INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Antigenic stimulation through the T cell receptor (TCR) causes peripheral T cells to enter the cell cycle and produce interleukin 2 (IL-2) (Kabelitz et al., 1993) but can also induce deletion of T cells through apoptosis, termed "activation-induced cell death" (AICD) (Jones et al., 1990; Rocha and von Boehmer, 1991; Kabelitz et al., 1993). In mature peripheral T cells that have been stimulated to expand with IL-2, further antigenic stimulation through the TCR causes apoptosis. This has been termed "propriocidal regulation" (Lenardo, 1991) and has been postulated to be a feedback mechanism by which T cell responses are shut off at the end of a specific immune response. The molecular mechanism responsible for this apoptosis appears to be the induction of Fas ligand (FasL) and activation through the Fas receptor (belonging to the tumor necrosis factor [TNF] and TNF receptor families, respectively) (Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995; Zheng et al., 1995). Accordingly, lpr and gld mice, defective in Fas and FasL, respectively, have impaired deletion of mature peripheral T cells and suffer from autoimmune manifestations (Cohen and Eisenberg, 1991), as do humans with autoimmune lymphoproliferative syndrome (Drappa et al., 1996; Le Deist et al., 1996).

The observation that cycling is a prerequisite for AICD has led to the hypothesis that susceptibility to apoptosis induced by TCR ligation is controlled by the cell cycle, so that cells in different phases of the cell cycle are differentially susceptible to AICD. However, the numerous studies that have addressed this question have produced conflicting results. Although initial studies of T cell hybridomas suggested an involvement of the G1 phase of the cell cycle in AICD (Ashwell et al., 1987; Cotter et al., 1992), others described a preferential sensitivity of cells in the G2-M phase (Fotedar et al., 1995). Studies in cloned cells and normal or leukemic T cell lines using pharmacological inhibitors of the cell cycle found that cells in S phase are preferentially sensitive to AICD (Boehme and Lenardo, 1993; Zhu and Anasetti, 1995; Radvanyi et al., 1996), but whether progression through the cell cycle was necessary remained unclear (Boehme and Lenardo, 1993; Radvanyi et al., 1996). Most recently Lissy et al. (1998) used centrifugal elutriation as a noninvasive means of synchronizing cells to demonstrate that AICD in the leukemic T cell line Jurkat requires progression through the cell cycle and occurs from a late G1 checkpoint in a pRb-dependent manner. Because the interaction of FasL with the Fas receptor has been implicated in AICD, the direct relationship between FasL-induced apoptosis and the cell cycle has also been studied. Although in leukemic cells G1-S transition was required for susceptibility to Fas (Komada et al., 1995; Komada and Sakurai, 1997), in peripheral blood lymphocytes (PBLs) either no dependency on G1-S transition (Fournel et al., 1996) or S phase resistance (Dao et al., 1997) has been reported. Given these conflicting results, the relationship between the cell cycle and Fas-mediated AICD remains obscure. Furthermore, there are a number of caveats in the interpretation of these findings. First, many studies relied on T cell hybridomas or leukemic cell lines, in which control of the cell cycle is abnormal. Second, the variety of pharmacological reagents that were used to synchronize cells in different phases of the cell cycle, such as mimosine, aphidocolin, or nocodazole, possess cell cycle-specific toxic effects of their own (Kuwakado et al., 1993; Bumbasirevic et al., 1995; Jha et al., 1995; Ji et al., 1997). Finally, it is not clear how closely the combination of ionophores and phorbol esters, commonly used to induce activation or cycling of peripheral T cells, mimics the normal response to activation through the TCR.

