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* Institute of Experimental Immunology, University of Zürich, Zürich, Switzerland;
Department of Immunology, Toho University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within lymphoid organs, induction and maintenance of antiviral immune responses depend on correctly formed lymphoid compartments. Early trapping of virus in the marginal zone leads to the initial extrafollicular induction of antiviral B cells against T cell-independent Ags. Marginal zone macrophages have also been shown to be involved in induction of antiviral CTL responses, as it has been shown that only weak, nonprotective antiviral CTL responses could be initiated after depletion of marginal zone macrophages (9). The environment of germinal centers is required for the maintenance of antiviral B cell memory in mice (10), and patients with X-linked hyperIgM-syndrome lacking germinal centers due to an impaired CD40-CD40 ligand interaction show hypogammaglobulinemia and insufficient B cell and CTL activation leading to increased susceptibility to infections (11, 12).
The localization of lymphocytes within secondary lymphoid organs is controlled by constitutive chemokines differentially expressed in the B and T cell zones (13). Primary B cell follicles, most probably follicular dendritic cells (DCs), produce CXC chemokine ligand 13 (B lymphocyte chemoattractant; Ref. 14), which attracts mature resting B cells and a T cell subpopulation recently described as follicular Th cells via CXCR5 (15). T cell zone stromal cells express both the serine isoform of CC chemokine ligand (CCL)21-Ser/secondary lymphoid organ chemokine-Ser and CCL19/EBI1-ligand chemokine (16, 17). These chemokines act via the CCR7 receptor and are capable of attracting naive T cells and mature DCs, and thus coordinate their interaction within the T cell zone (18, 19). T cells down-regulate CCR7 after TCR triggering and concomitant to their evasion to the periphery (20), suggesting that the CCR7-CCL19/CCL21 interaction may be involved in T cell priming (21, 22). However, only little is known about the role of constitutive chemokines in the modulation of antiviral immune responses.
The paucity of LN T cells (plt) mutation, which arose as a spontaneous recessive mutation in mice (23), was recently mapped to the chemokine locus chromosome 4 and results in loss of both the only functional CCL19/ELC and the CCL21-Ser/SLC-Ser genes and in an aberrantly formed lymphoid T cell zone (24, 25, 26). Therefore, these mice are an excellent model to investigate the induction and maintenance of immune responses in a situation where the recruitment of naive T cells and DC to the T cell zone is defective (27).
Infection of mice with the lymphocytic choriomeningitis virus (LCMV) is a well-characterized model system for the investigation of antiviral T and B cell responses in vivo. LCMV can infect various cells of the immune system, such as B cells, macrophages, and DC. In particular, the tropism of different LCMV strains to DC determines whether the virus infection results in induction of protective CTL responses and clearance, or leads to exhaustion of virus-specific CTL and establishment of persistent infection (28, 29). In this study, we used the LCMV system and other well-established virus infection models, vesicular stomatitis virus (VSV) and vaccinia virus (VV), to investigate the impact of defective DC and T cell homing on the induction and maintenance of antiviral T and B cell responses in plt/plt mice. We found that T and B cell priming in plt/plt mice occurred mainly in the marginal zone of the spleen and in superficial cortical areas of LNs. Furthermore, both antiviral T and B cell responses were comparable in plt/plt and plt/+ control mice, suggesting that the coordinate interaction of DC and T cells in the lymphoid T cell zone may be less important in virus infections than seems necessary for nonreplicating Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B6-plt/plt mice were bred at the Institut für Labortierkunde (University of Zürich, Zürich, Switzerland). They were back-crossed to C57BL/6 5–7 times, and used for experiments in sex-matched groups with heterozygous littermates at the age of 8–12 wk. plt/plt mice were typed by PCR using the D4 Mit286 and D4 Mit237 primer pairs as described (30). B6PL-Thy1.1. mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred to the LCMV-gp33-specific TCR transgenic line 318 (31). Heterozygous F1 animals (318 x Thy1.1.) served as donors of CD8+ T cells in adoptive transfer experiments.
