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The Journal of Immunology, 2008, 181, 5857 -5864
Copyright © 2008 by The American Association of Immunologists, Inc.
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NF-{kappa}B Modulates Sensitivity to Apoptosis, Proinflammatory and Migratory Potential in Short- versus Long-Term Cultured Human {gamma}{delta} Lymphocytes

Marina Ferrarini*, Fanny Delfanti1,{dagger}, Monica Gianolini{ddagger}, Chiara Rizzi§, Massimo Alfano§, Adriano Lazzarin{dagger},¶ and Priscilla Biswas2,{dagger}

* Laboratory of Tumor Immunology, Department of Oncology, {dagger} Laboratory of Clinical Immunology, Department of Infectious Diseases, {ddagger} Unit of Human Virology and § AIDS Immunopathogenesis Unit, DIBIT, Department of Infectious Diseases, Università Vita-Salute San Raffaele, San Raffaele Scientific Institute, Milan, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
V{gamma}9V{delta}2 T lymphocytes are involved in the immune response against hematological malignancies and certain pathogens through the recognition of nonpeptidic Ags expressed by tumors and infected cells. Being equipped with proinflammatory chemokine receptors, they participate to the early phases of inflammation acting as both effector and connector cells between innate and adaptive immunity. We show in this study that after initial TCR triggering short- and long-term cultured {gamma}{delta} lymphocytes differ in their susceptibility to activation-induced apoptosis and proinflammatory phenotype. Activation-induced apoptosis was triggered by anti-CD95 mAbs or by the {gamma}{delta}TCR stimuli isopentenyl pyrophosphate and pamidronate, the latter in the presence of monocytes. In particular, short-term cultured cells are resistant to apoptosis and characterized by expression of anti-apoptotic cellular FLIP molecules and partial spontaneous caspase-8 activation. Linked to this behavior, short-term {gamma}{delta} cells display constitutive activation of the transcription factor NF-{kappa}B, which is functionally related to their apoptosis-resistant phenotype. Finally, they spontaneously secreted elevated amounts of the NF-{kappa}B-regulated chemokines CCL3, CCL4, and CCL5, which likely contributed to down-modulation of the inflammatory CCR5 receptor. Conversely, long-term cultured apoptosis-sensitive {gamma}{delta} cells displayed uncleaved caspase-8 and no constitutive NF-{kappa}B activation; moreover, they secreted CC chemokines only upon TCR triggering coupled to the re-expression of CCR5. The expression of members of the TNF receptor family, including CD30 and TNFRII, also varied according to the time in culture. Altogether our data support a link between resistance to apoptosis and a proinflammatory phenotype in {gamma}{delta} T lymphocytes, unraveling the crucial role of NF-{kappa}B in regulating the switch from resistance to apoptosis susceptibility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The V{gamma}9V{delta}2 T lymphocytes, which represent the major fraction of peripheral human {gamma}{delta} cells, have long been implicated in the immune response against certain pathogens and malignancies. Differently from {alpha}β T lymphocytes, V{gamma}9V{delta}2 T lymphocytes recognize in a TCR-dependent, MHC-unrestricted manner nonpeptidic phospho-Ag derived through the isoprenoid biosynthetic pathway (1, 2). In particular, {gamma}{delta} lymphocytes recognize with the highest affinity compounds derived from the microbial nonmevalonate synthetic pathway, and less efficiently intermediates of the mammalian mevalonate pathway, including the prototypic Ag isopentenyl pyrophosphate (IPP)3 (2, 3, 4). The latter can also accumulate within cells as a result of malignant transformation (5); moreover, drugs such as aminobiphosphonates, including pamidronate, activate {gamma}{delta} T cells by inducing IPP accumulation through a blockade in the mevalonate pathway (2, 6, 7). Recognition of these protease-resistant phospho-Ags induces V{gamma}9V{delta}2 lymphocyte activation leading to production of proinflammatory cytokines and chemokines and killing of the target cells (3).

V{gamma}9V{delta}2 lymphocytes also differ significantly from {alpha}β T lymphocytes in their migratory properties, in that peripheral {gamma}{delta} cells lack the lymph node homing chemokine receptor CCR7 (8) and instead express receptors for inflammatory chemokines, such as CXCR3 and CCR5 (9). Moreover, chemokine receptor expression is driven and modulated by TCR triggering (8, 9). Due to these features, {gamma}{delta} T lymphocytes have the potential to promptly respond to inflammation providing both direct effector anti-tumor and anti-infectious responses (2, 10, 11) and acting as a bridge between innate and adaptive immunity (4, 12, 13).

