Download Cytotoxic T Cells Sufficient to Induce Primary and Memory Soluble

Soluble Antigen and CD40 Triggering Are
Sufficient to Induce Primary and Memory
Cytotoxic T Cells
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of August 3, 2017.
Leo Lefrançois, John D. Altman, Kristina Williams and Sara
Olson
J Immunol 2000; 164:725-732; ;
doi: 10.4049/jimmunol.164.2.725
http://www.jimmunol.org/content/164/2/725
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References
Soluble Antigen and CD40 Triggering Are Sufficient to Induce
Primary and Memory Cytotoxic T Cells1
Leo Lefrançois,2* John D. Altman,† Kristina Williams,* and Sara Olson*
T
he long-term outcome of an immune response is determined by multiple factors, some of which remain undefined. Whether a T cell response results in induction of
immune memory or in deletion or tolerance of T or B cells is
determined in part by the form of Ag used for immunization (1, 2).
In the case of CD8⫹ CTL, viral infections generally result in production of long-term memory cells residing in the secondary lymphoid tissues, such as the spleen and lymph nodes (LN)3 (3, 4). In
contrast, when the cognate antigenic peptide is used for immunization, a primary proliferative response is followed by deletion or
anergy of Ag-specific CD8 T cells (5, 6). One of the factors governing the distinction between a productive CTL response induced
by viral infection and a nonproductive response induced by tolerogenic forms of Ag may be the type or level of costimulation received during primary Ag recognition (7). Professional APC such
as dendritic cells (DC) constitutively express the costimulatory
molecules CD40 and B7 (8). DC activation, such as may occur
in viral or bacterial infections, results in up-regulation of these
molecules, which may be required for induction of a productive
immune response (9). Processing by DC of noninflammatory
forms of Ag, as in the case of soluble proteins, may not result
*Division of Rheumatic Diseases, University of Connecticut Health Center, Farmington, CT 06037; and †Emory Vaccine Center and Department of Microbiology and
Immunology, Emory School of Medicine, Atlanta, GA 30322
Received for publication September 7, 1999. Accepted for publication November
4, 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 U.S. Public Health Service Grants DK45260 and
AI41576, and by an American Cancer Society Faculty Research Award to L.L.
2
Address correspondence and reprint requests to Dr. Leo Lefrançois, Department of
Medicine, University of Connecticut Health Center, MC1310, 263 Farmington Avenue, Farmington, CT 06030. E-mail address: [email protected]
3
Abbreviations used in this paper: LN, lymph node; DC, dendritic cell; CD40L,
CD40 ligand; IEL, intraepithelial lymphocyte; LP, lamina propria; MLN, mesenteric
lymph node; PLN, peripheral lymph node; sOVA, soluble OVA; APC,
allophycocyanin.
Copyright © 2000 by The American Association of Immunologists
in modulation of costimulators and therefore does not induce
complete immunity.
Recently, the concept of effective help for CD8 T cell responses
has been explored with regard to the involvement of CD40-CD40L
interactions (10 –14). CD40 engagement induces a prolonged CD8
T cell response in a model of graft-vs-host disease (14) and also
delays superantigen-mediated deletion of CD4 and CD8 cells (15).
In addition, in certain CD8 T cell responses that require CD4 T
cells to prime for CTL induction, CD40-CD40L interactions are
involved. CD40L is up-regulated following activation of CD4
(16 –18) and at least some CD8 T cells (19 –21). In this scenario,
an Ag-specific CD4 T cell interacts with a DC and delivers a signal
via CD40L to the DC, which allows that APC to become competent to drive CTL responses. A subsequent encounter of an Agspecific CD8 T cell with the empowered DC will result in CTL
priming (11). The factors that empower the DC are unknown, but
it is known that CD40 triggering can up-regulate costimulatory
molecules and inflammatory cytokines such as IL-12 (22).
The essential costimulatory requirements for generation of T
cell memory are not defined. Although CD28-B7 interactions are
necessary for primary T cell activation (7, 23, 24), and therefore
generation of memory, the signals that distinguish nonproductive
primary activation from induction of long-term immunity remain
unclear. For example, primary activation of OVA-specific TCR
transgenic CD8 T cells by soluble Ag requires CD28/B7-2 interaction, yet this reaction does not result in production of memory T
cells (23). Similar results have been obtained in studies of activation of CD4 T cells (25, 26). Thus, additional costimulatory signals
appear to be requisite for memory T cell induction. Considering
the importance of CD40-CD40L interactions in development of
B cell memory (17, 18), we wished to determine whether CD40
signaling was effective in induction of CD8 T cell memory.
Using a T cell adoptive transfer system as well as visualization
of endogenous Ag-specific T cells using MHC/peptide tetramer
reagents, we demonstrate in this study that CD40 triggering
along with soluble Ag immunization is sufficient for induction
of CD8 memory T cells in secondary lymphoid tissues and in
mucosal effector sites.
0022-1767/00/$02.00
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The signals directing induction of tolerance rather than immunity are largely unknown. The CD8 T cell response to soluble
Ags generally results in deletional tolerance following transient, costimulation-dependent activation. We demonstrated that
CD40 signaling reversed the outcome of this response. Adoptive transfer of OVA-specific CD8 T cells followed by soluble
OVA immunization resulted in induction of lytic activity and optimal clonal expansion only when CD40 was triggered via
an agonistic mAb. Activation of CD8 T cells by CD40 signaling was indirect, because CD40 expression by host cells was
required. CD40 signaling along with soluble Ag immunization also induced expansion of secondary lymphoid and intestinal
mucosal endogenous OVA-specific CD8 T cells as detected by MHC tetramer reactivity. When CD40 activation was included,
long-lived secondary lymphoid and mucosal memory CD8 cells were generated from adoptively transferred and endogenous
CD8 T cells. Mucosal and peripheral CD8 memory cells exhibited constitutive Ag-specific lytic activity, with mucosal memory
cells being 10-fold more lytic than splenic or lymph node memory cells. These results demonstrated that CD40 signaling
during a response to a poorly immunogenic soluble Ag was necessary and sufficient for CTL and memory T cell
induction. The Journal of Immunology, 2000, 164: 725–732.