Viruses and peptides
LCMV, WE strain, originally obtained from Dr. F. Lehmann-Grube (Heinrich-Pette Institut, Hamburg, Germany), was propagated on L929 cells at a low multiplicity of infection and was plaqued as previously described (32). VSV Indiana strain (VSV-IND, Mudd-Summers isolate), was originally obtained from D. Kolakofsky (Department of Genetics and Microbiology, University of Geneva School of Medicine, Geneva, Switzerland). VSV-IND was propagated on baby hamster kidney-21 cells and plaqued on Vero cells. For some experiments, UV-inactivated VSV-IND was obtained using UV irradiation (7UV 15W; Philips, Eindhoven, The Netherlands) for 5 min in a thin layer of liquid in a 60-mm petri dish. The VV recombinant for the glycoprotein of the LCMV (LCMV-GP; VV-G2) was originally obtained from Dr. D. Bishop (Institute of Virology, Oxford, U.K.), and was propagated on BSC40 cells (33). The LCMV-GP peptide KAVYNFATM (gp33) and the nucleoprotein (np) of the LCMV (LCMV-NP) peptide FQPGNGQFI (np396) were purchased from Neosystem (Strasbourg, France).
Cytotoxic T cell response
Specific cytotoxicity was determined ex vivo in a standard 51Cr release assay as described (33). Briefly, cell suspensions were prepared from spleens or LNs of immunized mice at the indicated time point after priming. EL4 cells were labeled with gp33 (10-6 M) and 250 µCi 51Cr for 1.5 h at 37°C. A total of 104 target cells/well were incubated for 4 h in 96-well round bottom plates with 3-fold serial dilutions of spleen effector cells, starting at an E:T ratio of 90:1. EL-4 cells without peptide served as controls. The supernatants of the cytotoxicity assay cultures were counted in a Cobra II gamma counter (Canberra Packard, Downers Grove, IL). Percentage of specific lysis was calculated as (experimental release - spontaneous release)/(total release - spontaneous release) x 100. Spontaneous release was always <20%.
CTL were restimulated on peptide-labeled, irradiated (25 Gy) spleen cells as previously described (34), and tested in a conventional 51Cr release-assay using gp33-labeled EL-4 target cells.
LCMV-NP-specific ELISA
The LCMV-NP-specific ELISA has been described previously (35) using LMCV-NP expressed by Spodoptera frugiperda 9 cells after infection with a recombinant baculovirus.
VSV-specific serum neutralization test
Neutralizing Ab titers of sera were determined as described (36). Sera were prediluted 40-fold in supplemented MEM and heat-inactivated for 30 min at 56°C. Serial 2-fold dilutions were mixed with equal volumes of virus diluted to contain 500 PFU/ml. The mixture was incubated for 90 min at 37°C in an atmosphere containing 5% CO2. A total of 100 µl of the serum-virus mixture was transferred onto Vero cell monolayers in 96-well plates and incubated for 1 h at 37°C. The monolayers were then overlaid with 100 µl DMEM containing 1% methyl cellulose. After incubation for 24 h at 37°C, the overlay was removed and the monolayer was fixed and stained with 0.5% crystal violet. The highest dilution of the serum that reduced the number of plaques by 50% was taken as the neutralizing titer. To determine IgG titers, undiluted serum was first pretreated with an equal volume of 0.1 M 2-ME in saline.
Footpad swelling reaction
The indicated amounts of LCMV-WE were injected in a volume of 50 µl in balanced salt solution into both hind footpads in experimental groups of three mice. The footpad thickness was measured at the indicated time points with a spring-loaded caliper (37).
Construction of tetrameric class I-peptide complexes and flow cytometry
MHC class I (H-2Db) tetramers complexed
with gp33 were produced as previously described (38).
Briefly, H2-Db and human
2-microglobulin molecules were recombinantly
expressed in Escherichia coli (the plasmids were kindly
provided by J. Altman, Emory University, Atlanta, GA). Biotinylated
H2-Db peptide complexes were purified using an
Äkta Explorer 10 chromatography system (Pharmacia, Uppsala,
Sweden) and tetramerized by addition of streptavidin-PE (Molecular
Probes, Eugene, OR). At the indicated time points after immunization,
animals were bled and single-cell suspensions were prepared from spleen
and LNs. Aliquots of 5 x 105 cells or three
drops of blood were stained using 50 µl of a solution containing
tetrameric class I-peptide complexes at 37°C for 10 min followed by
staining with anti-CD8-FITC (BD PharMingen, San Diego, CA) at 4°C
for 20 min. Erythrocytes in blood samples were lysed with FACS lysis
solution (BD Biosciences, Mountain View, CA), and the cells were
analyzed on a FACScan flow cytometer (BD Biosciences) after gating on
viable leukocytes. For the determination of absolute cell counts, the
number of total viable leukocytes was assessed in an improved Neubauer
chamber. For blood, the number of total viable leukocytes was
automatically determined in an Advia counter (Bayer AG, Wuppertal,
Germany) in the Central Hematology Laboratory of the University
Hospital Zürich (Zürich, Switzerland).
