Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gorak-Stolinska, P.
Right arrow Articles by Noble, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gorak-Stolinska, P.
Right arrow Articles by Noble, A.
(Journal of Leukocyte Biology. 2001;70:756-766.)
© 2001 by Society for Leukocyte Biology

Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-{alpha}

Patricia Gorak-Stolinska, Jean-Philip Truman, David M. Kemeny and Alistair Noble

Guy’s, King’s and St. Thomas’ School of Medicine, Rayne Institute, London, United Kingdom

Correspondence: Alistair Noble, Department of Immunology, Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, U.K. E-mail: alistair.noble{at}kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human primary effector T cells were analyzed for their susceptibility to anti-CD3-induced activation-induced cell death (AICD). Th1 and Tc1 cells were more susceptible to AICD than their type 2 counterparts. Type 1 and type 2 subsets were also found to be differentially susceptible to CD95-mediated apoptosis, although cell-surface expression of CD95 and CD95L was at similar levels on all subsets. A role for CD95 in AICD was confirmed by the addition of anti-CD95L antibodies that partially abrogated AICD. Residual apoptosis could not be accounted for by TNF-{alpha}/TNFR interactions because although type 1 cells secreted more TNF-{alpha} than type 2 cells, the addition of TNFR:Fc fusion protein did not inhibit AICD. Instead, a reduction in AICD was observed in the presence of EGTA or concanamycin A. The inhibition of apoptosis by a granzyme B inhibitor z-AAD-CMK in Tc1 cells further indicated an involvement of the granule exocytosis mechanism in AICD.

Key Words: apoptosis • CD4+ • CD8+ • TNFR • perforin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Most studies on T-cell death have focused on CD4+, T-helper cell subsets. These cells are broadly divided into T helper type 1 (Th1) cells, producing the cytokines interleukin (IL)-2, lymphotoxin, and interferon-{gamma} (IFN-{gamma}), and Th2 cells, producing IL-5, IL-6, IL-10, IL-13, and IL-4 [1 , 2 ]. The pattern of cytokines produced reflects the type of immune response initiated. Type 1 cytokines tend to induce a cellular immune response with inflammation, the activation of macrophages for anti-microbial action, and delayed type hypersensitivity (DTH). Type 2 cytokines tend to induce a humoral response, providing B-cell help for antibody production and enhancing allergic responses with eosinophilia and immunoglobulin (Ig)E formation [2 , 3 ]. CD8+ T cells of type 1 (Tc1) and type 2 (Tc2) cytokine-secreting phenotype have been increasingly described in rodent and human models [4 , 5 ]. In the human system, Tc1 and Tc2 cells have been isolated ex vivo from peripheral blood and draining lymph nodes of individuals with chronic infectious diseases or allergies [3 , 6 ]. Their cytokine patterns were found to be similar to their CD4+ counterparts, although Tc2 cells are rare. CD4+ and CD8+ subsets have been shown to have distinct functions and are important in many aspects of disease resolution and pathology [7 ].

In addition to cytokine production, the most striking distinction between type 1 and type 2 T-cell subsets is their susceptibility to activation-induced cell death (AICD), a specific form of apoptosis initiated in previously activated T cells following restimulation via the CD3/T-cell receptor (TCR) complex. This form of apoptosis also requires the interaction of specific receptors, termed death receptors, with their ligands. For example, CD95 (Fas)/CD95 ligand (CD95L) interactions are the major mechanism for the AICD of murine CD4+ T cells [7 8 9 10 ]. Another mechanism using tumor necrosis factor {alpha} (TNF-{alpha}) and the TNF receptor (TNFR) has been described as important in AICD of murine CD8+ cells [11 , 12 ]. Moreover, mechanisms other than those mediated by death receptors have also been found to be important in T-cell apoptosis. Recently, a role for perforin in AICD of murine T cells has been shown [13 14 15 ]. In addition to the critical importance of the perforin/granzyme pathway in the induction of apoptosis in target cells by cytotoxic CD8+ T cells [16 , 17 ], the pathway may also contribute to homeostatic control of activated T cells by AICD of effectors themselves via suicidal or fratricidal mechanisms [18 19 20 ].

A comprehensive appreciation of how AICD is controlled in human, primary effector cells is lacking. Therefore, our aim in this study is to assess the differential susceptibility of CD4+ and CD8+ T-cell subsets to AICD. We identify in each subset the relative contributions of three major mechanisms of AICD: CD95, TNF-{alpha}, and perforin/granzyme B. We demonstrate that Th1 and Tc1 cells are more susceptible to AICD than their type 2 counterparts; CD95/CD95L interactions play a partial role in AICD; and TNF-{alpha} is not involved in CD4+ or CD8+ T-cell death. We also show a clear involvement of perforin/granzyme B in AICD of CD8+ cells, demonstrating that the perforin/granzyme cytotoxic pathway contributes to the high levels of apoptosis observed in Tc1 T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
T-cell preparation and subset differentiation
Peripheral blood mononuclear cells (PBMCs) from healthy volunteers or blood bank donors (South Thames Blood Transfusion Centre, London) were obtained by Lymphoprep (Nycomed, Birmingham, UK) separation of whole peripheral blood collected into sodium citrate solution (Sigma Chemical Co., Poole, UK).

CD8+ and CD4+ T cells were separated from PBMCs by positive selection using anti-CD8 or anti-CD4 Dynabeads (Dynal, Cheshire, UK), respectively, as previously described in our laboratory [21 ] and by others [22 ]. Positively selected cells were resuspended in 2% fetal calf serum (FCS)/phosphate-buffered saline (PBS) and released from beads by the addition of DETACHaBEADTM antibody (Dynal), as described in the manufacturer’s instructions. CD4+ and CD8+ T cells were diluted to 2 x 106 cells per ml in complete medium: RPMI 1640 with glutamine (Gibco BRL, Paisley, UK; 0.3 g/l), sodium pyruvate (Sigma Chemical Co.; 110 g/l), 2-mercaptoethanol (Gibco BRL; 0.05 M), gentamycin (Sigma Chemical Co.; 0.1 mg/ml), 1% modified Eagle’s medium (MEM) nonessential amino acids (Gibco BRL), and 10% AB human serum (Serotech, Oxford, UK). This method has been shown not to activate the cells [23 ].

Culture plates were coated with anti-CD3 (BD Pharmingen, Oxford, UK; 5 µg/ml) and anti-CD28 (CLB Amsterdam, Netherlands; 2 µg/ml) in PBS for 1 h at 37°. The wells were washed with complete medium prior to the addition of the purified T cells. Cells were added to the culture wells with equal volumes of culture medium containing recombinant (r)IL-12 (R&D Systems, Abingdon, UK; 500 U/ml) for the generation of type 1 cells or rIL-4 (R&D Systems; 200 U/ml) and neutralizing anti-IL-12 (R&D Systems; 10 µg/ml) for the generation of type 2 cells. Cytokine-containing medium was replaced on day 2 of culture, then with medium alone as required until day 6 in order to provide short-term primary effector cells. Cells were cultured at 37°C in 5% CO2.

Induction of apoptosis
AICD of short-term primary effector T-cell subsets was induced by stimulation of cells with immobilized anti-CD3 (BD Pharmingen; 5 µg/ml) in the presence of 100 U/ml recombinant human (rh)IL-2 (R&D Systems) unless otherwise stated. Controls included cells cultured in the presence of rIL-2 alone with no anti-CD3 antibody.

Alternatively, apoptosis was induced by direct ligation of CD95 with an anti-CD95 antibody (CH11; Upstate Biotechnology, Lake Placid, NY). As a control, cells were incubated with an IgM control antibody (Caltag Laboratories, South San Francisco, CA).

