Role of the CD5 molecule on TCR 
T cell-mediated immune functions: development of germinal centers and chronic intestinal inflammation
Atsushi Mizoguchi1,2,4,
Emiko Mizoguchi1,2,4,
Ype P. de Jong2,3,5,
Hiroko Takedatsu1,4,
Frederic I. Preffer1,4,
Cox Terhorst2,3,5 and
Atul K. Bhan1,2,4
1 Department of Pathology and 2 Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, MA 02114, USA 3 Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02115, USADepartments of 4 Pathology and 5 Immunology, Harvard Medical School, Boston, MA 02115, USA
Correspondence to: A. Mizoguchi, Immunopathology Unit, Warren 501, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA. E-mail: amizoguchi{at}partners.org
Transmitting editor: R. S. Geha
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Abstract
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Although CD4+ T cells form a major subset of TCR
ß T cells, only a small number of TCR
T cells express CD4. Factors contributing to the down-regulation of CD4+ TCR
T cells have not been identified. The CD5 molecule is expressed on most TCR
T cells in the spleen, whereas only a few intestinal intraepithelial TCR
T cells express this molecule in wild-type mice and TCRß mutant (ß/) mice. Unexpectedly, in the present studies, the lack of CD5 led to a remarkable increase of a CD4+ TCR
T cell subset in CD5/ß/ mice. The CD4+ TCR
T cells were also detectable in MHC II/CD5/ß/ triple-mutant mice. This CD4+ TCR
T cell subset provided help in Mycobacterium-induced germinal center (GC) formation and showed a Th-like cytokine profile. In contrast, CD5+ TCR
T cells suppressed the CD4+ TCR
T cell-mediated GC formation, presumably by eliminating this CD4+ subset. Unlike intraepithelial 
T cells, >30% of TCR
T cells in the colonic lamina propria (LP) expressed CD5. The lack of CD5 also led to increased numbers of CD4+ TCR
T cells in the colonic LP and increased susceptibility to development of chronic colitis in ß/ mice. Cell transfer studies suggest that CD5+ TCR
T cells are capable of selectively eliminating CD4+ TCR
T cells in the intestine. The CD4+ TCR
T cells possess immune functions similar to CD4+ TCR
ß T cells.
Keywords: CD4, immune deficiency, inflammatory bowel disease, regulatory, TCR ß knockout mice
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Introduction
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T cells can be divided into two subsets based on the surface expression of TCR heterodimer, as either TCR
ß or TCR
(1). TCR
T cells are an evolutionary conserved immune subset, and possess characteristic properties including a specialized anatomical distribution, unique antigen specificity and broad spectrum of cellcell interactions (29). Therefore, TCR
cells can effectively participate in immune responses (29).
The CD4 surface glycoprotein is an important molecule in T cell development and activation (10), and is required for Th2 differentiation (11). The expression of the CD4 molecule is controlled by a complicated network in which transcriptional control elements including at least the promoter, three enhancers and a silencer cooperate (12). CD4+ T cells are a major subset of TCR
ß T cells. In contrast, CD4+ TCR
T cells are present as a small population in human as well as mouse (4,7,13,14). Recently, the CD5 molecule has been proposed to negatively regulate the differentiation of CD4-commited cells (15,16). However, it is not know whether the CD5 molecule is involved in the development, differentiation and/or expansion of CD4+ TCR
T cells.
The CD5 molecule, a highly conserved receptor during evolution, is involved in natural immunity and several ligands for CD5 have been reported (1721). The large cytoplasmic domain of CD5 contains three potential tyrosine phosphorylation sites including an immunoreceptor tyrosine-based activation motif, an immunoreceptor tyrosine-based inhibition motif and multipotential Ser/Thr phosphorylation sites (22,23). Therefore, CD5 behaves as a dual-function receptor providing either positive or negative signals, depending on both the cell type and the stage of activation/differentiation of the cell (17,23). With regard to TCR ligation, co-ligation of CD5 up-regulates TCR-mediating signaling in peripheral TCR
ß T cells (24). In contrast, CD5 negatively regulates TCRCD3-mediated signaling in TCR
ß T cells (25) and also suppresses BCR-mediated signaling in a B cell subset (termed B-1a cell) which expresses CD5 (26). Although CD5 expression has been demonstrated in most of the TCR
T cells in the spleen (27), only a few TCR
+ intraepithelial lymphocyte (IEL) of the small intestine express CD5 (3). However, the functional role of the CD5 molecule in TCR
T cells is still unknown.
In the present studies, we have explored the role of the CD5 molecule in TCR
T cell-mediated immune responses.
