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IMMUNOBIOLOGY
From the Department of Microbiology and Immunology,
University of Miami School of Medicine, Miami, FL.
Experimental allogeneic bone marrow transplantation (BMT) models
using cytotoxic single-deficient (perforin/granzyme or Fas ligand [FasL]) and cytotoxic double-deficient (cdd) CD4+
donor T cells have previously demonstrated roles for both effector pathways in graft-versus-host disease (GVHD). In the present study, the
role of CD4-mediated antihost cytotoxicity in a GVH response is further
examined across a complete major histocompatibility complex class I/II
mismatch. As predicted, a double cytotoxic deficiency resulted in a
clear delay in GVH-associated weight loss, clinical changes, and
mortality. Interestingly, analysis of donor T-cell presence in 5.5-Gy
recipients soon after BMT demonstrated that the double cytotoxic
deficiency resulted in a marked decrease in donor CD4 numbers.
Transplantation of singularly perforin- or FasL-deficient donor
CD4+ T cells demonstrated that the absence of FasL was
responsible for the markedly diminished CD4 number in recipient lymph
nodes and spleens soon after BMT. However, increasing recipient
total body irradiation conditioning (11.0 Gy) abrogated the
decrease in FasL-defective B6-cdd and B6-gld CD4 numbers. Thus, the
decrease was not a result of inherent CD4 defects, but was probably
attributable to host resistance. Consistent with these observations,
transplantation into 11.0-Gy recipients resulted in identical GVH
lethality by equal numbers of B6 wild-type, B6-cdd, and B6-gld
CD4+ T-cell inoculum. In total, the findings indicate that
aggressive host conditioning lessens the requirement for donor
CD4+ cytotoxic function in GVH responses soon after BMT.
The present results thus support the notion of a role for cytotoxic
effector function in donor CD4+ T cells prior to
GVH-induced tissue injury.
(Blood. 2001;98:390-397) Donor antihost responses following allogeneic
bone marrow transplantation (BMT) can lead to acute and/or chronic
graft-versus-host disease (GVHD), a complex characterized by multiorgan
involvement that is often life threatening.1,2 The
principal targeted tissues, ie, lymphohematopoietic, liver, skin, and
gastrointestinal tract, exhibit a number of histologically identifiable
alterations, including mononuclear infiltrates, architectural
disruption, and cell death.1 T cells play a central role
in the development of this disorder. It is therefore important to
elucidate the molecular pathways responsible for GVHD-induced injury to
enable attempts to minimize/abrogate the tissue damage in order to
design approaches that selectively favor antitumor activity. A number
of laboratories, including our own, have been investigating the
involvement of donor-mediated cytotoxicity in the GVH
process.3-7 Previous work has shown that the absence of
granule-dependent cytotoxicity impairs the ability of donor T cells to
effect GVHD-induced weight loss, mortality, and histological
damage.3,4,6,7 Notably, the impairment has been manifested
by delayed kinetics of onset in comparison with equal numbers of
cytotoxically normal donor T cells.3,6,7 The use of
cytotoxically deficient T-cell subsets subsequently confirmed the
importance of perforin for optimal GVHD induction.7,8
Additionally, depending on the model system employed for
transplantation, the absence of CD95L-dependent cytotoxicity can
diminish GVH responses in the liver, skin, and
marrow.3,5,9
Several reports have examined the GVH capacity of donor cells
that are simultaneously impaired in their ability to mediate granule-
and CD95L-dependent cytotoxic killing.8,10-12 The results have been conflicting in that both the inability and the ability of
cytotoxically doubly impaired cells to mediate GVHD have been reported.8,10-12 It is clear from such studies that
cytotoxic double-deficient (cdd) T cells can mediate GVHD when
recipients are aggressively prepared with lethal total body irradiation
(TBI) prior to transplantation.11,12 The present
experiments were designed to investigate the importance of cytotoxic
effector function early in the GVH process induced by CD4+
T cells in complete major histocompatibility complex (MHC) class I/II-mismatched recipients. The results show that soon after
transplantation, host conditioning dictates the need for cytotoxic
