Prevention of acute graft-versus-host disease by humanizedanti-CD26 monoclonal antibody

Ryo Hatano,1 Kei Ohnuma,1

Junpei Yamamoto,1 Nam H. Dang,2

Taketo Yamada3 and Chikao Morimoto1

1Department of Therapy Development and Inno-

vation for Immune disorders and Cancers, Grad-

uate School of Medicine, Juntendo University,

Tokyo, Japan, 2Division of Hematology/Oncology,

University of Florida, Gainesville, FL, USA, and3Department of Pathology, Keio University

School of Medicine, Tokyo, Japan

Received 22 January 2013; accepted for

publication 3 April 2013

Correspondence: Dr Kei Ohnuma, Department

of Therapy Development and Innovation for

Immune disorders and Cancers, Graduate

School of Medicine, Juntendo University,

2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421,

Japan.

E-mail: kohnuma@juntendo.ac.jp

Summary

CD26 (DPP4) is a T cell costimulatory molecule as well as T cell activation

marker, and CD26+ T cells are accumulated in inflamed tissues, such as

rheumatoid synovitis and autoimmune thyroiditis. In the present study, we

found accumulation of CD26+ T cells in graft-versus-host disease (GVHD)

target organs. To expand our in vitro findings to an in vivo system, we

examined CD26-dependent organ injury in a xenogeneic GVHD (x-GVHD)

murine model. Following intraperitoneal injection of human peripheral

blood mononuclear cells into non-obese diabetic severe combined immu-

nodeficiency/cc�/� mice (hu-PBL-NOG mice), the mice exhibited the onset

of GVHD symptoms associated with the presence of CD26high human lym-

phocytes in the peripheral blood and GVHD target tissues. Administration

of humanized anti-human CD26 monoclonal antibody (mAb) decreased

x-GVHD severity and prolonged survival in hu-PBL-NOG mice without

loss of engraftment of human T cells, while increasing doses of

CTLA4- immunoglobulin fusion protein diminished engraftment of human

lymphocytes. Importantly, anti-CD26 mAb treatment preserved the graft-

versus-leukaemia effects in studies using cotransplantation of P815 murine

leukaemic cells. In addition, CD26+ lymphocytes infiltrated the GVHD

patients’ target tissues. Altogether, our data indicate a role for CD26 in the

regulation of GVHD and point to CD26 as a novel target for therapeutic

intervention in this disease.

Keywords: CD26 (DPP4), graft-versus-host disease, T cell costimulation,

haematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD) is a severe complication

and major cause of morbidity and mortality following alloge-

neic haematopoietic stem cell transplantation (alloHSCT)

(Pasquini et al, 2010; Giralt, 2012). GVHD results from an

immunobiological attack on target recipient organs by donor

allogeneic T cells that are transferred along with the allograft

(Shlomchik, 2007; Blazar et al, 2012). The pivotal role of

donor-derived T cells in acute GVHD (aGVHD) is supported

by the complete abrogation of GVHD following T cell deple-

tion from the graft (Giralt, 2012; Tsirigotis et al, 2012). This

approach remains the most effective in preventing aGVHD.

However, complications such as graft rejection, loss of graft-

versus-leukaemia (GVL) effect or increased opportunistic

infections can occur. Although new approaches are being

developed in the clinical setting for the prevention or

treatment of GVHD (Blazar et al, 2012), in-depth under-

standing of the precise cellular mechanisms of human GVHD

is necessary for more effective and less immunocompromis-

ing strategies to improve the clinical outcome of alloHSCT.

The important role of costimulatory pathways in trans-

plant biology has been established (Briones et al, 2011). The

most extensively studied pathways involve interactions

between CD28 and the B7 molecules CD80 and CD86 (Rudd

et al, 2009). Initial studies focused on the in vivo blockade of

interactions between CD28 or cytotoxic T lymphocyte anti-

gen 4 (CTLA4) and their B7 ligands, CD80 and CD86, using

a CTLA4-immunoglobulin fusion protein (CTLA4-Ig) or

B7-specific antibodies (Lenschow et al, 1992; Saito, 1998).

While CD28 is widely expressed on T cells, the CD28�CD8+

T cell population is a well-recognized subset of na€ıve or

ª 2013 John Wiley & Sons Ltd First published online 21 May 2013British Journal of Haematology, 2013, 162, 263–277 doi:10.1111/bjh.12378

research paper

highly-antigen experienced (late-differentiated) cells (Rudd

et al, 2009; Strioga et al, 2011). Meanwhile, blockade of

CD28 pathways may lead to profound immunosuppression

(Blazar et al, 1997).

CD26 (DPP4) is also associated with T cell signal trans-

duction processes as a costimulatory molecule, as well as

being a marker of T cell activation (Fox et al, 1984; Morim-

oto et al, 1989; Dang et al, 1990; Tanaka et al, 1992; Morim-

oto & Schlossman, 1998; Ohnuma et al, 2008). In fact,

patients with autoimmune diseases, such as multiple sclero-

sis, Grave disease, and rheumatoid arthritis, have been found

to have increased numbers of CD4+CD26+ T cells in

inflamed tissues as well as in their peripheral blood (Hafler

et al, 1985; Eguchi et al, 1989; Gerli et al, 1996; Mizokami

et al, 1996), with enhancement of CD26 expression in these

autoimmune diseases correlating with disease severity

(Eguchi et al, 1989; Muscat et al, 1994; Gerli et al, 1996). In

addition, we previously demonstrated that T cells migrating

through endothelial cell monolayers in vitro express

high levels of CD26 (Masuyama et al, 1992). These findings

imply that CD26+ T cells play an important role in the

inflammation process and subsequent tissue damage in such

diseases, and suggest that CD26+ T cells appear to be effector

T cells. However, little is known about the effectiveness of

CD26-targeting therapy on aGVHD.

In the current study, we utilized non-obese diabetic severe

combined immunodeficiency (NOD/SCID)/cc�/� (NOG)

mice inoculated with human peripheral lymphocytes devel-

oped xenogeneic GVHD (x-GVHD) by human lymphocytes

(Ito et al, 2002). We demonstrated that donor-derived

human lymphocytes expressed high level of human CD26 in

the peripheral blood of hu-PBL-NOG mice developing

x-GVHD, and human CD26+ lymphocyte levels were

enriched in the liver of mice undergoing x-GVHD. We dem-

onstrated that a humanized anti-CD26 monoclonal antibody

(mAb) was effective against x-GVHD-associated pathology

and mortality of hu-PBL-NOG mice. As compared to the

widely used costimulation blocking agent CTLA4-Ig, both

increasing doses of anti-CD26 mAb and CTLA4-Ig clearly

prolonged survival of hu-PBL-NOG mice with less manifes-

tation of x-GVHD physical symptoms. However, increasing

doses of CTLA4-Ig decreased engraftment of human lym-

phocytes and lowered proliferation of human CD4+ T cells

in hu-PBL-NOG mice, while such immune impairment after

inoculation was not observed in hu-PBL-NOG mice treated

with increasing doses of anti-CD26 mAb. More importantly,

anti-CD26 mAb preserved the GVL effect in hu-PBL-NOG

transplanted with tumour cells. Finally, we found that

human CD26+ lymphocytes infiltrated GVHD target organs

obtained from patients with aGVHD. These results provide

proof of principle that human CD26+ T cells are involved in

the pathophysiology of GVHD, including infiltration of

GVHD target organs, and that humanized anti-CD26 mAb

appears to be a promising novel therapy for the clinical

control of GVHD.

Materials and methods

Preparation of human T cells

Human peripheral blood mononuclear cells (PBMCs) were

collected from healthy adult volunteers after the documented

informed consent and institutional review board approval

were obtained. Using the magnetic-activated cell sorting

(MACS) cell separation system (Miltenyi, Bergisch Gladbach,

Germany), CD3+, CD4+ or CD8+ T cells were purified from

PBMC. The purity of CD3+, CD4+ or CD8+ T cells were

� 99%, � 97% or � 95%, respectively, as confirmed by flow

cytometry.

Flow cytometry and antibodies

Human-specific antibodies used for flow cytometry: fluores-

cein isothiocyanate (FITC)-labelled CD8 (clone HIT8a), FITC-

labelled CD14 (clone M5E2), FITC-labelled CD19 (clone

HIB19), phycoerythrin (PE)-labelled CD16 (clone 3G8), PE-

labelled CD56 (clone B159), peridinin chlorophyll- cyanin 5�5(PerCP-Cy5�5)-labelled CD4 (clone SK3), allophycocyanin

(APC)-labelled CD3 (clone UCHT1), APC-labelled CD8

(clone RPA-T8), and APC-labelled CD45 (clone HI30) were

purchased from BD Biosciences (San Jose, CA, USA). PE-

labelled CD26 antibody (clone 5K78), which recognizes

human CD26 even in the presence of humanized anti-CD26

mAb, was purchased from Santa Cruz Biotechnology (Dallas,

TX, USA) (Fig S1). Humanized anti-CD26 mAb was gener-

ously provided by Y’s Therapeutics (Tokyo, Japan). The

humanized anti-CD26 mAb employed in this study was gener-

ated by utilizing the complementarity determining regions of

murine anti-human CD26 mAb, 14D10, which has no crossre-

activity to murine CD26 (Dong et al, 1998). CTLA4-Ig (abata-

cept) and human polyclonal IgG (venilon-I) were purchased

from Alfresa Corporation (Tokyo, Japan). This CTLA4-Ig is

composed of the Fc region of human immunoglobulin IgG1

fused to the extracellular domain of human CTLA4, which also

reacts to murine CD80/86 molecules (Lenschow et al, 1992),

and is able to block interaction between human T cells and

murine antigen presenting cells in mice. For detection of cellu-

lar proliferation in x-GVHD mice, human PBMC (7 9 106/

body), labelled with 4 lmol/l of carboxy fluorescein diacetate

succinimidyl ester (CFSE; Molecular Probes, Carlsbad, CA,

USA), were inoculated peritoneally into NOG mice. Spleen

cells from mice were harvested on day 7, acquisition was per-

formed using FACSCalibur and data were analysed using a

FLOWJO software (TreeStar, Ashland, OR, USA).

