By
From the * Department of Medicine and the Department of Microbiology/Immunology, and the Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California
94143; and the § Veterans Administration Medical Center, San Francisco, California 94121
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Abstract |
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Interferon (IFN-
) has been implicated in T helper type 1 (Th1) cell development through
its ability to optimize interleukin 12 (IL-12) production from macrophages and IL-12 receptor
expression on activated T cells. Various systems have suggested a role for IFN-
derived from
the innate immune system, particularly natural killer (NK) cells, in mediating Th1 differentiation in vivo. We tested this requirement by reconstituting T cell and IFN-
doubly deficient
mice with wild-type CD4+ T cells and challenging the mice with pathogens that elicited either
minimal or robust IL-12 in vivo (Leishmania major or Listeria monocytogenes, respectively). Th1
cells developed under both conditions, and this was unaffected by the presence or absence of
IFN-
in non-T cells. Reconstitution with IFN-
-deficient CD4+ T cells could not reestablish control over L. major, even in the presence of IFN-
from the NK compartment. These
data demonstrate that activated T cells can maintain responsiveness to IL-12 through elaboration of endogenous IFN-
without requirement for an exogenous source of this cytokine.
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Introduction |
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The development of CD4+ T cell subsets remains an
important topic in understanding mechanisms by
which protective or detrimental T effector cell populations
arise (1, 2). Critical cytokines that promote the appearance
of Th1 cells include IL-12 and IL-18, which provide important growth and survival signals for this subset of cells
(3). The canonical Th1 cytokine, IFN-, may also contribute to Th1 development, since mice deficient in IFN-
(IFN-
/
) aberrantly developed Th2 cells when confronted with pathogens that normally engender Th1 responses (6).
IFN- can influence Th1 development by several mechanisms. IFN-
mediates IL-12R
2 chain expression (9)
and promotes IL-12 secretion from macrophages (10).
Since both IL-12 and a functional IL-12 receptor are required for the development of fully competent Th1 cells
(3, 11, 12), the requirement for IFN-
may be indirect.
Because naive T cells do not produce IFN-
until some time after activation, it is possible that IFN-
derived upon activation of NK cells (13) may prime Th1 development
through its ability to confer a competent IL-12 response to
the T cells. IFN-
derived from Th1 cells can also negatively regulate the growth of Th2 cells (14). Such studies
predict that T cells conditioned in vivo in the absence of a
source of exogenous IFN-
might display defective Th1
development.
To examine the relative contributions of NK cell- and T
cell-derived IFN- in Th1 development, we have generated mice with a selective deficiency of IFN-
in either the
NK cell or T cell compartments. The mice were challenged with pathogens that in the early stages of infection
elicit either little (e.g., Leishmania major; reference 15) or
robust (e.g., Listeria monocytogenes; reference 16) amounts of
IL-12 in vivo, and were assayed for their capacity to develop Th1 cells.
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Materials and Methods |
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Mice.
C57BL/6 mice, and IFN-CD4+ T Cell Purification and Adoptive Transfer.
Lymph nodes and spleens were collected from designated mice and dispersed through a 0.75-µm nylon mesh filter to produce single cell suspensions. After washing and counting, suspensions were depleted of B, dendritic, and CD8+ T cells by treatment with mAbs against heat-stable antigen (J11d; American Type Culture Collection [ATCC], Rockville, MD), MHC class II (BP107; ATCC), and CD8 (3.155; ATCC), followed by treatment with rabbit and guinea pig complement (Cedarlane Labs., Hornby, Ontario, Canada). After removal of dead cells over a Ficoll gradient, cells (routinely 75-85% CD4+ T cells) were labeled with FITC-conjugated anti-Thy 1.2 (5a-8; Caltag Labs., S. San Francisco, CA), PE-conjugated anti-NK1.1 (PK136; PharMingen, San Diego, CA), and TriColor-conjugated anti-CD4 mAbs (CT-CD4; Caltag Labs.), and then sorted using flow cytometry (FACStar PLUS®, Becton Dickinson, Mountain View, CA) to collect CD4+Thy1.2+NK1.1Leishmania Infection.
