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Departments of
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Microbiology and Immunology and
Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß in the
absence of any potential costimulatory molecules. Using T cells from
two different TCR transgenic mice in vitro, we find that TCR engagement
by peptide/MHC tetramers is sufficient for the activation of naive
CD8+ T cells. Furthermore, these T cells proliferate,
produce cytokines, and differentiate into cytolytic effectors. Under
the conditions where anti-CD28 is able to enhance proliferation of
normal B6 CD4+, CD8+, and TCR transgenic
CD8+ T cells with anti-CD3, we see no effect of
anti-CD28 on proliferation induced by tetramers. The results of
this experiment argue that given a strong signal delivered through the
TCR by an authentic ligand, no costimulation is
required. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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alone) are able to
overcome the lack of signal 2. The data for CD8+ T cells regarding the requirement of costimulation for activation are limited and contradictory. In vivo studies of tumor rejection have demonstrated that nonimmunogenic tumor cells could induce protection against a subsequent challenge if the immunizing tumor cells were transfected with CD80 (21, 22, 23, 24, 25). Treatment of mice with CTLA-4/Ig fusion protein designed to interrupt the interaction of CD28 with its ligands prolonged the survival of xenogeneic islet transplants (26) and cardiac allografts and was found to induce donor-specific tolerance (27, 28).
Other experiments suggest that costimulation is not required for CD8 activation. Some CD80/CD86-expressing tumor cells do not induce anti-tumor immune responses upon immunization (29, 30, 31, 32). While CD28-deficient mice infected with vesicular stomatitis virus have impaired anti-virus Ab production, their primary CTL response against lymphocytic choriomeningitis virus (LCMV)3 is intact (17). CD28-deficient mice can still reject allografts (33). The disruption of the CD28 gene in NOD mice does not prevent T cell-mediated destruction of pancreatic ß-cells (34). Therefore, the role of a costimulatory signal in the activation of naive CD8+ cells remains inconclusive.
MHC class I tetramers are molecules comprised of four identical class I heavy chains, each bound to ß2-microglobulin, and a single peptide. The class I heavy chains are biotinylated and added to avidin, which forms a stable, but noncovalent, complex. These complexes bind to T cells bearing TCRs specific for the peptide/MHC complex in the tetramers (35). We used these class I tetramers to determine whether a costimulatory signal is required for the activation of naive CD8+ T cells. Naive CD8+ T cells isolated from two different TCR transgenic (tg) mice, P14 (specific for LCMV gp33–41) and HY-TCR, both restricted by H2Db, were stimulated with their cognate peptide/MHC tetramers. Our results demonstrate that naive CD8+ T cells could be activated by the engagement of TCR with only the correct peptide/MHC complexes. Following stimulation, these CD8+ T cells proliferated and differentiated into functional effector cells. The gp33-specific CTL activity of CD8+ cells activated in vitro with gp33/Db tetramers was comparable to that of spleen cells isolated from P14 TCR transgenic mice primed in vivo by LCMV infection. The activation of CD8+ T cells by tetramers required engagement of the CD8 coreceptor. Addition of anti-CD8 mAb in the cultures inhibited the activation of CD8+ T cells induced by tetramers.
The results from the present study indicate that engagement of TCR with peptide-MHC complex alone provides a sufficient signal for naive CD8+ cells to differentiate into cytotoxic effector cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) or Charles River (Raleigh, NC). Tg mice, (B6,D2-TgN(Tcr-LCMV)327Sdz), expressing the P14 TCR specific for gp33 peptide (aa 33–41 of the LCMV glycoprotein) restricted by H2Db (36) were obtained from The Jackson Laboratory and subsequently backcrossed an additional four times to C57BL/6J mice. These mice are referred to as P14 in this manuscript. B6-TgN(Tcr-HY) mice that carry a transgene specific for male Ag were obtained from the National Institute of Arthritis and Infectious Diseases via Taconic Laboratories (Germantown, NY). These mice are referred to as HY-TCR. After backcrossing to C57BL/6 four times, the TCR tg mice were further bred to C57BL/6J-rag-1tm1 recombinase-deficient mice purchased from The Jackson Laboratory (37) to produce mice that express only the transgenic TCR. In some experiments transgenic rag-/- were used, in others the backcross stock were used. No differences were seen between responses. All animals used in this study were maintained under specific pathogen-free conditions in the American Association of Laboratory Animal Care-accredited University of North Carolina Department of Laboratory Animal Medicine Facilities.
