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T Cells1
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells has been known for over 15 years,
but their significance in innate immunity to virus infections has not
been determined. We show here that T cells are well suited to
provide a rapid response to virus infection and demonstrate their role
in innate resistance to vaccinia virus (VV) infection in both normal
C57BL/6 and 
TCR knockout (KO) mice. VV-infected mice deficient in
T cells had significantly higher VV titers early postinfection
(PI) and increased mortality when compared with control mice. There was
a rapid and profound VV-induced increase in IFN--producing T
cells in the peritoneal cavity and spleen of VV-infected mice beginning
as early as day 2 PI. This rapid response occurred in the absence of
priming, as there was constitutively a significant frequency of
VV-specific T cells in the spleen in uninfected TCR KO mice,
as demonstrated by limiting dilution assay. Also, like NK cells,
another mediator of innate immunity to viruses, T cells in
uninfected TCR KO mice expressed constitutive cytolytic activity.
This cytotoxicity was enhanced and included a broader range of targets
after VV infection. VV-infected TCR KO mice cleared most of the
virus by day 8 PI, the peak of the T cell response, but
thereafter the T cell number declined and the virus recrudesced.
Thus, T cells can be mediators of innate immunity to viruses,
having a significant impact on virus replication early in infection in
the presence or absence of the adaptive immune
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T and B cell responses
often being essential for clearance of the pathogen and establishment
of immunity. Although the existence of T cells has been known
for over 15 years, their significance in this paradigm of protective
immunity is still being determined. Some work has suggested that
T cells may play a role in the control of parasitic infections such as
malaria and Eimeria veriformis and bacterial infections such
as Listeria monocytogenes and Klebsiella
pnuemonia (2, 3, 4, 5, 6, 7, 8, 9, 10, 11), but information about these cells
in viral infections remains rudimentary (12, 13, 14, 15, 16, 17, 18, 19).
Increases in T cell number appear in the peripheral blood of
individuals infected with EBV and HIV infections (13, 14),
and some human T cell clones preferentially lyse targets
infected with vaccinia virus
(VV)3 or HSV
(15). In localized ocular HSV-1 infection of mice, the
absence of T cells have been correlated with enhanced
neurological disease late in infection; however, the role for T
cells in controlling HSV titers is hidden, as the antiviral function of
T cells can be substituted for and masked by T cells
(10, 16, 17); both TCR knockout (KO) and TCR
KO mice clear HSV, but this infection is fatal in + TCR KO
mice. These observations are in contrast with many other viruses such
as VV, lymphocytic choriomeningitis virus (LCMV), and influenza, which
the host is unable to clear in the absence of T cells.
Infections with the parasite E. veriformis also demonstrate
a T cell effector function that is masked by T cells, as
+ TCR KO mice are more susceptible to primary infection with
this pathogen than TCR KO mice, but there is no difference in
clearance of this parasite in -deficient mice when compared with
normal controls (7, 10).
T cells accumulate in the lung late after influenza infection of
mice, but their function in murine influenza is unknown. It has been
suggested that they may target heat-shock protein-displaying
macrophages and play a housekeeping role during the resolution phase of
the inflammatory response (18, 20, 21). T cells may
also have a role in the pathogenesis of various autoimmune diseases and
diseases of unknown etiology, as elevated levels of T cells have
been reported in rheumatoid arthritis (22), multiple
sclerosis (23, 24), pulmonary sarcoidosis
(25), inflammatory bowel disease (26), and
polymyositis (27). Some of these diseases might be
precipitated or exacerbated by viral infections, and recent evidence
has indicated that T cells may contribute to myocarditis in mice
infected with encephalomyocarditis virus (19).
Although we previously have predicted, based on a few preliminary
observations, that T cells may participate in protection against
VV infection (28), in no viral infection to date has a
clear role for T cells in innate immunity to virus infection
been definitively documented. Here we report that T cells not
only were constitutively cytolytic, but VV-specific T cells were
present at significant levels in the unprimed host, rapidly expanding
in number, secreting IFN-, and having enhanced cytotoxic activity on
VV infection. All of these features make them well fitted for providing
early resistance to VV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 (H-2b), TCR KO, TCR
KO, + TCR KO, and SCID mice all on the C57BL/6 background were
purchased from The Jackson Laboratory (Bar Harbor, ME) and used at
2–26 mo of age. The 129 x C57BL/6 TCR KO and +/- controls
(6) were donated to us by P. Mombaerts and S. Tonegawa
(Whitehead Institute, Boston, MA).
