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Originally published online as doi:10.1189/jlb.0306231 on October 19, 2006

Published online before print October 19, 2006
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(Journal of Leukocyte Biology. 2007;81:176-185.)
© 2007 by Society for Leukocyte Biology

Intervention of MAdCAM-1 or fractalkine alleviates graft-versus-host reaction associated intestinal injury while preserving graft-versus-tumor effects

Satoshi Ueha*, Masako Murai*, Hiroyuki Yoneyama*, Masahiro Kitabatake*, Toshio Imai{dagger}, Takeshi Shimaoka*,{ddagger}, Shin Yonehara{ddagger}, Sho Ishikawa* and Kouji Matsushima*,1

* Department of Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan;
{dagger} Kan Research Institute, Kyoto, Japan; and
{ddagger} Graduate School of Biostudies and Institute for Virus Research, Kyoto University, Kyoto, Japan

1Correspondence: Department of Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: koujim{at}m.u-tokyo.ac.jp

ABSTRACT

Coincidence of the beneficial graft-vs.-tumor (GVT) effects and the detrimental graft-vs.-host disease (GVHD) remains the major obstacle against the widespread use of allogeneic bone marrow transplantation (BMT) as tumor immunotherapy. We here demonstrate that intervention of MAdCAM-1 (mucosal vascular addressin cell adhesion molecule-1) or fractalkine/CX3CL1 after the expansion of allo-reactive donor CD8 T cells selectively inhibits the recruitment of effector donor CD8 T cells to the intestine and alleviates the graft-vs.-host reaction (GVHR) associated intestinal injury without impairing GVT effects. In a nonirradiated acute GVHD model, donor CD8 T cells up-regulate the expression of intestinal homing receptor {alpha}4ß7 and chemokine receptors CXCR6 and CX3CR1, as they differentiate into effector cells and subsequently infiltrate into the intestine. Administration of anti-MAdCAM-1 antibody or anti-fractalkine antibody, even after the expansion of alloreactive donor CD8 T cells, selectively reduced the intestine-infiltrating donor CD8 T cells and the intestinal crypt cell apoptosis without affecting the induction of donor derived anti-host CTL or the infiltration of donor CD8 T cells in the hepatic tumor. Moreover, in a clinically relevant GVHD model with myeloablative conditioning, these antibodies significantly improved the survival and loss of weight without impairing the beneficial GVT effects. Thus, interruption of {alpha}4ß7-MAdCAM-1 or CX3CR1-fractalkine interactions in the late phase of GVHD would be a novel therapeutic approach for the separation of GVT effects from GVHR-associated intestinal injury.

Key Words: chemokines • cell trafficking • mucosa • adhesion molecule • tumor

INTRODUCTION

Despite of its limitations and toxicity, allogeneic bone marrow transplantation (allo-BMT) is now widely used as effective therapy for hematologic malignancies [1 , 2 ]. The therapeutic benefits of allo-BMT appear to be twofold: not only can higher doses of chemo- and radiotherapy be given, but also there is the potential for graft-mediated immune response to tumors, so-called graft-vs.-tumor (GVT) effects [3 , 4 ]. On the other hand, acute graft-vs.-host disease (GVHD) remains a serious and often fatal complication of allo-BMT. Unfortunately, the beneficial GVT effects are closely associated with adverse GVHD, because both normal tissue injuries and anti-tumor effects are mediated by donor-derived anti-host/tumor cytotoxic T lymphocytes (CTL). Administration of immunosuppressive drugs or T cell-depleted BMT improves the severity and incidence of GVHD, but these treatments often lead to the loss of anti-host/tumor CTL, resulting in the relapse of leukemia [4 5 6 ]. These observations suggest that allo-BMT would be a safe and effective immunotherapy for tumors if anti-host/tumor CTL are fully induced while normal tissues are selectively protected from anti-host/tumor CTL.

In the early phase of allo-BMT, naive donor CD8 T cells migrate to secondary lymphoid tissues, where they proliferate and differentiate into anti-host effector T cells. In the late phase, alloactivated anti-host effector donor CD8 T cells recirculate and infiltrate into the peripheral tissues [7 , 8 ]. It is probable that effector donor CD8 T cells, which infiltrate into the skin, liver, and intestine trigger GVHD, while those infiltrate into the tumor induce GVT effects. Therefore, selective inhibition of infiltration of effector donor CD8 T cells to the target tissues would reduce tissue specific GVHD without impairing GVT effects.

Lymphocyte trafficking is tightly regulated by the expression of particular adhesion molecules and chemokine receptors on the surface of lymphocytes, combined with the expression of ligands for these receptors by the target tissues [9 10 11 ]. It has become clear that several trafficking-associated molecules are actively involved in the development of GVHD [12 , 13 ]. In the early phase of allo-BMT, circulating naïve donor T cells rapidly enter into the host secondary lymphoid tissues through adhesion molecule L-selectin, {alpha}4 integrin, {alpha}4ß7, MAdCAM-1, and chemokine receptor CCR5, and preventive intervention of these molecules inhibit the development of anti-host CTL and ameliorate GVHD [14 15 16 ]. In the late phase, it has been reported that anti-host CTL infiltrate into liver via LFA-1-ICAM-1 interaction and CCR5-MIP-1{alpha} interaction and into the intestine via CXCR3–CXCR3 ligand interaction [17 18 19 20 ]. However, molecular interactions essential for the development of tissue-specific GVHD are not fully established, because severity of GVHD induced by CCR5–/– donor cells depends on the conditioning regimen [21 ], and GVHD to MHC is not reduced in recipients of CXCR3–/– donor cells [20 ]. These studies suggest that further spacio-temporal understanding of donor CD8 T cell dynamics is required for the separation of GVHD and GVT effects by targeting trafficking-associated molecule.

