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*
Institute for Immunology, University Hospital, Hamburg, Germany;
The Jackson Laboratory, Bar Harbor, ME 04609; and
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143
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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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subunit of heterotrimeric G proteins
(3, 4, 5). Ample biochemical evidence has shown that
endogenous mono-ADP-ribosylation reactions also occur in animal
tissues. Recent findings suggest that this posttranslational protein
modification may be used to control important physiological functions
such as the induction of long-term potentiation in the brain, terminal
muscle cell differentiation, and the cytotoxic activity of T cells
(6, 7, 8). In the mouse, ADP-ribosylation of cell surface
proteins by a GPI-anchored ADP-ribosyltransferase
(ART)3 activity has
been shown to inhibit important T cell functions such as cell
proliferation, target cell binding, and cytolytic activity
(9). LFA-1 and CD8 have been identified as key target
proteins of ADP-ribosylation on the T cell surface
(10, 11, 12). Multiple ecto-ADP-ribosyltransferase (ART)-encoding genes have been identified in mice (13, 14, 15, 16, 17). A duplicated pair of genes on mouse chromosome 7, designated Art2a and Art2b, are homologues of the single RT6 locus in the rat (13, 18, 19, 20). There are two alloantigenic forms of RT6 expressed in rats as 25–35-kDa GPI-anchored maturation markers on the surface of T cells (21, 22). RT6-expressing T cells exert a regulatory role in rat models for autoimmune insulin-dependent diabetes mellitus (23, 24). Although the two alloantigenic forms of RT6 degrade NAD+, it is unclear whether they are capable of ADP-ribosylating T cell surface proteins other than themselves (25, 26, 27). The recombinant proteins expressed from mouse Art2a and Art2b genes (ART2.1 and ART2.2, respectively) ADP-ribosylate a variety of synthetic substrates (28, 29).
As mentioned above, ADP-ribosylation of integral T cell membrane proteins is associated with CTL down-regulation (9). The lack of mAbs against the mouse ART gene products has heretofore made it difficult to establish whether the T cell activities were indeed encoded by the RT6 homologues or by another member of the ART gene family (30, 31). In this study, we describe the production and staining characteristics of a rat mAb, Nika102, that reacts with the mouse ART2.2 ortholog of rat RT6. In this first report of a mAb against mouse T cell surface-expressed ART, we describe the use of this Ab for monitoring the Ag expression during postnatal T cell development and during T cell activation. We further demonstrate considerable variation among inbred strains in the percentages of T cells expressing this Ag, and we show that activation of T cells induces rapid Ag release from the cell surface, while other GPI-anchored proteins such as Thy-1 (CD90) and Ly-6A/6E are not affected.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wistar rats were from the animal facility of the University Hospital (Hamburg, Germany). ALS/Lt, ALR/Lt, NOD/Lt, NOR/Lt, NOcCB-1/Lt, NZO/Lt, and C3H/HeJBir mice were obtained from a research colony (E.H.L.); C57BLKS/J, C57BL/6J, NZB/BINJ, BXSB/MpJ, BALB/cByJ, Cast/Ei, CBA/J, C57BL/6.Tcrbtm1Mom, 129/SvJ, DBA/2J, (NZB x NZW)F1/J, C57BL/6.scid, and NZW/LacJ (f/8) mice were obtained from the Animal Resources Unit of The Jackson Laboratory (Bar Harbor, ME); CTS/Shi mice were purchased from TGC (Tokyo, Japan) with the sale stipulation that they could not be distributed by the purchaser. For dexamethasone-mediated depletion of thymocytes, mice received a single injection i.p. of 0.5 mg dexamethasone (Sigma, St. Louis, MO) in 0.5 ml PBS or 0.5 ml PBS alone.
Antibodies
mAbs used in this study for immunofluorescence staining and
activation assays include anti-CD3
(145-2C11), anti-CD4
(GK1.5), anti-CD5 (53-7.3), anti-CD8 (53-6.72), anti-CD24
(J11d), anti-CD25 (7D4), anti-CD62L (Mel-14), anti-CD69
(H1.2F3), anti-CD90 (HO13.4), anti-
TCR (GL3), anti
Ly-6A/E (Sca-1, E13-161), and anti-Ly-6C (AL-21). Biotin, PE, and
FITC conjugates were purchased from PharMingen (San Diego, CA).
Streptavidin-Alexa350 was purchased from Molecular Probes (Eugene, OR).
Polyclonal rabbit antiserum K48 was raised against a peptide derived
from the deduced amino acid sequence of ART2.2 (residues 48–59), as
described previously (13). GK1.5 (anti-CD4) was
labeled with Cy3 using a commercially available kit (FluroLink Cy3
Reactive Dye; Amersham, Piscataway, NJ).
