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*
Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322;
Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520; and
Department of Surgery, Emory University School of Medicine, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2) and Th2 (IL-4) responses. An in vivo functional
consequence of this Th cell defect was the inability of
CD40L-/- mice to control a chronic lymphocytic
choriomeningitis virus infection. This study highlights the importance
of CD40-CD40L interactions in generating virus-specific CD4 T cell
responses and in resolving chronic viral
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-herpesvirus, and lymphocytic
choriomeningitis virus
(LCMV)4 in mice induce
CD8 expansion (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). These CD8 T cells are responsible
for controlling the infection. They recognize virally infected cells
via TCR engagement with MHC class 1-peptide complexes and kill the
infected cells by secreting hole-punching perforin molecules and
granzymes and attenuate viral spread by secreting IFN- and other
cytokines (14, 18, 21, 22). This initial expansion of CD8
T cells is not only responsible for eliminating the primary infection,
but it may shape the memory T cell repertoire found in immune mice,
since activated T cells and memory T cells share similar TCR
repertoires (18). Therefore, it is important to understand
the mechanisms by which these cells are activated.
CD4 T cells also contribute to viral clearance by producing IL-2, which
facilitates CD8 T cell activation and expansion, and by secreting
IFN- and TNF to activate macrophages and inhibit viral replication.
In addition to secreting cytokines, CD4 Th cells mediate help to B
cells by direct cell-to-cell interaction involving CD40L (expressed on
activated CD4 T cells) and CD40 (expressed on B cells). This
interaction is required for B cell proliferation, Ab class
switching, and memory B cell development (16, 23, 24, 25, 26, 27, 28).
Signaling through CD40 also activates macrophages to produce TNF-
,
nitric oxide, and IL-12 and to express costimulatory molecules
(29, 30, 31, 32, 33, 34). Some CTL responses are dependent upon CD4 T
cells, and recently, several reports documented the role of CD40-CD40L
interaction for priming these Th-dependent CTL responses
(35, 36, 37).
Protective viral immunity requires Ag-specific T cells be activated sufficiently to proliferate and eliminate the infection. In the conventional model of T cell activation, peripheral T cells require two signals: 1) the first signal is delivered by the cognate interaction of the TCR complex with peptide-bearing MHC, resulting in up-regulation of CD28 and induction of CD40L expression; 2) the second signal is delivered by the interaction of CD28 with B7 and of CD40L with CD40. The purpose of these secondary (costimulatory) signals is 2-fold. First, they mediate changes in the CD40-expressing APC such as up-regulation of MHC class II and B7 molecules to make it a more effective APC (38, 39, 40, 41, 42). Secondly, these interactions strongly activate Ag-specific T cells to proliferate and secrete cytokines (43, 44, 45, 46, 47, 48). The role of CD40-CD40L interactions in driving T cell responses has been characterized in experimental models of autoreactive CD4 T cells and T cell responses against Leishmania infections, where it was found to be important for activating these cells and eliminating the infection (44, 46, 47, 48). However, the primary CD8 CTL response to LCMV is normal in CD40L-deficient mice, and these mice resolve the acute LCMV infection as well as +/+ mice (16, 23, 24), so it is unclear whether the rules that govern CD4 activation are the same as those responsible for CD8 activation.
The CD8 T cell response to LCMV is relatively CD4 independent, as
CD4-deficient mice are able to mount potent CD8 responses to LCMV
(49, 50, 51). The CD8 response is also not affected by defects
in the B cell response, since B cell-deficient
(µIg-/-) mice generate normal CD8 responses
(15). These features make acute LCMV infection a useful
system for addressing the role of the CD40-CD40L interaction in
inducing T cell responses, because the effect of this interaction on
CD8 T cells can be distinguished from its effects on CD4 T cells. In
addition, recent advances in the quantitation of T cell responses at
the single cell level (ELISPOT and intracellular staining for
cytokines) allow simultaneous quantitation of CD4 and CD8
responses, so not only are the effects of CD40-CD40L interaction
measured in the same animal, but they can be analyzed using the same
assays. Following infection of CD40L-deficient mice with LCMV, we found
that there was a differential requirement for CD40-CD40L interaction
for CD8 and CD4 T cells. CD40L-deficient mice generated large numbers
of IFN-secreting CD8 T cells, which were specific to dominant and
subdominant epitopes during acute infection, but anti-viral CD4 T
cell expansion was 10-fold lower than that in +/+ mice. This deficiency
was found for IL-2-IFN-, and IL-4-secreting CD4 T cells, indicating
that both Th1 and Th2 responses were weaker in CD40L-deficient mice. An
important consequence of this deficiency in the CD4 response was that
CD40L-deficient mice were unable to resolve a chronic LCMV infection.
