Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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INTRODUCTION |
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One such event is creation of an anergic state that occurs when T cells are treated with a single reagent that results in Ca2+ influx rather than with a costimulatory signal involving activation of the T cell receptor (9). One of the defects in an anergic cell is blocked transcription of the IL-2 gene. This state can be mimicked by transfection of a constitutively active form of Ca2+/calmodulin-dependent protein kinase II (CaMKII) into the Jurkat T cell line (10, 11). These data suggest that CaMKII is unlikely to represent a target of Ca2+ that is required for T cell activation. That such a target must exist is demonstrated by the fact that, in Jurkat cells, activation of reporter genes containing AP-1-binding sites such as those required for activation of fos and jun family member genes occurs in the presence of immunosuppressive drugs and yet still requires Ca2+ (12). Activation of AP-1 genes requires members of the cAMP response element binding protein (CREB) family (13), and these proteins must be phosphorylated on a specific serine residue (Ser-133 in the case of CREB) (14) to become competent as transcriptional activators. The importance of CREB to T cell activation was revealed by Barton et al. (15), who used the CD2 promoter to target expression of a mutant form of CREB in which Ser-133 was changed to Ala specifically to T cells in transgenic mice. Cells from these animals could not be activated by costimulation through the T cell receptor and produced neither immediate early gene products nor IL-2. The interpretation of these results was that the mutant form of CREB would dimerize with CREB or other binding partners. However, since the mutant form could not be phosphorylated, dimers containing this protein could not function as transactivators. Because phosphorylation is not required for dimerization or DNA binding, the mutant protein appeared to act on transcriptional activation in a dominant/negative manner.
Because T cell activation requires CREB and CREB must be phosphorylated on Ser-133 to function as a transcriptional activator, identification of the physiologically relevant protein kinase would be of considerable interest. Because transcription mediated by AP-1 sequences requires CREB and Ca2+, we suspected that one enzyme responsible for CREB phosphorylation in T cells might be a Ca2+-dependent protein kinase. A logical candidate for this enzyme is the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). This enzyme is present in T cells (16, 17), is localized in the nucleus (18), and has been shown to phosphorylate CREB specifically on Ser-133 both in vitro and when overexpressed in cultured cell lines (19, 20, 21). In addition, CaMKIV will activate reporter gene constructs that are driven by AP-1 sequences in Jurkat cells (22, 23). Support for this contention comes from the work of Bito et al. (24), who suggested that CaMKIV mediates CREB phosphorylation that is required for the delayed phase of long-term potentiation in cultured hippocampal neurons.
To test the possibility that CaMKIV catalyzes CREB phosphorylation in T lymphocytes, we generated transgenic mice that express a catalytically inactive form of CaMKIV only in T cells when resident in the thymus. Thymic lymphocytes from these animals exhibit striking developmental and T cell receptor-mediated signaling defects. The thymic T cells show dramatic impairment in the ability to phosphorylate CREB, induce transcription of the immediate early gene Fos B, and produce IL-2. Since the transgene is driven by the murine proximal lck promoter and this promoter has been silenced in T cells that exit the thymus (25, 26), splenic T cells do not contain the mutant hCaMKIV protein and regain the ability to signal normally. These data provide compelling evidence that CaMKIV may be an important Ca2+ target required for activation of T cells.
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RESULTS |
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Western blot analysis was used to confirm that the appropriate protein
was produced from the respective transgenes. The left panel
of Fig. 1C shows the results obtained when an antibody is used that
recognizes both the endogenous mouse and transgenically produced human
CaMKIV. T cell protein from two transgenic mice flank a similar
preparation from a control mouse. It can be seen that the expression
level of the transgenic protein is higher in the right lane
(Line I) than in the left lane (Line K). Densitometry was
used to quantify the difference in protein expression. We find that the
amount of immunoreactive CaMKIV in the Line I lane is 4-fold greater
than in the control lane. Thus the T cells from Line I produce about a
3-fold molar excess of the mutant hCaMKIV relative to endogenous levels
of the authentic enzyme. Animals from Line I were used in all
subsequent experiments described in this manuscript. We also prepared
an antibody to a synthetic peptide containing the C-terminal 17 amino
acids of hCaMKIV that, under our experimental conditions, is specific
for the human form of the protein. As can be seen in the far
left panel of Fig. 1C
, only T cells resident in the thymus of the
transgenic mouse contained the human protein. The fact that no
transgenic product is present in T cells isolated from the spleen of
the identical transgenic mouse is consistent with the fact that the
proximal lck promoter is not active in T cells that are
exported from the thymus (26). Additionally, the absence of the mutant
hCaMKIV in splenic T cells implies that this protein must be unstable
in these cells.
