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CUTTING EDGE |
-Chain (c) Reveals an Unexpected Role for CD4+ T Lymphocytes in Early IFN--Dependent Resistance to Toxoplasma gondii
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Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, and
Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain
(c) gene exhibit defective development of NK cells
and CD8+ T cells and greatly diminished production of
IFN-. Because resistance of SCID mice to Toxoplasma
gondii requires IL-12-dependent IFN- production by NK cells,
we expected that c-deficient mice would succumb rapidly
to the parasite. Surprisingly, however, most c-deficient
mice survived the acute phase of T. gondii infection. As in
wild-type mice, this resistance required IL-12 and IFN-;
nevertheless, whereas wild-type mice depleted of CD4+ T
cells survived, anti-CD4+ treated
c-deficient mice displayed diminished production of
IFN- and all succumbed to acute infection. These data not only
reveal a role for CD4+ T lymphocytes in IFN--dependent
host defense but also establish SCID and c-deficient
mice as powerful complementary tools for assessing the function of NK
vs CD4+ T cells in immunopathophysiologic responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, or
c,2 is a
component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15
(1, 2, 3). In humans, mutations of c can result in X-linked
severe combined immunodeficiency (XSCID) (4), a disease characterized
by profoundly diminished numbers of T cells and NK cells (1, 2, 3, 4).
Without successful bone marrow transplantation, patients with XSCID
typically die from opportunistic infections. Like humans with XSCID,
mice in which the c gene has been targeted by homologous
recombination exhibit a profound immunodeficiency (5, 6, 7, 8). The number of
thymocytes is approximately 1 to 5% of normal; interestingly, although
splenocytes are also diminished in c-deficient mice up
to 3 wk of age, there is a subsequent age-dependent accumulation of
CD4+ peripheral T cells that exhibit an activated-memory
phenotype (9). The accumulation of these cells appears to result from
defective peripheral deletion (10). In contrast, CD8+ T
lymphocytes are essentially absent at all ages. Interestingly, no
NK1.1+ cells were detected, and c-deficient
splenocytes failed to produce IFN- when stimulated with anti-CD3
plus anti-CD28 (6).
IFN- production by NK cells is a major mechanism of innate defense
against intracellular pathogens (11). Because
c-deficient mice are deficient in NK cells and defective
in IFN- synthesis (6), they provide an excellent in vivo model
system for evaluating the importance of the NK/IFN- pathway in host
defense. We now have assessed the resistance of
c-deficient mice to Toxoplasma gondii, an
intracellular protozoan that is a major cause of opportunistic
infection in immunocompromised individuals (12). In immunocompetent
hosts, early growth of T. gondii is controlled by IFN-
produced largely by IL-12-stimulated NK cells (13, 14). In contrast,
later in infection, CD8+ T lymphocytes appear to be the
major effectors of adaptive resistance, and their protective function
is also thought to result from IFN- synthesis (15, 16). Since
c-deficient mice have been reported to lack NK and
CD8+ T cells, we predicted that they would be highly
susceptible to T. gondii. Unexpectedly, most
c-deficient mice survived the acute phase of infection.
This NK/CD8--independent control of pathogen growth is
dependent on CD4+ T cells and requires host synthesis of
IFN- and IL-12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-deficient mice were generated as
previously reported (6, 9, 10) and back-crossed to C57BL/6 for more
than five generations. Five- to twelve-week-old male and female mice
were used for experiments.
Parasites and experimental infection
Cysts of the avirulent ME49 strain (initially provided by Dr. J. Remington, Palo Alto Research Foundation, Palo Alto, CA) were harvested from the brains of C57BL/6 mice 1 mo following i.p. inoculation with approximately 20 cysts. For experimental infections, mice received 20 ME49 cysts or PBS i.p. Control inoculations with normal brain suspensions failed to elicit detectable inflammatory responses, NK cytotoxicity, or significant increases in cytokine levels (data not shown). Soluble tachyzoite Ag was prepared as described (17).
In vivo assessment of acute infection
Acute tachyzoite growth was assessed using cytocentrifuge smears of peritoneal cells as previously described (18). Differential analyses, including assessment of intracellular T. gondii infection, were performed on 400 to 500 cells per animal.
Cell cultures and serum preparation
Single-cell suspensions were prepared from spleen and peritoneal
cells. Peritoneal cells were cultured at 4 x 105
cells and spleen cells at 8 x 105 per well in 200
µl in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented
with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml),
L-glutamine (2 mM), HEPES (10 mM), and 2-ME (5 x
10-5 M) in the presence or absence of soluble tachyzoite
Ag (5 µg/ml). Supernatants were harvested 72 h later for
determination of levels of IFN- and IL-12.
Blood was collected from mice at the time of sacrifice, allowed to clot at room temperature for 2 h, centrifuged for 5 min at 5000 rpm, and serum was assayed for cytokine levels.
IFN- and IL-12 measurements
IFN- and IL-12 levels were assayed by two-site ELISAs, as
previously described (18), and quantitated by comparison with standard
curves generated with rIFN- or rIL-12 (provided by Genentech, San
Francisco, CA, and Genetics Institute, Cambridge, MA,
respectively).
