Download IFN-g from CD4 T Cells Is Essential for Host Survival and Enhances

IFN-γ from CD4 T Cells Is Essential for Host
Survival and Enhances CD8 T Cell Function
during Mycobacterium tuberculosis Infection
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J Immunol 2013; 190:270-277; Prepublished online 10
December 2012;
doi: 10.4049/jimmunol.1200061
http://www.jimmunol.org/content/190/1/270
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References
Angela M. Green, Robert DiFazio and JoAnne L. Flynn
The Journal of Immunology
IFN-g from CD4 T Cells Is Essential for Host Survival and
Enhances CD8 T Cell Function during Mycobacterium
tuberculosis Infection
Angela M. Green, Robert DiFazio, and JoAnne L. Flynn
T
uberculosis continues to be a global health crisis, causing
∼1.7 million deaths per year. Lack of an effective vaccine and long treatment regimens with multiple chemotherapeutic agents make it imperative that research focuses on
understanding the immune response to infection. The proinflammatory cytokine IFN-g promotes the development of a Th1
T cell response (1). In addition, it synergizes with TNF to activate macrophages (Mf), promoting the induction of NO synthase 2 (NOS2), which participates in killing of Mycobacterium
tuberculosis (2). Humans with loss-of-function genetic mutations in either IFN-g or its receptor are very susceptible to
mycobacterial infections (3). Mice that lack IFN-g or NOS2 are
two of the most vulnerable strains, failing to control M. tuberculosis and succumbing within weeks of challenge (4, 5). Thus,
IFN-g is required for containment of M. tuberculosis infection
and host survival.
CD4 T cells are a primary source of IFN-g during the adaptive
immune response to M. tuberculosis infection and are required
for host survival during both the acute and the chronic stages of
infection (6, 7). CD42/2 and MHCII2/2 mice are unable to control
bacterial growth and succumb to infection significantly sooner
than wild-type (WT) counterparts (6), yet these mice survive at
Department of Microbiology and Molecular Genetics, University of Pittsburgh
School of Medicine, Pittsburgh, PA 15261
Received for publication January 6, 2012. Accepted for publication October 25,
2012.
This work was supported by National Institutes of Health Grant 2RO1AI50732-06A2
(to J.L.F.) and Grant T32 AI060525-02/03 (to A.M.G.).
Address correspondence and reprint requests to Prof. JoAnne L. Flynn, Department
of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine,
200 Lothrop Street, W1144 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail
address: [email protected]
Abbreviations used in this article: CTLAT, control group lacking CD4 T cells;
GammaAT, CD4 T cells from IFN-g2/2 mice; GAPtet, GAP tetramer; IBD, inflammatory bowel disease; Mf, macrophage; MFI, mean fluorescence intensity; NOS, NO
synthase; p.i., postinfection; WT, wild-type; WTAT, CD4 T cells from WT mice.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200061
least twice as long as those that lack IFN-g or NOS2 (4–6, 8).
Lack of CD4 T cells during initial infection results in delayed
IFN-g and NOS2 production, but eventually levels comparable
with WT are reached, because other cells can produce this cytokine. However, this did not rescue the CD4 T cell–deficient mice
from the infection (6). When CD4 T cells are depleted during
chronic infection, exponential bacterial growth and eventual host
death occur, despite maintenance of predepletion levels of IFN-g
and NOS2 (7).
As Th1 cells, CD4 T cells also produce IL-2 and TNF, interact
with dendritic cells to help with T cell priming, and provide T cell
help to B cells (9). CD8 T cells, as well as other cells, can and do
produce IFN-g during M. tuberculosis infection. Recent work by
Gallegos et al. supports that CD4 T cells have IFN-g–independent
mechanisms of controlling M. tuberculosis infection in vivo (10).
Taken together, these data suggest that CD4 T cells have roles in
addition to IFN-g production. These data lead to the hypotheses
that IFN-g from sources other than CD4 T cells is sufficient for
bacterial containment, and that CD4 T cells have functions in
addition to IFN-g production.
For decades, knockout and transgenic animals, as well as Ab
depletion, have been powerful tools for determining which immune
factors are necessary for control of M. tuberculosis. However, these
techniques are limited by studying the global effect of these immune mediators, and it can be difficult to determine which specific
function of a cell type, for example, is causing the in vivo phenotype in a murine model. To determine whether IFN-g from
sources other than CD4 T cells were sufficient to contain bacterial
growth, a new model system was needed. An adoptive transfer
model was designed to permit manipulation of the CD4 T cell
population. The data from this novel adoptive transfer system
indicate that CD4 T cells are necessary as a source of IFN-g, as
well as influencing the function of CD8 T cells in the immune
response against M. tuberculosis. The adoptive transfer systems
developed provide an opportunity to manipulate the immune response to identify specific factors important in control of M. tuberculosis infection.
