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Experimental Therapeutics, Preclinical Pharmacology |
Division of Cellular Immunology, Tumor Immunology Program, German Cancer Research Center, 69120 Heidelberg, Germany [S. M., V. U., V. S.]; Glaxo Wellcome, Stevenage, Hertfordshire, United Kingdom [M. R.]; and Department of Immunology, Erasmus University Rotterdam, 3000 DR Rotterdam, the Netherlands [J. D. L.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSTUNEL AssayIn Vivo Blocking of...RESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayIn Vivo Blocking of...RESULTSDISCUSSIONREFERENCES |
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Several recent studies indicate that the CD40-CD40L interaction plays a
critical role in the humoral immune response (e.g., isotype
switching, development of B-cell memory and germinal centers; Ref.
8
) as well as in T-cell mediated immunity
(e.g., viral target lysis, parasite clearance, and antitumor
responses; Refs. 9, 10, 11, 12
). CD40 is a member of the TNF
receptor family, is expressed constitutively in low levels on B cells,
monocytes/macrophages, and DCs, and can be further up-regulated by
IFN-
(9
, 11, 12, 13)
. CD40L (CD154) belongs to the TNF
family and is expressed on activated T cells, predominantly on
activated CD4 cells (8
, 9) . Triggering of CD40 on APCs
enhances the levels of adhesion (intercellular adhesion molecule-1) and
costimulatory molecules (CD80 and CD86; Refs. 9
, 14, 15, 16, 17
),
leading to enhanced antigen presentation, thereby facilitating
activation of T cell-mediated immune responses. Recent publications
showed that the CD40-CD40L interaction is also involved in the
secretion of NO by macrophages and DCs in vitro (9
, 18
, 19)
. NO is a highly effective molecule that can exert direct
cytotoxic effect toward microorganisms and tumor cells (8
, 20
, 21)
or that can act indirectly via stimulating the production of
inflammatory mediators like TNF-
and IL-1 (20)
.
Murine macrophages have been shown to express iNOS, which catalyzes the
production of NO from L-arginine in
vitro and in vivo (20
, 22
, 23)
. NO is able
to induce apoptosis in several cell types including tumor cells
(24)
. In the present study, we investigated a possible
role of CD40-CD40L interactions in our murine GvL model system. The
results provide in situ and in vivo evidence for
the involvement of the CD40-CD40L interaction in the therapy effect.
This interaction stimulates the up-regulation of iNOS expression
predominantly by SER+ macrophages. The increased NO synthesis in livers
of ADI-treated mice is associated with apoptosis in tumor cells early
after cell transfer as well as in liver-infiltrating T cells at later
stages. We suggest that the CD40-CD40L interaction contributes to the
effectiveness of the antitumor response as well as to the clearance of
T cells from the liver after completion of the antitumor therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayIn Vivo Blocking of...RESULTSDISCUSSIONREFERENCES |
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Tumor Cell Lines
ESb cells represent a spontaneous highly metastatic variant of the
chemically induced lymphoma L5178Y (Eb) of DBA/2 mice. The subline
ESb-MP is a plastic-adherent variant of the ESb lymphoma cells.
In vivo, ESb-MP cells grow progressively but show a less
aggressive phenotype, metastasizing more slowly than ESb and involving
multiple organs including the liver (2)
.
Adoptive Cellular Immunotherapy (ADI)
DBA/2 mice were anesthesized with Rompun (0.1%; Parke, Davis &
Company, Berlin, Germany): Ketanest (0.25; 1% Bayer, Leverkusen,
Germany): PBS at 1:1:3 (vol) to inject 2 x
105 ESb-MP tumor cells intradermally into the
dermis of the shaved flank. To generate allogeneic tumor immune
effector cells, B10.D2 mice were inoculated i.v. with 1 x
105 ESb-MP cells (1)
. Seven days
later, spleen cells were isolated from the donor and transferred i.v.
(2 x 107 cells/200 µl PBS; Life
Technologies, Inc., Eggenstein, Germany) into 5 Gy
(60Co source Gammatron F 80 S; Siemens,
Munich, Germany) sublethally irradiated DBA/2 mice that carried tumors
(>1 cm in diameter). The transfer of the antitumor immune spleen cells
was made 3 weeks after intradermal tumor cell inoculation. As a control
group, sublethally irradiated tumor-bearing DBA/2 mice remained
untreated. To analyze GvH responses, non-tumor-bearing DBA/2 mice were
irradiated and inoculated with the tumor-immune donor cells.
