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The Journal of Immunology, 2004, 173: 2324-2330.
Copyright © 2004 by The American Association of Immunologists

Regulated Expression of the Receptor-Like Tyrosine Phosphatase CD148 on Hemopoietic Cells1

Joseph Lin2,*,{dagger}, Jing W. Zhu2,*,{ddagger}, Jeanne E. Baker*,{ddagger} and Arthur Weiss3,*,{dagger},{ddagger}

* Departments of Medicine, and Microbiology and Immunology, {dagger} Biomedical Sciences Graduate Program, and {ddagger} Howard Hughes Medical Institute, Rosalind Russell Medical Research Center for Arthritis, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD148 is a receptor-like protein tyrosine phosphatase expressed on a wide variety of cell types. Through the use flow cytometry and immunofluorescence microscopy on tissue sections, we examined the expression of CD148 on multiple murine hemopoietic cell lineages. We found that CD148 is moderately expressed during all stages of B cell development in the bone marrow, as well as peripheral mature B cells. In contrast, CD148 expression on thymocytes and mature T cells is substantially lower. However, stimulation of peripheral T cells through the TCR leads to an increase of CD148 expression. This up-regulation on T cells can be partially inhibited by reagents that block the activity of src family kinases, calcineurin, MEK, or PI3K. Interestingly, CD148 levels are elevated on freshly isolated T cells from MRL lpr/lpr and CTLA-4-deficient mice, two murine models of autoimmunity. Together, these expression data along with previous biochemical data suggest that CD148 may play an important regulatory role to control an immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Also known as density-enhanced phosphatase-1, protein tyrosine phosphatase-{eta} (PTP-{eta}),4 PTP receptor type J, and {beta}-like tyrosine phosphatase, CD148 is a receptor-like PTP expressed on a wide range of cell types including fibroblasts, endothelial cells, hemopoietic cells, and many others (1, 2, 3). Most studies have described a role for CD148 in the regulation of cell growth and the cell cycle using fibroblasts or tumor cell lines (2, 3, 4, 5). Malignant tumors and transformed cell lines exhibit reduced CD148 expression compared with normal cells and overexpression of CD148 inhibits tumor cell growth (6, 7). CD148 is thought to inhibit cell cycle progression by increasing the stability of the cyclin-dependent kinase inhibitor p27Kip1 (8). Targeted disruption of the catalytic activity of CD148 in mice results in an early embryonic lethality due to vascular development defects providing further evidence that CD148 regulates cell growth and cell cycle (9). Together, these previous findings implicate the CD148 expression level as a critical regulator of cell growth and cell cycle.

Although CD148 is expressed on the surface of hemopoietic cells, very little is known about the expression pattern and function of CD148 in the murine immune system. A few studies examining the expression of CD148 in human lymphocytes, especially T cells, have been published (1, 10). These studies showed that CD148 is expressed at low levels on resting T cells, but is up-regulated following activation (1, 10). TCR-cross-linking with a mAb combined with an anti-CD148 mAb augmented T cell proliferation when compared with TCR-cross-linking alone, however, the mechanism by which anti-CD148 mAb effects responses has not been clearly determined (10). The role of CD148 in B cells remains elusive. One study implicated high CD148 expression as a marker of human memory B cells, however, limited functional data have been reported (11).

Experiments in T cell lines have demonstrated that CD148 is a negative regulator of TCR signaling. For example, when CD148 was expressed in T cell lines, the activation of the TCR-induced NFAT was reduced (12, 13, 14). Expression of CD148 also decreased TCR-induced phosphorylation of the adaptor protein linker for the activation of T cells and the enzyme phospholipase C-{gamma}1 (PLC-{gamma}1), two molecules critical for T cell activation (12, 13). In addition, immunofluorescence microscopy examining CD148 localization on the cell surface revealed that CD148 is excluded from the site of contact between a T cell and an APC, similar to another transmembrane phosphatase CD45 (13, 15). This observation supports the notion that localization can influence the function of CD148 as a negative regulator (13).

