| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115;
Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215;
Department of Pathology, University of Florida College of Medicine, Gainesville, FL 32610; and
§
Department of Pathology, Thomas Jefferson Medical Center, Philadelphia, PA 19107
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B6). We found a significantly
higher morbidity and mortality from GVHD compared with control B6
recipients. In contrast, B6.lpr recipients had very
little hepatic GVHD, although all other specific GVHD target organs
(skin, intestines, and thymus) were more severely affected than in the
control B6 recipients. B6.lpr recipients with GVHD
demonstrated intact donor lymphoid engraftment and an increase in
expansion of donor T cells and monocytes/macrophages compared with
control B6 recipients. Serum levels of IFN-
and TNF-
were higher
in B6.lpr recipients than in control B6 recipients, and
monocytes/macrophages in B6.lpr recipients appeared more
sensitized. B6.lpr recipients had more residual
peritoneal macrophages after BMT, and peritoneal macrophages from
B6.lpr mice could induce a greater proliferative
response from C3H.SW splenocytes. This study demonstrates that the
expression of Fas in the recipient is required for GVHD of the liver,
but shows unexpected consequences when host tissues lack the expression
of Fas for the development of GVHD in other organs and systemic
GVHD. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The pathology of acute GVHD in the recipient is caused by a combination of host tissue damage resulting from the conditioning regimen (radiation or chemotherapy), direct cytotoxicity of host target cells by donor CTL (or NK cells), and indirect cytotoxicity by cytokines (such as TNF) and NO from CTL, NK cells, or monocytes/macrophages. T cell depletion of the donor BM graft remains one of the most effective ways to prevent GVHD, which illustrates the absolute requirement for donor T cells in the initiation of GVHD.
CTL are important effector cells in GVHD and they seem to
exert their cytolytic activity through two lytic pathways: Fas/Fas
ligand (FasL) and perforin/granzyme (2, 3). FasL on the
CTL cell surface can trimerize Fas on the target cell membrane and
initiate the Fas cell death pathway. The release of perforin from
intracellular stores in the CTL results in the formation of pores in
the target cell membrane, which allows the proteolytic activity of
granzymes, which are released with perforin, to induce apoptosis in the
target cell. Finally, activated CTL can express or secrete TNF-,
which could contribute to CTL cytotoxicity (4).
Fas (FasR, Apo-1, CD95) is a 45-kDa type I integral membrane protein that belongs to the TNF-R family (5). Fas is expressed on many tissues, including the characteristic GVHD target organs: skin, liver, intestine, and thymus (6). Moreover, Fas expression can be increased during inflammation due to the exposure to proinflammatory cytokines (7).
FasL (CD95L) is a 36-kDa type II integral membrane protein and belongs to the TNF superfamily. Initially, it was thought that FasL expression was restricted to activated T lymphocytes, macrophages, and neutrophils (8, 9); however, several studies have demonstrated that FasL is also expressed on nonhemopoietic tissues, including lung, intestines, testis, eye, prostate, and uterus, and can be up-regulated during inflammation (5, 7).
lpr (lymphoproliferative response) mice have a spontaneous mutation in the Fas receptor that is associated with defects in peripheral tolerance and activation-induced cell death, lymphoid hyperplasia (adenopathy and splenomegaly), autoantibody production, and the accumulation of CD3+4-8- B220+ T cells (5, 6). Gld (generalized lymphoproliferative disease) mice express a defective Fas ligand and develop a similar phenotype (10). Fas mutations in humans result in a similar lpr-like inherited autoimmune lymphoproliferative syndrome (or Canale-Smith syndrome), which is also associated with lymphadenopathy and autoimmunity (11).
The Fas/FasL system plays an important regulatory role in a variety of processes, including peripheral deletion of autoimmune cells, activation-induced cell death, cytolysis of target cells by cytotoxic T cells, regulation of inflammatory responses, protection of immune-privileged sites (such as the eye, testis, and tumors), and as a costimulatory pathway in the activation of T cells (5, 12, 13).
Previous studies have analyzed the role of the Fas/FasL and
perforin/granzyme pathways in GVHD by using T cells from FasL-defective
gld mice, perforin-deficient (-/-) or granzyme B-deficient
(-/-) mice as donor T cell inoculum (14, 15, 16, 17, 18, 19), or by in
vivo treatment with anti-FasL Abs (19, 20). These
studies demonstrated the importance of these cytolytic pathways for
GVHD, but had different results regarding the role of the Fas/FasL
pathway in survival from GVHD and target organ pathology (14, 15). These discrepancies could be due to differences in donor T
cell populations, mouse strains, BMT models
(PF1 or allogeneic BMT), conditioning regimen
(lethal or sublethal radiation or no conditioning regimen), antigenic
disparity (MHC class I and/or II or mHAg), or method (defective T cell
infusion or in vivo Ab treatment). We therefore designed experiments
with Fas-deficient lpr recipients to further clarify the
role of the Fas/FasL pathway in GVHD.
In this study, we describe a significant increase in GVHD morbidity and
mortality in Fas-deficient B6.lpr recipients after
MHC-matched mHAg-mismatched allogeneic BMT, which is associated with a
significant increase in donor T cell expansion, serum levels of IFN-
and TNF-, and sensitization of monocytes/macrophages, and with a
4-fold higher number of residual host peritoneal macrophages in
B6.lpr recipients. In contrast, we found a remarkable lack
of hepatocyte apoptosis and liver destruction in B6.lpr
recipients, which otherwise displayed more GVHD-associated pathology in
other specific target organs of GVHD (skin, intestines, and thymus)
compared with B6 control recipients.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The EL-4 (H-2b) lymphoma cell line was
kindly provided by Dr. James Ferrara (University of Michigan, Ann
Arbor, MI). Anti-CD16/CD32 Fc block (2.4G2), anti-CD90.2 PE
(anti-Thy-1.2), anti-Ly-9.1 FITC, anti-CD4 FITC and PE
(RM4-5), anti-CD8 FITC and PE (53-6.7), and anti-CD3
FITC
or PE (145-2C11) were all obtained from PharMingen (San Diego, CA).
F4/80-PE was purchased from Caltag Laboratories (San Francisco, CA).
Murine GM-CSF was provided by Dr. Glenn Dranoff (Dana-Farber Cancer
Institute, Boston, MA). Tissue culture medium consisted of RPMI 1640
supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100
µg/ml streptomycin, and 2 mM L-glutamine.
