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Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-4, IL-5) within 12–24 h of Ag exposure and did not require multiple
cycles of cell division to do so. Memory cells proliferated at lower Ag
concentrations than did naive cells, were less dependent on
costimulation by B7 molecules, and independent of costimulation by
CD40. Furthermore, effector cytokine production by memory cells also
occurred in the absence of either B7 or CD40 costimulation. Lastly,
memory cells were resistant to tolerance induction. Together, these
findings suggest that the threshold for activation of memory
CD4+ cells is lower than that of naive cells. This would
permit memory cells to rapidly express their effector functions in vivo
earlier in the course of a secondary immune response, when the levels
of Ag and the availability of costimulation may be relatively
low. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the earliest features shown to distinguish memory and naive T cells was their migration pattern. Naive T cells preferentially migrate to peripheral lymph nodes, presumably due to high levels of L-selectin expression, whereas memory cells migrate to peripheral sites of inflammation (2, 4, 5, 6). Additionally, T cells exhibiting a "memory" phenotype can be identified both in the peripheral circulation and spleen (2, 7). While several studies have examined the homing of naive vs memory cells, these were all based on identification of populations by phenotypic markers (CD45RB and L-selectin) rather than Ag specificity (2, 6, 8, 9). Recent work suggests that such markers may be influenced by the state of activation of the cell, the manner in which the memory population was generated, and the presence of Ag, and thus may not be reliable for distinguishing naive, effector, and memory populations (10, 11, 12, 13, 14, 15, 16, 17, 18).
The functional capacities of memory cells are also not definitively
established. It is generally accepted that memory responses are more
rapid than primary responses, and this rapidity forms the basis for
protective immunity. Memory CD8+ lymphocytes,
induced by virus infection, can be readily induced to secrete cytokines
and become active CTLs upon Ag re-encounter (3, 19, 20, 21, 22, 23).
Unlike their naive counterparts, memory CD4+
cells produce effector cytokines such as IFN- and IL-4 upon Ag
encounter, thus promoting vigorous phagocyte and B cell responses and
elimination of the pathogen (2, 17, 24). The basis for
this difference in response kinetics between naive and memory cells is
unclear. By comparing responses of naive and memory T cells to Ags
presented by different APCs, it has been proposed that memory cells are
less costimulator dependent than naive cells, and that they may require
lower doses of Ag for activation (2, 25, 26). Other
characteristics of memory cells, such as their susceptibility to
tolerance induction, are also unclear as different studies suggest that
memory cells are more or less sensitive to anergy than naive cells
(27, 28, 29, 30).
We have previously demonstrated that adoptive transfer of in vitro-activated CD4+ T cells derived from the DO.11 TCR transgenic mouse into syngeneic BALB/c mice leads to the development of a stable population of OVA323–339-specific long-surviving cells that can be identified by staining with a clonotypic Ab (KJ1-26) (17). These long-lived KJ1-26+ cells possess characteristics believed to be typical of memory cells (17). Therefore, we used this adoptive transfer system to analyze the functional capacities of Ag-specific memory CD4+ T cells. Our results demonstrate that memory cells localize to both the spleen and peripheral lymph nodes of mice, and that memory cells present in the spleen are rapidly activated to migrate away from the spleen after subcutaneous Ag exposure. Furthermore, we show that memory cells produce effector cytokines in the absence of cell cycling, and proliferate at Ag concentrations significantly lower than those required by naive cells. Lastly, our data demonstrate that memory cells are less dependent on costimulation by B7 molecules than naive cells and, consequently, less sensitive to tolerance induction in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice, 6–8 wk of age, were purchased from The Jackson
Laboratory (Bar Harbor, ME). Transgenic mice expressing the DO.11.10
TCR (DO.11), specific for the chicken OVA peptide
OVA323–339 in the context of the MHC class II
molecule I-Ad, were obtained from Dr. D. Loh
(Hoffmann-La Roche, Nutley, NJ) (31). Mice deficient in
B7.1 and B7.2 on the BALB/c background were obtained from Dr. Arlene
Sharpe (Brigham and Women’s Hospital, Boston, MA) (32).
CD40-deficient mice on the BALB/c background were obtained from The
Jackson Laboratory (33). These mice were bred in our
pathogen- and viral Ab-free facility in accordance with the guidelines
of the Committee on Animals of the Harvard Medical School, and those
prepared by the Committee on Care and Use of Laboratory Animals of the
Institute of Laboratory Resources, National Research Council
(Washington, D.C). The mice were typed for the DO.11 TCR by staining
peripheral blood cells with Abs against CD4 and Vß8 (present in the
transgenic TCR). In the DO.11 mouse, 
90% of the
CD4+ T cells express the transgene-derived Vß8
gene compared with the wild-type syngeneic mouse (BALB/c), in which
only 5% of the CD4+ T cells are
Vß8+. In the DO.11 mouse, only 40–60% of the
CD4+ T cells are recognized by the clonotypic Ab
(KJ1-26) due to pairing of the transgenic Vß8 chain with endogenous
(nontransgenic) 
-chains. The B7-deficient mice were typed by
Southern blotting and PCR (32).
Adoptive transfers and FACS analysis
All adoptive transfers were performed by injection of cells via
the tail vein. Naive DO.11 T cells were prepared by harvesting
peripheral lymph nodes from DO.11 mice and purifying
CD4+ cells using Dynabeads (Dynal, Oslo, Norway).
