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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, the concept of effective help for CD8 T cell responses has been explored with regard to the involvement of CD40-CD40L interactions (10, 11, 12, 13, 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, 17, 18) and at least some CD8 T cells (19, 20, 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 Ag-specific 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
This method was adopted from Kearney et al. (30). A total of 2 x 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 µg of anti-CD40 mAb (clone 3/23) (31) or rat Ig as control. Each experiment was performed a minimum of three times.
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 µg
anti-CD40 mAb or 100 µg 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 mAb-coated 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 x 106-1 x
107 cells/ml, followed by incubation at 4°C for
30 min with 100 µl 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 µg/ml of the OVA-derived peptide SIINFEKL. Serial dilutions of effector cells were incubated in 96-well round-bottom microtiter plates with 2.5 x 103 target cells for 6 h at 37°C. Percent specific lysis was calculated as: 100 x [(cpm released with effectors) - (cpm released alone)]/[(cpm released by detergent) - (cpm released alone)].
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µg 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. 1
A). 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 µg 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 anti-CD40 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).
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). 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% 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.
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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 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, 12, 13).
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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 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.
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). 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.
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The eventual outcome of activation of OT-I cells via immunization
with sOVA in the absence of CD40 triggering was deletion of the 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, 39, 40, 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 µg 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).
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). CD11a expression, which is increased
on CD8 memory cells, was also analyzed. In mice that had been immunized
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.
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). However, unlike naive OT-I
cells, the long-lived 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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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 CD28-mediated
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, 48, 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, 12, 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, 52, 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 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, 56, 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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Footnotes |
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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:
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.
Received for publication September 7, 1999. Accepted for publication November 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nature 333:855.[Medline]
+ intraepithelial lymphocytes. Science 243:1716.v5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.This article has been cited by other articles:
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A. Th. den Boer, L. Diehl, G. J. D. van Mierlo, E. I. H. van der Voort, M. F. Fransen, P. Krimpenfort, C. J. M. Melief, R. Offringa, and R. E. M. Toes Longevity of Antigen Presentation and Activation Status of APC Are Decisive Factors in the Balance Between CTL Immunity Versus Tolerance J. Immunol., September 1, 2001; 167(5): 2522 - 2528. [Abstract] [Full Text] [PDF]
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M. S. Rolph and S. H. E. Kaufmann CD40 Signaling Converts a Minimally Immunogenic Antigen into a Potent Vaccine Against the Intracellular Pathogen Listeria monocytogenes J. Immunol., April 15, 2001; 166(8): 5115 - 5121. [Abstract] [Full Text] [PDF]
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 M. Sherritt, L. Cooper, D. J. Moss, N. Kienzle, J. Altman, and R. Khanna Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters Int. Immunol., March 1, 2001; 13(3): 265 - 271. [Abstract] [Full Text] [PDF]
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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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J. M.M. den Haan, S. M. Lehar, and M. J. Bevan Cd8+ but Not Cd8- Dendritic Cells Cross-Prime Cytotoxic T Cells in Vivo J. Exp. Med., December 18, 2000; 192(12): 1685 - 1696. [Abstract] [Full Text] [PDF]
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) or control rat Ig (rIg, •). 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.
, PLN cells from OT-I-transferred plus
rIg-treated mice; •, spleen cells from OVA plus 
, spleen cells from OVA plus rIg-treated 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%.
, 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%.
,