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Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, 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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- and CD8+
DC subsets. These data suggest that a major effect of adjuvants is to
promote mobilization of Ag-bearing DC to the T areas of lymphoid
tissue, and possibly also to enhance Ag processing by these DC. Thus,
microbial products promote T cell immunity not only through DC
activation for cosignaling, but through improvement in signal 1
delivery. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, that
can act directly on activated Ag-specific T cells to sustain clonal
expansion (3, 4, 5). Activation of APC constitutes another level at which microbial stimuli influence T cell immunity (6, 7, 8). APC have a profound influence on the fate of T cells responding to foreign Ags. The number and quality of costimulatory molecules on APC and other factors, such as the cytokines they produce, not only determine T cell clonal expansion but can also control differentiation toward Th1 or Th2 effector phenotypes (1). Microbial organisms can act on APC directly by binding to pattern-recognition receptors or, indirectly, through inflammatory cytokines or complement activation (7, 8). This can result in modulation of MHC expression, changes in Ag-processing function, increase in expression of costimulatory molecules, and/or alteration of APC localization (7, 8).
All of these observations suggest that microbial stimuli influence how Ags are presented to T cells. However, direct studies of Ag presentation in vivo have been limited in the past by the lack of availability of specific reagents that identify processed Ag bound to MHC molecules in the same form that is recognized by T cells. To address this issue, we have recently made mAbs that specifically recognize processed forms of the model Ag hen egg lysozyme (HEL)4 bound to the murine MHC class II molecule I-Ak (9). One such Ab, C4H3, has already been used to identify B cells lacking antilysozyme Igs as the principal APC that carry processed HEL on their surface shortly after in vivo systemic administration of soluble protein (10). Here, we extend those studies to compare presentation of HEL by B cells and dendritic cells (DC) in inflammatory and noninflammatory conditions of Ag challenge. We find that after systemic administration of HEL, resident splenic interdigitating DC (IDC) do not process and present the Ag at measurable levels. The appearance of a significant number of splenic DC in the T cell areas bearing a high proportion of HEL-loaded I-Ak seems to follow an influx of Ag-bearing IDC precursors that have captured the Ag outside splenic T cell areas. This colocalization of processed Ag-bearing DC with T cells in lymphoid tissue is dependent on the response of the host animal to endotoxin in the Ag preparation. In contrast, the proportion of HEL-loaded I-Ak molecules on B cells is much less influenced by endotoxin. These results are discussed in the context of a model in which microbial stimulation during antigenic challenge promotes immunity by enhancing T cell exposure in secondary lymphoid tissues to activated DC bearing high levels of antigenic peptides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BSA, HEL, and LPS were purchased from Sigma (St. Louis, MO). HEL was conjugated to FITC by coincubation in Na2CO3 (pH 9.0), followed by extensive dialysis against PBS. Proteins were tested for the presence of endotoxin using a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) as recommended by the manufacturer, using purified Escherichia coli LPS as a standard.
Experimental animals
Female CBA, B10.BR, C3H/HeN, and C3H/HeJ mice were obtained from the Division of Cancer Treatment, National Cancer Institute (Frederick, MD) or from The Jackson Laboratory (Bar Harbor, ME). All animals were housed in specific pathogen-free conditions and were used at 6–16 wk of age. CBA, B10.BR, and C3H/HeN mice are normal responders to LPS, whereas C3H/HeJ are hyporesponsive (11).
Mice were injected i.v. through the tail vein or i.p. with HEL, BSA, or LPS in PBS. Proteins were either used at 0.5 µmol/mouse or 8 mg/mouse. Either dose represents virtually equivalent amounts of HEL.
