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Department of Pathology, University of Massachusetts, Worcester, MA 01655
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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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Naive CD8 T cells undergo many cycles of division and expansion in cell number after only a brief contact with their cognate ligand expressed on an appropriate APC (9, 10, 11). This has been shown in vitro and in vivo by labeling cells with CFSE, a cytoplasmic dye that is diluted equally among daughter cells on cell division, and by following the expansion in cell number and the loss of CFSE label (12, 13). It appears that a multiple cycle proliferative expansion is a programmed event that does not require a continuous exposure of the naive T cells to their Ag (9, 10, 11). How then do memory T cells respond to homologous Ag, to potentially cross-reactive heterologous Ags, and to cytokines induced by viral infections or by an IFN inducer such as poly(I:C)? Additionally, when a host is infected with a heterologous virus expressing many potential T cell epitopes, how does the combination of viral Ags and virus-induced cytokines influence the activation and division of the host’s memory T cells?
We address these issues here by using mice adoptively reconstituted with CFSE-labeled splenocytes derived from lymphocytic choriomeningitis virus (LCMV)3-immune mice bearing a Thy alloantigen that allows for distinguishing donor from host T cells. We then examine the fate of the LCMV-specific memory T cells in these mice after exposure to poly(I:C), LCMV, or the heterologous viruses Pichinde (PV) and vaccinia (VV). We have previously documented that mice immune to LCMV are partially resistant to PV and VV and that splenocytes from LCMV-immune mice can be transferred into naive mice and provide resistance to PV and VV. This resistance is lost if CD8 T cells are depleted from these donor populations (7). We also have recently identified two cross-reactive CD8 T cell epitopes between LCMV and PV (LCMV-NP205 and PV-NP205) (36) and have evidence that VV may be cross-reactive with two LCMV epitopes (NP205 and gp33) (8, 14) (our unpublished data). Heterologous immunity is not necessarily reciprocal, given that immunity to VV does not provide mice with resistance to LCMV and PV. Therefore, we are poised to examine memory CD8 T cell activation under conditions where effective heterologous immunity occurs and where it does not occur. We find here that heterologous viruses have the capacity to induce a many cycled proliferative expansion of memory CD8 T cells, altering the Ag-specific hierarchies of T cells specific to previously encountered pathogens. In contrast, poly(I:C) drives a limited cell division without increases in cell number or changes in hierarchies. Further, we show that heterologous viruses stimulate memory T cells poorly when heterologous immunity is weak.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LCMV, strain Armstrong, an RNA virus in the Old World arenavirus
family, was propagated in BHK21 baby hamster kidney cells
(15). The WR strain of VV, a DNA virus in the poxvirus
family, was propagated in NCTC 929 cells (16), and the
AN3739 strain of PV, an RNA virus in the New World arenavirus family
only distantly related to LCMV, was propagated in BHK21 cells
(15, 17). Mice were inoculated i.p. with 5 x
104 PFU of LCMV, 2 x
107 PFU of PV, or 1 x
106 PFU of VV. These doses were selected based on
our previous experience in our studies on heterologous immunity between
these viruses (7). The IFN-
inducer,
poly(I:C) (Sigma-Aldrich, St. Louis, MO), was injected i.p. at a dose
of 200 µg/200 µl HBSS (Life Technologies, Gaithersburg, MD) per
mouse, 1 day after adoptive cell transfer (18).
Splenocytes were harvested from host mice 6 days after poly(I:C)
injection or virus infection, unless indicated otherwise, and assessed
for donor cell number, cell division, and peptide-specific IFN-
production.