Peptides
LCMV gp33 peptide (KAVYNFATM), Y4A peptide (KAVANFATM), and HY peptide (KCSRNRQYL) were synthesized by the University of North Carolina microchemical facility, purified by HPLC, and tested for purity by mass spectroscopy.
Tetramer preparation
Recombinant protein was prepared as previously described by Garboczi et al. (38). The plasmid encoding the complete wild-type Db (provided by Dr. Stanley Nathenson, Albert Einstein College of Medicine, Bronx, NY) was modified to encode a BirA recognition sequence at the C-terminus (35). Later, another modified Db clone was provided by Dr. Altman (Emory College of Medicine, Atlanta, GA) that encodes a more efficient biotinylation signal for BirA. Proteins were expressed in Escherichia coli strain BL21-pLyss and induced with isopropyl ß-D-thiogalactoside (IPTG). Inclusion bodies were purified, and the heavy chain was folded with mouse ß2-microglobulin and the appropriate peptide. Peptide/Db complexes were purified by HPLC size exclusion chromatography. Purified complexes were biotinylated following the methods provided by the manufacturer (Avidity, Denver, CO). Excess biotin was removed by size exclusion chromatography using Sephadex G-25, and the complexes bound to PE-labeled avidin (Leinco, St. Louis, MO). The extent of biotinylation was determined by incubating the protein with streptavidin (Sigma, St. Louis, MO) and analyzing by SDS-PAGE without boiling or reducing agent. Properly biotinylated material supershifted with streptavidin on the gel. In some experiments the tetramers were further purified by size exclusion chromatography.
Purification of naive CD8+ TCR transgenic T cells from spleen
Cell suspensions were prepared from the spleens of TCR transgenic mice and RBC lysed with ACK lysis buffer (0.15 M NH4Cl4, 1 mM KHCO3, and 0.1 mM Na2EDTA in water). Cells were incubated at 37°C for 1 h in flasks to eliminate adherent cells before purification. CD8+ T cells were purified using the MACS magnetic separation system according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Cells were resuspended at a concentration of 107 cells/90 µl in PBS containing 0.5% FBS. CD4+ and MHC class II+ cells were depleted by incubating with 10 µl of anti-CD4 (clone GK1.5) and anti-MHC class II Ab-conjugated microbeads/107 cells (clone M5/114.15.2) at 4°C for 15 min. After incubation, cells were washed and resuspended at 2 x 108 cells/ml in PBS with 0.5% FBS. LS+/VS+ columns (Miltenyi Biotec) were used for the selection of CD8+ T cells. In our initial studies we found that CD8+ T cells selected positively with anti-CD8 mAb-conjugated beads did not proliferate in response to tetramer stimulation. Therefore, the CD8+ T cells used in all experiments were purified by depletion of other cells.
Proliferation assay
Purified CD8+ T cells (4 x 105) were stimulated with tetramers at different concentrations in 200 µl of RPMI 1640 medium plus 10% FBS, antibiotics, glutamine, and 50 µM 2-ME in 96-well flat-bottom plates. The cultures were incubated for 48 h, and 1 µCi [3H]thymidine was added to each well for the final 10 h of culture. Cells were harvested using a multiple sample harvester (Otto Hiller, Madison, WI), and incorporation of [3H]thymidine was measured by scintillation counting using a Beckman LS5000 counter (Palo Alto, CA). All data represent the average counts per minutes of duplicate determinations. All proliferation experiments were repeated at least three times.