Intracellular cytokine staining
Spleens were removed at the indicated time points after
infection with LCMV. Single-cell preparations of 1 x
106 splenocytes were incubated for 5 h at
37°C in 96-well round bottom plates in 200 µl culture medium
containing 25 U/ml IL-2 and 5 µg/ml brefeldin A (Sigma-Aldrich, St.
Louis, MO). Splenocytes were stimulated with PMA (50 ng/ml) and
ionomycin (500 ng/ml) as a positive control or left untreated as a
negative control. For analysis of peptide-specific responses,
106 splenocytes were stimulated by adding
10-6 M gp33. After stimulation, splenocytes were
surface stained with anti-CD8-PE (53-5.8; BD PharMingen) in
FACS-buffer (PBS + 2% FCS + 20 mM EDTA + 0.03%
NaN3) for 1 h at 4°C. Splenocytes were
washed once with FACS buffer, fixed with 100 µl 4% paraformaldehyde
in PBS for 5 min at 4°C, and permeabilized with 2 ml of
permeabilization buffer (FACS buffer + 0.1% saponin) for 5 min at
4°C. Cells were then stained intracellularly with
anti-IFN-
-FITC (AN18; Ref. 39) in permeabilization
buffer for 30 min at 4°C. Cells were washed twice with
permeabilization buffer, and the percentage of IFN--producing cells
was determined after gating on CD8+ cells using a
FACScan flow cytometer.
Fluorescence microscopy and immunohistochemistry
Spleens and LNs of infected or noninfected animals were removed at the indicated time points, immersed in HBSS, and snap-frozen in liquid nitrogen. Six-micrometer cryostat sections were fixed in acetone for 10 min and dried for 30 min. After blocking of FcRs with 10 µg/ml 2.4G2 (BD PharMingen) Ab in PBS 1% PBS for 30 min, LCMV-NP was detected using FITC- or tetramethylrhodamine isothiocyanate (TRITC) labeled clone VL-4 Ab (32). Adoptively transferred Thy1.1.+CD8+ T cells from 318 x Thy1.1 mice cells were detected using an anti-CD90.1-FITC Ab (BD PharMingen). CD11c-positive cells were detected using N418 hybridoma supernatant (40) and rat-anti-hamster-TRITC second stage Ab (The Jackson Laboratory). VL4-TRITC, VL4-FITC, anti-CD90.1-FITC, and rat-anti-hamster-TRITC were used at 10 µg/ml in PBS 1% FCS; the N418-supernatant was used at a 1/5 dilution in PBS 1% FCS. The slides were treated for 1 h with each Ab and washed extensively with PBS between incubations. The fluorescence was monitored on a Zeiss Axiophot microscope (Carl Zeiss, Feldbach, Switzerland) with a JVC KYF70 camera (Spitzer Electronic, Oberwil, Switzerland), using the Analysis software (Soft Imaging System, Münster, Germany).
For immunohistochemistry, cryostat sections were fixed in acetone for 10 min and subsequently incubated with anti-mouse N220 (RA3-3A1/6.1; American Type Culture Collection, Rockville, MD) or VL-4. Alkaline-phosphatase-labeled, species-specific goat-anti-donkey Abs (Tago Scientific, Burlingame, CA) were used as secondary reagents. The substrate for the red color reaction was AS-BI phosphate/New Fuchsin. Sections were counterstained with hemalum.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After immunization with LCMV-WE, CD8+ T
cells directed against the immunodominant epitope gp33 undergo a
vigorous systemic clonal expansion. To test the effect of defective DC
and T cell homing on the initiation of LCMV-specific T cell responses,
we used MHC class I tetramers complexed with the immunodominant CTL
epitope gp33 derived from the LCMV-GP (tetramer (tet)-gp33)
(38, 41). At different time points after i.v. infection
with 200 PFU LCMV-WE, the proportion of tet-gp33-positive cells in the
CD8+ T cell pool (Fig. 1
, A–C), total cell
numbers of CD8+ T cells (Fig. 1, D–F), and total cell numbers of gp33-specific CTL
(CD8+tet-gp33+, Fig. 1, G–I) were assessed from blood, spleen, and inguinal
LNs. As expected, naive plt/plt mice showed a significant
paucity of total CD8+ T cell numbers in LNs (Fig. 1F). Following LCMV infection, the expansion of
CD8+ T cells and the gp33-specific CTL response
in both plt/plt and plt/+ mice peaked on day 8.
