Regulatory role of mature B cells in a murine model of inflammatory bowel disease
Emiko Mizoguchi,
Atsushi Mizoguchi,
Frederic I. Preffer and
Atul K. Bhan
Department of Pathology, Massachusetts General Hospital, Harvard Medical School, 100 Blossom StreetCox 5, Boston, MA 02114, USA
Correspondence to:
A. K. Bhan
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Abstract
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The spontaneous chronic colitis in TCR
mutant (TCR
/) mice mediated by CD4+ TCR
ß+ T cells is more severe in the absence of mature B cells, suggesting a suppressive role of B cells and Ig in the development of chronic colitis. To investigate the direct role of B cells in the suppression of this colitis, cell transfer studies were performed in TCR
/ x Igµ/ (
µ/) double-knockout mice. The chronic colitis was markedly attenuated in
µ/ mice after the adoptive transfer of peripheral B cells from TCR
/ mice into 3- to 4-week-old
µ/ mice prior to the development of colitis. Furthermore, transfer of mature B cells from TCR
/ mice markedly decreased the number of pathogenic colonic CD4+ TCR
ß+ T cells in
µ/ mice with established colitis. This B cell effect required the presence of functional co-stimulatory molecules CD40 and B7-2 (CD86) but not B7-1 (CD80). These results indicate that mature B cells play an important role in the development of chronic colitis in TCR
/ mice by directly regulating the pathogenic T cells (CD4+ TCR
ß+ T cells).
Keywords: adoptive transfer, B cells, colitis, knockout mice, TCR
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Introduction
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The adaptive immune responses are generated by the complex interactions between cells of the immune system (1). The regulatory T cells are considered a critical population to maintain immunological balance, resulting in prevention of development of autoimmune disorders (26). However, the regulatory role of B cells in immunological responses has received limited attention.
The T and B cell interactions are an important part of establishment of adaptive immune responses. The T and B cell interactions via CD40/CD154 molecules induce survival of B cells and establishment of the humoral immune responses (79). However, the fate of T cells after this interaction is unknown. Although B cells can act as antigen-presenting cells (APC), it is generally believed that dendritic cells (DC) rather than B cells are the primary APC in T cell responses (10). B cells can act as a secondary APC and prevent experimental autoimmune encephalomyelitis (EAE) (11,12), raising a possibility that B cells may play a regulatory role in immune responses.
TCR
/ mice spontaneously develop chronic colitis resembling human ulcerative colitis (UC) (1315). The development of colitis in TCR
/ mice is mediated by a unique T cell population, CD4+ TCR
ßlow T cells, which express TCR ß chain without TCR
chain on cell surface (14,16). In addition, like human UC, autoantibodies including anti-tropomyosin (17), anti-neutrophil cytoplasmic antibodies (ANCA) (18), anti-histone (18) and anti-DNA (19) are frequently detected in TCR
/ mice with colitis. The suppressive role of Ig-including autoantibodies has been suggested in the studies carried out in TCR
/ mice crossed with Igµ/ mice (
µ/) (20) that develop a more severe form of colitis than TCR
/ mice.
In the present study, we investigated the regulatory role of B cells in chronic colitis by performing cell transfer studies in
µ/ mice with or without the presence of colitis. The role of co-stimulatory molecules CD40, B7-2 and B7-1 in the regulation of pathogenic CD4+ TCR
ß+ T cells by B cells was also explored.
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Methods
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Mice
TCR
/ (14,21) and Igµ/ (Igh 6 mutant) (22) mice of C57BL/6 strain (H-2b) background were purchased from Jackson Laboratories (Bar Harbor, ME), crossed to generate the double mutant (
µ/) mice as described previously (20). CD40/ of C57BL/6x129 background (H-2b) (5) were kindly provided by Dr R. F. Geha (Boston, MA), and B7-1/ (23) and B7-2/ (24) of C57BL/6 background (H-2b) by Dr. A. H. Sharpe (Boston, MA). These mice were maintained under specific pathogen-free conditions at Massachusetts General Hospital (Boston, MA).
FACS analysis
Cells (2x105) obtained from the spleen and mesenteric lymph nodes (MLN) were pre-incubated in staining buffer (0.1% sodium azide and 0.2% BSA/PBS) containing 10% normal rat and hamster serum (Jackson ImmunoResearch, West Grove, PA) and unconjugated CD16/CD32 (2.4G2; PharMingen, San Diego, CA) before staining. After washing with staining buffer, cells were stained using anti-CD3
FITC (145-2C11; PharMingen) and TCRßphycoerythrin (PE) (H57-597; PharMingen) or TCR
FITC (GL3; PharMingen) at 4°C for 30 min. After washing with staining buffer, cells were analyzed on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) (25).