Cytotoxicity assay
CTL activity was assessed in a standard 4-h
51Cr release assay as previously described
(39). Briefly, naive CD8+ T cells
were stimulated as described above with tetramers at the concentrations
indicated. Following stimulation for 48 h, cells were harvested,
washed, and used as effector cells. EL4 target cells
(H2b) were 51Cr labeled,
pulsed with different concentrations of peptide for 1 h at 37°C
in RPMI with 10% FBS, and washed. After 4-h incubation at 37°C in
5% CO2, with effector cells, the supernatant was
harvested on filters using a Skatron harvesting system (Skatron
Instruments, Sterling, VA), and 51Cr release was
counted in a Packard Cobra Auto
Counter (Downers Grove, IL). All
assays were performed in triplicate, the percent specific lysis was
calculated as follows: [(experimental release - spontaneous
release)/(maximum release - spontaneous release)] x 100.
Spontaneous release was defined as counts per minute released from
target cells in the absence of effector cells, and maximum release was
defined as counts per minute released from target cells lysed with
2.5% Triton X-100.
Flow cytometric analysis
The directly conjugated Abs used for cell surface staining in
this study, anti-CD4 (H129.19), anti-CD8 (53-6.7),
anti-CD25 (7D4), anti-CD69 (H1.2F3), anti-CD62L (MEL-14),
anti-V2 (B20.1), and anti-Vß8.1, 8.2 (MR5-2), were
purchased from PharMingen (San Diego, CA). Two- and three-color
stainings were performed using standard methods. List mode data were
collected and analyzed on a FACScan (Becton Dickinson, Mountain View,
CA) using Cyclops software (Cytomation, Ft. Collins, CO).
Cytokine production
Naive CD8+ T cells were stimulated with
various concentrations of tetramers as described above. Supernatants
were taken at 40 h, and the production of IL-2 and IFN- was
measured. IL-2 was measured using [3H]thymidine
uptake by the IL-2-dependent cell line CTLL-2. The assay was
standardized using recombinant murine IL-2 (PharMingen). IFN- levels
in supernatants were determined using an ELISA kit (R&D Systems,
Minneapolis, MN) according to the manufacturer’s instructions.
Costimulation assay
CD4+ and CD8+ T cells from the spleens of C57BL/6 mice and TCR transgenic CD8+ T cells from the spleens of P14 TCR transgenic mice were purified by negative selection as described above. The cells were resuspended at a concentration of 2 x 106 cells/ml in RPMI 1640 with 10% FBS, antibiotics, glutamine, and 50 µM 2-ME.
For costimulation experiments, 96-well flat-bottom microtiter plates were coated with 10 µg/ml of anti-CD28 mAb (37.51; PharMingen) alone or together with the indicated concentrations of anti-CD3 mAbs (145-2C11; PharMingen) in 50 µl of sodium bicarbonate buffer (0.1 M NaHCO3, pH 8.2) at 37°C for 2 h. Hamster IgG was used as control Ab. Following incubation, the plates were washed twice with PBS and blocked with complete RPMI 1640 with 10% of FBS. Cells (2 x 105) were added to each well in triplicate. The gp33/Db tetramers at various concentrations were added to stimulate TCR transgenic CD8+ cells along with anti-CD28. Cells were incubated at 37°C in 5% CO2 for 48 h. [3H]thymidine (1 µCi) was added to each well for the final 10 h of culture. Cells were harvested and counted as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the requirement of costimulation for the activation
of naive CD8+ T cells, mice transgenic for either
a TCR specific for LCMVgp33 or the HY male Ag were used as source of
naive CD8+ T cells. CD4+
and class II-expressing cells were depleted with microbeads conjugated
to anti-CD4 and anti-class II mAbs. The resulting cells were
analyzed by two-color staining with mAbs specific for CD8 and MHC class
II to determine the purity. As shown in Fig. 1
A, >93% of cells were
CD8+ and MHC class II negative. To determine the
activation status of the purified T cells, expression levels of the
early activation markers, CD25 and CD69, and the late activation
markers, CD44 and CD62L, were assessed by flow cytometry. In each
experiment the purified T cells were negative for CD69 and CD25 and
expressed a low level of CD44 but a high level of CD62L, consistent
with a naive phenotype (Fig. 1B). In addition, spleen cells
from unmanipulated P14 mice did not lyse gp33-pulsed target cells (Fig. 1C). Thus, there was no evidence that the cells were
preactivated in uninfected mice.
