onment of LN of viremic FIV-infected cats.

Reports have described increased expression of

B7.1 and B7.2, as well as MHC class II molecules

on murine or human T cells following anti-CD3 or

antigen stimulation (Azuma et al., 1993; Sansom and

Hall, 1993; Wyss-Coray et al., 1993; Hathcock et al.,

1994). These activated T cells are capable of acting as

APC, but instead of inducing proliferation, transduce

signals for anergy and apoptosis of other activated T

Fig. 3. Co-localization of B7.1 costimulatory molecules on B7.2 or CTLA4 expressing CD4þ and CD8þ cells. PBMC were stimulated in vitro

with 2 mg/ml ConA and analyzed by three-color flow cytometric analysis after 24 and 48 h of culture. After 48 h of culture the majority of the

B7.2 positive or CTLA4 positive CD4þ and CD8þ cells also expressed B7.1 (A–D). The mean of the data � standard deviation obtained from

three independent experiments analyzed after 24 and 48 h are shown (E).


cells (Lombardi et al., 1994; Greenfield et al., 1997).

To reproduce this activation response in feline PBMC,

we utilized ConA, as anti-CD3 antibodies for the

feline TCR are unavailable. ConA binds the CD3

component of the TCR and shares most stimulatory

properties of anti-CD3 or anti-TCR monoclonal

antibodies on human lymphocytes (Chilson and

Kelly-Chilson, 1989) thereby mimicking the physio-

logical TCR ligand represented by the MHC-peptide

complex (Pani et al., 2000; Weiss et al., 1986). We

therefore believe that the results obtained with our in

vitro model are representative of TCR signaling and a

Fig. 3. (Continued ).


relevant model for in vivo T cell activation in FIV-

infected cats. ConA or I/PMA stimulation of PBMC or

purified CD4þ and CD8þ T cells resulted in an

increased expression of B7.1, B7.2 and CTLA4.

Within 48 h of stimulation the majority of B7.2þ

and CTLA4þ T cells co-expressed B7.1. This unusual

T cell phenotype closely resembles B7þCTLA4þ acti-

vated CD4þ and CD8þ LN T cells that is a character-

istic of chronically FIV-infected cats (Tompkins et al.,

2002) suggesting that this T cell activation model

may be relevant to the activation process occurring

in LN of FIV-infected cats. In support of this, three-

color flow cytometric analysis of the in vitro stimulated

T cells revealed a strong correlation between B7.1,

B7.2 and CTLA4 expressing T cells and T cell apop-

tosis (Fig. 4).

To determine the possibility of B7–CTLA4 mediated

T–T cell interactions mediated apoptosis, antibody-

blocking studies were performed. Treatment of stimu-

lated PBMC with anti-B7.1 antibodies significantly

reduced apoptosis of both CD4þ and CD8þ cells.

Inhibition of apoptosis was even more pronounced

0

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B7.

1 -

B7.

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2 -

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LA4-

CT

LA4+

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LA4-

CT

LA4+

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LA4

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LA4

+

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CD4+ cellsCD8+ cells

Unstimulated 24 h ConA stimulation 48 h ConA stimulation

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Fresh 24h Culture w/oConA

24h Culture withConA

Per

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CD4+ cellsCD8+ cells

(B)

(A)

Fig. 4. Increased numbers of apoptotic CD4þ and CD8þ cells upon stimulation in vitro with ConA. CD4þ and CD8þ cells were analyzed fresh

or after 24 h of cell culture in the presence or absence of 2 mg/ml ConA (A). The frequency of apoptotic CD4þ and CD8þ cells was determined

using the TUNEL assay and two-color flow cytometric analysis. The mean of the data � standard deviation obtained from three independent

experiments is shown. Increased frequency of apoptosis in CD4þ and CD8þ cells expressing B7.1, B7.2 and CTLA4 relative to those lacking

these costimulatory molecules (B). T cells were analyzed fresh and after 24 and 48 h of stimulation with 2 mg/ml ConA by three-color flow

cytometry to determine the percent of cells expressing B7.1, B7.2 or CTLA4 and the frequency of apoptosis among the different cell

phenotypes. Both, CD4þ and CD8þ cells showed an increased percent of TUNEL positive cells in populations expressing B7.1, B7.2 or

CTLA4 relative to those lacking these surface molecules.


in purified CD4þ and CD8þ T cell cultures suggesting

that other cell types expressing B7 molecules, such as

monocytes and B cells were not contributing to the T

cell apoptosis. Lewis et al. (1999) also reported that

spontaneous CD8þ T cell apoptosis in HIV-infected

individuals could be partially (�50%) inhibited by

combined antibodies to B7.1 and B7.2. T cell apop-

tosis was also partially inhibited by depletion of

monocytes, leading them to conclude that T cell

apoptosis was mediated by B7 positive monocytes.

