clonality and longevity of cd4 cd28null t cells are associated with defects in apoptotic pathways

Because in vivo expanded CD4⫹CD28⫹ and CD4⫹CD28null T cell clones generally have an activated phenotype 26, we exam-ined whether their differential sensitivity to AICD could be related t

of March 7, 2015.

This information is current as

Apoptotic Pathways Cells Are Associated with Defects in

T null CD28 +

Clonality and Longevity of CD4

and Jörg J Goronzy Abbe N Vallejo, Michael Schirmer, Cornelia M Weyand

http://www.jimmunol.org/content/165/11/6301 doi: 10.4049/jimmunol.165.11.6301

2000; 165:6301-6307; ;

J Immunol

References

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Immunologists All rights reserved.

Copyright © 2000 by The American Association of

9650 Rockville Pike, Bethesda, MD 20814-3994.

The American Association of Immunologists, Inc.,

is published twice each month by

The Journal of Immunology

Clonality and Longevity of CD4 CD28 T Cells Are

Abbe N Vallejo,2,3 Michael Schirmer,2,4 Cornelia M Weyand, and Jo¨rg J Goronzy3

CD4ⴙCD28 null

T cells are oligoclonal lymphocytes rarely found in healthy individuals younger than 40 yr, but are found in high frequencies in elderly individuals and in patients with chronic inflammatory diseases Contrary to paradigm, they are functionally active and persist over many years Such clonogenic potential and longevity suggest altered responses to apoptosis-inducing signals In this study, we show that CD4ⴙCD28 null

T cells are protected from undergoing activation-induced cell death Whereas CD28ⴙT cells underwent Fas-mediated apoptosis upon cross-linking of CD3, CD28 null

T cells were highly resistant CD28 null

T cells were found to progress through the cell cycle, and cells at all stages of the cell cycle were resistant to apoptosis, unlike their CD28ⴙcounterparts Neither the activation-induced up-regulation of the IL-2R ␣-chain (CD25) nor the addition of exogenous

IL-2 renders them susceptible to Fas-mediated apoptosis These properties of CD28 null

T cells were related to high levels of Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein, an inhibitor of Fas signaling that is normally degraded in T cells following activation in the presence of IL-2 Consistent with previous data showing protection of CD28 null

cells from spontaneous cell death, the present studies unequivocally show dysregulation of apoptotic pathways in CD4ⴙCD28 null T cells

that favor their clonal outgrowth and maintenance in vivo The Journal of Immunology, 2000, 165: 6301– 6307.

T he aging immune system and chronic inflammatory

syn-dromes such as rheumatoid arthritis and acute coronary

artery disease are characterized by high frequencies of

CD4⫹T cells that are deficient in CD28 expression (1–3)

Com-pared with their CD28⫹counterparts, they produce significantly

higher levels of IFN-␥ (3, 4), giving them the ability to function as

proinflammatory cells Moreover, CD4⫹CD28null

T cell clones persist for years in circulation (5) Longevity of these cells appears

to be related to their relative resistance to spontaneous cell death

even in the absence of IL-2 (6) This phenomenon is associated

with low levels of expression of the ␣-chain of the IL-2R

(IL-2R␣), despite their ability to produce large amounts of IL-2, and an

increased expression of the anti-apoptotic molecule Bcl-2 (4, 6)

Inasmuch as CD4⫹CD28null

T cells are highly oligoclonal (7), we examined their susceptibility to activation-induced cell death

(AICD).5

In the normal immune system, AICD is a mechanism to

delete activated T cells upon resolution of Ag-driven responses (8)

Although the antigenic basis of T cell oligoclonality during aging

and in chronic inflammatory diseases is not known, persistence of

CD4⫹CD28null

T cell clones in vivo suggests perturbation of ap-optotic pathways

AICD is one of the best-characterized systems of apoptosis Subsequent to activation, T cells up-regulate Fas ligand (FasL), which then interacts with the Fas receptor on the same or on neigh-boring T cells Fas-FasL interaction generates an apoptotic signal via the phosphorylation of the Fas receptor death domain, resulting

in a cascade of activation events of caspases that ultimately lead to cell death (9) Molecules that are collectively referred to as inhib-itors of apoptosis may, however, prevent Fas-mediated cell death One of these is the Fas-associated death domain-like IL-1-convert-ing enzyme inhibitory protein (FLIP), also known as Casper, CLARP, FLAME-1, or MRIT (10 –14) FLIP inhibits apoptosis by directly interacting with Fas-associated death domain, or with caspases 8 and 10, resulting in the interruption of signal transduc-tion from the Fas receptor This regulator of apoptosis can also interrupt signaling of other death receptors, particularly members

of the tumor necrosis receptor family (11, 15)