We were interested in the possibility that susceptibility to AICD in nontransformed human T cells maintained continuously cycling ex vivo in IL-2 is controlled by the cell cycle. We further hypothesized that if an apoptotic response to TCR signaling is regulated by the cell cycle, other functional activities stimulated through the TCR would also be differentially controlled. Because such cells have the potential of eradicating tumors (Rosenberg et al., 1988) or preventing viral diseases (Walter et al., 1995) when adoptively transferred to patients, the cell cycle could potentially be manipulated to a more active or less vulnerable phase before transfer, in an attempt to increase their in vivo efficacy. To test the involvement of the cell cycle in the functional activation and death induced by TCR signaling, we studied a panel of human melanoma tumor-reactive CD8+ T cell lines, which had been grown out of melanoma tumor-infiltrating lymphocytes (TILs) and which react with known antigens on melanoma tumors. To control for the variability inherent to a T cell line, all assays were repeated with an HER-2/neu-reactive CD8+ T cell clone, which had been cloned from PBLs of an immunized patient. To be better able to compare our results with previously published studies, the leukemic T cell line Jurkat was analyzed concurrently. Cells were separated by centrifugal elutriation into fractions highly enriched in G0-G1, S, and G2-M phases of the cell cycle. Short-term (intracellular Ca²⁺ flux) and middle-term (cytolytic activity and FasL induction) effects of TCR activation as well as apoptosis induced by human recombinant FasL or anti-CD3 mAb were compared between fractions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials

OKT-3 antibody was kind gift from Ortho Diagnostic Systems (Raritan, NJ). Recombinant FasL was used according to the manufacturer's recommendations (Alexis, San Diego, CA). Anti-Fas mAbs were obtained from PharMingen (San Diego, CA).

Cell Lines and Clone

T cell lines previously derived from TILs (Topalian et al., 1987) of human leukocyte antigen (HLA)-A2+ patients and that recognize the melanoma-associated antigen gp100 or melanoma antigen recognized by T cells 1 (MART-1) in the context of HLA-A2 were maintained in AimV (Life Technologies, Gaithersburg, MD) supplemented with 10 nM glutamine, 250 U/ml penicillin-streptomycin (Biofluids, Rockville, MD) (complete media [CM]), 5% human AB serum (Gemini Bio Products, Calabasa, CA), and 6000 IU of IL-2 (TECIN; Hoffmann-La Roche, Nutley, NJ) at 37°C in a humidified incubator with 5% carbon dioxide. These cytotoxic T lymphocytes (CTLs) were predominantly CD8+ (Zaks et al., 1999).

The CD8+ T cell clone L2.3w.1 recognizes the HLA-A2 restricted p369-377 epitope from HER-2/neu and was cloned by limiting dilution from the PBLs of a patient who had been immunized with the peptide in incomplete Freund's adjuvant, as has been previously described (Zaks and Rosenberg, 1998). Clonality was verified by TCR PCR (Zaks and Rosenberg, 1998). It was maintained in CM supplemented with 10% human AB serum (Gemini Bio Products), and 300 IU of IL-2 (TECIN; Hoffmann-La Roche) and expanded every 2-3 wk by stimulation with 300 Gy-irradiated autologous or allogeneic peripheral blood mononuclear cells in the presence of 30 ng/ml Ortho-anti-CD3 (Riddell and Greenberg, 1990; Zaks and Rosenberg, 1998).

Tumor cell lines that had previously been established and HLA typed in our laboratory were maintained in CM consisting of Iscove's medium (Biofluids) supplemented with 10 nM glutamine, 250 U/ml penicillin-streptomycin, and 10% fetal calf serum (Life Technologies).

L1210 murine lymphoblastoma cells transfected with hFas (L1210Fas) (Rouvier et al., 1993) were a kind gift from Dr. P. A. Henkart (National Cancer Institute). Jurkat cells were obtained from American Type Culture Collection (Manassas, VA). Jurkat and L1210Fas cells were maintained in CM.

Centrifugal Elutriation

Cells were concentrated by centrifugation, and 2.5 × 10⁸ cells were gently resuspended in 10 ml of elutriation buffer (HBSS without calcium and magnesium supplemented with 5% fetal bovine serum, 2 mM glutamine, and 100 U/ml penicillin). To ensure monodispersion, cells were passed through an 18-gauge needle followed by filtration trough a 35 µM mesh cap (Falcon, Oxnard, CA).

