REVIEW
Biology of Chronic Graft-versus-Host Disease:
Implications for a Future Therapeutic Approach
Paul J. Martin

Division of Clinical Research, Fred Hutchinson Cancer Research Center,
Department of Medicine, University of Washington, Washington, USA(Received for publication on March 29, 2008)(Accepted for publication on May 15, 2008)

Abstract. Hematopoietic cell transplantation (HCT) is frequently complicated by graft-versus-host disease (GVHD). During the past three decades, experimental studies and clinicalobservations have elucidated the pathophysiology of acute GVHD, but the biology of chronic
GVHD is much less well understood. Recommendations of the NIH Consensus Development
Project on Criteria for Clinical Trials in Chronic GVHD have begun to standardize the diagnosis and clinical assessment of the disease. These criteria have emphasized the importanceof qualitative differences, as opposed to time of onset after HCT, in making the distinction between acute and chronic GVHD. Experimental studies have generated at least four
theories to explain the pathophysiology of chronic GVHD. These theories include 1) thymic
damage and defective negative selection of T cells generated from marrow progenitors after HCT, 2) aberrant production of transforming growth factor-ß, 3) auto-antibody production, and 4) deficiency of T-regulatory cells. Recent studies in humans have corroborated a
possible role for each of these mechanisms in humans. No animal model fully replicates allof the features of chronic GVHD in humans, and it appears likely that multiple biologicalmechanisms account for the diverse features the disease. Chronic GVHD may represent a“syndrome” with diverse causes among individual patients. In the future, it might become
possible to tailor specific therapeutic interventions for patients as individually needed foreach distinct pathophysiologic mechanism involved in development of the disease. (Keio J
Med 57 (4) : 177－183, December 2008)
Key words: graft-versus-host disease, hematopoietic cell transplantation,

Clinical Presentation and
Significance of Chronic GVHD
Chronic GVHD is a pleiomorphic syndrome with onset
generally occurring between 3 and 24 months after allogeneic hematopoietic cell transplantation (Table 1).1 The
highly variable clinical manifestations of chronic GVHD
frequently involve the skin, liver, eyes, mouth, upper respiratory tract, esophagus, and less frequently involve
serosal surfaces, lower gastrointestinal tract, female genitalia, and fascia.2 The biological mechanisms leading tochronic GVHD are not as well understood as those leading to acute GVHD. Although acute GVHD has beenrecognized as a risk factor for chronic GVHD, not allcases of acute GVHD evolve into chronic GVHD, andchronic GVHD can develop in the absence of any priorovert acute GVHD. In the skin, the initial phase ofchronic GVHD is characterized by an intense mononuclear inflammatory infiltrate with destructive changes atthe dermal-epidermal junction, accompanied by irregularacanthosis, hyperkeratosis or atrophy, progressing to dermal fibrosis and sclerosis.3 Other hallmarks include destruction of tubuloalveolar glands and ducts in the skin,salivary and lacrimal glands and respiratory epithelium,and destruction of bile ducts in the liver.

Presented at the 1547th meting of the Keio Medical Society in Tokyo, January 22, 2008.
Corresponding author: Paul J. Martin, MD, Fred Hutchinson Cancer Research Center, D2-100, P.O. Box 19024, Seattle, WA 98109-1024, Email:pmartin@fhcrc.org
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Table 1 Overview of Chronic GVHD
・Pleiomorphic syndrome
・Resembles autoimmune diseases
・Onset at 3-24 months after transplant
・40-60% incidence among 3-month survivors
・Involvement of multiple organs
・Immune dysfunction with risk of infections
・30-50% risk of transplant-related mortality
・Reduced risk of recurrent malignancy

Immune-mediated Mechanisms of Fibrosis
Insight regarding the pathophysiology and immunobiology of chronic GVHD is limited. A wide variety of experimental models have indicated an association between
type-2 polarized immune responses and the developmentof fibrosis,4 and donor type 2 immune responses are required for induction of skin GVHD in mice.5 Complement factor 5 (C5) has been identified as a quantitative
trait that modifies liver fibrosis in mice and humans,6 andC5b-9 complexes are deposited in the skin, liver, lungand kidney in mice with GVHD.7 C3 is deposited at thedermal-epidermal junction in humans with chronic
GVHD,8 but deposition of C5b-9 complexes has notbeen described.
Animal Models of Chronic GVHD
At least four different hypotheses regarding the pathogenesis of chronic GVHD have emerged from studies
with animal models. One hypothesis posits that chronic
GVHD results from thymic damage caused by acute
GVHD, resulting in failure to delete nascent T cells thatrecognize antigens on donor or recipient cells. A second
hypothesis implicates a central role for TGF-β in thepathogenesis of the disease. A third hypothesis implicates
B cells and antibody-mediated mechanisms in certain
manifestations of the disease. Lastly, deficiency in thenumbers or function of T-regulatory cells might contribute to the development of chronic GVHD.
The literature is confusing because the term “chronic
GVHD” has been used to describe a syndrome of antibody-mediated glomerulonephritis that occurs when recipient B cells are activated by donor CD4 cells after
transplantation of spleen cells from certain parental
strains into non-irradiated F1 mice. The resulting nephrotic syndrome is more characteristic of lupus nephritis as opposed to chronic GVHD, although case reports
have occasionally described nephrotic syndrome in patients with chronic GVHD.
Failure of negative selection in the thymus. Cutaneouschanges of acute and chronic GVHD occur in H-2 b
MHC-identical transplants between LP and C57BL/6(B6) mice. Parkman9 showed that clones from B6 recipi-