Having exerted their effector functions, T lymphocytes undergo feedback mechanisms mainly aimed to limit their expansion, including activation-induced cell death (AICD). Members of the TNFR family, particularly CD95, initiate AICD upon engagement by their specific ligands, leading to activation of a regulated cascade of caspases and ultimately to apoptosis (14, 15). CD95 trimerization induced by CD95 ligand (CD95L) binding induces the intracellular formation of a death-inducing signaling complex (or DISC) that involves the C-terminal portion of CD95 containing the death domain, the adapter molecule FADD, and caspase-8. This results in caspase-8 cleavage, which represents the first step in the cascade of caspase activation (16, 17). CD95-induced apoptosis needs to be tightly regulated, and cellular FLIP (cFLIP), an endogenous caspase-8-like molecule lacking enzymatic activity, represents the major counteracting molecule by binding to the death-inducing signaling complex and blocking caspase-8 activation (18, 19). Depending upon intracellular concentrations of cFLIP, two scenarios can be foreseen: complete autoprocessing of caspase-8 resulting in apoptosis, and limited processing of caspase-8 and processing of cFLIP leading to generation of the p43 and p22 (20) fragments and to NF-{kappa}B activation (17, 19). NF-{kappa}B is a pivotal transcription factor that has long been recognized as a key regulator of immune and inflammatory responses (21). Later, a set of studies unraveled its role in the regulation of apoptosis (22).

We have previously reported that V{gamma}9V{delta}2 T cell clones undergo AICD following TCR triggering upon induction of CD95L expression and subsequent autocrine/paracrine cell death (23, 24). Our in vitro model of {gamma}{delta} T cell clones is characterized by cyclic restimulations via TCR engagement and, notably, sensitivity to CD95-induced AICD typically developed 3–4 wk after each restimulation. A differential sensitivity to CD95-mediated apoptosis has previously been reported for primary {alpha}β T lymphocytes, but with much shorter kinetics encompassing a period of 5–8 days after stimulation (25, 26). The precise mechanisms tuning T lymphocyte transition from an apoptosis-resistant to an apoptosis-sensitive status are still a matter of active debate. In this context, a number of cellular factors, in addition to cFLIP and including NF-{kappa}B, have been recently implicated (15, 17, 27). Given the extended activation period following each restimulation of V{gamma}9V{delta}2 T cell clones, we took advantage in this study of this in vitro model to dissect molecular correlates of susceptibility to apoptosis and proinflammatory behavior in short- vs long-term cultured cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
{gamma}{delta} T cell clones and {gamma}{delta} cell lines

{gamma}{delta} T cell clones were generated by limiting dilution and subsequently propagated by cyclic restimulation (every 2–3 wk) with irradiated allogeneic PBMC and PHA plus rIL-2 (50 U/ml), as described in Ref. 23 . {gamma}{delta} cell lines were generated upon stimulation of PBMC from healthy donors with specific {gamma}{delta}-inducing stimuli, IPP (16 µM; Sigma-Aldrich), and pamidronate disodium (16 µM; Novartis Pharmaceuticals) and were propagated in culture with rIL-2 (50 U/ml). To assess the contribution of monocytes/macrophages to {gamma}{delta} T cell activation by pamidronate, PBMC from healthy donors were seeded in 24-well plates at 2 x 106/ml for 2 h, then nonadherent cells were removed, and adherent cells were incubated or not with mevastatin (25 µM; Sigma-Aldrich) for an additional 2 h to block the mevalonate pathway upstream and thus prevent IPP accumulation after pamidronate treatment (5, 28). At the end of the incubation monocytes were extensively washed, and nonadherent cells were re-added and stimulated with either IPP or pamidronate (both at 16 µM). PBMC were cultured in IL-2-containing medium for 8 days, then cell counts and percentages of TCR {gamma}{delta}+ cells were determined. Expression of the V{gamma}9V{delta}2 {gamma}{delta} TCR was assessed by staining with the Ti{gamma}A mAb, which specifically recognizes the V{gamma}9 epitope, and with the BB3 mAb, which recognizes the V{delta}2 epitope (gifts from T. Hercend (Sanofi-Aventis, Vitry sur Seine, France) and E. Ciccone (University of Genoa, Genova, Italy)). Assessment of susceptibility to apoptosis, expression of surface markers and apoptosis-related molecules, and cytokine and chemokine production, was performed at weekly intervals following restimulation, as described below.

Assessment of apoptosis

Apoptosis induction with an overnight incubation with the cross-linking IgM anti-CD95 mAb CH11 (50 ng/2 x 105 cells; Medical and Biological Laboratories) was assessed by propidium iodide (PI) staining and FACS analysis, as previously described (24). In a separate set of experiments, apoptosis of pamidronate-generated {gamma}{delta} cell lines was evaluated following treatment with pamidronate (16 µM) in the presence or absence of autologous monocytes. The latter were obtained by adherence of PBMC on plastic and by the removal of nonadherent cells. Cell viability upon overnight incubation with the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC; 1–1000 µM; Sigma-Aldrich) was also determined through PI exclusion. As control, the effect of PDTC was also evaluated in PBMC from normal donors. Caspase 3 activity in {gamma}{delta} lymphocytes was evaluated by FACS analysis using the CaspGLOW Red Active Caspase-3 Staining Kit (BioVision) according to the manufacturer’s instructions. In brief, {gamma}{delta} lymphocytes (1 x 106/ml) were stimulated with IPP (16 µM) or pamidronate (16 µM) plus autologous monocytes or none for 3–4 h at 37°C, then the cell permeable sulf-rhodamine-conjugated DEVD-fluoromethyl ketone caspase 3 substrate (Red-DEVD-fmk) was added for an additional hour. Cells were then washed, stained with an FITC-conjugated anti-TCR {gamma}{delta} mAb (BD Biosciences), and analyzed by FACS. Activated caspase 3 in {gamma}{delta} lymphocytes undergoing apoptosis was visualized as red fluorescence (FL2). The NO donor S-nitrosoacetylpenicillamine (SNAP; Sigma-Aldrich), which we have previously shown to protect {gamma}{delta} cells from CD95-mediated apoptosis (29), was used at 300 µM.

