Unusual cytotoxic activities of thymus-independent, self-antigen-specific CD8+ T cells

Hisakata Yamada1–,3, Goro Matsuzaki2, Yukihide Iwamoto1 and Kikuo Nomoto2

1 Department of Orthopedic Surgery, Graduate School of Medical Sciences and
2 Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan

Correspondence to: H. Yamada, Section for Medical Inflammation Research, Department of Cell and Molecular Biology, Sölvegatan 19, 223 62 Lund, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We compared the cytotoxic activities of thymus-dependent and thymus-independent CD8+ T cells. Thymus-dependent CD8+ T cells, which are foreign antigen specific, acquired cytotoxic activity to tumor cells with a basal dose of the antigen peptides and to hybridoma cells expressing anti-TCR mAb only after differentiation into effector cytotoxic T lymphocytes (CTL). In contrast, thymus-independent CD8+ T cells, which have been shown to be self-antigen specific, never showed cytotoxic activity to the target cells with a basal dose of the self-antigen peptide, while they could lyse hybridoma cells expressing anti-TCR mAb even without prior antigenic stimulation. Furthermore, the ex vivo cytotoxic activity of thymus-independent CD8+ T cells was also observed against the target cells with high doses of the antigen peptides, which were not lysed by freshly isolated thymus-dependent CD8+ T cells. Thus it is revealed that thymus-independent, self-antigen-specific CD8+ T cells already acquire mature CTL functions in situ but have an increased threshold of TCR-mediated signaling for activation. These differences in cytotoxic activities between thymus-dependent and thymus-independent CD8+ T cells suggest distinct roles of the two subsets of CD8+ T cells in vivo.

Keywords: autoimmunity, cytotoxic T cells, nude mice, transgenic mice


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cytotoxic T lymphocytes (CTL) play important roles in host defense against intracellular pathogens and tumor cells. CTL activities are mainly attributed to CD8+ T cells which recognize viral or tumor antigen peptides on MHC class I molecules. Although naive CD8+ T cells are not equipped with efficient CTL functions, they mature into effector CTL which can effectively lyse virally infected cells or tumor cells after antigenic stimulation in an appropriate environment (13). It is also supposed that CTL specific for self-antigens can cause autoimmune diseases, once they arise.

Since immature thymocytes expressing self-antigen-specific TCR are deleted during development, self-antigen-specific CTL seem to hardly arise. However, recently, we have revealed that self-antigen-specific CD8+ T cells can develop via a thymus-independent pathway, by analyzing male H-Y antigen-specific TCR transgenic mice (H-Y TCR transgenic mice) (4). We also found that male antigen-specific T cells (T3.70+CD8+) in female transgenic mice developed only via a thymus-dependent pathway. Therefore, it is suggested that all of thymus-independent T cells are self-antigen specific, while thymus-dependent T cells are foreign antigen specific. Thymus-independent CD8+ T cells are also found in normal mice and are distinguishable from thymus-dependent ones by the expression of IL-2Rß chain (5). Numerous studies showed that T cells expressing self-specific TCR are frequently found among thymus-independent T cells in normal mice (610), which is consistent with our results of the TCR transgenic mice.

The thymus-independent, self-antigen-specific CD8+ T cells in the TCR transgenic mice showed neither proliferation nor cytokine production in response to antigenic stimulation (4). This may account for the absence of autoimmunity in the transgenic mice as well as normal mice. Nevertheless, they did proliferate and produce IFN-{gamma} after stimulation with anti-TCR mAb (4,11), suggesting that they are not simply in an anergic state but can function in certain situations, although the mechanisms of this discrepancy are still unclear. Moreover, CTL activities of the thymus-independent, self-antigen-specific CD8+ T cells have not been fully addressed yet.