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Methods
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Mice
ß/ mice (C57BL/6), CD5/ mice (B6/129) and RAG-1/ mice (C57BL/6) were purchased from Jackson Laboratory (Bar Harbor, ME). MHC II/ mice (C57BL/6) were obtained from Taconic Farm (Germantown, NY). The screening was performed as previously described (28,29). After screening ß/CD5+/ mice among pups from ß+/CD5+/ pairs, ß/CD5+/ mice were further backcrossed with ß/ mice 5 times. The CD5/ß/ mice were developed from the backcrossed ß/CD5+/ mouse pairs. The MHC II/ß/ double-mutant mice were generated by crossing ß/ mice with MHC II/ mice. The MHC II/CD5/ß/ triple-mutant mice were obtained by crossing MHC II/ß/ double-mutant mice with CD5/ß/ double-mutant mice. All mice were maintained under specific pathogen-free facilities at the Massachusetts General Hospital.
Detection of germinal centers (GC) and cell transfer
Mice (34 months of age) were immunized by i.p. injection of 100 µg Mycobacterium tuberculosis H37Ra (Difco, Sparks, MD). The spleen and serum were obtained from mice 7, 10 or 14 days after immunization. For the cell transfer, cells were extracted from the pooled spleens of donor mice 10 days after immunization with 100 µg H37Ra. CD19 (1D3)-, CD11b (M1/70)- and pan-NK cell (DX5)-positive cells were depleted by using MACS (Miltenyi, Auburn, CA). All antibodies were purchased from PharMingen (San Diego, CA). After examining the purity of TCR
T cells by flow cytometric analysis, 1 x 107 enriched 
T cells (CD3
+ TCR
+ = 93.8 ± 0.9%) were i.v. injected into ß/
/ mice. In some experiments, the enriched TCR
T cells were stained with FITCanti-CD4 (RM4-5) or anti-CD5 (53.7.3) mAb. Subsequently, cells were sorted by flow cytometry to further separate CD4+, CD4 or CD5+ TCR
+ T cell subsets as previously described (30) and 2 x 106 purified cells (purity >98.5%) were injected into ß/
/ mice. The recipient ß/
/ mice were then immunized twice (days 0 and 14 after cell transfer) with 100 µg H37Ra and were sacrificed at 28 days after the cell transfer. Frozen tissue specimens of the spleen were subjected to two-color immunohistochemical analysis using biotinylated anti-IgD mAb (PharMingen) and biotin-labeled peanut agglutinin (PNA; Vector, Burlingame, CA) as previously described (31). The area of GC (PNA+) was measured by light microscopy using a scaled field.
Detection of 5-bromo-2'-deoxyuridine (BrdU)-incorporated cells
Purified CD4+ 
T cells (5 x 106) were i.v. transferred with or without 5 x 106 purified CD5+ 
T cells into ß/
/ mice. The ß/
/ recipient mice were immunized with 100 µg H37Ra 7 days after the cell transfer and then administrated with BrdU by adding 0.8 mg/ml BrdU in drinking water (32). After 3-day administration, spleen cells were isolated and stained with biotinylated anti-TCR
mAb following incubation with PerCPstreptavidin. The cells were stained with phycoerythrinanti-CD4 or anti-CD5 mAb again and fixed and permeabilized with Cytofix buffer following with Cytoperm plus buffer (PharMingen). After re-fixation with Cytofix buffer, the cells were treated with 50 U/ml DNase I (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at 37°C, stained with FITCanti-BrdU mAb (PharMingen) and subjected to flow cytometric analysis.
Detection of colitis
Mice were sacrificed at 7 months of age. The severity of colitis was grossly and histologically estimated according to the previously described criteria (33,34). The disease score (grade 06) was estimated by a combination of both gross (grade 03) and histological findings (grade 03). The gross score was rated as 0, presence of normal beaded appearance; 1, absence of beaded appearance of colon; 2, focal thickened colon; 3, marked thickness of the entire colon. The histological score was based upon the extent of intestinal wall thickening and lamina propria (LP) infiltration. All the slides were evaluated in a blinded fashion by A. K. B.