Analysis of cell death
Apoptosis was measured by flow cytometry using Annexin V-FLUOS (Roche Molecular Biochemicals, Mannheim, Germany) and propidium iodide (PI; Sigma Chemical Co.) staining [24 ]. Cells were washed and incubated for 15 min in 100 µl Annexin V labeling solution prepared in incubation buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) with 10 µl PI (50 µg/ml). The test volume was made up to 500 µl with incubation buffer, and the samples were analyzed within 1 h of preparation using a FACScalibur flow cytometer and Cellquest software (BD Pharmingen). Annexin V single-positive cells were considered apoptotic and Annexin V/PI, double-positive necrotic. For anti-CD3-induced AICD, apoptosis was expressed as % specific apoptosis [25 ]:

Induced apoptosis is defined as the apoptosis induced with anti-CD3 in the presence of rIL-2. Control apoptosis is the apoptosis measured in the presence of rIL-2 alone with no cross-linking antibody present.

Blockade of AICD
AICD was blocked with the anti-CD95L antibody NOK1 (BD Pharmingen; 10 µg/ml) [26 ], TNFR:Fc fusion protein (kindly provided by Immunex Corp., Seattle, WA) [27 ], the granzyme B inhibitor z-AAD-CMK (Calbiochem, Nottingham, UK) [28 29 30 ], or the caspase 8 inhibitor boc-AEVD-CHO (Bachem, Saffron Waldon, UK) [31 ]. In addition, EGTA, a calcium chelator classically used to inhibit the exocytosis of lytic granules from cytotoxic cells, was used to inhibit the exocytosis of lytic granules from the T-cell subsets [32 ]. Concanamycin A (CMA, Sigma Chemical Co.), an inhibitor of the proton pump that maintains acidity of endosomes and lytic granules [33 ], was used to prevent the processing of the inactive (70 kDa) precursor form of perforin to its active (60 kDa) form [34 , 35 ]. T cells were induced to undergo AICD and cultured in the presence or absence of the inhibitors. Where appropriate, the background levels of apoptosis were measured in the presence of isotype-matched control antibody or Me2SO. Additional controls included culture in the presence of rIL-2 alone and medium alone.

Mixed lymphocyte reaction (MLR)
CD4+ T cells (105) were co-cultured with 5 x 104 allogeneic antigen-presenting cells (APC) in a final volume of 200 µL in 96-well plates, as described by Becher and colleagues [36 ]. APC were prepared by negative selection, and CD4+ and CD8+ cells were removed using Dynabeads (Dynal). The remaining cells were irradiated at 5000 rads prior to co-culture with CD4+ T cells. T cells and allogeneic APC were co-cultured for 5 days, and the culture supernatant was harvested for detection of IFN-{gamma} by specific cytokine enzyme-linked immunosorbent assay (ELISA).

Flow cytometric analysis
For the analysis of cell-surface expression of CD95L and TNF-{alpha}, cells were restimulated in the presence of matrix metalloprotease inhibitor KB8301 (BD Pharmingen) at 20 µM for 2 h. Cells (5x105) were then washed and stained with anti-CD95L (Alexis, Nottingham, UK) or anti-TNF-{alpha} mAb11 (BD Pharmingen) for 20 min at 4°C in 100 µl wash buffer (PBS, 2% FCS). Cells were analyzed using a FACScalibur flow cytometer and Cellquest software (BD Pharmingen).

For analysis of cell-surface expression of CD95, cells were restimulated and stained with anti-CD95 antibody DX2 (BD Pharmingen) or the appropriate isotype-control antibody. For the analysis of cell-surface expression of TNFR1 and TNFR2, cells were restimulated, and samples were taken for staining at intervals until 24 h post-stimulation. Cells (5x105) were stained with anti-TNFR1 (mouse IgG1; anti-human TNFR p60, Genzyme, Cambridge, MA) or anti-TNFR2 (rat IgG2b; anti-human TNFR p80, Genzyme) for 20 min at 4°C in 100 µl wash buffer. Cells were washed and incubated with the appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibody using anti-mouse Ig or anti-rat Ig (BD Pharmingen).

For intracellular cytokine analysis, T-cell cultures were restimulated with immobilized anti-CD3 and anti-CD28 for 6 h in the presence of Brefeldin A (Sigma Chemical Co.; 5 µg/ml). Cells were harvested, washed, and fixed for 20 min in 4% formaldehyde. After an additional wash (200 g, 10 min) and resuspension in permeablization buffer [PBS, 1% bovine serum albumin (BSA), 0.5% saponin; Sigma Chemical Co.], cells were incubated at room temperature for 20 min. After washing, all subsets were incubated with anti-IL-4 PE (BD Pharmingen) or IgG2a (isotype control) and anti-IFN-{gamma} FITC (BD Pharmingen) or IgG2b (isotype control) for 30 min at 4°C. Cells were washed twice before analysis.

Cytokine ELISA
TNF-{alpha} and IFN-{gamma} were measured using commercially available antibody pairs, a coating antibody anti-TNF-{alpha}, mAb1 (5 µg/ml), and the biotinylated anti-TNF-{alpha}, mAb11 (1/100 dilution), both from BD Pharmingen. For the IFN-{gamma} ELISA, the coating antibody anti-IFN-{gamma}, NIB42 (2 µg/ml), and the biotinylated anti-IFN-{gamma}, 4S.B3 (1 µg/ml), both from BD Pharmingen, were used. The ELISA was performed according to the manufacturer’s instructions using the substrate p-nitrophenyl phosphate (Sigma Chemical Co.). The ELISA was read at 405 nm using an Emax microplate reader, and data were analyzed using Softmax software (Molecular Dynamics, Sunnyvale CA).

Western blotting
Cell lysates were prepared by resuspending 2 x 106 cells in 40 µl hot lysis buffer [2% sodium dodecyl sulfate (SDS), 62.5 mM Tris, pH 6.8, 10% glycerol, 770 mM 2-mercaptoethanol]. The lysate was boiled for 2 min and then passed through a fine gauge needle to disrupt DNA. The cell lysates were resolved on SDS-polyacrylamide gel electrophoresis (PAGE) gels (10%) and blotted onto Hybond nitrocellulose membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) using a Trans-blot SD, semi-dry transfer cell (Biorad, Hertfordshire, UK). Proteins were detected with anti-CD95L (Transduction Laboratories, Lexington, KY) followed by goat anti-mouse Ig coupled to horseradish peroxidase and detected using the ECL Western blot detection system (Amersham Pharmacia Biotech).

Statistical analysis
Data were analyzed using Student’s t-test with P values <0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T-cell purity
T-cell purity was determined by flow cytometry. CD4+ T cells were found to be >99% CD3+CD4+. CD8+ T cells were found to be >95% CD3+CD8+ with contaminants consisting of 2% CD4+ cells, <=2.7% CD8+CD56+, 0.9% CD19+, and 0.5% CD14+. Positive selection of T cells with Dynabeads did not induce expression of the early activation marker CD69 on CD4+ cells (0.5% CD69+) or CD8+ cells (2% CD69+). On day 6 of culture, Th1 and Th2 cells remained >99% CD3+CD4+, and Tc1 and Tc2 cells were >97% CD3+CD8+ with contaminants consisting of 2% CD4+ cells, 1% CD56+ cells, and <1% CD19+ or CD14+ cells.

