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Immunobiology Department, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have generated a mAb (A3) that has been clustered as CD148 (13).
CD148 (also known as human protein tyrosine phosphatase-
or
density-enhanced protein tyrosine phosphatase-1) is a recently
identified receptor-type protein tyrosine phosphatase (14, 15). The
cloned product is a 180- to 250-kDa polypeptide with 8 to 10
fibronectin type III motifs in its extracellular domain, a single
transmembrane domain, and one catalytic phosphatase domain within the
intracellular domain (13, 14). CD148 mRNA and/or protein have been
detected in fibroblasts (14) and some hemopoietic cell lines (14, 16),
yet its expression in lymphoid cells remains uncharacterized. Using the
anti-CD148 mAb, we have analyzed the distribution of CD148 on
leukocyte populations and assessed the role of this protein tyrosine
phosphatase in T cell activation. Our results demonstrate that CD148 is
an inducible molecule expressed by activated T cells and that ligating
CD148 on anti-CD3 mAb-activated T cells induces high levels of
proliferation. Proliferation was even observed for highly purified T
cells that did not respond to anti-CD3 mAb alone. These data
suggest that the phosphatase activity of CD148 may be involved in the
regulation of T cell activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hybridoma cell line secreting the A3 mAb was generated by
fusing NS-1 cells with splenocytes from BALB/c mice that had been
repeatedly immunized with PHA-stimulated human PBMC. The isotype of A3
was IgG1 
. A3 was labeled with FITC using standard protocols. The
following mAb were used in this study: fluorochrome-conjugated mAb and
unconjugated mAb specific for CD3, CD4, CD8, CD14, CD16, CD19, CD20,
CD28, CD45RA, CD45RO, CD56, and CD57 (Becton Dickinson, San Jose, CA);
goat anti-mouse Ig (Jackson ImmunoResearch Laboratories, West
Grove, PA); Spv-T3b (anti-CD3) (17); control IgG1 (MOPC-31;
PharMingen, La Jolla, CA); and neutralizing rat anti-human IL-2
(17H12, IgG2a) and a control rat IgG2a mAb (anti-ß-galactosidase;
provided by J. Abrams, DNAX, Palo Alto, CA). All mAbs used for
functional studies were purified as previously described (12). IL-2 and
IL-15 were purchased from R&D Systems (Minneapolis, MN); PHA was
purchased from Sigma (St. Louis, MO).
Cells
T cells were isolated from the peripheral blood of healthy donors (Stanford Blood Bank, Stanford, CA) by negative selection using mAb specific for CD14, CD16, CD19, CD20, and CD56 and sheep anti-mouse Ig Dynabeads (Dynal, Oslo, Norway) (18). The recovered cell population was typically 85 to 97% T cells. Highly purified peripheral blood T cells were obtained by negative cell sorting using a FACS Vantage or a FACStarPlus (Becton Dickinson) after staining the cells with PE-labeled anti-CD14, anti-CD16, anti-CD19, anti-CD20, and anti-CD56 mAbs. On reanalysis, the sorted T cells were >97.5% CD3+. Peripheral blood CD4+ and CD8+ T cells were also obtained by negative cell sorting after staining the cells with the same PE-labeled mAbs as those described above and including either PE-anti-CD4 or PE-anti-CD8 mAb. The sorted T cells were >96% CD4+ and >85% CD8+. Human T and NK cell clones were generated as previously described (19). The cell lines used in this study included EBV-transformed B cell lines JY, MoB, and 721.221; the Burkitts lymphoma cell line Ramos; the monocytoid cell line U937; the immature NK leukemic cell line YT2C2; the erythroid leukemic cell line K562; the promyelocytic leukemic cell line HL60; and the human T cell lines Jurkat and HPB-ALL. All cell lines were cultured at 37°C in 5% CO2 in RPMI 1640 tissue culture medium (JRH Biosciences, Lenexa, KS) supplemented with 10% FBS, penicillin, streptomycin, and L-glutamine, hereafter referred to as complete medium.
