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Center for Immunology and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455
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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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Several of the surface proteins that have altered expression on memory cells are also likely to contribute to the distinct recirculation pathways of the two cell types, with memory cells selectively trafficking from blood to peripheral tissues while naive cells selectively traffic from blood to lymph nodes (10, 11, 12). This broader immune surveillance by memory cells could contribute to more rapid responses, particularly to Ag challenge at peripheral sites. Finally, recent reports have indicated that at least some memory CD8+ T cells either retain active effector function or very rapidly reacquire it upon exposure to Ag (13, 14), in contrast to the 2 to 3 days of differentiation required for naive cells to gain these functions. Whether the greater speed and efficiency of a memory response is simply due to increased precursor frequency, or is influenced by the qualitative differences between naive and memory cells, has remained controversial. An evaluation of the extent to which these factors contribute to enhanced memory responses requires in vivo examination of the Ag-specific cells during the response.
Adoptive transfer of TCR transgenic T cells into normal recipients, coupled with use of an anti-TCR mAb to identify the transferred cells, provides a powerful means of studying Ag-specific cells in vivo (5). The locations, numbers, and surface phenotype of the cells can be readily determined by flow cytometry during the course of a response, while the relatively small number of transgenic cells in the recipient does not significantly skew the normal immune response. Using this approach to study the CD8+ T cell memory response to lymphocytic choriomeningitis virus (LCMV) infection, Zimmerman et.al. (13) have characterized the surface phenotype of the memory cells and demonstrated that at least some of the cells are directly cytolytic when isolated from the recipients. A substantial fraction of the memory cells were found to be undergoing cell division, as assessed by BrdU labeling; whether persistent viral Ag was involved in stimulating this was not examined. Because systemic infection with lymphocytic choriomeningitis virus results in widely disseminated Ag, and Ag is presented to CD8+ T cells by host APC, issues of T cell trafficking would be difficult to study in this model.
Adoptive transfer of CD8+ T cells transgenic for the 2C TCR (15), specific for Ld alloantigen, allows the in vivo response to challenge with allogeneic tumor in the peritoneal cavity to be visualized using the 1B2+ mAb specific for the 2C TCR (16). Since the Ag recognized by the 2C receptor is native H-2Ld complexed with the p2Ca peptide (17), re-presentation to the 2C cells by host APC cannot occur. Thus, the location of stimulatory Ag is limited and can be determined by flow cytometry, allowing study of the trafficking of the Ag-specific cells and their site(s) of activation. When adoptive transfer recipients are challenged by i.p. injection of P815 allogeneic tumor, a primary response occurs that involves both endogenous host alloantigen-specific CD8+ T cells and the transferred 2C cells, with the ability to identify the 2C cells making it possible to track Ag-specific cells during the response. The response is characterized by initial clonal expansion of the 1B2+ 2C cells in the peritoneal cavity beginning on days 5 and 6 and peaking on day 8 (18). As expansion occurs in the peritoneal cavity; 1B2+ cells that have been activated but are no longer proliferating migrate into the peripheral lymphoid organs and blood. The total number of 1B2+ cells then decreases as tumor is eliminated and reaches a steady state level by about day 20 at a higher number than was present before antigenic challenge.