Antibodies and Other Reagents
The following rat mAbs were used as culture supernatants:
antimouse SER (clone SER4; Ref. 5
), antimouse B220 (clone
GB2; Ref. 25
), and antimouse CD40 (clone 3/23;
Instruchemie, Hilversum, the Netherlands). The rat antimouse N418
(clone N418), which was kindly provided by Dr. B. Kyewski (German
Cancer Research Center, Heidelberg, Germany), and the hamster antimouse
CD40L mAbs (clone MR1; Ref. 26
) were biotinylated.
In vivo injected anti-CD40L mAb was unconjugated and
immunohistologically detected with a goat antihamster IgG (Dianova,
Hamburg, Germany). iNOS was detected by a polyclonal rabbit antimouse
iNOS serum (Calbiochem, Bad Soden, Germany). SER and B220 were
visualized by using polyclonal donkey antirat IgG (H+L) second
antibodies linked either to PO or to AP (Dianova). Anti-N418 and
anti-CD40L were detected by ExtrAvidin linked to AP (Sigma,
Deisenhofen, Germany). Anti-CD40 was visualized by consecutive
incubation with a rabbit antirat IgG linked to PO and a swine
antirabbit IgG linked to PO (both DAKO, Hamburg, Germany). The rabbit
antimouse iNOS antibody was detected via a biotinylated donkey
antirabbit second antibody (Amersham, Braunschweig, Germany), and
avidin was linked to PO (Sigma). Endogenous biotin was blocked by the
biotin blocking system from DAKO.
Immunohistochemistry
Tissue Preparation.
Livers from DBA/2 mice were removed at different time points
after therapy and snap-frozen in liquid nitrogen. Six-µm-thick
consecutive cryostat sections were mounted on uncovered glass slides,
air-dried overnight at room temperature, fixed in acetone (Merck,
Darmstadt, Germany) for 10 min at room temperature, and air-dried again
for 1 h.
Single Staining.
After drying, the slides were washed in PBS three times for 5 min. To
avoid nonspecific binding, sections were treated with 1% normal mouse
serum for 15 min, followed by incubation with the first antibody for 45
min. After washing three times in PBS, the sections were treated with
the second antibody, and in the case of CD40, afterward with the third
antibody. Before the substrate reaction for PO or AP was performed, the
slides were washed three times in PBS for 5 min. When using a
biotinylated antibody, the slides were incubated with ExtrAvidin for 30
min. After staining, the sections were washed with water and mounted
with glycerol-gelatin (Merck). The same protocol was performed for
negative controls in which either the first or the second antibody was
omitted. All steps were performed in a humid chamber and at room
temperature, except for incubations with the first antibody for CD40,
CD40L, and iNOS, which were done overnight at 4°C.
Double Staining.
The staining procedure represents a combination of consecutive
stainings for each antigen. Every single staining was completely
finished with the substrate reaction before starting with the second
staining. For double staining of CD40 and CD40L, sections were
incubated with the two first antibodies in parallel.
Development of Enzyme Reactions.
PO reactivity was revealed by incubating the sections in a solution
containing 6 mg of 3-amino-9-ethylcarbazole (Merck), which was
dissolved in 1.5 ml of N,N-dimethylformamide (Merck), 15
µl of 30% hydrogen peroxide, and 28.5 ml of 0.1
M acetate buffer (pH 5.0). The substrate for the
development of AP consisted of 6.3 µl of 5% Neufuchsin (Sigma) or 2
mg of Fast Blue (Sigma) in 16 µl of 4% sodium nitrite (Fluka, Buchs,
Switzerland), 2 mg of naphthol-As-Bi-phosphate (Sigma) in 20 µl of
N,N-dimethylformamide (Merck), and 3 ml of 0.05
M/l Tris-HCl buffer (pH 8.7) containing 1
mM levamisole (Sigma). The freshly prepared
solutions were filtered through a 0.22 µm filter (Millipore,
Eschborn, Germany) and added to the sections. Development lasted about
3–10 min, with regular checking of the staining intensity by
microscope. Immunohistochemical results were evaluated by counting
positively stained cells per liver lobule from three to six
lobuli/mouse. The means and SDs of the data obtained from two to three
mice/time point and from different experiments were calculated and
presented in graphs.