Although IL-2 is a T cell growth factor, it can also potentiate Fas-mediated AICD (16) Subsequent to Ag recognition by T cells, IL-2 production and IL-2R␣ expression result in the progression of

cells through the cell cycle as well as the up-regulation of FasL and

a concomitant suppression of FLIP expression (17, 18) Conse-quently, activated T cells become susceptible to Fas-induced cell death as they proceed through the cell cycle (19, 20) Induction of FasL and the down-regulation of FLIP expression are IL-2-depen-dent processes (16 –18) Because CD4⫹CD28null

T cells unstably express IL-2R␣ (4, 6), we examined whether there is differential

susceptibility between CD4⫹CD28⫹and CD4⫹CD28null

T cells Studies described in this work examined the interrelationship, if any, between the levels of IL-2R␣ expression, cell cycle

progres-sion, and Fas-induced cell death Inasmuch as CD4⫹CD28null

T cells represent a highly oligoclonal subset of lymphocytes found during aging and in chronic disease states (1, 3, 7, 21–23), these studies permitted the evaluation of the hypothesis that T cell oli-goclonality may be the result of persistent immune activation and the prevention of AICD Alteration of cell death programs could account for the accumulation of CD4⫹CD28null

T cells in vivo

Departments of Medicine and Immunology, Mayo Clinic and Foundation, Rochester,

MN 55905

Received for publication May 24, 2000 Accepted for publication September 7, 2000.

The costs of publication of this article were defrayed in part by the payment of page

charges This article must therefore be hereby marked advertisement in accordance

with 18 U.S.C Section 1734 solely to indicate this fact.

1 This work was supported by the Mayo Foundation, the Austrian Research Fund

(Schroedinger Grant J01194), and grants from the National Institutes of Health

(RO1-AR41974, RO1-AR42527, and RO3-AR45830).

2 M.S and A.N.V contributed equally to this work and must be regarded as co-first

authors.

3 Address correspondence and reprint requests to Dr Abbe Vallejo (vallejo.abbe@

mayo.edu) or Dr Jo¨rg Goronzy (goronzy.jorg@mayo.edu), Mayo Clinic, 200 First

Street SW, Rochester, MN 55905.

4 Current address: Department of Medicine, University of Innsbruck, Austria.

5 Abbreviations used in this paper: AICD, activation-induced cell death; 7-AAD,

7-amino actinomycin D; FasL, Fas ligand; FLIP, Fas-associated death domain-like

IL-1-converting enzyme inhibitory protein; PerCP, peridinin chlorophyl protein.

Materials and Methods

Cell culture

Short-term human T cell lines and CD4⫹CD28null T cell clones were

established from fresh PBMC, as described previously (1, 6) Short-term

cell lines were established from PBMC that were initially stimulated with

immobilized anti-CD3 (OKT3; American Type Culture Collection,

Man-assas, VA) for 12 h and maintained at densities of 0.5–2⫻ 106cells/ml

Cells were passaged every 5–7 days in RPMI 1640 (BioWhittaker,

Walk-ersville, MD) containing 10% FCS (Summit Biotechnology, Fort Collins,

CO), 2 mML-glutamine, 50 U/ml penicillin, 5␮g/ml streptomycin (Life

Technologies, Grand Island, NY), and 10 U/ml human rIL-2 (Proleukin;

Chiron, Emeryville, CA) Feeder cells consisting of␥-irradiated

neuramin-idase-treated EBV-transformed B lymphoblastoid cells were also added to

the cultures Cells were maintained in a humidified 7.5% CO2tissue culture

incubator

T cell clones were established by limited dilution cloning of peripheral

blood CD4⫹T cells (7) Clonality was determined by standard seminested

PCR for TCR BV-BJ elements, size fractionation, and sequencing T cell

clones were maintained on feeder cells of ␥-irradiated,

neuraminidase-treated EBV-transformed B lymphoblastoid cells in the presence of 20

