Dendritic Cells in the Induction of Protective
and Nonprotective Anticryptococcal
Cell-Mediated Immune Responses
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J Immunol 2000; 165:158-167; ;
doi: 10.4049/jimmunol.165.1.158
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References
Sean K. Bauman, Kasie L. Nichols and Juneann W. Murphy
Dendritic Cells in the Induction of Protective and
Nonprotective Anticryptococcal Cell-Mediated Immune
Responses1
Sean K. Bauman, Kasie L. Nichols, and Juneann W. Murphy2
S
ince the first description of dendritic cells (DC)3 in 1973
(1), it has become increasingly apparent that they are a
heterogeneous population of APC (2). Most investigations
examining the phenotype in parallel with the function of DC have
been performed on splenic DC despite the fact that most immune
responses to infectious agents are initiated in draining lymph nodes
(3, 4). Splenic DC can be divided phenotypically and functionally
into two distinct populations, CD8␣⫺ and CD8␣⫹ DC. The
CD8␣⫺ cells are myeloid DC (MDC), and the CD8␣⫹ cells are
lymphoid DC (LDC) (5–10). Although both populations can induce CD4⫹ T cell proliferation in vitro, MDC are better inducers
of CD4⫹ T cell proliferation (7). An additional DC subset that is
efficient at stimulating cell-mediated immune (CMI) responses is
Langerhans cells, which are found in lymph nodes, but not spleens,
of naive mice (11, 12). In contrast, LDC from the spleen play a role
in maintenance of peripheral tolerance through induction of apoptosis in activated, Fas-expressing CD4⫹ T cells (7). Based on the
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190
Received for publication November 5, 1999. Accepted for publication April 20, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health Grants AI15716,
HL59852, and T32AI07364-07.
2
Address correspondence and reprint requests to Dr. Juneann Murphy, Department of
Microbiology and Immunology, University of Oklahoma Health Sciences Center,
P.O. Box 26901, BMSB 1053, Oklahoma City, OK 73190. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cells; CMI, cell-mediated immune;
MDC, myeloid DC; LDC, lymphoid DC; CneF, cryptococcal culture filtrate Ag;
HKC, heat-killed cryptococci; DTH, delayed-type hypersensitivity; FSC, forward
scatter.
Copyright © 2000 by The American Association of Immunologists
present understanding of the functional characteristics of the DC
subsets, a reasonable hypothesis is that the development of different types of immune responses involving CD4⫹ T cells, such as a
protective vs a nonprotective response to an infectious agent,
might be the result of the dominance of one DC subset over another during the induction phase of the response.
Cryptococcus neoformans is a yeast-like organism that causes
frequently fatal meningitis in immunocompetent and immunocompromised humans (reviewed in Ref. 13). CMI responses against
C. neoformans have been extensively studied (14 –18). Because
DC are the most effective APC for inducing CMI responses (19 –
21), they most likely have a critical role in the initiation of the
anticryptococcal CMI response. Yet, the involvement of DC in
CMI responses to C. neoformans has not been assessed. We have
previously shown that one can induce either protective or nonprotective CMI responses to C. neoformans in mice depending on the
immunogen (22). Immunization with the soluble cryptococcal culture filtrate Ag (CneF) emulsified in CFA induces activated CD4⫹
Th1 cells (23, 24) that provide protection against C. neoformans
infection (22). In contrast, immunization with heat-killed cryptococci (HKC) in CFA induces different functionally defined T cell
populations (24 –27) that do not provide protection (22) and can
even exacerbate the disease (28) (J. W. Murphy, unpublished observations). Both immunizations induce anticryptococcal CMI responses detectable by footpad swelling, although the response induced by HKC-CFA is generally half that induced by CneF-CFA
(27).
The present studies used a murine immunization model to test
whether different subsets of DC are associated with protective vs
nonprotective CMI responses to C. neoformans Ags. Our data
show that MDC and Langerhans cells are increased in draining
0022-1767/00/$02.00
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Dendritic cells (DC) can be divided into three subsets, Langerhans cells, myeloid DC (MDC), and lymphoid DC (LDC), based upon
phenotypic and functional differences. We hypothesized that different DC subsets are associated with the development of protective vs nonprotective cell-mediated immune (CMI) responses against the fungal pathogen, Cryptococcus neoformans. To test this,
mice were immunized with protective and/or nonprotective immunogens, and DC subsets in draining lymph nodes were assessed
by flow cytometry. The protective immunogen (cryptococcal culture filtrate Ag-CFA), in contrast to the nonprotective immunogen
(heat-killed cryptococci-CFA), the nonprotective immunogen mixed with the protective immunogen (cryptococcal culture filtrate
Ag ⴙ heat-killed cryptococci-CFA), or controls, stimulated significant increases in total leukocytes, Langerhans cells, MDC, LDC,
and activated CD4ⴙ T cells in draining lymph nodes. The protective immune response resulted in significantly increased levels of
anticryptococcal delayed-type hypersensitivity reactivity and activated CD4ⴙ T cells at the delayed-type hypersensitivity reaction
site. Draining lymph node LDC:MDC ratios induced by the protective immunogen were significantly lower than the ratios induced
by either immunization in which the nonprotective immunogen was present. In contrast, mice given the nonprotective immunogen
had LDC:MDC ratios similar to those of naive mice. Our data indicate that lymph node Langerhans cells and MDC are APC
needed for induction of the protective response. The predominance of LDC in mice undergoing nonprotective responses suggests
that lymph node LDC, like splenic LDC, are negative regulators of CMI responses. In addition to showing DC subsets associated
with functional differences, our data suggest that the LDC:MDC balance, which can be modulated by the Ag, determines whether
protective or nonprotective anticryptococcal CMI responses develop. The Journal of Immunology, 2000, 165: 158 –167.
The Journal of Immunology
159
lymph nodes undergoing the protective CMI response. By comparison, LDC are the predominant APC present during induction
of the nonprotective anticryptococcal CMI response. Moreover,
the protective response led to increased numbers of activated
CD4⫹ T cells in the draining lymph nodes and at an anticryptococcal DTH reaction site, whereas the nonprotective response did
not. The addition of the nonprotective immunogen to the protective
immunogen (CneF⫹HKC-CFA) resulted in 1) similar DC populations in the draining lymph nodes as observed in HKC-immunized mice, 2) reduced numbers of activated CD4⫹ T cells in the
draining lymph nodes and at the anticryptococcal DTH reaction
site compared with the protective immunogen, 3) a reduction in the
magnitude of the anticryptococcal DTH response compared with
the protective immunogen, and 4) a reduction in the survival time
after challenge with C. neoformans compared with the protective
immunogen. These combined data indicate that the DC subset balance relates to the type of CMI response (protective vs nonprotective) that develops in the draining lymph nodes during immune
response induction and that the Ag can modulate this balance.