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IFN-g is necessary in both humans and mice for control of Mycobacterium tuberculosis. CD4 T cells are a significant source of IFNg during acute infection in mice and are required for control of bacterial growth and host survival. However, several other types of
cells can and do produce IFN-g during the course of the infection. We sought to determine whether IFN-g from sources other than
CD4 T cells was sufficient to control M. tuberculosis infection and whether CD4 T cells had a role in addition to IFN-g production.
To investigate the role of IFN-g from CD4 T cells, a murine adoptive transfer model was developed in which all cells were capable
of producing IFN-g, with the exception of CD4 T cells. Our data in this system support that CD4 T cells are essential for control of
infection, but also that IFN-g from CD4 T cells is necessary for host survival and optimal long-term control of bacterial burden. In
addition, IFN-g from CD4 T cells was required for a robust CD8 T cell response. IFN-g from T cells inhibited intracellular
replication of M. tuberculosis in macrophages, suggesting IFN-g may be necessary for intracellular bactericidal activity. Thus,
although CD4 T cells play additional roles in the control of M. tuberculosis infection, IFN-g is a major function by which these
cells participate in resistance to tuberculosis. The Journal of Immunology, 2013, 190: 270–277.
The Journal of Immunology
Materials and Methods
Animals
Eight- to 12-wk-old Helicobacter pylori–free RAG12/2 (B6.129S7Rag1tm1Mom/J), Thy1.1 (B6.PL-Thy1a/CyJ), IFN-g2/2 (GKO) (B6.129S7Ifngtm1Mom/J), IFN-gR2/2 (B6.129S7-Ifngrtm1agt/J), and WT C57BL/6 mice
were obtained from Jackson Laboratories (Bar Harbor, ME) or maintained
in an in-house breeding facility. Mice were maintained in a biosafety level
3 facility in microisolator cages and free fed a diet of mouse chow and
autoclaved water. All animals were maintained as per the University of
Pittsburgh Institutional Animal Care and Use Committee.
Probiotic treatment of RAG 12/2 mice
Initially, we observed development of reconstitution-induced inflammatory bowel disease (IBD) (11–13) in our RAG 12/2 mice after T cell
transfer but before M. tuberculosis challenge. After investigating several
possible factors responsible for this syndrome, we treated RAG 12/2 mice
prophylactically with probiotics, which eliminated the signs of IBD. Before adoptive transfer, mice received one scoop (∼0.25 g) of Bene-Bac
Powder (25 million CFU/g, Lactobacillus fermentum, Enterococcus faecium,
Lactobacillus plantarum, Lactobacillus acidophilus; Pet Ag, Hampshire, IL)
in 350 ml fresh, autoclaved water every day for 14 d. Water bottles were
changed daily.
Thy1.1 mice were pretreated with 1 mg/mouse anti-CD4 Ab (clone GK1.5;
National Cell Culture Center, Minneapolis, MN) 7 d before splenocyte
harvest. GKO and WT mice did not receive pretreatment with Ab. Naive
FIGURE 1. Experimental design. (A) RAG12/2
mice were reconstituted with CD4-depleted
splenocytes (Thy1.1) in the presence or absence (CTLAT) of CD4 T cells from either
C57BL/6 (WTAT) or IFN-g–deficient (GammaAT) mice. (B) Before adoptive transfer,
RAG12/2 mice were treated with probiotics to
prevent development of IBD (see Materials and
Methods). Cells were adoptively transferred and
mice allowed to reconstitute for 2.5 wk. Mice
were infected via aerosol with low-dose M. tuberculosis and harvested at serial time points. The
experiments were repeated at least three times.
(C) Representative flow cytometry plots of CD4
and CD8 T cells are shown for each experimental
group (side scatter [SSC] versus CD4 or CD8).
Representative plots for CD4 versus IFN-g and
CD8 versus IFN-g after stimulation with antiCD3/CD28 in the presence of monensin. (D) The
frequency of CD4 and CD8 T cells within the live
cell gate. (E) The frequency of Mfs (CD11b+
CD11cdim), dendritic cells (CD11c+CD11b2),
and neutrophils (GR-1+) within the live cell gate.
*p , 0.05, **p , 0.01, ***p , 0.001, Student
t test.
donor mice were anesthetized by isoflurane and euthanized by cervical
dislocation. Spleens were isolated under sterile conditions. A single-cell
suspension was obtained by crushing each spleen individually through
a 40-mm cell strainer in Dulbecco’s PBS (Sigma-Aldrich, St. Louis, MO)
with the back of a sterile 5-ml syringe. Erythrocytes were lysed with RBC
lysis buffer (90 ml 0.16 M NH4, 10 ml 0.17M Tris pH 7.65) for 2 min at
room temperature. After erythrocyte lysis and wash, remaining splenocytes
from like animals were combined. Individual cell populations were isolated via Miltenyi MACS bead separation (Miltenyi Biotech, Auburn, CA)
as per manufacturer’s instructions. CD4+ cells were positively selected
from either WT or GKO mice, and naive Thy1.1 mice were depleted of
CD4 T cells by Ab neutralization; then CD4 T cells were positively selected T cells by magnetic bead separation as per manufacturer’s protocol
before adoptive transfer. Recipient RAG12/2 mice received purified
splenocytes (1.8 3 108 cells total 1:20 CD4 T cells to CD4-depleted
splenocytes) via a tail vein injection in 300 ml PBS per mouse.