U/ml IL-2

The T cell lines and clones used in the present study were derived from

patients with rheumatoid arthritis as well as from healthy donors The

phenotypic characteristics of CD4⫹CD28nullT cells, such as the lack of

CD40 ligand, elevated expression of IFN-␥, oligoclonality, etc (1–5, 7),

were generally very similar among the cells examined regardless of the

source donor

Jurkat cells (American Type Culture Collection) were maintained in

RPMI 1640 medium (as indicated above) in the absence of IL-2 They were

maintained at a density of 5⫻ 106cells/ml in a humidified 5% CO2tissue

culture incubator

Flow cytometry

Cell surface staining of T cells was performed using mAb to CD4, CD28,

IL-2R␣ (CD25), and IL-2R␤ (CD75) conjugated to the appropriate

fluo-rochrome (Becton Dickinson, San Jose, CA) Briefly, 2⫻ 105to 1⫻ 106

cells were incubated with mAb or Ig isotype control (Simultest; Becton

Dickinson) for 25 min on ice, washed with cold PBS, and fixed with 1%

paraformaldehyde for 60 min at 4°C

For immunostaining of the Fas receptor, cells were incubated with

un-conjugated anti-CD95 (CH11; Beckman Coulter, Miami, FL), followed by

FITC-conjugated anti-mouse Ig (BD-PharMingen, San Diego, CA) As

controls, cells were also incubated in IgG instead of anti-CD95 Cells were

subsequently stained with PE-conjugated anti-CD28 and peridinin

chloro-phyl protein (PerCP)-conjugated anti-CD4 (Becton Dickinson), washed,

and fixed with 1% paraformaldehyde Live cells were gated by forward and

side scatter Cells with reduced forward scatter were excluded, and Fas

expression was determined as FITC fluorescence on either CD4⫹CD28⫹or

CD4⫹CD28nullcells

FasL expression was determined by intracellular immunostaining Cells

were activated with the appropriate Ab or pharmacologic agent in the

pres-ence of 10 ␮g/ml brefeldin A (Epicentre Technologies, Madison, WI)

They were stained with PerCP-conjugated anti-CD4 and PE-conjugated

anti-CD28 mAb, fixed with paraformaldehyde, and subsequently

perme-abilized with 0.05% Tween 20 (Sigma, St Louis, MO) for 15 min at 37°C

Cells were washed, resuspended in biotin-conjugated anti-FasL mAb

(NOK-2; BD-PharMingen) for 25 min on ice, followed by

FITC-conju-gated streptavidin As control, cells were stained in a similar manner with

anti-Bcl-2 mAb (Dako, Carpenteria, CA)

Flow cytometry was performed on either a FACSCalibur or

FACSVan-tage flow cytometer (Becton Dickinson) Cell populations were analyzed

using the WinMDI program (Joseph Trotter, The Scripps Research

Insti-tute, La Jolla, CA)

Apoptosis and cytotoxicity assays

About 1⫻ 106T cells were cultured in 96-well plates coated with either

anti-CD3 mAb (OKT3), anti-CD95/Fas mAb (CH11), or an IgG isotype at

50␮g/ml After 18 h, cells were harvested and immunostained for CD4 and

CD28, fixed in paraformaldehyde, and permeabilized with 0.05% Tween

20 Subsequently, 2.5 ␮g/ml 7-amino-actinomycin D (7-AAD;

Calbio-chem, San Diego, CA) was added and incubated for 30 min at room

tem-perature The proportion of 7-AAD⫹subdiploid cells was determined by

flow cytometry IL-2R and FasL expression of parallel cultures were also

examined Cells were immunostained for IL-2R␣ and IL-2R␤, as described

above Apoptosis assays were conducted using T cell lines and clones 7–10

days after the last passage and stimulation with EBV-transformed B cell feeders

The biological activity of soluble FasL was examined CD28⫹CD28null and CD28⫹CD28⫹T cells were activated with either plate-immobilized anti-CD3 or a cocktail of 1␮g/ml PMA and 10 nM ionomycin (Sigma) for

24 h Culture supernatants were harvested and added to 1⫻ 106Jurkat cells Cells were cultured for 18 h in the presence or absence of 5␮g/ml

of the anti-FasL mAb NOK-2 Viability of Jurkat cells was determined by trypan blue exclusion

Immunoblotting

Western blots were performed as described previously (6) A total of 2⫻

106T cells was lysed in a hypotonic buffer and centrifuged at 12,000⫻ g

for 10 min at 4°C, and protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA) Detergent-solu-bilized protein at 10␮g/lane was separated on 12% SDS-polyacrylamide

gels using a mini-gel system and transferred onto Sequi-Blot polyvinyli-dene difluoride membranes (Bio-Rad) Membranes were incubated with 4% BSA in TBS, followed by a 1/500 dilution of a mAb to the short form

of FLIP (F-20), and subsequently incubated in a 1/1000 dilution of HRP-conjugated anti-mouse Ig (Santa Cruz Biotechnology, Santa Cruz, CA) Blots were developed using ECL chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to radiographic films (BIOMAX-MS; Kodak, New Haven, CT) Equal protein loading was as-certained by staining the membrane with GELCODE Blue reagent (Pierce, Rockford, IL)