The center panel of Fig. 1C shows that T cells removed from
the thymus of one line of transgenic mice produce considerable amounts
of calspermin. As there are no amino acids present in calspermin that
are not also present in CaMKIV, it has not been possible to produce an
antibody that recognizes calspermin but not CaMKIV. In addition, the
isoelectric pH of calspermin is 4.0, and consequently this protein does
not transfer quantitatively under the conditions usually employed for
Western blots (29). However, examination of the blots suggests that the
amount of calspermin present is in considerable excess to the amount of
endogenous CaMKIV. Unquestionably, the ratio is greater than 3:1.
Therefore we feel justified in using these cells to control for any
nonspecific effects, such as sequestration of
Ca2+/calmodulin, due to the presence of the mutant
hCaMKIV.
The Presence of Mutant hCaMKIV in T Cells Results in a
Developmental Defect
The most obvious phenotypic consequence resulting from the
expression of the mutant hCaMKIV was a large reduction in the size and
weight of the thymus (data not shown). This gross phenotype was
confirmed by analysis of the number of T cells resident in the thymus.
As shown in Fig. 2A (left panel) the total
number of T cells present in the thymus of transgenic mice is only
about 16% of that found in the thymus of nontransgenic littermates or
in transgenic mice that express calspermin (n = 14,
P < 0.01). The deficit observed in the number of T
cells in the thymus is completely normalized when the number of splenic
cells is quantified. As shown in the left panel of Fig. 2B
, no statistically significant difference exits between the number of
splenocytes in control, transgenic hCaMKIV mutant
[lck-CaMKIV Kin(-)], or transgenic calspermin
(lck-calspermin) mice. This normalization in the spleen is
entirely consistent with the fact that splenocytes cease expression of
the hCaMKIV transgene.
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The Presence of Mutant hCaMKIV Results in a T Cell-Signaling
Defect
It has been shown that CaMKIV is activated within seconds after
engagement of the T cell receptor (17, 23, 32). Similarly, CREB is
rapidly phosphorylated in response to a T cell-activating signal (15).
Because CaMKIV phosphorylates CREB on Ser-133 (19, 20, 21), we examined
whether the T cells that expressed the mutant hCaMKIV might have an
altered ability to phosphorylate CREB. The results of a representative
experiment are shown in Fig. 4. As can be seen in the
top left panel, addition of phorbol ester [phorbol-12
myristate-13 acetate (PMA)] and ionomycin (shown as P/I as in the
figure) to thymic T cells from normal mice resulted in robust
phosphorylation of CREB by 1 min. This signal was maintained for at
least 30 min. The Ser-133-phosphorylated form of CREB was selectively
examined by the use of an antibody that only recognizes this
phosphorylated epitope (33). Similar results were obtained when the
cells were stimulated by addition of the anti-CD3 antibody that
produces the signal through engagement of the T cell receptor
(
-CD3). The location of the phospho-CREB signal is depicted by an
arrow. Note that a faster migrating protein also shows an
increased phosphorylation in response to addition of P/I or
-CD3.
This protein has been provisionally identified as activating
transcription factor-1 (ATF1), another member of the CREB family of
transcription factors because: 1) the phosphorylated form of ATF1 is
known to be recognized by the phospho-CREB antibody; 2) it is the same
size as ATF1 based on migration in an SDS gel; and 3) ATF1 has also
been shown to be present in T cells (15) and can be phosphorylated and
activated by CaMKIV (34). The third autoradiogram on the
left shows that there was no change in the total amount of
CREB during the 30 min of the experiment by use of an antibody that
recognizes both phosphorylated and unphosphorylated CREB
(
-CREB).
