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The Journal of Immunology, 2008, 181, 7380 -7389
Copyright © 2008 by The American Association of Immunologists, Inc.
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Breaking of CD8+ T Cell Tolerance through In Vivo Ligation of CD40 Results in Inhibition of Chronic Graft-versus-Host Disease and Complete Donor Cell Engraftment1

Juyang Kim*, Keunhee Park{dagger}, Hyun J. Kim{dagger}, Jiyoung Kim{ddagger}, Hyun-A Kim{dagger}, Daehee Jung{dagger}, Hye J. Kim{dagger}, Hye-Jeong Choi§, Suck-Young Choi{ddagger}, Kwang W. Seo{dagger},¶, Hong R. Cho2,*,|| and Byungsuk Kwon2,*,{dagger}

* Biomedical Research Center, Ulsan University Hospital, School of Medicine; {dagger} Department of Biological Science; {ddagger} Department of Food Nutrition; and § Department of Pathology, Department of Internal Medicine, and || Department of Surgery, Ulsan University Hospital, School of Medicine, University of Ulsan, Ulsan, Republic of Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the DBA/2 -> unirradiated (C57BL/6 x DBA/2)F1 model of chronic graft-vs-host disease (cGVHD), donor CD4+ T cells play a critical role in breaking host B cell tolerance, while donor CD8+ T cells are rapidly removed and the remaining cells fall into anergy. Previously we have demonstrated that in vivo ligation of GITR (glucocorticoid-induced TNF receptor-related gene) can activate donor CD8+ T cells, subsequently converting the disease pattern from cGVHD to an acute form. In this study, we investigated the effect of an agonistic mAb against CD40 on cGVHD. Treatment of anti-CD40 mAb inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis. The inhibition of cGVHD occurred because anti-CD40 mAb prevented donor CD8+ T cell anergy such that subsequently activated donor CD8+ T cells deleted host CD4+ T cells and host B cells involved in autoantibody production. Additionally, functionally activated donor CD8+ T cells induced full engraftment of donor hematopoietic cells and exhibited an increased graft-vs-leukemia effect. However, induction of acute GVHD by donor CD8+ T cells seemed to be not so apparent. Further CTL analysis indicated that there were lower levels of donor CTL activity against host cells in mice that received anti-CD40 mAb, compared with mice that received anti-GITR mAb. Taken together, our results suggest that a different intensity of donor CTL activity is required for removal of host hematopoietic cells, including leukemia vs induction of acute GVHD.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the parent-into-F1 graft-vs-host disease (GVHD)3 model, the genetic background of donor strains is critical in determining the outcome of GVHD (1). In a striking example, the infusion of donor T cells from the DBA/2 strain into an unirradiated (C57BL/6 x DBA/2)F1 (BDF1) mouse induces chronic GVHD (cGVHD), whereas the infusion of T cells of the other parent, C57BL/6, induces acute GVHD (aGVHD) (2). CD4+ T cells of the DBA/2 strain activate and expand host B cells with an autoreactive potential, resulting in systemic lupus erythematosus-like symptoms such as autoantibody production and glomerulonephritis (3). In aGVHD, not only do donor CD8+ T cells eliminate host hematopoietic cells, particularly host B cells, to induce massive engraftment of donor cells, but also donor CD8+ T cells attack solid organs together with donor CD4+ T cells, resulting in loss of body weight (4). It seems that donor CD8+ T cell anergy is a restriction factor for the development of cGVHD (5). Even though donor CD4+ T cells have the ability to break host B cell tolerance in both cGVHD and aGVHD, donor CD8+ T cells have a different fate after transfer into the host; that is, donor CD8+ T cells are rapidly eliminated from the host and the remaining cells fall into anergy in cGVHD (5), whereas they are differentiated into effector T cells that have the ability to delete host B cells, including potential autoreactive B cells in aGVHD, thus depriving the host of the opportunity to produce autoantibody (6, 7, 8, 9). Therefore, blocking the activity of both CD4+ and CD8+ T cells is needed to inhibit the disease progression in these clinical settings of GVHD.

Interactions of CD40 on dendritic cells (DCs) and CD40L on CD4+ T cells induce the maturation of DCs and potentiate their ability to activate Ag-specific CD8+ T cells (9, 10, 11, 12). Since Ag presentation by immature DCs induces and maintains peripheral tolerance to various Ags, blockade of CD40/CD40L has been used to inhibit allograft rejection and autoimmune diseases (13). On the other hand, in vivo ligation of CD40 using agonistic anti-CD40 mAbs has been shown to break CD8+ T cell tolerance (14) as well as to promote CD8+ T cell immunity in a number of settings, including vaccination and the treatment of tumors (15, 16, 17, 18). It is assumed that licensing of DCs to prime CD8+ T cells by anti-CD40 mAb requires expression of cytokines, cell adhesion molecules, and costimultory molecules on DCs (19, 20, 21, 22, 23, 24). Recently it has been shown that CD27 costimulation is needed for DC-driven accumulation of antitumor CTLs following anti-CD40 mAb (25).

