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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4375-4386
Sialoadhesin-Positive Host Macrophages Play an Essential Role in
Graft-Versus-Leukemia Reactivity in Mice
By
Susanne Müerköster,
Marian Rocha,
Paul R. Crocker,
Volker Schirrmacher, and
Victor Umansky
From the Division of Cellular Immunology, Tumor Immunology Program,
German Cancer Research Center, Heidelberg, Germany; and The Wellcome
Trust Building, Department of Biochemistry, University of Dundee,
Dundee, UK.
 |
ABSTRACT |
We recently established an effective immune T-cell-mediated
graft-versus-leukemia (GVL) murine model system in which complete tumor
remissions were achievable even in advanced metastasized cancer. We now
describe that this T-cell-mediated therapy is dependent on host
macrophages expressing the lymphocyte adhesion molecule sialoadhesin
(Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive
immunotherapy (ADI) or the treatment of these animals with anti-Sn
monoclonal antibodies led to complete or partial inhibition of the
immune T-cell-mediated therapeutic effect. Furthermore,
Sn+ host macrophages in livers formed clusters during ADI
with donor CD8 T cells. To test for a possible antigen presentation
function of these macrophages, we used as an in vitro model the antigen  -galactosidase for which a dominant major histocompatibility complex
(MHC) class I Ld-restricted peptide epitope is
known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL).
We demonstrate that purified Sn+ macrophages can process
exogenous -galactosidase and stimulate MHC class I
peptide-restricted CTL responses. Thus, Sn+ macrophages,
which are significantly increased in the liver after ADI, may process
tumor-derived proteins via the MHC class I pathway as well as via the
MHC class II pathway, as shown previously, and present respective
peptide epitopes to CD8 as well as to CD4 immune T cells, respectively.
The synergistic interactions observed before between immune CD4 and CD8
T cells during ADI could thus occur in the observed clusters with
Sn+ host macrophages.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SOME MOUSE TISSUE macrophages, located in
bone marrow, spleen, liver, and lymph nodes, express a lectin-like
receptor, sialoadhesin (Sn), that mediates divalent cation-independent
binding but not phagocytosis of sheep erythrocytes.1-3 Sn
has been shown also to function as an adhesion molecule for
lymphocytes.4
We previously described a highly effective adoptive cellular
immunotherapy (ADI) protocol in the well-defined ESb
T-lymphoma model of DBA/2 (H-2d,
Mlsa) mice.5,6 The adoptive transfer of in
situ-activated tumor immune spleen lymphocytes from ESb
tumor-resistant, major histocompatibility complex (MHC)
identical but superantigen different B10.D2 (H-2d,
Mlsb) mice into tumor-bearing DBA/2 mice led to a rejection
of primary tumors (1.5 cm in diameter) from the skin and to eradication
of liver metastases. This therapy requires only relatively few specific T cells and involves synergistic interactions between CD4 and CD8
tumor-immune T cells.5 B10.D2 anti-ESb cytotoxic T
lymphocyte (CTL) responses were made up of a strong response to DBA/2
minor histocompatibility (H) antigens and a weaker one to ESb-specific MHC class I (Kd)-restricted tumor-associated antigen
(TAA).7 The frequency of B10.D2 CTL precursors for H
antigen was about 1:3,700, whereas the frequency of CTL precursors for
TAA was about 1:17,000.8
After immunohistological staining of livers from ADI-treated mice with
a monoclonal antibody (MoAb) against Sn, we observed (1) an increase in
Sn+ host macrophages and (2) a close physical association
between the latter and CD4 T lymphocytes.9 Sn+
macrophages that were isolated from the liver of ADI-treated animals
could directly restimulate in vitro CD8 T lymphocytes primed against
the tumor cells, suggesting that they could process and present
exogenous TAA to T lymphocytes.9 Furthermore, we demonstrated that Sn+ macrophages were capable of
processing and presenting human C-reactive protein via MHC class II to
a CD4 T-helper clone specific for an identified MHC II-associated
C-reactive protein-derived peptide.10
The classical pathways of antigen presentation to T cells involve (1)
endogenous proteins that are presented via MHC class I molecules and
(2) exogenous proteins that are presented via class II
molecules.11,12 However, a number of recent publications demonstrate that this classification is neither absolute nor exclusive. Thus, CD8 T-cell responses were observed in vivo in the absence of de
novo antigen synthesis within host antigen-presenting cells (APC) after
immunization with purified proteins13 or in cross-priming experiments in which CD8-mediated responses to donor cell antigens were
restricted to MHC molecules expressed only on recipient
cells.14 The APC involved in this type of antigen
presentation were identified as macrophages or dendritic
cells.15-19 Recent studies have suggested different
mechanisms for presentation of exogenous antigens via MHC class I
molecules in macrophages.20-23 It is at present not clear
whether some macrophage subpopulations can process and present exogenous soluble antigen (eg, TAA) via MHC class I molecules to CD8
T lymphocytes and thus be a part of host antitumor response.
In this report, we provide evidence that a subset of macrophages
bearing the lymphocyte adhesion molecule Sn can stimulate in vitro a
CD8 CTL response against an MHC class I-restricted model antigen.
Furthermore, Sn+ Kupffer cells will be shown to form
clusters with donor CD8 T cells in the livers of tumor bearing mice
upon adoptive cellular immunotherapy. The depletion of Kupffer cells or
treatment with anti-Sn MoAbs in vivo after ADI decreased the efficiency
of this therapy. We therefore suggest that Sn+ macrophages
are important for processing exogenous TAA from metastases and
presenting it, not only via MHC class II to CD4 cells, but also via
class I to CD8 immune donor T lymphocytes, and contribute thereby to
the eradication of lymphoma liver metastases during ADI.
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MATERIALS AND METHODS |
Animals and tumor cell lines.
DBA/2 mice and Balb/c nude mice (nu/nu) were obtained from IFFA Credo
(Lyon, France) and B10.D2 mice were obtained from Olac (Bicester, UK).
All animals were used at 6 to 12 weeks of age. ESb cells represent a
spontaneous highly metastatic variant of the chemically induced
T-lymphoma L5178Y (Eb) of DBA/2 mice. ESbL cells transduced with the
bacterial lacZ gene (clone L-CI.5s) were cultured as
described.24 The ESb-MP subline is a plastic adherent
variant of ESb lymphoma cells that have reduced malignancy and altered
metastatic capacity in vivo.25 P815 mastocytoma cells of
DBA/2 mice and P13.1 cells, a lacZ-transduced variant of the
P815 mastocytoma,26 were used in the cytotoxicity assay. All media used for tumor cell culture and inoculation as well as for
cell isolation were free of endotoxins. DBA/2 mice were anesthesized
with Rompun (0.1%):Ketanest (0.25%):phosphate-buffered saline (PBS)
at 1:1:3 (vol) and 2 × 105 ESb-MP cells in 100 µL
PBS were injected intradermally (ID) into the dermis of the shaved flank.
