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R. W. Johnson Pharmaceutical Research Institute, San Diego, CA 92121
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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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In this study, we investigated the requirement for LFA-1 in T cell activation and effector function, using CD8+ T cells isolated from LFA-1 knockout mice that are also transgenic for the 2C TCR (23, 29). Our data indicate that in this model system, LFA-1 plays a critical role during T cell activation and proliferation, and that it is required for efficient cytolysis of target cells by differentiated CTLs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2C TCR transgenic mice and gene-targeted mice lacking cell surface expression of LFA-1 have been described previously (23, 29, 30). The two strains were crossed and maintained under specific pathogen-free conditions. Other mouse strains used, BALB/cByJ (BALB/c), C57BL/6J-H2bm3/Eg (bm3), B6.C-H2bm11/KhEg (bm11), and C57BL/6J (B6), were obtained from The Jackson Laboratory (Bar Harbor, ME).
Media
All proliferation and cytolytic assays were performed using RPMI 1640 (Life Technologies, Gaithersburg, MD) with 20% heat-inactivated FCS, supplemented with L-glutamine, penicillin, streptomycin, and 50 µM 2-ME at 37°C in the presence of 5% CO2. Cell lines used as cytolytic targets were maintained in exponential growth phase in similar medium, but containing only 10% FCS.
Cell lines
P815 mastocytoma cells and EL-4 thymoma cells were obtained from American Type Culture Collection (Manassas, VA). RMA-S.Ld cell line, a transfectant of the parental RMA-S line expressing the MHC class I molecule Ld, was kindly provided by Dr. Zeling Cai (R. W. Johnson Pharmaceutical Research Institute, La Jolla, CA).
Peptides
Peptides p2Ca (LSPFPFDL), QL9 (QLSPFPFDL), dEV-8 (EQYKFYSV), and SIYR (SIYRYYGL) were synthesized on a 431 A synthesizer (Applied Biosystems, Foster City, CA) and purified with C18 reverse-phase HPLC at the peptide laboratory of The R. W. Johnson Pharmaceutical Research Institute. Peptide concentrations were determined by quantitative amino acid analysis.
Purification of CD8+ cells
Single cell suspensions prepared from murine spleens and lymph nodes were treated with a mixture of mAbs plus complement for 60 min at 37°C, essentially as described (31). The following mAbs were used in the Ab mixture: RL172 (anti-CD4), J11d (anti-HSA), and 28-16-8s (anti-I-Ab).
Proliferation assays
Purified nontransgenic CD8+ T cells from LFA-1+/- or LFA-1-/- mice (H-2b) were cultured with irradiated (2000 rad) allogeneic BALB/c (H-2d) or syngeneic B6 splenocytes in round-bottom 96-well plates at 200 µl/well. Responder cells, maximally 2 x 105/well, were 2-fold serially diluted. Stimulator cells were plated at 5 x 105/well. Three days later, a 50-µl aliquot of supernatant was replaced in each well with medium containing 1 µCi of [3H]thymidine. Following a 6-h incubation, proliferation was measured by [3H]thymidine incorporation using a Topcount Microplate Scintillation Counter (Packard, Downers Grove, IL). For studies of proliferation kinetics, CD8+ LFA-1+/- or LFA-1-/- 2C T cells were plated at 1.25 x 104 cells/well with 2.5 x 105 cells/well irradiated allogeneic BALB/c, bm3, or bm11 splenocytes in round-bottom 96-well plates at 200 µl/well. Peptide pulsing was performed by preincubating stimulators with 10 µM antigenic peptides at 25°C for 2 h, and then cells were plated out without removal of peptides. In some cultures, exogenous human IL-2 was supplemented at a final concentration of 20 U/ml. On days 2 to 7, 2C T cell proliferation was assessed by [3H]thymidine incorporation, as outlined above.
IL-2 production
IL-2 levels in supernatants of 2C T cell cultures were assessed using the commercially available IL-2 ELISA kit (Genzyme, Cambridge, MA).