726
Materials and Methods
Mice
C57BL/6J (Ly-5.1) mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). C57BL/6TacfBr-[KO]A␤b mice (27) were purchased
from Taconic (Germantown, NY). C57BL/6-Ly-5.2 mice were obtained
from Charles River (Wilmington, MA) through the National Cancer Institute animal program. The OT-I mouse line was generously provided by
W. R. Heath (WEHI, Parkville, Australia) and F. Carbone (Monash Medical School, Prahran, Victoria, Australia) (28) and was maintained as a
C57BL/6-Ly-5.2 or C57BL/6-Ly-5.1 line on a RAG⫺/⫺ background.
C57BL/6-CD40⫺/⫺ mice (29) were generously provided by Dr. Hitoshi
Kikutani (Osaka University, Osaka, Japan) via Dr. Nancy Philips (University of Massachusetts Medical Center, Worcester, MA).
Adoptive transfer
Detection of OVA-specific primary and memory CD8 T cells
with MHC tetramers
Mice were immunized by i.p. injection of 5 mg OVA with 100 ␮g antiCD40 mAb or 100 ␮g control rat Ig. At the indicated times, lymphocytes
were isolated and OVA-specific CD8 T cells were detected using H-2Kb
tetramers containing the OVA protein-derived peptide SIINFEKL (32) or
the vesicular stomatitis virus N protein-derived peptide RGYVYQGL.
MHC tetramers were produced essentially as previously described (33, 34).
Briefly, H-2Kb containing the biotin-protein ligase-dependent biotinylation
substrate sequence was folded in the presence of human ␤2-microglobulin
and the OVA peptide. Biotinylation was performed with biotin-protein
ligase (Avidity, Denver, CO). Tetramers were then produced from biotinylated HPLC-purified monomers by addition of streptavidin-allophycocyanin (APC) (Molecular Probes, Eugene, OR).
Isolation of lymphocyte populations
EL and LP cells were isolated as described previously (35, 36). For cytotoxicity assays, panning of Percoll-fractionated IEL on anti-CD8 mAbcoated plates was performed to remove contaminating epithelial cells. LN
and spleens were removed and single cell suspensions were prepared using
a tissue homogenizer. PLN included brachial, axillary, and superficial inguinal nodes. The resulting preparation was filtered through Nitex, and the
filtrate was centrifuged to pellet the cells.
Immunofluorescence analysis
Lymphocytes were resuspended in PBS/0.2% BSA/0.1% NaN3 (PBS/BSA/
NaN3) at a concentration of 1 ⫻ 106-1 ⫻ 107 cells/ml, followed by incubation at 4°C for 30 min with 100 ␮l of properly diluted mAb. The mAbs
were either directly labeled with FITC, PE, Cy5, APC, or were biotinylated. For the latter, avidin-PE-Cy7 (Caltag Laboratories, Burlingame, CA)
was used as a secondary reagent for detection. For tetramer staining, cells
were first reacted with PE-labeled anti-CD8␣ (Caltag Laboratories) and
FITC-labeled anti-CD11a (PharMingen, San Diego, CA). After staining, the
cells were washed twice with PBS/BSA/NaN3 and fixed in 3% paraformaldehyde in PBS. Relative fluorescence intensities were then measured with a
FACSCalibur (Becton Dickinson, San Jose, CA). Data were analyzed using
WinMDI software (Joseph Trotter; Scripps Clinic, La Jolla, CA).
Measurement of cytolytic activity
Cytolytic activity was measured using 51Cr sodium chromate-labeled EL4
cells (an H-2b thymoma) with or without the addition of 10 ␮g/ml of the
OVA-derived peptide SIINFEKL. Serial dilutions of effector cells were
incubated in 96-well round-bottom microtiter plates with 2.5 ⫻ 103 target
cells for 6 h at 37°C. Percent specific lysis was calculated as: 100 ⫻ [(cpm
released with effectors) ⫺ (cpm released alone)]/[(cpm released by detergent) ⫺ (cpm released alone)].
Results
CD40 triggering induces primary expansion of CD8 T cells
The adoptive transfer system allows the determination of the effect
of CD40 triggering on Ag-specific clonal expansion and lytic activity on a per cell basis in vivo. Activation of OVA-specific OT-I
TCR transgenic T cells with soluble OVA (sOVA) in the absence
of adjuvant results in clonal expansion in the periphery (23). To
determine whether CD40 signaling could affect this proliferation,
we tracked OT-I cells in PBL of cohorts of mice that received 100
␮g of an agonistic anti-CD40 mAb (31) or a control Ab simultaneous with 5 mg sOVA by i.p. injection. PBL from nonimmunized
mice contained on average 0.15% OT-I T cells. Interestingly, 24 h
after immunization, irrespective of whether anti-CD40 mAb was
administered, OT-I cells disappeared from the circulation. This
finding resembles a phenomenon in which alloreactive lymphocytes disappear from thoracic duct lymph after injection of allogeneic cells (37) and may reflect sequestration of Ag-specific cells
in secondary lymphoid organs. Between 48 and 72 h, OT-I cells
reappeared in the blood (Fig. 1A). At 72 h after immunization,
percentages of OT-I cells had increased 20-fold in control mice
(3.1 ⫾ 1.1) and 31-fold in anti-CD40 mAb-treated mice (4.7 ⫾
0.9). However, by day 4, an exceptional increase in OT-I cells to
29.3 ⫾ 6.6% of PBL had occurred in mice treated with anti-CD40
mAb as compared with control mice in which PBL contained
2.9 ⫾ 1% OT-I cells (Fig. 1B). As little as 12.5 ␮g of anti-CD40
mAb was sufficient to induce this increase (data not shown). The
proliferative response reached apogee on day 5, with 49.1 ⫾ 5.6%
of PBL made up by OT-I T cells. By day 6, the response in antiCD40 mAb-treated and control mice had begun to decline and
OT-I cells were essentially undetectable in control mice by day 18
(⬍0.1% of PBL). However, PBL from the anti-CD40 mAb-treated
mice contained detectable OT-I cells for 30 days (4.4 ⫾ 2.2%) and
longer (see below). This response required the presence of Ag
because, in the absence of sOVA, anti-CD40 mAb had no effect on
OT-I T cells (data not shown).