Immunofluorescent staining
PBMC were incubated with PE-anti-CD3, PE-anti-CD4, PE-anti-CD8, PE-anti-CD14, PE-anti-CD20, or PE-anti-CD56 mAb and FITC-anti-CD148 or FITC-IgG1. The expression of CD148 on subsets of PBMC was determined by gating on the PE-positive cells and assessing the fluorescence of the population of cells incubated with FITC-anti-CD148 compared with the fluorescence of cells incubated with FITC-IgG1. The expression of CD148 on T cell subsets was determined by three-color immunofluorescence using PE-anti-CD28, PerCp-anti-CD4 or -anti-CD8, and FITC-IgG1 or anti-CD148 or using PerCp-anti-CD3, PE-anti-CD45RA or anti-CD45RO, and FITC-IgG1 or anti-CD148. Expression of CD148 was assessed by gating on the PerCp-positive cells. Surface staining was measured on a logarithmic scale. Five to ten thousand events were collected per sample, and the data were analyzed using the CellQuest software program (Becton Dickinson).
Cell cultures
Ninety-six-well round-bottom culture plates (Costar, Cambridge,
MA) were coated with goat anti-mouse Ig (5 µg/ml, in 0.05 M
carbonate buffer, pH 9.6) at 37°C for 4 h. Anti-CD148 mAb or a
control IgG1 mAb (10 µg/ml) were added to the wells and incubated
overnight at 4°C. Anti-CD3 and anti-CD28 mAbs were added at a
final concentration of 1 µg/ml. In some experiments, soluble control
IgG1 mAb, soluble anti-CD148 mAb (10 µg/ml), neutralizing
anti-human IL-2 mAb, a control rat IgG2a mAb (20 µg/ml), or CsA
(Calbiochem, San Diego, CA) were added to the wells. For proliferation
studies, 2.5 to 5 x 104 T cells were added to each
well of a 96-well plate in a total volume of 200 µl of complete
medium and then cultured for 2 to 8 days at 37°C in 5%
CO2. Proliferation was determined by assessing the
incorporation of [3H]thymidine (1 µCi/well; Amersham,
Arlington Heights, IL) by triplicate or quadruplicate cultures of T
cells during the final 18 h of the culture period. Incorporation
of [3H]thymidine was measured as counts per minute by
liquid scintillation counting with a beta counter
(Pharmacia-LKB-Wallac, Turku, Finland). For cytokine secretion,
105 sort-purified peripheral blood T cells were cultured in
200 µl of Yssel’s medium (20) supplemented with 10% FBS,
penicillin, and streptomycin with anti-CD3 mAb (1 µg/ml) in the
presence or absence of immobilized control IgG1, immobilized
anti-CD148 mAb (10 µg/ml), or soluble anti-CD28 mAb (1
µg/ml). Supernatants were harvested from five replicate wells after
48 h of culture, and the levels of secreted IL-2, IL-10,
granulocyte-macrophage CSF, TNF-
, and IFN-
were determined by
ELISAs, as previously described (18, 21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the expression of CD148, mononuclear cells from human
peripheral blood were incubated with FITC-anti-CD148 mAb in
combination with PE-labeled mAb specific for T cells, B cells, NK
cells, and monocytes and were analyzed by flow cytometry. CD148 was
expressed at different densities on all leukocyte populations examined
(Fig. 1
). Monocytes expressed the highest
level of CD148, whereas expression was lowest on CD4+ T
cells. Expression on B cells, CD8+ T cells, and NK cells
was intermediate to that on CD4+ T cells and monocytes. The
differential expression of CD148 on CD4+ and
CD8+ T cells was consistently observed for all donor PBMC
samples and explains the broad staining pattern observed for total T
cells (Fig. 1). Expression of CD148 did not differ on subsets of
peripheral blood T cells defined by the expression of CD45RA, CD45RO,
or CD28 (data not shown). CD148 expression on CD4+ T cells,
CD8+ T cells, NK cells, and monocytes was similar
regardless of whether the mononuclear cells were obtained from
peripheral blood, spleen, or cord blood (data not shown). CD148 was
expressed at elevated levels on CD4+ T cell clones,
CD8+ T cell clones, and NK cell clones (Table I). CD148 was also expressed on the U937,
YT2C2, K562, and HL60 cell lines. In contrast, this molecule was either
absent from or was expressed at only very low levels on transformed B
and T cell lines (Table I).