These previously challenged adoptive transfer recipients have a stable population of Ag-specific memory cells that can be identified using the 1B2 mAb and have made it possible to examine the CD8+ T cell memory response with respect to memory phenotype, trafficking, sites of activation, and development of effector function. The results demonstrate that, while a higher frequency of Ag-specific cells contributes to a more efficient memory response, qualitative differences in trafficking to the site of Ag and development of effector function make major contributions to memory cells being more rapid and effective than naive cells in mounting a response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice expressing the 2C TCR (15), a gift from Dr. Dennis Loh (Hoffman-LaRoche, Nutley, NJ), were bred to wild-type C57BL/6 mice to generate mice heterozygous for the 2C TCR transgene, and these were used as the source of TCR transgenic CD8+ T cells in all of the experiments. C57BL/6NCr/Br mice from Charles River Laboratories (Wilmington, MA) were used as recipients for the adoptive transfer at 4 to 6 wk of age. Mice were housed in a specific pathogen-free environment at all times
Antibodies
The 1B2 mAb specific for the 2C TCR (16) (a gift from Dr. Herman
Eisen, Massachusetts Institute of Technology, Boston, MA) was purified
from culture supernatant of the 1B2 hybridoma using a protein
A-Sepharose column with elution by citric acid at pH 3.5. The purified
mAb in PBS was biotinylated by incubation for 4 h at
room temperature with 0.15 mg/ml biotin and 1 mg/ml mAb. Other Abs used
were Mel14, Mel14-FITC, anti-CD44-FITC (Pgp1), anti-
4-FITC,
anti-CD25-FITC, anti-CD8-phycoerythrin
(PE)3,
anti-CD8-cychrome, goat-anti-rat-FITC,
streptavidin-phycoerythrin (SA-PE), streptavidin-APC (SA-APC),
SA-PerCP, anti-H-2Kb-biotin,
anti-H-2Kd-FITC, and rat- and mouse-FITC isotype
controls. All conjugated Abs were purchased from PharMingen (San Diego,
CA), with the exceptions of the goat-anti-rat-FITC and isotype
controls that were purchased from Jackson ImmunoResearch (West Grove,
PA). SA-PerCP was purchased from Becton Dickinson (San Jose,
CA).
Cell lines
The mastocytoma P815 (H-2d), lymphoma RDM4 (H-2k), the thymoma EL4 (H-2b), and H-2Ld-transfected EL4 cells (EL4-Ld), were grown in vitro in RPMI 1640 media (Cellgro, Herndon, VA) with 10% FCS, 0.2% glutamine, 0.1% pen/strep, 0.1% HEPES, 0.1% nonessential amino acids, 0.01% sodium pyruvate, and 0.05% ß-mercaptoethanol. Cultures were always 80 to 90% viable (by trypan blue exclusion), and cells were washed with PBS before use for i.p. injections or in vitro chromium release assays.
Adoptive transfer of TCR transgenic cells and Ag priming
Lymph node cells (axillary, brachial, cervical, inguinal, periaortic, mediastinal, and mesenteric) from heterozygous 2C transgenic mice were removed, homogenized, and washed three times with PBS. 1B2+CD8+ cells (2–3 x 106) were transferred into sex-matched naive C57BL/6 mice by tail vein injection. Recipient mice were rested for a period of 1 to 5 days before priming by i.p. injection of 5 x 106 P815 tumor cells. Where noted, 1B2+ cells and P815 tumor were injected simultaneously i.p. The primary response was analyzed over 2 to 20 days following this initial priming. Memory responses were analyzed in mice that had been primed with P815 tumor 40 to 60 days previously and were then rechallenged by i.p. injection of 5 x 106 P815 or RDM4 tumor cells, and the response was analyzed from 12 h to 30 days after rechallenge.
Flow cytometric analysis of primary and memory responses
Mice were sacrificed at varying times after priming or rechallenge, and the LN and spleen were removed, homogenized, and ammonium chloride treated to lyse red blood cells. Brachial, axillary, and cervical LN were pooled as distant peripheral nodes, and periaortic, mediastinal, and mesenteric nodes as draining LN. Peripheral blood was drawn from the heart using a heparin-loaded syringe, and the red blood cells were lysed by ammonium chloride treatment. The peritoneal cavity was washed with 50 ml of PBS, and the resulting peritoneal exudate lymphocytes (PEL) were adherence depleted for 90 min in complete media at 37°C. Each cell population was counted for total cell number using trypan blue to exclude dead cells.
Cells (1–2 x 106) isolated from each site were
stained with 1B2-biotin mAb, PE-conjugated anti-mouse CD8 mAb,
and one of the following FITC-conjugated mAb: CD25, CD44, 4 (VLA-4),
or Mel14. After washing, SA-PerCP or SA-APC were added for detection of
1B2-biotin. Stained cells were fixed with 1% formaldehyde and analyzed
by three- or four-color flow cytometry using the CellQuest software
package (Becton Dickinson). Cytometer settings were identical for all
time points within a given experiment. For determination of the total
number of 1B2+CD8+ cells, 50 to 100 x
103 events were analyzed (see Fig. 2
A),
and the percent of 1B2+CD8+ cells was
multiplied by the total number of cells recovered from the site. This
reliably detects the 2C cells. Staining and detecting in this way shows
a background of less than 0.01% 1B2+CD8+ cells
in LN from normal C57BL/6 mice that have not received 2C cells (Ref.