In vitro generation of CD4+ and CD8+ T cells secreting type 1 or type 2 cytokines
Th1 and Th2 or Tc1 and Tc2 primary effector T cells were generated from human peripheral blood by stimulating CD4+ and CD8+ T cells, respectively. T cells were stimulated with immobilized anti-CD3 and anti-CD28 in the presence of recombinant IL-12 or neutralizing anti-IL-12 plus recombinant IL-4 to generate type 1 or type 2 phenotypes, respectively. After 6 days of culture, polarization of the populations to type 1 or type 2 phenotype was assessed by intra-cytoplasmic staining for IFN-{gamma} and IL-4 (Fig. 1 ). Small amounts of IFN-{gamma} were produced by CD4+ and CD8+ cells cultured in type 2 conditions (to a maximum of 2%); this has been shown previously in CD8+ cells [37 , 38 ]. In addition, type 1 cells produced very little IL-4. There were very few cells producing IL-4 and IFN-{gamma}, which suggests only minor contamination from Th0 or Tc0 cells.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 1. CD4+ and CD8+ T cells secreting type 1 or type 2 cytokines can be generated in vitro. CD4+ and CD8+ T cells were purified from human peripheral blood and activated with immobilized anti-CD3 and anti-CD28 in the presence of recombinant IL-12 or neutralizing anti-IL-12 plus recombinant IL-4 to generate type 1 or type 2 cells, respectively. After 6 days of culture, the cytokine-secreting phenotype of the cells was determined by flow cytometric analysis. Cells were stained for IFN-{gamma} (x-axis) and IL-4 (y-axis) or the appropriate isotype controls to set quadrant markers. The dot plot shows representative data from a single individual. Similar data were obtained in six further experiments.

 
Differential susceptibility to anti-CD3- initiated AICD
T cells from each of the four subsets were induced to undergo AICD in the presence of anti-CD3 and recombinant IL-2 (100 U/ml) with AICD measured at intervals (Fig. 2A ). The time course showed that AICD was initiated rapidly after the subsets were reactivated. Apoptosis reached a maximum between 6 and 8 h postinduction, which was also when differences between subsets were greatest. After 8 h post-induction, the percentage of apoptotic cells declined (unpublished results). Type 1 cells were more susceptible to AICD than their type 2 counterparts, with Tc1 cells being more susceptible to AICD than Th1 cells. We observed that apoptosis in type 1 cells was significantly different from that seen in type 2 cells for CD4+ and CD8+ cells (Fig. 2B) . Furthermore, this AICD was not dependent on cell-to-cell contact, because the addition of an anti-CD18 antibody (1B4 [39 ]) did not inhibit AICD (unpublished results).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Type 2 effector cells are resistant to AICD. T-cell subsets were induced to undergo AICD by stimulation with immobilized anti-CD3 in the presence of rIL-2 (100 U/ml) or IL-2 alone. (A) AICD of Th1 ({blacklozenge}), Th2 ({blacksquare}), Tc1 ({blacktriangleup}), and Tc2 (x) subsets was measured by Annexin V/PI staining at timed intervals following restimulation and expressed as percentage specific apoptosis. The graph is representative of four independent experiments. (B) AICD of T-cell subsets at 6 h post-stimulation showing pooled data from 16 donors. Bars indicate mean ± SE. Data were analyzed using Student’s t-test, with ** representing P < 0.006 and * representing P < 0.03.

 
Role of CD95-CD95L-mediated apoptosis in AICD of CD4+ and CD8+ T-cell subsets
Cell-surface expression of CD95 and CD95L was similar for all subsets. The CD95 receptor was present on all subsets prior to the induction of AICD (Fig. 3A ). CD95L was up-regulated to similar levels on the surface of all subsets following activation in the presence of a matrix metalloprotease inhibitor (Fig. 3B) . The matrix metalloprotease inhibitor prevents cleavage of CD95L from the cell surface [26 ]. In addition, expression of CD95L protein (37 kD) was measured by Western blot and found to be similar in all subsets, prior to and following reactivation (Fig. 3C) . An additional smaller molecular-weight band was visible below the transmembrane band and was believed to be a result of nonspecific binding of the antibody. This nonspecific band had similar intensity in all wells, confirming equal loading of samples on the gel.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 3. T-cell subsets express equal levels of CD95 and CD95L and are susceptible to CD95-induced apoptosis. (A) CD95 expression was measured on all subsets by flow cytometry using an anti-CD95 antibody (DX2, solid histogram) compared with an isotype control (open histogram). (B) CD95 ligand (solid histogram) is up-regulated on all subsets following activation with anti-CD3 and IL-2 in the presence of a matrix metalloprotease inhibitor (KB8301) compared with isotype control (open histogram). The percentages refer to the number of CD95- or CD95L-positive cells in the solid histograms. (C) Cellular extracts of un-activated (un) and anti-CD3 re-activated (re) T cells were resolved on a 10% SDS-PAGE gel, and CD95L protein was detected by Western blot analysis using an anti-CD95L antibody. The lower molecular weight band is nonspecific and is shown to verify that the total protein concentration per well was similar. (D) A time course of apoptosis induced by direct ligation of CD95 with an anti-CD95 antibody CH11 (1 µg/ml). The results show representative data from one of three experiments. Apoptosis was measured in Th1 ({blacklozenge}), Th2 ({blacksquare}), Tc1 ({blacktriangleup}), and Tc2 cells (x) by Annexin V staining. Differences between subsets were not statistically significant.

 
A dose response of anti-CD95-induced apoptosis in T-cell subsets revealed that maximal apoptosis occurred at 1 µg/ml CH11 (unpublished results). At this concentration of antibody, type 1 and type 2 subsets from CD4+ and CD8+ cells were susceptible to anti-CD95 ligation. All subsets showed similar rates of induction and decline in apoptosis with peak levels of apoptosis occurring at 4 h post-induction (Fig. 3D) . Although not statistically significant, differences in susceptibility were observed among subsets, with Th1 being more susceptible than Th2 cells and Tc1 being more susceptible than Tc2 cells (Fig. 3D) .

To further assess the involvement of the CD95/CD95L pathway in apoptosis, AICD was initiated in each of the four subsets from five different donors, in the presence or absence of the anti-CD95L antibody NOK1. This antibody is shown to block CD95/CD95L interaction [26 ]. AICD was partially but significantly inhibited in Th1 and Tc1 subsets (P<0.02 and P<0.05, respectively) but not in Th2 or Tc2 subsets (Fig. 4 ). Similar results were obtained using a Fas:Fc fusion protein (BD Pharmingen) at 50 µg/ml (unpublished results).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Blockade of CD95/CD95L-dependent AICD of Th1, Th2, Tc1, and Tc2 cells with an anti-CD95L antibody NOK1. AICD was induced with immobilized anti-CD3 in the presence of rIL-2 (100 U/ml). The T cells were incubated in the presence (0.1–10 µg/ml) or absence of anti-CD95L (NOK1). Apoptosis was measured by Annexin V staining 6 h after reactivation. AICD was expressed as percentage specific apoptosis. The data show the percentage of specific apoptosis of cells from five individuals; each donor is represented by the same symbol in each graph. Data were analyzed using a paired Student’s t-test between the lowest (0 µg/ml) and the highest (10 µg/ml) concentration of NOK 1 used for each subset. P values <0.05 were considered significant.

 
Role of TNF-{alpha}-mediated apoptosis in AICD
TNF-{alpha} was detected in culture supernatant from cells restimulated with anti-CD3 in the presence of rIL-2. It was observed that Th1 and Tc1 cells secreted more TNF-{alpha} than their type 2 counterparts (Fig. 5A ). This is consistent with studies of differential TNF-{alpha} secretion in type 1 and 2 murine T cells [2 , 40 , 41 ]. When restimulated cells were cultured in the presence of matrix metalloprotease inhibitors, TNF-{alpha} was found to be expressed on the surface of all T-cell subsets, but a greater number of cells in the type 1 populations expressed the membrane-bound form of the cytokine (unpublished results).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 5. Evaluation of TNF-{alpha} secretion and TNFR expression on T-cell subsets. (A) TNF-{alpha} production by the T-cell subsets Th1 ({blacklozenge}), Th2 ({blacksquare}), Tc1 ({blacktriangleup}), and Tc2 (x) was evaluated by specific cytokine ELISA of culture supernatants collected at timed intervals following restimulation with anti-CD3 and rIL-2. Data points show the mean of triplicate samples ± SD values. TNFR1 (B) and TNFR2 (C) expression was evaluated by flow cytometry. (B and C) Cells were sampled at timed intervals, and receptor expression was determined by staining cells with primary antibody, anti-TNFR1 (p60), anti-TNFR2 (p80), or the appropriate isotype control. The cells were then incubated with the appropriate FITC-conjugated secondary antibody. Note that Th2 cells have the lowest expression of TNFR1 and the lowest secretion of TNF-{alpha}. Results are representative of two independent experiments.