Adoptive cellular immunotherapy.
To generate allogeneic immune effector cells, ESb-MP lymphoma cells
were inoculated intravenously (IV) at a dose of 105 cells
into B10.D2 mice.5 Seven days later, spleen cells were isolated and transferred IV (2 × 107 cells/200 µL
RPMI-1640 medium; GIBCO BRL, Eggenstein, Germany) into 5 Gy
(60Co source Gammatron F 80S; Siemens,
Braunschweig, Germany) sublethally irradiated DBA/2 mice
that carried tumors (>1 cm in diameter). This adoptive transfer of
antitumor immune spleen cells (ISPL) was made 3 weeks after ID tumor
cell inoculation. Sublethally irradiated tumor-bearing DBA/2 mice of
the control group remained untreated. In some experiments, 2 × 107 ISPL were injected IV into nu/nu mice 3 weeks after ID
inoculation of ESb-MP cells.
To generate syngeneic antitumor immune cells, DBA/2 mice were primed by
injection into the ear pinna of 5 × 104 ESb lymphoma
cells and challenged 7 days later intraperitoneally with
1.5 × 107 100 Gy irradiated ESb cells.27
Three days after challenge, 5 × 106 in situ activated
lymphocytes from peritoneal exudate (PEC) were transferred IV into 5 Gy
irradiated DBA/2 mice that were IV injected 1 day before with
104 ESb lymphoma cells.
Antibodies and other reagents.
The following rat MoAbs were used as culture supernatants: antimouse
Thy-1.2 (clone AT83A),28 anti-CD8 (clone
53-6-72),29 specific for mouse B cells (clone
B220),30 for mature mouse macrophages
(F4/80),31 and for Sn (SER-4 and 1C2).4 In
addition, SER-4 MoAb was labeled with digoxigenin. Hamster-antimouse
B7.1 MoAb (clone 16-10A)32 was used as culture supernatant.
Rat-antimouse B7.2 MoAb (clone GL1) was purchased from PharMingen
(Hamburg, Germany). Rat-antimouse MHC class II MoAb (clone AMS32-1;
PharMingen) was biotinylated. Hamster-antimouse dendritic cell marker
N418 MoAb was labeled with digoxigenin and kindly provided by Dr B. Kyewski (German Cancer Research Center, Heidelberg, Germany). R-Phycoerythrin-conjugated streptavidine (GIBCO BRL) and
antidigoxigenin-fluorescein F(ab')2 antibody
(Boehringer Mannheim, Mannheim, Germany) were used as second reagents.
The mouse MoAbs T19.191 and K9-18,33 hybridoma supernatants
directed against H-2Ld and H-2Kd MHC class I,
respectively, were kindly provided by Dr B. Arnold (German Cancer
Research Center) and used in blocking experiments in vitro. The rat
MoAbs were visualized by using polyclonal donkey antirat IgG (H+L)
antibodies linked to horseradish peroxidase (PO) and to alkaline
phosphatase (AP; both from Dianova, Hamburg, Germany). -gal protein
(coded by the lacZ gene) and its derived MHC class I-restricted
peptide (TPHPARIGL), which were kindly provided by Dr H.J. Schild
(Eberhard-Karls-University, Tübingen, Germany), were used in
functional assays. Dichloromethylenebisphosphonate (chlodronate,
Cl2MBP) was kindly provided by Boehringer Mannheim.
Isolation of Sn+ macrophages from the spleen.
Single-cell suspensions from spleens of normal DBA/2 mice were prepared
by mechanical dissociation and macrophages isolated by adherence to the
plastic culture dishes were resuspended in PBS without divalent cations
(Biochrom KG, Berlin, Germany). The Sn+ macrophage
subpopulation was isolated by means of a rosetting technique using
unopsonized sheep erythrocytes, as described previously.1 Briefly, a 0.5% erythrocyte suspension was added to the macrophage suspension (at a ratio of 100:1). After thorough mixing, centrifugation at 200g for 15 minutes, and subsequent incubation on ice for 30 minutes, the percentage of macrophages binding greater than 4 erythrocytes was assessed by phase-contrast microscopy. Rosetted cells
were separated from unbound erythrocytes and nonrosetted macrophages
using Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient
centrifugation at 400g for 20 minutes. Then unbound
erythrocytes were removed by lysis with NH4Cl hypotonic solution.
Isolation of dendritic cells (DCs) from the spleen.
Spleen DCs were isolated according to van Voorhis et al.34
Briefly, after erythrocyte lysis and digestion with collagenase IV
(Boehringer Mannheim) and dispase (Interchemie, Munich, Germany), spleen cells were depleted of T lymphocytes by treatment with MoAb AT83A, rabbit complement (Camon, Wiesbaden, Germany), and DNase
(Sigma Chemical Co, Munich, Germany), followed by dBSA (Biomex, Mannheim, Germany) density gradient centrifugation and depletion of B
lymphocytes using MoAb B220 and MACS column (Miltenyi, Bergisch Gladbach, Germany).
Depletion of Kupffer cells.
Depletion of Kupffer cells was performed using the liposome-mediated
intracellular delivery of Cl2MBP that was entrapped in liposomes and kindly provided by Dr N. van Roojien (Vrije Universiteit, Amsterdam, The Netherlands).35 DBA/2 mice (10 animals per experimental group) were injected ID with ESb-MP cells and
3 weeks later were treated IV with immune spleen cells as described
above. Two hundred microliters of Cl2MBP-liposomes was
injected IV into tumor-bearing DBA/2 mice twice at 2 days before and 4 days after transfer of immune spleen cells. Another group of
tumor-bearing mice was treated with PBS at the same time points. It has
been described that, after treatment with Cl2MBP-liposomes,
the liver remains selectively depleted of macrophages for about 5 to 6 days, before repopulation starts, which is completed within 10 to 14 days.35 Other liver cell populations (eg, DCs, lymphocytes,
endothelial cells, etc) were shown to be unaffected by
chlodronate-containing liposomes.35-37
Treatment with MoAbs against Sn.
