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The Journal of Immunology, 2007, 178: 1542-1552.
Copyright © 2007 by The American Association of Immunologists, Inc.

B7-Deficient Autoreactive T Cells Are Highly Susceptible to Suppression by CD4+CD25+ Regulatory T Cells1

Kenneth F. May, Jr.*, Xing Chang*,{dagger}, Huiming Zhang*,{dagger}, Kenneth D. Lute*, Penghui Zhou{dagger}, Ergun Kocak*, Pan Zheng*,{dagger} and Yang Liu2,*,{dagger}

* Department of Pathology, Divisions of Cancer Immunology and Dermatology, Ohio State University Medical Center and Comprehensive Cancer Center, Columbus, OH 43210; and {dagger} Department of Surgery, Division of Immunotherapy, Comprehensive Cancer Center and Program of Molecular Mechanism of Diseases, University of Michigan, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4+CD25+ regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4+CD25+ T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is well established that CD4+CD25+ regulatory T cells (Tregs)3 are potent inhibitors of a variety of immune responses, including innate immune responses (1) and T cell responses to infection (2, 3, 4), tumors (5, 6), and perhaps most importantly autoantigens (7, 8, 9, 10). Numerous studies have demonstrated that the effector function of Tregs is manifested at various aspects of T cell activation, including cytokine production, proliferation, and inflammation of target tissues (11, 12, 13, 14). Although several studies have demonstrated that cognate interactions between regulatory and effector T cells are essential for immune suppression, the critical molecular interaction for T-T suppression has yet to be identified (15, 16).

The interaction between B7 and CTLA-4 has attracted considerable interest for several reasons. First, Tregs express high levels of the costimulatory molecule CTLA-4, and it has been suggested that CTLA-4 may play an essential role in the suppressor function of Tregs (17, 18). Second, T cells express considerable levels of cell surface B7-1 and/or B7-2, especially after activation (19). The function of B7-1 and B7-2 has not been adequately explained. Third, and perhaps more intriguing, are the recent reports that CTLA-4 can induce a "reverse signaling" via B7-1/2 expressed on dendritic cells (DC) (20, 21). In this regard, it is worth noting that B7-2 and possibly a B7-1 splicing variant have a long cytoplasmic tail with potential phosphorylation sites (22). Gavin and Rudensky (23) proposed that interaction between CTLA-4 on Tregs and B7 on effector T cells may be the major suppressor bridge. In refuting this hypothesis, Tang et al. (24) showed that CTLA-4 is not required for the development and function of Tregs. In contrast, Paust et al. (25) reported that expression of B7-1/2 on effector cells is required for susceptibility to Treg function. Therefore, the importance of B7-CTLA4 interaction in Treg function remains unsettled.

We recently observed that B7-deficient T cells cause multiorgan inflammation in vivo in syngeneic mice. The availability of activated autopathogenic T cells provided us with an opportunity to test whether expression of B7-1 and B7-2 on the effector T cells is essential for their susceptibility to Tregs. In this study, we report our systematic analysis of the effect of Tregs on the activation and pathogenicty of B7-deficient autoreactive T cells. Our results demonstrate that in both in vivo and in vitro models, B7-deficient T cells are highly susceptible to suppression by wild-type (WT) Tregs. Our results are consistent with the data that CTLA-4 is unnecessary for Treg function. Taken together, these data do not support the hypothesis that interaction between CTLA-4 on Tregs and B7 on effector T cells is essential for execution of immune regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Experimental animals

RAG-1–/– (26) and B7-1–/– B7-2–/– mice (27), which have been backcrossed to the C57BL/6J strain for at least 10 generations, were purchased from The Jackson Laboratory. C57BL/6J mice were purchased from the Charles River Animal Facility. All mice were maintained in the University Laboratory Animal Research Facility at the Ohio State University under specific pathogen-free conditions.

Flow cytometry

Cell surface markers, including CD4, CD8, CD25, CD44, and CD62L, and intracellular cytokines, including IFN-{gamma} and IL-4 and FoxP3, were analyzed with conjugated mAbs purchased from BD Pharmingen. Cell samples were analyzed on a BD FACSCalibur.

Treg purification

WT CD4+CD25+ cells were purified from pooled spleens and lymph nodes of normal C57BL/6J mice in two steps. First, CD4+ cells were isolated by negative selection using a mixture of Abs that react to CD8, FcR, Mac-1, CD11c, and B220, followed by anti-rat IgG-coated Dynabeads (Dynal Biotech), according to manufacturer’s instruction. CD25+ cells were then isolated from the CD4+ population by positive selection using PE-conjugated or allophycocyanin-conjugated anti-CD25 Ab (BD Pharmingen) followed by anti-PE or anti-allophycocyanin MACS beads (Miltenyi Biotec). In some cases, the CD4+CD25– fraction was used for proliferation assays.

Proliferation assay

CD4+CD25+ cells were purified from pooled spleens and lymph nodes of normal C57BL/6J mice as described above to use as suppressor cells. The CD4+CD25– fraction was used as responder cells. CD4+CD25– cells from healthy B7-deficient mice showing no signs of alopecia or skin disease were purified in the same manner. CD4+CD25– responder cells from either WT or B7-1–/–B7-2–/– mice were cultured at 5 x 104 cells/well with 1 µg/ml soluble anti-CD3 mAb and 1 x 105 irradiated spleen cells from WT mice. CD4+CD25+ cells were added at 2-fold titrations. Cells were cultured for 69–72 h, and [3H]TdR was added at 1 µCi/well for the last 6 h of culture. Cells were harvested and counted on a beta counter. Suppressive activity was calculated using the following formula: 100% x (cpmresponder – cpmresponder + suppressor)/cpmresponder.

