The Journal of Immunology
Lung Neutrophils Facilitate Activation of Naive
Antigen-Specific CD4+T Cells during Mycobacterium
Robert Blomgran* and Joel D. Ernst*,†,‡
Initiation of the adaptive immune response to Mycobacterium tuberculosis occurs in the lung-draining mediastinal lymph node and
requires transport of M. tuberculosis by migratory dendritic cells (DCs) to the local lymph node. The previously published
observations that 1) neutrophils are a transiently prominent population of M. tuberculosis-infected cells in the lungs early in
infection and 2) that the peak of infected neutrophils immediately precedes the peak of infected DCs in the lungs prompted us to
characterize the role of neutrophils in the initiation of adaptive immune responses to M. tuberculosis. We found that, although
depletion of neutrophils in vivo increased the frequency of M. tuberculosis-infected DCs in the lungs, it decreased trafficking of
DCs to the mediastinal lymph node. This resulted in delayed activation (CD69 expression) and proliferation of naive M. tuber-
culosis Ag85B-specific CD4 T cells in the mediastinal lymph node. To further characterize the role of neutrophils in DC migration,
we used a Transwell chemotaxis system and found that DCs that were directly infected by M. tuberculosis migrated poorly in
response to CCL19, an agonist for the chemokine receptor CCR7. In contrast, DCs that had acquired M. tuberculosis through
uptake of infected neutrophils exhibited unimpaired migration. These results revealed a mechanism wherein neutrophils promote
adaptive immune responses to M. tuberculosis by delivering M. tuberculosis to DCs in a form that makes DCs more effective
initiators of naive CD4 T cell activation. These observations provide insight into a mechanism for neutrophils to facilitate
initiation of adaptive immune responses in tuberculosis.The Journal of Immunology, 2011, 186: 7110–7119.
in diverse professional phagocytes in the lungs where it uses
strategies, such as preventing phagosome maturation and sub-
version of host cell-death pathways, to survive and replicate (1).
Effective immunity against M. tuberculosis requires CD4+Th1
and CD8+T lymphocyte responses to M. tuberculosis Ags (2–5).
Compared with other lower respiratory tract infections, such as
influenza A (6), for which the peak in naive T cell proliferation
occurs 4 d postinfection, the onset of the CD4+response against
M. tuberculosis is delayed until 10–12 d after aerosol infection
(7–9), giving the bacterium time to expand and establish a niche
that allows it to resist eradication.
Polymorphonuclear neutrophils are abundant, motile cells in-
volved in the innate immune response and form an early line of
defense against microbial pathogens. These professional phag-
ocytes are crucial in the defense against extracellular bacterial and
espite the availability of drugs to treat it, tuberculosis
(TB) remains a major burden to human health. Myco-
bacterium tuberculosis infects via inhalation and resides
fungal infections. Although parasites, such as Leishmania, have
evolved to exploit neutrophils to establish and promote disease
(10), neutrophils play a protective role against certain other in-
tracellular pathogens (11–14). In an in vivo intranasal M. bovis
bacille Calmette–Gue ´rin (BCG) infection model, neutrophils were
suggested to have a dual role in acute infection, a direct antimi-
crobial activity counterbalanced by anti-inflammatory properties
(15). Furthermore, innate immune responses to M. tuberculosis in
RAG-deficient mice revealed a compensatory function for neu-
trophils in keeping the bacterial burden in check in the absence of
IFN-g (16). In addition to a direct bactericidal or immunomodu-
latory effect, neutrophils readily undergo apoptosis, and phago-
cytosed microbe-containing apoptotic neutrophils can have a
stimulatory effect on macrophages (17) and dendritic cells (DCs)
(18). Additionally Davis and Ramakrishnan (19) clearly showed
that spread of bacteria through apoptotic cells is a major mecha-
nism by which macrophages obtain virulent mycobacteria in vivo.
Although neutrophils were shown to contribute to innate pro-
tection against mycobacteria (15, 16, 20–23), data to the contrary
are similarly compelling (15, 24–26). Other than the neutrophil’s
capacity to produce chemokines/cytokines (27–30), in vivo evi-
dence for a role of neutrophils in modulating adaptive immunity
during M. tuberculosis infections has not been reported.
Evidence for one or more roles of neutrophils in human im-
munity to TB includes the observation that the risk for TB infection
among household contacts is inversely associated with peripheral
blood neutrophil count, and killing of M. bovis BCG in a whole-
blood in vitro assay was significantly impaired by neutrophil de-
pletion (20). Moreover, humans exhibit a transcriptional signature
in peripheral blood that indicates a role for neutrophils and/or
a related myeloid cell that occurs in response to active pulmo-
nary TB (31). Consequently, a greater understanding of the roles
that neutrophils play in the innate and adaptive immune responses
to M. tuberculosis is needed.
