Inhibition of the Death Receptor Pathway by cFLIP Confers Partial Engraftment of MHC Class I-Deficient Stem Cells and Reduces Tumor Clearance in Perforin-Deficient Mice1 Mesha Austin Taylor,2*† Preet M. Chaudhary,‡ Jennifer Klem,*† Vinay Kumar,§ John D. Schatzle,† and Michael Bennett† NK cells mediate acute rejection of MHC class I-deficient bone marrow cell (BMC) grafts. However, the exact cytotoxic mechanisms of NK cells during acute BMC graft rejection are not well defined. Although the granule exocytosis pathway plays a major role in NK cell-mediated rejection, alternative perforin-independent mechanisms also exist. By analyzing the anti-apoptotic effects of cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein (cFLIP) overexpression, we investigated the possible role of death receptor-induced apoptosis in NK cell-mediated cytotoxicity. In the absence of perforin, we found that cFLIP overexpression reduces lysis of tumor cells by NK cells in vitro and in vivo. In addition, perforin-deficient NK cells were impaired in their ability to acutely reject cFLIP-overexpressing TAP-1 knockout stem cells. These results emphasize the importance of NK cell death receptor-mediated killing during BMC grafts in the absence of perforin. The Journal of Immunology, 2001, 167: 4230 – 4237.
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atural killer cells play a crucial role in innate immune surveillance through the elimination of infectious agents and tumors (1). They also are the main effector lymphocytes mediating the acute rejection of allogeneic and MHC class I-deficient bone marrow cell (BMC)3 grafts as predicted by the missing self-hypothesis (2, 3). NK cells can use various cytotoxic pathways to lyse targets, such as the calcium-dependent release of perforin and granzymes and the interaction of ligands with death receptors belonging to the TNF superfamily (4 –10). However, to date all of the critical effector components of NK cell cytotoxicity leading to target cell apoptosis during acute BMC graft rejection are not well defined. The granule exocytosis (perforin and granzymes) pathway has been shown to play a major role in NK cell-mediated rejection of MHC class I-deficient BMC grafts. 129:B6 mice that are deficient in perforin, either by genetic manipulation or by drug treatment, are unable to reject TAP-1 knockout (KO) BMC grafts, while C57BL/6 (B6) perforin-deficient mice are able to reject them (11, 12). These data emphasize the importance of perforin as a cytotoxic mechanism involved in the rejection of MHC class I-defi-
*Graduate Program in Immunology, and Departments of †Pathology, and ‡Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; and § Department of Pathology, University of Chicago, Chicago, IL 60637 Received for publication March 6, 2001. Accepted for publication August 10, 2001. 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 This work was supported by National Institutes of Health Grants CA36922, CA70134, and AI38938. 2 Address correspondence and reprint requests to Dr. Mesha Austin Taylor, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9072. E-mail address:
[email protected] 3 Abbreviations used in this paper: BMC, bone marrow cell; FLIP, Fas-associated death domain-like IL-1-converting enzyme inhibitory protein; cFLIP, cellular FLIP; KO, knockout; FADD, Fas-associated death domain protein; LAK, lymphokine-activated killer; rm, recombinant murine; J.cFLIP, cFLIP-transfected Jurkat; GFP, green fluorescent protein; TRAIL, TNF-related apoptosis-inducing ligand; PKO, perforin KO; UdR, deoxyuridine.