Briefly, sponges were cut into 17 ⫻ 18 ⫻ 10-mm blocks before rehydrating
with sterile HBSS containing 100 U of penicillin/ml and 100 mg of streptomycin/ml. Three days after immunization with CneF-CFA, HKC-CFA,
CneF⫹HKC-CFA, or control treatment, mice were anesthetized, and
sponges were implanted s.c. through an incision on the animals’ shaved
backs. The incisions were closed with wound clips. Four days after implantation, one sponge was injected with 0.1 ml of CneF, and the other was
injected with 0.1 ml of saline. The sponges were removed at 24 h after
injection.
Materials and Methods
Flow cytometric analysis
Mice
Portions of the lymph node single-cell suspensions from individual mice
(three mice per group) were immunolabeled with the designated mAbs. For
the isotype control, another portion of each cell suspension was pooled
within a treatment group before undergoing the staining procedure. Individual cell suspensions and pooled cell suspensions were treated with antiCD16/32 (HB197, American Type Culture Collection, Manassas, VA) for
30 min at 4°C to block Fc receptors and thereby reduce nonspecific staining
of cells. After centrifugation, cells were incubated for 30 min at 4°C in
wash buffer (PBS, 0.1% NaN3, and 0.1% BSA) containing fluorochromelabeled mAbs or isotype control mAbs. After washing, the cells were fixed
with 1% paraformaldehyde. The mAbs used in these studies included
DEC205-FITC (NLDC-145, American Type Culture Collection), 33D1
(American Type Culture Collection) visualized by goat anti-rat-PE (Caltag,
South San Francisco, CA), CD11c (N418, American Type Culture Collection) visualized by goat anti-hamster-PE (Caltag), CD8␣-biotin (CT-CD8a,
Caltag) visualized by ultraAvidin-APC (Leinco, Ballwin, MO), CD11bTriColor (M1/70, Caltag), CD40-PE (3/23, Caltag), CD80-PE (RMMP-2,
Caltag), CD86-PE (GL1, PharMingen), I-Ak-PE (14V.18, Caltag),
FasL-PE (MFL3, PharMingen), CD4-TriColor (CT-CD4, Caltag), CD45R/
B220-biotin (RA3-6B2, Caltag), macrophage-TriColor (F4/80, Caltag),
CD4-FITC (CT-CD4, Caltag), CD45RB-PE (16A, PharMingen), and fluorochrome-labeled isotype control mAbs (Caltag). After fixing, 100,000 –
250,000 cells were analyzed using a FACSCalibur flow cytometer in the
core facility and WinMDI 2.8 software. The percentage of positive cells
was the percentage of the designated cell population with fluorescent intensity above the fluorescent intensity of cells treated with isotype control
mAbs. The total number of positive cells in each sample was determined
by multiplying the percent positive cells for the sample by the total number
of leukocytes in the sample, which was assessed by hemocytometer counts.
Maintenance of endotoxin-free conditions
All reagents for injection into animals were tested for endotoxin content
and were not used if the endotoxin level was detectable by the Limulus
amebocyte chromogenic assay (BioWhittaker, Walkersville, MD; minimal
detectable level of endotoxin, 0.1 ng endotoxin/ml). Sterile tissue culture
plasticware was used whenever possible. All glassware was baked at 180°C
for 3 h to destroy endotoxin.
Cryptococcal Ags, immunization, and infection
CneF from C. neoformans 184A was prepared as previously described
(23). HKC were prepared by incubating C. neoformans isolate 184A for 1 h
at 60°C (26). Mice were injected s.c. at two sites at the base of the tail with
0.2 ml of a 1:1 emulsion of CneF in CFA, sterile physiological saline in
CFA, 107 HKC in CFA, or CneF plus 107 HKC in CFA. As controls for
each immunization, five additional mice were immunized to assess the
level of delayed-type hypersensitivity (DTH) responsiveness induced. For
survival studies, 7 days after immunization mice were injected i.v. with
9 ⫻ 104 viable 184A C. neoformans cells in 0.2 ml of saline, and the
percent survival was followed.
Detection of anticryptococcal DTH responsiveness
Mice were euthanized before surgical removal of sponges. Sponges were
put in Stomacher bags (Tekmar, Cincinnati, OH) with enzyme cocktail
(400 U of collagenase/ml; Sigma, St. Louis, MO), then homogenized with
three 10-s pulses on a Stomacher 80 Lab Blender (Tekmar) at 15-min
intervals (29). During 15-min intervals, the sponge homogenates were incubated at 37°C. Following the disaggregation step, the sponge homogenates were filtered through 390-␮m pore size nylon screens followed by
passage through 140-␮m pore size nylon screens and washed with HBSS.
The erythrocytes in the sponge homogenates were lysed by treatment with
Tris-NH4Cl (17 mM Tris and 139.7 mM NH4Cl), and the remaining cells
were washed once with HBSS. Viable cell counts were made using trypan
blue dye exclusion and a hemacytometer.
At 7 days after immunization, the hind footpads of the mice were measured
and then injected with 30 ␮l of saline in the left footpad and 30 ␮l of CneF
in the right footpad. The footpads were measured 24 h after the challenge
injection. The increase in footpad thickness was calculated by subtracting
the difference in swelling in the 0 and 24 h measurements of the salineinjected footpad from the difference in swelling between the 0 and 24 h
measurements of the CneF-injected footpads.
Statistical analysis
Preparation of single-cell suspensions
Lymph node cells from naive CBA/J mice were divided into four
major populations based on forward scatter (FSC) and DEC205
labeling
Lymph nodes (superficial inguinal, lumbar, and popliteal) were removed at
the indicated times and pushed through a wire mesh screen to prepare
single-cell suspensions. The screens and cell suspensions were incubated
for 30 min on ice in 100 U/ml collagenase D (Roche, Indianapolis, IN) in
HBSS/5% FCS. The cell suspensions were washed and resuspended in
HBSS/5% FCS. This method of DC isolation was selected because the
procedure extracts all subsets of DC (9, 11). Furthermore, we avoided
culture steps that might change the DC surface to a more activated
phenotype.