Although we have observed no unusual effects of OT-I transfer
and activation, it was possible that the large number of activated
cells was influencing clonal expansion. To test whether CD40 triggering could induce growth of endogenous OVA-specific CD8 T
cells, we used MHC class I/OVA peptide tetramers to monitor the
anti-OVA CD8 T cell response. Normal B6 mice were immunized
with sOVA with or without the addition of anti-CD40 mAb. Lymphocytes were isolated 5 days later and stained with H-2Kb/SIINFEKL tetramers (Fig. 2). Immunization with sOVA did not result
in the obvious appearance of tetramer⫹ CD8 T cells. It is possible
that a very small number of cells were Ag specific, but the staining
profile in this case was indistinguishable from that of unimmunized mice (Fig. 2 and data not shown). In contrast, including
anti-CD40 mAb in the immunization protocol induced expansion
of a significant population of tetramer-reactive cells that comprised
0.6% of total splenocytes. With anti-CD40 mAb included, appearance of tetramer⫹ cells was detectable at a dose of 0.5 mg OVA
(data not shown). OVA-specific CD8 T cells also appeared in the
intestinal mucosa, a site that normally contains high percentages of
activated T cells. IEL and LP lymphocytes contained 0.9% and
1.9% tetramer⫹ cells, respectively. When CD8⫹ cells were gated
and analyzed for CD11a expression, all of the Ag-specific cells
expressed high levels of CD11a. Furthermore, this analysis indicated that ⬃10% of the CD8␤⫹ cells in spleen and LP and ⬃5%
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This method was adopted from Kearney et al. (30). A total of 2 ⫻ 106
pooled CD8 LN cells from OT-I-RAG⫺/⫺ (Ly-5.1 or Ly-5.2) mice were
injected i.v. into C57BL/6 (Ly-5.1 or Ly-5.2) mice. Two days later, 5 mg
of OVA (grade VI; Sigma, St. Louis, MO) was administered by i.p. injection. Lymphocytes were isolated at the indicated times and analyzed for the
presence of transferred cells by flow-cytometric detection of Ly-5 differences. Ab treatments were performed by i.p. injection of 100 ␮g of antiCD40 mAb (clone 3/23) (31) or rat Ig as control. Each experiment was
performed a minimum of three times.
MEMORY CTL INDUCTION BY CD40 ACTIVATION
The Journal of Immunology
727
FIGURE 1. Effects of CD40 triggering on activation of transferred OT-I
cells. A total of 2 ⫻ 106 CD8 OT-I LN cells (Ly-5.2) were transferred to
B6 (Ly-5.1) mice. Two days later, mice were immunized with 5 mg OVA
by i.p. injection with the inclusion of 100 ␮g of anti-CD40 mAb (f) or
control rat Ig (rIg, F). At the indicated times after immunization, PBL
were analyzed for the presence of donor OT-I cells by fluorescence flow
cytometry. A, OT-I response from 0 to 3 days. B, OT-I response from
0 to 30 days. Each point indicates the average value from five to nine
mice ⫾ the SD.
similar in naive CD40⫺/⫺ mice (data not shown). Naive OT-I cells
expressed heterogenous amounts of CD44 (Fig. 3). Five days after
immunization of B6 mice without addition of anti-CD40 mAb,
OT-I cells comprised 1.2% and 1.4% of MLN cells and PBL,
respectively, and all of the cells had high CD44 levels. However,
much larger populations of OT-I cells were present in both sites
when sOVA-immunized B6 mice were treated with anti-CD40
mAb with 11% of MLN cells and 50% of PBL bearing Ly-5.2 and
high levels of CD44. In contrast, immunization with anti-CD40
mAb treatment of CD40⫺/⫺ mice harboring OT-I cells did not
result in an increase in OT-I cells as compared with immunization
with sOVA alone. Therefore, the observed effect of anti-CD40
mAb on CD8 T cells was not due to direct effects of the mAb on
the CD8 T cells, but rather on host cells, most likely Ag-bearing
APC, as previously proposed (11–13).
of the CD8␤⫹ IEL were OVA specific. Interestingly, the endogenous response was reduced in the presence of transferred OT-I
cells, presumably due to competition in the response by the much
larger number of Ag-specific transferred cells (data not shown).
These results along with those from the adoptive transfer studies
indicated that CD40 signaling delivered a powerful proliferative
signal to CD8 T cells.
CD40 expression by host cells is required for CD8 T cell
expansion
Because CD8 T cells can express CD40 (31), it was important to
determine whether the anti-CD40 mAb treatment had a direct or
indirect effect on Ag-induced OT-I T cell expansion. To test this,
OT-I cells were transferred to CD40⫺/⫺ mice. Two days after
transfer, the mice were immunized with sOVA with or without
anti-CD40 mAb treatment. PBL and mesenteric LN (MLN) cells
were then analyzed for the presence of OT-I cells. Without immunization in B6 mice, OT-I cells made up 0.4% and 0.1% of MLN
cells and PBL, respectively (Fig. 3), and these percentages were
FIGURE 3. Host CD40 expression is required to induce OT-I cell expansion by anti-CD40 mAb. A total of 2 ⫻ 106 CD8 OT-I LN cells (Ly5.2) were transferred to B6 (Ly-5.1) or to CD40⫺/⫺ (Ly-5.1) mice. Two
days later, mice were left untreated (naive) or were immunized with 5 mg
OVA by i.p. injection with the inclusion of 100 ␮g of anti-CD40 mAb or
control rat Ig (B6 panel). Five days after immunization, PBL and MLN
cells were analyzed for the presence of donor OT-I cells and CD44 expression by fluorescence flow cytometry.
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FIGURE 2. CD40 activation induces expansion of endogenous OVAreactive CD8 T cells. B6 mice were immunized i.p. with 5 mg sOVA with
the inclusion of 100 ␮g of anti-CD40 mAb or control rat Ig (rIg). Six days
later, lymphocytes were isolated from the indicated tissues, and three-color
flow cytometry was performed using APC-labeled SIINFEKL/H-2Kb tetramers, anti-CD8 PE, and anti-CD11a FITC. The right-hand panels show
analysis of gated CD8⫹ cells. The percentage of tetramer⫹ IEL is based on
the percentage of TCR␣␤ CD8␤⫹ cells in the total population, as determined in a separate sample.