To block the function of Sn+ macrophages in host liver
after ADI, we used anti-Sn MoAbs (1C2 and SER-4 clones directed against different epitopes of Sn). Tumor-bearing mice (6 animals per
experimental group) were treated IV twice (1 and 3 days after ADI) with
the mixture of IC2 and SER-4 MoAbs containing 0.5 mg of ammonium
sulfate-precipitated protein. Control tumor-bearing ADI-treated mice
were injected with normal rat Ig (0.5 mg of protein) on the same days
as anti-Sn MoAbs. Data were expressed in terms of animal survival.
Tissue preparation and staining.
Livers were removed and snap-frozen in liquid nitrogen.
Five-micrometer-thick consecutive cryostat sections were mounted on uncovered glass slides. After drying overnight at room temperature, the
sections were fixed in acetone for 10 minutes at room temperature and
air-dried. After the fixation, the slides were washed in PBS three
times for 5 minutes. To avoid nonspecific binding, the sections were
incubated for 30 minutes with 1% normal rat serum followed by rat MoAb
treatment for 45 minutes. After washing with PBS, the sections were
incubated for another 45 minutes with the second antibody, washed
again, and treated with the substrate for PO or AP. The sections were
then washed with water, counterstained with haemalaun (Merck,
Darmstadt, Germany), and mounted with glycerylgelatine (Merck).
Negative controls were incubated as described above, but either with
omission of the first antibody or treated with rat MoAbs directed to
irrelevant antigens. All steps were performed at room temperature.
The double-staining procedure represented a combination of consecutive
single stainings for Sn and CD8 molecules, as described above.
PO-linked secondary antibody and the corresponding enzyme substrate was
used in the first reaction, followed by an AP-mediated reaction for the
second staining. No counterstaining with haemalaun was performed. PO
activity was shown by immersing the sections in a solution containing 6 mg 3-amino-9-ethylcarbasole dissolved in 1.5 mL N,N-dimethylformamide
(Merck), 15 µL 30% hydrogen peroxide, and 28.5 mL of 0.1 mol/L acetate buffer, pH 5.0.38 The substrate for the
development of AP consisted of 6.3 µL 5% Neufucsin (Sigma) or 2 mg
Fast Blue (Sigma), 16 µL 4% sodium nitrite (Fluka, Buchs, Switzerland), 2 mg naphthol As-Bi-Phosphate (Sigma), 20 µL
N,N-dimethylformamide, and 3 mL of 0.05 mol/L Tris-HCl buffer, pH 8.7. The freshly prepared solution was filtered into the staining jar
containing the sections. Development lasted approximately 3 to 5 minutes, with regular checking of the reaction intensity by microscopy.
Immunohistochemical results were evaluated by counting positively
stained cells and relating them to the liver lobuli. The means and
standard deviations of the data obtained from the number of mice and
experiments indicated were then calculated and presented in graphics.
Antibody staining of Sn+ macrophages.
Spleen Sn+ macrophages (1 × 106) were
washed in PBS supplemented with 5% fetal calf serum (FCS), stained
with digoxigenin-labeled SER-4 first antibody and
antidigoxigenin-fluorescein F(ab')2 second antibody,
and analyzed by flow cytometry using a FACScan analyzer with CELLQuest
software (Becton Dickinson, Heidelberg, Germany). Control cells were
incubated with PBS/FCS instead of the first-step antibody before
staining with the second-step reagent. To study the possible
contamination of these macrophages with DCs, we stained them with MoAbs
for DC marker N418 and rat-antimouse MHC class II. Double-positive
cells were considered as DCs.
Functional assay for Sn+ macrophage-mediated antigen
processing and presentation through MHC class I.
To generate CD8+ CTLs specific for lacZ-transduced
ESbL cells, a subtumorigenic dose of live ESbL-lacZ cells (5 × 104 cells/50 µL PBS) was injected into the ear
pinna of DBA/2 mice. Nine days later, immune spleen lymphocytes were
isolated and depleted of macrophages and DCs by plastic
adherence.34 These lymphocytes containing -gal-specific
CTL precursors were then incubated for 5 days with the following
stimulator cells (2 × 107 responder and 2 × 106 stimulatory cells): (1) Sn+ spleen
macrophages isolated from normal DBA/2 mice; and (2) spleen cells (SPL)
containing all APC from normal DBA/2 mice. Before use, stimulator cells
were  -irradiated with 100 Gy (Gammacell 1000, Ottawa, Ontario,
Canada). The incubation mixture contained also protein antigen (2.2 µg/mL -gal protein) or a derived peptide (0.1 µg/mL of class
I-restricted peptide of the -gal-TPHPARIGL) that represents a major
epitope of antigen-specific CTLs. To exclude the possible contamination
of the -gal solution with peptides, we purified this solution by
ultrafiltration through low adsorption hydrophilic membrane using the
manufacturer's instructions (Amicon, Beverly, MA). This purification
results in the removal of all peptides with a molecular weight less
than 10 kD. In some experiments, anti-H-2Ld (6 µg/mL) and anti-H-2Kd (6 µg/mL) MHC class I MoAbs,
normal rat IgG (6 µg/mL), or the mixture of 1C2 and SER-4 MoAbs (12 µg/mL) directed against different epitopes of Sn were also added to
the culture to interfere with the generation of cytotoxic CD8 T cells.
Negative controls included responder and stimulatory cells without the
antigen or responder cells and antigen without APC.
To test for cytotoxic activity of generated effector T lymphocytes in
vitro, the following target cell lines were used: P13.1, a
lacZ-transduced variant of the P815 (H-2d)
mastocytoma cell line (as a positive control), and the parental P815
cells (as a negative control). For labeling, 1 × 106
target cells were incubated for 90 minutes at 37°C with 0.2 µCi 51Cr sodium chromate (Amersham, Braunschweig, Germany) in
RPMI-1640 medium with 30% FCS. After extensive washing with the
medium, cells were resuspended at a final concentration of 5 × 104/mL. The restimulated effector cells containing -gal
specific CTLs were resuspended at a concentration of 2.5 × 106/mL RPMI-1640 medium. One hundred microliters of
effector (E) cells was mixed with 100 µL of target (T) cells (E:T
cell ratio = 50:1, 25:1, 12:1, or 6:1) in 96-well round-bottom plates
(Renner, Dannstadt, Germany). After centrifugation to promote cell
contact, the plates were incubated for 4 hours at 37°C in 5%
CO2. To assess target cell death, supernatants (100 µL)
were harvested, the radioactivity released was measured in a gamma
counter (LKB-Wallac, Turku, Finland), and the percentage of specific
lysis was calculated from the mean of triplicate cultures according to
the following formula: percentage of specific lysis = 100 × (experimental release  spontaneous release)/(maximal release spontaneous release).
| |
RESULTS |
Depletion of host Kupffer cells in vivo results in breakdown of the ADI
therapy effect.
