By
From the * Department of Medicine, The outcome of murine infection with Leishmania major is regulated by major histocompatibility complex class II-restricted T helper cells. Invariant chain-deficient (Ii
Experimental infection of inbred strains of mice with
Leishmania major remains an exceptional model for
analysis of CD4+ subset differentiation in vivo (1). Control
of disease is dependent on class II-restricted Th type 1 (Th1)
cells and their production of IFN- Leishmania replicates productively only in host macrophages
within an endolysosomal-like compartment that contains
MHC class II molecules, some of which are devoid of invariant chain (10, 11). Infection of macrophages in vitro is associated with diminished MHC class II-dependent presentation of exogenous antigens (12, 13). Although it is unclear
whether this is due to degradation or inappropriate trafficking of MHC class II/peptide complexes from the parasitophorous vacuole (14), competent MHC class II molecules
reach the cell surface as demonstrated by immunofluorescent, functional, and biochemical studies (12, 13, 15). Since invariant chain is involved in both targeting newly synthesized MHC class II molecules to peptide-generating compartments,
and in protecting the peptide cleft during transit from the
endoplasmic reticulum (16), we expected significant impairment in host immune responses to L. major using invariant
chain-deficient (Ii Mice.
Ii L. Major Infection.
Mice were inoculated in both hind footpads with 5 × 105 metacyclic promastigotes of L. major
(WHOM/IR/-/173), maintained and purified as described (22).
Designated mice received 2 mg of mAB XMG1.2 (neutralizing
anti-IFN- Cytokine and Lymphoproliferation Analyses.
Popliteal lymph nodes
draining the site of infection were collected 6-8 wks after infection. Single-cell suspensions were made and triplicate aliquots
were distributed to 96-well round-bottom plates for determinations of cytokine production (106 cells/well) and proliferation
(1.5 × 105 cells/well) in the presence of media or media plus 100 µg/ml soluble Leishmania antigen (SLA). For cytokine analysis,
supernatants were collected after 48 h incubation and quantitated
for IL-2, -4, or IFN- Department of Molecular Genetics and Cell Biology, University of
Chicago, Chicago, Illinois 60637; and § Departments of Medicine and Microbiology & Immunology,
University of California, San Francisco, California 94143
/
) mice have impaired ability to present major histocompatibility complex class II-restricted antigens, and reduced numbers of CD4+ T cells. Despite these deficits, C57BL/6 Ii
/
mice controlled L. major infection comparably to wild-type mice. As assessed by mRNA analysis and in vitro antigen restimulation for IFN-
, Ii
/
mice had normal induction of Th1 subset differentiation
even though antigen-dependent proliferation of their lymph node cells was substantially compromised. In addition, BALB/c Ii
/
mice exhibited a progressive course of infection and
Th2 effector cell development that were comparable to that seen in wild-type BALB/c mice.
We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro. In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-
-or IL-4-secreting T helper
cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic. These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation
signals may facilitate the full differentiation of T helper subsets during limiting presentation of
antigenic peptides.
which is required to
activate macrophages to restrain intracellular replication of
the organism. Studies in T cell- (2) and IFN-
-deficient
(5) mice have confirmed the critical requirements for these
elements in host immunity. MHC class II-deficient mice
from a genetically resistant background are completely susceptible to infection (6, 7), while MHC class I-deficient mice
from a genetically resistant background retain resistance to
infection (8). In contrast to most strains of mice, BALB
animals are unable to contain L. major due to the development of an aberrant Th type 2 (Th2) response during
infection. The absence of class I does not impact Th2 development or susceptibility in BALB/c mice (9).
/
) mice. Unexpectedly, both Th1 and
Th2 responses were maintained in mice on genetically resistant or susceptible backgrounds, respectively, emphasizing the
capacity of the immune system to sustain T cell effector development even under conditions of suboptimal stimulation.
/
mice (17), class II
/
mice (18),
2m
/
mice (19), BALB/c (Jackson Laboratory, Bar Harbor, ME), and
C57BL/6 (Jackson Laboratory) mice were bred and maintained
in the University of Chicago animal facilities. Double mutant Ii
/
2m
/
mice were generated by interbreeding. Mice
were screened by fluorescent cytometry using appropriate monoclonal antibodies for levels of MHC class I, class II, CD4+ and
CD8+ T cells to confirm genotypes. Most mice used in these experiments were fourth generation C57BL/6, or fourth generation
BALB/c. Mice on a 129 × C57 (L. major-resistant) background,
but congenic for H-2d and H-2k, were used where designated.