Intestinal GVHD is one of the most important complications arising with acute GVHD because the damaged intestinal epithelium allows spread of endotoxins into the systemic circulation, which amplifies subsequent systemic GVHD [22 23 24 25 ]. In this study, we first performed kinetic studies in a well-established graft-vs.-host reaction (GVHR) model to determine the trafficking-associated molecules involved in the intestinal infiltration of effector donor CD8 T cells during allogeneic reaction, and then explored whether inhibition of effector donor CD8 T cell infiltration into the intestine would segregate GVT effects from GVHR-associated intestinal injury.

MATERIALS AND METHODS

Mice and cell lines
C57BL/6 Ly5.2 (B6; CD45.2, H-2b), (C57BL/6 x DBA/2) F1 (BDF1; CD45.2, H-2bxd) mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). C57BL/6 Ly5.1 (CD45.1) was provided from Dr. Ishikawa (Keio University, Tokyo, Japan). All animal procedures described in this study were performed according to the guidelines for animal experiments of The University of Tokyo. P815 (H-2d) and EL4 (H-2b) cell lines were provided from Dr. Abe (Nipponkayaku, Tokyo, Japan). GFP-expressing tumorigenic P815 cell line was generated by retroviral transduction using pMY vector [26 ].

GVHD induction and tumor induction
For the GVHR model, 6 x 107 splenocytes from either B6 or BDF1 mice were injected intravenously into nonirradiated BDF1 mice on day 0. In GVT experiments in GVHR model, recipient mice were intravenously injected with 2 x 105 P815 or EL4 tumor cells on day –1. For the irradiated GVHD model, recipient mice received 13 Gy total body irradiation, split into two doses that were separated by 3 h on day –1. Bone marrow cells were prepared from the femurs and tibias of donor mice, followed by depletion of Ter-119 positive red blood cells and Thy1.2 positive mature T cells by MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). Mature T cells were prepared from lymph nodes and spleen by negative selection with antibodies against CD11b, B220, and NK1.1 using MACS. The purity of selected T cells was at least 92%. Cell mixtures of 5 x 106 bone marrow cells supplemented with 5 x 106 T cells were then injected intravenously into irradiated recipient mice on day 0. In GVT experiments in irradiated GVHD model, recipient mice were intravenously injected with 1 x 104 P815 or EL4 tumor cells 2 h before transplantation.

Anti-mouse fractalkine monoclonal antibody
The anti-mouse fractalkine mAb was generated from Armenian hamsters immunized with recombinant mouse fractalkine by a standard method, and inhibited migration of mouse CX3CR1-transfected B300.19 pre-B cells induced by mouse fractalkine (data not shown).

In vivo treatments
For the neutralizing experiments in GVHR model, 200 µg/100 µl PBS of anti-fractalkine mAb or 500 µg/ 100 µl PBS of anti-CXCL16 mAb [27 ] were administered i.p. into BDF1 mice on day 6, day 8, and day 10 or 100 µg/200 µl PBS of anti-MAdCAM-1 mAb (MECA367, BD PharMingen, San Diego, CA) was administered i.p. on day 7 after GVHR induction. Normal hamster IgG or rat IgG was used as a control antibody. In the GVHD model, the same dose of anti-fractalkine mAb and anti-CXCL16 mAb were administrated every 4 days and anti-MAdCAM-1 mAb was administrated every 7 days from day 4 to day 32 after GVHD induction.

Cell preparation, antibodies, flow cytometry, and cell sorting
Intestinal and hepatic mononuclear cells were isolated according to the methods described previously [28 , 18 ]. In brief, the small intestine was removed and Peyer’s patches were excised from the intestine. The inverted intestine was then cut into four segments, and the segments were transferred to a 50 ml conical tube containing 45 ml RPMI-1640, 5% FBS, 25 mM HEPES. The tube was shaken at 37°C for 45 min. For the preparation of intestinal intraepithelial lymphocytes (IEL), cell suspensions were collected and passed through a nylon-wool column to deplete cell debris and sticky cells. Subsequently, the cells were subjected to 44/70% Percoll (Sigma-Aldrich, St. Louis, MO) gradient, and IEL were recovered at the interphase. For the preparation of lamina propria lymphocytes (LPL), residual intestinal pieces were digested with 50 U/ml collagenase (Type VIII, Sigma-Aldrich), and supernatants were subjected to percoll density gradient as described above. We mainly examined the IEL compartment as intestine-infiltrating lymphocytes, because the epithelium is the major target of intestinal GVHD. LPL compartment was only used in chemotaxis assay. For the preparation of hepatic lymphocytes, livers were perfused with PBS and pressed through stainless-steel mesh and suspended in 5% FBS-DMEM. The cell suspensions were treated with 33% Percoll containing 100 U/ml heparin and were centrifuged at 800 g for 10 min at room temperature. The pellets were resuspended in ACK lysing buffer, washed 2 times in DMEM, and resuspended in 10% FBS-DMEM. The mAbs specific for mouse Fc{gamma}R (2.4G2), CD8 (53-6.7), CD45.1 (A20), CD62L (MEL-14), and {alpha}4ß7 (DATK32) were purchased from BD PharMingen, and anti-mouse CCR9 mAb were purchased from R&D Systems (Minneapolis, MN). After incubation with anti-mouse Fc{gamma}R mAb, cells were stained with appropriate concentrations of mAbs and then analyzed by EPICS ELITE ESP cell sorter (Beckman Coulter, Hialeah, FL) with EXPO32 software. Dead cells were excluded on the basis of forward and sidescatter profiles, and propidium iodide staining. Donor CD8 T cells were purified by cell sorting, and the sorted cells showed >98% purity. For IFN-{gamma} detection, cells were stimulated with 5 ng/ml PMA and 0.5 µg/ml ionomycin for 5 h in the presence of 10 µg/ml brefeldin A followed by intracellular staining using Cytofix/Cytoperm Kit (BD PharMingen), according to the manufacturer’s directions.