ents with chronic GVHD were all CD4+ and all showed
IL-2-dependent proliferative responses specific for MHC
class II I-Ab antigens expressed by both the donor andrecipient. The observation that CD4+ clones from recipients with chronic GVHD showed specificity for I-A b
suggested that these cells had emerged from marrow progenitors that escaped negative selection in the thymus,
and the observation that similar clones could be detectedin mice with acute GVHD suggested that the processes
responsible for generating such autoimmune clones begin early after transplantation.
Acute GVHD causes severe histopathological damage
in the thymus, including injury to medullary epithelial
cells, effacement of the corticomedullary junction, disappearance of Hassal’s corpuscles and depletion of
CD4+CD8+ cells.10 In the thymus, developing T cells that
express receptors with high affinity for peptide-MHC
complexes or “self ”-antigens on adjacent cells are deleted. This process of negative selection occurs in the medulla and is mediated by marrow-derived dendritic cells
and thymic medullary epithelial cells. Negative thymic
selection among T cells developing in mice with GVHD
is impaired,11 and the T cells developing in mice with
GVHD are pathogenic. Zhang et al.1 2 showed that dendritic cells were depleted in the thymus of B6 mice withacute GVHD caused by donor C3H.SW CD8 cells. The
resulting thymic damage allowed the development of
CD4 cells that responded vigorously to recipient B6 alloantigens. These CD4 cells caused chronic GVHD when
they were adoptively transferred into irradiated secondary B6 recipients. Administration of keratinocyte growth
factor (KGF) at the time of the transplant enhanced reconstitution of dendritic cells in the thymus, and the CD4cells that emerged from KGF-treated recipients did notcause GVHD in secondary B6 recipients.12
Zhang et al.12 also showed that donor-derived C3H.SW
CD4 cells from B6 recipients with GVHD caused acutehepatic and intestinal GVHD when they were adoptivelytransferred into irradiated secondary C3H.SW recipients.
Further evidence for “autoimmune” recognition among T
cells developing in mice with GVHD was reported byTivol et al.13 in experiments where adoptively transferredspleen cells from B6 → BALB/c chimeras with GVHD
were adoptively transferred into nonirradiated secondary
immunodeficient B6 or BALB/c recipients. Cells fromthe chimeras caused colitis in secondary B6 recipients
but not in BALB/c recipients. In a different approach,several groups have shown that transplantation of MHCclass II-deficient marrow into wild-type recipients of thesame strain causes autoimmune damage in the skin,1 4liver and intestines.1 5 Thymectomy prevented the disease, and adoptive transfer of CD4+ cells caused acute
GVHD in irradiated secondary recipients of the samestrain.
In a variation of the same approach, Sakoda et al.1 6

Keio J Med 2008; 57 (4): 177－183

found that irradiated C3H (H-2k) recipients reconstituted
with T cell-depleted marrow from MHC-class II-deficient B6 (H-2b) donors developed a disease with clinicaland histopathological features characteristic of chronic
GVHD, including epidermal atrophy, follicular dropout,fat loss, dermal fibrosis, bile duct loss, with inflammation, atrophy and fibrosis of acinar tissue in the salivary
glands. Thymectomy prevented the disease, and the chimeric CD4 cells proliferated in response to donor-type
B6 antigen-presenting cells (APCs) but not in responseto recipient-type C3H APCs. Adoptive transfer of chimeric CD4 cells in the presence of B6 APCs caused chronic
GVHD in irradiated secondary C3H recipients. In irradiated secondary B6 recipients, however, the adoptivelytransferred CD4 cells caused acute GVHD.
These results are consistent with those of an earlier
study suggesting that CD4 cells cause acute GVHD
when they recognize antigens expressed by recipient epithelial tissues but cause chronic GVHD when they recognize antigens on marrow-derived cells but not on epithelial tissues.1 7 In these models, the acute or chronic
nature of GVHD appears to be dictated respectively by
the presence or absence of the recognized antigens onepithelial cells of the recipient. Similar experiments havebeen done with B6 donors and MHC-mismatched
BALB/c or MHC-matched BALB.B recipients. Irradiated secondary BALB/c recipients developed acute
GVHD,1 8 while secondary BALB.B recipients developed chronic GVHD1 9 after adoptive transfer of CD4
cells from chimeric donors with GVHD. Hence, an absence of the recognized antigens on recipient epithelial
cells does not entirely explain the development of chronic GVHD.
Further work is needed to define the B6 antigens that
stimulate proliferation of donor CD4 cells in the model
described by Sakoda et al.16 In principle, the CD4 cells
that develop in the chimeras are positively selected bythymic cortical epithelial cells of the C3H recipient andnegatively selected by MHC class I-positive B6 APCs
and by recipient C3H epithelial cells in the thymic medulla. Although the donor-derived CD4 cells that escapeMHC-class II-specific negative selection respond to wild
type B6 APCs in vitro, the B6-derived APCs in the primary recipients do not express MHC-class II molecules
and would not be expected to stimulate donor-derived
CD4 cells in vivo. Evidence from at least one study hassuggested that the CD4 cells escaping negative selectionin the thymus respond to MHC class I antigens.20
Taken together, the experiments with mice demonstratethat acute GVHD impairs negative selection of T cells in
the thymus and that CD4 cells recognizing donor or recipient alloantigens can cause a syndrome with remarkable similarity to chronic GVHD. Further work is needed
to determine whether these observations have relevancefor chronic GVHD in humans. As reported by Tsoi et