We previously reported that adoptive transfer of antitumor immune T
lymphocytes from B10.D2 mice into ESb-MP tumor-bearing animals led to
complete regression of liver metastases and survival in 50% to 100%
of treated mice.5,6 To investigate the significance of host
liver macrophages in this ADI therapy, we now performed the selective
Kupffer cell depletion in vivo by liposomes containing chlodronate
according to a protocol that caused a selective elimination of liver
macrophages for about 5 to 6 days without any effect on other liver
cell populations.35-37 Preliminary experiments showed that
chlodronate entrapped in liposomes did not induce any toxicity both in
normal mice and in ESb-MP tumor-bearing animals (data not shown).
Adoptive transfer of tumor-immune spleen cells on day 21 into
tumor-bearing mice resulted in complete tumor regression and survival
in 50% of treated mice (Fig 1, ADI). A
single injection of liposomes containing chlodronate 2 days before ADI
led to the death of all animals, despite the transfer of immune spleen
cells (Fig 1, LIP+ADI; P < .05). A similar result was
obtained in a group of animals treated twice with liposomes: 2 days
before and 4 days after ADI (Fig 1, LIP+ADI+LIP; P < .05).
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| Fig 1.
Effect of host liver macrophage depletion in vivo on the
efficiency of ADI therapy in tumor-bearing DBA/2 mice. To generate
antitumor immune effector cells, ESb-MP lymphoma cells were inoculated
IV at a dose of 105 cells into B10.D2 mice. One week later,
spleen cells were isolated and transferred IV into sublethally
irradiated ESb-MP tumor-bearing DBA/2 hosts (ADI, d0). Kupffer cells
were depleted from the host by IV injection of
Cl2MBP-liposomes. Each experimental group contained 10 mice. Chlodronate entrapped in liposomes did not induce any toxicity
both in normal mice and in ESb-MP tumor-bearing animals (data not
shown). ( ) Control (nontreated tumor-bearing mice); ( )
ADI-treated tumor-bearing mice; ( ) tumor-bearing mice injected with
Cl2MBP-liposomes 2 days before ADI; ( ) tumor-bearing
mice injected twice with Cl2MBP-liposomes 2 days before and
4 days after ADI. One representative experiment of three is shown.
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The number of Sn+ Kupffer cells is elevated during
immunotherapy of liver lymphoma metastasis.
It is known that approximately 50% of normal liver macrophages are
Sn+.1 With the help of the MoAb
SER-4,2 these macrophages could be mainly detected in liver
periportal areas. We found that the intensity of cell staining was
clearly stronger in ADI-treated mice in comparison either with normal
mice or with tumor-bearing animals receiving irradiation (which had no
antitumor effect) or without irradiation. As shown in
Fig 2A, the number of Sn+
Kupffer cells in irradiated tumor-bearing animals showed an early increase (with a peak of 57 cells per liver lobule 2 days after irradiation) and then sharply decreased throughout further development of liver metastases. The number reached 37 cells per liver lobule 8 days after irradiation, which corresponds to 30 days after tumor cell
inoculation. At this time point, tumor-bearing animals started to die;
therefore, further observations in this group were not possible.
Interestingly, nonirradiated tumor-bearing mice also showed an increase
in the number of Sn+ Kupffer cells (with a maximum of 42 cells per lobule). In contrast, in ADI-treated mice, Sn+
liver macrophages reached a maximum of 94 cells per liver lobule at day
8 (P < .05; Fig 2A). Thereafter, their number slowly
decreased (to 51 at day 28 after ADI; P < .05). These data
confirmed our previous observations.9 Although we observed
an elevation in the total number of Kupffer cells (stained with MoAbs
against the general tissue macrophage marker F4/80; Fig 2) upon ADI
treatment, most of these (up to 92% at day 8) were Sn+
macrophages. Livers of untreated tumor-bearing mice contained only
around 50% of Sn+ macrophages among Kupffer cells.
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| Fig 2.
Kinetics of Sn+ macrophage accumulation in
livers of tumor-bearing DBA/2 mice under ADI. Frozen tissue sections
were single-stained for total Kupffer cells (F4/80+) and
Sn+ Kupffer cells (A) or double-stained for
F4/80+/B7.1+ and for
Sn+/B7.1+ (B) with corresponding MoAbs. Six
lobules per liver were analyzed under the microscope. (A) The number of
Sn+ () and F4/80+ ( ) Kupffer cells in
ESb-MP tumor-bearing untreated (d 1, 5 Gy; d0, no immune cells) mice
and the number of Sn+ ( ) and F4/80+
() Kupffer cells in ADI-treated (d 1, 5 Gy; d0, 2 × 107 B10.D2 immune spleen cells) animals are shown. (B) The
number of Sn+ (),
Sn+/B7.1+ ( ), F4/80+
(), and F4/80+/B7.1+ () in
ADI-treated mice are shown. Data present the means and standard
deviations from two experiments with 2 to 3 animals per time point.
Data points without SD represent mean values for which the SD was
smaller than 1.
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We next studied whether Sn+ Kupffer cells expressed
costimulatory molecules for T-cell activation such as B7.1 (CD80) and
B7.2 (CD86). As shown in Fig 2B, the maximum of B7.1-expressing
Sn+ macrophages occurred at days 8 and 12 after ADI (36 and
35 cells per liver lobule, respectively). At day 12, 49% of host
Sn+ liver macrophages were B7.1+. These results
were in agreement with our previous findings.39 There was
also a clear-cut increase in the number of
F4/80+B7.1+ Kupffer cells at day 2 after ADI,
but then this population of double-positive cells gradually decreased,
and at day 28, all B7.1+ liver macrophages were
Sn+. In untreated tumor-bearing mice, the numbers of
Sn+B7.1+ and
F4/80+B7.1+ liver macrophages were
significantly lower than in treated animals (not more than 15 and 27 cells per liver lobule, respectively; P < .05; data not
shown). In contrast, the expression of B7.2 molecule on Sn+
and F4/80+ macrophages was very low both in untreated and
ADI-treated tumor bearing mice (data not shown).
