Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0306218 on February 20, 2007

Published online before print February 20, 2007
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(Journal of Leukocyte Biology. 2007;81:1297-1302.)
© 2007 by Society for Leukocyte Biology

Contrasting effects of FLIPL overexpression in human T cells on activation-induced cell death and cytokine production

Jehad Charo1 and Paul F. Robbins

Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

1 Correspondence at current address: Max-Delbruck-Center for Molecular Medicine, Robert-Rossle-Strasse 10, 13092, Berlin, Germany. E-mail: j.charo{at}mdc-berlin.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There have been disparate findings about the role of FLIP in the survival of mouse T cells and human tumor cell lines. The role of cellular FLIP in human T cell activation and function needs to be clarified further. To study this role, we have overexpressed long transcript FLIP (FLIPL) in primary T cells, including self-antigen-reactive, melanoma-specific T cells. We found that FLIPL overexpression protects human T cells from activation-induced cell death and enhances their prolifertive capacity but suppresses the ability of these cells to produce the proinflammatory cytokines IL-2 and IFN-{gamma} in response to CD3 or antigen-specific stimulation. The multiple effects of FLIPL indicate that this protein may influence T cell responses to antigenic stimulation.

Key Words: tumor • autoimmunity • apoptosis • cellular activation • tolerance


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cell survival is controlled by the outcome of competing pro- and antiapoptotic proteins. FLIP is an early stage inhibitor of apoptosis, which binds to Fas-associated death domain (FADD), preventing it from binding to caspase-8, thereby preventing the initiation of the apoptosis cascade and thus, acting as a dominant-negative caspase-8 [1 , 2 ]. It exists as a cellular and viral gene product, and the cellular FLIP is transcribed as short and long (FLIPL) transcripts [3 ]. FLIP overexpression in T cells correlates with autoimmunity in mice and humans and with resistance to FAS ligand (FAS-L)-mediated cell death [4 5 6 7 ]. To examine the functional consequences of FLIP overexpression, several studies have investigated the role of FLIP in mouse cells and in human tumor cell lines, including T cell lymphomas. Conflicting results were observed in these studies, which made it difficult to extrapolate the role of endogenous FLIP expression in a primary human T cell response [3 , 4 , 8 9 10 11 12 13 14 ]. For example, one study using FLIP-transgenic mice has reported that FLIP-overexpressing T cells can proliferate more rapidly than T cells from wild-type mice in response to CD3 stimulation [8 ], and another has shown the opposite [10 ]. In an earlier study, no effect of FLIP overexpression on the proliferative capacity of T cell was seen [4 ]. Investigators, who have examined the effects of FLIP on the ability of T cells to produce cytokines, also obtained contradictory results. In some studies, increased cytokine production was observed [8 , 11 ], whereas in others, decreased production [12 ] or no effect [4 ] of FLIP overexpression was observed. Similar confusion exists surrounding the significance of FLIP for cell survival, as resistance [4 , 10 ] increased death [15 ], or no effect [8 ] on T cell sensitivity to activation-induced cell death (AICD) was reported. To understand the role of FLIPL overexpression in human T cell survival and function, we have transduced primary or tumor-specific human T cells with a FLIPL-expressing retrovirus. Our results demonstrate that FLIPL overexpression can protect T cells from AICD and enhance the proliferative capacity of these cells and at the same time, reduce the ability of the cells to secrete Th1-associated cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
T cell lines used in this study have been described in a recent study [16 ]. Tumor-infiltrating lymphocyte (TIL) Lines 1749 and 1931-2F4, reactive with MART-1:27-35 peptide, were grown in complete medium (CM; RPMI 1640 supplemented with 10% FBS, L-Glu, ß-ME, and antibiotics, all from Gibco, Invitrogen, Carlsbad, CA, USA), containing 3000 IU human IL-2/ml (CM-2; IL-2 was kindly supplied by Chiron, Emeryville, CA, USA). Cryopreserved PBMC were collected from healthy donor blood donations (Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD, USA) by centrifugation on Ficoll-Hypaque gradients (Lymphocyte Separation Medium, Organon Teknika, Durham, NC, USA). PBMC were stimulated with 10 ng/ml CD3 (Orthoclone OKT3, Ortho Biotech, Bridgewater, NJ, USA) and 600 IU IL-2/ml. Three days later, cells were transduced as described below.