Adoptive transfer of virus-immune splenocytes
Congenic male C57BL/6 (B6, Thy-1.2) and B6.PL Thy-1a/Cy
(Thy-1.1) mice were purchased from The Jackson Laboratory (Bar Harbor,
ME) at 4–5 wk of age and maintained under specific-pathogen-free
conditions within the Department of Animal Medicine at the University
of Massachusetts Medical School. These Thy-1.1 and Thy-1.2 mice are
genetically identical mice differing only in their expression of the
Thy-1 allele. Thy-1.1 (or Thy-1.2) mice infected i.p with LCMV
(19) were considered LCMV immune after 6 wk or longer of
infection. Either Thy-1.1 or Thy-1.2 LCMV-immune splenocytes pooled
from four to five mice were adoptively transferred via the tail vein
into the congenic strain at 2 or 4 x 107
cells in a 200-µl volume of HBSS. Both strain combinations were used
in these studies based on the availability of LCMV-immune mice, and the
results were comparable. Flow cytometry analysis of donor cells before
transfer revealed that the LCMV-immune donor lymphocyte pool was
composed of 
10% CD8 T cells and 20% CD4 T cells. The CD8 T cell
compartment was composed of 60% CD44high and
40% CD44low subpopulations. Further analysis via
peptide-induced intracellular IFN- assays showed that 15–20% of
donor CD8 T cells were LCMV specific and had the usual hierarchy of
epitope-specific responses to the five known LCMV-specific peptides
(gp33 
NP396 > gp276 > NP205 > gp92) (8, 36). VV-immune mice were generated in a similar manner by
infecting mice with VV. Although VV epitopes are not known, the
frequencies of VV-specific CD8 T cells in the memory state are
comparable with that of LCMV (20). This type of technique
was adapted from previous studies (21, 22, 23). Adoptively
reconstituted mice were challenged with virus or poly(I:C) between 24
and 48 h after transfer. Data presented in the figures are from a
single representative individual host mouse.
CFSE labeling
Splenocytes from donor mice were harvested and isolated as previously described (7). The donor splenocytes were labeled with CFSE using a modification of previously described techniques (12, 13). Briefly, the splenocytes were suspended in HBSS at 2.5 x 107 cells/ml and incubated in 2 µM CFSE (Molecular Probes, Eugene, OR) solution for 15 min at 37°C. After incubation, donor cells were washed twice with HBSS.
Flow cytometry and intracellular IFN- staining
Single-cell lymphocyte suspensions were prepared from spleens
and the peritoneal cavity. The erythrocytes were lysed using a 0.84%
NH4Cl solution. After preincubation with 1 µl
of Fc Block (2.4G2) in 96-well plates containing 100 µl of FACS
buffer (HBSS, 2% FCS, 0.1% NaN3), the cells
were stained for 30 min at 4°C with combinations of fluorescently
labeled mAbs specific for CD8 (53-6.7, FITC, or PerCP), Thy-1.1
(OX-7, PE), and IFN- (XMG1.2, APC), all purchased from BD PharMingen
(San Diego, CA). LCMV peptide-specific, IFN--secreting
CD8+ T cells were detected using the
Cytofix/Cytoperm Kit Plus (with GolgiPlug; BD PharMingen), as
described previously (24). Briefly, cells were incubated
with 5 µM synthetic peptide, 10 U/ml human rIL-2 (BD PharMingen), and
0.2 µl GolgiPlug for 5 h at 37°C. Freshly stained samples were
analyzed using a BD Biosciences FACSCalibur and CellQuest software (BD
Biosciences, San Diego, CA). Peptides used here were originally defined
by Whitton et al. (25) and van der Most et al.
(26). They include NP396–404 (FQPQNGQFI), gp33–41
(KAVYNFATC), gp276–286 (SGVENPGGYCL), NP205–212 (YTVKYPNL), and
gp92–101 (CSANNSHHYI) and were purchased from American Peptide
(Sunnyvale, CA) at 90% HPLC purity.
