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Relative Contributions of NK and CD8 T
Cells to IFN- γ Mediated Innate Immune
Protection against Listeria monocytogenes
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of June 15, 2017.
Rance E. Berg, Emily Crossley, Sean Murray and James
Forman
J Immunol 2005; 175:1751-1757; ;
doi: 10.4049/jimmunol.175.3.1751
http://www.jimmunol.org/content/175/3/1751
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References
The Journal of Immunology
Relative Contributions of NK and CD8 T Cells to IFN-␥
Mediated Innate Immune Protection against Listeria
monocytogenes1
Rance E. Berg, Emily Crossley, Sean Murray, and James Forman2
T
he innate immune response to the Gram-positive intracellular bacterium, Listeria monocytogenes (LM)3, is a complex network involving cytokines, bactericidal effector
mechanisms, and multiple cell types such as neutrophils, macrophages, and NK cells (1, 2). TLR recognition of bacterial products
results in secretion of cytokines that recruit other cells, as well as
directly activate innate effectors. Specifically, TLR2 and TLR5
recognize LM through interactions with bacterial products such as
lipoteichoic acid, peptidoglycans, and flagellin (3, 4). Downstream
signaling through TLRs is mediated through the adapter molecule,
myeloid differentiation factor 88 (MyD88), and mice deficient in
this molecule are highly susceptible to LM infection (5, 6). However, MyD88-independent innate and adaptive immune responses
do exist (7, 8).
IL-12 and IL-18, produced mainly by activated macrophages,
are both important mediators in the immune response against LM,
with the primary function of inducing IFN-␥ secretion from responding immune cells (9 –12). However, an IFN-␥-independent
role in controlling LM has also been proposed for IL-18 (13).
IL-12-deficient mice show an early impaired resistance to LM in-
Center for Immunology, University of Texas Southwestern Medical Center, Dallas,
TX 75390
Received for publication November 13, 2004. Accepted for publication May
16, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI45764 (to J.F.)
and a National Institutes of Health postdoctoral fellowship (to R.E.B.).
2
Address correspondence and reprint requests to Dr. James Forman, Center for
Immunology, University of Texas Southwestern Medical Center, 6000 Harry
Hines Boulevard, Dallas, TX 75390-9093. E-mail address: James.Forman@
UTSouthwestern.edu
3
Abbreviations used in this paper: LM, Listeria monocytogenes; MyD88, myeloid
differentiation factor 88; VV/OVA, vaccinia virus expressing full-length OVA protein; VSV/OVA, vesicular stomatitis virus expressing full-length OVA protein;
PALS, periarteriolar lymphoid sheath.
Copyright © 2005 by The American Association of Immunologists, Inc.
fection but are able to eliminate low doses of the bacteria, suggesting that IL-12 is less important in generating adaptive immunity (14, 15). In addition, other recently described IFN-␥-inducing
cytokines such as IL-21, IL-23, and IL-27 could play overlapping
and/or redundant roles in the innate immune response against LM
(16 –18).
IFN-␥ secretion plays an important role during both innate and
acquired immunity to intracellular bacteria by enhancing Th1-type
immune responses through the activation of macrophages, the increase of MHC class I and class II expression, and the inhibition
of proliferation of Th2 cells (19 –21). IFN-␥ is produced by multiple cell types in response to LM or a combination of IL-12 and
IL-18, including NK cells (22), NKT cells (23), macrophages (24),
B cells (25), dendritic cells (26), ␥␦ T cells (27), CD8 T cells (28),
and primed CD4 T cells of the Th1 phenotype (29, 30). Evidence
that IFN-␥ is critically important in the innate immune response
comes from experiments using mice deficient in either the cytokine
or its receptor. These animals rapidly succumb when infected with
extremely low doses of LM (31, 32). SCID mice, which lack T and
B lymphocytes, show increased susceptibility to LM when they are
depleted of either IFN-␥ or IL-12 (9, 19). Providing IFN-␥ to IL12-depleted SCID mice can reverse this effect, thereby indicating
the importance of IL-12 for IFN-␥ production in response to LM
(9). Impaired macrophage activity is one probable mechanism for
the increased susceptibility of animals lacking the IFN-␥R (21).