Mice and BMT
Female C57BL/6 (H-2b), lpr/C57BL6 (H-2b), and C3H.SW (H-2b) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and used in BMT experiments between 8 and 10 wk of age. BMT protocols were approved by the Dana-Farber Cancer Institute Animal Care and Use Committee. Bone marrow (BM) cells were removed aseptically from femurs and tibias. Donor BM was T cell depleted twice by repeated incubation with anti-Thy-1.2 for 30 min at 4°C, followed by incubation with low-TOX-M rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) for 1 h at 37°C. Splenic T cells were obtained by purification over a nylon wool column, followed by red cell removal with ammonium chloride red cell lysis buffer (Sigma, St. Louis, MO). Cells (2.5 x 106 BM cells with or without 10 x 106 splenic T cells) were resuspended in Leibovitz’s L-15 medium (Life Technologies, Grand Island, NY) and transplanted by tail vein infusion (0.25 ml total vol) into lethally irradiated recipients on day 0. Recipients received before transplantation on day 0 1100 cGy total body irradiation (137Cs source) as split dose with 3 h between doses (to reduce gastrointestinal toxicity). Mice were housed in sterilized microisolator cages and received normal chow and autoclaved hyperchlorinated drinking water (pH 3) for the first 2 wk post-BMT and filtered water thereafter.
Assessment of GVHD
The severity of GVHD was assessed with a clinical GVHD scoring system, as first described by Cooke et al. (21). Briefly, ear-tagged animals in coded cages were individually scored every week for five clinical parameters on a scale from 0 to 2: weight loss (grade 1, 10–25%; grade 2, >25%), posture (1, kyphosis only at rest; 2, severe kyphosis impairs movement), activity (1, stationary >50% of the time; 2, stationary unless stimulated), fur (1, ventral and/or slight back ruffling; 2, ruffling entire body), and skin (1, erythema or scaling paws/tail/anus, ear shriveling; 2, multiple open lesions). A clinical GVHD index was generated by summation of the five criteria scores (0–10). Survival was monitored daily.
Histopathological analysis
Skin (ear), liver, terminal ileum, and ascending colon were preserved in Formalin (10%) and embedded in paraffin, and tissue sections were stained with hematoxylin and eosin. Slides were coded and examined in a blinded fashion by one individual (J. M. Crawford for liver and intestinal pathology, and G. F. Murphy for cutaneous pathology) using a semiquantitative scoring system (22, 23). Liver, small bowel, and large bowel were scored for 20 parameters associated with GVHD on a scale from 0 to 4 (0, normal; 0.5, focal and rare; 1, focal and mild; 2, diffuse and mild; 3, diffuse and moderate; and 4, diffuse and severe), as previously published (22, 24). Skin was evaluated for number of dyskeratotic cells per linear mm ("dyskeratotic index") as well as for number of positively labeled TUNEL-positive cells/linear mm. Dyskeratotic cells were identified by shrunken hypereosinophilic cytoplasm and pyknotic and fragmented nuclei (23). A minimum of 10 linear mm from multiple tissue levels was evaluated for each experimental animal.
Detection of apoptotic cells using the TUNEL technique
Apoptotic cells in sections of murine ear tissue were detected with the TUNEL technique, as previously described (25), and using an Apoptag peroxidase kit (Intergen, Purchase, NY) with minor modifications of the kit instructions. Briefly, paraffin sections were deparaffinized in three changes of xylene, rehydrated in decreasing concentrations of graded ethanols, and then washed in PBS before treatment with 20 µg/ml proteinase K (Boehringer Mannheim, Indianapolis, IN) at room temperature for 30 min. After proteinase K treatment, the sections were washed in distilled water (4x), treated with 2% hydrogen peroxide (Sigma) for 5 min to quench endogenous peroxidase, and washed in PBS. The sections were then preequilibrated and reacted with either TdT in reaction buffer containing digoxigenin-labeled UTP or buffer alone, followed by peroxidase-labeled anti-digoxigenin Ab (Apoptag kit). Labeled dsDNA breaks were then detected using 3-amino-9-ethylcarbazole (Biogenex, San Ramon, CA), followed by light counterstaining using Gill’s hematoxylin no. 1 (Fisher Scientific, Malvern, PA).
Cells and serum
As described before, splenic T cells were obtained by purification over a nylon wool column, followed by red cell removal with ammonium chloride red cell lysis buffer, which resulted in >85% purity. Peripheral blood was obtained by retroorbital or cardiac puncture of anesthetized animals. PBMC were obtained by layering over Lymphocyte Separation Medium (Organon Teknika, Durham, NC) and centrifugation for 10 min at 1000 x g. Cells at the interface were collected and washed twice with medium.
LPS stimulation assay
Splenocytes were treated with ammonium chloride red cell lysis
buffer to remove red cells and subsequently cultured at 2 x
105 cells/well in flat-bottom 96-well plates
(Falcon, Lincoln Park, NJ) with 1 µg/ml LPS (Escherichia
coli serotype 026:B6; Sigma) at 37°C and 5%
CO2. After 4 h, supernatants were collected
for TNF- ELISA assays.
Immunophenotyping
Splenocytes, thymocytes, and PBMC were washed in FACS buffer (PBS/2% BSA/0.1% azide), and 106 cells/ml were incubated for 30 min at 4°C with CD16/CD32 Fc block. Subsequently, cells were incubated for 30 min at 4°C with primary Ab/Abs (1 µg/ml), washed twice with FACS buffer, and, in some cases, incubated with appropriate secondary Ab for 30 min at 4°C. The stained cells were again washed twice with FACS buffer, resuspended in FACS buffer, and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Cytokine ELISAs
Abs for the IL-1ß, IL-4, IL-12 p40, and IFN- assays were
purchased from PharMingen, and anti-TNF- was obtained from
Genzyme (Cambridge, MA). Assays were performed according to the
manufacturer’s protocol (Genzyme). Briefly, samples were diluted at
1/3 to 1/24 and incubated in wells that had been coated with specific
anti-cytokine Ab. After several washes, wells were incubated with
secondary Ab coupled to HRP (for TNF-) or to biotin (for IL-1ß,
IL-4, IL-12 p40, and IFN-). The biotin-labeled assays were developed
with streptavidin and substrate (Kirkegaard & Perry Laboratories,
Gaithersburg, MD) and read at 450 nm with a microplate reader (Bio-Rad
Labs, Hercules, CA). Recombinant murine TNF- (Genzyme) and IL-1ß,
IL-4, IL-12 p40, and IFN- (PharMingen) were used as standards.