Viruses
The WR strain of VV was propagated in vero cells obtained
from the American Type Culture Collection (ATCC, Manassas, VA; Ref.
29). LCMV, strain Armstrong, was propagated in BHK21 baby
hamster kidney cells (29). These viruses were purified
over sucrose gradients and diluted in PBS. Purified viruses were used
to prevent activation of T cells by tissue culture contaminants.
Control uninfected mice were either left uninjected or injected with
mock-infected culture supernatant sedimented on a sucrose gradient. The
control mice were always age-matched to the experimental group and
housed under the same pathogen-free conditions as the experimental
group for the identical time period. All mice used were healthy, with
no evidence of any underlying disease. For acute virus infections, mice
were inoculated i.p. with 4 x 104 PFU of
LCMV or 1–2 x 106 PFU of VV.
Cell lines
KO (H-2b), a SV40-transformed kidney cell line derived from a C57BL/6 mouse (30) and provided to us by Satvir Tevethia (Pennsylvania State Medical Center, Hershey, PA), was propagated in DMEM (Life Technologies, Rockville, MD) supplemented with 20 µM 2-ME. MC57G (H-2b), a methylcholanthrene-induced fibroblast cell line from C57BL/6 mice, was propagated in Eagle’s MEM (Life Technologies). NCTC929 cells (ATCC) are a variant of L cells derived from C3H mouse liver (H-2k). YAC-1 cells are a Moloney virus-transformed T lymphocyte line from strain A mice, classically used as an NK-sensitive target in cytotoxicity assays. KO and MC57G cells were infected with LCMV at a multiplicity of infection of 0.01–0.02 PFU/cell and incubated for 2 days at 37°C. KO, MC57G and NCTC929 cells were infected with VV at a MOI of 4 for 2–4 h at 37°C. KO cells were infected with HSV at a multiplicity of infection of 4 and incubated overnight at 37°C. All cell lines were cultured in medium supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 2 mM L-glutamine, 10 mM HEPES, and 10% heat-inactivated (56°C, 30 min) FBS (Sigma, St. Louis, MO).
FACS staining
Freshly isolated splenocytes, peritoneal exudate cells (PEC),
and T cells derived from limiting dilution assays (LDA) were stained
for fluorescence, as described previously (29). The
following Abs obtained from BD PharMingen (San Diego, CA) were used:
anti- TCR biotin (clone GL3), anti- TCR biotin (clone
H57-597), anti-V2 biotin (clone UC3-10A6), anti-V4 biotin
or FITC-conjugated (clone GL2), anti-CD3
FITC-conjugated (clone
145-2C11), anti-NK1.1 biotin or PE-conjugated (clone PK136),
anti-CD19 FITC-conjugated (clone 1D3), anti-CD4 FITC-conjugated
(clone H129.19), anti-CD8 FITC-conjugated (clone 53-6.7), and
anti-Thy1.2 FITC-conjugated (clone 53-2.1). Secondary stains used
included PE- and FITC-conjugated streptavidin (Becton Dickinson, San
Jose, CA) and tricolor-conjugated streptavidin (Caltag,
Burlingame, CA).
Depletion of NK or T cells in vivo
A carefully titrated dose of antiserum to asialo
GM1 (5 µl; Wako Chemical, Dallas, TX) that
depleted NK cells but not CTL activity was injected i.p. in a
volume of 0.1 ml 2 days before harvesting spleens from naive uninfected
mice (31, 32). Anti- TCR mAb (clone UC7-13D5; 100
µg; BD PharMingen) was injected i.p. in a volume of 0.1 ml at day 0
and day 2 of VV infection. The control group received a hamster IgG
isotype mAb (clone A19-3; Jackson ImmunoResearch Laboratories, West
Grove, PA) at the same times.
LDA for VV-specific CTL precursors (pCTL)
Adult mice were inoculated i.p. with VV, and at the indicated times postinfection (PI), spleens were harvested. The VV-specific pCTL frequency per splenocyte was quantified by LDAs of unsorted cells, as adapted from a method described previously (29, 33). Briefly, splenic lymphocytes from uninfected or VV-infected mice were harvested and titrated into 96-well U-bottom plates with 48 replicates at each dilution. They were stimulated with irradiated VV-infected PECs (3–4 x 104/well) and with a hybridoma line producing anti-CD3 (1 x 103/well; 145-2C11; ATCC). They also were supplemented with irradiated splenic feeders (1–2 x 105/well) and growth factors provided by using culture supernatant from IL-2-secreting gibbon lymphoma tumor cell line MLA144 (ATCC) at 16% concentration (34) and 10 U/ml of IL-2 (Cetus, Emeryville, CA). The VV-infected PECs were generated by infecting thioglycollate-injected mice with 1 x 106 PFU of VV in 0.1 ml i.p. 12 h before plating. To these culture wells was added neutralizing polyclonal antisera to VV at 1/100 dilution to inactivate the highly lytic VV.