Extraction of cells and cell transfer
IEL of small intestine were isolated as previously described (35). Colonic LP cells were extracted from the large intestine (excluding the cecum) according to the method described previously (36). After flushing luminal contents with HBSS, the colon was turned inside out. The inverted colon was incubated with RPMI containing 4% FBS and 2 mg/ml dispase (Gibco/BRL, Gaithersburg, MD) for 30 min at 37°C to remove the epithelial compartment. The colon was then cut into small pieces and incubated in RPMI containing 4% FBS, 1 mg/ml collagenase type 2 and 1.5 mg/ml dispase (Gibco/BRL) for 45 min at 37°C with occasional vortexing. After washing with PBS, the cells were suspended in RPMI containing 5% FBS and passed through a glass-wool column. The colonic LP cells were then separated using 40/72% Percoll gradient centrifugation. TCR
T cells from the mesenteric lymph nodes (MLN) and colonic LP of ß/CD5/ mice (7 months of age) were enriched by negative sorting on a MACS system using a cocktail mix of biotinylated anti-CD19 (1D3), CD11b (M1/70), pan-NK cell (DX5) and Gr-1 (RB6-8C5), followed by staining with streptavidinMACS beads. Of the obtained cells, 91.8 ± 2.3% were TCR
+, and the enriched TCR
T cells contained 58 and 53% of the CD4 subset and 42 and 47% of the CD4+ subset in the first and second experiments respectively. The cells (5 x 106) were i.p. injected into RAG-1/ mice that have no T and B cells. An additional transfer of CD5+ TCR
T cells (2.5 x 106, CD5+ TCR
+ > 87%) that were negatively enriched from the MLN of ß/ mice (7 months of age) was performed into some RAG-1/ mice 21days after the first cell transfer. The recipient RAG-1/ mice were sacrificed at day 84. Statistical analysis was performed using the MannWhitney U-test.
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FACS
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Between 2 x 105 and 4 x 105 cells were washed with FACS buffer (PBS containing 0.2% BSA and 0.1% sodium azide), blocked with 10% normal rat and hamster serum (Jackson ImmunoResearch, West Grove, PA) and 1 µg of anti-Fc
II/III (2.4G2; PharMingen) mAb at 4°C for 20 min, and incubated with FITC-, phycoerythrin-, PerCP- and/or allophycocyanin-conjugated mAb at 4°C for 30 min (37). After washing with FACS buffer, cells were analyzed using a FACS (Becton Dickinson, Mountain View, CA).
RNase protection assay (RPA)
Total RNA was extracted from splenic cells excluding red blood cells as previously described (34). The RPA was performed using 10 µg of total RNA with the RiboQuant multi-probe RPA system (PharMingen) as previously described (38). Briefly, RNA was hybridized overnight with the [
-32P]UTP-labeled probe which is in vitro translated by T7 RNA polymerase. After hybridization, samples were treated with RNase A followed by proteinase K. After phenolchloroform extract and ethanol precipitation, the protected fragments were resolved by electrophoresis on a 5% acrylamideurea gel.
RT-PCR
After the purification of TCR
T cell subsets (CD4+ and CD4 TCR
+ T cells; the purity of both subsets > 98%) by FACS sorting, total RNA was extracted from the 1 x 105 cells by using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturers instructions. cDNA was synthesized using (dT)17 primer and Omniscript reverse transcriptase (Qiagen) in 20 µl of reaction buffer. Aliquots of 1 µl of reverse transcription reactions were amplified using specific primers for IL-4 (30 cycles) and ß-actin (30 cycles) (34).
T cell hybridoma
Splenic cells from ß/CD5/ mice (4 months of age) were cultured in vitro with plate-coated anti-TCR
mAb (UC-7, 10 µg/ml) in the presence of 20 ng/ml IL-7 (R & D Systems, Minneapolis, MN) and 25 ng/ml stem cell factor (Genzyme, Cambridge, MA) for 3 days. After washing, the activated cells were fused with TCRBW thymoma cell line in the presence of polyethylene glycol, mol. wt 1500 (Sigma, St Louis, MO) (39). Positive clones were selected with aminopterin-containing medium (Sigma). The CD4+ and CD4 TCR
clones were selected by flow cytometry, and further subcloned twice by using 1.5% methylcellulose medium (Fisher Scientific, Fair Lawn, NJ). The selected hybridomas (1 x 105/150 µl) in DMEM/F-12 containing 1.5% FBS, 1 x SITE-3 (Sigma), 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol and 2 mM L-glutamine were stimulate with plate-coated anti-TCR
(UC7, 10 µg/ml) mAb for 48 h. After collection of the supernatants, the serial diluted culture supernatants were subjected to cytokine Opti-ELISA (PharMingen) according to the manufacturers instruction.