Copyright © 2001 by The American Association of Immunologists
cient BMC by NK cells. However, because B6 perforin-deficient mice can still efficiently reject incompatible BMC, an alternative perforin-independent pathway of NK cell-mediated BMC rejection must also exist. A possible mechanism is the transmission of apoptotic signals to target cells through the TNF superfamily of death receptors. Currently, five different death receptors are known, TNFR-1 (13, 14), Fas (7, 8), TNF receptor-related apoptosis mediated protein (TRAMP) (9, 15–18), and TNF-related apoptosis-inducing ligand (TRAIL)-R1 and -R2 (19 –22). The best characterized member is Fas, which initiates an apoptotic signal in target cells when activated by its ligand, FasL, on the effector cell (7, 8). Ligand binding induces the recruitment of the adaptor protein, Fas-associated death domain protein (FADD), which in turn leads to procaspase8/Fas-associated death domain-like IL-1-converting enzyme/recruitment (23–26). Autoproteolytic activation of caspase-8 triggers a subsequent caspase cascade leading to cell death (27–29). Blocking death receptor-induced apoptosis during signal transduction is one way to determine the involvement of the death receptors in acute BMC graft rejection mediated by NK cells. The anti-apoptotic protein, cellular Fas-associated death domain-like IL-1-converting enzyme inhibitory protein (cFLIP), is an enzymatically inactive homologue of caspase-8, which can bind to the death effector domains of FADD and caspase-8 (30 –35). This prevents the recruitment of caspase-8 to FADD and ultimately inhibits apoptosis through the death receptors (36). cFLIP inhibits activationinduced cell death in T cells and Fas-dependent CTL-mediated target cell lysis (37). Moreover, overexpression of cFLIP in tumor cells results in escape from T cell immunity and promotes tumor progression (38, 39). cFLIP is also capable of interacting with TNFR-associated factor 1 and TNFR-associated factor 2, which are components of TNF receptor signaling complexes, suggesting its importance in TNF signaling. Mouse embryonic fibroblasts from cFLIP KO mice are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activity (40). The strong inhibitory activity of cFLIP overexpression on death receptor signaling allowed us to study the role of death receptors in NK cell-mediated cytotoxicity, using transfected tumor cells and 0022-1767/01/$02.00
The Journal of Immunology retrovirally infected stem cells. We found that cFLIP overexpression reduces lysis of tumor cells by B6 perforin KO (PKO) NK cells in vitro. In addition, B6 PKO mice were unable to clear cFLIP-overexpressing tumor cells from the lungs and showed a decrease in their ability to acutely reject TAP-1 KO stem cells overexpressing cFLIP.
Materials and Methods Reagents and Abs Anti-Fas ligand mAb (clone MFL3) was purchased from BD PharMingen (San Diego, CA). Anti-asialo-GM1 antiserum was purchased from WAKO (Richmond, VA). Anti-FLAG M2 Ab was purchased from Sigma-Aldrich (St. Louis, MO). Human rIL-2 was obtained from Chiron (Emeryville, CA).
Cell culture, retroviral vector, and constructs Lymphokine-activated killer (LAK) cells were cultured in 500 U/ml human rIL-2 as described (41). cFLIP-transfected Jurkat (J.cFLIP) tumor cells were transfected by electroporation (300 V, 960 F) with 20 g of MigR1 plasmid DNA encoding murine FLAG-tagged cFLIP and green fluorescent protein (GFP). Jurkat and J.cFLIP tumor cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 1% L-glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 100 U/ml penicillin, and 100 g/ml streptomycin.
Mice TAP-1 KO, B6, and B6 PKO mice (all H-2b) were obtained from The Jackson Laboratory (Bar Harbor, ME).
Flow cytometry analysis Fas expression on Jurkat and J.cFLIP tumor cells was evaluated with biotinylated anti-human Fas mAb (clone APO-1-1) (Kamiya Biomedical, Seattle, WA) or an isotype-matched control followed with streptavidin red 670 (Life Technologies, Rockville, MD). Purified TAP-1 KO progenitors were incubated with FITC-labeled anti-mouse Fas mAb (clone Jo2) (BD PharMingen) or an isotype-matched control. TRAIL-R2 expression on purified TAP-1 KO progenitors was analyzed with purified rabbit anti-human/ mouse TRAIL-R2 (R&D Systems, Minneapolis, MN) followed by incubation of cells with FITC-labeled goat anti-rabbit IgG Ab (Jackson ImmunoResearch Laboratories, West Grove, PA).
Stem cell purification, infection, and transplantation BMC were harvested from TAP-1 KO mice and stained with a biotinylated-Lineage mixture (Gr1, Mac-1, B220, CD2, NK1.1, and Ter119) (BD PharMingen) followed by incubation with streptavidin-conjugated microbeads. Using negative selection, BMC were passed through a MACS separation column (Miltenyi Biotec, Auburn, CA) for collection of c-kit⫹Lin⫺ progenitors. These progenitors were cultured for 4 days at 3 ⫻ 105 cells/ml with 20 ng/ml recombinant murine (rm)IL-3, 50 ng/ml rmIL-6, and 50 ng/ml rmSCF (Biosource International, Camarillo, CA) in DMEM containing 10% FCS. After 48 and 72 h, transduction of stem cells was performed by spinfection (as reported in Ref. 42) with control (containing GFP only) and FLIP-expressing (plus GFP) retroviruses that were generated as described (43). Most recipient mouse groups contained at least five mice. Recipient mice were lethally irradiated and infused with 8.5 ⫻ 105 infected progenitors. Seven days later, spleen cells were harvested and the percentage of GFP-positive cells was analyzed by flow cytometry. Values significantly different ( p ⬍ 0.05) from another group by nonparametric and parametric analyses are indicated in the figure legends.