Investigation of the CD8 response in chronically infected mice revealed
an absence of virus-specific CD8 T cell responses. These results show
that the activation requirements of CD4 T cells are different from
those of CD8 T cells and highlight a role for CD4 T cells and
CD40-CD40L costimulation in resolving chronic viral infections.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 x 129 (F2) and C57BL/6 (H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The CD40L-deficient mice used in these experiments were generated by targeted gene disruption, which abrogates surface expression of the CD40L molecule (27, 28). The C57BL/6 carrier mice used in these experiments were bred at Emory University (Atlanta, GA).
Virus
Mice were infected i.p. with 2 x 105 PFU of the Armstrong CA 1371 strain of LCMV (52). LCMV variants, clone 13 (52) and t1b (53), were injected i.v. in the tail vein of mice at 2 x 106 PFU to cause chronic infection. Infectious virus was quantitated by plaque assay (52).
CD4 enrichment
CD4 enrichment was performed using CD4 enrichment columns. Mouse CD4 Subset Column Kits were purchased from R & D Systems (Minneapolis, MN), and the manufacturer’s suggested protocol was used.
Quantitation of virus-specific IFN--secreting CD8 and CD4 T
cells by ELISPOT
Virus-specific CD8 and CD4 T cell responses were measured by
IFN- ELISPOT assay as described previously (14, 54).
For stimulation, LCMV carrier mouse spleen cells (congenitally infected
mice that express virus protein in the context of MHC class I and MHC
class II), purified LCMV peptides that bind to MHC class I and
stimulate CD8 T cell responses specifically (55), or LCMV
peptides that bind to MHC class II (NP309–328
and GP61–80 of Armstrong) and stimulate only
I-Ab-restricted CD4 T cell responses
(56) were used. Peptides NP396–404,
GP33–41, GP276–296,
NP205–212, and GP92–101
were used at 0.1 µg/ml final concentration and
GP61–80 and NP309–328
were used at a 1.0 µg/ml final concentration. The results shown for
the CD8 response as measured by ELISPOT assay are lower than recently
reported (14). The number of Ag-specific CD8 T cells shown
in Fig. 2
is about 2- to 5-fold lower than what we have reported
recently for LCMV-infected C57BL/6 mice using MHC class I tetramers and
intracellular cytokine staining. The main reason for this discrepancy
is that several of the experiments in the present study were performed
with C57BL6 x 129 mice. These mice show some variability and, in
general, exhibit slightly lower responses than C57BL/6 mice. Also, the
experiments shown in Fig. 2 were performed at a time when our IFN-
ELISPOT assay had not been fully optimized. This systematic difference
in the magnitude of the response due to variation in the assay does not
affect the comparisons between +/+ and CD40L-deficient CD8 responses,
as they were measured at the same time by the same assay.
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The method for intracellular IFN- staining has
been described previously (14, 54). Spleen cells
(106 cells/well in 96-well flat-bottom plates)
were stimulated in vitro with medium or GP61–80
(for CD4 T cells) or NP396–404 (for CD8 T cells)
for 5 h in vitro with brefeldin A (Golgistop, PharMingen, La
Jolla, CA). They were then harvested, washed once in FACS buffer,
stained with allophycocyanin-conjugated monoclonal anti-CD4 (clone
RM4-5, PharMingen) or allophycocyanin-conjugated monoclonal
anti-CD8 (clone 53-6.7, PharMingen), and stained for intracellular
IFN- using the Cytofix/Cytoperm staining kit (PharMingen) according
to the manufacturer’s recommended protocol. FITC-conjugated monoclonal
rat anti-mouse IFN- (clone XMG1.2) and its control isotype Ab
(rat IgG1; PharMingen) were used for intracellular IFN- staining.
Intracellular staining for CD40L was performed in the same fashion
using PE-conjugated anti-CD40L (clone MR1, PharMingen).
Quantitation of virus-specific IL-2- and IL-4-secreting CD4 T cells
ELISPOT assays for measuring IL-2- or IL-4-secreting CD4 T cells
were performed in the same fashion as the IFN- ELISPOT assay and
have been described previously (54). For the IL-2 ELISPOT,
the capture Ab was clone JES6-1A12, and the detection Ab was clone
JES6-5H4. For the IL-4 ELISPOT, the capture Ab was clone BVD4-1D11, and
the detection Ab was clone BVD6-24G2. All Abs were purchased from
PharMingen. ELISPOT plates were purchased from Millipore (Bedford,
MA).
Cytokine ELISAs
Cytokine ELISAs were conducted using cytokine-specific ELISA kits purchased from Genzyme Diagnostics (Cambridge, MA) and were performed and analyzed as recommended by the manufacturer. The ELISAs were read using a Bio-Rad Microplate Reader 3550 (Bio-Rad, Hercules, CA) using appropriate filters.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The level of T cell activation in the spleen was analyzed
following infection of +/+ and CD40L-deficient mice with LCMV-Armstrong
(Fig. 1). Consistent with previous
observations (16, 25, 27, 28), there were no intrinsic
differences between uninfected +/+ and CD40L-deficient mice in the
percentages of CD8 and CD4 T cells in the spleen. CD8 T cells became
strongly activated after infection in both +/+ and CD40L-deficient
mice. In contrast, CD4 T cell activation was lower in CD40L-deficient
mice than in +/+ mice. The ratio of activated
(CD44high) to resting
(CD44low) CD4 T cells was approximately 0.5 in
both groups of uninfected mice. On day 8 after the infection, the ratio
of activated CD4 T cells changed to 1.8 in +/+ mice, but remained
relatively unchanged (0.4) in CD40L-deficient mice. These data
indicated that there may be a difference in the requirement of
CD40-CD40L interaction for activation of virus-specific CD8 and CD4 T
cells. To address this, virus-specific CD8 and CD4 T cell responses
were further characterized.