We have demonstrated that there are various phenotypic changes following in vivo ligation of costimulatory molecules in the DBA/2 -> BDF1 cGVHD model. For example, treatment of anti-CD137 mAb triggers the induction of activation-induced cell death (AICD) of donor CD4+ T cells but has no distinguishable effect in breaking CD8+ T cell anergy (26). The overall consequence of anti-CD137 mAb treatment is the complete prevention of cGVHD. Surprisingly, anti-CD137 mAb is potent in the reversal of already established cGVHD in a minor histocompatibility Ag-mismatched cGVHD model that is similar to human cGVHD (27). In this model, anti-CD137 mAb is effective in inhibiting lethal GVHD mediated by both CD4+ and CD8+ T cells (27). These results indicate that anti-CD137 mAb can delete pathogenic CD8+ T cells as well as pathogenic CD4+ T cells in a less severe inflammatory condition where the conditioning effect of total body irradiation dissipates. On the other hand, in vivo ligation of glucocorticoid-induced TNF receptor-related gene (GITR) induces aGVHD in mice that are genetically susceptible to cGVHD (5). The anti-GITR-mediated conversion of cGVHD to aGVHD is due to breaking of donor CD8+ T cell anergy and their subsequent functional recovery of the anti-host CTL activity. Consistently, accumulating evidence has been provided that in vivo ligation of GITR breaks CD8+ T cell anergy to tumor Ags and induces their activation and expansion (28, 29). Since agonistic anti-CD40 mAbs have an immunosuppressive effect on clinical settings of various immunological diseases (30, 31, 32, 33, 34), we wanted to investigate the immunomodulatory effect of anti-CD40 mAb on cGVHD. Our results showed that treatment of anti-CD40 mAb blocks the development of cGVHD by activating donor CD8+ T cells, which otherwise fall into anergy after transfer into the host. Functionally activated donor CD8+ T cells induce the deletion of host hematopoietic cells. This phenomenon is associated with inhibition of autoantibody production, enhanced donor cell engraftment, and an elevated graft-vs-leukemia (GVL) effect. However, there are no such symptoms of aGVHD seen in mice treated with anti-GITR mAb. Our results suggest that CD40 has a distinct in vivo function in T cells that is different from that of other costimulatory molecules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female DBA/2 (H-2d) and BDF1 (H-2b/d) mice, 7–8 wk of age, were purchased from Orient Bio. All mice were maintained in pathogen-free conditions. These studies were approved by an institutional animal care committee.

Abs and reagents

Anti-CD40 (FGK45) and anti-IFN-{gamma} (R4-6A2) mAbs were purchased from BioExpress. The anti-GITR mAb (DTA-1) was described previously (28) and was purified from ascites. Control rat Ig was purchased from Sigma-Aldrich. The following FITC-, PE-, PerCP-, or biotin-conjugated mAbs to mouse proteins were purchased from BD Pharmingen: B220, CD4, CD8, CD62L, H-2Kb, IL-2, IL-4, IL-5, and IFN-{gamma}. HRP-conjugated rat anti-mouse IgG1 was also purchased from BD Pharmingen.

Induction of cGVHD

Single-cell suspensions in PBS were prepared from spleens and lymph nodes of normal DBA/2 parental donors, filtered through a sterile mesh (BD Falcon), and washed. After the erythrocytes were lysed in hemolysis buffer (144 mM NH4Cl and 17 mM Tris-HCl (ph 7.2)), the remaining cells were resuspended at 8 x 107 cells/0.2 ml in PBS. cGVHD was induced by transfer of 8 x 107 of DBA/2 cells into the tail vein of normal, unirradiated BDF1 mice. Thereafter, 200 µg of anti-CD40 mAb or control Ig was administered i.p. on days 0, 2, 4, 11, and 18. The anti-GITR mAb (500 µg/mouse) was injected one time immediately after disease induction as described previously (5). For neutralization of IFN-{gamma}, 500 µg of anti- IFN-{gamma} mAb was treated on days –2, 0, 2, and 7. In some experiments, CD4+ or CD8+ T cells were removed by anti-CD4- or anti-CD8-conjugated magnetic beads (Miltenyi Biotec) from DBA/2 spleen/lymph node cells. The remaining cells (8 x 107) were transferred into BDF1 mice to induce cGVHD.

ELISA

Mice were bled from the tail vein, and serum titers of anti-DNA IgG1 were assessed by ELISA. Plates (96-well) were incubated overnight at 4°C with 100 µl salmon sperm DNA (Sigma-Aldrich) at a concentration of 10 µg/ml. After blocking with 2% BSA, the plates were incubated with 100 µl serially diluted serum samples for 1 h at room temperature. They were washed three times with PBS containing 0.1% Tween 20, and HRP-conjugated anti-mouse IgG1 was added to each well and the plates were kept at room temperature for 1 h. They were washed again with the same solution and color was developed in 100 µl 3,5,3',5'-tetramethylbenzene substrate (Pierce) for 10–15 min and stopped by adding 100 µl of 1 N HCl. The plates were then read at 450 nm with a Wallac Victor 1420 multilabel counter (EG&G Wallac). OD values at a 1/10 dilution of sera were expressed as arbitrary units (AU) relative to a standard positive serum derived from a C57BL/6 mouse pool with cGVHD induced by B6.C-H2bm12 splenocytes (2 wk after disease induction).