Proliferation assay
TCR
ßlow T cells from the spleen of
µ/ mice were enriched by negative selection with a cocktail of biotinylated mAb [anti-TCR
(GL3), CD19 (1D3), NK1.1 (PK136) and CD11b (M1/70); PharMingen] followed by staining with streptavidin-labeled microbeads on a MACS system. Syngeneic colonic epithelial cells were isolated from
µ/ mice as previously described (20). After irradiation at 10,000 rad, the epithelial cells were used as a source of antigenic stimulus. Spleen and MLN B cells were isolated from TCR
/ mice by negative selection using a cocktail of biotinylated anti-CD3
(2C11) and anti-TCR
(GL3) mAb. For the purification of precursor B cells, mature B cells were depleted by using anti-CD38 mAb in a MACS system. CD38 precursor B cells and total B cells were irradiated at 2000 rad before culture. The enriched TCR
ßlow T cells and APC (7.5x104 cells/well) were cultured with epithelial cells (4.0x104 cells/well), with or without mature B cells (7.5x104 cells/well) in the presence of rIL-2 (10 U/ml; BioSource, Camarillo, CA) and rIL-4 (20 U/ml; BioSource). After culture for 4 days, the proliferative responses of these cells were analyzed by [3H]thymidine (NEN, Boston, MA) uptake and by cell cycle analysis. For the [3H]thymidine uptake, after a pulse with 1 mCi for 16 h, [3H]thymidine was measured by scintillation counting. All conditions were performed in triplicate 3 times.
Cell cycle analysis by flow cytometry
Cells were stained with CD4FITC (RM4-5; PharMingen) and TCRßPE (H57-597; PharMingen) mAb for 15 min on ice. After two washes in PBS, cells were simultaneously fixed and permeabilized in 5 µg/ml ToPro-3 (Molecular Probes, Eugene, OR) and 1 µg/ml RNase (Sigma). After 15 min incubation, cells were run on a FACScan flow cytometer utilizing standard techniques (25). Cells identified as either CD4+ TCRß+ or CD4 TCRß were examined for their proliferative fraction (S + G2/M) utilizing ModfitLT software (Verity Software, Topsham, ME) with a mathematical algorithm appropriate for the sample type. Cell debris were eliminated with forward versus side scatter analysis and doublets eliminated with pulse area versus width analysis.
Histologic and immunohistochemical analysis
All tissue samples were obtained from three different (proximal, middle and distal) parts of the colon and fixed in 3% buffered formalin. Paraffin-embedded tissue sections were stained with hematoxylin & eosin using standard techniques. The severity of colitis was graded (grade 0 to 3) based on histologic examination (26).
Fresh tissue samples of the colon were frozen in OCT compound (Ames, Elkhart, IN) and stored at 80°C until use. Frozen tissue sections (4 µm thick) were air-dried for 2 h, fixed in acetone for 10 min, air-dried again for 20 min and stained by the avidinbiotin complex method as described (13,26) using the following antibodies: biotinylated anti-IgM (R6-60.2; PharMingen), purified anti-CD4 (RM4-5; PharMingen) and purified anti-BrdU (Harlan Sera-Lab, Loughborough, UK).
Cell transfer studies
Purified B cells from spleen and MLN of TCR
/ mice were obtained by negative sorting using a mixture of biotinylated mAb [anti-CD4 (RM4-5), -CD5 (57-7.3), -TCR
(GL3), -NK-1.1 (PK136) and -Mac-1 (M1/70) (PharMingen)], followed by incubation with streptavidin-labeled microbeads on a magnetic cell sorting system MACS (Miltenyi Biotech, Auburn, CA). After the negative sorting, >95% of cells were B220+ B cells by flow cytometry. For the transfer of bone marrow B cells, adherent cells were removed by culture in complete media at 37°C for 2 h. T cells were then removed by two rounds of treatment with anti-Thy1.2 (30-H12), anti-CD4 (RM4-5) and anti-CD8 (53-6.7) for 30 min on ice followed by rabbit complement (Accurate Chemical, Westbury, NY) for 45 min at 37°C. Bone marrow recipient animals were pre-conditioned with 5-fluorouracil (50100 mg/kg) 48 h prior to bone marrow transplantation. Colonic lamina propria lymphocytes (LPL) were extracted as previously described (14).