Mice and x-GVHD experiments

NOD/SCID/cc�/� NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic mice

(NOG mice) were purchased from The Central Institute

for Experimental Animals (Kawasaki, Japan) and housed in a

specific pathogen-free facility in micro-isolator cages. Mice

R. Hatano et al

264 ª 2013 John Wiley & Sons LtdBritish Journal of Haematology, 2013, 162, 263–277

were used at 8–12 weeks. The animal protocol was approved

by Institutional Animal Care and Use Committee.

In the in vivo x-GVHD model, NOG mice were injected

intraperitoneally with human PBMC (10 9 106). Mice were

assessed for survival daily and weighed thrice weekly. In

GVHD prevention experiments, after 1 day of inoculation

of human cells, mice were injected intraperitoneally with

humanized anti-CD26 mAb, CTLA4-Ig, or control human

IgG at indicated doses in 200 ll of sterile phosphate-

buffered saline (PBS), and then were injected thrice weekly

for a total of 10 doses. To document human lymphocyte-

associated peripheral T cell expansion, mice were bled (10–

40 ll), red blood cells lysed, and PBMC subsets were

enumerated by flow cytometry. In addition, sera were

collected on day 21, serum alanine transaminase (ALT)

activity was assayed using an ALT Activity Kit (BioVision,

Milpitas, CA, USA). In GVL experiments, NOG mice

received H2-Kd MHC background P815 mastocytoma cells

(2 9 104) on the day of transplantation in addition to

human PBMCs. Tumour volumes were measured along

three orthogonal areas (a, b, and c) by digital vernier

calipers (Mitutoyo Corporation, Kanagawa, Japan), and

calculated as tumour volume = abc/2 (mm3). Treatments

with humanized anti-CD26 mAb, CTLA4-Ig, or control

human IgG were conducted as described above. Recipients

were monitored for tumour-related morbidity and mortality,

and death with leukaemia was determined if autopsy

identified hepatosplenomegaly, or if histopathology showed

leukaemic cell infiltration in spleen or liver.

Immunohistochemistry (IHC) staining and pathologydata

CD26-expressing cells were quantified in adult patients after

undergoing alloHSCT for acute myeloid leukaemia (AML)

at the Keio University Hospital. All patients had biopsies

performed as diagnostic procedures for dermal or digestive

GVHD symptoms. Clinical grading of acute GVHD was

scored using the Consensus scoring system (Przepiorka

et al, 1995), and tissue specimens were scored using histo-

pathological grading of skin or gastrointestinal (GI) GVHD

(Sale et al, 1977; Washington & Jagasia, 2009). This study

was approved by the Keio University Hospital ethical

review board and the permission was obtained from

patients. Human tissues were fixed in 10% formalin con-

taining PBS, and embedded in paraffin. Serial 5-lm sec-

tions from each tissue were stained with haematoxylin and

eosin (H&E). IHC staining for CD3, CD4, CD8 or CD26

was performed as described previously (Aoe et al, 2012).

Histopathological findings were evaluated and verified inde-

pendently by two pathologists. Images were captured with

an Olympus digital camera DP21 attached to an Olympus

BX43 microscope using CELLSENS software (Olympus, Tokyo,

Japan). The number of CD26+ MNC was counted and pre-

sented as per square millimetre.

To identify human CD26-expressing cells in x-GVHD

mice, specimens from skin, colon, spleen and liver of NOG

mice transplanted with or without human PBMC were fixed

in 10% formalin containing PBS, and embedded in paraffin.

Serial 5-lm sections from each tissue were stained with

H&E. Images were captured as described above. Slides were

analysed by two independent pathologists who were blinded

to the pathological results when quoting the intensity of the

staining and number of MNCs stained.

Quantitative real-time reverse transcription polymerasechain reaction (qRT-PCR) assay

In experiments assessing expression of mRNA of effector mole-

cules, human CD4+ or CD8+ T cells were purified from human

PBMCs, spleen or liver of hu-PBL-NOG mice, using human

CD4 or CD8 microbeads (Miltenyi). Quantification of mRNA

was performed as described previously (Hatano et al, 2013).

Expression levels of mRNA were calculated on the basis of stan-

dard curves generated for each gene and hypoxanthine phos-

phoribosyltransferase 1 (HPRT1) mRNA was used as an

invariant control. Sequences of primers used in qRT-PCR anal-

ysis are shown in the Table SI.

Statistics

Data were analysed by the analysis of variance (ANOVA) test

for multiple comparison test or Student’s t test for two

group comparison. P values � 0�05 were considered statisti-

cally significant. Survival rates were analysed by log-rank test

using Kaplan–Meier method.

Study approval

Human study protocols were approved by the Ethics Commit-

tees at Juntendo University, at the Institute of Medical Science,

University of Tokyo, and at Keio University. Informed consent

was obtained from all patients and healthy volunteers. All

studies on human subjects were carried out according to the

principles set out in the Declaration of Helsinki. Animal experi-

ments were conducted following protocols approved by the

Animal Care and Use Committees at Juntendo University and

at the Institute of Medical Science, University of Tokyo.

Results

Infiltration of human CD26+ T cells in the liver GI tractand skin of murine recipients with x-GVHD

To determine whether human CD26+ T cells play a role in the

animal model of inflammatory diseases by human effector

lymphocytes, we employed a xenogeneic model of GVHD

resulting from the adoptive transfer of human PBMCs into

NOG mice (hu-PBL-NOG mice) (Ito et al, 2002). We first

conducted a pathological analysis of x-GVHD target organs,

Humanized Anti-CD26 mAb Prevents Acute GVHD

ª 2013 John Wiley & Sons Ltd 265British Journal of Haematology, 2013, 162, 263–277

such as the skin, colon or liver, in hu-PBL-NOG mice.

Control mice without x-GVHD (treated with PBS alone)

showed no GVHD pathology in the liver, GI tract or skin

(Fig 1A), while the liver, colon and skin of hu-PBL-NOG

mice were infiltrated with human CD3+ mononuclear cells

(MNCs), with associated organ destruction (Fig 1B, panels a,

b, d, e, g, h). Moreover, human CD3+ MNCs reactive to anti-

human CD26 Ab were readily visible in all evaluated samples

in the liver, colon or skin of x-GVHD mice (Fig 1B, panels c,

f, i). The infiltrated CD26+ cells were confirmed to be human

CD3+ T cells by co-staining analysis with flow cytometry (Fig.

S2). In contrast to the hu-PBL-NOG mice, human CD3+ or

CD26+ MNCs were not detected in all evaluated samples of

the liver, colon, or skin of NOG mice without x-GVHD

(Fig 1A, panels b, c, e, f, h, j). These results suggest that

donor-derived human CD26+ cells play a role in the patho-

genesis of x-GVHD in our hu-PBL-NOG murine model.

Humanized anti-CD26 mAb treatment significantlyreduces x-GVHD-associated lethality and weight loss inmurine model

As shown above, CD26+ lymphocytes might function as effec-

tor cells and be an appropriate target of x-GVHD treatment.

(A)

(B)

Non-x-GVHD (PBS alone)

x-GVHD (human PBMC inoculation)

H&E anti-human CD3Staining :

H&E anti-human CD3

Liver

Colon

Skin

anti-human CD26

anti-human CD26Staining :

Liver

Colon

Skin

(c)(b)(a)

(f)(e)(d)

(i)(h)(g)

(c)(b)(a)

(f)(e)(d)

(i)(h)(g)

Fig 1. NOD/SCID/cc�/� mice receiving human

peripheral blood mononuclear cells (hu-PBL-

NOG mice) develop manifestation of x-GVHD

in target organs. (A) Specimens were obtained

from control NOG mice [non-x-GVHD, phos-

phate-buffered saline (PBS) alone]. Representa-

tive serial sections of liver (panels a–c), colon(panels d–f) or skin (panels g–i) were stainedwith haematoxylin and eosin (H&E), with

anti-human CD3 polyclonal antibody (pAb),

or with anti-human CD26 pAb, counterstained

with haematoxylin (original magnification,

2009). Non-specific staining with anti-CD3

pAb was observed in a part of colonic mucosal

epithelia (panel e) and epidermal basal area

(panel h). Staining with anti-CD26 pAb was

observed in bile canaliculi of the liver (panel c),

colonic mucosal epithelia (panel f) and

epidermal basal layer (panel i), due to the cross-

reactivity of goat anti-human CD26 pAb against

to murine CD26 antigen. Similar results were

observed in independent experiments using

mice receiving PBS alone (n = 13). (B) The

specimens were obtained from hu-PBL-NOG

mice (x-GVHD, 4 weeks after inoculation of

human PBMCs). Representative serial sections

were stained by the same methods as in (A)

(original magnification, 2009). Human

CD3+CD26+ lymphocytes infiltrated the peri-

portal area of the liver (panels a–c), the crypticabscesses of the colon (panels d–f), and the

upper dermis with subepidermal clefting (panels

g–i). Similar results were observed in indepen-

dent experiments using mice receiving human

PBMCs (n = 20).

R. Hatano et al

266 ª 2013 John Wiley & Sons LtdBritish Journal of Haematology, 2013, 162, 263–277

Therefore, we attempted to determine whether treatment with

humanized anti-CD26 mAb could ameliorate disease progres-

sion and severity in our x-GVHD murine model. As compari-

son, the clinically available T cell costimulation blocking

agent CTLA4-Ig (abatacept) was utilized (Gribben et al, 1996;

Genovese et al, 2005). As shown in Fig 2A, anti-CD26 mAb

(orange or red lines) or CTLA4-Ig (green or dark green lines)

treatment of hu-PBL-NOG mice significantly increased over-

all survival, as compared with control IgG treatment (blue

line). This life-prolonging effect of anti-CD26 mAb was

observed at the same low dose as CTLA4-Ig (orange or green

lines in Fig 2A, respectively). Moreover, GVHD-associated

weight loss for up to 4 weeks post-cell inoculation was not

significantly different between anti-CD26 mAb and CTLA4-Ig

treatment groups (orange or green lines in Fig 2B). These

data strongly suggest that anti-CD26 mAb treatment is a

promising novel therapeutic agent for x-GVHD, with efficacy

comparable to CTLA4-Ig.