The accumulation of Sn+ Kupffer cells was also observed
after other types of immunotherapy. Thus, adoptive transfer of
syngeneic immune PEC from DBA/2 mice into irradiated ESb
lymphoma-bearing DBA/2 mice resulted in an increase in the number of
Sn+ liver macrophages from 42 at day 0 to 63 at day 5 after
immunotherapy (P < .05). Similar findings were observed after
injection of allogeneic immune spleen cells from B10.D2 mice into
ESb-MP tumor-bearing nu/nu mice (from 40 cell per liver lobule at day 0 to 82 cells at day 5; P < .05). An increase in the number of
Sn+ Kupffer cells correlated in both cases with
graft-versus-leukemia (GVL)-mediated effects that led to complete
prevention of liver metastases and survival of treated mice.
Sn+ liver macrophages form clusters with CD8 T
lymphocytes after ADI.
Complete tumor regression under ADI therapy, which consists of
sublethal (5 Gy) host irradiation followed by B10.D2 antitumor immune
spleen cell (ISPL) transfer, was shown before to depend on synergistic
interactions between transfered immune CD4 and immune CD8 T
cells.5 In addition, donor CD4 T lymphocytes were detected
close to host liver Sn+ macrophages after ADI.9
We investigated here the localization of CD8 T lymphocytes in the
livers in relation to the position of this subset of macrophages.
Immunohistochemical double-stainings showed a close physical
association between CD8 T lymphocytes and host
Sn+ Kupffer cells. Such cell clusters (defined as
containing 1 or more cells of each cell type) were mainly located in
periportal areas (Fig 3A). High
magnification showed direct contacts between the membranes of the two
interacting cell types. Figure 3B shows the kinetics of the formation
of such cell clusters at different time points after ADI. The number of
clusters steadily increased in a time-dependent manner and reached the
maximum of 10 per liver lobule (at day 28 after treatment). The total
number of CD8 T cells infiltrating the liver of tumor-bearing mice
after ADI showed an increase with a peak of 45 cells per lobule at day
20 (P < .05). Already at day 5, 50% of all CD8 T lymphocytes
detected in the liver were in close contact with macrophages that
expressed the general marker F4/80 and Sn. The comparison of
double-stainings for CD8 and F4/80 as well as for CD8 and Sn markers
showed that almost all CD8 T cells were associated with Sn+
but not with Sn macrophages (Fig 3B). Four weeks
after treatment, there were still 84% of CD8 T cells in clusters
closely associated with Sn+ host macrophages.
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| Fig 3.
Kinetics of CD8-Sn+ cluster formation in
livers of tumor-bearing DBA/2 mice under ADI. Frozen tissue sections
were stained for CD8 T lymphocytes and Sn+ Kupffer cells
with corresponding MoAbs. Six lobules per liver were analyzed under the
microscope. (A) Immunohistochemical picture of sections, double-stained
for CD8 (brown) and Sn (blue) in livers of tumor-bearing mice 12 days
after ADI treatment. Show is the periportal area in which CD8 T
lymphocytes and Sn+ Kupffer cells are mainly located and
form cell clusters (original magnification × 400). pv, portal vein.
(B) Double-staining of CD8 T lymphocytes and Sn+
macrophages in mouse livers after ADI therapy. () Total number of
CD8 T cells; () the number of CD8 cells associated with
F4/80+ cells in clusters; () the number of CD8 cells
associated with Sn+ cells in clusters; ( ) the number
of CD8 cell-Sn+ cell clusters. Data show the means and
standard deviations from two experiments with 2 to 3 animals per time
point. Data points without SD represent mean values for which the SD
was smaller than 1.
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Interestingly, the immunotherapy of ESb lymphoma liver metastasis in
DBA/2 mice with syngeneic antitumor immune PEC from DBA/2 mice also led
to the formation of clusters between CD8 T cells and Sn+
macrophages in the liver. Similar to the ADI with allogeneic immune
spleen cells, 48% of all CD8 T cells detected in the liver at day 5 after treatment were associated with Sn+ macrophages, and 3 cell clusters per liver lobule were found at this time point.
Thus, the transfer of antitumor immune spleen cells caused an increase
in host Sn+ macrophages and in donor CD8 T
lymphocytes in the liver of tumor-bearing mice. The kinetics of
liver infiltration by these two cell types was quite similar, and there
was a remarkable physical interaction between them as shown by
specifically double-stained cell clusters. The formation of cell
clusters was shown not only during immunotherapy with allogeneic immune
lymphocytes, but also with syngeneic ones.
Inhibition of the ADI therapy effect caused by anti-sialoadhesin
MoAbs.
To determine the role of host Sn+ Kupffer cells during ADI,
we performed antibody blocking experiments in vivo. Mice were treated twice (1 and 3 days after ADI) with a mixture of 1C2 and SER-4 rat-antimouse MoAbs directed against different epitopes of Sn to block
the function of Sn+ macrophages in host livers. Whereas in
the positive control
(Fig 4A, ADI) 100%
of ADI-treated tumor bearing mice were alive at the end of the
experiment (at day 60 after tumor cell inoculation), only 33% survived
in the ADI-group treated with antisialoadhesin MoAbs (Fig 4A,
ADI+anti-Sn MoAbs; P < .05). Injection of normal rat IgG
containing Ig with the same isotype as the anti-Sn MoAbs did not affect
the immunotherapeutic effect of transferred immune spleen cells (Fig
4A, ADI+control Abs). We next tested whether 1C2 and SER-4 MoAbs were
able to bind to Sn+ macrophages in the liver or even to
deplete these cells from the organ. In mice treated only with ADI,
Sn+ cells could be detected predominantly in liver
periportal areas with the help of the MoAb
SER-42 (Fig 4B). Livers from corresponding
mice treated also with rat-antimouse Sn MoAbs were
immunohistochemically stained with AP-linked goat-antirat second antibody. The IV inoculated anti-Sn MoAbs were detectable in
these livers at the sites of Sn+ Kuppfer cells 8 days after ADI and 3 days after the last injection of MoAbs
(Fig 4C). These MoAbs thus were able to target the
macrophages in vivo. Targeting was not associated with depletion,
because the number of Sn+ Kupffer cells in this group and
in mice treated only with ADI were similar (82 and 79 cells per liver
lobule, respectively; P > .05). The MoAb-mediated decrease in
protective immunity could be interpreted as a blocking effect.
Immunohistological stainings under these conditions showed a
significant decrease in the numbers of CD8 T lymphocytes infiltrating
the livers (Fig 4D). At day 14 after ADI and at day 9 after the second
injection of the blocking anti-Sn MoAbs, the total number of CD8 T
cells was 1.8 times lower than in mice treated only with immune spleen
cells (18 and 33 cells per liver lobule respectively; P < .05). At this time point, the number of CD8 T lymphocytes associated
with Sn+ macrophages in clusters was also reduced (Fig 4D).
