(Received for publication, March 16, 1995; and in revised form, August 25, 1995)
From the
The CD45 family of transmembrane protein-tyrosine phosphatases plays a crucial role in the regulation of lymphocyte activation by coupling activation signals from antigen receptors to the signal transduction apparatus. Multiple CD45 isoforms, generated through regulated alternative mRNA splicing, differ only in the length and glycosylation of their extracellular domains. Differential distribution of these isoforms defines subsets of T cells having distinct functions and activation requirements. While the requirement for the intracellular protein-tyrosine phosphatase domains has been documented, the physiological role of the extracellular domains remains elusive. Here we report the generation of CD45-antisense transfected Jurkat T cell clones that lack CD45 or have been reconstituted to uniquely express either the smallest, CD45(0), or the largest, CD45(ABC), isoform. These cells exhibited marked isoform-dependent differences in IL-2 production and tyrosine phosphorylation of cellular proteins, including Vav after anti-CD3 stimulation. These results demonstrate that the distinct CD45 extracellular domains differentially regulate T cell receptor-mediated signaling pathways. Furthermore, these findings suggest that alterations in CD45 isoform expression by individual T cells during thymic ontogeny and after antigen exposure in the periphery directly affects the signaling pathways utilized.
Activation of resting T lymphocytes through the T cell receptor
(TCR) ()requires expression of the CD45 family of
transmembrane protein-tyrosine phosphatases (PTPases) (1, 2) . CD45 has been shown to regulate the basal
activity of the Fyn and Lck protein-tyrosine kinases (PTKs) by
dephosphorylation of their respective regulatory carboxyl-terminal
tyrosine
residues(3, 4, 5, 6, 7) .
However, it is not clear that these are CD45's sole functions.
For example, new evidence suggests that CD45 can also dephosphorylate
certain PTK substrates, such as the TCR
chain (8) and the
32-kDa CD45-associated phosphoprotein, LPAP(9) . Thus, the
precise functions of the CD45 phosphatase in signal transduction are
incompletely understood.
While the requirement for the intracellular PTPase domains has been documented(10, 11, 12, 13) , the function of the CD45 extracellular domain in lymphocyte signal transduction remains a major unresolved issue. In humans, five CD45 isoforms, ranging in size from 180-220 kDa, are generated by the regulated alternative mRNA splicing of three exons, encoded by a single gene(14, 15, 16) . The alternatively spliced exons, commonly referred to as A, B, and C, are located near the 5` end of the gene and give rise to isoforms that differ only in their extracellular regions. Individual lymphocytes simultaneously express more than one CD45 isoform(17, 18) . However, the expression of certain isoforms is highly regulated, resulting in their differential expression on lymphocytes of different lineage (e.g. T versus B cells), as well as on distinct functional subsets of T cells(19, 20, 21, 22) . Furthermore, individual T cells alter their isoform expression in a highly regulated manner during thymic selection and upon antigen exposure in the periphery(18, 23, 24, 25, 26) . The tight regulation of CD45 isoform expression by lymphocytes having distinct functions argues that these differences are likely to be of biologic importance. However, attempts to study the role of individual CD45 isoforms in signaling have been severely hampered by great difficulty in re-expressing different single intact CD45 isoforms into the same cellular background.
Recent studies have clearly
demonstrated that TCR-mediated signaling can be reconstituted in
CD45 mutants by transfection of chimeric molecules
containing the conserved PTPase domains, but lacking the CD45
transmembrane or extracellular regions (11, 12, 13) . However, these results do not
exclude a potentially important role for the CD45 extracellular domain,
since the various extracellular domains could superimpose distinct
regulatory constraints upon the cytoplasmic domain. Our present
findings strongly support this hypothesis. Utilizing a unique model
system, we now demonstrate that the expression of different individual
CD45 isoforms is associated with differences in IL-2 production, as
well as differences in the activation-related phosphorylation of
cellular proteins including Vav. These findings demonstrate the
preferential utilization of different signaling pathways by distinct
CD45 isoforms.
Rabbit polyclonal Ab to Vav was developed by immunization of rabbits with a synthetic peptide containing residues 575-594 of (murine) Vav. Immunoprecipitation of a 95-kDa band with the antiserum was specifically blocked by addition of the immunizing peptide to the lysate mixture (data not shown).
