Suicide induced by cytolytic activity controls the differentiation of memory CD8+ T lymphocytes

Joseph T. Opferman1,4, Bertram T. Ober2,4, Ramya Narayanan4 and Philip G. Ashton-Rickardt1,4

1 Committee on Immunology,
2 Department of Pathology and
3 Ben May Institute for Cancer Research, University of Chicago, Chicago, IL 60637, USA
4 Gwen Knapp Center for Lupus and Immunology Research, Chicago, IL60637, USA

Correspondence to: P. G. Ashton-Rickardt, R414, Gwen Knapp Center for Lupus and Immunology Research, 924 East 57th Street, Chicago, IL 60637, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cytotoxic T lymphocytes (CTL) confer protection against intracellular pathogens, yet the mechanism by which some escape activation induced cell death (AICD) and give rise to long-lived memory cells is unclear. We studied the differentiation of transgenic TCR CD8+ cells into CTL and memory cells using a novel system that allowed us to control cytolytic activity. The perforin/granzyme granules used to lyse targets induced the apoptosis of CTL in a fratricide-independent manner. After adoptive transfer to antigen-free mice, the ability of CTL to give generate memory cells was determined. We found that the extent of cytolysis by a common pool of CTL controlled the differentiation into memory cells, which were only generated under conditions of minimal cytolytic activity. Thus, the differentiation of naive CD8+ cells into memory cells may not depend on the presence on a subset of committed CTL precursors, but rather is controlled by the extent of granule-mediated cytolysis.

Keywords: apoptosis, cytotoxic T lymphocytes, immunological memory


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The engagement of TCR on naive CD8+ cells by antigen peptide–class I MHC (pMHC) leads to the proliferation and differentiation into cytotoxic T lymphocytes (CTL) (1). Cytolysis is initiated by the formation of antigen-specific conjugates that result in the lysis of pMHC-presenting target cells by the action of apoptosis-inducing molecules stored in cytoplasmic granules such as perforin and granzymes (1,2). After the effector phase, a period of death ensues during which activated T cells undergo activation-induced cell death (AICD) (3). The third phase of the T cell response is characterized by the appearance of memory cells that persist for many years and have accelerated responses seen upon re-exposure to antigen. This is due to both an increase in the frequency of antigen-specific T cells and to qualitative changes that allow them to respond to antigen more effectively than naive cells (antigen hyperactivity) (4).

We recently showed that post-effector CD8+ cells are the precursors of memory cells (5). However, how some CTL escape AICD and differentiate into memory cells remains unclear. Recent evidence has suggested that the cytolytic effector molecule perforin may play a role in the homeostasis of CTL (68). These studies demonstrate that perforin-deficient mice and humans have an impaired ability to clear activated CD8+ cells. However, it remains unclear whether the failure to down-regulate CD8+ cell immune responses is due to impaired effector cell suicide or to a failure to clear antigen in the perforin-deficient hosts (7,9). Furthermore, these studies were unable to discern whether perforin induced the suicide of CTL or was required for fratricide by fellow CTL during the clearance of activated CD8+ cells (10). In the absence of any definitive marker, the presence of memory CD8+ cells must be assessed functionally. This therefore precludes the use of effector-function-deficient animals to study the role of effector molecules during cell fate decisions leading to the generation of memory.

In this paper we test the hypothesis that cytolytic activity induces the apoptosis of CTL and controls subsequent differentiation into memory cells. We analyzed the differentiation of naive CD8+ cells into CTL and memory cells by using transgenic mice carrying the male-specific, anti-H-Y TCR B6.2.16 (11). We demonstrate that contact-mediated cytolytic activity of B6.2.16 CTL induces their apoptosis through the activation of the cytoplasmic perforin/granzyme granules. We found no evidence that B6.2.16 CTL are sensitized for lysis by fellow CTL (fratricide) during target cell lysis. The progeny of cytotoxic T cells that performed the least target cytolysis gave rise to memory CD8+ cells after adoptive transfer to antigen-free mice, whereas maximal cytolytic activity prohibited the differentiation into memory cells. We conclude that CTL suicide, induced by granule-mediated lysis of targets, determines effector cell fate and subsequent memory cell development.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Recombinant H-2Db molecules
Inclusion bodies of human ß2-microglobulin or mouse H-2Db heavy chain were recovered from Escherichia coli as described previously (12). Correctly folded H-Y/H-2Db complexes [H-Y peptide sequence: Lys-Cys-Ser-Arg-Asn-Arg-Gln-Tyr-Leu (13)] were purified using a conformation-specific, anti-H-2Db mAb (B22-249R1) (14). All purified complexes were subjected to analytical size exclusion fast pressure liquid chromatography and showed a homogeneous peak (>95% purity) with the retention time of an ~45 kDa protein. The half-life of the H-Y/H-2Db complexes was 45 ± 9 h at 37°C.

Mice
CD8+ cells were purified from B6.2.16 TCR transgenic recombination activation gene-1 (RAG1)-deficient mice (129/SvxC57BL/6) (11) and F5 TCR transgenic RAG1-deficient mice (129/SvxC57BL/6) (15). Recipients for adoptive transfer experiments were female RAG1-deficient mice (C57BL/6) (16). All mice were maintained and bred under specific pathogen-free conditions.

