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T Cell Subset Can Increase Host Resistance to a Bacterial Infection1
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Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206 and Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262;
Department of Pathology, University of Vermont, Burlington, VT 05405; and
Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan
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Abstract |
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T lymphocytes have been shown to regulate immune responses
in diverse experimental systems. Because distinct T cell
subsets, as defined by the usage of certain TCR V genes, preferentially
respond in various diseases and disease models, we have hypothesized
that the various T cell subsets carry out different functions.
To test this, we compared one particular T cell subset, the
V1+ subset, which represents a major T cell type
in the lymphoid organs and blood of mice, to other subsets and to
T cells as a whole. Using Listeria monocytogenes
infection as an infectious disease model, we found that bacterial
containment improves in mice depleted of V1+ T
cells, albeit mice lacking all T cells are instead impaired in
their ability to control Listeria expansion. Our
findings indicate that V1+ T cells reduce the
ability of the innate immune system to destroy Listeria,
even though other T cells as a whole promote clearance of this
pathogen. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes is not well
understood, several studies have now shown that they influence the
development of inflammatory lesions in certain diseases. However, in
some experimental disease models in mice, T cells appear to
reduce inflammatory damage, whereas in others, results instead imply
that they exacerbate it. For example, whether T cells were
experimentally ablated by genetic manipulation or depletion with a
specific mAb, increased or accelerated inflammatory damage of host
tissue was seen in the liver of mice infected with Listeria
monocytogenes (1, 2), in the lungs of mice infected
with Mycobacterium tuberculosis (3), and in the
testes of mice with induced autoimmune orchitis (4),
implying that T cells normally limit inflammation. In contrast,
in coxsackievirus B3-induced myocarditis, the T cells appeared
to enhance the inflammation, because transfer of the T cells
infiltrating the heart of a susceptible infected mouse resulted in
heart inflammation in a normally myocarditis-resistant strain following
infection (5). In a mouse candidiasis model, T
cells also appeared to have proinflammatory effects, inducing NO
production by macrophages and contributing to clearance of the pathogen
(6). Conversely, two additional studies attributed both
pro- and anti-inflammatory effects to T cells within a
single disease model, dependent upon the time point at which the
T cells were examined. Specifically, in mice with collagen-induced
arthritis, removal of T cells at early stages of the disease
reduced the incidence and severity of the ensuing joint inflammation,
whereas removal of T cells later caused a rapid onset of severe
arthritis (7). A time-dependent dual effect of T
cells was also reported in a mouse model of spontaneous abortion (in
DBA-2-mated CBA/J mice), in which T cells were found to have a
proinflammatory role early in the pregnancy, infiltrating the uterus
and producing Th1 cytokines that promote spontaneous abortion, but an
anti-inflammatory effect later, switching to Th2-type cytokine
production and protecting against abortion (8).
Consistently, T cells have been shown to produce Th1
(proinflammatory) cytokines during infection with one type of pathogen,
the intracellular bacterium L. monocytogenes, but to produce
Th2 (anti-inflammatory) cytokines in mice with another,
Nippostrongylus brasiliensis (9). Together,
these observations show that one cannot simply regard T cells as
either pro- or anti-inflammatory; instead, they appear to be
capable of playing either role, depending upon unknown influences.
The preferential tissue localization of some T cells expressing
particular V and/or V genes and the finding that certain
V-V pair combinations frequently recur among nonclonal T
cells (reviewed in Ref. 10) have led to a tendency to
regard T cells as subsets based on their expression of certain
V and/or V genes. Although a number of T cell clones have
been identified that respond to certain specific Ags
(11, 12, 13, 14, 15), in many cases, the type of V gene expressed
corresponds to the cell’s ability to respond to certain Ags, even
though other components of the TCR may differ. For example, in the
mouse, members of the V5/V1 subset, which resides primarily in
the skin, typically respond to what appears to be an autoantigen
expressed by keratinocytes (16). In addition, we and
others have found preferential responses of the mouse
V6/V1+ subset at various anatomical
locations during inflammation induced in a variety of ways: by L.
monocytogenes infection (17, 18), autoimmune orchitis
(18, 19), experimental allergic encephalomyelitis
(20), and Escherichia coli infection
(21). Thus, the V6/V1+ subset
probably responds to an inflammation-induced host Ag as well. Such
responses by these two subsets may be predictable because they each
express invariant canonical TCRs. However, polyclonal responses have
also been seen among T cell subsets with more diverse TCRs, such
as the junctionally variable V9/V2+ blood
human T cells, which strongly proliferate in response to
mycobacteria during in vitro culture (reviewed in Ref.