 
The death domain possessing TNFR1 (p60) was up-regulated on Th1, Tc1, and Tc2 subsets following restimulation but not on Th2 cells (Fig. 5B) . The kinetics of TNFR1 up-regulation followed a similar pattern to the secretion of TNF-{alpha}. TNFR2 was not up-regulated on any of the subsets but remained at its preactivation expression levels (Fig. 5C) .

The effect of TNFR:Fc on AICD was studied to test whether AICD was mediated by TNF/TNFR death pathways (Fig. 6A ). TNFR:Fc binds to soluble and membrane-bound TNF-{alpha} and prevents its interaction with TNFR1 or TNFR2 [27 ]. T-cell subsets were restimulated with immobilized anti-CD3 in the presence of rIL-2, and levels of AICD were determined at 8 h (Fig. 6A) , 24 and 48 h (unpublished results) postinduction. The addition of the TNFR:Fc did not inhibit AICD in any of the T-cell subsets tested at any of the time points investigated. Addition of recombinant TNF-{alpha} in soluble or immobilized form did not induce apoptosis in any of the T-cell subsets (unpublished results).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. TNF:Fc fusion protein fails to block AICD of human T-cell subsets. (A) AICD of Th1 ({blacklozenge}), Th2 ({blacksquare}), Tc1 ({blacktriangleup}), and Tc2 (x) cells was induced by stimulation with immobilized anti-CD3 (BD Pharmingen) in the presence of rIL-2. Apoptosis was measured by Annexin V staining after 8-h restimulation in the presence of TNFR:Fc fusion protein. As a control, apoptosis in the presence of IL-2 was also determined. The values presented are the percentage of apoptotic cells, and the error bars represent ± SE. Results show representative data from one of three independent experiments. (B) Functionality of TNFR:Fc. TNFR:Fc fusion protein inhibits TNF-{alpha}-mediated IFN-{gamma} production in a five-day MLR in which CD4+ T cells were mixed with allogeneic APC and TNFR:Fc or an irrelevant human IgG. Supernatant from the MLR was analyzed by ELISA for IFN-{gamma}. Data presented here are mean values ± SE.

 
To test if the TNFR:Fc had functional activity, an MLR was performed in the presence of TNFR:Fc or an irrelevant human IgG control. It has been determined previously that TNFR:Fc inhibits TNF-{alpha}-dependent production of IFN-{gamma} in an allogeneic MLR [36 ]. Briefly, CD4+ T cells were co-cultured at a 2:1 ratio with allogeneic APC. TNFR:Fc or the irrelevant IgG control antibody was added to the reaction, and the cells were cultured for 5 days. IFN-{gamma} in the supernatant was measured by ELISA, and it was shown that IFN-{gamma} production was inhibited in a dose-dependent manner by the TNFR:Fc (Fig. 6B) . It was observed that IFN-{gamma} production was inhibited by 44.2%, confirming functionality of the TNFR:Fc fusion protein.

Chelation of extracellular calcium inhibits AICD
EGTA inhibition was used initially to investigate whether lytic granule-induced cell death played a role in AICD of T-cell subsets. A reduction in apoptosis was observed in CD4+ and CD8+ subsets in the presence of EGTA (5 mM). Higher levels of inhibition were observed in CD8+ T cells, but this effect was not statistically significant because of variability between donors (Fig. 7A ).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 7. AICD of T-cell subsets is inhibited by EGTA and Con A. (A) T-cell subsets were induced to undergo AICD by stimulation with anti-CD3 in the presence of IL-2 (100 U/ml), in the presence or absence of 5 mM EGTA. After 6 h, apoptosis was measured by Annexin V staining. Values are from independent repeats using five different donors with each individual represented by the same symbol in each graph. Data were analyzed using a paired Student’s t-test comparing AICD without EGTA and AICD with EGTA for each subset. P values <0.05 were considered to be significant. (B) T-cell subsets were pretreated with Con A (10 nM) for 16 h prior to the induction of AICD. T-cell subset apoptosis was measured by Annexin V staining 6 h after induction of AICD in untreated (solid bars) and pretreated (hatched bars) cells. The graph shows mean values from triplicate cultures ± SD. Results are representative of two independent experiments.

 
CMA inhibits AICD in CD8+ T-cell subsets
T cells were pretreated with CMA (10 nM) for 16 h before induction of AICD to determine if prevention of perforin processing in T-cell subsets inhibited AICD. A consistent reduction of AICD was observed in Tc1 (39%) and Tc2 (27%) subsets, but no significant reduction of AICD was observed in CD4+ subsets, Th1 and Th2 (Fig. 7B) .

Effect of the granzyme B inhibitor z-AAD-CMK on AICD
When primary effector cells were induced to undergo AICD in the presence of the specific granzyme B inhibitor z-AAD-CMK, AICD of Th1, Th2, and Tc2 cells was unaffected. However, statistically significant inhibition (P<0.05) of AICD was observed in the Tc1 subset (45.4%±12.6%) at 10 µM (Fig. 8 ) This indicated that granzyme B, a component of the lytic-granule mechanism, is involved in AICD of CD8+ T cells. Furthermore, the granzyme B inhibitor 2-AAD-CMK does not inhibit CD95-medrated apoptosis whereas boc-AEVD-CHO, a caspase 8 inhibitor, does (Fig. 8C) .



View larger version (13K):
[in this window]
[in a new window]
  		Figure 8. Granzyme B contributes to AICD of CD8+ but not CD4+ T cells. (A) Inhibition of AICD by the granzyme B-specific inhibitor z-AAD-CMK. AICD was induced in Th1 ({blacklozenge}), Th2 ({blacksquare}), Tc1 ({blacktriangleup}), and Tc2 (x) cells in the absence or presence of z-AAD-CMK (0.001–10 µM). Apoptosis was assessed by Annexin V staining 6 h after induction of AICD. Results are representative of three independent experiments. (B) AICD was induced in all subsets in the presence (hatched bars) or absence (solid bars) of the granzyme B inhibitor, z-AAD-CMK (10 µM). Control and test samples contained equal concentrations of Me2SO. Data from three different donors were analyzed by Student’s t-test with P < 0.05 considered significant. Bars represent mean ± SE. Results showed significant inhibition of granzyme B-mediated apoptosis in Tc1 cells (*, P<0.05). (C) Direct ligation of CD95 by the anti-CD95 antibody CH11 induces apoptosis in Th1 cells (solid bars). Background levels of apoptosis, in the presence of rIL-2 alone (200 µ/ml; hatched bars), remain unchanged by addition of the inhibitors. CD95-mediated apoptosis of Th1 cells is inhibited by the caspase-8 inhibitor boc-AEVD-CHO but is not inhibited by the granzyme B inhibitor z-AAD-CMK. Values presented are means ± SE from triplicate cultures and are representative of two experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Differential susceptibility to AICD has been observed in clones, hybridomas, and effector populations. The majority of studies with regard to the human system have used T-cell clones to interpret functional differences between T-cell subsets. It is well established that Th1 and Th2 T-cell clones stimulated with anti-CD3 show differential susceptibility to AICD. In such studies, Th2 cells were seen to be more resistant to death than Th1 cells. In most cases, resistance to death correlated with a reduced expression of CD95 or CD95L [8 , 42 , 43 ], but this has not always been the case [44 ]. Although the AICD of Th1 and Th2 primary effectors derived from transgenic mice has been investigated [45 ], the susceptibility of in vitro-generated human primary effector cells has not yet been demonstrated. Work with human Tc2 clones [21 ] and cultured PBMC from individuals with disorders associated with Tc2 cells [6 ] suggest Tc2 cells are more resistant to AICD than their Tc1 counterparts, and this has been correlated with high Bcl-2 expression [6 ].