Figure 2:
IL-2
secretion by Jurkat versus CD45 antisense-transfected Jurkat cell lines (J-AS), CD45(0)
transfectants (J[0]) (open symbols), and CD45(ABC)
transfectants (J[ABC]) (closed symbols), after
stimulation with various doses of anti-CD3 (cross-linked with GAM) (A), anti-CD28 (1:400) and anti-CD3 (1 µg/ml)
(cross-linked with GAM) (B), and ionomycin (1 µM)
plus PMA (1 or 5 ng/ml) (C). Each point represents the mean
values for two J-AS clones, two J[0] clones, and three
J[ABC] clones each examined in three independent experiments.
Data for each experiment were normalized to the response of Jurkat to
the maximal stimulus (100% response averaged 50 units/ml in A,
40 units/ml in B, and 70 units/ml in C, where 1 ng of
rIL-2 = 50 units). Vertical lines indicate
S.E.
Like peripheral T cells, the Jurkat human T cell leukemia
line normally expresses CD45 at high levels, and individual cells
express multiple isoforms
simultaneously(17, 18, 19) . To examine the
role of CD45 and its individual isoforms, free from the potential
confounding influences of unknown mutations, we directly targeted
endogenous CD45 expression by stable transfection of a plasmid
construct (AS-CD45) expressing an antisense RNA directed at a 270-base
pair region of genomic CD45 just upstream from the coding region. (Fig. 1A). Of the several independent CD45 colonies selected and subcloned, two, denoted J-AS-1 and J-AS-2,
were selected on the basis of CD4 expression comparable to that of
parental Jurkat. (Fig. 1B). Jurkat expresses high
levels of total CD45 and lower levels of both the smallest CD45 isoform
(CD45RO) and the largest (two) isoforms which contain exon A (CD45RA).
J-AS-1 and -2 lack detectable CD45 expression either on the surface or
in the cytoplasm (Fig. 1, B and C).
Figure 1:
A, schematic representation of DNA
inserts encoding antisense CD45 RNA (AS-CD45), CD45(0), and CD45(ABC)
isoforms. The arrow under each insert indicates the direction
of transcription once inserted into the respective expression vectors.
Sense and antisense constructs have minimal overlap. B,
representative immunofluorescence analysis of cell surface expression
of various markers on Jurkat, CD45 (J-AS-1), and
single CD45 isoform transfectants expressing either the CD45(0) isoform
(J[0]-1) or the CD45(ABC) isoform (J[ABC]-1).
Isotype-matched negative controls are depicted as dotted
lines. The x and y axes represent log
fluorescence and cell number, respectively. C, anti-CD45
immunoblotting of whole cell lysates from: Raji (human B cell line),
Jurkat, CD45
(J-AS-1), CD45(0) transfectants
(J[0]-1 and -2), and CD45(ABC) transfectants
(J[ABC]-1 and -2). 300-19 (mouse pre-B cell) is shown
as a negative control. Arrows on left indicate previously
established human isoforms at 220, 205, 190, and 180 kDa(17) .
Lower M
bands represent immature forms, not yet
glycosylated at O-linked sites(17) . Arrows at
right indicate nonspecific bands present in control 300-19
cells.
J-AS-1
was then stably transfected with CD45 cDNA constructs modified to
minimize overlap with AS-CD45 antisense RNA and encoding either the
smallest isoform, denoted CD45(0), lacking alternative exons, or the
largest isoform, denoted CD45(ABC), which includes all three
alternative exons. These isoforms best exemplify differential
distribution on T cell subsets having distinct functions and activation
preferences(19, 21, 23) . Each of the
CD45 clones arising expressed solely the transfected
CD45 isoform by both immunofluorescence and by immunoblotting. (Fig. 1, B and C). Three independent CD45(ABC)
transfected isolates (J[ABC]-1, -2, and -3) and two CD45(0)
transfected isolates (J[0]-1 and -2) were selected for
further study, based on CD45 expression and wild-type levels of CD3.