18; data not shown), while LN from mice that have received 3 x
106 transferred cells have about 0.23%
1B2+CD8+ cells in the LN. In contrast,
background in this quadrant is minimal in the transferred mice when the
1B2 mAb is replaced with an isotype control (data not shown).
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B. Replacing the specific Abs with isotype control Abs
resulted in almost no events falling into the high gate for each marker
(data not shown). Cells from all sites were separately stained for the presence of P815 tumor using anti-H-2Kb-biotin, with a second step SA-Cy or SA-APC, and anti-H-2Kd-FITC. This protocol stains all endogenous cells (H-2Kb) with cychrome (FL3) or APC (FL4) and any P815 tumor present (H-2Kd) with FITC (FL1). By excluding all FL3+ events, any tumor present can be easily detected down to a frequency as low as 0.01% of total cells (data not shown). The total number of tumor cells at the various sites was determined by multiplying the percent of tumor cells in the population by the total number of cells recovered from each site.
Chromium release assay and calculation of lytic units
PEL were assayed for lytic activity following depletion of adherent cells using a standard 4-h 51Cr release assay with EL4-Ld target cells. LU at 50% target cell lysis (LU50) were calculated, with 1 LU50 defined as the number of effector cells required to lyse 50% of the target cells. For some experiments, lytic activity is expressed based on the number of 1B2+ cells in the population (as determined by flow cytometry), rather than on total cells. EL4 cells were used as the syngeneic control in all lytic assays. Up to day 5 of the primary response, some lysis of EL4 targets by PEL could be detected, consistent with published results by Nishi et al. (19). By day 6 of the primary response, lysis of EL4 targets was always less than 5%. On days where release from control targets was higher than 5%, the value was subtracted from the percent lysis of the specific target to achieve a more appropriate estimate of specific target lysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To directly examine the activity of memory CD8+ T
cells in vivo, lymph node (LN) cells from 2C TCR transgenic mice were
injected i.v. into naive C57BL/6 recipients using 2 to 3 x
106 1B2+CD8+ cells/mouse. Cells
bearing the 2C TCR were detected in the recipients by staining with
anti-CD8 mAb and the 1B2 mAb specific for the 2C TCR (16), and
phenotype was characterized using a third Ab and gating on the
CD8+IB2+ cells. The 1B2+ cells
maintain a naive phenotype in the recipients, and the numbers in the
spleen, lymph nodes, and blood remain relatively constant for greater
than 2 wk, after which there is a gradual decline in the number of
1B2+ cells at all sites (Ref.18; Fig. 1).
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After day 8, the number decreases at all sites through day 20 and then
remains constant for at least 2 to 3 mo at a frequency 5- to 10-fold
above that of recipients that were not primed with Ag (Fig. 1), and
1B2+ cells remain easily detectable in primed mice for at
least 6 mo (data not shown). These long term memory cells remain
functional and respond rapidly to a secondary challenge with Ag as
evidenced by clonal expansion beginning as early as 2 days after
rechallenge (Fig. 1). All of the following experiments examining memory
cells were done using cells from mice that had been primed at least 40
days previously by i.p. injection of P815 tumor.
In comparison with naive recipients, 1B2+ cells in primed
mice account for a greater percentage of the total cells in the LN,
spleen, peritoneal cavity, and blood (Fig. 2A). This is
particularly the case for blood and for the peritoneal cavity, where
1B2+ cells are undetectable in naive recipients while a
small but easily detectable population is present in the primed mice.
The majority of 1B2+ cells at all sites expressed high
levels of CD44 and VLA-4 (Fig. 2B), a well-documented
memory phenotype (20, 21). Naive 1B2+ cells lose L-selectin
from the surface during the initial phase of a primary in vivo
response, but about half of the cells reacquire an
L-selectinhigh phenotype within a few days, while retaining
high levels of CD44 and VLA-4 (18). Consistent with this, about half of
the cells present in the spleen and peritoneal cavity of the primed
mice express high levels of L-selectin (Fig. 2B). The
fraction expressing high L-selectin is greater in LN, consistent with
earlier work suggesting that L-selectin reexpression would contribute
to the ability of memory cells to recirculate through LN (22). Although
1B2+ cells could not be detected in unimmunized recipients
by sixty days posttransfer (Fig. 1), some 1B2+ cells with a
naive phenotype (CD44low, VLA-4low) could be
seen in the primed mice (Fig. 2B), suggesting either
a reversion of some memory cells to a naive phenotype or an enhanced
survival of naive cells that had not been activated in the primary
response.