Chemotaxis assay
Chemotaxis assay was performed with ChemoTx plate (Neuro Probe, Gaithersburg, MD), according to the manufacturer’s instructions. In brief, untreated CD8 T cells from B6 spleen or intestine, infiltrating donor CD8 T cells from GVHD induced BDF1 mice were suspended at 2 x 106 cells/ml in RPMI 1640 containing 0.5% BSA and 20 mM HEPES. Twenty-five microliters of cell suspensions were loaded on the membrane plate and placed onto a flat-bottomed 96-well microtiter plate containing 29 µl of fractalkine (10–8 M), TECK (10–7 M), and SLC (10–8 M).

Immunofluorescence staining and TUNEL assay
Recipient mice of GFP-expressing P815 were perfused with 4% paraformaldehyde/PBS. Immunofluorescent staining of fresh or paraformaldehyde fixed frozen sections were performed as described previously [29 ]. In brief, cryosections were fixed in ice-cold acetone and were preincubated with Block Ace (Dainippon Pharmaceutical Co. Ltd, Tokyo, Japan). Subsequently, samples were incubated with primary antibodies or appropriate control antibodies, followed by incubation with Alexa-dye labeled appropriate secondary reagents (Invitrogen Japan K. K., Tokyo, Japan). Anti-smooth muscle actin mAb (clone 1A4), Anti-EpCAM mAb (clone G8.8), and ER-TR7 were purchased from Sigma-Aldrich (St. Louis, MO, USA), BD PharMingen and BMA Biomedicals (Augst, Switzerland), respectively. Anti-fractalkine pAb and anti-CXCL16 pAb were purchased from R&D Systems. The sections were analyzed by an Olympus IX-70 confocal laser-scanning microscope system (Olympus Optical, Tokyo, Japan). For the detection apoptotic cell death, horizontal sections of intestine were stained with in situ cell death detection POD kit (Roche, Mannheim, Germany), according to the manufacturer’s instruction. For the counts of apoptotic cells in the intestine, 10 fields (330 µmx330 µm) of horizontal sections were examined for each mice, and the average number of TUNEL-positive cells/field of three mice was calculated.

Real-time RT-PCR analysis
Real-time RT-PCR was performed as described previously with a set of primers and Taqman probes corresponding to CCR1, CCR2, CCR5, CCR7, CCR9, CXCR3, and GAPDH, as described previously [29 ]. The sense primer for CXCR6 was 5'-AAGCTGAGGACTCTGACAGATGTGT-3', the antisense primer was 5'-CCAAAAGGGCAGAGTACAGACAA-3', the probe was 5'-CTGCTGAACTTGCCC CTGGCTGAC-3'. The sense primer for CX3CR1 was 5'- CCGCCAACTCCATGAACAA-3', the antisense primer was 5'-CGTCTGGATGATGCGGAAGTA-3', the probe was 5'-CGTCACCCCAGTTCATGTTCACAAATAG-3'. PCR was run in triplicate for each primer/template set, and the quantity of target mRNA was normalized by the level of GAPDH in each sample.

Short-term migration assay
Mononuclear cells were collected from either spleen of normal B6 mice (untreated) or peripheral blood of GVHR-induced BDF1 mice on day 10 (allo-activated), and CD8 T cells were negatively enriched by MACS with antibody against CD11b, B220, NK1.1, CD4, and CD45.2 (The purity of CD45.1+ CD8+ cells >90%). Untreated or allo-activated donor CD8 T cells were then labeled with CFSE and transferred into normal BDF1 mice (3x106 cells/mice). Two hours later, mice were perfused with 50 ml of PBS, and vertical sections of small intestine were embedded in Tissue-Teck OCT compound. Twelve-micrometer frozen sections of small intestine were analyzed by AX80 fluorescent microscopy (Olympus Optical). For the evaluation of trafficking efficiency to the intestine, 100 arbitrary villi/representative sections that exhibited exactly the vertical profile were chosen, and the numbers of CFSE-positive cells were counted.

Cytotoxicity assay
Donor CD8 T cells were purified from spleen of normal B6 or liver of GVHR-induced mice treated with control- or anti-fractalkine- or anti-MAdCAM-1 antibody. Effector (E) donor CD8 T cells were incubated with 1 x 104 target (T) P815 or EL4 cells for 5 h at 37°C. In all cases, the starting E:T ratio was adjusted to obtain an identical ratio of donor CD8 T cells to target cells. Effectors were tested in triplicate at four E:T ratios. After the incubation, cytotoxicity against target cells was determined by LDH cytotoxicity detection kit (Takara Biomedicals, Tokyo, Japan). The percent-specific LDH release was calculated from (experimental release–spontaneous release)/(detergent release–spontaneous release) x100.

Biochemical analysis
The increase in serum alanine transferase (ALT) concentration, which is an indicator of liver damage, was determined with a Fuji DRY-CHEM 5500V (Fuji Medical Systems, Tokyo, Japan).