Assessment of cell proliferation

Proliferation of {gamma}{delta} T cell clones was assessed by means of CFSE staining and [3H]thymidine uptake. In brief, at weekly intervals from the initial restimulation, 3–4 x 106 {gamma}{delta} cells were washed in PBS, incubated with CFSE (Molecular Probes) 5 µM for 10' at 37°C, washed again, and cultured for 4 days in the presence or absence of IL-2 before determination of CFSE staining by FACS analysis. In a separate set of experiments, at weekly intervals from the initial restimulation, [3H]thymidine (1 µCi/well; GE Healthcare) was added to {gamma}{delta} T cell clones (50 x 103 cells/well) and uptake evaluated following an overnight incubation.

Western blot analysis

{gamma}{delta} cells were collected at days 7, 14, and 21 after restimulation, washed once in 1x PBS, and pelletted. Whole cell extracts (30) were prepared from 2 to 3 x 106 {gamma}{delta} cells, disrupted by three freeze-thaw cycles, kept on ice for 10 min, and centrifuged at 13,000 rpm for 5 min. Total protein concentration was determined by the Bio-Rad Protein Assay. Equal amounts (50 µg) were loaded and separated on 15% SDS-PAGE and transferred to Hybond ECL membranes (Amersham Pharmacia Biotech) using standard procedures. Membranes were blocked for 30 min at room temperature in 5% nonfat dry milk dissolved in 1x PBS. Blots were incubated with the primary Abs (diluted in PBS plus 5% nonfat dry milk) overnight at 4°C. After washing twice with 1x PBS, blots were incubated for 1.5 h at room temperature with the secondary Ab (goat anti-rabbit IgG conjugated to HRP; Amersham Pharmacia Biotech) diluted 1/2000 in 5% nonfat dry milk-PBS. After washing, positive bands were detected through a chemiluminescence method, following the manufacturer’s protocol (ECL Kit; Amersham Pharmacia Biotech). The primary anti-actin (Sigma-Aldrich), anti-FLIP (N terminus) and anti-caspase-8 (Upstate Biotechnology) Abs (all polyclonal rabbit IgG) were used at the final concentrations of 2.5, 1, and 2 µg/ml, respectively. To evaluate CD95L expression, {gamma}{delta} cells (2 x 106) were treated for 2–6 h with IPP (16 µM) or pamidronate (16 µM). In the latter case, {gamma}{delta} cells were coincubated with allogeneic monocytes obtained by plastic adherence. As a control, cells were cultured in tissue culture medium alone. At the end of the incubation, cells were recovered, washed, and lysed. Western blot analysis was then performed using an anti-CD95L mAb (clone G247-4, 250 ng/ml; BD Pharmingen).

EMSA

Whole cell extracts were prepared from day 7 and day 14 {gamma}{delta} cells, as previously described (30) and used at 15 µg in each lane. The NF-{kappa}B oligonucleotide probe (5'–3') GCT ACA AGG GAC TTT CCG CTG GGG ACT TTC CAG G was annealed to its complementary strand and end-labeled with [{gamma}-32P]ATP (Amersham Pharmacia Biotech) using polynucleotide kinase (New England Biolabs). One µl of labeled probe (0.5 ng) was incubated with each cell extract in a reaction mixture for 30 min at room temperature. The mixtures were run on 5% (29:1) acrylamide (Bio-Rad) gels in 1x TBE buffer. Gels were dried and subjected to autoradiography.

Assessment of cytokine and chemokine production

Cytokine and chemokine secretion was assessed in supernatants generated from {gamma}{delta} lymphocytes (5 x 105/ml) either unstimulated or stimulated overnight with IPP, at the indicated concentrations. CCL4/MIP-1β and CCL5/RANTES were measured by single ELISA kits (R&D Systems), whereas CCL3/MIP-1{alpha} (not shown), TNF-{alpha}, IFN-{gamma}, IL-6, and IL-10 were detected simultaneously by the Fluorokine MAP Multiplex Kit (R&D Systems) using a Luminex analyzer (Bioplex, Bio-Rad). Intracellular content of CCL5/RANTES and IFN-{gamma} was determined by FACS analysis as described in Ref. 31 . In brief, {gamma}{delta} cells (5 x 105) were stimulated or not with 50 ng/ml PMA and 1 µg/ml ionomycin (both from Sigma-Aldrich) for 4 h; for IFN-{gamma} determination, brefeldin A (Sigma-Aldrich) was added at 10 µg/ml during the last 2 h of culture to prevent cytokine secretion. Cells were then fixed with paraformaldehyde, permeabilized, and stained with either an FITC-conjugated anti-IFN-{gamma} mAb or a PE-conjugated anti-CCL5/RANTES mAb (both from R&D Systems).