function by these donor T cells. In summary, the present findings
demonstrate that in addition to participation in the tissue injury
occurring during GVHD, donor CD4 T-cell cytotoxic function plays an
immediate and crucial role after transplantation Mice
Bone marrow transplantation
CD4+ T-cell preparation Highly enriched CD4+ T cells were obtained by positive selection by means of the Miltenyi Macs System (Miltenyi Biotec, Auburn, CA). Donor spleen and lymph node cells were suspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) at 1 × 108/mL and labeled with anti-CD4-conjugated magnetic beads by incubating for 15 minutes at 4°C. The cells were then washed and resuspended in PBS/0.5% BSA and loaded onto a Macs separation column in a magnetic field. The unlabeled cells were removed by 3 washes with PBS/0.5% BSA, and CD4+ cells were then eluted from the column by the use of the same buffer outside the magnetic field. In some experiments, CD4+ T cells were positively enriched with Dynal Beads (Dynal Biotech, Oslo, Norway). Briefly, donor spleen and lymph node cells were incubated with anti-CD4-conjugated Dynal magnetic beads for 20 minutes. The cells binding to Dynal beads were then precipitated on a magnetic rack, and the cells in suspension were discarded. After 3 washes using PBS/0.5% BSA, the cells attached to the beads were freed by means of the releasing reagent. Enriched CD4+ cells were stained with phycoerythrin anti-CD4 (Pharmingen, San Diego, CA) and fluorescein isothiocyanate anti-CD8 (Pharmingen) mAbs and examined for purity by flow cytometric analysis with a FACScan (Becton Dickinson, Mountain View, CA). The percentage CD4+CD8 cells obtained following either
enrichment protocol was always greater than 95%.
Identification and determination of donor CD4+ T-cell numbers after BMT At 5 days following BMT, the recipients were killed and the mesenteric lymph node cells were harvested. The cells were then counted and stained with anti-H-2Kb, anti-Ly5.1, and anti-CD4 mAbs (Pharmingen). Donor CD4+ T-cells were identified as the H-2Kb+Ly5.1CD4+ population by
flow cytometry analysis with FACScan flow cytometer (Becton Dickinson).
The percentage of H-2Kb+Ly5.1CD4+
cells × the total number of nucleated lymph node cells was used to determine the number of donor CD4+ T cells.
GVHD assessment Recipients were monitored for changes in total body weight and overall survival. The clinical signs of GVHD were recorded for individual mice by means of a clinical GVHD scoring system modified from Cooke et al.16 Briefly, recipients were scored on a scale of from 0 to 2 for 5 clinical parameters: weight loss (0, less than 10%; 1, 10% to 25%; 2, greater than 25%); diarrhea (0, not detectable; 1, mild; 2, severe); fur texture (0, unremarkable; 1, slight back ruffling; 2, entire body ruffling); posture (0, normal; 1, hunching noted only at rest; 2, severe hunching and impaired movement); alopecia (0, unremarkable; 1, mild to moderate; 2, severe with obvious areas of denuded skin). The clinical score for a BMT group represents the average of at least 5 mice per group.Statistics Comparison of the survival rates of BMT groups were assessed by means of the Mantel-Cox log-rank test. Groups of transplant recipients were analyzed for weight change, cell numbers, and clinical scores by means of a Student paired t test. A P < .05 was considered significant.
GVH induced weight loss in complete MHC-mismatched recipients with the use of B6 wild-type and B6-cdd T cells To study the ability of T cells lacking functional FasL and perforin to induce GVHD, an MHC class I/II-mismatched acute GVHD model using BALB/c mice as recipients was employed. In this model, BALB/c recipients (H-2d) were conditioned with lethal TBI (8.5 Gy) 1 day before receiving TCD B6 (H-2b) bone marrow cells. BMT of 5 × 106 TCD B6 allogeneic bone marrow cells only resulted in the complete recovery of recipient body weight within the first month after BMT (Figure 1). T cells from B6-cdd or wild-type B6 donors were also infused together with the B6 marrow. As previously observed, unfractionated B6-cdd T cells, as well as B6 wild-type T cells, resulted in GVHD-induced weight loss in the model (Figure 1).12 Although significantly greater numbers of B6-cdd than B6 T cells were required to induce weight loss with comparable kinetics, lower numbers of cdd T cells also induced weight loss albeit with delayed (10 to 12 days) onset.