Adoptive transfer

A total of 20 x 106 spleen and lymph node cells from age-matched WT and B7-1–/–B7-2–/– mice was injected i.p. into RAG-1–/–B7+/+ C57BL/6J mice. Purified Tregs were injected at 2.7 x 106 cells/mouse in conjunction with 20 x 106 spleen and lymph node cells from B7-deficient mice into RAG-1–/–B7+/+ C57BL/6J mice. Recipient mice were sacrificed for histological analysis when clinical symptoms of morbidity appeared.

Intracellular cytokine production

To assess intracellular cytokine production, spleen cells were cultured for 4 h with 50 ng/ml PMA, 500 ng/ml ionomycin, and 2 µM GolgiStop (BD Pharmingen). Cells were stained for cell surface markers CD4 and CD8 followed by intracellular staining for IFN-{gamma}, IL-4, or IL-10 using a CytoFix/CytoPerm kit (BD Pharmingen).

T cell proliferation in lymphopenic host

Total spleen T cells were purified from B7–/– mice and labeled with CFSE. CFSE-labeled T cells (2 x 106) were mixed with 2 x 106 of CD4+CD25+ or CD4+CD25– T cells from WT mice and injected i.v. into WT or B7-deficient mice irradiated with 500 rad. The recipient mice were sacrificed at 5 days after adoptive transfer, and spleen cells were stained with Abs specific for CD4 and CD8 markers.

Histological analysis

Mouse organs were fixed with 10% buffered formalin and paraffin embedded. Tissue sections were stained with H&E and examined under a microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
T cells activated in B7-deficient mice cause multiorgan inflammation in syngeneic RAG-1-deficient mice

In the process of studying mice with targeted mutations of B7-1 and B7-2, we observed a high incidence of cutaneous abnormalities in aged mice (data not shown). This was usually preceded by a progressive enlargement and activation of spleen and lymph nodes (data not shown). To determine whether T cells in the B7-deficient mice were pathogenic, we isolated and transferred spleen cells from diseased B7-deficient and sex-matched C57BL/6J mice into RAG-1–/–B7+/+ C57BL6/J recipients. At ~7 wk after the adoptive transfer when some recipients started to show clinical signs of illness, including weight loss, hair loss, and wobbly gait, all recipient mice were sacrificed for immunological and histological analyses. With the exception of sporadic low-grade hair loss, which was seen occasionally in RAG-1–/– C57BL6/J mice that received no adoptive transfer, the recipients of the normal spleen cells showed no clinical signs during the entire 7-wk period observed.

In comparison to the spleen cells before the adoptive transfer (Fig. 1a), T cells from both WT and B7-deficient mice underwent further activation after adoptive transfer, as demonstrated by the increase of CD44+CD62L+ and CD44+CD62L– T cells (Fig. 1b). However, the CD8 T cells from B7-deficient donors underwent more extensive activation as judged by the increased accumulation of CD44+CD62L– T cells. We also compared T cells recovered from recipients for their cytokine production after short-term stimulation in vitro. As shown in Fig. 1c, the CD4 T cells from WT and B7-deficient donors consisted of comparable proportions of IFN-{gamma}-producing cells. However, a high proportion of CD4 T cells from B7-deficient mice gained the ability to produce IL-4.


Figure 1
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  		FIGURE 1. Enhanced activation of B7–/– spleen cells after transfer to RAG-1–/–B7+/+ recipients. Cell surface staining of spleen cells from healthy WT and diseased B7–/– mice (KO) before (a) and after (b) adoptive transfer into RAG-1–/–B7+/+ recipients. Both CD4 and CD8 subsets display increased activation after adoptive transfer, as indicated by the CD44+CD62L– population. However, CD8 T cells from recipients of B7–/– spleen cells have undergone more activation than CD8 T cells from recipients receiving WT spleen cells. c, Spleen cells from recipient RAG-1–/– mice were stimulated in vitro with PMA and ionomycin for 4 h and stained for intracellular cytokines. Solid lines in histogram depict stain with cytokine-specific mAb while dotted lines represent isotype controls. The numbers in the panels are percentage of cells within the gate after subtracting those stained by isotype control. Data shown were profiles of pooled cells from five mice in each group and have been repeated once.

 
Given the signs of morbidity observed in several mice, we examined whether internal organs were affected by the transferred spleen cells. As shown in Fig. 2 and Table I, mice that received spleen cells from WT mice had essentially no inflammation in any organs examined, including lung, liver, kidney, pancreas, heart, salivary glands, spinal cord, and intestine. In contrast, in mice that received spleen cells from B7-1–/–B7-2–/– mice, extensive inflammation was found in lung, liver, pancreas, and intestine, although no inflammation was observed in heart, salivary glands, or spinal cord tissues (data not shown). Destruction of pancreas and collapse of lung alveoli were observed frequently among the mice that received B7-deficient T cells.


Figure 2
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  		FIGURE 2. Spleen cells from diseased B7–/– mice cause multiorgan inflammation. Spleen cells (20 x 106) from diseased B7–/– mice (KO) or healthy WT mice were transferred into RAG-1–/–B7+/+ recipients. Seven weeks after transfer when mice began to demonstrate clinical symptoms, all mice were sacrificed. Organs were H&E stained and examined for inflammation. Data shown are representative of five mice in each group.