Sponge implantation and injection with Ag
Gelatin sponges (Gelfoam sterile absorbable gelatin sponge, Upjohn,
Kalamazoo, MI) were surgically implanted under aseptic conditions (23).
Means, SEMs, and ANOVA with the Newman-Keuls multiple comparison
posttest were used to analyze the data. Survival data were analyzed by
Kaplan-Meier survival statistics. Groups were considered statistically different if p ⱕ 0.05.
Results
Lymph node cells from naive mice immunolabeled with the
DEC205 mAb were analyzed by flow cytometry for fluorescence
and FSC (size). Four distinct populations of cells were observed;
they were, DEC205⫺FSClow (63%), DEC205highFSClow (30%),
DEC205highFSChigh (2.6%), and DEC205lowFSChigh (1.7%; Fig.
1). The DEC205⫺FSClow population was 78% CD4⫹ T cells and
8% B cells (B220⫹) and was negative for F4/80 (macrophage
marker). The DEC205highFSClow population was 95% CD8␣⫹ T
cells and 8% F4/80⫹ cells. As previously defined by Vremec et al. (9)
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Female CBA/J mice were purchased from The Jackson Laboratory (Bar
Harbor, ME) and maintained in the animal facility at the University of
Oklahoma Health Sciences Center. Three to five mice per group were used
in all experiments except the survival experiment, which had 10 –20 mice/
group. The mice were 7–10 wk of age at the beginning of each experiment.
Sponge retrieval and disaggregation
160
and Salomon et al. (11), DEC205highFSChigh and DEC205lowFSChigh
cells were considered to be two distinct populations of DC. The
DEC205highFSChigh and DEC205lowFSChigh cells combined made up
⬃5% of lymph node cells.
on their expression of MHC class II (Table I). The
DEC205highFSChighCD8␣low cells were MHC class IIhigh, and the
DEC205highFSChighCD8␣high cells were MHC class IIint (Table I).
Vremec and Shortman (9) found the expression of CD11b and
CD8␣ on splenic DC to be almost inversely related; however, the
markers were not completely mutually exclusive. When we analyzed
DC subsets for CD11b expression, we found that most
DEC205highFSChighCD8␣low cells (M1) were CD11b⫹ (78% positive; Fig. 2B), whereas about half of DEC205highFSChighCD8␣high
cells (M2) were CD11blow (47%; Fig. 2C). There was a broad range
of expression levels of CD11b on the DEC205highFSChighCD8␣high
cell population. The DEC205lowFSChigh population was CD8␣⫺
(Fig. 2D) and mostly CD11blow (Fig. 2E). Therefore, based on cell
surface staining patterns and the large size of the cells, the cell subsets, DEC205highFSChighCD8␣lowCD11b⫹, DEC205highFSChigh
CD8␣highCD11blow, and DEC205lowFSChighCD8␣⫺CD11blow, were
considered DC (9, 11, 30). Based on this, three distinct DC subsets
from the lymph nodes of naive CBA/J mice can be identified by flow
cytometry, myeloid DC (MDC, DEC205highFSChighCD8␣low
CD11b⫹), lymphoid DC (LDC, DEC205highFSChighCD8␣high
CD11blow), and Langerhans cells (DEC205lowFSChighCD8␣⫺
CD11blow). These definitions are in keeping with the observations
described by others (9, 11) and were used in the investigations
presented here for designating, MDC, LDC, and Langerhans cells.
Identification of three DC subsets within the lymph node
FSChigh populations from naive mice
Expression of costimulatory molecules, MHC class II, and
molecules associated with apoptosis on MDC, LDC, and
Langerhans cells from lymph nodes of naive mice
By gating on DEC205highFSChigh cells and analyzing for CD8␣
expression, we found that the DEC205highFSChigh population of
cells could be divided further into two subsets, CD8␣low (M1) and
CD8␣high (M2; Fig. 2A). These subsets could also be divided based
Cell surface receptors, such as MHC class II, CD40, CD80, and
CD86, which are necessary for effective Ag presentation to T cells,
were assessed in each DC population. All three DC populations
expressed the DC markers, 33D1 and CD11c, and MHC class II,
FIGURE 2. Cell surface phenotype reveals three distinct lymph node DC populations. After collagenase digestion of lymph nodes, the cell surface
phenotype of FSChigh cells was determined by three-color flow cytometric analysis. DEC205highFSChigh and DEC205lowFSChigh cells were gated based on
DEC205 and FSC staining and were further analyzed for CD8␣ and CD11b expression by analyzing 100,000 –250,000 events. Open histograms show
isotype-matched control staining. Cells were gated on DEC205highFSChigh cells and analyzed for CD8␣ expression (A). M1, low density expression of
CD8␣; M2, high density expression of CD8␣ (A). DEC205highFSChigh cells were gated based on CD8␣low (B) or CD8␣high (C) and were analyzed for
CD11b expression. DEC205lowFSChigh cells were analyzed for CD8␣ (D) and CD11b (E) expression. Results are representative of five independent
experiments with similar results.
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FIGURE 1. Identification of four distinct populations of cells in the
lymph nodes of naive mice. After collagenase digestion of lymph nodes,
cells were stained with the mAb DEC205 and analyzed by flow cytometry.
Cells (100,000 –250,000 events were collected) were analyzed for DEC205
expression and FSC (size). Percentages are of total cells. Results are representative of five independent experiments with similar results.