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CREB phosphorylation in thymic T cells is required for the
activation of the immediate early genes c-fos, Fra-2, and
Fos B. (15). Therefore we examined the appearance of Fos-B mRNA in
response to T cell stimulation. As shown in Fig. 5, stimulation of control T cells with
-CD3 resulted in transient
expression of Fos B mRNA. The time course of this response and the
presence of the two mRNA species are identical to the results reported
by Barton et al. (15). However, cells that express the
mutant hCaMKIV fail to demonstrate an increase in Fos B mRNA in
response to stimulation. The Northern blot was stripped and reprobed
with a glucose-3-phosphate dehydrogenase (GAPDH) cDNA. The
bottom autoradiogram in Fig. 5
shows that each lane did
contain equivalent amounts of RNA. Thus the presence of the mutant
hCaMKIV correlates with the inability to phosphorylate CREB and to
produce Fos B mRNA.
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DISCUSSION |
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The question we set out to answer was whether the target of Ca2+ responsible for the activation of AP1-mediated gene transcription that was independent of the action of the immunosuppressive drugs was CaMKIV. To evaluate this possibility, we chose to use a construct designed by Chaffin et al. (27) that used the proximal promoter of the murine lck tyrosine kinase gene to restrict expression to T lymphocytes resident in the thymus. This promoter was chosen for three reasons. First, it is known to be expressed in all double negative and double positive thymic T cells and has been identified as early as embryonic day 15 (26). This is at least 1 day earlier than the first appearance of CaMKIV (45). Therfore, if the mutant hCaMKIV protein was to act in a dominant/negative manner, the kinetics of appearance of the transgene product relative to the endogenous gene product should be optimal to produce an effect. Second, Alberola-Ila et al. (37) have used this vector to express a catalytically inactive form of the mitogen-activated protein kinase kinase, MEK-1, in T cells that effectively blocked the activation of mitogen-activated protein kinase. This dominant/negative approach resulted in the selective inhibition of positive selection. Third, the proximal lck gene promoter is silenced in single positive T cells and, consequently, is nonfunctional in all T cells that exit the thymus (26). Inasmuch as we had evidence that inactive forms of CaMKIV seemed to be unstable when expressed in mammalian cells in culture (23), we reasoned that the hCaMKIV might be turned over in peripheral T cells. If so, then we could determine whether any effects on T cell activation due to the presence of the mutant hCaMKIV would be reversible. As shown by our results, the vector was well chosen.
T cells that contain a 3-fold excess of mutant to endogenous CaMKIV fail to phosphorylate CREB in response to activating stimuli. Failure to phosphorylate CREB leads to the inability to produce Fos B mRNA. Fos B has been shown to be involved in activation of the IL-2 promoter (1, 2); thus, IL-2 production is also severely attenuated. Splenic T cells indeed no longer contain the mutant hCaMKIV based on evaluation of Western blots. These cells regain the ability to respond to activating stimuli by increasing CREB phosphorylation. They also produce normal levels of IL-2 (data not shown). These data suggest that the mutant hCaMKIV is acting in a dominant/negative manner although we cannot be certain as to the molecular mechanism responsible for the dominant/negative effect. As even higher levels of calspermin are without phenotypic consequence, it is unlikely that the mutant hCaMKIV is nonspecifically sequestering Ca2+/calmodulin that might be needed for other events required for T cell activation. One such event is known to be the activation of calcineurin (5). CaMKIV is found in the nucleus (18), so it is possible that the inactive protein might prevent nuclear entry of CaMKIV or competitively inhibit its ability to interact with other nuclear proteins (including, but not restricted to, substrates such as CREB). Finally, CaMKIV must be phosphorylated by a CaM kinase kinase to become activated (23, 28, 32, 38, 39, 40, 41, 42, 43, 44). Perhaps this reaction is stoichiometric and thus the presence of an excess of an inactive form of CaMKIV prevents activation of the endogenous enzyme by the relevant CaMKIV kinase. Regardless of the mechanism involved, our data implicate CaMKIV as the physiologically relevant CREB kinase in T cells. Inasmuch as a number of other enzymes present in T cells are capable of phosphorylating CREB, this is a remarkable finding. The physiological relevance of phosphorylation of CREB by CaMKIV has also been demonstrated by Bito et al. (24), who used an antisense RNA approach to study expression of immediate early genes in response to electrical stimulation of hippocampal neurons. They also concluded that the important Ca2+-dependent enzyme in this system was CaMKIV.