The kinetics of the relative numbers of tet-gp33+
CTL revealed marked differences in the anti-LCMV response between
plt/plt and control mice, particularly in peripheral blood
(Fig. 1, A–C). However, the total numbers of
tet-gp33+CD8+ CTL were
comparable in plt/plt and plt/+ mice (Fig. 1, G–I). This discrepancy may be explained by the fact that
the expansion of CD8+ T cells in the respective
compartments of plt/plt mice was slightly faster than in
plt/+ mice (Fig. 1, D–F), thus counterbalancing
the differences in relative numbers.
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-dystroglycan-dependent
DC-tropism of LCMV is critical for the strength of the CTL response;
and therefore, determines whether the virus is controlled by the immune
system or can persist (28, 29). To assess whether the
distinct DC-tropism of different LCMV strains affects the induction of
CTL responses when DC homing to secondary lymphoid organs is impaired,
we compared gp33-specific CTL responses in spleens of
plt/plt and plt/+ control mice after infection
with ARM, WE, and DOC (Fig. 2). The peak
response on day 8 after infection with 200 PFU of ARM or DOC was
comparable in plt/plt and plt/+ mice both in
percentages of tet-gp33+ CTL within the
CD8+ pool (Fig. 2A) and in total
numbers of CD8+tet-gp33+
CTL (Fig. 2B). The proportion of
tet-gp33+ CTL in the CD8+ T
cell pool of plt/+ mice after LCMV-WE infection was slightly
higher than in plt/plt mice (Fig. 2A,
middle panel); however, like in the previous
experiments, the total numbers of tet-gp33+ CTL
were comparable in plt/plt and plt/+ mice (Fig. 2B). Taken together, these data show that the clonal
expansion of gp33-specific CD8+ T cells in
plt/plt mice after infection with LCMV mice is not
impaired.
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To examine the functional status of LCMV-specific CTL in
plt/plt mice in more detail, the cytolytic activity of CTL
was assessed in a 51Cr release assay using cells
from spleens and mesenteric LNs on day 8 after LCMV infection. CTL
responses against the LCMV-NP-derived peptide np396 and the
LCMV-GP-derived peptide gp33 were comparable in both compartments (Fig. 3A). Virus dissemination and
clearance in different organs were also determined on days 4 and 8
after infection. As shown in Fig. 3B, virus titers in
various organs of plt/plt and plt/+ mice showed
no significant differences at either time point. Similar results were
obtained for LCMV-ARM and LCMV-DOC (data not shown).
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A),
also when LCMV-ARM or LCMV-DOC were used (data not shown).
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B); however, the overall kinetics were similar to
nonsplenectomized mice, suggesting that the aberrantly structured
draining LNs of plt/plt mice alone were capable to initiate
and maintain an efficient antiviral immune response. Although the T
cell numbers of the LNs of plt/plt mice are dramatically
reduced, splenectomized plt/plt mice were also capable of
mounting a good systemic LCMV-specific CTL response after peripheral
infection. This is shown by the fact that the expansion kinetics of
gp33-specific CTL in blood and inguinal LNs were equivalent in
splenectomized plt/plt and plt/+ mice (Fig. 4C). Overall, these results indicate that the functional
activity of antiviral CTL in plt/plt mice is normal and that
CTL responses mediating rapid protection against systemic and
peripheral LCMV infection can be generated in plt/plt mice,
even in the absence of the spleen. Antiviral CTL responses against cytopathic VV
To evaluate whether plt/plt mice are able to mount
protective antiviral CTL responses against a cytopathic virus, we
infected plt/plt and plt/+ mice with 2 x
106 PFU of LCMV-GP rVV (Vacc-G2). Seven days
later, spleens were harvested and cytotoxicity was determined after
restimulation for 5 days with irradiated, gp33-pulsed splenocytes. As
shown in Fig. 5, plt/plt mice
mounted a normal CTL response against the cytopathic Vacc-G2.
Furthermore, Vacc-G2 was cleared from ovaries in plt/plt and
plt/+ mice with similar kinetics (Fig. 5B),
indicating that the plt defect has no major impact on the
CTL-mediated control of a cytopathic virus.