Although cell line data indicate a potential negative regulatory function for CD148 in lymphocyte activation, little is known about its role in regulating the immune response in primary cells. To further characterize the role of CD148 in immune responses, we have generated a mAb to murine CD148. Using this mAb, we assessed the expression of CD148 on mature hemopoietic cells and developing lymphocytes. In addition, we analyzed the regulation of CD148 expression following antigenic stimulation of T and B cells. We determined that CD148 is expressed on most hemopoietic cell lineages, and that its regulated expression on T cells, in particular, may play an important role in controlling the immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

Anti-murine CD148 mAb (8A-1) was generated by footpad immunization of Syrian hamsters with 293T cells expressing murine CD148. Cells from the draining lymph nodes were then fused to a myeloma cell line using polyethylene glycol and plated to isolate single clones. Clones were selected for secretion of Abs staining cells transfected with murine CD148, but not untransfected cells, as determined by flow cytometry. Positive clones were subcloned and assayed again. mAbs to CD3{epsilon} (2C11), CD11b (M1/70), CD19 (MB19-1), CD5 (53-7.3), Gr-1 (RB6-8C5), B220 (RA3-6B2), CD4 (GK1.5), CD8 (53-6.7), NK1.1 (PK136), TCR {gamma}{delta} (UC7-13D5), CD69 (H1.2F3), Ter119 (Ter119) IgM (1B4B), and TCR {beta} (H57) were from e-Bioscience (San Diego, CA). Anti-CD28 ascite was generated using hybridoma 37.51. 7-Aminoactinomycin D (7AAD) was from BD Pharmingen (San Diego, CA) and sheep anti-IgD was from The Binding Site (Birmingham, U.K.). Primary mouse lymphocytes were cultured in D-MEM with 10% FBS, 50 µM 2-ME, 2 mM glutamine, nonessential amino acids, penicillin, and streptomycin.

Immunofluorescence staining

Splenic sections were cut from Tissue-Tek OCT compound (Sakura, Torrance, CA) embedded tissues at 8 µm and then fixed with acetone. Sections were blocked in PBS with 1% BSA and 5% normal serum from the species which generated the secondary Ab. Slides were then stained with the indicated primary Abs for 1 h at room temperature. After washing, the appropriate secondary Ab conjugated to either Alexa 488 (Molecular Probes, Eugene, OR), Cy3, or Cy5 (Jackson ImmunoResearch Laboratories; West Grove, PA) was used. Slides were visualized on a Marianas Turn-Key system from Intelligent Imaging (Denver, CO) and images were analyzed using SlideBook software (Intelligent Imaging) and exported in TIFF format.

In vitro cell stimulation

For B cell purification, splenocytes were labeled with anti-CD11b and anti-CD43 mAb-conjugated MACS beads (Miltenyi Biotec, Auburn, CA) and passed through a MACS LS column (Miltenyi Biotec) to deplete non-B cells. B cells were stimulated for the indicated times with anti-CD40 mAb (HM40-3) (1 µg/ml; BD Pharmingen) and goat anti-mouse IgM F(ab')2 (10 µg/ml; Jackson ImmunoResearch Laboratories) or LPS (10 µg/ml; Sigma-Aldrich, St. Louis, MO). For T cell purification, lymph node cells were passed through an R&D T cell enrichment column (R&D Systems, Minneapolis, MN) and negatively selected using the manufacturer’s protocol. T cells were stimulated with plate-bound anti-CD3 (1 µg/ml) and anti-CD28 (1:1000 ascites) or PMA (2 ng/ml) and ionomycin (0.5 µM).

Inhibition of CD148 up-regulation

Bulk lymph node cells were cultured in a 24-well plate (Costar, Corning, NY) at 4 x 106 cells/well in 1 ml of the media as described above. Inhibitors or vehicle only were added for 30 min before stimulation with soluble CD3 (1 µg/ml) and anti-CD28 (1:1000 ascites). Cells were harvested at 40–48 h following stimulation and stained with anti-CD69 mAb, anti-TCR {beta} mAb, and 7AAD. Inhibitors were from Calbiochem (San Diego, CA) and the following final concentrations were used: PP2 (20 µM), FK506 (100 ng/ml), LY294002 (5 µM), U0126 (10 µM).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of CD148 expression with the anti-mCD148 mAb (8A-1)

Anti-murine CD148 mAb was generated by immunizing Syrian hamsters in the footpad with 293T cells expressing murine CD148. Cells from the draining lymph node were then fused and clones secreting anti-mCD148 Ab screened. One clone, 8A-1, displayed specificity to 293T cells transfected with mouse CD148, but not cells transfected with CD8 as a control, when analyzed by flow cytometry (Fig. 1A).