Cells were separated into populations of progressively increasing cell sizes in a modification of previously described protocols (Donaldson et al., 1997) in a centrifuge J-6MI (Beckman Instruments, Palo Alto, CA) equipped with a JE-5.0 elutriation rotor and a standard chamber at 25°C. CTL lines were loaded at a rotor speed of 2100 rpm and an initial flow rate of 14 ml/min maintained by a Master Flex 7518-10 digital pump (Cole-Parmer, Chicago, IL). Jurkat and L1210Fas cells were loaded at a rotor speed of 2500 rpm at an initial flow rate of 24 ml/min. Fractions were obtained by increasing the flow rate of the elutriation buffer at 2-ml/min increments and collecting 100 ml of dispensed media after each increase. The quality of separation of Jurkat and L1210Fas cells was better than that of the CTL lines, because the latter tended to form small aggregates even in Ca²⁺- and Mn²⁺-free medium, and these partially contaminated S and G2-M phase fractions with cells in G0-G1 phase. Overall, fractions of CTLs enriched in S phase yielded 70% cells in S phase, and those enriched in G2 yielded 60% in G2-M phase.

Flow Cytometry and Cell Cycle Analysis

All analyses were carried out on a FACSCalibur using CellQuest Software (both from Becton Dickinson, Mountain View, CA). Cells were collected and washed twice with PBS. Cell pellets were resuspended in 100 µl of PBS, fixed in 1 ml of 70% ethanol/30% saline buffer, and stored at 20°C until analysis, when they were washed twice with PBS followed by incubation for 40 min in 0.5 ml of PBS containing 0.1% Triton X-100 and 50 µg of RNase (Boehringer Mannheim, Indianapolis, IN) at room temperature. Ten micrograms of propidium iodide (Sigma, St. Louis, MO) were added, and the suspension was incubated in the dark at room temperature for an additional 15 min, after which DNA content was determined by flow cytometry.

Measurement of Intracellular Calcium Concentrations

Cells were loaded with 2 µM fluorescent calcium-binding dye Fura-2 and 0.01% (wt/vol) Pluronic F-127 (Molecular probes, Eugene, OR) for 1 h in room temperature. Cells were washed twice in HBSS, pH 7.4 (Biofluids), to remove excess dye. Cell were resuspended in HBSS containing 1% BSA at 1 × 10⁶ cells/ml. Cells (1 × 10⁶) were placed in a continuously stirred cuvette at 37°C, and Fura-2 emission was detected at 510 nm using excitation at 340 and 380 nm in an LS50 luminescence spectrometer (Perkin Elmer, Norwalk, CT) before and after stimulation with 10 µg/ml OKT-3 mAb. Calibration was performed for each sample using 1 µM ionomycin to measure Ca²⁺ saturated Fura-2 (F_max), followed by addition of 30 mM EGTA, 75 mM Tris, pH 9.3, and 0.1% Triton X-100 to measure Ca²⁺-free fura-2 (F_min), and the intracellular Ca²⁺ concentration was determined using FL WinLab software (Perkin Elmer).

Cytotoxicity Assays

Target cells were preincubated in 200 µCi of ⁵¹Cr (Amersham Pharmacia Biotech, Uppsala, Sweden) for 90 min, washed, and plated at 3.5-5 × 10³ cells per well in triplicates or quadruplicates, and various numbers of effectors were added for 4 h at 37°C, after which supernatants were collected and counted on a -counter (Wizard Gamma Counter; Wallac, Gaithersburg, MD). The percentage of specific lysis was calculated as (sample counts  spontaneous counts)/(maximal counts  spontaneous counts) × 100%. Maximal release was obtained by incubating target cells with 2% SDS.

Analysis of Apoptotic and Necrotic Cell Populations

Cells were washed twice in HEPES buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂) at room temperature. Cells (5 × 10⁵-1 × 10⁶) were resuspended in 0.2 ml of HEPES buffer supplemented with 4 µl of Annexin-V-FLUOS (Boehringer Mannheim) and were incubated for 15 min at room temperature in the dark. Twenty microliters of 7-amino-actinomycin solution (7-AAD, Via-Probe; PharMingen) used for DNA staining were added, and the suspension was incubated for additional 15 min. The staining was analyzed by flow cytometry.

