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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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While the phenotypic characterization of memory cells is controversial,
virtually nothing is known concerning the requirements for the
generation of memory cells during a primary immune response, or the
factors that promote the survival of these cells once an immune
response has been terminated. Some studies indicate that memory cells
are long-lived in the absence of Ag exposure, whereas other studies
indicate that memory populations are maintained in a noncycling state
by continuous low-level stimulation by persistent or cross-reactive Ag
(4, 5, 18, 19, 20). While it now appears that CD8+ memory cells
do require the presence of MHC class I for their long-term survival
(21), it is not clear if MHC class II is required for the persistence
of memory CD4+ cells. Recent evidence also suggests that
cytokines such as IFN-
/ß and IL-15 may play a role in maintaining
CD8+, but not CD4+, memory cells (22, 23, 24). The
cytokines, if any, that play a role in sustaining memory
CD4+ cells have yet to be identified. The obvious
importance of these problems is that lymphocyte memory is the basis of
prophylactic immunization, and understanding the control and
maintenance of memory may be valuable for enhancing protective immunity
against microbial pathogens.
Detailed analysis of Ag-specific long-lived CD4+ cells is often hampered by their low numbers, and much of what is known about these cells is derived from limiting-dilution assays (4, 25, 26, 27, 28). Moreover, in conventional systems it is difficult to quantitatively follow a cohort of Ag-specific T cells in vivo with and without exposure to Ag. The availability of Ag receptor transgenic mice has now made it possible to more critically examine the fate of an Ag-specific lymphocyte population in vivo. We have utilized CD4+ cells from the DO.11 TCR transgenic mouse as a source of T cells specific for a known Ag, and for which a clonotypic Ab is available. This experimental system allows us to define the phenotypic and functional characteristics of T cells recovered from mice by staining with the clonotypic Ab and studying responses to the cognate peptide. Our results demonstrate that a population of memory CD4+ T cells can be generated by in vitro Ag priming. In this paper we describe the phenotype and cytokine secretion profiles of these cells. Using a novel assay for cell survival and cycling, we show that memory, but not naive CD4+ cells, are capable of cycling in response to IL-2 and IL-4. Therefore, these cytokines may play a role in the maintenance of CD4+ memory cells 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). Mice deficient in the IgM heavy chain (µ-/-, B cell deficient) on the BALB/c background were obtained from Dr. Steve Reiner (University of Chicago, Chicago, IL). 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. The B cell-deficient mice were typed by staining peripheral blood cells with Abs against CD4 and B220.
Adoptive transfers, immunization, and FACS analysis
Naive DO.11 T cells were prepared by harvesting lymph node and spleen cells pooled from DO.11 mice and running these over a nylon wool column to enrich for T cells before transfer. In vitro-activated cells were prepared by isolating CD4+ T cells from lymph node and spleen cells pooled from DO.11 mice using Dynabeads (Dynal, Oslo, Norway). The resultant CD4+ cells were cultured 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. The number of T cells expressing the DO.11 TCR was measured by staining with the clonotypic Ab, KJ1-26, and flow cytometry. Naive or in vitro-activated KJ1-26+ T cells (15 x 106) were adoptively transferred into unirradiated BALB/c or µ-/- recipients by tail vein injection. Before transfer, the activated cells were >75% KJ1-26+, and >95% CD4+. At various time points after transfer, recipients were either not immunized, or immunized with 100 µg of OVA peptide emulsified in IFA (Difco, Detroit, MI) by s.c. injection 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 3–4 days after immunization. For flow cytometry, the lymph node and spleen cell suspensions from each group were blocked with anti-CD16/CD32 (mouse Fc receptor), then stained with cytochrome c-labeled anti-CD4 mAb (PharMingen, San Diego, CA) and biotinylated KJ1-26 clonotypic Ab followed by streptavidin-FITC or streptavidin-phycoerythrin. Samples were then stained with phycoerythrin-labeled Abs to CD25, CD44, or L-selectin (PharMingen). Analyses were performed on a FACScan flow cytometer.