Flow cytometry

The spleens of cGVHD mice were harvested on the indicated days after parental cell transfer. After lysis of the erythrocytes, the splenocytes were preincubated in a blocking buffer (PBS containing 2.4G2 mAb/0.2% BSA/0.1% sodium azide) and then incubated with the relevant mAbs for 30 min at 4°C. Finally, they were washed twice with staining buffer (PBS containing 0.2% BSA/0.1% sodium azide) and analyzed by FACScan (BD Biosciences). For the measurement of IL-2-, IL-4-, IL-5-, or IFN-{gamma}-producing T cells, splenocytes were isolated from cGVHD mice and 1 x 106 splenocytes were cultured for 24 h in the presence of 5 x 106 BDF splenocytes, which received gamma irradiation (3000 rad). Protein secretion was blocked by 1 µg/ml GolgiPlug (BD Biosciences) added during the last 4 h culture. Intracellular cytokine staining was performed, according to the manufacturer’s protocol (BD Biosciences).

Per cell-based CTL assay

CTL assay was performed as described previously (5). In brief, splenocytes from cGVHD mice were stained with anti-H-2Kb and anti-CD8 to count donor CD8+ T cells. Splenocytes containing equal numbers of donor CD8+ T cells (1 x 104 to 5 x 105) were used as effector cells to compare the cytotoxicities of single cells between experimental groups. EL4 cells (1 x 104) were used as a target cell. A20 (H-2d) cells were used as a negative control. Cytolysis was measured with a standard 6-h chromium-release assay. Target cells were labeled with 100 µCi of Na251CrO4 for 1 h, washed, and dispensed into the wells of U-bottom 96-well plates. Different numbers of the effector cells (1 x 104 to 5 x 105) were added to generate different E:T ratios of 1:1 to 50:1. The radioactivity released into supernatants was measured in a scintillation counter. In all experiments, the level of spontaneous 51Cr release was <20% of that of maximum release. Negative controls (spontaneous release) were supernatants from 51Cr-labeled target cell culture without effector cells. Postive controls (maximum release) supernatants of 51Cr-labeled target cells were lysed by 0.2% Triton X-100. Percentage specific lysis was caculated by (specific release – spontaneous release)/(maximum release – spontaneous release) x 100.

RT-PCR

RNA was isolated from colons and livers using TRIzol reagent (Invitrogen). Subsequently, cDNA was synthesized with the Moloney murine leukemia virus reverse transcriptase for RT-PCR, according to the manufacturer’s instructions (Invitrogen). The following primers were used: GAPDH, forward primer, 5'-AGGGCTGCCTTCTCTTGTGAC-3', reverse primer, 5-TGGGTAGAATCATACTGGAACATGTAG-3'; IFN-{gamma}, forward primer, 5'-CTTCTTCAGCAACAGCAAGGCGAAAA-3', reverse primer, 5'-CCCCCAGATACAACCCCGCAATCA-3'; IL-1β, forward primer, 5'-CATGGGATGATGATGATAACCTGCT-3'; reverse primer, 5'-CCCATACTTTAGGAAGACACGGATT-3'; TNF-{alpha}, forward primer, 5'-GTTCTATGGCCCAGACCCTCACA-3', reverse primer, 5'-TCCCAGGTATATGGGTTCATACC-3'. Amplified DNA was isolated using 1.2% agarose gel and the density of DNA bands was analyzed by ImageGel program provided by National Institute of Mental Health (Bethesda, MD).

GVL model

BDF1 mice were inoculated with 1 x 107 or 5 x 106 of EL4 cells via the tail vein. Four to seven days later, cGVHD was induced by transfer of 8 x 107 of DBA/2 spleen/lymph node cells into the tail vein. Anti-CD40 mAb or control Ig (200 µg/mouse) was administered i.p. on days 0, 2, 4, 11, and 18 after disease induction. For negative control, mice received anti-CD40 mAb or control Ig without donor cell infusion.

Histology and immunohistochemistry

Formalin-fixed kidney, liver, and large intestine were embedded in paraffin, and 5-µm-thick sections were stained with H&E and evaluated by light microscopy. Slides for liver and large intestine were coded and further examined in a blinded fashion by one individual (H.J.K.), using a semiquantitative system for abnormalities known to be associated with aGVHD (35, 36). For immunohistochemistry, kidneys were embedded in optimal cutting temperature compound (Sakura Finetek) and snap-frozen in liquid nitrogen. Sections (8 µm) were air-dried, fixed with acetone, and stained with FITC-conjugated anti-mouse IgG (BD Biosciences). Fluorescence was examined by confocal microscopy (Olympus).

Statistical analysis

Student’s t test was used to determine the statistical significance of differences between experimental groups. Error bars represent SD of the mean. A log-rank (Mantel-Cox) test was used for survival curves.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Prevention of cGVHD by anti-CD40 mAb treatment

To investigate the immunomodulatory effect of stimulatory anti-CD40 mAb on cGVHD, we administered anti-CD40 mAb into BDF1 recipients that received DBA/2 speen/lymph node cells on days 0, 2, 4, 11, and 18 after donor cell transfer. Treatment of anti-CD40 mAb significantly blocked the production of anti-DNA IgG1, a predominant Ig isotype in cGVHD (5, 26). A decrease in anti-IgG1 levels was observed as early as week 2 and was maintained thereafter (Fig. 1A). Glomerulonephritis, the primary cause of mortality in mice afflicted with cGVHD, was also significantly inhibited by anti-CD40 mAb (Fig. 1B). Whereas most kidneys of control Ig-treated mice demonstrated severe glomerulonephritis involving global or segmental sclerosis (arrows in Fig. 1B), glomeruli of anti-CD40-treated mice had an intact architecture (arrowhead in Fig. 1B). The less severe glomerulonephritis induced by anti-CD40 mAb was correlated with lower deposits of immune complex (Fig. 1B). Consistent with these data, anti-CD40-treated mice had an increased survival rate (Fig. 1C). Thus, anti-CD40 mAb has an effect on cGVHD mortality as well as morbidity. Overall, our findings suggest that anti-CD40 mAb treatment prevents cGVHD by inhibiting the production of autoantibodies, and so avoiding the induction and development of glomerulonephritis.