Generation of chimeras TAP-1 KO c-kit⫹Lin⫺ progenitors were isolated and infected (as described above), then sorted for GFP-positive expression. 5 ⫻ 105 cFLIP-positive cells were infused into lethally irradiated syngeneic hosts. Eight weeks later, chimeras were checked for BMC reconstitution and cFLIP overexpression.
Lung clearance assay Details were described previously (44). Most groups contained at least five mice. Briefly, Jurkat or J.cFLIP target cells were labeled with 125I-labeled dexoyuridine (UdR) (Amersham, Arlington Heights, IL), then 5 ⫻ 105 cells were injected i.v. into each recipient mouse. After 4 h, the lungs were excised from the mice, and the amount of 125I remaining in the lungs was
4231 measured. Where indicated, some mice were treated with 300 g i.p. of anti-FasL mAb 1 day before challenging with tumor cells. Results are expressed as the geometric mean (95% confidence limits) of the percentage of injected radioactivity remaining in the lungs. The percentage of 125Ilabeled UdR retention is inversely related to NK cell lytic activity. A high percentage of retention denotes low NK cell lytic activity, whereas a low percentage of retention indicates high NK cell lytic activity. The values were subjected to parametric (Student’s t test) and nonparametric (Welch’s t test) statistical analyses, using the UTSTAT program provided by the University of Texas Southwestern Medical Center (Dallas, TX).
Cytotoxicity assay As described (44), target cells were radiolabeled with 100 –150 Ci of sodium chromate (51Cr) (Amersham) for 1.5 h at 37°C. At various E:T ratios, effectors and radiolabeled targets were added to each well in triplicates. Before addition of targets, 10 g/ml anti-FasL mAb was added to effectors. After a 4-h incubation, 100 l of supernatant was removed and the 51Cr release was counted in a liquid scintillation counter. Specific lysis was expressed as the mean ⫾ SEM and calculated as follows: percent specific lysis ⫽ 51Cr cpm, (ER ⫺ SR)/(MR ⫺ SR) ⫻ 100, where ER is the experimental 51Cr released in the presence of effectors, SR is the spontaneous 51Cr released in the presence of medium only, and MR is the maximum 51Cr released in the presence of 0.5% Triton X-100. The values were subjected to Welch’s t test for statistical analyses.
Western blot analysis Cells were washed once in PBS, resuspended at 107 cells/ml in lysis buffer (50 mM TrisCl, pH 8; 150 mM NaCl; 1 mM MgCl2; 2% Nonidet P-40), and incubated on ice for 30 min. Lysates were spun for 15 min at 14,000 rpm at 4°C and aliquots of 20 l were resolved by SDS-PAGE on gels of 10% acrylamide. Proteins were transferred to nitrocellulose and stained with Ponceau dye. Filters were blocked for 30 min in 5% nonfat dried milk in PBS, 0.1% Tween 20, then incubated with primary Ab (1/1000) diluted in PBS, 0.1% Tween 20 for 1 h at room temperature. Filters were washed three times in PBS, 0.1% Tween 20, then incubated with anti-rat IgG horseradish peroxidase-labeled secondary Ab (Amersham) for 1 h at room temperature. After washing, blots were developed with the SuperSignal chemiluminescence kit from Pierce (Rockford, IL).