To assess the effect of B cells in the development of colitis in
µ/ mice, spleen, MLN or bone marrow B cells (1x107), or total cells (5x107) were isolated from age-matched TCR
/ mice or other knockout mice and i.p. transferred into 3- to 4-week-old
µ/ mice and sacrificed at 10 weeks of age. To examine the B cell effect in mice with established chronic colitis, B cells from spleen and MLN of TCR
/ or from other knockout mice were i.p. transferred into 20-week-old
µ/ mice and the recipient mice were sacrificed 10 days after cell transfer.
For the blocking experiments, B cells from TCR
/ mice were incubated for 1 h on ice with purified anti-CD40 (HM40-3), -B7-1 (CD80, 1G10) and -B7-2 (CD86, GL1) mAb, and i.p. injected into the 20-week-old
µ/ mice. Anti-CD40, -B7-1 and -B7-2 mAb were obtained from PharMingen.
BrdU incorporation
To detect proliferating colonic epithelial cells in vivo, BrdU incorporation method, as previously described, was used (26). Briefly, BrdU was injected i.p. (100 µg/g in PBS) 1 h before the mice were sacrificed. Anti-BrdU antibody (Harlan Sera-Lab) was used to detect BrdU-incorporated cells in frozen tissue sections by the immunoperoxidase technique (26). Counting of BrdU-labeled colonic epithelial cells was done as previously described (26).
ELISA
ELISA was carried out as previously described (18). Briefly, a 96-well polyvinyl microtiter plate (Costar, Cambridge, MA) was coated with 0.2 µg of mouse Ig (Dako, Carpinteria, CA) per well in 100 µl of PBS, overnight at 4°C. After washing with PBS, the plates were blocked with 2% non-fat dried milk and 1% goat serum in PBS for 2 h at 37°C, then 100 µl of several mouse sera diluted (1:100) was added per well in triplicate and the plates incubated for 1 h at 37°C. After washing with PBS, the wells were incubated for 1 h with 50 µl of alkaline phosphatase polyclonal goat anti-mouse Ig (Southern Biotechnology Associates) at a 1:1000 dilution for 1 h at room temperature followed by addition of 100 µl of p-nitrophenylphosphate substrate (Sigma) in 0.01 M Tris buffer for 5 min at room temperature. The plates were read at 405 nm using an Auto Reader (Bio-Tec Instruments, Burlington, VT). Results were statistically analyzed using the Student's t-test.
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Results
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Reduction of colonic LP cells in
µ/ mice after adoptive transfer of B cells in TCR
/ mice
Sixty percent of TCR
/ mice spontaneously develop chronic colitis by 1820 weeks of age, whereas >80% of B cell-deficient TCR
/ (
µ/) mice develop severe chronic colitis by 810 weeks of age (20). Our previous findings indicate that the Ig including autoantibodies can attenuate colitis (20). However, B cells play important functions in adaptive immune responses, and may directly regulate pathogenic T cells and alter the course of colitis. To investigate the direct effects of B cells in the pathogenesis of this colitis, we transferred B cells from TCR
/ mice into
µ/ mice at 20 weeks of age after severe disease was established. The number of total colonic LP cells were significantly increased in both TCR
/ and
µ/ mice as compared to wild-type mice or Igµ/ mice. The number of colonic LP cells was markedly more in
µ/ mice as compared to TCR
/ mice; the average number of colonic LP cells was 124 ± 12 and 382 ± 36 in TCR
/ and
µ/ mice (P < 0.001) respectively (Fig. 1
). However, the transfer of B cells from TCR
/ mice led to decrease in the number of colonic LP cells in
µ/ mice within a 10 days observation period; the number of LP cells in
µ/ mice transferred with B cells (186 ± 36) was significantly less than
µ/ mice transferred with PBS (352 ± 24). Transfer of 2 mg of purified serum Ig from TCR
/ mice into
µ/ mice failed to decrease the number of colonic LP cells in
µ/ mice (349 ± 108) within a 10 days observation period.