Retroviral constructs and the isolation of high titer-producer clones
A retroviral construct, based on the modified murine stem cell virus (MSCV) vector [17 ], was kindly provided by Dr. Luk Van Parijs (Massachusetts Institute of Technology, Cambridge, MA, USA). This construct is designated MIG. It encodes the MSCV long-terminal repeat and a cDNA-encoding GFP, which was inserted downstream of an internal ribosomal entry site (IRES) sequence, referred to as pGFP. The second construct was also a MSCV-IRES-GFP construct (kindly provide by Dr. Arthur Nienhuis, St. Jude Children’s Research Hospital, Memphis, TN, USA), in which human FLIPL was cloned 5' to the IRES [18 ] (kindly provided by Dr. Alf Grandien, Stockholm University, Sweden). High-titer, virus-producer clones were selected as described previously [16 ]. One clone, which produced the GFP-expressing virus (MIG) with a titer of 2 x 108 transduction units (TU), and one that produced the GFP and FLIPL-expressing virus (FLIP-MIG) with a titer of 7 x 106 TU, were selected for producing viruses for T cell transduction.

T cell transduction
T cell transduction was performed by plating 2 x 105 cells in a single well of a 24-well plate, and 2 ml supernatant from the selected virus-producer clone was added, as well as IL-2 to a final concentration of 600 IU IL-2/ml. The transduction was repeated 24 h following the first transduction. Following transduction, the rapid expansion protocol (REP) was used to expand the T cells [19 ], and 1–3 weeks later, the percentage of the transduced cells was estimated by FACS analysis based on GFP expression using a FACScan (BD Biosciences, San Jose, CA, USA). This experiment was repeated with similar results. GFP+ cells were then sorted using a FACSVantage (BD Biosciences) cell sorter. Sorting efficiency was high for PBL but lower for TIL, as 80–90% of the PBL compared with 60–90% of the TIL remained GFP+ when tested during the course of this study. FLIP-transduced T cells were sorted two to three times to obtain the 60–90%, GFP-positive T cell populations. Sorted cells were used throughout this study. The T cells were maintained in CM-2 and by stimulation using REP every 3–4 weeks, and cells for different experiments were used 1–2 weeks post-REP.

Apoptosis induction and the estimation of T cell survival and proliferation
To measure the resistance of MIG or FLIP-transduced T cells to FAS-L-induced death, cells were cultured in the presence of FAS-L and an activator for 24 h, per the manufacturer’s instructions (Alexis, San Diego, CA, USA), followed by measurement of T cell survival using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction assay (Promega, Madison, WI, USA). This assay was performed twice with similar results. The induction of AICD was evaluated as described [20 ]. Briefly, transduced T cells activated with fresh IL-2 (600 IU/ml) were washed twice and then cultured in OKT-3 (10–1000 ng/ml)-coated wells in the presence of 600 IU/ml fresh IL-2. Cell death was estimated 24 h later by staining with PE-conjugated Annexin-V (AV; Alexis), according to the manufacturer’s instructions, and analyzed by FACS (FACScan, BD Biosciences). AICD assays were performed in duplicate and were repeated four times. Live, transduced T cells represent those cells that were detected in the GFP-positive and AV-negative quadrant derived from the FACS analysis.

Proliferation assay
To study the effects of FLIPL overexpression on T cell response to stimulation, MIG or FLIP-MIG-transduced T cells were cultured in triplicates (2x105) in CM in the presence or absence of IL-2 (600 IU/ml) in wells of flat-bottomed, 96-well plates, which were uncoated or coated with 1–1000 ng/ml OKT-3. Three to five days later, proliferation was measured by the MTS reduction assay (Promega) according to the manufacturer’s protocol. Relative T cell numbers were estimated using untreated T cell standards (104–107 cells/well in triplicate) prepared prior to MTS addition. The MTS reduction by 2 x 105 T cells was used as a control, and MTS reduction by stimulated T cells was then calculated as the proportion of this value. This assay was repeated four times.