Cell cycle analysis

T cells were synchronized by incubation with aphidicolin (Sigma); 5␮M

aphidicolin was empirically determined to synchronize ⱖ98% of both

CD4⫹CD28nulland CD4⫹CD28⫹T cells at the G1-S boundary within 30 h

of incubation (data not shown) Synchronization was verified by

5-bromo-3⬘-deoxyuridine labeling using an in situ proliferation kit (BrDU-FLUOS;

Roche Molecular Biochemicals-Boehringer Mannheim, Indianapolis, IN) combined with standard propidium iodide staining for DNA content and flow cytometry

Synchronized cells were washed extensively and cultured on plates coated with 50␮g/ml IgG, anti-CD3, or anti-Fas Ab for 18 h As indicated,

activation was conducted either in the presence or absence of 5␮g/ml

exogenous IL-2 Cells were subsequently immunostained for CD4, CD28, IL-2R␣, and IL-2R␤, as described above, and with propidium iodide

stain-ing for DNA content

Statistical analysis Quantitative analysis was conducted with Student’s t test, and, if

appro-priate, with the Wilcoxon signed rank test using the SigmaStat software (SPSS, Chicago, IL)

Results

CD4⫹CD28 null

T cells are protected from undergoing AICD

Differential sensitivity of CD4⫹CD28⫹and CD4⫹CD28null

T cells

to AICD was examined by incubation on plate-immobilized anti-CD3 Levels of apoptosis were determined by flow cytometric analysis of 7-AAD-stained cells Apoptotic cells were identified as sub-G0 cells characterized by reduced DNA-activated fluores-cence, which was indicative of fractional or subdiploid DNA

con-tent Depicted in Fig 1A is a comparison of DNA staining between

representative CD28⫹and CD28null

T cell clones before and after incubation with anti-CD3 These results show that a higher pro-portion of CD28⫹cells compared with CD28null

cells was sub-G0

even in absence of activation Among the CD28⫹cells, the per-centage of subdiploid cells markedly increased after incubation with anti-CD3 This was unlike the situation in CD28null

cells, which showed little or no detectable increase in the number of subdiploid cells in the presence of anti-CD3 Curiously, anti-CD3 elicited a noticeable decrease in the proportion of CD28null

cells in

G0-G1 and an increase in the proportion of cells in S-G2-M In contrast, there was little difference in the proportion of CD28⫹ cells in G0-G1 and S-G2-M between anti-CD3-treated cells and controls

6302 DEFECTS IN AICD OF INFLAMMATORY CD4 T CELLS

Depicted in Fig 1B are results from all T cell lines and clones

that we examined These show that there was a significantly higher

proportion of CD28⫹ cells that underwent apoptosis compared

with CD28null

cells ( p⬍ 0.001) Corroborating previous data (6),

the present data demonstrate the levels of spontaneous apoptosis,

i.e., cells incubated in IgG, were also higher for the CD28⫹cells

( p⬍ 0.01) Such difference in the susceptibility to AICD between

CD4⫹CD28⫹and CD4⫹CD28null

was consistently observed in all cell lines and clones examined

Induction of AICD in CD28⫹, but not CD28null

, T cells was most likely Fas mediated As shown in Fig 2, incubation of cells

on either immobilized anti-CD3 or anti-Fas receptor mAb resulted

in a significant increase of subdiploid CD28⫹cells compared with

those incubated with IgG isotype control ( p⬍ 0.001) There were

no significant differences in the levels of apoptosis of CD28⫹cells

incubated on either anti-CD3 or anti-Fas Ab Incubation of cells in

a mixture of both Ab also showed similar high levels of apoptosis

In contrast, the CD28null

cells did not show any appreciable

in-crease of subdiploid cells following incubation with CD3, anti-Fas, or both Ab over those cells incubated with IgG Compared with the CD28⫹ cells, they also maintained significantly lower

levels of spontaneous apoptosis ( p⬍ 0.01) as in Fig 1 The

re-sistance of CD28null

cells to Fas-induced apoptosis was observed

in all cell lines and clones examined

T cells have equivalent levels

of Fas and soluble FasL expression

The resistance of CD4⫹CD28null

T cells to Fas-induced apoptosis might be argued to be due to a lack of the Fas receptor Various cell lines and clones were, therefore, examined for Fas expression

FIGURE 2. Cross-linking of CD3 and Fas does not induce apoptosis of

CD4⫹CD28nullT cells CD4⫹T cell lines and clones were incubated for

24 h on plastic-immobilized IgG, anti-CD3 (aCD3), and/or anti-Fas (aFas)

The number of subdiploid cells was examined by flow cytometry, as in Fig

1 Data shown are representative of four CD4⫹T cell lines containing a

mixture of CD28⫹and CD28nullcells and five clones of each of the CD28

phenotypes Features of box plots are as in Fig 1

Table I Fas expression on CD4⫹CD28⫹and CD4⫹CD28 null T cells

Cell Line or Clonea CD28 Phenotype

Fas-Expressing Cellsb(%)