 

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  		Table I. Multiorgan inflammation induced by mononuclear cells from B7-deficient micea

 
To determine the subset of T cells involved in the development of multiorgan inflammation, CD4 and CD8 T cells isolated from either WT mice or diseased B7-deficient mice were transferred into RAG-1–/–B7+/+ mice. At 7 wk after the adoptive transfer when some of the recipient mice became moribund, all mice were sacrificed, and spleen and other organs were harvested. Despite the potential for homeostatic proliferation, mice that received CD4 T cells alone had very few CD8 T cells and vice versa (Fig. 3a). These results verified the purity of the T cells used for the adoptive transfer. Moreover, compared with the phenotype of the starting populations (Fig. 1a), both CD4 and CD8 T cell subsets had undergone further activation in the recipients as revealed by the increase of the CD44+CD62L– population regardless of whether the two subsets were cotransferred (Fig. 3b). Likewise, cytokine synthesis after short-term in vitro culture was not affected by cotransfer of the two subsets (Fig. 3c). Interestingly, only the group that received CD8 T cells exhibited pronounced lymphadenopathy (data not shown). These results demonstrated that CD8 T cells can be activated regardless of the presence of CD4 T cells and vice versa.


Figure 3
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  		FIGURE 3. T cells from diseased B7–/– mice display an activated effector phenotype in RAG-1–/–B7+/+ recipients. Seven weeks after transfer of 2 x 106 CD4, 3 x 106 CD8, or 5 x 106 CD4+CD8 T cells, spleens were harvested and stained for activation markers and intracellular cytokines. a, Purity of transferred T cells was maintained after 7 wk in vivo. As observed with total spleen cell transfer, most CD4 and CD8 T cells displayed an ex vivo-activated phenotype of CD44+CD62L– (b), and substantial fractions of these cells produce IFN-{gamma} after PMA and ionomycin stimulation in vitro for 4 h (c). Solid lines in histogram depict stain with cytokine-specific mAb while dotted lines represent isotype controls. The numbers in the panels are percentage of cells within the gate after subtracting those stained by isotype control. Data shown are from pooled cells from groups with four to five mice each.

 
Histologically, mice that received CD4 T cells alone and CD4+CD8 T cells developed inflammation in multiple organs, including lung, liver, pancreas, and intestine. The severity and pattern of inflammation were comparable between the two groups (Fig. 4 and Table I). In addition, extensive destruction of pancreas and lung were observed in both groups. In contrast, despite their enhanced activation phenotype (Fig. 3b), the CD8 T cell subset alone induced very limited inflammation in the RAG-1–/– recipients with notable absence of inflammation in the pancreas and intestine (Fig. 4 and Table I). The inflammation in the liver was very focal. These results demonstrate that the inflammation was mediated primarily by CD4 T cells, although some inflammation could be found in the group that received CD8 T cells alone.


Figure 4
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  		FIGURE 4. Purified T cells from diseased B7–/– mice cause multiorgan inflammation. T cells purified from spleen and lymph nodes were transferred into RAG-1–/–B7+/+ recipients, and organs were fixed and H&E stained 7 wk later. CD4 and CD4+CD8 T cell transfers caused substantial inflammation in lung, liver, pancreas, and intestine, whereas CD8 T cells alone caused no inflammation in pancreas and intestine and minimal inflammation in the lung. Data shown are representative of two independent experiments.

 
Tregs suppress multiorgan inflammation mediated by pathogenic B7-deficient T cells

Thus far, we have demonstrated that T cells from the spleens of B7-deficient mice are highly pathogenic when transferred into RAG-1–/–B7+/+ mice. As shown in Fig. 5a, T cells from unstimulated WT mice expressed B7-1 and, at a higher level, B7-2 on the cell surface. As shown in Fig. 5b, B7-deficient mice had a drastic reduction of CD25+FoxP3+ cells. In addition, while most of the WT CD4+CD25+ cells expressed FoxP3, a major proportion of the FoxP3+ cells from the B7-deficient mice lack CD25 expression. Therefore, we purified CD4+CD25+ T cells from WT mice to test their ability to suppress T cell function. As shown in Fig. 5c, the Treg preparation was >95% pure with few non-CD4 T cells (<2%), and most of the WT CD4+CD25+ cells expressed FoxP3 (Fig. 5b). Moreover, as has been previously reported (17, 18), a substantial proportion of Tregs express intracellular CTLA-4 (Fig. 5c). To test whether the addition of Tregs could blunt the autoimmune multiorgan inflammation caused by B7–/– cells, we cotransferred WT CD4+CD25+ T cells purified from WT C57BL/6J mice with B7-1/2–/– spleen and lymph node cells into RAG-1–/– mice. At 6 wk after transfer, when mice receiving B7-deficient cells began to show clinical symptoms of illness (weight loss and wobbly gait), all mice were sacrificed for analysis of T cell function and for the presence of inflammation in various organs.