IL-2 and IFN-{gamma} production assays
Cultures of cells, which were plated as described above for the proliferation assays, were assayed for IL-2 release 16 h following stimulation using a specific ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s protocol. IL-2 production assays were performed in triplicates and repeated four times. Melanoma-specific T cells (1–2.5x104) were cocultured with target cells (1x105) for 24 h in 200 µl CM per well of 96-well, flat-bottomed plates (Costar, Corning, NY, USA). IFN-{gamma} was assayed by carrying out an ELISA specific for human IFN-{gamma} (R&D Systems), as published earlier [16 ]. The IFN-{gamma} assays were performed in duplicate and were repeated two to four times. The CD25 expression level was detected by FACS using mouse antihuman CD25 antibody (Clone M-A251), APC-conjugated (BD Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T cell transduction
To study the effect of FLIP overexpression on T cell survival and function, T cells from healthy donor PBL (PT) and TIL from melanoma patients were transduced with MIG or FLIP-MIG retroviruses. Between 8% and 50% of MIG-transduced T cells expressed GFP, as judged by FACS analysis. Fewer T cells (2–8%) were transduced with FLIP-MIG, as estimated by FACS analysis of GFP expression in these T cells. The efficiency of transduction was similar in PT and TIL (data not shown). Transduced T cells were sorted based on GFP expression, and the resulting cell lines were designated PT-GFP (for PT transduced with the vector expressing GFP alone) and PT-FLIP (for PT transduced with the vector expressing FLIPL and GFP). Similarly, melanoma patient TIL 1931-2F4 and 1749 transduced with the GFP and FLIPL-expressing retroviruses were designated 1931-GFP, 1749-GFP, 1931-FLIP, and 1749-FLIP, respectively. Transduction with FLIP-MIG appeared to be less efficient than transduction with MIG, presumably as the increased size of the packaged virus particles resulted in a lower virus titer.

FLIP overexpression protects T cell from AICD
FLIPL-overexpressing T cells were sorted twice (PT-FLIPlow) or thrice (PT-FLIPhigh), based on the expression of the IRES-coupled GFP. The effect of FLIPL overexpression on the induction of AICD was evaluated by activating FLIPL and control, GFP-transduced T cells with plate-bound OKT-3 (10–1000 ng/ml) and IL-2. The results demonstrated that T cell death is correlated with the concentration of OKT-3 and that PT-GFP cells were more sensitive to AICD (as judged by the percentage of AV-positive T cells) than PT-FLIP cells (Fig. 1 ). When activated with 1000 ng OKT-3, only 2–11% of the control-transduced T cells were resistant to AICD and remained AV-negative, whereas FLIPL-transduced T cells were more resistant to AICD, as between 40% and 60% of these cells remained viable following stimulation (Fig. 1) . Activation with lower concentrations of OKT-3 resulted in lower levels of apoptosis in all three groups. Nevertheless, following activation with 100 ng/ml OKT-3, 50% of the PT-GFP and only 18% of PT-FLIP cells underwent apoptosis (Fig. 1) . Resistance to AICD correlated with the level of FLIPL overexpression, as PT-FLIPlow cells were more resistant than PT-GFP cells but less resistant than PT-FLIPhigh cells to AICD (Fig. 1) . The PT-FLIPhigh cells were also more resistant to FAS-L-induced death than PT-GFP (Fig. 2 ), as 65% of the FLIPL-transduced T cells were viable in the presence of 10 ng/ml-soluble FAS-L, whereas only 35% of the GFP-transduced T cells were viable under the same conditions.


Figure 1
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  		Figure 1. FLIPL overexpression confers resistance to OKT-3-mediated AICD. T cells were activated as described in Materials and Methods for 24 h followed by AV-PE staining and FACS. The percentage of live cells indicates the GFP-positive and AV-negative population.

 

Figure 2
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  		Figure 2. FLIPL overexpression increases T cell resistance to FAS-L-induced death. Cells were cultured for 24 h in the presence of FAS-L and an activator, followed by the addition of MTS. Percent live cells indicate the percentage of MTS reduction in different concentrations of FAS-L and activator as compared with reduction in cultures without added FAS-L.