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The number of virus-specific CD8 T cells was quantitated by
intracellular IFN- staining on day 8 postinfection. Consistent with
previous observations of CTL generation (16, 23, 24), high
numbers of virus-specific CD8 T cells were generated in CD40L-deficient
mice, and the virus was eliminated (Table I and data not shown). The CD8 T cell
response to the dominant LCMV epitopes NP396–404
and GP33–41 was normal in CD40L-deficient mice,
and both groups generated 5–7 x 106
specific cells/spleen on day 8 (Table I). Responses to subdominant
epitopes constitute only a minority of the CD8 T cell response
(14, 55, 57). Since these epitopes may bind to MHC class I
and/or TCR with lower affinity, initiating a CD8 T cell response to
them might require costimulatory interactions. However, there was no
defect in the response to GP276–296,
GP92–101, or NP205–212.
Both CD40L-deficient and +/+ mice contained 2 x
106 cells specific to
GP276–296, 4 x 105
cells specific to GP92–101, and 1 x
106 cells specific to
NP205–212 (Table I). Overall, there was no
deficit in responses to dominant or subdominant epitopes in the absence
of CD40-CD40L interaction.
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The IFN- ELISPOT assay was used to analyze the CD4 T cell
response in the same mice in which the CD8 response was measured. CD4 T
cells were purified from spleen cells of +/+ and CD40L-deficient mice
on day 8 after LCMV infection and were restimulated with syngeneic
LCMV-infected carrier spleen cells or with syngeneic uninfected spleen
cells. Fig. 2
shows that CD40L-deficient
mice that generated potent CD8 T cell responses lacked normal
anti-viral CD4 T cell responses. CD40L-deficient mice had 9- to
11-fold fewer IFN--secreting virus-specific CD4 T cells per spleen,
whereas they had in some cases more IFN--secreting CD8 T cells
(compare Expt. 1 and 2 in Fig. 2A with Expt. 1 and 2 in Fig. 2B). The frequency of virus-specific CD4 T cells was 7.7-,
9.4-, and 14.9-fold lower in Expt. 1, 2, and 3, respectively.
Fig. 3 shows intracellular-IFN-
staining, illustrating that the CD4 T cell response was defective in
CD40L-deficient mice, while the CD8 response was normal. The frequency
of peptide-specific CD4 T cells was 10-fold lower in the
CD40L-deficient mice compared with that in the +/+ mice. In eight mice
analyzed by this assay, CD40L-deficient mice had 1.9 ± 1.2
x 105 GP61–80-specific
cells, whereas +/+ mice had 1.7 ± 0.5 x
106. By the same assay the frequency of CD8
T cells that were specific to NP396–404 was not
significantly different from that found in the +/+ mice.
CD40L-deficient mice had 4.5 x 106 specific
cells/spleen, and +/+ mice had 7.5 x 106
specific cells/spleen.
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production by ELISA. As shown in Table II, there was no intrinsic defect in
IFN- production, and CD8 T cells from CD40L-deficient mice produced
similar levels of IFN- when stimulated with
NP396–404 peptide compared with CD8 T cells from
+/+ mice (18.9 vs 18.6 ng/ml). To analyze CD4 responses in these mice,
CD4 T cells were purified using a negative selection column and then
stimulated with LCMV carrier spleen cells for 24 h. As shown
in Table II, CD4 T cells from CD40L-deficient mice made less IFN-
than those from +/+ mice. Again, by the same assay, it can be seen that
there is a differential requirement for CD40-CD40L interaction for
inducing CD8 and CD4 T cell responses: CD40L is dispensable for
generating primary antiviral CD8 T cell responses, but is required for
LCMV-specific CD4 T cell responses.
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A). Table II shows that less
IL-2 was made by CD40L-deficient CD4 T cells as determined by ELISA. In
addition, the IL-4 ELISPOT assay (Fig. 4B) demonstrated that
there was also a defect in generating IL-4-secreting CD4 T cells in
mice lacking CD40L. There were 2–3 x 104
IL-4-secreting CD4 T cells/spleen in +/+ mice vs only 3–7 x
103/spleen in CD40L-deficient mice. Table II also
shows that CD4 T cells from CD40L-deficient mice made less IL-4 than
those from +/+ mice. Overall, CD4 Th1 and Th2 responses were
compromised in the absence of CD40-CD40L interaction.