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To assess whether the activation of naive
CD8+ T cells requires a costimulatory signal,
naive CD8+ T cells from either P14 or HY TCR tg
mice were stimulated in vitro with H2Db tetramers
folded with gp33 peptide (gp33/Db) or a peptide
derived from HY male Ag (HY/Db).
CD8+ T cells proliferated vigorously in a
dose-dependent manner when stimulated with the appropriate tetramers
(Fig. 2). The proliferation, however, was
not observed when the same CD8+ T cells were
stimulated with the Db tetramers bound to a
control peptide. These results indicate that the interaction between
TCRs and soluble peptide/MHC tetramers can induce naive
CD8+ T cells to proliferate in a peptide-specific
manner. Furthermore, these results demonstrate that preparations of
different tetramers do not contain contaminants that nonspecifically
activate T cells.
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shows the pooled results of two
such experiments. After 48 h 48% of the P14 single cells
stimulated with gp33/Db had divided at least
once, while only 4% of the P14 T cells stimulated with
HY/Db had divided (p <
10-5). This experiment demonstrates that nothing
more than the T cell itself and the class I tetramer are required for
the induction of cell division.
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A, left panels).
They also increased cell surface expression of CD69 (Fig. 4A, right panels) and CD25 and CD44 as well as
reduced expression of CD62L (data not shown). Incubation with the
HY/Db tetramers did not induce blast formation of
the T cells specific for gp33 or increase the expression of these
activation markers.
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-chain of P14 tg TCR. TCR
expression was decreased with increasing concentration of tetramers
(Fig. 4
B). These results indicate that cells are activated
in a manner identical with cells activated with peptide-pulsed APC (not
shown). Costimulatory molecules are not required for naive CD8+ T cells to differentiate into cytotoxic effector cells
The fact that incubation with the appropriate peptide/MHC
tetramers results in increased expression of activation molecules,
decreased expression of TCR, and robust proliferation suggests that
naive CD8+ T cells can be activated without the
involvement of costimulatory molecules. We asked whether tetramer
stimulation induces naive CD8+ T cells to
differentiate into functional effector cells. Purified
CD8+ T cells from either P14 or HY TCR tg mice
were stimulated in vitro with gp33/Db or
HY/Db tetramers for 2 days, and CTL activity was
determined using a standard 51Cr release assay.
Fig. 5 shows that
CD8+ T cells activated by the cognate tetramer
could efficiently lyse target cells pulsed with the corresponding
peptide, but not those pulsed with the incorrect peptide. The CTL
activity of purified CD8+ T cells induced by
tetramer stimulation is comparable to that of peptide-stimulated whole
spleen cells in which professional APCs were present during activation
(data not shown). Similar lysis was seen in splenocytes from mice
infected with LCMV for 7 days (data not shown).
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We next tested whether the CD8 coreceptor is necessary for
activation of naive CD8+ T cells stimulated with
the gp33/Db tetramers. CD8+
T cells isolated from P14 TCR tg mice were stimulated with the
gp33/Db tetramers in the presence of anti-CD8
mAb, and proliferation was assayed. Addition of anti-CD8 mAb
inhibited the proliferation of naive CD8+ T cells
stimulated with the gp33/Db tetramers in a
dose-dependent manner (Fig. 6A). In addition, the
requirement for CD8 coreceptor by activated CD8+
T cells for cytotoxic function was assessed. As shown in Fig. 6B, the CTL activity of T cells was inhibited by the
addition of anti-CD8 mAb, but not by isotype control mAbs. Our
results indicate that the CD8 coreceptor is required not only for
activation of naive CD8+ T cells but also for the
CTL function of effector CD8+ T cells.