Generation of anti-H-Y CTL
Lymphocytes from B6.2.16 RAG1 mice were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) as described before (5), and cultured (106 cells/ml, 2 ml/culture) with plate-bound H-Y/H-2Db (5 µg/ml) and recombinant human IL-2 (10 U/ml). To remove unbound soluble H-Y/H-2Db, the wells were washed 3 times with PBS after coating (1 h, 37°C). The number of cell divisions was `counted' after FACS analysis by measuring 50% decreases of green fluorescence and staining with anti-CD8–CyChrome (PharMingen, San Diego, CA). For cytolytic assays, Ficoll-purified cells were incubated with or without H-Y peptide (100 nM) and 51Cr-labeled RMA cells (H-2b, 5x103) in triplicate at effector (E) (B6.2.16 CTL) to target (T) ratios of 10:1. After 4 h at 37°C, the degree of cytolysis was determined and the percentage specific lysis calculated from the specific 51Cr release as follows: . The percentage specific lysis of the targets in the absence of H-Y peptide was never >3% and the level of spontaneous lysis never exceeded >6% of maximal lysis. CFSE-labeled, B6.2.16 CD8 cells were intracellularly stained with anti-perforin mAb (Kamiya Biomedical, Seattle, WA) (17) or isotype control (rat IgG2a; PharMingen) as described (5).

Analysis of B6.2.16 CTL apoptosis
Plate-bound H-Y/H-2Db-stimulated B6.2.16 CTL were cultured with CFSE-labeled target cells pulsed with or without H-Y antigen (100 nM) at a 1:1 E:T ratio. After 4 h, 106 cells were harvested and washed 2 times with Annexin V-binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 5 mM CaCl2). The pellet was resuspended in 100 µl of Annexin V Alexa568-labeling solution (Roche Molecular Biochemicals, Indianapolis, IN) and 10 µl of anti-CD8–allophycocyanin (APC), and incubated at room temperature for 20 min. Cells were washed then analyzed by FACS for percentage of Annexin V binding over background on CFSECD8+ effector cells. The staining procedure disrupts >98% of CTL–target cell conjugates, therefore Annexin V staining is measured on CTL alone.

B6.2.16 CTL were cultured at a 1:1 E:T ratio with target cells that were labeled with the red-fluorescent dye PKH26 (Sigma, St Louis, MO) according to the manufacturer's specifications. In brief, RMA target cells were washed in PBS then resuspended at 107 cells/ml in solution C. PKH26 dye was added to a final concentration of 2 µM, mixed and incubated at room temperature. After 5 min, the reaction was quenched with 3 volumes of FCS and the cells were washed an additional 3 times in RPMI/10% FCS medium. After 4 h, 106 cells were stained with anti-CD8–APC and then washed in ice-cold PBS. Cells were resuspended in 1 ml of 0.1 µM YOPRO-1 dye (Molecular Probes, Eugene, OR) dissolved in PBS and incubated on ice for 30 min. The percentage of YOPRO-1high cells was determined by flow cytometry on live gated PKH26CD8+ cells.

F5 CTL were generated by culturing splenocytes from F5 RAG1-transgenic mice on plate-bound IF68/H-2Db [IF68: Ala-Ser-Asn-Glu-Asn-Met-Asp-Ala-Met (18)] refolded as described and rIL-2 for 4 days. The F5 CTL lysed only IF68-pulsed target cells (23% specific lysis) and not H-Y-pulsed or unpulsed target cells (<4% specific lysis). F5 CTL were susceptible to cytolysis-induced apoptosis and displayed Annexin V binding when incubated with target cells pulsed with 100 nM IF68 (13% of F5 CTL were positive) but not with unpulsed targets (1% of F5 CTL were positive). B6.2.16 CTL were distinguished from F5 CTL in the mixed CTL assay, by staining for B6.2.16 TCR expression using a clonotypic mAb (T3.70–FITC) (19).

Conjugate experiments
RMA target cells were labeled with CFSE and cultured in cRPMI-10 for 3 days. Plate-bound H-Y/H-2Db-stimulated, B6.2.16 CTL were labeled with PKH26 and incubated at a 1:1 E:T ratio with CFSE-labeled RMA target cells that were pulsed with H-Y antigen peptide (100 nM) for 1 h and washed to eliminate free antigen. The cultures were plated in 96-well round-bottom plates at 37°C for 4 h. To visualize conjugates, cells were resuspended using a standard shear force of 20 aspirations in and out of a disposable tip of an automatic pipette set at 150 µl (20), and then directly analyzed by FACS.

Apoptosis inhibition studies
B6.2.16 CTL generated on plate-bound H-Y/H-2Db were cultured for 4 h with equal numbers of 100 nM H-Y pulsed, CFSE-labeled target cells with or without an inhibitor of caspase-8 (z-LETD-fmk, 50 µM; Enzyme Systems Products, Livermore, CA), a negative control a cathepsin B inhibitor (z-FA-fmk, 50 µM; Enzyme Systems Products) or an inhibitor of mitochondrial superoxide dismutase, manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP, 100 µM; A. G. Scientific, San Diego, CA). For Fas-blocking human recombinant Fas–Ig fusion protein (Alexis Biochemicals, San Diego, CA) was added (12.5 µg/ml). After 4 h of culture, cells were resuspended and stained with Annexin V.

