Differential Requirements by CD4?and CD8?T Cells for
Soluble and Membrane TNF in Control of Francisella tularensis
Live Vaccine Strain Intramacrophage Growth
Siobha ´n C. Cowley,1* Jonathon D. Sedgwick,†and Karen L. Elkins1*
During primary infection with intracellular bacteria, the membrane-associated form of TNF provides some TNF functions, but the
relative contributions during memory responses are not well-characterized. In this study, we determined the role of T cell-derived
secreted and membrane-bound TNF (memTNF) during adaptive immunity to Francisella tularensis live vaccine strain (LVS).
Although transgenic mice expressing only the memTNF were more susceptible to primary LVS infection than wild-type (WT)
mice, LVS-immune WT and memTNF mice both survived maximal lethal secondary Francisella challenge. Generation of CD44high
memory T cells and clearance of bacteria were similar, although more IFN-? and IL-12(p40) were produced by memTNF mice.
To examine T cell function, we used an in vitro tissue coculture system that measures control of LVS intramacrophage growth by
LVS-immune WT and memTNF-T cells. LVS-immune CD4?and CD8?T cells isolated from WT and memTNF mice exhibited
comparable control of LVS growth in either normal or TNF-? knockout macrophages. Although the magnitude of CD4?T
cell-induced macrophage NO production clearly depended on TNF, control of LVS growth by both CD4?and CD8?T cells did
not correlate with levels of nitrite. Importantly, intramacrophage LVS growth control by CD8?T cells, but not CD4?T cells, was
almost entirely dependent on T cell-expressed TNF, and required stimulation through macrophage TNFRs. Collectively, these data
demonstrate that T cell-expressed memTNF is necessary and sufficient for memory T cell responses to this intracellular pathogen,
and is particularly important for intramacrophage control of bacterial growth by CD8?T cells. The Journal of Immunology,
2007, 179: 7709–7719.
virulent strains of this organism are highly infectious when ac-
quired via the aerosol route, with an estimated mortality rate of
30–60% in untreated patients (2). For these reasons, this organism
has been classified as a category A bioterrorism agent. The atten-
uated live vaccine strain (LVS)2of F. tularensis was derived by
repeated passage of a virulent F. tularensis strain on agar (3); LVS
has been studied as an investigational product but it is not currently
licensed for use in humans in the United States. Therefore, the
development of a new F. tularensis vaccine, as well as better un-
derstanding of the unlicensed LVS strain, remain high priorities.
Furthermore, immunity to Francisella has much in common with
other more clinically important intracellular pathogens, such as
Mycobacterium tuberculosis (4). Thus, studies that identify the
correlates of protection for tularemia can be used to further our
understanding of immunity to intracellular pathogens in general.
rancisella tularensis is a small Gram-negative facultative
intracellular bacterium, and the causative agent of an
acute febrile illness known as tularemia (1). The most
Although LVS is avirulent for humans when administered via
most routes, it is highly virulent for laboratory mice and causes a
fulminant infection in the organs of the reticuloendothelial system
(5). The outcome of a primary LVS infection in mice is dependent
on the route of inoculation: the LD50for infections initiated via the
intradermal (i.d.) route is ?106bacteria in BALB/cByJ mice,
whereas the LD50for infections administered via the i.v. or i.p.
routes approaches a single bacterium (5, 6). Mice given a sublethal
i.d. LVS dose can typically survive and clear the infection, and
survive secondary i.p. challenge doses as high as 100,000 LD50(1,
5, 7). Consequently, LVS murine infection is a convenient model
in which to analyze immunity to tularemia.
As for many intracellular pathogens, TNF is essential for sur-
vival of Francisella infection: mice treated with neutralizing anti-
TNF Abs quickly succumb to sublethal doses of LVS during both
primary and secondary infections (8). TNF is initially produced as
a 26-kDa transmembrane protein that is cleaved by the TNF-?-
converting enzyme to generate a soluble 17-kDa molecule (9). The
membrane-bound form of TNF (memTNF) is biologically active,
and can mediate the cytotoxic or microbicidal activity of a variety
of cells, including NK cells, CD4?T cells, and monocytes (10–
13). Recently, mice that express only memTNF were generated by
replacing the endogenous TNF allele in wild-type (WT) C57BL/6
mice with the ?1–9K11E TNF allele (14). This mutation resulted
in complete loss of TNF-?-converting enzyme-mediated cleavage
of TNF, while maintaining normal cell surface expression and
function of memTNF (14). Importantly, anti-TNF therapies such
as infliximab and etanercept are currently used as effective treat-
ments for several chronic inflammatory diseases, leaving those
patients with diminished TNF responses. Increased complications
related to granulomatous infections have been reported for these
products, including M. tuberculosis and Listeria monocytogenes
(15, 16). Because the two classes of TNF blockers are known to
*Laboratory of Mycobacterial Diseases and Cellular Immunology, Center for Bio-
logics Research and Evaluation, U.S. Food and Drug Administration, Rockville, MD
20852; and†Eli Lilly and Company, Indianapolis, IN 46285
Received for publication April 20, 2007. Accepted for publication September
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.
1Address correspondence and reprint requests to Dr. Siobha ´n C. Cowley, or Dr.
Karen L. Elkins, Laboratory of Mycobacterial Diseases and Cellular Immunology,
Center for Biologics Research and Evaluation, U.S. Food and Drug Administration,
1401 Rockville Pike, HFM 431, Rockville, MD 20852; E-mail addresses:
email@example.com and firstname.lastname@example.org
2Abbreviations used in this paper: LVS, live vaccine strain; i.d., intradermal;
derived macrophage; cDMEM, complete DMEM.
The Journal of Immunology
differentially neutralize soluble and membrane TNF (17), it is im-
portant to define the relative contributions of soluble and mem-
brane TNF in intracellular infections, including Francisella.
We have recently shown that mice expressing only memTNF
are less susceptible to sublethal i.d. LVS infection than totally
TNF-deficient mice (TNF knockout (KO) mice), but significantly
more susceptible to LVS infection than WT mice (S. Cowley, M.