It should be noted that graft rejection results in the

absence of x-GVHD, with mice exhibiting normal appear-

ance and behaviour as they mature. We therefore analysed

circulating human lymphocytes in the peripheral blood of

hu-PBL-NOG mice receiving anti-CD26 mAb or CTLA4-Ig.

As shown in Fig 2C, starting at 2 weeks after inoculation,

human lymphocytes were detected in the peripheral blood

of mice receiving control IgG (blue line). On the other

hand, at 3 weeks after inoculation, the human lymphocyte

level was increased in peripheral blood of mice receiving

anti-CD26 mAb at both low dose and high dose (orange

and red lines of Fig 2C). In addition, a comparable number

of human lymphocytes was observed in x-GVHD mice trea-

ted with low dose CTLA4-Ig (Fig 2C, green line). On the

other hand, even at 6 weeks after inoculation, very few

human lymphocytes were detected in the peripheral blood

of mice receiving high dose CTLA4-Ig (Fig 2C, dark green

line), with all the mice in the group surviving (Fig 2A, dark

Per

cent

sur

viva

l

(C)

%H

uman

CD

45+

cells

%In

itial

wei

ght

(B)

Time after inoculation (weeks)

Drug injection

Contl. IgG (200µg/dose)

CTLA4-Ig (200µg/dose)CTLA4-Ig (2µg/dose)Anti-CD26 (200µg/dose)Anti-CD26 (2µg/dose)

0

20

40

60

80

100

(A)

80

90

100

110

Time after inoculation (weeks)

Drug injection

Contl. IgG (200µg/dose)

CTLA4-Ig (200µg/dose)CTLA4-Ig (2µg/dose)Anti-CD26 (200µg/dose)Anti-CD26 (2µg/dose)

Contl. IgG (200µg/dose)

CTLA4-Ig (200µg/dose)CTLA4-Ig (2µg/dose)Anti-CD26 (200µg/dose)Anti-CD26 (2µg/dose)

0

20

40

60

80

100

0 1 2 3 4 5 6 0 1 2 3 4 5 6

0 1 2 3 4 5 6Time after inoculation (weeks)

Drug injection

Fig 2. Anti-CD26 mAb treatment reduces x-GVHD-related lethality, and weight loss in hu-PBL-NOG mice without rejection of donor-derived

human lymphocytes. Hu-PBL-NOG mice were injected intraperitoneally with humanized anti-CD26 mAb, CTLA4-Ig, or isotype IgG control to

assess potency in preventing x-GVHD, according to the injection schedule as described in Materials and methods. (A) Kaplan–Meier survival

curves for mice receiving PBMC plus control IgG (200 lg/dose, blue line, n = 23), low dose anti-CD26 mAb (2 lg/dose, orange line, n = 24)

(P = 0�0001 vs. control IgG group), high dose anti-CD26 mAb (200 lg/dose, red line, n = 7) (P = 0�0006 vs. control IgG group), low dose

CTLA4-Ig (2 lg/dose, green line, n = 9) (P = 0�0005 vs. control IgG group), or high dose CTLA4-Ig (200 lg, dark green line, n = 6)

(P = 0�0008 vs. control IgG group). (B) Average weight (percentage � standard deviation (SD) of initial) for mice surviving on a given day for

different groups of mice as shown in (A). (C) Time course changes of average percentage (�SD) of human CD45+ lymphocytes in peripheral

blood in mice receiving PBMC plus control IgG (200 lg/dose, blue line, n = 23), low dose anti-CD26 mAb (2 lg/dose, orange line, n = 24), high

dose anti-CD26 mAb (200 lg/dose, red line, n = 7), low dose CTLA4-Ig (2 lg/dose, green line, n = 9), or high dose CTLA4-Ig (200 lg/dose,dark green line, n = 6).

Humanized Anti-CD26 mAb Prevents Acute GVHD

ª 2013 John Wiley & Sons Ltd 267British Journal of Haematology, 2013, 162, 263–277

green line) and showing no weight loss (Fig 2B, dark green

line). Therefore, in mice receiving high dose CTLA4-Ig,

most of the inoculated human PBMCs were rejected, which

is one of the main reasons all of the mice in this cohort

survived without developing x-GVHD. Taken together, these

data indicate that anti-CD26 mAb treatment for x-GVHD

was comparable with CTLA4-Ig treatment. Moreover, while

treatment with increasing doses of CTLA4-Ig resulted in the

absence of x-GVHD development, graft rejection did occur,

potentially analogous to findings in a clinical trial with a

new CTLA4-Ig agent, belatacept (Vincenti et al, 2010). On

the other hand, increasing doses of anti-CD26 mAb resulted

in no graft rejection in hu-PBL-NOG mice with prolonging

survival as compared to hu-PBL-NOG mice receiving

control IgG. Given that the effectiveness of anti-CD26 mAb

on x-GVHD was observed equally at low or high dose of

therapy, we conducted subsequent experiments at the 2 lg/dose level.

Anti-CD26 mAb treatment decreases level of CD26high

lymphocytes in x-GVHD mice while preserving CD4+

T cell proliferation

We next examined human CD26 expression level in the

peripheral blood and spleen of hu-PBL-NOG mice receiving

anti-CD26 mAb or CTLA4-Ig. To avoid antigen masking by

crossreaction with humanized anti-CD26 mAb, we used an

anti-human CD26 mAb recognizing a separate epitope

distinct from the humanized anti-CD26 mAb, as described in

Materials and methods. As shown in Fig 3A, higher expres-

sion level of human CD26 on both CD4+ and CD8+ human

lymphocytes in the peripheral blood of hu-PBL-NOG mice

receiving control IgG (panels a and b, blue lines) or CTLA4-

Ig (panels a and b, green lines) was observed from 1 to

3 weeks after inoculation. On the other hand, in

hu-PBL-NOG mice receiving anti-CD26 mAb, neither CD4+

nor CD8+ T cells expressed CD26 from 1 to 3 weeks (Fig 3A,

panels a and b, red lines). At 3 weeks after inoculation, very

weak expression of human CD26 was observed on CD4+ or

CD8+ cells in the peripheral blood of hu-PBL-NOG mice

receiving anti-CD26 mAb (Fig 3A, panels a and b, red lines).

These findings suggest that circulation of donor-derived

human CD26high lymphocytes might be associated with

x-GVHD development in hu-PBL-NOG mice. To examine

this issue in more detail, we analysed human CD26 expres-

sion level on mouse spleen cells. CD26highCD4+ or

CD26highCD8+ human lymphocytes were predominantly

observed in the spleen of hu-PBL-NOG mice receiving con-

trol IgG (Fig 3B, panels a and b, blue lines) or CTLA4-Ig

(Fig 3B, panels a and b, green lines) at 2 weeks after inocula-

tion. On the other hand, CD26dullCD4+ or CD26dullCD8+

human lymphocytes were predominantly observed in the

spleen of hu-PBL-NOG mice receiving anti-CD26 mAb

(Fig 3B, panels a and b, red lines) at 2 weeks after inocula-

tion. Taken together, these data suggest that the decreased

number of CD26high effector T cells may be responsible for

the relative absence of x-GVHD development in mice receiv-

ing anti-CD26 mAb.

Given that T cell activation is strongly associated with

CD26high lymphocytes (Morimoto et al, 1989), we next

addressed issues regarding the immunological status of

CD26dull lymphocytes in hu-PBL-NOG mice receiving

anti-CD26 mAb. To further characterize the activity of donor-

derived human lymphocytes in hu-PBL-NOG mice, we per-

formed cell proliferation assays. For this purpose, NOG mice

were inoculated with CFSE-labelled human PBMCs, and

treated with control IgG, anti-CD26 mAb or CTLA4-Ig, and

spleens were harvested on day 7. The isolated splenocytes were

gated for human CD4+ or CD8+ cells, and CFSE intensity was

analysed for cell division as a reflection of proliferative activity.

The human CD4+ or CD8+ cell population in the spleen of

hu-PBL-NOG mice receiving control IgG contained increased

numbers of multiply dividing cells (Fig 3C, blue bars). These

data indicate that inoculated human lymphocytes proliferated

extensively in mice developing x-GVHD. On the other hand,

the proliferative activity of human CD4+ or CD8+ cells was

significantly decreased in hu-PBL-NOG mice receiving anti-

CD26 mAb or CTLA4-Ig, in contrast to control IgG (Fig 3C,

*, **). However, compared to hu-PBL-NOG mice receiving

CTLA4-Ig, human CD4+ cell proliferation was greater in

hu-PBL-NOG mice receiving anti-CD26-mAb (Fig 3C, ***),while proliferation of human CD8+ cells was equally

suppressed [Fig 3C, not significant (NS)]. Taken together with

the data above, these findings indicate that anti-CD26 mAb

had a therapeutic effect on x-GVHD-associated manifestations

that was comparable to CTLA4-Ig, and that a decreased level

of CD26high donor cells after inoculation may be associated

with a reduction in x-GVHD severity. In addition, as CD4+

cell proliferative activity in hu-PBL-NOG mice receiving

anti-CD26 mAb was greater than that in hu-PBL-NOG mice

receiving CTLA4-Ig, treatment with anti-CD26 mAb might

avoid unwanted immunosuppressive effects, such as loss of

GVL effect or early graft rejection.

Humanized anti-CD26 mAb treatment ameliorates tissuedamage and decreased production of proinflammatorycytokines in the liver of hu-PBL-NOG mice with GVHD

Given that CD26high effector lymphocytes have high capacity

for migration into inflamed tissues (Hafler et al, 1985;

Eguchi et al, 1989; Masuyama et al, 1992; Gerli et al, 1996),

we examined GVHD target organs of hu-PBL-NOG mice

for donor lymphocyte infiltration. As patients with aGVHD

can have life-threatening damage to the liver or GI tract

(Ferrara et al, 2009), our study focussed on the examination

of liver tissues of hu-PBL-NOG mice. In x-GVHD mice

receiving control IgG, liver damage was observed with infil-

tration of human CD4+ or CD8+ MNCs (Fig 4A, panels b,

f, j), which were not found in the liver of control NOG

mice receiving PBS alone (panels a, e, i of Fig 4A).