Western blot
Heat killed H37Ra was further disrupted by shaking with 0.1-mm glass beads (180 s, 5000 r.p.m.) in a Mini-beadbeater (Biospec, Bartlesville, OK). After centrifugation, the soluble components were separated by 10% SDSPAGE under reducing conditions and transferred to ECL membranes (Amersham, Arlington, IL). After blocking with ECL blocking reagent (Amersham) at 37°C for 1 h, membranes were incubated with serum (1:100) from the immunized mice for 1 h at room temperature. After incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, membranes were developed using the ECL kit (Amersham) and exposed to ECL hyperfilm (Amersham).
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Results
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The role of the CD5 molecule in the differentiation of the CD4+ TCR
T cell subset
CD5 expression has been demonstrated in most of the TCR
T cells in the spleen (27), whereas only a few TCR
+ IEL of the small intestine express CD5 (3). As previously described (3), the expression of CD5 was detectable only in a few IEL 
T cells of wild-type mice (Fig. 1A). In contrast, most of the CD3+ TCR
T cells expressed CD5 in the spleen of these mice (27) (Fig. 1B). A majority (80.2 ± 3.2%) of these splenic CD5+ TCR
T cells were CD4CD8 double-negative (DN) (27) (Fig. 1C). Alternatively, CD4+ TCR
T cells were detectable as a minor subset (4.0 ± 0.9%) in the splenic TCR
T cells of wild-type mice (Fig. 1C). Since TCRß mutant (ß/) mice, which contain TCR
T cells but no TCR
ß T cells, provide a useful model to study TCR
T cells (46), we next analyzed ß/ mice. Most of the splenic TCR
T cells expressed CD5 and 78.2 ± 2.1% of these CD5+ TCR
T cells were DN in ß/ mice (Fig. 1D). Since the phenotype of TCR
T cells in the ß/ mice was similar to that of wild-type mice, CD5-deficient ß/ (CD5/ß/) mice were generated to define the functional role of the CD5 molecule in TCR
T cells. Surprisingly, the absence of CD5 led to a remarkable increase in a TCR
T cell subset which expresses CD4 on the cell surface in the spleen of ß/ mice (Fig. 1D and E). In contrast, there was only a slight increase of CD4+ TCR
T cells in the splenic TCR
T cells of CD5/ mice (6.1 ± 1.2%, n = 10) as compared to wild-type mice (4.0 ± 0.9%, n = 9), both of which possess TCR
ß T cells. Interestingly, an increase of CD4+ TCR
T cells has been observed in certain immunodeficient conditions such as HIV and autoimmune diseases (4,5,7).

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Fig. 1. Increase in MHC II-independent CD4+ T cells with the absence of CD5 molecules. (A) The expression of CD5 on the TCR + IEL of the small intestine from wild-type C57BL/6 mouse is shown. (B) CD19+ cells were depleted from the splenic cells of a C57BL/6 mouse (4 months of age) by using MACS and stained with four mAb (anti-CD3 , -TCRß, -TCR and -CD5 mAb). The CD3 + TCRß+ population was gated out. The figure shows CD5 expression on the TCR + cells. (C) CD19 splenic cells, described above, were stained with four mAb (anti-CD3 , -TCR , -CD4 and -CD8 mAb). The figure shows CD4 and CD8 expression on the gated CD3 + TCR + population. (D) Cells from the spleens of ß/ and CD5-deficient ß/ (CD5/ß/) mice (4 months of age) were stained with antibodies (anti-TCR , -CD5, -CD4 and -CD8 mAb). The expression of CD4 and CD8 on the CD5+ TCR + cells of ß/ mice and on CD5 TCR + cells of CD5/ß/ mice is shown. (E) The absolute number of splenic CD4+  T cells from ß/ and CD5/ß/ mice (4 months of age) is shown. (F) The CD4 and CD8 expression on the gated TCR + IEL isolated from ß/ (upper panel) and CD5/ß/ mice (lower panel) is shown. (G). The splenic cells were extracted from MHC II/CD5/ß/ triple-mutant mice (4 months of age) and stained with anti-I-Ab mAb or four mAb (anti-CD3 , -TCR , -CD4 and -CD8 mAb). I-Ab is not detectable in the MHC II/CD5/ß/ triple-mutant mouse. The expression of CD4 and CD8 on the splenic CD3 + TCR + cells of this mouse is shown.
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Unlike splenic TCR
T cells, IEL 
T cells mature extrathymically through intestinal crypt patches and represent either CD4CD8 DN or CD8
subsets (40,41). Therefore, we also examined IEL 
T cells. Interestingly, CD4 expression was not detected on the IEL 
T cells of both ß/ and CD5/ß/ mice (Fig. 1F). Therefore, it is possible that the CD5 molecule may selectively affect thymic-derived TCR
T cells capable of expressing CD4 on their surface.