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  		FIGURE 1. Using anti-mCD148 mAb (8A-1) to study the expression of CD148 on murine hemopoietic cells. A, 293 T cells were transfected with either murine CD8 as a control or murine CD148. Cells were stained with 8A-1 mAb and CD148 expression analyzed by flow cytometry. B, Mouse hemopoietic tissues were isolated from C57BL/6 mice and the various hemopoietic cell lineages classified by the indicated cell surface markers. The CD148 expression on the examined cell lineages are shown as the geometric mean fluorescence of CD148 staining. The Geo. Mean of fluorescence (FL) on the table in Fig. 1B is obtained by subtracting the MFI of isotype control from the MFI of CD148 on each cell lineage. C, Spleen sections from C57BL/6 mice were stained by immunofluorescence to show CD148 (green) expression in tissues. IgD (red) staining delineates the B cell zone whereas CD4 (blue) staining delineates the T cell zone.

 
To further characterize the expression of CD148 on various hemopoietic cell lineages, cells were isolated from mouse tissues. Anti-CD148 mAb conjugated to FITC was used in combination with the indicated mAbs and the stained cells were analyzed by flow cytometry. The CD148 expression levels of the examined cell lineages are summarized in Fig. 1B. In the lymphoid lineage, B cells had substantial levels of CD148. Interestingly, peritoneal B1 B cells had the highest level of CD148 in the cell types examined. In contrast, all types of T cells as well as NK cells displayed low levels of CD148 expression. In the myeloid lineage, bone marrow macrophages expressed high levels of CD148 whereas neutrophils expressed moderate CD148 levels. Cells from the erythroid lineage also display moderate CD148 expression. In summary, although CD148 is expressed on a variety of hemopoietic cells, its expression level is quite variable between cell lineages.

The expression pattern of CD148 was further analyzed by immunofluorescence microscopy. Spleen sections from C57BL/6 mice were stained with Abs to CD148, IgD, and CD4 to visualize the B and T zones within the follicle (Fig. 1C). Consistent with our analysis by flow cytometry and previous T cell data, the T cell zone did not stain brightly for CD148. The B cell zone stained substantially brighter than T cell zones, reflecting the higher resting levels of CD148 on B cells seen by flow cytometry. However, cells outside the follicle that stained the brightest for CD148 also costained with Gr-1 and CD11b indicating they are likely macrophages (data not shown).

CD148 expression on developing and mature B lymphocytes

B cells and other hemopoietic progenitors develop in the bone marrow. Cells that are at different stages of B cell development can be distinguished by the expression of characteristic cell surface markers. The earliest B cells are characterized as pro-B cells (B220low, CD43+, CD25–). In response to cytokines and initial Ig chain rearrangement, these cells mature to pre-B cells (B220+, CD43–, CD25+). Signals derived from the pre-BCR complex induce further maturation, eventually giving rise to immature B cells (B220+, IgM+, IgD–). These cells then migrate to peripheral lymphoid tissues, where they become mature B cells (B220+, IgM+, IgD+) (16).

We examined the expression of CD148 during these stages of B cell development by isolating bone marrow cells from C57BL/6 mice and staining the cells with anti-CD148 mAb and markers for B cell development. As shown in Fig. 2A, CD148 is expressed throughout all stages of B cell development in the bone marrow as well as in mature cells in the periphery. We were also interested in determining whether CD148 expression is regulated on peripheral B cells because B cell activation is tightly regulated by a number of kinases and phosphatases. These pathways lead to induction of downstream effector functions such as proliferation, cytokine release, and differentiation into Ab-secreting plasma cells. To determine whether B cell activation has an effect on CD148 expression, splenic B cells were purified from C57BL/6 mice and a time course of stimulation with anti-CD40 and anti-IgM Abs or LPS was performed (Fig. 2B). After each time point, B cells were harvested and stained with the activation marker CD69, or CD148. Following both anti-CD40 and anti-IgM mAb or LPS stimulation, CD69 was up-regulated rapidly demonstrating that cells did indeed respond to the stimuli (data not shown). However, the CD148 expression level did not change substantially following stimulation thereby suggesting that CD148 expression is not modulated by B cell activation (Fig. 2B).