Comparison of capacity of B6 normal and cytotoxically impaired CD4+ T cells to induce GVHD weight loss and lethality in BALB/c recipients Both CD4+ and CD8+ T-cell subsets have been shown to contribute to the GVHD process in class I/II-mismatched models.17,18 Since CD4+ T cells can secrete a diverse array of cytokines that contribute to GVHD, we examined the impact of a cytotoxic double deficiency on the capacity of CD4+ T cells to induce GVHD. Donor CD4+ T cells were produced by positive selection as described in "Materials and methods." Highly enriched CD4+ T-cell populations were prepared from suspensions of donor spleen and lymph node cells. A representative population from the enrichment procedure that uses the Macs system illustrates the purity of the CD4+ T-cell populations determined by staining with anti-CD4 and anti-CD8 directly conjugated fluorescent mAbs (Figure 2). Routinely, greater than 95% of the selected cells were CD4+ T cells, whereas typically fewer than 1% were contaminating CD8+ cells. Similar results were obtained with populations prepared by means of Dynal beads (data not shown).
To investigate the GVHD-inducing capacities of B6-cdd and wild-type B6 CD4+ T cells, varying doses of enriched CD4+ T cells were transplanted together with TCD B6-Ly5.1 bone marrow cells. Addition of 2.5 × 106 and 1 × 106 B6-cdd CD4+ cells to the marrow inoculum induced significant weight loss in BALB/c BMT recipients (Figure 2B). However, recipients of B6-cdd CD4+ T cells (ie, 2.5 × 106 and 1 × 106) exhibited a clearly delayed and less severe loss of weight compared with recipients of equal numbers of B6 wild-type (B6) CD4+ cells (Figure 2B). B6-cdd CD4+ T cells were also capable of inducing lethal GVHD (Figure 2C). All BALB/c recipients died within 9 weeks following BMT that was done by means of 2.5 × 106 B6-cdd CD4+ T cells (mean survival time [MST] = 41.1). However, the mortality induced by B6-cdd CD4+ T cells was significantly delayed (P = .0002) compared with mice that received equivalent numbers of wild-type B6 CD4+ T cells (MST = 19.6). Lower doses of B6-cdd CD4+ T cells induced much lower mortality. For example, the majority (80%) of recipients survived 80 days following injection of 1 × 106 B6-cdd CD4+ T cells, and all recipients survived transplantations with 0.5 × 106 of these cytotoxically defective T cells. In total, CD4+ T cells unable to mediate perforin- and CD95L-dependent cytotoxicity could induce both GVH-associated weight loss and lethality following BMT across complete MHC class I/II disparities. However, as previously observed with the use of unfractionated B6-cdd spleen cells, considerably greater numbers of cytotoxically impaired than normal CD4+ T cells were required for these effects.12 These findings demonstrate that CD4+ T cells lacking cytotoxic function possess a diminished capacity to mediate a class I/II-disparate GVHD. To more carefully compare the numbers of cytotoxically double-deficient
and normal CD4+ T cells necessary for GVH-induced weight
loss and mortality, 2 T-cell doses were selected: a lethal
(2.5 × 106) and a sublethal (0.25 × 106)
number (Figure 3A). These doses enabled
analysis of a 10-fold difference in cell numbers between
transplantation groups. BALB/c BMT recipients of
2.5 × 106 or 0.25 × 106 B6-cdd T cells
induced significantly delayed (P < .0001) weight loss
compared with the equivalent numbers of B6 CD4+ T cells
(Figure 3A). Notably, 2.5 × 106 B6-cdd CD4+
T cells resulted in weight loss almost indistinguishable in onset and
severity from the weight loss that resulted from 10-fold fewer (0.25 × 106) normal B6 CD4+ T cells.
Similarly, the lethality curves comparing recipients of
2.5 × 106 B6-cdd and 0.25 × 106 B6 donor
inocula were not statistically different (Figure 3B, P = .1369). In total, these findings demonstrated that (1)
B6-cdd CD4+ T cells can induce GVHD weight loss and
lethality and (2) greater numbers (approximately 10-fold) of B6-cdd
versus B6 CD4+ T cells were required to induce comparable
GVH-associated effects.