On day 5 of culture, individual wells were split 2- to 3-fold and
assayed for cytolytic function on uninfected or VV- or HSV-infected
syngeneic target cells (KO) with a modified 51Cr
release assay. 51Cr-labeled targets (5 x
103) were added to all wells, and after an 8
h of incubation at 37°C the supernatant was harvested. Positive wells
were defined as those wells in which 51Cr release
exceeded the mean spontaneous release by >3 SDs. Frequencies were
calculated using 
2 analysis according to
Taswell (35) on a computer program kindly provided by
Richard Miller (University of Michigan, Ann Arbor, MI).
Cytotoxicity assays
Cell-mediated cytotoxicity was determined by a standard microcytotoxicity assay (36). Varying numbers of effector leukocytes were plated in triplicate to achieve the desired E:T ratio. 51Cr-labeled MC57G or NCTC929 target cells (5 x 103), either uninfected or infected with virus, or uninfected NK cell-sensitive YAC-1 cells, were added to all wells, and after a 6-h incubation at 37°C, the supernatant was harvested and counted. Data are expressed as the percentage of specific 51Cr release = 100 x [(experimental cpm - spontaneous cpm)/maximum release cpm - spontaneous release cpm)]. The spontaneous release for each target used in these assays was <20%.
Sorting for T cells present in the peritoneal cavity
PECs were harvested and pooled from 6 to 8 VV-infected (day 8)
mice. These cells were fluorescent-labeled with FITC-conjugated
anti-CD3 and PE-conjugated anti-NK 1.1 and sorted on either
a FACS 440 (Becton Dickinson) or FACStarPlus
(Becton Dickinson) for the CD3+
NK1.1+ and CD3+
NK1.1- populations. The purities of these
populations were >90%. These enriched T cell populations then
were used in cytotoxicity assays. The particular anti-CD3 (clone
145-2C11) used for sorting did not inhibit or enhance lysis of targets
when directly placed in cytotoxicity assays with unsorted PEC
lymphocyte populations from day 8 VV-infected TCR KO mice.
Similar experiments with anti- TCR mAb resulted in
enhancement of lysis of certain targets, including VV-infected MC57G,
NCTC929, and VV-infected NCTC929. Therefore, anti- TCR mAb
could not be used for sorting experiments.
Intracellular IFN- staining
Splenocytes or PECs at 2 x 106 per
tube were stimulated with 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin
(Sigma) for 4 h at 37°C or left unstimulated as described
previously (37). The unstimulated cells were kept at room
temperature during processing, as this was found to help retain
spontaneous IFN- production after removal from the host. The cells
were stained with anti-NK1.1 biotin and tricolor-conjugated
streptavidin and FITC-conjugated anti-CD3 and fixed with 2%
paraformaldehyde. They then were permeabilized with 0.5% saponin
before adding PE-conjugated rat anti-mouse anti-IFN- mAb (BD
PharMingen, San Diego, CA) or control PE-conjugated rat IgG1 isotype.
They were analyzed on either a FACS 440 (Becton Dickinson) or
FACStarPlus (Becton Dickinson).
Virus titration
The virus load in organs was determined by plaque assays on vero cells with a 10% tissue homogenate taken from individual mice. Results were expressed as the geometric mean titers, i.e., the arithmetic averages of the log10 titers for four or five animals, plus or minus the SEM. Titers reported are log10 PFU per whole spleen, liver, or both abdominal fat pads.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells are more susceptible to VV-induced
mortality
The importance of T cells in protection against VV
infection was examined in T cell-deficient mice. At a high dose
of VV (5 x 106 PFU), the TCR KO mice
began to die much more rapidly (day 3) than normal C57BL/6 mice (day 7;
Fig. 1
A) and were all dead by
day 8. In contrast, 43% of the normal C57BL/6 mice survived the VV
infection. With the usual sublethal dose of VV (1 x
106 PFU), both normal C57BL/6 and TCR KO mice
survived. Studies comparing TCR KO to + TCR KO mice also
suggested that T cells played a role in decreasing mortality
after VV infection (Fig. 1B). At the sublethal dose of VV in
normal C57BL/6 mice (1 x 106 PFU), only
20% of the + TCR KO mice were still alive by 12 days after VV
infection as compared with 80% of the TCR KO mice. However,
without the presence of T cells, these mice all eventually died
by 17 days after VV infection. SCID mice, which lack both and
T cells as well as B cells, died more rapidly than the +
TCR KO mice after VV infection, suggesting that T cell-independent Abs
produced by B cells may also play a significant role in mice that lack
T cells, as has been shown previously with polyoma virus infections
(38).