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TUNEL Assay |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayIn Vivo Blocking of...RESULTSDISCUSSIONREFERENCES |
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In Vivo Blocking of the CD40-CD40L Interaction with mAb against CD40L |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayIn Vivo Blocking of...RESULTSDISCUSSIONREFERENCES |
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayIn Vivo Blocking of...RESULTSDISCUSSIONREFERENCES |
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. In normal livers of DBA/2 mice,
neither CD40 nor CD40L could be detected (data not shown). Fig. 1A
shows that in the liver of tumor-bearing DBA/2 mice,
which were only sublethally irradiated with 5 Gy but not treated with
immune cells (control), some CD40+ cells (maximum, four cells/LL) could
be detected. All animals from this group died until day 32 after tumor
inoculation because of the tumor burden and metastasis. Tumor-bearing
mice that were treated with tumor immune donor cells showed a steady
increase of CD40+ cells with a significant peak at day 20 after cell
transfer (35 cells/LL) and a subsequent decrease at day 28 (18
cells/LL). The number of CD40L+ cells was much lower than that of CD40+
cells (Fig. 1B)
. Maximal levels were observed at day 20
(three cells/LL), whereafter their number decreased to one cell/LL at
day 28. In non-tumor-bearing mice that received the same immune cells
(GvH mice), the number of CD40+ cells increased similar to the GvL
animals but only until day 12, reaching a peak level of 12/LL (Fig. 1A)
. CD40L+ cells were not detected in GvH animals (Fig. 1B)
. Collectively, the data show that there is a significant
increase in CD40 expression in the liver of tumor-bearing mice during
ADI.
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As shown in Fig. 2A,
ADI
caused a significant increase in the numbers of F4/80+ and SER+
single-positive cells in tumor-bearing mice. The number of CD40+ F4/80+
double-positive cells also increased in these animals. At day 20, when
the highest number of CD40+ cells was detected, there was also a peak
of CD40+ F4/80+ cells (22 cells/LL, which represent 23% of the total
number of F4/80+ cells), which represented 70% of the total CD40+
cells. The highest number of CD40+ SER+ double-positive cells was also
seen at day 20 (16 cells/LL, 22% of the SER+ cells). Thus, at this
time point, 72% of CD40+ macrophages in the liver were SER+.
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. Although
in GvH considerably fewer CD40+ cells were found, the maximal number of
CD40+ F4/80+ and CD40+ SER+ cells were also detected at day 12, like in
GvL (six and six cells/LL, respectively). As in GvL, the majority of
CD40-expressing macrophages (F4/80+) were SER+ in GvH as well.
Fig. 2C
shows that the number of B220-positive cells
(B cells) in liver sections is very low, both in GvL and GvH
animals. Therefore, practically no CD40+ B220+ double-positive cells
were found in these mice. This suggests that B cells do not play
any role in ADI and that they do not contribute to the CD40-CD40L
interactions in the liver.
As shown in Fig. 2D,
the number of DCs as defined by
expression of N418 increased in the GvL significantly from 0 to 28
cells/LL at day 20. At this time point, the maximal number of
CD40-expressing DCs was detected (13 cells/LL). This corresponded to
46% of the DCs and to 40% of the total number of CD40+ cells at this
time point. In GvH, the number of DCs also increased and reached a peak
at day 12 after the transfer of immune cells (13 cells/LL). At this
time point, 38% of DCs (five cells/LL) also expressed CD40.
In conclusion, macrophages and DCs are the two APC populations in the liver expressing CD40 during ADI, whereas B cells do not.
iNOS Expression by SER+ Macrophages during ADI.
It has been shown that the CD40-CD40L interaction is involved in
the production of NO by macrophages and DCs in vitro
(9
, 19) . Therefore, we analyzed iNOS expression in
situ in the liver of ADI-treated mice, in particular by SER+
macrophages. In normal, non-treated livers, no iNOS expression could be
found (data not shown). Fig. 3
shows that
the maximal number of iNOS-expressing cells in the liver of GvL animals
was detected at day 20 (25 cells/LL). A lower peak was already seen at
day 8 (14 cells/LL). At these time points, most of the iNOS-expressing
cells (>80%) were also SER+ (20 and 12 cells/LL, respectively). In
the liver of GvH mice, the number of iNOS-expressing cells was much
lower than in GvL animals with a maximum of eight cells/LL, and there
was no second increase such as in GvL. At day 8, 71% of
iNOS-expressing cells were SER+. Double staining for iNOS and N418
revealed that DCs also expressed iNOS, but the cell numbers were much
lower than those for SER+ macrophages. The maximum number of iNOS+ DCs
was seen at day 20 with six cells/LL (data not shown). Thus, it can be
concluded that SER+ macrophages represent the major population and DCs
only a minor population of iNOS-expressing cells during ADI.
|
In the liver of GvL animals (Fig. 4A)
, the maximal number of
iNOS-expressing cells (25 cells/LL) was detected at day 20 after donor
cell transfer, coinciding with the highest level of CD40-expressing
cells (33 cells/LL; Fig. 1
). At this time point, all iNOS-expressing
cells were CD40+. In GvH mice, maximal iNOS expression also correlated
with the presence of CD40 (similar to the GvL situation). The number of
double-positive cells was much lower than in GvL animals and reached a
maximum at day 8 (eight cells/LL). iNOS expression, therefore, seems to
be induced in CD40+ cells during ADI.