Cloning and expression of rART2.2
Vectors for expressing Art2b in Escherichia coli and mammalian cells were cloned by standard techniques (32), replacing sequences for the N-terminal leader and/or C-terminal GPI-signal sequence. In brief, PCR products generated from Art2b cDNA with fusiogenic primers derived from the published Art2b sequence (13) were restriction digested, gel purified, and cloned into pASK (Biometra, Göttingen, Germany), pCDM8.AP (kindly provided by Sandy Zurawski, DNAX Research Institute, Palo Alto, CA), and pME.CD8LF (kindly provided by Rob Kastelein, DNAX Research Institute). The pASK.ART2.2-HF construct encodes the OMP leader (E. coli outer membrane protein) in place of the ART2.2 leader and a chimeric His(6x)-FLAG tag (HF) (33) in place of the ART2.2 GPI-signal sequence. The pCDM8.ART2.2-AP construct retains the ART2.2 leader, but encodes AP in place of the GPI-signal sequence. The pME.CD8LF-ART2.2 construct encodes the CD8 leader, followed by a FLAG tag in place of the ART2.2 signal sequence, but retains the ART2.2 GPI-signal sequence.
C-terminally His6x/FLAG-tagged ART2.2-HF was expressed in E. coli periplasm, as described previously (34). In brief, 1 L cultures of pASK.ART2.2-HF-transformed E. coli NM522 cells were propagated at 24°C for 20 h, and cells were harvested by centrifugation and resuspended in 10 ml lysis buffer (20 mM Tris (pH 8) and 500 mM glucose) for 30 min at 4°C. Lysates were cleared by high speed centrifugation for 15 min at 4°C, and supernatants were stored at 4°C. For purification of rART2.2, 200 µl Talon-Matrix (Clontech, Palo Alto, CA) was added to 10 ml lysate. After overnight incubation at 4°C, matrix-bound protein was pelleted by low speed centrifugation and washed four times with PBS. For preparation of soluble ART2.2, the matrix was pelleted again and resuspended in 100 mM EDTA, pH 7, for 10 min at room temperature. The matrix was pelleted by centrifugation and the supernatant was dialyzed against PBS.
For the production of ART2.2-AP fusion protein, human kidney 293T cells were lipofectamine (Life Technologies, Grand Island, NY) transfected with pCDM8.ART2.2-AP (20 µg/107 cells). After 5 days of culture, supernatants were harvested, cleared by high speed centrifugation, and stored at 4°C. N-terminally FLAG-tagged ART2.2 was expressed in mouse EL4 lymphoma cells after electroporation with pME.CD8LF-ART2.2 (20 µg/107 cells) and selection of stable transfectants with G418.
Immunization and generation of mAbs
pME.CD8LF-ART2.2 was coated onto 1-µm gold particles (Bio-Rad, Hercules, CA). DNA-coated gold particles (1 µg DNA/mg gold) were injected ballistically into the abdominal skin of Wistar rats with the Helios gene gun (Bio-Rad, Munich, Germany) at a pressure setting of 400 psi (6 shots per animal at each immunization). Gene gun immunization was repeated three times every 3–5 wk. Five days before sacrificing, animals received 50 µg purified rART2.2-HF in 500 µl PBS i.v. and 50 µg rART2.2-HF attached to 50 µl Talon-agarose beads in 500 µl PBS i.p. Splenocytes were fused with the Sp2/0 myeloma cell line using standard polyethylene glycol fusion techniques with PEG 6000 (Boehringer Mannheim, Mannheim, Germany). A total of 100 µl of supernatant was collected from wells containing growing cells after 14 to 20 days in selective medium and was tested by ELISA for reactivity with rART2.2-AP fusion protein. Supernatants from positive wells were further tested for reactivity with ART2.2-transfected EL4 cells and for lack of reactivity with parental EL4 cells. Hybridomas from positive wells were cloned by limiting dilution in the presence of irradiated rat thymocyte feeder layers.
ELISA was performed on 96-well plates that had been coated overnight with goat anti-rat Ig (Pierce, Rockford, IL) at 10 µg/ml in PBS. After blocking of the anti-rat Ig-coated plates with 10% goat serum, hybridoma supernatants were added for 30 min at room temperature. Plates were washed and then incubated with 293T cell supernatants containing ART2.2-AP fusion protein (diluted 1/4 in PBS). After 30 min at room temperature, plates were washed again and incubated with p-nitrophenylphosphate (Pierce) for 30 min at 37°C.