Statistical analyses
Results were expressed as the arithmetic mean ± SEM. An unpaired Student t test was used for data analysis where indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously demonstrated that adoptively transferred
LCMV-immune splenocytes can provide hosts with a moderate degree of
protective immunity against the heterologous viruses, PV and VV
(7). We wished to analyze the fate of the adoptively
transferred donor T cells under these conditions of heterologous
immunity and compare it with their fate on rechallenge with homologous
LCMV Ags or on stimulation by cytokines induced by poly(I:C). To
examine this issue, splenocytes from naive or LCMV-immune mice were
adoptively transferred into Thy-congenic mice, in which donor and host
T cells could be antigenically distinguished. On adoptive transfer,
host mice were then inoculated with poly(I:C), LCMV, PV, or VV i.p.,
and donor and host cells were visualized on flow cytometry by staining
cells with the appropriate Thy mAb. By day 6 postinfection or
treatment, both homologous and heterologous viral infections elicited
substantial increases in the number of donor CD8 T cells when
compared with unchallenged mice (Fig. 1
, A and B, and Table I). Increases in the number of
donor cells were generally more substantial in
the peritoneal cavity (Fig. 1B and Table I) than in the spleen (Fig. 1A and
Table I). In contrast, poly(I:C) induced no apparent expansion and,
more often than not, caused a reduction of donor cells in the spleen.
Only a marginal increase of cells in the peritoneal cavity (PEC) was
detected on poly(I:C) treatment (Fig. 1, A and B,
and Table I).
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, the amount of expansion in the spleen on
homologous or heterologous virus challenge occurring in the host
population, which is composed of naive T cells, was much less
than that in the donor population obtained from LCMV-immune mice. In
fact, the expansions in the donor CD8 LCMV memory T cell populations
formed a considerable portion of these heterologous virus CD8 T cell
responses in both the spleen and the PEC.
Very different results were observed if donor T cells were from naive
mice that never had a virus infection (Fig. 1C). Neither
poly(I:C) treatment nor any virus infection caused apparent increases
in donor T cell number during this 6-day time period. These data
suggested that the LCMV-specific memory T cells are participating in
the responses to heterologous viruses.
Poly(I:C) and virus infections elicit distinctive patterns of CD8 T cell divisions in CFSE-labeled donor cells from LCMV-immune mice
Recent evidence has indicated that naive CD8 T cells undergo many
cycles of division on exposure to Ag, leading to substantial increases
in CD8 T cell number (9, 10, 11). We questioned whether a
similar type of expansion of memory CD8 T cells would occur in response
to homologous or heterologous Ags or to poly(I:C) stimulated cytokines.
Here, we used the CFSE labeling technique to assess donor cell
divisions (12, 13). This CFSE labeling technique enabled
us to assess the extent of cell divisions on a single-cell level,
albeit within a limited range (less than seven cycles). By day 6,
poly(I:C) treatment elicited in donor CD8 T cells several rounds of
cell division that were distributed between two and more than seven
cycles (Fig. 2A). Consistent
with previous findings (5), a phenotypic analysis revealed
that the cell divisions occurred almost exclusively within the memory
phenotype (CD44high) subset of the donor CD8 T
cells (data not shown). Of significance is that the donor cell
divisions elicited on poly(I:C) treatment did not result in an overall
increase in donor cell number (Table I).
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). These
accumulating donor CD8 T cells, which had lost CFSE fluorescence
intensity, were all CD44high and accounted for
the substantial increase in donor CD8 T cell numbers after virus
infections revealed in Fig. 1 and Table I. About 50–66% of the
undivided donor CD8 T cells observed in the spleen during viral
challenge had a naive CD44low phenotype.