Analysis of surface markers

Surface expression of TNF receptor family members and of chemokine receptors by {gamma}{delta} T lymphocytes was assessed throughout the culture period by flow cytometric analysis. The following mAbs were used: anti-CD30-FITC (clone BerH8), anti-CXCR3-PE, anti-CCR5-FITC (clone 2D7; BD Biosciences), anti-CCR5-FITC (clone no. 45531, R&D Systems). TNFR expression was assessed by indirect staining with the primary anti-TNFRI mAb (32) and the anti-TNFRII (rat IgG2b; Amgen) followed by a FITC-conjugated second step reagent (Southern Biotechnology Associates). Staining of {gamma}{delta} positive cells was then determined by flow cytometric analysis with either the FACStarPlus or the FACSCalibur instruments (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
{gamma}{delta} T cell clones undergo AICD depending upon the time in culture

Human {gamma}{delta} T cell clones were periodically restimulated with PHA and irradiated allogeneic feeder cells and propagated in IL-2-containing medium (23). As reported for {alpha}β T cells (26, 33), {gamma}{delta} T cell clones acquire the sensitivity to CD95-mediated apoptosis upon in vitro culture, albeit with slower kinetics. As shown in Fig. 1A, a progressively increased susceptibility to CD95-induced apoptosis was observed in restimulated {gamma}{delta} T cells kept in culture for 7, 14, and 21 days. Day 7 and day 14 to 21 cultured {gamma}{delta} T cell clones will hereafter be referred to as short-term vs long-term cultured cells, respectively.


Figure 1
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  		FIGURE 1. Long-term cultured {gamma}{delta} T cell clones are prone to AICD. A, Differential susceptibility of long- vs short-term cultured {gamma}{delta} T cell clones to CD95-mediated apoptosis. Day 7, 14, and 21 {gamma}{delta} T cells were untreated (open columns, NT) or treated (filled columns) with the cross-linking, apoptosis-inducing CH11 anti-CD95 mAb. After an overnight incubation {gamma}{delta} cell viability was assessed by staining with PI and FACS analyses. Results are expressed as percentage of PI+ cells and are mean values ± SD of four independent experiments. B, CD95L expression in both unstimulated (NT) and stimulated {gamma}{delta} T cells was evaluated by Western blot analysis. Stimuli were IPP and pamidronate (pam), both used at 16 µM final concentration. C, Flow cytometric analysis of caspase-3 activity in untreated and TCR-stimulated {gamma}{delta} T cell clones. Cells were incubated for 4–6 h with medium alone or TCR-triggering stimuli, pamidronate and IPP (16 µM). The activated cells were then pulsed with the cell membrane permeable caspase-3 fluorogenic substrate (Red-DEVD-fmk) for 1h at 37°C, and finally stained with the anti-TCR {gamma}{delta} mAb. The NO donor SNAP was used as an inhibitor of AICD (28 ). The numbers in the upper right panels indicate the percentage of double positive ({gamma}{delta}-caspase 3-positive) cells.

 
Upon TCR triggering, short-term (not shown) and long-term cultured {gamma}{delta} T cell clones (Fig. 1B) up-regulated CD95L; in particular, when the latter were treated with both IPP and pamidronate, increased CD95L expression was observed by Western blot analysis (Fig. 1B), peaking at 2 and 4 h, respectively, and decreasing thereafter (24 h and data not shown). The interaction between CD95 and CD95L in susceptible cells led to apoptosis (24) and activation of downstream effector caspase 3, as indicated by flow cytometric analysis with Red-DEVD-fmk (Fig. 1C). Red-DEVD-fmk irreversibly binds caspase 3 only when it is activated, thereby allowing for the identification of cells undergoing apoptosis. Both IPP and pamidronate strongly increased the percentage of caspase 3-positive cells in long-term cultured {gamma}{delta} T cell clones (Fig. 1C), but not in short-term cultured cells (data not shown). Addition of the NO donor SNAP to IPP-stimulated (and to pamidronate-stimulated; data not shown) cells was able to prevent caspase 3 activation (Fig. 1C), in agreement with the previously reported capability of NO to inhibit CD95-induced apoptosis (29). Taken together, these results indicate that AICD can account for the cell death of long-term cultured {gamma}{delta} T cells.