Aerosol infection
As previously described (5), M. tuberculosis strain Erdman was passed
through mice, grown in culture once, and frozen in aliquots in PBS 0.05%
Tween 80 with 10% glycerol. Immediately before infection, an aliquot was
thawed and diluted in PBS 0.05% Tween 80. Bacterial clumps were disaggregated by cup horn sonication. Mice were aerosol infected with low
dose (∼50 CFU, Erdman strain) using a nose-only exposure aerosolizer
(InTox Products, Albuquerque, NM) (5). Mice were exposed to aerosolized
M. tuberculosis for 20 min, then room air for 5 min. One day postinfection
(p.i.), lungs of one mouse per aerosol group were crushed in 5 ml PBS0.5% Tween 80 and plated neat on 7H10 or 7H11 plates (Difco Laboratories, Detroit, MI) to determine inoculum.
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Isolation of cells and adoptive transfer
271
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IFN-g FROM CD4 T CELLS CONTROLS M. TUBERCULOSIS INFECTION
Sample harvest
At serial time points p.i., mice were anesthetized with isoflurane and euthanized by cervical dislocation. Under sterile conditions, organs were
crushed in DMEM (Sigma-Aldrich) through a 40-mm cell strainer with the
plunger from a sterile 5-ml syringe. Dilutions of cellular homogenate were
plated on 7H10 agar plates for CFU determination. Cells were pelleted and
washed, erythrocytes were lysed as described previously (14), and cells
were resuspended for enumeration and immunologic assays.
Flow cytometry
Histology sections
At necropsy, one quarter of lung was fixed in 10% normal buffered formalin,
paraffin imbedded, and sectioned. Slides were stained with H&E. Granuloma formation was evaluated by two independent investigators in a blinded fashion.
Intracellular bactericidal assays
Bone marrow–derived Mfs were obtained by culturing bone marrow from
the long bones of mice with 25% L929-cell supernatant containing media for
5 d in 10-cm culture dishes (2.5 3 106 cells/plate in 10 ml media). Cells by
this time had formed a monolayer of Mfs. Mfs were removed by incubation
with PBS on ice for 20 min followed by vigorous pipetting to dislodge any
remaining adherent cells. Cells were pelleted, resuspended, counted, and
diluted to a concentration of 2.5 3 107 cells/ml with Mf infection media
(DMEM, 1% FBS, 1% sodium pyruvate, 1% L-glutamine, 1% nonessential
amino acids). Mfs were plated at 100ml/well in a 96-well plate and allowed
to adhere for 45 min. Subsequently, Mfs were infected with M. tuberculosis
at a multiplicity of infection #1 for 4 h at 37˚C 5% CO2. p.i., supernatant
from three wells from both WT and IFN-gR2/2 Mfs was saved and the Mfs
in all wells washed. Three wells per each Mf type were lysed with 1%
saponin, and the saved supernatant and Mf lysis were diluted and plated on
7H10 plates to determine the “input” bacterial numbers.
T cells were obtained from the lungs of WT mice infected with M.
tuberculosis for 4 wk for culture with infected Mfs. Infected mice were
anesthetized with isoflurane and euthanized by cervical dislocation. Lungs
were retrieved under sterile conditions and crushed through a 40-mm cell
strainer with the back of a sterile 5-ml syringe. Once a single-cell suspension was obtained and erythrocytes lysed, CD4 and CD8 T cells were
purified by magnetic bead separation (Miltenyi) as per manufacturer’s
instructions. T cells were incubated in wells with infected Mfs at a 1:1
ratio of T cell to Mf (n = 3 wells per condition). Medium without T cells
in wells with infected Mfs was used as a control for growth of M. tuberculosis. As a positive control, wells were treated with IFN-g (250 U/ml;
Invitrogen) and LPS (3 mg/ml; Sigma-Aldrich). Cells were incubated for
72 h at 37˚C 5% CO2. After incubation, supernatant was removed and
saved, and cells were lysed with 1% saponin. Supernatants and cell lysates
were serially diluted and plated on 7H10 agar plates for determination of
bacterial burden per well. Data are reported as a percentage killed intracellular bacteria ([output CFU/input CFU] 3 100).
Results
Adoptive transfer model
An adoptive transfer model was developed so that the presence or
absence of CD4 T cells from IFN-g2/2 or WT mice could be
FIGURE 2. Granuloma in lungs of reconstituted mice. Four weeks
p.i., one quarter of the lung was fixed, paraffin embedded, and sectioned
for H&E staining. Representative sections for each group are shown at
original magnification 34 and 320.