Decrease in the number of CD4+ TCR
ßlow T cells by B cell transfer
Although, both CD4+ TCR
ßlow and TCR
T cells increase in the colon of TCR
/ mice with colitis as compared to the mice without colitis (26), the studies so far indicate that CD4+ TCR
ßlow T cells, but not TCR
T cells are the critical cells involved in the pathogenesis of colitis of TCR
/ mice (14,16,26). Therefore, the T cell phenotype (TCR
ß+ versus TCR
+) of LP cells was analyzed by flow cytometric analysis (Fig. 2
). CD4+ TCR
ßlow T cells are markedly increased in
µ/ mice as compared to TCR
/ mice. Ten days after the adoptive transfer of peripheral B cells from TCR
/ mice into
µ/ mice, the number of TCR
ßlow T cells in the colon are significantly reduced (14.8 % among the total T cells) as compared to the control
µ/ mice without B cell transfer (42.7 %) (Fig. 2
). In contrast, there was no significant change in the number of TCR
ßlow T cells following transfer of purified Ig into TCR
/ mice (Fig. 2D
) within a 10 days observation period. The total number of TCR
+ T cells were similar in
µ/ mice before and after B cell or Ig transfer from TCR
/ mice (data not shown).
Role of CD40 and B7-2 molecules on B cells in the elimination of pathogenic T cells
The CD40CD154 (gp39) pathway plays an important role in TB cell interactions. CD154 expression was detectable on the freshly isolated TCR
ß+ T cells, but not the TCR
+ß+ T cells from the colon. Therefore, the involvement of CD40CD154 and the other co-stimulatory pathways including B7-1 and B7-2 molecules in the elimination of pathogenic T cells by B cells was investigated by blocking these molecules on B cells with mAb. The treatment of B cells (from TCR
/ mice) with anti-CD40 or anti-B7-2 mAb before transfer failed to decrease the number of colonic LP cells of the recipient mice. In contrast, like transfer of B cells from TCR
/ mice without treatment, B cells treated with anti-B7-1 mAb also reduced the number of LP cells in
µ/ mice. In addition, the marked reduction of LP TCR
ßlow T cells (from TCR
ßlow/TCR
ratio of 1.18 ± 0.12 to 0.41 ± 0.01) in
µ/ mice following transfer of B cells was not observed in
µ/ mice transferred with B cells treated with anti-CD40 or anti-B7-2 mAb (Figs 3 and 4
). In contrast, treatment of B cells with anti-B7-1 mAb did not alter the elimination of TCR
ßlow T cells by B cells (Fig. 4
).
Protective effect of transferred B cells in the development of colitis in
µ/ mice
To observe the effect of B cells on the development of colitis, B cells were transferred into
µ/ mice at a time (3 weeks of age) when the mice do not have colitis. The mice were sacrificed at 10 weeks of age. Transfer of mature B cells not only led to decreased numbers of colonic TCR
ßlow T cells, but also in the suppression of colitis in
µ/ mice. Figure 5
(A) shows the gross appearance of the colon from
µ/ mice (10 weeks old) with (Fig. 5A
, right) and without (Fig. 5A
, left) B cell transfer from TCR
/ mice. The colon from the
µ/ mouse shows a markedly thickened wall. In contrast, the
µ/ mouse transferred with peripheral B cells shows a normal thickness of the colonic wall and a beaded appearance of the stools. Histologically, chronic colitis in
µ/ mice was characterized by a thickened colonic wall with marked elongation of the crypts and presence of mixed inflammatory cell infiltrate in the LP (Fig. 5B
). In contrast, after the adoptive transfer of cells from spleen and MLN cells from TCR
/, the severity of colitis was markedly attenuated in
µ/ mice (Fig. 5C
). The protective effect of colitis in
µ/ mice by adoptive transfer of total cells (5x107) or purified B cells (1x107) isolated from either peripheral lymphoid organs (spleen and MLN) or bone marrow of TCR
/ mice is summarized in Table 1
. The reconstitution of the transferred B cells in the recipient mice was confirmed by two-color immunohistochemical analysis of spleen (Fig. 5D and E
). Although Ig were detectable in
µ/ mice reconstituted with B cells from TCR
/ mice, the level of Ig was significantly lower than that in TCR
/ mice (Table 1
). Next, the effect of co-stimulatory molecules on B cells in the development of colitis was analyzed. This required that the mice be followed for a longer time (67 weeks) after the B cell transfer than in the studies designed to investigate the elimination of pathogenic T cells (Figs 14


). Therefore, B cells from knockout mice (CD40/, B7-1/ and B7-2/) were transferred rather than using B cells treated with specific mAb.