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It has been speculated that a high affinity interaction between
TCR and the peptide/MHC complex may abolish the need for a
costimulatory signal for T cell activation. It has been asserted that
substitution of tyrosine by alanine at position 4 (Y4A) in gp33–41
decreases the affinity of TCR to the Y4A/Db
complex without decreasing the peptide affinity to
Db (40, 41, 42). We therefore determined
whether Y4A/Db tetramers could activate the naive
CD8+ T cells. Y4A/Db
tetramers were able to induce the proliferation of naive
CD8+ TCR tg T cells, but required a 10-fold
higher concentration of the tetramer compared with
gp33/Db (Fig. 7A).
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B). If the lower affinity of the receptor required costimulation, then increasing the concentration alone should not have abolished that requirement, because it would only increase the fraction of receptor occupied, not the average time of occupancy. Thus, these data do not support a qualitative difference in the requirement for costimulation based on affinity.
We were interested in determining the cytokines produced, since perhaps
only some of the functions of CD8+ effector cells
were induced by tetramers. We expected production of IFN-, but
wondered whether there was autocrine production of IL-2. We tested
supernatants of CD8+ T cells stimulated with
wild-type gp33/Db or Y4A/Db
tetramers. As shown in Fig. 8, both
gp33/Db and Y4A/Db
tetramers induced significant amounts of both IFN- and IL-2
production by CD8+ T cells from P14 tg mice. IL-4
was not detectable by intracellular staining (not shown). The secretion
of IL-2 and IFN- correlated with both the proliferation and the CTL
activity of CD8+ T cells induced by both types of
tetramers (Fig. 8), although, again, more Y4A/Db
tetramers were needed. These results demonstrate that naive
CD8+ T cells from P14 TCR tg mice can be
efficiently activated by both wild-type gp33/Db
and Y4D/Db to produce needed cytokines in the
absence of apparent costimulation.
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The data presented above clearly show that naive
CD8+ T cells can differentiate into effector
cells following activation by tetramers without involvement with
costimulatory signal. It is important to determine whether provision of
costimulation would enhance the response to tetramers, either by
shifting the dose response to lower levels of tetramer or by increasing
the maximum response. It has been shown that cross-linking of CD28 on T
cells by specific mAb can trigger a costimulatory signal and results in
an optimal T cell proliferation (12). We examined the role
of costimulation in tetramer-induced activation of naive
CD8+ T cells. As reported previously, the
presence of costimulation provided by immobilized anti-CD28 mAb
significantly increased the proliferation of not only
CD4+ (Fig. 9A) and
CD8+ (Fig. 9B) T cells from B6 mice,
but also P14 transgenic CD8+ T cells (Fig. 9C) T cells, when stimulated with anti-CD3 mAb. This is
evidenced by a shift to lower anti-CD3 doses. Strikingly, the
enhanced proliferation by anti-CD28 mAb costimulation was not
observed when the same TCR transgenic CD8+ T
cells (Fig. 9D) were stimulated with tetramers. This further
demonstrates that in vitro tetramer-induced activation of naive
CD8+ T cells does not depend on costimulation,
and further, that it differs from anti-CD3-induced
activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or CTL activity was observed
(data not shown). Single, naive CD8+ T cells from TCR tg mice were activated with soluble peptide/MHC tetramers, and therefore, involvement of any other costimulation was excluded. In support of our data, it has been shown that CD8+ T cells can be activated by fibroblasts, which express the appropriate Ag for T cells, but not CD80/CD86 molecules (43, 44). In addition, mice deficient for the CD28 gene are able to mount an efficient CD8+ T cell-mediated cytotoxic response following LCMV infection (17) despite the fact that CD4+ helper function and Ab class switching are severely impaired in these mice. However, the findings from these studies could not rule out the possibility that unknown molecules might act as costimulators in the absence of CD28 engagement (11). Cell surface expression, cytokine secretion, and cytotoxic T cell assays all show that naive CD8+ TCR tg T cells activated by tetramers are indistinguishable from T cells activated by peptide-pulsed APC.