Monoclonal Abs
C4H3 is a rat IgG2b mAb specific for the HEL 46-61 peptide bound to the MHC class II molecule I-Ak and can be used to detect naturally processed HEL (9, 10). It also cross-reacts to a limited extent with I-Ak naturally loaded with certain unidentified peptides that are present in most mouse cells (9). E5H is a rat IgG2a mAb that was isolated during screening for Abs specific for HEL 34-45:I-Ak complexes but that behaves as a pan-anti-I-Ak Ab. 10-3.6 (PharMingen, San Diego, CA) is a mouse IgG2a specific for I-Ak (12).
N418 (13) and HL3 (PharMingen) (14) are hamster IgG mAbs against CD11c.
MOMA-1 (Serotec, Raleigh, NC) is a rat IgG2a against a marker present
on marginal zone metallophilic macrophages (15). 53-6.7 (PharMingen) is
a rat IgG2a anti-CD8 (16). RA3-6B2 (PharMingen) is a rat IgG2a
anti-B220 (17). Irrelevant isotype-matched controls for all Abs
were purchased from PharMingen and used in preliminary experiments to
validate the specificity of staining.
Immunohistochemistry
Frozen spleen sections were processed and stained with the indicated Abs, as previously described (10, 18). Stained sections were photographed on a Zeiss (Thornwood, NY) Axiophot compound microscope using Kodachrome 25 film (Eastman Kodak, Rochester, NY). 35-mm slides were digitized using a SprintScan 35-slide scanner (Polaroid, Cambridge, MA), and the scanned images were assembled using Adobe Photoshop 3.0.5 (Adobe Systems, Mountain View, CA) and ClarisDraw (Claris, Santa Clara, CA) software on a PowerCenter 150 or a PowerCenter Pro 210 computer (Power Computing, Round Rock, TX).
Flow cytometry
Two-color staining of live spleen cell suspensions with C4H3 and
anti-B220 was performed as described (10). For four-color staining,
spleen cell suspensions prepared by collagenase digestion (19) were
washed with PBS/5 mM EDTA, fixed in 1% paraformaldehyde in PBS/EDTA
for 10 min, washed, and kept overnight in washing solution (WS;
PBS/EDTA containing 1% FCS). The next day, cells were stained with
intact C4H3 or C4H3 Fab followed by biotin-conjugated mouse
F(ab')2 anti-rat IgG (Jackson ImmunoResearch, West
Grove, PA) and/or biotin-conjugated mouse anti-rat Ig
(PharMingen). Duplicate samples were stained with biotinylated-10-3.6.
All samples then received a mixture of FITC-conjugated HL3,
PE-conjugated 53-6.7, allophycocyanin-conjugated RA3-6B2, and
TriColor-conjugated streptavidin (Caltag, San Francisco, CA) diluted in
WS with 25 µg/ml rat IgG and 2.4G2 (anti-Fc
RII/III) (20). Ab
incubations ranged from 30 to 60 min on ice; washes and reagent
dilutions were in WS. For intracellular staining, 0.1% saponin was
included in WS. A total of 300,000–500,000 events were collected on a
FACScalibur cytometer (Becton Dickinson, Mountain View, CA) using a
scatter gate on live cells and were analyzed using FlowJo software
(TreeStar, San Carlos, CA). The median C4H3 fluorescence was calculated
for each spleen APC population in each sample. This median fluorescence
was then divided by the corresponding median fluorescence for
I-Ak staining calculated from the sample stained with
10-3.6. This normalization takes into account different levels of
I-Ak expression among different spleen APC and also
controls for any differences in total I-Ak expression in
different mice. It has the added advantage that various APC with
different total surface MHC class II levels can be compared directly
for the proportion of I-Ak loaded with HEL 46-61. For
analysis of HEL presentation, data are often displayed as a percentage
of the normalized C4H3 fluorescence of control mice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that B cells lacking specific Ig
receptors for HEL are able to process and present this Ag in vivo after