In addition, we found a decrease in the number of these CD8 T
cell-Sn+ macrophage clusters (from 7 to 3 clusters per
liver lobule at day 14 after ADI; P < .05).
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| Fig 4.
Effect of the treatment with anti-Sn MoAbs in vivo on the
efficiency of ADI therapy in tumor-bearing DBA/2 mice. (A)
Tumor-bearing mice (6 animals per experimental group) were treated IV
with the mixture of anti-Sn MoAbs (1C2 and SER-4 directed against
different epitopes of Sn) 1 and 3 days after ADI. () Control
(nontreated tumor-bearing mice); () ADI-treated tumor-bearing mice;
() tumor-bearing mice injected with anti-Sn MoAbs 1 and 3 days after
ADI; () tumor-bearing mice injected with normal rat IgG (control
Abs). One representative experiment of three is shown.
(B and C) Immunohistochemical pictures of
frozen liver sections from tumor-bearing DBA/2 mice (at day 8 after
ADI) treated with immune cells only (B) or in combination with the
mixture of anti-Sn MoAbs (1C2 and SER-4) (C). Shown are periportal
areas in which most of the Sn+ macrophages are located.
(B) shows the staining for Sn (pink) and (C) shows the staining for
anti-Sn MoAbs (pink) using goat antirat second antibody (original
magnification × 200). pv, portal vein. (D) Presence of CD8 T
lymphocytes in the livers of mice treated with ADI and anti-Sn MoAbs.
Total number of CD8 T cells in ADI-treated mice () and in animals
injected with anti-Sn MoAbs 1 and 3 days after ADI (). Number of CD8
T cells associated with Sn+ Kupffer cells in clusters in
mice treated with ADI only ( ) or with ADI in combination with
anti-Sn MoAbs ( ). Data represent the means and standard deviations
from two experiments with 2 to 3 animals per time point. *Statistically
significant difference when compared with the respective control.
|
|
Host Sn+ liver macrophages thus appear to be of real
importance in the GVL effect mediated by donor immune T cells.
Sn+ macrophages process and present exogenous antigen via
MHC class I to CD8 T cells.
We have previously reported that Sn+ macrophages can
process and present MHC class II-restricted antigen to CD4
cells.9 To test for a possible function of Sn+
macrophages as APC for CD8 lymphocytes, we used a well-defined antigen,
bacterial -gal, as a model in which a dominant peptide epitope is
known to be recognized by anti--gal CD8 CTL.40 T lymphocytes were enriched from APC-depleted -gal immune spleen cells. Specific CTL responses were generated in cultures to which either unprocessed -gal protein or derived dominant peptide together with either purified Sn+ macrophages or total SPL from
normal mice were added. The purity of spleen Sn+
macrophages isolated via a rosetting procedure was more than 95% and
the contamination by DCs in this cell preparation was 1.8%, as shown
by double-staining with MoAbs for DC marker N418 and MHC class II (data
not shown). The CTL activity generated was determined in a 4-hour
51Cr release assay using lacZ gene-transfected
P13.1 mastocytoma as target cells. In positive controls, target cell
lysis mediated by CD8 T lymphocytes stimulated with SPL (containing all
APC) and -gal-derived peptide TPHPARIGL ranged from 82% (E:T cell ratio = 50:1) to 5% (6:1), whereas effector cells generated by -gal
peptide without APCs caused only background lysis of 2% (Fig 5A). The addition of syngeneic
Sn+ macrophages as APC and the peptide to the cultures
resulted in the generation of specific CTL activity with 50% (50:1)
down to 19% (6:1) target cell lysis (Fig 5A). When effector
cells were generated with SPL as APC and unprocessed whole -gal
protein (extensively dialysed to remove any peptides as potential
antigens), target cell killing was significantly lower as compared with
stimulation with APC plus peptide (Fig 5B). When Sn+
macrophages were used as APC for -gal protein, a low but significant CD8 CTL response of 18% (50:1) and 10% (25:1) target cell lysis was
generated (Fig 5B).
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| Fig 5.
Sn+ macrophage-mediated presentation of MHC
class I-restricted peptide of -gal (A) and processing of -gal
protein and presentation of MHC class I-restricted peptide (B) to CD8 T
lymphocytes. Immune spleen lymphocytes (responder cells) from DBA/2
mice primed 9 days before by intrapinna injection of a subtumorigenic
dose of lac Z gene-transfected ESb-L cells were depleted of
macrophages and DCs by plastic adherence and incubated with SPL (),
Sn+ spleen macrophages (; 2 × 107
responder and 2 × 106 stimulatory cells), or DCs
( ; 2 × 107 responder and 2 × 104 stimulatory cells). The incubation mixture also
contained -gal whole antigen (B) or its derived MHC class
I-restricted peptide (TPHPARIGL) (A). In some cultures, anti-Sn
blocking MoAbs (1C2 and SER-4) were added ( ). Negative controls
included responder cells and Sn+ cells without the
antigen () or antigen with responder cells only (). Five days
later, the stimulation cultures were harvested and the cytotoxic
activity of generated effector cells (E) was tested in a 4-hour
51Cr release assay using as target cells (T) the lac
Z-transduced P815 mastocytoma cell line, P13.1. Means and standard
deviations from 4 independent experiments are shown. Data points
without SD represent values for which the SD was smaller than
1.
|
|
We next investigated whether the CTL response could be blocked by
anti-Sn MoAbs (SER-4 and IC2) when added to the stimulation cultures
containing Sn+ macrophages, T cells, and whole -gal
protein or its derived peptide. Treatment with these rat-antimouse
MoAbs resulted in a diminished CTL response against -gal-derived
peptide (Fig 5A; P < .01) and against the whole protein
(P < .008 at E:T cell ratio 50:1; Fig 5B). Addition of normal
rat IgG containing Ig with the same isotype as the anti-Sn MoAbs caused
no inhibition of CD8 CTL response (data not shown).
Taking into account that the isolated Sn+ macrophages
contained about 2% DCs with the potential to present MHC class
I-restricted antigens to CD8 T cells,16,18,19 we purified
spleen DCs and studied their capacity to process and present -gal
and/or derived peptides under our above-mentioned conditions. When 4 × 104 DCs (which correspond to 2% from 2 × 106 Sn+ macrophages) were added
into stimulatory cultures containing either -gal protein or peptide,
almost no CTL activity against P13.1 target cells could be generated
(Fig 5).