Statistical analysis
Statistical comparisons between groups were evaluated using the Student’s t-test, except for survival data. Data were presented as means with 95% confidence intervals (CIs). Differences in survival among groups of mice were evaluated with a log-rank test of the Kaplan-Meier survival curves. P < 0.05 was considered to be statistically significant.

RESULTS

Development of effector donor CD8 T cells and intestinal injury during GVHR
Infiltration of anti-host CTL into intestine is thought to be a trigger of intestinal injury in GVHD. To confirm this notion, we first performed the kinetic studies in GVHR model, in which intestinal injury is mediated solely by allogeneic reaction. The number of donor CD8 T cells in the spleen and mesenteric lymph nodes (MLN) increased from day 2 after GVHR induction, peaked on day 10, then decreased thereafter. Unlike secondary lymphoid tissues, in the small intestine, donor CD8 T cells were rarely detected during the first 8 days and rapidly increased from day 8 to day 14 with CD11ahi CD44hi IFN-{gamma} producing effector cell phenotype (Fig. 1A 1B 1C ). Immunohistological analysis revealed that donor CD8 T cells were barely detectable in the intestine during the first 6 days of GVHR, but they rapidly infiltrated the lamina propria, which located adjacent to crypt epithelium with only a few in the villous epithelium on day 8 (Fig. 1D) . Crypt cell apoptosis is a typical feature of intestinal GVHD [30 ]. Consistent with the kinetics of intestine infiltrating effector donor CD8 T cells, TUNEL-positive apoptotic crypt epithelial cells were rarely detected before day 6; however, they rapidly increased by day 8 and remained high over the following days (Fig. 1E 1F) . These results suggest that the infiltration of effector donor CD8 T cells into the intestine, which take place during the late phase of GVHR (i.e., days 8-14), is closely associated with the development of intestinal injury.


Figure 1
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  		Figure 1. Kinetics of donor CD8 T cells and development of intestinal injury during GVHR. CD45.2+ recipient BDF1 mice were inoculated with CD45.1+ B6 donor splenocytes. (A) Absolute number of donor CD8 T cells in the spleen, mesenteric lymph nodes (MLN), and intestine were analyzed by flow cytometry at several time points post-GVHR induction. Mean values with 95% confidence intervals (n=6 mice/time points, pooled data from two independent experiments). Cell surface markers (B) and IFN-g production (C) of intestine-infiltrating donor CD8 T cells on day 10. Dotted histogram indicates background staining with isotype control, open histogram indicates untreated donor CD8 T cells, and solid histogram indicates intestine-infiltrating donor CD8 T cells. The number in the quadrant represents the percentage of IFN-{gamma}+ cells/donor CD8 T cells after in vitro stimulation (unstimulated control were <1%). (D) Vertical sections of small intestine were prepared on day 0 and day 8 and costained with anti-CD45.1 Ab (green) and anti-CD8 Ab (red). (E) Horizontal sections of intestine were prepared on day 0 and day 8, and apoptotic cells in the crypt epithelium were detected by TUNEL reaction. Apoptotic cells are shown in brown, and mononuclear cells are counterstained with hematoxylin (blue). Scale bars: 50 µm (B), 25 µm (C). Reproducible results were obtained from two independent similar analyses. (F) Crypt cell apoptosis was quantified as described in Materials and Methods. Data are expressed as the number of TUNEL-positive cells in crypt area (/mm2). Mean values with 95% confidence intervals (10 fields/mice, n=6 mice/time points, pooled data from 2 independent experiments). *, P < 0.001 vs. day 0.

 
Developmental changes of trafficking property of donor CD8 T cells during GVHR
It is well established that the migration properties of T cells critically depend on their differentiation state. On the basis of this concept, we hypothesized that intestinal infiltration of donor CD8 T cells in the late phase is induced by developmental changes of trafficking property of donor CD8 T cells during GVHR. To compare migration properties of primary circulating donor CD8 T cells (i.e., untreated donor CD8 T cells) and allo-activated recirculating donor CD8 T cells (i.e., donor CD8 T cells recovered from peripheral blood of GVHR induced mice on day 10), we performed the short-term migration assay. Two hours after adoptive transfer, untreated donor CD8 T cells preferentially migrated into host lymphoid tissues, including lymph nodes, spleen, and Peyer’s patches but did not migrate to the intestinal lamina propria. In contrast, allo-activated donor CD8 T cells preferentially migrated into nonlymphoid tissues, including intestine and liver, but did not migrate to lymphoid tissues (Fig. 2A 2B and data not shown). The migration of lymphocytes to intestinal nonlymphoid compartment is highly dependent on the expression of intestinal homing receptor {alpha}4ß7 [31 ]. Thus, we next analyzed the kinetics of {alpha}4ß7 expression on donor CD8 T cells to examine whether the expression of {alpha}4ß7 correlate with intestinal homing property. Allo-activated donor CD8 T cells migrate from secondary lymphoid tissues to the intestine via peripheral blood. During the first 5 days of GVHR, most of the circulating peripheral blood donor CD8 T cells expressed high levels of CD62L, a homing receptor for the peripheral lymph nodes [32 ], whereas only a minority of these cells expressed {alpha}4ß7. The ratio of CD62Lhi cells among the circulating donor CD8 T cells gradually decreased with concurrent increase of {alpha}4ß7+ cells from day 5 to day 9. In addition, the majority of the intestine-infiltrating donor CD8 T cells also expressed {alpha}4ß7 (Fig. 2C) .