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T cells
Table I shows that at day 3 PI there
was a significant 2.5- to 3-fold higher VV titer in three organs
(spleen, fat pads, and liver) of the TCR KO mice in comparison
to normal mice. This increased to a 10- to 79-fold difference at day 4
and a 5- to 20-fold difference at day 5. There was a 10- to 63-fold
increase in VV titers 3 days PI in another T cell-deficient
strain of mouse, 129 x C57BL/6, when compared with a +/-
(129 x C57BL/6) control. VV titers were elevated when T
cells were depleted by using anti- TCR mAb in vivo in C57BL/6
mice. There was a 25-, 10-, and 6-fold higher VV titer in the spleen,
fat pad, and liver, respectively, of the T cell-depleted mice 4
days after VV infection. In contrast, it should be noted that the
titers of LCMV in TCR KO mice on day 4 after LCMV infection were
the same as in control C57BL/6 mice, suggesting that T cells do
not play an overt role in protection against LCMV infection.
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+ TCR KO mice had 25- and 8-fold higher VV titers and on day 8,
25- and 158-fold higher VV titers in the spleen and liver, respectively
than the TCR KO mice (Table I
). The fat pads were not titrated at
day 8, as the fat pads in the + TCR KO mice had virtually
disappeared, a phenomenon that we have observed previously with very
high dose VV infections in normal mice.
Recruitment and activation of T cells by VV infection in
C57BL/6 mice
There was a moderate but significant increase in T cells in
the spleen of normal C57BL/6 mice starting 2 days after VV infection,
increasing from 2.1% before infection to 5.0% by 6 days PI (Fig. 2A, i). This represents a
gradual increase in total T cell number of 4-, 6.2-, and
9.5-fold at days 2, 4, and 6 PI, respectively (Fig. 2C, ii).
In normal C57BL/6 mice, the percentage of T cells in the
peritoneal cavity, the initial site of virus replication, increased
from 1.2% to 9.1% at 2 days PI, and remained still elevated at
7.2% and 4.9% at 4 and 6 days, respectively. Because the total
number of leukocytes increased, this represents a dramatic 31-,
81-, and 63-fold increase in total T cell number at days 2,
4, and 6 PI, respectively, compared with uninfected mice (Fig. 2Ci).
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(39).
Therefore, we examined whether T cells were activated by VV
infection to produce IFN-. In C57BL/6 mice, by day 2 of VV
infection, 12% of the T cells in the peritoneal cavity produced
IFN- on PMA and ionomycin stimulation as compared with 1.0% in
uninfected mice (Fig. 2B, i). This increased level of
T cells capable of producing IFN- persisted even at day 6 of
infection. In fact, by day 2, there had been a 300-fold increase in the
total number of T cells producing IFN- after stimulation, and
they continued to increase at day 4 and day 6 of VV infection (Fig. 2C, i). This treatment with PMA and ionomycin measures the
potential of cells to make IFN-, but to determine whether there were
signals in their environment to elicit IFN-, we tested cells in the
absence of PMA and ionomycin and showed at 2 days PI that 5.6% of the
peritoneal T cells produced IFN- spontaneously (Fig. 2B, ii). Comparable results were seen in the spleen after
PMA and ionomycin stimulation, but with a more gradual increase in
T cells capable of producing IFN- to 5% by day 6 (0.5% in
uninfected mice) PI (Fig. 2A, ii and C, ii).
Recruitment and activation of T cells by VV infection in
TCR KO mice
In the above experiments shown with C57BL/6 mice, we could not
rule out that some IFN--producing T cells may have
contaminated the T cell population, especially at day 6, and
confound our results. Because of this, and because TCR KO mice
demonstrated an ability to control VV better than + TCR KO mice
(Table I), we also examined expansion and activation of T cells
in TCR KO mice. Fig. 3A,
i demonstrates a representative example of the gradual
percentage increase in T cells in the peritoneal cavity at
consecutive time points after VV-infection in TCR KO mice.