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The rapid induction of neutralizing antiviral Abs is crucial for
protection against infection with cytopathic viruses. It appears that
viral pathogens have the common characteristic to be T cell-independent
for IgM production, whereas isotype switching to IgG is mainly
dependent on cognate help delivered by the CD4+
Th cell subset (42). In the following set of experiments,
the induction of neutralizing IgM and IgG Abs was assessed in
plt/plt mice and plt/+ controls after i.v.
infection with either 2 x 106 PFU of live
VSV-IND (Fig. 6A), or with
1 x 108 PFU UV-inactivated VSV-IND (Fig. 6B). Both the replication competent (Fig. 6A) and
the inactivated VSV-IND (Fig. 6B) elicited comparable
neutralizing Ab responses in both groups of mice. Similarly, after i.v.
infection with 200 PFU LCMV-WE, anti-np Abs of the IgM (Fig. 6C) and IgG class (Fig. 6D) were induced in
plt/plt mice and plt/+ controls to a similar
extent.
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Maintenance of functional CTL memory responses in plt/plt mice
The notion has been put forward that memory T cell population
cells can be distinguished by their level of CCR7 expression
(43). In this study, we tested whether a disturbance of
the CCR7/CCL19/CCL21 system by the absence of CCL19 and CCL21-Ser has
an influence on the recirculation of memory CTL through secondary
lymphoid organs and their activity. Fig. 7A shows that LCMV-specific
CTL memory in plt/plt mice was not impaired even >300 days
after infection with 200 PFU LCMV-WE. The total numbers of
CD8+tet-gp33+ CTL (Fig. 7A, upper panel) and gp33-specific
IFN--producing CTL (Fig. 7A, lower panel) in
different lymphoid compartments were comparable in plt/plt
and plt/+ controls. These results indicate that the numbers
of virus-specific memory CTL, their distribution in lymphoid
compartments and peripheral blood, and their ability to differentiate
rapidly into IFN--producing effector cells is not affected by the
plt mutation.
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B, the clonal expansion of
gp33-specific CTL was comparable in plt/plt and
plt/+ mice. The weaker clonal expansion observed in
mesenteric LN shortly after Vacc-G2 infection may be explained
by a higher residual activity of the recently primed memory population
facilitating the elimination of LCMV; and therefore, eliciting only a
minor expansion of gp33-specific CTL. Localization of initial CTL activation in plt/plt mice
The above experiments established that the chemokine-driven
interaction of DC and naive T cells in lymphoid T cell zones in
plt/plt mice is not an exclusive prerequisite for the
induction of rapid protective antiviral immune responses. To determine
the site of primary activation of antiviral T cell responses in
plt/plt mice, we visualized the colocalization of
LCMV-specific CTL and virus-infected DC in lymphoid organs. To this
end, 1.5 x 107 MACS-sorted
Thy1.1-positive gp33-specific TCR transgenic CD8+
T cells were adoptively transferred into plt/plt mice.
Twenty-four hours later, the mice were i.v. infected with 2 x
104 PFU LCMV-WE, and spleens were removed and
processed for immunohistochemistry 3 days postinfection. In the spleens
of both plt/plt and control mice, LCMV Ag was largely
confined to the marginal zone (Fig. 8, A and B). Double-staining for virus Ag and the DC
marker CD11c revealed that a significant proportion of the marginal
zone DC in plt/plt mice were infected with LCMV (Fig. 8A, inset, arrowhead), whereas in
plt/+ mice, marginal zone DC (Fig. 8B,
right inset, arrowhead) as well as T cell zone DC (Fig. 8B, left inset, arrow) harbored LCMV Ag.
LCMV-specific 318 x Thy1.1. CD8+ T cells
were mainly localized in the red pulp and in the marginal zone of
plt/plt mice (Fig. 8C). These virus-specific CTL
formed foci in close contact with infected cells exclusively at the
marginal zone of plt/plt mice (Fig. 8C,
inset, arrowhead). However, in plt/+ mice,
virus-specific cells homed preferentially to the T cell zone and had
contact with virus-infected cells both from the side of the T cell zone
(Fig. 8D, inset, arrow) and from the side of the
marginal zone (Fig. 8D, inset, arrowhead).