It is generally thought that NK cells play a crucial role in the
innate control of LM infection due to secretion of IFN-␥ induced
by IL-12 and IL-18 (22). Indeed, depletion of NK cells before s.c.
infection with LM led to higher burdens of bacteria in both the
hind footpad and the draining lymph node (33). In contrast, other
studies have found that depleting NK cells led to decreased splenic
and liver LM burdens in B6 mice after either i.v. (34) or i.p. (35)
infection. Studies using mice deficient in the common cytokine
receptor ␥-chain (lacking NK cells) indicated that IFN-␥ was produced early after LM infection and that the most probable source
of this cytokine was ␣␤ T cells (36, 37). We and others (28, 38, 39)
0022-1767/05/$02.00
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During the innate immune response to Listeria monocytogenes (LM), the secretion of IFN-␥ is crucial in controlling bacterial
numbers. We have shown recently that CD8 T cells have the ability to rapidly secrete IFN-␥ independent of Ag, in response to
IL-12 and IL-18, during a LM infection. In the current study, we compared the relative abilities of NK and CD8 T cells to provide
innate immune protection. Upon transfer of either NK or memory OT-I T cells (specific for the OVA protein) into IFN-␥-deficient
hosts that were infected subsequently with wild-type LM, both cell types were found in the spleen and had the ability to secrete
IFN-␥. However, the OT-I T cells were more effective at providing innate immune protection as determined by spleen and liver
LM burdens. We used immunocytochemistry to demonstrate that upon infection with LM, marginal zone macrophages were
localized to the T cell area of the splenic follicle. Transferred memory OT-I T cells were also found in the T cell area of the spleen,
colocalizing with the LM and macrophages. In sharp contrast, NK cells were found predominantly in the red pulp region of the
spleen. In addition, memory OT-I T cells were also found to be associated with LM lesions in the liver. These results highlight the
importance of CD8 T cells in innate immune responses to LM and suggest that their increased protective ability compared with
NK cells is the result of their colocalization with LM and macrophages. The Journal of Immunology, 2005, 175: 1751–1757.
1752
have shown that CD8 T cells respond to cytokines, primarily IL-12
and IL-18, by secreting IFN-␥ rapidly after infection with intracellular bacteria, including LM. These reports suggest that CD8 T
cells are capable of contributing to the innate immune response to
LM, especially with respect to IFN-␥ production. In addition, we
have shown that memory CD8 T cells can provide innate immune
protection from a LM infection independent of cognate Ag (39).
The present study was performed to determine the relative abilities
of both NK and CD8 T cells to provide innate immune protection
against a LM infection.
Materials and Methods
CD8 T CELL MEDIATED INNATE IMMUNITY TO LM
lowed by Streptavidin Alexafluor 594. The liver sections were stained with
the following Ab combinations: purified anti-CD90.2 (Thy1.2) (53-2.1)
and Difco Listeria O polyserum. The Thy1.2 was developed with anti-rat
Alexafluor 488 (Molecular Probes), and the Listeria O polyserum was developed as above. All experiments were also performed with isotype control Abs to assure there was no background staining. Stained spleen and
liver sections were then visualized on a Zeiss Axiovert 100M digital light
microscope. Pictures were taken with a Hamamatsu Orca digital gray scale
camera. Image J Software, from the National Institutes of Health, was used
to analyze the data and give false colors to the images shown. The distribution of Thy1.2⫹ T cells in the liver sections in relation to LM lesions was
determined by dividing microscopic fields with ⬃100 grids, each ⬃37 ⫻
37 ␮m. T cells were scored as being in the same grid as LM, a grid adjacent
to LM, or a grid not associated with LM.
Mice
Statistical analyses
C57BL/6J (B6), C57BL/6.PL-Thy1a/Cy (B6.Thy1.1), B6.129S7-Ifngtm1Ts
(IFN-␥⫺/⫺), B6.129S7-Ifngr1tm1Agt/J (IFN-␥R⫺/⫺), B6.129S7-Rag1tm1Mom/J
(RAG-1⫺/⫺), and OT-I TCR transgenic mice were either purchased from The
Jackson Laboratory or bred and maintained at the University of Texas Southwestern Medical Center animal facility under the approval of the Institutional
Animal Care and Use Committee.
Statistical significance for Figs. 1 and 2 was measured using a Student’s
two-tailed t test. Statistical significance for Table I was measured using
Fisher’s exact test. Statistical significance for Fig. 5d was measured using
a goodness-of-fit ␹2 analysis.