*Division of Infectious Diseases, Department of Medicine, New York University
School of Medicine, New York, NY 10016;†Department of Pathology, New York
University School of Medicine, New York, NY 10016; and‡Department of Micro-
biology, New York University School of Medicine, New York, NY 10016
Received for publication January 4, 2011. Accepted for publication April 15, 2011.
This work was supported by National Institutes of Health Grant R01 AI051242, the
Fulbright Commission in Sweden visiting scholarship, the Swedish Heart Lung Foun-
dation, and the Swedish Research Council.
Address correspondence and reprint requests to Dr. Joel D. Ernst, New York Univer-
sity School of Medicine, 550 First Avenue, Smilow 901, New York, NY 10016.
E-mail address: email@example.com
Abbreviations used in this article: BALF, bronchoalveolar lavage fluid; BCG, bacille
Calmette–Gue ´rin; BMDC, bone marrow dendritic cell; DC, dendritic cell; Flt3L, Flt3
ligand; mDC, myeloid dendritic cell; MDLN, mediastinal lymph node; MOI, multi-
plicity of infection; TB, tuberculosis.
DCs are potent APCs that prime naive T cells in the lung-
draining lymph node (mediastinal lymph node [MDLN]) follow-
ing M. tuberculosis infection (32, 33). Initial activation of naive
M. tuberculosis-specific CD4+T cells in the MDLN depends on
DC transport of bacteria from the lungs to the MDLN (9), in
an IL-12p40 homodimer- (32) and temporally CCR7-dependent
manner (34). Furthermore, when characterizing the cells harboring
M. tuberculosis following aerosol infection of mice, we found that
neutrophils were a transiently dominant population of lung cells
infected early in infection (35). The observation that the peak
number of infected neutrophils immediately preceded the peak of
infected DCs in the lungs suggests at least two competing hy-
potheses: acquisition of M. tuberculosis by neutrophils transiently
sequesters the bacteria and delays their acquisition by DCs, or
infected neutrophils interact with DCs to promote DC acquisition
of the bacteria and bacterial Ags. To test these hypotheses and to
characterize the role of neutrophils in the initiation of adaptive
immune responses to M. tuberculosis, we depleted neutrophils
in vivo using a mAb against the neutrophil-specific Ag Ly6G
(clone 1A8) (15, 36). We found that neutrophils were necessary
for timely initiation of the adaptive immune response by sup-
porting DC migration and trafficking of M. tuberculosis to the
local lymph node.
Materials and Methods
C57BL/6 mice were bred and housed in a specific pathogen-free envi-
ronment in New York University School of Medicine animal facilities or
purchased from The Jackson Laboratory (Bar Harbor, ME). P25TCR-Tg
mice, whose CD4+T cells express a transgenic T cell Ag receptor that
recognizes peptide 25 (aa 240–254) of M. tuberculosis Ag85B bound to
I-Ab, were on a C57BL/6 background (CD45.2) or on a Rag12/2back-
ground (when specified), as previously described (37), and were bred in the
New York University School of Medicine animal facilities. CD45.1 mice
were either bred in New York University School of Medicine animal fa-
cilities or purchased from Taconic Farms. Genotypes of mice were con-
firmed by PCR testing of tail genomic DNA. All procedures conducted on
mice were in accordance with the conditions specified by the New York
University School of Medicine Institutional Animal Care and Use Com-
Abs, FACS staining, and acquisition
All Abs were purchased from BD Pharmingen, unless otherwise stated.
Anti-CD11c PerCP (H3L) (1:200) was custom conjugated from BD
Pharmingen, and other Ab conjugates used were anti-CD45.2 PerCP
(1:200), anti-CD4 Alexa Fluor 647 (1:200), anti-CD69 PE (1:200), anti-
CD11b PE or Pacific Blue (1:1500), anti-CD40 Alexa Fluor 647 or PE
(1:200), anti-CD86 allophycocyanin or PE (1:200), anti-CD80 Alexa Fluor
647 or PE (1:200), anti-MHC class II Alexa Fluor 647 or PE (1:1500),
CCR7 Alexa Fluor 647 or PE (1:200), anti-Ly6C FITC or PE (1:600), anti-
Ly6G Alexa Fluor 647 (1:600), and Gr-1 allophycocyanin (1:1500).
Staining for surface markers was done by resuspending up to 1 3 106cells
in 100 ml FACS buffer (PBS supplemented with 1% heat-inactivated FBS,
0.1% NaN3, and 1 mM EDTA) containing Abs and incubating at 4˚C for
25 min (or at 37˚C for CCR7). Cells were washed twice and fixed over-
night in PBS/1% paraformaldehyde at 4˚C. Data were acquired using
a FACSCalibur or LSR II flow cytometer, depending on the experiment.