Pamidronate-derived bulk {gamma}{delta} T cell lines acquire sensitivity to AICD with time in culture: requirement for monocytes

To validate our clonal model with a system that might better mimic the behavior of in vivo-activated {gamma}{delta} cells, we generated primary {gamma}{delta} cell lines by stimulating ex vivo PBMC with specific {gamma}{delta} TCR Ags, IPP and pamidronate (Fig. 2A). In agreement with a previous report (6), stimulation of PBMC from five healthy donors with both stimuli resulted in large and comparable expansions of {gamma}{delta} T lymphocytes. We observed that susceptibility to CD95-induced AICD of both pamidronate-derived (Fig. 2B) and IPP-derived (not shown) primary {gamma}{delta} T cell lines was clearly acquired with time in culture, similarly to our {gamma}{delta} clonal model. Because it has been previously shown that activation of primary human {gamma}{delta} T cells by aminobiphosphonates requires presentation by monocytes (34), we investigated whether monocytes were necessary for both {gamma}{delta} cell proliferation (Fig. 2C) and AICD by pamidronate (Fig. 2D). To this purpose, we evaluated {gamma}{delta} cell expansion in the presence of autologous monocytes pretreated with mevastatin, which inhibits the mevalonate pathway upstream to IPP generation and thus prevents pamidronate-induced IPP accumulation (5, 28). As shown in Fig. 2C, pretreatment with mevastatin abated pamidronate-induced, but not IPP-induced, {gamma}{delta} cell expansion. Similarly, only in the presence of autologous monocytes was pamidronate able to induce AICD in long-term cultured primary {gamma}{delta} cell lines (Fig. 2D). We can conclude that primary {gamma}{delta} T cell lines behave similarly to {gamma}{delta} T cell clones in terms of resistance/susceptibility to Fas/CD95-induced cell death, and that monocytes are required not only for pamidronate-induced {gamma}{delta} activation, but also for pamidronate-induced AICD.


Figure 2
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  		FIGURE 2. Pamidronate-stimulated primary {gamma}{delta} lymphocytes proliferate and undergo AICD in the presence of monocytes. A, Primary CD3+ {gamma}{delta} cells were derived from PBMC of five healthy donors upon stimulation with IPP and pamidronate (pam) (16 µM) and propagated for 2 wk in IL-2. Percentage of {gamma}{delta} T cells was determined by FACS analyses at the different time points indicated. B, Short-term (day 9) and long-term (day 18) cultured pamidronate-stimulated primary {gamma}{delta} cells were untreated (NT; open columns) and treated (filled columns) with apoptosis-inducing CH11 anti-CD95 mAb. Percentage of {gamma}{delta} apoptotic cells was evaluated by {gamma}{delta} TCR and PI staining by flow cytometry. Results are mean values ± SD of four donors. C, Expansion of {gamma}{delta} cells from PBMC treated with IPP or pamidronate with or without pretreatment of autologous monocytes with mevastatin (25 µM). {gamma}{delta} lymphocyte counts within the bulk culture were obtained upon assessment of percentage of {gamma}{delta} TCR+ cells by flow cytometry. One representative experiment of three performed is shown. D, Pamidronate-derived long-term cultured primary {gamma}{delta} cells were stimulated with pamidronate in the presence or absence of monocytes. {gamma}{delta} cell death was determined by flow cytometry through staining with anti-{gamma}{delta} TCR mAb and PI. Data from one donor of four are depicted.

 
Intrinsic molecular correlates associated to differential susceptibility to apoptosis in short-term vs long-term {gamma}{delta} clones

Two major isoforms of cFLIP exist, the 55-kDa cFLIP long (cFLIPL) and the 26-kDa cFLIP short (cFLIPS), both acting as dominant negative inhibitors of caspase-8 (18, 35). In apoptosis-resistant cells cFLIPL forms a heterodimer with caspase-8 leading to proteolytic activation of caspase-8; this results in both an autoprocessing with release of the p43 caspase-8 and the cleavage of cFLIPL with the generation of p43 cFLIP. The latter takes part of a signaling complex, ultimately leading to NK-{kappa}B activation (17).

Both cFLIP and caspase-8 expression were evaluated in day 7, 14, and 21 cultured {gamma}{delta} T cells by Western blot analysis in the absence of any further stimulation (Fig. 3A); the housekeeping protein β-actin was used as an internal control (Fig. 3A, lowest panel). Day 7 cells display both cFLIPL and cFLIPS, as well as the cleaved p43 cFLIPL. Along with an evidence of proteolytic activity of caspase-8 independent of death receptor triggering, day 7 cells show caspase-8 mostly in the cleaved p43 form (Fig. 3A, middle panel). Conversely, day 21 cells lack the expression of cFLIPS, have a decreased amount of cFLIPL, and caspase-8 is almost exclusively present in the full-length p55 form, indicating lack of active proteolysis. Day 14 cultured {gamma}{delta} T cells show an intermediate profile in the expression of both cFLIP and caspase-8. Taken together these features resemble those reported for short- vs long-term activated {alpha}β primary T cells and are responsible for their differential susceptibility to apoptosis (26).