Figure 2
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  		Figure 2. Changes in the trafficking properties of donor CD8 T cells and expression of trafficking-associated molecules during acute GVHR. (A) In vivo migration of untreated- or allo-activated donor CD8 T cells. CFSE-labeled donor CD8 T cells from normal B6 mice (untreated) or peripheral blood of GVHR-induced mice (allo-activated) were transferred into untreated BDF1 mice. Two hours after transfer, CFSE-positive cells in the lymph nodes and intestine were analyzed by a confocal laser-scanning microscopy. Scale Bar: 50 µm. Representative images from three independent experiments are shown. (B) Migration of untreated or allo-activated donor CD8 T cells to lymph nodes and intestine of untreated BDF1 mice were quantified by flow cytometry or confocal laser-scanning microscopy, respectively. Migration efficiency to intestine are shown in the number of CFSE-positive cells/100 villi. Mean values with 95% confidence intervals (n=3 mice/groups). *, P < 0.001. (C) Expression of CD62L and {alpha}4ß7 on circulating or intestine-infiltrating donor CD8 T cells. Open histogram in the lower panel indicates background staining with isotype control. Representative profiles of three mice are shown. (D) Levels of chemokine receptor mRNA in the intestine-infiltrating donor CD8 T cells on day 10. Data are shown in fold increase relative to CD8 T cells from normal B6 mice. Mean values with 95% confidence intervals (n=3 mice/groups, representative data from two independent experiments). *, P = 0.046, **, P < 0.001 vs. CD8 T cells from normal B6 mice. (E) CCR9 expression on untreated- and intestine infiltrating-donor CD8 T cells was analyzed by flow cytometry. Open histogram indicates background staining with isotype control. (F) Untreated donor CD8 T cells were prepared from normal B6 spleen, and intestine-infiltrating lymphocytes were prepared from intestinal intraepithelial lymphocyte compartment (IEL) and lamina propria lymphocyte compartment (LPL) of GVHR-induced mice on day 10. Chemotaxis response of untreated and intestine-infiltrating donor CD8 T cells to fractalkine (10–8 M), TECK (10–7 M), and SLC (10–8 M) were evaluated using a Boyden chamber. Data represent the percentage of migrated cells of triplicates. Representative data from two independent experiments. *, P = 0.049, **, P < 0.001 vs. background. (G) Vertical section of small intestine was prepared from normal BDF1 mice and stained with control goat IgG or anti-CXCL16 Ab (Red). Costaining with anti-smooth muscle actin (SMA) Ab indicates smooth muscle cells and ER-TR7 (green) indicates reticular fibroblast or reticular fiber. Some mice were injected intravenously with fluorescein-conjugated tomato lectin (LEA) before sacrifice to identify luminal surface of vascular endothelium (blue). Arrows indicate specific signal of CXCL16. (H) Vertical sections of intestine were prepared from GVHR-induced mice on day 7 or day 10. Day 7 section was costained with anti-MAdCAM-1 Ab (red) and anti-CD45.1 Ab (green). Day 10 sections were stained with control goat IgG or anti-CXCL16 Ab or anti-fractalkine Ab (Red). Costaining with anti-SMA Ab or anti-EpCAM Ab (green) indicates smooth muscle cells or epithelial cells, respectively. Scale bars: 50 µm. Arrows indicate specific signal of MAdCAM-1 (left), CXCL16 (center), and fractalkine (right), respectively. Representative image from 3 independent experiments are shown.

 
We next analyzed the relative chemokine receptor expression in allo-activated donor CD8 T cells to untreated donor CD8 T cells, because chemokine receptor expression closely associated with the intestinal homing property. Real-time PCR analysis revealed that untreated donor CD8 T cells expressed high levels of mRNA for the chemokine receptor CCR7, CCR9, and relatively low levels of those for CCR1, CCR2, CCR5, CXCR3, CXCR6, and CX3CR1. In contrast, donor CD8 T cells isolated from GVHR-induced intestine on day 10 of GVHR showed down-regulated expression of CCR7 and CCR9, but up-regulated expression of CXCR6 and CX3CR1 (Fig. 2D) . The down-regulation of CCR9, one of the intestinal homing receptor, and up-regulation of CX3CR1 were confirmed by flow cytometry and chemotaxis assay to TECK or fractalkine (Fig. 2E 2F) . Lymphocyte migration requires the expression of ligands for homing receptors by target tissues. To examine the expression of MAdCAM-1 ({alpha}4ß7 ligand [33 ]), CXCL16 (CXCR6 ligand [34 ]), and fractalkine/CX3CL1 (CX3CR1 ligand [35 ]), we performed the immunohistological analysis. CXCL16 has been reported to be expressed on dendritic cells, macrophages, smooth muscle cells and splenic red pulp [34 , 36 , 37 ], but its distribution in intestine has not been established. Therefore, we first examined the CXCL16 expression in the normal intestine. As shown in Fig. 2G , CXCL16 was expressed on the smooth muscle actin-positive smooth muscle cells in the lamina muscularis mucosa and the muscular coat of normal intestine. CXCL16 expression was also detected in a part of smooth muscle actin negative cells but was not detected on tomato-lectin labeled vascular endothelium (Fig. 2G) . The expression of CXCL16 in intestine was not changed during GVHR (Fig. 2H) . MAdCAM-1 is constitutively expressed on a part of the endothelium in the intestinal lamina propria, known as the lamina propria venules, and donor cells were located within or adjacent to the MAdCAM-1+ lamina propria venules on day 7 of GVHR (Fig. 2H) . It has been reported that fractalkine is expressed on intestinal epithelial cells [38 , 39 ]. Consistent with previous reports, fractalkine was detected on the intestinal epithelium, with the villous epithelial cells more strongly stained than the crypt epithelial cells (Fig. 2H) . The expression of MAdCAM-1, CXCL16, and fractalkine remained almost constant during GVHR in both mRNA and protein level (data not shown). Importantly, we could not detect the protein expression of MAdCAM-1 and fractalkine in the liver during GVHR (data not shown).