Goldberg, A. Ho, and K. Elkins, submitted for publication). The
memTNF mice succumbed during the first several days of a pri-
mary i.d. LVS infection in a dose-dependent manner; mice that
survived lower doses of LVS through the early innate immune
response ultimately cleared the infection. Thus, although memTNF
can contribute to resistance to LVS infection, it is not sufficient to
compensate for all of the functions of soluble TNF during the
innate immune response. Because TNF is also used by T cells
during secondary adaptive immune responses to control LVS in-
tracellular growth, in this study, we investigate the role of
memTNF and soluble TNF in the function of LVS-immune T cells,
by focusing on the ability of LVS-immune T cells to inhibit LVS
growth in macrophages.
Materials and Methods
F. tularensis LVS (ATCC 29684; American Type Culture Collection) was
grown and frozen as previously described (7, 18). Viable bacteria were
quantified by plating serial dilutions on Mueller Hinton agar plates.
Animals and infections
Male specific pathogen-free C57BL/6J mice and TNFR 1/2 KO mice were
purchased from The Jackson Laboratory. TNF KO and memTNF mice
were obtained via material transfer agreement with Schering-Plough Bio-
pharma (formerly DNAX Research), and bred at the Center for Biologics
Research and Evaluation, U.S. Food and Drug Administration (CBER/
FDA). All animals were housed in a barrier environment at CBER/FDA,
and procedures were performed according to approved protocols under
animal care and use committee guidelines. Throughout, “LVS-immune”
refers to splenocytes obtained from mice given a sublethal priming dose of
102LVS i.d. 1–3 mo before sacrifice. For in vivo secondary LVS chal-
lenges, all mice were given a sublethal priming dose of 102LVS i.d. 4 wk
before a 105LVS i.p. secondary inoculation. All materials, including bac-
teria, were diluted in PBS with ?1 endotoxin units/ml endotoxin
In vitro assessment of control of intracellular bacterial growth
in bone marrow-derived macrophages (BMM?)
The in vitro culture systems used, and validation of the culture system’s
abilities to reflect known parameters of T cell activities during in vivo
control of bacterial growth, have been described in detail elsewhere (19,
20). Briefly, BMM? were used as the target cells. Bone marrow was
flushed from femurs of healthy adult mice with DMEM (Invitrogen Life
Technologies) supplemented with 10% heat-inactivated FCS (HyClone),
10% L-929 conditioned medium, 0.2 mM L-glutamine (Invitrogen Life
Technologies), 1 mM HEPES buffer (Invitrogen Life Technologies), and
0.1 mM nonessential amino acids (Invitrogen Life Technologies; complete
DMEM (cDMEM)). Cells were washed, a single-cell suspension was pre-
pared, and plated at 1 ? 106viable cells/well in 48-well plates, in cDMEM
supplemented with 50 ?g/ml gentamicin (Invitrogen Life Technologies),
and incubated at 37°C in 5% CO2. After 1 day, the medium was replaced
with antibiotic-free cDMEM, and incubated for an additional 6 days at
37°C in 5% CO2. The medium was replaced with fresh, gentamicin-free
cDMEM every 2–3 days during the 7-day incubation.
Following the 7-day culture period, the BMM? formed a confluent
monolayer, and the concentration of BMM? was estimated to be 5 ? 106
cells/well in 48-well plates. BMM? were then infected with F. tularensis
LVS according to the following protocol: bacteria were diluted from frozen
stocks in cDMEM and added at a multiplicity of infection of 1:20 (LVS)
(bacterium-to-BMM? ratio). A low multiplicity of infection was chosen to
mimic initial in vivo exposure to relatively small numbers of organisms,
and permit controlled infection of the macrophage monolayer over 3 days.
This time point was selected based on previous data indicating the peak of
bacterial growth that coincided with maximal impact of cocultured immune
cells (19, 20). LVS was coincubated with BMM? at 37°C in 5% CO2for
2 h and then washed three to five times with PBS (Cambrex). The LVS-
infected monolayers were then incubated for 45 min–1 h in cDMEM sup-
plemented with 50 ?g/ml gentamicin to eliminate extracellular bacteria,
and infected BMM? were washed extensively. Following the last wash,
PBS was replaced with 1 ml/well cDMEM, and spleen cells or separated
subpopulations were added to the indicated wells for coculture. Also at this
time, anti-TNF Abs or isotype control Abs were added to the cultures at a
concentration of 25 ?g/ml. TNF neutralization by addition of the anti-TNF
Abs to the cultures was confirmed by measuring TNF levels in the super-
natants by ELISA following 72 h of coculture. Cultures were incubated at
37°C in 5% CO2for the remainder of the experiment. To determine bac-
terial uptake, some BMM? were lysed with sterile distilled water for 2–3
min immediately after infection and washing with PBS. Culture lysates
were serially diluted in PBS as necessary, and plated on Mueller Hinton
agar plates. Plates were incubated for 2–3 days at 37°C in 5% CO2and
colonies were counted. Growth of bacteria in BMM? was further moni-
tored by lysing cultures at the indicated time points after coculture, plating
lysates, and counting colonies as described above.
Viability of the macrophage monolayer was routinely assessed during
this time using both trypan blue exclusion and light microscopy. Macro-
phage monolayers at the end of the 3-day incubation period exhibited mor-
phological changes consistent with infection; however, the majority of the
cells in the monolayers were still intact, viable, and adherent.
Spleens used for coculture were aseptically removed from selected mice
and disrupted with a 3-ml syringe plunger. A single-cell suspension was
prepared, and erythrocytes were lysed with ammonium chloride. Cells
were washed, viability was assessed by exclusion of trypan blue, cells were
resuspended in Dulbecco’s PBS–2% FCS to the appropriate concentration,
and added cells to BMM? cultures. Unless otherwise stated, 2.5 ? 106
splenocytes (or their separated subpopulations) were added to the 48-well
cultures (?1 splenocyte to 2 BMM?).