179

Table 2 Responses of T Cells from patients*
Response after
stimulation
By donor alone, n (%)
By recipient alone, n (%)
By both, n (%)
Neither, n (%)

Chronic GVHD
Yes (n = 22)
No (n = 12)
1 (5)
0 (0)
8 (36)0 (0)
6 (27)1 (8)
7 (32)0 (0)*adapted from reference 21

al.21 in 1980, donor-derived T cells from patients showed
proliferative responses after stimulation with cells from
HLA-identical sibling recipients in 14 of 22 (64%) caseswith chronic GVHD and in only 1 of 12 cases without
chronic GVHD (Table 2). Responses after stimulation
with recipient cells could not be explained by progeny ofmature T cells in the graft, since T cells from patientswith acute GVHD did not respond after stimulation withrecipient cells. The presence of alloreactive T cells inthese patients suggests an impairment of negative selection by thymic medullary epithelial cells of the recipient.
Responses after stimulation with donor cells were observed 7 of 22 cases (32%) with chronic GVHD and in
only 1 of 12 cases without chronic GVHD. In all but oneof these 8 cases, responses were also observed after
stimulation with recipient cells. These results suggestthat the primary defect associated with chronic GVHD in
humans is an impairment of negative selection mediatedby medullary thymic epithelial cells, with or without
concomitant impairment of negative selection mediatedby donor-derived dendritic cells. It seems unlikely that
this mechanism can account for all cases, however, sincedonor-derived cells from 7 of the 22 patients with chronic GVHD showed no measurable proliferative response
after stimulation with either recipient or donor cells.
Role of TGF-β. A syndrome characteristic of chronic
GVHD develops after transplantation of B10.D2 lymphoid cells into irradiated BALB/c recipients. 2 2 Skinchanges include a mononuclear infiltrate deep in the dermis, loss of dermal fat, increased collagen deposition,and “dropout” of dermal appendages such as hair follicles, but in distinction to findings in acute GVHD, apoptosis of basal epithelial cells at the dermal-epidermal
junction does not occur. Skin changes begin as early as
day 11, and cutaneous fibrosis is apparent as early as day
21. Deposits of IgG, IgA and IgM appear at the dermal
epidermal junction in this model.2 3 Additional featuresof the disease in this model include inflammation and fibrosis in salivary and lacrimal glands, sclerosing cholangitis, progressive renal and gastrointestinal fibrosis, anddevelopment of anti-Scl-70 antibody.24
Naïve donor CD4 cells initiate the disease in this strain
combination,2 5, 2 6 and the dermal infiltrate is comprisedof T cells, monocytes and macrophages.2 7 Antigen pre-

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Table 3 GVHD phenotypes, according to differences in MHC
Donor
B10.D2B10.BR
B10
B10 x B10.D2

Recipient
BALB/c
BALB.K
BALB.B
BALB.B x BALB/c

MHC
H-2d
H-2k
H-2b
H-2b/dTNF-α
Production
Low
High
High
Intermediate

GVHD Phenotype
Skin
Systemic
Chronic
None
Modified* Present
None
Present
Chronic
Present*less severe, especially for hair loss; adapted from reference 32