In the present study, we compared the cytotoxic activities of thymus-dependent and thymus-independent CD8+ T cells in H-Y transgenic mice. The former is foreign antigen specific, while the latter is self-antigen specific. We found striking differences in CTL activities between thymus-dependent and thymus-independent CD8+ T cells, which suggest distinct roles of the two subsets of CD8+ T cells in vivo.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
The mice bearing transgenic TCR specific for male H-Y antigen peptide on H-2Db (H-Y transgenic mice) (12) were kindly provided by Dr T. W. Mak (AMGEN Institute, Toronto, Canada). C57BL/6 mice and BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). BALB/c nu/nu mice were purchased from Japan Crea (Tokyo, Japan). These mice were maintained in specific pathogen-free conditions and used at 10–14 weeks of age, with the exception of the BALB/c nu/nu mice which were used at 20 weeks of age.

Flow cytometric analysis
mAb used for flow cytometric analysis comprised FITC-conjugated T3.70 mAb (specific for transgenic TCR {alpha} chain, a kind gift from Dr T. W. Mak) (13), allophycocyanin-conjugated anti-CD8 mAb (53-6.7) (PharMingen, San Diego, CA), phycoerythrin (PE)-conjugated anti-TCR ß chain mAb (H57.597) (PharMingen) and FITC-conjugated anti-IL-2Rß (CD122) mAb (TM-ß1) (PharMingen). Freshly isolated or cultured cells were incubated with various combinations of mAb before being analyzed by a FACSCalibur flow cytometer with the CellQuest program (Becton Dickinson). Just before being run on the cytometer, we added of 50 µg/ml of propidium iodide (PI) to the cell suspensions to detect and exclude dead cells.

In vitro proliferation assay
Nylon wool non-adherent lymphocytes from naive female transgenic mice were adjusted to contain 1x104 T3.70+CD8+ T cells/well in 96-well plates. They were cultured with 5x105 irradiated spleen cells of male C57BL/6 mice or those of female mice with various doses of H-Y antigen peptide [sequence K-C-S-R-N-R-Q-Y-L (3)] (Genemed Synthesis, South San Francisco, CA). After 4 days, cells were harvested and live cells were analyzed by a flow cytometer by gating out PI+ cells. The numbers of surviving T3.70+CD8+ cells were calculated after the FACS analysis.

In vitro CTL generation
Nylon wool non-adherent spleen cells (5x105/ml) of female or male H-Y transgenic mice were cultured with 2.5x106/ml irradiated (30 Gy) spleen cells of male C57BL/6 mice in the presence of 20 U/ml of recombinant human IL-2 (a kind gift from Takeda Pharmaceutical, Osaka, Japan) in 24-well plates. After 5 days cells were harvested and used for cytotoxic assays.

Measuring cytotoxic activities
Cytotoxic activities were measured by a conventional 51Cr-release assay. Freshly isolated or 5-day cultured T cells of female or male H-Y transgenic mice were adjusted to contain the same number of T3.70+CD8+ cells/ml of cell suspensions after FACS analysis. They were incubated with 51Cr-labeled T3.70 hybridoma cells or 51Cr-labeled EL-4 lymphoma cells (H-2b) with or without H-Y peptide in triplicates at various effector:target ratios. H57.597 hybridoma cells were used as the target cells for freshly isolated T cells from C57BL/6 mice, BALB/c mice or BALB/c nu/nu mice. In some experiments, CD8+ cells or CD122+ cells were depleted immediately before the cytotoxic assay using Dynabeads M450 sheep anti-rat IgG (Dynal, Oslo, Norway) combined with anti-CD8 (2.43, rat IgG) or anti-CD122 (TM-ß1, rat IgG) mAb respectively. After 4 h, radioactivity of the supernatants was measured by a {gamma} counter. Cytotoxicity was estimated as percentage of specific lysis calculated as follows: specific lysis (%) = [(experimental release – spontaneous release)/(maximum release – spontaneous release)]x100. Maximal or spontaneous 51Cr release was obtained by incubation of target cells with 1% Triton X or medium only respectively. The data were expressed as the mean value of triplicate samples ± SD.

