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* Departments of Medicine and Pediatrics and
Institute for Human Gene Therapy, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and
Department of Medicine, Yale University, New Haven, CT 06510
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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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Cell-mediated cytotoxicity is critical to the tissue injuries in GVHD, although damage to the gastrointestinal tract can amplify disease in other tissues (1, 2, 3, 9, 10, 11, 12, 13, 14). Initial studies indicated that the infiltration of allogeneic CD8 T cells into the livers of recipient mice began as early as 4 days and peaked by 14 days following allo-BMT (15, 16, 17). Inhibition of CD8 T cell migration into the livers of recipient mice could significantly reduce the hepatic damage as well as the severity of acute GVHD in recipient mice (15, 17). Interestingly, the accumulation of donor CD8 T cells in the skin began by day 14 and peaked between days 28 and 35 after allo-BMT, in parallel with the occurrence of clinical signs of cutaneous GVHD (18). A recent study by Dickinson et al. (19) demonstrated in an ex vivo skin-explant model that the degree of CTL infiltration correlated directly with the severity of graft-vs-host reactions. In contrast, necrosis of the individual cells in intestinal crypts was the first change detected by light microscopy, and there was no apparent increase in lamina propria lymphoid cells in the vicinity of crypt necrosis (20). Thus, T cell-mediated toxicity is particularly critical to the injuries in the liver and skin (1, 2, 3, 20). Therefore, identifying the mechanism(s) by which alloreactive T cells are recruited into the liver and skin is important for understanding how T cells selectively target specific tissues in the recipients with acute GVHD.
Several lines of evidence suggest that tissue-resident APCs, such as macrophages and dendritic cells (DCs), are related to the development of T cell immunity. Depletion of macrophages and DCs from the livers and spleens of mice could inhibit T cell-mediated lysis of viral-infected hepatocytes. Specific elimination of macrophages from skin but not from drainage lymph nodes significantly decreased the number of locally infiltrating T cells and DCs, which resulted in the regression of cutaneous inflammation in mice of an autoimmune disease model (21). In experimental GVHD models, direct contact between dermal DCs and allogeneic T cells has been observed in the skin from recipient mice with allo-BMT (18, 22). Furthermore, in an ex vivo skin-explant assay, preincubation of skin sections with hematopoietic cells could significantly enhance the infiltration of CTLs in the skin sections (19). Taken together, these data suggest that residual hematopoietic APCs in skin may be essential for the development of cutaneous GVHD (19). However, it remains unclear whether resident liver macrophages and DCs are essential for the development of hepatic GVHD. Moreover, whether local tissue APCs prime naive allogeneic T cells to host Ags, or whether their major role is to recruit activated allogeneic T cells into target tissues, is unknown.
In this study, using miHA-mismatched donor C3H.SW and recipient C57BL/6 mice, we now report that tissue-specific depletion of hepatic and splenic APCs by systemic administration of liposomal clodronate (lipo-clodronate) significantly reduces the recruitment of activated allogeneic CD8+ T cells into the livers of B6 recipient mice. This selective APC depletion also results in the inhibition of acute GVHD, but this effect is largely confined to the liver, where resident APCs were depleted. Partial APC depletion and GVHD amelioration result in partially improved survival of lipo-clodronate-treated mice. These results indicate that tissue-resident APCs play an important role in directing activated allogeneic CD8+ T cells to the target organs of acute GVHD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B6 (H-2Db, CD45.2 +) or B6/SJL (H-2Db, CD45.1+) recipient mice, and miHA-mismatched C3H.SW (H-2Db, CD45.2+, and Ly9.1+) donor mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in sterile conditions. Drinking water of bone marrow transplantation (BMT) recipients was supplemented with neomycin sulfate and polymyxin B (Sigma-Aldrich, St. Louis, MO) from 2 days before irradiation or systemic administration of mice with lipo-clodronate until 3 wk posttransplant.