Recently, it was shown that TCR ligation with Ab in the absence of CD28 ligation results in activation-induced cell death, but in these experiments, anti-CD28 prevents cell death (45). Thus, although we do not see enhancement of proliferation, it is possible that we might see inhibition of cell death. Preliminary experiments suggest that most cells stimulated by tetramers undergo apoptosis. In this case ligation with anti-CD28 might block cell death without enhancing proliferation.
Delon et al. (46) have demonstrated that Ca2+
mobilization in CD8+ T cells can be triggered
with soluble peptide/MHC class I complex in the absence of a
costimulatory signal, and Goldstein et al. showed proliteration of ZC
allospecific tg mice with soluble secreted Ld. However,
this study did not address the differentiation of naive
CD8+ T cells into effector cells. Two other
studies have also demonstrated that CD8+ T cells
from other TCR tg strains are activated and differentiate into
functional cytotoxic T cells when stimulated with agonist peptide/MHC
complex without costimulation. However, in these studies, exogenous
IL-2 was required (47, 48). In our study, no exogenous
growth factors were needed, and our CD8+ T cells
produced significant amounts of IL-2 as well as IFN- (Fig. 8). One
possible explanation for the discrepancy between the previous study and
ours is that CD8+ T cells were positively
purified with anti-CD8 mAbs in the former study. We have found that
CD8+ T cells positively selected with
anti-CD8 mAb-coupled beads do not proliferate when stimulated with
tetramers. This is probably due to the blockade of the interaction
between MHC and CD8 coreceptor (Fig. 6) during culture.
It has been proposed that the affinity between the TCR and the
peptide/MHC complex determines the requirement for a costimulatory
signal in activation of CD8+ T cells
(42). Using CD8+ T cells from
CD28-deficient mice, Bachmann et al. (41) have reported
that decreasing the affinity of interaction between the TCR and the
peptide/MHC complex results in an increasing dependence on
costimulation for CD8+ T cell activation. We have
several lines of evidence that argue against this interpretation. In
our experiments, Y4A/Db tetramers caused the
equivalent proliferation and differentiation of
CD8+ T cells, although
Y4A/Db required a 10-fold higher concentration.
No added IL-2 was needed, and Y4A induced the production of both
IFN- and IL-2 from P14 cells, which differs from the findings of
Bachmann (41). In addition, our data (R. Maile and J.
A. Frelinger, unpublished observations) show that the
HY/Db complex has a low affinity for the
transgene-encoded TCR. In this paper we show that the
HY/Db tetramer was effective at stimulating the T
cells, although a higher concentration was required than for
gp33/Db tetramers. Thus, our in vitro results
argue that the dependence on costimulation is not determined by the
affinity of TCR for its ligand.
Additionally, it is important to note that the use of tetramers for the purification of peptide-specific T cells has been proposed. Our studies suggest that the binding of the tetramers to T cells is not a neutral event, and that investigators should be aware of the potential activation of T cells by their binding.
We have been able to clearly show that naive CD8+ T cells are able to be completely stimulated and differentiate into cytolytic effectors with only the signal generated from the interaction between TCR and peptide/MHC class I complex without apparent involvement with costimulatory molecules. This suggests that the rules for CD4+ and CD8+ T cell activation may indeed be different. The report that a monomeric form of peptide/MHC complex can activate CD8+, but not CD4+, T cells (46, 49) (our unpublished observations), supports this idea. This makes some sense given the role of CD8+ CTL in infectious disease, where the ability to respond to infected cells, which are typically not professional APC, might well provide an advantage.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeffrey A. Frelinger, Department of Microbiology and Immunology, University of North Carolina, CB#7290 MEJ, Chapel Hill, NC 27599-7290. E-mail address:
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; tg, transgenic.