Generation of CTLs and 51Cr release assays
CTL effectors were generated in 10-ml cultures of six-well plates by culturing CD8+ LFA-1+/- or LFA-1-/- 2C T cells at 5 x 105/ml with irradiated (2000 rad) BALB/c splenocytes at 2.5 x 106/ml for 4 days. To maintain their viability, LFA-1+/- T cells were diluted 2-fold on day 3 with fresh medium supplemented with human IL-2 at a final concentration of 20 U/ml. Some cultures of LFA-1-/- 2C T cells were supplemented with 20 U/ml of human IL-2 at the onset of stimulation. Some cultures of LFA-1-/- 2C T cells were supplemented with 20 U/ml of human IL-2 as well as pretreated with 10 µM QL9 peptide, as described above. On the day of the cytolytic assays, target cells were labeled with 51Cr (100 µCi per 1–2 x 106 cells) at 37°C for 90 min. P815 mastocytoma (H-2d) and EL-4 thymoma (H-2b) target cells were then incubated with or without 10 µM QL9 or SIYR peptide, respectively. These targets were plated at 104 cells/well mixed with serially diluted effector cells in round-bottom 96-well plates in a total volume of 200 µl/well. E:T ratios were maximally 100:1, with 3-fold serial dilutions. Anti-LFA-1 mAb M17/4 (PharMingen, San Diego, CA) was used at a final concentration of 2.5 µg/ml. 51Cr-labeled RMA-S.Ld target cells were plated at 104 cells/well onto round-bottom 96-well plates containing varying concentrations of QL9 peptide. Following this treatment, effector cells were added at 105 cells/well to obtain an E:T ratio of 10:1 in a final volume of 200 µl/well. All plates were centrifuged for 5 min at 150 x g before a 4-h incubation at 37°C. Subsequently, plates were centrifuged for 5 min at 300 x g, and the radioactivity in the supernatants was counted using a gamma counter. Maximum lysis was obtained by counting detergent-lysed target cells; spontaneous release was obtained by incubating target cells in the absence of effector cells. Percent specific lysis was calculated as [(measured 51Cr release - spontaneous 51Cr release)/(maximum 51Cr release - spontaneous 51Cr release)] x 100.
Flow-cytometric analysis
For analysis of cell surface expression of T cell-specific or activation markers, 106 cells were stained with mAb for 30 min at 4°C in 100 µl of PBS containing 2% FCS and 0.1% sodium azide. Cells were then washed, and live cells (propidium iodide negative) were analyzed on a FACScan (Becton Dickinson, San Jose, CA). mAbs used were specific for CD8, CD11a, CD25, CD69 (PharMingen), or the transgenic 2C TCR (clonotypic mAb 1B2) (32).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bulk cultures of LFA-1-deficient splenocytes, LN cells, and
enriched T cells have been shown to exhibit defective T cell
proliferation in response to alloantigen stimulation (23, 27). To determine whether and to what extent
CD8+ T cells contribute to this defect, the
capacity of purified CD8+ T cells from
LFA-1-/- mice to respond to alloantigens was
assessed in proliferation assays. LFA-1+/- mice
that were littermates of the LFA-1-/- mice were
used as controls in our studies, because prior analysis of the
heterozygous phenotype revealed that it was not significantly different
from that of wild-type cells (23).
CD8+ T cells from either
LFA-1+/- or LFA-1-/-
mice were enriched by complement depletion of
CD4+ T cells and B cells. Enriched
CD8+ T cells from either
LFA-1+/- or LFA-1-/-
mice (H-2b) were stimulated with irradiated
allogeneic BALB/c splenocytes (H-2d).
Proliferation was assessed 3 days later by
[3H]thymidine incorporation. As shown in Fig. 1
, LFA-1-/-
CD8+ T cells exhibited a profound reduction in
their ability to proliferate at all cell densities tested, in contrast
to the prominent proliferation observed in control
LFA-1+/- CD8+ T cells. The
result suggests an important role for LFA-1 in
CD8+ T cell activation.
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To investigate the role of LFA-1 in CD8+ T
cell activation, LFA-1 knockout mice were bred onto the 2C transgenic
TCR background (29, 30) to obtain mice expressing a
monoclonal TCR in the CD8+ T cell population.
Clonotypic CD8+ T cells isolated from
LFA-1-expressing or LFA-1-deficient 2C TCR transgenic mice were then
used in all subsequent in vitro proliferation and cytolytic studies.