Since the colonic epithelial cell proliferation detected by in vivo BrdU incorporation is a sensitive method for the detection of colitis in TCR
/ mice (15,26), we used this method to evaluate the suppressive effect of B cells in these mice (Fig. 6
). B cells (1x107) isolated from wild-type, TCR
/, CD40/, B7-1/ and B7-2/ mice were transferred into 3- to 4-week-old
µ/ mice, which were sacrificed at 10 weeks of age. The
µ/ mice transferred with B cells from TCR
/ mice showed a markedly decreased colonic epithelial cell proliferation (BrdU index 11.8 ± 0.4, P = 0.005) as compared to
µ/ control mice (26.1 ± 3.3) (Fig. 6
). The BrdU index in the
µ/ mice transferred with B cells from wild-type mice (18.5 ± 0.9, P = 0.026) or from B7-1/ mice (18.6 ± 0.9, P = 0.031) also showed a relative decrease in colonic epithelial cell proliferation as compared to
µ/ control mice (Fig. 6
). In contrast, there was no significant difference in the BrdU index of colonic epithelial cells between the
µ/ control mice (26.1 ± 3.3) and the
µ/ mice transferred by B cells from CD40/ mice (28.3 ± 2.8, P = 0.614) or B7-2/ mice (23.1 ± 0.6, P = 0.405). These results indicate that the CD40CD154 interactions and B7-2CD28 or CTLA-4 interactions can influence the B cell-mediated regulatory effect in the development of chronic colitis in TCR
/ mice.
Suppression of proliferative responses of CD4+ TCR
ß+ T cells by mature B cells in vitro
To further confirm the in vivo findings, the effect of B cells on the proliferative responses of CD4+ TCR
ß+ T cells was analyzed in vitro. TCR
ß+ T cells (enriched by negative selection; see Methods) and APC were cultured with or without B cells from TCR
/ mice in the presence of irradiated epithelial cells (as antigens) and rIL-4 and rIL-2 for 4 days. The enriched CD4+ TCR
ß+ T cells from the spleen of
µ/ mice showed proliferative responses against epithelial antigens (c.p.m. 22,878 ± 2589) (Fig. 7A
). Since the negatively selected population also contains other cells such as APC, the cells proliferating in this culture were analyzed by FACS. CD4+ TCR
ß+ T cells were found to be the predominant proliferating cells in the culture; 71.3 % of these cells were in growth phase (Fig. 7B
). Addition of total B cells to this culture inhibited the proliferative responses (c.p.m. 6809 ± 1514, P = 0.006) (Fig. 7A
). In contrast, precursor B cells (CD38) failed to inhibit the proliferative responses in this culture (c.p.m. 21,149 ± 2031). These findings strongly support in vivo findings that mature B cells possess an ability to suppress the expansion of self-reactive T cells.
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Discussion
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The spontaneous colitis in TCR
/ mice is mediated by the unique population of CD4+ TCR
ßlow T cells, but not B cells (20). The chronic colitis in TCR
/ mutant mice is more severe and develops more rapidly when the TCR
/ mice are depleted of mature B cells by crossing TCR
/ mice with Igµ/ mice (20). Previous studies indicate that the increased severity of colitis in
µ/ mice is not due to the presence of pathogenic flora, but due to the absence of B cells. The colitis of TCR
/ mice is associated with presence of autoantibodies (anti-tropomyosin, anti-nuclear, ANCA), which may play a role in the removal of potentially pathogenic self-antigens released by apoptotic cells (18). This possibility is supported by the increased numbers of detectable apoptotic cells in the colon and lymphoid tissue of
µ/ mice. Interestingly, passive transfer of Ig (long-term effect) from TCR
/ mice leads to a decrease in the detectable apoptotic cells and a decrease in the severity of colitis. However, the direct effect of B cells on the pathogenic T cells was not explored in the previous study.
In this study, B cell transfer from TCR
/ mice to
µ/ mice clearly indicate that B cells not only suppress the colitis when transferred prior to the development of colitis, but directly regulate the pathogenic TCR
ßlow T cells in the diseased colon when transferred to mice with established colitis. The latter effect can be observed within 10 days of cell transfer (short-term effect). The co-stimulatory molecules CD40 and B7-2, but not B7-1, are involved in the suppression of colitis and regulation of expansion of TCR
ßlow T cells. This regulatory effect of B cells was not mediated through antibodies because transfer of Ig from TCR
/ mice to
µ/ mice did not lead to a decrease in the number of TCR
ßlow T cells.