L. major (strain WHOM/IR/-/173) was grown in M199 medium supplemented with 30% FCS and antibiotics. For infection, metacyclic promastigotes were purified from stationary phase cultures by negative selection with peanut lectin-coated beads (Sigma Chemical Co., St. Louis, MO) as previously described (15). Designated groups of four to eight mice were inoculated with 4 × 105 organisms in each hind footpad. Footpad swelling was quantitated weekly using a metric caliper. At various times, mice were killed and the footpads and spleens were collected for quantitation of parasites as previously described (11). In brief, single cell suspensions were prepared and diluted serially 10-fold in triplicate microtiter wells in M199 medium with 30% FCS and antibiotics. After incubation for 2 wk at 26°C, motile promastigotes were identified using inverted microscopy.Listeria Infection.
L. monocytogenes (provided by D. Portnoy, U.C. Berkeley, Berkeley, CA) was maintained as frozen stock and grown in Luria-Bertani medium. Mice were infected intraperitoneally with 104 organisms in 100 µl of PBS. Mice were killed after 8 d and the spleens were collected for analysis of cytokines (see below).ELISPOT Assays for Cytokine Production.
Spleens from mice infected with L. major or Listeria were dispersed into single cell suspensions in RPMI with 10% FCS and antibiotics (culture medium). Production of IL-4 and IFN-Flow Cytometric Analysis and Intracellular Cytokine Detection.
Spleen cells collected from designated mice were analyzed for the numbers of CD4+TCR+NK1.1Serum IgE Analysis.
Serum prepared at the time of death was quantitated for total IgE using a mAb-based sandwich ELISA as previously described (7).NK Cytolytic Assay.
YAC-1 tumor targets (106 cells) were labeled at 37°C for 1 h with 200 µCi 51Cr-sodium chromate (Amersham Pharmacia Biotech, Arlington Heights, IL) in RPMI 1640 supplemented with 10% FCS, antibiotics, 5 × 10 ![]() |
Results |
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Prior studies of murine L. major infection,
including in C57BL/6 mice, have suggested a role for
IFN- derived from NK cells in establishing Th1 cytokine
responses necessary for control of disease (21, 22). To assess
this directly, T cell-deficient mice were created on the
IFN-
/
background by crossing and selecting C57BL/6
mice doubly deficient for RAG-1 and IFN-
. As expected,
these mice, and singly deficient RAG-1
/
mice, expressed
no B or T lymphocytes. Analysis of spleens from such mice
revealed that the NK compartment, as assessed by NK1.1+
TCR-
/
, -
/
cells, comprised ~2.6 ± 0.4 × 106 cells
in Rag-1
/
mice and 2.8 ± 0.7 × 106 cells in RAG-1
/
IFN-
/
mice, as compared with 2.5 ± 0.3 × 106 splenic
NK cells in wild-type C57BL/6 mice. Thus, despite the substantial decrease in spleen size in RAG-1
/
and RAG-1
/
IFN-
/
mice, the numbers of NK cells were comparable to wild-type mice.
The capacity of CD4+ T cells to reconstitute host responses to L. major in immunodeficient mice (23) prompted
us to reconstitute such doubly deficient mice with 106
highly purified CD4+ T cells from wild-type C57BL/6 animals. These adoptive transfers thus establish mice in which
the IFN--competent T cell compartment is donor derived and the IFN-
/
NK cell compartment is recipient
derived. Mice were infected 2 d after reconstitution. In
three independent experiments, all reconstituted mice were
capable of controlling infection with L. major, as assessed by
lesion size over time and by the numbers of parasites recovered from the footpads and spleens after 8 wk (Fig. 1 A). In
contrast, nonreconstituted mice were highly susceptible to
L. major infection (log10 parasites in footpads
1012 in both
and in spleens, 107.1 and 106.6 in RAG-1
/
and doubly deficient mice, respectively), as expected based on prior studies in T cell-deficient and IFN-
/
mice (7, 11, 23).