For purification of mAbs, Ab was precipitated from hybridoma supernatants with an equal volume of saturated ammonium sulfate (35). Precipitates were washed, and resuspended in and dialyzed against PBS. Ab was further purified by passage over a gel filtration column (molecular mass cutoff, 1 x 106 kDa; Bio-Rad, Hercules, CA). Purified Ab was conjugated to FITC (Sigma) following standard protocols (35). mAb isotypes were determined by Ouchterlony assay using a commercially available rat Ig isotyping kit (The Binding Site, Birmingham, U.K.).
Preparation of cells and immunofluorescence analyses
For preparation of PBLs, animals were bled retroorbitally using heparinized capillary tubes and blood was diluted 10-fold with HBSS containing 5 mM EDTA. In case of thymus, extraneous blood and parathymic lymph nodes were carefully removed before tissue disruption. Single cell suspensions from thymus, spleen, and lymph nodes were prepared in HBSS/2% FCS by passage through Nitex membrane (110 µm mesh; Tetko, Kansas City, MO). Erythrocytes in blood and spleen cell suspensions were lysed by incubation in two washes of Gey’s buffer for 5 min at 4°C, followed by a wash in FACS buffer. Cells (1 x 106) were incubated with saturating amounts of Abs for 30 min at 4°C and were then washed once in 2 ml HBSS. After washing, cells were resuspended in 250 µl FACS buffer (PBS, 1% BSA, 0.05% NaN3) and 10 µl propidium iodide (20 µg/ml). Cells were analyzed by flow cytometry on a FACStar or FACScan (Becton Dickinson, Mountain View, CA) using viable cell gating through exclusion of propidium dye.
Stimulation of cells
PMA (100 ng/ml) and ionophore A23187 (200 ng/ml) (Sigma) were added to cells (106/ml) suspended in HBSS. Cells were incubated for 2 h at 37°C and subjected to immunofluorescence analyses, as described above, with the exception that NaN3 was omitted from FACS buffer. For kinetic analyses, cells were prestained with appropriately labeled Abs before addition of PMA and A23187. For analysis of coexpression of CD25 and ART2.2, splenocytes (2 x 106 cells/ml) from 12-wk-old C57BL/6J males were cultured in RPMI medium supplemented with 10% FCS, L-glutamine, HEPES, and antibiotics for 20 h at 37°C with or without 2 µg/ml anti-CD3 mAb. Then the cells were collected, washed in PBS/1% BSA/0.05% sodium azide, and stained with biotin-conjugated A102 mAb and anti-CD25 FITC, followed by incubation with PE-labeled streptavidin, and analyzed by flow cytometry, as described above.
Treatment of cells with PI-PLC
Cell suspensions were washed with PBS, resuspended at 108 cells/ml in RPMI medium with or without 1 U PI-PLC (Molecular Probes), and incubated on a roller for 1 h at 37°C. Cells were pelleted by centrifugation, and supernatants were harvested and stored at -20o. Cells were washed once with PBS and subjected to immunofluorescence analyses, as described above.
Immunoprecipitation and Western blotting
E. coli lysates and supernatants of PI-PLC-treated cells were cleared by high speed centrifugation. After addition of Abs, samples were rotated for 20 h at 4°C. Protein G-Sepharose was then added to bind Abs and immune complexes. After rotation for 60 min at 4°C, the matrix was pelleted by centrifugation in an Eppendorf centrifuge, washed four times with TBS, 1% Triton X-100, and resuspended in SDS-PAGE sample buffer. All samples were heated for 5 min at 95°C and cleared by centrifugation before loading onto precast 10–12% polyacrylamide gels (Novex, Frankfurt, Germany). Gels were run in Tricine buffer under reducing conditions. Fractionated proteins were blotted onto nitrocellulose or polyvinylidene difluoride membranes and membranes were blocked with 10% goat serum in TBS.
Blots were incubated for 2–16 h at 4°C with anti-ART2.2 peptide rabbit antiserum K48 (13) diluted 1/2000 in TBS, 0.5% Tween 20 (TBST), and 10% goat serum, and washed extensively in TBST. Secondary reagent for detection of bound K48 Abs was peroxidase-conjugated goat anti-rabbit Ig (1:5000) (Amersham). After extensive washing in TBST, bound Ab was detected with the enhanced chemiluminescence (ECL) system, according to the manufacturer’s instructions and by exposure to ECL film (Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because no Abs suitable for detecting ART2.2 by fluorocytometry
were available at the time, we engineered a FLAG tag onto the N
terminus of ART2.2 to make the protein detectable by the anti-FLAG
tag mAb, M2 (33). To this end, we constructed the
expression vector pME.CD8LF-ART2.2, in which the presumptive N-terminal
ART2.2 leader was replaced by the CD8 leader, followed by a FLAG tag,
and in which the presumptive C-terminal GPI-anchor signal peptide of
ART2.2 was retained. Fig. 1
shows FACS
analyses of EL4 cells stably transfected with this expression vector.