To inhibit granular cytolysis, B6.2.16 CTL that were generated on plate-bound H-Y/H-2Db were cultured with or without 10 nM concanamycin A (CMA; Kamiya Biomedical Company) for 1 h prior to CTL assay. The CTL were then tested (1:1 E:T) for their ability to lyse CFSE-labeled targets (H-Y-pulsed or unpulsed) in the presence or absence of 10 nM CMA. After 4 h of killing, B6.2.16 CTL were harvested and stained with Annexin V.

Fratricide experiments
Fratricide experiments were performed according to a procedure described before (10). B6.2.16 CTL were labeled with 51Cr and cultured with RMA cells (unpulsed or pulsed with 100 nM H-Y peptide) at a ratio of 1:1 and 3:1 for 1 h at 37°C. Then fresh, unlabeled B6.2.16 CTL were added (final 10:1 and 30:1 E:T ratios of fresh effectors to primary effector-targets). For maximum lysis conditions H-Y peptide (100 nM) was added to half of the wells. After an additional 4 h of culture, the percentage of specific lysis of the B6.2.16 CTL targets was determined.

Quantitation of anti-H-Y memory cells
B6.2.16 CTL, stimulated on plate-bound H-Y/H-2Db, were incubated with various ratios of H-Y antigen-pulsed to unpulsed RMA target cells (antigen-pulsed:unpulsed 1:0, 1:1 and 0:1) in a 4 h CTL assay (5x104 effectors:5x103 targets). In parallel, B6.2.16 CTL were also cultured with the same ratios of 51Cr-labeled RMA targets to determine the amount of specific lysis. B6.2.16 CTL were positively sorted with anti-CD8 microbeads (Miltenyi Biotec, Auburn, CA) then adoptively transferred (106 live cells/mouse) by lateral tail vein injection to female RAG1-deficient recipients. Naive B6.2.16 CD8 cells were purified from the lymph nodes of B6.2.16 RAG1-deficient mice (>95% B6.2.16 CD8 cells) and adoptively transferred (106/mouse). After 10 or 21 days, the number of B6.2.16 CD8 cells in mixed spleen and lymph nodes cell suspensions from recipients was determined by staining with T3.70–FITC, anti-CD8–APC and anti-CD44–phyoerythrin (PE) mAb, and then analyzed by FACS.

Limiting dilutions of cells were performed to determine the number of anti-H-Y CTL precursors per mouse. B6.2.16 CD8 cells were quantitated and seeded in limiting dilutions with H-Y antigen (100 nM) and rIL-2 (10 U/ml), and then the CTL precursor frequency determined after 7 days as described (5).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of anti-H-Y CTL in the absence of cytolysis
To examine whether cytolysis determines the fate of CTL and memory cells, we generated CTL from RAG1-deficient transgenic mice that express an H-2Db-restricted TCR (B6.2.16) specific for a male antigen (H-Y) (11). Activation of B6.2.16 CD8 cells by H-Y-pulsed antigen-presenting cells gives rise to CTL (13). After adoptive transfer to antigen-free hosts, the progeny of B6.2.16 CTL persist as long-lived memory cells (5,13,21,22). In order to generate anti-H-Y CTL that had not participated in target cell lysis, we developed a novel system using plate-bound H-Y/H-2Db molecules to induce the differentiation of naive B6.2.16 CD8 cells into anti-H-Y CTL labeled with CFSE (Fig. 1Go).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1. Generation of B6.2.16 CTL on plate-bound antigen–MHC. (A) CFSE-labeled B6.2.16 CD8 cells were activated with plate-bound H-Y/H-2Db (5 µg/ml) or with soluble H-Y peptide (100 nM) for 4 days after which the division status of the cells was analyzed by FACS. The percentage of cells that had divided >5 times in vitro is noted beside the gate. (B) The ability of the cells to lyse 100 nM antigen-pulsed RMA targets (10:1 E:T) was determined. At various time points during culture an aliquot of cells was harvested and the median generation number (the median number of times that the cells had divided beyond the parental generation at each time point) was determined by FACS. (Inset) Intracellular perforin staining (open histogram) and isotype control (closed histogram) of CFSE-labeled, activated B6.2.16 CD8 cells from generation number 5 (arrow).

 
The number of cell divisions over 4 days was `counted' by measuring 50% dilutions of green fluorescence by flow cytometric analysis. The addition of soluble H-Y peptide or plate-bound H-Y/H-2Db induced the proliferation of B6.2.16 CD8 cells. On day 1 of culture few B6.2.16 CD8 cells had divided, by day 2 cells had divided 1 or 2 times, by day 3 the cells had divided 3–5 times and by 4 days the cells had divided 6–10 times (data not shown). After 4 days, activation of B6.2.16 CD8 cells by either soluble H-Y peptide (89%) or plate-bound H-Y/H-2Db (87%) gave rise to the same proportion of cells that had divided alt least 5 times (Fig. 1AGo). Thus plate-bound H-Y/H-2Db was equally effective as soluble HY peptide in driving B6.2.16 CD8 cell division.

As had been observed with soluble H-Y peptide (5), activation by plate-bound H-Y/H-2Db induced the proliferation and differentiation of B6.2.16 CD8 cells into anti-H-Y CTL (Fig. 1BGo). Measurement of the cytoplasmic, cytolytic protein perforin revealed that after 4 days every B6.2.16 CD8 cell, which had divided >5 times, had differentiated into an effector (Fig. 1BGo, inset). After 4 days, the low proportion (~10%) of B6.2.16 CD8 cells present that had not divided were unlikely to be CTL because differentiation of naive B6.2.16 CD8 cells into anti-H-Y CTL requires cell division (Fig. 1BGo) (5). This procedure allowed us to generate a pool of cytolytically competent CD8+ effectors that had not killed.