Enrichment of T cell subpopulations for in vitro coculture
CD4?and CD8?T cells were enriched using positive selection via MACS
beads (MACS magnetic cell sorting system; Miltenyi Biotec). All sepa-
rated subpopulations were analyzed by flow cytometry to determine the
purity of the cells. The purified cells were routinely ?80–90% of the
intended cell type; the identity of the other contaminating cells were always
accounted for, and were B cells, macrophages, neutrophils, or granulocytes
that do not have antibacterial activity on the BMM? monolayer in the in
Ex vivo culture of spleen cells harvested from LVS-infected mice
for quantification of cytokine and NO levels during secondary
At the indicated times following LVS infection, infected spleens were asep-
tically removed from selected mice and prepared as described above. Cells
were resuspended in DMEM (Invitrogen Life Technologies) supplemented
with 10% heat-inactivated FCS (HyClone), 0.2 mM L-glutamine (Invitrogen
Life Technologies), 1 mM HEPES buffer (Invitrogen Life Technologies), and
0.1 mM nonessential amino acids (Invitrogen Life Technologies) at a concen-
tration of 2 ? 107splenocytes/ml. One milliliter of splenocytes was cultured
overnight, and supernatants were collected for quantification of cytokine and
nitrite levels as described.
Quantitation of cytokines and NO in BMM? culture
Culture supernatants were assayed for IFN-?, IL-12(p40), and TNF by
standard sandwich ELISAs, using reagents obtained from BD Pharmingen
and quantified by comparison to recombinant standards, as per the manu-
facturer’s instructions. NO was detected in culture supernatants by the
Griess reaction as previously described, using commercial Griess reagent
(Sigma-Aldrich). Limits of detection for IFN-?, IL-12(p40), and TNF
ELISAs were 100, 78, and 100 pg/ml, respectively. The limit of detection
for the Griess reaction was 1.5 ?M nitrite/ml.
Flow cytometry analyses
Single-cell suspensions were prepared and stained for a panel of murine
cell surface markers and analyzed using a LSR II flow cytometer (BD
Biosciences) and FACS Diva or FlowJo software essentially as previously
described (18–21). Clones used included RM4-5 (anti-CD4), 53-6.7 (anti-
CD8a), GL3 (anti-?/anti-? TCR), PK136 (anti-NK1.1), 53-2.1 (anti-Thy1.2),
H57-597 (anti-TCR-? chain), 17A2 (anti-CD3), M1/70 (anti-CD11b), RA3-
7710T CELL memTNF CONTROLS Francisella INTRAMACROPHAGE GROWTH
above and Fc block were obtained from BD Pharmingen, and optimal con-
centrations were determined in separate experiments for use in three- to six-
color staining protocols as required using appropriate fluorochrome-labeled
isotype control Abs.
memTNF-transgenic mice survive a secondary lethal LVS
challenge, but produce increased levels of IL-12(p40) and
IFN-? as compared with WT mice
To determine the capacity of immunized memTNF mice to with-
stand maximal secondary LVS challenge, LVS-immune WT and
memTNF mice were given a secondary LVS infection. Previous
results have shown that WT mice that survive a primary sublethal
LVS infection are solidly immune to secondary lethal challenges
of ?106LVS i.p. (this dose is ?105LD50for a naive WT mouse)
(22). Furthermore, the majority of naive memTNF mice can sur-
vive and clear a primary 102LVS i.d. infection (Table I). Thus,
memTNF and WT mice were given a sublethal 102LVS i.d. pri-
mary infection. Four weeks after the initial primary infection, mice
were challenged with 105LVS i.p., and the LVS burdens in the
spleens and livers were enumerated during the first 4 days after
challenge. As shown in Fig. 1, A and B, the numbers of LVS in the
spleens and livers of the WT mice peaked on days 1–2 postchal-
lenge, and diminished to around the limit of detection by day 4
(?30 CFU/spleen and ?100 CFU/liver). Similarly, the LVS CFUs
in the spleens and livers of the memTNF mice were highest on
days 1 and 2 after challenge, and the memTNF mice cleared the
infection by day 4 after challenge. The slightly higher LVS organ
burden in the memTNF mice on day 1 after challenge was ob-
served in repeated experiments, but it was never significantly dif-
ferent from WT mice. All LVS-immune WT and memTNF mice
given a maximal secondary 5 ? 105LVS i.p. challenge survived
and cleared the infection within 2 wk, whereas LVS-naive WT and
memTNF mice given a primary 5 ? 105LVS i.p. dose succumbed
to infection within 1 wk (Table I). The same 5 ? 105LVS dose
given to mice via the i.d. route was sublethal for WT mice, but
100% lethal for memTNF mice (Table I).
To characterize the cytokine environment within the spleen dur-
ing a 105LVS i.p. secondary challenge, spleen cells from the LVS-
challenged mice were harvested on selected days and cultured
overnight ex vivo, and the levels of IFN-?, IL-12(p40), and NO
were measured in the culture supernatants. As shown in Fig. 1, C
and D, spleen cell production of IFN-? and p40 was significantly
higher in the memTNF mice than the WT mice on days 1 (p40) and
3 (IFN-? and p40) after infection (p ? 0.01). In contrast, no
significant differences in NO production were observed between
the memTNF and WT mice (Fig. 1E). To further examine the
nature of the responding T cells during in vivo secondary chal-
lenge, the numbers of activated CD44highCD4?and CD8?T cells
in the spleens of infected mice were enumerated on day 3 follow-
ing a 105i.p. secondary infection. As shown in Fig. 1F, in both the
memTNF and WT mice, the numbers of CD44highCD4?and
CD8?T cells present in the spleens increased ?3-fold by day 3
after secondary challenge, as compared with naive WT mice. There
the numbers of CD44highCD4?and CD8?T cells present in the
spleens on day 3 after secondary challenge (p ? 0.05). Thus, dur-
ing secondary lethal challenge, memTNF mice recruit WT levels
of activated CD4?and CD8?T cells, and control and clear a
secondary LVS challenge similar to WT mice; however, spleno-
cytes from the memTNF produce significantly higher levels of
IFN-? and IL-12(p40) as compared with WT mice.