Results cFLIP overexpression in Jurkat cells inhibits NK cell activity in vitro Previous evidence suggests that elevated cFLIP levels may correlate with resistance to TRAIL-induced apoptosis (37, 45). Furthermore, tumor targets with increased cFLIP expression resist Fasdependent, but not perforin-dependent, CTL killing in vitro (10). Therefore, we hypothesized that NK cell-mediated cytotoxicity may also be inhibited by the same mechanism. To analyze this directly, we stably transfected Jurkat cells, a human T cell leukemia that is sensitive to both perforin- and Fas-mediated killing (10, 12), with FLAG-tagged cFLIP (long form, ⬃55 kDa). Specifically, the cDNA for FLAG-tagged cFLIPL was introduced into the retroviral vector, MigR1 (46). This vector expresses a bicistronic mRNA encoding both the test protein and GFP, which serves as a marker of transfection. Stable transfectants were shown to express GFP and the exogenous FLAG-tagged cFLIP protein (Fig. 1, A and B). Both untransfected Jurkat cells and J.cFLIP cells express relatively similar levels of Fas receptor (Fig. 1C) and TRAIL-R2 (our unpublished data), indicating that transfection did not diminish Fas or TRAIL-R2 levels in J.cFLIP cells. The effect of cFLIP overexpression on NK cell cytotoxicity was assessed by a 4-h chromium release assay. IL-2-activated B6 LAK cells are able to lyse Jurkat and J.cFLIP targets similarly (Fig. 2A). Overexpression of cFLIP does not affect NK cell-mediated cytotoxicity in the presence of perforin. The addition of anti-FasL mAb reduces the lysis of Jurkat cells slightly, presumably because lysis is primarily mediated through perforin killing. Lysis of J.cFLIP is not lowered. Assessing Fas-mediated killing, B6 PKO LAK cells are also able to lyse Jurkat cells, although killing is reduced when
4232
cFLIP OVEREXPRESSION INHIBITS NK CELL-MEDIATED REJECTION lung retention between NK cell-depleted and untreated mice can be detected at 4 h. Similar to B6 mice, B6 PKO mice are able to eliminate Jurkat tumor targets from the lungs (Fig. 3B, left panel). The administration of anti-FasL mAb diminished the clearance of Jurkat tumor targets from the lungs of B6 PKO mice. However, this clearance is not as defective as that of B6 mice treated with anti-asialo-GM1 Ab, suggesting that other perforin- and Fas-independent mechanisms (such as TRAIL-R/TRAIL) may contribute to lung clearance of Jurkat cells. Moreover, the elimination of Jurkat tumor cells by B6.gld (FasL-deficient) mice was similar to that observed by B6 PKO mice treated with anti-FasL mAb (data not shown). Together, these data indicate that in perforin-deficient mice Fas/FasL interactions are involved in NK cell-mediated lung clearance of Jurkat tumor cells. Lung clearance of J.cFLIP tumor cells by B6 mice treated with anti-asialo-GM1 Ab is significantly less than that of untreated B6 mice (Fig. 3B, right panel). Interestingly, B6 PKO mice as well as B6 PKO mice treated with antiFasL mAb are similar to NK cell-depleted B6 mice in their inability to clear J.cFLIP tumor cells from their lungs. Therefore, in the absence of perforin, increased cFLIP expression in Jurkat tumor targets prevents clearance by NK cells via death receptor-induced apoptosis. Similar results were obtained in additional experiments. For example, the geometric mean values of the B6 PKO group challenged with Jurkat tumor cells were 0.07 (experiment 1) or 0.23 (experiment 2), while the B6 PKO group challenged with J.cFLIP tumor cells were 0.30 (experiment 1) or 0.44 (experiment 2) ( p ⬍ 0.05). In another experiment, the geometric mean values of the untreated B6 PKO group and B6 PKO group treated with
FIGURE 1. Expression of exogenous cFLIP and Fas in Jurkat and Jurkat transfected with cFLIP. A, Jurkat tumor cells were transfected with cFLIP-expressing (cFLIP-transfected) expression construct or were not transfected (Untransfected). cFLIP-transfected cells showed green fluorescence when analyzed by flow cytometry. B, The levels of exogenous cFLIP protein expressed by untransfected or cFLIP-transfected Jurkat tumor cells were determined by Western blot analysis. Cell lysates were resolved by SDS-PAGE under reducing conditions and immunoblotted with an Ab to the flag epitope present at the C terminus of the retrovirus-encoded cFLIP molecule. The 55-kDa band corresponds to cFLIPL form. C, The expression of Fas receptor on untransfected Jurkat (solid line) and J.cFLIP (dotted line) was determined using an Ab to Fas. Isotype control Ab is denoted by the filled histogram.