ART2.2-transfected cells are stained by M2 (Fig. 1a), while
parental cells are negative (not shown).
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a). These results are consistent with the
interpretation that M2 recognizes FLAG-tagged, GPI-anchored ART2.2 on
these transfectants. Production of mAb Nika102 to mouse ART2.2
Having shown that pME-CD8LF-ART2.2 directs cell surface expression
of ART2.2, we reasoned that this vector might be useful also for
raising ART2.2-specific antisera by DNA immunization. To this end,
Wistar rats were gene gun immunized with pME.CD8LF-ART2.2. Sera were
tested for reactivity with ART2.2-transfected EL4 cells. Spleen cells
from animal R8, whose immune serum contained ART2.2-specific Abs (Fig. 1b), were fused with the mouse myeloma cell line Sp2/0.
Supernatants of growing hybridoma cultures were screened by ELISA for
reactivity with ART2.2-AP fusion protein produced by appropriately
transfected 293T cells (not shown). Supernatants from positive wells
were further tested for reactivity with pME.CD8LF-ART2.2-transfected
cells and for lack of reactivity with mock-transfected cells. One
hybridoma that showed particularly bright staining with
ART2.2-transfected cells (Fig. 1c) was selected for further
analyses. The mAb produced by this hybridoma, Nika102, was determined
to be of the IgG2a isotype by Ouchterlony assay (data not shown).
Nika102 and an anti-ART2.2 peptide antiserum react with the same molecule
To further corroborate the specificity of Nika102 for ART2.2, we
next attempted to immunoprecipitate rART2.2 with Nika102. As shown in
Fig. 2, mAb Nika102 specifically
immunoprecipitates proteins with apparent molecular mass of 30 kDa
(lane 4) and 45 kDa (lane 10) from
lysates of ART2.2-transformed E. coli and ART2.2-transfected
EL4 cells, respectively, but not from mock-transfected cells
(lane 6). The same proteins are detected by
anti-ART2.2 peptide antiserum K48, which reacts with denatured
ART2.2 in Western blot analyses (13). These results
demonstrate that Nika102 recognizes ART2.2.
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Rat RT6 is expressed by the majority of peripheral helper and
cytotoxic T cells, but not by B cells or thymic lymphocytes, and is
down-regulated on activated T cells (22, 37). To assess
the distribution of ART2.2 on mouse T cells, we performed
flow-cytometric analyses on C57BL/6J mouse lymphocytes using
FITC-conjugated Nika102 in combination with Abs against the T cell
markers CD3, CD4, CD8, and CD25 (IL-2R). As shown in Fig. 3 and Table I, the majority of peripheral
CD3+ cells (Fig. 3, b and
c), but only a small fraction of CD3+
thymocytes (Fig. 3a) express ART2.2. In this mouse strain,
ART2.2 is expressed by 93% of peripheral CD8+
and by 66% of peripheral CD4+ T cells (Fig. 3e). Note that use of Cy3-conjugated anti-CD4 together
with PE-conjugated anti-CD8 vs FITC-conjugated Nika102 allows
direct comparison of ART2.2 expression levels on
CD4+ and CD8+ peripheral T
cells in the same plots (Fig. 3, d and e). Note
also that ART2.2 is expressed by few if any CD3-
cells (Fig. 3, a–c), and that expression of ART2.2 and CD25
is reciprocal (Table I).
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Considering that rat thymocytes are negative for RT6 and,
furthermore, that peripheral T cells express RT6 soon after emigrating
from the thymus (22, 38), we suspected that the small
fraction of ART2.2+ thymocytes might represent
mature cells and set up a series of experiments to test this. First, we
performed triple fluorescent staining analyses of C57BL/6J mouse
thymocytes (Fig. 4a). The
results reveal that neither
CD4-/CD8- double-negative
nor the immature CD4+/CD8+
double-positive cells contain any sizable fractions of
ART2.2-expressing cells (gates 1 and 2). In contrast, subpopulations of
mature CD4+ and CD8+
single-positive cells in the thymus do express ART2.2, with much larger
percentages of CD8 single-positive than CD4 single-positive thymocytes
expressing ART2.2 (gate 4 vs gate 3).