Antibodies
Abs for immunofluorescence and immunohistochemistry staining and
cell separation, including anti-CD8 (clone RM4-5), biotinylated
anti-mouse IFN-
, biotinylated anti-CD11b (clone M1/70),
biotinylated anti-CD11c (clone HL3), biotinylated anti-NK1.1,
and biotinylated anti-B220, were obtained from BD PharMingen (San
Diego, CA). Biotinylated anti-F4/80 was purchased from Serotec
(Oxford, U.K.). FITC-anti-CD11a, FITC and PE-conjugated
streptavidin, PE-anti-CD11c, CyChrome-anti-CD8, and
CyChrome-streptavidin were also obtained from BD PharMingen. All
anti-CD4, anti-CD8, anti-B220, anti-CD11b, and
anti-CD11c Abs conjugated with microbeads and the streptavidin
conjugated with microbeads were purchased from Miltenyi Biotec
(Auburn, CA).
Preparation of lipo-clodronate
Lipo-clodronate was prepared as described by van Rooijen et al. (23) with slight modification (24). Briefly, lipids composed of 86 mg of egg phosphatidylcholine (Sigma-Aldrich) and 19 mg of cholesterol (Sigma-Aldrich) were dissolved in 10 ml of chloroform. The organic solvent was removed under reduced pressure in a rotary evaporator and the resulting film was desiccated overnight. The thin, dry film was redehydrated in a suspension of 10 ml of PBS (pH 7.5) containing 2.0 g clodronate (Sigma-Aldrich). The liposomes were sonicated for 3 min at 20°C in a water bath sonicator. Lipo-clodronate was purified by three centrifugation washes at 4,000 x g for 20 min, and lipo-clodronate was resuspended in 4 ml of PBS. Lipo-clodronate can be selectively taken in by macrophages and myeloid DCs in the spleen and liver (23, 25). The phospholipid bilayers of liposomes are disrupted by lysosomal phospholipases, and clodronate is intracellularly released to inhibit the cellular metabolism, which induces cell suicide. In contrast, the extracellular free clodronate cannot easily cross cell membranes and has an extremely short t1/2 in circulation and therefore is not toxic to cells (23).
Previous studies have determined that i.v. administration of mice with
200 µl of lipo-clodronate prepared in this way can deplete
macrophages and DCs in the spleen and liver (23, 25). In
preliminary studies, we repeated these measurements, following i.v.
injection of 25–400 µl of lipo-clodronate/mouse, and found that
depletion of DCs and macrophages peaked and plateaued between 100 and
200 µl. Doses >250 µl of lipo-clodronate were toxic to the mice
and increased immediate peri-BMT mortality (we detected swollen
kidneys, but are otherwise unsure of the mechanism underlying this
high-dose toxicity), without any additional increase in APC depletion.
Therefore, we chose to treat each mouse with 150 µl of
lipo-clodronate, containing 
0.75 mg of clodronate (23),
to ensure that we were well within the dose-response plateau without
risking non-APC-related toxicity.
Cell preparations
Donor bone marrow cells were prepared from C3H.SW mice as previously described (6). T cell-depleted bone marrow (T-BM) cells were isolated with anti-CD4 and anti-CD8 Abs conjugated with magnetic microbeads. Donor CD8+ T cells were purified from C3H.SW mice by one of two protocols. On the first, donor CD8+ T cells were positively selected from spleens and lymph nodes of C3H.SW mice by anti-CD8 Ab conjugated with magnetic microbeads. Alternatively, donor CD8+ T cells were negatively selected by depletion of CD11b, NK1.1, B220, and CD4 cells using magnetic cell sorting (MiniMACS; Miltenyi Biotec). Because murine DCs express CD8 Ag (26), CD11c+ cells were also completely removed by anti-CD11c Ab conjugated with microbeads before purifying donor CD8+ T cells based on magnetic separation. The purity of isolated donor CD8+ T cells was always >95%, as reanalyzed by flow cytometry.