i.v. administration of HEL protein to mice (10). To extend these
observations, a more detailed kinetic analysis of B cell presentation
of HEL was performed by ex vivo staining of spleen cells from
HEL-injected mice with C4H3, an mAb specific for I-Ak
containing the 46-61 fragment of HEL (9, 10). As shown in Fig. 1
, an increase in C4H3 staining over
background could be detected on B220+ cells as early as
2 h after systemic administration of HEL protein. As reported
(10), the staining was unimodal, reflecting MHC class II presentation
of HEL by essentially all splenic B cells, regardless of receptor
specificity (Fig. 1A). The level of C4H3 staining increased
rapidly and peaked at 8–12 h after HEL administration (Fig. 1, A and B). During the same time period, total
surface I-Ak levels increased modestly and in much smaller
proportion, as compared with C4H3 staining (Fig. 1B). This
indicated that most of the rise in C4H3 reactivity reflected the
accumulation of HEL 46-61-loaded I-Ak molecules on the B
cell surface and was not due just to an increase in overall MHC class
II expression that included self peptide-containing molecules
cross-reactive with this reagent. C4H3 staining of B cells then
declined progressively from 12 to 48 h after HEL administration
(Fig. 1, A and B), despite the fact that total
I-Ak levels remained relatively constant (Fig. 1B), reflecting the specific loss of HEL-loaded molecules.
Similar kinetics of HEL presentation by B cells were seen in other
lymphoid organs (data not shown, and 10).
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During these studies, we became aware of possible endotoxin
contamination of commercial protein preparations, such as the HEL used
here. Because endotoxin is a known activator of B cells that can
enhance MHC class II molecule expression (21) and Ag presentation (22),
we examined the HEL and control proteins used in our studies for the
presence of LPS, finding substantial levels in all batches examined
(Table I). To address the effect of
endotoxin contamination on HEL presentation by B cells in vivo, we
compared the presentation of two batches of HEL, HEL 2
and HEL 3, that contain a high or low degree of
endotoxin contamination, respectively (Table I). A total of 0.5 µmol
of either batch was injected into mice, and spleens were isolated
4 h later, a time point at which C4H3 staining of B cells is
half-maximal (Fig. 1
, A and B) and, thus, at
which any potentiating or diminishing effects of LPS on HEL
presentation should be easily detectable. Purified E. coli
LPS was used to control for the possible effect of endotoxin on
presentation of the self-peptides that generate C4H3 cross-reactivity.
As additional controls, each Ag was administered in parallel to C3H/HeN
and C3H/HeJ mice, two related strains that are normal- or
low-responders to endotoxin, respectively (11). B cells from all
HEL-injected mice showed higher surface C4H3 staining than B cells from
PBS-injected controls (Fig. 1C). The increase in staining
was HEL-specific because it was not seen with purified endotoxin (Fig. 1C). Importantly, the increase in B cell staining was
independent of the batch of HEL that was used, and the difference in
staining between C3H/HeN and C3H/HeJ mice was not very pronounced (Fig. 1C). These results suggest that endotoxin contamination has
relatively little effect on the proportion of B cell MHC class II
molecules loaded with HEL after in vivo exposure to the Ag. In
agreement with this conclusion, the deliberate addition of high amounts
of exogenous LPS to HEL also did not affect theproportion of HEL-loaded
I-Ak molecules on the surface of B cells after in vivo
challenge, although it increased the absolute level of I-Ak
expression (data not shown).