In our study, polarized subsets were generated after only a single polyclonal stimulus in the presence of polarizing cytokines, not by multiple stimulations as is the case for T-cell clones. Evidence suggests that the number of cytokine-producing cells depends on proliferation and cell-cycle progression. A strong relationship has been found between cell division and cytokine gene expression. Frequency of IFN-{gamma} production was found to increase with each subsequent cell division, and IL-4 expression required a threshold of three cell divisions [46 ]. These authors suggest that during the cell cycle, there is the opportunity for the "derepression of epigenetically silenced cytokine genes." In other words, cytokine genes are reactivated, and their expression is stabilized during each subsequent passage through the cell cycle. Therefore, our system would be expected to have fewer cytokine-producing cells than a clonal model that has had multiple rounds of stimulation.

The polarization of T-cell subsets was promoted by IL-12 and IL-4, which acted as growth factors for type 1 and type 2 cells, respectively. Whether the polarizing cytokines IL-12 and IL-4 selected cells with predetermined phenotypes and promoted their growth, enriching the population with subsets already present, remains a contentious issue and has been recently reviewed by Reiner [47 ]. This type of polarization, termed "selection," whereby cells have already been programmed by the actions of transcription factors such as T-bet in Th1 cells and Gata-3 in Th2 cells into type 1 or type 2 cells. It is therefore possible that our effector populations could have been derived from memory or precursor cells. Alternatively, cytokines may instruct cells to take on a specific phenotype, termed "instruction." A large body of evidence supports a selective [48 49 50 51 ] rather than an instructive mechanism [46 ] for T-cell differentiation. However, it is still possible that cytokines can have both effects on T cells [47 , 52 , 53 ].

In our study, human primary effector CD8+ cells behaved in an analogous way to CD4+ cells. Type 1 CD8+ T cells were found to be more susceptible to AICD than their type 2 counterparts. Although it is well established that CD95/CD95L is involved in AICD of CD4+ and CD8+ cells, we have demonstrated a role for CD95/CD95L interaction in AICD of Th1 and Tc1 cells. However, susceptibility to death was independent of the level of CD95L expression, suggesting other regulatory mechanisms are involved in CD95/CD95L-dependent AICD. Equally, differences in AICD of subsets were not a result of expression of CD95, because this was expressed at equivalent levels. The signaling function of CD95 may play a role in differences in susceptibility, because after direct ligation of CD95 with anti-CD95, slight differences in apoptosis of T-cell subsets were observed. It is possible that differences in signaling pathways of polarized T-cell subsets affect apoptosis. TCR-induced signals are initiated on CD3 ligation and have been implicated in the regulation of apoptosis. For example, MEK 1 mitogen-activated protein (MAP) kinase and extracellular-regulated kinase (ERK) activity have been shown to be important in regulation of CD95-dependent AICD [54 , 55 ]. The p38 kinase is also required for protection against TNF-mediated apoptosis [56 ]. It has been demonstrated that calcium signaling of type 1 cells is stronger than type 2 cells for CD4+ clones [57 , 58 ] and CD8+ clonal populations [59 ]. Calcium signals are important in T-cell activation and induction of apoptosis and may further contribute to differential susceptibility to AICD.

Differential secretion of TNF{alpha} by type 1 T-cell subsets suggests a potential role of TNF-{alpha} in type 1 cell AICD. However, because TNFR:Fc fusion protein or anti-TNF-receptor antibodies failed to block AICD, and recombinant TNF-{alpha} did not induce apoptosis, we concluded that TNF-{alpha} did not play a significant role in AICD of primary effector cells generated in vitro. This may have been caused by a failure of primary effector cells to up-regulate TNFR2 on reactivation. Human primary effector cells show expression of TNFR1 on Th1, Tc1, and Tc2 cells and are similar to Jurkat cells in that they express TNFR1 but not TNFR2 [60 ] and are insensitive to TNF-induced apoptosis [61 ]. Our results confirmed the observation that TNFRI is the TNFR predominantly expressed on T cells [62 ]. A mechanism for TNFR1 and TNFR2 cooperation has been shown to be necessary to transduce a competent signal in PC60 cells [63 ]. TNFR2 has been shown to facilitate TNFR1-mediated apoptosis in T cells, thymocytes, HeLa transfectants, and T-cell hybridomas [64 65 66 ] by stimulating production of membrane-bound TNF-{alpha}. Also, "ligand passing" has been described, where TNFR2 cooperates with TNFR1 by passing bound TNF-{alpha} to TNFR1 [67 ]. Therefore, absence of TNFR2 may result in the lack of sensitivity to death via TNFR1 [68 ]. This is in contrast to studies with murine cells where TNF-{alpha} was shown to mediate AICD of CD8+ T cells via the p75 TNFR (TNFR2) [11 ]. These authors showed that CD95 alone accounted for almost all the AICD of CD4+ murine T cells and suggested that the CD95L mechanism is primarily used by CD8+ cells to kill target cells (including CD4+ cells), and the TNF-{alpha} pathway is used in auto-regulatory apoptosis (AICD) of the same CD8+ populations.

Because TNF-{alpha} was not shown to be involved in AICD of CD4+ or CD8+ T cells, an alternative mechanism was required to account for the considerable AICD not attributable to CD95/CD95L interactions. Perforin has been shown previously to have a role in AICD of murine T cells [13 14 15 ] and along with granzyme B, is a component of the lytic granules produced by cytolytic T lymphocytes. Like CD95L and TNF-{alpha}, granzyme B can also be expressed following T-cell stimulation via a diverse array of stimuli, e.g., anti-CD3, Con A, phorbol 12-myristate 13-acetate, and allogeneic stimulation [69 ]. In the same way that CD95L is preformed, granzyme B and perforin are stored in preformed lytic granules ready for release on activation of the effector cell. By using EGTA to chelate extracellular calcium and prevent lytic granule formation [32 ], we were able to show a partial but variable inhibition of AICD in CD4+ and CD8+ T cells. We then showed that components of lytic granules were involved in AICD with granzyme B, important in the AICD of Tc1 cells. It has been shown that a small, sustained Ca2+ influx is sufficient and required for CD95L up-regulation as well as CD95/CD95L killing [70 ]. There have been contrasting studies indicating that Ca2+ is also required for CD95L up-regulation but that CD95/CD95L killing is calcium-independent [71 72 73 ]. Because perforin/granzyme are predominantly produced by CD8+ T cells, inhibition of AICD in CD4+ T cells by EGTA suggests inhibition of other calcium-dependent mechanisms of AICD. Therefore, it is possible that the effects of EGTA on CD4+-cell AICD that we observed were a result of interference in CD95/CD95L-mediated apoptosis. This may also be true for calcium-dependent expression of other death ligands. Other mediators of apoptosis, e.g., caspases and cytochrome c release, are also calcium-dependent [74 ].

Another inhibitor of perforin is CMA, which is an inhibitor of the vascular type H+ ATPase, which acts as a proton pump to maintain acidification of endosomes and lytic granules [33 ]. This inhibitor was used to inhibit perforin-dependent AICD. Perforin is synthesized as an inactive precursor form that is cleaved to the active form only on entry into an acidic compartment. It is only the cleaved form that is stored in granules ready for secretion. Therefore, CMA is known to block selectively and completely the perforin pathway without affecting the CD95 pathway [34 ]. In the current study, we show that CMA inhibits AICD in CD8+ subsets, confirming a role for the granule exocytosis mechanism in AICD of CD8+ T cells.