CFSE labeling
Purified C3H.SW donor CD8+ T cells were resuspended at a concentration of 1 x 107 cells/ml in 2.5% FBS in PBS. CFSE (Molecular Probes, Eugene, OR) was added to a final concentration of 5 µM. Labeling was quenched with ice-cold PBS after 15 min of incubation at 37°C. Cells were washed twice in PBS and resuspended in PBS for transplantation.
BMT and CD8+ T cell transplantation
Mice underwent allo-BMT as previously described (6). Briefly, B6 recipients were irradiated with 9.5 Gy administered in two fractions from a 137Cs source. C3H.SW CD8+ T cells (2 x 106), mixed with or without C3H.SW T-BM cells (7 x 106), were transplanted into B6 recipients via tail vein injection (five to eight mice per group per experiment) immediately after irradiation. Recipient mice were weighed twice weekly and monitored for the clinical signs of acute GVHD and survival. The clinical grading criteria for the cutaneous inflammation of acute GVHD were followed as established.
Isolation of hepatic lymphocytes
Hepatic lymphocytes were isolated as previously described (27). Briefly, livers from recipient B6 mice were minced and suspended in PBS containing 1 mM EDTA. Percoll was added to the cell suspension (Amersham Pharmacia Biotech, Piscataway, NJ) to a final concentration of 33%. After centrifugation for 30 min at room temperature, hepatic lymphocytes were collected from the bottom and contaminating RBCs were lysed.
Cytokine assays
Assays of intracellular IFN- in donor T cells were performed
as previously described with slight modification (28).
Briefly, splenic and hepatic lymphocytes were cultured in medium
without stimulation or in the presence of precoated CD3 Ab and MC57SV
cells (H-D2b) irradiated with 30 Gy in MicroWell
plate for 16 h. GolgiStop containing monensin (BD PharMingen) was
added into the cultures at the final 6 h to block intracellular
transporter of IFN-. Cells were collected and stained with
CyChrome-anti-CD8 Ab combined with or without FITC-anti-CD45.2.
After fixation and permeabilization using Cytofix/Cytoperm containing
formaldehyde and saponin (BD PharMingen), cells were washed, stained
with biotinylated anti-mouse IFN- and then PE-conjugated
streptavidin, and then analyzed by flow cytometry.
Flow cytometry analysis
Immunofluorescence analyses were performed as previously described (29, 30). In tri-color analyses, mononuclear cells from spleen, PLN that includes popliteal, superficial inguinal, axillary, and salivary lymph nodes, MLN, liver, and peripheral blood were stained with FITC-conjugated anti-Gr-1, PE-conjugated anti-CD11c Abs, and biotinylated anti-CD11b or biotinylated anti-F4/80 followed by revealing with CyChrome-streptavidin. In other experiments, CFSE-labeled CD8+ T cells were further stained with CyChrome-anti-CD8 coupled with biotinylated anti-CD45.2 and PE-conjugated streptavidin. Cells were analyzed by FACScan (BD Biosciences, Mountain View, CA).
Pathologic examination of tissues
Mice were sacrificed and specimens of liver, skin, and intestine were taken for histopathologic analysis. All samples were placed in 10% neutral buffered formalin (Sigma-Aldrich), embedded in paraffin, sectioned, and stained with H&E for histopathologic assessment for acute GVHD (2, 31). The murine cutaneous GVHD was graded according to the criteria established by Kao and coworkers (32).
Immunohistochemistry and fluorescence staining of tissues
Recipient spleens were frozen in O.C.T. embedding medium (Sakura Finetek, Torrance, CA). Six-micrometer frozen sections were cut, air-dried, fixed in acetone, and rehydrated in PBS. Spleen sections were incubated with biotinylated hamster anti-mouse CD11c Ab (BD PharMingen) followed by PE-conjugated streptavidin (Vector Laboratories, Burlingame, CA). Nonspecific binding of biotin and avidin was blocked by addition of excess avidin and biotin using the blocking kit (Vector Laboratories). Isotype-matched rat IgG (BD PharMingen) was used as a negative control. After staining, the slides were coverslipped using Vectashield H-1200 (Vector Laboratories) and observed under fluorescent microscope (Nikon Microphot-FXA; Nikon, Melville, NY).