In vitro-activated cells were prepared by culturing naive
CD4+ DO.11 T cells with mitomycin C-treated
splenocytes from BALB/c mice (as APCs) and OVA peptide at 1 µg/ml in
24-well plates for 96 h in RPMI 1640 supplemented with 1 mM
L-glutamine, penicillin, streptomycin, nonessential amino
acids, sodium pyruvate, HEPES (all from Life Technologies, Grand
Island, NY), 5 x 105 M 2-ME, and 10% FBS
(Sigma, St. Louis, MO). The cells were then harvested and ficolled
(lymphocyte separation medium; Oregon Teknika, Durham, NC), before
transfer. For adoptive transfer of naive cells into BALB/c recipients,
lymph node and spleen cells were harvested from DO.11 mice. The number
of T cells expressing the DO.11 TCR in the naive and activated DO.11
populations was measured by staining with the clonotypic Ab, KJ1-26,
and flow cytometry. Next, 5–15 x 106 naive
KJ1-26+CD4+ T cells (in
30–60 x 106 whole spleen/lymph node
DO.11 mouse-derived cells) were transferred into BALB/c recipients, or,
to generate memory cells, 20–25 x 106 in
vitro-activated DO.11 T cells were transferred into BALB/c recipients.
For the in vitro analyses of memory responses, the lymph nodes and
spleens were collected from recipients of activated cells 6–7 mo after
transfer and the CD4+ cells were purified using
Dynabeads. For flow cytometry, the cell suspensions were blocked with
anti-CD16/CD32 (mouse Fc receptor), then stained with CyC-labeled
anti-CD4 mAb (both from PharMingen, San Diego, CA) and biotinlyated
KJ1-26 clonotypic Ab followed by streptavidin-PE. Some samples were
then stained with FITC-labeled Abs to CD25, CD44, CD45RB, CD40L, CD62L,
IL-2Rß, B7.1, or B7.2 (PharMingen). Analyses were performed on a
FACScalibur flow cytometer (Becton Dickinson, San Jose, CA).
In vitro proliferation and cytokine assays
To analyze proliferative responses of the naive and memory
cells, 5000 CD4+KJ1-26+ naive cells (purified from
DO.11 lymph nodes) or memory cells (purified from transfer recipients)
were cocultured with 2.5 x 105 mitomycin
treated BALB/c, B71/2-/- or
CD40-/- APCs in 0.2 ml of RPMI 1640
supplemented as described above in 96-well plates. Cells were
stimulated with 0–1 µg/ml of OVA peptide. After 24, 48, 72, or
96 h, cultures were pulsed for 6 h with 1 µCi
[3H]thymidine (New England Nuclear, Boston, MA), and
incorporated radioactivity was measured in a Betaplate scintillation
counter (LBK Pharmacia, Piscataway, NJ). To determine cytokine
production, 5 x 104
CD4+KJ1–26+ cells were
cultured with 2.5 x 106 mitomycin C treated
BALB/c APCs in 1 ml of medium in the presence of 0–1 µg/ml of OVA
peptide. Supernatants were collected after 0, 24, 48, and 72 h,
and levels of IL-2, IL-4, IL-5 and IFN- were assayed by ELISA as
previously described (34). Proliferative responses of
untreated, immunized or tolerized naive and memory cells were assessed
as above by culturing 5 x 105 total lymph
node cells in each well in the 96-well plate without additional APCs.
To measure cytokine responses of untreated, immunized, or tolerized
naive and memory cells, 4 x 106 total lymph
node cells were cultured in 24-well plates without or with OVA peptide
at 1 µg/ml, and cytokine levels in the supernatants were assayed as
above.
Fluorochrome labeling of cells and FACS analysis
To accurately assess cycling, naive cells from the lymph nodes of DO.11 mice or memory cells harvested from transfer recipients were labeled with chloromethylfluorescein diacetate succinimidyl ester (CFSE;4 Molecular Probes, Eugene, OR) (35). Cells were suspended at 10 x 106/ml in RPMI 1640 with no FCS, and the CFSE was added to a final concentration of 1 µM. The cells were incubated for 12 min at 37°C, at which time the labeling was terminated by the addition of cold RPMI 1640. CFSE is lipophilic and passively enters the cell where it is converted to the fluorescent 5-chloromethylfluorescein by esterase hydrolysis and can no longer diffuse out of the cell. With repeated cell division, the fluorochrome is gradually lost from the cell, thus serving as an indicator of cell cycle status. At 12, 36, 60, and 84 h after culture, the cells were collected, stained with biotinylated KJ1-26 followed by streptavidin PE, and analyzed by flow cytometry.