Anti-fractalkine or anti-MAdCAM-1 antibodies selectively inhibit intestinal infiltration of effector donor CD8 T cells and reduce intestinal injury
The up-regulation of {alpha}4ß7, CXCR6, and CX3CR1 in effector donor CD8 T cells suggest that {alpha}4ß7-MAdCAM-1, CXCR6-CXCL16, and CX3CR1-fractalkine interactions are involved in the recruitment of effector donor CD8 T cells to intestine and promote the intestinal injury. To test this, we administered neutralizing antibodies against MAdCAM-1, CXCL16, and fractalkine to GVHR-induced mice. In the neutralization experiments, we administered antibodies after the expansion of donor CD8 T cells to exclude the effects on the development of effector cells in the early phase [16 ]. As shown in Fig. 3 , administration of anti-fractalkine Ab significantly decreased the number of donor CD8 T cells in the intestine by 61% (P=0.001) and reduced the number of apoptotic crypt cells by 43% (P<0.001) compared with control antibody-treated group, whereas it had no significant effects on the number of donor CD8 T cells in the liver or serum ALT levels. Administration of anti-MAdCAM-1 Ab also decreased the number of donor CD8 T cells in the intestine by 85% (P<0.001) and reduced the number of apoptotic cells by 75% (P<0.001), whereas we did not see any significant changes in the liver. Treatment with anti-CXCL16 Ab decreased the number of CXCR6 positive donor CD8 T cells in the liver (30-35% of donor CD8 T cells in the liver of GVHR-induced mice on day 14, data not shown) but did not significantly affect overall pathology of intestinal- or liver-GVHR (data not shown).


Figure 3
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  		Figure 3. Anti-fractalkine or anti-MAdCAM-1 antibodies selectively inhibit intestinal infiltration of donor CD8 T cells and reduce intestinal injury. Anti-fractalkine Ab or anti-CXCL16 Ab were administered i.p. into BDF1 mice on day 6, day 8, and day 10, or anti-MAdCAM-1 Ab was administered i.p. on day 7 of GVHR. Normal hamster IgG or rat IgG was used as control antibody. (A) The number of intestine- and liver- infiltrating donor CD8 T cells on day 14. The horizontal lines mark the mean value. The number of crypt epithelial cell apoptosis (B) and serum ALT levels (C) on day 14. Mean values with 95% confidence intervals (n>6, pooled data of 3 independent experiments. *, P=0.001, **, P<0.001 vs. control).

 
Anti-fractalkine Ab or anti-MAdCAM-1 Ab treatments preserve GVT effects
We previously demonstrated that pretreatment of recipient mice with anti-MAdCAM-1 Ab inhibits the development of anti-host CTL activity during GVHR [16 ]. Preservation of the anti-host CTL activity is necessary for optimal GVT effects. Therefore, we examined the effects of the anti-fractalkine Ab or anti-MAdCAM-1 Ab treatments on the development of anti-host CTL activity. As shown in Fig. 4A , untreated donor CD8 T cells from normal B6 spleen showed only background lysis against Host-type P815 cells, whereas liver-infiltrating allo-activated donor CD8 T cells from day 14 after GVHR induction showed specific lysis against P815 but only background lysis against donor-type EL4 leukemia cells. Liver-infiltrating allo-activated donor CD8 T cells from anti-fractalkine antibody- or anti-MAdCAM-1 antibody-treated groups exhibited comparable lysis against P815 to those from the control antibody-treated group, suggesting that anti-fractalkine and anti-MAdCAM-1 antibody treatment does not impair the induction of anti-host CTL activity in our protocol. Next, we examined the effects of the antibody treatments on the infiltration of allo-activated donor CD8 T cells to hepatic tumor. As shown in Fig. 4B , GVHR-induced mice with GFP-expressing P815 tumor cells and control antibody treatment showed massive infiltration by donor CD8 T cells in the hepatic tumor on day 10 of GVHR. The extent of donor CD8 T cell infiltration was similar in the recipient mice treated with anti-MAdCAM-1 Ab or anti-fractalkine Ab (data not shown). To examine the mechanisms of the segregation of GVT effects and GVHD by the antibody treatments, we analyzed the expression of fractalkine and MAdCAM-1 in the GVHR-induced liver with hepatic tumor. As shown in Fig. 4C , fractalkine and MAdCAM-1 mRNA expression in the liver is relatively low compare with that of intestine. Furthermore, we could not detect the expression of MAdCAM-1 and fractalkine in tumor vasculature by immunohistological analysis (data not shown). These results suggest that administration of anti-fractalkine Ab or anti-MAdCAM-1 Ab after the expansion of donor CD8 T cells preserves induction of anti-host CTL activity and infiltration of CTL in the hepatic tumor.


