Naive CD8+ T Cells Do Not Require
Costimulation for Proliferation and
Differentiation into Cytotoxic Effector Cells
This information is current as
of June 15, 2017.
Bo Wang, Robert Maile, Roberta Greenwood, Edward J.
Collins and Jeffrey A. Frelinger
J Immunol 2000; 164:1216-1222; ;
doi: 10.4049/jimmunol.164.3.1216
http://www.jimmunol.org/content/164/3/1216
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References
Naive CD8ⴙ T Cells Do Not Require Costimulation for
Proliferation and Differentiation into Cytotoxic Effector Cells1
Bo Wang,* Robert Maile,* Roberta Greenwood,* Edward J. Collins,*† and
Jeffrey A. Frelinger2*
N
aive T lymphocytes need to be activated and subsequently differentiate into effector cells to perform their
immune functions. The current model of T cell activation postulates that full activation of T lymphocytes requires two
signals (1, 2). The first signal is postulated to be elicited by the
engagement of TCR by the peptide/MHC complex. The second,
costimulatory signal is provided by the interaction between accessory molecules, such as CD28, on the surface of T cells (3, 4) and
their ligands, such as CD80 and CD86, on APCs (5– 8). The role
of costimulation has been studied extensively in vivo and in vitro
in the activation of CD4⫹ T cells (9 –11). Most studies have shown
that a costimulatory signal is required for the activation of CD4⫹
cells. For example, in the absence of costimulation, engagement of
the TCR on CD4⫹ T cells by Abs to CD3 (12), Abs to TCR (13),
or Ags presented by MHC class II on APCs (14 –16) generally
leads to functional inactivation. Blocking the interaction between
CD28 and CD80 or CD86 molecules in vivo severely inhibits the
primary Ab responses to T cell-dependent Ags and impairs Ab
class switching (17–20). These data are consistent with a failure to
activate CD4⫹ Th cells in the absence of costimulation. There are
many reports that very efficient signals via the CD3/TCR complex
(for example, by anti-CD3⑀ 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
Departments of *Microbiology and Immunology and †Biochemistry and Biophysics,
University of North Carolina, Chapel Hill, NC 27599
Received for publication June 28, 1999. Accepted for publication November
15, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI20288, AI29323,
and AI41580. These experiments were initiated while one of the authors (J.A.F.) was
on leave from the National Institute of Arthritis and Infectious Diseases, the laboratory of Drs. Jack Bennink and Jonathan Yewdell.
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: [email protected]
Copyright © 2000 by The American Association of Immunologists
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 (21–25). 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 –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 ␤-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 ␤2-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
3
Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; tg,
transgenic.
0022-1767/00/$02.00
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Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR
by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as
CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8ⴙ T cells
can be activated via the signal generated through the TCR-␣␤ 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 CD8ⴙ 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 CD4ⴙ, CD8ⴙ, and TCR transgenic
CD8ⴙ 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. The Journal of
Immunology, 2000, 164: 1216 –1222.
The Journal of Immunology
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.
Materials and Methods
Animals
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 ⫻ 105) were stimulated with tetramers at different
concentrations in 200 ␮l of RPMI 1640 medium plus 10% FBS, antibiotics,
glutamine, and 50 ␮M 2-ME in 96-well flat-bottom plates. The cultures
were incubated for 48 h, and 1 ␮Ci [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)]
⫻ 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.
Peptides
Flow cytometric analysis
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.
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-V␣2 (B20.1), and anti-V␤8.1, 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).
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 ␤-D-thiogalactoside (IPTG). Inclusion bodies were purified, and the heavy chain was folded with mouse
␤2-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 ␮l in PBS containing 0.5% FBS. CD4⫹
and MHC class II⫹ cells were depleted by incubating with 10 ␮l of antiCD4 (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 ⫻ 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
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 ⫻ 106 cells/ml in RPMI 1640 with 10%
FBS, antibiotics, glutamine, and 50 ␮M 2-ME.
For costimulation experiments, 96-well flat-bottom microtiter plates
were coated with 10 ␮g/ml of anti-CD28 mAb (37.51; PharMingen) alone
or together with the indicated concentrations of anti-CD3 mAbs (1452C11; PharMingen) in 50 ␮l 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 ⫻ 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 ␮Ci)
was added to each well for the final 10 h of culture. Cells were harvested
and counted as described above.