8/V
4
TCR expanded early after infection by L. major (20), were established using standard methods, and are characterized elsewhere
(Reiner, S., manuscript in preparation). T cells from these mice
recognize an 18-amino acid peptide epitope restricted by I-Ad
from an immunodominant antigen, Leishmanial receptor for activated protein kinase (LACK)1, that is expressed in both the promastigote and amastigote forms of the parasite (21). Thymic selection but not peripheral activation, of the transgene clonotype
occurs on the mismatched H-2k background.
, rat IgG1) intraperitoneally at the time of infection.
Disease progression was monitored weekly by measuring the
footpads with a metric caliper. After 6-8 wks, the popliteal lymph
nodes draining the lesions were collected for the analysis of cell
phenotypes, number, and cytokine production (below). Aliquots
of the single-cell suspensions were analyzed for numbers of CD4+
and CD8+ T cells by fluorescent flow cytometry after staining
with the appropriate mAb's. The footpads were washed in ethanol, rinsed in HBSS, and homogenized in 3 ml M-199 medium.
The spleens were homogenized in 3 ml M-199 medium. Aliquots
were serially diluted into flat-bottom 96-well microtiter plates,
and the plates were sealed with parafilm and incubated at 26°C
for 2 wks. Wells were examined for the presence of the motile
promastigotes by inverted microscopy to determine the tissue
parasite burdens.
using ELISA (PharMingen, San Diego,
CA). For proliferation analysis, wells were pulsed after 72 h with
1 µCi [3H]thymidine, and harvested 16 h later.
which were also present in the competitor
plasmid. The ratio of the competitor to the authentic cytokine
transcripts was used to quantitate mRNA production in vivo.
Antigen Presentation Assays.
Bone marrow cells were flushed
from the femurs of H-2d Ii +/ and
/
littermates, and adherent cells allowed to differentiate for 5-7 d in media containing
30% L929 cell supernatant. The adherent macrophages were harvested after incubation for 30 min in PBS with 2% glucose,
washed, counted, and redistributed to wells containing fresh media, or media containing SLA, viable metacyclic promastigotes, or
freshly thawed and washed amastigotes isolated from infected mice
and stored in 10% DMSO/FCS at
70°C (Amastigotes retain infectivity when stored in this fashion.) Infection was monitored in
concomitantly inoculated monolayers established on tissue-culture chambered slides which were fixed and stained with DifQuik (Dade Diagnostics, Aguada, P.R.). Infection of 5 × 104
macrophages with 2.5 × 105 parasites typically resulted in ~50%
of the cells infected with 1-3 parasites/macrophage. After 20 h, the
antigen- or parasite-pulsed monolayers were washed extensively,
and designated numbers of T cells from TCR transgenic mice
(H-2k) lymph nodes were added to the wells. Supernatants were
harvested after 48 h and assayed for IL-2 and IFN-
production
by ELISA. In designated experiments, splenocytes from Ii +/
or
/
littermates were irradiated and used as APCs. There were
no measured alloreactive responses from the use of H-2k-derived
TCR transgenic T cells with H-2d-derived APCs.
C57BL/6 Ii /
and Ii +/
mice were
challenged with L. major, and the course of infection monitored by measuring the size of the local lesions. Both
groups of mice, in contrast to a concomitantly inoculated
cohort of susceptible BALB/c mice, controlled infection and limited the size of the footpad lesion (Fig. 1 A). As in
immunocompetent mice (24), control of the parasite was
dependent on the production of IFN-
, because treatment
of mice with anti-IFN-
neutralizing mAb abrogated the
resistance phenotype. Delayed-type hypersensitivity responses to injected Leishmania antigens were also comparable in Ii
/
and +/
mice (data not shown). Mice on
two additional MHC backgrounds (H-2d, and H-2k) with
the Ii
/
genotype also maintained resistance to L. major (data not shown).