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PBMC were cultured with PHA, anti-CD3 mAb, or the cytokines
IL-2 or IL-15, and the expression of CD148 was assessed on
CD4+ and CD8+ T cells at different times
following activation in vitro. Expression of CD148 on either
CD4+ or CD8+ T cells remained unchanged when
the cells were cultured without any exogenous stimuli (Fig. 2). Expression of CD148 was up-regulated
on all T cells following activation with PHA or anti-CD3 mAb (Fig. 2a). Kinetic studies indicated that CD148 expression was
increased 6 to 12 h after activation and that CD148 expression
continued to increase until maximum expression levels were observed
after 72 to 96 h (data not shown). Expression of CD148 on
CD4+ and CD8+ T cells was also increased
following culture with IL-2 or IL-15 (Fig. 2b). Exposure to
IL-2 for 4 days up-regulated CD148 expression on 20.8 ± 7.1% of
CD4+ T cells and 45 ± 8.5% of CD8+ T
cells (mean ± SD of four independent experiments). Culture with
IL-15 had a similar effect as IL-2, increasing CD148 expression on
17.6 ± 6.4% of CD4+ T cells and 47.1 ± 14.3%
of CD8+ T cells (Fig. 2b).
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To determine the role of CD148 in T cell activation, peripheral
blood T cells were cultured with anti-CD3 mAb in the absence or the
presence of soluble control IgG1, soluble anti-CD148 mAb,
immobilized control IgG1, or immobilized anti-CD148 mAb. As a
comparison, T cells were also cultured with anti-CD3 and
anti-CD28 mAbs. T cells activated with anti-CD3 mAb alone
exhibited a low level of proliferation, which was unaffected by the
presence of soluble control IgG1, soluble anti-CD148 mAb, or
immobilized control IgG1 mAb (Fig. 3).
However, proliferation induced by anti-CD3 mAb was significantly
enhanced in the presence of immobilized anti-CD148 mAb (Fig. 3).
The magnitude of the proliferative response induced by anti-CD3
plus anti-CD148 mAb was comparable to that induced by anti-CD3
plus anti-CD28 mAb. However, the T cell proliferation induced by
anti-CD3 plus anti-CD148 mAb differed from that induced by
anti-CD3 plus anti-CD28 mAb. In the presence of anti-CD28
mAb, an increase in the proliferation of anti-CD3 mAb-activated T
cells was evident after 2 days of in vitro culture, was greatest after
5 to 6 days, and declined at time points thereafter. In the presence of
anti-CD148 mAb, proliferation of anti-CD3 mAb-activated T cells
was only mildly increased (
2-fold) after 3 days, but was greatest
after 8 days, when proliferation was up to 50-fold higher than that
induced by anti-CD3 mAb alone. Furthermore, high levels of T cell
proliferation were maintained at these later times points following
activation by the coligation of CD148 and CD3 (Fig. 3). These results
indicate that concomitant ligation of CD148 not only enhances
TCR-induced T cell proliferation but also maintains high levels of
proliferation of anti-CD3-activated T cells by delaying the decline
in proliferation that is observed with anti-CD3 mAb alone. Although
anti-CD28 mAb and immobilized anti-CD148 mAb could comparably
enhance the proliferation of anti-CD3-activated T cells, the
combination of anti-CD3 mAb with optimal concentrations of both
anti-CD28 mAb and immobilized anti-CD148 mAb did not cause a
further increase in T cell growth (data not shown).