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  		FIGURE 2. Characterization of CD148 expression on developing, mature, and activated B cell subsets. A, Bone marrow cells and splenocytes were isolated from C57BL/6 mice and stained with Abs to identify the different stages of B cell development: pro- (B220low, CD43+, and CD25–), pre- (B220+, CD43–, CD25+), immature (B220+, IgM+, IgD–), and mature (B220+, IgM+, IgD+). The expression of CD148 was determined by staining with anti-CD148 mAb (8A-1). Shaded histograms represent staining with an isotype control. B, Splenocytes were isolated and B cells were purified by negative selection with anti-CD11b and anti-CD43 MACS beads. Purified B cells were then stimulated with anti-IgM (10 µg/ml) + anti-CD40 (1 µg/ml) mAbs or LPS (10 µg/ml) for the indicated times. Cells were then harvested and the CD148 expression was analyzed by flow cytometry. Shaded histograms represent an isotype control. C, Splenocytes were isolated and stained with anti-CD148, anti-B220, anti-CD21, and anti-CD23 mAbs. CD148 expression on MZ B cells (CD19+, CD21+, CD23–) is shown compared with follicular B cells (CD19+, CD21+, CD23+). Light gray shaded histogram is isotype control for follicular B cell; the dark-gray shaded histogram is isotype control for MZ B cell. The MFI for each population are included. D, To look at MZ B cells by immunofluorescence, spleen sections from C57BL/6 mice were stained with anti-IgM, anti-IgD, and anti-CD148 mAbs. Follicular B cells were defined as IgM med, IgD+, and MZ B cells as IgMhigh, IgDlow. The white dashed line indicates the MZ.

 
B cells can be further classified into subsets of B cells such as follicular B cells, marginal zone (MZ) B cells, or peritoneal B1 B cells. Development into these subsets is dependent on various cytokines and/or antigenic stimuli (17). Because peritoneal B1 and MZ B cells are thought to have BCR repertoires that are biased toward self-reactive Ags, we hypothesized that these cell types would require higher constitutive levels of negative regulators such as CD148. To examine CD148 expression on peritoneal B1 cells and MZ B cells, cells from C57BL/6 mice were isolated and analyzed by flow cytometry. Peritoneal B1 cells exhibit the highest levels of CD148 out of all cell types tested (Fig. 1B). Also, MZ B cells express higher levels of CD148 compared with follicular B cells (mean fluorescence intensity (MFI) 54.0 vs 18.9) (Fig. 1C). To further investigate CD148 expression on MZ B cells, spleen sections from C57BL/6 mice were processed and stained with Abs to CD148, IgM, and IgD. The majority of B cells in the spleen are follicular B cells. MZ B cells localize at the outer edges of the follicle and express high levels of IgM and low levels of IgD. Indeed, MZ B cells (IgMhigh, IgDlow) stain brighter than follicular B cells as indicated by the bright colocalization (yellow) of CD148 (green) with IgM (red) at the edges of the follicle in the merged image (Fig. 2D).

CD148 expression on developing and mature T lymphocytes

T cell development occurs in the thymus where immature T cells proliferate and mature by passing through a series of developmental stages that can be identified by characteristic expression of various cell surface proteins. The most immature cell population in the thymus is CD4–, CD8– (double negative (DN)). During the maturation process, these DN cells up-regulate both CD4 and CD8 (double positive (DP)). Most DP cells die in the thymus because they never encounter an Ag; however, those that do survive the selection process become either CD4+ or CD8+ (single positive (SP)) (18, 19). To address whether CD148 is expressed during this developmental process, we isolated thymocytes from C57BL/6 mice and stained with mAbs to CD4, CD8, and CD148. Unlike developing B cells, which express moderate levels of CD148, thymocytes express very low levels during development (Fig. 3A). Similarly, expression of CD148 on mature T cells in the periphery was also low and there was no difference between CD4 vs CD8 T cells (Fig. 1B).