Barton et al. (15) produced mice that expressed a mutant
form of CREB (Ser-133 to Ala) specifically in T cells using the CD2
promoter. Two phenotypic consequences of the presence of the
nonphosphorylatable form of CREB are similar to those we find in the
mice that express the mutant hCaMKIV. First, the distribution of cells
in the thymus is normal in both lines of transgenic mice. Second, both
types of cells fail to induce the expression of Fos-B mRNA or produce
IL-2 in response to a variety of activating signals. It seems likely
that both CaMKIV and CREB are involved in the pathways responsible for
activation. However, differences also exist in the phenotypes that
result from the expression of the mutant forms of CaMKIV and CREB. Even
though the mutant CREB-containing cells cannot produce IL-2, they do
up-regulate the expression of the -chain of the IL-2 receptor, CD25,
in a manner similar to control T cells (15). As shown in Fig. 6B
, this
is not the case for cells containing mutant hCaMKIV, which do increase
CD25 expression, but only to about 30% of normal. This indicates that
initial cell-surface signaling events are impaired and implicate
another role for CaMKIV in T cell activation in addition to its
function as a CREB kinase. Perhaps the most obvious difference between
the two types of transgenic mice is the degree of thymic cellularity.
Although the number of T cells is normal in mice containing mutant
CREB, expression of mutant hCaMKIV leads to a 20% reduction
compared to control animals. As mentioned above, although the
cellularity is reduced, the distribution between CD4-CD8- (double
negative, DN), CD4+CD8+ (double positive, DP), CD4+ single positive
(SP), or CD8+ SP cells is not disturbed. Also cells expressing mutant
hCaMKIV exhibit a markedly decreased rate of survival when placed in
culture and incubated under conditions that do not favor activation,
whereas cells expressing mutant CREB do not differ from control cells
in this assay. The defect that results in reduced thymic cellularity
and decreased rate of survival in culture might be linked. An
interesting avenue for future investigation is to determine the
mechanism responsible for this defect.
Krebs et al. (45) have reported that CaMKIV does not exist in rat brain or thymus at embryonic day 15 (E15) but the mRNA and protein are found 1 day later at E16. At least in culture, the timing of this expression can be advanced by addition of thyroid hormone to the culture medium. The majority of cells in the thymus at E15 are DN (46, 47). Since DP cells begin to accumulate at E16 and this corresponds to the presence of CaMKIV, it is tempting to speculate that the CaMKIV gene is activated as one of the events required to produce DP from DN cells. Hanissian et al. (17) have shown that both DP and SP T cells contain CaMKIV. Moreover the highest concentration is present in DP cells. However, since the murine proximal lck promoter is functional in DN cells both during embryogenesis and in adult thymus (26), the mutant hCaMKIV is also likely to be present in these cells. If this is the case, perhaps it prevents the effects of CaMKIV that could be involved in generation of DP cells. This scenario could result in slowing the conversion of DN to DP cells, which could in turn lead to an increased rate of apoptosis of this population. A general decrease in the number of DN cells would lead to a decrease in the number of DP cells, which would in turn reduce production of SP cells. A sequence of events such as this is one of the few that we can envision that would lead to a decrease in cellularity but no change in the distribution of the remaining T cells, based on the CD4 and CD8 cell surface markers. Alternatively, it is possible that the presence of the mutant hCaMKIV in DN cells might result in a decreased rate of proliferation, and this effect might be independent of a normal physiological role of CaMKIV. Certainly a decreased proliferative capacity of DN cells could also result in decreased cellularity without altering the maturation of the remaining T cells.