For infection of mice, log-phase cultures of LM 10403 serotype 1 were
washed twice and diluted in PBS to the desired concentration. LM was
injected in the lateral tail vein at the indicated dosage. Vaccinia virus expressing full-length OVA protein (VV/OVA) was injected in the lateral tail
vein at a dosage of 106 PFU for a primary response. Vesicular stomatitis
virus expressing full-length OVA protein (VSV/OVA) was injected in the
lateral tail vein at a dosage of 106 PFU for a primary response.
Abs and cell staining for flow cytometry
For cell staining experiments, the following Abs from BD Pharmingen
were used: anti-CD8␣ (53-6.7), anti-CD90.2 (Thy1.2) (53-2.1), anti-NK1.1
(PK136), and anti-IFN-␥ (XMG1.2). Secondary streptavidin-conjugated
reagents were used to reveal biotinylated primary Abs. In experiments
designed to test the direct ex vivo activity of NK and T cells, splenocytes
were cultured for 3 h in complete RPMI 1640 medium supplemented with
10% FCS (without added cytokines or Ags). Intracellular staining, data
acquisition, and data analysis were performed as described previously (28).
NK and T cell transfers
For generation of the memory OT-I T cell populations, RBC-depleted
splenocytes from OT-I TCR transgenic mice were passed over nylon wool
columns to enrich for T cells. Approximately 2 ⫻ 106 cells were then
injected i.v. into the lateral tail vein of B6.Thy1.1-recipient mice, which
were challenged subsequently with VV/OVA or VSV/OVA. At ⬎4 wk
postinfection, the mice were sacrificed, and RBC-depleted splenocytes
were purified on nylon wool columns. The resulting cells were stained for
CD8 and Thy1.2 and sorted using a MoFlo high-speed sorter (DakoCytomation) for expression of these molecules. For the NK transfer experiments, RBC-depleted splenocytes from RAG-1⫺/⫺ mice were stained for
NK1.1 and sorted for expression of this molecule. For each of the sorted
populations, the purity of the cells was ⬎95% as determined by flow cytometry postsorting (data not shown). CFSE (Molecular Probes) labeling of
splenocytes was performed at a final concentration of 1 ␮M. After CFSE
labeling, the indicated numbers of sorted cells were injected i.v. into IFN␥⫺/⫺ or IFN-␥R⫺/⫺ hosts, which were immediately infected with
⬃10,000 –20,000 wild-type LM.
Immunocytochemistry and microscopy
Immunocytochemistry of spleens was performed by making 5-␮m sections
of frozen spleens from B6, IFN-␥⫺/⫺, and IFN-␥⫺/⫺ mice transferred with
CFSE-labeled NK or memory OT-I T cells using a Leica CM 1850 cryostat. Five-micrometer liver sections were made from OT-I-transferred,
VSV/OVA-primed mice that were ⬎4 wk post primary infection. Spleen
and liver sections were then acetone fixed before staining. The Ab combinations used to stain splenic sections were as follows: purified anti-CD3⑀
(145-2C11), purified CD45R/B220 (RA3-6B2), purified CD11b (M1/70)
(all from BD Pharmingen), and Difco Listeria O polyserum (Fisher Scientific). The CD3⑀ was developed with anti-hamster biotin (Jackson ImmunoResearch Laboratories) followed by Streptavidin Alexafluor 594
(Molecular Probes). The CD45R/B220 and CD11b Abs were developed
with anti-rat Alexafluor 350 (Molecular Probes). The Difco Listeria O
polyserum was developed with anti-rabbit biotin (BD Pharmingen) fol-
Results
Memory OT-I T cells and NK cells secrete IFN-␥ in response to
wild-type LM when transferred into IFN-␥-deficient hosts
Our previous results have shown that memory CD8 T cells specific
for OVA (OT-I T cells) have the ability to provide innate immune
protection from a wild-type LM infection when transferred into
IFN-␥⫺/⫺ hosts (39). This protection is mediated by the secretion
of IFN-␥ in response to IL-12 and IL-18, which is produced during
the LM infection. Other studies have suggested a protective role
for NK cells during the innate immune response to LM infection,
once again citing their ability to rapidly secrete IFN-␥ (22, 33).