The Ii /
mice have an expanded population of
CD8+ T cells that, in some instances, have been shown to
exert immunologic activity in experimental leishmaniasis
(25, 26). Since
2m
/
mice have been demonstrated to
control primary L. major infection comparably to wild-type
mice (8), these mice were bred into the Ii
/
background, and the experiment was repeated to rule out a
contribution by the CD8+ T cell compartment. Again,
Ii
/
mice controlled the local lesion as well as Ii +/
or +/+ mice in the presence or absence of
2m (Fig. 1 B).
To confirm that measurements of the local lesions correlated with tissue parasite burdens, dilutions of footpad and
spleen were incubated in vitro and the numbers of recovered parasites were quantitated microscopically (Fig. 2).
Although there was some variability among experiments,
Ii /
mice had comparable or even lower numbers of
recovered organisms than Ii +/
mice in both tissues.
Both Ii
/
and +/
mice, however, had significantly lower numbers of parasites than genetically susceptible
BALB/c mice. The capacity of the Ii
/
mice to control
L. major contrasted sharply with the inability of class II
/
mice to resist infection. These latter mice demonstrated
unrestrained progression of the local lesion and large numbers of parasites in the footpad and spleen after 8 wk of infection (Fig. 2).
Ii
Popliteal lymph node cells
draining the lesions of infected mice were restimulated in
vitro with soluble parasite antigens to assess the qualitative
cytokine response. IFN- was recovered from supernatants
of stimulated cells from infected Ii
/
and
2m
/
Ii
/
mice at levels comparable to or greater than cells from Ii +/
mice (Fig. 3 A). Production of IFN-
in vitro
was reduced to less-than-detectable levels if anti-class II
monoclonal antibody, but not an isotype control antibody,
was included during the period of antigen stimulation (data
not shown). IL-4, which was readily recovered by stimulation of cells from susceptible BALB/c mice, was not detected. Similar results were obtained analyzing the expression of IFN-
and IL-4 transcripts in lymph node cells immediately after removal from infected animals (Fig. 3 B).
Taken together, these data suggest that the deficiency in Ii
expression does not impede the development of a class II-
dependent type 1 immune response to L. major. Th1 effector development was comparable in Ii
/
mice on H-2b,
H-2k, and H-2d backgrounds, despite the differences in efficient assembly of these different class II molecules in the
absence of Ii (27).
T Cell Proliferation and IL-2 Production Induced by L. major Is Ii Dependent.
The unusual intracellular compartment
occupied by Leishmania amastigotes raised the possibility that
peptides from the organisms might normally access class II
in an Ii-independent manner. However, we found that antigen-induced proliferation of lymph node cells taken from
infected animals was significantly impaired in Ii /
, as compared to I +/
, mice, demonstrating substantial Ii-dependence for optimal antigen presentation (Fig. 4). Assays using the same groups of cells showed comparable levels of
antigen-induced IFN-
, revealing dissociation of these two
lymphocyte activities under the conditions used (Fig. 4).
To further characterize the deficiency in presentation of
parasite-derived antigens by Ii /
cells, bone marrow-
derived APCs were established from H-2d Ii
/
and +/
mice and used to measure activation of I-Ad-restricted
TCR transgenic T cells that recognize an epitope from an
immunodominant antigen of L. major designated LACK
(21). After preincubating APCs for 20 h with the 18-
amino acid peptide epitope, production of IL-2 in the supernatant (Fig. 5) was consistently enhanced using Ii
/
,
as compared to Ii +/
, cells, in agreement with prior experiments (17, 28, 29). Processing and presentation of the
LACK epitope from SLA, viable promastigotes, or intracellular amastigotes, however, revealed that only Ii +/
APCs
could induce the transgenic T cells to release significant
levels of IL-2. Thus, optimal activation of these transgenic
T cells by processed antigen is Ii dependent.
IFN-
The striking preservation of the IFN-
response in vivo and in vitro suggested that costimulatory
signals triggered by the organisms might compensate for
the Ii
/
defect in antigen presentation. IL-12, induced
by the intracellular forms of L. major (22), is required for
optimal IFN-
production and control of infection (30),
and is required for Th1 effector cell development (33, 34).
Stimulation of transgenic T cells (Fig. 6) with LACK peptide at levels >0.1 µM presented by normal Ii + APCs, allowed the ready recovery of IL-2, but not IFN-
, from
culture supernatants. Below this concentration of peptide,
neither IL-2 nor proliferation was induced. Using suboptimal concentrations of the peptide, however, the addition
of recombinant IL-12 (rIL-12) induced the dose-dependent production of IFN-
by transgenic T cells not accompanied by IL-2 recovery in the supernatants (Fig. 6).