memTNF mediates control of LVS intramacrophage growth in
vitro, but maximal NO production requires soluble TNF
To directly assess the role of memTNF in control of LVS intram-
acrophage growth, we compared the ability of LVS-immune
spleen cells harvested from memTNF and WT mice to control
LVS growth in BMM? in an in vitro system. Splenocytes were
obtained from LVS-immune WT or memTNF mice that were
primed 4 wk earlier with a sublethal i.d. LVS infection. Immune
splenocytes were cocultured with LVS-infected WT or memTNF
BMM? monolayers, and control of LVS growth was assessed after
3 days in the two different BMM? monolayers. As seen in Fig. 2A,
addition of LVS-immune, but not naive, WT or memTNF spleno-
cytes to their cognate infected BMM? monolayers resulted in
readily measurable and significant (p ? 0.01) inhibition of LVS
growth compared with cultures containing either naive splenocytes
or no splenocytes. LVS replicated to a similar extent in both the
WT and memTNF BMM?, and growth was not significantly af-
fected by the addition of naive splenocytes to the cultures. Cocul-
ture of LVS-immune WT splenocytes with WT BMM? resulted in
a 2.65 log10reduction in growth of the bacteria, while coculture of
memTNF splenocytes with memTNF BMM? resulted in a similar
2.79 log10reduction of LVS growth. To assess the contribution of
memTNF to the observed inhibition of LVS growth in the
memTNF cultures, we tested the impact of abrogation of TNF
activity on the control of LVS growth in the coculture system. As
shown in Fig. 2A, addition of neutralizing anti-TNF Abs to both
WT and memTNF cocultures resulted in a significant reversal of
control of LVS growth as compared with cultures containing iso-
type control Ab, demonstrating that TNF functions to inhibit LVS
intracellular growth when it is available only in the membrane-
Several in vitro studies have previously shown that TNF-
dependent anti-LVS activity is mediated primarily through in-
duction of macrophage NO (5, 23). To determine whether
memTNF also acts primarily through induction of NO, we com-
pared the levels of nitrite (as a surrogate marker of NO) in the
supernatants of the memTNF and WT cultures. As seen in Fig.
2B, both the WT and memTNF cocultures with immune spleen
cells contained measurable levels of NO, and this NO produc-
tion was greatly reduced by addition of anti-TNF Abs, but not
isotype control Abs. However, the memTNF cultures consis-
tently produced significantly less NO than the WT cultures
(p ? 0.01). Thus, although memTNF can facilitate macrophage
Table I. Survival of primary and secondary LVS infection by
C57BL/6J5 ? 105
1 ? 102
5 ? 105
1 ? 102
1 ? 102
5 ? 105
5 ? 105
5 ? 105
5 ? 105
5 ? 105
TNF KO 10/10
aC57BL/6J wild-type, TNF KO, or memTNF male mice were administered the
indicated doses of LVS i.d. and monitored for survival; the dose of infection was
confirmed by simultaneous plate counts and is expressed as total CFU. One month
after primary infection, mice were administered the indicated dose of LVS i.p. and
monitored for survival for 1 mo. Surviving mice were sampled to confirm clearance
of infections. N/A, not applicable. These data are representative of two to three ex-
periments of similar design.
bMean time to death for C57BL/6 mice was 5.5 ? 1.0 days.
cMean time to death for memTNF mice was 5.2 ? 1.3 days.
dMean time to death for TNF KO mice was 5.0 ? 2.1 days.
7711The Journal of Immunology
production of NO, the levels produced in the absence of soluble
TNF were significantly lower.
CD8?T cells require macrophage TNFRs to control LVS
We next sought to identify the T cell subpopulations that use
memTNF to control LVS intracellular growth. First, we assessed
the ability of CD4?and CD8?T cells isolated from LVS-immune
memTNF mice to inhibit LVS growth in memTNF BMM?, as
compared with similar cultures containing WT T cells and WT
BMM?. As shown in Fig. 3A, control of LVS growth was not
significantly different between the WT CD4?and CD8?T cells,
and the memTNF CD4?and CD8?T cells (p ? 0.05). Addition
of neutralizing anti-TNF Abs to these cultures resulted in a sig-
nificant reversal in the control of LVS growth by CD4?and CD8?
T cells from both WT and memTNF mice. In repeated experi-
ments, the magnitude of this reversal of control of growth by ad-
dition of anti-TNF Abs was consistently less for the memTNF cells
as compared with the WT cells. This suggests that either the anti-
TNF Abs are less efficient at neutralizing the activity of memTNF,
or conversely, that the memTNF cells use additional compensatory
mechanisms to control LVS growth in the absence of soluble TNF.
Nevertheless, these results clearly demonstrate that memTNF con-
tributes to the control of LVS intracellular growth mediated by
both CD4?and CD8?LVS-immune T cells, and that CD8?T
cells are particularly dependent on TNF activity.
The IL-12-IFN-? axis is a tightly regulated feedback loop that
includes macrophage production of IL-12 and TNF, followed by T
cell production of IFN-?, culminating in macrophage activation
and control of bacterial intracellular growth (24). Because we
vive and clear maximal lethal secondary LVS
challenge. Normal C57BL/6 and memTNF-
transgenic mice were given 102LVS i.d.; 1 mo
later, mice were challenged with 5 ? 105LVS
i.p. Numbers of CFU in organs (four mice per
group) were assessed for bacterial burdens at
the indicated time points in the spleen (A) or
liver (B). Values shown are the mean numbers
of CFU per organ ? the SEM of viable bac-
teria. Limit of detection is 30 CFU/spleen and
100 CFU/liver (dotted lines). To assess the
challenge, spleen cells were harvested from in-
fected mice at the indicated time points and
cultured overnight. Culture supernatants were
tested for levels of IFN-? (C), IL-12(p40) (D),
and NO (E). Values shown are the mean val-
ues ? the SEM of triplicate cultures. To assess
memory T cell responses, total numbers of
CD44highCD4?and CD8?T cell populations
three infected mice per strain were measured
by flow cytometry on day 3 postsecondary
challenge (F). Asterisks (?) indicate p values
?0.01 as compared with WT cultures. These
data are representative of three experiments of
memTNF-transgenic mice sur-
7712T CELL memTNF CONTROLS Francisella INTRAMACROPHAGE GROWTH
observed higher levels of both IFN-? and IL-12(p40) production in
ex vivo-cultured splenocytes harvested from memTNF mice dur-
ing secondary challenge, we also assessed the coculture superna-
tants for cytokine production. Interestingly, both IFN-? and IL-
12(p40) production in the memTNF cocultures were significantly
greater than that of the WT cocultures (Fig. 3, B and C); this was
true for cocultures containing whole memTNF spleen cells, as well
as purified memTNF CD4?and CD8?T cells as compared with
their WT counterparts. The lower NO levels (Fig. 2B) coupled
with higher IFN-? and IL-12 production found in the memTNF
cultures suggest that, although control of LVS growth was equiv-
alent to that of WT cells, there is a defect in the regulation of the
IL-12-IFN-? axis in the memTNF cultures as compared with the
WT cultures. Thus, soluble TNF is required to regulate IL-12 and
IFN-? production and/or use appropriately.