Regardless of the mechanism involved, the currently available data would support a role for CaMKIV in DP cells that is independent of its role in the activation of SP cells. Resting DN T cells from adult mice do not contain T cell receptors or normally produce any cytokine. However, cytokine production can be induced by treatment with phorbol ester and ionophore (47). Inasmuch as these cells do not contain CaMKIV, the Ca2+ signal must not involve this enzyme. Even if CaMKII is present in DN cells, activation of this enzyme inhibits cytokine production (10), presumably because it phosphorylates two sites on CREB, and the second site (SER-142) is both dominant and inhibitory (19). Of the other pathways known to be responsive to phorbol ester and ionophore, protein kinase C does not phosphorylate CREB on Ser-133. However, this combination of agents does stimulate signaling through the MAP kinase cascade, and this cascade has been suggested to be Ca2+ sensitive because it is impacted by the CaMKIV pathway. Enslen et al. (44) show that the activation of the CaMKIV cascade can, in turn, activate the MAP kinases ERK-2, JNK-1, and p38 in NG108 cells. This pathway leads to the transcription of Elk-1, c-jun, and ATF-2. Chen et al. (48) have generated mice deficient in c-jun and find that there is a marked decrease in thymic cellularity, a phenotype in common with the mice that express mutant hCaMKIV. Finally, The MAP kinase pathway has been shown to be involved in the differentiation from DN to DP T cells (49). Perhaps, the presence of mutant hCaMKIV prevents the activation of the MAP kinase pathway in DN cells even though CaMKIV is not normally a component of these cells. One could envision that binding of mutant hCaMKIV to a component of this cascade could prevent its activation by another mechanism. This could result in a dominant/negative effect that would be independent of the activation of CREB by CaMKIV. At any rate, many of these suggestions may be tested experimentally, and the availability of transgenic mice expressing mutant hCaMKIV should provide a very useful model system for future research.
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MATERIALS AND METHODS |
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Transgene Construction and Generation of Mice
Targeted expression of the transgenes to murine thymic T cells
was accomplished by utilizing the shuttle vector p1017 [a gift from R.
Perlmutter, University of Washington, Seattle] (25). This vector
contains the sequence for the mouse proximal lck promoter
plus additional sequence derived from the human GH gene (hGH) and has
been shown to initiate high level expression of cDNAs specifically in
thymic T cells of transgenic mice (27). A 1.5-kb cDNA for the human
form of Ca2+/calmodulin-dependent protein kinase type IV
(hCaMKIV) that had been mutated to result in a catalytically inactive
protein (23) and a 0.5-kb cDNA for the rat form of calspermin (29) were
isolated from plasmids by the appropriate restriction digests and
subcloned separately into a BamHI site between the
lck promoter and the hGH sequences of the p1017 vector as
represented diagrammatically in Fig. 1A. After confirming that the
cDNAs were in the correct orientation by DNA sequencing, the transgenes
were linearized and isolated by restriction digestion with
NotI (Boehringer Mannheim, Indiana, IN) followed by
electrophoresis in a 1% agarose gel (FMC Corp., Rockland, ME). Both
transgenes were purified from the agarose using Prep-a-Gene (Bio-Rad,
Richmond, CA), aliquoted in 10 mM Tris and 0.1
mM EDTA buffer and stored at -20 C for subsequent
microinjection.
B6SLF1/J mice (Jackson Laboratories, Bar Harbor, ME) were used to generate the transgenic mice, because they produce excellent yields of viable eggs for microinjection (C. Bock, personal communication). Ovulation of female mice, male stud service, collection of eggs, microinjection of DNA, and reimplantation of fertilized eggs were done following standard methods previously described (50) and was performed by the Duke Comprehensive Cancer Center Transgenic Core Facility. Mice that expressed the correct DNA fragment as determined by Southern blot analysis were then bred to nontransgenic B6SLF1/J mice to establish viable lines for analysis. All mice were housed at the Duke Cancer Center Isolation Facility under a 12-h light, 12-h dark cycle. Food and water were provided ad libitum, and all care given to the mice was in compliance with the NIH guidelines for the care and use of laboratory animals.