About 15 to 30% of the memory 1B2+ cells expressed high
CD25 and were blasts as indicated by a high forward scatter (Fig. 2B). In contrast, naive transferred cells have at
most a few percent of 1B2+ cells with this phenotype. This
is in agreement with other studies showing that a fraction of memory
cells are in cycle, as assessed by BrdU labeling (13, 23).
Trafficking, clonal expansion, and acquisition of lytic activity in response to rechallenge with Ag
The number of 1B2+ cells in the peritoneal cavity
increases about 10-fold within 12 h of rechallenge with P815 tumor
i.p. (Fig. 3A) and
continues to increase through day 3 (Fig. 3B). As
this is occurring, the 1B2+ cells are decreasing in number
in the spleen and blood through the first 1 to 2 days of the response
(Fig. 3B). The rapid increase in the peritoneal
cavity occurs too quickly to be accounted for by proliferation of the
small number of 1B2+ cells present at this site at the time
Ag is injected. Rather, it appears that the majority of these are cells
migrating into the peritoneal cavity from the spleen and blood. A
similar pattern of decreasing numbers of Ag-specific cells in the
spleen and blood as cells appear in the peritoneal cavity is seen in
the primary response of 2C cells in adoptive transfer recipients
challenged i.p. with P815 (18). The primary and memory responses differ
dramatically, however, with respect to the timing of these events.
1B2+ cells increase in number in the peritoneal cavity
within 12 h in a memory response (Fig. 3, A and
B), but not until day 5 after challenge in a primary
response (18). After day 2 to 3, the number of 1B2+ cells
in the peritoneal cavity begins to decrease. During this time, however,
there is a large increase in the numbers of 1B2+ cells in
the spleen and blood (Fig. 3B). At longer times,
1B2+ cells persist, primarily in the spleen and blood, at
higher numbers than were present in the memory population before
rechallenge (data not shown).
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A low level of Ld-specific lytic activity can be detected
for cells from the peritoneal cavity and spleen of previously primed
adoptive transfer recipients before rechallenge with Ag (Fig. 4). By 12 h after rechallenge,
Ld-specific lytic activity has increased dramatically in
the peritoneal cavity. Thus, it appears that the memory population
includes some cells that may have a low level of lytic activity in the
absence of Ag stimulation and that lytic activity can increase very
rapidly upon stimulation. Although tumor was no longer detectable by 12
to 36 h, lytic activity persisted in the peritoneal cavity and
even increased in some experiments, through days 4–5, before returning
to background levels. In the experiment shown in Figure 4B,
there was a transient decrease in lytic activity at 36 h, and this
was seen to varying degrees in three independent experiments (e.g., see
Fig. 7C). This may result from loss of the rapidly
activated subset of effectors from the peritoneal cavity (through
death or migration from the site), followed by differentiation to
effector status of a different subset of memory cells that behave more
like naive cells in that they require 2 to 3 days to develop lytic
function.