R. Hatano et al

268 ª 2013 John Wiley & Sons LtdBritish Journal of Haematology, 2013, 162, 263–277

Moreover, slight inflammation of the portal duct areas in

the liver was observed in mice receiving CTLA4-Ig (Fig 4A,

panels d, h, l). On the other hand, infiltration of human T

cells in the liver was barely detected in mice receiving

anti-CD26 mAb (Fig 4A panels c, g, k). In addition to the

pathological changes seen in the liver, serum ALT activity

was examined as another method of detecting GVHD-asso-

ciated liver damage. Significant elevation of serum ALT

activity was observed in mice receiving control IgG or

CTLA4-Ig, while that of mice receiving anti-CD26 mAb was

found to be near normal levels (Fig 4B, *). These data

suggest that liver damage due to GVHD was suppressed in

(A)

MFI

of C

D26

(a) Human CD4+ T cells in PB (b) Human CD8+ T cells in PB

Human CD26

+ T cells in SP (B)

Rel

ativ

e ce

ll nu

mbe

r

Contl. IgG

CTLA4-Ig

Isotype contl.Human PBMC

Splenocytesin hu-PBL-NOG mice receiving:

(a) Human CD4 (b) Human CD8+ T cells in SP

(C)

Perc

enta

ge o

f CFS

E-ne

gativ

e (p

rolif

erat

ed) c

ells

Contl. IgGAnti-CD26CTLA4-Ig

Treatment:

Pre-inoculation cells:

CD4+ T cells CD8+ T cells0

20

40

60

80

100

Human T cells in spleen

NS

0

50

100

150

200

250

300

Time after inoculation (weeks)

Drug injection

0

30

60

90

120

150

180

0 1 2 3 4 5 6 0 1 2 3 4 5 6Time after inoculation (weeks)

MFI

of C

D26

Drug injection

Human CD26

CTLA4-Ig

Contl. IgGAnti-CD26

Anti-CD26

CTLA4-Ig

Contl. IgGAnti-CD26

100

80

60

40

20

0

100

80

60

40

20

0100 101 102 103 104 100 101 102 103 104

Fig 3. Anti-CD26 mAb treatment decreases CD26 expression of engrafted human lymphocytes in hu-PBL-NOG mice and decreases in vivo prolif-

eration of CD8+ T cells with preservation of CD4+ T cell proliferation. Hu-PBL-NOG mice receiving humanized anti-CD26 mAb, CTLA4-Ig, or

control IgG were bled every week. Red blood cells were then lysed, and PBMC subsets were analysed by flow cytometry. (A) Time course changes

of mean fluorescence intensity (MFI) of human CD26 of peripheral blood (PB) in NOG mice receiving human PBMC plus control IgG (2 lg/dose, blue line, n = 19), anti-CD26 mAb (2 lg/dose, red line, n = 24), or CTLA4-Ig (2 lg/dose, green line, n = 12). MFI of human CD26 expres-

sion gated for human CD4+ cells (panel a), or for human CD8+ cells (panel b) is shown as average � SD. (B) Representative histogram of

human CD26 expression on day 14 of spleen cells (SP) in hu-PBL-NOG mice receiving control IgG (2 lg/dose, blue line, n = 10), anti-CD26

mAb (2 lg/dose, red line, n = 10), or CTLA4-Ig (2 lg/dose, green line, n = 6). Panel (a) shows representative human CD26 expression gated for

human CD4+ cells in murine splenocytes, and panel (b) shows human CD26 expression gated for human CD8+ cells in splenocytes. Grey or

brown line shows histogram of isotype control Ig staining, or human CD26 expression of human PBMC before inoculation, respectively. Similar

results were observed in independent experiments using NOG mice receiving human PBMC plus control IgG (n = 19), anti-CD26 mAb (n = 24),

or CTLA4-Ig (n = 12). (C) To analyse in vivo cell proliferation in hu-PBL-NOG mice, CFSE-labelled human PBMC were inoculated intraperito-

neally into NOG mice, followed by injection of control IgG (2 lg/dose, blue bars, n = 7), anti-CD26 mAb (2 lg/dose, red bars, n = 8), or

CTLA4-Ig (2 lg/dose, green bars, n = 4). Spleen cells from mice were harvested on day 7, and acquisition was performed using flow cytometry.

Percentages of CFSE-negative, i.e., multiply dividing cells gated for human CD4+ or CD8+ cells, are shown as average � SD *, ** or *** indi-

cates P < 0�05, NS denotes not significant.

Humanized Anti-CD26 mAb Prevents Acute GVHD

ª 2013 John Wiley & Sons Ltd 269British Journal of Haematology, 2013, 162, 263–277

hu-PBL-NOG mice receiving anti-CD26 mAb, and that

treatment with anti-CD26 mAb provided an advantage over

CTLA4-Ig in preventing GVHD at similarly low doses.

Along with lymphocyte infiltration in the GVHD target

tissues, cytokine production level of donor-derived lympho-

cytes plays a role in tissue damages of the GVHD target

organs (Shlomchik, 2007; Ferrara et al, 2009; Blazar et al,

2012). We therefore analysed the mRNA level of effector

(A) NOG mice

Contl. IgG Anti-CD26 mAb CTLA4-Ig

hu-PBL-NOG mice

PBS

H&E

CD4staining

CD8staining

(b)(a) (c) (d)

(f)(e) (g) (h)

(k)(j)(i) (l)

Treatment :

(B)

1000

0

600

200

400

800

1200

Seru

m A

LT a

ctiv

ity (m

iu/m

l)

contl.IgG

Anit-CD26

CTLA4-Ig

Treatment:

CD8+ cells in Liver

(c)

(d) FASLG

(D)

(a) IL2

(c)

(d)

(b)

(C) CD4+ cells in Liver

Contl.IgG

Anti-CD26

CTLA4-IgTreatment:

Rel

ativ

e m

RN

A ex

pres

sion

00·20·40·60·81·0

00·20·40·60·81·0

00·20·40·60·81·0

00·20·40·60·81·0

0

0·5

1·0

1·5

2·0

00·20·40·60·81·0

00·20·40·60·81·0

00·20·40·60·81·0

(a) IL2

(b)

Contl.IgG

Anti-CD26

CTLA4-Ig

NS

NS

NS

IL17A

IL4 TNF

IFNG IFNG

R. Hatano et al

270 ª 2013 John Wiley & Sons LtdBritish Journal of Haematology, 2013, 162, 263–277

cytokines of donor-derived human CD4+ or CD8+ cells in

the recipient liver. In hu-PBL-NOG mice receiving anti-

CD26 mAb, levels of IL2, IFNG, IL4 and IL17A of human

CD4+ cells isolated from the mouse liver decreased

compared to those of hu-PBL-NOG mice receiving control

IgG (in of Fig 4C, panels a–d, *). Similarly, treatment of

hu-PBL-NOG mice with CTLA4-Ig resulted in lower levels

of IL2, IFNG, IL4 and IL17A of human CD4+ cells isolated

from mouse liver as compared to treatment with control

IgG (Fig 4C, panels a–d, **). For human CD8+ cells

isolated from hu-PBL-NOG mice receiving anti-CD26 mAb,

levels of IFNG, TNF and FASLG levels were decreased as

compared to those of hu-PBL-NOG mice receiving control

IgG (Fig 4D, panels b–d, **), while slight elevation of IL2

was observed (Fig 4D, panel a, *). On the other hand,

CTLA4-Ig-treated hu-PBL-NOG mice showed lower levels of

IFNG and TNF as compared to control IgG-treated mice

(Fig 4D, panels b and c,***), while IL2 and FASLG produc-

tion was at similar levels in the two groups (Fig 4D, panels

a and d, NS). Taken together with the above data, these

results indicate that anti-CD26 treatment in hu-PBL-NOG

mice ameliorated liver GVHD by decreasing production of

proinflammatory cytokines of donor-derived human

lymphocytes as well as inhibiting lymphocyte infiltration in

the liver, comparable to CTLA4-Ig.

GVL effect is maintained in hu-PBL-NOG micereceiving anti-CD26 mAb

Given that aGVHD and GVL effects are immune reactions

that are highly linked to each other (Zorn et al, 2002; Wu &

Ritz, 2009), we evaluated the potential influence of anti-

CD26 mAb treatment on GVL effect. Prior to performing in

vivo GVL experiments, we conducted mixed lymphocyte

reaction assays to determine the cytotoxicity of human

T cells on P815 leukaemic cells, and demonstrated that

human CD3+ T cells exerted in vitro cytotoxic effect on P815

cells (Fig S3A). For the subsequent animal studies, mice were

injected subcutaneously in the flank with P815 leukaemic

cells (H2d) on day 0, followed by inoculation of human

PBMCs and GVHD prophylaxis with anti-CD26 mAb or

CTLA4-Ig. All NOG mice transplanted with P815 cells suc-

cumbed to leukaemia by 6 weeks of inoculation with pro-

gressive tumour enlargement (Fig 5A,B, grey lines; Fig S3B,

panel a). Mice transplanted with P815 cells along with

human PBMCs and control IgG showed minimal signs of

tumour growth in the inoculated region, but all mice died

around 4 weeks after inoculation (Fig 5A, blue line). Necrop-

sies revealed that these mice died due to x-GVHD as mani-

fested by the infiltration of human MNCs into the liver,

spleen and lung (data not shown), suggesting that GVL effect

was exerted in the presence of severe GVHD. On the other

hand, mice inoculated with P815 along with human PBMC

and anti-CD26 mAb exhibited enhanced survival rate with

minimal evidence of GVHD (Fig 5A, red line). Importantly,

mice in this group showed significantly slow initial tumour

growth (Fig 5B, red line; Fig S3B, panel b), suggesting the

preservation of GVL effect more than CTLA4-Ig treatment

(Fig 5A, B, green lines).