Fluorochrome labeling of cells and FACS analysis
To determine whether memory or naive cells would proliferate in response to cytokines in vitro, memory cells were labeled with 5-chloromethylfluorescein diacetate (CMFDA;4 cell tracker green), and naive cells were labeled with BODIPY red (both from Molecular Probes, Eugene, OR). Cells were suspended at 10 x 106/ml in RPMI 1640 with no added FCS, and the respective fluorochromes were 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. CMFDA 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. BODIPY red is also lipophilic, but does not require chemical conversion to acquire fluorescence. With repeated cell division, the fluorochrome is gradually lost from the cell, thus serving as an indicator of cell cycle status. After 4 or 8 days, the cells were collected, stained with biotinylated KJ1-26 followed by streptavidin APC and anti-CD4 PerCP, and analyzed by flow cytometry.
In vitro proliferation and cytokine assays
To analyze proliferative responses of the adoptively transferred
cells, 5 x 105 lymph node cells collected from
transfer recipients before or after immunization were cultured in 0.2
ml of RPMI 1640 supplemented as described above in 96-well plates.
Cells were restimulated with 0–1 µg/ml of OVA peptide. After 48
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 (Pharmacia LKB, Piscataway, NJ). To determine cytokine
production, 4 x 106 lymph node cells collected from
transfer recipients were cultured in 1 ml of medium in the presence of
0 or 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 (29).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The experimental protocol we have used for generating long-lived T cells involves priming cells expressing a transgenic TCR in vitro, and transferring these primed cells into syngeneic mice. The advantages of such a system are that survival can be followed without overt Ag exposure or persistence, the Ag-specific cells can be identified with a clonotypic Ab and assayed for patterns of gene expression, and, in the future, selected mutations can be introduced into these cells.
To compare the survival of naive T cells vs activated T cells in vivo,
we adoptively transferred 15 x 106 naive or in
vitro-activated KJ1-26+ CD4+ T cells from DO.11
TCR transgenic donors into syngeneic BALB/c recipient mice. Transfer
recipients were assayed for the numbers of DO.11 T cells in lymphoid
tissues by staining and flow cytometry, without further exposure to Ag.
At 1 wk after adoptive transfer, there are comparable numbers of naive
and previously activated DO.11 T cells in the peripheral lymph nodes of
adoptive transfer recipients (Fig. 1
). In
this representative experiment, naive KJ1-26+ cells
represent 1.3% of the total lymph node population (3.9% of the
CD4+ T cells, 3.1 x 104
KJ1-26+ cells per lymph node), while the in vitro-activated
T cells are 1.8% of the lymph node population (5.2% of the
CD4+ T cells, 3.8 x 104
KJ1-26+ cells per lymph node). However, at 10 wk after
transfer, there are significantly fewer naive KJ1-26+ cells
(0.5% of total, 1.6% of CD4+ cells, 0.8 x
104 KJ1-26+ cells) compared with the surviving
previously activated KJ1-26+ cells (1.7% of total, 5.2%
of CD4+ cells, 4.5 x 104
KJ1-26+ cells). Results of multiple experiments summarized
in Table I confirm the prolonged survival
of previously activated T cells. Therefore, the process of activation
in vitro generates a population of cells that are stable for long
periods in vivo.
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-chain leading to
expression of a second TCR (30), it is possible that the long-term
survival of the in vitro-activated, transferred cells is due to
interaction of the second TCR on the transgenic cells with endogenous
Ags in the recipient BALB/c mice. To rule out this possibility, we
performed identical experiments with DO.11 mice bred onto the SCID
background. As these mice are unable to effectively rearrange both TCR
and Ig genes, all the T cells should express only the transgenic TCR.
As before, naive CD4+ cells were purified from the DO.11
SCID mice, activated in vitro, and adoptively transferred into BALB/c
recipients. At both 1 and 10 wk after transfer, comparable numbers of
SCID KJ1-26+ memory cells are found in the lymph nodes of
transfer recipients as were seen with wild-type KJ1-26+
memory cells (Table I
, Expts. 7 and 8). Therefore, expression of a
second TCR on a proportion of the memory cells derived from wild-type
DO.11 mice is not responsible for their long-term survival in the
BALB/c recipient mice. Surface phenotypes and cytokine profiles of long-lived, previously activated T cells
In mice, naive T cells are known to express high levels of CD45RB
and L-selectin and low levels of CD44 (1, 2, 6, 7, 17). Other markers,
such as CD25 and CD69, correlate with the state and timing of T cell
activation (7, 31). While the phenotype of naive cells is relatively
well characterized, that of long-lived memory cells has remained
controversial. To evaluate the phenotype of long-surviving
CD4+ T cells, lymph node cells from DO.11 transfer
recipients were stained for the expression of the clonotypic TCR and a
panel of surface markers and examined by flow cytometry. Before
adoptive transfer, the naive T cells were CD25low,
L-selectinhigh, and CD44low, while the
activated cells were CD25high, L-selectinlow,
and CD44high. Long-surviving T cells, at 6 or 10 wk after
adoptive transfer, became CD25low but maintained high
levels of CD44 (Fig. 2A).