Immunohistochemistry staining of the liver was performed as previously described (33). The cryosections of liver were stained with rat biotinylated anti-CD8 Ab (BD PharMingen) followed by HRP-conjugated goat-anti rat IgG (F(ab')2) (Vector Laboratories) staining. Sections were developed with diaminobenzidine substrate kit (Vector Laboratories). Isotype-matched rat IgG (BD PharMingen) was used as a negative control.
Statistical analysis
Survival data were analyzed by life-table methods using the
Mantel-Peto-Cox summary of 
2. Student’s
t test was used to analyze the difference of APC depletion
and T cell infiltration and migration in vivo. Values of
p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish a mouse model for investigating the role of host
tissue-resident macrophages and DCs in mediating organ-related acute
GVHD, lipo-clodronate (150 µl per injection per mouse) was
administered via tail vein at day -7 via tail vein and i.p. at day
-2. Control B6 mice received identical injections of empty liposomes.
Immunofluorescence staining was performed to monitor the depletion of
macrophages and DCs from the livers, spleens, PLN, MLN, and peripheral
blood by the day of allo-BMT. At day 0, the overall number of
mononuclear cells in the spleens of lipo-clodronate-treated mice was
reduced from 8.2 ± 1.1 x 107 to
1.9 ± 0.67 x 107 per spleen. Flow
cytometry analysis showed that up to 85% of
F4/80+ macrophages and 75% of
CD11c+ DCs in the spleens were eliminated in
these mice, compared with control B6 mice treated with empty liposomes
(Fig. 1
A). Lipo-clodronate
treatment also eliminated >75% of F4/80+
macrophages from the liver and peripheral blood (Fig. 1A).
CD11c+ DCs were detected in the liver only at
very low levels and were further depleted by lipo-clodronate. In
contrast, lipo-clodronate did not affect macrophages and DCs in the
PLN, MLN (Fig. 1A), or skin and other cell populations in
PLN and MLN (data not shown). Furthermore, immunofluorescence staining
showed that aggregation of CD11c+ DCs (red color)
and the apposition of CFSE-CD8 T cells (green color) next to
CD11c+ DCs were abrogated in the spleen of
lipo-clodronate-treated B6 recipients at 6 h following irradiation
and cell transplantation of C3H.SW T-BM cells
and CD8+ T cells (Fig. 1B). Taken
together, these results indicate that lipo-clodronate treatment creates
an organ-specific depletion of macrophages and DCs in the liver and
spleen.
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To assess whether depletion of macrophages and DCs might influence
the recruitment of donor CD8+ T cells into local
tissues, C3H.SW T-BM and CFSE-labeled
CD8+ T cells (CD45.2) were infused into
lipo-clodronate-treated B6 mice (CD45.1) or control B6 mice at day 0.
Lymphocytes were recovered from recipient livers, spleens, PLN, and MLN
at 1, 5, and 14 days following transplantation, enumerated, and stained
for flow cytometry analysis. When compared with T cell infiltration in
control B6 recipients, donor CD8+ T cell
infiltration was reduced in the livers of lipo-clodronate-treated
recipients by 6.8-fold at day 5 and 2.6-fold at day 14 following
allo-BMT. By 14 days, there was a 4-fold decrease in the number of
donor CD8+ T cells in the spleens from
lipo-clodronate-treated recipients (Fig. 2A). In contrast,
lipo-clodronate treatment had no effect on the recruitment of donor
CD8+ T cells into PLN and MLN (Fig. 2). Thus,
this organ-specific depletion of host APCs only reduced the recruitment
of donor CD8+ T cells into the tissues from which
APCs had been removed.