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For Ag-specific responses and intracellular cytokine staining, cells were
incubated with 1 ml of either ESAT61–20aa MHC class II multimer or GAP
MHC class I tetramer (Mtb32aa309–318, GAPINSATAM; National Institute
of Allergy and Infectious Diseases tetramer facility, Bethesda, MD) (15) per
100 ml at 37˚C 5% CO2 for 60 min in media, then washed. Once stained
with tetramers, cells were stimulated with either ESAT61–20aa peptide or
GAP peptide in the presence of monensin for 5 h (16). After staining with
GAP tetramer, anti-CD107 (1D4B) Ab was added during stimulation to
assess degranulation. After stimulation, cells were surfaced stained for
CD3, CD4 (clone L3T4), and CD8 (clone 53-6.7) at room temperature for
15 min in PBS 0.5% BSA 20% mouse serum, washed, and then fixed with
2% paraformaldehyde (Sigma-Aldrich) for 1 h. After fixation, cells were
permeabilized, then washed with PBS 0.5% BSA 0.2% saponin (SigmaAldrich). Cells were stained for intracellular cytokines IFN-g (clone
XMG-6.1) and TNF (clone MP6-XTT22) by incubating Abs in PBS 0.5%
BSA 0.2% saponin and 20% mouse serum for 15 min at room temperature.
All Abs were from BD Pharmingen unless otherwise noted. Cells were
washed and resuspended in 1% paraformaldehyde. Data were collected on
a FACSAria and analyzed using FloJo software 8.6.3 (Tree Star).
evaluated for ability to control M. tuberculosis infection. Three
experimental groups were compared: 1) a control group lacking
CD4 T cells (CTLAT), 2) a group in which CD4 T cells were from
IFN-g2/2 mice (GammaAT), and 3) a group in which CD4 T cells
were from WT mice (WTAT). In addition, the remainder of the
immune system components was capable of producing IFN-g.
RAG12/2 mice were reconstituted with and without CD4 T
cells from WT or IFN-g–deficient mice in conjunction with CD4depleted whole spleen equivalents. The whole spleen equivalents
were derived from CD4-depleted naive Thy1.1 mice and adoptively
transferred at a ration of 20:1 (CD42 splenocytes to CD4 T cells;
Fig. 1A). Reconstitution of lymphopenic mice often results in
homeostatic proliferation of the transferred cells and occasionally
mice develop IBD. Thus, distinguishing between M. tuberculosis–
specific responses and the aforementioned conditions can be difficult (13). Prophylactic probiotic treatment prevented the development of IBD (11, 12). Mice received cells 2.5 wk before aerosol
infection, enabling differentiation between homeostatic proliferation and the immune response to M. tuberculosis (Fig. 1B) (13).
To determine whether reconstitution of lymphopenic RAG12/2
mice was effective, we examined the lungs of mice in our experimental groups at 4 wk p.i. (6.5 wk posttransfer). Evaluation of the
cellular composition in infected lung showed both CD4+ and CD8+
T cells were detectable in the lungs of GammaAT and WTAT,
whereas only CD8 + T cells were found in the lungs of CTLAT
mice (Fig. 1C). When stimulated with anti-CD3 and anti-CD28 in
the presence of monensin, CD8 T cells were able to make IFN-g in
all groups, whereas only CD4 T cells in the WTAT group made
IFN-g (Fig. 1 C). In mice that received CD4 T cells, the frequency
of both CD4 and CD8 T cells (Fig. 1D) were comparable. CTLAT
mice (those that lack CD4 T cells) had a significantly higher frequency of CD8 T cells in the lungs compared with WTAT or
GammaAT (Fig. 1D). Finally, analysis of the monocyte populations
(Mfs, dendritic cells, and neutrophils; Fig. 1E) showed no significant differences in the frequency between reconstituted groups.
These data indicate that experimental animals were successfully
The Journal of Immunology
273
reconstituted and recruit a similar assortment of cell types to the
lung during infection.
Reconstituted mice form granulomas by 4 wk p.i.
Granuloma formation is the hallmark of M. tuberculosis infection,
as well as an integral part by which the host controls infection.
Lung sections from infected mice were evaluated at 4 wk p.i. for
granuloma formation by two independent investigators in a blinded fashion. Representative H&E sections found in Fig. 2 show
granulomas were present in all groups regardless of the presence
of CD4 T cells. Although WT mice had more circumscribed
granulomas, no differences were detected between the recon-
FIGURE 4. Adoptively transferred CD4 T cells produce cytokines in the lungs in response to M. tuberculosis Ags. Cells from lung homogenates were
stained with ESAT6 tetramer and stimulated with ESAT6 peptide (aa 1–20) for 5 h in monensin, then for CD4 and Thy1.2, and intracellular cytokines IL-2,
IFN-g, and TNF. (A) Representative plot: cells are gated on lymphocytes by size (forward and side scatter), then on Thy1.2, then on CD4 and expressed as
CD4 versus ESAT6 tetramer. (B) GammaAT and WTAT have similar frequencies of ESAT6 tetramer+ cells in lungs at 4 wk p.i. (C) Only WTAT mice
produce IFN-g from CD4 T cells. GammaAT and WTAT CD4 T cells produce TNF (D) and IL-2 (E). As expected, there are no CD4 T cells in the CTLAT
lungs. n = 4 mice/group.