Therefore, our first set of experiments was designed to ascertain
the IFN-␥-secreting potential of both NK and memory CD8 T cells
in response to wild-type LM in an IFN-␥⫺/⫺ setting. The NK cells
were isolated from spleens of RAG-1⫺/⫺ mice by sorting on the
NK1.1 molecule. The majority (⬃90%) of the NK1.1⫹ NK cells
also expressed DX5 before sorting (data not shown). Therefore,
this population of NK1.1⫹ cells isolated from mice deficient in
NKT or T cells represents a pure population of NK cells. The
memory OT-I T cells were isolated from OT-I transferred, VV/
OVA-primed B6.Thy1.1 mice by sorting on Thy1.2 and CD8 molecules. Our previous data established that these cells represent
memory CD8 T cells (39). Memory OT-I T or NK cells, which
were transferred into IFN-␥⫺/⫺ mice that were infected subsequently with wild-type LM, could be identified in the spleen (Fig.
1). These transferred cells were capable of secreting IFN-␥ at day
3 postinfection. In addition, our previously published data showed
that both NK and CD8 T cells were capable of secreting IFN-␥ at
day 1 postinfection with LM (28, 39).
Memory OT-I T cells provide more efficient innate immune
protection against LM than NK cells
The results presented in Fig. 1 suggest that both NK and memory
OT-I T cells should have the ability to provide protection against
a LM infection due to their abilities to secrete IFN-␥. However,
when we determined spleen and liver LM counts in transferred
IFN-␥⫺/⫺ recipients, we found that the memory OT-I T cells provided more efficient protection when compared with the NK cells
(Fig. 2). In fact, when we transferred four times more NK cells
than T cells, which resulted in a greater number of IFN-␥-secreting
NK cells compared with memory OT-I T cells, the T cells still
provided more innate immune protection against the LM infection.
We chose day 3 to analyze because our previously published data
indicated that before this time point there was no difference in LM
counts in B6 and IFN-␥⫺/⫺ mice (39). However, our previous data
(28, 39), as well as unpublished results, indicate that both NK and
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Bacteria and viruses
The Journal of Immunology
CD8 T cells are actively secreting IFN-␥ as early as day 1 postinfection with LM. To show that the IFN-␥ that is produced by the
transferred OT-I T cells is indeed the effector molecule responsible
for the protection from the LM infection, we used IFN-␥R⫺/⫺
mice as recipients for the transfer of memory OT-I T cells. Three
days postinfection with ⬃15,000 wild-type LM, the LM burdens
were measured in the spleens and livers (three mice per group).
The IFN-␥R⫺/⫺ mice that did not receive memory OT-I T cells
had an average of 1.87 ⫻ 109 LM/spleen and 9.96 ⫻ 108 LM/liver.
The memory OT-I-transferred IFN-␥R⫺/⫺ mice contained an average of 3.99 ⫻ 109 LM/spleen and 1.31 ⫻ 109 LM/liver. Although there was no protection offered by the memory OT-I T
cells, these transferred cells were visible (CFSE⫹) and were actively secreting IFN-␥ in the spleen (data not shown).
Splenic architecture and macrophage migration in LM-infected
IFN-␥⫺/⫺ and B6 mice are similar
To begin to dissect why memory OT-I T cells provide more efficient innate immune protection from a LM infection in IFN-␥⫺/⫺
mice, we sought to analyze the splenic architecture of these mice.
To this end, 5-␮m frozen sections from B6 (Fig. 3a) and IFN-␥⫺/⫺
(Fig. 3b) spleens were stained with Abs to visualize T cells, B
cells, and macrophages. The microscopy data reveals that in spleen
sections from both mice there are well-defined white pulp zones
that are surrounded by macrophages in the marginal zones. In the
next set of experiments, we infected B6 (Fig. 3c) and IFN-␥⫺/⫺
(Fig. 3d) mice with wild-type LM and then analyzed the location
of T cells, B cells, macrophages, and LM at day 1 postinfection.
After infection with LM, most macrophages are detected in the T
cell area of the periarteriolar lymphoid sheath (PALS) region of
the spleen, rather than the marginal zone in both strains of mice.
Previous studies have shown that LM are trapped by macrophages
in the marginal zone of the spleen and that these LM-infected
macrophages then migrate into the white pulp area within 12–24 h
postinfection (40, 41).