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  		FIGURE 3. Characterization of CD148 expression on developing, mature, and activated T cells. Thymocytes were isolated from C57BL/6 mice and stained with CD4 and CD8 to determine their stage of T cell development (DN CD4–, CD8–; DP CD4+, CD8+; SP CD4+, CD8– or CD4–, CD8+). These cells were evaluated for expression of CD148 by staining 8A-1 mAb. Shaded histograms represent an isotype control. B, Purified peripheral T cells were stimulated with anti-CD3 (10 µg/ml) + anti-CD28 (1:1000 ascites) mAbs or PMA (2 ng/ml) + ionomycin (0.5 µM) for the indicated time points. Cells were then analyzed for CD148 expression by flow cytometry. Shaded histograms represent an isotype control. Data are representative of at least three independent experiments.

 
Interestingly, in striking contrast to activation of peripheral B cells, stimulation of naive murine T cells induced the up-regulation of CD148. As shown in Fig. 3B, T cells purified from C57BL/6 mice stimulated for 0, 8, 24, 48, and 72 h with immobilized anti-CD3 and anti-CD28 mAbs or PMA and ionomycin displayed a rapid and substantial increase of CD148, peaking between 24 and 48 h. There is no obvious difference between CD4+ and CD8+ T cell subsets on the up-regulation of CD148 (data not shown). These data demonstrate that CD148 expression is regulated during T cell activation.

Inhibitors of CD148 up-regulation following T cell stimulation

To further investigate the signals required to up-regulate CD148 during T cell activation, we used various pharmacological inhibitors to block specific pathways required for T cell activation. To block the most proximal events of TCR-induced signal transduction, we treated the cells with the src family kinase inhibitor PP2. As expected, PP2 treatment potently blocked CD148 up-regulation in response to soluble anti-CD3 and anti-CD28 mAb stimulation (Fig. 4).



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  		FIGURE 4. Inhibition of signaling molecules involved in T cell activation blocks CD148 up-regulation. Lymph nodes were isolated from C57BL/6 mice and stimulated with soluble anti-CD3 (10 µg/ml) + anti-CD28 (1:1000 ascites) mAbs for 48 h in the presence of the indicated pharmacologic inhibitors: PP2 (20 µM), FK506 (100 ng/ml), Ly294002 (5 µM), U0126 (10 µM). Cells stimulated with PMA (2 ng/ml) + ionomycin (1 µM) were used as a control. CD148 expression was then analyzed on live T cells (TCR-{beta}+, 7AAD–) by flow cytometry. The MFI of the unstimulated sample was subtracted from all the stimulated samples. The MFI of carrier for each experiment was set as 100%, the samples with different inhibitors were presented as the percentage of carrier MFI. The result represents four independent experiments. Error bars indicate the SE. The paired Student t test was performed for the effect of inhibitors; for PP2, FK506, and U0126 p < 0.001, for Ly294002, p < 0.01.

 
To inhibit molecules further downstream of TCR stimulation in the signaling cascade, the inhibitor FK506, which blocks the activity of the protein phosphatase calcineurin, and the inhibitor U0126 which blocks MEK activation, were used. Both of these pharmacological agents inhibited CD148 up-regulation less than the proximal inhibitor PP2, but considerably more than a vehicle control (DMSO) (Fig. 4).

Stimulation with anti-CD3 mAb alone is much less effective at inducing CD148 up-regulation in purified T cells when compared with anti-CD3 and anti-CD28 mAb stimulation (data not shown). Because CD28 has a consensus YxxM motif in its cytoplasmic tail that has been demonstrated to recruit PI3K, we used a specific inhibitor of PI3K, LY294002, to assess whether PI3K is required for the up-regulation of CD148. Treatment of cells with Ly294002 also potently inhibited the up-regulation of CD148 on anti-CD3 and anti-CD28 mAb stimulated cells to levels similar to FK506 and U0126 treatment (Fig. 4). Thus, the PI3K signal is important in the up-regulation of CD148 following activation.