Virtually all of the CD8 T cells observed in the PECs were
CD44highCD8 T cells and had divided several times
on virus challenge, suggesting that only the activated CD8 T cells have
migrated into the PEC. Poly(I:C) treatment induces limited divisions of defined LCMV-specific memory CD8 T cells without an increase in cell number or hierarchical shift in the LCMV-specific T cell repertoire
Analysis of donor cells before transfer revealed that 30% of
the donor CD44highCD8 T cells represented bona
fide LCMV-specific memory CD8 T cells reactive to the five known
LCMV-specific peptides. Poly(I:C) treatment induced cell divisions
preferentially in the memory type CD44highCD8 T
cell compartment, consistent with the results of other investigators
(5) (data not shown). We questioned whether these
proliferating cells were bona fide LCMV-specific memory T cells. To
visualize the Ag-specific T cells, spleen leukocytes from a
reconstituted host were incubated with known LCMV-encoded peptides for
5 h and subsequently stained with anti-CD8, anti-Thy-1.1,
and anti-IFN- Ab. In FACS analyses, donor CD8 T cells were
gated, and cell division patterns were analyzed for the
IFN-+ Ag-specific CD8 T cells. Poly(I:C)
treatment induced limited cell divisions in all the LCMV
epitope-specific CD8 T cells examined (Fig. 3, A and B) and did
not result in an increase in the number of the Ag-specific CD8 T cells
(Fig. 3, A and B). The memory CD8 T cells
specific to each LCMV peptide had divided almost equally on poly(I:C)
treatment and, consequently, there were no hierarchical changes within
the LCMV-specific CD8 T cell repertoire (Fig. 3C). Also, the
CD8 T cells that divided the most after poly(I:C) treatment were not
specific to LCMV epitopes. This phenomenon is developed
elsewhere.4
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The fate of LCMV-specific CD8 T cells was then investigated during
the TCR-mediated recall response induced by a secondary challenge with
LCMV both in the spleen (Fig. 4B) and in the PEC (Fig. 5A). On LCMV infection, all
epitope-specific donor CD8 T cells divided approximately five times by
day 3 postinfection but with little increase in cell number (data not
shown). By day 6, all the LCMV-specific CD8 T cells had completely lost
CFSE, indicating that they had divided more than seven times and the
cell number had substantially increased (Fig. 4B). When the
frequencies of CD8 T cells specific to the known LCMV-specific epitopes
were counted at day 6, their percentage added up to 65 and 80% of the
expanded donor CD8 T cells in the spleen and the PEC, respectively, but
never reached 100% (Figs. 4B and 5A). We
consider this a very high frequency, because some of the response may
be directed against undefined epitopes, and cells in early
stages of apoptosis would not be expected to score as epitope positive
in the IFN- assays. Homologous challenge with LCMV tended to
stimulate a better NP396- than gp33-specific response in the spleen,
causing slight changes in the LCMV-specific T cell hierarchy in the
spleen (Fig. 4B) (27, 28). This result suggests
that an NP396 response has a slight advantage in the spleen on
secondary challenge with LCMV. The mechanism for this is presently
unclear given that there continued to be a dominant gp33 response in
the PEC (Fig. 5A).
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Having demonstrated marked differences in the pattern of memory T
cell division between secondary challenge with homologous LCMV Ags,
which caused a dramatic expansion of T cells specific to all LCMV
epitopes, and poly(I:C), which induced limited cell division without an
increase in number, we questioned what pattern of response would be
induced by heterologous viral infections. Infections with heterologous
viruses bring into the host new Ags that might cross-reactively
stimulate the memory T cells, and they induce high levels of cytokines
that might mimic the effects of poly(I:C). A history of an LCMV
infection provides a level of protective immunity against both PV and
VV (7), and Fig. 1 shows that both viruses caused
significant increases in the number of donor T cells from LCMV-immune
but not from naive mice (7). The expansion of LCMV
Ag-specific donor memory T cells was thus examined in the spleen (Fig. 4, C and D) and peritoneal cavity (Fig. 5, B, and C) in mice challenged i.p. with either PV
or VV. The spleen data were all derived from the same experiment with
the same source of donor T cells, and the hierarchies of the T cells
specific for LCMV epitopes after those responses are summarized in Fig. 6. The peritoneal cell data in Fig. 5
were derived from other representative experiments.