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FIGURE 3. CD4 T cells producing IFN-g are needed for long-term survival of M. tuberculosis infection. (A) Adoptively transferred mice were followed
for survival p.i. n = 5/group; p = 0.001, log-rank test. (B) Bacterial burden (CFU) in the lungs of adoptively transferred mice after M. tuberculosis infection,
week 6 (n 5 4/group/time point): ANOVA p = 0.002, Tukey’s multiple comparisons **p , 0.01 CTLAT versus GammaAT, *p , 0.01 CTLAT versus
WTAT; week 10: Student t test GammaAT versus WTAT, **p = 0.01. (C) Total number of cells in lungs at 4 wk p.i. Each symbol is one mouse from each
group. ANOVA p = 0.002, Tukey’s multiple comparisons ***p , 0.001, CTLAT versus GammaAT, ***p , 0.001 CTLAT versus WTAT. Experiments were
repeated at least three times.
274
IFN-g FROM CD4 T CELLS CONTROLS M. TUBERCULOSIS INFECTION
stituted groups when compared with each other. Thus, neither
CD4 T cells nor IFN-g from CD4 T cells was required for initial
granuloma formation.
IFN-g from CD4 T cells is necessary for control of bacterial
burden and host survival
In general, in M. tuberculosis–infected mice, higher bacterial
burdens result in larger numbers of cells in the lungs (17, 18), as
an indication that bacterial burden drives recruitment of cells to
the lung and detrimental pathology. To further characterize the
immune response, we focused on the peak of the immune response, 4 wk p.i. At 4 wk p.i., mice that lack CD4 T cells had
fewer total cells recovered from the lungs than those with CD4
T cells regardless of whether the CD4 T cells could produce IFN-g
(Fig. 3C), suggesting that CD4 T cells are needed to sustain the
local immune response.
Ag-specific CD4 T cells are functional in reconstituted mice
In addition to IFN-g production, Th1 CD4 T cells also produce
IL-2 and TNF. To determine whether CD4 T cells were functional
and produced cytokine in response to Ag-specific stimulation, we
assessed CD4 T cells from lungs for ESAT6-specific responses by
tetramer and intracellular cytokine staining (representative plots
shown in Fig. 4A). ESAT6tet+ CD4 T cells were detected in both
GammaAT and WTAT groups and, as expected, not in the group
that did not receive CD4 T cells (CTLAT; Fig. 4B). After in vitro
stimulation, similar frequencies of CD4 T cells from both GammaAT and WTAT groups produced TNF (Fig. 4D) and IL-2 (Fig.
4E). In addition, CD4+ESAT6tet+ cells produced similar amounts
of both IL-2 and TNF when mean fluorescence intensity (MFI)
was measured (data not shown). Only CD4 T cells from WTATreconstituted mice produced IFN-g (Fig. 4C). Thus, CD4 T cells
were functional in the adoptive transfer model and produced
cytokines in an Ag-specific manner.
FIGURE 5. CD8 T cell function is impaired in the absence of IFN-g from CD4 T cells. Lung homogenates at 4 wk p.i. were stimulated with GAPtet and
GAP peptide and CD107 in the presence of monensin. After stimulation, cells were stained for CD8, Thy1.1, and intracellular cytokines TNF and IFN-g.
(A) Representative plots: live cell gate, lymphocyte gate, Thy1.1, CD8+ gate, and GAPtet+ within the CD8+ gate. (B) WTAT lungs have more CD8+GAPtet+
cells compared with CTLAT lungs; ANOVA p = 0.01, Tukey’s multiple comparisons **p , 0.01 CTLAT versus WTAT. (C) Number of CD8+ GAPtet+IFNg+ T cells in lungs of WTAT, GammaAT, and CTLAT mice. ANOVA p = 0.015, Tukey’s multiple comparisons *p , 0.05 CTLAT versus WTAT. (D)
Number of CD8+GAPtet+CD107+ cells in lungs; ANOVA p = 0.008, Tukey’s multiple comparisons *p , 0.05 WTAT versus CTLAT and GammaAT versus
WTAT. (E) CD8+GAPtet+TNF+ cells in lungs; ANOVA p = 0.015, Tukey’s multiple comparisons *p , 0.05 CTLAT versus WTAT.