RNase Protection Assay

Total cellular RNA was prepared from 4-6 × 10⁶ cells using RNAzol B reagent (Tel-Test, Friendswood, TX). RiboQuant multiprobe human apoptosis set hAPO3 (PharMingen) was used as a template to direct the synthesis of ³²P-riboprobes (Riboprobe II system; Promega, Madison, WI) using [³²P]UTP (DuPont New England Nuclear, Boston, MA). Five to 15 µg of total RNA were hybridized overnight at 45°C, treated with RNase A, RNase T1, proteinase K, and phenol-chloroform, and precipitated. Protected probes were denatured, electrophoresed on an 8% polyacrylamide gel, and exposed to X-Omat AR film (Eastman Kodak, Rochester, NY) for 1-2 d.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Synchronization

To analyze the relationship between the cell cycle and cellular function, it is necessary to separate cells according to their phase in the cell cycle. As opposed to pharmacological synchronization of the cell cycle, we chose to use elutriational centrifugation to obtain fractions of cells highly enriched in G0-G1, S, and G2-M phases of the cell cycle. Lymphocytes are particularly suited to this type of analysis, because they grow as single cell suspension in-vitro. This method has the advantage of being minimally disruptive (Donaldson et al., 1997), and in our studies 95% of the cells remained viable for at least 5 h after elutriation. Once cells are separated, it is possible to compare various functions between cells enriched in different phases of the cell cycle and nonelutriated cells. Any analysis by synchronization is limited to short- and middle-term assays, because cells become desynchronized over time by advancing through the cell cycle. Separation of tumor-reactive T cell lines (Figure 1A) yielded fractions highly enriched in G0-G1, S, or G2-M. Although a small contamination of G1 appeared in S and G2 fractions (see Materials and Methods), a high level of enrichment was nevertheless achieved (70% of S phase and 60% of G2) in all nontransformed CTL lines. No such contamination appeared in the elutriate of the CD8+ T cell clone (Figure 1C), in which relatively pure fractions at all phases of the cell cycle were obtained, probably as a result of the increased homogeneity of the clonal population. Five hours after elutriation a small percentage of the cells in the G0-G1 fraction entered S phase, whereas most cells in the S fraction progressed within S-G2, and a significant proportion of cells in G2-M progressed to G0-G1. These results are in accordance with the relative length of each phase. Thus, cells not only remain viable but also remain functional and continue to cycle as would be expected. Similar results were obtained with all TIL-derived CTL lines examined (CTL 907 and 1235), with the transformed Jurkat cells (Figure 1B), with the HER-2/neu-reactive CD8+ T cell clone L2.3w.1 (Figure 1C), and with L1210.Fas lines (our unpublished results).

View larger version (32K): [in this window] [in a new window]   Figure 1.   Cell cycle progression of elutriated cells. CTL 1520 (A), Jurkat cells (B), and HER-2/neu-reactive CD8+ T cell clone (C) were elutriated, and fractions corresponding to G0-G1 (G1), S (S), and G2-M (G2) phases of cell cycle were fixed immediately and after 5 h. Cells were then analyzed for DNA content. Nonelutriated cells (NE) are shown for comparison.

Intracellular Ca²⁺ Responses to TCR Activation and the Cell Cycle

An early intracellular event after activation of T cells through the TCR is tyrosine phosphorylation of CD3 chains. This in turn leads to a number of second messengers, one of which is the increase in intracellular Ca²⁺ levels (Premack and Gardner, 1992). A widely used technique for studying the changes in intracellular Ca²⁺ concentration uses the incorporation of fluorescent Ca²⁺ chelators into the cell, the most commonly used being Fura-2 (Grynkiewicz et al., 1985). We used this method to analyze the immediate response of cells to TCR activation as a function of the position of the cell in the cell cycle. After elutriation, cells were loaded with Fura-2 for 1 h. Intracellular Ca²⁺ concentration was measured in the cell suspension in real time. In the nonactivated state, both Jurkat cells and the tumor-reactive CTLs have similar (~100 nM) intracellular Ca²⁺ concentrations. TCR activation by anti-CD3 mAb (OKT-3) ligation resulted in an immediate increase in intracellular Ca²⁺ concentration. Although Ca²⁺ fluxes were stronger in Jurkat cells when compared with the other T cell lines or the CD8+ T cell clone, no differences were observed in the amplitude or the curve shape between cells in different phases of the cell cycle and nonsynchronized cells within each line tested (Figure 2, A, CTL 1520, B, Jurkat cells, and C, clone L2.3w.1). The intracellular Ca²⁺ profile in response to activation in CTL 907 and 1235 was similar to that of CTL 1520 (our unpublished results). Thus, similar immediate Ca²⁺ fluxes in response to TCR activation occur in all phases of the cell cycle.