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T cells the kinetics
of the T cell response were altered. TCR KO mice had at
least 13- and 6-fold more T cells present in the uninfected
peritoneal cavity (1.0 x 105 vs 0.08
x 105) and spleen (3.8 x
106 vs 0.6 x 106),
respectively, than normal C57BL/6 mice. They also had 2-fold higher
numbers of T cells in the peritoneal cavity and spleen at the
peak of their response (Figs. 2C and 3B). In
TCR KO mice there also was a more gradual increase in T cells in
the peritoneal cavity after VV infection, with a 6-fold increase by day
5 PI and peaking at day 8 PI, with a 14-fold increase in T cell
number (Fig. 3B). This slight delay in T cell
increase as compared with normal C57BL/6 mice may relate to the already
high numbers of T cells in the peritoneal cavity of uninfected
mice, and the continued increase most likely relates to the greater
difficulty these mice have in completely clearing the virus. There was
a more moderate 2.5-fold increase in the total number of T cells
in the spleen, peaking at day 5 of infection.
In comparison to the C57BL/6 mice, a much greater percentage (50–60%)
of T cells recruited to the peritoneal cavity were capable of
producing IFN- in response to PMA and ionomycin, even in the
uninfected host (Fig. 3A, ii). This suggests that T
cells may be at a higher state of activation in the absence of T
cells. There was a 7-fold increase in IFN--producing T cells
in the peritoneal cavity at day 5 PI, ultimately peaking with a 20-fold
increase at day 8 PI (Fig. 3B, i). Day 5 of VV infection was
the peak of the T cell response in the spleen, with 
50% of
the T cells producing IFN-, representing a 4-fold increase in
total number of IFN--secreting T cells when compared with
uninfected mice (Fig. 3B, ii). Peritoneal T cells
after VV infection also spontaneously produced IFN- without in vitro
stimulation with PMA and ionomycin (Fig. 3B, iii). This
spontaneous production also appeared to peak slightly earlier, at day 5
of VV infection, rather than at day 8, which was seen with PMA and
ionomycin stimulation. Consistent with this increased ability of
T cells in TCR KO mice to produce IFN after VV infection was the
observation that serum of the TCR KO mice was shown by ELISA to
have 7-fold higher IFN- levels than + TCR KO mice at 8 days
after VV infection (2470 ± 931 vs 350 ± 50;
n = 4; p < 0.05, Student’s
t test). It should also be noted that there was very little
recruitment of T cells into the peritoneal cavity of TCR KO
mice infected with LCMV (data not shown), a virus whose replication in
vivo was not influenced by the presence or absence of T cells
(Table I).
Available variable chain Abs were used to examine the dynamics of
VV-induced T cell subpopulations in the TCR KO mice. In one
representative experiment at day 8 after VV infection, 5.8% of the
splenocytes were T cells, and of those, 28% (of the
CD3+, + population)
stained with V2 and 22% stained with V4. Only a very small
subpopulation (0.4%) of the CD3+,
+ cells expressed both V2 and V4. The
spleen showed comparable increases in both subsets on VV infection with
2-fold (day 0, 1.5 x 106 vs day 5, 2.6
x 106) and 3-fold (day 0, 0.7 x
106 vs day 5, 2.0 x
106) increases in V2 and V4, respectively,
at day 5, the peak of the splenic T cell response. There was a
more dramatic increase in both subsets in the peritoneal cavity by day
8 of VV infection with a 4-fold increase in V2 (day 0, 0.6 x
105 vs day 8, 2.4 x
105) and a 9-fold increase in the V4
subpopulations (day 0, 0.2 x 105 vs day 8,
1.8 x 105). These results suggest that
there was an of expansion of both of these two common peripheral
subsets of T cell populations on VV infection.
Cytotoxic activity of T cells
Potential cytotoxic lymphocyte populations in VV-infected TCR
KO mice.
To examine whether VV infection could also activate T cells into
cytotoxic effectors, we used TCR KO mice, which had high T
cell yields, and with which we would not have to distinguish T
cell from T cell cytotoxic activity. To design these experiments
we had to carefully consider the types of potentially cytotoxic
effector cells in the TCR KO mouse spleen. Fig. 4A demonstrates a
representative splenic lymphocyte population at day 8 of VV infection
in TCR KO mice. It consisted of 35% B cells
(CD19+), 21% NK cells
(NK1.1+ CD3--) and 16.7%
T cells
(TCR+CD3+). Some of
these T cells coexpressed molecules that are classically
associated with NK and T cells. Approximately 23% of the
T cells expressed NK1.1, and 18% expressed CD8. All T cells
coexpressed Thy1.2, which at day 8 of VV infection separated into a dim
and a bright population of Thy1.2 expression. The peritoneal cavity at
day 8 of VV infection showed very similar results (data not shown). The
T cell coexpression of NK1.1 and CD8 was the same in the spleen
and peritoneal cavity, whether or not the mouse was infected. Thus, the
presence of NK cells and T cells that coexpressed NK1.1 had to
be taken into consideration in designing our experiments to examine
T cell cytotoxic function in vivo.