compared with B6 LAK cells (Fig. 2B). However, lysis of J.cFLIP by B6 PKO LAK cells is significantly lowered. In the presence of anti-FasL mAb, NK cell lytic activity of Jurkat was further lowered, while that of J.cFLIP was abrogated completely. Consequently, cFLIP overexpression can inhibit Fas-mediated NK cell cytotoxicity of tumor targets in the absence of perforin. cFLIP overexpression prevents the clearance of Jurkat tumor cells from the lungs of PKO mice Given that both perforin- and Fas-dependent mechanisms of killing must be inhibited to reduce NK cell lysis in vitro, we subsequently wanted to determine the role of both cytotoxic pathways in vivo. Testing the ability of NK cells to rapidly clear tumor targets from the lungs is one alternative in vivo method that assays for NK cell cytotoxicity (47). Because different tumor targets have variable retention times in the lungs of mice, we used a time course lung clearance assay to determine the optimum retention time for Jurkat cells (Fig. 3A). B6 mice are able to rapidly clear Jurkat targets from the lungs because their NK cells possess normal lytic activity. However, B6 mice treated with anti-asialo-GM1 Ab are defective in lung clearance because their NK cells have been depleted, as previously reported (44). Thus, significantly different
FIGURE 2. cFLIP overexpression in Jurkat cells inhibits NK cell activity. B6 (A) or B6 PKO (B) IL-2-activated LAK cells were used as effectors in a 4-h 51Cr release assay against labeled Jurkat or J.cFLIP tumor targets at a 200:1 E:T ratio. 10 g/ml anti-FasL mAb were added to effectors before addition of targets, where indicated. A, B6 LAK cell killing of Jurkat without anti-FasL mAb is significantly different from killing of Jurkat in the presence of the mAb (p ⬍ 0.05). In the absence of anti-FasL mAb, lysis of J.cFLIP is not significantly different from lysis of Jurkat (p ⬎ 0.05). Likewise, lysis of J.cFLIP is statistically identical with or without anti-FasL mAb addition (p ⬎ 0.05). B, B6 PKO LAK cell lysis of Jurkat cells is statistically different from the lysis of J.cFLIP in the absence of anti-FasL mAb (p ⬍ 0.05). In the presence of anti-FasL mAb, lysis of Jurkat is significantly different from lysis of J.cFLIP (p ⬍ 0.05).
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FIGURE 3. cFLIP overexpression inhibits NK cell-mediated clearance of Jurkat tumor from the lungs of PKO mice. A, Where indicated, one day before infusion, B6 mice were injected i.p. with 15 l of anti-asialo-GM1 to deplete NK cells. Mice were infused with 5 ⫻ 105 125I-labeled UdR-labeled Jurkat tumor cells. Lung clearance capability was assessed at the indicated time points after injection by determining the percentage of 125I-labeled UdR retention. Results are expressed as geometric means. B, Histograms show the expression of retroviral genes as determined by GFP levels in untransfected Jurkat and cFLIP-transfected Jurkat. One day before infusion, B6 mice were injected i.p. with 15 l of anti-asialo-GM1 to deplete NK cells and B6 PKO mice were injected i.p. with 300 g of anti-FasL mAb to inhibit Fas/FasL interactions. Mice were infused with 5 ⫻ 105 125I-labeled UdR-labeled Jurkat (left panel) or J.cFLIP (right panel) tumor targets. Lung clearance capability was assessed 4 h after injection by determining the percentage of 125I-labeled UdR retention. This experiment is representative of three experiments performed. Results are expressed as geometric means. Geometric mean values in the B6 plus anti-asialo-GM1 group challenged with Jurkat or J.cFLIP tumor targets are significantly different (p ⬍ 0.05) from those of the other groups, except the untreated and anti-FasL mAb treated B6 PKO groups challenged with J.cFLIP tumor targets. When challenged with Jurkat tumor cells, the geometric mean values in the B6 PKO group treated with anti-FasL mAb are significantly different (p ⬍ 0.05) from those in the B6 or untreated B6 PKO groups.