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shows iNOS-positive cells and apoptotic cells in the
metastatic area at day 5 after transfer of donor cells. At day 20,
apoptotic cells were detected around portal veins, where clusters
containing CD4+, CD8+ T cells and SER+ macrophages were demonstrated
previously (6)
. These apoptotic cells might represent
liver-infiltrating T cells that had to be removed after successful
elimination of the metastases. Combination of the TUNEL assay and
immunohistological staining for the donor cell marker
ß2-microglobulin revealed that at day 20, 45% of the apoptotic cells
were donor cells, whereas at day 5, only 6% were of donor origin (data
not shown). At both time points, iNOS-expressing cells were clearly
found in close proximity to cells undergoing apoptosis (three and three
cells/LL at days 5 and 20, respectively), indicating that NO might
induce apoptosis in tumor cells at the early time points and in normal
T cells at later stages of ADI. In GvH animals, 3–5 apoptotic
cells/LL were also found at day 20 after cell transfer, of which some
were in close proximity to iNOS-expressing cells (data not shown).
Reduced ADI Effect Caused by in Vivo Treatment with
Anti-CD40L mAb.
To determine whether the CD40-CD40L interaction is crucial for the
therapeutic effect of ADI, we injected a blocking anti-CD40L mAb (MR1)
into tumor-bearing animals either at days 3 and 5 or at days 18 and 20
after ADI. One hundred % of ADI-treated, tumor-bearing animals that
were injected with normal hamster immunoglobulin were alive at the end
of the experiment (at day 60 after tumor cell inoculation; Fig. 5A,
ADI + control
Ig). In contrast, both groups that were injected with anti-CD40L
mAb showed reduced survival. Thus, only 49% of mice injected at days 3
and 5 after ADI and 37% of mice injected at days 18 and 20 (Fig. 5A)
were alive at the end of the experiment, which is
statistically significant in comparison to the control
(P < 0.0001). Animals that received neither ADI nor
antibody treatment were all dead at day 53 after tumor inoculation. The
injected anti-CD40L mAb could be detected with a goat antihamster
second antibody in the liver of the treated animals, indicating that
the injected antibodies were able to target CD40L+ cells. The observed
effect is most probably attributable to blocking of the receptor and
not to depletion of these cells. We further looked whether the
elimination of the effector T cells at late time points is abrogated by
the blocking of CD40-CD40L interaction. Interestingly,
immunohistological staining for CD4+ and CD8+ revealed that in livers
of mice injected with control immunoglobulin, the number of CD4+ and
CD8+ T cells decreased from day 7 to day 22 (from 36 to 29 CD4+ and
from 32 to 24 CD8+ T cells; Fig. 5B)
, whereas in mice
treated with the anti-CD40L mAb, the number of both T-cell subsets
increased (from 41 to 50 CD4+ and from 37 to 50 CD8+ T cells;
P < 0.05;
. At day 22, 
2-fold
more CD4+ and CD8+ T cells were found in the anti-CD40L mAb-treated
group than in the group injected with control immunoglobulin. The
CD40-CD40L interaction thus appears to be of importance at early as
well as at late time points of ADI.
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. In contrast, the expression of CD40 was
significantly lower in livers of mice injected with anti-CD40L mAb than
in ADI treated animals without antibody injection (four cells/LL at day
7 and 11 cells/LL at day 22; P < 0.05; Fig. 5C)
. Treatment with anti-CD40L mAb also caused a reduction
in the number of iNOS+ cells (from 9 cells/LL in the control group to 3
cells/LL at day 7, and from 20 cells/LL to 6 cells/LL at day 22;
P < 0.05; Fig. 5C)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayIn Vivo Blocking of...RESULTSDISCUSSIONREFERENCES |
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Maximal CD40 expression was detected at day 20 after immune cell
transfer. We showed previously that at this time point, the number of
SER+ macrophages also reached its maximum. These cells formed tight
clusters with CD4+ and CD8+ donor T cells (7)
. Ridge
et al. (27)
and Schoenberger et al.