Immunohistochemistry
A total of 15 x 106 naive or in vitro-activated KJ1-26+ T cells were adoptively transferred into unirradiated BALB/c recipients by tail vein injection. At 10 wk after transfer, recipients were either not immunized or immunized with 150 µg of OVA peptide emulsified in IFA (Difco, Detroit, MI) by s.c. injection in four to six sites along the back. The peripheral lymph nodes (submandibular, axillary, brachial, inguinal, and popliteal for nonimmunized; axillary, brachial, and inguinal for immunized) and spleens were harvested on day 0, 1, or 5 after immunization. The tissues were immersed in OCT (TissueTek; Miles, Elkhart, IN), flash frozen, cut into 6-µm thick sections, fixed in ice cold acetone for 10 min, and allowed to dry overnight. The sections were rehydrated in PBS with 0.1% Tween 20 for 10 min, then blocked with 5% BSA in PBS with anti-CD16/CD32 (mouse Fc receptor) added at 5 µg/ml for 60 min at room temperature. The sections were then incubated with FITC-labeled anti-B220 (PharMingen) at 5 µg/ml and biotinylated KJ1-26 at 2.5 µg/ml for 60 min at room temperature. The slides were washed in PBS with 0.1% Tween 20 for 15 min, then incubated with streptavidin-alkaline phosphatase (PharMingen) at a 1:1000 dilution and anti-FITC-HRP (Boehringer Mannheim, Indianapolis, IN) at a 1:100 dilution for 30 min at room temperature. The slides were washed with PBS for 15 min, and the alkaline phosphatase was developed first using the following mix: 5 mg napthol AS-MX phosphate in 250 µl N,N-dimethyl formamide was added to 40 ml 0.1 M Tris-HCl, pH 8.5; 10 mg fast blue base in 250 µl 2 N HCl and 250 µl 4% sodium nitrite was added to the Tris buffer, after which 10 mg of levamisole was added to inhibit endogenous alkaline phosphatase activity. The mixture was filtered, placed on the tissue sections, and the slides were incubated in the dark for 15–60 min. After the color had developed, the slides were washed in PBS for 15 min and the horseradish peroxidase was developed using the following mix: 4 mg of 3-amino-9-ethylcarbazole dissolved in 250 µl of N,N-dimethyl formamide was added to 9.75 ml of 0.05 M sodium acetate buffer, pH 5.0. This was filtered, 5 µl of 30% H2O2 was added, and the buffer was placed on the tissue sections. All chemicals for color development were obtained from Sigma. Color developing was terminated by a final wash in PBS, after which the slides were mounted using Biomedia Crystal Mount (Biomedia, Foster City, CA).
Tolerance induction
A total of 20–25 x 106 in vitro-activated DO.11 T cells or 5 x 106 naive cells were prepared as described above and adoptively transferred into BALB/c. At 6 wk or 9 mo after transfer of activated cells, or 1–2 days after transfer of naive cells, recipients were either not immunized, immunized with 150 µg of OVA peptide emulsified in IFA by s.c. injection along the back, or tolerized by tail vein injection of 300 µg of OVA peptide in PBS (this dose of OVA peptide has been previously shown to induce tolerance in the peripheral lymph node cells of adoptively transferred naive DO.11 T cells (34, 36, 37)). The peripheral lymph nodes (submandibular, axillary, brachial, inguinal, and popliteal for untreated and tolerized; axillary, brachial, and inguinal for immunized) were harvested 3 days after immunization. In vitro proliferation and cytokine assays were performed as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To provide a source of Ag-specific memory
CD4+ T cells, DO.11 T cells were activated in
vitro with peptide and syngeneic APCs and adoptively transferred into
syngeneic mice (BALB/c). This results in the development of a
long-lived population of quiescent cells that survive in the absence of
overt Ag exposure and can be detected as long as 9 mo after transfer,
without additional Ag exposure (Fig. 1
A and Ref. 17).
These cells express high levels of CD44 and low levels of CD25 (Fig. 1B), while naive KJ1-26+
CD4+ cells purified from the peripheral lymph
nodes of DO.11 mice express low levels of CD44 and CD25 (Fig. 1B). Additionally, we found that the memory
KJ1-26+ cells express identical levels of CD45RB,
IL-2Rß, and CD40L as those expressed by their naive counterparts
(Fig. 1B). L-selectin (CD62L) expression is biphasic on the
KJ1-26+ memory cells, with the majority of the
population having up-regulated expression to levels similar to those on
naive cells (Fig. 1B and Ref. 17).
Interestingly, memory cells express slightly higher levels of B7-1 and
B7-2 than naive cells. In summary, as with CD8+ memory cells
(38), it appears that, at least in our system, the most
reliable phenotype of CD4+ memory cells is
CD25low and CD44high.
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A). Each population was labeled with CFSE before culture
with APCs and Ag. At various times after culture initiation, the cells
were collected and analyzed by flow cytometry for CFSE expression in
the CD4+KJ1-26+ cells.
By 36 h of culture, a small amount of proliferation (as assessed
by loss of CFSE content) was noted in the memory
KJ1-26+ cells cultured with 1 µg/ml of OVA
peptide; no cycling was noted in the naive population (Fig. 2). At 60 h of culture, significant
cycling of the memory cells cultured with 1 µg/ml of OVA was evident;
at lower Ag concentrations (0.1 µg/ml), the memory cells showed much
more cycling than the naive cells. By 84 h, proliferation of
memory cells was noted at 0.01 µg/ml of OVA, and proliferation of
naive cells was noted at 0.1 µg/ml of OVA. These experiments indicate
that memory KJ1-26+ cells are capable of
proliferating more rapidly and at a log lower concentration of OVA than
their naive counterparts.
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). Interestingly, the memory cells also
began producing IL-4 at 12 h of Ag exposure at both 0.1 and 1
µg/ml of OVA. A small amount of IFN- was noted at 12 h when
memory cells were cultured with 1 µg/ml of OVA; this increased
dramatically by 36 h at both high and low peptide concentrations.