To confirm that rIL-12 could sustain IFN- production
under conditions where the numbers of class II/peptide
complexes might be limited, experiments were carried out
using the same preparations of normal Ii + APCs in which
either amounts of peptide, numbers of APCs, or numbers
of accessible class II/peptide complexes were titrated. For
the latter condition, dilutions of anti-class II monoclonal
antibody were included in the cultures during the period of
presentation to transgenic T cells. All of the experiments were performed in the presence of rIL-12. Supernatants
were collected after 48 h and analyzed for the presence of
IL-2 and IFN-
(Fig. 7). Under each set of conditions, the
amount of IL-2 recovered was directly related to the degree of class II-dependent stimulation. In contrast, under
these same conditions, the amount of IFN-
recovered was
essentially stable. Thus, as assessed using these transgenic T
cells, and as suggested by cells recovered from infected mice (Fig. 4), IL-12 preserves IFN-
production under
conditions suboptimal for inducing IL-2 or lymphoproliferation.
IL-12 Can Preserve IFN-
To confirm that rIL-12 could similarly
sustain IFN- production from T cells primed on Ii
/
APCs, Ii +/
or
/
APCs were incubated with varying
concentrations of the recombinant LACK antigen and transgenic T cells in the presence of rIL-12. After 48 h, supernatants were analyzed for IL-2 and IFN-
(Fig. 8 A). Consistent with the earlier results using normal APCs under
conditions of low antigenic stimulation, T cells primed on
Ii
/
APCs generated IFN-
that was comparable to
that generated by T cells primed on Ii +/
APCs, although only the latter generated IL-2 that could be recovered in supernatants. Recovery of IFN-
was dependent on both antigen and rIL-12; in the absence of either, no
cytokine could be recovered from transgenic T cells primed
on Ii
/
APCs. This experiment additionally confirms
that the LACK protein antigen, in contrast to the immunogenic LACK peptide (Fig. 5), requires Ii for optimal presentation.
The development of Th1 effector cells is mediated by
IL-12-dependent priming during the initial encounter of
naive T cells with the appropriate peptide/MHC complex (33). To confirm that the IL-12-induced responses
by T cells primed on Ii /
APCs extended to secondary responses, transgenic T cells were primed using antigen-pulsed Ii +/
or
/
APCs in the presence or absence of rIL-12. After 6 d, viable cells were purified and
redistributed to wells containing fresh antigen-pulsed Ii
+/
or
/
APCs in the absence of IL-12. After 48 h,
supernatants were collected and analyzed for IFN-
(Fig.
8 B). As assessed under these conditions, transgenic T cells stimulated with Ii
/
APCs displayed even greater production of IFN-
than T cells stimulated with Ii +/
APCs, and the production of IFN-
was dependent on
the presence of rIL-12 during the primary stimulation
with LACK.
The finding that Th1
development was maintained in Ii /
mice on a resistant
background might be partially explained by effects of low
antigen dose on Th subset development. Antigen dosage
effects on subset differentiation have been demonstrated using TCR transgenic T cells in vitro (35, 36), and have been
supported by observations in L. major infection. Thus, susceptible BALB/c mice have been rendered resistant by either administration of low numbers of parasites (37), or
through interventions such as anti-CD4 monoclonal antibody (38) or sublethal irradiation (39) that decrease the
numbers of responding T cells. The impaired antigen presentation and diminished CD4+ T cell numbers in Ii
/
mice might independently bias Th subset differentiation to
the Th1 phenotype.
To test this prediction, Ii /
mice were bred four
generations onto the susceptible BALB/c background, and
Ii
/
and +/
littermates were infected with L. major.
As assessed by monitoring of the local footpad lesions,
BALB/c Ii
/
suffered disease that was comparable to
Ii +/
littermates, and both differed markedly from the
course of infection in concurrently infected C57BL/6 Ii
/
or Ii +/+ mice (Fig. 9 A). Quantitation of parasites recovered from the footpads and spleen corroborated the lesion
measurements (data not shown). Lymph node cells recovered from infected animals and incubated with SLA in vitro
generated readily recovered IL-4 that was not obtained using lymph node cells from C57BL/6 mice (Fig. 9 B). Thus,
Th2 cell differentiation in vivo was also maintained in the
absence of invariant chain.