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, E and F) and the B cell
marker B220 (Fig. 8, G and H) revealed the
positioning of LCMV-infected cells in the marginal zone (Fig. 8, E and F, arrows), and for plt/+ mice,
also in the T cell zone. Similarly, an immunohistological analysis of mesenteric LNs of LCMV-infected plt/plt mice and plt/+ mice revealed that, in both groups of mice, LCMV-Ag-positive DC and gp33-specific antiviral CTL were in close contact in superficial cortical areas (data not shown). Taken together, these data suggest that the contact between virus-infected DC and naive virus-specific T cells in the splenic marginal zone and in superficial cortical areas of LNs of plt/plt mice was sufficient to generate potent antiviral immune responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Functional consequences of altered CCR7-SLC/ELC interaction for the induction of antiviral immune responses
It has been speculated (44, 45) that CCR7 and its ligand chemokines CCL19/ELC and CCL21/SLC not only influence T cell and DC homeostasis, but also have a fundamental impact on priming and maintenance of immune reactions. This hypothesis is supported by a number of studies describing the function of the CCR7-ELC/SLC system either in vitro (46) or based on correlations between the in vivo and in vitro migration pattern of T cells (21). In vivo, the ablation of CCR7 expression results not only in severely impaired migration of naive T cells and activated DC, but also in a reduced immune responsiveness such as decreased contact hypersensitivity (22). Furthermore, reduced responsiveness to hapten sensitization after in vivo blockade of CCL21/SLC suggested that selective interference with DC and naive T cell homing may down-modulate adaptive immune responses (47).
An earlier report on the in vivo significance of the CCL19/CCL21-driven DC and naive T cell migration for the induction of antiviral immunity showed an enhanced susceptibility of BALB/c-plt/plt mice to mouse hepatitis virus (27). BALB/c mice express the MHVR1 gene which renders them susceptible to MHV infection (48). However, the divergence in response to MHV among susceptible strains depends mainly on the susceptibility of macrophages (49, 50), which constitute the primary target of the virus. Because the F4/80+ macrophage compartment appears to be less prominent in spleens and LNs of plt/plt mice (T. Junt, unpublished observation), the increased susceptibility of plt/plt mice to mouse hepatitis virus may be at least partly due to differences in target cell availability. However, the altered lymphoid microenvironment of plt/plt mice did not influence the replication kinetics of LCMV, which is also a virus targeting macrophages.
Protection against LCMV (51) and VV (33) is
mediated primarily by CTL. In view of the fact that CTL also contribute
to the control of MHV infection (52, 53) and that
plt/plt mice showed increased susceptibility to MHV
infection, we expected a significant impairment of the protective
immunity in plt/plt mice after infection with the
noncytopathic LCMV or the cytopathic VV. However, both the kinetics of
virus-specific CTL, i.e., acute and memory responses, and their
function were not significantly altered in plt/plt mice. The
major difference between plt/plt and plt/+
control mice was found in the relative proportion of virus-specific CTL
among CD8 T cells (see Figs. 1 and 2). Nevertheless, these differences
were leveled out by a slight overshoot in the CTL expansion rate and
differences in the cellular distribution between lymphoid compartments.
The reduction in T cell numbers is much more severe in LNs than in the
spleen of plt/plt mice (23, 27). Therefore, it
is interesting to note that even after splenectomy, plt/plt
mice generated equivalent CTL responses compared with plt/+
controls (see Fig. 4). Furthermore, our study describes the quality of
humoral responses in plt/plt mice, showing that in addition
to the largely intact T cell responses, plt/plt mice
also generated normal antiviral B cell responses.
A recently published study by Mori et al. (54) investigated immune responsiveness in plt/plt mice in a model of contact hypersensitivity and described that the plt mutation results in fully functional T cell responses which only differ in their kinetics. Our data corroborate and significantly extend this previous study because we establish that the altered spatial organization of secondary lymphoid T cell zones due to defects in the CCR7-CCL19/CCL21 interaction in plt/plt mice is probably not crucial for initial priming and maintenance of protective T and B cell responses in the course of viral infections.
Where are antiviral immune responses induced when DC and naive T cell cannot meet in the T cell zone?
Stein et al. (55) have demonstrated that peripherally injected CCL21 may accumulate on high endothelial venules of plt/plt mice, and thus lead to an enhanced activation of lymphocyte transmigration. Because plt/plt mice express CCL21b outside lymphoid organs, it may well be that this peripherally expressed chemokine has an effect on T cell and DC migration into LNs, accounting for the small differences observed between plt/plt and plt/+ mice. In addition, CCL21b is still expressed at very low levels in LNs and spleen of plt/plt mice (56). Although the mechanism of the mildly impaired antiviral immune reactions remains elusive, it is surprising that the absence of a defined T cell zone had only rather mild effects on the generation of virus-specific immune responses.