CD28⫹ CD28 null

aCL1, CL2, DH-P, and DH-N were short-term CD4⫹T cell lines DH-P and DH-N were sorted sublines of CD28⫹and CD28 null cells, respectively.

bCells were cultured on plastic-immobilized anti-CD3 for at least 18 h, washed, and immunostained with the Fas mAb CH11 followed by FITC-conjugated anti-mouse Ab, and subsequently stained with PE-conjugated anti-CD28 and PerCP-con-jugated anti-CD4 Fas-expressing cells were distinguished by a distinct

FITC-fluo-FIGURE 1. CD4⫹CD28nullT cells are resistant to AICD Short-term T cell lines and T cell clones that were either CD28⫹or CD28nullwere cultured for 24 h on plastic-immobilized anti-CD3 (aCD3) or IgG Cells were washed and immunostained for CD4 and CD28, followed by 7-AAD, and analyzed

for DNA staining by flow cytometry Experiments were conducted on cells 7–10 days after the last stimulation A, Histograms are representative for

CD4⫹CD28⫹and CD4⫹CD28nullT cells, as indicated Apoptotic cells were recognized by their reduced DNA-associated fluorescence with sub-G0or subdiploid DNA content in the region marked M1 (indicated as percentage of total cells) Regions M2 and M3 represent the percentage of cells in S-G2-M and G0-G1, respectively B, Results from six CD4⫹T cell lines containing a mixture of CD28⫹and CD28nullcells and from five T cell clones for each

of the CD28 phenotypes are summarized as box plots depicting the median (bar), the 10th and 90th percentiles (whiskers), and the 25th and 75th percentiles (box)

As shown in Table I, CD28⫹and CD28null

T cells have equivalent levels of Fas expression In fact, the densities of Fas on the cell

surface are nearly identical between the two cell types (data not

shown) Fas-expressing cells were distinguished by FITC

fluores-cence shifts of one-half to two log intensities over the isotype

control At these levels of expression, the CD4⫹CD28null

, but not CD4⫹CD28⫹, T cells were consistently unresponsive to Ab

cross-linking of Fas (Fig 2)

The level of FasL expression was also examined Results of

flow-cytometric studies, however, showed low and widely variable

expression of either cytoplasmic or membrane FasL (data not

shown) Inasmuch as the reason for this variability is not known,

we examined the ability of either cell type itself to induce

Fas-mediated cell death, presumably through the production of

biolog-ically active soluble FasL CD28⫹and CD28null

T cells were ac-tivated either by incubation with anti-CD3 or with mitogenic

concentrations of PMA and ionomycin The culture supernatants

were harvested and assayed for cytotoxic activity on Jurkat cells

As shown in Fig 3, there was no significant spontaneous death of

Jurkat cells as indicated by the high survival rate of the cells in the

medium controls Addition of supernatants from either activated

CD28⫹or CD28null

cells was cytotoxic to Jurkat cells The levels

of cytotoxicity were equivalent for all of the supernatants tested

The addition of anti-FasL mAb resulted in the neutralization of the

cytotoxic activity of the supernatants The levels of biological

ac-tivity of soluble FasL-containing supernatants from either CD28⫹

or CD28null

T cells were similar for anti-CD3-activated or PMA/

ionomycin-treated cells

Resistance of CD4⫹CD28 null

T cells to AICD is associated with the induction of IL-2R ␣ expression

In addition to IL-2 being the major growth factor for both naive

and activated T cells (24, 25), it can also potentiate AICD (16, 17)

Because in vivo expanded CD4⫹CD28⫹ and CD4⫹CD28null

T cell clones generally have an activated phenotype (26), we exam-ined whether their differential sensitivity to AICD could be related

to the level of IL-2R␣ expression As depicted in Fig 4, the

con-stitutive levels of IL-2R␣ expression on CD4⫹CD28⫹T cells were significantly higher than on CD4⫹CD28null

cells ( p⬍ 0.001), as

we reported previously (4) Moreover, such high levels of expres-sion on CD28⫹cells were also found to further increase upon

incubation with anti-CD3 ( p⬍ 0.01) Such induction of IL-2R␣

with anti-CD3 was accompanied by a significant increase in the

number of subdiploid cells over the IgG controls ( p ⬍ 0.001)

Neither the combination of anti-Fas and anti-CD3 nor anti-Fas by itself significantly changed the observed levels of apoptosis and IL-2R␣ expression in CD28⫹cells