The 2C TCR specificity and its interaction with the peptide/MHC class I
complex have been characterized substantially by others. 2C
clonotype-positive CD8+ T cells display
alloreactivity to Ld and certain mutated
Kb variants (29, 30, 33). The 8-mer
peptide p2Ca, derived from the Krebs cycle enzyme 2-oxoglutarate
dehydrogenase, is the predominant endogenous peptide recognized by the
2C TCR when complexed to Ld
(34, 35, 36, 37, 38). p2Ca has an intermediate affinity for
Ld (Kd 
4 x 106 M-1) and,
when complexed to Ld, high affinity for the 2C
TCR (Kd 2 x
106 M-1). The 9-mer QL9, a
synthetic derivative of p2Ca that is extended at the amino terminus by
one amino acid, has very high binding affinity for
Ld (Kd
4 x 108 M-1) and,
when bound, is recognized by 2C TCR with high affinity
(Kd 2 x
107 M-1).
Kbm3 and Kbm11 are two of
the Kb mutants that are alloantigenic for 2C TCR
(31, 33). The 8-mer dEV-8, most likely derived from
another Krebs cycle enzyme termed MLRQ, is known to be the peptide
presented by Kbm3 that generates an alloresponse
(39). The 8-mer SIYR, which was identified from a
synthetic peptide library, effectively renders 2C reactive to
Kb (38).
We routinely obtained LFA-1+/- and
LFA-1-/- T cell populations in which 85% of
the cells or more expressed the clonotypic 1B2 TCR as well as CD8 (Fig. 2). 2C T cells were activated by plating
at 1.25 x 104 cells/well with irradiated
allogeneic splenocytes at 2.5 x 105
cells/well in 96-well plates. Proliferation of 2C T cells was measured
by [3H]thymidine incorporation daily from day 2
through 7. LFA-1+/- 2C T cells responded
maximally on day 3, with the greatest proliferation (254,820 cpm) when
exposed to BALB/c splenocytes pulsed with QL9 (Fig. 3A). In contrast, minimal
[3H]thymidine uptake by
LFA-1-/- 2C T cells was indicative of the
severely diminished proliferative response toward BALB/c stimulators.
Although this proliferation (810 cpm) was above that seen with
syngeneic stimulators (<400 cpm), it never attained a significant
level, even following peptide pulsing of stimulators with either p2Ca
or QL9. When plated at a higher cell density (5 x
104 cells/well), LFA-1-/-
2C T cells underwent a slightly increased proliferation on day 3
(13,000 cpm), but the enhancement by QL9 pulsing was still minimal
(27,109 cpm). Consistent with the defective proliferation, IL-2 levels
in culture supernatants of LFA-1-/- cells
remained low throughout day 2–4 of proliferation (Table I). With the addition of exogenous IL-2
(20 U/ml), LFA-1-/- 2C T cells did mount a
considerable proliferative response toward BALB/c stimulators (Fig. 3A). The magnitude and timing of peak proliferation appear
to correlate with the TCR avidity for the peptide/MHC complex. Without
peptide loading, LFA-1-/- alloresponse was
maximal on day 5, but less than a quarter of that seen on day 4 for
control cells. When pulsed with 10 µM p2Ca peptide, the response was
approximately half that of control cells on day 3 and also delayed by 1
day. With the high affinity QL9 peptide, LFA-1-deficient 2C T cells
proliferated as vigorously and as rapidly as control responders, but
the duration of the response was not sustained for the additional day.
Proliferation studies using APCs from bm3 or bm11 strains of mice
pulsed with dEV-8 peptide or supplemented with IL-2 give complementary
results (Fig. 3, B and C).
LFA-1-/- 2C T cells again could not mount a
significant proliferative response in the absence of exogenous IL-2,
and only a compromised response with the addition of IL-2,
characterized by reduced and delayed proliferation. Peptide pulsing
alone had no impact on proliferation of
LFA-1-/- 2C T cells, but did potentiate the
effects of IL-2. Taken together, these results indicate that LFA-1 is
essential for normal T cell activation and proliferation in response to
alloantigens. Furthermore, increased TCR signaling by pulsing APCs with
high affinity peptides does not significantly reconstitute normal
proliferation of LFA-1-deficient T cells in the absence of exogenous
IL-2, and only partially rectifies defective proliferation with IL-2
treatment.