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A shows spleen cells from
LCMV-infected mice that were stimulated with
NP396–404. The
NP396–404-specific T cells that made IFN-
were identified and analyzed for intracellular levels of CD40L as
depicted in Fig. 5B. Among the Ag-specific CD8 T cells,
18-56% expressed CD40L, and these had a mean fluorescence
intensity (MFI) of 86. For comparison, resting CD8 T cells from
uninfected mice were predominantly CD40L negative, with a MFI of 
10.
GP61–80-specific CD4 T cells were also
identified as illustrated in Fig. 5C, and their level of
CD40L expression is shown in Fig. 5D. While resting CD4 T
cells did not show any staining, almost all Ag-specific CD4 T cells
(98%) expressed CD40L, and the MFI of the CD40L staining for these
cells was 507. These results demonstrate that Ag-specific CD4 T cells
contain much higher levels of CD40L than Ag-specific CD8 T cells.
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The results presented so far have shown that CD40L-deficient mice
exhibit a selective defect in generating CD4 T cell responses during
acute LCMV infection. However, these CD40L-deficient mice generate a
potent virus-specific CD8 T cell response that is capable of
controlling infection by the LCMV-Armstrong strain (16, 23). In contrast to the LCMV-Armstrong strain that is eliminated
within a week by both +/+ and CD40L-deficient mice, infection of +/+
adult mice with macrophage tropic strains of LCMV, such as clone 13,
results in a disseminated infection that can last for several months or
longer (58). We next examined the ability of
CD40L-deficient mice to control infection by the more virulent LCMV
clone 13 (see Fig. 6). Groups of +/+ and
CD40L-deficient mice were infected with LCMV clone 13, and levels of
virus in the serum were monitored over time. High levels of virus were
present in the sera of both groups of mice (>105
PFU/ml) for the first 2–3 wk following infection. However, the level
of virus dropped following this period in +/+ mice, but not in
CD40L-deficient mice. The serum levels of virus in +/+ mice eventually
dropped to below detection by plaque assay (<50 PFU/ml) by day 77, but
the level of virus in CD40L-deficient mice never decreased, and on day
77 there remained between 5 x 104 to
105 PFU/ml of clone 13 in the serum. Similar
results were seen after another LCMV variant, clone t1b. Both +/+ and
CD40L-deficient mice initially showed high levels of virus in the serum
(3–10 x 104 PFU/ml) and in other tissues
on day 8. However, +/+ mice controlled the infection in approximately 1
mo in the serum and liver, whereas CD40L-deficient mice became
chronically infected.
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. In +/+ mice, 9% of CD8 T cells were
specific to GP33–41 as measured by intracellular
cytokine staining for IFN-. In contrast, CD40L-deficient mice did
not show any staining above the background level. Taken together, the
results presented in Figs. 6 and 7 show that CD40L-deficient mice are
unable to control a chronic LCMV infection and that under these
conditions of persistent antigenic stimulation, there is also a
functional loss of virus-specific CD8 T cell responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that CD40-CD40L interaction is not required
for CTL generation as measured by CTL killing of virally infected
target cells, but these CTL assays primarily measured the CD8 response
to the dominant MHC class I binding epitopes of LCMV (16, 23, 24). Here, we extend these studies and show that CD8 responses
to subdominant epitopes are also normal. This is important, as it has
been shown that vaccination with subdominant epitopes can confer
protection against viral challenge (55, 57, 59), and these
results indicate that CD40 costimulation is not necessary to
activate these cells. In contrast to CD8 T cells, virus-specific CD4 T
cell responses were severely compromised (
90% inhibition) in
CD40L-deficient mice. This requirement for CD40-CD40L costimulation was
seen for both Th1 (IFN-, IL-2) and Th2 (IL-4) CD4 responses.