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B). Although the speed of acquisition of lytic
function, and rate of tumor clearance, are dramatically increased in
the secondary response, the maximal lytic activity that develops is
significantly reduced in comparison to the primary response (Fig. 4B). However, equivalent levels of lytic activity are
found when the lytic activities of primary and memory effector
populations are compared on the basis of the number of 1B2+
cells (Fig. 4C) (the assumption being that the
relative numbers of 2C cells are proportional to the total number of
Ag-specific CTL), i.e., the primary and memory CTL have comparable
lytic activities at the level of the individual cells. Thus, it appears
likely that the greater activity generated in the primary response can
be accounted for simply by the more extensive clonal expansion that
occurs, probably as a result of the tumor Ag reaching a much higher
level by the time naive cells gain entry into the peritoneal cavity and
begin expanding. Sites of clonal expansion of memory cells
During a primary response in adoptive transfer recipients, the
only location where large numbers of 1B2+ cells that
are CD25+ and blasts can be detected is within the
peritoneal cavity during days 5 to 7 (Fig. 5A; 18 , suggesting that
the majority of clonal expansion occurs there as opposed to within the
secondary lymphoid tissue. In contrast, during days 2 to 3 of the
memory response a significant percentage of 1B2+ cells at
almost all sites are blasts, reaching as high as 50% of the
1B2+ cells in the draining LN, spleen, and peritoneal
cavity (Fig. 5A). This suggests that some clonal
expansion probably occurs at these sites, but the possibility of
migration of blasts from the peritoneal cavity to these sites cannot be
ruled out. Despite this increase in activated cells in the draining LN
and spleen, there is little increase in lytic function of the cells at
either of these sites at this time or any time thereafter (Fig. 5B; data not shown). This is again in contrast to the
primary response where, by day 6 and later, 1B2+ cells are
increasing in the spleen as they exit the peritoneal cavity after
having undergone activation there. These cells are no longer blasts but
retain potent lytic activity (Fig. 5B). Thus,
following the initial response in the peritoneal cavity, expanded
numbers of nonblasting but lytically active 1B2+ cells are
present in the spleen in the primary response, while expanded numbers
of blasting 1B2+ cells with little lytic activity are
present in the spleen in the memory response.
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Specificity of the memory response: trafficking and activation
Memory 1B2+ cells have access to the peritoneal
cavity in the absence of any inflammation or Ag (Fig. 2A) and rapidly traffic to that site when
rechallenged with Ag (Fig. 3B). We were interested in
determining whether the rapid trafficking of memory 1B2+
cells required Ag or whether it might occur in response to just an
inflammatory stimulus. Adoptively transferred recipients that had been
previously primed with P815 were therefore rechallenged with a
third-party allogeneic tumor, the RDM4 lymphoma of AKR
(H-2Kk) origin that is not cross-reactively recognized by
the 2C receptor (data not shown). Challenge by i.p. injection of RDM4
resulted in a substantial influx of 1B2+ cells into the
peritoneal cavity by day 3 (Fig. 6A). Concomitant with
this increase in 1B2+ cells in the peritoneal cavity, the
number of 1B2+ cells decreased by 3 to 5 x
104 in the spleen, and there was a decrease in the
percent 1B2+ cells in the blood (data not shown). While
significant, the increase in number of 1B2+ cells was about
10-fold less than when the mice were challenged with P815
tumor.
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B). This suggested that the 1B2+
cells were able to migrate to the peritoneal cavity in response to the
nonspecific inflammatory stimulus provided by the RDM4 tumor but were
not becoming further activated in the absence of specific Ag.
Consistent with this, although 1B2+ cells were present in
significant numbers, there was very little Ld-specific
lytic activity in comparison to that in mice rechallenged with the
specific Ag (Fig. 6B). The contribution of precursor frequency to the memory response
Determination of the extent to which qualitative differences
between naive and memory cells contributes to the more rapid and
efficient memory response would require that differences in frequencies
of the specific cells be eliminated as a factor. Simply varying the
number of 2C cells transferred into the recipients cannot accomplish
this, since the majority of the CD8 response to the tumor is by
endogenous host T cells. Attempting to adjust the size of the memory
population by varying the number of tumor cells injected for the
primary challenge would also be difficult, since the tumor grows very
rapidly for several days before being rejected. We found, however, that
injection of naive recipient mice with P815 tumor and 2C
CD8+ T cells simultaneously into the peritoneal cavity
results in a lower frequency of memory 1B2+ cells at the
conclusion of the response than does i.v. injection of 2C cells and
i.p. tumor challenge. This usually results in a frequency of memory
cells in the spleen, LN, and blood that is comparable to the frequency
of naive 1B2+ cells in mice receiving the transfer by i.v.
injection (Fig. 7A).
The lower frequency probably results from less clonal expansion
occurring because the tumor Ag is more rapidly eliminated when 2C cells
are placed in the peritoneal cavity simultaneously with tumor (data not
shown). It would be expected that the endogenous host memory population
would be correspondingly reduced in frequency for the same reason. This
provided a means of comparing primary vs memory responses where the
precursor frequencies are comparable.