Figure 5
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  		FIGURE 5. Phenotypic and functional characterization of effector and regulator cells. a, Basal expression of B7-1 and B7-2 by unstimulated T cells from WT but not B7-deficient mice. Solid lines represent B7-1 or B7-2 staining, whereas dashed lines represent isotype control stains. Plots represent spleens pooled from four mice per group and has been repeated at least three times. b, Reduced number and altered phenotype of Tregs in B7-deficient mice. The profiles of CD4 and CD8 T cell subsets are shown in the left panel; the FoxP3+CD4+ subsets and the FoxP3+CD4+CD25+ subsets are shown in the middle panels, whereas the summary data from three mice per group were shown in the right panels. Data shown represent those from three independent experiments. c, Purity of Tregs used for adoptive transfer. Spleen and lymph nodes from 12 mice were pooled. The CD4 T cells were first purified by negative selection. PE-anti-CD25 Ab was added to CD4 T cells and those that bound to the Abs were isolated by MACS beads. After two rounds of selection, the purified cells were restained with FITC-anti-CD4 Ab, and purity was determined by flow cytometry. Expression of intracellular CTLA-4 by naive Tregs was measured flow cytometry. Solid line in the histogram represents intracellular CTLA-4 staining, whereas the dashed line represents isotype control stain. Plot represents Treg purified from spleens and lymph nodes of four C57BL/6J mice. d, Tregs decreased lymphoproliferation of B7-1/2–/– spleen and lymph node cells transferred into RAG-1–/– recipients. Bars represent the number of CD4 and CD8 T cells per lymph node. Data shown are derived from pools of 8–10 anatomically matched lymph nodes from four to five mice. These results are representative of two independent adoptive transfer experiments.

 
As shown in Table II and Fig. 6, all mice receiving spleen and lymph node cells from diseased B7-deficient mice had extensive inflammation in multiple organs, particularly in the lung, liver, and intestine. Mice receiving B7–/– cells plus Tregs had minimal organ inflammation, similar to the amount observed in control mice receiving spleen and lymph node cells from healthy WT mice (Fig. 3 and Table I). These results demonstrate that B7-deficient autoreactive T cells are highly susceptible to suppression by Tregs from WT mice.


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  		Table II. Tregs protect RAG-1–/– recipients from multiorgan inflammation mediated by B7-deficient spleen and lymph node cellsa

 

Figure 6
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  		FIGURE 6. Tregs suppress multiorgan inflammation mediated by pathogenic B7-deficient T cells. A total of 20 x 106 spleen and lymph node cells from diseased B7-1/2–/– mice or healthy WT mice was transferred into RAG-1–/–B7-1/2+/+ recipients. One group of recipients of the B7-deficient cells also received 2.7 x 106 CD4+CD25+ T cells isolated from spleens and lymph nodes of WT B7-1/2+/+ C57BL/6J mice. Six weeks after transfer when the mice began to demonstrate clinical symptoms, all mice were sacrificed. Organs were H&E stained and examined for inflammation. Lung, liver, and intestine from B7-1/2–/– cell recipients had numerous inflammatory foci compared with WT cell recipients. Inflammatory infiltration was observed mainly surrounding bronchi and blood vessels of the lung, the portal tracts, and central venules of the liver and in the submucosa of the intestine. Mice receiving Tregs in addition to B7-1/2–/– cells showed minimal inflammation, similar to WT cell recipients. These results are representative of two independent adoptive transfer experiments.

 
Tregs blunt the lymphoproliferation, activation, and cytokine production by pathogenic B7-deficient T cells

When mice were sacrificed for histological analysis, lymph nodes and spleens were harvested and pooled from all mice in each group. The lymph nodes from mice treated with B7-1/2–/– cells were grossly larger and had 5-fold greater total cellularity on average than nodes from mice receiving B7–/– cells plus Tregs. Lymph nodes from B7–/– recipients had 7-fold more CD4 and 3.5-fold more CD8 T cells on average than Treg recipients (Fig. 5d) with a reversal of CD4/CD8 ratio from 1.3:1 in B7–/– recipients to 0.65:1 in Treg recipients. This greater reduction of CD4 T cell numbers suggests that Tregs are more efficient in suppressing the expansion and proliferation of the CD4 T cell subset than the CD8 T cell subset.

To further evaluate the effect of WT Tregs on B7-deficient cells in vivo, both spleen and lymph node cells were analyzed for the distribution of activation markers. Judged by percentages of CD44+CD62L– cells, lymph node cells from B7–/– cell recipients showed increased activation among both CD4 and CD8 T cells subsets (Fig. 7a, left and middle panels). Addition of WT Tregs substantially reduced activation of both subsets of B7-deficient T cells (Fig. 7a, middle and right panels). Interestingly, a major population of CD4 T cells express intermediate levels of CD44 but high levels of CD62L in the group that received both B7–/– cells and Tregs (Fig. 7a, right panel). Spleen cells showed a similar reduction in activation status among CD4 and CD8 T cells with Treg transfer, but with all cells exhibiting higher activation levels overall (Fig. 7b).


Figure 7
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  		FIGURE 7. Tregs diminished the enhanced activation status and cytokine production of B7-1/2–/– T cells transferred into RAG-1–/– recipients. Cell surface staining of lymph node (a) and spleen cells (b) from RAG-1–/– recipients 6 wk after receiving either WT spleen and lymph node cells or B7-1/2–/– spleen and lymph node cells with or without cotransfer of WT Tregs. c, Cytokine profiles of CD4 T cells recovered from T cell-reconstituted RAG-1–/– mice. The RAG-1–/– mice were adoptively transferred with either WT, B7-deficient cells, or B7-deficient cells plus WT Tregs. Six weeks later, the spleen cells from the recipients were stimulated with PMA and ionomycin for 4 h in vitro in the presence of Golgi blocker and stained for intracellular IFN-{gamma} and IL-4. Note that the CD4 T cells from RAG-1–/– mice receiving B7–/– cells show significant IL-4 production (11%), which is virtually absent in WT mice and in mice receiving Tregs in addition to B7–/– cells. Solid lines in histograms depict stain with cytokine-specific mAb, whereas dotted lines represent isotype controls. Numbers shown in the panels represent the percentage of positive cells after subtracting those stained with isotype controls. Data shown are profiles of pooled spleen cells from four to five mice per group and are representative of two independent experiments.