Why are CD4 T cells but not CD8 T cells affected by the absence of
CD40-CD40L interaction? There are several possibilities. 1) One
possibility is that the amount of Ag that CD8 T cells
encounter is sufficient to activate them, whereas CD4 T cells
encounter less Ag during the viral infection. One study that
examined the number of TCRs required to activate T cells found that
about 8000 TCR molecules had to be engaged to achieve activation, but
if the T cells were also stimulated through CD28, then the number of
TCRs that had to be engaged was reduced to approximately 1500/cell
(60). It is possible that a large number of MHC class I
molecules contain viral peptides, so a sufficient number of TCR
molecules may be engaged for CD8 T cell activation. Also, most cells
express MHC class I molecules. Fewer cells (namely B cells,
macrophages, and dendritic cells) express MHC class II, and it is
possible that Ag density per cell (i.e., MHC class II molecules
presenting viral peptides) is not sufficient to activate CD4 T cells in
the absence of CD40-CD40L costimulation. Hence, CD4 T cells may not
reach the activation threshold without additional costimulatory
signals. 2) The strength of the TCR signal for CD8 T cells could
obviate the need for costimulation (61, 62). The TCR
signal delivered by the LCMV peptides in association with MHC class I
along with any contribution by the CD8 coreceptor may be strong enough
to activate CD8 T cells. In contrast, the MHC class II-restricted
peptides of LCMV may not deliver as strong a TCR signal or the CD4
coreceptor is too weak, so that costimulation is needed to activate CD4
T cells. In particular, CD4 T cells may require more CD40L signaling,
and circumstantial evidence for this is the fact that CD4 T cells
contained higher levels of CD40L than CD8 T cells (Fig. 5). 3) Lymphoid
architecture could be more important for activating CD4 T cells than
CD8 T cells. Histological analysis of spleens from CD40L-deficient mice
show that the architecture of these spleens is abnormal, and there is
little or no migration of B cells and B7.1+ cells
into the follicles after infection with LCMV (our unpublished
observation). B7 signaling may play a role in priming CD4 T cells
(41, 45, 63), and since APC and CD4 T cells are not parked
next to each other, CD28-B7 interactions would not occur. In contrast,
CD8 T cells are localized outside of the follicles in the marginal zone
and red pulp, and therefore are less affected by follicular
architecture. 4) Another possibility is that there may be additional
molecules used by CD8 T cells. 41BB-41BBL interaction has been shown to
be important for alloreactive CD8 T cells (64), and it may
also be important for antiviral CD8 T cells (J. T. Tan, J. K.
Whitmire, R. Ahmed, T. C. Pearson, and C. P. Larsen,
manuscript in preparation).
Why is CD40-CD40L interaction required for clearance of chronic viral
infection? Initially after chronic infection, CD40L-deficient mice
generate normal anti-viral CD8 responses (J. K. Whitmire and
R. Ahmed, manuscript in preparation), but with time there is a loss of
these virus-specific responses until none can be detected (Fig. 7 and
unpublished observations). These CD8 T cells become nonresponsive with
regard to IFN- production or cytotoxic activity, or they are deleted
(J. K. Whitmire and R. Ahmed, manuscript in preparation). This
most likely reflects the requirement for sufficient numbers of primed
CD4 T cells. When mice have a deficiency in CD4 responses due to an
absence of CD40-CD40L interaction (this study), an absence of B7-CD28
interaction (unpublished observation), or a deletion of CD4 T cells
(58, 65), then they will be impaired in their ability to
resolve chronic viral infections even though their CD8 response to
acute viral infection is normal. CD4 T cells provide IL-2 and other
growth factors that prevent T cell death (66, 67).
Exposure to high levels of Ag over an extended period of time induces
CD8 T cells to die by apoptosis, and this can be prevented in vitro by
the addition of IL-2 to cultures. IL-2 may play a similar role in vivo
during a persisting viral infection, and it remains to be seen how
administration of IL-2 will affect virus-specific CTL during these
infections. CD4 Th cells could also affect APC. Since CD8 and CD4 T
cells may interact with a common APC that expresses MHC class I and MHC
class II, CD4 T cells could activate this shared APC so that it, in
turn, maintains effector CTL (68). One effect that T cells
could have through the CD40-CD40L pathway is the induction of IL-12 by
dendritic cells, which could be important in activating T cells
(40). Recently, it has been shown that CD40L-CD40
interaction between activated CD4 T cells and Ag-bearing dendritic
cells is important for priming CD8 CTL (35, 36, 37). These
reports show that signaling through CD40 "licenses" APC in some way
to prime Th-dependent CTL responses, perhaps by increasing MHC
expression or costimulatory molecules. While the CTL response against
LCMV can be generated in the absence of CD4 T cells or CD40-CD40L
interaction, an analogous role for APC licensing may exist during
chronic viral infection when CTL must be maintained for extended
periods of time so that they do not undergo activation-induced cell
death or become functionally exhausted (65). This
licensing could also manifest itself in the preservation of APC that
might otherwise be deleted during chronic LCMV infection. Some strains
of LCMV, such as those mentioned in these studies, result in
immunopathology with altered splenic architecture and a loss of
some cell types, including APC (69, 70). CD40-CD40L
interaction may preserve these APC or maintain the proper
microenvironment so that they can interact with and maintain
CTL.
Protracted HIV infection leads to a loss of virus-specific CD4 T cells, followed by the loss of anti-viral CD8 T cell responses over time (71). The results reported here suggest a role for CD40-CD40L interaction and CD4 T cells in preventing chronic viral infections. Help from CD4 Th cells likely plays a pivotal role in driving and maintaining the CTL responses under conditions of a protracted viral infection (58, 65, 72). Future investigations should resolve how CD4 T cells assist CTL, whether it is through production of growth factors such as IL-2 or through modification of APC. Dissecting the mechanisms that are important for generating and maintaining CD8 and CD4 T cell responses will lead to improved strategies for the prevention and treatment of chronic viral infections.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990.