Figure 3
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  		FIGURE 3. Apoptotic-related correlates in short-term vs long-term cultured {gamma}{delta} T cell clones. Day 7, 14, and 21 {gamma}{delta} T cells were analyzed for molecular apoptotic-related molecules (A) and proliferation (B and C). A, Lysates of {gamma}{delta} T cells were analyzed in Western blot with anti-FLIP, anti-caspase-8, and anti-actin Abs (upper, middle, and lower panels, respectively). The three bands detected by anti-FLIP Ab correspond to cellular (c) FLIP long (L) and short (S) isoforms and to the p43 cleavage product of cFLIPL. The anti-caspase-8 Ab identifies the isoforms a and b of procaspase-8 (p55/54) and its p43/41 cleavage products (55 ). B, [3H]Thymidine incorporation by {gamma}{delta} T cell clones. Results are expressed as mean + SD of five independent experiments. C, CFSE staining of {gamma}{delta} cells cultured for 4 days after CFSE loading in the presence or absence of IL-2.

 
Because the presence of cleaved p43 caspase-8 has been linked to T cell cycling (17, 35), we analyzed proliferation of short- vs long-term {gamma}{delta} T cell cultures (Fig. 3, B and C). Long-term cultures (day 21) displayed negligible [3H]thymidine incorporation, whereas day 7 and 14 {gamma}{delta} T cell clones showed elevated and intermediate levels of proliferation (Fig. 3B). In agreement, CFSE dilution, and hence proliferation, was observed only in day 7 and 14 cultures (Fig. 3C). These data suggest a link between cell cycling and spontaneous caspase-8 cleavage also in our experimental model.

NF-{kappa}B activation discriminates between short- and long-term {gamma}{delta} T cell survival

We therefore evaluated NF-{kappa}B activation via EMSA in short-term (day 7) vs long-term (day 14) cultured {gamma}{delta} T cells (Fig. 4A). Short-term cells displayed a clear upper band corresponding to specific NF-{kappa}B binding, comparable to that of the positive control represented by extracts from TNF-{alpha}-activated U937-clone 10 cells (30). Conversely, the band was absent in day 14 cultured cells. To functionally link the expression of NF-{kappa}B to the intrinsic susceptibility of {gamma}{delta} cells to apoptosis, we treated short- vs long-term {gamma}{delta} cells with serial dilutions of the NF-{kappa}B inhibitor PDTC (Fig. 4B). The highest concentrations of PDTC (100 and 1000 µM) dramatically increased the percentage of dead (PI-positive) cells in the short- (open squares), but not in the long-term (open circles) cultured cells. Notably, the cell death was not due to a toxic effect of the drug because it was not detected in PBL from healthy donors treated with the same serial dilutions of PDTC (open triangles). Of note, PDTC treatment did not further augment sensitivity to CD95-induced apoptosis in long-term cultured {gamma}{delta} T cell clones (data not shown), thus ruling out a role for NF-{kappa}B in protection from apoptosis in those cells.


Figure 4
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  		FIGURE 4. Differential NF-{kappa}B binding in short-term vs long-term cultured {gamma}{delta} T lymphocytes affects their proneness to apoptosis. A, The EMSA displays the binding of the heterodimeric NF-{kappa}B complex (p50/p65) in short-term (day 7) and long-term (day 14–21) activated cells. The negative controls are represented by the probe alone and by extracts obtained from irradiated APC kept in culture for 7 days; the positive control is whole cell extract from TNF-{alpha}-activated U937 clone 10 (30 ). B, Short-term (day 7; squares) and long-term (day 14–21; circles) activated {gamma}{delta} T lymphocytes were treated overnight with PDTC at the indicated concentrations, then their viability was assessed by PI staining and FACS analyses. PBL (triangles) treated with the same concentrations of PDTC were used as control. Data are expressed as mean ± SEM of four {gamma}{delta} T cell clones and PBL from three different healthy donors.

 
Spontaneous release of CC chemokines by short-term, but not long-term, {gamma}{delta} T cell clones as compared with inducible secretion of cytokines

Among the many factors regulated by the transcriptional activity of NF-{kappa}B are cytokines such as TNF-{alpha} and a number of CC chemokines (36, 37). Specifically in V{gamma}9V{delta}2 T lymphocytes NF-{kappa}B activation has been shown to mediate production of CCL3/MIP-1{alpha}, CCL4/MIP-1β, and CCL5/RANTES (38), whereas TNF-{alpha} was under the control of the p38-kinase and ERK-2 pathways (39). Thus, we evaluated the secretion of CCL4/MIP-1β and CCL5/RANTES in short-term vs long-term cultured {gamma}{delta} T cells in both unstimulated and IPP-stimulated conditions. As shown in Fig. 5A, day 7 stimulated cells displayed spontaneous release of chemokines that was not further increased by addition of IPP. Conversely, untreated day 14/21 {gamma}{delta} T cells failed to spontaneously secrete CCL4/MIP-1β and CCL5/RANTES, which were instead induced by IPP. Of note, residual contaminating, irradiated APC were unlikely to contribute to the constitutive secretion of chemokines in short-term cultured {gamma}{delta} cells as suggested by the negligible and not modulated secretion of IL-6 and IL-10, cytokines known to be produced by APC (Table I). The peculiar pattern of chemokine secretion was not shared by all soluble factors produced by {gamma}{delta} lymphocytes in that IFN-{gamma} and TNF-{alpha} were highly inducible in both short-term and long-term cultured cells (Table I).