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T cells from TCR KO mice constitutively lyse sensitive
targets.
Although T cells have often been shown to display cytotoxic
activity after several days of in vitro stimulation with various Ags,
it has been difficult to show that freshly isolated T cells are
cytotoxic (2). By using naive resting C57BL/6
(H2b) TCR KO mice, we showed that an
allogeneic target, NCTC929 (H2k), was very
sensitive to lysis, and its sensitivity was further augmented after
infection with VV. Fig. 4B, i shows that splenocytes from
naive TCR KO mice lysed targets in the hierarchy of NCTC929 +
VV > YAC-1 > NCTC929. Because NK cells in uninfected mice
are constitutively cytotoxic and capable of lysing YAC-1 cells
(40), we needed to determine whether NK cells in TCR
KO mice contributed to the constitutive lysis of these three targets.
Because a significant portion of T cells was shown to express
NK1.1 (Fig. 4A) we were not able to use anti-NK1.1,
which is commonly used to deplete NK cells in C57BL/6 mice. Instead, we
treated mice with a dose of anti-asialo GM1
that we have routinely used to selectively deplete NK cells but not T
cells in C57BL/6 mice and in mouse strains that do not express the
NK1.1 molecule (31, 32). Treatment of uninfected TCR
KO mice with anti-asialo GM1 resulted in the
selective loss of YAC-1 killing and in the retention of killing of
VV-infected and uninfected NCTC929 cell targets (Fig. 4B,
ii). There was no lysis of the syngeneic target MC57G
(H2b) or VV-infected MC57G by lymphocytes from
these uninfected mice (Fig. 4B). These results suggested
that T cells in TCR KO C57BL/6 mice
(H2b) constitutively lysed NCTC929 cells
(H2k) and that VV infection of these cells
further enhanced their lysis. It should be noted that it is possible
that T cells may not be in this activated state in the presence
of T cells in the normal host.
Splenic cytotoxic activity against all tested targets was greatly
elevated by day 8 after VV infection (data not shown), but when
VV-infected TCR KO mice were treated with anti-asialo
GM1 there was a significant decrease in the
cytotoxic activity against all of the targets (data not shown).
Residual activity resembled that of an uninfected mouse treated with
anti-asialo GM1 (Fig. 4B). FACS
staining demonstrated that activated T cells increased their
expression of asialo GM1 (data not shown), a
phenomenon that previously has been described for whole spleen cell
populations after viral infections (31). Thus, in
VV-infected mice, depletions with this Ab could not be used to
distinguish the effector populations.
Sorted peritoneal T cells from VV-infected TCR KO mice
are cytolytically active directly ex vivo.
Cytolytic activity in VV-infected mice was more potent with PEC than
with splenocytes (Fig. 5A).
The cytolytic activity of splenocyte populations diminished rapidly in
vitro, making it difficult to analyze the populations after cell
sorting, but we were able to analyze by cell sorting the more
cytolytically active, higher frequency peritoneal T cells, which
required less cell preparation time (i.e., there was no mincing of
tissue and NH4Cl treatment) before staining with
a mAb to CD3 and sorting. In three experiments, the proportion of the T
cells from day 8 VV-infected TCR KO mice that costained with
anti-CD3 and anti-NK1.1 ranged from 22 to 62%, with a mean of
38% ± 12. As so few cells could be obtained from sorted PECs even
from large numbers of mice (six to eight mice), only selected targets
were tested as shown in Fig. 5B. Sorted
CD3+ NK1.1+ T cells
mediated dramatic levels of cytotoxicity on allogeneic VV-infected
NCTC929 cells and on syngeneic VV-infected MC57G cells (Fig. 5B). There was 20% lysis of VV-infected NCTC929 cells at
an E:T ratio of 1:1. Sorted CD3+
NK1.1- T cells also effectively lysed
VV-infected NCTC929 cells. These results from Fig. 5 demonstrate that
cytolytically active T cells that preferentially lyse
VV-infected targets are elicited in the peritoneal cavity after VV
infection. They coexist in this environment with NK cells, shown
previously by us to be induced during VV infection
(41).