View larger version (34K): [in this window] [in a new window]   Figure 2.   Ca2+ fluxes in T cells after TCR activation. Basal intracellular free calcium levels in elutriated and nonelutriated CTL 1520 (A), Jurkat cells (B), and CD8+ T cell clone (C) loaded with Fura-2 were monitored spectrophotometerically as described in MATERIALS AND METHODS. Cells were stimulated with 10 µg/ml soluble OKT-3 mAb at the times indicated by arrows. Cell fractions are indicated as in Figure 1. Data are representative of at least two independent experiments.

Lytic Function in Different Phases of the Cell Cycle

In addition to the calcium signal, other signaling pathways are activated after the engagement of the TCR by cognate major histocompatibility complex (MHC)-peptide complexes, ultimately leading to cytolytic activity. The T cell lines used in this study were chosen not only as a model of normal nontransformed T cells but also for their specific antitumor reactivity mediated by recognition of the melanocyte differentiation antigens gp100 (CTL 1520) and MART-1 (CTL 1235). We thus examined the lytic activity toward cognate tumor targets as a function of the cell cycle. Cells were elutriated and immediately plated with MHC class I-matched and -mismatched tumor targets expressing the relevant antigen in a standard ⁵¹Cr release assay. Cells in all phases of the cell cycle had the same ability to lyse cognate tumor targets at effector-to-targer (E:T) ratios of 40:1-0.6:1 and were similar to nonelutriated cells. Results for CTL 1235 and 1520, shown for clarity at one representative E:T ratio of 10:1, are depicted in Figure 3. Thus, tumor-reactive CTLs maintain a similar capacity of lytic recognition of cognate target cells throughout the cell cycle.

View larger version (33K): [in this window] [in a new window]   Figure 3.   Tumor-specific CTLs are capable of tumor lysis in all phases of the cell cycle. CTLs recognizing gp100+/MART-1+ tumor lines in the context of HLA-A2 were plated with recognized (HLA-A2+) and mismatched (HLA-A2) tumor targets at different E:T ratios. Results are shown for CTL 1520 versus melanoma line 624 (A) and CTL 1235 versus melanoma line 526 (B) at an E:T ratio of 10:1. Similar results were obtained at all E:T ratios and were repeated at least twice.

Induction of FasL mRNA Occurs in Response to TCR Activation at All Phases of the Cell Cycle

The above results demonstrated that functional activation of T cells does not appear to depend on their position in the cell cycle. However, as noted, previous studies had pointed to a dependency of AICD on the cell cycle. Because AICD could result from FasL up-regulation and interaction with the Fas receptor (Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995), we hypothesized that this induction in response to TCR stimulation could be cell cycle dependent. We thus analyzed the induction of FasL mRNA in response to anti-CD3 stimulation in the different cell cycle fractions by an RNase protection assay. As shown in Figure 4, low basal levels of FasL mRNA were detected in the tumor-reactive CTLs but not in Jurkat cells. Up-regulation of FasL mRNA was observed after 2.5 h of incubation on plate-bound anti-CD3 mAb. The response by the tumor-reactive CTLs was much stronger than that of the Jurkat cell line (Figure 4, A for CTL 1520 and B for Jurkat). Results similar to those obtained with CTL 1520 were seen in CTL 907 and 1235 (our unpublished results). Thus, the functional induction of FasL in response to TCR activation is similar to the other responses studied (intracellular Ca²⁺ increases and lytic activity), in that there are no detectable differences between cells at different phases of the cell cycle.