In vivo cell and lymphokine depletion experiments
For cytokine depletion, mice were injected i.p. 1 day before
infection with 1 mg anti-IFN- mAb XMG6 (rat IgG1) or with 1 mg
anti-IL-12p40 mAb C17.8 (rat IgG2a) (cell lines provided
respectively by Drs. R. Coffman, DNAX, Palo Alto, CA, and G.
Trinchieri, Wistar Institute, Philadelphia, PA). To deplete
CD4+ and/or CD8+ T cells, mice were
treated i.p. with 1 mg of GK1.5 mAb (19) and/or 1 mg 2.43 mAb (20),
respectively, 2 days before and on the day of parasite challenge.
Ascites for the various mAbs were produced by Harlan Bioproducts for
Science (Indianapolis, IN); the Abs were partially purified by ammonium
sulfate precipitation. NK cells were depleted by treating mice with
rabbit anti-asialo-GM1 (Wako Bioproducts, Richmond, VA)
4 days before injection of parasites (1.5 mg i.p.) and on the day of
infection (0.5 mg i.p.). Depletion (as assessed by FACS) was 
90% in
each group.
Flow cytometric analysis
Splenocytes were stained and analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA) using CellQuest software (9). Conjugated anti-CD4 Cy-Chrome (H129.12), anti-CD8 FITC (53–6.7), and anti-NK1.1 PE (PK136) mAb were from PharMingen (San Diego, CA). Before staining, Fc receptors were blocked with anti-CD16/32 (clone 2.4G2, PharMingen).
Statistical analyses
Statistical analyses were performed using an unpaired, two-tailed Student’s t test.
Measurement of NK cell functional activity
To evaluate in vitro NK cell activation, splenocytes were cultured with or without 10 ng/ml (5.6 x 106 U/mg) of rIL-12 (Genetics Institute) and/or 60 IU/ml rIL-2 (Cetus Corp., Emeryville, CA) at 8 x 105 cells/well in triplicate wells in round-bottom 96-well plates. After 24 h, 51Cr-labeled YAC-1 cells (8 x 103/well) were added, and cytotoxicity was assessed as previously described (18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-deficient mice are partially resistant to
infection with T. gondii
Wild-type or c-deficient mice were infected
with T. gondii, and cumulative mortalities were monitored.
As expected, wild-type animals were resistant and exhibited long term
survival (Fig. 1
, A and
B, solid lines). Because of the absence of NK cells and
IFN- production in c-deficient mice (6), we
hypothesized that these animals, which typically live longer than 1 yr,
would rapidly succumb, analogous to IFN--deficient mice, which
routinely die within 2 wk after infection with T. gondii
(18). Surprisingly, although almost all c-deficient mice
died by 140 days following infection (Fig. 1A, broken line),
>50% of them survived for at least 28 days (Fig. 1B,
broken line). Moreover, when the presence of intracellular parasites
was assayed at the site of infection (peritoneal cavity), few (<1%)
infected cells were observed at 7 days following infection in both
wild-type and c-deficient mice, in contrast to the high
percentage of infected cells (>25%) typically observed in
IFN--deficient mice (18). Thus, c-deficient mice
display relatively normal control of early T. gondii
infection. Nevertheless, their increasing mortality at later time
points indicated impaired resistance to chronic infection. This
conclusion is supported by the observation that 30 days after
infection, the surviving c-deficient mice had 
10-fold
more cysts in the brain than did wild-type mice (data not shown). We
hypothesize that this defective resistance to chronic infection relates
to the virtual absence of CD8+ T cells, which are required
for adaptive immunity to T. gondii (15).
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c-deficient mice to acute
T. gondii infection is dependent on IL-12 and IFN-
To clarify the mechanism by which c-deficient
mice control T. gondii infection, we evaluated the synthesis
of the host-protective cytokines, IL-12 and IFN-. When examined at
day 5 postinfection, levels of IL-12 in c-deficient and
wild-type mice were similar in serum and in the supernatants of
splenocytes and peritoneal exudate cells (Fig. 2A). Consistent with a
previous report by Cao et al. (6), minimal IFN- was produced by
splenocytes in most infected c-deficient mice (Fig. 2B). Surprisingly, however, substantial quantities of
IFN- were detected in these mice in serum and at the site of
infection (peritoneal cavity).
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in the control of acute
T. gondii infection in wild-type mice (Fig. 3A), administration of
Abs to IL-12 or IFN- resulted in 100% mortality of
c-deficient animals by day 13 (Fig. 3B), demonstrating that the IL-12/IFN- pathway is
functional and able to limit parasite growth in these mice.