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b, gate 2), while the
ART2.2+ cells in the subpopulations of mature
CD4+ and CD8+
single-positive thymocytes (gates 3 and 4) are largely dexamethasone
resistant.
Third, we examined ART2.2 expression on thymocytes of the CTS/Shi
mouse, in which a genetic defect prevents the emigration of mature
thymocytes (39), so that mature cells accumulate in the
thymus. The results show a very high percentage of
ART2.2+ cells among the
CD4+ and CD8+
single-positive but not in the CD4/CD8 double-negative and
double-positive thymocyte subpopulations (Fig. 4c),
consistent with an accumulation of mature
(ART2.2+) thymocytes in this mouse strain.
Finally, we compared expression of ART2.2 with expression of other cell
surface markers known to be either up (CD3, CD5)- or down-regulated
(CD24, CD69, Ly-6C) during the final stages of thymocyte maturation.
Fig. 5 shows double stainings of C57BL/6J
thymocytes for ART2.2 and a series of other cell surface markers. The
results show that ART2.2+ cells are significantly
enriched in the following subpopulations: CD3high
(Fig. 5a), CD5high (Fig. 5b), CD69low (Fig. c),
CD24low (Fig. d), and
Ly-6Clow (Fig. 5e). Note, in
particular, that almost all of the CD24low cells,
which include the most mature thymocytes (40), are
ART2.2+ (Fig. 5d). Cumulatively, these
results support the interpretation that ART2.2 is a marker for the most
mature population of thymocytes.
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T cells, but only by
few if any NK cells
Next, we set out to determine whether ART2.2, like RT6 in the rat
(41), is expressed also on T cells and NK cells. To
this end, we performed dual staining analyses of ART2.2 vs T cell
and NK cell markers (the TCR and NK1.1, respectively) on
splenocytes of C57BL/6.Tcrbtm1Mom (denoted
Tcrb-/-),
C57BL/6J-Prkdcscid (denoted
C57BL/6.scid), and C57BLKS/J mice (Fig. 6).
Tcrb-/- mice lack TCR-/ß T cells, but
still contain TCR-/ T cells and B cells (42). As
shown in Fig. 6e, about one-half of the TCR-/
splenocytes in these animals express ART2.2. C57BL/6.scid
mice lack mature T and B cells and are known to contain a high
proportion of NK1.1+ splenocytes
(43). These NK1.1+ splenocytes
evidently do not express ART2.2 (Fig. 6i). However, a small
fraction of NK1.1+ cells in the
Tcrb-/- mice appears to express ART2.2 (Fig. 6h).
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To assess whether ART2.2, like RT6 in the rat (22, 44), is developmentally regulated during postnatal
ontogeny, expression of ART2.2 by thymic and splenic lymphocytes
obtained from C57BL/6J mice of different ages was assessed by flow
cytometry. As shown in Fig. 7, the
proportion of ART2.2-expressing CD4+ and
CD8+ cells increases steadily during the early
postnatal ontogeny. ART2.2 expression levels reach a maximum at 6–8 wk
of age and then fall off again.
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To determine whether ART2.2, like RT6 in the rat (22, 44), is expressed in different extents by different inbred
strains, we compared different strains of mice for ART2.2 expression by
flow cytometry (Fig. 8). Strains having
high proportions of ART2.2-expressing splenocytes include C57BL/6J,
C57BLKS/J, NOR/Lt, and NOD/Lt. In contrast, DBA2/J, C3H/HejBir, CBA/J,
NZO/Lt, and (NZB x W)F1/J mice have low
proportions of ART2.2-expressing splenocytes. In general, the mean
fluorescent intensity of ART2.2-expressing cells was relatively low in
mice with low percentages of ART2.2-expressing splenocytes. ART2.2 is
not detectable in NZW mice in which the ART2.2 gene is
deleted (45). ART2.2 is also not detectable in T
cell-deficient C57BL/6.scid mice (see also Fig. 6).
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The deduced amino acid sequence of ART2.2 contains a hydrophobic
C-terminal signal sequence characteristic of GPI-anchored proteins
(13). To assess whether the predicted GPI anchor of native
ART2.2 on mouse T cells, like that of rART2.2 (Fig. 1), is accessible
to PI-PLC, spleen cells were incubated with PI-PLC for 45 min at 37°C
before flow cytometry. As shown in Fig. 9, staining for ART2.2 is abolished by
treatment of cells with PI-PLC (Fig. 9b). In contrast,
staining for the type I membrane proteins CD4 and CD8 is resistant to
treatment with PI-PLC (Fig. 9b).