728
CD40 triggering is sufficient to induce CTL against soluble Ag
In light of our results showing that activation of peripheral OT-I
cells with sOVA induced proliferation but not lytic activity (23),
we tested whether CD40 triggering would allow induction of CTL
activity in this situation. As shown in Fig. 4, A and C, sOVA
immunization resulted in expansion of OT-I cells in PLN, but minimal CTL induction by 3 days after immunization. E:T ratios are
based on the actual number of OT-I cells. Even at later time points
(4 to 10 days) after immunization, CTL activity was low in peripheral lymphoid organs (data not shown). Injection of the agonistic anti-CD40 mAb during immunization did not greatly affect
the percentage of transferred OT-I cells at this time point (Fig. 4A).
However, CD40 triggering resulted in a remarkable induction of
CTL activity (Fig. 4C) that was attributable to the transferred OT-I
FIGURE 5. MHC class II-restricted CD4 T cells are not required for
␣CD40 mAb-induced OT-I cell proliferation. A total of 2 ⫻ 106 CD8 OT-I
LN cells (Ly-5.2) were transferred to B6 (Ly-5.1) or MHC class II⫺/⫺
(Ly-5.1) mice. Two days later, mice were immunized with 5 mg OVA by
i.p. injection with the inclusion of 100 ␮g of anti-CD40 mAb or control rat
Ig (control). Four days after immunization, lytic activity of MLN cells was
measured by 51Cr release from SIINFEKL-pulsed EL4 (H-2b) cells. f,
MLN cells from OT-I-transferred plus ␣CD40 mAb-treated MHC class
II⫺/⫺ mice; 䡺, MLN cells from OT-I-transferred plus rIg-treated MHC
class II⫺/⫺ mice; F, MLN cells from OT-I-transferred plus ␣CD40 mAbtreated B6 mice; ‚, MLN cells from OT-I-transferred plus anti-CD40
mAb-treated MHC class II⫺/⫺ mice with EL4 targets in the absence of
peptide. E:T ratios are based on the percentage of OT-I cells or tetramer⫹
cells in the respective populations. Specific lysis in the absence of immunization was ⬍5%. Spontaneous 51Cr release was ⬍10%.
cells. In addition, when immunization of unmanipulated mice with
sOVA included anti-CD40 mAb, OVA-tetramer⫹ CD8 T cells
were present in the spleen 6 days later, but were not detectable
without anti-CD40 mAb treatment (Fig. 4B). These OVA-specific
endogenous CD8 T cells were also potent effectors. On a per cell
basis, endogenous OVA-specific CTL had lytic activity comparable with that of OT-I cells (Fig. 4C). Thus, CD40 activation functioned not only at the level of clonal expansion, but at the level of
CTL differentiation.
Because CD4 T cells are required to provide help for some CD8
responses, we wished to determine whether CD4 cells were involved in the anti-CD40 mAb-induced activation of OT-I cells. To
this end, OT-I cells were transferred to mice lacking MHC class
II-restricted CD4 T cells by virtue of a lack of expression of the
I-Ab protein. Two days later, mice were immunized with sOVA
with or without the addition of anti-CD40 mAb. Three days later,
lymphocyte populations were analyzed for donor cell numbers and
for lytic activity. The absence of MHC class II had no effect on the
expansion of OT-I cells in control mice or in mice treated with
anti-CD40 mAb at this or later time points (data not shown). Similarly, high levels of lytic activity were detected in MLN cells from
anti-CD40 mAb-treated B6 or MHC class II-deficient mice (Fig.
5). These results indicated that the induction of proliferation and
lytic activity of primary activated OT-I cells by CD40 triggering
did not require MHC class II-restricted CD4 T cells.
Soluble Ag and CD40 activation are sufficient to generate CD8
memory T cells
The eventual outcome of activation of OT-I cells via immunization
with sOVA in the absence of CD40 triggering was deletion of the
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FIGURE 4. CD40 signaling and soluble Ag immunization induce lytic
activity in OVA-specific CD8 T cells. A, 2 ⫻ 106 CD8 OT-I LN cells
(Ly-5.1) were transferred to B6 (Ly-5.2) mice. Two days later, mice were
immunized with 5 mg OVA by i.p. injection with the inclusion of 100 ␮g
of anti-CD40 mAb or control rat Ig (control). Three days after immunization, PLN cells were analyzed for the presence of donor OT-I cells and
CD8 expression by fluorescence flow cytometry. B, B6 mice were immunized i.p. with 5 mg OVA with the inclusion of 100 ␮g of anti-CD40 mAb
or control rat Ig (control). Six days later, spleen cells were isolated and
three-color flow cytometry was performed using APC-labeled SIINFEKL/
H-2Kb tetramers, anti-CD8␣ PE, and anti-CD11a FITC. Analysis of gated
CD8⫹ cells is shown. C, Lytic activity of the cell populations shown was
measured by 51Cr release from SIINFEKL-pulsed EL4 (H-2b) cells. f,
PLN cells from OT-I-transferred plus ␣CD40 mAb-treated mice; 䡺, PLN
cells from OT-I-transferred plus rIg-treated mice; F, spleen cells from
OVA plus ␣CD40 mAb-treated mice; E, spleen cells from OVA plus rIgtreated mice. E:T ratios are based on the percentage of OT-I cells or tetramer⫹ cells in the respective populations. Specific lysis in the absence of
immunization was ⬍5%. Spontaneous 51Cr release was ⬍10%.
MEMORY CTL INDUCTION BY CD40 ACTIVATION
The Journal of Immunology
cells. By day 14 after immunization, few, if any, OT-I cells were
detectable in PBL (Fig. 1). However, when anti-CD40 mAb was
included in the immunization regimen, OT-I cells were present in
PBL after 30 days (Fig. 1). We tested whether CD40 triggering in
the presence of Ag was sufficient to generate long-term CD8 memory cells in secondary lymphoid tissues (Fig. 6). We also determined whether OT-I memory cells were present in the intestinal
mucosa because many cells in this site phenotypically resemble
memory cells (38 – 41). At ⬃10 wk after OT-I transfer and immunization with sOVA, donor cells were not detectable in the spleen
or the IEL population. In striking contrast, a substantial population
of OT-I cells was present in the spleen and IEL compartment of
mice immunized with sOVA and treated with a single 100 ␮g
injection of anti-CD40 mAb (Fig. 6). In several mice tested, the
percentage of OT-I memory cells varied from ⬃0.8 –5% of spleen,
LN, or intestinal LP lymphocytes, or of IEL (data not shown).