Comparison of the cells at 1 and 10 wk after transfer also shows that
memory cells are CD44high (Fig. 2B).
Interestingly, the memory KJ1-26+ cells present in lymph
nodes gradually become L-selectinhigh, and this is lost
upon Ag challenge (Fig. 2B). Thus, as has been observed with
CD8+ T cells (32), the expression of L-selectin varies with
the state of activation of CD4+ cells, down-regulating upon
Ag exposure and up-regulating upon entering a state of quiescence.
Conversely, CD44 expression remains high on activated cells, even after
they have resided in vivo for long periods of time (Fig. 2, A and B). Therefore, as with CD8+
memory cells, the most reliable phenotype for CD4+ memory
cells appear to be CD25low and CD44high.
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, at both 1 and
10 wk after transfer, the in vitro-activated T cells produce a mix of
cytokines, including IL-2, IFN-, IL-4, and IL-5, identical to those
produced after in vitro activation and before adoptive transfer (data
not shown). In contrast, the naive KJ1-26+ T cells produce
IL-2 and IFN- at 1 wk after transfer and low levels at 10 wk after
transfer; no IL-4 or IL-5 was detected. Therefore, the effector
phenotype of the in vitro-activated T cells is stable, even after
prolonged residence in vivo.
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Recent work suggests that CD8+ memory cells require
the presence of MHC class I molecules for survival, as memory cells do
not persist in TAP-1 deficient mice (21). It has been postulated that
CD4+ memory cells may be maintained by persistent Ag that
is presumably trapped in the form of Ag-Ab complexes, permitting TCR on
memory cells to intermittently encounter their cognate MHC class
II-peptide complexes (4). It is therefore possible that B cells are
responsible for this intermittent representation, as follicular
dendritic cell-derived Ag/Ab complexes are thought to serve as a
source for positive selection of B cells emerging from germinal
centers. To determine whether B cells and/or immune-complexed Ag were
required for the survival of memory cells, equal numbers of in
vitro-activated KJ1-26+ CD4+ cells were
adoptively transferred into B cell-deficient (µ-/-) mice and normal
BALB/c mice. Mice were analyzed at 6 and 10 wk after transfer and found
to have comparable numbers of memory KJ1-26+ cells
remaining in the lymph nodes of recipients (Fig. 4). The memory cells remaining in the B
cell-deficient mice were functionally competent, proliferating in
response to Ag stimulation in vitro, and rapidly producing the effector
cytokines IFN- and IL-4 (data not shown). Therefore, B cells and
immune-complexed Ag are not required for the maintenance of
CD4+ memory cells in vivo.
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In the next series of experiments, we wished to identify cytokines
that induced the survival or proliferation of CD4+ memory
cells. Studies with CD8+ memory cells have demonstrated
that they express high levels of the IL-2R ß-chain and proliferate in
response to IL-15 in the absence of Ag (24). In vitro-activated
KJ1-26+ cells were adoptively transferred into BALB/c
recipients and at 6 or 10 wk after transfer, CD4+ cells
were purified from the lymph nodes and spleens of recipient mice. For
comparison, naive KJ1-26+ cells were obtained by
purification of CD4+ cells from DO.11 SCID mice, or from
peripheral lymph nodes (only) of wild-type DO.11 mice. The percentages
of naive and memory KJ1-26+ cells in the respective
purified CD4+ cells were determined by FACS analysis (data
not shown). As expected, the memory KJ1-26+ population
expressed high levels of CD44, while the corresponding naive population
expressed the expected low levels of CD44, and both populations
expressed similar low levels of CD25 when compared with activated cells
(Fig. 2A).