22). In addition, previous studies of our own and of
others involving the mouse V1 subset (23, 24, 25, 26, 27), which
expresses a junctionally diverse V1-J4-C4 chain generally
together with either V6 or V4, revealed an apparent
autoreactivity. Specifically, V1+ T
cells spontaneously release cytokine at low to moderate levels when
simply grown by themselves in culture medium, in the absence of any
deliberately added Ag or accessory cells. Moreover, transfection of a
TCR-negative T cell line with a construct containing genes encoding a
V1-J4-C4-containing TCR gave rise to new cell line that
spontaneously released cytokine, indicating that the V1 TCR confers
this response (28). Recently, we have found that reducing
or eliminating serum in the medium greatly increases this response,
possibly indicating that the hybridomas respond to a stress-induced
self protein (C. T. Cady and W. K. Born, unpublished
observations), which may be related to the mycobacterial
stress-inducible protein heat shock protein-60, that augments the
responses of this subset (12, 23).
The responses of different T cell subsets have been shown to
occur at specific time points during infection (29, 30) or
to vary depending upon the severity of inflammation induced
(31). These findings taken together with the ability of
T cells to respond to Ags as entire subsets led us to propose
that the function of a T cell may be dictated by the TCR it
bears. Studies previously demonstrating that T cells can reduce
or prevent host inflammatory damage (1, 2, 4, 7) moreover
predict that certain T cell subsets might actually hinder the
host response to an infectious agent. In this study we present evidence
showing that whereas clearance of L. monocytogenes in
infected mice is negatively affected if all types of T cells are
depleted, removing only the V1+ subset is, in
contrast, beneficial in this disease model. (Note that the nomenclature
for the murine V genes in this paper follows that set forth by S.
Tonegawa’s laboratory (32).)
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both male and female mice were used in these experiments at
8–12 wk of age. For any given experiment, age- and sex-matched mice
were used, with three to five mice per group except as noted in Fig. 4
and Table I. C57BL/10J, CB.17/SCID, and
C57BL/6-TCR
gene-targeted mice were obtained from The Jackson
Laboratory (Bar Harbor, ME) and either used directly or bred in our
facility. C57BL/6 mice and C3H/HeN mice were obtained from Harlan
Sprague-Dawley (Indianapolis, IN) and bred in our facility. The
TCR-V4/6 gene-targeted mice (33) were back-crossed
three times with C57BL/10 mice, then interbred, and offspring
homozygous for the V4/6 null allele were identified by Southern
blotting. V4/6-/- mice on the C3H/HeN
background were similarly generated after five backcrosses. Mice
homozygous for the V4/6 null allele were then interbred to generate
the V4/6-/- mice used in this study. Mice
lacking functional TCR-C genes and having the C57BL/10 background
were also generated at the same time from
C57BL/6-TCR-/- progenitors (bred in our
facility from Jackson Laboratory stock), as controls, after four
backcrosses onto the C57BL/10 background.
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L. monocytogenes, strain EGD, was freshly grown from
frozen stocks on a shaker in tryptose phosphate broth overnight at
37°C and used the same day. Mice were infected by an i.v. injection
of 
1/10th the 50% lethal dose of L. monocytogenes by
inoculating 0.2 cc of bacteria diluted in sterile HBSS into the tail
vein (for exact doses in individual experiments, see figure legends).
An i.v. rather than an i.p. route of infection was chosen for these
experiments because it gave less variability in the number of bacteria
in individual infected mice than did i.p. infection. Mice were
sacrificed on day 3 of the infection, and the spleen and in some
experiments a section of the main liver lobe were removed from each.