P25TCR-Tg CD4+T cell isolation and labeling
P25TCR-Tg mice, between 8–16 wk of age, were killed according to
approved laboratory animal procedures, and naive P25TCR-Tg CD4+
T cells from lymph nodes and spleen were isolated, as previously de-
scribed (9). For proliferation assays, CD4+T cells were labeled with CFSE
Adoptive transfer and aerosol infection
CD45.1 mice routinely received 2–3 3 106CFSE-labeled P25TCR-Tg
CD4+T cells (CD45.2), by tail vein or retro-orbital injection, in 100 ml
sterile PBS. Three to twenty-four hours postcell transfer, mice were
infected by the aerosol route using an Inhalation Exposure Unit (Glas-Col).
The infectious dose was confirmed by euthanizing four or five mice and
plating homogenized lungs within 24 h of infection, as previously de-
Tissue processing and CFU determination
Mice were euthanized at designated time points, and tissues were used to
prepare single-cell suspensions and to determine the bacterial loads by
plating, as previously described (9, 35).
Phenotyping and quantitation of lung cells
To avoid epitope masking in mice treated with the neutrophil-depleting Ab
to Ly6G, 1A8, neutrophils were defined and quantitated as CD11bhi/Gr-1hi/
Ly6Cint/CD11clo/neg(35, 36, 38). For identification of lung macrophage
and DC subsets, neutrophils were first gated out. Based on previous
functional and morphological characterization, lung cell subsets were
designated as alveolar macrophages (CD11blow/CD11chigh), myeloid
DCs (mDCs; CD11bhigh/CD11chigh), recruited macrophages (CD11bhigh/
CD11cintemediate), or monocytes (CD11bhigh/CD11cnegative) (35, 39).
In vivo neutrophil depletion
The purified Ly6G-specific Ab 1A8 (36) was used to deplete neutrophils
in vivo, and purified 2A3 (Rat IgG2a) was used as isotype control Ab; both
were obtained from BioXcell (West Lebanon, NH). Single-dose treatment
(300 mg administered i.p.) was used to prevent the confounding effects of
an immune response toward the depleting Ab, which can be seen with
multiple treatments in vivo (25). Furthermore, 1A8 had no effect on Ly6C+
cells in spleen or lungs 2 d after administration, at which time neutrophils
were fully depleted (data not shown) (12).
Bacterial strains, treatment, and in vitro infection
M. tuberculosis (H37Rv) and FACS-optimized GFP-H37Rv (under the
control of the Mycobacterium bovis BCG Hsp60 promoter) were prepared
for in vivo and in vitro use, as previously described (35). For in vitro use,
log-phase bacteria (OD580= 0.5–0.9) were washed, resuspended in 15 ng/
ml mouse GM-CSF–supplemented RPMI 10 complete medium (referred to
as GM-CSF medium) and gravity filtered through a 5-mm filter to obtain
single-cell bacteria. Multiplicity of infection (MOI) was calculated,
depending on the OD at 580 nm, and validated through serial dilutions and
plating. DCs were infected at MOI = 5 for 19 h, resulting in infection of
60–70% of bone marrow DCs (BMDCs). To optimize uptake of M. tu-
berculosis by neutrophils and synchronize the assay, log-phase GFP-
H37Rv bacteria were opsonized using 50% pooled AB human serum in
RPMI 1640 without additives for 30 min at 37˚C, before addition to
neutrophils (MOI = 5), allowing for 40 min of phagocytosis at 37˚C
(routinely yielding 70–80% GFP+neutrophils, according to flow cytom-
etry). Infected cells were washed thrice, treated for 40 min with 200 mg/ml
amikacin, and washed twice more before being used. Neutrophils were
additionally labeled with CMTMR (CellTracker Orange; Invitrogen) for
BMDCs and neutrophil isolation
Bone marrow from C57BL/6 mice was cultured in GM-CSF medium at
37˚C, 5% CO2. Fresh GM-CSF medium was added on days 3 and 6. The
floating cell fraction was collected at day 7 and used as source of BMDCs
and neutrophils by positive selection, using magnetic beads coupled to
anti-CD11c mAb (N418) and anti–Ly-6G, respectively, and sorted by
AutoMACS, according to the manufacturer’s instructions (Miltenyi Biotec,
Auburn, CA). Cells were maintained in GM-CSF medium throughout the
experiment to prevent cytokine withdrawal-induced cell death. GM-CSF
medium is RPMI 1640 with 10% heat-inactivated FBS, 2 mM L-glutamine,
1 mM sodium pyruvate, 13 b-ME, 10 mM HEPES, and 15 ng/ml mouse
GM-CSF; culture supernatants from GM-CSF–producing melanoma cells
were quantified using a mouse GM-CSF ELISA kit (BioSource Interna-
tional) and stored as aliquots at 280˚C until use.