View larger version (23K): [in this window] [in a new window]   Figure 4.   FasL mRNA induction after TCR activation. Fractions of elutriated and nonelutriated CTL 1520 (A) and Jurkat cells (B) were plated on plates precoated with OKT-3 mAb (+) or control immunoglobulin G () for 2.5 h, after which total mRNA was extracted for analysis by an RNase protection assay. FasL- and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-protected bands are shown. Data are representative of at least two independent experiments.

AICD, FasL-induced Apoptosis, and the Cell Cycle

Although induction of FasL mRNA was not found to depend on the cell cycle, apoptosis induced downstream of Fas has been previously reported to be cell cycle dependent in other cell systems (Komada et al., 1995; Beletskaya et al., 1997; Dao et al., 1997). Such a dependency could be accounted for by a difference in the surface expression levels of the Fas receptor or by differences downstream of Fas signaling. Because similar levels of Fas surface expression were found on cells in different phases of the cell cycle (our unpublished results), apoptosis directly induced by soluble human recombinant FasL (rFasL) was analyzed in elutriated and nonelutriated cells. rFasL mimicks the induction of apoptosis by membrane-bound FasL both in vitro (Tanaka et al., 1995) and in vivo (Rensing-Ehl et al., 1995). Apoptosis was determined by Annexin-V binding, which measures the translocation of phosphatidylserine to the outer plasma membrane, one of the earliest detectable events in the induction of apoptosis (van Engeland et al., 1998). The specificity of FasL-induced apoptosis induction was also confirmed on the L1210 murine lymphoblastoid line transfected with human Fas (Zaks et al., 1999). In initial kinetic studies apoptosis could be detected in sensitive lines as early as 3 h after induction and, once cells became Annexin+, proceeded irrevocably to necrosis (Annexin+/7-AAD+) within a few hours (our unpublished results). The specificity of detection of apoptosis with Annexin-V staining was confirmed by subdiploid DNA content analysis. We thus studied the viability of Jurkat cells and tumor-reactive T cell lines and clone 5 h after inducing apoptosis with soluble rFasL, when synchrony of the different cell cycle fractions was still apparent (see Figure 1) and apoptosis could be detected in most of the cell types studied. A representative analysis of apoptosis induction is shown for the most rFasL-sensitive tumor-reactive T cell (CTL 907; Figure 5), and results of similar assays done with the remaining lines are summarized in Table 1. Background levels of apoptosis in all untreated cells were minimal (Figure 5A and Table 1). Although there were some differences in the sensitivity to FasL-induced apoptosis between lines (Jurkat cells being most sensitive), no cell cycle dependency was observed within any line (Figure 5B and Table 1).

View larger version (68K): [in this window] [in a new window]   Figure 5.   Induction of apoptosis by incubation with soluble FasL or by TCR activation. Elutriated and nonelutriated CTL 907 were plated with (B) or without (A) 50 ng/ml soluble rFasL and 1 µg/ml enhancer or on plates precoated with OKT-3 mAb (C). After 5 h cells were collected and stained with Annexin-V-FITC (x-axis) and 7-AAD (y-axis) to discriminate between viable (double-negative), apoptotic (Annexin-V-positive, 7-AAD-negative) and necrotic (double-positive) cells. Results are representative of two independent experiments.