Figure 1
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  		FIGURE 1. Anti-CD40 mAb prevents cGVHD. cGVHD was induced by transferring 8 x 107 DBA/2 spleen/lymph node cells into BDF1 mice. Control Ig or anti-CD40 mAb was administered on days 0, 2, 4, 11, and 18. A, Serum samples were collected every 2 wk and assayed in duplicate by ELISA for IgG1 anti-DNA autoantibody. Arbitrary units (AU) are shown as mean ± SEM (n = 10/group) of 10-fold dilution of each sample. B, Kidneys were harvested at 12 wk after disease induction. Representative kidney sections are shown for H&E staining (upper panels) and IgG immunostaining (lower panels). Notice glomeruli with sclerosis indicated by arrows in the control Ig kidney section (upper left panel). In contrast, glomeruli indicated by arrowheads show a normal architecture in the section of the anti-CD40 group (upper right panel). C, Anti-CD40 mAb treatment prevented death due to cGVHD (n = 18–19/group). **, p < 0.01 between the two groups. Data shown are representative of more than three independent experiments.

 
Treatment of anti-CD40 mAb enhances donor cell engraftment by increasing anti-host CTL activity

To address the mechanism underlying the inhibition of cGVHD by anti-CD40 mAb, we first examined the lymphocyte population in cGVHD mouse spleens. We found that treatment of anti-CD40 mAb led to a marked reduction in the number of total splenocytes (Table I). This lymphopenic phenotype was due to the elimination of host B cells, host CD4+ T cells, host CD8+ T cells, and other host cells. Since host CD4+ T cells and host B cells are required for the evolution of cGVHD (e.g., autoantibody production) (37, 38), deletion of host CD4+ T cells and host B cells following anti-CD40 mAb treatment might be an adequate explanation for anti-CD40-mediated inhibition of cGVHD. On the other hand, donor cells were almost completely engrafted into the host spleen 12 wk after anti-CD40 mAb treatment (Table I). Since the pattern of donor cell engraftment of anti-CD40-treated mice is observed in aGVHD mice, we hypothesized that in vivo ligation of CD40 during the induction phase of cGVHD can trigger the donor CTL response to alloantigens expressed on host hematopoietic cells, making niches for donor cell engraftment. Indeed, a per cell-based cytotoxicity assay demonstrated increased levels of anti-host CTL activities of donor CD8+ T cells in mice treated with anti-CD40 mAb (Fig. 2). In anti-CD40-treated mice, the significantly elevated CTL activities of donor CD8+ T cells were observed ex vivo even without an in vitro restimulation period at 2 wk after disease induction (Fig. 2A) and thereafter. Specific lysis of donor CD8+ T cells at 12 wk after disease induction was shown in Fig. 2B. As expected, minimal levels of cytotoxic activities were detected in mice treated with control Ig (Fig. 2). Our results indicate that anti-CD40 mAb drives accumulation of alloreactive CD8+ T cells.


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  		Table I. Anti-CD40 mAb increases donor cell engraftmenta

 

Figure 2
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  		FIGURE 2. Anti-CD40 mAb enhances anti-host CTL activity of donor CD8+ T cells. A, Splenocytes were harvested 3 wk after disease induction. After counting the numbers of donor CD8+ T cells, equal numbers of donor CD8+ T cells were set up in a conventional 6-h 51Cr-release assay with EL4 cells as a target cell, as described in Materials and Methods. A20 cell was used as a negative control. Data are presented as means ± SEM (from n = 4/group). ***, p < 0.001 and **, p < 0.01 between the two groups at the indicated time point. B, Splenocytes were harvested at 12 wk after disease induction. Percentage specific lysis was shown as mean ± SEM at the E:T ratio of 10:1 (n = 5–7/group). ***, p < 0.001 between the two groups. Similar experiments were repeated more than three times at different time points after disease induction.

 
Donor CD8+ T cells are required for anti-CD40-mediated inhibition of autoantibody production and donor cell engraftment

To directly assess the involvement of donor CD8+ T cells in the inhibition of cGVHD by anti-CD40 mAb, cGVHD was induced by transferring parental spleen/lymph node cells depleted of CD8+ T cells. In this model, donor CD4+ T cells are sufficient to induce cGVHD, and donor B cells are rapidly removed from the host after cell transfer and do not play a role in disease induction. Elimination of donor CD8+ T cells completely abrogated the effects of anti-CD40 mAb on deletion of host cells, donor cell engraftment, and inhibition of autoantibody production (Table II). Therefore, we confirmed that donor CD8+ T cells are prerequisite to the inhibition of cGVHD by anti-CD40 mAb.