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Considering that RT6 in the rat is down-modulated upon T cell
activation (37) and that cell surface expression of ART2.2
and the IL-2R (CD25) is inversely correlated on unstimulated
splenocytes (see above, Table I), we analyzed the expression of ART2.2
upon activation of T cells with anti-CD3 Abs or with direct
activation of protein kinase C with PMA. As shown in Table I, CD25
expression on splenic T cells is up-regulated following 20 h of
anti-CD3 stimulation, whereas this activation elicits a reciprocal
down-regulation in ART2.2 expression.
It has previously been shown that an ADP-ribosyltransferase activity is
released from mouse T cells upon treatment with the protein kinase C
activator PMA (46). To assess whether ART2.2 is released
from the cell surface by T cell activation, splenocytes were first
stained with fluorescently labeled Abs and then activated by the
addition of phorbol ester (100 ng/ml PMA) and
Ca2+-ionophore (200 ng/ml A23187) (Fig. 9, c and d). Staining for ART2.2 is reduced more
than 10-fold within 60 min after PMA activation (Fig. 9d).
In contrast, staining for the type I membrane proteins CD4 and CD8 is
resistant to treatment with PMA under these conditions (Fig. 9d). In a second experiment, splenocytes were first
incubated for 90 min in the presence or absence of PMA and A23187 and
then stained for surface expression of ART2.2 and other cell surface
proteins (Fig. 9, e–l). Again, ART2.2 expression is reduced
more than 10-fold in PMA treated vs control cells (Fig. 9, f
vs e). In contrast to GPI-anchored ART2.2, neither the
GPI-anchored membrane proteins CD90 (Thy-1) (Fig. 9, g and
h) and Ly-6A/E (Fig. 9, i and j) nor
the type I membrane proteins CD3 (data not shown) and CD8 (Fig. 9, e–l) are affected by treatment of cells with PMA/A23187.
Expression of the type I membrane protein CD62L, which is known to be
cleaved by a metalloprotease under these conditions (47, 48), is reduced more than 10-fold (Fig. 9, l vs
k). Staining for CD4, however, which is known to be
endocytosed under these conditions (49), is decreased
2-fold after 2 h of PMA/A23187-induced cell activation (Fig. 9, f, h, j, and l vs
e, g, i, and k). Note that
when cells are prestained with Ab before T cell activation
(panels c and d), CD4 down-modulation is
no longer visible, whereas down-modulation of ART2.2 (Fig. 9d) and CD62L (data not shown) proceeds unabated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c). Moreover, Nika102 specifically immunoprecipitates a
45-kDa protein from ART2.2-transfected EL4 cells that is not present in
mock-transfected cells (Fig. 2, lane 10 vs lane
6). Nika102 also specifically immunoprecipitates a 30-kDa protein
from periplasma lysates of ART2.2-transformed E. coli cells,
but not from control E. coli cells (Fig. 2, lane
4). The proteins precipitated by Nika102 are recognized in Western
immunoblot analyses by K48, a rabbit antiserum produced against a
peptide deduced from the sequence of mouse Art2b cDNA
(13). These results demonstrate that we have identified a
mAb specific for the product of the mouse Art2b
gene.
Based on the Art2b cDNA sequence and the ART2.2 amino acid sequence
deduced from it, we had previously predicted that ART2.2, like its rat
RT6 homologue (36), is expressed as a cell surface protein
anchored within the cell membrane via a GPI anchor (13).
We now corroborate this prediction by visualizing ART2.2 on the T cell
surface by FACS analyses with Nika102 and by demonstrating that ART2.2
disappears from the cell surface after treatment of cells with PI-PLC
(Figs. 1a and 9b).
The apparent m.w. of ART2.2 from E. coli (Fig. 2, lane
4) approximates the m.w. of native polypeptide deduced from the
cDNA sequence (29.3 kDa). The higher apparent m.w. of ART2.2 from EL4
cells (Fig. 2, lane 10) most likely reflects
posttranslational modification of ART2.2 in these cells. It is
possible, for example, that EL4 cells glycosylate ART2.2 at one or both
of the potential N-linked glycosylation sites deduced from
the Art2b cDNA sequence (13). The fact that
Nika102 immunoprecipitates both unglycosylated ART2.2 from E.
coli and posttranslationally modified ART2.2 from EL4 cells,
indicates that ART2.2 recognizes an epitope on the peptide backbone of
ART2.2 and, furthermore, that this epitope is not blocked by
posttranslational modification(s) in EL4 cells.