Analysis of the reconstituted mice at the conclusion of the
experiment confirmed the purity of the CD4+ T cell reconstitution; CD8+ T cells (<1%),
/
T cells (<1%), and
serum IgE (<1.2 µg/ml) were at or below the levels of detection in nonreconstituted RAG-1
/
mice. The cytolytic
capacity of NK cells taken from the infected T cell-reconstituted IFN-
+/+ or
/
mice was comparable, as assessed
by the ability to kill labeled YAC-1 targets in vitro (Fig. 2).
Thus, wild-type CD4+ T cells conferred a phenotypically
normal healer response to L. major in mice containing an
NK compartment unable to contribute the cytokine IFN-
.
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|
To
assess whether IFN- contributed from NK cells could impart any control over L. major, we reconstituted either
RAG-1
/
or doubly deficient mice with highly purified
CD4+ T cells from IFN-
/
mice. Reconstituted RAG-1
/
mice have intact IFN-
production from the NK cell
compartment, unlike the doubly deficient mice, which allows us to compare the course of infection in the two cohorts of animals. In two separate experiments, both reconstituted groups of mice were unable to control L. major,
and no significant differences were apparent among the
groups in either lesion size or parasite burdens (Fig. 1 B).
Indeed, susceptible BALB/c mice controlled infection better than either cohort of reconstituted mice. Thus, IFN-
derived from the NK cell compartment alone could not
sustain control of L. major in the absence of CD4+ T cell-
derived IFN-
.
Control of L. major among healer mice, including
C57BL/6 mice, is dependent upon the development of Th1
cells that produce IFN-. As assessed by both ELISPOT
assays (data not shown) and intracellular cytokine determinations (Fig. 3 A) in individual CD4+ T cells, Th1, but not
Th2, cells developed effectively in doubly deficient mice
reconstituted with wild-type CD4+ T cells. In contrast,
mice reconstituted with IFN-
/
CD4+ T cells developed
IL-4-producing cells, and such cells developed in both
RAG-1
/
mice (990 ± 95 IL-4-producing cells per 106
spleen cells) or doubly deficient mice (2,050 ± 960 IL-4-
producing cells per 106 spleen cells) in numbers comparable
to infected susceptible BALB/c mice (1,250 ± 170/106
spleen cells) and greater than those in resistant C57BL/6
mice (225 ± 10/106 spleen cells). Thus, under these conditions, Th1 cell development was unimpeded and Th2 development was not apparent in mice containing IFN-
/
NK cells. Conversely, development of IL-4-producing
cells occurred when IFN-
/
CD4+ T cells were used in
the reconstitutions despite the absence or presence of IFN-
in non-T cells.
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L. major is a relatively subacute infection that develops over weeks. It is possible that some kinetic deficit in Th1 development was missed in the doubly deficient mice due to the relatively slow course of infection. To address this issue, we compared the development of Th1 cells in doubly deficient mice reconstituted with wild-type CD4+ T cells and then inoculated with L. monocytogenes, a gram-positive bacterium that is capable of inducing strong Th1 responses (16). As assessed by both ELISPOT (data not shown) and intracellular cytokine determinations (Fig. 3 B), Th1 cells also developed in these mice, indicating that the results seen with L. major were not limited to that organism.
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Discussion |
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These studies demonstrate that Th1 cells can differentiate
in vivo without exposure to IFN- from other cells, such
as NK cells. Differentiated cells were functionally competent, as assessed by their capacity to maintain control over a
parasite, L. major, which is crucially dependent upon Th1
development (24). Finally, we were unable, at least under
these conditions, to define a role for IFN-
derived from
NK cells in modulating, either positively or negatively,
Th1 effector differentiation. These data suggest that IFN-
derived from CD4+ T cells can autonomously modulate
responsiveness to IL-12 and Th1 competence.
The role of IFN- in priming Th1 cell development has
been demonstrated using transgenic T cells in vitro (25-
28), and presumably reflects the capacity of IFN-
to promote IL-12R
2 chain expression and the appearance of
competent IL-12 receptors on activated CD4+ lymphocytes (9, 29). IFN-
also induces IL-12 p40 production by
macrophages (10), thus establishing an autocrine loop for the amplification of type 1 immunity. The importance of
this pathway was revealed by the profound Th1 deficiency
in mice with genetic deletion of members of the IL-12 signaling cascade, including IL-12, IL-12R
1, and stat4 (3,
12, 30, 31). Interestingly, IFN-
has also been implicated
in Th1 development in vivo in some systems (6, 32),
consistent with effects mediated indirectly through IL-12.