Finding no relationship between either T cell function or FasL-induced apoptosis and the cell cycle, we asked whether AICD of the tumor-reactive CTLs was dependent on the cell cycle. TCR activation was induced by cross-linking with anti-CD3 mAb, a commonly used model of AICD (Dhein et al., 1995) and a more physiological stimulus than the treatment with a combination of phorbol esthers and ionomycin. Moreover, previous results in our laboratory indicated that the tumor-reactive T cell lines are susceptible to both anti-CD3 and cognate tumor-induced AICD and are Fas/FasL dependent (Zaks et al., 1999). Five hours of OKT-3 treatment resulted in prominent apoptotic death regardless of the cell position in the cell cycle (as well as in nonelutriated unsynchronized cells; Figure 5C). Similar results were obtained in all tumor-reactive CTLs studied (Table 1), although anti-CD3 stimulation caused only a marginal effect on Jurkat cells within 5 h (Table 1). Thus, these tumor-reactive T cells were highly susceptible to anti-CD3-induced AICD in all phases of the cell cycle.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study the relationship between the cell cycle and functional and apoptotic responses to TCR stimulation in tumor-reactive CTLs explanted from human melanoma tumors or cloned from the PBLs of immunized patients were investigated. These T cells are nontransformed, have a limited life span in vitro, which is dependent on exogenous IL-2, and are susceptible to Fas-mediated AICD (Zaks et al., 1999). In the case of the HER-2/neu-reactive clone, in vitro survival also depends on periodic restimulation in the presence of "feeder" lymphocytes (Riddell and Greenberg, 1990). Not only do these cells represent a more physiological model than hybridomas or leukemic T cell lines for the study of the relationship between the cell cycle and responses to TCR stimulation, but understanding their physiology could lead to an increased antitumor clinical efficacy. The ability of these cells to expand to large numbers ex vivo enabled us to separate them by centrifugal elutriation according to their size and density (Donaldson et al., 1997), avoiding the use of pharmacological synchronization with its inherent toxic effects (Merrill, 1998) and allowing the progression of enriched fractions through the cell cycle to be monitored and analyzed. It should be noted that the cells used in the present study, whether T cell lines or a CD8+ T cell clone, are mature cycling T cells, which depend on exogenous IL-2 for growth and survival, and are thus different from "resting" fresh human PBLs. Fresh PBLs are resistant to FasL (Boise and Thompson, 1996). Moreover, the same TCR stimulation (e.g., OKT-3), which will stimulate their proliferation (Algeciras-Schimnich et al., 1999), induces AICD in cycling cells.

A relationship between the cell cycle and signaling events downstream of TCR activation has not been previously established. If TCR-triggered AICD were cell cycle dependent, other signaling events downstream of the TCR might also be expected to be cell cycle dependent. We thus began by analyzing the increase in intracellular Ca²⁺ after TCR stimulation, because this is one of the earliest detectable events, in fractions enriched for the different phases of the cell cycle. The expected immediate and late phase intracellular Ca²⁺ responses (Sakano et al., 1996) were found in the nontransformed T cell lines, a CD8+ T cell clone, and in the leukemic Jurkat line, but there were no differences between fractions enriched in G0-G1, S, or G2-M. Knowing the antigen specificity of the tumor-reactive CTLs enabled us to analyze the lytic capability of the cells in relation to their cell cycle phase. Previous studies had shown that the mixed lymphocyte reaction can proceed independent of the cell cycle (MacDonald, 1978), and that murine T cell clones could lyse targets with equal efficiency in all phases of the cell cycle (Sekaly et al., 1981). In accordance with those results and with the intracellular Ca²⁺ responses, there were no significant differences in the lytic function of the CD8⁺ CTLs between nonelutriated cells and those elutriated and enriched for the different phases of the cell cycle. In cycling T cells (as well as in Jurkat cells) AICD is dependent on the induction of FasL and binding to the Fas receptor (Brunner et al., 1995; Dhein et al., 1995). However, lysis of tumor cells is mediated solely by the granzyme-perforin pathway (Sarin et al., 1997), which can proceed independently of the induction of FasL mRNA (Esser et al., 1998). Thus, the specific induction of FasL mRNA might be dependent on the cell cycle and consequently explain a differential sensitivity to AICD. However, a strong induction of FasL occurred in response to TCR stimulation in all tumor-reactive T cells without any differences between fractions enriched in different phases of the cell cycle or between elutriated and nonelutriated fractions.

Although AICD is not always dependent on signaling through the Fas receptor (Van Parijs et al., 1996) and can occur by signaling through the p75 TNF receptor as well (Zheng et al., 1995), previous results (Zaks et al., 1999) have shown that in these tumor-reactive CD8+ T cells, AICD is solely dependent on Fas. When the relationship between susceptibility to soluble rFasL-induced apoptosis and the cell cycle was directly examined, differences in baseline susceptibilities were found between different T cell lines. However, in no case was susceptibility or resistance to apoptosis induced by soluble rFasL dependent on a particular phase of the cell cycle. This is in accordance with the rapidity and lack of requirement for de novo protein synthesis of apoptosis induced by Fas activation (Weis et al., 1995; Graves et al., 1998). These findings do not support a correlation between sensitivity to Fas-induced apoptosis and any specific phase of the cell cycle in actively cycling, mature, IL-2-dependent human T cells.