senting cells of either the donor or recipient are sufficient
to initiate the disease, and costimulation of donor T cells
through CD80 or CD86 on APCs is necessary in order toinduce chronic GVHD.28 In this model, costimulation ofdonor CD4 cells through CD40 on APCs is necessary to
induce intestinal disease but not skin disease. T cells andmacrophages in the skin express TGF-β1 but not TGF-β
2 or TGF-β3 mRNA.29 Microarray analysis also showed
upregulated expression of type 1 (interferon-γ) and type
2 (IL-6, IL-10 and IL-13) cytokines, chemokines, and avariety of growth factors and cell adhesion molecules inrecipients with chronic GVHD as compared to recipients
without chronic GVHD.30 Administration of a neutralizing antibody against TGF-β prevented or reduced virtually all cutaneous manifestations of chronic GVHD, including cellular infiltration, immune cell activation,
thickening and fibrosis.2 7 In contrast, neutralization of
TGF-β by administration of latency-associated peptide
prevented thickening and fibrosis, but did not prevent influx of T cells and monocytes or immune cell activationin the skin.31
Cutaneous manifestations of chronic GVHD develop
after transplantation of B10.D2 spleen cells into MHCmatched (H-2d) BALB/c recipients but not after transplantation of B10 spleen cells into MHC-matched (H-2b)
BALB.B recipients or after transplantation of B10.BR
spleen cells into MHC-matched (H-2k) BALB.K recipients (Table 3).32 In each of these combinations, the background genes of the donor are of B10 origin, while thoseof the recipient are of BALB origin, and the combinations differ from each other only in the MHC. These observations were interpreted as indicating that H-2d MHC
class II molecules (I-Ad or I-Ed) could present minor antigen peptides derived from BALB skin to CD4 cells of
B10 donors, while H-2 b and H-2 k class II molecules
could not. Another explanation for this observation could
be related to a polymorphism in the TNF-α gene, which
is located in the MHC.33 Expression of TNF-α is reducedin H-2d mice as compared to H-2b or H-2k mice. Since
TNF-α is a potent inhibitor of fibrosis induced by TGF-β,
it has been proposed that the fibrosis observed with
B10.D2 donors and BALB/c recipients might be related to
an inability of cutaneous CD4 cells to produce TNF-α.34

Attempts to prevent or treat chronic GVHD through direct or indirect manipulation of TGF-β may encounterunexpected complexity. For example, Asai et al. 3 5
showed that activated donor NK cells could attenuate theseverity of acute GVHD through a TGF-β-dependent
mechanism. TGF-β has a non-redundant, essential role inlimiting T cell and NK cell responses, and TGF-β-deficient mice develop an early onset lethal autoimmune disease.36 The ability of TGF-β neutralization to prevent or
treat chronic GVHD may depend on the context in which
the disease develops. Treatment of B10.D2 donors with
G-CSF exacerbates the severity of skin GVHD in sublethally irradiated BALB/c recipients. Donor T cells are
required for the development of GVHD, but the severityof cutaneous sclerosis is determined by the non-T cell
fraction of grafts from G-CSF-treated donors.3 7 In thismodel, neutralization of TGF-β from day 0 - 42 had noeffect on manifestations of GVHD in the skin, liver or
gastrointestinal tract, but neutralization of TGF-β beginning on day 14 appeared to attenuate progression of thedisease to some extent in the skin and gastrointestinal
tract, but not the liver.3 8 Neutralization of TGF-β after
transplantation exacerbated acute GVHD by interferingwith the regulatory effects of TGF-β on proliferation ofdonor T cells stimulated by recipient alloantigens. Finally, since TGF-β induces Foxp3 expression and T-regulatory function in CD4+CD25- precursors,3 9 TGF-β neutralization could exacerbate GVHD by interfering with
development of T regulatory cells.
The role of TGF-β in human chronic GVHD has notbeen defined. Results of one study showed elevated serum levels of TGF-β in patients with chronic GVHD as
compared to patients without chronic GVHD.4 0 The interpretation of these results is complicated, since assays
were carried out not with plasma, but with serum, whichcontains large amounts of TGF-β released from platelets
during clotting. Gene expression studies have shown that
increased TGF-β signaling in CD4 cells and CD8 cells
was associated with a reduced risk of chronic GVHD in
humans.4 1 These data might appear to conflict with results from experiments with B10.D2 donors and BALB/crecipients, but 4 of the 5 TGF-β pathway genes that were
examined also showed increased expression associatedKeio J Med 2008; 57 (4): 177－183

with a decreased risk of acute GVHD. The association ofincreased TGF-β activity with a reduced risk of chronic
GVHD might result from a decreased risk of acute
GVHD, since acute GVHD is a well-recognized risk factor for chronic GVHD.
Established chronic GVHD induced by B10.D2 T cells
in BALB/c recipients can be reversed by administrationof an agonistic CD137-specific antibody, but administration of this antibody at the time of the transplant exacerbated the severity of acute GVHD.4 2 Administration ofagonistic CD137-specific antibody has been used successfully to treat CD4-mediated autoimmune diseases invariety of experimental models. Clinical trials will beneeded to determine the safety and efficacy of this approach for treatment of chronic GVHD in humans.
Role of B cells. Deposition of antibody at the junction
between the dermis and epidermis has been demonstrated in both murine models23 and in humans8 with chronic
GVHD. Both euthymic and athymic BALB/c recipientsdeveloped high levels of double strand DNA-specific
IgG1 and IgG2a autoantibodies in the serum, cutaneoussclerosis, and glomerulonephritis with proteinuria, beginning within the first 2 weeks after transplantation of
spleen cells from DBA/2 (H-2d) donors.4 3 Induction of
disease required both donor CD4+CD25- T cells and donor B cells. In the absence of donor B cells, donor CD4
cells caused acute GVHD without cutaneous sclerosis or
glomerulonephritis. The relevance of this model for human chronic GVHD could be questioned, since doublestrand DNA-specific autoantibodies, immune complex
glomerulonephritis and proteinuria are characteristic ofsystemic lupus but rarely occur in patients with chronic
GVHD.44
Several lines of evidence suggest that B cells are likely
to have some role in the pathogenesis of chronic GVHD
in humans. First, anecdotal experience and phase 2 studies have shown clinical improvement in some patientswith chronic GVHD after administration of a CD20-specific antibody.4 5 Second, biomarker studies have shown
enhanced CD86 expression after TLR9 stimulation of B
cells from patients with chronic GVHD, as compared tothose from controls. 4 6 Third, patients with chronic
GVHD have high levels of B cell activating factor,
which promotes the survival and differentiation of activated B cells.4 7 Finally, agonistic antibodies against
platelet-derived growth factor receptor (PDGFR) weredetected in serum from each of 22 patients with clinicalextensive chronic GVHD but not in serum from any of
17 patients without chronic GVHD.4 8 These antibodies
induce tyrosine phosphorylation of PDGFR, accumulation of reactive oxygen species, and stimulation of type 1
collagen gene expression by fibroblasts, through a Ras
and ERK1/2-dependent signaling pathway. These results
suggest novel approaches for treatment of chronic
GVHD, since ligand-induced phosphorylation of the181