Received for publication June 28, 1999. Accepted for publication November 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1: lack of germinal centers correlated with poor affinity maturation and class switching despite normal priming of CD4+ T cells. J. Exp. Med. 179:819.ß T cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
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J. Buslepp, S. E. Kerry, D. Loftus, J. A. Frelinger, E. Appella, and E. J. Collins High Affinity Xenoreactive TCR:MHC Interaction Recruits CD8 in Absence of Binding to MHC J. Immunol., January 1, 2003; 170(1): 373 - 383. [Abstract] [Full Text] [PDF]
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B. Kovacs, M. V. Maus, J. L. Riley, G. S. Derimanov, G. A. Koretzky, C. H. June, and T. H. Finkel Human CD8+ T cells do not require the polarization of lipid rafts for activation and proliferation PNAS, November 12, 2002; 99(23): 15006 - 15011. [Abstract] [Full Text] [PDF]
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E. Schott, N. Bertho, Q. Ge, M. M. Maurice, and H. L. Ploegh Class I negative CD8 T cells reveal the confounding role of peptide-transfer onto CD8 T cells stimulated with soluble H2-Kb molecules PNAS, October 15, 2002; 99(21): 13735 - 13740. [Abstract] [Full Text] [PDF]
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A. Lev, H. Novak, D. Segal, and Y. Reiter Recruitment of CTL Activity by Tumor-Specific Antibody-Mediated Targeting of Single-Chain Class I MHC-Peptide Complexes J. Immunol., September 15, 2002; 169(6): 2988 - 2996. [Abstract] [Full Text] [PDF]
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B. Wang, A. Sharma, R. Maile, M. Saad, E. J. Collins, and J. A. Frelinger Peptidic Termini Play a Significant Role in TCR Recognition J. Immunol., September 15, 2002; 169(6): 3137 - 3145. [Abstract] [Full Text] [PDF]
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G. Demirci, W. Gao, X. X. Zheng, T. R. Malek, T. B. Strom, and X. C. Li On CD28/CD40 Ligand Costimulation, Common {gamma}-Chain Signals, and the Alloimmune Response J. Immunol., May 1, 2002; 168(9): 4382 - 4390. [Abstract] [Full Text] [PDF]
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E. Y. Kim and H.-S. Teh TNF Type 2 Receptor (p75) Lowers the Threshold of T Cell Activation J. Immunol., December 15, 2001; 167(12): 6812 - 6820. [Abstract] [Full Text] [PDF]
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A. Hombach, A. Wieczarkowiecz, T. Marquardt, C. Heuser, L. Usai, C. Pohl, B. Seliger, and H. Abken Tumor-Specific T Cell Activation by Recombinant Immunoreceptors: CD3{zeta} Signaling and CD28 Costimulation Are Simultaneously Required for Efficient IL-2 Secretion and Can Be Integrated Into One Combined CD28/CD3{zeta} Signaling Receptor Molecule J. Immunol., December 1, 2001; 167(11): 6123 - 6131. [Abstract] [Full Text] [PDF]
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H.-W. Mittrucker, M. Kursar, A. Kohler, R. Hurwitz, and S. H. E. Kaufmann Role of CD28 for the Generation and Expansion of Antigen-Specific CD8+ T Lymphocytes During Infection with Listeria monocytogenes J. Immunol., November 15, 2001; 167(10): 5620 - 5627. [Abstract] [Full Text] [PDF]
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U. M. Padigel, P. J. Perrin, and J. P. Farrell The Development of a Th1-Type Response and Resistance to Leishmania major Infection in the Absence of CD40-CD40L Costimulation J. Immunol., November 15, 2001; 167(10): 5874 - 5879. [Abstract] [Full Text] [PDF]
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 K. Y. Tsang, M. Zhu, J. Even, J. Gulley, P. Arlen, and J. Schlom The Infection of Human Dendritic Cells with Recombinant Avipox Vectors Expressing a Costimulatory Molecule Transgene (CD80) to Enhance the Activation of Antigen-specific Cytolytic T Cells Cancer Res., October 1, 2001; 61(20): 7568 - 7576. [Abstract] [Full Text] [PDF]