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C and
5B). In contrast, 1% of host CD8 T cells were specific
to NP205. PV-induced responses to NP396, gp33, or gp276 were not above
background levels (Figs. 4C and 5B). In some but
not all experiments, PV also induced a strong response to the LCMV
subdominant epitope gp92, and this usually was more striking in the
peritoneal cavity than in the spleen, as shown in the representative
experiment in Fig. 5B. In contrast to either poly(I:C) or
homologous LCMV challenge, the PV infection caused a substantial change
in the hierarchy of LCMV-specific T cells, consistent with an
Ag-specific expansion of T cells reactive with some epitopes but not
others (Fig. 6). In several experiments, the frequency of the
PV-expanded donor T cells that could be accounted for as LCMV specific
ranged from 30 to 40%. We could account for another 6% of these donor
cells as specific for three other newly identified PV immunodominant
epitopes (36), which in the same mouse accounted for 65%
of the host CD8 T cells at this same time point (data not shown). This
makes it unlikely that there is a large primary acute response to PV in
the donor cell population. The specificity of the other cells was not
determined, although it should be pointed out that in the memory pool
there are probably T cells specific for many undefined subdominant or
cryptic epitopes the cross-reactive amplification of which would not be
detected.
VV induced a lower level amplification of CD8 donor T cells from
LCMV-immune mice, but this was a true proliferation, in contrast to the
division without cell number increase seen after poly(I:C) treatment
(Figs. 1 and 2). In three experiments 5–14% of the VV-expanded
donor cells could be accounted for as LCMV-specific (Figs. 4D and 5C). VV did not induce as massive an
increase in T cells specific to LCMV epitopes as seen with homologous
LCMV challenge or with PV for the NP205 epitope, but a closer look at
the frequencies of the Ag-specific spleen T cells before (Fig. 4A) and after (Fig. 4D) VV infection showed a
marked alteration of the hierarchy (Fig. 6). In general, VV, like PV,
induced an expansion of NP205-specific cells. Fig. 4D shows
that VV induced a 65-fold increase in the total number of
NP205-specific T cells over the untreated control levels. In several
experiments, we found that VV often induced preferential expansions in
NP205- and gp33-specific T cells, but it is noteworthy that individual
animals varied in the relative amount of gp33-specific vs
NP205-specific T cells stimulated. Fig. 4D shows a mouse in
which only the NP205 response was significantly amplified, but Fig. 5C shows PEC data from another mouse in which the gp33
response was marginally higher than the NP205 response and considerably
higher than the normally strong NP396 response; Fig. 5C also
shows that some of the gp33-specific T cells divided more than seven
times, but others underwent fewer divisions, similar to that seen
during a poly(I:C)-type response. Despite this mouse to mouse
variation, it was clear that VV consistently induced a hierarchical
change in the LCMV-specific response (Fig. 6), consistent with
cross-reactive expansions and significantly different from the pattern
observed with poly(I:C).
Lack of reciprocity of memory T cell proliferation in response to heterologous viruses
An interesting aspect of heterologous immunity is that
it is not necessarily reciprocal (7). For example, a
history of an LCMV or PV infection protects a host against VV, but a
history of a VV infection does not protect a host from LCMV or PV. No
explanation for this has been forthcoming. We therefore questioned
whether LCMV could elicit the expansion of VV-induced memory T cells as
effectively as VV could elicit the expansion of LCMV-induced memory
cells. Fig. 7 shows that if mice were
adoptively reconstituted with CSFE-labeled T cells from VV-immune mice
and then challenged with the homologous VV, there was a dramatic
increase in the number of donor cells, which had divided more than
seven times. However, if these mice were challenged with LCMV, there
was a loss rather than an increase in donor T cell number (Fig. 7A), reminiscent more of a cytokine response than of an
Ag-specific amplification. Therefore, LCMV does not stimulate the
proliferation of VV-immune cells, even though VV stimulates the
proliferation of LCMV-immune cells.
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, B and C). This indicates that memory CD4 T
cells, like memory CD8 T cells, can be visualized with CSFE to undergo
many cycles of division and proliferative expansion during a secondary
challenge. VV induced several cycles of division of donor T cells from
LCMV-immune mice and a slight increase in their number (Fig. 7B). In contrast, LCMV caused a pronounced decrease in
number of donor CD4 T cells from VV-immune mice (Fig. 7C).