Production of IL-5 by memory cells appeared later and correlated with
cell cycling, as IL-5 was not detected until 60 h of
culture. No IFN-, IL-4, or IL-5 was found in supernatants from the
naive cell cultures. These results indicate that memory cells produce
effector cytokines before they begin to cycle, and thus can initiate an
effector response without the need for multiple rounds of clonal
expansion.
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Based on experiments using CD45RBlowCD4+ T cells cultured on dendritic cells or B cells as APCs, it has been postulated that memory cells are less reliant than naive cells on costimulation for activation, presumably because their threshold for activation is lower than that of naive cells (25, 26). However, the role of defined costimulators in the activation of memory cells has not been examined. To directly test this, we generated populations of CD4+ cells containing equal numbers of naive and memory KJ1-26+ cells (as described above) and analyzed their proliferative responses to costimulator-deficient APCs. We chose to use APCs deficient in B7.1/B7.2 or CD40, as the B7:CD28 and CD40:CD40L pathways of costimulation are known to be critical in activation of naive cells. The naive or memory cells were cultured with the various APCs at a ratio of 50:1 (APC:KJ1-26+ T cell) and analyzed at day 1–4 of culture for proliferation and cytokine production.
By 24 h of culture, a small amount of
[3H]thymidine incorporation was noted in the
memory population stimulated with all APCs, although this required a
log higher peptide concentration on B7-deficient APCs (Fig. 4). No significant incorporation was seen
in the naive population. At 48 h, the naive cells were beginning
to proliferate on the wild-type APCs, while minimal proliferation was
noted on B7- or CD40-deficient APCs. In contrast, the memory cells were
proliferating well on all APCs, although again, the B7-deficient APCs
required a log higher dose of peptide. These trends were maintained at
both 72 and 96 h of culture: proliferation of the naive cells was
severely impaired on both B7- and CD40-deficient APCs, while
proliferation of memory cells occurred on all APCs. Thus, the memory T
cells proliferate normally in the absence of CD40, and also proliferate
in the absence of B7 at high Ag concentration, while naive cells were
significantly impaired. Similar results were seen at APC:T cell ratios
of 25:1 and 5:1, although all responses were lower at reduced APC
numbers (data not shown). A small amount of proliferation was noted in
the memory cells cultured with peptide in the absence of APCs. This is
most likely attributable to the presence of a small number of
contaminating APCs, as the purified CD4+ cells
contain 2–4% non-CD4+ T cells. Although the
purified naive population also contained a similar number of
contaminating cells, no proliferation was noted in this group when OVA
peptide was added without APCs. This supports the conclusion that the
threshold for activation of memory cells is much lower than that of
naive cells, as they are capable of proliferating in the presence of
extremely small numbers of APCs.
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). These results
support the notion that the effector function of memory cells is not
dependent on costimulation through CD28:B7 or CD40:CD40L
interactions.
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The tissue localization of memory cells in vivo has not been clearly defined. While flow cytometric analysis of tissues containing Ag-specific memory cells is useful for quantitation, it provides no information about the anatomic compartmentalization of the population. To examine the location of memory cells in vivo, we used immunohistochemical analysis of lymphoid tissues to identify KJ1-26+ cells in mice 6 to 10 wk after adoptive transfer of in vitro-activated DO.11 T cells. For comparison, tissues from mice that had received naive KJ1-26+ cells were similarly analyzed. To follow the responses of the naive or memory cells to Ag, transfer recipients were immunized with OVA peptide in IFA by s.c. injection and lymphoid tissues were collected at day 0, 1, and 5 after Ag exposure.
At 10 wk after transfer, the in vitro-primed DO.11 lymphocytes (memory
cells) were detectable in the T cell zones of the lymph nodes and
spleen (Fig. 6) as well as circulating in
the blood (data not shown). Upon Ag challenge, there was a rapid
expansion of the T cells in the regional lymph nodes, beginning 24
h after immunization and increasing significantly by 5 days
postchallenge. Strikingly, in the spleen, the memory cells began
expanding 24 h after immunization, but then decreased in number
between days 3 and 5 postchallenge. This is most likely due to rapid
migration from the spleen to the site of Ag exposure and probably to
the lymph nodes draining the site of Ag challenge. At the same time (10
wk posttransfer), naive DO.11 cells were virtually undetectable by
immunohistochemistry. However, they are present, because they too could
be expanded by Ag challenge (Fig. 6). Interestingly, at the time when
memory cells had left the spleen (day 5), the naive cells (now
activated) begin expanding in the spleens of recipient mice.
These results indicate that the more rapid activation of memory
cells seen in vitro is mirrored in their responses to Ag challenge in
vivo. The rapid migration of memory cells from the spleen may be due to
the fact that these cells express adhesion molecules that permit
immediate entry into the vasculature upon activation.
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The question of whether or not memory cells can be tolerized is
largely unresolved. The system of adoptively transferring TCR
transgenic T cells is well-suited to studies of tolerance induction in
vivo. Previous work from several laboratories has demonstrated that the
administration of aqueous OVA peptide by i.v. injection to adoptive
transfer recipients of naive DO.11 T cells leads to functional
unresponsiveness in these T cells (34, 36, 37, 40, 41).
This is manifested by an abortive expansion of
KJ1-26+ cells in lymph nodes, and failure of
these cells to enter B cell follicles. Functionally, the tolerized
cells respond poorly to Ag restimulation in vitro and fail to produce
either IL-2 or IFN- (34, 36, 37, 40, 41).