The exquisite sensitivity of BALB/c mice to L. major infection has been proposed to develop due to premature downregulation of competent IL-12 receptors on activated
CD4+ T cells (33, 34). Such a defect might be secondary to
the decreased IFN-
produced after activation of BALB/c
CD4+ T cells as compared with cells from other inbred
mouse strains (35).
The earliest expression of IFN- after infection with L. major was shown to arise not in CD4+ T cells, but in the
NK cell population (15, 21, 22, 36). Despite these observations, the studies here suggest that IFN-
derived from the
NK cell population was neither required for Th1 cell differentiation nor for the control of L. major. Conversely, we
could discern no control of infection through IFN-
derived from the NK cell population in the absence of CD4+
T cell-derived IFN-
, although this may reflect the smaller
contribution made by NK cells in C57BL/6 mice (21).
The functional consequences of NK cell-depletion or activation that have been described in murine experimental
leishmaniasis may reflect contributions from NK cells that
are independent of IFN-
(21, 22). These findings contrast
with those in some viral systems, in which a critical role of
NK cell-derived IFN-
has been demonstrated (37), and may reflect differences among CD4+ and CD8+ effector
cells. Further studies will be required to elucidate how applicable such studies are to other classes of pathogens. We
could discern no impact on Th1 development in the absence of NK cell IFN-
after challenge with Listeria, a
more acute infectious disease than Leishmania. In L. major
infection, the earliest IFN-
and NK cell responses were
shown to be dependent on the induction of interferon
/
by the parasite and the consequent induction of type 2 nitric oxide synthase (38). As shown here in mice with intact
interferon
/
and type 2 nitric oxide synthase genes, these
early responses were unable to establish protective immunity in the absence of IFN-
from CD4+ T cells. This endogenous source of IFN-
is presumably necessary and sufficient to downregulate TGF-
activation that was felt to
contribute to early and rapid dissemination of the parasite (38, 39). However, the consistently higher proportion of
Th1 cells that developed in the reconstituted doubly deficient mice as compared with wild-type or reconstituted
RAG
/
mice (Fig. 3, A and B) suggests that the total
amounts of IFN-
required for a given biologic response
may be regulated, suggesting that IFN-
derived from
non-T cells may have functional activity independent of effects on Th development.
These results suggest that IFN-, in a cell-autonomous
manner, is required and sufficient to enable CD4+ T cells
to respond to exogenous signals, such as IL-12 and IL-18, to develop into functional Th1 effector cells. Similar conclusions have been reached regarding Th2 development, in
which autocrine IL-4 derived from T cells themselves was
sufficient to regulate this differentiative process (40). It
will be important to establish whether signals exogenous to
the T cells are required for Th2 development, akin to the
roles for IL-12 and IL-18 in Th1 development (3, 4). Such
insights will be critical in gaining understanding over the final pathways that regulate these differentiative pathways in
vivo during allergic and inflammatory diseases, so that cell autonomous and cell extrinsic regulatory molecules can be
efficiently targeted during immune intervention.
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Footnotes |
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Address correspondence to R.M. Locksley, UCSF, Box 0654, C-443, 521 Parnassus Ave., San Francisco, CA 94143. Phone: 415-476-9362; Fax: 415-476-9364; E-mail: rich_locksley{at}quickmail.ucsf.edu
Received for publication 10 July 1998 and in revised form 3 August 1998.
The authors thank E. Weider for assistance with cell sorting, A. O'Garra for assistance with the intracellular cytokine staining protocol, and R. Coffman (DNAX Research Institute, Palo Alto, CA) for critical reagents.
This work was supported by National Institutes of Health grant AI26918, a grant from the Crohn's and Colitis Foundation, a Hefni Scholars Award to A.E. Wakil, and a Juvenile Diabetes Foundation International Fellowship to D.J. Fowell.
Abbreviation used in this paper
RAG-1/
, recombinase activating gene-
deficient mice.
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