Samples and standards were run in duplicate, and the sensitivity of the
assays was 10–20 pg/ml for IL-1ß, IL-4, IL-12 p40, and TNF-, and
0.063 U/ml for IFN-.
51Cr release assays
EL-4 (H-2b) lymphoma target cells were labeled with 100 µCi of 51Cr at 2 x 106 cells/ml for 2 h at 37°C and 5% CO2. After three washes, labeled targets were plated at 104 cells/well in U-bottom plates (Costar, Cambridge, MA). Splenic T cells (prepared as described above) were added at various E:T ratios in a final volume of 200 µl to quadruplicate wells and incubated for 4 h at 37°C and 5% CO2. Subsequently, 100 µl of supernatant was removed from each well and counted in a gamma counter to determine experimental release (Cobra, Meriden, CT). Spontaneous release was obtained from wells receiving target cells and medium only, and total release was obtained from wells receiving 1% Triton X-100. The spontaneous release was <15% of the total release. Percent cytotoxicity was calculated by the following formula: percent cytotoxicity = 100 x [(experimental release - spontaneous release)/(total release - spontaneous release)].
Proliferation assays
C3H.SW splenic T cells (3 x 106 cells/well; prepared as described above) were initially cocultured with 5 x 106 irradiated (2000 cGy) splenocytes (from B6 or B6.lpr mice) as stimulators in 24-well plates (Costar). After 6 days, 4 x 105 responding cells were harvested and incubated for 3 days with 2 x 105 irradiated (2000 cGy) B6 or B6.lpr peritoneal macrophages in 96-well plates (Nunc, Roskilde, Denmark). Cultures were pulsed during the final 18 h with 1 µCi/well [3H]thymidine, and DNA was harvested on a Harvester 96 (Tomtec, Camden, CT).
Statistics
All values are expressed as mean ± SEM. Statistical analysis of clinical GVHD index scores, cytokine data, histology, and cell numbers was performed with the nonparametric unpaired Mann-Whitney U Test, whereas the Mantel-Cox log rank test was used for survival data. A p value <0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To evaluate the effects of Fas deficiency in recipient animals on
BM engraftment and GVHD, we employed a well-described MHC-matched
(H-2b) mHAg-mismatched murine model for GVHD:
C3H.SWC57BL/6 (26, 27). Recipients of donor BM + T
cells develop late onset acute GVHD (mortality from day 25 to 50), and
CD8+ T cells are required in the donor T cell
inoculum. We used lpr mice on a C57BL/6 (B6) background.
These mice have a milder phenotype than originally described for
lpr mice on a MRL background, which is a mouse strain with a
natural tendency to autoimmune disease (28).
B6.lpr mice do not develop any arthritis or arteritis, do
not develop splenomegaly and lymphadenopathy until 8 mo of age (whereas
MRL.lpr mice develop lymphoid hyperplasia at 4 mo of age),
and have a delayed 50% mortality at 12 mo of age (whereas
MRL.lpr mice exhibit 50% mortality at 5 mo of age and
succumb to glomerulonephritis due to accumulation of autoantibodies)
(28). All of our experiments were completed before the
animals reached 5 mo of age, and we detected no splenomegaly or
lymphadenopathy in any animals.
In previous studies, MRL.lpr mice were found to be more radioresistant than their normal MRL counterparts (29). Therefore, we determined first the dose of radiation that would be lethal without BMT. At 900 cGy (split dose with 3-h interval), only three of five B6.lpr animals and four of five C57BL/6 animals died. However, at 1100 cGy (split dose), all B6.lpr (4/4 animals) and all C57BL/6 (4/4 animals) recipients died within 22 days. We therefore used 1100 cGy (split dose) in all experiments.
We performed all of our experiments with six groups of recipients: 1)
B6 (BM + T cells) B6 (syngeneic BMT), 2) C3H.SW (BM only)
B6 (allogeneic BMT without GVHD), 3) C3H.SW (BM + T cells) B6
(allogeneic BMT with GVHD), 4) B6.lpr (BM + T cells)
B6.lpr (syngeneic BMT), 5) C3H.SW (BM only)
B6.lpr (allogeneic BMT without GVHD), and 6) C3H.SW (BM + T
cells) B6.lpr (allogeneic BMT with GVHD). All BM
cells were T cell depleted, and 2.5 x 106
BM cells/mouse were injected. Splenic T cells (10 x
106 cell/mouse) were added in groups 1, 3, 4,
and 6.
Perkins et al. (29) found that MRL.lpr
recipients of T cell-depleted allogeneic BM exhibit a resistance
against donor lymphoid engraftment (T and B cells). We therefore
evaluated engraftment and chimerism in all recipients at 4 wk post-BMT,
when the first B6.lpr recipients of donor BM + T cells began
to die. We found in all recipients of donor BM + T cells full donor
lymphoid engraftment as measured by expression of Ly-9.1 (not expressed
on host B6 or B6.lpr cells) on thymocytes and PBL-T cells
(Fig. 1
).
|
). However,
at 7 wk post-BMT, B6.lpr recipients of donor BM still had
only 35 ± 6% donor-derived PBL, whereas donor-derived PBL in
regular B6 recipients had increased to 70 ± 1.2%. These results
suggest that either host B6.lpr T cells persist after BMT or
B6.lpr T cells continue to be generated through an
extrathymic pathway. At 7 wk, most B6.lpr recipients in
group 6 had died, but the levels of donor lymphocyte chimerism in the
remaining animals again did not differ significantly from group 3:
91 ± 2% vs 98 ± 2% (p = 0.1).
These results confirm the resistance against lymphoid engraftment in
B6.lpr recipients of donor T cell-depleted BM, as described
previously by Perkins et al. (29). However, the addition
of donor T cells to the donor BM can overcome this resistance and
results in full donor lymphoid chimerism in the B6.lpr
recipient. lpr recipients undergo lethal GVHD after allogeneic BMT
After BMT, we followed all recipients daily for mortality of GVHD
and weekly for weight loss and GVHD morbidity by a clinical GVHD index.
The clinical GVHD index evaluates animals by weight, mobility, fur,
posture, and skin (see Materials and Methods) and has been
shown in some cases to be more sensitive than weight alone in
monitoring nonlethal GVHD (21, 24). B6.lpr
recipients of donor BM + T cells developed progressive GVHD by week 3
post-BMT with a clinical GVHD index score >2 (Fig. 2A) and by week 4 post-BMT
significant weight loss (Fig. 2B). From week 4 post-BMT on,
the clinical GVHD index score remained significantly higher
(p < 0.05) for B6.lpr recipients of
donor BM + T cells compared with any of the other groups. By day 44
post-BMT, 67% (8/12 animals) of B6.lpr recipients of donor
BM + T cells had died (Fig. 2C). In contrast, B6 recipients
of donor BM + T cells developed only mild nonlethal GVHD (Fig. 2, A–C). This result was repeated in four independently
performed experiments.
|
To determine specific GVHD target organ pathology, we performed a histopathological analysis on tissue sections of GVHD target organs (skin, liver, and intestines) at 4 wk post-BMT from animals in all BMT groups. At the time of harvest, only the B6.lpr recipients of donor BM + T cells had significant clinical GVHD, as determined by a clinical GVHD index score of 4.4 ± 1, whereas B6 recipients of donor BM + T cells had a GVHD index of only 0.7 ± 0.4.