Figure 4
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  		Figure 4. Anti-fractalkine or anti-MAdCAM-1 antibodies preserve GVT effects. (A) Anti-P815 (H-2d) CTL activity of liver-infiltrating donor CD8 T cells from control antibody- (open squares) or anti-fractalkine Ab- (open triangles) or anti-MAdCAM-1 Ab- (open circles) treated GVHR-induced mice on day 10. Anti-EL4 (H-2b) CTL activity of those from control Ab-treated group (closed triangles) and anti-P815 (H-2d) CTL activity of CD8 T cells from B6 mice (closed diamonds) indicate background lysis. Mean values with 95% confidence intervals of three mice. Representative data of two independent experiments. (B) Infiltration of donor CD8 T cells in the tumor site. GVHR-induced mice were inoculated with GFP-expressing P815 on day –1. Tumor-infiltrating liver sections were stained with anti-CD45.1 Ab (red) and anti-CD8 Ab (blue). GFP-bearing P815 was shown in green. Scale bars: 50 µm. Representative images from three independent experiments are shown. (C) Fractalkine and MAdCAM-1 mRNA expression in the intestine and liver of GVHR-induced mice bearing hepatic tumor were analyzed by real-time PCR. Mean values with 95% confidence intervals (n=6, pooled data of 2 independent experiments). *, P < 0.01.

 
Anti-fractalkine Ab or anti-MAdCAM-1 Ab treatments reduce severity and mortality of GVHD without affecting GVT-associated survival advantage
We finally addressed the clinical applicability of anti-fractalkine or anti-MAdCAM-1 antibody treatments using a GVHD model with myeloablative conditioning. Lethally irradiated recipient mice of allogeneic bone marrow and T cells were treated with control Ab or anti-fractalkine Ab or anti-MAdCAM-1 Ab as described in Materials and Methods. As shown in Fig. 5A , administration of anti-fractalkine Ab or anti-MAdCAM-1 Ab exhibited a significant survival advantage and reduced body weight loss compared with control antibody-treated group (Survival: P<0.001 and P=0.004 compared with control antibody-treated group, respectively, Body weight loss: P<0.05 (days 10–20) and P<0.05 (days 10–20) compared with control antibody-treated group, respectively). In the GVT experiments, recipient mice of syngeneic BMT and P815 died from tumor infiltration in the liver by day 18 (median survival=16.6, n=10), whereas recipients of allogeneic BMT and P815 with control antibodies died from GVHD, identified by the absence of tumor nodules in the liver, by day 34 (median survival=27.4 and 27.0, P<0.001 and P<0.001, n=10, respectively), suggesting a GVT-associated survival advantage. Allogeneic recipients treated with antifractalkine Ab or anti-MAdCAM-1 Ab also died from GVHD but exhibited significant survival advantage (P<0.001 and P<0.001 compared with control antibody-treated groups, respectively), suggesting a preservation of GVT effects (Fig. 5B) . Taken together, these results suggest that intervention of {alpha}4ß7-MAdCAM-1 and CX3CR1-fractalkine interactions selectively inhibits intestinal infiltration of effector donor CD8 T cells, which results in the alleviation of GVHD without impairing GVT effects.


Figure 5
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  		Figure 5. Anti-fractalkine or anti-MAdCAM-1 antibodies alleviate GVHD without impairing GVT effects. Lethally irradiated recipients received syngeneic (n=10, solid squares) or allogeneic BMT. Recipients of allogeneic BMT were treated with anti-fractalkine Ab (n=16, open triangles) or hamster IgG (n=12, open circles) every 4 days, or anti-MAdCAM-1 Ab (n=16, solid triangles) or rat IgG (n=12, solid circles) every 7 days from day 4 to day 32 after BMT. (A) Survival and body weight change. Body weight changes are shown in mean values with 95% confidence intervals. Results represent pooled data of 2 independent experiments. *, P < 0.05 vs. control antibody-treated groups, from day 10 to day 20, **, P < 0.001 vs. control antibody-treated groups. (B) Lethally irradiated recipients received syngeneic or allogeneic BMT with tumor cells and antibody treatments. Survival in the recipients of P815 and allogeneic donor cells with hamster IgG- (open triangles), rat IgG- (open circles), anti-MAdCAM-1 Ab- (solid circles), anti-fractalkine Ab- (solid triangles) treatment, recipients of P815 and syngenic donor cells (solid squares), recipients of EL4 and allogeneic donor cells (open squares). n = 10 mice/group, pooled data of two independent experiments. *, P < 0.001 vs. recipients of P815 and syngenic BMT, **, P < 0.001 vs. control antibody treated groups.

 
DISCUSSION

It is widely accepted that myeloablative conditioning in BMT is closely associated with the severity of GVHD. However, myeloablative conditioning in animal models hinders the understanding of allo-specific immune response, as systemic irradiation induces systemic inflammation and lymphopenia, which drives antigen-nonspecific homeostatic proliferation of donor T cells [40 ]. Using a well-established GVHR model, we demonstrated that allo-reactive donor CD8 T cells redistribute from secondary lymphoid tissues to the peripheral target tissues during GVHR, as they differentiate into the effector cells. Also, intestinal infiltration of effector donor CD8 T cells was closely associated with the crypt epithelial cell apoptosis. Our results from nonirradiated GVHR model suggest that the infiltration of donor CD8 T cells not only amplifies but also triggers the intestinal injury during GVHD.