We chose to use CD4+ cells purified from intact DO.11 or DO.11 SCID mice as a source of naive cells rather than those present in an adoptive transfer recipient (i.e., naive cells into BALB/C mice) primarily due to the fact that the percent of naive KJ1-26+ cells is much higher in the CD4+ cell population from intact DO.11 mice. Moreover, SCID DO.11 mice have reduced numbers of CD4+ cells (despite the presence of the TCR transgene), precluding adoptive transfer into BALB/c mice unless extremely large numbers of donor mice are used. We and others have performed many experiments in which naive DO.11 T cells are adoptively transferred into BALB/c recipients, and the responses of these transferred cells to stimulation with Ag in vitro or in vivo have been analyzed. We have found no difference in the behavior of naive KJ1-26+ cells harvested directly from the peripheral lymph nodes of intact DO.11 mice when compared with those present in adoptive transfer recipients (29, 33, 34, 35).
To follow the survival and proliferation of the naive and memory cell
populations, we used a novel method in which the cells are labeled with
different lipophilic fluorescent dyes and cultured with or without
cytokines. Harvested cells are then stained for expression of CD4 and
the DO.11 TCR and analyzed by four-color flow cytometry. This assay
distinguishes cell survival from proliferation, allows us to compare
two populations cultured together, and also tells us if the effects of
the cytokines are preferentially on the DO.11 T cells or equally on
bystander cells. The memory cells were labeled with CMFDA (tracker
FITC), and the naive cells were labeled with BODIPY red. Equal numbers
of both memory and naive KJ1-26+ cells were mixed together
(Fig. 5A) and placed in each
tissue culture well, and no cytokine, IL-2 (10, 20, or 50 U/ml), IL-4
(100 or 500 U/ml), or IL-15 (1, 10, or 100 pg/ml; 1, 10, or 50 ng/ml)
was added to the wells. In these cultures, the frequency of tracker
FITC positive cells is higher than BODIPY red positive cells. This is
due to the fact that CD4+ cells were purified from the
lymph nodes and spleens of adoptive transfer recipients to provide a
source of memory cells. In this population, only 10–20% of the cells
are KJ1-26+. Conversely, naive KJ1-26+ cells
were obtained by purifying CD4+ cells from the peripheral
lymph nodes of intact DO.11 mice, and the resultant population is
50% KJ1-26+. Therefore, fewer BODIPY-labeled cells are
added to the cell suspension to obtain a final mix of equal numbers of
naive (BODIPY red) and memory (tracker FITC) KJ1-26+ cells.
Additionally, the presence of the nontransgenic (KJ1-26-)
cells in the cultures serves as a critical internal control, allowing
us to separately quantitate the cytokine responsiveness of an
Ag-specific population of memory cells, an Ag-specific population of
naive cells, and a population of cells with unknown Ag specificity.
Cells were collected at 4 and 8 days after culture and analyzed for the
percent of remaining memory or naive KJ1-26+ cells, as well
as for the extent of cell cycling by measuring loss of tracker FITC or
BODIPY red fluorescence.
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B). In contrast, cycling was not observed in
the naive population of KJ1-26+ cells, or in the population
of BODIPY red or tracker FITC-labeled KJ1-26- cells, in
the presence of any cytokine. At 8 days of culture, the majority of the
memory cells had undergone cell division in the presence of IL-2 and
IL-4 (Figs. 5B and
6A). Furthermore, some
proliferation was noted in the presence of IL-15, especially at higher
concentrations (50 ng/ml, data not shown), although this was
significantly less than that seen with the other cytokines. The numbers
of remaining naive cells were reduced by day 8 of culture, most likely
due to passive cell death. A very small amount of proliferation was
noted in the tracker FITC-labeled KJ1-26- population at
day 8 when cultured in the presence of IL-2 and IL-4. However, these
cells were purified from both the lymph nodes and spleens (as opposed
to only lymph nodes for the naive cells) of adoptive transfer
recipients. It is probable that there are a small number of
CD4+ KJ1-26- memory (or activated) cells
present within this population that respond to the added cytokine. We
have also performed similar experiments in which the naive and memory
cells were both labeled with tracker FITC and cultured in separate
wells with the various cytokines. As in the studies described above in
which the populations were cocultured, when cultured separately, only
the KJ1-26+ memory cells were found to proliferate in both
IL-2 and IL-4 (data not shown). We and others have utilized
fluorochrome cytoplasmic labeling agents to follow the proliferation of
naive cells in response to Ag both in vivo and in vitro (36, 37, 38). It is
clear that these agents do not interfere with the activation and
subsequent cycling of naive cells. Therefore, the lack of proliferation
we observed in the naive KJ1-26+ population is not due to
an effect of the BODIPY red labeling procedure.