Inhibition of cell-to-cell contact with an anti-CD18 antibody suggested that cell contact was not required for AICD. However, this does not preclude a role for granzyme in AICD, because although granzyme B has been shown to require perforin for entry into cells, a putative granzyme B receptor has also been described [75 76 77 ]. In addition, a role for granzyme in a cell-autonomous mechanism of apoptosis is possible, whereby death may be a result of internal trafficking of perforin after T-cell activation, which would initiate granzyme activity [14 , 78 ].

It is evident from the data presented here that CD4+ and CD8+ T cells share common mechanisms of AICD but that some events remain distinct between these subsets. The susceptibility of subsets to AICD may be linked to their function. Tc2 cells that are resistant to AICD may regulate their proliferation and clonal expansion by mechanisms other than apoptosis. They may kill their stimulating APC, thereby limiting their own stimulation, or reduce T-cell-growth factor secretion, thereby down-regulating the immune response [16 ]. T cells might exhibit reduced AICD because of differential expression of regulatory molecules such as Bcl-2 and Bcl-xL, but these have not been shown to be important in AICD of murine cells [79 , 80 ]. Some studies have indicated that the CD95-associated protease FAP-1 can block CD95-mediated cell signaling. This protects some cells from AICD, particularly Th2 cells where FAP-1 up-regulation corresponds to apoptosis resistance [45 , 81 , 82 ].

Our study demonstrates that human CD8+ T cells are highly susceptible to AICD and that their cytotoxic function contributes to auto-regulatory apoptosis, an important mechanism of self-tolerance. Tc2 cells, like their Th2 counterparts, are relatively resistant to AICD despite being susceptible to CD95-mediated death. In contrast to murine T cells, we show that human CD8+ cells do not use TNF-{alpha} as an inducer of AICD. The lack of such regulatory pathways in Tc2 cells may therefore contribute to the survival of cells producing type 2 cytokines. This may occur after chronic exposure to antigen in the lungs of asthmatics [83 ] or in immune disorders, for example in AIDS, hyper-IgE/hypereosiniphilia, and idiopathic hypereosinophilic syndrome [6 ], where type 2 cells predominate. Therefore, so that appropriate treatments for the control of immunopathology are developed, it is important that mechanisms involved in AICD of different T-cell subsets are elucidated.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from The King’s Medical Research Trust and the Biotechnology and Biological Sciences Research Council. We thank Dr. Elaine Thomas of Immunex Corp., Seattle, WA, for supplying TNFR:Fc fusion protein.