3 Address correspondence and reprint requests to Dr. Rafi Ahmed, Emory Vaccine Center, Emory University School of Medicine, G211 Rollins Research Building, 1510 Clifton Road, Atlanta, GA 30322. E-mail address:
4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; CD40L, CD40 ligand; ELISPOT, enzyme-linked immunospot assay.
Received for publication December 23, 1998. Accepted for publication July 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. Fang, T. Miwa, H. Shen, and W.-C. Song Complement-Dependent Enhancement of CD8+ T Cell Immunity to Lymphocytic Choriomeningitis Virus Infection in Decay-Accelerating Factor-Deficient Mice J. Immunol., September 1, 2007; 179(5): 3178 - 3186. [Abstract] [Full Text] [PDF]
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S. Fuse, S. Bellfy, H. Yagita, and E. J. Usherwood CD8+ T Cell Dysfunction and Increase in Murine Gammaherpesvirus Latent Viral Burden in the Absence of 4-1BB Ligand J. Immunol., April 15, 2007; 178(8): 5227 - 5236. [Abstract] [Full Text] [PDF]
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M. G. H. Hernandez, L. Shen, and K. L. Rock CD40-CD40 Ligand Interaction between Dendritic Cells and CD8+ T Cells Is Needed to Stimulate Maximal T Cell Responses in the Absence of CD4+ T Cell Help J. Immunol., March 1, 2007; 178(5): 2844 - 2852. [Abstract] [Full Text] [PDF]
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M. Wuthrich, P. L. Fisette, H. I. Filutowicz, and B. S. Klein Differential Requirements of T Cell Subsets for CD40 Costimulation in Immunity to Blastomyces dermatitidis J. Immunol., May 1, 2006; 176(9): 5538 - 5547. [Abstract] [Full Text] [PDF]
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C. C. Kemball, E. D. H. Lee, E. Szomolanyi-Tsuda, T. C. Pearson, C. P. Larsen, and A. E. Lukacher Costimulation Requirements for Antiviral CD8+ T Cells Differ for Acute and Persistent Phases of Polyoma Virus Infection J. Immunol., February 1, 2006; 176(3): 1814 - 1824. [Abstract] [Full Text] [PDF]
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 T. L. Stephen, M. Niemeyer, A. O. Tzianabos, M. Kroenke, D. L. Kasper, and W. M. Kalka-Moll Effect of B7-2 and CD40 Signals from Activated Antigen-Presenting Cells on the Ability of Zwitterionic Polysaccharides To Induce T-Cell Stimulation Infect. Immun., April 1, 2005; 73(4): 2184 - 2189. [Abstract] [Full Text] [PDF]
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 C. Beadling and M. K. Slifka Differential regulation of virus-specific T-cell effector functions following activation by peptide or innate cytokines Blood, February 1, 2005; 105(3): 1179 - 1186. [Abstract] [Full Text] [PDF]
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M. J. Montfort, H. G. A. Bouwer, C. R. Wagner, and D. J. Hinrichs The Development of Functional CD8 T Cell Memory after Listeria monocytogenes Infection Is Not Dependent on CD40 J. Immunol., September 15, 2004; 173(6): 4084 - 4090. [Abstract] [Full Text] [PDF]
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D. Kreisel, A. M. Krasinskas, A. S. Krupnick, A. E. Gelman, K. R. Balsara, S. H. Popma, M. Riha, A. M. Rosengard, L. A. Turka, and B. R. Rosengard Vascular Endothelium Does Not Activate CD4+ Direct Allorecognition in Graft Rejection J. Immunol., September 1, 2004; 173(5): 3027 - 3034. [Abstract] [Full Text] [PDF]
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M. Xu, A. J. Lepisto, and R. L. Hendricks CD154 Signaling Regulates the Th1 Response to Herpes Simplex Virus-1 and Inflammation in Infected Corneas J. Immunol., July 15, 2004; 173(2): 1232 - 1239. [Abstract] [Full Text] [PDF]
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 E. J. Wherry and R. Ahmed Memory CD8 T-Cell Differentiation during Viral Infection J. Virol., June 1, 2004; 78(11): 5535 - 5545. [Full Text] [PDF]
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M. J. Nathan, J. E. Mold, S. C. Wood, K. Csencsits, G. Lu, E. J. Eichwald, and D. K. Bishop Requirement for Donor and Recipient CD40 Expression in Cardiac Allograft Rejection: Induction of Th1 Responses and Influence of Donor-Derived Dendritic Cells J. Immunol., June 1, 2004; 172(11): 6626 - 6633. [Abstract] [Full Text] [PDF]
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A. D. H. Doody, J. T. Kovalchin, M. A. Mihalyo, A. T. Hagymasi, C. G. Drake, and A. J. Adler Glycoprotein 96 Can Chaperone Both MHC Class I- and Class II-Restricted Epitopes for In Vivo Presentation, but Selectively Primes CD8+ T Cell Effector Function J. Immunol., May 15, 2004; 172(10): 6087 - 6092. [Abstract] [Full Text] [PDF]