FIGURE 2. Protection from LM infection by memory OT-I T cells is more
efficient than NK cells. The same mice analyzed in Fig. 1 for the presence of
transferred IFN-␥-secreting cells were also analyzed for the presence of LM in
both spleen and liver at day 3 postinfection with ⬃20,000 wild-type LM. In
addition, B6 and IFN-␥⫺/⫺ mice that were infected at the same time were also
analyzed for LM burdens in their spleens and livers. Homogenates from the
organs of the infected mice were diluted and then plated on brain-heart infusion medium plates. One day later, the number of bacterial colonies was determined by counting plates and multiplying by the dilution factor. Log CFU
protection was calculated by subtracting the average log bacterial burden in the
spleens and livers of transferred IFN-␥⫺/⫺ mice from the average log bacterial
burden in the same organs of untransferred IFN-␥⫺/⫺ mice. Data presented are
derived from two independent experiments with two to three mice in each
group per experiment. ⴱ, p ⬍ 0.05.
Splenic localization of transferred NK and memory OT-I T cells
in LM-infected IFN-␥⫺/⫺ mice
To establish a mechanism to explain why transferred NK and
memory OT-I T cells provide differential protective ability, experiments were performed to visualize the localization of these cells
in LM-infected IFN-␥⫺/⫺ mice. A representative splenic section
shows that the transferred memory OT-I T cells reside in a T cell
area of the PALS at day 1 after LM infection (Fig. 4a) and that the
LM and macrophages are also found in this area (Fig. 4b). Previous results from T cell transfer experiments have shown that bulk
CD8 splenocytes (42), as well as memory CD8 T cells (43), preferentially localize in white pulp regions of uninfected spleens. In
contrast to the OT-I T cells, transferred NK cells did not localize
into the T cell areas of LM-infected IFN-␥⫺/⫺ mice (Fig. 4c). The
NK cells are found mainly in the red pulp, which is not where the
LM and macrophages are found in a consecutive spleen section
(Fig. 4d). In support of this result, previous studies analyzing the
location of endogenous NK cells or transferred NK cells have
shown that they are predominantly found in the red pulp of uninfected spleens (44 – 46). Our data suggest that even during a LM
infection, NK cells are not induced to migrate toward the infected
foci in the spleen. Therefore, in combination, the previous results
suggest that memory CD8 T cells preferentially home to T cell
areas of the spleen, which is also the area where LM and macrophages migrate to during infection (Fig. 4, a and b). NK cells, in
contrast, do not localize to the T cell areas containing LM and
macrophages (Fig. 4, c and d). Table I summarizes data from experiments analyzing the location of transferred NK and memory
OT-I T cells. At day 1 after LM infection, the location of the
transferred cells was determined easily due to the intact splenic
architecture at this time. The data once again demonstrates that at
day 1 the transferred memory OT-I T cells are found in the T cell
areas of the spleen, whereas the NK cells are found predominantly
in the red pulp distant from the foci of LM. However, at day 3
postinfection, which is the time at which differences in spleen and
liver LM counts are seen between B6 and IFN-␥⫺/⫺ mice, the LM
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FIGURE 1. Innate IFN-␥ production by NK and memory OT-I T cells.
Memory OT-I T cells were sorted from spleens of OT-I transferred, VV/
OVA-primed B6.Thy1.1 mice based on expression of CD8 and Thy1.2.
NK cells were sorted from spleens of RAG-1⫺/⫺ mice based on expression
of NK1.1. The indicated numbers of purified cells were then labeled with
CFSE and transferred into IFN-␥⫺/⫺ recipients, which were infected with
⬃20,000 wild-type LM. At day 3 postinfection, the mice were sacrificed,
and splenocytes were cultured in vitro for 3 h in the presence of GolgiPlug
containing brefeldin A to inhibit protein transport. The cultures were harvested and stained for CD8, NK1.1, and intracellular IFN-␥. In addition,
the transferred cells were identified by their expression of CFSE. The number of cells was determined by multiplying the percentage of transferred
cells or transferred cells secreting IFN-␥ by the total number of splenocytes. Data presented are derived from two independent experiments with
two to three mice in each group per experiment. ⴱ, p ⬍ 0.05.