 
To assess cytokine production by the cells, pooled spleen cells from RAG-1–/– recipients were subjected to short-term stimulation with PMA and ionomycin and stained for intracellular cytokines. A significant proportion of CD4 T cells from B7–/– donors produced IFN-{gamma}, although the proportion is lower than WT counterparts (Fig. 7c, top left and middle panels). The percentage of IFN-{gamma}-producing B7-deficient cells was reduced by ~25% by the cotransferred WT Tregs (Fig. 7c, top middle and right panels). However, CD4 T cells from mice receiving B7–/– cells displayed substantially increased IL-4 production compared with mice receiving WT cells, which showed minimal IL-4 production (Fig. 7c, lower left and middle panels). This increase was completely eliminated by WT Tregs (Fig. 7c, lower middle and right panels).

Tregs can suppress the proliferation of B7-deficient T cells in vitro

To quantitatively compare the effect of suppression by Tregs on B7+/+ and B7–/– T cells, we isolated CD4+ T cells from spleens and lymph nodes of WT and B7-deficient mice, as well as Tregs from WT mice. We tested the proliferation of B7+/+ and B7–/–CD4+CD25– T cells with anti-CD3 mAb stimulation in vitro in the presence of WT Tregs isolated from WT mice (Fig. 8a). As has been reported by others (28), B7-deficient T cells have better proliferation than WT T cells. Nevertheless, both WT and B7-deficient T cells were strongly suppressed by WT Tregs, and on a cell-to-cell basis, targeted mutation of B7-1 and B7-2 in T cells had no effect on their susceptibility to Treg-mediated immune suppression when one considers the percentage of suppression (Fig. 8b).


Figure 8
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  		FIGURE 8. Tregs can suppress the proliferation of B7-deficient T cells in vitro. CD4+CD25– T cells from WT or B7-deficient mice were used as responders and stimulated with anti-CD3 mAb (1.0 µg/ml) and irradiated syngeneic spleen cells (105/well) in the presence of varying numbers of WT Tregs. Tregs were titrated to responder cells at 1:2, 1:4, 1:8, and 1:16 ratios. Cells were cultured for 69 h with tritiated thymidine (1 µCi/well) added for the last 6 h of culture. a, T cell proliferation as measured by [3H]TdR incorporation. Data shown are means and SD of triplicate samples and are representative of two independent experiments. Accessory cells (AC) alone, irradiated spleen cells alone. b, B7-deficient and WT T cells are equally susceptible to inhibition by Treg. Suppressive activity was determined by the following formula: 100% x (cpmresponder – cpmresponder + suppressor)/cpmresponder.

 
WT Tregs did not suppress homeostatic proliferation of B7-deficient T cells regardless of B7 expression in the host APC

Since T cells transferred into the RAG-1-deficient recipients undergo extensive homeostatic proliferation, an interesting question is whether the Tregs suppress autoimmune inflammation by preventing homeostatic proliferation. We tested whether the homeostatic proliferation of B7-deficient T cells can be suppressed by WT Tregs. We first transferred B7-deficient T cells into either WT or B7-deficient host, in conjunction with either CD4+CD25+ or CD4+CD25– T cells from WT mice to equalize the total number of T cells transferred. As shown in Fig. 9a, upon adoptive transfer into irradiated WT recipient, B7-deficient T cells undergo homeostatic proliferation, although CD8 T cells divided substantially faster than CD4 T cells. Importantly, even at a 1:1 ratio, WT Tregs had no more effect than the non-Tregs on the proliferation of B7-deficient T cells. A significant, although perhaps somewhat reduced homeostatic proliferation was also observed in the B7-deficient host. However, the rate of proliferation was again the same regardless of which group of WT T cells were cotransferred.


Figure 9
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  		FIGURE 9. Homeostatic proliferation of B7-deficient T cells is resistant to suppression by CD25+CD4+ T cells. a, CFSE-labeled T cells from B7-deficient mice were transferred into either WT (upper panels) or B7-deficient (lower panels) (2 x 106/mouse), in conjunction with 2 x 106 of either CD4+CD25– (shaded area) or CD4+CD25+ (open lines) spleen cells from WT mice. At 3 days after the adoptive transfer, the spleen cells were analyzed by flow cytometry. Data shown are profiles of gated CFSE+ CD4 (left panels) or CD8 T cells (right panels) and are representative of two experiments with two mice per group. b, Expression of B7 on T cells did not enable suppression of homeostatic proliferation by WT Treg. A total of 2 x 106 CFSE-labeled WT or B7–/– OT-1 CD8 T cells was transferred into RAG-1–/– mice in the presence or absence of 2 x 106 purified CD4+CD25+ cells from WT mice. Seven days after the transfer, spleen cells were harvested and analyzed by flow cytometry. Data shown are profiles of gated CD8+TCR+OT-1 cells and represent those from two mice per group.