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We next examined the kinetics of HEL processing and
presentation and the tissue localization of the positive
cells using preparations of HEL administered to CBA, C3H/HeN, and
C3H/HeJ mice. This analysis was directed at comparing B cell to DC
presentation, which is complicated by the observation that in sections
of lymphoid tissues from various control H-2k animals not
exposed to HEL, C4H3 stains a subpopulation of IDC that appears to
express elevated levels of I-Ak loaded with self-peptides
that generate C4H3 cross-reactivity (Fig. 2A, and C.R.S., unpublished
observations). In animals injected only 4 h previously with HEL,
C4H3 clearly delineated the B cell areas of the spleen (Fig. 2, B and C), yielding a follicular staining pattern
similar to that seen after staining with 10-3.6, an
anti-I-Ak Ab whose binding is peptide-independent (Fig. 2D). The extensive follicular C4H3 staining was
HEL-dependent because it was not seen with spleen sections from mice
injected with a control protein, BSA (Fig. 2A) and is in
agreement with the flow cytometric data reported above (Fig. 1). In
sharp contrast to these follicular areas, however, C4H3 did not stain
cells within the periarteriolar lymphoid sheaths (PALS) in the same
sections beyond the background level (Fig. 2B; outlined),
despite the presence of numerous strongly I-Ak+ IDC in
these areas (Fig. 2D; outlined). The lack of an increase in
the extent or intensity of IDC staining at 4 h after HEL
administration was surprising considering that there is no anatomical
barrier to entry of soluble blood-borne Ags into the PALS (23). We
confirmed that there was no limitation of IDC access to HEL in these
experiments by documenting uniform staining for fluorescein throughout
the spleen as early as 10 min after i.v. injection of FITC-conjugated
HEL (Fig. 2, G and H). Thus, despite having high
surface MHCclass II expression and access to the Ag, the bulk of IDC in
the inner PALS do not present an immunochemically detectable level of
HEL peptide:I-Ak complexes at a time when such presentation
by most of the B cells in follicles is easily observed by the same
method.
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, B and
C, arrows). These large C4H3+ cells were found
primarily in the outer PALS or within follicular areas (Fig. 2, B and C, arrows). Double staining with C4H3 and
anti-TCRß revealed that several of these cells straddled the
boundary between the T cell and B cell areas (Fig. 2C,
arrows). Many were positive for CD11c, a DC marker (data not shown) but
differed from the subset of IDC responsible for HEL-independent
staining (see above) in that the latter are clustered near the central
arterioles (compare Fig. 2A with 2B).
A noticeably different staining pattern was seen 24 h after HEL
injection. B cell staining was faint or not detectable, in accord with
the flow cytometric data (Fig. 1), and the large C4H3+
cells in the outer PALS or follicles were no longer conspicuous. In
contrast, C4H3 staining of cells within the PALS was now prominent
(Fig. 2E), in the form of compact accumulations
("nests") of C4H3+ cells with dendritic morphology
(Fig. 2E, inset) located around the central
arterioles, where resident IDC are typically found. These nests of
C4H3+ cells seen after HEL challenge were more prominent
and much more extensive than those seen in sections from BSA-injected
controls (compare Fig. 2E with 2A), suggesting
that they were not just resident IDC showing the HEL-independent
staining noted above.
To confirm the identification of these C4H3+ cells as IDC
and to further refine our analysis of the kinetics of their
accumulation, serial spleen sections from CBA mice injected with HEL at
different times before sacrifice were double stained with C4H3 and
MOMA-1, a marker for metallophilic macrophages (15), or C4H3 and CD11c
(N418) as a DC marker (13). As mentioned above, C4H3 stained a fraction
of IDC in sections from control animals (compare Fig. 3A with 3B).
However, compared with the controls, a much greater proportion of DC in
the PALS was positive for C4H3 at 8 h after HEL injection (Fig. 3, C and D). In addition, occasional
C4H3+ DC were found in aggregates interrupting the marginal
zone (Fig. 3, C and D, arrowhead), possibly
corresponding to splenic immature DC (13, 24).
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, E and F). These
cells were now found primarily in the inner PALS and corresponded to
the nests previously seen by single staining with C4H3 (Fig. 2E). Thus, it appears that presentation of HEL by DC in vivo
is an asynchronous process. Cells bearing processed Ag are first found
in the outer PALS/follicular regions and, with time, accumulate in the
inner PALS. This is in contrast to presentation by B cells, which
proceeds in a synchronous fashion in follicular areas.