Results
CD8⫹ T cells isolated from P14 TCR tg mice display a naive
phenotype
To investigate the requirement of costimulation for the activation
of naive CD8⫹ T cells, mice transgenic for either a TCR specific
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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(TcrLCMV)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.
1217
1218
DIRECT STIMULATION OF NAIVE T CELLS BY MHC CLASS I TETRAMERS
FIGURE 2. Tetramer stimulation induces proliferation of naı̈ve CD8⫹
T cells. Naive CD8⫹ T cells purified from P14 TCR tg (A) and HY TCR
tg (B) mice were stimulated with gp33/Db (F) and HY/Db (E) tetramers at
the indicated concentrations for 48 h. Cells were incubated with 1 ␮Ci of
[3H]thymidine for the last 10 h of culture. Incorporation of [3H]thymidine
was measured, and the average counts per minute of duplicates is shown.
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. 1A, ⬎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.
Activation of naive CD8⫹ T cells in the absence of
costimulation
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
FIGURE 3. The gp33 tetramer can stimulate the proliferation of purified
single gp33-reactive CD8⫹ T cells in the absence of accessory cells. CD8⫹
T cells were purified from P14 spleens and plated in Terasaki plates at an
average distribution of 0.4 cells/well. Wells containing single cells were
marked. After incubation with gp33 tetramer or irrelevant HY tetramer for
48 h, cells in the marked wells were recounted. The percentage of marked
wells containing more than a single cell was calculated. Actual numbers
were gp33/Db tetramer, 36/76; and HY/Db tetramer, 2/53.
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FIGURE 1. CD8⫹ T cells purified from P14 TCR tg mice display a
naive phenotype. A and B, Spleen cells from P14 TCR tg mice were depleted of CD4⫹ and MHC class II⫹ cells using magnetic microbeads conjugated with anti-CD4 (GK1.5) and anti-MHC class II (M5/114.15.2). Purified cells were stained with mAbs anti-IAb and anti-CD8 to show purity
(A) and with anti-CD69 and anti-CD62L to show lack of activation markers
(B). C, CTL activity of spleen cells from P14 TCR tg mice was assessed in
a standard 4-h 51Cr release assay at different E:T cell ratios. EL4 cells were
pulsed with 10 ␮M gp33 peptide and used as target cells. Specific lysis of
EL4 cells by spleen cells from noninfected animals (E) or, as a positive
control, spleen cells from a P14 TCR tg mouse infected with LCMV 7 days
previously (F) is shown.
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.
To stringently test both the lack of costimulation and the absence of requirements for other cells, we stimulated single purified
CD8⫹ T cells (purified by negative selection as described above)
with tetramers. Purified T cells were distributed into wells of microculture (Terasaki) plates, and each well was examined. Only
wells with a single cell immediately after distribution were analyzed. P14 cells were stimulated with either gp33/Db or HY/Db
tetramers. Fig. 3 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.
To determine the activation state of tetramer-stimulated cells,
purified CD8⫹ T cells from P14 TCR tg mice were stained with
mAbs specific for CD8, CD69, CD25, CD44, and CD62L and
analyzed by flow cytometry. Following stimulation, CD8⫹ T cells
exhibited blast cell morphology based on forward scatter (Fig. 4A,
The Journal of Immunology
1219
FIGURE 5. Naive CD8⫹ T cells differentiate into cytotoxic effector
cells in vitro following stimulation with soluble peptide/MHC tetramers.
Naive CD8⫹ T cells were purified via negative selection from P14 (A) and
HY (B) TCR tg mice. Cells (4 ⫻ 105) were stimulated with 0.5 ␮g/ml
gp33/Db or HY/Db tetramers for 48 h, and CTL activity was determined
using EL4 target cells pulsed with 10 ␮M gp33 (F) or HY (E) peptides.
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.
We next asked whether TCR down-regulation on the surface of
CD8⫹ T cells occurred following activation with the appropriate
tetramers. Purified naive CD8⫹ T cells were stimulated with
gp33/Db tetramers at different concentrations for 48 h. After stimulation, cells were stained with mAb specific for the V␣-chain of
P14 tg TCR. TCR expression was decreased with increasing concentration of tetramers (Fig. 4B). 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
Differentiation of naive CD8⫹ T cells into effector cells is CD8
coreceptor dependent
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.