PDGFR is susceptible to inhibition by certain tyrosinekinase inhibitors, including imatinib.
Role of T regulatory cells. Acute GVHD not only impairs negative selection of T cells in the thymus, as discussed above, but also impairs development of T regulatory cells,18 which might otherwise prevent the development of chronic GVHD. Anderson et al.4 9 showed that
donor or recipient-derived T regulatory cells could prevent GVHD caused by B10.D2 CD4 cells in BALB/c recipients, and Zhang et al.43 similarly showed that donorderived T regulatory cells could prevent GVHD caused
by DBA/2 CD4 cells and B cells in BALB/c recipients.
More impressively, Fujita et al.50 showed that recipientderived regulatory dendritic cells cultured in medium
containing GM-CSF, IL-10, and TGF-β and then matured by exposure to lipopolysaccharide were not only
able to prevent GVHD caused by B10.D2 CD4 cells in
BALB/c recipients but were also able to reverse previously established GVHD.
Conflicting results have been reported with respect tothe role of T-regulatory cells in preventing the development of chronic GVHD in humans. Results of at least
two studies suggested that the presence of increased
numbers of T regulatory cells in the blood was associated with a decreased risk of chronic GVHD,51, 52 but theseresults were not confirmed by two other studies.53, 54 The
most persuasive results emerged from a study by Rieger
et al.,5 5 who showed that colonic mucosa from patientswith acute or chronic GVHD contained very low numbers of T regulatory cells relative to the numbers of CD8
cells, whereas specimens from patients with cytomegalovirus infection or diverticulitis contained much higher
numbers of T regulatory cells relative to the numbers of
CD8 cells.
Future Prospects
The results summarized above raise more questions
than answers and leave the impression that no animalmodel fully replicates all of the features of chronic
GVHD in humans, and more importantly, that no single
biological mechanism is likely to account for the diversefeatures of chronic GVHD in humans. In the future, it
might become possible to tailor treatment for specificabnormalities that contribute to the disease in each individual patient. The disease is clearly initiated by donor T
cells that recognize recipient alloantigens, since the incidence of chronic GVHD can be decreased by exhaustive
depletion of T cells from the graft. Evidence from animalmodels has clearly demonstrated that acute GVHD cancause defects in thymic negative selection, allowing thedevelopment of alloreactive T cells that cause chronic
GVHD. Preliminary results have suggested that administration of KGF could potentially be used to prevent or
treat chronic GVHD that results from this type of mecha-

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Martin PJ: Biology of Chronic Graft-versus-host Diseasenism. T cells and B cells can also contribute to pathogenesis of the disease through mechanisms that do not necessarily depend on prior acute GVHD or thymic dysfunction. It seems likely that binding of agonistic antibodies to cell surface molecules other than PDGFR
might account for certain manifestations of chronic
GVHD. If so, then a range of small molecule inhibitorsof intracellular signal transduction could one day be used
to treat the disease. More importantly, elucidation of themechanisms responsible for generating pathogenic autoantibodies could provide a basis for development ofmethods to prevent chronic GVHD. Finally, development of methods for ex-vivo expansion of T regulatory
cells or for improving the generation, survival and function of these cells in vivo could be used to prevent or reverse chronic GVHD caused by deficits in the numbers
or function of T regulatory cells.
Acknowledgements
This work was supported by grant CA 98906 from the
National Cancer Institute, National Institutes of Health,
U.S. Department of Health and Human Services.
References
1.
2.

3.

4.
5.
6.

7.

8.