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  		Table II. Donor CD8+ T cells are required for anti-CD40-mediated inhibition of autoantibody production and donor cell engraftmenta

 
Donor CD4+ T cells and anti-CD40 mAb have a synergistic effect on donor cell engraftment

We next examined whether donor CD4+ T cells have an influence on the ability of donor CD8+ T cells to induce the phenotypic changes followed by treatment with anti-CD40 mAb. For this purpose, BDF1 mice received DBA/2 spleen/lymph node cells depleted of CD4+ T cells. Mice treated with anti-CD40 mAb showed significantly increased levels of donor cell engraftment even in the absence of donor CD4+ T cells compared with mice treated with control Ig (Table III). However, anti-CD40 mAb had a modest effect on deletion of host cells in mice that received donor cells depleted of CD4+ T cells, as compared with mice that received donor cells without depletion of donor CD4+ T cells. Interestingly, depletion of donor CD4+ T cells, regardless of whether anti-CD40 mAb or control Ig was administered, induced significant levels of host cell deletion, in particular, host B cells but not donor cell engraftment. This phenomenon might occur because of the absence of donor regulatory CD4+ T cells that regulate donor CD8+ T cell anergy in cGVHD (39). As expected, basal levels of autoantibody were produced in mice that received donor cells depleted of CD4+ T cells (Table III). These data suggest that help from donor CD4+ T cells maximizes the efficiency of anti-CD40 mAb to activate donor CD8+ T cells.


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  		Table III. Donor CD4+ T cells and anti-CD40 mAb have a synergistic effect on donor cell engraftmenta

 
Treatment of anti-CD40 mAb does not induce an evident form of aGVHD

As shown in Table I, the pattern of donor cell engraftment in mice treated with anti-CD40 mAb is typically found in mice afflicted with aGVHD. Furthermore, donor CD8+ T cells of anti-CD40-treated mice had the ability to kill host cells. Nonetheless, mice that received anti-CD40 mAb did not experience loss of body weight, a hallmark of aGVHD (Fig. 3A). Anti-CD40-treated mice also did not manifest other clinical signs of aGVHD such as profuse diarrhea and hunched posture. By contrast, histopathological analysis showed significant tissue injury in the large intestine and liver of mice that received either control Ig or anti-CD40 mAb. Overall pathological scores for the aGVHD target organs were similar between the two groups (Fig. 3B). Taken together, our results indicate that breaking of CD8+ T cell anergy by anti-CD40 mAb is not strong enough to induce full-blown aGVHD.


Figure 3
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  		FIGURE 3. aGVHD induced by anti-CD40 mAb is not so evident. cGVHD was induced by transferring 8 x 107 DBA/2 spleen/lymph node cells into BDF1 mice. Control Ig or anti-CD40 mAb was administered on days 0, 2, 4, 11, and 18. A, Changes of body weight. B, Pathological scores of colons and livers harvested at 11 wk after disease induction. There was no significant difference between the two groups (n = 3 for the control Ig group and n = 7 for the anti-CD40 group).

 
The overall phenotypic changes triggered by anti-CD40 mAb are in contrast with those seen in mice that received anti-GITR mAb (anti-GITR mAb shifts the GVHD disease pattern from a chronic form to an acute form, including loss of body weight; see Ref. 5). To search for the reason why even though anti-CD40 mAb elicits accumulation of donor CD8+ T cells exhibiting distinguishable levels of lytic activity against host hematopoietic alloantigens, those alloreactive donor CD8+ T cells do not cause visible symptoms of aGVHD, we wanted to examine the hypothesis that the magnitude of the alloantigen-specific donor CD8+ T cell pool determines the degree of donor cell engraftment vs damage to aGVHD target organs. We found that anti-GITR mAb was much more potent in increasing the ability of donor CD8+ T cells to delete host cells, as compared with anti-CD40 mAb (Fig. 4A). Indeed, per cell-based cytotoxicity assay showed that anti-GITR mAb is superior to anti-CD40 mAb in elevating the CTL capacity of donor CD8+ T cells at a single cell basis (Fig. 4C). Considering the number of total donor CD8+ T cells existing in the host spleen (Fig. 4D), the pool of donor CD8+ T cells exhibiting a CTL activity against host cells was much greater in anti-GITR-treated mice than in anti-CD40-treated mice (Fig. 4E). Paradoxically, however, pathological scores showed that there was significant tissue damage in the colons and livers of either anti-GITR-treated or anti-CD40-treated mice, as compared with control Ig-treated mice (Fig. 4F). Since anti-CD40 mAb has a toxicity to aGVHD target organs (40), it is thought that tissue injury could originate in the toxic effects of anti-CD40 mAb rather than in the elevated CTL activity of donor CD8+ T cells (also see Figs. 5 and 6). Consistent with this hypothesis, our long-term observation showed that 100% of mice (30 out of 30) that received anti-CD40 mice survived until 10 wk after disease induction, whereas 45% of mice (9 out of 20 mice) that received anti-GITR mAb were dead by that time. Further studies will be needed to clarify this issue. Taken together, our results indicate that the CTL capacity of alloreactive donor CD8+ T cells is the major engine to drive elimination of host hematopoietic cells (thus facilitating donor cell engraftment) and development of aGVHD.