Contact-mediated target cell lysis mediates apoptosis
To determine if antigen recognition on targets by anti-H-Y CTL stimulated on plate-bound H-Y/H-2Db induces effector cell death, apoptosis was measured in two ways. The expression of phosphatidylserine on the outer membrane of effector cells was measured by staining with Annexin V (23) and chromosomal DNA condensation measured using the dye YOPRO-1 (24). To avoid the effects of cognate peptide binding to CTL, which could result in fratricidal killing (25), we used RMA target cells pulsed with antigen and washed extensively. To distinguish between target and effector cell apoptosis we labeled target cells with CFSE or PKH26 and stained for the expression CD8 to identify B6.2.16 CTL (Fig. 2A and CGo). Concomitant with the lysis of H-Y-pulsed targets, B6.2.16 CTL underwent apoptosis as evidenced by an increase in the number of Annexin V+ (Fig. 2BGo) and YOPRO-1+ (Fig. 2DGo) cells. A summary of more than five independent experiments is shown (Fig. 3A and BGo).



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 2. B6.2.16 CTL undergo apoptosis during cytolysis. CFSE-labeled target cells (A) unpulsed or (B) pulsed with H-Y antigen were cultured with B6.2.16 CTL activated on H-Y/H-2Db. After 4 h, the cells were stained with anti-CD8 mAb and the binding of Annexin V on B6.2.16 CTL (CD8+CFSE) was determined by FACS. The percentage of Annexin V+ cells is indicated. PKH26-labeled target cells (C) unpulsed or (D) pulsed with H-Y antigen were cultured with B6.2.16 CTL. After 4 h, the cells were stained with anti-CD8 mAb and YOPRO-1. The percentage of YOPRO-1high B6.2.16 CD8 cells (CD8+PKH26) was determined by FACS. The percentage of YOPRO-1+ cells is indicated.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. B6.2.16 CTL undergo contact-mediated apoptosis during cytolysis. (A) Induction of B6.2.16 CTL apoptosis during cytolysis as measured by Annexin V binding. The level of phosphotidylserine expression on the membranes of cells was detected by Annexin V binding as described in Fig. 2Go(A and B). The mean values are from five independent experiments ± SEM. (B) Induction of B6.2.16 CTL apoptosis during cytolysis as measured by YOPRO-1 binding. The degree on chromosomal DNA condensation was detected by YOPRO-1 staining as described in Fig. 2Go(C and D). The mean values are from five independent experiments ± SEM. (C) Flow cytometric measurement of the percentage CFSECD8+ Annexin V+ effector cells when both F5 and B6.2.16 CTL were incubated with CFSE-labeled targets (pulsed or unpulsed with H-Y antigen). Cell were then stained with T3.70 (anti-B6.2.16 TCR clonotypic) anti-CD8, and the binding of Annexin V on B6.2.16 CTL (B6.2.16 TCR+CD8+CFSE) and F5 CTL (B6.2.16 TCRCD8+CFSE) was determined by FACS.

 
To determine if direct contact with H-Y-pulsed targets was required to induce the apoptosis of B6.2.16 CTL, we performed a mixed CTL experiment in which plate-bound pMHC-stimulated B6.2.16 (anti-H-Y/H-2Db) and influenza-specific F5 (anti-IF68/H-2Db) (18) CTL were together cultured with CFSE-labeled targets. To distinguish between F5 and B6.2.16 CTL we stained for the expression of the B6.2.16 TCR using a clonotypic mAb (T3.70) and CD8. B6.2.16 CTL lysed H-Y-pulsed targets (22% specific lysis) and underwent apoptosis (Fig. 3CGo). However, F5 CTL (B6.2.16 TCRCD8+) did not lyse H-Y-pulsed targets (4% specific lysis) nor undergo apoptosis when cultured with H-Y-pulsed targets (Fig. 3CGo). Therefore, effector cell apoptosis was not due to CTL- or target-derived soluble factors, but rather was triggered by contact-mediated cytolysis (26,27).

Effector cell apoptosis requires active cytolytic granules
To examine whether B6.2.16 CTL apoptosis was caused by effector molecules that induce target lysis, we used an inhibitor of vacuolar type H+-ATPase, CMA. It has been previously shown that CMA inhibits lytic granule-mediated cytolysis without inhibiting the formation of CTL and target conjugates (28,29). Furthermore, CMA selectively blocks perforin/granzyme mediated cytotoxicity and does not effect Fas-mediated cytotoxicity (29). The formation of conjugates between B6.2.16 CTL and H-Y-pulsed targets was uneffected by 10 nM CMA (Fig. 4AGo). However, we found that CMA inhibited 76% of the lysis of H-Y-pulsed target cells. The specific lysis of H-Y-pulsed target cells without CMA was 33 ± 2% (n = 11 experiments) and with CMA was 8 ± 1% (n = 11 experiments). Furthermore, CMA substantially decreased B6.2.16 CTL apoptosis, thus demonstrating a role for perforin/granzyme in the induction of CTL apoptosis during cytolysis (Fig. 4BGo).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4. B6.2.16 CTL apoptosis is mediated by lytic granules. (A) Analysis of the effect of CMA on CTL–target cell conjugate formation. PKH26-labeled B6.2.16 CTL were incubated with equal numbers of CFSE-labeled RMA targets (100 nM H-Y peptide pulsed or unpulsed) in the presence or absence of 10 nM CMA, then the percentage of cells in conjugates (indicated by the conjugate gate) was determined by direct FACS. (B) Flow cytometric analysis of Annexin V binding to B6.2.16 CTL when incubated with or without 10 nM CMA and CFSE-labeled target cells [H-Y pulsed (100 nM) or unpulsed] during a 4-h CTL assay (1:1 E:T). Open bars represent B6.2.16 CTL (CFSECD8+) and filled bars represent targets (CFSE+CD8), and error bars are SEM of eight independent experiments. The level of Annexin V on H-Y-pulsed target cells in the absence of CMA is defined as 100% maximum Annexin V binding which was 14 ± 1% (n = 8 experiments) of total CTL.