Figure 5
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  		FIGURE 5. Differential production of CC chemokines by short-term and long-term cultured {gamma}{delta} T cells. A, CCL4/MIP-1{alpha} and CCL5/RANTES secretion by unstimulated (open bars) and IPP-stimulated (50 µM; filled bars) was determined by ELISA at days 7, 14, and 21 of culture. B, Intracellular content of CCL5/RANTES and IFN-{gamma} was assessed by FACS analysis in day 14 cultured {gamma}{delta} cells either untreated (bold continuous line) or PMA plus ionomycin-treated (continuous line). Brefeldin A was added only to the IFN-{gamma} sample to prevent cytokine release. The dashed and dotted lines represent cells stained with isotype control Abs in untreated and treated conditions, respectively.

 

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  		Table I. Cytokine production by short- and long-term cultured {gamma}{delta} lymphocytes

 
Long-term cultured {gamma}{delta} T cell clones released preformed CCL5/RANTES from intracellular stores upon TCR stimulation, as indicated by intracellular staining (Fig. 5B, upper panel); instead, de novo synthesis of IFN-{gamma} was activated by TCR triggering (Fig. 5B, lower panel). As previously reported (40), the rapid release of stored RANTES protein upon TCR activation resembles a feature of memory {alpha}β CD8+ T lymphocytes (41) and is in agreement with a predominant memory phenotype of peripheral V{gamma}9V{delta}2 lymphocytes in adults (42).

Modulation of surface receptor correlates by time in culture

Certain members of the TNF R family have been found to be expressed on {gamma}{delta} T cell clones, such as CD95 (23) and CD30 (43). We here show that short-term cultured {gamma}{delta} T cell clones expressed high levels of CD30 that progressively declined with time in culture (Fig. 6, upper left panel). A similar pattern was observed for the expression of TNFRII/CD120b, whereas TNFRI/CD120a was undetectable throughout the culture period (Fig. 6, lower and middle left panels). Also, a number of chemokine receptors are expressed by {gamma}{delta} T cells and modulated upon activation, including CCR1, CC2b, CCR5, CCR7, and CXCR3 (8, 31, 44, 45, 46). CXCR3 was always expressed throughout the culture period, but differently from CD30 and CD120b, its expression increased with time reaching a maximum at about 2 wk of culture (Fig. 6, upper right panel). Expression of CCR5, substantially undetectable early upon culture (day 7), became evident at day 14 and was subsequently maintained up to day 21 (Fig. 6, middle and lower right panels), as assessed by means of flow cytometric analyses with the anti-CCR5 mAb 2D7 and 45531 mAbs which recognize two different epitopes of CCR5.


Figure 6
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  		FIGURE 6. Phenotypic characterization of short-, mid-, and long-term cultured {gamma}{delta} T cells. Untreated day 7 (bold continuous line), 14 (dashed line), and 21 (continuous line) cultured {gamma}{delta} T cells were stained with Abs targeting members of the TNFR (CD30, TNFRI, TNFRII) and chemokine R (CXCR3, CCR5) families. Expression of CCR5 was assessed by two different mAb, clone 2D7 and clone 45531. Because day (d) 7, 14, and 21 histograms of cells stained with control Ab were substantially overlapping, only one is shown (dotted line).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our in vitro model of sequential restimulation of V{gamma}9V{delta}2 T cell clones allows for an in-depth comparison of early and late events following {gamma}{delta} TCR-triggering. Short-term {gamma}{delta} cultures present spontaneous caspase-8 and NF-{kappa}B activation associated with spontaneous β-chemokine release and lack of CCR5 expression as compared with long-term {gamma}{delta} cultures. This peculiar phenotype of short- vs long-term {gamma}{delta} T cell cultures is correlated with their resistance and susceptibility, respectively, to CD95-induced apoptosis and may underlie a differential proinflammatory and migratory potential.

In the absence of CD95 triggering, short-term cultured {gamma}{delta} cells are characterized by the expression of cFLIPS and spontaneous activation of caspase-8, leading to both partial autoprocessing and cleavage of cFLIPL with generation of the p43 cFLIPL fragment. Further processing of p43 cFLIPL to generate the p22 fragment (20) could not be ruled out by the experimental conditions used in our study. This issue deserves further investigation given the recent interesting observation that cFLIPS inhibits caspase-8 activation, thus reducing NF-{kappa}B activity (47). Both cFLIPL and its alternative splice variant cFLIPS are major determinants in the transition between apoptosis resistant and susceptible status in T lymphocytes (18) and are up-regulated in T cells shortly after activation (26, 35). Accordingly, the expression of cFLIPL decreased and cFLIPS was undetectable in apoptosis-sensitive long-term cultured {gamma}{delta} T cells.