Figure 4
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  		FIGURE 4. Comparison of alloreactive donor CD8+ T cell pools generated by anti-CD40 or anti-GITR mAb. Anti-CD40 mAb or control Ig was injected on days 0, 2, 4, and 11 after cGVHD induction, and anti-GITR mAb (500 µg/mouse) was administered one time on day 0. Splenocytes were harvested at 2 wk after disease induction. A, Total numbers of splenocytes (n = 10/group). B, Percentage of donor CD8+ T cells in the spleen (n = 10/group). C, Per cell-based cytotoxicity assay. CTL assays were performed and data are presented as described in the legend of Fig. 2 (n = 3–5/group). D, The number of donor CD8+ T cells per spleen (n = 5–7/group). E, Specific lysis per spleen was calculated by considering the number of donor CD8+ T cells present in the spleen (n = 5–7/group). F, Pathological scores for colons and livers harvested at 2 wk after disease induction (n = 3–7/group). ***, p < 0.001 and **, p < 0.01 (n = 10/group). Experiments were repeated more than three times and similar data were obtained.

 

Figure 5
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  		FIGURE 5. Cell-based analysis of donor CD8+ T cells in the spleen and analysis of inflammatory cytokine expression in aGVHD target organs. Anti-CD40 mAb or control Ig was injected on days 0, 2, 4, and 11 after cGVHD induction, and anti-GITR mAb (500 µg/mouse) was administered one time on day 0. Splenocytes were harvested at 2 wk after disease induction. A, Percentage of activated donor CD8+ T cells. Splenocytes were stained with anti-H-2Kb and anti-CD8 in conjugation with anti-CD62L. CD62L-negative cells were counted in the gate of H-2Kb-negative donor CD8+ T cells (n = 10/group). B, Intracellular staining of donor CD8+ T cells after in vitro restimulation with host splenocytes. MLR and intracellular staining were done as described in Materials and Methods. **, p < 0.01 beween the indicated groups (n = 3–5/group). C, Expression of inflammatory cytokines in colons and livers at 2 wk after disease induction. RNA was extracted from colons and livers, and RT-PCR was performed. Data are presented as means ± SEM of the density ratio of cytokine/GAPDH. *, p < 0.05 (n = 3–7/group). Experiments were repeated more than three times.

 

Figure 6
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  		FIGURE 6. Reduction of the CTL activity of donor CD8+ T cells by neutralization of IFN-{gamma}. Anti-CD40 mAb or control Ig was injected on days 0, 2, 4, and 11 after cGVHD induction, and anti-GITR mAb (500 µg/mouse) was administered one time on day 0. Each group of mice was divided into two groups, and one group was treated with anti-IFN-{gamma} mAb (500 µg/mouse) on days –2, 0, 2, and 9. Splenocytes were harvested at 2 wk after disease induction. A, Per cell-based cytotoxicity assay. CTL assays were performed and data are presented as described in the legend of Fig. 2. Data are shown as mean ± SEM at the E:T ratio of 10:1. Upper panel, Data for EL4 targets. Lower panel, Data for A20 targets. **, p < 0.01 (n = 5–10/group). B, Total numbers of splelnocytes. **, p < 0.01 and *p < 0.05 between the indicated groups (n = 5–10/group).

 
IFN-{gamma} is critical in determining the CTL activity of donor CD8+ T cells

As shown in Fig. 5A, in vivo engagement of CD40 or GITR resulted in similar extents of activation in most donor CD8+ T cells in the DBA/2 -> BDF1 cGVHD setting when the down-regulation of CD62L expression was examined. In contrast, the generation of donor CD8+ T cells producing IFN-{gamma} was significantly greater in anti-GITR-treated mice than in CD40-treated mice (Fig. 5B). Even though donor CD8+ T cells producing other cytokines such as IL-2, IL-4, and IL-5 have a trend to be more numerous after in vitro restimulation with recipient splenocytes in anti-GITR-treated mice than in anti-CD40-treated mice, there was no statistical significance between the two groups. RT-PCR analysis showed that higher levels of proinflammatory cytokines including IL-Iβ, TNF-{alpha}, and IFN-{gamma} were expressed in the colons and livers of anti-GITR- or anti-CD40-treated mice compared with control Ig-treated mice (Fig. 5C). No significant difference was observed in levels of cytokine expression except for IFN-{gamma} in the colon between anti-GITR-treated and anti-CD40-treated groups. Again, the pattern of cytokine expression in the aGVHD target organs may reflect the toxicity of anti-CD40 mAb. Taken together with the CTL data shown in the Fig. 4, these results indicate that the pool of alloreactive donor CD8+ T cells generated by anti-GITR mAb obtains the capacity to produce a high amount of IFN-{gamma} that is a requirement for intestinal inflammation (41).

IFN-{gamma} has been shown to be important for CTL maturation in aGVHD (42). Consistently, in vivo neutralization of IFN-{gamma} markedly reduced the anti-host CTL activity of donor CD8+ T cells induced by anti-GITR mAb (Fig. 6A). Reduction in the CTL activity was correlated with decreased numbers of host cells in the spleen of anti-GITR-treated mice (Fig. 6B). The reducing extent of the anti-host CTL activity of donor CD8+ T cells after neutralization of IFN-{gamma} is smaller in anti-CD40-treated mice as compared with anti-GITR-treated mice. Since neutralization of IFN-{gamma} did not completely abrogate the CTL activity induced by engagement with either GITR or CD40, there must be other mechanisms underlying the CTL activity controlled by the GITR or CD40 costimulatory pathway.