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CD4 T cells are the primary source of IFN-g during early M.
tuberculosis infection (14). However, other cells, including CD8
T cells and NK cells, can and do produce IFN-g. For this study,
because T cells are the main source of IFN-g during M. tuberculosis infection, we chose to focus on production of IFN-g by
T cells on host survival and bacterial control. To determine
whether IFN-g from sources other than CD4 T cells was sufficient
to control M. tuberculosis infection, we followed the reconstituted
mice for bacterial burden in the lungs and monitored them for
survival (i.e., mice were euthanized when moribund). Mice
reconstituted with WT CD4 T cells (WTAT group) survived longer
than either the CTLAT or GammaAT groups (∼210 versus 45 and
135 d p.i., respectively; Fig. 3A). In the absence of CD4 T cells,
CTLAT mice failed to control bacterial burden (Fig. 3B) and did
not survive longer than mock reconstituted RAG12/2 mice (data
not shown). WTAT mice maintained the lowest lung bacterial
burden for the duration of the experiment (Fig. 3B). Interestingly,
when CD4 T cells were present but unable to produce IFN-g
(GammaAT), infected animals were able to control bacterial
burden and survive for a longer period of time, that is, more than
twice as long as animals that did not have CD4 T cells. Taken
together, these data suggest that, although IFN-g from CD4 T cells
is necessary for long-term control of infection and host survival,
CD4 T cells may have roles in addition to IFN-g production that
contribute to the protective immune response to M. tuberculosis.
Lack of CD4 T cells results in fewer cells recovered from the
lungs p.i.
The Journal of Immunology
Quality of the CD8 T cell response is truncated in the absence
of IFN-g from CD4 T cells
Studies of M. tuberculosis infection in CD42/2 and MHCII2/2
mice have shown that CD8 T cells can be a significant source of
IFN-g (6, 14). Previously work published by our laboratory has
275
shown that the cytotoxic function of CD8 T cells is not as robust
in the absence of CD4 T cells (14). In the adoptive transfer model,
we sought to determine whether the quality of the CD8 T cell
response generated was directly influenced by the presence of
CD4 T cells. Ag-specific CD8 T cells were identified by staining
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FIGURE 6. In the absence of IFN-g, the M. tuberculosis–specific CD8 T cell response is truncated. (A) Experimental design: RAG12/2 mice were
reconstituted with CD8-depleted Thy1.1 splenocytes in the presence of CD8 T cells from IFN-gR2/2 mice and compared against WTAT (previously
described in Fig. 1) at 4 wk p.i. RAG12/2 mice received naive CD8 T cells from either WT or IFN-gR2/2 mice in combination with Thy 1.1 CD8-depleted
naive splenocytes. CD8 T cells from experimental design shown in (A) were evaluated for (B) IFN-g (Student t test, ***p , 0.0001), (C) CD107+ (Student t
test, **p , 0.001), and (D) TNF (Student t test, ***p , 0.0001) production. The experiment was repeated as shown in the bottom panel in (A). RAG2/2
mice received CD8 T cells from either WT or IFN-gR2/2 mice in combination with CD8-depleted Thy1.1 naive splenocytes. CD8 T cells from experimental design shown in the bottom panel in (A) were analyzed for (E) IFN-g, (F) CD107, and (G) TNF. Experiments were completed at least two times.
Student t test, ***p , 0.001.
276
IFN-g FROM CD4 T CELLS CONTROLS M. TUBERCULOSIS INFECTION
IFN-g from CD4 enhances the quality of the CD8 T cell
responses
In the absence of IFN-g from CD4 T cells, fewer CD8 T cells were
present, and those present were less capable of producing cytokine
or exhibiting makers of cytotoxicity. Recent work has shown that
CD4 T cells are necessary for sustained IFN-g production during
M. tuberculosis infection (20). We hypothesized that IFN-g directly affected development of CD8 T cell responses, as has been
shown in a viral infection model (21). To test this hypothesis, we
added an additional experimental group in tandem with the previously described adoptive transfer groups. In the additional group,
RAG12/2 mice were reconstituted with CD8-depleted Thy1.1
splenocytes and CD8 T cells from IFN-gR2/2 mice and compared
with the Ag-specific CD8 T cell responses observed in the previously described WTAT group (Fig. 6A). The CD8 T cell response
was analyzed at 4 wk p.i. as previously described in Fig. 5. In the
absence of IFN-gR, fewer Ag-specific CD8 T cells produced IFN-g
(Fig. 6B), TNF (Fig. 6D), and CD107+ (Fig. 6C), suggesting that
IFN-g directly affects the quality of the CD8 T cell response during
M. tuberculosis infection. To confirm these results of the experiment and control for any effect CD8 T cell manipulation may
present, we reconstituted RAG12/2 mice with CD8-depleted Thy1.1
splenocytes and CD8 T cells from either WT or IFN-gR2/2 mice
(Fig. 6B). Interestingly, the trend remained the same. When CD8+
GAPtet+ T cells were unable to respond to IFN-g signaling, fewer
cells produced IFN-g (Fig. 6E), TNF (Fig. 6G), or were CD107+
(Fig. 6F), thus suggesting that IFN-g may act directly on CD8
T cells to enhance the quality of the response.