It is equally possible that the residual significant level

of T cell apoptosis in the monocyte-depleted human

PBMC cultures is due to B7þ–CTLA4þ mediated T–T

interactions. In support of this, we observed that anti-

B7.1 plus anti-B7.2 antibodies partially blocked apop-

tosis of mitogen stimulated enriched CD4þ and CD8þ

cell subsets, suggesting that monocytes or other APCs

were not involved. Failure to completely block apop-

tosis in 24 h stimulated PBMC may be due to the fact

that a significant fraction of these stimulated T cells

are already primed for apoptosis, which cannot be

reversed. This would be particularly true of the LN T

cells from FIV-infected cats and PBMC from HIV-

infected patients (Lewis et al., 1999), as these cells

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140

CD4+ cells CD8+ cells

Per

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UN

EL

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Rabbit serum

Anti B7.1

Anti B7.2

Anti CTLA4

Anti B7.1+B7.2

**

**

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120

140

CD4+ cells CD8+ cells

Per

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UN

EL

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**

**

(A)

(B)

Fig. 5. Effect of antibody blockade of B7.1, B7.2 and CTLA4 on apoptosis of ConA stimulated PBMC. PBMC were cultured for 24 h with

2 mg/ml ConA. After 24 h, anti-B7.1 antibodies, anti-B7.2 antibodies, or anti-CTLA4 antibodies or the combination of anti-B7.1 and anti-B7.2

antibodies were added to the cultures and T cells were assayed for apoptosis 24 h later. The TUNEL assay was used to determine the frequency

of apoptosis in CD4þ and CD8þ cells in PBMC (A) and enriched (B cells and monocytes depleted) CD4þ and CD8þ cell cultures (B).

Significant inhibition of T cell apoptosis was observed following the addition of anti-B7.1 antibodies (CD4þ, P ¼ 0:008; CD8þ, P ¼ 0:03) or

the combination of anti-B7.1 and anti-B7.2 antibodies (CD4þ, P ¼ 0:002; CD8þ, P ¼ 0:002) to PBMC cultures. Similarly, significant

inhibition of T cell apoptosis was observed following the addition of anti-B7.1 antibodies (CD4þ, P ¼ 0:02; CD8þ, P ¼ 0:03) or the

combination of anti-B7.1 and anti-B7.2 antibodies (CD4þ, P ¼ 0:01; CD8þ, P ¼ 0:007) to enriched T cell cultures. The mean of the data �standard deviation obtained from six independent experiments with the treatment groups normalized to the rabbit serum control (100%) are

shown.


received no further stimulation in culture and cells

progressing through apoptosis would have already

been programmed in vivo. Alternatively, we cannot

exclude that Fas/FasL, TNFa/TNFaR interactions are

also involved in T cell apoptosis. It is also possible that

failure to completely block apoptosis with anti-B7

antibodies might be due to the antibody concentration,

low density of surface receptors or the specificity of

the antibodies.

The observation that anti-B7.1 antibodies are much

more effective than anti-B7.2 antibodies in blocking

apoptosis is also of interest. While both are rabbit

polyclonal antibodies, the epitopes that they recognize

may be quite different. It is also interesting that anti-

B7.1 antibodies have a significant inhibitory effect on

T cell apoptosis even when B7.2 is not blocked. Both

of these observations suggest that B7.2 may not ligate

CTLA4 in this T cell model or may not transduce a

signal for apoptosis. While data suggest that B7.1 and

B7.2 costimulatory functions are overlapping (Free-

man et al., 1993; Linsley et al., 1991), there is also

evidence for a preferential role for B7.1. Pertinent to

our studies, Razi-Wolf et al. (1992) have shown that T

cell proliferation to ConA or alloantigen is solely

dependent on B7.1 even when B7.2 is present. Simi-

larly Fleischer et al. (1996) demonstrated that T cell

activation by monocytes is inhibited by anti-B7.1 but

not anti-B7.2 antibodies even when B7.2 is expressed

at a higher level. Gajewski (1996) similarly reported

that B7.1 but not B7.2 transfectants costimulated

CD8þ activation, and anti-B7.1 antibodies inhibited

costimulation. The molecular basis for these differ-

ences is not known, but studies demonstrated a higher

avidity or duration of interaction between B7.1 and

CTLA4 than between B7.2 and CTLA4 (Walunas

et al., 1994; Gajewski, 1996; Linsley et al., 1994).