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pCTL in unprimed
TCR KO mice as quantified by LDA
To examine the presence of VV-specific CTL clones in
TCR KO mice we stimulated splenocytes from uninfected and VV-infected
mice in vitro in LDAs. Consistent with the cytotoxicity data, we were
able to detect a significant frequency of VV-specific pCTL in
the unprimed uninfected spleen, as 1/3781 splenocytes was able to lyse
a VV-infected target (Fig. 6A,
i). Because a naive spleen has 4.0 ± .5% (n =
7) T cells, this means 1/151 of the T cells was
able to lyse a VV-infected target. Thus, unlike VV-specific T
cells, there is constitutively a high frequency VV-specific T
cells in the unexposed naive host, and it was further increased after
VV infection (Fig. 6A, ii).
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B shows that these pooled cells were virtually
all CD3+, Thy 1.2+,
TCR-expressing cells, with levels of coexpression of NK1.1 and CD8
similar to that of the in vivo splenic and peritoneal T cells.
We were unable to detect the expression of TCR, or the B cell
marker CD19 on these cells, nor were there significant numbers of
NK1.1+ cells not expressing CD3 or TCR.
These cells also were stained with Abs for the TCR variable
chains to examine subpopulations of VV-induced T cells (Fig. 6C). There were similar proportions of the two common
peripheral T cell subsets, V2 (30%) and V4 (17.4%).
There also was a small fraction (5%) of T cells that express
both V2 and V4.
Kinetics of VV clearance in TCR KO mice
Young (4 mo) and older (8 mo) TCR KO mice were examined for
their abilities to control VV infection. Both groups of mice were able
to bring the virus transiently under control by day 8 of VV infection.
The clearance of VV from the spleen, liver, and fat pads varied
somewhat, dependent on the age of the mice (Fig. 7). Younger mice (4 mo) had virus titers
that were high at day 4, lower at day 6, and, except in the fat pad,
very low at day 8, at the peak of the T cell response. This
indicates that a host innate response mechanism almost completely
cleared the virus in the absence of a normal T cell response.
However, Fig. 7 shows that there was a recrudescence in viral titers at
day 12, in parallel with the decline in the T cell number, as
presented in Fig. 1. In these younger mice we were unable to titrate
the virus in the fat pads at day 12, as the fat pads had virtually
completely disappeared. The older mice (8 mo) initially had much lower
virus titers than the younger mice, had almost completely cleared virus
by day 6, and had undetectable virus in all organs at day 8; however,
viral titers started to recrudesce by day 12. Young TCR KO mice
died at about day 14 of VV infection, whereas older mice survived for
3–6 mo before dying. This indicates that VV was transiently brought
under control without the participation of T cells, but when the
T cells declined in number, the virus recrudesced. These results
clearly indicate that these mechanisms of innate immunity can have a
significant impact on virus load in the absence of the adaptive T cell
immune response and may in part be age dependent.
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T cell and Ab-producing B cell
clones, which together usually clear the infection and provide lasting
immunity. Cytotoxic IFN--secreting lymphocytes are uniquely suited
for the control of virus infections, and innate immunity against some
viruses, such as murine CMV, has been shown by us to be
effectively mediated by NK cells activated by type I IFNs early in
infection (1). Recently, we also have reported that memory
T cells specific to previously encountered pathogens can
infiltrate virus-infected tissue early in infection and play protective
and/or immunopathological roles in antiviral immunity in response to
heterologous viruses (42). Here we provide evidence that a
third class of cytotoxic, IFN--producing lymphocytes, T
cells, can in immunologically naive mice rapidly respond to a viral
infection and curtail titers by 3 to 5 days PI. Hence, T cells
can be effectors of innate immunity to viruses.
Our results suggest that the T cells control VV infection early
but transiently, and that T cells have the capacity to
permanently clear the virus. TCR KO mice had much higher VV titers
than normal control mice early in infection, but they did ultimately
clear the virus. VV infection was gradually brought under control in
mice lacking T cells by day 8 of infection, but it then
recrudesced, leading to death of the hosts. In contrast, in other
reports of T cells altering the course of bacterial, parasitic,
or viral infections, the protective role of T cells could be
substituted for and masked by T cells. For instance, mice
deficient in T cells clear ocular HSV-1, L.
monocytogenes, K. pneumonia, and E.
veriformis as efficiently as normal mice (6, 7, 11, 16). Only in the absence T cells could a protective role
for T cells be identified in ocular HSV-1 (16) and
E. veriformis infection (7); mice deficient in
both and T cells were more susceptible to either
infection than were T cell-deficient mice. Interestingly, in
K. pneumonia infection, both T cell- or +
T cell-deficient mice were more susceptible than normal intact
or T cell-deficient mice (11). Our results with VV
infection were clearly different, as T cells could not
substitute for T cells, and T cells could not substitute
for T cells.