In vitro cytokine production assay
T cells in H-Y TCR transgenic mice were adjusted to contain 2x104 T3.70+CD8+ cells/well in 96-well plates and were cultured with 5x105 irradiated spleen cells of female C57BL/6 mice with various doses of H-Y antigen peptide. The culture supernatants were harvested after 24 or 96 h and IFN-{gamma} secreted in the supernatants was measured by an ELISA assay using mouse IFN-{gamma} DuoSeT (Genzyme, Cambridge, MA).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lack of cytotoxic activity of thymus-independent, self-antigen-specific CD8+ T cells to the self-antigen
In our previous report (4), we have shown that H-Y antigen-specific T cells (T3.70+CD8+) in female H-Y transgenic mice developed thymus dependently, while those in male mice developed thymus independently and the latter could neither proliferate nor produce IFN-{gamma} in response to male antigen. In this study, we first compared CTL activities of these two subsets of CD8+ T cells to male antigen. For the target cells, we used syngeneic EL-4 lymphoma cells (H-2b) pulsed with 0.1 µM of H-Y antigen peptide, since the cytotoxic activity of effector CD8+ T cells in female H-Y transgenic mice had been clearly demonstrated using syngeneic tumor cells pulsed with 0.1 µM of H-Y antigen peptide as target cells (3). Moreover, we also confirmed that <0.1 µM of H-Y antigen peptide pulsed on syngeneic female stimulator cells elicited comparable levels of proliferative response from T cells in female H-Y transgenic mice with those elicited by male stimulator cells (Fig. 1Go). Therefore, the amounts of H-Y antigen peptides presented on male stimulator cells may not be >0.1 µM. As shown in Fig. 2Go(A), freshly isolated T cells in both female and male H-Y transgenic mice did not show any CTL activity to male antigen. However, after in vitro culture with male stimulator cells for 5 days, T cells of female mice showed apparent CTL activity to male antigen, while those of male mice did not (Fig 2BGo). These data clearly showed that thymus-independent, self-antigen-specific CD8+ T cells failed to acquire CTL activity to the self-antigen even after in vitro stimulation.



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Fig. 1. Proliferative response of T cells in female H-Y TCR transgenic mice to male spleen cells or male antigen peptide. T cells from naive female H-Y transgenic mice were cultured with spleen cells of male C57BL/6 mice ({circ}) or female spleen cells with various doses of male H-Y antigen peptide (•). After 4 days, the numbers of T3.70+CD8+ cells were calculated after FACS analysis.

 


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Fig. 2. Cytotoxic activities of T cells in H-Y transgenic mice to male antigen. Freshly isolated (A) or 5-day cultured (B) T cells from female (•) or male ({circ}) H-Y transgenic mice were adjusted to contain equal numbers of T3.70+CD8+ cells/ml of cell suspensions after FACS analysis. They were incubated with 51Cr-labeled EL-4 lymphoma cells (H-2b) and 0.1 µM of H-Y antigen peptide in triplicates at various effector:target ratios as indicated. The radioactivity of the supernatants was measured after 4 h and the cytotoxic activity was calculated. Data are expressed as the mean ± SD.

 
Ex vivo CTL activity of thymus-independent, self-antigen-specific CD8+ T cells to anti-TCR mAb
Since we showed in the previous report that, although male antigen-specific T cells in male H-Y transgenic mice did not respond to male antigen, they could proliferate and produce IFN-{gamma} after anti-TCR mAb stimulation (4). Therefore, we next examined CTL activity of these cells by a redirected CTL assay (14) using T3.70 hybridoma cells which express the transgenic TCR-specific mAb as the target cells (Fig. 3Go). CD8+ T cells in female H-Y transgenic mice acquire cytotoxic activity to T3.70 hybridoma cells only after in vitro culture with male antigen, similar to the case of CTL activity to the target cells with male antigen. In contrast, freshly isolated CD8+ T cells in male H-Y transgenic mice showed significant CTL activities to T3.70 hybridoma cells as well as in vitro cultured CD8+ T cells in male H-Y transgenic mice. This indicates not only that thymus-independent, self-antigen-specific CD8+ T cells have a potential to mature into effector CTL, but rather that they had already acquired CTL functions in situ.