DC IN ANTICRYPTOCOCCAL CMI RESPONSES
The Journal of Immunology
161
Table I. Cell-surface phenotype of three lymph node DC populations from naive micea
MFI of Designated Markersb
MDC
LDC
Langerhans
cells
MFI of Isotype
Controls
99 ⫾ 6
628 ⫾ 7
7⫾1
15 ⫾ 2
25 ⫾ 3
5⫾1
250 ⫾ 3
712 ⫾ 14
1336 ⫾ 205
17 ⫾ 1
22 ⫾ 1
52 ⫾ 2
16 ⫾ 1
507 ⫾ 3
2⫾0
3⫾0
8⫾1
8⫾1
3⫾1
NA
3 ⫾ 0.6
3⫾0
3 ⫾ 0.3
3 ⫾ 0.3
1276 ⫾ 311
69 ⫾ 11
600 ⫾ 76
3 ⫾ 0.3
8⫾1
4⫾0
41 ⫾ 2
3 ⫾ 0.3
3 ⫾ 0.3
3 ⫾ 0.3
Cell Surface Expression
DC markers
DEC205
FSChigh
CD8␣
CD11b
33D1
CD11c
MHC class II
I-Ak
Costimulatory molecules
CD40
CD80
CD86
Apoptosis
FasL
19 ⫾ 2
12 ⫾ 2
166 ⫾ 32
5⫾1
4⫾1
100 ⫾ 7
7⫾1
high
high
low
⫹
48 ⫾ 4
4⫾1
3 ⫾ 0.3
DEC205 FSC CD8␣ CD11b , DEC205 FSC CD8␣ CD11b , and DEC205 FSC CD8␣⫺CD11blow
cells were identified as MDC, LDC, and Langerhans cells, respectively. MDC, LDC, and Langerhans cells were gated and
analyzed for additional cell-surface markers. A total of 100,000 –250,000 events were analyzed using four-color flow cytometric
analysis.
b
Mean-fluorescence intensity (MFI) ⫾ SEM.
a
high
Kinetics of leukocyte influx into draining lymph nodes induced
by protective and nonprotective cryptococcal immunizations
Before assessing the levels of various DC populations in the lymph
nodes of mice immunized with C. neoformans Ags, we determined
the total numbers of cells in draining lymph nodes. This was done
by immunizing mice with the protective immunogen (CneF-CFA)
or the nonprotective immunogen (HKC-CFA) or by treating mice
with saline-CFA or nothing (naive) as controls. CneF-CFA-immunized mice had significantly more leukocytes in their draining
lymph nodes than did HKC-CFA-immunized ( p ⬍ 0.01), salineCFA-treated ( p ⬍ 0.05), or naive ( p ⬍ 0.01) mice by 12 h after
immunization (Fig. 3). The differences were even more pronounced by 18 h after immunization, when CneF-CFA-immunized
mice had almost twice as many leukocytes as either HKC-CFAimmunized ( p ⬍ 0.001) or saline-CFA-treated ( p ⬍ 0.001) mice
and 4 times as many leukocytes as naive mice ( p ⬍ 0.001; Fig. 3).
HKC-CFA-immunized mice ( p ⬍ 0.001) and saline-CFA-treated
( p ⬍ 0.001) mice had significantly more leukocytes than naive
mice (Fig. 3). HKC-CFA-immunized and saline-CFA-treated mice
had similar numbers of leukocytes in their draining lymph nodes at
all times assessed (Fig. 3). Based on these data, the 18 h point was
selected for additional experiments to gain an understanding of the
cell populations in draining lymph nodes of mice undergoing the
two different anticryptococcal CMI responses.
high
low
low
high
MDC and LDC in draining lymph nodes during the induction of
protective and nonprotective anticryptococcal CMI responses
Because MDC and LDC from the spleen have been described to
have stimulatory and regulatory functions, respectively, we were
interested in determining whether the draining lymph node DC
populations were differentially associated with protective vs nonprotective anticryptococcal CMI responses. We found that CneFCFA-immunized and saline-CFA-treated mice had similar percentages of MDC (Fig. 4A) and similar percentages of LDC (Fig. 4B).
In contrast, HKC-CFA-immunized mice had a significantly reduced percentage of MDC (Fig. 4A) and a significantly increased
percentage of LDC (Fig. 4B) compared with saline-CFA-treated
( p ⬍ 0.001) or CneF-CFA-immunized ( p ⬍ 0.001) mice. Considering that the CneF-CFA-immunized mice had significantly
more cells in their draining lymph nodes than control-treated or
HKC-CFA-immunized mice, it is not surprising that the number of
MDC (Fig. 4C) in CneF-CFA-immunized mice was significantly
FIGURE 3. Kinetics of leukocyte influx into draining lymph nodes in
response to cryptococcal immunizations. Draining lymph nodes were removed at 3, 12, and 18 h from naive or saline-CFA-treated mice or mice
immunized with CneF-CFA or HKC-CFA. After collagenase digestion, the
number of leukocytes was assessed by hemocytometer counts. Results are
representative of five independent experiments with similar results. Error
bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences (ⴱ, p ⬍ 0.05).
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but at different levels, as indicated by the intensities of staining
(Table I). MDC and Langerhans cells displayed high density expression of MHC class II, whereas LDC had an intermediate level
of MHC class II expression. MDC expressed a high density of
CD86, with intermediate levels of CD40 and CD80. LDC and
Langerhans cells displayed intermediate levels of CD86, but low
levels of CD40 and CD80. To determine which DC population
may be able to induce apoptosis, we also determined the levels of
expression of FasL (CD95L) on the surface of cells in each population. LDC had more FasL than either MDC or Langerhans cells
(Table I). MDC, LDC, and Langerhans cells comprised 18, 44, and
37%, respectively, of the FSChigh cells in the lymph nodes from
naive CBA/J mice.
high
162
DC IN ANTICRYPTOCOCCAL CMI RESPONSES
increased compared with that in saline-CFA-treated controls ( p ⬍
0.01) or HKC-CFA-immunized mice ( p ⬍ 0.01). In addition,
CneF-CFA-immunized mice had significantly more LDC (Fig. 4D)
than saline-CFA-treated controls ( p ⬍ 0.01) or HKC-CFA-immunized mice ( p ⬍ 0.01). The percentages of MDC- and LDC-expressing costimulatory molecules (CD40, CD80, and CD86) were
similar in immunized and control-treated mice (data not shown),
whereas HKC-CFA-immunized mice had significantly higher percentages ( p ⬍ 0.006) of LDC-expressing FasL (15.7 ⫾ 0.8) than
CneF-CFA-immunized (12.1 ⫾ 0.5) or control-treated (12.3 ⫾
0.1) mice. The percentages of MDC-expressing FasL were similar
in immunized and control-treated mice (data not shown).