Because both macrophages and T cells express TNFRs and can
respond to TNF, we next determined the role of macrophage TNF
stimulation in the control of LVS growth. To this end, we cocul-
tured LVS-immune CD4?and CD8?T cells purified from both
WT and memTNF mice with BMM? derived from mice geneti-
cally deficient for TNFRs 1 and 2 (TNFR1/2 KO). Here, CD4?
and CD8?T cells can respond to TNF while the LVS-infected
BMM? cannot, permitting measurement of the relative importance
of macrophage TNF stimulation, as compared with TNF T cell
stimulation, in control of LVS intracellular growth. As seen in Fig.
4, a trend toward a small reversal of control of LVS growth was
evident for all cell types tested in both the WT and memTNF
cocultures containing TNFR1/2 KO BMM?, as compared with
their control BMM? cocultures. However, in repeated experi-
ments, reversal of control of growth was consistently significant
only for the CD8?T cell cocultures. Elimination of macrophage
TNFRs resulted in an almost complete loss of the ability of WT
and memTNF CD8?T cells to control LVS intracellular growth.
Together with the results of TNF blockade (Fig. 3A), these results
indicate that CD8?T cells control LVS intracellular growth pri-
marily via stimulation of macrophage TNFRs, and that CD8?T
cell memTNF mediates this activity.
Given the importance of TNF stimulation in the induction of
macrophage NO, we further measured the nitrite levels in the WT
and memTNF spleen cell cocultures containing TNF R1/2 KO
macrophages. As shown in Fig. 5, A and B, macrophage NO pro-
duction was severely diminished in all cultures containing
TNFR1/2 KO macrophages, regardless of whether a significant
effect on control of LVS growth was observed or not. This reduc-
tion was evident in both the memTNF and WT spleen cell cultures,
confirming that memTNF is sufficient to induce macrophage NO
production; however, as observed earlier (Fig. 2B), the levels of
NO produced in the memTNF cocultures were significantly less
than that of the WT cultures. Furthermore, cocultures containing
immune CD8?cells from both WT and memTNF mice exhibited
only low levels of nitrite, and these levels were not significantly
diminished further by the absence of macrophage TNFRs. Con-
versely, NO production by the TNFR1/2 KO BMM? in the CD4?
similarly to WT spleen cells, but exhibit defective NO production. A, Con-
trol of LVS intramacrophage growth by LVS-immune memTNF spleno-
cytes. BMM? from WT and memTNF mice were infected with LVS and
cocultured with splenocytes from either uninfected mice (naive spleen),
mice infected i.d. with LVS 4 wk previously (primed spleen), or no spleen
cells (LVS ? macrophages). Immediately following LVS infection of the
BMM?, splenocytes were added to the indicated wells at a ratio of 1:2
(splenocyte:BMM?). In the indicated wells, either anti-TNF Abs or control
IgG Ab (25 ?g/ml) were added to the cultures at the time of addition of the
splenocytes. Seventy-two hours after infection, the BMM? were washed,
memTNF spleen cells control LVS intracellular growth
lysed, and plated to determine the levels of intracellular bacteria. B, Se-
cretion of NO into culture supernatants following 72 h coculture of LVS-
infected BMM? and immune splenocytes. Values shown are the mean
numbers of CFU per milliliter ? the SEM of viable bacteria (A) or mean
micromoles per milliliter of nitrite (B) (triplicate samples). Asterisks (?)
indicate p values ?0.01 as compared with cocultures containing control
Ab. Symbols (?) indicate p values ?0.05 between WT and memTNF
cocultures. These data are representative of five experiments of similar
7713The Journal of Immunology
T cell cocultures was diminished almost to background levels;
however, this had only a small and insignificant effect on LVS
growth control by CD4?T cells (Fig. 4). Thus, the high levels of
NO detected in the CD4?T cell cocultures are mediated via stim-
ulation of macrophage TNFRs, although it does not significantly
affect the ability of CD4?T cells to inhibit LVS growth. However,
because the ability of CD4?T cells to control LVS growth was
significantly reduced by the addition of anti-TNF Abs (Fig. 3A),
CD4?T cells clearly possess a TNF-dependent anti-LVS activity.
To evaluate the contributions of IFN-? and IL-12 to NO pro-
duction, the coculture supernatants were assessed for cytokine pro-
duction. As shown in Fig. 5, C and D, significantly higher levels of
IFN-? (Fig. 5, C and D), but not IL-12 (Fig. 5, E and F), were
detected in all the TNFR1/2 KO BMM? cocultures containing
either WT or memTNF CD4?and CD8?T cells, as compared
with cocultures with WT BMM?. Thus, in the absence of macro-
phage TNFRs, IFN-? levels in the culture supernatants were in
excess. This indicates that the ability of macrophages to express
TNFRs is essential to maintain NO production and proper regula-
tion of IFN-? production and/or use.
T cell-derived, but not macrophage-derived, TNF is required for
control of LVS intramacrophage growth in vitro
Because macrophage TNFRs are a critical component in the ef-
fective control of LVS intracellular growth by CD8?T cells, and
macrophage-derived TNF is known to operate in an autocrine fash-
ion, we investigated whether macrophage TNF expression is a crit-
ical component of control of LVS intracellular growth by either
CD4?or CD8?T cells. To this end, LVS-immune CD4?and
CD8?T cells were purified from WT and memTNF mice, and
cocultured in vitro with LVS-infected BMM? derived from total
TNF KO mice. Thus, the only available TNF is derived from the
T cells in these cocultures. As shown in Fig. 6A, control of LVS
growth by WT CD4?and CD8?T cells was not significantly
different in cocultures containing either WT or TNF KO BMM?