GVHD-associated clinical changes in BALB/c recipients of B6 and B6-cdd T cells GVHD-associated clinical changes of recipients following BMT by means of highly enriched CD4+ T cells were also examined. Clinical changes associated with experimental GVHD include diarrhea, skin changes, hair loss, and kyphosis.2 In this BMT model, B6-cdd CD4+ T cells were able to induce all of these clinical signs, although with delayed kinetics compared with recipients of cytotoxically normal CD4+ T cells. Both B6-cdd and B6 CD4+ T cells caused changes in the skin of recipients. Skin changes (ruffled fur, tail scaling), haunched posture, and hair loss were observed in both high- and low-dose experimental groups with the use of B6 or B6-cdd T cells. The recipients of high doses of B6 and B6-cdd CD4+ T cells (2.5 × 106) started to develop these changes at approximately the same time (Table 1). However, comparison of recipients of low doses (0.25 × 106) of CD4+ T cells demonstrated a greater delay in the onset of ruffled fur, haunched posture, and hair loss. In contrast, no difference in onset times of diarrhea between recipients of B6 and B6-cdd T cells were detected (Table 1).
We assessed 5 clinical changes to compare BALB/c recipient groups of
different CD4+ T-cell doses (Figure
4). During the first 2 weeks after BMT, the clinical signs of groups that received equivalent doses
(2.5 × 106 or 0.25 × 106) of either B6 or
B6-cdd CD4+ T cells exhibited essentially similar kinetic
onset. However, following this time period, the recipients of both
high-dose (2.5 × 106) and low-dose
(0.25 × 106) B6-cdd groups displayed a slower
development in the pathological process. In the recipients of low, but
not high, T-cell doses, the severity of clinical changes decreased
somewhat during the second week following BMT and then began to
re-emerge during the third week. In total, these findings demonstrated
that B6-cdd CD4+ T cells were able to induce clinical
changes with a pattern similar to that of normal B6 CD4+ T
cells, but with delayed kinetics.
The importance of cytotoxic function by donor CD4+ T cells early after BMT Since 10-fold greater numbers of B6-cdd CD4+ T cells were required to induce GVH effects at kinetics comparable to that of wild-type B6 T cells, the numbers of donor cells present in recipient lymphoid tissues soon after BMT of normal and cytotoxically defective T cells were examined. Spleens and lymph nodes from BALB/c recipients of 1 × 106 highly enriched CD4+ donor T cells were collected, and the mean numbers of nucleated cells per tissue obtained. Aliquots of spleen or lymph node cells from groups receiving cytotoxically impaired or normal donor CD4+ T cells were stained for H-2b and Ly5.2 (donor) and CD4 expression to determine the numbers of these cells (Table 2). The total numbers of B6-cdd CD4+ T cells calculated (percentage of H-2Kb+Ly5.1CD4+ × total
cell number) were at least 3- to 5-fold lower compared with the numbers
of wild-type CD4+ T cells in recipient spleens and the
mesenteric lymph nodes at both time points examined during the first
week after BMT. These findings indicate that as early as 120 hours
following transplantation of equivalent cell numbers, there were
significantly lower numbers of donor B6-cdd CD4+ T cells
present in the spleens and lymph nodes of recipients who subsequently
demonstrate a markedly delayed onset of GVHD.
In order to compare the capacity of B6-cdd to B6 T cells to
proliferate in response to BALB/c alloantigens, a mixed lymphocyte response (MLR) was performed with unfractionated spleen cells or purified (greater than 95%) CD4+ T cells (Figure
5). No differences were detected in the
proliferative responses by cultures containing B6-cdd versus B6
responding cells. Thus, the diminished numbers of donor B6-cdd T cells
detected soon after BMT was not a result of their inherently diminished capacity to expand in response to recipient BALB/c antigen stimulation. Since donor B6-cdd CD4+ T-cell numbers were diminished
compared with B6 CD4+ T cells in 8.5-Gy-TBI BALB/c
recipients (Table 2), groups of BALB/c mice received lower (5.5 Gy) and
higher (11.0 Gy) doses of TBI to investigate the effect of conditioning
on the observed differences (Table 3). At
5 days after BMT of highly purified donor CD4+ T cells
transplanted into 5.5-Gy recipients, the percentage and numbers
(6-fold) of B6-cdd versus B6 CD4+ T cells were again
markedly diminished in the mesenteric lymph nodes (Table 3). In
contrast, the numbers of B6-cdd CD4+ T cells at this time
after BMT were not found to be diminished in 11.0-Gy-conditioned
BALB/c recipients. These findings indicate that under the latter
conditions, B6-cdd CD4+ T cells can efficiently expand in
recipients after BMT.