Cutaneous GVHD was worse in B6.lpr recipients of donor BM +
T cells with more dermal inflammation (4+ in
B6.lpr recipients vs 1–2+ in B6
recipients) and twice as many dyskeratotic keratinocytes in the
epidermis (1.7–2 cells/mm in B6.lpr recipients vs 0.5–1
cells/mm in B6 recipients) (Fig. 3).
Follicular dyskeratosis, a feature previously shown to be
characteristic of cytotoxic injury in murine GVHD (30),
was also frequently detected. Dyskeratotic cells were confirmed to be
apoptotic by TUNEL staining (Fig. 3), which we have previously shown to
be a reliable indicator of epidermal target cell injury in murine GVHD
(25). By this method, B6.lpr recipients of
donor BM + T cells had 0.63 (0.46–1.12) TUNEL-positive cells/mm, as
compared with 0.15 (0.06–0.32) TUNEL-positive cells/mm in B6
recipients of donor BM + T cells and 0.06 (0–0.12) TUNEL-positive
cells/mm in B6.lpr recipients of donor BM only.
|
A). However, although
B6.lpr recipients of donor BM + T cells had extensive
periportal infiltrates and endothelialitis, which are both
characteristic pathologic findings in hepatic GVHD (22),
no significant hepatocellular destruction was observed and the
parenchymal architecture was intact (Fig. 3F). In contrast,
B6 recipients of donor BM + T cells had moderate periportal infiltrates
and endothelialitis, but extensive apoptosis of hepatocytes and
destruction of the parenchymal architecture with signs of
hepatocellular regeneration (Fig. 3E). This discrepancy
between infiltration/inflammation and parenchymal destruction is
further illustrated by the difference in specific pathology score for
acidophil bodies (Fig. 4B), which reflect apoptosis of
hepatocytes. This score was higher in B6 recipients than in
B6.lpr recipients (1.5 ± 0.6 vs 0.2 ± 0.2,
p = 0.09). Moreover, in four of six B6 recipients,
acidophil bodies could be found in the liver, whereas these could only
be detected in one of six B6.lpr recipients.
|
I and 4C), whereas
B6.lpr recipients developed significant colon GVHD pathology
(score: 11 ± 0.6; B6 vs B6.lpr, p <
0.02) (Fig. 4C) with characteristic submucosal infiltrates,
flattening of the villi, and apoptosis of crypt cells (Fig. 3J). Significant epithelial apoptosis (Fig. 4D) was detected in B6.lpr recipients of donor BM
+ T cells (B6.lpr recipients (1 ± 0.3) vs B6
recipients (0 ± 0), p = 0.02) and, to a lesser
extent, in the other B6.lpr recipient groups. No epithelial
apoptosis was seen in colon sections of B6 recipients. Significant
small intestine GVHD pathology was also only observed in
B6.lpr recipients (Fig. 3, G and H),
which resulted in significantly higher small intestine GVHD pathology
scores (data not shown). B6.lpr recipients of donor BM + T cells develop significant thymic GVHD
The thymus is a target organ of GVHD, and we and others have found
that this results in a severe loss in thymic cellularity that is
primarily due to a loss of
CD4+8+ thymocytes
(16). We assessed thymic GVHD at 4 wk post-BMT by analysis
of thymic cellularity and phenotype. We found a significant loss in
thymic cellularity in B6.lpr recipients of donor BM + T
cells (Fig. 5A), and
phenotypic analysis of thymocytes demonstrated a significant decrease
in the percentage of CD4+8+
thymocytes (Fig. 5B) in the same group, whereas B6
recipients of donor BM + T cells displayed only a slight decrease in
thymic cellularity and the percentage of
CD4+8+ thymocytes.
|
Donor T cell activity is essential in the development of GVHD, and
several studies have indicated that donor T cell expansion during GVHD
occurs in the first weeks after BMT with maximal donor T cell expansion
in the spleen by day 10 to 14 post-BMT (31). We therefore
compared the expansion of donor T cells in B6 or B6.lpr
recipients at 14 days post-BMT by flow-cytometric analysis of
splenocytes with anti-Thy-1.2 (or anti-CD3) and
anti-Ly-9.1. We found a significantly (2-fold) higher number of
donor splenic T cells in B6.lpr recipients receiving donor
BM + T cells than in B6 recipients: 19.6 (±3.2) x
106 vs 9.9 (±0.6) x
106 cells, p < 0.02 (Fig. 6A). However, CTL activity
against host Ags, as determined in a 51Cr release
assay of splenic T cells against EL-4 lymphoma cells (H-2 matched with
B6 recipient), was comparable (when adjusting for comparable T cell
numbers) in B6 and B6.lpr recipients at 14 days post-BMT
(Fig. 6B). To further establish the importance of the
increased donor T cell expansion in vivo, we determined in one
experiment donor T cell expansion at day 14 post-BMT and cytolytic
activity of these cells against host Ags in a
51Cr assay. We then calculated the splenic
LU20 (defined as the number of splenocytes
required to achieve 20% cytolysis of 104 target
cells) and found a 4.3-fold increase in B6.lpr recipients
compared with B6 recipients (79.6 vs 18.6). This clearly demonstrates
the significantly greater anti-host donor T cell activity in
B6.lpr recipients.
|
In addition, we measured the numbers of donor and host monocytes/macrophages in the B6 and B6.lpr recipients by flow-cytometric analysis of splenocytes with F4/80 Ab (which recognizes murine monocytes/macrophages) and anti-Ly-9.1. We found significantly higher numbers of donor splenic monocytes/macrophages in B6.lpr recipients of donor BM + T cells than in B6 recipients: 16.5 ± 1.8 vs 9.8 ± 1 (p = 0.02), whereas host splenic monocytes/macrophages were present in equal numbers in both groups: 5.6 ± 0.8 vs 4.6 ± 0.5 (p = 0.3).