Flt3 ligand-expanded in vivo DCs
To increase the number of in vivo DCs, C57BL/6 mice were injected s.c.
with 3–6 3 106Flt3 ligand (Flt3L)-producing melanoma cells at 80%
confluence, and splenocytes were isolated 7–9 d later. Spleens were ex-
cised and forced through a 70-mm nylon cell strainer (BD Falcon), and
RBCs were removed using ACK lysis buffer. These in vivo-derived DCs
were purified by CD11c positive selection using magnetic beads coupled to
anti-CD11c mAb, which resulted in $96% DC purity. To generate mature
DCs, cells were incubated overnight in GM-CSF medium at 37˚C, 5%
CO2. Mature DCs were CD40hi, CD80hi, CD86hi, and MHC class IIhi. By
The Journal of Immunology7111
44. Seiler, P., P. Aichele, S. Bandermann, A. E. Hauser, B. Lu, N. P. Gerard,
C. Gerard, S. Ehlers, H. J. Mollenkopf, and S. H. Kaufmann. 2003. Early
granuloma formation after aerosol Mycobacterium tuberculosis infection is
regulated by neutrophils via CXCR3-signaling chemokines. Eur. J. Immunol. 33:
45. Meijer, A. H., A. M. van der Sar, C. Cunha, G. E. Lamers, M. A. Laplante,
H. Kikuta, W. Bitter, T. S. Becker, and H. P. Spaink. 2008. Identification and
real-time imaging of a myc-expressing neutrophil population involved in in-
flammation and mycobacterial granuloma formation in zebrafish. Dev. Comp.
Immunol. 32: 36–49.
46. Tobin, D. M., J. C. Vary, Jr., J. P. Ray, G. S. Walsh, S. J. Dunstan, N. D. Bang,
D. A. Hagge, S. Khadge, M. C. King, T. R. Hawn, et al. 2010. The lta4h locus
modulates susceptibility to mycobacterial infection in zebrafish and humans.
Cell 140: 717–730.
47. Peters, N. C., N. Kimblin, N. Secundino, S. Kamhawi, P. Lawyer, and
D. L. Sacks. 2009. Vector transmission of leishmania abrogates vaccine-induced
protective immunity. PLoS Pathog. 5: e1000484.
48. Tvinnereim, A. R., S. E. Hamilton, and J. T. Harty. 2004. Neutrophil in-
volvement in cross-priming CD8+ T cell responses to bacterial antigens. J.
Immunol. 173: 1994–2002.
49. Ravindran, R., L. Rusch, A. Itano, M. K. Jenkins, and S. J. McSorley. 2007.
CCR6-dependent recruitment of blood phagocytes is necessary for rapid CD4
T cell responses to local bacterial infection. Proc. Natl. Acad. Sci. USA 104:
50. Hinchey, J., S. Lee, B. Y. Jeon, R. J. Basaraba, M. M. Venkataswamy, B. Chen,
J. Chan, M. Braunstein, I. M. Orme, S. C. Derrick, et al. 2007. Enhanced priming
of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis.
J. Clin. Invest. 117: 2279–2288.
51. Velmurugan, K., B. Chen, J. L. Miller, S. Azogue, S. Gurses, T. Hsu,
M. Glickman, W. R. Jacobs, Jr., S. A. Porcelli, and V. Briken. 2007. Myco-
bacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of
infected host cells. PLoS Pathog. 3: e110.
52. Miller, J. L., K. Velmurugan, M. J. Cowan, and V. Briken. 2010. The type I
NADH dehydrogenase of Mycobacterium tuberculosis counters phagosomal
NOX2 activity to inhibit TNF-alpha-mediated host cell apoptosis. PLoS Pathog.
53. Chen, M., M. Divangahi, H. Gan, D. S. Shin, S. Hong, D. M. Lee, C. N. Serhan,
S. M. Behar, and H. G. Remold. 2008. Lipid mediators in innate immunity
against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of
macrophage death. J. Exp. Med. 205: 2791–2801.
54. Divangahi, M., D. Desjardins, C. Nunes-Alves, H. G. Remold, and S. M. Behar.
2010. Eicosanoid pathways regulate adaptive immunity to Mycobacterium tu-
berculosis. Nat. Immunol. 11: 751–758.
The Journal of Immunology7119