The lack of FasL function results in diminished CD4+ donor T-cell presence soon after BMT The lack of functional FasL and perforin resulted in lower numbers of donor CD4+ T cells in host lymph node and spleen soon after transplantation. To investigate the importance of each cytotoxic pathway during early donor CD4+ T-cell expansion, 5 × 106 TCD B6-Ly5.1 bone marrow cells were injected together with highly enriched CD4+T cells from B6-cdd, B6-gld, B6-pko, and B6 donors into BALB/c mice conditioned with nonmyeloablative TBI (5.5 Gy) 24 hours before transplantation. The numbers of donor cells in host lymph nodes were assessed 5 days following BMT. Compared with recipients of wild-type B6 CD4+ T cells, donor CD4+ cell numbers in recipients of B6-cdd and B6-gld CD4+ were both significantly reduced. In contrast, the donor CD4+ T-cell numbers in recipients of B6-pko donors were similar to those receiving B6 cells (Figure 6A). However, when a lethal dose of irradiation (11 Gy) was used to condition recipients, donor CD4+ T-cell numbers in each recipient group increased, and the difference in donor CD4+ numbers between each recipient group was essentially eliminated (Figure 6B). To confirm the correlation between early donor T-cell numbers in the host lymph node and the development of GVHD, weight changes and survival of recipient groups were monitored following BMT. High-dose irradiation (Figure 7) effectively resulted in the diminishment of differences in GVHD onset time of weight loss and lethality between recipients of B6 and B6-cdd CD4+ T cells. Additionally, recipients of FasL-defective B6-gld donor cells also exhibited the same GVHD onset. In total, the data indicate the FasL-dependent function is important during the process of early donor CD4+ expansion after transplantation.
Alloreactive donor T cells rapidly expand following recognition of antigen in the host after BMT, a process required for the subsequent development of GVHD. The present studies have examined the involvement of cytotoxic pathways in CD4+ donor T cells following a BMT across a full donor/recipient MHC class I/II disparity. The results demonstrate that CD4+ T cells lacking the ability to effect both perforin/granzyme- and FasL-dependent cytotoxicity are severely impaired in their ability to induce GVHD. The demonstration that increased numbers of cytotoxically impaired B6-cdd compared with wild-type CD4+ T cells are necessary to induce comparable GVHD-associated weight loss and lethality supports previous findings that donor T-cell-mediated cytotoxicity provides an important GVH component after transplantation.3-7 The complex nature of GVHD makes it difficult to define precisely when and how following BMT donor-mediated cytotoxicity is involved. Notably, examination of donor CD4+ T cells soon after transplantation revealed that their inability to express functional FasL resulted in markedly diminished numbers compared with cytotoxically normal CD4+ donor cells. Therefore, the present findings have demonstrated another role for CD4+ donor-mediated cytotoxic function, ie, prior to GVHD-associated tissue pathogenesis. CD4+ T cells possess the capacity to produce a diverse
array of cytokines. Since many of these, including IL-1,
interferon- Interestingly, a prior investigation reported that unfractionated
B6-cdd spleen cells together with normal B6 marrow failed to induce
GVHD lethality in the identical full MHC class I/II-mismatched (B6-cdd To more carefully assess the requirement of functional cytotoxic
effector pathways for successful expansion of donor T cells soon after
transplantation, CD4+ populations with single cytotoxic
deficiencies were examined. The numbers of CD4+ B6-gld, but
not B6-pko, T cells were also diminished following transplantation into
low- (ie, 5.5 Gy), but not high- (11.0 Gy), conditioned BALB/c
recipients. These results support the notion that under conditions when
host resistance is intact, a cytotoxic impairment in CD4+
donor T cells is responsible for their diminished numbers early after
transplantation. Moreover, the diminished numbers were associated with