Livers were directly homogenized in sterile water with a motor-driven
pestle, whereas spleens were first dispersed in HBSS on stainless steel
screens and divided into two equal portions, one for determining
bacterial content and the other for flow cytometry (see below). The
portion reserved for determining bacterial levels was then further
homogenized with a motorized pestle. Spleen and liver homogenates were
diluted sequentially in sterile water, and 100 µl of selected
dilutions were plated on trypticase soy agar plates. Following
overnight incubation at 37°C, colonies were counted, and the number
of bacteria per spleen, or for liver per gram of wet tissue, was
calculated. In some experiments a single animal contained
Listeria levels that were not representative of the others
within the same experimental group (2 or more SD above or below the
mean). Such outliers are probably caused by unknown health differences
in the starting population, and these were eliminated from the
analysis. All experiments were conducted at least twice; figures show
results from a representative experiment. In some experiments,
hematoxylin/eosin-stained sections of liver were also examined.
Ab-mediated depletion
Three to 5 days before infection, mice to be depleted of certain
T cells by mAb treatment were injected with 250 µg of the
appropriate purified mAb in a volume of 0.2 cc in sterile HBSS via the
tail vein. Three different Abs were used: anti-V1 (clone 2.11)
(34), anti-V4 (clone UC3) (35), and
anti-C (clone GL3) (36). Note that mAb 2.11 does
not stain hybridomas expressing a TCR containing the closely related
V2 chain (data not shown) and therefore appears to be specific for
V1. The mAbs were produced either by ascites tumor growth in
Pristane-primed C.B-17/SCID mice (anti-V4 and anti-C) or
by in vitro cell culture (anti-V1) and were purified by column
chromatography using either protein A (anti-C) or protein G
(anti-V1 and anti-V4)-Sepharose (Pharmacia, Piscataway,
NJ). All these mAbs were originally generated in the hamster and are
IgG mAbs. Thus, undepleted control mice were similarly sham-treated
with 250 µg of purified normal hamster serum IgG (Ham
IgG;3 Jackson ImmunoResearch Laboratories, West
Grove, PA). To remove potential Ab aggregates in the preparations, just
before injection the Abs were incubated for 10 min at 37°C, then spun
at 14,000 rpm in a microfuge for 10 min at room temperature, and all
but the bottom 50 µl were transferred to a fresh sterile tube.
Depletions of the relevant cells are usually apparent using these mAbs
within 24 h and remain effective for at least 2 wk (data not
shown; longer time periods were not tested).
Flow cytometry
Anti-CD3 mAb KT3 (37) as well as anti-V1,
-V4, and -C mAbs were purified as described above and conjugated
to biotin or, for anti-C, to fluorescein. PE-streptavidin (Tago
Immunologics BioSource, Camarillo, CA) was used as a secondary reagent
to detect binding of the biotin-labeled mAbs. Precalibrated amounts of
each preparation were used to examine splenic T cell populations in
mice previously injected with mAbs to deplete certain subsets, using
two-color flow cytometry. The staining was conducted essentially as
previously described (38); in brief, cell suspensions from
half of each spleen were treated with Gey’s solution to lyse the RBC,
then passed over nylon wool columns to enrich for T cells. For each
staining, between 105 and
106 cells/well in a 96-well plate were first
incubated with unlabeled mAb 2.4G2 (39) to reduce
nonspecific mAb binding via Fc receptor, then washed, incubated with a
biotinylated mAb, washed again, and incubated with an FITC-labeled mAb
together with PE-streptavidin. After final washing, the cells were
analyzed on a Coulter XL (Coulter, Miami, FL) to confirm the efficacy
of the depletion. Spleen cells from at least one mouse in each
experimental group were examined to verify depletion in every
experiment. This was necessary because the depletion requires intact Ab
(40), and the inadvertent use of a partially degraded or
denatured Ab stock could result in false negatives. Fig. 1 shows typical results from mice treated
in vivo with anti-V1, -V4, and -C together with results
from sham-treated controls. As shown, although the depletion is
probably never 100% complete, the few positive cells that escape
depletion generally show quite low levels of TCR and are thus
presumably functionally impaired. The absence of
V4+ cells in the
V4/6-/- mice is also documented in Fig. 1D. A lack of V6+ cells in the same
mice cannot be similarly verified because an anti-V6 mAb is not
yet available.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1+ T cell subset increases
the ability of mice to clear Listeria
C57BL/10 mice depleted of TCR-+ cells
by prior i.v. injection of anti-C mAb showed some impairment in
their ability to clear Listeria on day 3 of the infection as
previously reported (2, 41). Consistently in these
experiments, the degree of impairment was smaller than that in a
previous study (2), possibly due to the choice of an i.v.
rather than an i.p. route of infection. However, in contrast, mice
similarly depleted of V1+ T cells only
showed an improvement in bacterial clearance (see Fig. 2). The improvement ranged from a 3-fold
to 16-fold reduction in various experiments. In contrast, depletion
of V4+ T cells had no apparent
effect.