Complexes of virus-infected cells and specific T cells were found in plt/plt mice only in the marginal zone, whereas these clusters were distributed throughout the white pulp and in the splenic marginal zone of control mice. This suggests that priming of antiviral T cells in plt/plt mice occurred in the marginal zone. Mori et al. (54) have shown that immunization of plt/plt mice with OVA was followed by a remodeling of lymphoid organs indicating that T cell responses against nonreplicating proteinaceous Ags may also be elicited efficiently in the splenic marginal zone. Furthermore, our data are in line with a recent study by Ciavarra et al. (57) showing that after selective depletion of phagocytic marginal zone DCs, the remaining interdigitating DCs were able to trap Ag but failed to prime T cell responses against VSV.
It remains to be resolved whether splenic marginal zone DCs and their LN equivalents alone contribute to the priming of antiviral immune responses in plt/plt mice. We found that LCMV Ag in the marginal zone of the spleen and in the superficial cortex of the LNs was also associated with CD11c-negative cells. It may well be that marginal zone macrophages not only function as "Ag trapping structures" and transmit virus to adjacent DCs, but they may also contribute directly to the priming of antiviral T cells. This notion is supported by previous findings showing that functional marginal zone macrophages are crucial for the induction of anti-LCMV T cell responses (9). Overall, our study supports the concept that the splenic marginal zone and its LN equivalent are crucial structures for the rapid generation of antiviral immune responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Tobias Junt, Department of Pathology, Institute of Experimental Immunology, University of Zürich, Schmelzbergstr. 12, CH-8091 Zürich, Switzerland. E-mail address: cayenne{at}pathol.unizh.ch
3 Abbreviations used in this paper: LN, lymph node; DC, dendritic cell; LCMV, lymphocytic choriomeningitis virus; np, nucleoprotein; LCMV-GP, glycoprotein of the LCMV; LCMV-NP, nucleoprotein of the LCMV; plt, paucity of LN T cells; VSV, vesicular stomatitis virus; VSV-IND, VSV Indiana strain; VV, vaccinia virus; CCL, CC chemokine ligand; TRITC, tetramethylrhodamine isothiocyanate; tet, tetramer.
Received for publication January 10, 2002. Accepted for publication April 16, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dystroglycan correlate with viral tropism and disease kinetics. J. Virol. 75:448. interferon. Proc. Natl. Acad. Sci. USA 81:4515.This article has been cited by other articles:
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  P. A. Lang, L. Cervantes-Barragan, A. Verschoor, A. A. Navarini, M. Recher, M. Pellegrini, L. Flatz, A. Bergthaler, K. Honda, B. Ludewig, et al. Hematopoietic cell-derived interferon controls viral replication and virus-induced disease Blood, January 29, 2009; 113(5): 1045 - 1052. [Abstract] [Full Text] [PDF]
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 M. S. Zinkernagel, B. Bolinger, P. Krebs, L. Onder, S. Miller, and B. Ludewig Immunopathological Basis of Lymphocytic Choriomeningitis Virus-Induced Chorioretinitis and Keratitis J. Virol., January 1, 2009; 83(1): 159 - 166. [Abstract] [Full Text] [PDF]
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 H. Unsoeld, K. Mueller, U. Schleicher, C. Bogdan, J. Zwirner, D. Voehringer, and H. Pircher Abrogation of CCL21 chemokine function by transgenic over-expression impairs T cell immunity to local infections Int. Immunol., November 1, 2007; 19(11): 1281 - 1289. [Abstract] [Full Text] [PDF]
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 J. Rangel-Moreno, J. E. Moyron-Quiroz, L. Hartson, K. Kusser, and T. D. Randall Pulmonary expression of CXC chemokine ligand 13, CC chemokine ligand 19, and CC chemokine ligand 21 is essential for local immunity to influenza PNAS, June 19, 2007; 104(25): 10577 - 10582. [Abstract] [Full Text] [PDF]
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B. Xu, K. Aoyama, M. Kusumoto, A. Matsuzawa, E. C. Butcher, S. A. Michie, T. Matsuyama, and T. Takeuchi Lack of lymphoid chemokines CCL19 and CCL21 enhances allergic airway inflammation in mice Int. Immunol., June 1, 2007; 19(6): 775 - 784. [Abstract] [Full Text] [PDF]