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Alloantigen-stimulated T cells acquire a blasting phenotype with
characteristically enlarged cell size, a polarized shape, and increased
expression of various activation markers. To evaluate the extent and
kinetics of T cell activation in the absence of LFA-1, we assessed the
expression of a number of cell surface activation Ags on
LFA-1-deficient 2C T cells over the course of 3 days following
stimulation with allogeneic BALB/c splenocytes (Fig. 4). Within 24 h of stimulation,
50% of CD8+ LFA-1+/-
2C T cells expressed high levels of activation markers CD25 and CD69.
Expression of these markers peaked on day 2 with 75% of cells
showing high levels, and subsided somewhat by day 3. This expression
profile was not significantly altered in the presence of exogenous
IL-2. LFA-1-/- 2C T cells exhibited a delay in
expression of these markers, but by day 3, attained levels comparable
with those of control cells. Indeed, at this time point, the two
populations of T cells were also qualitatively similar in several other
phenotypic markers, including CD8, CD28, CD44, CD62L, CD71, and CD95
(data not shown). Addition of IL-2 to LFA-1-deficient cells at the
onset of stimulation also did not affect the level and the delayed
kinetics of expression of these CD Ags, even though the extent of the
proliferation of these cells was greatly enhanced.
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Our previous study revealed that following MLC of bulk
splenocytes, LFA-1-deficient CD8+ effector cells
exhibited impaired CTL activity (23). We therefore studied
the effector function of LFA-1-/- 2C T cells to
investigate the role of LFA-1 in cytolytic effector function. CTLs were
generated by culturing CD8+ 2C T cells at 5
x 105 cells/ml with irradiated BALB/c
splenocytes at 2.5 x 106 cells/ml, similar
to the setups in flow cytometry studies in the preceding section.
LFA-1-/- 2C T cells, activated with or without
IL-2, were used as effector cells. Following 4 days of stimulation,
cytotoxic activity of T cells toward allogeneic P815 mastocytoma cell
targets was assessed using a 51Cr release assay.
LFA-1-/- 2C effectors generated in the absence
of exogenous IL-2 exhibited a barely perceptible cytolytic activity
toward allogeneic target cells at 100:1 E:T ratio (Fig. 5). Target cell lysis was somewhat
improved when the CTLs were generated in the presence of exogenous
IL-2. In contrast, control LFA-1+/- effectors
exhibited a highly efficient cytolytic capability, even at low E:T
ratios. Pulsing of target cells with 10 µM QL9 peptide before the
51Cr release assay enhanced the ability of both
untreated and IL-2-treated LFA-1-/- 2C cells to
lyse P815 targets, but their lytic activity remained far below that of
LFA-1+/- cells for the same E:T ratios. These
results indicate that the cytolytic activity of
LFA-1-/- 2C CTLs can be improved by IL-2
supplementation during their generation, by high affinity peptide
pulsing of target cells, and synergistically by both. However, even the
combination of the two treatments results in
LFA-1-/- CTLs that exhibit a severely
compromised effector function.
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To determine whether increased TCR signaling during activation
would enhance cytolytic activity of LFA-1-/- T
cells, 51Cr release assays were also performed
with LFA-1-deficient CTLs that were generated in response to QL9-pulsed
BALB/c splenocytes. CTL activities of IL-2-supplemented
LFA-1-/- effectors generated by either pulsed
or nonpulsed stimulators were equally defective toward P815 targets
(Fig. 6). Furthermore, both sets of
effectors exhibited an identical increase in cytolytic activity toward
QL9-pulsed targets, which still remained substantially below that
of control LFA-1+/- CTLs. This indicates that
addition of exogenous IL-2 alone is sufficient to drive
LFA-1-/- CTL generation, and that increased TCR
signaling due to increased TCR/MHC interaction cannot induce
functionally more competent LFA-1-deficient CTLs.
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The reduced cytolytic activity observed for LFA-1-deficient CTLs
could originate from defective differentiation of T cells into
competent effector cells, or impaired cytolytic function of effector
cells, or both. To address this issue, we performed
51Cr release assays in the presence of the
LFA-1-blocking mAb M17/4. As expected, the two
LFA-1-/- CTL populations (generated with and
without QL9 pulsing of stimulators in the presence of exogenous IL-2)
were unaffected by the addition of the mAb (Fig. 7). In contrast, control
LFA-1+/- cytolytic activity was reduced to the
level of LFA-1-/- effectors in lysing p815
target cells pulsed with or without the high affinity peptide QL9.