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1+ subset affects
Listeria clearance differently in various strains
Fig. 3 shows the results of similar
experiments conducted with several different mouse strains. Both
C57BL/10 and C57BL/6 mice were examined because, despite their many
similarities, these two strains carry different TCR- gene alleles,
which render slight differences in the V1 chains they express
(25). As shown, however, depletion of
V1+ T cells was beneficial for
clearance of Listeria in both of these strains. (Note that
in Fig. 3b, the difference between the Ham IgG sham-treated
group and the anti-V1-treated group is significant,
p 
0.10.) Both the C57BL/10 and C57BL/6 strains are
relatively resistant to infection by Listeria; we therefore
examined in addition two Listeria-sensitive strains to test
whether V1+ T cells play a similar
role. As shown in Fig. 3, for C3H/HeN mice the beneficial effect of
V1+ cell removal was still evident, although
for BALB/c mice we could not demonstrate it. The reason for
Listeria sensitivity in the C3H/HeN strain is not known, but
in BALB/c mice it may be due to the inherent bias of these mice to
overproduce IL-4 and generate Th2-type immune responses
(42). Because C3H/HeN and BALB/c are different in this
regard, it is perhaps not surprising that they also respond
differentially to the removal of V1+
cells.
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1+ subset similarly impairs
bacterial containment in mice unable to generate an adaptive immune
response to Listeria (TCR-/- mice)
Mice infected with Listeria mount an 
T
cell-mediated adaptive immune response that is only first weakly
detectable on the third day of infection (43). The
detrimental effect of removing V1+ cells
during a Listeria infection at an early time point thus
suggested that they primarily influence components of the innate immune
response. Thus, we tested whether mice incapable of producing
Listeria-immune T cells
(TCR-/- mice) also showed an improvement in
bacterial clearance in the absence of this subset. As shown in Fig. 4
, a decrease in susceptibility to
Listeria was likewise evident in mice lacking T
cells.
Although depletion of the V4 subset does not affect
Listeria containment, it reverses the effect of V1
cell depletion
We had observed that removing V4+
T cells had, in contrast to removing V1+
cells, no effect on listerial clearance (see Figs. 2 and 3a). We next depleted C57BL/6 mice simultaneously of both
V1 and V4+ subsets (thus eliminating or
inactivating 85% of the T cells present in blood and spleen)
and tested the effect of this on Listeria clearance. As
shown in Fig. 5, the benefit of removing
V1+ cells largely disappeared when
V4+ cells were also removed. Thus, the
V4+ subset appears to play a role as well, one
that seems to have an outcome opposite that of the
V1+ subset. However, the effect of eliminating
both these subsets is to return bacterial clearance to approximately
the wild-type level. We had expected that removal of both subsets would
instead exacerbate the infection, because removing all T cells
has this effect, and very few T cells remain after depleting
both these subsets. This finding implies that removing another, less
abundant T cell subset must also occur to produce a detrimental
effect. We have previously noted a preferential increase in the
percentage of V6+ T cells in the liver
of mice with experimental listeriosis (17) and therefore
suspected that the V6+ T cell subset
might be the one in question.
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4+ and
V6+ T cells show a decrease in ability to contain
Listeria during infection
Because an anti-V6 mAb is not yet available, we could not
simply deplete mice of V6+ cells to examine
the role of this subset. However, mice incapable of producing either
V4 or V6+ T cells were previously
generated by gene targeting (33). We therefore backcrossed
these mice onto the C57BL/10 and C3H/HeN backgrounds to generate lines
with nearly homogenous backgrounds and tested them for their ability to
clear Listeria. C57BL/10 background,
TCR-/- mice, which contain no T
cells, were similarly infected at the same time as controls. As shown
in Fig. 6, for mice with the C3H/HeN
background, removing both the V4 and V6+
subsets had a small, but clearly detrimental, effect on
Listeria containment in spleen and liver (the
V4/6-/- mice, on the average, contained 3
times more bacteria in spleen and 7 times more in liver than did
wild-type mice; Fig. 6a). However, on the C57BL/10
background, V4/6-/- mice were
indistinguishable from wild-type mice in the numbers of bacteria
remaining in spleen or liver.