 
As an alternative approach, we generated B7+/+ and B7–/– OT-1 transgenic mice that have TCR specific for an OVA peptide presented by MHC class I. The CD8 T cells were purified, labeled with CSFE, and transferred into syngeneic RAG-1–/– hosts, either alone or in conjunction with WT Tregs. Seven days later, the proliferation of OT-1 cells were compared. As shown in Fig. 9b, regardless of B7 expression on the OT-1 cells, Tregs did not suppress homeostatic proliferation. Taken together, our analysis demonstrated that WT Tregs did not inhibit homeostatic proliferation of T cells regardless of B7 expression on the effector T cells.

These results suggest that the Tregs suppress autoimmune diseases by mechanisms other than preventing homeostatic proliferation. The lack of suppression by Tregs for the lymphopenia-driven proliferation of polyclonal B7-deficient T cells corresponds to previous observations with WT T cells (29, 30).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Accumulating evidence demonstrates that much like effector T cells, the development, activation and effector function of Tregs are regulated by both Ag and costimulatory molecules. Tregs are generated in the thymus, perhaps as a result of exposure to self-Ag (31). Several studies suggest that CD28 and perhaps CTLA-4 may be involved in the development and/or function of Tregs (17, 18, 32, 33). Developmentally, it has been shown that mice with targeted mutations of B7-1 and B7-2 have decreased numbers of CD4+CD25+ T cells in the spleen (32, 34). Recent studies also suggest that costimulation may also control survival of Tregs in the periphery (34).

Although it has been suggested that costimulatory molecules also control the effector function of Tregs (17, 18), the molecular mechanism has not been defined. A major hypothesis is that the interaction between B7-1/2 expressed on activated T cells and CTLA-4 on Tregs mediates the immune suppression (23). A critical prediction is that expression of B7-1/2 on effector T cells is essential for their susceptibility to Tregs. Our current study tested this prediction. In this adoptive transfer model, T cells from B7-deficient mice expanded more rapidly than the T cells from normal mice. In addition, a high proportion of the B7-deficient T cells gained the ability to produce IL-4, although the percentage of cells that produced IFN-{gamma} did not increase. Furthermore, the T cells acquired activation markers and caused inflammatory responses in multiple organs. Thus, this model offered an opportunity to test in vivo, the suppression of proliferation, acquisition of cytokine-producing ability and other markers of activation, and inflammatory responses to multiple organs. Our results demonstrated that each of these steps could be suppressed in the absence of B7-1/2 expressed on T cells.

At the proliferation stage, Tregs reduced the number of CD4 T cells in the lymph node by >10-fold. Quantitative analysis of Treg function in an in vitro proliferation assay revealed an essentially identical susceptibility of WT and B7-deficient T cells to Tregs. Nevertheless, since the in vitro suppression may not necessarily reflect the mechanism of immune suppression in vivo, the possibility that expression of B7 on the effector T cells may quantitatively increase the susceptibility to Treg suppression in vivo has not been completely ruled out in the current study. Interestingly, while Tregs suppressed IL-4-producing cells, they had no effect on the IFN-{gamma}-producing cells. The ability of Tregs to suppress Th2 cells is consistent with previous reports (35, 36). Further studies are needed to determine whether B7-1/2 on T cells is required for the suppression of IFN-{gamma}-producing cells in vivo. Perhaps the most impressive evidence for functional competence of Tregs over B7-deficient autopathogenic T cells is the near complete suppression of inflammation in all organs. Thus, despite the high levels of CTLA-4 expressed on Tregs, no known CTLA-4 ligand on effector T cells is required for immune suppression. Therefore, the simplest model in which CTLA-4 expressed by Tregs suppresses T cell function by reverse signaling via B7-1/2 on T cells (23) is refuted by our work.

Several points deserve consideration. First, since we have analyzed the Treg function only, our results do not exclude a role for B7-1/2 on effector T cells in suppression by other cell types less well characterized than Tregs. In this regard, Taylor et al. (28) reported that B7 expression by effector T cells in a graft-vs-host disease model may mediate suppression by CTLA-4 expressed by T cells that are distinct from traditional Tregs.

Second, our data do not refute the role of CTLA-4 in the function of immune regulation, particularly if one considers the fact that chimera mice consisting of WT and CTLA-4-deficient bone marrow do not develop lethal autoimmune disease, whereas those reconstituted with CTLA-4-deficient bone marrow do (37). However, it is worth pointing out that the essential role for CTLA-4 immune suppression is also being questioned (38). If further studies settle the debate and confirm a role for CTLA-4 in immune suppression, one can consider an alternative scenario that implicates reverse signaling through B7-1/2 expressed on DC by ligation with CTLA-4 on Tregs (20, 21). This interaction up-regulates tryptophan catabolism in DC, which has been shown to suppress T cells (39, 40). Although the in vivo significance of the model is difficult to test due to the requirement for B7-1/2 for the development of multiple organ inflammation, this idea that T-T suppression is mediated by APC is attractive as it is symmetrical to T-T help mediated by APC (41, 42).

Third, several groups have reported that mice with defective B7-CD28 costimulation have reduced number of Tregs (32, 34). Since the current studies demonstrate that Tregs can suppress the autoreactive T cells, defective Treg development and survival, together with higher burden of autoreactive T cells in these mice could explain higher pathogenicity.