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anti-FasL mAb challenged with Jurkat tumor cells were 0.21 and 0.30, respectively ( p ⬍ 0.05); the geometric mean values of the untreated B6 PKO group and B6 PKO group treated with antiFasL mAb challenged with J.cFLIP tumor cells were 0.15 and 0.16, respectively ( p ⬎ 0.05). cFLIP overexpression in TAP-1 KO stem cells facilitates engraftment in perforin-deficient mice Previously, we have observed that B6 PKO mice maintain the ability to reject TAP-1 KO BMC (11, 12). Because Jurkat tumor cells overexpressing cFLIP can escape lysis by perforin-deficient NK cells, we also wanted to test whether retrovirally mediated expression of cFLIP in TAP-1 KO stem cells could inhibit acute NK cell-mediated rejection in perforin-deficient mice. To determine whether stem cells express any of the receptors necessary for death receptor-induced apoptosis, we purified c-kit⫹Lin⫺ progenitors from TAP-1 KO mice. After culturing for 4 days in rmIL-3, rmIL-6, and rmSCF, TAP-1 KO progenitors expressed high levels of Fas receptor and low levels of TRAIL-R2 (Fig. 4). Subsequently, we retrovirally infected purified TAP-1 KO progenitors in vitro with the MigR1 vector containing FLAG-tagged cFLIP and GFP or control vector containing GFP only and determined transduction efficiency (Fig. 5A). Approximately 30% of the cells were transduced with control retrovirus (GFP only) or cFLIP and GFP (cFLIP) retrovirus. The exogenous cFLIP expression was also detected by performing a Western blot using anti-FLAG Ab (Fig. 5B). GFP-only- or cFLIP-transduced TAP-1 KO progenitors were then injected into various lethally irradiated mice to test for acute rejection by NK cells (Fig. 5C). After 7 days, the spleens of recipient mice were analyzed by flow cytometry to assay for hematopoietic reconstitution of donor cells. Syngeneic TAP-1 KO hosts were reconstituted with the same percentage of retrovirally infected stem cells (cFLIP or GFP alone) that were injected, indicating high stem cell activity and expression of retrovirus-encoded genes. B6 hosts rejected both cFLIP- and control vector-infected TAP-1 KO stem cells, presumably via perforin-mediated cytotoxicity. Rejection of control vector-infected TAP-1 KO stem cells was also observed by B6 PKO hosts. However, B6 PKO recipients failed to completely reject cFLIP-expressing TAP-1 KO stem cells. Therefore, cFLIP overexpression in TAP-1 KO progenitors can decrease the rejection capacity of B6 PKO mice by preventing NK cell-mediated cytotoxicity through death receptor-induced apoptosis. To further support the idea that increased expression of cFLIP can render BMC resistant to NK cell-induced apoptosis, we analyzed the ability of various lethally irradiated hosts to reject whole donor BMC from GFP-only-expressing (⬃21%) or cFLIP-expressing (⬃54%) TAP-1 KO radiation BMC chimeras (Fig. 6, A and B, respectively). After 7 days, the recipient spleens were harvested and absolute numbers of GFP-only- or cFLIP-positive donor BMC were determined. As expected, only syngeneic control recipients were engrafted with donor-derived BMC containing GFP only, while B6 and B6 PKO mice were not engrafted (Fig. 6A). These findings are consistent with previously observed data (11, 12). In Fig. 6B, the spleens of syngeneic control mice had 100-fold more cFLIP-expressing donor-derived cells than B6 PKO recipients. This finding suggests that other killing pathways may exist or that the levels of cFLIP overexpression were not optimal for blocking rejection. Reconstitution of cFLIP-expressing TAP-1 KO donor BMC was 50-fold higher in B6 PKO mice than in B6 mice. These data confirm that, in the absence of the granule exocytosis pathway, the partial engraftment of TAP-1 KO BMC can occur with cFLIP overexpression. Moreover, this impairment in
FIGURE 4. Expression of Fas and TRAIL-R2 by purified c-kit⫹Lin⫺ TAP-1 KO progenitors. After culturing in rmIL-6, rmIL-3, and rmSCF for 4 days, the expression of Fas receptor (top histogram) or TRAIL-R2 (bottom histogram) on purified c-kit⫹Lin⫺ TAP-1 KO progenitors (solid line) was determined using an Ab to Fas or TRAIL-R2, respectively. Isotype control Ab is denoted by the filled histogram.
rejection capacity is a direct result of blocking the signal transduction pathway of death receptor-induced apoptosis.