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T cell subsets in
spleens of infected V4/6-/- vs wild-type
mice (see Table I) and found that cells from mice of the C3H/HeN
background were quite differently affected than those of the C57BL/10
background. Specifically, the percentage of
V1+ cells increased in C3H/HeN, but decreased
in C57BL/10, a pattern that was also seen in both strains in the
absence of the V4 and V6 subsets. The
V4+ subset showed exactly the opposite
pattern, decreasing in percentage in C3H/HeN, but increasing in
C57BL/10. Cells expressing V genes other than 1, 4, and 6 (V
other in Table I) in the V4/6-/- mice
behaved similarly to the V4+ subset in
wild-type mice, decreasing in relative abundance on the C3H/HeN
background, but increasing on the C57BL/10 background. These changes in
percentage of V1 and V4 subsets in the wild-type mice were
similar to our results in a previous study comparing the same two
strains (44), and the increase in the percentage of
V1+ cells in C3H/HeN might by itself explain
why this strain is adversely affected by the lack of
V4+ and V6+ cells,
whereas C57BL/10 is seemingly not.
However, when we looked at the effect of infection on the actual
numbers of V1 and V4 subsets, an additional difference between
the strains was revealed; whereas the V1+ T
cells in C3H/HeN mice increased in number, those in C57BL/10 mice
decreased markedly (10-fold; see Table II). Induction of apoptosis in the
lymphocytes of C57BL/6 and BALB/c mice has been previously noted using
high Listeria doses early in infection (45),
and in our hands, splenic T cell yields, on the average, decrease to
about 25% of the normal level in C57BL/6 and C57BL/10 mice, whereas
they increase slightly in C3H/HeN under the experimental conditions
used in this study (data not shown). Whether C3H/HeN mice given
comparable doses of Listeria would also show T cell
apoptosis is not known and could not be examined here because this
Listeria-sensitive strain would die before day 3 of
infection if given doses at the levels used here for C57BL/10 mice. In
both strains, however, we found that the V4+
cells behaved differently from the V1+ cells,
showing no increase in numbers in C3H/HeN, and in C57BL/10 mice a
lesser tendency to disappear than V1+ cells
(about equal to that of T cells in general). Surprisingly, the
abundance of the V1+ subset after infection
depended in both strains on whether they were
V4/6-/-, because C3H/HeN
V4/6-/- mice showed virtually no increase in
V1+ cells, whereas C57BL/10
V4/6-/- mice showed an even greater loss of
the V1+ subset than did the wild-type
controls. In contrast, cells expressing V genes other than 1, 4, and
6 in V4/6-/- mice increased in C3H/HeN
background mice and decreased in V4/6-/-
mice of the C57BL/10 background. Thus, deletion of both the
V4+ and V6+ subsets
appears to affect the two strains differently not only in terms of
bacterial clearance, but also in the responses evoked in the other
T cell subsets. Whether T cell subset response
differences can explain the bacterial clearance difference remains to
be determined; certainly, other factors dictated by genetic disparities
between the strains may be involved as well.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1+ T cells,
even though depleting all T cells has a detrimental effect.
Although such findings are not unprecedented (46), this is
surprising given that removing a particular lymphocyte subset from a
mouse might be regarded as impairing its immune system. A trivial
explanation for this finding would be that the anti-V1 mAb used
to remove the V1+ cells cross-reacts with the
Listeria bacteria and causes their elimination. This is
unlikely not only because Listeria largely propagate
intracellularly and thus escape humoral defense mechanisms, but also
because the effect was absent in BALB/c mice (Fig. 3), and joint
depletion of the V4+ subset at the same time
reversed the beneficial effect of depleting
V1+ cells (Fig. 5). At this time we do not
understand how the V1 cell-dependent effect is mediated. It may be
due to an alteration in Th1 vs Th2 cytokine profiles in the course of
the infection, and indeed others have shown that at least some
V1+ T cells preferentially produce IL-4
(47) and might in this way alter the type of T cell
response that is elicited. Because mice that cannot produce T
cells are similarly affected, it seems more likely that the
V1+ cells themselves directly influence the
ability of macrophages and/or neutrophils to kill Listeria.