B6.lpr recipients of donor BM + T cells have
increased serum levels of TNF- and IFN-
Cytokines appear to play a significant role in the pathophysiology
of GVHD (32). We therefore analyzed serum levels of
IL-1ß, IL-4, IL-12, IFN-, and TNF- at day 5 to 7 and day 14
post-BMT. We could not detect any serum levels of IL-1ß or IL-4 at
any time after BMT (days 5, 7, or 14 post-BMT) in any of the
recipients. We measured IL-12 p40 (instead of p70), which has been
shown to correlate closely with levels of the biologically active IL-12
p70 during GVHD (32). We found no serum levels of IL-12
p40 at 5 days post-BMT, but did detect at 14 days post-BMT IL-12 p40 in
the serum of B6 and B6.lpr recipients of donor BM + T cells
(Fig. 7A). However, there was
no significant difference in serum IL-12 p40 levels between B6 and
B6.lpr recipients. At day 5 and 14 post-BMT, we detected low
(but significant) levels of IFN- only in B6.lpr
recipients of donor BM + T cells (Fig. 7B): 2.1 ± 0.4
U/ml at day 5 post-BMT and 4.1 ± 0.8 U/ml at day 14 post-BMT (vs
other groups, p = 0.006). We found only low serum
TNF- levels at day 7 post-BMT in B6 and B6.lpr
recipients; however, at day 14 post-BMT, B6.lpr recipients
of donor BM + T cells had 3-fold higher serum TNF- levels (Fig. 7C): 185 ± 29 pg/ml in B6.lpr recipients vs
61 ± 22 pg/ml in B6 recipients, p = 0.01.
Finally, we evaluated whether monocytes and macrophages in recipient
animals have been sensitized to secrete TNF- after in vitro
stimulation with LPS. As shown in Fig. 8,
splenic monocytes/macrophages from B6.lpr recipients of
donor BM + T cells produced more TNF- in a 4-h LPS stimulation assay
than B6 recipients (1443 pg/ml vs 588 pg/ml).
|
|
To explain the remarkable greater expansion of donor T cells in
B6.lpr recipients of donor BM + T cells, we directed our
attention to the role of the Fas pathway in host APCs. Several studies
have demonstrated that the Fas pathway is important for the regulation
of apoptosis of APCs during Ag presentation (33, 34, 35). We
therefore hypothesized that residual Fas-deficient B6.lpr
host macrophages, which are important for the Ag presentation to and
activation of donor T cells, would be resistant to apoptosis during Ag
presentation. To test this hypothesis, we determined the number of
residual host peritoneal macrophages in recipients of allogeneic BM
+/- T cells at 14 days after BMT. We found indeed a statistically
significant 4-fold higher number of
Ly-9.1-/F4/80+ residual
host peritoneal macrophages in B6.lpr recipients of
allogeneic BM + T cells compared with B6 recipients
(p = 0.03) (Fig. 9A). This was not due to a
constitutively higher number of peritoneal macrophages in
B6.lpr mice to begin with, because B6.lpr
recipients of syngeneic BM (data not shown) or allogeneic BM (Fig. 9A) had comparable numbers of residual host peritoneal
macrophages as B6 recipients (p = 0.19).
|
B). This enhanced proliferative response to
B6.lpr stimulator cells was consistently found in four
independently performed experiments. We repeated primary MLRs with C3H.SW splenic T cells as responders and irradiated BM-derived dendritic cells as stimulators. BM-derived dendritic cells from B6 or B6.lpr mice were obtained by 10-day in vitro culture with murine GM-CSF. Subsequently, C3H.SW splenic T cells were incubated for 7 days with varying concentrations of dendritic cells. We found again that B6.lpr stimulators resulted in a significantly greater proliferative response (stimulation index of 21) for C3H.SW effectors with B6.lpr stimulators compared with C3H.SW effectors with B6 stimulators (stimulation index of 4). These results suggest that the greater proliferative response of allogeneic T cells in B6.lpr recipients could be due to a higher number of residual host APCs with greater stimulatory capacity.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and
NO. Previous studies regarding the role of the Fas/FasL pathway in BMT
used either 1) allogeneic FasL-defective gld donor T cells
into normal hosts (14, 15, 16, 17, 18, 19), or 2) syngeneic BM or T
cell-depleted allogeneic BM into B6.lpr recipients
(29, 38), but the effects of the Fas-deficient
lpr host environment on the development of GVHD had never
been determined. Depending on the acute GVHD model used (allogeneic BMT
with lethal irradiation or parent into F1 spleen
cell transfer), Ag disparity (mHAg, MHC class I or II), or intervention
(FasL-defective gld donor T cells or anti-FasL Ab
injections), various roles have been suggested for the Fas/FasL pathway
in GVHD. The interpretation of studies using T cells from gld mice as donor T cells is complex, because a large fraction (which increases with age) of mature T cells from gld mice belongs to a unique subpopulation of CD3+4-8- B220+ cells. In addition, CD4+ T cells in gld mice have been shown to contain an increased percentage of CD44+ memory T cells, suggesting polyclonal activation in vivo. Preactivated memory T cells have been shown to be more susceptible to Fas-dependent apoptosis (39). Indeed, Via et al. (18) found that gld donor T cells at day 2 after parental cell transfer exhibited a higher percentage of memory T cells (Pgp-1 high/lymphocyte endothelial cell adhesion molecule low): 45% vs 25%. These differences in donor T cell population, which go beyond the lack of FasL, could have had impact on the GVHD-effector function of these cells. Only in the studies of Baker et al. (15, 16) were donor T cells depleted of CD3+4-8- B220+ cells before infusion, but this still leaves the increased CD4+ memory cell population intact.
Several studies in spleen cell transfer models (parent into F1 or B6 into BALB/c) using either FasL-defective gld donor T cells or neutralizing anti-FasL Abs have suggested that the Fas/FasL pathway is important for anti-host T cell cytolytic activity (18), host lymphoid depletion and myelosuppression (18, 19), expansion of donor T cells (18), weight loss (20), and mortality (14, 20). These studies were all performed in spleen cell transfer model in which no cytotoxic conditioning regimen and ablation of host hemopoietic system were used before transplant, which makes direct extrapolation to clinical BMT difficult.
Baker et al. (15, 16) used a MHC-matched mHAg-mismatched allogeneic BMT model (B6 into C3H.SW) in which lethally irradiated recipients received donor BM with a lethal dose of donor T cells from either perforin-deficient (B6.perforin 0/0), FasL-defective (B6.gld), or normal (B6) mice, and found that the FasL-defective donor T cells resulted in diminished cutaneous and hepatic GVHD, but had no effect on the development of severe cachexia and mortality. Perforin-deficient donor T cells resulted in delayed onset of GVHD, but had no effect on GVHD target organ pathology and mortality. The same experiment was repeated with a nonlethal dose of FasL-defective gld donor T cells, which resulted in a significant reduction in GVHD-associated lymphoid hypoplasia (measured as numbers of B cells in BM and total number of splenocytes or thymocytes) and intact in vitro B cell function (LPS proliferation assay), whereas transplantation of perforin-deficient or normal B6 T cells was associated with severe lymphoid hypoplasia and reduced splenic B cell proliferative response to LPS. Also, recipients of FasL-defective donor T cells exhibited mixed lymphoid chimerism (as determined by Ly-5.1/Ly-5.2), which suggests that Fas-mediated cytotoxicity is required for clearance of residual hemopoietic host stem cells that persist after lethal radiation.