Proliferative responses of nonfluorochrome-labeled naive, memory, and
activated KJ1-26+ cells to the various cytokines were also
evaluated at day 4 by [3H]thymidine incorporation. As
expected, the memory cells proliferated well in response to IL-2 and
IL-4, with lower levels of proliferation seen in the presence of IL-15
(Fig. 6B). Interestingly, the in vitro-activated cells
proliferated much more vigorously in response to IL-2 than IL-4,
exhibiting no response to IL-15. These results confirmed the data
presented in Fig. 5, demonstrating that, unlike naive cells, memory
cells retain the ability to respond to IL-2 and IL-4, and to a lesser
degree IL-15.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table I). This system enables us to define the
phenotypic characteristics of long-surviving T cells and evaluate the
signals required for their survival and functional responses to Ag
challenge.
We have chosen to use the in vitro activation and adoptive transfer
system for several reasons. Our experience, and that of many other
investigators, is that intact TCR transgenic mice cannot be primed
normally due to apoptosis of activated cells secondary to the high
frequency of responding cells and the subsequent replacement of cells
with new thymic emigrants (naive cells). The only other option is to
transfer naive DO.11 T cells into one set of recipients, prime these,
recover the in vivo-activated KJ1-26+ T cells, and transfer
these into a second set of recipients. We have actually tried such
experiments, but it is logistically impossible to do these routinely,
especially for many of the in vitro cytokine sensitivity experiments.
Furthermore, the issue of Ag persistence in the immunized mice may lead
to carry-over of Ag into the in vitro restimulation experiments. Note
that key to our studies is the ability to study T cell survival in the
absence of Ag exposure, and this can only be accomplished using the in
vitro activation and adoptive transfer system we have employed. The
long-lived cells generated by in vitro activation and adoptive transfer
possess several characteristics believed to be typical of memory cells:
1) by 10 wk after transfer, a stable population of DO.11 T cells
is generated after in vitro activation, whereas similar numbers of
adoptively transferred naive DO.11 T cells are markedly reduced (Fig. 1); 2) the surviving DO.11 T cells express markers typical of memory T
cells (CD25lowCD44high, Fig. 2); and 3) the
surviving DO.11 T cells exhibit functional responses believed to be
characteristic of memory cells including rapid effector cytokine
production upon restimulation (Fig. 3) and localization to the spleen
with rapid migration out of the spleen upon Ag exposure (data not
shown). Indeed, other investigators have successfully used the method
of in vitro activation and adoptive transfer (39). In conclusion, the
approach we have chosen is the only practical way of doing these
experiments in a quantitative, reproducible manner.
Phenotypic analysis of the long-surviving T cells shows that by 10 wk
after adoptive transfer, they express high levels of L-selectin,
similar to naive T cells, and high levels of CD44, which distinguishes
them from naive cells. Upon Ag stimulation the cells rapidly
down-regulate L-selectin (Fig. 2). Similar findings have been
reported in models of CD8+ T cell memory, in which
virus-specific CD8+ T cells gradually return to a
CD45RBhigh, L-selectinhigh state within 3
mo after virus challenge and recovery (26, 32). Our results support the
notion that L-selectin is a surface marker that reflects the
state of activation of T cells, rather than a naive or memory
phenotype. As previously reported (39), the long-surviving DO.11 T
cells also retain the cytokine profiles that were induced by in vitro
priming (Fig. 3). We have not addressed the question of whether these
cells are irreversibly committed to one effector program, or are
capable of differentiating into either Th1 or Th2 subset upon
subsequent Ag challenge.