Activation of CD4⫹ T cells by immunization with protective and
nonprotective cryptococcal immunogens
Langerhans cell migration into draining lymph nodes as a result
of cryptococcal immunization
Considering that MDC have stimulatory function and their numbers were highest in the lymph nodes from CneF-CFA-immunized
mice, we would predict that lymph nodes from those mice would
have a greater number of CD4⫹ T cells with an activated phenotype (CD4⫹CD45RBlow) (31, 32) than would lymph nodes from
HKC-CFA-immunized or control-treated mice. We stained draining lymph nodes from immunized and control groups of mice with
mAbs to detect activation markers on CD4⫹ T cells. We found, as
expected, that the lymph nodes from CneF-CFA-immunized mice
had significantly more ( p ⬍ 0.001) activated CD4⫹ T cells
(1.33 ⫾ 0.08 ⫻ 106) than those from HKC-CFA-immunized
(0.80 ⫾ 0.04 ⫻ 106), saline-CFA-treated (0.75 ⫾ 0.07 ⫻ 106), or
naive (0.35 ⫾ 0.02 ⫻ 106) mice. When CD4⫹ T cells were analyzed for CD69 expression, which is another activation marker for
In our immunization model it is possible that Langerhans cells may
be taking up Ag because Ags were injected in close proximity to
the epidermis. If this is happening, we would expect to see increased numbers of Langerhans cells in draining lymph nodes
from CneF-CFA-immunized mice compared with those in HKCCFA-immunized or control-treated mice. To assess this, the cells
in draining lymph nodes from the various groups of mice were
immunolabeled for Langerhans cells. We found that draining
lymph nodes from CneF-CFA-immunized mice had significantly
increased numbers of Langerhans cells compared with draining
lymph nodes from saline-CFA-treated ( p ⬍ 0.001) or HKC-CFAimmunized ( p ⬍ 0.001) mice (Fig. 5), whereas HKC-CFA-immunized and saline-CFA-treated mice had similar numbers of Langerhans cells in their draining lymph nodes (Fig. 5). The
percentages of Langerhans cells in draining lymph nodes were
similar for all treatment groups, ranging from 40 –50% of the
FSChigh cells. Also, the percentages of Langerhans cells expressing costimulatory molecules (CD40, CD80, and CD86) and FasL
were similar in immunized and control-treated mice (data not
shown).
FIGURE 5. Langerhans cell migration into draining lymph nodes in response to cryptococcal immunizations. Draining lymph nodes were removed from mice at 18 h after immunization with CneF-CFA or HKCCFA or treatment with saline-CFA. After collagenase digestion, cells were
stained for Langerhans cells as described previously, and 100,000 –250,000
events were analyzed by three-color flow cytometric analysis. Results are
representative of five independent experiments with similar results. Error
bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences (ⴱ, p ⬍ 0.05).
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FIGURE 4. Effects of cryptococcal immunizations on lymph node MDC and LDC
populations. Draining lymph nodes were removed from mice at 18 h after immunization
with CneF-CFA or HKC-CFA or treatment
with saline-CFA. After collagenase digestion, cells were stained for MDC and LDC as
described previously, and 100,000 –250,000
events were analyzed by three-color flow cytometric analysis. The percentages of MDC
(A) and LDC (B) of DEC205highFSChigh cells
present in draining lymph nodes are shown.
The total number of MDC (C) and LDC (D)
present in draining lymph nodes are indicated. Results are representative of five independent experiments with similar results. Error bars represent SEMs, and ANOVA with
the Newman-Keuls multiple comparison
posttest was used to determine significant differences (ⴱ, p ⬍ 0.05).
The Journal of Immunology
FIGURE 6. LDC:MDC ratio. The total number of LDC was divided by
the total number of MDC from draining lymph nodes of immunized or
control mice. Results are representative of five independent experiments
with similar results. Error bars represent SEMs, and ANOVA with the
Newman-Keuls multiple comparison posttest was used to determine significant differences (ⴱ, p ⬍ 0.05).
LDC:MDC ratio in draining lymph nodes of mice undergoing
protective and nonprotective anticryptococcal CMI responses
The draining lymph nodes from CneF-CFA-immunized mice also
had high numbers of LDC. In fact, CneF-CFA-immunized mice
had higher numbers than HKC-CFA-immunized or control-treated
mice. Based on data derived using splenic DC, it has been postulated that if both MDC and LDC present the same Ag, then Ag
presentation by MDC is dominant over presentation by LDC (34).
If this were true, then as the ratio between LDC and MDC is
reduced, one would expect an increase in effective Ag presentation. In addition to the absolute numbers of incoming DC, the ratio
of LDC:MDC also may be an important factor in determining the
developmental pathway of T cells. With this in mind we calculated
the LDC:MDC ratios for each of the experimental groups. Our
prediction proved to be true. Mice undergoing induction of a protective anticryptococcal CMI response (CneF-CFA immunized)
displayed a significantly reduced LDC:MDC ratio compared with
naive mice ( p ⬍ 0.001) or HKC-CFA-immunized mice ( p ⬍
0.001; Fig. 6). Further support was provided by the saline-CFA
control group. Mice treated with saline-CFA develop an antiMycobacterium CMI response (35), and their LDC:MDC ratio was
significantly reduced compared with that in naive mice ( p ⬍
0.001). The LDC:MDC ratio in the saline-CFA group was equivalent to the reduced ratio in the CneF-CFA group (Fig. 6). In
contrast, the mice immunized with the nonprotective immunogen
(HKC-CFA) had an LDC:MDC ratio that was not significantly
reduced from the ratio in naive mice.
We further reasoned that the HKC-CFA, which preferentially
induces more LDC than MDC in their draining lymph nodes,
should have an effect on the ratio of LDC:MDC in the CneF-CFAimmunized mice. We predicted that HKC added to the CneF-CFA
immunization would prevent the reduction in the LDC:MDC ratio.
If this proved true, then we would expect there also to be reduced
numbers of activated CD4⫹ T cells in mice that were given HKC
emulsified with CneF-CFA. As shown by the data in Fig. 6, when
HKC were added to the CneF-CFA immunization (CneF⫹HKCCFA), the LDC:MDC ratio was similar to that in lymph nodes of
naive mice or HKC-CFA-immunized mice. Having observed that
the HKC prevented the reduction in the LDC:MDC ratio normally
stimulated by CneF-CFA, we next wanted to know whether the
FIGURE 7. Effects of cryptococcal immunizations on activation of
CD4⫹ T cells in draining lymph nodes. Draining lymph nodes were removed from mice at 18 h after immunization with CneF-CFA, HKC-CFA,
or CneF⫹HKC-CFA or treatment with saline-CFA. A total of 100,000 –
250,000 events were analyzed by two-color flow cytometric analysis for
activated CD4⫹ T cells, identified as falling into the lymphocyte light
scatter gate and CD4⫹CD45RBlow. Results are representative of at least
three independent experiments with similar results. Error bars represent
SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest
was used to determine significant differences (ⴱ, p ⬍ 0.05).