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, B and C, donor C3H.SW
CD8+ T cells in the spleens, PLN, and MLN
recovered from control B6 recipient mice at 5 days following T cell
infusion included CD8+ T cells that had undergone
as many as six divisions in vivo. However, pretreatment of B6
recipients with lipo-clodronate inhibited donor C3H.SW
CD8+ T cell proliferation in the spleens, but not
PLN and MLN, increasing the number of splenic
CD8+ T cells limited to a single division by
3.3-fold (Fig. 2, B and C). In contrast, >90%
of donor CD8+ T cells that were recruited into
the liver, in both lipo-clodronate-treated and control B6 recipients,
had undergone at least four divisions (Fig. 2, B and
C). Taken together, these results suggest that DCs and
macrophages in the secondary lymphoid tissues are critical for the
activation and proliferation of donor CD8+ T
cells, whereas resident hepatic APCs are required for the recruitment
of previously activated donor CD8+ T cells into
the liver. Local depletion of host macrophages and DCs reduces CD8 T cell-mediated hepatic inflammation and improves the survival of recipients with acute GVHD
We next asked whether the decreased recruitment of activated donor
CD8+ T cells into the liver is reflected in
clinical improvement of acute GVHD. Histological examination of tissues
from the recipients of C3H.SW
T-BM+CD8+
T cells at 14 days after the transplant demonstrated that the
accumulation of mononuclear cells was significantly decreased in the
portal areas of the livers from lipo-clodronate-treated B6 as compared
with those of the controls (Fig. 3, A and B). Notably, infiltration of
CD8+ T cells into hepatic sinusoids, where
Kupffer cells normally reside, was completely blocked in
lipo-clodronate-treated mice (Fig. 3A). Furthermore, the
number of effector CD8+ T cells producing IFN-
was also decreased significantly in the livers and spleens from
lipo-clodronate-treated B6 recipients (Fig. 3C). In
parallel, there was marked reduction of the serum ALT levels (Fig. 3D) and less weight loss (Fig. 4A) in lipo-clodronate-treated
B6 recipients of C3H.SW T-BM and
CD8+ T cells. Consistent with these observations,
21.1% (n = 19) of lipo-clodronate-treated recipients
of C3H.SW T-BM and CD8+ T
cells died from acute GVHD vs 60.7% (n = 23) of
control B6 recipients (p < 0.01, Fig. 4A).
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, B and
C). These results indicate that the decreased recruitment of
activated donor CD8+ T cell into the liver and
spleen caused by lipo-clodronate treatment significantly inhibits
hepatic GVHD, whereas cutaneous GVHD is not affected. Migration of activated CD8+ T cells into target tissues is regulated by resident host macrophages and DCs
These results suggest that host APCs may play an important role in
the effector phase of acute GVHD, independent of their role in
triggering the activation and proliferation of allogeneic T cells at
the early phase. To test this hypothesis directly, we used adoptive
transfer to distinguish early T cell activation from the subsequent
effector phase of acute GVHD. CFSE-labeled C3H.SW
CD8+ T cells (CD45.2) and
T-BM cells were first transplanted into lethally
irradiated primary B6 recipients (CD45.1) to induce the activation and
proliferation of T cells. Six days later, activated donor
CD8+ T cells were prepared from the spleens, PLN,
MLN, and livers of these primary B6 recipients by depletion of
CD4+, CD11c+,
CD11b+ and CD45.1+ cells.