Discussion BMC graft rejection is a major clinical concern. For years, NK cells have been known to be capable of mediating graft rejection of incompatible BMC in mouse models. However, the cytolytic mechanisms used by murine NK cells during BMC graft rejection are not well defined. Previously, we have shown that the presence of perforin may be critical for class I-negative BMC rejection by NK cells (11, 12). However, our prior data also indicated that other cytotoxic mechanisms exist. Therefore, we determined the involvement of death receptors during BMC graft rejection by murine NK cells. In this study, we have demonstrated that cFLIP can function as an inhibitor of death receptor-induced apoptosis by protecting tumor cells from NK cell lysis and by facilitating engraftment of MHC class I-deficient stem cells in the absence of perforin. These results provide additional insight into the cytotoxic effector mechanisms of NK cells, implicating both the granule exocytosis and the death receptor pathways. Although it has been suggested that strong allogeneic resistance is maintained in FasL-deficient mice (48), we have demonstrated that, in the absence of perforin, death receptors are involved in the acute rejection of MHC class I-deficient BMC grafts. Using mice doubly deficient in perforin and a death receptor ligand (such as FasL) as recipients of incompatible BMC grafts would precisely determine the involvement of these cytotoxic components in acute BMC rejection. However, previous studies have shown that these double-deficient mice (i.e., perforin/FasL) can become extremely ill and die early (49). Our experiments, which test the ability of B6 PKO mice to reject MHC class I-negative BMC overexpressing cFLIP, reveal the contribution of perforin- and death receptor-mediated pathways of cell death executed during acute NK cell-mediated rejection. Because rejection of allogeneic BMC is weaker than that of MHC class I-negative BMC (50), experiments exploring the resistance of allogeneic BMC overexpressing cFLIP by perforin-deficient mice should also be completed.
The Journal of Immunology
4235 Additional experiments will be required to address which specific death receptors mediate rejection by NK cells in the absence of perforin. There are several death receptor candidates. Although the use of the perforin/granzyme pathway appears to predominate, NK cells have been shown to use both Fas/FasL (7, 12) and TRAIL-R/TRAIL (9, 51) interactions to mediate cytotoxicity of tumor cells. Signaling through the TNF receptor can also be a potent NK cell cytotoxic component in the lysis of virally infected cells (52) and tumor cells (53). These findings illustrate the importance of these death receptors in target cell apoptosis by NK cells. Therefore, it is feasible that these same death receptors may also be involved in the acute rejection of MHC class I-deficient BMC by NK cells. Our studies implicate the granule exocytosis and death receptor pathways as the most potent cytolytic components of NK cellmediated rejection. However, cytokines may serve as an alternative regulator. Directly stimulating or suppressing NK cells, bystander cell types can secrete cytokines that alter the expression of genes encoding other cytokines, transcription factors, or cytotoxic mediators. Some cytokines that influence NK cell cytotoxicity are IFN␣, IL-2, IL-12, IL-15, and IL-18 (54 –58). Alternatively, cytokines secreted by NK cells can modulate hematopoiesis. NK cells can produce TNF-␣ and IFN-␥, which are potent inhibitors of hemopoietic stem cell colony-forming ability (59, 60). IFN-␥ can also increase the levels of Fas on the surface of target cells, which may encourage Fas-induced apoptosis (61). NK cells also secrete TGF, which is capable of inducing apoptosis in hematopoietic precursor cells (62, 63). Furthermore, TGF can down-modulate the expression of c-kit, the receptor for stem cell factor in hematopoietic precursor cells, which deprives the cells of stem cell factor survival signaling (64). Thus, it is possible that cytokines acting on or secreted by NK cells could be partially responsible for the rejection of class I-deficient BMC by NK cells in this study. We have demonstrated that, in the absence of NK cell-mediated perforin/granzyme release, incompatible BMC rejection can be reversed by inhibiting death receptor-induced apoptosis. Clinically, these results might be simulated by the use of anti-perforin drug treatment, such as chloroquine, and the transduction of the donor BMC with an anti-apoptotic molecule, such as cFLIP. Therefore, the expression of anti-apoptotic molecules in stem cells using retroviral gene therapy is a possible approach to render donor BMC resistant to rejection by host NK cells. However, a better understanding of the individual involvement of various death receptors in NK cell-mediated BMC rejection is required for the development of completely effective prevention of NK cell-mediated rejection. These data set a precedent for an improved immunoregulation of NK cells.