We can thus conclude that Sn+ macrophages can principally
function as APCs capable of processing and presenting exogenous protein via the MHC class I pathway towards MHC class I/peptide-specific CD8
CTL precursors.
Requirement for restricting MHC class I molecules for antigen
presentation by Sn+ macrophages.
To demonstrate selective involvement of restricting MHC class I
molecules on Sn+ macrophages in the presentation of
-gal-derived antigenic epitopes to CTL, we used MHC class
I-Ld blocking MoAbs, because it was previously shown that
CTL specific for the dominant epitope are restricted by
H-2Ld molecules.40
Figure 6 shows that anti-Ld
MoAb exerted strong inhibitory effects on the generation of -gal peptide-specific CTL activity via either whole -gal protein or -gal peptide (95% and 67% of inhibition of kill, respectively, at
E:T cell ratio of 50:1). No suppression of CTL activity was observed
with an anti-Kd MHC class I antibody used as control (Fig
6A and B). If anything, there was an augmentation of generated CTL
response. When SPL were used instead as a source of APCs, the lysis of
tumor cells was also inhibited by anti-Ld but not by
anti-Kd antibody (90% inhibition in the presence of whole
-gal and 69% with the peptide; E:T cell ratio of 50:1).
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| Fig 6.
Selective inhibitory effect of anti-Ld
antibodies on Sn+ macrophage-mediated presentation of
-gal-derived MHC class I-restricted peptide (A) and on processing
of -gal protein and peptide presentation (B) to CD8 T lymphocytes.
Responder cell priming, restimulation, and kill assays were performed
as described in Fig 4. Cultures for responder lymphocyte restimulation
contained Sn+ spleen macrophages and antigen (). In
some cultures, anti-H-2Ld () or control
anti-H-2Kd () MoAbs were added. Negative controls
included antigen (-gal or its peptide) and responder cells only
(). Means and standard deviations from 3 independent assays are
shown. Data points without SD represent values for which the SD was
smaller than 1.
|
|
The difference between the effects obtained with antibodies against the
restricting and a nonrestricting MHC class I molecule in this system
clearly demonstrates that restricting MHC class I Ld
molecules on the surface of Sn+ macrophages function as
-gal peptide-binding molecules and are responsible for the
stimulatory capacity detected in this model of MHC class I-restricted
antigen processing and presentation.
| |
DISCUSSION |
In the present study, we investigated the involvement of a
well-characterized subpopulation of host tissue macrophages bearing the
sialic acid-receptor Sn1-4 in a GVL reactivity and in the presentation of exogenous antigen via MHC class I molecules. To address
this question, we used a well-defined protocol of adoptive cellular
immunotherapy of ESb lymphoma liver metastasis in DBA/2 mice
(H-2d, Mlsa).5 This therapy
requires only relatively few tumor-immune CD4 and CD8 T cells, works
even in late-stage disease, and results in the rejection of primary
tumors (1.5 cm in diameter) from the skin and in eradication of
established liver metastases.
We found that the depletion of Kupffer cells with liposomes containing
chlodronate in tumor-bearing mice during immunotherapy led to complete
prevention of the immunotherapeutic effect. It has been previously
reported by us and others that the treatment with chlodronate entrapped
in liposomes selectively affects liver macrophages but not other liver
cells (eg, lymphocytes, DCs, endothelial cells, etc).35-37
Furthermore, the depletion of macrophages with liposome-encapsulated
chlodronate did not result in cytokine release.37
It is known that approximately 50% of Kupffer cells in normal livers
express Sn.1 We found a dramatic increase in the number and
staining intensity of these cells in ADI-treated mice. In addition,
Sn+ Kupffer cells expressed also B7.1 and, to a lesser
extent, B7.2, costimulatory molecules involved in T-cell
activation.32 The observed increase in absolute numbers and
staining intensity is probably due to recruitment of monocytes from the
blood followed by differentiation into Sn+ macrophages in
the environment of the metastatic liver undergoing immunotherapy. Their
recruitment may be caused by chemotactic factors released by immune
cells. These factors can also be produced by tumor cells, because we
observed an increase in the number of Sn+ Kupffer cells in
nonirradiated tumor-bearing mice. Proliferative expansion from a
liver-derived precursor population is unlikely, because the cells do
not express proliferation-associated markers.39 Interestingly, irradiation itself can to some extent induce the accumulation of Sn+ macrophages in the liver. However,
transferred antitumor immune lymphocytes are the main stimulators of
this process, because we observed a much higher increase in
Sn+ Kupffer cells both in a syngeneic system (injection of
immune PEC from DBA/2 mice into irradiated ESb tumor-bearing DBA/2
mice) or in an allogeneic system without irradiation (transfer of
immune spleen cells from B10.D2 mice into ESb-MP tumor-bearing nu/nu mice). These types of immunotherapy also caused complete regression of
liver metastases and survival of treated animals.
We previously reported that these macrophages are of host origin, are
located in close association with metastatic lymphoma cells, and form
clusters with CD4 donor T lymphocytes in the livers.5,9 Using a well-established model of the proliferative response of a
T-helper clone recognizing a dominant MHC class II-associated peptide
of human C-reactive protein, we found that Sn+ macrophages
could both process and present this class II-restricted antigen.10 Finally, ex vivo isolated Sn+
Kupffer cells from ADI-treated tumor-bearing mice were found to
directly activate (without addition of TAA) CD8 T lymphocytes primed
against the ESb tumor cells,9 suggesting that they
presented TAA in association with MHC class I.
After ADI, an increase in the quantity of Sn+ host cells
was correlated with the elevation in the number of donor CD8 T
lymphocytes in metastatic livers. Most of the CD8 cells were localized
in close association with Sn+ host liver macrophages in the
form of clusters, the number of which was increased with
the time after immunotherapy. The presence of direct contacts between
the membranes of the two interacting cell types was confirmed by
transmission electron microscopy.39 Because
Sn Kupffer cells hardly formed clusters with CD8 T
lymphocytes, we conclude that this macrophage-lymphocyte interaction is
a special property of Sn-expressing macrophages.