(28)
presented a new dynamic model of CTL
activation. CD4+ helper T cells expressing CD40L engage and
"condition" APCs (e.g., DCs) via CD40-CD40L interaction,
which leads to the up-regulation of CD40 and other costimulatory and
adhesion molecules (9
, 11
, 17
, 27
, 29)
. It has been shown
that after immunization with keyhole limpet hemocyanin, few CD40L+
cells are enough to activate antibody-producing B cells (1 CD40L+
cell per 10–12 B cells; Ref. 30
). It is thus likely that
the few CD40L+ cells detected after immune cell transfer represent
donor helper T cells. These might activate host APCs, which then are
able to activate CD8+ T cells to become cytotoxic. The observed
clusters during ADI, thus, could be sites of T cell-macrophage/DC
interactions, which lead to the activation of cytotoxic and helper T
cells. Our data on the proliferation of CD4+ and CD8+ donor T
cells after ADI support this model (7)
.
It has been shown that cytotoxic CD8+ T cells can induce apoptosis (31) . We have demonstrated recently that the endogenous viral superantigen 7 (Mlsa) encoded by the mouse mammary tumor virus provirus Mtv-7 is expressed in DBA/2 mice and also in the ESb-MP tumor cells thereof derived but not in B10.D2 mice (32) . In DBA/2 mice, viral superantigen 7 causes deletion of superantigen-reactive T cells with certain Vß chains (e.g., Vß6) from their repertoire, whereas in B10.D2 tumor-resistant mice, Vß6+ T cells are present. These particular T cells can infiltrate the liver metastases during ADI (7) . We showed that Vß6+ T cells are able to kill superantigen-expressing ESb-MP tumor cells by apoptosis in vitro.4 It is also likely that T cells with specificities against other tumor antigens contribute to the antitumor response. Apoptosis mediated by CTLs apparently does not involve NO because T cells do not express iNOS.
The CD40-CD40L interaction is not only involved in priming,
differentiation, and activation of CD4+ and CD8+ T cells but also in
the mediation of effector functions of DCs and macrophages such as the
production of inflammatory cytokines (e.g., TNF- and
IL-1) or NO (9
, 11
, 18) . Ligation of CD40 on macrophages
and DCs leads to secretion of IL-12 and other inflammatory cytokines
that are critical for the differentiation of Th1 cells and therefore
for the induction of antitumor immunity (29
, 33
, 34)
.
NO is known to be produced by activated macrophages, DCs, and endothelial cells in vitro by iNOS (19 , 21 , 24) and can induce apoptosis in tumor cells including the ESb-MP cell line (21 , 22 , 24) . iNOS was predominantly expressed by SER+ macrophages and to a lower extent by DCs (data not shown). iNOS expression was observed in the liver close to metastases at early time points and at late time points close to portal veins, where the clusters containing CD4+, CD8+, and SER+ macrophages and DCs are located (6 , 7) . The question thus arises whether in this model, NO may function as a cytotoxic and/or as a regulatory molecule.
Different regulatory functions of NO have been described. Marcinkiewicz
et al. (21)
suggested a positive feedback
mechanism in which NO enhances the synthesis of TNF- and IL-1.
Both cytokines are known to stimulate NO generation and function as
cytotoxic and inflammatory molecules. This kind of feedback mechanism
could occur at the early time points of the therapy, when metastases
should be eradicated from the liver. At day 5 after donor cell
transfer, large metastases could still be seen in the liver. In the
central areas of the metastases, apoptotic cells could be detected in
high numbers (data not shown). Because metastases were completely
eradicated at day 12 after ADI and iNOS+ cells were seen before this
time point in close association with apoptotic cells in areas of
metastases, these findings suggest a cytotoxic role for NO in the
eradication of metastases during this early time period of ADI.
Additionally, ESb-MP tumor cells could be killed by
NO5
in
vitro, which supports the latter suggestion.