During GVHR, circulating donor CD8 T cells gradually up-regulate the adhesion molecule {alpha}4ß7 as they differentiate into effector cells. {alpha}4ß7 is an important intestinal homing receptor for both the naive and effector/memory T cells, and {alpha}4ß7-expressing lymphocytes interact with MAdCAM-1-bearing high endothelial venules in Peyer’s patches and MLN, as well as with the lamina propria venules in the intestinal lamina propria [31 , 33 ]. We previously demonstrated that administration of neutralizing antibody against MAdCAM-1 before GVHR induction successfully inhibits the migration of donor CD8 T cells to Peyer’s patches and prevents induction of anti-host CTL [16 ]. Petrovic et al. reported that {alpha}4ß7– donor T cells induced less GVHD morbidity and mortality compared with {alpha}4ß7+ donor T cells [15 ]. However, segregation of GVT effects and intestinal GVHD by preventive intervention of {alpha}4ß7-MAdCAM-1 interaction is thought to be difficult, because {alpha}4ß7-MAdCAM-1 interaction is required for the induction of anti-host/tumor CTL and {alpha}4ß7– naïve CD8 T cells can differentiate into {alpha}4ß7+ effector CD8 T cells. Our data demonstrate that the administration of anti-MAdCAM-1 Ab, even after the expansion of allo-reactive donor CD8 T cells, selectively reduced the numbers of intestine-infiltrating donor CD8 T cells without affecting induction of anti-host/tumor CTL. Thus, we suggest that therapeutic intervention of {alpha}4ß7-MAdCAM-1 interaction may be effective for the segregation of GVT effects from intestinal injury.

In addition to {alpha}4ß7, intestine-infiltrating donor CD8 T cells up-regulated the chemokine receptor CXCR6 and CX3CR1. It has been reported that CX3CR1 is expressed on terminally differentiated effector CD8 T cells [41 ]. Consistent with the previous reports, intestine-infiltrating donor CD8 T cells exhibited typical effector T cell phenotype. In general, chemokine/chemokine receptor interaction induces the firm adhesion of lymphocytes to endothelial cells and extravasation of lymphocyte into the tissues. However, we could not detect the expression of fractalkine on the intestinal endothelium. Among the chemokine family, fractalkine has unique structural and functional features, as it is expressed on the cell surface where it acts as an adhesion molecule, and when soluble fractalkine is released from the cell surface by proteolysis, it can act as a chemoattractant [35 ]. So, the possible role of fractalkine-CX3CR1 is that soluble fractalkine may be released from the epithelial cells and recruit CX3CR1-positive effector cells to the epithelial cells, while transmembrane fractalkine expressed on the epithelial cells may promote the firm adhesion between CX3CR1-positive effector cells and epithelial cells and, through this interaction, enhance the retention of effector donor CD8 T cells in epithelium and the contact-dependent cytotoxic pathway. Also, Niess et al. reported that CX3CR1-fractalkine interaction is important for intestinal DCs to sample luminal antigens [42 ]. Therefore, CX3CR1-fractalkine interaction may promote the induction of allo-MHC restricted luminal antigen-specific donor CD8 T cells in draining lymph nodes and/or induce activation of intestine infiltrating donor CD8 T cells through host-derived luminal antigen-bearing intestinal DCs. In addition, CX3CR1-fractalkine interaction has been reported to be involved in cell survival and T cell costimulation [43 , 44 ]. CX3CR1-fractalkine interaction may promote the survival and activation of intestine infiltrating donor CD8 T cells. Further studies will reveal the physiological significance of these possibilities.

It has been reported that CXCR6-deficient donor CD8 T cells showed partly reduced infiltration into the liver of GVHR-induced mice on day 7 [45 ]. Consistent with the previous report, anti-CXCL16 treatment blocked the liver infiltration of CXCR6+ donor CD8 T cells but did not change the overall immunopathology in the liver. In our data, CXCR6 is expressed on a subset of liver-infiltrating donor CD8 T cells on day 14. It remains to be determined which subset of liver-infiltrating donor CD8 T cells express CXCR6 and what role CXCR6+ donor CD8 T cells play during GVHR.

In this study, we demonstrated that the inhibition of the intestinal infiltration of effector donor CD8 T cells could segregate beneficial GVT effects from adverse GVHD. Kim et al. reported that the administration of FTY720, an inhibitor of the sphingosine-1-phosphate receptor, inhibits egression of donor T cells from the lymph nodes, which results in the inhibition of effector donor T cell infiltration into the target organs and alleviates GVHD without impairing GVT effects against lymphoma [46 , 47 ]. However, leukemia and lymphoma cells are not only located in the lymph nodes but are also found in the bone marrow or nonlymphoid tissues. Thus, the egression of effector donor CD8 T cells from secondary lymphoid tissues is thought to be indispensable for optimal GVT effects. In this respect, our approach that interferes with the tissue-specific infiltration of effector donor CD8 T cells could be adapted to a wide range of tumor patients.

In summary, intervention of MAdCAM-1 or fractalkine alleviates intestinal injury and severity of systemic GVHD without hampering GVT effects, suggesting a novel therapeutic approach for the separation of GVT effects from GVHD. It is not claimed that this approach could substitute for the immunosuppressive regimens; however, when used in conjunction with reduced immunosuppressive regimens, it may contribute to make allo-BMT a safe and effective tumor immunotherapy.

ACKNOWLEDGEMENTS

We are very grateful to Dr. T. Ezaki (Tokyo Woman’s Medical University School of Medicine) for helpful advice, to Drs. S. Hashimoto, K. Kakimi and M. Kurachi for scientific discussions, and to S. Fujita, T. Sato, S. Hontsu, A. Nakano, E. Toda, S. Takao, and S. Shawkat for their kind assistance. This work was supported, in part, by Solution-Oriented Research for Science and Technology (SORST), by the Japan Science and Technology Corporation (JST).

Received March 31, 2006; revised August 24, 2006; accepted September 13, 2006.

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