To confirm that the threshold for Th2 development
could be maintained in the absence of invariant chain,
LACK-specific transgenic T cells were primed using antigen-pulsed, irradiated spleen cells from Ii + or Ii /
mice in the presence or absence of rIL-4. After 6 d, viable
cells were washed extensively, counted, and redistributed
with freshly prepared, antigen-pulsed, irradiated spleen
cells from the same Ii backgrounds, but in the absence of
rIL-4. After 48 h, the supernatants were analyzed for IL-4 (Fig. 10). Recovery of IL-4 was substantially enhanced
during the secondary stimulation when cells were primed
using Ii
/
APCs, but only when rIL-4 was included
during the primary incubation with LACK.
Infection with L. major is a well-characterized model in
which differentiation of class II-restricted T cells into the
two mature helper subsets is required for expression of the
resistant and susceptible disease phenotype. Ii is required
for stable expression of surface class II molecules and, as
predicted, cells from Ii /
mice have substantially lower
amounts of surface class II that do not assume the compact
conformation that characterizes stable peptide binding (17,
28, 29). The major immunologic consequences are twofold: a severely compromised ability to present processed
antigens via the class II pathway, and a quantitatively and
qualitatively altered CD4+ population due to aberrant selection by thymic epithelial cells unable to present self peptides in a normal manner (40, 41). Despite this drastic effect
on the class II-dependent immune response, we could discern little consequence to the host in generating either Th1
or Th2 responses to L. major. How might we explain this
unpredicted outcome?
One possibility is that effector T cells producing IFN-
or IL-4 are responding to Ii-independent antigens, whereas
proliferating (or nonproliferating, in the case of Ii
/
mice) T cells are responding to Ii-dependent antigens. Although it is not possible to dismiss this hypothesis completely, we think it unlikely for several reasons. First, we
could demonstrate that an immunodominant L. major antigen, LACK, was presented optimally only by cells that
contained Ii and, in studies of infected mice, expansion of
LACK-reactive cytokine-producing T cells can be directly
documented (20). Secondly, our in vitro studies demonstrated the possibility of achieving effector differentiation in
the absence of discernible IL-2 production or proliferation.
Dissociation of effector function from proliferation has been
recently described among CTL clones that can be triggered
to kill targets at doses of peptide which are several orders of
magnitude below doses required for proliferation (42). Lastly,
a recent study of antiviral immunity demonstrated that the
same antigen which required Ii to elicit IL-2 production from
a hybridoma could elicit specific T helper-dependent antibody from infected or immunized Ii
/
mice (43). We,
therefore, consider it unlikely that distinct epitopes underlie proliferative and cytokine-effector responses.
The unique location of L. major within the class II pathway may also influence the ability of Ii /
mice to generate strong anti-parasite immunity. Infection of macrophages
does not limit surface expression of class II, and peptides
eluted from surface class II exhibited comparable diversity
to those from uninfected cells as well as parasite-specific
complexes (15). Comparable experiments have not been
done using infected Ii
/
macrophages, but analysis of
the fine specificity of peptides presented by such cells has
indicated that a varied range of epitopes might be presented
by Ii
/
, as compared to Ii +/+ APCs (44). The route
by which class II complexes exit the ER or cis-Golgi to transit to the surface in Ii
/
cells is not defined (17, 28, 29). Parasite-derived peptides could stabilize class II in the endoplasmic reticulum, as described for some endogenous
class II-dependent antigen presentation pathways (45), or
could load class II via a compartment that recycles from the
cell membrane, as described for influenza hemagglutinin
(46). The ability of the parasite to load class II with peptides
or the potential recruitment of additional epitopes may
contribute to an effective immune response in the absence
of Ii. Our direct examination of an epitope known to be
presented in vivo, however, demonstrated that parasitespecific class II complexes are functionally reduced in Ii
/
infected APCs (Fig. 5).