HKC added to CneF-CFA would also have an effect on the numbers of activated CD4⫹ T cells as we had predicted.
Influence of HKC on activation of CD4⫹ T cells in draining
lymph nodes induced by CneF-CFA
As we had observed earlier, there were significant increases in the
numbers of activated CD4⫹ T cells in the draining lymph nodes
from CneF-CFA-immunized mice compared with those in the
lymph nodes from HKC-CFA-immunized ( p ⬍ 0.001) or salineCFA-treated ( p ⬍ 0.001) mice (Fig. 7). Addition of HKC to the
CneF-CFA immunogen (CneF⫹HKC-CFA) resulted in a significant reduction in the numbers of activated CD4⫹ T cells in the
draining lymph nodes compared with numbers of activated CD4⫹
T cells in the draining lymph nodes of mice given CneF-CFA
alone ( p ⬍ 0.001). The total number of activated CD4⫹ T cells in
the draining lymph nodes of the CneF⫹HKC-CFA group was similar to the total number of that cell population in the draining
lymph nodes from HKC-CFA-immunized or saline-CFA-treated
mice (Fig. 7).
Influence of HKC on the level of anticryptococcal DTH
reactivity induced by CneF-CFA
Because fewer activated CD4⫹ T cells were produced in the draining lymph nodes of mice that were immunized with CneF⫹HKCCFA than were produced in the draining lymph nodes of mice that
received CneF-CFA, we hypothesized that the level of DTH reactivity in the Ag-injected footpad of the CneF⫹HKC-CFA-immunized mice would be reduced compared with the footpad responses
in CneF-CFA-immunized mice. The data in Fig. 8 show that
indeed mice immunized with the mixture of HKC and CneF-CFA
expressed weaker anticryptococcal DTH reactivity than mice
immunized with CneF-CFA alone ( p ⬍ 0.001). The combined
immunogens (CneF⫹HKC-CFA) induced anticryptococcal DTH
responses that were significantly elevated over reactions in the
HKC-CFA group ( p ⬍ 0.001; Fig. 8).
Influence of HKC on the numbers of activated CD4⫹ T cells at
a DTH reaction site induced by CneF-CFA
To further show that there is a direct relationship between the
induction and expression phases of the anticryptococcal DTH response, we determined the numbers of activated CD4⫹ T cells that
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CD4⫹ T cells (33), we found that there were more CD4⫹ T cells
that were CD69⫹ in CneF-CFA-immunized mice than in any other
group (data not shown).
163
164
infiltrated into a DTH reaction site in mice that had received either
CneF-CFA, HKC-CFA, or the combined immunogen. The prediction was that when more activated CD4⫹ T cells are produced in
the draining lymph nodes, there should be more activated CD4⫹ T
cells at the DTH reaction site. To assess this we used a sponge
model. Sponges implanted in immunized mice when injected with
the recall Ag act as surrogate DTH reaction sites and allow one to
study the cells and cytokines involved in the expression or efferent
phase of an anticryptococcal CMI response (23, 36 –39). Gelatin
sponges were implanted into the backs of immunized mice, and
after a sufficient time (4 days) for the sponges to become vascularized, one sponge was injected with CneF, and the other sponge
was injected with saline. Twenty-four hours after sponge injection
the sponges were removed and disaggregated. The cells were immunolabeled and evaluated by flow cytometry. Previous studies
using this sponge model have shown that mice immunized with the
protective immunogen (CneF-CFA) have significantly more CD4⫹
Th1 cells than control-treated mice (saline-CFA) (23). In the
present study, mice immunized with the protective immunogen
(CneF-CFA) had significantly more activated CD4⫹ T cells
(CD4⫹CD45RBlow at the site of an ongoing anticryptococcal DTH
reaction than did HKC-CFA-immunized ( p ⬍ 0.01), CneF⫹HKCCFA-immunized ( p ⬍ 0.05), or saline-CFA-treated ( p ⬍ 0.01)
mice (Fig. 9). In contrast, the numbers of activated CD4⫹ T cells
at the anticryptococcal DTH reaction site in mice immunized with
HKC-CFA or CneF⫹HKC-CFA or treated with saline-CFA were
not significantly different from each other (Fig. 9). When CD4⫹ T
cells were analyzed for CD62L (L-selectin) expression, which is
lost on activated T cells (40), we found that there were more CD4⫹
T cells that were CD62L⫺ in CneF-CFA-immunized mice compared with any other group (data not shown).
FIGURE 9. Effects of protective and nonprotective anticryptococcal
CMI responses on the influx of activated CD4⫹ T cells into an anticryptococcal DTH reaction site. Sponges implanted into control and immunized
mice were injected with saline or CneF at 7 days after immunization, and
24 h after sponge challenge the leukocytes were obtained from the disaggregated sponges and immunolabeled for flow cytometry. A total of
125,000 events were analyzed by two-color flow cytometric analysis for
activated CD4⫹ T cells, identified as falling into the lymphocyte light
scatter gate and CD4⫹CD45RBlow. Results are represented as the mean
increase in activated CD4⫹ T cells, which was calculated by subtracting
the number of activated CD4⫹ T cells in the saline-injected sponge from
the number of activated CD4⫹ T cells in the CneF-injected sponge. Results
are representative of at least two independent experiments with similar
results. Error bars represent SEMs, and ANOVA with the Newman-Keuls
multiple comparison posttest was used to determine significant differences
(ⴱ, p ⬍ 0.05).
survival over a 30-day period after immunizing mice and then
infecting them 7 days after immunization i.v. with 9 ⫻ 104 viable
C. neoformans cells (Fig. 10). CneF-CFA-immunized mice survived significantly longer (mean survival time, 26.5 ⫾ 1.2 days)
than mice immunized with CneF⫹HKC-CFA (mean survival time,
21.9 ⫾ 1.6 days; p ⬍ 0.02), HKC-CFA (mean survival time,
16.1 ⫾ 0.4 days; p ⬍ 0.0001), or treated with saline-CFA (mean
survival time, 14.6 ⫾ 1.1 days; p ⬍ 0.0001). Mice immunized with
CneF⫹HKC-CFA survived significantly longer than HKC-CFAimmunized ( p ⬍ 0.0001) or saline-CFA-treated ( p ⬍ 0.001) mice,
whereas HKC-CFA-immunized and saline-CFA-treated mice had
similar survival times. The results of this survival study with
CneF-CFA-immunized, HKC-CFA-immunized, and saline-CFAtreated mice are similar to our previous observations (22).