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Because the cell preparations used in the above studies contained
only about 90% CD3+ cells, the ability of anti-CD148
mAb to enhance the proliferation of anti-CD3 mAb-activated T cells
could be an indirect effect, resulting from the activation of other
CD148+ cells, such as monocytes. To assess this
possibility, highly purified T cells (>97.5% CD3+)
obtained by negative cell sorting were cultured with anti-CD3 mAb
in the absence or the presence of an immobilized control IgG1 mAb or
anti-CD148 mAb. Anti-CD3 mAb alone or anti-CD3 plus control
IgG1 mAb failed to induce the proliferation of purified T cells (Fig. 4). However, ligation of CD148 by
immobilized anti-CD148 mAb induced high levels of proliferation of
anti-CD3 mAb-activated T cells (Fig. 4). It is well known that
activation via CD3 and CD28 is capable of inducing maximal T cell
proliferation (1, 2, 3). Comparison of the ability of cross-linking CD28
or CD148 to induce T cell proliferation indicated that proliferation of
highly purified anti-CD3 mAb-activated T cells mediated by
immobilized anti-CD148 mAb was about 25 to 75% of that mediated by
anti-CD28 for the five different donors examined. These results
indicate that the combination of anti-CD148 mAb and anti-CD3
mAb is a potent activator of proliferation of highly purified T cells.
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Expression of CD148 was higher on resting CD8+ T cells
than on CD4+ T cells. Additionally, a higher percentage of
CD8+ T cells up-regulated CD148 expression in response to
IL-2 and IL-15 than did CD4+ T cells. To assess whether
these differences played a role in the in vitro response of T cells,
CD4+ and CD8+ T cell subsets were purified from
peripheral blood and were cultured with anti-CD3 mAb in the absence
or the presence of immobilized control IgG1 mAb, immobilized
anti-CD148 mAb, or anti-CD28 mAb. Purified CD4+ and
CD8+ T cells exhibited minimal or only a low level of
proliferation in response to anti-CD3 mAb alone or in the presence
of immobilized control IgG1 mAb (Fig. 5).
However, anti-CD3 mAb-activated CD4+ and
CD8+ T cells proliferated when CD148 was ligated by the
immobilized anti-CD148 mAb (Fig. 5). Although both T cell
populations proliferated, the response of CD8+ T cells was
consistently several-fold greater than that of CD4+ T
cells. As a comparison, the proliferation of CD4+ T cells
in response to anti-CD3 plus anti-CD148 mAb was only about 10%
of that induced by anti-CD3 plus anti-CD28 mAb, while the
proliferation of CD8+ T cells activated with anti-CD3
plus anti-CD148 mAb was 50 to 100% of the proliferative response
induced by anti-CD3 plus anti-CD28 mAb. Thus, the peripheral
blood cell type that predominates in the response to anti-CD3 plus
anti-CD148 mAb appears to be CD8+ T lymphocytes.
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To determine whether ligation of CD148 increased T cell
proliferation by an IL-2-dependent or a CsA-sensitive mechanism,
peripheral blood T cells were activated in the presence of neutralizing
anti-IL-2 mAb or CsA. In the presence of immobilized anti-CD148
mAb, T cell proliferation induced by anti-CD3 was increased up to
30-fold (data not shown). However, when anti-IL-2 mAb was included
in the culture, the enhanced levels of proliferation of anti-CD3
mAb-activated T cells induced by the anti-CD148 mAb were reduced by
73 ± 11.5% (Fig. 6; mean ±
SEM; n = 4). In contrast, culture in the presence of an
isotype control rat IgG2a mAb had no effect on the T cell response.