Lee SJ, Vogelsang G, Flowers MED: Chronic graft-versus-hostdisease. Biol Blood Marrow Transplant 2003; 9: 215－233
Filipovich AH, Weisdorf D, Pavletic S, Socie G, Wingard JR, Lee
SJ, Martin P, Chien J, Prezepiorka D , Couriel D, Cowen EW,
Dinndorf P, Farrell A, Hartzman R, Henslee-Downey J, Jacobsohn
D, McDonald G, Mittleman B, Rizzo JD, Robinson M, Schubert
M, Schultz K, Shulman H, Turner M, Vogelsang G, Flowers
MED: National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and Staging Working Group report. Biol Blood
Marrow Transplant 2005; 11: 945－956
Shulman HM, Kleiner D, Lee SJ, Morton T, Pavletic SZ, Farmer
E, Moresi JM, Greenson J, Janin A, Martin PJ, McDonald G,
Flowers MED, Turner M, Atkinson J, Lefkowitch J, Washington
MK, Prieto VG, Kim SK, Argenyi Z, Diwan AH, Rashid A, Hiatt
K, Couriel D, Schultz K, Hymes S, Vogelsang GB: Histopathologic diagnosis of chronic graft-versus-host disease: National Institutes of Health consensus development project on criteria forclinical trials in chronic graft-versus-host disease: II. Pathology
Working Group report. Biol Blood Marrow Transplant 2006; 12:
31－47
Wynn TA: Fibrotic disease and the T(H)1/T(H)2 paradigm (Review). Nature Reviews Immunology 2004; 4: 583－594
Nikolic B, Lee S, Bronson RT, Grusby MJ, Sykes M: Th1 andTh2 mediate acute graft-versus-host disease, each with distinct
end-organ targets. J Clin Invest 2000; 105: 1289－1298
Hillebrandt S, Wasmuth HE, Weiskirchen R, Hellerbrand C, Keppeler H, Werth A, Schirin-Sokhan R, Wilkens G, Geier A, Lorenzen J, Kohl J , Gressner AM, Matern S, Lammert F: Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans. Nat Genet 2005; 37: 835－843
Niculescu F, Niculescu T, Nguyen P, Puliaev R, Papadimitriou
JC, Gaspari A, Rus H, Via CS: Both apoptosis and complement
membrane attack complex deposition are major features of murine acute graft-vs.-host disease. Exp Mol Pathol 2005; 79: 136－
145
Tsoi M-S, Storb R, Jones E, Weiden PL, Shulman H, Witherspoon

R, Atkinson K, Thomas ED: Deposition of IgM and complement
at the dermo-epidermal junction in acute and chronic cutaneousgraft-vs-host disease in man. J Immunol 1978; 120: 1485－1492
9. Parkman R: Clonal analysis of murine graft-vs-host disease. I.
Phenotypic and functional analysis of T lymphocyte clones. J Immunol 1986; 136: 3543－3548
10. Ghayur T, Seemayer TA, Xenocostas A, Lapp WS: Complete sequential regeneration of graft-vs.-host-induced severely dysplastic
thymuses. Implications for the pathogenesis of chronic graft-vs.host disease. Am J Pathol 1988; 133: 39－46
11. Morohashi T, Ogasawara K, Kitaichi N, Iwabuchi K, Onoe K:
Abrogation of negative selection by GVHR induced by minor
histocompatibility antigens or H-2D antigen alone. Immunobiology 2000; 202: 268－279
12. Zhang Y, Hexner E, Frank D, Emerson SG: CD4+ T cells generated de novo from donor hemopoietic stem cells mediate the evolution from acute to chronic graft-versus-host disease. J Immunol
2007; 179: 3305－3314
13. Tivol E, Komorowski R, Drobyski WR: Emergent autoimmunity
in graft-versus-host disease. Blood 2005; 105: 4885－4891
14. Marguerat S, MacDonald HR, Kraehenbuhl JP, van Meerwijk JP:
Protection from radiation-induced colitis requires MHC class II
antigen expression by cells of hemopoietic origin. J Immunol
1999; 163: 4033－4040
15. Teshima T, Reddy P, Liu C, Williams D, Cooke KR, Ferrara JL:
Impaired thymic negative selection causes autoimmune graft-versus-host disease. Blood 2003; 102: 429－435
16. Sakoda Y, Hashimoto D, Asakura S, Takeuchi K, Harada M, Tanimoto M, Teshima T: Donor-derived thymic-dependent T cells
cause chronic graft-versus-host disease. Blood 2007; 109: 1756－
1764
17. Jones SC, Murphy GF, Friedman TM, Korngold R: Importanceof minor histocompatibility antigen expression by nonhematopoietic tissues in a CD4+ T cell-mediated graft-versus-host diseasemodel. J Clin Invest 2003; 112: 1880－1886
18. Chen X, Vodanovic-Jankovic S, Johnson B, Keller M , Komorowski R, Drobyski WR: Absence of regulatory T cell control
of TH1 and TH17 cells is responsible for the autoimmune-mediated pathology in chronic graft versus host disease. Blood 2007;
110: 3804－3813
19. Zhang Y, Joe G, Hexner E, Zhu J, Emerson SG: Alloreactive
memory T cells are responsible for the persistence of graft-versushost disease. J Immunol 2005; 174: 3051－3058
20. Trobonjaca Z, Leithauser F, Moller P, Bluethmann H, Koezuka Y,
MacDonald HR, Reimann J: MHC-II-independent CD4+ T cells
induce colitis in immunodeficient RAG-/- hosts. J Immunol 2001;
166: 3804－3812
21. Tsoi M-S, Storb R, Dobbs S, Medill L, Thomas ED: Cell-mediated immunity to non-HLA antigens of the host by donor lymphocytes in patients with chronic graft-vs-host disease. J Immunol
1980; 125: 2258－2262
22. Jaffee BD, Claman HN: Chronic graft-versus-host disease(GVHD) as a model for scleroderma. I. Description of model systems. Cell Immunol 1983; 77: 1－12
23. Claman HN, Jaffee BD, Huff JC, Clark RA: Chronic graft-versus-host disease as a model for scleroderma. II. Mast cell depletion with deposition of immunoglobulins in the skin and fibrosis.
Cell Immunol 1985; 94: 73－84
24. Ruzek MC, Jha S, Ledbetter S, Richards SM, Garman RD: A
modified model of graft-versus-host-induced systemic sclerosis(scleroderma) exhibits all major aspects of the human disease.
Arthritis Rheum 2004; 50: 1319－1331
25. Korngold R, Sprent J: Variable capacity of L3T4 ＋ T cells tocause lethal graft-versus-host disease across minor histocompatibility barriers in mice. J Exp Med 1978; 165: 1552－1564
26. Anderson BE, McNiff J, Yan J, Doyle H, Mamula M, Shlomchik
MJ, Shlomchik WD: Memory CD4+ T cells do not induce graftversus-host disease. J Clin Invest 2003; 112: 101－108