The effects of Ii deficiency on the T cell compartment
must also be considered in understanding the remarkable
preservation of subset effector differentiation. Peripheral
CD4+ T cells are reduced to ~25% of normal numbers in
Ii /
mice. CD4
/
mice have even lower numbers
of class II-restricted T cells in the periphery, yet readily
control L. major infection (6). Acutely decreasing the number of CD4+ T cells can confer protection on otherwise
susceptible BALB/c mice (38). Thus, it may not be surprising that Ii
/
mice have sufficient numbers of T cells to
mount an adequate Th1 response. The reduction in the
T cell compartment of Ii
/
mice is probably not an essential compensatory mechanism that allows for Th1 differentiation in C57BL/6 Ii
/
mice, however, since BALB/c
Ii
/
mice readily develop Th2 responses despite similarly reduced numbers of CD4+ T cells. In addition to decreased numbers, CD4+ T cells from Ii
/
mice display an
altered phenotype resembling prior activation (CD45RBlo,
CD44hi, and L-selectinlo, with slightly diminished TCR
expression [17] that is probably due to "incomplete" positive selection by abnormal class II/peptide complexes on
thymic epithelial cells (41). The "activated" phenotype of
CD4+ T cells in Ii
/
mice could contribute to a lower
threshold of activation. Resting T cells activated in the
presence of costimulatory ligands responded to lower antigen doses in a manner comparable to activated T cells
(47). Even in the absence of Ii-deficient thymic education,
however, transgenic T cells could be induced to secrete
IFN-
or IL-4 under conditions of limited peptide dose that was incapable of supporting proliferation or IL-2 production.
The finding that IFN- production by Th1 cells (and
IL-4 production by Th2 cells) was comparable or greater
from Ii
/
T cells, despite even fewer class II-restricted
T cells in the lymph nodes, demonstrates striking preservation of T cell effector function in the setting of suboptimal
antigen presentation. Titrating down the number of class
II/peptide complexes on normal APCs in vitro to levels at
which lymphoproliferation could not be sustained was accompanied by no diminution in IFN-
production; similar findings could be demonstrated using Ii
/
APCs as
compared to Ii +/
APCs, again consistent with retention
of effector function at levels of class II/peptide that could
not support IL-2 production. Just as the levels of cytokines
produced in Ii
/
mice would sometimes exceed those
seen in wild-type mice, activation of transgenic T cells in
vitro often yielded slightly higher IFN-
or IL-4 production when antigen was presented by Ii
/
APCs. These
results suggest that weaker antigenic stimulation may favor,
rather than limit, effector cytokine production at some
ranges of antigen dose. The mechanism for this finding is
unclear, but may also underly prior observations on the influence of peptide dose in T helper lineage commitment
(35, 36).
For Th1 development, the preservation or augmentation
of effector function was revealed only in the presence of
rIL-12 and in a dose-dependent manner. The ability of
IL-12 to augment IFN- production in response to antigen
is well known, and was confirmed in IL-12 p40
/
mice, in which IFN-
production was severely impaired
but IL-2 production and lymphoproliferation were unaffected (34). The studies reported here suggest that IL-12, at
least for Th1 development, may be one of the signals implicated in "tuning" the threshold for activation by differing levels of TCR engagement (47). This activity is distinct
from the costimulatory effect of IL-12 on committed Th1
cells, which includes a role in both proliferation and IFN-
production (48). The ability of IL-4 to support robust Th2
development of parasite-specific T cells primed on Ii
/
cells in vitro suggests a similar role for IL-4 in affecting the
threshold for Th2 effector cell function. The efficient development of Th1 and Th2 responses in Ii
/
mice, at
least in this system, implies that the cellular sources for IL-12 and IL-4 in infected mice remain unperturbed by the
loss of Ii.
It is intriguing to speculate that signals produced by cells of the innate immune system, such as IL-12, might be involved in lowering thresholds for distinct types of T cell effector responses to levels below those required for clonal expansion. This might enable maximal numbers of potentially protective naive T cells to be recruited at very low levels of peptide/MHC complex formation, thus limiting the spread of infection and allowing the sampling of multiple peptide/MHC complexes before clonal expansion and competition for growth factors and lymph node niches can occur. The production of inflammatory mediators by microbial pathogens has a major impact on T cell survival (49), so perhaps it should not be surprising that they greatly influence T cell effector functions at low thresholds for activation. In this manner, the immune system can focus attention on the few peptides presented in an inflammatory context, rather than the many peptides presented innocuously.
Address correspondence to S. Reiner, Gwen Knapp Center, University of Chicago, 924 E. 57th St., R420, Chicago, IL 60637-5420.
Received for publication 14 March 1996
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