Presentation of HEL by IDC in vivo involves the
CD8- and CD8+ subsets
Flow cytometry was used to phenotype more accurately the splenic
APC bearing processed HEL after systemic Ag delivery and to examine
levels of surface Ag display. We used B220 as a marker for B cells and
CD11c as a marker for DC (Fig. 4A). The latter were further
subdivided into the CD8- and CD8+
subsets (25, 26, 27), which, in untreated C3H mice, were present at a ratio
of 
5 CD8- to 1 CD8+ DC (Fig. 4A). DC express higher levels of I-Ak than B
cells, as expected (Fig. 4A). To account for this difference
in expression and to compensate for any treatment-induced changes in
total levels of I-Ak expression, all flow cytometric
analyses of C4H3 staining after HEL injection were conducted after
normalizing to the staining obtained with an anti-I-Ak
Ab (see Materials and Methods for details).
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2-fold more C4H3
staining than B cells from PBS-injected controls after compensating for
total I-Ak expression (Fig. 4B, 4 h). C4H3
staining could also be seen on CD11c+ cells at this time
point, but the relative increase in normalized fluorescence compared
with PBS-injected mice was lower for DC than for B cells (Fig. 4B, 4 h), as reported (10). Interestingly, the relative
increase in C4H3 staining of DC was more noticeable in the
CD8+ than the CD8- subset (Fig. 4B, 4 h).
A different picture emerged at 24 h after HEL injection (Fig. 4B, 24 h). As expected from the immunohistochemical
analysis, there was a decrease in B cell staining and a parallel
increase in bulk DC staining as compared with PBS-injected controls.
This was especially noticeable in the CD8+ DC subset:
CD8+ DC from HEL-injected mice had 3.5-fold more
surface C4H3 staining than the same subset from PBS-injected controls,
after accounting for differences in total I-Ak expression
(Fig. 4B, 24 h). The increase in staining over the
controls was always greater for CD8+ DC than for
CD8- DC, although the extent of the difference between
the two subsets was variable and, in some cases, small (see Fig. 5). Virtually all of the HEL-loaded
I-Ak molecules in both subsets appeared to be at the
cell surface because a comparison of intact and permeabilized cells did
not show evidence for the presence of a substantial intracellular
pool of C4H3 epitopes (data not shown).
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Because the HEL preparations used in these experiments contained
LPS, we assessed whether the striking delayed accumulation of
C4H3+ IDC in the PALS depended on the presence of endotoxin
in the Ag preparation. Collections of C4H3+ cells, similar
to those shown in Fig. 2E, were seen using
endotoxin-contaminated HEL 24 h after challenge of several
H-2k mouse strains that have a normal susceptibility to LPS
(Fig. 2E, and data not shown). Nests of C4H3+
IDC were also seen after injection of LPS alone, but they were
generally smaller and less intensely stained than those seen after HEL
challenge (data not shown; see Fig. 5) and may represent DC with
increased levels of MHC class II (28). In marked contrast to these
LPS-responder animals, immunization of LPS-hyporesponsive C3H/HeJ mice
with HEL resulted in a pattern of C4H3 staining at 24 h that
differed little from that seen in uninjected or BSA-injected controls
(Fig. 2F). There were occasional groups of C4H3+
IDC, but they were much smaller than those seen in normal LPS-responder
controls given endotoxin-containing HEL (compare Fig. 2F
with 2E).