Since TCR
ß T cells expressing CD4 are usually restricted by MHC class II (42), we examined the constraint of CD4+ TCR
T cells to MHC class II molecules by generating MHC II/CD5/ß/ triple-mutant mice. The CD4+ TCR
T cells were clearly detectable in a MHC II/CD5/ß/ triple-mutant mouse (Fig. 1G). These findings indicate that, unlike CD4+ TCR
ß T cells, MHC II molecules are not required for the differentiation and/or maintenance of CD4+ TCR
T cells.
GC development in CD5/ß/, but not ß/, mice
The CD4+ TCR
ß T cells play a critical role in GC formation where B cell affinity maturation and memory is developed by cellcell interactions (43,44). Among TCR mutant mice, GC formation has been detected in TCR
/ mice (45) which possess TCR
T cells and a unique population of CD4+ T cells expressing the TCR ß chain without the TCR
chain (46,47). In contrast, ß/ mice do not contain this unique population and do not develop GC following single immunization with several antigens (4850). To examine whether the CD4+ TCR
T cells are functionally capable of compensating for the absence of CD4+ TCR
ß T cells, CD5/ß/ and ß/ mice were immunized with a conventional antigen, ovalbumin (OVA), in complete Freunds adjuvant (CFA). As previously described (4850), ß/ mice did not develop GC after immunization with OVA in CFA. In contrast, this immunization led to the development of GC in CD5/ß/ mice (data not shown). However, CD5/ß/ mice developed neither humoral reactivity to OVA nor a proliferative in vitro response of splenic cells to OVA (data not shown). In addition, immunization of OVA with incomplete Freunds adjuvant did not induce GC formation in CD5/ß/ mice (data not shown). Therefore, GC are likely to be formed due to a response to M. tuberculosis H37Ra (H37Ra), a component of CFA, rather than OVA. Indeed, immunization with H37Ra alone induced GC formation in the spleen of CD5/ß/ mice, but not the ß/ mice (Fig. 2 and Table 1). Western blot analysis clearly demonstrated that the sera from immunized CD5/ß/ mice, as well as wild-type mice, recognized a dominant band migrating at
70 kDa; the sera from ß/ mice failed to recognize this band (Fig. 2G). Although ß/ mice do not develop GC following immunization (4850), repeated parasitic infections have been reported to result in GC formation in these mice (50). Therefore, it is possible that repeated infections with certain pathogens may directly or indirectly overcome the regulatory function of the CD5 molecule-related signaling cascades.

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Fig. 2. Development of GC in the spleen of CD5/ß/, but not ß/, mice. C57BL/6 (A and D), ß/ (B and E) and CD5/ß/ (C and F) mice were immunized with H37Ra alone and sacrificed 10 days after immunization. The spleen, stained to show PNA binding as red in (A)(C), and the binding of PNA (blue) and anti-IgD (red) in (D)(F), at x10 and x20 objective magnification respectively. (G) The sera were obtained from C57BL/6, ß/ and CD5/ß/ mice 14 days after immunization with H37Ra. After the transfer of H37Ra antigens into Hybond ECL membrane, the diluted serum (1:100) was added to the membrane. After incubation with HRPgoat anti-mouse IgG, the reactivity of the serum was detected by using the ECL detection system (Amersham).
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Contribution of the CD4+ TCR
T cell subset and suppressive role of the CD5+ TCR
T cell subset in the development of GC
To further determine whether CD4+ TCR
T cells can induce GC, CD4+ TCR
T cells from CD5/ß/ mice pre-immunized with H37Ra were transferred into TCRß/ x TCR
/ (ß/
/) mice; these mice were immunized with H37Ra after the cell transfer. CD4+ TCR
T cells were able to generate GC in ß/
/ mice; the size of GC was relatively small (Fig. 3A and D). Marked reconstitution of spleen of the recipient ß/
/ mice with CD4+ TCR
T cells was found (Fig. 3B). In contrast, the transfer of CD5+ TCR
T cells from pre-immunized ß/ mice failed to induce GC in ß/
/ mice. Of note, the co-transfer of CD4+ TCR
T cells from CD5/ß/ mice and CD5+ TCR
T cells from ß/ mice impaired the development of GC in ß/
/ mice (Fig. 3A and E). Although there was marked reconstitution of CD5+ TCR
T cells in the spleen of ß/
/ mice, only few CD4+ TCR
T cells were detectable in the spleen (Fig. 3C). In vivo proliferative CD4+ TCR
T cells as judged by BrdU incorporation was detectable in the spleen of recipient mice transferred with CD4+ TCR
T cells alone (Fig. 3F). In contrast, the co-transfer of CD4+ TCR
T cells and CD5+ TCR
T cells led to reduced numbers of proliferative CD4+ TCR
T cells in the recipient spleen. These findings indicate that CD4+ TCR
T cells are directly involved in the generation of GC and the CD5+ TCR
T cells negatively regulate the development of GC by suppressing the expansion of CD4+ TCR
T cells.