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Fig. 3. Redirected cytotoxic activities of T cells in H-Y transgenic mice to anti-TCR mAb. Cytotoxic activities of freshly isolated (A) or 5-day cultured (B) T cells from female (•) or male ({circ}) H-Y transgenic mice to T3.70 hybridoma cells expressing the transgenic TCR-specific mAb were measured. The CD8-depleted T cells in male mice ({square}) were adjusted to contain equal numbers of T3.70+CD8 cells/ml of cell suspensions as the whole T cells in male mice. Data are expressed as the mean ± SD.

 
Ex vivo CTL activity of thymus-independent CD8+ T cells in normal mice to anti-TCR mAb
We next addressed whether the ex vivo CTL activity observed in male antigen-specific CD8+ T cells in male H-Y transgenic mice is a common feature of thymus-independent CD8+ T cells. It has been reported that expression of IL-2Rß chain (CD122) is a suitable marker for thymus-independent T cells (5). Male antigen-specific CD8+ T cells in male H-Y transgenic mice are also CD122+ (4). So, we examined CTL activities of freshly isolated spleen cells in normal C57BL/6 mice depleted or not depleted of CD122+ cells (Fig. 4AGo). Spleen cells of C57BL/6 mice showed cytotoxic activities to H57-597 hybridoma expressing anti-TCR {alpha}ß mAb, but the cytotoxic activities was lost when CD122+ cells were depleted. CD122+ cells mediating the ex vivo CTL activity express CD8, because spleen cells of C57BL/6 mice depleted of CD8+ cells failed to lyse H57-597 hybridoma. It has been reported that CD44high `memory phenotype' CD8+ T cells in naive mice are CD122+ (15), thus it is possible that CD8+CD122+ cells mediating the ex vivo cytotoxic activity are memory CD8+ CTL, but not thymus-independent CD8+ T cells. To confirm the thymus-independent origin of CD8+CD122+ cells with ex vivo cytotoxic activity, we compared redirected CTL activity of spleen cells of athymic nude mice to anti-TCR mAb expressing-hybridomas with that of normal mice. As shown in Fig. 4Go(B), TCR{alpha}ß+ cells in BALB/c nude mice showed significant ex vivo CTL activities to H57-597 hybridoma cells, which was ~4-fold stronger than those of euthymic BALB/c mice. Interestingly, only one-fifth of TCR{alpha}ß+CD8+ cells in euthymic BALB/c mice express IL-2Rß, while all of TCR{alpha}ß+CD8+ cells in athymic nude mice are IL-2Rß+ (data not shown). Similar to the case of CTL activity of CD122+ T cells in euthymic mice (Fig. 4AGo), the ex vivo CTL activity of the splenocytes of nude mice was lost when they were depleted of CD8+ T cells, indicating that the CTL activity was mediated by CD8+ cells (data not shown). These results revealed that ex vivo CTL activity to anti-TCR mAb stimulation is a common feature of thymus-independent CD8+ T cells but not a special feature of self-specific TCR transgenic T cells.



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Fig. 4. Redirected cytotoxic activities of T cells in normal mice to anti-TCR mAb. (A) Cytotoxic activities of freshly isolated whole (•), CD4-depleted ({circ}), CD8-depleted ({blacksquare}) and CD122-depleted ({square}) T cells from C57BL/6 mice to H57.597 hybridoma cells expressing anti-TCR{alpha}ß mAb were measured. (B) Cytotoxic activities of freshly isolated T cells from BALB/c mice (•) and BALB/c nude mice ({circ}) to H57.597 hybridoma cells were measured. Data are expressed as the mean of triplicates ± SD.