Additional studies will be needed to resolve these

questions in our in vitro T cell activation model.

Recent studies indicate that B7 molecules may be

up-regulated on T cells activated in vitro and on a

subset of CD4þ and CD8þ cells in patients with

autoimmune disease (Folzenlogen et al., 1997; Ran-

heim and Kipps, 1994; Vervilghen et al., 1994) or HIV

infection (Wyss-Coray et al., 1993; Kochli et al., 1999;

Wolthers et al., 1996). Others have recently reported a

significantly higher numbers of CTLA4 expressing

CD4þ cells in HIV-infected patients (Steiner et al.,

1999; Leng et al., 2002). Vervilghen et al. (1994)

reported that a fraction of CD4þ and CD8þ cells in

the synovial fluid of patients with rheumatoid arthritis

expressed B7.1 and CTLA4 on their surface. None of

these studies determined if the B7 and CTLA4 recep-

tors were co-expressed on the same cell. With the

exception of the studies performed by Lewis et al.

(1999), there is no evidence that T cells from HIV-

infected individuals or persons with autoimmune con-

ditions participate in T–T induced apoptosis.

However, such a speculation is consistent with

reports that T cells expressing B7 molecules were

capable of engaging other activated T cell and transdu-

cing a signal for anergy (Greenfield et al., 1997; Chai

et al., 2000). Chai et al. (2000) were the first to propose

Activated CD4+/CD8+T cell

B7/CTLA4 mediatedinhibition of TCRsignaling

Activated CD4+/CD8+ T cell

MHC II

CD3/TCR

CTLA4

B7.1/B7.2

CD28

Antigen

B7/CTLA4 mediatedinhibition of TCR

signaling

No IL-2 production

Anergy/Apoptosis

Fig. 6. Proposed model for bi-directional negative signaling

between activated T cells. A small fraction of LN CD4þ and

CD8þ T cells co-express B7.1, B7.2 and CTLA4 receptors on their

surface. As binding of B7.1 and B7.2 for CTLA4 is 20–100�greater than for CD28 (Greenfield et al., 1998) negative signaling

dominates on activated CTLA4 expressing T cells. We propose that

the unique B7þCTLA4þ T cells play a role in terminating immune

responses by B7–CTLA4 induction of direct T–T anergy and

apoptosis. FIV infection induces a marked expansion in numbers of

these activated CD4þ and CD8þ cells (Tompkins et al., 2002)

enabling frequent B7–CTLA4 mediated T–T interactions resulting

in bi-directional IL-2 suppression, anergy and apoptosis. The

chronic activation of these LN T cells, as seen in lentivirus

infections would result in a progressive loss of T cells, as well as

premature termination of immune responses to unrelated antigens

and ultimately immune dysfunction.


that activated T cells expressing B7 receptors and

CTLA4 were capable of T–T interactions resulting in

bi-directional cell killing. To the best of our knowledge

our published studies (Tompkins et al., 2002) and the

data presented here are the first to demonstrate that

chronic activated T cells co-express B7 and CTLA4

receptors and that these cells are capable of T–T inter-

actions resulting in apoptosis.

These data support the hypothesis that in vivo B7–

CTLA4 mediated T–T cell interactions may contri-

bute to the high frequency of apoptosis observed in LN

of FIV-infected cats and HIV-infected humans (Fig. 6).

The cat FIV model provides unique resources to better

understand the immune pathology associated with

lentiviral immunsuppression. The development of this

in vitro model of T–T cell interactions will allow us to

further examine the mechanisms of B7.1/B7.2–

CTLA4 interactions and to investigate the role of

virus, as well as the possible MHC restriction and

antigen specificity of the T–T induced apoptosis.

These studies should provide significant insights into

the cellular and molecular mechanisms leading to

immunodeficiency in FIV-infected cats, and by infer-

ence in HIV-infected patients.

Acknowledgements

The work was supported in part by National Insti-

tute of Health grants AI38177 (MBT) and AI43858

(WAT). We thank Debra Anderson for her excellent

technical assistance and dedication to the cats.

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B7+CTLA4+ T cells engage in T–T cell interactions that mediate apoptosis: a model for lentivirus-induced T cell depletion