Although ß/
/ mice possess B1a cells that express CD5 (51), the expansion of CD4+ TCR
T cells was not impaired in these mice without co-transfer of the CD5+ TCR
T cells. Therefore, it is likely that the inhibitory effect of CD5 in the expansion of CD4+ TCR
T cells is dependent on the T cells expressing this molecule.
Role of CD5 in development of chronic intestinal inflammation
TCR
T cells are preferentially located in the intestinal mucosa. Most of the studies so far have focused in the study of TCR
T cells in the small intestine (3,5,35,52). Although an increased numbers of TCR
T cells in the colonic LP were found in patients with ulcerative colitis (5,53), there is only limited information available regarding the role of TCR
T cells in the colonic LP, an immune activation site in diseased states (46,54,55). As stated above, in contrast to the splenic TCR
T cells, most of which express CD5 (17) (Fig. 1B), the 
IEL do not express CD5 (3) (Fig. 1A). In the present study, we demonstrate that CD5 expression in the LP TCR
T cells is between that of splenic and intraepithelial TCR
T cells, with 30.4 ± 3.6% (n = 3) of C57BL/6 wild-type and 37.6 ± 2.8% (n = 6) of ß/ colonic LP 
T cells expressing CD5 (Fig. 4A).

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Fig. 4. Development of chronic colitis in CD5/ß/ mice. (A) The cells extracted from colonic LP of C57BL/6, ß/ and CD5/ß/ mice at 7 months of age were stained with anti-CD3 , -TCR and -CD5 mAb. The CD5 expression on CD3 + TCR + cells is shown. (BD) Representative H & E sections of the large intestine from ß/ (B, x10 objective) and CD5/ß/ (C, x10 objective; D, x20 objective) mice (7 months of age) are shown. (E) The severity of colitis, which was graded from 0 (normal) and 1 (borderline) to 6 (most severe colitis), is shown. A dot represents the severity of colitis in an individual C57BL/6, CD5/, ß/ and CD5/ß/ mouse at 7 months of age. (F) The absolute number of mononuclear cells extracted from the colonic LP of C57BL/6, ß/ and CD5/ß/ mice (7 months of age) is shown. (G) The cells extracted from the colonic LP of C57BL/6 and ß/ and CD5/ß/ mice with and without colitis were stained with anti-CD3 , -TCR and -CD4 mAb. The CD4 expression on CD3 + TCR + cells is shown.
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Although an initial study indicated spontaneous development of colitis with late onset in ß/ mice of mixed background (37), only 6.7% of the inbred ß/ mice of C57BL/6 background (7 months of age) maintained in the specific pathogen-free facility developed colitis (grade >2) recognizable by both gross and histological examination (Fig. 4BE). In contrast, 58.5% of CD5/ß/ mice at 7 months of age developed chronic colitis (grade >2) which was relatively mild as compared to the colitis mediated by CD4+ TCR
ß+ T cells in other models of inflammatory bowel disease (46,54,55). Known pathogenic organisms including Helicobacter species were not detectable in these mice. The absolute number of mononuclear cells isolated from colonic LP significantly (P < 0.005) increased in CD5/ß/ mice as compared to ß/ mice (7 months of age) and the increase in the absolute number of LP cells was associated with the development of colitis in CD5/ß/ mice (Fig. 4F). In contrast to a very small percentage of CD4+ cells present in the colonic LP CD3+ TCR
+ population of C57BL/6 wild-type (< 5%; n = 3) and ß/ (<5%; n = 6) mice, the proportion of CD4+ cells in the colonic LP CD3+ TCR
+ population was increased in the CD5/ß/ (21.4 ± 2.7%; n = 12) mice without colitis (Fig. 4G). A markedly increased percentage (38.3 ± 4.4%; n = 15) of CD4+ TCR
+ T cells was associated with the development and severity of colitis in CD5/ß/ mice; 58.9% of colonic LP TCR
+ T cells were CD4+ in a severe case of colitis (grade 5) (Fig. 4G). These findings indicate that the CD5 molecule negatively regulates the pathogenesis of chronic intestinal inflammation in the absence of functional TCR
ß T cells.