T cell activation by a low affinity interaction between class
I/peptide complex and TCR is not dependent on costimulation
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 – 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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FIGURE 4. Tetramer stimulation induces activation of naive CD8⫹ T
cells. A, CD8⫹ T cells isolated from P14 TCR tg mice were stimulated with
gp33/Db or HY/Db tetramers as described above, stained with PE-conjugated anti-CD8 mAbs and FITC-conjugated mAbs specific for CD69, and
analyzed by flow cytometry. The left panel shows cell size measured by
forward scatter, and the right panel shows the expression of CD69. B, The
expression of tg TCR on the CD8⫹ T cells was evaluated after stimulation.
Cells were stained with mAbs specific for the TCR tg V␣2-chain and CD8.
Events shown were gated on CD8⫹ cells.
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).
1220
DIRECT STIMULATION OF NAIVE T CELLS BY MHC CLASS I TETRAMERS
To examine the ability of lower affinity ligands to induce CTL
differentiation, we stimulated purified CD8⫹ T cells from P14
mice with 0.5 ␮g of gp33/Db or Y4A/Db tetramers. The CTL resulting from both Y4A/Db and gp33/Db were identical (Fig. 7B).
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
FIGURE 8. Stimulation with soluble tetramers induces production of
cytokines by CD8⫹ T cells. Naive CD8⫹ T cells from P14 TCR tg mice
were stimulated with various concentrations of wild-type gp33/Db (F) or
Y4A/Db (E) tetramers. After 40-h incubation, supernatants were assessed
for cytokine production. IFN-␥ (A) was determined via ELISA, and IL-2
(B) was determined via CTLL bioassay. No results are shown for HY/Db
tetramers, because too few cells survive 48 h to make any analysis feasible.
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.
Activation of naive CD8⫹ T cells induced with tetramers is not
enhanced by costimulatory signal
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.
Discussion
FIGURE 7. Lower affinity tetramers induce full activation of naive
CD8⫹ T cells. A, Naive P14 T cell proliferate with lower affinity Y4D/Db
tetramers. Naive CD8⫹T cells isolated from P14 TCR tg mice were stimulated with gp33/Db (F), Y4A/Db (E), or HY/Db (䡺) 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 ␮g of Y4A/Db tetramers (open symbols)
or gp33/Db (filled symbols) for 48 h, as described in Fig. 5. EL4 target cells
were pulsed with 10 ␮M gp33 (circles) or HY peptides (squares).
In this study we examined the ability of soluble peptide/MHC
tetramers to activate naive CD8⫹ T cells in vitro from two different
TCR tg mice, in the absence of apparent costimulation or exogenous growth factors. The data demonstrate that naive CD8⫹ T cells
can be fully activated and differentiate into cytotoxic CD8⫹ T cells
without costimulation. When CD8⫹ T cells were stimulated with
our tetramers in the presence of anti-CD28 mAbs, no enhancement
of proliferation (Fig. 9) or CTL activity was observed (data not
shown).
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 6. CD8 coreceptor is required for activation and effector function of CD8⫹ T cells. A, Naive CD8⫹ T cells (4 ⫻ 105) prepared from P14
TCR tg mice were stimulated with 0.5 ␮g/ml of gp33/Db tetramers for 48 h
in the presence of different concentrations of anti-CD8 mAbs (E) or isotype control mAbs (F). Cultures were pulsed with 1 ␮Ci of [3H]thymidine
for the final 10 h, and incorporation of [3H]thymidine was measured. Each
data point represents the average counts per minute of duplicate determinations. B, Naive CD8⫹ T cells purified from P14 TCR tg mice were stimulated
as described in Materials and Methods. EL4 target cells were pulsed with
different concentrations of gp33 peptide. CTL assays were performed in the
presence of 2.5 ␮g/ml of anti-CD8 or isotype control mAbs.
The Journal of Immunology
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 10fold 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.
Acknowledgments
We thank Katherine Midkiff, Timothy Broderick, and Brian Cox for technical assistance. We thank Dr. Roland Tisch for critical review of the
manuscript, and the members of the Frelinger and Collins laboratories for
many helpful discussions.
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