(p ? 0.05). Similarly, memTNF CD4?and CD8?T cells con-
trolled LVS growth equally well in memTNF and TNF KO
BMM? (Fig. 6B). Loss of macrophage TNF had a small effect on
the ability of the WT whole primed spleen cocultures to produce
NO (Fig. 6C). However, this reduction varied in magnitude and
significance in repeated experiments, and was not observed in the
WT cultures using CD4?and CD8?enriched T cells. Further-
more, the memTNF cocultures did not exhibit a significant reduc-
tion in NO production during coculture with TNF KO BMM? as
compared with memTNF BMM? (Fig. 6D). Thus, CD4?and
tramacrophage growth, but exhibit dysregulation of IFN-? and IL-12 pro-
duction in the absence of soluble TNF. A, Effects of TNF neutralization on
the ability of different T cell subsets to control LVS growth in WT and
CD4?and CD8?T cells use memTNF to control LVS in-
memTNF-transgenic cocultures. Infected BMM? were cocultured with
splenocytes obtained from either uninfected mice (naive spleen), whole
primed splenocytes from immune WT or memTNF mice (primed spleen),
or the indicated T cell subsets enriched from immune WT or memTNF
mice (CD4?cells, CD8?cells). All splenocyte populations were added at
a 1:2 ratio (splenocyte to BMM?). Anti-TNF Abs (25 ?g/ml) were added
to the wells at the time of addition of the splenocytes to the infected
BMM?. Seventy-two hours after infection, the BMM? were washed,
lysed, and plated to determine the levels of intracellular bacteria. Culture
supernatants harvested at 72 h were tested for levels of IFN-? (B) and
IL-12(p40) (C). Values shown are the mean numbers of CFU per millili-
ter ? the SEM of viable bacteria (A) or mean nanograms per milliliter of
IFN-? and IL-12(p40) (B and C) (triplicate samples). Asterisks (?) indicate
p values ?0.01 as compared with cognate cocultures containing no Ab.
Hatches (#) indicate CFU p values ?0.01 as compared with cognate co-
cultures containing naive spleen cells. Symbols (?) indicate p values
?0.05 between WT and memTNF cocultures. These data are representa-
tive of three experiments of similar design.
7714T CELL memTNF CONTROLS Francisella INTRAMACROPHAGE GROWTH
CD8?T cell-derived TNF, even when present only in the mem-
brane-bound form, is sufficient to mediate control of LVS intra-
cellular growth in the absence of macrophage production of TNF.
Examination of IFN-? and IL-12(p40) production (Fig. 6, E–H)
in the TNF KO BMM? cultures revealed a mild CD4?T cell-
specific defect in the IL-12-IFN-? axis in the absence of macro-
phage-derived TNF. Significantly higher levels of IFN-?, but not
IL-12, were present in both the memTNF and WT cocultures con-
taining CD4?T cells. This effect was not observed consistently, or
to a significant extent, in the CD8?T cell cocultures. The increase
Macrophages + LVS
+ Naive spleen
+ Primed memTNF spleen
+ memTNF CD4
+ memTNF CD8
Macrophages + LVS
+ Naive spleen
+ Primed WT spleen
+ WT CD4
+ WT CD8
TNFR1/2 KO BMMOs
TNFR1/2 KO BMMOs
macrophage TNFRs for control of LVS intramacrophage growth. Ability of
different T cell subsets isolated from LVS-immune WT (A) and memTNF (B)
B) BMM?. Infected BMM? were cocultured with splenocytes obtained from
either uninfected mice (naive spleen), whole primed splenocytes from immune
immune WT or memTNF mice (CD4?cells, CD8?cells). All splenocyte
populations were added at a 1:2 ratio (splenocyte to BMM?). Seventy-two
hours after infection, the BMM? were assessed for levels of intracellular LVS
growth. Values shown are the mean numbers of CFU per milliliter ? the SEM
of viable bacteria (triplicate samples). Asterisks (?) indicate p values ?0.01 of
cocultures containing TNFR1/2 KO BMM? as compared with cocultures con-
taining either WT (A) or memTNF (B) BMM?. These results are representa-
tive of three experiments of similar design.
LVS-immune CD8?, but not CD4?, T cells are dependant on
IFN-? regulation in WT and memTNF cocultures. Secretion of cytokines
and NO into culture supernatants following coculture of LVS-infected
TNFR1/2 KO BMM? with WT and memTNF LVS-immune splenocytes.
Culture supernatants were collected from the WT, memTNF, and TNFR1/2
KO BMM? cocultures described in Fig. 4, after 72 h, and tested for NO
(A and B), IFN-? (C and D), and IL-12(p40) (E and F). Values shown are
the mean micromoles per milliliter of nitrite (A and B) or the mean nano-
grams per milliliter ? the SEM of the indicated cytokine (C–F) (triplicate
samples). Asterisks (?) indicate p values ?0.01 of cocultures containing
TNFR1/2 KO BMM? as compared with their cognate cocultures contain-
ing either WT (A) or memTNF (B) BMM?. These results are representative
of three experiments of similar design.
Macrophage TNFRs are required for NO production and
7715 The Journal of Immunology
in IFN-? production was not as pronounced as that seen in the
TNFR1/2 KO BMM? cultures (Fig. 5, C and D), and varied in
magnitude from 2- to 3-fold between different experiments. How-
ever, this consistent increase in IFN-? suggests a small but strict
role for macrophage-derived TNF in the regulation of IFN-? and
IL-12 production and/or uptake in the CD4?T cell cultures.
Development of effective vaccines against intracellular pathogens
is limited by an incomplete understanding of the mechanisms that
contribute to protective immunity. For an intracellular pathogen
such as F. tularensis, a critical aspect of protective immunity oc-
curs during the interaction of Ag-specific T cells with infected host
cells. To study this interaction in greater detail, we have previously
developed an in vitro coculture model system to assess the mech-
anisms used by LVS-immune T cells to limit Francisella intracel-
lular growth inside macrophages (19). This in vitro system is de-
signed to reflect T cell activities that operate during secondary
exposure to Ag. Here, we have applied this in vitro system to study
the relative contributions of macrophage and T cell-derived solu-
ble TNF, as well as memTNF, to the control of F. tularensis LVS
intramacrophage growth. Using this system, we demonstrate that
both CD4?and CD8?T cells can efficiently use memTNF to
control LVS intracellular growth. Furthermore, we show that, un-
like LVS-immune CD4?T cells, LVS-immune CD8?T cells rely
heavily on the presence of TNF to mediate LVS growth control.