1753
1754
CD8 T CELL MEDIATED INNATE IMMUNITY TO LM
FIGURE 3. Immunocytochemistry of uninfected and
LM-infected B6 and IFN-␥⫺/⫺ mice. B6 (a), IFN-␥⫺/⫺
(b), day 1 LM-infected B6 (c), and day 1 LM-infected
IFN-␥⫺/⫺ (d) mice were sacrificed, and 5-␮m splenic
sections were stained for T cells, B cells, macrophages,
and LM. The mice in c and d were infected with
⬃20,000 wild-type LM and were sacrificed at 16 –20 h
postinfection. For each of the slides, B cells are shown
in blue, T cells in red, macrophages in cyan, and LM in
yellow. Original magnification was ⫻100. Data are representative of at least 10 sections from two to three independent mice.
Localization of memory OT-I T cells in the livers of LM-infected
mice
Previous studies have shown that T cells can be found within and
around LM lesions in the livers of LM-infected mice (47). We
sought to determine whether memory OT-I T cells that were not
specific for wild-type LM could be found in the livers of infected
mice. Our previous data suggests that there is a preferential localization of memory T cells to peripheral organs such as the liver and
importantly that these T cells can respond to the LM-induced cy-
tokines IL-12 and IL-18 (39). Therefore, we used B6.Thy1.1 mice
that had been transferred with OT-I T cells and primed ⬎4 wk
previously with VSV/OVA. Upon challenging these mice with
⬃20,000 LM, we were easily able to identify LM lesions at day 3
postinfection (Fig. 5, a and b). Importantly, memory OT-I T cells
could be visualized and were preferentially located in or surrounding the LM lesions. Analysis of memory OT-I T cells located in the
livers of mice that were not rechallenged with LM revealed cells
that were scattered randomly throughout the liver section (Fig. 5c).
Similar results were observed on day 1 before lesions of LM were
detectable in the liver at the LM dosage used for infection (data not
shown). To enumerate the localization of the memory OT-I T cells
in the liver of day 3 infected mice, we used a grid system that
divided the microscopic fields into ⬃100 grids that were ⬃37 ⫻
37 ␮m in size. For each T cell counted, a designation of the proximity of LM was assigned. Indeed, the vast majority of the T cells
counted at day 3 after LM infection either had LM residing within
FIGURE 4. Memory OT-I T cells preferentially localize with macrophages and LM in the T cell areas of
the spleen whereas NK cells do not. Sorted memory
OT-I T cells (7 ⫻ 105) (a) and NK cells (2 ⫻ 106) (c)
were purified as previously described, CFSE labeled,
and transferred into IFN-␥⫺/⫺ mice, which were infected subsequently with ⬃20,000 wild-type LM. At
day 1 postinfection, the mice were sacrificed, and 5-␮m
spleen sections were stained for B cells (blue) and T
cells (red). CFSE-labeled cells were identified as well
(green). Consecutive sections showing the macrophages
(magenta) and LM (yellow) are shown for the transferred memory OT-I T cell experiment (b) and the transferred NK cell experiment (d). Green arrows identify
either the transferred memory OT-I T cells (a) or the
transferred NK cells (c). Yellow arrows identify the LM.
Original magnification was ⫻100. Data are representative of at least four sections from two independent transfer experiments.
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infection has progressed to the point where the conventional anatomy of the spleen is altered (data not shown). Therefore, it was
difficult to assign exact locations to the identifiable transferred
populations of NK and memory OT-I T cells. Nonetheless, as previously demonstrated, the transferred memory OT-I T cells did
provide more efficient innate immune protection at day 3 postinfection compared with the NK cells (Fig. 2).
The Journal of Immunology
1755
Table I. Location of transferred memory OT-I T cells and NK cells 1
day postinfection with LMa
Location of Cells, No. (%)
Cells Transferred
Memory OT-Ib
NKb
PALS
B cell zone
Marginal zone
Red pulp
85 (69.7)
0 (0.0)
9 (7.4)
3 (1.8)
11 (9.0)
25 (14.7)
17 (13.9)
142 (83.5)
a
Results are from two combined, independent experiments.
Statistically significant ( p ⬍ 0.0001) difference exists between the localization
of memory OT-I T and NK cells in either the PALS or the red pulp.
b
the grid they were located within or in a surrounding grid (Fig. 5d).
Very few memory OT-I T cells were found to be independent of
LM lesions in the liver. Therefore, this data strongly suggests that
in the liver, as well as the spleen, memory CD8 T cells that are not
LM specific can preferentially localize with LM lesions and provide a protective effect by secreting IFN-␥.