T cell:Mf cell-to-cell contact–mediated killing of intracellular
bacteria is enhanced by IFN-g signaling
T cells that produce IFN-g are capable of interacting with Mfs,
via cytokine production (i.e., IFN-g) and cell-to-cell contact. IFNg can synergize with TNF to activate the Mf and mobilize
mechanisms of intracellular killing. A recent report by Gallegos
et al. (10) indicates that CD4 T cells have IFN-g–independent
mechanisms of controlling bacterial growth. However, that report
was based on bacterial burden in vivo and was unable to distinguish between intracellular bacterial killing and inhibition of
bacterial growth. We sought to determine whether T cell–Mf
interactions were sufficient in the absence of IFN-g signal to activate the Mf to kill intracellular M. tuberculosis. We hypothesized that one of three outcomes was possible in the absence of
IFN-g signal from T cell to Mf: 1) cell-to-cell contact would be
sufficient to activate bactericidal activity; 2) in the absence of
IFN-g signal, bacterial growth would be inhibited (bacteriostatic),
or 3) IFN-gR–deficient Mf would fail to slow intracellular bacterial growth. To test our hypotheses, we used an in vitro intracellular bacterial killing assay. Bone marrow–derived Mfs from
either WT or IFN-gR2/2 mice were infected with M. tuberculosis
and incubated with media only, LPS/IFN-g, CD4 T cells, or CD8
T cells isolated from the lungs of M. tuberculosis–infected WT
mice for 72 h. After incubation, bacterial numbers per well were
determined via plating for CFU, and we compared initial input
CFU to calculate a percentage of bacteria killed. In the presence
or absence of IFN-g signaling, WT T cells (either CD4 or CD8
T cells) were able to induce killing of the intracellular bacteria
(Fig. 7). When IFN-g signaling was intact and CD4 T cells
present, there was a significant increase in intracellular bacteria
killed (p , 0.05; Fig. 7). Taken together, these data suggest that
IFN-g signal enhances T cell:Mf cell-to-cell contact to induce
bactericidal Mf activation.
Discussion
We sought to investigate the role of IFN-g from CD4 T cells
during M. tuberculosis on host survival and overall immune response. We hypothesized that IFN-g from cells other than CD4
T cells would be sufficient to control M. tuberculosis infection;
however, our data instead supported that IFN-g must be produced
by CD4 T cells for optimal protection. Using a novel adoptive
transfer model in which lymphopenic RAG1-deficient mice were
reconstituted with CD4-depleted naive splenocytes with naive
CD4 T cells that either could or could not produce IFN-g, we
demonstrated that CD4 T cells are essential for the control of M.
tuberculosis infection and host survival. Although the presence of
IFN-g–deficient CD4 T cells significantly increased host survival,
long-term control of infection was not achieved when compared
with mice reconstituted with cells including WT CD4 T cells.
These data agree with published literature from both knockout and
Ab depletion studies (6, 7), as well as with Gallegos et al. (10),
FIGURE 7. IFN-g signal is required to induce intracellular bacterial
killing in Mfs. Infected bone marrow–derived Mfs from C57BL/6 or
IFN-gR–deficient mice were cultured for 72 h with either media, IFN-g
and LPS, CD4 T cells, or CD8 T cells isolated from the lungs of M. tuberculosis–infected mice. Data are reported as a percentage killing ([CFU
output/CFU input] 3 100). *p , 0.05, Mann–Whitney U test.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
samples with GAP tetramer (GAPtet) (19) stimulated with GAP
peptide in the presence of monensin. GAPtet is a fluoroflor-linked
MHC I molecule that holds the peptide GAPINSATAM, from
Rv0125 (Mtb32A309–319), which has been demonstrated previously to stimulate 3–10% of lung CD8 T cells after M. tuberculosis infection of C57BL/6 mice (15, 16) (representative plots
shown in Fig. 5A). Animals that lack CD4 T cells had a reduced
frequency of overall CD8 T cells in the lung (Fig. 5B). Fewer CD8
T cells from CD4-deficient animals produce either IFN-g (Fig.
5C) or TNF (Fig. 5E), as well as a reduced frequency of CD107+
cells (Fig. 5D). These data are consistent with recently published
literature reporting that the presence of CD4 T cells directly affected the production of IFN-g by CD8 T cells (20). When CD4
T cells could not make IFN-g (GammaAT), the total frequency of
CD8 T cells was decreased by 20% compared with those with WT
CD4 T cells. In addition, the lack of IFN-g from CD4 T cells
resulted in fewer CD8 T cells capable of producing IFN-g (Fig.