From the data presented here, it is possible to draw several con-
clusions regarding the role of TNF in the interaction of LVS-in-
fected macrophages with immune T cells. First, TNF is essential
for maximal control of LVS growth mediated by both LVS-im-
mune CD4?and CD8?T cells (Fig. 3A); furthermore, both cell
types can inhibit LVS intracellular growth when TNF is available
only in the membrane-bound form (Figs. 3A, 4, and 6, A and B).
However, the role of TNF in this process differs significantly be-
tween CD4?and CD8?T cells. CD8?, but not CD4?, T cells are
almost entirely dependent on the activity of TNF to control LVS
intracellular growth (Figs. 3A and 4). This is evidenced by the
finding that only CD8?T cells strictly require interaction with
macrophage TNFRs to mediate LVS growth control (Fig. 4). In
contrast, the ability of CD4?T cells to control LVS intramacroph-
age growth was only partially dependent on TNF (Fig. 3A), and
CD4?T cell function was not significantly diminished in the ab-
sence of macrophage TNFRs (Fig. 4). Although the magnitude of
CD4?T cell-induced macrophage NO production was clearly de-
pendent on TNF, control of LVS growth by both CD4?and CD8?
T cells did not correlate with the levels of NO measured in the
cocultures (Figs. 4 and 5, A and B). In particular, CD8?T cell
cocultures with TNFR1/2-deficient macrophages resulted in loss of
LVS growth control but no change in NO production, strongly
suggesting that TNF-mediated control of LVS growth by CD8?T
TNF KO MΦ
TNF KO MΦ
/ e t i r t i n s e l o
o r c i
TNF KO MΦ
TNF KO MΦ
Macrophages + LVS
+ Naive spleen
TNF KO MΦ
F I g
TNF KO MΦ
E IFN- γ γ
+ Primed whole spleen
F IFN- γ γ
Macrophages + LVS
+ Naive spleen
+ Primed whole memTNF spleen
+ memTNF CD4
+ memTNF CD8
TNF KO MΦ
macrophage growth or NO production. Ability of various T cell subsets
isolated from LVS-immune WT (A) and memTNF (B) mice to control LVS
growth in WT (A), memTNF (B), or TNF KO (A and B) BMM?. Infected
BMM? were cocultured with splenocytes obtained from either uninfected
mice (naive spleen), whole primed splenocytes from immune WT or
memTNF mice (primed spleen), or various T cell subsets enriched from
immune WT or memTNF mice (CD4?cells, CD8?cells). All splenocyte
populations were added at a 1:2 ratio (splenocyte to BMM?). Seventy-two
hours after infection, the BMM? were assessed for levels of intracellular
Macrophage TNF is not required for control of LVS intra-
LVS growth. Values shown are the mean numbers of CFU per milliliter ?
the SEM of viable bacteria (triplicate samples). C–H, Secretion of NO and
cytokines into culture supernatants following coculture of LVS-infected
TNFR1/2 KO BMM? with WT and memTNF LVS-immune splenocytes.
Culture supernatants were collected from the WT, memTNF, and TNF KO
BMM? cocultures after 72 h, and assessed for NO (C and D), IFN-? (E and
F), and IL-12(p40) (G and H). Values shown are the mean micromoles per
milliliter of nitrite (C and D) or the mean nanograms per milliliter ? the
SEM of the indicated cytokine (E–H) (triplicate samples). Asterisks (?)
indicate p values ?0.05 of cocultures containing TNF KO BMM? as com-
pared with their cognate cocultures containing either WT (C and E) or
memTNF (F) BMM?. These results are representative of three experi-
ments of similar design.
7716T CELL memTNF CONTROLS Francisella INTRAMACROPHAGE GROWTH
cells may act via mechanisms other than NO. Cell surface TNF has
been shown to mediate cytotoxic activities by CD4?T cells, as
measured via L929 fibroblast lysis assays (25) and thus can act via
mechanisms other than up-regulation of NO. Furthermore, TNF in
combination with IFN-? can synergistically activate a diverse
number of other macrophage antimicrobial activities, such as ex-
pression of the tryptophan-limiting enzyme indolamine 2, 3-oxy-
genase (26, 27), or induction of apoptosis (28). The combined
action of these two cytokines may also redirect the intracellular
trafficking of LVS to a compartment that does not support repli-
cation of the organism. The identity of other possible macrophage
mechanisms that limit LVS intracellular growth will be the subject
of ongoing studies.
The in vivo role of memTNF during primary infection has re-
cently been explored in the M. tuberculosis, L. monocytogenes,
and F. tularensis LVS murine infection models. In all cases, al-
though memTNF contributed to resistance to infection, mice ex-
pressing only memTNF were more susceptible to infection than
WT mice. The increased susceptibility of the memTNF mice to
Listeria and Mycobacterium infections was attributed to a require-
ment for soluble TNF to regulate chemokine expression and co-
ordinate an effective inflammatory response (29–31). In the pres-
ence of only memTNF, there was significant overproduction of
chemokines during primary infection. Transient overproduction of
IL-12(p40) was also observed in M. tuberculosis-infected
memTNF mice (32, 33). This dysregulated inflammatory response
was the likely cause of increased inflammatory lesions containing
neutrophils and necrotic tissue destruction in the lung (Mycobac-
terium) or liver (Listeria) of infected animals, resulting in mortal-
ity. Collectively, these studies indicate that soluble TNF has a
regulatory function that limits excessive TH1 proinflammatory re-
sponses during primary intracellular infections.
The role of memTNF during Mycobacterium and Listeria
secondary challenges has also been explored (29, 30). Similar to
the results observed here (Fig. 1), Listeria-primed memTNF
mice were fully protected against a lethal secondary Listeria chal-
lenge (29). Furthermore, in both the Mycobacterium and Listeria
murine models, adoptive transfer of immune WT or memTNF-
expressing T cells to TNF KO mice was sufficient to confer pro-
tection against challenge (29, 30), indicating that, similar to our in
vitro observations, macrophage-derived TNF is not essential for T
cell-mediated control of bacterial intracellular growth (Fig. 6, A
and B). Thus, in all three of the Mycobacterium, Listeria, and
Francisella murine infection models, memTNF is capable of me-
diating full resistance to secondary challenges. Here, we further
demonstrate that there is a defect in the regulation of the IFN-?-
IL-12 axis, as evidenced by overproduction of both cytokines in
the memTNF mice as compared with the WT mice. This defect
was observed in vivo (Fig. 1, C and D), as well as in vitro (Fig. 3,
B and C), and was not specifically explored in either the Myco-
bacterium or Listeria secondary challenge memTNF murine stud-
ies. The results presented here demonstrate that soluble TNF is an
important component in the regulation of the IL-12-IFN-? axis
during secondary LVS infection.