The clones were then sorted to obtain stable populations expressing
similar levels of CD4 and CD45. When matched for their surface
expression, J[0] and J[ABC] clones expressed
identical levels of CD45 by immunoblotting, indicating no inherent
differences in the relative distribution of intracellular and
extracellular CD45 (data not shown). Although total CD45 expression was
lower in the transfectants, the expression of individual CD45(0) and
CD45(ABC) isoforms by J[0] and J[ABC] cells,
respectively, was similar to their expression in wild-type cells. The
expression of CD3, CD2, and CD28 was nearly identical in each of the
cell lines (Fig. 1B and data not shown).
Current
evidence indicates that CD45 regulates the activity of proximal
components of the signaling apparatus such as the Src family PTKs, Lck
and Fyn, and, presumably, their
substrates(3, 4, 5, 6) . First,
TCR/CD3-induced IL-2 secretion, which depends on the coordinated
activation of multiple transcription factors(34) , was examined
as an integrated measure of such signaling events. The dose-response
curve to anti-CD3 (Fig. 2A) reveals that, in contrast
to Jurkat, the CD45 (J-AS) cell lines secreted
minimal IL-2 in response to all doses of anti-CD3 tested. Furthermore,
no enhancement was seen after co-stimulation by cross-linking anti-CD3
and anti-CD4 (data not shown). Reconstitution with the CD45(0) isoform
resulted in wild-type levels of IL-2 secretion after stimulation with
anti-CD3 (1.0 µg/ml). In contrast, the CD45(ABC) transfected cell
lines produced significantly less IL-2 than either Jurkat or the
CD45(0) transfectants at both 0.05 and 1.0 µg/ml anti-CD3,
secreting at maximum, 30% of wild-type levels. Increasing the anti-CD3
dose to 5 µg/ml had no additional effect on IL-2 secretion by any
of the cell lines (data not shown). However, at lower doses of anti-CD3
(0.005 µg/ml), transfectants expressing either individual isoform
secrete much less IL-2 than Jurkat, possibly owing to the lower levels
of overall CD45 expression. Stimulation with anti-CD2 gave overall
results similar to those observed above (not shown).
Similar responses by J-AS-1 and each of its single isoform-reconstituted derivatives after stimulation by Ab-mediated cross-linking of CD3 and CD28 (Fig. 2B), or with PMA plus ionomycin (Fig. 2C), documents similar inherent capacity of each cell line to secrete IL-2 when the proximal signaling machinery, or the requirement for CD45(30) , are bypassed, respectively.
Thus, after stimulation with anti-CD3 (at 0.05 to 1 µg/ml), IL-2 secretion by J[0] transfectants is not significantly different from wild-type cells, despite 6-7-fold lower CD45 expression. Nonetheless, it is possible that decreased IL-2 secretion by J[ABC] cells compared to J[0] cells is due to their somewhat lower levels of CD45 expression. To rule out this possibility, we sorted J[ABC] clones to obtain CD45 expression equal to that of J[0] cells and then compared IL-2 secretion by these cell populations after anti-CD3 stimulation (see Fig. 3). As before, clones expressing CD45(0) secrete wild-type levels of IL-2. As shown, increased CD45 expression by J[ABC] transfectants did not augment IL-2 secretion. Both sorted and unsorted J[ABC] populations averaged just 24% of the wild-type levels of IL-2.
Figure 3: A, representative immunofluorescence analysis of CD45 expression on J[ABC] transfectants both before (solid line) and after fluorescence activated cell sorting (dotted line) to obtain J[ABC] cells expressing CD45 at levels equal to those expressed by J[0] transfectants (upper panel). Log fluorescence of isotype-matched negative controls for each clone was in the first decade (not shown). B, IL-2 secretion by Jurkat, J[0] transfectants, and J[ABC] transfectants before and after cell sorting for increased CD45 expression. Cell populations depicted in A (plus wild-type Jurkat) were stimulated with anti-CD3 at doses of either 0.5 µg/ml or 1 µg/ml, and 24-h supernatants were assessed for IL-2 secretion, as described under ``Materials and Methods.'' IL-2 secretion was normalized to the response of Jurkat cells. Anti-CD45 treatment itself had no effect on IL-2 secretion by J[ABC] clones (not shown). Data depicted are the average of four experiments ± S.E.
Given differential anti-CD3-induced IL-2 secretion by these
cell lines, more proximal signaling events were next examined.