T cells that are stimulated to secrete IFN- have been shown
to enhance the ability of macrophages to kill Candida
albicans (6), so it may be that IL-4-producing
V1+ cells stimulated during a
Listeria infection, in contrast, down-regulate macrophage
function. Alternatively, or as well, the V1+
cells might indirectly influence macrophage activity by causing other T
cells to produce IL-4 through secreting other cytokines. This could be
brought about by the chemokine monocyte chemoattractant protein-1,
which has been shown to promote IL-4 production in T cells cultured in
vitro (48), because T cells were previously found
to be necessary for the production of monocyte chemoattractant
protein-1 during Listeria infection (49). In a
recent study by Egan and Carding, activated macrophages during
Listeria infection were found to stimulate mouse T
cells, particularly those expressing V1, to acquire cytolytic
activity and then to lyse the activated macrophages (50),
which suggests yet a third scenario by which
V1+ cells might decrease macrophage
activity.
In any case, if V1+ cells indeed down-regulate
macrophage function, one might predict that in other situations, their
depletion would instead be counterproductive and could lead to
inflammatory damage. In fact, we have already found that in
coxsackievirus B3 infection, depletion of V1+
T cells does exactly this, leading to an exacerbation of the
myocarditis evoked by infection with the virus (57). In contrast,
removal of the V4 subset reduces inflammatory damage in
coxsackievirus B3-induced myocarditis. Our observation that concomitant
V4+ cell depletion reverses the beneficial
effect of depleting V1+ cells in listeriosis
also supports the idea that the two subsets carry out opposing roles.
Thus, if V1+ cells decrease macrophage
activity, the V4+ cells, in contrast, might
increase it, and if these two subsets normally balance one another out,
deleting both would be expected to restore the status quo, as we indeed
observed. Although we did not see a decrease in bacterial killing in
V4 cell-depleted mice, as might be expected if they indeed
up-regulate macrophage activity, the enhancing effect of
V4+ cells might be substantially less
important in bacterial killing than is the down-regulatory effect of
the V1+ cells, at least in the
Listeria system. Further study will be needed to
determine this.
Whether the function of the V6 subset differs from that of the V1
and V4 subsets is still uncertain. Although we found that C3H/HeN
V4/6-/- mice showed an impairment in
bacterial clearance compared with wild-type controls (Fig. 6a), this was not true of V4/6-/-
mice on the C57BL/10 background. We did note differences between these
mice within the remaining T cells during infection (Table I); in
particular, although V1+ cells in C3H/HeN
V4/6-/- mice were present in comparable
numbers in both infected and infected mice, in C57BL/10
V4/6-/- mice the infection resulted in a
20-fold reduction in V1+ cells. Further study
will be necessary to determine whether this alone can explain the
difference in relative resistance of V4/6-/-
mice of the C3H/HeN vs the C57BL/10 background.
In mice lacking all T cells, not only bacterial containment but
also an increase in the frequency of necrotic liver lesions have been
previously reported by both ourselves (2) and others
(1). In the present study alterations in histopathology
were rarely seen in animals depleted of all types of T cells via
in vivo mAb injection, although these were clear when using
TCR-/- mice. Perhaps this is due to our
inability to completely eliminate each cell type with mAb-mediated
depletion or was less obvious due to our choice of an i.v. rather than
an i.p. route of infection as used in the previous studies. In support
of the former argument, we, in fact, in general find that the effect of
removing all T cells by disruption of the C gene has a
stronger effect than does depleting T cells with an mAb (for
example, see Fig. 6b, in which
TCR-/- mice show 27 times more bacteria
in liver and 4 times more in spleen compared with wild-type mice),
perhaps because genetic inactivation is 100% effective. We also failed
to note any histological differences in the livers of mice depleted of
V1+ or V4+ T
cells only or among V4/6-/- mice on either
the C57BL/10 or C3H/HeN background. Thus, it is possible that this
phenomenon depends on the absence of several different T cell
subsets.