High basal expression of CD148 in autoreactive T cells

Because CD148 is up-regulated following T cell activation, we hypothesized that CD148 could play a role regulating prolonged signals downstream from receptor stimulation. To see whether CD148 expression is disregulated in T cells from mice exhibiting profound autoimmunity, we studied lymphoproliferation (lpr) mice. The lpr defect in mice causes a profound lymphoproliferative disorder on the MRL background due to a defect in the death receptor Fas (20). Sera from these mice contain anti-nuclear Abs, a hallmark of autoimmune disease. The majority of the T cells populating the MRL lpr/lpr lymph nodes have uncharacteristic expression of T cell surface markers in that they are identified by CD4–, CD8–, CD3+, and B220+ (20). Freshly isolated lpr/lpr T cells expressed higher basal levels of CD148 compared with that of T cells from a wild-type mouse on the MRL background supporting our hypothesis that CD148 is an important negative regulator (Fig. 5A).



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  		FIGURE 5. Increased CD148 expression on T cells from autoimmune mice. A, Lymphocytes were isolated from lymph nodes of wild-type MRL mice and MRL lpr/lpr mice with overt lymphadenopathy and analyzed for the expression of CD148 by flow cytometry. Anti-CD3 and anti-B220 mAb staining was used to determine cell lineages. Overlays represent CD148 expression levels for indicated cell lineages. B, Lymphocytes were isolated from lymph nodes of wild-type and CTLA-4–/– mice and analyzed for the expression of CD148 as above. Data are representative of at least two independent experiments.

 
We also examined the expression of CD148 on mice deficient in CTLA-4 because these mice also display profound lymphadenopathy and have several characteristics of autoimmune disease (21). Lymph node T cells from 3- to 4-wk-old CTLA-4–/– mice displayed higher expression of CD148 when compared with littermate controls (Fig. 5B). Interestingly, CTLA-4–/– T cells from the spleen displayed normal CD148 expression, whereas T cells from the spleen of lpr/lpr mice were similar to lymph node T cells. Thus, lymph node T cells from MRL lpr/lpr mice and CTLA-4-deficient mice expressed a higher level of CD148 implicating a role in T cell regulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To characterize the expression of CD148 in the immune system, a mAb to murine CD148 was generated to investigate the expression pattern of CD148 on resting and stimulated primary lymphocytes. In summary, B cells express CD148 throughout development and in the periphery, whereas T lymphocytes only express low levels through development. However, when peripheral T cells are stimulated, they up-regulate CD148, whereas B cells do not dramatically alter expression. Reagents that mimic downstream TCR signals, such as PMA and ionomycin, also up-regulate CD148 in T cells. Our analysis of murine CD148 expression is similar to published results examining human lymphocytes in that B cells constitutively express CD148 and that T cells express low levels of CD148 until activated (1, 10).

Although there are several similarities between human and murine CD148, there are also some important differences. In humans, resting CD8+ T cells have higher levels of expression compared with resting CD4+ T cells, whereas in mice there is no discernable difference (Fig. 1B) (10). This difference is interesting because on human CD8+, but not CD4+, T cells, ligation with immobilized anti-CD148 mAb could substitute for anti-CD28 costimulation to induce proliferation (10). Although, the effects of CD148 ligation with the mAb toward human CD148 is not known, attempts to induce functional effects with the mAb 8A-1 described here did not give dramatic, reproducible results in either T cell or B cell proliferative responses (data not shown). We have also tested the kinetics for up-regulation of CD148 on CD4 vs CD8 T cells following activation, and did not observe obvious difference (data not shown).

The observation that CD148 expression is elevated on cells from the MRL lpr/lpr mouse suggests that CD148 may be up-regulated as part of an inhibitory feedback loop. In another report, T cells from the autoimmune prone c-cbl and cbl-b double knockout were noted to have higher levels of CD148 on T cells in the periphery (22). These cells appeared chronically activated and contribute to an autoimmune phenotype because these mice also have higher levels of anti-dsDNA Abs. Interestingly, the T cells from these mice exhibit a selective decrease in linker for the activation of T cells and PLC-{gamma}1 phosphorylation following TCR stimulation, a phenotype similar to that seen in Jurkat T cells stably expressing CD148 (12, 13). The CD148+ Jurkat cell line has a decrease in Ca2+ mobilization, Erk activation, and subsequent NFAT activation in response to TCR stimulation. A similar defect in TCR-mediated signal transduction has been reported for MRL lpr/lpr T cells (20). These data support the prediction that mice with a targeted deletion of CD148 would develop hyperreactive T cell responses or even autoimmunity. Unfortunately, mice deficient in CD148 die during embryogenesis due to angiogenic defects, therefore generating a conditional knockout of CD148 in T cells would be an ideal means of testing this hypothesis (9).