When competent T cells are transferred into the recipients,

allogeneic T cells have normal GVL capacity to eliminate

leukaemic cells within 2 weeks after transplantation, while

symptoms of GVHD become clinically obvious 3–4 weeks

after transplantation (Meguro, 2010). Therefore, to further

characterize the potential mechanisms involved in the preser-

vation of GVL effect observed in hu-PBL-NOG mice receiv-

ing anti-CD26 mAb, we examined the expression level of

effector cytokines of human CD8+ T cells isolated from the

spleens of hu-PBL-NOG mice at 2 weeks after transplanta-

tion. As shown in Fig 5C, the levels of IL2 (panel a), IFNG

(panel b), and FASLG (panel d) of human CD8+ T cells was

at comparable levels between hu-PBL-NOG mice receiving

anti-CD26 mAb (red bars) and control IgG (blue bars), and

the TNF level was increased in anti-CD26 mAb group

compared to control IgG group (Fig 5C, panel c, *). On the

other hand, CTLA4-Ig treatment suppressed the production

Fig 4. Anti-CD26 mAb treatment in hu-PBL-NOG mice reduces liver infiltration of human lymphocytes with reduction of proinflammatory

cytokines. (A) Representative examples of the liver on day 28 from NOG mice receiving PBS alone (negative control, panels a, e, i), or hu-

PBL-NOG mice receiving control IgG (panels b, f, j), low dose anti-CD26 mAb (panels c, g, k), or low dose CTLA4-Ig (panels d, h, l). Slides were

stained with H&E (panels a–d), anti-human CD4 (panels e–h), and anti-human CD8 (panels i–l). All slides are at 2009 magnification. Similar

results were observed in independent experiments using mice receiving PBS alone (n = 13), or hu-PBS-NOG mice receiving control IgG (n = 6),

anti-CD26 mAb (n = 14), or CTLA4-Ig (n = 10). (B) Serum alanine transaminase (ALT) activity on day 21 in hu-PBL-NOG mice receiving

control IgG (2 lg/dose, blue dots, n = 10), anti-CD26 mAb (2 lg/dose, red dots, n = 14), or CTLA4-Ig (2 lg/dose, green dots, n = 10). Each

dot indicates an individual value and horizontal bars indicate mean value. * indicates P < 0�05. NS denotes ‘not significant’. (C) Human CD4+ T

cells were isolated from the liver of hu-PBL-NOG mice receiving control IgG (Contl. IgG, blue bars, n = 6), anti-CD26 mAb (red bars, n = 6), or

CTLA4-Ig (green bars, n = 6) at 4 weeks after inoculation of human PBMCs. mRNA expression of human IL2 (panel a), IFNG (panel b), IL4

(panel c), or IL17A (panel d) was quantified by real-time RT-PCR, as described in Materials and methods. Each expression was normalized to

hypoxanthine phosphoribosyltransferase 1 (HPRT1) and relative expression levels compared with the sample of control IgG treatment (indicated

as dashed grey lines) were shown. * or ** indicates P < 0�05, compared to control IgG. (D) mRNA levels of human CD8+ T cells isolated from

the liver of hu-PBL-NOG mice receiving control IgG (Contl. IgG, blue bars, n = 6), anti-CD26 mAb (red bars, n = 6), or CTLA4-Ig (green bars,

n = 6) at 4 weeks after inoculation of human PBMCs are shown as in (C). mRNA expression of human IL2 (panel a), IFNG (panel b), TNF

(panel c), or FASLG (panel d) was quantified by real-time RT-PCR, as described in Materials and methods. The bar graphs are demonstrated as

in (C). *, ** or *** indicates P < 0�05, compared to control IgG. NS denotes ‘not significant’, compared to control IgG.

Humanized Anti-CD26 mAb Prevents Acute GVHD

ª 2013 John Wiley & Sons Ltd 271British Journal of Haematology, 2013, 162, 263–277

of IL2, IFNG, TNF and FASLG of human CD8+ T cells in

hu-PBL-NOG spleen as compared to control IgG or anti-

CD26 mAb treatment (Fig 5C, ** and ***). These results

suggest that the GVL effect of cytotoxic effector function

occurring at the early time period prior to manifestation of

x-GVHD was preserved with the associated increase in the

production of IL2, IFNG, TNF or FASLG in hu-PBL-NOG

mice receiving anti-CD26 mAb. On the other hand, in

hu-PBL-NOG mice receiving CTLA4-Ig, proinflammatory

cytokines produced from donor-derived CD8+ cells sup-

pressed any potential GVL effect at the early time period

prior to x-GVHD development. These results suggest that

CTLA4-Ig treatment suppressed the development of GVL as

well as x-GVHD in hu-PBL-NOG mice, whereas effective

GVL function was maintained with anti-CD26 mAb therapy.

CD26+ MNCs infiltrate the skin or GI tract of alloHSCTpatients with acute GVHD

Although treatment with a murine antibody against human

CD26 exerted the therapeutic effect of controlling GVHD in

patients with steroid-resistant acute GVHD following

alloHSCT (Bacigalupo et al, 1985; de Meester et al, 1993),

the precise cellular basis of human GVHD, as well as the role

of CD26 in this process, has not yet been clearly delineated.

To address this issue, we examined human GVHD tissue

biopsies in alloHSCT recipients with or without active

GVHD. Although the liver is one of the GVHD targeting

organs, liver tissues are rarely obtained from patients with

active hepatic GVHD in the clinical setting due to the risks

of liver biopsies. Pathological diagnosis is typically conducted

(A)

Time after transplantation (weeks)

Per

cent

sur

viva

l(B)

Time after transplantation (weeks)

Tum

our v

olum

e (m

m3 )

(C)

60

1000

2000

3000

0

0·5

1·0

1·5

00·20·40·60·81·0

00·20·40·60·81·01·21·4

00·20·40·60·81·0

(a) IL2

(b) IFNG

(c) TNF

(d) FASLG

Contl.IgG

Anti-CD26

CTLA4-IgTreatment:

P815 transplanted into :

hu-PBL-NOG + CTLA4-Ig

hu-PBL-NOG + Contl. IgGhu-PBL-NOG + Anti-CD26

NOG

0

100

80

60

40

20

3 4 50 1 2 3 4 5 6

Drug injection

P815 transplanted into :

hu-PBL-NOG + CTLA4-Ighu-PBL-NOG + Anti-CD26NOG

NS

NS

NS

Rel

ativ

e m

RN

A ex

pres

sion

Fig 5. Anti-CD26 mAb treatment does not

eliminate GVL activity. P815 leukaemic cells

(2 9 104/body) were inoculated subcutane-

ously into the shaved flank of NOG mice. On

the next day, human PBMCs (1 9 107/body)

were inoculated intraperitoneally, followed by

treatment with humanized anti-CD26 mAb,

CTLA4-Ig, or control IgG (each, 2 lg/dose 9 10 doses). (A) Kaplan–Meier survival

curves for mice receiving P815 alone (grey line,

n = 4), P815 plus PBMC plus control IgG

(blue line, n = 3), P815 plus PBMC plus anti-

CD26 mAb (red line, n = 4) or P815 plus

PBMC plus CTLA4-Ig (green line, n = 3). (B)

Tumour volume of P815 transplanted into the

flank of NOG mice (grey line, n = 4), or hu-

PBL-NOG mice receiving anti-CD26 mAb (red

line, n = 4), or CTLA4-Ig (green line, n = 3).

All mice receiving P815 plus PBMC plus con-

trol IgG (blue line of Fig 5A) died without

tumour formation, therefore tumour volume

of this group is not shown in the graph. *indicates P < 0�01 of anti-CD26 mAb group

versus P815 alone or control IgG groups. (C)

Human CD8+ T cells were isolated from the

spleen of hu-PBL-NOG mice receiving control

human IgG (Contl. IgG, n = 4), anti-CD26

mAb (n = 4), or CTLA4-Ig (n = 3) on day 14

after inoculation of human PBMCs. mRNA

expression of human IL2 (panel a), IFNG

(panel b), TNF (panel c), or FASLG (panel d)

was quantified by real-time RT-PCR. Each

expression was normalized to HPRT1 and rela-

tive expression levels compared with the sam-

ple of control IgG treatment (indicated dashed

grey lines) were shown. *, ** or *** indicates

P < 0�05. NS denotes ‘not significant’.

R. Hatano et al

272 ª 2013 John Wiley & Sons LtdBritish Journal of Haematology, 2013, 162, 263–277

with endoscopic GI tract mucosal biopsy for GI GVHD

(Kreisel et al, 2011), as well as skin biopsy for the diagnosis

of skin GVHD (Sale et al, 1977). Therefore, we evaluated

specimens from skin biopsies and endoscopic samples of

upper GI (duodenal) mucosa harvested from patients with

active GVHD patients. Thirty-four adult patients (median

age, 38 years; range, 22–60 years) with AML who underwent

alloHSCT at the Keio University Hospital were included in

this study. GVHD prophylaxis was ciclosporin and metho-

trexate for related donor transplantation, or tacrolimus and

methotrexate for unrelated donor transplantation. The demo-

graphic profiles of the biopsied patients are shown in

Table I. All 34 patients had clinical skin GVHD (Stage I,

n = 16; Stage II, n = 11; Stage III, n = 7). Skin biopsies from

34 patients were analysed for CD26+ MNC infiltration. Stain-

ing of human skin biopsies for CD26 revealed that all

patients with active GVHD had CD26+ MNC infiltration in

the skin (Fig 6A, panels a–c). To determine that CD26+

MNCs are CD3+ T cells, co-staining of a representative skin

specimen revealed that CD3+CD26+ MNCs infiltrated the

skin of patients with aGVHD (Fig. S4). Fig 6B demonstrates

data correlating the absolute number (per square millimetre)

of CD26+ MNCs in the skin with the GVHD pathological

grade, and reveals that the level of CD26+ MNCs constitu-

tively infiltrating the skin of patients with aGVHD did not

correlate with disease severity. Furthermore, among patients

who had biopsies of the upper GI tract mucosa performed as

a diagnostic procedure for digestive symptoms, 32 patients

had clinical GI GVHD (Stage I, n = 10; Stage II, n = 14;

Stage III, n = 7; Stage IV, n = 1). Staining of human duode-

nal mucosal biopsies for CD26 revealed that all patients with

active GVHD had CD26+ MNC infiltration in the duodenal

mucosa (Fig 6C, panels a–c). In addition, the absolute num-

ber (per square millimetre) of CD26+ MNCs constitutively

infiltrating the duodenal mucosa of patients with aGVHD

did not correlate with disease severity as indicated by GVHD

pathologic grade (Fig 6D). However, in patients with severe

GVHD, CD26+ MNCs were found to infiltrate into the stro-

mal tissue of the duodenum, and were not only limited to

the surface mucosa (Fig 6C, panel c). Taken together, these

results strongly suggest that CD26+ MNCs play an important

role in the inflamed tissues of aGVHD patients, consistent

with previous data demonstrating that CD26+ lymphocytes

migrated to inflamed tissues with effector functions (Eguchi

et al, 1989; Gerli et al, 1996; Hafler et al, 1985; Mizokami

et al, 1996), suggesting that CD26 is a logical therapeutic

target in aGVHD.