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c-deficient mice
During acute T. gondii infection, NK cells are believed
to play a major role in IFN--mediated resistance. For example, SCID
mice (which have NK cells but not T or B cells) survive for at least 20
days following infection with T. gondii (21, 22). Because
c-deficient mice lack NK1.1.+ cells and NK
cytolytic activity (6), we hypothesized that another cell type must be
important for IFN--dependent resistance in these animals. To
identify the relevant cell population, wild-type and
c-deficient mice were injected 2 days before and on the
day of infection with Abs to CD4, CD8, CD4 + CD8, or
asialo-GM1 (which recognizes NK cells). These treatments
substantially depleted each targeted population (Fig. 4, C vs A,
D vs B, G vs E, and
H vs F) but did not significantly reduce
the survival of wild-type mice (Fig. 3
C), suggesting
that multiple cell types normally can mediate IFN--dependent host
defense. Nevertheless, depletion of either CD4+ or
CD4+ + CD8+ lymphocytes diminished resistance
of c-deficient mice (Fig. 3D) to a
similar extent to that observed when the mice were treated with
anti-IFN- or anti-IL-12 (Fig. 3B). In
contrast, survival of c-deficient mice was not
substantially reduced following depletion of the CD8+ or NK
subpopulations (Fig. 3D), cells that are present at
very low levels in c-deficient mice even before
treatment with Abs to CD8 or asialo-GM1 (6) (Fig. 4, B and F). The diminished resistance of
mice depleted of CD4+ T cells correlated with a loss of
IFN- production. This was demonstrated by showing that, at day 9,
anti-CD4 treatment of c-deficient mice resulted in a
>10-fold decrease in IFN- (mean = 2.33 ng/ml in untreated vs
0.15 ng/ml in anti-CD4-treated mice, p < 0.02). In
contrast, wild-type mice exhibited no decrease in IFN- production
following CD4+ T cell depletion (mean = 7.42 ng/ml in
untreated vs 9.33 ng/ml in anti-CD4-treated mice).
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c-deficient mice (6), it was conceivable that
T. gondii might induce the activation of NK cells. We
therefore measured cytolytic activity in splenocytes of naive and
infected wild-type and c-deficient mice. Activity was
detected in infected wild-type but not c-deficient mice
(Fig. 5A). Moreover,
the addition of IL-2, IL-12, or both of these cytokines did not enhance
cytolytic activity by c-deficient splenocytes (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-deficient mice would
also be highly susceptible to opportunistic pathogens and have now
investigated host defense in these animals. The resistance of
c-deficient mice to acute T. gondii infection
was unexpected and demonstrated their capacity to display protective
immunity despite the unresponsiveness of their T and NK cells to IL-2,
IL-4, IL-7, IL-9, and IL-15. CD4+ T lymphocytes are known
to accumulate with age in c-deficient mice and to
exhibit an activated-memory phenotype (9). We now demonstrate that
these cells can mediate IFN--dependent effector activity in response
to host infection, further arguing that they represent a functional
lymphocyte population. The identification of the signals responsible
for the growth and activation of c-deficient
CD4+ T cells could lead to the development of strategies
for inducing cell-mediated immunity in certain disease settings,
including immunodeficiency states, in which conventional IL-2-dependent
activation is defective.
Previous studies employing SCID mice indicated that NK cells, through
production of IFN-, can mediate defense against acute intracellular
infection when T and B lymphocytes are lacking (11). We now demonstrate
that, in c-deficient mice, which lack NK and
CD8+ T cells, CD4+ T cells emerge as important
mediators for IFN--dependent resistance. Because SCID and
c-deficient mice have abnormal immune systems, it is
conceivable that their mechanisms of control of T. gondiiinfection differ from those operating in wild-type animals.
Nevertheless, it was striking to observe that selective depletion of
either NK cells or of CD4+ + CD8+ T cells in
wild-type mice did not significantly diminish resistance. This suggests
that immunity in wild-type mice infected with T. gondii can
be redundantly subserved by more than one cell lineage and that, early
in infection, both CD4+ T and NK cell populations may serve
as dual effectors of host defense.
Taken together with previous studies of host resistance to acute
T. gondii infection, the present report demonstrates how
c-deficient and SCID mice can provide complementary
tools for genetically isolating and identifying the immunologic
activities of NK and CD4+ T cells. We suggest that the
combined use of both of these immunodeficient mouse strains may provide
a powerful general strategy for assessing the respective contributions
of these cell lineages in immune responses.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: c, -chain; XSCID, X-linked SCID.
Received for publication December 15, 1997. Accepted for publication January 16, 1998.
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References |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu. Rev. Immunol. 14:179.[Medline]
chain mutation results in X-linked severe combined immunodeficiency in human. Cell 73:147.[Medline]
chain. Proc. Natl. Acad. Sci. USA 92:377. chain. Immunity 2:223.[Medline]
chain. Blood 87:956. chain in cytokine signaling and lymphoid development. Immunol. Rev. 148:97.
chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185:189. chain (c): defective Fas ligand expression in c-deficient mice. J. Immunol. 159:4737.[Abstract]
: the major mediator of resistance against Toxoplasma gondii. Science 240:516. synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153:2533.[Abstract]
antibody on the protective effect of lyt-2+ immune T cells against toxoplasmosis in mice. J. Immunol. 144:1954.[Abstract]
, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157:4045.[Abstract]
interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor 
. Infect. Immun. 62:2818. by an intracellular parasite and induces resistance in T-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115.This article has been cited by other articles:
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