5C), TNF (Fig. 5E), or CD107 (Fig. 5D). Despite fewer Agspecific CD8+GAPtet+ cells present in the absence of either
CD4 T cells or IFN-g from CD4 T cells, CD8+GAPtet+ T cells in
all groups produced similar amounts of both IFN-g and TNF
when measured by MFI (data not shown). Of note, the observed
truncated CD8 T cell response was restored with as little as 3%
contamination with CD4 T cells from a WT donor (data not
shown). These data indicate that CD4 T cells are required for a
quality CD8 T cell response that is consistent with recent literature
(20). In contrast, our data suggest that IFN-g from CD4 T cells
may act directly on CD8 T cells to boost the frequency of cells
capable of producing cytokine.
The Journal of Immunology
ment of this adoptive transfer model opens opportunities for asking
questions about the specific origin and function of cytokines from
cellular sources. It provides a powerful tool to move beyond
global systemic neutralization or loss of function and begin to
discover when and from where the factors required for an effective
immune response against M. tuberculosis originate.
Acknowledgments
We thank Dr. P. Ling Lin for helpful discussions, Amy Frasier for help
during early development of the model, and Judy Dininno for wonderful
animal care.
Disclosures
The authors have no financial conflicts of interest.
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indicating that CD4 T cells contribute to initial control of M. tuberculosis infection. In addition, mice reconstituted without CD4
T cells had a truncated or abortive immune response in which
greater bacterial burden failed to elicit an expected dramatic increase in cellular infiltrate. These data indicate that cell types
other than CD4 T cells are capable of producing enough IFN-g to
initially impair growth of the bacteria, but in the long term, IFN-g
from CD4 T cells is necessary for survival.
In contrast with our findings, a recent publication by Gallegos et al.
(10) showed that CD4 T cells can inhibit M. tuberculosis growth
in vivo at 3 wk p.i. independent of the Th1 transcription factor T-bet
and Th1 cytokines IFN-g or TNF production. Although these data
are very intriguing, the study did not address the long-term role of
IFN-g from CD4 T cells on host survival, as well as whether CD4
T cells in the absence of IFN-g are able to induce Mfs to kill
intracellular bacilli. The traditional approaches of knockout and
transgenic mice, as well as Ab-mediated depletion, have only provided information about the global effects of loss or gain of the
cellular subset and have failed to evaluate the individual contribution of each cell type. Other immune cells can and do produce IFN-g
over the course of infection, and as previously reported, CD8 T
cells are the major source of IFN-g in CD4-deficient mice (14).
Mice that lack CD4 T cells were deficient in cytotoxic CD8
T cells when measured by CD107 expression after stimulation.
These findings are consistent with previously published data in
knockout mice that measured cytotoxicity by limiting dilution
assay (14). In addition, unexpectedly, when CD4 T cells were
present but unable to make IFN-g, CD8 T cells also exhibited
a decreased frequency of IFN-g, TNF, and CD107 expression,
indicating that IFN-g from CD4 T cells may act directly on the
CD8 T cell. We confirmed this by modifying the adoptive transfer
method and reconstituting animals with CD8-depleted splenocytes
+/2 CD8 T cells from either WT or IFN-gR–deficient mice and
saw a similar trend. When CD8 T cells were unable to respond to
IFN-g, Ag-specific responses were less robust. Thus, IFN-g from
CD4 T cells may be necessary to enhance CD8 T cell responses in
our adoptive transfer model. Similar effects of direct IFN-g signaling have been reported in murine viral models (21), but these
data are, to our knowledge, the first to show that IFN-g directly
improves CD8 T cell functions during M. tuberculosis infection.
We were surprised by the data suggesting that IFN-g from CD4
T cells may act directly on the CD8 T cells to enhance the response to M. tuberculosis infection in our model. These data are in
contrast with two previous studies using knockout animals in
which there were no observed differences in CD8 T cell cytokine
production during M. tuberculosis infection (14, 20). One possibility is that the previous studies assessed the global CD8 T cell
response, whereas we were able to follow Ag-specific responses.
In addition, studies reported by Bold et al. (20) assessed the
responses of CD8 T cells primed in the presence of CD4 T cells.
Based on the data in our model, IFN-g on CD8 T cells may enhance responses and, therefore, a key role of CD4 T cells during
the initial response is to help CD8 T cell induction and function.
Finally, we evaluated the ability of CD4 T and CD8 T cells from
the lungs of infected mice to activate Mfs and kill intracellular
pathogens in the presence and absence of an IFN-g signal. In this
study, the data suggest that T cell:Mf cell-to-cell contact is sufficient, regardless of IFN-g signaling, to kill intracellular bacteria
in vitro, which leads naturally to questions of mechanism that are
the focus of future studies. However, IFN-g signaling was required
for maximal intracellular bacterial killing.
The data presented in this article provide novel data about the
role of IFN-g from CD4 T cells and its effects on survival, bacterial burden, and quality of the CD8 T cell response. Develop-
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