To generate a population of memory cells for the tolerance experiments,
20–25 x 106 in vitro-activated DO.11 T
cells were transferred into BALB/c recipients. These cells were allowed
to remain quiescent in the recipient mice for 6 wk or 9 mo. Three days
before tolerance induction, an additional group of BALB/c mice was
transferred with 5 x 106 naive
KJ1-26+ cells. The mice were either left
untreated or given OVA peptide in IFA by s.c. injection (immunized) or
OVA peptide in PBS by tail vein injection (tolerized). After 3 days,
the peripheral lymph nodes from the mice were collected and analyzed
for the remaining percent of KJ1-26+ cells.
Untreated recipients of naive and memory cells had similar numbers of
KJ1-26+ cells in the lymph nodes (Fig. 7A). Upon encounter with Ag in
adjuvant, the naive and memory KJ1-26+ cells
expanded equivalently.
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B). In striking contrast, the memory T cells showed no
significant defect after exposure to tolerogenic Ag, and this was
seen even in memory cells recovered 9 mo after adoptive transfer of in
vitro-activated T cells. Therefore, these studies suggest that memory
cells are resistant to tolerance induced by a large dose of
aqueous Ag. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data indicate that memory cells enter the cell cycle upon Ag
stimulation much more rapidly than do naive cells and produce effector
cytokines such as IFN- and IL-4 before cycling (Figs. 2 and 3). This
suggests that memory T cells require little or no cycling to produce
effector cytokines; this is in direct contrast to naive cells, which
require at least four cell divisions to produce IL-4 (42).
As effector cytokine production by naive cells has been demonstrated to
be dependent on changes in the chromatin, including alterations in
methylation status and accessibility for transcription factors
(42), our results suggest that the chromatin changes
induced during T cell activation may be maintained during memory. Thus,
as activated cells progress into quiescence and memory, the status of
the effector cytokine genes may not be significantly altered (e.g.,
methylated), allowing rapid cytokine gene expression upon reactivation.
This would permit rapid effector function upon Ag challenge, without a
need for clonal expansion.
It has been suggested that memory cells require less costimulation than
do naive cells for activation upon Ag encounter. This conclusion is
based on comparing different APCs for their ability to activate
effector and/or memory cells (2, 25, 26). Thus, the role
of costimulation has been inferred from the expected properties of the
APCs. We have examined this question directly, using APCs from knockout
mice in which the two major costimulatory pathways for T cells are
deleted. Our data indicate that memory cells are less dependent on B7
costimulation than their naive counterparts (Fig. 4). Furthermore, the
activation of memory cells does not require the presence of CD40 on
APCs (Fig. 4). Our results also demonstrate that memory cells are
activated at lower peptide concentrations than naive cells (Figs. 2 and 4). These characteristics of memory cells would permit the induction of
a memory response in the presence of limiting Ag and/or inflammation,
as may be the case very early after pathogen encounter. The rapid
subsequent effector response would serve to eliminate the pathogen.
Immunohistochemical analysis of memory T cells shows that contrary to
previous reports, memory cells do localize to the lymph nodes (Fig. 6).
This is likely because memory T cells re-express L-selectin when they
reach a stage of quiescence (Fig. 1B and Ref.
17). Indeed, recent experiments on memory cells in rats
demonstrated that CD4+ T cells expressing a
memory phenotype entered lymph nodes across the high endothelial
venules as well as via afferent lymphatics (43, 44). It is
also clear that memory cells are capable of mobilizing rapidly in
response to distant Ag, as the KJ1-26+ memory
cells present in the spleens of transfer recipients were rapidly
activated and left the spleen within 3–5 days of s.c. Ag exposure
(Fig. 6).
Finally, our data demonstrate that memory cells are resistant to
tolerance induction (Fig. 7). Anergy induction in naive cells in vivo
is believed to occur when these cells encounter Ag under conditions of
limited costimulation (i.e., low levels of B7 and absence of
inflammation) (34, 37). In this situation, B7:CTLA-4
interactions would predominate over B7:CD28 interactions, leading to
inhibition of IL-2 production and cell cycling (45, 46).
As memory cells are less dependent on B7 costimulation, the
differential in B7 expression normally important in controlling naive T
cell responses would be less consequential for memory cells. The reason
for a decreased dependence on B7 costimulation for the activation of
memory cells is not known. It is possible that memory cells differ from
naive T cells in TCR-induced signaling pathways, resulting in a lower
activation threshold. The rapid production of IFN- or IL-4 by memory
cells without cycling would also serve to limit the capacity for anergy
induction in this population.