Incubation of CD4⫹CD28null

T cells with anti-CD3 also re-sulted in significant up-regulation of IL-2R␣ expression compared

with unstimulated cells ( p⬍ 0.001) In fact, the levels of IL-2R␣

induction were equivalent to those seen on their CD28⫹ counter-parts However, there was no perceptible change in the number of subdiploid CD28null

cells despite such increases in IL-2R␣

expres-sion during activation with anti-CD3 The addition of anti-Fas to the CD28null

T cell cultures did not lead to further increases in IL-2R␣ expression nor did it increase the level of apoptosis

More-over, anti-Fas by itself did not induce IL-2R␣ expression and had

no perceptible effect on apoptosis over that of the unstimulated controls

There were equivalently high levels of IL-2R␤ expression on

both cell types (data not shown) In contrast to the results with IL-2R␣, incubation of cells with anti-CD3 did not affect the levels

of IL-2R␤ expression

Resistance of CD4⫹CD28 null

T cells to AICD is independent of cell cycle progression

The resistance of CD28null

T cells to apoptosis despite induction of IL-2R␣ following activation raised the question whether this was

due to their inability to progress through the cell cycle In normal

T cells, IL-2/IL-2R signaling commits cells to enter the cell cycle

FIGURE 3. CD4⫹CD28⫹and CD4⫹CD28nullT cells produce

equiva-lent amounts of cytotoxic soluble FasL T cell clones were incubated with

either anti-CD3 (circles) or a combination of PMA and ionomycin

(trian-gles) for 18 h Culture supernatants were collected and added to triplicate

cultures of Jurkat cells Survival of Jurkat cells was monitored after 24 h

Cytotoxicity of supernatants due to soluble FasL was examined by the

addition of anti-FasL Ab during the bioassay (gray symbols) Data shown

are representative of three clones for each of the CD28 phenotypes

FIGURE 4. Resistance of CD4⫹CD28nullT cells to Fas-mediated apo-ptosis is independent of IL-2R␣ expression Duplicate cultures of T cells were incubated overnight on plastic-immobilized IgG, anti-CD3 (aCD3),

or anti-Fas (aFas) Cells were washed and immunostained for CD4 and CD28, followed by 7-AAD staining, and analyzed by flow cytometry Par-allel cultures were analyzed for IL-2R␣ expression Data shown were based on the analysis of four short-term CD4⫹T cell lines containing both CD28⫹and CD28nullsubsets and six sublines sorted for CD28 Features of box plots are as in Fig 1

6304 DEFECTS IN AICD OF INFLAMMATORY CD4 T CELLS

(24, 25) Subsequently, they become susceptible to Fas-mediated

apoptosis (18), which peaks during the S phase of the cell cycle

(20) Thus, we examined the relative proportions of T cells at the

different stages of the cell cycle following activation CD28⫹and

CD28null

T cells were synchronized with aphidicolin and

incu-bated in anti-CD3 with or without anti-Fas, and the numbers of

cells in G0-G1and S-G2-M were determined As shown in Figs 1A

and 5, a significant proportion of CD4⫹CD28null

T cells was in S-G2-M following incubation with anti-CD3 compared with those

incubated with IgG ( p⬍ 0.03) Furthermore, the anti-CD3-induced

response was significantly higher than that seen with their CD28⫹

counterparts ( p⬍ 0.001)

As indicated in Fig 4, even under conditions of increased

IL-2R␣ expression due to activation by anti-CD3, there was no

per-ceptible increase in apoptosis among CD28null

cells, unlike CD28⫹cells, which underwent high levels of Fas-mediated AICD

The addition of anti-Fas to the cultures did not elicit any significant

change in relative proportion of cells in S-G2-M over those in

G0-G1phase of the cell cycle (Fig 5) Also, anti-Fas did not affect

the overall levels of anti-CD3-induced apoptosis in either

CD4⫹CD28⫹or CD4⫹CD28null

T cells (Fig 4)

High levels of FLIP expression are maintained in

CD4⫹CD28 null

T cells following activation

The similar levels of cell surface expression of Fas as well as the

production of biologically active FasL by CD28⫹and CD28null

T cells suggested that the resistance of the latter to Fas-mediated

death may be a perturbation in Fas signaling The role of the

ap-optosis inhibitor FLIP was evaluated because it has been shown to

inhibit proximal signals emanating from Fas-FasL interaction, and

its down-regulation subsequent to activation has been associated

with AICD (17, 18) Indeed, Western blotting experiments

re-vealed that incubation of CD4⫹CD28⫹T cell clones with

anti-CD3 resulted in the loss of FLIP expression (Fig 6) In contrast,

CD4⫹CD28null

T cell clones maintained high levels of FLIP

ex-pression in the presence of anti-CD3 Neither the presence nor

absence of exogenous IL-2 in CD28null

T cell cultures affected the

levels of FLIP expression (data not shown) In all CD28null

clones examined, there was a negligible difference between activated and unstimulated cells