When the apoptotic responses of the T cell lines to TCR stimulation were analyzed directly, no dependency on the cell cycle was found. In contrast to previous studies, we did not see S phase (Boehme and Lenardo, 1993; Radvanyi et al., 1996), or G2-M susceptibility (Fotedar et al., 1995). Moreover, a high percentage of cells were induced to undergo apoptosis within 5 h in both the elutriated and nonelutriated cells, ruling out a possible dependency on progression through the G1-S checkpoint (Lissy et al., 1998). Although a small percentage of contaminating G1 cells in the S and G2 fractions of the melanoma-reactive CTLs might theoretically be preferentially activated and kill bystander cells in other phases, the similar results obtained with a purer separation of the HER-2/neu-reactive CD8+ T cell clone as well as the lack of any noticeable difference between fractions highly enriched for different phases in the melanoma reactive CTLs make this explanation unlikely. We believe that two main reasons account for the differences between our present findings and earlier reports. First, as noted by Lissy et al. (1998), pharmacological cell cycle inhibitors might introduce artifacts because of cell cycle-specific effects even when used at sublethal concentrations. Second, many of the previous studies had used T cell hybridomas, leukemic T cell lines, such as Jurkat, or PBLs activated by pharmacological agents. Thus, our results do not address the requirement for progression through a late G1 phase cell cycle checkpoint for phorbol ester- and ionomycin-triggered AICD reported by Lissy et al. (1998) in Jurkat cells. Our results are in agreement with those of Fournel et al. (1996), who found that T cells require IL-2 but not G1-S transition to be susceptible to Fas-mediated apoptosis. This also correlates with the lack of dependency of AICD on p53 (Bates and Vousden, 1996) in nontransformed lymphocytes reported by Boehme and Lenardo (1996).

We propose that the susceptibility to AICD is not controlled by the cell cycle per se but is dependent on the number of cell cycles T cells go through as they differentiate from naïve precursors to mature T cells, similar to TCR  chain rearrangement (Tourigny et al., 1997), acquisition of a stable cytokine secretion profile (Gett and Hodgkin, 1998), and immunoglobulin G class switch in B cells (Hodgkin et al., 1996). This would explain why peripheral T cells activated for 1 d are resistant to Fas-mediated apoptosis but become sensitive by day 6 (Klas et al., 1993). Moreover, other mechanisms (such as decrease in FLICE inhibitory protein levels) might be linked to the initial TCR stimulation of resting PBLs and correlate with cell cycle progression initially (Algeciras-Schimnich et al., 1999), a correlation that might be lost upon further cycling and expansion. The availability of nontransformed T cell lines and clones combined with the ability to identify and follow low quantities of antigen-specific T cells in early phases of their differentiation with MHC class I-peptide tetrameric complexes (Altman et al., 1996) should enable a better understanding of the processes controlling antigen-specific responses and apoptosis in normal, nontransformed human T cells. IL-2 by itself is necessary for susceptibility to Fas (Fournel et al., 1996) and decreases FLICE inhibitory protein levels in activated PBLs (Algeciras-Schimnich et al., 1999). Moreover, IL-2 has been reported to induce Fas/FasL-mediated cytotoxicity in influenza-specific CD8+ and CD4+ T cell clones (Esser et al., 1997). However, this is clearly not the case in the tumor-reactive CTL lines or clone used here, whose growth is dependent on IL-2 and which respond by apoptosis to the same concentration of anti-CD3, which induces proliferation in resting fresh PBLs (Algeciras-Schimnich et al., 1999). It is not known whether the susceptibility to Fas-mediated AICD seen in TILs maintained ex vivo with high concentrations of IL-2 is a function of unique previous in vivo activation at the tumor site, the IL-2, or both. Progression through the cell cycle, by itself, has not been found to influence either the activity or the susceptibility to AICD of tumor-specific cycling CTLs either derived from the tumor or cloned from PBLs.