T cells appear to have features that make them well suited for
mediating innate immune responses to VV. For instance, T cells
were rapidly expanded and recruited to the site of virus replication
and activated to produce IFN-, to which VV is extremely sensitive
(39). The activation of IFN--secreting T cells
has been observed in a number of parasite and bacterial models such as
with Plasmodium chabaudi or Salmonella
choleraesuis (3, 4) but IFN- production by
T cells had not been observed previously in viral infections. The
finding that T cell expansion peaks early during VV infection
and then decreases sharply is a feature common to T cell
behavior in other antigenic systems, such as infection with L.
monocytogenes or E. veriformis (10). The
rapid increase of T cells in the peritoneal cavity on VV
infection, where there is such a low frequency of T cells, may
relate to both recruitment and expansion. T cells, much like NK
cells and memory CD8 T cells appear to be constitutively in a partially
activated state and therefore are easily stimulated (28).
It is possible that most T cells exist de facto as circulating
memory cells after activation by foreign- or self-ligands early in the
host’s development, and hence are able to respond rapidly. However,
there are no clear high-affinity, pathogen-specific T cells with
recall ability defined (10). Because their specificity is
broad, it also would be important that they be under stringent
immunological control and be rapidly down-regulated when they have
accomplished their function.
We also observed a second characteristic of T cells that would
be important in mediators of innate immunity. T cells from naive
TCR KO mice were constitutively cytolytic against a sensitive
allogeneic target, and this target’s sensitivity to T cells
increased on VV infection. This type of cytolysis by cells from a
resting naive mouse is analogous to NK cell lysis of YAC-1 cells and
suggests that the cytolytic machinery is partially turned on
constitutively and is further enhanced with virus challenge. Studies in
human infections with viruses such as EBV and HIV have focused
predominantly on demonstrating that the primary function of T
cells after a period of in vitro stimulation is to kill and eliminate
virus-infected targets (12, 43, 44, 45). However, freshly
isolated T cells are usually reported to be devoid of cytotoxic
activity (2, 44, 46), with the exception of one report of
freshly isolated T cells from healthy uninfected donors never
previously infected with tuberculosis being able to lyse monocytes
infected with Mycobacterium tuberculosis (47).
Experiments assessing fresh ex vivo cytotoxic activity are difficult
studies to do, as T cells are low in number and appear to lose
their cytolytic activity very quickly after harvest.
The presence of constitutively active cytotoxic T cells capable
of lysing VV-infected targets suggests that VV-specific CTL are present
at a high frequency without any priming with VV, and this would be
another important feature for mediating innate immunity. In fact, we
were able to directly quantify by LDA that there was a high frequency
(1/150) of T cells in the unprimed uninfected host capable of
lysing VV-infected targets. The further activation and expansion of
these cells by VV infection would make them even more useful in the
control of VV. This observation is compatible with the concept that
T cells have been reported to recognize very ubiquitous Ags
shared by different pathogens or host proteins induced by infection or
cell damage. Some of the Ags so far identified include allo-Ags
(48, 49, 50), heat-shock proteins (51, 52), and
microbial Ags such as phosphoantigens (2, 10, 53, 54, 55).
TCR recognition is reported to be more Ig-like than
TCR-like, suggesting that T cells can respond to Ags that would
not be recognized by T cells (2, 10, 56). T
cells can also mediate rapid cellular immune functions, as they don’t
appear to require Ag processing and presentation (2, 10, 56). Although at this time it is unclear what specific Ags are
involved in the activation and cytotoxicity of T cells during VV
infection, the rapidity and broad specificity of the response are
consistent with the above predicted T cell Ag recognition
paradigm and implicate T cells as potentially important
effectors of innate immunity.
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Acknowledgments |
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TCR KO and +/- controls. We also thank James Brien and Brian
Sheridan for their excellent technical assistance and J. Kang and
S. K. Kim for their useful discussions and advice on the
manuscript. |
Footnotes |
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2 Address correspondence to Dr. Raymond M. Welsh, Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: Raymond.Welsh{at}umassmed.edu
3 Abbreviations used in this paper: VV, vaccinia virus; KO, knockout; LCMV, lymphocytic choriomeningitis virus; PEC, peritoneal exudate cell; LDA, limiting dilution assay; pCTL, precursor CTL; PI, postinfection.
Received for publication February 26, 2001. Accepted for publication March 27, 2001.
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