In conclusion, the studies presented in this paper provide direct evidence that memory cells are capable of inducing rapid effector responses in the presence of limiting Ag and costimulation. Furthermore, unlike the case with naive cells, production of effector cytokines by memory cells does not require multiple rounds of cell division. These properties of memory cells ensure an expeditious and effective response to Ag challenge. The relative costimulator independence and tolerance resistance of memory T cells also raises the possibility that strategies for tolerance induction in naive cells, such as costimulator blockade in transplantation, may not be as effective for established immune responses.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Surgical and Radiological Sciences, University of California School of Veterinary Medicine, 2112 Tupper Hall, One Shields Avenue, Davis, CA 95616. E-mail address:
3 Address correspondence and reprint requests to Abul K. Abbas at the current address: Department of Pathology, University of California School of Medicine, S534, 513 Parnassus Avenue, San Francisco, CA 94143. E-mail address:
4 Abbreviation used in this paper: CFSE, chloromethylfluorescein diacetate succinimidyl ester.
Received for publication June 25, 1999. Accepted for publication October 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 W. Elyaman, P. Kivisakk, J. Reddy, T. Chitnis, K. Raddassi, J. Imitola, E. Bradshaw, V. K. Kuchroo, H. Yagita, M. H. Sayegh, et al. Distinct Functions of Autoreactive Memory and Effector CD4+ T Cells in Experimental Autoimmune Encephalomyelitis Am. J. Pathol., August 1, 2008; 173(2): 411 - 422. [Abstract] [Full Text] [PDF]
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A. E. Anderson, B. L. Sayers, M. A. Haniffa, D. J. Swan, J. Diboll, X.-N. Wang, J. D. Isaacs, and C. M. U. Hilkens Differential regulation of naive and memory CD4+ T cells by alternatively activated dendritic cells J. Leukoc. Biol., July 1, 2008; 84(1): 124 - 133. [Abstract] [Full Text] [PDF]
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N. Perez, S. Karumuthil-Melethil, R. Li, B. S. Prabhakar, M. J. Holterman, and C. Vasu Preferential Costimulation by CD80 Results in IL-10-Dependent TGF-{beta}1+-Adaptive Regulatory T Cell Generation J. Immunol., May 15, 2008; 180(10): 6566 - 6576. [Abstract] [Full Text] [PDF]
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 T. J. Kenna, R. Thomas, and R. J. Steptoe Steady-state dendritic cells expressing cognate antigen terminate memory CD8+ T-cell responses Blood, February 15, 2008; 111(4): 2091 - 2100. [Abstract] [Full Text] [PDF]
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S. Fuse, W. Zhang, and E. J. Usherwood Control of Memory CD8+ T Cell Differentiation by CD80/CD86-CD28 Costimulation and Restoration by IL-2 during the Recall Response J. Immunol., January 15, 2008; 180(2): 1148 - 1157. [Abstract] [Full Text] [PDF]
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A. B. Borowski, A. C. Boesteanu, Y. M. Mueller, C. Carafides, D. J. Topham, J. D. Altman, S. R. Jennings, and P. D. Katsikis Memory CD8+ T Cells Require CD28 Costimulation J. Immunol., November 15, 2007; 179(10): 6494 - 6503. [Abstract] [Full Text] [PDF]
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P. Soroosh, S. Ine, K. Sugamura, and N. Ishii Differential Requirements for OX40 Signals on Generation of Effector and Central Memory CD4+ T Cells J. Immunol., October 15, 2007; 179(8): 5014 - 5023. [Abstract] [Full Text] [PDF]
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S. K. O'Neill, Y. Cao, K. M. Hamel, P. D. Doodes, G. Hutas, and A. Finnegan Expression of CD80/86 on B Cells Is Essential for Autoreactive T Cell Activation and the Development of Arthritis J. Immunol., October 15, 2007; 179(8): 5109 - 5116. [Abstract] [Full Text] [PDF]
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S. L. Shiao, N. C. Kirkiles-Smith, B. R. Shepherd, J. M. McNiff, E. J. Carr, and J. S. Pober Human Effector Memory CD4+ T Cells Directly Recognize Allogeneic Endothelial Cells In Vitro and In Vivo J. Immunol., October 1, 2007; 179(7): 4397 - 4404. [Abstract] [Full Text] [PDF]
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N. Singh, P. R. Chandler, Y. Seki, B. Baban, M. Takezaki, D. J. Kahler, D. H. Munn, C. P. Larsen, A. L. Mellor, and M. Iwashima Role of CD28 in fatal autoimmune disorder in scurfy mice Blood, August 15, 2007; 110(4): 1199 - 1206. [Abstract] [Full Text] [PDF]
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 A. Valujskikh and X. C. Li Frontiers in Nephrology: T Cell Memory as a Barrier to Transplant Tolerance J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2252 - 2261. [Full Text] [PDF]
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L. K. Selin and M. A. Brehm Frontiers in Nephrology: Heterologous Immunity, T Cell Cross-Reactivity, and Alloreactivity J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2268 - 2277. [Abstract] [Full Text] [PDF]
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 T. Kallinich, K. C. Beier, U. Wahn, P. Stock, and E. Hamelmann T-cell co-stimulatory molecules: their role in allergic immune reactions Eur. Respir. J., June 1, 2007; 29(6): 1246 - 1255. [Abstract] [Full Text] [PDF]