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J. C. Sun and M. J. Bevan Cutting Edge: Long-Lived CD8 Memory and Protective Immunity in the Absence of CD40 Expression on CD8 T Cells J. Immunol., March 15, 2004; 172(6): 3385 - 3389. [Abstract] [Full Text] [PDF]
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K. Benlhassan-Chahour, C. Penit, V. Dioszeghy, F. Vasseur, G. Janvier, Y. Riviere, N. Dereuddre-Bosquet, D. Dormont, R. Le Grand, and B. Vaslin Kinetics of Lymphocyte Proliferation during Primary Immune Response in Macaques Infected with Pathogenic Simian Immunodeficiency Virus SIVmac251: Preliminary Report of the Effect of Early Antiviral Therapy J. Virol., December 1, 2003; 77(23): 12479 - 12493. [Abstract] [Full Text] [PDF]
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B. O. Lee, J. Moyron-Quiroz, J. Rangel-Moreno, K. L. Kusser, L. Hartson, F. Sprague, F. E. Lund, and T. D. Randall CD40, but Not CD154, Expression on B Cells Is Necessary for Optimal Primary B Cell Responses J. Immunol., December 1, 2003; 171(11): 5707 - 5717. [Abstract] [Full Text] [PDF]
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 F. G. Lakkis and M. H. Sayegh Memory T Cells: A Hurdle to Immunologic Tolerance J. Am. Soc. Nephrol., September 1, 2003; 14(9): 2402 - 2410. [Full Text] [PDF]
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B. Samten, B. Wizel, H. Shams, S. E. Weis, P. Klucar, S. Wu, R. Vankayalapati, E. K. Thomas, S. Okada, A. M. Krensky, et al. CD40 Ligand Trimer Enhances the Response of CD8+ T Cells to Mycobacterium tuberculosis J. Immunol., March 15, 2003; 170(6): 3180 - 3186. [Abstract] [Full Text] [PDF]
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M. A. Williams, A. B. Adams, M. B. Walsh, N. Shirasugi, T. M. Onami, T. C. Pearson, R. Ahmed, and C. P. Larsen Primary and Secondary Immunocompetence in Mixed Allogeneic Chimeras J. Immunol., March 1, 2003; 170(5): 2382 - 2389. [Abstract] [Full Text] [PDF]
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L. G. Thebeau and L. A. Morrison Mechanism of Reduced T-Cell Effector Functions and Class-Switched Antibody Responses to Herpes Simplex Virus Type 2 in the Absence of B7 Costimulation J. Virol., February 15, 2003; 77(4): 2426 - 2435. [Abstract] [Full Text] [PDF]
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M. Suresh, H. Molina, M. S. Salvato, D. Mastellos, J. D. Lambris, and M. Sandor Complement Component 3 Is Required for Optimal Expansion of CD8 T Cells During a Systemic Viral Infection J. Immunol., January 15, 2003; 170(2): 788 - 794. [Abstract] [Full Text] [PDF]
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 J. D. Ahlers, I. M. Belyakov, M. Terabe, R. Koka, D. D. Donaldson, E. K. Thomas, and J. A. Berzofsky A push-pull approach to maximize vaccine efficacy: Abrogating suppression with an IL-13 inhibitor while augmenting help with granulocyte/macrophage colony-stimulating factor and CD40L PNAS, October 1, 2002; 99(20): 13020 - 13025. [Abstract] [Full Text] [PDF]
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 C. Bourgeois, B. Rocha, and C. Tanchot A Role for CD40 Expression on CD8+ T Cells in the Generation of CD8+ T Cell Memory Science, September 20, 2002; 297(5589): 2060 - 2063. [Abstract] [Full Text] [PDF]
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H. Wedemeyer, X.-S. He, M. Nascimbeni, A. R. Davis, H. B. Greenberg, J. H. Hoofnagle, T. J. Liang, H. Alter, and B. Rehermann Impaired Effector Function of Hepatitis C Virus-Specific CD8+ T Cells in Chronic Hepatitis C Virus Infection J. Immunol., September 15, 2002; 169(6): 3447 - 3458. [Abstract] [Full Text] [PDF]
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 J. E. Christensen, J. P. Christensen, N. N. Kristensen, N. J. V. Hansen, A. Stryhn, and A. R. Thomsen Role of CD28 co-stimulation in generation and maintenance of virus-specific T cells Int. Immunol., July 1, 2002; 14(7): 701 - 711. [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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A. L. Marzo, V. Vezys, K. Williams, D. F. Tough, and L. Lefrancois Tissue-Level Regulation of Th1 and Th2 Primary and Memory CD4 T Cells in Response to Listeria Infection J. Immunol., May 1, 2002; 168(9): 4504 - 4510. [Abstract] [Full Text] [PDF]
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D. Montagna, R. Maccario, F. Locatelli, V. Rosti, Y. Yang, P. Farness, A. Moretta, P. Comoli, E. Montini, and A. Vitiello Ex vivo priming for long-term maintenance of antileukemia human cytotoxic T cells suggests a general procedure for adoptive immunotherapy Blood, December 1, 2001; 98(12): 3359 - 3366. [Abstract] [Full Text] [PDF]