Received December 12, 2000; revised June 22, 2001; accepted June 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A., Coffman, R. L. (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins J. Immunol. 136,2348-2357[Abstract]
  2. Mosmann, T. R., Coffman, R. L. (1989) Heterogeneity of cytokine secretion patterns and functions of helper T cells Adv. Immunol. 46,111-147[Medline]
  3. Romagnani, S. (1994) Regulation of the development of type 2 T-helper cells in allergy Curr. Opin. Immunol. 6,838-846[Medline]
  4. Mosmann, T. R., Coffman, R. L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties Annu. Rev. Immunol. 7,145-173[Medline]
  5. Kelso, A., Troutt, A. B., Maraskovsky, E., Gough, N. M., Morris, L., Pech, M. H., Thomson, J. A. (1991) Heterogeneity in lymphokine profiles of CD4+ and CD8+ T cells and clones activated in vivo and in vitro Immunol. Rev. 123,85-114[Medline]
  6. Carbonari, M., Tedesco, T., Del Porto, P., Paganelli, R., Fiorilli, M. (2000) Human T cells with a type-2 cytokine profile are resistant to apoptosis induced by primary activation: consequences for immunopathogenesis Clin. Exp. Immunol. 120,454-462[Medline]
  7. Russell, J. H. (1995) Activation-induced death of mature T cells in the regulation of immune responses Curr. Opin. Immunol. 7,382-388[Medline]
  8. Ramsdell, F., Seaman, M. S., Miller, R. E., Picha, K. S., Kennedy, M. K., Lynch, D. H. (1994) Differential ability of Th1 and Th2 T cells to express Fas ligand and to undergo activation-induced cell death Int. Immunol. 6,1545-1553[Abstract/Free Full Text]
  9. Brunner, T., Mogil, R. J., LaFace, D., Yoo, N. J., Mahboubi, A., Echeverri, F., Martin, S. J., Force, W. R., Lynch, D. H., Ware, C. F. (1995) Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas Nature 373,441-444[Medline]
  10. Ju, S. T., Panka, D. J., Cui, H., Ettinger, R., el-Khatib, M., Sherr, D. H., Stanger, B. Z., Marshak-Rothstein, A. (1995) Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation Nature 373,444-448[Medline]
  11. Zheng, L., Fisher, G., Miller, R. E., Peschon, J., Lynch, D. H., Lenardo, M. J. (1995) Induction of apoptosis in mature T cells by tumour necrosis factor Nature 377,348-351[Medline]
  12. Sarin, A., Conan-Cibotti, M., Henkart, P. A. (1995) Cytotoxic effect of TNF and lymphotoxin on T lymphoblasts J. Immunol. 155,3716-3718[Abstract]
  13. Kagi, D., Vignaux, F., Ledermann, B., Burki, K., Depraetere, V., Nagata, S., Hengartner, H., Golstein, P. (1994) Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity Science 265,528-530[Abstract/Free Full Text]
  14. Spaner, D., Raju, K., Radvanyi, L., Lin, Y., Miller, R. G. (1998) A role for perforin in activation-induced cell death J. Immunol. 160,2655-2664[Abstract/Free Full Text]
  15. Spaner, D., Raju, K., Rabinovich, B., Miller, R. G. (1999) A role for perforin in activation-induced T cell death in vivo: increased expansion of allogeneic perforin-deficient T cells in SCID mice J. Immunol. 162,1192-1199[Abstract/Free Full Text]
  16. Mosmann, T. R., Li, L., Sad, S. (1997) Functions of CD8 T-cell subsets secreting different cytokine patterns Semin. Immunol. 9,87-92[Medline]
  17. Shresta, S., Pham, C. T., Thomas, D. A., Graubert, T. A., Ley, T. J. (1998) How do cytotoxic lymphocytes kill their targets? Curr. Opin. Immunol. 10,581-587[Medline]
  18. Walden, P. R., Eisen, H. N. (1990) Cognate peptides induce self-destruction of CD8+ cytolytic T lymphocytes Proc. Natl. Acad. Sci. USA 87,9015-9019[Abstract/Free Full Text]
  19. Su, M. W., Walden, P. R., Eisen, H. N., Golan, D. E. (1993) Cognate peptide-induced destruction of CD8+ cytotoxic T lymphocytes is due to fratricide J. Immunol. 151,658-667[Abstract]
  20. Atkinson, E. A., Barry, M., Darmon, A. J., Shostak, I., Turner, P. C., Moyer, R. W., Bleackley, R. C. (1998) Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B J. Biol. Chem. 273,21261-21266[Abstract/Free Full Text]
  21. Vukmanovic-Stejic, M., Vyas, B., Gorak-Stolinska, P., Noble, A., Kemeny, D. M. (2000) Human Tc1 and Tc2/Tc0 CD8 T-cell clones display distinct cell surface and functional phenotypes Blood 95,231-240[Abstract/Free Full Text]
  22. Birkhofer, A., Rehbock, J., Fricke, H. (1996) T lymphocytes from the normal human peritoneum contain high frequencies of Th2-type CD8+ T cells Eur. J. Immunol. 26,957-960[Medline]
  23. Friedl, P., Noble, P. B., Zanker, K. S. (1995) T lymphocyte locomotion in a three-dimensional collagen matrix. Expression and function of cell adhesion molecules J. Immunol. 154,4973-4985[Abstract]
  24. Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V J. Immunol. Methods 184,39-51[Medline]
  25. Watanabe, N., Arase, H., Kurasawa, K., Iwamoto, I., Kayagaki, N., Yagita, H., Okumura, K., Miyatake, S., Saito, T. (1997) Th1 and Th2 subsets equally undergo Fas-dependent and -independent activation-induced cell death Eur. J. Immunol. 27,1858-1864[Medline]
  26. Kayagaki, N., Kawasaki, A., Ebata, T., Ohmoto, H., Ikeda, S., Inoue, S. K., Okumura, K., Yagita, H. (1995) Metaloproteinase-mediated release of human Fas ligand J. Exp. Med. 182,1777-17783[Abstract/Free Full Text]
  27. Mohler, K. M., Torrance, D. S., Smith, C. A., Goodwin, R. G., Stremler, K. E., Fung, V. P., Madani, H., Widmer, M. B. (1993) Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists J. Immunol. 151,1548-1561[Abstract]
  28. Odake, S., Kam, C. M., Narasimhan, L., Poe, M., Blake, J. T., Krahenbuhl, O., Tschopp, J., Powers, J. C. (1991) Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins Biochemistry 30,2217-2227[Medline]
  29. Gong, B., Chen, Q., Endlich, B., Mazumder, S., Almasan, A. (1999) Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases Cell Growth Differ 10,491-502[Abstract/Free Full Text]
  30. Yamamoto, T., Yoneda, K., Ueta, E., Doi, S., Osaki, T. (2000) Enhanced apoptosis of squamous cell carcinoma cells by interleukin-2-activated cytotoxic lymphocytes combined with radiation and anticancer drugs Eur. J. Cancer 36,2007-2017
  31. Garcia-Calvo, M., Peterson, E. P., Leiting, B., Ruel, R., Nicholson, D. W., Thornberry, N. A. (1998) Inhibition of human caspases by peptide-based and macromolecular inhibitors J. Biol. Chem. 273,32608-32613[Abstract/Free Full Text]
  32. Henkart, P. A., Millard, P. J., Reynolds, C. W., Henkart, M. P. (1984) Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granular lymphocyte tumors J. Exp. Med. 160,75-93[Abstract/Free Full Text]
  33. Drose, S., Bindseil, K. U., Bowman, E. J., Siebers, A., Zeeck, A., Altendorf, K. (1993) Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases Biochemistry 32,3902-2906[Medline]
  34. Kataoka, T., Yamada, A., Suzuki, H., Hamada, S., Magae, J., Nagai, K. (1996) Identification of low molecular weight probes on perforin- and Fas-based killing mediated by cytotoxic T lymphocytes Biosci. Biotechnol. Biochem. 60,1726-1728[Medline]
  35. Uellner, R., Zvelebil, M. J., Hopkins, J., Jones, J., MacDougall, L. K., Morgan, B. P., Podack, E., Waterfield, M. D., Griffiths, G. M. (1997) Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain EMBO J 16,7287-7296[Medline]
  36. Becher, B., Blain, M., Giacomini, P. S., Antel, J. P. (1999) Inhibition of Th1 polarization by soluble TNF receptor is dependent on antigen-presenting cell-derived IL-12 J. Immunol. 162,684-688[Abstract/Free Full Text]
  37. Fowler, D. H., Breglio, J., Nagel, G., Hirose, C., Gress, R. E. (1996) Allospecific CD4+, Th1/Th2 and CD8+, Tc1/Tc2 populations in murine GVL: type I cells generate GVL and type II cells abrogate GVL Biol. Blood Marrow Transplant. 2,118-125[Medline]
  38. Cerwenka, A., Carter, L. L., Reome, J. B., Swain, S. L., Dutton, R. W. (1998) In vivo persistence of CD8 polarized T cell subsets producing type 1 or type 2 cytokines J. Immunol. 161,97-105[Abstract/Free Full Text]
  39. McMichael, A. J. (1987) Leucocyte Typing III White Cell Differentiation Antigens Oxford University Press Oxford, New York, Tokyo.
  40. Conboy, I. M., DeKruyff, R. H., Tate, K. M., Cao, Z. A., Moore, T. A., Umetsu, D. T., Jones, P. P. (1997) Novel genetic regulation of T helper 1 (Th1)/Th2 cytokine production and encephalitogenicity in inbred mouse strains J. Exp. Med. 185,439-451[Abstract/Free Full Text]
  41. Li, L., Sad, S., Kagi, D., Mosmann, T. R. (1997) CD8Tc1 and Tc2 cells secrete distinct cytokine patterns in vitro and in vivo but induce similar inflammatory reactions J. Immunol. 158,4152-4161[Abstract]
  42. Oberg, H. H., Lengl-Janssen, B., Kabelitz, D., Janssen, O. (1997) Activation-induced T cell death: resistance or susceptibility correlate with cell surface fas ligand expression and T helper phenotype Cell Immunol 181,93-100[Medline]
  43. Accornero, P., Radrizzani, M., Delia, D., Gerosa, F., Kurrle, R., Colombo, M. P. (1997) Differential susceptibility to HIV-GP120-sensitized apoptosis in CD4+ T-cell clones with different T-helper phenotypes: role of CD95/CD95L interactions Blood 89,558-569[Abstract/Free Full Text]