These enriched, activated donor CD8+ T cells were
transplanted, along with C3H.SW T-BM cells, into
lipo-clodronate-treated or control secondary recipients that had been
lethally irradiated 3 days before the adoptive transfer. The migration
of these in vivo-activated donor CD8+ T cells
into the local tissues of the secondary recipients was examined 20
h following adoptive transfer. As shown in Fig. 5, the number of activated donor
CD8+ T cells in lipo-clodronate-treated secondary
recipients was markedly decreased in the livers and spleens by 88 and
86%, respectively, but relatively increased in the PLN by 55%,
compared with the tissue-specific infiltration in control B6
recipients. Thus, even when appropriately primed by normal APCs,
activated allogeneic CD8+ T cells require
additional, local signals from tissue-resident APCs for their
accumulation in local tissues. In particular, APCs in the liver are
critical for initiating the migration of activated allogeneic
CD8+ T cells into the liver.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that both DCs and macrophages are critical for eliciting T cell-mediated immunity in various animal models (21, 35, 36, 37). Single dose injection of lipo-clodronate via tail vein inhibited the induction of CTL responses against liposome-encapsulated Ags in the spleen (36) and viral Ags in the livers of immune-competent mice (33). In nonobese diabetic mice, depletion of local tissue macrophages by lipo-clodronate resulted in a deficit of T cell activation and a decrease in Th1 immune response, but an increase in Th2 immune response (37). We found that depletion of macrophages and DCs in the spleen by lipo-clodronate treatment significantly inhibited the activation and proliferation of donor CD8+ T cells in the spleen, but not in PLN and MLN. We have previously demonstrated that host DCs are rapidly eliminated from the recipients during the first week following preparative irradiation. Delayed infusion of C3H.SW donor CD8+ T cells into B6 recipients at 5 days following preparative irradiation significantly decreases the proliferation of donor CD8+ T cells in the recipients (7). These observations show that host APCs, particularly DCs, are essential for triggering the proliferation of allogeneic CD8+ T cells. In the present study, depletion of APCs, including DCs and macrophages, from the spleen, but not from the PLN and MLN, of lipo-clodronate-treated mice leads to the abrogation of local presentation of antigenic and costimulatory signals for T cell activation and proliferation. This may account for the inhibited proliferation of CD8+ T cells in the spleen, but not in PLN and MLN, of lipo-clodronate-treated mice. Thus, the decreased recruitment of allogeneic CD8+ T cells into the livers, which we observed in lipo-clodronate-treated mice, could theoretically have been caused by a reduction of T cell activation and proliferation. However, adoptive transfer of fully activated CD8+ T cells into lipo-clodronate-treated secondary recipients still resulted in greatly reduced hepatic infiltration. Therefore, resident tissue APCs have an essential role in the evolution and development of GVHD beyond T cell activation, at the level of T cell recruitment into target tissues.
The molecular mechanisms by which tissue-resident macrophages and DCs
attract and retain allogeneic T cells in GVHD target tissue are not
known. It has been shown that liver allogeneic
CD8+ T cells up-regulate their surface expression
of chemokine receptors CCR1, CCR4, and CXCR3 at day 7 and CCR5 at day
14 after being transplanted into the recipients (15).
Blocking the activity of either macrophage inflammatory protein-1
(MIP1) or its receptor CCR5 by neutralizing Abs can significantly
decrease the trafficking of effector CD8+ T cells
into the liver at day 14 after the transplant (15). A
recent study (17) indicates that activated donor T
cell-derived MIP1 itself may play a critical role in chemoattracting
more effector CD8+ T cells, suggesting a positive
feedback loop leading to accumulation of effector T cells in the liver.
However, allo-BMT can also induce the production of MIP1 in several
other organs, such as the kidneys and pancreas, which are apparently
not the main targets of acute GVHD (1, 2, 15, 17, 34, 38).
Therefore, the data available to date do not identify the primary
initiator(s) of inducing the entry of activated
CD8+ T cells into the liver.
An intriguing aspect of our findings is that activated allogeneic
CD8+ T cells recruited into the recipient liver
had already undergone as least four divisions. This indicates that
allogeneic CD8+ T cells recruited into the
recipient liver had been activated and undergone proliferation,
probably in lymphoid tissues, before their migration into GVHD target
organs. These data imply that the primary role of liver APCs in the
development of GVHD may be as a cellular magnet, attracting activated
allogeneic T cells specific for host tissue Ags. Although there is
still a controversy as to whether hepatic APCs might themselves be
capable of inducing the allo-specific responses (39, 40),
it is clear that activated Kupffer cells can produce high levels of
IL-6, TNF- (41, 42, 43, 44, 45, 46), and chemokines such as MIP1
(15). Thus, hepatic macrophages could direct the
trafficking of activated allogeneic CD8+ T cells
into the liver by the means of the chemokines secreted either by
hepatic macrophages themselves or by other cells in the liver via the
proinflammatory stimulation of macrophage-derived IL-6 and TNF-,
leading to hepatic GVHD. In addition, it is possible that APCs in the
liver might provide a survival signal for these intrahepatic CD8 T
cells to persist in the liver.