The homogenous avidity of a single TCR, as is the case with
OT-I cells, could influence the outcome of the response against
OVA. To determine whether CD40 signaling could drive a heterogenous memory CD8 T cell response to OVA, we immunized
normal B6 mice with OVA and simultaneously administered 100
␮g of agonistic anti-CD40 mAb. The presence of OVA-specific
memory cells was then determined using H-2Kb-OVA peptide tetramers (Fig. 7). CD11a expression, which is increased on CD8
memory cells, was also analyzed. In mice that had been immu-
FIGURE 7. CD40 signaling and soluble Ag immunization induce endogenous OVA-specific CD8
memory cells. B6 mice were immunized i.p. with 5
mg sOVA with the inclusion of 100 ␮g of antiCD40 mAb or control rat Ig (rIg). Lymphocytes
were isolated at the indicated times from the spleen
or LP, and three-color flow cytometry was performed using APC-labeled SIINFEKL/H-2Kb
(OVA-tet-APC) or RGYVYQGL/H-2Kb (N-tetAPC) tetramers, anti-CD8␣ PE, and anti-CD11a
FITC. The plots show analysis of gated CD8⫹ cells.
FIGURE 8. CD40 activation generates typical CD8 memory cells. A
total of 2 ⫻ 106 CD8 OT-I LN cells (Ly-5.1) were transferred to B6 (Ly5.2) mice. Two days later, mice were left untreated (naive) or were immunized with 5 mg OVA by i.p. injection with the inclusion of 100 ␮g of
anti-CD40 mAb. Five days (naive or activated) or ten weeks (memory)
after immunization, spleen cells were analyzed for the presence of donor
OT-I cells and CD8 and CD44 expression by fluorescence flow cytometry.
The data shown are from gated Ly-5.1⫹ CD8⫹ cells. FSC, forward light
scatter. Filled histograms, memory cells; bold open histograms, naive cells;
thin-lined open histograms, activated cells.
nized with OVA and treated with control Ig, OVA-specific CD8
cells could not be found in spleen or LP. In contrast, a population
of tetramer⫹, CD11ahigh, CD8 cells was readily detectable in
spleen and LP of mice treated with anti-CD40 mAb and sOVA.
The number of memory cells did not change appreciably from 30
to 142 days after immunization, indicating the stability and longevity of the memory pool. No staining was observed using a
control tetramer containing a vesicular stomatitis virus N peptide
(Fig. 7). Thus, CD40 activation alone was able to drive endogenous memory cell induction against a poorly immunogenic soluble
protein.
To support the contention that the long-lived OT-I cells from
CD40-activated mice were bona fide memory cells, we examined
their phenotype and size. Although primary activated OT-I cells
were significantly larger than naive OT-I cells, splenic OT-I cells
10 wk after immunization were essentially the same size as naive
OT-I cells (Fig. 8). However, unlike naive OT-I cells, the longlived OT-I cells expressed high levels of CD44, as did primary
activated OT-I cells. Similar results were obtained from analysis of
endogenous tetramer⫹ memory cells (data not shown). These results indicated that CD40 triggering in the presence of soluble Ag
resulted in induction of typical long-term CD8 memory cells in
peripheral and mucosal tissues.
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FIGURE 6. CD40 signaling and soluble Ag immunization induce OT-I
memory cells. A total of 2 ⫻ 106 CD8 OT-I LN cells (Ly-5.1) were transferred to B6 (Ly-5.2) mice. Two days later, mice were immunized with 5
mg OVA by i.p. injection with the inclusion of 100 ␮g of anti-CD40 mAb
or control rat Ig (sOVA panels). Ten weeks after immunization, spleen
cells or IEL were analyzed for the presence of donor OT-I cells and CD8
expression by fluorescence flow cytometry.
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Peripheral and mucosal memory CD8 T cells are directly
cytolytic ex vivo
To examine the functional characteristics of OT-I memory cells
generated via CD40 activation, we tested their lytic activity ex
vivo without restimulation in culture (Fig. 9). The adoptive transfer system allows comparison of the lytic activity of the memory
cells from different tissues on a per cell basis, because the number
of OT-I donor cells can be used to calculate precise E:T ratios.
Immunization with OVA in the absence of anti-CD40 mAb did not
induce detectable lytic activity in spleen cells, MLN cells, or IEL.
In contrast, memory cells from all tissues examined exerted substantial Ag-specific lytic activity (Fig. 9). Interestingly, splenic and
MLN memory cells had similar lytic activity on a per cell basis,
while IEL memory cells exhibited ⬃10-fold higher lytic activity,
further supporting the concept that the intestinal mucosa is a proactive site for CTL development (23).
Discussion
The results presented defined the outcome of CD40 signaling on
CD8 T cell activation. Using the T cell adoptive transfer system,
we demonstrated that CD40 activation in the presence of a poorly
immunogenic soluble Ag provided a powerful proliferative signal
to CD8 T cells. Similarly, as assessed by MHC tetramer staining,
an impressive increase in endogenous OVA-specific CD8 T cells
was observed when immunization with sOVA included an agonistic anti-CD40 mAb. The effect of anti-CD40 mAb was indirect in
that this treatment had no effect on OT-I cells transferred to
CD40⫺/⫺ mice. This is an important result because CD8 T cells
express CD40 (21, 31), and further supports the concept that APC
activation is essential to bring the CD8 T cell response to fruition.
Significantly, CD40 activation also provided potent signals to
drive induction of CD8 T cell cytotoxic activity. Thus, this system
allowed direct visualization of the CD8 T cell response in the
presence or absence of CD40 activation.