This was an absolute reduction in cell number, and not a dilutional
effect caused by expansion of LCMV-specific CD8 T cells, given that no
significant LCMV-specific expansion had occurred at this early time
point (Table I). Overall, these experiments indicate that VV is much
more capable of inducing the proliferation of LCMV-immune T cells than
LCMV is of stimulating VV-immune cells. This is true for both CD4 and
CD8 T cells and is reflective of the nonreciprocal nature of
heterologous immunity between these two viruses. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20% of the seeded CD8 T cells could be shown to be specific for one
of the LCMV epitopes, caused a multicycled (more than seven divisions)
expansion of T cells specific for each of the LCMV epitopes (Figs. 4B and 5A). This resulted in a >300-fold
increase in donor CD8 T cell number in the spleen, and >65% of those
cells could be accounted for as being LCMV-specific (Table I and Figs. 4B and 5A). Thus, like the primary T cell
response, in which T cells undergo many division cycles in a
preprogrammed manner (9, 10, 11), the memory T cell response
also involves a multicycled expansion rather than a more limited
division pattern as has previously been seen by other investigators
(29). A similar conclusion for stimulating influenza
virus-specific memory CD8 T cells has recently been made by Turner et
al. (23). The high frequency of definable LCMV-specific T
cells in this expanded donor population argues against an extensive
expansion of bystander T cells not specific for the virus, consistent
with studies published previously for the acute LCMV response
(30). The few cells not recorded as LCMV specific may
either have been dying cells that would not produce IFN- in response
to peptides or possibly to T cells reactive with undefined subdominant
or cryptic epitopes present in the memory pool at low frequency. Even
though T cells specific for all LCMV epitopes underwent sufficient
division to lose all of their CSFE intensity, there was a slight
hierarchical shift in the frequency of T cells specific for the
immunodominant peptides, with the NP396:gp33 ratio increasing somewhat.
This disproportional but reproducible increase of NP396-positive T
cells has been observed previously in LCMV-immune mice receiving a
secondary challenge (27, 28) and may represent a further
selection of the repertoire, as also demonstrated by TCR analyses
(28, 31). Interestingly, CD8 T cells in the PEC did not
demonstrate this same shift (Fig. 5A), and the reason for
that discrepancy is under investigation.
This massive Ag-specific expansion of memory T cells after homologous
viral challenge stood in marked contrast to the bystander stimulation
of T cells elicited by the IFN inducer, poly(I:C) (Fig. 3B
and Table I). Poly(I:C) induced a cell division pattern that did not
result in an increase in spleen donor cell number. Poly(I:C) did cause
a slight increase in donor cells in the peritoneal cavity, the site of
the poly(I:C) inoculation, but these were considerably small numbers
compared with the spleen. It remains possible that some of these cells
migrated out to other organs, but our previous work showed reductions
in host memory CD8 T cell numbers in all the organs tested at day 1
post-poly(I:C) treatment (18). Bona fide memory T cells defined by
their specificity for LCMV epitopes underwent a limited number of
divisions in response to poly(I:C), and an undefined
CD44highCD8+ T cell
population experienced more cell divisions and lost the CSFE label.
This may mean that CD44+CD8 T cells distinct from
true memory cells are better responders than bona fide memory cells to
bystander stimulation; that hypothesis is currently under investigation
in our laboratory. The phenomenon of poly(I:C)-induced division without
an increase in cell number in part reflects the recent observation that
poly(I:C) (and IFN type I) induces apoptosis in memory CD8 T cells
(18). This apoptosis offsets the cell division. Thus,
memory T cell division with an increase in number is characteristic of
an Ag-specific stimulation, whereas division with no increase or loss
in number is characteristic of cytokine-induced stimulation. The
poly(I:C)-induced division of the different LCMV-specific T cells all
appeared the same, and the hierarchy of the T cell response to the LCMV
peptides was similar before and after poly(I:C) treatment (Fig. 3C).