 
Since CMA did not inhibit conjugate formation between B6.2.16 CTL and H-Y-pulsed targets (Fig. 4AGo) we conclude that the ligation of surface molecules such as Fas or TCR contributed little to B6.2.16 CTL apoptosis during antigen recognition on targets. The lack of Fas involvement during the apoptosis of B6.2.16 CTL in vitro is further implied by two other observations. First, a Fas–Ig fusion protein had no effect on B6.2.16 CTL apoptosis during cytolysis at a concentration (12.5 µg/ml) that has been shown to inhibit Fas-mediated apoptosis in other systems (30,31). In the presence of Fas–Ig B6.2.16 CTL were 10.3% Annexin V+ and in the absence were 10% Annexin V+. Second, inhibition of caspase-8 activity, which normally transduces signals from Fas leading to cell death, also had no effect on either target or B6.2.16 CTL apoptosis. B6.2.16 CTL were 6.7 ± 1.4% Annexin V+ with control inhibitor (z-FA-fmk, 50 µM) and 6.7 ± 1% Annexin V+ in the presence of a caspase-8 inhibitor (z-LETD-fmk) at a concentration (50 µM) that has been shown to inhibit Fas-mediated cell death (32).

Fratricide plays little role in effector cell apoptosis
Recently it has been shown that CD8+ cells can internalize and re-express surface pMHC complexes from antigen-presenting cells by TCR-mediated endocytosis (10). This process was shown to sensitize the CD8+ cells to peptide-specific lysis by neighboring CTL. We wanted to determine the contribution of this pathway of CTL fratricide during the B6.2.16 CTL apoptosis in our system.

Target cells were cultured with 51Cr-labeled B6.2.16 CTL that we generated on plate-bound H-Y/H-2Db. After 1 h of cytolysis, fresh, unlabeled B6.2.16 CTL were added for an additional 4 h of cytolysis. To determine the maximum extent of CTL fratricide, soluble H-Y peptide was added (100 nM) to half of the wells. Incubation with H-Y-pulsed targets did not sensitize B6.2.16 CTL to lysis by fresh B6.2.16 CTL over the level observed for effectors incubated with unpulsed targets (Fig. 5Go). However, B6.2.16 CTL were not innately resistant to fratricidal killing because the addition of exogenous H-Y peptide sensitized effectors to lysis by B6.2.16 CTL (Fig. 5Go). These data demonstrate that although the perforin/granzyme pathway is necessary for the induction of B6.2.16 CTL apoptosis during cytolysis, fratricide mediated by re-expression of target-derived antigen played no detectable role.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5. Sensitization of B6.2.16 CTL to fratricide. B6.2.16 CTL were labeled with 51Cr and then cultured with target cells (100 nM H-Y pulsed or unpulsed) for 1 h. Then fresh, unlabeled B6.2.16 CTL were added and to half of the wells soluble peptide also added (100 nM H-Y). After 4 h, the mean percentage of specific 51Cr release from labeled B6.2.16 CTL was determined. The values for effector cell-specific 51Cr release are ± SEM (error bars) from 18–24 separate wells.

 
Memory cells are derived from cytolytically inactive effectors
We wanted to determine if the development of CD8+ memory cells depends upon the presence of a subset of predetermined CTL precursors or is influenced by cytolytic behavior. B6.2.16 CTL stimulated on plate-bound H-Y/H-2Db were allowed to perform differing amounts of cytolysis by controlling the availability of H-Y-pulsed targets. The ability of B6.2.16 CTL to give rise to memory cells 10 or 21 days after adoptive transfer of live cells (106) to antigen-free mice was then determined. In parallel, the same number of live naive B6.2.16 CD8 cells (106) were adoptively transferred and their ability to give rise to anti-H-Y CTL after re-challenge was compared with the progeny of transferred effectors. Memory B6.2.16 CD8 cells are defined as those that retain expression of the CD44 activation marker and give rise to more anti-H-Y CTL after re-challenge than the same number of naive cells (antigen hyper-reactive) (5,13). The progeny of cytolytically active B6.2.16 CTL (specific lysis >10%) did not generate any more anti-H-Y CTL than adoptively transferred naive cells (Fig. 6Go). However, B6.2.16 CTL that had killed little (<10% specific lysis) gave rise to a greater number of anti-H-Y CTL than adoptively transferred naive cells after re-challenge.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6. CTL effector function controls memory cell development. Numbers of anti-H-Y CTL precursors (CTLp) were quantitated by limiting dilution analysis from female RAG1 mice adoptively transferred with B6.2.16 CTL (106 cells) that had performed various amounts of cytolysis or with naive B6.2.16 CD8 cells (N). Each filled diamond represents the number from an individual mouse and the horizontal line denotes the mean number of anti-H-Y CTLp per mouse. (A) After 10 days, only B6.2.16 CTL that performed <2% specific lysis gave more anti-H-Y CTLp (mean = 10.1x103, n = 7) than naive cells (mean = 4.6x103, n = 2) (0.1 > P > 0.05), whereas B6.2.16 CTL that generated >19% specific lysis (mean = 0.8x103, n = 14) gave fewer anti-H-Y CTLp than naive cells (P < 0.001). (B) After 21 days, B6.2.16 CTL that had performed <2% specific lysis (mean = 7.5x103, n = 4) gave more anti-H-Y CTLp than naive cells (mean = 2.9x103, n = 2) (P < 0.001). B6.2.16 CTL that had generated >19% specific lysis gave less anti-H-Y-CTL (mean = 0.4x103, n = 8) than naive cells (P < 0.001).