Although previous studies have demonstrated that i.v. injection of lipo-clodronate is not toxic to lymphocytes in vivo (23, 25, 36), the total numbers of T, B, and NK cells were indeed reduced in the spleen, but not PLN and MLN, by day 5 following lipo-clodronate treatment alone in our studies, in proportion to the measured overall decline in cellularity. However, no depletion of T, B, or NK cells was observed at 24 or 48 h, in our studies or in published reports (23, 25, 36). Inasmuch as we have shown that activation of donor T cells in the spleen by host APCs is complete by 24 h following allo-BMT (7), this relatively late, but accelerated, decline in host lymphoid cells is unlikely to impact on allo-activation of donor T cells. Moreover, it is well known that lethal irradiation itself can completely eliminate lymphocytes in the secondary lymphoid organs and peripheral tissues, which still support the essential role of APCs in allogeneic T cell activation and effector T cell recruitment. Thus, it is unlikely that the reduced host lymphocytes in vivo are responsible for either the rapid recruitment of allogeneic CD8 T cells into GVHD target organs, such as liver and spleen, or the failure of this process in lipo-clodronate-treated mice.
In this study, lipo-clodronate treatment reduced the recruitment of activated donor CD8+ T cells into the liver, resulting in decreased hepatic inflammation and improved survival. These findings contrast with those of Everse et al. (47), who found that lipo-clodronate treatment did not decrease acute GVHD in mice with allo-BMT. Although the explanation for these disparate results is unclear, certain differences between the studies are noteworthy. We used highly purified naive C3H.SW CD8+ T cells to induce acute GVHD in B6 recipients, whereas Everse et al. (47) used unfractionated donor C3H.SW T cells, containing CD8+ and CD4+ T cells and DCs as well. Most importantly, in Everse’s study (47), most of the recipient mice that were treated with lipo-clodronate died from secondary infection in the peri-BMT period. Thus, effects on organ-specific GVHD may have been masked or overwhelmed by severe infection and inflammation in thesemice. In addition, we have found that treating the mice with lipo-clodronate several days before BMT is essential for blocking hepatic GVHD. In the present study, we did not observe an increasing infection in lipo-clodronate-treated recipients, and we found a clearly beneficial effect of depletion of host tissue-resident macrophages and DCs on local inflammation in the target organs and in improving the severity of acute GVHD.
In conclusion, our data suggest that the resident APCs in the liver and spleen are critical for recruiting activated donor CD8+ T cells into the liver during the effector phase of acute GVHD. Although host DCs are eliminated from the secondary lymphoid organs during the first week following the preparative irradiation (7), most of host tissue macrophages such as Kupffer cells and alveolar macrophages may not be as rapidly replaced by donor bone marrow cells (48). Previous studies by Teshima et al. (8) demonstrated that host APCs that survive irradiation and still reside in target organs or secondary lymphoid tissue are critical to the induction of T cell-mediated GVHD. In this study, our findings further provide a mechanistic explanation for the requirement of host APCs in GVHD target tissues, i.e., such radio-resistant tissue APCs may play an essential role in recruiting activated allogeneic T cells into target tissues, resulting in tissue-specific GVHD. Of note, which APC subsets (e.g., macrophages, DCs) play the dominant role in recruiting/retaining activated CD8+ T cells to GVHD target tissues, and by what molecular mechanisms this recruitment/retention process occurs, is not yet known. Further understanding the molecular basis by which host tissue APCs interact with and focus allogeneic CD8+ T cell activities to mediate acute GVHD in the target tissues may identify promising targets for the development of novel strategies to prevent acute GVHD.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen G. Emerson, Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. E-mail address: emersons{at}mail.med.upenn.edu
3 Abbreviations used in this paper: GVHD, graft-vs-host disease; PLN, peripheral lymph node; MLN, mesenteric lymph node; BMT, bone marrow transplantation; allo-BMT, allogeneic BMT; miHA, minor histocompatibilty Ag; lipo-clodronate, liposomal clodronate; T-BM, T cell-depleted bone marrow; MIP1, macrophage-inflammatory protein-1.