Our findings suggest a mechanism by which tolerance to soluble, noninflammatory Ags may be mediated. However, the mechanism by which sOVA enters the MHC class I Ag processing and
presentation pathway is not clear. In other studies of CD8 priming
by sOVA, whole spleen cells loaded with OVA were used (12, 42),
and so it is possible that Ag was acquired by APC after phagocytosis of dying cells (43, 44). We have previously shown that the
peripheral OT-I proliferative response to sOVA requires CD28mediated costimulation via B7-2 to induce proliferation (23), indicating that professional APC are acquiring soluble Ag. Nevertheless, as shown in this study, this interaction does not lead to
induction of lytic activity in secondary lymphoid tissues, but to
deletion (23). Therefore, provision of the classical signal 2 (45, 46)
results in tolerance via deletion in this example (23). However, the
outcome of the response appears to be the result of a lack of CD40
triggering. Induction of anergy rather than deletion may also occur
in the absence of CD40 ligation, although in the system described
in this work, this was not observed. The necessity for inflammatory
signals, such as CD40 activation, to drive productive CD4 and Ab
responses has been previously demonstrated (47– 49). In the case
of those CD8 T cell responses that require CD4 T cell help, it is
known that CD40-mediated activation of APC or virus infection of
APC can bypass the CD4 T cell requirement (11–13). Indeed, the
results presented (Fig. 5) showed that MHC class II-restricted CD4
T cells were not required to induce lytic activity or proliferation of
transferred OT-I cells. Thus, although the in vivo signals for proliferation and induction of lytic activity can be separated in our
system, both can be provided by CD40 activation. This finding
provides the basis for considering CD40 agonists as potential adjuvants to amplify weak or nonproductive CD8 responses to soluble, poorly immunogenic compounds.
In addition to generation of a productive CD40-triggered peripheral primary immune response to soluble protein, a significant
population of Ag-specific CD8 T cells was also detected in the
intestinal mucosa. This response was visualized by reactivity with
H-2Kb/SIINFEKL tetramers and provided direct identification for
the first time of endogenous Ag-specific CD8 T cells in the LP and
in the intestinal epithelium during an immune response in vivo.
This finding indicated that systemic activation resulted in an ongoing CD8 T cell response in the mucosa and, most likely, in other
tertiary tissues. This scenario makes good sense in that the goal of
the immune response is to seek and destroy incoming pathogens
and, in the absence of a localized Ag, the system takes a buckshot
approach to mounting a response. Although it has been suggested
that immunization via a mucosal route is needed to generate a
mucosal response (50), as shown in this study, this is clearly not
the case for all CD8 T cell responses.
The cellular and molecular factors required for generation of
long-term immunological memory are largely unknown. As we
and others have shown, costimulation-dependent primary CD4 or
CD8 responses to soluble or noninflammatory Ags generate poor
memory (23, 26). The addition of inflammatory signals in the form
of microbial infection or adjuvants provides the necessary milieu
to drive the response to completion and generate memory cells
(51–53). Our results identified CD40 signaling as a means to generate long-term CD8 memory to poor immunogens. Memory in
this case also extended to the intestinal mucosa. Secondary lymphoid memory CD8 cells generated via CD40 triggering physically
(size) and phenotypically resembled memory cells generated via
virus infection (3, and Lefrançois, unpublished). Furthermore,
CD8 memory cells induced by CD40 activation exhibited direct ex
vivo lytic activity, as has been shown for antiviral CD8 memory
cells (3, 54). However, memory cells in the intestinal epithelium
exhibited ⬃10-fold higher levels of lytic activity than peripheral
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FIGURE 9. OT-I memory cells generated by CD40 triggering and
sOVA are constitutively cytolytic. A total of 2 ⫻ 106 CD8 OT-I LN cells
(Ly-5.1) were transferred to B6 (Ly-5.2) mice. Two days later, mice were
immunized with 5 mg OVA by i.p. injection with the inclusion of 100 ␮g
of anti-CD40 mAb (filled symbols) or control rat Ig (open symbols). Thirty
days later, the lytic activity of spleen cells (F, E), IEL (f, 䡺), or MLN
cells (Œ, ‚) was measured by 51Cr release from SIINFEKL-pulsed EL4
(H-2b) cells. E:T ratios are based on the percentage of OT-I cells in the
respective populations. Specific lysis in the absence of immunization was
⬍5%. Spontaneous 51Cr release was ⬍10%.
MEMORY CTL INDUCTION BY CD40 ACTIVATION
The Journal of Immunology
memory cells, indicating that the intestinal mucosa is a proactive
site for CD8 memory cells, which most likely make up a significant portion of CD8 cells in LP and IEL of normal mice and
humans. Other studies have shown that in the absence of CD40
signaling, less CD8 memory to LCMV is induced, and this is apparently due to a reduced primary response (55–57). The primary
LCMV immune response is CD28 independent, however (58, 59),
so it is unclear whether this result can be generalized with regard
to CD40 involvement in induction of CD8 memory. In any case,
the ability to bypass tolerance induction via CD40 ligation will
provide a system that will allow eventual definition of the factors
required for memory CD8 T cell generation.
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22.
23.
24.
25.
26.
References
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1. Van Parijs, L., and A. K. Abbas. 1998. Homeostasis and self-tolerance in the
immune system: turning lymphocytes off. Science 280:243.
2. Goodnow, C. C. 1996. Balancing immunity and tolerance: deleting and tuning
lymphocyte repertoires. Proc. Natl. Acad. Sci. USA 93:2264.
3. Zimmermann, C., K. Brduscha-Riem, C. Blaser, R. M. Zinkernagel, and
H. Pircher. 1996. Visualization, characterization, and turnover of CD8⫹ memory
T cells in virus-infected hosts. J. Exp. Med. 183:1367.
4. Ahmed, R., and D. Gray. 1996. Immunological memory and protective immunity:
understanding their relation. Science 272:54.
5. Kyburz, D., P. Aichele, D. E. Speiser, H. Hengartner, R. M. Zinkernagel, and
H. Pircher. 1993. T cell immunity after a viral infection versus T cell tolerance
induced by soluble viral peptides. Eur. J. Immunol. 23:1956.
6. Zinkernagel, R. M., D. Moskophidis, T. Kundig, S. Oehen, H. Pircher, and
H. Hengartner. 1993. Effector T-cell induction and T-cell memory versus peripheral deletion of T cells. Immunol. Rev. 133:199.