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 E. Scandella, K. Fink, T. Junt, B. M. Senn, E. Lattmann, R. Forster, H. Hengartner, and B. Ludewig Dendritic Cell-Independent B Cell Activation During Acute Virus Infection: A Role for Early CCR7-Driven B-T Helper Cell Collaboration J. Immunol., February 1, 2007; 178(3): 1468 - 1476. [Abstract] [Full Text] [PDF]
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M. Ato, A. Maroof, S. Zubairi, H. Nakano, T. Kakiuchi, and P. M. Kaye Loss of Dendritic Cell Migration and Impaired Resistance to Leishmania donovani Infection in Mice Deficient in CCL19 and CCL21 J. Immunol., May 1, 2006; 176(9): 5486 - 5493. [Abstract] [Full Text] [PDF]
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T. Schreiber, S. Ehlers, S. Aly, A. Holscher, S. Hartmann, M. Lipp, J. B. Lowe, and C. Holscher Selectin Ligand-Independent Priming and Maintenance of T Cell Immunity during Airborne Tuberculosis J. Immunol., January 15, 2006; 176(2): 1131 - 1140. [Abstract] [Full Text] [PDF]
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T. Junt, K. Fink, R. Forster, B. Senn, M. Lipp, M. Muramatsu, R. M. Zinkernagel, B. Ludewig, and H. Hengartner CXCR5-Dependent Seeding of Follicular Niches by B and Th Cells Augments Antiviral B Cell Responses J. Immunol., December 1, 2005; 175(11): 7109 - 7116. [Abstract] [Full Text] [PDF]
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J. Rangel-Moreno, J. Moyron-Quiroz, K. Kusser, L. Hartson, H. Nakano, and T. D. Randall Role of CXC Chemokine Ligand 13, CC Chemokine Ligand (CCL) 19, and CCL21 in the Organization and Function of Nasal-Associated Lymphoid Tissue J. Immunol., October 15, 2005; 175(8): 4904 - 4913. [Abstract] [Full Text] [PDF]
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K. S. Lang, M. Recher, A. A. Navarini, S. Freigang, N. L. Harris, M. van den Broek, B. Odermatt, H. Hengartner, and R. M. Zinkernagel Requirement for Neutralizing Antibodies to Control Bone Marrow Transplantation-Associated Persistent Viral Infection and to Reduce Immunopathology J. Immunol., October 15, 2005; 175(8): 5524 - 5531. [Abstract] [Full Text] [PDF]
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 M. Kursar, U. E. Hopken, M. Koch, A. Kohler, M. Lipp, S. H.E. Kaufmann, and H.-W. Mittrucker Differential requirements for the chemokine receptor CCR7 in T cell activation during Listeria monocytogenes infection J. Exp. Med., May 2, 2005; 201(9): 1447 - 1457. [Abstract] [Full Text] [PDF]
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P. Krebs, E. Scandella, B. Odermatt, and B. Ludewig Rapid Functional Exhaustion and Deletion of CTL following Immunization with Recombinant Adenovirus J. Immunol., April 15, 2005; 174(8): 4559 - 4566. [Abstract] [Full Text] [PDF]
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T. Junt, E. Scandella, R. Forster, P. Krebs, S. Krautwald, M. Lipp, H. Hengartner, and B. Ludewig Impact of CCR7 on Priming and Distribution of Antiviral Effector and Memory CTL J. Immunol., December 1, 2004; 173(11): 6684 - 6693. [Abstract] [Full Text] [PDF]
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 K. R. Pilkington, I. Clark-Lewis, and S. R. McColl Inhibition of Generation of Cytotoxic T Lymphocyte Activity by a CCL19/Macrophage Inflammatory Protein (MIP)-3{beta} Antagonist J. Biol. Chem., September 24, 2004; 279(39): 40276 - 40282. [Abstract] [Full Text] [PDF]
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A. Martin-Fontecha, S. Sebastiani, U. E. Hopken, M. Uguccioni, M. Lipp, A. Lanzavecchia, and F. Sallusto Regulation of Dendritic Cell Migration to the Draining Lymph Node: Impact on T Lymphocyte Traffic and Priming J. Exp. Med., August 18, 2003; 198(4): 615 - 621. [Abstract] [Full Text] [PDF]
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) and plt/+ mice (
) were
infected i.v. with 200 PFU LCMV-WE. The proportion of tet-gp33-positive
cells in the CD8+ T cell pool (A–C), total
cell numbers of CD8+ T cells (D–F), and
total cell numbers of CD8+tet-gp33+ CTL were
assessed for blood, spleen, and inguinal LNs at the indicated time
points by FACS analysis. Given values represent mean ± SD
(n = 6–8 per data point)
) and plt/+ mice (