The distribution of ART2.2 on mouse T cells shows many similarities to
that of its structural orthologue in the rat. Like rat RT6 (22, 44), mouse ART2.2 is expressed by peripheral
CD4+ and CD8+ T cells, but
not by CD3- cells in the adult mouse (Fig. 3).
Similar to RT6 in the rat, expression of ART2.2 by T cells is low in
the newborn mouse and increases during postnatal ontogeny to reach a
peak at 6–8 wk of age (Fig. 7). Like immature rat thymocytes that are
RT6- (22, 44, 50, 51), immature
mouse CD4+/CD8+
double-positive thymocytes are ART2.2- (Fig. 4, a–c, gate 2). One notable difference between rat and mouse
regards expression of ART2.2 by mature thymocytes (Fig. 4, a–c, gates 3 and 4, and Fig. 5). Mature rat thymocytes also
are RT6- (22, 44, 50, 51). In
contrast, the mouse thymus contains a small but distinctly
ART2.2+ population of
CD3highCD4+/CD8-
and
CD3highCD4-/CD8+
cells (Figs. 4 and 5).
The expression of ART2.2 in the thymus is of particular interest
because it appears to identify primarily the most mature thymocytes:
i.e., Nika102 reacts with <1% of
CD4+/CD8+ double-positive
cells, but stains 15% of CD4+ and 58% of
CD8+ single-positive cells (Fig. 4a).
ART2.2+ cells expressed high levels of CD3 (Figs. 3a and 5a) and CD5 (Fig. 5b), but low
levels of CD69 (Fig. 5c), which is transiently expressed
during positive selection (52), and low levels of CD24
(Fig. 5d), which is down-regulated during the final stages of thymocyte
maturation (40). Moreover, ART2.2+
thymocytes are resistant to dexamethasone-induced apoptosis (Fig. 4b). Cumulatively, these data are consistent with ART2.2
being expressed late in thymic development after the most intense phase
of positive selection. This interpretation is also supported by the
finding that ART2.2+ cells are enriched in the
thymi of CTS/Shi mice (Fig. 4c) in which a genetic defect
prevents the emigration of mature cells from the thymus
(39). Thus, ART2.2 appears to be a useful marker for the
most mature thymocytes.
RT6-expressing T cells exert a regulatory role in the BB rat model for
autoimmune insulin-dependent diabetes mellitus (23, 24).
Moreover, it has been shown that T cells from diabetic BB rats can
transfer disease to nondiabetic rats only if the cells are stimulated
before the transfer with phorbol ester or mitogen (53, 54). In this context, it may be of interest to point out that
ART2.2 and the IL-2R (CD25) are reciprocally expressed (Table I). The
results indicate that ART2.2 is down-modulated from the cell surface
and/or that ART2.2-negative cells preferentially expand upon
anti-CD3-mediated T cell activation. It will be of interest to
analyze the potential immunoregulatory role of ART2.2-expressing cells
and of ART2.2 itself in murine models of autoimmune disease. The
results presented in this study indicate that Nika102 holds promise as
a new experimental tool to address these questions.
Using CTLs induced in a 10-day mixed lymphocyte reaction, Dennert and
coworkers have previously observed that CTL functions such as
proliferation, target cell binding, and cytotoxicity can be
down-regulated by ecto-NAD+
(10, 11, 12), and further, that this is due to the action of a
GPI-anchored cell surface ADP-ribosyltransferase (8, 9).
These authors also noted that ADP-ribosyltransferase activity is
released from the cell surface upon stimulation of CTLs with PMA
(46). In accordance with these findings is our observation
that mouse ART2.2 disappears from the surface of lymph node and spleen
cells upon stimulation of these cells with PMA and ionophore (Fig. 9, c–f) or anti-CD3 (Table I).
Disappearance of an Ag from the cell surface can, in principle, be
caused by shedding or endocytosis of the Ag. Precedences for both types
of mechanisms have been reported for other GPI-anchored cell surface
proteins (55, 56). Our results are more compatible with
shedding rather than with endocytosis of ART2.2. PMA-induced
disappearance of ART2.2 from the cell surface more closely resembles
that of CD62L than that of CD4 in both magnitude and kinetics. CD62L is
known to be shed from the cell surface, whereas CD4 is known to be
endocytosed under these conditions (47, 49). CD4 staining
levels remain unaltered when cells are prestained with Abs before
stimulation with PMA, as expected for an Ag that is endocytosed
together with its bound fluorescently labeled Ab (Fig. 9, c
and d). In contrast, like CD62L (not shown), disappearance
of ART2.2 from the cell surface can still be observed under these
conditions. Whatever the mechanism of PMA-induced loss of ART2.2 from
the cell surface may be, it is of interest to note that expression
levels of other GPI-anchored cell surface proteins such as CD90 and
Ly-6A/E are not similarly affected (Fig. 9, g–h). The
finding that ART2.2 disappears from the cell surface of PMA-stimulated
T cells raises interesting questions to be addressed in future
investigations, such as: 1) how is ART2.2 released from the cell
surface, e.g., by a phospholipase acting on the GPI anchor or by a
protease acting on the polypeptide backbone; 2) how is T cell function
affected by shedding of ART2.2; and 3) is ART2.2 released in an
enzymically active form?