Graubert et al. (17) studied a modified allogeneic BMT model with lethal irradiation, syngeneic BM grafts, and allogeneic donor T cells with MHC class I or II disparity. With the use of allogeneic donor T cells from FasL-deficient (gld), granzyme B-deficient, perforin-deficient, or FasL/perforin-deficient mice, they found that the Fas system was important for GVHD across a class II barrier, whereas the perforin/granzyme pathway was essential for class I-restricted GVHD. However, the interpretation of this data is (somewhat) confounded by the use of a syngeneic (instead of an allogeneic) BM graft in this GVHD model.
Based upon the previous studies using FasL-defective donor T cells or anti-FasL Ab treatment, we would have expected to see an ameliorating effect of Fas deficiency in the recipient on GVHD. However, we observed increased GVHD morbidity and mortality in B6.lpr recipients of donor BM + T cells. GVHD only occurred in B6.lpr recipients of BM + T cells and not in recipients of syngeneic BM + T cells or donor BM only. This rules out an underlying increased sensitivity of the B6.lpr recipients to the radiation of the conditioning regimen or failure of engraftment. The development of lethal GVHD in the B6.lpr mice is dependent on donor T cells in the inoculum. We therefore hypothesize that the increased expansion of donor T cells, which we observed in the B6.lpr recipient, is essential for the increased GVHD morbidity and mortality.
A number of studies have provided evidence for the important role of
IFN- in the development of acute GVHD (reviewed in Ref.
32). IFN- is thought to be important in the development
of a Th1-type response during acute GVHD and is secreted by donor
CD4+ T cells (32). Also, IFN-
increases mucosal permeability by altering tight junction integrity,
which could allow LPS to enter the bloodstream, and in vivo treatment
with anti-IFN- Abs can prevent intestinal GVHD
(40).
In B6.lpr recipients, we detected only low (but significant)
levels of IFN- at 5 and 14 days post-BMT, which suggests a stronger
activation of donor T cells and initiation of a type I immune response
in B6.lpr recipients compared with control B6 recipients.
These higher serum levels of IFN- may contribute to the increased
GVHD morbidity and mortality in these mice as compared with B6
mice.
IL-12 is secreted by APC and has been shown to induce a Th1/type 1 CD8+CTL (Tc1) response (32). Neutralizing IL-12 during the development of GVHD polarizes the immune response toward a Th2 cytokine profile and reduces GVHD (41). We found comparable serum levels of IL-12 in B6 and B6.lpr recipients of donor BM + T cells, which seems to indicate that polarization toward a type 1 response is unaffected by the lack of Fas expression in host animals.
TNF- has been implicated as an important effector of GVHD, based
upon the following findings (reviewed in Ref. 32): 1)
TNF- can cause cachexia, which is a characteristic feature of GVHD
in murine models; 2) patients with GVHD after allogeneic BMT had
elevated serum TNF- level (42); 3) anti-TNF- Ab
treatment can reduce skin GVHD (43), weight loss, and
improve survival in a murine model for acute GVHD (44); 4)
TNF receptor p55-deficient recipients of allogeneic T cells displayed
reduced mortality, although TNF- release and T cell proliferation
and cytotoxicity were similar to control recipients (45);
and 5) patients with refractory GVHD had some benefit in phase I/II
studies from treatment with anti-TNF- mAbs (46).
TNF- is secreted by non-T and T effector cells during acute GVHD,
and besides cachexia, can also induce target cell destruction,
especially in intestinal epithelium (40). The essential
role of TNF- in intestinal GVHD was also seen in the phase I-II
trial of anti-TNF treatment in patients with GVHD, which
demonstrated most improvement in intestinal GVHD (46).
Finally, in vivo treatment with anti-TNF Abs can prevent intestinal
GVHD in experimental models (44).
We found significantly higher serum levels of TNF- in
B6.lpr recipients at day 14 post-BMT and demonstrated that
monocytes/macrophages in B6.lpr recipients are more
sensitized to produce TNF- upon LPS stimulation. We postulate that
the significantly higher levels of IFN- and TNF- both can induce
damage to the intestinal mucosa (20, 44, 47), which
results in the entry of LPS into the circulation, and the LPS in turn
can stimulate monocytes/macrophages, which have been primed by Th1
cytokines (especially IFN-), to secrete TNF-
(48).
We found no evidence that the conditioning regimen (high dose total
body irradiation) induced the release of proinflammatory cytokines
(IL-1 and TNF-), as described by Hill et al. (24). It
seems therefore that the onset of GVHD in our experiments is primarily
related to the alloantigen response of donor T cells. Our data suggest
that the Fas-deficient lpr environment during the
development of GVHD provides a stronger allostimulation (with more
donor T cell expansion), increased cytokine release, and macrophage
sensitization, resulting in increased GVHD morbidity and mortality.
Evidence has been accumulating that the Fas pathway plays an important
role in the regulation of apoptosis of APC during Ag presentation and
may regulate the duration of T cell expansion, cytokine production, and
the termination of the immune response: 1) Macrophages undergo
activation-induced cell death (49), 2) macrophages express
Fas on their cell surface (9, 33, 50, 51) and can undergo
Fas-mediated apoptosis (33, 50, 51, 52, 53, 54), 3) Fas expression on
macrophages can be up-regulated by IFN- (6, 33, 52) and
TNF- (33), 4) TNF- renders peritoneal macrophages
sensitive to Fas-mediated apoptosis (33), 5)
CD4+ Th1 T cells can kill activated macrophages
from normal but not from Fas-deficient mice (33, 34), 6)
preexposure to IFN- renders Ag-pulsed macrophages sensitive to
Fas-mediated lysis by CD4+ T cells (33, 35), 7) blood-derived or cord blood-derived dendritic cells can
undergo Fas-mediated apoptosis (55, 56), and 8) T cell
activation can trigger apoptosis of dendritic cells
(39).