Down-regulation of the CD4+ TCR
T cell subset by the CD5+ TCR
T cell subset in the intestine
To determine whether CD4+ TCR
T cells directly participate in the development of colitis in the CD5/ß/ mice, purified CD4+ TCR
T cells from CD5/ß/ mice were transferred into RAG-1/ mice that have no T cells and B cells. After a single or repeated injections (3 times) of the purified CD4+ TCR
T cells (5 x 105), the donor T cells were able to reconstitute in the colonic LP of RAG-1/ mice, but the absolute number (<1.5 x 105) of the CD4+ TCR
T cells in the colonic LP was markedly less than in CD5/ß/ mice with colitis (9.4 ± 1.9 x 105). Consequently, the transfer of CD4+ TCR
T cells failed to induce colitis in RAG-1/ mice within a 12-week period of observation. In contrast, relatively low numbers (4 x 105) of CD4+ TCR
ß+ T cells have been demonstrated to expand and induce colitis in the immune-deficient recipient mice (55). Therefore, unlike colitic CD4+ TCR
ß+ T cells, CD4+ TCR
T cells expand poorly in the colonic LP of RAG-1/ mice, perhaps due to the lack of factors necessary for CD4+ TCR
T cell expansion.
To examine whether CD4+ TCR
T cell expansion in the colonic LP is selectively down-regulated by the CD5+ TCR
T cell subset, TCR
T cells (from CD5/ß/ mice) that contain both CD5CD4+ and CD5CD4 TCR
subsets were transferred into RAG-1/ mice. Both CD4+ and CD5CD4 TCR
T cell subsets were equally reconstituted in the colonic LP of RAG-1/ mice 12 weeks after the cell transfer (Fig. 5). These findings indicate that the CD5 TCR
subset does not affect CD4+ TCR
T cell expansion in vivo. To examine whether CD5+ TCR
T cells are able to affect the reconstituted CD4+ TCR
T cells in the colonic LP of the recipient mice, RAG-1/ mice were first reconstituted with both CD4+ TCR
and CD5CD4 TCR
subsets (5 x 106). Three weeks after the transfer, CD5+ TCR
T cells (2.5 x 106) from ß/ mice were then transferred into the reconstituted RAG-1/ mice and these mice were sacrificed 9 weeks after the CD5+ TCR
T cell transfer. The CD5+ TCR
T cell transfer led to a marked reduction of CD4+ TCR
T cells, but did not affect the CD5CD4 TCR
T cell subsets; both CD5CD4 and CD5+CD4 TCR
T cell subsets were clearly detected, whereas the proportion of the CD4+ TCR
T cell subset was markedly reduced in the colonic LP of the recipient RAG-1/ mice (Fig. 5). Therefore, the presence of the CD5+ TCR
T cell subset leads to a selective elimination of CD4+ TCR
T cells in the colonic LP.
The CD4+ TCR
T cell subset shows a Th-like cytokine profile
The TCR
ß T cells expressing CD4 are divided into Th subsets based on the cytokine profile (56). The CD4 molecule has been implicated in the production of IL-4 (11) and CD4 expression confers an ability to produce IL-4 by TCR
T cells (14). Therefore, we analyzed the cytokine profile by RT-PCR and RPA to examine whether CD4+ TCR 
T cells show a cytokine production pattern similar to that seen in CD4+ TCR
ß T cells. RPA showed that IL-4 mRNA expression is reproducibly detectable in the spleen of CD5/ß/, but not ß/, mice at 4 months of age (Fig. 6A). After separation of CD4+ and CD4 TCR
T cell subsets from the spleen of CD5/ß/ mice, RT-PCR clearly demonstrated IL-4 mRNA expression in the CD4+ TCR
, but not in the CD4 TCR
, T subset (Fig. 6B). To further characterize the cytokine profile of TCR
T cell subsets at the single-cell level, we generated CD4+ and CD4 TCR
T cell hybridomas by fusing splenocytes of CD5/ß/ mice with the TCRBW thymoma line. After TCR ligation, a variety of cytokine production patterns described in Th0, Th1 and Th2 subsets of TCR
ß T cells were found in the CD4+ TCR
hybridomas generated from CD5/ß/ mice (Fig. 6C). Therefore, it is possible that, like CD4+ TCR
ß T cells, CD4+ TCR
T cells may contain Th-like subsets with distinct cytokine profiles.
 |
Discussion
|
---|
Although CD4+ T cells form a major subset in TCR
ß T cells, CD4 is normally expressed by only a small population of human and mouse TCR
T cells (9,13,14) (Fig. 1C). Interestingly, the increase of the CD4+ TCR
T cell subset has been reported in certain conditions (including HIV) in which normal TCR
ß T cell functions are impaired (4,5,7). Here, we demonstrate that the CD5 molecule is either directly or indirectly involved in the down-regulation of CD4+ TCR
T cells in vivo under immunodeficient conditions present in TCRß/ mice. Since the absence of CD5 does not induce expansion of CD4+ TCR
T cells in the presence of TCR
ß T cells, it is likely that, in addition to CD5, TCR
ß T cells also participate in the suppression of CD4+ TCR
T cell expansion in vivo.