Because CD148 expression was augmented in murine models of autoimmunity, we did attempt to study CD148 expression in murine models of tolerance. We examined mice carrying a BCR transgene specific to hen egg lysozyme, but found no difference of CD148 expression on tolerized B cells compared with normal B cells (data not shown) (23). We also studied T cells from mice carrying a transgenic TCR specific to the endogenous male H-Y Ag (24, 25). When peripheral T cells from mice carrying the H-Y TCR transgene were isolated and analyzed, there was no difference in CD148 expression between male and female mice (data not shown). These data indicate that CD148 expression levels do not seem to regulate immune tolerance in these transgenic systems.

The role of CD148 in B cells has not been as well studied as T cells. One study did imply that CD148 is a good maker for memory cells on human splenic B cells along with CD27; however, functional data in both human and mouse B cells are lacking (11, 26). If we assume that the substrates of CD148 in T cells are similar to that of B cells, one would predict that potential B cell substrates could be molecules such as linker for the activation of B cells/non-T cell activation linker, B cell linker protein, or PLC-{gamma}2 (27, 28, 29). The expression of CD148 in B cells, however, is constitutive and does not change substantially following stimulation, suggesting that the function or regulatory mechanism in the respective cell types may be different. Similarly, a conditional knockout in the B cell lineage would address the role of CD148 in B cells.

Interestingly, both MZ and peritoneal B cells express higher levels of CD148 in the basal state compared with follicular B cells. These data support the model that higher levels of negative regulators may be required to keep the cells from becoming pathologically active because the BCR repertoire of MZ and peritoneal B cells are biased toward low affinity, self-reactive receptors (17, 30). In addition, MZ and peritoneal B cells are faster and more efficient at becoming Ab-secreting plasma cells. This again supports the model that these B cell subsets require more of a negative regulatory influence because they are in a more "primed" state (17, 31, 32).

Most studies of CD148 in nonhemopoietic cells have focused on its role in cell cycle. However, our data along with previously published results support an alternative role for CD148 as a negative regulator in hemopoietic lineages (5, 8, 12, 13, 14). Because CD148 expression does not change in response to mitogenic B cell stimulation, it is unlikely that it plays a role in cell cycle control in B cells. It is unclear whether CD148 affects cell cycle control in thymocytes or mature T cells. Because levels of CD148 are low in the thymus and do not change markedly during thymocyte development, CD148 does not seem to be involved in thymic proliferation during TCR repertoire selection. In peripheral T cells, CD148 up-regulation begins by eight hours after TCR stimulation, whereas stimulated T cells do not divide until ~30 h later. Thus, CD148 expression could either be terminating the signals emanating from the TCR, or CD148 could be acting on downstream events involved in cell cycle control.

Much is still not known about the role CD148 plays in the hemopoietic lineage, but these data provide important information regarding the relative expression of CD148 in the various hemopoietic cell lineages. Its regulated expression following T cell activation, higher expression on MZ and peritoneal B1 cells, and constitutive expression on autoimmune T cells support the model that CD148 plays a negative regulatory role in lymphocyte signaling.


   Acknowledgments
 
We thank Stan Grell for excellent technical assistance, S. Chikuma, and J. A. Bluestone for kindly providing CTLA-4 knockout mice, R. Reinhardt for helping us process the OCT sections and all members of the Weiss Lab for helpful ideas and suggestions. We are especially grateful to Susan Levin for critical reading of this manuscript.


   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 in part by research funds from the state of California. Back

2 J.L. and J.W.Z. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Arthur Weiss, Department of Medicine, University of California, 533 Parnassus Avenue, Room U-330, Box 0795, San Francisco, CA 94143-0795. E-mail address: aweiss{at}medicine.ucsf.edu Back

4 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; PLC, phospholipase C; 7AAD, 7-aminoactinomycin D; MZ, marginal zone; MFI, mean fluorescence intensity; DN, double negative; DP, double positive; SP, single positive. Back

Received for publication February 17, 2004. Accepted for publication June 10, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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