In the experiment shown in Figure 7, three sets of mice were examined.
These included mice that received 2C cells 3 days previous to challenge
("Primary"), mice 60 days after 2C transfer by i.v. injection and
priming by i.p. injection of P815 ("Hi Memory"), and mice 40 days
after simultaneous i.p. injection of 2C and P815 cells ("Low
Memory"). The number of 1B2+ cells in the spleens of low
memory mice was comparable to that in the primary mice, while the hi
memory mice had about sevenfold more 1B2+ memory cells
(Fig. 7A). When the mice were challenged by i.p.
injection of P815 cells, tumor was eliminated from the hi memory mice
within 12 h and from the primary mice by day 7 or 8 (Fig. 7B). Tumor elimination in the low memory mice was not
as rapid as in the hi memory mice but was nevertheless substantially
more rapid than in the primary mice.
Development of lytic activity in the peritoneal lymphocyte population
showed comparable differences in timing. Hi memory mice had detectable
lytic activity at 12 h, followed by a decline at day 2 and a
second peak at day 3, similar to the results shown above (Fig. 4B), while primary mice did not develop detectable
activity until day 7. Again, low memory mice developed lytic activity
less rapidly than the hi memory mice, but still several days sooner
than the primary mice. The peak level of lytic activity showed an
inverse relationship to the rate of tumor clearance and development of
the activity. Hi memory mice had the lowest activity, primary mice the
highest, and low memory mice were intermediate, probably reflecting the
more limited proliferation and differentiation that occur when the Ag
load is reduced early in the response.
Trafficking of 1B2+ cells in the low memory response is
similar to the hi memory and primary responses, the differences again
being the timing (Fig. 7, D and E). For
all responses, 1B2+ cells decrease in the spleen (Fig. 7E) and blood (not shown) for the first 2 to 3 days
after challenge, as they begin to appear in the peritoneal cavity (Fig. 7D). Here too, the increase in the number of cells in
the peritoneal cavity in the low memory response is 1 to 2 days slower
than for the hi memory response, but still 4 to 5 days more rapid than
the primary. Interestingly, the maximum level of expansion of
1B2+ cells in the peritoneal cavity and spleen in the low
memory response is more similar to that seen in the primary response
than in the hi memory response (Fig. 7, D and
E). The relationship between the level of Ag, the
timing of its elimination, and the extent of clonal expansion that
occurs is clearly complex and almost certainly influenced by factors
other than just the amount of Ag available for the T cells to interact
with. It is clear from these results, however, that memory CD8 T cells
can respond much more rapidly and clear Ag sooner than naive cells are
able to, even when comparable numbers of precursors are present at the
time of challenge with Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). These memory cells are
long-lived, remaining present for at least 6 mo following priming, and
provide identifiable Ag-specific cells within the memory population
that includes endogenous alloantigen-specific CD8+ T cells.
Persistent Ag is very unlikely to be involved in maintenance of the
memory pool. Only the P815 tumor can present the native Ld
alloantigen recognized by the 2C receptor, so a long-lived pool of Ag
associated with host APC would not be present, and tumor becomes
undetectable by flow cytometry within 8 to 10 days of the primary
challenge (18). Although we have not used more sensitive detection
methods, it is very unlikely that any tumor cells remain present long
term, given the very aggressive growth of the P815 tumor. While it
appears that Ag persistence may be important for maintenance of memory
CD4+ T cells (24), its importance in maintaining
CD8+ T memory cells has been more controversial (25, 26, 27, 28, 29).
Our results support the hypothesis that Ag persistence may not be
critical for CD8+ T cell memory. Recent experiments of
Tanchot et al. (30) have suggested that maintenance of memory
CD8+ T cells requires TCR-MHC class I interactions but that
a nonspecific class I is sufficient for this and Ag is not
required.
Despite the absence of persistent Ag, a substantial fraction of the
1B2+ memory cells express CD25 and are in cycle, as
indicated by high forward light scatter (Fig. 2). This is consistent
with previous studies that have used BrdU labeling and have shown that
a significant fraction of memory cells are dividing (10, 13, 23). The
potential role of persistent Ag could not be addressed in those
experiments, however, either because the Ag stimulus was viral
infection (10, 13, 23), which could allow low level persistence in the
host, or because the activating Ag was not known (10, 23). The presence
of CD25high 1B2+ blasts at long times after
priming (Fig. 2) indicates that Ag persistence is not necessary to
drive the continuing division of at least some of the memory cells.