Anti-CD40 mAb has an elevated GVL effect

Our data presented so far suggest that we can separate donor cell engraftment from GVHD by regulating the CTL activity of donor CD8+ T cells. Since donor cell engraftment induced by anti-CD40 mAb was associated with deletion of host hematopoietic cells in our GVHD model, we further wanted to examine if anti-CD40 mAb has the ability to elevate a GVL activity in vivo. BDF1 mice received DBA/2 spleen/lymph node cells together with anti-CD40 mAb at 1 wk after inoculation of high doses of T lymphoma cells (1 x 107 of EL4 cells per mouse). Anti-CD40 mAb had no visible effect on survival in the absence of donor T cells (Fig. 7A). Similarly, infusion of donor T cells plus control Ig was not effective in increasing survival. However, anti-CD40 mAb significantly increased survival only in the presence of donor T cells, even though anti-CD40 mAb plus donor lymphocyte infusion did not completely block death that was caused by inoculation with leukemia cells. A combination of treatment with anti-CD40 mAb and infusion of donor T cells had a more dramatic GVL effect when the inoculation number of T lymphoma cells was reduced in half (5 x 106/mouse) (Fig. 7B). Overall, our results suggest that the pool of alloreactive donor CD8+ T cells is a determining factor for segregation of donor cell engraftment and a GVL effect from GVHD.


Figure 7
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  		FIGURE 7. Anti-CD40 mAb elevates a GVL effect. BDF1 mice were inoculated i.v. with 1 x 107 (A) or 5 x 106 (B) EL4 cells. One week later, mice were divided into two groups. One group of mice was infused with parental cells and the other group of mice was not. The two groups of mice received either control Ig or anti-CD4 mAb on days 0, 2, 4, 11, and 18 after donor cell infusion. Survival of mice was monitored daily until termination of the experiment (n = 10/group). *, p < 0.05 between the indicated group and the other three groups.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Alloreactive T cells respond in a complex and sometimes unpredictable manner when transferred into an allogeneic environment. Following transfer, massive alloreactive T cell expansion is observed as well as extensive T cell anergy, apoptosis, and repertoire selection (43). It is common for donor CD8+ T cells to become anergic after transfer into the host in cGVHD models. We have demonstrated that most donor CD8+ T cells underwent a short burst of expansion, followed by AICD and the development of anergy in the residual population in the DBA/2 -> BDF1 model (5). Similarly, donor CD8+ T cells do not play a significant role in the pathogenesis of cGVHD in minor Ag-mismatched irradiated cGVHD models (27, 44, 45, 46). Since transfer of a high dose of donor CD8+ T cells can induce lethal GVHD only in the presence of donor CD4+ T cells or strong costimulation, it seems that donor CD8+ T cells would fall into anergy without CD4+ T cell help (27, 46). Visualization of alloreactive TCR transgenic CD8+ T cells into Ag-bearing hosts has shown that they follow a similar course in noninflammatory conditions as seen in DBA/2 polyclonal CD8+ T cells in unirradiated BDF1 recipients (47, 48, 49, 50, 51).

The parent-into-unirradiated GVHD model is unique among GVHD models in that the development of GVHD is initiated only by alloreactivity under no inflammatory environment. This property provides less complication in interpreting experimental data and finds its usefulness in an experimental tool to dissect a variety of issues related to immune tolerance to alloantigens. Using the DBA/2 -> unirradiated BDF1 GVHD model, we previously provided evidence showing that donor CD8+ T cell anergy is a restriction factor in the diverging checkpoint of cGVHD vs aGVHD (5). When the activity of donor CD8+ T cells, which are genetically destined for an anergic status, is fully recovered by strong costimulation via GITR, recipient mice show manifestations of aGVHD, such as complete donor cell engraftment and target organ damage. In this study, we further demonstrated this model as an experimental system that can be used to explore the issue regarding how donor cell engraftment plus a GVL activity can be segregated from GVHD. Costimulation via CD40 was phenotypically shown to result in the inhibition of both types of GVHD. The inhibition of cGVHD by CD40 stimulation was associated with a donor CD8+ T cell’s CTL activity against host hematopoietic cells, because activated donor CD8+ T cells followed by CD40 stimulation efficiently deleted host CD4+ T cells and host B cells that are responsible for autoantibody production. It seems to be without exception that donor cell engraftment was followed after removal of host hematopoietic cells by donor CD8+ T cells. Donor CD8+ T cells activated by CD40 stimulation also exhibited a heightened GVL effect on inoculated leukemia. On the other hand, although there was no doubt that CD40 stimulation activated donor CD8+ T cells to the extent that they killed host hematopoietic cells including leukemia, its strength to activate donor CD8+ T cells was not sufficient to facilitate the induction of aGVHD. In comparison with GITR stimulation, which leads to distinguishable clinical signs of aGVHD (5), CD40 stimulation generated a smaller size of alloreactive donor CD8+ T cell pool. Therefore, the resistance or susceptibility to aGVHD was correlated with the magnitude of the alloreactive donor CD8+ T cell pool generated by costimulation. It might be possible that a limited number of donor CD8+ T cells reactive to target organ Ags for aGVHD are present in their pool. Without local inflammation in the aGVHD target organs, this size of alloreactive donor CD8+ T cells may be unable to induce aGVHD (52). In this context, note that subclinical aGVHD proceeds in the target organs, including the colon and the liver, presumably by being triggered by the toxicity of anti-CD40 mAb. However, it seems that full-blown aGVHD would not present with the involvement of a threshold number of pathogenic donor CD8+ T cells. However, further study is needed to clarify this issue. Nonetheless, our results suggest that it is possible to separate a GVL activity from GVHD by regulating the size of the alloreactive donor CD8+ T cell pool.