Keio J Med 2008; 57 (4): 177－183
27. McCormick LL, Zhang Y, Tootell E, Gilliam AC: Anti-TGF-beta
treatment prevents skin and lung fibrosis in murine sclerodermatous graft-versus-host disease: a model for human scleroderma. J
Immunol 1999; 163: 5693－5699
28. Anderson BE, McNiff JM, Jain D, Blazar BR, Shlomchik WD,
Shlomchik MJ: Distinct roles for donor- and host-derived antigen-presenting cells and costimulatory molecules in murine
chronic graft-versus-host disease: requirements depend on target
organ. Blood 2005; 105: 2227－2234
29. Zhang Y, McCormick LL, Desai SR, Wu C, Gilliam AC: Murine
sclerodermatous graft-versus-host disease, a model for humanscleroderma: cutaneous cytokines, chemokines, and immune cell
activation. J Immunol 2002; 168: 3088－3098
30. Zhou L, Askew D, Wu C, Gilliam AC: Cutaneous gene expression by DNA microarray in murine sclerodermatous graft-versushost disease, a model for human scleroderma. J Invest Dermatol
2007; 127: 281－292
31. Zhang Y, McCormick LL, Gilliam AC: Latency-associated peptide prevents skin fibrosis in murine sclerodermatous graft-versus-host disease, a model for human scleroderma. J Invest Dermatol 2003; 121: 713－719
32. Kaplan DH, Anderson BE, McNiff JM, Jain D, Shlomchik MJ,
Shlomchik WD: Target antigens determine graft-versus-host disease phenotype. J Immunol 2004; 173: 5467－5475
33. Freund YR, Sgarlato G, Jacob CO, Suzuki Y, Remington JS:
Polymorphisms in the tumor necrosis factor alpha (TNF-alpha)gene correlate with murine resistance to development of toxoplasmic encephalitis and with levels of TNF-alpha mRNA in infected
brain tissue. J Exp Med 1992; 175: 683－688
34. Askew D, Zhou L, Wu C, Chen G, Gilliam AC: Absence of cutaneous TNFalpha-producing CD4+ T cells and TNFalpha may allow for fibrosis rather than epithelial cytotoxicity in murine
sclerodermatous graft-versus-host disease, a model for humanscleroderma. J Invest Dermatol 2007; 127: 1905－1914
35. Asai O, Longo DL, Tian ZG, Hornung RL, Taub DD, Ruscetti
FW, Murphy WJ: Suppression of graft-versus-host disease andamplification of graft-versus-tumor effects by activated naturalkiller cells after allogeneic bone marrow transplantation. J Clin
Invest 1998; 101: 1835－1842
36. Rubtsov YP, Rudensky AY: TGF-beta signaling in control ofT-cell-mediated self-reactivity (Review). Nat Rev Immunol 2007;
7: 443－453
37. MacDonald KP, Rowe V, Filippich C, Johnson D, Morris ES,
Clouston AD, Ferrara JL, Hill GR: Chronic graft-versus-host disease after granulocyte colony-stimulating factor-mobilized allogeneic stem cell transplantation: the role of donor T-cell dose anddifferentiation. Biol Blood Marrow Transplant 2004; 10: 373－
385
38. Banovic T, MacDonald KP, Morris ES, Rowe V, Kuns R, Don A,
Kelly J, Ledbetter S, Clouston AD, Hill GR: TGF-beta in allogeneic stem cell transplantation: friend or foe? Blood 2005; 106:2206－2214
39. Fu S, Zhang N , Yopp AC, Chen D, Mao M, Chen D, Zhang H,
Ding Y, Bromberg JS: TGF-beta induces Foxp3 + T-regulatory
cells from CD4 + CD25 - precursors. Am J Transplant 2004; 4:
1614－1627
40. Liem LM, Fibbe WE, van Houwelingen HC, Goulmy E: Serumtransforming growth factor-beta1 levels in bone marrow transplant recipients correlate with blood cell counts and chronic graftversus-host disease. Transplantation 1999; 67: 59－65
41. Baron C, Somogyi R, Greller LD, Rineau V, Wilkinson P, Cho
CR, Cameron MJ, Kelvin DJ, Chagnon P, Roy DC, Busque L,
Sekaly RP, Perreault C: Prediction of graft-versus-host disease in