It has been recently demonstrated that B cells and Ag-Ab complexes are
not essential for the maintenance of CD8+ CTL memory (40, 41). Our data indicate that B cells are not required for the survival
of the KJ1-26+ memory cells. Therefore, if MHC class II is
necessary for intermittent stimulation of memory cells, it must be
provided by other professional APCs, such as macrophages and dendritic
cells. However, persistent Ag is unlikely to play a role in memory cell
maintenance for several reasons. It is known that class II MHC/peptide
complexes on APCs are rapidly recycled/internalized unless the APCs are
activated (mature) in an inflammatory environment, after which time the
complexes may remain on the cell surface for over 100 h (42). This
process serves to allow APCs to efficiently present foreign Ags to T
cells during an immune response. In the noninflammatory environment,
any remaining persisting Ag in class II MHC complexes would likely be
rapidly recycled, thus limiting the chances of memory cells to
encounter the relevant APC. Furthermore, Ag remaining after challenge
would be expected to persist complexed to Ab. As the µ-/- mice do
not have circulating Ab, Ag-Ab complexes cannot form. Therefore, the
survival of the KJ1-26+ memory T cells cannot be due to
either Ag presentation by B cells or Ag in the form of immune
complexes. Since the TCR transgenic T cells we have examined are
restricted by the I-Ad allele, we cannot study their
survival in mice lacking class II MHC molecules (I-Ab
deficient) (43) in which the cells would be incapable of recognizing
any Ag.
The possibility that memory cells are maintained by exposure to
cytokines is an interesting one. Recent work has demonstrated that
IL-15 can induce the proliferation of CD8+ memory cells in
vitro, and administration of IFN-/ß to mice can induce their
proliferation in vivo (22, 23, 24). Our results show that IL-15 has little
effect on memory CD4+ cells except at high (50 ng/ml)
concentrations. However, both IL-2 and IL-4 are able to induce the
survival and proliferation of memory cells, to a much greater extent
than that of naive cells. It is interesting that memory cells express
almost the same level of CD25 as do naive cells (Fig. 2B),
yet only the memory cells respond to IL-2. This raises the possibility
that cytokine responsiveness is determined by factors in addition to
the levels of receptor expression.
Finally, whereas in vitro-activated T cells show prolonged survival in vivo, the population of adoptively transferred naive T cells that homes to the lymph nodes of adoptive transfer recipients gradually decreases over time. Therefore, the process of in vitro activation must induce a genetic program capable of supporting long-term survival in the absence of proliferation or overt Ag exposure. These results also indicate that previously activated T cells possess survival signals that are not present in naive cells. We do not know the nature of the change induced in the T cells as a result of in vitro priming which results in their enhanced survival. By isolating T cells expressing the specific Ag receptor at various times after adoptive transfer, it should be possible to define the genetic and biochemical characteristics of long-surviving T cells and the features that distinguish them from naive cells. Such studies are currently in progress.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, MA 02114.
3 Address correspondence and reprint requests to Dr. Abul K. Abbas, Brigham and Women’s Hospital and Harvard Medical School, LMRC-521, 221 Longwood Avenue, Boston, MA 02115. E-mail address:
4 Abbreviation used in this paper: CMFDA, 5-chloromethylfluorescein diacetate.
Received for publication August 6, 1998. Accepted for publication October 6, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N. R. Thirumalapura, H. L. Stevenson, D. H. Walker, and N. Ismail Protective Heterologous Immunity against Fatal Ehrlichiosis and Lack of Protection following Homologous Challenge Infect. Immun., May 1, 2008; 76(5): 1920 - 1930. [Abstract] [Full Text] [PDF]
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D. Duffy, S. M. Sparshott, C.-p. Yang, and E. B. Bell Transgenic CD4 T Cells (DO11.10) Are Destroyed in MHC-Compatible Hosts by NK Cells and CD8 T Cells J. Immunol., January 15, 2008; 180(2): 747 - 753. [Abstract] [Full Text] [PDF]