 
FLIPL overexpression enhances T cell proliferation
To evaluate the effects of FLIPL overexpression on T cell proliferation, PT-GFP and PT-FLIPhigh cells were then stimulated with varying concentrations of OKT-3 for 4 days, in the presence or absence of IL-2. Cells, which were cultured in the absence of IL-2, demonstrated a net loss in cell number over this time, whereas cell numbers increased in the PT-GFP and PT-FLIPhigh groups when stimulated with 1 ng/ml OKT-3 in the absence of IL-2 (Fig. 3 ). Increasing concentrations of OKT-3 did not appear to result in a significant increase in cell numbers over that observed with 1 ng/ml OKT-3 in the PT-FLIPhigh group but led to a decline in cell numbers in the PT-GFP group below the level observed with 1 ng/ml OKT-3. In the absence of OKT-3 stimulation, IL-2 addition led to enhanced survival of PT-GFP and PT-FLIPhigh cells with a statistically significant increase (P=0.005) in the proliferative capacity of FLIPL-overexpressing T cells, as compared with the starting number, and to that number of cells in the PT-GFP culture under similar conditions. Increasing concentrations of OKT-3 resulted in no additional accumulation of PT-FLIPhigh cells, whereas PT-GFP cells stimulated with increasing concentrations of OKT-3 led to the recovery of lower cell numbers. The increased accumulation of PT-FLIPhigh cells was not a result of the potential overexpression of CD25 on these cells as compared with that on PT-GFP, as both cell lines expressed similar levels of CD25 in the absence or presence of CD3 stimulation (Fig. 3B) .


Figure 3
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  		Figure 3. Proliferative response of GFP and FLIPL-transduced cells (A) to stimulation by IL-2 alone or together with OKT-3. Fold of proliferation is calculated using MTS reduction by the starting cell number (indicated by 100%) of 2 x 105 T cells as a control, and MTS reduction by stimulated T cells was then calculated as the proportion of this value 96 h after the initiation of the experiment. Similar CD25 expression level (B) on GFP and FLIPL-transduced cells is detected by FACS analysis.

 
FLIP overexpression decreases T cell capacity to produce cytokines
PT-GFP and PT-FLIPhigh cells were then evaluated for their ability to secrete cytokines in response to plate-bound OKT-3 stimulation. In the absence of stimulation, no IL-2 could be detected in the culture supernatants of PT-GFP or PT-FLIPhigh cells. Following stimulation using between 1 and 1000 ng/ml OKT-3, significant levels of IL-2 were released from PT-GFP and PT-FLIPhigh cells. PT-GFP-stimulated T cells produced significantly (P<0.005) higher levels of IL-2 than PT-FLIPhigh cells at all concentrations tested (Fig. 4A ). This was not a result of an increase in CD25 expression on PT-FLIPhigh cells, as CD25 expression on PT-GFP and PT-FLIPhigh cells was indistinguishable (Fig. 3B) . The TIL 1931-GFP and 1749-GFP cultures, which recognized the HLA-A2-restricted MART-1:27-35 melanoma antigen peptide, released relatively high levels of IFN-{gamma} in response to T2 target cells pulsed with the MART-1 peptide and HLA-A2-positive melanoma cells, whereas significantly lower levels of IFN-{gamma} were produced by 1931-FLIP and 1749-FLIP TIL cultures in response to these target cells (Fig. 4B and 4C) .


Figure 4
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  		Figure 4. FLIPL-transduced PT (A) and TIL (B and C) produce less IL-2 or IFN-{gamma} in response to stimulation. PT cells were cultured in wells coated with OKT-3, and supernatants were assayed for IL-2 using ELISA 16 h later. TIL were cultured with the indicated target and assayed for IFN-{gamma} using ELISA 24 h later.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented in this report indicate that FLIPL overexpression protects nontransformed, primary human T cells from death induced by FAS-L and AICD and at the same time, leads to decreased cytokine release in response to TCR stimulation.

Overexpression of FLIPL in mouse T cells as a result of transgene expression has been reported to have no effect or render cells less sensitive to AICD than nontransgenic T cells [4 , 8 , 10 ]. The variable effects of FLIPL overexpression on cell sensitivity to death were reported to be a result of its expression level, and high expression levels appeared to be cytocidal in some tumor cell lines [9 , 14 ]. Although these results might indicate that the response to FLIPL overexpression may be dependent on the cell type being analyzed, it is also possible that the results vary depending on the level of FLIPL expression, which presumably differs among studies.