DNA and RNA Analysis
Genotyping of founder mice and subsequent generations of mice
was done by Southern blot analysis as previously described (51). In
this procedure, 10 µg genomic DNA were digested with SpeI
(Boehringer Mannheim) for 12 h at 37 C electrophoresed through a
1% agarose gel and blotted onto Zeta-Probe membranes (Bio-Rad,
Richmond, CA). 32P randomly labeled probes were used to
confirm the presence of the transgene and to ascertain copy number. For
the lck-hCaMKIVkin(-) mice a 500 bp
BamHI-BglII fragment derived from the hCaMKIV
cDNA 3'-end was used as the probe. In the case of the
lck-calspermin transgene, we used the entire 522-bp cDNA
encoding rat calspermin. It should be noted that the SpeI
restriction site used for Southern analysis was engineered into the
p1017 plasmid at both the extreme 5'- and 3'-ends (25, 27) and does not
exist in the wild type locus for either CaMKIV or calspermin. Hence,
while the cDNA probes also hybridized to the wild type locus, only a
single band was detected in Southern blots because the wild type band
was substantially larger than the transgene. Total RNA was isolated
from tissue after extraction with Utraspec total RNA isolation reagent
(Biotex Laboratories, Houston, TX). RNA was subjected to
electrophoresis through 1.5% agarose-formaldehyde gels (5 µg/lane)
and blotted onto Zeta-Probe membranes (Bio-Rad, Richmond, CA). RNA
blots were then hybridized to a 32P randomly labeled cDNA
fragment corresponding to the appropriate mRNA. The Fos B probe was
made from a l.6-kb HindII fragment of murine Fos B. The
GAPDH probe was from a 780-bp PstI/XbaI fragment
of human GAPDH. Hybridization was carried out in 500 mM
sodium phosphate (pH 7.2), 7% SDS, and 1 mM EDTA at 65 C
for 18 h. Washes were done at 65 C in 40 mM sodium
phosphate and 1% SDS until background level radioactivity was
undetectable. Visualization of blots was accomplished by exposure of
the blot to Kodak XAR film (Eastman Kodak, Rochester, NY) at -70 C for
up to 24 h.
Primary Cell Culture
Primary cultures of thymic or splenic T cells were established
from mice between the ages of 21 and 30 days of age for all
experiments. Mice were killed by cervical dislocation, and the thymus
or the spleen was harvested. Cells were teased from the organs by
placing the organs between two pieces of 40xx nytex nylon mesh (Tetko,
Kansas City, MO) submerged in RPMI-1640 (GIBCO) supplemented with 10%
FBS, 0.5% penicillin, 0.5% streptomycin, 1x nonessential amino
acids, and 0.001 M 2-mercaptoethanol at 4 C and gently
pushing down on top of the mesh to rupture the organs. The cells were
collected from the supernatant fluid, and the remaining tissue was
removed and discarded. This procedure resulted in a T cell purity of
>95% from the thymus as assessed by flow cytometry. Splenic T cells
were differentiated from B cells based upon the size differential when
counting by trypan blue exclusion compared with T cells present in the
thymus. This method yielded consistent results, and flow cytometry was
employed to confirm the presence of equal numbers of T cells. After two
successive washes in RPMI-1640 media, the cells were counted on a
hemocytometer by trypan blue exclusion to determine both the number of
cells as well as the fraction representing live cells before aliquoting
them for experiments.
Survival assays were carried out on aliquots of cells from spleen or thymus. Cells were collected by centrifugation at 1000 rpm for 5 min at 4 C and resuspended in DMEM (GIBCO) supplemented with 10% FBS, 0.5% penicillin, and 0.5% streptomycin. Cells were then recounted on a hemocytometer by trypan blue exclusion, and 4 x 106 live cells were plated in pentuplicate in 24-well Falcon brand plates (Becton Dickinson, San Jose, CA) containing 2 ml DMEM. The cells were then incubated at 37 C in 95% O2/5% CO2. After 30 h the cells were harvested and counted on a hemocytometer by trypan blue exclusion to determine the rate of survival.
Fluorescence-Activated Cell Sorter (FACS) Analysis
All FACS analyses were carried out at the Duke Comprehensive
Cancer Center Flow Cytometry Core Facility. Experiments were analyzed
within 24 h after completion using a Becton-Dickinson model
FACScan with data analysis done using the Cell Quest program
(Becton-Dickinson).
CD4/CD8 determinations for thymic and splenic T cells were carried out as previously described (52). Briefly, 1 x 106 T cells were placed into 10 x 75 mm tubes (Becton Dickinson) on ice. The cells were washed two times with PBS containing 5% FBS (FACS buffer). The supernatant fluid was aspirated and 100 µl FACS buffer were added containing either PE-conjugated anti-CD4 (diluted 1:50) or FITC-conjugated anti-CD8 (diluted 1:50) or both phycoerythrin (PE)-CD4 and FITC-CD8. The reaction was allowed to proceed 20 min at 4 C before the reaction was terminated by the addition of fresh FACS buffer. Cells were then briefly washed with fresh FACS buffer and pelleted at 1000 rpm two times. The cells were then fixed with PBS containing 1% formaldehyde.