Discussion

CD4⫹CD28null

T cells are characterized by oligoclonality and lon-gevity (26 –28) Although their high frequencies of up to 50% of the total CD4 compartment (1–3) suggest chronic activation by persistent, but yet undefined Ag(s), it may be argued that this phe-nomenon may also be explained by perturbation of apoptotic path-ways Indeed, the present data demonstrate their resistance to AICD Incubation of CD4⫹CD28null

T cells with anti-CD3, a con-dition that renders normal T cells susceptible to Fas-mediated cell death (29), did not result in apoptosis (Figs 1 and 2) Furthermore, neither the cross-linking of Fas by itself nor the cocross-linking of CD3 and Fas promoted apoptosis in these cells This is unlike the situation in CD4⫹CD28⫹T cells, which are highly susceptible to apoptosis following incubation with Ab to CD3 or Fas These data support the notion that CD4⫹CD28null

T cells have a survival ad-vantage over their CD28⫹counterparts (27) Aberrations in AICD could confer such advantage and contribute to the phenomenal restriction of the T cell repertoire found during aging and in chronic inflammatory diseases (28 –35)

The insensitivity of the CD4⫹CD28null

T cells to Fas-mediated cell death is not due to defective FasL expression CD28null

cells produce soluble FasL that is as functionally active as that secreted

by CD28⫹cells (Fig 3) Because CD28null

and CD28⫹cells ex-press equivalent levels of Fas (Table I), the resistance of CD28null

, but not CD28⫹, cells to AICD suggests a perturbation of Fas sig-naling Additionally, CD4⫹CD28null

T cells did not display the proapoptotic effects of IL-2, as have been reported for normal ac-tivated T cells (16, 17) Cross-linking of CD3 on CD28null

cells induces IL-2R␣ expression with concomitant cell cycle

progres-sion without perceptible increase in Fas-mediated apoptosis (Figs

4 and 5)

The notion of defective Fas signaling in CD4⫹CD28null

T cells

is supported by the finding that these cells maintain high levels of the anti-apoptotic molecule FLIP subsequent to activation (Fig 6) Normal resting T cells express large amounts of FLIP, which binds

to either the death domain of Fas or to one of the effector caspases, thereby effectively interrupting transduction of death signals (36) Activating signals through the TCR-CD3 complex, however, result

in the degradation of FLIP (17) Thus, activated T cells become sensitive to Fas-mediated apoptosis, as seen with CD4⫹CD28⫹

T cells, but not with CD4⫹CD28null

T cells (Figs 1, 2, and 4) This protection of CD28null

T cells from Fas-mediated AICD is clearly related to the accompanying high levels of FLIP Such relationship between FLIP expression and resistance to apoptosis

FIGURE 5. Cross-linking of Fas does not interfere with cell cycle

pro-gression of CD4⫹CD28nullT cells Aphidicolin-synchronized cells were

incubated overnight with plastic-immobilized IgG or anti-CD3 (aCD3) in

the presence or absence of anti-Fas (aFas) All cultures contained

exoge-nous IL-2 Cells were washed and immunostained for CD4 and CD28,

followed by propidium iodide staining for DNA content Cell cycle

ysis was examined by flow cytometry Data shown are based on the

anal-ysis of 10 T cell lines and clones as in Fig 4 Features of box plots are as

in Fig 1

FIGURE 6. CD4⫹CD28nullT cells maintain high levels of FLIP expres-sion following activation T cells were incubated on plastic-immobilized IgG (⫺) or anti-CD3 (⫹) for 24 h Total cell lysates were prepared and

subjected to SDS-PAGE and Western blotting for FLIP Addition of ex-ogenous IL-2 did not alter FLIP expression of CD28null cells (data not shown) Immunoblots shown are representative of eight clones for each of the CD28 phenotypes examined

has been reported for various cell types, including T lymphocytes

(17, 18, 37, 38)