Influence of HKC on the survival of mice immunized with
CneF-CFA
Having observed that fewer activated CD4⫹ T cells were present
at the anticryptococcal DTH reaction site in mice immunized with
CneF⫹HKC-CFA than in mice immunized with only the protective immunogen (CneF-CFA), we predicted that mice immunized
with CneF⫹HKC-CFA would have decreased survival times compared with mice that received the protective immunogen alone
(CneF-CFA). Indeed, this is what we saw when we monitored
FIGURE 10. Effects of protective and nonprotective cryptococcal immunogens on the survival of infected mice. Ten to 20 mice per group were
immunized with CneF-CFA, HKC-CFA, or CneF⫹HKC-CFA or were
treated with saline-CFA. Seven days after immunization the mice were
infected i.v. with 9 ⫻ 104 viable 184A C. neoformans cells, and the percent
survival was followed. Kaplan-Meier survival analysis was used to determine significant differences (ⴱ, p ⬍ 0.05).
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FIGURE 8. DTH responsiveness induced by different cryptococcal immunogens. DTH responsiveness was elicited by CneF in mice immunized
8 days earlier with CneF-CFA, saline-CFA, HKC-CFA, or CneF⫹HKCCFA. Results are representative of seven independent experiments with
similar results. Error bars represent SEMs, and ANOVA with the NewmanKeuls multiple comparison posttest was used to determine significant differences. ⴱ, p ⬍ 0.001 for the CneF-CFA group compared with all other
groups. ⴱⴱ, p ⬍ 0.001 for the HKC-CFA group compared with the
CneF⫹HKC-CFA and saline-CFA groups. ⴱⴱⴱ, p ⬍ 0.001 for the
CneF⫹HKC-CFA group compared with the saline-CFA group.
DC IN ANTICRYPTOCOCCAL CMI RESPONSES
The Journal of Immunology
Discussion
nodes from naive mice. In fact, immunization with the nonprotective immunogen resulted in significant increases over controls in
the percentage of LDC in the lymph nodes, and concomitant with
this, levels of activated CD4⫹ T cells did not exceed those in
lymph nodes from control mice. The activated CD4⫹ T cells that
were induced by the protective immunogen could be attracted to
the site of cryptococcal Ag injection in the periphery, as shown by
the increase in numbers of activated CD4⫹ T cells at the DTH
reaction site in mice immunized with the protective immunogen. In
mice given the nonprotective immunogen, the small numbers of
activated CD4⫹ T cells produced in the lymph nodes resulted in
low numbers of activated CD4⫹ T cells at the DTH reaction site.
By immunizing mice with the nonprotective immunogen mixed
with the protective immunogen, we were able to shift the responses
induced by the protective immunogen toward the direction of the
response induced by the nonprotective immunogen. The shifts in
the parameters were based, as would be predicted, on our interpretation presented above. The mixture of nonprotective and protective immunogens simultaneously affected several parameters.
Addition of the nonprotective immunogen to the protective immunogen resulted in 1) down-regulation of the total numbers of
cells in the draining lymph nodes, 2) inhibition of the reduction in
the LDC:MDC ratio, 3) reduction in the numbers of activated
CD4⫹ T cells in the draining lymph nodes as well as at the DTH
reaction site, and 4) down-modulation of the protective immune
response, as shown by the inability of mice immunized with the
combination of immunogens to survive as long as mice immunized
with the protective immunogen alone.
Based on the current understanding of cell-mediated immunity
(3, 44) and our data, we propose a model for the induction of a
protective anticryptococcal CMI response. The model consists of
the following sequence of events: 1) Ag (CneF) with the adjuvant
(CFA) cause the production of proinflammatory cytokines by resident tissue cells, establishing an inflamed microenvironment (reviewed in Refs. 12, 41, and 45); 2) Langerhans cells take up Ag at
the site of Ag deposition (i.e., epidermis and s.c. spaces) (reviewed
in Refs. 12 and 41); 3) uptake of Ag and exposure to proinflammatory cytokines induce Langerhans cell to undergo maturation
(i.e., increased MHC class II expression, increased costimulatory
molecule expression, etc.) (reviewed in Refs. 12 and 41) with the
acquisition of a phenotype similar to that of MDC (11); 4) concomitant with maturation, Langerhans cells would migrate to the
lymph nodes via the afferent lymphatics (reviewed in Refs. 12 and
41); 5) once inside the draining lymph node, MDC (i.e., mature
Langerhans cells) would present the acquired Ags to naive CD4⫹
T cells; 6) when naive CD4⫹ T cells recognize Ag in the class II
MHC plus costimulatory molecules, they become activated and
proliferate, then migrate out of the lymph nodes; and 7) APC
would then probably die by apoptosis (46) to effectively terminate
the induction of new CD4⫹ T cells.