Paust et al. (25) reported similarly higher pathogenicity of the CD4 T cells from B7-deficient mice. However, these authors failed to observe suppression of B7-deficient T cells by Tregs. Although the difference in this conclusion is difficult to reconcile at this point, several interesting differences are noteworthy. Our studies used mice backcrossed to C57BL/6J background, whereas Paust et al. (25) used mice of BALB/c background. Since B7 blockade leads to increased accumulation of viral superantigen-reactive T cells in the H-2d mice (43), it is likely that B7-deficient T cells are more pathogenic and therefore can be more resistant to suppression in vivo. In addition, Paust et al. (25) have used 5- to 10-fold less effector and Tregs in their adoptive transfer into immune-deficient mice. It can be anticipated that their effector T cells will undergo more vigorous homeostatic proliferation and yet encounter a lower concentration of Tregs in vivo. Furthermore, while we and Taylor et al. (28) found that B7-deficient T cells are hyperreactive to anti-CD3-induced proliferation, Paust et al. (25) reported poorer proliferation of B7-deficient T cells. The lowered proliferation makes it difficult to record suppression. Thus, although our quantitative analysis has not revealed any difference in susceptibility of B7–/– and B7+/+ effectors to Tregs, it is possible that some quantitative difference may exist between these effectors under certain conditions. Nevertheless, it should be pointed out that, since Paust et al. (25) and Tang et al. (24) showed that both CTLA-4 and CD28 on Tregs are unnecessary for Treg-mediated suppression, one would have to postulate an elusive new receptor expressed by Tregs for B7 on effector T cells, if B7 is an essential ligand for Treg function.


   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 This work was supported by National Institutes of Health Grants AI51342 and CA12001 and a grant from the U.S. Army. K.F.M. was supported by the Ohio State University College of Medicine and Public Health Medical Scientist Program. Back

2 Address correspondence and reprint requests to Dr. Yang Liu, Division of Immunotherapy, Department of Surgery, Comprehensive Cancer Center and Program of Molecular Mechanism of Diseases, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109. E-mail address: Yangl{at}umich.edu Back

3 Abbreviations used in this paper: Treg, CD4+CD25+ regulatory T cell; DC, dendritic cell; RAG-1, recombinase-activating gene 1; WT, wild type. Back