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J. J. Bell, J. S. Ellis, F. B. Guloglu, D. M. Tartar, H.-H. Lee, R. D. Divekar, R. Jain, P. Yu, C. M. Hoeman, and H. Zaghouani Early Effector T Cells Producing Significant IFN-{gamma} Develop into Memory J. Immunol., January 1, 2008; 180(1): 179 - 187. [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. Mostbock, M. Catalfamo, Y. Tagaya, J. Schlom, and H. Sabzevari Acquisition of antigen presentasome (APS), an MHC/costimulatory complex, is a checkpoint of memory T-cell homeostasis Blood, March 15, 2007; 109(6): 2488 - 2495. [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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V. R. Moulton, N. D. Bushar, D. B. Leeser, D. S. Patke, and D. L. Farber Divergent Generation of Heterogeneous Memory CD4 T Cells J. Immunol., July 15, 2006; 177(2): 869 - 876. [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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H. Dooms, E. Kahn, B. Knoechel, and A. K. Abbas IL-2 Induces a Competitive Survival Advantage in T Lymphocytes J. Immunol., May 15, 2004; 172(10): 5973 - 5979. [Abstract] [Full Text] [PDF]
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M. Iwata, Y. Eshima, and H. Kagechika Retinoic acids exert direct effects on T cells to suppress Th1 development and enhance Th2 development via retinoic acid receptors Int. Immunol., August 1, 2003; 15(8): 1017 - 1025. [Abstract] [Full Text] [PDF]
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G. F. Debes, U. E. Hopken, and A. Hamann In Vivo Differentiated Cytokine-Producing CD4+ T Cells Express Functional CCR7 J. Immunol., June 1, 2002; 168(11): 5441 - 5447. [Abstract] [Full Text] [PDF]
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A. Y. Park, B. Hondowicz, M. Kopf, and P. Scott The Role of IL-12 in Maintaining Resistance to Leishmania major J. Immunol., June 1, 2002; 168(11): 5771 - 5777. [Abstract] [Full Text] [PDF]
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C. M. Porter and N. A. Clipstone Sustained NFAT Signaling Promotes a Th1-Like Pattern of Gene Expression in Primary Murine CD4+ T Cells J. Immunol., May 15, 2002; 168(10): 4936 - 4945. [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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Y.-C. Lim, H. Xie, C. E. Come, S. I. Alexander, M. J. Grusby, A. H. Lichtman, and F. W. Luscinskas IL-12, STAT4-Dependent Up-Regulation of CD4+ T Cell Core 2 {beta}-1,6-n-Acetylglucosaminyltransferase, an Enzyme Essential for Biosynthesis of P-Selectin Ligands J. Immunol., October 15, 2001; 167(8): 4476 - 4484. [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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P.-J. Linton, J. Harbertson, and L. M. Bradley A Critical Role for B Cells in the Development of Memory CD4 Cells J. Immunol., November 15, 2000; 165(10): 5558 - 5565. [Abstract] [Full Text] [PDF]
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K. Lukin, M. Cosyns, T. Mitchell, M. Saffry, and A. Hayward Eradication of Cryptosporidium parvum Infection by Mice with Ovalbumin-Specific T Cells Infect. Immun., May 1, 2000; 68(5): 2663 - 2670. [Abstract] [Full Text] [PDF]
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R. Merica, A. Khoruts, K. A. Pape, R. L. Reinhardt, and M. K. Jenkins Antigen-Experienced CD4 T Cells Display a Reduced Capacity for Clonal Expansion In Vivo That Is Imposed by Factors Present in the Immune Host J. Immunol., May 1, 2000; 164(9): 4551 - 4557. [Abstract] [Full Text] [PDF]
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P. R. Rogers, C. Dubey, and S. L. Swain Qualitative Changes Accompany Memory T Cell Generation: Faster, More Effective Responses at Lower Doses of Antigen J. Immunol., March 1, 2000; 164(5): 2338 - 2346. [Abstract] [Full Text] [PDF]
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C. A. London, M. P. Lodge, and A. K. Abbas Functional Responses and Costimulator Dependence of Memory CD4+ T Cells J. Immunol., January 1, 2000; 164(1): 265 - 272. [Abstract] [Full Text] [PDF]
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M. Ahmadzadeh, S. F. Hussain, and D. L. Farber Effector CD4 T Cells Are Biochemically Distinct from the Memory Subset: Evidence for Long-Term Persistence of Effectors In Vivo J. Immunol., September 15, 1999; 163(6): 3053 - 3063. [Abstract] [Full Text] [PDF]
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H. Xie, Y.-C. Lim, F. W. Luscinskas, and A. H. Lichtman Acquisition of Selectin Binding and Peripheral Homing Properties by CD4+ and CD8+ T Cells J. Exp. Med., June 7, 1999; 189(11): 1765 - 1776. [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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