Comparison of Lck and Fyn activities by immune complex kinase assays
failed to reveal isoform-dependent differences (data not shown). T cell
activation is associated with alterations in the tyrosine
phosphorylation of a number of cellular proteins. Therefore, we
compared the tyrosine phosphorylation of cellular proteins in each cell
line before, and at various time points after, anti-CD3 stimulation (Fig. 4). Under basal conditions, J-AS-1 consistently revealed
hyperphosphorylation of a limited set of bands at 70-76 kDa
and decreased tyrosine phosphorylation of several other bands
(
105,
95, and
50-52 kDa) when compared to Jurkat (Fig. 4A).
Figure 4: Comparison of tyrosine phosphorylation of cellular proteins in Jurkat and J-AS-1 (A) and representative single-isoform transfectants J[0]-1 and J[ABC]-1 (B) before and after anti-CD3 stimulation for the times indicated. Whole cell lysates were immunoblotted with anti-Tyr(P). Brackets and arrows to the right indicate bands exhibiting the most consistent differences between cells in each panel. Approximate molecular mass of bands indicated (from top to bottom): A, 95, 70-75, 60, 52, and 38-40 kDa; B, 95, 58-60, and 32 kDa.
After anti-CD3 stimulation of Jurkat, there was rapid phosphorylation (peaking at 30 s to 1 min) and subsequent dephosphorylation of a number of bands. Although many of the same bands were ultimately phosphorylated (within 5-10 min) after stimulation of J-AS-1 cells, the kinetics were significantly slowed. Furthermore, once phosphorylated, these bands did not undergo dephosphorylation, consistent with decreased action of the CD45 PTPase and perhaps of other cellular PTPases whose activities depend on regulated tyrosine phosphorylation (35, 36).
Re-expression of either
the CD45(0) or the CD45(ABC) isoforms generally restored basal and
activation-related tyrosine phosphorylation, although the kinetics were
somewhat prolonged compared to wild-type Jurkat (Fig. 4B). This may reflect the lower overall levels of
CD45 expression in these cells. More importantly, direct comparison
reveals clear isoform-dependent differences in the relative
phosphorylation of several bands. For example, J[ABC] cells
consistently exhibited relative hyperphosphorylation of a band at
95 kDa when compared to J[0] cells.
This prompted a
comparison in our cells of the tyrosine phosphorylation of p95 (Vav) which is rapidly and transiently phosphorylated on tyrosine
after ligation of the TCR(33, 37) , CD28(38) ,
or upon the binding of IL-2 to its receptor(39) . While the
exact function of this proto-oncogene product in signal transduction is
unclear, gene ablation studies document the important role of Vav in
the activation and proliferation of mature lymphocytes as well as in
the normal developmental expansion of lymphocyte precursors in the
marrow and thymus(40, 41) .
Basal Vav tyrosine
phosphorylation was minimal but detectable in each of our cell lines (Fig. 5A). Anti-CD3 stimulation consistently induced
significantly greater tyrosine phosphorylation of Vav within 1 min, in
Jurkat and particularly in all three J[ABC] transfectants
compared to either of the two J[0] transfectants or the
CD45 J-AS-1 cells. Reprobing the same membrane with
anti-Vav antisera confirmed similar loading of Vav protein in each lane (Fig. 5B). These differences are not secondary to
altered kinetics, since the same pattern is observed 4 min after
anti-CD3 stimulation, at which time phosphorylation of Vav in Jurkat
and single-isoform transfectants is decreasing (data not shown and (33) ).
Figure 5: A, tyrosine phosphorylation of Vav in Jurkat, J-AS-1, and single isoform expressing transfectants expressing either CD45(ABC) or CD45(0) isoforms before (0 min) or after (1 min) stimulation with anti-CD3. Vav was immunoprecipitated from each cell line and immunoblotted with anti-Tyr(P). C (first lane) indicates control immunoprecipitation from lysates of stimulated Jurkat cells using rabbit pre-immune serum. B, reprobing the same membrane with anti-Vav antisera to demonstrate similar amounts of Vav protein/lane.