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M. Suresh, J. K. Whitmire, L. E. Harrington, C. P. Larsen, T. C. Pearson, J. D. Altman, and R. Ahmed Role of CD28-B7 Interactions in Generation and Maintenance of CD8 T Cell Memory J. Immunol., November 15, 2001; 167(10): 5565 - 5573. [Abstract] [Full Text] [PDF]
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S. E. Hamilton, A. R. Tvinnereim, and J. T. Harty Listeria monocytogenes Infection Overcomes the Requirement for CD40 Ligand in Exogenous Antigen Presentation to CD8+ T Cells J. Immunol., November 15, 2001; 167(10): 5603 - 5609. [Abstract] [Full Text] [PDF]
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M. A. Williams, J. T. Tan, A. B. Adams, M. M. Durham, N. Shirasugi, J. K. Whitmire, L. E. Harrington, R. Ahmed, T. C. Pearson, and C. P. Larsen Characterization of Virus-Mediated Inhibition of Mixed Chimerism and Allospecific Tolerance J. Immunol., November 1, 2001; 167(9): 4987 - 4995. [Abstract] [Full Text] [PDF]
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R. Xiang, F. J. Primus, J. M. Ruehlmann, A. G. Niethammer, S. Silletti, H. N. Lode, C. S. Dolman, S. D. Gillies, and R. A. Reisfeld A Dual-Function DNA Vaccine Encoding Carcinoembryonic Antigen and CD40 Ligand Trimer Induces T Cell-Mediated Protective Immunity Against Colon Cancer in Carcinoembryonic Antigen-Transgenic Mice J. Immunol., October 15, 2001; 167(8): 4560 - 4565. [Abstract] [Full Text] [PDF]
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J. D. Ahlers, I. M. Belyakov, S. Matsui, and J. A. Berzofsky Mechanisms of cytokine synergy essential for vaccine protection against viral challenge Int. Immunol., July 1, 2001; 13(7): 897 - 908. [Abstract] [Full Text] [PDF]
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S. M. Ensminger, B. M. Spriewald, H. V. Sorensen, O. Witzke, E. G. Flashman, A. Bushell, P. J. Morris, M. L. Rose, A. Rahemtulla, and K. J. Wood Critical Role for IL-4 in the Development of Transplant Arteriosclerosis in the Absence of CD40-CD154 Costimulation J. Immunol., July 1, 2001; 167(1): 532 - 541. [Abstract] [Full Text] [PDF]
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 Y. Qian, F. Boisgerault, G. Benichou, and M. R. Dana Blockade of CD40-CD154 Costimulatory Pathway Promotes Survival of Allogeneic Corneal Transplants Invest. Ophthalmol. Vis. Sci., April 1, 2001; 42(5): 987 - 994. [Abstract] [Full Text]
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D. Masopust, J. Jiang, H. Shen, and L. Lefrancois Direct Analysis of the Dynamics of the Intestinal Mucosa CD8 T Cell Response to Systemic Virus Infection J. Immunol., February 15, 2001; 166(4): 2348 - 2356. [Abstract] [Full Text] [PDF]
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K. H. Edelmann and C. B. Wilson Role of CD28/CD80-86 and CD40/CD154 Costimulatory Interactions in Host Defense to Primary Herpes Simplex Virus Infection J. Virol., January 15, 2001; 75(2): 612 - 621. [Abstract] [Full Text]
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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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 R. S. Kornbluth The emerging role of CD40 ligand in HIV infection J. Leukoc. Biol., September 1, 2000; 68(3): 373 - 382. [Abstract] [Full Text] [PDF]
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 Y. Wan, J. Bramson, A. Pilon, Q. Zhu, and J. Gauldie Genetically Modified Dendritic Cells Prime Autoreactive T Cells through a Pathway Independent of CD40L and Interleukin 12: Implications for Cancer Vaccines Cancer Res., June 1, 2000; 60(12): 3247 - 3253. [Abstract] [Full Text]
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J. T. Tan, J. K. Whitmire, K. Murali-Krishna, R. Ahmed, J. D. Altman, R. S. Mittler, A. Sette, T. C. Pearson, and C. P. Larsen 4-1BB Costimulation Is Required for Protective Anti-Viral Immunity After Peptide Vaccination J. Immunol., March 1, 2000; 164(5): 2320 - 2325. [Abstract] [Full Text] [PDF]
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J. T. Tan, J. K. Whitmire, R. Ahmed, T. C. Pearson, and C. P. Larsen 4-1BB Ligand, a Member of the TNF Family, Is Important for the Generation of Antiviral CD8 T Cell Responses J. Immunol., November 1, 1999; 163(9): 4859 - 4868. [Abstract] [Full Text] [PDF]
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10-fold lower in CD40L-deficient mice (open bars).
Purified CD4 T cells were analyzed by IFN-