This conclusion was also reached in experiments where HY-specific
memory CD8+ T cells were transferred into female recipients
(30). The memory cells continued to divide despite the lack of HY Ag in
the recipients. Continuing division of memory cells in the absence of
specific Ag may be driven by the interaction with nonspecific class I
that appears necessary for their survival (30), by environmental growth
factors (31, 32), or both.
The majority of 1B2+ cells in previously primed recipients
have a surface phenotype characteristic of CD8+ memory
cells; they express high levels of CD44 and VLA-4. In contrast, a
smaller fraction express low L-selectin levels, and this varies
depending on the site examined (Fig. 2B). Although
activated CD8+ T cells express low L-selectin, it is again
up-regulated on a significant fraction of cells following the response
(22, 33, 34). During the primary response to P815 in the peritoneal
cavity, virtually all of the responding 1B2+ cells
(>90%) convert to an L-selectinlow phenotype at the peak
of the response on day 8. By day 11, however, about half have converted
back to L-selectinhigh while remaining CD44high
and VLA-4high (18). A small fraction of the
1B2+ cells in previously primed recipients have a phenotype
characteristic of naive cells, expressing low levels of CD44 and VLA-4
and high L-selectin (Fig. 2B). These could
potentially be naive cells that did not respond to the initial
challenge. This appears unlikely, however, since naive cells do not
remain present at detectable levels beyond about day 30 in recipients
that have not been primed with Ag (18). Thus, it appears more likely
that these are cells that responded during the initial challenge but
retain a naive phenotype, consistent with evidence from Tough and
Sprent (23) indicating that some memory T cells, and particularly
CD8+ cells, retain a naive phenotype.
Heterogeneous expression of surface receptors involved in migration and
homing is likely to promote more effective immune surveillance by the
memory population, allowing recirculation through both the lymph nodes
and peripheral tissue. Consistent with this possibility is the finding
that a greater number of 1B2+ cells express high L-selectin
and low VLA-4 and CD44 levels in the lymph nodes than at other sites
(Fig. 2B). Furthermore, the distribution of the
memory 1B2+ cells is very different from that of naive
cells (Fig. 2A). Memory cells are present in much
higher proportion in the blood, which would allow for rapid migration
into peripheral sites of inflammation. Unlike naive cells, they are
also found in small numbers in the peritoneal cavity, consistent with
the ability of memory cells to traffic through peripheral tissue in the
absence of any apparent inflammation (10, 11, 20).
Memory 1B2+ cells respond to Ag challenge much more rapidly than do naive cells with respect to both the speed of migration to the site of challenge and the acquisition of effector function. In a primary response, 1B2+ cells do not become detectable in the peritoneal cavity until days 5 to 6 after challenge. P815 tumor is detectable in spleen and draining LN by days 2 to 4, and the fraction of 1B2+ cells expressing low L-selectin and high VLA-4 increases at these sites during this time, although CD25 up-regulation and blast transformation are not occurring. During days 4 to 6, the number of 1B2+ cells is declining in the spleen, and they are becoming detectable in the peritoneal cavity where they express high CD25 and are blasting. These observations suggest that efficient migration of naive cells to the peripheral site of Ag may require interaction with Ag in the spleen and draining LN to promote altered expression of homing receptors and that migration does not occur until Ag has reached these sites. This model is consistent with earlier studies showing that VLA-4 is up-regulated on CD8+ T cells in response to viral infection or contact Ags and implicating this integrin in being involved in migration to peripheral sites (35, 36, 37).