Although DC have been suggested to play a critical role in the initiation of primary immune response as a professional APC; B cells also play an important role as a secondary APC (11,12,27,28). It has been postulated that there are some differences in antigen-processing mechanisms by DC and B cellsB cells may process wide variety of determinants as compared to DC (12,27,29).
B cells have also been shown to be involved in the regulation of immune responses in infections and autoimmune diseases (11,30,31). B cell-deficient (µ/) mice fail to down-regulate the late stage of CD4+ T cell-mediated granulomatous pathology in Schistoma mansoni mansoni infection, indicating a suppressive role of B cells in inflammatory diseases mediated by CD4+ T cells (32). In addition, B cells can tolerize antigen-specific CD8+ T cells (30).
CD40CD154 interactions provide a critical signal between T and B cells to regulate the adaptive immune responses (9). In this study, we show that the B cells isolated from CD40/ mice as well as B cells treated with anti-CD40 mAb lost their ability to regulate the number of pathogenic TCR
ßlow T cells in colon of
µ/ mice. This result strongly suggests that direct contact between CD40 and CD154 was critical for the B cell-mediated T cell regulation in the pathogenesis of colitis in TCR
/ mice. In addition, our results suggest that the CD86 (B7-2)-, but not CD80 (B7-1)-mediated pathway is also involved in the regulation of pathogenic T cells by B cells in TCR
/ mice. The suppressive effect of B cells from TCR
/ mice was significantly more than B cells from wild-type and B7-1/ mice. The B cells in TCR
/ mice are highly activated (15) and have unique reactivity against the self components (1719). Therefore, the increased suppressive effect of B cells in TCR
/ may be due to their higher level of activation as compared to those in wild-type mice. In human, the specific TB cell interaction is mediated by the B cell-produced cytokines such as IL-12 and IL-6 (12). Whether murine B cells produce these kinds of cytokines has not been determined yet. After CD40CD154 interaction, the stimulated B cells may secrete the regulatory cytokines such as IL-10 and/or transforming growth factor-ß, and suppress the activation and the proliferation of pathogenic T cells (4,33). It is also possible that direct interaction between B cells and activated T cells leads to apoptosis of T cells by a Fas-mediated pathway. A recent study suggests that thymic B cells, which express higher levels of CD40 and B7-2 than splenic B cells, may play an important role in the induction of T cell negative selection and elimination of autoreactive thymocytes (34). Further studies are required to define precise mechanisms involved in B cell-mediated T cell regulation in chronic colitis.
Recent studies in EAE indicate that the lack of endogenous TCR
chain alters the differentiation and/or development of regulatory T cells (3,4). The lack of regulatory T cells may be important in the pathogenesis of colitis of TCR
/ mice (35). Therefore, it is possible that in the T cell-deficient state such as in mice lacking TCR
chain, the regulatory function of B cells may be enhanced. Our present study indicates that the function of mature B cells includes direct regulation of pathogenic T cells via the CD40 and B7-2 pathway.
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Acknowledgments
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This work was supported by NIH grant DK47677 (A. K. B. and E. M.) and by the Massachusetts General Hospital/Center for the Study of Inflammatory Bowel Disease (DK43351 to A. K. B. and E. M.). A. M. is supported by a Career Development Award from the Crohn's & Colitis Foundation of America, Inc. We are grateful to Drs Arlene H. Sharpe and Raif S. Geha for providing us with the mutant mice, Drs Ramnik Xavier, Scott B. Snapper and Faith Young for helpful discussion, and Miss Cheryl A. Nason for her preparation of the manuscript. We also would like to acknowledge the expert technical assistance of Mr David Dombkowski.
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Abbreviations
|
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µ/ B cell deficient TCR deficient |
ANCA anti-neutrophil cytoplasmic antibodies |
APC antigen-presenting cell |
DC dendritic cell |
EAE experimental autoimmune encephalomyelitis |
LP lamina propria |
LPL lamina propria lymphocytes |
MLN mesenteric lymph node |
PE phycoerythrin |
TCR / TCR chain knockout |
UC ulcerative colitis |
 |
Notes
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Transmitting editor: C. Terhorst
Received 18 October 1999,
accepted 10 January 2000.
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