defective FasL, not perforin, function. A defect in the ability of
B6-cdd or B6-gld cells to expand could also be proposed as contributing
to the diminished numbers of these cells detected. A defect in the
ability of FasL-defective CD4+ donor T cells to expand
after BMT was noted in a P The early elimination/deletion of transplanted donor T cells
would have effects similar to decreasing the size of the donor inoculum
and would therefore be expected to reduce the GVH potential. Strong
radio-resistant host antigraft responses are mediated against donor
cells following BMT across complete MHC class I and II
disparities.25-27 Both natural killer and
CD8+ T cells function as a barrier that inhibits
engraftment by donor progenitor populations contained in the inoculum
and eliminates T cells contained in the transplant.27-30
Martin and colleagues11,31 have convincingly demonstrated
that donor-mediated cytotoxicity is crucial for overcoming allogeneic
host resistance leading to successful engraftment. It was demonstrated
that perforin-deficient CD8+ T cells, including B6-cdd T
cells, were greatly diminished in their capacity to overcome host
resistance.11 Although CD4+ donor cells cannot
directly recognize class II-negative host resistance elements,
FasL-impaired CD4+ T cells may be particularly unable to
defend themselves from HVG effector attack. Thus, under BMT conditions
that permit significant HVG responses, ie, low conditioning (5.5 Gy)
(Braun et al10 and the present study) and/or that involve
the addition of HVG activity (eg, inclusion of syngeneic, ie, recipient
bone marrow) at time of transplantation,8 it may not be
surprising that diminished numbers of B6-cdd and B6-gld, compared with
wild-type T cells, survive the early period after BMT. In contrast,
under conditions of weak/diminished HVG resistance (P
We thank Dr Dingding Xiong for his help with screening of B6-cdd mice; Mrs Emma Weaver for animal care and technical assistance; Monica Jones for overseeing the breeding colonies of the mice used in this study; and the Sylvester Cancer Center for support of the Flow Cytometry Facility for the phenotypic analysis of cell populations used in these experiments.
Submitted July 28, 2000; accepted March 19, 2001.
Supported by National Institutes of Health (NIH) grants 1RO1 RR11576 and 5RO1 HL52461 (R.B.L.) and by NIH grants CA39201, CA57904, and CA80228, US Department of Defense grant DAMD17-98-1-8317 (E.P.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Robert B. Levy, University of Miami School of Medicine, Department of Microbiology and Immunology, PO Box 016960 (R-138), Miami, FL 33101; e-mail: rlevy{at}med.miami.edu.
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3.
Baker MB, Altman NH, Podack ER, et al.
The role of cell-mediated cytotoxicity in acute GVHD after MHC-matched allogeneic bone marrow transplantation in mice.
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1996;183:2645-2656 4. Levy RB, Baker M, Podack ER. Perforin-deficient T cells can induce acute GVHD following transplantation of MHC matched or MHC disparate allogeneic bone marrow. Ann N Y Acad Sci. 1995;770:366-367[CrossRef][Medline] [Order article via Infotrieve].
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Hattori K, Hirano T, Miyajima H, et al.
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Braun YM, Lowin B, French L, et al.
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Martin P, Akatsuka Y, Hahne M, et al.
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Top
Abstract
Introduction
prior to
pathogenesis. We speculate that under nonmyeloablative conditioning,
this cytotoxic function is required by these donor T cells to overcome
host resistance.
, 0.5 × 106 B6
T cells; 
, 5.0 × 106 B6-cdd T cells; 
,
0.5 × 106 B6-cdd T cells.
, 2.5 × 106 B6-cdd;
, 2.5 × 106 B6; 
, 0.25 × 106 B6 donor CD4+ T cells.
) were not significantly different
(P = .1369). 
,
0.25 × 106 B6-cdd; 
, 2.5 × 106 B6; 
) or B6-cdd (
), B6-pko (
, and tumor necrosis factor-
are known to contribute
to the induction and inflammation processes that underlie
GVHD,
BALB/c) model examined in the present studies.