We have focused in this study solely on V gene expression, ignoring
the impact of the coexpressed V. This decision, based on our
observations of common response patterns among T cells
expressing V1 without apparent limitation from V, could
nonetheless be biasing our findings. In particular, Pereira et al. have
previously reported a largely V1+ subset that
in the DBA/2 strain usually coexpresses a V6.3 allele, is
Thy-1low in the thymus, displays restrictions in
TCR diversity, and has a tendency to produce IL-4 (45, 47, 51). Therefore, in a preliminary experiment we attempted to
deplete only this portion of the V1 subset using a V6.3 mAb
(52) (which should be possible, because virtually all
V6.3 cells in C57BL/10 mice coexpress a V1 chain
(53)); this had a slightly detrimental effect on bacterial
clearance, a result opposite to what we expected (data not shown).
However, at this time it is unclear whether the properties of the
V6.3+ cells described by Pereira et al. are
peculiar to DBA/2 mice and may be absent or less pronounced in the
C57BL/10 strain.
The technique of cell depletion via mAb injection is often considered
to be less stringent than genetic inactivation, particularly when using
anti-TCR mAbs, because the treatment also usually transiently
activates the cells of interest. Cytokines elicited by anti-TCR mAb
treatment generally fall to normal levels in the serum within 48 h
after injection, however (54), so to circumvent this
problem, we injected mice with mAb 3–5 days before infecting them with
Listeria. As well, all the mAbs used, anti-V1, -4,
and TCR-, are hamster-derived IgG Abs, and thus any Fc
receptor-driven side effects they have should be similar. Thus, even if
transient activation rather than depletion produced the effects seen in
the study described here, our observations indicate that stimulation of
different T cell subsets results in the production of different
cytokines. We believe that the effect is instead due to cell depletion,
because in a related study using coxsackievirus B3 infection in mice
(57), we found that reconstituting TCR-/-
mice with T cell populations first depleted in vitro of certain
subsets produced effects in accordance with those obtained using direct
in vivo depletion. Because cytokine production would not be triggered
using the reconstitution protocol, the simplest explanation is that the
depletion is responsible for the observed effect in both cases. We are
now in the process of generating V1 and V6 gene-inactivated mice
to both verify and expand our analyses.
In a recent report from Nakamura et al. (55) in which
T cell function in Listeria-infected C3H/HeN mice was
examined, the depletion of V1+ cells was found
to be detrimental to bacterial clearance, a result in exact opposition
to that presented here. Because the mouse strain was the same as one of
the strains used here, differences in protocols used in the two studies
(i.e., Nakamura et al. used a much lower infectious dose and a
different route of infection and examined bacterial levels at a later
time point) could have implications for the actual timing and/or
stimulation of T cell regulatory responses.
The findings presented here support the hypothesis that the type of TCR
borne by a T cell predisposes it toward a particular function.
Because many of the TCRs that arise on T cells are
developmentally predetermined at least to some degree, the idea that
their function might be "hardwired" along with the TCR is actually
consistent. The ligands recognized by TCRs remain in most cases
a matter of speculation, but several lines of evidence implicate host
molecules elicited by infection or inflammation. In this way a limited
set of inducible host molecules might serve as switches that alter
immune mechanisms by stimulating the responses of particular T
cell subsets. Our findings also suggest that T cell subsets that
control inflammatory tissue damage come at a cost: decreased host
resistance to a bacterial infection. In this context we are intrigued
by observations (56) (A. Mukasa et al., manuscript in
preparation) suggesting that certain T cells subsets, during the
course of infection with agents such as Mycobacterium
tuberculosis and L. monocytogenes, are naturally
depleted via a Fas ligand-dependent apoptotic pathway, a normal
mechanism that could lead to increased host resistance.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rebecca L. O’Brien, National Jewish Medical and Research Center, Department of Immunology, 1400 Jackson Street, Denver, CO 80206.
3 Abbreviations used in this paper: Ham IgG, normal hamster serum IgG; V4/6-/- mice, gene-targeted mice homozygous for induced mutations inactivating both the V4 and V6 genes; TCR-/- mice, gene-targeted mice homozygous for an inactivating mutation in TCR-C; TCR-/- mice, gene-targeted mice homozygous for a deletion in the TCR-C locus.
Received for publication June 8, 2000. Accepted for publication August 31, 2000.
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