In contrast to naive cells, the decrease in memory 1B2+
cells in the spleen and LN and entry into the peritoneal cavity begin
within 12 h of rechallenge (Fig. 3B). Although a
small number of 1B2+ cells are present in the peritoneal
cavity before rechallenge, it is unlikely that proliferation of these
cells could account for the substantial increase in the number of
1B2+ cells at this site by 12 h. The memory
1B2+ cells enter the peritoneal cavity well before tumor
cells become detectable in the spleen or LN. In fact, tumor has become
almost undetectable by 12 h in the peritoneal cavity and is never
found in the spleen or LN during a secondary response. This suggests
that rapid migration of memory cells to the site of Ag does not depend
on Ag recognition in lymphoid organs. This conclusion is supported
by the fact that a substantial number of 1B2+ memory cells
migrate into the peritoneal cavity at early times in response to
challenge with RDM4 tumor (Fig. 6A), although they
fail to develop lytic activity in the absence of Ag that can be
recognized by the 2C receptor. In contrast, naive 1B2+
cells cannot be found in the peritoneal cavity at these times when
challenge is with the RDM4 tumor (18). Thus, these results strongly
support the conclusion that memory cells rapidly traffic to an
inflammatory site in an Ag-independent manner, while trafficking of
naive cells is much slower (by several days) and is likely to require
interaction with Ag in lymphoid organs before migration.
Rapid entry alone appears unlikely to account for the increased speed
of the memory response. When naive 1B2+ cells are injected
directly into the peritoneal cavity of a naive mouse along with P815
tumor, so that naive 1B2+ cells are present at the site of
Ag in greater numbers than in the memory response, tumor is not cleared
until day 4 or 5 (data not shown). In addition to much more rapid
migration into the peritoneal cavity, the memory populations present in
the spleen and peritoneal cavity exhibit a low level of cytolytic
activity before rechallenge, and potent cytolytic activity develops in
the peritoneal cavity within 12 h of rechallenge (Fig. 4A). Virus-specific memory populations have recently
been demonstrated to have immediate effector function without a
requirement for Ag-dependent differentiation, as measured by target
cell lysis (13, 38) and IFN-
production (14), and a population of
human effector CD8+ T cells that have cytolytic activity
without in vitro stimulation has been identified (39). Given the
likelihood that P815 tumor is not present in the adoptive transfer
recipients at long times after a primary challenge, it appears that Ag
persistence is not necessary to maintain this cytolytic effector
function. Our results also suggest that the memory population may
include a subset of cells with immediate effector function, accounting
for the lytic activity detected at 12 h in the peritoneal cavity,
as well as a subset that must undergo Ag-dependent differentiation to
acquire effector function, accounting for the second peak of lytic
activity seen on day 3 after rechallenge (Figs. 4 and 7). If so, this
second population also migrates very rapidly to the peritoneal cavity
since the lytic activity peaks on day 3, in contrast to the primary
response where lytic activity peaks on day 8, i.e., 3 days after the
naive cells enter the peritoneal cavity (18).
Rapid trafficking of memory cells to the site of Ag, together with
immediate cytolytic effector function of at least a subset of the
memory cells, might be expected to contribute substantially to the
greater speed and efficiency of a memory response in comparison with a
primary response by naive cells. This was directly demonstrated by
experiments comparing the primary response of 1B2+ cells to
the memory response when the number of 1B2+ cells present
at the time of challenge with P815 was comparable (Fig. 7). Despite
similar numbers of precursors, entry of 1B2+ cells into the
peritoneal cavity, development of cytolytic effector function, and
elimination of tumor all occurred about 4 days sooner in the previously
primed mice than in naive adoptive transfer recipients, and the P815
tumor never reached as high a number in the primed mice as in the naive
mice. Precursor frequency clearly does make some contribution, however,
since mice with a higher memory cell frequency developed detectable
cytolytic activity in the peritoneal cavity and cleared tumor even more
rapidly (Fig. 7). Thus, increased precursor frequency, more rapid
trafficking to peripheral sites, and the immediate effector function of
at least some of the cells all contribute substantially to making a
memory response to antigenic challenge at a peripheral site more rapid
and efficient than a primary response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, University of Minnesota, Box 334 Mayo, 420 Delaware Street, S.E., Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: PE, phycoerythrin; SA, streptavidin; LN, lymph node; PEL, peritoneal exudate lymphocytes; VLA, very late Ag.
Received for publication December 19, 1997. Accepted for publication March 18, 1998.
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References |
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during the differentiation of cytotoxic T lymphocytes induced by allografts. J. Immunol. 148:2348.[Abstract]
4 integrin directs virus-activated CD8+ T cells to sites of infection. J. Immunol. 154:5293.[Abstract]
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