We have recently shown that the endogenous viral superantigen 7 (vSAG-7; Mlsa) encoded by mouse mammary tumor virus (MMTV)
provirus Mtv-7 is expressed in DBA/2 but not in B10.D2
mice.41 In DBA/2 mice, vSAG7 causes deletion of
SAG-reactive T cells with certain V chains (eg, V6) from their
repertoire,42 whereas in B10.D2 tumor-resistant mice,
V6+ T lymphocytes are present. We showed previously that
these cells can infiltrate ESb liver metastasis in DBA/2 mice after ADI
with immune spleen cells from B10.D2 mice.39 To exclude the
influence of the vSAG7 on the observed clustering of donor CD8 T cells
with host Sn+ Kupffer cells, we injected antitumor immune
PEC from DBA/2 mice (which contained no V6+ T
lymphocytes) into ESb lymphoma-bearing DBA/2 mice. Also under these
experimental conditions, which led to complete protection, CD8
T-cell-Sn+ macrophage clusters were detected in the
livers. These data suggest that CD8 T-cell clustering with
Sn+ macrophages involves cognitive interactions of T-cell
receptors with MHC class I-associated peptides from processed tumor
antigens. Moreover, upon transfer of both allogeneic or syngeneic
antitumor immune cells, we found a significant increase in the number
and size of clusters formed by three cell types: donor CD4 and CD8 T
lymphocytes and host Sn+ macrophages (data not shown). To
clarify directly the role of host Sn+ Kupffer cells during
immunotherapy in vivo, ADI-treated mice were injected with a mixture of
MoAbs directed against different epitopes of Sn on macrophage surfaces
(SER-4 and 1C2). We observed a significant inhibition of the
immunotherapeutic effect. Interestingly, anti-Sn MoAbs were found to
target to liver macrophages but did not delete the cells. Their
inhibitory effect in vivo may be due to a blocking activity. As a
consequence, we observed (1) a decrease in the number of CD8 T
lymphocytes infiltrating the liver after ADI and (2) a decrease in the
quantity of clusters formed between CD8 T cells and host
Sn+ Kupffer cells.
Taking all of these observations with Sn+ macrophages into
consideration, we suggest that this subpopulation of host tissue macrophages plays a role as APC in the liver during ADI and performs cognitive interactions with both CD4 and CD8 antitumor immune T cells.
To address the question of the functional significance of
Sn+ macrophage-CD8 T-cell clusters in MHC class I-mediated
antigen presentation, we used a well-defined model antigen (-gal) to stimulate MHC class I peptide-specific CTL.26,40 The fact
that we showed approximately 20% of specific lysis by CD8 T
lymphocytes stimulated via Sn+ macrophages and -gal
whole protein suggests that Sn+ cells can process this
exogenous protein and present derived peptide via MHC class I to
-gal-primed CTL precursors. The CTL response generated by
Sn+ macrophages in vitro could be partially affected (but
not totally blocked) by anti-Sn MoAbs added into the stimulation
cultures containing Sn+ macrophages, T lymphocytes, and
whole -gal protein or its derived peptide. This inhibitory effect
was specific, because the treatment by control antibodies (normal rat
IgG with the same phenotype as anti-Sn MoAbs) did not negatively affect
the CTL response. We suggest that the lymphocyte adhesion molecule Sn
may facilitate Sn+ macrophage-CD8 T-lymphocyte interactions.
It is known that the liver not only contains macrophages, but also DCs
with potential APC function for CD4 and CD8 T
cells.16,19,20 Using immunohistological staining with MoAb
N418 specific for DCs, we observed that the latter can also form
clusters with donor CD8 T lymphocytes in livers of ADI-treated mice
(unpublished observations). However, it has been recently
reported that mouse DCs are negative for Sn, as shown by
immunofluorescence and flow cytometry with MoAb SER-4.43
Thus, DCs cannot bind sheep erythrocytes, as do Sn+
macrophages, a property that is used for their isolation. Nevertheless, we found that the preparation of Sn+ macrophages contains a
small number of DCs (<2%), so we had to exclude the possiblity that
these were responsible for the observed APC effect. When highly
enriched DCs were added as APC to stimulation cultures in numbers
corresponding to this contamination (2%), almost no CTL activity
against -gal target cells could be generated. Therefore, the
contaminating DCs in the purified Sn+ macrophage population
could not be responsible for the observed MHC class I-restricted
antigen stimulatory capacity. Interestingly, the inhibitory effect of
anti-Sn MoAbs in vivo was significant but not 100% (Fig 1B). This
leaves the possibility for other APC (including DCs) to participate in
the observed GVL effect.
Our data raise the question about the mechanism(s) of Sn+
cell-mediated presentation of exogenous soluble antigens in association with MHC class I molecules. It has recently been reported that macrophages take up bead-bound or soluble ovalbumin by phagocytosis or
macropinocytosis and transfer the intact protein to the
cytosol.21,44 Another mechanism includes the uptake and
digestion of particulate antigens in phagolysosomes followed by binding
of produced peptides to MHC class I molecules directly on the cell
surface or inside the phagosome.22,45 The third mechanism
involves the capture of chaperones such as gp96 that may carry
proteasome-derived peptides to MHC class I molecules.23,46
We are currently investigating which mechanism(s) among those listed
above is operative in the case of MHC class I-restricted antigen
presentation mediated by Sn+ cells.
In conclusion, we demonstrate that Sn+ host macrophages
play an important role in a highly effective T-cell-mediated GVL
immunotherapy of advanced metastasized cancer. We suggest that
Sn+ macrophages expressing costimulatory molecules for
T-cell activation (such as B7.1 and B7.2) may represent a new type of
professional APC in the liver and perhaps in other tissues. They can
process and present exogenous TAA via MHC class I to immune CD8 cells and via MHC class II to immune CD4 cells10 and form
clusters with T lymphocytes in target organs of metastasis that may be facilitated by Sn molecules. These characteristics appear to enable them to focus CD4 and CD8 T lymphocytes at their cell surfaces, thus
allowing synergistic T-T-cell interactions.
| |
ACKNOWLEDGMENT |
The authors thank Dr B. Kyewski for critical reading of the manuscript,
Dr A. Benner for help with biostatistics, Dr N. van Roojien for
providing the liposomes containing chlodronate, Dr B. Arnold for
providing MoAbs directed against H-2Ld and
H-2Kd MHC class I molecules, and Dr H.J. Schild for
providing the MHC class I-restricted peptide of -gal.
| |
FOOTNOTES |
Submitted November 12, 1998; accepted February 13, 1999.
Supported by a grant from the Dr. Mildred Scheel Stiftung (no.
10-0980-Schi2, V.U. and M.R.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Victor Umansky, PhD, Division of Cellular
Immunology, Tumor Immunology Program, German Cancer Research Center,
D-69120 Heidelberg, Germany; e-mail: V.Umansky{at}dkfz-heidelberg.de.
| |
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