Given the recent observations of unanticipated T cell cross-reactivity
between viruses and the fact that the pathogenesis of viral infections
is greatly altered by prior exposures to heterologous viruses
(heterologous immunity) (7, 8, 16, 32, 36), we wished to
examine how memory T cells specific for one virus responded during
heterologous viral infections. A history of an LCMV infection provides
a degree of protective immunity to both PV and VV, and this
heterologous immunity is at least in part due to CD8 T cells
(7). CTL generated against either LCMV or PV do not lyse
targets infected with the heterologous virus, indicating very little
cross-reactivity between these viruses (17, 33). However,
CTL generated in response to PV infection of LCMV-immune mice lyse
targets infected with either virus, and limiting dilution assays have
indicated that a low frequency of cross-reactive T cells is amplified
by the second viral infection (16, 17). We have recently
found that PV encodes a weak subdominant epitope (PV-NP205) that shares
7 of 9 aa with the LCMV NP205 epitope (36). We questioned,
then, what effect PV infection would have on the LCMV memory pool in
adoptively reconstituted mice. The results indicated that PV caused a
substantial increase in the number of T cells specific for NP205 (Figs. 4C and 5B). This resulted in a strong skewing of
the hierarchy of T cells recognizing LCMV-encoded epitopes (Fig. 6). In
some but not all experiments, we found a significant increase in T
cells specific for gp92 (Fig. 5B). About 30–40% of the T
cells expanded by PV could be shown to be LCMV specific. It was not
clear whether the other expanded cells were cross-reactive with
undefined LCMV epitopes or were the products of bystander expansion,
but PV caused a decline rather than an increase in cell number of donor
cells from nonimmune mice (Fig. 1 and Table I), arguing for a
cross-reactive mechanism. We cannot rule out that the cross-reactive T
cell response may liberate cytokines capable of nonspecifically
stimulating an increase in number of non-cross-reactive T cells.
Keeping in mind that we could account for the great majority of
expanded cells in the homologous LCMV system as being LCMV-specific, we
favor the explanations that the unknown population of PV-expanded cells
may have been memory cells specific for undefined LCMV epitopes
cross-reactive with PV or else of low affinity to the defined epitopes.
Regardless of that interpretation, this experiment shows that during a
heterologous virus challenge, a cross-reactive epitope can induce a
multicycled expansion and dramatic increase in number of cross-reactive
memory T cells specific for a heterologous virus.
Vaccinia virus is a large DNA virus very different from LCMV, but it
encodes over 200 proteins, thereby creating the possibility of many
cross-reactive epitopes (16, 33). VV infection induces an
expansion of CD8 T cells similar to that of LCMV and PV infection.
Although immunodominant epitopes for VV are not defined, data from
limiting dilution assays (33) and intracellular IFN-
assays indicate that the frequencies of VV-specific and LCMV-specific
memory CD8 T cells are comparable. (20). VV infection of
mice seeded with LCMV-immune T cells caused only a moderate expansion
in donor CD8 T cell number that was 10-fold less than that
stimulated by PV (Table I). Nevertheless, this VV-induced expansion of
T cells from LCMV-immune mice was considerably greater than that
elicited from a nonimmune donor population (Fig. 1). The question we
posed was whether the VV stimulation resembled an Ag-specific or a
poly(I:C)-like bystander stimulation. The expansion of the LCMV
epitope-specific T cell population was more subtle than that driven by
homologous LCMV challenge or by PV, which encodes a strongly
cross-reactive epitope, but VV did significantly alter the hierarchy of
the LCMV-specific repertoire and in most cases tended to provoke a
moderate expansion in T cells specific to some (NP205, gp33) but not
other epitopes. For example, the spleen data depicted in Fig. 4A (no challenge) and Fig. 4D (VV challenge)
showed that VV-induced an elevation of NP205-specific T cells from 700
to 13,000, a 2-fold loss in T cells specific for gp276, and about
comparable numbers for NP396 and gp33. In the experiment shown in Fig. 5C, in PEC, substantial expansions of gp33-specific and
NP205-specific T cells were noted. In general, we find that VV induced
expansion of gp33- and NP205-specific T cells is common but that the
relative amounts of expansion vary from mouse to mouse. This might
reflect the fact that TCR usage is different between individuals for a
specific epitope (28, 31). We had previously reported that
VV infection of LCMV-immune mice elicited CTL that preferentially lysed