To obtain quantitative information on the effects of endotoxin
contamination on HEL presentation by DC, we repeated the experiment
reported in Fig. 1C and measured C4H3 staining of splenic DC
subsets at 24 h after Ag administration. In C3H/HeN mice, the
HEL 2 batch (high LPS content) reproducibly gave an increase
in C4H3 epitopes as a proportion of total I-Ak, relative to
PBS-injected controls (Fig. 5). This increase was observed for both
CD8- and CD8+ DC and was not simply due
to an LPS-induced accumulation of DC presenting self-peptides that
generate C4H3 cross-reactivity, because injection of an equivalent dose
of purified LPS into the same mice either had no effect
(CD8+ DC) or increased the C4H3:10-3.6 ratio only
slightly (CD8- DC; Fig. 5). In contrast to C3H/HeN
mice, the same batch of HEL administered to age- and sex-matched
C3H/HeJ mice resulted in no significant increase in C4H3 staining of
either DC subset, suggesting that HEL presentation by DC depends on
responsiveness to endotoxin (Fig. 5). Cells from C3H/HeN mice injected
with the HEL 3 batch of Ag, containing less LPS, showed only
slightly higher normalized C4H3 staining than the PBS-injected
controls, and this effect was lost in the C3H/HeJ mice, again
indicating that DC staining by C4H3 was favored by the effect of
endotoxin (Fig. 5). This was further supported by the observation that
the deliberate addition of significant amounts of purified LPS to
HEL 3 allowed presentation by DC, even in C3H/HeJ mice (data
not shown). Thus, in contrast to B cells, the ability of DC in lymphoid
tissues to display a significant proportion of Ag-loaded MHC class II
molecules on their surface after in vivo Ag challenge depends on the
presence of endotoxin in the Ag preparation and on the responsiveness
of the mice to this inflammatory stimulus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), as recently reported for collagen
(34). Being unable to adequately remove this contamination, we opted to study presentation of different batches of HEL in mice susceptible or resistant to the effects of LPS. We show here a dramatic effect of this microbial product on presentation of peptide:MHC class II molecule complexes by DC in the T cell areas (PALS) of the spleen. The number of IDC bearing a high proportion of processed HEL peptide:I-Ak complexes on their surface increased markedly in LPS-responder mice injected with HEL contaminated with endotoxin. The appearance of DC displaying processed HEL was not observed using Ag with low endotoxin levels and LPS-hyporesponsive mice. In contrast, substantial levels of HEL peptide:I-Ak complexes were found on virtually all B cells in follicular areas of the white pulp, irrespective of LPS effects. Thus, for MHC class II Ag presentation, B cell display of processed Ag lacked a clear dependence on signals arising from an infectious process, whereas the exposure of T cells to DC with high ligand density required such stimulation.
HEL presentation by the vast majority of B cells lacking anti-HEL
surface Ig occurred rapidly and synchronously and then was no longer
detectable by 48 h after challenge. The transient nature of HEL
presentation by B cells is likely to reflect the natural turnover of
MHC class II molecules on the surface of these APC following bolus
administration of Ag and the ensuing transient loading of newly
synthesized MHC class II molecules with processed Ag (35, 36). The 20-h
average half-life of HEL-loaded MHC class II molecules on B cells
lacking anti-HEL Ig in LPS-sensitive mice is consistent with
measurements in vitro made in the presumed absence of LPS (36). LPS did
not noticeably increase the proportion of B cell I-Ak
molecules loaded with the HEL 46-61 determinant (Fig. 1C),
although it did lead to an increase in total MHC class II levels and,
therefore, in the absolute number of HEL-loaded molecules. Remarkably,
4 h after HEL injection, at a time when virtually all B cells
displayed a significant proportion of HEL-loaded complexes, C4H3
staining of PALS regions was conspicuously absent, despite the presence
of numerous I-Ak+ IDC in those areas (Fig. 2). Because
FITC-HEL easily diffuses into the PALS immediately after i.v. injection
(Fig. 2), the lack of IDC staining is not likely to be due to poor
access of the cells to Ag. Instead, it may reflect a markedly reduced
ability of DC in the PALS to endocytose and/or process intact Ags,
consistent with the traditional model proposing that IDC are the
product of a differentiation process that converts Ag-capturing
immature DC in peripheral tissues or outside the splenic PALS into
Ag-presenting mature cells in T cell areas (37, 38).