It is well established that IFN-␥ is required during the innate immune response to LM (31, 32, 39, 48). However, the population of
cells responsible for secreting IFN-␥ is open for debate. Reports
suggest that multiple cell types have the ability to rapidly secrete
IFN-␥ in response to infection with LM. Therefore, dissecting
which cell or cells are actually able to control the LM by secreting
IFN-␥ has been difficult. Our recent data has shown that memory
CD8 T cells secrete IFN-␥ in response to IL-12 and IL-18, which
are produced by LM-infected macrophages (28, 39). Indeed, when
small numbers of memory OT-I T cells (specific for the OVAderived peptide, SIINFEKL) are transferred into IFN-␥-deficient
mice, these lymphocytes can provide innate protection from wildtype LM in the absence of cognate Ag (Fig. 2) (39). Our current
report now determines the relative contribution of NK and memory
CD8 T cells in providing protection from a LM infection. Surprisingly, we have found that memory OT-I T cells are more efficient
at providing protection against a LM infection than are NK cells.
Why were the memory OT-I T cells more effective than NK
cells at reducing LM burdens? Several hypotheses were considered
to explain this finding, including: 1) increased survival of the
FIGURE 5. Memory OT-I T cells preferentially localize with LM lesions in the liver. OT-I T cell-transferred B6.Thy1.1 mice were infected with VSV/OVA
and allowed to rest for ⬎4 wk to generate memory OT-I
T cells. Liver sections day 3 postinfection with LM (a
and b) or from uninfected memory OT-I mice (c) are
shown with LM identified with red and the memory
OT-I T cells identified with green. b, The inset box
shown in a. d, Cumulative data assigning memory OT-I
T cells to one of three areas: within a grid containing
LM, within a grid that has LM in a surrounding grid, or
within a grid neither containing nor surrounded by LM.
Data are representative of two liver sections. Original
magnification was ⫻100. A value of p ⬍ 0.001 as determined by ␹2 test for a random assortment of T cells in
the three defined areas.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Discussion
memory OT-I T cells compared with the NK cells; 2) increased
production of IFN-␥ by the memory OT-I T cells; or 3) localization of the memory OT-I T cells, but not the NK cells, with the
LM-infected areas of the spleen and liver. An experiment designed
to count the number of viable cells remaining in the spleen at days
1 and 3 postinfection with LM revealed that both NK cells and
memory OT-I T cells suffer considerable losses (unpublished results). In addition, Figs. 1 and 2 suggest that even when more NK
cells are present during the LM infection, they are still less efficient
at reducing the bacterial burden. Therefore, we believe that increased survival of the memory OT-I T cells does not account for
their increased protective ability. Our previous data (28), as well as
data presented here (Fig. 1), indicate that both NK and memory
CD8 T cells have the ability to rapidly secrete IFN-␥ after infection with LM. Therefore, a difference in IFN-␥-secreting ability is
unlikely to account for the difference in protective ability between
the two cell types.
With the above information in mind, we focused on the localization of the two different cell types to ascertain why T cells were
more effective at providing innate immune protection from LM
when compared with the NK cells. Prior studies showed that the
majority of NK cells reside in the red pulp region of the spleen in
uninfected mice (44 – 46). T cells, in contrast, reside in the PALS
region of the spleen in uninfected mice (42, 43). Upon infection
with LM, the splenic marginal zone macrophages are responsible
for the uptake of the bacteria (40). At ⬃6 –24 h postinfection (depending upon the dose of LM), the LM can be found within the
white pulp area of the PALS (Refs. 41 and 49; Fig. 3). However,
the molecular basis for this macrophage migration is unknown.
CCL21 and CCL19 play a role in the maintenance of the localization of macrophages in the marginal zone of the spleen, and
furthermore, infection with Leishmania donovani results in the loss
of stromal cells secreting CCL21 and CCL19 (50). Infections that
deplete the stromal cell subset responsible for secreting CCL21
and CCL19 were suggested to result in selective loss of the marginal zone macrophages. Future studies will be required to determine whether this scenario controls the loss of marginal zone macrophages during LM infection. However, even if the loss of
CCL21 and CCL19 does occur, this does not explain why the LM,
1756
Acknowledgments
We thank Angie Mobley of the Dallas Cell Analysis Facility for cell sorting and J. Marshall Haynie for technical assistance.
Disclosures
The authors have no financial conflict of interest.
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of the white pulp. Our data shows that transferred memory OT-I T
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