 
Increased threshold of antigen doses for CTL activities of thymus-independent, self-antigen-specific CD8+ T cells
Recently, it was reported that self-antigen-specific CD8{alpha}{alpha} iIEL T cells have an increased threshold of TCR-mediated signaling for activation (16). It is of note that CD8{alpha}{alpha} iIEL T cells have been shown to develop thymus independently, like CD8{alpha}ß T cells in liver or spleen (17,18). This raises a possibility that the different CTL activity of male antigen-specific CD8+ T cells in spleens of male H-Y transgenic mice to male antigen and anti-TCR mAb may result from the differences in the strength of TCR-mediated signaling. Consistent with this hypothesis, both freshly isolated and 5-day cultured CD8+ T cells in male mice could lyse the target cells with a high dose of male antigen peptides (1–10 µM) (Fig. 5Go). There was no difference in the activation threshold of peptide doses between fresh and cultured male T cells. Therefore, the difference in the stimulating potential between male spleen cells and anti-TCR mAb may be attributable to the difference in the strength of TCR-mediated signaling. Importantly, freshly isolated T cells in female mice did not lyse the target cells with various dose of male antigen peptide, while 5-day cultured effector T cells from female mice required lower doses of male antigen peptides to lyse the target cells than male T cells (Fig. 5Go). Both female and male T cells also produced IFN-{gamma} after stimulation with the antigen peptide (Fig. 6Go). However, similar to the case of cytotoxic activity, the female T cells required several days to produce IFN-{gamma}, while male T cells rapidly produced significant amounts of IFN-{gamma} within 24 h after stimulation, although they required higher doses of the antigen peptide than female T cells. These results indicated that thymus-independently-developed, self-antigen-specific CD8+ T cells which posses effector functions in situ have an increased threshold of TCR stimulation for activation than thymus-dependently-developed, foreign antigen-specific CD8+ T cells.



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Fig. 5. Cytotoxic activities of T cells in H-Y transgenic mice to various doses of male antigen peptide. Cytotoxic activities of freshly isolated (A) or 5-day cultured (B) T cells from female (•) or male ({circ}) H-Y transgenic mice to EL-4 lymphoma cells with various doses of H-Y antigen peptide were measured. The effector:target ratio was 10 in all groups. Data are expressed as the mean ± SD.

 


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Fig. 6. IFN-{gamma} production of T cells in H-Y transgenic mice to various doses of male antigen peptide. T cells from female (•) or male ({circ}) H-Y transgenic mice were cultured with various doses of H-Y antigen peptide. Culture supernatants were harvested after 24 (A) or 96 (B) h. Amounts of IFN-{gamma} in the supernatants were measured by ELISA.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present study, we revealed several differences in cytotoxic activities between thymus-dependent CD8+ T cells and thymus-independent CD8+ T cells. The first is that thymus-dependent CD8+ T cells require prior antigenic stimulation to mature into effector CTL, while freshly isolated, thymus-independent CD8+ T cells already have mature CTL functions in situ. Consistent with this result, freshly isolated T3.70+CD8+ cells in male but not female H-Y transgenic mice express perforin mRNA (data not shown). Interestingly, we and others have shown that thymus-independent T cells express high levels of CD44 similar to activated/memory T cells (4,5). Additionally, thymus-independent CD8+ T cells in both TCR transgenic mice and normal mice rapidly produce IFN-{gamma} after TCR cross-linking with anti-TCR mAb without prior priming (4,19). Thus, thymus-independent CD8+ T cells not only phenotypically but also functionally resemble activated/memory T cells. However, these T cells may not be memory T cells, since thymus is indispensable in generating antigen-specific T cell responses, indicating the thymic origin of memory T cells (20). Lack of antigen-specific T cell response of athymic mice may partly be due to the self-specificity of the T cells in these mice. This also suggests the thymic origin of memory T cells since memory T cells are theoretically foreign antigen specific. Furthermore, if thymus-independent CD8+ T cells are derived from conventional naive CD8+ T cells, it seems unreasonable that athymic mice lack naive phenotype T cells (5).

Interestingly, CTL activity to hybridomas expressing anti-TCR mAb and expression of perforin mRNA have been shown in freshly isolated iIEL T cells expressing CD8{alpha}{alpha} homodimer, which develop thymus independently (14,17,18). The ex vivo cytotoxic activity of TCR {alpha}ß+ or {gamma}{delta}+ iIEL T cells in athymic nude mice was also observed (21,22). Furthermore, from the analysis of TCR transgenic mice, CD8{alpha}{alpha} iIEL was shown to be positively selected by self-antigen (23,24), similar to thymus-independently-developed CD8+ T cells in spleen and liver (4), although the latter express CD8{alpha}ß heterodimer. Additionally, self-super antigen-specific CD8 T cells are frequently found in iIEL in normal mice (9,10). Therefore, mature CTL function is a common feature of thymus-independent, self-antigen-specific CD8+ T cells.