Such maturation-related loss of processing activity provides one
possible explanation for the gradual increase in staining of IDC over
the next 20 h, namely slower processing of HEL protein by these
more mature IDC as compared with B cells or DC in the peripheral PALS/B
cell area. It is also possible that immigrant DC simply act as a source
of cell-bound Ag for the IDC already in the T cell area, in accord with
a recent report (39). However, the dramatic redistribution of
N418+ cells within the spleen after HEL administration and
the sequential appearance of C4H3+ cells first in the outer
PALS and then the T areas raise the intriguing possibility that most of
the C4H3 staining of IDC observed 24 h after HEL administration is
due to an influx of IDC precursors that acquired the Ag at an immature
stage elsewhere. Maturational signals, such as provided by endotoxin,
may relieve a block in invariant chain degradation, allowing loading of
MHC class II with peptides and the transport of these complexes to the
plasma membrane of the arriving DC (40, 41, 42). DC present in vascularized
organs can travel via the blood to the spleen (43), and DC efflux from
nonlymphoid organs is accelerated by inflammatory stimuli, such as LPS
(44, 45). Cells that captured the Ag outside the spleen would enter
this lymphoid organ via terminal arterioles opening at the boundary
between the white and the red pulp. Similarly, resident immature DC in
the spleen are also found in this location, in nests interrupting the
marginal zone (13, 24). Centripetal migration of either population from
the red/white pulp boundary into the PALS would explain the fact that
C4H3+ DC are first seen in the outer PALS/follicular
regions and only later in the inner PALS (Figs. 2 and 3). Migration
from the red pulp/marginal zone to the PALS is actively promoted by
microbial stimuli (18, 28), explaining why endotoxin-containing batches
of HEL work best in allowing detection of HEL-loaded I-Ak
molecules on IDC (Fig. 5). This possible centripetal migration of DC to
the PALS is entirely consistent with our prior observation that
IL-12-producing DC are found in the outer PALS at early times after
challenge with microbial products, before being seen in clusters around
the central arterioles (18).
Endotoxin also causes loss of IDC following redistribution to the PALS, through induction of apoptosis (28, 46). Thus, LPS may act as a double-edge sword, allowing enhanced T cell interaction with HEL-presenting IDC, but also causing the rapid disappearance of these presenting cells. The dramatic effects of endotoxin contaminants on DC presenting function may help explain the discrepancies in the literature regarding ex vivo presentation to T cells by DC exposed to model Ags in vivo (47, 48), because the extent of endotoxin contamination of those Ags is not known. Although the precise mechanism by which LPS induces DC migration and maturation has not been addressed in the present study, previous work has suggested that the induction of TNF and IL-1 release (44) may be involved. The latter cytokines have been clearly documented to enhance migration of DC from various tissue sites (44, 49, 50).
The CD8+ DC subset showed the highest proportion of
I-Ak molecules loaded with HEL peptides after injection of
HEL protein containing endotoxin. These cells have been postulated to
belong to a special lymphoid lineage of DC that is involved in
induction of T cell tolerance rather than activation (51, 52, 53). It has
recently been proposed that CD8+ "lymphoid" DC do
not have the capacity to internalize and process foreign proteins (53),
in marked contrast to the results presented here. However,
CD8+ DC also include cells that respond rapidly to
microbial stimulation in vivo and that are the predominant APC in mouse
spleen producing IL-12 after in vivo challenge with an extract of
Toxoplasma gondii (18). This same challenge and other
microbial stimuli also lead to dramatic up-regulation of B7-1 and B7-2
molecules on CD8+ DC (C.R.S., unpublished observations).
Overall, this phenotype suggests a role of some CD8+ DC
in induction of immunity rather than tolerance. This notion is entirely
consistent with the ability of CD8+ DC to load a
significant proportion of MHC class II molecules with peptides from
endotoxin-contaminated HEL, which can be viewed as a model microbial
Ag. It is possible, therefore, that CD8+ DC normally act
to tolerize T cells against peripheral Ags in uninflamed tissue, but
that at least some of these cells or their precursors can differentiate
in response to infection into APC able to promote effective immune
stimulation.