  44. Varadhachary, A. S., Perdow, S. N., Hu, C., Ramanarayanan, M., Salgame, P. (1997) Differential ability of T cell subsets to undergo activation-induced cell death Proc. Natl. Acad. Sci. USA 94,5778-5783[Abstract/Free Full Text]
  45. Zhang, X., Brunner, T., Carter, L., Dutton, R. W., Rogers, P., Bradley, L., Sato, T., Reed, J. C., Green, D., Swain, S. L. (1997) Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis J. Exp. Med. 185,1837-1849[Abstract/Free Full Text]
  46. Bird, J. J., Brown, D. R., Mullen, A. C., Moskowitz, N. H., Mahowald, M. A., Sider, J. R., Gajewski, T. F., Wang, C. R., Reiner, S. L. (1998) Helper T cell differentiation is controlled by the cell cycle Immunity 9,229-237[Medline]
  47. Reiner, S. L. (2001) Helper T cell differentiation, inside and out Curr. Opin. Immunol. 13,351-355[Medline]
  48. Ouyang, W., Ranganath, S. H., Weindel, K., Bhattacharya, D., Murphy, T. L., Sha, W. C., Murphy, K. M. (1998) Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism Immunity 9,745-755[Medline]
  49. Finkelman, F. D., Morris, S. C., Orekhova, T., Mori, M., Donaldson, D., Reiner, S. L., Reilly, N. L., Schopf, L., Urban, J. F. (2000) Stat6 regulation of in vivo IL-4 responses J. Immunol. 164,2303-2310[Abstract/Free Full Text]
  50. Jankovic, D., Kullberg, M. C., Noben-Trauth, N., Caspar, P., Paul, W. E., Sher, A. (2000) Single cell analysis reveals that IL-4 receptor/Stat6 signaling is not required for the in vivo or in vitro development of CD4+ lymphocytes with a Th2 cytokine profile J. Immunol. 164,3047-3055[Abstract/Free Full Text]
  51. Noble, A., Thomas, M. J., Kemeny, D. M. (2001) Early Th1/Th2 cell polarization in the absence of IL-4 and IL-12: T cell receptor signaling regulates the response to cytokines in CD4 and CD8 T cells Eur. J. Immunol. 31,2227-2235[Medline]
  52. Coffman, R. L., Reiner, S. L. (1999) Instruction, selection, or tampering with the odds? Science 284,1283-1285[Free Full Text]
  53. Reiner, S. L., Seder, R. A. (1999) Dealing from the evolutionary pawnshop: how lymphocytes make decisions Immunity 11,1-10[Medline]
  54. van den Brink, M. R., Kapeller, R., Pratt, J. C., Chang, J. H., Burakoff, S. J. (1999) The extracellular signal-regulated kinase pathway is required for activation-induced cell death of T cells J. Biol. Chem. 274,11178-11185[Abstract/Free Full Text]
  55. Walker, L. S., McLeod, J. D., Boulougouris, G., Patel, Y. I., Ellwood, C. N., Hall, N. D., Sansom, D. M. (1999) Lack of activation induced cell death in human T blasts despite CD95L up-regulation: protection from apoptosis by MEK signalling Immunology 98,569-575[Medline]
  56. Roulston, A., Reinhard, C., Amiri, P., Williams, L. T. (1998) Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor alpha J. Biol. Chem. 273,10232-10239[Abstract/Free Full Text]
  57. Gajewski, T. F., Schell, S. R., Fitch, F. W. (1990) Evidence implicating utilization of different T cell receptor-associated signaling pathways by TH1 and TH2 clones J. Immunol. 144,4110-4120[Abstract]
  58. Sloan-Lancaster, J., Steinberg, T. H., Allen, P. M. (1997) Selective loss of the calcium ion signaling pathway in T cells maturing toward a T helper 2 phenotype J. Immunol. 159,1160-1168[Abstract]
  59. Noble, A., Truman, J. P., Vyas, B., Vukmanovic-Stejic, M., Hirst, W. J., Kemeny, D. M. (2000) The balance of protein kinase C and calcium signaling directs T cell subset development J. Immunol. 164,1807-1813[Abstract/Free Full Text]
  60. Ting, A. T., Pimentel-Muinos, F. X., Seed, B. (1996) RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis EMBO J 15,6189-6196[Medline]
  61. Pimentel-Muinos, F. X., Seed, B. (1999) Regulated commitment of TNF receptor signaling: a molecular switch for death or activation Immunity 11,783-793[Medline]
  62. Ware, C. F., Crowe, P. D., Vanarsdale, T. L., Andrews, J. L., Grayson, M. H., Jerzy, R., Smith, C. A., Goodwin, R. G. (1991) Tumor necrosis factor (TNF) receptor expression in T lymphocytes. Differential regulation of the type I TNF receptor during activation of resting and effector T cells J. Immunol. 147,4229-4238[Abstract]
  63. Vandenabeele, P., Declercq, W., Vanhaesebroeck, B., Grooten, J., Fiers, W. (1995) Both TNF receptors are required for TNF-mediated induction of apoptosis in PC60 cells J. Immunol. 154,2904-2913[Abstract]
  64. Declercq, W., Denecker, G., Fiers, W., Vandenabeele, P. (1998) Cooperation of both TNF receptors in inducing apoptosis: involvement of the TNF receptor-associated factor binding domain of the TNF receptor 75 J. Immunol. 161,390-399[Abstract/Free Full Text]
  65. Welsh, R. M., Lin, M. Y., Lohman, B. L., Varga, S. M., Zarozinski, C. C., Selin, L. K. (1997) Alpha beta and gamma delta T-cell networks and their roles in natural resistance to viral infections Immunol. Rev. 159,79-93[Medline]
  66. Grell, M. (1995) Tumor necrosis factor (TNF) receptors in cellular signaling of soluble and membrane-expressed TNF J. Inflamm. 47,8-17[Medline]
  67. Tartaglia, L. A., Pennica, D., Goeddel, D. V. (1993) Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor J. Biol. Chem. 268,18542-18548[Abstract/Free Full Text]
  68. Grell, M., Zimmermann, G., Gottfried, E., Chen, C. M., Grünwald, U., Huang, D. C., Wu Lee, Y. H., Dürkop, H., Engelmann, H., Scheurich, P., Wajant, H., Strasser, A. (1999) Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1 activation by endogenous membrane-anchored TNF EMBO J 18,3034-3043[Medline]
  69. Prendergast, J. A., Helgason, C. D., Bleackley, R. C. (1992) Quantitative polymerase chain reaction analysis of cytotoxic cell proteinase gene transcripts in T cells. Pattern of expression is dependent on the nature of the stimulus J. Biol. Chem. 267,5090-5095[Abstract/Free Full Text]
  70. Esser, M. T., Haverstick, D. M., Fuller, C. L., Gullo, C. A., Braciale, V. L. (1998) Ca2+ signaling modulates cytolytic T lymphocyte effector functions J. Exp. Med. 187,1057-1067[Abstract/Free Full Text]
  71. Anel, A., Buferne, M., Boyer, C., Schmitt-Verhulst, A. M., Golstein, P. (1994) T cell receptor-induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclosporin A Eur. J. Immunol. 24,2469-2476[Medline]
  72. Vignaux, F., Golstein, P. (1994) Fas-based lymphocyte-mediated cytotoxicity against syngeneic activated lymphocytes: a regulatory pathway Eur. J. Immunol. 24,923-927[Medline]
  73. Vignaux, F., Vivier, E., Malissen, B., Depraetere, V., Nagata, S., Golstein, P. (1995) TCR/CD3 coupling to Fas-based cytotoxicity J. Exp. Med. 181,781-786[Abstract/Free Full Text]
  74. Mariani, S. M., Krammer, P. H. (1998) Surface expression of TRAIL/Apo-2 ligand in activated mouse T and B cells Eur. J. Immunol. 28,1492-1498[Medline]
  75. Shi, L., Mai, S., Israels, S., Browne, K., Trapani, J. A., Greenberg, A. H. (1997) Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization J. Exp. Med. 185,855-866[Abstract/Free Full Text]
  76. Froelich, C. J., Orth, K., Turbov, J., Seth, P., Gottlieb, R., Babior, B., Shah, G. M., Bleackley, R. C., Dixit, V. M., Hanna, W. (1996) New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis J. Biol. Chem. 271,29073-29079[Abstract/Free Full Text]
  77. Trapani, J. A., Jans, P., Smyth, M. J., Froelich, C. J., Williams, E. A., Sutton, V. R., Jans, D. A. (1998) Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction Cell Death Differ 5,488-496[Medline]
  78. Pinkoski, M. J., Hobman, M., Heibein, J. A., Tomaselli, K., Li, F., Seth, P., Froelich, C. J., Bleackley, R. C. (1998) Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis Blood 92,1044-1054[Abstract/Free Full Text]
  79. Van Parijs, L., Ibraghimov, A., Abbas, A. K. (1996) The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance Immunity 4,321-328[Medline]
  80. Inaba, M., Kurasawa, K., Mamura, M., Kumano, K., Saito, Y., Iwamoto, I. (1999) Primed T cells are more resistant to Fas-mediated activation-induced cell death than naive T cells J. Immunol. 163,1315-1320[Abstract/Free Full Text]
  81. Sato, T., Irie, S., Kitada, S., Reed, J. C. (1995) FAP-1: a protein tyrosine phosphatase that associates with Fas Science 268,411-415[Abstract/Free Full Text]
  82. Janssen, E. M., van Oosterhout, A. J., van Rensen, A. J., van Eden, W., Nijkamp, F. P., Wauben, M. H. (2000) Modulation of Th2 responses by peptide analogues in a murine model of allergic asthma: amelioration or deterioration of the disease process depends on the Th1 or Th2 skewing characteristics of the therapeutic peptide J. Immunol. 164,580-588[Abstract/Free Full Text]
  83. Ying, S., Humbert, M., Bakans, J., Corrigan, C. J., Pfister, R., Menz, G., Larche, M., Robinson, D. S., Durham, S. R., Kay, A. B. (1997) Expression of IL-4 and IL-5 mRNA and protein prduct by CD4+ and CD8+ T cells, eoinophils, and mast cells in bronchial biopsies obtained from atopic and nonatopic (intrinsic) asthmatics J. Immunol. 158,3539-3544[Abstract]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Y. Zhang, G. Xu, L. Zhang, A. I. Roberts, and Y. Shi
Th17 Cells Undergo Fas-Mediated Activation-Induced Cell Death Independent of IFN-{gamma}
J. Immunol., July 1, 2008; 181(1): 190 - 196.
[Abstract] [Full Text] [PDF]