Received for publication July 24, 2002. Accepted for publication October 10, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is critical to the recruitment of CD8+ T cells to the liver, lung, and spleen during graft-versus-host disease. Blood 96:2973. in vitro from lineage phenotypes-negative c-kit+ murine hematopoietic progenitor cells. Blood 90:4842. prevents graft-versus-host disease after allogeneic bone marrow transplantation in mice. J. Immunol. 151:6451.[Abstract]
B activation and cytokine production. J. Leukocyte Biol. 69:622.This article has been cited by other articles:
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R. Zeiser, S. Youssef, J. Baker, N. Kambham, L. Steinman, and R. S. Negrin Preemptive HMG-CoA reductase inhibition provides graft-versus-host disease protection by Th-2 polarization while sparing graft-versus-leukemia activity Blood, December 15, 2007; 110(13): 4588 - 4598. [Abstract] [Full Text] [PDF]
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R. Chakraverty and M. Sykes The role of antigen-presenting cells in triggering graft-versus-host disease and graft-versus-leukemia Blood, July 1, 2007; 110(1): 9 - 17. [Abstract] [Full Text] [PDF]
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J. Gorski, X. Chen, M. Gendelman, M. Yassai, A. Krueger, E. Tivol, B. Logan, R. Komorowski, S. Vodanovic-Jankovic, and W. R. Drobyski Homeostatic expansion and repertoire regeneration of donor T cells during graft versus host disease is constrained by the host environment Blood, June 15, 2007; 109(12): 5502 - 5510. [Abstract] [Full Text] [PDF]
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M. M. Hauri-Hohl, M. P. Keller, J. Gill, K. Hafen, E. Pachlatko, T. Boulay, A. Peter, G. A. Hollander, and W. Krenger Donor T-cell alloreactivity against host thymic epithelium limits T-cell development after bone marrow transplantation Blood, May 1, 2007; 109(9): 4080 - 4088. [Abstract] [Full Text] [PDF]
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 M. Bogunovic, F. Ginhoux, A. Wagers, M. Loubeau, L. M. Isola, L. Lubrano, V. Najfeld, R. G. Phelps, C. Grosskreutz, E. Scigliano, et al. Identification of a radio-resistant and cycling dermal dendritic cell population in mice and men J. Exp. Med., November 27, 2006; 203(12): 2627 - 2638. [Abstract] [Full Text] [PDF]
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N. Durakovic, K. B. Bezak, M. Skarica, V. Radojcic, E. J. Fuchs, G. F. Murphy, and L. Luznik Host-Derived Langerhans Cells Persist after MHC-Matched Allografting Independent of Donor T Cells and Critically Influence the Alloresponses Mediated by Donor Lymphocyte Infusions J. Immunol., October 1, 2006; 177(7): 4414 - 4425. [Abstract] [Full Text] [PDF]
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S. Vodanovic-Jankovic, P. Hari, P. Jacobs, R. Komorowski, and W. R. Drobyski NF-{kappa}B as a target for the prevention of graft-versus-host disease: comparative efficacy of bortezomib and PS-1145 Blood, January 15, 2006; 107(2): 827 - 834. [Abstract] [Full Text] [PDF]
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C. M. Bosio and S. W. Dow Francisella tularensis Induces Aberrant Activation of Pulmonary Dendritic Cells J. Immunol., November 15, 2005; 175(10): 6792 - 6801. [Abstract] [Full Text] [PDF]
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 C. H. Schimmelpfennig, S. Schulz, C. Arber, J. Baker, I. Tarner, J. McBride, C. H. Contag, and R. S. Negrin Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation Am. J. Pathol., November 1, 2005; 167(5): 1321 - 1331. [Abstract] [Full Text] [PDF]
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