Thus, CD148-mediated proliferation of anti-CD3 mAb-activated T
cells is dependent on endogenous production of IL-2. Similarly, culture
in the presence of 10 ng/ml CsA reduced the proliferative response by
84.0 ± 9.0% (mean ± SEM; n = 3), while 100
ng/ml CsA abolished T cell proliferation (Fig. 6). This indicates that
the effect of ligating CD148 on T cell activation is mediated via the
calcineurin-dependent signaling pathway and is, therefore, distinct
from that delivered via CD28, which is insensitive to the effect of CsA
(22). The inhibitory effect of CsA and the anti-IL-2 mAb was not
due to toxicity, as these reagents did not affect T cell proliferation
induced by PMA in combination with anti-CD28 mAb (data not shown).
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The effect that cross-linking CD148 had on cytokine secretion was
assessed using sort-purified T cells from three separate donors.
Activation with anti-CD3 mAb alone induced a low level of secretion
of IL-2, IL-10, IFN-, TNF-, and granulocyte-macrophage CSF (Table III). Interestingly, this response was
unaffected by cross-linking CD148 on the activated T cells. In
contrast, costimulation with anti-CD28 mAb increased the level of
secreted cytokines by 3- to 20-fold (Table III). The inability of the
anti-CD148 mAb to influence cytokine secretion was not due to
inefficient activation because the cells responded with high levels of
proliferation when assessed after 5 days of in vitro culture (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that expression of CD148 is increased on T cells following activation suggested that this molecule may be involved in the functional regulation of activated T cells. Indeed, ligation of CD148 on T cells by immobilized, but not soluble, anti-CD148 mAb greatly augmented the in vitro proliferative response induced by ligation of the TCR by anti-CD3 mAb. More importantly, anti-CD148 mAb induced proliferation of highly purified, FACS-sorted T cells that had been activated with anti-CD3 mAb, which was unable to drive T cell proliferation in the absence of costimulatory signals. The combination of anti-CD3 plus anti-CD148 mAb often induced a proliferative response in highly purified T cells that approximated that induced by anti-CD3 plus anti-CD28 mAb. This indicates that the combination of anti-CD3 plus anti-CD148 mAb is a potent T cell activator and suggests that CD148 engagement can initiate T cell proliferation in the absence of CD28 costimulation. CD148-mediated T cell proliferation was also found to be dependent on the production of endogenous IL-2 and was sensitive to treatment with CsA. Analysis of T cell subsets indicated that the response of CD8+ T cells to activation with anti-CD3 and anti-CD148 mAb was much greater than that of CD4+ T cells. These differences in proliferation of CD4+ and CD8+ T cells were even more impressive when it was considered that the CD8+CD28- T cell subset, which can comprise up to 40% of total CD8+ T cells (26), failed to proliferate in response to anti-CD3 plus anti-CD148 mAb (data not shown). CD4+ T cells and CD8+ T cells have previously been reported to preferentially respond to activation via CD40 ligand (11) and Fas ligand (27), respectively. Similarly, CD27 is a costimulatory molecule for CD45RA+ T cells, but not CD45RO+ T cells (9, 28). These differences in responses of T cell subsets correlated with the differential surface expression of such molecules. This is consistent with our observation that expression of CD148 is greater on unstimulated CD8+ T cells than on CD4+ T cells, and that more CD8+ T cells up-regulate CD148 expression in response to IL-2 and IL-15.
Cell surface molecules previously found to enhance the proliferation of Ag receptor-activated T cells are often also capable of enhancing cytokine secretion by the activated T cells (9, 11, 12). It was therefore surprising that there was no detectable increase in the level of cytokine secreted by T cells activated with anti-CD3 plus anti-CD148 mAb. This, however, is inconsistent with the finding that the increased proliferation mediated by anti-CD148 mAb was dependent on the production of endogenous IL-2. Autocrine consumption of cytokines by the proliferating T cells may at least in part account for the inability to detect an increase following ligation of CD148. The ability of anti-IL-2 mAb to reduce CD148-induced proliferation suggests that at least IL-2 production is enhanced by anti-CD3 mAb-activated T cells in the presence of immobilized anti-CD148 mAb.