FIGURE 5. Engraftment in perforin-deficient mice of purified c-kit⫹Lin⫺ TAP-1 KO progenitor expressing exogenous cFLIP. A, Purified c-kit⫹Lin⫺ TAP-1 KO progenitors were cultured in rmIL-6, rmIL-3, and rmSCF for 4 days and infected with cFLIP-expressing (cFLIP) retrovirus or control (GFP only) retrovirus. Infected cells showed green fluorescence when analyzed by flow cytometry. B, The levels of cFLIP protein expressed by purified and cultured c-kit⫹Lin⫺ TAP-1 KO progenitors that had not been infected (Uninfected) or had been infected with a cFLIPexpressing (cFLIP-infected) retrovirus were determined by Western blot analysis. Cell lysates were resolved by SDS-PAGE under reducing conditions and immunoblotted with an Ab to the flag epitope present at the C terminus of the retrovirus-encoded cFLIP molecule. The 55-kDa band corresponds to cFLIPL form. C, Lethally irradiated hosts were infused with 8.5 ⫻ 105 purified c-kit⫹Lin⫺ TAP-1 KO progenitors that had been infected with a cFLIP-expressing (cFLIP) or control (GFP only) retrovirus in vitro. Host spleens were harvested 7 days after injection of donor progenitors to assess splenic hematopoietic cell repopulation. Growth of infected
donor cells is represented as the percentage of GFP-expressing spleen cells. The data shown represent the repopulation of one mouse of five mice with similar repopulation. The percentage of repopulation in the TAP-1 KO recipients receiving GFP-only-expressing stem cells is similar to the percentage of repopulation in the TAP-1 KO group receiving cFLIP-expressing stem cells (p ⬎ 0.05). The percentages of repopulation in the TAP-1 KO recipients receiving GFP-only- or cFLIP-expressing stem cells is significantly different from the B6 and B6 PKO groups receiving stem cells infected with GFP-only or cFLIP retrovirus (p ⬍ 0.05). The percentage of reconstitution in B6 or B6 PKO mice challenged with GFP-only-expressing stem cells is similar to the percentage of repopulation in the B6 group receiving cFLIP-expressing stem cells (p ⬎ 0.05). The percentage of reconstitution in B6 PKO mice challenged with control retrovirus-infected stem cells is significantly less than the percentage of repopulation in the B6 PKO group challenged with cFLIP-expressing stem cells (p ⬍ 0.05).
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Acknowledgments We express our appreciation to Dr. Maria Johansson for the critical reading of this manuscript, Margaret A. Morris for the use of BMC from a transduced chimera, and Silvio and Maria Pen˜ a for the maintenance of the animal facilities.
References
FIGURE 6. Engraftment in perforin-deficient mice of TAP-1 KO donor BMC expressing exogenous cFLIP. Lethally irradiated TAP-1 KO mice were infused with 5 ⫻ 105 purified and sorted c-kit⫹Lin⫺ syngeneic progenitors that had been infected with a control (GFP only) (A) or cFLIPexpressing (cFLIP) (B) retrovirus. The percentage of GFP expression (histogram) in the bone marrow of one chimera was determined by flow cytometry. Lethally irradiated hosts were then infused with 8 ⫻ 106 GFPonly-expressing (A) or cFLIP-expressing (B) donor BMC. Host spleens were harvested 7 days after injection of the indicated donor BMC to assess splenic hematopoietic cell repopulation. The mean ⫾ SEM absolute numbers of GFP-expressing donor BMC were determined. The percentages of donor-derived BMC reconstituted in the host groups (five mice per group) are presented in parentheses. A, The mean value of the B6 and B6 PKO groups are similar (p ⬎ 0.05). The mean value of the TAP-1 KO group is statistically different from the B6 and B6 PKO groups (p ⬍ 0.05). B, The mean value of the B6 group is significantly less (p ⬍ 0.05) than the B6 PKO and TAP-1 KO groups, and the B6 PKO group is significantly less (p ⬍ 0.05) than the TAP-1 KO group.
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