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V. Mirenda, S. J. Jarmin, R. David, J. Dyson, D. Scott, Y. Gu, R. I. Lechler, K. Okkenhaug, and F. M. Marelli-Berg Physiologic and aberrant regulation of memory T-cell trafficking by the costimulatory molecule CD28 Blood, April 1, 2007; 109(7): 2968 - 2977. [Abstract] [Full Text] [PDF]
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M. Shi, S. Hao, T. Chan, and J. Xiang CD4+ T cells stimulate memory CD8+ T cell expansion via acquired pMHC I complexes and costimulatory molecules, and IL-2 secretion J. Leukoc. Biol., December 1, 2006; 80(6): 1354 - 1363. [Abstract] [Full Text] [PDF]
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M. P. Ndejembi, J. R. Teijaro, D. S. Patke, A. W. Bingaman, M. R. Chandok, A. Azimzadeh, S. G. Nadler, and D. L. Farber Control of Memory CD4 T Cell Recall by the CD28/B7 Costimulatory Pathway J. Immunol., December 1, 2006; 177(11): 7698 - 7706. [Abstract] [Full Text] [PDF]
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K. D. Klonowski, A. L. Marzo, K. J. Williams, S.-J. Lee, Q.-M. Pham, and L. Lefrancois CD8 T Cell Recall Responses Are Regulated by the Tissue Tropism of the Memory Cell and Pathogen J. Immunol., November 15, 2006; 177(10): 6738 - 6746. [Abstract] [Full Text] [PDF]
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A. H.-M. Tan, S.-C. Wong, and K.-P. Lam Regulation of Mouse Inducible Costimulator (ICOS) Expression by Fyn-NFATc2 and ERK Signaling in T Cells J. Biol. Chem., September 29, 2006; 281(39): 28666 - 28678. [Abstract] [Full Text] [PDF]
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 M. P. Watson, A. J. T. George, and D. F. P. Larkin Differential effects of costimulatory pathway modulation on corneal allograft survival. Invest. Ophthalmol. Vis. Sci., August 1, 2006; 47(8): 3417 - 3422. [Abstract] [Full Text] [PDF]
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P. M. Gray, S. L. Reiner, D. F. Smith, P. M. Kaye, and P. Scott Antigen-Experienced T Cells Limit the Priming of Naive T Cells during Infection with Leishmania major J. Immunol., July 15, 2006; 177(2): 925 - 933. [Abstract] [Full Text] [PDF]
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 M. Shi and J. Xiang CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation Int. Immunol., June 1, 2006; 18(6): 887 - 895. [Abstract] [Full Text] [PDF]
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 M. MacLeod, M. J. Kwakkenbos, A. Crawford, S. Brown, B. Stockinger, K. Schepers, T. Schumacher, and D. Gray CD4 memory T cells survive and proliferate but fail to differentiate in the absence of CD40 J. Exp. Med., April 17, 2006; 203(4): 897 - 906. [Abstract] [Full Text] [PDF]
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S. Yang, J. W. Hodge, D. W. Grosenbach, and J. Schlom Vaccines with Enhanced Costimulation Maintain High Avidity Memory CTL J. Immunol., September 15, 2005; 175(6): 3715 - 3723. [Abstract] [Full Text] [PDF]
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 R. Irons, P. Pinge-Filho, and K. L. Fritsche Dietary (n-3) Polyunsaturated Fatty Acids Do Not Affect the In Vivo Development and Function of Listeria-Specific CD4+ and CD8+ Effector and Memory/Effector T Cells in Mice J. Nutr., May 1, 2005; 135(5): 1151 - 1156. [Abstract] [Full Text] [PDF]
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L. S. van Rijt, S. Jung, A. KleinJan, N. Vos, M. Willart, C. Duez, H. C. Hoogsteden, and B. N. Lambrecht In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma J. Exp. Med., March 21, 2005; 201(6): 981 - 991. [Abstract] [Full Text] [PDF]
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 A Davidson, B Diamond, D Wofsy, and D Daikh Block and tackle: CTLA4Ig takes on lupus Lupus, March 1, 2005; 14(3): 197 - 203. [Abstract] [PDF]
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T. N. J. Bullock and H. Yagita Induction of CD70 on Dendritic Cells through CD40 or TLR Stimulation Contributes to the Development of CD8+ T Cell Responses in the Absence of CD4+ T Cells J. Immunol., January 15, 2005; 174(2): 710 - 717. [Abstract] [Full Text] [PDF]
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A. L. Marzo, V. Vezys, K. D. Klonowski, S.-J. Lee, G. Muralimohan, M. Moore, D. F. Tough, and L. Lefrancois Fully Functional Memory CD8 T Cells in the Absence of CD4 T Cells J. Immunol., July 15, 2004; 173(2): 969 - 975. [Abstract] [Full Text] [PDF]
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G. Chalasani, Q. Li, B. T. Konieczny, L. Smith-Diggs, B. Wrobel, Z. Dai, D. L. Perkins, F. K. Baddoura, and F. G. Lakkis The Allograft Defines the Type of Rejection (Acute versus Chronic) in the Face of an Established Effector Immune Response J. Immunol., June 15, 2004; 172(12): 7813 - 7820. [Abstract] [Full Text] [PDF]
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Y. Chen, P. S. Heeger, and A. Valujskikh In Vivo Helper Functions of Alloreactive Memory CD4+ T Cells Remain Intact Despite Donor-Specific Transfusion and Anti-CD40 Ligand Therapy J. Immunol., May 1, 2004; 172(9): 5456 - 5466. [Abstract] [Full Text] [PDF]