Caspases are a family of aspartate-specific cystein-dependent proteases that have long been recognized as major mediators of the apoptotic program; nevertheless, accumulating evidence link caspases to an array of nonapoptotic functions (17). In particular, in apoptosis-resistant T cell cultures TCR triggering results in limited caspase-8 activity, i.e., cleavage of cFLIP with incomplete autoprocessing, leading to NF-{kappa}B activation. NF-{kappa}B has early been associated with intracellular signaling pathways leading to protection from TNF-induced cell death (48, 49, 50). More recently, cleavage products of cFLIP have been shown capable of activating the NF-{kappa}B transcriptional factor, thus preventing cells from apoptosis (17, 19, 20). Importantly, short-term {gamma}{delta} cultures were characterized by sustained NF-{kappa}B activation, which was functional as demonstrated by the enhanced cell death induced by the NF-{kappa}B inhibitor PDTC. These data, in agreement with a previous report showing that the NF-{kappa}B inhibitor curcumin caused apoptosis of IPP-stimulated {gamma}{delta} T lymphocytes (38), point to NF-{kappa}B as a major determinant in resistance to apoptosis.

A broad array of genes are known to be under the transcriptional control of NF-{kappa}B, including proinflammatory chemokines such as CCL4/MIP-1β and CCL5/RANTES (51). In our experimental model short-term activated {gamma}{delta} lymphocytes with elevated basal NF-{kappa}B binding were characterized by spontaneous secretion of the above mentioned CC chemokines, whereas in long-term cultured {gamma}{delta} cells secretion of CCL4/MIP-1β and CCL5/RANTES was induced only upon TCR-mediated triggering. Accordingly, it was previously reported that curcumin inhibited the production of CCL4/MIP-1β and CCL5/RANTES in V{gamma}9V{delta}2 T cells (38). Promptness in releasing inflammatory chemokines may represent a key function of {gamma}{delta} lymphocytes in recruiting other effector cells and thus contributing to the amplification of the immune response.

At variance with the CC chemokines, a smaller difference in TNF-{alpha} production existed between short- and long-term cultures. This could be accounted for by the fact that TNF-{alpha}, generally known to be transcriptionally activated by NF-{kappa}B, was shown to be under the control of the p38-kinase and ERK-2 pathways in {gamma}{delta} T lymphocytes (39).

The functional activation state of short-term cultured {gamma}{delta} T cell clones, i.e., spontaneous NF-{kappa}B binding and chemokine release, could be reflected by a peculiar pattern of surface receptors. In this context, constitutive levels of NF-{kappa}B binding were correlated with surface expression of a member of the TNFR family, CD30, in lymphocytic and monocytic cell lines (52). We previously reported expression of the costimulatory molecule CD30 in {gamma}{delta} T cell clones (43); in the present study expression of CD30, as well as TNFRII, was elevated in short-term cultured {gamma}{delta} T cells and decreased with time in culture, following the pattern of NF-{kappa}B activation. Conversely, CCR5 was substantially absent in short-term cultures and became evident in long-term {gamma}{delta} T cell clones. It is known that elevated levels of CC chemokines may account for internalization of CCR5 (45). NF-{kappa}B, which transcriptionally controls CC chemokines, may be involved in the regulation of CCR5 expression not directly, but through the differential constitutive secretion of its ligands. TNF-{alpha}, which was shown to decrease CCR5 expression through the up-regulation of its ligands via interaction with TNFRII (53), may also contribute to our findings.

In conclusion, our in vitro {gamma}{delta} cell culture model recapitulates molecular and functional features that may occur in {gamma}{delta} T lymphocytes in vivo, in particular the modulation of migratory and proinflammatory potential of {gamma}{delta} T lymphocytes following TCR triggering. Fresh peripheral {gamma}{delta} lymphocytes are equipped with proinflammatory chemokine receptors, i.e., CCR5 (8, 9), which drive them to sites of inflammation. Once activated they release chemokines and switch to expression of the lymph node homing CCR7 (4, 54), thereby providing an amplification of the inflammatory reaction and bridging the innate with the adaptive immune response. Finally, we identified a central role for NF-{kappa}B as an anti-apoptotic and proinflammatory transcription factor in {gamma}{delta} T lymphocytes.


   Acknowledgments
 
We thank Clara Sciorati (DIBIT, San Raffaele Scientific Institute, Milan, Italy) for help in the flow cytometric assessment of apoptosis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Current address: Ospedale Maggiore di Lodi, Lodi, Italy. Back

2 Address correspondence and reprint requests to Dr. Priscilla Biswas, San Raffaele Scientific Institute, Department of Infectious Diseases, Via Stamira d’ Ancona 20, 20127 Milan, Italy. E-mail address: biswas.priscilla{at}hsr.it Back

3 Abbreviations used in this paper: IPP, isopentenyl pyrophosphate; AICD, activation-induced cell death; CD95L, CD95 ligand; cFLIP, cellular FLIP; PI, propidium iodide; PDTC, pyrrolidine dithiocarbamate; Red-DEVD-fmk, sulf-rhodamine-conjugated DEVD-fluoromethyl ketone caspase 3 substrate; SNAP, S-nitrosoacetylpenicillamine; cFLIPL, cFLIP long; cFLIPS, cFLIP short. Back

Received for publication March 17, 2008. Accepted for publication August 14, 2008.


   References
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 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosures
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