The down-regulation of FLIP in T cells is IL-2 dependent (17)

and coincides with cell cycle progression (18) These findings

in-dicate that FLIP is a convergence point of Fas and IL-2 signaling

pathways They also suggest that the metabolic fate of FLIP is a

critical factor in determining whether the transduction of IL-2/

IL-2R signals will ultimately lead to apoptosis (16, 17) or the

completion of cell division (25, 39) In the present study,

CD4⫹CD28null

, but not CD4⫹CD28⫹, T cells have high levels of

FLIP expression (Fig 6), which are accompanied by the

up-reg-ulation of IL-2R␣ subsequent to activation (Fig 4) Instead of

apoptosis, such induction of IL-2R␣ expression on CD4⫹CD28null

T cells following incubation with anti-CD3 is associated with the

progression of cells through the cell cycle (Fig 5) In fact, the

number of CD28null

cells that are in S-G2-M is not diminished by the cocross-linking of CD3 and Fas These data are in marked

contrast to previous studies showing that normal T cells undergo

apoptosis as they progress through the cell cycle (19, 40), with

predominance of apoptosis during the S phase (20) Collectively,

these results indicate that the molecular machinery responsible for

FLIP down-regulation is disconnected from IL-2 signaling in

CD4⫹CD28null

T cells Rather than apoptosis, the

growth-promot-ing effects of IL-2 signal transduction appear to be a default

path-way in these cells

Although the pathway that directly links IL-2 to FLIP remains to

be elucidated, previous studies have demonstrated that FLIP

deg-radation can be prevented by cyclosporin A and rapamycin, which

selectively inhibit IL-2 production and IL-2 signal transduction,

respectively (18) Inasmuch as IL-2 production and the induction

of IL-2R␣ expression are intact in CD4⫹C28null

T cells (3, 4, 6, 23) (Fig 4), the maintenance of FLIP in these cells suggests a

defect in the IL-2-dependent regulation of this anti-apoptotic

mol-ecule This is reinforced by the observation that the addition of

exogenous IL-2 altered FLIP expression following activation of

CD28⫹, but not of CD28null

, T cells (data not shown) Although earlier studies have suggested a role for transcriptional repression

in mouse T cells (17), recent studies with human T cells have

shown that IL-2 does not affect the steady state levels of FLIP

transcripts (18) IL-2, therefore, appears to influence the

transla-tional control of FLIP and/or targeting of the FLIP protein to

deg-radation pathways Whether or not such pathways regulating FLIP

expression are defective in CD4⫹CD28null

T cells remains to be examined

Recent studies have implicated STAT-5 in the proapoptotic

ef-fects of IL-2 (41) Gene reconstitution experiments with IL-2R␤

and STAT-5 knockout mice demonstrated that IL-2 conferred

sus-ceptibility to apoptosis in activated mouse T cells through IL-2R

␤-coupled activation of STAT-5, which led to the up-regulation of

FasL expression This confirms that activation invariably results in

FasL induction (16 –18), which subsequently triggers cell death

upon its interaction with the Fas receptor In the present work, it is

clear that CD28null

T cells do not significantly differ from their CD28⫹counterparts in the levels of Fas expression (Table I) and

the production of biologically active FasL (Fig 3) Moreover, both

cell types express equivalent levels of IL-2R␤ (data not shown)

Thus, it is unlikely that protection of CD28null

T cells from AICD could be due to a dissociation of STAT-5-dependent events from

FasL expression

It should also be mentioned that STAT-5 has been reported to be

an inducer of IL-2R␣ (42) Thus, activation-induced up-regulation

of IL-2R␣ on T cells, as shown in Fig 4, would be predicted to

sustain IL-2 signaling, which would consequently promote FLIP

down-regulation and susceptibility to Fas-mediated apoptosis As

already discussed, this is clearly not the case for CD4⫹CD28null

T cells, which up-regulate IL-2R␣ following activation and yet

maintain FLIP expression A peculiar feature of these cells, how-ever, is that IL-2R␣ is particularly unstable (4) Although clearly

inducible (Fig 4), the high level of IL-2R␣ drops precipitously

after 24 h (4 and data not shown) Although the molecular basis for IL-2R␣ instability on CD4⫹CD28null

T cells is not yet known, their resistance to Fas-mediated cell death is curiously reminiscent

of mouse T cells that are deficient in IL-2R␣, which also do not

undergo AICD (43, 44)

In conclusion, the present data provide strong evidence for the dysregulation of apoptotic pathways in CD4⫹CD28null

T cells Unlike their CD28⫹ counterparts, they maintain high levels of FLIP following activation Such persistence of FLIP occurs despite the induction of IL-2R␣, and neither the cross-linking of CD3 nor

Fas affects cell cycle progression, clearly indicating the absence of proapoptotic effects of IL-2 in these cells Although these FLIP-expressing CD4⫹CD28null

T cells are a major component of the T cell repertoire in several human autoimmune diseases (2, 28, 45, 46), it is important to note that overexpression of FLIP in mice results in lymphoproliferative autoimmune disorders (47) Because many CD4⫹CD28null

T cells have autoreactive properties (31, 46, 48), the role of FLIP in the differentiation of T cell effector func-tions is a provocative proposition

Acknowledgments

We thank Dr Alicia Algeciras-Schimnich for technical advice and discus-sions, and James Fulbright for assistance in the preparation of this manuscript

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