Discussion

In the current study, we show that CD26+ T cells play an

important role in GVHD mediated by human lymphocytes,

and that anti-CD26 mAb is an effective treatment for GVHD

in hu-PBL-NOG mouse model. The effect of anti-CD26 mAb

on GVHD is shown to be comparable to the clinically avail-

able costimulation blocking agent, CTLA4-Ig (abatacept).

Importantly, anti-CD26 mAb therapy appears to be superior

to CTLA4-Ig treatment in that it preserves engraftment and

the GVL effect. Finally, our work demonstrates significant

infiltration of CD26+ MNCs in GVHD target tissues obtained

from patients with aGVHD.

Our present work indicates the infiltration of human

CD26+ lymphocytes in the x-GVHD lesions of hu-PBL-NOG

mice (Fig 1B) and the presence of human CD26high T cells

in the peripheral blood or spleen of hu-PBL-NOG mice

that developed x-GVHD (Fig 3A,B). When x-GVHD of

hu-PBL-NOG mice was diminished by anti-CD26 mAb treat-

ment, CD26high T cells were not detected in the peripheral

blood and spleen (Fig 3A,B). A potential explanation for this

finding may be internalization of surface CD26 expression on

the inoculated human lymphocytes induced by humanized

anti-CD26 mAb therapy (Dang et al, 1990). The decreased

CD26high lymphocyte level, with resultant decline in CD26-

mediated costimulation and migration, might contribute to

lowered xenogeneic activation and decreased tissue infiltra-

tion of GVHD target organs. In fact, a low level of lympho-

cyte infiltration in the liver was shown in hu-PBL-NOG mice

receiving a low dose of CTLA4-Ig (Fig 4A, panels d, h, l),

while lymphocyte infiltration in the liver was barely detected

Table I. Patients characteristics and clinical status at time of biopsy

for diagnosis of acute GVHD.

Disease (n)

AML 34

1st or 2nd CR 24

Advanced 10

Age (years)

Median 38

Range 22–60

Gender (n)

Male 20

Female 14

Allo-graft source (n)

BM 30

PB 1

CB 3

Conditioning (n)

Full 25

Reduced 9

Matching (n)

Related 8

Unrelated 26

*aGVHD clinical grade (n)

0 6

I 10

II 9

III 8

IV 1

*aGVHD, acute graft-versus-host disease; AML, acute myeloid leu-

kaemia; BM, bone marrow; CB, cord blood; CR, complete remission;

PB, peripheral blood.

Humanized Anti-CD26 mAb Prevents Acute GVHD

ª 2013 John Wiley & Sons Ltd 273British Journal of Haematology, 2013, 162, 263–277

in hu-PBL-NOG mice receiving anti-CD26 mAb (Fig 4A,

panels c, g, k). These observations might result from the fact

that CTLA4 has no direct effect on lymphocyte migration,

whereas the CD26 molecule is strongly associated with

lymphocyte migration into inflamed tissues (Hafler et al,

1985; Eguchi et al, 1989; Masuyama et al, 1992; Gerli et al,

1996; Mizokami et al, 1996). It is also possible that anti-

CD26 mAb treatment could affect other functional program-

ming of inoculated human lymphocytes, such as cell cycle

regulation. We have previously reported that binding of anti-

CD26 mAb to CD26 on T cell surface inhibits human T cell

growth and proliferation in both CD26-transfected Jurkat

T-cell lines and human T cell clones by inducing G1/S arrest,

which is associated with enhancement of p21Cip1 (CDKN1A)

expression (Ohnuma et al, 2002). In this regard, the suppres-

sion of x-GVHD by anti-CD26 mAb shown here might result

from a direct effect on cell cycle inhibition mediated by anti-

CD26 mAb. In addition to the preventive effect on x-GVHD

of anti-CD26 mAb, we observed that systemic manifestations

of x-GVHD were greatly reduced by administration of anti-

CD26 mAb when x-GVHD symptoms developed after

4 weeks of human PBMCs inoculation (data not shown).

Taken together, our results indicate that anti-CD26 mAb

treatment appears to affect T cell functions of inoculated

human lymphocytes in hu-PBL-NOG mice.

Costimulatory pathways are necessary to induce T cell

proliferation, cytokine secretion and effector function follow-

ing antigen-mediated T cell receptor activation. Given that

CD28 is widely expressed on T cells and CD28�CD8+ T cells

are well-recognized as na€ıve or highly-antigen experienced

(late-differentiated) cells (Rudd et al, 2009; Strioga et al,

2011), blockade of CD28 pathway may lead to profound

suppression of T cell function. In fact, our current study

showed that T cell proliferation or cytokine production was

inhibited at a greater level by CTLA4-Ig than by anti-CD26

mAb (Figs 3C, 4C, 4D or 5C). In this regard, immunosup-

pression by CTLA4-Ig not only diminished the engraftment

of donor-derived human lymphocytes in NOG mice

Stage I Stage II Stage III

(C)

GI

Clinical GI GVHD

Stage I Stage II Stage III

(A)

Skin

Clinical Skin GVHD

(a) (b) (c)

(a) (b) (c)

Cells/mm 2(B)

(D)

CD

26+ M

NC

s

0

10

20

30

Skin GVHD (pathological grade)

Grade 0 Grade 1 Grade 2 Grade 3-4

Cells/mm2

CD

26+ M

NC

s

0

10

20

GI GVHD (pathological grade)

Grade 1 Grade 2 Grade 3-4Grade 0

Fig 6. CD26+ MNCs infiltrate into the skin or duodenal mucosa of alloHSCT patients with acute GVHD. Histopathological evaluation of

representative skin and duodenal mucosa samples biopsied from transplant patients and stained with anti-human CD26 polyclonal antibody,

counterstained with haematoxylin, and then analysed in a blinded fashion, as described in Materials and methods. (A) Representative examples of

skin obtained from patients with clinical Stage I (panel a), Stage II (panel b), or Stage III (panel c) of acute skin GVHD. Original magnification

1009. (B) Graphs comparing the relative amount of CD26+ MNCs in the skin with acute GVHD pathologic grade 0 (n = 6), 1 (n = 10), 2

(n = 9), or 3–4 (n = 9). Each dot indicates individual value. The horizontal lines in the scattergrams indicate each mean value, and error bars

indicate SD. (C) Representative examples of duodenal mucosa obtained from patients with gastrointestinal (GI) clinical Stage I (panel a), Stage II

(panel b), or Stage III (panel c) of acute GVHD. Original magnification 1009. CD26+ MNCs infiltrated locally in the mucosal epithelia, while for

patients with Stage III, increasing number of CD26+ MNCs was observed in the interstitial tissue as well as in the mucosal epithelia. (D) Graphs

comparing the relative amount of CD26+ MNCs in the duodenal mucosa with acute GVHD pathological grade 0 (n = 4), 1 (n = 11), 2 (n = 8),

or 3–4 (n = 9). Each dot indicates individual value. The horizontal lines in the scattergrams indicate each mean value, and error bars indicate

SD.

R. Hatano et al

274 ª 2013 John Wiley & Sons LtdBritish Journal of Haematology, 2013, 162, 263–277

(Fig 2C), but also decreased the GVL effect in hu-PBL-NOG

mice (Fig 5A,B, green lines). These data strongly suggest

that while CTLA4-Ig exhibits strong suppressive effect on

x-GVHD, it also has several adverse effects, such as graft

rejection, inhibition of GVL reactivity or enhancement of

opportunistic infections.

Our previous studies showed that CD26-mediated costi-

mulation in human CD4+ T cells exerted an effect on

production of TH1 type proinflammatory cytokines such as

IL2 or IFNG (Morimoto & Schlossman, 1998), and that

soluble anti-CD26 mAb treatment decreased IL2 production

of human CD4+ T cells (Ohnuma et al, 2002). Our present

data indicate that anti-CD26 mAb treatment suppressed the

generation of IL2 and IFNG of donor-derived human CD4+

cells in the liver of hu-PBL-NOG mice (Fig 4C, panels a, b),

and that this suppression might result in the inhibition of x-

GVHD. In addition to TH1 type cytokines, the TH2 type

cytokine IL4 was found to be abundant in GVHD liver

lesions in human studies, and a high level of IL4 production

in the GVHD liver tissue appears to contribute to its patho-

genesis (Lai et al, 2012). In agreement with previous studies

of patients with aGVHD (Lai et al, 2012), we observed the

reduction of IL4 production of donor-derived human CD4+

T cells in the liver of hu-PBL-NOG mice receiving

anti-CD26 mAb or CTLA4-Ig (Fig 4C, panel c). Recently, it

was reported that CD26high T cells contain TH17 cells, and

that CD26high TH17 cells are enriched in the inflamed tissue

of patients with hepatitis and inflammatory bowel disease

(Bengsch et al, 2012). We observed decrease in the produc-

tion of IL17A in hu-PBL-NOG mice treated with anti-CD26

mAb or CTLA4-Ig (Fig 4C, panel d). Our present data on

cytokine production suggest that, due to the paradoxical and

variable effects resulting from targeting TH1-, TH2- and

TH17-type cytokines, such approaches alone may not be suf-

ficient to fully treat or prevent GVHD in the clinical setting,

but they may serve as useful adjunct therapeutic strategies to

reduce GVHD-associated tissue injuries.