Fas deficiency in lpr mice results in the persistence and
accumulation of activated macrophages with higher expression of MHC
class II and greater production of inflammatory cytokines. Mice that
are deficient for Fas or FasL are unable to delete macrophages during
Ag presentation, which could explain the increased numbers of
macrophages, enhanced T cell proliferation, increased inflammatory
cytokine production, enhanced inflammation, and increased
susceptibility to certain diseases in lpr mice. Studies with
lpr mice have demonstrated that: 1) lpr mice have
significantly more peritoneal macrophages (57) and
increased serum levels of CSF-1 (58), 2) macrophages from
lpr mice have an enhanced MHC class II expression (36, 37), 3) peritoneal macrophages from lpr mice secrete
constitutively greater levels of IFN-, TNF-, IL-1ß, IL-6, and
IL-10, and produce more TNF upon incubation with SEB (37, 59), 4) Fas-mediated apoptosis is important for the
down-modulation of TNF- in LPS-activated macrophages
(60), 5) lpr mice develop significantly more
autoimmune disease (arthritis, nephritis, autoantibodies), weight loss,
and increased proinflammatory cytokine levels in response to SEB
(37, 61), 6) lpr mice have increased
susceptibility to SEB-induced septic shock, which can be inhibited by
anti-TNF- Abs (62), 7) lpr or
gld mice fail to heal lesions induced by
Leishmania major, which requires the Fas-mediated
lysis of infected APC by CD4+ T cells (34, 54), 8) lpr mice are more susceptible to toxic shock
syndrome toxin-1, which requires T cell proliferation and TNF
production (63), 9) lpr mice have an inability
to control retrovirus-induced murine AIDS (64), and 10)
mice deficient for FasL and perforin die early from severe
pancreatitis, which is accompanied by infiltration of activated
macrophages and can be reversed by in vivo inactivation of macrophages
(65).
We did not find evidence for an increased number of peritoneal macrophages in B6.lpr mice at baseline. This discrepancy between our findings and a previous study (57) is most likely due to differences in the genetic background of the strains tested, age differences, and exposure to lethal irradiation. We studied relatively young (2–3 mo) animals with the lpr mutation on a B6 background. These B6.lpr mice develop their lymphoproliferative phenotype significantly later and survive longer than MRL.lpr mice that were used in the referenced study. Moreover, we analyzed relatively young mice (2–3 mo) that had received lethal irradiation, whereas the previous study describes older mice without radiation treatment.
A recent study by Shlomchik et al. (66) demonstrated that
at 4 mo post-BMT, 3–7% of splenic macrophages and 15–30% of
macrophages in lymph nodes remained of host origin. Our data regarding
the presence of host monocytes/macrophages in the spleen and peritoneal
fluid at 14 days post-BMT are in agreement with this study. Although we
have not specifically addressed this question, we hypothesize therefore
that the increase of activated persisting host monocytes/macrophages in
Fas-deficient lpr mice can contribute to non-T cell-mediated
GVHD activity, such as the elevated serum TNF- levels.
An alternative hypothesis for the increased GVHD in B6.lpr recipients would involve nonspecific Fas-mediated antidonor activity by FasL-expressing residual host cells. Several studies have described a constitutively increased expression of FasL in a specific population of CD3+4-8- B220+ T cells of lpr mice (67, 68). This population of FasL-expressing lpr T cells has been held responsible for the GVHD-like wasting syndrome that occurs after transplantation of MRL.lpr BM cells into lethally irradiated MRL+/+ recipients (38, 69). However, our study provides no convincing evidence to propose a role for residual FasL-expressing T cells of host origin in the development of GVHD.
Several studies have provided evidence for the role of the three effector mechanisms (FasL, perforin/granzyme, and TNF) in specific GVHD target organ damage. These studies demonstrated that the Fas/FasL pathway was associated with cutaneous and hepatic GVHD and lymphoid hypoplasia (15, 20) and TNF with cutaneous and intestinal GVHD and lymphoid hypoplasia (20, 44, 46). The perforin/granzyme pathway could not be related to specific GVHD target organ damage (15).
Our study confirms the essential role of the Fas/FasL pathway in hepatic GVHD in a dramatic way: Fas-deficient B6.lpr recipients that are undergoing lethal acute GVHD do not develop any hepatocyte apoptosis or destruction of the parenchymal architecture, whereas control B6 recipients, which develop only mild nonlethal GVHD, display significant hepatic GVHD with hepatocyte apoptosis and destruction of the parenchymal architecture. Interestingly, B6.lpr recipients did develop characteristic periportal infiltrates, suggesting that the role of the Fas/FasL pathway in liver GVHD is restricted to target cell apoptosis.
Other studies have also demonstrated the importance of the Fas/FasL
pathway for hepatic apoptosis and liver injury. Kondo et al. found that
blocking FasL prevented liver injury in transgenic mice expressing
hepatitis B Ag and in Corynebacterium parvum-primed animals
that were treated with LPS (70). Injection of an
anti-Fas Ab in mice resulted in the development of fulminant lethal
hepatitis within 6 h (71). Moreover,
Fas-/- mice that have no expression of Fas, in contrast
to the leaky mutation of Fas in lpr mice, develop apart from
lymphoproliferation and the autoimmune syndrome also substantial liver
hyperplasia. This suggests that the Fas system might also play a role
in the homeostasis of the normal liver (72). However,
Ksontini et al. (73) found that Con A-induced hepatitis,
which involves T cells and results in the expression of TNF- and
FasL in the livers of Con A-challenged mice, is primarily TNF-
dependent. In our study, liver GVHD was ameliorated by the use of
Fas-deficient recipients in the context of increased serum TNF-
levels, which indicates that in this model system the Fas/FasL pathway
(and not TNF-) is essential for hepatic GVHD.
Finally, we confirmed the resistance to lymphoid engraftment of
lpr mice, as described by Perkins et al. (29),
when T cell-depleted allogeneic donor BM alone was transplanted into
lethally irradiated B6.lpr recipients. However, we found
that in all lethally irradiated B6.lpr recipients of donor
BM (with or without donor T cells), the thymus at 4 wk post-BMT
contained only thymocytes of donor origin (Fig. 1). This suggests that
the resistance to lymphoid engraftment in B6.lpr recipients
involves only mature host peripheral lymphocytes and not host T cell
precursors or thymocytes. This could be due to the persistence of host
T cells that have decreased sensitivity to radiation-induced apoptosis.
Reap et al. (74) have shown a significant difference in
apoptosis of T and B cells after total body irradiation in
B6.lpr mice (25%) compared with B6 control mice (73%).
Moreover, radiation increased Fas expression on B6 splenocytes, and
radiation-induced apoptosis could be inhibited by a Fas-Fc fusion
protein.