One limitation of the technical approach used here involving Ab
reagents directed against specific peptide:MHC molecule complexes
involves cross-reactions with MHC molecules associated with peptides
other than the desired fragment of the model Ag. For example, C4H3
binds to a subset of I-Ak molecules loaded with certain
self-peptides (9) and, therefore, the absolute background of C4H3
staining is influenced by the total amount of I-Ak present
on the APC. To account for any differences in total I-Ak
levels, C4H3 staining measured by flow cytometry (Figs. 1C,
4, and 5) was normalized against the staining obtained with an Ab
against I-Ak that is peptide-independent in its binding
(see Materials and Methods). Dividing the normalized
fluorescence of APC from HEL-injected mice by that of the same APC in
control mice further accounts for any differences in unspecific
staining between APC types and/or mouse strains. The data presented in
this manner represent C4H3 epitopes as a proportion of total
I-Ak rather than total C4H3 epitopes and presumably reflect
the efficiency of HEL processing by each APC type.
For related technical reasons, the lack of enhanced C4H3 staining of IDC in noninflammatory conditions also does not mean that MHC class II presentation of exogenous Ags by DC in vivo occurs only in the presence of these stimuli. Presentation may take place continuously in the T cell areas of lymphoid tissues by incoming DC bearing processed peripheral Ags and even possibly by Ag processing at low efficiency by resident IDC themselves. However, the small number of cells involved, what is probably a much lower absolute level of processing, and the slow rate of turnover of IDC in the PALS together prevent us from visualizing this process with C4H3 until it is markedly accelerated by inflammatory stimuli. Nonetheless, even a few undetectable Ag-bearing DC immigrating into lymphoid organs in noninflammatory conditions of Ag administration could have a significant impact on Ag-reactive T cells, consistent with the ability of T cell responses to be initiated by very few of these APC (38, 54, 55).
Many studies in the past have focused attention on the role of inflammatory signals in promoting conversion of "tolerogenic" presenting cells into "immunogenic" presenting cells through up-regulation of costimulatory proteins. Others have emphasized the role of microbial signals in production of cytokines regulating effector phenotype (6, 7, 8). More recently, evidence has accumulated that these agents promote maturation of the MHC class II-related processing machinery of DC (40, 41, 42). The data reported here add to these several effects of microbial signals or "adjuvants" by documenting a dramatic increase in the number of DC bearing high levels of processed Ag in the T areas of the spleen upon coadministration of soluble HEL and endotoxin. This effect may arise through enhanced migration of DC into the PALS, and possibly also via activation of the processing machinery of these precursors of IDC. These findings imply that one major mechanism by which adjuvants enhance immunity is by promoting the colocalization of Ag-bearing DC with potentially responsive T cells within secondary lymphoid tissues. This results in a dramatic increase in the access of T cells to ligands for their receptors, which can complement the costimulatory and cytokines signals also provided by these activated presenting cells, together resulting in effective induction of immunity. Together with previous data showing up-regulated CD80/86 levels on these cells (28) and their production of IL-12 (18), these findings suggest that signals from infectious agents coordinately enhance both costimuli (signal 2) (6) and Ag display (signal 1) for T cells, resulting in strong immune responses of the appropriate effector class.
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Acknowledgments |
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Footnotes |
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2 Current address: Immunobiology Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K.
3 Address correspondence and reprint requests to Dr. Ronald N. Germain, Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1892. E-mail address:
4 Abbreviations used in this paper: HEL, hen egg lysozyme; DC, dendritic cells; IDC, interdigitating DC; PALS, periarteriolar lymphoid sheath.
Received for publication December 15, 1998. Accepted for publication March 18, 1999.
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