Recently, it has been reported that CD8+ T cells specific for

influenza express high levels of CD26, and that this subset

could produce IL2 following stimulation, while CD8+ T cells

specific for chronic infection with persistent antigens, such as

cytomegalovirus (CMV), Epstain-Barr virus (EBV) or human

immunedeficiency virus (HIV), lack CD26 expression (Ibegbu

et al, 2009). The authors concluded that high CD26 expres-

sion among CD8+ T cells may be a marker of effective long-

term memory T cell formation and that the lack of CD26

expression among CD8+ T cells in CMV, EBV or HIV infec-

tion may indicate defective memory T cells (Ibegbu et al,

2009). It is therefore a concern that anti-CD26 mAb therapy

may potentially cause increased infection. However, CD8+ T

cells in hu-PBL-NOG mice treated with anti-CD26 mAb

exhibited increased IL2 production (Fig 4D, panel a; Fig 5C,

panel a). These results strongly suggest that IL2-producing

CD8+ T cells persist despite humanized anti-CD26 mAb

therapy, which may lead to the formation of effective memory

T cells against potential opportunistic infections following

alloHSCT. In addition, IL2 is necessary for the proliferation of

regulatory T cells (Treg), which can ameliorate GVHD

(Koreth et al, 2011). Therefore, our data that IL2-producing

CD8+ T cells still remain following humanized anti-CD26

mAb therapy may indicate the existence of immune tolerance

via Treg proliferation in the GVHD target organs.

Results from murine GVHD models have not always

translated to human disease and discrepancies between mur-

ine and human samples have been reported. While the

CD26 amino acid sequence has 85% amino acid identity

with the mouse CD26 (Marguet et al, 1992), mouse CD26

exerts no costimulatory function in mouse T cells, and is

not an activation marker of T cells nor an adenosine deami-

nase-binding protein (Marguet et al, 1992; Yan et al, 2003).

On the other hand, caveolin-1 (CAV1), a costimulatory

ligand for CD26 in humans, has 95% amino acid identity

with the mouse caveolin-1 (Engelman et al, 1998), and the

binding regions of the mouse caveolin-1 for human CD26

are well conserved. Therefore, human T cells were activated

even in NOG mice via CD26-caveolin-1 interaction. Another

important point to consider when interpreting our data is

the fact that, in our studies, mice were analysed at a time

when they were moribund, in contrast to the samples

obtained from patients that, for the most part, were obtained

at the time of GVHD diagnosis or shortly thereafter. More-

over, first-line therapies for clinical GVHD, such as metho-

trexate or calcineurin inhibitors, may have an effect on T

cell costimulatory signalling, cytokine production or expres-

sion of cell surface molecules.

Specific inhibition of DPP4 enzyme via Diprotin A

enhanced bone marrow engraftment in certain murine BMT

models (Christopherson et al, 2004). Although DPP4 enzy-

matic activity is required for CD26-mediated T cell costimu-

lation (Morimoto & Schlossman, 1998), it is unclear whether

DPP4 enzyme inhibition has an effect on GVHD develop-

ment. Regarding this point, we note that the humanized

anti-CD26 mAb used in the present study does not inhibit

DPP4 enzymatic activity of human CD26 (data not shown).

In conclusion, CD26-mediated T cell activation appears to

play a significant role in human GVHD. As full suppression

of x-GVHD with interventional therapies is currently a

difficult challenge, our data demonstrating that control of

x-GVHD can be achieved by modulating CD26high T cells

with anti-CD26 mAb are potentially important clinically.

Our work also suggests that anti-CD26 mAb treatment may

be a novel therapeutic approach for GVHD in the future.

Acknowledgements

The authors thank Ms Kaoru Komoriya for excellent assis-

tance with animal husbandry. This work was supported by

Grant-in-Aid of The Ministry of Education, Science, Sports

(K.O. and C.M.) and Culture, Ministry of Health, Labour,

and Welfare, Japan (C.M.).

Humanized Anti-CD26 mAb Prevents Acute GVHD

ª 2013 John Wiley & Sons Ltd 275British Journal of Haematology, 2013, 162, 263–277

Authorship contributions

Contributions: R.H., and J.Y. performed the experiments,

interpreted the data and assisted with the paper, K.O. and

C.M. designed the research, interpreted the data and wrote

the paper, N.H.D. interpreted the data, assisted with the

paper, and proof-read the manuscript, and T.Y. provided the

clinical data and samples, performed the experiments, analy-

sed pathological results, interpreted the data and assisted

with the paper.

Conflict-of-interest disclosure

The authors declare no competing financial interests.

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig S1. Mouse anti-human CD26 mAb clone 5K78

recognizes human CD26 even in the presence of humanized

anti-CD26 mAb.

Fig S2. Human CD3+CD26+ T cells infiltrated the liver of

hu-PBL-NOG mice developing x-GVHD.

Fig S3. GVL experiments in hu-PBL-NOG mice.

Fig S4. CD3+CD26+ MNCs infiltrate the skin of alloHSCT

patients with acute GVHD.

Table SI. The primer sequences for RT-PCR.

References

Aoe, K., Amatya, V.J., Fujimoto, N., Ohnuma, K.,

Hosono, O., Hiraki, A., Fujii, M., Yamada, T.,

Dang, N.H., Takeshima, Y., Inai, K., Kishimoto,

T. & Morimoto, C. (2012) CD26 overexpression

is associated with prolonged survival and

enhanced chemosensitivity in malignant pleural

mesothelioma. Clinical Cancer Research, 18,

1447–1456.

Bacigalupo, A., Corte, G., Ramarli, D., van Lint,

M.T., Frassoni, F. & Marmont, A. (1985) Intra-

venous monoclonal antibody (BT 5/9) for the

treatment of acute graft-versus-host disease. Acta

Haematologica, 73, 185–186.

Bengsch, B., Seigel, B., Flecken, T., Wolanski, J.,

Blum, H.E. & Thimme, R. (2012) Human Th17

cells express high levels of enzymatically active

dipeptidylpeptidase IV (CD26). Journal of

Immunology, 188, 5438–5447.

Blazar, B.R., Taylor, P.A., Boyer, M.W., Panoskalt-

sis-Mortari, A., Allison, J.P. & Vallera, D.A.

(1997) CD28/B7 interactions are required for

sustaining the graft-versus-leukemia effect of

delayed post-bone marrow transplantation

splenocyte infusion in murine recipients of mye-

loid or lymphoid leukemia cells. Journal of

Immunology, 159, 3460–3473.

Blazar, B.R., Murphy, W.J. & Abedi, M. (2012)

Advances in graft-versus-host disease biology and

therapy. Nature Reviews Immunology, 12, 443–458.

Briones, J., Novelli, S. & Sierra, J. (2011) T-cell

costimulatory molecules in acute-graft-versus

host disease: therapeutic implications. Bone

Marrow Research, 2011, 976793.

Christopherson, K.W. II, Hangoc, G., Mantel, C.R.

& Broxmeyer, H.E. (2004) Modulation of hema-

topoietic stem cell homing and engraftment by

CD26. Science, 305, 1000–1003.

Dang, N.H., Torimoto, Y., Sugita, K., Daley, J.F.,

Schow, P., Prado, C., Schlossman, S.F. & Mor-

imoto, C. (1990) Cell surface modulation of

CD26 by anti-1F7 monoclonal antibody.

Analysis of surface expression and human T cell

activation. Journal of Immunology, 145,

3963–3971.

Dong, R.P., Tachibana, K., Hegen, M., Scharpe, S.,

Cho, D., Schlossman, S.F. & Morimoto, C.

(1998) Correlation of the epitopes defined by

anti-CD26 mAbs and CD26 function. Molecular

Immunology, 35, 13–21.

Eguchi, K., Ueki, Y., Shimomura, C., Otsubo, T.,

Nakao, H., Migita, K., Kawakami, A., Matsuna-

ga, M., Tezuka, H., Ishikawa, N., Ito, K. &

Nagataki, S. (1989) Increment in the Ta1+ cells

in the peripheral blood and thyroid tissue of

patients with Graves’ disease. Journal of Immu-

nology, 142, 4233–4240.

Engelman, J.A., Zhang, X., Galbiati, F., Volonte,

D., Sotgia, F., Pestell, R.G., Minetti, C., Scherer,

P.E., Okamoto, T. & Lisanti, M.P. (1998)

Molecular genetics of the caveolin gene family:

implications for human cancers, diabetes,

Alzheimer disease, and muscular dystrophy.

American Journal of Human Genetics, 63,

1578–1587.

Ferrara, J.L., Levine, J.E., Reddy, P. & Holler, E.

(2009) Graft-versus-host disease. Lancet, 373,

1550–1561.

Fox, D.A., Hussey, R.E., Fitzgerald, K.A., Acuto,

O., Poole, C., Palley, L., Daley, J.F., Schlossman,

S.F. & Reinherz, E.L. (1984) Ta1, a novel 105

KD human T cell activation antigen defined by

a monoclonal antibody. Journal of Immunology,

133, 1250–1256.

Genovese, M.C., Becker, J.C., Schiff, M., Luggen,

M., Sherrer, Y., Kremer, J., Birbara, C., Box, J.,

Natarajan, K., Nuamah, I., Li, T., Aranda, R.,

Hagerty, D.T. & Dougados, M. (2005) Abatacept

for rheumatoid arthritis refractory to tumor

necrosis factor alpha inhibition. New England

Journal of Medicine, 353, 1114–1123.

Gerli, R., Muscat, C., Bertotto, A., Bistoni, O.,

Agea, E., Tognellini, R., Fiorucci, G., Cesarotti,

M. & Bombardieri, S. (1996) CD26 surface

molecule involvement in T cell activation and

lymphokine synthesis in rheumatoid and other

inflammatory synovitis. Clinical Immunology

and Immunopathology, 80, 31–37.