NO can also act by a negative feedback mechanism, inhibiting the
secretion of the Th1 cytokines IFN- and IL-2 (35)
. It
is further able to limit Th1 cell activity by supporting
down-regulatory IL-4 production and by inhibition of MHC II expression
(35
, 36) . This mechanism may be operative at a later stage
of ADI, when local T-cell responses have to be terminated and the liver
has to be cleared from undesired immune reactive cells. At these time
points, iNOS-expressing cells as well as apoptotic cells were detected
around periportal veins, where the clusters containing CD8+ and CD4+ T
cells are also located (7)
. Some of these CD8+ and CD4+ T
cells were shown to be apoptotic.5 Moreover, at
this time point, 45% of the apoptotic cells were of donor origin
(positive for the donor cell marker ß2-microglobulin; data not
shown), indicating a role for apoptosis in elimination of donor cells
from the liver. After day 20, the numbers of CD8+ and CD4+
liver-infiltrating T cells decreased markedly (7)
.
Double staining for iNOS and CD40 revealed that at day 20, when the maximum of NO production and CD40 expression was reached, all iNOS-expressing cells were positive for CD40. It has been described that macrophages are able to secrete NO in vitro after interaction with CD4+ T cells or plasma membranes of T cells activated with anti-CD3 antibodies and that this effect is mediated by CD40-CD40L interaction (18 , 23) . It was also shown for DCs that ligation of CD40 in vitro induces a significant elevation in NO synthesis (19) . Our in situ observations of CD40/iNOS coexpressing cells suggest that the CD40-CD40L interaction is also involved in the induction of iNOS in the situation of GvL reactivity. Furthermore, the in vivo blockade of CD40L during ADI led to a marked inhibition of the therapeutic effect. In the livers of anti-CD40L mAb-treated animals, iNOS and CD40 expressions were reduced significantly. Moreover, in vivo experiments in which iNOS activity was blocked by the inhibitor N-(3-(aminomethylbenzyl)acetamidin (1400W) at early and at late time points after ADI treatment also showed a marked reduction of the survival,5 supporting the importance of the induced NO production for the therapeutic effect.
Interestingly, the number of CD4+ and CD8+ T cells in the liver of mice
treated with anti-CD40L mAb at days 18 and 20 after ADI was not
decreased 22 days after ADI, such as in animals treated with control
immunoglobulin, but rather increased (Fig. 5B)
. The same
phenomenon was observed when we suppressed iNOS activity by a specific
inhibitor in vivo.5 Because at these
later time points the metastases were already eradicated, the animals
that received anti-CD40L mAb injections died not from metastases but
more likely from GvH disease. GvH disease can be caused by donor T
cells that were not eliminated via HvG reactivity from the liver, which
is likely to be the standard physiological ADI situation. The blockage
of the NO induction may have led to reduced HvG reactivity so that
fewer donor cells were eliminated and that their proliferative
expansion was not inhibited. Such expanding T-cell clusters may involve
HvG- and GvH-reactive T cells, such as in mixed lymphocyte reactions.
This study demonstrates that at early and at late stages of the therapy, CD40-CD40L interactions are important for the GvL therapy effect of the ADI. The resultant production of NO would contribute to the destruction of liver metastases at early time points and also at a later stage to the removal of the liver-infiltrating T cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant 10-0980-Schi2 from the Dr.
Mildred Scheel Stiftung (to V. U. and M. R.). 
2 To whom requests for reprints should be
addressed, at Division of Cellular Immunology, Tumor Immunology
Program, German Cancer Research Center, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany. Phone: 49-6221-423708; Fax: 49-6221-423702;
E-mail: s.mueerkoester{at}dkfz-heidelberg.de
3 The abbreviations used are: ADI, adoptive
immunotherapy; SER, sialoadhesin; TNF, tumor necrosis factor;
DC, dendritic cell; APC, antigen-presenting cell; IL, interleukin;
iNOS, inducible NO synthase; GvH, graft-versus-host;
GvL, graft-versus-leukemia; mAb, monoclonal antibody;
PO, horseradish peroxidase; AP, alkaline phosphatase; TUNEL,
TdT-mediated dUTP nick end labeling; LL, liver lobule.
Received 7/22/99; revised 1/31/00; accepted 2/16/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayIn Vivo Blocking of...RESULTSDISCUSSIONREFERENCES |
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. J. Exp. Med., 179: 1109-1118, 1994., endotoxin, and interaction with allogeneic T cells. J. Immunol., 157: 3577-3586, 1996.[Abstract]
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), and of
normal 5 Gy pretreated mice that received donor immune cells
(GvH; 
). B, CD40L expression in the
liver of either control mice (•), GvL (
,
no surviving animals.
), CD40+ F4/80+
double-positive cells (
), SER+ macrophages (
, B220+ CD40+) and in GvH mice (
) and GvH
(
and 
)
C, expression of CD40 in livers of mice treated with
control hamster immunoglobulin (