Our data suggest that the nonprotective immunogen disrupts the
induction of the protective CMI response at the level of maturation
of Langerhans cells into MDC and their subsequent migration into
the draining lymph nodes. This is supported by the fact that mice
given the nonprotective immunogen either alone or with the protective immunogen have reduced numbers of Langerhans cells and
MDC in their draining lymph nodes compared with mice given the
protective immunogen alone. The inability of the nonprotective
immunogen to induce increased numbers of Langerhans cells and
MDC above control levels (saline-CFA-treated mice) suggests that
at the site of Ag deposition the nonprotective immunogen has
pleiotropic antagonistic effects on the maturation of Langerhans
cells into MDC and the migration of Langerhans cells into draining
lymph nodes. The mechanisms that possibly play a role in blocking
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Most of what is known about DC phenotypes and functions comes
from in vitro studies on DC from the spleen (5–10). DC are considered to be the major APC in the induction of CMI responses (3,
4, 41), and most CMI responses to infectious organisms are initiated in draining lymph nodes (3, 4). Consequently, information on
the phenotypes and functions of DC in regional lymph nodes is of
great importance, yet there is only a limited amount of information
available. Other investigators (9, 11) have defined, through phenotypic studies, three different lymph node DC subsets in DBA/2
and C57BL/6J mice. Our observations on lymph nodes from CBA/J
mice confirm the presence of three different DC subsets with the
following phenotypes: Langerhans cells (DEC205lowFSChigh
CD8␣⫺CD11blow), MDC (DEC205highFSChighCD8␣lowCD11b⫹),
and LDC (DEC205highFSChighCD8␣highCD11blow). All three subsets
express the DC markers 33D1 and CD11c. The MDC and Langerhans
cells found in the lymph nodes would be expected to be the main
APC during the induction of activated T cells because they express
18- and 9-fold, respectively, more surface MHC class II molecules
needed for Ag presentation than do LDC. Furthermore, of the DC
subsets, the MDC have the highest levels of costimulatory molecules (CD40, CD80, and CD86), which are necessary for providing
the second signal to T cells during the activation process (42, 43).
In contrast, LDC express 7- and 12-fold more Fas ligand than
MDC or Langerhans cells, respectively, making them more likely
to stimulate apoptosis in Fas-bearing cells (7), a process that would
appear to be a down-modulatory or regulatory function. Based on
data described by Salomon et al. (11), Langerhans cells and MDC
are probably the same cell type at different stages of maturation
(i.e., mature Langerhans cells are MDC). Our data would support
this based on MDC having higher levels of MHC class II and
costimulatory molecules than Langerhans cells. However, because
Langerhans cells express high levels of MHC class II and low to
intermediate levels of costimulatory molecules, Langerhans cells
could be functioning, in addition to MDC, as APC inducing T
lymphocyte activation. Considering what is known about the function of splenic DC and the expression of surface markers on the
DC subsets of the draining lymph nodes, it is reasonable to predict
that lymph node DC subsets have functional differences. The
lymph node MDC and Langerhans cells most likely present Ag
and stimulate activation of Ag-reactive CD4⫹ T cells, whereas
lymph node LDC probably serve to regulate CD4⫹ T cell
activation.
With these ideas in mind, we studied the DC subsets in lymph
nodes draining sites of immunization with cryptococcal Ags that
induce either protective or nonprotective anticryptococcal CMI responses. The protective immunogen not only induced a significant
increase in the total number of cells in the draining lymph nodes,
it also preferentially induced more MDC and Langerhans cells that
have the capability of presenting Ag and activating T lymphocytes.
The ratio of LDC:MDC in the draining lymph nodes of the mice
immunized with the protective immunogen compared with the
LDC:MDC ratio in naive mice or mice immunized with the nonprotective immunogen was significantly reduced, indicating that
the MDC had been preferentially increased. We must assume that
the MDC and Langerhans cells in the draining lymph nodes performed their expected function because we also found a significant
increase in CD4⫹ T cells with an activated phenotype in the lymph
nodes draining the site of the protective immunogen. This same
scenario was not true for the lymph nodes draining the site of
immunization with the nonprotective immunogen. In the latter
case, there was not a significant increase in the total numbers of
cells, nor was the LDC:MDC ratio changed from that in lymph
165
166
Acknowledgments
We greatly appreciate the assistance of Dr. Doug Drevets in the critical
evaluation of this manuscript, Fredda Schafer for technical assistance with
animal procedures, and Jim Henthorn for technical assistance with flow
cytometric analyses. Flow cytometric analyses were performed at the Flow
Cytometry and Cell Sorting Core Facility for Molecular Medicine and St.
Francis Research Institute, University of Oklahoma Health Sciences
Center.
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maturation and trafficking of Langerhans cells are 1) lack of or
reduced induction of proinflammatory cytokines at the site of Ag
injection, 2) minimal levels of chemokine induction (47), 3) defective chemokine receptor expression (48), 4) HKC could be toxic
to Langerhans cells, and/or 5) the possibility that NK cells interacting with HKC could result in direct suppression or elimination
of Langerhans cells (49 –53). Any of these mechanisms would ultimately result in inefficient maturation and migration of Langerhans cells and ultimately inefficient activation of CD4⫹ T cells.
Further studies are required to define the mechanism(s) responsible
for the reduced numbers of DC moving into the draining lymph
nodes of mice injected with the nonprotective immunogen.
The differences in the immunogen’s ability to induce activated
CD4⫹ T cells in the draining lymph nodes directly correlates with
the differences seen in the levels of DTH responsiveness, the numbers of activated CD4⫹ T cells at the DTH reaction site, and the
level of protection when mice are challenged with C. neoformans.
In accordance with this, the protective immunogen induces higher
concentrations of IFN-␥ at the site of a DTH reaction than does the
nonprotective immunogen (K. L. Nichols, S. K. Bauman, and J. W.
Murphy, manuscript in preparation) or control treatments (23). Increased numbers of IFN-␥-producing-activated CD4⫹ T cells lead
ultimately to enhanced activation of natural effector cells (reviewed in Ref. 54). IFN-␥-activated natural effector cells more
efficiently kill C. neoformans (55–57), resulting in increased protection to the host upon subsequent infection. In fact, mice that
received the protective immunogen survived significantly longer
than mice that received either the nonprotective immunogen alone
or the nonprotective immunogen mixed with the protective
immunogen.
In summary, this is the first report of studies performed in vivo
to associate function with the different DC populations in draining
lymph nodes during the induction of CMI responses to a human
pathogen. The data show that increases in stimulatory MDC and
Langerhans cells in the lymph nodes draining the site of Ag deposition are needed to induce a protective anticryptococcal CMI
response that functions to increase the number of activated CD4⫹
T cells in the draining lymph nodes, at an anticryptococcal DTH
reaction site, and presumably at the infection sites. By comparison,
high levels of regulatory LDC are associated with a nonprotective
anticryptococcal CMI response and stimulate no measurable increase in numbers of activated CD4⫹ T cells. Thus, the balance of
stimulatory vs regulatory DC has a profound effect on the developing CMI response. In addition, the nature of the Ag can affect
the balance between these two disparate DC populations and,
ultimately, whether a protective CMI response develops against
C. neoformans.
DC IN ANTICRYPTOCOCCAL CMI RESPONSES
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