Also note that the DBA/2 -> BDF1 cGVHD model has been questioned with regard to its relevance to human cGVHD (53). Although autoantibodies are not uncommonly observed in human cGVHD patients, they are derived from engrafted donor B cells. Furthermore, disease symptoms observed in the DBA/2 -> BDF1 cGVHD model such as splenomegaly and glomerulonephritis are not a component of human cGVHD. Also, most human transplantation occurs between MHC-matched individuals using some form of recipient conditioning. In this respect, our data presented herein should be carefully interpreted.

Although it has been well documented that GVHD and a GVL activity are closely linked, several strategies are being developed to separate GVHD from a GVL activity (54). These strategies are mainly focused on decreasing the toxicity of GVHD while retaining the GVL activity. First, it is possible to block activated donor effector T cells to exit lymphoid tissues and traffic to mucosal sites and parenchymal target organs (55). Recent studies suggest that these approaches save the GVL activity of donor T cells while suppressing their GVHD activity (56). Recruitment of activated T cells to host target tissues or sites of leukemic infiltration could be likely mediated by different combinations of adhesion molecules and chemokine receptors on leukocytes and the ligands for these receptors on host cells. In this context, it is desirable to reduce the intensity of conditioning or to neutralize inflammatory cytokines, since local inflammation in target organs followed by conditioning is critical for the recruitment of alloreactive T cells to target organs. Second, selective removal of alloreactive donor T cells is an effective way to suppress GVHD. Although most CD8+ T cells expanding during GVHD are directed to a few immunodominant minor histocompatibility Ags (57), it seems that polyclonal donor T cells are required for full GVHD manifestations (58). However, it is plausible that those alloreactive CD8+ T cells are not sufficient but are necessary to elicit GVHD, making this approach applicable to clinical situations. Our laboratory has been seeking an in vivo method to delete alloreactive T cells but to enhance antitumor activity. CD137 is a target molecule suitable for this approach. In vivo ligation of CD37 has been shown to be highly effective in removing alloreactive T cells by inducing AICD (26, 27), while eradicating a variety of established tumor cells (59, 60). Our preliminary data demonstrate that these two seemingly contradictory functions of CD137 originated in its tendency to overactivate those T cells reactive to strong Ags such as alloantigens, followed by AICD, and to activate or break tolerance of T cells reactive to weak Ags such as tumor Ags (J.K. and B.K., unpublished data). Third, recent work by Negrin’s group has demonstrated that CD4+CD25+ regulatory T cells inhibit GVHD without impairing GVL activity (61). The cellular basis of this differential control by regulatory T cells has not been known. One possibility is that effector T cells mediating GVHD and those mediating a GVL effect may be different in sensitivity to regulatory T cell-dependent control (62). Taken together, it seems that there exist different temporal and spatial microenvironments for the development of GVHD and a GVL activity that can be changed by immunoregulatory interventions.

Our results suggest that in vivo engagement of costimulatory molecules may be used as a GVHD prophylaxis in the parent-into-unirradiated F1 GVHD setting. Since it is possible to predict the behavior of donor CD8+ T cells after cell transfer into the host in an in vitro MLR (5), anti-CD40 mAb may be proved to be effective in inhibiting GVHD without impairing a GVL activity and donor cell engraftment in a combination of the recipient and donor where donor CD8+ T cells have a tendency to become anergic. On the other hand, anti-CD137 mAb has been shown to effectively delete alloreactive donor T cells in the parent-into-unirradiated F1 GVHD model (26). In recipient mice that receive a low dose of irradiation as preconditioning in this clinical setting, anti-CD137 mAbs do not induce GVHD with preserving donor cell engraftment and a GVL effect (J.K. and B.L., unpublished data), further supporting anti-CD137 mAb as a candidate for a GVHD prophylaxis. In conclusion, this and other studies by our laboratory demonstrate that agonistic mAbs against costimulatory molecules are useful in addressing issues regarding how donor T cells can differentially regulate GVHD and a GVL activity. Furthermore, costimulation would be considered to be a GVHD prophylaxis particularly in the parent-into-F1 hematopoietic transplantation.


   Acknowledgments
 
We express our gratitude to the Immunomodulation Research Center, University of Ulsan, for allowing us to use its facilities, as well as members of our laboratory for their help.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Korea Research Foundation (C00088, E00010, and J00302) and the Korea Health 21 Research and Development project of the Korean Ministry of Health and Welfare (A040004). Back

2 Address correspondence and reprint requests to Dr. Byungsuk Kwon and Dr. Hong R. Cho, Department of Biological Science, University of Ulsan, San 29 Mukeo-dong Nam-ku, Ulsan, Ulsan 680-749, Republic of Korea. E-mail addresses: bkwon{at}mail.ulsan.ac.kr and hrcho{at}mail.ulsan.ac.kr Back

3 Abbreviations used in this paper: GVHD, graft-vs-host disease; BDF1, (C57BL/6 x DBA/2)F1; cGVHD, chronic GVHD; aGVHD, acute GVHD; DC, dendritic cell; AICD, activation-induced cell death; GITR, glucocorticoid-induced TNF receptor-related gene; GVL, graft-vs-leukemia. Back

Received for publication December 21, 2007. Accepted for publication September 3, 2008.


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 Introduction
 Materials and Methods
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