Our data indicate that this resistance to lymphoid engraftment in B6.lpr recipients can be completely corrected by the addition of donor T cells to the donor graft. This result could be explained by the persistence of radioresistant host mature T cells in the B6.lpr recipient, which is responsible for the resistance against lymphoid engraftment. Assuming that this is the case, then one can hypothesize that the Fas/FasL pathway is not essential for donor activity against radioresistant lymphocytes in the recipient. This hypothesis is supported by a recent study that demonstrated that resistance to graft rejection is dependent on the presence of an intact perforin system in the donor T cells, but was not affected by the absence of FasL on the donor T cell (75).
In conclusion, we have explored the role of the Fas/FasL pathway in GVHD and its specific target organ pathology. We have observed greater GVHD morbidity and mortality in the B6.lpr recipient, and our data implicate increased donor T cell and monocyte/macrophage expansion and inflammatory cytokines as the cause of this unexpected result.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Marcel R.M. van den Brink, Department of Medicine, Box 111, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail address:
3 Abbreviations used in this paper: GVHD, graft-vs-host disease; BM, bone marrow; BMT, BM transplantation; mHAg, minor histocompatibility Ag; SEB, staphylococcal enterotoxin B; FasL. Fas ligand.
Received for publication June 30, 1999. Accepted for publication October 14, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
antibodies on acute graft-versus-host disease pathologies. Blood 91:4051.. J. Immunol. 157:1758.[Abstract]
modulation of epithelial barrier function: time course, reversibility, and site of cytokine binding. J. Immunol. 150:2356.[Abstract]
precede major complications of bone marrow transplantation. Blood 75:1011. antibody for the treatment of refractory severe acute graft-versus-host disease. Blood 79:3362. prevent immunologically mediated intestinal damage in murine graft-versus-host reaction. Immunology 68:18.[Medline]
during graft-versus-host disease. J. Exp. Med. 175:405.. J. Exp. Med. 181:127. and tumor necrosis factor- by stimulated CD4+ T cells. J. Immunol. 146:4138.[Abstract]
and Fas ligand in concanavalin A-induced hepatitis. J. Immunol. 160:4082.This article has been cited by other articles:
|
|
 |
|
  J. A. Rotolo, B. Stancevic, S. X. Lu, J. Zhang, D. Suh, C. G. King, L. W. Kappel, G. F. Murphy, C. Liu, Z. Fuks, et al. Cytolytic T cells induce ceramide-rich platforms in target cell membranes to initiate graft-versus-host disease Blood, October 22, 2009; 114(17): 3693 - 3706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Carlson, M. L. West, J. M. Coghill, A. Panoskaltsis-Mortari, B. R. Blazar, and J. S. Serody In vitro-differentiated TH17 cells mediate lethal acute graft-versus-host disease with severe cutaneous and pulmonary pathologic manifestations Blood, February 5, 2009; 113(6): 1365 - 1374. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Wysocki, Q. Jiang, A. Panoskaltsis-Mortari, P. A. Taylor, K. P. McKinnon, L. Su, B. R. Blazar, and J. S. Serody Critical role for CCR5 in the function of donor CD4+CD25+ regulatory T cells during acute graft-versus-host disease Blood, November 1, 2005; 106(9): 3300 - 3307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Askenasy, E. S. Yolcu, I. Yaniv, and H. Shirwan Induction of tolerance using Fas ligand: a double-edged immunomodulator Blood, February 15, 2005; 105(4): 1396 - 1404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. E. Anderson, J. M. McNiff, C. Matte, I. Athanasiadis, W. D. Shlomchik, and M. J. Shlomchik Recipient CD4+ T cells that survive irradiation regulate chronic graft-versus-host disease Blood, September 1, 2004; 104(5): 1565 - 1573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Wysocki, S. B. Burkett, A. Panoskaltsis-Mortari, S. L. Kirby, A. D. Luster, K. McKinnon, B. R. Blazar, and J. S. Serody Differential Roles for CCR5 Expression on Donor T Cells during Graft-versus-Host Disease Based on Pretransplant Conditioning J. Immunol., July 15, 2004; 173(2): 845 - 854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. B. Sugerman, S. B. Faber, L. M. Willis, A. Petrovic, G. F. Murphy, J. Pappo, D. Silberstein, and M. R. M. van den Brink Kinetics of Gene Expression in Murine Cutaneous Graft-versus-Host Disease Am. J. Pathol., June 1, 2004; 164(6): 2189 - 2202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, G. Joe, J. Zhu, R. Carroll, B. Levine, E. Hexner, C. June, and S. G. Emerson Dendritic cell-activated CD44hiCD8+ T cells are defective in mediating acute graft-versus-host disease but retain graft-versus-leukemia activity Blood, May 15, 2004; 103(10): 3970 - 3978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Socie, J.-Y. Mary, M. Lemann, M. Daneshpouy, P. Guardiola, V. Meignin, L. Ades, H. Esperou, P. Ribaud, A. Devergie, et al. Prognostic value of apoptotic cells and infiltrating neutrophils in graft-versus-host disease of the gastrointestinal tract in humans: TNF and Fas expression Blood, January 1, 2004; 103(1): 50 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Jung, J. E. Foley, A. A. Erdmann, M. A. Eckhaus, and D. H. Fowler CD3/CD28-costimulated T1 and T2 subsets: differential in vivo allosensitization generates distinct GVT and GVHD effects Blood, November 1, 2003; 102(9): 3439 - 3446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ichiba, T. Teshima, R. Kuick, D. E. Misek, C. Liu, Y. Takada, Y. Maeda, P. Reddy, D. L. Williams, S. M. Hanash, et al. Early changes in gene expression profiles of hepatic GVHD uncovered by oligonucleotide microarrays Blood, July 15, 2003; 102(2): 763 - 771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Schmaltz, O. Alpdogan, S. J. Muriglan, B. J. Kappel, J. A. Rotolo, E. T. Ricchetti, A. S. Greenberg, L. M. Willis, G. F. Murphy, J. M. Crawford, et al. Donor T cell-derived TNF is required for graft-versus-host disease and graft-versus-tumor activity after bone marrow transplantation Blood, March 15, 2003; 101(6): 2440 - 2445. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.B. Sugerman, N.W. Savage, L.J. Walsh, Z.Z. Zhao, X.J. Zhou, A. Khan, G.J. Seymour, and M. Bigby THE PATHOGENESIS OF ORAL LICHEN PLANUS Critical Reviews in Oral Biology & Medicine, July 1, 2002; 13(4): 350 - 365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Alpdogan, C. Schmaltz, S. J. Muriglan, B. J. Kappel, M.-A. Perales, J. A. Rotolo, J. A. Halm, B. E. Rich, and M. R. M. van den Brink Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease Blood, October 1, 2001; 98(7): 2256 - 2265. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