humans by donor gene-expression profiling. PLoS Medicine
2007; 4: e23183
42. Kim J, Kim HJ , Park K, Kim J, Choi HJ, Yagita H, Nam SH, Cho
HR, Kwon B: Costimulatory molecule-targeted immunotherapyof cutaneous graft-versus-host disease. Blood 2007; 110: 776－
782
43. Zhang C, Todorov I, Zhang Z, Liu Y, Kandeel F, Forman S,
Strober S, Zeng D: Donor CD4+ T and B cells in transplants induce chronic graft-versus-host disease with autoimmune manifestations. Blood 2006; 107: 2993－3001
44. Rouquette-Gally AM, Boyeldieu D, Prost AC, Gluckman E: Autoimmunity after allogeneic bone marrow transplantation. Transplantation 1988; 46: 238－240
45. Cutler C, Miklos D, Kim HT, Treister N, Woo SB, Bienfang D,
Klickstein LB, Levin J, Miller K, Reynolds C, Macdonell R, Pasek M, Lee SJ, Ho V, Soiffer R, Antin JH, Ritz J, Alyea E: Rituximab for steroid-refractory chronic graft-versus-host disease.
Blood 2006; 108: 756－762
46. She K, Gilman AL, Aslanian S, Shimizu H, Krailo M, Chen Z,
Reid GS, Wall D, Goldman F, Schultz KR: Altered Toll-like receptor 9 responses in circulating B cells at the onset of extensive
chronic graft-versus-host disease. Biol Blood Marrow Transplant
2007; 13: 386－397
47. Sarantopoulos S, Stevenson KE, Kim HT, Bhuiya NS, Cutler CS,
Soiffer RJ, Antin JH, Ritz J: High levels of B-cell activating factor in patients with active chronic graft-versus-host disease. Clin
Cancer Res 2007; 13 : 6107－6114
48. Svegliati S, Olivieri A, Campelli N, Luchetti M, Poloni A, Trappolini S, Moroncini G, Bacigalupo A, Leoni P, Avvedimento EV,
Gabrielli A: Stimulatory autoantibodies to PDGF receptor in patients with extensive chronic graft-versus-host disease. Blood
2007; 110: 237－241
49. Anderson BE, McNiff JM, Matte C, Athanasiadis I, Shlomchik
WD, Shlomchik MJ: Recipient CD4+ T cells that survive irradiation regulate chronic graft-versus-host disease. Blood 2004; 104:
1565－1573
50. Fujita S, Sato Y, Sato K, Eizumi K, Fukaya T, Kubo M, Yamashita N, Sato K: Regulatory dendritic cells protect against cutaneous
chronic graft-versus-host disease mediated throughCD4+CD25+Foxp3+ regulatory T cells. Blood 2007; 110:3793－
3803
51. Miura Y, Thoburn CJ, Bright EC, Phelps ML, Shin T, Matsui EC,
Matsui WH, Arai S, Fuchs EJ, Vogelsang GB, Jones RJ, Hess AD:
Association of Foxp3 regulatory gene expression with graft-versus-host disease. Blood 2004; 104: 2187－2193
52. Zorn E, Kim HT, Lee SJ, Floyd BH, Litsa D, Arumugarajah S,
Bellucci R, Alyea EP, Antin JH, Soiffer RJ, Ritz J: Reduced frequency of FOXP3+ CD4+CD25+ regulatory T cells in patientswith chronic graft-versus-host disease. Blood 2005; 106: 2903－
2911
53. Meignin V, de Latour RP, Zuber J, Regnault A, Mounier N, Lemaitre F, Dastot H, Itzykson R, Devergie A, Cumano A, Gluckman E, Janin A, Bandeira A, Socie G: Numbers of Foxp3-expressing CD4+CD25high T cells do not correlate with the establishment of long-term tolerance after allogeneic stem cell transplantation. Exp Hematol 2005; 33: 894－900
54. Arimoto K, Kadowaki N, Ishikawa T, Ichinohe T, Uchiyama T:
FOXP3 expression in peripheral blood rapidly recovers and lacks
correlation with the occurrence of graft-versus-host disease after
allogeneic stem cell transplantation. Int J Hematol 2007; 85: 154－162
55. Rieger K, Loddenkemper C, Maul J, Fietz T, Wolff D, Terpe H,
Steiner B, Berg E, Miehlke S, Bornhauser M, Schneider T, Zeitz
M, Stein H, Thiel E, Duchmann R, Uharek L: Mucosal FOXP3+
regulatory T cells are numerically deficient in acute and chronic
GvHD. Blood 2006; 107: 1717－1723