Quantification of the murine IL-2 receptor on T cells was accomplished
by taking an aliquot of 4 x 106 live cells, dividing
them in half, and plating the paired samples containing 2 x
106 live cells each into 24- well Falcon Brand plates
(Becton-Dickinson) containing fully supplemented RPMI-1640. The final
volume of each well was then adjusted to 2 ml. One half of each paired
cell sample was left unstimulated while the other half was stimulated
with PMA (10 ng/ml) and ionomycin (0.5 µg/ml). After 30 h the
cells were harvested, washed in FACS buffer as described above, and
immunostained for FACS analysis using FITC-conjugated anti-CD25
antibody (GIBCO) which recognizes the IL-2 receptor -chain.
Protein and Cytokine Analysis
Detection of proteins on Western blot was accomplished by either
standard radioactivity methods or by chemiluminescence. Expression of
protein from the transgene was examined by first extracting protein
from 5 x 106 T cells isolated from the thymus or
spleen in Laemmli buffer (53). These protein samples were then heated
to 95 C, loaded onto a 10% polyacrylamide SDS-containing gel, and run
at 100 V for 2 h or until the dye front reached the bottom. The
protein was transferred electrophoretically to Immobilon Membranes
(Millipore, Bedford, MA). Membranes were blocked for 1 h at room
temperature using Tris-buffered saline (TBS) with 5% dried milk and
0.01% Tween 20. Application of the primary antibody for either hCaMKIV
or calspermin was then carried out for 2 h at room temperature in
TBS followed by six washes in TBS containing 0.01% Tween 20 (TBSt)
lasting 10 min each; 1 x 106 cpm/ml of
[125I] protein-A was then applied for 1 h in TBS
containing 5% dried milk and 0.01% Tween 20 followed by six washes in
TBSt for 10 min each. Blots were exposed on Kodak XAR film overnight
for visualization. CREB and phospho-CREB detection was accomplished by
stimulating 5 x 106 cells with PMA (10 ng/ml) and
ionomycin (0.5 µg/ml) or anti--CD3 monoclonal antibody (16
µg/ml) that had been immobilized to 24-well tissue culture plates at
37 C in 95% O2/5% CO2. At various time
points, T cells were harvested and lysed in Laemmli buffer as described
previously. Proteins were then fractionated by SDS-PAGE on a 10% gel
and subjected to Western blotting as described above using commercial
antibodies from Upstate Biotechnology Inc. ECL chemiluminescent
detection (Amersham) was substituted for [125I] protein-A
following the established manufacturers protocols. Blots were
quantified by scanning densitometry on a 445 SI PhosphorImager equipped
with a personal densitomiter SI (Molecular Dynamics, Sunnyvale, CA).
Digital analysis of the scans was completed using the program Image
Quant v1.1 (Molecular Dynamics).
To measure production of IL-2, 2 x 105 T cells were
placed into a flat bottom 96-well plate (Corning-Costar, Cambridge, MA)
containing the RPMI-1640 media previously described. Well volumes were
then adjusted to 250 µl, and the cells were stimulated at 37 C in a
humidified chamber with PMA (10 ng/ml) and ionomycin (0.5 µg/ml) or
16 µg/ml of the anti--CD3 monoclonal antibody that had been
immobilized onto the plate. After 48 h the media were removed for
analysis. ELISA measurements were done following the standard protocol
distributed with the ELISA components (Pharmingen, San Diego, CA) for
measurement of IL-2. Plates were then quantified on a Multiskan MS
96-well plate reader (Lab Systems, Needham Heights, MA)
Statistical Analysis
Initial statistics were performed by a two-way ANOVA utilizing a
95% confidence level. Post-hoc analysis was completed using either the
Student-Newman-Keuls test when analyzing data from all three types of
mice or a two-tailed Students t test for data derived from
just two types of mice. All data are presented as the mean ±
SD. A level of significance was accepted when
P < 0.05
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These authors contributed equally to this work.
This work was supported by NIH Grant HD-07503 (to A.R.M.). K.A.A. is recipient of a National Research Service Award from the NIH, and M.I. is recipient of a fellowship from Federico II University, Naples, Italy.
Received for publication February 27, 1997. Revision received March 18, 1997. Accepted for publication March 19, 1997.
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REFERENCES |
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