These results suggest that the addition of IL-2 during alloantigen
stimulation of LFA-1-/- 2C T cells will result
in adequately differentiated and armed CTL effectors whose defective
cytolytic activity most likely results from the absence of LFA-1
function during target cell lysis.
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The extent to which exogenously administered peptide can be loaded
onto MHC molecules is largely dependent on its ability to displace the
endogenous peptide repertoire. Consequently, it is difficult to
estimate the level of exogenous Ag presentation by typical APCs.
Controlled loading of peptides can be attained through the use of
TAP-2-deficient RMA-S cells that are unable to load intracellular
peptides onto class I molecules. This results in newly synthesized
class I molecules that are empty, and while they do reach the cell
surface, especially at lower temperature, they are unstable and rapidly
fall apart unless stabilized with exogenously applied peptides
(40, 41). We used an Ld-transfected
RMA-S cell line pulsed with various concentrations of QL9 peptide as
lytic targets for 2C effectors to determine whether high ligand density
abrogates the requirement for LFA-1. LFA-1+/- 2C
CTLs achieved a 50% lysis of RMA-S targets at a peptide concentration
of 0.1 µM (Fig. 8). This percentage was
maintained over a 2-log increase of QL9 concentration, indicating that
the target cells had reached a maximal level of peptide/MHC
presentation. The lower susceptibility of these cells to lysis compared
with P815 targets may stem from differences in cell surface expression
of Ld and/or lower levels of costimulatory
molecules. In contrast, maximal cytolytic activity of
LFA-1-/- 2C effectors was 10% or less of
target cells, even when the targets were loaded with 10 µM QL9
peptide. Clearly, high affinity peptides, at a concentration as high as
100 times that needed for maximum cytolytic function of
LFA-1-expressing effector cells, cannot rectify the cytolytic defects
in LFA-1-/- CTLs.
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To determine whether LFA-1 deficiency also critically affects the
ability of CTLs to lyse self targets presenting foreign epitopes,
effector cells generated against BALB/c stimulators were used in
51Cr release assays with syngeneic EL-4 targets
that had been pulsed with 10 µM concentration of the synthetic
peptide SIYR. This peptide effectively renders 2C TCR reactive to
Kb (38). Forty to 50% of the
maximal lysis of EL-4 targets was attained by
CD8+ T cells expressing LFA-1 at E:T ratios of
10:1 (Fig. 9). In contrast,
LFA-1-/- 2C effectors failed to appreciably
lyse any significant level of SIYR-pulsed EL-4 cells, even at the
highest ratio tested. Generation of LFA-1-/- 2C
effectors with QL9-pulsed stimulators did not improve their cytolytic
capacity. These results indicate that the
LFA-1-/- 2C CTLs are defective in their
cytolytic ability toward a wide range of target cell types.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytolytic performance of LFA-1-/- CTLs generated in the absence of exogenous IL-2 was found to be virtually absent even at high E:T ratios. The same cells when treated with exogenous IL-2 at the onset of alloantigen stimulation exhibited a somewhat increased but still poor CTL activity. Antigenic peptide loading of target cells greatly improved cytolytic activities of LFA-1-deficient CTLs, indicating the presence of at least some functional CTLs that had undergone sufficient activation, differentiation, and acquisition of cytolytic capability. The reduced cytolytic activity seen in the LFA-1-/- CTLs could be due to incomplete differentiation of T cells into effector CTLs, or defective cytolytic function of effector cells in the absence of LFA-1. Our data support the latter possibility, because cytolytic activities of wild-type CTLs decreased to levels comparable with those of LFA-1-/- CTLs when blocked with an anti-LFA-1 mAb. Furthermore, activation markers displayed by LFA-1-/- T cells by day 3 of stimulation were comparable with that of wild-type T cells. We also showed that IL-2 supplementation alone at the onset of alloantigen stimulation is sufficient to propel LFA-1-deficient CD8+ T cells into functionally effective CTLs. Antigenic peptide pulsing of stimulators during the generation of LFA-1-/- CTLs did not further improve their cytolytic activity above that attained with IL-2 supplementation, suggesting that TCR signaling in LFA-1-/- cells is adequate for CTL differentiation when supplemented with exogenous IL-2. Thus, increase in TCR signaling by peptide pulsing enhances T cell proliferation, but does not further improve differentiation of LFA-1-/- T cells into effector CTLs. Concomitantly, increased peptide/MHC density on P815 target cells enhances their susceptibility to lysis by LFA-1-/- effectors, but does not entirely restore normal CTL function.