 
The activation status of cells derived from B6.2.16 CTL that had killed little (<10% specific lysis) was compared with adoptively transferred naive B6.2.16 CD8 cells. Transfer of naive B6.2.16 CD8 cells into RAG1-deficient recipients did not result in an increase in CD44 expression or cell size, indicating that they retained a naive phenotype. These findings are consistent with earlier reports which showed that the adoptive transfer of naive B6.2.16 CD8 cells to lymphopenic recipients did not lead to their expansion and subsequent activation of clonotypic T cells (21,22). The progeny of B6.2.16 CTL retained the expression of CD44 but reverted to smaller cell size (Table 1Go). Furthermore, the progeny of B6.2.16 CTL that had killed little but not naive B6.2.16 CD8 cells displayed the phenotype of antigen hyper-reactivity after adoptive transfer (Fig. 6Go). Taken together we conclude that memory B6.2.16 CD8 cells were generated from CTL that had killed little.


View this table:
[in this window]
[in a new window]
 
Table 1. Phenotype of B6.2.16 CD8 cells after adoptive transfer
 
The differences in anti-H-Y-CTL precursor frequency after antigen re-challenge may have been due to the presence of functionally impaired cells derived from cytolytically active B6.2.16 CTL. However, after adoptive transfer, the progeny of B6.2.16 CTL that had performed the least cytolysis gave rise to both the most B6.2.16 CD8 cells and anti-H-Y CTL precursors (Table 2Go).


View this table:
[in this window]
[in a new window]
 
Table 2. Numbers of B6.2.16 CD8 cells recovered from RAG-1-deficient recipient mice
 
It is possible that differential homing to sites other than lymph nodes and spleen may in part account for differences in cell recovery. However, during cytolysis a superoxide dismutase mimetic (MnTBAP, 100 µM) (31) inhibited 78% of B6.2.16 CTL apoptosis and gave rise to a 2.3-fold increase in B6.2.16 CD8 cells 21 days after adoptive transfer, thereby demonstrating a direct role for CTL apoptosis in determining cell recovery after adoptive transfer.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have shown previously that memory CD8+ cells develop from CTL (5). However, how a subset of CTL escapes cell death and generates memory cells is not known. In this paper, we examined the role of cytolytic behavior on the cell fate decision made by functionally competent CTL. We show that suicide of B6.2.16 CTL during cytolysis is induced by the action of effector molecules stored in lytic granules (Figs 2–4GoGoGo). Our findings demonstrate that the perforin/granzyme pathway plays a role in inducing CTL AICD.

We tested the role of suicide induced by cytolytic activity on CD8+ memory cell development by controlling access to pMHC on targets. After adoptive transfer to antigen-free mice we observed that memory B6.2.16 CD8 cells were only generated under conditions of low cytolytic activity when access to antigen was limited (Fig. 6Go and Table 2Go). This implies that the CTL that had killed little were less likely to undergo apoptosis and therefore more likely to be able to give rise to memory cells. The ratio of CTL to antigen-expressing targets may effect the probability of cytolytic activity and thus the extent of CTL apoptosis. While the exact permissible number of targets a CTL can still kill without dying is at present not known, our findings suggest that those CTL that give rise to memory cells are likely to have performed less cytolysis than those that undergo AICD.

We have shown that the action of effector molecules stored in granules induces CTL apoptosis. However, it has been reported that CTL appear to be more resistant to cytolysis than experimental targets in vitro (33,34). A possible mechanism for ensuring CTL survival during cytolysis may involve the action of endogenous serine-protease inhibitors (serpins) such as human PI-9 (35,36) and murine SPI6 (37) that have been identified in CD8+ cells. These serpins may protect CTL from misdirected granzymes, and thereby play a role in determining CTL cell fate and memory cell development. However, the mechanism by which perforin/granzyme reaches the cytoplasm of CTL to induce apoptosis is at present unclear.