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 M. A. Aronica, S. McCarthy, S. Swaidani, D. Mitchell, M. Goral, J. R. Sheller, and M. Boothby Recall Helper T Cell Response: T Helper 1 Cell-resistant Allergic Susceptibility without Biasing Uncommitted CD4 T Cells Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 587 - 595. [Abstract] [Full Text] [PDF]
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H. M. Finney, A. N. Akbar, and A. D. G. Lawson Activation of Resting Human Primary T Cells with Chimeric Receptors: Costimulation from CD28, Inducible Costimulator, CD134, and CD137 in Series with Signals from the TCR{zeta} Chain J. Immunol., January 1, 2004; 172(1): 104 - 113. [Abstract] [Full Text] [PDF]
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S.-A. Younes, B. Yassine-Diab, A. R. Dumont, M.-R. Boulassel, Z. Grossman, J.-P. Routy, and R.-P. Sekaly HIV-1 Viremia Prevents the Establishment of Interleukin 2-producing HIV-specific Memory CD4+ T Cells Endowed with Proliferative Capacity J. Exp. Med., December 15, 2003; 198(12): 1909 - 1922. [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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J.C. Kips, G.P. Anderson, J.J. Fredberg, U. Herz, M.D. Inman, M. Jordana, D.M. Kemeny, J. Lotvall, R.A. Pauwels, C.G. Plopper, et al. Murine models of asthma Eur. Respir. J., August 1, 2003; 22(2): 374 - 382. [Abstract] [Full Text] [PDF]
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K. Kimachi, K. Sugie, and H. M. Grey Effector T cells have a lower ligand affinity threshold for activation than naive T cells Int. Immunol., July 1, 2003; 15(7): 885 - 892. [Abstract] [Full Text] [PDF]
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E. Ozkaynak, L. Wang, A. Goodearl, K. McDonald, S. Qin, T. O'Keefe, T. Duong, T. Smith, J.-C. Gutierrez-Ramos, J. B. Rottman, et al. Programmed Death-1 Targeting Can Promote Allograft Survival J. Immunol., December 1, 2002; 169(11): 6546 - 6553. [Abstract] [Full Text] [PDF]
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M. Munder, E. Bettelli, L. Monney, J. M. Slavik, L. B. Nicholson, and V. K. Kuchroo Reduced Self-Reactivity of an Autoreactive T Cell After Activation with Cross-reactive Non-Self-Ligand J. Exp. Med., November 4, 2002; 196(9): 1151 - 1162. [Abstract] [Full Text] [PDF]
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H.-M. Hu, H. Winter, J. Ma, M. Croft, W. J. Urba, and B. A. Fox CD28, TNF Receptor, and IL-12 Are Critical for CD4-Independent Cross-Priming of Therapeutic Antitumor CD8+ T Cells J. Immunol., November 1, 2002; 169(9): 4897 - 4904. [Abstract] [Full Text] [PDF]
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M. M. Mazanet and C. C. W. Hughes B7-H1 Is Expressed by Human Endothelial Cells and Suppresses T Cell Cytokine Synthesis J. Immunol., October 1, 2002; 169(7): 3581 - 3588. [Abstract] [Full Text] [PDF]
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B. Pantenburg, F. Heinzel, L. Das, P. S. Heeger, and A. Valujskikh T Cells Primed by Leishmania major Infection Cross-React with Alloantigens and Alter the Course of Allograft Rejection J. Immunol., October 1, 2002; 169(7): 3686 - 3693. [Abstract] [Full Text] [PDF]
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 N. Hayashi, D. Liu, B. Min, S. Z. Ben-Sasson, and W. E. Paul Antigen challenge leads to in vivo activation and elimination of highly polarized TH1 memory T cells PNAS, April 30, 2002; 99(9): 6187 - 6191. [Abstract] [Full Text] [PDF]
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 D. T. Deurloo, B. C.A.M. van Esch, C. L. Hofstra, F. P. Nijkamp, and A. J.M. van Oosterhout CTLA4-IgG Reverses Asthma Manifestations in a Mild but Not in a More ""Severe"" Ongoing Murine Model Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 751 - 760. [Abstract] [Full Text] [PDF]
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H. Gudmundsdottir and L. A. Turka A Closer Look at Homeostatic Proliferation of CD4+ T Cells: Costimulatory Requirements and Role in Memory Formation J. Immunol., October 1, 2001; 167(7): 3699 - 3707. [Abstract] [Full Text] [PDF]
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S. Mirshahidi, C.-T. Huang, and S. Sadegh-Nasseri Anergy in Peripheral Memory Cd4+ T Cells Induced by Low Avidity Engagement of T Cell Receptor J. Exp. Med., September 17, 2001; 194(6): 719 - 732. [Abstract] [Full Text] [PDF]
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M. Ahmadzadeh, S. F. Hussain, and D. L. Farber Heterogeneity of the Memory CD4 T Cell Response: Persisting Effectors and Resting Memory T Cells J. Immunol., January 15, 2001; 166(2): 926 - 935. [Abstract] [Full Text] [PDF]
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Z.-Q. Wu, A. Q. Khan, Y. Shen, J. Schartman, R. Peach, A. Lees, J. J. Mond, W. C. Gause, and C. M. Snapper B7 Requirements for Primary and Secondary Protein- and Polysaccharide-Specific Ig Isotype Responses to Streptococcus pneumoniae J. Immunol., December 15, 2000; 165(12): 6840 - 6848. [Abstract] [Full Text] [PDF]
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N. Hayashi, D. Liu, B. Min, S. Z. Ben-Sasson, and W. E. Paul Antigen challenge leads to in vivo activation and elimination of highly polarized TH1 memory T cells PNAS, April 30, 2002; 99(9): 6187 - 6191. [Abstract] [Full Text] [PDF]
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