Giralt, S. (2012) Graft-versus-host disease: have we

solved the problem? Journal of Clinical Oncology,

30, 3160–3161.

Gribben, J.G., Guinan, E.C., Boussiotis, V.A., Ke,

X.Y., Linsley, L., Sieff, C., Gray, G.S., Freeman,

G.J. & Nadler, L.M. (1996) Complete blockade

of B7 family-mediated costimulation is necessary

to induce human alloantigen-specific anergy: a

method to ameliorate graft-versus-host disease

and extend the donor pool. Blood, 87, 4887–

4893.

Hafler, D.A., Fox, D.A., Manning, M.E., Schloss-

man, S.F., Reinherz, E.L. & Weiner, H.L. (1985)

In vivo activated T lymphocytes in the periph-

eral blood and cerebrospinal fluid of patients

with multiple sclerosis. New England Journal of

Medicine, 312, 1405–1411.

Hatano, R., Ohnuma, K., Yamamoto, J., Dang,

N.H. & Morimoto, C. (2013) CD26-mediated

co-stimulation in human CD8+ T cells provokes

effector function via pro-inflammatory cytokine

production. Immunology, 138, 165–172.

Ibegbu, C.C., Xu, Y.X., Fillos, D., Radziewicz, H.,

Grakoui, A. & Kourtis, A.P. (2009) Differential

expression of CD26 on virus-specific CD8+ T

cells during active, latent and resolved infection.

Immunology, 126, 346–353.

Ito, M., Hiramatsu, H., Kobayashi, K., Suzue, K.,

Kawahata, M., Hioki, K., Ueyama, Y., Koyanagi,

Y., Sugamura, K., Tsuji, K., Heike, T. & Nakah-

ata, T. (2002) NOD/SCID/ccnull mouse: an

excellent recipient mouse model for engraftment

of human cells. Blood, 100, 3175–3182.

Koreth, J., Matsuoka, K., Kim, H.T., McDonough,

S.M., Bindra, B., Alyea, E.P. 3rd, Armand, P.,

Cutler, C., Ho, V.T., Treister, N.S., Bienfang,

D.C., Prasad, S., Tzachanis, D., Joyce, R.M.,

Avigan, D.E., Antin, J.H., Ritz, J. & Soiffer, R.J.

(2011) Interleukin-2 and regulatory T cells in

graft-versus-host disease. New England Journal of

Medicine, 365, 2055–2066.

Kreisel, W., Dahlberg, M., Bertz, H., Harder, J.,

Potthoff, K., Deibert, P., Schmitt-Graeff, A. &

Finke, J. (2011) Endoscopic diagnosis of acute

intestinal GVHD following allogeneic hematopoi-

etic SCT: a retrospective analysis in 175 patients.

Bone Marrow Transplantation, 47, 430–438.

Lai, H.Y., Chou, T.Y., Tzeng, C.H. & Lee, O.K.

(2012) Cytokine profiles in various graft-versus-

R. Hatano et al

276 ª 2013 John Wiley & Sons LtdBritish Journal of Haematology, 2013, 162, 263–277

host disease target organs following hematopoietic

stem cell transplantation. Cell Transplantation, 21,

2033–2045.

Lenschow, D.J., Zeng, Y., Thistlethwaite, J.R.,

Montag, A., Brady, W., Gibson, M.G., Linsley,

P.S. & Bluestone, J.A. (1992) Long-term survival

of xenogeneic pancreatic islet grafts induced by

CTLA4Ig. Science, 257, 789–792.

Marguet, D., Bernard, A.M., Vivier, I., Darmoul,

D., Naquet, P. & Pierres, M. (1992) cDNA clon-

ing for mouse thymocyte-activating molecule. A

multifunctional ecto-dipeptidyl peptidase IV

(CD26) included in a subgroup of serine prote-

ases. Journal of Biological Chemistry, 267, 2200–

2208.

Masuyama, J., Berman, J.S., Cruikshank, W.W.,

Morimoto, C. & Center, D.M. (1992) Evidence

for recent as well as long term activation of T

cells migrating through endothelial cell mono-

layers in vitro. Journal of Immunology, 148,

1367–1374.

de Meester, I., Scharpe, S., Vanham, G., Bosmans,

E., Heyligen, H., Vanhoof, G. & Corte, G.

(1993) Antibody binding profile of purified and

cell-bound CD26. Designation of BT5/9 and

TA5.9 to the CD26 cluster. Immunobiology, 188,

145–158.

Meguro, A. (2010) Lack of IL-21 signal attenuates

graft-versus-leukemia effect in the absence of

CD8 T-cells. Bone Marrow Transplantation, 46,

1557–1565.

Mizokami, A., Eguchi, K., Kawakami, A., Ida, H.,

Kawabe, Y., Tsukada, T., Aoyagi, T., Maeda, K.,

Morimoto, C. & Nagataki, S. (1996) Increased

population of high fluorescence 1F7 (CD26)

antigen on T cells in synovial fluid of patients

with rheumatoid arthritis. Journal of Rheumatol-

ogy, 23, 2022–2026.

Morimoto, C. & Schlossman, S.F. (1998) The

structure and function of CD26 in the T-cell

immune response. Immunological Reviews, 161,

55–70.

Morimoto, C., Torimoto, Y., Levinson, G., Rudd,

C.E., Schrieber, M., Dang, N.H., Letvin, N.L. &

Schlossman, S.F. (1989) 1F7, a novel cell surface

molecule, involved in helper function of CD4

cells. Journal of Immunology, 143, 3430–3439.

Muscat, C., Bertotto, A., Agea, E., Bistoni, O.,

Ercolani, R., Tognellini, R., Spinozzi, F., Cesar-

otti, M. & Gerli, R. (1994) Expression and func-

tional role of 1F7 (CD26) antigen on peripheral

blood and synovial fluid T cells in rheumatoid

arthritis patients. Clinical and Experimental

Immunology, 98, 252–256.

Ohnuma, K., Ishii, T., Iwata, S., Hosono, O.,

Kawasaki, H., Uchiyama, M., Tanaka, H.,

Yamochi, T., Dang, N.H. & Morimoto, C.

(2002) G1/S cell cycle arrest provoked in human

T cells by antibody to CD26. Immunology, 107,

325–333.

Ohnuma, K., Dang, N.H. & Morimoto, C. (2008)

Revisiting an old acquaintance: CD26 and its

molecular mechanisms in T cell function. Trends

in Immunology, 29, 295–301.

Pasquini, M.C., Wang, Z., Horowitz, M.M. & Gale,

R.P. (2010) 2010 report from the Center for

International Blood and Marrow Transplant

Research (CIBMTR): current uses and outcomes

of hematopoietic cell transplants for blood and

bone marrow disorders. Clinical Transplants,

2010, 87–105.

Przepiorka, D., Weisdorf, D., Martin, P., Klinge-

mann, H.G., Beatty, P., Hows, J. & Thomas,

E.D. (1995) 1994 consensus conference on acute

GVHD grading. Bone Marrow Transplantation,

15, 825–828.

Rudd, C.E., Taylor, A. & Schneider, H. (2009)

CD28 and CTLA-4 coreceptor expression and

signal transduction. Immunological Reviews, 229,

12–26.

Saito, K. (1998) Involvement of CD40 ligand-

CD40 and CTLA4-B7 pathways in murine acute

graft-versus-host disease induced by allogeneic T

cells lacking CD28. J. Immunol., 160, 4225–4231.

Sale, G.E., Lerner, K.G., Barker, E.A., Shulman,

H.M. & Thomas, E.D. (1977) The skin biopsy in

the diagnosis of acute graft-versus-host disease in

man. American Journal of Pathology, 89, 621–636.

Shlomchik, W.D. (2007) Graft-versus-host disease.

Nature Reviews Immunology, 7, 340–352.

Strioga, M., Pasukoniene, V. & Characiejus, D.

(2011) CD8+CD28- and CD8+CD57+ T cells and

their role in health and disease. Immunology,

134, 17–32.

Tanaka, T., Camerini, D., Seed, B., Torimoto, Y.,

Dang, N.H., Kameoka, J., Dahlberg, H.N.,

Schlossman, S.F. & Morimoto, C. (1992)

Cloning and functional expression of the T cell

activation antigen CD26. Journal of Immunology,

149, 481–486.

Tsirigotis, P., Or, R., Resnick, I.B. & Shapira, M.Y.

(2012) Immunotherapeutic approaches to

improve graft-versus-tumor effect and reduce

graft-versus-host disease. Immunotherapy, 4,

407–424.

Vincenti, F., Charpentier, B., Vanrenterghem, Y.,

Rostaing, L., Bresnahan, B., Darji, P., Massari,

P., Mondragon-Ramirez, G.A., Agarwal, M., Di

Russo, G., Lin, C.S., Garg, P. & Larsen, C.P.

(2010) A phase III study of belatacept-based

immunosuppression regimens versus cyclospor-

ine in renal transplant recipients (BENEFIT

study). American Journal of Transplantation, 10,

535–546.

Washington, K. & Jagasia, M. (2009) Pathology of

graft-versus-host disease in the gastrointestinal

tract. Human Pathology, 40, 909–917.

Wu, C.J. & Ritz, J. (2009) Revealing tumor immu-

nity after hematopoietic stem cell transplanta-

tion. Clinical Cancer Research, 15, 4515–4517.

Yan, S., Marguet, D., Dobers, J., Reutter, W. &

Fan, H. (2003) Deficiency of CD26 results in a

change of cytokine and immunoglobulin secre-

tion after stimulation by pokeweed mitogen.

European Journal of Immunology, 33,

1519–1527.

Zorn, E., Wang, K.S., Hochberg, E.P., Canning, C.,

Alyea, E.P., Soiffer, R.J. & Ritz, J. (2002)

Infusion of CD4+ donor lymphocytes induces

the expansion of CD8+ donor T cells with

cytolytic activity directed against recipient

hematopoietic cells. Clinical Cancer Research, 8,

2052–2060.

Humanized Anti-CD26 mAb Prevents Acute GVHD

ª 2013 John Wiley & Sons Ltd 277British Journal of Haematology, 2013, 162, 263–277