The differences in cell recoveries observed between the PT-FLIP and PT-GFP groups in the presence of OKT-3 and exogenous IL-2 may at least in part have resulted from the resistance of FLIPL-overexpressing T cells to AICD, as described above. This is particularly true in conditions where cells were stimulated with OKT-3 and exogenous IL-2, as resistance of PT-FLIP cells to AICD would contribute to the increase in cell number. Other factors might contribut to this difference, such as the decreased IL-2 production by PT-FLIP cells. FLIPL-overexpressing T cells accumulate in autoimmune disease lesions [5 6 7 ]. What is the causative relationship between these two events? The ability of FLIPL-overexpressing cells to escape death may lead to the accumulation of autoimmune T cells that overexpress it in the affected lesion. Alternatively, the accumulation of FLIPL high cells in the affected tissues may be coincidental and can be explained as a direct outcome of FLIPL overexpression, which enhances T cell proliferation and may not necessarily be related to the specificity of these cells.

The lower levels of IL-2 production by FLIPL-overexpressing human T cells resembles that observed in T cells isolated from caspase-8-deficient individuals [21 ], along with a recent observation demonstrating the involvement of caspase-8 in NF-{kappa}B activation [22 ] and Th2 cytokine response [23 ], suggesting that a common mechanism might be responsible for this phenomenon. IL-2 and IFN-{gamma} are involved with the induction of AICD [2 , 17 , 20 , 24 , 25 ], and the ability of FLIPL to suppress these two proapoptotic cytokines suggests that FLIPL may protect cells from AICD, not only in a direct manner by acting as a dominant-negative FADD or caspase-8 analog but also indirectly, by suppressing the production of these cytokines. This suppressive effect could be mediated by the ability of FLIPL to decrease the level of the transcription factor T-bet, which has been shown to enhance the transcription of Th1 cytokines in mouse T cells [26 ]. Together with these previously published reports, our data suggest that a negative feedback loop may exist between IL-2 and IFN-{gamma} production on one hand and the FLIPL level of expression on the other.

Using FLIP-transgenic or -knockout mice, three groups have analyzed steady-state or inducible CD25 expression on T cells. Dohrman et al. [27 ] have reported about the increased level of CD25 expression on FLIP-overexpressing T cells, Zhang and He [28 ] have described the opposite, and Chau et al. [29 ] did not find such a difference. This has prompted us to explore whether the increase in the proliferative capacity, which is associated with decreased IL-2 detection in the supernatant of the PT-FLIP culture as compared with that in the PT-GFP culture, can be explained by a different level of CD25 expression on these two cell lines. As no such difference was found between PT-GFP and PT-FLIP CD25 expression levels, another mechanism would account for these differences in human T cells.

A lack of apoptosis has been implicated in the etiology of a variety of autoimmune diseases [6 , 7 ]. Although animal models have been useful in studying the mechanisms of these diseases, the role of molecules involved in the processes of apoptosis and immunity may differ between mice and humans [30 ]. For example, caspase-8 deficiency leads to embryonic lethality in mice, whereas in humans, it is associated with the development of autoimmunity [21 ]. Our data suggest that FLIPL overexpression in human T cells results in increased resistance to AICD and enhanced proliferation and at the same time, reduces their ability to produce proapoptotic and proinflammatory cytokines. These effects of FLIPL overexpression resemble those seen following the stimulation of T cells with partial agonist peptides, which have also been implicated in autoimmunity [31 ]. In a recent study, adoptively transferred T cells from FLIPL-transgenic mice protected recipient mice from developing experimental autoimmune encephalomyelitis [32 ]. The role of cytokines in autoimmunity is complicated, however, as IL-2 and IFN-{gamma} were reported to enhance or inhibit the development of autoimmune diseases [24 , 33 ]. Further studies are necessary to delineate the role of FLIPL in determining the outcome of T cell responses to antigenic stimulation.


   ACKNOWLEDGEMENTS
 
We thank Drs. Steven A. Rosenberg and Thomas Blankenstein for their support, Dr. John R. Wunderlich and the TIL laboratory for providing the T cells and melanoma cell lines used for this study, Mr. Arnold Mixon and Mr. Shawn Farid for providing assistance with FACS analysis and sorting, and Mrs. Mona El-Gamil, Mrs. Linda L. Parker, Mrs. Jennifer A. Westwood, Mr. Yong F. Li, Ms. Stephanie Kupsch, and Mr. Markus Hensel for providing technical assistance.

Received March 23, 2006; revised December 15, 2006; accepted January 18, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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