gp33-coated targets over NP396-coated targets (14). More
recently, we have shown the selective expansion of gp33 and
NP205-specific memory CD8 T cells in the lung of LCMV mice challenged
with VV (8). We also have identified three VV-encoded
peptides, with homology to NP205, when coated onto target cells will
sensitize them to lysis by T cells from LCMV-immune mice challenged
with VV (our unpublished observations). Thus, the fact that VV causes
an increase in donor cell number and a change in the hierarchy of
LCMV-specific T cells is consistent with its acting more like an
Ag-specific than a bystander stimulus. Only 5–10% of the donor T
cell population after VV infection could be accounted for as LCMV
specific. Some of the stimulation could be nonspecific bystander, but
again, the other expanded cells may have had reactivity with undefined
minor LCMV epitopes. In contrast to our results, others have concluded
that VV infection does not increase the frequency of LCMV-specific T
cells in the spleen or peripheral blood (21, 27). It is
clear from the results reported here, however, that the influence of VV
infection on the LCMV-specific memory pool is subtle, with loss of some
T cells occurring while others are expanding in frequency. Certainly,
VV infection causes substantial alterations in the LCMV-specific
repertoire, and sometimes this can be seen more strikingly at sites of
infection such as the lung (8) or the PEC.
One of the perplexing aspects about heterologous immunity is that it is
not necessarily reciprocal. For example, whereas both LCMV and PV
infections can provide immunity to VV, VV infection does not protect a
host against either LCMV or PV (7). Fig. 7A
shows that this lack of protective immunity can be explained by the
failure of VV-immune CD8 T cells to proliferate in response to LCMV,
even though there is a strong expansion in response to homologous VV
infection. Similarly, VV induces some division of donor CD4 T cells
from LCMV-immune mice, but LCMV infection caused a complete loss of
donor CD4 T cells from VV-immune mice (Fig. 7, B and
C) Hence, there appear to be few VV-specific memory T cells
for LCMV to call on. It could be that a virus like VV, that encodes
many proteins, may more likely encode some epitopes that will
cross-react with some T cells in a preexisting memory pool than would a
virus, like LCMV or PV, that encodes only four proteins. Recent work by
Turner et al. (23) showed that influenza B did not expand
T cells specific to influenza A virus, so strong expansions in T cells
are not induced between all virus combinations. It is likely that large
viruses that encode many proteins would need to interfere with class I
Ag presentation, as many of them do, to keep them from being eliminated
by cross-reactive T cell responses (34, 35).
These results indicate that, in response to natural stimuli, memory CD8 T cells may either undergo a limited number of cell divisions or else divide many (more than seven) times and lose their CSFE label. They also show that undergoing a series of cell division events may either result in a marked increase in cell number, as would be expected, or else lead to no increase or even to a decrease in number. This less expected result is probably accounted for by IFN-induced apoptosis of memory T cells (18) and by the fact that there may be something fundamentally different between an Ag-independent cytokine-mediated homeostatic cell division and an Ag-dependent stimulation that results in the proliferation of T cells in the true sense of the word, i.e., an increase in cell number. It is important to make the distinction between true proliferation and division without an increase in number. Heterologous viral infections have the capacity to drive both an Ag-specific proliferation, as a consequence of cross-reactive epitopes, or a cytokine-mediated homeostatic division, as a consequence of their abilities to induce IFN, IL-15, and other cytokines. Either way, some form of memory cell stimulation is likely to occur whenever a host becomes infected by pathogen.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Raymond M. Welsh, University of Massachusetts, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: Raymond.welsh{at}umassmed.edu
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; PV, Pichinde virus; VV, vaccinia virus; PEC, peritoneal cavity leukocyte.
4 C. D. Peacock, S.-K. Kim, and R. M. Welsh. Reduced capacity of bona fide memory CD44hiCD8+ T cells to respond to homeostatic and poly(I:C)-induced proliferation. Submitted for publication.
Received for publication March 6, 2002. Accepted for publication April 29, 2002.
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