Although T cell proliferation induced by anti-CD3 mAb in combination with anti-CD148 mAb was comparable to that induced by anti-CD3 plus anti-CD28 mAb, it appears that the responses of anti-CD3 mAb-activated T cells to these two different mAbs are distinct. Differences were evident for 1) the kinetics of the T cell response; 2) the sensitivity of proliferation induced by anti-CD148 mAb, but not anti-CD28 mAb (22), to CsA; 3) the inability of anti-CD148 mAb to have any apparent effect on cytokine secretion after 2 days of in vitro culture; and 4) the ability of CD8+ T cells to exhibit greater levels of proliferation in response to anti-CD148 mAb.
To date, the best characterized protein tyrosine phosphatases identified to play important roles in Ag receptor-mediated signal transduction are CD45 and SH2-containing protein tyrosine phosphatase-1, protein tyrosine phosphatases that are constitutively expressed by all hemopoietic cells. Analysis of cell lines and of mice deficient in these phosphatases have indicated that CD45 is necessary and sufficient for initiation of Ag receptor-mediated signal transduction in both B and T cells (29, 30, 31, 32, 33, 34). In contrast, SH2-containing protein tyrosine phosphatase-1 can negatively regulate Ag receptor-mediated signal transduction (35, 36, 37, 38, 39). Thus, phosphatases can act to either initiate or terminate cellular responses. Presently, the mechanism by which anti-CD148 mAb amplifies the proliferative response of anti-CD3 mAb-activated T cells is unknown. Ligating the molecule with anti-CD148 mAb, which may mimic the interaction with its physiologic ligand, may deliver a positive signal. Alternatively, ligating CD148 may inhibit a negative signal by inducing dimerization of the phosphatase domain of CD148 (40, 41). This would be consistent with the ability to inhibit signal transduction mediated via CD45 by ligand-induced dimerization of its extracellular domain (42). The outcome of either delivering a positive or inhibiting a negative signal would be reflected by an elevated T cell response. Elucidation of the mechanism is presently under investigation. In conclusion, CD148 is a receptor-type protein tyrosine phosphatase expressed on all leukocytes that is up-regulated following in vitro stimulation. Thus, CD148 is a T cell activation molecule. The phosphatase activity of CD148 may be involved in regulating the expansion of in vivo-activated T cells, particularly CD8+ T cells. The finding that this molecule is expressed at elevated levels on monocytes as well as on resting and activated NK cells suggests that CD148 may have a role in the biologic responses of different populations of mononuclear cells and that its primary function is not restricted to T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. S. G. Tangye, DNAX Research Institute, 901 California Ave., Palo Alto, CA 94304. E-mail address:
3 Current address: Novartis Research Institute, Brunner Strasse 59, A-1235 Vienna, Austria.
4 Abbreviations used in this paper: SLAM, signaling lymphocytic activation molecule; PE, phycoerythrin; PerCp, peridinin chlarophyll; CsA, cyclosporin A.
Received for publication March 23, 1998. Accepted for publication May 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or in the presence of soluble control IgG1 mAb (10 µg/ml; 
),
soluble anti-CD148 mAb (10 µg/ml; •), immobilized control IgG1
mAb (10 µg/ml; 
), immobilized anti-CD148 mAb (10 µg/ml;
), or anti-CD28 mAb (1 µg/ml; 
). Proliferation was assessed
at the times indicated by determining the incorporation of
[3H]thymidine during the final 18 h of culture. Each
point represents the mean ± SD of quadruplicate samples. This
result is representative of five independent experiments.
), immobilized
anti-CD148 mAb (10 µg/ml; 
), or soluble anti-CD28 (1
µg/ml; 