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R. Maile, B. Wang, W. Schooler, A. Meyer, E. J. Collins, and J. A. Frelinger Antigen-Specific Modulation of an Immune Response by In Vivo Administration of Soluble MHC Class I Tetramers J. Immunol., October 1, 2001; 167(7): 3708 - 3714. [Abstract] [Full Text] [PDF]
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C. A. Piccirillo and E. M. Shevach Cutting Edge: Control of CD8+ T Cell Activation by CD4+CD25+ Immunoregulatory Cells J. Immunol., August 1, 2001; 167(3): 1137 - 1140. [Abstract] [Full Text] [PDF]
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B. Wang, C. C. Norbury, R. Greenwood, J. R. Bennink, J. W. Yewdell, and J. A. Frelinger Multiple Paths for Activation of Naive CD8+ T Cells: CD4-Independent Help J. Immunol., August 1, 2001; 167(3): 1283 - 1289. [Abstract] [Full Text] [PDF]
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A. Hombach, D. Sent, C. Schneider, C. Heuser, D. Koch, C. Pohl, B. Seliger, and H. Abken T-Cell Activation by Recombinant Receptors: CD28 Costimulation Is Required for Interleukin 2 Secretion and Receptor-mediated T-Cell Proliferation but Does Not Affect Receptor-mediated Target Cell Lysis Cancer Res., March 1, 2001; 61(5): 1976 - 1982. [Abstract] [Full Text]
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H. Ito, J. Kurtz, J. Shaffer, and M. Sykes CD4 T Cell-Mediated Alloresistance to Fully MHC-Mismatched Allogeneic Bone Marrow Engraftment Is Dependent on CD40-CD40 Ligand Interactions, and Lasting T Cell Tolerance Is Induced by Bone Marrow Transplantation with Initial Blockade of this Pathway J. Immunol., March 1, 2001; 166(5): 2970 - 2981. [Abstract] [Full Text] [PDF]
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J. M. Grayson, K. Murali-Krishna, J. D. Altman, and R. Ahmed Gene Expression in Antigen-Specific CD8+ T Cells During Viral Infection J. Immunol., January 15, 2001; 166(2): 795 - 799. [Abstract] [Full Text] [PDF]
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M. A. Williams, J. Trambley, J. Ha, A. B. Adams, M. M. Durham, P. Rees, S. R. Cowan, T. C. Pearson, and C. P. Larsen Genetic Characterization of Strain Differences in the Ability to Mediate CD40/CD28-Independent Rejection of Skin Allografts J. Immunol., December 15, 2000; 165(12): 6849 - 6857. [Abstract] [Full Text] [PDF]
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D.-G. Lim, K. Bieganowska Bourcier, G. J. Freeman, and D. A. Hafler Examination of CD8+ T Cell Function in Humans Using MHC Class I Tetramers: Similar Cytotoxicity but Variable Proliferation and Cytokine Production Among Different Clonal CD8+ T Cells Specific to a Single Viral Epitope J. Immunol., December 1, 2000; 165(11): 6214 - 6220. [Abstract] [Full Text] [PDF]
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Y. Zhan, A. J. Corbett, J. L. Brady, R. M. Sutherland, and A. M. Lew CD4 Help-Independent Induction of Cytotoxic CD8 Cells to Allogeneic P815 Tumor Cells Is Absolutely Dependent on Costimulation J. Immunol., October 1, 2000; 165(7): 3612 - 3619. [Abstract] [Full Text] [PDF]
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) or, as a positive control, spleen cells from
a P14 TCR tg mouse infected with LCMV 7 days previously (•) is
shown.
) tetramers at various concentrations for 48
h, and proliferation was measured by incorporation of
[3H]thymidine. B, Naive P14 T cells
stimulated with a lower affinity tetramer can effectively lyse target
cells. Naive CD8+ T cells from P14 TCR tg mice were
stimulated with 0.5 µg of Y4A/Db tetramers (open symbols)
or gp33/Db (filled symbols) for 48 h, as described in
Fig. 5