Interestingly, we have observed significant differences in the
percentage of peripheral T cells expressing ART2.2 as a function of
inbred strain (Fig. 8). T cells from NZW/BinJ mice, unable to express
ART2.2 because the Art2b gene is deleted in this strain
(45), did not react with Nika102, nor did splenocytes from
T cell-deficient C57BL/6J.scid mice (Figs. 5 and 8). High
ART2.2-expressing strains include C57BL/6J, C57BLKS/J, NOR/Lt, and
NOD/Lt; low expressors include DBA/2J, C3H/HeJBir, CBA/JLt, and NZO/Lt.
In general, ART2.2 cell surface expression levels as detected by
Nika102 correlate well with results of previous studies showing marked
differences in ART2.2-specific mRNA, as detected by RT-PCR in different
strains of mice (13, 57, 58). The strain-specific
differences in ART2.2 expression may also be of relevance for graft
rejection across the H1 minor histocompatibility barrier. The ART2.2
gene maps near the H1 minor histoincompatibility locus and ART2
haplotypes correlate with H1 allotypes (59). We note that
H1a mice (DBA/2J, C3H/HeJBir, CBA/JLt) all are low ART2.2 expressors,
whereas H1c mice (C57BL/6J, C57BLKS/J) are high ART2.2 expressors. It
is conceivable that differences in ART2.2 expression levels contribute
to graft rejection across the H1 barrier. It remains to be determined
whether these strain-specific differences in ART2.2 expression on T
cells are compensated by expression of other
ecto-ADP-ribosyltransferases on these cells, e.g., ART1 (15, 16, 60) or ART2.1 (13, 28, 29). In case of C57BL/6J and
BXSB/MpJ mice, in which the closely linked ART2.1 gene is inactivated
by a premature stop codon (57, 58), it is possible that
ART2.2 is up-regulated to high expression levels in compensation for
loss of ART2.1.
Abs to the other ecto-ARTs are necessary to complete the picture. The strategy described in this study for raising mAb Nika102 against ART2.2 by ballistic DNA immunization may be an attractive alternative to traditional protein immunization strategies for raising mAbs to other ARTs. Indeed, reports from other laboratories (61, 62, 63) lend support to the idea that DNA immunization is generally applicable for raising mAbs against molecularly cloned cell surface proteins.
In conclusion, we have demonstrated that gene gun technology is an efficient means to raise mAbs against a T cell surface protein. We have used one such mAb, Nika102, to show that mouse T cells express ecto-ART ART2.2 as a GPI-anchored cell surface molecule. In the thymus, ART2.2 is not expressed by immature CD4+/CD8+ double-positive cells, but is expressed by mature CD4+ and CD8+ single-positive cells. In peripheral lymphoid organs, ART2.2 is expressed at high levels on CD8+ cells, and at slightly lower levels on CD4+ cells. ART2.2 expression levels are low in the newborn mouse, but increase during the early postnatal ontogeny. ART2.2 is rapidly down-modulated from the cell surface after PMA-induced T cell activation. Thus, the ART2.2-specific mAb Nika102 described in this study should be of use as new differentiation marker of thymic and postthymic T cells in the mouse and as a tool to further elucidate the function of this intriguing cell surface enzyme in the mouse.
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Acknowledgments |
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and 2;
T.D., M.N., and F.K.-N. those described in Figs. 3, 4, and 6–9; N.K. that in Fig. 5; and V.A. that in Table I. We thank Drs. David Serreze, Robert Graser, and
Heinz-Günter Thiele for critical reading of the manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Friedrich Koch-Nolte, Professor of Immunology and Molecular Biology, Institute for Immunology, University Hospital, D20246 Hamburg, Germany. E-mail address:
3 Abbreviations used in this paper: ART, ADP-ribosyltransferase; AP, alkaline phosphatase; HF, chimeric His(6x)-FLAG tag; PI-PLC, phosphatidylinositol-specific phospholipase C; NZW, New Zealand White.
Received for publication May 28, 1999. Accepted for publication September 8, 1999.
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) and CD4+ (
) cell
populations.