Dysregulation of the IL-12-IFN-? circuit can have deleterious in
vivo effects, artificially inflating the proinflammatory response. In-
deed, overproduction of these cytokines, in the presence and ab-
sence of TNF, can affect survival during infection: depletion of T
cells from TNF KO mice in the Mycobacterium bovis bacillus
Calmette-Gue ´rin infection model prolonged survival by limiting
excessive Th1-type inflammatory responses (34, 35). Acute toxo-
plasmosis is accompanied by overproduction of TH1 cytokines
that is ultimately lethal (35). Similarly, it is well-known that pa-
tient therapies using excessive IL-12 treatment can be detrimental
to the host (36). Additionally, TNFR1 KO mice were acutely sus-
ceptible to Mycobacterium avium infection in a manner that was
dependent on T cells and excessive production of IL-12 (37). Most
of these examples describe primary exposures to infectious agents,
where the levels of IFN-? and IL-12 are significantly higher than
those observed here during secondary LVS challenge. Thus, be-
cause the secondary LVS infection was quickly resolved in the
memTNF mice, and the levels of IFN-? measured were low as
compared with primary LVS infections, the observed disruption of
the IL-12-IFN-? circuit was not detrimental to the host. Although
we did not examine the effect of IL-12-IFN-? dysregulation on
pathology in this study, it clearly did not have an impact on opti-
mal protection against LVS secondary challenge. The results pre-
sented here underscore the differential requirements for key cyto-
kines such as IFN-?, IL-12, and TNF during primary and
secondary intracellular infections, and the need to explore both
types of infections for a complete understanding of immunity to
TNF has a critical role in activating macrophages to control
LVS intramacrophage growth. For example, in vitro, the addition
of neutralizing anti-TNF Abs to LVS-infected macrophages abol-
ished the ability of IFN-? treatment to induce NO production and
control intracellular growth (5, 23). Similar in vitro observations
have been made for other intracellular pathogens, including Leish-
mania, Listeria, and Mycobacterium species (38–40). Thus, one
important role of TNF in intracellular infections is the direct con-
trol of bacterial intramacrophage growth via NO production. Here,
we demonstrate that although memTNF can induce macrophage
production of NO through stimulation of macrophage TNFRs, it is
not as efficient as soluble TNF (Figs. 2B and 5, A and B). Previous
studies have shown both reduced and normal levels of inducible
NO synthase activity in the spleens of memTNF mice given a
primary i.v. bacillus Calmette-Gue ´rin infection (32, 33). Other
studies have demonstrated overproduction of inducible NO syn-
thase mRNA in memTNF mice during Listeria primary infection
(29). This is the first study to show directly that T cell-derived
memTNF is less efficient at activating macrophages to produce NO
in the absence of soluble TNF during intracellular infection.
The differential abilities of memTNF and soluble TNF to control
intracellular infections has important implications for anti-TNF
treatment therapies that are currently on the market. Treatment
regimens that target TNF function have been highly successful in
improving chronic inflammatory diseases such as rheumatoid ar-
thritis and Crohn’s disease. However, patients receiving these
treatments have an increased occurrence of granulomatous infec-
tions such as M. tuberculosis, L. monocytogenes, and Histoplasma
capsulatum (41). Tularemia is not included in this list, likely
because it is not a common public health problem. However, F.
tularensis is an intracellular pathogen that causes granulomatous
lesions in humans, and the results obtained here are likely repre-
sentative of other similar intracellular pathogens. Infliximab is a
chimeric anti-TNF mAb that binds both soluble and membrane
TNF (42). In contrast, etanercept is a dimeric fusion protein con-
sisting of the extracellular portion of TNFRp75 linked to the Fc
domains of human IgG1, binding mostly soluble trimeric TNF, and
exhibiting low avidity for memTNF (42). As shown here, complete
neutralization of TNF with an anti-TNF mAb significantly dimin-
ishes the ability of LVS-immune CD4?and CD8?T cells to con-
trol Francisella infection. However, cultures containing only
memTNF still maintain control of LVS intracellular growth in
vitro. In keeping with these observations, the highest incidences of
infectious complications are associated with infliximab, which
binds both soluble and membrane TNF (43). Although LVS-im-
mune T cells expressing memTNF can control LVS growth in vitro
7717The Journal of Immunology
and in vivo, the observed dysregulation of IFN-? and IL-12 pro-
duction could lead to unexpected complications in vivo, even when
memTNF is available.
Identification of correlates of protection during T cell-mediated
protective immune responses to intracellular pathogens is a major,
and thus far elusive, goal. Although IFN-? is often proposed as a
correlate, the bulk of data accumulated to date suggest that vac-
cine-induced IFN-? production may be a necessary, but not suf-
ficient, indicator of secondary protection (44). Frequently, the
presence of IFN-? either in sera or in stimulated culture superna-
tants does not reliably predict immune status. The studies pre-
sented here clearly demonstrate that T cell, but not macrophage,
expression of memTNF is critical to the ability of immune T cells
to control and eliminate intracellular bacterial growth, particularly
for CD8?T cells. Whether memTNF is typically used by T cells
to control LVS intracellular growth, or whether it is only func-
tionally important in the absence of soluble TNF, remains to be
determined. Production of TNF has also been proposed as a po-
tential alternate or complementary T cell correlate (45). The find-
ings presented here have important implications for the application
of TNF as a correlate; measurement of memTNF is much more
difficult technically than measurement of secreted TNF (46). Se-
creted TNF quantities may not reflect the most relevant T cell
activities, but thus far attempts to measure cell surface TNF on
activated LVS-immune T cells using flow cytometry have been
inconclusive (data not shown). Therefore, future studies will be
directed at efforts to quantitate each form of this important medi-
ator, with a view toward facilitating better understanding of the
relative importance of membrane and soluble TNF, and determin-
ing its use as a practical correlate of protection for intracellular
The authors have no financial conflict of interest.
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