In contrast to the redirected CTL activities to anti-TCR mAb, thymus-independent, self-antigen-specific CD8+ T cells never showed CTL activity to conventional levels of antigenic stimulation (<0.1 µM of peptides) which can activate the thymus-dependently-developed effector CD8+ T cells of the same TCR specificity. However, the self-antigen-specific CD8+ T cells can lyse the target cells with higher amounts of the self-antigen peptides. We also showed that they produce IFN-{gamma} in response to a high dose of the antigen peptides (Fig. 6Go). Thus, it is speculated that the increased threshold of TCR-mediated signaling for activation works as a mechanism preventing autoimmunity caused by the self-antigen-specific CD8+ T cells. This is consistent with the absence of autoimmunity in male H-Y transgenic mice, as well as normal mice which also harbor thymus-independent, self-antigen-specific CD8+ T cells. It is also supposed that the thymus-independent self-antigen-specific CD8+ T cells become autoaggressive, if the expression level of the self-antigen peptide–class I complexes are unusually up-regulated. Actually, CTL activities of self-antigen-specific T cells have also been suggested to be involved in the autoaggressive pathology observed in several infection models which induce up-regulation of class I molecules (25,26). Interestingly, it was reported that T cells specific for tyrosinase-associated protein-2 (TRP-2), which is expressed in melanocytes and thus is regarded as a self-antigen, were detected in spleens of naive mice, only when they are stimulated with high doses of the antigen peptides (27). Furthermore, we have also found that the T cells reactive to high doses of TRP-2 peptide are detected in spleens of naive athymic mice (M. Harada et al., manuscript submitted). Therefore, It is suggested that the increased threshold of TCR-mediated signaling is a common characteristic of thymus-independent, self-antigen-specific T cells, but is not a special feature of the TCR transgenic T cells.

From a point of view of host defense, thymus-independent CD8+ T cells may work as a first line of host defense against infection, because they can rapidly exert effector functions without prior priming when the expression levels of self-antigen peptide–class I complexes are unusually up-regulated. Up-regulation of class I molecules were observed at various sites of infection, which are induced directly by pathogens themselves or indirectly via inflammatory cytokines (2831). It is important to note that, in these conditions, thymus-independent T cells may work in an antigen-independent manner of the invading pathogen, since they express self-antigen-specific TCR. Furthermore, we have recently reported that thymus-independent CD8+ T cells are activated by some inflammatory cytokines in an antigen-independent manner (32). Therefore, these T cells may play roles as a component of the innate immune system, which can rapidly respond to some inflammatory stimuli elicited by infection. On the other hand, thymus-dependent, foreign antigen-specific CD8+ T cells can work specifically to the invading pathogens after clonal expansion and differentiation. Thus, they should play important roles as an effector of acquired immune response but can only work at a relatively late stage of infection and also at the time of the re-infection of the same pathogen. It is important to clarify the in vivo relevance of the thymus-independent CD8+ T cells in host defense, which is now under investigation.


    Acknowledgments
 
This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. We are grateful to Dr T. W. Mak for providing us with H-Y transgenic mice and hybridoma T3.70.


    Abbreviations
 
CTL cytotoxic T lymphocyte
H-Y TCR mice transgenic for male H-Y antigen-specific transgenic mice TCR
iIEL intestinal intraepithelial lymphocytes
PE phycoerythrin
PI propidium iodide

    Notes
 
3 Present address: Section for Medical Inflammation Research, Department of Cell and Molecular Biology, Sölvegatan 19, 223 62 Lund, Sweden Back

Transmitting editor: H. R. MacDonald

Received 24 May 2000, accepted 24 August 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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