It has been shown that the re-expression of target-derived pMHC induces the fratricidal killing of CTL (10). However, our data demonstrates that this fratricidal mechanism of CTL apoptosis does not play a role in inducing B6.2.16 CTL AICD during cytolysis (Fig. 4Go). It is conceivable that differences between the B6.2.16 and 2C TCR transgenic systems used in each set of experiments may explain the differing contributions of fratricide. For example, the affinity of the 2C TCR for QL9/H-2Ld may be significantly higher than the B6.2.16 TCR for H-Y/H-2Db allowing considerably fewer transferred pMHCs to be needed to induce 2C CD8+ cell apoptosis. Also there may be differences in the stability of the pMHCs in each system that allowed QL9/H-2Ld to be internalized and re-expressed while the H-Y/H-2Db may be degraded.

The role of cytolytic activity in CTL AICD implies that the generation of memory cells may be based upon the access to antigen over time. The binding of high-affinity pMHC by the TCR of naive CD8+ cells drives the proliferation and differentiation into CTL that express TCR specific for pMHC. Our findings would argue that access to pMHC would then control CTL fate. Those CTL that arrive at the site of infection early are confronted with high antigen levels on target cells, perform cytolysis and succumb to apoptosis. However, CTL that arrive at the site of infection later, when antigen levels have waned, will not perform cytolysis and differentiate into memory cells (38). This model provides an internal homeostatic mechanism by which the immune system can eliminate the majority of effector cells from the site of infection, while preserving a pool of CTL that may develop into protective memory cells.


    Acknowledgments
 
We thank Chyung-Ru Wang, Marcus Peter and Jim Miller for helpful comments on the manuscript, and Stan Nathenson for providing H-2Db-expressing E. coli. The work was supported by a grant from the NIH to P. G. A.-R. (AI45108).


    Abbreviations
 
AICD activation-induced cell death
APC allophycocyanin
CFSE carboxyfluorescein diacetate succinimidyl ester
CMA concanamycin A
CTL cytotoxic lymphocyte
CTLp CTL precursor
FSC forward light scatter
MnTBAP superoxide dismutase mimetic
pMHC peptide–MHC
RAG1 recombination activation gene-1
PE phycoerythrin
z-LETD-fmk caspase-8 inhibitor
z-FA-fmk control cathepsin B inhibitor.