As previously described (14,45,50), CD4+ but not CD4 TCR
T cells can help GC formation. Interestingly, the CD4+ TCR
T cell-mediated GC formation results from the response to mycobacterial antigens present in CFA, but not a conventional T cell-dependent antigen, OVA. Indeed, our present study demonstrates that the CD4+ TCR
T cells are MHC class II independent as indicated by the presence of this subset in the absence of MHC class II. Since GC plays an important role in the generation of B cells producing high-affinity antibodies (43,44), it is possible that CD4+ TCR
T cells may be involved in helping the clearance of certain pathogens by facilitating production of high-affinity antibodies to non-classical antigens (present in pathogens) which are not recognized in a MHC class II-dependent fashion.
Interestingly, CD4+ TCR
T cell-mediated GC formation is inhibited by the other TCR
T cell subset expressing CD5. The interactions between TCR
ß T cells and TCR
T cells and between peritoneal B-1 cells and TCR
T cells have been shown to regulate certain immune responses (7,35,5759). The coordinated immune functions by TCR
T cell subsets have also been proposed to play a role in linking between innate and adaptive immunity (60). In addition, a recent report suggests that TCR
T cells may present certain peptide antigens (61). Therefore, it is possible that the interactions between functionally distinct TCR
T cell subsets characterized by either CD5 or CD4 expression control certain immune responses especially under immune-deficient conditions such as HIV infection and autoimmune diseases.
The CD4 molecule has been implicated in the production of IL-4 (11) and CD4 expression confers an ability to produce IL-4 by TCR
T cells (14). Indeed, our present study suggests that CD4+ TCR
T cells possess functionally distinct subsets with the cytokine production pattern resembling Th (CD4+ TCR
ß) subsets. However, as this conclusion is based on results obtained from hybridomas generated by fusion with the thymoma cell line, a more direct proof is needed to confirm these observations.
Most splenic TCR
T cells express CD5, whereas CD5 is detectable in only a few 
IEL (3,27). Here, we demonstrate that CD5 expression in the LP TCR
T cells is between that of splenic and intraepithelial TCR
T cells. The lack of CD5 also leads to an increase of CD4+ TCR
T cells in the colonic LP and results in the development of colitis in CD5/ß/ mice. Therefore, it is possible that CD4+ TCR
T cells possess a potential ability to induce chronic intestinal inflammation. However, cell transfer studies failed to provide direct support for this hypothesis. A recent study has also demonstrated that intestinal TCR
T cells are involved in the exacerbation of intestinal inflammation (62). The transfer of these cells also did not induce colitis in immune-deficient recipient mice (62). Therefore, it is possible that some additional factors may be required to fully bring out the pathogenic potentiality of the CD4+ TCR
T cells in the recipient mice.
In the thymus, the CD5 molecule negatively regulates the differentiation of the CD4-committed lineage (15,16). In addition, our present studies suggest that CD5 molecules are involved in CD4+ TCR
T cell expansion and/or differentiation. The CD4+ TCR
T cells may play a role in immune responses, especially under immune-deficient conditions.
 |
Acknowledgements
|
---|
We thank Dr M. B. Brenner (Brigham and Womens Hospital) for his critical comments and A. Kelliher for her help with FACS analysis. This work has been supported National Institutes of Health (NIH) grant DK47677 (A. K. B.), by the Center for the Study of Inflammatory Bowel Disease at the Massachusetts General Hospital (NIH DK43351), and First Award (A. M.), the Crohns and Colitis Foundation of America, Inc.
 |
Abbreviations
|
---|
ß/TCR ß knockout
BrdU5-bromo-2'-deoxyuridine
CD5/ß/CD5-deficient TCRß double-knockout
CFAcomplete Freunds adjuvant
DNdouble negative
GCgerminal center
H37RaMycobacterium tuberculosis H37Ra
HRPhorseradish peroxidase
IEL intraepithelial lymphocyte
LPlamina propria
MLNmesenteric lymph nodes
PNApeanut agglutinin
OVAovalbumin
RPARNase protection assay
 |
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