7. Lenschow, D. J., T. L. Walunas, and J. A. Bluestone. 1996. CD28/B7 system of
T cell costimulation. Annu. Rev. Immunol. 14:233.
8. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of
immunity. Nature 392:245.
9. Bhardwaj, N. 1997. Interactions of viruses with dendritic cells: a double-edged
sword. J. Exp. Med. 186:795.
10. Mackey, M. F., J. R. Gunn, C. Maliszewsky, H. Kikutani, R. J. Noelle, and
R. J. Barth, Jr. 1998. Dendritic cells require maturation via CD40 to generate
protective antitumor immunity. J. Immunol. 161:2094.
11. Ridge, J. P., F. Di Rosa, and P. Matzinger. 1998. A conditioned dendritic cell
can be a temporal bridge between a CD4⫹ T-helper and a T-killer cell. Nature
393:474.
12. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, and
W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40
signalling. Nature 393:478.
13. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, and C. J. Melief.
1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L
interactions. Nature 393:480.
14. Buhlmann, J. E., M. Gonzalez, B. Ginther, A. Panoskaltsis-Mortari, B. R. Blazar,
D. L. Greiner, A. A. Rossini, R. Flavell, and R. J. Noelle. 1999. Sustained expansion of CD8⫹ T cells requires CD154 expression by Th cells in acute graft
versus host disease. J. Immunol. 162:4373.
15. Maxwell, J. R., J. D. Campbell, C. H. Kim, and A. T. Vella. 1999. CD40 activation boosts T cell immunity in vivo by enhancing T cell clonal expansion and
delaying peripheral T cell deletion. J. Immunol. 162:2024.
16. Noelle, R. J., M. Roy, D. M. Shepherd, I. Stamenkovic, J. A. Ledbetter, and
A. Aruffo. 1992. A 39-kDa protein on activated helper T cells binds CD40 and
transduces the signal for cognate activation of B cells. Proc. Natl. Acad. Sci. USA
89:6550.
17. Van den Eertwegh, A. J., R. J. Noelle, M. Roy, D. M. Shepherd, A. Aruffo,
J. A. Ledbetter, W. J. Boersma, and E. Claassen. 1993. In vivo CD40-gp39
interactions are essential for thymus-dependent humoral immunity. I. In vivo
expression of CD40 ligand, cytokines, and antibody production delineates sites of
cognate T-B cell interactions. J. Exp. Med. 178:1555.
18. Foy, T. M., D. M. Shepherd, F. H. Durie, A. Aruffo, J. A. Ledbetter, and
R. J. Noelle. 1993. In vivo CD40-gp39 interactions are essential for thymusdependent humoral immunity. II. Prolonged suppression of the humoral immune
response by an antibody to the ligand for CD40, gp39. J. Exp. Med. 178:1567.
19. Roy, M., T. Waldschmidt, A. Aruffo, J. A. Ledbetter, and R. J. Noelle. 1993. The
regulation of the expression of gp39, the CD40 ligand, on normal and cloned
CD4⫹ T cells. J. Immunol. 151:2497.
20. Hermann, P., C. Van-Kooten, C. Gaillard, J. Banchereau, and D. Blanchard.
1995. CD40 ligand-positive CD8⫹ T cell clones allow B cell growth and differentiation. Eur. J. Immunol. 25:2972.
21. Sad, S., L. Krishnan, R. C. Bleackley, D. Kagi, H. Hengartner, and
T. R. Mosmann. 1997. Cytotoxicity and weak CD40 ligand expression of CD8⫹
27.
type 2 cytotoxic T cells restricts their potential B cell helper activity. Eur. J. Immunol. 27:914.
Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, and
G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high
levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via
APC activation. J. Exp. Med. 184:747.
Kim, S. K., D. S. Reed, S. Olson, M. J. Schnell, J. K. Rose, P. A. Morton, and
L. Lefrançois. 1998. Generation of mucosal cytotoxic T cells against soluble
protein by tissue-specific environmental and costimulatory signals. Proc. Natl.
Acad. Sci. USA 95:10814.
Kearney, E. R., T. L. Walunas, R. W. Karr, P. A. Morton, D. Y. Loh,
J. A. Bluestone, and M. K. Jenkins. 1995. Antigen-dependent clonal expansion of
a trace population of antigen-specific CD4⫹ T cells in vivo is dependent on CD28
costimulation and inhibited by CTLA-4. J. Immunol. 155:1032.
Perez, V. L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, and
A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires
CTLA-4 engagement. Immunity 6:411.
Pape, K. A., R. Merica, A. Mondino, A. Khoruts, and M. K. Jenkins. 1998. Direct
evidence that functionally impaired CD4⫹ T cells persist in vivo following induction of peripheral tolerance. J. Immunol. 160:4719.
Grusby, M. J., R. S. Johnson, V. E. Papaioannou, and L. H. Glimcher. 1991.
Depletion of CD4⫹ T cells in major histocompatibility complex class II-deficient
mice. Science 253:1417.
Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and
F. R. Carbone. 1994. T cell receptor antagonistic peptides induce positive selection. Cell 76:17.
Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu,
N. Yoshida, T. Kishimoto, and H. Kikutani. 1994. The immune responses in
CD40-deficient mice: impaired immunoglobulin class switching and germinal
center formation. Immunity 1:167.
Kearney, E. R., K. A. Pape, D. Y. Loh, and M. K. Jenkins. 1994. Visualization
of peptide-specific T cell immunity and peripheral tolerance induction in vivo.
Immunity 1:327.
Hasbold, J. C., C. J. Johnson-Leger, C. J. Atkins, E. A. Clark, and G. G. B. Klaus.
1994. Properties of mouse CD40: cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies. Eur. J. Immunol. 24:1835.
Van Bleek, G. M., and S. G. Nathenson. 1990. Isolation of an endogenously
processed immunodominant viral peptide from the class I H-2Kb molecule. Nature 348:213.
Altman, J. D., P. A. H. Moss, P. J. R. Goulder, D. H. Barouch,
M. G. McHeyzer-Williams, J. I. Bell, A. J. McMichael, and M. M. Davis. 1996.
Phenotypic analysis of antigen-specific T lymphocytes. Science 274:94.
Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac,
J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T
cells: a reevaluation of bystander activation during viral infection. Immunity
8:177.
Goodman, T., and L. Lefrancois. 1988. Expression of the ␥-␦ T-cell receptor on
intestinal CD8⫹ intraepithelial lymphocytes. Nature 333:855.
Laky, K., L. Lefrançois, and L. Puddington. 1997. Age-dependent intestinal lymphoproliferative disorder due to stem cell factor receptor deficiency. J. Immunol.
158:1417.
Sprent, J., and J. F. Miller. 1976. Effect of recent antigen priming on adoptive
immune responses. III. Antigen-induced selective recruitment of subsets of recirculating lymphocytes reactive to H-2 determinants. J. Exp. Med. 143:585.
Lefrançois, L., and T. Goodman. 1989. In vivo modulation of cytolytic activity
and Thy-1 expression in TCR-␥␦⫹ intraepithelial lymphocytes. Science
243:1716.
Huleatt, J. W., and L. Lefrançois. 1995. Antigen-driven induction of CD11c on
intestinal intraepithelial lymphocytes and CD8(⫹) T cells in vivo. J. Immunol.
154:5684.
Fuss, I. J., M. Neurath, M. Boirivant, J. S. Klein, C. de la Motte, S. A. Strong,
C. Fiocchi, and W. Strober. 1996. Disparate CD4⫹ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. J. Immunol. 157:1261.
Lundqvist, C., S. Melgar, M. M. W. Yeung, S. Hammarstrom, and
M. L. Hammarstrom. 1996. Intraepithelial lymphocytes in human gut have lytic
potential and cytokine profile that suggest T helper 1 and cytotoxic functions.
J. Immunol. 157:1926.
Moore, M. W., F. R. Carbone, and M. J. Bevan. 1988. Introduction of soluble
protein into the class I pathway of antigen processing and presentation. Cell
54:777.
Albert, M. L., S. F. Pearce, L. M. Francisco, B. Sauter, P. Roy, R. L. Silverstein,
and N. Bhardwaj. 1998. Immature dendritic cells phagocytose apoptotic cells via
␣v␤5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp.
Med. 188:1359.
Albert, M. L., B. Sauter, and N. Bhardwaj. 1998. Dendritic cells acquire antigen
from apoptotic cells and induce class I-restricted CTLs. Nature 392:86.
Lafferty, K. J., and A. J. Cunningham. 1975. A new analysis of allogeneic interactions. Aust. J. Exp. Biol. Med. Sci. 53:27.
Matzinger, P. 1994. Tolerance, danger, and the extended family. Annu. Rev.
Immunol. 12:991.
Khoruts, A., A. Mondino, K. A. Pape, S. L. Reiner, and M. K. Jenkins. 1998.
A natural immunological adjuvant enhances T cell clonal expansion through
a CD28-dependent, interleukin (IL)-2-independent mechanism. J. Exp. Med.
187:225.
Pape, K. A., A. Khoruts, A. Mondino, and M.K. Jenkins. 1997. Inflammatory
cytokines enhance the in vivo clonal expansion and differentiation of antigenactivated CD4⫹ T cells. J. Immunol. 159:591.
732
49. Mackey, M. F., R. J. Barth, Jr., and R. J. Noelle. 1998. The role of CD40/CD154
interactions in the priming, differentiation, and effector function of helper and
cytotoxic T cells. J. Leukocyte Biol. 63:418.
50. Mcghee, J. R., J. Mestecky, M. T. Dertzbaugh, J. H. Eldridge, M. Hirasawa, and
H. Kiyono. 1992. The mucosal immune system: from fundamental concepts to
vaccine development. Vaccine 10:75.
51. Liu, Y., and C. A. Janeway, Jr. 1991. Microbial induction of co-stimulatory
activity for CD4 T-cell growth. Int. Immunol. 3:323.
52. De Smedt, T., B. Pajak, E. Muraille, L. Lespagnard, E. Heinen, P. De Baetselier,
J. Urbain, O. Leo, and M. Moser. 1996. Regulation of dendritic cell numbers and
maturation by lipopolysaccharide in vivo. J. Exp. Med. 184:1413.
53. Malvey, E. N., M. K. Jenkins, and D. L. Mueller. 1998. Peripheral immune
tolerance blocks clonal expansion but fails to prevent the differentiation of Th1
cells. J. Immunol. 161:2168.
54. Selin, L. K., and R. M. Welsh. 1997. Cytolytically active memory CTL present
in lymphocytic choriomeningitis virus-immune mice after clearance of virus infection. J. Immunol. 158:5366.
MEMORY CTL INDUCTION BY CD40 ACTIVATION
55. Borrow, P., A. Tishon, S. Lee, J. C. Xu, I. S. Grewal, M. B. A. Oldstone, and
R. A. Flavell. 1996. CD40L-deficient mice show deficits in antiviral immunity
and have an impaired memory CD8(⫹) CTL response. J. Exp. Med. 183:2129.
56. Whitmire, J. K., M. K. Slifka, I. S. Grewal, R. A. Flavell, and R. Ahmed. 1996.
CD40 ligand-deficient mice generate a normal primary cytotoxic T- lymphocyte
response but a defective humoral response to a viral infection. J. Virol. 70:8375.
57. Borrow, P., D. F. Tough, D. Eto, A. Tishon, I. S. Grewal, J. Sprent, R. A. Flavell,
and M. B. Oldstone. 1998. CD40 ligand-mediated interactions are involved in the
generation of memory CD8⫹ cytotoxic T lymphocytes (CTL) but are not required
for the maintenance of CTL memory following virus infection. J. Virol. 72:7440.
58. Shaninian, A., K. Pfeffer, K. P. Lee, T. M. Kundig, K. Kishihara, A. Wakeham,
K. Kawai, P. S. Ohashi, C. B. Thompson, and T. W. Mak. 1993. Differential T
cell costimulatory requirements in CD28-deficient mice. Science 261:609.
59. Kundig, T. M., K. Kawai, H.-W. Mittrucker, E. Sebzda, M. F. Bachmann,
T. W. Mak, and P. S. Ohashi. 1996. Duration of TCR stimulation determines
costimulatory requirement of T cells. Immunity 5:41.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017