Our results fundamentally differ from the conclusions reached by Bachmann et al. (28), but are in agreement with an earlier report (26) regarding the role of LFA-1 during T cell activation as well as effector function. We have demonstrated in this study a critical requirement for LFA-1 in T cell activation and cytolytic function, which could not be fully compensated by increased TCR signaling resulted from peptide pulsing. Using LFA-1-deficient mice on a different transgenic TCR background, Bachmann et al. showed that defective T cell activation and target cell lysis due to the absence of LFA-1 were apparent only at low Ag peptide concentrations, and defects could be corrected at high peptide concentrations (10 µM). Bachmann et al. therefore concluded that LFA-1 does not play a costimulatory role, but facilitates TCR signaling by promoting T cell/APC adhesion. The use of different transgenic TCR models on the LFA-1 knockout background, our 2C TCR system vs the TCR specific for a viral LCMV glycoprotein studied by Bachmann et al. (28), possibly is the major source of the discordant observations. Inherently, our model system of T cell activation involved alloantigenic stimulation, in contrast to antigenic peptide being presented by syngeneic APCs. In our studies, pulsing of stimulators or cytolytic targets with peptides did not rectify the defective proliferation and cytolysis by LFA-1-/- T cells. All three alloantigenic stimulators (BALB/c, bm3, and bm11 splenocytes) elicited similarly defective LFA-1-/- proliferative responses, and all three different target cells (P815, RMA-S.Ld, and EL-4) were poorly lysed by LFA-1-/- CTLs. Our use of EL-4 targets pulsed with SIYR peptide is reminiscent of the in vitro experimental setup of Bachmann et al., in which EL-4 cells were pulsed with the LCMV peptide. In our studies, pulsing of EL-4 targets with comparable concentrations of the respective peptides did not reconstitute cytolytic activities of LFA-1-/- CTLs.
The major finding presented in this work is that in the 2C transgenic TCR system, LFA-1 is absolutely essential for a normal T cell immune response. First, LFA-1 is critically required during T cell activation in order for a potent proliferative response to occur. This requirement for LFA-1 can be partially circumvented by the addition of exogenous IL-2, but not by increased TCR signaling. Second, functionally differentiated CTLs require LFA-1 for efficient target cell lysis. This dependence on LFA-1 in cytolysis may be reduced but not eliminated by increased TCR signaling. Our results indicate that LFA-1 has a requisite functional role for optimal CD8+ T cell activation and effector function, which cannot be overcome entirely by TCR or IL-2-mediated signaling events.
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Acknowledgments |
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Footnotes |
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Received for publication March 5, 1999. Accepted for publication August 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Q.-W. Zhang, D. D. Kish, and R. L. Fairchild Absence of Allograft ICAM-1 Attenuates Alloantigen-Specific T Cell Priming, But Not Primed T Cell Trafficking into the Graft, to Mediate Acute Rejection J. Immunol., June 1, 2003; 170(11): 5530 - 5537. [Abstract] [Full Text] [PDF]
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 P. J. Kilshaw and J. M.G. Higgins Alpha E: No More Rejection? J. Exp. Med., October 7, 2002; 196(7): 873 - 875. [Full Text] [PDF]
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C. Abraham and J. Miller Molecular Mechanisms of IL-2 Gene Regulation Following Costimulation Through LFA-1 J. Immunol., November 1, 2001; 167(9): 5193 - 5201. [Abstract] [Full Text] [PDF]
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 D. Bouvard, C. Brakebusch, E. Gustafsson, A. Aszodi, T. Bengtsson, A. Berna, and R. Fassler Functional Consequences of Integrin Gene Mutations in Mice Circ. Res., July 30, 2001; 89(3): 211 - 223. [Abstract] [Full Text] [PDF]
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) T cells to allogeneic BALB/c or syngeneic
B6 stimulators were assessed on day 3. Data represent averages of
triplicates.
-chain (CD11a) was analyzed by flow cytometry.
Numbers represent the percentage of cells expressing high levels of
specified marker.
) or without (