Received for publication August 22, 2006. Accepted for publication November 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Maloy, K. J., L. Salaun, R. Cahill, G. Dougan, N. J. Saunders, F. Powrie. 2003. CD4+CD25+ T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms. J. Exp. Med. 197: 111-119. [Abstract/Free Full Text]
  2. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, D. L. Sacks. 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420: 502-507. [Medline]
  3. Montagnoli, C., A. Bacci, S. Bozza, R. Gaziano, P. Mosci, A. H. Sharpe, L. Romani. 2002. B7/CD28-dependent CD4+CD25+ regulatory T cells are essential components of the memory-protective immunity to Candida albicans. J. Immunol. 169: 6298-6308. [Abstract/Free Full Text]
  4. Suvas, S., U. Kumaraguru, C. D. Pack, S. Lee, B. T. Rouse. 2003. CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses. J. Exp. Med. 198: 889-901. [Abstract/Free Full Text]
  5. Sutmuller, R. P., L. M. van Duivenvoorde, A. van Elsas, T. N. Schumacher, M. E. Wildenberg, J. P. Allison, R. E. Toes, R. Offringa, C. J. Melief. 2001. Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25+ regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J. Exp. Med. 194: 823-832. [Abstract/Free Full Text]
  6. Onizuka, S., I. Tawara, J. Shimizu, S. Sakaguchi, T. Fujita, E. Nakayama. 1999. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor {alpha}) monoclonal antibody. Cancer Res. 59: 3128-3133. [Abstract/Free Full Text]
  7. Lafaille, J. J., K. Nagashima, M. Katsuki, S. Tonegawa. 1994. High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice. Cell 78: 399-408. [Medline]
  8. Olivares-Villagomez, D., Y. Wang, J. J. Lafaille. 1998. Regulatory CD4+ T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis. J. Exp. Med. 188: 1883-1894. [Abstract/Free Full Text]
  9. Green, E. A., Y. Choi, R. A. Flavell. 2002. Pancreatic lymph node-derived CD4+CD25+ Treg cells: highly potent regulators of diabetes that require TRANCE-RANK signals. Immunity 16: 183-191. [Medline]
  10. Asano, M., M. Toda, N. Sakaguchi, S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184: 387-396. [Abstract/Free Full Text]
  11. Thornton, A. M., E. M. Shevach. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188: 287-296. [Abstract/Free Full Text]
  12. Powrie, F., J. Carlino, M. W. Leach, S. Mauze, R. L. Coffman. 1996. A critical role for transforming growth factor beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlowCD4+ T cells. J. Exp. Med. 183: 2669-2674. [Abstract/Free Full Text]
  13. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190: 995-1004. [Abstract/Free Full Text]
  14. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330-336. [Medline]
  15. Piccirillo, C. A., J. J. Letterio, A. M. Thornton, R. S. McHugh, M. Mamura, H. Mizuhara, E. M. Shevach. 2002. CD4+CD25+ regulatory T cells can mediate suppressor function in the absence of transforming growth factor beta1 production and responsiveness. J. Exp. Med. 196: 237-246. [Abstract/Free Full Text]
  16. Nakamura, K., A. Kitani, W. Strober. 2001. Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor beta. J. Exp. Med. 194: 629-644. [Abstract/Free Full Text]
  17. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192: 295-302. [Abstract/Free Full Text]
  18. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192: 303-310. [Abstract/Free Full Text]
  19. Azuma, M., H. Yssel, J. H. Phillips, H. Spits, L. L. Lanier. 1993. Functional expression of B7/BB1 on activated T lymphocytes. J. Exp. Med. 177: 845-850. [Abstract/Free Full Text]
  20. Grohmann, U., C. Orabona, F. Fallarino, C. Vacca, F. Calcinaro, A. Falorni, P. Candeloro, M. L. Belladonna, R. Bianchi, M. C. Fioretti, P. Puccetti. 2002. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3: 1097-1101. [Medline]
  21. Fallarino, F., U. Grohmann, K. W. Hwang, C. Orabona, C. Vacca, R. Bianchi, M. L. Belladonna, M. C. Fioretti, M. L. Alegre, P. Puccetti. 2003. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212. [Medline]
  22. Borriello, F., G. J. Freeman, S. Edelhoff, C. M. Disteche, L. M. Nadler, A. H. Sharpe. 1994. Characterization of the murine B7-1 genomic locus reveals an additional exon encoding an alternative cytoplasmic domain and a chromosomal location of chromosome 16, band B5. J. Immunol. 153: 5038-5048. [Abstract]
  23. Gavin, M., A. Rudensky. 2003. Control of immune homeostasis by naturally arising regulatory CD4+ T cells. Curr. Opin. Immunol. 15: 690-696. [Medline]
  24. Tang, Q., E. K. Boden, K. J. Henriksen, H. Bour-Jordan, M. Bi, J. A. Bluestone. 2004. Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function. Eur. J. Immunol. 34: 2996-3005. [Medline]
  25. Paust, S., L. Lu, N. McCarty, H. Cantor. 2004. Engagement of B7 on effector T cells by regulatory T cells prevents autoimmune disease. Proc. Natl. Acad. Sci. USA 101: 10398-10403. [Abstract/Free Full Text]
  26. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68: 869-877. [Medline]
  27. Borriello, F., M. P. Sethna, S. D. Boyd, A. N. Schweitzer, E. A. Tivol, D. Jacoby, T. B. Strom, E. M. Simpson, G. J. Freeman, A. H. Sharpe. 1997. B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity 6: 303-313. [Medline]
  28. Taylor, P. A., C. J. Lees, S. Fournier, J. P. Allison, A. H. Sharpe, B. R. Blazar. 2004. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions. J. Immunol. 172: 34-39. [Abstract/Free Full Text]
  29. Prlic, M., B. R. Blazar, A. Khoruts, T. Zell, S. C. Jameson. 2001. Homeostatic expansion occurs independently of costimulatory signals. J. Immunol. 167: 5664-5668. [Abstract/Free Full Text]
  30. Hagen, K. A., C. T. Moses, E. F. Drasler, K. M. Podetz-Pedersen, S. C. Jameson, A. Khoruts. 2004. A role for CD28 in lymphopenia-induced proliferation of CD4 T cells. J. Immunol. 173: 3909-3915. [Abstract/Free Full Text]
  31. Apostolou, I., A. Sarukhan, L. Klein, H. von Boehmer. 2002. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3: 756-763. [Medline]
  32. Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12: 431-440. [Medline]
  33. Akiyama, Y., N. Shirasugi, N. Uchida, K. Matsumoto, M. Kitajima, H. Bashuda, H. Yagita, K. Okumura, O. Aramaki, M. Niimi. 2002. B7/CTLA4 pathway is essential for generating regulatory cells after intratracheal delivery of alloantigen in mice. Transplantation 74: 732-738. [Medline]
  34. Lohr, J., B. Knoechel, S. Jiang, A. H. Sharpe, A. K. Abbas. 2003. The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens. Nat. Immunol. 4: 664-669. [Medline]
  35. Cottrez, F., S. D. Hurst, R. L. Coffman, H. Groux. 2000. T regulatory cells 1 inhibit a Th2-specific response in vivo. J. Immunol. 165: 4848-4853. [Abstract/Free Full Text]
  36. Suri-Payer, E., A. Z. Amar, R. McHugh, K. Natarajan, D. H. Margulies, E. M. Shevach. 1999. Post-thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti-H/K ATPase-reactive T cells. Eur. J. Immunol. 29: 669-677. [Medline]
  37. Bachmann, M. F., G. Kohler, B. Ecabert, T. W. Mak, M. Kopf. 1999. Cutting edge: lymphoproliferative disease in the absence of CTLA-4 is not T cell autonomous. J. Immunol. 163: 1128-1131. [Abstract/Free Full Text]
  38. Shevach, E. M.. 2002. CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2: 389-400. [Medline]
  39. Mellor, A. L., B. Baban, P. Chandler, B. Marshall, K. Jhaver, A. Hansen, P. A. Koni, M. Iwashima, D. H. Munn. 2003. Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion. J. Immunol. 171: 1652-1655. [Abstract/Free Full Text]
  40. Munn, D. H., M. D. Sharma, A. L. Mellor. 2004. Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J. Immunol. 172: 4100-4110. [Abstract/Free Full Text]
  41. Guerder, S., P. Matzinger. 1992. A fail-safe mechanism for maintaining self-tolerance. J. Exp. Med. 176: 553-564. [Abstract/Free Full Text]
  42. Wu, Y., Y. Liu. 1994. Viral induction of co-stimulatory activity on antigen-presenting cells bypasses the need for CD4+ T cell help in CD8+ T cell responses. Curr. Biol. 4: 499-505. [Medline]
  43. Gao, J. X., H. Zhang, X. F. Bai, J. Wen, X. Zheng, J. Liu, P. Zheng, Y. Liu. 2002. Perinatal blockade of B7-1 and B7-2 inhibits clonal deletion of highly pathogenic autoreactive T cells. J. Exp. Med. 195: 959-971. [Abstract/Free Full Text]




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