Our results are the first to demonstrate that signaling pathways utilized by the TCR are differentially regulated by the extracellular domain of distinct CD45 isoforms. Stimulation of the TCR leads to the phosphorylation of a number of cellular proteins including Vav. Although the signaling pathways involving Vav have not yet been clarified, Vav contains an array of signaling and DNA-binding motifs, including SH2 and SH3 domains, a Dbl domain, and a helix-loop-helix, which all appear to be involved in the generation of downstream signals (33, 42, 43, 44, 45) . Activation-related tyrosine phosphorylation directs SH2-mediated interactions between Vav and several other signaling molecules. Thus, Vav has been shown to associate with Shc, Grb2, ZAP-70, phosphatidylinositol 3-kinase (p85), CD19, VAP-1, and several other uncharacterized bands through SH2 and/or SH3-mediated interactions after activation of B or T lymphocytes(37, 46, 47, 48) .
How
different CD45 isoforms might regulate this pathway remains
speculative. Particular CD45 isoforms might directly dephosphorylate
Vav or could differentially regulate the activity of the PTK(s)
responsible for Vav phosphorylation. However, preferential
dephosphorylation of Vav by a particular isoform is difficult to
envision given that Vav phosphorylation is decreased in both
CD45 cells and those expressing CD45(0), yet
increased in wild-type cells (which express both CD45(0) and CD45(ABC)
isoforms) and in cells expressing CD45(ABC). Activation-related
phosphorylation of Vav clearly does not directly correlate with
absolute levels of CD45 expression. Our results are more consistent
with augmented activity of the PTK responsible for Vav phosphorylation
by cells expressing the CD45(ABC) isoform. However, the PTK(s)
responsible for the in vivo phosphorylation of Vav are
presently unknown. Although Lck is capable of phosphorylating Vav in vitro, IL-2 mediated phosphorylation of Vav occurs in the
absence of Lck(39) . CD28 ligation results in Itk
phosphorylation followed temporally by that of Vav, suggesting a
possible link(38) . Recently, it was reported that ZAP-70 can
physically associate with the Vav-SH2 domain after T cell activation,
although it is unknown whether Vav serves as a substrate for this
PTK(47) . Further analysis of these PTKs and Vav-associated
molecules in our single isoform transfectants may help delineate those
pathways relevant to CD45.
As mentioned above, chimeric PTPase molecules lacking the CD45 transmembrane or extracellular domains are able to restore nearly normal patterns of tyrosine phosphorylation and calcium flux(11, 12, 13) . In general agreement, we showed that either the CD45(0) or CD45(ABC) isoforms are capable of reconstituting activation-related tyrosine phosphorylation. However, isoform-specific differences in IL-2 production and the tyrosine phosphorylation of cellular proteins indicate that the extracellular domain can superimpose regulatory influences on a ``constitutive'' cellular PTPase requirement. Thus, even though many of the signaling pathways are conserved, a subset (that includes Vav) appears subject to differential regulation by the various CD45 extracellular domains. Differences in the utilization of these pathways can lead to rather substantial isoform-dependent differences in IL-2 secretion as demonstrated in our model and in the mouse thymocyte model of Novak et al.(49) .
Exactly how the CD45 extracellular domains may regulate the PTPase domains is unknown. One long-standing hypothesis supported by our findings is that the distinct extracellular domains of the various CD45 isoforms interact with different molecules on the surface of the same or other cells, thereby directing the cytoplasmic phosphatase domains toward distinct substrates. In this regard, the co-capping studies of Dianzani et al.(50) support the notion that differential interactions between CD45 isoforms and other molecules on the surface of the same cell can occur.
In conclusion, our results indicate that the regulated expression of distinct CD45 isoforms on different developmental and functional subsets of T cells may impose preferential utilization of particular TCR-mediated signaling pathways. Alterations in CD45 isoform expression by individual T cells in response to thymic selection or peripheral antigen exposure, may consequently allow that cell to respond to TCR ligation using a different subset of signals. We speculate that the delivery of these different signals to the cell nucleus might have a significant influence on cell differentiation, the expression of functional repertoire, or in allowing T cells to ``fine-tune'' their responsiveness. A more complete understanding of these differences is likely to have important implications for signal transduction and for the interpretation of the highly regulated expression of CD45 isoforms in lymphocytes.