    Notes
 
Transmitting editor: C. B. Thompson

Received 31 August 2000, accepted 11 December 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Zinkernagel, R. M. and Doherty, P. C. 1974. Restriction of in vitro T cell mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semi-allogeneic system. Nature 248:701.[ISI][Medline]
  2. Kagi, D., Ledermann, B., Burki, R. M., Zinkernagel, R. M. and Hengartner, H. 1996. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14:207.[ISI][Medline]
  3. Razvi, E. S. and Welsh, R. M. 1995. Apoptosis in viral infections. Adv. Virus. Res. 45:1.[ISI][Medline]
  4. Ahmed, R. and Gray, D. 1996. Immunological memory and protective immunity: understanding their relationship. Science 272:54.[Abstract]
  5. Opferman, J. T., Ober, B. T. and Ashton-Rickardt, P. G. 1999. Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science 283:1745.[Abstract/Free Full Text]
  6. Spaner, D., Raju, K., Radvanyi, L., Lin, Y. and Miller, R. G. 1998. A role for perforin in activation-induced cell death. J. Immunol. 160:2655.[Abstract/Free Full Text]
  7. Stepp, S. E., Dufourcq-Lagelouse, R., Le Deist, F., Bhawan, S., Certain, S., Mathew, P. A., Henter, J.-I., Bennett, M., Fischer, A., de Saint Basile, G. and Kumar, V. 1999. Perforin gene defects in familial hemophagocytic lymphohistocytosis. Science 286:1957.[Abstract/Free Full Text]
  8. Kagi, D., Odermatt, B. and Mak, T. W. 1999. Homeostatic regulation of CD8+ T cells by perforin. Eur. J. Immunol. 29:3262.[ISI][Medline]
  9. Kagi, D., Ledermann, B., Burki, K., Seiler, P., Odermatt, B., Olsen, K. J., Podack, E. R., Zinkernagel, R. M. and Hengartner, H. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31.[ISI][Medline]
  10. Huang, J.-F., Yang, Y., Sepulveda, H., Shi, W., Hwang, I., Peterson, P. A., Jackson, M. R., Sprent, J. and Cai, Z. 1999. TCR-mediated internalization of peptide–MHC complexes acquired by T cells. Science 286:952.[Abstract/Free Full Text]
  11. Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M. and von Boehmer, H. 1988. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature 333:742.[ISI][Medline]
  12. Young, A. C., Zhang, W., Sacchettini, J. C. and Nathenson, S. G. 1994. The three-dimensional structure of H-2Db at 2.4 Å resolution: implications for antigen-determinant selection. Cell 76:39.[ISI][Medline]
  13. Markiewicz, M. A., Girao, C., Opferman, J. T., Sun, J., Hu, Q., Agulnik, A. A., Bishop, C. E., Thompson, C. B. and Ashton-Rickardt, P. G. 1998. Long-term T cell memory requires the surface expression of self-peptide/major histocompatibility complex molecules. Proc. Natl Acad. Sci. USA 95:3065.[Abstract/Free Full Text]
  14. Townsend, A., Ohlen, C., Bastin, J., Ljunggren, H. G., Foster, L. and Kara, K. 1989. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 340:443.[ISI][Medline]
  15. Mamalaki, C., Norton, T., Tanaka, Y., Townsend, A. R., Simpson, E. and Kioussis, D. 1992. Thymic deletion and peripheral activation of class I major histocompatibility complex-restricted T cells by soluble peptide in T-cell receptor transgenic mice. Proc. Natl Acad. Sci. USA 89:11342.[Abstract]
  16. Mombaerts, P., Iacomini, J., Johnson, R. S., Herrup, K., Tonegawa, S. and Papaioannou, V. E. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68:869.[ISI][Medline]
  17. Kawasaki, A., Shinkai, Y., Kuwana, Y., Furuya, A., Iigo, Y., Hanai, N., Itoh, S., Yagita, H. and Okumura, K. 1990. Perforin, a pore forming protein detectable by monoclonal antibodies, is a functional marker for killer cells. Int. Immunol. 7:677.
  18. Townsend, A., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. and McMicheal, A. J. 1986. The epitopes of influenza nucleoprotien recognized by cytotoxic T cells can be defined with short synthetic peptides. Cell 44:959.[ISI][Medline]
  19. Teh, H. S., Kishi, H., Scott, B. and Von Boehmer, H. 1989. Deletion of autospecific T cells in T cell receptor (TCR) transgenic mice spares cells with normal TCR levels and low levels of CD8 molecules. J. Exp. Med. 169:795.[Abstract]
  20. Denizot, F., Wilson, A., Battye, F., Berke, G. and Shortman, K. 1986. Clonal expansion of T cells: a cytotoxic T-cell response in vivo that involves precursor cell proliferation. Proc. Natl Acad. Sci. USA 83:6089.[Abstract]
  21. Bruno, L., Kirberg, J. and von Boehmer, H. 1995. On the cellular basis of immunological T cell memory. Immunity 2:37.[ISI][Medline]
  22. Tanchot, C., Lemonnier, F. A., Perarnau, B., Freitas, A. A. and Rocha, B. 1997. Differential requirements for survival and proliferation of CD8 naive or memory T cells. Science 276:2057.[Abstract/Free Full Text]
  23. Vermes, I., Haanen, C., Steffens-Nakken, H. and 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.[ISI][Medline]
  24. Idziorek, T., Estaquier, J., DeBels, F. and Ameisen, J.-C. 1995. YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J. Immunol. Methods 185:249.[ISI][Medline]
  25. Walden, P. R. and Eisen, H. N. 1990. Cognate peptides induce self-destruction of CD8+ cytolytic T lymphocytes. Proc. Natl Acad. Sci. USA 87:9015.[Abstract]
  26. Green, L. M., Reade, J. L., Ware, C. F., Devlin, P. E., Laing, C. M. and Devlin, J. J. 1986. Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. II. A novel CTL-produced cytotoxin that is antigenically distinct from tumor necrosis factor and {alpha}-lymphotoxin. J. Immunol. 137:3488.[Abstract/Free Full Text]
  27. Schmid, D. S., Tite, J. P. and Rude, N. H. 1986. DNA fragmentation: manifestation of +target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant. Proc. Natl Acad. Sci. USA 83:1881.[Abstract]
  28. Kataoka, T., Takaku, T., Magae, J., Shinohara, N., Takayama, H., Kondo, S. and Nagai, K. 1994. Acidification is essential for maintaining the structure and function of lytic granules of CTL. J. Immunol. 153:3938.[Abstract/Free Full Text]
  29. Kataoka, T., Shinohara, N., Takayama, H., Takaku, K., Kondo, S., Yonehara, S. and Nagai, K. 1996. Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J. Immunol. 156:3678.[Abstract]
  30. Ju, S., Panka, D. J., Cui, H., Ettiniger, R., El-Khatib, M., Sherr, D. H., Stanger, B. and Marshak-Rothenstein, A. 1995. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature 373:444.[ISI][Medline]
  31. Hildeman, D. A., Mitchell, T., Teague, T. K., Henson, P., Day, B. J., Kappler, J. and Marrack, P. C. 1999. Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity 10:735.[ISI][Medline]
  32. Garcia-Calvo, M., Peterson, E. P., Leiting, B., Ruel, R., Nicholson, D. W. and Thornberry, N. A. 1998. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273:32608.[Abstract/Free Full Text]
  33. Kranz, D. M. and Eisen, H. H 1987. Resistance of cytotoxic T lymphocytes to lysis by a clone of cytotoxic T lymphocytes. Proc. Natl Acad. Sci. USA 84:3375.[Abstract]
  34. Zanovello, P., Cerundolo, V., Bronte, V., Giunta, M., Panozzo, M., Biasi, G. and Collavo, D. 1989. Resistance of lymphokine-activated T lymphocytes to cell-mediated cytotoxicity. Cell. Immunol. 122:450.[ISI][Medline]
  35. Sun, J., Bird, H., Sutton, V., McDonald, L., Coughlin, P. B., De Jong, T. A., Trapani, P. and Bird, I. 1996. A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier a is present in cytotoxic lymphocytes. J. Biol. Chem. 271:27802.[Abstract/Free Full Text]
  36. Bird, C. H., Sutton, V. R., Sun, J., Hirst, C. E., Novak, A., Kumar, S., Trapani, J. A. and Bird, P. I 1998. Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol. Cell. Biol. 18:6387.[Abstract/Free Full Text]
  37. Sun, J., Ooms, L., Bird, C. H., Sutton, S. R., Trapani, J. A. and Bird, P. I. 1997. A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9). J. Biol. Chem. 272:15434.[Abstract/Free Full Text]
  38. Sprent, J. 1994. T and B memory cells. Cell 76:312.